■■■■I v'1' '' PRINCIPLES OF SOIL MICROBIOLOGY PRINCIPLES OF SOIL MICROBIOLOGY BY LT) ru _D m r-=1 ru □ o m o □ SELMAN A. WAKSMAN Associate Professor of Soil Microbiology, Rutgers University, and Microbiologist of the New Jersey Agricultural Experiment Stations LONDON BAILLIERE, TINDALL AND COX 8 HENRIETTA STREET, COVENT GARDEN, W. C. 2 1927 ALL RIGHTS RESERVED, 1927 PRINTED IN AMERICA COMPOSED AND PRINTED AT THE WAVERLY PRESS BALTIMORE, MD., U. 8. A. In the years of their seventy-fifth and seventieth anniversaries respectively, this book is dedicated to Professors M. W. BEIJERINCK and S. WINOGRADSKY the investigators who have thrown the first UgJd upon some of the most important soil proc- esses and whose contributions can well be considered first and fore- most in the science of Soil Microbiology PREFACE Although the biochemical processes in the soil as well as the nature of the microorganisms present there have received considerable atten- tion from various points of view and although an extensive literature has accumulated, not only dealing with soil processes in general but even with certain specific activities of the organisms, our present knowledge of the soil microflora and microfauna and of the numerous transforma- tions that they bring about has not advanced beyond a mere beginning of a systematic study. The isolation of numerous microorganisms from the soil, their identification and cultivation upon artificial media is very important but such data do not tell what role they play in the soil. A knowledge of the activities of certain organisms isolated from the soil is certainly necessary, but that is not a knowledge of the extent to which these processes take place in the soil itself. A book on soil microbiology should include a study of the occurrence of microorganisms in the soil, their activities and their role in soil processes. It is this last phase which has been studied least and where the information available is far from satisfactory in explaining what is taking place in the soil. This is due largely to the limitations of the subject which depends for its advance on botany, zoology, bacteriology, chemistry, including biological and physical, and especially upon the advance of our understanding of the physical and chemical conditions of the soil. There are various kinds of audiences to which a book on soil micro- biology may appeal. There is the scientific farmer who may search for a better understanding of the processes taking place in the soil, those processes which control the growth of his crops and indirectly influence the growth of his animals. There is the agronomist, who is interested in the fundamental reactions controlling soil fertility, by reason of the need of directing such processes towards a greater utilization of the nutrients added to the soil or stored away in the soil organic matter. There is the investigator, the soil chemist or the soil microbiologist, who, in attacking problems dealing with the occurrence of microorganisms in the soil, their activities, and especially with the relation of these activities to the physical and chemical soil conditions, seeks for specific or general information. These investigators may deal with organisms Vlll PREFACE or processes which could be better understood when correlated with the other soil organisms and the numerous other processes. An attempt has been made to compile a book which will be of service not only to the investigators in soil science, but also to workers in allied sciences, especially botany, plant physiology, plant pathology and bacteriology, as well as to the general student in agriculture. This book is a collection of known facts concerning microorganisms found in the soil and their activities; it is a study of the literature dealing with the science in question; it is an interpretation of the facts already presented; it indicates the various lines of investigation and notes where further information is especially wanted. Soil microbiology is a science which is at the very base of our understanding of agricultural processes and the practice of agriculture; it comprises a number of sciences. The book may, therefore, be looked upon more as an introduction to further research rather than as an ordinary text-book; as of help to those work- ing in the allied sciences, who are desirous of obtaining some information concerning the soil population and its activities. If this volume will help to disclose to the reader some of the numerous interrelated processes in the soil, if it will present in a clearer light to the chemist, the physiologist, the botanist, the bacteriologist and the zoologist the nature of the many scientific and practical problems awaiting the investigator, if it contributes in a small measure toward making soil science an exact science, the author will feel that he has been amply rewarded. The author is greatly indebted to his various colleagues for reading and criticizing the different chapters of the book and for the many helpful suggestions generously offered, especially to Dr. H. J. Conn, of the New York Agricultural Experiment Station, for reading Chapters I and VI; to Dr. B. M. Bristol Roach, of the Rothamsted Experimental Station, and Dr. G. T. Moore, of the Missouri Botanical Garden, for reading the Chapter on Algae; to Dr. Ch. Thorn of the Bureau of Chemistry, for reading the Chapter on Fungi; to Dr. M. C. Rayner, of Bedford College, London, for reading the section dealing with Mycor- rhiza Fungi; to Dr. A. T. Henrici, of the University of Minnesota, for reading the Chapter on Actinomyces; to Dr. W. M. Gibbs, of the Idaho Agricultural Experiment Station, for reading the Chapter on Nitrifying Bacteria; to Dr. A. L. Whiting, of the University of Wis- consin and to Dr. L. T. Leonard of the Bureau of Plant Industry, for reading the Chapter on Nodule Bacteria; to Dr. R. Burri, of Liebefeld, Switzerland, and to Dr. I. C. Hall, of the Colorado Medical School, PREFACE IX for reading the Chapter on Anaerobic Bacteria; to Mr. D. W. Cutler, Mr. H. Sandon, of the Rothamsted Experimental Station, and Prof. C. A. Kofoid, of the University of California, for reading the Chapter on Protozoa; to Dr. N. Cobb and Dr. Steiner, of the Bureau of Plant Industry, for reading the Chapter on Soil Invertebrates; to Dr. O. Meyerhof, of the K. Wilhelm Institute, Berlin, for reading the Chapter on Energy Transformation; to Dr. T. B. Osborne, of the Connecticut Agricultural Experiment Station, for reading the Chapter on Protein Transformation; to Mr. A. Bonazzi, of Cuba, for reading the Chapter on Non-symbiotic Nitrogen Fixation; to Dr. R. L. Jones, of the University of Wisconsin, for reading Chapter XXX; to Dr. E. B. Fred, of the Uni- versity of Wisconsin, for reading the Chapter on Nitrate-reducing Bacteria; to Prof. D. R. Hoagland and Dr. W. P. Kelley, of the Uni- versity of California for reading Chapters 24 and 25 respectively; to the members of the Soil Microbiology Division of the New Jersey Ag- ricultural Experiment Station, especially to Dr. J. G. Lipman and Dr. R. L. Starkey, for reading various parts of the book, and to all those who have generously allowed the use and reproduction of the various illustrations in the text. Selman A. Waksman. August 25, 1826. New Brunswick., N. J., U. S. A. A CLASSIFIED LIST OF BOOKS FOR REFERENCE IN SOIL MICROBIOLOGY CLASSIFICATION OF ORGANISMS Bacteria Bergey, D. H. A manual of determinative bacteriology. Williams & Wilkins Co., Baltimore. 1923. Buchanan, R. E. General systematic bacteriology. History, nomenclature, groups of bacteria. (Monogr. on systematic bacteriology, vol. 1.) Williams & Wilkins Co., Baltimore. 1925. Chester, F. D. A manual of determinative bacteriology. Macmillan Co., New York and London. 1901. FlUgge, C. Die Mikroorganismen. 2 vols., 3rd Ed. Leipzig. 1896. Lehmann, K. B., and Neumann, R. O. Atlas und Grundrisz der Bakteriologie. 6th Ed. Teil. I, Atlas. Teil II, Text. J. F. Lehmann. Miinchen. • 1920. Matzuschita, T. Bakteriologische Diagnostik. Jena. 1902. Migula, W. System der Bakterien. Jena. Bd. I, 1897; Bd. II. 1900. Winslow, C. E. A., and Winslow, A. R. The systematic relationships of the Coccaceae. J. Wiley & Sons, New York. 1908. Fungi Brefeld, O. Botanische Untersuchungen iiber Schimmelpilze. 1872. Clements, F. E. The genera of fungi. Minneapolis. 1909. Coupin, H. Fungi (champignons). Album Gen. Cryptogames. 1921. DeBary, A. Comparative morphology and biology of the fungi, mycetozoa and bacteria. (Tr. Gainey, H. E. F. and Balfour, I. B.) Clarendon Press, Oxford. 1887. Engler, A., and Prantl, K. A. Die natiirlichen Pflanzenfamilien. Teil I, Abt. I. Engelmann, Leipzig. 1900. Fischer, E. Pilze. Handwort. Naturwiss. V. 7. Jena. 1912. Gaumann, E. Vergleichende Morphologie der Pilze. G. Fischer, Jena. 1926. Lindau, G. Fungi imperfecti. Hyphomycetes. Rabenhorst's Kryptogamen Flora. Vols. 8 and 9. 1907-1910. Lindau, G. K. Kryptogamenflora fur Anfanger. 2 (1) Die mikroskopischen Pilze (Myxomyceten, Phycomyceten und Ascomyceten). 2nd. Ed. J. Springer, Berlin. 1922. Saccardo, P. A. Sylloge Fungorum. Patavia. 1882-1913. Wettstein, R. Handbuchder systematischen Botanik. 3d Ed. Vol.1. Wien. 1923. Zopf, W. Die Pilze in morphologischer, physiologischer, biologischer und systematischer Beziehung. Breslau. 1890. Xll CLASSIFIED LIST OF BOOKS Algae Chodat, R. Monographie d'Algues en culture pure. Bern. 1913. Cotjpin, H. Les algues du globe. V. 1. Paris. 1912. Engler, A., and Prantl, K. A. Die naturlichen Pflanzenfamilien. Teil I, Abt. la. Leipzig. 1897. Heurck, H. J. Traite des Diatom6es. Anvers. 1899. Lindau, G. Kryptogamenflora fur Anfanger. Bd. IV, 1, 2 and 3. Die Algen. J. Springer, Berlin. 1914-1916. Oltmanns, Fr. Morphologie und Biologie der Algen. 3 vols. 2nd Ed. G. Fischer, Jena. 1922. Pascher, A. Die Susswasserflora Deutschlands, Osterreichs und der Schweiz. Jena. H. 4 to H. 12. G. Fischer. 1915-1925. Tilden, J. E. Minnesota algae. Minneapolis. 1910. de Toni, G. B. Sylloge algarum omnium hucus que cognitarum. 1889-1907. Padua. West, G. S. Algae. Cambridge Botanical Handbooks. I. Cambr. Univ. Press. 1916. Yeasts Chapman, A. C, and Baker, F. G. C. An atlas of the saccharomycetes. Lon- don. 1906. Jorgensen, A. Die Mikroorganismen der Giirungsgewerbe. 5th Ed. Guillermond, A. The yeasts. (Trans. F. W. Tanner.) J. Wiley & Sons, New York. 1920. Henneberg, W. Garungsphysiologisches Praktikum. Berlin. 1909. Klocker, A. Die Giirungsorganismen in der Theorie und Praxis der Alkohol- garungsgewerbe. Max Waag. 2nd Ed. Stuttgart. 1906. Kohl, F. G. Die Hefepilze. Quelle und Meyer. Leipzig. 1908. Lindner, A. Saccharomycetineae. In Kryptogamenflora der Mark Branden- burg. Bd. 7, H. 1. Leipzig. 1905. Protozoa Butschli, O. Protozoa. In Bronn's Thierreich. 1882-1887. Calkins, G. N. The biology of the protozoa. Lea & Febiger, Philadelphia. 1926. Cash, J. The British freshwater Rhizopods and Heliozoa. Roy. Soc. London. 1905-1921. Doflein, F. Lehrbuch der Protozoenkunde. 4th Ed. Jena. G. Fischer. 1916. Lister, A. A monograph of the mycetozoa. 3rd Ed. Rev. G. Lister, Brit. Museum, London. 1925. MacBride, T. H. North American slime molds. 2nd Ed. Macmillan. 1922. Minchin, E. A. An introduction to the study of the protozoa. E. Arnold, London. 1912. Pascher, A., and Lemmermann, E. Die Susswasserflora Deutschlands, Oster- reichs und der Schweiz. H. 1 to H. 3. G. Fischer, Jena. 1913. Wenyon, C. M. Protozoology. 2 vols. Bailliere, Tindall and Cox. London. 1926. CLASSIFIED LIST OF BOOKS Xlll Schaeffer, A. A. Taxonomy of the Amebas with description of thirty-nine new marine and fresh-water species. Vol. 24, Carnegie Inst. Wash., Dept. Marine Biol. 1926. Nematodes Baylis, H. A., and Daubney, R. A synopsis of the families and genera of Nematoda. Brit. Museum, London. 1926. De Man, J. G. Nouvelles recherches sur les nematodes libres terricoles. M. Nijhoff, Hague. 1922. Micoletzky, H. Die freilebenden Erd-Nematoden. Arch. Naturges. 87 (Abt. A). 1922. Yorke, W., and Maplestone, P. A. The nematode parasites of vertebrates. P. Blakiston's Son & Co., Philadelphia. 1926. Ward, H., and Whipple, G. C. Fresh Water Biology. J. Wiley & Sons, New York. 1918. THEORETICAL AND APPLIED MICROBIOLOGY General Microbiology Baumgartel, T. Grundriss der theoretischen Bakteriologie. J. Springer, Berlin. 1924. Benecke, W. Bau und Leben der Bakterien. Teubner, Leipzig. 1912. Conn, H. W., and Conn, H. J. Bacteriology. Williams & Wilkins Co., Balti- more. 1923. Ellis, D. Practical bacteriology. London. 1923. Fischer, A. Vorlesungen iiber Bakterien. Jena. 1903. Hiss, P. H., and Zinsser, H. A text book of bacteriology. 5th Ed. Appleton & Co., New York. 1922. Jordan, E. O. A text-book of general bacteriology. 7th Ed. Philadelphia, 1922. Kendall, A. J. Bacteriology, general, pathological and intestinal. 2d Ed. Philadelphia. 1921. Kolle, W., and Wassermann, A. Handbuch der pathogenen Mikroorganismen. 2d Ed. Jena. 1913. Kruse, W. Allgemeine Mikrobiologie. Vogel, Leipzig. 1910. Kruse, W. Einfiihrung in die Bakteriologie. W. de Gruyter, Berlin. 1920. Meyer, A. Die Zelle der Bakterien. Jena. 1912. Omeliansky, W. L. Principles of microbiology (Russian). U. S. S. R. Leningrad. 5th Ed. 1924. Park, W. H., Williams, A. W. and Krumwiede, C. Pathogenic microorgan- isms. Eighth Ed. Lea & Febiger, Philadelphia. 1924. Agricultural Microbiology Baumgartel, T. Vorlesungen iiber landwirtschaftliche Mikrobiologie. P. Parey, Berlin. 1925-1926. Buchanan, R. E. Agricultural and industrial bacteriology. New York. 1922. Chudiakov, H. H. Agricultural Microbiology (Russian). Moskau. 1926. XIV CLASSIFIED LIST OF BOOKS Dcclaux, E. Traits de Microbiologic. Masson et Cie, Paris. Vols. I-IV. 1898-1901. Fuhrmann, F. Vorlesungen Uber technische Mykologie. G. Fischer, Jena. 1913. Gkeaves, J. E. Agricultural bacteriology. Lea and Febiger, Philadelphia. 1922. Janke, A. Allgemeine technische Mikrobiologie. I. Steinkopff. Dresden and Leipzig. 1924. Kayser, E. Microbiologic appliquee a la fertilisation du sol. J. B. Bailliere, Paris. 1921. Kossowicz, A. Einfiihrung in die Agrikulturmykologie. Teil I, Bodenbak- teriologie. Borntraeger, Berlin. 1912. Lafar, F. Handbuch der technischen Mykologie. 5 Vols. G. Fischer, Jena. 1904-1913. Lipman, J. G. Bacteria in relation to country life. The Macmillan Co., New York. 1911. Lohnis, F. Handbuch der landwirtschaftlichen Bakteriologie. Borntraeger, Berlin. 1910. Lohnis, F., and Fred, E. B. Agricultural bacteriology. McGraw-Hill, New York. 1923. Russell, Sir John, and others. The microorganisms of the soil. Long- mans, Green & Co., London. 1923. Marshall, C. E. Microbiology. Blakiston, Philadelphia. 3rd Ed. 1922. Russell, H. L., and Hastings, E. G. Agricultural bacteriology. 1915. Rossi, G. de. Microbiologia agraria e technica. Unione Tip, Torino. 1921— 1926. Smith, E. F. Bacteria in relation to plant diseases. Vol. I, 1905; vol. II, 1911; Vol. Ill, 1914. Stoklasa, J., and Doerell, E. G. Handbuch der physikalischen und biochem- ischen Durchforschung des Bodens. P. Parey, Berlin. 1926. Tanner, F. W. Bacteriology and mycology of foods. New York. 1919. Manuals of Bacteriologic Technic Abderhalden, E. Handbuch der biologischen Arbeitsmethoden. Abt. XI. 2nd Ed. 1924-1926. Abel, R. Bakteriologisches Taschenbuch. C. Kabitzsch, Leipzig. 26th Ed. 1923. American Public Health Association standard methods for the examination of water and sewage. 1915. Barnard, J. E., and Welch, F. V. Practical photo-micrography. 2nd Ed. Longmans, Green & Co., New York. 1925. Besson, A. Technique microbiologique et s6rothe>apique. 3 vol. 1921-1923. Burgess, P. S. Soil bacteriology laboratory manual. 1914. Emich, F. Lehbach der Mikrochemie, Munich. 1926. Ehringhaus, A. Das Mikroskop, seine wissenschaftlichen Grundlagen und seine Anwendung. Teubner, Leipzig. Ehrlich and Weigert. Encyclopedic der mikroscopischen Technik. Vol. I & II. 1910. CLASSIFIED LIST OF BOOKS XV Eyre, J. W. H. Bacteriological technique. 2nd Ed. 1913. Fred, E. B. A laboratory manual of soil bacteriology. Blakiston Co., Phila- delphia. 1916. Gage, S. H. The microscope. 14th Ed. Comstock, Ithaca, N. Y. 1925. Giltner, W. Laboratory manual in general microbiology. 3rd Ed. J. Wiley and Sons, New York. 1926. Hager, H. Das Mikroskop und seine Anwendung. 13th Ed. by F. Tobler. J. Springer, Berlin. 1925. Heinemann, P. G. A laboratory guide in bacteriology. 3rd Ed. 1915. Hewlett, R. T. A manual of bacteriology. London. 1921. Koch, A. Mikrobiologisches Praktikum. J. Springer, Berlin. 1922. Kraus, R., and Uhlenhuth, P. Handbuch der mikrobiologischen Technik. 3 vols. Urban and Schwarzenberg, Berlin. 1923-1924. Kuster, E. Kultur der Mikroorganismen. 3rd Ed. Teubner, Leipzig. 1921. Langeron, M. Precis de microscopie. 4th Ed. Masson et Cie, Paris. 1925. Laubenheimer. Lehrbuch der Mikrophotographie. 1920. Lee, A. B. The microtomist's Vade-Mecum. 8th Ed. Churchill, London. 1921. Lipman, J. G., and Brown, P. E. Laboratory guide in soil bacteriology. 1911. Lohnis, F. Landwirtschaftlich-bakteriologisches Praktikum. 2nd Ed. Born- traeger, Berlin. 1920. Mace, E. Traite pratique de bact6riologie. Atlas de Microbiologic Bailliere. Paris. 1913. Meyer, A. Praktikum der botanischen Bakterienkunde. G. Fischer, Jena. 1903. Prowazek, S. V. (V. Jollos). Taschenbuch der mikroskopischen Technik der Protistenuntersuchung. 3rd Ed. J. A. Barth, Leipzig. 1922. Sieben, H. Einfuhrung in die botanische Mikrotechnik. Fischer, Jena. 1913. Schneider, A. Bacteriological methods in food and drug laboratories. Blakis- ton, Philadelphia. 1915. SOILS AND PLANTS The Physics and Chemistry of Soils and Manures Airman, C. M. Manures and principles of manuring. London. 1910. Cameron, F. K. The soil solution, the nutrient medium for plant growth. Easton, Pa. 1911. Clarke, F. W. The data on geochemistry. Bui. 770, U. S. Geol. Survey. 1924. Ehrenberg, P. Die Bodenkolloide. 3rd Ed. Steinkopff, Dresden and Leipzig. 1920. Emerson, F. V. Agricultural geology. J. Wiley & Sons, New York. 1920. Fraps, G. S. Principles of agricultural chemistry. The Chemical Publishing Co., Easton, Pa. 1913. Glinka, K. D. Soils of Russia and adjoining countries (Russian). Gosizdat, Moskau. 1923. Hall, A. D. The soil, an introduction to the scientific study of the growth of crops. 3rd Ed. London. 1920. Hinkle, S. F. Fertility and crop production. Sandusky, Ohio. 1925. XVI CLASSIFIED LIST OF BOOKS Lyon, T. L., and Buckman, H. C. The nature and properties of soils. Mac- millan, New York. 1922. Heiden, E. Lehrbuch der Dungerlehre. 2 parts. Hannover. 1879-1887. Hilgard, E. W. Soils, their formation, properties, composition and relations to climate and plant growth. Macmillan, New York. 1912. Hoering, P. Moornutzung und Torfverwertung mit besonderer Berucksichti- gung der Trockendestillation. J. Springer, Berlin. 1915. Honcamp, F., and Nolte, O. Agrikulturchemie. T. Steinkopff, Dresden and Leipzig. 1924. Hopkins, C. G. Soil fertility and permanent agriculture. Ginn & Co., Boston. 1910. Kober, L. Der Bau der Erde. Borntraeger, Berlin. 1921. Mayer, A. Die Dungerlehre. 7th Ed. C. Winters, Heidelberg. 1924. Merrill, G. P. Rocks, rock weathering and soils. Macmillan, New York. 1897. Mitscherlich, E. A. Bodenkunde fur Land- und Forstwirte. 3rd Ed. P. Parey, Berlin. 1920. Murray, J. A. The science of soils and manures. 3rd Ed. D. VanNostrand Co. 1925. Puchner, H. Der Torf. F. Enke, Stuttgart. 1920. Ramann, E. Bodenkunde. 3rd Ed. J. Springer, Berlin. 1911. Russell, J. Soil conditions and plant growth. Longmans, Green & Co. 4th Ed. London. 1921. Van Slyke, L. L. Fertilizers and crops. O. Jodd Co., New York. 1912. Warington, R. Lectures on some of the physical properties of soil. Oxford. 1900. Wheeler, H. J. Manures and fertilizers. New York. 1913. Wiley, H. W. Principles and practice of agricultural analysis. Vol. I. Soils. 3rd Ed. Chemical Publ. Co., Easton, Pa. 1926. The Soil Environment and Higher Plants Brenchley, W. E. Inorganic plant poisons and stimulants. Cambridge. 1914. Clements, F. E. Aeration and air content; the role of oxygen in root activity. Carnegie Inst. Wash. Publ. No. 315. 1921. Czapek, F. Biochemie der Pflanzen. 2te Aufl. 3 vols. Jena, vol. 1, 1913; vol.2, 1920; vol. 3, 1921. Hahn, J. Handbuch der Klimatologie. 3 vols. Stuttgart. 1908-1911. Jost, L. Plant physiology. Tr. R. J. H. Gibson. Oxford. 1907-1913. Kolkwitz, R. Pflanzenphysiologie. G. Fischer, Jena. 1922. Kostytschew, S. Pflanzenatmung. J. Springer, Berlin. 1925. Lundegardh, H. Klima und Boden in ihrer Wirkung auf das Pflanzenleben. G. Fischer, Jena. 1925. Palladin, V. I. Plant physiology. Trans, by B. E. Livingston. 2nd Ed. Blakiston, Philadelphia. 1923. Pfeffer, W. The physiology of plants, a treatise upon the metabolism and sources of energy in plants. Tr. A. J. Ewart. 3 vols. Oxford. 1900- 1906. CLASSIFIED LIST OF BOOKS XV11 TREATISES IN GENERAL SCIENCES General Biology, Physiology and Physiological Chemistry Abderhalden, E. Handbuch der biologischen Arbeitsmethoden. 2nd Ed. Urban & Schwarzenberg, Berlin. 1920-1926. Abderhalden, E. Biochemiscb.es Handlexikon. 11 vols. Berlin. 1911-1924. Bayliss, W. M. Principles of general physiology. 4th Ed. London. 1924. Bayliss, W. M. The nature of enzyme action. London. 1925. Cohnheim, O. Enzymes. 1912. Effront, J. Enzymes and their applications. Trans. S. C. Prescott, New York. 1902. Biochemical catalysts in life and industry. New York. 1917. Euler, H. Chemie der Enzyme. 2nd Ed. Bergmann, Munchen. 2 vols. 1922-1924. Etjler, H. Grundlagen und Ergebnisse der Pflanzenchemie, nach der schwedis- chen Ausgabe bearbeitet. I Teil, Das chemische Material der Pfian- zen. Braunschweig. 1908. II Teil, Die allgemeinen Gesetze des Pflanzenlebens. Ill Teil, Die chemischen Vorgange im Pflanzen- korper. Braunschweig. 1909. Fowler, G. J. Bacteriological and enzyme chemistry. Longmans, Green & Co., New York. 1911. Haas, P., and Hill, T. G. An introduction to the chemistry of plant products. 3rd Ed. London. 1921. Hartmann, M. Allgemeine Biologie. Jena. 1925. Henry, T. A. The plant alkaloids. Philadelphia. 1913. Hober, R. Physikalische Chemie der Zelle und der Gewebe. 5th Ed. Leipzig and Berlin. 1924. Loeb, J. The dynamics of living matter. New York. 1906. Loeb, J. The mechanistic conception of life: biological essays. Chicago. 1912. Loeb, J. The organism as a whole, from the physicochemical viewpoint. New York and London. 1916. Loeb, J. Proteins and the theory of colloidal behavior. 2nd Ed. McGraw-Hill, New York. 1924. Mathews, A. P. Physiological chemistry. 3rd Ed. New York. 1920. Oppenheimer, C. Handbuch der Biochemie der Menschen und der Tiere. 2nd Ed., 5 vols. G. Fischer, Jena. 1924-1926. Oppenheimer, C. Die Fermente und ihre Wirkungen. 5th Ed., 2 vols. G. Thieme, Leipzig. 1925-1926. Robertson, T. B. Principles of biochemistry. Lea & Febiger, Philadelphia. 1920. Robertson, T. B. Physical chemistry of the proteins. Longmans, Green & Co., New York and London. 1918. Schorger, A. W. Chemistry of cellulose and wood. McGraw-Hill, New York. 1926. Thatcher, R. W. The chemistry of plant life. McGraw-Hill, New York. 1924. Verworn, M. Allgemeine Physiologie, ein Grundriss der Lehre vom Leben. 6th Ed. Jena. 1915. XV111 CLASSIFIED LIST OF BOOKS Wiesner, J. V. Die Rohstoffe des Pflanzenreiches. 3rd Ed., 3 vols., Engel- mann, Leipzig. 1921. Wohlgemuth, J. Grundrisz der Fermentmethoden. Berlin. 1913. Physics and Chemistry, as applied to Biology Bechhold, H. Colloids in Biology and Medicine. New York. 1919. (Tr. J. G. M. Bullowa.) Clark, W.M. The determination of hydrogenions. 2ndEd., Williams & Wilkins Co., Baltimore. 1922. Cohen, E. Physical chemistry for physicians and biologists. (Tr. M. Fischer.) New York. 1903. Eichwald, E., and Foder, A. Die physikalisch-chemischen Grundlagen der Biologic Berlin. 1919. Findlay, A. Osmotic pressure. 2nd Ed. London. 1919. Freundlich, H. Kapillarchemie, eine Darstellung der Chemie der Kolloide und verwandter Gebiete. Leipzig. 1923. Hatschek, E. An introduction to the physics and chemistry of colloids. 4th Ed., London and Philadelphia. 1922. Hedin, S. G. Grundziige der physikalischen Chemie in ihrer Beziehung zur Biologic J. F. Bergmann, Munchen. 1924. Jellinek, K. Lehrbuch der physikalischen Chemie. 2 vols. Stuttgart. 1914— 1915. Kolthoff, I. M., and Furman, N. H. Indicators. J. Wiley & Sons, New York. 1926. Lewis, G. N., and Randall, M. Thermodynamics and the free energy of chem- ical substances. McGraw-Hill, New York. 1923. Lewis, W. C. McC. A system of physical chemistry. 3 vols., 3d and 4th Ed. Longmans, Green & Co., London and New York. 1920-1925. Lotka, A. J. Elements of physical biology. Williams & Wilkins Co., Baltimore, Md. 1926. McClendon, J. F., and Medes, G. Physical chemistry in medicine. W. B. Saunders Co., Philadelphia. 425 p. 1925. Nernst, W. Theoretische Chemie vom Standpunkte der Avogadroschen Regel und der Thermodynamik. 10th Ed., Enge. Stuttgart. 1921. Philip, J. C. Physical chemistry: its bearing on biology and medicine. Long- mans, Green & Co., New York and London. 3d Ed. 1925. Taylor, W. W. The chemistry of colloids and some technical applications. 3d Ed. London. 1921. Waksman, S. A., and Davison, W. C. Enzymes. Williams & Wilkins Co., Baltimore. 1926. Washburn, E. W. An introduction to the principles of physical chemistry from the standpoint of modern atomistics and thermodynamics. 2d Ed. New York. 1921. Willows, R. S., and Hatchek, E. Surface tension and surface energy and their influence on chemical phenomena. 3d Ed. London. 1923. Zsigmondy, R., Spear, E. B., and Norton, J. F. The chemistry of colloids. New York. 1917. CLASSIFIED LIST OF BOOKS XIX Mathematics Davenport, C. B. Statistical methods, with special reference to biological variation. 3d Ed. New York. 1914. Fischer, R. A. Statistical methods for research workers. Oliver and Boyd, Edinburgh. 1925. Mellor, W. J. Higher mathematics for students of chemistry and physics, with special reference to practical work. 4th Ed. London. 1913. Nernst, W., and Schoenflies, A. Einfiihrung in die mathematische Behand- lung der Naturwissenschaften. Berlin. 1919. CONTENTS PART A. OCCURRENCE AND DIFFERENTIATION OF MICRO- ORGANISMS IN THE SOIL Chapter I NUMBERS OF DIFFERENT GROUPS OF MICROORGANISMS FOUND IN THE SOIL AND METHOD OF DETERMINATION The occurrence of microorganisms in the soil. Proof of microbial activities in the soil. Methods of study. Direct microscopic method. Organisms found in the soil by the direct microscopic method. Cultural methods for demonstrating the kinds of organisms active in the soil. Cultural methods for the determination of numbers of microorganisms in the soil. Culture media. Sampling of soil. Treatment of soil samples and preparation of plates. Incubation of plates and counting of organisms. Mathematical interpretation of results. Comparison of plate and microscopic methods. Numbers of bacteria in the soil. Bacterial numbers in manure. Numbers of bacteria in the soil during different seasons of the year. Distribution of bacteria at various depths. Numbers of specific physiological groups of bacteria. Numbers of actinomyces in the soil. Numbers of fungi in the soil. Methods of counting protozoa. Numbers of protozoa in the soil. .. . 3 PART B. ISOLATION, IDENTIFICATION, AND CULTIVATION OF SOIL MICROORGANISMS Chapter II PURE CULTURE STUDY AND CLASSIFICATION OF SOIL BACTERIA Pure culture study. Differentiating characters of bacteria. Life cycles of bacteria. Classification of soil bacteria based upon their physiological activities 53 Chapter III AUTOTROPHIC BACTERIA The nature of autotrophic bacteria. Bacteria deriving their energy from nitrogen compounds. Solid media for the isolation and cultivation of the nitrite forming organisms. Morphology of the nitrite forming organisms. Nitrate forming organisms (Nitrobacter). Occurrence of nitrifying bac- teria in the soil. Bacteria deriving their energy from the oxidation of sulfur and its compounds. Classification of sulfur bacteria. Oxidation of selenium and its compounds. Bacteria oxidizing iron compounds. Bac- 3038 XX11 CONTENTS teria obtaining their energy from the oxidation of simple carbon com- pounds. Methane bacteria. Bacteria oxidizing carbon monoxide. Bac- teria oxidizing hydrogen 61 Chapter IV BACTERIA FIXING ATMOSPHERIC NITROGEN Nitrogen fixation in nature. Classification of nitrogen-fixing bacteria. Iso- lation of anaerobic bacteria. Morphology of the anaerobic bacteria. Distribution of anaerobic nitrogen-fixing bacteria in the soil. Physiology of anaerobic nitrogen-fixing bacteria. Non-symbiotic nitrogen fixing aerobic bacteria. Description of species of Azotobacter. Morphology and life cycle of Azotobacter. Physiology of Azotobacter. Other non- symbiotic nitrogen-fixing bacteria. Symbiotic nitrogen fixation by nodule bacteria. Historical. Nomenclature. Media. Nodule formation. Iso- lation of organism from nodules. Isolation from soil. Colony appearance. Morphology and life cycle of organism. Motility. Physiology of nodule bacteria. Specific differentiation. Nodule formation by non-leguminous plants. Nodule formation in the leaves of some plants 103 Chapter V HETEROTROPHIC, AEROBIC BACTERIA REQUIRING COMBINED NITROGEN General classification. Spore-forming bacteria. Classification of spore- forming bacteria. Occurrence of spore-forming bacteria in the soil. Non- spore-forming bacteria. Classification. Occurrence of non-spore-forming bacteria in the soil. Thermophilic bacteria. Mycobacteria. Myxobacteria 141 Chapter VI ANAEROBIC BACTERIA Oxygen tension in the growth of bacteria. Methods of isolation of anaerobic bacteria from the soil. Cultivation of anaerobes. Classification of soil anaerobes. Physiological activities of anaerobic bacteria. Soil processes in which anaerobic bacteria take an active part 160 Chapter VII BACTERIA REDUCING NITRATES AND SULFATES General classification of nitrate reducing bacteria. Organisms reducing nitrates to nitrites. Organisms reducing nitrates to ammonia. Bacteria reducing nitrates to atmospheric nitrogen. Description of some typical denitrifying bacteria. Bacteria reducing sulfates to hydrogen sulfide. . . . 180 Chapter VIII BACTERIA CAPABLE OF DECOMPOSING CELLULOSES AND OTHER COMPLEX CARBOHYDRATES AND HYDROCARBONS IN THE SOIL Microorganisms concerned in the decomposition of celluloses in nature. Anaerobic bacteria. Aerobic bacteria. Decomposition of cellulose by CONTENTS XX1U denitrifying bacteria. Thermophilic bacteria. Pectin decomposing bac- teria. Bacteria decomposing hydrocarbons and benzene ring compounds. . 190 Chapter IX BACTERIA DECOMPOSING UREA, URIC, AND HIPPURIC ACIDS Organisms decomposing urea. Methods of isolation. Occurrence of urea bacteria. Classification and description. Bacteria decomposing calcium cyanamide. Uric and hippuric acid bacteria 206 Chapter X SOIL ALGAE Introductory. Methods of isolation of impure cultures of algae. Isolation of pure cultures. Cultivation of soil algae. Distribution of algae in the soil. Occurrence of algae in the soil. Biochemical activities of algae. Role of algae in the soil 215 Chapter XI SOIL FUNGI Occurrence of fungi in the soil. Methods of demonstrating the occurrence and abundance of fungi in the soil. Methods of cultivation of soil fungi. Isolation of single spore cultures. Classification of fungi with special refer- ence to those occurring in the soil. Occurrence of specific fungi in the soil. Activities of fungi in the soil. Influence of reaction upon the growth of fungi. Cellulose decomposition by fungi. Decomposition of nitrogenous substances (ammonia formation). Utilization of nitrogen compounds by fungi. Nitrogen fixation. Mycorrhiza Fungi. Nature of mycorrhiza formation. Plants forming mycorrhiza. Organisms responsible for mycorrhiza formation. Role of mycorrhiza in the nutrition of plants. . . . 236 Chapter XII SOIL ACTINOMYCES General considerations. General description of the genus Actinomyces. Terminology and systematic position. Species differentiation. Methods of study. Nature of growth on artificial media. Vegetative mycelium. Spore bearing mycelium. Utilization of carbon compounds by actinomyces as sources of energy. Nitrogen utilization. Oxygen requirement. Influ- ence of temperature, drying and radiation. Influence of reaction and salt concentration. Influence of poisons. Reduction of nitrates and other compounds. Production of odor. Pigment formation. Variability. Species differentiation. Key to the identification of species of soil actinomyces. Importance of actinomyces in the soil , , , 285 XXIV CONTENTS Chapter XIII SOIL PROTOZOA General morphology of protozoa. Physiology of protozoa. Media for the cultivation of protozoa. Isolation of pure cultures of protozoa. Staining of protozoa. Life history of protozoa. Occurrence of trophic and encysted protozoa in the soil. Classification and occurrence of protozoa in the soil. Importance of protozoa in the soil 311 Chapter XIV THE NON-PROTOZOAN FAUNA IN THE SOIL Animal ecology as a whole and classification of soil forms. Methods of study. Flatworms. Nematoda. Rotatoria. Annelida. Tartigrada. Arthropoda. Arachnida. Myriapoda. Insecta. Mollusca. Influence of environmental conditions on the invertebrate fauna of the soil. Economic importance of the invertebrate fauna of the soil 341 PART C. CHEMICAL ACTIVITIES OF MICROORGANISMS Chapter XV GENERAL PRINCIPLES OF MICROBIAL METABOLISM Metabolism as a whole. Chemical reactions in the microbial cell. Enzymes of microorganisms. Reaction velocity. Growth, life and death of micro- organisms. Chemical composition of the microbial cell 367 Chapter XVI ENERGY TRANSFORMATIONS IN THE METABOLISM OF MICROORGANISMS Life and energy. Energy transformations by autotrophic bacteria. Energy utilization from the oxidation of nitrogen compounds. Energy utilization from the oxidation of sulfur and its compounds. Energy utilization from the oxidation of iron compounds. Energy utilization from the oxidation of hydrogen. Energy utilization from the oxidation of simple carbon com- pounds. Heterotrophic utilization of energy by microorganisms. Aerobic utilization of energy. Anaerobic utilization of energy. Efficiency of energy utilization by heterotrophic microorganisms. Reduction of nitrates and sulfates and energy utilization. Comparative amounts of energy liberated by microbiological processes. Energy transformation in syn- thetic processes. Energy transformation in the soil 384 Chapter XVII CHEMISTRY OF DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER BY SOIL MICROORGANISMS Composition of vegetable organic matter. Chemistry of celluloses. Mech- anism of decomposition of cellulose by microorganisms. Decomposition of cellulose by anaerobic bacteria. Decomposition of cellulose by aerobic CONTENTS XXV bacteria. Decomposition of cellulose by thermophilic bacteria. Decom- position of cellulose by denitrifying bacteria. Cellulose decomposition by actinomyces. Cellulose decomposition by filamentous fungi. Cellulose decomposition in manure. Importance of cellulose decomposition in the soil. Influence of soil conditions upon cellulose decomposition. Chemistry of hemicelluloses. Decomposition of hemicelluloses by micro- organisms. Lignins, ligno-celluloses and their decomposition. Pectins, mucilages and gums and their decomposition by microorganisms. Starches and their decomposition by microorganisms. Decomposition of fats and waxes. Decomposition of paraffins, aliphatic hydrocarbons and benzene ring compounds in the soil. Decomposition of glucosides and monosac- charides. Decomposition of organic acids 427 Chapter XVIII DECOMPOSITION OF PROTEINS AND OTHER ORGANIC NITROGENOUS COMPOUNDS BY SOIL MICROORGANISMS Physical and chemical properties of proteins. Chemistry of protein hydrol- ysis. Protein decomposition by microorganisms. Chemistry of ammonia formation in the decomposition of proteins by microorganisms. Decom- position of organic nitrogenous compounds of a non-protein nature. Am- monia formation by bacteria. Ammonia formation by fungi and actinomy- ces. Rate of ammonia formation by microorganisms and methods of determinination. Nitrogen transformation in the rotting of manure. Nitrogen transformation in the decomposition of organic matter in the soil. Influence of nitrogenous decomposition products on the growth of plants and microorganisms 470 Chapter XIX INFLUENCE OF AVAILABLE ENERGY UPON THE TRANSFORMATION OF NITROGENOUS COMPOUNDS BY MICROORGANISMS Carbon and nitrogen transformation by microorganisms. Influence of non- nitrogenous organic compounds upon the decomposition of nitrogenous compounds and upon the amounts of ammonia liberated. Decomposition of organic substances of varying carbon-nitrogen ratio. Different groups of microorganisms as affecting the carbon nitrogen ratio in the medium. Influence of straw and plant residues upon the growth of cultivated plants . . 504 Chapter XX OXIDATION PROCESSES — NITRATE FORMATION Oxidation-reduction phenomena. Oxidation processes in the soil. Source of nitrates in the soil. Mechanism of ammonia oxidation. Mechanism of nitrite oxidation. Nitrate formation from inorganic salts and from organic nitrogenous compounds. Influence of reaction on nitrate formation. Influence of organic matter upon nitrate formation. Influence of salts. Influence of soil gases. Nitrate formation in solution and in soil. Influ- ence of soil treatment upon nitrification in the soil. Oxidation of sulfur and other minerals in the soil. Oxidation of organic compounds in the soil. 520 XXVI CONTENTS Chapter XXI REDUCTION PROCESSES — NITRATE REDUCTION Reduction processes in the soil. Transformation of nitrates by micro- organisms. Nitrate assimilation. Utilization of nitrates by micro- organisms as sources of oxygen. Reduction of nitrates to gaseous nitro- gen and oxides of nitrogen. Formation of nitrogen gas from organic com- pounds. Denitrification in the soil. Importance of nitrate-reduction in the soil. Reduction of other substances in the soil 542 Chapter XXII FIXATION OF ATMOSPHERIC NITROGEN BY MICROORGANISMS Non-symbiotic fixation of nitrogen. Source of energy. Chemistry of de- composition of carbohydrates. Respiration and nitrogen fixation. Pro- tein synthesis by Asiotobacter. Chemistry of process of non-symbiotic nitrogen fixation. Influence of available nitrogen compounds upon nitro- gen fixation. Influence of salts upon nitrogen fixation. Influence of organic matter upon nitrogen fixation. Influence of reaction. Influence of moisture and temperature upon nitrogen fixation. Soil cultivation and nitrogen fixation. Importance of non-symbiotic nitrogen-fixation proc- esses in the soil. Symbiotic Nitrogen Fixation. Relation between the bacteria and the host plant. Chemistry of nitrogen-fixation by symbiotic bacteria. Production of gum by legume bacteria. Influence of reaction on the growth of Bad. radicicola and nodule formation. Nodule formation and nitrogen fixation. Influence of environmental conditions. Importance of symbiotic nitrogen fixation in the soil. Associative action of legumes and non-legumes 558 Chapter XXIII TRANSFORMATION OF SULFUR BY MICROORGANISMS Sources of sulfur in the soil and processes of transformation. The nature of oxidation of sulfur and its compounds in the soil. Reduction of sulfur and its compounds. Formation of H2S in the decomposition of organic matter. Sulfur oxidation and transformation of minerals 600 PART D. SOIL MICROBIOLOGICAL PROCESSES AND SOIL FERTILITY Chapter XXIV THE SOIL AS A MEDIUM FOR THE GROWTH AND ACTIVITIES OF MICROORGANISMS The soil as a culture medium. Soil composition and microbiological activi- ties. The mineral composition of the soil. The physico-chemical role of organic matter in the soil. Colloidal condition of soils and microbiological " activities. Soil solution. Soil reaction and microbiological activities. The soil atmosphere. Soil temperature. Growth of microorganisms in soil in pure and mixed culture. The idea of a soil population 619 CONTENTS XXV11 Chapter XXV TRANSFORMATION OF MINERALS IN THE SOIL Nature of mineral transformation by microorganisms. Decomposition of rocks and rock constituents by microorganisms. Nature of phosphorus compounds in the soil. Decomposition of organic phosphorus compounds by microorganisms. Transformation of insoluble tri-calcium phosphates into soluble forms by microorganisms. Transformation of insoluble phos- phates by inorganic and organic acids formed by microorganisms. Trans- formation of potassium in the soil by microorganisms. Transformation of calcium in the soil. Transformation of magnesium in the soil. Transfor- mation of manganese in the soil. Transformation of zinc. Transformation of iron. Transformation of aluminum in the soil. Role of minerals in bacterial metabolism 644 Chapter XXVI TRANSFORMATION OF ORGANIC MATTER IN THE SOIL Nature of soil organic matter. Decomposition of organic matter added to the soil. Transformation of the various constituents of the organic matter added to the soil. Evolution of carbon dioxide as an index of decomposition of organic matter in the soil. Formation of ammonia (and nitrate) as an index of decomposition of organic matter in the soil. Formation of "humus" as an index of decomposition of organic matter in the soil. Nature of soil "humus." Chemistry and classification of humus compounds. Soil organic matter and the activities of microorgan- isms. Carbon-nitrogen ratio in the soil 669 Chapter XXVII MICROBIOLOGICAL ANALYSIS OF SOIL AS AN INDEX OF SOIL FERTILITY Soil fertility and microbiological activities. Methods for determining the microbiological condition of the soil. Numbers of microorganisms in the soil. Nitrifying capacity of the soil. Evolution of carbon dioxide. Cellulose decomposing capacity of the soil. Nitrogen fixing and mannite decomposing capacity of the soil. The catalytic action of the soil. Oxi- dizing and reducing power of the soil 708 Chapter XXVIII SOIL MICROBIOLOGICAL EQUILIBRIUM; INFLUENCE OF AIR DRYING AND PARTIAL STERILIZATION UPON THE ACTIVITIES OF MICROORGANISMS IN THE SOIL Microbiological equilibrium in the soil. Influence of air drying of soil upon the microbiological equilibrium. Influence of caustic lime upon soil processes. Partial sterilization of soil. The use of heat as an agent of partial sterilization. Influence of volatile antiseptics upon bacterial ac- tivities in the soil. Protozoan theory. Agricere and bacteriotoxin theory. Destruction of selective groups of organisms. Interrelationships of micro- organisms in the soil 738 XXV111 CONTENTS Chapter XXIX INFLUENCE OF ENVIRONMENTAL CONDITIONS, SOIL TREATMENT, AND PLANT GROWTH UPON MICROORGANISMS AND THEIR ACTIVITIES IN THE SOIL Influence of organic matter upon the soil population. Influence of stable manure. Influence of temperature. Influence of moisture. Influence of soil cultivation. Influence of salt concentration upon the activities of microorganisms in the soil. Influence of calcium oxide and carbonates of calcium and magnesium. Influence of growing plants upon soil micro- organisms and their activities 767 Chapter XXX SOIL AS A HABITAT FOR MICROORGANISMS CAUSING PLANT AND ANIMAL DISEASES Influence of saprophytic soil microorganisms upon plant growth. Sapro- phytism and parasitism among soil microorganisms. Animal and plant diseases caused by bacteria that may be found in the soil. Plant diseases caused by fungi found in the soil. Plant and animal diseases caused by species of actinomyces. Plant and animal diseases caused by invertebrate animals found in the soil. Relation of soil environment to plant infection. Influence of reaction upon the growth of plant pathogenic organisms in the soil. Methods of control SOI Chapter XXXI SOIL INOCULATION Beneficial and injurious microbiological processes in the soil. Introduction of certain useful microorganisms into the soil. Legume inoculation. Use of soil for inoculation of legumes. Commercial cultures and their prepara- tion. Biological types of legume bacteria. Importance of legume inocula- tion. Inoculation of non-leguminous plants with nodule bacteria. Inocu- lation of soil with non-symbiotic nitrogen-fixing bacteria. Soil inoculation with autotrophic bacteria. Inoculation of soil with heterotrophic, non- nitrogen-fixing microorganisms 817 Chapter XXXII HISTORY OF SOIL MICROBIOLOGY, ITS PAST, PRESENT AND FUTURE Beginnings of soil microbiology. Soil microbiology as an independent science. Recent advances of the science. Present outstanding problems in soil microbiology 834 PART A OCCURRENCE AND DIFFERENTIATION OF MICROORGANISMS IN THE SOIL ". . . le role des infiniments petits m'apparaissait infiniment grand . . — Pasteur. CHAPTER I Numbers of Different Groups of Microorganisms Found in the Soil and Methods of Determination The occurrence of microorganisms in the soil. The microorganisms present in the soil belong, in an uneven proportion, to the plant and animal kingdoms, the former including the large majority both in numbers and in kinds. Chart 1 gives a visual representation of the relationships of the various groups of soil microorganisms. The relative importance in the soil, however, both as to numbers and physiological activities, varies with the different groups. The animal world is represented in the soil by the protozoa, nema- todes, rotifers, earthworms and various other worms as well as insects. The nematodes occur abundantly in all soils, but especially in green- house soils and certain infested field soils. Large numbers as well as numerous species of amoebae, ciliates and flagellates represent the protozoa in the soil. The microscopic plant world is represented in the soil by the algae, fungi and bacteria, named in the order of their increasing importance of numbers and activities. Among the algae, the Cyanophyceae and Chlorophyceae are best represented in the soil. The soil fungi can be subdivided further into three groups : 1. Yeasts and yeast-like fungi, like the Monilia and Oidia (these two groups may, however, be classed with the true fungi). 2. Molds and other true fungi. Here we find the Mucorineae repre- sented by the extensive genera Rhizopus, Mucor, Zygorhynchus and other Phycomycetes; various Ascomycetes, including the genus Chae- tomium and other genera; Hyphomycetes represented by the Mucedi- naceae (Aspergillus, Penicillium, Sporotrichum, Botrytis, Trichoderma, Verticillium, etc.), Dematiaceae, Stilbaceae and Tuberculareaceae. The Basidiomycetes are probably represented abundantly in the soil by the sterile mycelium as well as by some of the mycorrhiza fungi. 3. Actinomyces. Ten to 50 per cent of the colonies developing from a soil on the common agar or gelatin plate belong to this important group of soil organisms. They are generally classified by bacteriologists with 3 4 PRINCIPLES OF SOIL MICROBIOLOGY the bacteria; actually they belong to the fungi and are so far known to be represented in the soil by one extensive genus Actinomyces. Bacteria predominate, in numbers and in the variety of activities, over all the other groups of microorganisms. This was the reason why the earlier microbiologists named the whole science of soil microbiology "soil bacteriology." It has long been recognized, however, that the soil population consists of various microorganisms other than bacteria, so that the more comprehensive term is fast coming into general use. Since the bacterial activities in the soil do not coincide with their taxonomic groupings, these organisms may be classified on the basis of their physiology for the sake of convenience in treatment. As a major division, the bacteria can be separated into two large groups: Chart I. The microflora and microfauna of the soil (1) autotrophic, and (2) heterotrophic forms. Living organisms that require for their nutrition substances which have been built up by other organisms are called heterotrophic. The heterotrophic saprophytic bacteria consume, for their energy and for the building up of their protoplasm, the organic compounds of plant and animal bodies. Organ- isms like the green plants and certain bacteria that can thrive on purely inorganic substances and obtain their carbon from the carbon dioxide of the atmosphere are called autotrophic. But while the green plants derive their energy photosynthetically, the autotrophic bacteria derive their energy from the oxidation of purely inorganic substances, or chemosynthetically. The autotrophic group of bacteria is represented in the soil by smaller numbers and by much fewer species than the heterotrophic group, but it includes forms which are of greatest impor- NUMBERS OF MICROORGANISMS 5 tance in the physiological processes in the soil, namely the organisms which oxidize ammonium salts to nitrites, nitrites to nitrates, sulfur and sulfur compounds to sulfates, and a few other less important groups. The heterotrophic bacteria are further subdivided on the basis of their nitrogen utilization: (1) Those bacteria that are able to fix atmospheric nitrogen in the presence of sufficient carbohydrates as sources of energy. This division is again only secondary in numbers, but its three representative groups play an important part in the soil economy, namely in the increase of the combined nitrogen of the soil. They are the symbiotic nitrogen-fixing, or nodule bacteria; the non- symbiotic aerobic nitrogen-fixing bacteria and the non-symbiotic anaerobic nitrogen-fixing bacteria. (2) Those bacteria which depend, for their metabolism, upon the nitrogen of the soil, in organic or inor- ganic forms. The heterotrophic non-nitrogen-fixing bacteria can be further subdivided, using as a basis either the need of free or com- bined oxygen or spore formation. The heterotrophic, non-nitrogen- fixing, aerobic bacteria are usually the organisms which are found on the plates, when an analysis of numbers of bacteria in the soil is made by the common agar or gelatin plate method. In addition to the microscopic forms, ultramicroscopic microorganisms capable of passing through bacterial filters have been reported1 as present in the soil. These have been only insufficiently studied. We may be dealing here with certain stages of other organisms, as sug- gested by Lohnis for gonidia. A certain relation was observed between the ultrafilterable microbes and microbial enzymes and other cell constituents. Attention may be called here to the extensive literature concerning the nature of the bacteriophage; investigators do not agree as yet whether these are ultramicroscopic organisms or are of the nature of enzymes. An attempt to study the physiological activities of the invisible soil microorganisms has been made2 but without any success. Proof of microbial activities in the soil. The food requirements of the various groups of soil microorganisms are so distinctly different that no single artificial culture medium could be devised on which all of them could be studied. A large number of microorganisms, to which some of the most important soil forms belong, will grow only under very special conditions, such as selective media or selective environments. 1 Melin, E. Ultramikroskopische Mikroben im Waldboden. Ber. deut. Bot. Gesell. 40: 21-25. 1922. See also Miehe, H. Biol. Centrbl. 43: 1-15. 1923. 2 Rossi, G. Preliminary note on the microbiology of the soil and the possible existence therein of invisible germs. Soil Sci. 12: 409-412. 1921. 6 PRINCIPLES OF SOIL MICROBIOLOGY Various media and different methods have to be used for the study of the different groups. In some cases, special enrichment culture media favor- ing the development of particular organisms have to be devised, so that the growth of these will take place in preference to that of all the other organisms. We thus often create artificial conditions which are distinctly different from those of the soil and conclusions, based on the results of growth of the organisms under such artificial conditions, often do not hold true for the soil. To be able to grow the organisms in pure culture in the soil, the latter must be first sterilized. No method of sterilization has yet been devised which would not modify, in a funda- mental manner, the chemical conditions of the soil. What will hold true for sterilized soil, then, may not hold true for unmodified soil. Again, the various organisms exist in the soil in large numbers, with a number of associative and antagonistic influences at work (both by living microorganisms and their products). Each organism has adapted itself to its environmental conditions and to the other organisms and may be, so to speak, in a condition of "unstable equilibrium." When this same organism is cultivated, in pure culture, upon a favor- able medium, its activities are very likely to be different from those in the normal soil. Before we can conclude that a microorganism is active in the soil and that certain chemical transformations are produced by this organism under ordinary soil conditions, certain requirements must be satisfied. The following postulates, applied by Koch to pathogenic bacteria, and modified by Conn3 in their application to soils should hold true for soil microorganisms: (1) The organism must be shown to be present in the soil in an active form when the chemical transformation under investigation is taking place. (2) The organism must be shown to be present in larger numbers in such soil than in similar soil in which the chemical change is not taking place. (3) The organism must be isolated from the soil and studied in pure cul- ture. (4) The same chemical change must be produced by the or- ganism in experimentally inoculated soil, making the test, if possible, in unsterilized soil. (5) The organism must be found in the inoculated soil. Methods of study. The methods generally employed for the study of soil bacteria can be divided into those of direct microscopic observation and cultural methods. The former have been suggested by Conn and further developed by Winogradsky. The latter have been used by the 3 Conn, H. J. The proof of microbial agency in the chemical transformation of soil. Science. N. S. 46: 252-255. 1917. NUMBERS OF MICROORGANISMS 7 great majority of other soil microbiologists. Artificial culture media are employed, or at least artificial conditions are created. In many in- stances, therefore, no direct evidence is furnished as to what is actually taking place in the soil, under natural conditions. The results obtained under laboratory conditions often have to be interpreted as to their bearing upon actual field results. The information obtained from the study of soil microbiology by the use of the different methods can throw light upon three groups of phenomena: (1) the numbers and kinds of microorganisms occurring in the soil; (2) the activities of soil microorganisms; (3) the bearing of these activities upon soil fertility. Direct microscopic method. The method consists in preparing; a suspension of soil in a dilute fixative solu- tion, then spreading one or two drops of the suspension upon a clean slide, drying and staining with an acid dye. For qualitative purposes, about 0.5 to 1 gram of soil is placed4 in a test tube; 6 to 8 cc. of a fixing solution, consisting of 0.04 per cent sterile gelatin in water, are then added and the mixture well shaken. Two loopfuls of the suspension are placed upon clean slides; after drying, the slides are stained with a 1 per cent solution of rose bengal in 5 per cent phenol-water mixture. The preparation is heated on a steam bath until most of the liquid has evaporated and the excess of stain is removed by dipping the slide in water. The preparation is then dried on the steam bath and examined microscopically. The gelatin fixative can be omitted5 and the films fixed to the slide by flooding, after drying, with a very dilute solution of collodion in ether and alcohol. The method was modified and improved by Winogradsky,6 who found that the presence of large yellow grains of inorganic soil material hinders the proper examination of the field under the microscope. The soil samples are well mixed and powdered. One gram of the soil (on a dry basis) is then added to 4 cc. of distilled water and shaken vigorously for five minutes. After allowing to rest 30 seconds the suspension covering the large sedimented inorganic particles is poured off into a small tube of a hand centrifuge. Two 3-cc. portions of distilled water are then added to the residue, shaking each time one minute, allowing to rest 30 seconds and then pouring into the same tube of the centrifuge. Ten units of water are thus used for one unit of soil. After these three washings the 4 Conn, H. J. The microscopic study of bacteria and fungi in soil. N. Y. Agr. Exp. Sta. Tech. Bui. 64. 1918; An improved stain for bacteria in soil. Stain Technol. 1: 126-128. 1926. 5 Whittles, C. L. The determination of the number of bacteria in soil. Jour. Agr. Sci. 13: 18-48. 1923; 14: 346 369. 1924. 8 Winogradsky, S. Sur l'etude microscopique du sol. Compt. Rend. Acad. Sci. 179: 367-371. 1924; Etudes sur la microbiologic du sol. 1. Sur la methode. Ann. Inst. Past. 39: 299-354. 1925. 8 PRINCIPLES OF SOIL MICROBIOLOGY first sediment suspended in distilled water settles immediately. During these manipulations, which require about 10 minutes, a second sediment is formed in the tube of the centrifuge. About half of the suspension is carefully taken out and placed in another centrifuge tube; on centrifuging, a third sediment is formed. Preparations are then made from each sediment and from the non- centrifuged and centrifuged suspensions. One drop of the various preparations is placed upon a slide covering just 1 sq. cm. ; the preparations are dried in an oven and are rapidly covered with a very dilute agar solution. One per cent warm agar solution is best for the first two sediments and 0.1 per cent cold agar solution for the third sediment. For the suspensions, no fixative is necessary. When the agar is dried, several drops of absolute alcohol are used for fixing and the prepa- ration is stained by means of a solution of an acid dye in 5 per cent phenol solution. Rose bengal may be used, but its action is prolonged, followed by a drop of acetic acid, then washed. Extra erythrosine in 5 per cent phenol solution is superior. The bacterial cells are colored, but not the capsules and mucus; this is especially true of the compact colonies as those of Nitrosomonads and other soil forms which so readily over-color with basic dyes; the colloids are only faintly colored; the agar is readily discolored by the process of washing with cold water. The dye is allowed to act 5 to 15 minutes in the cold or on slight warming, then washed a few seconds in water. The preparations from the first sediment are usually free from bacteria, except in soils rich in organic matter, when some of the particles are not removed by three washings. The second preparation shows on examination the same mi- crobes, qualitatively and quantitatively, as the third sediment, where conditions for examination are most favorable. The fourth preparation made from the suspension is usually most instructive. The living cells only take the stain, while the spores stain only very faintly or not at all and can be seen only when present in large numbers. Protozoan cysts are recognized by their intense coloration and can easily be counted. Winogradsky suggested to use always for comparison a control soil, which had no addition of fresh organic matter for a considerable period of time. A normal arable soil contains a native or autochtonous flora consisting of short bacteria with rounded ends and of cocci, 1 to 1.5/z in diameter. Often larger forms, 1 to 3/x in diameter, resembling Azoto- bacter are found. They group into rounded colonies consisting of about 100 cells in a compact mass with a common capsule, but occa- sionally with as few as a dozen individuals (PI. I). The field between is completely devoid of microbes. The colonies are situated on the soil colloidal matter. This is the reason why the centrifuged suspension is practically free from colonies which are carried down by the flakes of organic matter. Spore-bearing bacilli, filamentous bacteria, spirals, mycelial filaments, actinomyces, and protozoan cysts are absent or are very rare. The presence of these indicates that the soil is in an active state of fermentation, due to recent addition of organic matter. PLATE I • • ; V # * 3 1. The bacteria grow in the soil, in the form of zooglea-like masses, upon the colloidal material surrounding the inorganic soil particles, as shown by the direct microscopic method, X 1200 (from Winogradsky) . 2. Zooglea-like mass of bacteria in soil, as shown by the direct microscopic method, X 1200 (from Winogradsky). 3. Large cells of bacteria in Texas sandy soil (Azotobacter?), as shown by direct microscopic examination, X 1200 (from Winogradsky). 4. The distribution of organic matter and bacteria in the soil (Russian tshernoziem) (after Winogradsky). NUMBERS OF MICROORGANISMS 9 The application of the direct microscopic examination to the study of occurrence and distribution of microorganisms in the soil gives more direct evidence as to the presence and relative abundance of specific groups of microorganisms. The direct microscopic examination has been used7 for counting bacteria in animal feces; it can also be used for counting various microorganisms in culture. To determine the numbers of microorganisms quantitatively by the use of the direct microscopic method, various difficulties are encountered: 1. Some of the microorganisms, like the protozoa, will be destroyed in the process of staining. 2. Others, like the fungi, may prove too large, for the very small quantity of soil that can be used for the examination. 3. The bacteria themselves are found in clumps upon the colloidal film and not in the soil solution. Not only is it difficult to count the bacteria in the film, but the variability is so great that it would take a large number of counts to obtain reliable results The same procedure is followed as for qualitative determinations. The soil is diluted by means of a weak solution of gelatin (0.15 gram gelatin in 1000 cc. of hot water and kept sterile in a cotton plugged flask) using one part of soil to 3 to 10 parts of solution depending on the soil type, heavier soils requiring a higher dilution. The smear is prepared from 0.1 cc. of the infusion measured out from a thin graduated pipette, to cover 1 sq. cm. on a clean slide, previously rinsed in alcohol; the smears are allowed to dry over a steam bath. For staining either rose bengal (1 gram in 100 cc. of 5 per cent phenol solution) or erythrosine can be employed. The stain is allowed to act 1 to 3 minutes, then washed and dried. With the rose bengal stain the bacteria are found deep pink or red, the mineral particles uncolored, some of the dead organic matter light pink but most of it yellow or unstained. The preparations are examined with an oil immersion objective and a high-power eye-piece. By means of a simple equation, the number of organisms can then be determined. It is advisable not to count the entire field, but to mark off the central portion.8 A disc with circles and cross lines is placed in the eye-piece. Conn suggested to use a circle of such a size as to cover an area on the slide either 80 or 113 microns in diameter. Every organism in the area will represent two and one millions respectively per cubic centimeter, using a 1.9 (TV inch) fluorite objective CN.A. 1.32) with a 12.5 X ocular. This quantity is multiplied by the dilution of the soil to give the number of bacteria per gram of soil. However, the uneven distribution of the bacteria in the soil, causing great irregularities and the difficulty of distinguishing bacterial cells from 7 Klein, A. Die physiologische Bakteriologie des Darm-Kanals. Arch. Hyg. 45: 117. 1902. 8 Breed, R. S., and Brew, J. D. Counting bacteria by means of the microscope. N. Y. Agr. Exp. Sta., Tech. Bui. 49. 1916. 10 PRINCIPLES OF SOIL MICROBIOLOGY soil particles, especially in case of clay soils, and of separating living from dead bacteria make accurate counts impossible. The method can, therefore, not be used as yet for quantitative work, but is quite applica- ble for qualitative purposes, to show the types of microorganisms which exist in the soil in an active form. The microscopic method may be used for counting bacteria in culture media, especially in a liquid form, but even here the total volume of microbial cells may prove9 a better index of the activities of the organisms than their numbers. Organisms found in the soil by the direct microscopic method. Conn demonstrated that the actual number of bacteria found in the soil, by the use of the microscope, is probably five to twenty times as great as that indicated by the culture plate method. This discrepancy is due to that fact that a large number of soil bacteria do not grow on the plates. By far the greatest number of microorganisms found in the soil, by the use of the microscope, consists of the minute non-spore- forming rods and cocci. The large spore-forming bacteria (as Bac. megatherium and Bac. cereus) have been found in normal soil only in the form of spores, which make up a very small proportion of the total bacterial flora of the soil. Filaments of actinomyces have also been found, but to a lesser extent than the spores of these organisms. Fungus mycelium was not found in any soil, except when an unusual amount of organic matter is present. The spore-forming bacteria become10 active in the soil only when a great excess of easily decomposable organic matter has been added or when the moisture content of the soil is high. The minute non-spore-forming rods and cocci are considered11 to form the autochtonous microflora of the soil. Other investigators found that the microscopic examination of soil bacteria allows the differentiation of three distinct groups,12 namely (a) cocci and short rods, (b) typical large cells of Azotobacter and (c) bacillary forms. The first two groups are largely connected with the 9 Skar, O. Mikroskopische Zahlung und Bestimmung des Gesamtkubikin- haltes der Mikroorganismen in festen und fliissigen Substanzen. Centrbl. Bakt., II, 57 : 327-344. 1922. Fries, K. A. Eine einfache Methode zur genauen Bestim- mung der Bakterienmengen in Bakteriensuspensionen. Centrlbl. Bakt. I (Orig.), 86: 90-96. 1921. 10 Winogradsky, S. Sur la microflore autochtone de la terre arable. Compt. Rend. Acad. Sci. 178: 1236-39. 1924. 11 Joffe, J. S., and Conn, H. J. Factors influencing the activity of spore form- ing bacteria in soil. N. Y. Agr. Exp. Sta. Bui. 97. 1923. 12 Richter, A. A. and B. A. To the question of microscopic soil investigation. (Russian). Utchonie Zapiski, Saratov Univ. 4: No. 1. 1925. NUMBERS OF MICROORGANISMS 11 colloidal soil particles, in the form of zooglea, surrounded by slimy capsules; the third group is found mostly in the soil solution, but the representatives of this group occur frequently also in the form of clumps, especially on decomposing organic matter. A comparative study of the occurrence of these three groups at different depths of soil has given the following results : TYPE OF SOIL Forest soil. Brown loam soil. Sandy soil. Surface 10 cm. 20 cm. Surface 10 cm. Surface 10 cm. 20 cm. NUMBERS OF BACTERIA. IN MILLIONS PER GRAM YEAST CELLS Cocci Azoto- bacter cells Bacilli 1,379 991 281 870 569 519 407 269 156 82 188 188 184 155 112 51 1,212 466 169 376 106 192 153 139 millions per gram 1 31 84 1 79 23 8 PIECES OF FUNGUS MYCELIUM millions per gram 47 34 7 3 19 3 The non-spore forming bacteria are thus found to be most abundant, the large rods or bacillary forms coming next, especially in soils rich in decomposing organic matter. Fungus mycelium is also abundant in such a soil. Cultural methods for demonstrating the kinds of organisms active in the soil. The cultural methods for the study of soil microorganisms are divided into methods for (a) quantitative study of soil microorganisms, (6) qualitative studies, and (c) for the study of microbiological activities, both in pure culture and in the soil. Winogradsky13 suggested to use the silica gel plate, to which a specific substance is added, for demonstrating the existence of specific organisms in the soil. Pure, colorless potassium silicate is dissolved in water to a specific gravity of 1.06 (6 to 8 Beaume). A dilution of HC1 equivalent to a specific gravity of 1.10 (13 Beaume) is also prepared. An equal volume of the silicate is poured into the 13 Winogradsky, S. Sur une methode pour apprccier le pouvoir fixateur de l'azote dans les terres. Compt. Rend. Acad. Sci. 180: 711-716. 1925. 12 PRINCIPLES OF SOIL MICROBIOLOGY acid solution and both are well mixed. The mixture is then distributed into Petri dishes and these are allowed to rest over night. A firm gel is obtained. The uncovered dishes containing the gel are then placed in flowing water for at least twenty-four hours, until no reaction is given with methyl red or brom cresol purple and with AgN03. A solution containing the minerals and specific sub- stance, either in solution or as an insoluble suspension, may then be poured over the surface of the gel and the dishes placed in an oven at 60° to 65°C, until the excess moisture has dried off. The gel in the dish is inoculated with small particles of soil, the dish is covered and placed in an incubator. After a few days, the specific organism, if present in the soil, will develop on the gel surrounding the particles of soil. By this method the presence of Azotobacter in soil can be readily demonstrated, provided mannite and CaC03 are employed in addition to the minerals. Nitrite-forming bacteria will develop in an ammonium salt medium, nitrate-forming organisms in a nitrite medium, etc.14 However, the cultural methods, largely the enrichment media de- veloped by Winogradsky and Beijerinck, and the common gelatin and agar plate have been used most extensively for establishing the presence and abundance of specific organisms in the soil. Cultural methods for the determination of numbers of microorganisms in the soil. The earliest investigations in soil bacteriology were car- ried out, 15-16.17 by the use of methods developed in medical bacteriology. Soils were diluted with sterile soil, then plated out with gelatin and numbers determined after a certain incubation period. Later, sterile water was used for making the dilutions. In some cases small quantities of soil were weighed directly for the preparation of the plates. The method itself was imperfect and the results unrepresentative, and no relation was established between numbers and soil productivity. Hiltner and Stormer18 suggested the use of the dilution method, with the hope of doing away with the plate method, but here again the hetero- trophic bacteria were determined by their growth on agar or gelatin 14 The gel may also be prepared by methods described elsewhere (p. 196). 16 Koch, R. Zur Untersuchung von pathogenen Organismen: Bodenunter- suchung. Mitt. K. Gesundheitsamt. 1: 34-36. 1881. 16 Proskauer, B. Uber die hygienische und bautechnische Untersuchung des Bodens auf dem Grundstucke der Charite und des sogen. "Alten Charitd- Kirchhofes." Bakteriologisches Verhalten des Bodens. Ztschr.Hyg.il: 22-24. 1882. 17 Frankel, C. Untersuchungen iiber das Vorkommen von Mikroorganismen in verschiedenen Bodenschichten. Ztschr. Hyg. 2: 521-582. 1887. 18 Hiltner, L., and Stormer, K. Studien fiber die Bakterienflora des Acker- bodens,mit besonderer Beriicksichtigung ihres Verhaltens nach einer Behandlung mit Schwefelkohlenstoff und nach Brache. Arb. Biol. Abt. Land. r. Forstw., K. Gesundheitsamt. 3: 445-545. 1903. NUMBERS OP MICROORGANISMS 13 media, while the autotrophic and nitrogen-fixing organisms were found not to be able to develop readily in high dilutions. Each of these two methods (plate and dilution) for determining the number of microor- ganisms in the soil has certain advantages and disadvantages. The plate method consists in diluting the soil with sterile tap water, making a series of dilutions, so that 1 cc. of the final dilution, when plated out with nutrient agar or gelatin, will allow 40 to 200 colonies to develop on the plate. The dilution method consists of diluting the soil first with sterile water, as with the plate method, but transferring 1 cc. of several of the final dilutions into special sterile nutrient media adapted for the growth of particular groups of microorganisms. The number of microorganisms will be found to lie between the two highest dilutions, one of which gives positive and the other negative growth. This allows us to determine approximately the number of organisms belonging to each group and present in the particular soil.19 The latter method is rather cumbersome, since it involves the preparation of a large number of media and the use of a number of containers for the development of various physiological groups of organisms for making the various dilutions, also, it involves great variability in the results.20 The plate method is convenient, but its chief limitation is the fact that it allows the development of only the heterotrophic, non-nitrogen fixing, aerobic bacteria and of yeasts, molds and actinomyces. The dilution method can be used for the study of practically all known soil forms. The two methods may then be used each for its particular purpose, particularly in view of the fact that, for those microorganisms that develop on the common culture plate, the dilution method was not found18 to give higher results than the plate method. The latter method should, therefore, be utilized for a general study of the numbers of microorganisms in the soil, keeping in mind its limitations, while the dilution method should be used for the determination of the abundance of special groups of microorganisms which do not develop on the plate. Culture media. With the introduction by Koch in 1881 of the gelatin plate for counting bacteria in general, an impetus was also given to the study of soil bacteria. But, unfortunately, Koch himself and prac- tically all the bacteriologists following him for the next fifteen years were medical men interested particularly in the possible presence of patho- 19 Lohnis, F. Zur Methodik der bakteriologischen Bodenuntersuchung. Centrbl. Bakt. II, 14: 1-9. 1905. 20 Fischer, H. Zur Methodik der Bakterienzahlung. Centrbl. Bakt. II, 25: 457-459. 1910. 14 PRINCIPLES OF SOIL MICROBIOLOGY genie bacteria in the soil and their importance as carriers of infection. They quite properly, from their point of view, used the methods of medical bacteriology. But even the excellent and stimulative researches of Koch and those following him21 could not lay a proper foundation of soil microbiology, due primarily to the lack of proper methods. The meat-extract-peptone agar or gelatin, found so valuable in patho- genic bacteriology, is entirely inappropriate for soil work, for various reasons, chief among which is the fact that the medium is not standard in composition and that it allows a rapid development of a few organ- isms which readily overgrow the plate and thus may prevent entirely the development of others. The distinct inferiority of bouillon agar or bouillon gelatin can be seen from the results of Engberding,22 who found that a soil giving 99 colonies with Heyden agar, gave 39 with bouillon agar and only two with bouillon gelatin. But even the Heyden agar is not definite in composition, although it is often used for counting soil bacteria. The media used for the determination of numbers of microorganisms in the soil (those that develop on the plate) should allow the development of the greatest possible number of organisms and should be standard in composition, so that every batch made up in the same laboratory or at any other laboratory will be like every other batch. This means that inorganic salts should be used. If organic substances are necessary, they should be pure, stable, and standard if possible, as in the case of the carbon and nitrogen sources. Various sugars or organic acids used as sources of carbon can be obtained in a standard form. Nitrogen substances should also be as standard as possible and used in as small amounts as possible. Agar in itself should not serve as a nutrient and should be, therefore, as pure as possible. The objection to gelatin is that it serves also as a nitrogen source for many microorganisms, thus making the medium not standard. It should, therefore, be used only in qualitative work or in special instances, as in the study of the number of gelatin-liquefying organisms in the soil. To hold in check the development of certain rapidly growing organisms, which prevent the growth of the numerous but slow growing bacteria in the soil, the organic matter content of the media had to be reduced to a minimum. 21 Houston, A. C. Chemical and bacteriological examination of soils. Local Gov't. Board, Rept. 27: 251-296. 1898. "Engberding, D. Vergleichende Untersuchungen liber die Bakterienzahl im Ackerboden in ihrer Abhangigkeit von aussern Einfiiissen. Centrbl. Bakt. II, 23: 569 642. 1909. NUMBERS OF MICROORGANISMS 15 The first important modifications in the composition of the medium for a quantitative determination of soil bacteria were made by the introduction of the soil infusion agar,23 and later by the elimination even of the soil extract,24 using a synthetic agar, with only 0.05 gram of peptone per liter. The soil extract media do not, however, meet the qualification of being "standard in composition," since the soil infusion varies with the soil used for making the infusion. The synthetic medium was further modified25 by the substitution of egg-albumin and casein for peptone. Among the other synthetic media suggested for the quantitative estimation of soil bacteria and actinomyces, sodium asparaginate agar,26 asparaginate-mannite agar,27 and urea nitrate agar28 should be mentioned. For the estimation of fungi, special acid media have to be used. For the protozoa, the dilution method still remains the most reliable, and nutrient agar or special liquid media can be employed for the development of the organisms in the final dilutions. Composition of synthetic media I. Fischer's soil extract agar: Soil extract 1000 cc. Agar 12 grams K2HPO« 2 grams The soil extract is prepared by heating soil for half an hour at 15 pounds pres- sure with an equal weight of a 0.1 per cent solution of Na2C03. 23 Fischer, H. Bakteriologisch-chemische Untersuchungen. Bakteriologis- cher Teil. Landw. Jahrb. 38: 355-364. 1909. !4 Lipman, J. G., and Brown, P. E. Media for the quantitative estimation of soil bacteria. Centrbl. Bakt. II, 25: 447-454. 1910. 25 Brown, P. E. Media for the quantitative determination of bacteria in soils. Centrbl. Bakt. II, 38: 499-506. 1913; also Iowa Agr. Exp. Sta., Res. Bui. 11, 396-407. 1913. Waksman, S. A. Microbiological analysis of soils as an index of soil fertility. II. Methods of the study of numbers of microorganisms in the soil. Soil Sci. 14: 283-298. 1922; Waksman, S. A., and Fred, E. B. A tentative outline of the plate method for determining the number of microorganisms in the soil. Ibid. 14:27. 1922. 26 Conn, H. J. Culture media for use in the plate method of counting soil bacteria. N. Y. Agr. Exp. Sta. Tech. Bui. 38. 1914. 27 Thornton, H. G. On the development of a standardized agar medium for counting soil bacteria, with especial regard to the repression of spreading colonies. Ann. Appl. Biol. 9: 241-274. 1922. 28 Cook, R. C. Quantitative media for the estimation of bacteria in soils. Soil Sci. 1: 153-161. 1916. 16 PRINCIPLES OF SOIL MICROBIOLOGY II. Lipman and Brown's synthetic agar: Distilled water 1000 cc. Glucose 10 grams Agar 20 grams MgS04-7H20 0.20 gram Peptone 0.05 gram K2HP04 0.50 gram III. Albumin agar: Distilled water 1000 cc. MgS04-7H20 0.20 gram Agar 15.00 grams K2HP04 0.50 gram Powdered egg al- Fe2(S04)3 trace bumin 0.25 gram Glucose 1.00 gram The albumin is suspended in 5 to 10 cc. of water, then 1.0 cc. of 0.12V NaOH solution is added, so as to convert it into sodium albuminate; the albuminate is added only to the filtered medium. IV. Casein agar. Same as medium III, only 1.0 gram of purified casein is used in place of the albumin. The casein is dissolved in 8 cc. of 0.12V NaOH. V. Asparaginate agar: Distilled water 1000 cc. NH4H2P04 1.5 grams Agar 12.0 grams CaCl2 0.1 gram Sodium asparaginate. 1.0 gram KC1 0.1 gram Glucose l.Ogram FeCl3 trace MgS04-7H20 0.2 gram 10 cc. of 1.02V NaOH solution added per liter to bring the desired reaction. VI. Asparagine-mannite agar: Distilled water 1000 cc. NaCl 0.1 gram Agar 15 grams FeCl3 0.002 gram K2HPO4 l.Ogram KN03 0.5 gram MgS04-7H20 0.2 gram Asparagine 0.5 gram CaCl2 0.1 gram Mannite l.Ogram The mannite is added after the agar and other constituents have been dis- solved and medium filtered. The reaction is adjusted to pH 7.4. VII. Urea ammonium nitrate agar: Distilled water 1000 cc. Glucose 10.0 grams Agar 15.0 grams Urea 0.05 gram K2HP04 0.5 gram Ammonium nitrate. . . 0.1 gram MgS04-7H20 0.2gram Fe2(S04)3 trace Reaction is about pH 7.0. In addition to these media, soil extract and tap water gelatin recommended by Conn and used chiefly for qualitative purposes can also be mentioned: VIII. Tap water gelatin29 Tap water 1000 cc. Gelatin (Gold Label) 200 grams These media are about equally favorable to the development of aerobic, heterotrophic bacteria, deriving their nitrogen from inorganic 19 Medium is clarified by means of white of egg; reaction is adjusted to 0.5 per cent normal acid to phenolphthalein, which requires 20-30 cc. 1.0 N NaOH; when Bacto-gelatin is used, only 10 oc. of alkali is required and no clarification. NUMBERS OF MICROORGANISMS 17 M CO ffl ,o «5i is .5 « 2 ■a a I* o. o as,™ O O O O CO o o o o o o IO M M ooNHHooaoo^sioioto 8o o o o o © o © © o © o © © lOOlOOOLQO©^*! © o © o o © © © © o lO CO lO 00 © © © © to © oo lOiQHOOtOTjIOJKliOlOOOl Q O © © D o a © p 8 O o o c o © © -■> © 8 c C : c s © © o <-.- lO © t~ m C O lO o © lO CO t- i-H CI T-l O Y-i 35 o w l> © © 8©©©©oo©© ©©©©©O©© iO»OCO©iOOOOO a* o oo CO © ^ © O iO >-o co CO~ d <» r^ o CM co oo CO I- u 1-1 1-1 *~i o CO CM co e co co pQ "s- o o o o © © © © © 1~ © 7_l CO fa. o o o © 5 © o o lO a c © Q o o o >o © CO o CO oo I - t^ J o 3 H o o t- oo © © IM © oo id o o co r- io CM t- »o I— 1 „ o> »o oo eo i-i u 2 ^ < © o K < o o o o c © o © t^ 1^ o •"# o o o o CI © © o © eo 1- 00 o o o iO l> © to lO CO »o © CO H ^2 a g o o co co ^H lO oo lO CI CO CO m d s o O CM oo co £ eo ~ c 00 s~ o o © © oc © © o CO CO 1- 00 «s © t^ o o © b- lO OO iO CI »o •5* i> ■<*< 1 o OO CO CO © CO "« o oo CM lO CO "S co CM O o 03 > .. . "& tf: g 'S, ©.2 +3 CD o3 > .°3. a ^ c3 03 .5 03 73 03 EC ^ t»-i' r3 o ^ ^? 28: 461-473. 1910. 101 Schroeder, H. The bacterial content of coal. Centrbl. Bakt. II, 41: 460^69. 1914. 102 Lohnis, 1910, p. 542. 103 Beijerinck, M. W., and Van Delden, A. tjber eine farblose Bakterie, deren Kohlenstoffnahrung aus der atmospharischen Luft herrnhrt. Centrbl. Bakt. II, 10: 33-47. 1903. 104 Kaserer, H. Die Oxydation des Wasserstoffes durch (Mikroorganismen. Centrbl. Bakt. II, 16: 681-696, 769-775. 1906. 108 Lantzsch, 1923 (p. 298). 106 de Saussure, Th. Action de la fermentation sur le melange des gaz oxygene et hydrogene. Mem. Soc. phys. Hist. Nat. Geneve, 8: 163-190. 1839. 107 Niklewski, B. Ein Beitrag zur Kenntnis wasserstoffoxydierenden Mikro- organismen. Centrbl. Bakt. II, 20: 469-473. 1908; Niklewski, B. tlber die Wasserstoffoxydation durch Mikroorganismen. Jahrb. Wiss. Bot. 48: 113-142. 1910. Nabokich, A. J., and Lebedeff , A. F. t)ber die Oxydation des Wasserstoffes durch Bakterien. Centrbl. Bakt. II, 17: 350-355. 1906; Biochem. Ztschr. 7: 1-10. 1908. AUTOTROPHIC BACTERIA 99 means of a single polar flagellum. The gelatin colonies were yellow, smooth, rarely greenish; the gelatin was not liquefied. Yellow to greenish growth on agar. Kaserer suggested that both the methane and hydrogen oxidation phenomena are of great importance in the soil, due to the fact that these substances, which are produced in the subsoil by anaerobic proc- esses, are thus oxidized and made available to the soil. Kaserer's medium consisted of: K2HPO4 0.5gram NaHC03 0.5 gram MgS04 0.2 gram FeCl3 Trace NH4CI 1.0 gram Water 1000 cc. The organism growing on this medium developed poorly under auto- trophic conditions, the oxidation of hydrogen becoming prominent in the presence of small amounts of soluble organic matter.108 A non- motile bacterium, 1.4 by 0.5/x in size, was isolated, different from the H ■pantotropha of Kaserer. The greatest amount of hydrogen was oxidized in the presence of 0.01 to 0.03 per cent peptone, nutrose or sodium asparaginate. In association with certain bacteria, the organism was much more active. Niklewski used a medium containing: NH4CI l.Ogram NaCl 0.2 gram KH2P04 l.Ogram FeCl3 0.0001 gram MgS04-7H20 0.2 gram Agar 15.0 grams NaHCOj l.Ogram Water 1000 cc. The cultures were placed in a bell-jar, through which purified hydrogen was passed, at 38° to 35°C. The cultures developed in 3 to 4 days. Two organisms were isolated: Hydrogenomonas vilrea formed a pellicle on the surface of the liquid medium. Small yellow subsurface colonies were formed on the agar. On the surface the colonies were transparent, folded. The cells are 2/i long. Obligate autotrophic. No motility observed. Hydrogenomonas flava formed shining yellow colonies on the surface of the agar, not spreading as rapidly as the H. vitrea, surface smooth, edge entire; microscopically, the cells were found to be somewhat smaller (1.5/* long). No pellicle formation on liquid media. Obligate autotrophic. No motility observed. By further study, Niklewski109 isolated an organism (H. agilis) which can oxi- 108 Harrison, W. H., and Aiyer, P. A. S. The gases of swamp rice soils. III. A hydrogen-oxidizing bacterium from these soils. Mem. Dept. Agr. India, Chem. Ser. 135-148. 1916. 109 Niklewski, B. Uber die Wasserstoffaktivierung durch Bakterien unter besonderer Beriicksichtigung der neuen Gattung Hydrogenomonas agilis. Kos- mos, Lemberg. 1923. (Centrbl. Bakt. II, 40: 430-433. 1914.) 100 PRINCIPLES OF SOIL MICROBIOLOGY radsk rder em radsk rder em -o '3 13 0 M '* 03 S£ i- ** c .a .3 M e » h 09 O o3 03 O c3 03 .a .SDK .SOW m -3 h5 H fe R g 55 m 0] 0 3 O tf X T o «5 ^H «- o A a & Mbd2 O H S H S P g H ^ 3 .■§ 55 W '3 55 S ■ E 03 fl 5 3 -o O ,<, c _ T3 a o es 0 0 rf o 1 ■< fc 5 w & Ho® 6 « •- s C i a> j, o^ 5*. -a | 3 CD ^J A H O 43 o O 03 o o (J i-" 3 s 50 00 u U P fc O H W 'u CO to oS w ITS n o o C .5 o » ff -4 >/ - B C 42 BACTERIA FIXING ATMOSPHERIC NITROGEN 123 origin from the atmosphere." Boussingault94 was the first to carry out a series of systematic studies on the nitrogen nutrition of leguminous and cereal plants. He established the fact that, in the cultivation of clover in unmanured soils, there is a definite gain, not only of carbon, hydrogen and oxygen, but also of large quantities of nitrogen; wheat, however, under the same conditions shows no gain or loss in nitrogen. Boussingault definitely expressed his opinion that nitrogen belongs to those elements which leguminous plants (clover, peas) can assimilate from the atmosphere, while cereal plants (wheat, oats) cannot do so. In attempting to repeat these experiments under more carefully controlled conditions, Boussingault ignited the sand (thus killing the bacteria) and found that neither cereals nor legumes were capable of assimilating atmospheric nitrogen.95 In an elaborate series of experiments begun in 1857 at the Rothamsted Experimental Station, Lawes, Gilbert and Pugh96 were so careful to eliminate any possibility of the plants obtaining any combined nitrogen from the atmosphere, that they destroyed the organism fixing the nitrogen symbiotically with leguminous plants; they thus failed to become the discoverers of this symbiotic relationship, since, in the absence of the bacteria, the leguminous plants behaved like the cereals and could not utilize the atmospheric nitrogen. Breitschneider97 demonstrated in 1861 that legumes do not fix any nitrogen when the soil is ignited but do so in unignited soil. Schulz-Lupitz98 grew lupines for fifteen consecutive times, without the application of nitrogen fertilizer and without diminishing yields; cereals following lupines gave much higher yields than on the same land not preceded by the leguminous crop; the nitrogen content of the soil was actually found to increase. The presence of nodules on the roots of leguminous plants was re- corded by Malpighi99 as early as 1687, but he, as well as others, con- 94 Boussingault. Recherches chimiques sur la vegetation enterprises dans le but d'examiner si les plantes prennent de l'azote de l'atmosphere. Compt. Rend. Acad. Sci.6: 102-112. 1838; 7: 889-892; Ann. Chim. et phys. (2), 67: 1-54 1838; 69: 353-367. 1838; Compt. Rend. Acad. Sci. 38: 580-607. 1854; 39: 601-613. 96 Ville. Note sur l'assimilation de l'azote de l'air par les plantes. Compt. Rend. Acad. Sci. 31: 578. 1850; 35: 464-468, 650-654. 1852; 38: 705-709, 723- 727. 1854;43:143-148. 1856. 96 Lawes, Gilbert and Pugh, 1861 (p. 106). 97 Breitschneider. Kann der freie Stickstoff zur Bildung der Leguminosen beitragen? Jahresber. Agr. Chem. 4: 123. 1861. 98 Schultz, L. Reinertrilge auf leichtem Boden, ein Wort der Erfahrung, zur Abwehr der wirtschaftlichen Noth. Landw. Jahrb. 10: 777-848. 1881. 99 Malpighi. Opera omnia. Anatomia plantarum, Pars II. De gallis. 1687, 126. 124 PRINCIPLES OF SOIL MICROBIOLOGY sidered them as root galls. Lachmann100 observed, in 1858, that motile bacteria cause the formation of the nodules and he suggested that the nodules are the organs of nitrogen fixation. In 1866 Woronin101 found the nodules to consist of bacteria, but even he considered these nodules as pathological outgrowths. Frank102 demonstrated in 1879 that the formation of nodules can be prevented by the sterilization of the soil. Frank's view as well as that of other investigators103 was that the nodules are caused by outside infection. Hellriegel and Wilfarth104 and Atwater105 • 106 finally demonstrated in 1884-1886 that the nodules on the roots of leguminous plants are due to bacterial infection, that this is beneficial, since it is within these nodules where the bacteria fix the atmospheric nitrogen. When nodules were formed, the plants could be grown on artificial soils containing but traces of combined nitrogen, provided the mineral elements necessary for the nutrition of the plant were present. In the absence of nodules, the plants were unable to utilize the atmospheric nitrogen for its growth. When sterilized soil was treated with fresh soil infusion, nodule formation took place and the plants grew normally. The growth of the Gramineae de- pended, however, on the nitrate content of the soil. These results were soon confirmed by Lawes and Gilbert107 and others. 100 Lachmann. tlber Knollchen der Leguminosen. Landw. Mitt. Zeitschr* K. Lehranstalt. u. Vers. Sta. 1858, p. 37. 101 Woronin, M. Observations sur certaines excroissances que presentent les racines de l'aune et du lupin des jardins. Ann. Sci. Nat. Bot., ser. 5, 7: 73-86. 1867; also Mem. Acad. Imp. Sci. St. Petersberg, 7 ser., 10: 1-13. 1866. 102 Frank, B. Uber die Parasiten in der Wurzelanschwellung der Papiliona- ceen. Bot. Ztg. 37: 377-388, 393-400. 1879. 103 Ward, M. On the tubercular swellings on the roots of Vicia faba. Phil. Trans. Roy. Soc. London 178, 1887. 104 Hellriegel, H. Welche Stickstoffquellen stehen der Pflanze zu Gebote? Tagebl. Natforsch. Vers. Berlin, 1886, p. 290; Chem. Centrbl. 1886, 871; Landw. Vers. Sta. 33: 464-465. 1886. Hellriegel, H., and Wilfarth, H. Untersuchungen uber die Stickstoff-Nahrung der Gramineen und Leguminosen. Beilageheft Ztschr. Ver. Riibenzuckerind. 1888, 1-234. 105 Atwater, W. O. On the assimilation of atmospheric nitrogen by plants. Rpt. Brit. Assn. Adv. Sci. 54: 685. 1884. 106 Atwater, W. O., and Woods, C. D. The acquisition of atmospheric nitrogen by plants. Amer. Chem. Jour. 6: 365. 1885; also 8: 398-420. 1886; Conn. (Storrs) Agr. Exp. Sta. Bui. 5, 1889; Conn. (Storrs) Agr. Exp. Sta. Ann. Rpt. 1889. 11-51. 107 Lawes, J., and Gilbert, J. New experiments on the question of fixation of free nitrogen. Proc. Roy. Soc. London 47: 85-118. 1890. BACTERIA FIXING ATMOSPHERIC NITROGEN 125 The causative organism was isolated in 1888, in pure culture, by Bei- jerinck,108 who named it Bacillus radicicola. Beijerinck described three stages in the development of the organism. 1 . The organism is present in the soil in the form of small rods which can penetrate the root hairs of the leguminous plants and from there it is transferred to the "infectious tissue." 2. The organism changes into a motile bacillus. 3. It changes into the bacteroid form which functions as the sym- biotic organism. The organism was soon grown, on artificial culture media, by a number of investigators.109 The mechanism of root infection by pure cul- tures of bacteria was worked out by Prazmowski in 1889. 109a Schloesing and Laurent110 demonstrated that the nitrogen is actually obtained by the bacteria in the form of nitrogen gas from the atmosphere. Legumi- nous plants were grown in sterile glass cylinders containing sterile sand and watered with sterile water. When the composition of the gas in the cylinder was determined, it was found that, while the uninoculated plants showed a gain of only 0.6 mgm. of nitrogen and no nodule forma- tion, inoculated plants showed a gain of 34.1 and 40.6 mgm. of nitrogen and abundant nodule formation. Nobbe and Hiltner111 concluded that the fixation of nitrogen by leguminous plants is closely related to the formation of bacteroids in the nodules. Nomenclalure. The causative organism of the nodules on the roots of leguminous plants is referred to by different names, depending on the particular system of classification. As pointed out above, the discoverer 108 Beijerinck, 1888 (p. 103). See also Prazmowski. Das Wesen und die biologische Bedeutung der Wurzelknollchen der Erbse. Bot. Centrbl. 39: 356- 362. 1889; Landw. Vers. Sta. 37: 161-238. 1890. 109 For a review of earlier literature see Voorhees and Lipman, 1907 (p. 491). Hiltner, 1904 (p. 128). Lohnis, 1910. Burrill and Hansen, 1917 (p. 126); Miiller, A., and Stapp, C. Beitriige zur Biologie der Leguminosenknollchenbakterien mit besonderer Beriicksichtigung ihrer Artverschiedenheit. Arb. Biol. Reich- sanst. L. u. Forstm. 14: 455-554. 1925. loss Prazmowski, A. Die Wurzelknollchen der Erbse. Landw. Vera. Sta. 37: 161; 38: 5. 1890. 110 Schloesing, Th., and Laurent, E. Recherches sur la fixation de l'azote libre par les plantes. Compt. Rend. Acad. Sci. Ill : 750-754. 1890; 113 : 776-778, 1095-1060. 1891; 115: 1017. 1892; Ann. Inst. Past. 6: 65-115, 824-940. 1892. 111 Nobbe, F., and Hiltner, L. Wodurch werden die knollchenbesitzenden Leguminosen befahigt, den freien atmospharischen Stickstoff fur sich zu ver- werten. Landw. Vers. Sta. 42: 459-478. 1893. 126 PRINCIPLES OF SOIL MICROBIOLOGY of the organism, Beijerinck, termed it Bacillus radicicola. In view of the fact that this is a non-spore forming organism and it is destroyed at 60° to 70°C, Prazmowski changed its name to Bacterium radicicola. The fact that a number of races produce only a single polar fiagellum led various investigators112 ,u3 to classify the organism with the genus Pseudomonas, under the name of Pseudomonas radicicola. E. F. Smith114 and the Committee of the Society of American Bacteriologists (p. 58) decided that the organism described by Frank115 in 1879 as Schinzia leguminosarum was the nodule forming organism and deserves priority; the name of Bacterium leguminosarum or Rhizobium leguminosarum was therefore suggested. It is doubtful, however, whether Frank ever saw the nitrogen-fixing, nodule-forming organism.116 According to Lohnis and Hansen,117 the nodule bacteria do not represent a special genus Rhizobium, but are closely related to Bad. radiobacter, Bact. lactis viscosum, Bact. pneumoniae and Bact. aerogenes, the last three being immotile and the first motile. The species differ only to a slight extent, in their physiological and morphological characters; the branched cell forms (so-called "bacteroids") are common to all mem- bers of the group of capsule bacteria, when tested adequately. These closely related forms are well distributed in the soil and Bact. radio- bacter may actually be present in the root nodules of leguminous plants. On account of its resemblance to Bact. radicicola, it has been mistaken for the nodule-producing organism in the cowpea-soybean group, since it grows rapidly on the plates made from the nodules; however, it can be differentiated from the latter by its brown growth on potato. Media. A number of media have been suggested, at various times, for the cultivation of the organism causing the nodules on leguminous 112 Moore, G. T. Bacteria and the nitrogen problem. Yearb. U. S. Dept. Agr. for 1902, 333-342. 113 Burrill, T. J., and Hansen, R. Is symbiosis possible between legume bac- teria and non-legume plants? 111. Agr. Exp. Sta. Bui. 202: 115-181. 1917. (Complete bibliography to 1915). 114 Smith, E. F. Bacteria in relation to plant diseases. Washington, 2: 97- 146. 1921. 118 Frank, B. Uber den gegenwartigen Stand unserer Kenntnis der Assimila- tion elementaren Stickstoffs durch die Pflanze. Ber. deut. bot. Gesell. 7: 234- 247. 1889; Landw. Jahrb. 19: 523-640. 1890; also Frank, 1879 (p. 124). 116 Kellerman, K. F. The present status of soil inoculation. Centrbl. Bakt. II, 34: 42-50. 1912. 117 Lohnis, F., and Hansen, R. Nodule bacteria of leguminous plants. Jour. Agr. Res. 20: 543-556. 1921. BACTERIA FIXING ATMOSPHERIC NITROGEN 127 plants. In addition to various organic media, extracts of carrots, of leaves and of seeds of leguminous plants, a number of inorganic media have been suggested. Of these, several may be selected: 1. Wood ash medium:118 Wood ash extract (15 grams ashes to 1 liter of tap water) . . 1000 cc. Sucrose 10 grams KH2P04 3 grams 2. Ashby's mannite solution (p. 113J. 3. Conn's asparaginate solution (p. 16) 4. Glucose medium:119 Distilled water 1000 cc. NaCl Trace Glucose 20 grams FeS04 Trace KH2P04 l.Ogram MnS04 Trace MgS04-7H20 O.lgram CaCl2 Trace 5. Sucrose medium: Tap water 1000 cc. KH2P04 1.0 gram Sucrose 10 grams MgS04 0.5 gram 6. Mannite medium:120 Mannite 10 grams CaC03 l.Ogram NaCl 0.2 gram Yeast water 100 cc. K2HP04 0.5 gram Distilled water 900 cc. MgSO 4.7H20 0.2 gram Washed agar 15 grams CaS04-2H20 0.1 gram The yeast water is prepared121 by stirring starch-free yeast with ten times its weight of tap water, steaming for 1 to 2 hours, then sterilizing and, after allowing to stand 24 hours, siphoning off the clear brown liquid. Various legume extract and tomato extract media are also employed: A decoc- tion of 100 grams material of the green plants and roots in 1000 cc. of water, to which 1 per cent glucose is added and some CaC03 to make the reaction neutral.1'22 For solid media, 1.2 to 1.5 per cent of agar is used; for gelatin media, 12 per cent of gelatin is used. When the reaction is adjusted by the hydrogen-ion con- centration method, it should be brought to pH 6.8 to 7.5. 118 Harrison, F. C, and Barlow, B. The nodule organism of the Leguminosae— its isolation, cultivation and commercial application. Centrbl. Bakt. II, 19: 264-272, 426^41. 1907; Trans. Roy. Soc. Can. Ser. (2), 12: 157-237. 1907. 119 Fred, E. B. A physiological study of the legume bacteria. Va. Agr. Exp. Sta. Ann. Rpt. 1911, 145-174. 120 Wright, W. H. The nodule bacteria of soybeans. I. Bacteriology of strains. Soil Sci. 20: 95-120. 1925. 121 Fred, E. B., Peterson, W. H., and Davenport, A. Fermentation character- istics of certain pentose destroying bacteria. Jour. Biol. Chem. 42: 175. 1920. 122 Nobbe, F., and Hiltner, L. Kunstliche Ueberfuhrung der Knollchenbak- terien von Erbsen in solche von Bohnen (Phaseolus). Centrbl. Bakt. II, 6: 449- 457. 1900. 128 PRINCIPLES OF SOIL MICROBIOLOGY Nodule formation. The bacteria usually enter the plant through the root hairs, being attracted through the secretion of soluble carbohydrates or organic acids by the plant. On entering the root, the bacteria multiply forming a thread of infection, similar to a fungus hypha, which enters the root and branches out into the parenchymatous cells of the plant. In some cells, the thread breaks up into individual cells which, on multiplication, fill the whole protoplasm of the cell; the bacteria give rise at the same time to branching forms, commonly referred to as "bacteroids." The size, form and position of nodules vary with the nature of the plant, soil in which it is grown and virulence of the bacteria, as shown by Hiltner,123 who explained nodule formation by his theory of immunity discussed elsewhere (p. 589). According to Bryan, ui nodule formation is greatly influenced by the reaction of the soil : alfalfa and clover produce maximum growth and number of nodules at pH 7.8, alsike and red clover at pH 5.6; the critical pH values for nodule formation are 4.0 and 9.0 to 10.0. Nodules will be formed at all temperatures at which the plant can make a growth that is at all vigorous.125 The presence of nitrates or other available nitrogen compounds in the soil depresses nodule formation. Isolation of organism from nodules. Harrison and Barlow126 describe in detail the method of isolation and cultivation of the organism. A medium sized nodule, appearing young and sound, is selected. It is cut off so as to leave 2 to 3 mm. of the root on both sides to permit handling it with forceps. The nodule is then washed, rinsed in distilled water and dropped into a sterilizing liquid containing 1 gram HgCl2 and 2.5 cc. c. p. HC1 in 500 cc. of water. The nodule is well shaken in the solution for 3 to 4 minutes, then washed three times in sterile distilled water. It is then covered with about 1 cc. of sterile distilled water and crushed with a sterile, heavy glass rod. Two or three drops of the cloudy suspension are placed into a test tube of the agar medium, which has previously been liquefied and cooled to 45°C. A second tube of agar is then in- oculated with five drops from the first; a third tube is inoculated from the 123 Hiltner, L. Die Bindung von freiem Stickstoff durch das Zusammenwirken von Schizomyceten und von Eumyceten mit hoheren Pflanzen. Lafar's Handb. techn. Mykol. 3: 24-70. 1904. 124 Bryan, O. C. Effect of reaction on growth, nodule formation and calcium content of alfalfa, alsike clover and red clover. Soil Sci. 15: 23-35. 1923. 126 Jones, F. R., and Tisdale, W. B. Effect of soil temperature upon the de- velopment of nodules on the roots of certain legumes. Jour. Agr. Res. 22: 17-31. 1921. 1,6 Harrison and Barlow, 1907 (p. 127). BACTERIA FIXING ATMOSPHERIC NITROGEN 129 second and a fourth tube from the third; the plates are poured and incubated at 20° to 25°C. The organism is isolated upon sterile agar slants or liquid media from a typical colony upon the plate, using the third and fourth plates and discarding the first two. The lens-shaped and pin-head colonies should be selected rather than the giant colonies. In case of questionable plates, replating may be necessary from the culture isolated. To keep the cultures in stock, one of the above agar media (ash or mannite agar) may be used. Isolation from soil. The Bad. radicicola can readily spread through the soil127 and persist there for a long period of time. The bacteria move in the soil at a definite rate.128 Bad. radicicola can also be cul- tivated from the soil, although the specificity of the forms isolated by Nobbe and Hiltner and Gage129 has not been sufficiently demon- strated. The results of Greig-Smith concerning the great abundance of Bad. radicicola in the soil were not confirmed. The numbers of each strain in the soil depend upon the reaction of the soil, an acidity greater than pH 5.4 being detrimental to the development of most strains; at a favorable reaction (pH 5.4-6.8, depending on strain), as many as 100,000 to 1,000,000 cells of different strains may be found per gram of soil.130 Kellermann and Leonard131 could isolate the organism only from soils sterilized and previously inoculated. Lip- man and Fowler132 isolated Bad. radicicola from soil, in which le- gumes have previously been grown, and demonstrated its ability to cause the formation of nodules on the roots of leguminous plants. Two media were employed: (1) 1000 grams of water, 10 grams maltose, 1 gram K2HP04, 1 gram MgS04, 2 to 3 drops each of 10 per cent solution of NaCl, FeCl3, MnS04, and CaCl2 and 15 grams of agar. (2) Soil extract, obtained by boiling one part of soil with three parts of water for one hour, then filtering and adding 15 grams of agar and 10 grams of maltose to 1 liter of the extract. A soil in which 127 Ball, O. M. A contribution to the life history of Bacillus (Ps.) radicicola Beij. Centrbl. Bakt. II, 23: 47-59. 1909. 128 Kellerman, K. F., and Fawcett, E. H. Movements of certain bacteria in soils. Science, 25: 806. 1907. 129 Nobbe, Hiltner and Schmid, 1895 (p. 134); Gage, G. E. Biological and chemical studies on nitrosobacteria. Centrbl. Bakt. II, 27: 7-48. 1910. 130 wnSon, J- K. Legume bacteria population of the soil. Jour. Amer. Soc. Agron. 18:911-919. 1926. 131 Kellerman, K. F., and Leonard, L. T. The prevalence of Bacillus radici- cola in soil. Science, n. s. 38:95-98. 1913. 132 Lipman, C. B., and Fowler, L. W. Isolation of Bacillus radicola from soil. Science, N. S. 41: 256-259, 725. 1915. 130 PEINCIPLES OF SOIL MICROBIOLOGY Vicia sicula has been grown a year before was used for plating out on these media. The capacity of the colonies developing on the plate to inoculate plants obtained from disinfected seed grown in sterile soil was then tested, and it was found that nearly half of the colonies were those of the organism in question. Vogel and Zipfel133 demonstrated by agglutination tests, using highly potent immune serum, that the nodule bacteria can be readily isolated from the soil; this method is even more reliable than the direct inocula- tion test, since, with the latter method, negative inoculation results are often obtained. Colony appearance. The colonies appearing on the plate are either surface or deep colonies. The first are drop-like, watery, mucilaginous in appearance, gray-white to pearly white in color, glistening, and semi- translucent to opaque. The edges are smooth and even ; they frequently attain a size of 1 cm. or more in diameter. The deep colonies are small, lens or spindle shaped, with smooth and even edges, opaque, granular in structure, and cream colored to chalky white. They slowly increase in size, eventually appearing on the surface, when growth becomes rapid. When first isolated, colonies may not appear before 6 to 14 days. Some races grow much faster than others, as in the case of Pisum, Vicia, Lupinus, Trifolium, Melilotus, and Medicago. To the slow growers belong the Vigna (cowpea), Glycine (Soja, soybean), and others (No. 46, PI. VIII). Morphology and life cycle of organism. The organism varies greatly in size and shape in the nodule. Many small, oval forms, described by Beijerinck as swarmers, and normal rods are found together with a few large club-shaped or branching forms (bacteroids) in the young nodules. In the old, decomposing nodule, the branching forms are extremely vacuolated, showing small, oval, deep staining bodies within.134 These bodies may be the motile swarmers or the branching form dividing into bacilli. In pure cultures, the organism forms minute short rods, motile when young by means of flagella.135 The bacteroids may be produced also 133 Vogel, J., and Zipfel, H. Beitrage zur Frage der Verwandtschaftsverhalt- nisse der Leguminosenknollchenbakterien und deren Artbestimmung mittels serologischen Untersuchungsmethoden. Centrbl. Bakt. II, 54: 13-34. 1921. 134 de Rossi, G. Uber die Mikroorganismen welche die Wurzelknollchen der Leguminosen erzeugen. Centrbl. Bakt. II, 18: 289-314, 481^89. 1907. 136 Barthel, Chr. Die Giesseln des Bacterium radicicola (Beij.). Ztschr. Gar- ungsphys. 6: 13. 1917. BACTERIA FIXING ATMOSPHERIC NITROGEN 131 on artificial culture media in the presence of acid phosphate,136 sodium succinate and glycerol,137 caffeine138 and cumarine.139 According to Barthel,140 caffeine and other vegetable alkaloids, like guanidine, pyridine and chinoline, will stimulate the formation of involution forms in pure culture; he suggested, therefore, that the formation of these so-called bacteroids in root nodules is due to the presence of alkaloids in the plant. The bacteroids are never so large and numerous on the artificial culture media as in a young nodule; they are produced, either in the medium, or in the nodule due to specific nutrition or to unfavorable conditions; in that stage they are hardy and multiply rapidly. According to Zipfel, the branching forms are not degeneration forms, but may be looked upon as a normal and necessary stage in the life of the organism with specific biological functions; they are formed from rods and change again into rods when inoculated into proper media. Five stages in the life cycle of the Bad. radicicola, through which it passes under cultural conditions, were recognized.141 1 . Non motile, pre-swarmer form, obtained in 4 to 5 days when a culture of the organism is placed in a neutral soil solution. 2. Larger, non-motile coccus. The pre-swarmer coccoid changes in the pres- ence of saccharose, certain other carbohydrates and phosphates, by increasing in size until the diameter has doubled. 3. Motile, swarmer stage, when the cell becomes ellipsoidal and develops high motility. 4. Rod-form, as a result of the further elongation of the swarmer, with decreas- ing motility. 5. Vacuolated stage. When available carbohydrates become exhausted or the organism is placed in a neutral soil extract, the cell becomes highly vacuolated and the chromatin divides into a number of bands. Finally these bands become rounded off and escape from the rod as the coccoid pre-swarmer. The pre- swarmer stage is usually formed from normal rods in calcareous soils, when 136 Stutzer, A. Die Bildung von Bakteroiden in kiinstlichen Nahrboden. Centrbl. Bakt. II, 7: 897-912. 1901. 137 Buchanan, R. E. The bacteroids of Bacillus radicicola. Centrbl. Bakt. II, 23: 59-91. 1909. 138 Zipfel, H. Beitrage zur Morphologie und Biologie der Knollchenbakterien der Leguminosen. Centrbl. Bakt. II, 32: 97-137. 1912. 139 Fred, 1911 (p. 127). 140 Barthel, C. Contribution a la recherche des causes de la formation des bacteroides chez les bacteries des Legumineuses. Ann. Inst. Past. 35: 634-647. 1921. 141 Bewley, W. F., and Hutchinson, H. B. On the changes throughwJufllLthe nodule organism (Ps. radicicola) passes under cultural conditionfl^JtyWr./&gr>\ Sci. 10: 144-162. 1920. /<^Oor' *o VN » -ft &\ ujl LIBRARY 3QJ 132 PRINCIPLES OF SOIL MICROBIOLOGY calcium or magnesium carbonates are added to the medium, or under anaerobic conditions. Acid soils cause the production of highly vacuolated cells and eventually kill the organism. These studies need further confirmation. Motility. In young agar slants, the organisms are found to be very motile. Owing to the slime produced by the organism, the demonstra- tion of flagella is very difficult; this was the reason for considerable disagreement among the different investigators. It has come to be recognized,142 however, that the nodule bacteria possess two types of flagellation: peritrichous and monotrichous. Differences, however, have been reported even for a single strain. The soybean organism was reported143 by some as possessing peritrichic flagellation, but by most other workers144 as monotrichous. The differences thus obtained were due either to the fact that cultures of various ages were employed or different types of bacteria exist, even for the same plant (as Soja max), in different parts of the world.145'146 For staining of flagella, the following modification of the Loeffler's stain may be used : Solution A parts Ferric chloride (1 : 20 aqueous solution) 1 Saturated aqueous solution of tannic acid 3 This solution improves with age; it should be at least a week or two old and should be filtered before using. Solution B parts Anilin oil 1 95 per cent alcohol 4 The bacterial suspension is allowed to air-dry on a clean cover glass. About 5 drops of solution A are then placed on the cover glass, followed immediately by 1 to 2 drops of solution B. The combination is allowed to act at room tem- perature for 2 minutes and is then washed in distilled water. The stain (30 parts of saturated alcoholic solution of methylene blue, 13 parts of solution B as mord- ant and 100 parts of 1 : 10,000 KOH solution) is applied for 2 minutes. 142 Hansen, R. Note on the flagellation of the nodule organisms of the Legum- inosae. Science. N. S. 50: 568-569. 1919. 143 Wilson, J. K. Physiological studies of Bacillus radicicola of soybean {Soja max Piper) and of factors influencing nodule production. Cornell Univ. Exp. Sta. Bui. 386. 1917. 144 Wright, 1925 (p. 127). 145 Shunk, I. V. Notes on the flagellation of nodule bacteria of leguminosae. Jour. Bact. 6: 239-246. 1921; Ibid. 5: 181-187. 1920. 140 Fred and Davenport, 1918 (p. 582). BACTERIA FIXING ATMOSPHERIC NITROGEN 133 Lohnis and Hansen and Shunk observed the two distinct types of flagellation referred to above. In the single flagellate types (monotri- chous) , the flagellum is not strictly polar but is usually attached to the corner. However, organisms obtained from nodules of different species of plants belonging to one genus have the same type of flagellation. Physiology of nodule bacteria. The different strains of Bad . radicicola are strictly aerobic. Maltose, sucrose, glucose and mannite offer the best sources of carbon; lactose, dextrin and glycerol can also be utilized. According to Beijerinck, separate carbon and nitrogen sources (asparagine, ammonium sulfate, sodium or potassium nitrate) are required. Laurent147 first showed that the organism can be cultivated on nitrogen- free media, containing 0.1 per cent KH2P04, 0.01 per cent MgS04 and 5 to 10 per cent of an available energy source. When grown on such a medium, it will fix atmospheric nitrogen.148 The presence of nitrates in the medium and in the soil diminishes nitrogen-fixation by the organism. This has been demonstrated by Nobbe and Richter149 and others, and it was found to be due not to any injurious influence of the nitrate but to the fact that the plant, capable of obtaining its nitrogen from the soil, represses the development of the nodules. A condition is found here very similar to the influence of nitrates upon nitrogen fixation by non-symbiotic bacteria. Prucha150 found that the addition of KN03, Ca(N03)2, NH4C1, or peptone to sandy soil, at the rate of 0.25 gram of the salts to 300 grams air-dry soil, had an inhibiting effect on nodule development of Canada field pea, while MgS04, KH2P04, Ca (H2P04)2 and tannic acid, especially in low concentrations, had a beneficial effect. The optimum reaction for the growth of the bacteria is pH 5.5 to 7.0, depending on the nature of the plant, with limiting reactions of pH 3.2 to 5.0 on the acid side, and pH 9.0 to 10.0 on the alkaline. The optimum temperature is 25° to 28°C. with 0° and 50° as the limits. 147 Laurent, E. Sur le microbe des nodosit6s des Lcgumineuses. Compt. Rend. Acad. Sci. Ill: 754. 1890; Ann. Inst. Past. 4: 722. 1890; 5: 105-139. 1891. 148 Fred, 1910 (p. 120). 149 Nobbe, F., and Richter, L. Uber den Einfluss des Nitratstickstoffs und der Humussubstanzen auf den Impfungserfolg bei Leguminosen. Landw. Vers. 56: 441-448. 1902; 59: 167-174. 1904. 150 prucha, M. J. Physiological studies of Bacillus radicicola of Canada field pea. Cornell Univ. Agr. Exp. Sta. Mem. 5, 1915. 134 PRINCIPLES OF SOIL MICROBIOLOGY The nodule bacteria can be modified in their ability to grow under unfavorable conditions; a character, such as tolerance to dyes, may be modified relatively quickly (Burke and Burkey).151 However, the character which has been lost as a result of cultivation on artifi- cial media is quickly regained when the culture is returned to the soil. Specific differentiation. Three groups of methods are usually em- ployed for the specific differentiation of the nodule bacteria: (1) plant inoculation, (2) morphological and cultural studies, (3) serological and immunological reactions. Although Nobbe, Hiltner and Schmid152 came to the conclusion that the bacteria in the nodules of all legumes are strains of the same organism, the fact was soon brought to light that not all the bacteria obtained from the nodules of various plants can cross-inoculate and produce nodules on the roots of other leguminous plants. These plants could readily be divided into several closely re- lated groups, the plants belonging to each group having their own specific organism, with cross inoculation taking place only by the mem- bers of each group. Hiltner and Stdrmer153 came to recognize, on the basis of morphological and cultural studies, two groups of nodule bacteria: (1) Bad. radicicola on Pisum, Vicia, Lathyrus, Phaseolus, Trifolium, etc., and (2) Bact. beijerinckii on Lupinus, Ornithopus, Glycine. The former grows well on certain gelatin media and readily produces branching forms, while the latter grows poorly on gelatin media. It was soon found that a further subdivision would have to be made, Pisum, Trifolium, Medicago and Lupinus bacteria being taken as representative types. Zipfel154,155 made use of agglutination tests and concluded that nodule bacteria were not varieties of the same species, but that distinct species existed. Six groups were thus distinguished: (1) Lupinus, (2) Trifolium, (3) Medicago, (4) Pisum, (5) Faba, and (6) Phaseolus. 151 Burke, V., and Burkey, L. Modifying Rhizobium radicicolum. Soil Sci. 20: 143-146. 1925. 162 Nobbe, F., Hiltner, L., and Schmid, E. Versuche iiber die Biologie der Knollchenbakterien der Leguminosen, insbesondere liber die Frage dei Arteinheit derselben. Landw. Vers. Sta. 45: 1-27. 1895. 153 Hiltner, L., and Stormer, K. Neue Untersuchungen iiber die Wurzelknoll- chen der Leguminosen und deren Erreger. Arb. k. Gesundhtsamt., Biol. Abt. 3: 151-307. 1903. 154Zipfel, 1912 (p. 131). 165 Vogel and Zipfel, 1921 (p. 130). BACTERIA FIXING ATMOSPHERIC NITROGEN 135 On the basis of serological investigation, Klimmer and Kruger156 formed nine groups of legume bacteria: (1) Lupinus and Ornithopus, (2) Melilotus, Medicago, and Trigonella, (3) Vicia (V. sativa), (4) Pisum, (5) Vicia faba, (6) Trifolium pratense, (7) Phaseolus vulgaris, (8) Soja hispida, and (9) Onobrychis sativa.157 Other serological studies158 confirmed the general conclusion that the nodule bacteria include more than one organism. The agar test-tube method may be used for the study of nodule forma- tion on the roots of legumes by different strains of bacteria.159 On the basis of the cultural method, the nodule bacteria were divided into the following groups: (1) alfalfa organism inoculating also Medicago lupu- lina, M. denticulata and Melilotus, (2) clover organism inoculating all species of Trifolium, (3) vetch and garden pea, (4) cowpea, (5) soybean, (6) garden bean. Burrill and Hansen160 demonstrated, by cross-inocula- tion studies, eleven kinds of bacteria divided into three groups, namely: (1) thin, scant, slow growth on ash-agar slant; little gum formed, flagella easily demonstrated — Vigna, Cassia, Acacia, Glycine, etc.; (2) more rapid and more abundant growth; glistening, opaque and pearly white; considerable gum formed which interferes with attempt of staining flagella — Melilotus, Medicago, Trigonella; (3) very fast, spreading growth; watery and semi-translucent; very slimy and sticky, due to excess of gum — Vicia, Pisum, Lens, Lathyrus, Trifolium, Phaseolus and Strophostyles. Lohnis and Hansen161 divided the bacteria of the leguminous plants into two groups, the representatives of which differ both morphologi- cally and physiologically. The first group shows all the features of Bad. radicicola; it is peritrichic, grows relatively fast on agar plates and 156 Klimmer, M., and Kruger, R. Sind die bei den verschiedenen Legumino- sen gefundenen Knollchenbakterien artverschieden? Centrbl. Bakt. II, 40: 256-265. 1914; Klimmer, M. Zur Artverschiedenheit der Leguminosen-Knoll- chenbakterien festgestellt auf Grund serologischer Untersuchungen. Centrbl. Bakt. II, 55: 281-283. 1922. 157 Simon, J. Uber die Verwandtschaftsverhaltnisse der Leguminosen-Wurzel- bakterien. Centrbl. Bakt. II, 41: 470-479. 1914. 168 Stevens, J. W. Can all strains of a specific organism be recognized by agglu- tination? Jour. Inf. Dis. 33: 557. 1923; A study of various strains of Bacillus radicicola from nodules of alfalfa and sweet clover. Soil Sci. 20: 45-66. 1925. 169 Garman, H., and Didlake, M. Six different species of nodule bacteria. Ky. Agr. Exp. Sta., Bui. 184: 343-363. 1914. 160 Burrill and Hansen, 1917 (p. 126). 161 Lohnis and Hansen, 1921 (p. 126). 136 PRINCIPLES OF SOIL MICROBIOLOGY changes milk characteristically; it produces nodules on the roots of clover, sweet clover, alfalfa, vetch, pea, navy bean, lupine, black locust, Amorpha and Strophostyles. The second group is characterized by monotrichic flagellation, comparatively slow growth on agar plates, and inability to cause a marked change in milk. It has been isolated from the soybean, cowpea, lima bean, peanut, beggarweed, Acacia, Genista Cassia and Amphicarpa. However, they do not suggest sepa- rating the organism into two new species before the complete life his- tory of the two groups is known. Bergey,162 following the system proposed by the Society of American Bacteriologists placed the Bad. radicicola in a separate genus "Rhizobium," and separated the different forms into two species: (1) Rh. leguminosum Frank, inoculating Pisum, Vicia, Lathyrus, etc., (2) Rh. radicicolum Beij. of Trifolium, Phaseolus, etc. The following is a list of leguminous plants, divided on the basis of inter- inoculation.163 The different members in any one group are those which can be inoculated by the strain of the Bact. radicicola specific for that group. Group I: Group III: Trifolium pratense, red clover Vigna sine7isis, cowpea Trifolium hybridum, alsike clover Cassia chamaecrista, partridge pea Trifolium alexandrinum, bersem Arachis hypogoea, peanut clover Lespedeza striata, japan clover Trifolium incarnalum, crimson clover Mucuna utilis, velvet bean Trifolium repens, white clover Baptisia linctoria, wild indigo Trifolium medium, zigzag, or cow Desmodium canescens, tick trefoil clover Acacia armata, acacia Group II: Genista tinctoria, dyer's greenwood Melilotus alba, white sweet clover Phaseolus lunatus, lima bean Melilotus officinalis, yellow sweet Group IV: clover Pisum sativimi arvense, Canada field Mcdicago sativa, alfalfa pea Medicago hispida, bur clover Vicia villosa, hairy vetch Medicago lupulina, black medick, or Vicia sativa, spring vetch yellow trefoil Vicia faba, broad bean Trigonella foenum-graccum, fenu- Lens esculenta, lentil greek Lathyrus latifolius, sweet pea 162 Bergey, 1923 (p. x). 163 Hansen, R. Symbiotic nitrogen-fixation by leguminous plants with special reference to the bacteria concerned. Scientific Agriculture (Canada) 1: 59-62. 1921; Whiting, A. L., Fred, E. B., and Helz, G. E. A study of the root nodule bacteria of wood's clover (Dalea alopecuroides). Soil Sci. 22: 467-476. 1926. BACTERIA FIXING ATMOSPHERIC NITROGEN 137 Group V: Group IX: Glycine hispida (Soja max), soybean Amorpha canescens, lead plant Group VI: Group X: Phaseolus vulgaris, garden bean Strophostyles helvola, trailing wild Phaseolus mullifiorus, scarlet runner bean Group VII: Gr°UPXI: „ Lupinus percnnis, lupine Robtma pseudo-acacia, black or com- Ornithopus saliva, seradella mon locust Group VIII: Group XII: Amphicarpa monoica, hog peanut Dalea alopecuroidcs, wood's clover An interchangeability between the soy bean and cowpea has been demonstrated164 in the laboratory, however. Field tests from other sources do not indicate such relationship. Various explanations for the specificity, based on soil reaction, climate, etc., have been proposed. Burrill and Hansen properly suggested that it may be a case of specific enzymes produced by the bacteria or of differences in the root-sap, which cannot be detected by chemical methods. So far we have to depend on cross inoculation and serological tests for the specific separa- tion. No morphological differences have yet been established, ex- cept the division into two groups suggested by Lohnis and Hansen; we do not know whether we are dealing here with different species or mere biological races. The application of serological reactions has brought out the fact that various strains of bacteria may form nodules on the same plant, but only one serological type is found in the same nodule.165 Other investigators166,167 also found that not all strains of Bad. radicicola of one leguminous plant are identical. This suggested the existence of various biotypes even for the same plant. The existence of two general types of the organism which can form nodules on the soy bean, identical morphologically but different physiologically and especially serologi- cally, has been demonstrated.167 These results are probably due to the fact that a bacterial culture is actually a population in which the different cells have variable proper- ties. Although morphology may not be sufficient to demonstrate any 164 Leonard, L. T. Nodule production kinship between the soybean and cow- pea. SoilSci. 15: 277-283. 1923. 165 Bialosuknia, W., and Klott, C. Badania nad Baklerium radicicola. Roczn. Nauk. Rolniczych. 9: 288-335. 1923. 166 Stevens, 1923-25 (p. 135). 167 Wright, 1925 (p. 127). 138 PRINCIPLES OF SOIL MICROBIOLOGY differences between the members of the population, physiological reac- tions and the even more sensitive serological reactions can bring out these variations. This explains the modification of a strain when grown on artificial culture media or as a result of repeated passage through the host plant. It also suggests the possibility of improving or deteriorating a strain by the proper selection of the types of cell. This phenomenon explains the increase in activity and fixation of nitro- gen by repeated passage through plants.168 The process of adaptation to a particular host plant is longer in case of vegetatively weak organ- isms than for vegetatively strong organisms. A detailed study of the chemistry of nitrogen fixation by nodule bacteria (588) and the artificial inoculation of soil with bacterial cul- tures (817) will be discussed elsewhere. Nodule formation by non-leguminous plants. In addition to the legumes, a number of non-legumes are found possessing nodules on their roots. Of these, most attention has been paid to Ceanothus (red-root) , Elaeagnus (silver berry), Alnus (alder), Podocarpus, Cycas and Myrica (sweet gale). At first these nodules were thought to be of fungus origin. The nodules of Alnus, Elaeagnus and Ceanothus were found169-171 to be caused by bacteria belonging to the Bad. radicicola group and capable of fixing nitrogen. In some plants at least (Myrica) the organism is of the nature of an Actinomyces.172 Coriaria japonica produces nodules similar to those produced by the Alder, due probably to an Actinomyces (Act. myricae according to Peklo) in both cases.173 In the roots of cycads, Bad. radicicola, Azotobacter and an alga ( Anabaena) were demon- 168 Wunschik, H. Erhohung der Wirksamkeit der Knollchenerreger unserer Schmetterlingsblutler durch Passieren der Wirtpflanze. Centrbl. Bakt. II, 64: 395-445. 1925. 169 Hiltner, L. Uber die Bedeutung der Wurzelknollchen von Alnus glutinosa fur die Stickstoffernahrung dieser Pflanze. Landw. Vers. Sta. 46: 153-161. 1896. 170 Kellerman, K. F. Nitrogen-gathering plants. Yearb. Dept. Agr. U. S. A., 1910, 213-218. 171 Bottomley, W. B. The root nodules of Ceanothus americanus. Ann. Bot. 29: 605-610. 1915. 172 Arzberger, E. G. The fungous root-tubercles of Ceanothus americanus, Elaeagnus argentea, and Myrica cerifera. Mo. Bot. Gard. 21 Ann. Rpt. : 60-103, 1910. 173 Shibata, K., and Tahara, M. Studien uber die Wurzelknollchen. Bot. Mag. Tokyo, 31: 157-182. 1917. BACTERIA FIXING ATMOSPHERIC NITROGEN 139 strated.174 Burrill and Hansen175 came to the conclusion that the root-nodules of Ceanothus (C. americanus), Alnus, Cycas revoluta, and Myrica are not caused by Bad. radicicola. The evidence that Elaeagnus and Podocarpus nodules are caused by B. radicicola is not conclusive. It is still questionable whether nitrogen fixation by any of these plants takes place,176 although it is claimed176* that some plants (like Casua- rina) are thus able to grow readily in very poor sandy soil. The ques- tion of symbiosis with fungi (mycorrhiza formation) is discussed else- where. It is of interest to point out, in this connection, that there are legumi- nous plants, which do not form any nodules. These include Gymno- cladus, Carcis, Gleditsia and the Cassias of the subfamily Caesal- pinaceae. Nodule formation in the leaves of some plants. A condition similar to nodule formation by bacteria on the roots of leguminous plants has been observed on the leaves of certain tropical plants, namely the Myrsina- ceae, such as Ardisia, certain Rubiaceae, such as Pavetta and Grumilea. Koorders177 demonstrated the presence of either bacteria or fungi in the bloom bud hydathodes of nineteen species of tropical plants, repre- senting six genera; a symbiotic relation was found to exist between the host plant and the microorganisms. Zimmermann178 was the first to show that the nodules on the leaves of the Rubiaceae (four species examined) are filled with bacteria. He also found nodules on the upper side of the leaf of Pavetta lanceolota and on the under side of P. angusti- folia. The bacteria were present in chains and as longer forms. P. indica had even a greater number of nodules scattered over the whole surface of both sides of the leaf and formed dark green spots. The bacteria do not penetrate the cell but are found in the intra-cellular 174 Spratt, E. R. The formation and physiological significance of root nodules in the Podocarpineae. Ann. Bot. 26: S01-814. 1912; The root nodules of the Cycadaceae. Ibid. 29: 619-626. 1915. 178 Burrill and Hansen, 1917 (p. 126). 176 Miehe, H. Anatomische Untersuchung der Pilzsymbiose bei Casuarina equisetifolia nebst einigen Bemerkungen liber das Mykorhizenproblem. Flora, 111-112: 431-449. 1918. 176aRao, R. A. Casuarina root nodules and nitrogen fixation. Madras Agr. Dept. Yearbook, 1923, 60-67. 177 Koorders, S. H. Uber die Bluthenknospen Hydathoden einiger tropischen Pflanzen. Ann. Jard. Bot. Buitenzorg, 14: 354-477. 1897. 178 Zimmermann, A. tlber Bakterienknoten in den Blattern einiger Rubiaceen. Jahrb. wiss. Bot. 37: 1-11. 1902. 140 PRINCIPLES OF SOIL MICROBIOLOGY spaces. An organism belonging to the mycobacteria (My cob. rubia- cearum) was isolated179 from the leaf nodules. The same organism was also isolated from Pavetta and other plants. Miehe180 isolated a rod- shaped organism, Bac. foliicola, active in forming nodules on the leaves of Ardisia. It is a motile rod (1-2.5 by 0.4-0.5ju) with peritrichic fiagella and later changes into a branching form. These "bacteroids" may be found in the cells of the leaves and also on special media. The bacteria are already present in the seeds, between the embryo and the endosperm, so that the plants do not have to be inoculated anew with each new growth. In this respect they are similar to the endotrophic mycorrhiza of the Ericaceae which are considered elsewhere. When the young plants grow, the bacteria follow the growing tip to the new parts of the plant, as they develop. The bacteria are eventually found in the entire plant, where they develop in masses in the intracellular spaces. With the development of the fruit, the bacteria are enclosed in the embryo sack and remain with the seed. Miehe concluded that Bac. foliicola fixed nitrogen; he recognized this phenomenon as one of hereditary symbiosis. The bacteria forming nodules on the leaves of Pavetta indica and Chomelia asiatica enter the stomata of the leaf, live there and fix the nitrogen from the air. The bacteria are found at all the life stages of the plant, symbiosis being developed to a much greater extent than in the Leguminosae and being hereditary in nature. Plants freed from bacteria, by warming the seed for 25 minutes at 50°C, develop very slowly and suffer from lack of nitrogen. The bacteria are aerobic, rod-shaped cells.181 The presence of nitrogen-fixing bacteria in the swollen glands on the points of the leaves of Dioscorea macroura has also been demonstrated.182 179 von Faber, F. C. Die Bakteriensymbiose der Rubiaceen. Jahrb. wiss. Bot. 54: 243-264. 1914; also Ibid. 51: 285-295. 1912. 180 Miehe, H. Weitere Untersuchungen liber die Bakteriensymbiose bei Ardisia crispa. Jahrb. wiss. Bot. 53: 1-54. 1913; 58: 29. 1917. Ber. Bot. Gesell. 29: 156. 1911; 34: 576. 1916. 181 Rao, K. A. A preliminary account of symbiotic nitrogen fixation in non- leguminous plants, with special reference to Chomelia asiatica. Agr. Jour. India, 18: 132-143. 1923. 182 Orr, M. V. Nitrogen fixation in leaf glands. Notes from the Roy. Bot. Gard., Edinburgh, 14: 57-72. 1924. CHAPTER V Heterotrophic, Aerobic Bacteria Requiring Combined Nitrogen General classification. The heterotrophic bacteria requiring combined nitrogen comprise the large numbers of organisms developing on the common plate used for counting bacteria and probably a still greater number of organisms, which develop very slowly or do not develop upon the plate at all. Morphologically they consist of spore-forming and non-spore forming rods, cocci and spirilli. Physiologically they take part in numerous soil processes, especially in the decomposition of both simple and complex organic substances in the soil including pro- teins, their derivatives, and other nitrogen compounds ; celluloses, pen- tosans, and other complex and simple carbohydrates; fats and various other ingredients of natural organic matter. Morphology alone is an insufficient basis for the classification of these organisms. Just as in the general classification, one must consider the various physiological processes in which these bacteria are concerned. The system used here is far from satisfactory, due to insufficient knowledge concerning the organisms themselves. This system is bound to change with the advance of our knowledge. The cellulose-decomposing bacteria, the nitrate and sulfate-reducing bacteria, and the urea-decomposing organisms are treated separately, because of their specific physiology and the special methods which are essential for their isolation, cultivation and study. Some of these, especially some of the urea-decomposing forms and nitrate-reducing bacteria, are no doubt modifications of the more general groups consid- ered here. As a general basis of classification, the following one may be used conveniently: I. Aerobic bacteria: 1. Spore-forming rods 2. Non-spore forming rods 3. Cocci 4. Spirilli II. Anaerobic bacteria The difference between the aerobism and anaerobism of soil bacteria is largely one of degree and not of kind, as will be shown later. The 141 142 PRINCIPLES OF SOIL MICROBIOLOGY anaerobic bacteria, especially the obligate forms, will be treated sepa- rately because of their peculiar physiology. Of the two groups of aero- bic rod-shaped bacteria, the non-spore formers are more numerous than the spore-formers. The latter usually become very active when fresh organic matter, rich in proteins, is added to the soil but they soon sporulate and generally remain in the soil in that condition until another favorable period arrives. The non-spore forming bacteria and cocci, living upon the colloidal film surrounding the inorganic soil particles, make up the bulk of the numbers of the soil population. Most of these organisms have not yet been described at all or only very insufficiently. Their physiological activities are also insufficiently studied and their role in soil processes is little understood. Spore-forming bacteria. The spore-forming, aerobic, heterotrophic bacteria have been studied more completely than the non-spore formers or the anaerobic bacteria. This is due to the fact that they readily develop on the common gelatin and agar media, forming large charac- teristic colonies. When a short period of incubation is used, they are found to be among the most numerous organisms developing on the plate. Houston1 found in 1898 four common spore-forming bacteria in the soil: Bac. mycoides, Bac. subtilis (which was, according to Conn,1 Bac. cereus), a "granular bacillus," equivalent to Bac. megatherium, and Bac. mesentericus representing a group composed of a number of ill- defined, small spore-forming organisms.2 Houston states that Bac. mycoides is present in the vegetative stage and as spores. Others3 found the spore-forming bacteria to be present in the soil only in the 1 Houston, 1898 (p. 14). 2 Conn, H. J. Soil flora studies. III. Spore-forming bacteria in soil. N. Y. Agr. Exp. Sta. Tech. Bui. 58. 1917. 3 Conn, H. J. Are spore-forming bacteria of any significance in soil under normal conditions? N. Y. Agr. Exp. Sta. Tech. Bui. 51. 1916. PLATE VIII 43. Bacterium phlel: A, colony of organism on washed agar containing inor- ganic salts, with petroleum vapor as the only source of energy; B, colony on agar with inorganic salts and 1 per cent glycerol (after Sohngen and de Rossi). 44. Deep agar colonies of anaerobic bacteria: colonies of Bac. perfringens in nitrate glucose agar (from Weinberg and Seguin). 45. Deep agar colonies of anaerobic bacteria: Bac. pulrificus in glucose agar, 48 hours old (from Weinberg and Seguin). 46. Ash-agar plate showing the organism forming nodules on the roots of a, Genista tincloria, 25 days old; b, Pisurn sativum, 7 days old (from Burrill and Hansen). PLATE VJII 43 # 0, 46 HETEROTROPHIC BACTERIA REQUIRING COMBINED NITROGEN 143 form of spores. The spores vegetate in the presence of a large amount of organic matter and an excess of moisture.4 A detailed study of the spore-forming bacteria has been made by various investigators5-8 and recently by Ford9 and associates, whose work is used as a basis for the following classification. Classification of Spore-forming Bacteria9 Group I. Subtilis group Small, homogeneous, sluggishly motile organisms measuring 0.4 by 1.5 to 2.5ju. No threads on glucose agar. Central or excentric spores, oval, measuring 0.5 by 0.75 to 0.88;u, often retaining terminal tags of protoplasm. Growth on solid media hard and penetrating, with tenacious scums on fluid media. Bacillus subtilis Cohn Bacillus subtilis-viscosus Chester (Characterized by viscosity) Group II. Mesenlericus group Small, homogeneous, actively motile organisms measuring 0.5 by 2 to 4^. They often produce long threads on glucose agar. The spores measure 0.5 by 1 to 1.12/i, oval and retaining terminal tags of protoplasm. The growth on solid media is a soft pultaceous mass with tendency to wrinkle; on fluid media, growth is in the form of a friable easily-broken scum. Bacillus vulgatus (Flugge) Trevisan (Bacillus mesentericus vulgatus Flugge) Bacillus mesentericus (Flugge) Migula (Bacillus mesentericus fuscus Flugge) Bacillus aterrimus Lehmann & Neumann (Bacillus mesentericus niger Lunt) Bacillus globigii Migula (Bacillus mesentericus ruber Globig) 4 Winogradsky, 1924 (p. 542). 6 Gottheil, O. Botanische Beschreibung einiger Bodenbakterien. Centrbl. Bakt. II, 7: 430^35, 449^65, 481^97, 529-544, 582-591, 627-637, 680-691, 717-730. 1901. 6 Neide, E. Botanische Beschreibung einiger sporenbildenden Bakterien. Centrbl. Bkt. II, 12: 1-32, 161-176, 337-352, 539-554. 1904. 7 Chester, F. D. Observations on an important group of soil bacteria. Organ- isms related to Bacillus subtilis. Del. Agr. Exp. Sta., Rept. 15: 42-96. 1904. A review of the Bacillus subtilis group of bacteria. Centrbl. Bakt. II, 13: 737- 752. 1904. 8 Holzmiiller, K. Die Gruppe des Bacillus mycoides Flugge. Centrbl. Bakt. II, 23: 304-354. 1909. 9 Ford, W. W., Lawrence, J. S., Laubach, C. A., and Rice, J. L. Studies on aerobic spore-bearing non-pathogenic bacteria. Jour. Bact. 1: 273-320, 493-534. 1916. 144 PRINCIPLES OF SOIL MICROBIOLOGY Bacillus niger Migula {Bacillus lactis niger Gorini) Bacillus mesentericus var. flavus Bacillus panis Migula (Bacillus mesentericus panis viscosus Vogel) (Motility lost by capsule formation) Group III. C ohaer ens-simplex group Motile organisms somewhat larger than either Bacillus subtilis or Bacillus mesentericus, measuring 0.37 to 0.75 by 0.75 to 3^. Thicker and longer forms on glucose agar. Involution and shadow forms are common and appear early. The spores are cylindrical, measuring 0.56 to 0.75 by 1 to 1.5^. A soft mass is formed on solid media; turbidity with little or no scum on liquid media. Bacillus cohaerens Gottheil Bacillus simplex Gottheil Bacillus agri Ford and associates Bac. asterosporus and Bac. teres A. M. and Neide belong also to this group. Group IV. Mycoides group Large organisms with square ends growing in long chains. Single cells measure 0.5 by 3 to 6fi. On glucose agar, the organisms are thicker and longer and are made up of globular bodies. Tendency for the organisms to grow in curves or spirals. The spores are central or excentric, round or oval to cylindrical, measur- ing 0.75 to 1 by 1 to 2 fi. Dry and penetrating growth on solid media; firm tenacious scum on liquid media. Bacillus mycoides Flugge Bacillus prausnitzii Trevisan (Bacillus ramosus liquefaciens Prausnitz) Bacillus adhaerens Ford and associates (No motility) Group V. Cereus group Large, motile organisms with rounded ends, measuring 0.75 by 2.25 to 4fi. Tend to grow in short chains. Thicker and longer on glucose agar, where protoplasm is converted into globular bodies. Central or excentric spores, cylindrical, meas- uring 0.5 to 0.75 by 1.12 to 1.5/x. Spores retain protoplasm at one or both ends, often resembling enlarged subtilis or mesentericus spores. A soft pultaceous mass is formed on solid media, with tendency to fold or wrinkle; thick friable scum on liquid media. Bacillus cereus Frankland (The Bac. ellenbachensis often referred to as an important soil organism belongs here). Bacillus albolactus Migula Bacillus cereus var. fluorescens Ford and associates Group VI. Megatherium group Very large, actively motile organisms, measuring 0.75 to 1.25 by 3 to 9p. Long forms are often produced ; these spread out, lose their cytoplasm and show peculiar HETEROTROPHIC BACTERIA REQUIRING COMBINED NITROGEN 145 aggregations of protoplasm at the periphery. The protoplasm is rapidly con- verted into peculiar globular, highly refractile bodies, particularly on glucose agar. Shadow and transparent forms appear early. The spores are central, excentric or sub-terminal, oval to cylindrical, measuring usually 0.75 to 1.12 by 1.5 to 2ju. Spores vary greatly in shape, being sometimes round, sometimes rectangular, often reniform. Growth on solid media as thick pultaceous mass, on liquid media as turbidity with little or no scum formation. Bacillus megatherium De Bary Bacillus petasites Gottheil Bacillus ruminatus Gottheil Group VII. Round terminal spored group Small, actively motile organisms, measuring 0.5 to 0.75 by 1.5 to 3ju, often forming long threads in old cultures. Protoplasm homogeneous. Spores sub- terminal or terminal, round, thicker than the organisms from which they spring, measuring 1 to 1.5ju in diameter. Bacillus pseudotetanicus (Kruse) Migula (Bacillus pseudotetanicus var. aerobius Kruse) Bacillus fusiformis Gottheil Group VIII. Cylindrical terminal spored group Small, thin, actively motile organisms, measuring 0.37 to 0.5 by 2.5 to 4/z. Slightly larger on glucose agar but no change in character of protoplasm. Spores terminal, cylindrical, measuring usually 0.75 by 1.12 to 1.5/*. Bacillus circulans Jordan Bacillus brevis Migula Bacillus terminalis Migula Group IX. Central spored group L^ng, actively motile organisms with pointed ends, measuring 0.37 to 0.5 by 1.12 to 4ju. Slightly larger on glucose agar, but no change in character of proto- plasm. The spores develop in the middle of the rods, which become spindle- shaped. The spores are large, cylindrical, measuring 0.6 to 0.8 by 1.12 to 1.5/x. Bacillus centrosporus Ford and associates Bacillus laterosporus Ford and associates A summary of the characteristic points of the spore-forming bacteria, recognized by A. Meyer and his associates is given in table 13. 10 Occurrence of aerobic, spore-forming bacteria in the soil. By the use of gelatin plates the three most common spore-forming bacteria in the soil can be readily recognized by speed of gelatin liquefaction and type of colony. Except for the non-spore forming Bad. fluorescens, Bac. mycoides is the most rapid liquefier; it produces large filamentous to rhizoid colonies. Bac. cereus liquefies gelatin almost as rapidly as Bac. 10Stapp, 1920 (p. 213). 146 PRINCIPLES OF SOIL MICROBIOLOGY 23 0> t-H -3 a a J3 $ a ■£ m c a £ OS 2 a 2; * — S 'S ■< a a. 03 02 ^3 Lai S tfi -s -o js -a £ a :£ c o cj o cs a a i s £ 2 ■a 2 * Si a •S ° il .2 Si a fc « « £ Z + I + I + 5 S5 £2 I + + + + I Si!* P a o SgB H 0. o° fc a; H o 3 H ,_, a) !> O o < i I i 00 1 oo T 1 00 1 1 o i o o pj o N -^ o e i *7 "71 T 1 1 T "j1 ? o •* t~ •* < K M go O —I rt O O •-! O0 rt ■ I OO •"" CO OcJ, ~ o w a : a S 03 Oq 03 03 03 03 HETEROTROPHIC BACTERIA REQUIRING COMBINED NITROGEN 147 .£! 5 ,3 5 "3 3 "^ 3 "° ^ ft '3 IS '3 ^ a '3 > _g £ fc 0 £ w pq i3 W » WWW £> -a" ^ -a ts J c 5 3 01 »"c ■$ § 2 a * O S J3 S O S •v ? +s 3 ~v 3 *£ =3 £ ^ O P> o£ J ai >■ o ■>■ i> !> o w O pq £ a £ a c a S S 1 g S a g °E5 £ .2 Ml a © - ^ CN 00 f. Y CO CO o CD »-H ■^ © oo Th o CN « o cji a> ~ oo *-l co x co o r~ ©■<*<© > © >, !**! S H £ U O pq w O K P£ u w Pi P C c :- Pi 3 s K •3 g a ^r J "3 1 "3 s 5 i 1 1 "ci 1. § c 1. f | i ■2 fit 05 03 a: « 05 «5 a K BS a « « K fit a oa 148 PRINCIPLES OF SOIL MICROBIOLOGY H H < ■« 3 H 5 < H a I fa w H O H Ph ^ O •< o t> 5 ^ 55 6 H O SO M CO *"H K to O h-l a m & OS & X cp b- *^ o «5 lO —i o • o IH —" —I —1 IM N o .J? o 3 05 09 cq 05 05 1 05 j, •= -b ■< * .9 0 B '"H '3 SkHSf — « o it! •- •3-2(3 2 & o S fl 3 » EH cc -d '3 T3 ^ 73 a) o 5 8 fl o m 3 a 2 S g 3 -a a rt . <- .2 *2 M & - — * — * £ -a 2 ^ m >-j « — a ~ > © - 1-1 3 2 9 S^'O 3 ■H . . ,»> -9 £ M "2 ^aaH^^SS SSSslffeJi 0 O g :£ - - r 3 . J "fl 1H - O rf T3 fl J J 47 47. Life cycle of Azotobacter chroococcum: a, formation of symplasm by regenerative bodies on potato, in 9 days; b, regenerative units starting to grow, on beef gelatin, in 4 weeks; c, regenerative bodies growing from symplasm, on beef agar, in 4 weeks; d, formation of new cells by agglomeration of regenerative units on mannite soil extract, in 4 days; X 600 (from Lohnis and Smith). 48. Influence of composition of medium upon the morphology of Bact. ■pneumoniae: A, on beef agar, 1 day at 37°C; B, on egg agar, 1 day at 19°C; C, on starch agar, 1 day at 19°C; X 660 (from Scales). 49. Some typical soil bacteria, as shown by the India Ink method" A, short non-spore forming rods (bacteria); B, long, non-spore forming rods (bacteria); C, spore-forming rods (bacilli) (from Kursteiner). HETEROTROPHIC BACTERIA REQUIRING COMBINED NITROGEN 159 inorganic source of nitrogen and inoculated with a soil suspension, if the cultures are placed under a bell jar together with a dish of benzene or petroleum, and incubated at 30°C. The final isolation and purification of the organisms can be accomplished by means of ordinary bacteriological methods. Spore formation takes place by contraction of the cell contents, in a manner similar to that of the actinomyces (p. 294), giving coccus-like fragments. The colonies on solid substrates have a certain thread-like structure (No. 43, PI. VIII). The organisms readily utilize various hydrocarbons as sources of energy.54 They do not form any ammonia from proteins;53 most of them reduce nitrates to nitrites (similar to actinomyces). Their role in the soil seems to consist largely in the decomposition of certain organic compounds. Myxobacteria. Mycobacteria occur abundantly in manure and probably take a part in the decomposition of certain constituents of natural organic materials. To demonstrate the presence of Myxo- bacteria in the soil, balls of rabbit manure, previously moistened with water and sterilized in the autoclave, are placed on the surface of the particular layer of soil. Frequently 7-10 species are thus obtained from one soil sample.56 5S Krzemieniewsky, H. and S. Die Myxobakterien von Polen. Acta Soc. Bot. Poloniae. 4: 1-54. 1926. CHAPTER VI Anaerobic Bacteria Oxygen tension in the growth of bacteria. Pasteur1 was the first to demonstrate that there are organisms, among them yeasts, which can live in the presence of only small traces of oxygen. Since the growth of the microorganisms is so abundant that the small amount of oxygen present is rapidly used up, it can be assumed that the greater part of their development takes place in the absence of free oxygen. Pasteur has further shown that, in the case of yeasts, growth in the absence of oxygen takes place only in the presence of sugar utilizable by these organisms. Those organisms which are able to grow both in the pres- 1 Pasteur, L. Animalcules infusoires vivant sans gaz oxygene libre et deter- minant des fermentations. Compt. Rend. Acad. Sci. 52: 360. 1861; Experi- ences et vues nouvelles sur la nature des fermentations. Ibid., 1260; 56 : 416, 1189- 1863; 75: 784. 1872; 80: 1875. PLATE X Heterotrophic Aerobic and Anaerobic Bacteria 50. Bac. mycoides, X 660 (from Conn). 51. Bac. cereus, X 660 (from Conn). 52. Bac. megatherium, X 660 (from Conn). 53. Bac. simplex, X 660 (from Conn). 54. Bad. vulgare, X 660 (after Omeliansky). 55. Bad. pyocyaneum, X 660 (after Omeliansky). 56. Bad. fluorescens, X 600 (after de Rossi). 57. Bac. butyricus: a, non-spore forming; b, spore forming, X 660 (from Omeliansky). 58. Bac. sporogenes: a, 24 hour culture upon glucose bouillon; b, flagella, stained by Loeffler's method (from Weinberg and Seguin). 59. Bac. pulrificus, 48 hour old colony in deep glucose agar (from Weinberg and Seguin). 60. Bac.probatus: A, non-sporulating bacilli of a fresh agar culture; B, sporu- lating bacilli of an agar culture 4-8 days old; C, spores with adhering membrane of a 2 to 3 week old culture upon potato, X 1300 (after Viehoever and de Rossi). 61. Sarcina ureae, X 660 (after Omeliansky). 62. Bac. nitroxus, 3-day old culture, grown at 30°, X 480 (after Beijerinck and Minkmann and de Rossi). 63. Spirillum desulfuricans, X 660 (after Beijerinck and Omeliansky). 160 PLATE X o fl o o o G 0 50 CR>a*g» d =tccooo 11 «•* <«■ G0000J0 52 ""' ^""T* 53 51 V \ V\J ' r i * \ 54 55 / A- -/ v / 56 / *-,/ ^ ^ 5S 59 57 , 7 „= ' 6Z 60 v / I ANAEROBIC BACTERIA 161 ence and absence of free oxygen were termed by Liborius2 "facultative anaerobes." It has also been observed by Pasteur that certain butyric acid bacteria grow abundantly in the liquid medium, through which a current of carbon dioxide is passed, but are destroyed, when a current of air is passed for 2 hours through the liquid. Those organisms, which are unable to thrive under partial oxygen pressure and cannot withstand even small amounts of oxygen, were termed by Liborius "obligate an- aerobes." Beijerinck3 divided the bacteria into two groups, according to their oxygen need: (1) "aerophile," or those requiring a high oxygen tension, including the aerobes and facultative anaerobes, which can grow in ordinary atmosphere; and (2) "microaerophile," or those organ- isms that require a more or less low oxygen tension and do not grow readily in ordinary atmosphere. The influence of oxygen on some bac- teria was illustrated by the accumulation of the cells in a hanging drop preparation; the aerophiles gathered in the outer zone, while the micro- aerophiles massed together where the oxygen tension was least. The spirillum type was intermediate. Burri4 could not agree with this divi- sion and suggested that the terminology of Liborius is much more appropriate. Not only obligate anaerobic bacteria, but also the facul- tative forms were able to live in the complete absence of oxygen for a number of generations without being injured. No general minimum oxygen tension could be found for all obligate anaerobic bacteria, but the various anaerobic forms varied in the limit of this tension :5 the oxygen limit for the blackleg bacillus (Bac. chauvoei) is 1.04 per cent oxygen in the atmosphere, 0.65 per cent for Bac. tetani, 0.27 per cent for Clostridium butyricum and 0.13 per cent for Bactridium butyricum; the obligate anaerobic bacteria could be so adapted as to withstand some amounts of oxygen. Even many species which usually grow in the complete absence of oxygen, such as Bac. amylobacter, can thrive in the presence of oxygen. A typical obligate anaerobe has no minimum oxygen tension limit, it is characterized by the existence of a very low maximum oxygen tension and it can grow in the total absence 2 Liborius, P. Beitrage zur Kenntnis des Sauerstoffbediirfnisses der Bakterien. Ztschr. Hyg. 1: 115. 1886. 3 Beijerinck, M. W. Ueber Atmungsfiguren beweglicher Bakterien. Centrbl. Bakt. 14: 827-845. 1893; also Arch. Neerland., Ser. II, 2: 397. 1899; Pheno- menes de reduction produits par les microbes. Ibid. 9: 131. 1904. 4 Burri. R. Intramolekulare Atmung, Anaerobiose und Mikroaerophilie. Centrbl. Bakt., II, 17: 804. 1907. 5 Chudiakow, N. Zur Lehre von der Anaerobiose. Moskau. 1896 (Centrbl. Bakt. II, 4: 389-394. 1898). 162 PRINCIPLES OF SOIL MICROBIOLOGY of oxygen. We do not know of any true anaerobes which grow only in the complete absence of oxygen. Small quantities of free oxygen will even act as stimuli to obligate anaerobes. The oxygen need of an organism was characterized6 by the "cardinal points" for growth and spore formation, namely: minimum, optimum, and maximum, so that there is a gradual transition between aerobes and anaerobes. The following cardinal points for spore formation charac- terize a series of typical bacteria, atmospheric air at 18° and 750 mm. pressure containing 276 mgm. of oxygen per liter: Bac. amylobacter. Bac. asterosporus Bac. fusiformis. . Bac. mycoides Bac. simplex Bac. subtilis Bac. lactis MINIMUM OPTIMUM MAXIMUM mgm. mgm. mgm. 0 10 (?) About, 25 0 100 5,600 6.8 70 1,061 4.3 70 1,336 6.8 276 1,263 4.3 400 4,317 20.0 400 1,336 A high maximum does not necessarily correspond to a high minimum. The first generation of anaerobes is more sensitive to oxygen than the following generations, which may even thrive better in the presence of a limited oxygen supply than in its complete absence.7 This points to adaptation in course of time. Even in the case of a single generation, the organism can withstand greater concentrations of oxygen after the growth of the culture has somewhat advanced than in the beginning. It has been claimed8 that the growth of even obligate anaerobic bacteria 6 Meyer, A. Apparat fur die Kultur von anaeroben Bakterien und f tir die Bestimmung der Sauerstoffminima fur Keimung, Wachstum und Sporenbildung der Bakterienspecies. Centrbl. Bakt., II, 15: 337. 1906. Bemerkungen uber Aerobiose und Anaerobiose. Centrbl. Bakt. I, 49: 305 316. 1909; also Ibid. II, 15: 1905; 16: 386, 481-488, 577-588, 673-687. 1906. Wund, M. Feststellung der Kardinalpunkte der Sauerstoffkonzentration. Centrbl. Bakt. 42: 97-101, 193- 202, 289-296, 385-393. 1906. 7 Burri, 1907 (p. 161); Kiirsteiner, J. Beitrage zur Untersuchungstechnik obligat anaerober Bakterien, sowie zur Lehre von der Anaerobiose iiberhaupt. Centrbl. Bakt. II, 19: 1-26, 97-115, 202-220, 385-399. 1907; Burri, R. and Kiir- steiner, J. Ein experimentaler Beitrag zur Kenntnis der Bedeutung des Sauer- stoffentzugs f tir die Entwicklung obligat anaerober Bakterien. Ibid. 21: 289- 307. 1908; Landw. Jahrb. d. Schweiz. 1909, 422. 8 Fermi, C, and Bassu, E. Untersuchungen iiber die Anaerobiosis. Centrbl. Bakt. I, 35: 563-568, 714-722. 1905; 38: 138-145, 241-248, 369-380. 1905. ANAEROBIC BACTERIA 163 is greatly injured in the complete absence of oxygen; however, Kiir- steiner7 demonstrated that both obligate and facultative anaerobes will thrive well for a number of generations in atmospheres free from oxygen. Free oxygen exerts an injurious effect upon obligate anaerobic bacteria, as pointed out already by Pasteur, the degree of injury depending on temperature, age and abundance of cells.9 In the following pages, the term "anaerobe" will be applied only to the so-called "obligate anaerobes." The presence of suspended particles, especially in case of colloidal suspensions, favors the growth of anaerobic bacteria possibly through their oxygen absorption.10 The more recent studies on oxidation-reduction processes in the growth of microorganisms have brought out the fact that only those bacteria are capable of growing anaerobically, which are cap- able of activating some constituent of the medium as a hydrogen acceptor. Some bacteria, like B. vulgar e, can activate nitrate and can, therefore, grow anaerobically in the presence of nitrate and certain hydrogen donators ; Bad. coli and Bad. prodigiosum can activate nitrate, fumarate, malate and aspartate and can grow anaerobically, in the presence of any of these substances, and with glycerol as a hydrogen donator.11 Recent important contributions point to the lack of cata- lase formation by anaerobic bacteria.12 Peroxides are formed in the aerobic growth of bacteria and these peroxides would become injurious to the organisms if not for the catalase which is formed and which rapidly breaks up the peroxide into inactive oxygen and water. The anaerobic bacteria, which] are unable to form catalase are thus subject to the destructive action of the peroxide when grown under aerobic conditions. A number of indicators are employed for measuring anaerobiosis or 9Bachmann, 1912 (p. 164). 10 Hata, S. Uber eine einfache Methode zur aerobischen Kulti vie rung der Anaeroben mit besonderer Beriicksichtigung ihrer Toxinproduktion. Centrbl. Bakt. I, 46: 539-554. 1908; v. Lennep, R. Folia Microb. 1: No. 3. 1913. 11 Quastel, 1925 (p. 469). 12 McLeod, J. M., and Gordon, J. Catalase production and sensitiveness to H202 among bacteria; with a scheme of classification based on these properties. Jour. Path. Bact. 26: 326-331, 332-343. 1923; The relation between the reducing powers of bacteria and their capacity for forming peroxide. Ibid. 28: 155-164, 147-153. 1925. 164 PRINCIPLES OF SOIL MICROBIOLOGY determining the end point of free oxygen.13 However, various difficul- ties are found in an attempt to use indicators, such as methylene blue, as criteria in anaerobiosis. For the existence of even obligate anaerobes in the soil we need not imagine a soil atmosphere free from atmospheric oxygen, but simply that anaerobic conditions, favorable for the activities of these organisms, are produced due to the active utilization of the oxygen and production of CO2 by aerobic organisms, which results in a reduction of the oxygen tension. This can be imitated artificially in the laboratory, when anaerobes are grown readily under ordinary conditions, in the pres- ence of rapidly growing aerobic bacteria, like Bac. sabtilis. Another illustration of this phenomenon is the growth of the two nitrogen- fixing organisms, the anaerobic Bac. amylobacter and the aerobic, rapidly growing Azotobacter. Exposure to oxygen has, however, an injurious effect upon anaerobic organisms, vegetative cells being de- stroyed in 10 minutes and spores in 8 days;14 in the case of Bac. amylo- bacter; the injurious effect of air exposure upon the vegetative cells sets in only after 40 minutes, while the spores are not injured even after 3 hour exposure. ^5 Methods of isolation of anaerobic bacteria from the soil. There are a number of methods available for the isolation of anaerobic bacteria.16 These bacteria have to be separated not only from aerobic organisms, but often also from other facultative or obligate anaerobic bacteria. The anaerobes, just as the aerobic bacteria, vary greatly in their food requirements and manner of growth, and the methods of isolation have to be adapted to the particular organism in question. There is a large number of species of anaerobes in the soil and it is insufficient to depend on microscopic examinations alone for demonstrating the existence of specific forms. In all cases, the isolation and demonstration of the different species must be undertaken. In case the nature of an organ- ism that is looked for is known, the development of a proper culture 13 Hall, I. C. Chemical criteria of anaerobiosis with special reference to methylene blue. Jour. Bact. 6: 1-42. 1921. Kadisch, E. Centrbl. Bakt. I, Orig. 90:462-468. 1923. Clark, W. M., Cohen, B., and Gibbs, H. D. Studies on oxidation-reduction. VIII. Methylene blue. U. S. Publ. Health Serv. Publ. Health Repts. Repr. no. 1017. 1925. 14 Bachmann, F. Beitrag zur Kenntnis obligat anaerober Bakterien. Cen- trbl. Bakt. II, 36: 1-41. 1912. 15Dorner, 1924 (p. 165). 16 Heller, H. H. Principles concerning the isolation of anaerobes. Jour. Bact. 6: 445-470. 1921. ANAEROBIC BACTERIA 1G5 medium is simplified. An enriched culture is first prepared either by adding some soil to a specific medium kept under specific conditions, or the specific substance is added to the soil itself. An attempt is then made to obtain a culture of the specific bacterium free from accom- panying non-spore-forming and spore-forming aerobic and anaerobic organisms. For the separation of spore-forming organisms from non-spore- formers, whether aerobes or anaerobes, the soil is heated at 75° to 80°C, by placing 2 grams of soil in 10 cc. of sterile water and keeping in a water bath for 10 minutes. This leads to the destruction of all the vegetative cells, while bacterial spores are not injured. The soil is then inoculated into a proper medium, favorable for the development of the specific organism, which will develop under proper cultural conditions; the cul- ture is then transferred repeatedly upon the selective medium and grown under strict anaerobic conditions. To purify anaerobes from aerobes, the method of Dorner17 can be used. The deep agar tube, inoculated with the organisms, is allowed to cool and the agar to solidify. Two cubic centimeters of melted agar containing 0.2 per cent of mercury bichloride is then poured on the surface of the cooled agar and the tubes are closed with rubber stoppers. The aerobes are thus completely eliminated. However, neither of these methods will separate the facul- tative anaerobes from the obligate anaerobes. To separate anaerobes from spore-forming aerobes, use is made of three procedures: (1) Strict anaerobic methods of cultivation. (2) The inhibitive action of gentian- violet on aerobic growth;18,19 a 1 : 100,000 to 1:400,000 dilution of the dye in the agar medium is sufficient to render cultures of anaerobic bacteria free from spore-forming aerobes. (3) Anaerobic organisms are less sensitive than aerobes to pyrocate- chin, chinon, sodium formate, and sodium sulphindigotate.20,21 The most difficult process, often involving a complicated technic, is 17 Dorner, W. Beobachtungen liber das Verhalten der Sporen und vegetativen Formen von Bac. amylobacter A. M. et Bredemann bei Nachweis- und Reinzucht- versuchen. Landw. Jahrb. Schweiz. 1924, 1-28. 18 Churchman. The selective bactericidal action of gentian violet. Jour. Exper. Med. 16: 2, 221, 1912. 19 Hall, I. C. Practical methods in the purification of obligate anaerobes. Jour. Inf. Dis. 27: 576-590. 1920. 20 Kitasato, S., and Weyl, Th. Zur Kenntniss der Anaeroben. Zeitschr. Hyg. 8: 41, 404. 1890. 21 Rivas, D. Ein Beitrag zur Anaerobenzllchtung. Centrbl. Bakt. 32: 831- 842. 1902. 166 PRINCIPLES OF SOIL MICROBIOLOGY the separation of spore-forming anaerobes from other spore-forming anaerobes. The improper separation has led to exaggerated claims for the nature and activities of the organisms. All or some of the following procedures are utilized for this separation: 1. Heating the soil so as to kill the vegetative forms, then introducing various diutions of the heated soil suspension into the proper medium and making transfers from the culture at different stages of develop- ment (heating the culture every time a new transfer is made). The various spore-forming anaerobes sporulate at different periods of their development: some, like the saccharolytic bacteria, sporulate early; others, like most proteolytic forms, sporulating late. 2. Use of selective media stimulating the predominant development of the organism sought. This method has been of great help in the isolation of some important soil anaerobes. It is sufficient to mention that by the use of selective media and proper environmental conditions, such organisms as the anaerobic nitrogen-fixing forms, thermophilic and cellulose-decomposing forms and others were isolated. The specific medium is inoculated with an infusion of soil or manure, which may be previously heated, if the organism in question forms spores, and incubated at the desired temperature. The adjustment of the medium to specific reactions may often be sufficient to separate one group of organisms from another, often even anaerobic forms from one another. For instance, the adjustment of the nitrogen-free glucose media to a pH of 5.5 will not only favor the development of the nitrogen-fixing Clostridium pastorianum, but will also prevent the development of the proteolytic organisms, which usually accompany it.22 For the enrich- ment of cellulose decomposing anaerobic organisms, the use of a specific liquid medium or of a silica gel plate with cellulose as the only source of energy is recommended (p. 196). For the decomposisition of hemicel- lulose, physiological salt solution containing cubes of potato has been used,23 while, for starch splitting organisms, media containing 1 per cent peptone broth and 5 per cent starch have been suggested.24 3. The use of aniline dyes for the elimination of certain species of organisms. 4. Selective temperatures for the enrichment of various organisms, 22Dorner, 1924 (p. 165). 23 Ankersmit, P. Untersuchungen iiber die Bakterien im Verdauungskanal des Rindes. Centrbl. Bakt. I, Orig, 39: 359-574, 687. 1905; 40: 100-118. 24 Choukevitch, J. Etude de la flore bacte>ienne du gros intestin du cheval. Ann. Inst. Past. 25: 247. 1911. ANAEROBIC BACTERIA 167 developing preferably at the different temperatures, as in the case of thermophilic bacteria. 5. Use of high dilutions for the separation of organisms before plating.25 6. Isolation of the individual colony. This can be accomplished either (a) by the picking of surface colonies from agar or gelatin plates or slants in large tubes, kept under anaerobic conditions; (6) by picking colonies from deep agar tubes,26 the last procedure being the easiest and most reliable in the process of separation of pure cultures of anaerobic bacteria from all accompanying forms. 7. Finally the isolation of single cells either by the India ink method,27 by the method of Barber, or by one of the microscopic methods.28 A detailed study of the various methods used for the isolation from surface colonies is given elsewhere.29,30 In general, plates or large agar slants containing the proper culture media are streaked out and placed either in vacuo, in hydrogen, carbon dioxide, or in an atmosphere from which the oxygen is removed by means of sodium pyrogallate.31 To produce discreet colonies, the agar plates or slants must be dried before inoculating, but too much drying of the medium is injurious. The slants or plates are streaked out with a loop- ful of the material taken from the enriched culture or using a dilution of it. The plates are immediately placed in the atmosphere of the neu- tral gas; the agar may also be placed into the upper part of a Petri dish, which is then covered directly with the sterile inverted lower half of the dish and the whole covered with a larger Petri dish.32 26 Stoddard, J. L. Points in the technic of separating anaerobes. Jour. Am. Med. Assn. 79: 906. 1918. 26 Burri, R. Zur Isolierung der Anaeroben. Centrbl. Bakt. II, 8: 533-537. 1902. 27 Burri, 1909 (p. 55); also in Krause-TJhlenhut's Handbuch der mikrobiolo- gischer Technik. 2: 329. 1923. 28 Barber, 1911-1920 (p. 56). Kendall, A. I., Cook, M., and Ryan, M. Methods of isolation and cultivation of anaerobic bacteria. Jour. Inf. Dis. 29: 227-234. 1921. Holker, J. Micro- and Macro-methods of cultivating anaerobic organ- isms. Jour. Path. Bact. 22: 28. 1919; 23: 192-195. 1920. 29 von Hibler, E. Untersuchungen liber die pathogenen Anaeroben. Jena. 1908. 30 Besson, A. Practical bacteriology, microbiology and serum therapy. Lon- don, 1913. 31 Lentz, O. In Friedberger und Pfeiffer's Lehrbuch der Mikrobiologie. Jena, 1919, p. 370. 32 Marino, F. Mcthode pour isoler les anaerobes. Ann. Inst. Past. 21: 1005. 1907; also Ogata, M., and Takenouchi, M. Einfache Plattenkultur- methode der anaeroben Bakterien. Centrbl. Bakt. I, 73: 75-77. 1914. 168 PRINCIPLES OF SOIL MICROBIOLOGY fill However, the deep colony procedure, used first by Liborius for the isolation of anaerobic bacteria, has been preferred by a number of workers. The selection of a suitable medium for this purpose is essen- tial; the medium should be clear and transparent and enough dilution tubes should be used. Some actively growing anaerobes will grow through the agar as if it was a broth; this "permeat- ing growth" will contaminate the other colonies. The deep tubes of sterile agar are placed in boiling water till the agar is melted, tubes are shaken to remove air, and agar cooled down to 45°. Long boiling is inadvisable, since the cotton becomes saturated with moisture. Three tubes are employed for ordinary pur- poses of dilution, but for new material or for weakly growing organisms among rapidly growing forms, more tubes may be used. Tube 1 is inoculated with one loopful of the enriched culture or soil suspension. The tube is then shaken, and transfer is made by means of a sterile pipette (a Pasteur pipette may be used), pre- viously flamed, into tube 2. The inoculum is placed throughout the length of the agar, while withdrawing the pipette, taking care not to blow air into the agar in the tube, the latter being then shaken. The pipette is flamed and, by means of it, some of the agar from tube 2 is transferred to tube 3, which is also shaken. The tubes are plugged with cotton, as ordinary aerobic tubes, and incubated aerobically at 25° to 28°. For actively growing species, 12 to 24 hours' incubation are sufficient; for slow growing forms, such as Bac. amylo- bacter, 4 to 8 days may be required. The colonies are examined, by means of a hand lens, for permeating growth and aerobic organisms. Final isolation is made from the colonies of the mixed culture. The tube and colonies to be transferred are selected. A plain glass or metal rod, steri- lized in the flame and cooled, may be used to pierce the agar to the bottom of the tube, so as to admit air and allow the expulsion of the unbroken agar from the tube upon a sterile half of a Petri dish. The agar tube may also be placed for a second or two into warm water so as to separate the agar from the walls of the tube. The Fig. 9. Buchner tube for the anaerobic cultivation of bacteria: p, the alkaline pyrogallol solution; inner tube contains culture of or- ganism (after Omeliansky). ANAEROBIC BACTERIA 169 agar cylinder is then cut up into fine slices by means of a sterile scalpel; the desired colony is selected, either with the naked eye or using the microscope, and the agar is carefully cut away from it. A transfer is then made by pricking the colony with a fine sterile platinum needle and inoculating deep tubes with sterile agar or slants and liquid media, which are then incubated in an oxygen-free atmosphere. When single cells are separated from one another to obtain pure cul- tures, it is better to isolate the spores rather than vegetative cells, since these give a much larger number of successful cultures (Barber). A medium somewhat more acid than the optimum (as pH 6.0) is more favorable for the germination of the spores. Semi fluid media (con- taining 0.1 to 0.2 per cent agar) are preferable to liquid media, since the presence of a colloid greatly hastens the germination of the bacterial spores.33 Further information on the isolation of anaerobic bacteria is given elsewhere.34-39 Cultivation of anaerobes. There are a number of methods available for the cultivation of anaerobes, these methods being largely concerned with the reduction of the oxygen tension; some of these have been re- ferred to already previously. I. Cultivation in the absence of oxygen: 1. Mechanical protection against the atmospheric oxygen. The use of large volumes of freshly-boiled liquid media placed at a high level; also the process of covering the media with a laj-er of liquid petrolatum or other inert oil has been known since Pasteur. A layer of solid medium can be placed in a Petri dish, then inoculated with anaerobic bacteria and covered with a solution of agar (1.2 to 1.5 per cent) in distilled water. This layer of agar, in covering the medium, prevents sufficiently the admission of oxygen. The solid medium may also be 33 Lantzsch, 1921 (p. 620). 34 Kursteiner, 1907 (p. 162). 36 Veillon, A., and Maz6, P. De l'emploi des nitrates pour la culture et l'isole- ment des microbes anaerobies. Compt. Rend. Soc. Biol., 68: 112. 1910. 36 Northrup, Z. A simple apparatus for isolating anaerobes. Jour. Bact. 1: 90-91. 1916. 37 Hort, E. C. The cultivation of anaerobic bacteria from single cells. Jour. Hyg. 18: 361. 1920. 38 Fuhrmann, F., and Pribram, E. Die wichtigsten Methoden beim Arbeiten mit Bakterien. Abderhalden's Handb. biol. Arb. Methoden. XII: 483-702. 1924. 39 Lowi, E. Zur Technik der Anaerobenkultur mittels des Pyrogallolverfah- rens. Centrbl. Bakt. I, Orig., 82: 493-496. 1919. 170 PRINCIPLES OF SOIL MICROBIOLOGY placed in the upper part of a Petri dish, then covered with the lower part, placed into the upper part. Solid medium may also be placed in deep layers in ordinary containers, then inoculated with a long platinum loop reaching to the bottom of the container (Liborius). The agar can be taken out from the deep tube, by stabbing to the bottom a sterile glass or metallic tube, 2 mm. in diameter, so as to admit air.40 2. Cultivation of anaerobes in vacuo. This method was also proposed by Pasteur and consists in placing the medium in a tube with a capillary end, inocu- lating, pumping out the air, then sealing the end. Petri dishes can also be placed in an ordinary desiccator, from which the atmosphere is then pumped out. The method described by Meyer41 can be used for the cultivation of bacteria at differ- ent partial oxygen tensions. 3. Absorption of oxygen from the atmosphere. The most common method of absorption of oxygen from the atmosphere is carried out by means of alkaline pyrogallate solution introduced by Buchner.42 A mixture of equal portions of 10 per cent solutions of pyrogallol and KOH are often used, or 5 per cent solution of the first and 12.5 per cent of the second. Buchner used 1 gram of pyrogallic acid and 10 cc. of 10 per cent solution of KOH for every 100 cc. of air space.42 The method of Buchner was modified for liquid media :44 the sterile cotton plug is pushed into the tube; 1 cc. of 20 per cent pyrogallic acid and 1 cc. of 20 per cent KOH are placed upon it, the tube is then closed with a rubber stopper. An alkaline pyro- catechin FeS04 solution to be used as a sensitive reagent for determining traces of oxygen has been suggested.45 The following method is very convenient:45 About 15 to 20 cc. of agar medium is placed in a large tube, about 1 inch in diameter, the tube is plugged with cotton and sterilized, then slanted. Imme- diately after inoculation, the cotton plug is pressed deeply into the tube, about 1 to 2 inches above the tip of slant. One cubic centimeter of a 20 per cent solu- tion of pyrogallic acid (or a tabloid containing 0.13 gram of the acid) and 0.25 cc. of a 40 per cent solution of KOH are poured upon the plug, the tube closed with a rubber stopper, turned upside down and placed in the incubator. In the case of Petri dishes, Omeliansky used a combination of evacuation and absorption of oxygen.43 Ten per cent solution of KOH is poured upon the bottom of a desiccator and an open Petri dish containing dry pyrogallol is placed in it. The dishes containing fresh medium and inoculated are then placed into the 40 Burri, R., Staub, W., and Hohl, J. Si'issgriinf utter und Buttersaure- bazillen. Schweiz. Milchztg. 45: nos. 78, 83. 1919. "Meyer, 1905 (p. 162). 42 Buchner, H. Eine neue Methode zur Kultur anaerober Mikroorganismen. Centrbl. Bakt. 4: 149. 1888. 43 Omeliansky, W. L. Ein einfacher Apparat zur Kultur von Anaeroben im Reagenzglase. Centrbl. Bakt. II, 8: 711-714. 1902. 44 Wright, J. H. A method for the cultivation of anaerobic bacteria. Centrbl. Bakt. I, 29: 61. 1901. 46 Binder, K., and Weinland, R. F. liber eine neue scharfe Reaktion auf ele- mentaren Sauerstoff. Ber. deut. chem. Gesell. 46: 255-259. 1913. 46 Buchanan, R. M. An inset absorption appliance for the test-tube culture of Anaerobes. Centrbl. Bakt. I, Orig., 74: 526-527. 1914. ANAEROBIC BACTERIA 171 desiccator. The latter is covered and evacuated. The desiccator is then care- fully turned so as to mix the alkali with the pyrogallol. Since this takes place in the presence of traces of oxygen it browns only slightly. If the cover is not tight, the admission of oxygen is readily indicated by the rapid browning of the mixture. A beaker with water may be placed in the desiccator to prevent the rapid drying out of the media.47 Various other methods for the physical or chemical absorption of the oxygen from the atmosphere have been used; they are based upon the addition of organic or inorganic substances, possessing a strong reducing power, to the medium or outside of the medium in a gas-tight vessel. These include ferrous sulfate, sodium sulfide, ammonium sulf-hydrate, sodium sulfite, ferro-ammonium sulfate, phos- phorus; glucose, sodium formate, pyrocatechin, indigo-carmin; metallic iron and zinc; various tissues, pieces of potato, carrot, fresh yeast, etc. These treat- ments are often accompanied by a partial vacuum. The plates or tubes may be placed in a container to which a quantity of freshly cut potatoes is added, then covered with a bell jar. 4. Replacement of air by an indifferent gas. Hydrogen, carbon dioxide, nitro- gen, and other inert gases may be used for this purpose. A tube,48,49 flask or desiccator50,51 supplied with a two-holed rubber stopper can be used for this purpose. When all the air is replaced by the inert gas, the tubes are sealed.62 II. Cultivation in the presence of oxygen: 5. Cultivation of anaerobes in the presence of aerobic organisms. This method approaches nearest to what takes place in nature than any of the other methods. By cultivating an anaerobic spore forming organism with an aerobic non-spore former, like Bad. -prodigiosum, it is easy to obtain a pure culture of the former by pasteurization. This method has only a limited application in the study of pure cultures. Beijerinck53 employed obligate aerobic bacteria to eliminate the last traces of oxygen from the atmosphere. A combination of two of the above processes may be used. The media used for the isolation and cultivation of anaerobic bacteria depend 47 Rockwell, G. E. An improved method for anaerobic cultures. Jour. Inf. Dis. 35: 581-486. 1924. 48 Fraendel, C. tlber die Kultur anaerober Mikroorganismen. Centrbl. Bakt. 3: 735, 763. 1888. 49 Petri, R. J., and Maaszen, A. Ein bequemes Verfahren fur die anaerobe Zuchtung in Flussigkeiten. Arb. K. Ges. Amt. 8: 314. 1893. 60 Botkin, S. Eine einfache Methode zur Isolierung anaerober Bakterien. Ztschr. Hyg. 9: 383. 1890. 81 Novy, F. G. Die Plattenkultur anaerober Bakterien. Centrbl. Bakt. 16: 566. 1894. 62 Richardson, A. C, and Dozler, C. C. A safe method for securing anaero- biosis with hydrogen. Jour. Inf. Dis. 31: 617-621. 1922. 63 Beijerinck, M. W. Oidium lactis, the milk mould, and a simple method to obtain pure cultures of anaerobes by means of it. Proc. Sec. Sci. K. Akad. Wettensch. Amsterdam, 21: 1219-1226. 1919. 172 PRINCIPLES OF SOIL MICROBIOLOGY upon the specific organisms. Certain special methods may also be used. Among these, gelatin and milk have played an important part. By inoculating milk with a small quantity of soil, a certain type of butyric acid bacteria can be readily demonstrated. This is not a medium for enrichment of anaerobes, as for differ- ential purposes. Various protein (egg-albumin) and glucose media can be used. The medical bacteriologists have made extensive use of brain and blood agar media. To demonstrate the presence of certain organisms, specific media may have to be used. To demonstrate the presence of Bac. amylobacter, nitrogen-free g'.ucose (2 per cent) agar placed in a deep tube is inoculated with a soil sus- pension; if quantitative results are wanted, various dilutions are employed (the soil suspension may be previously heated, in a water bath, at 80° for 10 minutes, whereby only the number of spores is obtained). The tubes are closed with rubber stoppers (when the culture is to be isolated, a surface layer of sublimate agar is used) and incubated at 30°. Gas formation will take place on the second day, breaking up the medium. This and the production of butyric acid will indicate the presence of the organism ; the colonies are lens-shaped. Microscopic examina- tion of the culture can be made by staining with Lugol's reagent. The method of Burri can be used for determining the number of anaerobic bacteria in the soil, not only by establishing the presence of growth in the final dilution, but by actually counting the colonies in the deep tube. Classification of soil anaerobes. Various systems for the classification of anaerobic bacteria and their relation to aerobes have been proposed at different times.54,55 But even at the present time, a proper classifica- tion of anaerobes, especially of soil forms is lacking. The idea that anaeobic bacteria vary greatly has served further to increase the exist- ing confusion. This led to various exaggerations, such as the existence of only a few anaerobic forms which change into one another, or the making of new genera on the basis of minor physiological differences.56 The following system of classification of soil anaerobes may be sug- gested here merely as a tentative working basis : I. Bacteria acting primarily upon carbohydrates: 1. Bacteria utilizing largely simple carbohydrates and starches as sources of energy, often referred to as saccharolytic. Here belong the buty- ric acid bacteria, often classified as one species, Bac. amylobacter A. M. et Bred. These decompose sugars with the formation of butyric acid and gas : (a) Nitrogen-fixing bacteria — ■Clostridium pastorianum Winogradsky (Bac. amylobacter von Tieghem, Bac. amylocyme Perdrix, Bac. butyricus Botkin, Granulobacler saccarobutyricum Beij., Bac. orthobutylicus Grimbert, Clostridium americanum Pring- " Hibler, 1908 (p. 167); Bredemann, 1909 (p. 109). 65 Jungano, M., and Distaso, A. Les anaerobies. Paris. 1910. "Heller, H. H. Classification of the anaerobic bacteria. Bot. Gaz. 73: 70-79. 1922; Jour. Bact. 7: 1-38. 1922. ANAEROBIC BACTERIA 173 sheim, Bac. amylobacter A. M. Bred.) and allied forms found in great abundance in practically all soils. This organism or group was classified by Bergey as CI. butyricum Prazmowski and by Lehman and Neumann as Bac. pastorianus (Winograd- sky).57 Its physiology and occurrence in the soil is discussed elsewhere (p. 110). (b) Bac. welchii Migula (Bac. aerogenes capsulalus Welch and Nutall, Bac. perfringens Veillon and Zuber, Bac. enteritidis sporogcnes Klein), a short rod 4 to 8 by 1 to 1.5/u, single or in pairs; non- motile, forming oval, central or excentric spores; encap- sulated (No. 44, PI. IX). Found repeatedly in the soil and in sewage.58 2. Bacteria decomposing pectins. (a) Bac. amylobacter group, which includes the Clostridium pastor i- anum (same as la). The forms causing the retting of flax have been described under various names. Here belong the Plectridium of Fribes and Winogradsky, the Clostridium of Behrens, the Plectridium pectinovorum of Stormer, the Granulobacter pectinovorum of Beijerinck and van Delden.19 (b) Bac. felsineus Carbone. 3. Bacteria decomposing celluloses: (a) Anaerobic bacteria decomposing celluloses at ordinary temper- atures. Here belong the hydrogen and methane organisms of Omeliansky and the Bac. cellulosae dissolvens Khouvine. (b) Thermophilic cellulose decomposing bacteria — Clostridium ther- mocellum Viljoen, Fred and Peterson. The occurrence and isolation of these organisms is described elsewhere (p. 202). II. Bacteria acting primarily upon proteins: 1. Strongl y proteolytic forms: (a) Bac. sporogenes Metchnikoff (No. 58, Pi. X), a motile, flagellated, gram positive bacillus, with rounded ends, 3 to 7 by 0.6 to 0.8m,* one of the strongest proteolytic bacteria known; it de- composes proteins with the formation of gas, a darkening of the medium and production of a pronounced odor; the sub- terminal spores are formed readily. Found abundantly in the soil, manure, street dust and sewage. 67 Further information on the classification of the anaerobic bacteria acting primarily upon carbohydrates is given by Donker, H. J L. Bijdrage tot de kennis der Baterzuur — , Butylalcohol — en Acetongistingen. Delft. 1926. 68 Klein and Houston. Rept. Med. Officer, Local Govt. Board, London, 1898- 1899, 318; Greer, F. E. Anaerobes in sewage. Amer. J. Publ. Health, 15: 860-867. 1925. 69 Ruschmann, G., and Ravendamm, W. Zur Kenntnis der Rosterreger Bacil- lus fehineus Carbone und Plectridium pectinovorum [Bac. amylobacter A. M. et Bredemann). Centrbl. Bakt. II, 64: 340-394. 1925. 174 PRINCIPLES OF SOIL MICROBIOLOGY (b) Bac. oedematis maligni Koch (Vibrion septique Pasteur), found in the intestines of man and in the soil.60 (c) Bac. putrificus Bienstock, a motile, flagellated bacillus, forms terminal oval spores and has weak saccharolytic and strong proteolytic properties. Milk is gradually digested, without rapid coagulation (No. 45, Pi. IX). (d) Bac. histolyticus Weinberg and Seguin, 3.0 to 5.0 by 0.5 to 0.7/u, occurring singly or in pairs, motile by peritrichous flagella; spores oval excentric. This organism has been isolated from the soil by Peterson and Hall.61 (e) Bac. botulinus van Ermengem, large rods with rounded ends; oval, subterminal spores. The natural habitat of this organism has been found in virgin and cultivated soils, mountain and forest soils,62-64 throughout the world. 2. Weakly proteolytic organisms: (a) Bac. bijermentans Tissier and Martelly, non-motile bacillus, with large central, oblong to oval spores. (b) Bac. telani Nicolaier; 4 to 8 by 0.4 to 0.6/x," motile by means of peritrichic flagella; unable to utilize carbohydrates, intro- duced into the soil with the manure.65 Its occurrence in the soil has been demonstrated64,66 in many of the samples examined. Various other anaerobic bacteria which are weakly pro- teolytic, but are capable of attacking different carbohydrates, with the formation of gas have been isolated either directly from the soil or from other sources, which may indicate a soil habitat, such as Bac. chauvoei. A detailed study of the various anaerobic bacteria, including Bac. sporogenes, Bac. histolyticus and others, secured from wound infections and 60 Gt. Britain National Health Ins. Joint Comm., Medical Research Com- mittee. Special Reports, Series No. 39. Reports of the Committee upon anae- robic bacteria and infections. 1919. 61 Peterson, E. C, and Hall, I. C. The isolation of Bacillus histolyticus from soil in California. Proc. Soc. Exp. Biol. Med. 20: 502-503. 1923. 62 Tanner, F. W., and Dack, G. M. Clostridium botulinum. Jour. Inf. Dis. 31: 92-100. 1922. 63 Dubovsky, B. J., and Meyer, K. F. An experimental study of the methods available for the enrichment, demonstration of B. botulinus in specimens of soil, etc. Jour. Inf. Dis. 31: 501-540, 541-555, 556-558, 559-594, 595-599, 600-609, 610-613. 1922. 64 Hall, I. C, and Peterson, E. C. The detection of Bacillus botulinus and Bacillus tetani in soil samples by the constricted tube method. Jour. Bact. 9: 201-209. 1924. 66 Noble, W. Experimental study of the distribution and habitat of the tet- anus bacillus. Jour. Inf. Dis. 16: 132-141. 1915. 66 Dubovsky, S. J., and Meyer, K. F. The occurrence of B. tetani in soil and on vegetables. Jour. Inf. Dis. 31: 614-616. 1922. ANAEROBIC BACTERIA 175 probably coming in most cases originally from the soil has been made by Weinberg and Seguin.67 III. Bacteria obtaining their oxygen from inorganic salts: 1. Bacteria reducing nitrates. 2. Bacteria reducing sulfates. Both of these groups are described in detail elsewhere (p. 180). Anaerobic organisms may occur in the soil in great abundance; Ucke68 found a garden soil to contain 13| million cells of anaerobic bac- teria and 500,000 spores per 1 gram of soil. In some cases individual species are found in the soil in great abundance. Kiirsteiner, for exam- ple, found as many as 1 million and more cells of Bac. putrificus per 1 gram of soil. Bac. amylobacter was found by Bredemann to be present in practically every soil examined, both in the surface layer and in the subsoil, in cultivated soils, in primeval forests and in pure sand; the organism occurred only irregularly in acid peat soil. Out of 200 sam- ples of Swiss soils examined, only seven did not contain this organism.69 The number of colonies formed on artificial media are considerably less than the actual number of organisms actually present in the soil; this is brought out by the results of Dorner,69 who found that out of 1000 spores present in a medium, only 3 germinated and developed into colonies, while out of 1000 vegetative cells, 45.1 produced colonies. By the use of the dilution and selective culture method, Duggeli70 found 1000 to 1,000,000 anaerobic butyric acid bacteria per gram of soil, 0 to 1000 anaerobic cellulose-decomposing bacteria, 100 to 1,000,000 anaerobic nitrogen-fixing bacteria, from 100 to 1,000,000 anaerobic protein-decomposing bacteria and 100 to 1,000,000 pectin-decomposing bacteria. By the deep tube method, only between 19,000 and 900,000 anaerobic bacteria were found per gram of soil. This is due to the fact that no single solid medium can be devised which would be favorable for the development of all anaerobic bacteria. Various anaerobic bacteria take an active part in the composting of manure in the heap, whenever there is an insufficiency of aeration. The so-called phenomenon of "putrefaction" is chiefly a result of the decomposition of protein substances under anaerobic conditions, due to 67 Weinberg, M., and Seguin, P. La gangrene gazeuse. Masson & Cie. Paris. 1918. 68 Ucke, A. Ein Beitrag zur Kenntnis der Anaeroben. Centrbl. Bakt. I, 23: 996-1001. 1898. "Dorner, 1924 (p. 165). 70 Duggeli, 1921 (p. 39). 176 PRINCIPLES OF SOIL MICROBIOLOGY incomplete oxidation as a result of insufficient aeration. The absence of air in the deeper piles of manure, the slightly alkaline reaction and the presence of large amounts of undecomposed substances make conditions favorable for the development of anaerobic bacteria.71 Various anaero- bic urea bacteria7^ and thermophilic organisms73 also find conditions in the composting manure heap favorable for their development. Well rotted horse manure contains spore-forming, anaerobic thermophilic bac- teria;74 the limiting temperature for their growth was found to be 60° to 65°C. and the thermal death point 110° to 120°C. Some of these or- ganisms were found to be actively proteolytic. No growth took place at room temperature. Various anaerobic spore-bearing bacteria are no doubt brought into the soil with the feces in great abundance; a number of these organisms have actually been demonstrated in intes- tinal secreta.75 Physiological activities of anaerobic bacteria. It is impossible to dis- cuss the physiological activities of the various obligate anaerobic bac- teria, since they differ greatly in the nature of their metabolism. Those that obtain their energy from cellulose, those that can obtain their nitrogen from the elementary form, those that can utilize nitrate or sulfate oxygen, and those that produce foul odors from complex proteins have a distinct physiology from one another and cannot be considered under one heading, merely because they are similar in their requirements of oxygen tension. They usually have an optimum range of hydrogen- ion concentration at pH 6.0 to 8.2 with a limiting range of pH 5.0 to 9.0; the spores germinate better at a higher acidity, with an optimum at pH 6.0 to 7.2.76 While aerobic bacteria produce largely carbon dioxide among the volatile gases, the anaerobic bacteria are characterized by the production 71 Severin, S. A. Die im Miste vorkommende Bakterien und deren physiolo- gische Rolle bei der Zersetzung derselben. Centrbl. Bakt. II, 1: 799-817. 1895; 3: 628-633, 708. 1897. Zhur. Opit. Agron. (Russian), 1: 463-489. 1920. "Geilinger, 1917 (p. 210). 73 Veillon, R. Sur quelques microbes thermophiles strictement anadrobies. Ann. Inst. Past. 36: 422-438. 1922. 74 Damon, S. R., and Feiber, W. A. Anaerobic sporulating thermophiles. Jour. Bact. 10: 37-46. 1925. 75 Kahn, M. C. Anaerobic spore-bearing bacteria of the human intestine in health and in certain diseases. Jour. Inf. Dis. 35: 423-478. 1924. 76 Dozier, C. C. Optimum and limiting hydrogen-ion concentrations for B. botulinus and quantitative estimation of its growth. Jour. Inf. Dis. 35: 105- 133. 1924. ANAEROBIC BACTERIA 177 of a number of other gases. It is sufficient to mention hydrogen and methane, as a result of decomposition of carbohydrates, hydrogen sul- fide as a result of reduction of sulfates, elementary nitrogen and oxides of nitrogen as a result of reduction of nitrates, and various amines, elementary nitrogen and oxides of nitrogen, hydrogen sulfide, mercap- tans and thioether as a result of decomposition of proteins. It is neces- sary to be able to measure these and determine them quantitatively, especially since they are often of great economic importance when a soil is water-logged for a longer or shorter period of time. The bacteria are grown on suitable media (specific for the various organisms) under anaerobic conditions, in tubes or bottles connected with a manom- eter. The tubes may also be placed in a Novy jar used as a respiratory chamber.77 The growth may be carried on in an atmosphere of pure gas, such as N2, H2, C02. By using a compensation manometer, the pressure changes taking place within the culture tube or jar can be observed constantly, these changes indicating the periods of active growth followed by the cessation of growth and respiration. The samples of gas are withdrawn directly into a burette or first into a sampler, then into a modified Henderson-Haldane or other suitable apparatus. The volume of the gas to be analyzed is first measured; the gas is then passed back and forth into 10 per cent KOH solution to absorb the C02, which is determined by difference in the volume of gas. The latter, freed from C02, is passed into an alkaline pyrogallate solution (or sticks of yellow phosphorus in water) to absorb the oxygen; the latter is determined also by the difference in volume of the gases. The estimation of hydrogen, methane and other combustible gases is carried on in a combustion chamber over heated platinum, in the presence of oxygen (or air as a source of oxygen). By measuring the amount of C02 formed in combustion, it is possible to calculate the amount of methane and other hydrocarbons present in the gas mixture; the amount of hydrogen is then determined by the difference between the loss due to combustion and the methane present. The amount of oxygen absorbed in the combustion is obtained by calculation or by the differ- ence between the oxygen added and that remaining, as determined by absorption in the pyrogallate solution. The C02 present in the medium (liquid) is aerated into standard Ba(OH)2 solution, then titrated. 77 Novy, F. G., Roehm, H. R., and Soule, M. H. Microbic respiration. I. The compensation manometer and other means for the study of microbic respira- tion. Jour. Inf. Dis. 36: 109-167. 1925. 178 PRINCIPLES OF SOIL MICROBIOLOGY Oxides of nitrogen are determined by combustion in the platinum spiral before oxygen (or air) is admitted, in the presence of hydrogen. The contraction in volume serves as an index of N20 (N20 + H2 — * H20 + N2). The oxides of nitrogen may be absorbed from 100 cc. sample of gas in 200 cc. m/50 KOH solution, then oxidized to nitrate by adding 5 cc. of 30 per cent hydrogen peroxide. The solution is evapo- rated to dryness on a water bath and nitrates determined by the phe- noldisulphonic acid method.78 Volatile amines and mercaptans do not occur in great abundance among the decomposition products in the soil, but are found largely in the anaerobic decomposition of manure:79,80 H2S gas can be determined by absorption with acetates of lead and cad mium, or ammoniacal cadmium chloride solution, then titrating the CaS with iodine in acid solutions.81 Among the gases formed by the proteolytic bacteria, like Bac. sporo- genes, we find largely C02 and some hydrogen; the odoriferous gases consist largely of H2S; some elementary nitrogen and N20 are also formed. The saccharolytic organisms, like Bac. welchii, produce a large amount of hydrogen, often as much as 50 per cent of the gases.81 The ratio between the C02 and hydrogen depends largely upon the environmental conditions of growth. Anaerobic bacteria form various acids (acetic, butyric, lactic), alco- hols (ethyl, butyl), and in some cases acetone. Often closely related or- ganisms vary greatly in their metabolic products. For example, while different members of the Bac. amylobacter group (Clostridia, Plectridia, Granulobacter) produce butyric acid, the closely related Bac. felsineus does not do so. Soil processes in which anaerobic bacteria take an active part. Atten- tion has already been called to a number of important physiological processes in the soil, in which anaerobic bacteria take an active part. It is sufficient to indicate that such processes as decomposition of cellu- loses, pectins and proteins, and the fixation of nitrogen non-symbioti- cally are as active anaerobically as aerobically. Ammonia formation 78 Allison, V. C, Parker, W. L., and Jones, J. W. Determination of oxides of nitrogen. Tech. Paper No. 249, U. S. Bureau of Mines. 1921. 79 Guggenheim, M. Die biogenen Amine. 1920. 80 Hirsh, P. Die Einwirkung von Mikroorganismen auf die Eiweisskorper. Borntraeger. Berlin. 1918. 81 Anderson, B. G. Gaseous metabolism of some anaerobic bacteria. XIX. Methods. Jour. Inf. Dis. 35: 213-243. 1924. ANAEROBIC BACTERIA 179 from proteins is very active under anaerobic conditions.82-84 Two maxima were found for nitrogen-fixation in the soil, one under aerobic and another under anaerobic conditions;83-86 a higher fixation may actually be obtained anaerobically.87 The decomposition of cellulose under anaerobic conditions is carried on entirely by bacteria. The phenomena of reduction under anaerobic conditions, especially that of nitrates, may become an important economic factor. It is important to point out, in this connection, the active role which anaerobic bacteria play in the rotting of manure. As a matter of fact, the lowest loss of nitrogen and the most efficient conservation of the important elements of the manure is accomplished by keeping it com- pact and moist, so as to prevent the action of aerobic fungi and bacteria and stimulate the action of anaerobic bacteria. As far back as 1889, Schloesing88 pointed out that under anaerobic conditions there is no loss of nitrogen. The gases were found to consist of equal volumes of methane and carbon dioxide, when the manure is incubated at 52°C. Water takes part in the reaction supplying some oxygen for the forma- tion of C02 and some hydrogen for the methane. The amount of gas produced per hour rapidly increases until it reaches a maximum on the 6th day, when it begins to diminish. At 42°C, 850 cc. of gas collected from the decomposition of 100 gm. of manure consisted of 713.6 cc. C02, 97.6 cc. methane and 38.8 cc. hydrogen. Further information on the decomposition of proteins and carbo- hydrates under anaerobic conditions and on the nature of soil gases is given elsewhere (p. 638). »2L6hnis, 1905 (p. 120). 83 Traaen, A. E. Uber den Einfluss der Feuchtigkeit auf die Stickstoff um- setzungen im Erdboden. Centrbl. Bakt. II, 45: 115. 1916. 84 Murray, T. J. The oxygen requirements of biological soil processes. Jour. Bact. 1: 597-614. 1916. 85 Greaves, J. E. Azofication. Soil Sci. 6:163-218. 1918. 86 Lipman and Sharp, 1915 (p. 584). 87 Panganiban, E. H. Rate of decomposition of organic nitrogen in rice paddy soils. Phillip. Agriculturist, 12: 63. 1923; Temperature as a factor in nitrogen changes in the soil. Jour. Amer. Soc. Agron. 17: 1-31. 1925. 88 Schloesing, 1889 (p. 62). CHAPTER VII Bacteria Reducing Nitrates and Sulfates General classification of nitrate reducing bacteria. A large number of organisms, including numerous bacteria and actinomyces, fungi, yeasts and higher plants, but especially the first two groups, are capable of reducing nitrates to nitrites; this often serves as the first step in the process of assimilation of nitrate nitrogen. Some organisms, chiefly fungi and certain bacteria, but also higher plants, are capable of reduc- ing the nitrate to ammonia. However, only specific bacteria are cap- able, under certain conditions, of reducing the nitrate and the nitrite to elementary nitrogen and oxides of nitrogen, in which form the nitro- gen escapes into the atmosphere. Under anaerobic conditions, the nitrate and nitrite may serve as sources of oxygen for these bacteria, which enables them to oxidize the available sources of energy.1 The last process is usually referred to as complete or direct denitrification and the bacteria concerned in this process are spoken of as denitrifying bacteria. These bacteria can be further subdivided into (a) those which use as a source of energy inorganic substances, notably sulfur, and (6) those that use organic carbon compounds as sources of energy. Com- plete denitrification is generally favored by the presence of nitrate, suitable sources of energy (usually carbon compounds), absence of free oxygen and proper reaction. The bacteria, which reduce nitrates only to nitrites or to ammonia, but not to nitrogen gas (elementary form and oxides), may be best spoken of as nitrate reducing bacteria, reserving the term denitrifying bacteria for the other organisms. Organisms reducing nitrates to nitrites. The reduction of nitrates in the soil has been demonstrated in the first part of the 19th century. 1 Weissenberg, H. Studien uber Denitrifikation. Arch. Hyg. 30:279-290. 1897; Jensen, H. Das Verhaltnis der denitrifizierenden Bakterien zu einigen Kohlenstoffverbindungen. Centrbl. Bakt. II, 3: 622-627, 689-698. 1897; Bei- trage zur Morphologie und Biologie der Denitrifikationsbakterien. Ibid. 4: 401-411, 449-460. 1898; Pakes, W. C. C, and Jollyman, W. H. The collection and examination of the gases produced by bacteria from certain media. Jour. Chem. Soc. I, 79: 322-329. 1901. 180 BACTERIA REDUCING NITRATES AND SULFATES 181 This process was found to be brought about by various groups of micro- organisms, capable of reducing nitrates to nitrites, first by Schonbein2 in 1868, then by others, especially by Gayon and Dupetit.3 In addition to various bacteria,4 certain yeasts, filamentous fungi,5 and actinomyces6 are capable of reducing nitrates to nitrites. The composition of the medium is important in this respect, particularly the nature of other sources of nitrogen and of the energy source. The presence of carbo- hydrates, glycerol and organic acids, in addition to peptone, was found to stimulate the reduction of nitrate to nitrite, while an abundance of oxygen injured it. Frankland7 called attention to the fact that certain bacteria {Bac. ramosus and Bac. pestifer) are specifically concerned in this process. The reduction was favorably influenced by increasing the organic matter content of the solution, especially the peptone. Anaerobiosis or lack of sufficient aeration greatly favors nitrite formation.8-10 Nitrite-forming bacteria are well distributed in the soil.11,12 Such soil forms as Bac. megatherium13 and Bad. vulgareu are found among the 2 Schonbein, C. F. tJber die Umwandlung der Nitrate in Nitrite durch Confer- ven und andere organische Gebilde. Jour, prakt. Chem. 105: 208-214. 1868. 3 Gayon, U., and Dupetit, G. Sur les fermentations des nitrates. Compt. Rend. Acad. Sci. 95 : 644-646. 1882; Sur la transformation des nitrates en nitrites. Ibid., 1365-1367; Recherches sur la reduction des nitrates par les infiniments petits. Nancy. 1886; Mem. Soc. Sci. phys. Nat. Bordeaux. 1886; Ann. Sci. Aeron. 1: 226-325. (1885) 1886. 4 Maassen, A. Die Zersetzung der Nitrate und Nitrite durch die Bakterien. Arb. K. Gesundheitsamt, 18: 21-77. 1901. 5 Wolff, K. Denitrifikation und Garung. Hyg. Rundschau, 91: 538. 1899. 6 Waksman, 1919 (p. 299). 7 Frankland, P. J. The action of some specific microorganisms on nitric acid. Chem. News, 57: 89. 1888; Uber einige typische Mikroorganismen im Wasser und im Boden. Ztschr. Hyg. 6: 373. 1899. 8 Laurent, E. Experiences sur la reduction des nitrates par les vegetaux. Ann. Inst. Past. 4: 722-744. 1890. 9 Kiihl, H. Beitrag zur Kenntnis des Denitrifikationsprozesses. Centrbl. Bakt. II, 20: 258-261. 1908. 10 Caron, H. V. Untersuchungen uber die Physiologie denitrifizierender Bakterien. Centrbl. Bakt. II, 33: 62-116. 1912. 11 Jensen, 1897 (p. 180). 12 Klaeser, M. Die Reduktion von Nitraten zu Nitriten und Ammoniak durch Bakterien. Centrbl. Bakt. II, 41: 365-430. 1914: Ber. deut. bot. Gesell. 32: 58. 1914. 13Stoklasa, 1898 (p. 104). 14 Horowitz, A. Contribution a l'£tude du geare Proteus vulgaris. Ann. Inst. Past. 30: 307-318. 1916. 182 PRINCIPLES OF SOIL MICROBIOLOGY nitrite formers. Out of 109 species of bacteria tested by Maassen,15 in a solution containing 5 per cent peptone and 0.5 per cent sodium ni- trate, 85 were found capable of reducing nitrates to nitrites, especially Bad. pyocyaneum; 46 reduced the nitrite to ammonia and 4 liberated atmospheric nitrogen. Out of 28 species of bacteria studied by Klae- ser,12 all but one were found capable of reducing nitrates. Many strict aerobic bacteria are capable of acting anaerobically in the pres- ence of nitrates. Intensive aeration inhibits the process of nitrate re- duction. The reaction of the medium has an important influence in determining whether nitrates are reduced to nitrites or ammonia; an alkaline reaction favors the first process and an acid reaction the second. Klaeser used a medium having the following composition : KN08 2 grams NaCl 0.1 gram Glucose 10 grams MgS04 0.3 gram K2HP04 1 gram FeCl3 0.01 gram CaCl2 0.1 gram Other media, with and without peptone, but containing nitrates, can also be used for demonstrating nitrate reduction by bacteria. The formation of nitrites from nitrates has been suggested as a test in characterizing bacteria.16 The following organisms can be recorded as capable of reducing nitrates to nitrites: Bad. coli, Bad. vulgare and allied species, Bad. prodigiosum, Bad. putidum, Bad. fluorescens, Bad. pyocyaneum, Bad. herbicola, Bac. subtilis and allied species, Bac. vulgatus, Bac. mycoides, Micr. pyogenes, Mycobad. phlei and other mycobacteria, B. porticensis and others. Some of these organisms, such as Bad. coli, are also cap- able of forming hydrogen.17 The products formed from the reduction of the nitrate depend largely upon the composition of the medium and oxygen tension. Organisms reducing nitrates to ammonia. Marchal18 was one of the first to demonstrate that certain bacteria (Bac. mycoides) are capable of reducing nitrates to ammonia, with the intermediate formation of ni- 15 Maassen, 1901 (p. 181). 16 Conn, H. J., and Breed, R. S. The use of the nitrate-reduction test in char- acterizing bacteria. Jour. Bact. 4: 267-290. 1919. 17 Maze, P. Les phdnomenes de fermentation sont les actes de digestion nou- velle demonstration apport£e par l'etude de la devitrification dans le regne vegetal. Ann. Inst. Past. 25: 289-312, 369-391. 1911. 18 Marchal, E. The production of ammonia in the soil by microbes. Agr. Sci. 8: 574. 1S94; Centrbl. Bakt. II, 1: 758. 1895. BACTERIA REDUCING NITRATES AND SULFATES 183 trites. Beijerinck and van Delden19 found that various bacteria, like Bac. subtilis and Bac. mesentericus vulgatus, are capable of producing both ammonia and nitrite from nitrates, but no ammonia from nitrites; Azotobacter chroococcum, however, produced ammonia from nitrates and nitrites. The reduction process takes place in the presence of carbo- hydrates and organic acids as sources of carbon.20 These bacteria undoubtedly include the "protein-forming bacteria" described by Gerlach and Vogel,21 capable of transforming nitrate into protein nitro- gen with an intermediate reduction to ammonia nitrogen. Kruse22 called attention to the fact that those microorganisms, which cannot bring about "fermentation of the nitrate" (complete reduction to nitrogen), are capable of reducing it to ammonia. This seems to be the natural process, when microorganisms are assimilating nitrates and nitrites, to reduce them first to ammonia, as shown for a number of bacteria and fungi.23 Bacteria reducing nitrates to atmospheric nitrogen. The formation of gaseous nitrogen in the process of decomposition of organic matter in the soil was first observed by Davy.24 This was ascribed to a chemical interaction between nitrites and amino acids in the soil, resulting in the formation of gaseous nitrogen.25 Gayon and Dupetit26 pointed out in 1882 that bacteria were responsible for this process and that the free nitrogen originated from the nitrates. Deherain and Maquenne27 demonstrated that nitrate decomposition in the soil takes place only in the absence of atmospheric oxygen and in the presence of an abundance 19 Beijerinck, M. W., and van Delden, A. tJber die Assimilation des freien Stickstoffs durch Bakterien. Centrbl. Bakt. II, 9: 3-43. 1902. 20 Stoklasa, J., and Vitek, E. Beitrage zur Erkenntnis des Einflusses verschie- dener Kohlenhydrate und organischer Sauren auf die Metamorphose des Nitrats durch Bakterien. Centrbl. Bakt. II, 14: 102-118. 1905. 21 Gerlach and Vogel. tlber eiweissbildende Bakterien. Centrbl. Bakt. II, 7: 609-623. 1901. 22 Kruse, 1910 (p. xii). 23 Kostyschew, S., and Tswetkowa, E. Uber die Verarbeitung der Nitrate in organische Stickstoffverbindungen durch Schimmelpilze. Ztschr. physiol. chem. Ill: 171-200. 1921. 24 Davy, 1814 (p. 122). 26 Dietzell, B. E. Ueber die Entbindung von freien Stickstoff bei der Faulnis. Ztschr. Landw. Ver. Bayern. 72: 186 201. 1882. (Biederm. Centrbl. Agrik. Chem. 11: 417-420. 1882). 26 Gayon and Dupetit, 1882 (p. 181). 27 Deh6rain, P. P., and Maquenne. Sur la reduction des nitrates dans la terre arable. Compt. Rend. Acad. Sci. 95: 691-693, 732-734, 854-856. 1882. 184 PRINCIPLES OF SOIL MICROBIOLOGY of organic matter. The process is checked by heating the soil or treat- ing it with chloroform, which results in the destruction of the bacteria responsible for the reduction of the nitrates.28 In the decomposition of organic nitrogenous compounds, free from nitrates, both in the presence and absence of oxygen, nitrogen gas is not produced; when nitrates are present, an active reduction takes place in the absence of oxygen, with the formation of gaseous nitrogen and various oxides of nitrogen.29 This reduction diminishes with an increase in the amount of oxygen present but does not stop entirely. Even those investigators who believed at first that denitrification is a purely chemical process, carried out by means of the soil colloids, were convinced by later studies that nitrate reduction is not of a chemical nature.30 Bacteria may bring about the formation of nitrogen gas from nitrates in two different ways: (a) indirectly and (b) directly. The nitrite which is formed in the process of reduction of nitrate by Bad. coli, Bad. vulgare, Bad. prodigioswn, Bac. vulgatus, may interact chemically with the amino nitrogen of the peptone molecule or the various amino acids formed from the decomposition of peptone, liberating gaseous nitrogen. The various oxides of nitrogen formed from the reduction of nitrate may also interact with the ammonia nitrogen formed from the peptone and result in free nitrogen gas: NH4N02 = 2H20 + N2 These indirect processes play only a questionable role in the soil. How- ever, in addition to these bacteria, which in themselves are unable to produce nitrogen gas directly from nitrates, the soil harbors various specific bacteria capable of reducing the nitrate molecule directly to atmospheric nitrogen. Breal31 found that a nitrate solution to which straw is added liberates a great deal of gaseous nitrogen. Similar results have been obtained on inoculating a nitrate solution with horse 28 Ehrenberg, A. Experimentaluntersuchungen iiber die Frage nach dem Frei- werden von gasformigen Stickstoff bei Fiiulnissprocessen. Ztschr. physiol. Chem. 11: 145-178, 438-471. 1886. 2D Tacke, Br. tJber die Entwicklung von Stickstoff bei Fiiulniss. Landw. Jahrb. 16: 917-939. 1888. 30 Vogel, J. tiber das Verhalten von Nitrat im Ackerboden. Centrbl. Bakt. II, 34: 540. 1912; Landw. Vers. Sta. 78: 265-301. 1912; 82: 159-160. 1913. 31 Breal, E. De la presence dans la paille d'un ferment a^robie r<5ducteur de l'acide nitrique. Ann. Agron. 18: 181-195. 1892. Compt. Rend. Acad. Sci. 114: 681-684. 1892. BACTERIA REDUCING NITRATES AND SULFATES 185 manure. Wagner32 then attempted to draw, on insufficient ground, broad generalizations concerning the reduction of nitrates to gaseous nitrogen by denitrifying bacteria in manure, even when added to the soil. Gayon and Dupetit33 isolated from the soil, in 1886, two anaerobic bacteria (B. denitrificans a and jS) capable of reducing nitrates to gaseous nitrogen. The two organisms were cultivated upon a medium having the following composition:34 1. Distilled water 250 cc. 2. Distilled water 500 cc. KN03 2 grams Citric acid 5 grams Asparagine 1 gram KH2PO 4 2 grams MgS04 2 grams CaCl2 0.2 gram FeCl3 Trace Solution 2 is neutralized with a 10 per cent solution of NaOH or KOH, with phenolphthalein as an indicator. The two solutions are mixed and made up to 1000 cc. with distilled water. For the isolation of denitrifying organisms, various other media can be used: (1) 1000 cc. water, 10 grams glucose, 6 grams NaN03, 6 grams NaCl, 0.02 gram Ca3(P04)2.35 (2) 100 cc. water, 0.5 to 1.5 grams NaN03, 20 to 50 grams glycerol, 7 grams malic acid (neutralized with sodium carbonate), 0.5 gram sodium phos- phate, 0.5 gram NaCl, 0.5 gram Na2C03, 0.1 gram MgS04.36 (3) 1000 cc. water, 20 grams of calcium tartrate, citrate or malate, 10 to 20 grams KN03, 0.5 gram K2HP04. Under anaerobic conditions, practically all the nitrate nitrogen can be transformed into gaseous nitrogen. When asparagine is replaced by sugar, the ammonia otherwise produced from the asparagine is not formed. In the reduction of nitrate to gaseous nitrogen (so-called "ni- trate fermentation"), there is an abundant accumulation of alkali, till the process is stopped when the alkali concentration is equivalent to 1 per cent sodium carbonate.37 When the alkali is neutralized by means 32 Aeby, J., Dorsch, R., and Matz, Fr., and Wagner, P. Forschungen iiber den relativen Dungewart und die Konservierung des Stallmistsickstoffs. Landw. Vers. Sta. 48: 247-360. 1897. 33 Gayon and Dupetit, 1886 (p. 181). 34 Giltay, E., and Aberson, G. Denitrifizierende Organismen im Boden. Arch. Neerland. 25: 341. 1892. 36 Ampola and Ulpiani. Gazz. chim. ital. 1898, 410. 36Maassen, 1901 (p. 181). 37 Burri, R., and Stutzer, A. Uber Nitrat zerstorende Bakterien und den durch dieselben bedingten Stickstoffverlust. Centrbl. Bakt. II, 1: 257-265, 350-364, 392-398, 422-432. 1895; 2: 473-474. 1896. 186 PRINCIPLES OF SOIL MICROBIOLOGY of an acid, nitrate reduction continues further, until all the nitrate has disappeared.38 The organisms are very sensitive to free acids. The optimum reaction for the reduction of nitrates is pH 7.0 to 8.2; the limit- ing reactions are pH 5.5 and pH 9.8. The optimum reaction for the reduction of nitrites is pH 5.5 to 7.0. The reduction of nitrates to atmospheric nitrogen may be a result of associative action of two bacteria, one (Bad. coli) reducing the nitrate to nitrite and the other (Bad. denitrificans I) reducing the nitrite to atmospheric nitrogen.37 In case of associative growth, the aerobic form removes the free oxygen, thus enabling the other organism to be- come the denitrifier. Some organisms reduce only nitrates to nitrogen. The four species found by Maaszen capable of reducing nitrate to gaseous- nitrogen were Bad. fluorescens liquefaciens, Bad. fluorescens from blood, Bad. pyocyaneum and Bad. praepollens. These results were confirmed by other investigators,39-41 who found Bad. pyocyaneum, Bad. hartlebii and fluorescent bacteria among the most active denitrifying organisms. Among the forms capable of reducing nitrates completely to gaseous- nitrogen, we may also include various organisms isolated from horse manure,42 from cattle excreta (Bad. denitrificans agilis)i3 and from the soil.44-46 Van Iterson47 demonstrated the presence in the soil of various bacteria, namely Bad. stutzeri, Bad. denitrofluorescens and Bad. vul- 38 Zacharowa, T. M. Process of denitrification as dependent upon the reaction of the medium. Trans. Institute of Fertilizers, No. 15, 1923, Moskau. 39 Sewerin, S. A. Zur Frage fiber die Zersetzung von salpetersauren Salzen durch Bakterien. Centrbl. Bakt. II, 3: 504-517, 554-563. 1897; 22: 348-370. 1909; 25: 479-492. 1909. 40 Christensen, H. R. Zwei neue fluoreszierende Denitrifikationsbakterien. Centrbl. Bakt. II, 11: 190-194. 1904. 41 Fred, E. B. Eine physiologische Studie iiber die nitratereduzierenden Bakterien. Centrbl. Bakt. II, 32: 421-449. 1911. 42 Schirokikh, J. Uber einen neuen Salpeter zerstorenden Bacillus. Centrbl. Bakt. 11,2: 204-207. 1896. 43 Ampola, G., and Garino, E. Ueber die Denitrifikation. Centrbl. Bakt. II, 2: 670-676. 1896; 3: 309-310. 1897. 44 Jensen, 1897-8 (p. 180). 45 Hoflich, C. Vergleichende Untersuchungen iiber die Denitrifikationsbakter- ien des Mistes, des Strohes und der Erde. Centrbl. Bakt. II, 8: 245-248, 273-278, 305-308, 336-339, 361-367, 398-406. 1902. 46 Cingolani, M. Recherche intorno al processo della denitrificazione. Staz. Sper. Agr. ital. 41: 521-538. 1908; Ann. Staz. Chim. Agr. Spes. Roma (2), 2: 274. 1908. (Centrbl. Bakt. II, 23: 238. 1909.) 47 van Iterson, C. Anhaufungsversuche mit denitrifizierenden Bakterien. Centrbl. Bakt. II, 12: 106-116. 1904. BACTERIA REDUCING NITRATES AND SULFATES 187 pinus, which reduce nitrates to gaseous nitrogen, in the presence of small quantities of organic matter. In the same soil, where nitrification takes place under aerobic conditions, denitrification will take place in the absence of free oxygen. The following authentic organisms capable of reducing nitrates to atmospheric nitrogen have been isolated and described (some of these are probably only varieties of other species which do not denitrify) : Bact. denitrificans (= Bact. denitrificans I Burri and Stutzer, Pseud, stutzeri Mig.) L and N (1.5 to 3 by 0.7/i), a motile, non-spore forming, aerobic organism. Bact. stutzeri (= Bact. denitrificans II Burri and Stutzer, Bact. nitrogenes Mig.) L and N (2 to 4 by 0.7 to 0.8ju), a motile, non-spore forming, facultative anaerobic organism, isolated from straw and horse manure.48 Bact. kunnemanni (= Bac. denitrificans III Kunnemann), a motile, non-spore forming organism. Bact. denitrificans agilisi9 (1 to 1.5 by 0.1 to 0.3/z), a motile, peritrichic, non- spore forming organism; gram-negative, facultative anaerobic, granulated and developing slow; according to Lohnis this is a denitrifying variety of Bact. radio- bacier. Bact. ulpiani (= Bac. denitrificans VI Ampola et Ulpiani), a motile, non-spore forming, gram-negative organism. Vibrio denitrificans**0 (2 to 4 by 0.5^), a motile, non-spore forming organism. Bac. schirokikhi,&1 a motile, spore-forming, proteolytic, aerobic organism. Bact. praepollens,™ a small, non-motile, obligate aerobic organism, reducing only nitrites. Bac. nitroxusb3 (No. 62, PI. X) comprising bacilli of variable dimensions, globous, pyriform, filiform; they take the form of Clostridia at the time of spore formation, giving an intense glycogen reaction; facultative anaerobic; on repeated transfer under aerobic conditions may lose faculty of reproduction; gelatin is liquefied. In addition to these and the above mentioned bacteria, we may also call attention to a few other denitrifying forms which were isolated, such as Bact. ful- vum,bi Bact. hartlebii,hh Bact. centropunctatum, Bact. nitrovorum, B. porticensis, etc. Most of these organisms are strict aerobes, some being capable of decomposing proteins actively. Most of them grow on nitrate (0.2 to 1.0 per cent) media, with 48 Kunnemann, O. tJber denitrifizierende Mikroorganismen. Landw. Ver- suchsta. 50: 65-113. 1898. 49 Ampola and Garino, 1896-1897 (p. 186); Kuntze, VV. Beitriige zur Mor- phologie und Physiologie der Bakterien. Centrbl. Bakt. II, 13: 1-12. 1904. "Sewerin, 1897 (p. 186). 61 Jensen, 1898 (p. 180). 62Maassen, 1899 (p. 181). 63 Beijerinck and Minkman, 1910 (p. 546). 54 Bierema, S. Die Assimilation von Amnion-, Nitrat- und Amidstickstoff durch Mikroorganismen. Centrbl. Bakt. II, 23: 672-726. 1909. 66 Jensen, 1898 (p. 180). 188 PRINCIPLES OF SOIL MICROBIOLOGY the formation of a gas (largely N, some C02) and nitrite. In the absence of free oxygen, these organisms can exist anaerobically in the presence of nitrate. A thermophilic denitrifying bacillus (3.5 to 7 by 1 to 1.8/x), facultative anaerobic, reducing nitrates with the formation of gas and growing at high temperatures (52°C.) has also been described.56 Several organisms reducing nitrates are capable of obtaining their energy from inorganic compounds. Thiob. denitrificans Beij. oxidizes sulfur and reduces nitrates to nitrogen gas. This organism, or rather group of organisms, is widely distributed in the soil.57-58 Thiosulfate can be oxidized by the organism under anaerobic conditions only in the presence of nitrate as a source of oxygen.59 The utilization of the energy obtained by the oxidation of hydrogen gas for the reduction of nitrates has been pointed out by Niklewski60 for H. agilis. The decomposition of cellulose in the soil may be carried on by the symbiotic action of two bacteria, one reducing nitrate to atmospheric nitrogen and the other decomposing the cellulose; the decomposition products of the cellulose are used by the nitrate reducing organism as a source of energy, which enables it to reduce the nitrate, while the oxygen thus liberted is utilized by the cellulose decomposing organism, under anaerobic conditions.61 Bacteria reducing sulfates to H2S. A detailed study of the formation of hydrogen sulfide in nature is given elsewhere (p. 600). It is sufficient to call attention here to the bacteria capable of producing this sub- stance as a result of reduction of sulfates and other oxygen-rich sulfur compounds (like thiosulf ates) . Microspira desulfuricans (No. 63, PI. X), capable of bringing about this reduction, was first studied by Beijerinck,62 then obtained in pure culture by Van Delden.63 It was isolated on the following medium: K2HPO4 0.5gram MgS04 or CaS04 1.0 gram Sodium lactate 5.0 grams FeSC>4 Trace Asparagine 1.0 gram Tap water 1000 cc. 88 Ambroz, 1913 (p. 158). 57 Lieske, 1912 (p. 86). 68Gehring, 1914 (p. 87). 69 Trautwein, 1921 (p. 87). 60 Niklewski, 1914 (p. 99). 61 Gerretsen, 1921 (p. 736). 62 Beijerinck, M. W. tlber Spirillum desulfuricans als Ursache von Sulfatre- duktion. Centrbl. Bakt. II, 1: 1-9, 48-59, 104-114. 1895. 63 Van Delden, A. Beitrag zur Kenntnis der Sulfatreduktion durch Bak- terien. Centrbl. Bakt. II, 11: 81-94, 113-118. 1904. BACTERIA REDUCING NITRATES AND SULFATES 189 The medium is filled to the neck of the bottles, then inoculated and incubated at 25°C. Sulfur reduction becomes evident by a change in color due to formation of H2S. The bacterium can be isolated from the soil when some sodium sulfite is added to the medium. The presence of organic substances as sources of energy and anaerobic conditions are required for the action of the organism. For the isolation of pure cultures, 10 per cent gelatin or 2 per cent agar is added to the above medium; in place of FeS04 a trace of FeSO^NH^oSO^GIi^O together with some sodium carbonate is used. In 3 to 6 days small black colonies appear. Sulfur is deposited in the colony, on the solid medium, among the bacterial cells, due to the incomplete reduction of the sulfate. The organism is a very motile spirillum, 4 by lju in size and is strictly anaerobic. Another organism {Microspira aestuarii) was isolated from sea water. Various thermophilic bacteria (Vibrio thermo desulfuricans) are capable of reducing sulfates.04 These three forms are closely related to one another and have the ability, apart from all other bacteria, to utilize sulfates and thiosulfates as sources of oxygen under anaerobic conditions. An actinomyces (A. pelogenes) capable of reducing sulfates to sulfides and forming iron sulfide was also isolated.65 These organisms occur in great abundance in certain lakes and seas, and especially in the black curative muds; their reducing properties under these conditions keep the sulfur in the process of constant transformation,66 as discussed in detail elsewhere (p. 611). 64 Elion, L. A thermophilic sulfur-reducing bacterium. Centrbl. Bakt. II, 63: 58-67. 1924. 65 Sawyalow, W. tJber Schwefelwasserstoffgiirung im schwarzen Heilsch- lamme. Centrbl. Bakt. II, 39: 440-447. 1913. 66 Nadson, G. A. On the hydrogen sulfide fermentation in the Weissovo Lake and the participation of microbes in the formation of the black mud. 1903. St. Petersburg. (Russian.) CHAPTER VIII Bacteria Capable of Decomposing Celluloses and Other Complex Carbohydrates and Hydrocarbons in the Soil Microorganisms concerned in the decomposition of celluloses in nature' Among the microorganisms concerned in the decomposition of different constituents of plant and animal tissues, those capable of breaking down celluloses have attracted considerable attention, due to the fact that these materials make up a large part of the bulk of the organic matter added to the soil, but chiefly because the organisms concerned are more or less specific in nature. Many bacteria are capable of existing only with celluloses as a source of energy and some cannot even utilize any other source of energy. Organisms capable of decomposing celluloses are found among various groups of fungi, among the actino- myces and among the bacteria. However, under anaerobic conditions, the fungi and actinomyces do not thrive and bacteria alone are entirely concerned in the process. The cellulose-decomposing bacteria can be divided into two groups, (1) the aerobic and (2) the anaerobic forms. Certain special groups of these forms may be concerned in the process, namely (3) the ther- mophilic bacteria, probably active in the decomposition of celluloses in manure and also in the soil under certain conditions, and (4) the denitrifying bacteria, active only in the presence of nitrates and under certain specific conditions.1 Anaerobic bacteria. Mitscherlich2 observed in 1850 that, in the rot- ting of potatoes in water, the cell walls are destroyed while the starch accumulates at the bottom of the container. He ascribed this action to 1 See Pringsheim, H. Die Polysaccharide. 2 Aufl. Springer. Berlin. 1923; Karrer, P. Einfiihrung in die Chemie der polymeren Kohlenhydrate. Akad. Verlagsges. Leipzig. 1926. Rippel, A. Der biologische Abbau der pflanz-, lichen Zellmembrannen. Ztschr. angew. Bot. 1: 78-97. 1919; Waksman, S. A. and Skinner, C. E. The microorganisms concerned in the decomposition of celluloses in the soil. Jour. Bact. 12: 57-84. 1926. 2 Mitscherlich. Zusammensetzung der Wand der Pflanzenzelle. Monatschr. K. Akad. Wiss. Berlin. 1850, 102-110. 190 BACTERIA DECOMPOSING CELLULOSES 191 vibrios which were present in abundance in the water. Van Tieghem3 described in detail a species of Amylobacter previously found to occur in decomposing plant tissues and staining blue with iodine; it decom- posed young plant tissues with the formation of butyric acid, carbon dioxide and hydrogen. However, this organism was not a species in the true sense of the word, but a collective form; it is doubtful whether it decomposed pure cellulose at all, so that it could hardly deserve the term "cellulose organism."4 Since cellulose forms an important con- stituent of manure, attention has been directed chiefly towards cellulose decomposition in the rotting of manure. It has been found, for ex- ample that the atmosphere at different depths of the manure pile con- sists of various gases. The content of carbon dioxide and especially of methane increases and the nitrogen content decreases with depth. Oxygen is entirely absent at the lower depths of the pile. Omeliansky5 was the first to establish definitely the connection be- tween the activities of microorganisms and the decomposition of cellulose. The following medium was employed: K2HP04 l.Ogram NaCl trace MgS04 0.5 gram Distilled water 1000 cc. (NH4)2S04or\ (NH4)2HP04 f • 10gram The ammonium salt may be replaced by 0.5 per cent asparagine or 0.1 per cent peptone. Some chalk and pure filter paper were placed in long-necked bottles, which were then filled with the medium to the stopper. The flasks were inocu- lated with horse manure or river mud and incubated at 34° to 35°. After a con- siderable period of incubation (usually more than a week), gas production set in. The paper became covered with specks; these were the places where the decom- position of the cellulose began. At the end of the growth period (active fermenta- tion), which is accompanied by abundant gas formation, there remained only a part of the paper, half rotted and entirely changed in appearance. This residue fell apart at the slightest touch. The white color of the paper had changed into yellow-brown, the medium also was colored, and the odor of the medium was that of rotten cheese. When precipitated cellulose was used in place of filter paper, the reaction was more rapid. 3 Van Tieghem, P. E. L. Sur le Bacillus amylobacter et son role dans la putre- faction des tissus veg^taux. Compt. Rend. Acad. Sci. 68: 205-210; 89: 5-8> 1102-1104. 1879; Bull. Soc. Bot. France, 24: 128-135. 1877; 26: 25. 1879; 28: 243-245. 1887. 4 Omeliansky, W. L. Die Cellulosegarung. Lafar's Handb. tech. Mykol. 3: 245-268. 1904. 6 Omeliansky, W. Ueber die Garung der Cellulose. Centrbl. Bakt. II, 8: 193-201, 225-231, 257-263, 289-294, 321-326, 353-361, 385-391, 1902; 11: 369-377, 1904; 36: 472-473. 1913. 192 PRINCIPLES OF SOIL MICROBIOLOGY Omeliansky found that the mixture of gases contained hydrogen or methane, these two gases being produced by two different organisms. When the inoculum was added without preliminary heating, methane formation took place; when the inoculum was heated for 15 minutes at 75°, conditions favored the development of the bacteria which produced hydrogen in the decomposition of cellulose. The spore of the methane- forming organism was found to germinate earlier than that of the hy- drogen form. When the culture was transferred, the former organism predominated and the latter could be finally entirely eliminated. By heating the inoculum or a young culture, the vegetative cells pro- duced from the spores of the methane form, which had already germi- nated, were killed, while the ungerminated spores of the hydrogen form survived and proceeded to develop. If this process of heating the cul- ture at an early stage of development was repeated several times, the hydrogen form could be obtained free from the methane form. PLATE XI Cellulose and Pectin-decomposing Bacteria 64. (x), Bac. cellulosae hydrogenicus syn Bac. fossicularum L & N, and (y), Bac. cellulosae methanicus syn Bac. methanigenes L & N: a, young cells; b, spore formation; c, ripe spores, X 660 (from Omeliansky). 65. Bac. cellulosae dissolvens, showing bacteria attached to the cellulose fiber, by their non-sporulating extremities (from Khouvine). 66. Holes in paper produced by Spirochaeta cytophaga, grown in Petri dish culture upon NaN03 — mineral salt agar with filter paper superimposed, natural size (from Hutchinson and Clayton). 67. Spirochaeta cytophaga, young culture on filter paper placed in tube; typical incurvation of thread forms (from Hutchinson and Clayton). 68. Sp. cytophaga, formation of pre-sporoid stage with double granules (from Hutchinson and Clayton). 69. Bad. fimi, 15-day old colonies on cellulose agar plate, at 30°C. (from McBeth and Scales). 70. Bact. fimi, vegetative cells from 24-hour culture on nutrient agar, stained with carbol-fuchsin, X 660 (from McBeth and Scales). 71. Bac. cytaseus, 15-day old colonies on cellulose agar plate, at 30°C. (from McBeth and Scales). 72. Bac. cytaseus, nine day old culture at 30°C, showing spore formation; aqueous fuchsin stain, X 660 (from McBeth and Scales). 73. Clostridium thermocellum, a thermophilic cellulose decomposing bacillus. A 48 starch-lagar culture at 65°C. stained with carbol fuchsin for 5 minutes, at 100°C, showing free spores, sporangia and vegetative rods (from Viljoen, Fred and Peterson). 74. Granulobacter pectinovorum (after Beijerinck). PLATE XI 64 X n. 64Y ■ n# ^ y / 72 A % .1 \ 73 < H\ \ 74 BACTERIA DECOMPOSING CELLULOSES 193 The hydrogen organism (Bac. fossicularwn L. et N.) was found to form thin, straight rods (4 to 8 by 0.5/i) in young cultures. With age of culture the cells become longer until they reach a length of 10 to 15^, without increasing in thickness and without forming chains. The cells are often slightly curved, sometimes even spiral-like, especially on precipitated cellulose. At a later stage, one end of the cell swells up gradually and takes the appearance of an oblong and then of a round body. A perfectly round spore develops in this swelling, fills all the space, and has a diameter not exceeding 1.5ju when ripe. After some time, the spore is liberated by the breaking up of the mother cell. Old cultures, in which the decomposition of the paper is well advanced, show only spores with a slight admixture of vegetative forms which are usually in the stage of forming spores. The spore-containing cultures may be stained with a double stain of carbol fuchsin and methylene blue. The organism is never colored blue with iodine and, therefore, lacks the important characteristics of Amylobacter. By repeated transfers on enrichment culture media, a microscopically pure culture of the organ- ism can be obtained, especially if the inoculum is heated for 20 minutes at 90°C. to kill all non-spore forming contaminations. But all repeated attempts to cultivate the organism on solid media failed. This pre- vented a detailed study of its metabolism. The methane organism (Bac. methanigenes L. et N.) is quite similar to the hydrogen organism, but is even thinner and more gently con- toured. By several transfers and on heating the inoculum, a culture is obtained which seems microscopically pure. Chains are never formed in a young stage and the cells have a tendency to curve slightly. The spores are smaller than those of the hydrogen form, being 1/x in diameter. Iodine does not give a blue color. Morphologically both organisms may be classified as one species, while, physiologically, they are distinctly different. Attempts to cultivate this organism on solid media and obtain a pure culture also failed. Kellerman and associates6 could not confirm the results of Omeliansky. They even succeeded in isolating from Omeliansky's cultures an aerobic cellulose-decomposing organism. They suggested, therefore, that the cellulose was decomposed by aerobic bacteria in Omeliansky's cultures, while the accompanying anaerobic forms produced gas from the products of decomposition of the cellulose by the former. However, Khouvine7 6 Kellerman et al., 1912-1914 (p. 197). 7 Khouvine, Mme. Y. Digestion de la cellulose par la flore intestinale de l'homme. Cour D'Appel. Paris. 1923. 194 PRINCIPLES OF SOIL MICROBIOLOGY succeeded in isolating from the intestine of man an obligate anaerobic organism, Bac. cellulosae dissolvens, capable of decomposing cellulose very vigorously, especially in mixed culture. The organism was 2.5 to 12.5^ long, and did not form any flagella; the spores were 2.5 by 2n in size. It was cultivated upon a medium containing fecal mat- ter as a source of nitrogen. The spores were killed only on boiling for 45 to 50 minutes. The organism decomposed cellulose at 38° to 51°C, without any distinct optimum. When the oxygen tension of the atmos- phere was above 12 mm. mercury, no growth took place. The organism clung to the paper, so that the contaminating forms could be removed by washing the paper with sterile salt solution. Sixty per cent of the cel- TABLE 14 Summary of characteristics of two anaerobic cellulose decomposing bacteria Size of rods Size of spore Flagella Nutrient broth with or with- out glucose Nutrient agar Potato slant Milk Carbohydrates decomposed BACILLUS CELLULOSAE DISSOLVENS— KHOUVINE 2.0x2 to 12 ix 2.0x2.5^ Absent No growth No growth No growth No growth Only cellulose CLOSTRIDIUM THEPMOCELLU V, P AND P 5.0x0.4M 0.9x0.6^ Peritrichous Ring, pellicle and sediment; acid and gas with glucose Small surface and subsurface colonies on starch agar Yellow growth, potato browned Acid curd in three days Hemicellulose, starch, various hexoses and pentoses lulose decomposed was accounted for by the carbon dioxide, hydrogen, ethyl alcohol, acetic and butyric acids, and a brown pigment. The presence of other bacteria greatly stimulated the power of this organism to decompose cellulose. The organism was found to be also abundantly distributed in the soil, occurring in all soil types, under various conditions. The results of other investigators8 also point to the anaerobic nature of cellulose decomposing bacteria in the digestive tract of horses. Under anaerobic conditions, cellulose decomposition is carried out entirely by bacteria; the nature of the processes involved being different 8 Hosslin, A., and Lesser, liber die Zersetzung der Zellulose durch den Inhalt des Coecums des Pferdes. Ztschr. Biol. 54: 47. 1910. BACTERIA DECOMPOSING CELLULOSES 195 from that of cellulose decomposition under aerobic conditions.9 The thermophilic Clostridium thermocellum Vil., Fred and Peterson, described later, is also an anaerobic organism, decomposing cellulose very actively. Aerobic bacteria. The first attempt to study cellulose decomposition under aerobic conditions was made by Van Iterson,10 who described a non-spore-forming organism, Bacillus ferrugineus, which decomposed cellulose under aerobic conditions, in symbiosis with a yellow micro- coccus, the latter not decomposing any cellulose when alone. The following medium was used: Tap water 100 cc. K2HPO< 0.05 gram Filter paper 2 grams CaC03 2.0 grams NH4Cl(orKN02,KN02, MgNH4P04, pep- tone) 0.1 gram The medium was placed in Erlenmeyer flasks to a depth of 0.5 to 1 cm., inoculated and incubated at 28° to 35°. Cellulose decomposition was also demonstrated by placing two pieces of filter paper and some powdered ammonium magnesium phosphate in a dish, moistening with a 0.05 per cent solution of K2HPO4 and in- oculating with some soil. Yellowish brown spots were produced on the paper in 4 to 5 days; the paper soon became pulpy, and the individual fibers became en- veloped in a "micrococcus mucilage." Pure cultures of the organism could never decompose the paper. Merker11 described two bacteria, Micrococcus cytophagus and M. melanocyclus, neither of which was isolated in pure culture, but which were found to be accompanied by a rod-like organism; they decomposed paper partly immersed in the medium with the formation of transparent yellowish spots. A similar organism was studied by Bojanovsky12 on silica gel media, but he also failed to separate the coccus-like form from the rod-shaped form. The organism (Spirochaeta cytophaga) isolated from the soil by Hutchinson and Clayton13 was found to develop first as a sinuous 9 VVaksman and Skinner, 1926 (p. 190). 10 Van Iterson, C. Die Zersetzung von Cellulose durch aerobe Mikroorganis- men. Centrbl. Bakt. II, 11 : 689-698. 1904. 11 Merker, E. Parasitische Bakterien auf Blattern von Elodea. Centrbl. Bakt. II, 31: 578. 1912. 12 Bojanowsky, R. Zweckmiiszige Neuerungen fiir die Herstellung eines Kieselsaure Nahrbodens und einige Beitrage zur Physiologie aerober Zellu- loseloser. Centrbl. Bakt. II, 64: 222-233. 1925. 13 Hutchinson, H. B., and Clayton, J. On the decomposition of cellulose by an aerobic organism (Spirochaeta cytophaga n. sp.). Jour. Agr. Sci. 9: 143-173. 1918. 196 PRINCIPLES OF SOIL MICROBIOLOGY filamentous cell (3 to 10 by 0.3 to 0.4/z), which appears to go through a number of phases terminating in the production of a spherical body (sporoid). This differs in a number of respects from the true spores of bacteria; germination of the sporoid again gives rise to the filamentous form which possesses perfect flexibility and is feebly motile although no flagella were observed. The organism is aerobic, with an optimum at 30°C, and is destroyed when kept at 60°C. for ten minutes. It does not grow in nutrient agar or gelatin, and is injuriously affected by concentrations of peptone above 0.025 per cent. As sources of nitrogen, ammonium salts, nitrates, amides and amino acids can be used, while cellulose is the only source of carbon. The soluble carbohydrates are more or less toxic. The following medium was used for the isolation and cultivation of the organism: K2HP04 1.0 gram Fe2Cl6 0.01 gram CaCl2 0.1 gram NaN03 2.5 grams MgS04 0.3 gram Cellulose 10. 0 grams NaCl 0.1 gram H20 1000 cc. The same organism was found to occur in the soil and to decompose cellulose readily.14 By the use of the silica gel method suggested by Winogradsky, the Sp. cytophaga can be shown to be very abundant in the soil, especially soils receiving applications of farmyard manure and straw.15 The gel is prepared by placing a mixture of equal parts of a normal solution of HC1 and its equivalent of potassium silicate solution into Petri dishes. After the gel has formed, the dishes are placed in running tap water for 24 hours, then several times in boiled distilled water until free from chlorides. Five grams of ground filter paper are then suspended in 100 cc. of medium containing 5 grams (NH4)2HP04, 1 gram MgS04, 1 gram KC1, 0.02 gram FeS04, in 1000 cc. distilled water; about 2 cc. portions of the suspension are spread over the surface of each plate and a small amount of CaC03 is dusted on. The plates are then placed in the thermostat at 60°C, until the surface of the gel becomes free from excess of liquid. Small particles of soil can then be inoculated directly upon the plate which is then covered and incubated. After 2 to 4 days, yellow and orange growth will be found to develop from the soil into the medium. Transfers are then made into flasks containing 1 gm. of filter paper and 25 cc. of above solution. The organisms will begin to develop in the form of yellow specks, forming a yellow slimy mass all over the paper in 2 to 4 days. By repeated dilutions, the organism can be isolated pure. 14 Lohnis, F., and Lochhead, G. Experiments on the decomposition of cellu- lose by aerobic bacteria. Centrbl. Bakt. II, 58: 430^34. 1923. 15 Waksman, S. A., and Carey, C. On the use of the silica gel plate for the isolation of cellulose-decomposing bacteria. Jour. Bact. 12: 87-95. 1926. BACTERIA DECOMPOSING CELLULOSES 197 The abundant distribution in the soil and the rapid decomposition of cellulose by this organism is thus established and can be demonstrated without difficulty. In addition to the Sp. cytophaga, however, there are other bacteria found in the soil which are capable of decomposing cellu- lose under aerobic conditions. Extensive work has been carried out, in this connection, by Keller- man and associates.16 A special culture medium was developed, for which the cellulose was prepared in the following manner: One liter of ammonium hydroxide, specific gravity 0.90, is poured into a glass- stoppered bottle; 250 cc. of distilled water and 75 grams of pure copper carbonate are added; the solution is shaken vigorously until all the copper is dissolved (from 10 to 15 minutes is ordinarily required). Fifteen grams of high grade, sheet filter paper is then added to the copper-ammonium solution and mixture is shaken vigorously, at intervals of 10 minutes, for one-half hour. The solution is examined carefully to see that the paper is completely dissolved. If any parti- cles of paper remain in the solution, the shaking must be continued until the solution is perfectly clear. The ammonium-copper-cellulose solution (250 cc.) is diluted to 10 liters with cap water; a weak hydrochloric acid solution prepared by adding 500 cc. of con- centrated acid to 10 liters of tap water is slowly added, with frequent shaking. The addition of the acid is continued until the blue color disappears: a slight excess of acid is added. The mixture is shaken vigorously and allowed to stand a few minutes. The finely precipitated cellulose will rise to the top, due to the large quantity of free hydrogen liberated in the precipitation process. The solu- tion is shaken vigorously, at intervals of a few minutes, to dislodge the hydrogen. As soon as the free hydrogen has escaped, the cellulose will settle rapidly. The cellulose is washed through repeated changes of water until free from copper and chlorine. After the washing is complete, the cellulose in the solution is brought up to 0.5 per cent, by allowing to settle a few days and siphoning off the clear solution or by evaporation. The nutrient salts together with 1 per cent of thoroughly washed agar are then added and heated in autoclave or boiled, until the agar is dissolved. The medium is then tubed and sterilized in the usual way. Another method for the preparation of cellulose has been suggested by Scales:17 Concentrated sulfuric acid (100 cc.) is diluted with 60 cc. of distilled water in a 2-liter Erlenmeyer flask and cooled to 60° or 65°C. Five grams of filter paper, 16 Kellerman, K. F,, and McBeth, I. G. The fermentation of cellulose. Cen- trbl. Bakt. II, 34: 485-494. 1912; Kellerman, K. F., McBeth, I. G., Scales, F. M., and Smith, N. R. Identification and classification of cellulose dissolving bac- teria. Ibid. 39: 502-522. 1914. McBeth, I. G., and Scales, F. M. The destruc- tion of cellulose by bacteria and filamentous fungi. U. S. Dept. Agr., Bur. PI. Ind., Bui. 266, 1913. McBeth, I. G. Studies on the decomposition of cellulose in soils. Soil Sci. 1: 437-487. 1916. 17 Scales, F. M. A new method of precipitating cellulose for cellulose agar. Centrbl. Bakt. II, 44: 661-663. 1915. 198 PRINCIPLES OF SOIL MICROBIOLOGY sufficient for one liter of cellulose agar, is moistened with water and added to the acid which is then vigorously agitated till the cellulose is dissolved. The flask is then quickly filled with cold tap water. The whole process of dissolving the paper and filling the flask requires about one minute. The precipitate is now filtered through paper and washed until the filtrate no longer gives any test for sulfuric acid. When the volume of the suspension is drained to about 200 cc, a hole is punched in the bottom of the filter and the whole precipitate washed out and made up to 500 cc. The cellulose may also be obtained from plant tissues by extracting well ground material with 2 per cent KOH solution, washing, chlorinating, extracting again with boiling 2 per cent NaOH for 30 minutes and washing. Cellulose suspension (500 cc.) prepared by any of the above methods is now added to 500 cc. of a solution containing 0.5 gram K2HP04, 0.5 gram MgS04, 0.5 gram NaCl, 1 gram (NH4)2S04) 1 gram CaC03 and 500 cc. of tap water. Ten grams of agar are dissolved in the mixed solutions and medium tubed and ster- ilized as usual. When soil suspensions are directly inoculated upon poured plates with cellulose agar, filamentous fungi will develop and tend to overgrow the plate, making difficult the isolation of the bacteria. The sample of soil or manure is added to sterile flasks containing the cellulose broth or peptone cellulose broth and, as soon as the cellulose shows signs of dis- integration, transfers are made upon fresh sterile flasks. This avoids the development of fungi. After several preliminary cultivations upon the liquid enrichment media, the cultures are plated out on the cellu- lose agar. The colonies of cellulose-decomposing organisms developing on the plates show a translucent area due to the decomposition of the cellulose in the agar, as well as neutralization of the CaC03 by the acids formed from the ammonium sulfate and cellulose decomposition. Starch agar and finally nutrient agar may be used for the final cultivation of the organisms. The cellulose-destroy- ing bacteria studied by Kellerman and associates were found to grow more rapidly under aerobic conditions, although some anaerobic development has also been observed. They show a more vigorous growth on media containing organic nitrogen (peptone) than inorganic nitrogen. They usually reduce nitrates to nitrites, attack various carbohydrates, and do not form any gaseous products from cellulose or the lower carbohydrates. The paper is disintegrated into fine fibres and a small amount of organic acids is formed. The following liquid medium was used for demonstrating the dissolution of the filter paper: Soil extract 500 cc. Peptone 2.0 grams Distilled water 500 cc. CaC03 excess K2HP04 0.5 gram Reaction pH 7.0 (NH4)2S04 1.0 gram All the forms studied were rod-shaped organisms varying in length from 0.8 to 3.5ju. Only in three species were involution forms observed. Five species produced spores. Twenty-seven of the thirty-six species isolated were motile. BACTERIA DECOMPOSING CELLULOSES 199 These organisms are widely distributed in the soil. When kept under laboratory conditions, for any length of time, especially on nutrient agar media, they undergo marked physiological changes which may include loss of cellulose-decomposing power. Transfers made from the clear zone around the colony of the cellulose-decomposing bacterium growing on the plate decomposed cellulose readily; when the culture was transferred upon nutrient agar, the organism lost its cellulose- decomposing power. Lohnis and Lochhead18 suggested that the thread- like organism, described above, plays a prominent role in the aerobic decomposition of cellulose and is accompanied by numerous other cellulose-decomposing bacteria of lower efficiency. This led Omeli- ansky19 and Pringsheim20 to suggest that the organisms isolated by Kellerman, McBeth and Scales were not in themselves cellulose decom- posing forms but were present as contaminations. When isolations were attempted on the agar plate, it was these contaminations that were isolated, while the true cellulose decomposing forms were lost. In general, a large number of aerobic bacteria capable of decomposing cellulose have been isolated from the soil, but the identity of many of them is doubtful.21-24-26 Gray and Chalmers25 isolated from the soil an aerobic organism capable of decomposing cellulose and liquefying agar {Microspira agar-liquefaciens); this 18 Lohnis and Lochhead, 1923 (p. 196). 19 Omeliansky, 1913 (p. 191). 20 Pringsheim, H., and Lichtenstein, S. Zur vermeintlichen Reinkultur der Zellulosebakterien. Centrbl. Bakt. II, 60: 309-311. 1923. 21 Sack, J. Zellulose-angreifende Bakterien. Centrbl. Bakt. II, 62:77-80. 1924. 22 Epstein, A. Un nouvel agent destructeur des polysaccharides complexes, Pseudomonas jwlysaccharidarum n. sp. Bull. Soc. Bot. Geneve (2), 11: 191-198. 1920. 23 Distaso, A. Sur un microbe qui desagrege la cellulose (Bacillus cellulosae desagregans n. sp.). Compt. Rend. Soc. Biol. 70: 995-996. 1911. 24 Hopfe, A. Bakteriologische Untersuchungen liber die Celluloseverdauung. Centrbl. Bakt. I, 83: 374-386. 1919. 25 Gray, P. H. H., and Chalmers, C. H. On the stimulating action of certain organic compounds on cellulose decomposition by means of a new aerobic micro- organism that attacks both cellulose and agar. Ann. Appl. Biol. 11: 324-338. 1924. 26 Gescher, N. tlber cellulosezersetzende Bakterien. Faserforschung, 2: 28-40. 1922. 200 PRINCIPLES OF SOIL MICROBIOLOGY bacterium is 2 by 0.5 to 0.7^ with coccoid forms of 0.5 to 0.7^, motile with a single flagellum. The addition of dextrin, lignin, xylose and certain sugars stimulated cell development.26 Decomposition of cellulose by denitrifying bacteria. Certain bacteria are able to decompose cellulose and utilize the energy for the reduction of nitrates to nitrites. A medium containing 0.25 gram KN03, 0.05 gram K2HP04 and 2 grams of cellulose in the form of filter paper in 100 cc. of tap water can be used.27 This medium is placed in a glass stoppered flask up to the neck, inoculated with canal slime and incu- bated anaerobically at 35°. Cellulose decomposition begins in a week and is accompanied by reduction of nitrate to nitrite. The nitrite also disappears in 15 days. On renewing the medium, the process of nitrate reduction is greatly hastened. The cellulose becomes orange yellow and of a slimy consistency. It is broken down into fibers which finally disappear. The gases consist of a mixture of nitrogen and carbon diox- ide. No organism responsible for the process of cellulose-decomposi- tion could be isolated from the mixture of various bacteria and protozoa. Groenewege,28 using a medium which consisted of 20 grams filter paper, 2.5 grams KN03 and 0.5 gram K2HP04 in 1000 cc. water and inoculated with soil, found that the process of cellulose decomposition by denitrify- ing bacteria is carried on by the symbiotic action of two groups of organisms, one of which decomposes the cellulose and the other of which uses the products formed as a source of energy for the reduction of nitrates. By the symbiotic action of the two organisms the cellulose disappears much more rapidly than by the action of the cellulose organ- ism alone. Gas formation began to take place on the second day, accompanied by a reduction of nitrate to nitrite and NO with a gradual dissolution of the paper. The process was especially active when the culture solution was renewed by decantation. To stimulate the development of cellulose-decomposing organisms, Van Iterson buried filter paper in the soil and after four weeks found that it became almost entirely decomposed and covered with orange and black spots. The black spots consisted of fungi. Using this material for inoculation, a complete reduction of 0.6 per cent KN03 took place in three days. On replacing the solution by decantation, complete denitrification may take place in twenty-four hours. The organisms responsible for the process were obtained in pure cultures by the use of 27 Van Iterson, 1904 (p. 195). 28 Groenewege, J. Untersuchungen liber die Zersetzung der Zellulose durch aerobe Bakterien. Bull. Jard. Bot. Buitcnzorg. 2, f. 3: 261-345. 1920. BACTERIA DECOMPOSING CELLULOSES 201 nutrient agar plates. A small piece of the decomposing cellulose is thoroughly disintegrated on the plate, by means of a sterile spatula. If a large piece of paper is used, it should be previously washed in salt solution, to wash off the rapidly growing organisms not taking part in the processes of cellulose decomposition and nitrate reduction. These organisms develop only slowly as minute colonies and the plates have to be incubated for four to six days at room temperature, before trans- fers can be made. In the presence of rapidly growing organisms the minute colonies usually fail to develop. The bacteria isolated in the processes of cellulose decomposition and nitrate reduction were divided28 into three groups: (1) those which effected denitrifica- tion in nitrate bouillon but not in the cellulose-KN03 (0.1 per cent) medium; (2) those which did not reduce nitrates in the nitrate bouillon but caused re- duction in the cellulose nitrate medium; (3) those which did not reduce nitrate in either medium. A combination of the first two groups or of all three brought about active reduction of nitrate and decomposition of cellulose. Group 1 was found to consist of two bacteria, namely Bad. opalescens and Bad. viscosum; group 2 consisted of three strains of Bad. cellar esolv ens (a very fine, aerobic, rod- shaped organism), all of which grew very slowly and formed minute colonies on nutrient agar. The cellulose decomposing organism was obligate aerobic. B. cellar esolv ens attacked cellulose; the products of the decomposition (acetic, butyric and lactic acids) could serve as food for the denitrifying organisms, B. opalescens and B. viscosum. The complete process is one of symbiosis. For a quantitative study of cellulose decomposition, Groenewege used a medium consisting of: Filter paper 200 mgm. CaC03 400 mgm. NH4C1 40 mgm. Tap water 40 cc. K2HP04 20 mgm. The medium was inoculated with pure cultures of the organisms and mixtures of the cellulose decomposing and denitrifying forms. After three weeks, 10 cc. of 10 per cent HC1 was added, flasks filled to half with water and after the cellulose settled, the liquid was taken off. This was repeated until the liquid was free from acid. The residue was then centrifuged, dried and weighed. The cellulose de- composing bacteria alone decomposed 17 to 135 mgm. of the cellulose (depending on strain) and 22 to 151 mgm. in the presence of the denitrifying bacteria. Sim- ilar results were obtained with asparagine as a source of nitrogen, indicating that the favorable influence of the B. opalescens on B. cellar esolv ens is due to a sym- biotic action. Thermophilic bacteria. MacFayden and Blaxall29 were the first to demonstrate the presence in the soil of organisms which are able to decompose cellulose at 60° to G5°C. The process was accelerated under 29 Macfayden, A., and Blaxall, F. R. Thermophilic bacteria. Trans. Jenner. Inst. Prevent. Med. Ser. 2: 162-187. 1899. 202 PRINCIPLES OF SOIL MICROBIOLOGY anaerobic conditions. They believed that the process is carried out by the combined action of several organisms. Kroulik30 used a medium, similar to that employed by Omeliansky, consisting of 1 gram (NH4)3P04, 1 gram K2HP04, 0.5 gram MgS04, trace of NaCl, 1000 cc. distilled water, 1 to 2 per cent cellulose and 0.5 to 1 per cent MgC03. He demon- strated the common occurrence of bacteria able to decompose cellulose at 60° to 65°C, particularly in places where cellulose is present in abun- dance. Both aerobic and anaerobic forms have been demonstrated. Two aerobic forms were found in great abundance during the early stages of decomposition, but when isolated on nutrient agar media, they did not decompose cellulose further. Two anaerobic bacteria were isolated which did not grow upon agar.31 Various other thermophilic cellulose- decomposing bacteria have also been isolated from the soil.32"-34 For this purpose a medium consisting of 2 grams NaNH4HP04 H20, 1 gram KH2P04, 0.3 gram CaCl2,5grams peptone, 15 grams cellulose, 1000 cc. of tap water and excess of CaC03is inoculated with infusions of rapidly de- composing manure and the cultures are incubated at 65°C. Gas bubbles begin to rise after 18 to 24 hours and the formation of H2S becomes evi- dent.34 After 30 to 36 hours, the cellulose pulp is raised to the surface, loses its fibrous structure, and turns brownish-yellow in color. Further transfers do not form any more H2S. The cellulose begins to decompose after 18 hours and the process is completed after 6 to 8 days. When inoculations from individual colonies are made upon glucose agar, the isolated cultures do not decompose cellulose. Pure cultures are ob- tained by means of deep agar-cellulose tubes. The organism does not grow at 28 to 37°C, makes some growth at 43° to 50°C, grows best at 65°C. The spores are destroyed at 115° in 37 minutes. When grown on common agar media, the power of cellulose decomposition is lost. This is probably due to the loss of some highly oxidizable com- ponent, during the process of plating, which is needed to initiate the process of decomposition. A comparative summary of this thermophilic organism {Clostridium 30 Kroulik, 1913 (p. 157). 31 See also Noack, 1912 (p. 300). 32 Pringsheim, H. tjber die Vergarung tier Zellulose durch thermophilc Bakterien. Centrbl. Bakt. II, 38: 513-516. 1913. 33 Langwell, H., and Lymn, A. Discussion on the action of bacteria on cellu- losic materials. Jour. Soc. Chem. Ind. 42T: 280-287. 1923. 34 Viljoen, J. A., Fred, E. B., and Peterson, W. H. The fermentation of cellu- lose by thermophilic bacteria. Jour. Agr. Sci. 16: 1-17. 1926. BACTERIA DECOMPOSING CELLULOSES 203 thermocellum V. F. and P) and the anaerobic organism of Khouvine is given in table 14. The Bac. cellulosae clissolvens approaches the Spirochaeta cytophaga in its cellulose-decomposing capacity, in being- unable to grow on any media except those containing cellulose. CI. thermocellum can grow with other sources of energy. One must keep in mind, however, the fact that when this organism is grown on other sources of energy it looses its power to decompose cellulose. Pectin decomposing bacteria. Pectins, like celluloses, are decomposed by (1) fungi,35 (2) aerobic bacteria, and (3) anaerobic bacteria. The aerobic bacteria capable of decomposing pectins include Bac. astero- sporus and Bac. mesentericus.ZG In addition to these organisms, there are various other aerobic bacteria, such as Bac. subtilis, Bad. fluores- ceins,^ Bac. macerans37 and Pectinobacter amylophilum3* which were found to be able to decompose pectins. The Bac. comesii and Bac. kramerii of Rossi39 are merely species of Bac. mesentericus or Bac. asterosporus.40 Mention should also be made of the bacteria causing soft rots of vegetables, especially Bac. carotovorus,41 which are capable of breaking down pectins readily. The anaerobic bacteria capable of decomposing pectins include two organisms: (a) Bac. amylobacter and (b) Bac. felsineus. The former42 35 Hauman, L. Etude microbiologique et chimique du rouissage aerobic du lin. Ann. Inst. Past. 16: 379-385. 1902; Behrens, J. Uber die Taurotte von Flachs und Hanf. Centrbl. Bakt. II, 10: 524-530. 1903; Die Pektingarung. Lafar's Handb. techn. Mykol. 3: 269-286. 1904. 36 Beijerinck, M. W., and van Delden, A. Over de bacterien, welke bij het roten van vlas werdzaam zijn. Kon. Akad. Wetensch. te Amsterdam. Dec. 19: 673. 1903. 37 Schardinger, F. Ueber die Bildung kristallisierter, Fehlingsche Losung nicht reduzierender Korper (Polysaccharide) aus Starke durch mikrobielle Tatigkeit. Centrbl. Bakt. II, 22: 98-103. 1909. 38 Makrinov, I. A. Sur un nouveau microorganisme provoquant la fermenta- tion de l'amidon et des matieres pectiques. Arch. Sci. Biol. 18: No. 5. 1915. 39 Rossi, G., and Guarnieri, G. II bacillus comesii e le sue proprieta. Primi tentativi di macerazione di fibre tessili con fermenti selezionati. R. Sc. Agr. Portici. 1906; Industrial retting of textile plants by microbiological action. Inter. Inst. Agr. Bur. Intel. PI. Dis. 7: 635. 1916. 40 Ruschmann, G., and Ravendamm, W. Die Flachsroste mit Plectridium peclinovorum (Bac. amylobacter A. M. Bredemann) und Bacillus felsineus Carbone. Centrbl. Bakt. II, 65: 43-58. 1925. 41 Jones, L. R. Pectinase, the cytolytic enzyme produced by Bacillus caroto- vorus and certain other soft-rot organisms. N. Y. Agr. Exp. Sta. Tech. Bui. 11, 289-368. 1909. 42 Bredemann, 1909 (p. 109). 204 PRINCIPLES OF SOIL MICROBIOLOGY comprises a variety of forms described under different names, including Plectridium,43 Clostridium44 and Granulobacter.45 However, not all forms of Bac. amylobacter are capable of retting flax. Bac. felsineus Carbone46 is 3 to 5 by 0.3 to 0.4ju and forms free spores 3 by 1.5 to 2/x in size. It is similar in morphology and general physiology to Bac. amylobacter A. M. et Bred., but varies from the latter by being unable to produce butyric acid.40 Bacteria decomposing hydrocarbons and benzene ring compounds. Petroleum, paraffin oil and other hydrocarbons can be readily used by a number of soil bacteria as sources of energy.47 To obtain an enriched culture, the following medium was used: Tap water 1000 cc. CaC03 trace K2HPO 4 0.5 gram One of the NH4C1 0.5 gram paraffins about 10.0 grams Petroleum, benzin and paraffin oil can be used directly. The paraffin is first melted by warming and the solution is then vigorously shaken. The medium is inoculated with soil and incubated. At 20°, fluorescent and fat-splitting organ- isms develop; at 38°, Mycobacteria (4 to 10 by 0.5 to 1.5/x) and Micr. parajflnae develop. Sohngen found that 4 to 8 mgm. of petroleum were oxidized in 24 hours at 28°C. for every square centimeter of surface of solution. Pure cultures of the organisms were obtained on a medium consisting of: Washed agar 20 grams Distilled water 1000 cc. K2HPO4 0.5 gram Paraffin vapor MgS04 0.5 gram The number of bacteria capable of oxidizing paraffin in the soil are very large, reaching 50,000 to 200,000 per gram of garden soil. Among the species isolated we find Bad. fluorescens liquefaciens and non-U quefaciens, Bad. pyocyaneum, B. stutzeri. B. lipolyticum a, (3, 7 and 8, Micr. paraffinae, and all fat-splitting forms. Tausz and Peter48 isolated three organisms from the soil capable of decomposing hydrocarbons: Bad. aliphaticum, Bad. aliph. liquefaciens and Paraffi nbaderium . The first was isolated 43 Winogradsky, S., and Friebes, V. Sur le rouissage du lin et son agent microbien. Compt. Rend. Acad. Sci. 121: 242. 1895. 44 Stormer, K. Ueber die Wasserroste des Flaches. Mitt. deut. Landw. Gesell. 18: 193. 1903; Chem. Centrbl. 76: 41. 1905. 45 Beijerinck and Van Delden, 1902 (p. 183). 46 Carbone, D., and Tobler, F. Die RSste mit Bacillus felsineus. Faserforsch. 2: 163-184. 1923. 47 Sohngen, N. L. Benzin, Petroleum, Paraffinol und Paraffin als Kohlen- stoff- und Energiequelle fur Mikroben. Centrbl. Bakt. II, 37: 595 609. 1913. 48 Tausz, J., and Peter, M. Neue Methode der Kohlenwasserstoffanalyse mit Hilfe von Bakterien. Centrbl. Bakt. II, 49: 497-554. 1919. BACTERIA DECOMPOSING CELLULOSES 205 on inorganic and organic media to which a few drops of n-hexan was added; it is 2 by 1.5/* in size, motile by means of peritrichic fiagella, Gram-negative and grows well with aliphatic hydrocarbons as the only source of carbon and energy. The second organism was isolated on media containing naphthenes as cyclohexan and 1-3 dimethylcyclo- hexan. It is similar to the first organism, but is strongly proteolytic. The third organism was isolated on selective media consisting of inor- ganic solutions and paraffin oil. In liquid media, the organism forms motile rods, 4 to 6 by 2/z, developing into long chains. Rapid spore formation is characteristic and it is, therefore, related to B. subtilis. Various bacteria capable of oxidizing phenol to C02, pyrocatechin to oxychinon, benzol to fatty acids and C02, and capable of decomposing toluol, xylol and guajacol were isolated from the soil.49 Aerobic Clostridia, Bad. fluorescens group and Mycobacteria, are also concerned in the decomposition of phenols, cresols and related compounds in the soil. 49 Wagner, R. fiber Benzol-Bakterien. Ztschr. Garungsphysiol. 4: 289-319. 1914. CHAPTER IX Bacteria Decomposing Urea, Uric and Hippuric Acids Urea, uric and hippuric acids are the products of protein decomposi- tion in the animal organism and form the most important nitrogen con- stituents of the liquid part of the manure. These compounds are not directly available as sources of nitrogen to higher plants. They have to be first decomposed by various groups of microorganisms existing in manure and in the soil and transformed into ammonia and other com- pounds. The chemical processes concerned are discussed in detail else- where (p. 486). Organisms decomposing urea. Pasteur1 was the first to recognize in 1860 that ammonia formation from urea is brought about by a living organism, namely Torula ammoniacale. It was later found that organ- isms capable of decomposing urea are found in most families of bacteria, actinomyces and fungi, but certain specific bacteria, whose metabolism is closely connected with the transformation of this substance, are termed urea bacteria. These are divided into cocci and bacilli. The former are usually destroyed at 60° to 70°, while the latter, due to their ability to form endospores, can withstand heating at 90° to 95° for several hours. The optimum temperature for the action of these organ- isms is about 30°C. They usually thrive best in media containing urea (2 per cent), particularly when made alkaline with ammonium carbon- ate (2 to 3 grams per liter). The accumulation of ammonium carbon- ate from the hydrolysis of the urea is so great, in many instances, as to kill the organisms themselves. A rapid urea decomposition does not necessarily accompany a rapid growth. The urea bacteria differ in their oxygen tension; most of them are aerobic, although the amount of oxygen required may be rather small. Many of these organisms are probably varieties of some of the common soil bacteria. The urea splitting bacteria are commonly found in great abundance in soil, manure, dust and water. Miquel2 found urea organisms in the canal 1 Pasteur, L. De l'origine des ferments. Nouvelles experiences relatives aux generations dites spontances. Compt. Rend. Acad. Sci. 50: 849-854. 1860. 2 Miquel, P. Die Vergarung des Harnstoffes, der Harnsaure und der Hip- pursaure. Lafar's Handb. techn. Mykol. 3: 71-85. 1904. (References to earlier work.) 206 BACTERIA DECOMPOSING UREA 207 waters of Paris, fifty-two forms in the sewage waters and sixty-six in the drainage of the privy closets. The urea bacteria of the surface soil were found to form 1 to 2 per cent of the total number of bacteria. Manure and urine contained 10 per cent of their flora as urea bacteria. The air in Paris was found to contain one urea splitting organism for every sixty-seven other forms. Urea bacteria are very abundant in the soil (p. 37). Frequently the urea is so rapidly decomposed as to lead to actual losses of ammonia. This can be prevented, however, by a proper mixture of the urea in the soil.3 Methods of isolation. The isolation and cultivation of the urea bac- teria does not present great difficulty. The selective and enrichment cultivation4 can be readily utilized for this purpose. For the purposes of isolation, two solutions have been suggested:5 1. Tap water 1000 cc. 2. Tap water 1000 cc. K2HPO4 0.5 gram K2HP04 0.5 gram Calcium citrate or Ammonium tartrate 10.0 grams malate 10.0 grams Urea 30.0 grams Urea 50.0 grams One hundred cubic centimeters of either solution is inoculated with 2 grams of soil and incubated at 23° or 33°; in 36 to 48 hours, the medium becomes well inoculated with urea bacteria. After two or three transfers, the organisms are readily obtained in pure culture. A medium consisting of 50 grams of urea, 0.5 gram K2HPO4, 100 cc. of soil extract and 900 cc. of tap water may be used.6 Peptone gelatin containing 2 to 5 per cent of urea was found to be a very good solid medium.7 A few days after inoculation, often only after twenty-four hours, most of the visible colonies will be found surrounded with a halo. This is com- posed of dumb-bell shaped crystals insoluble in water and consisting of carbonate and phosphate of calcium which were precipitated from the medium as a result of the formation of ammonium carbonate from the urea. The stronger the action of the bacteria, the wider is the zone. The halo of crystals either surrounds the colony to a width of several millimeters or rapidly covers, in 24 hours, the whole plate. The urea organisms are thus readily recognized and are then transferred upon the specific media. 3 Littauer, F. Zersetzung des Harnstoffs im Boden. Ztschr. Pflanzenernahr. Diing. 3A: 65. 1925. 4 Beijerinck, M. W. Anhiiufungsversuche mit Ureumbakterien. Ureumspal- tung durch Urease und durch Katabolismus. Centrbl. Bakt. II, 7: 33-60. 1901. Duggeli. Naturw. Wochschr. 14: 305. 1915. 5 Sohngen, N. L. Ureumspaltung bei Nichtvorhandesein von Eisweisz. Cen- trbl. Bakt. II, 23: 91-98. 1909. 6L6hnis, 1905 (p. 120). 7Miquel, 1904 (p. 206). 208 PRINCIPLES OF SOIL MICROBIOLOGY Ordinary bouillon, with or without peptone, yeast water, peptone solution, which have been made alkaline and to which 0.1 to 0.2 per cent of urea has been added are quite suitable for the cultivation of the organisms. To study the urea splitting power of the organisms, various quantities of urea may be em- ployed (usually 2 per cent), but the greater the amount of urea, the greater is the danger of the destructive action of the ammonium carbonate. Viehoevers used, for the isolation of Bac. probalus and other urea bacteria, a medium consisting of : Water 1000 cc. Nad 0.1 gram K2HP04 1 gram FeCl3 0.01 gram CaCl2 0.1 gram Urea 20.0 grams MgSO i 0.3 gram Liebig's beef ex- tract 5.0 grams Agar is added to this solution when a solid medium is wanted for the isolation of the bacteria from colonies. Ammonium carbonate or urea agar are prepared by adding 3 grams of the first or 3 to 30 grams of urea to a medium consisting of 1500 parts of water + 30 agar + 6 peptone + 4 Liebig's beef extract + 1 NaCl -I- 5 glucose. Classification and description. The urea bacteria include both anaero- bic and aerobic forms. Miquel divided all these bacteria into two general groups: (1) Urococci and (2) Urobacilli. Among the cocci he observed as many as thirty different species representing four genera: Urococcus, Urosarcina, Micrococcus and Planosarcina, while the Uro- bacilli were also divided into various genera. The urea decomposing organisms can thus be divided into four groups: I. Spore-bearing cocci. Planosarcina vreae Beij. forms packets on solid and liquid media, of 4 to 8 cells (0.7 to 1.2/*), and is motile by means of long flagella. The spherical endospores (0.6m) can withstand 80°C. for ten minutes. II. Non-spore bearing cocci. Urococcus van tieghemi Miquel (Syn. Torula ammoniacale Pasteur, Mic. ureae Cohn), 1 to 1.5^ in diameter, occurring in twos, often in chains; Mic. ureae liquefaciens Flugge, Uros. hansenii Miquel and a num- ber of other Urosarcinae and Micrococci described by Miquel, Rochaix and Du- fourt,9 as well as the common species Micr. pyogenes and Strept. pyogenes. III. Spore-bearing bacilli. These include the Urobacillus pasteurii (Miquel) Beij., 1 to 1.2 by 2.5^, single or short chains, motile, forming egg-shaped endo- spores, persisting in dry soil for many years, decomposing urea very actively; Urob. duclauxii, Urob. freudenreichii, Urob. maddoxii and other species described by Miquel; Urob. leubii Beij., Bac. probatus Vieh., Bac. ureae II and III Burri. According to Lohnis, Bac. mycoides and Bac. megatherium, which decompose urea, 8 Viehoever, A. B. Botanische Untersuchung harnstoffspaltender Bakterien mit besonderer Berucksichtigung der Spezies diagnostisch verwertbaren Merk- male und des Vermogens der Harnstoffspaltung. Centrbl. Bakt. 39: 209-359. 1913. 9 Rochaix and Dufourt. Contribution a l'etude des urobacteries. Compt. Rend. Soc. Biol. 69: 312-314. 1910. BACTERIA DECOMPOSING UREA 209 belong here, as well as other spore-bearing bacteria (so-called putrefactive forms), such as the anaerobic Bac. putrificus, and Bac. perfringeus. IV. Non-spore bearing bacteria. Bact. ureae Leube, Bad. ureae I Burri, Urobact. schutzenbergii Miquel, Urobad. miquelii Beij., Urobad. jackshii Sohn- gen, Urobact. beijerinckii Christ., as well as a number of common bacteria like Bad. coli, Bad. prodigiosum, Ps. jiuorescens, Bad. vulgare, etc. A large number of spore-forming and non-spore-forming bacteria were isolated from manure.10 According to Lohnis, the urea-decompos- ing power of the various urea bacteria quickly ceases when these organ- isms are kept in culture. They cannot, therefore, be considered as representatives of separate genera, but merely as varieties of common species, like Bact. vulgare, Bact. coli, Bact. ■prodigiosum, Bad. fluores- cens, Bad. erythrogenes. Most of the Urococcus species, which are dis- tinguished only by degree of pigment formation and gelatin liquefaction, belong chiefly to the Microc. pyogenes Rosenb. The same may be true of Microc. ureae Cohn. Bad. ureae Leube, named by Miquel Urobacil- lus leubii, belongs to the Bact. vulgare group; the same is true of Urob. miquelii Beij. and other Urobacilli (like Urob. jakschii Sohngen). This assumption is justified in view of the fact that a number of common soil bacteria, including various cocci, non-spore-forming and spore-forming bacteria, are capable of decomposing urea. Sohngen11 described two new non-spore-forming bacteria decompos- ing urea in the absence of proteins. An aerobic non-spore-forming, rod-shaped organism (Urob. beijerinckii), 1| by f to lp in size, was found to utilize urea both as a source of carbon and nitrogen, in the complete absence of other organic substances.12 Glucose cannot be utilized and may even injure the urea-splitting power of the organism. Humic acid was found to have a favorable influence upon the decom- position of urea. Viehoever13 found that most of the common urea bacteria, such as Urobacillus pasteurii, Urob. leubii and probably also Urob. maddoxii, Bac. ureae II and III, Bact. ureae could be combined into one species, Bac. probatus (No. GO, PI. X). This organism was obtained by heating some soil at 100°C, then inoculating into a medium containing 1 gram K2HPO.i, 0.1 gram CaCl2, 0.3 gram MgS04, 10 Lohnis and Kuntze, 1908 (p. 32). "Sohngen, 1909 (p. 207). 12 Christensen, H. R. tjber den Einflusz der Humustoffe auf die Ureumspal- tung. Centrbl. Bakt. II, 27: 337-362. 1910. 13 Viehoever, 1913 (p. 208). 210 PRINCIPLES OF SOIL MICROBIOLOGY 0.1 gram NaCl, 0.01 gram FeCl3, 20 grams urea and 5 grams Liebig's beef extract per liter. On incubating for three weeks at 28°, the culture was found to contain rod-shaped organisms 10/i long which formed spores l/i in size. A small amount of the culture was boiled for one minute at 100° and the organism was obtained in pure culture by the dilution method, using the above medium with the addition of agar. The maximum acid tolerance was found to be two drops N H2SOi per 5 cc. of agar medium (| concentration of nutrients). The maximum alkalinity was expressed by 2 per cent dehydrated Na2C03 (optimum 0.2 per cent) or 22 to 25 per cent of ammonium carbonate (optimum 0.3 per cent). The maximum concentration of urea tolerated by the organism was 30 to 40 per cent, the opti- mum was 3 per cent. Bacillus probatus A. M. et Viehoever is motile, with peritrichic flagella and rounded ends; forming on ammonium carbonate or urea agar chains of 2 to 3 cells. The cells vary greatly in size, usually 1 by 0.4 to 0.8/t, reaching on some media a size of 3 to 10 by 0.7/*. The organism is gram-positive, spore-forming, aerobic. The size of the spores on the ammonium carbonate agar ranges from 0.5 to 1 by 0.6 to 1.2/* to almost spherical, 0.8 to 0.9/x in diameter. The spores ap- pear in 3 to 4 days on the ammonium carbonate agar at 28°C., more or less at the end of the cell, as drum-shaped or spindle-shaped, swollen sporangia (PI. X). Development is weak on common nutrient and nutrient-glucose agar. White to grayish-opalescent. Colonies are formed in three days on the car- bonate or urea agar. In peptone broth containing 0.2 per cent ammonium carbonate, indol, H2S and trimethylamine are formed. No gas is formed from sugars, acid only from glucose. Ammonia is formed from nitrites. Crystals of calcium carbonate and phosphate are formed on urea agar. The enzymes cata- lase, urease, reductase are formed, but not oxidase or amylase. The minimum temperature for growth is 3° to 5°, optimum 28° to 35°, maximum for spore- formation 42° to 43°, for spore germination 45° to 47°, for growth 44° to 45°. Minimum oxygen tension per liter for spore germination and growth is 4 mgm., for spore formation 10 mgm. Maximum oxygen tension for spore germination is about 10 atmospheres, for growth 5 atmospheres, and for spore formation 1 to 5 atmospheres. The spores are killed in 11.5 to 12.5 minutes at 100°C, in 9 to 10 hours at 80°C. Geilinger14 made a detailed study of the biology of urea-decomposing bacteria, with a view of preventing the rapid loss of nitrogen due to the decomposition of urea in the manure pile. Only 5.6 per cent of the urea organisms isolated from soil and manure were able to live and de- compose urea in the absence of oxygen. Some organisms were found to be obligate anaerobes and were able to thrive in the presence of a mere trace of residual oxygen. A series of bacteria capable of decomposing 14 Geilinger, H. Beitrag zur Biologie der Harnstoff vergarenden Mikro- organismen, mit besonderer Berucksichtung der Anaerobiose. Centrbl. Bakt. II, 47: 245-301. 1917. BACTERIA DECOMPOSING UREA 211 urea at low temperatures, even below 0°C, were isolated from water and curative muds.15 Bacteria decomposing calcium cyanamide. Calcium cyanamide is decomposed with the formation of ammonia by Bad. erythrogenes, Bart, kirchneri as well as other non-spore-forming bacteria present in the soil.16 For the isolation of the organisms, the following media can be used: meat peptone gelatin, soil extract gelatin (10 per cent gelatin, reaction alkaline) or cyanamide solution with the addition of 10 per cent gelatin, then made alkaline after boiling. Certain bacteria can use dicyandiamide as a source of nitrogen, in the presence of glucose. The amide, however, is decomposed to a very inappreciable extent and no ammonia is formed.17 Uric and hippuric acid bacteria. Uric acid, which is an important constituent of the manure of birds and snakes and is also present to a slight extent in the urine of mammals, is also decomposed by bacteria, urea being the chief product.18 A solution of uric acid inoculated with putrid urine was found to be completely decomposed in a few days to ammonium carbonate with the formation of CC>2.19 When the process was interrupted before the uric acid was completely decomposed, urea could be demonstrated. Bart, ureae and Bart, fluorescens were found to be responsible for the process. Gerard20 found that the decomposition of uric acid goes on in two stages. The uric acid is first decomposed into urea and tartronic acid, the urea is then hydrolized to ammonium carbonate. Ulpiani and Cingolani21 isolated from chicken excreta an aerobic, motile, slime-forming streptococcus, not producing any spores 15 Rubentschik, L. Tiber die Lebenstiitigkeit der Urobakterien bei einer Temperatur unter 0°C. Centrbl. Bakt. II, 64: 116-174. 1925; 66: 161-180. 1926. 16 Lohnis, F., and Sabashnikoff, A. Ueber die Zersetzung von Kalkstickstoff und Stickstoffkalk. II. Centrbl. Bakt. II, 20: 322-332. 1908. 17Perotti, R. Uber die Dicyandiamidbakterien. Centrbl. Bakt. II, 21: 200-231. 1908. 18 Lex, R. Uber Fermentwirkungen der Bakterien. Centrbl. Med. Wiss. 10: 292, 513. 1872. 19 Sestini, F. and L. Tiber die ammoniakalische Garung der Harnsiiure. Landw. Vers. Sta. 38: 157-164. 1890. 20 Gerard, E. Fermentation de l'acide urique par les microorganismes. Compt. Rend. Acad. Sci. 122: 1019-1022. 1896; 123: 185-187. 21 Ulpiani, C. Uber das Bakterium der Harnsaure. Cingolani, M. Chem- ische Gleichung der Garung der Harnsiiure. Ref. Chem. Centrbl. 2: 1287. 1903; Gaz. Chim. Ital. II, 33: 93-98, 98-124. 1903; Atti Roy. Accad. Lincei, Roma, (5) II. 12: 236-240. 1903. 212 PRINCIPLES OF SOIL MICROBIOLOGY and hydrolizing uric acid into ammonium carbonate and carbon dioxide. The optimum temperature was 29° to 42° and at 50° the organism was killed. Bad. vulgare can also decompose uric acid.22 Liebert23 isolated from the soil Bad. acidi urici, an incompletely described species, 5 by 0.7/x in size, spore forming and anaerobic. It decomposed uric acid, under anaerobic conditions, with the formation of C02, ammonia and acetic acid. The aerobic decomposition of uric acid is also carried out by Bad. fluorescens liquefaciens and non-liquefaciens, by Bad. calco-aceticum and Bad. pyocyaneum in neutral and acid media, and by Bad. odoratum in alkaline media. These transform uric acid into C02 and NH3 with the intermediate production of allan- toin, urea, and oxalic acid. On a medium consisting of 2000 parts of water, 6 parts uric acid, 1 part K2HP04, various organisms (aerobic and anaerobic) capa- ble of utilizing uric acid both as a source of nitrogen and carbon can be isolated from the soil. Bad. stutzeri and Bad. pyocyaneum were found capable of utiliz- ing uric acid as a source of carbon for denitrification. Various Radiobacter strains are able to decompose uric acid with the formation of ammonia.21 By using a medium consisting of five grams NaCl, 0.2 gram MgSO,}, 0.1 gram CaCl2, 1.0 gram K2HP04, 30.0 grams of glycerol and 0.2 gram of uric acid in 1000 cc. of distilled water, an organism, Aerobader aerogenes, capable of decom- posing uric acid was isolated.25 In addition to bacteria, certain fungi and yeasts also decompose uric acid.26 Hippuric acid abundantly produced by herbivorous animals is trans- formed by bacteria into glycocoll and benzoic acid.27 It can be used by a large number of bacteria both as a source of nitrogen and carbon, with the formation of ammonia and carbon dioxide. Among the bacteria described by the earlier investigators, we may mention: Microc. ureae, Microc. pyogenes, Bad. prodigiosum,2* Bad. erythrogenes, Bad freu- denreichii, Bac. vulgatus, Bac. mesentericus, etc. A medium con- taining 1 per cent sodium hippurate, 0.2 per cent K2HP04 and 0.1 per 22 Nawiasky, P. Uber die Umsetzung von Aminosauren durch Bac. proteus vulgaris. Arch. Hyg. 66: 241. 1908. 23 Liebert, F. The decomposition of uric acid by bacteria. Akad. Weten- schap. Amsterdam. May 6, p. 61. 1909. 24 Bierema, 1909 (p. 187). 28 Morris, J. L., and Ecker, E. E. Destruction of uric acid by bacteria and molds. Jour. Inf. Dis. 34: 592-598. 1924. 26 Kossowicz, A. Die Zersetzung von Harnstoff, Harnsiiure, Hippursaure und Glykokoll durch Schimmelpilze. Ztschr. Garungsphysiol. 1: 60-62. 1912. 27 Van Tieghem. Recherches sur la fermentation de Puree et de l'acide hip- purique. Compt. Rend. Acad. Sci. 58: 210-264. 1S64. 28 Crisafulli, G. La reazione rossa del legno di pino per la ricerca dello indolo nelle culture in brodo dei microorganismi. Roma. 1905. BACTERIA DECOMPOSING UREA 213 cent MgS04 was used to demonstrate that microorganisms, capable of transforming hippuric acid, are present in the surface layer of the soil in larger numbers and are more active than in the subsoil.29 A medium containing 1500 cc. water, 5 grams hippuric acid, 2 grams K?HP04 and 1 gram MgS04, neutralized with sodium carbonate, was also used30 for the study of hippuric acid bacteria. Thirty-four species were iso- lated from soil, manure, urine, etc.; of these, twenty-eight decomposed hippuric acid and the others decomposed urea and uric acid. All those bacteria that decomposed urea also decomposed uric acid and vice versa; but those that decomposed hippuric acid did not necessarily decompose urea and uric acid, and vice versa. Stapp31 employed, for the isolation of uric and hippuric acid bacteria, the following two media: I. Uric acid 0.5 gram II. KH2P04 0.50 gram Na2HP04 3.0 grams MgS04 0.25 gram Mineral solution 50 cc. Sodium hippurate 1.25 grams Water 450 cc. Water 500 cc. The mineral solution used in Medium I was that of A. Meyer: KH2P04 l.Ogram NaCl O.lgram CaCl2 O.lgram Fe2Cl6 0.01 gram MgSO^H^O 0.3 gram H20 1000 cc. Portions of the solutions (50 cc.) were placed into 200 cc. Erlenmeyer flasks and inoculated with soil or feces of various animals. Six species of bacilli were carefully described. 1. Bac. cobayae A. M. & S. a non-motile, spore-forming organism. The cells attain a size up to 5.5/t long (usually 4/*) and 1 to 1.2/* in diameter. The ellip- soidal to cylindrical spores, usually with convex poles, are 1.4 by 0.8/*. The organ- ism forms diastase, protease, also H2S, tryptophane and skatol; nitrates are reduced. 2. Bac. capri A. M. & S. is without flagella, up to 6.2/* long and 1 to 1.1/* in diameter; ellipsoidal or egg shaped spores are 1.4 by 0.8/*; reduces nitrates and forms diastase. 3. Bac. guano A. M. & S. is a motile rod, with peritrichic flagellation, up to 5.4/* long and 0.7/t in diameter (2.8 to 3.4 by 0.6 to 0.7/*); the ellipsoidal spores are 1.4 by 0.8/*; weak reducing power, no diastase formation; gelatin is liquefied. 4. Bac. musculi A. M. & S., with peritrichic flagellation, is 4.5 to 5 by 1 to 1.2/iJ 29 Yoshimura, K. Note on the behavior of hippuric acid in soils. Bull. Coll- Agr. Tokyo. Imp. Univ. 2: 221-223. 1895. 30 Schnellmann, H. Uber die hippursaure-vergarenden Bakterien. Diss. Gottingen. 1912. 31 Stapp, C. Botanische Untersuchungen einiger neuer Bakterienspezies, welche mit reiner Harnsaure order Hippursaure als alleinigem organischen Nahrstoff auskommen. Centrb], Baktr II, 51: 1-71. 1920. 214 PRINCIPLES OF SOIL MICROBIOLOGY spores 1 to 2.2 by 0.6 to 1.2^ (1.8 by 0.8/x) ; weak reducing power; diastase is formed; gelatin is liquefied. 5. Bac. hollandicus A. M. & S., with peritrichic flagellation, is 6 by 0.7 to 0.8^; spores 1.6 X 0.8^; weak reducing power; diastase not formed, gelatin is liquefied. 6. Bac. carolarum Koch. Ulpiani and Cinglani32 isolated from pigeon manure a bacterium capable of decomposing guanin and guanidin, but not uric acid. The chemical processes involved in the transformation of urea, uric and hippuric acids are discussed elsewhere (p. 487). 32 Ulpiani, C, and Cingolani, M. Sulla fermentazione della guanina. Atti R. Accad. Line. Rend. CI. Sci. fis. Mat. et Nat. (5), 14, pt. 2: 596-600. 1905. CHAPTER X Soil Algae and Their Activities Introductory. The microscopic chlorophyll-containing forms of the great plant division Thallophyta, the Algae, are represented in the soil by three large groups: the Cyanophyceae, Chlorophyceae and Bacil- lariaceae. The first contain, in addition to chlorophyll, also the pig- ments phycocyanin and carotin and are, therefore, blue-green to violet or brown in color; the second usually contain only chlorophyll, but some- times also xanthophyll, and are, therefore, grass-green or yellow-green; the third contain, in addition to chlorophyll, also carotin and xantho- phyll, and are golden brown in color. The chlorophyll-bearing micro- scopic forms of life are also represented in the soil by the Eugleneaceae and the Cryptomonadaceae (or Flagellata and Dinoflagellata), com- monly classified with the Protozoa (Flagellata), and by the filamentous moss protonema, which belongs to the higher group of plants, the Eryophyta. Algae and the autotrophic groups of bacteria are the only micro- organisms in the soil that can synthesize organic matter from inorganic materials, the fungi and the heterotrophic bacteria depend for their energy upon organic matter synthesized by other forms of life. The autotrophic bacteria obtain their energy chemosynthetically, using in- organic substances as a source of energy; the algae obtain their energy photosynthetically, using the energy of the rays of the sun. Algae are universally distributed on the surface of the soil, wherever moisture and light are available. It is sufficient to moisten the soil with water and expose it to the light to obtain in a short time an abund- ant vegetation. However, algae may also be living below the surface of the soil, not exposed to the direct rays of the sun and under more uniform temperature and moisture conditions. The algae, as well as the other groups of soil microorganisms grow in the soil in mixture and, for purposes of identification and particularly for physiological investiga- tions, they have to be isolated and cultivated upon artificially prepared media. Some of the algae are isolated readily from the soil and others only with difficulty. For morphological studies and classification, it is suffi- 215 216 PRINCIPLES OF SOIL MICROBIOLOGY cient to separate the various forms and to cultivate them under artificial conditions, even if they are contaminated with fungi or bacteria; but for physiological studies, especially in the assimilation and transforma- tion of various elements, as in organic matter decomposition, nitrogen fixation and symbiotic action, it is important to obtain them free from any contaminating organisms. Methods of isolation of impure cultures of algae. The methods of isolation of algae from the soil fall into enrichment culture and pure culture methods. The enrichment culture methods consist in making conditions favorable for an abundant development of algae, for iden- tification purposes; the pure culture methods deal with the processes of obtaining the organisms free not only from fungi and bacteria, but also from other algae, for physiological studies as well as for a more care- ful study of their morphological characteristics.1 The enrichment cul- ture is also the preliminary step in the isolation of the pure culture. Since these organisms require light for the autotrophic assimilation of carbon dioxide (photosynthetically), this need of light is utilized for enrichment purposes. A small quantity of soil added to a large flask containing 0.02 per cent K2HP04 in tap water and exposed to light will soon allow 1 Wettstein, F. von. Zur Bedeutung und Technik der Reinkultur fur die Systematik und Floristik der Algen. Osterreich Bot. Ztg. 70: 23-28. 1921. PLATE XII Soil Algae 75. Pleurococcus (from Robbins). 76. Mostoc commune (from Robbins). 77. Microcoleus vaginatus (from Robbins). 78. Phormidium species (from Robbins). 79. Anabaena sp. (from Robbins). 80. Nodularia sp. (from Robbins). 81. Chlamydomonas communis: 1-3, motile vegetative cells; 4, resting cell in which division is about to take place; 5-6, longitudinal fission into four zoo- gonidia; 7, oblique fission, X 960 (from Bristol). 82. Ulothrix tenuissima X 550 (from Bristol). 83. Bumillaria exilis: a and b, vegetative filaments showing variable number of chloroplasts, X 550; c and d, filaments showing stages in formation of zoo- gonidia, X 960 (from Bristol). 84. Cylindrospermum muscicola: a, typical filament in different stages of spore-formation; b, spnre formed in an irregular position, X 550 (from Bristol). 85. Some typical soil diatoms: 1-2, Navicula borealis; 3-5, N. balfouriana; 6-9, N. intermedia; 10-12, N. brebissonii, var. diminuta; 13, N. elliptica, var. oblongella; 14, N. elliptica, var. minima; 15-17, N. terricola, X 960-1150 (from Bristol). PLATE XII SOIL ALGAE 217 the development of various blue-green algae; dry soil will give the spore-forming Nostococaceae, such as Anabaena and Cylindrospermum.2 When a large quantity of soil is placed in tap water, various small diatoms will always develop. When proteins are added to the soil and covered with water, various Volvocineae, such as Chlamydomonas, Karteria, Chlorogonium, Spondylomorum are obtained.3 For the pur- pose of isolating the algae, Robbins4 used 500 cc. Florence flasks filled, to their greatest diameter, with ground quartz, previously washed free from all suspended matter. The flasks are plugged with cotton and sterilized at 120°C. for 30 minutes. The soil is then shaken, for 5 minutes, with sterile water; an amount (25 cc.) equivalent to 10 grams of soil is evenly distributed, with a sterile pipette, over the ground quartz surface. The flasks are tipped to one side so as to offer both a moist sand and a free water surface for the algae to grow on. The flasks are kept in the greenhouse in a sunny place, then in a shady place. After growth of algae has taken place, they are transferred to 1 per cent agar medium with soil extract as a base. Some of the algal material is shaken up in a test tube with a few cubic centimeters of sterile water, then transfers are made, with a platinum loop, to tubes of liquefied agar cooled to 42°C. The tubes are shaken and the agar is poured into sterile Petri dishes. Growth of algae will appear in 2 or 3 weeks. The cultures may then be transferred to insure purity. Moore and Karrer5 placed about l\ inches of sand in pint milk bottles, to which 150 cc. of a culture solution had been added. The bottles were plugged with cotton and sterilized at 8 to 10 pounds pressure for one-half hour. Because of the soluble material present in the sand, the culture solution was one-half the strength of a modified Beijerinck solution used by Moore.6 2 Beijerinck, M. W. t)ber oligonitrophile Mikroben. Centrbl. Bakt. II, 7: 561-582. 1901. 3 Jacobsen, H. C. Kulturversuche mit einigen niederen Volvocaceen. Ztschr. Bot. 2: 145-188. 1910. 4 Robbins, W. W. Algae in some Colorado soils. Colorado Agr. Exp. Sta. Bui. 184. 1912. 5 Moore, G. T., and Karrer, J. L. A subterranean algal flora. Ann. Mo. Bot. Gard. 6: 281-307. 1919. 6 Moore, G. T. Methods for growing pure cultures of algae. Jour. Appl. Microsc. 6: 2309-2314. 1903. 218 PRINCIPLES OF SOrL MICROBIOLOGY The composition of the undiluted solution is as follows: NH4NO3 0.5gram CaCl2 0.1 gram KH2PO4 0.2gram FeS04 trace MgS04-7H20 0.2 gram Distilled water 1000 cc. The bottles are inoculated with about 10 grams of soil taken at the desired depth. To lessen the amount of evaporation, waxed paper covers are placed over the cotton plugs. The sand is slanted in the bottle so as not to be wholly submerged, giving various moisture conditions. The bottles are placed so as to get good light for at least part of the day. The water lost by evaporation is replaced from time to time with sterile water. The following two media are also recommended for the isolation of algae : Bristol's solution7 Detmer's solution NaN03 0.5 gram Ca(N03)2 1.0 gram KH2P04 0.5 gram KH2PO, 0.25 gram MgS04-7H20 0.15 gram KC1 0.25gram CaCl2 0.05 gram MgS04-7H20 0.25 gram NaCl 0.05 gram Tap water 1000 cc. FeCl3 0.005 gram Distilled water 1000 cc. Detmer's medium is diluted to one-third of its strength and 0.01 per cent FeCl3 is added. Distilled water is always prepared in a silver or glass still. Sand is placed into wide-mouth culture bottles to a depth of about 1.5 inches and moist- ened with one of the above media; the bottles are plugged and sterilized, then inoculated with a suspension of the soil in a sterile mineral salt solution. The soil may also be packed in a Petri dish to a depth of about 1 cm., well moistened with sterile distilled water, and the surface covered with a piece of pure filter paper. The cultures are kept in diffuse light, preferably at a tem- perature of 20° to 25°C The paper is moistened from time to time with sterile distilled water. After 2 to 60 days, various blue-green algae are found to grow through the pores of the paper to the light.8 The mixed cultures are transferred to sterile culture solutions or proper agar media for the isolation of the individual species. Isolation of pure cultures. The separation of various species of algae can be done either mechanically by the use of a loop and the micro- scope, or culturally by the use of solid media.9 The Barber pipette can also be employed as a mechanical means of separation. 7 Bristol, B. M. On the algal-flora of some desiccated English soils: an im- portant factor in soil biology. Ann. Bot 34: 35-79. 1920. 8 Esmarch, F. Untersuchungen liber die Verbreitung der Cyanophyceen auf und in verschiedenen Boden. Hedwigia, 55: 224-273. 1914; Diss. Kiel. 1914. 9 Pringsheim, E. G. Algenkultur. Abderhald. Handb. biol. Arbeitsmeth. Abt. XI, T. 2, 377-406. 1924. SOIL ALGAE 219 Beijerinck10 was the first to isolate algae in pure culture, using a medium consisting of ditch water, to which 10 per cent gelatin had been added. The liquid media given above are well suitable for the cultivation of algae. An alkaline reaction is most favorable, since algae are usually injured in acid media. The agar plate method for the isolation of pure cultures of algae has been successfully employed.11,12 The broken-up mass of algal material can be streaked out several times upon the surface of a solidified agar plate, so that each streak carries less of the inoculum than the preceding one. The inoculum may also be placed in a tube of melted and cooled agar; then a series of successive transfers are made into other tubes, so as to obtain a series of dilutions. These agar tubes inoculated with successively decreasing portions of material are poured into sterile Petri dishes. The streak method allows the development of surface colonies and the tube method of deep colonies. The plates are then exposed to light, so as to stimulate the development of the algae and to prevent the growth of other organisms. When the colonies have developed sufficiently they are transferred into liquid media. Algae are usually provided with a more or less highly dev- veloped exterior mucilaginous investment, either in the form of a sheath or of a mere gelatinization. They also develop much more slowly than fungi. Both of these factors contribute to the difficulties encountered in pure culture work with algae.13 When the life-history of the organism is known, the best period for obtaining it free from bacteria can be readily determined. Schramm13 used, for the isolation of algae, the medium recommended by Moore with the addition of 10 gm. of agar. The latter is carefully washed first in tap water, then in distilled water,14 so that the medium can be cooled down to about 10 Beijerinck, M. W. Kulturversuche mit Zoochlorellen, Lichengonidien, und anderen niederen Algen. Bot. Ztg. 48: 725-785. 1890; Also Centrbl. Bakt., 13: 368-373. 1893. 11 Tischutkin, N. Ueber Agar-Agarkulturen einiger Algen und Amoben. Centrbl. Bakt. II, 3: 183-188. 1897. 12 Ward, H. M. Some methods for use in the culture of algae. Ann. Bot. 13: 563-566. 1899. 13 Schramm, J. R. Some pure culture methods in the algae. Ann. Mo. Bot. Gard. 1: 23-45. 1914. 14 Richter, O. Reinkulturen von Diatomeen. Ber. deut. bot. Gesell. 21 : 493-506. 1903; Die Ernahrung der Algen. Monograph. Abhandl. Int. Rev. Hydrobiol. Hydrogr. 2: 31. 1911. 220 PRINCIPLES OF SOIL MICROBIOLOGY 34.5° to 35°C, without solidification. Six to eight cubic centimeters of agar are placed in a Petri dish 10 cc. in diameter. If the alga is filamentous, it is first washed in sterilized nutrient solution; if it is a unicellular form, it is diluted, the extent of dilution depending on the abundance of organisms. The material is added to the tube of liquid agar, which is then vigorously shaken so as to separate the adhering bacteria, and the contents are poured into a Petri dish. The plates are allowed to cool, then they are turned upside down, so as to prevent the moisture from spreading bacteria over the surface, and are placed in the light of a north window, preferably in a glass case. The plates are examined frequently and, if rapidly spreading bacteria and fungi are found, they are dissected out. The algal colonies usually appear in from three to four weeks. If the inverted plates are examined from time to time with the compound microscope (12 m. objective), the algal colonies may be located in the very early stages of develop- ment. The colonies are marked with a glass pencil and are transferred, by means of a platinum loop, to sterile agar slants. The purity of the culture ma}' be further tested by transferring it to media suitable for bacterial growth. This method may have to be modified for particular forms of algae : in some instances the method of Barber is used;15 in other cases the fact is utilized that certain species readily produce zoospores or other free endogenous spores; in some species the vegetative cells are either free from bacteria or can be rendered so by mechanical means. Pure cultures of various algae, particularly of Chlorophyceae, were thus iso- lated. The Cyanophyceae presented more difficult problems of isola- tion, since the gelatinous investments are all impregnated with bac- teria, which cannot be removed even by most vigorous washing. By the use of silicic acid gel, one species of Oscillatoria and one Microcoleus were isolated. However, as soon as these two organisms are completely separated from bacteria, the media, otherwise favorable, seem to become unfavorable and the organisms eventually die. By repeated transfer to sterile silicic acid gel plates, a species of Nostoc was isolated10 in pure culture. Another method17 consists in growing the organisms in a dilute mineral salt solution (Detmer's), either placed in flasks or impregnated in silica gel. Subcultures are made for enrichment purposes. Dilute suspensions of the algae, well shaken for the separation of the cells, are then inoculated into flasks 15 Barber, 1907 (p. 55). 16 Pringsheim, E. G. Kulturversuche mit Chlorophyllfi'ihrenden Mikroor- ganismen. I. Die Kultur von Algen auf Agar. III. Zur Physiologie der Schizo- phyceen. Reitr. Biol Pflanz. 11. 1912; 12: 49-108. 1913. 17 Chodat, R. fitude critique et experimentale zur le polymorphisme des algues. Geneve. 1909; Monographic d'algues en culture pure. Mat6riaux pour la flore cryptogamique Suisse. Vol. IV, Fasc. 2, 1913. Berne. SOIL ALGAE 221 containing melted and cooled (42°C.) agar that has been well shaken. The colonies are allowed to develop in the solid agar in bright sunlight, and are then cut out as^eptically and transferred to fresh media. The process may have to be repeated.18 Cultivation of soil algae. For the cultivation of soil algae, the above three solutions, either in liquid form or with 1.5 per cent agar, can be used. A medium containing 1.475 gram of Ca(N03)2-4H40, in place of 0.5 gram NH4N03, per liter has been used19 with good results. For the cultivation of diatoms, a modification of Miquel's20 medium, con- sisting of the following two solutions has been found21 to give satisfac- tory results. A. 20.2 grams KN03 in 100 cc. of distilled water. B. 4 grams Na2HP04 in 40 cc. water + 2 cc. concentrated HC1 + 2 cc. FeCl3 (melted at 45°C.) + 4 grams CaCl2 dissolved in 40 cc. water. Forty drops of A and 10 to 20 drops of B are added to 1 liter of distilled water. In addition to the above media, the following two solutions are very favorable for the growth of algae :22 Pringsheim solution Benecke23 solution NH.,MgP04 1.0 gram Ca(N03)2 0.5 gram K2S04 0.25 gram MgS04-7H20 0.1 gram Fe2(P04)2 trace K2HP04 0.2 gram Water 1000 cc. FeCl3 trace Water 1000 cc. The following solid medium can be used for the cultivation of algae : Chodat2i medium Ca(N03)2 1.0 gram FeS04 trace K2HP04 0.25 gram Distilled water 1000 cc. MgS04-7H20 0.25gram Washed agar 10 grams KC1 0.10 gram pH 5.3-5.5 18 See also Bristol Roach, 1926 (p. 225). 19 Wann, F. B. The fixation of nitrogen by green plants. Jour. Bot. 8: 1-29. 1921. 20 Miquel, P. De la culture artificielle de3 diatom6es. Ch. I, Le Diatomiste. 1: 93-99. 1890. 21 Ailen, E. J., and Nelson, E. W. On the artificial culture of marine plankton organisms. Jour. Marine Biol. Assoc. 8: No. 5. 1900. 22 Pringsheim, 1924 (p. 218). 23 Benecke, W. tJber Kulturbedingungen einiger Algen. Bot. Ztg. 56: 83- 97. 1898. 24 Chodat, R., and Grintzesco, J. Sur les m6thodes de culture pure des algues vertes. Actes Congr. Intern. Bot. Paris. 1910, 157. 222 PRINCIPLES OF SOIL MICROBIOLOGY The KC1 may be replaced by CaCl2 and the reaction adjusted to pH 7.3.25 Organic media may also be employed. Various proteins, including peptone, can be used as sources of nitrogen; sugars (glucose, fructose), higher alcohols (mannite, glycerol), and organic acids (in the form of neutral salts) can be used as sources of carbon. Various decoctions of hay, manure, peas and soil extracts can also be used, especially when the N and P content of the latter is increased by the addition of inorganic salts. Of the various organic media suggested, mention may be made here of two :26 (a) Cane sugar 10 grams (b) Malt extract 890 grams Asparagine 2 grams Glucose 29 grams Peptone 8 grams Peptone 0.5 gram Gelatin 80 grams Asparagine 0.5 gram Water 900 cc. Gelatin 80 grams To obtain inorganic solid media, add 15 to 20 grams of washed agar to one of the above solutions. In addition, soil, peat, sand and gypsum blocks can be used very readily, the last two moistened with a nutrient solution. To eliminate all traces of organic matter silica gel can be employed. Distribution of algae in the soil. The soil is a favorable medium for the growth of algae, which require only a relatively small amount of moisture to replace that lost by the protoplast in drying.27 In view of the fact that algae can develop on organic media also in the dark, their existence in the soil even below the sufacee is made possible. The occurrence of algae in the soil, particularly that of diatoms, has been referred to by a number of earlier writers.28,29 Esmarch30 attempted to determine the distribution of algae on the surface of the soil, their presence in the subsurface, and whether cultivation influenced their distribution. Four types of uncultivated soils were used : sandy 24 Fred and Peterson, 1925 (p. 378). 26 Beijerinck, M. W. Over gelatine culturen van eencellige groenwieren. Verh. Prov. Utrechtsch. Genootsch. Kunst en Wetensch. 1S89, 35-52. (Centrbl. Eakt. 8: 460-462. 1890.) 27 Fritsch, F. E., and Haines, F. M. The moisture relations of terrestrial algae. Ann. Bot. 148: 683-728. 1923. 28Ehrenberg, 1837 (p. 92). 29 Gregory, W. On the presence of Diatomaceae, Philolithoria, and sponge spicules in soils which support vegetation. Amer. Jour. Sci. Arts, II, 21: 434- 437. 1856. 80 Esmarch, 1914 fp. 218); see also Esmarch, F. Beitrag zur Cyanophyceen- flora unserer Kolonien. Hamburg, wiss. Anst. 28: 63-S2. 1910. SOIL ALGAE 223 heathland containing only traces of organic matter, marshy bog, forest humus, and moist sand. The various samples were often taken from quite different localities having the same type of soil. Only 3 out of 34 samples of the sandy heathland showed the presence of Cyanophyceae on the surface. Thirty-five samples of the marshy bog soil showed no blue green algae, but contained a few diatoms and grass-green algae. Only 5 samples out of 40 of the forest humus soil contained blue green algae and only 5 species were obtained altogether from soils of this type. The moist sandy soils indicated numerous blue-green algae on the surface. Subsurface samples from below the uncultivated soils were destitute of algae except in the moist sandy soils where they were fairly extensive in distribution. A larger number of blue-green algae was found in cultivated soils. A clay soil, for example, contained 23 species of blue-green algae in 35 out of 37 samples and 29 out of 45 samples of sandy soil contained 12 different kinds of algae. In general, cultivated soils were found to contain a greater number of blue-green algae than uncultivated, possibly because of the difference in moisture and mineral content. Grass land was richer in species than arable land. Subsurface samples were obtained at a depth of 10 to 25 cm. and, in some cases, at 30 to 50 cm., in a manner to prevent surface con- tamination. Only a few of the samples, coming from soils where there were no surface forms, contained no blue-green algae. Eighteen sepa- rate species were isolated, the number of algae decreasing with depth. In all, 45 species were described, of which 34 belonged to the OsciUatori- aceae and Nostococaceae. Esmarch ascribed the occurrence of subsurface forms to their being carried down by soil cultivation and by seepage of surface waters, as well as by earthworms and other soil organisms. By growing blue- green algae in the dark, or burying algae in the soil, then examining microscopically at various intervals of time, the filaments were found to become discolored, finally changing to a yellow color; the filaments disintegrated leaving only spores and heterocysts behind. On moisten- ing and exposing these to light, blue-green growth again appeared. The conclusion was reached, therefore, that blue-green algae cannot persist beneath the surface for any length of time, because of the absence of light and the destructive influence of the soil itself. Acid soils were reported31 to contain a different algal flora from that commonly found in alkaline or neutral soils. Twenty-four species 31 Petersen, J. B. Danske aerofile alger. Danske Vidensk. Selsk. Skrifter. 7 Raekke, Naturv og Mathem. Afd. 12: 7. 1915. 224 PRINCIPLES OF SOIL MICROBIOLOGY and varieties of diatoms were found in field and garden soils, 5 in marshy soils, and comparatively few or none at all in forest and heath- land soils. Robbins32 sampled several Colorado soils which were very rich in nitrate, by removing first the loose debris on the surface, then taking samples from the upper 3 to 4 inches. Out of 21 different species recorded, there were 18 Cyanophyceae, 1 diatom, and only 1 unicellular organism belonging to the Chlorophyceae. The Nostococaceae were best represented. The most prevalent species were Phormidiwm tenue, Nostoc sp., Anabaena sp., Nodularia harveyana, and Stigonema sp. A distinct subterranean algal flora independent of the nature of the soil and the locality was found by Moore and Karrer.33 Some species multiplied even when buried at a depth of one meter. In view of the fact that these algae were found in compact undisturbed soil, the pos- sibility suggested itself that algae are present in the soil in a vegetative condition and actually grow there and play a definite function in soil transformations. The following list contains the algae found in the soil by Moore and Karrer and the greatest depth at which they occurred: Chlorococcum humicola (Nag.) Rab 100 cm. Hantzschia amphioxys (Ehr.) Grun 100 cm. Navicula atomoides Grun 100 cm. Trochiscia ? 80 cm. Stichococcus bacillaris Nag 70 cm. Oscillaloria amphibia Agardh 70 cm. Cladophora sp 60 cm. Anabaena sp 20 cm. Nitzschia kutzingiana Hilse 20 cm. Nostoc muscorum Ag 20 cm. Oscillaloria chlorina Kutz 20 cm. Oscillatoria sublilissima Kiitz 20 cm. Scytonema hofmanni Ag 20 cm. Oscillaloria anoema (Kutz) Gomont surface Oscillaloria formosa Bory surface Oscillatoria splendida Greville surface An extensive study of the algal flora of desiccated soils has been made by Bristol.34 Forty-four samples of soil desiccated from 4 to 26 weeks and from widely separated localities were examined; a widely distributed ecological plant formation consisting of moss protonema 32 Robbins, 1912 (p. 217). 13 Moore and Karrer, 1919 (p. 217). 34 Bristol, 1920 (p. 218). SOIL ALGAE 225 and algae was present in cultivated soils. In these soils, 64 species and varieties of algae consisting of 24 species of Cyanophyceae, 20 Chlorophyceae and 20 Bacillarieae (diatoms) were found. The most important species in the plant-formation are Hantzschia amphioxys, Trochiscia aspera, Chlorococcum humicola, Bumilleria exilis and, to a less degree, Ulothrix subtilis var. variabilis; moss protonema was universally present. There seemed to be an association between three blue-greens; namely, Phormidium tenue, Ph. autumn ale and Plectonema battersii, two of which were found together in 16 of the samples and all three in 7 samples. Soils rich in blue-greens contained only a few species of diatoms, and vice versa; the first occurred more frequently in arable soils and the second in old garden soils. The resting forms could survive desiccation for a long period of time;35 9 species of blue-green algae, 4 grass-greens and 1 diatom were isolated from soils stored for about 40 years; the Nostoc muscorum and Nodularia harveyana retained vitality for the longest period of time. Most of the algae, except a few diatoms, are severely affected by frost, so that their numbers and activities usually reach a minimum in Febru- ary. As soon as the snow melts a rapid development takes place, followed again by a minimum growth in the late summer.36 With an increase in moisture, there is an increase in the numbers and activities. However, this phenomenon is not absolute. Observations by West and others indicate that different species of fresh water algae attain their maximal growth at different periods in the year. Soil algae show a similar variability. The determination of numbers of algae in the soil is even less accurate than in the case of bacteria, fungi and protozoa, largely because of the layer of mucilage with which the cells of various species are surrounded. The dilution method may be used, in a manner similar to the determina- tion of numbers of protozoa;37 a nutrient solution (p. 221) is used for the preparation of the different dilutions, and 5-cc. portions of the final dilutions are added to test-tubes containing 15-gram portions of 35 Bristol, B. M. On the retention of vitality of algae from old stored soils. New Phyt. 18, 1919, Nos. 3 and 4; 1920 (p. 92-107). 36 France, 1921 (p. 642). Magedeburg, P. Vergleichende Untersuchung der Hochmoor-Algenflora zweier deutscher Mittelgebirge. Hedwigia. 66: 1-26. 1926. 37 Bristol Roach, B. M. Methods for studying soil algae. Abderhald. Handb. Biochem. Arb. Meth. Abt. XI, T. 3. 1926. 226 PRINCIPLES OF SOIL MICROBIOLOGY «« Sf'2 O -3 o *-• ° ta « o a 2<5 © C3 _. •S.2-C.2 3 I. 0) — so 3 a g « a ot3 oq tn Q°» d an « 8 i-l c CQ &> ri j a ot>£ 2 M2^ IS G- o C o •7 O ID 8 a S a. o ° O rfS n ss, h o a y 8 8 :*> *5 w O O - « o -C -sS O g a 2 < a a « O -c 93 -C < e 8 rO c JS j S O 8 u §■ g ^ O fl« "^ «* iH o »-»• < o £ a < w o u 811 SOIL ALGAE 227 * # * * * * * * # » 1 **# ******** * * * * * # * * * * * * * * * * # * # * * * - * * * * * * * * * * * * * a a E 0) 0 m > os S u c ~5 o O X DC • s u B i-Q S DC '5 03 P a 5 5 5 v. eg Q .e > , a h : < < 1 £ 2 z -2 o u a ^ CO e s e c CO H < . H • < C h g z S o .5 o 5 CO a. ^> -2 a I w • •3 i : co : w •J : i— i h a Ph < s tj as *S a | s W a g 'ca U CO 0 a •~. •ea CO O CO _a '3 03 P Cfi > P « a ■<, -» H .« a' ■oa BS < a ■*«a =J £ 13 »b'2? Si o Si u u i» © © "S - * ,2 5 o 0^^000,OQO^^^Ctili)^^(^^^OClH SOIL ALGAE 229 ******* * * * * < H o e .2 230 PRINCIPLES OF SOIL MICROBIOLOGY cleaned, sterilized sand. The inoculated tubes are placed in closed glass vessels and exposed to sunlight. The presence and type of growth are determined at the end of a month by microscopic examination. Occurrence of algae in the soil. Several heterogeneous groups of Thallophytes are commonly included among the algae; they all contain chlorophyll, which is often accompanied by other pigments. Phylo- genetically, the algae belong to different groups of organisms: the Cyanophyceae (Myxophyceae) or blue-green algae are related to the bacteria or Schizomycetes. F. Cohn united both of these groups into the Schizophyta, characterized by lack of a nucleus. The Flagellata and Dinoflagellata (forming thick-walled resting cysts) are related to the protozoa and are often included with these organisms. The Bacil- lariales, characterized by their shells and auxospore formation, stand apart also. It is also difficult to establish the relationship of the Charo- phyta, Phaeophyceae, and Rhodophyceae. Without going into a detailed discussion of the classification of algae, it is sufficient to indicate that comparatively little work — a few isolated investigations — has been done on their occurrence in the soil. To indicate the very incomplete nature of the records, it is sufficient to call attention merely to the representatives of each group which have been demonstrated to be present in the soil (Table 15). Biochemical activities of algae.42 Algae are able, in the light, to 38 For a classification of the blue-green algae, see Tilden, J. E. Minnesota algae. I. Myxophyceae. Minn. Bot. Survey. 1910; Tilden, J. E. Synopsis of the blue-green algae -Myxophyceae. Trans. Amer. Microscop. Soc. 36, 1917, 179-266; Forti-Sylloge Myxophycearum. See also the general texts of de Toni, Engler and Prantl, and West; Collins, F. S. A working key to the genera of North American Algae. Tufts College Studies, 4: No. 8. 1918. 39 For a classification of diatoms see vol. x by Schonfeldt of Pascher's series, and van Heurck, Traite des Diatom<5es. 40 For a classification of the grass-green algae see West, Pascher, De Toni and Collins. 41 Moore, G. T., and Carter, N. Further studies on the subterranean algal flora of the Missouri Botanical Garden. Ann. Mo. Bot. Gard. 13: 101-140. 1926. 42 The following contributions should also be consulted on the carbon utiliza- tion of algae: Artari, A. Zur Ernahrungsphysiologie der griinen Algen. Ber. deut. bot. Gesell. 19: 7. 1901; Jahrb. wiss. Bot. 52: 410-466. 1913; 53: 527-535. 1914; Chodat, 1913 (p. 220) ; Bokorny, Th. Zur Kenntnis der physiologischen Fa- higkeiten der Algengattung Spriogyra und einiger anderen Algen. Vergleich mit Pilzen. Hedwigia, 59: 340-393. 1918; Dangeard, P. A. Observation sur une algue cultivee a l'obscurite depuis huit ans. Compt. Rend. Acad. Sci. 172: 254- 260. 1921; Grintzesco, J. Contribution a l'etude des Protococcoidees: Chlorella vulgaris Beijerinck, Rev. Gen. Bot. 15: 5-19, 67-82. 1903; Ternetz, C. Beitrage SOIL ALGAE 231 synthesize their protoplasm from C02 and from water containing inor- ganic nitrogenous and mineral compounds. Some algae, however, can also utilize organic materials, the extent depending on the species; some may thus develop in the complete absence of light, leading a hetero- trophic existence. Under those conditions, the chlorophyll may be either completely lost or retained. Some species can even utilize or- ganic nitrogenous compounds, and may bring about decomposition of proteins (Chodat). In general, however, algae prefer nitrates as a source of nitrogen; ammonium salts are less favorable. Of the nitrates, Ca(N03)2 is best, followed by KN03 and NaN03. The secondary am- monium phosphate is preferable to the other salts of ammonium, for, when the ammonium is used up, the secondary phosphate will be changed to the primary, which is only slightly acid, but the sulfate and chloride will leave the reaction of the medium acid. The preferential utilization of certain nitrogen sources may be due to a large extent to secondary reactions brought about by the residual ions. As different species behave differently toward the different sources of nitrogen, until further work has been done with a large number of species, no general conclusions can be drawn. Nitrites are not favorable, but can be utilized under proper conditions of reaction and concentration (Pringsheim) . In addition to water, carbon dioxide and nitrogen source, algae require for nutrition K, Fe, Mg, P, S, and in most cases also Ca. These are best added in the form of potassium phosphate and magnesium sulfate ; a trace of iron is added in the form of chloride; and calcium, as sulfate, if it is not used as nitrate. The salts are used only in very dilute solu- tions43: Ca(N03)2 as 0.1 per cent, MgS04 as 0.01 per cent and K2HP04 as 0.02 per cent. A faintly alkaline reaction, as given by secondary phosphate and alkali bicarbonate, is best. A biochemical process believed to be carried out by algae, namely, their ability to fix atmospheric nitrogen attracted considerable attention. As most of the earlier work in this connection has been carried out with impure cultures contaminated with various bacteria, results obtained under these conditions were not reliable. The negative results could be depended upon more than the positive results. Frank44 suggested in zur Morphologie und Physiologie der Euglena gracilis Klebs. Jahrb. wiss. Bot. 51: 435-514. 1912; Bristol Roach, B. M. On the relation of certain soil algae to some soluble carbon compounds. Ann. Bot. 40: 149-201. 1926. 43Richter, 1911 (p. 219). 44 Frank, B. Ueber den experimentallen Nachweis der Assimilation freien Stickstoffs durch erdbewohnende Algen. Ber. deut. Bot. Gesell. 7: 34-42. 1889. Untersuchungen iiber die Ernahrung der Pfianze mit Stickstoff und iiber den Kreislauf desselben in der Landwirtschaft. Landw. Jahrb. 17: 421-453. 1888. 232 PRINCIPLES OF SOIL MICROBIOLOGY 1889 that algae are able to fix atmospheric nitrogen; his results were substantiated by other workers, who used impure cultures of algae, Kossowitch,45 however, who was the first to use pure cultures of algae, namely a species of Cystococcus (Nageli) and Chlorella vulgaris Beij., demonstrated that algae do not fix any atmospheric nitrogen, and that some of the bacteria are the only organisms capable of doing that. In general, whenever pure cultures of algae were employed, nitrogen fixa- tion was found to be negative.43'46 But even if algae do not fix any nitrogen by themselves, they were found to exert a favorable effect on the process of nitrogen-fixation by non-symbiotic bacteria. Kossowitsch, therefore, suggested that the algae work in a manner symbiotically with nitrogen-fixing bacteria, furnishing them, through photosynthetic activity, with carbohydrates. These results were confirmed by other investigators,47 especially in the case of Azotobacter and algae.48-51 Beijerinck considered those organisms that can live on media to which no nitrogen has been added (without being, however, free from combined nitrogen), to be able to fix atmospheric nitrogen; he con- cluded, therefore, that various Cyanophyceae are able to fix atmospheric nitrogen, since they grew in media almost free from combined nitrogen. These media were not free from nitrogen-fixing bacteria, however, and no analytical data were presented. In a series of carefully controlled experiments with pure cultures of several grass-green algae, Schramm52 46 Kossowitsch, P. Untersuchungen iiber die Frage, ob die Algen freien Stick- stoff fixieren. Bot. Ztg. 52: 97-116. 1894. 46 Charpentier, P. G. Alimentation azot£e d'une algue, le Cystococcus humi- cola. Ann. Inst. Past. 17: 321-334, 369-420. 1903. 47 Kriiger, W., and Schneidewind, W. Sind niedere chlorophyllgrune Algen imstande den freien Stickstoff der Atmosphaere zu assimilieren und den Boden an Stickstoff zu bereichern? Landw. Jahrb. 29: 776-804. 1900. 48 Reinke, J. Symbiose von Volvox and Azotobacter. Ber. deut. bot. Gcsell. 21: 481. 1903. Fischer, H. Uber Symbiose von Azotobacter mit Oscillarien. Centrbl. Bakt. II, 12: 267-268. 1904. 49 Heinze, B. Einige Beitrage zur mikrobiologischen Bodenkunde. Centrbl. Bakt. II, 16: 640-663, 703-711. 1906; Uber die Stickstoffassimilation durch nie- dere Organismen. Landw. Jahrb. 35: 889-910. 1906. 60 Nakano, H. Untersuchungen fiber die Entwicklungs- und Erniihrungs- physiologie einiger Chlorophyceen. Jour. Coll. Sci., Tokyo Univ. 40: 66. 1917. " Pringsheim, 1913 (p. 220). 62 Schramm, J. R. A contribution to our knowledge of the relation of certain species of grass-green algae to elementary nitrogen. Ann. Mo. Bot. Gard. 1: 157-184. 1914. SOIL ALGAE 233 came to the conclusion that Chlamydomonas pisiformis Dill, forma minor Spargo, Protosiphon botryoides (Kiitz) Klebs, Chlorococcum humi- cola (Nageli) Rabenh., Chlorella vulgaris Beij., Stichococcus bacillaris Nag., Chlorella sp. and Kirchneriella sp. are unable to fix free atmos- pheric nitrogen in the complete absence of combined nitrogen. Wann,53 however, working in Schramm's laboratory claims to have found that seven species of algae exhibited the ability to fix atmospheric nitrogen, when grown in pure cultures on mineral nutrient agar media containing either ammonium nitrate or calcium nitrate, as a source of nitrogen, and a small amount of glucose. A gain of 1 to 12.5 mgm. of nitrogen per flask was obtained. In the absence of glucose, growth and nitrogen- fixation were only slight. When urea, glycocoll, asparagine or am- monium sulfate was supplied as a source of nitrogen, either with or with- out glucose or mannite, no fixation took place; one species caused a loss of nitrogen. Positive nitrogen-fixation by algae has also been claimed recently by Moore and Webster.54 The fact, however, that bacteria were present in the cultures and that these were exposed to the air, from which traces of ammonia could be absorbed, would tend to invalidate these results. Bristol and Page,55 in a series of carefully controlled experiments, repeated Wann's work, using four different species of algae, each growing on six different media selected from among those used by Wann. They found no evidence to indicate any fixation of atmospheric nitrogen. In the presence of combined nitrogen, good growth was obtained, but only the original nitrogen was recovered even where it had originally been present in the form of nitrate. Bris- tol and Page pointed out a serious source of error in the chemical method used by Wann for the determination of the initial nitrogen content of media containing nitrates and suggested that his apparent fixation of nitrogen was probably the outcome of a faulty chemical technic since they completely failed to corroborate his results. They also suggested that the results of Moore and co-workers was of doubtful validity, since their cultures of algae were probably not free from bacteria. Bacteria can develop in the gelatinous sheaths of algae and need, therefore, not cause any turbidity of the medium. "Wann, 1921 (p. 221). 64 Moore, B., and Webster, T. A. Studies of photosynthesis in fresh-water algae. I. The fixation of both carbon and nitrogen from the atmosphere to form organic tissue by the green plant cell. Proc. Roy. Soc. B. 91: 201-215. 1920; also 92: 51-60. 1921. 56 Bristol, B. M., and Page, H. J. A critical inquiry into the alleged fixation of nitrogen by green algae. Ann. Appl. Biol. 10: 378-408. 1923. 234 PRINCIPLES OF SOIL MICROBIOLOGY Rdle of algae in the soil. It is impossible to generalize concerning the role that algae may play in soil processes. Although it seems to be definitely established that algae are unable to fix atmospheric nitrogen, they may be able to do so by living symbiotically with nitrogen-fixing bacteria. They may also accumulate organic matter in the soil, but since they need available nitrogen, they may compete with higher plants for the soluble minerals and available nitrogen compounds in the soil. Gautier and Drouin56 exposed samples of artificial soil, free from organic material and containing only ammoniacal nitrogen, in a sheltered posi- tion for a considerable period of time. The soil became, in course of time, covered with algae (Pleurococcus vulgaris, Protococcus viridis, etc.). This resulted in a loss in total nitrogen, a still greater loss in am- monia nitrogen, and a gain in organic nitrogen. The ammonia nitrogen was converted into organic nitrogen by the algae; with an increase in growth, there was a decrease in the loss of the total nitrogen. The algae thus play also a part in preventing the loss of ammonia nitrogen, as well as the leaching out of nitrates from the soil. The probable role of algae may thus consist in accumulating organic matter in newly formed soils.57'58 It has been suggested59 that algae, by taking in C02 and giving off oxygen, make swamp soils suitable for the growth of the rice plant. The roots of rice plants are typical land roots and possess no special adaptations to growth under swamp conditions. The large supply of dissolved oxygen in the swamp water produced by the photo- synthetic activity of the algae enables the rice plants to grow under these artificial conditions.60 The fact that algae are present in the soil in considerable numbers, that they can grow even in the subsoil and in the dark, that they re- tain their vitality for very long periods, even after prolonged drought, that they can store large quantities of energy thus making them avail- able for other organisms, all point to their probable importance in 66 Gautier, A., and Drouin, R. Recherches sur la fixation de l'azote par Ie sol et les vegetaux. Compt. Rend. Acad. Sci. 106: 754-7, 863-6, 944-7, 1098-1101, 1174-6,1232-4. 1888;113:820-825. 1891. " Treub, M. Notice sur la nouvelle flore de Krakateu. Ann. Jard. Buiten- zorg. 7: 213-223. 1888. " Fritsch, F. E. The role of algal growth in the colonization of new ground and in the determination of scenery. Geogr. Jour. 30: 531-548. 1907. 69 Harrison, W. H., and Aiyer, S. Gases of swamp rice soils. Pusa Mem., Chem. Series, 3: 65-106. 1913; 4: 1-17. 1914. 60 Brizi, U. Richerche sulla malattia del Rizo detta "Bruzone." Ann. dell Instit. Agr. Dott, A. Buti. 5: 79-95. 1904;6:61-103. 1905;7:104-174. 1908. SOIL ALGAE 235 the soil. Algae also exert a solvent action upon insoluble calcareous materials (Chodat). In this respect, algae together with certain auto- trophic bacteria play an important role in the disintegration of rocks and in the formation of soils. The possible role of algae in the deposition of limonite has also been suggested.61 61 Steinecke, F. Limonitbildende Algen der Heide-Flachmoore. Bot. Archiv, 4: 403-405. 1923. CHAPTER XI Soil Fungi Occurrence of fungi in the soil. The chlorophyll-free microscopic plants are divided into slime-molds (Myxomycetes) , bacteria (Schizo- mycetes or fission fungi) and true fungi {Eumycetes) . The slime-molds are characterized by the formation of a Plasmodium, as a result of fusion of separate individuals. They have been studied only to a very limited extent from the point of view of their occurrence and activities in the soil. All the available information is either limited to the forms occurring on decomposing wood or to those causing plant diseases. Forms like potato wart (Synchitrium endobioticum) or the club- root of cabbage and other cruciferae (Plasmodiophora brassicae) once intro- duced into the soil persist there for a considerable period of time. How- ever, little is known concerning their role in soil processes. The Eumycetes or true fungi cannot derive their energy from the oxidation of inorganic substances; they are heterotrophic, depending for their energy supply on the decomposition of plant and animal sub- stances; their existence in the soil is thus closely connected with the decomposition of the organic matter added to the soil. Two distinct groups of fungi are found in the soil: (1) the ordinary filamentous fungi or molds, living freely in the soil, and (2) those capable of forming mycorrhiza with higher plants. The higher or mushroom fungi are found both among the free living forms and among those forming mycorrhiza. The soil also harbors various fungi capable of causing plant diseases, which are considered in detail elsewhere (p. 801). It has been known for some time that fungi occur abundantly in the soil, particularly in soils rich in organic matter and acid in reaction. But in view of the fact that fungi are present in the soil both in the form of vegetative mycelium and as reproductive spores, it is rather difficult to estimate their abundance; it is even less possible to find a basis for comparing the relative abundance of fungi and bacteria in the soil and their capacity for causing a certain amount of transfor- mation in the soil. It has been recognized1 that, under certain condi- 1 Moore, G. J. Microorganisms of the soil. Science, 36: 609-615. 1912. 236 SOIL FUNGI 237 tions, particularly in uncultivated soils and below the layer containing humus, fungi may be as abundant as, if not more so than, bacteria. The earlier workers2 emphasized the fact that fungi predominate in acid soils, and bacteria in neutral soils. This is true only to a certain extent. Fungi can, as a rule, withstand greater concentrations of acidity than bacteria and actinomyces, so that, at a pH of 4.0, the soil may contain only small numbers of the last two groups of organisms, while the fungi may still be present in abundance. Such a reaction is also inhibitive to the growth of most higher plants. At less acid reac- tions, when conditions are favorable for cultivated plants (pH 4.6 to 6.5), bacteria occur most abundantly, whereas the numbers of fungi will depend on the soil reaction, on the amount of organic matter, and on the abundance of water in the soil. As far back as 1886,3 attempts were made to isolate fungi from the soil and to give them names and descriptions; it is only since 1902, however, that the subject began to be treated in a systematic manner. Oudemans and Koning4 published a paper in 1902, which was the first real attempt made at a systematic study of the occurrence of fungi in the soil and their proper classification. In 1908 appeared the excellent contributions of Hagem5 and Lendner6, on the Mucorales of the soil. These contributions were soon followed by those of other investigators7-9 who made extensive studies of the occurrence of fungi in various types of soil, under different climatic and other environmental conditions. In addition to these, a number of other contributions have been made dealing in one way or another with one or more groups of soil fungi. 2 Ramann, E. Bodenkunde. Berlin, Springer. 1920. 3 Adametz, L. Untersuchungen iiber die niederen Pilze der Ackerkrume. Inaug. Diss. Leipzig, 1886, 78 p., 2 pi. 4 Oudemans, C. A. J. A., and Koning, C. J. Prodrome d'une flore mycolo- gique, obtenue par la culture sur gelatin preparee de la terre humeuse du Span- derswoud pres de Russum. Arch. Neerland. Sci. Exact, et Nat. (2), 7: 266-298. 1902. 5 Hagem, O. Untersuchungen iiber norwegische Mucorineen, Vidensk. Selsk., I Math. Naturw. Klasse, 7: 1-50. 1907; 10: 1-52. 1910; Ann. Mycol. 8: 265- 286. 1910. 6 Lendner, A. Les Mucorinees de la Suisse. Bern, 1908. 7 Jensen, C. N. Fungus flora of the soil. Cornell Univ. Agr. Exp. Sta. Bui. 315, 1912. 8 Dale, E. On the fungi of the soil. Ann. Mycol. 10: 452-477. 1912; 12: 33-62. 1914. 9 Waksman, S. A. Soil fungi and their activities. Soil Sci. 2: 103-155. 1916; 3: 565-589. 1917. 238 PRINCIPLES OF SOIL MICROBIOLOGY These were either limited to the isolation of a few forms for bio- chemical purposes, or they dealt with an important group of soil organ- isms like the Mucors,10 or with representatives of various groups in the study of one important soil process, like nitrogen fixation11 or cellulose decomposition.12'13 A number of papers and monographs are available which are devoted to certain groups of fungi, some of which were not isolated from the soil although they are of common occurrence in the soil. These contributions are of much assistance in the study and iden- tification of the soil forms. Reference must be made here to the work of Wehmer14 and Thorn15 on the genus Aspergillus; of Thorn,16 Westling,17 Sopp18 and Biourge19 on Penicillium; of Hanzawa20 on Rhizopus; of Butler21 on Pythium; of Chivers22 on Chaetomium; of Sherbakoff23 on Fusarium; and of Berkhout24 on Monilia and allied forms. The composition of the fungus flora of the soil changes with a change in the nature of the soil, both quantitatively and qualitatively. Hagem, 10 Povah, A. H. W. A critical study of certain species of Mucor. Bull. Tor- rey. Bot. Club, 44: 241-259, 287-313. 1917. "Goddard, 1913 (p. 259). 12 Daszewska, 1913 (p. 265). 13 Traaen, A. E. Untersuchungen iiber Bodenpilze aus Norwegen. Nyt. Magaz. Naturw. Christiania, 52: 21-121. 1914. 14 Wehmer, C. Die Pilzgattung Aspergillus. Geneve. 1901 ; Morphologie und Systematik der Familie der Aspergillaceen. Lafar's Handb. Techn. Mykol. 4: 192-238. 1905-7. 16 Thorn, C. and Church, M. B. The Aspergilli. The Williams & Wilkins Co., Baltimore, Md. 1926. 16 Thom, C. Cultural studies of species of Penicillium. U. S. Dept. Agr. Bur. Anim. Indus. Bui. 118, 1910; also Mycologia, 7: 134-142. 1915. 17 Westling, R. Uber die grunen Species der Gattung Penicillium. Inaug. Dissert. Upssala. Ark. Bot., 11: 1-156. 1911. 18 Sopp, J. O. Monographic der Pilzgruppe Penicillium. Vidensk. Selskr. I. Mat. Naturv. Kl., No. 11, Kristiania. 1912. 19 Biourge, Ph. Les moississures du groupe Penicillium Link. La cellule. 33: 1st. fasc. 1923. Louvain. 20 Hanzawa, J. Studien uber einige Rhizopus Arten. Mycol. Centrbl., 1: 406^09. 1912; 5: 230. 1915. 21 Butler, E. J. An account of the genus Pythium and some Chytridiaceae. Mem. Dept. Agr. India, Bot. Ser. 5, 1: 1-160. 1907. 22 Chivers, A. H. A monograph on the genera Chaetomium and Ascotricha. Mem. Torrey Bot. Club, 14: 155-240. 1915. 23 Sherbakoff, C. D. Fusaria of potatoes. Cornell Univ. Agr. Exp. Sta. Mem., 6: 85-270. 1915. 24 Berkhout, C. M. De Schimmelgeslachten Monilia, Oidium, Oospora en Torula. Diss. Univ. Utrecht. 1923. SOIL FUNGI 239 for example, has shown that cultivated soils have a distinctly different population of Mucorales than pine forest soils. The influence of reac- tion on the fungus population of the soil can be seen from the following example : A soil receiving manure year after year, in addition to minerals (pH 5.5), had 79,000 fungi; the same soil receiving lime in addition to manure (pH 6.7) had only 10,000 fungi per gram. The soil receiving no manure or fertilizer (pH 5.1), had 87,000 fungi; the same soil limed (pH 7.0) had only 16,000 fungi. The soil receiving ammonium sulfate and mineral (pH 4.2) had 129,000 fungi; the same soil limed (pH 5.2) had 32,000. Methods of demonstrating the occurrence and abundance of fungi in the soil. The methods of studying the occurrence of fungi in the soil can be divided into two groups; (1) Those methods which demonstrate the presence of particular fungi in the soil, without any reference to the question whether these occur there only in the form of spores or also in the form of vegetative mycelium. (2) Those methods which tend to demonstrate the occurrence of fungi in the soil in the form of vege- tative mycelium. The first method is usually carried out as follows: Soil samples are taken under aseptic conditions into sterile containers. There is greater danger of exposing the soil to air contamination, in the study of fungi than of bacteria. Various fungus spores are very abundant in the ordinary bac- teriological laboratory, and because of the smaller number of fungi than bacteria in the soil (or cells developing into colonies) this error introduced will be greater in the case of fungi. The presence of dust fungi will lead also to misstatements in reference to the types of fungi present in the soil. A definite amount of soil is diluted with a definite amount of sterile tap water, and sufficiently stirred to separate the spores and pieces of mycelium from the soil particles. One-cubic-centimeter portions of the desired dilutions are then plated out with agar of definite composition and the plates are allowed to incu- bate for 48 to 72 hours at 25° to 30°C. Ordinary bacteriological media can be used for this purpose but acid media, having a reaction of pH = 4.0, are preferable for this first step of the isolation of fungi. The acidity prevents the bacteria from developing and the fungi can be isolated free from bacterial contamina- tions. A lower dilution can be employed, than would be the case with media upon which bacteria are able to develop; this allows the development of greater numbers and, therefore, of a greater variety of fungi. The medium described above (p. 19) can be used for this purpose. Any other sugar medium well adapted for the growth of fungi, to which some citric acid is added (about 1 per cent) can be used.25 For the isolation of yeast, a medium containing saccharose and 1.2 to 1.5 per cent citric acid is often recommended. Lactic acid can also be 26 Piettre, M., and de Souza, G. Milieux acides pour l'isolement des champig- nons. Compt. Rend. Soc. Biol. 86: 336-337. 1922; Isolement des levures en milieux acides. Ibid., 338-340. 240 PRINCIPLES OP SOIL MICROBIOLOGY used. When fruit extracts, like raisin or plum extracts, are used as a base for the medium, no acid is required, since the natural acidity of the fruit is sufficient to prevent the development of bacteria. After the organism has been isolated, it is often necessary to obtain a single spore culture; especially when the organism is wanted for the study of hereditary characteristics or for physiological in- vestigations. For the demonstration of fungi present in the soil in the form of vegetative mycelium, the direct inoculation method26 and the direct microscopic method27 are available. According to the first method, lumps of soil, about 1 cc. in diameter, are placed, with a sterile forceps, into a sterile plate containing 10 cc. of sterile solidified agar medium. The plates are allowed to incubate for 24 hours at 22°C. This period of time is not sufficient for spores of the majority of soil fungi to germinate and form a mycelium visible to the naked eye, whereas the organisms actually living in the soil and forming a mycelium develop at once from the soil, so that the mycelium becomes visible earlier. After 24 hours' incubation the mycelium is transferred from the plates into sterile slants of fresh agar medium (Czapek's), care being taken to isolate the mycelium, which has grown away from the soil. The organisms thus isolated can now be cultivated, purified if necessary, and identified. According to the second method, a small crumb (10 mgm. or less) of soil is placed upon a microscopic slide and mixed with two or three drops of water. A drop of methylene blue solution (saturated aqueous or Loeffler solution) is then added by means of a glass rod, well mixed with the soil suspension and covered with a cover slip. The preparation appears blue to the naked eye. If too much stain has been added, it is diluted by a drop of water. Examination is made with a dry lens and a highpower eye piece. By this method, fungus filaments can be demon- strated in all the soils examined. Some soils contain only 4 to 5 fila- ments in a preparation (comprising 5 to 10 mgm. of soil), while others, especially soils rich in undecomposed organic matter, contain fungus mycelium in great abundance. The microscopic method, however, gives no means of identifying the particular species of fungi present in the soil as vegetative mycelium. This would be rather difficult, since the very nature of the growth of 26 Waksman, S. A. Do fungi actually live in the soil and produce mycelium? Science, N. S., 44: 320-322. 1916; The growth of fungi in the soil. Soil Sci., 14: 153-158. 1922. 27 Conn, H. J. A microscopic method for demonstrating fungi and actin- omycetes in soil. Soil Sci., 14: 149-152. 1922. SOIL FUNGI 241 fungi in the soil and on culture media is different.28 The first method is also not without fault, since some fungi, especially those forming a long mycelium, like the Mucorales, make a more extensive growth upon the plate, than other fungi. Not only are the morphological characters of the organisms different in the soil and on culture media, but they may vary with different media.29 The same is true of course of the physiological activities of the fungi; freshly isolated organisms behave differently from those kept in culture on artificial media; young cultures from spores act differently from fully developed mycelium. Methods of cultivation of soil fungi.30 Fungi are cultivated to facili- tate the study of their morphology, their reaction to environmental conditions, and their general physiology. The organisms, therefore, must be first isolated from the plate and grown in pure culture. This can be accomplished much more readily than in the case of algae or bacteria, since fungi grow rapidly, are aerobic, produce aerial spores abundantly, and can withstand comparatively large concentrations of acid. The media for the cultivation of fungi may be designated as natural and artificial. Among the natural media, solid substrates including soil, hay, manure, fruits, bread, and branches are largely used for the growth of fungi. These are either kept at an optimum moisture or are previously steri- lized, then inoculated with the organism. In the case of acid media, heating at 100° for 20 minutes may be sufficient for purposes of sterili- zation, but in the case of soil or hay, 1^ hours at 15 pounds pressure or 30 minutes at 100° on seven consecutive days is required. Fruit ex- tracts, as well as manure extracts, can also be used as nutrient solutions. In the preparation of artificial media a nitrogen source, a carbon source, and minerals must be provided. Ammonium salts, nitrates and organic nitrogen compounds can be used as sources of nitrogen.31 28 Church, M. B., and Thom, C. Mold hyphae in sugar and soil compared with root hairs. Science, N. S., 54: 470-471. 1921. 29 Brierly, W. B. Some concepts in mycology — an attempt at synthesis. Trans. Brit. Mycol. Soc, 6: pt. 2. 1919. 30 The cultivation of fungi is described in detail by O. Brefeld — Untersuch- ungen auf dem Gesamtgebiete der Mykologie, H. 14: 60. 1908; Kiister, E. Kultur der Microorganismen. 3d Ed., 1921; Lafar's Handb. d. tech. Mykol. 1, 1904- 1907; E. G. Pringsheim— Pilzkultur. Abder. Handb. Biochem. Arb. Meth. Abt. XI, T. 2: 407-444. 1921; E. Pribram. Die wichtigsten Methoden beim Arbeiten mit Pilzen. Ibid. XII, H. 3: 461-482. 1924. 31 Brenner, \V. Die Stickstoffnahrung der Schimmelpilze. Centrbl. Bakt., II, 40: 555-647. 1914. 242 PRINCIPLES OF SOIL MICROBIOLOGY The ammonium salts can be used in the form of phosphate, sulfate, chloride and salts of organic acids, like acetic, tartaric and citric, in concentrations of 0.1 to 0.5 per cent. ■ Ca, K, and Na nitrates can be used by almost all Aspergillaceae and various other fungi.32 Among the organic nitrogenous compounds, peptone and amino acids (as- paragine, leucine, etc.), followed by amides, amines, and alkaloids,33 are found to be favorable sources of nitrogen. Carbohydrates and higher alcohols are the best sources of carbon; of these, glucose comes first, followed by other hexoses and pentoses.34 Sucrose is utilized only by fungi which can produce invertase; when added to an acid medium, it is inverted in the process of heating. Starch is utilized only by fungi which can produce diastase. It is employed either in the form of a paste or as soluble starch. Pectins are also used as sources of energy by various fungi.35 Celluloses can be decomposed by certain fungi, hence mold activity is of great importance in the decomposition of organic matter in the soil. Of the alcohols, glycerol and mannite are used most readily; the lower alcohols only in dilute solutions. Of the organic acids, those having more carbon atoms, like tartaric, citric, and malic, are best. Some fungi can utilize fats as sources of energy.36 Of the mineral elements, K, Mg, S, and P are necessary and cannot be replaced by others. If required Ca, Na, CI need be present only in traces; Fe as well is sufficient in mere traces, when needed. Cu, Zn and Fe can act as stimulants. The following media can be used for the cultivation of the great majority of soil fungi: 32 Blochwitz, A. Vergleichende Physiologie der Gattung Aspergillus. Centrbl. Bakt. II, 39: 499-502. 1913; Kossowitz. Biochem. Ztschr., 67: 400. 1914. 33 Ehrlich, F. t)ber einige chemische Reaktionen der Mikroorganismen und ihre Bedeutung fur chemische und biologische Probleme. Mitt, landw. Inst. Breslau, 6: 705-713. 1912; (Centrbl. Bakt. II, 41: 245-246. 1914). 34 Peterson, W. H., Fred, E. B., and Schmidt, E. G. The fermentation of pentoses by molds. Jour. Biol. Chem., 55: 19-34. 1922. 36 Hauman, 1902 (p. 203); Behrens, 1903 (p. 203). 36 Spieckermann, A. Mykologie der Kraftfuttermittel. Lafar's Handb. techn. Mykol., 2: 361-388. 1907; Ztschr. Unters. Nahr. Genuszm., 27: 83. 1914; Rahn, O. Die Zersetzung der Fette. Centrbl. Bakt.. II, 15: 422-429. 1906. SOIL FUNGI 243 Czapek's solution consisting of: Distilled water 1000 cc. MgS04-7H20 0.5 gram Cane sugar 30.0 grams KC1 0.5 gram NaN03 2.0 grams FeS04 0.01 gram K2HP04 1.0 gram (Sterilized at 15 pounds for 15 minutes) Since this medium contains cane sugar as a source of energy, and this is un- favorable for the development of the majority of Mucorales, another medium containing glucose as a source of energy should be employed for the isolation and cultivation of these forms. This medium has the following composition: Cook's No. II medium:37 Distilled water 1000 cc. MgS04-7H20 0.25 gram Peptone 10.0 grams K2HP04 0.25 gram Glucose 20.0 grams Agar 15.0 grams Povah38 employed the following medium for the isolation of Mucors: NHUNO3 1.0 gram Cane sugar. . 5.0 grams K2HP04 0.5 gram Agar 13.0 grams per liter MgS04-7H20 0.25 gram To prepare solid media, 10 or more per cent of gelatin or 1.5 to 2.0 per cent of agar is added to the above solutions. In the case of acid media (at pH 4.0), 3 per cent agar is required. In addition to the media mentioned above, Povah used another medium for stock cultures of Mucors: Peptone 1.0 gram Dry malt ex- Glucose 20.0 grams tract 20.0 grams Agar 20.0 grams per liter For the cultivation of the wood-destroying Basidiomycete, Merulius lacrymans, the following medium may be used:39 NH 4N03 10 .0 grams .Lactic acid 2.0 grams K2HP04 5.0 grams Water 1000 grams MgSO-4-7H20 1.0 gram 50 cc. of this solution is added to 10 gm. of filter paper, the latter being used as a source of energy. Species of Coprinus can be cultivated upon sterile horse manure or manure decoction agar. Agaricus may be grown upon bread or bread mixed with sawdust. Fungi will tolerate rather high concentrations of nutrients. Asp. niger has its optimum at 20 to 30 per cent cane sugar, its maximum 37 Cook, M. T. The relation of parasitic fungi to the contents of the cells of the host plants. Del. Agr. Exp. Sta. Bui. 91, 1911. 38 Povah, 1917 (p. 238). 39 Tubeuf, C. V. Beitriige zur Kenntnis des Hausschwammes Merulius lacry- mans. Centrbl. Bakt. II, 9: 127-135. 1902; Wehmer, C. Hausschwammgu- tachten. Jahresb. Ver. angew. Bot., 8: 178-198. 1911. 244 PRINCIPLES OF SOIL MICROBIOLOGY in a solution containing 53 per cent glucose. The limiting osmotic pressure, when salts are used, is 17 to 21 atmospheres for Asp. niger and for certain green Penicillia.10 Fungi grow readily, in pure cul- ture, at a wide range of reaction (see p. 261) and are not injured by high acidity as readily as bacteria; acid reactions, including acid soils, will, therefore, favor the development of fungi, in crude culture. With carbohydrates as sources of energy, the reaction of the medium becomes acid as a result of the growth of many fungi. With proteins and nitrates, however, the reaction will tend to become alkaline.41 Fungi, with the exception of certain yeasts and certain Mucorales and Dema- tium, are strictly aerobic; certain Mucors are capable of developing anaerobically, especially in the presence of available carbohydrates. Aeration will greatly stimulate the activities of most fungi, because of their strict aerobiosis. The optimum temperature lies at 20 to 30° for the majority of species, in some cases {Asp. niger, Mucor. pusillus) going up to 37°. Some fungi (Penicillium expansion, Botrytis cinerea, Alternaria sp.) germinate slowly at 0°, others (Fusarium radicicola, Cephalothecium roseum) germinate slowly only at 5°, whereas Aspergillus niger will germinate only above 10°.42'43 In the case of certain 40 Pringsheim, E. G. tJber den Einflusz der Nahrstoffmenge auf die Entwick- lung der Pilze. Ztschr. Bot., 6: 577-624. 1914. 41 Bach, M. Variations de la concentration en ions hydrogenes sous 1'influ- ence de l'assimilation des nitrates par {'Aspergillus repens DeBary. Compt. Rend. Acad. Sci., 178: 520-522. 1924. 42 Brooks, S., and Cooley, J. S. Temperature relations of apple rot fungi. Jour. Agr. Res., 8: 139-164. 1917. 43 Brown, W. The germination and growth of fungi in various temperatures and in various concentrations of oxygen and CO2. Ann. Bot., 36: 257-283. 1922. PLATE XIII Soil Fungi— Mucorales 86. Absidia glauca, showing the runners with sporangiophores, X 3.5 (from Hagem). 87. Absidia glauca, columellae, X 200 (from Hagem). 88. Rhizopus nigricans, showing sporangiophores, rhizoids, and columellae, X 40; spores X 360 (from Jensen). 89. Rhizopui arrhizus: 1, runners with sporangiophores, X 35; 2, swollen sporangiophore, X 35; 3, abnormally divided sporangiophores, X 35; 4, collumel- lae, X 120; 5, spores, X 660 (from Hagem). 90. Zygorhynchus molleri, showing zygospore formation, X 200 (from Hagem). 91. Different forms of branching of Mucors (from Lendner). 92. Different forms of columellae of Mucors: a, spherical; e, spherical with persisting collarette; c, oval; d, oval depressed; e, pyriform; /, panduriform; g, conical; h, cylindro-conical; i, manniform; k and I, spinescent (from Lendner). 93. Cunninghamella echinulata (from Lendner) . PLATE XIII SOIL FUNGI 245 fungi, like Pen. expansum, once growth has started at ordinary tempera- tures, the mycelium will continue to develop at 0°. Oxygen pressure has little effect upon the germination and growth of various fungi. Increased carbon dioxide pressure has a retarding effect, espe- cially at low temperatures.44 Heat has a destructive effect upon fungi; the spores of Botnjtis cinerea are destroyed in ten minutes at 50.3° ;45 the spores are, however, rather resistant to the action of sun- light.46 Heating for thirty minutes at 62.8°C. is sufficient to destroy the conidia of most fungi, except certain species of Aspergillus.47 The morphology of the fungi is appreciably affected by the composition of the medium. The nature of the mycelium, the rapidity of spore formation and the color of the culture will often depend, to a greater or less extent, upon the different constituents of the medium, its concentration, and the environmental conditions, such as temperature or aeration. For this reason, synthetic media and standard conditions should be used in the study of the morphology and classification of such fungi as will thrive upon them. (The same is true, of course, of other microorganisms as well.) Isolation of single spore cultures. In the study of fungi, especially their physiology, pure cultures from a single spore are prerequisites for any investigation. This was pointed out by Hagem and others. Some spore material is transferred by means of a platinum needle, to a flask containing about 30 cc. of sterile water. After vigorous shaking to separate the spores, a few cubic centimeters of the suspension is poured into a second flask containing sterile water. This is repeated once more, and 2 cc. of the final dilution is poured into a Petri dish con- taining solid nutrient material, moistening the whole surface of the plate; the excess water is then poured off. The plates are allowed to incubate for 2 to 3 days and are examined under the microscope for isolated growth derived from a single spore. This examination can be carried out by removing the cover from the Petri dish; also by 44 Kostytschew, S., and Afanassiewa, M. Die Verarbeitung verschiedener organischer Verbindungen durch Schimmelpilze bei Sauerstoffmangel. Jahrb. Wiss. Bot., 60: 628-650. 1921. 45 Smith, J. H. The killing of Botrytis cinerea by heat, with a note on the determination of temperature coefficients. Ann. Appl. Biol., 10: 335-347. 1923. 46 Weinzirl, J. The resistance of mold spores to the action of sunlight. Univ. Wis. Studies in Science. 1921, No. 2, 55-59. 47 Thorn, C. T., and Ayers, S. H. Effect of pasteuriza.tiQU on mold spores. Jour. Agr. Res., 6: 153-166. 1916. 246 PRINCIPLES OF SOIL MICROBIOLOGY examining the inverted dish under the microscope, using the low power and marking with a colored pencil the spot where an individual spore has germinated. If such a growth is found, it is transferred with a small amount of substrate, by means of a sterile platinum loop, to a new dish or sterile agar slant. Povah sprayed a spore dilution upon a poured plate by means of capillary pipettes, then proceeded as before. Within twenty-four hours, after the spore was removed, transfers were made from the edge of the growth to a fresh tube; the possibility of contamination through a neighboring spore delayed in germination was thus avoided. The following procedure may also be followed : A small amount of the spore material is well shaken in 50 to 100 cc. of sterile water in an Erlenmeyer flask; a sterile platinum loop is then dipped into this suspension and carefully streaked out 3 or 4 times over the solidified agar in a Petri dish. The spores drop heavily at first, then singly, so that the third or fourth streak will have only single spores separated from one another. The plate is incubated for 36 to 48 hours, then examined in an inverted condition with the low power of the microscope. The streaks make easier the location of the spores. Where single spores have been dropped, they can be readily recognized, marked, and transferred with small blocks of agar into fresh dishes with agar or slants. By transferring again, within 24 hours, from the edge of the colony, and repeating the whole process when spore development takes place, single spore cultures can be assured. Of course, when the Barber43 pipette can be employed, there is greater certainty of obtaining single spore cultures. This is, however, not so necessary, in the case of fungi, as in the case of bacteria.49 Blakeslee50 devised a procedure for the isolation of two strains of Mucorineae, plus and minus strains. This consisted in teasing out an immature zygospore and placing it in a nutrient medium favorable for growth. In some cases, growth occurred from both suspensors in sufficient amount so that they could both be transferred to a fresh culture. CLASSIFICATION OF FUNGI, WITH SPECIAL REFERENCE TO THOSE OCCURRING IN THE SOIL EUMYCETES. Vegetative tissues deprived of chlorophyll, unicellular or multicellular, with a typical apical growth and formation of ramified my- celium. Reproduction sexual and asexual. No locomotion in developed cells. 48 Barber, 1907 (p. 55). 49 Roberts, J. W. A method of isolating single spores. Phytopathol., 13: 558-560. 1923. 60 Blakeslee, A. F. Sexual reproduction in the Mucorineae. Proc. Amer. Acad. Arts. Sci., 40: 205-315. 1904. SOIL FUNGI 247 A. PHYCOMYCETES61 (algal fungi). Mycelium unicellular, unseptated, branched profusely, sexual reproduction by zygospore or oospore. I. Oomycetes. Conjugating cells differing in appearance and function and consisting of a large oogonium and small antheridium. 1. Saprolegniales (water fungi), unicellular, abundantly branched vegetative mycelium, asexual reproduction by means of conidia or swarm spores, produced in separate spor- angia; sexual reproduction by means of an oogonium. Aphanomyces laevis has been found to live saprophytically in the soil;62 the same is true of Pythium de baryanum. 2. Peronosporales (downy mildews) : Species of Pythium are found abundantly on rotting manure at the first stages of decomposition of organic matter. Butler53 isolated 6 species of Pythium from the soil, including P. de baryanum and P. intermedium. Various species of Phytophthora can live in the soil saprophytically. II. Zygomycetes: Sexual reproduction by fusion of terminal cells of branches of mycelium similar in appearance but different in sex. The most important group is the order Mucorales. 1. Reproduction asexually by spores contained in sporangia, sub- order Sporangiophorae: 1'. Sporangia generally only of one kind, spherical or pyri- form with a membrane that dissolves or fractures easily. The septum separating the sporangiophore from the sporangium curves into the interior of the latter to form a columella. Sporangioles with persistent membranes occur very rarely and in such cases are disposed without order along the principal sporangiophore. Zygospores naked or surrounded by appendages, but never with a complete envelope. Mucoraceae. l". Sporangiophores arising from stolons (runners): (a) Sporangiophores produced from the nodes of the stolons; spores often striated longitu- dinally; sporangia globose. Rhizopus. This genus is represented in the soil by five or more species, only three of which (Rh. nigricans (88, PI. XIII), Rh. nodosus and Rh. arrhizus (89, PI. XIII)) have been iso- lated in different parts of the world by Hagem, Lendner, Dale and others. (b) Sporangiophores produced from the inter- node of the stolon, sporangia pyriform. 61 Fischer, A. Pilze. In Rabenhorst's Kryptogamen Flora. I, 4: 1, 5. 1892; Lendner, 1908 (p. 237); Hagem, 1908 (p. 237). 62 Busse, W., Peters, L., and Ulrich, P. Uber das Vorkommen von Wurzel- branderregernimBoden. Arb. K. Biol. Anst. Land- u. Forstw., 8: 260-302. 1911. " Butler, 1907 (p. 238). 248 PRINCIPLES OF SOIL MICROBIOLOGY Absidia (Nos. 86-87, PI. XIII). This genus is fairly well represented in the soil, although not very common; 12 species of Absidia have been described by Lendner. A number of species have been isolated by Hagem (A. orchidis, A. glauca, A. spinosa). Other species have been isolated by Oude- mans and Koning, Dale, Waksman and others. 2". No stolons are formed by the mycelium. (a) Heterothallic, occasionally homothallic, but, in the latter case, the zygophores generally arise from comparatively distant parts of the mycelium, never formed between branches of a single aerial hypha, and are usually equal. Mucor (Nos. 91-92, PI. XIII). This genus is one of the most common in the soil and is the largest in the number of species found in the soil. It has been studied in detail by Lendner, Hagem, Povah, Jensen and others, several of whom having isolated some 25-30 species from different soils, in different parts of the world. (b) Homothallic zygospores produced early and abundantly. Zygophores arise close to- gether, always originating from a single aerial hypha; they are usually unequal. Zygorhynchus (No. 90, PI. XIII). This genus is represented in the soil only by two species,64 but these are among the very few most common soil fungi. They have been isolated by Hagem, Namyslowski, Jensen, and have been found in every soil examined from all parts of the world. They are found especially in sandy sub- soils poor in organic matter. 2'. Sporangia similar to those of the Mucoraceae, but of two kinds: one kind is multispored, the membrane breaking up, leaving only a naked columella; the other kind of sporangia (sporangioles) contains few spores, which have persistent membranes and are often without colu- mellae. They are disposed at the extremity of branched sporangiophores, which are themselves arranged at regular intervals on the principal sporangiophore. 64 Namyslowski, B. Zygorrhynchus Vuilleminii, une nouvelle Mucorinee isol£e du sol et cultivee. Ann. Mycol., 8: 152-155. 1910. SOIL FUNGI 249 Thamnidiaceae. Species of Thamnidium (T. elegans) have been isolated from the soil by Jensen, Dale, Pratt and others. 3'. Sporangia of one kind only, multispored : the membrane is for the major part solid, persistent, of a very dark blackish color, or is swelling only toward the base. Sometimes the sporangium dissolves simply, leaving the columella, while at other times it is thrown off at the same time as the columella and opens only after swelling of the membrane. Pilobolaceae. Species of Pilobolus have been isolated by Oudemans and Koning from soil and Povah from horse manure. 4'. Sporangia without a columella, with a diffluent dis- appearing membrane, as in the case of the Mucoraceae. Zygospores enclosed singly in a carposporium. Mort- ierellaceae. Several species of Mortierella have been found in the soil by Oudemans and Koning. The last 3 genera are not of common occurrence in the soil; however, they are found abundantly on rotting manure and are thus readily introduced into the soil. 2. Reproduction asexually by conidia produced either solitary or in chains, sporangia not produced, suborder Conidio- phorae. 1'. Conidia solitary, spherical or oval, borne on conidio- phores swollen in the middle or at the extremity. Chaetocladiaceae. (a) Conidiophores branched dichotomously in bunches or arranged irregularly. Round or oval conidia are borne around a spherical head. Cunning- hamella (No. 93, PI. XIII). C. elegans has been isolated from the soil by Lendner, Povah, and others. (b) Conidiophores verticiliately branched, swollen into small heads furnished with sterile threads. Chaetocladium. 2'. Conidia in chains: (a) Conidiophores not swollen at tip. Piptocephalis. (b) Conidiophores swollen at apex: (a') Conidiophores not branched. Syncephalis. (b') Conidiophores branched. Syncephalaslrum. The last 4 genera are only rarely found in the soil. B. ASCOMYCETES. Mycelium multicellular. The group is characterized by the formation of an ascus or sac which usually contains eight spores; these asci are assumed to represent a "perfect" stage, in some cases certainly developed subsequent to fertilization; the fruiting masses con- taining the asci are very variable. 250 PRINCIPLES OF SOIL MICROBIOLOGY I. PROTOASCI, without ascogenic hyphae. This group includes the yeasts, which reproduce vegetatively by budding:65 65 A detailed study of the classification of yeasts and yeast-like fungi is given by Guilliermond — Tanner. The Yeasts; by Anderson, H. W. Yeast-like fungi of the human intestinal tract. Jour. Inf. Dis., 21: 341-381. 1917; Kohl, F. G. Die Hefepilze. 1908. PLATE XIV Soil Fungi — Ascomycetes and Hyphomycetes 94. Chaelomium olivaceum: A, perithecium, X 40; B, mature and immature asci, X 160; C, ascospores, X 360 (from Jensen). 95. Trichodermalignorum (from Jensen). 96. Trichoderma koningi: a, hyphae with conidiophores, X 56; b-c, conidio- phores with sterigmata; e, conidia, X 250 (from Goddard). 97. Sporotrichum poae (from Schwartze). 98. Acrostalagmus cinnabarinus: a-b, hyphae bearing conidiophores X 56; c, conidiophore with sterigmata; d, conidia, X 250 (from Goddard). 99. Monilia koningi: b, conidial fructification, X 56; d, same, X 100;/, sterig- mata, X 250 (from Goddard). 100. Cladosporium herbarum: a, portion of colony grown in a hanging drop, showing mode of branching; b, part of the same to show arrangement of spores, X270 (from Dale). 101. Fusarium oxysporum: showing conidia {A, B, C, D), chamydospores (E, F, I) and conidiophores (G, H, J) (from Sherbakoff). 102. Typical green Penicillium, Pen. chrysogenum: c, d, branching of conidial fructification, X 600; k, j, m, sketches of conidial fructifications, X 90 (from Thorn). 103. Penicillium purpurogenum: b, typically verticilliate branching at the apex of the conidiophore; e, conidia bearing cell or sterigmae; g, diagrammatic repre- sentation of the entire conidial apparatus (from Thom). 104. Penicillium, soil series: colonies pale green, velvety at border, but more or less floccose in center with under side of mycelium rose to dark-red, conidia becoming glubose, 2 to 3^ in diameter (from Thom). 105. Asp. terreus: a, semidiagrammatic sketch of vesicle and sterigmata: b, c, d, primary and secondary sterigmata, X 1000; e, conidia, X 1000;/, diagram of stalk and base of calyptrate conidial mass (from Thom and Church). 106. Asp. nidulans: a, diagrammatic section of vesicle with two sterigmata; b, c, primary and secondary sterigmata, X 1000; d, conidia X 1000; e, f, g, h, j, diagrams of stalks and heads; k, perithecium surrounded by sterile hyphae; m, group of ascospores (from Thom and Church). 107. Allernaria humicola, showing a portion of branching chains of spores, X 150 (from Dale). 108. Cephalothecium roseum, showing conidiophore and conidia (from Schwartze). 109. Botrytis vulgaris (from Schwartze). 110. Dematium: a, showing intercalary dark cells; b, Torula-like type showing terminaldark cells on lateral branches, X 270 (from Dale). PLATE XIV SOIL FUNGI 251 1. Cells not forming at once a surface membrane on sugar media: (a) Ascospores having a single membrane, cells not fusing in pairs before formation, spore germination by ordinary budding. Saccharomyces. (b) Ascospores having two membranes. Saccharomycopsis . 2. Cells forming a surface membrane at once on sugar media: Ascospores lemon shaped or with pointed ends. Willia. The presence of yeasts in the soils of orchards and vineyards has been established by Hansen56 and others. Wine yeasts have been found in such soils even at a depth of 20 to 30 cm. below the surface, but not at 40 cm. depth.67 Soils rich in humus, such as peat soils, offer a favorable habitat for these organisms.68 The greatest number of yeasts isolated from the soil59 are wild yeasts, including also white and red species of Torula. An examination of eighty-seven soils from dif- ferent parts of the United States revealed the presence of yeasts only in forty-five per cent of the soils; only two soil samples gave more than one species.60 II. EUASCI, with ascogenic hyphae. This group is divided into a number of subgroups, some of which contain some important soil forms. It is sufficient to mention: 1. DlSCOMYCETES, 2. Plectoascineae, containing the genera Aspergillus and Pcni- cillium. 3. Pyrenomycetineae. These contain the following 2 common soil genera: (a) Chaetomium*1 (No. 94, PI. XIV). Various species of this genus have been isolated from the soil by Jensen, Traaen, Waksman and others. (b) Sordaria and various other ascomycetes (Sporormia) have frequently been isolated from the soil. 66 Hansen, E. C. Experimental studies on the variation of yeast cells. Ann. Bot., 9: 549-560. 1895. Neue Untersuchungen fiber den Kreislauf der Hefen- aften in der Natur. Centrbl. Bakt. II, 10: 1-8. 1903; 14: 545-550. 1905. Compt. Rend. Trav. Lab. Carlsberg., 9: 61-69. 1911. 67 Mliller-Thurgau, H. Nachweis von Saccharomyces cllipsoides im Wein- bergsboden. Centrbl. Bakt. II, 14: 296-297. 1905. 68 Lemmermann, Fischer, et al., 1909 (p. 260). 69 Klocker, A. Deux nouveaux genres de la famille des saccharomycetes. Compt. Rend. Lab. Carlsberg., 7: 273-278. 1909. De Kruyff, E. Unter- suchungen uber auf Java einheimische Hefearten. Centrbl. Bakt. II, 21: 616- 619. 1908. 60 Starkey, R. L., and Henrici, A. T. The occurrence of yeast in soil. Soil Sci., 23: 33-46. 1927. 61 Chivers, 1915 (p. 251). 252 PRINCIPLES OF SOIL MICROBIOLOGY C. FUNGI IMPERFECTA no sexual spore formation known. I. HYPHOMYCETES.62 Hyphae septate, hyaline or dark colored, separated from one another or united into coremia. Conidia formed either as oidia, by the breaking up of hyphae, or are formed on little differentiated branches of the mycelium or on special conidiophores. The latter are simple or much branched. The order is divided into 4 families, according to shape of conidio- phore and structure of mycelium. 1. Conidia produced on single conidiophores, more seldom in the form of oidia. Vegetative hyphae as well as conidia and conidophores hyaline, pale or light colored, not dark, Mucedi- naceae Link: 1'. Spores one-celled: I". Conidiophores never sharply differentiated from mycelium, sometimes lacking; conidia may de- velop by the breaking up of hyphae into oidia: 1'". Conidia oval or spherical, never spindle- shaped : (a) Conidiophores very short, hardly distin- guished from the mycelium: (a') Conidia produced on short side branches singly or one after another, Myceliophthora. Isolated from the soil by Goddard. (b') Conidia large, with a thick membrane, Coccospora. Isolated from the soil by Goddard. (b) Conidia developing as oidia by the breaking up of hyphae or as chains on short, not sharply differentiated branches, Oidium (Oospora). Isolated frequently from the soil. (c) Conidia on definite branches; mycelium usually well developed and compact, Monilia (No. 99, PI. XIV). Various species of Monilia have been isolated from the soil. 2"'. Conidiophores well defined, erect, short; conidia in chains, short cylindrical, truncate at both ends, Geotrichum. Seldom found in the soil. 2". Conidiophores sharply differentiated from the my- celium: 62 In the study of this group of fungi the system used by Lindau (Fungi im- perfecti: Hyphomycetes. Rabenhorst's Krypt. Flora, Abt. 8 and 9. 1907- 1910) has been followed. This work as well as that of Engler, A., and Prantl, K. (Die naturlichen Pflanzenfamilien. Leipzig. 1897-1907) will be found to be of great assistance in the identification of most representatives of the Hyphomy- cetes, except in the case of the genera Aspergillus, Penicillium and Fusarium, where special monographs are available. SOIL FUNGI 253 I'". Conidiophores unbranched, or slightly branched, forming a head of branches and conidia: 1"". Conidia single, not in chains: (a) Conidiophores unbranched, with swollen tip: (a') Surface of terminal swelling definitely divided into hexagonal areas, Rhopalomyces. Found in the soil by Beckwith. (b') Surface of terminal swelling not so divided, Oedocephalum. Rarely found in the soil. (b) Conidiophores simple, but not with swollen tip, or branched: (a') Conidiophores unbranched, seldom divided, conidia adjoined at tip, one after another, but all remaining united into a head: (a") Conidia embedded in slime, Hyalopus. Rarely found in soil. (1)") Conidia not embedded in slime, Ceph- alosporium. Frequently found in the soil. (I)') Conidiophores branched: (a") Conidiophores tapering to a point bearing a head, Trichoderma (Nos. 95-96, PI. XIV). One of the most common groups of soil fungi. Very active in the decomposition of celluloses in the soil. Species of this organism have been isolated from the soil in different parts of the world, (b") Conidiophores with three or more fine spines, each of which bears a head, Botryospo- rium. Rarely found in the soil. 2"". Conidia born in chains: (a) Conidiophores swollen at apex, Aspergillus (Nos. 105-106, PI. XIV). Common in the soil, represented by several species. (b) Conidiophores not swollen at apex: (a') Conidiophores branched, branches more or less unequal and not radiating: (a") Conidia not embedded in slime, Penicillium (Nos. 102-104, PI. XIV). One of the few most common genera in the soil; represented by many species, some of which are specifi- cally soil forms. (b") Conidia embedded in slime, Gliocladium. Isolated from the soil by Dale. (b') Branches of conidiophore terminal, approxi- mately equal and radiating, A?nblyosporium. Rarely found in the soil. 254 PRINCIPLES OF SOIL MICROBIOLOGY 2'". Conidiophores unbranched or branched, but branches and conidia not forming a terminal head: V" . Conidia born on simple or branched, but not whorled hyphae: (a) Conidia produced irregularly on the myce- lium, or on short lateral branches, Sporo- trichum (No. 97, PI. XIV). Commonly found in the soil, but insufficiently studied. (b) Conidia produced on definitely differentiated erect conidiophores, which are usually much branched: (a') Conidia single, terminal, Monosporium. Isolated from the soil by Koning, Dale and others, (b') Conidia are usually loosely grouped at tip, Botrytis (No. 109, PI. XIV). Represented in the soil by a number of species, some of which (B. cinerea) are cosmopolitan. 2"" . Conidiophores branched in whorls: (a) Conidia-bearing branches thick and flask- shaped; conidiophores with long sterile tips, Pachybasium. Isolated from the soil by Goddard. (b) Conidiophores without sterile tip, conidia not produced on flask-shaped branches: (a') Conidia not forming chains: (a") Conidia not embedded in slime, Verticillium. Represented in the soil by various species, some of which possess a strong cellulose de- composing power. Species of Geomyces, related to Verticillium, have been isolated from the soil by Traaen. (b") Conidia embedded in slime, Acrostalagmvs (No. 98, PI. XIV). Frequently found in the soil. (1)') Conidia in terminal chains, Spicaria. Vari- ous species of this genus were isolated from the soil. 3". Conidia born on differentiated intercalary cells of the conidiophore: (a) Cells, bearing conidia, with raised points for attachment of conidia, Gonatobotrys. Rarely found in the soil. (b) Cells, bearing conidia, smooth, Nemato- gonium. Isolated from the soil by Koning and Dale. SOIL FUNGI 255 2'. Spores two-celled, conidia solitary: (a) Conidia with both cells smooth: (a') Conidia born on sides of conidiophores usually on inflated cells, not terminal, Arthobotrys. Rarely found in the soil. (b') Conidia produced at tips of conidiophores, not lateral, conidia solitary or in heads, pear shaped, Trichothecium. Isolated fre- quently from the soil. (b) Terminal cell of conidium enlarged and roughened, Mycogone. Isolated frequently from the soil. 2. Vegetative hyphae either short, almost unnoticeable, often breaking up into spores, or as abundant moldy growth, septated, usually dark, seldom light colored (with dark spores). Conidiophores short, upright, simple or branched, dark colored. Conidia unseptated or variously septated, always dark colored, light only in exceptional cases (the my- celium and conidiophores are then dark). Dematiaceae. 1'. Conidia unicellular: 1". Mycelium little developed and breaking up into oidia, or conidia formed on short lateral hyphae that are not well differentiated from the remainder of the mycelium; conidia in chains easily broken up, Torula. Various species of Torula were found in the soil by Koning, Dale, Pratt and others. 2". Mycelium definitely developed, with well differen- tiated conidiophores: (a) Conidia not in chains: (a') Conidia in terminal heads: (a") Conidia developing directly from conidiophore or with very short sterigmata, Syn- sporium. Isolated from the soil by Dale, (b") Conidia on thick, long sterig- mata, Stachybolrys. Two species isolated from the soil by Jensen, (b') Conidia not in terminal heads ; conidia prickly, Zygodesmus. Isolated from the soil only rarely, (c') Single conidia produced on branches, irregularly produced on the sides of the mycelium, Acremoniella. Rarely found in the soil. (b) Conidia in chains: (a') Conidiophores unbranched, lateral, with terminal chain of spores, Dema- 256 PRINCIPLES OF SOIL MICROBIOLOGY Hum (No. 110, PL XIV). Fre- quently found in the soil, (b') Conidiophores with branched chains of conidia, Cladosporium (Hormo- dcndrum). Common in the soil, various species having been isolated by a number of investigators (No. 100, PI XIV). 2'. Conidia two-celled: (a) Conidiophores short, not well differentiated from the mycelium: (a') Conidia solitary, Dicoccum. Rarely found in the soil, (b') Conidia in chains, Dispora. Rarely found in the soil. 3'. Conidia more than two-celled: (a) Septa of conidia perpendicular to long axis of spore, all parallel; conidia not in chains, Hel- minthosporium,. Frequently found in the soil. (b) Septa of conidia both longitudinal and crosswise; conidiophores well differentiated: (a') Conidia solitary and apical: (a") Conidiophores decumbent, formed in lateral branches of mycelium, Stemphylium. Various species have been isolated from the soil, (b") Conidiophores straight, more erect, conidium terminal, Macros porium. Found in the soil by Dale and Pratt, (b') Conidia in chains, Alternaria (No. 107, PI. XIV). Represented in the soil by sev- eral species. 3. Vegetative hyphae mostly invisible, septated, branched, hyaline or dark colored. Conidiophores uniting in parallel strands to form upright coremia. From the separation of hyphae, at the top of the coremium, the conidiophores either form a head or are slightly radiating, Stilbaceae: 1'. Hyphae, coremium, and conidia hyaline or light colored; conidia one-celled; coremium with a more or less defi- nite head, conidia not born along entire side: (a) Conidiophores scarcely diverging at top, Ciliciopo- dium. Rarely found in the soil. (b) Conidiophores divergent at top; each coremium with lateral heads as well as terminal, Tilachli- dium. Found in the soil by Koning. 2'. Hyphae, coremium and conidia usually all dark: (a) Conidia not in chains, ovoid to oblong, hyaline, Graphium. Rarely found in the soil. SOIL FUNGI 257 (b) Conidia in chains, Stysanus. Found in the soil by Koning, Goddard and others. 4. Mycelium consists of branched, septated hyphae, growing in or on the medium; characteristic fructification; growth mostly of a waxy or slimy constituency, often quite tough, Tuberculariaceae Ehrenberg. (a) Conidia and hyphae hyaline and light colored; sickle shaped conidia, both ends more or less pointed, Fu- sarium (No. 101, PI. XIV). One of the most common groups of soil fungi; very active in cellulose decomposition.63 Various species of Fusarium were isolated from soils of different parts of the world. (b) Conidia or hyphae dark or gray; conidiophorcs very short, conidia netted or prickly, Epicoccum. Rarely found in the soil. II. Melanconiales, Melanconiumu is rarely found in the soil. III. Sphaeropsidales: 1. Chaetomella. Found in the soil by Koning and Pratt. 2. Sphaeronema. Rarely found in the soil. IV. Sterile mycelium: 1. Sclerotia formed: (a) Sclerotia abundant, mycelium occupies secondary place, Sclerotium. Frequently found in the soil. (b) Sclerotia seldom formed, Rhizoclonia. Various Rhizoc- tonia, especially Rh. solani, are frequently found in the soil. 2. No sclerotia formed; hyphae united in strands, Ozonium. Often found in the soil (Dale). D. Basidiomycetes, characterized by the formation of a basidium, produc- ing four sterigmata, each bearing a single spore. Among the various subgroups it is sufficient to mention the I. USTILAGINALES II. Uredinales III. Hymenomycetes and other groups of mushroom fungi. These include various organisms (Mcrulius, Boletus, Russula, etc.) which form mycorrhiza with higher plants65 and other forms, like Psilocybe, which may become agents in the decomposition of organic matter in the soil.66 63 Sherbakoff, 1915 (p. 238); Pratt, O. A. Soil fungi in relation to diseases of the Irish potato in Southern Idaho. Jour. Agr. Res., 13: 73-99. 1918; Taylor, 1917 (p. 259). 64 Edgerton, C. W. The Melanconiales. Jour. Amer. Microscop. Soc, 31: No. 4. 1912. 65 Peyronel, B. Nuovi casi di rapporti micorizici tra Basidiomiceti e Fanero- game arboree. Bull. Soc. Bot. Ital., No. 1, p. 3-10. 1922. 66 Thorn, C, and Lathrop, E. C. Psilocybe as a fermenting agent in organic debris. Jour. Agr. Res., 30: 625-628. 1925. 258 PRINCIPLES OF SOIL MICROBIOLOGY AX Q K d 6D 3 ! * 1 * 1 1 1 * * * * * 1 1 * 1 * 1 a o o s * * # # * * « < S _o3 d a § a I a .3 3 03 3 ^ a a -s a -g Willis* w ^, -u -^ tn t-< V >> SOIL FUNGI 259 Occurrence of specific fungi in the soil. Of the various genera of fungi found in the soil, the most common, both in number of species and in frequency of occurrence, are Zygorhynchus, Penicillium, Tri- choderma, Fusarium, Mucor, Aspergillus and Rhizopus. This is clearly demonstrated in table 16, where an asterisk designates that a particular genus is represented by one or more species in one or more soils. Brierly67 tabulated systematically all the fungi which have been recorded and described in soil investigations and found 56 species of Phycomycetes belonging to 11 genera; 12 species of Ascomycetes belonging to 8 genera; 197 species of Fungi imperfecti including Actino- myces, but not sterile mycelia, belonging to 62 genera. Fungi are not limited in the soil to any particular depth, but occur at all depths, at least to a depth of four feet or more even in soils in humid regions. The numbers usually drop below the surface (upper six inches) , but in the subsoil there does not seem to be a rapid diminu- tion with greater depth. Some genera, like Zygorhynchus, occur abun- dantly in the subsurface soils. The distribution of fungi in the soil is be- lieved to depend upon the amount of moisture and the character of the soil.77 The distribution of certain organisms in the soil, like Fusarium, has been ascribed to earthworms.78 Various fungi are found in the soil at a depth of 1 to 44 inches (Trichoderma koningi most abundantly); practically the same species were isolated from the intestinal canals of grubs and worms picked out from the soil and properly washed; 67 Brierly, W. B. The occurrence of fungi in the soil. In The microorganisms of the soil, by Sir John Russell. 1923, 118-130. e8Oudemans and Koning, 1902 (p. 237). 69 Dale, 1912 (p. 237). 70 Jensen, 1912 (p. 237). 71 Goddard, H. M. Can fungi living in agricultural soil assimilate free nitro- gen? Bot. Gaz., 66: 249-305. 1913. 72 McLean, H. C., and Wilson, G. W. Ammonification studies with soil fungi. N. J. Agr. Exp. Sta. Bui., 270: 39. 1914. "Waksman, 1916 (p. 240). 74 Rathbun, A. E. The fungus flora of pine seed beds. Phytopathol., 8: 469-483. 1918. 76 Pratt, 1918 (p. 257). 76 Takahashi, R. On the fungus flora of the soil. Ann. Phytopathol. Soc. Japan, 12: 17-22. 1919 (Bot. Abstr. 6: 92. 1920). 77 Beckwith, T. D. Foot and culm infections of wheat by soil fungi in North Dakota. Phytopathol., 1: 169-176. 1911. 78 Taylor, M. W. Preliminary note on the vertical distribution of Fusarium. Phytopathol., 7: 374-378. 1917. 260 PRINCIPLES OF SOIL MICROBIOLOGY it was, therefore, concluded that the grubs and earthworms are the car- riers of spores of soil fungi.79 It is interesting to note that Takahashi isolated the species Zygorhynchus molleri and Trichoderma koningi in Japan at lower depths, while just below the surface he found Aspergilli, Penicillia, and other organisms, like Mucor racemosus, Stemphylium, and Chaetomium. In addition to the more than GO genera of fungi reported to be found in the soil, probably as many more could be demonstrated, but to a comparatively more limited extent. Our methods are not sufficiently developed as yet as to allow the direct isolation of certain organisms like the Basidiomycetes. The presence of a certain organism in large numbers need not indicate its great abundance in the soil, but may be due to abundant spore formation or to local development. Repeated isolation of an organism from different soils and from various parts of the same soil is essential, before any claim can be laid to its active part in soil transformations. Activities of fungi in the soil. Fungi require for their development the following elements: C, H, O, N, K, P, Mg, S, Fe. They obtain these either from organic or from inorganic (outside of carbon) sources. This points to their role in the soil, where they take part in at least two important processes: (1) rapid decomposition of complex organic substances; (2) assimilation of soluble inorganic nitrogen compounds and minerals, especially in the presence of available energy, thus removing them temporarily from the soil solution. The addition of fresh stable manure rich in straw, of green manures, and of other plant residues to the soil greatly stimulates the development of fungi ; the nature of the organ- isms developing most abundantly depends to a large extent upon the constituents of the organic matter added. The addition of stable manure was found to stimulate the development of Penicillia and especially of Mucorales and of actinomyces.80 The -addition of pure cellulose, especially in the presence of available nitrogen, brings about an extensive development of various fungi, such as Trichoderma, Fusaria, Verti- cillia, Monosporia, certain Penicillia, and other cellulose decomposing organisms. The reaction of the soil, the moisture content, and the nature and amount of the available nitrogen greatly modify the types of fungi developing in the soil as a result of the addition of celluloses or cellulose-rich materials. Plant substances are commonly added to the "Rathbun, 1918 (p. 259). 80 Lemmermann, O., and Fischer, H., Kappen, H., and Blanck, E. Bac- teriologisch chemische Untersuchungen. Landw. Jahrb., 38: 319-364. 1909. SOIL FUNGI 261 soil in the form of residues or green manures, which contain only a very small amount of nitrogen (0.3 to 2.0 per cent). Fungi rapidly decom- pose practically all the constituents of the organic matter added to the soil, with the possible exception of the lignins. The fungi are very economical in this process, assimilating as much as 30 to 50 per cent of the carbon for the synthesis of cell substance.81 The latter contains 3.5 to 8.0 per cent of nitrogen. In other words the minimum nitrogen content of the fungus mycelium is twice as much as the maximum nitro- gen content of green manure. If one part of fungus mycelium is synthe- sized for every three parts of green manure and plant stubble decomposed, and if the former contains three or more times as much nitrogen as the latter, this element will be completely reassimilated by the fungi; they may even assimilate, under certain conditions (when the Infl uence of TABLE 17 reaction upon the growth of fungi ORGANISM CRITIC AL pH VALUES Mucor giomerula Asp. oryzae 3.2-3.4 to 8.7- 9.2 1.6-1.8 to 9.0- 9.3 Asp. terricola 1.6-1.8 to 9.0- 9.3 Pen. italicum 1.9-2.2 to 9.1- 9.3 Pen. variabile 1.6-1.8 to 10.1-11.1 Fus. bullattim 2.0-2.2 to 9.2-11.2 Fas. oxusporum 1.8-2.0 to 9.2-11.1 plant material is low in nitrogen), the available nitrogen compounds in the soil (p. 515). The role of fungi in the growth of higher plants may thus be both beneficial and injurious, depending upon conditions. This can be illustrated well by the phenomenon of formation of "fairy rings." When the spores of Agaricus germinate in the soil, small circular areas are formed and the native grasses are stimulated. The mycelium begins to spread in all directions as fungi usually do in culture media. The outward growth is slow — about 12 cm. a year. The sod is at first stimulated by an increase in the available nitrogen resulting from the decomposition of the organic matter in the soil, then killed by insufficient soil moisture in the area of dense mycelium. When the mycelium in its 81 Waksman and Heukelekian, 1924 (p. 443) ; Waksman and Skinner, 1926 (p. 190). 262 PRINCIPLES OF SOIL MICROBIOLOGY turn begins to decompose, the native grasses again invade the soil and develop luxuriantly, because of the abundant supply of readily available nitrogenous materials.82 Influence of reaction upon the growth of fungi. A detailed discussion of the various phases of the physiology of the fungi is out of place here, since it is given in the standard texts on plant physiology. Attention may be called to only some physiological properties of fungi which are important from the point of view of the growth and activities of these organisms in the soil. It has been pointed out above that acid soil conditions favor the de- velopment of fungi. It need not, however, be construed that fungi grow only under acid conditions or even that they have their optimum growth at distinctly acid reactions; they have a rather wide range of reaction optimum, as shown in table 17.83 Fungi are thus shown to be much more resistant to acidity than the other groups of soil microorganisms. On the alkaline side, however, they are not more resistant than the bacteria. On the acid side there will, therefore, be no competition for the available plant food. The reaction of the medium has an important influence upon the germination of fungus spores84 and upon the respiration of the organ- isms.85 Increasing acidity favorably influences the germination of the spores; a maximum of germination is exhibited by the majority of the spores tested at a pH of 3.0 to 4.0. Inhibition of germination is evi- denced only at pH 1.5 to 2.5; the alkaline limits vary with the organism and with the medium. Fungi as a rule modify greatly the reaction of the medium, by the production of organic acids from available carbohydrates, by the consumption of organic acids (leaving the medium less acid), or by the formation of ammonia from proteins.86 82 Shanz and Piemeisel, 1917 (p. 282). 83 Johnson, H. W. Relationships between hydrogen ion, hydroxyl ion and salt concentrations and the growth of seven soil molds. Iowa Agr. Exp. Sta. Res. Bui. 76. 1923. 84 Webb, R. W. Germination of the spores of certain fungi in relation to hydro- gen-ion concentration. Ann. Mo. Bot. Gard., 8: 283-341. 1921. 84 Molliard, M. Influence de la r6action du milieu sur la respiration du Sterigmatocystis nigra. Compt. Rend. Soc. Biol., 83: 50-51. 1920. 86 Butkewitsch, Wl. Umwandlung der Eiweisstoffe durch die niedere Pilze. Jahrb. wiss. Bot., 38: 147-240. 1903. SOIL FUNGI 263 Cellulose decomposition by fungi. Koning87 was the first to point out the great abundance of fungi in forest soils, where colorless and brown, septated and non-septated mycelia are found to penetrate the whole mass of organic matter. Koning suggested that fungi play an important role in the soil in decomposing the organic matter and in transforming it into humus. That under favorable conditions and in the presence of avail- able energy, fungi grow very rapidly in the soil and produce a greater amount of C02 than do bacteria,88,89 indicates a greater energy utiliza- tion. This led Neller to assume that fungi exist in the soil not merely in the form of spores, but are active there, since the C02 production by pure cultures of fungi approached more that of a normal soil than the C02 produced under similar conditions by bacteria. The decomposition of celluloses and of allied compounds in the soil by fungi is of great importance in soil fertility. This accounts for the abundance of fungi in soils rich in organic matter and for the great increase in numbers when stable manure and green manure are added to the soil. It has been found,90 for example, that the addition of 1 per cent of cellulose to the soil in the form of pure filter paper resulted, in two weeks, in an increase in the number of fungi from 50,000 to 1,250,000 per gram. It has been further found that when a soil to which cellulose is added is treated with a volatile antiseptic, in sufficient amounts to kill the fungi, cellulose decomposition is greatly reduced. This process was found to take place under aerobic conditions, parallel with the development of fungi ; in other words, these organisms were found to be important agents in the breaking down of the most abundant constituent of natural organic matter. Students of plant diseases observed in the sixties of the nineteenth century that fungus hyphae grow in plant tissues, thereby penetrating cell walls. Hartig91 found that, in trees affected by fungi, all tissues 87 Koning, C. J. Contributions a la connaissance de la vie des champignons humicoles et des ph^nomenes chimiques qui constituent l'humification. Arch, neerland. Sci. Exact. Nat., Ser. II, 9: 34-107. 1904. 88 Neller, J. R. Studies on the correlation between the production of carbon dioxide and accumulation of ammonia by soil organisms. Soil Sci., 6: 225-241. 1918. 89 Potter, R. S., and Snyder, R. S. The production of carbon dioxide by molds inoculated into sterile soil. Soil Sci., 5: 359-377. 1918. 90 McBeth and Scales, 1912-1915 (p. 197) ; Waksman and Starkey, 1924 (p. 770). 91 Hartig. Untersuchungen fiber die Zersetzungserscheinungen des Holzes. Berlin, 1878. 264 PRINCIPLES OF SOIL MICROBIOLOGY disappear partly or entirely. De Bary92 was the first, however, to demonstrate that Botrytis vulgaris can decompose cellulose; actually he established that this organism can dissolve the middle lamella, which was not distinguished from cellulose by the older anatomists. Behrens93 first used filter paper as a nutrient for fungi and established the fact that Botrytis cinerea, Sclerotinia libertiana, Botrytis vulgaris, and a pseudo-Dematophora were able to derive their energy from pure cellulose. However, only about 10 per cent of the paper was decomposed, which led Schellenberg94 to suggest that only the impurities or hemicellulose-like compounds were decomposed. Van Iterson95 inoculated, with soil or humus, filter paper moistened with a solution of 0.05 per cent ammonium nitrate and 0.5 per cent of KH2P04 in tap water. He isolated 35 fungi including the genera Sporotrichum, Chaetomium, Botrytis, Stachobotrys, Cladosporium, Trichocladium, Mycogone, which are capable of decomposing cellulose. Not more than 4 to 14 per cent of the paper was decomposed. These results were subject to criticism, since tap water, which may contain various impurities, was used. The comparatively small loss in the weight of the cellulose was ascribed to the hemi celluloses present in the cellulose source used. Fusarium vasinfectum and other species of Fusarium were found96 to transform as much as 50 to 80 per cent of the cellulose, in the form of filter paper, into soluble forms; the medium used consisted of 10 gm. of paper and 50 cc. of a synthetic solution (KN03, KH0PO4, MgS04) placed in Erlenmeyer flasks. As late as 1908, however, both Schellenberg and Froehlich97 claimed that, with the possible exception of Botrytis, Fusarium and wood destroying fungi, it has not been demonstrated as yet that fungi are capable of decom- 92 de Bary, A. Uber einige Sclerotinien und Sclerotinienkrankheiten. Bot. Ztg., 44: 377, 420. 1886. 93 Behrens, J. Untersuchungen uber den Wurzelschimmel der Reben. Centrbl. Bakt. II, 3: 584, 639, 743. 1897; also Ibid., 4: 514, 547, 577, 635, 700, 739, 770. 1898. 94 Schellenberg, H. C. Untersuchungen liber das Verhalten einiger Pilze gegen Hemizellulosen. Flora, 98: 257-308. 1908. 95 van Iterson, C. Die Zersetzung von Cellulose durch aerobe Mikro- organismen. Verslagen d. k. Akad. Wetensch., 9: 807-820. 1903; Centrbl. Bakt. II, 11: 689-698. 1904; van Iterson, C. J. and Koning, C. J. La connais- sance de la vie des champignons humicoles. Arch, neerland. 2 ser. 1904, 34. 96 Appel, O. and Schikhorra. Beitrage zur Kenntnis der Fusarien und der von ihnen hervorgerufenen Pflanzenkrankheiten. Arb. K. biol. Anst. Land. u. Forstw., 5: 155-188. 1906. 97 Froehlich, H. Stickstoffbindung durch einige auf abgestorbenen Pflanzen haufige Hyphomyceten. Jahrb. wiss. Bot., 45: 256-302. 1908. SOIL FUNGI 265 posing cellulose. It remained for the more recent investigators98-101 to demonstrate definitely that not only do fungi decompose cellulose, but that pure cultures of fungi will decompose quantitatively within 3 to 4 weeks 50 per cent or more of the cellulose added in the form of filter paper. The addition of cellulose to the soil brings about an extensive development of fungi, most of which possess a very strong cellulose decomposing power. These include various species of Penicillium, Aspergillus, Trichoderma, Sporotrichum, Fusarium, and other forms which were found to be able to decompose cellulose. McBeth102 sug- gested that, in moist soils, particularly in humus soils, the fungi play a much more important part than in dry soils. Daszewska" found Verticillium celhclosae, V. glaucum, Sporotrichum olivaceum and various other Sporotricha, Fusaria, Monosporia, Alternaria and Monilia among the strongest cellulose decomposing fungi in the soil. She also con- cluded that the Hyphomycetes play a much more important part than the bacteria in the decomposition of cellulose in the soil, the color of the humus being due to the color of the mycelium and the spores of fungi. Sugars and alcohols were formed as intermediary products. Otto101 investigated a series of soil fungi and found the following to be able to decompose true celluloses actively: Stemphylium, Mycogone, Strachybotrys, Trichoderma, Cladosporium (Hormodendrum), and certain species of Penicillia. The cellulose was decomposed by the fungi by means of hydrolytic enzymes, which are produced only in the presence of cellulose in the medium. None of the Phycomycetes in- vestigated could decompose cellulose. Further information on cellulose decomposition by fungi is given elsewhere.103-105 98 Koning, C. J. Beijdrage tot de kennis van het leven der humicole fungi en van de scheidkundige Processen, welche bijd. humificatie hebben. Verle- sungen v. de gewone Vergad. d. Wis. e. nat. Afdelling. November, 1912. 99 Daszewska, W. Etude sur la dcsagregation de la cellulose dans la terre de bruyere et la tourbe. Bull. Soc. Bot. Geneve., ser. 8, fas. 8, 255-316. 1913. 100 McBeth, I. G., and Scales, F. M. The destruction of cellulose by bac- teria and filamentous fungi. U. S. Dept. Agr. Bur. Plant Indus. Bui. 266, 1913; Scales, F. M. Some filamentous fungi tested for cellulose destroying power. Bot. Gaz., 60: 149-153. 1915. 101 Otto, H. Untersuchungen fiber die Auflosung von Zellulosen und Zell- wanden durch Pilze. Inaug. Diss. Berlin. Borntraeger. 1916. 102 McBeth, 1916 (p. 197). 103 Heller, F. Die Zersetzung der Zellulosen durch Pilze. Inaug. Diss. Rostock. 1917. 104 Hopfe, A. Bacteriologische Untersuchungen tiber die Celluloseverdauung. Centrbl. Bakt. I, 83: 374-386, 531-537. 1919. 104Traaen, 1914 (p. 238). 266 PRINCIPLES OF SOIL MICROBIOLOGY The Mucorales are unable to decompose celluloses and most hemi- celluloses.106 The ability of fungi to decompose different celluloses does not depend on the solubility of the latter in acids, but on the chemical composition of the substances in question.107 Certain Mucorales, like Rhizopus and Mucor stolonifer, are able to decompose pectins but not celluloses, whereas Botrytis and other fungi decompose the fiber itself.108 According to the earlier investigators fungi are the proper humus builders in the soil. The fallen leaves, at the end of the vegetative period in the fall, are found to be penetrated with fungus mycelium, which decomposes the leaves readily, with the production of humic substances. These accumulate, because they cannot serve as a source both of carbon and of nitrogen109'110 but, in the presence of available sources of carbon, they can be used as sources of nitrogen by fungi. More recent infor- mation tends to show that, although fungi decompose most of plant residues (with the exception of lignins) completely, they synthesize extensive protoplasm, which is an important part of the soil organic matter. Decomposition of nitrogenous substances by fungi (ammonia formation) . Just as in the decomposition of celluloses and allied compounds, fungi play an important role in the decomposition of organic nitrogenous compounds. In the presence of available carbohydrates, the fungi utilize the nitrogen compounds only as sources of nitrogen; in the com- plete or relative absence of available carbohydrates, they utilize the nitrogenous substances as sources of carbon and of nitrogen. In view of the fact that the energy requirements of the fungi are greater than their nitrogen requirements, a great deal more of the protein molecule will be broken down to supply the necessary carbon. The excess of nitrogen present in the protein molecule over that required by the fungus for the building up of its own proteins will be left as a waste product, in the 106 van Iterson, 1904 (p. 264); Hagem, 1910 (p. 237) ; Waksman and Heukelekian, 1924 (p. 443); Waksman and Skinner, 1926 (p. 190). 107 Schellenberg, 1908 (p. 264). 108Behrens, J. Taurote von Flachs und Hanf. Centrbl. Bakt. II, 10: 524- 530. 1903. 109 Reinitzer, F. Ueber die Eignung der Huminsubstanzen zur Erniihrung von Pilzen. Bot. Ztg., 58: 59-73. 1900. 110 Nikitinsky, J. Uber die Zersetzung der Huminsiiure durch physikalisch- chemische Agentien und durch Mikroorganismen. Jahrb. Wiss. Bot., 37: 365- 420. 1902. SOIL FUNGI 267 form of ammonia.111 In general, fungi play an important part in the mineralization of the organic matter, whereby the nitrogen compounds and minerals are liberated in inorganic forms; a part of these is used by the fungi for the synthesis of fungus proteins. Miintz and Coudon112 and Marchal113 pointed out, in 1893, the abun- dant formation of ammonia by fungi, the latter even ascribed the ammonia production in soils (particularly acid soils) chiefly to the ac- tion of fungi. The decomposition of proteins with the formation of amino acids and ammonia has been pointed out by Kosyachenko and others.114 Cyanamide is decomposed, with the formation of ammonia,115 as are urea, uric acid, and glycocoll.116 According to McLean and Wilson,117 filamentous fungi are capable of producing a greater accumu- lation of ammonia from proteins than bacteria. All the organisms studied, including representatives of the families of Mucoraceae, Asper- gillaceae, Moniliaceae and Dematiaceae, were found to be capable of producing ammonia from dried blood and from cottonseed meal. The Moniliaceae were most active. In 8 to 10 days, Trichoderma koningi liberated as ammonia over half of the nitrogen in dried blood (1 per cent in sterile soil). The Aspergillaceae formed the least amounts of ammonia from proteins. The addition of soluble phosphate stimulated in most cases the amount of ammonia accumulated. Most fungi were capable of allowing greater accumulations of ammonia from dried blood than from cottonseed meal. This is probably due to the fact that the latter is richer in available carbon compounds, which will allow a greater synthesis of fungus proteins with the decomposition of propor- tionally less protein of the cottonseed meal. Utilization of nitrogen compounds by fungi. Soil fungi may assimilate 111 Waksman, S. A. The influence of available carbohydrates upon ammonia accumulation by microorganisms. Jour. Amer. Chem. Soc, 39: 1503-1512. 1917. 112 Miintz, A., and Coudon, H. La fermentation ammoniacale de la terre. Compt. Rend. Acad. Sci., 116: 395-398. 1893. 113 Marchal, E. Sur la production de l'ammoniaque dans le sol par les mi- crobes. Bui. Acad. Roy. Sci. Belg., 25: 727-771. 1893. 114 Kosyachenko, I. S. The influence of A. niger on the transformation of albu- minoids in peas. Zhur. Opitn. Agron., 4: 439-449. 1903. 115 Kappen, H. Uber die Zersetzung des Cyanamids durch Pilze. Centrbl. Bakt. II, 26: 633-643. 1910. 116 Kossovicz, A. Die Zersetzung von Harnstoff, Harnsiiure, Hippursaure und Glykokoll durch Schimmelpilze. Ztschr. Garungsphysiol., 1: 60. 1912. 117 McLean and Wilson, 1914 (p. 259). 268 PRINCIPLES OF SOIL MICROBIOLOGY the readily available nitrogen compounds of the soil, in the presence of favorable sources of energy, thus exerting a very unfavorable action upon the growth of higher plants. Rothe118 stated that the fungi exceed the bacteria and actinomyces, in acid as well as in neutral media, in the assimilation of available nitrogen and in storing it away as micro- bial organic matter; in the presence of CaC03, large quantities of nitro- gen added to the soil in the form of ammonium salts are transformed by these organisms into very insoluble nitrogen compounds. The com- petition between fungi and higher plants for the available nitrogen, under certain conditions, was also pointed out by Hall and associates.119 Hagem120 found that Mucorales will readily assimilate ammonium salts and transform them into microbial proteins. As pointed out above, with cellulose or other carbohydrates as sources of energy, the fungi may reassimilate 30 to 40 per cent of the carbon of the substrate decomposed. This necessitates a parallel assimilation of nitrogen; about one unit of available nitrogen is transformed into microbial protein for every 30 units of cellulose decomposed. This leads to a considerable reduction of the available nitrogen in the soil. Ehrenberg121 stated that fungus protein is much less available for further decomposition than bacterial protein, the fungus spores con- taining a large quantity of nitrogen stored away in an unavailable form, to some extent in the form of chitin, not readily subject to de- composition.122 Other investigations seem to point, however, that a large part at least of the cell substance syntherized by fungi is as rapidly decomposed as organic substances of animal origin.1223 The disappear- ance of the available nitrogen added to the soil in the form of ammon- ium salts and nitrates is to be looked for more in the development of fungi than of bacteria. Particularly is that true when these nitro- 118 Rothe. Untersuchungen liber das Verhalten einiger Mikroorganismen des Bodens zu Ammonium Salze und Natriumnitrat. Inaug. Diss. Konigsberg. 1904. 119 Hall, A. D., Miller, N. H., and Gimmingham, C. T. Nitrification in acid soil. Proc. Roy. Soc. (London), B, 80: 196-211. 1908. 120 Hagem, 1908 (p. 237). 121 Ehrenberg, P. Die Bewegung des Ammoniakstickstoffs in der Natur. Mitt. Landw. Inst. Breslau, 4: 47-300. 1907. 122 Wettstein, F. Das Vorkommen von Chitin und seine Verwertung als sys- tematisch-phylogenetisches Merkmal im Pflanzenreiche. Sitz. Ber. Akad. Wiss. Wien, Math. Nat. Kl. (I), 130: 3-20. 1921 (Centrbl. Bakt. II, 58: 329. 1923). 122B Starkey, 1924 (p. 684). SOIL FUNGI 269 genous fertilizers are added together with large quantities of manure or straw, since the available energy introduced into the soil will allow a rapid growth of the fungi, with the result that available nitrogen compounds are used up by them, to the detriment of the growth of higher plants. This action of the soil fungi has also a favorable side, namely the temporary storing of the available nitrogen salts in an in- soluble form, thus preventing their leaching by drainage and irrigation. The favorable and unfavorable actions depend upon the presence or absence of higher plants. The nutritive value of nitrogen compounds for fungi depends on the rapidity with which they can be transformed into amino acids, according to some investigators.123'124 Other investigators125-127 are, however, of the opinion that the amino acids and nitrates are reduced to ammonium salts before they are assimilated by fungi. The ability of an organism to assimilate ammonium salts is in direct relation to its ability to with- stand the mineral acid liberated.128 Assimilation of nitrates by fungi goes through the reduction of nitrates to nitrites and ammonia. Organ- isms, like certain Mucorales, that are incapable of reducing the nitrate molecule cannot assimilate this source of nitrogen.129 123 Czapek, 1901-1902 (p. 502). 124 Puriewitsch, K. Untersuchungen liber die Eiweisssynthese bei niederen Pflanzen. Biochem. Ztschr., 38: 1-13. 1912. 128 Raciborski, M. I. tJber die Assimilation der Stickstoffverbindungen durch Pilze. Anz. Akad. Wiss. Krakau, Math. Naturw. Kl., p. 733. 1906. 126 Hagem, 1910 (p. 237). 127 Abderhalden, E., and Rona, P. Die Zusammensetzung des "Eiweiss" von Aspergillus niger bei verschiedener Stickstoffquelle. Ztschr. physiol. Chem., 46: 179-186. 1910. 128 Ritter, G. Ammoniak und Nitrate als Stickstoffquelle fur Schimmel- pilze. Ber. deut. bot. Gesell., 25: 255; 27: 582-588; 29: 570-577. 1908-1911. 129 A detailed study of the nitrogen utilization by fungi is found in a paper by Brenner, while the influence of environmental conditions on the activities of soil fungi has been reviewed by Coleman. Further information on the physiol- ogy of fungi including curves of growth, influence of temperature, reaction and concentration is given by Mtiller. The antagonistic action of fungi to one another was studied by Nadson and Zolkiewicz and Porter. Brenner, 1914 (p. 241); Coleman, D. A. Environmental factors influencing the activity of soil fungi. Soil Sci., 2: 1-66. 1916; Mtiller, K. O. Untersuchungen zur Entwick- lungsphysiologie des Pilzmycels. Beitr. Allg. Bot. 2: 276-322. 1922; Nadson, G. A., and Zolkiewicz, A. I. Spicaria purporogenes n. sp. On the question of antagonism among microbes. Bull. Jard. Bot. Rep. Russe., 21: suppl. 1. 1921; Porter, C. L. Concerning the characters of certain fungi as exhibited by their growth in the presence of other fungi. Amer. Jour. Bot., 11: 168-188. 1924. 270 PRINCIPLES OF SOIL MICROBIOLOGY Nitrogen-fixation . Various claims have been put forth, at different times, that fungi are able to assimilate atmospheric nitrogen. In most cases the quantities fixed were very small, amounting to a few milli- grams, so that doubt might arise whether this was not due merely to ex- perimental errors. In some cases the mere fact that fungi grew on agar free from nitrogen compounds was taken as an index of positive nitrogen- fixation, the fact being overlooked thereby that some of these organisms can readily assimilate traces of ammonia present in the atmosphere and that various chemicals may contain, as impurities, small amounts of nitrogen. The more careful studies of recent investigators130-132 have definitely established the fact that common soil fungi are unable to fix atmospheric nitrogen. The only possible exceptions to this rule may be in the case of certain mycorrhiza fungi,133 especially organisms belong- 130 Goddard, 1913 (p. 259). 131 Chambers, C. O. The fixation of free nitrogen by certain fungi. Plant World, 19: 175-194. 1916. 132 Duggar, B. M., and Davis, A. R. Studies in the physiology of the fungi. I. Nitrogen fixation. Ann. Mo. Bot. Gard., 3: 413-437. 1916. 133 Peklo, J. Neue Beitrage zur Losung des Mykorrhizaproblems. Ztschr. Garungsphysiol., 2: 275-289. 1913. PLATE XV Mycorrhiza Fungi 111. Apparatus for rooting cuttings under controlled conditions: w, cotton,' c, cutting; s, sand; r, glass rod; h, rain water; p, potash tube (from Rayner). 112. Vessel for study of mycorrhiza formation in pure culture (from Melin). 113. Hyphae of Tricholoma flavobrunnea, grown in pure culture in symbiosis with birch tree, X 50 (from Melin). 114. Oblong section of hyphae radiating from mycorrhiza-root, X 500 (from Melin). 115. Beginning of infection of epidermis of young wheat root by phycomycoid endophyte; p, points of entrance of mycelium into the root; attention is called to the growth of the mycelium between the cells, X 130 (from Peyronel). 116. Stages of evolution, showing the process of tuber formation as a result of symbiosis: A, Solanum tuberosum; B, Orobus tuberosus; C, Ficaria ranunculoides ; D, plantlet of Bletitta hyacinthina inoculated with attenuated Rhizoctonia repens; E, plantlet of Bl. hyacinthina inoculated with an active Rh. repens; F, embryo tuber of Cattleya; t = tubers (after N. Bernard and Magrou). 117. Longitudinal section of a potato root, showing an early stage of fungus infection; to, coiled mycelium; n, cellular nuclei; n', fungus nuclei (from Magrou). 118. Two infected cells of a potato root, the lower cell showing large bodies resulting from disintegration by phagocytosis and the upper cell showing non- disintegrated mycelium which attacks the cell (from Magrou). 119. Mycorrhiza cells from young root of seedling of Calluna vulgaris showing, at right, "clumping" at early stage of digestion and, at left, digestion process (from Rayner). 112. Oidium — > Hyphomycetes , . Z1 Actinomyces bacillary forms — > Mycobacteria — > Corynebacteria — > Bacteria The fact seems to be definitely established that trre actinomyces are not bacteria. But they cannot be classified unreservedly with the fungi, "Bergey, 1923 (p. x). 16 Savageau and Radais. Sur les genres Cladothrix, Streptothrix, Actinomy- ces. Ann. Inst. Past., 6: 242-273. 1892. 16 Claypole, E. On the classification of the streptothrices, particularly in their relation to bacteria. Jour. Exp. Med., 17: 99-116. 1913. 17 Waksman, S. A., and Curtis, R. E. The actinomyces of the soil. Soil Sci., 1: 99-134. 1916; also Waksman, 1919 (p. 288). 290 PRINCIPLES OF SOIL MICROBIOLOGY particularly with the Hyphomycetes which is a rather loose conglomer- ate of various forms. They should be looked upon as a group of fungi, to be classified separately from the other groups, till their exact syste- matic position has been definitely established. However, the property of acid-fastness correlated with a certain type of pathogenicity (forma- tion of tubercles) and with cross immunity reactions with the acid fast bacteria, points to a certain relation, of at least some pathogenic forms, to the bacteria. Species differentiation. Large numbers of actinomyces have been de- scribed by various investigators.9,17 The larger the number of forms collected, the more difficult is the division into species. The variability of these organisms is such that out of a dozen isolations from a plate not more than two or three may be alike. Even forms recognized to be alike on one medium will be found to be different when grown on another medium. Sometimes two forms found to be alike on several media, will be found to show distinctive characters on further study.18 This is especially true of the cultural and biochemical characters and to some extent even of the morphology of the organisms. It is important first to obtain an absolutely pure culture of the organ- ism. Even then, an attempt to designate a species of actinomyces by the sum total of its morphological and physiological characters may not give very satisfactory results. It is best to classify the organ- isms into groups, defined by a sum total of certain definite morphological and physiological characters. The amount of variability within the group and the amount of overlapping between two groups is something that cannot be definitely established and must be left to the judgment of the investigator. The species differ primarily in the length of the mycelium, type of aerial mycelium, absence or presence of spores, method of spore forma- tion, shape and color of colony, formation of soluble pigment, oxygen requirement, production of diastatic and proteolytic enzymes and a number of other morphological and physiological characters. These vary in quantity as well as quality, not only under the influence of various environmental conditions but even on continued cultivation under the same conditions. Not only the soluble pigment may be lost or changed in color, but the color of the aerial mycelium may change and even the very property of forming aerial mycelium may be lost. An important advance in the study of this group has been the intro- duction of synthetic media. A great deal of the variability mentioned 18 Conn, H. J. The use of various culture media in characterizing acti- nomycetes. N. Y. Agr. Exp. Sta. Tech. Bui. 83, 1921. SOIL ACTINOMYCES 291 by Lieske was due to the use of non-synthetic nutrient agar, which is variable in composition. Two media made up exactly alike, but differing merely in the amount of boiling, period of sterilization, a slight change in ratio between the carbon and nitrogen sources, or in reaction and concentration of nutrients, will show distinctive differences for the various organisms. By growing these on synthetic media, with due allowance to variability, certain definite characters may be established. Methods of study. For determining the abundance of actinomyces in the soil, the general media used for the determination of numbers of bacteria can be employed (p. 16). For the study of cultural and morphological characters, several media have been suggested: Glucose agar:5 10 grams glucose 0.5 gram K2HP04 0.5 gram asparagine 15 grams agar 1000 cc. distilled water Malate-glycerin agar:5,7 10 grams calcium malate 0.5 gram NH4C1 0.5 gram K2HPO« 10 grams glycerin 15 grams agar 1000 cc. distilled water Reaction adjusted by means of NaOH to pH = 7.0 Citrate-glycerin agar:7 Same as 2, except that calcium citrate is used in place of malate 4. Czapek's agar:6 2 grams NaN03 1 gram K2HP04 0.5 gram MgS04 0.5 gram KC1 0.01 gramFeSO, 30 grams sucrose 15 grams agar 1000 cc. distilled water 5. Starch agar:6 10 grams starch is suspended in 800 cc. of water and boiled down to 500 cc. 500 cc. water to which are added 1 gram K2HP04, 1 gram MgS04, 1 gram NaCl, 2 grams (NH4)2S04, 3 grams CaC03, 10 grams agar. The two solu- tions are mixed and tubed. 6. Gelatin: 15 per cent Gold Label gelatin in distilled water. The first four media are used for the study of general cultural characters of the organisms and are well suited for morphological studies; the starch and gelatin media supply information on two of the most important physiological properties of the organisms, namely the diastatic and proteolytic. For microscopic examinations, one of the two following methods may be used: 1. Method of Henrici:19 Melted and cooled agar is inoculated with the specific organism and spread in a thin film on flamed slides, which are then incubated in 19 Personal communication. 292 PRINCIPLES OF SOIL MICROBIOLOGY a sterile moist chamber. After growth has taken place, the slides are dried, fixed in alcohol and stained. The entire colony, with both vegetative and aerial mycelium, can thus be examined in an undisturbed condition. 2. Method of Drechsler: The organism is grown on a synthetic medium and, when the culture is fully developed, the whole colony is cut from the agar and removed, as carefully as possible, from the tube or plate. A slide smeared with albumin fixative is now brought into firm contact with the surface mycelium and then separated from it, precautions being taken to avoid any sliding of the two surfaces on each other. If the growth is not too young, the upper portions of the aerial mycelium will be left adhering to the slide without any series dis- arrangement; killing and fixation may be effected at once. The material is then stained and mounted in balsam. Preparations, in which the spore chains have commenced to disintegrate, are impaired by the large masses of free spores. The most convenient fixative agent is 95 per cent alcohol. As a stain, Maiden- hain's iron-alum haemotoxylin is good for protoplasmic structures. Delafiold s haemotoxylin, allowed to act for 24 hours with the proper degree of decoloriza- tion, yields deeply stained, clear preparations showing distinctly the vacuoles, metachromatic and nuclear structures, and septa. Nature of growth on artificial media. The term colony is used incor- rectly in designating a mass of growth of an actinomyces, since it is merely a mass of mycelium developing out of a single spore, and not a colony in the sense of bacterial growth. Each spore or piece of mycelium separated from the colony is capable of individual existence, develop- ing into a new colony. The single-celled colony of an actinomyces is characteristic and is easily distinguished from that of bacteria or fungi. It is usually round and develops in the form of a semi-circle into the medium (No. 120, PI. XVII). The colonies are mostly compact, leathery, adhering to the medium, the surface being either flat or elevated; the outer zone is smooth, round as seen with the naked eye, and has a fringe of minute hyphae projecting for a short distance into the medium when observed under the low power. The surface is usually dry and often presents a conical appearance; it is either free from any aerial mycelium, or covered with a chalky (mealy, mildewy) white, drab or grey aerial mycelium, or with an abundant cottony, fuzzy, white, red or grey aerial mycelium. The subsurface growth of different organisms is little differentiated from one another, being usually of white-greyish or yellowish color ; but the surface growth and the subsurface growth which may develop up to the surface, have a characteristic appearance on synthetic media. The growth of some organisms presents a smooth surface, while others have a much folded, or lichnoid surface; still others form a fine network on the surface. These characters are not constant but change with the composi- tion of the medium and age of culture even on artificial culture media. SOIL ACTINOMYCES 293 Another characteristic of the growth of some species is the formation of "fairy rings" consisting of concentric spore-bearing rings and spore- free rings disposed in zones; the zones are also formed in the spore-free colony. It has been suggested20 that ring formation by fungi is a result of diffusion of injurious substances present or formed in the medium or due to the action of light, which produces a change in transpiration and temperature. No ring formation by actinomyces could take place as a result of changing periodically the environmental conditions. Vegetative mycelium. The vegetative growth consists of a mycelium composed of profusely branching hyphae (Nos. 121-122, PL XVI), the terminal growing portions of which are densely filled with pro- toplasm. The vacuoles increase in size towards the center of the thallus. They are possibly associated with the presence of metachro- matic granules which are often mistaken for micrococci or bacterial en- dospores. The actinomyces may be divided into two groups on the basis of the length of the hyphae: (1) those forming long, abundantly branching hyphae and (2) those which show on the slide only short unbranching pieces of mycelium or rods. According to Lieske, the aerobic forms, both spore-forming and non-spore forming, growing fast into the substratum, belong to the first group, while the anaerobic pathogenic forms are included in the second. The composition of the medium greatly influences the length of the hyphae so that, with 20 per cent of cane sugar in the medium or in strongly alkaline media, a long mycelial species was found to form a very short mycelium. The vegetative mycelium is usually infrequently and irregularly septated. While in some forms transverse walls appear with somewhat greater frequency, there are none in which septation approaches any pronounced degree of regularity or closeness. The branches are formed by the elongation of lateral buds arising some distance back from the growing point of an axial filament. The branch thus produced gives rise to secondary branches by lateral proliferation.21 In addition to the typical monopodial branching, true dichotomy may occasionally occur.22 In old cultures, certain swellings of the terminal ends of the hyphae are observed; these may also be formed under abnormal growth conditions, as in concentrated media or in the presence of substances like caffeine. 20 Alunk, M. Bedingungen der Hexenringbildung bei Schimmelpilzen. Centrbl. Bakt. II, 32: 353-375. 1912. Biol. Centrbl., 34: 621. 1914. 21 Mace, E. Sur les caracteres de culture du Cladothrix dicholoma. Compt. Rend. Acad. Sci., 106: 1622. 1888. 22 Neukirch, H. Uber Strahlenpilze. II, 1902. 294 PRINCIPLES OF SOIL MICROBIOLOGY These swellings are to a degree similar to the tubercles formed by patho- genic actinomyces in the animal body. These formations (so-called involution forms) cannot serve as a criterion for the separation of the organisms. The acid-fast staining reaction cannot serve for the differentiation of soil actinomyces. In the case of pathogenic forms, the property seems to be quite constant as long as the organisms are growing in the tissues of the infected animals. Spore bearing mycelium. Most actinomyces produce an aerial myce- lium on suitable substrates either in the form of a mat of fructifications or numerous erect sporodochia (coremia). In any case each individual fructification represents a well characterized sporogenous apparatus, consisting of a sterile axial filament bearing branches in an open racemose or dense capitate arrangement. The primary branches may function directly as sporogenous hyphae, or may produce branches of the second and higher orders. In the latter case sporogenesis is confined to the terminal elements and the hyphal portions below the points of attach- ment of branches remain sterile. The morphology of the spore-bearing hyphae of the various actino- myces exhibits distinct individuality and can readily serve as a basis for specific differentiation. The specialized, sporogenous hyphae are distinguished from the sterile hyphae of the aerial mycelium at an early stage of their development. While the diameter of the sterile mycelium which arises through the elongation of the growing filament tip shows very little subsequent increase in thickness, the sporogenous hyphae are in the beginning thinner than the axial hyphae from which they are derived. Increase in thickness of the sporogenous hyphae follows after the final linear extension has been attained. The final diameter of the sporogenous hyphae may be less or appreciably more than that of the vegetative hyphae. Orskov suggested the division of actinomyces into three groups. 1. Sporogenic fungi. The spores develop into a unicellular substrate my- celium that does not divide spontaneously. Out of this mycelium arises an aerial mycelium which later divides by the breaking up of the protoplasm into regularly sized parts. These are separated from one another by constriction of the thread membrane between the individual elements. They grow in the form of flakes in liquid media, usually at the bottom. The name Cohnistrepto- thrix was suggested for this group. 2. An initially undivided substrate mycelium is formed with an early de- velopment of aerial hyphae. Both substrate and aerial mycelia divide spon- taneously into segments. There is early surface growth on liquid media. It is suggested to reserve the name Actinomyces for this group. SOIL ACTINOMYCES 295 3. A unicellular delicate branching mycelium, at the extreme tips of which single oval spores are formed. There is growth in liquid media in the form of small firm grains at the bottom and along the glass. The name Micromonospora is suggested for this group. A classification found very convenient for the study of soil action- myces is tentatively suggested here. It is based on the formation of sporogenous hyphae. 1. Presence of substrate mycelium alone and no aerial mycelium formed on synthetic or organic media. 2. The aerial mycelium consisting of very long filaments, rarely branching and mostly sporogenous almost to the point of origin in the nutritive mycelium, without any coiling. This group can be subdivided into two sub-groups: (a) long mycelium and little branching (No. 123, Pi. XVI); (b) short mycelium and abundant branching (No. 124, PI. XVI). 3. Maturation of the sporogenous hyphae associated with the formation of characteristic spirals. A flexuous habit of the young filament early manifesting the tendency towards the coiling condition which becomes more definite with continued elongation. This group can be also divided into sub-groups on the basis of the obliquity of the spiral, diameter of turns, construction with ref- erence to the dextrorse and sinistrorse condition (constant characters, according to Drechsler). The spirals range from open (No. 127, PI. XVI), barely per- ceptible turns to strongly compressed spirals (No. 126), so that the adjacent turns are in continuous contact. The number of turns ranges from two or three to twenty or more. 4. Sporogenous hyphae formed in knot-like groups of three or four along a central hypha (No. 125, PI. XVI). Group 3 is most abundant in the soil and is, therefore, described in most detail. The representatives of this group form sporogenous hyphae ranging from the straight mycelium with a barely perceptible waviness of group 2 to the strongly compressed spirals, resembling a closed fist. The diameter of the spirals is usually somewhat in an inverse ratio to the number of turns characteristic of the species. It must be noted here, however, that the nature and composition of the medium greatly influ- ence the morphology of the organisms. Species belonging to group 4 (Act. reticuli) when grown on Czapek's agar, are found in group 3 when grown on nutrient agar or even certain inorganic media. By comparing the relation of the sporogenous branches to each other and the axial filaments, Drechsler noted two main types approaching each other in apparently intermediate forms, but quite distinct at the extremes : (a) erect dendroidic type in which the sequence of develop- ment of the sporogenous hyphae is successive; fructification starts from a single erect hypha with a spiral termination; sporogenesis commences 296 PRINCIPLES OF SOIL MICROBIOLOGY at the tip by the insertion of regularly spaced septa, and proceeds downward toward the base of the filament; (b) prostate, racemose type in which development of fructifications is more nearly simultaneous. In most species, however, both types are combined. The various species are usually characterized by clearly defined septation and have been separated by Drechsler into three different groups, on the basis of the disposition of their septa and development of their spores. In the first group, the cross walls in the sporogenous hyphae remain without any very pronounced change and continue to separate the adjacent cells until they have developed into a chain of mature contiguous spores. The insertion of these septa progresses from the tip toward the base and does not break the physiological continuity of the hyphae. Food material is, apparently, readily transported through these septa to the young spores at the termination, since the spores increase in size and may deposit a wall of measurable thickness. In the second group the septa apparently split into halves, which are then drawn apart by the longitudinal contraction of the individual protoplasts. In the third group the cross-walls first undergo a deep constriction which, by involving the ends of the young cylindrical spores, gives to the latter an elon- gated ellipsoidal shape. The constricted septum now gradually loses its stain- ing properties, and appears to become slightly drawn out in a longitudinal direc- tion. A preparation stained with Delafield's haematoxylin usually shows many old spore chains in which the individual spores are thus connected by hyaline isthmuses. Occasionally an isthmus may be found with a remnant of the old deep staining septum still unchanged in its center. Beyond these three types of sporulation another must at least be provisionally recognized, in which septa are either absent from the developing sporogenous hyphae or are not demonstrated by the use of ordinary stains. The protoplast appears to contract at regular inter- vals, yielding a series of non-contiguous spores, held together for a time by the connecting segments of evacuated filament wall. The germination of the spores takes place in dilute nutrient solutions. The spore first swells, then one to four germ tubes are produced. The number of germ tubes is more or less characteristic of the species. Utilisation of carbon compounds by actinomyces as sources of energy. Various species of actinomyces utilize a number of sugars and higher alcohols as sources of energy, with inorganic sources of nitrogen, es- pecially glucose, starch, maltose and glycerol. Lactose, sucrose and inulin are utilized to a less extent, depending upon the ability of the organism to form the corresponding enzyme. Arabinose, mannite SOIL ACTINOMYCES 297 and cellulose26 serve as good sources of carbon only for certain species.23 Various organic acids, such as succinic, malic, tartaric and citric, are well utilized, but formic, acetic, propionic, butyric, valerianic, lactic, benzoic and oxalic are not well utilized.24 Proteins and amino acids can readily be used as sources of carbon.25 Lieske27 employed a solution of 1 per cent urea, traces of K2HP04 and MgS04 and 2 per cent of the corre- sponding carbon source in distilled water. He found that the aerobic, saprophytic strains utilized maltose, lactose, levulose, dextrin, glucose, glycerin, glycogen, inulin, mannite, asparagine, human blood serum, and sucrose with decreasing efficiency in the order named. Only one of the two strains utilized starch and none utilized cellulose. Failure to obtain growth with cellulose is more often due to the fact that the proper method has not been employed. Utilization of cellulose should be tested on the agar plate28 with reprecipitated cellulose or by adding cellulose to sterile soil. The same is true, to a less extent, of the utilization of starch which was found to be one of the best sources of energy for the majority of actinomyces (except a few human patho- genes). Some strains utilize ethyl and methyl alcohol and, to a small extent, tannin. Amygdalin, caffeine, sodium soap and potassium ace- tate may also be assimilated.27 Actinomyces can multiply in sterile butter fat; growth is accompanied by a definite increase in acidity.29 By the use of a modification of Eij Io- nian's method, it was demonstrated that a large number of actinomyces are able to produce fat splitting enzymes.27 This was accomplished by adding 1-3 per cent of the fat to the molten agar, shaking well, pouring plates, then inoculating. On adding an indicator, such as litmus or brom phenol blue, no acidity could be indicated. The fatty acids are neutralized as can be readily demonstrated by the formation of crystals 23 Waksman, S. A. Studies in the metabolism of actinomycetes. II. Carbon metabolism. Jour. Bact., 4: 307-330. 1919. 24 Salzmann, P. Chemisch-physiologische Untersuchungen liber die Lebens- bedingungen von zwei Arten denitrifizierender Bakterien und der Streptothrix odorifera. Diss. Konigsberg. 1901. 25 Miinter, F. Uber Aktinomyceten des Bodens. Centrbl. Bakt. II, 38: 365-381. 1913. 26Krainsky, A., 1914 (p. 28S). On the decomposition of cellulose by micro- organisms. Zhur. Opit. Agron., 14: 255-261. 1913. 27 Lieske, 1921 (p. 288). 28 Scales, 1915 (p. 265). 29 Jensen, O. Studien iiber das Ranzigwerden der Butter. Centrbl. Bakt. II, 8: 171. 1902. 298 PRINCIPLES OF SOIL MICROBIOLOGY on the plate; the form of the crystals depends on the nature of the fat used. Some actinomyces can utilize rubber as a source of carbon.30 Lantzsch31 isolated an Actinomyces growing in distilled water contain- ing quartz or in a medium very poor in organic matter; this organism was found to be identical with Bac. oligocarbophilus of Beijerinck and it could assimilate CO as well as the higher aliphatic hydrocarbons, except benzol and xylol. Actinomyces are among the very few organisms in nature which are capable of utilizing lignins and soil "humus" as sources of carbon. This can be readily demonstrated both in liquid media and by the use of silica gel plates.32 Nitrogen utilization. Proteins form good sources of nitrogen, the same is true of peptones and various amino acids. Gelatin (15 per cent in distilled water, neutralized or reaction unadjusted) is liquefied in 3 to 5 days, at 18° to 20°C, but some species will produce only a very narrow liquefied zone after 30 to 40 days. Some do not form any pig- ment, others form a brown to purple-brown soluble pigment on the gela- tin. A number of species can also hemolyze red blood cells and produce a clear zone around the colony when grown on blood agar. The acti- nomyces can be divided into several groups on the basis of milk coagulation.33 1. Rapid coagulation of milk, followed by rapid liquefaction of the coagulum, so that the tube of milk becomes clear in 6 to 7 days. 2. Rapid coagulation of the milk followed by slow liquefaction. 3. Slow coagulation followed by a rapid liquefaction. 4. Digestion of the milk proteins without any previous coagulation. This tends to indicate that the rennet-like and proteolytic enzymes are distinct. Among the inorganic compounds, ammonium salts are utilized well, especially in the presence of sufficient buffer, especially silicates.34 The same is true of nitrates; they are reduced first to nitrites before they are assimilated. Nitrites themselves, when used in very low concen- 30 Sohngen, N. L., and Fol, J. G. Die Zersetzung des Kautschuks durch Mikroben. Centrbl. Bakt. II, 40: 87-98. 1914. 31 Lantzsch, K. Actinomyces oligocarbophilus (Bacillus oligocarbophilus Beij.), sein Formwechsel und seine Physiologic Centrbl. Bakt. II, 57: 309-319. 1922. 32 Unpublished data. 33 Waksman, S. A. Studies in the metabolism of actinomycetes. I. Jour. Bact., 4: 189-216. 1919. 34 Miinter, F. Uber Stickstoffumsetzungen einiger Aktinomyceten. Centrbl. Bakt. II, 39: 561-583. 1914. SOIL ACTINOMYCES 299 trations (0.01 per cent), are also utilized. Creatinine is readily utilized; urea and acetamide are assimilated only to a limited extent by the majority of organisms, while a few forms may utilize the substances readily.35 Lieske found that urea forms an excellent source of nitrogen, while asparagine was only scantily utilized ; uric and hippuric acids were not assimilated. The proteins are decomposed, with the formation of ammonia, even in the presence of available carbohydrates, such as glucose. In other words, we do not observe here that sharp sparing action of the carbo- hydrate upon the decomposition of proteins, as in the case of fungi or bacteria. Guittonneau36 demonstrated that certain actinomyces are capable of producing urea from proteins, both in the presence and ab- sence of glucose, in addition to ammonia. Oxygen requirement. The influence of oxygen tension on actinomyces is still an undecided question. There is no doubt that the majority of soil actinomyces are aerobic. The fact that some are able to grow deep into the medium would indicate ability to grow under semi-anaerobic conditions. Beijerinck37 classified the actinomyces as facultative anae- robes. Lieske, among others, found that the actinomyces acting as animal pathogenes are anaerobic, while the saprophytic forms are chiefly aerobic. A. scabies will not germinate in the absence of oxygen; the amount of available oxygen is the limiting factor both for germi- nation and growth.373 Other investigators38 pointed out that the recognition of strict aerobes or anaerobes is based upon errors of tech- nic, since in no instance could a strict aerobe or anaerobe be obtained. Influence of temperature, drying and radiation. Some actinomyces grow slowly at temperatures of 3° to 6°. Good growth of most species is obtained at 6° to 38°, 13° to 32°C. being the optimum for the majority of soil forms. Few organisms can thrive at 40-42° and none will grow at temperatures above 46°C. except the thermophilic forms. The temperature limits may be raised a few degrees by gradual adaptation. 86 Waksman, S. A. Studies in the metabolism of actinomycetes. III. Nitro- gen metabolism. Jour. Bact., 5: 1-30. 1920. 88 Guittonneau, G. Sur la production de l'uree au cours de rammonifica- tion par les Microsiphondes. Compt. Rend. Acad. Sci., 178: 1383-1385. 1924. 37 Beijerinck, M. W. Uber Chinonbildung durch Streptothrix chromogena und Lebensweise dieses Mikroben. Centrbl. Bakt. II, 6: 2-12. 1900. 37a Sanford, G. B. Some factors affecting the pathogenicity of Actinomyces scabies. Phytopath., 16: 525-548. 1926. 83 Musgrave, W. E., Clegg, M. T., and Polk, M. Streptothricosis with special reference to the etiology and classification of Mycetoma. Philip. Jour. Sci., B., Med. Sci., 3: 447-544. 1908. 300 PRINCIPLES OF SOIL MICROBIOLOGY Low temperatures (even 8° to 10°C.) do not injure the majority of actinomyces.27 High temperatures are very injurious. Some species are killed at 45°.39 The thermal death point of most actinomyces is 75°. The mycelium of some organisms may survive at 60°, but is killed in 20 minutes at 70°; the spores may survive 75° for 20 minutes but are killed in 30 minutes. Some soil organisms maybe able to withstand even somewhat higher temperatures. The actinomyces spores resemble in this respect the spores of other fungi in that they do not possess the degree of resistance of the bacterial spores and are readily destroyed at temperatures a few degrees (about 5°) above the destructive temperature of the mycelium. Some species are thermophilic in nature and have their optimum at much higher tempera- ture than the majority of actinomyces. The existence of actinomyces in the soil which can grow readily at 60° is established.40-41 These forms are especially abundant in the upper layers of forest and garden soils; they are also found abundantly in dry peat, grass, hay and straw. The thermophilic forms do not represent one species, but several distinctly different forms. Various investigators42'43 suggested that in the self- heating of straw and other plant materials, the thermophilic forms have a chance to develop and that the addition of straw or manure to the soil helps to spread the organisms. The thermophilic actinomyces multiply rapidly in the soil when the soil is warmed during the hot summer months by the direct rays of the sun. The fact that they are found in cold places where the minimum temperature (40°) for growth of thermophilic forms is never reached, such as forest soils, deep subsoils, and frozen soil, led Lieske to conclude that some of the common forms give out mutants which develop at the higher temperatures. This whole sub- ject deserves careful study. All actinomyces are very resistant to drying, as indicated by the fact that they are found abundantly on dry straw, hay and soil. Bere- stneff44 inoculated some ears of grain with a pure culture of A. violaceus 39 Foulerton, A. G. R., and Jones, C. B. On the general characteristics and pathogenic action of the genus streptothrix. Trans. Path. Soc. London, 53: 56-127. 1902. 40Globig. Uber Bakterienwachstuin bei 50-70°. Ztschr. Hyg., 3: 294. 1888. 41 Gilbert, tiber Actinomyces thermophilics und andere Aktinomyceten. Ztschr. Hyg., 47: 383-406. 1904. 42 Miehe, H. Die Selbsterhitzung des Heus. G. Fischer. Jena. 1907. 43 Noack, K. Beitriige zur Biologie der thermophilen Organismen. Jahrb. wiss. Bot., 51: 593-648. 1912. 44 Berestneff, N. M. Uber die Lebensfahigkeit der Sporen von Strahlenpilzen. Centrbl. Bakt. I, Ref., 40: 298. 1907. SOIL ACTINOMYCES 301 and found that he could obtain a pure culture of the organism from the dry material after preserving it for ten years. Lieske found that only the saprophytic spores and mycelium were viable after they had been preserved on filter paper in a desiccator for eighteen months. This would point to the fact that the pathogenic actinomyces are not carried over for any long time in dry soil or straw, as is often assumed. Direct sunlight does not exert any injurious effect upon the actino- myces and does not modify their growth. Exposure to ultraviolet rays for 10 minutes does not affect them; after one hour, the organism is definitely affected but not destroyed. Rontgen rays have no influence.27 Influence of reaction and salt concentration. It has been generally observed that alkalies and alkali substances favor the development of actinomyces, while acids and acid substances injure their activities. This is particularly true of inorganic acids, since the organic acids are utilized to some extent by the organisms and are thus broken down. The limiting acid reaction for the majority of soil actinomyces is pH 4.8 to 5.0, although some species may grow at as high an acidity as pH 3.0 to 4.0. The optimum is pH 7.0 to 8.5. On the alkaline side, the majority of organisms will still grow at pH 8.6 to 9.0, while some will grow even at more alkaline reactions. This fact can be uti- lized in adjusting the reaction of the soil so as to prevent the develop- ment of A. scabies causing potato scab. Gillespie45 was the first to point out the fact that soils having a reac- tion of pH 4.8 or less are free from scab, while those having a reaction more alkaline than pH 4.8 are apt to have scab. Various strains of A. scabies may behave somewhat differently: some may be inhibited in their development at pH 5.0, while others only at pH 4.6. The organ- isms are also able to withstand a somewhat greater acidity in the soil than in solution.46 The reaction of the medium is usually changed by the growing organism to less acid or more alkaline ;47 this is true of media containing proteins, amino acids and NaN03. The proteins and amino acid are decomposed with abundant formation of ammonia. With ammonium salts, the ammonium radical is rapidly used up, thus allow- 45 Gillespie, L. J. The growth of the potato scab organism at various hydro- gen-ion concentrations as related to the comparative freedom of acid soils from the potato scab. Phytopath., 8: 257-269. 1918. 46 Waksman, S. A. The influence of reaction upon the growth of actinomyces causing potato scab. Soil Sci., 14: 61-79. 1922. 47 Naslund, C.f and Dernby, K. G. Untersuchungen iiber einige physiolo- gische Eigenschaften der Strahlenpilze. Biochem. Ztschr., 138: 477. 1923. 302 PRINCIPLES OF SOIL MICROBIOLOGY ing the medium to become acid. No acids are formed from carbohy- drates. Certain actinomyces will grow in media containing 12 per cent glycerol or 7 per cent NaCl.48 In the presence of 18 per cent glycerol and 9 per cent NaCl, no growth was observed. Actinomyces were found49 to grow in the presence of 5 per cent of KC1, NaCl, KN03, NaN03, as well as mixtures of these, but spore formation is depressed. Ten per cent of the salts repressed the growth of all forms except one. Magnesium salts proved much more injurious. Small quantities of alkali earths stimulated, while larger quantities injured growth and spore formation. Difficultly soluble carbonates have little effect. AgN03 inhibited growth completely; 0.1 per cent Cu as CuS04 or CuCl2 was injurious, HgClo was less injurious. Lead nitrate and iron salts were least injurious. Lieske could not observe any diminution in growth of various actinomyces when 15 per cent cane sugar was added to nutrient bouillon. Slight growth was obtained in the presence of 20 per cent cane sugar; no further growth was observed in the presence of 30 per cent sugar. Actinomyces are found abundantly in nature on substances containing very little water and are very active in the soil in dry seasons. Influence of poisons. The addition of benzol to the soil was found to stimulate the development of actinomyces but they are injured by the application of carbon bisulfide.50 These organisms are not very sensi- tive towards chemical poisons but cannot resist the action of metallic salts. The limit of the action of a poison can be changed by gradual adaptation. Of the dyes, methylene blue, methyl violet and gentian violet are most toxic, preventing growth in 1:500,000 dilution in nutri- ent bouillon.27 Reduction of nitrates and other compounds. The majority of actino- myces species are able to reduce nitrates to nitrites, but not to ammonia nor to atmospheric nitrogen.51 By using the ordinary Czapek's solution, nitrite formation can be demonstrated to run parallel to the growth of the organism. Some starch solution and 0.5 per cent KN03 may be added to ordinary nutrient agar, which is then placed in Petri dishes, 48 Neukirch, 1902 (p. 293). 49 Miinter, F. tJber den Einflusz anorganischer Salze auf das Wachstum der Aktinomyceten. Centrbl. Bakt. II, 44: 673-695. 1916. 60 Stormer, K. tlber die Wirkung des Schwefelkohlenstoffs und ahnlicher Stoffe auf den Boden. Centrbl. Bakt. II, 20: 282-286. 1908. 51 Fousek, A. Die Streptothricheen und ihre Bedeutung in der Natur. Mitt, landw. Lehrkanz. K. K. Hochsch. Bodenk. Vienna, 1: 217-244. 1912. SOIL ACTINOMYCES 303 cooled and inoculated with the organism in question. After the organ- ism has developed for a few days, the plates are covered with a dilute solution of potassium iodide, to which some hydrochloric acid is added. The plates, in which the nitrate is reduced to nitrite, are colored blue since the nitrite liberates the iodine from the potassium iodide.27 This method, however, is not so reliable as the common method (a naphthala- mine + sulfonilic acid) of nitrite estimation, since the starch may be decomposed by the diastatic enzymes. Selenium and tellurium salts (0.01 per cent concentration) are reduced by numerous actinomyces to elementary selenium and tellurium; the colonies are colored deep red and deep black respectively, due to the fact that the metals are deposited within the mycelium. The phenome- non is intracellular. According to Husz,52 various actinomyces isolated from the soil can reduce organic arsenic compounds, similar to Pen. brevicaule, with the production of the characteristic garlic odor. These results could not be confirmed by Lieske. This may be due either to the difference in the organisms employed or difference in methods. Production of odor. Most actinomyces grown on organic or synthetic media produce a characterictic odor of freshly plowed soil. This is particularly true of the organisms possessing a mildewy aerial mycelium. The odor varies from earthy to musty. It was thought at first53 that this odor was formed by a specific organism. It was soon found that this is the property of a large number of species and the odor production is in itself a variable factor. The non-spore-forming organisms usually do not produce any odor. The intensity of the odor depends on the composition of the medium. Carbohydrates, particularly glycerol, stimulate the odor formation. The odoriferous substance has not been isolated yet. A few actinomyces, particularly thermophilic forms, produce a pleas- ant fruity odor. Pigment formation. The property of pigment formation is wide- spread amcng the actinomyces. No culture can be considered non- chromogenic until it has been studied on protein media and a variety of protein-free media. Three different kinds of pigment should be con- sidered: that of the vegetative mycelium, of the aerial mycelium, and 52 Husz, H. Zur Kenntnis der biologischen Zersetzung von Arsenverbin- dungen. Ztschr. Hyg., 76: 361. 1914. 63 Rullmann. Chemisch-bakteriologische Untersuchungen von Zwischen- decken-fiihlungen mit besonderer Beriicksichtigung von Cladothrix odorifera Diss. Miinchen. 1895. 304 PRINCIPLES OF SOIL MICROBIOLOGY pigment dissolved out into the medium. The aerial mycelium is usually colored white, gray or buff, sometimes red, yellow, brown, light green or bluish-green. The vegetative or substrate growth is usually colored gray, red, yellow, orange, brown or black. Among the soluble pigments, purple, brown, black and yellow are predominant; red, blue and green are also formed by some species. Soluble brown to purple pigments are very common on protein media. A slight difference in the composition of the medium has an important influence upon the pigment formation by actinomyces.54 Various attempts have been made to explain the nature of the dark brown pigments produced on organic media. Beijerinck55 suggested that the brown pigment is a result of quinone-formation, as shown by the fact that ferri-salts color the brown-colored gelatin black and the gelatin itself is made insoluble due to the action of the pigment. In the presence of HC1, iodine is liberated from potassium iodide. The for- mation of the pigment has also been ascribed56 to the action of an enzyme, as in the case of tyrosine media coloring black. But the organ- isms must be able to synthesize their own tyrosine and produce a brown or dark pigment since the pigment is produced also on tyrosine-free media. Some actinomyces form blue or green pigments, particularly when freshly isolated. The rapidity of gelatin liquefaction and pigment formation are utilized for diagnostic purposes. The characterization of an organism by the nature of the pigments formed has sometimes led to duplications, as in the naming of one organism, on the basis of formation of a red and blue pigment, A. violaceus ruber57 and A. tricolor.58 The pigment acts as a natural indicator. On media which are slightly acid (pH 6.0), the pigment is at first red, then, with a change of reaction of the medium to alkaline, the pigment becomes blue. The chemical nature of this pig- ment is yet to be investigated. Variability. The actinomyces show, in their morphological and physiological characteristics, greater variability than any other group of organisms. Size, shape and color of colonies, length of mycelium, abundance of mycelium and spore formation are determined largely 64 Conn, 1921 (p. 290). 65 Beijerinck, 1900 (p. 299). 66 Sano, K. Beitrage zur Kenntnis der Oxydasen, insbesondere bei Bak- terien. Diss. Wurzburg. 1902. 57 Waksman and Curtis, 1916 (p. 290). 68 Wollenweber, H. W. Der Kartoffelschorf. Arb. d. Forsschungsinst. f. Kartoffelhau. H. 2, Berlin. 1920. SOIL ACTINOMYCES 305 by the composition of the medium and age of culture. When or- ganisms are named merely on the basis of pigment production on complex media (-4. chromogenus, A. snlfureus, etc.), or on the basis of color of aerial mycelium (A. albus, A. niger), on the basis of ring formation in aerial mycelium (A. annulatus), we are merely utilizing variable properties of the organisms as some distinguishing charac- ters. When the organisms are transferred to other media, or even when cultivated continuously on the same medium, the pigment may change and rings may no longer be formed. The species thus lose their distinguishing characteristics and become other species. In addi- tion to using a group of morphological and physiological characteristics which are commonly employed for the species determination, one must also allow for the variability of the organisms. Observations should be made of the morphological and physiological characteristics on synthetic media for a large number of generations. Such characters as pigment production may change. A. verne produced, when originally isolated from the soil, a beautiful green pigment on Czapek's agar, but lost the property in a few months when grown on synthetic media; after several years, it gained the property of producing a brownish-purple pigment on the same medium. The property of producing aerial mycelium may be lost and the character of growth changed. A. halstedii, when freshly iso- lated from the soil, produced a dark growth with a mouse-gray aerial mycelium on Czapek's agar; on continued cultivation on artificial media, the property of forming an aerial mycelium was lost altogether, and the growth became dark brown and lichnoid in appearance. In some instances, organisms that lost the power of forming aerial mycelium regained it after cultivation in sterile soil. Similar variability is found in the rapidity of gelatin liquefaction, production of soluble pigment, action on milk, oxygen requirement, and odor production. One can readily observe in some actinomyces cul- tures the formation of sectors, differing in one respect or another from the rest of the growth. On transferring from such a sector to a fresh medium, an organism may be obtained which differs from the mother culture in some character such as color, presence of aerial mycelium, zone formation, pigment, etc. Lieske has thus isolated five new forms, in addition to the original, which were distinguished from one another by at least one character. Although the claim is put forth that pure spore cultures were used, no mention is made of the use of such a pro- cedure as the Barber pipette, which would absolutely insure a single- spore culture. 306 PRINCIPLES OF SOIL MICROBIOLOGY Species differentiation. For a study of cultural and biochemical characteristics of the organisms, a group of media may be recom- mended, which will help to bring out the salient features. 1. Synthetic media, as described above: (a) Modified Czapek's agar (b) Krainsky's glucose agar (c) Malate-glycerin agar (Conn) (d) Citrate-glycerin agar (Conn) Temperature of incubation 25°, period of incubation 7 to 15 days. 2. Gelatin, 15 per cent in distilled water, reaction unadjusted; temperature of incubation 16° to 18°; period of incubation 30 days. 3. Sterile skimmed milk; temperature of incubation 25° and 37°; observations made daily. 4. Potato plug, 25°, for 7 days. 5. Starch agar, 25°, 10 to 15 days (test for diastatic strength). 6. Nutrient peptone agar, 25°, 7 to 15 days. General morphology to be studied by direct examination of colony on plate by means of low power. For detailed study, the method of Drechsler to be used, magnification 1000 to 1200. The cultural characteristics of actinomyces in these media make pos- sible the suggestion of the following tentative key for their differentia- tion, until a more permanent one based on morphological studies can take its place. KEY TO THE IDENTIFICATION OF SPECIES OF SOIL ACTINOMYCES (Based chiefly on physiological characteristics) A. Formation of a soluble pigment on all media containing protein substances: I. Pigment deep brown (chromogenus types): 1. A brown pigment is produced on tyrosine agar: (a) Pigment dark brown; white to cream-colored growth on synthetic media; soluble brown pigment on synthetic media containing arabinose, glucose or lactose A. scabies A number of strains of Actinomyces were isolated from potato scab lesions; it was suggested that there is no justification for including all these organisms in one species.58* (b) Pigment faint brown; sulfur-yellow soluble pigment on creatinine solution; aerial mycelium on glucose agar is ocher to reddish ocher colored A. olivochromogenus &8a Millard, VV. A., and Burr, S. A study of twenty-four strains of Actino- myces and their relation to types of common scab of potato. Ann. Appl. Biol., 13:580-644. 1926. SOIL ACTINOMYCES 307 2. Growth and aerial mycelium on synthetic agar green to dark- green; soluble brown pigment on synthetic media with most carbohydrates A. viridochromogenus 3. Deep brown to black pigment on synthetic agar: (a) Weakly growing organisms; orange-red growth on potato plug; no visible aerial mycelium on synthetic agar A . purpeochromogenus (b) Vigorously growing organisms; brown to black growth on potato plug; abundant cottony aerial mycelium on synthetic agar A . pheochromogcnus 4. Usually no action on milk (37°), accompanied by the darkening of the milk; mouse-gray aerial mycelium on synthetic agar; ammonium salts used readily with different sources of carbon A. aureus 5. Brown pigment never produced on synthetic media: (a) Aerial mycelium on synthetic media has lavender shade A. lavendulae (b) Aerial mycelium on synthetic agar is abundant, of a water green color Actinomyces 218 (c) Whirl formation in aerial mycelium on synthetic agar: (a') Growth colorless and aerial mycelium white A. reticuli (b') Growth pink, aerial mycelium rose colored; nitrate reduction very abundant; fewer whirls A. reticulum-ruber (d) Growth on synthetic agar sulfur-yellow, with yellow aerial mycelium; barnacle-like, greenish-yellow growth on potato plug A. flavus (e) Growth on synthetic agar red colored, aerial mycelium abundant, orange colored; aerial hyphae usually do not form spirals A . ruber II. Soluble pigment on organic media faint brown, golden, yellow or blue: 1. Pigment blue, not always definite; soluble red pigment turning blue on synthetic agar A . violaceus -ruber 2. Pigment at first green on organic media and synthetic agar, property lost on continued cultivation, becoming brown on synthetic agar; aerial mycelium not produced on most media A. verne 3. Soluble pigment at first brown, property lost entirely on con- tinued cultivation; growth and aerial mycelium on synthetic agar abundant, white A. albus 4. Soluble pigment yellowish green; growth on synthetic agar penetrating into the medium is pink A. calif ornicus 5. Brown pigment produced only on certain protein media (usually gelatin and glucose broth, not nutrient agar) : (a) Growth on synthetic agar red to pink; no differentiated aerial mycelium or only scant white A. bobili 308 PRINCIPLES OF SOIL MICROBIOLOGY (b) Growth on synthetic agar colorless; aerial mycelium thin, rose-colored A. roseus (c) Growth on carrot and potato rapidly spreading, envelop- ing the whole plug and destroying it rapidly, plug becoming colored deeply brown A. griseolus (d) Red (vinaceous) soluble pigment on synthetic agar, often turning red-brown; white aerial mycelium A. erythreus B. No soluble pigment produced on gelatin or other protein media: I. Species strongly proteolytic: gelatin liquefied rapidly, milk clotted and peptonized rapidly. 2. Brown soluble pigment on synthetic agar; diastatic action very strong A . diastaticus 2. Rapid liquefaction of coagulated blood serum, strong hemolysis of blood (37°); aerial mycelium on synthetic agar has a tea- green tinge A. griseus 3. Yellowish green growth on starch plate with pinkish aerial my- celium; citron-yellow growth on synthetic agar A. citreus 4. Greenish-yellow growth on synthetic agar, gray powdery aerial mycelium, greenish-yellow soluble pigment A, flavovirens 5. Colorless growth on synthetic agar, white to grayish aerial my- celium, no spiral formation; thin reddish-brown growth on potato plug (purplish zone on plug) ; faint yellow pigment may develop on gelatin, blood and egg-media A. poolcnsis 6. Buff colored growth on glucose agar, violet-gray aerial my- celium; yellow growth on synthetic agar with light drab aerial mycelium; rapid destruction of potato plug A . olivaceus 7. Very scant colorless growth with scant white aerial mycelium on synthetic agar and on synthetic media containing NaNCh as a source of nitrogen; abundant brown growth with white aerial mycelium and soluble brown pigment on glucose agar; growth on potato plug greenish turning black.. .A. gelaticus II. Proteolytic action weak: 1. Soluble pigment produced on synthetic agar: (a) Pigment blue or blue-black A. violaceus-cacsari (b) Pigment brown to almost black on all synthetic media with NaN03 as a source of nitrogen A. exfolmtus 2. No soluble pigment on synthetic agar, although growth is colored : (a) Growth turning black, diastatic action very strong: (a') Growth on synthetic agar scant with abundant spirals in aerial mycelium, no invertase pro- duction A. rutgersensis (b') No spirals on synthetic agar, characteristic green colored growth on protein-glycerin media A. lipmanii (c') None or scant aerial mycelium on all media; growth abundant on synthetic agar (invertase positive); SOIL ACTINOMYCES 309 none or scant growth on blood agar and egg- media A. halstedii (b) Growth orange colored on most synthetic and organic media; aerial mycelium pink A. f radii (c) Growth yellowish on synthetic and glucose agars; pinkish to cinnamon-colored on calcium malate agar; no growth on blood serum and egg media; none or only very scant and late aerial mycelium on most media. A. alboflavus (d) Growth on synthetic media rose to red colored, aerial my- celium white, no visible action on milk. .A. albosporeus Over fifty species of actinomyces have been isolated from the soil and described.59 A much larger number, however, can readily be obtained from the soil. Some of them are of wide occurrence, as the A. chromo- genus types (A. viridochromogenus, A. pheochromogenus, etc.), A. aureus, A. rutgersensis, A. violaceus ruber.60 Importance of actinomyces in the soil. Actinomyces take an active part in the decomposition of organic matter in the soil, both of a nitrog- enous and non-nitrogenous nature. Some species are capable of de- composing celluloses very rapidly and there is no doubt that under conditions favoring their development, as in neutral or alkaline and arid soils or with insufficient moisture, actinomyces play an important part in this process. Krainsky even divided all the actinomyces into two groups: (1) the macro-actinomyces, forming oval spores and large colonies on agar and not decomposing cellulose or only to a very limited extent; and (2) the micro-actinomyces, forming spherical spores and minute colonies on agar and decomposing cellulose rapidly with the formation of pigments. Mace61 pointed out that actinomyces decom- pose proteins into amino acids and ammonia; he suggested that they may bring about the formation of humus (ulmic acids) in the soil. Ac- tive protein decomposition by actinomyces has also been recorded.62-64 In view of the fact that the amount of mycelium synthetized by this group of organisms is considerably smaller than that of fungi, only small amounts of nitrogen are assimilated and most of it is liberated free in "Krainsky, 1914 (p. 288). Waksman and Curtis, 1916 (p. 290). Conn, 1917 (p. 288). 60 A detailed description of various actinomyces found in the soil is given by Waksman, 1919 (p. 288) and Bergey, 1923 (p. x). 61 Mace, E. De la decomposition des albuminoides par les Cladothrix (Actino- myces). Compt. Rend. Acad. Sci., 141: 147. 1905. 62Fousek, 1913 (p. 40). "Munter, 1914 (p. 298). "Waksman, 1919 (p. 288). 310 PRINCIPLES OF SOIL MICROBIOLOGY the form of ammonia. Non-nitrogenous organic matter does not exert such a depressing effect upon ammonia accumulation as in the case of bacteria and other fungi; as a matter of fact, ammonia formation from proteins will take place even in the presence of available carbohydrates, as pointed out above. The accumulation of "humus" in the soil is an index of the great resistance of this group of organic substances to decomposition by microorganisms. Since this substance contains the larger part of the soil nitrogen, its decomposition is of great importance to soil fer- tility. Actinomyces seem to be among the very few organisms capable of attacking this resistant material and bring about its decomposition. Liming of soil and draining of swampy soil favors the development of actinomyces and also the decomposition of the soil organic matter. It is possible that a definite connection exists between these two phenom- ena. According to Fousek an increase in plant growth is obtained by adding actinomyces mycelium to soil, due to the increased decomposition of the organic matter thus brought about. We find, among the actinomyces, organisms causing important plant diseases, of which potato scab and sugar beet scab65 are known. There is considerable evidence that actinomyces may enter into certain asso- ciations with plants, as pointed out by Peklo,66 who cultivated an or- ganism, named A. alni, out of the nodules on the roots of Alnus and Myrica. Similar results were obtained by Lieske. In view of the fact that it has not as yet been possible to obtain the nodules by artificial inoculation, these results cannot be accepted as positive. 66 Kruger, F. Untersuchungen liber den Gtirtelschorf derZuckerruben. Arb. Biol. Anst. L. Forstwirt., 4: 254-318. 1905. 66 Peklo, J. Die pflanzlichen Aktinomykosen. Centrbl. Bakt. II, 27: 451- 479. 1910. CHAPTER XIII Soil Protozoa The protozoa form numerically the most abundant group of the animal population of the soil. It has been known since the work of Ehrenberg that the soil harbors a number of different protozoa, but the investigation of this subject has been greatly stimulated by the con- tributions of Russell and Hutchinson,1 who suggested that the limi- tation of bacterial activities in the soil is due to a factor of biological origin, probably the protozoa; this factor is less resistant than the soil bacteria and is readily destroyed by the treatment of soil with heat or disinfectants. This brings about a rapid development of the bacteria and the liberation of the combined nitrogen in a form available for plant growth, namely ammonia. The inference was that the soil protozoa, using the bacteria as food, limit the bacterial activities in the soil and, as a result of that, also limit soil fertility. On treating the soil with heat and disinfectants, the protozoa are destroyed, while the bacteria remain alive and begin to multiply very rapidly, their increased activities resulting in the liberation of the nitrogen and an increase of plant growth. Additional plausibility was given to this theory by suggestions made a few years previously and supported by a number of observations,2 that a similar function is performed by protozoa during the self-purifica- tion of water (i.e., the disappearance of pathogenic bacteria during storage). In fact, in view of the close similarity between many of the biochemical problems of water and sewage purification and those of the soil, the extensive investigations that have been made in the former fields are well worth careful consideration by the soil microbiologist.3 General ?norphology of protozoa. A detailed study of the morphology, 1 Russell, E. J., and Hutchinson, H. B. The effect of partial sterilization of soil on the production of plant food. Jour. Agr. Sci., 3: 111-144. 1909; 5: 152-221. 1913; Russell, E. J. Soil protozoa and soil bacteria. Proc. Roy. Soc. B., 89: 76-82. 1915. 2 Fehrs. Die Beeinflussung der Lebensdauer von Krankheitskeimen im Wasser durch Protozoen. Hyg. Rundsch., 16: 113-121. 1906 8 Buswell and Long. Activated sludge experiments. Illinois State water supply, Bui. 18, 1923. 311 312 PRINCIPLES OF SOIL MICROBIOLOGY classification, and physiology of protozoa is found elsewhere.4-8 Some of the information, necessary for a knowledge of the identification and study of activities of soil protozoa, is given here. Protozoa are unicellular organisms, varying in size from a few microns to 4 to 5 cm. Some protozoa may also form colonies consisting of numerous individuals. The majority of species, particularly the soil 4 Biitschli, O. Protozoa. In Bronn's Klassen und Ordnungen des Thier- reichs. 1, 1883. 6 Doflein, F. Lohrbuch der Protozoenkunde. 4th Ed., Jena, 1916. 6 Minchin, E. A. An introduction to the study of protozoa. Arnold, Lon- don. 1912. 7 Edmondson, C. H. The protozoa of Iowa. Proc. Davenport Acad. Sci., 12: 1-124. 1906. Schaeffer A. A. Taxonomy of the amebas, with descrip- tions of thirty-nine new marine and freshwater species. Dept. Marine Biol. Carnegie Inst., Washington, 24. 1926. 8 Calkins, G. N. The biology of the protozoa. Lea & Febiger, Philadelphia. 1925. PLATE XVII Soil Protozoa 128. Naegleria gruberi: a, small organism (8 to 3G>) with one broad blunt pseudopodium or sometimes several blunt ones, and one subcentral nucleus. When it enflagellates the karyosome sends out a chromatic process b, which tra- verses the nuclear membrane, forms a marginal blepharoplast, and emerges as two long flagella (c, d). The body assumes a rigid asymmetrically curved shape and the organism swims away in the typical spiral course. When it exflagellates the flagella shorten, thicken and retreat into the cytoplasm and the blepharo- plast returns to the karysome within the nucleus (from Kofoid). 129. Vahlkampfia soli, a Umax amoeba, from fresh fixed film (from Martin and Lewin). 130. Amoeba cucumis, a lamellipodian amoeba (from Martin and Lewin). 131. Euglypha sp., a thecamoeba (from Martin and Lewin). 132. Cyst of Amoeba cucumis (from Martin and Lewin). 133. Colpoda steinii, X 800 (from Goodey). 134. Balantiophorus elongatus, X 800 (from Goodey). 135. Pleurolricha grandis (?), X 410 (from Goodey). 136. Gonostomum affine, X 510 (from Goodey). 137. Vorticella microstoma, living stalked form, X 410 (from Goodey). 138. V. microstoma, free swimming, recently excysted form with aboral ciliary ring, X 510 (from Goodey). 139. Bodo caudatus (from Martin and Lewin). 140. Monas guttula (from Fellers and Allison). 141. Cercomonas crassicauda (from Fellers and Allison). 142. Bodo ovatus (from Fellers and Allison). 143. Pleuromonas jaculans (from Fellers and Allison). PLATE XVII > £SV- 151 m 129 v.. 132 128 133 130 138 139 134 136 135 137 SOIL PROTOZOA 313 forms, are microscopic and can be studied in detail only with the highest magnifications. The protoplasm is a colloidal liquid containing chro- matic or nuclear substance, generally forming nuclei readily distinguish- able from the protoplasmic body, which is either naked at the surface or enclosed by a cell membrane. Usually one or two nuclei are present, in some cases several of them; most infusoria contain a large macronu- cleus (vegetative functions and asexual division) and a small micronu- cleus (sexual reproduction). Contractile vacuoles, when present, are for the elimination of waste fluids or possibly for the adjustment of the osmotic pressure of the protoplasm (absent in marine forms). Green, yellow or brown chromatophores are present in the endoplasm of some Mastigophora. The most important constituents of the cell are the complex proteins, particularly nucleins and nucleo-proteins. In addi- tion to these, there are always present in the living cell carbohydrates, lipoids and enzymes. There are also found in the protozoa undigested food particles, waste materials or foreign elements, which take no part in the physiology of the organism; algae and bacteria may often be present in the endoplasm, possibly as a result of symbiotic relationship. Many species are subject to the attacks of minute parasitic organisms, either in the nucleus or in the cytoplasm. Reproduction is usually effected by fission, and, in the great majority of protozoa, a process of conjugation occurs at some stage in the life- cycle, the essential part in the process being fusion of the nuclear matter from distinct individuals. Locomotion is accomplished either by cilia, flagella, pseudopodia, or may be absent entirely, this serving as a basis for classification. Organs for the capture and assimilation of food may be present or entirely absent. The protozoa are classified on the basis of locomotion into: 1. Sarcodina or Rhizopoda, motility by means of pseudopodia, i.e., extensions (usually temporary) of the protoplasm of the cell body. The pseudopodia are either broad, blunt, finger-like or filiform, simple or branched. In some (Helio- zoa), the ray-like pseudopodia are usually supported by axial filaments. Some Sarcodina are naked, while others form shells; these are composed of materials secreted by the animals themselves, as chitin, silica, calcium carbonate, or are constructed from foreign particles, as diatoms, sand grains, clay particles, etc. Some shells (chitinous) are delicate, transparent, while others are composed of distinct plates, arranged more or less regularly. 2. Mastigophora or Flagellata, motility by means of flagella. These flexible, whip-like processes are usually attached at one end of the body. Either one or more flagella may be present; when single the flagellum is usually directed for- ward and draws the body forward by its movement. When more than one 314 PRINCIPLES OF SOIL MICROBIOLOGY flagellum is present, one or more may be directed backward. Some low flagel- lates can form pseudopodia. 3. Ciliata or Infusoria, motility by means of numerous cilia or short hair-like processes present during the entire existence of the protozoa or during their embryonic stage only. The cilia are either evenly distributed over the surface of the organisms or are restricted to certain regions. Large spine-like cirri or setae, or vibrating membranelles may be formed from fusion of cilia. Most are free swimming, some are attached by rigid or flexible stalks or pedicels. 4. Sporozoa, motility reduced by parasitism. The soil forms are found among the first 3 general groups. Physiology of protozoa. The physiology of the protozoa cannot be studied adequately, in a manner similar to higher organism or to micro- scopic plant organisms, as fungi, bacteria and algae. This is due largely to the fact that they cannot be cultivated yet in pure cultures, free from bacteria. An approach in this direction has been made by the cultivation of protozoa with single species of bacteria. Most of the protozoa are aerobic, obtaining their oxygen, necessary for oxidation purposes, from the air by absorbing it through the permea- ble membrane, without the aid of specialized respiratory organs. A few forms are anaerobic, obtaining the oxygen by reduction of oxygen rich substances. Oxidation is closely associated with nutrition and is followed by the excretion of the waste products, such as C02, urea and other products of metabolism. The optimum temperature for the development of protozoa is 18- 22°C. Excessive heat kills the protozoa, but excessive cold does not injure them beyond retarding their vital activities. The optimum reaction for the activities of protozoa was at first be- lieved to be at the neutral point. Good growth was also obtained in acid media.9 The limiting acid and alkaline reaction values for the growth of Paramoecium caudatum were found to be at pH 5.0 and 9.0 ;10 in other experiments Paramoecium was reported to develop normally at pH 6.4 to 8.0; beyond these limits, development is restricted and en- cystment may take place. Various soil ciliates, flagellates and amoebae were found to be able to live and reproduce in artificial media even at 9 Vahlkampf. Beitrilge zur Entwicklungsgeschichte von Amoeba Umax, einschliesslich der Ziichtung auf ktinstlichen Nahrboden. Arch. Protistenk., 6: 167. 1905. 10 Dale, D. On the action of electrolytes on Paramoecium. Jour. Physiol., 46: 129-140. 1913; Koffman, M. tJber die Bedeutung der Wasserstoffionen- konzentration fur die Encystierung bei einigen Ciliatenarten. Arch, mikrosp. Anat. Entwicklungs., 103: 168-181. 1924. SOIL PROTOZOA 315 pH 3.5-3.9 on the one hand and 9.75 on the other.11 Testaceous rhizopods are most numerous in acid peat soils and are very scarce in alkaline soils. Many of the common protozoa can tolerate semi-anaerobic con- ditions. Aeration of soil has, therefore, little direct influence upon their development. On the basis of their nutrition, the protozoa are divided into auto- trophic (or holophytic forms which synthesize food using the energy of sunlight) and heterotrophic forms; these are subdivided into holozoic, when solid food particles are taken in, and saprophytic, when nourish- ment is absorbed from soluble organic substances by diffusion through the body surface. The great majority of protozoa are holozoic in their nutrition. The autotrophic protozoa are usually found among the flagellates; they obtain their nutrition entirely from inorganic substances and the CO2 from the atmosphere ; they are often spoken of as Phytoflagellates and may even be considered with the algae. They contain colored substances (chromophyll), which enables them to utilize the energy of light, similarly to higher plants. These colored substances are seldom diffused throughout the cell, but are usually present in special bodies, termed chromatophores. The most common representative of this group is the genus Euglena. In some cases, protozoa are found to live symbiotically with green algae. Such protozoa have been demon- strated among the amoebae and flagellates. The holozoic protozoa, to which the majority of ciliates belong, use as food the various complex materials which form the constituents of liv- ing organisms (bacteria, algae). Some protozoa, like various amoebae, can feed on other protozoa, even on the larger ciliates.12 The whole organism or part of it is taken into the body of the protozoan, where it is subjected to the chemical action of digestion, with the production of organic substances utilized by the protozoan as food. The process of nutrition takes place in three stages, the taking in of the food, its decomposition, and the excretion of the unutilizable part. Digestion in all free-living protozoa is intracellular, by means of diastatic, proteo- 11 Nasir cited by Sandon, 1927 (p. 329); Fine, M. S. Chemical properties of hay infusions. Jour. Exp. Zool., 12. 1912. 12 Beers, C. D. The clearing of ciliates by amoeba. Science, 64: 90. 1926; Brit. Jour. Exp. Biol., 1: 325-341. 1924; Mast, S. O., and Root, F. M. Jour. Exp. Zool., 21: 33-49. 1916; Schaeffer, A. A. Quart. Rev. Biol., 1: 95-118. 1926. 316 PRINCIPLES OF SOIL MICROBIOLOGY lytic and other enzymes.13 In some, if not in the majority of protozoa, there seems to exist a certain selection for the food, some species of bac- teria being preferred to others. This may be due to the fact that some bacteria are digested more readily than others by the specific enzymes of the protozoan or to the formation by the particular bacteria of substances which are injurious to the protozoan.14 Some protozoa, especially cer- tain amoebae and flagellates, are capable of digesting complex proteins, starches and in some cases even celluloses.15 The autotrophic protozoa have no apparent relation to the bacteria in the soil; the heterotrophic forms may play an important part in the soil by using the bacteria as food. The latter utilize the soluble inor- ganic and organic compounds and serve, in their turn, as food for many rhizopods and ciliates. Many of the latter are specifically adapted to the feeding on bacteria. Some protozoa also feed on fungi, algae and smaller protozoa. Doflein divides the protozoa, which require complex organic substances as food, into 4 groups: those that feed on (1) bac- teria, (2) on waste products, (3) on plants (diatoms and other algae), and (4) on small animals. Most forms take in mixed food, feeding on bacteria and waste products, but the fact that the different protozoa differ from one another in their feeding habits and that some species have considerable power of selecting their food, makes a more detailed knowledge of their food requirements a necessity for the satisfactory elucidation of their role in the soil economy. When an infusion of hay, straw, or moss is prepared and allowed to incubate, bacterial development takes place immediately. This is soon followed by abundant growth of various species of protozoa, including the flagellates and amoebae, later followed by the ciliates; these feed on the bacteria in the infusion. The protozoa come into the infusions from the air, where they are present in the form of cysts; as many as 13 13 Mouton, H. Recherches sur la digestion chez les amibes et sur leur diastase intracellulaire. These. Paris, 1902, Charaire; Compt. Rend. Acad. Sci., 133: 244. 1901. Ann. Inst. Past,, 16: 457-509. 1902. 14 Hargitt, G. T., and Fray, W. W. The growth of Paramecium in pure cul- tures of bacteria. Jour. Exp. Zool., 22: 421-455. 1917; Phillips, R. L. The growth of Paramecium in infusions of known bacterial content. Ibid., 36: 135- 183. 1922. 15 Stole, A. Beobachtungen und Versuche liber die Verdauung und Bildung der Kohlenhydrate bei einem amobenartigen organismus — Pelomyxa palustris Greeff. Ztschr. wiss. Zool., 68: 625-668. 1900; Clevelend, L. R. The method by which Trichonympha campanula, a protozoon in the intestine of termites, in- gests solid particles of wood for food. Biol. Bui., 48: 282-287. 1925. SOIL PROTOZOA 317 species of protozoa have been demonstrated in the air in the form of cysts.16 The soil amoebae and closely related forms select their food instead of ingesting all smaller organisms indiscriminately. Micrococci and bacteria are eaten most readily, bacilli less readily, while yeasts and actinomyces are not consumed at all.16a Different species of amoebae may prefer different organisms, Gram-negative forms being consumed more readily than Gram-positive forms, young cells more readily than old cells (Oehler). When an amoeba is once accustomed to feed on a certain bacterium, it will continue to select the particular organism in preference to others. Facts are on record concerning the ability of various protozoa to utilize various soluble organic substances formed in the soil by bacterial action.17 This is true of flagellates and even of certain ciliates, which are said to have been cultivated free from bacteria, and to be able to derive their nutrients from various soluble organic and inorganic materials.18 Under natural conditions, however, Colpidium colpoda may feed entirely by phagocytosis, although, under artificial conditions, they may be able to obtain their food from solution.19 These results need further con- firmation, the evidence submitted being insufficient to be accepted. Media for the cultivation of protozoa. The media commonly employed in the cultivation of protozoa consist of complex organic substances. For the cultivation of chlorophyll-bearing protozoa, as well as many heterotrophic forms, purely synthetic media can be used. For the growth of Euglena, a medium consisting of 0.5 gram peptone, 0.5 gram glucose, 0.2 gram citric acid, 0.2 gram MgS04-7H20, 0.05 gram K2HP04 in 100 parts of water has been suggested.20 The concentration of citric acid and peptone may be doubled and 0.05 per cent NH4NO3 may be added. 16 Puschkarew, B. M. fiber die Verbreitung der Suszwasserprotozoen durch die Luft. Arch. Protistend., 28: 323. 1913. iea Severtzoff, L. B. Method of counting, culture medium and pure cultures of soil amoebae. Centrbl. Bakt. I, Orig., 92: 151-158. 1924. 17 Thornton, H. G., and Smith, G. On certain soil flagellates. Proc. Roy. Soc. B., 88: 151-165. 1914; Alexeiev, A. G. Protistic Coprology, as a special branch of protistology, and a description of several new species of protists — "coprocolae." Russ. Jour. Microb., 4: 97-134. 1917. 18 Peters, R. A. The substance needed for the growth of a pure culture of Colpidium colpoda. Jour. Physiol., 55: 7-32. 1917. 19 Lwoff, A. Sur la nutrition des infusoires. Compt. Rend. Acad. Sci., 176: 928-930. 1923. 20 Zumstein. Zur Morphologie und Physiologie der Euglena gracilis Klebs. Jahrb. wiss. Bot., 34: 149. 1900. 318 PRINCIPLES OF SOIL MICROBIOLOGY The green Paramoecium bursaria was cultivated21 on a medium consisting of: Ca(N03)2 0.20 gram NaCl 0.20 gram MgS04-7H20 0.02 gram FeS04 Trace K2HP04 0 .02 gram Water 1000 cc. Peters' glycerophosphate medium, described below, has been used successfully for the cultivation of ciliates. Colpidium and Oicomonas were grown on the following medium:22 Ammonium lactate. .. . 0.1 gram KC1 0.3 gram Glucose 0.4 gram MgS04-7H20 0.001 gram NH4CI 0.03gram CaCl2 0.02 gram Na2HP04 0.01 gram The reaction is adjusted to pH 7.0-7.4. Phenol red may be added to the cul- tures to follow changes in the acidity and alkalinity during the growth of the culture. Giltay solution and a synthetic glycerin medium may also be em- ployed.23 However, the majority of the media used for the isolation of protozoa are based upon a previous development of bacteria, which, either alive or dead, may serve as food for the protozoa. Such substances as soil, straw, hay, grass, lettuce, leaves, etc. cooked in water are favorable. Thirty to 40 grams of hay are cooked in one liter of water for thirty minutes;24 solution is filtered, made up to volume; to prepare a solid medium 15 grams of agar are added. 0.025 per cent meat extract in distilled water was found26 to be favorable for Paramoecium aurelia. Other media containing one to two per cent of nutrose, somatose, or peptone and 1.5 per cent agar are also favorable. Martin and Lewin26 used a medium prepared by boiling three lumps of horse dung in 500 cc. water for 1£ hours, filtering through cloth and adding 6 grams of agar. A small amount of water or dilute albumin added to the culture plates to a depth 21 Pringsheim, E. G. Die Kultur von Paramoecium bursaria. Biol. Centrbl. 36: 375. 1915; Zur Physiologie saprophytischer Flagellaten (Polytoma, Astasia, and Chilomonas). Beitr. Allg. Bot., 2: 88-137. 1921. 22 Cutler, D. W., and Crump, L. M. The rate of reproduction in artificial culture of Colpidium colpoda. Biochem. Jour., 17: 174-186, 878-886. 1923. 23 Cutler, D. W. Methods for the study of soil protozoa. Abderhald. Handb. biol. Arb. Method. Abt. XI, T. 3. 1926. 24 Further information on hay infusions is given by Woodruff, L. L. Observa- tions on the origin and sequence of the protozoan fauna of hay infusions. Jour. Exp. Zool., 12: 206-264. 1912; Fine, M. S. Chemical properties of hay infusions with special reference to the titratable acidity and its relation to protozoan sequence. Ibid., 12: 265-289. 1912. 26 Woodruff, L. L. Jour. Exp. Zool., 12: 205. 1912; 14: 575. 1913. 26 Martin, C. H., and Lewin, K. B. Some notes on soil protozoa. Phil. Trans. Roy. Soc. B, 205: 77-94. 1914; Jour. Agr. Sci., 7: 106-119. 1915. SOIL PROTOZOA 319 of 2 mm. favored the growth of protozoa. Meat extract agar may also be used. Bacterial growth takes place from those cells which are transferred with the protozoa. In some cases, special bacterial cultures are inoculated. To pre- vent the accumulation of injurious by-products, the protozoan culture must be frequently transferred. A rapid development of bacteria may lead to the de- struction of protozoa or may prevent their growth.27 Media containing 0.3 to 0.5 gram Liebig's beef extract, 0.3 to 0.5 gram NaCl and 20 grams of agar in 1000 cc. of water have commonly been employed. Beijerinck28 used yeasts for the cultivation of amoebae and ciliates. As a solid medium for the cultivation of amoebae, a mixture of 20 parts of agar, 100 parts of beef bouillon and 900 parts distilled water of a neutral or slightly alkaline reaction may be employed.29 The use of dead bacteria for the cultivation of amoebae has been sug- gested.30 Amoebae and ciliates were found31 capable of assimilating pure cultures of bacteria, utilizing dead cells as well as small particles of protein material. Gram negative bacteria were preferred to gram-posi- tive forms; young bacterial cultures in many cases were more assim- ilable than old cells. Successful cultures were obtained on bacteria smeared on the plate, then autoclaved at 130°, for 1 hour. Azo- tobacter was found32 to offer good food for amoebae, on a medium containing 10 grams of dextrin, 2 grams K2HP04, 0.2 gram MgS04, 0.2 gram CaC03, 10 grams agar in 1000 cc. water. Paramoecium was also grown in pure cultures of bacteria.33 Severtzoff34 cultivated an amoeba on a pure culture of Bad. coli; she found that the bacterium was destroyed by a small quantity of chlorine, while the protozoan cysts 27 Oehler, R. Wirkungen von Bakteriengiften auf Ciliaten. Centrbl. Bakt. I, Orig., 86: 494-500. 1921. 28 Beijerinck, M. W. Kulturversuche mit Amoben auf festem Substrat. Centrbl. Bakt. I, 19: 257-267. 1896; 21: 101-102. 1878. See also Mouton, 1902 (p. 316). 29 Wasielewski, T. V., and Kiihn. Untersuchungen iiber Bau und Teilung des Amobenkerns. Zool. Jahrb. Anat., 38: 253. 1914, also Heidelberg. Akad. Wiss Math. u. Naturw., 1: 1-31. 1913. 30 Tsujitani, J. Uber die Reinkultur der Amoebae. Centrbl. Bakt. I, 24: 666-670. 1898. 31 Oehler, R. Amobenzucht auf reinem Boden. Arch. Protistenk., 37: 175- 190. 1916; Flagellaten und Ziliatenzucht auf reinem Boden. Ibid., 40: 16. 1919; 41: 34. 1920; 49: 112-134. 1924; Gozony, L. Kultur von Flagellaten in festen Nahrboden. Centrbl. Bakt. I, Orig., 84: 565-566. 1920. 32 Welch, M. W. The growth of amoeba on a solid medium for class use. Trans. Amer. Micr. Soc, 36: 21-25. 1917. 33 Hargitt, F. T., and Fray, W. W. Paramoecium grown in pure cultures of bacteria. Anat. Rec, Philadelphia, 11: 516. 1916; Jour. Exp. Zool., 22: 421. 1917. 34 Severtzoff, 1924. 320 PRINCIPLES OF SOIL MICROBIOLOGY were left unaffected. As soon as the chlorine was removed, the amoebae excysted and began to feed rapidly on the dead bacterial cells ; when the food supply became exhausted, the protozoa encysted again.35 Robertson36 ascribed the autocatalytic phenomenon observed in the growth of protozoa to an accessory foodstuff, a soluble product of bac- terial metabolism. According to Cutler it seems very uncertain that autocatalysis occurs in protozoal growth, but, if the curve is autocatalytic in nature, this is due to an increased food supply (bacteria) and not to an accessory food as Robertson suggested. Certain species of bacteria, like Bad. fluorescens and Bad. coli, may produce substances injurious to the development of the protozoa. This injurious effect may be due to the consumption of oxygen by the bacteria, or to a change in reaction brought about by bacterial growth.37 Isolation of pure cultures of protozoa. By examining crude cultures of protozoa from time to time, it is found that there is usually in any given culture a more or less definite succession of animal forms. By selecting the time and method of culture, it is possible to isolate pure protozoan cultures. Several methods have been utilized38 for isolation of amoebae, the simplest of which consists in destroying the bacteria by means of heat or disinfectants (also 2 per cent HC1 over night or 20 per cent Na2C03 for 3 days) in encysted protozoan cultures; the more resistant cysts survive. The use of the Barber pipette or Chambers' apparatus has also been suggested. Oehler inoculated first a dish containing agar or gelatin medium with a pure culture of a certain bacterium, then a mixed protozoan culture was inoculated into the center of the dish. 35 Further information on the cultivation of amoebae is given by Schaeffer, A. A. Choice of food in amoeba. Jour. Anim. Behavior, Cambridge, 7: 220- 258. 1917; Arndt, A. Zur Technik der Amobenziichtung. Centrbl. Bakt. I, Orig., 88: 417. 1922. The influence of bacteria upon the growth of protozoa is discussed by Chatton, E., and Chatton, M. L'influence des facteurs bacteriens sur la nutrition, la multiplication et la sexualite des infusoires. Compt. Rend. Acad. Sci., 176: 1262-1265. 1923. The use of bacterial solutions for the cultiva- tion of protozoa was reviewed by Cunningham, A., and Lohnis, F. Studies on soil protozoa: I. The growth of protozoa on various media and the effect of heat on active and encysted forms. Centrbl. Bakt. II, 39: 596-610. 1914. Killer, J. Die Zahlung der Protozoen im Boden. Centrbl. Bakt. II, 37: 521- 534. 1913. A detailed review of the earlier literature on the cultivation of protozoa is given by Kopeloff, Lint and Coleman, 1917 (p. 328). 36 Robertson, T. B. The multiplication of isolated infusoria. Biochem. Jour., 15: 595-611, 612-619. 1921. 37 Oehler, 1921 (p. 319). 38 Gordon, C. E. A method for obtaining amoeba. Science, 46: 212. 1917. SOIL PROTOZOA 321 After three days, the protozoa reached the edge of the dish. The proc- ess was repeated until finally a culture of protozoa was obtained free from any bacteria except the species inoculated into the dish. A sample of the liquid containing the protozoa may be brought into a fine capillary tube, from which a single drop is ejected on a counting chamber (haemocytometer pattern) and immediately examined under the microscope. When a drop is obtained which contains a single organ- ism, a drop of sterile medium or a culture of the specific bacterium is placed upon a clean cover slip which is then put on the chamber so that both drops coalesce. The chamber is then placed in a Petri dish lined with moist filter paper, and incubated. By projecting the image of the drop upon a screen, the multiplication of the protozoan can be followed.39 It is sometimes essential to obtain cultures of protozoa free from living bacteria. This has been done by Peters,40 who isolated Colpidium colpoda from a hay infusion culture, in a drop of sterile medium, upon a sterile microscope slide. By the use of a capillary pipette made of drawn-out glass tubing and fitted with a rubber bulb the organism was transferred, through several changes of sterile medium, upon sterile microscope slides. After about six washings, the organism was transferred to a fresh drop of sterile medium, which was placed in a two inch depression block and covered with a small sterile cover-glass, both of which had been previously sterilized by heat. By a process of trial and error, organisms were finally obtained which divided well in the sterile medium. The culture was then transferred to a sterile tube plugged with cotton. Two media were used for the isolation and cultivation of the organism: per cent per cent I. NaCl 0.06 Glucose 0.03 KC1 0.0014 Histidine 0.01 CaCl2 0.0012 Arginine 0.01 Na2HP04 0 .0001 Ammonium lactate 0 .003 KH2P04 0.0001 FeCl3 Trace MgS04 0.001 KI Trace NaHC03 0.002 MnCl2 Trace Phenol red Trace Glass-distilled water 39 Cutler and Crump, 1923 (p. 318). 40 Peters, (p. 317). The results need confirmation and should not be con- sidered as conclusive evidence. 322 PRINCIPLES OF SOIL MICROBIOLOGY The constituents are sterilized separately and the final mixture sterilized at S0°C. on three consecutive days. The reaction of the medium is adjusted to pH 7.4. per cent per cent II. NaCl 0.06 MgS04 0.001 KC1 0.001 Ammonium glycerophos- CaCl2 0.002 phate 0.06 Phenol red Trace As a result of a series of studies, Peters demonstrated that glucose and ammonium lactate can serve as good sources of carbon and nitrogen for Colpidium. In addition to these, phosphates and chlorides, as well as potassium and magnesium are required. Amino acids can take the place of ammonium salts as sources of nitrogen. Carbon sources con- taining less than three carbon atoms in the molecule are not utilized; glycerate is used, but not lactate or citrate. Purdy and Butterfield41 found, however, by the use of pure, bacteria-free cultures of protozoa, that the latter cannot exist in culture solutions containing organic matter, but free from bacteria; these form the main food of the protozoa, which develop at the expense of the bacteria. The concentration of the medium, reaction and temperature are of great importance in the cultivation of protozoa. A ciliate and a bacte- rium were cultivated42 in a 0.1 per cent peptone solution, adjusted to pH 6.8, at 22° to 25°. At 24 hours, bacterial growth took place followed by that of protozoa. The protozoa could be separated from the bacteria electrolytically. Under the influence of the fall of potential, the protozoa travel to the cathode and the bacteria to the anode. This has to be repeated 6 times before cultures of protozoa free from bacteria are obtained. This procedure combined with the destruction of bacterial cells by heat were utilized by Oehler43 for the purification of amoebae and ciliates. By keeping dry cultures of protozoan cysts at 37° for 6 weeks, the bacterial vegetative cells were destroyed. Water and a culture of Saccharomyces were then added to the culture and the protozoa ex- cysted. The yeasts can be killed at 60° for 24 hours. By the use of a cataphoresis apparatus and unpolarized electrodes in 0.65 per cent NaCl solution, the ciliates were found to travel to the cathode. How- ever, the plate method was found to be best and the fact that protozoa 41 Purdy, W. C, and Butterfield, C. T. The effect of plankton animals upon bacterial death-rates. Amer. Jour. Public Health, 8: 499-505. 1918. 42 Amster. Ein neues Zi'ichtungsverfahren fur Protozoen. Centrbl. Bakt. I, Orig., 89: 166-168. 1922. "Oehler, 1924 (p. 319). SOIL PROTOZOA 323 travel faster than bacteria is utilized in the making of transfers. Bad. fluorescens was used to repress the accompanying bacteria. Staining of protozoal Whenever possible, protozoa should be ex- amined in a living condition, by suspending a drop of the medium or a suspension in water in a hanging drop. The motility of protozoa can be reduced as pointed out elsewhere (p. 45); various colloids (e.g. 5 per cent gelatin solution), narcotics (e. g. cocaine) or other poisons in subletal concentrations (e.g. tannic acid) can be used, varying with the species. Among the vital stains, 1 to 800-10,000 neutral red, 1 to 10,000 -100,000 Bismark brown, nigrosin, methylene blue, malachite green or 1 to 500,000 cyanin may be used. For the examination of finer structure, the protozoa are first fixed, while moist, then stained and finally dehydrated without allowing the preparation to dry. Mayer's albumin (50 cc. filtered egg-white + 50 cc. glycerol, + 1 gram sodium salicilate) may be first spread over the slide to prevent the washing away of the protozoa. A number of fixative agents have been suggested; of these a few may be mentioned: a. Sublimate fixative (Schaudinn's): 2 parts of a concentrated aqueous solu- tion of sublimate + 1 part of 96 per cent alcohol + 5 per cent acetic acid. Fix 10 to 30 minutes, wash in 60 per cent alcohol containing some iodine and potassium iodide, then in 70 per cent solution. b. Picric acetic acid: 95 cc. of concentrated aqueous solution of picric acid + 5 cc. of acetic acid. Fix for 10 minutes, then wash with 50 per cent alcohol, then in 70 per cent alcohol. c. Chromo-aceto-osmic acid (Flemming's): 15 cc. of 1 per cent chromic acid, 4 cc. of 2 per cent osmic acid, and 1 cc. of acetic acid. Fix for 30 minutes and wash with water. d. Picro-formol: 75 parts of an aqueous saturated picric acid solution, 25 parts of formaldehyde, 5 parts of acetic acid. For staining purposes a number of dyes have been recommended, a few of which are given here: a. Heidenhain's iron haemotoxylin. The preparation is first treated with 3 per cent solution of ferric alum (NH4)2Fe2(S04)4, for 30 minutes, then washed with water and placed in a 0.5 per cent aqueous solution of haemotoxylin for 1 to 14 hours, then washed with water and treated with 1.5 per cent solution of ferric alum. As soon as a satisfactory differentiation has been obtained, the prepara- tion is washed for 30 minutes in flowing water. 44 A detailed review of the fixing and staining or protozoa is given by Hargitt, Doflein and von Prowazek, while the staining of soil protozoa is given by Goodey. Hargitt, G. W. Methods of studying and mounting protozoa. Jour. Appl. Micros., 385-388. 1899; Goodey, T. A contribution to our knowledge of the protozoa of the soil. Proc. Roy. Soc. (London) B, 84: 165-180. 1911; 88: 437- 456. 1915; 89: 297-314. 1916. See also Bolles Lee. The microtomist's Vade mecum, and N. Hartmann. Praktikum der Protozoologie. Fischer. Jena. 324 PRINCIPLES OF SOIL MICROBIOLOGY b. Delafield's haemotoxylin: 4 grams of haemotoxylin crystals are dissolved in 20 cc. of 95 per cent alcohol and added to 400 cc. of a saturated solution of ammonia alum. The mixture is exposed for a few days, filtered, and 100 cc. of glycerol and 100 cc. of methyl alcohol are added. Solution is allowed to stand until dark, then filtered. The preparation is placed in a dilute solution of the stain in water for 30 minutes or more. When overstained, acid alcohol may be used to remove excess. c. Safranin light green. The preparation is placed for 1 to 24 hours in a con- centrated aqueous safranin solution, then washed in alcohol and placed in an alcoholic light green solution. Among the other stains, the Giemsa stain, Mann's methylene blue-eosin stain, gentian-violet and safranin may also be mentioned, while for ciliates simple staining in borax carmine is usually sufficient. After staining, the preparation is dehydrated by passing through increasing strengths of alcohol (after washing with water, a low concentration of alcohol is used at first) to 90 per cent, then in a mixture of 1 part of absolute alcohol + 1 part xylol, then in pure xylol. The preparation is then mounted in Canada balsam. Martin and Lewin46 examined the active soil fauna in fresh films by using picric alcohol (50 per cent saturated solution picric acid in water+50 per cent pure alcohol), or corrosive alcohol (50 per cent saturated solution of corrosive sublimate in water + 50 per cent pure alcohol). The soil is placed in a porce- lain dish and enough of the fixative is poured through a funnel to the bottom of the soil layer until the soil is just covered: the dish is then slightly shaken. A film is formed which contains protozoa in a fixed and stained condition. By floating coverslips on the surface of the liquid, the protozoa are removed and can be examined microscopically. Life history of protozoa. The life history of a protozoan in the soil consists of a period of activity, when the animal moves, feeds and repro- duces, and a period of rest, when a thick wall is secreted around the body and the cell (or cyst) becomes capable of resisting adverse conditions; the animal is distributed from place to place in the cyst state. When conditions become favorable, the wall is ruptured and the animal again becomes active. Sometimes, actual reproduction takes place within the cyst, as in Colpoda steinii. More seldom the cyst results from conjugation of two similar animals forming a large body known as the zygote.46 The physiology of encystment and excystation are obscure points concerning which a considerable literature is growing up. Earlier writers regarded cyst formation as a direct response to unfavor- able external conditions and excystation as occurring whenever a cyst found itself again in an environment suitable for active life. Experi- 45 Martin and Lewin, 1914-15 (p. 318). 46 Martin, C. H. A note on the protozoa from sick soils, with some account of the life h:story of a flagellate monad. Proc. Roy. Soc. B, 85: 393-400. 1912. SOIL PROTOZOA 325 mental work has however failed to support this attractive theory and it appears that internal causes may play at least as great a part as external factors.47 Cysts are more resistant than active protozoa to the action of enzymes; also to chemicals,48 the toxic order being CS2 > alcohol > acetone > benzol > ether > xylol > chloroform > carbon tetrachloride. The relative toxic effect of acids upon excystation is salicylic > butyric > oxalic > phosphoric > hydrochloric > sulfuric > acetic. Cysts and active organisms acclimatize to higher temperatures. The greater the degree of desiccation, the higher the resistance of cysts to higher tem- peratures and chemicals. The cyst of Colpoda was found49 to consist of a carbohydrate, which is digested by an enzyme secreted by the enclosed organism during the process of excystation. In other protozoa the structure of the cyst wall is different being siliceous in Monas and related flagellates. Excystation may take place by digestion of the wall or by its rupture or by the emergence of the organism through a preformed pore which is a very characteristic structure in some forms. Certain soil amoebae have also flagellate stages.50 Naegleria gruberi (No. 128, PL XVII) is a soil amoeba found in California soils to a depth of over 20 feet. This amoeba has a biflagellate phase, enflagellates and exflagellates rather quickly on slight provocation under the conditions of laboratory culture. It takes about 70 minutes for a culture of amoebae to change into the flagellate stage, while the reverse process is somewhat more prolonged and less uniform. The addition of water, of fresh culture medium (filtered and sterilized soil and manure infusions) or an excess of air tend to induce enflagellation, but exflagellation is less definite in response to opposite factors.51 Occurrence of trophic and encysted protozoa in the soil. The first detailed careful examination of the occurrence of active and inactive 47 Kofoid, C. A. The life cycle of the protozoa. Science, N. S., 57: 397- 408. 1923. 48 Bodine, J. H. Excystation of Colpoda cucullis. Some factors affecting excystation of C. cucullis from its resting cysts. Jour. Exp. Zool., 37: 115-125. 1923. 49 Goodey, T. The excystation of Colpoda cucullis from its resting cysts, and the nature and properties of the cyst membranes. Proc. Roy. Soc. B, 86: 427- 439. 1913. 60 Wilson, C. W. On the life-history of a soil amoeba. Univ. Cal. Publ. Zool., 16: 241-292. 1915. Kofoid, C. A. On the relative numbers of rhizopods and flagellates in the fauna of soils. Science, N. S., 42: 937-940. 1915. 61 A detailed discussion of natural history of protozoa is given by Dofflein, F. Studien zur Naturgeschirhte der Protozoen. Zool. Jahrb., 41: 1-112. 1919. 326 PRINCIPLES OF SOIL MICROBIOLOGY protozoa in the soil was carried out by Goodey52 who concluded that ciliates are present in the soil only in an encysted condition and can, therefore, not function as a factor limiting bacterial activity in the soil. Martin53 found that smaller amoebae and flagellates play the most im- portant part in the phenomena of sick soils, while the limiting factor as regards the activity of protozoa in the soil is the average quantity of water. Subsequent investigations54 demonstrated that a protozoan fauna normally occurs in the soil in a trophic state ; this trophic fauna is most readily demonstrated in moist soil well supplied with organic matter, like heavily manured soils, sewage soils and especially green- house "sick" soils; the forms predominating in the soil are not neces- sarily the same as those that develop on artificial media (hay infusions) inoculated with soil. A series of preparations of trophic amoebae, thecamoebae and flag- ellates were made54 by the methods described above. Flagellates were found55 to be the most numerous and the only active forms in moist soils; however, amoebae were not looked for. According to Crump and Cutler,56 flagellates, amoebae and thecamoebae are present in large numbers in a trophic condition in the soil; the fauna increasing with an increase in the content of organic matter in the soil. Various American investigators57 believed that the protozoa exist in the soil mainly in a non-trophic state. Koch concluded that protozoa do not exist in normal field soils or even in soils where the moisture con- tent is somewhat above normal; in greenhouse soils containing much organic matter and with a high moisture content, a few living protozoa were present. The protozoa were found to become active in the soil, whenever the moisture content rises considerably above the normal; both moisture and organic matter were found to be the principal limit- 52 Goodey, 1911-1915 (p. 323). 63 Martin, C. H. The presence of protozoa in soils. Nature (London). 1913, 111. " Martin and Lewin, 1914-1915 (p. 318); Goodey, 1916 (p. 323). 65 Waksman, S. A. Studies on soil protozoa. Soil Sci., 1, 1916, 135-152; 2: 363. 1916. 66 Crump, 1920 (p. 328); Cutler, D. W. Observations on soil protozoa. Jour. Agr. Sci., 9: 430-444. 1919. 67 Sherman, J. M. Studies on soil protozoa and their relation to the bacterial flora. Jour. Bact., 1: 35, 165. 1916; Koch, G. P. Soil protozoa. Jour. Agr. Res., 4: 511-559. 1915; 6: 477-4S8. 1915; Soil Sci., 2: 163. 1916; Fellers, C. R., and Allison, F. E. The protozoan fauna of the soils of New Jersey. Soil Sci., 9: 1-25. 1920. SOIL PROTOZOA 327 ing factors in the development of protozoa in the soil. According to Cutler,66 there is an intimate mechanical association between the proto- zoa and the soil particles which depends on a mutual surface action, and the capacity of various substances, such as sand, soil and clay, for re- taining these organisms is specific and constant. The difficulty of seeing living protozoa in the soil is, therefore, due to the fact that the organisms rigidly adhere to the soil particles, and, up to a certain limit, they can be completely removed from a suspension by shaking for a few minutes with soil. Only in exceptional cases can organisms be dislodged suffici- ently as to be recognized under the microscope. This is probably the reason why some investigators failed to observe living protozoa in the soil. It is beyond any doubt that protozoa lead a trophic existence in the soil; this is especially true of the small flagellates. Even if most of them encyst after a continuous dry period, the first rain that brings the moisture content of the soil to optimum will lead to a rapid excystation of the protozoa and to a longer or shorter period of activity. A method was described by which it is possible to estimate the number of protozoa present in the soil both in a trophic and in a cyst condition.58 The total number is first determined by the dilution method. A fresh portion of the soil is then treated with 2 per cent HC1 (specific gravity 1.15) over night, whereby all active forms are killed. A second count by the dilution method gives the number of protozoa present in the soil in the form of cysts. The difference between the first and second counts gives the number of active protozoa. In this connection reference may be made to the work of Cunningham and Lohnis,59 who found 60°C. to be the thermal death point of active protozoa (44° for flagellates, 48° for amoebae and 54° for ciliates), while 72°C. was found to kill the cysts. However, the temperature destructive to protozoa in the soil would be different from that of culture solutions. A temperature of 58°C. was used60 for distinguishing between cysts and active forms, bat it was soon found that many cysts are also killed at that temperature. Drying reduced the number of protozoa, particularly ciliates and flagellates, amoebae preferring a somewhat drier soil. The protozoa are widely distributed in the soil, comprising ciliates, flagellates, amoebae and thecamoebae. Some investigators reported 68 Cutler, D. W. A method for estimating the number of active protozoa in the soil. Jour. Agr. Sci., 10: 135-143. 1920. 69 Cunningham and Lohnis, 1914 (p. 320). 60 Cunningham, A. Studies on soil protozoa. Jour. Agr. Sci., 7: 49-74. 1915. 328 PRINCIPLES OF SOIL MICROBIOLOGY an abundance of ciliates and flagellates and few amoebae,61 others62 found amoebae and thecamoebae most prevalent. The discrepancy may be due to the difference in methods used, especially in view of the sensi- tiveness of the latter two groups to the composition of the medium. Water appears to be the most important controlling factor, favoring the development of the soil fauna.63 The largest numbers of protozoa are found in the soil in spring, after the thawing of snow or in summer, after heavy rainfall; only cysts are found in dry and semi moist soils.64 According to Crump, the protozoan fauna is largely confined to the top six inches of soil. The amoebae are influenced by variations in the water content and temperature of the soil and by the rainfall. The richer the soil is in organic matter the richer it is in protozoa, especially in amoebae and thecamoebae. A detailed study has been made of the presence of protozoa in peat soils,65 in Egyptian soils,66 in Italian soils,67 in German soils,68 in the soils of the United States,69 in Russian soils,70 and in English soils;71 an abundant fauna was found in various samples of moss and soil from Spitzbergen and in soils of various South Sea and Atlantic islands (Tristan da Cunha, Gough Islands, etc.).72 Most 61 Feuilletau and de Bruyn, W. K. H. t)ber die Verbreitung von Boden- protozoen in den Alpen. Centrbl. Bakt. II, 56: 12-13. 1922. 62 Crump, L. M. Numbers of protozoa in certain Rothamsted soils. Jour. Agr. Sci., 10: 182-198. 1920. 63 Coppa, A. Ricerche sui protozoi dei terreni delle acque ticinesit. Staz. sper. Agrar. ital., 54: 181-213. 1921. 64 Nowikoff, M. Die Bodenprotozoen und ihre Bedeutung fur die Bodenkultur. Heidelberg. 1923. C. Winter. 66 Scheffelt, E. Die Einzeller der siiddeutschen Moore. Mikrokosmos, 15: 113-118. 1922. 66 Ross, R., and Thomson, D. Egyptian sand amoebae. Proc. Soc. Med., Sect. Epidem., 9: 33. 1916. 67 Cauda, A., and Sangiorgi, G. Untersuchungen iiber die Mikrofauna der Boden aus Reisgegenden. Centrbl. Bakt. II, 42: 393-398. 1914. 08 Wolff, 1912 (p. 329) ; Oehler, 1916-1919 (p. 319). 69 Kopeloff, N., Lint, C, and Coleman, D. A. A review of investigations in soil protozoa and soil sterilization (complete bibliography up to 1917). Soil Sci., 3: 197-269. 1917. Fellers and Allison, 1918 (p. 326). 70 Yakimoff, M. L., and Zeren, S. Contribution a l'etude des protozoaires des sols de Russie. Centrbl. Bakt. II, 63: 33-57. 1924; Nowikoff, 1923. "Goodey, 1911-1915 (p. 323); Martin and Lewin, 1914-1915 (p. 318); Cutler and Crump, 1920 (p. 50); Sandon, 1927 (p. 329). 72 Sandon, H. Some protozoa from the soils and mosses of Spitzbergen. Linn. Soc. Jour., 35: 449-475. 1924; Sandon, H., and Cutler, D. W. Some pro- tozoa from the soils collected by the "Quest" Expedition. Linn. Soc. Jour., 36: 1-12. 1924. SOIL PROTOZOA 329 of the protozoa found in these far-away soils were identical with those found in soils in temperate climates such as English soils. A detailed study of numbers of protozoa in the soil is given elsewhere (p. 45). Classification and occurrence of protozoa in the soil. The majority of soil protozoa are cosmopolitan, since the species found throughout the world are, with some exceptions, identical, although not all the species are found in every soil. Protozoa were demonstrated in large numbers in the soil by Ehrenberg,73Greef,74 and Rosenberg-Lip insky,75 who considered them to be of importance in soil fertility. In the more recent contribu- tions to the subject, a study has been made of the occurrence of protozoa in the soil and of the influence of environmental conditions upon their distribution.76-81 Fellers and Allison78 found 17 species of rhizopods, thirty-four flagel- lates, and fifty-one ciliates in New Jersey soils, fertile soils containing more species and greater numbers of protozoa than unfertile soils. They concluded that the soil micro fauna consists principally of small, hardy protozoa able to withstand, by means of encystation or otherwise, such extremes of heat and cold, desiccation, aeration, etc., as are natural to their life in the soil. Practically all species identified from the soil have also been found in fresh water lakes, ponds, pools and streams of New Jersey, but not in the same relative abundance, while several of the most common plankton organisms are rarely found in the soil. Cutler and associates79 found six species of protozoa occurring con- 73 Ehrenberg, C. G. Die fossilen Infusorien und lebendige Dammerde. 1837. Berlin; Die Infusoriensthierchen als volkommene Organismen. 1839. 74 Greef, R. t)ber einige in der Erde lebende Amoben und andere Rhizopoden. Arch, micros. Anat., 2: 299-311. 1866. 76 Rosenberg-Lipinsky, Alb. v. Der praktische Ackerbau (etc.), 2: 27. 1869, Breslau. 76 Wolff, Max. Der Einfluss der Bewasserung auf die Fauna der Ackerkrume mit besonderer Berucksichtigung der Bodenprotozoen. Mitt. Kaiser Wilhelm Inst. Landw. Bromberg., I: 382-401. 1909; Centrbl. Bakt. II, 33: 314-320. 1912. 77 Martin and Lewin, 1914-1915 (p. 318). 78 Fellers and Allison, 1920 (p. 326). 79 Cutler, Crump and Sandon, 1922 (p. 32). 80 Fantham, H. B., and Taylor, E. Some protozoa found in certain South African soils. So. African Jour. Sci., 18: 373-393. 1922; 19: 340-371. 1922; 20: 437-492. 1923; 21: 445-479. 1924. 81 Sandon, 1924 (p. 328); Sandon and Cutler, 1924 (p. 328); Sandon, H. The composition and distribution of the protozoan fauna of the soil. Oliver and Boyd. Edinburgh and London. 1927. 330 PRINCIPLES OF SOIL MICROBIOLOGY stantly in the soil in sufficient numbers to admit the application of statis- tical methods to the results. These are: (1) Dimastigamoeba gruberi, (2) a small limax amoeba, (3) Heteromita sp. resembling Bodo repens, (4) a small soil flagellate, 3 to 6 by 2 to 3^; (5) Cercomonas sp. and (6) Oicomonas termo. Sandon found the following average number of species of protozoa in 107 soils examined: 7.2 flagellates, 3.4 ciliates, 2.45 amoebae and 2.0 testaceous rhizopods. While some species de- velop in all media employed, other forms develop only upon certain specific media. In all Sandon81 recorded 250 species of protozoa, a small number of which were found in every soil, often in very large numbers. The flagellates Heteromita globosus, Oicomonas termo and Cercomonas sp., the ciliates Colpoda cucullus and C. steinii, and the limax amoebae Ndegleria gruberi and Hartmanella hyalina were most common and most abundant. While most protozoa found in the soil are also found in various other habitats, such as standing and flowing fresh waters, sea water, plankton, etc., a few are found only in the soil. The genera and species of protozoa vary with locality and degree of soil cultivation. Fantham and Taylor80 found 1 to 22 species in each of a series of South African soils ; the flagellates were largest in total num- bers, while the ciliates showed the largest number of species; the amount of organic matter being the limiting factor; dark, heavy, humus rich soils contained more protozoa than sandy soils; the reaction of the soil was not found to have any effect upon the protozoan fauna. Sandon has shown that the extreme climate of arctic land is no;t in itself an obstacle to the abundant development of protozoa, provided the soil is well manured and in good condition; however, peat soils are decidedly unfavorable for the development of protozoa except for the testaceous forms. A close positive connection has also been observed78,81 between the numbers of protozoan species and bacteria in the soil. The protozoa reported to have been found in the soil can be classified as follows:82 82 The following letters can be used to designate the names of the investi- gators, who have demonstrated the presence of the specific protozoa in the soil: A — Wolff in Germany, B — France in Germany, C — Goodey in England, D — Martin or Martin and Lewin in England, E — Waksman in New Jersey, U. S. A., F — Fellers and Allison in New Jersey, U. S. A., G — Cutler, Sandon and Crump in England, H — Cutler and Sandon in Spitzbergen soils, etc., I — Fantham and associates in South African soils, J — Sandon working with soils collected through- out the world, K — Yakimoff and Zcren in Russia, L — Perey87 in France, M — Allison88 in England using American soils, N — Nowikoff in Russia. SOIL PROTOZOA 331 A. SARCODINA*3 I. Actinopoda, pseudopodia with axial filaments. Heliozoa, spherical, with fine radiating pseudopodia, with stiff axial rods; endoplasm surrounded by vacuolated ectoplasm. The fol- lowing representatives of this group have been found in the soil: Actinophrys sol (F, I, K, J), Raphidiophrys (F), Acanthocystis aculeata (I), Clathrulina elegans (F). II. Rhizopoda, pseudopodia without axial filaments. 1. Proteomyxa, pseudopodia fine and radiating, often anas- tomising or forming a net-work. Among the soil forms be- longing to this group are Biomyxa vagans (J), Nuclearia simplex (A, I, J, L, M), Gephyramoeba delicatula (C and J), Leplomyxa reticulata and L. flabellala(c), Vampyrella lat- erita (F). 2. Amoeboea, naked or with chitinous shells (tests). (a) Amoebida, naked rhizopoda, without any shells or sup- porting structures; pseudopodia blunt or pointed but never filamentous; may be reduced to wave-like ex- pansions of protoplasm. (a') Amoeba Umax group. Small amoebae with single rounded pseudopodium; sometimes several finger-like pseudopodia formed simultaneously. Naegleria gruberi, one of most common soil pro- tozoa recorded by Wilson, F and G. Hart- manella hyalina is also very abundant in the soil. Amoeba (Hyalodiscus) gutulla was found by A, B, F, I, J and K. (b') A. verrucosa group. Pseudopodia in the form of ridges or folds of the ectoplasmal pellicle. A. verrucosa (A. terricola?) was found in the soil by A, B, C, I, J, K and N. A. diploidea by D and J, A. striata by B and J. (c') A. lamellipodia group, similar to previous group, with less strongly developed pellicle, including: A. glebae (J), A. actinophora (J), A. goban- niensis (D). (d') A. proleus group. Large amoebae, changeable in shape, with numerous long, cylindrical pseudo- 83 For the identification of this group, consult, in addition to the general treatises and papers, also Edmondson, C. H. Amoeboid protozoa (Sarcodinia), in Ward and Whipple's Fresh-water biology. 1918, p. 210; Leidy, J. Fresh water rhizopods of North America, U. S. Geol. Surg., 12: 324. 1879; Cash, J., and Wailes, C. H. The British freshwater rhizopoda and heliozoa. Vols. 1-5, 1905-1921. Roy. Soc. London; Penard, E. La faune rhizopodique du bassin de Leman. Geneve. 1902; Poche, F. Arch. Protistenk., 30: 251-310. 1913; Ghosh, E. A new general classification of protozoa. Jour. Roy. Micr. Soc, 272: 327-329. 1925; Sandon, 1927 (p. 329). 332 PRINCIPLES OF SOIL MICROBIOLOGY podia. Representatives of this group were found in a number of soils by B, I and J. (e') Various other amoebae have been found in the soil by different investigators, such as A. radiosa (F, J, K) and A. albida (J), (b) Testacea, rhizopods with shells (tests), into which the whole body can be withdrawn. (a') Arcellidae, shells chitinous, pseudopodia lobose or simply branched. Arcclla vulgaris was found in the soil by A, B, C, F, I, J and N. A. dis- coides by A and J, Pseudochlamys patella by B, Corycia flava by B, Diflugiella by B and J, Hyalosphenia elegans, H. cuneata, H. pupilis and H. tincta by B, H. minuia by J. (b') Difflugidac, chitinous shells covered by foreign bodies. Representatives of this group found in the soil are Difflugia pyriformis (B, F, I, J), D. globulus (B, C, F, I, J), D. pcnardi (D. fallax) (J), D. lucida and D. craterella (B), D. urceolata (J), D. lobostoma and D. arcula (B and J), D. constricta (B and J), Centro- pyxis aculeata and C. laevigata (J), Phryganella acropodia and P. nidulus (B), Heleopera petri- cola, H. picta, H. rosea and 11. sylvalica (B). (c') Euglyphidae, chitinous shells with plates made by organism, including Euglypha tuberculata (J), E. mucronata (B), E. bryophila (J), E. strigosa (B and J), E. rotunda (J), E. laevus, E. ciliala (B and J), Placocysta spinosa (B), Nebela collaris and N. lageniformis (B and J), Quadrula symmetrica and Q. globulosa (B), Q. irregularis (J), Assulina muscorum (B and J), A. seminulum (B), Sphenoderia lenta (B), S. fissirostris (J), S. dentata (B and J), Campascus sp. (B), Tri- nema enchelys (A, B, C, J), T. lineare and T. complanatum (B and J), Corythion dubium (B and J). (d') Gromiidae, membranous shells, pseudopodia- reticulate, forming a network, including Lecy- thium (Pamphagus) hyalinum (Syn. Chlamydo- phrys stercorea) found by A, B, C, D, F, J, K and L, L. mutabile by B and F, Pseudodifflugia gracilis by B, Allogromia Uuvialis (Gromia terricola) by Midler,84 A and J, Microgromia socialis by F, J and K, Diplophrys archeri by F. 84 Muller, P. E. Studien liber die naturlichen Humusformen. Berlin. 1887. SOIL PROTOZOA 333 B. MASTIGOPHORA.™ The soil flagellates are found largely in the follow- ing groups. I. Pantostomatinae. Flagellates naked, colorless; food ingested, usually by means of pseudopodia, at all points of their surface; the organisms in this group possess one or more flagella and are usually more or less amoeboid. Actinomonas mirabilis was found by J in 11 soils collected from various parts of the world. Cercomonas crassicauda and C. longi- cauda are very common soil protozoa (F, G, J, K). Cercobodo was found by J to be represented in the soil by several species. Mastigamoeba and Mastigella, comprising organisms which closely resemble amoebae but possess a single flagellum, directed for- ward; the flagellum is connected with the nucleus in the case of the former, but not of the latter. Mastigamoebae have been found in the soil by F, I, J, L and N. II. Protomastiginae. Small flagellates, usually more or less amoeboid and having a fine periplast. Food taken in at one point, no chromatophores. Pseudopodia when present never acting as organs of locomotion. These include Codonosiga botrytis (C), Monosiga ovata (F and J), Salpingoeca convallaria (A), Phalan- sterium solilarium (J in 56 out of 148 soils examined), Bodo (Prowa- zekia) caudatus (A, D, J), B. edax (Kiihn, 86 G, J, K, N), B. saltans (A, C, F, J, N), Bodo terricola (D and others). A few other species of Bodo were reported in the soil by F, J and K. Col- ponema symmetrica (J), Dinomonas vorax (D), Heteromita com- pressus (J), H. globosa and H. lens are among the most common soil protozoa: these and H. obovata, H. ovata, H. repens were found in the different soils by H and J ; some species of this genus were also recorded by A, E, F. Phyllomitus undulans by A, E and J, P. amylophagus found by F, G and J, Pleuromonas jacu- lans by A, E, F and I. Sainouron mikroteron was found to be common in Rothamsted soils by J and also in 45 other soils, found also by L and M. Allantoin tachyploon was found in the soil by J and M. Phyllomonas contorta by A. Proleptomonas 85 Klebs, G. Flagellatenstudien. Ztschr. wissensch. Zool., 55, 1893; Lemmer- mann's Algen I. Flagellaten. In Kryptogamenflora der Mark Brandenberg und angrenz. Gebiete. v. 3; Pascher, A., and Lemmermann, E. Flagellatae in "Die Siisswasserflora und Fauna Deutschlands." H. 12, Jena. 1913-1914; Senn, G. Flagellata, in Engler and Prantl's "Die natiirlichen Pflanzen- familien." Bd. I, T. I, 1900; Conn, H. W., and Edmondson, C. H. Flagellate and ciliate protozoa, in Ward and Whipple's Fresh-water biology. 1918, p. 238. Keys for identification of soil flagellates. Sandon, 1927 (p. 329). 86 Kiihn, A. t)ber Bau, Teilung und Encystierung von Bodo edax Klebs. Arch. Prot., 35: 212-254. 1915. 87 Perey, J. F. Les protozoaires du sol. Ann. Sci. Agron., 39: 333-352. 1923. 88 Allison, R. V. A note on the protozoan fauna of the soils of the United States. Soil Sci., 18: 339-352. 1924. 334 PRINCIPLES OF SOIL MICROBIOLOGY faecicola is the only member of the Trypanosomaceae found by J as free-living in the soil. Spiromonas angusta was found by A, E, G, J and Cunningham and Lohnis. Spongomonas is com- mon in the soil (J), while Cladomonas was found in a Spitzbergen soil by J. Tetramilus roslratus was found by J and M, T. spiralis by C, J, L and M, T. pyriformis by J. F and K also recorded the presence of species of Tetramitus in the soil. Hexamitus inflatus was found by F, Spironema multiciliatum by C and J. III. Chrysomonadinae. Small flagellates; when not possessing chroma- tophores, resemble the Protomastiginae. Cuticle generally present, but is thin and does not prevent them from becoming amoeboid; 1 or 2 flagella. Cysts endogenous, wall being more or less impregnated with silica. This group includes the following forms found in the soil: Oicomonas termo (D, F, G, J, K), 0. granulata (K), Chrysamoeba radians (I), Mallomonas (E), Monas guttula (A, E, F, J, K, N, Koch, Cunningham and Lohnis), M. vivipara (E, F, N), C ephalothamnion cyclopum (J), Physomonas elongata (F), Polypseudopodius bacterioides (D). IV. Cryptomonadinae. Small forms, with two flagella, usually equal, arising behind the anterior end in a hollow which is usually the opening of a funnel running deep into the interior of the cell. Egg-shaped and more or less flattened; body enclosed in mem- brane and not amoeboid. One or two simple contractile vacuoles at anterior end. These include Chilomonas paramoecium (A, F, I, J, K), Cryptomonas (F, J, K), Cyathomonas truncata (Cun- ningham and Lohnis), Rhodomonas (I). V. Euglinidae, characterized by a complicated vacuole system situated at anterior end and consisting of one or more accessory vacuoles which, in contracting, empty their contents into a large main vacuole or reservoir, which communicates with the cytopharynx. Mostly with green chromatophores, enclosed in a membrane and with 1 or 2 flagella. Euglcna acus was found in the soil by F and K, E. deses, E. oxyuris and E. spirogyra by I, E. vclata by B, E. viridis by A, E, F, I, N, Eutreptia viridis by F, Phacus longicauda by F and I, Ph. pyrum by F and K, Trachelomonas volvocina and Cryptoglcna pigra by F. Species of Astasia were found in the soil by D, F, N. Distigma (Astasia) proteus by B andK. Clos- tenema (S phenomonas) socialis by F, Menoidium by J, Petalomonas angusta by J, P. mediocanellata by B and F, P. pleurosigma by I, Scytomonas pusilla by D, L and J, Peranema trichorophorum by B, F, I, K, Urceolus cyclostomus by K, Anisonema minus by J, Enlosiphon sulcatum by C, I and J, Hcleronema acus by FandN. VI. Phytomonadinae. Solitary or in colonies, enclosed in a cellulose wall; chlorophyll and stigma nearly always present; 1 or more simple contractile vacuoles at anterior end. Chlamydomonas sp. was found commonly in the soil by A, E, F, K, Polytoma uvella by A, K and J, Chlorogonium eucMorum by J and K, Pandorina morum by A. SOIL PROTOZOA 335 VII. Dinoflagellata are enclosed in a rigid lorica and possess 2 flagella, one of which lies in a transverse groove and moves with an un- dulating motion and the other lies in a longitudinal groove and is trailed behind. Only one form, Glenodinium pulvisculus, has been found in the soil by I. The last 5 orders are among the Phytoflagellata, the typical mem- bers of which possess chromatophores ; colorless species are also found in all orders and it is these which are largely found in the soil. C. INFUSORIA {CI LI AT A). ^ The common soil ciliates are found in the following groups: I. Holotricha, body uniformly covered with cilia; these are similar or slightly lengthened about the mouth; no adoral spiral zone. Various species of Holophrija were found in a number of soils by A, B, F, J, K. Urotricha farcla by N, U. agile by F; Enchelys is common in the soil, having been found by A, C, E, C, J, K, Koch, Cunningham and Lehnis. Spathidium spatula by C and K, Lacrymaria sp. by I and N, Prorodon teres by A, F and K, P. ovum by A, E, I and Koch, Choenia sp. by J, Coleps hirtus by I and K, Mesodinium sp. by F, Amphileptus cygnus and A. gigas by I, Lionotus fascicola by F and I, Loxophyllum flexilis and L. rostratum by I, Dileptus by F, J and K, Nassula elegans by A, E and I, Chilodon cucullulus by F and I, C. megalotrocha by F, C. uncinatas by J and K and Trochilia palustris by A and J. Uronema marina was found in the soil by A, F, J and K. Glaucoma scintillans, G. pyriformis by A, Colpidium colpoda by A, E and K and others, this being one of the most common soil ciliates. C. striatum was found by F and I. Colpoda cucullus and C. steinii, two of the most common soil ciliates, were recorded by most in- vestigators on soil protozoa. C. maupasii was found in the soil by F and J. F also recorded the presence in the soil of C. campyla, C. flavicans, C. helia and C. sapi ophila. Various species of Para- moecium have been found in the soil, although Sandon records the complete absence of this group in English and foreign soils. P. aurelia was found by I and N, P. bursaria by I, P. caudatum by F, I and N, P. putrinum by A and I, P. trichium by F, etc., Pleuronema chrysalis by A, F and I. Other species of Pleuronema were found by E, J and K. Cyclidium glaucoma was found by F, J and K, Balantiophorus elongatus by C, J and others, B. minutus by A, C and J, Lembus pusillus by F. II. Heterotricha. Body uniformly covered with cilia, forming cirri or stout cilia in spiral adoral zone; undulating membrane often 89 Conn and Edmondson, in Ward and Whipple's "Fresh-water biology"; Stokes, A. C. A preliminary contribution toward a history of the fresh water infusoria of the United States. Jour. Trenton Nat. Hist. Soc, 1: 71-344. 1888; Roux, J. Faune infusorienne des l'eaux stagnantes des environs de Geneve. Geneve, 1901. 336 PRINCIPLES OF SOIL MICROBIOLOGY inside mouth. The following organisms belonging to this group were found in the soil: Blepharisma ovata by F, JB. laterita by J, Metopus sigmoides and Metapides acuminata by F, Spirostomum ambiguum by I, Condylostoma sp. by Koch. III. Oligotricha. Spherical or conical, with adoral zone often form- ing a closed ring; cilia usually absent from other parts of body. The following forms were recorded as present in the soil: Strombidium sp. by E, F and I, Halteria grandinella by A, F, I and K. Other species of Halteria were found by B, E, J and N. IV. Hypotricha. Body flattened dorso-ventrically; cilia often fused to form larger appendages or cirri, usually limited to ventral surface; adoral zone of membranellae. This group is represented in the soil by Urostyla grandis (C, K), Stichotricha secunda (F), Uroleptus musculus (A, F, K and Koch), U. mobilis (J), U. piscis (I, J), U. dispar (F, I, N), Onychodromus grandis (J and other investigators), Gastrostyla steinii (C, J), Gonostomum (Plagio- tricha) affine (C, J, K, L, M), Oxytricha fallax (J), 0. bifaria (F), 0. pellionella (Cunningham and Lohnis, F, I, J); other species of Oxytricha were also found by different investigators of soil protozoa. Pleurotricha lanceolata and P. grandis (C, J), Sty- lonychia mytilus (B, F, I), S. pustulata (F, K), Euplotes carinata (F, J), E. charon (A, F, J), E. harpa and E. patella (I), Aspidisca coslata (F, I, K) and A. lyncaster (K). V. Peritricha. Body cup-like or cylindrical, often stalked, of a sedentary habit; cilia usually limited to adoral zone, the mem- branellae leading down to a vestibule, into which pharynx and contractile vacuoles open; a posterior ring of cilia may be tem- porarily present. This group is represented in the soil by Vorti- cella microstoma (C, F, J, K, N), V. citrina and V. globularia (F), V. nebulijera (K, N), V. putrina (A, F); other species of Vorticella have also been found by different other investigators. Epislylis coarctata (C), Cothurnia doliolum (B), Vaginicola ter- ricola (B). Importance of protozoa in the soil. No definite evidence has as yet been submitted as to the actual role of protozoa in the soil. We know, on the one hand, that certain groups of protozoa at least, particularly the ciliates and amoebae, are not only capable of ingesting bacteria, but some actually use this sort of food exclusively.9091 By determining the number of amoebae and bacteria in the soil, at daily intervals, an inverse relationship has been obtained between these two groups of organisms. 90 Huntemtiller, O. Vernichtung von Bakterien im Wasser durch Protozoen. Arch. Hyg., 54: 89-100. 1905; Calkins, 1926 (p. 312); Purdy and Butterfield, 1918 (p. 322) ; Cunningham, A. Studies on soil protozoa. Tour. Agr. Sci., 7: 49- 74. 1915. 91 Cutler and Crump, 1920 (p. 50). SOIL PROTOZOA 337 Cutler92 purified cultures of an amoeba and a flagellate, so that they contained only three species of common soil bacteria. These were isolated free from protozoa, and suspensions were prepared of the bac- teria alone and bacteria + protozoa, the latter in an encysted condition. These suspensions were sprayed on by a fine nozzle upon 100 grams of sterile soil in large sterile Petri dishes. The protozoa added per 1 gram of soil were: 25,000 for the Dimastigamoeba gruberi, 20,000 for Cercomonas crassicauda and 1 1 to 13 millions of bacteria. At the end of 15 days, the numbers of protozoa were: 230,000 amoebae and 420,000 flagellates per gram. The bacteria, in the protozoa free culture, reached a maximum of 214.4 millions in 6 days, then diminished to 169.2 mil- lions in 15 days (21 per cent decrease); in the presence of the amoebae, the maximum of bacteria (178.4 millions) was attained in 3 to 5 days, then the decrease was more rapid, falling down to 72.8 millions in 15 days (59 per cent decrease) ; in the presence of flagellates the maximum of 103 millions was reached in 7 days, dropping to 88 millions in 15 days (14.5 per cent decrease). Goodey93 has previously shown that amoebae of the limax group and other larger forms can lead an active existence in the soil and exert a depressing effect upon bacterial numbers. He suggested the probabil- ity that a certain point must be reached in protozoan development be- fore the depression in bacterial numbers is caused; this number seems to be about 30,000 cells of Amoeba Umax per gram of soil. On the other hand, certain suggestions have been made that protozoa can live in the absence of bacteria. Breal94 (1896) believed that Colpoda is active in the decomposition of plant constituents of the soil with the production of ammonia. Other investigators95,96 claimed that the pro- tozoa play an important role in the decomposition of organic matter in the soil. A number of protozoa are found97 to be saprophytic in nature and capable of obtaining their food by absorption. The same may be true of the soil flagellates, as evidenced by the work of Thornton and 92 Cutler, D. W. The action of protozoa on bacteria when inoculated into sterile soil. Ann. Appl. Biol., 10: 137-141. 1923. 93 Goodey, T. Further observations on protozoa in relation to soil bacteria. Proc. Roy. Soc, 89: 297-314. 1916. 94 Breal, E. Ann. Agron. 22: 362-375. 1896. 96 Muller, 1887 (p. 332), p. 15, 56, 167. 96 Hiltner, L. Tiber neuere Ergebnisse und Probleme auf dem Gebiete der landwirtschaftlichen Bakteriologie. Jahresb. Ver. angew. Bot. for 1907, 5: 200-222. 1908. 97 Minchin, 1912 (p. 312). 338 PRINCIPLES OF SOIL MICROBIOLOGY Smith and Alekseiev, and even ciliates, as shown by Peters. In most instances, of course, no direct evidence has been submitted to show that the protozoa took an active part in the decomposition of the organic matter; the statements were often based upon the fact that protozoa were found to occur in soils in which decomposition was taking place. Goodey has also shown that when various protozoa are added to the soil, bacterial activity has not been limited, as seen in table 18. This seems to be contrary to the latter results of Goodey98 mentioned previously; however, they are explained by the fact that soil treated with an anti- septic does not afford a suitable medium for the development of pro- tozoa. The drop in the numbers of bacteria follows the exhaustion of available plant food in the soil. TABLE 18 Bacteria in millions per gram INCUBATION, DATS Start 32 60 93 151 208 487 Untreated 14.4 9.2 11.3 4.5 3.0 27.0 2.3 10.3 73.0 49.0 371.0 285.0 247.0 500.0 13 60 61 292 185 214 341 11.4 61.0 43.0 296.0 141.0 227.0 311.0 12 40 19 56 74 196 181 8 49 45 64 90 104 151 12 Toluene treated 56 Toluene + untreated soil Toluene + ciliates 48 73 Toluene -f- amoebae 57 Toluene + flagellates 113 Toluene + bacteria 150 Few attempts were made to demonstrate whether protozoa actually injure important soil biological processes. Nasir" determined the in- fluence of the presence of protozoa (Colpidium colpoda) upon the fixation of nitrogen by Azotobacter in mannite cultures, both in solution and in sand. In 31 experiments out of 36, the presence of protozoa resulted in an increase in the amount of nitrogen fixed by Azotobacter. The feeding action of protozoa upon Azotobacter seems to stimulate the further development of the latter and thus maintain its nitrogen-fixing efficiency for a longer period."3 Very little soluble nitrogen was 98 Goodey, 1915 (p. 323). 99 Nasir, S. M. Some preliminary investigations on the relationship of pro- tozoa to soil fertility with special reference to nitrogen fixation. Ann. Appl. Biol., 10: 122-123. 1923. 99a Cutler, D. W., and Bal, D. V. Influence of protozoa on the process of nitrogen fixation by Azotobacter chroococcum. Ann. Appl. Biol. 13: 516-534. 1926. SOIL PROTOZOA 339 found100 in pure cultures of Azotobacter; this nitrogen would be pro- duced as a result of autolysis. However, in impure cultures of Azoto- bacter, considerable quantities of soluble nitrogen were found; this phe- nomenon was ascribed to the action of amoebae. While the results of Cutler and associates seem to demonstrate defi- nitely that protozoa are capable of reducing the number of bacteria in the soil, due to their phagocytic action (as determined by the plate method), there is very little information available concerning their influence upon the activities of microorganisms in the soil and upon soil processes in general. The activities of bacteria decomposing or- ganic nitrogenous compounds may not be influenced injuriously by the presence of protozoa and may even be favored, as indicated by an in- crease in the amount of ammonia liberated (Hill, Waksman) or nitro- gen fixed by Azotobacter (Nasir). Excessive development of bacteria may become harmful to the growth of protozoa in artificial culture media, although it remains to be seen to what extent this may take place in the soil. The fact that the protozoa destroy some soil bacteria need not indi- cate that they exert an injurious influence, but may result in a decided benefit to soil biological processes. Decomposition of organic matter as well as other biological activities are resultants of the multiplication and growth of the bacterial cells. By destroying the excess of bacteria, the protozoa may stimulate further bacterial development and, there- fore, further biological transformations in the soil. The protozoa themselves may become later a source of energy for bacteria. The phenomena observed by Russell and Hutchinson as a result of partial sterilization of soil may not be due at all to the destruction of protozoa, but to other factors, as shown elsewhere (p. 757). The protozoa may also take a part in some definite soil processes, such as the decomposition of certain organic substances. Cleveland102 found that protozoa living in the intestinal tract of termites feed on wood cellulose. When the protozoa are killed, the termites die on a wood 100 Moler, T. Ein Beitrag zur Kenntnis der Entbindung des durch Azotobacter fixierten Stickstoffes. Bot. Notiser. 1915, 163-175. (Centrbl. Bact. II, 47: 635-636. 1917.) 101 Hill, T. L. The relation of protozoa to certain groups of soil bacteria. Jour. Bact., 1: 423-433. 1916. 102 Cleveland, L. R. The physiological and symbiotic relationships between the intestinal protozoa of termites and their host, with special reference to Reticulitermes flavipes Kollar. Biol. Bull., 46: 177-225. 1924. 340 PRINCIPLES OF SOIL MICROBIOLOGY diet, since they themselves cannot utilize the cellulose, unless it has been previously decomposed by fungi. This symbiotic relationship exists for at least some protozoa. We do not know as yet whether some soil protozoa may be able to decompose complex organic substances; it is known, however, that they can readily assimilate such soluble organic and inorganic constituents as found in manure.103 103 Alexeiev, 1917 (p. 317). The relation of protozoa to reducion phenomena in the soil is discussed by von Wolzogen Kuhr, C. A. H. Protozoa and the phe- nomena of reduction in soil. Arch, voor de Suikerind. nederland. Indie., No. 27, 1125-1182. 1917. (Int. Rev. Sci. Pract. Agr., Agr. Int. and PI. Dis., 9: 788. 1918.) CHAPTER XIV The Non-Protozoan Fauna of the Soil Animal ecology as a whole and classification of soil forms. In addi- tion to protozoa, other groups of invertebrate animals inhabit the soil, namely, the rotifers, nematodes, annelid-worms, insects and others. Such animals which live in the soil can be generally divided into three groups : 1. Those that spend all their life in the soil, coming to the surface only oc- casionally or not at all. These include various worms and rotifers. 2. Those that spend only a part of their life cycle in the soil or on its surface, as in the case of various insects. 3. Those that find only their habitat in the soil, while they may spend a large part of their time on the surface of the soil. These include ants, termites and many insects. These organisms influence directly or indirectly various soil processes and plant growth: 1. They cause a change in the physical condition of the soil, by modifying the mechanical structure of the soil, through their continued motion or by pass- ing the soil through their bodies as in the case of earth worms. 2. They cause various chemical changes in the soil, either directly, in their digestive processes, or indirectly, by influencing the activities of the soil fungi and bacteria. 3. They bring about a more uniform distribution of various soil bacteria and other organisms. 4. They may devour other members of the soil flora and fauna, like algae, fungi and protozoa. In this way, the higher fauna also contributes to the complex system of numerous activities going on in the soil. 5. Damage may be done to crops by certain representatives of these groups, particularly by some of the nematodes, earthworms, insects, etc. The soil or terrestrial fauna, outside of the protozoa, includes mem- bers from the following systematic groups: I. Plathelminthes or Flatworms, represented in the soil, in moist environ- ments, by the (1) Turbellaria or flatworms and (2) Trematoda or flukes. II. Nemathelminthes or Roundworms, represented in the soil by the Nema- toda or true roundworms. 341 342 PRINCIPLES OF SOIL MICROBIOLOGY III. Trochelminthes or Trochalworms, represented in the soil by the Rotatoria or wheel animalcules. IV. Coelhelminthes (Annelida) or Segmented Worms are represented in the soil by the Oligochaeta, including the earthworms or Terricolae and the Enchytraeids or Limicolae, and the Tardigrada. V. Arthropoda are represented in the soil by (1) Crustaceae, especially Copepoda and Isopoda; (2) Arachnida, including the mites, ticks and spiders; (3) Myriapoda;. and (4) Insecta. VI. Mollusca, including the Gastropoda. VII. Chordata. The vertebrates are represented in the soil by the mice, moles, marmots, etc., but these are beyond our field of discussion. Methods of study. For the investigation of the soil fauna, Morris1 devised an apparatus, which consists of four iron plates, two 12 by 10 inches, one 12 by 9 and one 4 by 9 inches. Each plate has an iron bar 1 Morris, H. Observations on the insect fauna of permanent pasture in Cheshire. Ann. App. Biol., 7: 141-155. 1921; On a method of separating in- sects and other arthropods from soil. Bull. Entom. Res., 13: 197. 1922; The insect and other invertebrate fauna of arable land at Rothamsted. Ann. App. Biol., 9: 282-305. 1922. PLATE XVIII Soil Nematodes 144. The relative abundance of nematodes in each successive two inches of upper foot of soil; derived from a low-lying alluvial soil containing about 3,000,000,000 nematodes to the acre, most of which are in the upper 3 inches, around the plant roots (from Cobb). 145. Beneficial soil nematode, Mononchus papillatus Bastion: it feeds on other nematodes, showing remnants of several Tylenchuli (J, t) (from Cobb.) 146. Assymetrical nematode Bunonema, found in decomposing organic matter (from Cobb). 147. Iota, or scaly nematode, common in the soil; head and tail end of male and female (from Cobb). 148. Male and female parasitic nematode, very simple in structure in com- parison with free living nematodes (from Cobb). 149. Tylenchus devastatrix infecting onions and other bulbous plants (from Cobb). 150. Mononchus attacking Anguillula aceti (from Steiner and Heinly). 151. Sketch of the head-end of Mononchus attacking a larval Rhabditis (from Steiner and Heinly). 152. Schematic representation of the behavior of two different populations of Tylenchus dipsaci. The one population lived on Hyacinths, the other on Nar- cissus. Therefore, if left to choose, the first population will ignore the Narcissus, the second the Hyacinth, for each will attack only the host of its ancestors. (Slogterem, after Steiner). PLATE XVin Nff -1 ■7\ .■/ '; 1 j 1 \ s^U "4 y . 145 NON-PROTOZOAN FAUNA OF SOIL 343 fastened to it at the top, and each of the three larger plates has two projecting teeth at the bottom. The plates are driven into the ground down to the required depth to form a box 9 inches square, the small- est plate being on the side towards the outside of the plot. The plates enclose a cube of soil, with a side dimension of 9 inches, giving a total of 729 cubic inches. The soil is removed from the cube, in layers: the first sample contains only the upper inch of soil, the second and succeeding samples taken at a depth of 2 inches at a time, giving in all five samples for each cube. For making a census of the soil population, Cobb2 devised soil sam- pling tubes, which are open cylinders of thin metal (tin or galvanized iron) with an internal diameter of 72.1 mm. The rim of one end is reinforced and the other sharpened. The area of the internal cross - section of the tube is one-millionth of an acre. The tubes may be of any length; for counting nematodes, 6 to 9 inch lengths are sufficient; below that depth, only few nematodes occur in the soil. Since the ani- mal population is unevenly distributed in the soil, a number of samples are required, with a minimum of five. The various samples for one plot can be mixed and the census made. After the sampling tube is forced into the soil, enough earth is dug away to enable one to introduce a knife or saw-blade beneath the lower end of tube. The tube is then removed full of soil and capped at both ends. The samples of soil from one field are sifted and thoroughly mixed; wire sieves of \ to \ inch mesh may be used. Various methods of mixing and sampling of the soil are described by Cobb. An aliquot portion of soil is placed in an abundance of animal-free water, usually 10 to 20 times its volume. The soil is well suspended in the water by proper stirring with compressed air, carried on fast enough not to allow the particles to settle. The heavy particles are allowed to settle for about five seconds and the supernatant liquid is rapidly poured into another vessel. The residue is washed several times with clean water, so as to remove all adhering animals, the washings being added to the original liquid. The sand and gravel are discarded. The process may be repeated so as to remove another portion of the heavy inorganic material, being sure that it is free from animals. The liquid is then allowed to run through a series of superimposed sieves, ranging from 16 to 200 meshes per inch; the sieves, especially the finer ones, are agitated when the liquid is passed through them. The finer sieves may be made of mill- ers bolting silk. The nematodes will all pass through the 16 mesh sieve; the residual particles should be washed so as to remove the animals. Beginning 2 Cobb, N. A. Estimating the nema population of soil. Bur. PI. Ind., U. S. Dept. Agr., Agr. Tech. Circ. 1. 1918. 344 PRINCIPLES OF SOIL MICROBIOLOGY with the 20-mesh, the residual material should be examined carefully. To make sure that no animals remain in the liquid, the latter is passed several times (5 to 10) through the finest sieve. When a portion of the final liquid is examined and no nematodes are found, the liquid is finally discarded. The larvae of some animals, like those of Heterodera, are caught on the finest sieves. The animals are then washed away from the sieves (kept at a slightly inclined position) by a small amount of water. The washings with the animals are either mixed or kept in separate vessels.3 The separation of the animals from the clay portion of the soil which is kept in suspension, is based upon the fact that they will settle quicker than the clay. Care should be taken that no animals are floating on the sur- face of the liquid. After the latter is allowed to stand for 30 minutes, it is poured off and replaced by clean water. The floating animals can be made to sink by adding some alcohol to the run-off material (so as to make 20 to 30 per cent alcohol), shaking well and adding water immediately (fig. 10). This process can be modi- fied greatly depending on nature of soil. The mixture of inorganic soil particles and or- ganisms, lying in clear water, are then examined by using an ordinary dissecting microscope. Nem- atodes are fished out by slender, tapering, sharp needles free from grease. A small portion of the debris is placed in a watch glass, in clear water, about | inch in depth. When a nematode is lo- cated, the point of the needle is brought under it and it is floated to the surface, lifted on the point of the needle and transferred to a watch glass con- taining a few drops of clean water. The final sus- pension may be well mixed and only an aliquot portion examined in a graduated watch glass. Examination should be made soon after washing is completed. ig. .j pp Certain members of the animal, non-protozoan for the separation ol . . ' c nematodes from the population can also be isolated by the use of agar soil (after Cobb). media. Small worms can be isolated4 by the fol- lowing method: 3 See also Baunacke, W. Untersuchungen 7Air Biologie und Bekiimpfung des Riibennematoden Heterodera schachtii Schmidt. Arb. Biol. Reichsanst. Land. u. Forst, 11: 185-288. 1922. 4 Shaw, C. Zuchtungsversuche zur Gewinnung von Reinkulturen kleiner Wurmarten der Garten- und Ackererde. Centrbl. Bakt. II, 64: 41-45. 1925. NON-PROTOZOAN FAUNA OF SOIL 345 Finely cut agar (3.5 grams) is soaked over night in tap water; excess of water is poured off and agar is dissolved in 200 cc. of tap water containing 1.3 grams of NaCl. The agar is neutralized and filtered; 1.6 grams of powdered brown sugar or malt sugar is then added, the agar is sterilized and distributed into sterile Petri dishes. The soil is inoculated, in the form of a fine layer, over the centre of the plate. The living worms will move away from the soil and, after 24 hours, they will be found on the clear agar about 0.5 cm. away. These worms, es- pecially those bearing eggs, can be transferred to fresh agar plates, for the prep- aration of pure cultures. FLAT WORMS (PLATHELMINTHES) The Turbellaria or free living flatworms are represented in the soil by various species of Rhabdocoelae, Allocoelae and land Planarians. Over thirty species of Rhabdocoelae were isolated from the soil.5 Among the various genera found in the soil, it is sufficient to mention Archivortex, Ade?ioplea, Acrochordonoposita, Geocentrophora, Prorhyn- chus (P. stagnalis) and Planaria. The soil forms feed largely on diatoms, rotatorians, tardigrads, oligochaetes, upon one another and especially upon soil nematodes. The Trematoda are represented in the soil by the larvae of different river flukes. NEMATODA Adult nematodes are usually cylindrical or spindle shaped, the pos- terior end being often acutely pointed or modified in form. They are transparent, non-segmented organisms, 20 to 100 times or more as long as wide. When alive and active, they thrash about in pure liquid without making much progress. They do not change their length appreciably, being thus distinguished from earthworms and other elongated small organisms, which change their length while moving. Dead nematodes lie outstretched or in a slightly curved condition. Nematodes are found in all soils under different conditions, largely in the upper 6-8 inches, although they are often abundant even at lower depths6 (No. 144, PI. XVIII). They can adjust themselves to Further information on the artificial cultivation of free-living nematodes is given by H. Metcalf in Trans. Amer. Micr. Soc, 24: 89-102. 1903; and A. C. Chandler in Science, N. S., 60: August 29, 1924. s Reisinger, E. Turbellaria. Strudelwiirmer. L. 6, T. 4, Schulze's Bio- logie der Tiere Deutchlands. Borntraeger. 1923. 6 Godfrey, G. H. The depth distribution of the root-knot nematode, Heter- odera radicicola, in Florida soils. Jour. Agr. Res., 29: 93-98. 1924. 346 PRINCIPLES OF SOIL MICROBIOLOGY various habitats. They are distributed by the wind, by water, by moving animals, by various plant products, implements, etc. The eggs and larvae are sometimes very resistant to drying and other adverse conditions, and can survive for many years. Large numbers of parasitic, saprophytic, and free-living species inhabit the soil, making up a large numerical proportion of its population. The number of species alone reaches many thousands. Some of these are of wide distribution. The identification of soil nematodes may be carried out by using fixed material. Fleming's solution (p. 323) can be employed for this purpose. The organisms are placed in the solution from a few minutes to one or two hours. Should the cells become darkened, they can be bleached with H202. When the nematodes are dead and fixed, they are mounted, counted and identified, or are placed in a mixture of 5 per cent glycerol and 95 per cent water. After the water has evapo- rated, the animals remain in the glycerol. For careful identification and detailed study of morphology, the nematodes are placed in the middle of a glass slide in a small drop of water and covered with a cover glass. The edge of the cover glass can then be sealed to the slide by means of hot wax — paraffin which contains a certain proportion of beeswax. The slides are now examined with the compound micro- scope, very high power lenses being necessary. deMan7 (1884) was the first to make a careful study of the soil nematodes. He divided the organisms into three groups: (1) omnivagous species not bound to any particular soil (Dorylaimus obtusicaudatus, Monohystera Jilifor- mis, etc.); (2) meadow and field soil nematodes (Plectus cirratus, etc.); (3) sand nematodes (Mononchus parvus, etc.). Nematodes are generally found to be abundant in forest humus as well as in cultivated soils. They are parasitic on animals and plants, or are saprophytic and free living. Even the parasites may lead an independent existence in the soil at certain stages of their development. According to Steiner,8 the nematodes are represented in Swiss soils by 139 known species, but there might be still many more. Cobb9 found nematodes to occur in large numbers in every cultivated and uncultivated soil, including forms which are parasitic on plants or 7 de Man, 1922 (p. 348). 8 Steiner, G. Freilebende Nematoden aus der Schweiz. Arch. Hydrobiol. Planktonk., 9: 259 276. 1913; Zool. Anz., 46: 336-368. 1916. ' Cobb, N. A. Nematodes and their relationships. U. S. Dept. Agr. Year- book, 1914, 457-490. NON-PROTOZOAN FAUNA OF SOIL 347 animals and those that are entirely saprophytic. Different soils may be found to contain large and varying numbers of nematodes as shown in the following summary:10 CORN FIELD SOILS MINIMUM NUMBER OP NEMATODES PER ACRE, TOP 6 INCHES (15.2 CM.) Missouri 648,000,000 North Carolina 242,400,000 New Jersey 129,600,000 Rhode Island 610,800,000 New Hampshire . . 99,600,000 Minnesota 121,200,000 Vermont. 580,000,000 Kansas 278,400,000 Morris11 also found large numbers of nematodes in the soil, the great- est number occurring at a depth of two to three inches in manured soil and four to five in unmanured soil. Four to five times as many nematodes were found in the manured as in the unmanured soil. According to Micoletzky,12 the soil and fresh water nematodes embrace 75 genera and 525 species. Half as many nematodes were found in the winter as in the summer. The abundance of these or- ganisms in the soil is determined by moisture, abundance of plant life and plant residues, and other microorganisms. The different nematodes vary in their nutrition: 1. Some, like Rhabditis, Diplogastcr, Cephalobus, feed at least partly on de- composing organic matter which results from the activities of the bacteria, as well as upon the bacteria themselves, and also upon fungi and algae; this is also true of such forms as Monohystera and Bunonema (No. 146, PI. XVIII). 10 Steiner, G., and Heinley, H. The possibility of control of Heterodera radicicola and other plant injurious nemas by means of predatory nemas, es- pecially by Mononchus papillatus Bastian. Jour. Wash. Acad. Sci., 12: 367-386. 1922. "Morris, 1922 (p. 342). 12 Micoletzky, H. Die freilebenden Erdnematoden mit besonderer Beruck- sichtigung der Steiermark und der Bukowina, zugleich eine Revision samtlicher nicht mariner, freilebender Nematoden in Form von Genus-Beschreibung und Bestimmungsschliisseln. Arch. Naturg., 87: 1-650. 1921. Zur Kenntnis tro- pischer, freilebender Nematoden aus Surinam, Trinidad und Ostafrika. Zool. Anz., 64: 1-28. 1925. Die freilebenden Suszwasser- und Moornematoden Da- nemarks. D. Kgl. Danske Vidensk. Selsk. Skr. Afd. 8 R., x, 2. Kobenhavn. 1925. 348 PRINCIPLES OF SOIL MICROBIOLOGY 2. Some feed on the tissues and fluids of healthy or injured plants, thus be- coming injurious to higher plants; these include Tylenchus, Helerodera, Aphe- Icnchus (Nos. 148-149, PI. XVIII), some of which are truly parasitic and others are semi-parasitic (like Hoplolaimus) or facultative parasitic. 3. Some are parasitic on animals, especially invertebrate soil forms. 4. Some feed both on plants and animals, like Dorylaimus. 5. Some are carnivorous and predatory, feeding on other nematodes, like Ironus, Tripyla, and especially the numerous species of Mononchus. Mononchus papillalus, for example, can feed readily upon Heterodera radicicola10 (Nos. 150- 151, PI. XVIII). The maximum number of nematodes was found13 in Tyrol soils in August, reaching 320 per 10 cc. of soil; the number then dropped rapidly and reached the lowest point in November, with 23 animals per 10 cc. It remained at a low level during the winter months and began to increase again in February. On the average, 120 nematodes were found per 10 cc. of soil throughout the year. The different species do not reach their maximum and minimum at the same time, depend- ing on the moisture and the temperature resistance of the organism. The genera Dorylaimus, Tylenchorhynchus, Mononchus and Hoplolai- mus are almost the only organisms found during the winter months. The class Nematoda consists of numerous small forms which are usually free-living and non-parasitic. Among the free-living forms in the soil, the genus Mononchus composed of numerous species14 is particularly abundant. Many of these species are cosmopolitan. The Mononchs occur in all kinds of arable soil, sometimes in hundreds of millions per acre. They feed on living microzoa, including other nematodes.14-16 Among the nematodes attacking the roots of various plants, causing the formation of galls, we find the sugar-beet nematode Heterodera schachtii,17 the root-knot nematode Caconema radicicola, the wheat 13 Seidenschwartz, L. Jahreszyklus freilebender Erdnematoden einer Tiroler Alpenwiese. Arb. Zool. Inst. Univ. Innsbruck, 1: 37-71. 1923. 14 Cobb, N. A. The Mononchs, a genus of free-living predatory nematodes. Soil Sci., 3: 431-486. 1917. 15 De Man, J. G. Nouvelles recherches sur les nematodes libres terricoles. M. Njhoff. Hague. 1922. 16 See also Wiilker, G. Nematodes. Fadenwurmer. L. 11, T. 8, Schulze's Biologie der Tiere Deutchlands. Borntraeger. 1924. 17 Shaw, H. B. Control of the sugar-beet nematodes. Bur. PI. Ind., U. S. Dept. Agr. Farm. Bui. 772. 1916. Baunacke, 1922 (p. 344). NON-PROTOZOAN FAUNA OF SOIL 349 nematode Tylenchus tritici, and various others.18 Some of them attack a great variety of plants. C. radicicola, for example, attacks about five hundred kinds of plants. This organism flourishes best in high- sandy soils, which are moist and warm. It attacks cotton, beans, celery, eggplants, potatoes, lettuce, peas, tomatoes, cowpeas, soybeans, nursery stock, weeds, ornamental plants and various field crops. Some plants, however, are not attacked. Heterodera schachtii attacks pota- toes, sugar beets, etc., and may prove to be very injurious.19 Tylen- chus dipsaci attacks clover, alfalfa, and other crops, ornamental plants, etc.20 The nematodes are represented in the soil by a large number of genera.21 It is sufficient only to enumerate some of the most common : Iota (syn. Hoplolaimus) found in swamp and acid soils, on the roots of trees. Tylenchus, found in peat and moist soils, on plant roots; many parasitic species. Aphelenchus, found in various soils, many species parasitic. Isonchus, found on the roots of the cotton plant. Dorylaimus, omnivagus, very abundant in the soil. Actinolaimus, found in peat bogs and marshy soils. Ironus, occurs to a limited extent in very moist soil. Mononchus, predacious nematode, represented in the soil by many species, cosmopolitan. Diplogasier, found in moist soils. Cyatholaimus, found in moist soils. Plectus, omnivagous, well distributed in the soil. Rhabditis, cosmopolitan, some species microbivorous, well distributed in the soil. 18 Marcinowski, K. Parasitisch und semi-parasitisch an Pflanzen lebende Nematoden. Arl. Biol. Anst. Land. u. Forst., 7: 1-192. 1909. 19 Zimmermann, H. Nematodenbefall (Heterodera) an Kartoffeln. Ztschr. Pflanzenkrank., 30: 139-145. 1920. 20 Goodey, T. On the susceptibility of clover and some other legumes to stem-disease caused by the eelworm, fylenchus dipsaci, syn. devastatrix Kuhn. Jour. Agr. Sci., 12: 20-30. 1922. Ritzema-Bas, J. Les nematodes parasites des plantes cultivees. VI. Int. Congr. Inst. Agr., Paris, 2: 306-312. 1900. The cultivation of plant pathogenic nematodes is discussed by Byars, L. P. Preliminary notes on the cultivation of the plant parasitic nematode, Heterodera radicicola. Phytopath., 4: 323-326. 1914; Hilgermann and Weissenberg, R. Nematodenziichtung auf Agarplatten. Centrbl. Bakt., I, Orig., 80: 467-472. 1918; Berliner, E., and Busch, K. fiber die Ziichtung des Rtibennematoden {Heterodera schachtii Schmidt) auf Agar. Biol. Centrbl., 34: 349. 1914. 21 A detailed classification of nematodes is given by Cobb, N. A. Free-living nematodes. Fresh-water Biology by Ward and Whipple, 1918, p. 459-505. Micoletzky, 1921 (p. 347) and De Man, 1922 (p. 348). 350 PRINCIPLES OF SOIL MICROBIOLOGY Rhabdolaimus, found in moist soil. Cephalobus, commonly found in the soil, can be grown on decomposing or- ganic matter. Teratocephalous, omnivagous, found in moist soil. Bastiana, largely soil forms. Tripyla, found in soils rich in undecomposed organic matter, not very abundant. Alaimus, well distributed in the soil, especially in moist and forest soils. Prismatolaimus, represented in the soil by some species. Monohystera, omnivagous, found in moist soils, some feed on diatoms. Trilobus, seldom found in moist soils, feeds on diatoms and rotatorians. Various Mermithidae are also found in the soil. A number of other genera, like Bunonema, Tylopharynx, Archionchus, Euty- lenchus, etc., are found in the soil less abundantly. What is known of the nutrition of free-living soil nematodes has been reviewed in detail by Menzel.22 He found that Mononchus papil- latus, when brought together with Tylenchus sp., Plectus auriculatus, Tripyla media and Anguillula aceti, killed these forms either by swal- lowing them completely or by sucking out their contents. Steiner and Heinley grew the Mononchus papillatus in water containing some soil and placed in concave slides. It is important to use a small amount of soil free from excess of organic matter so as to prevent the rapid development of bacteria. The medium should be frequently renewed. Heterodera, Rhabditis and Anguillula were used for food. As many as 83 Heterodera radicicola were killed in one day by one Mononch; during a life time of about 12 weeks, one animal killed 1332 nematodes. It is possible that this number may be much larger under natural condi- tions (No. 151, PI. XVIII). Steiner and Heinley, therefore, brought further weight to the sug- gestion of Cobb that the predatory Mononchs could be used to control the plant parasitic forms, when the latter are still free in the soil. How- ever, we must keep in mind that the mere introduction of an organism into the soil is not sufficient to insure its development; the soil should be treated in such a manner as to favor the development of the bene- ficial organism and discourage the development of the injurious forms.23 The role of nematodes in the soil may, therefore, consist of the fol- lowing processes: 1. Consuming and destroying cultivated plants, often causing considerable damage. 22 Menzel, R. Uber die Nahrung der freilebenden Nematoden und die Art ihrer Aufnahme. Ein Beitrag zur Kenntnis der Erniihrung der Wurmer. Ver- handl. Naturf. Gesell. Basel., 36: 153-188. 1920. "Baunacke, 1922 (p. 344). NON-PROTOZOAN FAUNA OF SOIL 351 2. Consuming soil bacteria and fungi. 3. Consuming soil protozoa. 4. Destroying other nematodes (predatory forms). 5. Distributing bacteria and fungi throughout the soil. 6. Taking an active part in the transformation of the soil organic matter. 7. Improving soil aeration. ROTATORIA24 Rotatoria or Rotifera, commonly known as wheel animalcules, are minute, chiefly microscopic, animals. They are mostly characterized by the presence of a ciliated area, or corona, at or near the anterior end of the body, which serves both for locomotion and for bringing food to the mouth. Cilia are lacking on other parts of the body; in exceptional cases, they may be present at the posterior end. The corona may, in a few cases, be lacking. The body is usually somewhat elongated, with the corona at the anterior end and a tail-like appendage at the posterior end beyond the cloacal opening. The sexes are separate, the male being a minute, degenerate form, without an alimentary canal. They are commonly found in swamps and marshes, as well as in moss and forest leaves. France found the following species in the soil: Rotifer tarligradus Callidina papillosa Rotifer vulgaris Callidina ehrenbergii Philodina erythrophthalma Callidina mullispinosa Philodina aculeata Habrotrocha angusticollis Philodina vorax Diaschiza semiaperta Adineta vaga Chaelonotus macrotrichus A number of other forms may occur, but they have not yet been made the subject of special study. ANNELIDA The annelids are represented in the soil by the earthworms (Oligo- chaeta-Terricolae), whose whole life cycle is passed in the soil, and by the white worms or Enchytraeids (Oligochaeta-Limicolae), which are usually abundant in moist soils, especially those rich in organic matter. Oligochaeta-Terricolae.2b The earthworms are characterized by their flexible segmented bodies, with four rows of bristles or setae. They 24 Harring, H. K. Synopsis of the rotatoria. U. S. Natl. Museum, Bui. 81, 1913; Harring, H. K., and Myers, F. J. Rotifer fauna of Wisconsin. Trans. Wiscon. Acad. Sci., 21: 415-549. 1924. 25 Michaelsen, W. Oligochaeta. Das Tierreich. No. 10, 1900. 352 PRINCIPLES OF SOIL MICROBIOLOGY have a well-defined body cavity and are hermaphroditic. The setae aid in locomotion. Various families are found in the soil. The occurrence of earthworms in the soil has been of common knowledge since the work of Darwin and Hensen.26 They are especially abundant in forest soils and soils rich in organic matter27 while they are almost absent in sandy soils. Heimburger28 suggested that a cor- relation exists between the degree of moisture of the soil and species of earthworms inhabiting it. The worms react definitely to atmos- pheric moisture but less sharply than to contact with moist substrate. To determine the numbers of earthworms in the soil, a certain volume of it is spread in a thin layer on a flat surface; when the soil begins to dry, the animals begin to move rapidly and can be counted. Moist soil may also be covered with a solution of sugar or powdered KHSO4, which will bring the worms to the surface. Morris29 found 1,010,101 earthworms per acre of manured soil and 457,912 per acre of unmanured soil. The greatest numbers occurred at a depth of two and three inches. The following species of earthworms were found in the soil by France: Eisenia rosea, Lumbricus terrestris, Lumbricus rubellus, Allolobophora aporata, Helodrillus octaedrus. It was estimated that between 200 and 1000 pounds of earthworms are present in an acre of soil. Thompson30 found eighteen individuals in a nine inch cube of the upper three inches of a pasture soil. Soil reaction has an influence upon the development of earthworms.31 The worms feed not only upon plant residues, but also on soil organisms. France found various algae, fungus mycelium, protozoa and yeasts in the excreta of earthworms. The animals pass earth through their bodies, grinding it in the 26 Darwin, Ch. Vegetable mould and earthworms. London. J. Murray. 1881 ; D. Appleton, 1900. Hensen, V. Uber die Fruchtbarkeit des Erdbodens in ihrer Abhiingigkeit von den Leistungen der in der Erdrinde lebenden Wiirmer. Landw. Jahrb., 11: 661-698. 1882. 27 Remele, E., Schellhorn, and Krause, M. Anzahl und Bedeutung der niederen Organismen in Wald und Moorboden. Ztschr. Forst. u. Jagdwes., 31: 575-606. 1899. 28 Heimburger, H. V. Reactions of earthworms to temperature and atmos- pheric humidity. Ecology, 5: 276-282. 1924. 29 Morris, H. M. Rothamsted Station Rept. for 191S-1920, p. 20. 30 Thompson, M. The soil population. An investigation of the biology of the soil in certain districts of Aberystwyth. Ann. Appl. Biol., 11: 349-394. 1924. 31 Arrhenius, O. Influence of soil reaction on earthworms. Ecology, 2: 255-262. 1921. NON- PROTOZOAN FAUNA OF SOIL 353 gizzard into fine particles and decomposing some of the organic matter which may be present. The earth is then passed out of the body and deposited as castings at the surface of the burrows. The soil is thus well mixed with the organic matter and brought from the lower layers to the surface. According to Darwin, ten tons of earth for each acre of land are passed through the bodies of the earthworms every year in some cases. This mechanical action of the worms upon the structure of the soil is of great importance. Wollny32 considered that the worms were concerned with the decomposition of the nitrogenous compounds in the soil; a soil containing worms was found to have a higher ammonium content than the same soil free from worms. Soils containing earth- worms and upon which grass was growing was found to contain more ammonia, nitrate and total nitrogen than soils without worms and grass, but this is probably due more to the grass than to the worms.33 It was suggested34 that earthworms increase plant growth by increas- ing the surface of soil due to excreta, thus affecting the water-holding capacity of the soil and movement of water. These organisms are commonly considered to exert an important influence upon the mechan- ical transformation in soil as well as soil productivity as a result of their effect upon nitrogen transformation.35 Russell,36 however, pointed out that earthworms do not appear to have any marked effect on the production of plant food; their chief work is to act as cultivators, loosen- ing and mulching the soil, facilitating aeration and drainage by their borrows. Oligochaeta-Limicolac or Enchytraeidae. This family is character- ized by their whitish appearance and presence of more than two straight setae in some of the bundles. Moist soils, especially those rich in organic matter, will contain large numbers of these organisms. Thomp- son found as many as 86 forms in a 9-inch cube of the upper 3 inches of 32 Wollny, E. Die Zersetzung der organischen Stoffe. 1897, p. 39. 33 Blanck, E., and Giesecke, F. Uber den Einfluss der Regenwurmer auf die physikalischen und biologischen Eigenschaften des Bodens. Ztschr. Pflanzener- nahr. Dung., 3B: 198-210. 1924. 34 Kohswitz, H. G. Untersuchungen Uber den Einfluss der Regenwurmer auf Boden und Pflanze. Bot. Archiv., 1: 315-331. 1922. 35 Aichberger, R. V. Untersuchungen uber die Ernahrung des Regenwurmes. Arb. Biol. Inst. Miinchen. No. 4, Kleinwelt. 1914; Heymons, R. Der Einflusz der Regenwurmer auf Beschaffenheit und Ertragsfahigkeit des Bodens. Ztschr. Pflanzenernahr. Dung., 2A: 97-129. 1923. 36 Russell, E. J. The effect of earthworms upon soil productiveness. Jour. Agr. Sci., 3: 346. 1910. 354 PRINCIPLES OF SOIL MICROBIOLOGY pasture soil, including several species of Fredericia and Enchytraeus (E. albidus). France found species of Enchytraeus, Fredericia and Anachacta in the soil. According to Jegen,37 these are capable of neutralizing the injurious effect of certain nematodes in the soil; he also claimed that they play a role in the formation of humus in the upper soil layers. They are very sensitive to drying and to lack of oxygen. Their relative abundance is indicated by the following numbers found in one square meter of different soils, at different seasons of the year. They were practically absent in heavy clay soils, as shown in the fol- lowing summary: SOIL TYPE Spring Summer (dry). . Summer (moist) Autumn Winter 60- 100 28- 75 70- 300 100- 450 800-1600 SANDY SOIL 6,900- 9,000 2,000- 4,900 6,500- 8,500 7,000-11,000 6,800- 9,400 HUMUS SOIL 30,000- 70,000 11,800- 16,000 28,000- 50,000 60,000-150,000 50,000-120,000 Tardigrada. The tardigrads are a group of Annelids, although they are often wrongly classified with the Arachnids and Arthropoda.38 The organs of locomotion are unarticulated, with more or less retract- ible parapodia. The body is 1 to 10 mm. long, cylindrical, often al- most worm-like. Some are without eyes, some have compound eyes in the form of black or red spots. The sexes are separate. During a period of dryness, they hibernate. Hibernation my last for years without injury to the organism. The tardigrads can withstand con- siderable heat and cold. Under unfavorable conditions, they encyst; regeneration of the organs follows this stage. Various species are found in the soil. France- found this group represented by the genera Macrobiotus and Milnesium. ARTHROPODA Crustacea. The crustaceans are represented in the soil by the Copepoda39 (family Harpacticidae) and the Isopoda,40 or higher crus- 37 Jegen, G. Bedeutung der Enchytraeiden fur die Humusbildung. Landw. Jahrb. Schweiz, 34: 55-71. 1920. 38 Richters, F. Tardigrada. Handworterbuch der Naturwissenschaften., 9. 1913. 39 Van Douwe, C, and Neresheiner, E. Copepoda. Die Susswasserfauna Deutschlands. H. 11, 1909. 40 Richardson. H. A monograph on the Isopods of North America. U. S. Nat. Mus. Bui. 64. 1905. NON-PROTOZOAN FAUNA OF SOIL 355 taceans. The Harpacticidae do not have the cephalothorax and ab- domen distinctly separated, so that the body is worm-like. France found in the soil one species of Moraria and six species of Canthocamptus. 33,700 to 80,000 Isopods were recorded per acre of soil by Morris. The presence of crustaceans in the soil was also reported by Thompson. ARACHNIDA The Arachnids are represented in the soil by the mites and ticks (Acarina41), which are chiefly carnivorous, free-living or parasitic, and by the carnivorous spiders (Areinida). Morris found the Acarina represented in the soil by the Amystidae, Tarsonemidae and Tyro- glyphidae. The presence of various members of the Trombidiidae and Oribatidae in the soil was also reported by Thompson. Por- rhomma, Robertus, Oedothorax, Linyphia, and others are the genera of Areinida found in the soil by Morris. The greatest numbers of Aca- rina were found in the upper one inch of soil, the total number being 531,986 per acre of manured soil and 215,488 per acre of unmanured soil. MYRIAPODA Among the myriapods present in the soil, are the millipedes (Dip" lopoda) which attack various crops, the centipedes (Chilopoda), which are carnivorous, and the Sympkyla. The following species of myriapods were found in the soil :42 Glomeris hexasticha, G. frausalpina, Polydesmus sp., Craspedosoma rawlinsii, C. canestrinii, Chordeuma nodulosum, Ch. silvestre, Julus nigrofuscus, C. verhoeffi, Schizophyllum sabulosum and Lithobius forficatus. Morris found 1,781,143 myriapods in the upper nine inches of an acre of manured soil and 878,787 in the corresponding unmanured soil. They were distributed more or less uniformly with depth. The Diplopoda were represented by species of Brachydesmus, Cylindroiulus, Blaniulus and Archiboreoiulus; the Chilopoda by Lithobius, Geophilis and Geo- philomorph. Symphyla were also found in both soils. 41 Wolcott, R. H. A review of the genera of the water mites. Trans. Amer. Micr. Soc, 26: 161-243. 1905. 42 Diem, K. Untersuchungen liber die Bodenfauna in den Alpen. Jahrb. St. Gall. Naturw. Gesell. Vereinsjahr. 1901-1902. 356 PRINCIPLES OF SOIL MICROBIOLOGY INSECTA The term "soil insect" comprises all insects which, at one time or another in the course of development from the egg to the imago, spend some stage or stages of their life-histories either on or beneath the surface of the soil.43 A great many species of insects, found in most orders, are associated with the soil in one or more stages of their de- velopment. As a matter of fact, it has been stated that as many as 95 per cent of all insect species invade the soil at some stage of their development. Millions of insects are found in every acre of arable land. On the basis of their feeding habits, the soil insects can be divided into six groups:44 1. Those feeding on subterraneous parts of plants, as the larvae of Melolontha, Agriotes and Tipula. 2. Those living saprophytically in the soil, as Collembolla and larvae of Dip- tera and Coleoplera. 3. Those living on other members of the soil fauna, or predaceous species, as the Carabidae and many larvae of Diptera. 4. Parasitic species, as the Hymenoptera and the Tachinidae, which pass their larval stages on or within the bodies of other organisms. 5. Insects which find their habitat in the soil, without seeking a food supply there, as in the case of ants. G. Insects which only undergo pupation in the soil, as in the case of the Lepi- doptera. A survey of the insect fauna in cultivated and pasture lands revealed the fact that the distribution and numbers of the soil fauna are more stable on grass than on arable land.45 This is due to the fact that grassland bears a vegetative covering all the time, which offers food for the fauna. In grass land, hibernation can proceed normally. Cultiva- tion of land brings the fauna to the surface exposing it to harsh climatic conditions and to bird attack. As vegetative growth increases, there is a corresponding increase in the fauna in both arable and grass land. Conditions in winter and early spring are detrimental to the soil fauna. The fauna of arable land consists of species which have passed the winter in the soil and those which have migrated or are introduced 43 Cameron, A. E. Soil insects. Science Progress, No. 77: 92-108. 1925. 44 Imms, A. D. The invertebrate fauna of the soil (other than protozoa). In book by Sir E. John Russell et al. The microorganisms of the soil. Long- mans, Green & Co. 1923. 46 Buckle, P. A preliminary survey of the soil fauna of agricultural land. Ann. Appl. Biol., 8: 135-145. 1921. NON-PROTOZOAN FAUNA OF SOIL 357 during the growing season. There is no characteristic fauna in culti- vated land. Buckle isolated from the soil one species of Collembola, 35 species of Coleoptera, 6 Diptera, 2 Hymenoptora, 4 Chilopoda and Diplopoda. A detailed study of the insect fauna of the soil has also been made by Cameron, Morris and Thompson.46 Morris found 2,475,000 TABLE 19 Distribution of the invertebrate soil fauna in two plots of arable land at Rothamsted1 Insecta Nematoda Myriapoda: Diplopoda Chilopoda Symphyla Total Oligochaeta (Terricolae) Arachnida: Acarina Areinida Total Crustacea (Isopoda) . . . Mollusca (Pulmonata) . . Total invertebrata. . . . NUMBERS OF ORGANISMS PER ACRE OF SOIL Unmanured plot Manured plot 2,474,700 7,727,300 794,600 3,600,400 596,000 1,367,000 215,400 208,700 64,000 215,500 875,400 1,791,200 457,900 1,010,100 215,400 531,900 20,200 20,200 235,600 552,100 33,700 80,800 13,500 33,700 4,885,400 14,795,600 46 Cameron, A. E. General survey of the insect fauna of the soil within a limited area near Manchester. A consideration of the relationships between soil insects and the physical conditions of their habitat. Jour. Econ. Biol., 8: 1913, No. 3; The insect association of a local environmental complex in the dis- trict of Holmes Chapel, Cheshire. Trans. Roy. Soc. Edin., 52: pt. 1, No. 2. 1917; Morris, 1920 (p. 352); Thompson, 1924 (p. 352); See also Diem, 1901-1902; Holthaus, K. Die Siebetechnik zum Aufsammeln der Terricolfauna nebst Bemerkungen liber die Oekologie der im Erdboden lebenden Tierwelt. Ztschr. 358 PRINCIPLES OF SOIL MICROBIOLOGY insects in the upper nine inches of an acre of unmanured plot and 7,727,000 in a manured plot. The dominant groups in both plots were the Collembola and Formicidae; the Chironomidae larvae and Trichocera larvae were much more abundant in the manured plot. The Collembola were represented by 14 species: the Thysanura by 3 species, the Orthoptera and Thysanoptera by one each; the Hemiptera by 4; the Lepidoptera by unidentified larvae; the Coleoptera by 30 species; the Diptera by 7 species and various unidentified larvae; the Hymenoptera by 10 species. The greatest majority of the organisms were found in the upper three inches of soil. The wireworms attain a maximum at a depth of 5 to 7 inches. Manuring increases the total number of soil organisms about 200 per cent, but has no appreciable influence on the number of wireworms. Morris also found 3,586,088 insects in an acre of permanent pasture, the numbers of the different orders being Collembola- — 566,680, Rhyn- chota — 15,140, Thysanoptera- — 43,258, Lepidoptera- — 15,140, Coleop- tera—744,038, Diptera— 2,193,180, Hymenoptera— 8652. Among the injurious insects, the following were found per acre: Agriotes — 114,643 larvae and 8652 adults, Triphaena pronuba- — 4326 larvae and pupae, Tipula oleracea and T. paludosa 19,466 larvae. The family most represented was the Bibionidae, the species of this family making up 32.4 per cent of the total number of soil insects; Myceto- philidae was represented by 16.7 per cent and Staphyinidae by 12.2 per cent. The Coleoptera were represented by 29 species. According to Thompson, the orders Collembola and Acarina determine the trend of the total fauna curve, since they are the dominant groups. They persist throughout the year, while other groups like the Nematoda and Oligochaeta may be entirely missing for varying lengths of time. Cul- tivated land contains a considerably smaller population than pasture or grass land; the maximum population was found to occur in the winter months, due to sufficient moisture in the soil. Peat soils also contain a definite fauna of insects, as represented by the Collembola.47 M'Atee48 reported the presence of 1,216,880 animals belonging to In- wiss. Insektenbiol., 6: 1-4, 44-57. 1910; Adams, C. C. An ecological study of prairie and forest invertebrates. 111. State Lab. Nat. Hist. Bui. 11: Art. 2. 1915; Shelford, V. E. Animal communities in temperate America. Geogr. Soc. Chicago, Bui. No. 5; Univ. Chicago Press. 1913; Vestal, A. G An associational study of Illinois sand prairie. 111. State Lab. Nat. Hist. Bui. 10: Art. 1. 1913. 47 Handschin, E. Beitrage zur Kenntnis der Collembolefauna der Hoch moore Estlands. Beitr. Kunde Estlands., 10: 167-176. 1924. 48 M'Atee, W. L. Census of four square feet. Science, N. S., 26: 447-449. 1907. NON-PROTOZOAN FAUNA OF SOIL 359 secta, Arachnida and other Arthropoda, Annelida and Gastropoda in an acre of forest soil. A detailed study of the subterranean aphids has been made by Cut- right.49 MOLLUSCA The molluscs possess a soft body encased in a hard shell consisting either of one part (Gastropoda snails) or two parts (Lamellibranchiata) . The soil molluscs include the snails and the slugs, which frequently leave the soil which they inhabit for feeding purposes in the presence of sufficient moisture. Most of these forms usually consume vegetable matter, while some (Testacella) are carnivorous. France* found in the soil the genera Carychium and Helix.50 Influence of environmental conditions on the invertebrate fauna of the soil. Larvae of soil insects are very sensitive to evaporation of mois- ture, especially at 20°C. or over.51 They do not occur in dry exposed soils, but rather in most soils where the humidity is not far below saturation and the temperature seldom goes above 20° to 23°C. Below 8°C., most soil insects become inactive. Hibernating soil insects pos- sess a great capacity of resisting freezing temperatures. They migrate vertically, according to season, especially in cold climates.52 The invertebrate fauna of the soil is generally much more abundant in heavy than in light sandy soils because of the moisture conditions.53 According to Morris, the depth to which insects penetrate into the soil is due to depth of food, aeration, moisture and soil temperature; in- sects are found, therefore, at greater depths in arable soil than in pas- ture land. The addition of organic matter to the soil increases the moisture holding capacity of the soil and offers food material for many species. Different soil insects respond differently to varying degrees of humidity, 49 Cutright, C. R. Subterranean aphids of Ohio. Ohio Agr. Exp. Sta. Bui. 387: 175-238. 1925. 60 The influence of soil reaction upon snail development is discussed by Atkins, W. R. G., and Lebour, M. V. Soil reaction, water snails, and river flukes. Na- ture, 11: 83. 1923. 61 Hamilton, C. C. The behavior of some soil insects in gradients of evaporat- ing power of air, carbon dioxide and ammonia. Biol. Bull. 32: 159-182. 1917. 62 Griddle, N. The habits and control of white grubs in Manitoba. Agr. Gaz. Canada, 5: No. 5. 1918. 63 Ramann, E. Regenwurmer und Kleintiere im deutschen Waldboden. Int. Mitt. Bodenk., 1: 138-164. 1911. 360 PRINCIPLES OF SOIL MICROBIOLOGY some selecting light, sandy soils and others living only in soils saturated with water. The optimum soil habitat is determined by the ratio or balance between the amount of available oxygen and the amount of carbon dioxide which can be endured without injury.54 Loam soils have a more abundant insect fauna than clay and sandy soils.51 Table 19 shows the influence of manuring of soil upon the distribu- tion of the invertebrate fauna; these are not maximum numbers, but are in many cases, especially in the case of nematodes, far too low, as can be seen from the data recorded previously (p. 347). The dis- tribution of the organisms with depth of soil is greatly influenced by manuring and plowing of the soil, the greatest number of insects and most other invertebrates occurring within the upper 3 inches of soil. The great majority of free-living soil nematodes are "world wide" or are found everywhere when conditions are favorable for their develop- ment. To what extent this is true of the other members of the inver- tebrate fauna of the soil still remains to be investigated. Economic importance of the invertebrate fauna of the soil. The treble role of the invertebrate fauna in the soil may be included under the heads of (1) mechanical effect upon the soil, as discussed above; (2) a relation to the transformation of organic substances in the soil; (3) a relation to the growth of higher plants. The influence of the invertebrate fauna upon the bacteria, fungi, actinomyces and algae of the soil is still a matter of speculation. There is no doubt that the fauna feeds to some extent upon the soil microflora and thus influences its activities. The earlier idea of Pasteur that the distribution of anthrax bacteria in the soil is brought about by worms inhabiting the soil may hold true also for the true soil microflora. McColloch55 suggested that there is a reciprocal relation between the soil and its insect population : these utilize the soil for shelter, pro- tection, as an avenue for travel, and find there their food, moisture and atmosphere. The soil is benefited, on the other hand, by being well mixed by the mechanical separation of particles, improvement of aeration and drainage, and addition of organic matter. The me- chanical effect may be injurious as a result of the fact that the soil may become porous leading to an increase in evaporation and plant injury. Morris found that the nitrogen content of the invertebrate fauna 64 Adams, 1915 (p. 358). See also Hesse, R. Tiergeographie auf okologischer Grundlage. Jena. 1924. 65 McColloch, J. W. The role of insects in soil deterioration. Jour. Amer. Soc. Agr., 18: 143-159. 1926. NON-PEOTOZOAN FAUNA OF SOIL 361 varied from 4.88 per cent for the Myriapoda to 11.18 per cent for the Collembola, making a total nitrogen content of the fauna of an acre of manured ground 16.2 pounds and 7.5 pounds for unmanured ground. More than half of this nitrogen was in the bodies of the earthworms. The action of worms and insects in assisting the breaking down of vegetable matter, with the formation of amorphous "humus," was considered56 of importance. The soil insects are frequently classified57 into three groups: beneficial, noxious and innocuous. The first include such forms as species of Ichneumonidae and Braconidae, which are parasitic on cutworms, predatory Carabidae and scavenging Scarabaeidae. The noxious insects are found among the Elateridae, Noctuidae, some Scarabaeidae, Curculionidae and Tipulidae. The innocuous forms are those which find in the soil a temporary retreat for pupation ; even injurious species, like the potato-beetle, may be harmless during the soil phases. Among the most important insects whose larvae may become of great economic importance in the soil in injuring field crops, are the wireworms, white grubs and cutworms.58 The parents of the wire- worms are the so-called "click," "jumping" or "snapping" beetles. The adults deposit their eggs chiefly in grass land. The small wire- worms which hatch out feed on the roots of various plants or seeds before they are sprouted, especially when the crop is planted after sod. This continues until the insects are fully mature, which requires three to five years. The damage is usually most severe in spring. An abun- dance of wireworms in sandy soil frequently makes it necessary to abandon or "rest" the land. The parents of the white grubs are the June bugs (beetles) which also lay their eggs principally in sod land during June. The eggs hatch in about two weeks. The grubs feed on any plants available and go down seven to fourteen inches below the surface when cold weather approaches. With the coming of warm weather the following spring, they come up again towards the surface where they feed on plants throughout the '6 Kostytschew, P. Russian tschernoziems. Pctrograd. 1886, p. 165-191. Ann. Sci. Agr., 2: 1887; Recherches sur la formation et les qualites de l'humus. Ann. Agron., 17: 17-38. 1891. 47 Cameron, 1925 (p. 356). *8 Headlee, T. J. Soil-infesting insects. N. J. Agr. Exp. Sta. Cir. 26; Ander- son, G. M. The slender wireworm; its relation to soils. S. C. Agr. Exp. Sta. Bui. 204. 1920; Thomas, W. A. Corn and cotton wireworms. S. C. Agr. Exp. Sta. Bui. 155. 1911. 362 PRINCIPLES OF SOIL MICROBIOLOGY season.59 This is twice repeated; the common cycle being three years. This white grub may become a limiting factor in continuous wheat pro- duction in certain sections ; the infestation usually increasing with each generation, so that it becomes necessary to rotate with a cultivated crop. The green June beetle and muck beetle also (McCulloch) prefer soils receiving heavy applications of animal manures. The cutworms hatch in September or October and become very active during the following spring. They cut off young plants near the surface of the soil and lap up the exuding sap. This is usually done at night. Crop rotation, use of artificial fertilizers, fall plowing,60 the use of poisoned and other baits,61 soil fumigants (CS2) and insecticides (Paris green) ,62 as well as the direct mechanical protection of plants, are among the remedies suggested for these three pests. Insects may also inhibit the activities of certain specific soil organisms, such as legume bacteria, by feeding on the nodules. This was pointed63 out for the beet leaf larva Cerotoma trifurcata, and for the mealy bugs Pseudococcus maratinus.64 As many as fifteen bugs were seen on one soybean nodule. In addition to the invertebrate, there exists also a vertebrate fauna in the soil, including moles, blind mice, field mice, marmots, etc. France suggests that there exists an association in soil (Edaphon) con- sisting of bacteria, algae, fungi, diatoms, protozoa, rotatoria, nemato- des, worms, myriapods and insects. The bacteria and fungi liberate 59 McColloch, J. W., and Hayes, W. P. Soil temperature and its influence on white grub activities. Ecology, 4: 29-36. 1923; Osborn, H. The problem of permanent pasture with special reference to the biological factors. Proc. 39th Ann. Meet. Soc. Prom. Agr. Sci., 7-18. 1919. 60 Hyslop, J. A. Wireworms attacking cereal and forage crops. U. S. Dept. Agr. Bui. 156. 1915; Hunter, W. D. Relation between rotation systems and insect injury in the south. U. S. Dept. Agr. Yearbook, 1911: 201-210. 1912. 61 Treherne, R. C. Wireworm control. Entom. Branch, Dept. Agr., Canada, Pamphl. 33. 62 Davis, J. J. Miscellaneous soil insecticide tests. Soil Sci., 10: 61-76. 1920. 63 McConnell, W. R. A unique type of insect injury. Jour. Econ. Entom., 8: 261-267. 1915; Another nodule destroying beetle. Ibid., 8: 551. 1915; Leonard, L. T., and Turner, C. F. Influence of Ceratoma trifurcata on the nitrogen-gathering functions of the cowpea. Jour. Ajner. Soc. Agr., 10: 256- 261. 1918. 64 Leonard, L. T. Mealy bugs on the roots and nodules of legumes growing in the field. Science, N. S., 57: 671-672. 1923. See also Folsom, J. W. The insect pests of clover and alfalfa. 111. Agr. Exp. Sta. Bui. 134. 1909. NON-PROTOZOAN FAUNA OF SOIL 363 nitrogen for the algae; all three forms serve as food for rhizopods and together with these they serve as food for rotatoria and nematodes ; the latter are eaten by amoebae, myriapodes, insects, etc.; these are, in turn, decomposed by the fungi and bacteria. It is sufficient to call attention to the role of termites (Hodotermes) in certain soils, to obtain an idea as to the probable importance of the animal population in soil processes. Termites live in tropical and subtropical countries, not only in wooden structures, in which they cause active decomposition of organic matter of living and dead plants, but also in the soil itself. The underground termites are found in great abundance in dry countries and in deserts. They form a great abundance of nests, which are connected by a net of underground pas- sages. These termite nests make the soil very porous, with the result that there is a great increase in the amount of water required for satura- tion of the soil. The activities of the termites in the soil result in increases in the concentration of salts. These increases may be from 0.06 to 1.3 per cent. The termites, as a result of the numerous pas- sages formed in the soil, appreciably improve soil drainage.65 65 Dimo, N. A. The role and importance of termites in the life of soils and sub- soils of Turkestan (Russian). In Soil and Bot. Geogr. Investig. of the basins of Amu-Daria and Sir-Daria. Moskau, 2: 1-38. 1916. PART C CHEMICAL ACTIVITIES OF MICROORGANISMS "Fur alle Lebewesen ist ein nie fehlendes Kennzeichen der Energie- strom. Meist bezeichnet man den hier stattfindenden V or gang mit dem Namen Stoffwechsel. Dieses Wart trifft aber nicht die Hauptsache" — Wl. OSTWALD. CHAPTER XV General Principles of Microbial Metabolism Metabolism as a whole. To be able to understand the chemical processes taking place in the soil as a result of the activities of micro- organisms and to learn how to control these processes so as to produce conditions which make a soil productive and thus benefit the growth of higher plants, we must understand the metabolism of the various groups of soil microorganisms. The biological changes produced in the soil fall under the class of chemical reactions. However, the biolo- gist is dealing with dynamic phenomena, while the chemist considers chiefly static phenomena. This is the reason why a chemical analysis of a soil is far from sufficient to give us information as to productivity of the soil, or the rapidity with which the nutrients necessary for the growth of higher plants become available. We must consider not only chemical changes as such, but also the course or rate of change. This can be done and the information, subject to a host of variable factors, can be properly interpreted only when the metabolism of the organisms concerned is taken into consideration. The metabolism of the leading groups of soil microorganisms can be considered under the transformation of carbon, of nitrogen and of mineral compounds. From the point of view of soil productivity, various groups of soil microorganisms may be considered to play im- portant roles in certain specific transformations, depending on the nature of the organism and nature of the medium. The various transformations in the soil dovetail and, for a proper understanding of the resulting phenomena, metabolism should always be considered as a whole. The carbon source is used by the heterotrophic microorganisms as a source of energy and as a source of carbon for structural purposes, namely, for the building up of the microbial cell. In both cases the carbon is required in the form of complex organic compounds, such as carbohydrates, hydrocarbons, fats, fatty acids, proteins and their split products including amino acids and acid amides. Some organisms prefer one group of compounds and some another, while some can utilize a variety of substances as sources of carbon. A great many 367 368 PRINCIPLES OF SOIL MICROBIOLOGY of the known soil bacteria are more or less selective in their action (when grown, of course, upon artificial culture media) ; many soil fungi and actinomyces and a number of bacteria can derive their carbon, both for energy and structural purposes, from a great variety of substances. Bad. pyocyaneum, for example, can obtain its carbon not only from carbohydrates, but also from lactic and acetic acids, glycerol, ethyl and methyl alcohols, and other substances.1 Other bacteria like the cellulose decomposing Spirochaeta cytophaga require only very specific compounds, namely celluloses, as sources of energy. The autotrophic bacteria need no complex carbon compounds as sources of energy or for structural purposes. They can derive their carbon for the synthesis of their protoplasm from the carbon dioxide of the atmosphere or in solution. The facultative auto- trophic bacteria can obtain their carbon either from CO2 or from organic compounds. There is some evidence, however, that growth of heterotrophic organisms is also favorably affected by the presence of C02, as in the case of Bac. subtilis and Bad. vulgare, which could not grow when both oxygen and carbon dioxide were removed.2 The presence of carbon dioxide is essential not only for the growth of aerobic bacteria but also for the development of the anaerobic forms. In view of the fact that the microbial cells contain between 3 and 15 per cent of nitrogen, large quantities of this element have to be assimilated, particularly by organisms producing an extensive growth. Nitrogen is obtained from proteins and their degradation products or simple inorganic nitrogenous compounds, including the ammonium salts of organic and inorganic acids and nitrates. Some organisms, especially the heterotrophic bacteria, prefer and many even require complex proteins, albumoses or peptones as a source of nitrogen (and energy), while other microorganisms, especially the fungi and auto- trophic bacteria, will thrive just as well and sometimes even better upon simple compounds of nitrogen. Bad. pyocyaneum can obtain its nitrogen from amino compounds, amides, nitrates and nitrites, but these substances must be changed, either by hydrolysis or by reduc- tion, to ammonia before they are assimilated. The nitrogen-fixing 1 Supniewski, J. Untersuchungen iiber den Stoffwechsel der Kohlenstoff- verbindungen bei Bacillus pyocyaneus. Biochem. Ztschr., 154: 90-97, 98-103. 1924. 2 Rockwell, G. E. The influence of C02 on the growth of bacteria. Jour. Infec. Dis., 32: 98-104. 1923; 35: No. 6. 1924; 38: 92-100. 1926; Valley G., and Rettger, L. F. Preliminary report on the influence of carbon dioxide on bacterial growth. Abstr. Bact. (Proc), 9: 344-345. 1925. PRINCIPLES OF MICROBIAL METABOLISM 369 microorganisms, capable of utilizing free nitrogen gas, in the absence of available compounds of this element, stand as a group by themselves. The minerals, chiefly phosphates and potassium salts, but also iron, magnesium, sulfur, calcium and traces of other elements, are utilized by all microorganisms either in the form of simple inorganic compounds or are obtained from complex organic substances in the process of their decomposition. The minerals may often be obtained from insoluble inorganic materials, especially if the organism produces acids which tend to make them soluble. In the utilization of nutrients by heterotrophic microorganisms, two general stages are observed. (1) The dissimilation or decomposition stage, in which organic matter is broken down by the agencies of hy- drolysis, oxidation and reduction. (2) Assimilation stage, or syn- thesis, whereby the cells of microorganisms are built up out of the substances previously broken down. The metabolism of autotrophic bacteria consists only of the syn- thesizing stage so far as organic substances are concerned. The auto- trophic microorganisms utilize for their synthesis the products of dis- similation of the heterotrophic organisms, such as the various minerals, nitrogen compounds and even energy sources such as ammonia, hydro- gen sulfide, etc. The heterotrophic microorganisms utilize for their dissimilation stage the products of assimilation of the autotrophic forms, namely the complex organic substances synthesized by these cells. HjO"* NH3^ RjS COz organic acids other compounds Autotrophic organisms Heterotrophic organisms Only the autotrophic organisms actually produce work, in the true thermodynamic sense, as shown later, while the heterotrophic forms may simply build up the new protoplasm out of the constituents of the medium. This explains the considerably greater assimilation and the more extensive protoplasm produced for the same amount of energy available. Chemical reactions in the microbial cell. The microbial cell may be considered as an osmotic system. The absorption and liberation of substances by microorganisms leads to a series of chemical reactions 370 PRINCIPLES OF SOIL MICROBIOLOGY necessary for the continuation of life and characteristic of the living cell. Most of these reactions are carried on in the cell by the agency of organic catalysts or enzymes, which may also be secreted outside of the cell; this allows certain chemical reactions to take place outside of the cell. The chemical reactions depend on the presence of specific substances or substrates, the chemical and physical condition in which these substances are present, temperature, reaction, etc. The activities of the microorganisms will result in a change both in nature of the substrate as well as in the condition of the medium in which they work. Celluloses and proteins, substances of high molecular weight and low osmotic pressure, will be changed by processes of hydrolysis, to sugars and organic acids or to peptides and amino acids, substances of low molecular weight and high osmotic pressure. On the other hand, the absorption of soluble nitrogen salts and minerals and their syn- thesis into microbial protoplasm will bring about a reverse condition. The ionic exchange in the living cell, as the absorption of the base in the case of ammonium salts or absorption of the acid in the case of nitrates, will tend to leave the medium more acid or more alkaline respectively. The formation of organic acids, such as gluconic, citric, oxalic and fumaric by fungi, lactic, formic, propionic and acetic by bacteria, and inorganic acids, such as carbonic, nitrous, nitric and sulfuric, will also lead to a change in the hydrogen-ion concentra- tion of the medium. These acids will combine with the insoluble and soluble bases forming new salts. Some of these, such as the salts of organic acids, may be used as sources of carbon, liberating the bases which will combine with the carbon dioxide of the atmosphere to form carbonates, and again change the reaction of the medium from acid to alkaline. Others, like the nitrates and sulfates, may be again assimilated by microorganisms and higher plants. These may be washed from the soil in the drainage waters, they may be absorbed by the soil colloids, or they may form simple or complex salts with the various inorganic or organic soil constituents. All of these reactions bring about constant changes in the osmotic concentration and the reaction of the medium. This is further ac- centuated by the formation of electrolytes from non-electrolytes (am- monia and nitrates from proteins and amino acids, phosphates and sulfates from complex protoplasm) and vice versa. It is important, therefore, to gain knowledge of the osmotic concentration of the soil PRINCIPLES OF MICROBIAL METABOLISM 371 solution, as determined by the lowering of the freezing point,3 change in conductivity,4 or other convenient method ; also of the hydrogen-ion concentration, as determined by the electrometric or colorimetric method,5 and of the buffer content of the soil, as determined by the curves which show the relation between addition of acid and alkali and change in reaction. Enzymes of microorganisms. A large number of chemical reactions are carried on by the microbial cell by means of enzymes. These are either secreted outside of the cell (exo-enzymes) or remain within the cell and can be separated from it only with great difficulty (endo- enzymes). Among the different reactions, in which enzymes take an active part, we need mention but four: (1) Hydrolysis. This involves the transformation of various polysaccharides into sugars, of proteins into amino acids, of amino acids into ammonia and oxy-acids; the transformation of insoluble organic substances (starch, fibrin) into soluble forms, and finally glycolytic decompositions, as the trans- formation of sugar into lactic acid (C6H12O6 = 2C3H6O3). (2) Oxi- dation, resulting in the liberation of energy. Some of the oxidation processes, such as the formation of acetic acid from alcohol, citric and oxalic acids from glucose, are frequently referred to as fermenta- tions. These are distinguished from true fermentations, which result in the liberation of energy by the decomposition of complex organic compounds into simpler forms, without the intervention of free oxy- gen, as in alcoholic and butyric acid fermentations. (3). Reduction. Substances rich in oxygen are reduced to substances poor in oxy- gen, such as the reduction of nitrates, nitrites and sulfates, and the oxygen may be used for purposes of oxidation. The coupled reac- tions of oxidation and reduction, whereby the one substance is oxidized and the other reduced at the same time are common in microbio- logical reactions. (4) Synthetic reaction, including anhydride forma- tion and condensation. 3 Bouyoucos, G. J., and McCool, M. M. The freezing point method as a new means of measuring the concentration of the soil solution directly in the soil. Mich. Agr. Exp. Sta., Tech. Bui. 24, 1915; 27, 1916; 31, 1916; 37, 1917; 43, 1918. Hoagland, D. R. The freezing point method as an index of variation in the soil solution due to season and crop growth. Jour. Agr. Res., 12: 369. 1918. 4 Hibbard, R. P., and Chapman, C. W. A simplified apparatus for measuring the conductivity of electrolytes. Mich. Agr. Exp. Sta., Tech. Bui. 23, 1915. 6 Clark, W. M. The determination of hydrogen ions. The Williams & Wilkins Co., Baltimore. 1922. 372 PRINCIPLES OF SOIL MICROBIOLOGY Reaction velocity. Reactions between ions take place in homogeneous media with great velocity. In colloidal media, such as the living cell or the soil, the time factor is of great importance. The reactions be- tween molecules is much slower than between ions even in homogeneous media, but more so in such an heterogeneous medium as the soil. The kinetics of chemical reactions, which includes the decomposition of fats, carbohydrates, glucosides, proteins, as well as the various oxida- tion and reduction phenomena, is of prime importance in soil micro- biology. The course of change is determined by measuring at definite intervals of time the amount of change that has taken place and the results are calculated on a definite basis, such as moles per liter. The law of Wilhelmy,6 that the reaction velocity is at any given time pro- portional to the amount of unchanged substrate, applies to mono- molecular reactions, or where only one substance changes in concen- tration. The hydrolysis of sugars and glucosides belongs to this type of reaction, while saponification of esters is already bimolecular. The fundamental equation of the monomolecular reaction is dt It becomes, on integration, dx „ . . = K (a — x), K = \ln t a — x where a is amount of undecomposed substrate, x the amount decom- posed in t minutes. An understanding of the temperature coefficient is also important. In most reactions, with every increase in 10°C. tem- perature, the velocity is increased two to three times.7 This usually holds true at temperatures ranging from 0 to 35°. Above that tem- perature, the destructive effect of heat is greater than the stimulating action. The thermophilic bacteria will exist even at 55° to 65°C. and carry on their activities at such temperatures. Growth, life and death of microorganisms. The numbers of bacteria and other microorganisms in the soil vary greatly not only from day to day, but even within brief periods of time, as a result of the changes in the environmental conditions. The age of the bacteria may be only a few days, while they reproduce within twenty to thirty minutes. 6 Wilhelmy, L. Uber das Gesetz, nach welchem die Einwirkung der Siiuren auf Rohrzucker stattfindet. Pogg. Ann., 81: 413^99. 1850. 7 van't Hoff, J. H. Chemische Dynamik. 1898, p. 224. PRINCIPLES OF MICROBIAL METABOLISM 373 Growth of an organism can be continued indefinitely, when repeatedly- transferred upon fresh media. In the same medium, however, there is at first a decided increase in the numbers and activities of a micro- organism, soon coming to a maximum and this is followed by a rapid decrease. The limitations to further growth are the lack of nutrients and the formation of injurious waste products. Kruse reports that, after two days of growth on agar cultures, cer- tain bacteria are reduced to 10 per cent of the maximum; in three days, to 1 per cent and, in seven days, they are all dead. The curve shown in fig. II8 illustrates this process. Fig. 11. Rate of increase in numbers of a bacterium (from Buchanan) Different organisms grow at a different rate of rapidity and also disintegrate at a different rate. Growth of microorganisms in a limited amount of medium soon reaches a maximum due to the exhaustion of one or more nutrients in the medium or to the formation of injurious by-products, such as acids, alkalies or some toxic substances.9 While the older parts die off, the younger may still continue to grow and these may use the nutrients made available by the decomposition (autolysis) of the older parts. A microbial cell thus passes through a period of youth, full develop- ment and old age. These stages can be expressed by the autocatalytic 8 Buchanan, R. E. Life phases in a bacterial culture. Jour. Inf. Dis., 23: 109-125. 1918. 9 Chambers, W. H. Studies in the physiology of the fungi. XI. Bacterial inhibition by metabolic products. Ann. Mo, Bot. Card., 7: 249-289. 1920. 374 PRINCIPLES OF SOIL MICROBIOLOGY curve, suggested by Robertson.10 A difference exists in the physio- logical activities and in the physico-chemical condition of young and mature cells.11 A medium, in which growth of a certain organism has ceased as a result of accumulation of an injurious substance, may be treated so as to neutralize the accumulated acid or alkali, the limiting nutrient may be added, or finally the toxic substance may be destroyed by means of heat.12 The organism will then begin to grow again and 0 24 48 72 96 120 Time in hours Fig. 12. Rate of increase in numbers of a protozoan (after Cutler and Crump) pass through another cycle of activities. Limitations of growth and formation of certain substances (ammonia, nitrate) may thus be due 10 Robertson, T. B. On the nature of the autocatalyst of growth. Arch. Entw. Mech., 37: 497-508. 1913; Tables for the computation of curves of auto- catalysis, with especial reference to curves of growth. Univ. Cal. Publ. Physi- ology, 4: 211-228. 1915. 11 Sherman, J. M., and Albus, W. B. Physiological youth of bacteria. Jour. Bact., 8: 127-138. 1923. 12 Rahn, O. Uber den Einflusz der Stoffwechselprodukte auf das Wachstum der Bakterien. Centrbl. Bakt. II, 16: 417-429, 609-617. 1906. PRINCIPLES OP MICROBIAL METABOLISM 375 either to reactions of auto-catalysis, as 'suggested by Robertson, or perhaps more likely to the development of a certain balance between the reproduction of the cells and the accumulation of injurious products of metabolism.13 Miyake,14 using the results of Lipman and asso- ciates15 on ammonia accumulation and of Warington on nitrate accum- ulation in the soil, calculated that these processes are autocatalytic chemical reactions. The maximum rate occurs when the total amount / /nn jo -£ -ici S O .« S3 . /a 0 ■f<. '' r< < ■> C / J" J + S 6 7 8 i) /& 7/ / P Gijji^-t — | — r"i~ 1.7 1.6 1.5 1.4 . 13 12 1.1 1-0 0.9 Kur £x?6 ' o o 1 c o ■9 o » -f>m __, ^ » f- _ jLo 10 I— 0 __ ^ » jL_ _ 1 *> J~ 1 ol — I — I — I — L_J — I — I — I — I — EL ;.i 1.8 7.8 8.0 Fig. 17. Influence of reaction upon the respiration of nitrite-forming bacteria (after Meyerhof). For the study of respiration, Meyerhof grew the organism in the general medium till a good growth had taken place. A certain con- centration of the nitrite was introduced into a definite volume of cul- ture and the flask placed in thermostat. The change produced in a definite period of time (usually 4 to 6 hours) was taken as a measure of the respiratory power of the culture. Meyerhof demonstrated that, outside of the nitrite oxidation and carbon assimilation, no other reac- tion takes place in the process of energy transformation. The nitrate organism utilizes only the nitrite-ion as a source of energy and no other substance. With an increasing nitrate content, growth and respiration are ENERGY TRANSFORMATION 393 lessened (fig. 19). This injury is caused also by other salts in similar osmotic concentrations. A decrease in the concentration of oxygen lessens both growth and respiration, so that at TV atmosphere pressure, respiration is decreased by 66 per cent (fig. 20). This injury is reversible. Growth may be even more injuriously affected than respiration. The optimum and minimum reaction points for respiration lie at pH 5.6 and 10.3 respectively, with an optimum pH 8.3 to 9.3 (fig. 17). 17 For growth and respiration definite concentrations of 02, N02, N03, OH- and traces of H2C03 are required. The other salts and nutrients play only the part of buffering agents. 100 V o > o SO 50 1 ' 1 - — •— 0,1% 0,5% 1% 2% 3% 4% "Nitrite Concentration Fig. 18. Influence of nitrite concentration upon the oxidation velocity of nitrate forming bacteria (from Meyerhof). Winogradsky observed the interesting phenomenon that ammonium salts injuriously affect the growth of nitrate bacteria. This seemed rather strange in view of the fact that the nitrate bacteria are active side by side with the nitrite bacteria which use the ammonium salt as a source of energy. It was then'8 suggested that the two processes follow in two successive periods in the soil, nitrate formation beginning only after all the ammonium salt is converted into nitrite. On de- 17 As shown elsewhere (p. 528), the limiting and optimum reactions for the growth of the nitrate-forming organism are different from the optima of respiration. 18 Omclianski, W. tJber die Nitrifikation des organischen Stickstoffes. Centrbl. Bakt. II, 5: 473-490. 1899. 394 PRINCIPLES OF SOIL MICROBIOLOGY creasing the amount of Na2C03, which would lead to a lower alkalinity, Boulanger and Massol19 found that the injurious effect of ammonium salt is less and concluded, therefore, that the growth of nitrate bacteria is not injured by the salt but by free ammonia. This was confirmed by Meyerhof who found that the injurious influence of ammonia and its derivatives (aliphatic amines) consists in the penetration of the base into the cell (which does not take place in the case of ammonium salt) and in a specific action of the NH3 and NH2 group. Lipoid in- vzo no 90 d .2 80 ■3 •a o 60 50 — T~ ■ \ "<5 'a N, -*-o 0 12 3 4 5 6 Nitrate concentration, percent Fig. 19. Influence of nitrate concentration upon the growth ( — piration (— ■) of nitrate forming bacteria (from Meyerhof). -) and res- soluble amines, like the diamines, are not injurious. The injurious effect of amines and cations depends upon their ability to penetrate into the cell and upon the reaction of the media; respiration is usually less affected than growth. Iron salts are taken out by the organism from iron poor media. Whether or not iron plays the part of a catalyzer is still undecided. According to Ashby,20 nitrification takes place in the "Boulanger and Massol, 1905 (p. 65). 20 Ashby, S. F. Some observations on nitrification. 67. 1907-8. Jour. Agr. Sci., 2: 52- ENERGY TRANSFORMATION 395 presence of ferric hydroxide to a considerable extent. The important role of iron was also established for the growth and respiration of various other bacteria. As to the energy utilization of the bacteria oxidizing ammonium salts, the following results of Winogradsky are illuminating: N oxidized (from NHj to NOj), mgm 722.0 506.1 928.3 815.4 C assimilated (from CO.), mgm 19.7 15.2 26.4 22.4 N:Cratio 36.8 33.3 35.2 36.4 About 35 parts of nitrogen were thus found to be changed from am- monia to nitrite for every part of carbon assimilated from the CO2 of TABLE 25 Influence of organic matter upon the growth of nitrite and nitrate-forming bacteria NITRITE ORGANISM NITRATE ORGANISM SUBSTANCE Growth checked Growth stopped Growth checked Growth stopped Glucose ■per cent 0.025 0.025 0.025 More than 0.2 More than 0.2 0.5 per cent 0.05 0.2 0.3 More than 1 . 5 per cent 0.05 0.8 0.05 0.05 0.5 1.5 0.0005 per cent 0.2 Peptone 1.25 Asparagine 0.5 Glycerol 4.0 Urea 4.0 Sodium acetate Ammonia 3.0 0.015 the atmosphere. Similar results were obtained by Meyerhof. In the process of oxidation of ammonia to nitrite four times as much energy (78.4 Calories per mol) is liberated as in the process of oxida- tion of nitrite to nitrate (18.8 Calories per mol). But four times as much nitrogen is transformed in the latter processes (N:C = 135). The utilization of energy for chemosynthesis of carbon is, therefore, almost identical and is equivalent to about 5 per cent. With an active culture under proper conditions, the process of oxi- dation of ammonium salts is very rapid. Meyerhof found that, with a maximum aeration, 4 grams of ammonium sulfate are oxidized to nitrite in one liter of medium in 24 hours. Respiration decreases with a decrease in oxygen concentration, the reaction being reversible. The reaction optimum for respiration is at a pH 8.4 to 8.8, with mini- 396 PRINCIPLES OF SOIL MICROBIOLOGY mum points at 9.4 and 7.6. Warington21 believed that nitrification of ammonia could take place only with ammonium carbonate and that the function of carbonates in solution was to supply the ammonium carbonate by interaction with the other ammonium salts. This was, however, found to be incorrect by Bonazzi,22 and others. According to Bonazzi, the oxidation of ammonia can be considered to take place in two steps: (a) respiration, with resultant gain in energy and syn- chronous nitrogen absorption; (6) nitrogen assimilation (nitrification 100 90 80 "•>-1 70 V 0 G 50 o 3 40 13 • 1-1 3 30 20 10 /__ _._ . . - 0,1 0,3 0,4 0.6 0,7 0.8 1 atmosphere Fig. 20. Influence of oxygen pressure upon the oxidation of nitrite to nitrate (from Meyerhof). proper) whereby oxidation of the absorbed nitrogen takes place, the utilized portion goes to make up the following cell generations, ni- trous acid is split off and excreted as a non-utilizable product, and energy is liberated. 21 Warington, 1878-1891 (p. 63). 22 Bonazzi, A. On nitrification. IV. The carbon and nitrogen relations of the nitrite ferment. Jour. Bact., 6: 479-499. 1921. ENERGY TRANSFORMATION 397 The influence of organic matter on nitrite and nitrate-forming bac- teria has been studied in detail by Winogradsky and Meyerhof . These results indicate that soluble organic substances have a distinct in- jurious effect upon the respiration and growth of both the nitrite and nitrate-forming bacteria. The injurious effect of amines was found to be parallel to their "lipoid solubility." The importance of this phenom- enon in soil processes is discussed elsewhere. A detailed study of the influence of various ions and ion antagonism upon the respiration of these two groups of organisms has been made by Meyerhof. Energy utilization from the oxidation of sulfur and its compounds. Winogradsky23 was the first to demonstrate that certain bacteria living in sulfur springs, like Beggiatoa, Thiothrix and others, use H2S as a source of energy. This is first oxidized to sulfur and water: H2S + h (0)2 = H20 + S + 61.1 Cal. The reaction H2S = H2 + S is endothermic, but the simultaneous oxidation of the hydrogen makes the process exothermic. The sulfur accumulates within the cells and is further oxidized, in the presence of carbonates (CaC03). (S) rh + l\ (02) + H20 = H2 S04 + 142.2 Cal. H2S is a weak acid and undergoes hydrolytic dissociation, which increases with a decrease in the hydrogen-ion concentration: K! [H,S] = [H+] [HS-] in which Ki = 0.91 X 10 ~\ at 18°C. K2 [HS-] = [H+] [S— ] K2 is very small, about 10 ~15 Baas-Becking24 assumed that not the H2S as such, but rather the hydrosulfide is used by the sulfur bacteria as a source of energy, since the H2S has a lower energy value. This is seen by arranging the sul- fur compounds according to their free energy levels. 23 Winogradsky, 1887 (p. 80). 24 Baas-Becking, L. G. M. The source of energy of the sulfur bacteria. Proc. Soc. Exp. Biol. Med., 22: 127-130. 1924; Studies on the sulfur bacteria. Ann. Bot., 39: 613-650. 1925. 398 PRINCIPLES OF SOIL MICROBIOLOGY s-- | 20 Cal. HS~ | 3 Cal. S | 6.5 Cal. H2S aq. | 170 Cal. so — H2S itself is probably poisonous to Beggiatoa as it is to other bacteria. The hydrogen sulfide is produced by bacteria from the decomposition of organic matter, from the reduction of sulfates and other compounds of sulfur and by inorganic agencies (p. 600), and is used as a source of energy. The sulfur bacteria can be divided into three physiological groups on the basis of the sulfur compounds from which they derive their energy : 1. Sulfide bacteria. These organisms obtain their energy chiefly from the oxidation of hydrogen sulfide and other sulfides. They include the thread-form- ing, purple, and other bacteria accumulating sulfur within their cells. According to Keil,26 only Beggiatoa and Thiothrix are autotrophic bacteria, while the purple bacteria are not autotrophic.26 2. Thiosulfate or thionic acid bacteria. These obtain their energy primarily from the oxidation of thiosulfates. Some seem to be also capable of oxidation of elementary sulfur. They include Thiohacillus thioparus Beij. and the various strains of Th. denitrificans Beij. The first is obligate autotrophic while the second, at least some strains of it, is facultative autotrophic. According to Lieske,27 Th. denitrificans assimilated 10.9 mgm. of carbon for every gram of thiosulfate oxidized. One gram of thiosulfate is equivalent to 405 mgm. of sulfur. Therefore, thirty-seven parts of sulfur are oxidized to two parts of carbon assimi- lated chemosynthetically. Nathanson first suggested the following reaction for the process of trans- formation of thiosulfate, which results in the accumulation of sulfur in the medium : 3 Na2 S2 03 + 2K02) = 2 Na2 S04 + Na2 S4 06 The free sulfur, which was formed in the medium, was believed to originate by the chemical interaction of the tetrathionate with some of the thiosulfate. How- ever Beijerinck believed the sulfur to be produced directly from the thiosulfate: Na3 S2 03 + M02) = Na2 S04 + (S) 26 Keil, 1912 (p. 81). "Molish, 1907 (p. 83). 27 Lieske, 1912 (p. 86). ENERGY TRANSFORMATION 399 The organism isolated by Trautwein oxidized thiosulfate without the forma- tion of free sulfur. The results obtained tend to sustain the reaction suggested by Nathanson: 4 Na2 S- 03 + 6 02= 4 Na2 S2 06 3 Na2 Ss 03 + 2§(02) = Na2 S4 06 + 2 Na2 S04 Sulfate, dithionate and tetrathionate are formed, but no free sulfur and no sul- furic acid. In the oxidation of sulfur and reduction of nitrates by Th. denitrificans, the following reactions have been suggested: 5(S)rh + 6 KN03 + 2 H20 = K2S04 + 4 KHS04 + 3 N£ 5(S)rh + 6 KNO3 + 2 CaC03 = 3 KaS04 + 2 CaS04 4- 2 C02 + 3 N2 5 Na2S203 + 8 KNO3 + 2NaHC03 = 6Na2S04 4- 4K2S04 + 2C02 4- 4N2 4- H20 3. Sulfur bacteria. These organisms derive their energy primarily from the oxidation of elementary sulfur. They include Th. thiooxidans Waksman and Joffe. The oxidation takes place according to the reaction given above (p. 397). One mol of rhombic sulfur will thus liberate 142.2 Cal. Thiosulfate is oxidized by this organism to sulfate and sulfuric acid. Thiobacillus thioparus was found to derive its carbon only from the CO2 of the atmosphere or from bicarbonates and carbonates in solu- tion.28-30 Organic materials do not exert any injurious influence, nor stimulate growth, since they cannot take the place of C02. Th. denitrificans is capable also of developing readily on organic media, assimilating organic carbon ;29-31 in other words, this organism is facul- tative autotrophic. On thiosulfate media the carbon is derived either from C02 or bicarbonates but not from carbonates. Lieske,32 how- ever, could not find any injurious or stimulating action due to organic matter, Th. denitrificans deriving its carbon only chemosynthetically. Although small quantities of glucose are used up by Th. thiooxidans, neither the growth of the organism nor the sulfur-carbon ratio (sulfur oxidized \ 7^ : ., , , . : r — ) are appreciably affected.33 It re- C02 assimilated from atmosphere/ mains to be found, therefore, in what connection the glucose is used. 28 Nathanson, 1902 (p. 84). 29 Beijerinck, 1904 (p. 84). 30 Jacobsen, 1912 (p. 85). "Trautwein, 1921 (p. 87). 82 Lieske, 1912 (p. 86). 33 Starkey, R. L. Concerning the physiology of Thiobacillus thiooxidans, an autotrophic bacterium oxidizing sulfur under acid conditions. Jour. Bact., 135-164, 165-195. 1925. 400 PRINCIPLES OF SOIL MICROBIOLOGY According to Trautwein respiration does not depend on the oxygen tension; oxidation is not affected even at a pressure of f atmosphere. At iV atmosphere, however, respiration completely ceases. Th. deni- trificans can grow in the complete absence of oxygen when nitrates are present. The latter are reduced to atmospheric nitrogen and the oxygen is used for the purpose of oxidation. Sulfates, even up to 5.0 per cent concentration, do not injure sulfur oxidation. In this respect the thiosulfate bacteria behave differently from the Nitrosomonas and Nitrobacter, since the sulfur organisms are not injured by the oxidation products while the nitrite and nitrate bac- teria are. The chlorine ion exerts, however, an injurious influence: oxidation of sulfur is reduced by 38 per cent at 0.3 N concentration of NaCl. The optimum reaction for the activities of Th. denitrificans lies at a pH 7.9 to 9.1, with a minimum of pH 3.5. In acid media the reaction tends to become more alkaline as a result of the activities of the organ- ism, since no free acid is formed. The most extensive work on the respiration of sulfur-oxidizing bacteria has been done with Th. thiooxidans. This organism is capable of utilizing elementary sulfur, sulfide and thiosulfate as sources of energy. The carbon is derived only from carbon dioxide. The presence of glucose is not injurious to the growth and sulfur oxidizing capacity of the organism but it cannot serve either as a source of energy or as a source of carbon. No growth takes place when the cultures are placed in a C02-free atmosphere.34 Bicarbonates, particularly when the proper reaction of the medium is obtained by means of an acid, may take the place of C02 only to a small extent, as seen from table 26. The slight increase in acidity in the cultures of the ordinary medium and glucose medium grown in the C02-free atmosphere is due to the acidity introduced with the inoculum (3 drops per 100 cc. of culture medium). It may be of interest to point out, in this connec- tion, that the optimum reaction for the growth of the organism is pH 1.0 to 5.0. A modified medium may be used for this purpose, namely 0.2 gram (NH4)2S04, 0.1 gram MgS04, 0.01 gram FeS04, 0.25 gram CaCl2, 3.0 grams K2HP04, 10 grams sulfur in 1000 cc. water. The water is distilled twice, the second time using glass containers only and pre- 34 Waksman, S. A., and Starkey, R. L. Carbon assimilation and respiration of autotrophic bacteria. Proc. Soc. Exp. Biol. Med., 20: 9-14. 1922. ENERGY TRANSFORMATION 401 viously treating the water with KM11O4. The ratio between the sulfur oxidized and carbon assimilated from CO2 of the atmosphere (S:C) was found to be about 32. With thiosulfate as a source of energy the ratio was found to be about 64. The apparatus used for carbon de- termination is given in figure 16. About six and two-thirds per cent of the heat liberated in the oxida- tion of elementary sulfur is utilized for the chemosynthetic assimilation of carbon.35 However, when conditions are unfavorable for the de- velopment of the organism, the sulfur-carbon ratio may increase. For example, the presence of nitrates in the medium, which exerts a toxic effect upon the respiration of the organism, resulted in a wider sulfur- TABLE 26 Influence of carbon source upon the growth and sulfur oxidation of Thiobacillus thiooxidans ATMOSPHERE TREATMENT Ordi nary C02 -free Final pH Titre* PH Titre* Regular medium, control 4.2 1.2 3.0 1.2(-) 6.6 5.4 6.2 1.5 2.20 12.15 2.20 13.15 1.3 2.0 2.2 9.3 4.2 3.8 3.0 2.8 6.6 6.0 6.2 5.3 2.20 Inoculated 2.25 1 per cent glucose, control 2.2 Inoculated 2.3 0. 1 per cent NaHCOs, control 1.3 Inoculated 1.75 0.1 per cent NaHC03 + H3P04, control Inoculated 2.2 2.5 * Titre = cubic centimeter of 0.1N NaOH necessary to neutralize 10 cc. of medium, with phenolphthalein as indicator. carbon ratio. The sulfates were found to have no effect on the respira- tion of the sulfur oxidizing bacteria, except in very high concentrations. Nitrates are very toxic, especially in concentrations above 0.2 molar. Phosphates proved to be more toxic than sulfates, but less toxic than nitrates. As stated above, glucose was not found to be toxic, but peptone became toxic in concentrations of 0.05 per cent. The concen- tration of sulfur in the medium did not exert any important influence on sulfur oxidation; an increase in the quantity of sulfur led to an increase in oxidation. As to the influence of sulfuric acid or the prod- 35 Waksman, S. A., and Starkey, R. L. On the growth and respiration of sulfur oxidizing bacteria. Jour. Gen. Physiol., 5: 285-310. 1923. 402 PRINCIPLES OF SOIL MICROBIOLOGY uct of the reaction, it was found that normal growth took place even in concentrations of 0.25 molar, while 0.5 molar did not completely prevent growth. In general, the metabolism of the Thiobacillus thio- oxidans approached, in its energy utilization, very closely that of Nitro- somonas and Nitrobacter. Energy utilization from the oxidation of iron compounds. Certain bacteria are able to utilize the energy obtained from the oxidation of certain iron compounds and live autotrophically. According to Lieske,36 Spirophyllum ferrugineum can grow in inorganic media free from or- ganic matter and oxidize ferrous carbonate to ferric hydroxide. The process supplies the organism with the necessary energy for the chemo- synthetic assimilation of CO2: 2 Fe C03 + 3 H20 + £(02) = Fe2(OH)6 + 2 C02 + 29.8 Cal. One mol of ferrous carbonate thus liberates only about 15 Calories, and 1 gram of the substance oxidized liberates 0.12 Cal. The amount of energy obtained is small in comparison with that liberated in the processes of oxidation of the nitrogen and sulfur compounds. The organism has to oxidize, therefore, large quantities of iron to obtain enough energy to assimilate the necessary carbon chemosynthetically. This results in the accumulation of large quantities of iron hydrate in the bacterial bodies; to assimilate one part of carbon, about 750 parts of hydrate must be formed. This figure is, however, far from accurate, as stated by Lieske. Other ferrous salts will not take the place of the carbonate. Much organic matter impairs growth and may finally stop it altogether. Some organisms, like species of Leptothrix, can live without iron compounds, but can utilize them either in the form of ferrous carbonate or as soluble organic iron salts. Other bacteria will use certain soluble organic iron compounds, but cannot utilize inorganic iron salts. We have here a series of transition stages from pure autotrophy to pure heterotrophy. Harder37 divides the iron bacteria, as regards their phys- iological activities, into three groups: 1. Those that precipitate ferric hydroxide from solutions of ferrous bicar- bonate and use the carbon dioxide liberated and the energy produced during oxidation for their life activities (autotrophic). 36 Lieske, 1911 (p. 95). 37 Harder, E. C. Iron depositing bacteria and their geologic relations. Prof. Paper 113, U. S. Geological Survey, Washington. 1919. ENERGY TRANSFORMATION 403 2. Those which do not require ferrous bicarbonate for their life processes but which deposit ferric hydroxide when either inorganic or organic iron salts are present (facultative autotrophic). 3. Those which attack organic iron salts, using the organic acid radical as a nutrient and precipitating ferric hydroxide or basic ferric salts which are gradu- ally changed to ferric hydroxide. Inorganic iron salts, however, are not utilized as sources of energy and this group is heterotrophic. Energy utilization from the oxidation of hydrogen. The oxidation of hydrogen by bacteria is accompanied by the liberation of large quanti- ties of heat, according to the following reaction: H2 + KOj) = H20 -f- 6S.4 Cal. Thus 1 gram of hydrogen gives more than eight times as much heat of combustion (34.2 Cal.) as 1 gram of starch (4.1 Cal.). Some of the hydrogen bacteria {Hydrogenomonas pantotropha) can grow on purely inorganic media, utilizing the energy obtained from the oxidation of hydrogen for the assimilation of carbon chemosynthetically from the C02 of the atmosphere.38 Kaserer suggested that the mechanism of hydrogen transformation will depend on the nature of the organism. Hydrog. pantotropha changes the hydrogen and carbon dioxide directly into the first substance necessary for the synthesis of organic matter, namely to formaldehyde. H2 CO, + 2 H2 = C H20 + 2 H20; CH20 + 02 = H2C03 The oxidation of hydrogen is thus found to lead to two processes, assimilation of carbon dioxide and oxidation of the hydrogen. For every volume of C02 assimilated, two volumes of hydrogen gas are oxidized. For every two volumes of hydrogen oxidized one volume of oxygen disappears. The formation of formaldehyde as an inter- mediary product was not confirmed by Lebedeff . The bacteria (Hydrog. vitrea and Hydrog. flava) isolated by Niklewski39 also grew in an inorganic solution with an atmosphere of H2, O2, and C02. Organic compounds were formed from the hydrogen and carbon dioxide; those were then oxidized to carbon dioxide and water during respiration. The auto- trophic nature of the process has been definitely established by Lebe- 88 Kaserer, 1906 (p. 97). See Klein and Svolba, Ztschr. Bot. 19: 65. 1926. 39Niklewski, 1910 (p. 98). <0 Lebedeff, A. F. tiber die Assimilation des Kohlenstoffes bei wasserstoff- oxydierenden Bakterien. Ber. deut. bot. Geaell., 27: 598-602. 1909. Nabokich and Lebedeff, 1907 (p. 98). 404 PRINCIPLES OF SOIL MICROBIOLOGY deff,40 who found that the growth of Bac. hydrogenes in an inorganic medium resulted in an exchange of gases (reduced to 0° and 760 mm.) with the H2:02 varying from 2.00 to 2.62. 588 to 1860 cc. H2 are oxi. dized .for every 100 cc. C02 assimilated. The oxidation of H2 is not necessarily connected with the reduction of C02. The ratio H2:C02 de- pends upon the age of the culture. Since the organisms utilize the Fig. 21. Culture vessel for study of hydrogen utilization by bacteria (from Iluhland). energy obtained from the oxidation of the H2 for the reduction of C02, the H2:C02 ratio is wider in older than in younger cultures. The energy process was believed to be independent of the process of assimi- lation of carbon and could be expressed by the equation: 2 H2 + 0-2 = 2 H20 ENERGY TRANSFORMATION 405 The gas exchange in the autotrophic assimilation of carbon by non- chlorophyll-containing bacteria is thus similar to the gas exchange of green plants. On organic media, the organism grows in a manner similar to heterotrophic bacteria. In the presence of organic matter the organism oxidizes H2 considerably less than in its absence. Fig. 22. Reaction of medium and oxidation of hydrogen by bacteria (Ruhland) The organisms oxidizing hydrogen are thus found to be facultative autotrophic, i.e., they are able to develop heterotrophically in the absence of hydrogen. Whenever hydrogen is present, however, they are cap- able of developing autotrophically. The presence of iron (when only 2.4 x 10-5 mgm. Fe are present in 50 cc. no growth or hydrogen oxidation takes place) and a proper pH value (6.8 to 8.7 opt.) were found to be essential for the respiration of organisms oxidizing hydro- 406 PRINCIPLES OF SOIL MICROBIOLOGY gen.41 On the acid side, growth diminishes due to the disappearance of HCO,3~ (in favor of CO2) and to the direct action of the hydrogen-ion on the hydrogen oxidation. On the alkaline side, growth diminishes due to an increasing diminution of the iron. Organic carbon sources exert a certain protective action upon the oxidation of the hydrogen due to the production of organic acids and, therefore, a reaction un- favorable to the bacteria. Organic substances, which do not result in formation of acid, as well as small amounts of sugar, tend to slow down the oxidation of hydrogen at first, but then accelerate it. The reaction is carried on to completion, so long as there are any traces of H2 and 02 in the atmosphere. Different C02 pressures influence growth by influencing the reaction. The hydrogen-oxygen (elemental) ratio is usually greater than two to one, reaching 2.78 because of the formation of free oxygen in the reduction of C02. The most efficient utilization of the energy of the reaction for chemosynthetic purposes is at a neutral reaction when 1 cc. C02 is assimilated for 8 cc. H2 oxi- .„,.... „ C02 assimilated .. , dized. Ihe utilization quotient Qn = — lies between H« oxidized 0.01 and 0.148. As much as 20 per cent of the energy liberated may be used for the chemosynthetic assimilation of C02. Partial pressure of H2 has no influence on the energy utilization and C02 assimilation. Nitrate is reduced to nitrite (not to atmospheric nitrogen), but the oxygen cannot be used for the anaerobic oxidation of hydrogen. Narcotics (urethane, HCN) exert an injurious effect upon oxidation. Energy utilization from the oxidation of simple carbon compounds. Methane is oxidized by certain bacteria which use the energy thus obtained for the chemosynthetic utilization of C02. CH4 -f 2 02 = C02 + 2 H20 + 218 Cal. This process is carried out, under autotrophic conditions, by Metha- nomonas methanica.42 Mlinz43 found these bacteria developing readily in an atmosphere rich in methane with an optimum concen- tration of oxygen at 2 per cent. Nitrogen is utilized in inorganic and organic forms. The organism is facultative autotrophic, being 41 Ruhland, W. Aktivierung von Wasserstoff und Kohlensaureassimilation durch Bakterien. Ber. deut. Bot. Gesell., 40: 180-184. 1920-1922; also Ruhland, 1924 (p. 101). 42 Sohngen, 1906 (p. 96). 43 Miinz, E. Zur Physiologie der Methanbakterien. Diss. Halle. 1915. ENERGY TRANSFORMATION 407 able to develop heterotrophically. The methane cannot be replaced by hydrogen or carbon monoxide, but can be replaced by various organic compounds, such as alcohols, carbohydrates, and salts of organic acids. The utilization of carbon monoxide as a source of energy by bacteria was demonstrated by Kaserer: CO + K02) = C02 + 74 Cal. Closely related processes to the autotrophic utilization of inorganic compounds are the oxidation of simple organic compounds, of which we need mention only the oxidation of alcohol to acetic acid, and the utilization of hydrocarbons as sources of energy. The CO2 assimila- tion of these as well as of the methane bacteria is not established. The physiology of these organisms is little understood. The acetic acid bacteria are divided,44 on the basis of their carbon and nitrogen utilization, into two groups: (1) haplotrophic, those which obtain their energy from the oxidation of alcohol, and (2) symplo- trophic, or those which require organic compounds. The energy utili- zation of the former is of interest here: CH3-CH2-OH + 02 = CH3COOH -f H20 + 112 Cal. 1.47 Cal. are thus liberated for every gram of alcohol oxidized. These bacteria do not need any other organic matter, either as sources of energy or carbon. The tendency was, therefore, to classify these organisms with the autotrophic bacteria. According to Neuberg,45 however, in the case of the acetic acid bacteria the ethyl alcohol is first oxidized to acetaldehyde; this is either further oxidized to acetic acid or is used for the synthesis of the microbial protoplasm. These or- ganisms, therefore, are not autotrophic since they do not derive their carbon from carbon dioxide and, in regard to energy and carbon utili- zation, may both be classified with the heterotrophic organisms. CH3 ■ CH2 • OH CH3 • CH2OH CH3 • CH, • OH -> CH3 • CHO CH3 • CHO \ \ CH3 • COOH CH3 • COOH 44 Janke, A. Forschungsergebnisse auf dem Gebiete der Essigbakteriologie und Fortschritt der Garungsessigindustrie. Centrbl. Bakt. II, 53: 81-124. 1921. 46 Neuberg, C, and Windisch, F. Vom Wesen der Essiggrirung und von verwandten Erscheinungen. Die Naturwiss., 13: 993-996. 1925; Biochem. Ztschr., 166: 454-481. 1925.< 408 PRINCIPLES OF SOIL MICROBIOLOGY Energy is liberated in the oxidation of the ethyl alcohol to acetaldehyde, but only one-third of that energy is liberated in the oxidation-reduction of the acetaldehyde. The nature of energy utilization by bacteria capable of oxidizing hydrocarbons and amorphous carbon46 still remains to be studied. Heterotrophic utilization of energy by microorganisms. The great majority of soil microorganisms, both in respect to numbers and species, have to depend for their energy supply upon complex organic or car- bonaceous substances. These may be simple or complex carbohy- drates, fats, or proteins and their derivatives. The carbohydrates contain about 40 to 45 per cent carbon, the proteins about 50, the lignins about 60, and the fats about 75 per cent. In the combustion of the carbon and the hydrogen, energy is liberated. The nutrient consumption by heterotrophic microorganisms follows three courses; viz., energy, structural or reserve purposes and residual substances. The energy goes to produce work against the exterior and to elevate the chemical potential of the substances formed.47 The organism may derive its energy and material needed for the synthesis of the protoplasm from the same nutrient or from two different nu- trients. Fats form only a rather small part of the constituents of the plant residues and manures added to the soil. Our chief attention may, therefore, be devoted to the other two groups of substances. Both carbohydrates and proteins may be utilized as sources of energy by most of the aerobic and anaerobic heterotrophic microorganisms. The decomposition products will differ. Under aerobic conditions a carbohydrate is usually decomposed to C02 and H20 or to various organic acids, C02 and H20. Under anaerobic conditions, it is de- composed into H2 or CH4, or both, and into different acids, C02 and H20. The amount of energy made available depends upon the nature of the decomposition taking place. Under aerobic conditions the proteins are decomposed to amino acids, NH3, C02, and H20; under anaerobic conditions, to different products of putrefaction, such as various amines, mercaptans, etc., NH3 and C02. 46 Tausz, J., and Peter, M. Neue Methode der Kohlemvasserstoffanalysemit Hilfe von Bakterien. Centrbl. Bakt. II, 49: 497-554. 1920. Any possible action of bacteria upon amorphous carbon is still a matter of conjecture. See Potter, M. C. Bacteria as agents in the oxidation of amorphous carbon. Proc. Roy. Soc. B, 80: 239-259. 1908. 47 Terroine, E. F., and Wurmser, R. L'energie de croissance. I. Le devel- oppement de l'Aspergillus niger. Bull. Soc. Chim. Biol., 4: 519-567. 1922; Wurmser, R. Ibid., 5: 506-528. 1923. ENERGY TRANSFORMATION 409 When carbohydrates are used as sources of energy, the organisms have to obtain their nitrogen from another source, either inorganic or organic. In the case of proteins, both the energy and nitrogen are obtained from the same source. In view of the fact that the proto- plasm synthesized by microorganisms has about the same carbon con- tent as the original protein and usually a lower nitrogen content and since only a part of the energy liberated is utilized by the organism and often a larger part converted into kinetic energy, the use of pro- teins as sources of energy results in a liberation of inorganic nitrogen, usually in the form of ammonia. Two methods are commonly employed for determining the energy transformation of microorganisms, (1) the differential method which determines the heat of combustion of a medium before and after the growth of microorganisms; (2) the direct method which measures the heat formed during the life processes of the microorganisms. Rub- ner48 found that both of these methods gave comparable results. A third method is also possible. It is based upon indirect calorimetry or the calculation of energy utilization from the chemical changes produced in the metabolism. This method has been used largely in the study of energy changes by autotrophic bacteria where the chemical substances involved are relatively simple in composition. In the case of the reactions brought about by heterotrophic bacteria, we are still in the dark as to a number of chemical and thermochemical reactions involved. Most nutrients are richer in oxygen than the microbial protoplasm. This is particularly true when nitrate forms the nitrogen source. Syn- thesis of protoplasm can, therefore, be looked upon in heterotrophic processes more as a process of reduction rather than of oxidation. The intake of oxygen, or respiration, serves the purpose of energy transformation, rather than for synthetic purposes. Oxygen oxidizes the nutrients available, the degree of oxidation determining the final products. The complete oxidation of carbohydrates gives car- bon dioxide and water; in the case of nitrogenous substances ammonia (or nitrate) and sulfuric acid are also produced. In the case of sub- stances low in oxygen (hydrogen-oxygen ratio more than 1), such as fats, fatty acids and alcohols, the amount of oxygen necessary for oxidation is so great that only aerobic organisms, such as fungi, are capable of using them as sources of energy. It must be emphasized 48Rubner, 1903 (p. 419). 410 PRINCIPLES OF SOIL MICROBIOLOGY in this connection that there is a distinct difference in the usage of the terms "supply, expenditure, utilization and transformation" of energy in the case of autotrophic bacteria on the one hand, and hetero- trophic on the other. In the case of the autotrophic organisms, a part of the energy goes to produce work in the true thermodynamic sense. In the case of the heterotrophic organisms, we can speak of a "supply and exchange of energy" only in specific cases, as in the syn- thesis of fats from carbohydrates, where thermodynamic work takes place. When bacteria or fungi growing in carbohydrate media, store away carbohydrates (hemicelluloses, cellulose or chitin), this is not work in the thermodynamic sense and is carried out with practically no energy transformation. One should differentiate between true syntheses which are also reductions and a mere assimilation of nu- trients. However, the formation of carbohydrates by microorganisms involves only a small loss of energy when carbohydrates are used as a substrate; a greater loss of energy with fats and a very considerable loss of energy when carbohydrates are formed from proteins as a substrate.49 Aerobic utilization of energy. Taking glucose as a starting point, the following products of oxidation are produced, depending on the degree of oxidation:50 C6H1206 + §02 = C6H1207 + ? Cal. Gluconic acid (1) 06H1206 + 02 = C6H10O7 + H20 + ?Cal. (2) Glucuronic acid C6H1206 + H02 - C6H807 + 2 H20 + 199 Cal. Citric acid (3) C6H1206 + 30, = C3H603 + 3 COl + 3 H20 Lactic acid (4) CaH120, + 4^02 = 3 C2H20, + 3 H20 + 493 Cal. Oxalic acid (5) C6H1206 + 602 = 6 C02 + 6 H20 + 676 Cal. (6) *9 Terroine, E. F., Bonnet, R., Jacquot, R., and Vincent, G. Rendements £nerg6tiques compares dans le d^veloppement de moissures aux depens d'hy- drates de carbone ou de protdiques et action dynamique sp£cifique. Compt. Rend. Acad. Sci., 177: 900-902, 1923. 178: 869, 1488. 1924. 60Kruse, 1910 (p. xii). ENERGY TRANSFORMATION 411 All reactions, except the fourth one, actually take place and the prod- ucts of oxidation can be determined from the amount of sugar and oxygen consumed. The ratio of C02 to 02, or the respiratory quotient, is equal to zero in most of the above equations. When the quotient is less than 1, the oxidation is incomplete. With complete oxidation, C02:02 = 1. In the oxidation of alcohol to acetic acid, C02:02 = 0, but with complete combustion, C02:02 = 0.67, C2H6OH + 3 02 = 2 C02 + 3 H20 + 326 Cal. In the oxidation of tartaric acid, the C02:02 = 1.6, C4 H6 Oe + 2| 02 = 4 C02 + 3 H20 + 262 Cal. In the oxidation of palmitic acid, the C02:02 = 0.7, Ci6 H32 02 + 23 O2 = 16 C02 + 16 H20 + 236 Cal. The oxidation of nitrogen compounds gives quotients smaller or greater than 1. C6Hi3NC>2 + 7\ O2 = 6 CO2 -f 5 H20 + NH3 + 775 Cal. Leucine C02:02 = 0.8 C2H5NO2 + 1J 02 = 2 C02 + H20 + NH3 + 152 Cal. Glycocoll C02:02 = 1.33 The oxidation of proteins, which consist of the different amino acids, will, therefore, give a quotient falling between the two above equations, becoming 1 with complete oxidation. Usually, it is less than 1 and may even be 0 when oxidation stops at the oxalic acid stage. Some of the oxygen may be used up for the oxidation of nitrogen and sulfur compounds, as in the case of the action of the autotrophic bacteria when the respiratory quotient equals 0. Puriewitsch51 calculated the respiratory quotient for Asp. niger, using different sources of energy: RESPIRATORY QUOTIENT SUBSTANCE OXIDIZED Calculated accord- ing to formula for chemical oxidation Found on analyz- ing the gas formed Tartaric acid 1.60 1.00 1.00 0.85 0.92 1.62 Glucose 0.95 Lactic acid 0.85 Glycerol 0.75 Mannite 0.65 61 Puriewitsch, K. Physiologische Untersuchungen iiber Pflanzenatmung. Jahrb. wiss. Bot., 35: 572-610. 1900. 412 PRINCIPLES OF SOIL MICROBIOLOGY In this particular case, tartaric acid and glucose have undergone complete oxidation while for the other three substances it is only partial. For a quantitative determination of respiration of microorganisms the methods suggested by Bieling,52 Warburg,53 and Osterhaut54 can be used. The last is based upon the time required to produce definite changes in the acidity. The hydrogen-ion concentration has no in fluence upon the absolute respiration of fungi or upon the ratio between, growth and respiration, as shown by Gustafson55 for Pen. chrysogenum and Lundegardh56 for Fusaria. Phosphate was found to have a favorable effect on respiration. It is of interest to note that phosphate has a specific action in bringing about the auto-oxidation of fructose.53 Anaerobic utilization of energy. According to Pasteur,57 fermentation is the activity of cells in the absence of air or free oxygen, which results in the liberation of energy necessary for the growth and other vital activities of the cells in a manner similar to aerobic respiration in the presence of oxygen. In the absence of air, cells obtain the oxygen from the carbohydrate itself, whereby one half of the carbohydrate molecule is reduced and the other oxidized. In the case of alcoholic fermentation by yeast, alcohol is the reduction product and C02 the oxidation product. C6H1206 = 2 CH3 • CHOH • COOH + 25 Cal. Lactic acid C6H1206 = 2 CH3 • CH2 • OH + 2 C02 + 27 Cal. Alcohol C6H1206 = CH3 • CH2 • CH2 • COOH + 2 C02 + 2 H2 + 15 Cal. Butyric acid 62 Beiling, R. Eine Methode zur quantitativen Bestimmung der Atmung von Mikroorganismen und Zellen. Centrbl. Bakt. I, O, 90: 49-52. 1923. 63 Warburg, O., and Negelein, E. Uber die Reduktion des Salpetersaure in griinen Zellen. Biochem. Ztschr., 110: 66-115. 1920; 142: 317-333. 1923; Warburg, O., and Yabusoe, M. Uber die Oxydation von Fructose in Phosphat- losungen. Biochem. Ztschr., 146: 380-386. 1924. 64 Osterhaut, W. J. V. Comparative studies on respiration. Jour. Gen. Physiol., 1: 171-179. 1918. 66 Gustafson, F. G. The effect of hydrogen ion concentration on the respira- tion of Penicillium chrysogenum. Jour. Gen. Physiol., 2: 617-626. 1920. 66 Lundegardh, H. Der Einflusz der Wasserstoffionenkonzentration in Gegen- wart von Salzen auf das Wachstum von Gibber ella saubinetii. Biochem. Ztschr., 146: 564-572. 1924. 67 Pasteur, L. Etudes sur la biere. Paris. 1876. ENERGY TRANSFORMATION 413 The heat liberated in the fermentation reactions is calculated as follows: Heat of combustion of glucose 674.0 Cal. Heat of combustion of alcohol 326.2 Cal. Pleat of solution of glucose 2.1 Cal. Heat of solution of alcohol 2.25 Cal. (674 + 2.1) - 2(326.2 - 2.25) = 28.2 Cal. glucose alcohol Since in the reaction of alcoholic fermentation, some other reaction products are formed, the amount of heat produced by a mol of sugar converted into alcohol is about 27 cal. Rubner found that actually 24 cal. were liberated in this re- action. Some of the anaerobic fermentations are not accompanied by the production of CO2, as in the lactic and acetic acid fermentations. C6 H12 Oe = 3 CH3 COOH + 44 Cal. In the anaerobic decomposition of cellulose, whereby hydrogen and methane are formed (whether by pure cultures or mixed cultures) the following reactions may be suggested: 3 C6H1206 = 3 CH3 • CH2 • CH2 • COOH + CH3 • COOH + 4 C02 +2 H20 + 2 H2 butyric acid acetic acid 3 C6H1206 = 2 CH3 • CH2 • CH2 • COOH + 2 CH3 • COOH + 4 C02 + 2 H20 + 2 CH4 When the heat of reaction of oxidation is compared with that of "fermentation," the former is found to be much greater than the latter: 1 mol of sugar liberates 676 Cal. when it is completely oxidized (to C02 and H20) and only about 25 Cal. in the alcoholic or lactic acid "fer- mentation," and even less in the butyric acid fermentation. Since the amount of growth of the cells of microorganisms is in direct pro- portion to the production of energy, much larger quantities of sub- strate have to be decomposed in the "fermentations" than in the oxi- dations by free oxygen in order to liberate the same amount of heat and allow the same amount of work or equal growth of the cells to take place. Some of the products of the "fermentation" processes may be used for the synthesis of the cellular substance. This is par- ticularly true in the decomposition of proteins and carbohydrates.58 When organic acids, hydrogen and methane are the products of decomposition they may be used as sources of energy by other micro- 68 See Meyerhof, O. Uber den Einflusz des Sauerstoffs auf die alkoholische Giirung der Hefe. Biochem. Ztschr., 162: 43-86. 1925. 414 PRINCIPLES OF SOIL MICROBIOLOGY organisms, if conditions are made favorable, as by the addition of free oxygen. 2 CH3 • CHOH • COOH + 2 02 = 2 CH3COOH + 2 C02 + 2 H20 + 240 Cal. Lactic acid acetic acid 2 CH3COOH + 3 02 = 2 HCOOH + 2 C02 + 2 H20 + 294 Cal. Formic acid 2 HCOOH + 02 = 2 C02 + 2 H20 + 126 Cal. The more recent conceptions of oxidation-reduction processes (p. 520), tend to explain the mechanism of energy utilization under "anaerobic" conditions. Quastel and associates59 have shown that anaerobic growth of an organism can be expected to take place when a pair of organic compounds, neither of which supports anaerobic growth, fulfill the following conditions: (1) both are "activated" by the organism so that simultaneous oxidation and reduction may occur; (2) energy necessary for growth is liberated in the interaction; (3) as a result of such an in- teraction some substance is produced which is capable of entering into the synthetic processes of the cell. These conditions are fulfilled for Bad. coli by lactic acid or glycerol, on the one hand, and nitrate or fumerate, on the other, the former contributing hydrogen and the latter utilizing it. CH2 OH-CH OH-CH2OH + COOH -CH:CH- COOH = CH3CH OH -COOH + COOH • CH2 • CH2 • COOH + 33 Cal. glycerol Fumaric acid CH2 OH • CH2OH + 2COOH • CH:CH • COOH = CH3 • CO COOH + 2COOH • CH2 • CH2 • COOH + 50 Cal. Malic and aspartic acids can also serve as weak hydrogen acceptors for Bad. coli. In some cases, the same substance can serve both as the hydrogen donator and acceptor, as brought out by the Cannizzaro reaction, especially in the case of pyruvic acid: CH3 • CO • COOH + H20 + CH3 • CO • COOH = pyruvic acid pyruvic acid CH3 • CHOH • COOH + CH3 • COOH + C02 + 15 Cal. lactic acid acetic acid 69 Quastel, H., Stephenson, M., and Whetham, M. D. Some reactions of resting bacteria in relation to anaerobic growth Biochem. Jour., 19: 304-317. 1925; Quastel, J. H., and Stephenson, M. Further observations on the anaerobic growth of bacteria. Ibid., 660-666. ENERGY TRANSFORMATION 415 According to Aubel,60 glucose is decomposed under anaerobic condi- tions by Bad. coli by a reaction of internal coupling, one half molecule (lactic acid) giving an exothermic reaction and the other half molecule (pyruvic acid, used as a starting point for synthetic processes) giving an endothermic reaction: C6H1206 = CH,.CHOH.COOH + CH3-COCOOH + H2 0.48 gram of carbon of the sugar decomposed gave 0.216 gram as lactic acid, 0.081 gram as pyruvic acid, 0.042 gram as acetic acid, 0.044 gram as alcohol, and 0.052 gram as C02. From the point of view of energy utilization, "aerobiosis" and "anaerobiosis" mean simply the liberation of energy by free oxygen or by an intramolecular rearrangement. The transformation of urea into ammonium carbonate is a purely hydrolytic process, which yields a small amount of energy.61 NH2CONH2 + 2 H20 = (NH4)2C03 + 7 Cal. Efficiency of energy utilization by heterotrophic microorganisms. When the various microorganisms are compared in the amount of nutrients transformed and in the synthesis of cellular material, fungi are found to consume, under favorable conditions, 1| to 2 times as much nu- trient as is necessary for the building of the cells. Raulin,62 for example, found that A. niger will synthesize 1 gram of mycelium for every 2.30 grams of sugar decomposed, i.e., the coefficient of utilization of sugar by this organism was 44 per cent. According to Wehmer,63 the growth of A. niger (weight of mycelium) is parallel to the energy value of the nutrient. This is clearly illustrated in table 27. 64 Peptone is the exception. This is probably due to the fact that a large part of the energy was left unutilized in the form of protein-split products. Ter- roine55 has also shown that while 100 Cal. in the form of glucose will 60 Aubel, E. Sur l'origine de l'energie permettant au Bacterium coli, de se deVelopper aux depens du glucose. Compt. Rend. Acad. Sci., 181: 571-573. 1925. 61 Berthelot, M., and Petit, P. Sur la chaleur animale et sur les chaleurs de formation et de combustion de l'ur6e. Ann. chim. Phys. (6), 20: 13-20. 1890. 62 Raulin, J. Etudes chimiques sur la vegetation. Ann. Sci. Nat. Bot. (5), 11: 93. 1869. 63 Wehmer, C. Entstehung und physiologische Bedeutung der Oxalsaure ini Stoffwechsel einiger Pilze. Bot. Ztg., 41: 337. 1891. "Kruse, 1910 (p. xii). "Terroine, Bonnet, Jacquot, and Vincent. 1923-1924 (p. 410). Bull. Soc. Chim. Biol., 7: 351-379. 1925; Terroine, E. F., and Wurmser, P. L'utilisation des substances ternaires dans la croissances de V Aspergillus niger. Compt. Rend. Acad. Sci., 174: 1435-7. 1922; 175: 228-230. 416 PRINCIPLES OF SOIL MICROBIOLOGY allow the synthesis of 58 Cal. of fungus mycelium, 100 Cal. in the form of protein will allow the synthesis of only 39 Cal. of fungus mycelium. There is also a large loss of energy in the process of deaminization. Fungi generally waste (give out) about as much energy as they assimilate in their bodies. Under unfavorable conditions, the amount of energy assimilated by the body is much smaller in comparison with that utilized. The coefficient of utilization was found to decrease with age of culture, due to the fact that, after the nutrients have been used up, the cells of the organism begin to undergo autolysis and its con- stituents are used as sources of energy Terroine and Wurmser determined, for Asp. niger, "the utilization quotient" of sugars by comparing the dry weight of the mycelium pro- duced with the weight of the sugar consumed. It was found to be TABLE 27 Influence of energy source upon the growth of fungi SOURCE OF ENERGY Tartaric acid Citric acid . . Glucose Glycerol Peptone .... Olive oil ... . 1.5 GRAMS FUNGUS MYCELIUM OF MATERIAL calories gram 2.6 0.155 3.7 0.240 5.6 0.278 6.5 0.475 6.8 0.162 14.0 0.810 43 to 44 per cent for glucose, levulose, saccharose, maltose, arabinose xylose. The concentration of the nitrogenous substance, varying from 0.5 to 4 per cent, does not influence the quotient of utilization, which was found to be lower with sodium and aluminum nitrate as a source of nitrogen (0.34 to 0.35) than with ammonium sulfate or nitrate, urea or guanidine (0.42). The addition of sulfuric acid to the sodium nitrate medium resulted in an increase in the quotient. The utiliza- tion of an energy source by A. niger was calculated as follows. The heat of combustion of mycelium was 4.8 Cal. per gram and that of glucose 3.76 Cal. The energy utilization is, therefore, (44 parts of mycelium X 4.8) -J- (100 parts of sugar X 3.76) = 56 per cent. The combustion of glucose in the culture medium was, however, not complete. A combustion of the medium, made before and after the cultivation of A. niger, gave 6.46 and 0.51 respectively. The difference of these figures, 5.95, represents the metabolizable energy U for ENERGY TRANSFORMATION 417 the particular culture. The mycelium from this culture gave on combustion 3.55 Cal. = U'. The ratio of the energy stored in the mycelium to the metabo- lizable energy U is thus found to be 3.55 -5- 5.95 = 59.6 per cent, or very near the above figure, calculated from other data. When fully developed mycelium is placed in Czapek's solution, it does not grow further but forms C02; this permits the calculation of a minimum value for maintenance energy. The consumption of glucose by Asp. niger is at any instant the sum of two terms: (1) a term pro- portional to the rate of growth, which represents the amount of glucose which should disappear to form the substance of the mycelium TABLE 28 Relation of metabolic products of fungi to one another and to sugar consumed (pentoses) Days of incubation _ _ . weight of CO2 Respiration coefficient or — — . . . weight of dry fungus _ . _ . weight of sugar consumed Economic coefficient or weight of dry fungus . . . , carbon of C02 X 100 Respiration equivalent or . . . carbon of sugar _,, M . . . carbon of fungus X 100 Plastic equivalent or .... carbon of sugar Asp. niger 1.5 3.5 41 47 28 2.5 2.4 55 36 Pen. glaucum 2.5 3.2 54 39 28 4.5 4.4 70 28 and (2) a term proportional to the weight of mycelium already formed at that instant, which represents the consumption of sugar for maintenance.66 There is a definite relation between utilization of sugar, growth of fungi and evolution of C02, as shown in table 28.67 The "economic 60 Further information on energy utilization by fungi and yeasts, in the absence of an excess of oxygen, is given by Kostytschew, S., and Afanasjeva, M. Die Verarbeitung verschiedener organischer Verbindungen durch Schimmelpilze bei Sauerstoffmangel. Jahrb. wiss. Bot., 60: 628-650. 1921; Rona, P., and Grassheim, K. Studien zur Zellatmung. I. Beitriige zur Atmung der Hefezel- len. Biochem. Ztschr., 134: 146-162. 1922; Meyerhof, 1925 (p. 413). 67 Peterson, Fred and Schmidt, 1922 (p. 242). 418 PRINCIPLES OF SOIL MICROBIOLOGY coefficient" of storage-rot fungi was found68 to vary from 3.86 to 22.86, while the respiration coefficient varied from 2.9 to 11.4. The respiration of thermophilic fungi is less than that of common fungi. Penicillium sp. produces at 15° in 24 hours enough C02 to correspond to 67 per cent of dry weight; at 25°, 133 per cent. By applying the van 't Hoff formula for the temperature coefficient, Noack69 calculated that, at 45°, 532 per cent C02 would be produced by Penicillium, while the thermophilic Thermoascus produced only 310 per cent C02 at 45°. The temperature quotient is 1.7-1.9 for 10° temperature difference for Thermoascus and 2-3 for common fungi.70 Bacteria utilize for synthetic purposes a smaller amount of the energy made available than do fungi. The utilization is largest with aerobic bacteria. This is further accentuated by the fact that bacteria usually form a larger quantity of intermediary products than fungi. The energy utilization by the various bacteria depends upon the nature of the organism and mechanism of decomposition of substrate, ranging from 37 to 38 per cent of the energy made available in the case of Bac. subtilis and aerobic yeasts to only 0.5 to 7.0 per cent of the energy for urea, acetic, and nitrite bacteria. The lower the relative amount of energy made available the greater is the transformation of matter and energy for the same amount of cell-protoplasm synthesized. The fact that heterotrophic bacteria are capable of synthesizing a large part of the cell constituents by merely assimilating the constituents of the substrate without producing much work, while the urea and autotrophic bacteria have to do work, also explains the lower energy utilization and, therefore, the larger amount of matter and energy transformation in the case of the last group. Arnaud and Charrin71 cultivated Bad. pyocyaneum on a medium 68 Harter, L. L., and Weimer, J. L. Respiration of sweet potato storage rot fungi when grown on a nutrient solution. Jour. Agr. Res., 21: 211-226. 1921. 69 Noack, K. Der Betriebstoffwechsel der thermophilen Pilze. Jahrb. wiss. Bot., 59: 413-466. 1920. 70 Further information on the influence of temperature upon life processes is given by Kanitz, A. Temperature undLebensvorgange. Borntraeger. Berlin. 1915; Arrhenius, S. Quantitative laws in physical chemistry, 1915; Crozier, W. J., On biological oxidations as function of temperature. Jour. Gen. Physiol., 7: 189-216. 1924. 71 Arnaud, A., and Charrin, A. Recherches chimiques sur les secretions microbiennes. Compt. Rend. Acad. Sci., 112: 755-758, 1157-1160. 1891. ENERGY TRANSFORMATION 4l9 containing 0.5 per cent asparagine, as a source of carbon and nitrogen. These two elements were accounted for as follows: Assimilated by the bacteria. Given off as CO* or NH3 Non- volatile products CARBON NITROOEN per cent per cent 13.8 4.66 72.5 91.0 13.5 4.04 Kruse calculated from these data that 19 per cent of the energy that was made available from the decomposition of the asparagine had been utilized by the bacteria for the synthesis of bacterial protoplasm. According to Rubner,72 the relative energy utilization of bacteria is independent of temperature. The total amount of energy transformed depends, however, largely on the temperature of growth, as shown in table 29. The data were obtained by growing Bad. vulgare in 500 cc. portions of 6 per cent meat extract solution. Rubner found that aerobic bacteria are capable of utilizing 30.8 to 11.6 per cent of the energy transformed, pathogenic bacteria the least. The thermophilic bac- teria utilized 24.9 per cent of the energy. Linhart73 calculated that only about 1 per cent of the total available energy in mannite is utilized by Azotobacter for the fixation of nitrogen. This was based upon the assumption that Azotobacter fixes 10 grams of nitrogen for every gram of mannite decomposed and that all the mannite is converted into C02. No allowance was made for the forma- tion of intermediary products or the synthesis of microbial protoplasm. When amino acids are used as sources of energy, the process of synthesizing microbial protoplasm is a very economical one in which little energy is wasted. This is true for fungi74 and bacteria. Bad. coli was found76 to be able to utilize the energy of amino acids three to eight times more effectively than that of glucose. According to Czapek 72 Rubner, M. Energieverbrauch im Leben der Mikroorganismen. Arch. Hyg., 48: 260-311. 1903; Die Beziehungen zwischen Bakterienwachstum und Konzentration der Nahrung (Stickstoff und Schwefelumsatz). Ibid., 67: 161— 192. 1906; Energieumsatz im Leben einiger Spaltpilze. Ibid., 57: 193-243. 73 Linhart, G. A. The free energy of biological processes. Jour. Gen. Physiol., 2: 247-251. 1920. 74 Czapek, 1902 (p. 502). 76 Shearer, C. On the amount of heat liberated by B. coli when grown in the presence of free amino acids. Jour. Physiol., 55: 50-60. 1921. 420 PRINCIPLES OF SOIL MICROBIOLOGY this may be due to the direct assimilation of the amino acids for the synthesis of microbial protoplasm. In summarizing the energy utilization by different groups of micro- organisms, it may be said that fungi transform usually 30 to 60 per cent of the substrate into mycelium, aerobic bacteria 10 to 20 per cent, while anaerobic bacteria may transform only 1 per cent or less of the nutrients into synthesized cells. Reduction of nitrates and sulfates and energy utilization. In addi- tion to respiration by the agency of free oxygen (autotrophic and heterotrophic) and intramolecular respiration or "fermentation," there is another form of respiration whereby the oxygen in certain inorganic compounds, namely nitrates and sulfates, is utilized. Just as the oxidation phenomena are accompanied by the liberation of TABLE 29 Influence of temperature upon growth and energy utilization of Bad. vulgar e INCUBA- . TEMPER- ATURE NITROGEN CONTENT OF BACTERIAL GROWTH CALORIFIC VALUE OF DRY CELLS ENERGY OF MEDIUM METABO- LIZABLE ENERGY TOTAL ENERGY TRANS- FORMED ENERGY TION Before growth After growth TION days 7 14 21 30 37 36.0 14.5 14.5 14.5 14.5 gram 0.135 0.037 0.032 0.045 0.067 4.00 1.72 1.44 2.06 2.82 84.22 84.22 84.22 84.22 84.22 69.28 79.32 78.38 77.73 72.44 14.94 4.90 5.84 6.49 11.78 18.94 6.62 7.28 8.55 14.60 per cent 21.2 25.9 19.9 24.1 20.5 energy, so are the reduction phenomena accompanied by the consump- tion of energy. This energy is, however, returned even to a greater extent in the accompanying oxidation. The oxygen rich compounds are reduced and the oxygen is utilized for the oxidation of other sub- stances, the latter process supplying the energy for the former. Nitrification, which is #n oxidation process, is accompanied by the reduction of C02, which is used as a source of carbon; denitrification, which is a reduction process, is accompanied by oxidation phenomena (oxidation of carbohydrates, etc.), in which the combined oxygen is used to produce energy. The oxygen obtained from the nitrate or the sulfate is utilized for the oxidation of carbohydrates, organic acids or certain inorganic substances. This may be accomplished by one organism, as in the case of the denitrifying sulfur-oxidizing organism, or by the symbiotic ENERGY TRANSFORMATION 421 action of two organisms, as in the case of cellulose oxidation and de- nitrification.76 Trautwein77 pointed out that Thiob. denitrificans, which oxidizes thiosulfate aerobically, can carry on this process in the absence of atmospheric oxygen but in the presence of nitrates. When tartaric acid is oxidized by atmospheric oxygen or by reduction of nitrates, nearly equal amounts of energy are liberated, since the reduc- tion of nitrates to atmospheric nitrogen does not consume a large amount of energy. C4H606 + 2§ (02) = 4 C02 + 3 H20 + 282 Cal. Tartaric acid C4H606 + 2 HN03 = 4 C02 + 4 H20 + N2 + 253.4 Cal. Attention should also be called to the reduction of nitrate in the as- similation of nitrate by the chlorophyll-bearing algae73 and to the role of nitrate as hydrogen acceptor in the growth of certain anaerobic bacteria. The first is carried through the following series of reactions: N07+ H20 + 2 H+ = NHt + 2 02 - 68 Cal. 2 02 + 2 C = 2 C02 + 230 Cal. or N07 + H20 -j-2H+ + 2C = NHf + 2 C02 + 162 Cal. of free energy Out of a total of 230 Cal. liberated, only 68 Cal. or about 30 per cent is utilized for the reduction of the nitrate. The question of nitrate reduction by bacteria will be discussed in detail elsewhere (p. 545). 79 The reduction of sulfate to sulfide requires a large expenditure of energy and accounts for the distinct difference in energy gain when oxidation takes place by means of atmospheric oxygen or when the oxygen is derived from the reduction of sulfates. H2S04 = H2S + 2 02 - 135 Cal. 2 C3H603 + 6 02 = 6 CO, + 6 H20 + 659 Cal. lactic acid 2 C3H603 + 3 H2S04 = 6 C02 + 6 H20 + 3 H2S + 254 Cal. 76 Groenewege, 1920 (p. 200). 77 Trautwein, 1921 (p. 87). 78 Warburg, O., and Negelein, E. Uber den Energieumsatz bei der^Kohlen- saureassimilation. Ztschr. physik. Chem., 102: 235-266. 1922. 79 Jensen, Orla. Die Hauptlinien des natiirlichen Bakteriensystems. Centrbl. Bakt. II, 22: 97-98, 305-346. 1909. 422 PRINCIPLES OF SOIL MICROBIOLOGY The amount of energy thus liberated is only about 38 per cent of the energy liberated in oxidation of lactic acid by free oxygen. Comparative amounts of energy liberated in microbiological processes. The different nutrients develop the following number of calories as listed here. The final products, when not C02, are given in parenthesis. I. Oxidation by means of free oxygen (Kruse) 1 ORAM OP NUTRIENT 1 MOL. OF NUTRIENT Nitrous acid (nitric a cid). . . Calories 0.4 0.68 1.4 1.37 1.88 2.39 2.48 2.5 3.51 2.54 2.90 2.8 3.49 3.13 3.66 3.72 3.74 3.96 4.18 4.37 4.5-5.0 4.6 4.96 5.96 6.53 5.92 6.4 7.1 8.0 9.2 34.6 Calories 61.0 Glucose (to citric aci d) 62.9 280.0 320.0 475.0 Asparagine (complete combu stion) 464.1 153.0 386.0 Glucose (to oxalic ac d) . 209.6 235.0 326.0 588.0 674.0 1,354.0 388.0 Ammonia (to nitrous 367.0 525.0 856.0 1,072.0 H2S(H2S04) . IT. Oxidation by means of oxygen from oxygen-rid i compounds (Kruse) Tartaric acid by means of de Lactic acid by means of Spii nitrifying bacteria '. desulfuricans 1.6 1.0 ENERGY TRANSFORMATION 423 III. Intramolecular respiration "fermentation" Urea (to ammonia) Glucose (to acetic acid-anaerobic). Glucose (to alcohol) Glucose (to lactic acid) Glucose (to butyric acid) Acetic acid (to methane) Energy transformation in synthetic processes. The energy transfor- mation in the synthetic action of microorganisms depends upon the nature of the organism and of the substances present as nutrients. The autotrophic bacteria and algae build up their carbohydrates from the C02; this phenomenon is one of reduction and involves, therefore, a great expenditure of energy. 6 C02 + 6 H20 = C6Hi206 + 6 02 - 676 Cal. This energy is obtained from the oxidation of ammonia, nitrous acid, thiosulfate, sulfur, etc. (and, in the case of algae, photosynthetically). Because of the large amount of energy required for synthetic purposes and since work in the true sense is to be produced, large quantities of these substances have to be oxidized. When carbohydrates are synthesized from organic compounds or, in general, when an organism grows in a medium containing organic substances as sources of energy, much less energy is consumed in the synthesis of the protoplasm, as seen by reversing several of the above reactions. 3 C2H402 = C6H,20« - 44 Cal. 2 C3H603 = C6H1206 - 2S Cal. The energy required in this case is obtained by aerobic microor- ganisms from oxidation processes and by anaerobic bacteria by means of "fermentations" or reactions of oxidation-reduction. The pro- duction of polysaccharides from the monosaccharide requires only a small expenditure of energy. 2 C6Hi206 - H20 = CijH220,i - 3.3 Cal. n(C6H1306) - H20 = (C8HioOo)n - (4.3 Cal.)n The building up of fats from carbohydrates or glycerol is a reduc- tion phenomenon, it involves work in the thermodynamic sense and requires a large expenditure of energy. This can, therefore, take 424 PRINCIPLES OF SOIL MICROBIOLOGY place only when accompanied by active oxidation whereby a large amount of energy is made available. In the synthesis of protein compounds by microorganisms, nitrates are first reduced to ammonia and, if proteins or amino acids form the nitrogen source, they are hydrolized to ammonia and the correspond- ing oxy-acids. The combination of the ammonia with the various oxy- acids, either synthesized by the autotrophic organisms from C02 or produced by the heterotrophic organisms from the complex organic substances in the medium, results in the formation of amino acids, from which the microbial proteins can be formed: C3H602 + NH3 = C3H7NO2 + H2. Propionic acid Alanine The propionic acid is derived from lactic acid or from glucose, so that glucose and ammonia can be used directly for the synthesis of alanine :80 C(H,806 + 2NH3 = 2C3H7N02 + 2H20 + 63 Cal. Alanine CflH1206 + NH3 = C6H13N02 + H20 + 30 - 101 Cal. Leucine C6H1206 + 3NH3 = 3C2H5N02 + 3H2 + 11 Cal. Glycocoll 2C6Hi208 + 3NH3 = 3C4H7N04 -K6H2 + 31 Cal. Asparagine When proteins or amino acids form the only sources of both carbon and nitrogen, the oxy-acids produced from the amino acids may be utilized for the building of the protein molecule.81 In the case of carbohydrates, pyruvic acid and acetaldehyde are important sub- stances in the synthesizing activities of microorganisms (Neuberg). Once the amino acids are synthesized, the protein molecule is formed from them by the union of a carboxyl and an amino group with the elimination of a molecule of water. The amount of energy required is much smaller in comparison with that necessary for the synthesis of the amino acids.82 80Kruse, 1910 (p. xii). 81 Czapek, 1902 (p. 502). 82 Further information on the transformation of energy in synthetic processes is given by Nathanson, A. tlber kapillarelektrische Vorgange in der lebenden Zelle. Kolloid-chem. Beihefte., 11: H. 10-12. 1919. ENERGY TRANSFORMATION 425 Energy transformation in the soil. The microbiological processes in the soil can be considered from the point of view of energy transfor- mation,83 by determining the calorific value of the organic matter present in the soil. "When organic matter is added to the soil, in the form of manures, green manures, plant stubble, etc., a certain amount of potential energy is introduced. A part of this is liberated as heat and dis- sipated into space as a result of the activities of microorganisms, a part is stored in the microbial cells, and a part is left in the form of unde- composed organic matter or various intermediary products. The energy stored away in the microbial cells and that left in the unde- composed or partially decomposed organic matter go to increase the energy content of the soil. This energy is only gradually liberated as a result of the activities of microorganisms. TABLE 30 Relation between COi and energy liberation in some typical microbiological processes 1 GRAM OP CO: LIBERATES IN Butyric acid "fermentation" Oxidation of sugar to oxalic acid. Complete oxidation of sugar Alcoholic "fermentation" Methane fermentation CALORIES RATIO 0.29 1 0.69 2.3 2.55 8.5 3.71 12.3 4.80 16.5 The calorific value of the soil organic matter represents the minimum energy available for the activities of microorganisms, since the auto- trophic bacteria can obtain their energy also from inorganic substances in the soil. Since the organic matter added to the soil has a calorific value of 4.6 to 5.0 Cal. per gram, the addition of 3 tons of dry ash-free organic matter, in the form of manure or green manure, per acre of soil, in- troduces 13,000,000 Cal. available for the activities of microorganisms. Considering the average content of organic matter in the soil as 2 to 3 per cent, the potential energy available in one acre of soil (12 inches deep) is about 200,000,000 Cal., equivalent to about twenty-four tons of anthracite coal. The freshly introduced organic matter begins to decompose immediately in the soil, at first rapidly, then more and more slowly, until a certain equilibrium is established, as shown by the 83 van Suchtelen, F. H. H. Energetic und die Mikrobiologie des Bodens. Centrbl. Bakt. II, 58: 413^30. 1923. 426 PRINCIPLES OF SOIL MICROBIOLOGY evolution of carbon dioxide. A part of the energy is stored away in the cells of the microorganisms, while a larger part is dispersed into space. The amount of C02 produced from the soil, as well as in an artificial culture, can be used as an index of the activities of soil microorganisms. There is no close correlation, however, between the amount of C02 formed and the energy liberated by microorganisms, since energy may be liberated without the production of C02 (oxidation- reduction processes) and C02 may be formed as a result of chemical interaction between acids and carbonates, without energy liberation. Different microorganisms bring about different reactions, each of which involves a different transformation of energy. The different amounts of energy liberated in different microbiological reactions are given in table 30. 83 The production of heat serves as a more reliable index of the energy transformation by microorganisms, since the latter are chiefly re- sponsible for the exothermic processes in the soil. However, most of our information on the decomposition of organic matter in the soil is limited to the determination of C02 as an index, as shown elsewhere (p. 681). CHAPTER XVII Chemistry of Decomposition of Non-nitrogenous Organic Matter by Soil Microorganisms Composition of vegetable organic matter. Vegetable organic matter is added to the soil in the form of green manure, stable manure, plant residues and roots of cultivated and wild plants. This organic matter consists largely of celluloses, hemicelluloses and pentosans, starches and other complex carbohydrates; lignins and compounds of these with carbohydrates; simple carbohydrates; complex proteins, protein degradation products and purine bases; fats, waxes, glu- cosides, tannins, pigments and mineral matter. A typical analysis of a series of native vegetable substances is given in table 31, in per cent of water-free substance.1 However, the composition of the organic materials varies with the degree of ripening of the plant, as shown2 in table 32 for oat straw. A decrease in nitrogen content and an increase in content of lignocel- luloses depresses the rapidity of decomposition of the plant materials in the soil, as shown later (p. 459). The elementary composition of some typical plant products, on the basis of several analyses is given in tables 33 and 34. The difference in analysis reported by different investigators is due to differences in the plant varieties, in the composition of the soil, age of plant, error of manipulation, etc. Chemistry of celluloses. The constituents of cell membranes can be generally classified into five groups:^4 1. True celluloses, or the condensation products of glucose, not acted upon by dilute acids. 1 Pringsheim, H. Die Polysaccharide. 2nd Ed. 1923, Berlin. 2 Berry, R. A. Composition and properties of oat grain and straw. Jour. Agr. Sci., 10: 358. 1920. 3 Tollens, B. "Dber Cellulose, Oxycellulose, Hydrocellulose, die Pektinkorper, sowie Traganth. Ber. deut. chem. Gesell., 34: 1434-1441. 1901. 4 Heuser, E. Lehrbuch der Cellulosechemie. 2 Aufl. Berlin, Borntraeger. 1923. 427 428 PRINCIPLES OF SOIL MICROBIOLOGY TABLE 31 The composition of a few natural organic materials Hay Oats straw Barley straw Corn cobs Corn fodder Rice straw Straw of winter cereals . CELLU- LOSE PENTO- SANS LIGNIN CRUDE PROTEIN GUMS AND WAXES 28.50 13.52 28.25 9.31 2.00 35.43 21.33 20.40 4.70 2.02 32.92 21.45 18.66 3.20 1.40 37.66 31.50 14.70 2.11 1.37 30.56 23.54 15.13 3.50 0.77 31.99 27.67 18.48 5.33 0.51 34.27 21.67 21.21 3.00 0.67 6.05 4.81 5.56 1.80 6.15 5.43 4.33 TABLE 32 Influence of age on the composition of oat straw Crude protein Crude fiber. . . Cellulose Total nitrogen Ash P*06 K20 JUNE 7 JUNE 21 JULY 5 JULY 19 AUGUST 3 16.5 10.6 7.0 4.7 2.8 14.1 21.6 24.7 24.7 24.8 12.4 19.0 20.8 21.1 0.515 5.5 0.296 1.03 21.4 AUGUST 16 1.4 28.3 23.6 0.200 6.6 0.164 0.98 TABLE 33 Elementary composition of a few plant products6 Dry pine needles Oak leaves Maple leaves Wheat straw Corn stover Grain stubble and roots Lupine Luzerne Clover c H N O 41.96 3.98 1.42 21.07 49.11 6.12 1.71 29.38 44.61 5.01 1.89 29.18 47.01 5.66 0.82 38.61 43.30 5.75 1.69 37.58 32.81 4.62 1.63 28.82 44.12 6.03 3.19 33.67 43.28 5.86 1.95 38.54 44.31 5.91 2.98 36.01 31.57 13.68 19.31 7.90 11.68 32.12 12.99 10.37 10.29 6 Dvorak, J. Studien fiber die Stickstoffaniiufung im Boden durch Mikro- organismen. Ztschr. landw. Vers. Sta. Oesterreich., 15: 1077-1121. 1912. DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER 429 2. Hemicelluloses, or the condensation products of hexoses (galactose, man- nose) and of pentoses (arabinose, xylose), readily hydrolyzed by dilute mineral acids. 3. Pectins, different in composition, but related to the hemicelluloses.7 4. Lignins, forming complex physical, absorption compounds or mechan- ical incrustations with celluloses, known as ligno-celluloses, which constitute the woody tissues of plants.8 5. Corky or cutinized lamellae, identified microscopically by the Sudan- glycerol reaction and not well defined chemically. The woody parts of plants or the plant cell membranes are usually- referred to as "crude fiber." This is made up of cell walls composed principally of celluloses; they are accompanied by various other sub- stances, depending upon the nature of the plant; even the carbon con- tent of celluloses of different origin varies.9 This is probably due, not to differences in the constitution of the celluloses, but to the differences TABLE 34 Mineral composition of a few plant products' Corn stover. Corn cobs . . Oat straw . . , Wheat straw Clover hay . . Timothy hay N 0.81 0.50 0.58 0.53 2.17 0.84 0.07 0.03 0.09 0.09 0.18 0.13 K 0.78 0.64 1.09 0.83 1.12 1.34 in the amount and kind of impurities present. In the case of cotton, flax and hemp, pectin forms the chief impurity. In the case of straw, wood and jute, lignin is the impurity. This gave rise to the terms pecto-celluloses and lignocelluloses. Celluloses of different origin are decomposed with different rapidity,10 probably as a result of differ- ences in the physical condition as well as impurities present. 6 Thorne, C. E. Farm manures. O. Judd. New York. 1914. 7 Ehrlich, F. Die Pektinstoffe, ihre Konstitution und Bedeutung. Chem. Ztg., 41: 197-200. 1917. 8 Riefenstahl, R. Der gegenwartige Stand der Ligninchemie. Ztschr. angew. Chem., 37: 169-177. 1924. 9 Konig, J. Bestimmung der Cellulose, des Lignins und Rutins in der Roh- faser. Z. Unters. Nahrungs u. Genussm., 12: 388. 1906; Ber. deut. chem. Gesell., 39: 3564-3570. 1906. 10 Ernest, A. Beitrag zur Kenntnis einiger Cellulosen. Ber. deut. chem. Gesell., 39: 1947-1951. 1906. 430 PRINCIPLES OF SOIL MICROBIOLOGY Chemically, the true celluloses are non-nitrogenous, amorphous polysaccharides, exhibiting a characteristic fibrous structure. They are insoluble in simple solvents and soluble in ammoniacal copper solution, in ZnCl2 and strong acids (H2S04), giving a dark brown to violet color with chlor-zinc iodide. They are very resistant to the action of plants, animals and majority of microorganisms, but they can be hydrolized by strong acids and certain specific microorganisms. They occur only in a natural state (plant tissues) and have not as yet been synthesized in the laboratory. Cotton contains 87 to 91 per cent celulose; wood of evergreens, 60 per cent; and cereal straw, 35 per cent. The empirical formula for cellulose is that of polysacchar- ides, namely (C6Hi0O5)n, the same as that of starch. It contains 44.42 per cent carbon and 6.22 per cent hydrogen. The ratio of oxygen to hydrogen is 8:1. On hydrolysis of cellulose, substances with an alde- hyde (d-glucose united by cellobiose linkages) grouping are obtained. Various formulae have been suggested to account for the chemical structure of the cellulose molecule. This is looked upon either as a polymerized molecule of cellobiose held together by strictly primary valencies or as a colloid molecule held together by secondary or residual valencies. When hydrolyzed by means of acetic acid anhydride and sulfuric acid, the cellulose swells and goes into solution in the form of a colloidal cellulose slime. On further hydrolysis, the disaccharide cellobiose is obtained (only about 36 per cent): r -° — — i CH2 OH • CHOH • CH • CHOH • CHOH • CHO CHOH • CHOH • CHOH • CH • CH • CH2 OH L o 1 This consists of two molecules of d-glucose and differentiates cellulose from starch, which on hydrolysis gives first maltose, then glucose.11 2(C6 H10 06)„ + nH20 = nC12 H22 On C12 H22 Ou + H20 = 2C6 Hi2 06 cellobiose glucose The existence of a reducing trisaccharide (procellose) as a possible intermediary product of hydrolysis between cellulose and cellobiose 11 Skraup, Z. H., and Konig, J. tlber Cellose, eine Biose aus Cellulose. Ber. deut. cbem. Gesell., 34: 1115-1118. 1901. DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER 431 has been suggested.12 On hydrolysis, it gives a molecule of glucose and one of cellobiose, the latter breaking down to two molecules of glucose.13 In the earlier literature, especially on the transformation of plant materials in the composting of manure and in soil, the conception of cellulose has frequently been confused with that of "crude fiber." However, the two are not identical, and it is essential to know how to measure quantitatively the well defined chemical complex, namely, the cellulose. For this purpose, the method of Charpentier14 is most suitable. Schweitzer's reagent is prepared by dissolving 200 grams-CuSC^ in hot water, then precipitating with a calculated amount of ammonia (23 grams NH3 or 99 cc. of ammonia water, specific gravity 0.90). The excess of ammonia is neutralized with sulfuric acid. The precipitate is washed 3-4 times by decantation, then filtered on a Buchner funnel through a hardened filter paper. The hardened pre- cipitate is dissolved in sufficient ammonia water (specific gravity 0.91), by shak- ing for 4 to 5 hours, so that 100 cc. of reagent contains 1.5 grams Cu (5 cc. of reagent is evaporated over sulfuric acid under a bell-jar, dried and heated to constant weight and weighed as CuO). Twenty grams of soil previously treated with finely divided cellulose are placed in a cylinder of 150 cc. capacity with 100 cc. of the Schweitzer reagent. The cylinder is closed and shaken, in a special shaking machine, for 30 minutes; the suspension is then allowed to settle. The solution is filtered through as- bestos in a Gooch crucible and 50 cc. of the filtrate are precipitated with 200 cc. of SO per cent alcohol. The precipitate is filtered through a Gooch crucible and 12 Bertrand, G., and Benoist, S. Sur un nouveau Sucre, le procellose, obtenu a partir de la cellulose. Compt. Rend. Acad. Sci., 176: 1583-1587. 1923. 13 Further information on the chemistry of celluloses is given by Schwalbe, C. G. Die Chemie der Cellulose unter besonderer Beriicksichtigung der Textil- und Zellstoffindustrien. Berlin, Borntraeger. 1911; Czapek, F. Biochemie der Pflanzen. Bd. 1, p. 645. Heuser, 1923 (p. 427). Pringsheim, 1923 (p. 427). Euler, A. C. Uber die Konstitution der Zellulose und der Zellobiose. Chem. Ztg., 46: 977-998. 1921. Hibbert, H. Studies on the chemistry of cellulose. I. The constitution of cellulose. Jour. Ind. Engin. Chem., 13: 256-260; 334- 342. 1921; Cross, C. F., and Bevan, E. J. Cellulose. Longmans, Green & Co., London. 1916; Karrer, P. Einfiihrung in die Chemie der polymeren Kohlen- hydrate. Akad. Verlagsgesellsch. Leipzig. 1925. 14 Charpentier, C. A. G. Studien uber den Einfluss des Rindvieh- und Pfer- destallmistes auf die Zersetzung der Zellulose in der Ackererde. Thesis. Univ. Helsingfors. 1921. Also Meddel. No. 205 Centralanst. forsoksv. jordbruck. Bakteriol. Avdel. No. 22, Stockholm. 1920. On the preparation of Schweitzer's reagent, see Dischendorfer, O. Uber das Zellulosereagens Kupferoxydammoniak. Ztschr. wiss. Mikrosk., 39: 97. 1922. General methods of cellulose deter- mination are given by Cross and Bevan. 432 PRINCIPLES OF SOIL MICROBIOLOGY is then washed with (I) dilute (1 per cent) hydrochloric acid, (2) distilled water, (3) dilute (2 per cent) KOH solution, (4) distilled water, (5) dilute HC1 solution, (6) distilled water, (7) alcohol, and finally (8) ether. The cellulose is then dried at 100° to 110° to constant weight, burnt off and the crucible is reweighed. The difference obtained gives the amount of cellulose in 10 grams of soil. Soils rich in organic matter (so-called "humus soils") have to be treated first with 10 per cent CaO, so as to neutralize the humus sub- stances, which would prevent the solution of the cellulose,15 or extracted with 2 per cent solution of sodium hydroxide, then washed with water, dilute acetic acid and water. To determine cellulose in straw, wood or other natural products added to the soil, the latter is first treated with sodium acid sulfite, and then extracted with Schweitzer's reagent;16 the natural plant material may also be extracted with 2 per cent sodium hydroxide solution, at 15 pounds pressure, for 30 to 60 minutes, then washed and boiled with 2 per cent solution of sulfuric acid and washed, and only then extracted with Schweitzer's reagent; the precipitation of the cellulose, filtering, washing and igniting is carried out as in the case of pure cellulose. Mechanism of decomposition of cellulose by microorganisms. Mits- cherlich17 was the first (1850) to attribute the fermentation of cellulose to the activities of microorganisms. Popoffls demonstrated in 1875 the connection between cellulose decomposition and methane formation; in the anaerobic decomposition of organic matter, the C02:CH4 ratio was found to be 1:1. Tappeiner19 established conclusively that micro- organisms are concerned in the decomposition of cellulose. In studying the bacterial changes which take place normally in the intestinal canal, Tappeiner introduced finely divided cotton or paper into flasks containing a 1 per cent neutral solution of beef extract. The flasks and contents were sterilized and then inoculated with small quantities of pancreatic juice and incubated at 35°C. They were so arranged that the gases could be collected and analyzed. The resulting product consisted of acetic acid, isobutyric acid, 15 Dmochowski, R., and Tollens, B. tlber eine neue Methode der quanti- tativen Zellulosebestimmung. Jour. Landw., 58: 1-20. 1910. 16Bengtsson, 1925 (p. 680). 17 Mitscherlich, E. tlber die Zusammensetzung der Wand der Pflanzenzelle. Ber. Bekanntmach. Verhandl. Konigl. Preuss. Akad. Wissensch. Berlin, 1850, 102-110. 18 Popoff, L. tlber die Sumpfgasgarung. Arch. Ges. Physiol., 10: 113-146. 1875. 19 Tappeiner, H. Uber Celluloseverdauung. Ber. deut. chem. Gesell., 15: 999-1002. 1882; Ztschr. Biol., 20: 52-134. 1884. DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER 433 acetaldehyde, methane and carbon dioxide. The last two were in the ratio of 1:7.2 at the beginning of the experiment and 1:3.4 at the close. In another set of experiments, an alkaline medium was used; the same qualitative but different quantitative results were obtained, there being a large amount of hydrogen evolved in the alkaline medium. Hoppe-Seyler20 placed 25.773 grams of filter paper into 1000 cc. flasks containing 700 cc. of water; this was inoculated with sewage and the gaseous products collected over mercury. The cultures were incu- bated at room temperature for four years. During the first year there was considerable gas evolved, but the evolution gradually became slower until, at the end of four years, the evolution of gas had practically ceased. The analysis showed that 15 grams of the cellulose had been decomposed with the formation of carbon dioxide and methane. Hoppe-Seyler was unable to find any of the true sugars, although he thought it possible that there were some dextrin compounds in solu- tion. When air was excluded, there was a greater production of me- thane and to a less extent carbon dioxide. Hoppe-Seyler suggested that the process of cellulose decomposition proceeds in two stages: the cellulose is first hydrated with the formation of glucose, ac- cording to the equation : (C6H10O5)„ + nH20 = nC6H,206 The glucose is then broken down to carbon dioxide and methane: C6H1206 = 3C2H402 = 3C02 4- 3CH4 This reaction depended on the supply of oxygen; the greater the amount of available oxygen the less CH4 and the more C02 was formed in the process. However, neither was glucose demonstrated as an inter- mediary product nor was there any evidence submitted that only C02 and CH4 are formed. Omeliansky21 distinguished methane and hydrogen fermentations of cellulose, both processes being anaerobic in nature; organic acids, alcohols and gases were also formed. Van Iterson22 was the first to demonstrate that cellulose decomposition may also take place under aerobic conditions. Aerobic and anaerobic decomposition of cellulose 20 Hoppe-Seyler, F. tlber Garung der Cellulose mit Bildung von Methan und Kohlensiiure. Ztschr. physiol. Chem., 10: 201-217; 401-440. 1886. 21 Omeliansky, 1902 (p. 191). 22 Iterson, G. van. Die Zersetzung von Cellulose durch aerobe Mikroor- ganismen. Centrbl. Bakt. II, 11: 689-398. 1904. 434 PRINCIPLES OF SOIL MICROBIOLOGY were two distinct processes, carried on by different organisms and under distinctly different conditions. The anaerobic processes of cellulose decomposition may be conve- niently divided into two groups. (1) In the absence of nitrate, the cellulose may undergo a hydrogen or methane fermentation by anaero- bic bacteria. (2) In the presence of nitrates, the cellulose is destroyed by aerobic denitrifying bacteria. Under aerobic conditions cellulose decomposition was found to take place in two ways. (1) If the medium is slightly alkaline, certain aerobic bacteria and actinomyces will be most active. (2) If the medium is acid, fungi are active in the decomposition of the cellulose. The microorganisms capable of decomposing cellulose can be divided into seven general groups: (1) anaerobic bacteria, (2) aerobic bac- teria, (3) thermophilic bacteria, (4) denitrifying bacteria, (5) actino- myces, (6) fungi, and (7) possibly also invertebrate animals.23 In normal cultivated soils, the aerobic bacteria, fungi, and actinomyces are largely concerned with the decomposition of celluloses.2425 Under anaerobic conditions, as in peat and cranberry bogs, anaerobic bacteria are responsible for the decomposition of celluloses; while, in the manure heap, thermophilic bacteria may be largely active. The true celluloses are digested by the herbivorous animals by means of the bacteria present in their intestinal tract.26 When stable manure is added to the soil, large quantities of cellulose decomposing bacteria are introduced. This has led to the claim that the addition of stable manure is equivalent to inoculations of soil with strong cellulose de- composing bacteria. It is doubtful, however, whether these organisms could become active in the soil, even if all normal soils were not already abundantly supplied with various types of organisms capable of de- composing celluloses. To be able to demonstrate the nature of the decomposition of cellu- lose by microorganisms, enzymes had to be obtained so as to allow the investigation of the hydrolytic nature of the phenomenon. Some 23 Younge, C. M. The digestion of cellulose by invertebrates. Sci. Progr. No. 78: 242-248. 1925. 24 Miitterlein, C. Studien liber die Zersetzung der Cellulose im Diinger und im Boden. Inaug. Diss. Leipzig. 1913;|Waksman and Skinner, 1926 (p. 190). 25 Pringsheim, H. Die Beziehungen der Zellulosezersetzung zum Stickstoff- haushalt im der Natur. Centrbl. Bakt. II, 37: 111-112. 1913. 26 Scheunert, A. Beitriige zur Kenntnis des Cellulosverdauung im Blindarm und des Enzymgehaltes des Coecalsecretes. Ztschr. physiol. Chem., 48: 9- 26. 1908. DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER 435 bacteria decompose celluloses with the formation of a clear zone around the colony, indicating the formation of an exo-cellular enzyme;27 other microorganisms, especially certain fungi and bacteria, can utilize cellu- lose without the production of a clear zone on the cellulose plate, in- dicating the endo-nature of the corresponding enzyme. Pringsheim28 allowed the decomposition of cellulose to proceed till a maximum has been reached, as indicated by gas formation. Bac- terial action was then quickly brought to a standstill by the introduc- tion of a proper antiseptic, which prevented the further development of the bacteria without injuring the enzyme cellulase. The products of hydrolysis (sugars) will then accumulate, due to the fact that the sudden stop of bacterial action does not prevent the hydrolytic en- zyme (cellulase) from breaking down the cellulose. The hydrolytic products cellobiose and glucose are demonstrated by the reduction of Fehling 's solution, and formation of corresponding osazones. Since the decomposition of cellulose is a comparatively slow proc- ess, especially at normal temperatures, the hydrolytic action of the enzyme is also very slow and does not lead to any abundant accumu- lation of products of hydrolysis. This, as well as the destruction of the hydrolytic enzymes by proteolysis, suggested the use of large quantities of media which are concentrated in vacuo, at a low tempera- ture, so as to obtain a solution with a sugar concentration sufficient for identification. In the methane, hydrogen, and denitrifying proc- esses, it takes two to seven days before reducing sugar can be demon- strated. The thermophilic bacteria, which are much more active, give a strong reduction of Fehling solution in 24 hours. Just as starch is hydrolyzed by amylase at first to the disaccharide maltose and then by a separate enzyme (maltase) to glucose, so is cellulose first hydrolyzed by the cellulase of bacteria to the disaccharide cellobiose, then by the enzyme cellobiase to glucose. The latter en- zyme has also been demonstrated in cellulose decomposing bacteria and fungi.29 "Kellerman and McBeth, 1912 (p. 197). Lohnis, F., and Lochhead, G. Experiments on the decomposition of cellulose by aerobic bacteria. Centrbl. Bakt. II, 58: 430-434. 1923. 28 Pringsheim, H. tlber den fermentativen Abbau der Zellulose. Ztschr. physiol. Chem., 78: 266-291. 1912. 29 Fischer, E., and Zemplen, G. Verhalten der Cellobiose und ihres Osons gegen einige Enzyme. Liebig's Ann. Chem., 335: 1-6. 1909: 372: 254-256. 1910; Bertrand, G., and Holderer, M. La cellase et le d^doublement diastatique du cellose. Compt. Rend. Acad. Sci., 149: 1385. 1909; 150: 230. 1910. 436 PRINCIPLES OF SOIL MICROBIOLOGY Cellulase acts at temperatures of 20° to 70°. When a culture of thermophilic bacteria acting at 55° is brought to room temperature, the action of the living bacterial cells upon cellulose and the production of gas stop but the enzyme action continues even at 20°. By a mere change in temperature, active growth accompanied by gas formation is discontinued; under these conditions enzyme action results in an accumulation of sugar, as indicated by reduction of Fehling 's solution and osazone formation. The cellulose is transformed into cellobiose and the latter partly to glucose by the enzyme cellobiase, which is still active at 20°. By raising the temperature to 67°, Pringsheim succeeded in repressing both the growth of bacteria and the action of cellobiase, leaving only cellulase active; this resulted in the accumulation of only cellobiose. Groenewege30 also demonstrated that bacteria hydrolyze cellulose, by means of an enzyme cellulase, into cellobiose and the latter is then hydrolyzed to glucose. In an alkaline medium, cellobiose is formed faster than it is hydrolyzed. It is much easier to demonstrate the formation of an enzyme decomposing celluloses by fungi.31 The glucose formed from cellulose by the action of the bacterial enzymes is rapidly broken down by the same organisms or by accom- panying forms to various organic acids, such as acetic, butyric and lactic or formic, acetic and valeric (Groenewege). These acids are decom- posed, either by the same bacteria or by a secondary flora, to carbon dioxide and water. In the decomposition of sugars by the anaerobic bacteria, methane, hydrogen and carbon dioxide are formed directly. In some cases, the transformation of the cellulose leads to the forma- tion of mucilages consisting of hemicelluloses. According to Neuberg and Cohn,32 acetaldehyde is formed as an intermediary product in the decomposition of cellulose by thermophilic bacteria. It is this ace- taldehyde which may serve as the building stone for the synthesis of the microbial protoplasm. The formation of pigments in the decom- position of cellulose is more characteristic of the organism than of the cellulose. 30 Groenewege, J. Untersuchungen liber die Zersetzung der Zellulose durch aerobe Bakterien. Bull. Jard. Bot. Buitenzorg (3), 2: 287. 1920; Meddel. Alg. Proefsta. Landbr. Dept. Nijv. Handel., 13: 1-23. 1923. 31 Kellerman, K. F. Formation of cytase by Penicillium pinophilum. U. S. Dept. Agr., Bur. PI. Ind. Circ. 113, 1912; Ellenberger, W. Zur Frage der Cellu- losverdauung. Ztschr. physioi. Chem., 96: 236-254. 1915; Kosin, N. I. On the aerobic decomposition of cellulose by fungi. Rpt. of the Physico-Chem. Lomonossov Soc, Moskau, 2: 57-98. 1921. 32 Neuberg, C, and Cohn, R. tJber Zwischenprodukte des bakteriellen Ab- baues von Cellulose. Biochem. Ztschr., 139: 527-544. 1923. DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER 437 In view of the fact that certain aerobic bacteria cannot utilize sugars as sources of energy, it still remains to be demonstrated just how the cellulose molecule is decomposed by these organisms. Decomposition of cellulose by anaerobic bacteria. The anaerobic decomposition of cellulose results in the formation of organic acids and gases consisting of carbon dioxide and methane or hydrogen. As pointed out above, Omeliansky3* found that the formation of methane or hydrogen in the anaerobic process is due to the fact that different organisms are active in either case. When cellulose was decomposed by the methane organism 50 per cent of the substrate was changed to gas (6.5 per cent methane and 43.5 per cent C02), and 50 per cent into fatty acids (acetic and butyric). When cellulose was decomposed by the hydrogen organism, about 33 per cent of the substrate was changed into gases (4 per cent hydrogen and 29 per cent C02) and 67 per cent into fatty acids (acetic, butyric, and small quantities of valerianic acid). The decomposition of cellulose is soon stopped by the rapid accumulation of acids unless CaC03 is added to keep the medium neu- tral. Even then the process of cellulose decomposition is very slow and it may take months before 5 to 10 grams of filter paper suspended in a liter of medium are fully dissolved. Omeliansky showed that it took the hydrogen organism thirteen months to form the following products from 3.347 grams of cellulose: gra m s Fatty acids 2.240 C02 0.972 H2 0.014 The loss of 0.121 gram was believed to be due to substances not determined, including valerianic acid, higher alcohols, aromatic sub- stances and dissolved hydrogen. The methane organism formed, out of 2 grams of cellulose decom- posed: grams Fatty acids 1 .022 C02 0 . 868 CH< 0.137 The methane and hydrogen processes go on simultaneously in mixed culture. A convenient apparatus for the study of cellulose decomposi- tion under anaerobic conditions is shown in fig. 23. 33 Omeliansky, 1904 (p. 191). 438 PRINCIPLES OF SOIL MICROBIOLOGY The anaerobic bacillus isolated by Khouvine34 from the human in- testinal tract was found to be capable of using cellulose as the only- source of energy; sugars could not be utilized. One gram of cellulose was decomposed in 16 days; but, in the presence of other bacteria, five Fig. 23. Apparatus for study of cellulose decomposition, under anaerobic conditions (after Pringsheim) . times as much cellulose was decomposed. Among the decomposition products, C02, H2, ethyl alcohol, acetic and butyric acids, traces of lactic acid, products precipitated by alcohol and a yellow pigment could be demonstrated. " Khouvine, 1923 (p. 193). DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER 439 The following equations were suggested35 to explain the anaerobic decomposition of celluloses: C6H10O6 + H20 -> 2 CH3 • CO • COOH + 2 H2 Pyruvic acid CH3 • CO • COOH + H2 -> CH3 • CHOH • COOH Lactic acid CH3 • CO • COOH -> CH3 • CHO + C02 acetaldehyde 2 CH3 • CHO + H20 -> CH3 • CH2 • OH + CH3 • COOH 2 CH3-CHrOH -> CH3COOH + 2 CH4 Lactic acid may also break down to give butyric acid 2 CH3 • CHOH • COOH -* CH3 • CH2 • CH2 • COOH + 2 C02 + 2 H2 Decomposition of cellulose by aerobic bacteria. Cellulose decomposi- tion by aerobic bacteria can be studied by suspending filter paper in a shallow layer of a medium containing an appropriate nitrogen source and the necessary minerals and inoculated with a pure or crude culture of bacteria, or with soil or manure. At room temperature, the cellu- lose will be found to be rapidly decomposed with the formation of a slime or mucilage which may be colored yellow or red. Carbon dioxide is formed abundantly but no other gas, so that no visible "fermenta- tion" is found to take place; small quantities of acids are formed in the medium. The cellulose is largely macerated and the fibers separated from one another and gradually reduced to a pulp. Some organisms are very active and others are very slow. With some species isolated by Kellerman and associates,36 the principal by-products were found to consist of formic and acetic acids, while others gave rise only to traces of fatty acids; no aldehydes, ketones or alcohols were formed. The aerobic bacterium Spirochaeta cytophaga uses cellulose as the only source of carbon and produces a pigment related to the carotin group, also some mucilage, which does not give rise to optically active com- pounds on hydrolysis, and small quantities of fatty acids, chiefly butyric. The mucilage is extracted in the crude "humus" fraction in soil analysis.37 Decomposition of cellulose by thermophilic bacteria. Thermophilic bacteria destroy cellulose very actively. A nutrient solution contain- 36 Langwell and Lymn, 1923 (p. 202). »6 Kellerman et al., 1914-1916 (p. 197). 17 Hutchinson and Clayton, 1919 (p. 195). 440 PRINCIPLES OF SOIL MICROBIOLOGY ing pure filter paper, inoculated with a small quantity of soil and incubated at 60°C, produces gas and a distinct odor. The filter paper is all broken up in 10 to 14 days, with the formation of C02 and CH4; formic and acetic acids are also demonstrated among the decom- position products.38 Kroulik39 obtained cellulose decomposition at 60°C, under aerobic and anaerobic conditions. When cellulose was decomposed aerobically, only C02 was formed; under anaerobic condi- tions, the cellulose was decomposed more completely, with the forma- tion of H2, C02 and even H2S. Among the decomposition products, considerable quantities of acetic acid, some butyric, formic and acetic acids were demonstrated. Organic nitrogen was found to be the best source of nitrogen. Pringsheim40 obtained, under anaerobic condi- tions, out of 3 grams of cellulose decomposed, 0.2125 gram formic acid, 1.15 gram acetic acid and a small quantity of lactic acid. Carbon dioxide made up 21.9 to 49.1 per cent of the gases, the rest was hydrogen. Ethyl alcohol, acetic, butyric and lactic acids, hydrogen and methane result from the decomposition of cellulose at higher temperatures. By changing the conditions of growth, the relative amounts of the products may be changed. Viljoen, Fred and Peterson41 found that, out of 60 grams of cellulose added to 4 liters of medium containing 20 grams of peptone, 42 grams were decomposed by thermophilic (anaerobic bac- teria) with the formation of the following products: YIELD CARBON CONTENT Acetic acid grams 21.6 10.3 11.94 gra m s 8.6 Alcohol 5.4 C02 3 0 Carbon of cellulose decomposed 18.6 grams Carbon of products accounted for 17.0 grams Among the gases, hydrogen was formed in considerable quantities, but no methane. The rapid heating of hay results from transformation of cellulose and other carbohydrates in the hay by microorganisms.42 Bad. colt, 18 MacFayden and Blaxall, 1899 (p. 201). "Kroulik, 1913 (p. 157). 40 Pringsheim, 1913 (p. 202). 41 Viljoen, Fred and Peterson, 1926 (p. 202). 42 Miehe, 1907 (p. 300); Uber die Selbsterhiztung des Heues. Arb. deut. landw. Gesell. H 196, 36 p. 1911; (Centrbl. Bakt. II, 34: 281-282. 1912). DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER 441 Oidium lactis and Bac. calfactor were found capable of heating up well packed hay and a mixture of two of these organisms could bring about a normal heating of moist hay. In addition to these, Miehe42 isolated an actinomyces and various fungi from the piles of heated hay. However, the actual role of these organisms in the heating of hay is still unclear. It has been suggested that either inflam- mable products are formed or that the initial reaction of raising the temperature is biological, followed by chemical processes. Decomposition of cellulose by denitrifying bacteria. In the decom- position of cellulose by denitrifying bacteria, the energy liberated is used for the reduction of nitrates to atmospheric nitrogen; the oxygen thus obtained is utilized for the oxidation of the cellulose. Carbon dioxide and water are the chief products formed in the decomposition of cellulose, while nitrogen gas is a product of the reduction of nitrates. The formation of carbonates, due to the reduction of the nitrates, leads to the alkalinization of the medium: 5 C«H1()06 + 24 KN03 = 24 KHC03 + 12 N2 + 6 C02 + 13 H20 C6Hio05 + 8 KNO3 = 4 KHCO3 + 2 K2CO3 + 4 N2 + 3 H20 Groenewege43 demonstrated that this process of cellulose decom- position is a result of the symbiotic action of two groups of bacteria, one of which decomposes the cellulose and the other reduces the nitrate utilizing the energy liberated by the former. By the combination of these two processes the cellulose is decomposed much more readily than by the cellulose bacteria alone. Under aerobic conditions, cellu- lose decomposition takes place readily with other sources of nitrogen, as seen from table 35. The cellulose decomposing organism produces an enzyme which hydrolyzes the cellulose into sugars. The latter are transformed by the organism into organic acids (acetic, butyric and lactic). The sugars and the acids are used as sources of energy by the denitrifying bacteria which decompose them into C02 and water. The intermediary products of cellulose decomposition can also be used as a source of energy by Az. chroococcum which is enabled thereby to fix nitrogen and form dark brown pigments in the soil. Cellulose decomposition by actinomyces. The capacity of decomposing cellulose and using it as a source of energy is well distributed among the actinomyces.44 This can be readily demonstrated either by the 43 Groenewege, 1920-1923 (p. 436). 44 Fousek, 1913 (p. 302). 442 PRINCIPLES OF SOIL MICROBIOLOGY cellulose-plate method or by adding cellulose, in the form of filter paper, to a medium containing the necessary inorganic salts and a source of nitrogen. Krainsky45 found that certain pigment-producing species, forming small, spherical spores (Act. melanocyclus and Act. melanosporeus) are particularly active in this connection, forming black or red rings on the paper and causing a decomposition of the cellulose. On the agar plate, clear rings are formed around the colony, due to the decomposition of the cellulose by an exo-enzyme. To study the process quantitatively, a definite amount of filter paper is placed in flasks containing a synthetic nutrient medium, with ammonium salt (using in the case of chloride or sulfate also calcium carbonate so as not to allow the reaction to become too acid, which would prevent the develop- ment of the organism) or nitrate as a source of nitrogen. The paper TABLE 35 Cellulose decomposed in 28 days by a mixture of a cellulose-decomposing and denitrifying organism NITROGEN SOURCE FILTER PAPER ADDED FILTER PAPER DECOMPOSED NH4NO3, 0. 1 per cent mgm. 231.0 225.8 231.5 226.5 mgm. 191.0 KNO3, 0 1 per cent. . . 107.3 NH4CI,* 0 1 per cent 209.5 198.0 * CaC03 was added with the ammonium chloride so as to keep the reaction alkaline. is allowed to dip partly in the medium, since the actinomyces are aerobic organisms and do not develop readily below the surface of the medium. After a definite period of incubation, the culture is filtered and residual paper washed and weighed. It is necessary to determine the nitrogen content of the washed paper, due to the presence of some of the mycelium of the organism. By allowing 10 per cent as the nitrogen content of the mycelium, the residual cellulose can be readily calculated. The liquid medium is analyzed for the residual inorganic nitrogen and various organic acids. It is found that a definite ratio exists between the amount of cellulose decomposed and nitrogen assimi- lated by the organism. Some of the organisms will decompose the paper to such an extent as to leave transparent mucilaginous strands *« Krainsky, 1913-1914 (p. 297). DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER 443 which fall to pieces when an attempt is made to lift them out of the medium. Some actinomyces produce red and black pigments through the paper. However, when compared with the bacteria and fungi capable of decomposing celluloses, the action of most of the actinomyces upon cellulose is only slow and limited. Cellulose decomposition by filamentous fungi. The destruction of hemicellulose in plant tissues is to be sharply differentiated from that of cellulose. Fungi act differently upon hemicelluloses of different plants, due probably to the difference in chemical composition of the plant tissues rather than to differences in solubility of the hemicellu- loses.46 It has been shown elsewhere (p. 263) that fungi possess a strong cellulose-decomposing power, especially certain Fusaria, Asper- gilli, Penicillia, Trichodermae and various Dematiaceae. It has even been suggested that in normal cultivated soils, these organisms play the most important role in this connection. The following method can be used for comparing the cellulose decomposing power of different microorganisms : Finely ground filter paper (1 gram) and 20 cc. of a nutrient solution [10 grams (NH4)2S04, (NH4)2HP04 or NH4NO3, 3 grams K2HP04, 2 grams MgS04, 1 gram NaCl, 1000 cc. of tap water, with or without 5 grams CaCOs] are added to 100 grams portions of sand or soil, placed in flasks, sterilized (for 2 hours at 15 pounds pressure) and inoculated. If possible, it is advisable to connect the flasks with an aeration apparatus and determine the evolution of CO2. When soil is used as a medium, a control without cellulose, sterilized and inoculated should also be employed. When CaCOs is added, allowance should be made for the CO 2 given off by the interaction of organic acids with the carbonate. The cultures are in- cubated for 3 to 6 weeks at 25° to 28°C. At the end of the incubation period, the amount of cellulose left in the 20 grams of the moist culture (allowing for the moisture content) is determined by the quantitative method of Charpentier. A series of results47 obtained by this procedure, with sand as a medium, are presented in table 36. When cellulose is the only source of energy available, one part of nitrogen is assimilated by fungi from inorganic sources added to the medium for every thirty parts of cellulose decomposed.48 This fact 46 Schellenberg, 1908 (p. 264). 47 Waksman, S. A., and Heukelekian, H. Cellulose decomposition by various groups of soil microorganisms. 4th Int. Cong. Pedology. Rome. 3: 216-228. 1924. 48 Heukelekian, O., and Waksman, S. A. Carbon and nitrogen transforma- tions in the decomposition of cellulose by filamentous fungi. Jour. Biol.Chem., 66: 323-342. 1925. 444 PRINCIPLES OF SOIL MICROBIOLOGY permits the measurement of nitrogen transformation as an index of cellulose decomposition, since they run parallel. There is good evidence that fungi are chiefly responsible for the decomposition of celluloses in acid, humid soils under aerobic conditions. When the soil is partially sterilized to eliminate the fungi, cellu- lose decomposition comes practically to a standstill. When cellu- lose is added to the soil, especially in the presence of an available source of nitrogen, the fungi develop much more abundantly than TABLE 36 Decomposition of cellulose by pvre cultures of microorganisms ORGANISM Trichoderma koningi Trichoderma koningi Fusarium 115 Aspergillus fumigatus Aspergillus glaucus Aspergillus wentii Aspergillus fuscus Penicillium of soil Group III . Mucor racemosus Z ygorhynclius molleri Cunninghamella elegans Actinomyces violaceus-ruber . . . . A ctinomyces cellulosae Actinomyces viridochromogenus Bacterium fimiia Bac. cereus Bac. vulgalus CELLULOSE INCUBATION DECOMPOSED days per cent 21 46.8 42 95.0 21 36.8 21 93.0 21 49.0 21 0 21 0 21 8.5 21 0 21 0 21 0 21 6.8 42 12.76 42 0 42 29.0 42 0 42 0 either the bacteria or actinomyces;50 this can be readily demonstrated both by the plate and direct microscopic methods. Cellulose decomposition in manure. Thirty to forty per cent of the dry weight of manure consists of cellulose and 20 to 30 per cent of pentosans.51 When manure undergoes decomposition, whether in the 49 This organism of Kellerman et al., was from stock culture; Spirochaeta cytophaga studied later was found to be much more active and decomposed al- most as much cellulose as the fungi themselves. 60 Waksman and Skinner, 1926 (p. 190). 61 Stoklasa, J. tTber die Wirkung des Stallmistes. Ztschr. landw. Versuchsw. Oesterr., 10: 440. 1907; Fuhling's landw. Ztg., 56: 411. 1907. DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER 445 compost heap or in the field, the celluloses and the pentosans are the first to disappear (following of course the immediate decomposition of the small amounts of sugars and starches that may be present in the manure). Deherain52 distinguished two processes of manure decom- position: (1) aerobic, when the temperature goes up to 65° to 70°; (2) anaerobic, at 30° to 35°. The composition of the gases given off in the decomposition of manure is given in table 37. Deherain believed that a large part of the cellulose of the straw is decomposed, with the formation of CH4 and C02. The residue left, after a part of the nitrogenous substances and the cellulose have been decomposed, forms the black matter of the manure. This is accom- panied by the transformation of ammonia (urea) nitrogen into protein nitrogen. A spore bearing bacillus was found to decompose the cellu- lose actively under anaerobic conditions with the formation of methane, TABLE 37 Composition of gases in different parts of the manure heap TOP LAYER OF HEAP MIDDLE LAYER OF HEAP BOTTOM LAYER OF HEAP Carbon dioxide per cent 21.6 0.0 0.0 78.4 per cent 31.0 0.0 33.3 35.5 per cent 37.1 Oxygen 0.0 Methane 58.0 Nitrogen 4.9 when a small amount of manure was inoculated into a synthetic me- dium. Omeliansky succeeded in demonstrating that both methane and hydrogen-cellulose fermentations take place in manure. In view of the fact that the decomposition of the organic matter in manure and in soil leads to a narrowing of the carbon-nitrogen ratio and forma- tion of dark-colored substances, Omeliansky53 suggested that the cellu- lose decomposition takes place according to the reaction: 2(C6H10O6) = 5 C02 + 5 CH4 + 2 C. lie states "It is possible that a general reaction of this sort forms the basis of the universal processes of humification, that is, the gradual 62 Deherain, P. P. Ann. Agron., 14: 97-133. 1888. (Deherain, 1902 (p. 793).) 53 Omeliansky, 1926, (p. xii) p. 309. No experimental evidence was submitted to justify this equation and it is doubtful whether it ever takes place in nature. Cellulose decomposition never results in the direct formation of "humus." 446 PRINCIPLES OF SOIL MICROBIOLOGY transformation of organic substances into a mixture of brown and black substances with a high content of carbon such as is characteristic of fossil coals. But whatever the mechanism of these transformations, the active participation of microorganisms in the latter cannot be denied." According to Pringsheim,54 thermophilic bacteria are also active in the decomposition of cellulose in manure, with the formation of acetic and formic acids, C02, hydrogen and methane. The pentosans may be acted upon even before the celluloses are decomposed. When fresh manure is allowed to compost, the bacteria and fungi transform the celluloses, pentosans and pectins of the straw into various organic substances and carbon dioxide. The organisms thereby absorb a great deal of the available nitrogen present in the urine for the synthesis of their protoplasm. When fresh manure is applied to the soil directly, it may not stimulate and may even injure plant growth. This is due to the assimilation of the available nitrogen in the manure and in the soil by the microorganisms which use the carbohydrates of the manure as sources of energy. The composting of the manure brings about a great reduction in the nitrogen-free compounds and the transformation of the available nitrogen into microbial proteins.55 Importance of cellulose decomposition in the soil. According to Mutterlein's56 calculation, 1 acre of soil in Germany receives yearly about 200 kgm. of cellulose in the form of manure. When we add to that the amount introduced by green manuring, plant stubble, root residues, weeds, etc., quite appreciable quantities are obtained. Some of the plant constituents are attacked more readily than others. The sugars and starches and, to a more limited extent, the proteins are acted upon first, then the pentosans and celluloses; then the oils, fats and lignins; and finally the waxes which may persist in the soil for a long time unaltered. Cellulose was considered by older investigators and even some re- cent ones57 to be the mother substance of soil "humus." On the other hand, Hoppe-Seyler5S and others have demonstrated that since 64 Pringsheim, 1913 (p. 202). 65 Heinze, B. tlber die Verrottung des Stalldungers. Centrbl. Bakt. II, 25: 503-504. 1910. 66 Mutterlein, 1913 (p. 434). 67 Marcusson, J. Torfzusammensetzung und Lignintheorie. Ztschr. angew. Chem., 38: 339. 1925. 48 Hoppe-Seyler, 1889 (p. 700). DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER 447 no dark colored substances are formed directly from the decomposition of celluloses in the soil, the sources of "humus" are to be looked for in the other plant constituents besides the celluloses (p. 691). How- ever, indirectly cellulose may contribute to the soil organic matter or "humus", due to the fact that it is a readily available source of energy and is decomposed in aerated soils largely through the activities of fungi and bacteria, resulting in the synthesis of an extensive mi- crobial protoplasm.59 Although celluloses cannot be used directly as sources of energy for nitrogen-fixing bacteria, they may form products which are available sources of energy for these organisms. Beijerinck60 found that a medium consisting of two parts of paper, two parts of chalk and 0.05 per cent K2HPO4 in 100 cc. of tap water was a favorable medium for nitrogen-fixation. At 25° to 30°, there developed chiefly the hydrogen- forming organism together with Bad. radiobacter and Azotobacter. For every gram of cellulose decomposed, 8 to 9 mgm. of nitrogen were fixed. Bac. amylobacter*1 and Azotobacter62 are unable to attack cellu- lose in pure culture. Only in mixture with cellulose decomposing organisms, are they able to fix nitrogen, at the expense of the cel- lobiose, the glucose or the fatty acids formed from the decomposition of the cellulose. In a mixture of the methane-forming organism and Clostridium americanum, Pringsheim61 obtained 12.1 mgm. nitrogen fixed for 1 gram of cellulose decomposed. The methane bacterium produced 10 grams fatty acid out of 20 grams cellulose ; but in the presence of nitro- gen-fixing organisms, only 0.064 gram fatty acid accumulated. Azoto- bacter fixed 4.5 mgm. of nitrogen out of 1 gram of cellulose, in sym- biosis with the methane organism; this is probably due to the fact that Azotobacter does not thrive well under semi-anaerobic conditions. "Waksman, S. A. Soil Sci., 22: 123-162, 221-232, 323-333, 395-406. 1926. 60 Beijerinck, M. W. L'influence des microbes sur la fertilite du sol et la croissance des vegetaux supe^rieurs. Arch. Neerland. Sci. Ex. Nat. Ser. II, 9: 8-36. 1904. 61 Pringsheim, H. Ueber die Verwendung von Cellulose als Energiequelle zur Assimilation des Luftstickstoffs. Centrbl. Bakt. II, 23: 300-304. 1909; 26: 222-227. 1910. Die Beziehungen der Zellulosezersetzung zum Stickstoff- gehalt in der Natur. Mitt. deut. landw. Gesell., 1912, 1913, p. 26, 43 (Centrbl. Bakt. II, 37: 111. 1913). 62 Koch, A., and Seydel, S. tlber die Verwertung der Zellobiose als Ener- giequelle bei der Stickstoffbindung durch Azotobacter. Centrbl. Bakt. II, 31: 567-577. 1911. 448 PRINCIPLES OF SOIL MICROBIOLOGY Under aerobic conditions, Koch63 obtained a more favorable nitrogen fixation by Azotobacter. Koch found stronger cellulose-decomposing bacteria in manure than in normal soil and, therefore, suggested that the beneficial effect of stable manure is due to the introduction of strong cellulose decomposing bacteria, as shown by the increased action of green manure when it is inoculated with a small amount of stable manure. The more rapid decomposition of the green manure results in an increased nitrogen fixation. Similar ideas on the favorable influence of small amounts of stable manure have been expressed by Lipman and associates.64 Their ideas were not confirmed by a careful analysis of the processes of cellulose decomposition. It was found65 that the favorable addition of small amounts of stable manure is due to the nutrients, especially the nitrogen that it contains. Nitrogen-fixation as a result of symbiotic action of nitrogen-fixing and cellulose-decomposing bacteria, with cellulose as the only source of energy, has also been demonstrated by other investigators.66 Peat is very resistant to the action of cellulose decomposing bacteria, but this resistance can be overcome by preliminary treatment of the peat, as boiling, steaming, or grinding. It is claimed that it may then become a source of energy for nitrogen fixing microorganisms.67 The importance of this process in increasing the supply of soil nitrogen is, however, still questionable. Influence of soil conditions upon cellulose decomposition. The exist- ence of thermophilic bacteria in the soil indicates that a high tempera- ture is not injurious to cellulose decomposition but may even be highly beneficial. According to Bertrand and Compton,68 46° is the 63 Koch, A. Die Luftstickstoffbindung im Boden mit Hilfe von Cellulose als Energiematerial. Centrbl. Bakt. II, 27: 1-7. 1910; Jour. Landw., 55: 355- 416. 1907. 64 Lipman, J. G., Blair, A. W., Owen, I. L., and McLean, H. C. The influ- ence of bacteria supplied in manure on the decomposition of green manure (legume and non-legume). N. J. Agr. Exp. Sta. 25th. Ann. Rpt.: 24S-260. 1912; 26th: 474-478. 1913; 27th: 223-226. 1914. 65 Barthel, Chr., and Bengtsson, N. Action of stable manure in the decom- position of cellulose in tilled soil. Soil Sci., 18: 1S5-200. 1924; Medd. No. 300, Centralanst. forsoks. jordbruks. Bakt. avd. 40. Stockholm. 1926. 66 Hutchinson and Clayton, 1918 (p. 195). Groenewege, 1920 (p. 436). 67 Schmidt, E. W. Torf als Energiequelle fiir die stickstoffassimilierende Bakterien. Centrbl. Bakt. II, 52: 281-289. 1920. 68 Bertrand, G., and Compton, A. Influence de la temperature sur l'activite de la cellase. Bull. Soc. chim. France, (4), 9: 100. 1911. DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER 449 optimum temperature for the action of cellobiase, while Pringsheim found that cellulose decomposition will take place at temperatures of 20° to 70°. As to the influence of soil reaction, Hellstrom69 demonstrated in 1899 that the action of lime on peat soils is not so much to serve as a nutrient for plants as to stimulate the decomposition of the organic substances in the peat and neutralize the acid products formed. Christensen70 also found that a basic soil shows a much higher cellulose decomposition than a base-free soil. The results of Charpentier and Barthel65 seem to prove that liming of soil has no favorable action at all upon cellulos Cellulose 6 8 0.i 0.6 O.i 0.* 0.3. Oi 04 0 Sandy soil Clay 50il I Clay soil 2 Peat soil 0 Pay; 60 90 120 210 Fig. 24. Influence of soil type upon the decomposition of cellulose (after Charpentier). decomposition. This is due largely to the fact that the process is carried out both by fungi and bacteria. The reaction of the soil no doubt influences the type of organisms taking an active part in cellu- lose decomposition, but not the process itself. Since various fungi and various bacteria decompose cellulose actively, a change in reaction will favor the development of either one group or another, while the actual amount of cellulose decomposed may be influenced only inappreciably. However, the decomposition of the organic matter of the soil itself, such as "humus" of mineral soils or peat and other decomposed or semi-decomposed organic materials may be greatly stimulated by the 69 Hellstrom, P. 1909. Ref. Charpentier, 1921 (p. 431). 70 Christensen, 1915 (p. 578). 450 PRINCIPLES OF SOIL MICROBIOLOGY addition of lime. Using C()2 evolution as an index of decomposition of organic matter, Potter and Snyder71 found that CaC03 accelerates the rate of decomposition of the organic matter present in the soil or added in the form of stable manure (10 to 50 tons per acre). Similar results were previously obtained by Lemmermann and associates.72 To stimulate cellulose decomposition in the soil, it may be sufficient to add the proper nutrients, such as phosphoric acid or nitrogen sub- stances. Charpentier7* found that the addition of 2 per cent of cow or horse manure will greatly stimulate cellulose-decomposition in the soil. The influence of the manure depends on the content of nutrients in the manure and in the soil. With a sufficient amount of moisture and a fav- orable reaction, the action of the manure is greater with a higher content Cei'ivlo&e i> 9.9 O.t V 3.--- ■■*--"|.1»w^« 1% Cellulose „ _ ., 27. stable manure . . — ♦''0.015?'. ammomium sulfate ^-"-3 ZhO 360 390 Fig. 25. Influence of nitrogen source upon the decomposition of cellulose in the soil (after Charpentier). of nutrients in the manure and a lower content in the soil. The greater the moisture content of the soil, the quicker will the cellulose be de- composed. Cellulose decomposition is active both in acid and alka- line soils, and lime merely plays the part of a regulator of soil reaction. The addition of equal amounts of nitrogen in the form of ammonium sulfate and cow manure brings about an equal stimulation of cellulose 71 Potter, R. S., and Snyder, R. S. Carbon dioxide production in soils and carbon and nitrogen changes in soils variously treated. Iowa Agr. Exp. Sta. Res. Bui. 39: 255-309. 1917. 72 Lemmermann, O., Aso, K., Fischer, H., and Fresenius, L. Untersuchungen liber die Zersetzung der Kohlenstoffverbindungen verschiedener organischer Substanzen im Boden, speziell unter dem Einfluss von Kalk. Landw. Jahrb., 41: 217-256. 1911. 73 Charpentier, 1921 (p. 431). DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER 451 decomposition in the soil. This points conclusively to the fact that the stimulating influence of cow and horse manure is due to its nitro- gen content. Tables 38 and 39 and figures 24 and 25 show the in- fluence of manure, soil reaction and nitrogen source on cellulose decomposition in various soils, as determined by Charpentier. The content of available nitrogen in the soil is thus found to be the most important factor controlling cellulose decomposition. The ratio between the cellulose decomposed and nitrogen required by the or- ganisms is about 30-35 to l.74 This ratio will depend of course upon the amount of available nitrogen. When the latter is in excess and is, TABLE 38 Composition of soils used for study of cellulose decomposition SOIL TYPE Clay soil I . Clay soil II Sandy soil. . Peat soil . . DRY MATTER PER CENT 82.0 82.0 92.0 40.0 ASH PER CENT OF DRY MATTER 94.8 93.5 97.8 19.9 CELLULOSE PER CENT OF DRY MATTER 0.06 0.01 0.04 0.03 pH 6.12 7.08 6.56 5.44 TABLE 39 Influence of cellulose (1 per cent) upon soil reaction (using clay soil I) NO LIME WITH LIME (0.5 PER CENT CaCOi pHat beginning pH at end pH at beginning pH at end Unmanured soil 6.12 6.12 5.54 630 7.73 7.73 7.37 2 per cent cow manure 7.77 therefore, not the limiting factor, the above ratio will hold true. When the amount of available nitrogen is low, it will be utilized by the or- ganisms over and over again, i.e., a part of the synthesized protoplasm of the microorganisms will be decomposed, liberating some of the nitrogen which is immediately again assimilated thus enabling the organisms to decompose more cellulose. The process will be con- tinuous, tending to give a higher ratio between the cellulose decom- position and apparent nitrogen assimilation. 74 Waksman and Heukelekian, 1924 (p. 443). Anderson, J. A. The influ- ence of available nitrogen on the fermentation of cellulose in the soil. Soil Sci., 21: 115-126. 1926. 452 PRINCIPLES OF SOIL MICROBIOLOGY Chemistry of hemicelluloses. The hemicelluloses are amorphous polysaccharides, but are distinguished from the celluloses by their easy solubility in dilute alkalies and in hot dilute acids, such as 1 per cent HC1. Some give the brown to black iodine reaction like true celluloses. On hydrolysis hemicelluloses give glucose, mannose, galac- tose, or mixtures of these, xylose and arabinose; the hemicelluloses are thus termed dextrans, mannans, galactans, mannogalactans, pentosans (xylans, arabinans), according to the constituent mono- saccharides. Hemicelluloses are present not only in higher plants, but also in algae, fungi and lichens (lichenin). Hemicelluloses of plant seeds are insoluble in water, diastase solution (differentiating them from starches), cold dilute KOH, but are more readily hydrolized by acids into sugars than the celluloses. In relation to their role in the plant tissues, celluloses are commonly believed to serve as pro- tective substances in plants and are unaffected by plant metabolism, while hemicelluloses are reserve materials in the plants, which must be brought into a soluble form by means of enzymes, before they can be utilized in plant nutrition; in some plants they serve for structural purposes holding the fibers together. The hemicelluloses thus comprise two different groups of substances: (1) the reserve celluloses (mostly mannans) of seed, and to some extent of grasses; (2) supporting substances, mostly galactans and pentosans, having a mechanical function. The reserve hemicelluloses and starches can take the place of one another, so that seeds poor in starch are rich in reserve celluloses and vice versa. The pentosans are present in the cell walls of all green plants, in the bark and woody fiber of trees, in mosses, fungi, seeds and fruits. They occur very abundantly in straw which usually contains 23 to 29 per cent pentosan. Corn cobs contain 32 per cent pentosan; pine needles 6.8 per cent, oak leaves 10.3 per cent. Older tissues contain larger quantities of pentosans than younger ones. According to Van Hulst, Peterson and Fred,75 the pentosan content of the corn plant increases from 7.4 per cent in the kernel to 31.8 per cent in the cob at maturity.76 The pentosans are probably not reserve materials but 75 Van Hulst, J. H., Peterson, W. H., and Fred, E. B. Distribution of pento- sans in the corn plant at various stages of growth. Jour. Agr. Res., 23: 655- 663. 1923. 76 The method of determination of pentosans is given by Pervier, N. C, and Gortner, R. A. The. estimation of pentoses and pentosans. Jour. Ind. Engin. Chem., 15: 1167, 1255. 1923. DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER 453 are of importance in the formation of wood and skeletal structure of various plants. The formula for pentosans is (C5H804)n, yielding, on hydrolysis, C5H10O5 or pentoses. The quantitative method of de- termining pentosan is based on the formation of a pentose from the pentosan, and of furfural from the pentose on further heating with acid. Pentosans are determined quantitatively by (1) the phloro- glucinol method,77 (2) the bromine titration method,78 or (3) by the colorimetric estimation of furfural.79 A weighed quantity of material (also soil or culture medium) is distilled with 12 per cent HC1 until the distillate gives no further coloration with aniline acetate. All connections in the distilling apparatus should be of glass. The distillate is made up to 500 cc. with 12 per cent HC1. Standard, approximately 0.1 N sodium bromide-bromate solution (25 cc.) is pipetted into each of four well-stoppered bottles, 200 cc. of the distillate is added to each of two bottles and 200 cc. of 12 per cent HC1 to the two other bottles. These are allowed to stand in the dark for one hour, and. after the addition of 10 cc. of 10 per cent KI solution, the liberated iodine is determined by titration with 0.1 iV sodium thiosulfate solu- tion. The number of cubic centimeters required by the sample is subtracted from the number required by the blank, the difference being a measure of the furfural present. One cubic centimeter of 0.1 N sodium thiosulfate = 2.4 mgm. furfural. Humus soils are rich in pentosans or substances which yield fur- fural on boiling with HC1.80 Forest soil, with 23.42 per cent humus, was found to contain 0.75 per cent pentosan; garden soil, with 9.85 per cent humus, contained 0.39 per cent pentosan; sandy soil, with 2.68 humus, contained only 0.04 per cent pentosan. Shorey and Lathrop81 found pentosans universally distributed in the soil, the pentosan car- bon ranging from 1.3 to 28.53 per cent of the total carbon. As much as 2.75 per cent pentosan was found in a North Dakota soil in which flax had been grown for a number of years. The presence of pentosans 77 Krober, E. Untersuchungen liber die Pentosanbestimmungen mittelst der Salzsaure-Phloroglucinmethode nebst einigen Anwendungen. Jour. Landw., 48: 357-384. 1900. 78 Powell, W. J., and Whittaker, H. The determination of pentosans in wood cellulose. Jour. Soc. Chem. Ind. Trans., 43: 35-36. 1924. 79 Youngburg, G. E., and Pucher, G. W. Studies on pentosan metabolism. I. A colorimetric method for the estimation of furfural. Jour. Biol. Chem., 61: 741-746. 1924. 80 Chalmot, G. de. Pentosans in plants. Amer. Chem. Jour., 16: 218-228. 1894; Note on pentosans in soils. Ibid., 229. 81 Shorey, E. C., and Lathrop, E. C. Pentosans in soils. Jour. Amer. Chem. Soc, 32: 1680-1683. 1910. 454 PRINCIPLES OF SOIL MICROBIOLOGY in the soil is a result of addition of plant residues, which have resisted decomposition, such as a part of the ligno-cellulose or is a product of the decomposition of complex compounds, such as nucleo-proteins. Fungus mycelium will contain about 1 per cent pentosan even when grown on a pentose-free medium. Decomposition of hemicelluloses by microorganisms. Many fungi are capable of decomposing hemicelluloses. These include various species of Aspergillus and other organisms.82 Certain lower animals are also capable of assimilating hemicelluloses.83 The hydrolysis of these polysaccharides is carried on by means of an enzyme cytase.u TABLE 40 Decomposition of pentosans by fungi and by a mixed floraSi ORGANISM Asp. flavus Asp. fumigatus. . . Asp. niger Asp. oryzae Asp. repens Pen. glaucum Cvnninghamella sp Rhizopus nigricans Soil suspension . . . Cow feces CORN STOVER PENTOSAN DESTROYED IN 142 DAYS RYE STRAW PENTOSAN DESTROYED IN 300 DAYS per cent per cent 40.1 38.1 53.0 35.1 44.5 33.9 38.0 31.0 33.0 36.6 42.5 29.5 35.1 33.2 Schmidt, Peterson and Fred85 tested the pentosan decomposing power of a number of different fungi, by adding 1 gram of corn stover or rye straw to 25 cc. of a nutrient mineral solution containing NH4N03 as the only source of nitrogen. Different organisms were found to vary in their ability to decompose pentosans. It is interesting to note 82 H6rissey, H. Recherches chimiques et physiologiques sur la digestion des mannanes et des galactanes par le seminase chez les vegetaux. These. Paris. 1903. Schellenberg, 1908 (p. 264). Otto, 1916 (p. 265). 83 Bierry, H., and Giaja, J. Untersuchungen liber die Mannane, Galaktine und Zellulosen-angreifenden Enzyme. Biochem. Ztschr., 40: 370. 1912. 84 Newcombe, F. C. Cellulose-enzymes. Ann. Bot., 13: 49-81. 1899. 86 Schmidt, E. G., Peterson, W. H., and Fred, E. B. The destruction of pento- sans by molds and other microorganisms. Soil Sci., 15: 479-488. 1923. Ver Hulst, Peterson and Fred, 1923 (p. 452). DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER 455 that a suspension of soil or of cow feces, containing a mixed flora, did not decompose any more pentosan than pure cultures of fungi. Wood pentosan (alder, poplar, birch) was not destroyed in solution, due to the presence of a substance inhibiting the growth of the fungi. When added to the soil, 37 to 72 per cent of the wood pentosan was decomposed. Pure cultures of bacteria were also found capable of de- composing pentosans in the corn, varying from 1.7 to 12.8 per cent. The maximum destruction of the pentosan (12.8 per cent) was made by Bac. flavigena, a cellulose decomposing organism. Among the other hemicelluloses decomposed by bacteria, we find also agar-agar.86 Pringsheim87 demonstrated that a trisaccharide is formed as an inter- mediary product of the decomposition of mannans by bacteria.88 It should be noted here that, in the transformation of organic matter added to the soil, the pentosans are decomposed more rapidly than the celluloses and both much more rapidly than the lignins (p. 460). Lignins, ligno-celluloses and their decomposition. Lignin, or the non- carbohydrate portion of the lignified tissues after it has been freed from fats, waxes, resins and tannins, is, next to celluloses and hemicelluloses, the most abundant constituent of plant tissues. Sphagnum moss contains 9 to 13 per cent lignin, cereal straw 18 to 22 per cent, saw grass, reeds and wood 28 to 37 per cent and nut shells up to 47 per cent. In the plant tissues, lignin is present in a free state only to a very inconsiderable extent, but largely in the form of compounds with celluloses. Ligno-celluloses are considered by some to be chemical complexes in the form of esters, by others as physical (incrustants) or adsorption compounds. These have a higher carbon content (47 to 50 per cent) than pure celluloses, due to the presence of the lignins, which contain 62 to 64 per cent carbon. The exact chemical nature of the lignin itself is still a matter of dispute. The cell wall of plants consists of practically pure cellulose in the early stages of growth, but this is changed into ligno-cellulose with advancing growth, the lignin is formed from carbohydrates originally 86 Gran, H. H. Studien iiber Meeresbakterien. II. Uber die Hydrolyse des Agar-agars durch ein neues Enzym, die Gelase. Bergens Mus. Aarborg, 1902, H. I. Biernacki, W. Bacterium nenckii Biern., ein neuer den Agar verflus- sigender Mikroorganismus. Centrbl. Bakt. II, 29: 166-169. 1911. Gray and Chalmers, 1925 (p. 199). 87 Pringsheim, H. Uber den fermentativen Abbau der Hemicellulosen. Ztschr. physiol. Chem., 80: 376-382. 1912. 88 See also Cramer. Inaug. Diss. Halle. 1910. 456 PRINCIPLES OF SOIL MICROBIOLOGY present in the cell wall. Pentosans are considered89 as intermediate products in the formation of lignin. The latter is believed to be made up of hydrosols of high molecular weight which are adsorbed from the sap by the cellulose fibers.90 Maximum lignification corresponds with maximum percentage of adsorbed material; this may be followed by chemical reactions, such as dehydration. The lignin content of rye straw was found91 to increase with age, the greatest increase occurring during the period preceding ear formation. In the ligno-cellulose complex the cellulose is linked with two non-cellulose substances one of which contains an aromatic nucleus, while the other is presumed to be a pentosan since it yields furfural on distillation with HC1. Two different processes are available for the preparation of lignin, based upon the fact that it is insoluble in concentrated acids and is soluble in alkalies, when heated under pressure; acid or alkali lignin are thus obtained. To prepare acid lignin, finely ground straw or wood is extracted with ether and then treated with a concentrated acid, using either 72 per cent H2SO4, or fuming HC1 solution (specific gravity 1.21), or a mixture of 1 vol. HC1, specific gravity 1.07, and 6 vol. 72 per cent H2S04.92 Alkali lignin can be prepared by extracting the plant material with NaOH. The actual amount of lignin thus obtained will depend on the concentration of the alkali used, temperature and period of extraction. The following method may be used :93 Ten parts of a 10 per cent NaOH solution are added to one part of finely ground straw, wood, or soil containing these materials, and the mixture is heated at 130° under 89 Rassow, B., and Zschenderlein, A. t)ber die Natur des Holzes des Hanfes. Ztschr. Angew. Bot., 34: 204-206. 1921. 90 Esselen, G. J. A few high spots in the chemistry of cellulose. Jour. Ind. Engin. Chem., 15: 306-307. 1923. 91 Beckmann, E., Liesche, D., and Lehmann, F. Lignin als Winterrogen- stroh. Ztschr. Angew. Bot., 34: 285-288. 1921; Biochem. Ztschr., 139: 491- 508. 1923. 92 Klason. Beitrage zur Kenntnis der chemischen Zusammensetzung des Fichtenholzes. Berlin. 1911; Willstatter, R., and Zeichmeister, L. Zur Kenntnis der Hydrolyse von Cellulose. I. Ber. deut. chem. Gesell., 46: 2401-2412. 1913; Hagglund, E., and Bjorkman, C. B. Untersuchungen fiber das Salzsiiure- Lignin. Biochem. Ztschr., 147: 74-80. 1924; Cellulosechemie, 4: 74-77. 1923; Schwalbe, H. Eine neue Methode zur Bestimmung des Lignins. Papierfabr., 23: 174-177. 1925; Wenzl, H. Papierfabr., 23: 305-306. 1925; Schorger, 1926 (p. xvi). 93 Powell, W. J., and Whittaker, H. The chemistry of lignin. I. Flax lig- nins and some derivatives. Jour. Chem. Soc. Trans., 125: 35-36. 1924. DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER 457 pressure, for one hour or more. Two volumes of water are then added to the digested mixture and the dark-colored solution containing the lignin is filtered off. The warm filtrate is acidified with hot hydrochloric acid, brought to boiling and the precipitated lignin is centrifuged or filtered off. To obtain pure lignin, this preparation is washed with hot dilute hydrochloric acid, dried, redissolved in a mixture of acetone and water, and reprecipitated by pouring into a mixture of hot hy- drochloric acid (20 per cent) solution. The lignin is now filtered off, washed with hot water and dried at 40 °C. The yield of lignin by alkali extraction is considerably lower than that obtained by treatment with strong acids. By raising the temperature of extraction to 180°C, a yield almost equivalent to that of acid lignin may be obtained.94 It is doubtful whether lignin is a single chemical compound, Ritter95 having shown that it can be separated even by mechanical means into two forms, one of which is located in the middle lamella of the tree and has a methoxyl content of 10.8 to 13.6 per cent and the other is cell wall lignin with a methoxyl content of 4.3 to 4.8 per cent. By treat- ment with alcohol or with (3 naphthol, lignin can also be separated into two fractions, one soluble and the other insoluble. A close relation- ship was found to exist between certain alcohol-soluble resins, tan- nins, and lignin. Various formulae have been suggested to account for the chemical composition of lignin. It is sufficient to give the formula of Klason: CH, 0 CH /\ C/\ H2C CO • CH3 Cl / o c/ N:— CH:CH-C / CH CH, v/ \ CO ■ CH, N^O • CH3 SH 94 Mehta, M. M. Biochemical and histological studies on lignification. I. The nature of lignin: its physiological significance and its estimation in timbers. Biochem. Jour., 19: 958-978. 1925. 96 Ritter, G. J. Distribution of lignin in wood. Jour. Ind. Engin. Chem., 17: 1194^1197. 1925. 458 PRINCIPLES OF SOIL MICROBIOLOGY Powell and Whittaker suggested the following formula for flax lignin: C4oH3o06(OCH3)4(OH)5-CHO For rye lignin the formula C40H44O15 has been suggested. The car- bon content of lignin is thus found to be about 63.0 per cent. The methods of determination of lignin in natural organic sub- stances are based either upon the destruction of all the carbohy- drates with concentrated acids, or upon the oxidation of lignin with chlorine dioxide or other oxidizing agent, or upon the determination of some chemical constituent of lignin, such as the methoxyl groups.953 However, none of these methods are very accurate. Alkalies ex- tract, under pressure, only a part of the lignin; but this method can be applied most readily to soils. For the study of its decomposition by microorganisms, lignin can be added, either in crude or pure form, to soil or to a solution containing a source of nitrogen and the necessary minerals. The growth of the organisms, the evolution of C02, or the disappearance of the lignin added can be taken as quantitative indices of decomposition. Pringsheim and Fuchs96 dissolved 10 grams of lignin (obtained by treating spruce wood with 11.5 per cent NaOH under pressure, then precipitating the lignin with HC1 and heating in the presence of an excess of 2 per cent acid) in NH4OH and then warmed the solution to drive off the excess of ammonia. The solution thus obtained was added to 5 liters of a nutrient medium containing 20 grams (NH4)2S04, 3 grams K2HP04, 2.5 grams MgS04 and 2 grams CaC08. The medium was inoculated with soil and incubated at 37°; decomposition was found to take place. It resulted in the complete disappearance of the pento- san content and in the reduction of the methoxyl content of the lignin. The ability of certain bacteria to decompose lignins has also been pointed out by Schrader.97 Attention must be called here to the fact that alkali lignin usually contains some pentosans and probably some proteins; it is these substances which frequently undergo decomposition 96a Schorger, 1926 (p. xvi); Schwalbe, C. G., Die chemische Untersuchung pflanzlicher Rohstoffe und der daraus abgeschiedenen Zellstoffe. Papier. Ztg. Bd. 13. 1920. 96 Pringsheim, H., and Fuchs, W. tJber den bakteriellen Abbau von Lig- ninsaure. Ber. deut. chem. Gesell., 56: 2095-2097. 1923. 97 Schrader, H. Uber das Verhalten von Cellulose, Lignin, Holz und Torf gegen Bakterien. Ges. Abh. Chem. der Kohle., 5: 553; Chem. Centrbl., 4: 1044. 1922; 3-4: 1649. 1923. DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER 459 and not the lignin itself, the latter being very resistant to the action of microorganisms.98 Very few organisms (largely actinomyces and certain bacteria) are capable of attacking lignins and use them as sources of energy. Celluloses and other carbohydrate constituents of straw, such as the pentosans, are completely digested by animals. However, the pres- ence of lignins which thoroughly impregnate the cellulose make the process of digestion more difficult. When the lignins are made soluble or removed by alkaline treatment, the straw becomes a more available source of energy and its digestibility is greatly increased. This re- moval of the lignin can be accomplished by treating the straw with various concentrations of NaOH or Ca(OH)2 at different temperatures and pressures. In the soil the celluloses and hemicelluloses are de- composed by microorganisms long before the lignins are appreciably acted upon. The very organisms concerned are different. Crude plant materials undergo in the soil a series of chemical trans- formations carried out by a number of different organisms; the sugars, starches and proteins are decomposed by some, the celluloses and pentosans by others and the lignins by still others. Conditions favorable and uninjurious to the decomposition of celluloses (such as acidity of medium) may be unfavorable or distinctly injurious to the decomposition of lignins. In addition to certain actinomyces and bacteria, higher fungi are capable of decomposing lignins to a limited extent, much less so than celluloses, and it is these organisms which are concerned largely with the rotting of wood. The mycelial filaments penetrate into the woody tissues and cause their decomposition. Most of these fungi be- long to the Basidiomycetes, chiefly Hymenomycetes." Czapek100 suggested that two enzymes (hydromase and cellulase) are active in the reaction. Certain filamentous fungi seem to exert some action upon lignocelluloses, as shown by Otto,101 who found that Trichothe- cium, Aspergillus and Mucor can dissolve out certain substances from 98 Fischer, 1923 (p. 693); Waksman, 1926 (p. 447). 99 Rudan, B. Vergleichende Untersuchungen iiber die Biologie holzerstor- enderPilze. Beitr. Biol. Pflanz., 13: 375. 1917; Hubert, E. E. Jour. Agr. Res., 29: 526. 1924. 100 Czapek, F. Zur Biologie holzbewohnender Pilze. Ber. deut. bot. Gesell. 1899; Ueber die sogenannten Ligninreaktionen des Holzes. Ztschr. physiol. Chem., 27: 141-166. 1899. 181 Otto, 1916 (p. 265). 460 PRINCIPLES OF SOIL MICROBIOLOGY the organic complexes.102 Among the higher fungi Merulius lacrimans occupies the leading place. The course of decomposition is as follows: The fungi first assimilate the sugars, then the dextrins and gums, later the hemicelluloses, and finally the celluloses are acted upon. The physi- cal properties are changed at the same time. The formation of so- called "humus" in the soil, especially in forest soils, is in close connection with the decomposition of wood ; the organic substance is first attacked by fungi; a mixed flora of bacteria and fungi then follows, finally a rich fauna of lower animals. Among the residual substances, which go to form the so-called humus and humic acids, lignin occupies a leading place. It may be added here that, as far as our present information is concerned, corky and cutinized lamellae are not acted upon to any extent by microorganisms.104 Pectins, mucilages and gums, and their decomposition by microorganisms. A number of constituents of the vegetable organic matter are character- ized by a colloidal slimy constituency; they are usually amorphous in nature and soluble in water. Some are of a pentosan nature, like the pectins; others are of a hexosan nature, like the levulosans. They can thus be classified with the hemicelluloses. A number of sub- stances synthesized by microorganisms belong here, namely the slime of cellulose-decomposing bacteria, of nitrogen-fixing and other bacteria, the mycodextrans and mycogalactans of fungi, etc. The intracellular substance (middle lamella) of parenchymatous tissues of plants consists of pectic substances, or pectose, insoluble in cold water. Heating for a few minutes in an acid solution transforms the insoluble pectoses into soluble pectins; these change into pectic acid on heating with alkali.105 As pointed out above, pectins are readily 102 Schellenberg, H. C. Die Holzersetzung als biologisches Problem. Vier- teljahrschr. Naturf. Gesell. Zurich, 65: 30-31. 1920. (Centrbl. Bakt. II, 55: 351. 1922); Ward, H. M. Ann. Bot. 12: 565. 1898. 103 Further information on the decomposition of wood and the organisms con- cerned is given by Tubeuf, C. F. V. Holzzerstorende Pilze und Haltbarmachung des Holzes. Lafar's Handb. techn. Mykol., 3: 286-333. 1904; Thaysen, A. C. The action of bacteria on cellulose and lignified vegetable tissues. Fuel., 2: 274-276. 1923. (Chem. Abstr., 18: 700. 1924.) 104 Miyoshi, M. Die Durchbohrung von Membranen durch Pilzfaden. Jahrb. Wiss. Bot., 28: 269-289. 1895; Otto, 1916 (p. 265). io6 Devaux, H. Sur la nature de la lamelle moyenne dans les tissus moux. Mem. Soc. Sci. phys. Nat. Bordeaux (6), 3: 89. 1903. (Bot. Centrbl., 96: 1. 1904) ; Compt. Rend. Acad. Sci., 162: 561-563. 1916; Conrad, C. M. A bio- chemical study of the insoluble pectic substances in vegetables. Amer. Jour. Bot. 13: 531-547. 1926. DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER 461 decomposed by various aerobic and anaerobic bacteria and fungi. Sugars are claimed to be the primary products formed from the hy- drolysis of pectins. Among the secondary products, we find volatile acids (acetic and butyric), hydrogen and carbon dioxide. Some organisms produce formic, lactic and succinic acids, in addition to C02, H2 and acetic acid. In the process of retting of flax and hemp, especially under anaerobic conditions, alcohols and acetone are also formed, in addition to the above products. Different species of fungi, like Rhizopus, both parasitic and non- parasitic forms, secrete an enzyme pectinase which dissolves the middle lamella of potatoes and of other plants.106 A similar enzyme is formed by Bac. carotovorus in the rotting of carrots.107 Microorganisms capable of decomposing pectins do not usually possess the ability of attacking celluloses, otherwise the retting process would be accompanied by the destruction of the celluloses of the fibers. Certain levulosans are not readily acted upon by microorganisms.108 Starches and their decomposition by microorganisms. Starches are predominantly reserve materials, forming 60 to 70 per cent of the dry weight of cereal grain. They are complex carbohydrates of the for- mula (C6Hl0O6)n-H2O but are more readily soluble than celluloses and give the characteristic blue color with iodine. They swell in hot water. They are much more readily acted upon by microorganisms than the celluloses, due to the fact that a large number of soil forms, including the bacteria, actinomyces and fungi, produce very active diastatic enzymes which hydrolyze the starches first into dextrins of different complexity, then into sugars (maltose and glucose). 2(C6H10O6)n + nH20 = nC«HMOu starch maltose C12H22O11 -f- H2O = 2CeHi20s maltose glucose The ability of certain bacteria and fungi to produce enzymes hydro- lyzing starch is so great that the processes have been utilized for various commercial purposes where diastatic enzymes are required. Certain bacteria, however, are capable of breaking down starches with the 106 Harter, L. L., and Weimer, J. L. A comparison of the pectinase produced by different species of Rhizopus. Jour. Agr. Res., 22: 371-377. 1921; Amer. Jour. Bot., 10: 127-132, 167-169. 1S95. 107 Jones, 1909 (p. 203). 108 Colin, H„ and Estienne, V. Utilisation de 16vulosanes par les organis- mes. Bull. Soc. China. Biol., 6: 431^35. 1924. 462 PRINCIPLES OF SOIL MICROBIOLOGY formation of acids, alcohols and acetone.109 In this, the action of bacteria upon starch may be distinct from that of diastatic enzymes, which give 100 per cent maltose, and from the acid hydrolysis of starch, which results in the formation of glucose. The number of organisms in the soil capable of hydrolyzing starch can be readily determined. The soil is diluted 1:1000 to 1:200,000. The final dilution is plated out on a medium which consists of 15 grams potato starch, 1 gram of an organic or inorganic source of nitrogen, 0.5 gram K2HPO4, 15 grams of agar and traces of MgS04 and FeCl3 in 1000 cc. of water. After a few days incubation (3 to 7), the plates are covered with a dilute solution of iodine and potassium iodide. The colonies of the microorganisms producing diastase will be surrounded with a clear zone; these colonies may then be counted. A number of bacteria are capable of decomposing starch, including various spore forming organisms, such as Bac. subtilis, Bac. mesentericus, Bac. cereus and other common soil bacteria, and also various species of Bac. amy- lobacter. Certain non-spore bearing bacteria, such as certain cellu- lose-decomposing organisms and others, are also capable of decom- posing starch. The ability to hydrolyze starch is widely distributed among fungi, such as Asp. oryzae, Asp. nigcr and Amijlomyces boidin. Formic, acetic and butyric acids, traces of lactic and succinic acids, various alcohols (ethyl and butyl), aldehydes and acetone, hydrogen and carbon dioxide have been obtained among the products of decom- position of starches by microorganisms. Bac. mesentericus, for example, breaks down starches into carbon dioxide, formic and valerianic acids. Bac. granulobacter pectinovorum growing in media rich in starch changes the starch into glucose by means of enzymes; the sugar passes into the cell and is oxidized to acetic and butyric acids, a part of which is re- duced to the corresponding alcohols.110 Inulin, similar in its properties to starch, but giving levulose on hydrolysis, can also be decomposed by various bacteria and fungi.111 109 Schardinger, F. Bacillus macerans, ein Aceton-bildender Rottebacillus. Centrbl. Bakt. II, 14: 772-781. 1905; Zur Biochemie des Bacillus macerans. Ibid., 19: 161-163. 1907; 22: 98-103. 1909; 188-197. 1911. For further in- formation on this subject, consult Pringsheim, 1923 (p. 427). 110 Speakman, H. B. Biochemistry of the acetone and butyl alcohol fermenta- tion of starch by Bacillus granulobacter 'pectinovorum. Jour. Biol. Chem., 41: 319-343. 1920. 111 Grafe, V., and Vouk, V. Das Verhalten einiger Saccharomyzeten (Hefen) zu Inulin. Ztschr. Garungsphys., 3: 327-333. 1913; Kiesel, A. L'influence de la reaction du milieu sur Taction de l'inulase de l'Aspergillus niger. Ann. Inst. Past., 28: 747-757. 1914. DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER 463 Decomposition of fats and waxes. Fats are widely distributed in the plant and animal residues added to the soil. They are also synthe- sized by the different groups of soil microorganisms. The amount of fat synthesized and the nature of the fat will depend upon the type of organisms and stage of growth. The fat is a reserve substance and is readily utilized by the organisms in the absence of other available sources of energy.112 Fats decompose only slowly in moist soils, and almost not at all in dry soils. According to Rubner,113 only 22.9 per cent of butterfat added to soil (4.5 grams of fat to 200 grams of soil) was decomposed during a period of one year and 38.1 per cent in twelve years; other fats were decomposed at a different rate. The fats are first hydrolyzed, according to the general reaction: CsHsOs-Rs + 3H20 = C3H803 + 3R-OH The glycerol or corresponding alcohols are readily utilized by various groups of microorganisms as sources of energy, while the fatty acids are decomposed further. A typical fat is decomposed in the soil as follows: CH2 • CO • Ci 7HS3 1 CH2OH 1 CHO • CO • C17H33 + 3H20 = CHOH + 3C17H33 • COOH 1 CHsO-CO-Ci7H„ CH2OH Oleic acid Triolein Glycerol CH3(CH2)7CH = CH(CH2)7 CO,H + H20 + h(02)-> Oleic acid CH3(CH2)7 • CHOH • CHOH(CH2)7 C02H Dioxystearic acid This last substance has been demonstrated in the soil114 and was also found in the cells of fungi. Fats are decomposed chiefly by fungi, with the possible formation of ketones, and by a number of aerobic 112 Dubaquie. Recherches sur les matieres grasses des vegetaux inferieurs. Mem. Soc. Sci. Phys. Nat. Bordeaux (6). 1910. 113 Rubner, N. Notiz liber die Zersetzung von Fetten im Boden. Arch. Hyg., 91: 290. 1922. 114 Schreiner, O., and Shorey, E. The isolation of harmful organic substances from soils. Bur. Soils, U. S. Dept. of Agr. Bui. 53. 1909. 464 PRINCIPLES OF SOIL MICROBIOLOGY bacteria, including Staph, pyogenes aureus, Bad. prodigiosum, Bad. pyocyaneum, Bad. fluorescens, Bad. lipolyticum.115 It is possible, however, that some fat may also be decomposed under anaerobic conditions. The chemical processes involved may be differ- ent, Bach and Sierp116 having shown that, under anaerobic conditions, CO2 is split off and the fatty acids change into hydrocarbons. This results in the formation of products of a lower saponification and higher iodine number than the original fat. Waxes are chemically related to the fats, being esters of higher alco- hols and fatty acids. For example, flax wax consists of phytosterol and ceryl alcohol, as well as of palmitic-, stearic-, oleic-, linolic-, and linoleic acids. These substances are even more resistant to decomposi- TABLE 41 Influence of age of culivre vpon the fat content of Asp. niger11- 350 cc. Raulin's solution containing 4.7 per cent invert sugar AGE OF CULTURE RESIDUAL SUGAR days gm. 1 8.6 2 1.9 3 0 4 0 7 0 12 0 DRY WEIGHT OF MYCELIUM mgm. 0.29 5.10 6.30 4.1 1.7 1.3 FAT CONTENT per cent 2.11 12.0 7.5 4.0 1.6 0.6 116 Schenker, R. Zur Kenntniss der Lipase von Aspergillus niger (van Tiegh). Biochem. Ztschr., 120: 164-196. 1921; Derx, H. G. Der oxydative Abbau der Fette durch Schimmel. Konigl. Akad. Wiss. Amsterdam, 33: 545-558. 1924; Sohngen, N. L. Lipase production of microbes. Konink. Acad. Wetenschap. Amsterdam., 19: 698. 1910; 20: 126. 1911; Eijkman, C. Uber Enzyme bei Bakterien und Schimmelpilzen. Centrbl. Bakt. I, 29: 841-848. 1901 ; de KruyfT, E. Les bacteries hydrolysant et oxydant les graisses. Bull. dept. agr. Ind. norland. IX. Buitenzorg. 1907. (Centrbl. Bakt. II, 20: 610-611. 1908); Huss, H. Eine fettspaltende Bakterie (Bactridiurn lipolyticum n. sp.). Centrbl. Bakt. II, 20: 474-484. 1908; Stephenson, M., and Whetham, M. D. Fat metab- olism of the timothy hay bacillus. Proc. Roy. Soc. B., 93: 262-280. 1922; Shibata, N. Zur Frage der Fettzersetzung einiger Saprophyten. Jour. Biochem. Tokyo, 1: 249-260. 1922. 118 Bach and Sierp. Untersuchungen iiber den anaeroben Abbau organischer Stoffe durch Bakterien des Klarschlammes. Centrbl. Bakt. II, 62: 24-76. 1924. A detailed review of the formation and decomposition of fats by microorganisms is given by Seliber, G. The formation and decomposition of fats by micro- organisms. Glavnauka. Leningrad. 1926. DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER 465 tion than the fats; they are acted upon under completely aerobic conditions by soil fungi and certain bacteria. It may be of interest to mention here that Greig-Smith117 attempted to explain soil exhaustion as a result of an accumulation of fats and waxes ("agricere"); when these are partly removed by the action of volatile antiseptics, further decomposition of the soil organic matter sets in (see p. 759). Decomposition of paraffins, aliphatic hydrocarbons and benzene ring compounds in the soil. According to Sohngen,118 various non-spore forming bacteria and Mycobacteria are capable of oxidizing paraffin, benzin, petroleum and paraffin oil. On adding 2 grams of paraffin to the medium, incubating one month at 28°, then extracting the remain- ing paraffin with petroleum ether, it was found that the following amounts were decomposed: mgm. Mycob. album 300 Mycob. rubrum 330 Micr. paraffinae 180 Bad. fl acetaldehyde \ . ., I acetic acid acetic acid -f- formic aldehyde — > formic acid Bad. coli changes glucose to ethyl alcohol, lactic and acetic acids, C02 and H2. 2 C6H1206 + H20 = 2 C3H603 4- C2H402 4- C2H6OH + H2 4- 2 C02 As an instance of the chemistry of decomposition of starch or sugar under anaerobic conditions, it is sufficient to illustrate the action of Bac. acetoethylicujn.12* CeHiuOe I H2 + C02 + CH3 • COH «- H2 4- CH3 • CO • COOH + H2 -> CH3 • CHOH • COOH + H20 CH3 • CH2 • OH CH3 • COOH 4- H • COOH 126 Falck, R., and Kiuyma, B. Methodisches und Prinzipielles zur Darstel- lung organischer Sauren auf biologischen Wege mit Hilfe von Fadenpilzen. Ber. deut. chem. Gesell., 57: 915-920, 920-923. 1924. 126 Butkewitsch, W. tlber die Bildung der Glukon-und Citronsaure in den Pilzkulturen auf Zucker. Biochem. Ztschr., 154: 177-190. 1924; Jahrb. wiss. Bot., 64: 636-650. 1925. See also Berhauer, K. Zum Problem der Saurebildung durch Aspergillus niger. Ibid., 153: 517-521. 1924. 127 Aubel, E. Attaque du glucose et du levulose par le bacille pyocyanique. Compt. Rend. Acad. Sci., 175: 1493-1495. 1921. 128 Northrop, J. H., Ashe, L. H., and Senior, J. K. Biochemistry of Bacillus acetoethylicum with reference to the formation of acetone. Jour. Biol. Chem., 468 PRINCIPLES OF SOIL MICROBIOLOGY The action of Bac. granulobacter pectinovorum upon starch, pentoses and hexoses results129 in the formation of acetone, butyl alcohol, hydrogen and carbon dioxide, with various acids (butyric, acetic, lactic) as in- termediary products. When aliphatic compounds with carboxyl groups are acted upon by Bact. pyocyaneum, they become alkaline as a result of the oxidation of the carboxyl groups. Compounds contain- ing — CHO or — OH = CO— groups become acid as a result of oxi- dation.130 The formation of butyric acid by the various butyric acid bacteria under anaerobic conditions can be represented as follows: C6 Hi, 06 = C4 H8 02 + 2 C02 + 2 H2 This process is much more complex than represented by the above reaction, since other acids and various alcohols are also formed. The acid itself may be formed as a result of the synthetic processes rather than by direct decomposition.131 C6Hi206 = 2 CH3 • CO • COOH + 2 H2. 2 CHS • CO • COOH = CH3 • COH • COOH COOH CH2 • CO • C Aldole CbHsOb = 2 C02 + C^HgOa. 2 CH3 • CO • COOH -> CH3 • CHO + C02 2 CH3 • CHO + H20 = CH3 • COOH + CH8 CH2 • OH. Decomposition of organic acids. Fatty-acids are thus formed from poly- and mono-saccharides, from proteins and their derivatives. When neutralized, these acids serve as good sources of energy for various bacteria and fungi. The great majority of heterotrophic bacteria can utilize malic, citric, fumaric, glyceric, succinic, formic, lactic, mucic, and tartaric acids; a small number utilize acetic, propionic, quinonic 39: 1-21. 1919; Speakman, H. B. The biochemistry of acetone formation from sugars by Bacillus acetoethylicum. Jour. Biol. Chem., 64: 41-52. 1921. 129 Speakman, H. B. Molecular configuration in the sugars and acid produc- tion by Bacillus granulobacter pectinovorum. Jour. Biol. Chem., 58: 395-413. 1923. 130 Supniewski, J. Recherches sur la transformation des combinaisons car- bones par le Bacille pyocyanique. Compt. Rend. Soc. Biol., 89: 1377-1379. 1923. 1,1 Neuberg, C, and Arinstein, B. Vom Wesen der Buttersiiure und Butyl- alkoholgiirung. Biochem. Ztschr., 117: 269-314. 1921. DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER 469 acids. Maleic, jS-oxy-butyric, a-oxy-butyric and oxalic acids are utilized only to a very limited extent. The decomposition of these acids results in the formation of alkali carbonates, which lead to an alkaline reaction of the medium: 2 H • COO Na + 2 H20 = Na2C03 + H20 + C02 + 2 H2 C2H402 Acetic acid CH4 + C02; or C2H402 + 2 02 = 2 C02 + 2 H20 2 C4H802 + 2 H20 = 5 CH4 + 3 C02; or C4H802 + 5 02 = 4 C02 + 4 H20 Butyric acid CH3 • CO • COOH = CH3 • CHO + C02 Pyruvic acid Acetaldehyde Fumaric acid is decomposed by Bad. pyocyaneum13- to the lower fatty acids, chiefly acetic; pyruvic acid may also be isolated. CHCOOH , 0 C(OH)COOH CO • COOH ,0 II -4 || ► J C HCOOH CH • COOH • CH2 • COOH CH3 • COOH + 2C02 The role of pyruvic acid in fermentation processes (anaerobic utiliza- tion of energy) suggested by Neuberg and associates found support in various investigations on the nutrition of bacteria.133 Aspartic acid Malic acid g*gg£jg» Glycol lie acid loxidation Tartaric acid I ' (dehydration J> Oxal- acetic acid- Acetic Succinic Furoartc acid oxidation acid oxidation acid A&°2^rGhcZri$ -lactic gSZ^0^ aldehyde l a <31-ycepol ■ Methyl Glyoxal Pyruvic -^Bacterial acid cells Alanine 132 Quastel, J. H. The fermentation of the unsaturated dicarboxylic acids. I. Fumaric acid. Biochem. Jour., 18: 365-380. 1924. 133 Quastel, J. H. On a possible role of pyruvic acid in bacterial growth. Biochem. Jour., 19: 641-644, 645-651, 652-659, 660-666. 1925. A detailed re- view of the subject of the transformation of the sugar molecule by bacteria is given by Schoen, M. Le probleme des fermentations. Masson et Cie. Paris. 1926. CHAPTER XVIII Decomposition of Proteins and Other Organic Nitrogenous Compounds by Soil Microorganisms Most of the nitrogen added to the soil by the plowing under of sod, plant stubble, green and stable manures, is in the form of proteins and their derivatives. The same is true of the organic nitrogenous fertilizers of plant and animal origin, such as dried blood, tankage, fish scraps and cottonseed meal. These substances cannot be assimilated by higher plants as such but have to be first broken down into simple compounds. This process is carried out in the soil by the agency of microorganisms, the final product of hydrolysis being chiefly ammonia. The latter is either used by the plants as such or is oxidized further to nitrates. Nitrates are either assimilated by plants or by microorganisms, reduced by denitrifying bacteria, or washed out in the drainage waters. The nitrogen content of cereal straw, corn cobs and leaves of trees varies from 0.40 to 0.80 per cent; of legume hay from 2 to 3 per cent; of cow manure, free from straw, about 3.5 per cent; horse manure, about 1.5 per cent; chicken manure, 2.1 per cent, on an air dry basis.1 When these substances are added to the soil they undergo a series of transformations, largely biological in nature, involving processes of hydrolysis, oxidation, reduction and synthesis. These transformations result in the liberation of nitrogen in an available form which may again be wholly or partly reassimilated by soil microorganisms, in the presence of available energy material. Physical and chemical properties of proteins. Proteins are complex substances, consisting of carbon, hydrogen, oxygen, nitrogen, sulfur, and in some cases of phosphorus and iron. The average composition of a typical protein is as follows: percent percent C 50.6-55.0 N 15.0-19.3 H 6.5-7.3 S 0.3-2.2 0 21.5-23.5 P 0-0.9 1 Thorne, C. E. Farm manures. O. Judd Co., New York. 1914. 470 DECOMPOSITION OF PROTEINS 471 No molecular formula can be ascribed to the proteins and their molecular weight is still a subject of study. Structurally, the proteins are characterized as condensation products of amino acids which are united chiefly by the peptide linkage (R — CO- NH— R), similar to the polypeptides. On hydrolysis by acids, alkalies, or specific enzymes, the proteins break up into the constituent amino acids. From 10 to 25 per cent of the nitrogen is present in the proteins in the unstable form of the amide linkage (R — CONH2). About 60 per cent of the nitrogen is assumed to be in the peptide linkage. The physical properties of the proteins vary very widely. When dried in the presence of moisture, when boiled with acids and alkalies, and when acted upon by some microorganisms, the proteins show a tend- ency to coloration. This is due to the formation of insoluble pigmented substances, called melanins, probably related to the so-called "humins." Most proteins are soluble in water or in dilute acids or alkalies; a few, like keratin from horn, are insoluble in water and require strong acids and alkalies to bring them into solution. The proteins are ampho- teric substances, being capable of combining with both acids and alkalies, neutralizing them, and causing a decrease in the hydrogen- or hydroxyl-ion concentration. Coagulation, precipitation and color reactions vary with the different proteins, depending on their consti- tution and state of purity. On hydrolysis by acids or enzymes, the proteins are broken down to proteoses, then to peptones and finally to amino acids, which are simple crystallizable substances. Some amino acids, like tyrosine, may appear in the early stages of hydrolysis. The proteoses and peptones consist of several groups of amino acids, these groups being smaller than the original protein. The majority of amino acids in the protein molecule are characterized by the fact that one hydrogen in the a position is replaced by NH2. In the case of two of the basic amino acids, viz., arginine and lysine, a second amino group is present. A third amino acid, histidine, contains an imidazol nucleus and is basic. Ace- tic acid, CH3COOH, gives amino-acetic acid, glycocoll or glycine, CH2NH2COOH. The general formula of a mono-ami no-monocar- boxylic acid is R — CH(NH2)COOH. When two hydrogens are re- placed by amino groups, we have di-amino acids. Due to the presence of amino groups, the amino acids possess both acid and basic properties, so that glycocoll can form salts both with bases (CH2NH2COOK) and acids (CH2NH2COOH • HC1) under proper conditions of hydrogen-ion concentration. The dicarboxylic acids, 472 PRINCIPLES OF SOIL MICROBIOLOGY /COOH the general formula of which is H2NRX , like aspartic acid \COOH (COOH-CH2-CHNH2-COOH) and glutamic acid, possess properties of stronger acids than the mono-carboxylic acids. One of the carboxyls in the dibasic acids is relatively strong, the other is of about the same strength as the carboxyl groups of the ordinary mono amino acids. The three basic amino acids, arginine, histidine and lysine, are fairly strong bases and only show acid properties at extremely small hydro- gen-ion concentrations, i.e., pH 12.0 to 14.0. When the hydroxyl group of a carboxylic acid is replaced by an amino group, an acid- amide, like CH3CONH2 (acetamide) is formed. A mixture of amino acids obtained by the hydrolysis of proteins can be analyzed according to Fischer's method. The diamino-acids are first separated from the mono-amino acids, by precipitation with phosphotungstic acid. Excess of HC1 is added and the mixture of mono-amino acids is then evaporated to a sirup in vacuo and dis- solved in ethyl alcohol; dry hydrochloric acid gas is passed into the solution to form the chlorides of the amino-acid esters. The esters must then be set free by neutralization of the HC1, and extracted with ether or chloroform. The esters are separated by fractional distillation in vacuo. When a knowledge of the total proportion of the various groups of amino acids is sufficient, the method of Van Slyke affords a good procedure. Fischer2 succeeded in combining amino acids into complex groups known as peptides, thus obtaining dipeptides (CH2NH2CO • NHCH2 COOH or glycyl-glycine) and other polypeptides, the more complex ones approaching native proteins, in their general properties. The investigations of Fischer and associates gave strong evidence support- ing the view that the protein molecule is built up of amino acids according to the following structure: NH2 • CHR • CO - (NH • CHR • CO)X-NH-CHR-COOH. The albumins, globulins, glutelins and gliadins (or prolamins) are the most important vegetable proteins.3 The chemical nature of the different proteins is determined by the quantitative relationship of the various amino acids and their arrange- ment in the molecule. 2 Fischer, E. Untersuchungen fiber Aminosiiuren, Polypeptide unci Proteine. Berlin. 1906. 3 Osborne, T. B. The vegetable proteins. Longmans Green & Co. 2nd ed. 1924. DECOMPOSITION OF PROTEINS 473 Chemistry of protein hydrolysis. Protein decomposition by micro- organisms includes a group of processes; namely, (1) hydrolysis of proteins to albumoses, peptones and amino acids, (2) deaminization resulting in the formation of ammonia, (3) formation of secondary decomposition products, such as amines, (4) completion of decomposi- tion of proteins involving phenomena of oxidation and reduction with the formation of C02, H20, H2S and NH3. When the proteins are hydrolyzed by acids and alkalies, the resulting products are amino acids and some ammonia. The latter is probably liberated as a result of the action of the acid or alkali on the amide union (CO-NH2) in the protein molecule, and in the case of the alkali treatment as a result of the destruction of arginine. The larger portion of the nitrogen is present, after hydrolysis, in the form of amino groups. However, in the protein molecule, the greater por- tion of the nitrogen is present in imino groups (NH), with the excep- tion of one of the two amino groups of lysine (w group) which exists free.4 This is also true of a part of the nitrogen in histidine, arginine and tryptophane. This co group of lysine accounts for the entire amount of amino nitrogen found in the native protein molecule on treating with nitrous acid.5 The a-amino groups, which con- stitute the larger portion of the protein nitrogen, are present only condensed into peptide linkages. On hydrolysis, the free amino nitro- gen increases and the peptide linkages (R— CO-NH — R) become separated into amino and carboxyl groups. The measure of this increase in amino nitrogen can serve as an index of the process of pro- tein hydrolysis. Some proteins are easily hydrolyzed, while others are acted upon with great difficulty. This is very important from the point of view of the availability of nitrogen for plant growth. The action of chemi- cal reagents and moist heat will bring about a complete hydrolysis of the proteins to amino acids and ammonia. Proteolytic enzymes usually do not break down the protein molecule completely. Some, like pepsin, split up the protein chain at one or more junctures, with- 4 Van Slyke, D. D., and Birchard, F. J. The nature of the free amino groups in proteins. Jour. Biol. Chem., 16: 539-547. 1913. 5 Van Slyke, D. D. The analysis of proteins by determination of the chem- ical groups characteristic of the different amino acids. Jour. Biol. Chem., 10: 15-55. 1911; Quantitative determination of alipathic amino groups, Ibid., 12: 275-284. 1912; Improvement in the method for analysis of proteins by de- termination of the chemical groups characteristic of the different amino acids. Ibid., 16: 539-547. 1913; 22: 281-285. 1915. 474 PRINCIPLES OF SOIL MICROBIOLOGY out forming free amino acids, but form groups (albumoses, peptones, peptides) of lower amino acid content. Other enzymes, like trypsin and erepsin, split the protein molecule more completely, bringing about the formation of free amino acids. Still other enzymes (desamidases, deaminases) act upon the amino acids and acid amides liberating ammonia. The degradation of proteins by microorganisms proceeds along the general lines of that produced by acids or proteolytic enzymes. A further transformation of the protein derivatives takes place, however, with the production of various secondary decomposition products, such as ammonia and carbon dioxide, as well as amines, fatty acids, alcohols, aldehydes, methane, phenol, indol, skatol, hydrogen sulfide, etc. Am- monia which is so important, both from the point of view of the me- tabolism of microorganisms and soil fertility, is usually formed as a secondary decomposition product of the proteins; usually the amino acid is used thereby as a source of energy. In some cases proteins form compounds with nucleic acids, giving nucleo-proteins (or protein nucleates). These compounds are present to a limited extent in all plants, animals and microorganisms, and are thus introduced into the soil. On hydrolysis, a nucleo-protein is transformed into an albumin (histone) and nuclein; the nuclein is further hydrolyzed to albumin and nucleic acid. The protein, or albumin, is decomposed by the microorganisms into albumoses, pep- tones, amino acids and ammonia. The presence of these substances in the soil has actually been demonstrated.6 Often several groups of organisms take part in the process; some break down the protein to amino compounds and others utilize the latter and form ammo- nia, as shown later. This is again comparable to the action of the different groups of enzymes. The nucleic acids consist of C, H, O, N and P, in various propor- tions, depending on the source of the acids. The composition of nucleic acid from wheat is given as C4JL31O31N16P4; that of yeast, C36H4803oNi4P4. The dissociation products vary with the source of the acid; those of plant origin are phosphoric acid, guanine, adenine, cyto- • Walters, E. H. The presence of proteoses and peptones in soils. Jour. Ind. Eng. Chem., 7: 860. 1915; Lathrop, E. C. The organic nitrogen com- pounds of soils and fertilizers. Jour. Frankl. Inst., 183: 169-206, 303-321, 465- 498. 1917; Shorey, E. C. The isolation of creatinine from soils. Jour. Amer. Chem. Soc, 34: 99-107. 1912; Nucleic acids in soils. Science, 35: 390. 1912; Some organic soil constituents. Bur. Soils, U. S. Dept. Agr. Bui. 88. 1912. DECOMPOSITION OF PROTEINS 475 sine, thymine and laevulinic acid. Laevulinic acid is formed from a hexose group in the molecule of the nucleic acid. The decomposition of the nucleic acids takes place as follows:7 Zoo or Fhytonucleic acids Nucleotides Phosphoric acid /\ Nucleosides Carbohydrate / \iBase and carbohydrate) complex J Base (Purine or pyrimidine) Phosphoric acid Carbohydrate (Pentose or hexose) Protein decomposition by microorganisms. The course of protein decomposition by microorganisms can be followed in three different ways. 1. The disappearance of the protein. The residual protein is pre- cipitated by means of an acid or alkali, by alcohol or other precipitating agents or by heat. The protein is determined either by weighing the dry precipitate or determining the total nitrogen in it. In the case of peptone, the biuret test may be used8 as a measure of its decomposition. 2. The formation of intermediary products, such as peptones or amino compounds. The first can be determined quantitatively by the biuret reaction and the second by the Van Slyke method,9 the formol titration method,10 or the Folin method.11 3. The formation of ammonia as the final product of protein decomposition. Each of these three methods has its advantages and disadvantages. By the first method, we determine the absolute amount of protein decomposed, but we do not know how far the decomposition has pro- 7 Levene, P. A. Partielle Hydrolyse der Nucleinsauren. Abderhald. Haadb. Biochem. Arbeitsm., 2: 605-609. 1911; 5: 489-499. 1911. 8 Berman, N., and Rettger, L. F. The influence of carbohydrates on the nitrogen metabolism of bacteria. Jour. Bact., 3: 3S9-402. 1918. 9 Van Slyke, 1913-14 (p. 473). 10Sorensen, S. P. L. Enzymstudien. I. Biochem. Ztschr., 7:45. 1907. 11 Folin, O. A new colorimetric method for the determination of amino acid nitrogen in blood. Jour. Biol. Chem., 51: 377-391. 1922. 476 PRINCIPLES OF SOIL MICROBIOLOGY ceded, whether to soluble polypeptide molecules or to amino acids and ammonia. The methods of precipitation are also different for the various proteins and involve differences in procedure. The second method enables one to follow the course of protein de- composition by the increase in the amino nitrogen.12 The great dis- advantage of this method is that the various microorganisms will show different increases of amino nitrogen with the same amount of protein decomposed. This is due to the fact that the different organisms, even decomposing equal amounts of proteins, will transform the in- termediary products with different rapidity. The fungi, for example, will hardly allow any great increase in amino nitrogen, but will rapidly transform the intermediary products to ammonia, especially in the absence of available carbohydrate. In the presence of carbohydrate, the protein will be decomposed only to a limited extent. The bacteria and actinomyces, however, will allow a much greater accumulation of amino-nitrogen and a correspondingly lower accumulation of ammonia. The fact that the amino compounds are only intermediate products and that their accumulation depends on the presence of carbohydrates indicates that, at best, this index can be only approximate. The third method has the advantage of measuring a final product and not an intermediate one. The fact that various organisms form ammonia from proteins with various speeds, some breaking down the protein completely and others incompletely, is an outstanding dis- advantage of this method. In the presence of available carbohydrates, the ammonia may also be reassimilated by the organism as a source of nitrogen for the synthesis of its protoplasm, so that a mistaken impression may be had that no protein is decomposed. The study of protein decomposition would be incomplete without mentioning the so-called processes of putrefaction, or decomposition of proteins in the absence of oxygen or in the presence of a limited amount of it, with the production of evil smelling gaseous products.13 This subject has been least studied from the point of view of trans- formation in the soil; most of the work was done in connection with pathogenic anaerobic bacteria. Putrefaction was often differentiated 12 Sears, H. J. Studies in the nitrogen metabolism of bacteria. Jour. Inf. Dis., 19: 105-137. 1916; Itano, A. The relation of hydrogen ion concentration of media to the proteolytic activity of Bacillus subtilis. Mass. Agr. Exp. Sta. Bui. 167. 1916; Waksman, 1918 (p. 495); Debord, J. J. Certain phases of nitrog- enous metabolism in bacterial cultures. Jour. Bact., 8: 7-45. 1923. is Fliigge. Die Mikroorganismen. Leipzig. Vogel. 1896. v. I, p. 254. DECOMPOSITION OF PROTEINS 477 from "decay;" the latter (often referred to as "eremacausis") was used to designate the decomposition of nitrogenous organic substances in the presence of oxygen, marked by the volatilization of organic constituents while the non- volatile mineral constituents are left behind.14 However, both of these phenomena (putrefaction and decay) were not sufficiently understood by the old chemists and bacteriologists and did not have a proper biological or chemical basis. With the advance of our knowledge of the chemistry of proteins, particularly when it was found that ammonia and the "ill-smelling gaseous prod- ucts" were by-products of secondary reactions of protein hydrolysis, the difference between "decay" and "putrefaction," as indicating activities of special groups of bacteria, disappeared. Both of these terms may just as well be dropped from the bacteriological vocabulary, since they do not designate definite chemical processes. Like all chemical reactions brought about by biological agencies, the final products of protein decomposition are a result not only of specific microorganisms, but of various environmental conditions, such as oxygen supply and presence of non-nitrogenous substances, which determine the secondary reactions involved after the hydrolysis of the proteins has taken place. The designation of a process by the nature of these secondary reactions, as a result of environmental condi- tions, led to a great deal of confusion and a lack of proper understand- ing of the processes involved. The first systematic study of the chemical reactions accompanying the decomposition of proteins by bacteria (in the so-called process of putrefaction) was made by Nencki.15 He found that, in the decom- position of fibrin, albumin and gelatin by bacteria, various decomposi- tion products are formed, including leucine, tyrosine, glycocoll and in- dol. When gelatin was decomposed for four days at 40°C. there were formed, for every 100 parts of gelatin, 9.5 parts of ammonia, 24.2 vola- tile fatty acids, 12.2 glycocoll, 19.4 peptone and 6.5 carbon dioxide, 71.8 per cent in all. Quantities of gas were also produced in the proc- ess. Nencki concluded that the decomposition of proteins takes place in two stages; viz., processes of hydrolysis and reduction and 14 Wollny, E. Die Zersetzung der organischen Stoffe. Heidelberg, Winter. 1897. 15 Nencki, M. t)ber die Harnfarbstoffe aus der Indigogruppe und uber die Pankreasverdauung. Ber. deut. Chem. Gesell., 7: 1593-1600. 1S74; 8: 336- 338. 1875. 478 PRINCIPLES OF SOIL MICROBIOLOGY oxidation. Jeannert,16 studying the decomposition of proteins under anaerobic conditions, demonstrated among the decomposition prod- ucts of gelatin, C02, NH3, H2S, acetic-, butyric-, and valeric acids, glycocoll and leucine. These substances, except glycocoll, were also formed from albumin, in addition to hydrogen, hydrogen sulfide, tyrosine and amido-valeric acid. These investigations were followed by numerous others with crude and pure cultures of bacteria, whereby amino acids, fatty acids and certain gases, including NH3, C02 and H2S, were demonstrated as products of hydrolysis. In the case of the so-called putrefactive organisms, indol, skatol, phenol, and other substances were also demon- strated. Bac. subtilis was found17 to produce from cotton-seed meal in five weeks: albumoses, peptones, phenyl-acetic and phenyl-pro- pionic acids, ammonia, mercaptan, basic amines, H2S, and C02; after three months, valerianic and indol-acetic acids, indol, skatol, and phenol were also demonstrated in the culture. The presence of specific amino acids in the protein molecule is neces- sary for the formation of some of the final products. It is sufficient to mention tryptophane as a source of indol, cysteine and other sulfur compounds as a source of H2S, and tyrosine as a source of cresol and phenol. A number of bacteria are unable to attack pure proteins; only the degradation products are acted upon. The presence of simple nitro- gen compounds may be required to start the development of the or- ganism.18 In many instances, however, even native proteins will be acted upon after the organism starts to grow and the proper enzymes are formed. Under natural conditions, the proteins are usually always accompanied by small amounts of their derivatives or by simple nitro- gen compounds, especially when added to the soil. The nature of the products formed will depend not only upon the 16 Jeannert, J. Untersuchungen liber Zersetzung von Gelatine und Eiweiss durch die geformten Pankreasfermente bei Luftausschluss. Jour, prakt. Chem. N. F., 15: 353-389. 1877. 17 Konig, J., Spieckermann, A., and Olig, A. Die Zersetzung pflanzlicher Futtermittel durch Bakterien. Centrbl. Bakt. II, 10: 535-549. 1903. 18 Bainbridge, F. A. The action of certain bacteria on proteins. Jour. Hyg., 11: 341-355. 1911; Sperry, J. A., and Rettger, S. F. The behavior of bacteria towards purified animal and vegetable proteins. Jour. Biol. Chem., 20: 445-459. 1915; Berman, N., and Rettger, L. F. Bacterial nutrition: further studies on the utilization of protein and non-protein nitrogen. Jour. Bact., 3: 367-388. 1918. DECOMPOSITION OF PROTEINS 479 environmental conditions but also upon the organisms concerned. In the great majority of cases, especially in the study of the activities of soil microorganisms, the measurement of ammonia was used as an index of protein decomposition.19 But even in the case of protein decomposition in soil, where one group of organisms readily acts upon the products formed by another, various protein derivatives are found, in addition to ammonia. Lathrop,20 for example, found that histidine, hypoxanthine, cytosine, xanthine, nucleic acid, creatinine, cyanuric acid are of common occur- rence in the soil; arginine, lysine, adenine, choline, trimethylamine occur only infrequently in the soil. This leads to the assumption that a part of the proteins and other organic nitrogenous compounds are accumulating in the soil, originating from plant residues, stable manure, green manure, organic fertilizers and the bodies of microorganisms. Lathrop analyzed, by the Van Slyke method, soils to which proteins (dried blood) had been added, at the beginning of the experiment and at the end of various periods of incubation. Even after a 240-day period of decomposition of dried blood in the soil, proteins, or protein- like complexes, insoluble in distilled water, but extractable by dilute alkaline solution, were found to be present in the soil. It is not known, however, whether these proteins are residues from the dried blood which have resisted decomposition by the soil microorganisms, or whether they are synthesized materials or constituents of the bodies of the latter. Evidence was obtained to indicate that a formation of new protein material takes place in the soil in the course of decom- position of proteins and this new protein is perhaps somewhat resistant to decomposition. Protein-like bodies giving reactions for proteoses and peptones have also been isolated from the soil.21 Miyake22 found that fatty amino compounds seem to be transformed into ammonia more easily than aromatic compounds; aromatic imino compounds are decomposed with greater difficulty than the aromatic amino compounds. The nature of the other group in the molecule 19 A detailed review of the extensive literature on ammonia formation in the decomposition of organic matter, up to 1910, is given by Voorhees and Lipman, 1907 (p. 491) and Lohnis, 1910 (p. xiii). 20 Lathrop, 1917 (p. 474). 21 Walters, 1915 (p. 474). 22 Miyake, K. Influence of the chemical structure of the compounds to be ammonified upon the rate of ammonification. Jour. Amer. Chem. Soc, 39: 2378-2382. 1917. 480 PRINCIPLES OF SOIL MICROBIOLOGY does not seem to have any influence upon the rate of transformation of imino nitrogen into ammonia nitrogen. These results tend to indicate that a simple observation of the amount and rate of ammonia formation need not necessarily indicate the course of protein decomposition. A certain set of conditions will lead to the formation of one group of compounds by bacteria from a certain pro- tein, while another group of compounds will be formed from the same protein under different conditions. It was also generally assumed23 that the rapid oxidation of proteins may result in the liberation of elementary nitrogen, according to the reaction: 4 NH3 + 3 02 - 6 H20 + 2 N2 However, the work of Ehrenberg24 pointed to the negligible loss of nitrogen from this source. Appreciable losses may occur either through direct volatilization of ammonia or by leaching of nitrates. Chemistry of ammonia formation in the decomposition of proteins by microorganisms. Mtintz25 was the first to demonstrate in 1890 that organic matter is decomposed by organisms with the formation of ammonia, which is only then nitrified. In soils in which nitrification has been stopped by the use of heat or disinfectants, ammonia accu- mulates, indicating that this treatment was sufficient to kill the organ- isms oxidizing ammonia to nitrate, but not those that produce ammonia from proteins. Muntz and Coudon26 have further shown that no ammonia was formed during two and one-half years in sterilized soil, while the unsterilized soil produced, in sixty-seven days, 41 to 100 mgm. of ammonia per 100 grams of soil. These investigations were followed by those of Marchal27 and numerous others which pointed to the importance of ammonia formation in the soil and the role of microorganisms in its formation from proteins. This was found to be not a specific property of certain bacteria, but a function of a large number of microorganisms. n Voorhees and Lipman, 1907 (p. 491), p. 49. "Ehrenberg, 1907 (p. 268). 26 Mtintz, A. Sur la decomposition des engrais organiques dans le sol. Compt. Rend. Acad. Sci., 110: 1206-1209. 1890. "Miintz, A., and Coudon, H. La fermentation ammoniacale de la terre. Compt. Rend. Acad. Sci., 116: 395-398. 1893. 27 Marchal, E. Sur la production de Pammoniaque dans le sol par lea microbes. Bull. Acad. Roy. Sci. Belg., (3), 25: 728-738; 27: 71-103. 1895; Centrbl. Bakt. II, 1: 753-758. 1895. DECOMPOSITION OF PROTEINS 481 When proteins are hydrolyzed by means of acids or enzymes, only 10 per cent of the total nitrogen in casein and 25 per cent in gliadin is liberated as ammonia, as a result of the breaking of the acid amide ( — CO-NH2) linkages. When proteins are acted upon by microor- ganisms, especially when those are used as sources of energy, large quantities of ammonia will be produced as a waste product. Seventy- five per cent or more of the protein-nitrogen can be found to accumulate in the soil in the form of ammonia within a few days, with proteins as the only source of energy. The ammonia is produced by a series of chemical changes which depend upon the nature of the organism, presence of nutrients other than amino acids (such as available carbo- hydrates), oxygen tension, and other conditions under which the reactions take place. Ammonia formation from amino acids may involve processes of hydrolysis, oxidation or reduction, or a combination of two or all, resulting in the splitting of the amino group or the carboxyl group or both. The various reactions may be summarized, as follows: 1. Hydrolytic decomposition: NH • C(NH2) • NH • (CH2)3 • CH(NH2) • COOH + H20 = arginine CH2(NH2) ■ CH2 • CH2 • CH(NH2) • COOH + CO(NH2)2 ornithine urea The hydrolysis of an amino acid may result in the formation of a lower fatty acid and ammonia, of an alcohol, C02 and ammonia, or of an aldehyde, lower acid and ammonia, as shown by the general formulae: RCH-NH2-COOH + H20 = RCHOH-COOH + NH3 (1) RCH CH2OH + C02 COOH COOH Amino acid Hydroxy- Alcohol fatty acid CH, • CHNH2 • COOH + H20 = CH, ■ CH2OH + NH, + C02 Alanine CH,\ >CH • CH2 • CH • NH2 • COOH + H20 - NH, + CH/ Leucine CH,\ + Hj >CH • CH2 • CHOH • COOH I_J CH/ H20+ 3NCH-CH2-CH2C00H--^H20 + 3\cHCH2 • COCOOH CH/ CH/ Caproic acid CH,. . H CH,X C02 + >CH • CH2 • CHO — " >CH • CH2 • CH2OH -> CH/ CH/ iso-amylalcohol 4- Oi CH,^ 1-4 H30 + >CH • CH2 • COOH CH, iso-valerianic acid. 2. Decarboxylation: R-CH-NHrCOOH = R-CH2-NH2 + C02 (4) R-CH2-NH2 + H20 = RCH2-OH + NH, (5) This process of amino acid decomposition through the amine stage, with the formation of alcohol and ammonia, has been described for yeasts and fungi.31 The first part of the process, namely the formation of amines, is characteristic of the so-called putrefaction processes.32 The transformation of amino acids into nitrogen bases is found to take place in the formation of para-oxy-phenyl-ethyla- mine from tyrosine, of pentamethyl-diamine (cadaverine) from lysine, of tetra- methylenediamine (putrescin) from ornithine, etc. 31 Ehrlich, F., and Pistchimuka, P. Uberftihrung von Aminen in Alkohole durch Hefe und Schimmelpilze. Ber. deut. chem. Gesell., 45: 1006-1012. 1912. 3S Rettger, L. F. Studies on putrefaction. Jour. Biol. Chem., 2: 71-96. 1906; 4: 45-55. 1907; 13: 341-346. 1912. DECOMPOSITION OF PROTEINS 483 OH • C6HHN:C:0 + NH, v NH, Urea (cylic) Cyanic acid form) Cyanic acid is hydrolyzed, in the presence of water: HN : C : O + H20 = NH3 + C02. 43 A detailed study of the chemistry of urea is given by E. A. Warner. The Chemistry of Urea. Longmans, Green & Co., New York. 1923. 44 Yamazaki, E. Chemical reaction of the system urease-urea. Jour. Tokyo Chem. Soc, 39: 125-184. 1918; Sci. Rept. Tokoku Imp. Univ., 9: 97, 136. 1920. 45 Fearon, W. R. Urease. I. The chemical changes involved in the zymol- ysis of urea. Biochem. Jour., 17: 84-93. 1923; Physiol. Rev., 6: 399-439. 1926. 488 PRINCIPLES OF SOIL MICROBIOLOGY However, this still remains to be confirmed. According to Sohngen,46 urea offers an exclusive source of energy to the urea bacteria but not a source of carbon, so that a carbohydrate is also necessary to insure a growth of the organisms. The maximum hydrolysis of urea, however, accompanies a minimum oxidation of organic compounds. B. erythro- genes, for example, hydrolyzes 500 mgm. urea for every 20 mgm. of carbon assimilated, while Urobac. jakschii hydrolyzes 1800 mgm. of urea for 10 mgm. of carbon assimilated. ^I^^jjfc/awaiamiae 7 21 42 9 A days Fig. 26. Accumulation of ammonia from cyanamide and dried blood, as in- fluenced by the presence of diacyanodiamide (from Cowie). Cyanamide readily breaks down in the soil yielding ammonia which is then nitrified practically quantitatively. Cyanamide may first be decomposed in the soil into urea by a purely chemical process,47 under the influence of catalyzers, or it may polymerize into dicyanodiamide (especially in the presence of catalyzers such as ZnCl2). Dicyanodi- amide does not nitrify and is even toxic to nitrifying bacteria, but is 46 Sohngen, N. L. Ureumspaltung bei Nichtvorhandensein von Eiweiss. Centrbl. Bakt. II, 23: 91-98. 1909. 47 Cowie, G. A. Decomposition of cyanamide and dicyanodiamide in the soil. Jour. Agr. Sci., 9: 113-136. 1919; 10: 163-176. 1920. DECOMPOSITION OF PROTEINS 489 not toxic to ammonia forming organisms.48 No ammonia is formed from cyanamide in sterile soil, but considerable amounts of ammonia are produced on the addition of urease; this indicates the presence of urea. The urea is, of course, decomposed in the soil by various organisms.49 Among the other nitrogenous substances which are gradually decomposed in the soil by microorganisms, we find various alkaloids, such as cocaine, strychnine, morphine, etc.80 Chitin is found among the synthesized constituents of the cells of microorganisms, especially fungi, and is constantly added to the store of soil organic matter. It is a polymer of mono-acetyl-glucosamine (Ci4H26N2Oio)„, giving acetic acid and glucosamine upon hydrolysis. It gives a violet color with chlor-zinc iodide and a brown-red color with a solution of iodine and potassium iodide. Certain bacteria and actinomyces decompose chitin in the soil by means of an enzyme chitinase.51 Chitin can be used by these organisms both as a source of carbon and nitrogen, in the presence of K2HP04 and MgS04. To be able to understand, how the decomposition of proteins and other nitrogenous substances by microorganisms in the soil results in the formation of ammonia, how different organisms bring about differ- ences in the accumulation of the ammonia and how the latter is in- fluenced by the soil environmental conditions, a knowledge of the action of the different organisms upon proteins, under different con- ditions, is essential. Ammonia formation by bacteria. The earlier investigators of bacterial metabolism, like Hoppe-Seyler, Bienstock, Hauser and others, found that mixtures and pure cultures of bacteria, like Bad. vulgare, Bac. sub- 48 Norris, R. V., Vismanath, B., and Aiyer, C. V. R. A preliminary note on the decomposition of calcium cyanamide in South Indian soils. Mem. Dept. Agr. India, Pusa., 7: 55-75. 1923; Ulpiani, C. Evoluzione chimica e biochimica della calciocianamide nel terreno agrario. Gaz. Chim. Ital., 40: 613-666. 1910. 49 Further information on ammonia formation from cyanamide is given by Perotti, R. Uber den mikrobiochemischen Prozess der Ammonization im Acker- boden. Centrbl. Bakt. II, 20: 514-518. 1908; tlber die Stickstoffernahrung der Pflanzen durch Amidsubstanzen. Ibid., 24: 373-382. 1909; Lohnis, 1910 (p. xiii), p. 590. 50 Lavialle, P. Destruction of alkaloids in the soil. Bull. Sci. pharmacol., 30: 321-325. 1923; (Chem. Abstr., 17: 2732). 81 Benecke, \V. t)ber Bacillus chitinovorus, einen Chitin zersetzenden Spalt- pilz. Bot. Ztg., 63: 227-272. 1905; Folpmers, T. Die Zersetzung des Chitins und des Spaltungsproduktes desselben, des Glucosamins, durch Bakterien. Chem. Weekbl., 18: 249. 1921; (Centrbl. Bakt. II, 57: 97-98. 1922). 490 PRINCIPLES OF SOIL MICROBIOLOGY tilis, Bad. prodigiosum, Bac. putrificus, Bad. fiuorescens liquefaciens, are capable of breaking down proteins with the formation of various end products, one of which was ammonia. Proteins of both plant and animal origin were found to be decomposed by a number of bacteria giving a great variety of products.52 The investigations of Mlintz and Coudon and Marchal53 called attention to the existence of large numbers of bacteria and fungi in the soil, capable of decomposing proteins with the formation of am- monia. A solution containing 1.5 per cent nitrogen, in the form of egg albumin made insoluble by means of 0.01 per cent ferric sulfate, was inoculated with various bacteria; ammonia was determined after 20 days' incubation at 30° by distilling with MgO. TABLE 42 Protein nitrogen transformed into ammonia Bac. mycoides Proteus vulgaris Bac. m,esentericus vulgatus Sarcina lutea Bac. subtilis Bac. janthinus Bad. fiuorescens pulidum. PER CENT 46 36 36 27 23 23 22 Bac. arborescens Bad. fiuorescens liquefaciens C ephalothecium roseum Asp. terricola Botryotrichum piluliferum. . . Stemphylium Actinomyces PER CENT 19 16 37 32 24 5 21 The various strains of Bac. mycoides derived from different sources varied in their power to produce ammonia from proteins. In the case of one strain of Bac. mycoides, Marchal obtained a transformation of 58 per cent of egg-albumin nitrogen into ammonia, accompanied by a marked change of the reaction of the medium to alkaline. The more dilute the solution of the protein the greater was the transforma- tion. Of the individual amino acids, 66 per cent of the nitrogen of tyrosine in a 0.4 per cent solution, in the presence of some sugar and salts, was transformed into ammonia, with 40 per cent of the leucine and 37 per cent of asparagine in a 1 per cent solution. Only 9 per cent of the crea- tine was transformed into ammonia. In addition to C02 and ammonia, peptone, leucine, tyrosine, some formic, propionic and butyric acids were demonstrated among the products of the digestion of albumin by 62 Olig. Die Zersetzung pflanzlicher Futter und Nahrungsmittel durch Bak- terien. Diss. Miinster (Berlin. Springer). 1903. "Miintz and Coudon, 1890-1893 (p. 267); Marchal, 1893 (p. 267). DECOMPOSITION OF PROTEINS 491 bacteria. For every milligram of ammonia formed 8.9 mgm. C02 were liberated. Marchal concluded that Bac. rmjcoides is one of the most common soil organisms and one that attacks proteins most energeti- cally. It is favored by a temperature of 30°, complete aeration, slightly alkaline medium and a slight concentration of nitrogenous substance in solution. The work of Marchal was confirmed and further extended by numer- ous investigators.54 The great majority of soil organisms developing on the plate were found to produce ammonia from proteins. The gelatin-liquefying bacteria were found to be capable of inducing a greater protein decomposition with a more abundant ammonia forma- tion.55 Since these organisms form at times more than 15 per cent of the total number of soil bacteria (developing on the plate), they were believed to do the initial work in rendering soluble the protein nitro- gen in the soil, so that it might be further decomposed by the same or other soil organisms.56 Lipman and Burgess57 tested a series of pure cultures of bacteria for their ammonia-producing power, using various nitrogenous substances in various soils. Bac. tumescens was found to be the most efficient organism of all tested, although in some cases greater efficiency was obtained for Bac. mycoides and Sarcina lutea. Usually 20 to 30 per cent of the protein nitrogen was transformed into ammonia in twelve days. Conn,58 however, found that, in manured soil, the non-spore forming bacteria are much more active than the spore forming organisms. According to Waksman and Lomanitz,59 Bac. cereus rapidly decomposes proteins to amino acids while Bad. fluorescens acts largely upon amino " Severin, S. A. Die im Miste vorkommenden Bakterien und deren physi- ologische Rolle bei der Zersetzung derselben. Centrbl. Bakt. II, 1: 97-104, 160-168, 799-817. 1895; 628-633. 1897; 7: 369-386. 1901; 13: 616-631. 1904; Chester, F. D. The bacteriological analysis of soils. Delaware Sta. Bui. 65. 1904; Lohnis, 1905 (p. 120); Lipman, J. G. Chemical and bacteriological factors in the ammonification of soil nitrogen. N. J. Agr. Exp. Sta. 19th Ann. Rpt. 1906, 119-188. M Gage, S. D. Contribution to the biochemistry of sewage purification; the bacteriolysis of peptones and nitrate. Jour. Amer. Chem. Soc, 27: 327-363. 1905. M Voorhees, E. B., and Lipman, J. G. A review of investigations in soil bac- teriology. Bui. 194, Office Exp. Sta., U. S. Dept. Agr. 1907. s7 Lipman, C. B., and Burgess, P. S. Studies on ammonification in soils by pure cultures. Univ. Cal. Publ. Agr. Sci., 1: 141-172. 1914. " Conn and Bright, 1919 (p. 41). " Waksman and Lomanitz, 1925 (p. 379). 492 PRINCIPLES OF SOIL MICROBIOLOGY acids. In the presence of a mixture of the two organisms, the pro- tein is rapidly changed to ammonia. Thus there is a possibility that various organisms take an active part in the various stages of the process; organisms like Bac. cereus may be active in the first stages of hydrolysis and organisms like Bad. fluorescens, in the latter stages leading to the formation of ammonia (fig. 27). This confirmed the earlier investigations of Tissier and Martelly,60 who found that the action of Bad. colt, various micrococci and Bad. filiformis aerobius upon natural proteins was nil, or almost nil; but they acted very readily ;V~j£Z '-Sfisa-^ Fig. 27. Course of accumulation of amino- and ammonia-nitrogen from casein by Bac. cereus and Bad. fluorescens (from Waksman and Lomanitz). upon the hydrolytic products of proteins. The same was true of certain anaerobic bacteria and even the action of Bad. vulgare upon pure proteins has been doubted. However, various spore-forming bacteria, especially anaerobes, like Bac. gracilis putidus, Bac. putrificus and also Bac. perfn'ngens, Bac. bifermentans and Bac. sporogenes rapidly attacked native proteins. They suggested, therefore, to divide the bacteria into two groups, on this basis. The number of soil bacteria capable of forming ammonia from pro- 60 Tissier, H., and Martelly. Recherches sur la putrefaction de la viande de boucherie. Ann. Inst. Past., 16: 865-903. 1902. DECOMPOSITION OF PROTEINS 493 teins is very large; when tested in pure culture upon native proteins, the spore-forming bacteria are most active. When protein derivatives, like peptone, amino acids, and urea, are used, various non-spore form- ing bacteria will be found to play an important role in the process. In the soil itself, all of those organisms probably contribute to a greater or less extent to this process, depending upon the nutrients available and environmental conditions. The rapidity of ammonia formation from proteins by bacteria depends not only upon the nature of the organism but also upon the kind of protein. The process of ammonia formation is completed in a few days in the case of casein, while it continues, even after a month, from gliadin.61 The amino-nitrogen content of the gliadin and casein media was 0.57 and 0.68 mgm. before hydrolysis; 42.56 and 99.31 mgm. after acid hydrolysis, and 17.03 and 46.00 after hydrolysis with Bac. sub- tilis. All the nitrogen forms of the protein molecule are changed more or less by the action of bacteria, the end product being ammonia; in no case, however, is one form of nitrogen completely destroyed. A similarity was found in the chemical change produced by acid hydroly- sis and bacteria. Ammonia formation by fungi and actinomyces. The actinomyces develop on artificial culture media and in the soil slower than the fungi and, when a short period of incubation is used, their intense ac- tivity in breaking down proteins and forming ammonia may be over- looked. When a long period of incubation (30 days or more) is used, they are found to be very active in this respect.62 The important point in this connection is that these organisms are capable of allowing a large accumulation of ammonia even in the presence of available carbohydrates; in other words, they prefer proteins to carbohydrates as sources of energy. According to Guittonneau,63 actinomyces pro- duce, from proteins, not only ammonia, but also urea, both in the presence and absence of dextrose. Fungi can decompose proteins very vigorously. Different species vary greatly in this respect, and the nature of the protein, reaction of 81 Robinson, R. H., and Tartar, H. V. The decomposition of protein sub- stances through the action of bacteria. Jour. Biol. Chem., 30: 135-144. 1917. 82 Fousek, 1912 (p. 302); Mace, E. De la decomposition des albuminoides par les Cladothrix (actinomyces). Compt. Rend. Acad. Sci., 14: 147-148. 1905; Waksman, 1920 (p. 299). 63 Guittonneau, G. Sur la production de l'uree au cours de rammonifica- tion par les Microsiphonees. Compt. Rend. Acad. Sci., 178: 1383-5. 1924. 494 PRINCIPLES OF SOIL MICROBIOLOGY medium and presence of available carbohydrates also affect the proc- ess. A large part of the nitrogen may be left in the form of inter- mediary products. Organisms like Asp. niger, which produce large amounts of acid (oxalic and citric) from carbohydrates and even from proteins and which are thus enabled to neutralize the ammonia, ac- cumulate only very small amounts of amino acids in artificial cultures; at the same time appreciable quantities of ammonia are formed in the medium. But when the oxalic acid is neutralized with CaC03, or when the formation of both oxalic acid and ammonia is prevented by means of insufficient aeration, an accumulation of amino acids will take place, as with the other fungi.64 When the protein is the only source of carbon during the develop- ment of fungi on the protein media, a definite parallelism is found between the growth of the mycelium and the production of ammonia. Different protein derivatives are not utilized alike and their nitrogen is not liberated alike in the form of ammonia. Asp. niger, for example, grows best with leucine, followed by peptone, asparagine and glycocoll. The difference in the nature of the carbon compounds either accompany- ing the proteins or the protein carbon itself accounts for the difference in the amount of fungus growth and ammonia formation. This is due to the fact that, in the absence of available carbohydrates, the organism uses the protein both as a source of energy and as a source of nitrogen; the amount of nitrogen liberated as ammonia will depend not only upon the nitrogen content of the protein, but largely upon the availability of the carbon; the nitrogen is then either liberated as a waste product, ammonia, or is reassimilated and changed into micro- bial protein. McLean and Wilson65 concluded that fungi, rather than bacteria, are responsible for the large accumulations of ammonia in soil rich in organic nitrogenous substances and that this depends upon the chemical and physical composition of the soil, quality of the organic matter present and presence of soluble phosphates. The period of maximum formation and accumulation of ammonia from protein substances by 64 Butkewitsch, W. Umwandlung der Eiweissstoffe durch die niederen Pilze im Zusammenhange mit einigen Bedingungen ihrer Entwicklung. Jahrb. wiss. Bot., 38: 147-240. 1903. Ammonia as a product of protein transformation by fungi and conditions of its formation (Russian). Rec. d'articles dedies au Prof. C. Timiriazeff. 1916, 457-499. 66 McLean, H. C, and Wilson, G. W. Ammonification studies with soil fungi. N. J. Agr. Exp. Sta., Bui. 270. 1914. DECOMPOSITION OF PROTEINS 495 pure cultures of various fungi was found66 to depend on the type of organism used ; Monilia sitophila reached its maximum in 3 to 4 days, Mucor plumbeus reached it in 6 to 10 days. Rate of ammonia formation by microorganisms and methods of deter- mination. In the decomposition of proteins by pure cultures of bacteria TABLE 43 Ammonia formation by soil fungi Pen. intricatum. Pen. intricatum. Pen. chrysogenum . Pen. chrysogenum Asp. fumigatus Asp. fumigatus Mucor hiemalis. Mucor hiemalis . Rhiz . nigricans . Rhiz. nigricans . Zyg. vuilleminii , Zyg. vvilleminii . Monilia sitophila . Monilia sitophila . Trich. koningi. Trich. koningi. SOURCE OF NITROGEN* MILLIGRAMS OF NH|N X>. B. 20.45-11.26 C. S. M. 4.65- 3.85 D. B. 21.81 C. S. M. 16.82 D. B. 12.16 C. S. M. 7.91 D. B. 12.75-29.94 C. S. M. 18.09-25.89 D. B. 12.65 C. S. M. 25.40 D. B. 28.73 C. S. M. 31.43 D.B. 19.51 C. S. M. 40.23 D. B. 76.48-66.16 C. S. M. 42.60-30.63 * To 100 grams of soil were added 155 mgm. of nitrogen in the form of D. B. Dried blood or of C. S. M. = cottonseed meal. or fungi, as well as in the soil itself, the rate of ammonia accumulation is that of an autocatalytic chemical reaction.67 However, the nature of the protein and the presence of non-nitrogenous organic matter 66 Waksman, S. A., and Cook, R. C. Incubation studies with soil fungi. Soil Sci., 1: 375-384. 1916. 87 Miyake, 1916 (p. 375); Waksman, S. A. Studies on proteolytic activities of soil microorganisms with special reference to fungi. 1918. Jour. Bact., 3: 475^92. 496 PRINCIPLES OF SOIL MICROBIOLOGY will influence greatly the rate of the reaction. Gainey68 observed a remarkable similarity in the rates of formation of ammonia and car- bon dioxide from dried blood and cotton seed meal. At first there was a rapid increase which soon reached a maximum and then decreased rapidly. Insufficient aeration and moisture resulted in a decrease in ammonia formation. Unfavorable conditions had a more detri- mental effect on the latter than on C02 evolution (fig. 28). An in- teresting correlation was found69 between the amounts of ammonia Fig. 28. Rate of decomposition of cottonseed meal in soil, as shown by the evolution of CO2 and accumulation of NH3; 1 and 2 were aerated continuously, S and 4 were aerated 30 minutes daily (from Gainey) . formed and the numbers of bacteria, as a result of addition of organic matter to the soil. The literature on the subject of ammonia formation from the de- composition of nitrogenous organic substances added to the soil is very extensive. Unsuccessful attempts were even made to determine 88 Gainey, 1919 (p. 685). 89 Beckwith, T. D., Vass, A. F., and Robinson, R. H. Ammonification and nitrification studies of certain types of Oregon soils. Ore. Agr. Exp. Sta. Bui. 118. 1914. DECOMPOSITION OF PROTEINS 497 the productive capacity of a soil by its ammonia producing power, as will be shown later. Among the methods used for determining ammonia in the soil and in solution, only three need be mentioned: (1) the direct distillation of the soil or solution with magnesium oxide; (2) the aeration method; (3) the extraction of ammonia with KC1 solution (followed by distilling the ammonia with magnesium oxide). The first is more rapid, but may bring about the liberation of some ammonia from amides and perhaps from other simple nitrogenous substances. The aeration method can be used in determining ammonia in liquid culture, but requires a long time for a complete extraction of the am- monia from soils.70 It consists in placing 25 to 50 cc. of the culture or soil suspension in large heavy glass tubes or flasks, adding some heavy oil, 2 to 3 grams of NaCl and 2 grams of Na2C03, then aerating for 2 to 3 hours. The ammonia is absorbed in a standard solution of sulfuric acid to which a proper indicator has been added (like sodium alizarine sulfonate). On placing the tubes in a water bath, at 55 to 60°C, the process is carried out more rapidly and completely. Where heat is used, there is, of course, always some danger of hydrolysis of undecomposed proteins or their derivatives. The extraction of the ammonia from soil by a KC1 solution is based upon the fact that the ammonium base is replaced in its adsorbed condition in the soil by another base when added in excess to the soil (in this case by the potassium of the KC1 solution). The process is usually carried out by extracting 25 grams of the soil successively with five to seven 100-cc. portions of approximately 4 per cent potassium chloride solution (or until the filtrate gives no test for ammonia with Nessler's reagent). Peat soils should be extracted two or three times more. Alkaline soils should be first neutralized with ammonia-free hydrochloric acid. The combined filtrates are then distilled with MgO into standard O.bV H2S04. When distillation is finished, the carbon dioxide is removed from the distillate by boiling, before the acid is titrated back with a standard alkali.71 70 Potter, R. S., and Snyder, R. S. The determination of ammonia in soils. Jour. Ind. Engin. Chem., 7: 221. 1915; Gibbs, W. M., Neidig, R. E., and Batchelor, H. W. Aeration method for determining ammonia in alkali soils. Soil Sci., 15: 260-268. 1923. 71 Tarassoff, B. On the methods of determining ammonia in the soil (Rus- sian). Zhur. Opit. Agron., 15: 118-138. 1914; Bengtsson, N. The determina- tion of ammonia in soil. Soil Sci., 18: 255-278. 1924. 498 PRINCIPLES OP SOIL MICROBIOLOGY Nitrogen transformation in the rotting of manure. About half of the nitrogen in manure is in the form of ammonia (and urea) and half is in the form of proteins and other complex nitrogen compounds. The first part of the nitrogen rapidly changes into nitrates before the micro- organisms, using the constituents of the straw and other undecom- posed materials, have a chance to absorb it and change it into organic complexes. The second part has to be gradually decomposed before the nitrogen can be made available; for this a considerable period of time is required.72 The final product in the decomposition of the proteins, of the urea and of the other nitrogen compounds of the manure is chiefly ammonia. A small part of it may be lost to the atmosphere TABLE 44 Transformation of nitrogen in the composting of manure under aerobic and anaerobic conditions7^ NHrN NO3-N NO2-N AMIDE-N OTHER N COM- POUNDS (proteins) Anaerobic conditions Start 6°C for 50 days, 26°C for 50 days Start 15°C 26°C per cent per cent per cent per cent per cent 28 8 64 29 9 57 44 7 47 per cent 100 95 98 Aerobic conditions 33 8 59 11 11 8 42 5 2 7 55 100 72 69 through volatilization73 but a much greater part is retained in the manure, as such, by physical or chemical agencies. A part of the ammonia is nitrified and a part is reabsorbed by the bacteria and fungi in the manure in the process of their metabolism for which the straw of the manure is used as a source of energy. Another source of loss, in the decomposition of manure, is the evolution of gaseous nitrogen, either through denitrification or through the chemical interaction of 72 Barthel, Chr. Neuere Untersuchungen iiber die Ausniitzung des Stall- miststickstoffs im Ackerboden. Fortschr. der Landwirtsch., 1: H. 2. 1926. 73 Ehrenberg, P. Die Bewegung des Ammoniakstickstoffs in der Natur. P. Parey. Berlin. 1907. DECOMPOSITION OF PROTEINS 499 amino acids with nitrous acid, but this loss is comparatively small. Niklewski74 found that the greatest loss of nitrogen from the stable manure is a result of the action of the nitrifying bacteria. Only about 3 per cent of the nitrogen was lost, largely as ammonia, when manure was kept for 255 days free from nitrifying bacteria. In the presence of a large amount of urine, 5 to 12 per cent of the nitrogen was lost. Manure inoculated with nitrifying bacteria lost about 20 to 24 per cent of its nitrogen. This is due chiefly to the fact that the nitrates are reduced rapidly as soon as formed and elementary nitrogen is lost into the atmosphere. Fig. 29. Apparatus for study of ammonia formation in the decomposition of manure (from Conn and Collison). According to Russell and Richards,75 there is a distinct difference in the transformation of nitrogen compounds in the manure when stored under anaerobic or aerobic conditions. There was practically no loss of nitrogen under anaerobic conditions; the proteins were de- composed with the formation of ammonia, particularly at the higher temperature. Under aerobic conditions, there was a great loss of nitrogen, the amide form practically disappeared, while most of the ammonia was transformed into nitrites or nitrates. In general, under anaerobic conditions, the loss of dry matter and nitrogen is at a mini- 74 Niklewski, B. t)ber die Bedingungen der Nitrifikation im Stallmist. Centrbl. Bakt. II, 26: 388-442. 1910; Rocznikow Nauk Rolniczych, 9: 1-18. 1923. 75 Russell, E. J., and Richards, E. R. The changes taking place during the storage of farmyard manure. Jour. Agr. Res., 8: 495-563. 1917. 500 PRINCIPLES OF SOIL MICROBIOLOGY mum; the gases formed consist of C02, CH4, H2 and NH3. Under aerobic conditions, there is a much greater loss of dry matter with a much greater decomposition of the nitrogenous compounds. Hydro- gen and methane are not found in the gases. There is no ammonia accumulation, but nitrates are formed in the outer layers; with in- complete aeration, gaseous nitrogen is formed. Various groups of bacteria and fungi are active in the process of formation of ammonia in manure, but not with equal rapidity. According to Conn and Collison,76 the strong proteolytic and gelatin liquefying organisms, like Bac. cereus and Bad. fluorescens, are not able to give off ammonia in quantities comparable to those given off from unsterilized manure. Conn found that a minute, gram-negative, non-motile, non-spore forming rod, Bad. parvulum was able to give off from manure, in pure culture in the laboratory, amounts of ammonia, equal to or greater than those obtained from unsterilized manure. The decomposition of manure in the pile may not necessarily be carried on by the same organisms as in the soil. Bad. fluorescens, which was found to be relatively less active in the manure pile, was very active in the forma- tion of ammonia from manure in the soil.77,78 Nitrogen transformation in the decomposition of organic matter in the soil. When nitrogenous organic substances are added to the soil, a group of complex reactions will result as far as the nitrogen is concerned : (1) The hydrolysis of the proteins into polypeptides and amino acids, with the liberation of some ammonia. (2) This is followed by the decomposition of the amino acids and other products of protein hydrolysis, with a further liberation of ammonia. (3) Synthesis of microbial protoplasm, which will lead to a stor- ing away of a part or the whole of the ammonia nitrogen; the greater the quan- tity of available non-nitrogenous organic matter accompanying the nitrogenous substances, the greater will be the synthesis of microbial protoplasm, leading to a greater assimilation of the nitrogen and to a smaller accumulation of am- monia. (4) Various soil conditions, as well as differences in the composition of 76 Conn, H. J., and Collison, R. C. A study of certain bacteria involved in the ammonification of manure. N. Y. Agr. Exp. Sta. Bui. 494. 1922. 77 Conn, H. J., and Bright, J. W. Ammonification of manure in soil. N. Y. Agr. Exp. Sta. Tech. Bui. 67. 1919. 78 Further information on the conservation of manure and the changes taking place in manure during storage is given by Lohnis, F., and Smith, J. H. Die Veranderungen des Stalldiingers wahrend der Lagerung und seine Wirkung im Boden. Fiihling's landw. Ztg. 1914, 153-167; Lemmermann, O., and Weissmann, H. Untersuchungen iiber die Konservierung der Jauche durch verschiedene Zusatzmittel. Landw. Jahrb., 52: 297-341. 1918. DECOMPOSITION OF PROTEINS 501 the nitrogenous and the accompanying non-nitrogenous organic substances, will lead to the development of different microorganisms capable of decom- posing the nitrogenous materials; the carbon-nitrogen metabolism of these micro- organisms is different; this leads, therefore, to differences in the amounts of ammonia liberated in a free state. These various reactions lead to a transformation of a larger or smaller part of the nitrogen of the organic complexes into ammonia, which, either as such or after it has been oxidized to nitrates, is available as a source of nitrogen for the growth of cultivated plants. In view of the fact that the liberation of this ammonia is of such great economic importance, numerous contributions have been made to the subject, known as "ammonification.'' These studies were chiefly limited to adding about 1 gram of the nitrogenous organic material to 100 <:,rams of soil, mixing, placing in tumblers, then bringing the moisture content of the soil to optimum (CO per cent saturation), incubating for 4 to 14 (usually 7) days, then measuring the amount of ammonia present in the soil by distilling with MgO. These studies resulted in a most extensive literature. It was found,79 for example, that organic nitrogenous materials of different origin liberated ammonia with a different degree of rapidity as follows: NATURE OF MATERIAL AMMONIA FORMED, PER CENT OF NITROGEN ADDED 4 days 7 daya Blood 18.24 32.35 49.07 32.28 Tankage 38.52 Fish 55.39 The difference in rapidity of decomposition of the organic nitrogen compounds with the liberation of ammonia is due to the nature of the nitrogen complex,80 accompanying non-nitrogenous substances (see p. 505) and environmental conditions, which favor the develop- ment of specific organisms and, therefore, specific processes. Influence of nitrogenous decomposition products on the growth of plants 79 Lipman, J. G., Blair, A. W., Owen, I. L., and McLean, H. C. The avail- ability of nitrogenous materials as measured by ammonification. N. J. Agr Exp. Sta. Bui. 246. 1912. 80 Jodidi, S. L. Amino acids and acid amides as sources of ammonia in soils. Iowa Agr. Exp. Sta. Res. Bui. 9. 1912; Lathrop, E. C. Protein decomposition in soils. Soil Sci., 1: 509-532. 1916. 502 PRINCIPLES OF SOIL MICROBIOLOGY and microorganisms. As a result of the activities of microorganisms, a large number of substances are formed from the decomposition of proteins. The most important of these is ammonia. This is either assimilated without change by plants or microorganisms or is first converted into nitrates by the nitrifying bacteria. In addition to ammonia, other nitrogenous compounds formed from the decomposi- tion of proteins by microorganisms are beneficial to the growth of higher plants.81 It has even been suggested that substances such as nucleic acid, hypoxanthine, guanine, histidine, arginine and creatinine, are absorbed directly by the plant, without first being transformed into ammonia and nitrates. Collectively these compounds were found to be more beneficial than when used singly.82 It is possible that these substances, especially the nucleic acids, are not used as nutrients directly, but play a role in the growth of plants and microorganisms similar to that played by vitamines in the growth of higher plants. Some of the decomposition products may have a harmful effect upon plant growth,83 as in the case of the various nitrogenous and non-nitrogenous substances that can be isolated from the soil, includ- ing pyridine and its derivatives, dihydroxystearic acid, etc. Bacteria utilize a wide range of substances as sources of nitrogen, including proteins and their derivatives which they tend to break down further with the liberation of ammonia.84 The same is true of fungi.85 According to Emmerling, fungi utilize the a-amino acids quite 81 Schreiner, O., Reed, H. S., and Skinner, J. J. Certain organic constituents of soils in relation to soil fertility. Bur. Soils, U. S. Dept. Agr. Bui. 47. 1909; Schreiner, O., and Lathrop, E. C. The chemistry of steam heated soils. Jour. Amer. Chem. Soc, 34: 1242-1259; Skinner, J. J. Effects of creatinine on growth and absorption. Bur. Soils, U. S. Dept. of Agr. Bui. 83. 1911; Hutchinson, H. B., and Miller, N. H. J. The direct assimilation of inorganic and organic forms of nitrogen by higher plants. Centrbl. Bakt. II, 30: 513-547. 1911. 82 Schreiner, O., and Skinner, J. J. Nitrogenous constituents and their bear- ing on soil fertility. Bur. Soils, U. S. Dept. Agr. Bui. 87. 1912. 83 Schreiner, O., and Shorey, E. C. 1909. The isolation of harmful organic substances from soils. Bur. Soils, U. S. Dept. of Agr. Bui. 53. 1909; Chem- ical nature of soil organic matter. Ibid., Bui. 74. 1910. 84 Bierema, S. Die Assimilation von Ammon-Nitrat und Amidstickstoff durch Mikroorganismen. Centrbl. Bakt. II, 23: 672-726. 1909; Nawiasky, P. Ueber die Ernahrung einiger Spaltpilze in peptonhaltigen Nahrboden. Arch. Hyg., 64: 33-61. 1908; Uber die Umsetzung von Aminosauren durch Bac. proteus vulgaris. Arch. Hyg., 66: 209-243. 1908. 86 Czapek, F. Untersuchungen iiber die Stickstoffgewinnung und Eiweiss- bildung der Pflanzen. Beitr. Chem. Physiol. Path., 1: 538-560. 1902; 2: 557- 590. 1902; 3: 47-66. 1902; Emmerling, O. Aminosauren als Nahrstoff fur DECOMPOSITION OF PROTEINS 503 readily, but seem to be unable to attack the /3-amino acids. Czapek found that Asp. niger could not utilize all the various nitrogenous com- pounds tested but grew very well on a large number of them. Amino acids were used most economically, which led Czapek to suggest that the fungi assimilate the nitrogen in that form and are spared the trouble of synthesizing the amino acids needed for their protoplasm. Hagem86 however, maintains that ammonia is the starting point in the synthesis of protoplasm by microorganisms. Fungi can utilize urea, uric acid, glycocoll, guanidine, guanine, nitrates, nitrites and ammonium salts as sources of their nitrogen, while uric acid, gycocoll and hippuric acid may serve as a source of carbon as well.87 The whole process of protein transformation and protein synthesis in the soil is very complex and is constantly in a dynamic condition. The net result is fertility or infertility, depending on which set of factors predominates in the soil at any one time. A mere determina- tion of the amount of ammonia formed after adding to the soil a definite amount of a certain organic fertilizer, like dried blood or cottonseed meal, cannot solve the question of the availability of the nitrogen in the particular fertilizer; it does not indicate the amount of intermediate compounds formed by the decomposition of the organic matter or fertilizer in the soil and the character of the action of these compounds on plant growth. niedere Pflanzen. Ber. deut. chem. Gesell., 36: 2289-2290. 1902; Butkewitsch, 1903 (p. 494); Brenner, W. Die Stickstoffnahrung der Schimmelpilze. Centrbl. Bakt. II, 40: 555-640. 1914. " Hagem, 1910 (p. 237). «TKossowicz. (Rev. Lathrop, 1917 (p. 474).) CHAPTER XIX Influence of Available Energy upon the Transformation of Nitrogenous Compounds by Microorganisms Carbon and nitrogen transformation by microorganisms. All micro- organisms require a certain amount of energy for the building up of their protoplasm as well as a certain minimum of nitrogen, phosphorus and other minerals. In the case of heterotrophic, non-nitrogen-fixing microorganisms, the energy is obtained either from nitrogen-free or- ganic compounds or from proteins and their derivatives. The nitrogen is obtained from inorganic nitrogenous salts, such as ammonium com- pounds and nitrates, or from complex organic compounds, such as proteins and their derivatives. Phosphates and other minerals are obtained from the inorganic or organic compounds present. When an organism has to derive both its carbon and nitrogen from proteins, only a small part of the nitrogen is reassimilated, while a larger part will remain as a waste product (ammonia). Several fac- tors contribute to this phenomenon: 1. Only 10 to 40 per cent of the carbon is reassimilated by the or- ganism and synthesized into protoplasm; a larger part is given off as C02 (in the process of energy utilization) or is left in the form of unde- composed material or in the form of intermediary products. The smaller the amount of carbon assimilated by the organism, the less is the amount of protoplasm synthesized and, therefore, the less is the amount of nitrogen assimilated and the more of it is left in the medium as a waste product (largely NH3). 2. The microbial protoplasm may contain a lower per cent of nitrogen than the original protein. This will tend further to diminish the amount of reassimilated nitrogen. The excess nitrogen will be liberated as ammonia or left in the form of various protein degradation products. In the presence of available carbohydrates, however, the micro- organisms will assimilate the available ammonia nitrogen and convert it into microbial protoplasm. The greater the quantity of carbohy- drate present for a given amount of protein or its derivatives the more of the nitrogen will be reassimilated by microorganisms and the less of it will be left as ammonia. The larger the ratio of the protein-free 504 ENERGY AND NITROGEN TRANSFORMATION 505 substances to the protein, the smaller is the amount of ammonia liber- ated. This has an important bearing upon the liberation of ammonia in the soil, since the great mass of organic matter usually added to ^he soil, in the form of manures and plant residues, contains a low per cent of nitrogen and a high per cent of energy-yielding material. The available nitrogenous plant food in the soil is also greatly affected by the conditions under which decomposition takes place, since, under different conditions, different organisms will take part in the process and will, therefore, bring about different sets of reactions. Microorgan- isms vary in the amount of carbon reassimilated, in the relative nitro- gen content, and in the nature of decomposition that they bring about. Influence of non-nitrogenous organic matter upon the decomposition of nitrogenous compounds and upon the amounts of ammonia liberated. The presence of non-decomposed or only partly decomposed non- nitrogenous organic matter in the soil modifies in various ways the decomposition of nitrogenous compounds by microorganisms, by influencing the amount and the nature of decomposition. Hirschler1 was the first to point out that the decomposition of pro- teins by microorganisms is modified by the presence of carbohydrates which prevent the formation of aromatic products of putrefaction. Indol, phenol, and oxy-acids were not formed in the decomposition of proteins by bacteria when sucrose, starch, dextrin, glycerol or lactic acid are present; i.e., the presence of an available source of energy modified the decomposition processes. It was later2 demonstrated conclusively that bacteria do not decompose large amounts of proteins in the presence of available carbohydrates; the amount of ammonia formed may also be greatly diminished. This is due to the fact that the organisms derive their energy preferably from carbohydrates and act upon the proteins only to an extent sufficient to obtain the nitrogen required for the synthesis of their protoplasm. Any ammonia that is produced, in this connection, may be reassimilated. In the absence of available carbohydrates, proteins are used also as sources of energy and large amounts of nitrogen are liberated as waste products in the 1 Hirschler, A. Uber den Einflusz der Kohlehydrate und einiger anderer Korper der Fettsaurereihe auf die Eiweissf iulniss. Ztschr. physiol. Chem., 10: 306-317. 1886. 2 Kendall, A. I. The significance and quantitative measurement of the nitroge- nous metabolism of bacteria. Jour. Inf. Dis., 30: 211. 1922; also Ibid., 17: 442^53. 1915; Jour. Amer. Chem. Soc, 35: 1201-1249. 1913; 36: 1937-1962. 1914. 506 PRINCIPLES OF SOIL MICROBIOLOGY form of ammonia. The presence of an available carbohydrate does not inhibit, but rather stimulates the multiplication of the bacteria; it lessens, however, the amount of protein to be utilized, and, therefore, the amount of ammonia accumulated. Doryland3 explained this by the fact that the organisms utilize the ammonia as a source of nitrogen and the carbohydrates as a source of energy. Thus, in the presence of available carbohydrates, two factors are at work: (1) less of the protein is decomposed since the bacteria and fungi may prefer the carbohydrate to the protein as a source of energy, (2) the ammonia that has been formed from the decomposition of proteins may be reassimilated by the microorganisms which utilize the carbohydrate as a source of energy. These organisms are, therefore, competing with higher plants, for the available nitrogen compounds in the soil. As a result of these studies, Doryland defined ammonification as "an expression of an unbalanced ratio for microorganisms, in which the nitrogen is in excess of the energy-nitrogen ratio." If the available energy material is equal to or is in excess of the energy-nitrogen ratio required by the flora, the coefficient of ammonia formation tends to approach zero; it tends to approach a maximum, if the available energy material is less than the energy-nitrogen ratio. Depending on the proportion of energy material to nitrogenous substances, "beneficial" bacteria may become "harmful." This is brought out in table 45. Bac. subtilis produced, in the absence of glucose, 1 mgm. of NH3 for every 49 mgm. casein decomposed. In the presence of glucose, 1874.1 mgm. of casein was decomposed; this should have produced 38.2 mgm. NH3, whereas only 11.9 mgm. were found. The difference between the actual amount of ammonia present in the glu- cose medium and the amount that would have accumulated, had the glucose been left out, is 26.3 mgm.; this quantity of ammonia must have been assimilated by the bacteria. At the same time, 1934 mgm. of glucose has disappeared or about 13 mgm. of NH3 for every 1 gram of glucose. The amount of nitrogen utilization by Bac. sub- tilis, with casein as a source of nitrogen, was found to be considerably greater than the nitrogen assimilated by this organism from inorganic salts in synthetic media, with glucose as a source of energy. This is due not only to the actually greater assimilation of nitrogen, but also because the organisms had at their disposal the energy that was made 3 Doryland, C. J. T. The influence of energy material upon the relation of soil microorganisms to soluble plant food. N. D. Agr. Exp. Sta. Bui. 116. 1916. ENERGY AND NITROGEN TRANSFORMATION 507 available from that part of the casein which has undergone de- composition.4 The various other bacteria behaved in a similar manner, differing, however, not only in the absolute amount of ammonia liberated, but also in the ratio between the glucose consumed, casein decomposed and ammonia liberated. Bac. mycoides liberated not only the largest absolute amount of ammonia, but consumed a smaller amount of TABLE 45 Influence of glucose on ammonia accumulation from casein in 400 cc. of synthetic solution in six days at room temperature ORGANISM B. subtilis B. proteus B. mycoides B .megatherium. . . B. vulgatus Sarcina lutea. . . . Casein Casein + glucose Casein Casein + glucose Casein Casein -f glucose Casein Casein -f- glucose Casein Casein + glucose Casein Casein + glucose NHa ACCUMU- LATED DIFFER- ENCE DUE TO GLU- COSE CASEIN CON- SUMED GLU- COSE CON- SUMED Total Per 1 mgm.of NHi mgm. mgm. mgm. mgm. mgm. 43.0 2109.1 49.0 11.9 31.1 1874. 1 1934 13.6 2054.4 151.0 2.6 11.0 1716.4 1771 64.9 1459.0 22.4 14.3 50.6 1001.0 1885 20.8 2055.2 98.8 10.5 10.3 2086.5 1864 32.0 1734.9 54.2 16.7 15.3 1773.1 1988 28.4 829.5 29.2 9.2 19.2 900.0 1565 BAC- TERIA millions 28.8 42.6 62.4 67.7 50.2 61.0 57.8 87.9 91.4 111.2 14.7 15.6 casein for a unit of ammonia liberated. Glucose brought about in all cases an increase in the numbers of bacteria, but a decrease in the amount of casein decomposed. 4 It can be easily demonstrated in the case of fungi that the nitrogen content of the organism is higher when the energy is obtained from proteins than when it is obtained from carbohydrates. Further information on the influence of the carbon source on nitrogen utilization by Bac. subtilis is found in a paper by Aubel: E. A. Aubel. Influence de la nature de l'aliment carbone sur l'utilisation de l'azote par le Bacillus subtilis. Compt. Rend. Acad. Sci., 171: 478^179. 1920. 508 PRINCIPLES OF SOIL MICROBIOLOGY A small amount of sugar (0.05 per cent) may even have a stimu- lating effect on the formation of ammonia from casein by causing an increase in the numbers of bacteria. After the sugar has all been decomposed, the increased numbers of bacteria will bring about a greater consumption of energy and, therefore, a greater decomposition of the protein and liberation of ammonia. A change from a depressing to a stimulating effect by the addition of a small amount of available carbohydrate upon the accumulation of ammonia from dried blood is TABLE 46 Influence of various concentrations of glucose on the formation of ammonia from casein2 INCUBA- TION c * C + 0.05 PER CENT G C + 0.1 PER CENT G C + 0.2 PER CENT G NHs Baot. NH3 Bact. NH, Bact. NH, Bact. days mgm. mill. mgm. ■mill. mgm. mill. mgm. mill. B. subtilis 1 2 4 6 15.7 25.1 49.5 2.9 11.4 49.0 13.0 20.0 55.0 4.6 22.8 54.7 12.0 15.9 48.7 4.9 30.9 54.3 13.2 13.0 17.0 4.0 33.0 59.0 B. proteus .... < 2 4 6 13.8 20.2 30.3 5.1 21.1 86.6 10.9 22.0 32.0 6.0 39.2 80.1 11.8 13.7 30.1 6.8 48.3 89.1 10.9 15.0 15.7 6.9 50.1 92.2 *"-"-■■{ 2 4 6 22.6 64.0 69.6 4.7 25.9 63.2 14.8 20.1 60.0 4.4 32.1 65.9 13.9 14.4 52.0 3.8 33.0 66.7 14.0 15.0 11.9 4.8 37.0 66.1 Sarcina lutea. . . A 2 4 6 12.9 22.4 26.1 1.9 5.9 7.9 11.2 10.2 20.0 2.0 7.6 19.0 11.9 11.9 11.3 2.8 6.1 10.7 10.8 12.0 11.0 3.1 7.4 8.9 C = casein, G = glucose; Bact. = bacteria in millions. illustrated in fig. 30. In the presence of undecomposed organic matter, the soluble nitrogen salts are transformed into insoluble proteins; these compounds will be decomposed later and make the nitrogen compounds available again.5 Since the formation of ammonia is a prerequisite to nitrate formation, one would expect from these results that an excess of available energy 8 Gerlach and Deusch. Uber den Einflusz organischer Substanzen auf die Umsetzung und Wirkung stickstoffhaltiger Verbindungen. Mitt. Kais. Wilh. Inst. Landw. Bromberg, 4: 259. 1912; (Centrbl. Bakt. II, 37: 296. 1913). ENERGY AND NITROGEN TRANSFORMATION 509 would repress nitrate formation in the soil. Nitrification was found6 to be checked when the carbon-nitrogen ratio in the soil is 13-15 to 1, but not when the ratio is 11-11.6 to 1. When molasses was added to the soil, nitrification was stopped when the ratio was about 11:1, but was not injured when the ratio was less. However, the addition of carbon sources not readily available, such as butyric acid and alcohol, did not injure nitrification at a ratio of 14:1, but did injure it at a higher ratio. This phenomenon is brought out clearly when cellulose is added to the soil. The organisms using the cellulose as a source of energy assimilate the soil nitrates, without injuring, however, the activities So o u o K) u "30 Z < < Number or ZiAys at "Room Te m vehatu'f.e / 2 3 4 'S lo 7 1 r' 1 1 1 1 1 j Dw iedBlood Useltd= iSS M$S- M. No 2)exmos,£. ,, ,, ,, T 4^,5- ,, .■> 3o ,, >> Fig. 30. Influence of glucose on ammonia accumulation from dried blood added to soil (after Lipman and associates). of the nitrifying bacteria. Only after all the celluloses have decom- posed, do nitrates begin to accumulate again, as shown in fig. 31. As long as there is free available energy, in excess of the available nutrients, there will be only a minimum accumulation of available plant food. When the energy approaches exhaustion the nutrients begin to accumulate, as shown in table 47. 7 6 Clark, H. W., and Adams, G. O. The influence of carbon upon nitrification. Jour. lnd. Engin. Chem., 4: 272-274. 1912. 7 Waksman, S. A. The influence of available carbohydrates upon ammonia accumulation by microorganisms. Jour. Amer. Chem. Soc, 39: 1503-1512 1917. 510 PRINCIPLES OP SOIL MICROBIOLOGY In sufficient time, however, there is a narrowing of the energy-nu- trient ratio in the soil organic matter, and an ultimate liberation of plant food takes place. The death of large numbers of microorganisms with a low energy-nutrient ratio leads to the same end. Succeeding generations of microorganisms have at their disposal the energy ma- terial of the decomposition products from the original organic matter, the dead cells of microorganisms and the original compounds which decompose only very slowly; all of these have a narrower energy-nu- Nitrale - nitrogen Mgm. Days Fig. 31. Influence of cellulose on nitrate accumulation in the soil. cellulose; — — — ■ nitrate nitrogen. 8-1 and 10-2 are the experiment numbers (from Anderson). trient ratio than the original organic matter added to the soil. A gradual accumulation of plant food takes place, which the microorgan- isms are unable to assimilate and which is left for the use of higher plants. Decomposition of organic substances of varying carbon-nitrogen ratio. The nature and composition of the organic matter greatly influence its decomposition. The ratio between the carbon and nitrogen of the material used is of especial importance in this connection. The same is true of the nature of the non-nitrogenous organic materials introduced into the soil in addition to the nitrogenous substances. ENERGY AND NITROGEN TRANSFORMATION 511 Table 48 has been compiled from the results of Lipman and associates,8 who added different organic nitrogenous materials to 100 gram por- tions of soil; the moisture content was brought to an optimum and the soils incubated for 7 days, when the ammonia was determined by TABLE 47 Influence of concentration of sugar upon the accumulation of ammonia from 2% peptone solution by Asp. niger INCUBATION SUGAR ADDED NHj-N in 100 cc. WEIGHT OP MYCELIUM SUGAR LEFT IN MEDIUM days per cent mgm. mgm. 5 0 44.80 200 0 5 1 40.74 280 + 5 3 14.14 1,304 ++ 5 5 1.26 1,500 +-f-+ 5 20 0 1,620 ++ + + 15 0 73.08 360 0 15 1 50.68 930 0 15 3 36.54 3,270 0 15 5 33.04 5,220 + 15 20 0 11,210 ++ TABLE 48 Influence of carbohydrates upon the accumulation of ammonia from nitrogenous organic materials TOTAL NITROGEN IN MATERIAL AMMONIA FORMED, MILLIGRAMS No carbo- hydrate Glucose Sucrose Starch 4 grams Rice flour mgm. 46.4 51.2 94.8 156.8 247.0 245.6 246.1 1.26 1.18 5.14 50.88 110.69 12964 123.63 2 grams 1.30 1.30 3.66 31.71 96 01 108.03 99.67 2 grams 1.48 1.04 5.84 28.57 60.73 94.88 97.23 t grams 0.87 Corn meal 0.69 Wheat flour 1.56 Cowpea meal 23.70 Linseed meal 63.34 Soybean meal 54.36 Cottonseed meal 54.54 distilling with MgO. Rice flour and corn meal, with a wide carbon- nitrogen ratio allowed no accumulation of ammonia at all either with or 8 Lipman, J. G., Blair, A. W., Owen, I. L., and McLean, H. C. Experiments on ammonia formation in the presence of carbohydrates and other non-nitroge- nous organic matter. N. J. Agr. Exp. Sta. Bui. 247. 1912. 512 PRINCIPLES OF SOIL MICROBIOLOGY without any additional carbohydrate. The substances rich in nitro- gen allowed an accumulation of almost 50 per cent of the nitrogen as ammonia, but this was considerably reduced when additional available energy in the form of carbohydrates was added. Table 49 illustrates the formation of ammonia from different organic materials when sufficient non-nitrogenous organic matter (starch) is added so as to introduce the same amount of fresh undecomposed organic matter. In these studies, the various amounts of the organic materials were added to 100 gram portions of soil and the ammonia determined after 9 days.9 When the accumulation of ammonia from the various nitrogenous substances is compared, it is found to be, with the exception of casein, in direct relation to the nitrogen content of TABLE 49 Per cent of organic nitrogen transformed into ammonia in soil SOURCE OF NITROGEN Casein Dried blood Soybean cake . . . Cottonseed meal Linseed meal . . . NITROGEN CONTENT per cent 12.40 13.29 8.28 5.10 5.00 1 GRAM OF EACH ORGANIC MATERIAL ADDED TO 100 GRAMS SOIL NHs-N mgm. 50.2 42.4 40.9 27.1 26.0 132.9 MGM. DF ORGANIC NITROGEN ADDED TO 100 GRAMS SOIL Aerobic Anaerobic conditions conditions NHs-N NH,-N mgm. mgm. 56.9 53.2 49.3 12.3 48.7 14.0 32.0 8.5 34.6 6.9 132.9 MGM. ORGANIC NITRO- GEN PLUS ENOUGH STARCH TO MAKE EQUIVALENT AMOUNTS OF CARBON NH3-N mgm. 31.4 18.9 34.1 34.0 34.1 the materials. When the same amount of nitrogen is added to the soil, the amount of ammonia accumulated depends upon the concen- tration of carbonaceous substances present. These may serve as sources of energy for the microorganisms, so that less of the protein is decomposed and more of the nitrogen used up by the organisms for the synthesis of their protoplasm. When enough starch is added to make the amount of carbon equal in all cases, the amount of ammonia accumulated will generally be constant. When the amount of nitrogen is very high as in the case of dried blood, the amount of starch added was very large; since this is a very readily available source of carbon, 9 Kelley, W. P. The biochemical decomposition of nitrogenous substances in soils. Hawaii Agr. Exp. Sta. Bui. 39. 1915. ENERGY AND NITROGEN TRANSFORMATION 513 its rapid decomposition is accompanied by a greater disappearance of the ammonia nitrogen. Casein contains more nitrogen in comparison to the carbon; therefore, the amount of nitrogen liberated as the waste product (ammonia) from the casein will be greater. When sufficient carbon is added to the casein, the amount of ammonia is the same as that of the cottonseed meal and linseed meal. The influence of the carbon content of the nitrogenous material itself upon ammonia formation can be readily studied with simple amino acids. According to Jodidi,10 the formation of ammonia from various amino acids differs with the composition of the amino-acid molecule. In the case of glycocoll, 80 per cent of the nitrogen was transformed into ammonia, while in the case of leucine only 49 per cent nitrogen was changed to ammonia, under the same conditions. This difference was ascribed to the inert paraffin character of the com- paratively long chain of the leucine molecule. However, the results presented11 in table 50 show that the greater the carbon content of the acid the more abundant is the growth of the organism and the less is the relative amount of nitrogen liberated as ammonia, i.e., the ammonia liberated from the decomposition of a definite amount of amino acid does not depend upon the absolute amount of nitrogen of the material decomposed, but upon the relative carbon-nitrogen content. The lower the ratio of carbon to nitrogen, the greater is the amount of ammonia liberated per unit of material decomposed. Different groups of microorganisms as affecting the carbon-nitrogen ratio in the medium. Fungi, as a rule, can readily obtain their energy from carbohydrates, using inorganic salts as sources of nitrogen, as well as from proteins and may even prefer the former. Actinomyces and heterotrophic bacteria, however, prefer as sources of energy proteins, peptones and certain peptides to carbohydrates, especially to the polysac- charides. This accounts for the difference in behavior of these organisms towards various organic compounds added to the soil. When ground alfalfa, which contains about 2.5 to 3.0 per cent nitro- gen, is added to the soil, sterilized and inoculated with fungi, only a slight accumulation of ammonia takes place, while the alfalfa is rapidly decomposed as indicated by the abundant C02 production. This is due to the fact that the fungi produce an abundant growth and use as much as 50 to 60 per cent of the carbon for structural purposes. Since the fungi contain about 4 to 5 per cent nitrogen in 10 Jodidi, 1912 (p. 501). 11 Waksman and Lomanitz, 1925 (p. 379). 514 PRINCIPLES OF SOIL MICROBIOLOGY their protoplasm (3 to 9 per cent as extremes), the nitrogen made available from the decomposition of the alfalfa may be just sufficient for the synthesis of the fungus protoplasm, without any excess left as ammonia. When the same amount of alfalfa (0.5 to 2 per cent) is added to soil, sterilized and then inoculated with actinomyces or bacteria, some ammonia will be readily formed. This is due to the fact that these organisms synthesize a considerably smaller amount of protoplasm than do the fungi. The nitrogen content of those organisms is higher than that of fungi, viz., 7 to 12 per cent, but because of the considerably lower carbon assimilation, a great deal of the nitrogen is liberated as ammonia. In a series of detailed studies on the decomposition of organic matter by pure cultures of microorganisms, Neller12 (figs. 32-33) found TABLE 50 Influence of composition of amino acid upon ammonia productionby microorganisms AMINO ACID Glycocoll Glycocoll Alanine Alanine Glutamic acid Glutamic acid Glutamic acid C/N 1.7 1.7 2.57 2.57 4.28 4.28 4.28 ORGANISM Trichoderma Actinomyces Trichoderma Actinomyces Trichoderma Actinomyces Bad. fluorescens DRT GROWTH OP CELLS NHj-N mgm. mgm. 50 24.28 59 30.46 80 21.98 126 39.17 218 29.12 169 28.36 128 28.50 NH.-N 2.0 2.0 3 6 3.2 7.5 5.9 4.5 that, under sterile conditions, the fungi bring about a much greater evolution of C02 than bacteria, the action of the pure cultures of fungi approaching that of the complex mixture of organisms found in a soil suspension. The amount of ammonia liberated by the fungi, particu- larly by the rapidly growing forms (Asp. niger), was negligible in com- parison with that liberated by the bacteria. In 12 days Trichoderma and Asp. niger liberated, from 1 per cent alfalfa, about 21 per cent of the carbon as C02 and a mere trace of the nitrogen as ammonia. Bac. subtilis liberated, in 8 days, from 2 per cent alfalfa, only 8.9 per cent of the carbon as C02, but 10.4 per cent of the nitrogen was 12 Neller, J. R. Studies on the correlation between the production of carbon dioxide and the accumulation of ammonia by soil organisms. Soil Soi., 5: 225- 241. 1918. ENERGY AND NITROGEN TRANSFORMATION 515 changed to ammonia. This tends to demonstrate that, with a sub- stance that has a C:N ratio of 16 (alfalfa meal), fungi require all the nitrogen for synthetic purposes while bacteria can liberate, as am- monia, an amount of nitrogen equivalent to the amount of carbon decomposed. Since fungi produced as great an amount of C02 as the complex soil suspension, Neller suggested that the fungi can be looked upon as organisms active in normal soil. However, if fungi were the predominating or only organisms in the soil, this state of affairs could hardly be considered beneficial to higher plants. The rapid decom- position of the organic matter by the fungi with a lack of ammonia 1 — Soil infusion 2 — Trlchoderma 6p. 3--Aspergill.ua nlg«r gatherium subtil le DAYS Fio. 32. Rate of decomposition of alfalfa meal by pure cultures of micro- organisms and by the mixed soil flora, as indicated by the daily evolution of C02 (from Neller). accumulation is true only of substances with a relatively wide carbon- nitrogen ratio, such as alfalfa meal; these substances are usually acted upon first of all by fungi and only later by bacteria. The fungi rapidly break down the complex carbohydrates and cause a narrowing of the carbon-nitrogen ratio. This leads to an abundant liberation of C02 but not of ammonia, since the proteins are decomposed to a limited extent and all the ammonia is reassimilated. The synthesized fun- gus mycelium is richer in nitrogen than the alfalfa and the fungi are efficient utilizers of the available energy. The bacteria first of all break down the proteins and liberate large quantities of ammonia as a waste product. Due to the limited utilization of the celluloses and to 516 PRINCIPLES OF SOIL MICROBIOLOGY the low nitrogen content of the bacteria, only a small amount of the ammonia is reassimilated. This is the reason why bacteria produce small amounts of C02 while considerable quantities of ammonia may accumulate. The amount of ammonia and of nitrate produced and accumulated in the soil will, therefore, depend upon the carbon- nitrogen ratio of the organic matter added. The influence of micro- organisms upon the carbon-nitrogen ratio of the soil itself is discussed elsewhere (p. 702). Influence of straw and plant residues upon the growth of cultivated plants. Higher cultivated plants may have to compete with micro- organisms for the available plant food, especially the nitrogen. When MGM. OF NH 3 „ -- 1-- 1— B. subtilla 3— B. megatherium 3- — Trichoderma 6p. — 1 rAYS Fig. 33. Rate of decomposition of alfalfa meal by pure cultures of micro- organisms and by the mixed soil flora, as indicated by the accumulation of ammonia (from Neller). large amounts of green manure or straw are plowed under and a crop is planted soon afterwards, distinct injury to the crop may set in. This injury is largely a phenomenon of nitrogen starvation. Kriiger and Schneidewind13 submitted definite evidence that the addition of cellu- lose to the soil stimulates the development of various soil organisms which reduce the soil nitrates and prevent the plants from obtaining sufficient nitrogen for their growth. The fact that leguminous plants 13 Kriiger, W., and Schneidewind, \V. Ursache und Bedeutung der Salpeterzer- setzung im Boden. Landw. Jahrb., 23: 217-252. 1899; Zersetzungen und Um- setzungen von Stickstoffverbindungen im Boden durch niedere Organismen und ihr Einfluss auf das Wachstum der Pflanzen. Landw. Jahrb., 39 : 633-64S. 1901. ENERGY AND NITROGEN TRANSFORMATION 517 grew readily in soil in the presence of materials rich in cellulose, since they are independent of the soil nitrogen, and the fact that no ill effects were observed in partially sterilized soil served to prove the above theory. Pfeiffer and Lemmermann14 pointed out that the injurious effect of straw upon plant growth was due not to denitrification but to the development of organisms which assimilated the nitrates and used the nitrogen for the synthesis of microbial protoplasm. It was later found15 that substances like hay and sugar caused a harmful effect the first year and a beneficial effect the second year; i.e. the applica- tion of materials rich in carbohydrate depressed crop growth when applied just previous to planting but stimulated crop growth when a considerable period elapsed.16 According to Rahn,17 easily assimilable carbon compounds are present in the soil only in minute quantities; ammonia and nitrate can, therefore, accumulate as a result of a gradual decomposition of the soil organic matter. The addition of straw and other plant substances rich in available carbon (energy) brings about an increase in the development of microorganisms as a result of which a nitrogen minimum may occur. This will last as long as easily de- composable carbon compounds are still left. The nitrogen minimum appears more quickly and lasts longer in nitrogen-poor than in nitrogen-rich soils. During this condition, the plants cannot obtain any nitrogen from the soil. The addition of available nitrogen overcomes the harmful results. The depressing effects upon plant growth follow- ing the application of straw has been reported by a number of other investigators.18 The results of Kellerman and Wright19 on the nutri- 14 Pfeiffer, Th., and Lemmermann, O. Denitrifikation und Stallmistwirkung. Landw. Ver. Stat., 54: 386-462. 1900. 15 Bredemann, G. Untersuchungen iiber das Bakterien-Impfpraparat "Heyl's Concentrated Nitrogen Producer." Landw. Jahrb., 43: 669-694. 1912. 16 Hutchinson, H. B. The influence of plant residues on nitrogen fixation and on losses of nitrate in the soil. Jour. Agr. Sci., 9: 92-111. 1918. 17 Rahn, O. Die schadliche Wirkung der Strohdiingung und desen Verhutung. Zeitschr. Tech. Biol., 7: 172-186. 1919. 18 Albrecht, W. A. Nitrate accumulation under straw mulch. Soil Sci., 14: 299-305. 1922; 20: 253-265; Hill, H. H. The effect of green manuring on soil nitrates under greenhouse conditions. Virginia Agr. Exp. Sta. Tech. Bui. 6. 1915; Murray, T. J. The effect of straw on the biological soil processes. Soil Sci., 12: 233-260. 1921; Scott, H. The influence of wheat straw on the accumu- lation of nitrate in the soil. Jour. Amer. Soc. Agron., 13: 233-258. 1921; Martin, T. L. Effect of straw on accumulation of nitrates and crop growth. Soil Sci., 20: 159-164. 1925. 19 Kellerman, K. F., and Wright, R. C. Relation of bacterial transformations 518 PRINCIPLES OF SOIL MICROBIOLOGY tion of seedlings and the results of Barthel and Bengtsson20 on the influence of manure upon cellulose decomposition definitely indicate that we are not dealing here with an injury of the process of nitrifica- tion but with the actual assimilation of the nitrate by the soil fungi and bacteria that use the celluloses as sources of energy. Collison and Conn21 concluded that two separate harmful factors are associated with the influence of straw and other plant residues upon plant growth : (1) a toxic chemical agent which acts upon the plants immediately after germination, the effect not being pronounced in soils; (2) a bio- logical factor as a result of the competition between soil microorganisms and plants for the available nitrogen. The results of Viljoen and Fred22 on the effect of wood and wood pulp upon the growth of oats TABLE 51 Influence of different kinds of wood on the growth of oats and red clover TREATMENT None 1.5 per cent coarse wood 3.0 per cent coarse wood 3.0 per cent fine wood 3.0 per cent wood burned and ash used YIELD OP OATS, AVERAGE grams 26.6 18.3 14.8 21.0 27.5 YIELD OF RED CLOVER, AVERAGE Uninocu- lated gra ms 27.5 26.5 21.5 24.5 60.0 Inocu- lated grains 44.0 37.3 43.0 41.5 56.0 and clover demonstrated definitely that the injurious effects are due to a lack of available nitrogen (table 51). A study of the decomposition of two natural organic materials, varying in nitrogen content, indicates that the decomposition of the material of a lower nitrogen content, like timothy residues, is extended of soil nitrogen to nutrition of citrous plants. Jour. Agr. Res., 2: 101-113. 1914. 20 Barthel, C, and Bengtsson, N. Action of stable manure in the decomposi- tion of cellulose in tilled soil. Soil Sci., 18: 185-200. 1924; Idem., 1926 (p. 448); also Anderson, 1926 (p. 451). 21 Collison, R. C, and Conn, H. J. The effect of straw on plant growth. N. Y. (Geneva) Agr. Exp. Sta. Tech. Bui. 114. 1925. 22 Viljoen, J. A., and Fred, E. B. The effect of different kinds of wood and wood pulp on plant growth. Soil Sci., 17: 199-208. 1924. ENERGY AND NITROGEN TRANSFORMATION 519 over a longer period of time than that of an organic material con- taining more nitrogen, like clover residues. The more rapid decom- position of the latter is accompanied by a more rapid increase in the number of microorganisms concerned in the process followed by a more rapid drop. The removal of nitrate from the soil solution will, therefore, not be as prolonged in the case of clover as in the case of timothy residues; this tends to explain the slight depression of the nitrate content of the soil as a result of addition of clover or other leguminous materials than that following the addition of timothy or other plant residues low in nitrogen.23 23 Wilson, B. D., and Wilson, J. K. An explanation for the relative effects of timothy and clover residues in the soil on nitrate depression. N. Y. (Cornell) Agr. Exp. Sta., Mem. 95. 1925. CHAPTER XX Oxidation Processes in the Soil — Nitrate Formation Oxidation-reduction phenomena. Oxidation-reduction processes have commonly been interpreted in terms of oxygen; "oxidation" designated a process whereby oxygen was added to a substance or hydrogen was removed; "reduction" was applied to reactions involving the removal of oxygen or addition of hydrogen. However, in certain processes of oxidation-reduction no oxygen or hydrogen participate, as in the following reaction: FeCl2 4- CI <=> Fe CI, Clark and associates1 were, therefore, led to consider these processes in the light of addition or withdrawal of electrons. Taking the oxida- tion and reduction of iron, the following reactions may be given: 2 FC+++ + H2 <=± 2 Fe++ + 2 H+ 2 Fe+++ + 20^2 Fe++ + O, Ye+++ + e <=± ¥&++ When the reaction proceeds from left to right, reduction is taking place; when it proceeds from right to left, oxidation is taking place. In the presence of methylene blue, nitrate or any other substance capable of acting as hydrogen acceptors, oxidation may take place even in the absence of atmospheric oxygen; this enables certain bacteria to live anaerobically. Processes of oxidation can thus be considered either as (1) aerobic processes, in which atmospheric oxygen acts as the hydrogen acceptor or the oxidizing agent and (2) anaerobic proc- esses, in which organic or inorganic compounds act as the hydrogen acceptors, both processes resulting in the liberation of energy. In addition to the action of unactivated molecular oxygen as a hydro- gen acceptor, there are at least three ways in which activation of oxygen can occur: (1), by means of iron, which acts as oxygen carrier;2 (2) by 1 Clark, W. M., et al. Studies on oxidation-reduction. Public Health Re- ports, 38: 443, 666, 933, 1669; 39: 381, 804. 1924. 2 Warburg, O. Uber Eisen, den sauerstoffiibertragenden Bestandteil des Atmungsferments. Biochem. Ztschr., 152: 479-194. 1924. 520 OXIDATION PROCESSES IN THE SOIL 521 the action of the sulfhydryl group;3 (3) by substances which are capable of peroxide formation.4 The oxygen liberated in synthetic processes is consumed directly for the liberation of energy as in the formation of amino acids from organic acids and ammonia, which is the first step in protein synthesis by heterotrophic microorganisms : 2 CH3 • CO • COOH + 2 NH3 = 2 CH3 • CH • NH2 ■ COOH + O, 1/6 (C6H1206) + O2 -» C02 + H20 The oxygen in the C02 molecule that has thus become liberated is not derived from free gas but from a molecule capable of reduction by glucose, a phenomenon distinctly different from that involving oxygen occurring in an external medium.5 In considering oxidation as the activation of hydrogen, atomic oxygen will be formed if the hydrogen of water is activated. In the presence of a substance, such as methyl- ene blue or nitrates, which readily absorbs hydrogen, oxidation be- comes possible as a result of the reduction of the active hydrogen. For example, acetic acid bacteria oxidize alcohol in the absence of oxygen but in the presence of methylene blue. CH, CH2OH = CH3COH + 2 H+ 2H+ + O ->■ HjO The role of oxygen consists in binding the hydrogen and its place can be taken by other hydrogen acceptors. The acetaldehyde is changed by the Cannizzaro reaction to alcohol and acetic acid, this process being accompanied by the liberation of energy: 2 CH3COH + H20 -* CH3 • COOH + CH3 • CH2OH The phenomena of oxidation and reduction by microorganisms are brought about in most instances by means of specific enzymes of the oxidase-peroxidase nature on the one hand and reductase or perhydridase nature on the other. The oxidation-reduction intensities of biological systems can be 3 Hopkins. Biochem. Jour., 19: 798. 1925. 4 See Quastel, J. H. Dehydrogenations produced by resting bacteria. IV. A theory of the mechanism of oxidations and reductions in vivo. Biochem. Jour., 20: 166-194. 1926. * Aubel, E., and Wurmser, R. Sur l'utilisation de l'Snergie Iib6r6e par les oxydations. Compt. Rend. Acad. Sci., 179: 848-851. 1924. 522 PRINCIPLES OF SOIL MICROBIOLOGY determined colorimetrically by the use of indigo-sulfonate or other appropriate indicators, or by means of an electrode of a noble metal. By immersing two electrodes in two liquids of different oxidation potential connected with a siphon, a current is formed and oxidation will occur in the solution about one electrode and reduction in the solution about the other. The intensity factor can thus be measured.6 There is a relation between the hydrogen-ion concentration and the oxidation-reduction potential of the cell.7 Oxidation processes in the soil. According to the older conception, oxidation of inorganic and organic compounds in the soil consists in the intake of oxygen and the transformation of the soil compounds into higher oxidized forms. According to the newer conceptions, it consists in the change of a substance from a higher potential to a sub- stance of a lower potential, with the liberation of energy. Although some of the oxidation processes in the soil may be purely chemical in nature or may be carried on by plant roots and their enzymes,8 the predominant role of oxidation in the soil is played by microorganisms. Liebig9 recognized that proper oxidation is essential for the decom- position of plant and animal residues added to the soil. Mulder10 called attention to the fact that oxidation as well as reduction proc- esses take place in the soil at the same time, with a certain equilibrium established between the two. Oxidation processes usually lead to the complete decomposition of organic substances (mineralization) in the soil (so-called "decay"), the oxidation of ammonium salts to nitrites and the latter to nitrates, the oxidation of elementary sulfur and sulfur compounds to sulfates, and the oxidation of hydrogen, methane, and other substances produced by processes of incomplete oxidation or reduction. Oxidation processes may be looked upon as beneficial processes in the soil. Reduction processes may often become harmful, since, with incomplete oxidation, substances may be formed which are directly injurious to plant growth. It is sufficient to indicate that the reduced forms of nitrates (nitrites), of sulfates (sulfites) and of phosphates (phosphites) are toxic to plant growth. 6 Gillespie, K. J. Reduction potentials of bacterial cultures and of water- logged soils. Soil Sci., 9: 199-216. 1920. 7 Needham, J., and Needham, D. M. Hydrogen-ion concentration and the oxidation-reduction potential of the cell interior; a micro-injection study. Proc. Roy. Soc. B, 98: 259-286. 1925. 8 Schreiner, O., and Reed, H. S. The role of oxidation in soil fertility. Bur. Soils, U. S. Dept. Agr. Bui. 56. 1909. 9 Liebig, J. Chemie in ihrer Anwendung auf Agrikultur. 4 Aufi., 1842. 10 Mulder. Die Chemie der Ackerkrume 1. 1863, Tr. by Midler. Berlin. OXIDATION PROCESSES IN THE SOIL 523 Deh^rain and Demoussy11 demonstrated that, in the process of oxidation of organic matter, oxygen is always taken up and carbon dioxide set free. They distinguished between microbial and chemical oxidation. Microbial oxidation is most active at normal temperatures and is favored by increased temperatures, 65° being the maximum; the quantities of oxygen absorbed and C02 produced are found to differ with the soil type, moisture, aeration. The volume of C02 produced is usually smaller than that of oxygen absorbed. Chemical oxidation is low at normal temperatures and increases with temperature eleva- tion, reaching a maximum at 110°. The C02 produced chemically often exceeds the oxygen absorbed. Russell12 found that oxidation increases with an increase in the fertility of the soil. The rate of oxidation increased with temperature, amount of water (up to a certain optimum) and amount of CaC03. Heating of soil to 100°C. or treatment with volatile antiseptics, which were subsequently re- moved, brought about a great increase in the oxygen absorbed. Schreiner and Sullivan13 used a solution of aloin (0.125 per cent) for the study of soil oxidation, the change in color being taken as an index of oxidation. As determined by this method, oxidation in soil was found to be non-enzymatic in nature and was considered a result of interaction between inorganic constituents and certain types of organic matter. The addition of salts of Mn, Fe, Al, Ca and Mg increased this type of oxidation. Factors decreasing oxidation in soils were found to bring about conditions which decrease soil productivity and vice versa. Oxidation of organic matter and the formation of nitrates are among the most important oxidation processes in the soil. The evolution of C02 is usually taken as an index of the first (p. 681). Source of nitrates in the soil. The decomposition of proteins and of other nitrogenous organic substances leads to the formation and often the accumulation of ammonia in the soil. Under favorable conditions, this is rapidly oxidized to nitrites and then to nitrates. Under certain conditions, when the nitrifying bacteria are killed, as 11 Deh£rain, P. P., and Demoussy, E. Sur l'oxydation de la matiere organique du sol. Ann. Agron., 22: 305-337. 1896. 12 Russell, E. J. Oxidation in soils, and its connection with fertility. Jour. Agr. Sci., 1: 261-279. 1905; Darbishire and Russell, 1908 (p. 752). 11 Schreiner, O., and Sullivan, M. X. Studies in soil oxidation. Bur. Soils, U. S. Dept. Agr. Bui. 73. 1910; Schreiner, O., and Reed, H. S. The role of oxidation in soil fertility. Ibid., Bui. 56. 1909. 524 PRINCIPLES OF SOIL MICROBIOLOGY in the partial sterilization of soil, or when conditions do not favor nitrification, as with excessive soil acidity, ammonia may accumulate in the soil. The intense accumulation of nitrates in places where large quantities of organic matter are allowed to decompose was explained by the chemists of the earlier part of the nineteenth century to be a result of chemical processes. Davy14 expressed the opinion that nitrates are formed at the expense of the ammonia of the soil and atmospheric oxygen; Liebig demonstrated that atmospheric nitrogen takes no part at all in the process of nitrification. The oxidation of ammonia to nitrate can be accomplished by chemi- cal means, especially at high temperatures and in the presence of catalysts,15 as in the case of the electrolytic oxidation of ammonia in the presence of copper oxyhydrate.16 Ammonia can be oxidized to nitrate to a limited extent in an atmosphere saturated with ammonia and in the presence of ferric hydrate.17 Ammonia is also oxidized to nitrite by ultra-violet radiation.18 According to Weith and Weber,1' hydrogen peroxide and ammonia react with each other giving rise to nitrous acid. The interaction between ozone and ammonia to give ammonium nitrate has been known for several decades: (NH3)2 + 4(0,) -» NH4N02 + H202 + 4(02) NH4NO2 + H202 -> NH4NO3 + H20 The quantities of nitrite and nitrate formed by chemical agencies are insignificant and of little importance in the soil. In the soil, 14 Davy, H. Elements of agricultural chemistry. 1814, p. 308. 16 Sestini, F. Bildung von salpetriger Saure und Nitrifikation als chemischer Prozess im Kulturboden. Landw. Vers. Sta., 60: 103-112. 1904; Mooser, W. Biologisch-chemische Vorgange im Erdboden. Beitrage zur Stickstofffrage. Landw. Vers. Sta., 75: 53-106. 1911. 18 Traube, W., and Biltz, A. Die Gewinnung von Nitriten und Nitraten durch elektrolytische Oxydation des Ammoniaks bei Gegenwart von Kupferhydroxyd. Ber. deut. chem. Gesell., 39: 166-178. 1906. 17 Russell, E. J., and Smith, N. On the question whether nitrites or nitrates are produced by non-bacterial processes in the soil. Jour. Agr. Sci., 1: 444-453. 1906. 18 Berthelot, D., and Gaudechon, H. La nitrification par les rayons ultra- violets. Compt. Rend. Acad. Sci., 152: 522-524. 1911. 19 Weith, W., and Weber, A. Ueber Bildung von Salpetrigsauren Ammoniak aus Wasser und N. Ber. deut. chem. Gesell., 7: 1745-1749. 1874. OXIDATION PROCESSES IN THE SOIL 525 nitrification is entirely biological in nature, as established by Schloesing and Miintz,20 Warington21 and others. Mechanism of ammonia oxidation. Various reactions have been suggested to explain the mechanism of oxidation of ammonia to nitrite by the nitrite forming bacteria. The following two reactions are most probable : 2 NH3 4- 3 02 -> 2 HN02 + 2 H20 (1) 2 NH3 + 302-> N203 + 3 H20 (2) Godlewski22 found that the ratio between the oxygen consumed in the process and the nitrogen changed from the ammonia to the nitrite 02 stage is 3, or R = — = 3. However, Schloesing23 recorded previously JN2 that this ratio varies from 4.57 to 5.57. The difference between these results may be due to the fact that Schloesing made his determinations in the soil as a medium while Godlewski carried out his studies in solution. In the first instance, some of the oxygen was no doubt con- sumed for other oxidation processes, in addition to nitrate formation. Meyerhof24 used the theoretical value of R = 3 for the calculation, from the amount of oxygen consumed, of the quantity of nitrogen oxidized from ammonia to nitrite. In a careful study of the gaseous exchange in the reaction of oxidation of ammonia to nitrite, Bonazzi25 demonstrated that the average value of R is 2.89 ± 0.08, which is very close to 3.0, thus justifying the above equations. The presence of the carbonate is essential both as a source of C02 (although the organism can also use free C02 present in the atmosphere), and for keeping the medium properly buffered, pre- venting it from becoming acid. Nitrites are formed also in an atmos- 20 Schloesing, Th., and Miintz, A. Sur la nitrification par les ferments or- ganises. Compt. Rend. Acad. Sci., 84: 301. 1877; 85: 1018. 1877; 86: 892. 1878; 89: 891, 1074. 1879. 21 Warington, R. On nitrification. Jour. Chem. Soc, 33: 44-51. 1878; 35: 429-456. 22 Godlewski, K. Uber die Nitrifikation des Ammoniaks und die Kohlen- stoffquellen bei der Ernahrung der nitrifizierenden Fermente. Centrbl. Bakt. II, 2: 458-462. 1896. 23 Schloesing, Th. Sur la nitrification de l'ammoniaque. Compt. Rend., 109: 423-428, 883-887. 1885. 24Meyerhof, 1916 (p. 390). 26 Bonazzi, A. On nitrification. V. The mechanism of ammonia oxidation. Jour. Bact., 8: 343-363. 1923. 526 PRINCIPLES OF SOIL MICROBIOLOGY phere free from C02 (but containing C02 in the medium), although at a slower rate. The cells of the organism are strongly catalytic and are capable of liberating oxygen from a peroxide. Ferric hydrate has a stimulating effect upon the oxidation of ammonia to nitrate.26 Since iron was found in active cultures partly in a ferrous state, Bonazzi suggested that it acts as a carrier of oxygen, thereby hastening the oxidation process; iron fulfills in these cultures the functions of the peroxide, due to its mechanism of auto-oxidation resulting in a com- bination with oxygen, while the cells liberate the oxygen thus bound. Fe" + O -> Fe"' • O Fe"' • O + peroxidase -f NH3 -> HN02 + peroxidase + Fe". In 100 cc. of medium and at 35°, 20 mgm. of nitrogen in the form of ammonium sulfate can be oxidized in 6 hours. Nitrite formation comes to a standstill, when the solution contains about 1.5 to 2.0 per cent N02. The concentration of the substrate which is most favor- able for the oxidation process lies at 0.005 M NH4, while with a tenth molar solution of ammonia oxidation is nil. The more recent theories tend to indicate that the oxidation of am- monia to nitrous acid goes through the hydroxylamine and hyponitrous acid stages.26* /H H /H N(-H + O -> N^-H + H20, \\H M)H \OH Hydroxylamine /H .H /H /H Nf-H + O -» N< , N/ + H20 -* N^-OH M)H ^O ^O X)H /H Jd \OH xOH Mechanism of nitrite oxidation. The oxidation of nitrite to nitrate takes place according to the following reaction: NaN02 + h (02) = NaN03 This was demonstrated to hold true by measuring the nitrite and oxy- gen consumption. With optimum concentration of the nutrients and 28Ashby, 1907 (p. 394). 26a Kluyver, A. J., andDonker, H. J. L. Die Einheit in der Biochemie. Chem. d. Zelle u. Gewebe., 13: 134-190. 1926. OXIDATION PROCESSES IN THE SOIL 527 proper aeration of the culture, the nitrate forming organisms, in liquid culture, may oxidize 4 to 5 grams NaN02 per liter in 24 hours. According to Miyake,27 nitrification in the soil obeys the law of autocatalysis; i.e., the reaction is at first slow, then becomes more rapid and finally comes to a standstill, as a result of accumulation of nitrates. The relation between nitrite oxidation and concentration of substrate is given in figure 18, which shows a rapid increase in oxida- tion with an increase in nitrite concentration up to 0.05 per cent. An optimum is reached with 0. 1 per cent of substrate, with a slow decrease to 0.3 per cent. This is followed by a gradual drop, so that in a 4 per cent solution of nitrite, oxidation is only 26 per cent of the optimum. A detailed discussion of the oxidation process, especially from the point of view of growth and respiration is given elsewhere (p. 388). Nitrate formation from inorganic salts and from organic nitrogenous compounds. Schloesing28 compared the formation of nitrates from various ammonium salts added to the soil and found that the following relative amounts of nitrogen (in milligrams) are nitrified per day: NH4C1 - 3.4, (NHO2SO4 - 9.0, (NH4)2 C03 - 4.0 Ammonium salts of organic acids are also nitrified rapidly.29 It was thought at first that organic matter can be nitrified directly. However, Miintz30 has shown that organic matter has to be decomposed first and ammonia liberated, before nitrates can be formed. Omeli- ansky31 later obtained negative results also for urea, asparagine, methyl- amine, dimethylamine and egg-albumin, so that he concluded that all forms of organic nitrogen have to be transformed first into ammonia be- fore they can be nitrified. The same was found to hold true for calcium cyanamide.32 When the processes of nitrate formation from ammo. 11 Miyake, K. On the nature of ammonification and nitrification. Soil Sci., 2: 481-192. 1916; Miyake, K., and Soma, S. Jour. Biochem. Tokio, 1: 123-9. 1922. 28 Schloesing, 1885 (p. 525). 29 Boullanger, E., and Massol, L. Etudes sur les microbes nitrificateurs. Ann. Inst. Pasteur., 18: 181-196. 1904; Lohnis, F., and Blobel, S. Die Ursachen der Wirkungsunterschiede von Schwefelsaurem Ammoniak und Chilesalpeter. Fuhlings landw. Ztg., 57: 385^02. 1908. 30 Miintz, A. Sur la decomposition des engrais organiques dans le sol. Compt. Rend. Acad. Sci., 110: 1206-1209. 1890. Jl Omelianski, \V. Uber die Nitrifikation des organischen Stickstoffes. Centrbl. Bakt. II, 5: 473^90. 1899; 9: 63-65. 1902. 32 de Grazia, S. Sulla nitrificazione della cianamide di calcio in diversi tipi di terreno. Staz. sper. agr. ital. Modena, 41: 241-257. 1908. 528 PRINCIPLES OF SOIL MICROBIOLOGY nium salts and from amino acids are compared, the latter is found to take place more slowly.33 This is probably due to the fact that the amino-acids have to be changed first to ammonia and also to the fact that some of the nitrogen will be stored away in the microbial cells which use the carbon of the amino compounds as a source of energy. According to Barthel and Bengtsson34 only the ammonia (and the urea, which is rapidly transformed into ammonia) of stable manure is readily nitrified; the other part of the nitrogen, present in the manure in the form of complex proteins, nitrifies only very slowly since it has to be decomposed first by various microorganisms, with the liberation of ammonia. Nitrite bacteria find conditions in stable manure very favorable for their development so long as aeration is favorable and the manure is not saturated with urine.35 Deep layers as well as compact manure contain only few nitrifying bacteria, since conditions are not very favorable for their development, under anaerobic or semi- anaerobic conditions. The nitrite bacteria cannot develop in urine, probably because of the presence of some injurious substances and the high concentration of the soluble organic matter; they develop readily in the solid portion of the manure. The free ammonia, especially in a medium of a high alkalinity, is also injurious to the nitrate-forming bacteria. Influence of reaction on nitrate formation. The optimum reaction for the respiration of the nitrite-forming bacteria was found36 to be at pH 8.4 to 8.8, with limiting reactions at pH 7.6 and 9.3. The optimum reaction for the respiration of the nitrate-forming bacteria was found to be at pH 8.3 to 9.3 and the limits at pH 5.6 and 10.3. The presence of NaHC03, which acts as buffer at pH 8.4, is, therefore, beneficial to the activities of these organisms, as shown in figure 30. This is true, however, only for the respiration of the organisms but not for their growth. The optimum reaction for the growth of the nitrite bacteria is at pH 7.8, with a minimum at pH 7.0 and maximum at pH 8.6; for nitrate 33 Batham, H. N. Nitrification in soils. Soil Sci., 20: 337-351. 1925. 34 Barthel, Chr., and Bengtsson, N. The nitrification of stable manure nitro- gen in cultivated soils. Medd. Centralanst. Forsokov. Jordbruksomradet, No. 211, 1920; also 1926 (p. 448). 36 Niklewski, B. Uber die Bedingungen der Nitrifikation im Stallmist. Centrbl. Bakt. II, 26: 388^42. 1910; Diminution du taux de l'azote dans le fumier sous l'influence des bacte>ies nitrifiantes. Bull. Soc. Chim. biol., 6: 491-500. 1923. "Meyerhof, 1916 (p. 390). OXIDATION PROCESSES IN THE SOIL 529 bacteria the optimum is at 7.1 with limiting reactions at 6.5 and 7.8. The kind of buffer used is of importance.37 According to Gerretsen,38 the limiting acid reaction is at pH 3.9 to 4.5, depending on the origin of the nitrifying bacteria; those isolated from acid soils are better adapted to acid conditions. The limiting alkali reaction was found to be at pH 8.9 to 9.0. Meek and Lipman39 demonstrated that the limiting reactions for the nitrite and nitrate-forming bacteria may- depend on the reaction of the soil from which they were isolated. By gradual adaptation, the organisms can be made to grow at reactions beyond their acid and alkaline range so that nitrate formation was even obtained at pH 13.0. When ammonium sulfate is used as a source of nitrogen for nitrate formation and the reaction of the soil is acid to begin with, there will be an increase in acidity in the absence of sufficient buffer or base, as a result of formation of nitric acid from the oxidation of the ammonia and the accumulation of the residual sulfuric acid. Nitrate accumulation will proceed until the reaction of the soil has reached a pH of about 4.0. The amount of nitrate formed under these conditions depends upon the initial reaction of the soil and its buffer and base content; the higher the buffer and base content of the soil, the larger will be the amount of nitrate formed for a certain change of reaction.40 The continuous use of ammonium sulfate as a fertilizer without the addition of lime will, therefore, lead to a gradual increase in soil acidity. How- ever, nitrates may be found even in very acid soils. This was explained by Hall and associates41 as due to the fact that, under acid conditions, nitrate formation takes place in films surrounding the small isolated particles of CaC03. The addition of CaC03 has, therefore, a decided stimulating effect on nitrate formation, particularly in acid soils.42 "Gaarder and Hagem, 1920-1923 (p. 77). 38 Gerretsen, 1921 (p. 736). 39 Meek, C. S., and Lipman, C. B. The relation of the reaction and salt concentration of the medium to nitrifying bacteria. Jour. Gen. Physiol., 5: 195-204. 1922. 40 Barthel and Bengtsson, 1920 (p. 528); Waksman, S. A. Microbiological analysis of soils as an index of soil fertility. V. Methods for the study of nitrifi- cation. Soil Sci., 15: 241-260. 1923. 41 Hall, A. D., Miller, N. H. J., and Gimingham, C. T. Nitrification in acid soils. Proc. Roy. Soc. B., 80: 196-212. 1908. 42 Fischer, H. Versuche iiber Stickstoffumsetzung in verschiedenen Boden. Landw. Jahrb., 41: 755-822. 1911; Vogel, J. Ammoniak und Salpeterassimila- tion durch Mikroorganismen. Centrbl. Bakt. II, 32: 169-179. 1911; Lemmer- 530 PRINCIPLES OF SOIL MICROBIOLOGY In alkaline soils which are deficient in humus, CaC03 may have the opposite effect since it tends to liberate from ammonium salts free ammonia, which retards nitrification. Lime does not stimulate the activities of the nitrifying bacteria43 so much as it serves as a base for neutralizing the acid formed from the oxidation of the ammonium salt. Nitrate formation takes place readily in peat and muck soils44 if the reaction is not too acid and if the soil is properly inoculated with the organisms. Acid peat soils do not generally offer a favorable medium for the development of the nitrate forming bacteria. When lime is added, these organisms become TABLE 52 Influence of application of lime upon nitrogen content and nitrification of a muck soil NITROGEN CONTENT OF SOIL NITRATE FORMATION REACTION APPLICATION OF LIMB Original soil Incu- bated soil Original soil Incu- bated soil None per cent 1.120 1.203 1.151 1.108 1.054 1.114 1.165 1.133 1.016 1.046 parts per million 28 38 37 35 37 30 35 35 40 35 parts per million 114 142 162 178 205 59 151 130 189 173 pH 3.67 4.15 4.37 5.02 5.46 3.88 4.06 4.70 5.40 6.13 pH 3.56 Different forms of lime, 1 ton . . Different forms of lime, 2 tons.. Different forms of lime, 3 tons.. Different forms of lime, 4 tons.. Fertilizer 3.80 4.24 4.80 5.24 3.48 Fertilizer -f- limestone, 1 ton. . . Fertilizer -J- limestone, 2 tons . . Fertilizer + limestone, 3 tons . . Fertilizer -f- limestone, 4 tons . . 3.85 4.32 4.93 5.44 mann, O., Fischer, H., and Husek, B. fiber den Einfluss verschiedener Basen auf die Umwandlung von Ammoniakstickstoff und Nitratstickstoff. Landw. Vers. Sta., 70: 317-342. 1909; Fred, E. B. A study of the formation of nitrates in various types of Virginia soils. Centrbl. Bakt. II, 39: 455-468. 1913; Mil- ler, F. Tiber den Einflusz des Kalkes auf die Bodenbakterien. Ztschr. Giirungs- physiol., 4: 194-206. 1914; White, J. W. Nitrification in relation to the reaction of the soil. Ann. Rpt. Penn. Agr. Exp. Sta. 1913-14, 70-84. 43 Stephenson, R. E. Nitrification in acid soils. Iowa Agr. Exp. Sta. Res. Bui. 58. 1920; Temple, J. C. Nitrification in acid or non-basic soils. Ga. Agr. Exp. Sta. Bui. 103. 1914. 44 Arnd, Th. Beitrag zur Kenntnis der Mikrobiologie unkultivierter und kultivierter Hochmoore. Centrbl. Bakt. II, 45: 554-574. 1916; Uber die Entstehungsweise sal peter und salpetrigsaurer Salze in Moorboden. Landw. Jahrb., 51: 297-328. 1918. OXIDATION PROCESSES IN THE SOIL 531 very active, leading often to a rapid diminution of available nitrogen,45 as shown in table 52.46 Influence of organic matter upon nitrate formation. It has been pointed out that small amounts of soluble organic matter are found to retard the activities of nitrite and nitrate forming bacteria. Glucose, which is so important for the activities of the majority of microorgan- isms, is injurious in concentrations of 0.025 to 0.05 per cent. In the soil, however, the organisms can stand high concentrations of organic matter.47 Miintz and Laine48 concluded that organic matter or humus in the soil may even be distinctly favorable to the activities of the nitrifying organisms. The greater the organic content of the soil, the more abundant is its bacterial flora and the more rapid will be the process of nitrification taking place. It was later found49 that, even in solution, impure cultures may be favored by soil extracts. According to Barthel,50 the easily soluble organic substances must be mineralized in the soil before nitrate formation takes place, if no in- jurious effect is to occur (figure 38). Difficultly soluble organic sub- stances have little effect on the process. Influence of salts upon nitrate formation. Winogradsky51 observed that ammonium salt had a distinctly injurious action upon nitrate- forming bacteria; this action was found to be due not to the salt itself but to free ammonia.52 At as low a concentration as 0.001 N of am- 45 Arnd, Th. tlber schadliche Stickstoffumsetzungen in Hoehmoorboden als Folge der Wirkung starker Kalkgaben. Landw. Jahrb., 47: 372-442. 1914; 49: 191-213. 1916; Tulin, A. F. The injurious action of high doses of lime on podsol soils, in connection with the nature of the biological processes in them (Russian). Trans. Institute Fertilizers No. 26, 1925. 46 Willis, L. G. Nitrification and acidity in the muck soils of North Carolina. Tech. Bui. 24, N. C. Agr. Exp. Sta. 1923. 47 Stevens, F. L., and Withers, W. A. Studies in soil bacteriology. IV. The inhibition of nitrification by organic matter, compared in soils and in solutions. Centrbl. Bakt. II, 27: 169-186. 1910. 48 Miintz, A., and Laine, E. Role de la matiere organique dans la nitrifica- tion. Compt. Rend. Acad. Sci., 142: 430-435. 1906. 49 Coleman, 1908 (p. 391); Maze— Compt. Rend. Acad. Sci., 152: 1625. 1911; Fremlin— Jour. Hyg., 14: 149. 1914; Wright, 1916 (p. 552); Barthel —Centrbl. Bakt. II, 49: 382. 1919; Greaves and Carter— Jour. Agr. Res., 6: 889. 1916; Makrinow, J— Centrbl. Bakt. II, 24: 415. 1919; Lohnis and Green, 1914 (p. 579). 60 Barthel, C. Die Einwirkung organischer Stoffe auf die Nitrifikation und Denitrifikation im Ackerboden. Ztschr. Garungsphysiol., 4: 11-48. 1914. 61 Winogradsky, 1890 (p. 389). 62Meyerhof, 1916 (p. 390). 532 PRINCIPLES OF SOIL MICROBIOLOGY monia, at a pH 9.5, the injury to nitrate-formation was equal to 70 per cent. Most inorganic alkali salts have only a slightly injurious effect, usually only at a concentration greater than 0.3 N; alkali earths are more injurious, 0.1 N causing an injury of 60 per cent (p. 394). The injurious effect of cations of alkali salts depends to a large extent on the OH concentration. Meek and Lipman53 found that nitrifica- tion takes place in solution in the presence of 10,000 parts per million of NaCl(0.1 N), but not in higher concentrations. Na2S04 was not injurious even in concentrations of 30,000 parts per million (0.42 N). Na2C03 was found to be most injurious.54 However, the nature and amount of the nitrogen source were found to modify the injurious effect of the salt.55 The ratio of the calcium to magnesium is not of great importance to the activities of the nitrate-forming bacteria, but the total concentration of magnesium in solution and its relations to the concentration of the other constituents are of great importance.59 According to Greaves and associates57 chlorides, nitrates, sulfates and carbonates of Na, K, Ca, Mg, Mn and Fe exert a toxic effect upon nitrate-formation in the soil, depending on the specific salt and not on the electro-negative ion. The quantity of a salt which can be applied to a soil without decreasing the nitrate-nitrogen accumulation in the soil varies with the salt. In the soil under investigation, the order of decreasing toxicity was found to be as follows: Na2S04, Na2C03, CaC03, K2S04, K2C03, Fe(N03)3, NaN03, MgS04, Fe2(S04)3, Ca(N03)2, KN03, KC1, Mg(N03)2, MnC03, MnCl2, MnS04, Fe2(C03)3, MgCl2, Mn(N03)2, FeCl3, MgC03, NaCl, CaCl2 and CaS04. Those com- pounds which become toxic in lower concentrations are not necessarily most toxic in higher concentrations, as the toxicity of some salts increases more rapidly than the toxicity of others. The common alkali salts are very toxic, including CaCl2, Na2S04, Na2C03, and the less common Ca(N03)2. All the salts, except Na2S04, Na2C03, CaC03, K2S04, K2C03 and Fe(N03)3, act as stimulants in some concentrations; the amount of stimulation depends on the salt, CaS04 and CaCl2 being most efficient. "Meek and Lipman, 1922 (p. 529). 64 Lipman, C. B. Toxic effects of "alkali salts" in soils on soil bacteria. II. Nitrification. Centrbl. Bakt. II, 33: 305-313. 1912. 66 Kelley, W. P. Nitrification in semi-arid soils. Jour. Agr. Res., 7: 417- 437. 1916. 86 Kelley, W. P. The lime-magnesia ratio: II. The effects of calcium and magnesium carbonates on nitrification. Centrbl. Bakt. II, 42: 577-582. 1914. 57 Greaves, J. B., Carter, E. G., and Goldthorpe, H. C. Influence of salts on the nitric-nitrogen accumulation in the soil. Jour. Agr. Res., 16: 107-135. 1919. OXIDATION PROCESSES IN THE SOIL 533 A marked antagonism was found58 to exist between the anions of Na2C03, Na2S04 and NaCl in respect to nitrification in soils. Com- bination of salts, each of which is toxic, may not only bring about normal nitrification but may even stimulate it. 0.2 per cent NaCl is antagonized by 0.05 per cent Na2S04 or 0.025 per cent Na2C03; 0.35 per cent Na2SC>4 is antagonized by 0.15 per cent NaCl or 0.02 per cent loo- s' a Fig. 34. Influence of dicyanodiamide upon nitrate production in the soil (from Cowie). Na2C03; 0.05 per cent Na2C03 is antagonized by 0.4 per cent Na2SC>4 or 0.2 per cent NaCl. These results, however, need to be interpreted in terms of physiological processes. 68 Lipman, C. B., and Burgess, P. S. Antagonism between anions as affecting soil bacteria. II. Nitrification. Centrbl. Bakt. II, 41: 430-444. 1913; Plant World, 17: 295. 1914. 534 PRINCIPLES OF SOIL MICROBIOLOGY It has been claimed that manganese59 and arsenic60 exert a stimu- lating effect upon nitrate formation. Montanari61 could not confirm this so far as arsenic is concerned. Heavy metals inhibit nitrate for- mation according to their protein-precipitating properties, Hg and Ag salts being most injurious. Copper, zinc, iron and lead may exert a stimulating effect.62 Ashby63 found that, in the presence of iron hydrox- ide, nitrification takes place even in the absence of carbonates; the catalytic effect of iron is very important in the growth and respira- tion of the organisms. The injurious influence of dicyanodiamide upon the activities of the nitrate forming bacteria is illustrated in figure 34. Influence of soil gases upon nitrate formation. A liberal supply of oxygen was found64 to be very favorable to nitrate formation. The mere stirring of the soil was found to greatly stimulate the process; this stimulating effect was looked upon65 as due to better aeration (fig. 35). It is known, however, that nitrate formation takes place as rapidly in compact clay soils as in coarser grained soils, when the available water is the same in both cases.66 This would tend to indi- cate67 that the amount of oxygen necessary for nitrate formation need not be abundant, so long as it is sufficient for the normal respiration of the organisms and the moisture supply is favorable. The optimum 69 Olaru, D. A. Role du manganese en agriculture. Son influence sur quel- ques microbes du sol. Paris. Bailleres. 1920. 60 Greaves, J. E. Some factors influencing ammonification and nitrification in soils. The influence of arsenic. Centrbl. Bakt. II, 39: 542-560. 1913; Biochem. Bui. 3: 2. 1913. 61 Montanari, C. Azione degli elementi oligodinamici sui batteri della nitri- ficazione. II. Staz. sper. agr. ital. Modena, 50: 69-72. 1917. 62 Lipman, C. B., Burgess, P. S. The effects of copper, zinc, iron and lead salts on ammonification and nitrification in soils. Univ. Cal. Publ. Agr. Sci., 1: 127-139. 1914. 63 Ashby, 1907 (p. 394). 64 Warington, R. Lectures on investigations at the Rothamsted Experi- mental Station. Exp. Sta. Rec, 3: 894-903. 1892. 65 King, F. H., and Whitson, A. R. Development and distribution of nitrates and other soluble salts in cultivated soils. Wisconsin Agr. Exp. Sta. Bui. 85. 1901; Bui. 93. 1902. 66 Schloesing, Th., Jr. Contribution a l'etude de la nitrification dans les sols. Compt. Rend. Acad. Sci., 125: 824-827. 1897; Fischer, H. Versuche iiber Stickstoffumsetzung in verschiedenen Boden. Landw. Jahrb., 41: 755-822. 1911. 67 Gainey, P., and Metzler, L. F. Some factors affecting nitrate-nitrogen accumulation in soil. Jour. Agr. Res., 11: 43-64. 1917. OXIDATION PROCESSES IN THE SOIL 535 concentration of oxygen for nitrate formation was found68 to be 35 per cent. Similar observations were made for the influence of CO2 con- centration.69 Some believed that a supply of this gas is very important both for the nitrite and nitrate forming organisms. Owen,70 however, found that C02 (above a certain concentration) has no effect on nitrate formation in the soil. In view of the fact that the C02 is used by the organism for the building up of its cells chemosynthetically, its presence is necessary for growth. But since the organism produces only a limited amount of growth, only small amounts of C02 are required Parts per million of nitrates 700 600 500 300 Percentage of oxygen 10 20 30 40 50 60 70 80 90 roo Fig. 35. Influence of oxygen tension upon nitrate formation in the soil (from Plummer). even for the maximum nitrification. Larger amounts seem to act merely as an inert gas. In general, while only small amounts of C02 are required, an excess of oxygen is essential and a lack of this gas will produce anaerobic conditions which will lead to nitrate reduction until all the nitrates are destroyed. ts Plummer, J. K. Some effects of oxygen and carbon dioxide on nitrification and ammonification in soils. N. Y. (Cornell) Univ. Agr. Exp. Sta. Bui. 384. 1916. "Coleman, 1908 (p. 391). 70 Owen, W. L. Effect of carbonates upon nitrification. Georgia Agr. Exp. Sta. Bui. 81. 1908. 536 PRINCIPLES OF SOIL MICROBIOLOGY Toluol, in strength of 0.1 cc. per 100 grams of soil, and CS2, in strength of less than 1 cc. per 100 grams of soil, do not exert any appreciable effect upon nitrate formation.71 Larger quantities (5 to 10 times) exert a temporary retarding effect; as a matter of fact, when these substances are used for partial sterilization of soil nitrate forming bacteria are killed, and it takes a long time before the soil becomes inoculated again. Nitrate formation in solution and in soil. Stevens and Withers72 were the first to call attention to the fact that nitrate formation in solution inoculated with a certain amount of soil is not the same as i ( 0 re 3 If i 9 liizent <. i ■cent Wat ? to 1 Daturstkrt 0 t >r in Sotf 3 ts vr 0 30 ? c 6 i 0 p 35 ST 9 39 7® 1 s 1 *r 1 -~~. --. / \ / i \ b0 i ■•' \ \ A / \ 1 / v ) / Q.10 y / r 1 r^:: t / i ID — -r, y V ! ^L \ u R, « 10 /* 13 St 26 to M 36 -S* 46 <* #» C8 Cc. Water per 1 00am. Soil Fig. 36. Influence of moisture content upon nitrate production in the soil (from Gainey). nitrate formation in the soil itself. Nitrates are formed in the soil in the upper layers, 90 per cent of the process being carried out in the upper 40 to 50 cm.73 This is due to the need of oxygen for the activities 71 Gainey, P. L. Effect of CS2 and toluol upon nitrification. Centrbl. Bakt. II, 39: 584-595. 1914. 72 Stevens, F. L., and Withers, W. A. Studies in soil bacteriology. I. Nitri- fication in soils and in solutions. Centrbl. Bakt. II, 23: 355-373. 1909; 34: 187-203. 1912. 73 Koch, A. Versuche iiber die Salpeterbildung im Ackerboden. Jour. Landw., 69: 293-315. 1911; MacBeth, I. G., and Smith, N. R. The influence of irrigation and crop production on soil nitrification. Centrbl. Bakt. II, 40: 24-51. 1914. OXIDATION PROCESSES IN THE SOIL 537 of the organisms. Even nitrate formation in solution is greatly stimu- lated by aeration.74 Deherain75 found that when the moisture content of the soil is 5 per cent the process of nitrate formation is very slight but it becomes appreciable with 10 per cent moisture and reaches a maximum with 15 to 20 per cent. Schloesing and Mlintz76 reported that nitrate forma- tion in soil is at a maximum with the highest moisture content which will not saturate the soil. When the soil approaches the saturation APXIL /~t/fr t/u/ve tec „ s" - - N PSj no / / *** *~^v / /' , / ?*t .. ■-13^ S ^ «=*** v«. *c = TT= rr=^ - — " — __ as. ***** >/.»£• r, V; /^, ■- --? ?ro £ OH ill " =:;:=i: =^s^ = — *p .— ■ /' ^ ^ saasS £ So- *h „ Fig. 37. Influence of crop upon nitrate content of the soil (after Lyon and Bizzell). point, the process of nitrate formation is greatly reduced and may disappear completely77 (figure 36). The nature of the nitrogen source was found to be important in this connection; the nitrogen in the 74 Barthel, Chr. Bodenbakteriologische Untersuchungen. Centrbl. Bakt. II, 25: 108-125. 1909. 76 Deherain, 1902 (p. 793). 76 Schloesing, Th., and Mlintz, A. Recherches sur la nitrification. Corapt. Rend. Acad. Sci., 89: 1075. 1879. 77 Traaen, A. E. tlber den Einflusz der Feuchtigkeit auf die Stickstoffum- setzungen im Erdboden. Centrbl. Bakt. II, 46: 119-135. 1916. 538 PRINCIPLES OP SOIL MICROBIOLOGY form of ammonium sulfate and cottonseed meal change into nitrates more rapidly in arid soils, while dried blood and the soil's own nitrogen change into nitrates with greater vigor in humid soils.78 Days o .■3 1000 a 3 "8 o S a 3 Fig. 38. Influence of organic matter upon the formation of nitrate in the soil: a, soil receiving 0.2 per cent ammonium sulfate, — — nitrate N, ammonia N; b, soil receiving 0.25 per cent peptone,—"— nitrate N, ammonia N; c, soil receiving 1 per cent peptone, nitrate nitrogen, ammonia N; d, soil receiving 1 per cent dextrose •••• nitrate N, ammonia N (after Barthel and de Rossi). Abundant nitrate formation and even extensive accumulation may take place in semi-arid soils.79 The amount and application of irriga- 78 Lipman, C. B., Burgess, P. S., and Klein, M. A. Comparison of nitrifying powers of some humid and some arid soils. Jour. Agr. Res., 7: 47-82. 1916. 79 Stewart, R. The intensity of nitrification in arid soils. Centrbl. Bakt. II, 36: 477-490. 1913; Sackett, \V. G. The nitrifying capacity of certain Colo- OXIDATION PROCESSES IN THE SOIL 539 tion water has an appreciable influence upon the process of nitrate formation in arid and semi-arid soils.80 Air drying has a favorable effect upon the formation of nitrates in the soil; this effect is noticeable even after spreading out the soil for twenty-four hours and then remoistening.81 Freezing of the soil in winter was also found to improve the nitrifying power of the soil.82 According to Mtintz and Gaudechon,83 maximum nitrifying activities take place in spring (March 28 to April 25). Conditions which tend to promote nitrate formation in the soil are:84 (1) temperature of 37.5°C, (2) an abundant supply of air (oxygen), (3) proper moisture supply, (4) a favorable reaction (pH greater than 4.6), (5) presence of carbonates or other buffering agents, and (6) absence of large quantities of soluble organic matter in the soil. The nature of the crop grown also influences the nitrate content of the soil, as shown in figures 37 and 38. Further information on the influence of soil treatment upon nitrate formation in the soil and on the correlation between nitrate formation and other microbiological activities in the soil is given elsewhere (p. 715). The formation of nitrates from organic and inorganic nitrogenous fertilizers can be used as an index of the availability of the nitrogen for the growth of higher plants.85 rado soils. Colo. Agr. Exp. Sta. Bui. 193. 1914; Science, 42: 452. 1914; Head- den, W. P. The excessive quantities of nitrates in certain Colorado soils. Jour. Ind. Engin. Chem., 6: 5S6-590. 1914; also Col. Agr. Exp. Sta. Bui. 155, 160, 178, 179, 183, 184, 186, 193; Kelley, 1916 (p. 532) ; Stewart, R., and Greaves, J. E. The production and movement of nitric nitrogen in soil. Centrbl. Bakt. II, 34: 115-147. 1912. 80McBeth and Smith, 1914 (p. 536). 81 Buddin, W. Note on the increased nitrate content of a soil subjected to temporary drying in the laboratory. Jour. Agr. Sci., 6: 452-455. 1914. 82 Lyon, T. L., and Bizzell, J. A. Some relations of certain higher plants to the formation of nitrates in soils. N. Y. (Cornell) Univ. Agr. Exp. Sta. Mem. 1. 1913. 83 Miintz, A., and Gaudechon, H. Le reveil de laterre. Compt. Rend. Acad. Sci., 154: 163-8. 1912. "Owen, 1908 (p. 535). 86 Withers, W. A., and Fraps, G. S. The rate of nitrification of some fertil- izers. Jour. Amer. Chem. Soc, 23: 31S-326. 1901; also Ibid., 28: 213-233. 1906; Lipman, C. B. Some observations on the present status of the subject of the availability of nitrogen in fertilizers. Jour. Ind. Engin. Chem., 9: 189. 1917: Cal. Agr. Exp. Sta. Bui. 260. 1915; Lipman, J. G., Brown, P. E., and Owen, I. L. Relative availability of nitrogenous materials as measured by nitrification. N. J. Agr. Exp. Sta., 31st Ann. Rept., 152-155. 1910. 540 PRINCIPLES OF SOIL MICROBIOLOGY Oxidation of sulfur and other minerals in the soil. The cycle of sulfur transformation in the soil is treated in detail elsewhere (p. 600). It is sufficient to indicate here that, when elementary sulfur is added to the soil, it is oxidized to a limited extent chemically and to a much greater extent biologically. A large number of organisms seem to be capable, in the presence of various organic substances, of oxidizing small amounts of sulfur, with the formation of various compounds. Certain specific groups of bacteria seem to be most active in the proc- ess, since these organisms utilize the sulfur as a source of energy. This is true also of sulfides; the speed of oxidation of these depends on their solubility. Hydrogen sulfide and alkali sulfides are oxidized very readily and rapidly; alkali earth sulfides are oxidized more slowly; while the biological oxidation of iron sulfide (iron pyrites) has not yet been demonstrated. A detailed study of the oxidation and reduction of arsenic compounds by microorganisms has been made by Van Zyl.86 Oxidation of organic compounds in the soil. Attention has been called previously to the fact that various organic compounds are formed in the soil as a result of the activities of microorganisms. These may become toxic to the growth of higher plants unless further oxidized. Conditions favoring oxidation processes stimulate the decomposition of these substances and make conditions in the soil more favorable for the growth of higher plants. Various oxidation processes are also essential for the liberation of a sufficient amount of energy for the activities of microorganisms. The maximum energy is liberated by organic substances only when they are completely oxidized. Oxidation of amino acids and oxidation of purine bases are important soil processes, especially in respect to the formation of ammonia.87 Iron plays an important part as a catalytic agent in the oxidation of various substances. In the auto-oxidation of cysteine, an inter- mediate cysteine-iron complex is formed which is auto-oxidizable; this process is inhibited by HCN.88 86 Van Zyl, J. P. Union S. Africa Dept. Agr. Repts. Div. Vet. Ed. and Res. 9-10: 727-808. 1923 (Exp. Sta. Reed., 82: 178). 87 Dakin, 1922 (p. 483). 88 Warburg, O., and Sakuma, S. Pflug. arch. ges. Physiol., 200: 203. 1923; Sakuma, S. Uber die sogenannte Autooxydation dea Cysteins. Biochem. Ztschr., 142: 6S-78. 1923; Harrison, D. G. The catalytic action of traces of iron on the oxidation of cysteine and glutathione. Biochem. Jour., 18: 1009- 1022. 1924. OXIDATION PROCESSES IN THE SOIL 541 The oxidation of hydrogen, methane and carbon monoxide, resulting from the incomplete decomposition of organic matter is treated in detail elsewhere (p. 403). Oxidation processes, which depend upon the presence of substances which act as hydrogen donators and hydro- gen acceptors are also treated elsewhere (p. 414). CHAPTER XXI Reduction Processes in the Soil- — Nitrate Reduction Reduction processes in the soil. Just as aerobic conditions in the soil favor oxidation processes, so do anaerobic conditions (exclusion of free oxygen) favor processes of reduction. Either organic or inorganic compounds may be formed, as a result of these processes, depending upon the constituents of the medium.1 It is not necessary for the soil to be saturated with water for the conditions to be anaerobic. Wino- gradsky2 demonstrated, by the development of anaerobic nitrogen- fixing bacteria (see p. 783), that when the soil contains water equivalent to only about 40 per cent of its moisture-holding capacity, anaerobic bacteria find conditions favorable for their development up to the surface of the soil. A soil possessing a reducing power will form naphthol-blue from a mixture of para-nitroso-dimethylaniline and a-naphthol but will not readily oxidize easily oxidizable substances, such as aloin.3 Other indicators, like p-nitromalachite green which is reduced to p-amino malachite green,4 can also be used to determine the oxidation-reduction potential of the soil. Reduction phenomena are also characterized by the reduction of inorganic salts rich in oxygen, especially nitrates and sulfates. In the absence of atmospheric oxygen, the organic matter of the soil is broken down with the formation of hydrogen which, in statu nascendi, brings about the reduction of the salts rich in oxygen. Forma- tion of H2S is thus a secondary phenomenon. Under aerobic con- ditions, however, the formation of H2S is primary since it results in the decomposition of proteins. Bad. coli, for example, decomposes glucose 1 Van Wolzogen Ktihr, C. A. Biochemical reduction processes in the soil. Arch. Suikerind, Nederlandsch. Indie, 23: 501-511. 1915. 2 Winogradsky, S. Sur l'etude de l'anaerobiose dans la terre arable. Compt. Rend. Acad. Sci., 179: 861. 1924. 3 Sullivan, M. X. Reduction processes in plant and soil. Science, 39: 95S. 1914. 4 Felton, L. D. A new indicator for testing reducing power of bacteria. Jour. Inf. Dis., 34: 414-419. 1924. 542 REDUCTION PROCESSES IN THE SOIL 543 under anaerobic conditions, with the formation of pyruvic acid and hydrogen.5 C6 Hu 06 = 2 CH3-CO-COOH + 2 H2 + 12 Cal. Nitrates and sulfates can act as the hydrogen acceptors and are reduced to nitrites, sulfides, etc. Reducing conditions in the soil have usually been recognized either by the absence of oxidation or by the presence of specific reducing sub- stances, as ferrous carbonate; soils acquire these conditions when water- logged for a few days. The H2S formed from the reduction of sulfates combines with iron to form insoluble iron sulfide. The ferrous com- pounds themselves act as reducing agents. The very presence of these compounds indicates the intense reducing power of the soil. Bacterial cultures themselves are normally reducing.6 Processes of reduction require sources of energy to enable the organisms to carry on their activities. In most cases, however, these are obtained from various organic compounds.7 The autotrophic bacteria, for example, use the energy obtained by chemosynthetic processes for the reduction of CO2. Various organic compounds may be reduced under anaerobic conditions, especially in connection with oxidation of other substances which result in the liberation of energy. The reducing power of bacteria has commonly been determined by the use of certain organic substances, especially dyes, and of certain inorganic substances, such as nitrates and sulfates, acting as hydrogen acceptors. The hydrogen obtained from the decomposition of organic matter is used by the bacteria for the reduction of the dye8 or the nitrate. In some cases aldehydes or purine bases (hypoxanthine, xanthine, adenine) are required as hydro- gen donators.9 Transformation of nitrates by microorganisms. The disappearance of nitrates in the soil as a result of activities of microorganisms may be 5 Aubel, E., and Salabartan, J. Mecanisme de la production d'hydrogene aux depens du glucose par le bacille coli. Compt. Rend. Acad. Sci., 180: 1183- 1186, 1784-1787. 1925. 8 Rubner, M. Reduktionswirkungen bei Bakterien. Arch. Hyg., 16: 62. 1893. 7 Beijerinck, M. W. Phenomenes de reduction produits par les microbes. Arch. Sci. Ex. Nat. Neerl. (II), 9: 131-157. 1904. 8 Carapelle, E. tlber die Reduktionerscheinungen bei Bakterien. Centrbl. Bakt. I, Orig., 47: 545-559. 1908. 9 Dixon, M., and Thurlow, S. Studies on xanthine oxidase. III. The reduc- tion of nitrates. Biochem. Jour., 18: 989-992. 1924. 544 PRINCIPLES OF SOIL MICROBIOLOGY due to three groups of phenomena: (1) direct utilization of nitrates by microorganisms as sources of nitrogen, in the presence of sufficient energy material, (2) reduction of nitrogen to nitrites and ammonia in the process of the nitrate assimilation, (3) utilization of nitrates as sources of oxygen (nitrates as hydrogen acceptors) . In the last process oxygen is used by the organism for the oxidation of carbon compounds or inor- ganic substances, such as sulfur. The energy thus derived is used for the reduction of the nitrate to the nitrite, free nitrogen gas, oxides of nitrogen or the ammonia stage. The formation of nitrogen gas from nitrate may be so rapid under favorable conditions that the gas can actually serve as a measure of the amount of nitrate reduced. The disappearance of nitrates in the soil due to the various processes of nitrate reduction and nitrate assimilation has often been referred to as "denitrification." However, the reduction of nitrates to nitrites and ammonia as well as their assimilation by microorganisms does not involve any losses of nitrogen, but merely indicates that the nitrates are for the moment taken out of circulation and transferred into forms from which nitrate can be again produced. The nitrates may even completely disappear without involving any loss of nitrogen, as in the case of their assimilation by fungi and various bacteria in the presence of available energy.10,11 The term denitrification (or complete denitri- fication) should designate the complete reduction of nitrates to atmos- pheric nitrogen and oxides of nitrogen, while the other processes involving disappearance of nitrates may be referred to as nitrate reduction and nitrate assimilation. Nitrate assimilation. Large numbers of microorganisms, including bacteria, actinomyces, fungi and algae are capable of utilizing .nitrates as a source of nitrogen. In the presence of a sufficient source of avail- able energy, the microorganisms rapidly assimilate the nitrate nitro- gen and transform it into proteins. The nature of the organism, the amount and nature of energy source, as well as the environmental con- ditions, influence the amount of nitrate thus assimilated. The fungi readily utilize nitrate-nitrogen, although often not to such an extent as ammonia nitrogen. For every 30 to 40 units of carbohy- drate decomposed, certain fungi assimilate one part of nitrogen. The nitrate is usually first reduced to ammonia before it is assimilated. Only certain groups of bacteria (so-called "nitrate" bacteria) are capable of utilizing this source of nitrogen. The actinomyces assimi- 10 Vogel, J. Ammoniak und Salpeterassimilation durch Mikroorganismen. Centrbl. Bakt. 11,^32: 169-179. 1912. "Bierema, 1909 (p. 486). A REDUCTION PROCESSES IN THE SOIL 545 late nitrate readily, but usually reduce it first to nitrites; carbon sources favoring growth also favor nitrate reduction; when nitrite is the sole source of nitrogen, particularly in low concentrations (0.01 to 0.05 per cent), it is assimilated very readily as such, but when present in the nitrate form it is first reduced to nitrites and then assimilated. The reduction of nitrate by microorganisms is usually accompanied by an increase in alkalinity of the medium, due to the fact that the reduced anion is assimilated and the cation is left. The amount of nitrate- nitrogen converted into microbial protoplasm will thus depend upon the nature of the organisms active in the process as well as upon the environ- mental conditions. Seiser and Walz12 demonstrated that a pure culture of a bacterium (Bad. putidum) assimilated, under anaerobic conditions, about ten per cent of the nitrate nitrogen in the medium, but, under aerobic conditions, nearly thirty-three per cent of the nitrogen was assimilated, due to the greater utilization of the energy (citric acid), under aerobic conditions. Utilization of nitrates by microorganisms as sources of oxygen. Certain bacteria are capable of reducing nitrates to nitrites, ammonia, and atmos- pheric nitrogen or oxides of nitrogen. Goppelsroder13 was the first to observe that nitrates are reduced in the soil to nitrites. This was at- tributed by Meusel14 to the action of bacteria. As already mentioned, microorganisms can utilize nitrates as sources of oxygen in the presence of inorganic or organic substances which serve as sources of energy (or as hydrogen donators). In the absence of free oxygen but in the presence of nitrate, various aerobic organisms are capable of existing anaerobically. Some organisms bring about complete denitrification; others reduce the nitrate to the nitrite stage only, with a smaller amount of oxygen becoming thereby available. 2 HN03 = 2 HN02 + 02 (- 36.6 Cal.) (1) If we assume that one molecule of oxygen can liberate 112 Calories, when carbohydrates are used as a source of energy (with complete oxidation to H20 and C02) a net gain in the above reaction is obtained: 112 - 36.6 = 75.4 Cal. 12 Seiser, A., and Walz, L. Stickstoffumsatz bei der Denitrifikation. Arch. Hyg., 95: 189-208. 1925. 13 Goppelsroder, F. Beitrage zum Studium der Salpeterbildungen. Poggens- dorf's Annallen., 115: 125. 1862. ■ 14 Meusel, E. Nitritbildung durch Bakterien. Ber. deut. chem. Gesell., 8: 1214, 1653. 546 PRINCIPLES OF SOIL MICROBIOLOGY When the nitrate is reduced to atmospheric nitrogen, 2 HN02 = N, + 1J O, + H20 (+ 6.8 Cal.) (2) The second reaction gives, therefore, a net gain of 1J X 112 + 6.8 - 174.8 Cal. In the reduction of nitrate to ammonia, the following reaction takes place:16 HNO, + H20 = NH, + 2 02 The more complete the reduction of the nitrate, the more oxygen be- comes available and, therefore, the greater is the amount of carbohy- drate that can be oxidized and the greater is the gain in energy. In some cases two organisms may participate in the same process, as in the decomposition of cellulose; one organism reduces the nitrate in the absence of atmospheric oxygen, and the other decomposes the cellu- lose. The second organism supplies energy to the first and utilizes the oxygen liberated by the first.16 Sulfur and thiosulfate may also be used under anaerobic conditions as a source of energy, with nitrate as a source of oxygen. This was first demonstrated by Beijerinck,17 who assumed that two organisms are concerned in the process, one reduc- ing the nitrate and the other oxidizing the sulfur chemosynthetically. But he18 later demonstrated that only one organism carries out the complete reaction: 6 KN03 + 5S + 2CaC03 = 3 K2S04 + 2 CaSO, + 2C02 + 3 N2 About 1 Calorie is produced per gram of nitrate reduced. When a mix- ture of sulfur (10 per cent), calcium carbonate and KN03 solution (up to 10 per cent) is inoculated with soil, spontaneous and intense gas production takes place, accompanied by slime formation. The gas consists of nitrogen and C02. In the absence of organic matter and with sulfur as the only source of energy, carbon dioxide of the atmosphere is 15 Warburg, O., and Negelein, E. tlber die Reduktion der Salpetersaure in griinen Zellen. Biochem. Ztschr., 110: 66-115. 1920. 16 Groenewege, 1920 (p. 436). 17 Beijerinck, 1904 (p. 84). 18 Beijerinck, M. W., and Minkman, D. C. J. Bildung und Verbrauch von Si ickstoffoxydul durch Bakterien. Centrbl. Bakt. II, 25: 30-63. 1910. REDUCTION PROCESSES IN THE SOIL 547 utilized for the synthesis of the microbial protoplasm. The soil was believed to act as a catalyzer which hastens the process since, on con- secutive transfer, the activities of the organism are weakened. According to Beijerinck19 the organisms concerned in the process may occur in two physiologically different modifications, which are heredi- tarily constant when the feeding conditions remain unchanged. One, an autotrophic form which is adapted to inorganic media (sulfur- or thiosulfate-carbonate-nitrate) and which shows chemosynthesis; the other, an heterotrophic form, requires organic food. The heterotrophic forms preserve the power of denitrification with organic food. Trautwein20 also demonstrated that some soil organisms are capable of oxidizing thiosulfate under aerobic conditions in the absence of nitrate; TABLE 53 Influence of nitrate upon the decomposition of sucrose in (4 per cent) nutrient bouillon, under anaerobic conditions23 Volume of gas produced C02 per 100 of gas H2 per 100 NO per 100 N20 per 100 N2 per 100 BACT. LACTIS AEROGENES Nitrate present 628.4 57.6 0 6.7 2.3 23.4 Nitrate absent 1715.15 64.7 33.4 0 0 PNEUMOBACILLUS OP FHIEDLANDER Nitrate present 548.9 69.3 0 1.2 0 29.5 Nitrate absent 1118.9 62.9 35.0 0 0 growth and autotrophic respiration took place anaerobically only when nitrate was present as a source of oxygen. Nitrate reduction can be brought about readily by a number of soil bacteria, under anaerobic conditions, when carbon sources are available as source of energy.21 Nitrates enable many facultative anaerobes to develop under anaerobic conditions, using sources of carbon which could otherwise not be utilized.22 19 Beijerinck, M. W. Chemosynthesis at denitrification with sulfur as source of energy. K. Acad. Wetenschappen. Amsterdam, 22: Nos. 9 and 10. 1920. 20 Trautwein, 1924 (p. 88). 21 Van Iterson, G. Anhiiufungsversuche mit denitrifizierenden Bakterien. Centrbl. Bakt. II, 12: 106-115. 1904. 22 Ritter, G. Beitrage zur Physiologie der fakultativ anaeroben Bakterien. Centrbl. Bakt. II, 20: 21-38. 1908. 548 PRINCIPLES OF SOIL MICROBIOLOGY According to MazeV3 nitrate reduction is caused by the hydrogen produced by anaerobic bacteria; however, not all hydrogen-forming organisms are capable of reducing nitrate, as in the case of butyric acid bacteria. Table 53 illustrates the role of nitrate in the decomposition of carbohydrate (4 per cent sucrose) under anaerobic conditions and in a nutrient bouillon; a much greater decomposition of the sugar and an abundant formation of hydrogen in the absence of nitrate points to a distinct difference in the mechanism of the decomposition of the substrate. Reduction of nitrates to gaseous nitrogen and oxides of nitrogen. The reduction of nitrates to atmospheric nitrogen always goes through the nitrite stage. The following reaction was at first suggested24 to explain the complete reduction of the nitrate molecule : 5 C6H1206 + 24 KN03 = 24 KHCO3 + 6 C02 + 18 H20 + 12 N2 The carbohydrates or organic acids of the media are decomposed with the formation of carbon dioxide and nascent hydrogen;25 the nitrate is then used by the organism as the hydrogen acceptor, which results in the reduction of the nitrate. The theories concerning the nitrate reduc- tion current about 1910 illustrate the reactions involved as follows: 2 KNO3 + C = 2 KN02 + C02 4 KN02 + 3C = 2N2 + 2 K2COa + C02 C designates the carbon source. In view of the fact that oxides of nitrogen are always produced in the complete reduction of the nitrate the above reactions had to be modified:26 2 KNO3 + C = 2 KN02 + C02 2 KN02 + C = N20 + KjCOs 2 N20 + C = 2 N2 + C02 An abundant formation of N20 takes place at a high nitrate concentra- tion of the medium and at a high temperature. The above reactions are altogether hypothetical and are not based upon sufficient experL 23Maz6, 1911 (p. 182). "Gayon and Dupetit, 1882 (p. 181); Dehe>ain, P. P., and Maquenne, L. Compt. Rend. Acad. Sci., 95: 691. 1882. 26 Stoklasa, J., and Vitek, E. Beitrage zur Erkenntnis des Einflusses verschiedener Kohlenhydrate und organischer Sauren auf die Metamorphose des Nitrats durch Bakterien. Centrbl. Bakt. II, 14: 102-118, 183-205. 1905. 26 Beijerinck and Minkman, 1910 (p. 546). KlDUCftOtf PROCESSES IN THE SOIL 540 mental evidence. However, the more recent ideas concerning the proc- esses involved in the reduction of nitrates can be best presented as follows:268 OH / /OH N=0 +2H^N{ +H20 \ ^0 O nitrate nitrite /OH 1 N< +2H ^O N— OH il + 2H20 /OH i N— OH N/ +2H ^O J hyponitrite NOH Nv || - ||>0 + H20 NOH N/ Nv N ||>0 + 2H->|||+H20 To explain the formation of hydroxylamine and ammonia, in the reduc- tion of nitrates, by the above theories, the following reactions may be suggested : 2 H2NOH N— OH + 2 HI H N— OH + 2 HJ H2NOH + 2 H -> NH3 + H20 With the exception of the organisms capable of oxidizing sulfur and the Bac. amylobacter group, all denitrifying bacteria reduce nitrate to nitrogen gas and N20, in varying proportions, Bac. nitroxus being par- ticularly active in the process. A 5 to 12 per cent solution of nitrate inoculated with soil gives, at 20° to 37°, a current of gas which is eighty per cent N20. Various other denitrifying bacteria, like Bad. pyocy- aneum and Bad. stutseri, give in solutions of nitrate (particularly NH4NO3) a gas rich in N20. Out of one hundred cultures of bacteria tested by Maassen,27 thirty-one were found capable of reducing nitrate *6a Kluyver and Donker, 1926 (p. 526); Blom, J. Studier over Nitrat, Nitrit og Hydroxylamin. N. J. F. Kongr. Oslo. 1926; Joss, E. J. The action of metals on nitric acid. Jour. Phys. Chem., 30: 1222-1275. 1926. 27 Maassen, 1901 (p. 181). 550 PRINCIPLES OF SOIL MICROBIOLOGY to nitrite; the latter is then reduced to atmospheric nitrogen and NO. This process was rather slow and independent of the oxygen supply. Tacke28 found that thirty-eight per cent of the gas mixture formed dur- ing the process of nitrate reduction by bacteria may consist of N20. The formation of nitric oxide in the reduction of nitrates has also been demonstrated by other investigators.29 The free nitrogen may, of course, be a result of the chemical interac- tion of the oxides of nitrogen with ammonia : NH4N02 = N2 4- 2 H20 Formation of nitrogen gas from organic compounds. To make the study of formation of nitrogen gas in microbiological reactions complete, attention should be called to the possibility of its formation in the decomposition of organic compounds, especially as a result of rapid oxidation of ammonia which is formed from those compounds: 4 NH3 + 3 02 = 6 H20 + 2 N2 2 NH3 4- 3 H202 - 6 H20 + N2 These two processes may play an important part causing a loss of nitro- gen in the decomposition of manure.30 Nitrogen gas may also be formed by the interaction of nitrites with amino compounds ; both of these may be formed in the decomposition of organic matter rich in nitrogen accompanied by the incomplete liberation of ammonia. -CH2-NH2 + HN02 = -CH2-OH 4- H20 + N2 Denitrification in the soil. Denitrification takes place in an entirely different manner in soil than in solution.31 In solution and in the presence 28Tacke, 1888 (p. 184). 29 Lebedeff, A. J. tlber die Bildung des Stickoxyds bei dem durch Bac. hartlebri eingeleiteten Denitrifikationprozess. Ber. deut. bot. Gesell., 29: 327- 329. 1911; Suzuki, S. Uber die Entstehung der Stickoxyde im Denitrifikations- prozess. Centrbl. Bakt. II, 31: 27-49. 1911; see also Acklin, O. Zur Biochemie des Bacterium pyocyaneum. Ein Beitrag zur Frage seines Stoffwechsels und dessen Beziehungen zur intramolecularen Atmung. Biochem. Ztschr., 164: 312-370. 1925. 30Pfeiffer, Th., Franke, F., Gotze, C, and Thurmann, H. Beitriige zur Frage uber die bei den Faulnis stickstoffhaltiger organischer Substanzen ein- tretenden Umsetzungen. Landw. Vers. Sta., 48: 1S9-245. 1897; Street, J. P. A review of the investigations concerning denitrification. N. J. Agr. Exp. Sta. 14th Ann. Rept.: 183-210. 1901; Lohnis, 1910, p. 488 (p. xiii). 31 Koch, A., and Pettit, H. Uber den verschiedenen Verlauf der Denitrifika- tion im Boden und in Fliissigkeiten. Centrbl. Bakt. II, 26: 335-345. 1910. REDUCTION PROCESSES IN THE SOIL 551 of the proper organic substances the bacteria may liberate practically all the nitrogen present in the nitrate form as free nitrogen gas, while in moderately moist soil only protein may be formed out of the nitrate. But if the soil is very moist and nitrates are present, denitrifying bacteria behave as in solution and liberate considerable quantities of free nitrogen gas. The minimum moisture content at which the complete reduction of nitrate may occur may depend upon the nature of the soil. Oelsner32 found that it takes place in soils containing 40 per cent moisture even when no additional carbon has been added. This is especially true of rice fields33 and of peat soils.34 The loss of available nitrogen as a result of liming acid peat soil was ascribed35 to the reduction of nitrate to nitrite and to atmospheric nitrogen. A decrease in aeration leads to an increase in denitrification; cultivation alone could not prevent the loss of nitrogen. The use of disinfectants for the destruction of the deni- trifying bacteria in the soil is often recommended. Denitrification is found to be at an optimum at 27° to 30°. However, it takes place abundantly in the soil even in the coldest seasons of the year when the temperature of the soil at a depth of 0.2 to 0.3 meters is about zero.36 The optimum reaction for denitrification is at pH 7.0 to 8.2; the process is greatly retarded at pH 5.2 to 5.8 and 8.2 to 9.O.37 The denitrifying bacteria (except the forms which obtain their energy from the oxidation of sulfur) require organic matter for their metabolism and are, therefore, favored by an addition of available organic matter. Various mono- and di-basic organic acids (except oxalic) can be utilized as sources of carbon.38 Glucose is one of the best sources of energy. Fresh straw is utilized to a more limited extent and composted straw 3J Oelsner, A. Uber Nitratereduktion in nassem Ackerboden ohne Zusatz von Energiematerial. Centrbl. Bakt. II, 48: 210-221. 1918. 33 Daikuhara, G., and Imaseki, T. Bui. Imp. Centrl. Agr. Exp. Sta. Japan, 1: 7. 1907. 34 Ritter, 1912 (p. 790); see also Lemmermann, O., and Wichers, J. L. Verlauf der Denitrifikation in Boden bei verschiedenem Wassergehalt. Centrbl. Bakt. II, 41: 608-625. 1914. 35Arnd, 1914-1916 (p. 531). 36 Barthel, 1909 (p. 537). 37 Zacharova, T. M. The influence of soil reaction upon denitrification, in connection with liming of soil (Russian). Institute of Fertilizers, Moskau. No. 29. 1925. 38 Jensen, 1897 (p. 180); Salzmann, P. Chemisch-physiologische Untersuchun- gen liber die Lebendauer zweier denitrifizierender Bakterien und der Strepto- thrix chromogena. Diss. Konigsberg. 1902. 552 PRINCIPLES OF SOIL MICROBIOLOGY even less so,39 due probably to a slow availability of the more insoluble constituents, since cellulose is not used to any extent as a source of energy by these organisms.40 A combination of an available source of energy and anaerobic conditions in the soil (either brought about by high moisture content or replacement of oxygen by hydrogen and C02) leads to most active denitrification. An increased nitrate content of a soil favoring conditions of complete denitrification will favor the process further. The addition of large quantities of straw manure and green manure41 has, therefore, an important influence upon the disappearance of nitrates, both through transformation into pro- tein and reduction to nitrites, ammonia and nitrogen gas. It has been found that even difficultly decomposable organic substances may also have a favorable influence upon denitrification in the soil.42 The fact that denitrifying bacteria are favored by an alkaline reac- tion and are injured by acids suggests the use of substances which would make the soil reaction acid. However, the final reaction should not be acid enough so as to injure the activities of useful bacteria, like the nitri- fying organisms. Applications of acid phosphate were found to be useful in the preservation of the manure by neutralizing the ammonia ; this brings about a change in reaction and tends, therefore, to lessen denitrification. Of the various nitrates, salts of alkalies and alkali earths are readily denitrified; the reduction of A1(N03)3 is doubtful.43 Iron, manganese, thorium, ythrium and silver nitrates give negative results, due to the toxic action of the cations. Ethyl nitrate is reduced, but not nitro-methane. The same bacteria that reduce ni- trates were found capable of reducing potassium chlorate, arsenate and potassium ferricyanide. Importance of nitrate reduction in the soil. Gayon and Dupetit and Deherain and Maquenne established in 1882 that there are bacteria in the soil capable of reducing nitrates to atmospheric nitrogen and oxides 39 Caron, H. V. Untersuchungen iiber die Physiologie denitrifizierender Bakterien. Centrbl. Bakt. II, 33: 62-116. 1912. 40 Wright, C. R. The influence of certain organic materials upon the trans- formation of soil nitrogen. Centrbl. Bakt. II, 46: 74-79, 1916; Albrecht, 1922- 1926 (p. 517). 41 Ferguson, M., and Fred, E. B. Denitrification: the effect of fresh and well- rotted manure on plant growth. Va. Agr. Exp. Sta. Rep. 1908, 134-149. 42 Nolte, O. t)ber Denitrifikation bei Gegenwart von schwerzersetzlichen organischen Substanzen. Centrbl. Bakt. II, 49: 182-184. 1919. 43 Ampola, G., and Ulpiani, C. Sull'azione riduttrice dei batteri denitrificanti. Gazz. chim. ital. Rome, 29, pt. 1: 49-72. 1899; 34, pt. 2: 301-315. 1904. REDUCTION PROCESSES IN THE SOIL 553 of nitrogen. Lawes, Gilbert and Warington pointed out the same year that considerable quantities of nitrogen may be given off when a soil receives heavy applications of manure and is saturated with water or is improperly aerated. Breal44 announced in 1892 that many substances of organic origin, especially straw, can serve as sources of energy which would enable the bacteria to liberate atmospheric nitrogen from nitrates. This is seen from table 54, where 2 grams of straw and sodium nitrate were added to 400 cc. of water. Breal emphasized the conclusion that denitrification is not of any importance in normal soils, but may become so in humus-rich forest soils. In 1895 Wagner45 reported that the addi- tion of manure to liquid cultures containing nitrates greatly increased denitrification; this observation led him to the conclusion that the same process takes place in the soil. He found confirmation in this in field experiments where organic nitrogen and nitrates were added simulta- TABLE 54 Influence of straw wpon denitrification in solution Nitrogen content of straw Nitrogen content of nitrate added Total nitrogen added Nitrogen found at end of experiment Nitrogen lost into the atmosphere . . . MILLIGRAMS 9.7 26.0 35.7 27.1 8.6 neously before the crop was planted. Wagner declared, on the basis of these experiments, that denitrification may take place extensively in cultivated soils; the application of manure (cow or horse) to the soil was believed to be often not only unprofitable but even harmful; this was believed to be due to the fact that manure carries microorganisms which destroy the nitrates in the soil, not only added as such, but even those formed by the nitrifying bacteria. These, as well as similar other investigations, created the impression that when nitrates are added to the soil denitrification sets in and may cause an injurious action by causing the transformation of the nitrate into gaseous nitrogen. It was soon found that these results were greatly "Breal. Compt. Rend. Acad. Sci., 114:681-683. 1892; Ann. Agron., 18: 181; 22: 32; Deherain, 1902 (p. 793). 45 Wagner, P. Die geringe Ausnutzung des Stallmiststickstoffs und ihre Ursachen. Deut. landw. Presse, 22: 98. 1895; see also 1897 (p. 185). 554 PRINCIPLES OF SOIL MICROBIOLOGY exaggerated.46 Losses of nitrogen were found possible only when con- siderable amounts of organic matter are added together with the nitrate, but this is not commonly done. Pfeiffer and Lemmermann" demon- strated that very little actual denitrification takes place in the soil as a result of addition of manure. The lack of nitrogen often observed is due to other causes rather than to the actual loss of nitrogen. Nitrate reduction sets in when the soil is saturated with water. Only in the presence of a great abundance of organic manures is there any fear of loss of nitrate-nitrogen from the soil in a gaseous form.48 Great losses of nitrogen may take place in a humid, hot climate;49 the rate of loss was found to be much increased by liming; bare fallows in rainy season were found to be especially wasteful because of the leaching of nitrates in drainage waters. There is little danger from denitrification in normal soils.50 The partial reduction of nitrates to nitrites and ammonia, which is more extensive and carried out by larger numbers of microorganisms does not involve any actual losses of nitro- gen. The nitrates may completely disappear from the medium without any actual loss of nitrogen.51 The products formed from the nitrates (nitrites and ammonia) can be further acted upon by nitrifying bacteria; the part of the nitrate assimilated by microorganisms is merely stored away in the soil in an organic form.52 46 Burri, R., Herfeldt, E., and Stutzer, A. Bakteriologisch-chemische Forsch- ungen fiber die Ursachen der Stickstoffverluste in faulenden organischen Stoffe, insbesondere im Stallmist und in der Jauche. Jour. Landw., 42: 329-384. 1894. 47 Pfeiffer, Th., and Lemmermann, O. tlber Denitrifikationsvorgange. Landw. Vers. Sta., 50: 115-142. 1898; Lemmermann and Wichers, 1914 (p. 551). 48 Stoklasa, J. Treten Stickstoffverluste im Boden ein bei Diingung mit Chilisalpeter? Centrbl. Bakt. II, 17: 27-33. 1906; Fischer, H. Versuche iiber Stickstoffumsetzung in verschiedenen Boden. Landw. Jahrb., 41: 755- 822. 1911. 49 Meggitt, A. A. Studies of an acid soil in Assam. II. Mem. Dept. Agr. India, 7: 31-53. 1923. 60 Voorhees, E. B. Investigations relative to the use of nitrogenous materials. N. J. Agr. Exp. Sta. Rpts., 12: 97-119. 1899; 13: 88-110. 1900; 14: 144-154, 183-211. 1901; 15: 133. 1902; 16: 148. 1903; 17: 191. 1904. 61 Lemmermann, O. Kritische Studien liber Denitrifikationsvorgange. (Hab- ilitationsschrift), Jena. 1900. 62 Gerlach and Vogel. Ueber eiweissbildende Bakterien. Centrbl. Bakt. II, 7: 609-623. 1901; Ueber Nitrifikation und Denitrifikation in der Ackererde. Centrbl. Bakt. II, 13: 706-715. 1904; Lohnis, F. Beitriige zur Kenntnis der Stickstoffbakterien. Ibid., 14: 5S2-604, 713-723. 1905. REDUCTION PROCESSES IN THE SOIL 555 Attention must be called here to the fact that many of the studies on denitrification were carried out in solution and not in soil. It is known that in very wet soils and in liquids which do not have a ready access to oxygen the bacteria utilize the oxygen from the nitrate molecule for oxidation purposes, while this does not occur in the presence of sufficient oxygen as in well aerated soils. The original suggestion of Winogradsky that the introduction of large quantities of manure favors denitrification while preventing nitrification has been proved erroneous. Both processes may take place side by side. The losses of nitrogen in the manure compost were found53 to be due largely to the presence of nitrate forming bacteria. When these bacteria are eliminated or conditions are made unfavorable for their action the losses are considerably reduced. The presence of the bacteria resulted in an increase in the loss of nitrogen from 6.28 to 23.75 per cent in the case of cow manure and from 0.73 to 11.66 per cent in the case of horse manure.54 It is often observed that the addition of large quantities of undecom- posed organic matter to the soil particularly rich in carbohydrates and poor in nitrogen injures crop growth. This is not due to denitrification, to which it has often been ascribed, but to the fact that, in the presence of an excess of available organic matter, the fungi, actinomyces, and vari- ous heterotrophic bacteria synthesize an extensive protoplasm. For this purpose, they assimilate the nitrates and ammonium compounds present in the soil and thus compete with higher plants. The conclusion may be reached that the phenomenon of denitrifica- tion is of no economic significance in well aerated, not too moist soils, in the presence of moderate amounts of organic matter or nitrate. How- ever, in the case of soils kept under water for some time, as rice soils, the addition of nitrates may even prove injurious due to the formation of toxic nitrite.55 The injury by denitrification in peat soils has been mentioned above. Reduction of other oxygen-rich compounds in the soil. Among the various inorganic, oxygen-rich compounds which can be readily reduced 63 Niklewski, 1923 (p. 499); Smirnow, V. G. Role of nitrifying bacteria in the process of decomposition of manure. Zhur. Opit. Agron., 16: 329-386, 1915. 64 A detailed review of the subject is given by Ldhnis, 1910 (p. xiii); see also Russell and Richards. Jour. Agr. Sci., 8: 495-563, 1917. 65 Nagaoka, M. Effect of nitrate of soda on paddy soils. Bui. Coll. Agr. Tokyo, 6: No. 3. 1904; Kelley, W. P. The assimilation of nitrogen by rice. Hawaii Agr. Exp. Sta. Bui. 24. 1911. 556 PRINCIPLES OF SOIL MICROBIOLOGY by microorganisms, the following may be mentioned in addition to nitrates and nitrites: sulfates,56 sulfur, sulfites, selenates, selenites, tellurates and phosphates. H2S is formed not only from sulfates but also from elementary sulfur, sulfites and polythionates.57 The reduction of selenium compounds,58 including selenious acid59 and various salts as well as tellurium salts, to their elements has been demonstrated for a number of bacteria60 and fungi. The amount of reduction was found61 to be proportional to the growth of the organisms. Selenic acid is re- duced in two stages, first to selenious acid and then to free selenium.62 The reduction of arsenic to arsine is carried out by Pen. brevicaule, an organism found in the soil,63 as well as by certain other organisms. Various bacteria are also capable of reducing organic64 and inorganic phosphorus compounds. According to Rudakov,65 mineral phosphates are reduced, under anaerobic conditions, to phosphites (H3P03) and hypophosphites (H3P02) as well as to phosphene. Pure cultures were obtained of a bacterium capable of bringing about the reduction of the phosphate. Different soils varied in their capacity of bringing about this reduction. The addition of KN03 and MgS04 to the medium led to a 66 Saltet, R. H. t)ber Reduktion von Sulfaten in Brackwasser durch Bakterien. Centrbl. Bakt. II, 6: 648-695. 1900; van Delden, A. Beitrag zur Kenntnis des Sulfatreduktion durch Bakterien. Centrbl. Bakt. II, 11: 81-94, 113-119. 1903; Kochmann, R. Uber Schwefelwasserstoff-bildung aus Sulfaten durch Faeces. Biochem. Ztschr., 112: 255. 1920. 67 A detailed study of sulfate reduction in the soil is given elsewhere (p. 610). 58 First reported by Japha, A. Experimenta nomulla de vi selenii in organis- num animalem. Diss. Halle. 1842. 69 Chabrie, C, and Lapicoque, L. Sur Taction physiologique de l'acide selenieux. Compt. Rend. Acad. Sci., 110: 152. 1890. 60 Scheurlen, E. Die Verwendung der selenigen und tellurigen Saure in der Bakteriologie. Ztschr. Hyg., 33: 135-136. 1900. 61 Klett, A. Zur Kenntnis der reduzierenden Eigenschaften der Bakterien. Ztschr. Hyg., 33: 137. 1900. 62 Levine, V. E. The reducing properties of microorganisms with special reference to selenium compounds. Jour. Bact., 10: 217-264. 1925. 63 Gosio, B. Azione di alcune muffe sui composti fissi d'arsenico. Riv. d'igiene, 3: 201. 1892; Abel, R., and Buttenberg, J. Uber die Einwirkung von Schimmelpilzen auf Arsen und seine Verbindungen. Ztschr. Hyg., 32: 449. 1899. 61 Barrenscheen, H. K., and Beckh-Widmenstetter, H. A. t3"ber bakterielle Reduktion organisch-gebundenen Phosphorsiiure. Biochem. Ztschr., 140: 279- 283. 1923. 65 Rudakov, K. I. The biological reduction of mineral phosphates (Russian). Viestnik Bact. Agron. Sta., 26: 171-188. 1926. REDUCTION PROCESSES IN THE SOIL 557 diminution of the reduction of phosphate, since the activities of the reducing microorganisms were directed towards the more readily reduca- ble compounds. Carbon compounds were used as sources of energy: 2 H3PO4 + C = 2 H3PO3 + C02 2 H3PO3 + C = H3PO2 + C02 H3PO2 + C = PH3 + C02 The reduction of various organic compounds in the soil, under anaerobic conditions, is very common, as pointed out previously. CHAPTER XXII Fixation of Atmospheric Nitrogen by Microorganisms Attention has been called to the fact that fixation of nitrogen in the soil is carried on largely by bacteria. The most active representatives of the non-symbiotic nitrogen-fixing bacteria are Azotobacter and Clos- tridium (Bac. amylobacter) groups, while the symbiotic nitrogen fixing bacteria are so far represented by one group, the Bad. radicicola. The great majority of investigations on the activities of the non-symbiotic bacteria, particularly on the energy utilization, mechanism of nitrogen fixation, and influence of various environmental conditions upon this process have been carried out with species of Azotobacter. The same principles may or may not apply to the CI. pastorianum, Bac. astcro- sporus and the other bacteria capable of fixing nitrogen non-symbio- tically. Special emphasis should be laid on the difference in the mechan- ism of energy utilization, nitrogen fixation and general principles of growth between the aerobic and anaerobic bacteria. The first decom- pose most of the sugar to carbon dioxide and water and utilize, there- fore, a large amount of the energy that can be made available from the decomposition of carbohydrates ; the anaerobic organisms break down the sugar largely to organic (butyric) acids and gases, and utilize only a small part of the energy (see p. 412). The difference in growth and nitrogen fixation between the aerobic and anaerobic bacteria is further emphasized by the difference in the influence of oxygen tension and resistance towards acidity and alkalinity. Non-symbiotic fixation of nitrogen. Berthelot1 was the first to recog- nize that the fixation of atmospheric nitrogen leading to an increase in the supply of combined nitrogen in the soil is due to the action of micro- organisms. When the soil was heated to 100°, the property was lost. By placing 50 kgm. of soil in pots, having a surface of 1500 sq. cm. and a perforated bottom, and allowing the pots to remain exposed to weather for seven months, he found an increase of 12.73 grams of nitrogen, tak- 1 Berthelot, M. Nouvelles recherches sur la fixation de l'azote atmospherinue par les microbes. Compt. Rend. Acad. Sci., 101: 775. 1885; 115: 569-574. 637, 737. 1892; Chimie vegetale et agricole. Paris, Masson et Cie. 1899. 558 FIXATION OF ATMOSPHERIC NITROGEN 559 ing into consideration the combined nitrogen brought down by the rain- fall and the nitrates washed out by the water. These results were confirmed in a series of other experiments by Berthelot as well as by a number of other writers.2 Although Berthelot did not succeed in isolat- ing any organisms, he found that the bacteria require combined carbon and hydrogen and enough combined nitrogen to promote their initial growth, and that, when the amount of combined nitrogen available is increased, the bacteria prefer to use this combined nitrogen rather than to fix atmospheric nitrogen. Berthelot demonstrated conclusively that in bare soil, free from vegetation, bacteria exist which are capable of fixing atmospheric nitrogen; these organisms, he found, act best at tem- peratures of 10° to 40°C, in the presence of sufficient oxygen and with an optimum amount of moisture (from 2-3 to 12-15 per cent) . This work led to the important contributions of Winogradsky and Beijerinck, as pointed out above. Winogradsky3 carefully freed the nutrient solutions from all traces of nitrogen and used only mineral salts with 4 per cent glucose as a source of energy. He obtained a gain of 24.68 mgm. and 28.87 mgm. of nitrogen per liter of medium, after 15 and 20 days' incubation. A slight addition of ammonium salt or nitrate stimulated the growth and butyric acid fermentation of the organism, but not nitrogen fixation. When more than 0.6 gram ammonia or ni- trate nitrogen were added per 100 grams of sugar, nitrogen-fixation ceased entirely; in other words, 6 parts of combined nitrogen for 1000 parts of sugar is just sufficient to prevent any fixation of gaseous nitro- gen. In the absence of combined nitrogen, 2.4 to 3 mgm. of nitrogen were fixed by the organism for every gram of glucose supplied. As a result of this evidence it was concluded that growth and nitrogen fixation are two distinctly separate phenomena; the nitrogen fixing bacteria grow as other organisms do in the presence of available energy and combined nitrogen, but, in the absence of combined nitrogen and in the presence of available energy, they are able to obtain their nitrogen from the atmosphere. At first Azotobacter was considered to be the most active nitrogen- fixing organism, since it fixed as much as 15 to 20 mgm. of nitrogen per gram of sugar, while the CI. pastorianum fixed only 2 to 3 mgm. of nitro- 2 For detailed review of earlier literature consult Voorhees and Lipman, 1907 (p. 491); Koch, A. Lafar's Handb. techn. Mykol., 3: 1. 1907; Lohnis, 1910 (p. xiii), Omeliansky, W. L. Monogr. 5 Russian Acad. Sci., Petrograd. 1923. 3 Winogradsky, S. 1893 (p. 107). 560 PRINCIPLES OF SOIL MICROBIOLOGY gen for the same amount of sugar. However, more recent work4 tends to show that CI. pastorianum is more abundant and more universally distributed in the soil ; also that it actually fixes far more nitrogen when the C02 formed is considered as the real index of energy consumption/' The activities of these two groups of organisms do not exclude, however, one another; this has been brought out by Omeliansky, who demonstrated that, by "symbiosis" or "commensalism" in reference to the oxygen tension, even larger quantities of nitrogen are fixed. Gainey6 compared the nitrogen fixing capacity of a large number of soils in which Azotobacter was present or absent. A total of 418 soils were examined, of which 199 contained Azotobacter and 219 did not. The presence of Azotobacter was noted by pellicle formation and microscopic examination of a culture prepared by inoculating some soil into 50 cc. of a medium consisting of: K2HPO4 0.5 gram CaCl2 Trace MgS04 0.2 gram Mannite. 20 grams NaCl 0.2 gram Distilled water 1000 cc. FeCl3 Trace This was neutralized to phenolphthalein by means of sodium hydroxide solution. A small quantity of sterile CaC03 was added to each flask before inoculation in order to insure sufficient base for the neutralization of the organic acids formed. The average amount of nitrogen fixed, in 50 cc. mineral solution con- taining 2 per cent mannite, in three weeks by 367 soils was 6.36 mgm. ; 174 soils containing Azotobacter fixed an average of 8.30 mgm. of nitro- gen and 193 soils not containing Azotobacter fixed only 4.61 mgm. Thus the quantity of nitrogen fixed by other microorganisms in the soil is practically one-half of that fixed when Azotobacter is present. The amounts of nitrogen fixed by pure cultures of the other bacteria is usually not greater than 1 to 2 mgm. per 1 gram of sugar consumed, although, in some cases, larger quantities were reported. These differ- ences in amounts of nitrogen fixed are undoubtedly due to the efficiency * Pringsheim, 1908 (p. 564); Omeliansky, 1923 (p. 559); Truffaut, G., and Bezs- sonofT, N. Sur la predominance de l'activite des fixateurs anaerobies d'azote dans le sol. Compt. Rend. Acad. Sci., 181: 165-167. 1925. 6 Bonazzi, A. The mineralization of atmospheric nitrogen by biological means. IVth. Intern. Soil. Sci. Conf. IIIB, No. 8, Rome. 1924, 42 p. 6 Gainey, P. L. Influence of the absolute reaction of a soil upon its Azotobacter flora and nitrogen-fixing ability. Jour. Agr. Res., 24: 907-938. 1923. FIXATION OF ATMOSPHERIC NITROGEN 561 with which the different organisms use the available energy, as will be shown later. Sources of energy. The process of nitrogen fixation is an endothermic reaction, which necessitates a large supply of energy for the organisms concerned. This energy is derived primarily from carbohydrates and allied compounds as well as from salts of organic acids. Glucose, TABLE 55 Nitrogen fixed per gram of energy material RESULTS SUBSTANCE ADDED Lohnis an d Pillaii° Krainsky" Hoffmann and No CaCOs CaCOa added Hammer12 ■mgm. 9.96 9.40 8.80 8.80 8.58 7.44 7.86 7.34 7.58 5.90 4.36 3.50 2.82 1.68 2.82 2.22 1.00 0.96 0.26 0.16 -1.10 mgm. 9.40 9.54 9.12 8.52 7.72 7.86 7.44 7.62 7.18 8.60 4.62 3.36 5.06 4.7s 2.96 2.49 1.42 1.10 0.12 0.02 -0.96 mgm. 5.70 0.80 5.55 5.80 0.67 2.80 0.60 1.20 0 1.35 0.40 2.40 mgm. 14.40 Xylose 4.55 7.20 Laevulo.se . . 10.30 10.85 Galactose . 7.35 Maltose Arabinose 10.00 Dextrin . . 13.40 Sucrose Glucose 11.70 8.95 Starch Sodium tartrate Glycerol Sodium succinate Calcium lactate 5.05 Sodium citrate Sodium proprionate Potassium oxalate Calcium butyrate Humus mannite and other simple carbohydrates and alcohols form the most available sources of energy for nitrogen-fixing bacteria. It has been found that polysaccharides, like celluloses, can also serve as valuable sources of energy if they are first partially broken down by cellulose de- composing organisms.7 However, these results need still further con- 7 McBeth, I. G. Cellulose as a source of energy for nitrogen-fixation. U. S. Dept. Agr. Bur. PI. Ind. Circ. 131: 25-34. 1913; Pringsheim, 1906-1912 (p. 110); 562 PRINCIPLES OF SOIL MICROBIOLOGY firmation; this is especially important since the bulk of the energy- material commonly added to the soil consists of celluloses and pen- tosans. It is claimed that the latter can be used as a source of energy by nitrogen-fixing bacteria.8 Table 55 contains a summary of the results obtained by different investigators concerning the relative availability of different sources of energy for nitrogen fixation; the results are rather variable and point to the unreliability of such criteria, which are subject to numerous influ- ences. By using different periods of incubation, media of different composition and different soils for inoculation (difference in mixed flora) , a different series of results would be obtained. As to the availability of natural organic materials as sources of energy for nitrogen fixing organisms, table 56 shows the amounts of nitrogen fixed for 100 grams of carbon by Az. chroococcum, as determined by TABLE 56 Nitrogen fixed by Az. chroococcum for 100 grams of carbon MILLI- GRAMS MILLI- GRAMS Pine needles 57.3 126.9 89.5 325.4 280.3 Plant roots and stubble Lupines Alfalfa 596.8 Oakleaves Maple leaves 711.5 319.5 Wheat straw Clover 1237.9 Corn stover Glucose 1456.5 Dvorak.9 He used a medium containing 1 gram K2HP04 and 1 gram CaC03 in 1 liter of tap water; 10 grams of organic matter and 250 cc. of medium were placed in liter flasks, which were then sterilized and inoculated. The favorable action of the last four substances preceding glucose is Bottomley, W. B. The assimilation of nitrogen by certain nitrogen-fixing bac- teria in the soil. Proc. Roy. Soc. B., 82: 627-629. 1910; 85: 466. 1912; Koch, A. Uber Luftstickstoffbindung im Boden mit Hilfe von Zellulose als Energie- materia'. Centrbl. Bakt. II, 27: 1-7. 1910; 31: 567-570. 1912; Stoklasa, J. Beitrag zur Kenntnis der chemischen Vorgange bei der Assimilation des elemen- taren StickstofTs durch Azotobacter und Radiobacter. Centrbl. Bakt., II, 21: 4S4-509, 620-632. 1908. 8 Stranak, Fr. Zur Assimilation des LuftstickstofTes durch im Boden freileb- enden Mikroorganismen. Zeitschr. Zuckerind. B>hmen 33: 599. 1909; (Centrbl. Bakt. II, 25: 320. 1909). • Dvorak, 1912 (p. 428). FIXATION OF ATMOSPHERIC NITROGEN 563 due to at least two factors: (1) the abundance of monosaccharides or readily hydrolyzable hemicelluloses in the leguminous plants and fresh materials; (2) the legumes were used fresh and might have exerted, therefore, a more stimulating effect upon the assimilating capacity of the cells, whereas the other substances were all used in a dry condition. Substances especially rich in lignin are not good sources of energy for the activities of the nitrogen fixing organisms. The amount of nitrogen fixed depends not oiJy upon the nature of the energy source, but also on the presence of available nitrogen, minerals, reaction and other environmental conditions, as well as upon the specific organisms.10-12 Some species utilize more readily one source of energy than another. The amount of nitrogen fixed depends upon the energy value of the particular compound as well as the nature of its decomposi- tion. Lipman13 recorded an increase in the amount of nitrogen fixed with an increase in molecular weight of fatty acids, in the form of sodium salts, namely, acetic, propionic, butyric; the next member of the homolo- gous series (valerianic acid) presented a poor source of carbon; the sodium salts of succinic and citric acids were not utilized at all. Mocke- ridge14 obtained 6.08 mgm. of nitrogen fixed with butyric acid and 1.47 mgm. with formic acid as sources of energy. . There was nearly a cor s':ant ratio between the amount of nitrogen fixed and heat of combustion of the fatty acids; the heat of combustion of butyric acid is 5.96 calories per gram and of formic acid 1.37 calories. Benzene derivatives and most glucosides seem to be unsuitable as sources of energy for Azotobncter.1' The amount of nitrogen fixed is usually calculated per unit of carbon source (sugar) consumed. This does not, however, give a true picture of the process. As pointed out above, some organisms (Azotobacter) break down the sugar chiefly to carbon dioxide and water; others (CI. 10 Lohnis, F., and Pillai, N. K. Tiber Stickstoff-fixierende Bakterien. Centrbl. Bakt. II, 20: 781-800. 1908. A nutrient solution was inoculated with 10 gm. of soil and incubated for 10 days. 11 Krainsky, A. Azotobacter chroococcum und seine Wirkung im Boden. Zhur. Opit. Agron., 9: 6S9. 1908; (Centrbl. Bakt. II, 20: 725-736. 1908). 12 Hoffmann, C, and Hammer, R. W. Some factors concerned in the fixation of nitrogen by Azotobacter. Wis. Agr. Exp. Sta. Res. Bui. 12. 1910; Centrbl. Bakt. II, 28: 127-139. 1910. Pure cultures used in Ashby's solution containing 1 per cent sugar and incubated 30 days. 13 Lipman, 1903 (p. 115), p. 217; 1904 (p. 112), p. 237. 14 Mockeridge, F. A. Some organic matter as culture media for Azotobacter. Biochem. Jour., 9: 272-283. 1915; also Ann. Bot., 26: 871-887. 1912. "Greaves, J. E. Azofication. Soil Sci., 6: 163-217. 1918. 564 PRINCIPLES OF SOIL MICROBIOLOGY pastorianum) produce largely organic acids. Different amounts of energy are thus made available for the organisms (see p. 412). The amount of nitrogen fixed should, therefore, be calculated on the basis of energy utilized and not on the basis of sugar consumed. By comparing two strains of Clostridia, one anaerobic and the other aerobic, Pring- sheim16 reported that the difference in the amount of nitrogen fixed per unit of sugar consumed is due to the relative amounts of acids and gases formed. The anaerobic organism produced 45 per cent acid and 55 per cent gas out of the sugar consumed. The aerobic strain produced only 33 per cent acid and 67 per cent gas because of the more complete decomposition of the organic matter; this strain fixed, therefore, more nitrogen. The ratio between the sugar changed into gas and the nitro- gen fixed was nearly the same for both organisms, namely, 23 and 21. The differences in the consumption of sugar by the different organisms cannot serve, therefore, as criteria. Azotobacter fixes as much as 3 to 20 mgm. of nitrogen per gram of sugar consumed, while CI. pastorianum fixes a maximum of 2 to 3 mgm. nitrogen for the same amount of sugar. However, when the actual energy liberated is compared, the latter organism may be found to be more efficient. Chemistry and decomposition of carbohydrates. The anaerobic nitro- gen-fixing bacteria decompose carbohydrates and their derivatives with the formation of various acids, chiefly butyric and acetic, and various gases. One liter of medium containing forty grams of glucose inoculated with the anaerobic organism and placed in a nitrogen atmosphere showed a gain of 53.6 mgm. of nitrogen in 20 days. All the sugar disappeared, giving rise to 3.714 grams acetic acid, 14.164 grams n-butyric acid, | cc. alcohol, chiefly iso-butyl, and traces of lactic acid. The amount of acids as well as the relation of the gases (C02:H2) were found to vary in the different experiments.17 The gases were found to make up 55 to 67 per cent of the sugar decomposed and to consist of 49 per cent carbon dioxide and of 51 per cent hydrogen.18 Azotobacter decomposes carbohydrates, higher alcohols and organic acids, without the formation of considerable amounts of intermediary products, such as various organic acids; C02 is the only gas formed; the reaction of the medium does not become more acid, but may even 16 Pringsheim, H. Uber die Verwendbarkeit verschiedener Energiequellen zur Assimilation des Luftstickstoffes und die Verbreitung stickstoffbindenden Bakterien auf der Erde. Centrbl. Bakt. II, 20: 248-256. 1908. 17 Winogradsky, 1893 (p. 107). 18 Omeliansky, 1923 (p. 559). FIXATION OF ATMOSPHERIC NITROGEN 565 become more alkaline because of the utilization of the organic acids present as sources of energy.19 Some aerobic bacteria may produce ethyl alchohol and acetic acid out of the sugar, in the process of nitrogen fixation.20 V- ' a. t\ *? a » /\ » » » » » \ % » \ V) i \ h K*^ 1 1 i / » St 1 1 / * 1 • / ' <: ** \ 1" X \ 4 <: \ "» \ v \ > # / X / X / X / As exr/?as£ ooa COONH4 + H2 -» COO • NH4 + |(Oa). CO • NH2 CH2 • NH2 It has also been suggested52 that the fixation of nitrogen by Azotobacter takes place by reduction by means of hydrogen with the formation of cyanides as intermediary products. Wieland53 considered that the ac- tion of the hydrogen acceptors formed in the cells of nitrogen-fixing bacteria does not depend upon oxygen for hydration, but upon the molecular nitrogen with which it forms ammonia, perhaps through the hydrazine stage in a manner similar to the Haber synthesis. The fixation of nitrogen by an oxidation process, similar to nitro- gen-fixation in moist air through the oxidation of organic matter, has also been suggested.54 The nitrogen is believed to be first oxidized to N203 with iron hydrate as a catalyst; the nitrous acid may then be assimilated by the organism.55 It has also been suggested56 that am- monium nitrite is first synthesized. However, neither nitrate nor 60 Kostytschew, S., and Ryskaltshouk, A. Les produits de la fixation de 1'azote atmosphyrique par V Azotobacter agile. Compt. Bend. Acad. Sci., 180: 2070-2072. 1925; Ztschr. physiol. Chem., 154: 1-17. 1926. 61 Loeb, W. tlber das Verhalten des Formamids unter der Wirkung der stil- len Entladung. Ein Beitrag zur Frage der Stickstoff- Assimilation. Ber. deut. chem. Gesell., 46: 684-697. 1913. 62 Stoklasa, 1908 (p. 562). 63 Wieland, H. tlber den Verlauf der Oxydationsvorgiinge. Ber. deut. chem. Gesell., 55: 3639-3648. 1922. 64 Gautier, A., and Drouin, R. Becherches sur la fixation de 1'azote par les sols et les vegetaux. Compt. Bend. Acad. Sci., 106: 754-757, 863, 944, 1174, 1232, 1605. 1888; 113: 820-825. 1891 ; also Berthelot, 1892 (p. 558). 65 Bonnema, A. Gibt es Bakterien, die den freien N assimilieren oder ist dies ein chemischer Frozesz? Chem. Ztg., 27: 148. 1903; Centrbl. Bakt. II, 10: 598-602. 1903. 66 Czapek, F. Der Stickstoff im Stoffwechsel der Fflanze. Ergeb. Physiol. I, 2: 638-672. 1903; Heinze, B. Centrbl. Bakt. II, 12: 364. 1904. 574 PRINCIPLES OF SOIL MICROBIOLOGY nitrite could be found in the cells of Azotobacter.57 Loew and Azo5S considered the reaction of nitrogen-fixation by Azotobacter to take place as follows: N2 + 2 H20 = NH4 N02 The ammonium nitrite is reduced to ammonia which will interact with the decomposition products of carbohydrates to give amino acids.59 CH3 • CO • COOH + NH3 = CH3 ■ CHN • COOH + H20 pyruvic acid CH3 • CHN • COOH -f H2 = CH, • CHNH2 • COOH alanine CHO • COOH + NH3 = H • CNH • COOH + H20 glyoxylic acid HCNH • COOH + H2 = CH2 • NH2 • COOH glycocoll By the process of condensation, the amino acids are built up into proteins. Beijerinck and van Delden60 originally thought that the nitrogen is first fixed in the form of an inorganic soluble compound which goes into solution. No soluble inorganic nitrogen compounds could be demon- strated in pure cultures, but in crude cultures the proteins may again be hydrolyzed into soluble forms of nitrogen. It was suggested,61 therefore, that the nitrogen is attached to the nitrogen-free organic compounds which are then changed to proteins. Soluble organic nitrogen com- pounds were found,62 however, not only in the filtrate of a phospho- tungstic acid precipitate of a dead Azotobacter culture, but also after the culture was filtered through a Chamberland filter. It was sug- gested63 that Azotobacter forms, in its early stage of development, a complex consisting of sugar and nitrate which is similar to the sugar- phosphoric acid complex which takes an active part in alcoholic fermea- 57 Kellerman, F., and Smith, N. P. The absence of nitrate formation in cultures of Azotobacter. Centrbl. Bakt. II, 40: 479. 1914. 68 Loew and Aso. On changes of availability of nitrogen in soils. II. Bull. Coll. Agr., Tokyo, 7: No. 5 (Centrbl. Bakt. II, 22: 452. 1909). 69Lipman, 1904-5 (p. 112). 60 Beijerinck and van Delden, 1902 (p. 105). 61 Gerlach and Vogel, 1903 (p. 379). 6- Lipman, 1903 (p. 115). 63 Bonazzi, 1921 (p. 567). FIXATION OF ATMOSPHERIC NITROGEN 575 tation. That complex is later used partly for energy and partly for synthesis of protoplasm. It was actually demonstrated64 that a large percentage of the total nitrogen fixed in the first few days of growth of Azotobacter consists of amino acid nitrogen. This proves that the elementary nitrogen goes through the simple organic forms before it is changed into protein. It also tends to prove that nitrogen is fixed by combination with hydrogen and not with oxygen, thus insuring much greater economy of energy. Influence of available nitrogen compounds upon nitrogen fixation. The nitrogen fixing bacteria do not depend entirely upon atmospheric nitro- gen for their need, but are capable of utilizing combined nitrogen present in the medium. This is a direct result of the distinct difference in growth and nitrogen-fixation pointed out above. Nitrates are readily utilized by Azotobacter as sources of nitrogen;65 accordingly the presence of nitrates in the medium inhibits the fixation of atmospheric nitrogen. Ammonium sulfate and peptone are also available nitrogen compounds.66 The injurious action of the nitrate upon the fixation of nitrogen was considered to be due either to (a) a direct toxic action of the salt upon the growth of the organism, (b) stimulation by nitrate of other organisms in a mixed culture which are antagonistic to Azotobacter, and (c) competition by such organisms with Azotobacter for the energy supply. By taking the number of organisms developing and the nitrogen fixed in the nitrate-free cultures as 100, it was found67 that the addition of 50 mgm. of NaN03 brought about an increase in the number of organisms from 100 to 3150, while the nitrogen fixed was increased 342 per cent in sterilized soil and 500 per cent in unsterilized soil. The presence of ni- trate was thus found to stimulate greatly the multiplication of the Azo- tobacter organism, while it reduced its physiological efficiency. Bon- azzi,68 therefore, suggested that Az. chroococcum may fix nitrogen in the 64 Waynick, D. D., and Woodhouse. By what steps does Azotobacter fix nitrogen? Cal. Agr. Exp. Sta. Ann. Rpt. 1918-19, p. 62-63. 65 Lipman, 1903 (p. 115); Pringsheim, H. Zur Stickstoffassimilation in Gegen- wart von Saltpeter. Centrbl. Bakt. II, 40: 21-24. 1914; Hanzawa, J. Einige Beobachtungen fiber Stickstoffbindung durch Azotobacter in stickstoffarmen und in stickstoffreichen Substanzen. Centrbl. Bakt. II, 41: 573-576. 1913. 66Krainsky, 190S-1912 (p. 566); Prazmowski, A. Azotobacter-Studien. II. Physiologie und Biologic Bull. Intern. Acad. Sci. Cracovie, Math. Nat. CI. Sc. B, No. 7: 855-950. 1912. 67 Hills, T. L. Influence of nitrates on nitrogen assimilating bacteria. Jour. Agr. Res., 12: 183-230. 1918. 68 Bonazzi, 1921 (p. 567). 576 PRINCIPLES OF SOIL MICROBIOLOGY absence of such fixed nitrogen and act as a "denitrifying" organism in the presence of nitrates. Except in high concentrations, however, the nitrogen of humus does not seem to exert any injurious influence upon nitrogen-fixation. This is probably due to the fact that its availability is only limited. As a matter of fact, it is even claimed69 that the soil organic compounds can be used as sources of energy by nitrogen-fixing bacteria. These results, however, need confirmation and elucidation. Influence of salts upon nitrogen fixation. In addition to an energy source, the presence of certain minerals in minimum concentrations is important for the activities of the nitrogen-fixing bacteria. Azotobac- ter was found70 to prefer soils containing calcium salts and races of the organism isolated from those soils were more active than those iso- lated from unlimed soils. Calcium was looked upon as serving a double purpose: (1) directly for metabolism (in the form of soluble phosphates, as the oxide, carbonate, and in the form of salts of organic and inorganic acids) and (2) for the purpose of neutralizing the acids formed in the metabolism (in the form of oxide, hydroxide or carbonate). Certain calcium salts, especially tricalcium phosphate and the chloride, cannot be utilized.71 Magnesium salts cannot take the place of calcium.72 The primary importance of calcium is due, however, not to its direct nutrient quality but chiefly to its buffering properties, and it may serve to some extent as a catalytic agent.73 According to Ashby,74 magnesium carbonate is very favorable for the development of Azotobacter in solution; its presence discourages also the development of foreign organisms, probably due to their supression by the magnesium. Potassium salts favor the development of Azotobacter, the minimum 69 Lipman, C. B., and Teakle, L. J. H. The fixation of nitrogen by Azotobacter in a displaced solution and in soil residue therefrom. Soil Sci., 19: 99-103. 1925. 70 Krzemieniewski, 1908 (p. 579). 71 Christensen, H. R. Uber das Vorkommen und die Verbreitung des Azoto- bacter chroococcum in verschiedenen Boden. Centrbl. Bakt. II, 17: 109-119. 1907; 19: 735-6. 1917. 72 Fischer, H. Ein Beitrag zur Kenntnis der Lebensbedingungen von stick- stoffsammelnden Bakterien. Centrbl. Bakt. II, 14: 33-34. 1905; 15: 235-236. 1906. 73 Vogel, J. Untersuchungen iiber das Kalibedilrfnis von Azotobacter. Centrbl. Bakt. II, 32: 411-442. 1912; Krzemieniewska, H. Zur Ernahrung des Azotobakters. Bui. Intern. Acad. Sci. Cracovie, CI. Sci. Math. Nat. No. 5: 445-448. 1908. 74 Ashby, 1907 (p. 113). FIXATION OF ATMOSPHERIC NITROGEN 577 quantity of this element corresponding to that of calcium. A minimum of 0.38 mgm. K, 0.36 mgm. Ca and 0.35 mgm. Mg was required by Azo- tobacter for every gram of sugar decomposed. When nitrogen- fixation takes place in the soil, the addition of small amounts of potas- sium is without any effect.75 In higher concentrations, potassium salts, as a rule, become more toxic than sodium salts. The latter do not seem to be indispensable for the growth of Azotobacter, although the addition of three per cent of NaCl does not injure the development of the organ- ism. There are claims in the literature,76 however, that alkalies, and particularly alkali carbonates, are injurious to nitrogen-fixation; the action of NaCl becomes apparent only when 0.5 to 0.6 part are present in 100 grams of dry soil. Na2S04 becomes injurious at 0.25 per cent concentration, while 0.4 to 0.5 per cent of Na2C03 inhibits growth of the nitrogen-fixing organisms completely. Phosphorus compounds greatly accelerate the activities of the nitro- gen-fixing organisms, since they play an important role in the metabo- lism of Azotobacter,77 which requires large quantities of the mineral for the synthesis of its cells. The organism utilizes particularly well those soluble phosphates which do not tend to make the soil reaction acid, as in the case of the di- and tri-sodium and potassium phosphates and di-calcium phosphates. The presence of mono-basic-phosphates which serve as buffers and tend to make the reaction of the medium more acid than the minimum for the growth of Azotobacter is not favorable. The difficultly soluble tri-basic calcium, iron and aluminum salts are available only according to the degree of their solubility. In the pres- ence of an excess of available energy a definite relation is found to exist between the growth of Azotobacter and the phosphorus content of the soil.78 It was suggested that information can be obtained on the presence of available plant food in the soil by determining the food requirements of bacteria. When a mannite solution free from phosphorus yields a good growth of Azotobacter, after inoculation with soil, it may be assumed that the soil is not deficient in phosphorus 76 Greaves, J. E., Carter, E. G., and Lund, Y. Influence of salts on azofication in soil. Soil Sci., 13: 481-499. 1922. 76 Lipman, C. B., and Sharp, L. T. The effects of alkali salts in soils on soil bacteria. III. Nitrogen fixation. Centrbl. Bakt. II, 35: 647-655. 1912. See also Greaves, Carter and Lund, 1922. 77 Dzierzbicki, A. Beitrage zur Bodenbakteriologie. Bui. Intern. Acad. Sci. Cracovie, B. 1910, 21-66. 578 PRINCIPLES OF SOIL MICROBIOLOGY so far as its availability to crops is concerned. Stoklasa78 found that 5.0 to 5.7 mgm. of atmospheric nitrogen are fixed for every mgm. of phosphorus used. The minimum need of phosphorus is 2.43 mgm. of P (or 5.46 mgm. P205) for every gram of glucose. Sulfur in the form of sulfates is essential for the growth of Azotobacter.79 Iron, in the form of its salts, has a definitely favorable influence upon the development of Azotobacter, by playing a part in its metabolism and exerting a favorable influence through its colloidal condition. When iron sulfate was added to the medium the amount of nitrogen fixed per gram of mannite was increased from 2.23 to 10.3 mgm.80 It has been suggested that the colloidal iron absorbs the nitrogen and oxygen of the air and thus brings them into more intimate contact with the cells of Azotobacter. In the presence of organic colloids, only small quantities of iron are effective. The favorable influence of soil infusion upon nitro- gen fixation by Azotobacter has been found81 to be due chiefly to their content of iron and partly to silicic acid. Inorganic colloids like the oxides of aluminum, iron and colloidal silicic acid greatly stimulate nitrogen-fixation by Az. chroococcum82 After inserting a strip of paper into the medium, Azotobacter grows exclusively in contact with the paper. This led Sohngen to conclude that microbial life in the soil takes place chiefly upon the colloids. The colloid is believed to absorb the nitrogen and oxygen thus making them more readily available to the organism. Iron has the same stimulating effect upon nitrogen-fixation as "humus" substances do, but much larger quantities of the inorganic colloid are required when it is present alone as when it is used together with the organic colloid. The iron is most active in the form of an or- ganic compound.81 78 Stoklasa, J. Biochemischer Kreislauf des Phosphat Ions im Boden. Centrbl. Bakt. II, 29: 385-419. 1911; Christensen, H. R. Studien iiber den Einflusz der Bodenbeschaffenheit auf das Bakterienleben und dem Stoffumsatz im Erdboden. Centrbl. Bakt. II, 43: 1-166. 1915; also Soil Sci., 15: 329-360. 1923; Greaves, 1918 (p. 563). 79 See also Koch, A. Ernahrung der Pflanzen durch frei im Boden lebende stickstoffsammelnde Bakterien. Ber. deut. landw. Gesell., 22: 117-121. 1907; Jour. Landw., 55: 355-416. 1907. 80 Rosing, G. Zusammenfassung der Ergebnisse von Untersuchungen uber die Stickstoffsammlung von Azotobacter chroococcum. Centrbl. Bakt. II, 33: 618-623. 1912. 81 Remy, Th., and Rosing, G. Uber die biologische Reizwirkung natiirlicher Humusstoffe. Centrbl. Bakt. II, 30: 349-384. 1911. 82 Sohngen, N. L. Einflusz von Kolloiden auf mikrobiologische Prozesse. Centrbl. Bakt. II, 38: 621-647. 1913. FIXATION OF ATMOSPHERIC NITROGEN 579 Some salts may act directly as stimulants to nitrogen-fixing bacteria. The optimum concentration of manganese for nitrogen-fixation by Azotobacter chroococcum in mannite solution was found to be83 1 to 2 nigra, per 100 gram of medium; further increases diminished nitrogen- fixation. One part of manganese per million parts of soil was the opti- mum for Bac. amylobacter. The addition of small amounts of arsenic (10 parts per million) exerted a stimulating effect upon nitrogen-fixa- tion in the soil and upon the more economic utilization of energy by Azotobacter.84 Aluminum has been looked upon85 both as a stimulant and as a retarding agent. Uranium salts exert a decided stimulating effect upon Azotobacter.86 The maximum amount of nitrogen fixed and maximum growth take place in the yellow or green rays; the least, in violet light. The maximum amount of nitrogen per gram of sugar decomposed is fixed in blue light ; the least, in yellow light. Influence of organic matter upon nitrogen fixation. The favorable influence of soluble soil organic matter, or so-called "humus," on nitro- gen-fixation was known for a long time;87 however, it was recognized that the nature of this action depends on the source of the organic matter. It was at first suggested that some of the constituents of the soil organic matter are used by Azotobacter as a source of energy.88 Lohnis and Green,89 however, working with various species of Azotobacter in mannite solution, found that the amount of nitrogen fixed in three weeks in 100 cc. of solution was 5.6 mgm. ; the addition of green manure increased it to 8.0 mgm., fresh stable manure to 9.8 mgm., and fresh straw to 10.0 mgm. When these substances were previously decom- 83 Olaru, D. A. Role du manganese en agriculture. Son influence sur quel- ques microbes du sol. Paris, Bailleres. 1920. 84 Greaves, J. E. Stimulating influence of arsenic upon the nitrogen-fixing organisms in the soil. Jour. Agr. Res., 6: 389-416. 1916. 86 Kaserer, H. Zur Kenntnis des Mineralstoffbedarfs von Azotobacter. Ber. deut. bot. Gesell., 28: 208-262. 1910. 86 Kayser, E. Influence des radiations lumineuses sur 1' Azotobacter. Compt. Rend. Acad. Sci., 171: 969-971. 1920; 172: 183-185, 491-493, 1133-1134. 1921; Ann. Inst. Nat. Agron. (2), 16: 11-43. 1922. 87 Krzemieniewski, S. Untersuchungen uber Azotobacter chroococcum Beij. Bull. Int. Acad. Sci. Cracovie, CI, Sci. Math. Nat., No. 9: 929-1051. 1908. 88Remy, 1909 (p. 569). 89 Lohnis, F., and Green, H. H. Uber die Entstehung und die Zersetzung von Humus, sowie uber dessen Einwirkung auf Stickstoff-Assimilation. Centrbl. Bakt. II, 41: 52-60. 1914. 580 PRINCIPLES OP SOIL MICROBIOLOGY posed, the fixation of nitrogen was even greater. By increasing the amount of stable manure added up to three per cent, there was90 a great increase in nitrogen-fixation. The stimulating effect is probably caused by the increase in available energy, due to the introduction of straw and its derivatives, and by the colloidal content of the material. A further increase in organic content may bring about a depression un- less the soil is well aerated and contains sufficient CaC03. The various plant residues, such as roots, leaves and stems will react similarly. Manure is also a carrier of various nitrogen-fixing organisms.91 The possible favorable influence of growth-promoting substances (vitamins, auximones) upon Azotobacter is still a subject of discussion.92 The beneficial action of humus is frequently ascribed to its inorganic constituents, particularly aluminum and silicic acid. This is confirmed by the fact that the so-called artificial humus has no such effect, while the source of the natural humus influences the degree of its beneficial action. The claim that the action of the humus is due to its inorganic constituents has been further substantiated by the fact that purified humates do not possess the stimulating effect.93 The role of the col- loid is probably chiefly due to its catalytic action and its protective action against poisons;94 the protective action of the colloid has also been ascribed to the distribution of the phosphorus and to the buffer- ing effect upon the reaction of the medium. 90 Hanzawa, 1913 (p. 575). 91 Tottingham, W. E. The increase of nitrogen in fermenting manures. Jour. Biol. Chem., 24: 221-225. 1916; Fulmer, H. L., and Fred, E. B. Nitrogen- assimilating organisms in manure. Jour. Bact., 2: 422-434. 1917. 92 Bottomley, W. B. Some accessory factors in plant growth and nutrition. Proc. Roy. Soc. B., 82: 627. 1910; 85: 466. 1912;88:237-247. 1914; 89: 102. 1915; The significance of certain food substances for plant growth. Ann. Bot., 28: 531-540. 1914 ; Mockeridge, F. A. Some effects of organic growth promoting substances (auximones) on the soil organisms concerned in the nitrogen cycle. Proc. Roy. Soc. B., 89: 508-533. 1915; Biochem. Jour., 19: 272-283. 1915; Allen, E. R. Some conditions affecting the growth and activities of Azotobacter chroococcum. Ann. Mo. Bot. Gard., 6: 1-AA. 1919; Itano, A. Physiological study of Azotobacter chroococcum. I. Influence of vitamine B (?) and nucleic acid on Azotobacter. Jour. Bact., 8: 483-486. 1923; Hunter, O. W. Production of a growth promoting substance by Azotobacter. Jour. Agr. Res., 23: 825- 830. 1923. 93 Kaserer, H. Uber die biologische Reizwirkung naturlicher Humusstoffe. Centrbl. Bakt. II, 31: 577-578. 1912. 94 Voicu, J. Influence de l'humus sur la sensibilite de V Azotobacter chroococ- cum vis-a-vis du bore. Compt. Rend. Acad. Sci., 175: 317-319. 1922. FIXATION OF ATMOSPHERIC NITROGEN 581 According to Voicu,94 organic matter will influence the sensitiveness of Azotobacter to poisons such as boron. Urea, glycocoll, formamide and allantoin depress nitrogen-fixation; this was attributed,95 not to a direct toxicity but to the fact that those substances furnish an available nitrogen source. Among the substances acting injuriously upon nitro- gen-fixing organisms, we find caffeine, alloxan, betaine, trimethyl- amine, legumin, cinnamic acid, aspartic acid, hippuric acid, creatine, creatinine, xanthine and hypoxanthine. The first two have a stimulating effect in dilute solutions. Influence of reaction upon the growth of non-sytnbiotic nitrogen- fixing bacteria. Lime exerts such a favorable influence upon the activities of Azotobacter in the soil that Christensen96 suggested using the presence of this organism as an index of the lime requirement of the soil. When the reaction of the soil is more acid than pH 5.7, Azotobacter is absent and the soil needs lime; when the pH of the soil is above 7.4, the soil does not need any lime. But when the pH of the soil is between 5.8 and 7.3, an Azotobacter test is made. A definite weight of soil (5 or 10 grams) is added to a definite amount of mannite solution free from calcium carbon- ate (50 or 100 cc.) ; the flasks are sterilized, inoculated with a fresh cul- ture of Azotobacter and incubated. The greater the lime or buffer content of the particular soil, the more abundant will be the growth of Azotobacter. The amount of pellicle development is an index of the buffer action of the soil and can yield information on its lime require- ments. Out of 100 soils used, the Azotobacter test for the lime require- ment of soils agreed in 90 per cent of the cases with the known soil condition, while the ammonium chloride and litmus tests agreed only in 50 and 40 per cent of cases respectively. The amount of CaC03 which should be added to the soil, to obtain maximum nitrogen fixation, varies with the soil. This is due to differences in the buffer content of soils, in addition to differences of the initial reaction. Soils of a different buffer content, even of the same initial reaction, will require different quantities 95 Reed, H. S., and Williams, B. The effect of some organic soil constituents upon nitrogen-fixation by Azotobacter. Va. Agr. Exp. Sta. Tech. Bui., 4: 81-95. 1915; Walton, J. H. Azotobacter and nitrogen-fixation in Indian soil. Mem. Dept Agr. India. Bact. Ser., 1: 97-112. 1915. 96 Christensen, 1907 (p. 576); 1915 (p. 578); Experiments in methods for deter- mining the reaction of soils. Soil Sci., 11: 115-178. 1917; Untersuchungen iiber einige neuere Methoden zur Bestimmung der Reaktion und des Kalkbediirf- nisses des Erdbodens. Intern. Mitt. Bodenk., 13: 111-146. 1923; Christensen, H. R., and Larsen, O. H. Untersuchungen iiber Methoden zur Bestimmung des Kalkbediirfnisses des Bodens. Centrbl. Bakt. II, 29: 347-380. 1911. 582 PRINCIPLES OF SOIL MICROBIOLOGY of lime to bring them to the same optimum reaction. Azotobacter is capable of existing in many soils which contain none or only mere traces of CaC03 and also in some soils reacting acid by the ordinary test.97 A reaction equivalent to about pH 6.0 is found to be, in most cases, the limiting acid reaction for Azotobacter. The optimum reaction for nitro- gen fixation by Azotobacter in pure culture seems to be closely associated with the optimum reaction for growth. Different strains of Azotobacter may vary in their sensitiveness to the limiting acid reactions, the mini- mum for growth having been reported to be in some cases pH 6.6 to 6.8.98 The optimum reaction for the development of Azotobacter is pH 7.0 to 7.8, while the limiting alkaline reaction was reported to be pH 8.8.99 Different species of Azotobacter may vary, however, also in their behavior to the optimum reaction.100 AZOTOBACTER SPECIES OPTIMUM pH LIMITING pH chroococcum 7.45-7.60 6.65-6.75 7.50-7.70 5.80 beijerinckii 5.80 vinelandii 5.90 The fixation of nitrogen in soils of a greater acidity than the limiting reaction for Azotobacter (pH less than 6.0) is due to the activities of Bac. amylobacter (CI. pastorianum) , which has its optimum at pH 6.9 to 7.3, but can still grow at an acidity greater than pH 5.7.101 As a matter of fact growth of this organism can be obtained at pH 5.0. Other nitrogen-fixing forms, like Bad. aerogenes and Radiobacter , can also grow at a higher acidity than Azotobacter. The nitrogen fixed under these conditions is much less than that fixed in soils supporting an Azo- tobacter flora. Figures 41 and 42 show the correlation between the hydrogen-ion concentration and nitrogen fixation by Azotobacter.102 Calcium carbonate stimulates nitrogen fixation and is never toxic to 97 Gainey, P. L. Soil reaction and the growth of Azotobacter. Science, N. S., 48: 139-140. 1922; Jour. Agr. Res., 14: 265-271. 1918. 98 Fred, E. B., and Davenport, A. Influence of reaction on nitrogen-assimilat- ing bacteria. Jour. Agr. Res., 14: 317-336. 1918. 99 Johnson, H. W., and Lipman, C. B. The effect of reaction on the fixation of nitrogen by Azotobacter. Univ. Cal. Publ. Agr. Sci., 4: 397-405. 1922. 100 Yamagato and Itano, 1923 (p. 117). 101 Dorner, 1924 (p. 165). 102 Gainey, P. L., and Batchelor, H. W. The influence of hydrogen-ion con- centration on the growth and fixation of nitrogen by cultures of Azotobacter. Jour. Agr. Res., 24: 759-768. 1923. FIXATION OF ATMOSPHERIC NITROGEN 583 Az. chroococcum at a concentration of 2 per cent in mannite solution.103 Magnesium carbonate is very toxic above 0.1 to 0.2 per cent. Calcium exerts a protective influence against the injurious effect of magnesium. Influence of moisture and temperature upon nitrogen- fixation. Nitro- gen-fixing bacteria are able to resist drying for a long period of time, depending upon the nature of the medium. The soil contains sub- stances which exert a protective influence upon bacteria subjected to desiccation; the bacteria will resist desiccation longer in a rich clay soil than in sand, probably because of the colloidal content of the clay.104 CO \ X so to 30 zo 1.0 -T-l i > 1 Y' < ", > Z i ' u X /*) f- t i i i i i i i i i i i i i / i i > £ XP ?R/M £*/r W.A f i /« > • £XP£ft/M£NT //0./7 > 1 > 4 L^ / < r /• — •— ' "■"""< r" "*" h— •< '$* £5 £6 £7 £d £9 6.0 6/ 62 63 6.4 65 6.6 67 68 6.9 7.0 7./ MWA&GS/V - /ON COA/C£/Vr/?A T/ON £XP/?£SS£D AS P# 7.2 Fig. 41 . Influence of reaction of medium upon nitrogen-fixation by Azotobacter (from Gainey and Batchelor). Soils air-dried for five to twenty years and kept in stoppered museum bottles were found to contain103 viable nitrogen-fixing bacteria. Nitro- gen-fixation takes place in the soil when its moisture content is only 2 103 Lipman, C. B., and Burgess, P. S. The protective action against magnesium carbonate of calcium carbonate for Azotobacter chroococcum. Jour. Agr. Sci. 6: 484-494. 1914; Gainey, P. L. On the use of calcium carbonate in nitrogen fixation experiments. Jour. Agr. Res., 24: 185-190. 1923. 104 Giltner, W., and Langworth, H. V. Some factors influencing the longevity of soil microorganisms subjected to desiccation with special reference to soil solution. Jour. Agr. Res., 5: 927-942. 1916. 105 Lipman, C. B., and Burgess, P. S. Studies on nitrogen-fixation and Azoto- bacter forms in soils of foreign countries. Centrbl. Bakt. II, 44: 481-511. 1915. 584 PRINCIPLES OF SOIL MICROBIOLOGY to 15 per cent.106 Soil with a higher content of organic matter will have higher moisture optima. An excess of water may stop the action of Azotobacter but may stimulate the action of the anaerobic bacteria. Using a loam soil with a maximum moisture holding capacity of 27.4 per cent, Traaen107 observed a fixation of 1.9 mgm. of nitrogen in 100 grams of soil with 5 to 10 per cent moisture, 13.2 mgm. with 17.5 per cent moisture, 16.6 mgm. with 25 per cent moisture, and 15.5 mgm. with 30 per cent moisture. At a temperature of 13°C, the amounts of nitrogen fixed were less, with a similar maximum. With the higher moisture contents, the anaerobic organisms play an important part in the fixation of nitrogen. Two maxima for nitrogen-fixation in relation 100 /-\ 90 to T73 80 o TO 70 ^6 60 ■ / P' t: 50 8 40 / U 30 P* 20 10 / i ■ i 5.8 5.8 6.0 6.2 6.4 6.6 6.8 pH .0 7.2 7.t '.li 7.8 Fig. 42. A correlation between soil reaction and the presence of Azotobacter: positive test; abundant growth of Azotobacter (from Christensen). to the water content of the soil are frequently recorded, depending on whether the conditions are favorable to the action of aerobic or anaerobic bacteria.108 The optimum temperature for the activities of nitrogen-fixing bacteria 106Krainsky, 1908 (p. 566). 107 Traaen, A. E. tlber den Einflusz der Feuchtigkeit auf die Stickstoffum- setzungen im Erdboden. Centrbl. Bakt. II, 45: 119-135. 1916. 108 Lipman, C. B., and Sharp, L. T. Effect of moisture content of a sandy soil on its nitrogen-fixing powers. Bot. Gaz., 59: 402-406. 1915; Greaves, J. E., and Carter, E. G. Influence of barnyard manure and water upon the bac- terial activities of the soil. Jour. Agr. Res., 6: 889-926. 1916; also 9: 293-341. 1917. FIXATION OF ATMOSPHERIC NITROGEN 585 lies at 28°C. (25° to 30°), with limits between 9° and 33°C.109 Koch110 obtained fixation of 3, 11, and 15.5 mgm. of nitrogen in 100 grams of soil at 7°, 15,° and 24° respectively. The maximum temperature for Azotobacter is111 55° to 60° and the minimum near zero. The organism can withstand heating at 45° to 50°C. for 15 minutes, but is destroyed in 30 minutes. The optimum temperature for the growth of CI. pastoria- num is 20° to 25°, according to Winogradsky, and 28° to 30°, according to Bredemann.112 At 30° to 35°, the action of the organism is retarded. m The organism can withstand a temperature of 75° for even 5 hours or more; the spores can be preserved in a dry state for 20 years without losing their power of germination and nitrogen fixation. Soil cultivation and nitrogen-fixation. It has been generally observed114 that fallowing leads to an increase in nitrogen-fixation, probably due to better aeration and moisture conditions. According to Hiltner,115 non-symbiotic nitrogen-fixation is stimulated by growing plant roots; the higher plants use up the available nitrogen in the soil and thus create a nitrogen-hunger for the non-symbiotic nitrogen-fixing bacteria. The plants supply the bacteria with available energy, in the form of rotting roots hairs, root tips, etc. Plant roots may also create a better physical environment for the nitrogen fixing organisms. In view of the fact that different cultural methods are used for the growth of different crops, the influence of the crops upon nitrogen-fixation will vary. A much higher nitrogen-fixing power was found116 in cultivated than in virgin soils; the fallowed soils show more nitrogen fixed than the cropped soils. Importance of non-symbiotic nitrogen- fixation in the soil. It should 109 Krzemieniewski, 1908 (p. 579); Lohnis and Westermann, 1908 (p. 115). 110 Koch, A. Ernahrung der Pflanzen durch frei im Boden lebende stick- stoffsammelnde Bakterien. Ber. deut. landw. Gesell., 22: 117-121. 1907. 111 Jones, 1913 (p. 114). 112 Bredemann, 1909 (p. 111). 113 Omeliansky, W. L. Sur la physiologie et la biologie des bacteries fixant l'azote. Arch. Sci. Biol. Petrograd, 19: 209-228. 1915. 114 Heinze, B. tlber die Stickstoffassimilation durch niedere Organismen Landw. Jahrb., 35: 889-910 1906. 116 Hiltner, L. Ueber neuere Erfahrungen und Probleme auf dem Gebiete der Bodenbakteriologie und unter besonderer Berticksichtigung der Grundting- ung und Brache. Arb. deut. landw. Ges. H. 98: 59-78. 1904 (Centrbl. Bakt. II, 14: 46-48. 1904). 116 Greaves, J. E. A study of the bacterial activities of virgin and cultivated soils. Centrbl. Bakt. II, 41: 444-459. 1914; Reed, H. S., and Williams, B. Nitrogen-fixation and nitrification in various soil types. Va. Agr. Exp. Sta. Tech. Bui., 3: 59-80. 1915. 586 PRINCIPLES OF SOIL MICROBIOLOGY not be assumed that the addition of available carbohydrates to various soils is always sufficient to induce non-symbiotic fixation of nitrogen. In the presence of available nitrogen in the soil, the addition of carbo- hydrates stimulates the development of various fungi and bacteria which use the added source of energy and transform the available nitrogen into microbial protein. In the presence of available nitrogen, the non- symbiotic nitrogen-fixing bacteria will act upon the carbohydrates like the other heterotrophic bacteria, merely synthesizing proteins. Only in the absence of available nitrogen is there a probability of nitro- gen fixation by non-symbiotic bacteria. But even when fixation of nitrogen takes place the process is usually a slow one in normal soils; in many cases, the actual amount of nitrogen fixed falls within the prob- able error for the determination of total nitrogen. There are undisputed claims in the literature that very porous soils of a moderately high water content can fix small amounts of nitrogen under sterile conditions.117 There is still more definite evidence that appreciable quantities of nitro- gen can be fixed both in the laboratory and in the field by non-symbiotic bacteria, when there is sufficient available energy.118 Remy found that considerable nitrogen fixation takes place as long as provision is made for the neutralization of the acids formed and a proper source of energy is present. The nitrogen fixed by the bacteria becomes a proper source of nitrogen for higher plants; it becomes available slowly, although not less so than the most active organic fertilizers. It has been sug- gested119 that the great economy with which the nitrogen fixing bacteria use the organic matter in the soil is due to their symbiotic action with algae. There is no doubt that the nitrogen content of sand or soil may be appreciably increased by the activity of Azotobacter, if sufficient energy is supplied.119 About 6 mgm. of nitrogen were fixed per 1 gram of plant residue, under laboratory experiments, and up to 9 mgm. in pot 117 Warmbold, H. Untersuchungen ilber die Biologie stickstoffbindender Bakterien. Landw. Jahrb., 35: 1-123. 1906. Centrbl. Bakt. II, 20: 121-126. 1907. 118 Koch, A., Litzendorff, J., Krull, F., and Alves, A. Die Stickstoffanreich- ung des Bodens durch freilebende Bakterien und ihre Bedeutung fur die Pflan- zenernahrung. Jour. Landw., 55: 355-416. 1907; 57: 269-286. 1909; Remy, Th. Untersuchungen liber die Stickstoffsammlungsvorgange in ihrer Beziehung zum Bodenklima. Centrbl. Bakt. II, 22: 561-651. 1909; Lohnis, F. Centrbl. Bakt., 15: 361. 1905; Schneidewind. Ibid., 21: 437. 1908; H. Fischer. Ibid., 22: 654. 1909. 119 Krainsky, A. Uber die Stickstoffanreicherung des Bodens. Centrbl. Bakt. II, 26: 231-235. 1910. FIXATION OF ATMOSPHERIC NITROGEN 587 experiments.120 Among the most important conditions required for non-symbiotic nitrogen fixation are: (1) a proper supply of energy material, (2) sufficient CaC03 to neutralize soil acidity and improve the physical condition of the soil, (3) available phosphorus, (4) proper temperature, and (5) aeration of the soil. According to statements usually made in the texts and found at Rothamsted and other stations, non-symbiotic nitrogen-fixing bacteria add, under favorable conditions, 15 to 40 pounds of available nitrogen to each acre of soil yearly and TABLE 58 Influence of sugar upon crop yield and nitrogen content of crop YEAR CONTROL OKA Ms OF DRY SUBSTANCE PER POT FOR 3-YEAR PERIOD Glucose, 360 grams Cane sugar, 360 grams Cane sugar, 720 grams 1905-07 91.3 111.6 113.2 157.6 1908-10 51.2 78.5 77.6 91.8 1911-13 76.1 79.8 85.6 89.9 1914-16 63.6 65.9 68.6 67.4 1917-19 73.0 80.9 75.6 69.6 1920-22 65.3 64.0 72.6 66.3 Total 420.5 480.7 60.2 492.2 72.7 542.6 Excess over control 122.1 765 MILLIGRAMS OF NITROGEN IN CROP 1905-07 984 982 1476 1908-10 519 785 808 883 1911-13 616 634 670 739 1914-16 463 613 540 548 Total 2363 2916 553 3000 637 3640 Excess over control 1283 usually not more than 10 pounds. In view of the fact that the energy added to the soil is not directly available to the nitrogen-fixing bacteria, that small amounts of available nitrogen are always present in the soil and that the error in the laboratory determination of total nitrogen by the Kjeldahl method is greater than the possible amount of nitrogen fixed by non-symbiotic bacteria, we are still unable to decide the ques- 120 Hutchinson, H. B. The influence of plant residues on nitrogen-fixation and on losses of nitrate in the soil. Jour. Agr. Sci., 9: 92-111. 1918. 588 PRINCIPLES OF SOIL MICROBIOLOGY tion definitely. Until our methods are more accurate, the question cannot be answered in a positive way. It has been stated121 that the apparent gain of nitrogen in the soil is often due to drifting dust and plant residues or to soil variability. Certainly the field results of A. Koch122 do not speak for any nitrogen fixation in the soil, following the addition of celluloses and even straw. Positive fixation was obtained only when soluble sugars were added, as seen from table 58. By determining the amount of nitrogen fixed in the soil per gram of sugar added, it was found that although 720 grams of cane sugar had been added per pot of soil and 9.75 mgm. of nitrogen has been fixed per gram of sugar added, the plants utilized only 1.78 mgm. of nitrogen per gram of sugar added. The larger part remained in the soil in a complex TABLE 59 Influence of cellulose upon crop yield CONTROL GRAMS OF DRY SUBSTANCES PER POT FOR 3-YEAR PERIOD 120 grams paper 120 grams paper + manure infusion Manure infusion alone 1911-13 1914-16 1917-19 1920-21 68.3 60.0 66.0 65.2 12.8 81.7 77.4 71.1 17.9 87.3 82.8 72.8 67.0 62.8 69.9 67.0 Total 259.5 243.0 260.8 266.7 form not readily utilized. The addition of cellulose exerted a decided injurious effect upon crop yield due to competition for the available nitrogen between the microorganisms and the plants. The following years the nitrogen is made available again; however, one cannot speak here of any nitrogen fixation. Similar results were obtained with straw. SYMBIOTIC NITROGEN FIXATION Relation between the bacteria and the host plant. "Virulence" in con- nection with nodule bacteria has been defined as the ability of the organ- ism to penetrate into the root tissues of the host plant, to multiply there, 121 Hopkins, C. Soil fertility and permanent agriculture. Gin & Co., New York. 1910. 122 Rippel, A. Versuche aus dem Nachl&sz von Alfred Koch. Jour. Landw., 72: 17-52. 1924. FIXATION OF ATMOSPHERIC NITROGEN 589 and to cause a certain benefit or injury. Various attempts have been made to study this physiological property by vegetation experiments. It was found1215 that the activities (including the ability of fixing nitrogen) of the bacteria that have already penetrated into the plants increase with an increase in the amount of nitrogen available to the plants. Hiltner124 observed an increased growth of leguminous plants (peas) when grown continuously upon the same soil; he ascribed this not only to an increase in the number of bacteria causing inoculation, but also to an increase in the virulence of the bacteria, similar to an increase in virulence of a pathogenic organism when passed through several animals. Assuming that the nodule bacteria increase in virulence by repeated symbiosis with plants, Hiltner planted peas repeatedly on the same soil, which was sufficiently provided with minerals; he found an increase in the infection by the organism from the first to the fourth generation, a period without change then followed, and finally the continued growth of peas gradually led to a diminution in plant growth. On the basis of these results Hiltner proposed the "immunity" theory, according to which substances are formed by the bacteria within the nodules which immunize the plant against further invasion of bacteria. The organism (1) may not get into the plant, (2) it may gain admission, but without producing nodules because of the greater resistance of the plant, (3) it may enter the plant and produce nodules but without fixing any nitrogen, (4) it may fix nitrogen which is assimilated by the plant, (5) the bacterium may become more efficient than the plant, which is then injured, or (6) the bacterium itself may even be killed. According to the "immunity" theory, active nodules impart to the plant an immunity against bacteria of lower or equal virulence than those already found in the plant; only bacteria of higher virulence are capable of penetrating into the plant. The above theory was not confirmed by subsequent investigations. Nodules were found to be transient on biennial and perennial legumes, depending somewhat on the climatic conditions; i.e., there are two crops of nodules in biennial legumes, one each year, while there are many crops on perennial legumes such as alfalfa. When a fresh culture is added to a leguminous plant growing on agar and having already formed nodules, more nodules are formed on the new roots that have grown since the first inoculation. Under 123 Remy, Th. Ueber die Steigerung des Stickstoffsammlungs-Vermogens der Hulsenfruchte durch bakterielle Hilfsmittel. Deut. landw. Presse, 29: 31-32, 37-38, 46-47. 1902. 124 Hiltner, 1904 (p. 128). 590 PRINCIPLES OF SOIL MICROBIOLOGY these conditions, one cannot speak of plant immunity against further invasion by bacteria.125 The "equilibrium" theory proposed by Siichting,126 as an explanation for the mutual relationship between the leguminous plant and nodule- forming bacteria, is more plausible and has many facts to support it. A state of equilibrium was considered to exist between the attacking power of the bacteria and resisting power of the plant, due perhaps to the fact that the bacteria produce a toxin and the plants an antitoxin. The degree of equilibrium determines the extent of nodule formation, the plant becoming immunized by an antibody and not by a substance produced by the bacteria and the nitrogen supply being regulated by the production of the antibody. When the leguminous plants are grown in soil containing plenty of nitrates, their resisting power to the infec- tion of the bacteria is greater than when grown on nitrogen free media. The bacteria may vary in virulence, depending on the media in which they are grown. Increasing virulence was also found to be directly correlated with a shortening of the vegetation period of the plant. The nitrogen is fixed by the bacteria present within the nodules and is made available for the growth of the host plants by the autolysis of these nodules, or through the production of a bacteriophage by the plant, or perhaps as a result of the action of enzymes produced by the plant. The plant obtains its carbon from the C02 of the atmosphere by photo- synthetic processes; a part of the carbohydrates thus synthesized is transferred to the roots and used by the bacteria as a source of energy. This enables the microorganism to fix atmospheric nitrogen much more efficiently than it can ever do in artificial media, or even more efficiently than any non-symbiotic nitrogen fixing organism. Wunschik127 based his idea of the relation between the bacterium and the host plant on the statement of Beijerinck that "when living plant cells have to derive help from another organism, an equilibrium between the growth of both must be reached." The equilibrium is in this case between the vegetative energy of the plant and of the nodule forming organism. Wunschik differentiated between the vegetative energy, or ability to penetrate into the roots of the plant, and nitrogen-fixinsj; capacity of the organism. The vegetative energy of the bacteria results in the removal from the host plant of a part of its nutrients, thus causing i" Whiting, A. L.: 111. Agr. Exp. Sta., Bui. 179. 1915. 128 Suchting, H. Kritische Studien iiber die Knollchenbakterien. Centrbl. Bakt. II, 11: 377-388, 417-441, 496-520. 1904. i" Wunschik, 1925 (p. 13S). FIXATION OF ATMOSPHERIC NITROGEN 591 injury; the nitrogen-fixation by the bacterium is beneficial to the plant and is, to a certain extent, correlated with the vegetative energy of the bacterium. This stimulates the growth energy of the plants. The equilibrium established is between the growth energy of the plant, which enables it to utilize the nitrogen made available by the bacterium, and the vegetative energy of the bacterium. When this stage is reached, the growth of the plant continues uninterrupted. The vegetative energy of the nodule bacteria is increased by repeated physiological adaptation to the host plant, namely by repeated passage through the host plant. Chemistry of nitrogen- fixation by symbiotic bacteria. In the presence of an abundance of available nitrogen in the soil, the leguminous plants utilize that nitrogen and do not depend on the activities of the bacteria.12S Alkali nitrates in concentrations of 1 : 10,000 and ammo- nium salts in 1:2,000 repress nodule formation.129 The addition of 5 mgm. nitrogen as KN03 per liter of medium was sufficient to prevent the penetration of the bacteria into the roots of the plants in water cultures; this action was much less in sand and hardly obtained in soil. In some cases small amounts of nitrogenous substances were found to stimulate plant growth and nodule formation.130 The average amount of nitrogen fixed by a good crop of a legume, under favorable conditions, may be taken as 200 pounds per acre. If the energy need of the organisms is 100 parts of carbohydrate for every part of nitrogen fixed, as in the «ase of the non-symbiotic bacteria, the symbiotic bacteria would require 20,000 pounds of carbohydrate per acre for the fixation of the favorable amount of nitrogen. This would have to be supplied by the growing plant which is hardly imaginable since it would amount to two to four times as much as the total crop itself. We must assume that the organism uses the energy supplied by the plant much more efficiently than the non-symbiotic bacteria or that the process of nitrogen-fixation by the legume bacteria is exo- thermic.131 In the second case, the energy liberated is so small that it would hardly be sufficient to cover the need of the bacteria for metabo- 128 Wohltmann and Bergene. Die Knollchen-Bakterien in ihrer Abhangigkeit von Boden und Dungung. Jour. Landw., 50: 377-395. 1902. 129 Marchal, E. Influence des sels mineraux nutritifs sur la production des nodosites chez le pois. Compt. Rend. Acad. Sci., 133: 1032-1033. 1901. 130 Hiltner, L. Uber die Ursachen, welche die Grosse, Zahl, Stellung und Wirkung der Wurzelknollchen der Leguminosen bedingen. Arb. biol. Anst, K. Ges. Amt., 11: 177-222. 1900. 131 Christiansen-Weniger, 1923 (p. 570). 592 PRINCIPLES OF SOIL MICROBIOLOGY lism alone. It was, therefore, suggested131 that the organism actually utilizes the energy derived from the exothermic nitrogen fixation for metabolic processes. Of the three possible processes by which the nitrogen can be fixed, namely, (a) reduction, (6) oxidation and (c) direct union with organic compounds, the first is the most plausible, especially in view of the fact that a great many microorganisms assimilate the nitrogen in the form of ammonia. Whiting132 could not demonstrate any ammonia, nitrites or nitrates within the plants, his work tends to confirm the direct organic combination theory. Some evidence was previously obtained133 on the direct union of the free nitrogen with some organic compound in- side the bacterial cell; this compound was believed134 to be glycogen and carbamic acid as the first product of combination. Once the nitrogen has been fixed in the bacterial cells, it may be trans- ferred to the host when the bacterial cells (so-called bacteroids) are decomposed and the contents absorbed by the plants,135 or when the ni- trogen has been secreted by the bacterial cells in a form which the plant then utilizes.136 This removal of the products of bacterial growth by the plant was believed to stimulate further nitrogen fixation. The compo- sition of the nitrogenous substance of the bacterial secretions is still unknown, except that it is believed to be protein-like in nature. The formation of a bacteriophage in the nodules of the leguminous plants has been established.137 This bacteriophage dissolves the bac- teria and thus makes their contents available to the plant. It was found not only in the nodules but also in the roots and stems of the plants, and not in the leaves, also in garden and field soils, but not in prairie soils. The bacteriophage is specific in its lytic action and attacks only those bacteria which form the nodules in the roots of the specific plants. The bacteriophage can resist, according to species, a temperature of 60° 132 whiting, A. L., and Schoonover, W. R. Nitrogen fixation by cowpeas and nodule bacteria. Soil Sci., 10: 411-420. 1920. 133 Gerlach and Vogel, 1903 (p. 379). 134 Heinze, B. tlber die Stickstoffassimilation durch niedere Organismen. Landw. Jahrb., 35: 889-910. 1906. 135 Nobbe and Hiltner, 1900 (p. 127). 136 Golding, J. The importance of the removal of the products of growth in the assimilation of nitrogen by the organisms of the root nodules of leguminous plants. Jour. Agr. Sci., 1 : 59-G4. 1905. 137 Gerretsen, F. C., Gryns, A., Sack, J., and Sohngen, N. L. Das Vorkom- men eines Bakteriophagen in den Wurzelknollchen der Leguminosen. Centrbl. Bakt. II, 60: 311-316. 1923. FIXATION OF ATMOSPHERIC NITROGEN 593 to 65°C, for fifteen minutes. It withstands drying and passes through a thin collodium membrane; it also resists ultra-violet light eight times as much as the corresponding bacteria. To obtain the bacteriophage, fresh nodules, previously sterilized on the surface, are ground up and are placed in a nutrient medium. After 5 days, the turbid solution is filtered through a Chamberland filter and a few cubic centimeters of the clear filtrate is added to a fresh medium previously inoculated with a corresponding pure culture of the nodule organism. This is repeated after 10 days, diminishing every time the amount of liquid used for infection. This results in an accumulation of the bacteriophage in the culture, and if a few cubic centimeters of such a culture are added to a culture of Bad. radicicola, the turbid culture of the latter will become transparent due to the dissolution of the bacteria. When some of the bacteriophage is placed upon an agar slant and a culture of the nodule organism is then inoculated, the latter will grow only where the bac- teriophage was absent. It is also possible that the plant produces bacteriolytic enzymes, which hydrolize the bacterial cell liberating the available nitrogen. The bacterium itself seems to produce a cellulose-dissolving enzyme, by means of which it enters the root hairs of the host plant, dissolving the cell wall;138 however, this still needs confirmation. The presence of oxidase in the slime of various nodule bacteria has also been demonstrated.139 Production of gum by the nodule bacteria. In artificial cultures, Bad. radicicola produces a gum which goes partly into solution and is partly held by the zoogleal masses of the organism. This gum is precipitated by alcohol, acetone, concentrated solutions of ammonium sulfate, magnesium sulfate or ammoniacal lead acetate. It does not reduce Fehling's solution, but, on heating with a dilute solution of sulfuric acid (2 per cent) at 120°C. for one hour, reducing sugars are formed, indicat- ing that it is of a hemicellulose nature. It contains no protein or other forms of nitrogen. The gum is formed with various sources of energy in the medium, such as cane sugar, glycerol, or legume extract,140 and 138 Hiltner, L. Uber die Bakteroiden der Leguminoseknollchen und ihre willkt'irliche Erzeugung ausserhalb der Wirtspflanzen. Centrbl. Bakt. II, 6: 273-281. 1900. 139 Fred, E. B. A physiological study of the legume bacteria. Va. Agr. Exp. Sta. Ann. Rpt. 1911-1912, 145-173. 140 Buchanan, R. E. The gum produced by Bacillus radicicola. Centrbl. Bakt. II, 22: 371-396. 1909; Greig-Smith, R. The slime or gum of Rhizobium leguminosarum. Centrbl. Bakt. II, 30: 552-556. 1911; Fred, 1911-12. 594 PRINCIPLES OF SOIL MICROBIOLOGY should be considered as a synthesized product. The bacteria of the monotrichcus type do not produce as much gum as those of the peri- trichous type.141 Different strains of the same organism vary greatly in the amount of gum formed.142 A further study of the nature of this substance and its role in the fixation of nitrogen is desirable. Influence of reaction on the growth of Bad. radicicola and nodule forma- tion. Symbiotic nitrogen-fixing bacteria have limiting reactions which are not so sharply defined, however, as in the case of Azotobacter and Clostridium. Maze143 was the first to call attention to the fact that there are acid resistant and acid sensitive types of nodule bacteria. A sharp line of demarcation was obtained between the reaction permit- ting growth and the one entirely preventing it.144 The nodule bacteria were divided, according to their sensitiveness towards acidity, into five groups: limiting pH 1. Alfalfa and sweet clover 4.9 2. Garden pea, field pea and vetch 4.7 3. Red clover and common beans 4.2 4. Soybeans and velvet beans 3.3 5. Lupines 3.15 Of the nodule bacteria, the alfalfa organism is most sensitive to acid- ity and the lupine organism the most resistant to acidity. It was sug- gested that a correlation exists between the acid resistance of bacteria and acid resistance of higher plants. The critical reaction values for nodule formation are pH 4.0 and 9.0 to 10.0, with an optimum at about pH 7.0; this optimum may be higher or lower depending on the legume. Different biological types of alfalfa and soybean organisms may vary in their limiting reactions.145 In the case of the soybean organism one strain was found to have its acid limit at pH 4.0 to 4.5 and the other at 4.5 to 5.0. The optimum was found to be at pH 4.8 to 6.5, depending also on strains; the alkali limit was not so sharp. Germination of alfalfa seed in the soil is practically the same at reac- tions ranging from pH 4.5 to 7.0; it is greatly reduced, however, at pH 141 Burrill and Hansen, 1917 (p. 126). H2 Wright, 1925 (p. 127). 143 Maze, M. Les microbes des nodosites des l£gumineuses. Ann. Inst. Past., 13: 145-185. 1899. 144 Fred and Davenport, 1918 (p. 582). 146 Stevens, 1925 (p. 135); Wright, 1925 (p. 127). FIXATION OF ATMOSPHERIC NITROGEN 595 less than 4.5.146 Alfalfa yields were found to show an increase with an increase in the pH value of the soil from 3.8 to 7.0. The plants experi- ence difficulty in becoming established in soil with low pH values, but, after becoming established, they make an excellent growth at as low a pH as 3.8. With increasing hydrogen-ion concentrations (decrease of pH), nodule formation is decreased. According to Bryan,147 alfalfa organisms do not survive a greater acidity than pH 5.0, red clover organisms pH 4.5 to 4.7 and soybean organisms pH 3.5 to 3.9. The optimum growth for all strains of the alfalfa organism was found to be at pH 7.0, with a limiting acid reaction at pH 5.5.145 Nodule formation and nitrogen fixation. When leguminous plants are grown on soil properly limed, containing the necessary amount of min- erals, and inoculated with the proper organism, considerable nitrogen is found in the plant. Most of this nitrogen comes from the atmo- sphere. The presence of nitrates in the soil will retard and may even prevent, if present in sufficient quantities, nodule formation by the plant.148 The injurious influence of nitrate upon nodule formation is due largely to the fact that it offers a source of available nitrogen to the plant; it may also have a specific action upon the plant juice. But other nitrogen compounds, such as ammonium salts or ammonia-producing substances, may also reduce or even inhibit nodule formation.149 This is probably due to the direct assimilation of the combined nitrogen by the plants rather than to any inhibition of bacteria from penetrating into the roots. Carbonaceous substances, such as carbohydrates, certain or- ganic acids and alkaloids stimulate nodule production in the soil. However, appreciable amounts of nitrogen will be fixed even in the pres- ence of considerable quantities of available nitrogen, including nitrates, in the soil.150 The mechanism of nitrogen fixation by the plant was a subject of early 146 Joffe, J. S. The influence of soil reaction on the growth of alfalfa. Soil Sci., 10: 301-307. 1920. 147 Bryan, O. C. Effect of acid soils on nodule forming bacteria. Soil Sci., 15: 37-^0. 1923. 148 Strowd, W. H. H. The relation of nitrates to nodule production. Soil Sci., 10: 343-356. 1920. 149 Wilson, J. K. Physiological studies of Bacillus radicicola of soybean (Soja max Piper) and of factors influencing nodule production. N. Y. (Cornell) Agr. Exp. Sta. Res. Bui. 386. 1917. 150 Albrecht, W. A. Symbiotic nitrogen fixation as influenced by the nitro- gen in the soil. Soil Sci., 9: 275-319. 1920. 596 PRINCIPLES OF SOIL MICROBIOLOGY controversy. It was shown conclusively151 that leguminous plants (cow- pea and soybean) fix atmospheric nitrogen through their roots and not through their leaves, as it had been assumed in some cases. In the earlier part of the growth of the plant, the roots contain the larger part of the nitrogen while, at the time of harvest, 74 per cent of the nitrogen of cowpeas and soybeans was found in the tops. The fixation of the nitrogen takes place in the early stages of growth of the seedling, some- times within fourteen days. A study of the composition of leguminous plants152 established the presence of various amino acids and amides. Inoculation was found to increase the protein content of the plant, often without even increasing the crop yield. TABLE 60 Influence of inoculation and fertilization vpon yield, alkaloid and nitrogen content of Lupinus angustifolius YIELD OF GRAIN YIELD OF STRAW Weight Alkaloid content Nitrogen content Weight Nitrogen content Sterile, uninoculated 100 244 186 200 151 144 100 1648 558 678 738 577 100 554 229 236 300 277 100 119 111 113 56 59 100 Sterile, inoculated (average of two good preparations) . . ... 266 Sterile, fertilized with (NH4)2S04. . . Sterile, fertilized with NaN03 Unsterilized, uninoculated 113 103 66 Unsterilized, inoculated 81 The formation of branching forms, or bacteroids, in the nodule is due largely to specific nutrition, especially to the presence of alkaloids. Plants depending largely upon the bacteria for their nitrogen show a high alkaloid content; plants which obtain their nitrogen from inorganic nitrogenous compounds, especially lupines, are poor in alkaloids.153 Table 60 shows that inoculation resulted in a large increase in the nitrogen content of the plant, but this is accompanied by a still larger increase in its alkaloid content. When sterile plants obtain their 161 Whiting, 1915 (p. 590). 152 Schulze, E. Ueber den Umsatz der Eiweissstoffe in der lebenden Pflanze. Ztschr. physiol. Chem., 24: 18-114. 1895; 30: 241-312; 48: 387, 396. 1906. 153 Weber, E. tlber den Einflusz der Stickstoffernahrung auf den Bitterstoff- gehalt der Lupine. Inaug. Diss. Leipzig. 1920. FIXATION OF ATMOSPHERIC NITROGEN 597 nitrogen from (NH^SCh or NaN03, not only is the alkaloid content lower, but the nitrogen content of the plant decreases accordingly. Influence of environmental conditions upon the growth of symbiotic nitrogen- fixing bacteria. The limiting temperatures for the growth of nodule bacteria are 3° and46°C. ; the thermal death point is at 60° to 62°; the optimum varies between 18° and 26°.154 Bact. radicicola is not in- jured by diffused sunlight and can readily withstand direct sunlight. Drying injures the organism,155 but does not destroy it completely even after two years.156 The numbers of Bact. radicicola are greatly dimin- ished as a result of direct and rapid drying, as determined by the plate method ; however, the number of cells that actually remain alive is much greater than the number germinating on the plate.157 In the soil it may persist for at least several years, even in the absence of the host plant.158 Importance of symbiotic-nitrogen fixation in the soil. In the case of non-symbiotic nitrogen fixation, the evidence as to actual amount of nitrogen fixed under field conditions is still of doubtful value ; however, in the case of symbiotic fixation of nitrogen, the evidence is undisputed. The amount of nitrogen added to the soil by the bacteria depends upon the relative abundance of available nitrogen in the particular soil, both in inorganic and organic forms. The poorer the soil is in available nitro- gen (for the growth of the leguminous plants) and the richer it is in lime, available phosphorus and potash, the greater will be the gain in nitrogen. In addition to this, the kind of legume and seasonal condi- tions affect the amount of nitrogen fixed. The maximum amount of nitrogen was found to be fixed a little before, or just at blossoming time. "Warington159 pointed out in 1891 that an approximate increase of 350 pounds of nitrogen per acre may be obtained as a result of the growth of inoculated legumes (clover). Since then, extensive data have been secured, all of which point to definite increases in soil nitrogen due to the 154 Zipfel, H. Beitrage zur Morphologie und Biologie der Knollchenbakterien der Leguminosen. Centrbl. Bakt. II, 32: 97-137. 1912. 165 Chester, F. B. The effect of desiccation on root tubercle bacteria. Del. Agr. Exp. Sta. Bui. 78. 1907. 156 Ball, O. M. A contribution to the life history of Bacillus (Ps.) radicocola Beij. Centrbl. Bakt. II, 23: 47-59. 1909. 157 Duggar, B. M., and Prucha, M. J. The behavior of Pseudomonas radici- cola in the soil. Centrbl. Bakt. II, 34: 67. 1912. 158 Lipman and Fowler, 1915 (p. 129). 169 Warington, R. The circumstances which determine the rise and fall of nitrogenous matter in the soil. U. S. Dept. Agr. Off. Exp. Sta. Bui. 8, 22-4U 1892. 598 PEINCIPLES OF SOIL MICROBIOLOGY growth of leguminous plants in the presence of the proper bacteria. Poor soils are usually found to give larger gains than rich soils. Soils to which lime and phosphorus compounds have been added show greater increases in combined nitrogen than soils where those minerals were lack- ing. Inoculated soils give better results than uninoculated, particularly if the legume in question or the related forms have not been grown pre- viously on the same soil. Hiltner,160 for example, obtained an increase of 1.7 to 31 times the yield for lupines and 15-80 times for serradella as a result of inoculation with the proper organism. On the average, there may be a gain of 50 to 100 pounds of nitrogen per acre of soil due to the growth of legumes. Lipman and Blair161 found a gain of 54 pounds annually over a period of seven years from the growth of legumes in rotation with corn, potatoes, oats and rye in cylinders. According to Hopkins,162 a 3-ton crop of cowpea hay adds 86 pounds of nitrogen per acre, a 25-bushel crop of soybeans with 2\ tons of straw adds 106 pounds, a 4-ton clover crop adds 106 pounds and a 4-ton alfalfa crop adds 132 pounds. At least two-thirds of the nitrogen in legumes grown on normally productive soils is obtained from the air. Under optimum conditions and on a relatively poor soil as much as 400 pounds of nitrogen may be added per acre per year.163 The net yearly gain per acre from the growth of clover on a light sandy soil was found to be 50 pounds of nitrogen.164 From 120 to 250 mgm. of nitrogen are fixed per plant of red clover and alfalfa.165 If the tops are removed, the nitro- gen content of the soil may not be increased, since the amount fixed may be just sufficient to fulfill the need of the tops. In the case of per- ennial legumes, like alfalfa, there may not be an actual increase in soil nitrogen, as compared with uncultivated soils, although the nitrogen is higher than in the same soils upon which grains are grown.166 160 Hiltner, 1904 (p. 128). 161 Lipman, J. G., and Blair, A. W. Cylinder experiments relative to the utilization and accumulation of nitrogen. N. J. Agr. Exp. Sta. Bui. 289. 1916. 162 Hopkins, C. Nitrogen bacteria and legumes. 111. Agr. Exp. Sta. Bui. 94. 1904. i6j Wheeler, H. J. Cooperative experiments in alfalfa culture. R. 1. Agr. Exp. Sta. Bui. 152. 1912. 164 Shutt, F. T. Nitrogen enrichment of soils. Experiment Farms Rpt., Ottawa. 1912, 144-146. 166 Brown, P. E., and Stallings, J. H. Inoculated legumes as nitrogenous fertilizers. Soil Sci., 12: 365-407. 1921. 166 Swanson, C. O., and Latshaw, W. L. Effect of alfalfa on the fertility elements of the soil in comparison with grain crops. Soil Sci., 8: 1-39. 1919. FIXATION OF ATMOSPHERIC NITROGEN 599 Associative action of legumes and non-legumes. When non-leguminous plants are grown together with legumes, the former will obtain from the soil a larger quantity of nitrogen; this has been known since earlier times and has been pointed out by La Flize167 in 1892. It was later confirmed by other investigators. By placing a small unglazed porous pot in which a non-leguminous plant was growing, inside of a large glazed pot, in which a legume was growing, Lipman16S observed a favorable influence of the legume upon the non-leguminous crop; he ascribed this to the diffusion of the nitrogen fixed, from the legume to the non-legume. Lyon and Bizzell169 found that timothy grown alone contained 12.75 per cent pro- tein and, when grown together with alfalfa, 15.56 per cent. The same was true of oats when grown together with peas; the non-legume con- tained a greater amount of nitrogen when grown with the legume than when grown alone. 167 La Flize, S. Experiences sur L6gumineuses. Ann. Sci. Agron., 1: 174- 178. 1892. 168 Lipman, J. G. A method for the study of soil fertility problems. Jour. Agr. Sci., 3: 297-300. 1909: The associative growth of legumes and non-legumes. N. J. Agr. Exp. Sta. Bui. 253. 1912. 169 Lyon, T. L., and Bizzell, J. A. Availability of soil nitrogen in relation to the basicity of the soil and to the growth of legumes. Jour. Ind. Engin. Chem. 2: 313-315, 1910; also N. Y. (Cornell) Univ. Agr. Exp. Sta. Bui. 294. 1911. CHAPTER XXIII Transformation of Sulfur by Microorganisms Sources of sulfur in the soil and processes of transformation. In addition to carbon and nitrogen, there are a number of elements which are of prime importance in the growth of plants and microorganisms. We need mention only sulfur, phosphorus, potassium, iron, calcium and magnesium. The transformation of sulfur by microorganisms will be discussed at this point not because this element is more important than the others, but because, next to carbon and nitrogen and except for oxygen and hydrogen, it can be used by certain organisms for energy purposes and is required by the majority of organisms for structural purposes. Certain forms of sulfur may also be used as sources of oxygen under anaerobic conditions. Sulfur is more similar to nitrogen than any other element in the many transformations that it enters and in the types of microorganisms which produce these transformations. One finds in the sulfur cycle apparent duplications of the processes associated with the nitrogen cycle. Sulfur occurs in the soil and may be introduced there in the form of organic and inorganic compounds. The latter comprise elementary sulfur, sulfides and sulfates. The organic matter added to the soil contains from 0.1 to 0.5 per cent of sulfur, as shown in table 61. 1 The sulfur is present in the plant, chiefly in the cystine group of the protein molecule. When large quantities of sulfates are present in the soil, the plant may also contain sulfur in the form of sulfate. Cer- tain plants contain various volatile sulfur compounds, including cer- tain glucosides, such as sinigrin /OSO,K C3H6N:< XS • C«Hn06 which is decomposed in the soil to mustard oil (C3H5NCS), glucose and potassium acid sulfate.2 1 Hart, E. B., and Peterson, W. H. Sulphur requirements of farm crops in relation to the soil and air supply. Wis. Agr. Exp. Sta. Res. Bui. 14. 1911. 2 Peterson, W. H. Forms of sulfur in plant materials and their variation with the soil supply. Jour. Amer. Chem. Soc, 36: 1290-1300. 1914. 600 TRANSFORMATION OF SULFUR 601 The amount of sulfur brought down yearly by rainfall may be as much as 45 pounds per acre.3 The sulfur content of the soil varies from 0.01 to 0.09 per cent, the upper 6 to 7 inches of soil usually containing 250 to 1000 pounds of sulfur per acre.4 According to Kossowitsch,5 the average sulfur content of the upper 30 cm. of soil is 0.1 per cent S03, while that of the following 70 cm. is 0.025 per cent S03. One-half this amount is sufficient for 285 cereal crops or 70 alfalfa crops (cereal grains containing 0.29 to 0.45 per cent S03; straw, 0.26 to 0.55 per cent S03; alfalfa hay, 0.50 per cent S03). Considerable quantities of sulfur are also brought down by rainfall; this amounts to 1.93 to 14.17 mgm. S03 per liter, or about 10 pounds per acre (Kossowitsch). The quantity is considerably higher near large cities. Drainage waters TABLE 61 Sulfur content of various organic materials MATERIAL SULFUR Alfalfa hay per cent 0.287 Barley straw 0.147 Oat straw 0.207 Rye straw 0.049 Wheat straw 0.140 Red clover 0.164 Corn stover 0.120 Cottonseed meal 0.487 Turnip tops 0.900 are richer in S03 than rainfall; the concentration depending on climate, topography, type of soil, fertilization, etc. Sulfur is also added to the soil in the form of gypsum, superphosphates and elementary sulfur in artificial fertilizers. 3 Johnson, E. M. Analyses of rainfall from a protected and an exposed gage for sulfur, nitrate nitrogen, and ammonia. Jour. Amer. Soc. Agron., 17: 589- 591. 1925. * Woodward, J. Sulphur as a factor in soil fertility. Bot. Gaz., 73: 81-109. 1922; Olson, G. A., and St. John, J. L. An investigation of sulfur as a plant food. Wash. Agr. Exp. Sta. Bui. 165. 1921; Joffe, J. S. The role of sulfur in agri- culture. N. J. Agr. Exp. Sta. Bui. 374. 1922. 6 Kossowitsch, P. C. On the cycle of sulfur and chlorine in the soil and on the importance of this process in the life of soils and in the plant world (Russian). Zhur. Opit. Agron., 14: 181-218. 1913. 602 PRINCIPLES OF SOIL MICROBIOLOGY The sulfur does not remain long in the form in which it is introduced. It undergoes in the soil a series of reactions involving the activities of a number of microorganisms, the nature of which depends on the nature of the compound containing the sulfur. If it is introduced into the soil in the form of an organic substance, the organic matter is first decomposed by various heterotrophic bacteria, fungi and actinomyces and the sulfur bearing fraction is liberated. This is either assimilated by microorganisms or it is decomposed by various bacteria, and the sulfur is finally liberated as H2S. The part of the sulfur utilized by the microorganisms for the synthesis of microbial protein has to be decomposed again before the sulfur is liberated and made available for higher plants. The H2S is oxidized by autotrophic and facultative autotrophic bacteria to sulfur and then to sulfuric acid, which combines with the soil bases to give sulfates. The latter are either assimilated by higher plants or microorganisms and again transformed into pro- teins or reduced to H2S by specific reducing bacteria under anaerobic conditions. The H2S is of course again oxidized. When unoxidized or partially oxidized inorganic forms of sulfur, such as thiosulfates, sulfides, including hydrogen sulfide, and elementary sulfur, are added to the soil they are at first oxidized, if the soil aeration and moisture are favorable. These substances may originate from the decomposition of organic matter in the soil, in sulfur springs, in river and sea waters, from the reduction of sulfates, from volcanic eruptions, from the burning of coal or sulfide ore smelters. The oxi- dation of sulfur may be both chemical and biological in nature result- ing in the formation of sulfates. When soil conditions favor anaer- obiosis, as in soils saturated with water, or when sufficient aeration is lacking, the sulfates as well as the elementary sulfur may be reduced to sulfides. Sulfates may be leached out from the soil into lakes and seas, where they are reduced by other microorganisms to hydrogen sulfide. The transformation of sulfur in the soil may thus be summarized under four headings: (1) oxidation, (2) reduction, (3) synthesis (into proteins), and (4) decomposition of proteins and protein derivatives containing sulfur. The nature of oxidation of sulfur and its compounds in the soil. The question of the chemical or biological oxidation of sulfur has been the subject of various investigations. There is no doubt that some quantities of sulfur, particularly in a finely divided or colloidal state, as well as small amounts of H2S and sulfides, may be oxidized by chemi- TRANSFORMATION OF SULFUR 603 cal agencies, especially in the presence of proper catalysts. The oxi- dation of thiosulfate to tetrathionate and even to sulfate can be carried out by means of inorganic catalysts, the iodine ion and peroxide being sufficient for the first reaction and molybdic acid and a peroxide for the second:6 2 S203= + H202 i> S406= + 2 OH+ Na2Mo04 S203 + 4H202 > 2 S04= + 2 H+ + 3H20 The purely chemical theory of the process of sulfur oxidation has been suggested.7 With quartz sand containing iron oxide, as the me- dium for the transformation of the sulfur used in the form of milk of sulfur or in the colloidal form, the quantities given in table 62 were oxidized. The oxidation of sulfides which took place very rapidly, TABLE 62 Formation of SO* from 200 mgm. of S in 100 grams of soil in six weeks KIND OF BOIL RHOMBIC SULFUR MILK OF SULFUR Quartz sand mgm. 3.69 3.81 16.45 10.92 mgm. 44.64 Sandy soil 144.22 Loam soil 227.58 Meadow soil 284.02 especially in the case of the more soluble forms, was believed to go through the sulfur stage, as shown by the following reactions: Fe203 + 3H2S = Fe2 S2 + S + 3H20 Fe2S2 + S + 30 + 3H20 = Fe203-3H20 + 3 S The elementary colloidal sulfur was rapidly oxidized to sulfate; the rhombic sulfur, only very slightly. Kappen and Quensell themselves have brought forth data to demonstrate that considerably larger quantities of sulfur are oxidized in unsterile than in sterile soils. When active sulfur oxidizing organisms are present, it is easy to demonstrate that this process is primarily biological in nature. 6 Abel, E. Uber katalytische Reaktionsauslese. Ztschr. Elektrochem., 18: 705. 1912; 19: 480. 1913. 7 Kappen, H., and Quensell, E. Uber die Umwandlungen von Schwefel und Schwefelverbindungen im Ackerboden, ein Beitrag zur Kenntnis des Schwefel- Kreislaufes. Landw. Vers. Stat., 86: 1-34. 1915. 604 PRINCIPLES OF SOIL MICROBIOLOGY Maclntire, Gray and Shaw8 have also attempted to prove that sulfur is oxidized chemically in the soil, but considerably more sulfur was found to be oxidized under unsterile than under sterile conditions. A careful comparison of the chemical and biological oxidation of sul- fur led to the conclusion that the process is chiefly biological in nature.9 Of the various compounds, the soluble sulfides and especially K2S and CaS are rapidly oxidized in the soil by chemical agencies.10 Elementary sulfur and various sulfides are, however, oxidized in the soil more actively by microorganisms. It was thought originally that this process is limited to certain specific groups of bacteria which are capable of utilizing the energy obtained in the process of oxidation for chemosynthetic purposes. We are coming more and more to recognize that the property of slow oxidation of sulfur or of incompletely oxidized sulfur compounds, such as sulfides, is probably widely dis- tributed among microorganisms. Not only various common hetero- trophic soil bacteria (Bac. mycoides, Bad. fluorescens) are capable of oxidizing small amounts of elementary sulfur, in nutrient solutions containing organic nitrogen and sources of energy (glycerol),11 but also various common soil fungi and even actinomyces are reported as oxidizing small amounts of sulfur in artificial media and in soil.12 Pen. luteum seems to be particularly active in this connection. However, neither the oxidation of sulfur by chemical agencies nor its transforma- tion by heterotrophic microorganisms can compare with the rapidity with which sulfur is oxidized, when used as a source of energy by autotrophic bacteria. The mechanism of assimilation of elementary sulfur by heterotrophic microorganisms is still unknown. It may be 8 Maclntire, W. H., Gray, F. J., and Shaw, W. M. The non-biological oxida- tion of sulfur in quartz media. Jour. Ind. Eng. Chem., 13: 310-313. 1921. 9 Boullanger, E., and Dujardin, M. Mecanisme de Taction fertilisante du soufre. Compt. Rend. Acad. Sci., 155: 327-329. 1912; Brioux, Ch., and Guerbet, M. L'action fertilisante du soufre. Son evolution dans le sol. Ann. Sci. Agr. (4), 2: 384-396. 1913; Evolution du soufre dans le sol; etude sur son oxydation. Compt. Rend. Acad. Sci., 156: 1476. 1913; Demolon, M. A. Recherches sur Taction fertilisante du soufre. Compt. Rend. Acad. Sci., 156: 725-728. 1913. 10 Brown, P. E., and Kellogg, E. H. The determination of the sulfofying power of soils. Jour. Biol. Chem., 21: 73-89. 1915. 11 Demolon, A. Sur le pouvoir sulfoxydant des sols. Compt. Rend. Acad. Sci., 173: 1408-1410. 1921. 12 Abbott, E. V. The occurrence and action of fungi in soils. Soil Sci., 16: 207-216. 1923; Rippel, A. tlber einige Fragen der Oxydation des elementaren Schwefels. Centrbl. Bakt. II, 62: 290-295. 1924; Guittonneau, 1926 (p. 609). TRANSFORMATION OF SULFUR 605 reduced by means of an enzyme, as in the case of yeasts,13 to hydrogen sulfide and the latter assimilated and utilized for protein synthesis. The mechanism of autotrophic oxidation of sulfur has been studied in detail and is well known. In considering the process of sulfur oxidation by autotrophic bac- teria, we must differentiate carefully between the nature of the or- ganism and the sources of sulfur. Of the different groups that have been enumerated as capable of oxidizing sulfur and its compounds (p. 79), only the Thiobacillus group is found in normal soils. The presence of larger forms belonging to the unbranched types, accumu- lating sulfur within their cells, is possible only in muds or in soils kept under anaerobic conditions, where the formation of hydrogen sulfide takes place. The Thiobacillus group is present abundantly in all soils but the most active forms are found in soils receiving applications of sulfur as a fertilizer, either in organic (sewage, etc.) or inorganic forms. This is due either to their direct introduction into the soil or to a response due to the addition of the specific nutrient. It was shown, for example, that soils receiving stable manure or green manure were capable of oxidizing sulfur more rapidly than the untreated soils poor in organic matter. By increasing soil aeration and keeping the moisture content at 50 per cent of the moisture holding capacity, favorable conditions are created for the oxidation of sulfur in the soil. Oxidation of sulfur in the soil may be followed by (1) an increase in acidity as expressed by a change in the pH; (2) an increase in the amount of sulfates in the soil; (3) the disappearance of elementary sulfur. The last condition can be determined by extracting the re- sidual sulfur from the soil with acetone.14 Oxidation of sulfur by microorganisms. The biological oxidation of sulfur has been studied in detail by Winogradsky.15 As a source of sulfur H2S was used or conditions were made favorable for its production. The reactions involved in the process were presented as follows: 2H2S + 02 = 2H20 + S2 S2 + 302 + 2H20 = 2H2S04 H2S04 + CaC03 - CaS04 + H20 + C02 13 Morison, C. B. The production of hydrogen sulphide by yeast. Science, 60: 482-483. 1924. 14 Simon, R. H., and Schollenberger, C. J. The acetone method of extracting sulfur from soil. Soil Sci., 20: 393-396. 1925. "Winogradsky, 1887 (p. 80). 606 PRINCIPLES OF SOIL MICROBIOLOGY Elementary sulfur was formed as an intermediary product and was actually demonstrated in the cells of the bacteria (Beggiatoa and Thiothrix). Some organ- isms like Thiobacillus thiooxidans oxidize the H2S directly to sulfuric acid without forming elementary sulfur, while others, like Thiobacillus thioparus, liberate free sulfur outside of their cells. Thiosulfate is oxidized by microorganisms, according to the following group of reactions, depending on the organism taking part in the process: 3Na2S203 + 2iQ2 = 2Na2S04 + Na2S406 (1) Free sulfur was found to be liberated in the process; Nathanson16 considered this to be due to the interaction of the Na2S406 and Na2S203. However, according to Beijerinck,17 the reaction actually takes place as follows: Na2S203 + O = Na2S04 + S (2) TABLE 63 Oxidation of elementary sulfur to sulfuric acid by Thiobacillus thiooxidans AMOUNT OF CONTROL FLASK INOCULATED FLASKS ELEMEN- TARY INCREASB INCUBATION CULTURE SULFUR IN IN FLASK Sulfur Sulfate* Sulfur Sulfate DISAP- PEARED SULFATE days CC. mgm. of S mgm. of S mgm. of S mgm. of S mgm. of S mgm. of S 15 100 1,001 86.4 788 302.1 213 215.7 30 100 992 90.5 735 354.0 257 263.5 15 300 3,002 112.2 2,496 633.0 506 520.8 30 300 2,997 126.5 1,974 1,168.0 1,023 1,041.5 * Milligrams of soluble sulfates as sulfur in flask ; averages of 3 flasks are given ; the concentration of sulfates in the small flasks was greater, due to the fact that, in these, a medium containing 2 gm. (jNH4)2SQ4 and 0.5 gm. MgS04 per liter was used. It was believed that the sulfur was separated in the process of direct oxidation. However, the precipitation of the sulfur is probably caused by some secondary reaction. Trautwein18 suggested that the oxidation of the thiosulfate by the organism that he isolated takes place according to the following reaction: 7Na2S203 + 8|02 = 4Na2S206 + Na2S406 + 2Na2S04 (3) No sulfur was precipitated, the reaction did not become acid. Th. thiooxi- dans oxidizes thiosulfate directly to sulfate as follows:19 Na2S203 + 202 + H20 = Na2S04 + H2S04 (4) 16 Nathanson, 1902 (p. 84). These reactions have been discussed in detail elsewhere, in reference to energy utilization. 17 Beijerinck, 1904 (p. 84). 18 Trautwein, 1921 (p. 87). 19 Waksman, S. A., and Starkey, R. L. On the growth and respiration of sulfur-oxidizing bacteria. Jour. Gen. Physiol., 5: 285-310. 1923. TRANSFORMATION OF SULFUR 607 Elementary sulfur is oxidized to sulfuric acid: 2S + 302 + 2H20 = 2H2SO« The sulfur is oxidized quantitatively without any intermediary reactions taking place (table 63). In the presence of. tricalcium phosphate, the sulfuric acid interacts giving first di-calcium phosphate, then mono-calcium phosphate and finally phosphoric acid (figs. 43, 44): Ca3(P04)2 + H2S04 + 2H20 = Ca2(HP04)2 + CaS04-2H20 Ca2(HP04)2 + H2S04 + 2H20 = Ca(H2P04)2 + CaS04-2H20 Ca(H2P04)2 + H2S04 + 2H20 = 2H3P04 + CaSO«-2H,0 1CK00 9600 SQj. in MG P205 in MG 0 5 10 15 20 25 30 PERIOD OF INCUBATION IN WEEKS Fig. 43. Course of accumulation of citrate-soluble P2Os and S04 in composts of soil-rock phosphate and sulfur (from Lipman, McLean and Lint). When the sulfur is oxidized by the denitrifying organism, the reaction may take place as follows: 5S + 6KN03 + 2CaC03 = 3K,S04 + 2CaS04 + 2C02 + 3N2 5Na2S203 + 8KNO3 + 2NaHC03 = 6Na,S04 + 4K2S04 + 2C02 + 4N2 + H20 608 PRINCIPLES OF SOIL MICROBIOLOGY Soluble Sulfates inlOOCC Mg. of SO 4 150 OH 2.4 3.4 120 100 4.4 70 5.4 60 l i ft. fc UPvz? — — " Ph OS, OtK ypu. 5 i\uP ve £>L 7fa P I "ur >ve \ / / \ /' \ «• / \ s~ \ / / / \ / / \ / \ 1 1 / \ J__ 77 \ i / ft / 1 it \\ / // / \ \ / I ll / \ / \ ll / \ y i / r \ * i / 1 s I / /.• * .s ■') y v — s y *— - ^>' Soluble Phosphates inlOOCC Mcj. of: p 240 200 160 120 80 40 Days 0 15 5 10 Incubation period Fig. 44. Course of sulfur oxidation and transformation of insoluble phosphate into soluble forms by Th. thiooxidans in liquid media (from Waksman and Joffe). The addition of CaC03 frequently stimulates sulfur oxidation in the soil;20 it also prevents the injurious effect of sulfur oxidation upon the 20 Brown, H. D. Sulfofication in pure and mixed cultures with special refer- ence to sulphate production, hydrogen-ion concentration and nitrification. Jour. Amer. Soc. Agron., 15: 350-3S2. 1923. TRANSFORMATION OF SULFUR 609 nitrifying bacteria. The influence of sulfates, elementary sulfur, and reaction upon the oxidation of sulfur by Th. thiooxidans is shown in figures 45 and 46. According to Guittonneau,21 the oxidation of elementary sulfur in the soil may be carried out by two different or- ganisms, one of which oxidizes the sulfur (S) to hyposulfite (S202) and the other oxidizes the hyposulfite to sulfate (S03). An outside source of energy, either in the form of organic acids, carbohydrates or H- 500 1 •a 8 |300 N / "•'00 / a o "2 3 ^ 1 Cm. o 10 20 30 40 tiO Sulfur per 100 cc. of medium Fig. 45. Influence of sulfur content of medium upon the oxidation velocity of sulfur by Th. thiooxidans (from Waksman and Starkey). amino acids, is required. The rapidity of the process depends on the nature of the carbon source and the active organisms. It has been suggested22 that different sulfides are acted upon by different groups of organisms. Among the iron sulfides, markazite and ferrous sulfide are oxidized more readily than pyrite. 21 Guittonneau, G. Sur la formation d'hyposulfites aux depens du soufre par les microorganismes du sol. Compt. Rend. Acad. Sci., 180: 1142-1144. 1925; 181: 261-262. 1925; 182: 661-663. 1926. 22 Gubin, B. M. On the oxidation of sulfur and sulfides by soil bacteria (Rus- sian). Viestnik Bakt. Agron. Sta., 24: 52-74. 1926. 610 PRINCIPLES OF SOIL MICROBIOLOGY Reduction of sulfur and its compounds. When sulfur is added to the soil in an elementary form it is subject to reduction processes, especially when it comes in contact with the living protoplasm of bacteria, fungi, or yeasts;23 thiosulfates, tetrathionates and pentathionates are also subject to reduction processes, with the formation of H2S. Rey- 1 l'> Soil. jy' I / 1 20 i « a) 15 »•>. ^ V f N V X / / V > 3 / / , \ \ i \ \ \ i \. V \ 1 \ \ 3 4 \ 1 \ V .V " f. 5 i 5 3 5 4 > i .5 6 .5 7 5 Initial pH Fig. 46. Influence of hydrogen-ion concentration of medium upon sulfur oxida- tion by Th. thiooxidans (from Waksman and Starkey). Pailhade24 suggested that this process is enzymatic in nature. When sulfates are added to the soil, they are either assimilated by the plants 23 Rubner, M. liber den Modus der Schwefelwasserstoffbildung bei Bakterien. Arch. Hyg., 16: 53-72. 1893. 24 Rey-Pailhade, J. de. Etudes sur les propi6t6s chimiques de l'extrait al- coolique de levure de biere; formation de l'acide carbonique et absorption d'oxy- gene. Compt. Rend. Acad. Sci., 118: 201-203. 1894. TRANSFORMATION OF SULFUR 611 and microorganisms and transformed into proteins, or are gradually washed out in the drainage waters, or are reduced under anaerobic conditions. This phenomenon was explained25 as due to the produc- tion of nascent hydrogen by the microbes. It was suggested26 that the oxygen of the sulfate obtained in the reduction process is used for the oxidation of organic matter. 3CaS04 + 2C,HiOjNa = 3CaC03 + Na2C03 + 2H20 + 2C02 + 3H,S This process is exothermic, resulting in the liberation of a small amount of energy. The sulfate is usually first reduced to the sulfide which is then transformed into hydrogen sulfide, according to the reaction: CaS + C02 + H20 = CaC03 + H2S The organisms concerned in this process are described elsewhere (p. 188). The Microspira desulfuricans is not very sensitive to the products of its metabolism, and can withstand as much as 246 mgm. of H2S per liter of medium. The use of sulfates as a source of oxygen is limited to a closely related group of organisms, of which only three have been described, including a thermophilic form isolated by Elion (Vibrio ther- modesulfuricans) .27 Both sulfate and thiosulfate can be used as a source of oxygen and salts of organic acids as well as other compounds as sources of carbon. In crude cultures, the H2S formed from the reduction of sulfate in the presence of organic matter and in contact with oxygen is again oxidized by the sulfur-oxidizing bacteria. In curative muds and cer- tain lakes there is an increase in the H2S content with depth, starting from none at the surface and reaching a concentration of 30 mgm. at a depth of 25 to 30 meters. The hydrogen sulfide formed in the lower layers of the mud is oxidized to sulfate on reaching the surface; the latter is then again reduced when reaching the lower layers.28 26 Petri, R., and Maassen, A. Beitriige zur Biologie der krankheitserregenden Bakterien inbesondere liber die Bildung von Schwefehvasserstoff durch dieselben unter vornehmlicher Beriicksichtigung des Schweinerothlaufs. Arb. K. Gesund. Amt., 8: 318, 490. 1893. 26 Beijerinck and VanDelden, 1904 (p. 188). 27 Elion, L. A thermophilic sulfate-reducing bacterium. Centrbl. Bakt. II, II, 63: 58-67. 1924. 28 A detailed review of this subject is given by Nadson, G. On the hydrogen sulfide fermentation in the Veissovo-Salt lake and the part played by the micro- organisms in the formation of black mud. 1903; St. Petersburg (Russian) ; and Duggeli, 1919 (p. 82). 612 PRINCIPLES OF SOIL MICROBIOLOGY The presence of sulfur-reducing bacteria in deep layers of earth has been demonstrated by Wolzogen Kt'ihr29 who found them at depths of 6 to 35 meters. One can readily imagine that processes, similar to those taking place in muds, may also occur in the soil. The require- ments for sulfate-reduction by Microspira desulfuricans are (1) absence of free oxygen, (2) presence of organic compounds as sources of energy, (3) presence of sulfate as a source of oxygen, and (4) presence of essen- tial inorganic elements in available forms. The following reactions will then take place: 4C3H603 + H2S04 = 4C2H402 + H2S + 4C02 + 4H2 C2H402 + H2S04 = 2C02 + H2S + 2H20 or 4C3H60s + 2H2S04 = 3C2H402 + 2H2S + 6C02 + 4H2 + 2H20 Certain actinomyces are also capable of reducing sulfates to hydro- gen sulfide.30 Formation of //2S in the decomposition of organic matter. It has been pointed out elsewhere that a large number of bacteria, including aerobic and anaerobic forms, are capable of forming hydrogen sulfide and other volatile sulfur compounds in the decomposition of organic matter containing sulfur. Proteins 'may contain as much as 1.5 per cent of sulfur; on hydrolysis, sulfur is liberated from them partly in the form of H2S. This process is carried out by a large number of bacteria, especially certain obligate (Bac. putrificus, Bac. sporogenes) and facul- tative (Bad. coli, Bad. vulgare, Staph, pyogenes aureus) anaerobes. The formation of H2S can be tested by the use of a piece of bibulous paper saturated with lead acetate to which a little glycerol has been added. The paper is suspended in the upper part of the container over the growing culture ; it will at first become brown, then black. To determine the numbers of organisms in the soil capable of forming H2S from a specific organic material, it is sufficient to incorporate some lead acetate into an agar medium; the formation of a dark color will indicate the ability of the organism to produce H2S.31 The differences in the amounts of H2S formed by various organisms 29 von Wolzogen Kuhr, C. A. H. Occurrence of sulfate-reduction in the deep layers of the earth. Proc. Roy. Soc. Amsterdam, 25: 188-198, 1922. (Chem. Abstr., 17: 47). 30Sawjalow, 1913 (p. 189). 31 Burnet, E., and Weissenbach, R. J. Valeur des renseignements founds par la culture en gelose a l'acetate de plomb, pour la differenciation des bacilles typhique, paratyphique A et paratyphique B. Compt. Rend. Soc. Biol., 78: 565-568. 1915. TRANSFORMATION OF SULFUR G13 are quantitative rather than qualitative in nature;32 they are largely determined by the nature of the medium and oxygen tension. Ac- cording to Rubner,33 Bad. vulgare formed 33 mgm. of H2S under anaerobic conditions, and only 4 to 5 mgm. under aerobic conditions in one liter of peptone-free bouillon. Quantitatively, the H2S is de- termined by the difference in the total sulfur content of the medium.34 The gases formed may be absorbed in standard iodine solution from which the sulfide is determined by titration with thiosulfate. When organic sulfur compounds are decomposed by the agency of micro- organisms, most of the sulfur may be assimilated and resynthesized, as shown by Rubner for Bad. vulgare grown in a liter of bouillon: BEFORE THE EXPERIMENT AT THE END OF THE EXPERIMENT DIFFERENCE Sulfate S mgm. 6.1 52.8 1.2 mgm. 1.5 28.1 25.3 mgm. -4.6 Organic S Iron precipitated S, in the bacterial cells. -24.7 +24.1 The loss of 5.2 mgm. is due to the formation of H2S, while the larger portion of the organic sulfur is transformed into microbial protoplasm. When proteins and other sulfur bearing organic materials are added to the soil they are hydrolized by the activities of microorganisms into the various constituent groups and the sulfur-bearing amino acid, cys- tine or di- cystine, or di-jS-thio-a-amino- propionic acid liberated. S— S— CH2 I CH • NH2 CH I NH2 • HC HOOC COOH Cystine 32 A detailed study of the organisms concerned in the formation of H2S from various sulfur compounds is recorded by Sasaki, T., and Otsuka, I. Experi- mented Untersuchungen fiber die Schwefelwasserstoffentwicklung der Bakterien aus Cystin und sonstigen Schwefelverbindungen. Biochem. Ztschr., 39: 208- 215. 1912; Myers, J. T. The production of hydrogen sulfide by bacteria. Jour. Bact., 5: 231-252. 1920. 33 Rubner, M. Die Wandlungen des Schwefels im Stoffwechsel. Arch. Hyg. 16: 78-100. 1893. 34 See Almy, L. H. A method for the estimation of hydrogen sulfide in pro- teinaceous food products. Jour. Amer. Chem. Soc, 47: 1381-1390. 1925. 614 PRINCIPLES OF SOIL MICROBIOLOGY Another source of sulfur in an organic form is bile which contains taurine. CH2 • S02OH CH, • NH, These substances are decomposed in the soil or in culture media by microorganisms. The sulfur is liberated as H2S or in the form of mercaptans,35 depending on the organisms and environmental condi- tions, especially oxygen supply. The production of mercaptan from Z-cystine by Bad. vulgare is not affected by the presence of glucose, lac- tose and glycerol. Bad. vulgare and B. coli are also capable of forming H2S and ethyl sulfide from Z-cystine, independent of the presence of the carbon sources just named. Mercaptans, either the ethyl or methyl form (C2H5'HS or CH3-HS) often accompany H2S as a decomposition product of proteins under anaerobic conditions. These are often produced in mere traces. A definite parallelism has been found be- tween the influence of carbohydrates on bacterial multiplication and on the production of H2S from proteins. The rate of formation of H2S even increases in the presence of glucose, although the formation of amino-nitrogen remains stationary.31 Taurine is very resistant to the action of microorganisms.37 Influence of sulfur oxidation upon the transformation of minerals in the soil. The acid produced from the oxidation of elementary sul- fur by microorganisms can be utilized (1) as a solvent for such difficultly soluble minerals, as phosphorus in rock phosphate, potassium in glau- conite, or green-sand marl; (2) for the neutralization of excess base in alkaline soils; (3) for the control of certain plant diseases. When a compost of soil, sulfur and insoluble calcium phosphate is made, the sulfuric acid formed from the oxidation of the sulfur acts upon the phosphate and makes it soluble. By comporting 100 grams of soil, 5 grams of flowers of sulfur and 15 grams ground rock phosphate, 85 per cent of the phosphate is made available after a period of 30 weeks.38 A compost consisting of 100 parts of soil, 120 parts of sulfur and 400 parts of rock phosphate is the most economical com- 35 Petri and Maassen, 1893 (p. 611); Kondo, M. Uber die Bildung des Mer- captans aus 1-Cystin durch Bakterien. Biochem. Ztschr., 136: 198-202. 1922. 36 Heap, H., and Cadness, B. H. E. The influence of carbohydrates on H2S production by Bacillus acrtrycke (Mutton). Jour. Hyg., 23: 77-93. 1924. 37 Sasaki and Otsuka, 1912 (p. 613). 38 Lipman, J. G., McLean, H. C, and Lint, H. C. Sulfur oxidation in soils and its effect on the availability of mineral phosphates. Soil Sci., 2: 499-538. 1916. TRANSFORMATION OF SULFUR 615 bination for the production of phosphoric acid.'59 However, for the maximum transformation of the phosphate, a compost of equal amounts of sulfur and phosphate and a large amount of soil is required. When the compost is inoculated, the reaction takes place much more rapidly. Abundant aeration and optimum moisture offer favorable conditions; small amounts of ferrous and aluminum sulfates exert a stimulating effect. The uncomposted mixture cannot be added as such to the soil since the phosphate will not become soluble under soil conditions. The reaction of the soil would have to be made acid, before the trans- formation would take place. Fig. 47. Relation between sulfur oxidation and water-soluble potassium in composts containing sulfur and greensand marl (from Rudolfs). The same reactions take place in sterile liquid media inoculated with a pure culture of Th. thiooxidans. By adding 1 gram of powdered sulfur and one gram of chemically pure tri-calcium phosphate to 100 cc. of a synthetic solution, sterilizing and inoculating with a pure cul- ture of the organism, the following curves are obtained (fig. 44) .40 Composts prepared from greensand and sulfur will allow the libera- tion of small amounts of potassium at pH 2.7 to 2.3 (fig. 47); this 39 McLean, H. C. The oxidation of sulfur by microorganisms in its relation Co the availability of phosphates. Soil Sci., 5: 251-290. 1918. 40 Waksman, S. A., and Joffe, J. S. The chemistry of the oxidation of sulfur by microorganisms to sulfuric acid and transformation of insoluble phosphates into soluble forms. Jour. Biol. Chem., 50: 35-45. 1922. 616 PRINCIPLES OF SOIL MICROBIOLOGY potassium when introduced into the soil is readily assimilated by plants.41 Another interesting process in which the oxidation of sulfur may be utilized is the neutralization of the alkalinity of black alkali soils. In view of the fact that the alkalinity of such soils is due not only to the presence of sodium carbonate but to the fact that sodium forms the saturating base in the zeolitic silicates, large quantities of sulfur have to be applied before any marked effect is observed. The sulfur is readily oxidized and the sulfuric acid changes the carbonates to bicarbonates and then to sulfates. However, when insufficient amounts of sulfur are used, the zeolitic sodium will soon form fresh carbonates and the reaction of the soil will again become markedly alkaline. The acid not only neutralizes the carbonates, but coagulates the colloids, thus making the soil more permeable and allowing leaching to take place.42 An increase in the acidity of the soil which results from the applica- tion of sulfur can be utilized for the control of organisms which cause plant diseases, such as Act. scabies causing potato scab. However, an increased acidity may prove injurious to microorganisms whose activities are essential for the normal biochemical soil processes, such as the bacteria concerned in the process of nitrification, etc.43 The practical application of sulfur must, therefore, always be accompanied by a careful study of the reaction of the soil. 41 Rudolfs, W. Sulfur oxidation in inoculated and uninoculated greensand mixtures and its relation to the availability of potassium. Soil Sci., 14: 307- 319. 1922. 42 This process was studied in detail by Hibbard, P. L. Sulfur for neutralizing alkali soil. Soil Sci., 11: 385-387. 1921; Rudolfs, W. Sulfur oxidation in "Black alkali" soils. Soil Sci., 13: 215-229. 1922; Joffe, J. S., and McLean, H. C. Alkali soil investigations. Soil Sci., 17: 395-409. 1924; 18: 13-30, 133-149, 237- 251. 1924. 43 The influence of sulfur oxidation upon microbiological activities in the soil is discussed in detail by Vogel, J. Die Einwirkung von Schwefel auf die bakteriellen Leistungen des Bodens. Centrbl. Bakt. II, 40: 60-83. 1914. PART D SOIL MICROBIOLOGICAL PROCESSES AND SOIL FERTILITY . ... la terre vegetate est regardee comme un support actif, une chose vivante . . . . — M. Berthelot CHAPTER XXIV The Soil as a Medium for the Growth and Activities of Microorganisms To be able to understand the processes resulting in the soil from the activities of microorganisms, we must understand the nature of the medium, the soil, in which these organisms live, act and reproduce. The soil as a culture medium. The soil is a complex system consisting of (a) mineral particles formed as a result of mechanical and chemical decomposition of the various mineral constituents of native rocks; (6) organic matter of plant, animal and microbial origin, in the process of decomposition or resisting further decomposition; (c) compounds formed as a result of interaction of substances produced in the decom- position of organic matter with materials resulting from the disinte- gration of the inorganic soil complexes; (d) soil moisture, containing in solution C02, oxygen and other gases, and various organic compounds and inorganic salts; and finally (e) soil atmosphere. The soil is regarded1 as a mineral framework, the particles of which are coated with a jelly-like layer of organic and inorganic materials present in a colloidal condition, the soil solution being present partly in the colloidal layer and partly in a free condition. The microbial population of the soil lives largely in the colloidal layer and partly in the soil solution. The activities of the microorganisms living in the soil as a medium are affected by the nature of the different ingredients of the medium and by the various conditions influencing them. The complex medium supplies to the various organisms inhabiting it the necessary nutrients, of an organic and inorganic nature, con- sisting of the elements oxygen, hydrogen, carbon, nitrogen, phosphorus, sulfur, potassium, magnesium, calcium and iron; and, to a less extent, chlorine, silicon, sodium, aluminum and manganese. The availability 1 Page, H. J. The part played by organic matter in the soil system. Trans. Faraday Soc, 17: 272-287. 1922. Bouyoucos recently called attention to the fact that the colloids in the soil do not exist entirely as a coating around the soil grains, but are also independent components; sand particles may not be covered with colloidal gel (Soil Sci., 21: 481^88. 1926.) 619 620 PRINCIPLES OF SOIL MICROBIOLOGY of these nutrients as well as their utilization will be greatly influenced not only by the (1) physical and chemical composition of the solid part of the soil, but also by the (2) composition of the soil atmosphere, (3) composition of the soil solution, (4) reaction of the soil and (5) soil temperature. When any one of these factors is changed there is a correlated change in the biological composition of the soil; i.e., any modification in the physical, chemical or physico-chemical conditions of the soil will greatly affect the biological flora and its activities.2 We possess only fragmentary information concerning the biological responses to modifications of these soil conditions. Soil composition and microbiological activities. The solid, liquid, and gaseous phases of the soil influence individually and collectively the distribution and activities of microorganisms. The mineral frame- work is of prime importance. It is made up of (1) mineral matter derived from the disintegration of rock materials; (2) calcium carbo- nate, calcium phosphate, and organic matter deposited in the soil by the various marine organisms during soil formation; and (3) the mineral substances (zeolites) present in a colloidal state. The organic soil constituents contributing to the soil as a medium for the growth of microorganisms are (1) undecomposed plant and animal residues of recent origin; (2) various intermediary substances, various more or less inert constituents of the original organic matter added to the soil, and final products of decomposition of plant and animal substances; and (3) the various dead and living cells of microorganisms and the products resulting from their decomposition. The mineral framework influences the activities of microorganisms by modifying the mechanical condition of the medium, by forming sub- stances (such as the zeolitic silicates) which, either themselves or by interaction with certain of the organic compounds, form the colloidal film in which most of the microbial transformations take place, by offer- ing direct mineral nutrients to the microorganisms, and by combining with various products of the metabolism of microorganisms, such as the organic and inorganic acids. The inorganic as well as the organic soil colloids give to the soil such desirable properties as its capacity for absorption and retention of water and bases and its buffering action, which regulates changes in 2 Lantsch, K. Bacillus amylobacter A. M. et Bred, und seine Beziehungen zu den Kolloiden. Centrbl. Bakt. II, 54: 1-12. 1921; Konig, J., and Hasenbaumer, J. Die Bedeutung neuer Bodenforschungen fur die Landwirtschaft. Landw. Jahrb., 55: 185-252. 1920. THE SOIL AS A CULTURE MEDIUM 621 reaction. They may also exert a direct influence upon the distribution and nature of action of microorganisms in the soil. The organic matter of the soil gives to it a brown to black color. The higher moisture holding capacity of the soil is due to a large extent to a higher content of organic matter. Carbon is present in the soil chiefly in the various organic substances collectively termed "humus" and in the form of carbonates. The nitrogen is present in the soil in the form of complex proteins and their derivatives3 as well as other complex nitrogenous compounds largely derived from the cells of micro- organisms inhabiting the soil; only about one per cent of the nitrogen of the soil is present there as ammonia and nitrates. The various minerals required by the microorganisms for their activities are present partly in the mineral framework, partly in the organic and inorganic soil colloids, partly in the soil solution, and partly in a precipitated form. The soil moisture4 is combined with the colloidal materials, forming the liquid phase of the complex, and is also in a free state. The amount of moisture that a soil can hold varies with the size of these particles and will be low in the coarse sandy soils and greater in the fine clay and especially in the peat soils, which consist largely of organic matter in a colloidal state. The minimum and optimum amounts of moisture for the activities of microorganisms will depend upon the nature of the soil and its colloidal content. Using the evolution of C02 from glucose as an index of the activities of microorganisms, Van Suchtelen5 found that when a loam soil contained only 4.4 per cent moisture, the activi- ties were at a standstill. When the moisture was increased to 6 per cent, 19 mgm. of C02 were formed, and with 15 per cent moisture, 208 mgm. were formed. Similar results were obtained when the number of bacteria developing on the plate was used as an index of microbio- logical activities.6 Optimum moisture conditions, for the activities of many soil microorganisms, are reached when about half the pore space 3 Jodidi, S. L. The chemical nature of the organic nitrogen in the soil. Iowa Agr. Exp. Sta. Res. Bui. 1. 1911; Potter, R. S., and Snyder, R. S. Jour. Agr. Res., 6: 61. 1916. 4 Soil sampling as well as moisture determination is discussed in detail by E. Heine. Die Praktische Bodenuntersuchung. Borntniger, Berlin, 1911; and J. Stoklasa. Methoden zur biochemischen Untersuchung des Bodens. Abder- hald. Handb. biochem. Arbeitsmeth. 5, Pt. 2, 843-910. 1925. * Van Suchtelen, 1910 (p. 684). 8 Engberding, 1909 (p. 14). 622 PRINCIPLES OF SOIL MICROBIOLOGY of the soil is filled with water. In the case of light sandy soils this con- dition obtains when the water content is about 8 to 10 per cent of its weight and, in heavy silt or clay soils, when the water content is 16 to 20 per cent or more. As the soil dries out the activities of the organ- isms are gradually reduced ; the larger forms, like the fungi, suffer most while the smaller and more resistant forms, like the actinomyces and spores of bacteria,7 suffer least. Excessive moisture may prove un- favorable to aerobic microorganisms by limiting the supply of oxygen, while anaerobic bacteria are favored Since the microorganisms live largely upon the colloidal film surround- ing the inorganic soil particles, the lower the colloidal content of a soil the smaller is the amount of water necessary to bring it into a condition favorable for the activities of microorganisms. An excess above the optimum amount of moisture will prove injurious to the ac- tivities of the aerobic organisms. The heavier the soil, the higher must be the water content to bring about equal decomposition of organic nitrogenous substances. Using the liberation of ammonia and nitrate from horn meal as an index of the activities of microorganisms, Miinter8 demonstrated (table 64) that in a sandy soil decomposition did not vary within the moisture range of 6 to 18 per cent (decreasing at greater moisture contents). The rapidity of decomposition reached its high- est point during the first three weeks in the sandy soil. In a loam soil, decomposition was twice as great at 16 per cent moisture as at 8 per cent, and was constant between 16 and 24 per cent. The in- fluence of the moisture content was still more marked in a clay soil. Only 0.63 per cent of the material was decomposed in three weeks at 8 per cent moisture, while at 18 and 28 per cent moisture and in the same period of time, 30.80 and 36.36 per cent of the material was de- composed respectively. After twelve weeks, 13.35, 63.21 and 57.94 per cent of the material was decomposed at the respective moisture contents. The effect of moisture is also shown in the composition of the soil flora and the nature of the processes occurring in the soil will conse- quently be materially affected. The decomposition of cellulose can readily illustrate this phenomenon. In the presence of sufficient avail- able nitrogen and minerals, cellulose is decomposed at a medium mois- 7 Hoffmann, C. Relation of soil bacteria to evaporation. Wis. Agr. Exp. Sta. Res. Bui. 23. 1912. 8 Miinter, F. Untersuchungen iiber chemische und bakteriologische Umsetz- ungen im Boden. Landw. Jahrb., 55: 62-138. 1920. THE SOIL AS A CULTURE MEDIUM 623 ture content by filamentous fungi and aerobic bacteria ; at a low mois- ture content, by filamentous fungi and actinomyces; when the soil is saturated with water, anaerobic bacteria are largely concerned with the decomposition of the cellulose. When sugar is added to the soil and the development of microorganisms is followed by direct micro- scopic examination, Azotobacter is found to develop under aerobic and Bac. amylobacter under anaerobic conditions. The latter condition TABLE 64 Influence of moisture upon the formation of ammonia and nitrate [from horn meal) in different soils SOIL TYPE MOISTURE CONTENT PERIOD OF INCUBATION AMMONIA AND NITRATE NITROGEN per cent weeks per cent ' 6 3 45.7 12 60.2 Sand \ 12 3 12 42.4 55.5 18 3 38.5 . 12 55.0 • 8 3 18.9 12 49.7 Loam ( 16 3 12 45.5 53.9 24 3 39.5 12 39.0 8 3 0.6 12 15 4 Clay < 18 3 12 29.1 54 9 28 3 36.9 12 24.2 may be reached below the surface even when the moisture content of the soil is only 40 per cent of saturation.9 The influence of moisture upon the activities of bacteria is due to two factors:10 (1) penetration of atmospheric oxygen through the medium; 9 Winogradsky, 1924 (p. 542). 10 Rahn, O. Die Bakterientatigkeit im Boden als Funktion der Nahrungs- konzentration und der unloslichen organischen Substanz. Centrbl. Bakt. II, 38: 484-494. 1913; Bacterial activity in soil as a function of the grain-size and moisture content. Mich. Agr. Exp. Sta. Tech. Bui. 16. 1912. 624 PRINCIPLES OF SOIL MICROBIOLOGY (2) rapidity of diffusion of nutrients. A high moisture content is more favorable for bacteria, but it diminishes aeration; when the soil is saturated with water, oxygen can penetrate only by solution, which is too slow for the development of aerobic organisms. When the moisture content of the soil is diminished, aeration increases. The larger the inorganic soil particles the greater are the possibilities for aeration. The smaller the inorganic soil particles, the greater is the amount of moisture required for the activities of microorganisms and the less is the soil aerated. TABLE 65 Comparative mineral composition of surface soil and of lithosphere COMPOUNDS Si02 AI2O3 Ti02 Fe203 MnO Na20 K20 CaO MgO P206 S03 N2 Organic matter Carbon AMERICAN SURFACE SOILS LITHOSPHERE per cent per cent 84.67 59.77 6.73 14.89 0.66 0.77 2.53 6.25 0.06 0.09 0.49 3.25 1.03 2.98 0.40 4.86 0.27 3.74 0.09 0.28 0.09 0.28 0.07 2.61 1.51 0.03 Aeration of the soil or the diffusion of the soil gases greatly influence the activities of microorganisms. Oxygen is needed for oxidation proc- esses, CO2 for the activities of autotrophic bacteria, and nitrogen for the nitrogen-fixing organisms.11 Oxidation favors the activities of ni- trate-forming and nitrogen-fixing bacteria, fungi, actinomyces, and other organisms which bring about the decomposition of organic matter. The processes of decomposition may be so rapid in well aerated soils that farmers must resort to rolling, marling and manuring in order to maintain the proper supply of organic matter in the soil. In hepvy fine-grained soils with insufficient aeration, the decomposition of or- 11 Russell, E. J., and Appleyard, A. The composition of the soil atmosphere. Jour. Agr. Sci., 7: 1-48. 1915; 8: 385^17. 1917. THE SOIL AS A CULTURE MEDIUM 625 ganic matter may be too slow and the farmer may use drainage, tillage, liming and manuring to intensify aeration. Insufficient aeration favors reduction processes. The mineral composition of the soil. While nitrogen and, to a large extent, sulfur are present in the soil almost entirely in organic form and the carbon utilized by the heterotrophic organisms is also of complex organic nature, the other elements are largely or entirely of inorganic origin. Phosphorus exists in the soil as apatite (Ca5(P04)3-Cl or Ca5(P04)3-F), calcium phosphate (Ca3(P04)2), iron and aluminum phos- phates, as well as in various organic combinations. Potassium occurs in the soil in orthoclase and microcline feldspar (KAlSi308), in mus- covite mica (KH2Al3Si30i2) and in hydrated and non-hydrated alum- inum silicates and to a less extent in organic combination. Calcium exists in the soil in various minerals such as calcite, plagioclase, feldspar, hornblende and augite, and in absorbed compounds with kaolinite, etc. Table 65 shows the composition of some typical American soils com- pared with the composition of the lithosphere.12 It is only seldom that the mineral constituents become limiting fac- tors to the development of microorganisms in normal soils. These minerals influence the activities of the organisms by modifying the soil reaction, the concentration of the soil solution and by serving as direct nutrients and in some cases even as sources of energy. The physico-chemical role of organic matter in the soil. Organic matter plays a manifold role in the soil. Most soil microorganisms find in it their source of energy and other nutrients. Its influence on soil texture is of especial importance. Organic matter helps to loosen a clay soil and add body to a sandy soil. It is best to apply only undecomposed organic matter to heavy soils, since the large quantities of C02 pro- duced in the process of decomposition tend to make the heavy soil porous. However, it is best to subject the organic matter first to partial de- composition before applying it to sandy soils, so as not to make the soil too open. The colloidal "humus" seems to have a cementing ef- fect upon the coarser soil particles. It also exerts a protective effect upon clay, so that greater concentrations of electrolytes are required for its flocculation. 12 Robinson, W. O. The inorganic constituents of some important American soils. U. S. Dept. Agr. Bui. 122. 1914. Methods of chemical and mechanical soil analysis are discussed by Russell and others (see list of texts). 626 PRINCIPLES OF SOIL MICROBIOLOGY Due to its large capacity for absorbing water, the soil organic matter causes a swelling of the soil when wetted. The influences of successive drying, moistening and freezing modify the nature of the organic mat- ter and make it more available to the activities of microorganisms with the production of larger amounts of C02 and nitrate. When a soil is well cultivated, the formation of spaces allows a rapid development of the fungi, actinomyces and aerobic heterotrophic bacteria. These decompose the organic matter, use the available oxygen, and form C02 as one of the chief products. The gases formed produce a certain pressure which brings about a further extension of the spaces between the soil particles and create a condition referred to in German as "Bodengare," or puffing up of the soil similar to the rising of bread. An improved tilth results. This condition is favored by fertilizing the soil with stable or green manure, by liming the soil and by thorough cultivation. The colloidal organic matter increases the water holding capacity of the soil and helps to warm up the soil more rapidly, due to a decrease in the evaporation of water, to better drainage and absorp- tion of the radiant heat of the sun. The organic colloids exert a definite buffer effect upon the reaction of the soil solution;13 they also regulate the composition and concentration of the solution. Colloidal condition of soils1* and microbiological activities. Colloids are characterized by a group of reactions which were believed originally to differentiate them sharply from crystalloids, and especially by the fact that they show no marked tendency to diffuse in solutions or to pass through semi-permeable membranes. With the advance of our knowledge on colloids, it has been found that no sharp line of demarca- tion can be drawn between them and crystalloids. Both conditions depend on the method of preparation, including the nature of the sol- 13 Oden, Sven. The application of physico-chemical methods to the study of humus. Trans. Faraday Soc, 17: 288-294. 1922. 14 The following texts give an excellent discussion of the subject of colloids. Ostwald, Wo. Colloid chemistry (Tr. Fischer). 1916; Zsigmondy, R. Kol- loidchemie. 5th Ed. Spamer. Leipzig. 1925; Taylor, \V. W. Chemistry of colloids. 3d. Ed. New York. 1915; Freundlich, H. Kapillarchemie. 2d Ed. Leipzig. 1922; Ehrenberg, P. Die Bodenkolloide. Steinkopff. Leipzig. 2d Ed. 1922; Bechhold, H. Colloids in biology and medicine (Tr. J. G. M. Bul- lowa). Van Nostrand Co. New York. 1919; Burton, E. F. The physical properties of colloidal solutions. Longmans. 2d Ed. 1921; Casuto, L. Der kolloidale Zustand der Materie. Steinkopff. 1913; Vv'iegner, G. Boden und Bodenbildung in kolloidchemischer Betrachtung. T. Steinkopff. 3rd Ed. Dresden. 1924; Bogue, R. H. The theory and application of colloidal beha- vior. 2 vols., McGraw-Hill. New York. 1924. THE SOIL AS A CULTURE MEDIUM 627 vent employed. We speak now of colloidal systems rather than of colloidal substances. Colloidal systems are not stable. Colloids are also characterized by extended surfaces which allows them to absorb water and dissolved substances from a solution. The attraction for water is both chemical and physical. Heat of wetting is characteristic of the soil colloids. An active soil colloid may be changed to an inac- tive one by changing its physical condition (ignition) and also to some extent by changing its chemical condition.15 Soil colloids consist largely of silica, alumina, iron oxides and or- ganic matter.16 The natural organic matter added to the soil consists largely of colloidal substances (proteins, celluloses, starches, lignins). When these substances are acted upon by microorganisms, they are partly transformed, either directly or through a series of stages, into crystalloids, and partly into new colloids, namely the soil organic matter (p. 6S0). The clay portion of the soil is in itself colloidal in nature. Colloids are usually separated from crystalloids by dialysis, ultra- filtration, and centrifuging. The osmotic pressure of colloidal solu- tion is small, due to the large molecular weight; diffusion is, therefore, also small ; the lowering of the freezing point is almost negligible. When the colloids are transformed by microorganisms into crystalloids, the molecular weight is decreased with a corresponding increase in osmotic pressure, diffusion and lowering of freezing point. A study of the chemi- cal properties of the proteins as organic colloids led to the conclusion17 that the theory of amphoteric colloids is in its general features identical with the theory of inorganic metal hydroxides. The colloidal properties of soil which are of special importance in the growth of microorganisms in the soil as a culture medium are (1) the absorption of substances from solution and their concentration upon the surface of the colloid, including substances used as nutrients by microorganisms and those which may be injurious to their activities; (2 1 ability to absorb water in large amounts; (3) flocculation and de- flocculation phenomena of colloids themselves and of bacterial cells con- sidered as colloids; (4) the "sol" and "gel" states of the colloids; (5) modification of soil conditions, such as reaction. 15 Anderson, M. S. The heat of wetting of soil colloids. Jour. Agr. Res., 28: 927-936. 1924; Bouyoucos, G. J. The chief factors which influence the heat of wetting of soil colloids. Soil Sci., 19: 477-483. 1925. 16 Robinson, W. O., and Holmes, R. S. The chemical composition of soil colloids. U. S. Dept. Agr. Bui. 1311. 1924. 17 Loeb, J. Proteins and the theory of colloidal behavior. McGraw-Hill. New York. 1922. 628 PRINCIPLES OF SOIL MICROBIOLOGY Colloids are coagulated, or flocculated, by means of electrolytes; the univalent ions have a less intensive effect than the bivalent and the latter less than the trivalent. Clay is an electro-negative colloid and can be flocculated by positively charged ions; at a certain point it is deflocculated by the negatively charged hydroxyl ion. This defloccu- lation may be due to the lessened solubility of the di- and tri-valent cations in the soil rather than to the direct effect of the hydrogen-ion concentration.18 Of particular importance is the action of calcium in flocculating colloids. Sodium salts, notably sodium carbonate, serve to deflocculate the soil colloids, although a sufficient concentration of sodium ions may even cause flocculation. The flocculation of soil particles is similar to the flocculation of suspensoid sols and is amenable to the isoelectric theory, except in the case of lime.19 Soil organic matter has a protective effect on the flocculation of clay.20 Calcium carrying a double positive charge precipitates the negative soil colloids, bringing about a change in the plastic properties of the soil. The soil structure is thus changed entirely; the resistance to penetration of moisture is reduced, an increase in pore space is brought about, and there is an increase in the water holding capacity of the soil. This improves the physical condition of the soil as a medium for the activities of microorganisms. Drying of the soil also causes a precipitation of colloids, but the change produced in the colloidal soil is reversible. The change produced by lime is not reversible. The soil organic compounds contain reversible and irreversible colloids. The addition of lime brings about an increase in the water- soluble carbon compounds of the soil which favorably influences bac- terial activities (in addition to favorable effect of reaction). This leads to a greater decomposition of the soil organic matter with the formation of C02, NH3, nitrates and soluble phosphates. Thus the application of lime leads to a neutralization of the soil acids, an in- 18 Dayhuff, W. C, and Hoagland, D. R. The electrical charge on a soil col- loid as influenced by hydrogen-ion concentration and by different salts. Soil Sci., 18: 401-408. 1924. 19 Comber, N. M. The mechanism of flocculation in soils. Trans. Faraday Soc, 17: 349. 1922; Jour. Agr. Sci., 10: 425-436. 1920; Mattson, S. E. Die Beziehungen zwischen Ausflockung, Adsorption, and Teilchenladung mit besond- erer Berucksichtigung der Hydroxylionen. Inaug. Diss. Breslau. 1922; Kol- loid Chem. Beihefte, 14: 227-313. 1922. 20 Wolkoff, M. I. Studies on soil colloids. I. Flocculation of soil colloidal solutions. Soil Sci., 1: 585-601. 1916; Oden, Sven. Die Koagulation der Tone und die Schutzwirkung der Humussiiure. Jour. Landw., 67: 177-208. 1919. THE SOIL AS A CULTURE MEDIUM 629 crease in the decomposition of the soil organic matter, greater libera- tion of C02, mineralization of the organic matter, absorption of bases and increase in colloidal matter.21 Van Bemmelen32 was the first to show that soil organic matter (humus) plays an important part in the absorption of both basic and acid radicals from the soil solution; this process of absorption was found to be similar in nature to that of an artificial calcium-aluminum silicate. However, different forms of absorption in the soil are often recognized:23 (1) biological absorption or the assimilation of the anions or cations by microorganisms; (2) mechanical absorption, or the mere mechanical retention of particles suspended in water; (3) physical, or surface ad- sorption, which may be positive or negative, depending on the fact as to whether the substance decreases or increases the surface tension of the dispersion medium; (4) physico-chemical, or adsorption in the narrow sense, which consists in the exchange of bases between the added salt and the zeolitic or alumino-silicate (and humic) complex of the soil ; and finally (5) chemical absorption, or the chemical interaction between two substances giving difficultly soluble compounds, as in the formation of calcium phosphate from the carbonate and soluble phos- phate. Certain investigators, however, do not take the view that any sharp differentiation exists between chemical and physical reactions, which may all be due to electrical forces differing only in degree; this applies especially to the exchange of bases in the soil. In proportion to their total mass, colloids exhibit a remarkable power of adsorption because of the large surface that they possess. Adsorp- tion increases with the concentration of the solute. The absorption of ammonia by soils or by calcium silicates (permutite) from a solution of an ammonium salt follows the laws of adsorption.24 The same was found to hold true for the adsorption of other bases. Of the acids, the phosphates alone are absorbed, or rather precipitated on interaction 21 Thaer, W. Der Einflusz von Kalk unci Humus auf die mechanische, physik- alische und chemische Beschaffenheit von Ton-, Lehm- und Sandboden. GJt- tingen. 1910 (Centrbl. Bakt. II, 32: 271-274. 1912). 22 van Bemmelen, J. M. Die Absorptionsverbindungen und das Absorptions- vermdgen der Ackererde. Landw. Vers. Sta., 35: 69-136. 1888; Die Absorp- tion. Dresen. Steinkopff. 1910. 23 Gedroiz, K. K. On the absorptive power of soils. Zhur. Opit. Agron., 19: 269-322. 1918; Leningrad. 1922. 24 Wiegner, G. Zum Basenaustaush in der Ackererde. Jour. Landw., 60; 111-150, 197-222. 1912. 630 PRINCIPLES OF SOIL MICROBIOLOGY with the soil bases (Ca, Mg). When bases are absorbed by the soil, they displace an equivalent amount of another base which is combined in the soil either with the inorganic zeolites or with the organic compounds. The adsorption of dyes by soils, which depends upon the surface of the soil, has been used for the estimation of colloids of the soil by as- suming that only the colloids in the soil take part in this process.25 Colloids play an important part in making the soil a favorable med- ium for the growth of microorganisms, by absorbing the soluble fer- tilizing elements added to or produced in the soil and by their buffering properties in preventing rapid changes of the soil reaction. The growth of bacteria was found26 to be a function of the soil surface; in culture media colloidal silicic acid and its compounds, as well as colloidal ferric and aluminum hydrates and humus stimulate nitrogen fixation by Azotobacter, possibly by absorbing nitrogen gas. A colloid (like soil extract, gelatin, etc.) quickly shortens the period necessary for germ- ination of the spores of Bac. amylobacter A. M. and Bred, in a nutri- ent solution.27 In the presence of 0.25 to 1.0 per cent gelatin, the period of incubation, from inoculation to beginning of fermentation, was reduced from fifty-one days in a nutrient solution free from colloids to three days. In the presence of a colloid, a clear zone is found to surround the spores; this zone is absent in a suspension of spores in a colloid-free solution. The shortening of the period neces- sary for spore germination depends on the dispersion of the colloid and is explained chiefly by adsorption phenomena.28 26 A detailed discussion of the subject of absorption is found in the following papers. Whitney, M., and Cameron, F. The chemistry of the soil as related to crop production. U. S. Dept. Agr. Bur. of Soils, Bui. 22. 1903; Bui. 23. 1904; Cameron, 1911 (p. xiv), p. 61; Wiegner, G. Die Festlegung des Stickstoffs durch sogenannte Zeolithe. Jour. Landw., 61: 11-58. 1913; Prescott, J. The phenomenon of absorption in its relation to soils. Jour. Agr. Sci., 8: 110-130. 1916; Gedroiz, 1922 (p. 629); Fischer, E. A. The phenomena of absorption in soils: a critical discussion of the hypotheses put forward. Trans. Farady Soc, 17: 305-316. 1922; Mattson, 1922 (p. 628). 26 Sohngen, N. L. Einflusz von Kolloiden auf mikrobiologische Prozesse. Centrbl. Bakt. II, 38: 621-647. 1913. "Lantzsch, 1921 (p. 620). 28 Further information on the influence of colloids upon the activities of micro- organisms is given by Plotho, O. Der Einflusz kolloidaler Metallosungen auf niedere Organismen und seine Ursachen. Biochem. Ztschr., 110: 33. 1920; Schade, H. Die Kolloide als Trager der Lebenserscheinungen. Die Naturw., 9: 89-92. 1921. THE SOIL AS A CULTURE MEDIUM 631 Bacteria are adsorbed by various colloids29 as well as by sand.30 Since soils contain an abundance of substances in a colloidal condition, it is but natural to expect a marked influence upon the bacteria. The following method can be used for the study of this phenomenon.31 One cubic centimeter of a bacterial culture is added to 9 cc. of water and the mixture placed in a flask containing 5 grams of soil. After shaking for one minute, the soil is allowed to settle for ten minutes. The number of bacteria is then determined in the suspension both by plating and by direct microscopic examination. It was found that pure sand has little adsorptive action. Some bacteria, like Bac. my- coides, Bact. prodigiosum and Staph, pyogenes, are adsorbed rapidly and completely (80 to 98 per cent) ; other bacteria, like Bad. coli, are only weakly adsorbed (10 to 20 per cent). Adsorption of the bacteria was found to lead to a diminution not only in numbers but also in their chemical activities. Decomposition of organic matter in the soil seems to be carried out largely by the unadsorbed bacteria, probably due to the lower oxygen tension upon the soil colloidal particles. Ad- sorption does not diminish the action of anaerobic bacteria upon or- ganic matter in the soil. The absorption of inorganic materials by microorganisms is quite marked,32 some bacteria and fungi possessing a greater absorptive power than higher plants per unit of cells.33 Soil solution. The water present in the soil and added through rainfall dissolves some of the soil constituents. If the soil conditions were stable the solution would soon become saturated. Constant evaporation, rainfall, change in weather conditions, development of 29 Eisenberg, P. Ueber spezifische Adsorption von Bakterien. Centrbl. Bakt. I, Orig., 81: 72-104. 1918. 30 Frey, W., and Erismann, H. Beitrage zur Theorie der Bakterienfiltration. Centrbl. Bakt. I, 88: 306-336. 1922. 31 Dianowa, E. W., and Woroshilowa, A. A. The adsorption of bacteria by soils and its influence upon microbiological activities (Russian). Nautchno- Agron. Zhur. No. 10. 1925; Chudiakow, N. N. Centrbl. Bakt. II, 68: 345-358. 1926. 32 Beijerinck, M. W. tTber die Absorptionserscheinung bei den Mikroben. Centrbl. Bakt. II, 29: 161-166. 1911; Stoklasa, J. tlber die biologische Absorp- tion der Boden. Chem. Ztg., 35: 1425. 1911; Labes, R. tTber die fcrdernde Wirkung von Kohlensuspensionen und anderen Korpern mit groszer Oberflach- enentwicklung, etc. Biochem., Ztschr. 130: 1-13. 1922. 33 Beard, E., and Cramer, W. Surface tension and fermentation action. Proc. Roy. Soc. B., 88: 575. 1915; 98: 584. 1915. 632 PRINCIPLES OF SOIL MICROBIOLOGY acids by microorganisms, absorption of inorganic elements by higher plants, and many other changing conditions, cause an unceasing fluctua- tion in the composition and concentration of the soil solution. The osmotic pressure of the soil solution varies34 from 0.1 to 1 atmosphere in most soils to 4.5 to 16.5 atmospheres in soils with low moisture content. In normal soils, the concentration of the soil solution ranges between 0.1 and 1 atmosphere, depending on the rainfall, fertilization and plant growth.35 The soil solution contains calcium nitrate and bicarbonates, some organic matter, Na, Mg, Si, CI, S04, small amounts of K, and traces of ammonia and phosphates. The following constituents were found in a soil solution obtained by the soil pressure method36 (table 66). It is from this solution that the microorganisms obtain a large part of their food and in it they leave their waste products. The colloidal TABLE 66 Composition of soil sohition NATURE OF SOIL MOISTURE IN SOIL PARTS PER MILLION OF SOIL SOLUTION K P04 Ca N Fine sand 29.74 37.80 24.50 132.90 24.1 71.1 44.8 50.1 5.2 12.2 4.6 2.5 30.6 68.2 42.9 183.8 3.1 Loam 3.2 Clay 6.1 Peat 17.1 nature of the soil has an important bearing upon the nature of the soil solution. The rates of solubility, decomposition of organic matter, carbon dioxide production, nitrification, absorption of the soluble con- stituents by plants, microorganisms and soil particles, all have an important bearing upon the nature and concentration of the soil solu- tion. At a given moisture content, the rate of formation of soluble material increases with the temperature.37 At higher temperatures, 34 Bouyoucos, G. J., and McCool, M. M. The freezing point method as a new means of measuring the concentration of the soil solution directly in the soil. Mich. Agr. Exp. Sta. Tech. Bui. 24. 1915; 27, 1916; 31. 1910. 35 Hoagland, D. R. The freezing point method as an index of variations in the soil solution due to season and crop growth. Jour. Agr. Res., 12: 369-395. 1918. 36 Morgan, J. F. The soil solution obtained by the oil pressure method. Soil Sci., 3: 531-545. 1917. 37 McCool, M. M., and Whiting, L. C. Some studies on the rate of formation of soluble substances in several organic soils. Soil Sci., 11: 233-247. 1921. THE SOIL AS A CULTURE MEDIUM 633 optimum moisture conditions tend to bring greater amounts of material into solution than are found under saturated water conditions; with lower temperatures, the opposite effect was observed. Below two feet, the muck soils are very inactive, the ability of producing soluble materials decreasing regularly from the surface to the water level, in- dicating that aeration greatly influences this process. A definite correlation was found38 between bacterial activities in the soil and the thickness of the moisture film. The optimum thickness of the film in the case of Bac. mycoides was found to be between 20 and 40 microns. This film was obtained in sand of 1 mm. diameter at a moisture content of about 10 per cent. In arable soils with a grain size not more than 0.1 mm., it would require more than 50 per cent of moisture to produce the optimum film thickness. Lowering of the freezing point and conductivity of the soil39 can also be used as indices of changes in the composition of the soil solu- tion. Pantanelli40 suggested the use of electrolytic conductivity of soils for studying the course of solubilization of soil constituents by microorganisms; this was found, in most cases, to vary with the bacterial content of the soil. It is doubtful whether the actual concentration of the soil solution can be determined by the electrical bridge, since in most of these measurements an excess of water is added. Soil reaction and microbiological activities. The nature and quantity of substances present in the soil in a colloidal condition, which act as buffers or are capable of combining with acids and bases, the nature and amount of bases present in the soil either in an absorbed condi- tion or in the form of carbonates, influence the reaction of the soil, the medium in which the microorganisms live and act. Soil acidity may be due either to free organic and inorganic acids, which liberate hydrogen- ions, or to a non-saturation of the soil organic and inorganic complexes with bases, which results in a replacement of the base by hydrogen.41 38 Rahn, 1913 (p. 623). 39 Davis, R. O. E., and Bryan, H. The electrical bridge for the determination of soluble salts in soils. U. S. Dept. Agr. Bur. Soils, Bui. 61. 1910; Konig, J., Hasenbiiumer, J., and Glenk, K. Uber die Anwendung der Dialyse und die Bestimmung der Oxydationskraft fur die Beurteilung des Bodens. Landw. Vers. Sta., 80: 491-534. 1913. 40 Pantanelli, E. Elektrolytische Bestimmung der biologischen Bodenauf- schlieszung. Centrbl. Bakt. II, 42: 439-443. 1915. 41 Gedroiz, K. K. Soils unsaturated with bases. Method of determining in soils the hydrogen present in an absorbed condition. Soil requirement of lime 634 PRINCIPLES OF SOIL MICROBIOLOGY When neutral salts are added to a soil, the cations are adsorbed, re- placing the hydrogen ions, thus making the soil even more acid.42 It has also been suggested43 that acidity in well aerated soils is due to the hydrolysis of silicates; the bases are removed by plants or soil water, while the acid silicates are left behind. Definite concentrations of free hydrogen-ions have actually been demonstrated in soils, and have been measured electrometrically44 and colorimetrically.45 A pH of 3.7 is the extreme value obtained for mineral acid soils, while values of pH 3.2 have been reported for peat soils; pH 9.7 to 10.0 were reported for alkaline soils (containing free sodium carbonate); fertile soils usually give a range of pH values of 6.0 to 7.5. Soils are usually well buffered over considerable ranges of hydrogen- ion concentrations.46 By adding acid or base to a soil and titrating the resulting hydrogen-ion concentrations, a linear titration curve is as a neutralizing agent (Russian). Zhur. Opit. Agron., 22: 3-27. 1924; see also Hissink, D. J., and Van der Spek, J. The acidity of the soil. Verslag. Land. Onderzoek, Rijksland., 27: 146-161. 1922 (Chem. Abstr., 16: 4293). 42 Cameron, F. K. The soil solution. Easton, Pa. 1911; Kappen, H. Zu den Ursachen der Aziditat der durch Ionenaustausch sauren Boden. Landw. Vers. Sta., 89: 39-80. 1916; 96: 277-307. 1920; Wrangell, M. Phosphorsaur- eaufnahme und Bodenreaktion. Landw. Vers. Sta., 96: 209-255. 1920; Harris, J. E. Soil acidity. Mich. Agr. Exp. Sta., Tech. Bui. 19. 1914; Jour. Phys. Chem., 18: 355. 1914. 43 Truog, E. Cause and nature of soil acidity with special regard to colloids and adsorption. Jour, physik. Chem., 20: 457-484. 1916. 44 Sharp, L. T., and Hoagland, D. R. Acidity and adsorption in soils as meas- ured by the hydrogen electrode. Jour. Agr. Res., 7: 123-145. 1916; Soil Sci., 7: 196-200. 1919. 48 Gillespie, L. The reaction of soil and measurement of hydrogen-ion con- centration. Jour. Wash. Acad. Sci., 6: 7-16. 1916; Soil Sci., 4: 313-319. 1917. An excellent theoretical discussion of the hydrogen-ion concentration of soils and culture media, methods for determination and applications to microbiological processes is given by L. Michaelis, Die Wasserstoffionenkonzentration. Berlin. 1922; Clark, 1922 (p. 371). The use of the quinhydrone electrode for determining the soil reaction is discussed in detail by H. Christensen and S. T. Jensen, Unter- suchungen bezuglich der zur Bestimmung der Bodenreaktion benutzten elek- trometrischen Methoden. Intern. Mitt. Bodenk., 14: 1-26. 1924; and Baver, L. D. The use of the quinhydrone electrode for measuring the hydrogen-ion concentration of soils. Soil Sci., 21: 167-180. 1926. 46 Charlton, J. The buffer action of some Burma soils. Mem. Dept. Agr. India, 7: 101-121. 1924; Jensen, S. T. Om bestemmelse af jordens stodpudevirk- ning. 177 Ber. Statens Forsogs. i. Plantenk, 1924; Arrhenius, O. Clay as an ampholyte. Jour. Amer. Chem. Soc, 44: 521-524. 1922; see also various papers in the Trans, second Comm. of the Intern. Soc. Soil Sci. Groningen. 1926. THE SOIL AS A CULTURE MEDIUM 635 obtained which can serve as an index of the buffer content of the soil. The slopes of the curve vary with different soils according to their buffer content. It has been suggested47 that, although there may be no correlation between the reaction of an acid soil and crop growth, there is a definite correlation between the latter and the buffer content of the soil. The concentration of carbon dioxide is of great importance in such a system of measurement, especially when a relatively poorly buffered soil extract is used. The actual soil solution surrounding the absorbing membrane of the plant roots may be slightly acid, although the soil suspension gives an electrometric measure of pH 7.0 and above. Titrati- ble acidity has much wider ranges of variation due to difference in buffer content. A peat or clay soil may have the same hydrogen-ion TABLE 67 Influence of different nitrogenous fertilizers upon the reaction of the soit 100 mgm. nitrogen added to 100 grams of soil INCUBATION Start . . 2 days 35 days 50 days 76 days UREA 220 MGM. (NH4)2S04 500 MGM. NaNOs 660 MGM. PH pH pH 6.45 6.45 6.45 7.60 6.90 6.85 6.25 6.20 6.80 5.70 5.40 6.60 5.35 5.10 6.55 concentration as a sandy soil, but the titration (or lime requirement) of the first two soils will be much higher due to the greater buffer con- tent. When nitrogen-poor organic matter is added to the soil in the form of green manure or plant stubble, the first stage in the decomposition results in the formation of various organic acids, particularly in the absence of free calcium carbonate.48 If aeration, temperature and reaction favor a further decomposition of the organic acids thus formed, there may be a change in reaction to alkalinity, due to the formation " Arrhenius, O. The potential acidity of soils. Soil Sci., 14: 223-232. 1922. A possible correlation between the fertility of rice soils and their titration curves. Ibid., 21-26. 48 Charpentier, 1921 (p. 431). 636 PRINCIPLES OF SOIL MICROBIOLOGY of C02 and carbonates.49 The reaction of the soil as a result of appli- cation of fertilizer may change, according to the nature of the biological transformation of the fertilizer. Urea, for example, causes the soil reaction first to become alkaline because of the formation of ammonia and then acid because of the oxidation of the ammonia to nitric acid, as shown in table 67. 50 The soil reaction is also influenced by the moisture content of the soil, application of fertilizers, green manures, stable manures, plants grown and other factors.51 The reaction of the soil has a definite influence upon the activities of various microorganisms52 and upon the very distribution of the microflora and microfauna in the soil. An acid soil favors the develop- ment of fungi and is distinctly injurious to development of certain groups of bacteria, like Azotobacter, which has a limiting reaction at pH 6.0. Nitrifying bacteria are limited in their activities to a maxi- mum acid range of pH 4.0 to 4.6; Bad. radicicola has its limiting acid reaction at pH 3.4 and pH 6.0. Actinomyces are inhibited in growth by reactions more acid than pH 4.8; this fact is utilized for the control of Act. scabies causing potato scab in the soil. The application of lime to an acid soil has a favorable influence upon the bacteriological 49 Coville, F. V. The formation of leafmold. Smithsonian Report for 1913: 333-343. 1914; Ayers, S. H., and Rupp, P. Simultaneous acid and alkali bac- terial fermentations from dextrose and the salts of organic acids respectively. Jour. Inf. Dis., 23: 188-216. 1924. 50 Brioux, Ch. Influence de l'uree employee comme engrais, sur la reaction du sol. Compt. Rend. Acad. Sci., 179: 914-917. 1924. 51 Plummer, J. K. Studies in soil reaction as indicated by the hydrogen elec- trode. Jour. Agr. Res., 12: 19. 1918; Knight, H. G. Acidity and alkalinity of soils. Jour. Ind. Engin. Chem., 12: 559. 1920; Hardy, F. Soil sourness — its meaning and significance. West Indian Bui. 19: 37-85. 1921; Salter, R. M., and Morgan, M. F. Factors affecting soil reaction. I. The soil-water ratio. Jour. Phys. Chem.. 27: 117-140. 1923; Fischer, E. A. Studies on soil reaction. Jour. Agr. Sci., 11: 19-44. 1921; Sci. Progr., 16: 408. 1922; Connor, S. D. Soil acidity as affected by moisture conditions in the soil. Jour. Agr. Res., 15: 321. 1918; Morse, F. W. Effect of fertilizers on hydrogen-ion concentration in soils. Jour. Ind. Engin. Chem., 10: 125. 1918; Atkins, W. R. G. Some factors affecting the hydrogen-ion concentration of the soil and its relation to plant dis- tribution. Sci. Proc. Roy. Dublin Soc, 16: 369-413, 414-426, 429-434. 1922; Kappen, H., and Zapfe, M. Uber Wasserstoffionenkonzentrationen in Auszligen von Moorboden und von moor- und rohhumusbildenden Pflanzen. Landw. Vers. Sta., 90: 321-374. 1917. 52 Adam, A. Uber die Bedeutung der Eigenwasserstoffzahl (des H-Ionen optimum) der Bakterien. Centrbl. Bakt. I, 87: 481-486. 1922. THE SOIL AS A CULTURE MEDIUM 037 TABLE 68 Optimum and limiting reactions for the activities of microorganisms ORGANISMS Nitrosomonas Nitrobacter Nitrification in soils Thiobacillus denilrificans . . Th. thiooxidans Bac. pycnoticus Bac. amylobacter Azotobacter Bad. radicicola of Medicago and Melilotus.. Pisum and Vicia Trifolium and Phaseolus. Soja Lupinus Bact. coli Bad. vulgare Bact. pyocyaneum Bact. stutzeri Bac. subtilis Bac. pulrificus Act. scabies Mucor glomerula Asp. terricola Pen. italicum Fus. oxysporum Asp. niger Gibberella saiibinetii . : Spore germination of fungi. Protozoa: Paramoccium Colpidium ACID MAXIMUM OPTIMUM pH pH 3.9 7.7-7.9 3.9 6.8-7.3 3.5 6. 5-7. 5 5.0 7.0-9.0 1.0> 2.0-4.0 5.2 6.8-8.7 5.7> 6.9-7.3 5.6-6.0 6.5-7.8 5.01 4.8 4.3 3.4 3.2 4.4 6.5 4.4 6.5 5.6 6.8 6.1 7.0-8.2 4.2 7.5-8.5 5.8 6.8 4.8-5.0 6.5-7.5 3.2-3.4 1.6-1.8 1.6-1.8 1.8-2.0 1.2 1.7-7.7 3.0 4.8-9.4 1.5-2.5 3.0-4.0 3.3-3 9 7.0-7.4 ALKALI MAXIMUM pH 9.7 13.0 >11.9 10.75 6.0(?) 9.2 8.8-9.2 11.0 7.8 8.4 8.0 9.6-9.* 9.4 8.5 8.7 8.7- 9. 9.0- 9. 9.1- 9. 9.2-11. 11.7 9.0 Gaarder and Hagem Meek and Lipman Gerretsen, Waks- man Trautwein Waksman and Starkey Ruhland Dorner Gainey, Johnson and Lipman, Yamagato and Itano, Stapp Fred and Daven- port, Fred and Loomis, Bryan Dernby Dernby Dernby Zacharowa Itano Dernby Gillespie, Waksman Johnson Terroine and Wurmser Maclnnes Webb Cutler and Crump 638 PRINCIPLES OF SOIL MICROBIOLOGY activities; the growth of microorganisms may even be stimulated more than that of higher plants grown upon the soil.53 Acids affect the activities of microorganisms not merely by creating a favorable or unfavorable hydrogen-ion concentration, but also through the undissociated part of the molecule.54 The optimum and limiting reactions of some typical soil organisms are indicated in table 68. The soil atmosphere. The soil atmosphere is a mixture of gases which change constantly in composition, chiefly because of biological activi- ties and also to some extent because of chemical processes. The com- position of this atmosphere depends upon the amount and nature of the organic matter and upon environmental conditions. During dry seasons, when oxidation of the organic matter is low, the soil gases are rich in oxygen and poor in C02. Soon after heavy rains, the oxygen content rapidly diminishes and the C02 content increases because of the active oxidation of the soil organic matter. The nitrogen content of the atmosphere of aerated soils does not vary appreciably and is not affected either by the assimilation of nitrogen by bacteria or by its liberation from the decomposition of the nitrogen compounds of the soil. In rice soils, which are kept under water, a large part of the nitro- gen may be derived from the soil organic matter. For collecting and analyzing the soil gases, the apparatus shown in figure 58 may be employed. The amount of carbon dioxide in fallow land is smaller than in soil which is vegetated. The atmosphere of soil freshly treated with farm manure or green manure contains a high proportion of C02 and a low proportion of oxygen. 54a The actual C02 content of the soil atmosphere thus depends upon a number of factors, including (1) C02 production in the soil from the decomposition of organic matter and interaction between carbonates and acids; (2) diffusion of the C02 in the soil at- mosphere; (3) assimilation of C02 by plants.55 A large part of the 63 Brown, P. E. Some bacteriological effects of liming. Centrbl. Bakt. II, 34: 148-172. 1912; 35: 234-248. 1912; Waksman, 1922 (p. 712). 64 Hall, I. W., and Fraser, A. D. The action of dilute acids upon bacterial growth in optimum hydrogen-ion concentration. Jour. Pathol. Bact., 25: 19-25. 1922. 84a Attention need only be called here to the early contribution of Boussin- gault and Lewy. Memoire sur la composition de 1'air confine' dans la terre veg^tale. Am. Chim. Phys. (3 ser.j. 37. 1853. 55 Romell, L. G. Die Bodenventilation als okologischer Faktor. Meddel. fran Statens Skogsforsoks. H. 19. 1922; Lundegardh, H. Der Kreislauf der Kohlensaure in der Natur. G. Fischer, Jena. 1924. THE SOIL AS A CULTURE MEDIUM 639 C02 is present in the soil solution. Diffusion of C02 into the atmos- phere and of oxygen into the soil is very rapid at a depth of six inches,56 as shown in table 69. The soil atmosphere shows much greater fluctuations in composition than atmospheric air. On the average, the soil air was found57 to contain 0.25 per cent C02 and 20.6 per cent oxygen. From November to May the curves for C02 follow closely those of the soil temperature; from May to November they follow rainfall and to a less extent the soil temperature curves. The favorable effect of rainfall is believed to be due58 to the dissolved oxygen brought down. While the decomposition of green manure in normal soils leads to the formation of carbon dioxide, the gases formed under anaerobic TABLE 69 Composition of gas in variously treated soils Nitrogen Oxygen Carbon dioxide Hydrogen Methane Argon N A AVERAGES OF SEVERAL DETERMINATIONS, IN PER CENT Fallow land Before rainfall 78.05 20.40 0.58 None None 0.977 80.0 After rainfall 78.83 19.26 0.95 None None 0.955 82.5 Gases near roots of corn 80.15 9.00 9.11 0.73 None 1.010 79.5 Green manured land 79.18 7.71 12.03 0.07 None 1.003 78.8 Swamp rice land 85.59 0.54 4.42 6.42 2.81 0.893 95.7 conditions, as in swamp rice soils, are largely methane, a small amount of nitrogen, carbon dioxide, and hydrogen.59 An increase in atmospheric pressure brings about first an increase 56 Leather, J. W. Soil gases. Mem. Dept. Agr. India, Chem. Ser., 4: 85- 134. 1915. 57 Russell, E. J., and Appleyard, A. The atmosphere of the soil; its com- position and the cause of variation. Jour. Agr. Sci., 7:1. 1915. 88 See also Potter, R. S., and Snyder, R. S. Carbon dioxide production in soils and carbon and nitrogen changes in soils variously treated. Iowa Agr. Exp. Sta. Res. Bui. 39. 1916. 69 Harrison, W. H., and Aiyer, P. A. S. The gases of swamp rice soils, their composition and relationship to the crop. Mem. Dept. Agr. India, Chem. Ser., 3: 65-106. 1913. 640 PRINCIPLES OF SOIL MICROBIOLOGY in the activities of certain microorganisms, such as the autotrophic bacteria, and then a slackening.60 The C02 of the soil atmosphere was found61 to be a more important source of carbon for the growth of plants than the C02 of the air. Plants thus depend entirely upon the activities of the microorganisms in the soil for their C02, which is liberated largely as a result of the decomposition of the soil organic matter. Soil temperature. Soil temperature is affected by climate, season of year, chemical and mechanical composition of soil, topography, and soil treatment. In the spring of the year, fine-grained soils containing a large amount of water warm up more slowly than coarse-grained soils containing a relatively small amount of moisture. The heat con- ductance of the specific soil constituents is of importance as well as the cultivation of the soil which influences the rate of evaporation. In general, sandy soils and sandy loams warm up more quickly in spring than heavy clay and clay loam soils; microbial activities are, there- fore, sooner accelerated in the spring in the first types of soil than in the second.62 The colloidal condition of the soil and the soil organic matter are modified in the temperate climates by the action of frost during the winter, so that, when the soil finally warms up in the spring and loses the excess moisture, a rise in biological activities takes place.63 In the summer months there is a drop to normal which is undoubtedly due to the fact that the available energy has been largely used up and the soil may lack in sufficient moisture. In the autumn there is another rise in biological activities which is probably due to the addition of plant residues. The drop in winter is due to low temperature. Bac- terial activities are not, however, entirely suspended at low tempera- tures. The activities of some of the most important soil organisms become marked at temperatures above 10°C. with an optimum at 25°. A detailed study of the influence of temperature upon the biological activities in the soil is given elsewhere (p. 774). 60 Berghaus, W. H. Uber die Wirkung der Kohlensaure, des Sauerstoffs und des Wasserstoffs auf Bakterien bei verschiedenen Druckhohen. Arch. Hyg., 62: 172-200. 1907; Chlopin, G. W., and Tammann, G. Einflusz hoher Drucke auf Mikroorganismen. Ztschr. Hyg., 45: 171-204. 1903. 61 Lundegardh, H. Klima und Boden. Fischer, Jena. 1925. 62 Lipman, J. G. Microbiology of soil. In Marshall's Microbiology. 3d Ed. 345-427. 1921. 63 Muntz, A., and Gaudechon, H. Le reveil de la terre. Ann. Sci. Agron. (4), 2: 1-15. 1913. THE SOIL AS A CULTURE MEDIUM 641 Growth of microorganisms in soil in pure and mixed culture. There is no method available for sterilizing the soil without changing its physical and chemical properties. The common method used at pres- ent for sterilizing the soil consists in heating it in flowing steam for thirty minutes on seven consecutive days,64 or at high pressure (15 pounds) for two hours. Both of these treatments cause a decided change in the physical and chemical condition of the soil which results in an increase in the available organic matter in the soil. Sterile soil forms an excellent medium for the development of various bacteria and other microorganisms.65 A number of soil organisms, such as Azotobacter or Bac. amylobacter, will regain their vigor of fixing nitrogen, when cultivated in sterile soil.66 However, from processes carried out by pure cultures of micro- organisms grown in sterile soil we cannot determine what actually occurs in normal soils. Not only is the nature and the composition of the culture medium completely changed by sterilization of soil, but the various antagonistic and associative influences which are active in normal soils are eliminated. This can be readily illustrated by the following instances. An organism belonging to the Bac. mesentericus group was found67 capable of dissolving and clarifying cultures of Bact. coli, Staphylococcus, and other bacteria, a phenomenon which may not take place in pure culture. The inhibitive effects of filamentous fungi, especially of ascomycetes, on the growth of microorganisms has been commonly observed;68 this may be due to exhaustion of nutrients or formation of some toxic products during growth.69 Various symbiotic processes take place in the soil, such as the symbiosis between the chlorophyll-bearing algae and the nitrogen fixing Azotobacter, between the anaerobic CI. pastorianum and the aerobic Azotobacter, between Azotobacter and cellulose-decomposing bacteria. Then we have proc- 64 Eckelmann, E. Uber Bakterien, welche die fraktionierte Sterilization lebend uberdauern. Centrbl. Bakt. II, 48: 140-178. 1918. 155 Barthel, C. Kulturen von Mikroorganismen in sterilisierter Erde. Centrbl. Bakt, II, 48: 340-349. 1918. 66Bredemann, 1909 (p. 111). 67 Kimmelstiel, P. tJber eine biologische Eigenschaft eines Wurzelbazillus. Centrbl. Bakt. I, Orig. 89: 113-115. 1922. 68 Porter, C. L. Concerning the characters of certain fungi as exhibited in their growth in the presence of other fungi. Amer. Jour. Bot., 11: 168-188. 1924. 69 Liesegang, R. Gegenseitige Wachstumshemmung bei Pilzkulturen. Centrbl. Bakt. II, 51: 85-86. 1920. 642 PRINCIPLES OF SOIL MICROBIOLOGY esses which depend upon the activities of 'other microorganisms: the nitrite-forming bacteria depend upon the heterotrophic bacteria and fungi for ammonia, the nitrate bacteria depend upon the nitrite formers for nitrite, the sulfur bacteria depend upon the heterotrophic organisms for the decomposition of the proteins and liberation of the H2S. The soil population. In the complex medium of the soil and under the influence of various physical, chemical and environmental conditions, soil microorganisms carry on their activities, not as individual forms or even as groups, but as a soil population. The most common soil bacteria, including the heterotrophic spore-formers (Bac. cereus, Bac. mycoides, Bac. mesentericus, etc.) and the non-spore formers (Bad. fluorescens, Bad. caudatum, Bad. radiobacter, etc.), the nitrogen-fixing Azotobacter and CI. pastorianum, the nitrifying bacteria, etc., are of universal occur- rence, limited only by specific soil conditions, such as reaction. They are reported from all soils, from East and West, North70 and South. The most common soil protozoa, including the amoebae, flagellates and ciliates, are also universally distributed in the soil.71 The common soil fungi, including species of Zygorhynchus, Trichoderma, Penicil- lium, etc., have also been isolated from various soils coming from dif- ferent parts of the world.72 The same is true of algae and other soil microorganisms. As a result of soil conditions this population shows quantitative, rather than qualitative differences. Excessive moisture, for example, stimulates the development of anaerobic bacteria73 but inhibits the development of aerobic forms like Azotobacter and fungi. Ex- cessive acidity and a high content of organic matter rich in carbo- hydrates greatly stimulate the development of fungi. Applications of lime and the addition of organic matter rich in protein stimulate the development of bacteria and actinomyces. We are thus fully justified in speaking of a soil population and may even accept the idea of an Edaphon as suggested by France^ 74 although his conclusion that the edaphon is an indicator of soil fertility may not be fully justified.75 The composition and activities of the soil popula- 70 Barthel, Chr. Recherches bactcriologiques sur le sol et sur les matures fecales des animaux polaircs du Groenland Septentrional. Saertr. Meddl. Gronland., 64. 1922. 71 Sandon, 1924-1927 (p. 329). 72 Waksman, 1917 (p. 237). 73 Winogradsky, 1924 (p. 542). 74 France, R. H. Das Edaphon, Stuttgart. 1921. 75Fischer, H. Gibt es ein Edaphon? Int. Mitt. Bodenk., 13: 192-200. 1923. THE SOIL AS A CULTURE MEDIUM 643 tion is controlled by the nature of the soil and other environmental conditions. The magnitude of the population is especially controlled by the supply of available energy. It may be controlled qualitatively by the nature of the available energy. Very little is known concerning the role of the rhizosphere, or the subterraneous part of the plant system, in controlling this population. The concept of this system and its possible influence upon the soil population was first introduced by Hiltner.76 It is known (p. 792) that plants actually secrete carbohydrates and phosphatides.77 The sloughed off portions of the root caps, the root hairs, etc., may influence greatly the nature of the population developing in their neighborhood. The nature of the gases formed by the plants also influences the nature of the organisms developing in the particular locality. A certain soil decomposes cellulose with varying rapidity according to the kind of plants which have been growing in it; also, the nature of the organisms taking part in the decomposition of the cellulose varies with the plants grown in the soil.78 Further studies in this direction are much needed. 76 Hiltner, 1904 (p. 128). 77 Hansteen Cranner, B. Zur Biochemie und Physiologie der Grenzschichten lebender Pflanzenzellen. Meld. Norges. Landbruks., 2. 1922. 78 Rokitzkaia, A. Aerobic fermentation of cellulose under the influence of soil- microflora in the root-zone of plants. Jour. Sci. Inst. Amelior. Leningrad., 13: 168-208. 1926. CHAPTER XXV Transformation of Minerals in the Soil Nature of mineral transformation by microorganisms. Among the mineral elements of plant food which are subject directly or indirectly to the action of microorganisms, the following may be included : phos- phorus, sulfur, potassium, calcium, magnesium, iron, sodium, manga- nese, also chlorine, aluminum, zinc and silicon. The transformation of the elements and compounds of carbon, nitrogen, hydrogen and oxygen, both in organic and inorganic combinations, are considered in detail elsewhere. The above minerals are transformed in the soil by different processes : 1. Mineral elements (S, Fe, Mn, etc.) or their inorganic compounds may be used by certain bacteria as sources of energy. 2. Certain salts (nitrates, sulfates, etc.), rich in oxygen, may be used, under conditions favoring anaerobiosis, as sources of oxygen. This usually leads to the reforming of substances which have been acted upon in the processes of oxidation. 3. The transformation of minerals present in the soil in the form of complex organic compounds. When the bodies of plants, animals and microorganisms are decomposed or mineralized by the soil microorganisms, a part of the minerals is liberated in the form of inorganic compounds and a part may be reassimilated. 4. The assimilation of minerals by microorganisms. In the presence of available energy, simple inorganic salts are converted into complex organic com- pounds; this is especially true of phosphates, potassium salts and sulfates and leads to a temporary removal of the soluble salts. Most of these materials are again made available upon the death and decomposition of the microbial cells, as described in the third process. 5. The indirect transformation of minerals in the soil by products of the metabolism of microorganisms. The action of carbon dioxide, organic and inorganic acids upon carbonates, phosphates and silicates is largely due to the change in the H+ or the OH- concentrations of the soil. This is even true of the interaction between insoluble phosphates and the "humic acids."1 Phosphates, sulfates and salts of potassium, calcium and magnesium (iron salts in smaller amounts), are the most important compounds in 1 Baumann and Gully, Mitt. K. Bayr. Moork. 4: 31-156, 1910; Niklas, N. Untersuchungen ilber den Einflusz von Humusstoffen auf die Verwitterung der Silikate. Diss. Miinchen 1912. 644 TRANSFORMATION OF MINERALS IN THE SOIL 645 the metabolism of all microorganisms. Large quantities of these are taken from the soil solution and synthesized into microbial protoplasm. Notwithstanding the fact that in the decomposition of plant residues and animal manures, phosphates and potassium salts are quite soluble, Kraw- kow2 found that when organic matter is decomposed in the soil by mi- croorganisms, calcium and magnesium are less soluble. The decomposi- tion and synthesis of organic matter in the soil takes place constantly and leads to constant changes in the amount of available minerals in the soil. It has been suggested3 that fungi take a more active part in the decomposition of mineral soil constituents than root secretions of higher plants. A great many bacteria also play important roles in the process. The fungi and algae as well as other groups of micro- organisms store away considerable quantities of soluble minerals in the form of microbial protoplasm due to the extensive growth of the organisms in the presence of available energy.4 Decomposition of rocks and rock constituents by microorganisms. Not only the mineral constituents of normal soils but rocks as well may undergo disintegration and degradation through the action of micro- organisms. Autotrophic bacteria obtain their carbon from the carbon dioxide of the atmosphere and their energy from inorganic substances of the soil or ammonia formed by electrical discharges and rainfall; algae utilize the photosynthetic energy of the sun. The various inor- ganic and organic acids formed by these organisms exert solvent action upon the rocks. The chemosynthetic assimilation of C02, fixation of nitrogen, and denitrification are considered5 as the three most primitive activities of microorganisms in the development of life upon this planet. It has been demonstrated6 that various algae, particularly the Cyanophy- ceae, exert a corroding effect upon stones. Diatoms transform alumi- 2 Krawkow, S. The processes of interaction of the soluble products of decom- position of organic matter with the soil constituents. Zhur. Opit. Agron., 9: 569-624. 1908; 10: 1-34. 1909. 3 Kunze, F. Tiber Sauereausscheidung bei Wurzeln und Pilzhyphen und ihre Bedeutung. Jahrb. wiss. Bot., 42: 357-391. 1906. 4 Pantanelli, 1915 (p. 633). 6 Fischer, II . Physiologische Leistungen primitivster Organismen in ihrer stammesgeschichtlichen Bedeutung. Centrbl. Bakt. II, 55: 1-5. 1921. 6 Jensen, P. R. Uber Steinkorrosion an den Ufern von Fureso. Intern. Rev. ges. Hydrob. Hydrogr. Leipzig, 2. 1909; Roux, M. Recherches biologiques sur le lac d'Annecy. Ann. biol. lacustre., 2. 1907. 646 PRINCIPLES OF SOIL MICROBIOLOGY num silicates into hydrated aluminum oxide.7 Miintz8 suggested in 1890 that bacteria are concerned in rock decomposition, their action being confined not only to the surface but often entering into the depth of the rock mass; nitrifying organisms were always demonstrated in decomposed rocks. Other investigators9 also suggested that bacteria are possible agents in the decomposition of rocks. Certain bacteria were found10 capable of deriving their necessary mineral nutrients from feldspars, bringing considerable quantities of undecomposed orthoclase into solution, probably by means of the carbon dioxide which is formed. The action of a number of bacteria, including B. extorquens, nitrate-forming and butyric acid or- ganisms, as well as yeasts, upon twelve different silicates and upon apatite was investigated. The bacteria were able, by means of their products of respiration, to dissolve considerable amounts of pulverized silicates; the formation of organic acids by Bac. amylobacter markedly influenced the solubility of silicates. The intensity of contact of the organism with the mineral to be acted upon was found to be of even greater importance than the other agents of solubility. Thus, B. extorquens, which produced only carbon dioxide but which formed a close and firm envelope around the mineral particles, had the strongest solvent action. Yeasts which do not form as close a contact, although they produce more carbon dioxide, brought about less solubility. Nitrite forming bacteria, as a result of the production of a strong in- organic acid (HNO2), caused a considerable solubility of the silicates. Minerals rich in alkaline earths were most readily acted upon. Apa- tite dissolves only to a limited extent in carbonic acid and only those bacteria which produce organic acids can bring about a considerable solubility. The chemical constituents of the minerals were found10 in the filtrates of the bacterial cultures, especially in case of B. extorquens. The alkalies came into solution first, followed by the alkaline earths and iron; silicic acid and aluminum oxide came into solution last. 7 Vernadsky, W. J. Sur le probleme de la decomposition du kaolin par les organismes. Compt. Rend. Acad. Sci., 175: 450-452. 1922; see also Stoklasa, J. tlber die Verbreitung des Aluminums in der Natur. G. Fischer, Jena. 1922. 8 Miintz, A. Chimie agricole. Sur la decomposition des roches et la forma- tion de la terre arable. Compt. Rend. Acad. Sci., 110: 1370-1372. 1S90. 9 Merrill, G. P. Disintegration of the granite rocks of the District of Colum- bia. Bui. Geol. Soc. Am., 6: 321-332. 1895. 10 Bassalik, K. Tiber Silikatzersetzung durch Bodenbakterien. Ztschr. Garungsphysiol., 2: 1-32. 1912; 3: 15-42. 1913. TRANSFORMATION OF MINERALS IN THE SOIL 647 Kamamura11 described an organism, Volcanothrix silicophila, found in the volcanic material in Japan at an altitude of 6600 feet, which formed a zoogloeal mass, the ash of which contained 8.873 per cent silica. 2.0 _ a 3 a 3.0 4.0 5.0 6.0 log Ch 5.0 4.0 3.0 2.0 Fig. 48. Influence of sulfuric acid upon the solubility of calcium silicate (from Wright). The presence of bacteria was also found to increase the etching power of the roots of plants.12 A certain relationship exists between the hydrogen-ions produced by 11 Kamamura (cited by Wright, 1922 (p. 648)). 12 Fred, E. B., and Haas, A. R. C. The etching of marble by roots in the pres- ence of bacteria. Jour. Gen. Physiol., 1: 631-638. 1919. 648 PRINCIPLES OF SOIL MICROBIOLOGY bacteria and the amount of bases brought into solution from the mineral, since the magnitude of the effect of bacterial end-products upon a mineral depends upon the equilibrium established.13 It was suggested that the action of bacterial end-products, acid in nature, upon minerals is explainable as a chemical reaction. Figures 48 and 49 show the action of mineral acids and bacterial cultures upon some typical sili- 4.0 5.0 logCh 4.0 3.0 Fig. 49. Influence of B. lactis acidi upon the solubility of orthoclase (from Wright). cates. The numbers along the ordinates represent the logarithms of the hydrogen-ion concentration, or log Ch. Those along the abscissae measure the logarithm of the concentrations, or log of Ca, of Mg, of Fe or K. is Wright, D. Equilibrium studies with certain acids and minerals and their probable relation to the decomposition of minerals by bacteria. Univ. Cal. Publ. Agr. Sci., 4: 247-337. 1922. TRANSFORMATION OF MINERALS IN THE SOIL 649 Definite information is available on the transformation of a few of the more important mineral elements by microorganisms; some of the rarer elements, such as Ni and Co,14 exist in the soil in small quantities and probably play a role in the activities of microorganisms. A detailed study of the transformation of sulfur in nature is given elsewhere (p. 600). Nature of phosphorus compounds in the soil. Phosphorus undergoes various changes in the soil as a result of activities of microorganisms. When organic matter is mineralized, the phosphorus is liberated in the form of inorganic salts; the latter may be reassimilated by the same or by other microorganisms and synthesized into microbial protoplasm, especially in the presence of available energy. Insoluble inorganic phosphates may be made soluble, as a result of the action of the prod- ucts of metabolism of microorganisms, including carbon dioxide and the various organic and inorganic acids. Phosphorus compounds may also be reduced by microorganisms.15 Phosphorus is present in normal soils in the form of inorganic and organic compounds. The inorganic compounds include mono-, di-, tri-, and tetra-phosphates of potassium, sodium, calcium, magnesium, aluminum, iron and manganese. The organic compounds comprise the phosphorus of plant and animal residues and that in the living or dead protoplasm of microorganisms : these include various compounds, such as nucleic acids,16 lecithin17 and phytin.18 Phosphorus is usually added to the soil in the form of superphosphates, insoluble tri- and tetra- calcium phosphates, and organic forms, both in plant residues and in organic fertilizers. When soluble phosphates, such as superphosphate, are added to the soil, they interact with the hydroxides, carbonates, silicates and tri-phosphates of calcium, magnesium, iron and aluminum to give insoluble precipitated phosphates. Superphosphates were found to change, in the presence of sufficient CaC03 in the soil, into 14 Bertrand, G., and Mokradnatz, M. Sur la presence simultanee du nickel et du cobalt dans la terre arable. Ann. Sci. Agron. 1921, 179-182. 15 Rudakov. Viestnik Bakteriol. Agron. Sta. Moskau. No. 26. 1926. 16 Shorey, E. Nucleic acid in soils. Science N. S., 35: 390. 1911; Biochem. Bull., 1: 104. 1911; Some organic soil constituents. U. S. Dept. Agr., Bur. Soils, Bui. 88. 1913. 17 Stoklasa, J. Biochemischer Kreislauf des Phosphat-Ions im Boden. Centrbl. Bakt. II, 29: 385-519. 1911. '8Auten, J. T. Organic phosphorus of soils. Soil Sci., 16: 281-294. 1923; see also Aso, K. On organic compounds of phosphorus in soils. Bui. Coll. Agr. Tokyo Imp. Univ., 6: 277-294. 1904. 650 PRINCIPLES OF SOIL MICROBIOLOGY insoluble phosphates, within 50 to 60 days. Normal soils contain 0.025 to 0.3 per cent of phosphorus, very little of which is soluble in pure water, but quite appreciable quantities are soluble in water con- taining carbon dioxide. In fertile soils, the phosphorus is present partly in the form of organic phosphorus compounds.18 A part or even all the organic phosphorus found in the soil may be in the form of bodies of microorganisms.19 The total phosphorus brought into the soil by a two ton crop of green manure (including roots) may amount to 20 to 50 pounds P205 per acre. The P205 content of straw is 0.15 to 0.30 per cent, of clover and timothy hay 0.50 to 0.55 per cent, of fresh horse manure (without straw) 0.34, cow manure 0.21, sheep manure 0.40, chicken manure 0.83 per cent.20 The following analysis of the dry matter of a few typical bacteria show that these organisms can store away considerable quantities of phosphorus,17 (table 70). Seventy-nine to 81 per cent of this phos- phorus was found to be in the form of nucleic acid and 7.6 to 8.6 per cent as lecithin. The ash of yeasts may consist of 60 per cent P2O5. The ash of fungi, however, contains a lower concentration of phosphorus than yeasts and bacteria, depending largely on the phos- phoric acid content of the medium. A more or less constant nitrogen- phosphorus ratio (N:P205 = 4.2%: 2.0%) was found21 in the dry mycelium of Asp. niger. A similar ratio is found in the cells of other organisms and in the soil organic matter, pointing to a definite c/p (organic) ratio in the soil. The presence of phosphorus is so important for the growth of micro- organisms that a direct correlation has been found between the amount of available phosphate in the medium and the growth, mannite de- composition and nitrogen fixation by Azotobacter (see p. 577). Decomposition of organic phosphorus compounds by microorganisms. A large number of microorganisms including various heterotrophic bac- teria, fungi and actinomyces, are capable of decomposing organic phosphorus compounds. Lecithin contains 9.39 per cent P205, 1.6 per cent N and 65.36 per cent C. It contains two fatty acid radicals, usually palmitic and stearic or oleic, which are rather poor sources of carbon for microorganisms. 19 Gortner, R. A., and Shaw, W. M. The organic matter in the soil. V. Some data on humus-phosphoric acid. Soil Sci., 3: 99-111. 1916. 20 Thome, 1914 (p. 429). 21 Schmi'icke, R. Der Phosphorstoffwechsel einiger Pilze, mit besonderer Berucksichtigung von Aspergillus niger. Biochem. Ztschr., 153: 372^423. 1924. TRANSFORMATION OF MINERALS IN THE SOIL 651 In the presence of available carbon and nitrogen sources, microorgan- isms will rapidly break down the lecithin and liberate the phosphorus. A transformation of G6 per cent of the lecithin phosphorus into soluble phosphate by different bacteria, in 60 days at 28° to 30°C, has been reported;17 the rest of the phosphorus was reassimilated by the bacteria. Phytin is an hexaphosphate, occurring abundantly in vegetable tissues, especially in seeds or grains.22 It contains (as phytic acid, C6H24027P6) about 26 per cent phosphorus. It is acted upon by fungi and bacteria by means of an enzyme, phytase,23 with the transforma- tion of the phosphorus into the inorganic form. Nucleo-proteins contain 7 to 9 per cent phosphorus and 13 to 14 per cent nitrogen. The resulting products of their decomposition are phos- phoric acid, a sugar, purine and pyrimidine bases.24 The nucleic acids are broken down by various soil microorgansims, both in the presence Phosphorus and 'potassium TABLE 70 content of some typical soil bacteria ASH TOTAL P2O5 TOTAL K2O Az. chroococcum . per cent 8.2-8.6 7.5 6.48 per cent 4.93-5.2 4.07 5.32 per cent 2.41-2.65 Bac. mycoides Bad. fluorescens liquefaciens 2.27 0.83 and absence of other nitrogen sources. Koch and Oelsner24 found that, in addition to some common soil organisms, a special group of bacteria (genus Nucleobacter) is specifically concerned in the decomposition of nucleins, through the nucleic acid stage, into phosphoric acid. 22 Anderson, R. J. Phytin and phosphoric acid esters of inosite. N. Y. (Geneva) Agr. Exp. Sta. Tech. Bui. 19. 1912; The organic phosphoric acid of cottonseed meal. Ibid., Bui. 25. 1912. 23 Dox, A. W., and Golden, R. Phytase in lower fungi. Jour. Biol. Chem., 10: 183-186. 1911; Plimmer, R. H. A. The metabolism of organic phosphorus compounds. Their hydrolysis by the action of enzymes. Biochem. Jour., 7: 43-71. 1913; Egorov, M. A. tlber das Verhalten von Schimmelpilzen (A. niger und P. glaucum) zum Phytin. Ztschr. physiol. Chem., 82: 231-242. 1912. 24 Iwanoff, N. Nuclease, forkommend in Schimmelpilzen. Ztschr. Garungs- physiol., 1: 60. 1912; also Ztschr. physiol. Chem., 39: 31-43. 1903; Schitten- helm, A., and Schroeter, F. Uber die Spaltung der Hefenukleinsaure durch Bakterien. Ztschr. physiol. Chem., 39: 203-207. 1903; 40: 62-69. 1903; 41: 283-292. 1903; 57: 21-27. 1908; Koch, A., and Oelsner, A. Uber Nucleopro- teidspaltende Bakterien und ihre Bedeutung fur die Erschliesung des Phosphor- Kaptials im Ackerboden. Biochem. Ztschr., 134: 76-96. 1922. 652 PRINCIPLES OF SOIL MICROBIOLOGY In addition to these compounds, other organic phosphorus com- pounds, such as that of wheat bran (inosite mono-phosphate — CeH^OgP) are added to the soil, in considerable quantities. Their decomposition is probably similar to that of phytin. Transformation of insoluble tri-calcium phosphates into soluble forms by microorganisms. Insoluble tri-calcium phosphates may be brought into solution by microorganisms in three different ways: (1) by the direct metabolism of microorganisms, perhaps through the formation of some enzyme or interaction with some synthesized substance, (2) by the action of carbon dioxide as well as various organic acids produced by soil microorganisms, (3) by the action of inorganic acids formed in the metabolism of the autotrophic nitrifying and sulfur oxidizing bacteria. Soil microorganisms were found capable of rendering insoluble tri- calcium phosphate soluble when it is present in culture media as the only source of phosphorus. Attempts have been made to explain this by a direct metabolism of the organism, involving the formation of specific enzymes. It has also been suggested25 that various fungi may directly assimilate the phosphorus from the tri-calcium phosphate: Asp. niger, Pen. brevicaule, Pen. glaucum assimilated, in 60 days at 22°C, one-fifth to one-third of the phosphate present as Ca3(P04)2 in liquid culture media. When the mycelium of the organisms is further de- composed by soil bacteria and fungi, the phosphorus goes into solution in an available form. Out of twenty-five bacteria isolated from the soil, twelve had a defi- nite solvent action on rock phosphate, bone, pure tri-calcium phos- phate, di-calcium phosphate and calcium carbonate, when supplied with some form of sugar in the nutrient medium.26 Both the acid formed by the bacteria and the carbon dioxide were found to be factors exerting the solvent action. The solubility of phosphates is influenced by the nature of carbon and nitrogen sources for the growth of micro- organisms;27 disaccharides are better sources of carbon than mono- 26 de Grazia, S., Gerza, U. Sull intervento dei microorganismi nella utiliz- zazione dei fosfati insolubili del suolo da parte delle piante superiori. Ann. R. Sta. Chem. Agr. Sper. Roma (II), 2. 1908; 3. 1909. 26 Sackett, W. G., Patten, A. J., and Brown, C. W. The solvent action of soil bacteria upon the insoluble phosphates of raw bone meal and natural raw rook phosphate. Centrbl. Bakt. II, 20: 688-703. 1908. 27 Perotti, R. tlber den biochemischen Kreislauf der Phosphosiiure im Acker- boden. Centrbl. Bakt. II, 25: 409-419. 1909. TRANSFORMATION OF MINERALS IN THE SOIL 653 saccharides and ammonium salts are better sources of nitrogen than other nitrogenous substances. The soluble bases (especially Ca) pre- vent the solubility of phosphates; iron oxide influences the process least. This would seem to indicate definitely that this process is rather secondary in nature and depends upon the action of the products of carbon and nitrogen metabolism upon the insoluble phosphate. The production of soluble phosphates was reported28 to be associated with the vital function of microorganisms. However, the existence of specific enzymes capable of bringing into solution insoluble phosphates has been questioned by Bazarevski,29 who submitted evidence to show that this process is chiefly a result of acid production by microorganisms. Even if the organism does not form any acid in the medium, a part of the phosphate may be assimilated by it, either by the action of carbon dioxide or as a result of basic exchange between the soluble products formed and certain soil constituents. The continuous removal of the soluble phosphate by the growing organism and its synthesis into or- ganic matter may sometimes bring about appreciable transformation. The greatest amount of dissolution of the phosphate has been obtained with ammonium salts and the least with nitrate ; this substantiates the claim that the acids formed in the metabolism of the organisms, rather than any enzymatic phenomena, are responsible for the process. In the case of ammonium salts, the organisms remove the ammonium as a source of nitrogen, leaving the S04 in the medium, while in the case of the nitrate, the acid-ion is removed and the alkaline is left.30 The growing organism is capable of forming large quantities of carbon dioxide and often appreciable quantities of organic acids, both under aerobic and anaerobic conditions. One gram of bacterial cells produces 0.25 to 0.5 mgm. of C02 in twenty-four hours and 1 gram of fungus mycelium produces 0.13 to 0.18 mgm. C02. As much as 6000 pounds of C02 may be given off in 200 days by one acre of normal soil, on a 2,000,000 pounds basis. The soil atmosphere may contain 0.6 28 Pozerski, E., and Levy, M. M. Sur l'exretion de composes phosphor's par les microbes. Compt. Rend. Soc. Biol., 87: 1157-1159. 1922. 29 Bazarevski, S. On the question of mobilization of phosphoric acid in the soil by the agency of microorganisms (Russian). Moskau. 1916. 30 Haselhoff, E. Untersuchungen iiber die Zersetzung bodenbildender Ges- teine. Landw. Vers. Sta., 70: 53-143. 1909; Stalstrom, A. Beitrag zur Kenntnis der Einwirkung steriler und in Garung befindlicher organischer Stoffe auf die Loslichkeit der Phosphorsiiure des Tricalciumphosphats. Centrbl. Bakt. II, 11: 724-732. 1904. 654 PRINCIPLES OF SOIL MICROBIOLOGY to 3.8 per cent of CO2. Czapek31 ascribed the most important role in bringing the soil minerals into solution to carbon dioxide. Carbon dioxide interacts with the different phosphates in the follow- ing manner: Ca3(P04)2 + 4H20 + 4C02 = Ca(H2P04)2-H20 + 2Ca(HC03)2 2(CaHP04-2H20) + 2C02 = Ca(H2PO*)2-H20 + Ca(HC03)2 + H20 Ca4P209 + 5H20 + 6C02 = Ca(H2P04)2 + 3Ca(HC03)2 One kilogram of soil containing 0.13 per cent P2O5, when extracted for twenty-five days with distilled water (five repeated extractions of five days each), yielded 0.002 gram P205, while 0.0085 grams P205 was extracted in the same period by water containing carbon dioxide.32 TABLE 71 Solubility of different phosphates PHOSPHATE P2O5 CONTENT SOLUBLE IN 0.5 PER CENT ACETIC ACID SOLUBLE IN 0.5 PER CENT FORMIC ACID SOLUBLE IN CO2 WATER DISSOLVED AND ASSIMI- LATED BY AZ. CHROO- COCCUM Di-calcium Tri-calcium . ... per cent 41.0 41.0 47.0 38.0 44.0 36.0 21.0 0.103 0.180 per cent 97.13 73.83 19.58 8.13 22.09 16.31 62.25 7.76 7.22 per cent 99.54 90.90 29.35 16.00 93.79 54.04 67.65 6.79 8.33 per cent 45.79 25.01 27.44 7.87 4.85 5.50 per cent 32.41 24.89 Mono-differi 40.94 Tri-ferri 9.05 Tri-aluminum 10.71 Florida rock 18.57 Steamed bone meal Dry granitic soil 49.02 Dry basalt soil 50.27 The various organic acids (butyric, lactic, acetic, propionic, formic, valerianic, gluconic, citric, oxalic, fumaric, etc.) formed by soil organ- isms interact with the phosphates, carbonates and silicates present in the soil, to give lactates, acetates, butyrates, citrates, etc. These are usually further decomposed by different microorganisms to carbon dioxide and carbonates. Table 71 shows the amounts of various phosphates made soluble when extracted by dilute solutions of organic acids (for 500 hours), by water saturated with carbon dioxide and by the growth of Azotobacter chroococcum, the particular phosphate being the only source of phosphorus in the medium. 31 Czapek, 1920-1921 (p. xv). 32 Stoklasa, 1911 (p. 649). TRANSFORMATION OF MINERALS IN THE SOIL 655 In the case of the bone meal, the solubility of the phosphate was found to depend on the fineness of division, amount and composition of fat and nitrogen in the keratin and collagen. The decomposition of organic matter in the soil is thus found to influence to a large extent the process of rendering insoluble soil phosphorus available.33 The presence of calcium carbonate represses the solubility of the phosphate. The activities of the soil microorganisms, however, do not always result in an increase of soluble phosphates, but may often even lead to an actual diminution.34 This takes place especially when a large amount of energy bearing materials is added to the soil without a corresponding addition of soluble phosphates. The available energy stimulates the activities of various soil microorganisms, the synthe- TABLE 72 Influence of soil sterilization upon the activities of microorganisms and transfor?na- tion of phosphorus SOIL TREATMENT CO2 FORMATION NITRATE FORMATION IN 100 GRAMS OF SOIL SOLUBLE PHOSPHATE NUMBERS OF BACTERIA 30 days 60 days PER GBAM Sterile soil mgm. 0.281 7.636 2.962 mgm. 0.418 10.854 4 640 mgm. of N2Os 48.3 114.0 12.3 per cent P2O6 0.0060 0.0040 0.0014* 0.0018 millions Sterile soil inoculated with fresh soil suspension Non-sterilized soil 22.8-26.7 3.2- 5.8 Upper figure found at beginning, lower at end of experiment. sizing processes of which result in a disappearance of the soluble phos- phates in the soil. The actual amount of available phosphorus in the soil was found to be a result of the sum total of the activities of the microbial soil population which includes acid formation, secondary reactions, and the synthesizing activities. Soil sterilized in the auto- clave was reinoculated with pure cultures of bacteria or with soil sus- 33 Krober, E. TJber das Loslichwerden der Phosphorsaure aus wasserunlcis- lichen Verbindungen unter der Einwirkung von Bakterien und Hefen. Jour. Landw., 57: 5-80. 1909; see also Koch, A., and Krober, E. Fuhlings landw. Ztg., 55: 225-235. 1906. 34 Sewerin, S. A. Die Mobilisierung der Phosphors;'iure des Bodens unter dem Einflusz der Lebenstiitigkeit der Bakterien. Centrbl. Bakt. II, 28: 561- 580. 1910; 32: 498-520. 1912; Viestnik Bakteriol.-Agron. Stan. (Russian), No. 21: 53-83. 1914. 656 PRINCIPLES OF SOIL MICROBIOLOGY pensions, and, after a certain period of incubation, the amounts of car- bon dioxide formed from 1,100 gm. of soil and the phosphorus brought into solution were determined,34 as shown in table 72. On inoculating sterilized soil with pure cultures of bacteria, Sewerin obtained a gain of 14 per cent of P2O5 soluble in acetic acid for Azoto- bacter -f- Bacterium sp.; a gain of 12.9 per cent for Bac. mesentericus vul- gatus; a gain of 8.0 per cent for Bad. radicicola + Azotobacter; a loss of 5.8 per cent for Bad. fluorescens liquefaciens; and a loss of 12.6 per cent for unsterilized soil. No correlation was found between the bacterial population of the soil and the soluble P205, and none between the latter and the energy of decomposition of the soil organic matter. When manure is added to the soil, the rapidly growing bacteria cause a definite decrease in the water-soluble phosphorus of the manure, and transform it into organic phosphorus. This is eventually re- leased in an available form as a result of the action of the bacteria on the dead microbial cells, after the available energy had been used up.35 The addition of green manure and stable manure to citrus soils in Cali- fornia was found to bring about a measurable increase in solubilty of phosphorus, calcium, magnesium and iron.36 The addition of carbohydrates to the soil brings about an increase in the number of microorganisms and a diminution in the amount of phosphoric acid soluble in 2 per cent acetic acid.37 The amount of soluble phosphate in the soil was found to depend not so much upon the numbers of microorganisms as upon their kind. As to the influence of season of year, an increase in soluble phosphate is usually found in the spring and fall and a decrease in the summer as a result of the activities of microorganisms. The amount of available phosphorus in the soil will thus depend on the total phosphorus in the soil, nature of the phosphorus compounds, soil reaction, presence of available energy and nitrogen, and kind of microorganisms. A bacteriological method for determining the amount of available phosphorus in the soil is described elsewhere (p. 577). Transformation of insoluble phosphates by inorganic and organic acids formed by microorganisms. Inorganic acids are formed in the soil by 35 Tottingham, W. E., and Hoffmann, C. Nature of changes in the solubility and availability of phosphorus in fermenting mixtures. Wis. Agr. Exp. Sta. Res. Bui., 29: 273-321. 1913. 36 Jensen, C. A. Effect of decomposing organic matter on the solubility of certain inorganic constituents of the soil. Jour. Agr. Res., 9: 253-268. 1917. 17 Bazarevski, 1916 (p. 653). TRANSFORMATION OF MINERALS IN THE SOIL 657 the nitrite-forming, nitrate-forming, and sulfur-oxidizing bacteria. The continued oxidation in normal soils of ammonium salts to nitrous and nitric acids results in the formation of appreciable quantities of these acids, 132 parts of ammonium sulfate giving, on complete oxidation, 126 parts of nitric and 196 parts of sulfuric acids. Even organic com- pounds rich in nitrogen, like urea, finally lead to a high acidity. CO(NH2)2 + 2H20-+(NH4)2 C03 + 402-^2 HN03 + C02 + 3H20 The acids may, under proper conditions of reaction, interact with the tri-calcium phosphate added to the soil and make it soluble. Ca3(P04)2 + 2 HN02 = 2 Ca HP04 + Ca(N02)2 Ca3(P04)2 + 4 HN02 = Ca(H2P04)2 + 2 Ca(N02)2 Ca3(P04)2 + H2S04 = 2 CaHPO, + CaS04 Theoretically 188 parts of nitrous acid mixed with 310 parts of pure rock phosphate should give 234 parts of acid phosphate and 264 parts of calcium nitrite. As the average of thirteen tests in liquid culture, Hopkins and Whiting38 found that 115 parts of phosphorus and 211 parts of calcium were made water soluble for every 56 parts of nitrogen oxidized by the nitrite forming bacteria. No further increase was obtained from the action of the nitrate bacteria, since the oxidation of the nitrite to nitrate does not bring about any further increase in acidity. However, the reactions taking place in the soil are not similar to those observed in liquid cultures, due to the fact that the nitrous acid combines in the soil with calcium and magnesium carbonate and salts of organic acids liberating rather weak organic acids, as shown in table 73.39 The acids resulting from the activities of the bacteria neutralize the carbonate in preference to the tri-calcium phosphate in the soil. When considerable acidity is produced, as in the presence of ammonium sulfate, some phosphate goes into solution. But the degree of acidity necessary for the transformation of the tri-calcium phosphate into soluble phosphates is so high (p. 659) that it may become distinctly injurious to crop growth. The amount of phosphate that goes into solution, as a result of the activities of the nitrifying bacteria is, there- 58 Hopkins, C. G., and Whiting, A. L. Soil bacteria and phosphates. 111. Agr. Exp. Sta. Bui. 190. 1916. 39 Kelley, W. P. Effect of nitrifying bacteria on the solubility of tricalcium phosphate. Jour. Agr. Res., 12: 671-683. 1918. 658 PRINCIPLES OF SOIL MICROBIOLOGY fore, very limited. Nitrification of organic nitrogenous compounds like dried blood does not increase the amount of citrate-soluble phosphate when rock phosphate is added to the soil. The nitrification of ammo- nium sulfate in normal soils does not have any appreciable effect upon the solubility of rock phosphate. However, the concentration of the water- soluble calcium is increased in both instances, due largely to the action of the acid upon the calcium present in the soil in the form of silicates.40 TABLE 73 Effect of nitrification on the solubility of tricalcium phosphate in soil* AFTER 28 DAY3 AFTER 57 DAYS AFTER 157 DAYS MATERIALS ADDED X, 0 c3 O d d p.p.m. 25.5 29.0 28.0 28.0 99.0 98.0 990 101.0 90.0 90.0 88.0 87.5 a O p.p.m. 50.6 70.8 58.8 70.1 225.4 270.5 229.6 230.4 113.9 140.2 117.7 138.1 d p.p.m. 11.0 13.2 25.0 22.4 19.4 7.4 38.0 13.9 10.0 11.5 22.2 18.3 d S3 p.p.m. O p.p.m. d Control p.p.m. 20.0 22.0 21.0 22.0 98.0 97.0 99.0 100.0 91.0 89.0 82.0 81.0 p.p.m. 45.0 56.5 53.5 59.1 219.4 254.4 217.7 253.4 107.7 107.2 111.7 118.2 p.p.m. 13.1 11.9 24.2 17.3 18.5 18.5 52.1 26.6 9.7 9.8 24.3 19.5 p.p.m. CaCO-3 Ca3(P04)2 CaCC-3 + Ca3(P04)2 (NHO2SO4 114.0 111.0 94.0 232.1 218.4 116.4 8.0 (NH4)2S04 -}- CaC03 (NH4)2S04 + Ca3(P04)2 (NH4)2S04 + CaCC-3 + Ca3(P04)2 30.0 Dried blood 5.7 Dried blood + CaCOs Dried blood + Ca3(P04)2. . . . Dried blood + CaCOs + Ca3(P04)2 * The ammonium sulfate was added at the rate of 0.01 gram of nitrogen per 100 grams of soil; an equal quantity of nitrogen was added in the form of dried blood; tri-calcium phosphate — 0.10 gram and calcium carbonate — 0.25 gram per 100 grams of soil. The transformation of rock phosphate into soluble forms by sulfuric acid formed from the oxidation of sulfur by microorganisms is very similar to its transformation by the nitrous acid. In pure culture or in composts, the transformation of the phosphate is rapid and almost complete. In the soil, the sulfuric acid tends to transform the calcium *° Ames, J. W. Solvent action of nitrification and sulfofication. Ohio Agr. Exp. Sta. Bui. 351. 1921. TRANSFORMATION OF MINERALS IN THE SOIL 659 and magnesium carbonates, the silicates and salts of organic acids, in preference to the phosphate. The reaction of the medium has to be distinctly acid (pH 3.0) for the phosphate to become soluble. Such acidity is injurious to plant growth. Figure 50 shows the correlation H2SO4 0 cc. 10 20 30 40 50 60 pH 52. 5.0 4.0 3.0 2.0 1.0' Fig. 50. Hydrogen-ion concentration at which rock phosphate becomes avail- able (from Rudolfs). between soil reaction and transformation of tri-calcium phosphate.41 When rock phosphate is used in place of tri-calcium phosphate, the reactions involved are more complicated, since it contains aluminum 41 Rudolfs, W. Influence of sulfur oxidation upon growth of soybeans and its effect on bacterial flora of soil. Soil Sci., 14: 247-262. 1922. 660 PRINCIPLES OF SOIL MICROBIOLOGY and iron phosphates in addition to calcium as shown by the following analysis of Florida rock: per cent -per cent P206 35.52 A1203 1.03 CaO 49.92 Si02 3.57 MgO 0.46 C02 1.64 Fe203 0.98 S03 0.21 H20 2.67 The reactions involved in the conversion of the rock phosphate (in- soluble tricalcium phosphate) into soluble forms (di- and monocalcium phosphate and phosphoric acid) by means of acid belongs to the type of reactions of heterogeneous systems. The rock phosphate minerals have no definite composition and the products formed are not always the same. The following factors control the reaction:42 (1) concentra- tion of the reacting mass; (2) temperature of the reacting medium; (3) the amount of contact of the reacting substances; (4) the speed of diffusion of the reacting substances; and (5) catalytic agents. In addition to these, other factors are of importance, including the chemical composition and the physical properties of the solid phase. These have a tremendous influence on the speed of the reac- tion and they are the least known, since the chemical makeup of the rock phosphate is still obscure. Schloesing43 showed in 1898 that the quantity of phosphoric acid dissolved in normal soils is a result of an equilibrium of very complex chemical processes tending, on the one hand, to take this acid out of solution and, on the other, to bring it into solution. It may only be added that these chemical processes are brought about by the activi- ties of microorganisms, and whatever will influence these activities will also influence the amount of phosphorus available in the soil at any given time. Transformation of potassium in the soil by microorganisms. Potas- sium is present in the soil in the form of organic compounds and in the form of various zeolitic and non-zeolitic silicates. The potassium added to the soil is either in a soluble inorganic form, an insoluble inor- ganic form (marl) , or an insoluble organic form (manures) . The K20 content of fresh manure ranges from 0.288 to 0.504 per cent.44 The ash <2 Kazakov, A. V. Moskau Inst. Agron., 9: 21^5, 57-68. 1913. 43 Schloesing fils, Th. Etude sur l'acide phosphorique dissout par les eaux du sol. Compt. Rend. Acad. Sci., July 25, 1898. "Thome, 1914 (p. 429). TRANSFORMATION OF MINERALS IN THE SOIL 661 content of bacterial cells contains 4.0 to 25.6 per cent K20 and of fungus mycelium 8.7 to 39.5 per cent. The activities of bacteria may lead to an increase in the available potassium, as in the decomposition of organic matter by microorganisms and in the formation of acids which liberate potassium from zeolites. Microbial activities may also lead to a decrease of the available potassium through processes of assimilation and transformation into organic materials. Orthoclase, for example, may interact with acids formed by microorganisms or with the calcium bicarbonate formed from insoluble calcium phosphate by the action of carbon dioxide, to give, in both cases, soluble potas- sium salts: Al203-K20-6Si02 + 4H2S04 = Al2(SO«)» + K2S04 + 6Si02 + 4H20 Al203-K20-6Si02 + Ca(HCOs), = Al203-CaO-6Si02 + 2KHC03 The process of replacement of basic ions in the zeolitic part of the soil is of common occurrence, the hydrogen ion acting also as a base. The solubility of the potassium, often ascribed to the action of acids, may be due merely to the replacement of the potassium in the zeolitic complexes by the calcium or magnesium salts of organic or inorganic acids added to the soil. However, when feldspar, glauconite or other silicates rich in potassium are composted with substances which result in the formation of acid (e.g., sulfur), a great deal of the potassium may go into solution (p. 615). By composting greensand, sulfur, manure and soil, 9.1 to 41.3 per cent of the total initial potassium present can be made soluble.45 The results of Wright cited previously on the action of organic acids upon silicates containing potassium tend to confirm this assumption. This process will take place only very slowly in normal soils. According to Ames,46 the liberation of potassium in the soil is brought about by the salts formed rather than by the direct action of acidity on insoluble potassium compounds, although he found that both nitrification of organic and inorganic nitrogen compounds and oxidation of sulfur in the soil increased the amount of water-soluble potassium. The available potassium compounds are also readily assimilated by the heterotrophic bacteria and fungi and stored away in their 45 McCall, A. G., and Smith, A. M. Effect of manure-sulfur composts upon the availability of the potassium of greensand. Jour. Agr. Res., 19: 239-256. 1920; also Jour. Assoc. Offic. Agr. Chem., 5: 133-136. 1921. « Ames, 1921 (p. 658). 662 PRINCIPLES OF SOIL MICROBIOLOGY mycelium.47 When this is decomposed the potassium again becomes available. However, only a part of the free potassium salt remains in the soil as such; it also replaces some of the zeolitic bases, such as Ca, Mg, Na. The available potassium in the soil at any given time de- pends not only on the total content of this element in the soil, but also on the form in which it is present in the soil, the degree of satura- tion of zeolitic compounds, soil reaction, available organic matter and activities of various groups of microorganisms. The method outlined on page 577 for determining the available phosphorus in the soil can also be utilized for determining the available potassium;48 30 cc. of water containing 2.5 grams glucose, 1 gram Na2HP04 and 0.05 gram MgCl2, is added to 100 grams of air-dry soil. The mixture is sterilized and inoculated with Azotobacter. The amount of nitrogen fixed, under these conditions, is an index of the available potassium (allowing that Azotobacter cells contain about 2.5 per cent K20 and 10 per cent nitrogen). Stoklasa thus studied a fertile soil with a total of 0.093 per cent K20, of which 27.4 per cent was available; a soil of medium fertility with a total of 0.27 per cent K20, of which 5.46 per cent was available, and a poor forest soil with a total of 0.137 per cent K20 of which only 2.18 per cent available. Transformation of calcium in the soil. In normal soils, calcium forms the chief base with which the soil zeolites and the organic complexes are saturated. If an alkali salt is added to the soil, the base is absorbed and an equivalent amount of calcium is replaced. The activities of the microorganisms will affect the transformation of calcium in the soil in various ways. (1) Calcium salts, particularly calcium carbonate, may be precipitated in the soil,49 as a result of the interaction of the soluble calcium salts (of organic acids or nitrates) with carbonic acid produced by the decomposition of organic matter. (2) Calcium car- bonate may be made soluble, as a result of activities of microorganisms 47 Kyropoulos. Uber die Festlegung von Kali durch Bodenbakterien. Ztschr. Giirungsphysiol., 5: 161. 1915. 48 Stoklasa, 1925 (p. 021). 49 Gimingham, C. T. The formation of calcium carbonate in the soil by bac- teria. Jour. Agr. Sci., 4: 145-149. 1911; Drew, G. H. On the precipitation of calcium carbonate by marine bacteria and on the action of denitrifying bacteria in tropical and temperate seas. Carnegie Inst. Washington, Dept. Marine Biology, Papers from Tortugas Labor, 5: 7-45. 1914; Kellermann, K. F., and Smith, N. R. Bacterial precipitation of calcium carbonate. Jour. Wash. Acad. Sci., 4: 40(M02. 1914. TRANSFORMATION OF MINERALS IN THE SOIL 663 in a manner similar to that of the phosphates. The calcium is made soluble much more easily than the phosphate, since organic and inor- ganic acids interact very readily with calcium carbonate and silicates in the soil with the formation of soluble calcium compounds. Nadson50 found that soil bacteria may bring about the formation of calcium carbonate by means of ammonium carbonate which is formed in the decomposition of organic matter: CaS04 + (NHO2CO, = CaC03 + (NH4)2 S04 (1) The formation of calcium carbonate was also observed in the de- composition of organic compounds containing calcium. Nadson even reported the formation of dolomite, or a mixture of calcium and magne- sium carbonates, in media containing bacterial mixtures or a pure cul- ture of Bad. vulgare. According to Molisch,51 various bacteria, yeasts and actinomyces are capable of causing the precipitation of calcium salts; but his use of the term "calcium bacteria" is not justified. Kellerman and Smith49 suggested that calcium carbonate precipita- tion takes place in any of the following ways: (1) Nitrates are reduced to nitrites and ammonia; the ammonia unites with C02 to form (NH4)2C03 which reacts with CaS04 to form CaC03. (2) Ammonia itself may act upon calcium bicarbonate and precipitate CaC03: Ca(HC03)2 + 2 NH4OH = CaC03 + (NH4)2 C03 + 2 H£0 (3) The bacteria utilize organic acids as source of energy; the calcium, with which the organic acids were combined in the form of salts, is thus liberated and reacts with the free C02 to give precipitated CaC03. The dissolution of calcium in the soiL whether present in the form of carbonates or as an absorbed base, is brought about by the various organic and inorganic acids formed by the activities of microorganisms. The calcium thus dissolved is either removed by the plant or is lost in drainage waters. The washing out of the calcium from the soil is sometimes so great that some soils of calcareous origin are practically 60 Nadson, G. Mikroorganismi kak geologitscheskie dieinteli. St. Peters- burg. 1903. 51 Molisch, H. tlber Kalkbakterien und andere kalkfallende Pilze. Centrbl. Bakt. II, 65: 130-139. 1925. 664 PRINCIPLES OF SOIL MICROBIOLOGY free from lime.52 The following reactions are involved in these proc- esses: 1. CaC03 + (NH4)2S04 = CaS04 + (NH4)2C03 2. (NH4)2S04 + 402 = 2H N03 + H2S04 + 2H20 CaC03 + H2S04 = CaS04 + H20 + C02 CaC03 + 2HN03 = Ca(N03)2 + H20 + C02 3. Ca(H2 P04)2 + CaC03 = 2CaHP04 + H20 + C02 4. CaC03 + C02 + H20 = Ca(HC03)2 The use of fertilizers which are directly acid or which lead to the for- mation of acids in the soil, such as ammonium sulfate, superphosphate and sulfur, leads to a depletion of the calcium content of the soil. Calcium is also used directly as a nutrient by various microorganisms and small amounts of it may be assimilated. However, its chief value lies in the neutralization of organic and inorganic acids in the soil and in replacing injurious soil bases (sodium in alkaline soils), thus pro- ducing a medium more favorable for the growth of plants and micro- organisms. Calcium salts also neutralize to some extent the injurious action of soluble magnesium salts, as shown by various studies515 on the influence of the lime-magnesia ratio upon crop growth. This ratio is not of great importance in bacterial action but the concentra- tion of magnesium in solution and its relation to the concentration of the other constituents are of great importance.54 The amount of available calcium in the soil can be determined by adding 5 to 10 grams of soil to 50 or 100 cc. of mannite solution free from calcium, inoculating with Azotobacter and determining the rela- tive amount of growth.55 Transformation of magnesium in the soil. Next to calcium magnes- ium is the most common base in humid soils with which the zeolites and humates are saturated. Magnesium is also introduced in considerable quantities into the soil in the various inorganic materials (dolomitic limestone, rock phosphate) and in the organic matter added to the soil. 42 Hall, A. D., and Miller, N. H. J. Effect of plant growth and of manures upon the retention of bases by the soil. Proc. Roy. Soc. (London), B., 77: 1-32. 1905. 83 Lemmermann, O., Einecke, A., und Fischer, H. Untersuchungen fiber die Wirkung eines verschiedenen Verhaltnisses von Kalk und Magnesia in einigen Boden auf hohere Pflanzen und Mikroorganismen. Landw. Jahrb., 40: 173- 254. 1911. Kelley, W. P. The lime-magnesia ratio. Centrbl. Bakt. II, 42: 519-526, 577-582. 1915. 84 See Greaves, J. E., and Carter, E. G. The action of some common soil amendments. Soil Sci., 7: 121-160. 1919. 66 Christensen, 1923 (p. 730). TRANSFORMATION OF MINERALS IN THE SOIL 665 In the metabolism of bacteria and especially of fungi, magnesium may- form a more important mineral nutrient than calcium. The transfor- mation of magnesium in the soil is very similar to that of calcium, al- though Ames56 found that magnesium compounds are less resistant than calcium salts to the action of nitric and sulfuric acids formed in the processes of nitrification and sulfur oxidation. Transformation of manganese in the soil. Beijerinck57 described several fungi and bacteria which are capable of oxidizing manganese carbonate to oxides of manganese, using cellulose or other carbohy- drates as sources of energy. It has been further shown58 that, in alka- line media, manganese salts are changed to manganese hydroxide, which is then oxidized by atmospheric oxygen to MnCV Microor- ganisms also oxidize manganese salts of organic acids to manganese carbonate. Manganic compounds may also be reduced to manganous compounds by biological processes. Mn02 + 2H2S = MnS + 2H20 + S M11SO4 + 2C + 2H20 = 2H2C03 + MnS MnS + H2C03 = H2S + MnC03 MnS + 2 02 = 2 MnS04 This process was utilized for the determination of the number of cellulose decomposing bacteria in the soil. Filter paper saturated with MnS04 solution is placed in a KMn04 solution. The Mn02 formed turns the paper black. The paper is then placed in petri dishes, steri- lized and moistened with a solution of inorganic salts in tap water. When this is inoculated with cellulose decomposing organisms, the oxy- and fatty acids formed from the decomposing cellulose reduce the manganese dioxide giving clear zones on the paper. When sulfur is oxidized in the soil, considerable quantities of man- ganese may go into solution, especially in soils rich in this element. The utilization of certain manganese compounds as sources of energy by some of the iron bacteria has been pointed out elsewhere (p. 59). Small quantities of manganese seem to act as a stimulant for various organisms,59 and especially for the nitrogen-fixing bacteria.60 56 Ames, 1921 (p. 658). 57 Beijerinck, M. \Y. Oxydation des Mangancarbonats durch Bakterien und Schimmelpilze. Folia Microb., 2: 123-135. 1913. 68 Scihngen, N. L. Umwandlungen von Manganverbindungen unter dera Einflusz mikrobiologischer Prozesse. Centrbl. Bakt. II, 40: 545-554. 1914. 59 Pietruszczynski, Z. The influence of manganese on the nitrification of ammonia. Rocz. Nauk Rolnicz., 9: 235-287. 1923. 60Olaru, 1920 (p. 534). 666 PRINCIPLES OF SOIL MICROBIOLOGY Transformation of zinc. Certain microorganisms are capable of oxidizing zinc sulfide (blende) to zinc sulfate. The transformation is favored by the presence of sulfur.61 Sulfur oxidizing bacteria can bring about the dissolution of natural silicates and carbonates of zinc by the sulfuric acid formed from the oxidation of the sulfur. Traces of zinc will greatly increase the growth of various organisms, especially fungi.62 Transformation of iron. A detailed discussion of the transformation of iron by specific bacteria is found elsewhere. Most of the so-called iron bacteria seem to be able to exist heterotrophically or without iron as a source of energy.63 The precipitation of iron salts seems to be merely a physiological phenomenon accompanying the activities of various bacteria growing in media rich in iron salts. Iron hydroxide and basic ferric salts are precipitated when organic iron compounds and inorganic salts of iron are added to the soil. The precipitation occurs also without the interaction of microorganisms but is much less. In addition to bacteria, some algae, fungi and protozoa are also capable of bringing about precipitation of iron in the form of ferric hydroxide, basic ferric salts, and ferrous sulfide. Indirectly, microorganisms may be of im- portance in depositing ferrous carbonate and ferrous silicate.64 Micro- organisms also play an important part in the transformation of the insoluble iron compounds into soluble forms by means of the carbon dioxide as well as organic acids formed in the metabolism of the or- ganisms. Small quantities of iron added to nutrient media have a catalytic effect upon the activities of various microorganisms. This is especially true of various autotrophic processes, such as oxidation of hydrogen and sulfur. The precipitation of small amounts of iron as a result of sterilization of an alkaline medium containing phosphate has been shown to stop the development of various organisms, such as the hydrogen bacteria. Transformation of aluminum in the soil. Aluminum forms one of the most abundant elements in the soil, especially in clay soils, where it occurs in combination with silicates and with organic substances. It 61 Rudolfs, W., and Hellbronner, A. Oxidation of zinc sulfide by micro- organisms. Soil Sci., 14: 459^64. 1922; also Compt. Rend. Acad. Sci., 174: 1378-1380. 1922. 62 Steinberg, R. A. A study of some factors in the chemical stimulation of the growth of Aspergillus niger. Amer. Jour. Bot., 6: 330-372. 1919; But- kewitsch, W., and Orlow, W. G. Zur Frage nach den "Okonomischen Koeffi- zienten" bei Aspergillus niger. Biochem. Ztschr., 132: 556-565. 1922. 63 Some exceptions are noted by Lieske, 1911 (p. 95). 64 A detailed study of these processes is given by Harder, 1919 (p. 93). TRANSFORMATION OF MINERALS IN THE SOIL 667 may even form definite compounds with some of the constituents of the soil organic matter.65 The transformation of organic and inorganic substances in the soil by microorganisms affects directly or indirectly the solubility and condition of aluminum in the soil; the oxidation of sulfur in the soil brings into solution considerable quantities of alu- per * lOOCC ID 60' 3 days ' growth 2 dolus1 growth 45 ■30 15 + — ♦ — • ^..^' .« -.._... •— • / / / t per 100 cc. 10 15 20 25 30 Fig. 51. Influence of KH2P04 on the formation of ammonia from peptone by bacteria (from Fred and Hart). minum, the same is true of the formation of nitrous and nitric acids from ammonia in soils deficient in bases.66 Role of minerals in bacterial metabolism. The various minerals studied here are more or less essential to the metabolism of microorganisms 65Niklas, 1912 (p. 644); Ostwald, W., and Steiner, A. Beitrage zur Kolloid- chemie von Humussiiure und Torf. Kolloidchem. Beih., 21: 97-170. 1925; Waksman, 1926 (p. 447). 66 Further information on the transformation of aluminum in the soil is given by'Stoklasa, 1922 (p. 646). 668 PRINCIPLES OF SOIL MICROBIOLOGY (fig. 51). Although microorganisms require small amounts of min- erals, they are capable of developing in media having considerable concentrations of the various salts.67 Investigators frequently differ- entiate between fixed and free salts, in regard to their influence upon bacteria.68 The presence of various minerals in the soil is essential not only to the nutrition of microorganisms, but also for the purpose of neutralizing unfavorable reactions, which result from the activities of various organisms. The soil bases, including iron and aluminum hydrates,69 are especially important in this respect. The maintenance of a proper reaction in the soil is very essential for the activities of such important organisms as nitrate-forming,70 nitrogen fixing and various cellulose decomposing bacteria.71 Microorganisms thus play a manifold part in the transformation of minerals in the soil. The activities of these organisms which result in a change of the minerals from one chemical state into another may be classified as follows: (1) Heterotrophic energy utilization of micro- organisms leads to a mineralization of the soil organic matter or to the liberation of minerals from their combination with organic compounds. (2) A part of the minerals thus liberated or added to the soil in the form of inorganic fertilizers may be reassimilated by various soil organisms and thus changed from a soluble into an insoluble condition. (3) The autotrophic bacteria, utilizing minerals as sources of energy, bring about a change in the chemical nature of the minerals in question. (4) The interaction between insoluble minerals in the soil with the products formed by the activities of microorganisms, especially organic and in- organic acids, results in an increase in solubility of these minerals. The stimulating effect of small amounts of various minerals, such as zinc, iron and copper, upon the activities of various specific organisms, and of arsenic upon certain soil processes in general has already been noted. The action of arsenic is probably of the nature of partial sterilization of soil.72 67 Sperlich, A. tJber Salztoleranz bezw. Halophilie von Bakterien der Luft, der Erde und des Wassers. Centrbl. Bakt. II, 34: 406-430. 1912. 68 Guillemin, M., and Larson, W. P. The relation between the fixed and free salts of bacteria. Jour. Inf. Dis., 31: 349-355. 1922. 69 Whiting, A. L. Inorganic substances, especially aluminum, in relation to the activities of soil organisms. Jour. Amer. Soc. Agron., 15: 277-289. 1923. 70 Ashby, 1907 (p. 394). 71 Further information on the influence of minerals and salts upon bacteria and their activities in the soil is given by Greaves, J. E. and Carter, E. G. The action of some common soil amendments. Soil Sci., 7: 121-160. 1919. 72 Greaves, J. E. Stimulating influence of arsenic upon the nitrogen-fixing organisms of the soil. Jour. Agr. Res., 6: 389-416. 1916. CHAPTER XXVI Transformation of Organic Matter in the Soil Nature of soil organic matter. The organic matter of the soil comprises a great mass of substances of plant, animal, and microbiological origin. Some of these substances are undecomposed and include the living or recently dead roots of plants and microorganisms, others are ingredients of the original organic matter resistant to decomposition, others are intermediary substances formed from the original constituents of the organic matter, and the remainder are products synthesized by the activities of the soil microorganisms. Soil organic matter is, therefore, complex in composition and varies according to the original materials, extent of their decomposition, environmental conditions under which the processes of decomposition are taking place, and the organisms con- cerned in these processes. The natural materials which contribute chiefly to the formation of the soil organic matter are plant roots, branches, stems, leaves, plant seeds, dead bodies of all sorts of plants and animals, manures, and the bodies of microorganisms. These natural organic substances consist of proteins and other nitrogenous compounds, of carbohydrates (celluloses, hemicelluloses, compound celluloses, starches, pectins, di- and mono- saccharides, glucosides) and their derivatives, of fats, waxes, lignins, tannins, resins, alkaloids and mineral matter. However, these various constituents of the original organic matter are found in the soil only to a limited extent. Most of the organic materials actually found in the soil consist of substances more or less resistant to decomposition and, to a less extent, of materials undergoing decomposition. Quantita- tively and qualitatively, this organic matter depends upon the activi- ties of the soil microorganisms, which are in their turn influenced by environmental soil conditions including moisture, aeration, soil reaction, and presence of available nitrogen and mineral elements. The average content of organic matter in the soils of the United States is 2.06 per cent and of the subsoils 0.83 per cent. Very little is known concerning the chemical composition of this organic matter, although a large number of chemical substances have been isolated from the soil. 669 670 PRINCIPLES OF SOIL MICROBIOLOGY Carbon makes up over 50 per cent of the elements in this organic matter. The ratio of soil nitrogen to soil carbon is in the case of most cultivated soils more or less constant, about 1 to 10, as shown later. Total organic matter in the soil is measured by ignition, or by multi- plying the total carbon found in the soil by 1.75. The carbon is deter- mined either (1) by dry combustion, (2) by the bomb method, or (3) by wet combustion, using a mixture of chromic and sulfuric acid or of KMnC>4 and sulfuric acid.1 The carbon dioxide is absorbed either in a weighed KOH or soda-lime tube or in standard alkali solution.2 The available organic matter may be determined by oxidation with KMn04,3 or with 6 per cent H202.4 A fraction of the soil organic matter known as "humus" or "humic acid," which comprises 50 to 80 per cent of the total soil organic matter, is frequently determined sepa- rately, by extracting the soil with alkalies, as shown later. The amount and nature of the organic matter in the soil varies with the kind of soil, type of vegetation, methods of fertilization and the numerous environ- mental conditions.5 Various attempts have been made to isolate definite chemical com- pounds from the soil organic matter. Without going into a detailed review of this extensive subject, it is sufficient to say that although the existence of a number of complexes in the soil has been established, it is still a question whether many of these have not been obtained as a result of the treatment of the soil with chemical reagents. By treating the soil with alkali solutions (NH4OH, NaOH, KOH), the organic matter can be divided into several distinct fractions: (1) a part insoluble in the alkali solutions, which includes some of the undecomposed natural organic material; (2) a part soluble in alkalies and precipitated by an excess of hydrochloric acid; (3) a part soluble in alkalies but not 1 Gehring, A. Beitrag zur Kliirung der Dtingewirkung organischer Sub- stanzen. Centrbl. Bakt. II, 57: 241-271. 1922; White, J. W., and Holben, F. J. Perfection of chromic acid method for determining organic carbon. Jour. Ind. Eng. Chem., 17: 83-85. 1925. 2 Waksman and Starkey, 1923 (p. 401). 3 Robinson, C. S., Winter, O. B., and Miller, D. J. Studies on the availability of organic nitrogenous compounds. J. Ind. Engin. Chem., 13: 933-936. 1921; Konig, 1926 (p. 676) Fallot, B. Humus et humification, sur une methode de dosage de l'humus dans les terres. Chimie et Ind. 2: 873-874, 1924. 4 Robinson, G. W., and Jones, J. O. A method for determining the degree of humification of soil organic matter. Jour. Agr. Sci., 15: 26-29. 1925. 6 Lipman, C. B., and Waynick, D. D. A detailed study of effects of climate on important properties of soils. Soil Sci., 1: 5^18. 1916. TRANSFORMATION OF ORGANIC MATTER 671 precipitated by an excess of acid.6 The second group has commonly been known as "humus" or "humic acid," and has been frequently fur- ther subdivided into a number of fractions by the use of various solvents, as the alcohol soluble fraction ("hymetomelanic acid" of Hoppe-Seyler) and the alcohol insoluble fraction ("humic acid" of Oden).7 By special manipulations, various definite chemical substances have been isolated from one or another of the above fractions. Some of these substances are not characteristic of soil organic matter but are constituents of the original materials added to the soil, while some are products of the cell substance, synthesized by the soil microorganisms. Among these isolated substances are those found by Schreiner and Shorey,8 namely pentosans, arginine (C6Hi402N4), histidine (C6H902N3), cytosine (C^HvONs-HoO), hypoxanthine (C5H4ON4), xanthine (C5H4O2N), picoline carboxylic acid (C7H702N), dihydroxystearic acid (C18H36O4) (found also in fungus mycelium), paraffinic acid (C24H48O2), lignoceric acid (C24H48O12), agroceric acid (C21H42O3), agrosterol (C2e H44OH2O), phytosterol (C26H44OH2O), glycerides, resin acids and resin esters. Some of these may become toxic to plants and micro- organisms and have been held responsible by some investigators for the injurious influence of straw upon plant growth.9 Some of the resins, pigments and other substances frequently found among the constituents of the plants added to the soil may be more or less detri- mental to the germination of seed.10 Various aldehydes,11 nitrogenous compounds,12 fats, waxes, and cho- lesterols13 are commonly found in the soil. The organic matter of peat and muck soils may contain 3.3 to 6.2 per cent of fats and waxes;13 6 Waksman, S. A. What is humus? Proc. Nat. Acad. Sci., 11:463-468. 1925. 7 Oden, S. Die Huininsauren. Kolloid Chem. Beihefte, 11: 75-260. 1919. 8 Schreiner, O., and Shorey, E. C. Chemical nature of soil organic matter. Bur. of Soils, U. S. Dept. Agr. Bui. 74. 1910. 9 Collison, R. C. The presence of certain organic compounds in plants and their relation to the growth of other plants. Jour. Amer. Soc. Agr.. 17: 58-68. 1925. 10 Sigmund, W. Uber die Einwirkung von Stoffwechsel-Endprodukten auf die Pflanzen. IV. Einwirkung N-freier pflanzlicher Stoffwechsel-Endprodukte auf die Keimung von Samen: Harze, Farbstoffe. Biochem. Ztschr., 154: 399- 422. 1924. 11 Skinner, J. J. Soil aldehydes. Jour. Frankl. Inst., 186: 165. 1918. 12Lathrop, 1917 (p. 474). 13 Ramann, E. Die von Postlischen Arbeiten liber Schlamm, Moor, Torf, und Humus. Landw. Jahrb., 17: 405^20. 1888. 672 PRINCIPLES OF SOIL MICROBIOLOGY mineral soils may contain as much as 0.154 per cent wax and 0.03 per cent fat.14 The high content of wax is due to its accumulation in the soil, since it is not readily decomposed by microorganisms. The so- called soil "humus" may contain 10 per cent of material soluble in ether and in alcohol (dihydroxystearic-, oxystearic-, lignoceric, and other acids), in the case of virgin soils rich in organic matter, and as much as 4:) to 50 per cent of ether and alcohol soluble substances in the case of soils poor in organic matter.15 The nitrogen of the soil organic matter is made up of complexes which give, on hydrolysis, amino acids and acid amides.16 By boiling the soil with hydrochloric acid, 75.8 per cent of the soil nitrogen was extracted in the form of these compounds, while boiling water alone extracted only 2.92 to 9.96 per cent of the total soil nitrogen. The amino acids and acid amides do not exist in the soil in a free state but in com- bination, probably as constituents of the cells of microorganisms, modified or unmodified by secondary processes of decomposition. When the soil "humus" is obtained from the alkali extract by precipita- tion with an excess of hydrochloric acid and is carefully washed with acid and water, it is found to contain about 3 per cent nitrogen. The nature of this nitrogen has been the subject of considerable controversy; some17 have claimed that it is an extraneous material (as protein nitrogen) and can be separated from the non-nitrogenous "humic acids," while others18 have claimed that it forms an important constituent of the soil "humus" or "humic acids." Various organic acids, such as formic, acetic, butyric and propionic, as well as alcohols, are often found in the soil, especially when the soil is kept under anaerobic conditions. These substances do not remain in the soil as such for a long time, but are used, under favorable conditions, 14 Reinitzer, 1900 (p. 266). See also Fraps, G. S., and Rather, J. B. The ether extract and the chloroform extract of soils. Texas Agr. Exp. Sta. Tech. Bui. 155. 1913. 15 Piettre, M. Recherches, au moyen de la pyridine, des matieres humiques et des matieres grasses du sol. Compt. Rend. Acad. Sci., 176: 1329-1336. 1923. 16 Jodidi, S. L. Organic nitrogenous compounds in peat soils. Tech. Bui. 4, Mich. Agr. Exp. Sta. 1909; The chemical nature of the organic nitrogen in the soil. Iowa Agr. E^p. Sta. Res. Bui. 1. 1911; Jour. Amer. Chem. Soc, 32: 396. 1910; 33: 1226. 1911; 34: 94. 1912. 17 Detmer, W. Die naturlichen Humuskorper des Bodens und ihre land- wirtschaftliche Bedeutung. Landw. Vers. Sta., 14: 248-300. 1871; Oden, 1919 (p. 671). 18 Eggertz, C. G. Studier och undersokninger ofver mullamnen i aker-och mossjord. Diss. Lund. Centrbl. Agrik. Chem., 18: 75-80. 1888. TRANSFORMATION OF ORGANIC MATTER 673 as sources of energy by various microorganisms. Even different alde- hydes and other compounds toxic to plant growth are decomposed in the soil, under favorable conditions.19 Some of these numerous compounds may be present in the soil in very low concentrations or as mere traces, or may be absent altogether. Most of them are probably formed in the processes of extraction of these materials from the soil and are not present in normal soil. The very nature of the larger part of the soil organic matter, which is com- monly referred to as "humus" and which is only very slowly decom- posed in the soil is still little understood, notwithstanding the extensive literature dealing with this subject, as shown later. To be able to understand the nature of the soil organic matter, it is essential to know the processes of decomposition of the various constituents of the natural organic materials added to the soil. Without a knowledge of these transformations, the phenomena leading to a change from the organic substances of plant or animal origin to a group of complex bodies in the soil, very resistant to decomposition, can never be explained. Decomposition of organic matter added to the soil. When a complex natural organic material is added to the soil, it is acted upon by various groups of microorganisms, including the fungi, actinomyces and bac- teria. There is no doubt that some of the protozoa and the various worms and insects present in the soil ingest the organic materials, utiliz- ing their various constituents as nutrients thereby causing various physical and chemical changes, the extent of which depends upon the nature of the organism and the environmental conditions. As a result of these activities, a number of the constituents of the organic tissues are broken down by processes of hydrolysis, oxidation, reduction, and condensation. These processes make available the locked up energy, which is utilized by the microorganisms for their activities of mineraliz- ing the organic matter and liberating the inorganic compounds of nitro- gen, phosphorus, potassium, magnesium, etc. in forms available for plant growth. The composition of plants varies at different stages of growth. Green plants are rich in soluble sugars and soluble nitrogenous compounds;20 19 Gardner, W. A. The decomposition of salicylic aldehyde by soil organ- isms. Science N. S., 60: 503; also p. 390. 1924; Robbins, W. J. The cause of the disappearance of cumarin, vanilin, pyridine and quinoline in soil. Ibid. 44:894-895. 1916. 20 Bogdanov, S. The culture of buckwheat. Selsk. Khoz. i. Liesovod., 193: 227-271. 1899 (Exp. Sta. Reed., 11 : 724) ; Singleton, G. H. Nitrogen availability studies on crops harvested at different stages of growth. N. J. Agr. Exp. Sta. Bui. 421. 1925. 674 PRINCIPLES OF SOIL MICROBIOLOGY mature plants are rich in pentosans, celluloses and lignins. This is true also of leaves.21 It is natural, therefore, to find22 that green plants decompose much more readily than mature plants and that the nitrogen in the former is changed much more rapidly into nitrates than the nitrogen of mature plants, due to differences in the relative composition, in the rates of decomposition and in the nature of the organisms bringing these processes about. A part of the organic matter, consisting of monosaccharides, pento- sans, hexosans, and of the proteins and their derivatives, is completely decomposed, with the formation of C02,H20, NH3 and various minerals, under aerobic conditions; CH4 and H2 are also formed under anaerobic conditions. A part of the materials decomposed is reassimilated by the organisms and synthesized into microbial protoplasm; under aerobic conditions this amounts to as much as 20 to 40 per cent of the decom- posed materials. A part of the original materials is left in the form of intermediate products, due either to the greater resistance of these to the action of the specific microorganisms, or to the formation, under certain conditions such as excessive moisture and excessive acidity, of products which hinder further development of the organisms. A part of the original organic matter, consisting largely of fats, waxes, tannins, resins, and lignins, is left undecomposed. This mass of undecomposed, partially decomposed and transformed materials makes up the soil organic matter, which is being modified constantly. A large part of this organic matter is soluble in alkalies and is commonly referred to as "humus" or the "humified" fraction of the organic matter. The chemical ingredients of the organic matter added to the soil are decomposed at various rates and to varying degrees. Of the non-nitrog- enous substances, the mono-saccharides are the first to disappear; these are followed by the starches and pectins and then by the celluloses and pentosans. The lignins, fats and waxes are decomposed only very slowly. The more ripe and mature the original plant from which the organic matter is derived, the greater is the degree of its lignification and the more slowly does the lignified portion decompose. The pentosans 21 Fricke, K. Beitr;'ige zur Kenntnis der Bestandteile einiger Laubholzblatter. Ztschr. physiol. Chem., 143: 272-289. 1925. 22 Martin, T. L. Decomposition of green manures at different stages of growth. N. Y. (Cornell) Agr. Exp. Sta. Bui. 406. 1921, 139-169; Whiting, A. L., and Schoonover, W. R. The comparative rate of decomposition of green and cured clover tops in soil. Soil Sci., 9: 137-149. 1920. TRANSFORMATION OF ORGANIC MATTER 675 disappear more rapidly than the celluloses, as has been observed in the numerous studies on the composting of manure. It is sufficient to cite the results obtained22* in composting horse manure at 35° to 37°C, under various conditions: Percentage loss of material AEROBIC CONDITIONS C02 ATMO- Moisture content 30 per cent 50 per cent 75 per cent 85 per cent Loss of dry matter 38.5 71.0 53 2 48.2 82.6 61.8 47.8 78.2 64 6 35.7 55.8 41.1 36 3 Loss of pentosans 61 1 Loss of celluloses 47 3 WUat Strew J)ayS Fig. 52. Course of decomposition of different organic materials in the soil, as indicated by the evolution of carbon dioxide (after Dvorak). The protein content of the manure increased as a result of composting. Another result of the composting of natural organic materials, or of their decomposition in the soil, is an increase in the carbon content of the re- sidual material, due to the fact that the pentosans and celluloses with a low carbon content (42 to 44 per cent) disappear and the liginins 22a Jegorow, M. Report of commission on decomposition of manure for 1910 (Russian). Moskau Agr. Inst., 17: 1-59. 1911. 676 PRINCIPLES OF SOIL MICROBIOLOGY (as well as the synthesized cell substance) with a high carbon content accumulate.225 50 40 . 30- 20- £ _ 10- 1 1 I 1 i I - - _, <* ALFALFA M3AL ^■" „<■* a it + UaNO; - 1 1 i i i s f S fS '' / ' / s / ' / s - / s / / - / f - / / // yS / / .^^ ■ ,' / yS 1 / J^ - I1 / '' y it y " // y - it y - /' y i y _ i y _ / / ^^^^ - f y ^^"^ 'i / ^^^ - 11 / s^^ II / ^^ - S /\s _ f /y ■" ^ y^ _ t y i ^^ _ i ^ - Is - 1 1 1 1 1 1 1 1 1 1 1 10 TU£B Fig. 53. Effect of nitrate upon the course of decomposition of rye straw and alfalfa meal (from Starkey). The proteins introduced into the soil with the organic matter are first hydrolyzed to amino acids; some of the amino acids are more rapidly 22b Konig, J. Die Formgebilde der Zellmembran und ihr Verhalten bei der Zersetzung derselben. Biochem. Ztschr., 171: 261-276. 1926. TRANSFORMATION OF ORGANIC MATTER 677 and completely decomposed than others. As a result of incomplete oxidation, substances may be formed which are more resistant than the original materials. In view of the fact, however, that constant synthe- sis of microbial protoplasm takes place, it is often difficult to establish how much of the protein has been decomposed and how much has been resynthesized. The nature and extent of decomposition depend largely Fig. 54. Effect of nitrate upon the course of decomposition of cellulose in soils of different fertility (from Starkey). on the organisms concerned, which in their turn are influenced by the environmental conditions. Under aerobic conditions, especially in aerated and acid soils, the fungi are active. Fungi attack the organic material rapidly, especially in the presence of sufficient available nitro- gen, and synthesize large quantities of mycelium; this is of course later decomposed by the bacteria, with the result that the nitrogen is liberated again as ammonia and rapidly changed to nitrate. The bacteria and 678 PRINCIPLES OF SOIL MICROBIOLOGY actinomyces, which develop in the less acid aerated soils, also attack the proteins readily, liberating ammonia even before all the carbohy- drates have been decomposed, a process unimaginable for fungi. Bac- teria leave a larger amount of the material in the form of intermediate products, including the various organic acids. The extent of decomposition of carbohydrates by microorganisms is found to depend not only on the nature of the organism, but also on the nature and amount of available nitrogen. For every unit of carbon decomposed as a source of energy, a certain amount of nitrogen Plant substances Aninfll substances Complex carbohydrates Proteins and other nitrogenous substances Organic Free COo Fig. 55. Decomposition of organic matter in the soil by microorganisms (original.) is assimilated, whether the latter is present in the form of proteins, simple protein degradation products or inorganic salts. In this respect the fungi differ distinctly from the bacteria. Fungi assimilate a great deal of the carbon decomposed and, although their nitrogen requirement is less than that of bacteria, the total amount of nitrogen assimilated is much greater because of the comparatively much smaller amount of protoplasm synthesized by the bacteria. A schematic presentation of the decomposition of carbohydrates and proteins, the two most important constituents of natural organic matter, with the formation of some intermediate and some final products is TRANSFORMATION OF ORGANIC MATTER 679 given in figure 55, while the transformation of a typical protein in the soil is given in figure 56.23 The decomposition of organic matter in the soil can be traced by determining one or more of the products of the reaction, at a definite set of environmental conditions, and with a definite knowledge of the ALIPHATIC BEw:rffE K . i COC\ EJBDS aaic CCKFJJHDS Cdt?0O3rS 1 1 i i U*thylaMn« ISr.ffinlc told 7o Llenoesrlo «eld Sj Suoolnio aotd Ot illUc soW On aria ioW TrithloUn«- CSQX.'JIC Arjlnln. Pleo a~l° . L) In. CtJO\iKIF Fig. 56. Decomposition of a complex protein in the soil by microorganisms (Schreiner). nature of the organisms which are active. These methods can be classi- fied into three groups. (1) Methods which are based upon measuring the amount of total original organic matter decomposed or only of one or more of its constituents, such as cellulose or pentosan. (2) Those methods which are based upon measuring the accumulation of one of 23 Schreiner, O. Changes in character, condition and amount of soil organic matter. Jour. Amer. Soc. Agron., 18: 115-126. 1926. 680 PRINCIPLES OF SOIL MICROBIOLOGY the final products of the reaction: viz., carbon dioxide, ammonia or nitrate. These are the easiest and most reliable methods; carbon diox- ide is the end product of energy utilization and respiration of microorgan- isms; NH3 is the product of the nitrogen metabolism of microorganisms; since some or all of the ammonia is rapidly oxidized in the soil to nitrates, both ammonia and nitrates should be determined in the latter case. (3) Methods for measuring the formation or accumulation of the less readily decomposable organic materials in the soil, namely the so-called "humus" and the related compounds. Decomposition of the various constituents of the organic matter added to the soil. In view of the fact that the most important constituents of natural organic materials added to the soil are the celluloses, pentosans, lignins, proteins and their derivatives, fats, waxes, and the lower sac- charides, the quantitative determination of these substances is sufficient to indicate the course of transformation of the organic matter added. The methods for making these determinations are described in another chapter. However, these methods have to be modified for certain of the complex natural organic materials, such as straw, corn stover, and forest products. Organic matter or the soil containing it is dried to con- stant weight, extracted with ether to remove the fats, waxes and resins. The material is then treated with cold and hot water to remove the pro- tein degradation products, the sugars and starches. This is followed by alkali extraction (2 to 5 per cent NaOH, for forty-eight hours in the cold or for thirty minutes at 15 pounds pressure), which removes the lignins, proteins, and, in the case of soil, the humus-like substances. The pen- tosans are determined in 1 gram of organic matter or in an aliquot portion of soil by distilling with 12 per cent hydrochloric acid, until a pink color is no longer given with aniline acetate paper, then precipitating as phloroglucide. The cellulose can be determined24 by treating a separate portion of the soil (20 grams) at 98° to 100°C. in flowing steam for 72 hours (for straw and manure) or 192 hours (for sawdust and moss) with 100 cc. of a solution which contains 80 grams NaHS03 and 200 cc. normal HC1 per liter, in flasks with patent rubber clamp stoppers; the material is filtered through hardened filter paper, then washed with water till colorless, dried at 50°C, and extracted with Schweizer's reagent, then precipitating the cellulose with hydrochloric acid, washing and drying as usual. Instead of this procedure, the soil containing the organic material may be extracted with ether, water, 2 per cent NaOH 24 Bengtsson, N. Bestamming av inkrusterad cellulosa i jord. Meddl. f. Centralanst. forsoksv. jordbruks. Bakt. Avd. No. 37. 1925. TRANSFORMATION OF ORGANIC MATTER 681 solution, at 15 pounds pressure for 30 minutes, and boiled with 2 per cent H2SO4 solution, washed and dried and extracted with Schweizer's reagent. These methods permit one to observe the processes of decomposition of the various constituents of natural organic substances added to soil or to sand media, the rapidity with which each is decomposed, and the nature of the constituents which contribute to the formation of the com- plex mass of soil organic matter or "humus." But when, in addition to the original organic substances, the end products of decomposition are also measured, a clearer picture is obtained of the mechanism of trans- formation of organic matter in the soil. In view of the fact that these can frequently be measured, as in the case of the evolution of C02, without disturbing the soil in which decomposition is taking place, they are especially important. Evolution of carbon dioxide as an index of decomposition of organic matter in the soil. In determining the power of the soil to decompose organic matter as indicated by the evolution of C02, the terms "oxida- tive capacity," "carbon dioxide producing capacity," "respiratory capacity of soils" are often used. They all designate the decomposition of organic matter in the soil by microorganisms, whereby energy is liberated. However, carbon dioxide may be formed without the libera- tion of energy, as in the decomposition of pyruvic acid : CH3-COCOOH -> CH3-COH + C02 pyruvic acid acetaldehyde On the other hand, energy may be liberated without the formation of carbon dioxide, as in the case of the anaerobic fermentations of sugars. C6H1206 = 2 CH3-CHOH-COOH Glucose Lactic acid C6H1206 = 3 CH3-COOH Acetic acid Some C02 undoubtedly also originates in normal soils from carbonates interacting with organic or mineral acids formed by biological agencies. The absorption of oxygen can be only a partial index of energy trans- formation, since some energy is liberated without the intervention of free oxygen, as in the case of anaerobic decomposition of organic matter. A more accurate index of energy transformation would be the calorific changes in the soil (the liberation of heat) as a result of the activities of microorganisms.25 "Van Suchetelen, 1923 (p. 425). 682 PRINCIPLES OF SOIL MICROBIOLOGY A detailed study of the carbon dioxide content of the soil atmosphere is given elsewhere (p. 640). A discussion of the evolution of C02 in the soil as an index of the availability of the soil organic matter is also discussed elsewhere (p. 717). It is sufficient to point out here that the evolution of C02 from the soil and its content in the soil atmosphere depend upon a number of other factors outside of the actual carbon content of the soil. Kissling and Fleischer,26 as far back as 1891, used C02 production of peat soils as an index of the rapidity of decomposi- tion going on in the soil. The addition of sand was found to stimulate oxidation greatly, while temperature was found to be one of the most important factors. As a measure of oxidation taking place in the soil, Deherain and Demoussy27 used the amount of C02 present in the atmosphere of a closed 100-cc. tube containing the soil. The tube of soil was kept at constant temperature for a certain period of time. They demonstrated that the production of carbon dioxide in basic soil (1) is due almost wholly to bacteria; (2) it increases with temperature to about 65°C, then decreases, and at higher temperatures (90°C.) in- creases again; (3) it increases with the amount of water present up to a certain point and then decreases, the optimum amount varying with the soil; and finally (4) it is greatly influenced by the state of division of the soil and aeration. Sterile soil produces small amounts of carbon dioxide, but, when reinoculated with soil extract, it forms twenty-five times as much C02.28 Sterilized and inoculated soil gives two to five times as much carbon dioxide as unsterilized and inoculated soil. When manure is treated with disinfectants (thymol, phenol, HgCl2), little C02 is produced; this proves that the process of decomposition of organic matter in the rotting of manure and the evolution of carbon dioxide are biological in nature. Since microorganisms are influenced in their activities by the temperature and moisture content of the medium, one would expect that a change in temperature and moisture, within certain limits, influences the evolution of C02. Decomposition of the organic matter was found to vary roughly with the amount of oxygen available, although some C02 is formed in the complete absence 26 Kissling, R., and Fleischer, M. Die Bodenluft in besandeten und nicht besandeten Hochmoor und Niederungsmoorboden. Landw. Jahrb., 20: 876- 889. 1891. 27 Deherain, P. P., and Demoussy, E. Sur l'oxydation de la matiere organ- ique du sol. Ann. Agron., 22: 305-337. 1896. 28 Sewerin, S. A. Die Mobilisierung der Phosphorsauren des Bodens unter dem Einflusz der Lebenstatigkeit der Bakterien. Centrbl. Bakt. II, 13:616; 28: 561. 1910; 32: 498. 1912. TRANSFORMATION OF ORGANIC MATTER 683 of oxygen.29 Under anaerobic conditions, the organic matter is not decomposed completely and a large part of the energy is left in the form of intermediary products; the amount of C02 liberated is small and cannot serve as good an index of decomposition as under aerobic con- ditions. When the amount of oxygen absorbed is used as an index of oxidation in soil, the rate of absorption is found to increase with the temperature, the amount of water (up to a certain point), and the amount of calcium carbonate; oxygen absorption is favored by con- ditions obtaining in the surface soil as opposed to those in the subsoil.30 When a soil is air-dried and then moistened, or when it is partially sterilized by means of heat or chemicals, there follows a decided increase in microbiological activities, resulting in the liberation of greater amounts of C02. When undecomposecl organic matter is added to the soil, it is decomposed with a rapidity depending not only on the nature of the sub- stance added, but also upon the presence of available nitrogen, soil re- action, moisture, aeration, etc. The amount of C02 produced from the decomposition of a certain organic substance depends also upon the nature of the organisms which are concerned in the process; different organisms attack the same substance, but yield different products. The nature and composition of the organic material added to the soil greatly influence the type of organism developing and the mechanism of the process of decomposition. Glucose decomposes in the soil very rapidly. The absence of available nitrogen does not be- come a limiting factor, since nitrogen-fixing organisms can use glucose as a source or energy and develop readily when large amounts of it are added to the soil. One per cent of glucose is rapidly decomposed in forty-eight hours. The decomposition of cellulose, however, is carried out by certain specific bacteria and fungi, which require an available source of nitrogen; the amount of the latter in the soil will, therefore, become the limiting factor in the decomposition of cellulose. Natural organic substances, which are poor in nitrogen, like cereal straws, corn stover, wood products, contain various water soluble substances. They decompose more rapidly than pure cellulose, but here also the nitrogen soon becomes the limiting factor. In the decomposition of natural organic substances, the monosaccharides and starches are decomposed first, followed by the pentosans, celluloses, pectins, and proteins; the strongly resistant carbonaceous residue (consisting largely of lignins and "Wollny, 1897 (p. 477). 30 Russell, E. J. Oxidation in soils and its connection with fertility. Jour. Agr. Sci., 1: 261-279. 1905. 684 PRINCIPLES OF SOIL MICROBIOLOGY waxes) is decomposed only very slowly.:u Organic matter containing sufficient nitrogen, like ground leguminous plants, cotton seed meal, dried blood, and fungus protoplasm, decompose even more rapidly; the nitrogen, sooner or later, depending on its concentration, becomes liberated as ammonia. Substances rich in proteins decompose at an entirely different rate than those composed principally of carbohydrates. Materials rich in oxygen and low in carbon decompose more quickly than those rich in carbon and poor in oxygen.32 The following amounts of various organic substances, on a per cent basis, were decomposed in 21 days, as indicated by the carbon dioxide, using 10 grams of material per 100 grams of soil: per cent Clover 59 .7 Glucose 42 . 1 Rice straw 29.0 Oak leaves 17 .7 Wheat straw 14.5 Cellulose 11 .8 Similar results were obtained33 when the decomposition of 0.5 per cent organic matter in the soil was studied. The addition of available nitro- gen (NaN03) to a nitrogen poor substance, like straw, stimulates its decomposition but not that of a substance containing sufficient nitrogen, like alfalfa meal (fig. 53). The influence of available nitrogen upon the composition of a nitrogen-poor substance, like cellulose, depends also upon the nature of the soil, as shown in figure 54, where the stimulating effect was greatest in a well manured, fertile soil (5A), least in an unma- nured, poor soil (7A), and medium in an unfertilized but limed soil (7B). These results confirm the earlier observations that the C02 content of the soil rises and falls with the amount of organic matter present and that the addition of manure to the soil stimulates the evolution of carbon dioxide. Fresh manure stimulates this evolution more than old decomposed manure, because of the introduction of both available energy and available nitrogen. Grinding of peat increases its rate of decomposition. 31 Van Suchtelen, F. H. H. tJber die Messung der LebenstJitigkeit der aero- biotischen Bakterien im Boden durch die Kohlensaureproduktion. Centrbl. Bakt. II, 28: 45-89. 1910. 32 Dvorak, 1912 (p. 428). " Starkey, R. L. Some observations on the decomposition of organic matter in soils. Soil Sci., 17: 293-314. 1924. TRANSFORMATION OF ORGANIC MATTER 685 It was suggested34 that C02 formation in soil, whether treated with organic matter or not, does not proceed in accordance with the growth law of bacteria. Under aerobic and constant environmental conditions, it proceeds in accordance with the equation x = ak tm in which x is the quantity of C02 produced in time t, a is the initial CO2 content of the soil, and k and m are constants. However, as experi- ments with peat and other soils have shown, the CO2 formation in the soil is not always necessarily parallel to the concentration of carbon. As to the method used for measuring the evolution of carbon dioxide, at first the air freed from C02 was passed through the soil placed in a container, and the C02 in the outgoing air measured.35 This method was later modified so that air, previously feed from C02, was passed continuously over the surface of the soil in containers. Under these conditions the soil more nearly approaches normal, since constant aeration greatly accelerates microbiological activities.36 Formation of ammonia {and nitrate) as an index of decomposition of organic matter in the soil. Nitrogen is present in the complex organic substances largely in the form of proteins. In the process of decom- position, these are first hydrolized to proteoses, peptones and poly- 34 Lemmermann, O., and Weissmann, H. Z. Pflanzenernahr. u. Dung., 2B: 387-395. 1924. 35 Pettenkofer, M. Ueber eine Methode die Kohlensaure in der atmosphar- ischen Luft zu bestimmen. Chern. Soc. Trans., 10: 292. 1858; Peterson, P. Ueber den Einfluss des Mergels auf die Bildung von Kohlensaure und Salpeter- saure im Ackerboden. Landw. Vers. Sta., 13: 155-175. 1870; Wollny, 1897 (p. 477); Stoklasa and Ernest, 1905 (p. 34); Lemmermann, K., Fischer, H., and Fresenius, L. Untersuchungen liber die Zersetzung der Kohlenstoffverbindungen verschiedener organischer Substanzen im Boden, speciell unter dem Einfluss von Kalk. Landw. Jahrb., 41: 217-256. 1911; Klein, M. A. Studies in the drying of soils. Jour. Amer. Soc. Agr., 7: 49. 1915; Gainey, P. L. Parallel formation of carbon dioxide, ammonia and nitrate in the soil. Soil Sci., 7: 293-311. 1919. 36 Fred, E. B., and Hart, E. B. The comparative effect of phosphates and sulfates on soil bacteria. Wis. Agr. Exp. Sta. Res. Bui. 35. 1915; Fraps, G. S. Oxidation of organic matter in soils. Texas Agr. Exp. Sta. Bui. 181. 1915; Potter, R. S., and Snyder, R. S. Carbon dioxide production in soils and carbon and nitrogen changes in soil variously treated. Iowa Agr. Exp. Sta. Res. Bui. 39. 1916; Merkle, F. G. The decomposition of organic matter in soils. Jour. Amer. Soc. Agron., 10: 281-302. 1918; Keller, J. R. Studies on the correlation be- tween the production of COo and the accumulation of ammonia by soil organ- isms. Soil Sci., 5: 225-239. 1918; Waksman and Starkey, 1923 (p. 739). 686 PRINCIPLES OF SOIL MICROBIOLOGY peptides, and finally to amino acids and acid amides. The latter, when used completely or incompletely as sources of carbon by microorganisms, liberate ammonia. This is oxidized in normal aerated soil to nitrites and then to nitrates.37 Some amino acids are probably split off in the first stages of protein hydrolysis. Some ammonia is also formed long before all of the protein is changed to amino acids; some of the latter are decomposed very readily, while others are more resistant. The amino acids may be directly assimilated by microorganisms or they may form condensa- tion products with carbohydrates. Ammonia and nitrates are the final steps in the transformation of nitrogen in the decomposition of proteins; their determination can serve as a good index of the course of decompo- sition of simple and complex organic nitrogenous compounds in the soil. In the case of some soils rich in organic matter, as in the fertile prairie soils, the transformation of the ammonia into nitrate goes on less rapidly than the formation of ammonia, hence the latter may accumulate.37"1 In the presence of carbohydrates or other carbon compounds, serving as sources of energy for microorganisms, the ammonia and nitrate are rapidly changed again to proteins. The nitrogen in stable manure is about evenly divided between the urea and ammonia nitrogen, on the one hand, and protein or complex organic nitrogen, on the other. When manure is composted or added to the soil, the urea and ammonia nitrogen readily changes to nitrate. The second part of the nitrogen has to be first liberated as ammonia and may again be reassimilated by microorganisms in the presence of considerable quantities of straw. Only after the excess of available carbohydrates has been decomposed, is the ammonia liberated and changed to nitrate.38 The sum total of ammonia and nitrate formed as a result of decompo- sition of a certain organic substance added to the soil can be used as an 37 A detailed study of the decomposition of nitrogenous substances in the soil is given by Lathrop, 1917 (p. 474) ; in forest soil by Si'ichting, H. Der Abbau der organischen Stickstoffverbindungen des Waldhumus durch biologische Vor- gange. Ztschr. Pflanzernahr. Di'mgung., 1: 113-154. 1922; Chemical nature of soil organic nitrogen is discussed by Jodidi, S. L. The chemical nature of the organic nitrogen in the soil. Iowa Agr. Exp. Sta. Res. Bui. 1. 1911. 37a Wyatt, F. A., Ward, A. S., and Newton, J. D. Nitrate production under field conditions in soils of Central Alberta. Sci. Agr., 7: 1-24. 1926. 38 Bright, J. W., and Conn, H. J. Ammonification of manure in soil. N. Y. Agr. Exp. Sta., Tech. Bui. 67. 1919. TRANSFORMATION OF ORGANIC MATTER 687 index of the availability of the nitrogen in the particular material, especially in respect to the particular soil.39 Different organic sub- stances decompose with different rapidity.40 Attempts have been made to differentiate between the fertility of different soils on the basis of the ammonia formed by a given quantity of soil from a given amount of protein, either in solution or in soil. When dried blood, cottonseed meal, urea or other organic material with a high nitrogen content and, therefore, with a narrow C:N ratio is added to normal soils, a large amount of nitrogen will accumulate as ammonia. A number of investigations ("ammonification studies") have been devoted to measuring this ammonia formed from an excess of protein material in different soils. The solution method was at first suggested.41 A 0.5 to 5.0 per cent sterile peptone solution was inocu- lated with a quantity of soil (usually 0.5 to 10 grams per 50 to 100 cc. of solution) , incubated for 2 to 8 days, then the ammonia was determined by distilling the solution with MgO. Certain differences were obtained in the amount of ammonia formed in various soils. It was soon found,42 however, that these differences may be due more to the difference in the quantity of minerals introduced with the various soils rather than to the differences in the kind or activity of the soil flora. It was then suggested43 to use a soil extract peptone solution, but this medium also shows variations as a result of differences in the composition of the ex- tract obtained from the various soils. Since the peptone is so rapidly de- composed in solution and since all soils contain large numbers of bacteria capable of forming ammonia from peptone, very little difference is obtained between different soils. These limitations of the solution method led to the introduction of the soil method (beaker or tumbler method).44 The method consists in 39 Lipman, J. G. Bacteriological methods for determining the available nitrogen in fertilizers. Jour. Indus. Engin. Chem., 2: 146-148. 1910. 40 Lohnis, F., and Green, H. H. Uber die Entstehung und die Zersetzung von Humus, sowie tiber dessen Einwirkung auf die Stickstoff Assimilation. Centrbl. Bakt. II, 40: 52-60. 1914. « Remy, 1902 (p. 710). 42 Fischer, H. Uber die physiologische Wirkung von Bodenauszugen. Centrbl. Bakt. II, 24: 62-74. 1909; Versuche uber Stickstoff umsetzung in verschiedenen Boden. Landw. Jahrb., 41: 755-S22. 1911. 43 Lohnis, F. Zur Methodik der bakteriologischen Bodenuntersuchung. Centrbl. Bakt. II, 12: 262-267, 448-463. 1904; 14: 1-9. 1905. 44 Vogel. Beitrage zur Methodik der bakteriologischen Bodenuntersuchung. Centrbl. Bakt. II, 27: 593-605. 1910; Lipman, J. G. Chemical and bacterio- logical factors in the ammonification of soil nitrogen. N. J. Agr. Exp. Sta. 27th Ann. Rpt. 1906, 119-187. PRINCIPLES OF SOIL MICROBIOLOGY adding a small amount of a certain organic substance (usually 1 per cent) rich in nitrogen, such as hoof meal, cottonseed meal, dried blood, or a pure protein, such as casein, to 100 grams of fresh soil, placing the mixture in a tumbler in the presence of sufficient water and incubating for 7 to 10 days; the ammonia formed is then determined. Even with this method, the differences obtained between the formation of ammonia in different soils were found45 to be due to a number of causes other than the microbial flora of the soil. This becomes self- evident when we consider the following factors: (1) a large number of soil organisms are capable of decomposing proteins with the formation of ammonia; (2) the amount of ammonia formed depends also upon the nature of the carbon of the organic matter added to the soil in the form of nitrogenous and non-nitrogenous substance; (3) a part of the ammonia changes into nitrates, depending upon the physico-chemical, chemical and biological condition of the soil; (4) the amount of ammonia that a soil can hold depends upon its initial reaction, nature of absorbed bases and buffer content. However, according to some investigators,46 no necessary fundamental difference exists between bacteriological processes in soil and solution media. Among the important factors in soil and in solution, the nature, quantity, and distribution of substrate, aeration, diffusion, absorption, destruction or evaporation of metabolic products, reaction of the medium, temperature and duration of experiment are all of importance. Ammonia accumulation can be readily used as an index of the activities of pure cultures of microorganisms or of the complex soil flora upon proteins or different organic materials. The course of ammonia accumulation and the course of C02 produc- tion from the same nitrogenous organic substance added to the soil were found47 to run parallel, tending to indicate that both can be taken as indices of the rapidity of decomposition of organic matter in the soil. However, when the curves of NH3 accumulation from dried blood and C02 evolution from another organic substance, like soy bean meal, were compared, even in the same soil, no parallelism at all was observed.48 46 Temple, J. C. The value of the ammonification test. Ga. Agr. Exp. Sta. Bui. 126. 1919. 46 Lohnis, F., and Green, H. H. Methods in soil bacteriology. VII. Ammoni- fication and nitrification in soil and solution. Centrbl. Bakt. II, 40: 457-479 1914. 47 Gainey, 1919 (p. 685). 48 Neller, 1918 (p. 685). TRANSFORMATION OF ORGANIC MATTER 689 This is due to the fact that different organic substances are attacked at different rates even by the same groups of microorganisms. The addi- tion of these different organic substances to the soil may stimulate the development of different groups of organisms, with the result that there is no basis for comparison between the decomposition of two different substances, even in the same soil, when two different indices of decom- position of organic matter (C02 and NH3 evolution) are employed. It was found49 that, for a certain amount of organic nitrogen, in the form of soy bean cake and herring cake, changed to ammonia, twice as much organic matter is changed to C02 in an acid soil as in a loam soil. By comparing two sources of organic matter, it was found that 1.5 times as much is changed to C02 in herring cake as in soy bean cake. The mechanical soil conditions influence markedly the relation between ammonia, nitrate and carbon dioxide production.50 A detailed study of the influence of straw upon utilization of nitrogen by plants is dis- cussed in detail elsewhere (p. 516). 51 Formation of "humus" as an index of decomposition of organic matter in the soil. The term "humus" has commonly been applied to a hetero- geneous mass of organic matter occurring in soils in different pro- portions. Not only did the methods of its determination vary, but the mere conception of it remained unclear. An alkaline solution, usually 4 per cent NH4OH or 2 to 4 per cent NaOH is commonly used to extract the soil. In some cases, an aliquot portion of the ammoniacal solution is evaporated to dryness and weighed, then ignited and weighed again; the loss in weight is calculated as "humus." In other instances, the alkaline solution is precipitated with hydrochloric acid and this precipitate is taken to be "humus;" this preparation is often referred to as "humic acid." Grandeau's52 original method consisted in leaching the soil first with a dilute acid to set the "humus" free from its combination 49 Miyake, K., and Nakamura, K. On the effect of calcium oxide and cal- cium carbonate upon the decomposition of soybean cake and herring cake in two different soils. Jour. Biochem. Tokyo., 3: 27-54. 1923. 50 Carpenter, P. H., and Rose, A. K. Ammonia, nitrate and C02 produc- tion in relation to the best mechanical soil conditions. Indian Tea Assoc. Sci. Dept. Quart. Jour. 1921, p. 103. 5 > See also May, F. V. tJber den Einflusz von Stroh auf die Ausnutzung organ- ischgebundenen Diingerstickstoffes. Mitt. Wien Hochschule f. Bodenkultur., 2: 433-454. 1913-14. 62 Grandeau, L. Recherches sur le role des maticres organiques du sol dans les phenomenes de la nutrition des vegetaux. Compt. Rend. Acad. Sci., 74: 988-991. 1872. 690 PRINCIPLES OF SOIL MICROBIOLOGY with alkaline earths ("humates"), removing the excess of acid by wash- ing with water, then adding ammonia to the soil and allowing this to stand a few hours; this results in a dark brown solution of the "humus" or "matiere noire." An aliquot portion of this solution was used for the determination of the "humus." This method served as a basis for all modern methods, with various modifications. Some investigators differentiated between the total alkaline solution and the hydrochloric acid precipitate. Schreiner and Shorey,53 for example, referred to a 2 per cent NaOH solution as the "humus" ex- tract and to the precipitate obtained by adding acid to this solution as "humic acid." However, it has been shown54 that not only does a 4 per cent sodium hydroxide solution not extract the same quantity and quality of substances as a 4 per cent ammonium hydroxide solution, but that the very "humus" extract of soils is not a typical soil product. It was also shown that the organic matter extracted by an alkaline solution consists of a black pigment, which contains a relatively small propor- tion of the nitrogen, and of colorless substances. It was frequently concluded, therefore, that the determination of "humus" by alkaline extraction is wholly without scientific justification. Other methods for determining "humus" in the soil are based upon the color of the alkaline extract,55 or upon extraction of the soil with pyridine.56 However, none of these methods were found to be free from criticism. When a soil is treated with an equal volume of 5 per cent solution of sodium hydroxide (or 2 consecutive portions of 2.5 per cent solution), either for 48 hours in the cold or for 30 minutes at 15 pounds pressure, a dark extract is obtained; an equal volume of water is added immediately to the mixture of soil and alkali and the solution filtered through paper; the soil is again treated with an equal volume of 2 per cent NaOH solution and the second filtrate and the washings of the soil with distilled water are added to the first. The combined solution (I) is treated with warm hydrochloric acid (1:1) until a flocculant precipitate is formed, adding a few cc. of acid in excess and shaking the flask. The precipitate (a) is filtered " Schreiner, O., and Shorey, E. C. The isolation of harmful organic sub- stances from soils. U. S. Dept. Agr. Bur. Soils Bui. 53. 1909. M Gortner, R. A. The organic matter of the soil. I. Some data on humus, humus carbon, and humus nitrogen. Soil Sci., 2: 395-441; 539-548. 1916. " Eden, T. A note on the colorimetric estimation of humic matter in mineral soils. Jour. Agr. Sci., 14: 469-472. 1924; Oden, 1919 (p. 671); A detailed review of the investigations dealing with the origin and nature of "humus" in the soil is given by Baumann and Gully, 1910 (p. 644); Oden, 1919 (p. 671); and Waks- man, 1926 (p. 447). " Piettre, 1923 (p. 672); Page, H. J. The part played by organic matter in the soil system. Trans. Faraday Soc, 17: 272-287. 1921. TRANSFORMATION OF ORGANIC MATTER 691 through a weighed Gooch crucible or filter paper; a brown to yellow brown solution (II) is obtained. The precipitate is washed with dilute acid, then with distilled water, dried and weighed; this precipitate or fraction a of the soil organic mat- ter is equivalent to the portion commonly referred to as "humus" or "humic acid." When properly washed with acid, the a fraction contains only 1 to 4 per cent ash and about 3.0 to 3.5 per cent nitrogen. The filtrate (II) from this fraction is then treated with 5 per cent NaOH solution, until the reaction reaches approximately pH 4.8. At this point a heavy precipitate (fraction /3) is formed. This is filtered off; the solution (III) should be straw to yellowish colored; if the solution is brownish, more alkali or acid solution should be added to adjust to the proper reaction and the precipitate is filtered off and added to the rest of fraction /?. This precipitate (J3) is well washed with distilled water, dried to constant weight and then ashed. This fraction is found to contain about 40 to 60 per cent ash and about 1 per cent of nitrogen. It consists of either a chemical or physical complex of aluminum hydroxide and organic materials. These two fractions contain 60 to 80 per cent of the soil organic matter (the undecomposed part is left in the soil and a part is left in the final solution III). The amounts of these two fractions vary with the soil and its treatment and can serve as an index of the amount and nature of the organic matter in the soil.56 Nature of soil "humus." Since the chemical nature of "humus" is not known and the very methods for measuring it are not exact, there is little wonder that a number of theories have been suggested to explain the formation of this more or less stable substance or group of substances in the soil. It was at first supposed that "humus" is derived chiefly from carbohydrates.56 Even at present various theories are frequently suggested to explain how celluloses may change, through the oxy-cellulose stage, into "humic acids."57 However, as far back as 1889, Hoppe-Seyler suggested that cellulose decomposition does not contribute to the formation of "humus" under anaerobic conditions. Cellulose was found to be decomposed completely to CO2 and CH4, according to the following tentative reactions: C6H1206 = 3C02 + 3CH4 The greater the amount of oxygen, the less methane and the more carbon dioxide was formed. The direct sources of "humus" in the soil are looked for among the lignins, proteins, tannins, chlorophyll, pig- ments, certain fats, and resins. The celluloses, hemi-celluloses, mono- and di-saccharides, glucosides, organic acids and various alcohols do not appear to form "humus" directly.58 The protoplasm of fungi 57 Marcusson. Zur Kenntnis der Huminsauren. Ztschr. angew. Chem., 31: 237-238; 34: 437; Ber. deut. Chem. Gesell., 54: 542. 1918-1021. 68 Trusov, A. G. Contributions to the study of soil humus. I. Processes of forr. ation of "humic acid." Materials on the study of Russian soils. XXVI- XXVII. 1-210. 1917. 692 PRINCIPLES OF SOIL MICROBIOLOGY and probably also of bacteria can serve as a source of "humus" in the soil; all organic substances utilized by microorganisms for nutrients can thus become indirect sources of "humus." Typical black "humus" was found to be formed only as a result of the participation of a group of substances: lignin, protein, pigments and tannins; a mixture of lignin and the latter alone, when acted upon for a considerable period of time, may suffice. Celluloses are decomposed completely by soil fungi without the direct formation of humus-like substances, except through the synthesized microbial protoplasm.59 Grave doubts were expressed60 as to whether a specific humification of plant material takes place in the soil, since the so-called humus extract of soils and peats is not a typical soil product formed in the soil by the action of microorganisms, but similar extracts can be obtained from unchanged vegetable material, i.e., the "humus" found in the soil was believed to depend entirely on the process of extraction employed. The formation of "humus" in the soil is thus found to be due either to chemical or microbiological agencies. The different theories explaining this phenomenon can be summarized as follows: 1. "Humus" is formed from the interaction of carbohydrates with amino acids or polypeptides, formed fr?>m the decomposition of plant proteins.61 Proteins were frequently considered62 to be the most im- portant sources of humus in the soil. Certain amino acids, like trypto- phane, may be concerned in the reaction which produces black insoluble humin, but this reaction cannot take place without the presence of some as yet unidentified component of the protein molecule.63 2. "Humus" is a result of oxidation of benzene ring compounds.64 The oxidation of quinone, hydroquinone and phenol results in the for- E9Waksman and Heukelekian, 1924 (p. 443); 1925 (p. 443). 60Gortner, 1916 (p. 690). 61 Maillard, L. C. La formation de matiere humique sans l'oxygene d'atmo- sphere. Compt. Rend. Acad. Sci., 154: 66. 1912; 155: 1554-1556. 1912; 156: 1159. 1913; Genese des matieres proteiques et des matieres humiques. Paris. 1913. 62 Benni. Uber die Entstehung des Humus. Diss. Gieszen. 1896. Suz- uki. Ref. Centrbl. Agr. Chemie., 37: 347. 1908. 63 Gortner, R. I., and Norris, E. R. The origin of the humin formed by the acid hydrolysis of proteins. Jour. Amer. Chem. Soc, 37: 1613. 1915; also 39: 24. 1917; 42: 821, 632, 2378. 1920; 45: 550. 1923; Jour. Biol. Chem., 26: 127. 1916. 64 Eller, W., and Koch, K. Synthetische Darstellung von Huminsauren. Ber. deut. Chem. Gesell., 53: 1469-1476. 1920; Ann. Chim. phys., 431: 133, 162, 177. 1923. TRANSFORMATION OF ORGANIC MATTER 693 mation of acids similar to natural humic acid, of the formula (C6H403)x. These compounds may condense with amino acids to give "humus;"65 the black coloration produced by Bac. mesentericus niger in nutrient media containing a carbohydrate was found to be due to components of the benzene series closely related to o- and p-dihydroxybenzenes which apparently form condensation products with the amino acids. Similar results were obtained for bacteria and fungi.66 3. "Humus" is formed from the polymerization of furfural, the latter being produced when acids are acting upon carbohydrates.67 A dark colored substance containing 75.1 per cent carbon results on heating furfural or oxy-methyl furfural with hydrochloric acid.68 A similar compound was demonstrated in rotting straw and in the soil, but not in the decomposition of cellulose by bacteria.69 These three theories can account only for a very small amount of "humus" in the soil. It must be kept in mind that very strong mineral acids are not found in normal soils and amino acids are not liberated in a free state to any considerable extent. Even the benzene ring compounds, which are not present in great abundance in natural organic substances, are decom- posed by certain soil microorganisms. 4. The formation of "humus" from lignins.70 Lignins are present in amounts ranging from 10 to 40 per cent in natural organic matter.71 While the constituent celluloses, pentosans, and proteins are decom- posed by microorganisms, more or less rapidly, when natural organic matter is added to the soil, the fats, waxes and lignins are acted upon only very slowly, and gradually accumulate in the soil. 5. The role of microorganisms, especially by synthesizing fresh cell 65 Muschel, A. Zur Chemie der Schwarzfarbung kohlenhydrathaltiger Nahr- boden durch den Bac. mesentericus var. niger. Biochem. Ztschr., 131: 570-590. 1922. 68 Perrier, A. Recherches sur la fermentation de quelques composes de la serie cyclique et sur la formation de la matiere noire de l'humus. Ann. Sci. Agron (4), 2: 321-350. 1913. 67 Roxas, M. L. The reaction between amino-acids and carbohydrates as a probable cause of humin formation. Jour. Biol. Chem., 27: 71-93. 1916. 68 Marcusson, J. Die Synthese der Humine und Huminsauren. Ber. deut. Chem. Gesell., 54: 542-545. 1921. 69 Beckley, V. A. The formation of humus. Jour. Agr. Sci., 11: 69-77. 1921. 70 Fischer, F. Was lehrt die Chemie liber die Entstehung und die chemische Struktur der Kohle. Die Naturwiss., 9: 958-965. 1921; Schrauth, W. tlber die chemische Struktur der Kohle. Brennstoffchemie., 4: 161-164. 1923. 71 Ritter, G. J. Distribution of lignin in wood. Jour. Ind. Engin. Chem., 17: 1194. 1925. 694 PRINCIPLES OF SOIL MICROBIOLOGY substance, in the formation of "humus."72 The black pigment of Azof, chroococcum is insoluble in chemical reagents but can be extracted by boiling with strong NaOH solutions, thus also contributing to the soil "humus." The same is true of a large number of fungi.73 To demonstrate the probable role of different groups of microorgan- isms in the formation of soil "humus," it is sufficient to cite the ideas of Falck74 on the origin of "humus" in forest soils. Several processes of transformation of organic matter leading to various types of "humus" formation are differentiated: 1. Mycocriny. This is the complete decomposition of organic matter by fungi, whereby the raw materials (broad leaves, pine needles, roots, branches) added every year to the soil are readily decomposed and there is no increased accumulation of "humus." The filamentous fungi and the higher or mushroom fungi (represented in the soil to a large extent also by the mycorrhiza fungi) can decompose the organic matter to the end, without forming any intermediate dark, humus-like substances. The carbon is either assimilated or given off as CO2, the nitrogen and minerals are largely transformed into fungus protoplasm; the latter forms an excellent forest fertilizer, since it is readily decomposed further. The woody and crude fibrous substances may be thereby destroyed or corroded. In destruction, the cellulose is decomposed completely to C02 through the organic acid stage, more or less resistant to decomposition. The residual substances are soluble in ammonia. In corrosion, the lignin crust is first attacked (by Merulius and other Basidiomycetes), then the cellulose (or both at the same time) is de- composed almost completely. 2. Anthracriny. This is the process of decomposition of organic matter inter- 72 Muller, P. E. Die natiirlichen Humusformen. Berlin. 1887. Kos- tytschew, M. Ann. Agron., 17: 17-38. 1891; Koning, C. J. Beitriige zur Kennt- nis des Lebens der Humuspilze und der chemischen Vorgiinge beiderHumusbild- ung. Archiv. Neerland. Sci. Exact. Nat. (2), 9: 34-107. 1904; Beijerinck, M. W. Uber Chinonbildung durch Strcptothrix chromogenus und Lebensweise dieses Mikroben. Centrbl. Bakt. II, 6: 2-12. 1900; Wehmer, C. Zum Abbau der Holzsubstanz durch Pilze. Ber. deut. chem. Gesell., 48: 130-134. 1915; Waksman, 1926. 73 Rippel, A., and Ludwig, O. Die Schwarzfarbung von Azolobacter chroococ- cum Beij. als Melaninbildung. Centrbl. Bakt. II, 64: 161-166. 1925; Further information on the formation of "humus" in the soil is given by Lohnis, 1910 (p. xiii); Heinze, B. Humusbildung und Humuszersetzung im Ackerboden. Centrbl. Bakt. II, 26: 682-3. 1910; Bottomley, W. B. Formation of humic bodies from organic substances. Biochem. Jour., 9: 260-268. 1915; Muller and Hansendorf. Humusstudien. Ztschr. Forst. u. Jagdwessen., 53: 789-S40. 1921; Waksman, S. A. On the nature and origin of the soil organic matter or soil "humus." I. Introductory and historical. Soil Sci., 22: 123-162. 1926. 74 Falck, R. Mykologische Untersuchungen und Berichte. Gebr. Gotthelft. Cassel., 2: 1923. TRANSFORMATION OF ORGANIC MATTER 695 rupted by lower invertebrates and bacteria. In this process, the fungi begin the decomposition of the organic matter, but cannot bring it to completion. Larvae of various insects and various lower invertebrates devour the mycelium and the complex cells of the original organic matter covered with mycelium and produce a dark mass of "humus." This is then attacked by bacteria, the activities of which result in the formation of nitrates. The process of "humus" formation thus consists here of three distinct biological stages: (a) growth of fungi, which attack the organic matter in a preparatory manner and only to a certain extent; (6) activities of insect larvae and worms, which break down the organic matter, surrounded with the fungus mycelium, and prepare it for the following stage; (c) "humification" proper, the bacteria beginning their action probably in the body of the larvae and then continuing outside. The nitrogen is changed to ammonia and then to nitrate and the ash content of the residue is increased, while appreciable quantities of CO2 are produced. A muck soil re- sults. A good forest soil consists of four layers: (1) an upper layer of unchanged residues, heavier in the autumn when the leaves have fallen; (2) a fungus layer especially active in spring and early summer; (3) a true humus layer consisting of dark-colored insect excreta, mixed with decomposed and powdered leaf residue rich in bacteria and animals; and (4) the first layer of mineral soil, containing extractives of the upper layer washed down by rain water. In the case of My- cocriny predominating, there is no dark muck layer, the unchanged mineral soil following soon after the fungus layer. In some cases, the fungus layer is at a minimum and the muck layer is more extensive. In the second layer the plant residues are all surrounded with the fungus mycelium (the abundance of the fungi under these conditions cannot be determined by the plate method, since the mycelium extends throughout the soil, representing one individual. Only the spores are counted by the plate method, which does not give a fair idea of the abundance of growth and the nature of the population. For such studies the direct microscopic method should be used). The fourth layer is ramified with the roots of trees, which may also penetrate into layers two and three. The two processes of decomposition of organic matter result in a constant evolution of carbon dioxide and later in a continuous formation of nitrate. The first is rapid at the start and then retarded; the second is at first slow, then gradually more rapid. Nitrate is used up in the forest soil as soon as formed; in beaker studies in the laboratory it accumulates only from layer 3. 3. Peat formation. When fungus development is lacking or is insufficient, the undecomposed leaves gradually accumulate on the surface of the soil. The material can be "humified" and broken up and can also pass through the bodies of animals. The chemical processes of peat formation are still unknown;75 it takes place when organic matter is decomposing in the presence of an excess of water; it consists chiefly in the preservation of the carbon, in the splitting off of water and the darkening of the material. The sugars, celluloses and the various other constituents are rapidly decomposed, while the lignins, fats and waxes are fully preserved, since the anaerobic and acid conditions do not allow the development of organisms which are capable of decomposing these substances. 75 For a full discussion of nature of humification process, see H. J. Page. Fuel. 1923, August. 232. 696 PRINCIPLES OF SOIL MICROBIOLOGY The solubility of the organic matter and the ease with which it can decompose decrease with an advance of this process. The further advanced the process of peat formation, the more resistant the materials become to the attack of microorganisms. When peat soils are brought under cultivation, they are at first drained, and if lime is lacking (high-peat or low-lime soils) they are limed, so as to produce favorable conditions for the development of the actinomyces and certain bacteria which are able to decompose the "humus" complexes and liberate the nitrogen in an available form. Since the constant washing of these soils results in the removal of the soluble minerals, the decomposition processes and especially the growth of higher plants are usually favored by the addition of phosphates and potassium salts. Inoculation of a newly drained (and limed if necessary) peat soil with a rich garden soil is essential, so as to introduce the organisms active in the processes of decomposition of organic matter and nitrate formation. Factors influencing the different processes of transformation of organic matter in the soil are (1) temperature (low temperature favors peat formation; thorough aeration and high soil temperature favor fungus development); (2) physical and chemical soil conditions (excessive moisture favors peat formation, low moisture and soil acidity with proper aeration and temperature favor fungus development, high lime content favors the processes of decomposition by neu- tralizing the acids formed by the fungi); (3) abundance of spores, and (4) the nature of plant products (thick needles are decomposed less readily than loose leaves, old needles are decomposed with greater difficulty than young ones). The various forms of "humus formation" are not distinct but may be followed one by another, depending on the soil conditions. Forest trees also thrive well in soils in which the process of decomposition is incomplete, due to the interaction of mycorrhiza fungi, usually the ecto- trophic types. 75a Chemistry and classification of humus compounds. While some in- vestigators consider all the soil organic matter as "humus," others designate by that name only that part of the soil organic matter which is soluble in a solution of an alkali. Still others designate by "humus" only that part of the organic matter which is precipitated from the alka- line solution on acidification; this substance, insoluble in acids and solu- ble in alkalies, was first regarded as an acid ("humic acid"). The decomposition of plant residues under aerobic or anaerobic conditions was considered to yield different groups of substances, to which different names and formulae were assigned: 7Ba A detailed study of the nature of organic matter and nitrate formation in forest soils has been recently made by Hesselman, H. Studien fiber die Humus- decke des Nadelwaldes, ihre Eigenschaften und deren Abhlingigkeit vom Wald- bau. Meddel. Statens Skagsfors., 22: No. 5. 1926. TRANSFORMATION OF ORGANIC MATTER 697 Portion soluble in alkali — Ulmic acid — C40H28Oi Substances formed , , . . .. (modern iormula) under aerobic condi- < _, , . . , , , ditions : Substances formed under anaerobic con- ditions: Portion insoluble in alkali — Ulmin — C40H32O14 (modern formula) Portion soluble in alkali — Humic acid — CtoH3lX)i5 (modern formula) Portion insoluble in alkali — Humin — C^H^Oio (modern formula) The ulmic bodies (in the brown humus) were considered as the first products of decomposition of the plant material in the soil and as occurring with the absorp- tion of oxygen, evolution of C02, and elimination of water. The humic bodies (in the black humus) were considered as the second stage in the decomposition of the plant material. The formation of apocrenic acid (C24H12O12) and crenic acid (C04H24O16) were considered as the third stage in the decomposition of organic matter in the soil. These acids were not precipitated on acidification of the alkaline solution. Crenic acid was a colorless substance and, on exposure to the air, was oxidized to apocrenic acid; the former could be obtained from the latter by reduction with zinc and HC1. The nitrogen was considered to be present in the soil humus in the form of ammonium or related compounds, since most of it was given off as ammonia on boiling with potassium hydroxide. All these theories and formulae were based of course on mere speculation and not on ex- perimental evidence. On the other hand, the physical chemist considered that the organic matter of the soil is not present there in the form of "humic acids" and "humates," or definite chemical compounds of calcium and magnesium, but in the form of complex colloidal systems which form adsorption compounds with the various cations present in the soil. The colloidal nature of "humus" was first recognized by Van Bemmelen76 in connec- tion with his studies on the absorptive properties of soils. "Humic acid" is a hydrosol and can be flocculated by concentrated electrolytes, especially polyvalent cations (calcium). Similar to silicic acid, it is electronegative, but the charge can be reversed by certain electrolytes. Freezing causes the flocculation of "humus" in soils. "Humus" is capable of absorbing large quantities of water and swelling up. It also absorbs nutrient minerals in the soil, particularly bases. The exact 76 Mulder, J. J. Die Chemie der Ackerkrume., 1: 308. 1863; Hoppe-Seyler, F. Uber Huminsubstanzen, ihre Entstehung und ihre Eigenschaften. Ztschr. physiol. Chem., 13: 66-121. 1889; Hermann, R. Untersuchungen fiber den Moder. Jour, prakt. Chem., 22: 65-81; 23: 375-386; 25: 189-206. 1841-1842; Van Bemmelen, J. M. Die Absorptionsverbindungen und das Absorptionsvermogen der Ackererde. Landw. Versuchst., 35: 69-136. 1888; Die Absorption, 81-144. Dresden. 1910. 698 PRINCIPLES OF SOIL MICROBIOLOGY nature of combination of the bases with the soil "humus" is insuffi- ciently understood, some considering them to form definite chemical compounds and some as forming absorption complexes.77 In general the fractionating of the soil organic matter can be repre- ented by the following scheme :78 Soil Treating with NH4OH (or NaOH) Black solution Adding acid I Insoluble humin (Identity of components unknown) r~ i Solution Precipitate (Mulder's apocrenic (Humus) acid or Oden's fulvic acid) (Contains dihydroxystearic acid, Dissolving in alcohol xanthine, hypoxanthine, cytosine, histidine, arginine, pentosan) r 1 Solution Residue (Hoppe-Seyler's (Humic acid of S. Oden) hymetomelanic acid) 1 J Treating with pyridine 1 r n r i inin Compounds Soluble Insoluble obtained by humic acid humic acid Schreiner and Shorey Oden79 submitted some evidence to indicate that "humic acid" is a definite chemical compound, with three or four replaceable hydro- gens, forming salts with alkalies and being electrically conductive. He believes with earlier investigators that the nitrogen is present in this "humic acid" as an impurity, since the content of nitrogen does not fit into the particular chemical formula. However, other investigators80 77 Gedroiz, K. K. The absorbing capacity of the soil and the zeolitic bases of the soil. Zhur. Opit. Agron., 17: 472-527. 1916; Contributions to our knowl- edge of the absorptive capacity of soils. Ibid., 19: 269-322. 1918; 20: 31-58. 1919. 78 Beckley, 1921 (p. 693). "Oden, 1919 (p. 671). 80 Schmuck, A. The organic substances of the soil. Trans. Kuban Agr. Inst. (Russian), I, No. 2- 1-92. 1923. TRANSFORMATION OF ORGANIC MATTER consider "humus" to be a nitrogen containing substance of an acid type, the acidity due partly to adsorption by the colloidal "humic acid" and partly to the presence of carboxyl groups. The salts of "humic acid" are not considered as true salts formed in stoichiometric propor- tions, but as complicated chemical and adsorption compounds. The nitrogen containing part of the "humic acid" is protein in nature and gives hydrolytic products as proteins do; these proteins are at least partly of microbial origin. The "humic acid" contains benzene ring compounds with hydroxyl groups in the form of side chains. "Humic acid" contains about 62 per cent carbon, 3.2 per cent nitrogen, and 4.2 per cent hydrogen, calculated on an ash-free basis. These and more recent81 investigations point definitely to the lignins as the mother sub- stances of these so-called "humic acids." In addition to the lignins, the cell substance synthesized by microorganisms is another source of the soil "humus." A complex mixture of these accompanied by vari- ous decomposed and partly decomposed constituents of plant and micro- bial origin form the soil organic matter or soil "humus." Some of these constituents are amphoteric in nature and increase the buffer content of the soil. The mycelium of fungi, for example, has an isoelectric point at pH 4.9 to 5.5.82 Soil organic matter and the activities of microorganisms. Organic matter influences the growth and activities of microorganisms in the soil by forming a more favorable physical and chemical environment and by offering a source of energy and other nutrients. The different con- stituents of the organic matter and the degree of their decomposition influence to a large extent the nature of the organisms which are capa- ble of developing. The favorable influence of small amounts of soil extract upon the activities of various bacteria, like Azotobacter, led to various assump- tions that we are dealing with certain vitamines (auximones).83 It was also suggested that the favorable influence of organic matter upon the growth of various microorganisms is due to the chemical improve- ment of the medium. Others84 consider soil "humus" as a reservoir of nutrients required by the soil organisms. 81 Waksman, 1926 (p. 694). 82 Robbins, W. J. Isoelectric points for the mycelium of fungi. Jour. Gen. Physiol., 6: 259-271. 1924; Jour. Agr. Res., 31: 385-399. 1925. "Bottomley, 1915 (p. 580); Mockeridge, 1915 (p. 580). 84 Kaserer, H. Einige neue Gesichtspunkte Liber die Rolle des Humus in der Ackererde. Inter. Mitt. Bodenk., 1: 367-375. 1911. 700 PRINCIPLES OF SOIL MICROBIOLOGY However, more careful studies have established the fact that soil "humus" cannot serve as an available source of energy for the great majority of soil microorganisms. Hoppe-Seyler85 believed that "humic" substances afford a habitat and substrate for the soil bacteria, fungi, algae and lower animals, but that "humus" itself cannot offer any food to plants, or animals and cannot be decomposed by bacteria. The very fact that "humus" may accumulate in the soil indicates the low avail- ability of these compounds as sources of energy for microorganisms. One could hardly expect that sources of available energy should exist in the soil in great abundance in the presence of the various microorganisms and not be attacked. The low availability of the soil organic matter as a source of carbon is further confirmed by the fact that the addition of a small amount of available nitrogen, such as NaN03 or (NH.O2SO4, does not greatly stimulate decomposition of the organic matter, as indi- cated by the evolution of carbon dioxide, except in soils with a wide carbon-nitrogen ratio. The nitrogen part of the soil organic matter seems to be more available for the activities of microorganisms than the carbon part. On adding available energy, even in the form of cellu- loses, various organisms are enabled to use some of the soil nitrogen. This has been also clearly demonstrated by various investigators,86 who cultivated fungi using purified "humus" as the only source of nitro- gen; the "humus" could not be utilized, however, as a source of carbon. The various claims put forth that "humus" can be used as a source of energy for nitrogen fixing bacteria, as well as for fungi and urea bacteria still need confirmation.87 Warmbold88 demonstrated previously that 85 Hoppe-Seyler, F. Uber Huminsubstanzen, ihre Entstehung und ihre Eigenschaften. Ztschr. physiol. Chem., 13: 66-121. 1889. 86 Reinitzer, F. Uber die Eignung der Huminsubstanzen zur Ernahrung von Pilzen. Bot. Ztg., 58: 59-73. 1900; Nikitinsky, J. Uber die Zersetzung der Huminsaure durch physikalisch chemische Agentien und durch Mikroorganismen. Jahrb. wiss. Bot., 37: 365-420. 1902. 87 It is sufficient to cite the work of Robertson, R. A., Irvine, J. C, and Dob- son, M. E. A contribution to chemistry and physiological action of the humic acids. Biochem. Jour., 2: 458-480. 1907; Christensen, H. Uber Ureumspalt- ung. Centrbl. Bakt. II, 24: 130. 1909; Krzemieniewski, 1908 (p. 579); Prings- heim, 1908-1912 (p. 564). The same is true of the results of Lipman and Teakle, on the use of displaced soil solution and residual soil as sources of energy by Azotobacter; Lipman, C. B., and Teakle, L. J. H. The fixation of nitrogen by Azotobacter in a displaced solution and in soil residue therefrom. Soil Sci., 19: 99-103. 1925. 88 Warmbold, H. Untersuchungen liber die Biologie stickstoffbindender Bakterien. Landw. Jahrb., 35: 1-123. 1906. TRANSFORMATION OF ORGANIC MATTER 701 "humus" cannot serve as a source of energy for nitrogen-fixing bacteria. Even crude "humic acids" (extracted with NaOH and precipitated with HC1) cannot be used as nutrients by the majority of microorganisms, but may stimulate the activities of various organisms in a physico- chemical way as accompanying mineral impur t'es wil do.89 It is true, however, that "humus" does decompose in well aerated and limed soils and that it does not accumulate under these conditions. The exact nature of the organisms concerned in these processes and the processes themselves still await investigation. When the soil is heated, treated with antiseptics, or air dried, its organic matter decomposes more readily than in the untreated soil. As a matter of fact the amount of C02 formed from sterilized soil inocu- lated with a suspension of bacteria or fungi may run parallel to the availability of the organic matter in the soil. Konig and Hasenbaumer90 determined the amount of C02 evolved from 500 grams of soil in seven weeks, after the soil was sterilized and inoculated with pure cultures of some microorganisms. co2 mgm. Sterile soil 60 .7 Bac. ramosus 179.2 Bac. vulgatus 273 .8 Act. chromogenus 156 .0 The evolution of C02 by a pure culture of an organism from the soil itself, to which no fresh organic matter has been added, varies not only with the organism but with the amount and availability of the organic matter in the soil. Trichoderma was found91 to produce in eight days, from 100 grams of sterile sandy loam manured with cow manure each year, 124.1 mgm. C02, while only 37.4 mgm. were produced from the same quantity of the corresponding unfertilized soil. These quantities are much larger than those obtained by Konig and Hasenbaumer because of the difference in composition of soil used and the greater activity of the fungus over the bacteria. The fact that a soil kept at an optimum mois- ture and temperature will give off a constant stream of C02 indicates that the soil organic matter is constantly decomposing, truly at a low 89 Ritter, G. A. Einige Versuche betrefend die physiologische Bedeutung der Humusstoffe des Bodens. Intern. Mitt. Boden., 2: 301-311. 1912. Centrbl. Bakt. II, 34: 577-666. 1912. 90 Konig and Hasenbaumer, 1920 (p. 620). •» Waksman and Starkey, 1924 (p. 723). 702 PRINCIPLES OF SOIL MICROBIOLOGY rate, but very definitely. Among the various soil organisms, certain actinomyces and various non-spore forming bacteria and cocci seem to be especially capable of attacking the soil "humus."92 Carbon-nitrogen ratio in the soil. According to Agafonoff,93 there is a definite equilibrium between the accumulation of organic matter and its decomposition in the soil. But there is no doubt that by constant addition of fresh organic matter, in the form of green manure or stable manure, the supply of soil organic matter can be increased, especially so under conditions which do not favor soil aeration and in the absence of sufficient bases. There is another equilibrium between the carbon and nitrogen of the soil. Analyses of numerous soils with differ- ent contents of organic matter, reveal a constant ratio between these two elements, this ratio varying in normal soils within narrow limits. Just what this ratio will be in any soil in an undisturbed condition depends on a number of factors, such as the physical and chemical conditions of the soil, which may cause the unequal development of the different groups of microorganisms. The ratio of C to N in the soil has been variously estimated as between 8:1 and 12:1; the ratio becomes narrower in the subsoil. Hutin94 was among the first to report that the average ratio between the carbon and nitrogen of soil organic matter (or "humus") is 11.4. Similar results have been reported by other investigators.95 This ratio varies also with the type of soil,96 as shown in figure 57. Although the first foot of soil in dry land agriculture contains 2.24 per cent organic matter and the second foot 1.62 per cent, the C:N is, in both cases, ll.97 It was suggested98 that the activities of microorgan- isms play an important role in establishing this definite relationship in the soil between the two most important elements. In decomposing 92 Nikitinsky, 1902 (p. 700); Winogradsky, 1924 (p. 10). 93 Agafonoff, V. Sur la limite d'accumulation de l'humus dans les sols a propos des observations des sols de la Nievre. Compt. Rend. Acad. Sci., 177: 828-830. 1923. 94 Hutin, A. Chem. Centrbl. II, 174. 1913. 96 Sievers, F. J. The maintenance of organic matter in soils. Science N. S., 58: 78-79. 1923; Sievers, F. J., and Holtz, H. F. The silt loam soils of eastern Washington and their management. Wash. Agr. Exp. Sta. Bui. 166. 1922; Bui. 176. 1923. 96 Brown, P. E., and O'Neil, A. M. The color of soils in relation to organic matter content. Iowa Agr. Exp. Sta. Res. Bui. 75. 1923. 97 Jones, J. S., and Gates, W. W. The problem of soil organic matter and nitrogen in dry-land agriculture. Jour. Amer. Soc. Agron., 16: 721-730. 1924. 98 Waksman, 1926 (p. 694). TRANSFORMATION OF ORGANIC MATTER 703 Surface. Soil 80.001 70000 60.000 JO.000 40.000 30.000 ^ hf \l \L V^ \\ ;/ .Ccibcn Subsoil R; \ % ? * t 4 ■^ « ^ £ i \ 1 | 5i 1 • ! i 1 s 7C.0CD> l ^ 1 1 L 1 4 Si •^ | 1 / i I , \ 1 \ 60000 /,' v>— // \\ / \ 1; SO OOO // \\ U \ 1! \ is \\ i ! a \ 40.000 i \\ '; \ 1 \i 1 1 i \ 50000 * i \ i 20.000 .flitroqen Carbon i ^ I 'i l s 1 £ i •o o to o ■g o O CO .g 3 > > '^3 -o 3 o T) o h-= o bC t- 03 Sj a 03 £ 43 >, i (- Si D h C O 0> 03 C o "5 CQ > P. Ph s "e o bt s~ a ^ sa. SI Tj4 O tC o Tti m io •<«» 4J E o UJ M K CO o CO ■g a 2 ts 5 &> a H • J t> 3 ^-N £ o e ■< o o a- os n. a ■ n< i- CN e z o 03 > _ ; — o ■< o> 55 >> •^ftd. .Eh^-o s < T» 3 © « O O CO 'r-» a 03 t-h CO i— 1 C? B o T5 > > > . . . ;*j H 3 o •-■ •- O ^ 3 5 '■g E E E 2 -—> -~> --^ fe: fcd Ph 5S ° c O h ^ > Ph Ph -3 U, M S-c o O O O (h o o o Ph Ph Ph Ph u c .— u S3 * C 0 co oo «c 05 CO CN .■S o> B 0 a & a S «! w Eh '5 O M *o aj > >H >> a a a •< 3 -a o lost product ntermediate oorest & H- 1 Ph 714 PRINCIPLES OF SOIL MICROBIOLOGY function for measuring the bacteriological condition of the soil and potential crop production, or soil fertility. The relative numbers of bacteria, actinomyces and fungi can further indicate the chemical condition of the soil, such as soil reaction, degree of decomposition of organic matter, etc. A comparison between the crop yields of a series of plots, fertilized in the same manner for a number of years, in which definite differences in fertility have been established, with bacterial numbers, is given in figure 62. The plots recorded in figure 62 have been fertilized as follows: The A plots were unlimed, the B plots limed, receiving two tons of ground lime- stone per acre every five years; plots 4 and 19 received minerals only (640 pounds acid phosphate and 320 pounds potassium chloride per acre); plot 5 received manure (32 tons per acre) and minerals; plot 7, no fertilizer; plot 9, sodium nitrate (320 pounds per acre) and minerals; plot 18, manure, minerals and sodium nitrate. The addition of lime was found to have a more stimulating effect upon the numbers of microorganisms than upon crop yield. It brought about, in the particular plots, a change in reaction from one below the acid limit for the development of Azotobacter and not very favorable for the development of actinomyces to a reaction very favor- able for the development of both groups of organisms. This, com- bined with the redistribution of the various groups of soil organisms, as a result of liming, may account for the lack of perfect parallelism between the unlimed and limed soils in regard to numbers and crop yields. Let us compare two plots, each ^ of an acre in size. Plot 9A re- ceived yearly applications of 320 pounds of sodium nitrate per acre, 640 pounds of acid phosphate and 320 lbs. of potassium chloride. Plot 11B received an equivalent amount of ammonium sulfate and the same minerals; two tons of lime were also applied every five years. The following results were obtained: Total bacteria and actinomyces Actinomyces, per cent Fungi (on acid medium) Reaction of soil, pH Total crop yield, pounds PLOT 9A PLOT 11 B 10,113,000 9,500,000 25 26 5 46,450 39,100 5.5 5.8 50,488 53,826 MICROBIOLOGICAL ANALYSIS OF SOIL 7l5 NITRIFYING CAPACITY OF THE SOIL Several methods are available for the study of nitrification: 1. Solution or sand method. A standard sterile solution containing a cer- tain amount of ammonium sulfate and CaCOs or MgCOs as a base, in addition to the necessary minerals, is placed in a series of flasks and inoculated with 10 per cent of the soil to be tested. The flasks are incubated at 28° to 30°C. for thirty days and the nitrates formed are determined by the phenol-disulfonic acid method. The results obtained by this method supply information as to the presence or absence of nitrifying bacteria, influence of stimulating substances present in the soil, etc. The solution method may be replaced by the sand method. One hun- dred-gram portions of pure washed sand, containing 210 mgm. CaCOs are placed in 250 cc. Erlenmeyer flasks; 15 cc. portions of a mineral solution (2 grams K2HP04, 1 gram MgS04, 0.4 gram FeS04 in 1000 cc. of water) are then added to each flask. The flasks are plugged and sterilized in the autoclave for 1 hour; 5 cc. portions of a sterile aqueous solution containing 30 mgm. of nitrogen, in the form of ammonium sulfate, are added after sterilization; these are inoculated with 10 grams of soil and incubated for 30 days at 28° to 30°C. The nitrates are then determined. 2. Nitrification of the soil's own nitrogen. This consists in incubating, for 30 days at 25° to 28°C, 100-gram portions of the soil to be tested. The soil is placed in covered tumblers and contains the optimum amount of water (50 to 60 per cent of saturation). The results obtained by this method indicate the con- dition of the forms of nitrogen present in the soil and the rapidity with which these are transformed into nitrate nitrogen. 3. Nitrification of ammonium sulfate. Thirty milligrams of nitrogen in the form of ammonium sulfate are added to 100-gram portions of soil, which are placed in tumblers or flasks and kept at optimum moisture for 30 days at 25° to 28°C. The nitrates formed supply information on the buffer content of the soil and on the maximum nitrate accumulation when no basic substances or buffering agents are added. Initial and final hydrogen-ion concentrations of the soil should be determined. 4. Thirty milligrams of nitrogen as ammonium sulfate and 210 mgm. CaCOs are added to the soil. The lime should be well mixed with the soil before the ammonium sulfate is added. This amount of carbonate is equivalent to an addition of the theoretical amount of base necessary for the complete neutraliza- tion of all the nitric and sulfuric acids formed from the complete oxidation of the 30 mgm. of nitrogen, in the form of ammonium sulfate. The results obtained by this method are more indicative of the nitrifying condition of the soil than any of the other methods, since nitrification is tested here with the reaction factor eliminated. Further work may, however, lead to a modification of this method. Initial and final hydrogen-ion concentration should always be de- termined. 5. Nitrification of organic nitrogenous materials. One-quarter per cent of organic matter with a high nitrogen content (10 to 12 per cent), such as dried blood, or 0.5 to 1.0 per cent of organic materials of a low nitrogen content (cot- tonseed meal, soy bean meal, alfalfa meal) should be employed. Nitrates are 716 PRINCIPLES OF SOIL MICROBIOLOGY determined at the end of 15 and 30 days incubation at 25° to 28°C. The nitrate content of the original soil should always be determined.11 At first the solution method, with or without soil extract, was used12 for meas- uring the nitrifying power of the soil. This method was later found13 entirely unreliable and the use of the soil method was suggested. Lohnis and Green14 called attention to the fact that many of the known critical factors in solution studies on nitrification were ignored by those who have criticized them severely. A definite correlation between the nitrifying power of a soil and its crop productivity has been observed by various investigators.15 In some cases a correlation was found to exist between the nitrogen con- tent of the soils and their nitrifying powers,16 as shown in figures 61 and 62. Some investigators reported that the nitrifying power of a soil may or may not correlate with its crop producing power and that continuous cropping, especially without fertilization, was found to reduce the nitrifying power of the soil.17 While nitrification is a valuable and essential asset in fertility, it probably does not, under normal conditions, become a limiting factor in productivity.18 This is suggested on the 11 Waksman, S. A. Microbiological analysis of soils as an index of soil fer- tility. V. Methods for the study of nitrification. Soil Sci., 15: 241-260. 1923. "Remy, 1902 (p. 710); Lohnis, 1904 (p. 687); Gutzeit, E. Einwirkung des Hederichs auf die Nitrifikation der Ackererde. Centrbl. Bakt. II, 16: 358- 381. 1906; Buhlert and Fickendey. Zur Methodik der bakteriologischen Bo- denuntersuchung. Centrbl. Bakt. II, 16: 399-405. 1906. 11 Stevens, F. L., and Withers, W. A. Studies in soil bacteriology. I. Nitri- fication in soils and in solutions. Centrbl. Bakt. II, 23: 355-373. 1909. l* Lohnis and Green, 1914 (p. 6S8). 16 Gainey, P. L. The significance of nitrification as a factor in soil fertility. Soil Sci., 3: 399^116. 1917; Lipman, C. B. The nitrifying power of soils as indices to their fertility. Proc. Soc. Prom. Agr. Sci. 35th Ann. Med. 1914, 33-39; Cal. Agr. Exp. Sta. Bui. 260, 107-127; Given, G. B. Bacteriology of the general fertilizer plots. Penn. Agr. Exp. Sta. Rpt. 1912-13, 204-206; Brown, P. E. Bacteriological studies of field soils. Centrbl. Bakt. II, 35: 234-272. 1912; Jour. Agr. Res., 5: 855-869. 1916; Burgess, P. S. Can we predict probable fertility from soil biological data? Soil Sci., 6: 449-462. 1918; Ashby, S. F. The comparative nitrifying power of soils. Trans. Chem. Soc, 85: 1158-1170. 1904; Kellerman, K. F., and Allen, E. R. Bacteriological studies of the soil of the Truckee-Carson Irrigation Project. U. S. Dept. Agr., Bur. PI. Industry, Bui. 211. 1911; Waksman, S. A. Microbiological analysis of soil as an index of soil fertility. VI. Nitrification. Soil Sci., 16: 55-67. 1923. 16 Fraps, G. S. Nitrification studies. Science, 42: 68. 1916. 17 Allen, E. R., and Bonazzi, A. On nitrification. Ohio Agr. Exp. Sta. Tech. Bui. 7. 1915. 18 Gainey, 1917 (p. 534). MICROBIOLOGICAL ANALYSIS OF SOIL 717 basis of the fact that all normal cultivated soils contain active nitrifying organisms, which transform ammonia into nitrate. There are other cases on record where the nitrate and ammonia formation in the soil and bacterial numbers are not correlated. When fresh organic matter, particularly of non-nitrogenous nature, is added to the soil, there is a rapid increase in the number of microorganisms. This is not accompanied by an immediate increase in the amount of ammonia or nitrate in the soil, but rather by a decrease, due to the fact that the microorganisms use up the available nitrogen compounds in the process of growth and multiplication. Russell and Appleyard also observed that the curve for nitrate always lags behind that of bacterial numbers. Also as a result of partial sterilization of soil, the bacterial numbers greatly increase without any corresponding increase in nitrates; the ammonia increases but not necessarily in proportion to the numbers. The lack of correlation between certain bacterial proc- esses, such as nitrogen changes, and soil fertility may be due to the fact that, in these cases, the latter is limited by some factor other than the nitrogen supply, such as moisture, temperature, aeration. Whenever plant growth is limited by the supply of compounds pro- duced by bacterial activities, the relationship between bacterial numbers and activities and plant growth is definite. Otherwise it may be accidental or it may not exist at all. CARBON DIOXIDE EVOLUTION The carbon dioxide produced by microorganisms from the decomposi- tion of organic matter has both a chemical and physical action upon the soil. It renders certain insoluble soil minerals soluble and it imparts to the soil, after spring plowing and cultivation, a condition of ripeness ("Gare" in German). An increased carbon dioxide production also stimulates plant growth. Since all heterotrophic aerobic microbiologi- cal processes are accompanied by the production of carbon dioxide, this can be readily taken as an index of the microbiological activities in the soil. After harvesting a crop of rye, oats, clover or alfalfa, considerable amounts of organic matter are left in the soil, so that the quantities of carbon dioxide formed, as a result of the decomposition of these residues in the soil, are quite appreciable. The larger part of the organic matter is decomposed in the first few days, the rate of decomposition soon falling off. In 1905 Russell19 pointed out that soil oxidation, when measured 19 Russell, 1905 (p. 683). 718 PRINCIPLES OF SOIL MICROBIOLOGY by the amount of oxygen absorbed, varied with fertility and suggested using the former as a measure of the latter. Oxidation was influenced by soil temperature and moisture and content of calcium carbonate. Stoklasa and Ernest20 placed 1-kgm. portions of sieved soil in a glass 1(6) Fig. 58. Apparatus for determining the COj-production in field soil: a, Zinc bell, in side view; b, arrangement for taking sample (R = Recipient) (from Lundegardh). 20 Stoklasa, J., and Ernest, A. Uber den Ursprung, die Menge und die Bedeu- tung des Kohlendioxyds irn Boden. Centrbl. Bakt. II, 14: 723-736; Ztschr. Zuckerind. Bohmen., 31: 291-401. 1911; Stoklasa, J. Methoden zur Bestim- mung der Atmungsintensitiit der .1'akterien im Boden. Ztschr. Landw. Ver- suchst. Oesterreich., 14. 1911, 1243-1279; also Chem. Ztg., 46: 681-683. 1922. MICROBIOLOGICAL ANALYSIS OF SOIL 719 cylinder through which a current of air was passing at the rate of 10 liters in 24 hours; for the study of anaerobic activities they substituted hydrogen for air. They suggested that the determination of carbon dioxide evolved by a soil under given conditions of moisture, tempera- ture and time, can furnish a reliable and an accurate method for the n Fig. 59. Gas analysis apparatus: B, gas burette; H, three way stop cock; A, potash apparatus (30 per cent KOH); i, index; N, mercury vessel; F, fine adjustment (from Lundegardh). determination of bacterial activities in the soil. The presence of organic matter and the temperature were found to be of greatest importance in the production of carbon dioxide. Evolution of carbon dioxide was found to occur most abundantly in neutral or slightly alkaline soil abundantly supplied with air and readily assimilable plant nutrients; 720 PRINCIPLES OF SOIL MICROBIOLOGY it was also found to run parallel with nitrification. Rahn21 used sugar solutions containing CaC03, so that he measured not only C02 formed by bacteria but also that produced from the interaction of the organic acids formed with the CaC03. Drying of soil was found to exert a decidedly favorable influence. Van Suchtelen22 placed upon the bottom of a jar pure sand and upon it 6 kgm. of soil. Through this he passed air, usually 16 liters in 24 hours. The intensity of C02 production was found to be much greater in the beginning of the experiment but it decreased rapidly after a short time. Carbon dioxide was measured until a definite intensity Fig. 60. Method for determining the ability of soil to decompose organic matter (from Waksman and Starkey). has been attained. The average production of carbon dioxide in a unit of time was used as a measure. It was concluded that the com- parison of the carbon dioxide production of different soils furnishes a better means for the estimation of their relative bacterial activities than the bacterial content. Soil cultivation was found to have a favorable influence upon the carbon dioxide evolution, well sieved 11 Rahn, O. Bakteriologische Untersuchungen liber das Trocknen des Bodens. Centrbl. Bakt. II, 20: 38-S1. 22 Van Suchtelen, F. H. H. tlber die Messung der Lebenstiitigkeit der aero- biotischen Bakterien im Boden durch die Kohlensiiureproduktion. Centrbl. Bakt. II, 28: 45-89. 1910. MICROBIOLOGICAL ANALYSIS OF SOIL 721 soils producing 177 per cent as much C02 as unsieved soils. An increase in aeration brought about an increase in CO2 production in soil to which organic matter had been or had not been added. Moisture was found to be one of the most important factors. The influence of salts upon the evolution of carbon dioxide is seen from the following table. CS2 was found to serve first as a check and then as a stimulant upon carbon dioxide production. TABLE 77 CO2 formed in 5 days, at 10° to 12°C.,from 6 kgm. of soil, with and without different salts TREATMENT Soil alone Soil + 90 grams MgSO* Soil + 6 grams CaO Soil + 30 grams ammonium sulfate. Soil + 6 grams superphosphate . . . MILLIGRAMS OF CO2 145 408 62 864 306 TABLE 78 Nmnbers of bacteria and evolution of CO2 in different soil types 801LTTPE Sandy soil Loam Clay Bacterial numbers 8,300,000 96 193 3179 9,800,000 109 188 3730 11,900,000 Milligrams of C02 produced in 24 hours, no glucose added 111 Milligrams of C02 produced in 3j days, no 434 Milligrams of C02 produced in 3| days, 0.1 per cent glucose added 3660 By using C02 as an index, it was found that similar soils behave the same under similar conditions. When glucose is added, the abundant production of CO2 tends to obliterate any differences, hence the briefest possible period (12 hours) should be used in this connection. A certain parallelism was obtained between the numbers of bacteria and the evolution of carbon dioxide in different soils, as shown in table 78. The curves for bacterial numbers, nitrate content and carbon dioxide in the soil air were found23 to be sufficiently similar to justify the view 23 Russell, E. J., and Appleyard, A. The atmosphere of the soil: its com- position and the cause of variation. Jour. Agr. Sci., 7: 1. 1915. 722 PRINCIPLES OF SOIL MICROBIOLOGY that all these phenomena are related. A rise in bacterial numbers was accompanied by a rise in the C02 in the soil air, and somewhat later by a rise in the nitrate content. The rate of decomposition of organic matter in the soil was, therefore, looked upon as a function of bacterial activity. It was further demonstrated24 that the principal factors affecting carbon dioxide production are, in order of importance, tempera- ture, moisture, dissolved oxygen and the growing crop. In all the investigations up to 1915, the air was either drawn through the soil, thus greatly accelerating microbiological activities, or inter- mittenly over the soil. Potter and Snyder25 found that field results can be most closely duplicated in laboratory studies when the air is passed continuously over the soil. The amount of air passing over the soil in the laboratory does not affect materially the amount of C02 evolved. When the soil is placed under optimum laboratory conditions, the initial rise in carbon dioxide formation is soon followed by a drop, which becomes nearly a straight line. Klein26 also found that the amount of CO2 given off by the soil when brought into the laboratory and the proper amount of moisture added is considerable for a short period. He attributed this phenomenon to the previous drying of the soil which made the soil a better medium, both physically and chemi- cally, for the growth of bacteria. This is no doubt correct, since when normally moist soil is used no such rapid drop is observed. Previous drying of the soil alters its colloidal condition to the extent of in- creasing the rate of oxidation. Because of this, rainfall may increase the carbon dioxide formation in field soils, due to the increase in moisture content more favorable for biological activities rather than for the reason of oxygen brought down by the rain, as suggested by some investigators. It was found, by the use of this method, that manure stimulated C02 production while sodium nitrate and ammonium sulfate did not. CaC03 did not indicate at first any increase of CO2 as a result of the decomposition of the organic matter (the abundant formation of C02 when CaC()3 is added to an acid soil is due to chemical interaction between the carbonate and the buffering sub- 24 Russell, E. J., and Appleyard, A. The influence of soil conditions on the decomposition of organic matter in the soil. Jour. Agr. Sci., 8: 3S5-417. 1917. 25 Potter, R. S., and Snyder, R. S. Carbon dioxide production in soils and carbon and nitrogen changes in soils variously treated. Iowa Agr. Exp. Sta. Res. Bui. 39. 1916. 26 Klein, M. A. Studies in the drying of soils. Jour. Amer. Soc. Agron., 7: 49. 1915. MICROBIOLOGICAL ANALYSIS OF SOIL 723 stances of the soil). After prolonged incubation, following the addition of CaC03, a definite favorable effect was evidenced. A marked correlation between crop yield, nitrate accumulation and bacterial numbers, but not between crop yield and ammonia accumu- lation, is shown in figure 61 where the bacterial activities of limed and unlimed soils are compared.27 The soil (1 kgm.) may be placed in a pot which is then covered with a bell-jar using the suction apparatus described elsewhere. One can differentiate between the formation of C02 in untreated soil and the formation of C02 from glucose or other available organic matter CROP YIELDS COj PRODUCTION NC3 ACCUMULATION BACTERIAL NUMBERS NH3 ACCUMULATION 7T3TT" TxTTZ 7TTT3 Unlimed Plots 11A and 21 Limed Plots 11B and 24 Fig. 61. The correlation between crop yields, C02-producing power of the soil, bacterial numbers, NH3 and N03 accumulation in soil from limed and unlimed plots (from Neller). added to the soil.28 In the first instance, a definite amount of soil is placed under optimum conditions of moisture and temperature and the amount of CO2 formed in a definite period of time is measured. This was found (Stoklasa) to depend upon (1) the number and kind of microorganisms present, (2) the amount of organic matter in the soil, (3) the composition of this organic matter and the degree of its decom- 27 Neller, 1920 (p. 712). 28 Waksman, S. A., and Starkey, R. L. Microbiological analysis of soils as an index of soil fertility. VII. Carbon dioxide evolution. Soil Sci., 17: 141— 161. 1924. 724 PRINCIPLES OF SOIL MICROBIOLOGY position, (4) soil aeration, (5) moisture content, (6) physical condition of the soil, (7) chemical composition (altered by fertilization), (8) soil reaction and (9) kinds of plants grown. The C02 thus determined is a measure of the rapidity with which the soil organic matter itself de- composes under the influence of the sum total of microbiological complexes. It can be determined in three different ways: 1. One-kilogram portions of fresh soil from a composite sample taken to a depth of 65 inches and put through a 3 mm. sieve are placed in pots. Enough water is then added to bring the moisture content of the soil to the optimum. The pots of soil are then placed in the respirator and the amount of C02 evolved in fourteen days is determined at various intervals. 2. One kilogram portions of air-dried, sieved soil, taken to a definite depth, are placed in proper containers; the necessary amount of water is added and the CO2 evolved in 24 hours is determined. By this method, Stoklasa29 found that an infertile soil, poor in organic matter, produced 8 to 14 mgm. CO2, while a good beet soil produced 56 to 68 mgm. 3. One hundred-gram portions of fresh soil, prepared as in the first method, are placed in 300-cc. flasks with long necks (A in figure 60). Cotton plugs are placed in the necks of the flasks and in the glass connections. After the proper amount of water is added (50 per cent of total moisture holding capacity), the flasks are sterilized for 1 to l-2- hours, on two consecutive days, at 15 pounds pres- sure. The soils are then inoculated with a culture of Trichoderma which was found to be one of the most active groups of soil fungi decomposing celluloses, proteins, pectins and other complex organic substances; a suspension of fresh cow manure may also be used for inoculation. The flasks are then connected with the Ba(OH)2 tubes in the respirator and the amount of C02 evolved is determined for 12 to 14 days. By this method, two soils, one, fertile and rich in organic matter, and another, infertile and poor in organic matter, were found to produce 124.08 and 37.40 mgm. of C02 respectively, in eight days. The decomposition of fresh organic matter added to the soil can be determined by a group of methods, which differ chiefly in the nature of the organic matter used. A few substances have been commonly employed : 1. Glucose. This substance is very readily decomposed in the soil; an ex- cess of the material, as well as a long period of incubation, may obliterate finer differences in the activities of the microorganisms in the different soils. It is best to add 500 mgm. of glucose to 100 grams of soil and determine the C02 evolved every 6 or 12 hours for a period of 48 to 72 hours. The resulting curves bring out distinctly the differences in the microbiological activities of the dif- ferent soils. Since glucose is used very readily as a source of energy not only 29 Stoklasa, J. Le role de l'acide carbonique degage par les microorganismes dans l'amelioration des terres culturales arables pour obtenir le meilleur rende- ment. Compt. Rend. Agr. France, 8: 594-596. 1922. MICROBIOLOGICAL ANALYSIS OF SOIL 725 by the soil fungi and actinomyces, but also by the great majority of hetero- trophic soil bacteria, including the nitrogen-fixing organisms, its rate of de- composition is very rapid. It is especially rapid when conditions are favorable for the latter group of organisms, which make them independent of a supply of available nitrogen. 2. Cellulose. The decomposition of cellulose in the soil is based upon the establishment of a nitrogen minimum. One gram of cellulose, in the form of ground filter paper, is added to 100 grams of soil and the C02 evolved is deter- mined; this indicates not only the ability of soil to form C02 from cellulose, but also the amount of available nitrogen and phosphate present in the soil. This is due to the fact that the cellulose is decomposed in the soil (with the exception of alkaline or partially sterilized soils) to a large extent by fungi. These rapidly growing organisms consume a great deal of nitrogen in the synthesis of their mycelium, and the nitrogen soon becomes a limiting factor; therefore, the greater the amount of available nitrogen in the soil the larger will be the quantity of cellulose decomposed. The distinctive differences in the curves of C02 evolu- tion from glucose and cellulose have been pointed out elsewhere (p. 675). In addition to glucose and cellulose, other substances, like straw, alfalfa meal and dried blood, may be used in the study of decomposition of organic matter in the soil. Cellulose may also be used together with sufficient nitrogen in the form of inorganic salts. CELLULOSE DECOMPOSING CAPACITY OF THE SOIL Christensen30 was the first to suggest that the cellulose decomposing power of a soil may serve as an index of soil fertility. A definite amount of the soil to be investigated was placed in Erlenmeyer flasks so as to cover four-fifths of the bottom of the flasks. Water was added from a pipette to the uncovered part of the bottom of the flask. A few strips of filter paper were then pressed upon the soil, the latter being kept moist during the period of incubation. Between 9 to 93 days were required for the complete decomposition of the paper. The physical condition of the soil and its reaction did not influence greatly the cellulose decomposing capacity of a particular soil.31 The presence of available minerals, primarily phosphates, as well as available nitrogen were found to be of first importance, and, in some cases, the microbial flora; i.e., the phenomenon of cellulose decomposition is influenced by the chemical and microbiological soil conditions. The amount of cellulose decomposed was governed by the available nitrogen and phosphates in the soil. Only in the case of certain peat soils, did the 30 Christensen, H. R. Ein Verfahren zur Bestimmung der zellulosezersetz- enden Fahigkeit des Erdbodens. Centrbl. Bakt. II, 27: 449-451. 1910. 31 Christensen, H. R. Studien iiber den Einflusz der Bodenbeschaffenheit auf das Bakterienleben und den Stoffumsatz im Erdboden. Centrbl. Bakt. II, 43: 1-166. 1915. 726 PRINCIPLES OF SOIL MICROBIOLOGY inoculation of the soil with cellulose cleccmposing bacteria have any effect. Mutterlein32 suggested placing one or two pieces of filter paper of a uniform weight (10 grams) at various depths of soil, then, after 2 to 3 weeks, removing the paper from the soil and weighing the residue; the loss in weight of the paper was taken as an index of the cellulose- decomposing capacity of the soil. Niklewski33 added cellulose to the soil and measured the carbon dioxide produced by a soil thus treated. In addition to cellulose, 1 gram K2HP04, 1 gram MgS04, 8 grams CaC03, and (NH4)2S04 were added to 8 kgm. of soil. He found that the decomposition of cellulose is chiefly controlled by the presence of available nitrogen in the soil. The greater the amount of cellulose added or present in the soil, the quicker does the nitrogen need set in. Nitrogen fixing organisms were thought to play only a secondary role in normal soils, since cellulose is very slowly decomposed in normal soils without the addition of avail- able nitrogen. This would not be the case if nitrogen-fixing organisms were active, as when glucose is added. When only 0.125 per cent cellulose was added to a loess soil, containing 0.15 per cent nitrogen, a nitrogen need could be observed. The greater the amount of cellulose added, the greater was the evolution of C02 up to a certain concentra- tion, 1.5 per cent giving at first less C02 than 1.0 per cent cellulose. When the available nitrogen is exhausted, the curve soon falls to a certain level depending upon the rapidity of decomposition of the nitrogenous substances in the soil and the rate with which the nitro- gen becomes available. The addition of 0.0125 per cent ammonium sulfate greatly stimulated cellulose decomposition; this amounted to about 1 gram of (NH4)2S04 in the case of soil, and 2 grams of the ammonium salt in the case of sand, for every 10 grams of cellulose. Larger amounts of nitrogen acted injuriously; this injurious action may be later overcome. On comparing the evolution of carbon dioxide, with and without the addition of a nitrogen salt, Niklewski suggested calculating the available nitrogen in the soil from the amount of cellulose decomposed, as indicated by the evolution of C02. In the case of loess soil, with a total of 0.150 per cent nitrogen, 0.040 per cent nitrogen was found to 32 Mutterlein, C. Studien ttber die Zersetzung der Zellulose im Dilngen und im Boden. Inaug. Diss., Leipzig. 1913. 33 Niklewski, B. Bodennakteriologische Beobachtungen als Mittel zur Beurteilung von Boden. Centrbl. Bakt., II, 32: 209-217. 1912. MICROBIOLOGICAL ANALYSIS OF SOIL 727 be active, while in the case of a sandy soil with a total of 0.015 per cent, all the nitrogen was active, or could be made readily available. One per cent of cellulose, in the form of finely divided or ground filter paper, is added to soil sieved through a 2-mm. sieve. After carefully mixing the paper with the soil the proper amount of moisture is added. At the end of the incubation period, the soil is air dried and the amount of cellulose left undecomposed determined by extract- ing with Schweizer's reagent (see p. 431). Stable manure was found to have a decided effect upon cellulose decomposition in the soil, es- pecially when the moisture content is satisfactory. The influence of reaction is not of great importance in cellulose decomposition and lime serves merely in adjusting the reaction. The favorable influence of manure is due to the nutrients present, especially the nitrogen. The greater the amount of nutrients (nitrogen and minerals) present in the manure, the greater is its favorable influence. The poorer the soil is, the greater is the influence of the manure. When ammonium sulfate and manure containing the same amount of nitrogen were added to the soil, the stimulating effect upon cellulose decomposition was found to be the same.34 These facts, namely that the cellulose decomposing power of a soil depends more upon the physical and chemical conditions of the soil, especially the available nitrogen, rather than upon a specific microbial flora can be readily explained when the activities of microorganisms concerned in cellulose decomposition in the soil are considered. As pointed out above, cellulose is decomposed in normal soils largely by various fungi and aerobic bacteria. Only in soils saturated with water do the anaerobic bacteria become active in cellulose decomposi- tion. The ratio between the amount of cellulose decomposed and the amount of nitrogen assimilated is about 30:1 in the case of fungi and aerobic bacteria; however, in the soil, when the cells of microorganisms freshly synthesized are constantly decomposed by other organisms, the ratio is 50-60:1. In other words, for every unit of nitrogen that can become available in the soil in a definite period of time, about 50 to 60 units of cellulose will be decomposed. If one gram of cellulose in the form of ground filter paper were added to 100 grams of soil, then 34 Charpentier, C. A. G. Studien fiber den Einflusz des Rindvieh und Pferde- stallmistes auf die Zersetzung der Zellulose in der Ackererde. Inaug. Diss. Helsingfors. 1921; Barthel, Chr., and Bengtsson, N. Bidrag till fragen om Stallgodselns verkningssi'itt vid cellulosasonderdelningen. 1. Akerjorden. Meddl. No. 248, Centralaust. Forsoksv. Jordbrucks. Bakt. Avdel. No. 29. 728 PRINCIPLES OF SOIL MICROBIOLOGY incubated at optimum temperature and moisture conditions for 30 or 60 days, and the amount of cellulose decomposed in that period of time were found to be 400 mgm., this would indicate that about 7 to 8 mgm. of nitrogen had become available in that period of time. TABLE 79 Influence of NaNOz upon the decomposition of cellulose, in the presence of CaCOi and minerals NaNOj ADDED TO 100 GRAMS OF SOIL EVOLUTION OF CO2 IN 14 DAYS CELLULOSE BOIL NUMBER Soil + cellulose Soil alone Due to addition of cellulose 100 GRAMS OF SOIL mgm. mgm. mgm. mgm. mgm. 5A 0 544.50 75.50 469.00 387 5A 25 778.25 75.50 702.75 567 5A 50 817.30 75.50 741.80 680 7A 0 364. 10 88.60 275.50 222 7A 25 526.35 88.60 437.75 437 7A 50 684.20 88.60 595.60 557 TABLE 80 Influence of different forms of nitrogen upon the decomposition of cellulose in soil3* (1 per cent filter paper added to 1 kilogram of soil) NITROGEN SOURCE Control 16 mgm. nitrogen, as manure (10 grams) 32 mgm. nitrogen, as manure (20 grams) 64 mgm. nitrogen, as manure (40 grams) 32 mgm. nitrogen, as (NH^SCU 16 mgm. nitrogen, as NH4NO3 32 mgm. nitrogen, as NH4NO3 CELLULOSE LEFT, PER CENT At start After 2 months After 4 months 0.87 0.53 0.48 0.80 0.51 0.31 0.85 0.38 0.24 0.80 0.27 0.18 0.85 0.35 0.26 0.83 0.34 0.27 0.81 0.22 0.13 Tables 79 and 80 bring out the influence of nitrogen in different forms upon the decomposition of cellulose in the soil. To measure the cellulose-decomposing power of the soil, three meth- ods are recommended.35 85 Waksman, S. A., and Heukelekian, O. Microbiological analysis of soil as an index of soil fertility. VIII. Decomposition of cellulose. Soil Sci., 17: 275-291. 1924. MICROBIOLOGICAL ANALYSIS OF SOIL 729 1 . One gram of finely cut or well ground filter paper is well mixed with 100 grams of fresh sieved soil. This is placed in a tumbler, brought to optimum moisture, covered, and incubated for 42 days, at 25° to 28°C. with frequent additions of water to keep at optimum moisture. The amount of residual cellulose is deter- mined by the method of Charpentier in the soil which is first air dried. The residual cellulose is then subtracted from the amount of cellulose originally present in the soil, which is determined by extracting 20 grams of the original soil to which 200 mgm. of the paper has been added. The amount of cellulose actually decomposed in the soil is thus obtained. 2. One gram of well ground filter paper and 100 mgm. of sodium nitrate are added to 100 grams of soil. The mass is then well mixed and placed in a tumbler, brought to optimum moisture, covered, and incubated for 15 days. The amount of cellulose decomposed is determined as in the case of the first method. 3. One hundred grams of soil, 200 mgm. of CaC03, 50 mgm. K2HP04, 25 mgm. MgS04, with and without one gram of ground dry filter paper are mixed in tumblers. These are placed in a respiratory apparatus and the amount of COi given off in fourteen days is determined. The excess of C02 produced in the soil containing the cellulose over that produced in the soil containing the minerals only, and the amount of cellulose decomposed will serve as an index of the cellulose decomposing power and, ipse facto, of the available nitrogen in the soil. For every milligram of nitrogen that is available in the soil or that can become available in the given period of time, about 50 mgm. of cellulose are decomposed. NITROGEN-FIXING AND MANNITE DECOMPOSING CAPACITY OF THE SOIL The principle of the various methods used for the study of nitrogen fixation can be summarized as follows. A readily available source of energy, chiefly mannite or glucose, is added to the soil or to a solution inoculated with soil ; the amount of available nitrogen in the soil is very limited, so that the fungi and heterotrophic non-nitrogen fixing bacteria, which would otherwise be capable of consuming the mannite or glucose, cannot do that extensively. The amount of glucose or mannite commonly used in the laboratory studies (1 to 2 per cent) is in great excess, so that the amount of available nitrogen is far from sufficient for supplying the requirements of the non-nitrogen fixing organisms. The bacteria, which are capable of obtaining nitrogen from the gaseous form, can readily utilize mannite or glucose as sources of energy. It has been shown elsewhere (p. 444) that the addition of celluloses and cellulose-rich substances to the soil greatly stimulates the development of fungi, especially in the presence of available nitrogen. The addi- tion of glucose to the soil does not affect greatly the development of these organisms, but brings about an abundant multiplication of bacteria, especially the nitrogen-fixing forms. In the presence of an available source of energy, the nitrogen-fixing 730 PRINCIPLES OF SOIL MICROBIOLOGY bacteria may become limited in their development by the lack of suffi- cient available phosphorus in the soil or in the medium. Since Azoto- bacter cells may contain as much as 2 to 5 per cent P2O5, the rapid development of this and other nitrogen-fixing bacteria, which produce an extensive growth in the presence of an excess of available energy, may be limited by the presence of this mineral. For every unit of nitrogen fixed or assimilated by Azotobacter and synthesized into microbial protein about half a unit of available phosphorus (P205) is required. The amount of phosphorus present in an available form can be calculated from the amount of nitrogen fixed. The latter may then become merely an index of the available phosphorus in the soil. Four methods may be suggested36 for measuring the nitrogen-fixing and mannite decomposing power of a soil: 1. The solution method consists of adding 1 or 5 grams of soil to 50 or 100 cc. of a standard mannite solution (20 grams mannite, 0.2 gram MgS04-7H20, 0.2 gram K2HPO4, 0.2 gram NaCl, 5.0 grams CaC03, in 1000 cc. distilled water and made neutral to phenolphthalein), incubating for 7 to 28 days, then determining the increase in total nitrogen above the control (original solution + original soil analyzed immediately for total nitrogen). This serves as an index of the activities of the nitrogen-fixing flora of the soil and thus also, to some extent, of the microbiological condition of the soil. 2. The sml -method consists of adding 1 or 2 grams of mannite to 100 grams of fresh sieved soil, bringing the latter to optimum moisture, incubating for 28 days, and determining the increase of nitrogen in the treated soil over the un- treated soil, incubated under similar conditions. 3. The -pure culture method31 consists of adding 10 grams of the particular soil to 100 cc. of a 2 per cent mannite solution, free from available phosphates, steril- izing and inoculating with a vigorous culture of Azotobacter. After incubating for 20 to 30 days, the increase in total nitrogen is determined. This can serve as an index of the available phosphate in the soil. This method can also be modified by adding to the above mineral solution, free from phosphorus, 10 per cent of soil and estimating, from the amount of Azotobacter growth or from the actual increase in total nitrogen, the amount of available phosphate in the soil. In view of the fact that the majority of field soils contain only small quantities of available phosphate, increasing quantities (0.0005 to 0.005 gram K2HPO4) can be added to a series of flasks, which are then inoculated with the soil. 4. The determination of residual mannite (or rather soluble organic matter in the soil).38 This consists of adding 2 per cent of mannite to air dry soil, 36 Waksman, S. A., and Karunaker, N. Microbiological analysis of soil as an index of soil fertility. IX. Nitrogen fixation and mannite decomposition. Soil Sci , 17: 379-393. 1924. 37 Niklewski, 1912 (p. 726); Stoklasa, 1925 (p. 621); Christensen, 1915 (p. 578). 38 Christensen, H. R. Influence of soil conditions on bacterial life and changes in the soil. II. Ability of soil to break down mannite. Soil Sci., 15: 329-360, 361-363. 1923. MICROBIOLOGICAL ANALYSIS OF SOIL 731 bringing to optimum moisture, incubating, and determining the residual man- nite every five days by oxidation with KAln04. This method can serve as an index of the activities of the nitrogen fixing flora of the soil, as well as of the amount of phosphorus available. The method of determination of soluble organic matter in the soil is carried out as follows. Five grams of soil is withdrawn and allowed to air-dry; the air- dry soil is then weighed again and extracted for two hours, with occasional shaking, with 200 cc. of water. The extract is filtered through paper and 10 cc, or an amount equivalent to 0.25 gram of soil, is placed in a 400-cc. beaker with 50 cc. of 0.05 N potassium permanganate solution and 3 cc. of dilute (6 :100) sulfuric acid. The beaker is placed in boiling water for twenty minutes, 50 cc. of 0.05 N oxalic acid is then added and the solution is titrated with 0.02 N potas- sium permanganate solution. The number of cubic centimeters of the latter expresses the amount of organic matter (residual mannite + soluble soil organic matter). TABLE 81 Nitrogen fixed in 100 cc. of mannite solution -f- 10 grams of soil SOLUTION STERILIZED BEFORE SOIL WAS ADDED SOLUTION STERILIZED AFTER SOIL WAS ADDED P2O6 in medium NoP2Osin medium P2O5 in medium NoP;06in medium None Medium mgm. 8.22 5.48 3.78 mgm. 5.35 4.08 1.67 mgm. 14.51 15.05 mgm. 2.85 1.95 Great 0.35 A definite correlation was found to exist between nitrogen fixation in mannite solution and soil fertility.39 A certain correlation was also reported40 between crop productivity of a soil and its ability of fixing nitrogen, when mannite is added to it (soil method). By adding a definite amount of soil (10 grams) to a mannite solution, free from phosphates, then inoculating with a culture of Azotobacter and deter- mining the amount of nitrogen fixed, after a definite period of incuba- tion, an approximate index of the presence or absence of available phosphorus in the soil can be "obtained,41 as shown in table 81. 39 Lohnis, F., and Pillai, N. K. tlber stickstofffixierende Bakterien. III. Beitrag zur Methodik der bakteriologischen Bodenuntersuchung. Centrbl. Bakt. II, 20: 781-795; Green, H. Investigation into the nitrogen metabolism of soil. Centrbl. Bakt. II, 41: 577-608. 1914; Burgess, 1918 (p. 716). 40 Brown, P. E. Bacterial activities and crop production. Iowa Agr. Exp. Sta. Res. Bui. 25. 1915. 41 Niklewski, 1912 (p. 726). 732 PRINCIPLES OF SOIL MICROBIOLOGY The available phosphorus in the soil may be calculated from the amount of nitrogen fixed. To 100 grams of soil, 30 cc. of water con- taining 2.5 grams of glucose, 0.2 gram K2S04 and 0.05 gram of MgCl2 were added. The soil was then sterilized and inoculated with Azoto- bacter. After 21 days incubation, the total nitrogen and phosphoric acid were determined in the soil. The following process was used for calculating the amount of available phosphorus. One hundred grams of soil contained 0.164 gram nitrogen in the inoculated and 0.110 gram in the uninoculated soil. The amount of nitrogen fixed was, therefore, 0.054 gram. Since Azotobacter cells contain 10 per cent nitrogen and 5 per cent P205, 0.027 gram of the latter was made avail- able in the given quantity of soil. The total P205 in 100 grams of soil was 0.103 gram, hence about 26 per cent of this phosphorus is readily available.42 A very fertile soil containing 0.084 per cent P205 has shown 48.8 per cent of it utilizable; a soil of medium fertility contained 26.21 per cent utilizable P205, and a poor forest soil only 11.66 per cent of the P205 utilizable. There is no doubt that all agricultural soils can be made to fix nitro- gen when an excess of an available source of energy is added. However, the reaction of the soil, which favors the development of specific nitro- gen fixing organisms, is of great importance in this respect, as pointed out above. The presence of available phosphorus in the soil and the soil reaction influencing the development of specific nitrogen-fixing bacteria, are the two factors controlling the amount of nitrogen fixed and mannite decomposed. Winogradsky43 recently suggested several new methods for determin- ing the nitrogen-fixing capacity of the soil, the results serving in a way as a measure of the fertility of the soil. 1. A silica gel plate, 9 cm. in diameter, is inoculated with a few grains of soil- In the presence of Azotobacter, the soil will be surrounded, after 48 hours, with the colonies of the organism. The relative abundance of the colonies will indi- cate the biological activities of the soil. 2. One-half gram of mannite is added to 50 grams of soil, which is incubated for 48 hours. The soil is then examined microscopically and the abundance of nitrogen-fixing bacteria serves as an index of the activity of the soil. 3. Five parts of fresh soil are added to 100 parts of starch and the mixture kneaded with sufficient water and placed in a Petri dish; an excess of water is avoided. After 48 hours incubation, minute colonies will be formed on the 42 Stoklasa, 1911 (p. 649). 43 Winogradsky, 1925 (p. 11). MICROBIOLOGICAL ANALYSIS OF SOIL 733 a >? c e3 -1- e Ph o 734 PRINCIPLES OF SOIL MICROBIOLOGY surface of the mixture; the abundance of the colonies serves as an index of the activity of the soil. 4 and 5. A large silica-gel plate, containing 2 grams of mannite is inoculated with 1 gram of soil. After 48 hours incubation, the number of colonies on the plate is determined; after 5 days incubation, the contents of the plate are ana- lyzed for total nitrogen. An active soil will show 2,000 to 3,000 colonies per 1 gram of soil and will fix 20 mgm. of nitrogen for the 2 grams of mannite. THE CATALYTIC ACTION OF SOIL The catalytic action, or the catalytic power, of a soil is its ability to produce oxygen from hydrogen peroxide; this has often been found to be an index of the fertility of the soil. This action can be determined by adding 5 grams of soil to 20 or 40 cc. of a 1.5 or 3 per cent solution of H202 and collecting the oxygen liberated; a 100 cc. gas-measuring tube filled with a dilute solution of NaOH or KOH and inverted into a bath of the same solution is used for collecting the gas. The soil is usually placed in a large test tube or in a 300-cc. Erlenmeyer flask and may be suspended in a little water, before adding the peroxide. One portion of soil is used untreated and another portion is previously sterilized in the autoclave so as to determine the role of the living or- ganisms in the process; a third portion of soil may be ignited and then used in the test, so as to determine the role of the organic matter in the process of H202 decomposition. The period of incubation is usually 5 to GO minutes and the temperature 17° to 37°C. An increase in concentration of substrate, temperature and period of incubation will all lead to an increase in the amount of oxygen liberated. Konig44 found that the decomposition of H202 by soil was due chiefly to the enzyme catalase produced by the soil microorganisms and plant materials, and to some extent to the inorganic part of the soil and to organic colloids. On sterilizing the soil by heat or treat- ment with chloroform, iodine, mercury bichloride and especially hydrocyanic acid, the liberation of oxygen was greatly diminished. The catalytic action was found to be further increased by a similar action of manganic oxide, iron and aluminum oxides. The forma- tion of oxygen by heated soils was ascribed to the increase in alkalinity due to the formation of CaO from CaC03. When heated soil is moistened and allowed to remain one day in a desiccator filled with C02, its catalytic power is greatly diminished. Konig found a 44 Konig, J., Coppenrath, E., and Hasenbaumer, J. Beziehungen zwischen den Eigenschaften des Bodens und der Nahrstoffaufnahme durch die Pflanzen. Landw. Vers. Sta., 66: 401-461. 1907; 53: 472-476. 1906. MICROBIOLOGICAL ANALYSIS OF SOIL 735 direct correlation between the humus content of the soil and its cata- lytic power. According to May and Gile,45 the catalytic action of a soil is a rough measure of the combined quantity of bacteria and organic matter present. Surface soils were found46 to be more active than subsoils, more fertile soils are more active than infertile ones. Since inorganic soil constituents are also capable of liberating oxygen from H202, Chouchack47 used the difference between the oxygen formed by normal soil and that formed by the same amount of sterilized soil as an index of the biological activities. By treating the soil with phosphates, potassium and nitrogen salts, then determining the increase in catalytic action, reliable information can be obtained on the practical value of such treatments. Osuga,48 however, confirmed the previous observations of Konig and associates that ferric oxide, manganese oxide and humus show marked catalytic action. He suggested that these substances may be the main constituents which react with the H2O2. Bacterial effect in soil catalysis was believed to be small. In this respect, he confirmed the earlier observations of Kappen49 that the catalytic action of the soil is due largely to the colloidal complexes of the soil. The catalytic action of the soil is thus found50 to be due to the inor-* ganic constituents of the soil; to certain organic soil compounds, such as benzol derivatives, and to the action of catalase formed by micro- organisms in the soil. Although a correlation exists between the catalytic action of the soil and the numbers of soil microorganisms as well as soil productivity, the phenomenon is very complex and cannot " May, D. W., and Gile, P. L. The catalase of soils. Porto Rico Agr. Exp. Sta. Circ. 9. 1909. 46 Sullivan, M. X., and Reid, F. R. Studies in soil catalysis. Bur. of Soils. U. S. Dept. Agr. Bui. 86. 1912. 47 Chouchack, D. L'analyse du sol par les bact6ries. Compt. Rend. Acad. Sci., 178: 1842^, 2001-2. 1924. 48 Osuga, C. On the catalytic action of soil. Ber. Ohara. Inst. Agr. Invest. Kuraschiki., 2: 197-218. 1922. 49 Kappen, H. Die katalytische Kraft des Ackerbodens. Fiihling's landw. Ztg., 62: 377-392. 1913; see also Smolik, L. Hydrogen-peroxide catalase of marsh soils. Proc. Intern. Soc. Soil Sci., 1: 6-21. 1925; A detailed review of the occurrence and action of catalase in general is given by Morgulis, 8. Die Kata- lase. Ergebn. Physiol., 23: 308-367. 1924. 60 Waksman, S. A., and Dubos, R. Microbiological analysis of soils as an index of soil fertility. X. The catalytic power of the soil. Soil Sci., 22: 407-422. 1926. 736 PRINCIPLES OF SOIL MICROBIOLOGY be used as a simple method of determining soil productivity, unless the various factors are determined individually. OXIDIZING AND REDUCING POWER OF THE SOIL Oxidation consists in the addition of oxygen or subtraction of hydrogen, the oxygen can be obtained either directly from the at- mosphere or from a peroxide (see p. 520). Soil fertility and the rate of oxidation were found51 to be influenced by the same factors and to the same extent, so that it was suggested that the latter could be used as a measure of the former. Oxidation was found to be greater in fertile than in unfertile soils, in surface soil than in subsoil. The oxi- dizing power of soils can be determined62 by shaking 5 grams of soil with 10 cc. of an alcoholic solution of gum guaiac and then allowing the soil to settle. The formation of a blue color indicates the degree of oxidation. When the blue color fades, it can be brought back by the addition of 0.5 cc. of a 2 per cent H202 solution. Another method of testing oxidation in soils consists in shaking 20 grams of soil with 50 cc. of 0.125 per cent aqueous solution of aloin for one hour; the soil is allowed to settle or the solution is centrifuged (if turbid, 50 cc. of 95 per cent alcohol is added to flocculate the soil and extract the oxidized aloin) ; the clear solution is poured off and the depth of color is determined by a colorimeter. It was found that soils known to be productive had a strong oxidizing power, and that the poorer soils had little or no oxidizing power; factors favoring oxidation also favor soil productivity. However, agreement has not been obtained in all cases. Gerretsen53 used, as an index of the oxidizing power of soils, the amount of iodine liberated when 100 grams of soil are treated with a dilute solution of potassium iodide acidified with sulfuric acid. The method was carried out as follows. Two grams of soil were ground in a mortar, then washed into an Erlenmeyer flask with water; 5 cc. of a 1 per cent solution of potassium iodide and 6 drops of a 1:1 solution of sulfuric acid were then added to the soil suspension. After five minutes, the suspension was centrifuged, filtered and titrated with 0.01 Ar Na2S203. When the moisture content of the soil is known, the oxidizing power of 100 grams of dry soil can be calculated. It was found that rich soils had 51 Russell, 1905 (p. 683). 62 Schreiner, O., and Sullivan, M. X. Studies in soil oxidation. Bur. of Soils, U. S. Dept. Agr. Bui. 73. 1910. 63 Gerretsen, F. C. Het oxydeerend vermogen van den bodem in verband met het uitzuren. Meddl. Proefsta. Java. Suikerind., 5: 317-331. 1915; Meddel. Proefsta. Java. Suik. No. 3. 1921. MICROBIOLOGICAL ANALYSIS OF SOIL 737 a high iodine (or HI) value; poor soils had a low value. These results were not confirmed, however, by Honing54 for soils of Deli, due to large quantities of organic matter present and to the irregular distribu- tion of the ferric iron. The oxidation of sulfur in the soil, the reduction of nitrates (denitri- fication) and formation of ammonia from proteins are often used for comparing the microbiological condition of different soils, but the value of these methods in throwing light upon the soil microbiological processes is often questioned. 64 Honing, J. A. The oxidizing power of some soils in Deli, Sumatra. Bui. Deli Proefsta, No. 8. 1917; Bui. Agr. Intel., 8: 838. 1917. CHAPTER XXVIII Soil Microbiological Equilibrium influence of air drying and partial sterilization upon the activities of microorganisms in the soil Microbiological equilibrium in the soil. The large numbers of micro- organisms harbored in the soil vary greatly morphologically and physio- logically. Conditions favoring the activities of one group of organisms may be distinctly injurious to others. An acid reaction, for example, may be favorable to the development of fungi but inhibitory to a number of bacteria and actinomyces. The presence of an excess of lime will favor bacteria, including nitrogen-fixing, nitrifying and other groups, but may depress the development of fungi. Aeration has a favorable influence upon some organisms but not upon others. The very metabolism of some organisms depends upon the activities of others which provide the substrate for them, as in the case of ammonia forming, nitrite and nitrate bacteria, etc. The soil flora is so complex and the resulting activities are so various that one group of processes can hardly be separated from another and studied by itself. The addition to the soil of nitrogen compounds, carbon compounds, or minerals does not only stimulate the activities of one or more groups of organisms, but may bring about a series of changes in the soil, the resultant or the end of which is hard to foresee. Not only are the various organisms affected in different ways by differ- ent soil treatment, but they themselves exert stimulating or injurious influences upon the activities of one another. Some aerobic organisms, for example, use up the excess of oxygen, and thus create in the soil conditions favorable for the activities of the anaerobic forms and, ipse facto, unfavorable for the activities of other aerobic organisms. Some break down complex carbon compounds, like the celluloses, making the energy available for other forms, like the nitrogen-fixing organisms; others may compete with the latter for this available energy in the presence of available nitrogen. Some break down the complex nitrogenous substances liberating the ammonia, which can be utilized by higher plants or can serve as a substrate for nitrifying bac- 738 SOIL MICROBIOLOGICAL EQUILIBRIUM 739 teria; others use up the ammonia for the building up of microbial protoplasm. Some produce soluble substances as intermediary or by- products of metabolism, which are either distinctly beneficial and stimulating to other organisms, or distinctly injurious. The soil also harbors a number of fungi, actinomyces and bacteria which are causative agents of plant diseases, although they may be able to live in the soil saprophytically. When a soil is left undisturbed for a long period of time the numbers and activities of the various groups of organisms come to a condition which may be termed unstable equilibrium. This equilibrium is not static but dynamic, in a chemical sense, especially under field condi- tions. Sunshine and rain, freezing and thawing, plowing and cultivating, fertilizing and manuring and a host of other factors which affect the soil will bring about a change in this equilibrium. If the numbers of bacteria and protozoa are determined daily for a period of time, constant fluctuations are found.1 The same is true of the numbers of fungi, nitrate and carbon dioxide content of the soil. However, when a soil is kept under constant optimum conditions and undisturbed, the daily variability is very small and there is a constant gradual diminution in the numbers and activities of the microorganisms, as shown2 in figure 63. This figure indicates that the rapid rise of the numbers of bacteria and evolution of carbon dioxide, as a result of moistening of an air-dry soil, was followed by a gradual drop for about 200 days, when the drop became hardly perceptible but was still present. The soil was kept in pots and the soluble products, resulting from the decomposition of the organic matter in the soil, were not removed by drainage, nor by growing plants, nor by any microorganisms, since no fresh sources of energy were added. For this reason nitrates continued to accumulate. This equilibrium in microbiological activities is not due to a lack of nitrogen, but to a lack of available energy. Rahn3 found that the addition of straw to such a soil will result in a rapid increase in the growth and develop- ment of microorganisms, lasting as long as the available energy does and followed again by a decline. The same increase in the activities 1 Cutler, et al., 1923 (p. 32). 2 Waksman, S. A., and Starkey, R. L. Partial sterilization of soil, microbio- logical activities and soil fertility. Soil Sci., 16: 137-156, 247-268, 343-357. 1923. ' Rahn, O. Die schadliche Wirkung der Strohdungung und deren Ver- hiitung. Ztschr. techn. Biol., 7: 172-186. 1919. 740 PRINCIPLES OF SOIL MICROBIOLOGY of microorganisms can also be obtained, however, by treatment of soil with various volatile antiseptics, heating the soil or even merely drying it. The resulting activities are similar to those following the addition of a fresh supply of energy, as shown in the following pages. Influence of air-drying of soil upon the microbiological equilibrium. The favorable effect of drying of soil upon the growth of higher plants has been reported from various sources.4 This effect was first attributed to the improvement in the physical condition of the soil, especially in Fig. 63. Course of biological activities in undisturbed soil (from Waksman and Starkey). case of fine grained soils, then it was ascribed to some chemical change in the soil as a result of drying. The numbers and activities of micro- organisms are found to be markedly influenced by the drying of the soil. When one part of the same soil is allowed to remain moist and the other part is air dried for some time and then moistened, a rise in the numbers of bacteria and an increase in the evolution of carbon 4 Lebedjantzev, A. N. Drying of soil, as one of the natural factors in main- taining soil fertility. Soil Sci., 18: 419-447. 1924. SOIL MICROBIOLOGICAL EQUILIBRIUM 741 dioxide and in the accumulation of nitrates is found to take place in the second soil. The rise in the numbers and activities of microorgan- isms is soon followed by a rapid fall until they approach those of the control soil. When the formation of acid in glucose solution and ammonia in urea and peptone solutions were used as indices, an air-dried soil was about 20 per cent more active than the corresponding as v. UJH-ETf 40 8AS8 . ,. ,. a6» / "N ' - IK v___ 1 / „,-■ 6 " \ / _. 18 X J' \ 6 150 o TUB Fig. 65. Influence of heating of soil upon the numbers and activities of its population (from Waksman and Starkey). decomposition of the bodies of microorganisms in the soil. The in- creased numbers of bacteria bring about an increase in the decomposition of the soil constituents, both of organic and inorganic nature. Heating of peat soil, at 100° for fifteen minutes, was found29 to stimulate greatly the biological activities, when the evolution of carbon dioxide (from 200 grams of soil) was used as an index of these activities. 29 Demolon, A., and Boischot, P. Sur l'activite" des phenomenes biologiques dans la tourbe. Compt. Rend. Acad. Sci., 177: 282-284. 1923. 748 PRINCIPLES OF SOIL MICROBIOLOGY This favorable influence of heating was found to be due not to the destruction of toxins or protozoa, but to a chemical modification of the peat. A stimulative effect of steam treatment of soil upon the numbers of bacteria in the soil has also been observed.30 The results of heating (65°C. for one hour) of a soil upon the numbers of fungi and bacteria, evolution of carbon dioxide and accumulation of available nitrogen (ammonia + nitrates) are given in figure 65. Heating a soil even to low temperatures seems to improve it as a medium for bacterial growth. It is interesting to mention, in this connection, the change brought about by heat in the physical and chemical condition of the soil. While treatment of soil at temperatures lower than 100°C. rendered the soil more fertile, temperatures higher than 100° rendered it less fertile.31 The lower temperatures bring about an increase in the soluble organic matter32 in the soil; treatment of soil at higher temperatures (especially above 10Q°C.) results in the formation of substances toxic to the growth of higher plants: among those guanine, arginine, dihydroxystearic acid have been recorded. There is also an increase in acidity, or rather ability of soil to neutralize bases.33 The toxic substances are unstable, gradually disappearing in the course of time, if the soil is kept moist and aerated. This is probably due to oxidation and other activities of microorganisms. The increase in soluble organic matter, as a result of heating, varies directly with the temperature to which the soil is subjected, particularly in the case of carbohydrates. It has also been observed that heating of soil brings about a greater solubility of the phosphorus and nitrogen compounds in the soil.34,35 Similar results were obtained by a number 30 Osmun, A. V. A comparison of the numbers of bacteria in sterilized and unsterilized soils. Mass. Agr. Exp. Sta. Rpt. 1905, 146-148. 31 Pickering, S. U. The activities of heat and antiseptics on soils. Jour. Agr. Sci., 3: 32-54, 258-276. 1910. 32 Schreiner, O., and Lathrop, E. C. The chemistry of steam heated soils. U. S. Dept. Agr. Bur. Soils Bui. 89. 1912; Jour. Amer. Chem. Soc, 34: 1142- 1159. 1912. 33 Robinson, R. H. Concerning the effect of heat on the reaction between limewater and acid soils. Soil Sci., 9: 151-157. 1920. 34 Liebscher, G. Welche Kulturpflanzen vermehren den Stickstoffvorrat der Wirtschaft. II. Die Stickstoffsammlung der Kulturpflanzen. Deut. Landw. Presse., 20: 975-976. 1893. 36 Kruger, W., and Schneidewind, W. Ursache und Bedeutung der Salpeter- zersetzung im Boden. Landw. Jahrb., 28: 217-252. 1899. SOIL MICROBIOLOGICAL EQUILIBRIUM 749 of other investigators.36-39 Bouyoucos40 and Wilson41 found an increase in the concentration of the soil solution as a result of heating of soil, using the freezing point method. This increase is greater in soils rich in organic matter than in mineral soils. Heat also effects a floecula- tion of the soil colloids, thus changing the physical condition of the soil. The increase in the soluble matter and the changes in the micro- biological population of the soil brought about by heating result in an increase in the numbers of bacteria; these in their turn decompose more organic matter, which results in a greater liberation of available nitrogen. This favors the growth of plants.42 Influence of volatile antiseptics upon bacterial activities in the soil. Volatile antiseptics, especially carbon bisulfide, were first applied to soils and plants for the destruction of insect and fungus pests. As far back as 1870 the observation was made that this disinfectant has a stimulating effect upon plant growth. Girard43 used CS2 to clear a piece of sugar-beet ground badly infested with nematodes and observed marked increases in the succeeding crops as a result of the treatment. 36 Whitney, M., and Cameron, F. K. The chemistry of the soil as related to crop production. U. S. Dept. Agr., Bur. Soils, Bui. 22, 1-71. 37 Lyon, T. L., and Bizzell, J. A. Effect of steam sterilization on the water- soluble matter in soil. N. Y. (Cornell) Agr. Exp. Sta. Bui. 275. 1910; Bui. 326. 1913. 38 Seaver, J. F., and Clark, F. D. Changes brought about by heating of soils and relation to the growth of Pyronema and other fungi. Mycologia, 2: 109- 124. 1910; Biocehm. Bui. 1: 413-427. 1912. 39 Gustafson, 1922 (p. 743). 40 Bouyoucos, G. The freezing point method as a new means of measuring the concentration of the soil solution directly in the soil. Mich. Agr. Exp. Sta. Tech. Bui. 24. 1915. 41 Wilson, A. Changes in soils brought by heat. Proc. Roy. Dublin Soc. N. S., 38: 513. 1915. 42 The favorable influence of heating of soil at temperatures less than 100° upon crop growth has been studied in detail by Russell, E. J., and Petherbridge, F. R. On the growth of plants in partially sterilized soils. Jour. Agr. Sci., 5: 248-287. 1913; The practice of burning soils is described by Mann, H. H., Joshi, N. V., and Kanitkar, N. V. The "rab" system of rice cultivation in Western India. Mem. Dept. Agr. India, Chem. Ser., 2: 141-192. 1912; Kelley, W. P., and McGeorge, W. The effect of heat on Hawaiian soils. Hawaii Agr. Exp. Sta. Bui. 30. 1913; Demolon, A. The partial sterilization of peat. Intern. Rev. Sci. Pract. Agr., 3: 431. 1924 (Chem. Abstr., 20: 1878). 43 Girard, A. Recherches sur l'augmentation des recoltes par l'injection dans le sol du sulfure de carbone a doses massives. Bui. Soc. Nat. Agr. France, 54: 356-363. 1894. 750 PRINCIPLES OF SOIL MICROBIOLOGY In 1894 Oberlin44 applied CS2 for the destruction of Phylloxera and noticed that the productiveness of the soil was greatly increased. He suggested, therefore, that soil sickness can be corrected by the appli- cation of the disinfectant and ascribed this favorable influence to the destruction of injurious microorganisms. This favorable effect of the disinfectant was ascribed to the direct stimulation of young plants.45-46 Nobbe and Richter47 obtained a definite increase in crop yield by treatment of soil with ether, chloroform or benzene. It was soon found that the stimulating effect applies to all soils and all plants.48 The antiseptics, like carbon bisulfide, carbon tetrachloride, toluol, 1 1 / J'qI z,1 /Vrv<--..r>.*«»* / — Act momy ce$ original / 1 ■*N 1 1 'too \ \\ \ 1 \ I \ / / / / / f \ ^ \ — — - . — ^ ' added '7- i2~ month Fig. 66. Influence of CS2 upon the numbers of bacteria and actinomyces in the soil (from Hiltner and Stormer). benzol, phenol, cresol, are in themselves directly poisonous to plants when added to water cultures. They were found to exert a decided 44 Oberlin. Bodenmiidigkeit und Schwefelkohlenstoff. Mainz. 1894. 46 Koch, A. Untersuchungen liber die TJrsachen der Bodenmiidigkeit. Arb. deut. landw. Gesell. H., 40. 1899, 44 p. 46 Egorov, M. A. On the influence of carbon bisulfide upon soils and plants (Russian). Zhur. Opit. Agron., 9: 34-95. 1908. 47 Nobbe, F., and Richter, L. tJber die Behandlung des Bodens mit Ather, Schwefelkohlenstoff, Chloroform, Benzol und Wasserstoffsuperoxyd und deren Wirkung auf das Wachstum der Pflanzen. Landw. Vers. Sta., 60: 433^48. 1904. 48 Stormer, K. Uber die Wirkung des Schwefelkohlenstoffs und ahnlicher Stoffe auf den Boden. Jahresb. ver. angew. Bot. for 1907, 5: 113-131. 1908; Centrbl. Bakt. II, 20: 2S2-28S. 1908. SOIL MICROBIOLOGICAL EQUILIBRIUM 751 stimulating effect when added to the soil some time before the crop is planted. This was attributed49 to a changed bacterial population. A bacterial flora of 9| millions per gram, as determined by the plate method, was depressed by the addition of the disinfectant (fig. 66); it soon increased rapidly, reaching, within a month after the evaporation of the disinfectant, 50 millions; the numbers then slowly fell but re- mained above the original number. The normal flora of the soil consisted of 75 per cent non-liquefying bacteria, 20 per cent actinomyces and 5 per cent gelatin liquefying forms. As a result of treatment with carbon bisulfide, the non-liquefying organisms were greatly increased, while the actinomyces were reduced and did not return to the original proportion for at least two years. The denitrifying bacteria were completely destroyed, while the pectin-fermenting organisms were reduced. As a result of these studies, Hiltner and Stormer came to the following conclusions: 1. By destroying the existing bacterial equilibrium in the soil, carbon bisul- fide opens the way for an entirely new bacterial development. This is achieved through the unequal retardation in the growth of the different groups of bac- teria. Hence certain groups become disproportionately prominent, while others are almost entirely suppressed. 2. The rapid increase in the numbers of the bacteria is followed by a more in- tense transformation of plant food substances. Decomposition and fixation processes result in an accumulation of readily available nitrogen compounds utilized by the crops. Hence the action of carbon bisulfide is in the nature of nitrogen action. 3. The initial suppression of the nitrifying species becomes of advantage in that the nitrogen compounds, simplified by other species, are prevented from being rapidly changed into nitrates and being leached out of the soil. 4. The more or less permanent suppression of the denitrifying organisms must be regarded as an additional factor favoring plant growth.60 Van Suchtelen51 found that CS2 stimulates the decomposition of organic matter, as indicated by the C02 production; 12 kgm. of soil 49 Hiltner and Stormer, 1903 (p. 12); Hiltner, L. Uber neuere Ergebnisse und Probleme auf dem Gebiete der landwirtschaftlichen Bakteriologie. Jahresb. Angew. Bot., 5: 200-222. 1907; Centrbl. Bakt. II, 38: 228. 1912; Stormer, 1907 (p. 750). 60 The observations of Hiltner and Stormer were confirmed by Moritz, J., and Scherpe, R. Uber die Bodenbehandlung mit Schwefelkohlenstoffe und ihre Einwirkung auf das Pflanzenwachstum. Arb. K. Gesundhsamt. Biol. Abt., 4: 123-156. 1904; also Arb. K. Biol. Anst. Land. Forstw., 7: 353-425. 1909; Centrbl. Bakt. II, 13: 573. 61 Van Suchtelen, 1910 (p. 720). 752 PRINCIPLES OF SOIL MICROBIOLOGY + 6 grams glucose gave 7,215 milligrams of C02 in nineteen days when treated with 170 grams of CSj, while the untreated soil gave 5,991 milligrams. Soil treated with heat or volatile antiseptics has a much greater oxidizing power, as indicated by the oxygen absorption by the soil.52 Stormer suggested that the disinfectants kill the larger soil organisms, such as worms, insects, fungi, algae, protozoa; these are then decom- posed by the surviving bacteria with the formation of ammonia. Bac- terial development and ammonia accumulation are a result of this decomposition. The total increase in ammonia nitrogen over the untreated soil is not more than 3 to 4 mgm. of nitrogen per 100 grams of soil; this quantity can be readily derived from the decomposed organisms. Garden soils may be very rich in nematodes which often do great damage to the crop.53 These nematodes are destroyed by the disinfectant. Among the injurious bacteria, which may also be destroyed, are the nitrate and sulfate reducing forms. According to Stoklasa,54 the increase in soil fertility due to treatment with CS2, chloroform, benzol, or ether is due to the destruction of a definite number of soil microorganisms; the surviving bacteria readily break down the dead organisms, liberating phosphate and other ions, which now become available for plant growth. It is to be noted that among the organisms which develop in great abundance in partially sterilized soils Clostridium pastorianum, the anaerobic nitrogen-fixing organism, occupies a prominent place, occurring as 100,000 or more per gram of soil.55 It thus became evident that the treatment of soil with antiseptics is equivalent to nitrogen fertilization. It was suggested56 that partial sterilization of soil renders a number of undecomposed plant residues, such as pectins and pentosans, more soluble; these are used as sources *2 Darbishire, F. V., and Russell, E. J. Oxidation in soils and its relation to productiveness. II. The influence of partial sterilization. Jour. Agr. Sci., 2: 305-326. 1908. 63 Emmerich, R. W., Graf zu Leiningen, and Loew,~0. t)ber Bodensaubei- ung. Centrbl. Bakt. II, 29: 668. 1911; 31: 466-477. 1911. 64 Stoklasa, 1911 (p. 649). 66 Truffaut, G., and Bezssonoff, N. Influence de la sterilisation partielle sur la composition de la flore microbienne du sol. Compt. Rend. Acad. Sci., 170: 1278-9. 1920; 171: 268-270. 1920; 172: 1319-1323. 1921. 56 Heinze, B. Einiges iiber den Schwefelkohlenstoff, dessen Wirkung auf niedere pflanzliche Organismen, sowie seine Bedeutung fiir die Fruchtbarkeit des Bodens. Centrbl. Bakt. II, 16: 329-357. 1906; 18: 56, 246, 462, 624, 790. 1907; Landw. Jahrb., 36: 418. 1907. SOIL MICROBIOLOGICAL EQUILIBRIUM 753 of energy by nitrogen-fixing bacteria; the subsequently more intense transformation of the bacterial proteids and of other nitrogenous organic substances into amino- and ammonium compounds places an abundant and uniform supply of soluble nitrogen compounds at the disposal of the plant. TABLE 84 Influence of CSi upon the development of bacteria in the soil68 TIME CONTROL SOIL 2 PER CENT CS2 2 PER CENT CS2, EVAPORATED 2 PER CENT CSj EVAPORATED + 5 PER CENT FRESH SOIL days 1 3 5 9 11,496,000 22,010,000 20,635,000 14,739,000 16,115,000 19,508,000 18,272,000 15,346,000 12,372,000 1,965,000 23,975,000 25,253,000 36,651,000 90,473,000 60,149,000 68,276,000 90,645,000 58,101,000 2,260,000 8,254,000 27,416,000 61,904,000 98,850,000 71,257,000 86,483,000 84,272,000 60,000,000 2,358,000 12,480,000 95,499,000 13 21 25 29 60 80,420,000 52,495,000 64,570,000 38,495,000 30,000,000 TABLE 85 Numbers of bacteria in untreated and partially sterilized soils63 AT START END OF FIRST PERIOD END OF SECOND PERIOD END OF THIRD PERIOD END OF FOURTH PERIOD 16 days 30 days 74 days Untreated CSii 27 millions 2 millions 13 millions 13 millions 11 millions 2 millions 10 millions 17 millions 10 millions 53 millions 45 millions 121 millions 15 days 110 days 170 days 200 days 65°C 9 millions 21 millions 4 millions 37 millions 9 millions 45 millions 12 millions 60 millions 40 days 100 days 160 days 500 days Toluene 16 millions 43 millions 9 millions 41 millions 13 millions 43 millions 6 millions 18 millions The original idea of Koch57 that increased crop growth due to the application of the disinfectant is a result of a direct stimulation of the plant by traces of the disinfectant or its decomposition products «' Koch, 1899 (p. 750). 754 PRINCIPLES OF SOIL MICROBIOLOGY found various adherents. Fred58 found that proper concentrations of ether, CS2 and CuS04 have a stimulative effect upon the growth of lower microorganisms; even nitrification, which was at first decreased, was later stimulated. CS2, in dilute solutions, was found to stimulate growth of plants, including that of fungi.59 According to Hiine,60 small doses of poisons may be directly stimulative to bacterial development. The influence of antiseptics upon the development of microorganisms in the soil, namely those that can be determined by the plate method, is brought out by tables 84 and 85. A large increase in soluble nitro- gen, as a result of treatment with CS2, is found both in the inoculated CHLOROFORM Ml 50 toM 100 5 40 0 U PARTS a. J PER -i MILL i O z -I: H O < CO U 10 20 .N.TI.U M/50 to M LARGER PROTOZOA KILLED AMMONIA and NITRATE PRODUCED DAYS. Fig. 67. Influence of different amounts of chloroform upon the bacteria, protozoa and available nitrogen in the soil (from Buddin). and uninoculated soils. The ammonia content of the soil was found to follow the curve of bacterial growth and later giving rise to nitrates. CS2 did not act alike on all soils and toward all crops. Russell and associates (figs. 67 and 68) attempted to correlate the de- 68 Fred, E. B. Uber die Beschleunigung der Lebenstiitigkeit huherer und niederer Pflanzen durch kleine Giftmengen. Centrbl. Bakt. II, 31: 185-245. 1912. 69 Oldenbusch, C. Stimulation of plants by CS2. Bull. Torrey. Bot. Club., 49: 375-390. 1922. 80 Hiine, Dr. Die begiinstigende Reizvvirkung kleinster Mengen von Bak- teriengiften auf die Bakterienvermehrung. Centrbl. Bakt. I, Orig., 48: 135- 140. 1907. SOIL MICROBIOLOGICAL EQUILIBRIUM 755 struction of protozoa following partial sterilization with the increase in the numbers of bacteria and their activities and subsequently soil 200 100 • Bacterial Numbers. 200 ■to^r-'- r--"'" 100 I UNTRtATED Soil NK3and Nitrate. Limit J^ .'''Untreated Soil 19 40 70 130 19 40 70 130 Days 250 150 Bacterial Numbers. Toluened Soil Untreated Soil 500 300 100 310 Fig. 68. Influence of toluene upon numbers of bacteria and available nitrogen (ammonia and nitrate) formed in the soil; upper figures from a soil in which small amounts of available nitrogen were initially present; lower figures from a soil in which large amounts of available nitrogen were initially present (after Russell). fertility. They suggested that the protozoa are responsible for keeping down bacterial numbers in an untreated soil and, therefore, affect 756 PRINCIPLES OF SOIL MICROBIOLOGY adversely the production of plant food. The partial sterilization of the soil results in the destruction of protozoa, thus removing the agent injurious to normal bacterial development. The various theories and hypotheses that have been proposed in explanation of the favorable influence of partial sterilization of soil upon its fertility can be summarized as follows: 1. Direct stimulation theory. Plant roots and microorganisms may be stimu- lated directly by small quantities of antiseptics;61 it has been suggested62 that the latter are used directly as a nutrient by microorganisms. o o h* J 8*0 36 SJO 30 *0 V eo" i 1 — n • • .COETROl f 0. • • .COFtBOL -M.TJGI KOS . , TRRATE3 . ■ • .C0H7H0L Tl • , corraoL 1 -4 / i s L. i 1 w \ i A \ | i 1<> - i ^ sT .__ _-_ "V / .._.' i ""■•s. _.-: Ll __ . ! ts r~ — i -i i i 1 ! a i 1. 1 i .. | | \ i 1 I Fig. 69. Influence of CS2 upon the numbers and activities of microorganisms in the soil (from Waksman and Starkey). 2. Indirect stimulation of bacteria. The organic matter in the soil may be modified in such a manner, as a result of partial sterilization, as to make it more 81 Maassen, A., and Behn, H. Das Verhalten der Bakterien, insbesondere der Bodenbakterien gegeniiber dem Schwefelkohlenstoff, und die Beeinfluszung des Pflanzenwachstums durch eine Schwefelkohlenstoffbehandlung des Bodens. Mitt. K. Biol. Anst. Land. u. Forstwirt., 12: 285-338. 1924. 82 Matthews, A. Partial sterilization of soil by antiseptics. Jour. Agr. Sci., 14: 1-5. 1924. SOIL MICROBIOLOGICAL EQUILIBRIUM 757 available for bacterial action ; this may be due either to the removal of the fats, to greater solubility of carbohydrates, nitrogen compounds, or phosphates; to the killing of worms, nematodes, protozoa, algae, fungi, which are then de- composed by the bacteria; or to all these combined. 3. Microbiological balance or equilibrium. Partial sterilization produces a change in the balance between the bacterial flora and the other groups of organ- isms, such as the fungi and actinomyces. 4. The protozoa are responsible for the limitation of bacteria in the soil; their removal by partial sterilization leads to increased bacterial development, greater decomposition of organic matter and, therefore, improved soil fertility. 5. Toxin theory. The soil is believed to contain toxins of biological origin. Partial sterilization of soil leads to their destruction, hence to improved fer- tility. 6. Destruction of fungi and bacteria which are causative agents of plant dis- eases. The repeated growth, year after year, of the same crop leads to an accumu- lation of fungi and insects injurious to the particular crop. Partial sterilization of soil brings about the destruction of these pests. 7. Increased nitrogen-fixation. Partial sterilization of soil is believed to render a greater amount of energy available to the nitrogen-fixing bacteria. Koch, however, maintains that nitrogen fixation by bacteria is decreased by partial sterilization. Only three of these theories need be discussed at greater length. Protozoan theory. The "protozoan theory of soil fertility" advanced by Russell and Hutchinson63 has met with severe criticisms. Accord- ing to this theory, the number of bacteria found in the soil, at any given time, is not merely a function of environmental soil conditions, but depends on the interrelationship between the bacteria and the pro- tozoa; partial sterilization does not bring about an improvement in the bacterial flora but makes the soil a better medium for the growth of bacteria, by eliminating the detrimental factor. It was suggested64 that the destruction of spores of disease producing fungi and bacteria have more to do with the final increase in produc- tiveness of heated soils than either the destruction of bacteriarloving protozoa or the increase in soluble plant food. The increase in ammonia formation in partially sterilized soils was believed to be due to fungi.65 "Russell and Hutchinson, 1909-1912 (p. 311). 64 Bolley, H. L. Interpretation of results in experiments upon cereal cropping methods after soil sterilization. Science, 33: 229-234. 1911; also 32: 529-541. 1910; 38: 48-50, 249-259. 1913; N. D. Agr. Exp. Sta. Bui. 107. 1913; Jachs- chewski, A. On the causes which determine infertility of the soil and loss of crops. Khoziastvo, Z. 1912, 1103-1108 (Intern. Inst. Agr. Bui. Bur. Agr. Inst. PI. Dis., 3: 2528). 65 Kopeloff, N., and Coleman, D. A review of investigations in soil protozoa and soil sterilization. Soil Sci., 3: 197-269. 1917. 758 PRINCIPLES OF SOIL MICROBIOLOGY The fact that fungi grow readily on soils subjected to dry or moist heat66 would tend to add further weight to this idea. The abundant O — « — Bacteria • Bacteria* Amoebae Bacteria* flagellates 3 4 5 6 7 8 15 16 17 U Kumlicr of days .aftci inoculation Fig. 70. Action of protozoa upon the development of bacteria (from Cutler) growth of fungi in soils treated with disinfectants, after a certain period has elapsed, is also quite marked. Figures 70 and 71 show that 66 Seaver and Clark, 1912 (p. 749). SOIL MICROBIOLOGICAL EQUILIBRIUM 759 the inoculation of soil with protozoa may lead to a decided depression in the numbers of bacteria;07 however, so far no definite proof has been submitted that the introduction of protozoa actually depresses bio- chemical processes in the soil important to soil fertility; the meagre 293 323 Fig. 71. Influence of protozoa upon the development of bacteria: U = un- inoculated;T -f- W = toluene treated soil plus distilled water; T + F = toluened soil plus hay-infusion, filtered through a Berkfeld filter; T + C - toluened soil plus ciliates in hay-infusion; T + M = toluened soil plus mixed culture of pro- tozoa and bacteria in hay-infusion (from Goodey). results available seem to point to the contrary, as shown elsewhere (p. 338). Agricere and Baderiotoxin theories. The results of Schreiner, Shorey and their associates seemed to indicate that the soil harbors certain 87 Cutler, 1923 (p. 32). 760 PRINCIPLES OF SOIL MICROBIOLOGY substances (dihydroxy-stearic acid, etc.) which are distinctly injurious to crop growth. Livingston08 regards the general hypothesis that unproductiveness of agricultural soils as due frequently to soil toxins as well established and generally accepted. According to Greig-Smith,69 no one phenomenon can explain the cause of the enhanced fertility of soils treated with volatile antiseptics. Bacteria growing in any culture medium produce injurious or toxic products, which check and inhibit their further growth. These toxins may be in the nature of lysins, acids, accumulated by-products, etc. Soil extract filtered through porcelain was found to have a destructive influence upon Bad. prodigiosum, while the same extract heated and treated by sunlight or allowed to stand in aqueous solution stimulated bacterial growth. Greig-Smith considered that, in addition to the toxic substances, the soil contains a mixture of fatty substances {agricere) derived from plant material. These fats are not readily acted upon by microorgan- isms and finally cover and inpregnate the residual organic matter. Volatile antiseptics, being fat solvents, dissolve the agricere, which is either carried toward the surface of the soil or is segregated upon the points and angles of the individual soil particles. This was believed to be the cause of the favorable influence of the action of antiseptics upon bacterial growth and activities. When the soil fats are removed or segregated, the soluble matter diffuses out more readily from the soil particles and serves as a source of food for microorganisms.70 Treatment with volatile antiseptics was found to induce an increased growth of bacteria in soils in which the protozoa have been destroyed by moist or dry heat at 65° to 75°C. It was suggested, therefore, that two factors influence bacterial activities in the soil, one of which is a bacteriotoxin and which is destroyed by heat, and the other a soil fat or wax, which is dissolved by volatile antiseptics. According to Hutchinson,70a the formation of toxic substances depends upon the existence of anaerobic conditions due to water logging; when organic 68 Livingston, B. E. Palladin's Plant Physiology. 1st Ed. 1918, p. 93. 69 Greig-Smith, R. The agricere and the bacteriotoxins of the soil. Centrbl. Bakt. I., 34: 224-226. 1912; Proc. Linn. Soc. N. S. Wales., 36: 679-699. 1911. 70 Greig-Smith, R. Contributions to our knowledge of soil fertility. Proc. Linn. Soc. N. S. Wales, 35: 808-822. 1910; 36: 679-699. 1911; 37: 238-243, 655- 672. 1912; 38: 725-746. 1913; 39: 839-850. 1914; 40: 631-645, 724-733. 1915; 42: 162-166. 1917; 43: 142-190. 1918. 70a Hutchinson, C. M. Causes of infertility in soils in relation to bacterial action. Agr. Jour. India, 21: 125-133. 1926. SOIL MICROBIOLOGICAL EQUILIBRIUM 761 matter is decomposed under these conditions, waxes and slimes are formed which coat the soil particle tending to block up its pores, thus interfering with aeration and drainage and protecting the organic particles from the further action of the bacteria. Greig-Smith admitted that protozoa may play a part in checking the multiplication of bacteria in the soil, but they were not believed to be alone responsible for this limitation. These results on the formation of a bacteriotoxin in the soil were not confirmed by other investigators.71 However, certain metabolic products are formed by at least some soil microorganisms, which are either toxic to themselves or to other organisms. The removal, destruction or modification of these products may bring about an increased activity of the microorganisms concerned. Treatment of soil by heat or antiseptics results in a number of very complex processes, which cannot be explained by a protozoan theory, toxin theory, presence of soil waxes, etc., although all of these may play a certain part in limiting bacterial development. A series of changes are brought about which lead to an increase in the soluble soil organic matter,72 and to a modification of the concentration of the soil solution and of the soil reaction. The physical condition (per- meability, capillarity, cohesion, surface tension) of the soil is also modified by the treatment, particularly the colloidal properties of the soil.72a Quantities of toluol and CS2, insufficient to modify the number and types of protozoa in the soil, were found73 to exert a stimulative effect upon bacterial activities in the soil. Destruction of selective groups of organisms. Different soil organisms are not affected alike by disinfectants.74 Some are injured but not completely destroyed. The degree of injury also depends upon the concentration and length of action of the disinfectant, on the soil moisture and aeration conditions. The gelatin liquefying bacteria (mostly spore-formers) are not affected at all or to only a very limited extent, while the actinomyces may be considerably reduced ; the same is true of other aerobic, especially non-spore-forming bacteria. When 71 Hutchinson, H. B., and Thaysen, A. C. The non-persistence of bacterio- toxins in soil. Jour. Agr. Sci., 9: 43-62. 1918. 72 Pickering, 1910, (p. 748). 72a Taylor, E. McK., and Burns, A. C. The effect of the summer fallow upon soil protozoa in Egypt. Egypt. Min. Agr. Tech. Sci. Serv. Bui. 52. 1924. 11 Gainey, P. L. The effect of toluol and carbon bisulfide upon the micro- flora and fauna of the soil. Mo. Bot. Gard. Ann. Rpt., 23: 147-169. 1912. 74 Hiltner and Stormer, 1903 (p. 12). 762 PRINCIPLES OF SOIL MICROBIOLOGY the disinfectant is evaporated, there is a rapid increase in the non-lique- fying forms; the activity of the actinomyces and gelatin liquefying bacteria lags very much behind. After the maximum of bacterial activities is attained, a gradual decrease follows and within about a year the flora of the treated soil has reached the level of the untreated soil. The most active bacteria, after partial sterilization, were found75 to be Bac. butyricus, Bac. mycoides, Bac. megatherium, Bac. arborescens, Microc. ochraceus luteus and, to a less extent, Bact. fluoresceins liquefaciens. Various other investigators found76 actinomyces to be decidedly injured by treatment with CS2; the Bac. mycoides group was less affected, the spore formers being especially resistant. Legume bacteria (peas) and denitrifying bacteria (B. stutzeri) are rapidly killed, within two and a half hours; Bact. coli, Bact. prodigiosum, Bact. vulgar e, Micr. ureae survive four hours. Staphylococci are very resistant, surviving forty-eight hours. Az. chroococcum survives in moist soil impregnated with fumes of CS2 for forty-eight hours, but it is destroyed after a longer treatment. The vegetative forms are destroyed in twenty- four hours, but the spores survive. A comparison of the influence of antiseptics upon bacteria and protozoa brought out the fact that smaller doses are required to kill the organisms in solution than in soil.77 The actual concentration of the antiseptic required to destroy amoebae in the soil is so large as to become unapplicable for purposes of partial sterilization, which has as its aim the destruction of the protozoa. Even 60 per cent CS2 did not kill the cysts of amoebae in the soil; the same was true of 15 per cent ether, 6 per cent chloroform, 25 per cent CaO, 30 parts per thousand of chlorine water, less than 15 per cent toluol and 5 per cent CaS. Bacterial spores were found to be more resistant to the antiseptic than the cysts of amoebae. Active amoebae have a lower resistance than non-spore-forming bacteria, but the latter are more readily destroyed than cysts of amoebae. The data did not justify any claim for an equilibrium between the numbers of amoebae and bacteria in the soil, the fluctuations of the numbers of 76 Truffaut and Bezssonoff, 1920-21 (p. 37); Compt. Rend. Acad. Agr. France, 4: 1049-1057, 1030-103S. 1918. 76 Maassen and Behn, 1924 (p. 756). 77 Sewertzoff, L. B. The effect of some antiseptics on soil amoeboe in par- tially sterilized soils. Centrbl. Bakt. II, 65: 278-291. 1925; Ibid I. Or., 92: 151-158. 1924. SOIL MICROBIOLOGICAL EQUILIBRIUM 763 amoebae and bacteria being due more to the successive drying and moistening of the test soil than to the action of amoebae on the bacteria. Various plant pathogenic fungi, particularly organisms like Fusarium, Phytophthora, Melanospora, Rhizoctonia, Neocosmopora vasinfecta, Cladosporium scabies, Sclerotinia, Synchytrium, spores of rusts and smuts, are readily destroyed by volatile antiseptics, such as toluol, carbon bisulfide, or by heat.78 A soil containing over 100,000 fungi (spores and pieces of mycelium) per gram becomes practically free from fungi after treatment with steam or volatile antiseptics. Partial sterilization of soil can thus correct the condition of soil sickness caused by the development of certain specific plant pathogens.79 Hiltner80 used a logical process of reasoning for demonstrating the destruction of fungi and actinomyces by antiseptics. When straw is applied to soil, the available nitrogen is stored away by the micro- organisms to the detriment of higher plants. When the soil is treated with carbon bisulfide, during or after the addition of straw, the in- jurious influence is not observed. This is due to the fact that, with straw fertilization, the soil nitrogen is stored away chiefly by fungi and actinomyces. The disinfectant brings about an appreciable reduction in the number of these organisms, thus leaving the soil nitrogen available for higher plants. The favorable influence of CS2 is due not merely to the destruction of the organisms directly injurious to higher plants but also to the destruction of those which have a passive effect by storing away the soil nitrogen. Parasitic nematodes, such as Heterodera radicicola and various other worms, are also killed by heating the soil to 60° to 90°C. or by volatile antiseptics like CS2. In view of the fact that actinomyces grow only very slowly and that the injurious factor in the soil is apparently something which is slowly growing, for an infection with 5 per cent of raw soil only begins to show a limiting action upon the fortieth day, Greig-Smith suggested that actinomyces are the limiting factor for bacterial activities. Not all the disinfectants, however, injure the actinomyces alike. Interrelationships of microorganisms in the soil. If the soil could be 78 Pathogenic fungi in soil and their treatment are described elsewhere; see P. Waget. Sterilization et disinfection du sol. La Revue d. prod, chimiques., 22: 655. 1920; 4: 115. 1921; 6: 183. 1921. 79 Kaserer, H. Versuche fiber Bodenmiidigkeit, besonders Leinmlidigkeit. Mitt. Wien. Hochschule f. Bodenkultur., 2: 375-410. 1913-14; Russell, E. J., and Petherbridge, F. R. Partial sterilization of soil for glasshouse work. Jour. Board Agr., 19. 1913, No. 10. 80 Hiltner, 1908 (p. 41). 764 PRINCIPLES OF SOIL MICROBIOLOGY imagined in an undisturbed condition, even for a very brief period of time, when the moisture content, aeration and temperature are not changed appreciably, one could speak of a condition of equilibrium. A microbiological equilibrium is distinctly different from that of a chemical reaction; in the latter, equilibrium is reached when the reaction goes both ways at an equal rate. A microbiological equilibrium in the soil may occur when the changes in the numbers and activities of the various groups of organisms are constant, possibly when as many new cells are formed in a given period as are destroyed in that time. An ideal condition of equilibrium may never be reached under field conditions more than for a few brief seconds. Under carefully controlled laboratory conditions, an equilibrium can be readily demonstrated, although it takes a long period of time before it is established. When a soil is brought into the laboratory and kept at constant optimum moisture and temperature, there is at first a rise in the num- ber of microorganisms, particularly if the soil has been partially or fully air dried before being placed in the incubator. The numbers of microorganisms and the rate of their activities, using the carbon dioxide production of the soil as an index, rise at first, soon drop rapidly and then more slowly, until the rate of change in the numbers and activities becomes constant. But even under soil conditions, one may speak roughly of a certain equilibrium which becomes established between various groups of microorganisms, in the competition for the available energy, as shown by Hiltner and Stormer. The quantitative and qualitative composi- tion of the soil flora and fauna were found to depend on the amount of energy and nitrogen available as well as the forms in which these are present in the soil. Any modification in the amount and form of energy and nitrogen brings about a modification not only in the number but also in the kind of microorganisms developing in the soil. Such modifications are brought about either by introducing fresh energy and nitrogen materials, air drying and cultivating of soil, partial sterili- zation, growth of plants, etc. These modifications can be looked upon as resulting in a shift in the condition of equilibrium. The differences in the energy and nitrogen metabolism of the various soil microorganisms combined with their relative resistance to the action of disinfectants and their rates of multiplication are the basic factors governing the phenomenon of soil microbiological equilibrium. The fungi consume a large amount of the available energy for struc- tural purposes. They produce, therefore, large quantities of carbon SOIL MICROBIOLOGICAL EQUILIBRIUM 765 dioxide and consume a great deal of nitrogen which they transform into microbial protein. They multiply rapidly when large quantities of energy material, like undecomposed organic matter, are added to the soil. They are also less resistant to the action of heat and anti- septics, but multiply very rapidly when reintroduced into partially sterilized soils. In such a case, the fungi, together with the bacteria, contribute the large amount of carbon dioxide formed, but they use up, for structural purposes, a part of the available nitrogen, which otherwise would remain in the soil as ammonia or nitrates. The actinomyces develop very slowly in the soil, but are more re- sistant to the action of antiseptics like toluene and air drying of soil. When the soil is kept under uniform conditions and the flora gradually comes to an equilibrium, there is generally an increase in the proportion of actinomyces. When the equilibrium is shifted either by air drying of soil, volatile antiseptics, heat, etc., the actual number of actinomyces may actually diminish only slightly, but after the disinfectant is re- moved, or when moisture is added to the air dry soil, the actinomyces regain their previous numbers only very slowly. In comparison with the rapid increase of the bacteria, they diminish rapidly so that, two years after treatment with carbon bisulfide, they have not fully regained their former numbers. They do not use up much available nitrogen unless an available source of energy is introduced, but a number of them produce substances distinctly toxic to certain bacteria and fungi. The bacteria are such an heterogeneous group of organisms that their activities cannot be classified together. The spore formers are very resistant to the treatments that result in partial sterilization of soil and may develop abundantly afterwards. The non-spore-forming bacteria are very sensitive to the treatment and are much diminished in numbers as a result of partial sterilization of soil; but soon afterwards they begin to multiply very rapidly and may bring up the numbers to hundreds of millions per gram. They use very little of the energy for structural purposes, and, therefore, consume only very little of the ammonia liberated from the decomposition of the proteins. The ammonia can thus accumulate in the soil, unless the fungi are reintro- duced. The temporary suppression of the nitrifying bacteria tends to intensify the accumulation of the ammonia. The soil protozoa probably also play some part in this group of processes. At least some of the protozoa consume bacteria as food, perhaps even large numbers of them; their selective feeding may affect only certain groups of bacteria. They may thus store away consider- 766 PRINCIPLES OF SOIL MICROBIOLOGY able quantities of energy in their bodies. Their direct role in definite soil processes has not been established as yet, although some information seems to point to the fact that by removing the old bacterial cells, they may even exert a beneficial effect. The possibility that some protozoa exist as saprophytes in the soil, taking perhaps a part in the decomposi- tion of the soil organic matter, is not excluded. We may conclude in the words of Miege81 that disinfection of soil still presents too many obscurities and uncertainties. It is certain that it cannot be presented as a panacea capable of remedying the ills from which agriculture suffers. A detailed review of the subject of soil disinfection, from theoretical and practical viewpoints, is given elsewhere.82 81 Miege, E. Les nouvelles theories de la fertilization des terres. Compt. Rend. Soc. Nat. Agr. France. March, 1914; also Compt. Rend. Acad. Sci., 164: 362-365. 1917. 82 Vogt, E. Methoden der Schadlingsbekampfung. III. Bodendesinfektion. Centrbl. Bakt. II, 61: 323-356. 1924. CHAPTER XXIX Influence of Environmental Conditions, Soil Treatment and Plant Growth upon Microorganisms and Their Activities in the Soil When a soil is kept at the same moisture and temperature for a considerable period of time the microorganisms are found to reach in that soil a certain condition of equilibrium, as pointed out elsewhere (p. 740). Winogradsky found that a change in the physical, chemical and physico-chemical condition of the soil brings about a change in the soil flora and fauna. The nature of the change depends upon the treatment. The addition of organic matter stimulates the development of certain specific groups capable of decomposing this particular organic substance. A change in the soil reaction brought about by liming, by addition of sulfur or of ammonium salts favors the development of organ- isms more adapted to that soil reaction. An excessive rainfall or reduc- tion of pore space by mechanical means modifies the composition of the soil atmosphere and stimulates the development of anaerobic organisms in preference to aerobes. Changes in temperature and aeration also lead to changes in the quantitative and qualitative relationships of the soil population. Whether we accept the idea of Winogradsky concerning an autoch- tonous or native soil flora or not, one thing is certain that a soil, under a given set of conditions, can liberate a definite amount of energy, which is sufficient for the activities of a definite number of microorgan- isms, the qualitative composition of the population depending upon the soil conditions. Any change in these conditions will modify the popu- lation both quantitatively and qualitatively. The changes in the soil population may take place from day to day and even from hour to hour, under field conditions.1 Samples of soil taken at long intervals of time may not give, therefore, a true picture of the actual soil popula- tion and its changes in the soil. It is important to study the soil flora and fauna at frequent intervals and establish the interrelationships 1 Cutler, D. W., and Crump, L. M. Daily periodicity in numbers of active soil flagellates. Ann. Appl. Biol., 7: 11-24. 1920; Cutler et al., 1923 (p. 32). 767 768 PRINCIPLES OF SOIL MICROBIOLOGY between the different members of the population and between the latter and the environmental conditions. Influence of organic matter upon the soil population. The addition of organic matter to the soil results in an increase in the numbers of various groups of soil microorganisms, of which some are directly concerned in the process of decomposition and some utilize the products formed by the first or attack the living or dead cells of those organisms themselves. Differences in the physical and chemical nature of the soil lead to the development of different groups of micro- organisms as a result of addition of the same organic substance. The composition of the organic matter is of prime importance in this con- nection, modifying largely the nature of the organisms which develop in preference to others. The addition of soluble sugars to the soil brings about an extensive development of bacteria, primarily nitrogen- fixing forms, such as Azotobacter, under aerobic conditions, and Clos- tridium under anaerobic conditions. The addition of substances rich in celluloses and poor in proteins, like cereal straw, stimulates largely the development of fungi; these decompose the celluloses and synthesize an extensive mycelium. This mycelium is immediately attacked by a large number of bacteria and actinomyces, leading to the formation of various products in addition to the synthesized cells of these micro- organisms, which in their turn along with the residual products of the organic matter and the fungus mycelium, serve as food for protozoa and other invertebrate animals. One group of organisms carries on a certain process and then gives way to another group, which carries on the process further; both may be active at the same time. The activities of these organisms lead to a gradual increase in the carbon content of the residual organic matter, which has become changed into a colloidal mass more or less constant in composition and less readily available as a source of energy. Finally those microorganisms (largely the minute non-spore forming bacteria), which require only a small amount of energy and nutrients, continue to act in the colloidal film surrounding the soil particles, slowly breaking down the residual lignins and the resistant synthesized materials of a protein nature. Engberding2 found that the addition of 2 per cent cane sugar to a soil brought about an increase of 1000 to 1500 per cent in the number of bacteria developing upon Heyden agar; 0.5 per cent glucose brought about an increase of only 300 to 400 per cent. This increase was soon 2 Engberding, 1909 (p. 14). INFLUENCE OF ENVIRONMENTAL CONDITIONS 769 followed by a decrease and in some cases there were no more bacteria in the treated soils than in the control soils after two and one-half months. The addition of glucose to the soil increases the number of bacteria but diminishes the number of fungi, both in the presence and absence of available nitrogen salts.3 It was suggested that this may be due to the formation of a dry pellicle of sugar on the surface of the soil preventing the admission of oxygen. Cellulose, however, greatly favors the development of fungi and also favorably influences the development of bacteria, especially in the presence of ammonium salts. By direct microscopic examination, it can be demonstrated4 that the addition of glucose or mannite to the soil brings about a rapid develop- ment of nitrogen-fixing bacteria; starch stimulates the development of actinomyces, the addition of cellulose brings about an extensive development of fungi, the addition of dried blood leads to an abundant growth of spore forming bacteria. These results indicate that bacteria are favored primarily by lower carbohydrates and protein-rich sub- stances, while the fungi and actinomyces are increased to a greater extent by celluloses and other polysaccharides and natural organic substances, especially those of a non-protein nature. The greater stimulative effect of sugars upon bacteria than upon fungi and actino- myces is due to several factors: 1. The majority of bacteria prefer glucose to higher carbohydrates and their derivatives, while many fungi readily decompose celluloses, pentosans, etc.; many actinomyces are capable of attacking the lignins in the natural organic materials. 2. Bacteria generally require much less nitrogen for the synthesis of their cells per unit of glucose decomposed than do the fungi, which produce an abun- dant mycelium requiring considerable nitrogen. 3. Among the bacteria, the nitrogen-fixing forms readily utilize sugars as sources of energy without requiring any combined nitrogen. The addition of glucose, in the presence of even a small amount of available nitrogen will, therefore, greatly stimulate the develop- ment of rapidly growing bacteria and may not affect at all the develop- ment of fungi, which require a large amount of available nitrogen, or of actinomyces, which develop only very slowly. The stimulative effect of celluloses upon the development of fungi in the soil is especially marked under aerobic conditions and in the presence of available nitrogen. An extensive bacterial development a Bazarevski, 1916 (p. 653). 4 Winogradsky, 1924 (p. 10). 770 PRINCIPLES OF SOIL MICROBIOLOGY may take place as a result of addition of celluloses to the soil. However, the true cellulose-decomposing bacteria are usually not determined, since they do not develop (as Spirochaeta cytophaga) on the ordinary plate. The actual increase in bacterial numbers resulting from addition of celluloses, as shown by the ordinary plate, may be a result of develop- ment of bacteria feeding upon synthesized or intermediary products, which result from the activities of fungi and aerobic cellulose decom- posing bacteria. Under anaerobic conditions, however, it is the bacteria which are greatly stimulated by the addition of celluloses. Pure cellu- lose may even depress the development of bacteria which develop on the ordinary plate.5 Straw, however, stimulates the development of bac- teria due to the presence of soluble carbohydrates and proteins. The stimulative effect of proteins upon the development of spore- forming and non-spore forming bacteria has been recorded by a number of observers. Urea stimulates the development of various bacteria, especially certain non-spore forming rods. Natural organic matter, like straw, plant stubble and green manures, as well as stable manures or organic fertilizers, consist of a number of various substances. The addition of this organic matter to the soil will stimulate the development of various groups of organisms.6 The greater the protein content of the organic materials added to the soil, the greater is the development of bacteria in preference to the fungi, as shown in table 86. It has been shown elsewhere (p. 517) that the addition of organic matter of a wide carbon-nitrogen ratio to the soil leads to a considerable reduction of the nitrate nitrogen, which results in a harmful effect upon plant growth. However, the following year a beneficial effect may be noted,7 due to the subsequent decomposition of the synthesized protoplasm. In some cases it has been claimed8 that the depressing effect of a straw mulch upon nitrate formation in the soil is due to the checked evaporation of the soil moisture which lowers the tempera- 5 Hill, H. H. The effect of green manuring on soil nitrates under greenhouse conditions. Va. Polyt. Inst. Agr. Exp. Sta. Tech. Bui. 6, 121-153. 1915. 6 Waksman, S. A., and Starkey, R. L. Influence of organic matter upon the development of fungi, actinomycetes and bacteria in the soil. Soil Sci., 17: 373-378. 1924. 7 Bredemann, G. Untersuchungen liber das Bakterien-Impfpraparat "Heyls concentrated nitrogen producer" (Composite Farmogerm). Landw. Jahrb., 43: 669-G94. 1913. 8 Albrecht, W. A., and Uhland, R. E. Nitrate accumulation under the straw mulch. Soil Sci., 20: 253-268. 1925. INFLUENCE OF ENVIRONMENTAL CONDITIONS 771 ture, prevents the normal air exchange, and creates an unfavorable environment for the formation of nitrates. No attempt, however, has been made to correlate these results with the microbiological activities of the vast soil population, outside of the nitrate-forming bacteria. Influence of stable manure. The introduction of stable manure affects chiefly the following physical, chemical and biological condi- tions of the soil: 1. Soil temperature. The amount of temperature change depends upon the kind and amount of manure added.9 The addition of 25 tons of manure per acre may give an average increase of five degrees Centigrade in the temperature of the soil.10 TABLE 86 Influence of various substances upon the development of fungi and bacteria (including actinomyces) in a poor soil TREATMENT Untreated 0. 5 per cent glucose Cellulose, 1 per cent Cellulose, 1 per cent + 0.1 per cent NaNOs Straw, 1 per cent Dried blood, 1 per cent INCUBATION FCNGI BACTERIA days 115,700 3,860,000 2 82,000 22,200,000 17 160,000 3,600,000 17 4,800,000 4,800,000 10 600,000 25,200,000 12 1,438,300 473,900,000 2. Soil moisture. A higher moisture holding capacity of the soil results from the addition of manure because of the accumulation of the soil organic matter. This affects bacterial activities favorably.11*12 3. Soil atmosphere. The rapid decomposition of manure added to the soil results in the formation of large quantities of C02, which will tend to improve the physical condition of the soil giving it a crumbly appearance. 9 Wagner, F. Uber den Einflusz der Dungung mit organischen Substanzen auf die Bodentemperatur. Forsch. Agr. Phys., 5: 373-402. 1882 (Centrbl. Agr. Chem., 12: 150. 1883). 10 Troop, J. The relation of barn manures to soil temperature. Ind. Agr. Exp. Sta. 8th Ann. Rpt., 18-19. 1895. 11 Engberding. D. Vergleichende Untersuchungen liber die Bakterienzahl im Ackerboden in ihrer Abhangigkeit von ausseren Einfliissen. Centrbl. Bakt. II, 23: 569-642. 1909. 12 King, W. E., and Doryland, C. J. T. The influence of depth of cultivation upon soil bacteria and their activities. Kans. Agr. Exp. Sta., Bui. 161, 211-242. 1909. 772 PRINCIPLES OF SOIL MICROBIOLOGY 4. Reaction and buffer content of the soil. The decomposition of available nitrogenous substances (in the liquid part of the urine) leading to the forma- tion of ammonia and nitric acid, on the one hand, and the decomposition of the carbohydrates which may lead to formation of some organic acids, on the other, are important in this connection. The buffering properties of the residual "humus" are considerable. 5. The introduction of large quantities of readily available energy, as well as of nitrogen and minerals, will in itself greatly stimulate bacterial activities. 6. Finally the introduction of large quantities of living bacteria in the manure may result in a change of the qualitative composition of the soil flora and fauna. A number of observations have been made concerning the increase in the numbers of bacteria in the soil as a result of addition of manure. The number of bacteria present in the soil was found13 to depend not only upon the manure added but also upon the cultural methods and crop grown. Fallowing of a soil leads to a decrease in numbers of bacteria as compared with the untreated soil, while manuring and fallowing lead to a decided increase.14 Chester15 stated in 1898 that "the greater the organic matter or humus in the soil the greater, pari passu, is the number of bacteria." The increase in bacterial numbers in the soil as a result of the addi- tion of stable manure has been explained as due to the introduction of large numbers of bacteria with the manure; or due to the introduction of readily decomposable organic matter which stimulates bacterial activities. The fertilizing effect of the manure, aside from the quanti- ties of fertilizer constituents contained within them, was believed to be due merely to the bacterial content of the manure.16'17 Frequent small applications of manure rather than large applications made at longer intervals were, therefore, recommended. The bacteria intro- duced into the soil, with small quantities of manure, were believed18 to be valuable in bringing about a more rapid decomposition of a green manure crop. 13 Caron, A. Landwirtschaftlich-Bakteriologische Probleme. Landw. Vers. Sta., 45: 401-418. 1895. 14 Hiltner and Stunner, 1903 (p. 12). 16 Chester, F. D. Soil bacteria and their relation to agriculture. Del. Agr. Exp. Sta. Bui. 40. 1898; The microbiological analysis of soils. Ibid. Bui. 65, 1904. 16 Hellstrom, P. On the effect of animal manures on marsh soils. (Exp. Sta. Rec, 11: 627. 1900.) 17 Stoklasa, J. Uber die Wirkung des Stallmistes. Chem. Centrbl. Jahrg., 78: (N. F. 11): 1702. 1907; also Stoklasa and Ernest, 1905 (p. 34). 18 Lipman, J. G., McLean, H. C, et al. The influence of mechanical composi- tion of the soil on the availability of nitrate of soda and dried blood. N. J. Agr. Exp. Sta. Bui. 268. 1914. INFLUENCE OF ENVIRONMENTAL CONDITIONS 773 By comparing the influence of manure with inorganic fertilizers, Temple19 found that the addition of sodium nitrate to the soil increased the number of bacteria from 6,500,000 to 8,480,000; the addition of a complete mineral fertilizer increased these numbers to 11,540,000, while stable manure brought about an increase to 23,310,000, on the average of several determinations. This increase continues over a considerable period. However, when the manure was previously sterilized, before adding it to the soil, the increase in the numbers of bacteria was even greater than in the case of unsterilized manure. Temple suggested, therefore, that the increase in the numbers of bacteria as a result of addition of manure is due to the addition of the organic matter (available energy) rather than to the actual introduction TABLE 87 Influence of coto manure (10 tons per acre) upon the number of bacteria per gram of dry soil (1 gm. of manure contained 625,000,000 bacteria) DATE OF INCUBATION NO MANURE MANURE days 2 2,227,000 2,227,000 6 3,780,000 6,000,000 13 6,540,000 13,600,000 20 6,750,000 11,690,000 28 7,700,000 24,200,000 33 3,630,000 6,330,000 40 4,270,000 6,330,000 90 3,800,000 7,850,000 of bacteria, as suggested by other investigators. A direct relationship between the organic matter added to the soil and the bacterial count was also observed.20 The work of Charpentier and Barthel (p. 450) on the decomposition of pure cellulose in the soil tends to confirm the observations that the fertilizing effect of manure is due entirely to its nitrogen and minerals and not to the bacteria introduced. No greater increase in the numbers of bacteria from the addition of stable manure with green manure to the soil was obtained than from the 19 Temple, J. The influence of stall manure upon the bacterial flora of the soil. Ga. Agr. Exp. Sta. Bui. 95. 1911; Centrbl. Bakt. II, 34: 204-223. 1912. 20 Briscoe, C. F., and Harned, H. H. Bacteriological effects of green manure. Miss. Agr. Exp. Sta. Bui. 168, 20 p. 1915. 774 PRINCIPLES OF SOIL MICROBIOLOGY green manure itself.21 The kind of manure greatly influences the change in microbial activities.22 A close correlation was found between the bacterial numbers, am- monifying power, nitrifying power and crop production of a soil receiv- ing no manure, 5 tons and 15 tons of manure per acre.23 The maximum activities were obtained from the addition of the largest amounts of manure; the increase from 20 inches of irrigation water was also most marked in soil receiving the largest quantity of manure. Table 88 shows that the addition of manure to the soil results in an immediate rapid increase in the numbers of microorganisms which reaches the maximum in a few days, and is soon followed by a precipitous decrease due to the rapid exhaustion of the available organic matter. TABLE 88 Influence of manure on the development of bacteria in the soil as determined by the plate method2™ (5 parts of manure per 100 parts of soil) INCUBATION TOTAL NUMBER OF ORGANISMS INCUBATION TOTAL NUMBER OP ORGANISMS days days 2 60,000,000 21 50,000,000 3 80,000,000 24 55,000,000 4 125,000,000 29 85,000,000 6 235,000,000 38 45,000,000 9 45,000,000 58 95,000,000 13 43,000,000 94 18,000,000 16 35,000,000 123 20,000,000 Influence of temperature. Soil microorganisms are often divided in respect to temperature, into three groups: 1. Thermophilic, or those which require a high temperature for their development, usually 45° to 65°C. 21 Lemmermann, O., and Einecke, A. tlber die Wirkung einer Beigabe von Stalldunger zur Griindungung. Mitt. deut. Landw. Gesell., 29 (Stiick 52): 702-704. 1914. 22 Emmerich, R., Graf zu Leiningen, W., and Loew, O. tlber schadliche Bakterientatigkeit im Boden und liber Bodensauberung. Centrbl. Bakt. Abt.. II, 29: 668-683. 1911. 23 Greaves, J. E., and Carter, E. G. Influence of barnyard manure and water upon bacterial activities of the soil. Jour. Agr. Res., 6: 889-926. 1916; also 9: 293-341. 1917. 23a Bright and Conn, 1919 (p. 41). INFLUENCE OF ENVIRONMENTAL CONDITIONS 775 2. Psychrophilic, or those which grow best at low temperatures (below 10°C). 3. Mesophilic, or those which grow best at 10° to 45°C. Attention has already been called to certain processes carried on by thermophilic bacteria and fungi, notably the decomposition of cellu- lose (p. 439). It is doubtful whether any specific group of "psychro- philic" bacteria exists in the soil, these organisms being more "psychro- tolerant." The presence of "thermophilic" groups of organisms is more definite, although a large number of these are probably also only "ther- motolerant." Most of the thermophilic microorganisms have their optimum at 50 to 70°C. with a minimum at 30° to 40° and a maximum at 70° to 74°C. Krohn even isolated bacteria having an optimum at 54° and a maximum at 89°C. The question of heating of hay called forth considerable discussion. Miehe found that by inoculating hay with a mixture of Bart, coli and Bac. calfactor or Oidium lactis and Bac. calfactor, heating of hay to 70°C. could be brought about. At that temperature, chemical processes take place in the hay which lead to the formation of substances which ignite on contact with air. The biological nature of the process was denied by some investigators, who ascribed the phenomenon to the action of oxidative and reducing enzymes.23b Higher temperatures usually never become limiting factors in the activities of microorganisms in the soil, except in very hot climates, where only the surface layer may be affected. The destructive action in this case may be due more to the lack of moisture at the surface of the soil. Most microorganisms are able to withstand dryng and this process exerts a decided stimulating effect upon the organisms after the soil is remoistened, as shown elsewhere. The thermophilic bacteria are not only able to withstand high temperatures (65° to 75°C), but they may take an active part in various processes of great importance in the soil (p. 157). It is not necessarily essential for the soil to be heated up to 45° to 50°C, before these organisms become active. They act in the soil even at lower temperatures, but to a more limited extent. The pe- culiar ability of these organisms to grow at such high temperatures is due to their adaptation to environment. The temperature optimum of soil bacteria depends upon the soil from which they were isolated, c5b Tschirch, A. Die Lokalisation der chemischen Arbeit in der Pflanze. Mitt. Naturf. Gesell. Bern., 138-152. 1917; Laupper, G. Die neuesten Ergeb- nisse der Heubrandforschung. Landw. Jahrb. Schweiz., 34: 1-54. 1920. 776 PRINCIPLES OF SOIL MICROBIOLOGY bacteria isolated from soils of colder climates being capable of growing at lower temperatures than bacteria isolated from soils of warm climates.24 SO 25 30 Percent of Water Fig. 72. Influence of moisture and potassium salts upon nitrate accumulation in the soil (from Greaves and Carter). Ammonia formation takes place between 15° and 60°C, the thermo- philic organisms being very active in this respect. Nitrification takes place at 15° to 40°C, the optimum being at 35° or a little higher (fig. 24 Mischustin, E. The analysis of temperature conditions of bacterial proc- esses in the soil in connection with the adaptation of bacteria to climate. Potch- voviedienie., 20: 43-67. 1925. INFLUENCE OF ENVIRONMENTAL CONDITIONS 777 73). Nitrate reduction takes place at 15° to 40°, with an optimum at 25° to 30°; the same is true of aerobic and anaerobic nitrogen-fixation.25 Ordinary soil bacteria can withstand very low temperatures, even 40°C. below zero.26 The action of frost upon the soil flora may be compared to that of partial sterilization. Prucha,27 for example, demonstrated that Bad. typhosum added to ice cream, kept constantly frozen, at first increases in numbers (due to the breaking up of the bacterial lumps), then gradually diminishes, but will no tbe completely destroyed, even when the ice cream is frozen for over two years; when the ice cream is allowed to thaw out, an increase in numbers takes place. Freezing of soil or even very low temperatures may bring about a change in the balance of soil microorganisms; this may be followed by a great increase in numbers, especially of those organisms which develop on the plate. This increase may be due both to the breaking up of the clumps of bacteria and to an actual multiplication when the soil is warmed up again. Freezing of soil causes the coagula- tion of soil colloids;28-29 heating of soil also modifies its physical condi- tions. Grinding30 of soil particles also exerts an important influence upon the numbers and activities of soil microorganisms.31 One may compare the soil flora found in very cold climates with that of temperate zones. Omeliansky32 examined the soil obtained from the frozen ground of Northern Siberia (72° 15' latitude and 142° longi- tude East) and found the microflora to consist of spore forming and 25 Panganiban, E. H. Temperature as a factor in nitrogen changes in the soil. Jour. Amer. Soc. Agron., 17: 1-31. 1925. 26 Harder, 1916 (p. 33). 27 Prucha, M. J., and Brannon, J. M. Viability of Bacterium typhosum in ice cream. Jour. Bact., 11: 27-30. 1926. 28 Czermak, W. Ein Beitrag zur Erkenntnis der Veranderungen der sog. physikalischen Bodeneigenschaften durch Frost, Hitze, und die Beigabe einiger Salze. Landw. Vers. Sta., 76: 75-116. 1912. 29 Nolte, O., and Hahn, E. Die Wirkung des Frostes auf den Boden. Jour. Landw., 65: 75-81. 1917. 30 Fred, E. B. Effect of grinding soil on the number of microorganisms. Science, 44: 282. 1916. 31 Further information on the influence of low temperatures upon soil bac- teria is given by Smith, E. F. Das Verhalten von Mikroorganismen gegen niedere Temperaturen. Centrbl. Bakt. II, 33: 335. 1912; Keith, S. C. Factors influencing the survival of bacteria at temperatures in the vicinity of the freez- ing point of water. Science, 37: 877. 1913. 32 Omeliansky, V. L. Etude bacteriologique du mammouth de Sanga Jourah et du sol adjacent. Arch. Sci. Biol. St. Petersburg, 16: No. 4. 1911. 778 PRINCIPLES OF SOIL MICROBIOLOGY non-spore forming bacteria, sarcina, actinomyces and fungi commonly associated with processes of decomposition of organic matter. How- Fig. 73. Influence of temperature upon nitrification in the soil (from Pan- ganiban). ever, nitrifying, denitrifying, nitrogen-fixing, cellulose decomposing and pathogenic bacteria were absent. A careful study of the flora of the soil of northern Greenland and of the island of Disko near Cape INFLUENCE OF ENVIRONMENTAL CONDITIONS 779 York has been made by Barthel,33 who found, in addition to various aerobic and anaerobic, spore-forming and non-spore forming bacteria, also actinomyces and fungi (Mucors, Torulae) . Nitrifying bacteria were present only in three out of fourteen soils of Disko, while Azotobacter was absent in all; Bac. amylobacter could not be demonstrated, although its presence originally was not excluded; Azotobacter and protozoa were found in the Greenland soils. Various investigations have been made concerning the temperature re- lations of fungi, especially in relation to fruit rot in storage. The great majority of these fungi grow at 4.5°C. and even develop slowly at 0°C.34 The minimum temperatures of germination of fungi vary from 1°C, for Monilia fructigena and Pen. digitatum, to 6° to 9° for Cephalo- thecium roseum.Zb Spores of Alternaria, Botrytis, Pen. expansum and Sclerotinia germinate slowly at zero; the spores of Ceph. roseum, Fu- sarium radicicola and others failed to germinate at zero but germinated slowly at 5°C.36 Some fungi, like Pen. expansum, after starting growth at ordinary temperatures, were able to continue growth at zero. The rate of growth increases with rise in temperature up to a certain limit, being a direct function of (t — t°) , where t is the particular temperature and t° is the minimum temperature.37 It is of interest to call attention, in this connection, to the changes in the numbers and activities of microorganisms in the soil with the season of the year, due to changes in soil temperature and moisture, and introduction of available organic matter, soil aeration, etc. Rus- sell and Appleyard38 found that there is very little activity in the soil during the winter months (November to March). Below 5°C, the changes taking place in the soil are very slow; above that temperature, bacterial numbers, C02 production, and nitrate accumulation all 33 Barthel, 1922 (p. 149). 34 Schneider-Orelli, O. Versuche iiber die Wachstumsbedingungen und Verbreitung der Faulnispilze des Lagerobstes. Centrbl. Bakt. II, 32: 161-169. 1912. " Ames, A. The temperature relations of some fungi causing storage rots. Phytopathol., 11: 19. 1915. 38 Brooks, C, and Cooley, J. S. Temperature relations of apple-rot fungi. Jour. Agr. Res., 8: 139-164. 1917. 37 Brown, W. On the germination and growth of fungi at various temperatures and in various concentrations of oxygen and of carbon dioxide. Ann. Bot., 36: 257-283. 1922. 38 Russell, E. J., and Appleyard, A. The influence of soil conditions on the decomposition of organic matter in the soil. Jour. Agr. Sci., 8: 385-417. 1917. 780 PRINCIPLES OF SOIL MICROBIOLOGY increase, the curves agreeing closely with the temperature curve. Un- der favorable temperature conditions, rainfall becomes the limiting- factor as well as the oxygen brought down by the rain. In general they have observed a period of spring activity, summer sluggishness, autumn activity, followed by winter inertness. Woitkiewicz39 also found bacterial numbers to be highest in spring and lowest in winter; the ratios of nitrogen-fixation in solution, in the seasons of winter, \ \ \\ V\ N\ \ ^ \ \\ :,— .X __\ "*""■-- "-'.\ Dry 15 Percent 30Pcrcertf 45 Percent SOPercent- 75PcV«"t • ■ Mi llijroros ot rtmmomo in Soil » P«pton«. __. __ dilhqrcirn ol A-VKtma in Soil *• B!o»d Meat. — — — . Milligrorns of Ammonia in Soil 'Lime — Milligrams of Ammyn\a in Normal Soil — . Bacteria in millions per lOq/ams ot soil containing Peptone. .. — -.„ Bacteria 111 million* per \0 qreme ot »oit contoinmg Blood Meal Fig. 74. Influence of moisture content of an Oregon soil upon ammonia for- mation and bacterial development from peptone and blood meal, 75 per cent moisture being that of full saturation (from Beckwith, Vass and Robinson). spring, summer, and fall were as l:2:2f:3. This may be due to the greater abundance of available energy in the fall of the year. Deni- trification was highest in fall and lowest in spring. By the use of the Remy method, it was40 found that the urea-decomposing, nitrifying, and 39 Woitkiewicz, A. Beitrage zur bakteriologischen Bodenuntersuchungen. Centrbl. Bakt. II, 42: 254-261. 1914. 40 Lohnis, F., and Sabaschnikoff, A. t)ber die Zersetzung von Kalkstickstoff und Stickstoffkalk. Centrbl. Bakt. II, 20: 322-332. 1908. INFLUENCE OF ENVIRONMENTAL CONDITIONS 781 nitrogen-fixing powers of the soil reached a maximum in the spring, a minimum in summer and a maximum again in autumn. Similar results were obtained in the study of the influence of season of year on ammonia formation from peptone in solution.41 Maximum nitrifying power of the soil was found in spring.42 Lemmermann and Wichers,43 however, found that the season of year, outside of influence of tempera- ture and other physical weathering conditions, has no appreciable influence on nitrification. The seasonal course of ammonia and nitrate formation, with all other factors alike, were found44 to be influenced primarily by the temperature course. The season of year, independent of temperature and other physical conditions, had no influence at all; the maximum activity is found in the spring and is due to outer influ- ences, primarily change in temperature, but is independent of inner causes. Influence of moisture. Some bacteria, like the nitrifying organisms, are very sensitive to drying, while others, like Azotobacter, can resist drying for a long period of time. The addition of moisture to a soil, which contains less water than is the optimum for biological activities, brings about an increase in numbers and activities of microorganisms. Most active nitrification takes place when the soil is allowed to become partially dry between the applications of water;45 a direct relationship was observed between the speed of nitrification and the moisture content of fallow soil. Every soil has an optimum for nitrification; higher or lower amounts of moisture reduce this optimum. Nitri- fication was found46 to be at its highest when the soil contains moisture 41 Moll, R. Beitrage zur Biochemie des Bodens. Diss. Leipzig. 1909. 42 Miintz, A., and Gaudechon, H. Le reveil de la terre. Compt. Rend. Acad. Sci., 154: 163-168. 1912; see also Lumiere, A. Le rythme saisonnier et le rdveil de la terre. Rev. Gen. Bot., 33: 545-557. 1921; Nitrate content of soil at different seasons of the year has been studied by Jensen, C. A. Seasonal nitrification as influenced by crops and tillage. U. S. Dept. Agr. Bur. PI. Ind. Bui. 173. 1910; Leather, J. W. Water requirements of crops in India. Mem. Dept. Agr. India, Chem. ser., 1: 133-184. 1910; 205-281; 2: 63-140. 1912. 43 Lemmermann, O., and Wichers, L. tJber den periodischen Einflusz der Jahreszeit auf den Verlauf der Nitrifikation. Centrbl. Bakt. II, 53: 33-43. 1920. 44 Schonbrunn, B. Uber den zeitlichen Verlauf der Nitrifikation, unter be- sonderer BeriAcksichtigung der Frage nach dem periodischen Einflusz der Jah- reszeit. Centrbl. Bakt. II, 56: 545-565. 1922. 45 Deherain, P. P. Sur la production des nitrates dans la terre arable. Ann. Agron., 13: 241-261. 1887; also 22: 515-523. 1896. 46 Fraps, G. S. The production of active nitrogen in the soil. Texas Agr. Exp. Sta. Bui. 106. 1908. 782 PRINCIPLES OF SOIL MICROBIOLOGY equivalent to 55.6 per cent of its water-holding capacity. Excessive quantities of water were found to be much more injurious than too small quantities, due to the fact that in the first case soil conditions become anaerobic and denitrification sets in. The water requirements of the microorganisms vary considerably with the soil. Maximum nitrification in a loam soil occurred with 16 per cent water; by reducing the water content to 10 per cent or increasing to 26 per cent, nitrifi- cation was greatly retarded.47 A definite favorable influence of increased moisture upon bacterial numbers was recorded.48 Moisture content of the soil was found49 to have a greater influence upon bacterial numbers than the tempera- ture. By increasing the moisture content of the soil from 6.5 to 14 per cent, there was an increase in bacterial numbers from 10 to 16.4 mil- lions per gram. By raising the moisture content of the soil from 6 to TABLE 89 Influence of moisture upon the evolution of COz from different soils MOISTURE CO2 EVOLUTION FROM LOAM SOIL MOISTURE CO2 EVOLUTION FROM HUMUS SOIL per cent 2.59 5.75 8.49 13.75 100 397 554 767 per cent 2.43 5.43 7.90 12.68 100 207 329 254 15 per cent, van Suchtelen obtained an increase in C02 production from 19 to 208 nigra., which led him to conclude that the latter was a more sensitive method for studying changes in microbiological activi- ties than determining numbers, as is shown elsewhere. Sixty to eighty per cent of the total water holding capacity of the soil is an optimum for bacterial activities.50 Excessive soil moisture reduces both the numbers of soil microorganisms and their activities.51 A definite rela- tion between moisture content of different soils and bacterial activities 47 Coleman, L. C. Untersuchungen uber Nitrifikation. Centrbl. Bakt. II, 20: 401-420, 484-513. 190S. 48 Fischer, H., Lemmermann, A., Kappen, H., and Blanek, E. Bakteriologisch- chemische Untersuchungen. Lanchv. Jahrb., 38: 319-364. 1909. 49 Engberding, 1909 (p. 771). 60 Lohnis, F. Untersuchungen uber den Verlauf der Stickstoffumsetzungen in der Ackererde. Mitt, landw. Inst. Leipzig, 7: 1-105. 1905. 61 King and Doryland, 1909 (p. 771). INFLUENCE OF ENVIRONMENTAL CONDITIONS 783 has been demonstrated.52 At the lower limits of moisture, less water is required to start nitrification in sand than in claj'' soils; at the higher limits of moisture, less water is required to stop nitrification in sand than in clay. The optimum for the two soils varies; a rise above the optimum is more injurious than an equal fall below the optimum. Table 89 gives the results obtained53 on the influence of moisture upon the decomposition of organic matter in the soil as measured by CO2 production. The nature of the soil is also found to be an important factor in modifying the influence of moisture upon microbiological activities in the soil, as shown in table 89 and in the following summary:54 Tenacious clay soil Diluvial loam Alluvial soil CARBON CONTENT OF SOIL per cent 1.68 2.12 1.73 CO: PRODUCED IN 24 HOURS, BY 1 KGM. OF FRESH SOIL mgm. 8.2 14.6 36.6 CO2 PRODUCED IN 24 HOURS BY 1 KGM. OF SOIL, STERI- LIZED AND INOCULATED mgm. 14.0 27.8 59.8 Winogradsky55 found that, in the case of a soil with a moisture holding capacity of 48 per cent, 15 per cent moisture favored the development of aerobic bacteria to a depth of 23 cm.; 19 per cent moisture to 18 cm.; 21 per cent moisture caused the anaerobic bacteria to develop a few centimeters below the surface, while 24 per cent moisture and above made the soil decidedly favorable for anaerobic bacteria. Nitrogen fixation was found56 to be at a maximum near the point of saturation; even higher nitrogen fixation was recorded under anaerobic than under aerobic conditions.57 In some cases two maxima were recorded58-59 for 62 Patterson, J. W., and Scott, P. R. The influence of soil moisture upon nitrification. Jour. Dept. Agr. Victoria, 10: 275-282. 1912. 63 Konig, J., and Hasenbaumer, J. Die Bedeutung neuer Bodenforschungen fur die Landwirtschaft. Landw. Jahrb., 55: 185-252. 1920. 54 Stoklasa, 1926 (p. XIII). 63 Winogradsky, 1924 (p. 542). 66 Traaen, A. E. tlber den Einflusz der Feuchtigkeit auf die Stickstoffum- setzungen im Erdboden. Centrbl. Bakt. II, 45: 119-135. 1916. 17 Panganiban, 1925 (p. 777). "Greaves and Carter, 1917 (p. 774). 69 Lipman, C. B., and Sharp, L. T. Effect of moisture content of a sandy soil on its nitrogen fixing power. Bot. Gaz., 59: 402-406. 1915. 784 PRINCIPLES OF SOIL MICROBIOLOGY nitrogen fixation, one under aerobic and the other under anaerobic conditions. Ammonia formation from proteins was found'50 to be at an optimum when the soil contained water equivalent to 60 per cent of its total moisture holding capacity. The formation of ammonia may also be very intensive even in saturated soils.61,62 This is due to active de- composition of proteins by anaerobic bacteria. The mechanism of ammonia formation is different of course under aerobic and anaerobic conditions.63 In arid regions, the application of irrigation water has64 a definite beneficial effect upon the number of organisms in fallow soils and upon the ammonifying and nitrifying capacities of both fallow and cropped soils. These activities result in an increase in soluble nitrogen. An excess of water may result in the washing out of the nitrates from the soil. Greaves and Carter,00 using the Briggs formula for the moisture equivalent and the wilting and hygroscopic coefficients, found that the following equations represent the water requirements for maximum bacterial activities: Mam = 0.6c = 0.942e + 12.6 = 1.74u> + 12.6 = 2.55^ + 12.6 Mn = 1.55c = 0.8525e + 11.55 = 1.472m; + 11.55 - 2.163/i + 11.55 Mn/ = 0.7c = 1.049e +14.7 = 1.947u> + 1.47 = 2.84SA + 14.7 c = moisture capacity as defined by Hillgard, w = wilting coefficient, e = the moisture equivalent, h — the hygroscopic coefficient, am = ammonification, n = nitrification, nf = nitrogen fixation: Influence of soil cultivation. Cultivation tends to conserve the soil moisture at a time of the year when it is most needed; it brings about a better aeration of the soil, it influences the soil temperature and 60 Greaves, J. E., and Carter, E. G. Influence of moisture on the bacterial activities of the soil. Soil Sci., 10: 361-387. 1920; 13: 251-270. 1922. 61 Lipman, J. G., and Brown, P. E. Report of the soil chemist and bacteri- ologists. 29th Ann. Rpt., N. J. Agr. Exp. Sta. 105-115. 1908. 62 Murray, T. J. The oxygen requirements of biological soil processes. Jour. Bact., 1: 547-614. 1916. 63 See also Miinter, F., and Robson, W. P. tJber den Einflusz der Boden und des Wassergehaltes auf die Stickstoffumsetzungen. Centrbl. Bakt. II, 39: 419-440. 1916; Rahn, O. Bacterial activities in soil as a function of grain size and moisture content. Mich. Agr. Exp. Sta. Bui. 16. 1912; Centrbl. Bakt. II, 35: 429-465. 1912; 38: 484. 1913. 64 Prescott, J. A. A note on the Sheraqi soils of Egypt. Jour. Agr. Sci., 10: 177-181. 1920. INFLUENCE OF ENVIRONMENTAL CONDITIONS 785 tends to improve the physical condition of the soil; it brings about a rapid drying of the surface layer of the soil and, when moistened by rainfall, bacterial activities are stimulated. Prolonged drought brings about similar results in a still more pronounced way.64 Greater num- bers of microorganisms were found in cultivated than in uncultivated soils.65'66 The efficiency of the soil for nitrate production is increased by cultivation and aeration.67 The following quantities of aerobic bacteria (growing on the common plate) were recorded68 in 1 gram of moist soil: Raw peat, undrained 138,500 Drained, but not cultivated 200,300 Freshly cultivated, treated with lime and sand 6,900,400 Cultivated for some time, limed, manured 6,224,500 Same, fallowed 7,801,600 In comparing the bacteria in soils from under corn and under alfalfa, greater numbers of organisms were found69 to be present in the first three feet of corn soil than in the same layer of alfalfa soil; this is prob- ably due to better aeration of the corn soil brought about by the soil treatment. The number of organisms in cultivated soils may be twice as large as in corresponding virgin soils, and higher in wheat land than in alfalfa land.70 Nitrification and nitrogen-fixation were also twice as active in land under cultivation. The beneficial effect of summer fallowing and disking was believed to be due, in part at least, to increased available plant food in cultivated soil, brought about by increased bacterial activities. Fallowing has a favorable influence on nitrate formation and rapidity of decomposition of the organic matter in the soil.71 Caron's results « Caron, 1895 (p. 772). 66 Houston, 1898 (p. 14). 67 Lyon, T. L. Intertillage of crops and formation of nitrates in soils. Jour. Amer. Soc. Agron., 14: 97-109. 1922. 08 Fabricius, O., and von Feilitzen, H. tlber den Gehalt an Bakterien in jung- friiulichem und kultiviertem Hochmoorboden auf dem Versuchsfelde des Schwe- dischen Moorkulturvereins bei Flahult. Centrbl. Bakt. II, 14: 161-168. 1905. 69 Waite, H. H., and Squires, D. H. A comparative study of the bacterial content of soils from fields of corn and alfalfa. Nebr. Agr. Exp. Sta., 24th Ann. Rpt., 160-177. 1911. 70 Greaves, J. E. A study of bacterial activities in virgin and cultivated soils. Centrbl. Bakt. II, 41: 444-459. 1914. 71 Ehrenberg, P. Uber den Stickstoffhaushalt dea Ackerbodens. Fiihlings landw. Ztg., 58: 241-246. 1909. 786 PRINCIPLES OF SOIL MICROBIOLOGY point to a stimulating effect of fallowing upon bacterial numbers, but those of Hiltner and Stormer do not. An increase in the numbers of bacteria followed later by a decrease in numbers, as a result of fallow- ing, has also been reported.72 Several times as much carbon dioxide was found73 in fallowed land than in corresponding land left under grass; however, in soils poor in inorganic matter carbon dioxide may soon become lower in the fallowed soil. Nitrate formation74 and to 3 o . c ■160 5» fallo*/ \ £ Q !40 O /— "" " \ ^3v ftl l u * s I. ti X *< Cr. / -C k 3 / ^ "2 U =1 100 /\ c£ Fallow/ O -C c o to l y~~~ o o : Cr°PPec/ V CQ i. -» -J \ O Q. .10 l\ -C [Cropped, / XN. o L_ /xfe. / '^- " .*; J y^^- / \cl. 5: ■Sff \ / *0 . 20-24 21.8 50.5 326.3 349.0 36.8 43.4 Total 811.8 960.4 1295.3 1098.3 780.9 876.6 Dry weight of crop 1280.8 2756.5 1442.5 1523.6 Ash in crop* 220.0 1080.8 1916.0 840.5 394.0 1048.5 424.2 Organic matter in crop 1099.4 CO2 used to produce organic mat- ter in crop 1747.4 1359.1 1695.4 1783.9 COa used to produce organic mat- ter of seedlings ... 26.9 26.9 69.5 69.5 CO2 fixed by plants during experi- ment 1702.8 1332.2 1295.3 1098.3 1625.9 2406.8 1714.4 Total C02 obtained from soil. . . . 2514.42292.6 2591.0 Average C02 obtained from soil. 2403.6 mgm. 1196.8 mgm. 2498.9 mgm. Average increase over check. . . 100.9 per cent 108.8 per cent * Including also the soil grains which adhered to roots. The growing crop may have a depressive effect upon the nitrate content of the soil (not necessarily nitrate-producing capacity of the 120 Kellerman, K. F., and Wright, R. C. Mutual influence of certain crops in relation to nitrogen. Jour. Amer. Soc. Agr., 6: 204-210. 1914. PLATE XIX 153 154 153. A view of the front or outlet side of the apparatus used to determine the influence of plants upon the oxidation activities in the soil. Twelve barium hydroxide towers were used for recovering carbon dioxide from air removed while soda lime in the long horizontal tube, lying on the table, freed the indrawn air from carbon dioxide. The six central jars contain barley and soybean plants (from Neller). 154. Influence of inoculation upon the growth of soy beans in sterilized water. A, Uninoculated; B, inoculated (from Garman and Didlake). INFLUENCE OF ENVIRONMENTAL CONDITIONS 799 soil).121 It was suggested122 that the lower nitrate formation in cropped land may be due to the adverse effect of the crop upon bacterial activi- ties or to some process of destruction of nitrates, at work in the cropped soil which does not take place in the fallow soil.123 The stimulative effect of the growing crop upon the activities of heterotrophic organ- isms (including decomposition of organic matter) and the injurious effect upon nitrate accumulation explain one another since one process is the cause of the other, the heterotrophic organisms, using the available energy of the fresh organic material of a wide carbon-nitrogen ratio, consume some of the nitrate. The growing crop brings about an increase in the carbon dioxide content of the atmosphere, as indicated by the fact that the carbon dioxide content in a cropped soil is much greater than in a corre- sponding bare soil.124 The excess of carbon dioxide was ascribed to the respiratory activity of the plants rather than to the decomposition of the root particles from the crop growing in the soil. This is in line with the previous observations of Barakov125 that plants produce much greater quantities of carbon dioxide in the soil than do the bac- teria; maximum carbon dioxide production was found to coincide with the maximum life activity of the plant. Leather126 also found greater quantities of carbon dioxide in the neighborhood of roots of crops than in fallow land. Neller127 obtained quantitative measurements of the total carbon dioxide liberated from oxidation processes taking place in the soil during plant growth (fig. 76, no. 153, PI. XIX); much more rapid 121 Voorhees, E. B., Lipman, J. G., and Brown, P. E. Some chemical and bac- teriological effects of liming. N. J. Agr. Exp. Sta. Bui. 210. 1907. 122 Russell, E. J. The nature and amount of fluctuations in nitrate contents of arable soils. Jour. Agr. Sci., 6: 18-57. 1914; also Ibid., 7: L45. 1915. 123 Burd, J. S. Water extracts of soils as criteria of their crop producing power. Jour. Agr. Res., 12: 297-310 (304). 1918. 124 Russell and Appleyard, 1915 (p. 721); Turpin, H. W. The carbon dioxide of the soil air. Cornell Univ. Agr. Exp. Sta. Mem., 32: 319-362. 1920. 126 Barakov, P. The carbon dioxide content of soils during different stages of growth of plants. Zhur. Opit. Agron., 11: 321-342. 1910. 126 Leather, J. W. Soil Gases. Mem. Dept. Agr. India, Chem. Series, 4: 85-132. 1915; see also Bizzell, J. A., and Lyon, T. L. The effect of certain fac- tors on the carbon dioxide content of soil air. Jour. Amer. Soc. Agron., 10: 97-112. 1918. 127 Neller, J. R. The influence of growing plants upon oxidation processes in the soil. Soil Sci., 13: 139-159. 1922. 800 PRINCIPLES OF SOIL MICROBIOLOGY oxidation was found to occur in a soil in which plants were growing than in the corresponding uncropped soil kept under the same condi- tions of moisture, aeration, temperature, etc. The growing roots were found to exert a direct influence upon the decomposition of organic matter in the soil. This will, of course, also bring about a greater liberation of available plant nutrients and thus stimulate further plant growth. A symbiotic relationship between the growing plant and the oxidizing organisms in the soil was, therefore, suggested. Further information on the influence of nature of crop upon the numbers of bacteria and evolution of C02from soil is given in tables91 192-128 93 and 94. m It is of interest to note here that the harmful effect of grass upon the growth of trees so commonly observed has been found129 to be due to the fact that the surface roots of the trees are deprived of combined nitrogen; by producing a soil atmosphere rich in CO2, the grass causes the surface roots to grow down and thus suffer from lack of oxygen; there was no evidence of the formation of a toxin by the grass. Berthelot130 recorded less nitrogen fixed by non-symbiotic organisms in cropped than in uncropped soil. Heinze,131 however, found that fallowing increased the nitrogen-fixing capacity of the soil. Definite information on this problem is difficult to obtain since our methods are not sensitive enough to detect a minute increase in the total nitrogen of the soil. In the case of leguminous plants, which offer a favorable substrate for the growth of the different froms of B. radicicola, the death of the plant and the decomposition of the roots lead to an increase in the numbers of the nodule organisms in the soil. 128 Stoklasa, J. Die modernen Ziele der biochemischen Forschung des Bodens. Chemie d. Zelle u. Gewebe, 12: 22-44. 1924. 129 Howard, A. The effect of grass on trees. Proc. Roy. Soc, 97B: 234-321. 1925. 130 Berthelot, 1S88 (p. 104). 131 Heinze, B. Bodenbakteriologische Untersuchungen. Landw. Jahrb. 39, Erganzungbd., 3: 314-343. 1910. CHAPTER XXX Soil as a Habitat for Microorganisms Causing Plant and Animal Diseases The majority of soil microorganisms carry on in the soil processes which are of prime importance to the growth of higher plants. Some- times the microorganisms compete with higher plants for specific nutri- ents; some of them may also produce substances which are directly injurious to higher plants. In addition to the injury which saprophytic soil microorganisms may cause to plants by carrying on certain physio- logical processes, the soil harbors organisms directly parasitic to plants or animals. Influence of saprophytic soil microorganisms upon plant growth. Microorganisms take part in four soil processes which directly affect the growth of higher plants. (1) They decompose the soil organic matter and liberate the nitrogen and minerals necessary for the growth of higher plants. They also produce considerable quantities of C02, which is essential for the growth of plants. (2) They oxidize and other- wise transform the various minerals introduced into the soil (ammonium salts, sulfur, etc.), or formed from the decomposition of the organic matter (as NH3, H2S, etc.), into forms readily available to plants. (3) They synthesize organic matter from inorganic compounds and thus compete with higher plants for the available nitrogen and minerals. This process may become useful in the absence of a growing crop, since the soluble materials are prevented from being leached out. (4) They reduce, under proper conditions, various oxidized substances like sulfates and nitrates to substances which may be directly toxic to higher plants. The growth of the extensive group of leguminous plants is directly affected by the symbiotic bacteria, so much so that these plants become almost independent of the soil nitrogen and, therefore, of all processes affecting the available nitrogen in the soil. The growth of a large number of trees and other plants depends to a large extent upon the fungi forming mycorrhiza on their roots, as shown elsewhere. What- ever the nature of the phenomenon, whether it is a case of symbiosis or of mutual parasitism, there is no doubt that the fungi favor in some way 801 802 PRINCIPLES OF SOIL MICROBIOLOGY the growth of the plants, both in the case of ectotrophic and endotrophic forms. Microorganisms affect the growth of higher plants not only directly, but also indirectly. The formation of carbon dioxide and various or- ganic acids brings about a greater solubility of the soil minerals, particularly the carbonates and phosphates, as well as to some extent the zeolitic materials. To this we must add, of course, the action of inor- ganic acids, namely nitrous, nitric and sulfuric, which result directly from the activities of microorganisms. The favorable influence of bacteria upon the activities of the roots of plants, by increasing their etching power, may also be referred to here.1 The favorable influence of legumes upon non-leguminous plants has also been noted above.2 When seeds are planted immediately after turning under a green man- ure crop, the seedlings may be injured. As a result of the decomposi- tion of the green manure, numerous fungi develop, some of which are destructive to the seedlings, especially in the case of oil seeds. The rapid evolution of C02 and utilization of oxygen produce conditions unfavorable to oxidation, which is essential for the seeds in the process of germination. However, when seeds are planted two weeks after the addition of the green manure, no serious injury is caused to their ger- mination.3 An attempt was made to explain unproductiveness of soils not by a lack of proper nutrients but by the presence of substances actually injurious to plant growth;4 these substances were presumably formed in the soil partly at least as a result of activities of microorgan- isms. The hypothesis that protozoa are concerned in the destruction of bacteria and, therefore, bring about the formation of "sick" and unpro- ductive soils, as well as other biological theories which attempt to explain this phenomenon, have been discussed above. 1 Fred and Hass, 1919 (p. 647). 2 See also Koch, A. Die Pflanzennahrstoffe des Bodens unter dem Einflusz der Bakterien. Chem. Ztg., 36: 726. 1911; Koch, A. "Uber die Einwirkung des Laub- und Nadelwaldes auf den Boden und die ihn bewohnenden Pflanzen. Centrbl. Bakt. II, 41 : 545-572. 1913; Gibbs, W. M., and Werkman, C. H. Effect of tree products on bacteriological activities in soil. I. Ammonification and nitrification. Soil Sci., 13: 303-322. 1922; Bondorff, A. The use of micro- organisms for the determination of the content in the soil of plant food available for higher plants. Den. Kgl. Verteriner. Z. Lanbohojskoles aarskrift. 1918, 339-362 (Physiol. Abstr., 6: 137). 3 Fred, E. B. Relation of green manures to the failure of certain seedlings. Jour. Agr. Res., 5: 1161-1176. 1916. 4 Schreiner, O., and Reed, H. S. Some factors influencing soil fertility. U. S. Dept. Agr. Bur. of Soils, Bui. 40. 1907. SOIL AS HABITAT FOR PATHOGENIC MICROORGANISMS 803 In other words a soil condition may be brought about, commonly referred to as "sickness," which may not be a result of the activities of microorganisms directly pathogenic to plants, but which is due to certain processes brought about by saprophytic soil microorganisms. Saprophytism and parasitism among soil microorganisms. Parasitism is a form of nutrition whereby an organism obtains its nutrients from the tissues or cells of another living organism. Symbiosis or mutual parasitism takes place when the host plant obtains nutrients from the invading organism. Parasitism is a matter of degree in the case of a number of microorganisms (especially fungi) found in the soil. (1) Some organisms are strictly parasitic and are brought into the soil by the growing plant, by wind or other agencies. They cannot grow in the soil, but remain there alive and capable of causing the disease only for a short time; this is true of most smuts and certain bacteria among plant pathogens; Bad. typhosum is a case of an animal pathogen. (2) Some organisms continue to live in the soil a season or more but, in the absence of the host plant, they soon die out. They may, however, be able to attack a number of hosts and thus become more or less perma- nent in the soil, until all the host plants are completely removed ; this is true of the club root organism, Plasmodiophora brassicae; (3) Some organisms are able to grow saprophytically in the soil for many years and become parasitic when the specific host plant is introduced. In this group there are various species of Fusaria (F. oxysporum, F. radicicola, F. lini, etc.), Act. scabies, certain Rhizoctonia (Rh. solani), and many other plant pathogens. Bac. tetani, Bac. anthracis, Bac. botulinus, numerous gas gangrene types as Bac. welchii, and Act. bovis are instances of organisms capable of causing animal diseases, which may find a more or less permanent habitat in the soil. (4) Finally we find organisms primarily saprophytic in nature and abun- dantly distributed in the soil. They are capable, however, of causing diseases of the growing plant or various storage rots, when conditions become favorable. This group includes Rh. nigricans, certain species of Fusarium, Penicillium, etc.5 In the interaction of plants and microorganisms, various gradations between strict parasitism and strict symbiosis are found, with mutual parasitism as an intermediary phenomenon.6 Mycorrhiza formation 6 See Strong, R. P. The relationship of certain "free-living" and saprophytic microorganisms to disease. Science N. S., 61: 97-107. 1925. 6 Bernard, N. Involution dans la symbiose. Ann. Sci. Nat. Bot. 9 me Ser., 1909, 9; Caullery, M. La symbiose chez les animaux. Bull. Inst. Past., 19: 569-583, 617-627. 1921. 804 PRINCIPLES OF SOIL MICROBIOLOGY by fungi fills in the gap between parasitism and symbiosis. Some of the mycorrhiza are probably more symbiotic; others are often looked upon as parasitic in nature; still others first attack the plant and then are di- gested by the plant juices. In the last instance we have a case of bal- anced parasitism; the root-tubercles of Arbutus unedo are caused by a fungus first ectotrophic, then endotrophic; the digestion of the fungus by the root tubercles confers immunity upon the root system as a whole.7 On the other hand we find cases, like the infection of the thallus of the liverwort Pellia epiphylla with a species of Phoma,8 where the fungus kills the protoplasmic contents of the infected cells. Since the plant can be grown without the fungus, the relationship is largely parasitic. The mycorrhiza of forest trees may be classed between these extremes. The relation of the soil organisms to the plant can thus be arranged schematically as follows: Obligate parasitism - (certain bacteria, smuts, etc.) ■ Balanced parasitism - (certain mycorrhiza) Largely parasitic, — > although plants may derive some benefit (certain mycorrhiza) Saprophytic microorgan- isms (organisms de- composing soil organic matter, etc.) Parasitism combined - with soil saprophy- tism (Fusaria, Rhiz- octonia) Symbiosis (legume - bacteria and legumi- nous plants, Pavetta indica and other plants with gall- forming bacteria, lichen-formations, etc.) The problem of host selection and host specialization has been studied carefully in the case of plant infesting nematodes.9 The chief species of these organisms attack a large number of host plants. However, different populations of a nema species may prefer different host plants; when different host plants are growing in a given soil area the soil nemas will attack the one preferred, leaving the others unattacked even though they are favored host plants of the particular species. If peas or oats are grown on a soil for a number of successive years, Heterodera schachtii becomes so adapted to this host that it will attack this plant in the 7 Rivett, M. F. The root tubercles in Arbutus Unedo. Ann. Bot., 38: 661- 677. 1924. 8Ridler, 1922-1923 (p. 277). 9 Steiner, G. The problem of host selection and host specialization of certain plant infesting nemas and its application in the study of nemic pests. Phyto- pathol., 15: 499-534. 1925. SOIL AS HABITAT FOR PATHOGENIC MICROORGANISMS 805 presence of a number of other plants, which may not be attacked at all. This observation was first made by Liebscher.10 Nemas are capable of locating their host plants at considerable distances, moving even against the water-flow.11 The nemas are even capable of distinguishing closely related plant species. This was explained by the fact that although different species of plant-parasitic nemas feed on a wide range of host plants, a given population of one species will, if possible, always attack first the kind of host plants that its ancestors lived on. If this host plant is not available, host plants of near relationship (taxonomical, chemical) are sought and attacked. If the ancestors of a given population lived on a number of host plants for many generations, the population is polyphagous. If the ancestors of a population lived for many generations on a single species or variety of plant, their de- scendants will always attack that host plant and, only in exceptional cases, other plants, unless the host is absent; such a population is monophagous. The growing plants seem to produce some root secretions which are carried by the soil water and act as stimuli upon the nemas. The latter perceive the stimuli by means of a special sense organ (amphid). The nema then moves towards the points of higher concentration of the stimulating fluid until the host plant is reached. A detailed review of the relation between parasite and host plant, especially in respect to rusts, is given elsewhere.12 Animal and plant diseases caused by bacteria that may be found in the soil. A number of bacteria capable of causing various animal dis- eases have been isolated from the soil, where they find a natural habitat or persist only for longer or shorter periods of time. It is sufficient to mention that Bac. anthracis (Pasteur, Koch), Bac. tetani (Nicolaier, Sanfelice), Bac. chauvoei (Arloing, Pellegrino), Bad. pestis (Jersin), Bad. typhi (Mace) will persist in the soil for many months or even years. Pathogenic bacteria were often found to be actually capable of developing in the soil. Pasteur showed that earthworms can spread 10 Liebscher, G. Beobachtungen iiber das Auftreten eines Nematoden an Erbsen. Jour. Landw. 40: 357-36S. 1892. « Baunacke, 1922 (p. 344). 12 Zimmermann. Sammelreferate iiber die Beziehungen zwischen Parasit und Wirtspflanze. Centrbl. Bakt., II, 65: 311—418. 1925. 13 Prausnik, \V. Die Hygiene des Bodens. Handb. der Hygiene (Rubner usw.), 1: 520-562. 1911 (Int. Mitt. Bodenk., 4: 239); Bail, O., and Breinl, F. Versuche iiber das seitliche Verdringen von Verunreinigungen im Boden. Arch. Hyg., 82: H. I. 1914. 806 PRINCIPLES OF SOIL MICROBIOLOGY anthrax organisms through the soil and even isolated the organism from the intestines of the worms. The soil may thus become a carrier of human disease, as demonstrated in the case of V. cholerae and Bad. typhosum.13 Bac. botulinus is commonly found in the soil; the presence of this organism has been demonstrated not only for soils infested with the organism, but also for various virgin mountain and forest soils.14 Bac. tetani appears to be also universally distributed in the soil, es- pecially in soils fertilized with animal manures and subject to the dust of the streets. Nicolaier15 demonstrated the presence of this organism in over fifty per cent of the soils examined. It was even suggested16 that the organism develops in rotting straw or manure, taking a part in processes of decomposition. The presence of this organism in the soil has also been ascribed to its presence in fecal secretions due to its development in the intestine. The soil harbors various bacteria capable of causing plant diseases; these include Bad. tumefaciens, Bad. solanacearum and Bac. phytoph- thorus.17 It has long been known18 that the mosaic disease of tobacco is caused by a filterable virus and this has since been found true of a large number of similar plant diseases of the type known as infectious chlorosis. The nature of this virus is problematical. It has been held that in some cases the virus may persist in the soil but this remains as a question deserving further critical study. Plant diseases caused by fungi found in the soil. Numerous fungi capable of causing plant diseases find their natural or temporary habitat in the soil. Such fungi have been isolated not only from cultivated soils where they might have been introduced, but also from virgin soils or from soils on which the particular host plant has never been grown before. Fungi, like F. radicicola and Rhizodonia solani known to be parasitic on the Irish potato, were isolated from Idaho soils never cropped 14 Meyer, K. F., and Dubovsky, B. J. The distribution of the spores of B. botulinus in California. Jour. Inf. Dis., 31: 41-55. 1923; also 56-58, 59-94 95-99, 100-109. 15 Nicolaier, A. Beitrage zur Aetiologie des Wundstarrkrampfen. Inaug. Diss. Gottingen. 1885; (Baumgart. Jahresber., 2: 270-272. 1886). 16 Vincent, H. Le bacille du tetanos se multiplie-t-il dans le tube digestif des animaux? Compt. Rend. Soc. Biol., 65: 12-14. 190S. 17 Smith, E. F. An introduction to bacterial diseases of plants. Sanders Co., Philadelphia, 1920. 18 Beijerinck, M. W. Ueber ein Contagiura vivium fluidum als Ursache der Fleckenkrankheit der Tabaksblatter. Centrbl. Bakt., II, 5: 27. 1899. SOIL AS HABITAT FOR PATHOGENIC MICROORGANISMS 807 with potatoes as well as from virgin desert lands.19 Disease-free seed planted on new lands yielded a diseased product. Land previously- planted to alfalfa, clover or grain is better adapted to the production of disease-free potatoes than virgin land. Various species of Phytoph- thora may persist in the soil for considerable periods of time and can stand the low winter temperatures without much injury; they can also resist some desiccation. Ph. infestans can live saprophytically in the soil, growing on old, partially decomposed plants. Their patho- genicity is not diminished by living in the soil.20 A number of these and other organisms are facultative parasites, in other words, they are capable of growing in the soil in the absence of the host plant. The spores of Sclerotinia trifoliorum, for example, were found21 to give rise to a mycelium which is at first saprophytic and then becomes faculta- tively parasitic. The spores appear to germinate on vegetable residues in the soil; the mycelium spreads over the soil at a rate which depends on the environmental conditions. These plant pathogenic fungi com- prise several distinct groups: 1. Various damping off fungi, including Pythium debaryanum, Sclerotinia libertiana, Phoma solani, Sclerotium rolfsii, Rhizoctonia solani (Corticium vagum), species of Fusarium and Colletotrichum.22 2. Root rots as well as other root infections comprising a number of fungi, such as Rh. solani and other species of Rhizoctonia.23 The constant culture of wheat on the same soil will bring about a condition of "wheat sickness." This is not a question of soil infertility or thu formation of toxins detrimental to wheat, but is due to the introduction of fungi which cause various wheat dis- eases, by blighting, rotting and destroying the roots. These fungi are capable of persisting in the soil, living on the decomposing straw. In this group are species of Macrosporium, Alternaria, Helminthosporium, Fusarium and Colletotrichum. 3. Wilts. Fusarium oxysporum has been isolated as a soil saprophyte.24 19 Pratt, O. A. Soil fungi in relation to diseases of the Irish potato in Southern Idaho. Jour. Agr. Res., 13: 73-100. 1918. 20 Bruyn, H. L. G. de. The saprophytic life of Phylophthora in the soil. Medd. Landbou. Wageningen., 24. 1922; The overwintering of Phytophthora in- festans (Mont.) DeBy. Phytopath., 16: 121-146. 1926. 21 Wadham, S. M. Observations on clover rot (Sclerotinia trifoliorum Eriks). New Phytol., 24: 50-56. 1925. 22 Hartley, C. Damping-off in forest nurseries. U. S. Dept. Agr. Bui. 934. 1921. 23 Bolley, H. L. Wheat: soil troubles and seed deterioration. N. D. Agr. Exp. Sta. Bui. 107. 1913; Beckwith, T. D. Root and culm infections of wheat by soil fungi in North Dakota. Phytopathol., 1: 169. 1911. 24 Goss, R. W. Relation of environment and other factors to potato wilt caused by Fusarium oxysporum. Nebraska Agr. Exp. Sta. Res. Bui. 23. 1923. 808 PRINCIPLES OF SOIL MICROBIOLOGY This organism may cause a potato wilt disease. F. lycopersicum, can also live as a saprophyte in the soil, upon the dead stems of the wilted tomato plants and on the soil organic matter; it can live in the soil several years retaining its virulence, even without the host plant.25 The same is true of F. conglutinans and F. lini. F. hyperoxysporwn and F. batatatis, causing the stem-rot of sweet potato, may also be generally disseminated in the soil; also other pathogenic Fusaria.26 Among the other plant pathogenic fungi which can find their habitat in the soil, we may include different species of Rhizoctonia.27 Rh. solani, for example, is abundant in cultivated land, where it lives on dead organic matter in the soil. When a proper host is introduced, the organisms may become active parasites, as in the damping-off of carnation cuttings, stem-rot and potato diseases; they can also attack a variety of weeds. Rh. solani attacks as many as 165 species of plants. Spongospora subterranea causes powdery scab of potatoes. Thielaina basicola causes root rot of tobacco, legumes and many other plants. Synchitrium endobiolicum produces the wart disease of the potato and its spores may re- main in the soil for two to eight years.28 Asterocystis radicis attacks flax. Urophlyctis alfalfae produces swellings on the roots of alfalfa. Aphanomyces laevis causes "Wurzelbrand" on beets. Pythiacystis citrophthora causes the brown rot of the lemon. Ozonium omnivorum produces root rot on cotton and alfalfa. Sclerotium rolfsii can propagate itself by mycelium in the soil, forming sclerotia under unfavorable conditions. Rosellinia necatrix and Melanospora29 must also be mentioned. Cercospora personata is capable of multiplying in the soil saprophyti- cally, preserving its virulence for eleven years.30 Various smuts are often found in the soil and may persist there for long periods of time ; however, the extent to which rusts may persist in the soil has not been established yet.31 Cabbage and tomato sick soils may show as many as forty thousand colonies (on plate) of the parasitic organisms per gram of soil.32 On land showing much root rot of corn, Fusarium moniliforme and a Cepha- losporium have frequently been found. Trichoderma koningi and Tr. lignorum, two of the most common saprophytic soil fungi, are the causes of storage rots of sweet potato; the former is also associated with the 25 Scott, 1924 (p. 811). 26 Harter, L. L., and Field, E. C. The stem-rot of sweet potato (Ipomea batatus). Phytopathol., 4: 279-304. 1914. 27 Peltier, G. L. Parasitic Rhizoctonias in America. 111. Agr. Exp. Sta. Bui. 189. 1916. 28 Schander and Richter. tJber den Nachweis von Dauersporen von Chryso- phlyciis endobiotica Schill. (Kartoffekrebs) in der den Kartoffeln anhaftenden Erde. Centrbl. Bakt., II, 58: 454-461. 1923. 29 Delacroix and Maublanc. Maladies parasitaires des plantes cultivces. Bailliere Ed. 30 Miege, E. Le disinfection du sol. Paris. 1918. 31 Klebahn, cited by Waget, P. Sterilization et disinfection du sol. Rev. Prod. Chim., No. 22. 1920, 655; No. 4. 1921, 115; No. 6, 183. 32 Manns, T. F. Soil bacteriology. Del. Agr. Exp. Sta. Bui. 133, 35-36. 1922. SOIL AS HABITAT FOR PATHOGENIC MICROORGANISMS 809 so-called "ring rot."33 The common soil organism R.hizopus nigricans is the cause of soft rot of sweet potatoes. Plant and animal diseases caused by species of actinomyces. Several plant pathogenic actinomyces have been found in the soil, including Act. scabies, the organism causing the common scab disease of potatoes and sugar beet. On comparing a large number of organisms isolated from scabby potatoes, we can readily recognize that we are dealing here not with one species, but with a whole group which can be readily subdivided into several sub-groups, not only on the basis of physiological charac- teristics, but also on the basis of morphology. As a matter of fact, as many as 30 species of actinomyces have been described,34 which are supposed to be causative agents of potato scab, the type of lesion being influenced by the species. The formation of pox on sweet potatoes may be due, to some extent at least, to an actinomyces35 found in the soil. The causative agents of human and animal actinomycotic diseases are often claimed to be brought about by soil organisms or forms harbored upon plants.36 Klinger37 called attention to the fact that the aerobic actinomyces commonly found on grasses and in straw infusions (also in soil) have never been isolated by him in any actinomycotic case. Only anaerobic forms were obtained from the latter; these develop on most media at temperatures above 30°C, and only seldom were cultures obtained which make a scant growth under aerobic conditions. Mixed infections consisting of anaerobes growing at body temperature together with aerobes are often obtained. We have to do here with species which have adapted themselves to a symbiosis with warm blooded animals, and which have almost nothing in common with aerobic sap- rophytes. However, there is no doubt that some aerobic actino- myces are capable of causing infections of men and animals. The actinomyces capable of causing plant diseases, such as potato 33 Cook, M. T., and Taubenhaus, J. J. Trichoderma koningi the cause of a disease of sweet potatoes. Phytopathol., 1: 184-189. 1911. 34 Wollenweber, H. W. Der Kartoffelschorf. Arb. Forsch. Inst. Kartoffelbau. H. 2. 1920; Millard, W. A., and Burr, S. Ann. Appl. Biol. 13: 580-644. 1926. 35 Taubenhaus, J. J. Pox, or pit (soil rot), of the sweet potatoes. Jour. Agr. Res., 13: 437-450. 1918. 36 Odermatt, W. Aetiologisches zur Aktinomykoseerkrankung. Schweiz. Med. Wochnschr., 50: 26-28. 1920. 37 Klinger, R. Zur Oetiologie der Aktinomykose. Centrbl. Bakt., I, Or., 85: 357-359. 1921. 810 PRINCIPLES OF SOIL MICROBIOLOGY and sugar-beet scab, are true soil organisms. Application of lime, which produces a favorable reaction, and of barnyard manure favor the development of scab. The addition of acid fertilizers (acid phosphate) or fertilizers which make the soil reaction acid (sulfur, ammonium salts) tends to decrease the development of scab, as shown elsewhere (p. 301). According to Millard,38 sufficiently [liberal dressings of green manure added to the soil will inhibit the disease; this is probably due to the temporary increase in soil acidity, as a result of the decomposition of the organic matter by the soil fungi, and to an increase in soil moisture (scab is much more prevalent in dry seasons; actinomyces are much less active in very moist soils) . Sanford39 suggested that the soil reaction may not be the import- ant factor in controlling the development of potato scab in the soil. Moisture was found to be directly or indirectly the main factor, a high moisture content controlling the disease, while abundant scab is formed in dry soils. The development of scab is influenced also by the tem- perature of the soil, the optimum for scab being 22°C.40 Plant and animal diseases caused by invertebrate animals found in the soil. Among the animal pests present in the soil, we find protozoa, nematodes and other worms, crustaceans, myriapods and insects. Among the nematodes, we find Heterodera schachtii causing the disease of mangels, Tylenchus tritici41 of wheat, Heterodera radicicola causing swellings or knots on roots of tomatoes, cucumbers, etc.42 Tylenchus dipsae (syn. devastatrix) causing the root knot on oats, tulip root, clover (one form of clover sickness), and Aphelenchus olesistus causing leaf blight. Relation of soil environment to plant infection. The soil environment, including temperature, moisture, reaction and composition, has an important controlling influence upon all plant parasites found in the soil, whether they are obligate parasitic or can also exist in the soil facultatively. These soil environmental factors may determine not 38 Millard, W. A. Common scab of potatoes. Ann. Appl. Biol., 9: 156-164. 1922; 10: 70-88. 1923. 19 Sanford, G. B. The relation of soil moisture to the development of common scab of potato. Phytopathol., 13: 231-236. 1923. 40 Jones, L. R., McKinney, H. H., and Fellows, H. The influence of soil tem- perature on potato scab. Wis. Agr. Exp. Sta. Res. Bui. 53. 1922. 41 Guenaud, C. Zoologie agricole et Entomologie et Parasitologic agricole. Bailliere Ed. 42 Bessey, E. Root-knot and its control. U. S. Dept. Agr., Bur. PI. Ind. Bui. 217. 1911. SOIL AS HABITAT FOR PATHOGENIC MICROORGANISMS 811 only the geographical distribution of the disease, but also its seasonal severity.43 The case of onion smut illustrates the possible importance of a specific factor of soil environment in determining the possible geographical range of soil parasites. This smut is a persistent soil born fungus, Urocystis cepulae, which is each year distributed throughout the United States on smutty onion sets. It infects the seedling onions only at low temperatures, being totally inhibited at the higher soil temperatures, 28°C. or above. As a result, although established in all the northern onion districts where onion seed is planted in cool soil in spring, it is unknown in the southern states, Texas and Louisiana, where the seed is planted in the autumn when soil temperature is so high as to inhibit infection. Different soil born parasites are affected very differently by environmental factors. Thus high soil temperatures stimulate the development of the Fusarium "yellows" disease of the cabbage and low temperatures inhibit it. By contrast the Thielavia root rot of tobacco is checked in warm soils and is seriously injurious only in cool soils. Jones further points out this seasonal contrast by citing evidence from two successive summers of which the one, 1915, was very cool, with a mid-summer soil temperature averaging about 5°C. lower than that of the succeeding summer. In the cool summer the Thielavia root rot of tobacco was unusually severe whereas the cabbage remained relatively free from disease. The succeeding year with its warm mid-summer period brought disaster to the cabbage crop because of the yellows disease, whereas the tobacco was free from root rot even on old "tobacco sick" soils. In general high soil temperatures favor the vascular Fusarium diseases, including flax wilt, F. lini,u tomato wilt, F. lycopersici,45 and cabbage yellows, F. conglulinans.™ High 43 Jones, L. R. The relation of environment to disease in plants. Amer. Jour. Bot., 11: 601-609. 1924; Soil temperature as a factor in phytopathology. Plant world, 20: 229-237. 1917; Experimental work on the relation of soil tem- perature to disease in plants. Trans. Wis. Acad. Sci., XX: 433. 1922. 44 Tisdale, W. H. Relation of soil temperature to infection of flax by Fusarium lini. Phytopathol., 6: 412. 1916. 45 Scott, I. T. The influence of hydrogen ion concentration on the growth of Fusarium lycopersici and on tomato wilt. Missouri Agr. Exp. Sta. Res. Bui. 64. 1924. 46 Gilman, J. C. Cabbage yellows and the relation of temperature to its occurrence. Ann. Mo. Bot. Card., 3: 25-S4. 1916; Tisdale, W. B. Influence of soil temperature and soil moisture upon the Fusarium disease in cabbage seed- lings. Jour. Agr. Res., 24: 55-86. 1923. 812 PRINCIPLES OF SOIL MICROBIOLOGY temperature (25-27°) also favors Sclerotium rolfsii and certain other plant pathogenic fungi. On the other hand, not only the onion smut as noted earlier, but certain grain smuts are favored by low soil temperatures, e.g., the stink- ing smut of wheat, Tilletia tritici, thrives at 9-12° according to some,47 and even at 5° to 10°, according to others.45 Rhizoctonia solani, which may attack potato and many other plants, is aggressive only at relatively low temperatures.49 Of the two fungi capable of causing tomato wilt or "sleepy disease," V erticillium albo-atrum operates only at a low soil temperature (21-23°) whereas Fusarium wilt as already noted is a high temperature disease (28-29°) .60 Soil moisture also exerts a potent influence on many plant parasites. As previously noted, Sanford found dry soils favorable and wet soils inhibitory to potato scab. More often soil fungi, especially of the "damping off" types, are favored by high moisture; wet soils, even to the saturation point, favor the club root parasite of cabbage, Plasmo- diophora brassicae.51 Spongospora subterranea (powdery scab) develops best in periods of damp, rainy, and cloudy weather and is favored by poor drainage.52 Hungerford recorded that there is a definite relation between the amount of moisture in the soil at seeding time and the amount of bunt or stinking smut which occurs in the resulting crop of wheat; the drier the soil at seeding time the less will be the amount of infection. When the soil is moist and cultivated frequently, the spores of Tille- tia tritici rapidly lose their power of infection. Of course in all such cases more than one variable factor is concerned. In the case of both cabbage club root and potato scab, it has long been known that soil reaction influences their occurrence. The above cited investigations, as well as the fact that high soil temperature, 22° or 47 Hungerford, C. W. The relation of soil moisture and soil temperature to bunt infection in wheat. Phytopathol., 12: 337-352. 1922. 48 Faris, J. A. Factors influencing the infection of wheat by Tilletia tritici and Tilletia laevis. Mycologia, 16: 259-282. 1924. 49 Richards, B. L. Pathogenicity of Corticium vagum on the potato as affected by soil temperature. Jour. Agr. Res., 21: 459-482. 1921. 50 Bewley, W. F. Sleepy disease of the tomato. Ann. Appl. Biol., 9: 116- 134. 1922; Jour. Ministry Agr., Great Britain, 30: 430-457. 1923. 61 Monteith, J. Relation of soil temperature and soil moisture to infection by Plasmodiophora brassicae. Jour. Agr. Res., 28. : 549-561. 1924. 62 Melhus, J. E., Rosenbaum, J., and Schultz, E. S. Sponjospora subterranea and Phorna tuberosa on the Irish potato. Jour. Agr. Res., 7: 213-254. 1916. SOIL AS HABITAT FOR PATHOGENIC MICROORGANISMS 813 above, favors potato scab,53 all point to the conclusion that when these potential parasites are present in the soil the occurrence and severity of the disease must be interpreted as a resultant of several variable en- vironmental factors operating simultaneously. The amount of organic matter present in the soil also influences plant infection, since it offers a source of energy for the saprophytic existence of the organisms. Thielavia basicola cannot infect the host plant in pure sand, but can do so in the presence of organic matter, which allows the mycelium to exist for some time.54 Clay soils are more favorable to infestation than sandy soils.55 The decay of the under- ground portion of the pea plant is largely due to four fungi :56 Fusarium martii var. pisi, Pythium debaryanum, Corticium vagum and a species of Aphanomyces. The Fusarium is not disseminated by the seed, but spreads through the soil and is especially favored by a high content of organic matter. Ozonium omnivorum Shear, the cotton and alfalfa root rot, spreads through the soil radially with a growth similar to fairy rings; it is favored by heavy soils, humid weather and dense cover crops.5758 The influence of soil and manure on plant diseases has been recorded elsewhere,59 as well as the influence of soil nutrients and soil structure upon plant infection.60 Influence of reaction upon the growth of plant pathogenic organisms in the soil. Some plant pathogenic organisms are readily affected by certain hydrogen-ion concentrations of the soil which are not injurious 53 McKinney, H. H. Influence of soil temperature and moisture on infection of wheat seedlings by Helminthosporium, sativum. Jour. Agr. Res., 26: 195-218. 1923. 64 Massee, C. E. A disease of sweet peas, asters, and other plants. Roy. Gard. Kew. Bui. Misc. Inform. No. 1. 1912, 44-52. 56 Johnson, J., and Hardman, R. E. Influence of 'soil environment on the root-rot of tobacco. Jour. Agr. Res., 17: 44-52. 1919. 56 Jones, F. R. Stem and root rot of peas in the United States caused by species of Fusarium. Jour. Agr. Res., 26: 459-476. 1923. 57 Duggar, B. M. The Texas root rot fungus and its conidial stage. Ann. Mo. Bot. Gard., 3: 11-23. 1916. 68 King, C. J. Habits of the cotton root-rot fungus. Jour. Agr. Res., 26: 405- 418. 1923. 69 Ehrenberg, P. Der Einfluss des Bodens und der Dungung auf Pflanzen- krankheiten. Fuhlings Landw. Ztg. 1917, 130-132; 1919, 401-412. 60 Levine, M. Studies on plant cancers. III. The nature of the soil as a determining factor in the health of the beet, Beta vulgaris, and its relation to the size and weight of the crown gall produced by inoculation with Bacterium lume- faciens. Amer. Jour. Bot., 8: 507-525. 1921. 814 PRINCIPLES OF SOIL MICROBIOLOGY to the growth of the host plant, as in the case of potato scab61-62 and wheat scab.63,64 Some organisms have a certain acid minimum or alkali maximum which permit methods of control. Act. scabies and most other actinomyces, for example, not do thrive well at pH less than 4.8; Plasm, brassicae is inhibited by an alkali reaction65 obtained by the addi- tion of lime. Bart, solanacearum causes serious infection in acid soils, but seldom in neutral or alkaline soils.66 F. lycopersici causes minimum infection at pH 6.4 to 7.0 (Scott) ; it has both an acid and an alkaline maximum. Method of control. To combat disease producing organisms, one has to know not only the life history of the pathogen, but frequently, as in the case of nemas, the host history of the organism. In this case crops susceptible to the particular species, but not the particular population, may be employed. The saturation of soil with formaldehyde to prevent spreading of disease has often been practiced. In addition to formaldehyde, various other soil fungicides and volatile antiseptics, like CS2 and toluol, have been frequently employed for the destruction of the plant pathogenic fungi.67,68 CS2 can be used with success against a number of fungi, such as Demalophora necatrix, Rhizoctonia, Synchitrium69 etc. Miege70 obtained the best results with toluene in controlling Sclcrotinia libcrtianat 61 Gillespie, L. J. The growth of the potato scab organism at various hydro- gen-ion concentrations as related to the comparative freedom of acid soils from the potato scab. Phytopathol., 8: 257-269. 1918; Gillespie, L. J., and Hurst, L. A. Hydrogen-ion concentration-soil type-common potato scab. Soil Sci., 6: 219-236. 1918. 62 Waksman, S. A. The influence of soil reaction upon the growth of actino- myces causing potato scab. Soil Sci., 14: 61-79. 1922. 63 Hopkins, R. F. The hydrogen-ion concentration in its relation to wheat scab. Amer. Jour. Bot., 9: 159-179. 1922. 64 Mclnnes, J. The growth of wheat scab organism in relation to hydrogen- ion concentration. Phytopathol., 12: 290-294. 1922. 65 Atkins, W. R. G. Hydrogen-ion concentration and club root. Sci. Proc. Roy. Dubl. Soc. N. S., 16: 369-413. 1922. 66 Arrhenius, O. A factor influencing the growth of tobacco wilt disease. Ark. Bot., 18: No. 1. 1922. 67 Halsted, B. D. Soil fungicides for potato and turnip diseases. N. J. Agr. Exp. Sta., Sp. Bui. S. 1900. 68 Johnson, J. The control of damping off disease in plant beds. Wis. Agr. Exp. Sta. Res. Bui. 31, 29-61. 1914. 69 Gimingham, C. T., and Spinks, G. T. Soil sterilization. Jour. Bat. and West and South Cont. Soc. 14, 126-130. 1920. T0 Miege, 1917 (p. 766). SOIL AS HABITAT FOR PATHOGENIC MICROORGANISMS 815 and Fusarium lycopersici.71 When soil is steamed, the fungi are readily- destroyed; but once a parasitic organism like Pythium debaryanum is introduced, it will readily develop in the treated soil and may even cause a larger amount of infection. This parasitic activity can be decreased by inoculating the treated soil with various saprophytic fungi. Treat- ment of soil with a disinfecting agent followed by inoculation with sapro- phytic fungi may prove to be most efficient in increasing the value of the treatment.72 In genera] the methods of control consist, on the one hand, of crop rotation, seed treatment and selection of resistant varieties, on the other hand, of soil sterilization by heat or by chemicals, change in soil reaction, or other chemical treatment. For the sterilization of greenhouse soil, the following temperatures are required:73 INFECTING ORGANISM Nematodes Pythium Rhizoclonia Sclerolinia Septoria lycopersici spores Anthracnose beans, spores. . . Anthracnose beans, mycelium Corn root rot73a TEMPERATURE OP CONTROL 18 hours Few minutes °C. °C. 40 60 60 80 SO 55 48 65 65 Further information on the steam disinfection of soil is given elsewhere.74 71 See Foex, E. Protection of seeds and young plants against diseases by soil sterilization. Jour. Soc. Natl. Horticult., France, 4, Ser. 22: 242-254. 1921. 72 Hartley, C. Damping off in forest nurseries. U. S. Dept. Agr. Prof. Paper Bui. 934. 1921. 73 Brown, H. D., Baldwin, I. L., and Conner, S. D. Greenhouse soil steriliza- tion. Purdue Univ. Agr. Exp. Sta. Bui. 266. 1922. 73a Valleau, W. D., Karraker, P. E., and Johnson, E. M. Corn root rot— a soil-borne disease. Jour. Agr. Res., 33: 453-476. 1926. 74 Hunt, N. R., O'Donnell, F. G., and Marshall, R. P. Steam and chemical soil disinfection with special reference to potato wart. Jour. Agr. Res., 31: 301-363. 1925; Beinhart, E. G. Steam sterilization of seed-beds for tobacco and other crops. U. S. Dept. Agr. Farm. Bui. 996. 1918; Byers, L. P., and Gilbert, W. W. Soil disinfection with hot water to control the root-knot nematode and parasitic soil fungi. II. S. Dept. Agr. Bui. 818. 1920. 816 PRINCIPLES OF SOIL MICROBIOLOGY For the control of potato scab, sulfur is used with success. Care must be exercised, however, in using the proper amounts, so as not to make the soil too acid. Sweet potato scurf and pox of sweet potatoes can also be checked by the application of sulfur. Organisms like Pythium (damping off of seedlings) and Spongospora subterranea are in- tolerant to acid, while the myxomycete Plasm odiophora brassicae (finger and toe disease) is kept in check by addition of lime. CHAPTER XXXI Soil Inoculation Beneficial and injurious microbiological processes in the soil. The growth of higher cultivated plants is usually taken as a criterion in determining whether a certain microorganism or a certain microbio- logical process is beneficial or injurious. But a careful study of these processes and the organisms concerned can hardly justify such a strict division in all cases. Some, like the nitrogen-fixing, the nitrifying and the sulfur oxidizing bacteria, the various organisms decomposing cellu- loses and proteins, are no doubt beneficial to the growth of higher plants. Plant pathogenic fungi, like the various Fusaria, Rhizoctonia, Pythium, etc., may, no doubt, become injurious, when environmental conditions are favorable. We may even call denitrifying and sulfur reducing bacteria harmful, although their action is indirect and depends entirely upon the soil conditions. However, as pointed out above, some organisms may carry on proc- esses in the soil which are both injurious and beneficial to the growth of higher plants; a certain process may be beneficial at one time and in- jurious at another. The question becomes then merely relative. A fungus, like Trichoderma or Asp. fumigatus, decomposes cellulose rapidly and is no doubt beneficial, but it also synthesizes considerable protoplasm and stores away large amounts of nitrogen and it becomes, therefore, temporarily injurious to higher plants. When a protein is decomposed by fungi, smaller amounts of ammonia are liberated than when it is decomposed by bacteria. When the protein is added, an acid soil favors the development of fungi, while a neutral or alkaline soil favor the development of bacteria. Facts like these as well as the conditions which may favor the development of the different groups of soil microorganisms and the presence or absence in the soil of the particular organism in ques- tion, must be known before we can make use of the principle of soil inoculation. This consists not merely in the introduction of useful organisms which may be lacking, but also in making soil conditions fa- vorable for the biological processes useful to the growth of higher plants. 817 818 PRINCIPLES OF SOIL MICROBIOLOGY Introduction of certain useful microorganisms into the soil. Among the useful microorganisms, which we may want to introduce into the soil are: (1) organisms, which carry on important processes beneficial to a specific plant or to plant growth as a whole ; (2) strains more vigorous than those already found in the soil; (3) organisms which destroy or injure the development of organisms directly injurious to higher plants. It is not merely sufficient to introduce the beneficial organisms, but the soil conditions should be made favorable for the development of these organisms. So far as our present knowledge of the soil biological processes is concerned, the microorganisms which may be lacking in the soil, or whose activities in the soil are to be stimulated are as follows: (1) symbiotic and non-symbiotic nitrogen fixing bacteria; (2) sulfur-oxidiz- ing bacteria; (3) nitrifying bacteria, and (4) microorganisms capable of vigorous decomposition of the soil organic matter. The favorable influence of small quantities of manure added to the soil is ascribed by some investigators to the inoculating power of various bacteria pres- ent in the manure, these organisms presumably decomposing the soil organic matter more vigorously than the native flora. However, this favorable action is probably due not to the organisms, but rather to the presence in the manure of certain inorganic substances, such as available nitrogen and phosphates, which stimulate the growth of higher plants or of certain soil organisms. Most of the bacteria capable of decomposing starches and celluloses in the intestinal tract of animals are specific inhabitants of the tract and are not found in great abundance outside of the animal.1 Among the organisms which may directly destroy or otherwise eliminate the activities of soil or- ganisms directly injurious to higher plants, the following may be mentioned: (1) the predacious nematodes, like Mononchus, which destroy the injurious nematodes, like Heterodera or Tylenchus; (2) saprophytic fungi which may act as a check to the development of pathogenic fungi. Among the soil conditions, which may have to be modified, so as to stimulate the development of organisms whose activities in the soil are favorable to the growth of higher plants, the following may be included: (1) a proper carbon-nitrogen ratio of the soil; (2) a favorable soil reac- tion and presence of sufficient bases; (3) presence of inorganic nutri- 1 Henneberg, W. Untersuchungen fiber die Darmflora des Menschen mit besonderer Beriicksichtigung der jodophilen Bakterien im Menschen- und Tier- darm sowie im Kompostd linger. Centrbl. Bakt., II, 55: 242-281. 1922. SOIL INOCULATION 819 ents, especially phosphates and potassium salts; (4) soil moisture and aeration. In addition to these, certain specific treatments may prove to be useful for the control of specific microorganisms, as presence of available carbohydrates for the stimulation of non-symbiotic nitrogen fixation; a certain reaction, for the control of specific plant diseases; soil sterilization, for the improvement of the physical and chemical conditions of the soil and the elimination of certain injurious microor- ganisms. When the soil is to be inoculated with certain microorganisms, one may choose between the use of (1) soil, in which the desired crop has been grown successfully; (2) pure cultures, or artificially prepared mixed cultures; (3) specific vigorous strains, which are more active than those already present in the soil. Legume inoculation.2 The first inoculation test on record is the experiment carried out in 1887 at the Bremen Experiment Station. It was found that a good stand of clover was obtained on newly drained heath or swamp soils inoculated with old soil in which clover was grown, provided the swamp soil was limed properly. In comparing the use of soil, in which the specific legume was grown, with pure cultures, for inoculation purposes, it is often found that soil gives better results. Hiltner3 suggested that this may be due to the fact that, when the seed germinates, certain toxic substances pass out from the embryo which seem to be toxic to the bacteria ; this danger can be obviated by inoculat- ing the soil directly rather than the seed. Atwater and Woods4 were among the first in America to show the favorable effect of inoculation on the growth and nitrogen content of alfalfa and peas. The gain in nitrogen was proportional to the number of nodules on the roots. Warington3 soon demonstrated that the growth of properly inoculated legumes resulted in an increase in the nitrogen content of the soil; part of a wheat field seeded to clover con- tained 0.156 per cent nitrogen and only 0.142 per cent was found in 2 A. detailed review of the earlier literature on legume inoculation is given by K. F. Kellerman. The present status of soil inoculation. Centrbl. Bakt., II, 34: 42-50. 1912. 3 Hiltner, L. tlber die Impfung der Leguminosen mit Reinkulturen. Deut. landw. Presse, 29: 15, 119. 1902. 4 Atwater, W. O., and Woods, C. D. The acquisition of atmospheric nitrogen by plants. Storrs Agr. Exp. Sta. Rpt. 1889-90, 11-51. 6 Warington, R. The circumstances which determine the rise and fall of the nitrogenous matter in the soil. U. S. Dept. of Agr. Off. Exp. Sta. Bui. 8, 22-41. 1891. 820 PRINCIPLES OF SOIL MICROBIOLOGY the part of a field seeded to barley. The nitrogen content of inoculated plants was found to be much higher than that of uninoculated plants, Nobbe and Richter6 reporting 4.29 per cent of nitrogen for the first as against 1.85 per cent for the second. These results were soon confirmed by a number of workers in Europe and in America, as shown later. In 1896 Nobbe7 suggested the use of pure cultures of B. radicicola for the inoculation of leguminous plants. A product placed on the market consisted at first of B. radicicola grown on gelatin. However, gelatin was found to be an unsuitable medium for the growth of this organism. It was then replaced by a liquid medium, namely a 2 per cent peptone solution or skimmed milk,8 and later by agar media. In 1902 the use of cotton cultures was introduced.9 Cotton was placed in a liquid culture of bacteria, then dried and placed in packages. This was accompanied by two packages of nutrient substances, the first con- taining sugar, K2HP04 and MgS04, and the second ammonium phos- phate. Before using, the cotton was placed in boiled water and the contents of the first package were added. After 24 hours at 20°, the contents of the second package were added and the culture allowed to incubate ; this culture was then used for moistening the seed or for in- oculation of a small amount of soil which was then spread over the field. Usually mixtures of the various legume bacteria were employed. The re- sult proved to be unsatisfactory, due to the fact that the bacteria could not withstand the process of drying on cotton.10 Various other preparations, consisting of liquid, semiliquid or solid cultures were soon introduced. However, the early beneficial results 6 Nobbe, F., and Richter, L. tlber den Einfluss des im Kulturboden vorhand- enen assimilierbaren Stickstoffs und die Aktion der Knollchenbakterien. Landw. Vers. Sta., 59: 167-174. 1903. 7 Nobbe, F. Einige neue Beobachtungen, betreffend die Bodenimpfung mit rein kultivierten Knollchenbakterien fur die Leguminosenkultur. Bot. Centrbl., 68: 171-173. 1896. 8 Hiltner, L., and Stormer, K. Arb. K. Gesundsamt. Biol. Abt. 3. 1903, 151. 9 Moore, G. T. Bacteria and the nitrogen problem. U. S. Dept. Agr. Year- book 1902-1903, 333-342; Soil inoculation for legumes; with reports upon success- ful use of artificial cultures by practical farmers. U. S. Dept. Agr., Bur. PI. Ind. Bui. 71. 1905. 10 Simon, J. Die Wiederstandsf;'ihigkeit der Wurzelbakterien der Legumino- sen und ihre Bedeutung fur die Bodenimpfung. Jahresb. ver. angew. Bot. 1907. SOIL INOCULATION 821 secured with these pure cultures by Nobbe and others11 were not con- firmed by investigators both in America12 and in Europe,13 who found soil to be superior to artificial cultures for inoculation purpose. The in- oculating value of some of these earlier preparations has been com- pared critically with the inoculating value of soil on which the particular legumes were grown.14 Use of soil for inoculation of legumes. Nodule production on plants is a result of chance contact; a large number of nodule-forming bac- teria must, therefore, be present in the soil so that maximum nodule formation may take place. After the first experiments on the inocula- tion of legumes, it was found that certain crops, like clover, peas and beans, did not benefit from inoculation; others, like alfalfa and soybeans, could not be grown successfully without inoculation of the soil with some soil in which these crops had been grown previously. It became a common practice to top dress fresh soil with some old soil for these crops. In most cases 300 to 500 pounds of soil, taken from the upper 6 inches of a field where the particular legume had been grown successfully, was spread over each acre of soil and disked or harrowed in before the planting of the seed. It was found15 that soils once inoculated for soy- beans and red clover did not need to be reinoculated when these crops were again grown in the four-year rotation. Dry soil stored for thirty months was as good for purposes of inoculation as fresh soil from the field. Further studies16 have shown that there is a considerable improve- ment in the growth of peas in an acid silt loam, in which peas had grown eleven years previously, as a result of inoculation with artificial cultures. An acid soil may lead to a disappearance of certain nodule bacteria, the destruction of the bacteria running parallel with increasing acidity. The nodule bacteria survived for fifteen years in soils which were limed, 11 Edwards, S. F., and Barlow, B. Legume bacteria. Ont. Dept. Agr. Bui. 164. 1908. 12 Stevens, F. L., and Temple, J. C. The efficiency of pure culture inocula- tion for legumes. N. C. Agr. Exp. Sta. 30th Ann. Rept., 48-57. 1908. 13 Barthel, C. Culture experiments with bacterial inoculations of lupine and alfalfa. Meddel. Centralanst. Forsoksv. Jordbruk., 95. 1914; K. Landbr. Akad. Handb. o. tdskr., 53: 251-280. 1914. 14 Feilitzen, H. V. Nitro-Bacterine, Nitragin oder Impferde? Centrbl. Bakt. II, 23: 374-378. 1909. 16 Albrecht, W. A. Viable legume bacteria in sun dried soil. Jour. Amer. Soc. Agron., 14: 49-51. 1922; Mo. Agr. Exp. Sta. Bui. 197. 1922. 14 Whiting, A. L. The relation of inoculation to quality and yield of peas. Jour. Amer. Soc. Agr., 17: 474-487. 1925. 822 PRINCIPLES OF SOIL MICROBIOLOGY but corresponding unlimed soils showed a deficiency of bacteria even when the host plant had been grown a year previously. Artificial inoculation of such a soil was found17 to lead to a considerable increase in nodule formation. The use of large quantities of soil for purposes of inoculation involves great expense and trouble in transportation and handling, aside from the introduction, with the old soil, of weed seeds and injurious microorgan- isms, such as the fungi causing various wilts and nematodes. This led again to the use of pure cultures. More reliable cultures are now produced, both on artificial media and in sterile soils, as a result of the increased knowledge on the cultivation of the organisms.18 Commercial cultures and their preparation. The commercial prepara- tions of nodule bacteria commonly found on the market are in the form of liquid, agar, or soil and peat cultures. The historical process of development of the artificial culture of these bacteria is as follows: Gelatin —> cotton — » liquid — * agar — » organic — * inorganic material.10 Two media are used at the United States Department of Agriculture, for the preparation of the legume cultures. One is a soil extract medium, made from 10 kgm. of field soil, 40 grams CaO and 100 liters ofjtap water. Ten grams of cane sugar and 0.5 gram K2HP04 are added for each liter of the extract. The reaction is adjusted to slight acidity to prevent the precipitation of the phosphate. The other medium is a modification of Ashby's medium for aerobic nitrogen assimilat- ing organisms: Cane sugar 2000 grams NaCl 20 .0 grams K2HP04 20.0 grams Calcium carbonate . . . 100 grams MgS04-7H20 20.0 grams Tap water 100 grams CaS04-2H20 10 0 grams The organism is grown in square bottles containing about 200 cc. of medium, which is the quantity used for one bushel of seed. The cultures are not kept for more than a month and their efficiency is tested by inoculating plants grown in sand cultures. 17 Wilson, J. K. Effect on nodulation of supplementing the legume bacteria of the soil with artificial cultures. Jour. Amer. Soc. Agron., 18: 280-294. 1926. 18 The movement of legume bacteria in the soil is discussed by Frazier, W. C, and Fred, E. B. Movement of legume bacteria in soil. Soil Sci., 14: 29-35. 1922. The application of inoculated soil to legume seed by Amy, A. C, and Mc- Ginnis, F. W. Method of applying inoculated soil to the seed of leguminous crops. Jour. Amer. Soc. Agron., 13: 289-303. 1921. A comparative study between the inoculating power of artificial cultures with inoculated soil has been made by v. Feilitzen, 1909 (p. 821) and Teisler, E. Azotogen, Nitragin oder Naturimp- ferde? Centrbl. Bakt. II, 34: 50-56. 1912; also Ktihn, A. Ibid., 30: 548. 1911. 19 Rural New Yorker, April 20, 1915. SOIL INOCULATION 823 As an agar medium, the following 20 may be employed: Mannite 10.0 grams CaC03 1.0 gram K2HP04 0.5 gram Distilled water 900 cc. NaCl 0.2 gram Agar 15.0 grams MgS04-7H20 0.2 gram Reaction pH G.8 CaS04-2H20 0.1 gram Sterile yeast water .. . 100 cc. The liquefied agar is allowed to solidify in the form of slants on the broad side of the flat square bottles; the solidified agar is then inoculated with a few drops of a vigorous liquid culture or a suspension of a solid culture in distilled water. The bottles are incubated at 28°. Each culture is sufficient for the inoculation of one acre. For the preparation of large quantities of medium, the following method may be employed:21 175 grams of agar are dissolved in 3000 cc. of water, by placing it in the autoclave at 10 to 15 pounds pressure; 2.25 pounds hard wood ashes are boiled in 1000 cc. of water and filtered; 0.5 gram KH2P04, 0.5 gram MgS04, 0.5 gram NaCl, 0.25 gram CaS04. 2H20 and 6.25 grams CaC03 are placed in 1000 cc. of hot water. The three solutions are mixed and 87.5 grams saccharose and 12.5 grams mannite are added. The medium is placed in 1.5 ounce bottles and sterilized at 10 pounds. The bottles are then inoculated with 2 cc. of a culture of the desired organism, incu- bated seven days, and then distributed. The use of good fertile soil, which has been previously sterilized, for the cultivation of Bad. radicicola was found22 to give very good results both for the propagation of the organism and as a culture for distribu- tion. The growth of the nodule-organism on nitrogen-rich media does not destroy the infecting power of the organism.23 A sandy soil to which some decomposed organic matter is added is air dried, then placed in ten pots and sterilized for two hours at 100°C. Water is then added to the pots to bring the soil to optimum moisture and the soil inocu- lated with a suspension of the culture in Ashby's solution. A few cc. are used for inoculating each pot. Soil cultures last much longer than 20 Fred, E. B., Peterson, W. H., and Davenport, A. Fermentation charac- teristics of certain pentose-destroying bacteria. Jour. Biol. Chem., 42: 175. 1920. 21 Harrison, F. C. Nitro-cultures and their commercial application. Trans. Roy. Soc. Canada, Ser. 3, 9: 219. 1915. 22 Simon, J. Mitt. Okon. Gesell. Sachsen., 13: 1-27. 1908; Kuhn, A. Azoto- gen, Nitragin und Impferde. Centrbl. Bakt. II, 30: 54S-552. 1911; Temple, J. C. Studies of Bacillus radicicola. Ga. Agr. Exp. Bui. 120. 1916. 23 Prucha, M. ,T. Physiological studies of Bacillus radicicola of Canada field pea. Cornell Univ. Agr. Exp. Sta., Mem. 5. 1915. 824 PRINCIPLES OF SOIL MICROBIOLOGY agar cultures. Each pot weighing about 680 grams can be used to inoculate one bushel of seed or one acre of land (Wilson) . Peat cultures of the nodule bacteria are also being used quite extensively.24 To test cultures of legume bacteria for the abundance and vitality of the particular organism, two methods are used: 1. The culture is diluted to 1: 10,000 or 1: 100,000, then 1 cc. of the final dilution is added to 9 cc. of agar medium (Temple used a medium consisting of 10 grams sucrose, 1 gram KH2P04, 15 grams agar, 1000 cc. tap water, pH = about 6.5 to 7.0) and plates prepared. These are incubated for 6 to 7 days at 25°C. ; the number of viable bacteria, as well as abundance of contaminations, can then be determined. It is fre- quently difficult to differentiate on the plate between Radiobacter and Bad. radicicola25 and it is also impossible to learn which particular groups of the organism are present in the soil. 2. To identify the strain, direct inoculation tests must be employed. Either bottles with sterilized sand containing 20 per cent moisture or tall cylinders containing sterile 0.75 per cent agar media must be used. The seeds are sterilized by treatment for fifteen minutes with 0.1 per cent corrosive sublimate, 1 per cent formaldehyde or 5 per cent hypo- chlorite, then rinsed in sterile water and germinated on moist filter paper in a moist chamber. The sprouted seeds are then removed with sterile forceps, dipped in the inoculating material and dropped upon the substrate, in which they are expected to grow. Controls should always be employed. The formation of nodules is an index of the activ- ity of the culture. The purity of culture can also be tested on sterilized potato, upon which nodule bacteria do not grow (some give some growth in 4 weeks), while common contaminations and Bad. radiobacter produce a growth in 5 to 7 days at 28°C.26 Bad. radicicola multiplies very rapidly in sterile soil and the develop- ment of the organism is greatly diminished when the sterile soil is mixed with non-sterile soil, indicating that normal soil is not a very favorable medium for their development.27 Biological types of legume bacteria. It has been pointed above that, 24 Earp-Thomas, G. H. Peat as a carrier for bacteria. Jour. Amer. Peat Soc, 16: 18-23. 1922. 25 Joshi, N. V. Studies on the root nodule organism of the leguminous plants. India Dept. Agr. Mem. Bact. Ser., 1: 219-276. 1920. 2«Lohnis and Hansen, 1921 (p. 126). 27 Duggar, B. M., and Prucha, M. J. The behavior of Pseudomonas radicicola in the soil. Centrbl. Bakt. II, 34: 67. 1912. SOIL INOCULATION 825 although so far all the bacteria capable of inoculating leguminous plants are classified under one species Bad. radicicola or Rhiz. legumino- sarum, different morphological, serological, and cultural differences are found between the forms inoculating different plants. Morphologically they are differentiated by the formation of peritrichous or monotrichous flagellation. Serologically and culturally they are differentiated into a number of groups (3 to 11), the different representatives of each group being capable of cross-inoculation. However, even one type of plant may be inoculated by strains of the organism which possess certain dis- tinct differences. It was found28 that (1) different strains of bacteria used in inoculating soybeans differ in their nitrogen-fixing efficiency; (2) different strains of bacteria used for soybean inoculation differ in their power of producing nodules on the roots of the plants, as shown by actual count both as to number and size of nodules; (3) different varieties of beans differ in their relative "susceptibility" of inoculation; (4) the efficiency of nitro- gen fixation varies with the soil composition and reaction. There is no difference in the morphology of the strains, but physiologically they may be different. This raises anew the question of the value of inocula- tion of soil, already inoculated, with vigorous strains of the organism. Importance of legume inoculation. The effect of inoculation upon the growth of legumes depends to a large extent upon the physical and chemical soil conditions, such as aeration, temperature, moisture, soil composition, reaction, etc. The effect of legume inoculation was found to consist in increasing the percentage of nitrogen in the tops and roots of the plants and the percentage of ash (excluding phosphorus) in the tops.29 Inoculation alone increased30 the yield of clover and alfalfa on a Colby silt loam 15.6 per cent; lime and inoculation gave an increase in yield of 49.7 per cent and in nitrogen content of 52.3 per cent. The addition of phosphorus and potassium to this soil did not give any large increase in yield. However, in the case of a poor soil, inoculation and lime, as well as applications of phosphorus and potassium, gave marked increases in crop yield; inoculation alone nearly doubled the crop yield, while 2« Wright, 1925 (p. 127). 29 Amy, A. C, and Thatcher, R. W. The effect of different methods of in- oculation on the yield and protein content of alfalfa and sweet clover. Jour. Amer. Soc. Agron., 7: 172-185. 1915; 9: 127-137. 1917. 30 Graul, E. B., and Fred, E. B. The value of lime and inoculation for alfalfa and clover on acid soils. Wis. Agr. Exp. Sta., Res. Bui. 54. 1922. 826 PRINCIPLES OF SOIL MICROBIOLOGY CaC03, in addition to inoculation, brought about an increase in yield of 182.8 per cent. Inoculation usually increased the percentage of nitro- gen in the roots. Alfalfa showed an average gain of 87.5 pounds of nitrogen on a poor soil and only 41.3 pounds on a rich soil; soybeans properly inoculated fixed about 108 pounds of nitrogen in an acid soil and about 129 pounds when half enough lime needed to neutralize the soil acidity was added. The use of pure cultures affords a quick and easy method for intro- ducing the bacteria which enable the leguminous plants to obtain nitro- gen from the atmosphere. Fresh inoculation of soil with specific nodule bacteria may be of direct benefit to the crop, even if the same plant has been grown previously.31 This is due to the fact that the organisms present in the soil itself may not be as vigorous as freshly introduced cultures and the small expense involved by fresh inoculation may be fully compensated by the more vigorous growth of the plants. Nobbe and Richter found that in some cases 93 to 96 per cent of the nitrogen in vetch was obtained from the atmosphere. The addition of available nitrogen to the soil brought about a decrease in the amount of nitrogen fixed. A fixation of 92 per cent of nitrogen in the alfalfa plants as a result of inoculation was recorded.32 Others 33 obtained a fixation of 15 pounds of nitrogen for alfalfa with soil as an inoculum and 35 pounds when a commercial culture was used for inoculation. In cylinder experiments with various legumes turned under as green manures, in a rotation of corn, potatoes, oats and rye, a gain of 54 pounds of nitrogen annually over a period of seven years, as a result of inoculation, was reported.34 Clover was found to contain at maturity an average of 27 per cent of its nitrogen in the roots; 46 per cent of the total nitrogen of alfalfa was also found in the roots.35 The nitrogen content of clover and especially of alfalfa inoculated with the proper bacteria is greatly increased as a result of inoculation. 31 Fred, E. B., and Bryan, O. C. The effect of nodule bacteria on the yield and nitrogen content of canning peas. Soil Sci., 14: 413-415. 1922. 32 Alway, F. J., and Pinckney, R. M. The nitrogen content of inoculated and uninoculated alfalfa plants. Neb. Agr. Exp. Sta. 25th Ann. Rpt. 1912, 5G. 33 Lipman, J. G. Tests of commercial cultures for soil inoculation. N. J. Agr. Exp. Sta. Bui. 227. 1910. 34 Lipman, J. G., and Blair, A. W. The yield and nitrogen content of soy- beans as affected by inoculation. Soil Sci., 1: 579. 1916. 36 Brown, P. E., and Stalling, J. H. Inoculated legumes as nitrogenous fertilizers. Soil Sci., 12: 365-307. 1921. SOIL INOCULATION 827 In some soils the increase was 171.2 per cent greater than that of the untreated control. On adding 2.5 tons of CaC03 per acre in addition to inoculation, the increased crop yield was 310.7 per cent more than the control. Inoculation also increased the nitrogen percentage in the roots and vines.36 In case of soybeans an average increase of 100 Fig. 77. Influence of inoculation and liming upon the growth and nitrogen content of alfalfa: checked columns denote uninoculated and dark columns inoculated soil (from Fred and Graul). pounds of nitrogen per acre from inoculation, in pot experiments, and 24 pounds in field experiments is recorded. Inoculation increased the 36 Graul and Fred, 1922 (p. 825) ; Fred, E. B. The fixation of atmospheric nitro- gen by inoculated soybeans. Soil Sci., 11: 469^177. 1921; Whiting, A. L., Fred, E. B., and Stevens, J. W. Inoculation increases yield and quality of peas for canning. Wis. Agr. Exp. Sta Bui. 372. 1925. 828 PRINCIPLES OF SOIL MICROBIOLOGY yield of soybeans 1787 pounds per acre, or more than threefold; it also resulted in a net gain of nitrogen of 57 pounds per acre, 87 per cent of which was in the tops. The residue left after the crop has been removed, also benefits the succeeding crop. An average increase in the total nitrogen content of the crop, as a result of inoculation, is given as 122 pounds per acre for American soil, and 200 pounds per acre for German soil. Inoculation of non-leguminous plants with nodule bacteria. Various attempts have been made to inoculate nodule bacteria upon non-legu- minous plants, with variable success. Burrill and Hansen,37 basing their conclusions on their own observations and reports of other inves- tigators, reported the results to be absolutely negative. Blunck,38 however, reported positive results. He grew the organisms first on a synthetic medium, then added to the medium an extract of the roots of the non-leguminous plant, then grew the organism on the sterile dead root of the plant, and finally on the living root. By this process of gradual adaptation, Blunck claims to have obtained positive results. However, in view of the fact that these results have not yet been con- firmed, nor has Blunck himself brought further evidence to substantiate his hypothesis, we must consider the results as doubtful. Various other claims to discoveries of cultures of symbiotic nitrogen-fixing bacteria adapted to non-leguminous plants are usually found to be worthless on careful study. As far as our present information is concerned, non- legumes cannot yet be inoculated with beneficial results.39 Inoculation of soil with non-symbiotic nitrogen fixing bacteria. It has long been known that a soil is capable of moderating in some way the losses of nitrogen due to removal in crop, drainage, etc. From various practical observations, it has been established that one hectare of soil of Central Europe is capable of fixing between 10 to 60 kilograms of nitrogen per year, independent of the leguminous plants.40 The most abundant and most active of the non-symbiotic nitrogen fixing organ- isms are the species of Azotobacter and Bac. amylobacter. As pointed out elsewhere, the most important factors influencing the activity of 37 Burrill and Hansen, 1917 (p. 126). 38 Blunck, G. Die Anpassung der Knollchenbakterien an Nichtleguminosen. Centrbl. Bakt. II, 51: 87-90. 1920. 39 Kordes, H. Kritische Besprechung der Frage "Impfung der Nichtlegu- minosen." Ztschr. Pflanzenenahr. u. Dung., 4B: 382-394. 1925. 40 Omeliansky, V. L. On the application of non-symbiotic nitrogen-fixing bacteria for soil fertilization. Russian Jour. Microb., 2: 125-139. 1915. SOIL INOCULATION 829 the nitrogen-fixing bacteria in the soil are (1) the degree of activity of the specific organisms; (2) amount of available carbohydrates and other carbon compounds in the soil, which could serve as source of energy for nitrogen-fixation; (3) presence of mineral substances, especially calcium, phosphates, potassium, iron, etc. ; (4) proper soil aeration favoring the activity of aerobic forms and not injuring the development of anaerobes (together with the aerobes); (5) presence of sufficient water; (6) proper soil temperature; and (7) favorable soil reaction, etc. These factors have been considered in detail elsewhere. It is sufficient to emphasize here that, in addition to the presence of vigorous nitrogen fixing bacteria, the soil must be in a proper physical and chemical condition, before nitrogen-fixation will take place. If soil conditions are made favor- able, the nitrogen fixing bacteria will develop rapidly, since they are present in sufficient abundance in all soils. In acid soils, no infection with Azotobacter is apt to occur soon after the reaction is cor- rected.41 It is also important to emphasize that a sufficient amount of energy is required for nitrogen fixation to take place, i.e., for every pound of nitrogen fixed by the non-symbiotic bacteria, about 100 pounds of available carbohydrate or other carbon compounds are consumed. However, when natural organic substances, such as straw and various plant residues, green manure, etc. are added to the soil, they are first of all attacked by the numerous saprophytic soil microorganisms, especially the fungi, which break them down rapidly with the libera- tion of carbon dioxide as the final product. In assimilating the available carbon compounds, the fungi and other microorganisms require available nitrogen to build up their body proteins, about 2 to 5 parts of nitrogen for every 100 parts of carbon available. This nitrogen is obtained from the available nitrogen in the soil, to the detriment of the growing plants, unless it is also introduced into the soil, as in stable manure. The fungi and other saprophytic organisms will stop growing when the available nitrogen is exhausted. It is then when the nitrogen-fixing bacteria become active. In the presence of some available carbohydrate or its derivatives, such as the various organic acids, and in the absence of available nitrogen, they may fix the nitrogen of the atmosphere. After all the available carbohydrate has been used up or transformed into unavailable forms, the fungus mycelium and bacterial cells, including those of the nitrogen-fixing bacteria, begin to be decomposed by the 41 Remy and Rosing, 1911 (p. 578); Gainey, 1925 (p. 832). 830 PRINCIPLES OF SOIL MICROBIOLOGY various soil microorganisms, especially the bacteria and actinomyces, with the result that the nitrogen is becoming available for higher plants. It is this group of phenomena which brought about confusion in an attempt to explain why the addition of available carbohydrates at first lowers crop yield,42 and why favorable results are obtained one year after the application of carbohydrates, as shown in the following summary : CROP YIELD, DRY MATTER TOTAL NITROOEN IN NITROGEN IN CROP SOIL, SPRING OP 1906 NITRATE N Oats, 1905 Beets, 1906 per cent grams per cent p.p.m. None 100.0 100.0 0.5914 0.093 10 Glucose, 2 32.8 186.0 0.6814 0.105 17 Sucrose, 2 33.3 179.0 0.6800 0.105 15 Sucrose, 4 37.7 283.0 1.0092 0.119 37 In various studies no effect has been noted as a result of application of carbohydrates and, in some cases, even an injurious effect has been observed. This may be due to the variability of the method for deter- mining the total nitrogen. The following example may be taken as an illustration: The addition of five tons of a pure carbohydrate (on a water-free basis), whether in the form of straw, hay, plant stubble, green manure, etc., is quite a large amount to add per acre of soil. Even assuming that such a quantity is added and that it is all utilized by the nitrogen-fixing bacteria as a source of energy (which is again doubt- ful), the maximum amount of nitrogen fixed under these conditions would be 0.5 part of nitrogen for every 100 parts of carbohydrate, or 50 pounds of nitrogen per acre. If the soil contains only 0.1 per cent nitrogen, this will form only about 2\ per cent of the nitrogen content of the soil, i.e., less than the error involved in the method for determining- nitrogen. By using 5 gm. of soil for total nitrogen determination, the difference will be only \ of a milligram. Those familiar with the method know how easily such an error is obtained. The question of soil reaction has been also discussed in detail else- 42 Kr tiger, W., and Schneidewind, W. Ursache und Bedeutung der Salpeter- Zersetzung im Boden. Landw. Jahrb., 29: 747-770. 1900; Gerlach, M., and Vogel, I. Versuche mit stickstoffbindenden Bakterien. Centrbl. Bakt. II, 9: 817, S80. 1902; 10: 636-643. 1903; Lipman, J. G. Soil inoculations with Azotobacter beijerincki. N. J. Agr. Exp. Sta. 21 Ann. Rep. 1908, 144-147. 43 Koch, Litzendorff, Krull and Alves, 1907-1909 (p. 586). SOIL INOCULATION 831 where. No introduction of Azotobacter will help to establish this or- ganism in the soil and bring about increased nitrogen fixation in an acid soil with a pH less than 6.0. However, the addition of lime to change the pH to above 6.0 will by itself bring about a development of an active nitrogen fixing flora, of course if other conditions are favorable. Good results from inoculation may be obtained in the case of freshly drained swamps, in which Azotobacter would be absent.44 But the reaction of the soil must first be adjusted by the use of lime. All attempts to inoculate normal soils with Azotobacter and other non-symbiotic nitrogen-fixing organisms failed on repeated study. From Caron's "alinit"45 in 1895 to Bottomley's46 "bacterized peat," all attempts to exploit commercially the nitrogen-fixing capacity of Azotobacter and other bacteria failed.47 The soil itself harbors suffi- cient organisms which become active when conditions and nutrients are favorable, as shown by Gainey for Azotobacter. Hiltner48 claimed to have obtained good results from inoculation of sugar beets with bac- teria; just what these bacteria do in the soil, has not been determined. The U-cultures of Kiihn,49 which are also used as an all-crop inoculant, have been found worthless by Barthel.50 Ehrenberg51 compared soil inoculation with symbiotic and non-symbio- tic nitrogen-fixing bacteria, with the following conclusions: 1. On comparing the abundance of the various bacteria living in the soil, hardly any change takes place as a result of artificial inoculation, since the bacteria 44Stoklasa, J. Deut landw. Presse 1908, No. 25-27; Stranak, Fr. Zur Assimilation des Luftstickstoffes durch im Boden freilebenden Mikroorganismen. Centrbl. Bakt. II, 25: 320-321. 1909. 45 Caron, A. Landwirtschaftlich-bakteriologische Probleme. Landw. Vers. Sta., 45: 401-418. 1895; see also Stoklasa, J. Biologische Studien uber Alinit. Centrbl. Bakt. II, 4: 39-41, 78-86, 119-130, 284-289, 507-513, 535-540. 1898; Deut. landw. Presse, 35: 274, 286-297. 1908; Heinze, B. Uber die Beziehungen der sog. Alinit bakterien (Bac. ellenbachiensis Caron) zu dem Bac. megatherium deBary bezw. zu den Heubacillen (B. subtilis Cohn). Centrbl. Bakt. II, 8: 391, 417, 449, 513, 545, 609, 663. 1902. 46 Bottomley, W. B. Rpt. Brit. Assn. Adv. Sci. 1911,607-608. 47 Russell, E. J. Report on humogen. Jour. Bd. Agr. (London), 24: 11-20. 1917. 48 Hiltner, L. Uber die Impfung der Putter und Zuckerruben. Mitt. Deut. Landw. Gesell., 26: 243. 1921; Engelmann, E. Mitt. deut. Landw. Gesell., 37: 560. 1922. 49 Ktihn. Deut. Landw. Presse, 44: 467. 1917. 60 Barthel, Chr. Forsok med Dr. A. Kuhns U— Kulturer. Meddl. No. 184, Centralanst. f. forsoksv. jordbruks. 1919; Deut. Landw. Presse, 50: 192. 1920. 61 Ehrenberg, P. Theoretische Hinweise zur Frage der Wirkung einer Boden- 832 PRINCIPLES OF SOIL MICROBIOLOGY from the commercial preparations rapidly succumb. The legume bacteria have the opportunity of penetrating the roots of the leguminous plants, whereby they are protected from competition with other bacteria. The protection is afforded also when the leguminous plants are dead since the nodules do not decompose so readily. 2. Although soil bacteria fix appreciable quantities of nitrogen under laboratory conditions and a definite success may be obtained on inoculating soil with such bacteria, the use of rather expensive substances like mannite or sugar make it rather prohibitive. The growth of algae was found to offer only questionable hopes.62 Soil inoculation with autotrophic bacteria. A number of organisms causing plant diseases can be affected in their growth by a proper control of the soil reaction ; this is especially true of those that are very sensitive to acidity, as in the case of diseases caused by actinomyces (potato scab, etc.). The addition of sulfur to the soil is used as a means of increasing the acidity of the soil to a point at which the development of the disease-producing organism is checked. But before this can take place, the sulfur has to be oxidized to sulfuric acid by proper bacteria. All soils contain organisms capable of oxidizing elementary sulfur; these may carry on the oxidation only very slowly, especially in certain soils. When strong sulfur oxidizing organisms are added, the oxidation of the sulfur may be hastened appreciably. This led to the development of a commercial product, which consists of sulfur inoculated with a crude culture of Thiobacillus thiooxidans. It still remains to be proved, however, how long the culture will survive on the dry sulfur and how efficient it may be, in comparison with the organisms present in ordinary soils. The inoculation of soil with nitrifying bacteria, especially in case of freshly drained swamps may also be of direct benefit. Inoculation of soil with heterotrophic, non-nitrogen-fixing microorgan- isms. Attention has already been called to Caron's first attempt to prepare a bacterial culture (alinit53) for soil inoculation, with the idea of stimulating the decomposition of organic matter in the soil. Although impfung mit freilebenden stickstoffsammelnden Bakterien. Fiihl. landw. Ztg., 69: 161-166. 1920; Centrbl. Bakt. II, 53: 409. 1921. 62 Further information on the inoculation of soil with Azotobacter is given by Emerson, P. Soil inoculation with Azotobacter. Iowa Agr. Exp. Sta. Res. Bui. 45. 1918; Omeliansky, 1923 (p. 559); Brown, P. E., and Hart, W. J. Soil inoculation with Azotobacter. Jour. Amer. Soc. Agr., 17: 456-473. 1925; Gainey, P. L. Inoculating soil with Azotobacter. Soil Sci., 20: 73-86. 1925. 13 Complete literature on alinit is given by Heinze, 1902 (p. 831). SOIL INOCULATION 833 the first attempts were unsuccessful, references are still found in recent literature concerning the use of similar preparations. It is claimed54 that B. ellenbachensis a and alinit-bacillus a will allow luxuriant growth of grain crops without the addition of nitrogenous fertilizers. Various other preparations (A. Kiihn's U-cultures, All-crop Inoculant, Inoculin) have been placed on the market for the inoculation of cultivated plants other than legumes. The results have so far proved negative. It still remains to be seen whether the inoculation of soil with strong cellulose-decomposing bacteria can stimulate the processes of decompo- sition of organic matter in the soil. We need also mention here again the results of Hartley55 showing that soils partially sterilized by means of heat or volatile antiseptics will benefit by the inoculation with saprophytic fungi. These fungi develop rapidly in the soil and thus prevent development of parasitic fungi causing the damping off of forest seedlings. If a soil is infested with injurious nematodes, it may be benefited by inoculation with predatory nematodes.56 64 Daude. Impfung von Feldern mit Bakterien. Blatt. Zuckerriiben., 25: 156. 1919; 26: 30, 45, 176. 1919; Centrbl. Bakt. II, 53: 408. 1921. "Hartley, 1921 (p. 000). " Steiner and Heinley, 1925 (p. 347). CHAPTER XXXII History of Soil Microbiology, its Past, Present, and Future "The history of a science is not merely a chronicle of discovery, but a study of the relation of methods and ideas in progress and the applica- tion of the conceptions thus gained to guide us in present and future work." — Henderson. Every science, especially every biological science, goes through, in the course of its development, a series of stages which can be briefly summarized as follows: 1. Ecological stage, including description and classification. Here we are largely interested with the description of the organisms, their morphology and taxonomy. 2. Physiological stage, or a study of the activities of the organisms in question. 3. Experimental stage, whereby changes in the physiology of the organisms are studied, as a result of experimental conditions. 4. Mathematical stage, when formulae are developed to express in exact language the mechanism of the physiological processes. Finally, we find that every science, when it reaches a certain stage of development, branches off into several new sciences. The age of a science is definitely indicated by the stage in which it is in. Soil microbiology is only a science in the making; but, while it has not left as yet the ecological stage and the very methods of study are still undergoing active change, it has already reached the stage when expressions are found for a correlation between the activities of the microorganisms and the environmental conditions. This science includes not only a study of soil microorganisms and their activities under experimental conditions, but also the resultant phenomena of the sum total of their activities in the soil. The fact that numerous groups of organisms, the activities of which may be supplementary or antagonistic, exist in a very complex medium, the soil, under very complex environmental conditions, tends to complicate the subject still further. The science of soil microbiology is also beginning to branch off into soil mycology, algology, protozoology, nematology, etc., as well as soil biochemistry. 834 HISTORY OF SOIL MICROBIOLOGY 835 Beginnings of soil microbiology. Each science has its roots and ante- cedents in the past and each is developed out of the materials of the past. This is true particularly of soil microbiology, which has de- veloped directly from the empirical practices in agriculture and as a result of the advances made by the science of bacteriology; it owes a great deal to the older sciences, botany and its daughter science my- cology, zoology and its offspring protozoology, chemistry, physics, and especially these sciences as applied to soil processes. Among the empirical practices, we need mention (1) the beneficial influence of the growth of legumes upon subsequent crops, (2) the composting of manure or various farm wastes, (3) the burning of the upper layer of soil to insure better crops, (4) the addition of fertile soil to soil newly prepared from bogs. The progress of physics resulted in the development of the microscope and balances. The progress of chemis- try resulted in a knowledge of the chemical composition of matter, a better understanding of the composition of complex proteins and car- bohydrates, and in the development of various methods used in or- ganic and inorganic analysis. The development of physical chemistry resulted in the progress of our understanding of the nature of colloids and surface phenomena, of the hydrogen-ion concentration of the medium, oxidation and reduction processes. All have contributed to the development of soil microbiology. The study of the microorganisms themselves dates back to the work of Kircher and Leeuvenhoek (1683), who made the first observations on the bacteria, followed by the investi- gations of 0. G. Miiller (Animalcula infusoria, 1786), Ehrenberg1 and T. Schwann.2 The last demonstrated that yeast was a living organism. The science of botany has contributed to a better knowledge of the mor- phology and physiology of fungi and algae. The science of zoology ad- vanced our understanding of the protozoa, nematodes and other inverte- brates found in the soil, especially in respect to their nutrition and relation to the other members of the soil population. Bacteriology, beginning with the work of Pasteur on microorganisms as chemical agents, has been one of the most fruitful fields in stimulating the development of soil microbiology. Both the medical and agricultural bacteriologists have made important contributions. It is sufficient to mention the methods of pure culture study of bacteria, finally leading to a differen- tiation of microorganisms on a physiological basis; the plate method for counting and isolating bacteria; the introduction of selective enrich- 1 Ehrenberg. Die Infusionstierchen als vollkommene Organismen. 1839. 1 Schwann, T. Gilbert's Ann. Phys. u. Chemie., 51: 1837. 836 PRINCIPLES OF SOIL MICROBIOLOGY ment and specific culture media, the anaerobic methods, etc., all of which were necessary steps in the development of the science. The develop- ment of media proceeded from the complex organic media, introduced by R. Koch, to the special inorganic media such as silica jelly, introduced by Winogradsky, and synthetic media introduced by Beijerinck. These artificial media finally led to the use of the soil itself as a culture medium for the growth and activities of microorganisms. Any modification of the physical or chemical condition of this medium, either as a result of addition of nutrients or stimulants, or as a result of change of en- vironmental conditions, leads to a change in the numbers and activi- ties of the microorganisms. Three distinct biological processes had been clearly outlined and partly understood by the middle of last century: (1) Decomposition of organic matter. This was known to give rise to humus which was believed to be one of the fundamental principles in soil fertility. Some investigators (Liebig, 1840) considered humus only as an intermediary product and not as a plant food; organic matter was believed to de- compose slowly by chemical oxidation ("eremacausis"). The work of Schloesing, Wollny and others finally led to a better understanding of the process. (2) Nitrification. The accumulation of nitrates in the soil as a result of decomposition of organic matter was known in 17th and 18th centuries, but only Boussingault connected this process with soil fertility. (3) Nitrogen fixation. The use of legumes for enrichment of the soil was known to the ancient Romans. Berthelot was the first to suggest that nitrogen fixation may be accomplished also by non-symbiotic bacteria. The isolation of the organisms con- cerned both in nitrification and nitrogen fixation took place only at the close of last century. Soil microbiology as an independent science. Three definite and often distinct conceptions are included in the science of soil micro- biology, namely: (1) a knowledge of the organisms occurring in the soil, their numbers, types and relationships; (2) the biochemical activi- ties of these organisms, under laboratory conditions and in pure culture ; (3) the role of these activities in the soil processes and their application to agriculture. Any advance in the science of botany, zoology, or bacte- riology, which throws light upon the nature of organisms which occur in the soil, such as the development of new methods, a better system of classification in bacteriology, the role of bacteria in the nutrition of protozoa, the question of the physiology and classification of filamentous fungi, the role of mycorrhiza in plant nutrition, etc. can be considered HISTORY OF SOIL MICROBIOLOGY 837 as contributing to the advance of soil microbiology. Any discovery in the field of chemistry or physics, which has a bearing upon soil formation, composition and processes, upon the chemistry of plant cells, as well as any new methods of analysis, are of direct importance to the development of soil microbiology. It is argued, however ( Winogradsky) , that, while a great deal of information has accumulated on the methods of isolation and culti- vation of certain organisms present in the soil, while a great many organisms have been isolated and described, while the biochemical activities of a number of these organisms are known, there is still lacking the science of soil microbiology proper, or the applied science. The beginnings of soil microbiology as an independent science date back to the sixth and seventh decades of the last century. There are two outstanding names in soil chemistry and bacteriology, whose theories were far from agreeing, but whose researches have dovetailed to give origin to the science; namely, those of Liebig (1840) and Pasteur (1860). Liebig 's theories of soil fertility fell short because he did not recognize the activities of microorganisms. Pasteur's work was not concerned directly with soil microorganisms, but his bacterio- logical investigations in general, and specifically the study of the various fermentations including that of urea and butyric acid, pointed the way to a new development. It remained for the practical agriculturist to combine the efforts of the chemist and bacteriologist and call attention to the importance of microorganisms in soil fertility. Kette3 (1865) deserves the credit for being the first to recognize this fact. He advanced the fermenta- tion theory, in which he stated that the importance of the addition of stable manure to the soil consisted in the fact that it cannot be replaced by nitrogen compounds and minerals as well as by purely vegetable matter, because the latter lack "a true vibrion fermentation." His views have found ardent adherents, as can be recognized from the work of Rosenberg-Lipinsky,4 who stated that "milliards of lower animals per acre are born every moment and die after a few days, sometimes after a few hours, serving others as food." The birth, or rather awakening, of medical bacteriology in the early part of the ninth decade of last century was also accompanied by a rapid development in soil bacteriology. The work of Koch on the gelatin plate method, of 3 Kette, W. Die Fermcntationstheorie gegenuber der Humus-Mineral und Stickstofftheorie. 2 Aufl. 1865. 4 Rosenberg-Lipinsky. Der praktische Ackerbau. 3 Aufl., 2: 27. 1S69. 838 PRINCIPLES OF SOIL MICROBIOLOGY Hellriegel and Wilfarth on the nodule bacteria and fixation of nitrogen by leguminous plants; the work of Frank and Beijerinck on the iso- lation of the organisms and their cultivation in pure culture, and finally the work of Winogradsky on the autotrophic bacteria were the contributions which transformed the science from its preparatory into the building period. With the introduction by Robert Koch, in 1881, of the gelatin plate for the study of bacteria, a stimulus was given to the systematic study of soil microorganisms, although the earliest bacteriologists were medical men and were more interested in public health and hygiene than in soil processes. They limited themselves entirely to a study of the numbers of bacteria and fungi in various soil layers, that would develop on the gelatin plate. Any organism that did not develop on the plate was not considered to be of importance. The occur- rence of specific organisms was studied chiefly from the point of view of finding out whether the soil contained pathogenic organisms. Here may be mentioned, in addition to Koch, Frankel in Germany and Houston in England. Of biochemical processes in the soil, the first to attract universal attention, as said before, were those of nitrification and nitrogen-fixa- tion. Pasteur suggested in 1862 that nitrification is a bacterial process and Schlosing and Miintz definitely proved that in 1877. This was soon followed by the work of Warington, who in a series of splendid contributions, beginning with 1878, established some of the most fundamental principles of the process of nitrification in the soil, outside of actual isolation of the organisms concerned. This was accomplished in 1890, by Winogradsky. In reference to nitrogen-fixation, universal attention was first centered upon the symbiotic process. Boussingault emphasized in 1838 that the favorable action of legumes upon the soil is due to their power of fixing atmospheric nitrogen. Frank demon- strated in 1879 that the nodules on the roots of the plants are formed as a result of inoculation with microorganisms. This was definitely demonstrated in 1886 by Hellriegel and Wilfarth. The organism Bad. radicicola was isolated and described by Beijerinck in 1888. The non-symbiotic nitrogen-fixing bacteria were isolated in 1893 by Wino- gradsky (Clostridium) and in 1901 by Beijerinck (Azotobacter). The names of Winogradsky and Beijerinck stand for the most fundamental work that has been done in building up the science of soil microbiology. While Winogradsky limited himself to the study of autotrophic and the anaerobic nitrogen-fixing organisms, Beijerinck 's HISTORY OF SOIL MICROBIOLOGY 839 contributions are distributed throughout the whole field of soil micro- biology. His studies embraced symbiotic and non-symbiotic nitro- gen-fixing bacteria, sulfur-oxidizing bacteria, nitrate and sulfate re- ducing bacteria, actinomyces, algae, etc. The bulk of Winogradsky 's work was limited, but the quality is of the highest, it stands for the most classical work in the science of soil microbiology. The study of decomposition of nitrogenous organic compounds in the soil is closely connected with the names of Pasteur (1863), Miintz and Coudon (1893) and Marchal (1893), who pointed out that various bacteria and fungi are capable of breaking down proteins with the rapid formation of ammonia. Here belongs the work of Gayon and Dupetit (1881) on nitrate reduction, of Deherain (1886) on the de- composition of farmyard manure, and Wollny (1897) who studied organic matter as a whole. The beginning of the study of cellu- lose decomposition by bacteria is closely connected with the name of Omeliansky, but neither the organisms nor the chemistry of the process were completely understood for a long time. Attention should also be called in this connection of the important contribu- tion of Ferdinand Cohn to the classification and description of a number of heterotrophic soil bacteria, followed by the work of A. Meyer and his associates on the spore-forming bacteria of the soil, as well as by Chester and others. The name of one other man should be mentioned here, that of Caron (1895) ; he was neither a chemist nor a bacteriologist, but a practical agriculturist who contributed a great deal to the stimu- lation of the theoretical and practical interest in the subject of soil microbiology. Caron demonstrated that any soil treatment which leads to an increase in the number of microorganisms also leads to an increase in crop productivity; fallowing of a heavy soil can be used in place of green manure. Although the practical agriculturists, by pointing out the great importance of microorganisms in soil processes and, therefore, in agriculture, often aroused great interest in the soil processes, the influence was frequently not far reaching. The practical men expected that soil microbiology would revolutionize agriculture just as medical bacteriology revolutionized medicine, but this did not materialize. Where this influence was strongest, especially in Germany and in the United States, some people came to believe that, outside of legume inoculation, there is nothing to the whole science of soil micro- biology. This attitude toward a science which lies at the very roots of all soil economy and will no doubt influence, in the future, the whole agricultural practice, could result only from a lack of sufficient knowl- edge concerning the problems under consideration. 840 PRINCIPLES OF SOIL MICROBIOLOGY The soil is a medium, more or less colloidal in nature, containing a great mass of microscopic, forms of life. These produce various physical and chemical changes in the soil which are of greatest im- portance to the growth of higher plants. The pathologist can study the action of his organisms in vivo; the microbiologist working on fermentation processes can sterilize his medium, without altering its composition greatly, and inoculate it with a pure culture of the organism concerned; the soil microbiologist, however, has great difficulties in attempting to learn just what the particular organism does in the soil. When the soil is sterilized, it is no longer, biologically and chemi- cally, a normal soil. In a pure culture, free from stimulating and competing influences of other microorganisms, an organism may manifest certain activities which would not take place in the soil, or vice versa. It is even possible that, in pure culture, different races develop from those present originally in the soil and it is quite probable that the biochemical action is often quite different. As a matter of fact, a very large number of soil organisms develop upon artificial media only with great difficulty and are often repressed there by other organisms which may be only occasional visitors in the soil. Recent advances of the science. During the first decade of the present century, the methods used in the study of soil biological processes have undergone various modifications. Some investigators centered their attention upon the study of the metabolism of specific soil micro- organisms, especially the mechanism of transformation of organic or inorganic substances as bearing upon soil processes. This was deter- mined either by adding a small amount of soil to a sterile solution containing the specific substance, then measuring the change that took place after a definite period of incubation; or by adding the specific substance to the soil, keeping it at optimum moisture and temperature for a definite length of time, and then measuring the change. In most of these studies, the organisms responsible for the change were not considered at all. In the study of protein decomposition, ammonia was usually taken as an index, without considering the fact that the process can be carried on by numerous types of organisms and various associations and combinations, each resulting in a different amount of ammonia accumulating. In the study of nitrogen fixation, the fact was usually left out of consideration that different organisms are active at different reactions and, therefore, different amounts of nitrogen are fixed under laboratory conditions, which may or may not hold true in the field. In the study of nitrification, the fact that the addition HISTORY OF SOIL MICROBIOLOGY 841 of large amounts of ammonium salts will soon result in a reaction (the degree depending on the buffer content of the soil) injurious to nitri- fication, while the addition of a large quantity of organic nitrogenous material may result in the formation of such large amounts of ammonia that the nitrifying bacteria will be injured, were usually left out of consideration. These investigators, often referred to as the physiological group, consisted of practical men, often insufficiently interested in microbiology, but primarily interested in the phenomena resulting from soil processes rather than in the organisms active in the soil itself. The first representatives of this group are Remy and Lohnis, later followed also by various workers in Germany, J. G. Lipman, Brown, Greaves, C. B. Lipman, and others in America, with Perotti in Italy, Christensen in Denmark, and others contributing more to one or another phase of the subject. The other group of investigators, often referred to as the botanical group, were more interested in knowing how many bacteria there are in the soil, what these bacteria are, and if physiological groups were studied, they wanted to know the numerical relation of one group to another. Hiltner and Stormer (1902) were the strongest advocates of this method of attack, followed by H. Fischer in Germany, Chester, H. J. Conn and others in this country, etc. In addition to these two groups of investigators interested in soil biological processes chiefly from the standpoint of the soil, a number of botanists, zoologists, general microbiologists, and chemists continued to make definite contributions to the science of soil microbiology, either by the study of one more group of soil organisms, including the soil bacteria (A. Meyer and associates, Ford et al.), fungi (Hagem, Lendner, Dale, Waksman, etc.), algae (Chodat, Bristol, Esmarch, etc.), actinomyces (Krainsky, Conn, Waksman, Drechsler), protozoa (Wolff, Goodey, Cutler et al.) and invertebrate animals (Cobb, Micoletzky), or by the study of one chemical process in the soil and the organisms concerned, such as cellulose decomposition (Kellerman and associates, Barthel, Pring- sheim, etc.), nitrogen fixation (Omeliansky, Bredemann, J. G. Lipman, Christensen, Gainey, Winogradsky, etc.), evolution of C02 (J. Russell, Stoklasa, van Suchtelen, Neller, etc.). The more outstanding recent contributions to the science of soil microbiology deal with microorganisms non-bacterial in nature. It is sufficient to mention the work of Russell and his associates on the occurrence of protozoa in the soil and on the phenomenon of partial sterilization; the occurrence and activities of algae, fungi, actinomyces and nematodes in the soil. It is also important to call attention to 842 PRINCIPLES OF SOIL MICROBIOLOGY the development of methods for the direct examination of microorgan- isms in the soil by H. J. Conn and recently by Winogradsky. We possess now also a better understanding of the organisms concerned in the oxidation of sulfur in the soil (Lipman and associates), while the role of microorganisms in the decomposition of cellulose and other polysaccharides in the soil (Hutchinson and Clayton, Fred, etc.) has been made clearer; a knowledge of the controlling influence of soil re- action upon the distribution and activities of soil microorganisms has influenced certain practices; the same is true of our increased knowl- edge of legume cultivation and inoculation (Hiltner, Whiting, etc.), of the use of green cover crops, fallowing and soil cultivation. Present outstanding problems in soil microbiology. The science of soil microbiology is in its mere infancy. New contributions open up broader and broader vistas, rich in reward both to the investigator and to practical men. The soil is the basis of all agricultural practice. The population of the soil makes the soil what it is and not a mass of debris containing all the elements necessary for plant growth in an unavailable form; sooner or later a study of this population will be recognized to be of most importance in the future advance of agriculture. We possess at the present time considerable information on the organisms inhabiting the soil and on the chemical processes of many of these organisms, under controlled laboratory conditions; but little is known of the processes carried on in the soil itself, by the numberless representatives of the soil flora and fauna. The transformation of organic matter, the availability of the mineral elements, the fixation and transformation of nitrogen, the best means for the preservation of the nitrogen already present in the soil or manure, these are a few of the processes which depend largely upon the activities of microorganisms and which control the growth of cultivated plants. Some of the outstanding problems in the science may be suggested here: 1. Microscopic and cultural methods in soil microbiology, especially those which tend to indicate the organisms active in the soil under field conditions and their, role in the transformations taking place in the soil. 2. The soil population, nature, extent and activities; the com- plexity of the population with its various associative and antagonistic influences. It is especially desirable to know what role the animal population, such as protozoa and nematodes, play in soil processes and how they influence bacterial activities, also the interrelation between the fungi and the bacteria and the r61e of actinomyces in the soil. HISTORY OF SOIL MICROBIOLOGY 843 3. Transformation of organic matter in the soil, both as to the chemical processes involved and organisms concerned, also the role of these transformations in soil fertility. 4. The energy balance in the soil as well as the balance between the soil constituents, largely the carbon and nitrogen. 5. A better understanding of the role of cultivated higher plants in soil transformations and the influence that they exert upon the activities of soil microorganisms. 6. Methods of modifying the soil population and its activities with a better understanding of the processes of partial sterilization, applica- tion of lime and fertilizing materials, soil inoculation, as well as soil fallowing. 7. The physical, chemical, and physico-chemical condition of the soil (reaction, buffer content, moisture holding capacity, temperature) and the occurrence and activities of soil microorganisms. These as well as a host of other problems to which attention has been called in the previous pages will not only throw light upon the different phases of soil microbiology, little understood at the present time, but will place the science where it should be, namely in the front rank of agricultural sciences. Depending as he does upon the contributions of the protozoologist, mycologist, bacteriologist, nematologist, etc., for a better understand- ing of the organisms inhabiting the soil and their activities, the soil microbiologist is in a position to correlate the sum total of the knowledge gained from these investigations and throw light upon the chemical processes in the soil. The soil physicist and the soil chemist do and will contribute definitely to the understanding of the nature of the medium in which these organisms act and of the soil solution, which receives, on the one hand, the waste products of their activities and which supplies nutrients to the plants and frequently to the micro- organisms. It is to the development of the science of soil microbi- ology as much as to any other science that we must look for the proper understanding of the soil as to its ability to supply the nutrients necessary for the growth of higher plants 5 5 Further information on the history and development of soil microbiology is found in the following papers and books: Fischer, 1909 (p. 712); Lohnis, 1910 (p. xiii); Lohnis, F. Ergebnisse amerikanischer, britischer und franzosicher Arbeiten auf dem Gebiete der landwirtschaftlichen Bakteriologie aus den Jahren 1915 bis 1920. Centrbl. Bakt. II, 54: 273-307. 1921; Winogradsky, S. La m^thode directe dans l'etude microbiologique du sol. Chimie et Industrie., 11: No. 2. 1924; Waksman, S. A. Soil microbiology in 1924; an attempt at an analysis and a synthesis. Soil Sci., 19: 201-249. 1925. INDEX OF AUTHORS Abderhalden, E., xiii, xvi, 269 Abel, E,, 603 Abel, R., xiii, 556 Aberson, G., 185 Aberson, Y. H., 788 Abbott, E. V., 270, 604 Ackermann, D., 485 Acklin, O., 550 Adametz, 28, 237 Adam, A., 636 Adams, C. C,', 358, 360 Adams, G. O., 509 Aeby, J., 185 Afanassiewa, M., 244, 417 Agafonoff, V., 702 Aichberger, R. V., 353 Aikman, C. M., xiv Aiyer, C. V. R., 489 Aiyer, P. A. S.,96, 97, 99, 234, 639 Albrecht, W. A., 517, 552, 595, 770, 821 Albus, W. B., 374 Alexeiev, A. G., 317, 340 Alilaire, E., 378 Allen, E. J., 221 Allen, E. Rr, 580, 716 Allison, F. E., 49, 326, 328, 329 Allison, R. V., 333 Allison, V. C, 178 Almy, L. H., 613 Alves, A., 586, 830 Alway, F. J., 826 Ambroz, A., 156, 158, 188 Ames, A., 779 Ames, J. W., 658, 661, 665 Ampola,G.,185, 186, 187,552 Amster, 322 Anderson, B. G., 178 Anderson, H. W„ 250 Anderson, J. A., 451, 518 Anderson, G. M., 361 Anderson, M.S., 627 Anderson, R, J., 651 Ankersmit, P., 166 Appel, O., 264 Appleyard, A., 624, 639, 721, 722, 779, 799 Arinstein, B., 468 Arnaud, A., 418 Arnd, Th., 530, 531, 551, 707 Arndt, A„ 320 Arny, A. C, 822, 825 Arrhenius, O. . 352, 634, 635, 814 Arrhenius, S., 418 Artari, A.,230 Arzberger, E. G., 138 Ashby, S. F., 113, 117, 394, 526, 534, 576, 668, 716 Ashe, L. H., 467 Aso, K., 450, 574, 649, 791 Atkins, W. R. Gj, 359, 636, 814 Atwater, W. O., 124, 819 Aubel, E., 415, 467, 507, 521, 543 Auten, J. T., 649 Ayers, S. H., 245, 636 B Baas-Becking, L. G. M., 387, 397 Bach, 464 Bach, Mi, 244 Bachmann, F., 163, 164 Bail, O., 805 Bainbridge, F. A., 478 Baker, F. G.C.,xi Bal, D. V., 338 Baldwin, I. L., 815 Ball, O. M., 129, 597 Barakov, P., 799 Barber, M. A., 55, 167, 220, 246 Barlow, B., 127, 128, 821 845 846 INDEX OF AUTHORS Barlow, P., 26 Barnard, J. E., xiii, 55 Barrenscheen, H. K., 556 Barthel, Chr., 104, 130, 131, 149, 154, 448, 498, 518, 528, 529, 531, 537, 551, 641, 642, 727, 779, 821, 831 Bassalik, K., 646 Bassu, E., 162 Batchelor, H. W., 497, 582 Batham, H.N.,528 Baumann, 644, 690 Baumgartel, T., xii Baunacke, W., 344, 348, 350, 805 Bavendamm, W., 82 Baver, L. D., 634 Baylis, H. A., xii Bayliss, W. M., xvi Bazarevski, S. V., 154, 653, 656, 769 Bear, F. E., 790 Beard, E., 631 Beauverie, J., 58 Bechhold, H., xvii, 626 Beckh-Widmenstetter, H. A., 556 Beckley, V.A.,693, 698 Beckmann, E., 456, 807 Beckwith, T. D., 259, 496 Beers, C. D., 315 Behn, 712, 756, 762 Behrens, J., 203, 242, 264, 266, 466 Beijerinck, M. W., v, 70, 76, 84, 87, 98, 103, 104, 105, 112, 115, 119, 121, 125, 160, 171, 183, 187, 188, 203, 204, 207, 217, 219, 222, 299, 304, 319, 383, 385, 391, 399, 447, 484, 543, 546, 547, 548, 571, 574, 606, 611, 631, 665, 694, 806 Beiling, R., 412 Beinhart, E. G., 815 Van Bemmelen, J. M., 629, 697 Benecke, W., xii, 221, 489 Bengtsson, N., 432, 448, 497, 518, 528, 529, 680, 727 Benni, 692 Benoist, S., 431 Bergey, D. H., x, 136, 158, 289, 309 Berestneff, N. M., 300 Bergene, 591 Berghaus, W. H., 640 Berhauer, K., 467 Berkhout, C. N., 238 Berkmann, M., 793 Berliner, E., 349 Berman, N., 475, 478 Bernard, N., 272, 276, 803 Berry, R. A., 427 Berthelot, D., 524 Berthelot, M., 104, 415, 558, 573, 800 Bertrand, G., 431, 435, 448, 649 Bessey, E., 810 Besson, A., xiii, 167 Bevan, E. J., 431 Bewley, W. F., 131, 812 Bezssonoff, N., 37, 110, 121, 560, 565, 569, 752, 762 Bialosuknia, W., 137 Bierema, S., 187, 212, 486, 502, 544 Biernacky, W., 455 Bierry, H., 454 Biltz, A., 524 Binder, K., 170 Biourge, Ph., 238 Birchard, F. J., 473 Bizzell, J. A., 539, 599, 705, 749, 795, 796, 797, 799 Bjorkman, C. B., 456 Blair, A. W., 375, 448, 501, 511, 598, 826 Blakeslee, A.F.,246 Blanck, E., 260, 353, 711, 782 Blau, O., 148, 156 Blaxall, F. R., 156, 201, 440 Blobel, S.,527 Blochwitz, A., 242 Blom, J., 549 Blunck, G., 828 Bodine, J. H., 325 Bogdanov, S., 673 Bogue, R. H., 626 Boischot, P., 747 Bojanowsky, R., 195 Bokorny, Th., 230 Bolley, H. L., 757, 807 Bonazzi, A., 66, 71, 73, 114, 396, ."2", 560, 566, 567, 572, 574, 575, 716 Bondorff, K. A., 121, 802 Bonnema, A., 573 Bonnet, R,, 410, 415 INDEX OF AUTHORS 847 Bornebusch, C. H., 117 Botkin, S., 171 Bottger, H., 789 Bottomley, W. B., 138, 562, 580, 694, 699, 831 Boulet, V., 283 Boulanger, E., 65. 394, 527, 694 Boussingault, J. B., 106, 122, 638, 786 Bouyoucos, G. J., 371, 619, 627, 632, 749 Brannon, J. M., 777 Brasch, W., 485 Breal, E., 184, 337, 553 Bredemann, G., 108, 109, 110, 111, 121, 172, 203, 517, 585, 641, 770 Breed, R. S., 9, 22, 57, 182, 288 Brefeld, O., x, 241 Breinl, F., 805 Breitschneider, 123 Btenchley, W. E., xv Brenner, W., 91, 117, 241, 269, 503 Brew, J. D., 9 Brierley, W. B., 239, 259 Bright, J. W., 41, 491, 500, 686, 774 Brioux, Ch.,604, 636 Briscoe, C. F., 773 Bristol-Roach, M. B., 50, 218, 221, 224, 225, 231, 233 Brizi, U.,234 Broadhurst, J., 58 Brooks, C., 779 Brooks, S., 244 Brown, C. W., 652 Brown, H.D., 91, 608, 815 Brown, P. E., xiv, 15, 32, 33, 35, 43, 112, 539, 598, 604, 638, 702, 711, 716, 731, 779, 785, 787, 791, 792, 799, 826, 832 Brown, W., 244 Brussoff, A., 94 Bruyn, H. L. G. de, 807 Bruyn, W. K. H. de, 328 Bryan, H., 633 Bryan, O. C, 128, 595, 826 Buchanan, R. E. , x, xii, 58, 131, 373, 593 Buchanan, R. M., 170 Buchner, H., 170 Buckle, P., 356 Buckman, H. C., xv Buddin, W., 539, 744 Buhlert, 716 Burd, J. S.,799 Burgeff, H., 276 Burgess, P. S., xiii, 116, 491, 533, 534, 538, 583, 716, 731, 789 Burke, V., 134 Burkey, L., 134 Burnet, Ei, 612 Burns, A. C., 761 Burr, S., 306, 809 Burri, R., 55, 115, 161, 162, 167, 170, 185, 554 Burrill, T. J., 125, 126, 135, 139, 156, 594, 828 Burton, E. F., 626 Busch, K., 349 Busse, W., 247 Buswell, 311 Buttenberg, J., 556 Butkewitsch, Wl., 262, 467, 494, 503, 666 Butler, E. J., 238, 247 Biitschli, O., xi, 312 Butterfield, C. T., 322, 336 Byars, L. P., 349, 815 C Cadness, B. H. E., 614 Calkins, G. N., xi, 312, 336 Cameron, A. E., 356, 357, 361 Cameron, F. K., xiv, 630, 634, 749 Carbone, D., 204 Carapelle, E., 543 Carey, C, 196 Car on, A., 34, 104, 772, 785, 794, 831 Caron, H. V., 181, 552 Carpenter, P. H., 689 Carter, E. G., 531, 532, 577, 584, 664, 668, 774, 783, 784, 789 Carter, N., 230 Cash, J., xi, 331 Casuto, L., 626 Cauda, A., 328 Caullery, M., 803 Chabrie, C, 556 Chalmers, C. H., 199, 455 Chalmot, G. de, 453 848 INDEX OF AUTHORS Chambers, C. O., 270 Chambers, R., 56 Chambers, W. H., 373 Chandler, A. C, 345 Chapman, A. C, xi Chapman, C. W., 371 Charaire, 316 Charlton, J., 634 Charpentier, C. A. G., 431, 449, 450, 635, 727 Charpentier, P. G., 232 Charrin, A., 418 Chatton, 320 Chen, C. C, 155 Chester, F. D., x, 35, 103, 143, 150, 491, 597, 710, 772 Chi vers, A. H., 238, 251 Chlopin, G. W., 640 Chodat, R., x, 220, 221, 230 Cholodny, N.,94, 385 Chouchack, D., 735 Christensen, H. R., 116, 186, 209, 449, 576, 578, 581, 634, 664, 700, 707, 709, 725, 730, 788, 792 Christiansen-Weniger, F., 570, 591 Christoph, H., 277 Chudiakow, N. N,, xii, 161, 631 Chukevitch, J., 166 Church, M.B., 238, 241, 744 Churchman, 165 Cingolani, M.,186,214 Clark, F. D., 749, 758 Clark, H. W., 509 Clark, W. M., xvii, 18, 164, 371, 520, 634 Clarke, F. W», xiv Claypole, E., 289 Clayton, L, 120, 195, 439, 448 Clegg, M,.T.,299 Clements, F. E., x, xv Cleveland, L. R., 316, 339 Cobb, N. A., 343, 346, 348, 349 Cobet, R„, 789 Cohen, B., 164 Cohen, E., xvii Cohnheim, O., xvi Cohn, F„ 80 Cohn, R., 436 Coleman, D. A., 269, 320, 328, 757 Coleman, L. C, 391, 531, 535, 782 Colin, H„ 461 Collins, F. S., 230 Collison, R. C, 500, 518, 671 Comber, N. M., 628 Compton, A., 448 Conn, H. J., xii, 6, 7, 10, 15, 23, 27, 32, 40, 41, 56, 142, 149, 150, 152, 182, 240, 288, 290, 304, 309, 491, 500, 518, 686, 774 Conn, H. W., xii, 333, 335 Connor, S. D., 636, 712, 815 Conrad, C. M., 460 Cook, M., 167 Cook, M. T., 243, 809 Cook, R. a, 15, 495 Cooley, J. S.,244, 779 Coppa, A., 328 Coppenrath, E., 734 Coudon, H., 267, 480, 490 Coupin, H., x, xi Coville, F. V., 636 Cowie, G. A.,488 Cramer, 80, 378, 380, 382, 455 Cramer, W., 631 Crisafulli, G., 212 Crozier, W. J., 418 Cross, C. F., 431 Crump-, L. M., 32, 49, 50, 318, 321, 326, 328, 329, 336, 767 Cubbon, M. H., 790 Cunningham, A., 45, 320, 327, 336 Curtis, R. E., 40, 290, 304, 309 Cutler, D. W., 32, 46, 47, 50, 318, 321, 326, 327, 328, 329, 336, 337, 338, 739, 759, 767 Cutright, C. R., 359 Czapek, F., xv, 269, 270, 379, 419, 424, 459, 502, 573, 654, 793 Czermak, W., 777 D Dachnowski, A. P., 707 Dack, G. M., 174 Daikuhara, G., 551 Dakin, H. D., 481, 483, 540 Dale, D., 314 INDEX OF AUTHORS 849 Dale, E., 237, 259 Damon, S. R,, 176 Dangeard, P. A.,230 Darbishire, F. V., 523, 752 Darwin, C, 352 Daszewska, 238, 265 Daude, 833 Davenport, A., 76, 127, 132, 582, 594, 823 Davenport, C. B,, xviii, 26, Davis, A. R., 270, 281 Davis, J. J., 362 Davis, R. O. E., 633 Davison, W. C, xvii Davy, H., 122, 183, 524 Dayhuff, W. C, 628 DeBary, A.,x,264 Debord, J. J., 476 Deherain, P. P., 183, 445, 523, 537, 548, 553, 682, 781, 786, 793 Delacroix, 808 DeMan, J. G., xii Demolon, A., 604, 747, 749 Demoussy, E., 523, 682 Dernby, K. G., 301 Derx, H. G., 464 Detemar, K., 277 Detmer, W., 672 Deusch, 508 Devaux, IE, 460 Dianowa, E. W., 631 Didlake, M., 135 Diem, K., 355, 357 Dietzell, B. E., 183 Dimo, N. AM 363 Dischendorfer, O., 431 Distaso, A., 172, 199 Dixon, M., 543 Dmochowski, R., 432 Dobson, M. E., 700 Doerell, E. G., xiii Doflein, F., xi, 312, 323, 325 Donker, H. J. L., 173, 526, 549 Dorner, W., Ill, 164, 165, 166, 175, 582 Dorsch, R., 185 Doryland, C. J. T., 35, 69, 506, 771, 782 Dotterer, W. D., 22 Dox, A. W., 382, 651 Dozier, C. C,., 171, 176 Drechsler, C., 288 Drew, G. H., 662 Drouin, R., 234, 573 Dubaquie, 463 Dubos, R., 735 Dubovsky, B. J., 174, 806 Duclaux, E., xiii Dufort, 208 Dufrenoy, J., 283 Dliggeli, M., 30, 39, 77, 82, 85, 110, 175, 611 Duggar, B. M., 270, 281, 597, 813, 824 Dujardin, M., 604 Dukes, C. E., 382 Dupetit, G., 181, 183, 185, 548 Dupont, C., 156 Dvorak, J., 428, 562, 684 Dzierzbicki, A., 577 E Earp-Thomas, G. H., 824 Eckelmann, E., 150, 641 Ecker, E. E.,212 Edgerton, C. W., 257 Eden, T., 690 Edmondson, C. H., 312, 331, 333, 335 Edwards, S. F., 821 Effront, J., xvi Eggertz, C. G., 672 Egorov, M. A;, 651, 750 Ehrenberg, A., 184 Ehrenberg, C. G., 92, 222, 329, 835 Ehrenberg, P., xiv, 268, 480, 498, 626, 710, 785, 813, 831 Ehringhaus, A., xiii Ehrlich, F., 242, 429, 481, 482, 483 Ehrlich, R., xiii Eichwald, E., xvii Eijkman, C., 464 Einecke, A., 664, 774 Eisenberg, P., 631 Elion, L., 189, 611 Ellenberger, W., 436 Eller, W., 692 Ellinger, A., 485 Ellis, D., xii, 93, 94 Elveden, V., 746 850 INDEX OF AUTHORS Emerson, F. V., xiv Emerson, P., 832 Emich, xii Emmerich, R. W., 752, 774 Emmerling, O., 502 Emoto, Y., 746 Engberding, D., 14, 21, 621, 712, 768, 771, 782, 788 Engelmann, E., 831 Engler, A., x, xi, 230, 252 Enlows, E.M.A.,59 Epstein, A., 199 Erismann, H., 631 Ernest, A., 34, 429, 685, 718, 772, 794 Esmarch, F., 218, 222 Esselen, G. J., 456 Esten, W. M., 30 Estienne, V., 461 Euler, A. C, 431 Euler, H., xvi Eyre, J. W. H., xiv Faber, F. C, 105, 140 Fabricius, O., 785 Falck, R., 467, 694 Fallot, B., 670 Fantham, H. B., 329 Faris, J. A., 812 Fawcett, E. H., 129 Fearon, W. B., 487 Fehrs, 311 Feiber, W. A., 176 von Feilitzen, H., 785, 821, 822 Fellers, C. R., 49, 326, 328, 329 Fellows, H., 810 Felton, L. D., 542 Ferguson, M., 552 Fermi, C, 162 Fernandes, J. F. S. A., 288 Feuilletau, 328 Fickendey, 716 Field, E. C, 808 Findlay, A., xvii Fine, M. S., 315, 318 Fischer, A., xii, 247 Fischer, E., x, 435, 472 Fischer, E. A., 630, 636 Fischer, F., 459, 693 Fischer, H., 13, 15, 105, 118, 121, 232, 251, 260, 450, 529, 530, 534, 554, 576, 586, 664, 685, 687, 711, 712, 743, 746, 782, 790, 791, 843 Fischer, Her., 642, 644 Fischer, R. A., xviii, 26, 46 Fleischer, M., 682 Floess, R., 26 Flugge, C, x, 149, 476 Fodof, A., xvii Foex, E., 815 Fol, J. G.,298 Folin, O., 475 Folpmers, T., 489 Folsom, J. W., 362 Ford, W. W., 143 Forti, 230 Foulerton, A. G. R., 300 Fousek, A., 40, 302, 309, 441, 493 Fowler, G. J., xvi, Fowler, L. W., 120, 129, 597 Fraendel, C., 171 France, 225, 642 Frank, B., 124, 126, 231, 271, 280 Franke, B., 745 Franke, F., 550 Frapkel, C., 1-2, 20, 34 Frankland, 64, 181 Fraps, G. S., xiv, 539, 672, 685, 716, 781 Fraser, A. D., 638 Fray, W. W., 316, 319 Frazier, W. C., 822 Frear, W., 788 Fred, E. B., xiii, xiv, 15, 76, 120, 121, 127, 131, 132, 133, 136, 157, 186, 202, 222, 242, 378, 379, 417, 440, 452, 454, 518, 530, 552, 580, 582, 593, 594, 647, 685, 754, 777, 788, 802, 822, 823, 825, 826, 827 Fremlin, 531 Fresenius, L., 450, 685, 791 Freudenreich, E. V., 110, 119 Freundlich, H., xvii, 626 Frey, W., 631 Fricke, K., 674 Friebes, V., 204 INDEX OF AUTHORS 851 Fries, K. A., 10 Fritsch, F. E.,222, 234 Froehlich, H., 264 Fuchs, W., 458 Fuhrmann, F., xiii, 169 Fulmer, H. L., 121, 580, 791 Furman, N. H., xvii G Gaarder, T., 77, 529 Gage, S. D., 491 Gage, S. H., xiv Gainey, P. L., 116, 496, 534, 536, 560, 582, 583, 685, 688, 716, 761, 787, 829, 832 Gallaud, I., 272 Galle, E., 98 Gardner, W. A., 673 Garino, E., 186, 187 Garman, H., 135 Gates, W. W., 702 Gaudechon, H., 524, 539, 640, 781 Gaumann, E., x Gautier, A., 234, 573 Gayon, U., 181, 183, 185, 548 Gedroiz, K. K., 629, 630, 633, 698 Gehring, A., 87, 188, 670 Geilinger, H., 176, 210 Georgevitsch, 105 Gerard, E., 211 Gerlach, M., 119, 183, 379, 508, 554, 574, 592, 830 Gerretsen, F. C, 188, 529, 592, 736 Gerza, U., 652 Gescher, N., 199 Ghosh, E., 331 Giaja, J., 454 Gibbs, H. D., 164 Gibbs, W. M., 66, 497, 787, 802 Gicklehorn, J., 83 Giesecke, F., 353 Gilbert, 300 Gilbert, J. H., 106, 122, 124, 795 Gilbert, W. W., 815 Gile, P. L., 735 Gillespie, L. J., 18, 301, 522, 634, 814 Gilman, J. C, 811 Giltay, E., 185 Giltner, W., xiv, 583 Gimmingham, C. T., 268, 529, 662, 814 Girard, A., 749 Given, G. B., 716 Glenk., K.,633,742 Glinka, K. D., xiv Globig, 155, 300 Goddard, H. M., 238, 259, 270 Godlewski, K., 525 Golden, R., 651 Golding, J. , 592 Goldthorpe, H. C, 532 Goodey, T., 323, 325, 326, 328, 337, 338, 349, 742 Goodfrey, G. H., 345 Goppelsroder, F., 545 Gordon, G. E., 320 Gordon, J., 163 Gortner, R. A., 452, 650, 690, 692 Gosio, B., 556 Goss, R. W., 807 Gottheil, O., 143, 14S Gotze, C, 550 Gozony, L., 319 Grafe, V., 382, 462 Gran, H. H., 455 Grandeau, L., 689 Grassheim, K., 417 Graul, E. B., S25, 827 Gray, F. J., 694 Gray, P. H. H., 199, 455 Grazia, S. de, 527, 652 Greaves, J. E., xiii, 36, 117, 179, 531, 532, 534, 539, 563, 577, 578, 579, 584, 585, 664, 668, 774, 783, 784, 785, 789 Greef, Rr, 329 Green, H. H., 531, 579, 687, 688, 716, 731 Greenfield, M., 155 Gregory, W., 222 Greig-Smith, R., 37, 465, 593, 742, 760 Griddle, N., 359 Griffith, B. M., 82 Grint esco, J., 221, 230 Groenewege, J., 117, 203, 421, 4^6, 444, 448, 546 Grohmann, G., 101 Gruber, Th., 29 852 INDEX OF AUTHORS Gryns, A., 592 Guarnieri, G., 203 Gubin, B. M., 609 Guenaud, C, 810 Guerbet, M., 604 Guggenheim, M., 178 Guillemin, M., 668 Guillermond, A., xi, 250 Guittonneau, G., 299, 493, 604, 609 Gully, 644, 690 Gustafson, A. F., 743, 749 Gustafson, F. G., 412 Gutzeit, E., 716 II Haas, A. R. C, 647, 802 Haas, P., xvi Hagem, O., 77, 237, 247, 266, 268, 269, 503 Hager, H., xiv Hagglund, F., 456 Hahn, E., 777 Halm, J.1, xv Haines, F. M., 222 Hall, A. D., xiv, 268, 529, 664, 793 Hall, I.C., 164, 165, 174 Hall, I. W., 638 Halsted, B. D., 814 Halversen, W. V., 33, 43 Hamilton, C.C., 359 Hammer, R. W., 563, 569, 570 Handschin, E., 358 Hansen, E. C.,251 Hansen, R., 125, 126, 132, 135, 136, 139, 594, 824, 828 Hansendorf, 694 Hansteen Craner, B.. 643 Hanzawa, J., 119, 238, 575, 580 Harder, E.G., 93, 402, 666 Harder, E. G., 33, 777 Harding, H. A., 56 Hardman, R. E., 813 Hardy, F., 636 Hargitt, G. T., 316, 319, 323 Harned, H. H., 773 Harring, H. K., 351 Harris, J. E., 634 Harrison, D. G., 540 Harrison, F. C, 823 Harrison, W. H., 96, 99, 234, 639 Harrison, F. C, 127, 128 Hart, E. B., 600, 685, 788 Hart, W. J., 832 Harter, L. L., 418, 461, 808 Hartleb, R., 77, 105 Hartley, C, 807, 815, 833 Hartmann, M., xvi, 323 Hartig, 263 Harz, CO., 288 Haselhoff, E., 110, 653 Hasenbiiumer, J., 620, 633, 701, 734, 742, 783, 791 Hastings, E. G., xiii Hata, S., 163 Hatschek, E., xvii Hauman, L., 203, 242 Hayes, W. P., 362 Headden, W. P., 539 Headlee, T. J.,361 Heap, H., 614 Hecker, F., 55 Hedin, G., xvii Heiden, E.,, xv Heimberger, H. V., 352 Heine, E., 621 Heinemann, P. G., xiv Heinicke, A. J., 793 Heinley, H., 347, 833 Heinze, B;, 121, 232, 446, 573, 585, 592, 694, 742, 752, 786, 788, 800, 831, 832 Hellbronner, A., 666 Heller, E., 265 Heller, H. H., 164, 172 Hellriegel, H., 124 Hellstrom, P., 449, 772 Helz, G. E., 136 Henneberg, W., xi, 818 Henrici, A. T., 58, 251, 291 Henry, T. A., xvi Hensen, V., 352 Herfeldt, E., 554 Herissey, H., 454 Hesse, R., 360 Hesselman, H., 696 Heraeus, W., 63 INDEX OF AUTHORS 853 Hermann, R., 697 Heukelekian, H., 261, 266, 443, 451, 692, 728 Heurck, H. J., xi, 230 Heuser, E., 427, 431 Hewlett, R. T., xiv Heymons, R., 353 Hibbard, P. L., 616 Hibbard, R. P., 371 Hibbert, H., 431 Hibler, E. V., 167, 172 Hilgard,E.W.,xv,707 Hilgermann, 349 Hill, H. H., 517, 706, 770 Hill, T. G., xvi Hill, T. L., 339 Hills, T. L., 575 Hiltner, L., 12, 21, 41, 47, 125, 127, 128, 129, 134, 138, 150, 337, 585, 589, 591, 592, 593, 598, 643, 744, 751, 761, 763, 772, 819, 820, 831 Hinkle, S.F.,xiv Hinze, G., 82 Hirschler, A., 505 Hirsch, P., 178 Hiss, P. H., xii, Hissink, D. J., 634 Hoagland, D. R., 371, 628, 632, 634 Hober, R., xvi Hoering, P., xv Hoflich, C, 186 Hoffmann, C, 39, 158, 563, 569, 570, 571, 622, 656, 794 Hoffmann, F., 30 Holben, F. J., 670, 791 Holderer, M., 435 Holker, J., 167 Holmes, R. S., 627 Holthaus, K., 357 Holtz, H. F., 702 Holzmiiller, K., 143, 149 Honcamp, F., xv Honing, J. A., 737 Hopfe, A., 199, 265 Hopkins, C. G., xv, 521, 588, 598, 657 Hopkins, R. F., 814 Hoppe-Seyler, F., 433, 446, 697, 700 Horowitz, A., 181 Hort, E. C, 55, 169 Hosslin, A., 194 Houston, A. C, 14, 142, 154, 173, 785 Howard, A., 800 Huber, B., 275 Hubert, E. F., 459 Hucker, G. J., 153 Htine, 754 Hungerford, C. W., 812 Hunt, N. R., 815 Huntemuller, O., 336 Hunter, O. W., 568, 571, 580 Hunter, W. D., 362 Hurst, L. A., 814 Husek, B., 530 Husz, H., 303, 464 Hutchinson, C. M., 117, 121, 760 Hutchinson, H. B., 120, 131, 195, 311, 439, 448, 502, 517, 587, 743, 746, 757, 761, 790 Hutin, A., 702 Huttermann, W., 30 Hyslop, J. A., 362 I Imaseki, T., 551 Imms, A. D., 356 Irvine, J. C., 700 Itano, A., 117,476,580, 582 van Iterson, 186, 195, 200, 264, 266, 433, 547 Iwanoff, N. L., 381, 651 Jachschewski, A., 757 Jacobsen, A., 481, 483 Jacobsen, H. C., 85, 217, 399 Jacquot, R., 410, 415 Janke, A., xiii,407 Janse, J. M., 272 Japha, A., 556 Jeannert, J., 478 Jegen, G., 354 Jegorow, M., 675 Jegunow, M., 83 Jellinek, K., xvii Jensen, C. A., 656, 781, 796 854 INDEX OP AUTHORS Jensen, C. N., 237, 259 Jensen, H., 180, 181, 186, 187, 551 Jensen, O., 59, 297, 421 Jensen, P. R., 645 Jensen, S. T., 634 Jodidi, S. L., 501, 513, 621, 672, 686 Joffe J. S., 10, 89, 595, 601, 615, 616 Johnson, E. M., 601, 815 Johnson, H. W., 262, 582 Johnson, J., 813, 814 Jollyman, W. H., 180 Jones, C. B., 300 Jones, D. H., 114, 116, 118, 585 Jones, F. R., 128, 275, 813 Jones, J. O., 670 Jones, J. S., 702 Jones, J. W., 178 Jones, L. R., 203, 461, 810, 811 Jordan, E. O., xii Jorgensen, A., xi Joshi, N. V., 749, 824 Jost, L., xv Jungano, M., 172 K Kadisch, E. 164 Kahn, M. C, 176 Kamamura, 647 Kanitkar, N.V., 749 Kanitz, A., 418 Kappen, H., 260, 267, 603, 634, 636, 711,735,782 Karraker, P. E,, 815 Karrer, J. L„ 217, 224 Karrer, P., 190, 431 Karunaker, N., 730 Kaserer, H., 97, 98, 403, 579, 580, 699, 763 Kauffman, C. H., 276 Kayser, E., xiii, 572, 579 Kazakov, A. V., 660 Keil, F., 81, 398 Keith, S. C, 777 Kellerman, K. F., 126, 129, 138, 193, 197, 435, 436, 439, 444, 517, 574, 662, 716, 798, 819 Kelley, W. P., 512, 532; 539, 555, 657, 664, 743, 749, 789 Kellogg, E/H., 604 Kendall, A. I., xii, 167, 505 Kette, W., 837 Khouvine, Y., 193, 438 Killer, J., 45, 320 Kimmelstiel, P., 641 King, C. J., 813 King, F. H., 122, 534, 795 King, W.E., 35, 771, 782 Kissling, R., 682 Kitasato, S., 165 Kiuyma, B.t 467 Klaeser, M., 148, 181 Klason, 456 Klebahn, 808 Klebs, G., 333 Klein, A., 9, 173 Klein, G., 88 Klein, M. A., 538, 685, 722 Klett, A., 556 Klimmer, M., 135 Klinger, R., 809 Klocker, A., xi, 251 Klott, C, 137 Kluyver, A. J., 526, 549 Knight, H.G., 636 Knudson, L., 281 Kober, L., xv Koch, A., xiv, 158, 447, 443, 536, 550, 559, 562, 567, 578, 585, 536, 651, 655, 711, 750, 753, 802, 830. Koch, G. P., 45, 326 Koch, K., 692 Koch, R., 12, 838 Kochmann, R., 556 Koffman, M., 314 Kofoid, C A., 325 Kohl, F. G., xi, 250 Kohswitz, H. G., 353 Kolkwitz, R., xv, 82 Kolle, W., xii Kolthoff, I. M., xvii Kondo, M., 614 Konig, J., 429, 430, 478, 620, 633, 670, 676, 701, 734, 742, 783, 791 Koning, C. J., 237, 259, 263, 234, 265, 694 Koorders, S. H., 139 Kopeloff, N., 320, 328, 757 INDEX OP AUTHORS 855 Kordes, H., 828 Koser, S. A., 153 Kosin, N. I., 436 Kossowicz, A., xiii, 212, 267, 270, 503 Kossowitsch, P., 232, 601 Kostytschew, P., 361 Kostytschew, S., xv, 183, 244, 417, 573 Kosyachenko, I. S., 267 Krainsky, A., 40, 288, 297, 309, 442, 563, 566, 569, 575, 584, 586 Kramer, E., 383 Kraus, R., xiv Krause, M., 353, 790 Krawkow, S., 645 Krober, E., 453, 655 Krohn, V., 158 Kroulik, A., 157, 202, 440 Kr tiger, F., 310 Kriiger, R., 135 Kriiger, W., 105, 232, 516, 746, 748, 786, 830 Krull, F., 586, 830 Krumwiede, C, xii, 58 Kruse, W., xii, 183, 378, 389, 410, 415, 424 de Kruyff, E., 157, 251, 464 Krzemieniewski, H., 159, 576 Krzemieniewski, S., 159, 576, 579, 585, 700 Ktihl, H., 181 Ktihn, A., 319, 333, 822, 823, 831 Kunnmann, O., 187 Kuntze, W., 32, 187 Kunze, F., 645 Ktirsteiner, J., 162, 169 Ktirsteiner, R., 30 Kusano, S., 284 Ktister, E., xiv, 241 Kyropoulos, 662 Labes, R., 631 Lachmann, 124 Lachner-Sandoval, V., 288 Ladd, E. F., 796 Lafar, F., xiii, 241 LaFlize, S.,599 Laine\ E,, 66, 531 Langeron, M., xiv Langwell, H., 202, 439 Langworth, H. V., 583 Lantsch, K., 98, 169, 298, 620, 630 Lapicoque, L., 556 Larsen, O. H., 581 Larson, W. P., 668 Lathrop, E. C, 257, 453, 474, 479, 501, 502, 503, 671, 686^ 748 Latshaw, W. L., 598 Laubach, C. A., 143 Laubenheimer, xiv Laupper, G., 775 Laurent, E., 125, 133, 181 Lavialle, P., 489 Lawes, J. B., 106, 122, 124, 795 Lawrence, J. S., 143 Leather, J. W., 639, 781, 799 Lebedeff, A. F., 98, 101, 403, 550 Lebedjantzev, A. N., 740 Lebour, M. V., 359 Lechair, C. A., 794 Lee, A. B., xiv, 323 Lehmann, F., 456 Lehmann, K. B., x, 56, 288 Leidy, J., 331 Leiningen, Graf zu, 752, 774 Lemmermann, E., xi, 333 Lemmermann, O., 251, 260, 450, 501, 517, 530, 551, 554, 664, 685, 711, 774, 781, 791 Lendner, A., 237, 247 Lennep, R. V., 163 Lentz, O., 167 Leonard, L. T., 129, 137, 362 Lesser, 194 Levene, P. A., 475 Levine, M., 813 Levine, V. E., 556 Levy, M. M., 653 Lewin, K. R., 43, 318, 324, 326, 328, 329 Lewis, G. N., xvii, 386 Lewis, W. C., McC, xvii LewV, 638 Lex, R., 211 Lichtenstein, S., 199 Liebert, F., 212, 486 856 INDEX OF AUTHORS Liebig, J., 522, 837 Liebscher, G., 748, 805 Liesche, D., 456 Liesegang, R., 641 Lieske, R., 86, 95, 188, 288, 297, 398, 399, 402, 666 Limburger, A., 88 Lindau, G. K., x, xi, 252 Lindner, A., xi Linhart, G. A., 419, 570 Lint, H. C, 88, 320, 328, 614 Lipman, C. B., 36, 77, 106, 116, 120, 129, 179, 491, 529, 532, 533, 534, 538, 539, 576, 577, 582, 583, 584, 597, 670, 700, 716, 783, 789 Lipman, J. G., xiii, xiv, 15, 88, 89, 92, 112, 115, 119, 125, 375, 448, 479, 480, 491, 501, 511, 539, 559, 563, 569, 570, 574, 575, 598, 599, 614, 640, 687, 711, 772, 784, 788, 791, 792, 799, 826, 830 Lissauer, M., 29 Lister, A., xi Littauer, F., 207 Litzendorff, J., 5S6, 830 Livingston, B. E., 760 Lochhead, A. G., 34, 41, 196, 199, 435 Loeb, J., xvi, 627 Loeb, W., 573 Loew, O., 381, 390, 574, 752, 774 Lohnis, F., xiii, xiv, 13, 28, 30, 32, 36, 55, 56, 94, 98, 114, 115, 119, 120, 126, 135, 179, 196, 199, 207, 209, 211, 320, 327, 435, 479, 489, 491, 501, 527, 531, 554, 555, 559, 563, 579, 585, 586, 687, 688, 694, 709, 711, 712, 716, 731, 780, 782, 824, 843 Lomanitz, S., 379, 491, 513 Long, 311 Lotka, A. J., xvii Lowi, E., 169 Ludwig, O., 694 Lumiere, A., 781 Lund, Y., 577 Lundegardh, H., xv, 412, 63S, 640 Lwoff, A., 317 Lymn, A., 202, 43,9 Lyon, T. L., xv, 539, 599, 705, 749, 785, 792, 793, 794, 795, 796, 797, 799 M Maassen, A., 171, 181, 182, 185, 187, 549, 611, 614, 712, 756, 762 MacBride, T. H., xi MacDougal, D. T., 275, 281 Mace, E., xiv, 293, 309, 493 Macfayden, A., 156, 201, 440 Maclntire, W. H., 604 Mackenzie, W. A., 26 Magdeburg, P., 225 Magistris, H., 382 Magrou, J., 273, 281 Maillard, L. C., 692 Makkus, W., 786 Makrinov, I. A., 71, 203, 531 Malone, R. H., 55 Malpighi, 123 Mameli, E., 106 de Man, J. G., 346, 348, 349 Mann, H.H., 749 Manns, T. F., 808 Maplestone, P. A., xii Maquenne, L., 183, 548 Marchal, E., 182, 267, 480, 490, 591 Marcinowski, K., 349 Marcusson, J., 446, 691, 693 Marino, F., 167 Marshall, C. E., xiii Marshall, R. P., 815 Martelly, 492 Martin, C. H., 48, 318, 324, 328, 329 Martin, T. L., 517, 674 Mason, C. J., 30 Massee, C. E., 813 Massol, L., 65, 394, 527 Mast, S. O., 315 M'Atee, W. L., 358 Mathews, A. P., xvi Matthews, A., 756 Mattson, S. E., 628, 630 Matz, F., 185 Matzuschita, T., x, 29 Maublanc, 808 May, D. W., 735 May, F. V., 6S9 Mayer, A., xv, 381 INDEX OF AUTHORS 857 Maze, P., 103, 169, 182, 378, 531, 548, 594, 793 McBeth, I. G., 197, 263, 265, 435, 536, 539, 561, 797 McCall, A.G., 661 McClendon, J. F., xvii McColoch, J. W., 360, 362 McConnell, W. R., 362 McCpol, M. M., 371, 632 McDougal, W. B., 283 McGeorge, W., 749 McGinnis, F. W., 822 Mclnnes, J., 814 McKinney, H. H., 810, 813 McLean, H. C., 88, 259, 267, 375, 448, 494, 501, 511, 614, 615, 616, 772 McLennan, E. I., 272 McLennan, K., 790 McLeod, J. M., 163 Medes, G., xvii Medical Research Committee, 174 Meek, C. S., 529, 532 Meggitt, A., 554 Mehta, M.M.,457 Melhus, J. E., 812 Melin, E., 5, 274, 276, 278, 279, 281, 793 Meller, R., 375 Mello, F., 288 Mellor, VV. J., xviii Mendel, L. B., 30 Menzel, R., 350 Merker, E., 195 Merkle, F. G, 685 Merrill, E. D., 288 Merrill, G. P., xv, 646 Metzler, L. F., 534 Meusel, E., 545 Meyer, K. F., 174, 806 Meyerhof, O., 385, 390, 413, 417, 525, 528, 531 Michaelis, L., 634 Michaelsen, W., 351 Micoletzky, H., xii, 347, 349 Miehe, II., 105, 139, 140, 156, 300, 440 Miege, E., 766, 808, 814 Migula, W., x, 67, 157, 707 Millard, W. A., 36, 306, 809, 810 Miller, D. J., 670 Miller, F., 530, 743, 792 Miller, N. H., 268, 502, 529, 664, 793 Minchin, E. A., xi, 312, 337 Minkmann, D. C. J., 187, 546, 548 Miquel, P., 206, 207, 221 Mischustin, E., 157, 776 Mitscherlich, E. A., xv, 190, 432 Miyake, K., 375, 479, 495, 527, 689, 791 Miyoshi, M., 460 Mockeridge, F. A., 377, 563, 580 Moeller, A., 158 Mokradnatz, M., 649 Moler, T., 339, 572 Molisch, H., 83, 94, 385, 39S, 663 Moll, R., 781 Mollard, M., 106 Moller, A., 275, 281 Molliard, M., 262 Montanari, C., 534 Monteith, J., 812 Moore, B., 233 Moore, G. T., 126, 217, 224, 230, 236, 820 Mooser, W., 524 Morgan, J. F., 632 Morgan, M. F., 636 Morgulis, S., 735 Morison, C.B.,605 Morris, H., 342, 347, 352, 357, 380 Morris, J. L., 212 Morse, F. W., 636 Mouton, H., 316 Mulder, J. J., 522, 697 Muller, A., 125 Muller, C., 80 Miiller, K. O., 269 Miiller, O. G., 835 Muller, P. E., 332, 337, 694 Muller, P. T., 45 Miiller-Thurgau, H., 251 Mulvania, 114 Mumford, E. M., 96 Munk, M., 293 Miinter, F., 297, 298, 302, 309, 622, 784 Munro, J. H. M., 63 Miintz, A., 62, 66, 267, 480, 490, 525, 527, 531, 537, 539, 640, 646, 781 Miinz, E., 97, 406 Murdoch, F. C., 116 858 INDEX OF AUTHORS Murray, J. A., xv Murray, T. J., 153, 155, 179, 517, 784 Muschel, A., 693 Musgrave, W. E., 299 Miitterlein, C, 434, 446, 726 Myers, F. J., 351 Myers, J. T., 613 N Nabokich, A. J., 98, 403 Nadson, G. A., 82, 83, 189, 269, 611, 663 Nagaoka, M., 555 Nakamura, K., 689, 791 Nakano, H., 232 Namyslowski, B., 248 Nasir, S. M., 315, 338 Naslund, C, 301 Nathanson, A., 84, 399, 424, 606 Nawiasky, P., 212, 481, 502 Needham, 522 Negelein, E.,412,421,546 Negre, L., 156 Neide, E., 143, 148 Neidig, R. E., 382, 497 Neller, J. R., 263, 514, 685, 688, 712, 723, 799 Nelson, E.W., 221 Nencki, M.,477, 484 Neresheiner, E., 354 Nernst, W., xvii Neuberg, C, 407, 436, 468, 485 Neukirch, H., 293, 302 Newcombe, F. C, 454 Newmahn, R. O., x, 56, 288 Newton, J. D., 686 Nicolaier, A., 806 Nicolle, M., 378 Nikitinsky, J., 266, 700, 702 Niklas, N., 644, 667 Niklewski, B., 98, 99, 188 385, 403, 499, 528, 555, 709, 726, 730, 731 Nikolsky, M., 378 Noack,K., 202, 300,418 Nobbe, F., 125, 127, 129, 133, 134, 592, 750, 820 Noble, W., 174 Nolte, O., xv, 552, 777 Norris, E. R., 692 Norris, R. V., 489 Norton, J. F., xvii, xviii Northrop, J. H., 467 Nortrup-Wyant, Z., 21, 169 Novy, F. G., 171, 177 Nowikoff, M., 328 Noyes, H. A., 21, 712 O Oberlin, 750 Oden, S., 626, 628, 671, 672, 690, 698 Odermatt, W., 809 O'Donnell, F. G., 815 Oehler, R., 319, 320, 322, 328 Oelsner, A., 551, 651 Ogata, M., 167 Olaru, D. A., 534, 579, 665 Oldenbusch, C, 754 Olig, A., 478, 490 Olson, G. A., 601 Oltmanns, Fr., xi Omeliansky, W. L., xii, 66, 68, 71, 82, 96, 10S, 109, 110, 113, 118, 121, 170, 191, 199, 393, 433, 437, 445, 527, 560, 564, 565, 566, 569, 571, 585, 777, 828, 832 O'Neil, A. M., 702 Oppenheimer, C, xvi Oprescu, V., 156 Orlow, W. G., 666 Orr, M. V., 140 Orskov, J., 287, 288 Osborn, H., 362 Osborne, T. B., 30, 472 Osmun, A. V., 748 Osterhaut, W. J. V., 412 Ostwald, Wo., 626, 667 Osuga, C, 735 Otsuka, I., 613, 614 Otto, H., 265, 454, 459, 460 Oudemans, C. A. J. A., 237, 259 Owen, I. L., 375, 448, 501, 511, 539, 791 Owen, W. L., 535, 539 INDEX OF AUTHORS 859 Page, H. J., 233, 619, 690, 695 Pakes, W. C. C, 180 Palladin, V. I., xv Panganiban, E, H., 179, 777, 783 Pantanelli, E., 633, 645 Park, W. H., xii Parker, W. L., 178 Parks, 387 Pascher, A., xi, 333 Pasteur, L., 62, 160, 206, 385, 412, 837 Patten, A. J., 652 Patterson, J. W., 783 Peck, S. S., 791 Peklo, J., 270, 275, 310 Peltier, G. L., 808 Penard, E., 331 Pennington, L.H., 276 Perey, J. F., 333 Perotti, R., 211, 489, 652 Perrier, A., 693 Pervier, N. C, 452 Peter, M., 97, 204, 408, 465 Peters, L., 247 Peters, R. A., 317, 321 Petersen, J. B., 223 Peterson, E. G., 174 Peterson, W. H., 127, 157, 202, 242, 378, 379, 417, 440, 452, 454, 600, 823 Peterson, P., 685 Petherbridge, F. R., 749, 763 Petit, P., 415, 711 Petri, R. J., 171,611,614 Pettenkofer, M., 685 Pettit, H., 550 Peyronel, B., 257, 274, 277 Pfeffer, W., xv, 270 Pfeiffer, Th., 517, 550, 554, 786 Philip, J. C., xvii Phillips, R. L., 316 Pickering, S. U., 748, 761 Piemeisel, H. L., 262, 282 Pietruszczynski, Z., 665 Piettre, M., 239, 672, 690 Pillai, N. K., 120, 563, 731 Pinckney, R. M., 826 Pistchimuka, P., 482 Plimmer, R. H. A., 651 Plinius, 122 Plotho, O. V., 388, 630 Plummer, J. K., 535, 636, 792 Poche, F., 331 Polk, M., 299 Pollacci, G., 106 Popoff, L., 432 Porter, C. L., 269, 641 Potter, R. S., 263, 450, 485, 497, 621, 639, 685, 722 Povah, A. H. W., 238 Pozerski, E., 653 Powell, W. J., 453, 456 Prantl, K., 252 Pratt, O. A., 257, 259, 807 Prausnik, W., 805 Prazmovski, A., 114, 125, 565, 575 Prescott, J., 630, 784 Pribram, E., 169, 241 Pringsheim, E. G., 218, 220, 221, 232, 241,244,318,377 Pringsheim, H., 54, 110, 120, 121, 157, 190, 199, 202, 427, 431, 434, 435, 440, 446, 447, 455, 458, 462, 560, 561, 564, 569, 575, 700 Proskauer, B., 12, 34 Prowazek, S. V., xiv, 323 Prucha, M. J., 133, 597, 777, 823, 824 Pucher, G. W., 453 Puchner, H., xv Pugh, E., 106, 122 Purdy, W. C, 322 Puriewitsch, K., 269, 411 Puschkarew, B. M., 317 Q Quastel, H., 163, 414, 469, 521 Quensell, E., 603 R Rabinowitsch, L., 156 Raciborski, M. I., 269 Radais, 289 Rahn, O., 45, 47, 56, 374, 465, 517, 623, 633, 720, 739, 741, 784 Raistrick, H., 483 860 INDEX OF AUTHORS Ramann, E., xv, 237, 359, 671, 790 Randall, M., xyii, 3S6 Rao, K. A., 139, 140 Rassow, B., 456 Rathbun, A. E„ 259, 260 Rather, J. B., 672 Raulin, J., 380, 415 Ravendamm, W., 173, 203 Rayner, M. C, 272, 274, 277, 2S1, 283 Razoomov, A. S., 37 Reed, H. S., 502, 522, 581, 585, 802 Reid, F. R., 735 Reimers, J., 34 Reinitzer, F., 266, 672, 700 Reinke, J., 105, 232 Reisinger, E., 345 Remele, E., 352, 790 Remy, Th., 23, 32, 569, 572, 578, 579, 586, 589, 687, 710, 716, 829 Rettger, L. F., 155, 368, 475, 478, 482 Reuter, C, 381 Rexhausen, L., 281 Rey-Pailhade, J., 610 Riccardo, S., 110, 117 Rice, J. L., 143 Richards, B. L., 812 Richards, E. R., 499, 555 Richardson, A. C, 171 Richardson, H., 354 Richter, 808 Richter, A. A., 10 Richter, L., 133, 750, 820 Richter, O., 54, 219, 231 Richters, F., 354 Rideal, E. K., 376 Ridler, W.F.F.,277, 804 Riefenstahl, R., 429 Rippel, A., 190, 270, 588, 604, 694 Ritter, G., 269, 547, 701, 707, 742, 790 Ritter, G. J., 457, 693 Ritzema-Bas, J., 349 Rivas, D., 165 Rivett, M. F., 274, 804 Robbins, W. J., 673, 699 Robbins, W. W., 217, 224 Roberts, J. W., 246 Robertson, R. A., 700 Robertson, T. B., xvi, 320, 374 Robinson, C. S., 670 Robinson, G. W., 670 Robinson, R. H., 493, 496, 748 Robinson, W. O., 625, 627 Robson, W. P., 784 Rochaix, 208 Rockwell, G. E., 368 Roehm, H.R., 177 Rogers, L. A., 58 Rakitzkaia, A., 643 Roman, 432 Rommel, L. G., 276, 638 Rona, P., 269, 417 Root, F. M., 315 Rose, A. K., 689 Rosenbaum, J., 812 Rosenberg-Lipinsky, A., 329, 837 Rosing, G., 578, 829 Ross, R., 328 Rossi, G., 5, 203 Rossi, G. de, xiii, 130, 381 Rothe, 268 Roux, J., 335 Roux, M., 645 Roxas, M, L., 693 Rubentschik, L., 211 Rubner, M., 409, 419, 543, 610, 613 Rubner, N., 463 Rudakov, K. I., 556, 649 Rudan, B., 459 Rudolfs, W., 616, 659, 666 Ruhland, W., 101, 406 Rullmann, W., 94, 303 Rupp, P., 636 Ruschmann, G., 120, 173, 203 Russell, E. J., xiii, xv, 311, 353, 499, 523, 524, 555, 624, 639, 683, 717, 721, 722, 736, 743, 744, 746, 749, 752, 757, 763, 779, 799, 831 Russell, H. L., xiii Ryan, M., 167 Ryskaltshouk, A., 573 S Sabaschnikoff, A., 780 Saccardo, P. A., x Sack, J., 77, 199, 592 INDEX OF AUTHORS 861 Sackett, W. G., 652 Sakuma, S., 540 Salabartan, J., 543 Salter, R. M., 636 Saltet, R. H., 556 Salzmann, P., 297, 551 Sames, T., 156 Sandon, H., 32, 49, 315, 328, 329, 333, 642 Sanford, G. B., 299, 810 Sangiorgi, G., 328 Sano, K., 304 Sasaki, T., 613, 614 Saussure, Th. de, 98 Savageau, 289 Sawyalow, W., 189, 612 Scales, F. M., 197, 263, 265, 297 Schade, H., 630 Schatz, 276 Schaeffer, A. A., xii, 312, 320 Schander, 808 Schardinger, F., 462 Scheffelt, E., 328 Schellenberg, H. C., 264, 266, 443, 454, 460 Schellhorn, 352, 790 Schenker, R., 464 Scherpe, R., 751 Scheunert, A., 434 Scheurlen, E., 556 Schikhorra, 264 Schillinger, A., 156 Shirokikh, J., 186 Schittenhelm, A., 651 Schloesing, Th., 62, 156, 179, 525, 527, 534, 537 Schloesing fils, Th., 125, 660 Schmid, E., 129, 134 Schmidt, D., 382 Schmidt, E. G., 242, 379, 417, 454 Schmidt, E. \V., 448 Schmuck, A., 698 Schmlicke, R., 650 Schneider, A., xiv Schneider-Orelli, O., 779 Schneidewind, W., 105, 232, 516, 586, 746, 748, 830 Schnellmann, II., 213 Schoen, M., 469 Schoenflies, A., xviii Schollenberger, C. J., 605 Schonbein, C. F., 1S1 Schonbrunn, B., 781 Schoonover, W. R., 592, 674 Schorger, A. W., xvi, 456, 45S Schorler, B., 93 Schrader, H., 458 Schramm, J. R., 219, 232 Schrauth, W., 693 Schreiner, O., 463, 502, 522, 523, 671, 679, 690, 736, 748, 802 Schroeder, H., 98 Schroeter, F., 651 Schulov, I. C., 793 Schultz, E. S., 812 Schultz, L., 123 Schulz, K., 707 Schulze, E., 596 Schwalbe, H., 456, 45S Schwann, T., 835 Scott, H., 517 Scott, I. T., 808, 811 Scott, R. P., 783 Sears, H. J., 476 Seaver, J.F.,749, 758 Seguin, P., 175 Seidenschwartz, L., 348 Seiser, A., 545 Seliber, G., 464 Sen Gupta, N. N., 466 Senior, J. K., 467 Senn, G., 333 Sestini, F., 211, 524 Sewerin, S. A., 154, 176, 186, 1S7, 491, 655, 682 Severtzoff, L. B., 317, 319, 762 Seydel, S., 447, 567 Shanz, H. L., 262, 282 Sharp, L. T., 179, 577, 584, 634, 783 Shaw, C., 344 Shaw, II . B., 348 Shaw, W. M„ 604, 650 Shearer, C., 419 Shelford, V. E., 358 Sherbakoff, C. D., 23S, 257 Sherman, J. M., 45, 47, 326, 374 &62 INDEX OF AUTHORS Shibata, K., 138, 281, 464 Shorey, E. C, 453, 483, 474, 502, 649, 671, 690 Shouten, S. L., 55 Shunk, I. V., 132 Shutt, F. T., 598 Sieben, H., xiv Sieber, H. O., 571 Sierp, 464 Sievers, F. J., 702 Sigmond, W., 671 Simon, J., 135, 820, 823 Simon, R. H., 605 Skar, O., 10 Skinner, C. E., 153, 155, 190, 195, 261, 266, 444 Skinner, J. J., 502, 671 Skraup, Z. H., 430 Smirnow, V. G., 555 Smith, A. M.., 661 Smith, E. F., xiii, 126, 777, 806 Smith, G., 317 Smith, G.H., 58 Smith, J. H., 30, 244, 501 Smith, N., 524 Smith, N. R., 18, 56, 114, 197, 536, 539, 574, 662, 797 Smith, R. E., 32, 35 Smolik, L., 735 Snow, L. M., 36 Snyder, R. S., 263, 450, 485, 497, 621, 639, 685, 722 Sohngen, N. L., 96, 97, 158, 204, 207, 209, 298, 406, 408, 409, 488, 578, 592 630, 665 Solounskoff, M., 108, 110 Soma, S., 527 Sopp, J. O., 238 Sorensen, S. P. L., 475 Soule, M.H., 177 Souza, G., 239 Speakman, H. B., 462, 468 Spear, E. B., xvii Sperlich, 668 Sperry, J. A., 478 Spieckermann, A., 242, 478 Spinks, G. T., 814 Spratt, E. R., 139 Squires, D. H., 785 Sseverowa, O. P., 113, 565 Stahl, E., 273, 280 Stalstrom, A., 653 Stallings, J. H., 826 Stapp, C, 120, 125, 145, 213, 571 Star key, R. L., 251, 263, 268, 399, 400, 401, 606, 670, 684, 685, 701, 723, 739, 744, 770 Statkewitsh, P., 45 Stearn, 382 Steinberg, R. A., 666 Steinecke, F., 235 Steiner, A., 667 Steiner, G., 346, 347, 804, 833 Stephenson, M., 414, 464 Stephenson, R. E., 530 Stevens, J. W., 135, 137, 594, 827 Stevens, F. L., 69, 531, 536, 711, 716, 821 Stewart, R., 538, 539 St. John, J. L., 601 Stockhausen, 54 Stoddard, J. L., 167 Stokes, A. C, 335 Stoklasa, J., xiii, 29, 34, 104, 114, 155, 181, 183, 381, 444, 548, 554, 562, 570, 573, 578, 621, 631, 649, 654, 662, 667, 685, 718, 724, 730, 732, 752, 772, 783, 788, 800, 831 Stole, A., 316 Stormer, K., 12, 21, 47, 97, 134, 150, 204, 302, 744, 750, 751, 772, 820 Stranak, Fr., 562, 831 Street, J. P., 550 Strong, R. P., 803 Strowd, W. H. H.,595 Stutzer, A., 77, 105, 131, 185, 554 van Suchtelen, F. H. H., 425, 621, 681, 684, 720, 751 Siichting, H., 590, 686 Sullivan, M. X., 523, 542, 735, 736 Supniewski, J., 368, 468, 485 Suzuki, S., 550 Swanson, C. O., 598 Szilcs, J., 789 INDEX OF AUTHORS 863 Tacke, B., 184, 550 Tahara, M., 138 Takahashi, R., 259 Takenouchi, M,, 167 Tammann, G., 640 Tangl, F., 386 Tanner, F. W., xiii, 174, 250 Tappeiner, H., 432 Tarassoff, B., 497 Tartar, H. V., 493 Taubenhaus, J. J., 809 Tausson, W. O., 465 Tausz, J., 97, 204, 408, 465 Taylor, E., 329 Taylor, E. M., 761 Taylor, J. K., 106 Taylor, M. W., 257, 259 Taylor, W. W., xvii, 626 Teakle, L. J. H., 576, 700 Teisler, E., 822 Temple, J. C, 69, 530, 688, 773, 821, 823 Ternetz, C, 270, 281 Terroine, E. F., 408, 410, 415 Thaer, W., 629 Thatcher, R. W., xvi, 825 Thaysen, A. C., 460, 761 Thorn, C, 238, 245, 257, 744 Thomas, W. A., 361 Thompson, M., 352, 357 Thomson, D., 328 Thorne, C. E., 429, 470, 650, 660 Thornton, H. J., 15, 26, 317, 466 Thurlow, S., 543 Thurmann, H., 550 van Tieghem, P. E. L., 191, 212 Tilden, J. E., xi, 230 Tischutkin, N., 219 Tisdale, 128, 811 Tissier, H., 492 Tokugawa, Y., 746 Tollens, B., 427, 432 deToni,G.C.,xi,230 Topley, W. W. C, 55 Tottingham, W. E., 580, 656 Traube, W., 524 Traaen, A. E., 179, 238, 265, 537, 584, 783 Transeau, E.N.,707 Trautwein, K., 87, 88, 385, 399, 421, 547, 606 Treherne, R. C, 362 Treub, M., 234 Troop, J., 771 Truffaut, G., 37, 110, 121, 560, 565, 569, 752, 762 Truog, E., 634 Trusov, A. G., 691 Tschirch, A., 775 Tsikilinsky, P., 156 Tsujitani, J., 319 Tswetkowa, E., 183 Tubeuf, C. V., 243, 460 Tulin, A.F.,531 Turner, C. F., 362 Turpin, H. W., 799 II Ucke, A., 175 Uhland, R. E., 770 Uhlenhuth, P., xiv Ulpiani, C., 185, 211, 214, 489, 552 Ulrich, P., 247 Vahlkampf, 314 Valleau, W. D., 815 Valley, G., 368 Van Delden, A., 98, 105, 115, 121, 183, 188, 203, 204, 556, 571, 574, 611 Vanderleck, J., 33 Van der Reis, V., 789 Van der Spek, J., 634 Van Douwe, C., 354 VanHulst, J.H.,452,454 Van Sly ke, D. D., 473, 475 Van Slyke, L. L., xv Van't Hoff, J. H. 372 Van Zyl, J. P., 540 Varro, 122 Vass, A. F., 33, 496 Veillon, A., 169, 176 864 INDEX OF AUTHORS Vernadsky, W. J., 646 Verworn, M., xvi Vestal, A. G., 358 Viehoever, A. B.,208, 209 Vierling, K., 158 Viljoen, J. A., 156, 202, 440, 518 Ville, 122 Vincent, G., 410, 415 Vincent, H., 806 Vismanath, B., 489 Vitek, E., 183, 548 Vogel, J., 119, 130, 134, 183, 184, 379, 529, 544, 554, 574, 576, 616, 687, 711, 830 Vogt, E., 766 Voigt, E., 21 Voorhees, E. B., 125, 479, 480, 491, 554, 559, 799 Voicu, J., 580 Vouk, V., 462 W Wade, H. W., 288 Wadham, S. M., 807 Waget, P., 763 Wagner, F., 771 Wagner, P., 185, 553 Wagner, R., 205 Wailes, C. H., 331 Waite, H.H.,785 Waksman, S. A., xvii, 15, 19, 20, 26, 40, 43, 47, 85, 89, 90, 92, 116, 181, 190, 195, 196, 237, 240, 259, 261, 263, 266, 267, 288, 290, 297, 298, 299, 301, 304, 309, 326, 376, 379, 400, 401, 443, 444, 447, 451, 459, 476, 491, 493, 495, 509, 513, 606, 615, 638, 642, 670, 671, 685, 690, 692, 694, 699, 701, 702, 712, 716, 723, 728, 730, 735, 739, 770, 814, 843 Walters, E.H., 474, 479 Walton, J. H., 581 Walz, L., 545 Wann, F. B., 221 Warburg, O., 412, 421, 520, 540, 546 Ward, H., xii Ward, H. M., 219, 640 Ward, M., 124 Warington, R., xv, 63, 67, 396, 525, 534, 597, 795, S19 Warmbold, H., 586, 700 Warner, E. A., 487 Washburn, E. W., xvii Wasielewski, T. V., 319 Waynick, D. D., 575, 670 Webb, R. W., 262 Weber, A., 524 Weber, E., 596 Weber, G.G.A.,33 Webster, T. A., 233 Wehmer, C., 238, 694 Weigert, xiii Weimer, J. L., 418, 461 Weinberg, M., 175 Weinzirl, J., 244 Weis, F., 117 Weissenbach, R. J., 612 Weissenberg, H., 180 Weissenberg, R., 349 Weissmann, H., 501, 685 Weith, W., 524 Werkman, C. H., 802 West, G. S., xi, 82, 230 Westermann, T., 115, 585 Westling, R., 238 Wettstein, F., 216, 268 Wettstein, R., x Weyl, Th., 165 Weyland, H., 282 Wheeler, H.J. , xv, 598 Whetham,M, D.,414,464 Whipple, G. C., xii White, J. W., 530, 670 Whiting, A. L., 136, 590, 592, 596, 657, 668, 674, 821, 827 Whiting, L. C., 632 Whitney, M., 630, 749 Whitson, A. R., 534, 795 Whittaker, H., 453, 456 Whittles, C. L., 7, 22 Wiechowski, W., 486 Wiegner, G., 626, 629, 630 Wieland, H., 573 Wiesner, J. V., xvii Wiley, H. W., xv INDEX OF AUTHORS 865 Wilfarth, H., 124,788 Wilhelmy, L., 372 Williams, A. W., xii Williams, B., 581, 585 Willis, L. G., 531 Willows, R. S., xvii Willstatter, R., 456 Wilson, A., 749 Wilson, B. D., 519, 705, 793 Wilson, C. W., 325 Wilson, G. S., 55 Wilson, G. W., 259, 267, 494 Wilson, J. K., 129, 132, 519, 595, 792, 794, 796, 822 Wimmer, G., 788 Windish, F., 407 Winogradsky, S., v, 7, 8, 10, 11, 19, 55, 61, 62, 64, 65, 66, 68, 71, 72, 74, 76, 80, 92,93, 104, 107, 113, 121, 143, 152, 204, 389, 397, 531, 542, 559, 564, 572, 605, 623, 642, 702, 732, 769, 783, 843 Winslow, C. E. A., x, 58 Winter, O. B., 670 Winterstein, E., 381 Wislouch, C. N., 82, 831 Withers, W. A., 531, 536, 539, 711, 716 Wohlgemuth, J., xvii Wohltmann, F., 591 Woitkiewicz, A., 780 Wolcott, R.H.,355 Wolff, K., 181 Wolff, M., 328, 329 Wolkoff, M. I., 628 Wollenweber, H. W., 304, 809 Wollny, E., 353, 477, 683, 786, 839 Wolzogen Kiihr, C. A. H., 340, 542, 612 Woodhouse, 575 Woodruff, L. L., 318 Woods, C. D., 124, 819 Woodward, J., 601 Worden, S., 18 Woronin, M., 124 Woroshilowa, A. A, 631 Wrangell, M., 634 Wright, C.R., 517, 531, 552 Wright, D., 647, 648 Wright, R. C, 798 Wright, W. H., 127, 132, 137, 594, 825 Wulker, G., 348 Wund, M„ 148, 162 Wunschik, H., 138, 590 Wurmser, R., 408, 415, 521 Wyatt, F.A.,686 Yabusoe, M., 412 Yakimoff, M. L., 328 Yamagata, U., 117, 582 Yamazaki, E., 4S7 Yoshimura, K., 213 Young, C. C, 155 Youngburg, G., 453 Younge, C. M., 434 Z Zacharowa, T. M., 186, 551 Zapfe, M., 636 Zeichmeister, L., 456 Zemplen, G., 435 Zeren, S., 328 Ziemiecka, J., 117 Zikes, H., 94 Zirnmermann, SOS Zimmermann, A., 139 Zipfel, H., 131, 134, 597 Zolkiewicz, A. I., 269 Zopf, W., x, 380 Zschenderlein, A., 456 Zsigmondy, R., xvii, 626 Zumstein, 317 INDEX OF SUBJECTS Absidia, 248, 258, 275 Acacia, nodule bacteria of, 135, 136 Acanthocystis aculeata, 331 Acarina, 355, 357, 358 Accumulative culture method, 54 Acetaldehyde, 439, 467, 469 Acetic acid, as a source of energy, 407 formation of, by bacteria, 178, 411, 414, 440 Achnanlh.es, 227 Achromatium, 79, 82, 83 Acid soils, occurrence of algae in, 223 Acidity of soil and development of actinomyces, 42, 301, 810 Acids and solubilization of phosphates, 656-660 formation of, in soil, 437-439, 635, 672 formation of, from monosaccharides, 466 organic, utilization of, by fungi, 242 Acremoniella, 255 Acrochordbnoposita, 345 Acrostalagmus, 254, 258 Actinolaimus, 349 Actinomonas, 333 Actinomycetes (see Actinomyces) Actinomyces, 285-310, 490, 514, 636 ammonia formation by, 493 causing plant and animal diseases, 809 cellulose decomposition by, 441 classification of, 306 description of, 285 importance of, in soil, 309 nitrogen fixation by, 105 occurrence of, in manure, 30 occurrence of, in soil, 39, 259, 260 reduction of nitrates by, 181 thermophilic forms, 157 Actinomyces albojlavus, 309 albosporeus, 309 albus, 305, 307 alni, 310 annulatus, 305 aureus, 307, 309 bobili, 307 bovis, 803 californicus, 307 cellulosae, 444 chromogenus, 305, 701 citreus, 308 diastaticus, 308 erythreus, 308 exfoliatus, 308 fiavovirens, 308 flavus, 307 fradii, 309 gelaticus, 308 griseolus, 308 griseus, 308 halstedii, 305, 309 lavendulae, 307 lipmanii, 308 melanocyclus, 442 melanosporeus, 442 myricae, 138 niger, 305 olivaceus, 308 olivochromogenus, 306 pelogenes, 189 pheochromogenus, 307, 309 poolensis, 308 purpeochromogenus, 307 reliculi, 295, 307 reticulus-ruber, 307 roseus, 308 ruber, 307 rutgersensis, 308, 309 scabies, 299, 301, 306, 616, 637, 803, 814 867 868 INDEX OF SUBJECTS Actinomyces sulfur eus, 305 tricolor, 304 verne, 305, 307 violaceus, 300 violaceus-caesari, 30S violaceus-ruber, 304, 307, 309, 444 viridochromogenus, 307, 309, 379, 444 Aclinophrys sol, 331 Actinopoda, 331 Adaptation of nodule bacteria to host plant, 138 Adenine in soil, 479 Adenoplea, 345 Adineta, 351 Adsorption, in soil, 629-630 of bacteria, 631 Aerobacter aerogenes, 212 Aerobic bacteria, 4, 112, 141, 145 utilization of energy bj', 410 Aerophile bacteria, 160 Agar plate method, for counting bac- teria, 13 for counting protozoa, 45 for isolation of algae, 219 Agar tubes, deep, use of, in the isola- tion of anaerobic bacteria, 164, 168 Agaricus, 243, 261 Agricere, in soil, 465 theory of soil fertility, 759-761 Agriotes, 356, 35S Agroceric acid, 671 Agrosterol in soil, 671 Air-drying of soil, influence of, on activities of microorganisms, 539, 740-743 Alaimus*, 350 Albumin agar, 16 Alcohol, formation by microorganisms, 412, 413, 440. oxidation of, by bacteria, 99 utilization by fungi, 242 Aldehydes in soil, 671 Alfalfa, nodule formation by, 136 Algae, in soil, 3, 50, 215-235 composition of, 378 corroding action of, 645 cultivation of, 218 distribution of, in soil, 222 Algae, isolation of, in pure culture, 221 nitrogen fixation by, 105, 121, 232-233 role of, in the soil, 234 Algal fungi (see Phycomycetes) Alinit preparation, 105, 831 Aliphatic hydrocarbons, decomposi- tion of, 465 Alkali soils, neutralization by sulfur oxidation, 616 occurrence of algae in, 224 Alkaline pyrogallol solution, 170 Alkaloids, content of, in leguminous plants, 596 use of, in stimulation of bacteroid formation, 131 Allantoin, formation of, in the decom- position of uric acid, 212, 486 Allantion tachyploon, 333 Allocoelae, 345 AUogromia fluvialis, 332 Alloloboplwra, 352 Alnus, bacteria in, 105, 138 Aloin, as indicator of oxidation in soil, 523 Alsike clover, nodule bacteria of, 136 Alternaria, 244, 256, 258, 265, 779, 807 Aluminum, transformation of, in soil, 666 Amanita, 276, 278 Atablyosjyorium, 253 Amides, decomposition of, 485 Amines, formation by bacteria, 178 Amino acids, as sources of energy, 419 formation of, in synthetic processes, 424 Amino nitrogen, as index of protein decomposition, 476 Ammonia formation, as index of pro- tein decomposition, 475, 501, 685 as influenced by presence of non- nitrogenous organic substances, 504 by bacteria, 178, 4S9 by fungi, 266,376,493 chemistry of, 480 from different organic materials, 511, 512 in the reduction of nitrates, 182 INDEX OF SUBJECTS 869 Ammonia formation, rate of, 375, 495, 688 factors influencing, 688-689 methods of determination, 497 Ammonia oxidation, by chemical agen- cies, 524 mechanism of, 525 Ammonification studies, 687 (see also Ammonia-formation) Ammonium salts, nitrification of, 715 Amoeba, 331, 332, 337 Amoebae, in soil, 2, 47, 315, 326, 328 cultivation of, 319 Amoebida, 331 Amorpha, nodule formation by, 137 A?nphicarpa, nodule formation by, 137 Amphileptus, 335 Amylobacter group of bacteria, 106 (see Bac. amylobacter) Amylomyces boidin, 462 Amystidae, 355 Anabaena, in soil, 217, 224, 227 in the roots of cycads, 138, 302 Anachaeta, 354 Anacystis, 226 Anaerobic bacteria, 4, 141, 160-179 classification of, 172 cultivation of, 169 decomposing celluloses, 166, 437 decomposing pectins, 203 facultative, 160 isolation of, 164 nitrogen-fixing, 107 numbers of, in soil, 38, 175 obligate, 163 physiology of, 176 Anaerobic conditions in soil, 164 utilization of energy, 412 Aniline dyes, use of, in the separation of soil bacteria, 166 Anguillula, 350 Animal diseases caused by, actinomyces in soil, 809 bacteria found in the soil, 805 Animal ecology, 341 Anisonema, 334 Ankistrodesmus, 228 Annelida in soil, 341, 351, 354, 359 Anorgoxydants, 61 Anthracnose of beans in soil, 815 Anthracriny, 694 Antiseptics, volatile, influence of, on bacterial activities, 744-745 749- 759 Aphanethece, 226 Aphanocapsa, 226 Aplianomyces, 247, 808, 813 Aphelenckus, 348, 349, 810 Apocrenic acid, 697, 698 Arachnida, 354, 355, 357, 359 Arachnis, nodule formation by, 136 Arbutus unedo, mycorrhiza formation by, 274, 804 Arcella, 332 Arcellidae, 332 Archiboreoiidus, 355 Archionchus, 350 Archivortex, 345 Ardisia, bacteria in, 105, 139 Areinida, 355, 357 Arid soils, distribution of bacteria in, 36 Arginine in soil, 479, 671 Armillaria, 276, 284 Arsenic compounds, oxidation of, 540 reduction of, 303, 540, 556 influence of, on bacterial activities, 534, 668 Arthobotrys, 255 Arthropodainsoil, 341, 354, 359 Ascomycetes, 3, 249, 250, 259, 277 Asparaginate agar, 16 Aspergillaceae, 242, 267 Aspergillus, in soil, 3, 238, 245, 251, 253, 258, 259, 260, 265, 443, 459 flavus, 454, 465 fumigatus, 444, 454, 495, 817 fuscus, 444 glaucus, 444 niger, 243, 244, 380, 382, 411, 415, 416, 417, 454, 462, 464, 467, 494, 503, 511, 514, 637, 650, 652 oryzae, 261, 454, 462 repens, 454 terricola, 261, 490, 637 wentii, 444 870 INDEX OF SUBJECTS Aspidisca, 336 Associative action of bacteria in the reduction of nitrate, 186 Associative growth of legumes and non-legumes, 599 Assulina, 332 Astasia, 334 Aslerocystis radicis, 278, 808 Atmosphere of soil, 638 Atmospheric nitrogen, fixation of, 103 Aulosira, 227 Autocatalytic reaction in the growth of microorganisms, 320, 373-376, 527 Autochtonous microflora of soil, 10, 151 Autotrophic bacteria, in soil, 4, 59, 61-102, 369 classification of, 388 Autotrophic protozoa, 315, 316 Auximones, 5S0, 699 Azolobacter, 59, 105, 106, 110, 111, 112-122, 138, 164, 447, 559, 560, 563, 564, 565, 575, 579, 581, 594, 623, 630, 637, 641, 642, 656, 662, 699, 708, 779, 828 agile, 105, 114, 117, 118, 572 beijerinckii, 115, 117, 118, 582 chroococcum, 105, 107, 112, 114, 116, 117, 118, 119, 155, 183, 441, 562, 566, 567, 572, 575, 578, 579, 582, 654, 694, 762 vinelandii, 115, 116, 117, 118, 572, 582 vilreum, 115, 117 Azolobacter, as food for amoebae, 319 composition of, 381, 570, 571 energy utilization by, 419, 561, 569 fixation of nitrogen by, in presence of protozoa, 338 life cycles of, 117 nitrogen-fixation by, 560, 567 numbers of, in soil, 37 occurrence in soil, 10, 11, 117 physiology of, 119 protein synthesis by, 570 species, description of, 114 B Bacillariaceae, 215, 225 Bacillariales, 227, 230 Bacilli in soil, 11 Bacillus, 58 aceloethylicum, 467 adhaerens, 144 aerogenes capsulatus, 173 agri, 144, 150 albolactus, 144 alvei, 146 amylobacter, 37, 59, 104, 106, 107, 110, 146, 161, 162, 164, 167, 172, 173, 175, 178, 191, 203, 204, 447, 462, 549, 558, 579, 582, 623, 630, 637, 641, 646, 708, 779, 828 (see also Clostridium pastorianum) amylocyme, 172 anthracis, 146, 803, 805 arborescens, 490, 762 asterosporus, 105, 120, 144, 146, 149, 162, 203, 383, 558 alerrimus, 143 azophile, 121 bifermentans, 174, 492 botulinus, 174, 803, 806 brevis, 145, 150 butyricus, 172, 762 calf actor, 157, 441, 775 capri, 146, 213 carotarum, 146, 149, 214 carolovorus, 203, 461 cellulosae dissolvens, 173, 194, 203 centrosporus, 145 cereus, 10, 27, 142, 144, 145, 149, 150, 382, 444, 462, 491, 500, 642 cereus, var. fluoresceins, 144, 150 chauvoei, 161, 174, 805 circulans, 145 cobayae, 146, 213 cohaereus, 144, 146, 149, 150 comesii, 203 danicus, 120 ellenbachensis, 104, 144, 146, 149, 833 enteritidis sporogenes, 173 felsineus, 173, 178, 203, 204 INDEX OF SUBJECTS 871 Bacillus ferru-gineus, 195 jlavigena, 455 foliicola, 140 fossicularum, 193 fusiformis, 118, 145, 146, 149, 150, 162 globigii, 143 gracilis pulidus, 492 granulobacter pectinovorum, 462, 468 graveolus, 146, 149 guano, 147, 213 histolyticus, 174 hollandicus, 147, 214 hydrogenes, 101, 404 janthinus, 490 kramerii, 203 lacticola, 147 Jactts, 147, 162 iach's niger, 144 laterosporus, 145 luteus, 147 macerans, 203 malabarensis, 120, 149 megatherium, 10, 104, 120, 142, 145, 147, 149, 150, 181, 208, 507, 515, 762 mesenlericus, 120, 142, 143, 149, 150, 155, 203, 212, 462, 641, 642 mesentericus var. flavus, 144, 150 mesenlericus fuscus, 143 mesentericus niger, 143, 693 mesentericus panis viscosus, 144 mesentericus ruber, 143, 150 mesenten'cws vulgatus, 143, 183, 490, 656 methanicus, 96, 100, 406 methanigenes, 193 musculi, 147, 213 mycoides, 105, 142, 144, 145, 147, 149, 150, 155, 162, 182, 208, 490, 491, 507, 508, 604, 631, 633, 642, 762 niger, 144 nitroxus, 187, 549 oedematis maligni, 174 oligocarbophilux, 98 orthobutylicus, 109, 172 oxalalicus, 147 panis, 144 parvus, 147 Bacillus pastorianus, 173 perfringens, 173, 209, 492 pestifer, 181 petasiles, 145, 147, 149, 150 phytophthorus, 806 prausnitzii, 144 probatus, 147, 208, 209, 210 pseudotetanicus, 145 pseudoletanicus var. aerobius, 145 pumilus, 118, 147, 149 putrificus, 111, 174, 175, 209, 490, 492, 612, 637 pycnoticus, 100, 101, 637 radicicola (see Bad. radicicola) ramosus, 181, 701 rawosus liquefaciens, 144 robustus, 147 ruminatus, 145, 149 schirokikhi, 187 silvalicus, 147 simplex, 144, 147, 149, 150, 162 sphaericus, 147 sporogenes, 173, 174, 178, 492, 612 sm6{i7{s, 96, 142, 143, 148, 149, 150, 162, 164, 182, 183, 203, 205, 368, 382, 383, 418, 462, 478, 489, 490, 506, 507, 508, 514, 637 subtilis-viscosus, 143 feres, 144, 148 lelom in soil, 148, 161, 174, 803, 805, 806 thiogenes, 82 tostus, 148 tumescens, 148, 149, 491 ureae, 208 vulgatus, 143, 149, 150, 182, 184, 212, 444, 507, 701 welchii, 173, 178, 803 Bacteria, composition of, 378 decomposing celluloses, 190, 195, 200, 201 fixing atmospheric nitrogen, 103 in leaves of plants, 139 in roots of legumes, 122 in roots of non-leguminous plants, 138 life cycles of, 57 numbers of, in soil, 28-29 872 INDEX OF SUBJECTS Bacteria, numbers of, in manure, 29- 31 oxidizing carbon compounds, 96 oxidizing hydrogen and its com- pounds, 98 oxidizing iron compounds, 92 oxidizing nitrogen compounds, 62 oxidizing sulfur and its compounds, 78 reducing nitrates to nitrites, 180 reducing nitrates to ammonia, 182 reducing nitrates to nitrogen gas, 183 requiring combined nitrogen, 141 Bacteriaceae, 58, 78 Bacterio-chlorin, 83 Bacteriophage in nodules of legumi- nous plants, 592 Bacterio-purpurin, 83 Bacteriotoxin theory, 759 Bacteriotoxins in soil, 859 Bacterium, 58 acidi lactici, 30, 155, 648 acidi urici, 212 aerogenes, 59, 119, 126, 153, 155, 382, 582 aliphaticum, 204, 465 aliphaticum liquefaciens, 204, 465 anlhracoides, 155 bovista, 82 calco-aceticum, 212 caudatum, 153, 154, 642 cellar esolvens, 201 centropunctatum, 187 cloacae, 155 coli, 153, 154, 155, 157, 163, 182, 184, 186, 209, 319, 440, 320, 382, 419, 492, 542, 612, 614, 631, 637, 641, 762, 775 denitrificans , 88, 185, 186, 187 denitrificans agilis, 186, 187 denitrofluorescens, 186 erythrogenes, 209, 211, 212, 488 extorquens, 646 filiformis aerobius, 492 fimi, 444 fluorescens, 27, 30, 107, 151, 154, 182, 186, 203, 205, 209, 320, 323, 379, 464, 491, 492, 500, 514, 604, 642 Bacterium Jluorescens liquefaciens, 97, 155, 186, 209, 212, 465, 490, 656, 762 Jiuorescens pulidum, 490 freudenreichii, 212 fulvum, 155, 187 guntheri, 30 hartlebii, 186, 187 herbicola, 30, 182 hexacarbovorum, 97 kirchneri, 211 kunnemanni, 187 lactis viscosum, 118, 120, 126, 154 leguminosarum, 126 lipolyticum, 204, 464 nilrovorum, 187 ochraceum, 154, 155 odoratum, 212 opalescens, 201 parvulum, 500 pcstis, 805 pneumoniae, 59, 120, 126, 155 porticensis, 182, 187 praepollens, 186, 187 prodigiosum, 120, 154, 163, 171, 182, 184, 209, 212, 383, 464, 490, 631, 760 proteus (see Bad. vulgar e) punctatum, 154, 155 putidum, 118, 155, 182, 545 pyocyaneum, 97, 182, 186, 204, 212, 368, 418, 464, 467, 468, 485, 549, 637 radicicola, 39, 120, 122-138, 139, 383,558, 593, 597, 637, 656, 708, 818, 823, 824 colony formation, 130 isolation of, from nodules, 128 isolation of, from soil, 129 media for cultivation of, 126 morphology and life cycles, 130 motility of, 132 nitrogen fixation by, 592, 597 nodule formation by, 128 nomenclature, 125 numbers of, in soil, 129 physiology of, 133 specific differentiation of, 134 stages of development, 125 staining of, 132 INDEX OF SUBJECTS 873 Bacterium radiobacter, 119, 120, 126, 447, 642 solanacearum, 806, 814 stutzeri, 186, 187, 204, 212, 549, 637, 762; tumefacier 806 typhi, 805 typhosum, 777, 803, 806 ulpiani, 187 umbilicatum, 154 ureae, 209, 211 violaceum, 154 viscosum, 201 vulgare, 121, 154, 155, 163, 181, 182, 184, 209, 212, 368, 485, 489, 490, 492, 507, 508, 612, 613, 614, 637, 663, 762 vulpinus, 186 zopfii, 154 Bacterized peat, 831 Bacteroids, 126, 130, 140 Bactridium butyricum, 161 Balantiophorus, 335 Baptisia tinctoria, nodule formation by, 136 Barber pipette method, for isolation of actinomyces, 305 for isolation of algae, 218, 220 for isolation of bacteria, 55 for isolation of protozoa, 320 for isolation of spores of sfungi, 246 Basidiomycetes in soil, 3, 243, 257, 260, 277, 278, 282, 459 Bastiana, 350 Beaker method, 687 Beetles in soil, 361 Beggiatoa, 79, 80, 81, 100, 397, 398, 606 Benzene ring compounds, as a source of humus in soil, 692 decomposition of, by bacteria, 204, 465 Benzoic acid, formation from hippuric acid, 212 Benzol, oxidation of, 97, 205 Bersem clover, nodule bacteria of, 136 Betaine, 486 Bibionidae, 358 Biochemical activities of algae, 230 Biomyxa vagans, 331 Biotypes in nodule bacteria, 137, 824 Bispora, 256 Black locust, nodule bacteria of, 137 Black medick, nodule bacteria of, 136 Blaniulus, 355 Blepharisma, 336 Blue-green algae (see Cyanophyceae) Bodo, 330, 333 Boletus, 257, 276, 278 Boron, influence of, on Azotobacter, 581 Botrydium, 229 Botryosporium, 229, 253 Botrytis, 3, 244, 254, 258, 264, 266, 779 Brackydesmus, 355 Braconidae, 361 Broad bean, nodule bacteria of, 136 Buffer content of soil, 635 Bumilleria, 225, 229 Bunonema, 347, 350 Bur clover, nodule bacteria of, 136 Butyl alcohol, formation of, by bac- teria, 178 Butyric acid bacteria, 106, 108, 155, 172, 175 in manure, 30 numbers of, in soil, 38 Butyric acid, formation of, by bacteria, 178,204,412,439 Caconema radicicola, 348, 349 Caffeine, use of, for stimulation of bacteroid formation, 131 Calcium, accumulation of, by bacteria, 663 as a bacterial nutrient, 664 influence of, on biological activities in soil, 689 (see also Lime) cyanamide, decomposition of, 211 influence of, on nitrogen-fixation, 576 oxide as an agent of partial steriliza- tion, 743 transformation of, in soil, 662-664 Callidina, 351 Calluna vulgaris, mycorrhiza formation by, 276, 277, 281,283 Calothrix, 227 874 INDEX OF SUBJECTS Campascus, 332 Canada field pea, nodule bacteria of, 136 Canthocamptus, 355 Carabidae, 356, 361 Carbohydrates, decomposition of, by microorganisms, 409, 564, 678 influence of, on protein decomposi- tion, 511 utilization of, by nitrogen-fixing bacteria, 561 Carbon bisulfide, influence of, on bi- ological activities in soil, 749, 754, 762 Carbon dioxide, content of soil at- mosphere, 640, 682 evolution, 376, 445, 515, 621, 681-685, 688-689, 701, 720, 721 and energy utilization, 425, 566 as an index of biological activities in soil, 717-725 Carbon monoxide as a source of energy for bacteria, 59, 98, 407 Carbon-nitrogen ratio, in medium, as affected by different groups of organisms, 513 in soil, 509, 510,702-707 of organic matter, 514, 687, 704, 770 Carbon, oxidation of, by bacteria, 98 sources for bacteria, 368 transformation by microorganisms, 504 utilization of, by actinomyces, 296 utilization of, by fungi, 242 Carboxydomonas oligocarbophila, 98 Cardinal points for growth and spore formation of obligate anaerobic bacteria, 162 Carotin in algae, 215 Carrots as reducing agents, 171 Carychium, 359 Cassia, nodule bacteria of, 135, 136 Casuarina, nodule formation by, 139 Catalase formation by anaerobic bac- teria, 163 Catalytic action of soil, 734-736 Ceanothus, nodule formation by, 105, 138, 139 Cellulase, 435, 436 Cellulose agar medium, 197, 297 Cellulose as a source of energy, 297, 448 for nitrogen-fixing bacteria, 447 Celluloses, chemistry of, 427-432 Cellulose decomposing bacteria, 27, 38, 60, 154, 175, 190, 448 decomposing capacity of soil, 725 decomposition and CO2 evolution, 725 decomposition and nitrogen assimi- lation, 727 decomposition by aerobic bacteria, 195-200, 434, 439 decomposition by anaerobic bacteria, 166,173,190-195,432,437 decomposition by denitrifying bac- teria, 188, 200-201, 441 decomposition by herbivorous ani- mals, 434 decomposition by thermophilic bac- teria, 173, 201-203, 435-436, 439- 441,445 decomposition by actinomyces, 441 decomposition by fungi, 242, 263, 443-444 decomposition by protozoa and other invertebrate animals in manure, 444 decomposition in soil, importance of, 446, 683 decomposition, influence of soil con- ditions on, 448 Cellulose, determination of, 431 influence of on soil microorganisms, 769 preparation of, 197 Centricae, 227 Centropyxis, 332 Cephalobus, 347, 350 Cephalosporium, 253, 258, 277, 808 Cephalolhamnion, 334 Cephalolhecium roseum, 244, 490, 779 Cercobodo, 333 Cercomonas, 330, 333, 337 Cercospora per sonata, 808 Cerotoma, 362 Chaetocladiaceae, 249 INDEX OF SUBJECTS 875 Chaetocladium, 249 Chaelomella, 257 Chaetomium, 3, 238, 250, 251, 258, 260, 264 Chaetonatus, 351 Chamaesiphonaceae, 226 Chamaesiphon, 226 Chambers apparatus for isolation of bacteria, 56 Charophyta, 230 Chemical activities of microorganisms, 369 agents, influence of, on protozoa, 325 composition of the microbial cell, 377 Chilodon, 335 Chilomonas, 334 Chilopoda, 355, 357 Chinon, action of, on bacteria, 165 Chironomidae, 358 Chitin in the microbial cell, 382, 489 Chitinase, 489 Chlamydomonas, 211, 228, 233, 334 Chlamydophrys, 332 Chlorella, 228, 232, 233 Chlorochytrium, 228 Chlorococcum, 224, 225, 228, 233 Chloroform as an agent of partial sterilization of soil, 754 Chlorogonium, 217, 334 Chlorophyceae, 3,215,220, 224, 225, 228 Chlorophyll in algae, 215 Choenia, 335 Cholesterols in soil, 671 Choline in soil, 479, 485 Chordatainsoil, 342 Chordeuma, 355 Chromatium cuculliferum, 83 Chroococcaceae, 226 Chroococcus, 226 Chrysamoeba, 334 Chrysomonadinae, 334 Ciliciopodium, 256 Ciliates, in soil, 3, 48-49, 314, 328, 335 cultivation of, 319 Citric acid, 410 Citrate-glycerin agar, 291, 306 Cladomonas, 334 Cladophora, 224, 228 Cladosporium, 155, 256, 258, 264, 265, 278, 763 Cladothrix, 287, 288 Classification, of actinomyces, 294-290 of anaerobic bacteria, 172 of autotrophic bacteria, 388 of soil animals, 341 of soil bacteria, 58-60, 141, 143, 151 of soil protozoa, 313, 329 Clathrulina elegans, 249, 331 Clostenema, 334 Clostridium, 59, 106, 107, 110, 115, 178, 204, 594 americanum, 110, 172, 447 butyricum, 161, 173 pastorianum, 37, 104, 108, 109, 110, 111, 112, 120, 166, 172, 173, 558, 559, 560, 564, 566, 569, 641, 642, 752 (see also Bacillus amylobacler) thermocellum, 173, 194, 195, 202, 203 Clover, composition of, 428 Club-root of cabbage, 236 Coccaceae, 58 Cocci in soil, 11, 141, 154 Coccomyxa, 228 Coccospora, 252 Codosiga botrytis, 333 Coelhelminthes in soil, 342, 488 Cohnistreptothrix, 294 Coleoptera, 356, 357, 358 Coleps, 335 Collembola, 356, 357, 358, 361 Colletolrichum, 807 Colloidal condition of soils, 33, 626 nature of soil humus, 697 ^ | Colloids, influence of, on the growth of microorganisms, 578, 622, 627, 630 properties of, 627-631 Colon group of bacteria in the soil, 155 (see also Bad. coli) Colony isolation, 167 Colpoda, 324, 330, 335 Colpidium, 317, 318, 321, 335, 338, 637 Colponema, 333 Commensalism, 560 Commercial cultures of nodule bac- teria, 822 876 INDEX OF SUBJECTS Composition, chemical, of the micro- bial cell, 377 of vegetable organic matter, 427 Composts, of greensand and sulfur, 661 of sulfur and phosphate, 614 Conductivity of soil, 633 Condylosloma, 336 Conidiophorae, 249 Conifers, mycorrhiza formation by, 272 Conjugatae, 229 Control of plant diseases, 814 Copepoda in soil, 342, 354 Coprinus, 243 Coriaria, nodules of, 105, 138 Corn cobs and fodder, composition of, 428 Corn root rot, temperature of control, 815 Corticium vagum, 807, 813 Cortinarius, 276, 278 Corycia, 332 Corynebacteria, 289 Corythion, 332 Coscinodiscus, 227 Cothurnia, 336 Cow clover, nodule bacteria of, 136 Cowpea, nodule bacteria of, 136 Craspedosoma, 355 Creatinine in soil, 479 Crenic acid, 697 Crenothrix, 93 Cresol, decomposition of, 205, 465 Crimson clover, nodule bacteria of, 136 Cropping of soil, influence of, on micro- biological activities, 712, 800 Cross-inoculation of nodule bacteria, 134 Cruciferae, mycorrhiza formation by, 273 Crude fibre, 429 Crustacea in soil, 342, 354, 357 Cryploglena, 334 Cryptomonadaceae, 215 Cryptomonadinae, 334 Cryptomonas, 334 Cultivation, of algae, 221 of soil, influence of, on biological activities, 743, 784-787 Cultivation, of soil, influence of, on nitrogen-fixation, 585 of soil, influence of, on numbers of microorganisms, 40 Cultural methods for study of soil bacteria, 11, 12 Culture media for counting soil bac- teria, 13-19 Cunninghamella, 249, 444, 454 Curculionidae, 361 Cutins, 460 Cutworms in soil, 362 Cyanamide, decomposition of, by bac- teria, 211, 487, 488 decomposition of, by fungi, 267 Cyanophyceae, 3, 215, 220, 223, 224, 225, 226, 230, 645 Cyanuric acid in soil, 479 Cyatholaimus, 349 Cyathomonas, 334 Cycas, nodule formation by, 138, 139 Cyclidium, 335 Cyclotella, 227 Cylindrocystis, 228 Cylindroiulus, 355 Cylindrospermum, 217, 227 Cymbella, 227 Cyperaceae, mycorrhiza formation by, 273 Cystine, 600, 613, 614 Cystococcus, 231 Cysts of protozoa, 46-50, 316, 325 Cytase, 454 Cytosine in soil, 479, 671 Czapek's solution, 243, 291 D Dactylococcus, 228 Dalea alopecuroides, nodule bacteria of, 137 Damping-off fungi in soil, 807 Deaminization, 473 Death of microorganisms, 372 Decarboxylation, 482 Decay, 477, 522 Decomposition, of manure, 496 of organic matter in soil, 500, 680 INDEX OF SUBJECTS 877 Deep colony procedure in isolation of bacteria, 168 Deflocculating agent in preparation of soil suspension, 22 Dematiaceae, 3, 255, 267, 443 Dematium, 244, 256 Dematophora, 264, '814 Denitrification, 180, 544, 550 Denitrifying bacteria, 60, 180, 185 cellulose decomposition by, 441 numbers of, in soil, 37, 38 Depths of soil, and distribution of actinomyces, 40 and distribution of bacteria, 34-36 and distribution of fungi, 259 and distribution of protozoa, 48-49 Desiccation of bacteria, 583 Desmidium, 228 Desmodium canescens, nodule bacteria of, 136 Desmobacteriaceae, 78 Dextrans in the microbial cell, 383 Diaschiza, 351 Diatoms, occurrence of, in soil, 223, 227, 645 Dicoccum, 256 Dicyanodiamide, decomposition by bacteria, 488 influence of, on nitrate formation, 533 Difflugia, 332 Difflugidae, 332 Diflugiella, 332 Dihydroxystearic acid in soil, 463, 671 Dileptus, 335 Dilution method for counting protozoa in the soil, 45-47 for determining numbers of soil bacteria, 13, 27 in the isolation of soil bacteria, 55, 167 Dilutions of soil in preparation of plates, 18, 21-23 Dimastigamoeba gruberi, 330, 337 Dinoflagellata, 215, 230, 335 Dinornonas, 333 Diplogaster, 347, 349 Diplophrys, 332 Diplopoda, 355, 357 Diptera, 356, 357, 358 Direct method of isolation, of nitrite- forming bacteria, 71 of soil bacteria, 55 Direct microscopic methods for study of soil bacteria, 7-11 Discomyces, 287, 288 Discomycetes, 251 Discorea macroura, bacteria in, 140 Disinfectants (see Antiseptics) Distigma, 334 Distribution of bacteria in soil, 34-36 Dorylaimus, 346, 348, 349 Drying, influence of, on actinomyces, 299 of soil and increase of soluble salts, 742 Dydymopsis, 278 Dyer's greenwood, nodule bacteria of, 136 Dyes, as indicators of oxidation-reduc- tion potential, 542 influence of, on the growth of acti- nomyces, 302 E Earthworms, as carriers of pathogenic bacteria, 805-806 occurrence of, in soil, 3, 342, 351-353 Economic coefficient, 417 Ectotrophic mycorrhiza, 271, 280, 281 Edaphon, 642 Egg-albumin agar, 16 Eisenia, 352 Elateridae, 361 Eleagnus, nodule formation by, 138, 139 Enchytraeids in soil, 342, 353 Enhytraeus, 354 Enchelys, 335 Endogone, 277 Endotrophic mycorrhiza, 140, 271, 280 Energy, of ionization, 387 liberation in microbiological proc- esses, 422 -nitrogen ratio, 506 Energy source for non-symbiotic nitrogen fixation, 561-553, 569 878 INDEX OF SUBJECTS Energy transformation in the metabo- lism of microorganisms, 384-426 transformation in the soil, 425 utilization by autotrophic bacteria, 384, 387 utilization by heterotrophic bacteria, 384, 704 utilization by nitrogen-fixing bac- teria, 566 utilization, efficiency of, 415 Enrichment culture method, 54, 215 Environmental conditions, influence of, on symbiotic nitrogen fixation, 597 Entosiphon, 334 Enzymes of microorganisms, 371, 436 Eparidaceae, mycorrhiza formation by, 271 Epicoccum, 257 Epistylis, . 336 Equilibrium, between carbon and ni- trogen in soil, 702, 704 microbiological, in soil, 6, 738-740, 757, 764, 767 theory of symbiotic nitrogen fixation, 590 Ericaceae, mycorrhiza formation by, 271, 272, 273, 276, 281, 283 Errors in plate counts of numbers of microorganisms, 24 Erythrosine stain for soil bacteria, 8 Ethyl alcohol, formation of, by bac- teria, 178 Euasci, 251 Euastrum, 229 Eubacteria, 58, 78, 92 Euglena, 315, 317, 334 Euglenaceae, 215 Euglypha, 332 Euglyphidae, 332 Eumycetes, 236, 246 Euplotes, 336 Eutreptia, 334 Eulylenchus, 350 Evolution in plants, 273 Exhausted soils, 709 Facultative anaerobic bacteria, 160 autotrophic bacteria, 59, 96, 101, 399 Fairy rings, 262, 293 Fat-splitting bacteria, 204 Fat utilization, by actinomyces, 297 by fungi, 242 Fats in soil, 463, 671 Fauna of soil, 341-363 Feces as a source of bacteria in soil, 29, 176 Feldspars, decomposition of, by bac- teria, 646 Fenugreek, nodule bacteria of, 136 Fermentation, 385, 413 Fementation of nitrates (see Denitri- fication) Ferrous sulfate, as reducing agent, 171 Fertilizers, influence of, on biological activities in soil, 733, 788 influence of, on soil reaction, 635 Filamentous fungi (see Fungi) Fixation of nitrogen, 103, 558 Fixative agents, 323 Flagellata, 215, 230, 313 Flagella staining, 132 Flagellates in soil, 3, 47-50, 326, 328 Flagellation of nodule bacteria, 132 Flatworms, 341, 345 Fleming's solution, 346 Fluctuation of bacteria in soil, 32 Flukes in soil, 341,345 Fluorescent bacteria in the soil, 154 Forest soils, formation of humus in, 694 occurrence of algae in, 224 Formaldehyde as intermediary prod- uct in CO2 assimilation, 403 Formic acid, formation of by bacteria, 414, 440 Formicidae, 358 Fragillaria, 227 Fredericia, 354 Free energy, 386 Free-living nematodes in soil, 348 INDEX OF SUBJECTS 879 Freezing of soil, influence of on bac- terial activities, 777 Frozen soil, numbers of bacteria in, 32 Fulvic acid, 698 Fumaric acid, formation of, by bac- teria, 414 decomposition of, 469 Fumigants of soil, 362 Fungi, activities of fungi in soil, 260 ammonia formation by, 266, 493 carbon and nitrogen assimilation by, 413 causing plant diseases, 806 cellulose decomposition by, 238, 260, 263, 443 composition of, 379 cultivation of, 241 decomposing uric acid, 212 decomposition of organic matter by, 708 destruction of, by partial steriliza- tion of soil, 757, 763 germination of spores and reaction of medium, 637 influence of reaction on, 237, 239, 262, 637 isolation of, 237, 245 media for isolation and cultivation, 19, 239-244 methods of studying soil fungi, 239 mycelium of, in soil, 11, 42 mycorrhiza fungi, 238 nitrogen-fixation by, 105, 238, 270 nitrogen utilization by, 267 numbers of, in soil, 42-44, 237 occurrence of, in soil, 3, 27, 236, 259 reduction of nitrates by, 181 relation of, to plant diseases, 806 spores of, in soil, 42 thermophilic, 157 Fungi imperfecti, 252, 259 Fungus agar medium, 19 mycelium in soil, 11, 268 Furfural as a source of humus, 693 Fusarium, 155, 238, 257, 258, 259, 260, 264, 265, 278, 443, 444, 763, 807 batatatis, 808 bullatum, 261 Fusarium conglulinans, 808, 811 hyperoxysporium, 808 lint, 803, 808, 811 lycopersici, 808, 811, 814, 815 martii ver. pisi, 813 moniliforme, 808 oxysporum, 261, 637, 803, 807 radicicola, 244, 779, 803, 806 vasinjeclum, 264 G Galactans in the microbial cell, 383 Gallionella, 93, 94 Garden bean, nodule bacteria of, 136 Gas illuminating, utilization of, by bacteria, 97 Gas, indifferent, for air replacement, 171 Gases of soil, composition of, 639 influence of, on nitrate formation, 534 Gasteromycetes, 275, 276 Gastrodia elata, 284 Gastropods in soil, 342, 359 Gastrostyla, 336 Gelatin liquefying bacteria, 14, 151, 153 Gelatin media, 16, 291, 306 Genista tinctoria, nodule bacteria of, 136 Gentian-violet, inhibitive action of, on the growth of aerobic bacteria, 165 Geocentrophora, 345 Geomyces, 254 Geotrichum, 252 Geophilis, 355 Geophilomorph, 355 Gephyramoeba delicatula, 331 Gibberella saubinetii, 637 Glaucoma, 335 Glenodinium, 335 Gliocladium, 253 Gloeococcus, 228 Gloeocopsa, 226 Gloeocystis, 228 Glomeris, 355 Gluconic acid, 410 Glucose, as a reducing agent, 171 decomposition of, in soil, 683, 725 880 INDEX OF SUBJECTS Glucose, influence of, on ammonia formation from proteins, 50S influence of, on development of microorganisms, 769 Glucose agar, 291, 306 Glucosides, decomposition of, 466 Glucuronic acid, 410 Glycerides in soil, 67 Glycine, nodule bacteria of, 130, 134, 137 Glycocoll, as asource of energy, 411 formation from hippuric acid, 212 Gunatobotrys, 254 Gongrosira, 228 Gonidia formation by bacteria, 57 Gonostomum, 336 Gramineae, mycorrhiza formation by, 274 Granulobacter, 95, 105, 106, 109, 119, 120, 178, 204 pectinovorum, 73, 110 saccharobutyricum, 172 Graphium, 256 Grass-green algae (see Chlorophyceae) Grass lands, insect fauna in, 356 Green manure, influence of, on seed germination, 802 Greensand-sulfur compost, 661 Gromia, 332 Gromiidae, 332 Growth, of microorganisms, 372 promoting substances, influence of, on nitrogen fixation, 580 Grubs in soil, 361 Grumilea, nodule formation by, 139 Guanidin, decomposition of, by bac- teria, 214, 486 Guanin, decomposition of, by bacteria, 214, 486 Gum formation by bacteria, 439, 593 Gums and their decomposition, 460 Gypsum block media, for isolation of nitrite-forming bacteria, 71 H Habrotrocha, 351 Hairy vetch, nodule bacteria of, 136 Halteria, 336 Hantzschia, 224, 225, 226 Hapalosiphon, 227 Harpacticidae, 354, 355 Hart marietta hyalina, 330, 331 Hay, composition of, 428 infusion medium, 318 Heat as an agent of partial steriliza- tion, 744, 745-749 Heat, destruction of fungi by, 244 formation of, in microbiological processes, 425 Heat, of dilution, 387 of reaction, 387 of solution, 387 Heating of soil, for separation of bacteria, 166 influence of on biological activities, 742 Heleopera, 332 Heliozoa, 331 Helix, 359 Helodrillus, 352 Hemicelluloses, chemistry of, 452 decomposition of, by microorganisms, 166, 454 Helminthosporium, 256, 807 Hemiptera, 358 Heterodera, 344, 348, 350, 818 radicicola, 348, 350, 763, 810 schachtii, 348, 349, 804, 810 Heterokontae, 229 Heteromita, 330, 333 Heteronema, 334 Heterotricha, 335 Heterotrophic bacteria, 4, 59, 141, 369 anaerobic, 60 classification of, 141 non-spore forming, 150 spore-forming, 142 Heterotrophic protozoa, 315 Heterotrophic utilization of energy, 408 Hexamitus, 334 Hibernation of insects in soil, 359 Hippuric acid, 206, 212 decomposition of, by bacteria, 212, 487 Histidine in soil, 479, 671 INDEX OF SUBJECTS 881 History of soil microbiology, 834 Hodotermes, 363 Hog peanut, nodule bacteria of, 137 Holophrya, 335 Holotricha, 335 Holozoio protozoa, 315 Hoplolaimus, 348 Hormodendrum, 265 Humic acids in soil, 671, 696-697, 698-699 Humid soils, distribution of bacteria in, 34 Humification, 695 Humin, 697, 698 Humus compounds, chemistry of and classification, 671, 696-699 Humus, decomposition of, 151, 298, 310, 449 formation of, in soil, 309, 447, 460, 681, 689-691, 692-696 influence of, on nitrogen fixation, 580 nature of, 691-696, 707 role of, in soil processes, 629 Hyalopus, 253 Hyalosphenia, 332 Hyalotheca, 229 Hydrocarbons, decomposition of, by bacteria, 159, 204, 465 Hydrogen bacteria, 59, 403 Hydrogen, energy utilization in the oxidation of, 403 Hydrogen formation, by bacteria, 177 in the decomposition of cellulose by bacteria, 193, 437, 440 Hydrogen oxidation by bacteria, 98, 99, 188 Hydrogen-ion concentration (see Re- action) Hydrogen peroxide, decomposition of in soil, 734 Hydrogen sulfide formation, in the decomposition of organic matter, 612, 613 in the reduction of sulfates, 602 Hydrogen sulfide, oxidation of, 78, 605 Hydrogenomonas, 99, 188, 403 Hydrolytic decomposition of proteins, 473, 481 Hydroxylamine, 526 Hymenomycetes, 257, 275, 276, 278, 459 Hymenoptera, 356, 357, 358 Hymetomelanic acid, 671, 698 Hypheothrix, 226 Hyphomycetes, 3, 250, 252, 265, 276, 289 Hypochnus, 278 Hypolricha, 336 Hypoxanthine in soil, 479, 671 Ichneumonidae, 361 Immunity theory and nodule forma- tion, 128, 589 Incubation of plates, 23 India-ink method of isolating bacteria, 55, 101 Indigo-carmin as reducing agent, 170 Infusoria, 314, 335 Inoculation, cross, of nodule bacteria, 136 Inoculation of soil with non-symbiotic bacteria, 105 Inoculation, principles of, 817-833 Insects in soil, 3, 50, 342, 356, 359 Insecticides, 362 Interpretation of results of plate counts, 24-27 Inulin, decomposition of, 462 Invertebrate fauna of soil, causing plant and animal diseases, 810 economic importance of, 360 occurrence of, 341-363 role of, in soil, 360 Invigoration of nitrogen-fixing bac- teria, 111 Iota, 349 Iron, as a catalytic agent, 540 as a reducing agent, 170 accumulation by bacteria, 94 bacteria, 59, 92-96, 402-403 compounds, energy utilization in the oxidation of, 402 influence of, on the growth of Azoto- bacter, 578 precipitation of, 402 transformation of, in soil, 666 Ironus, 348, 349 882 INDEX OF SUBJECTS Irrigation, influence of, on biological activities in soil, 784 Isocystis, 226 Isolation, of algae (impure cultures) from soil, 216 of algae (pure cultures) from soil, 216 of bacteria from nodules, 128 of nodule bacteria from the soil, 129 of protozoa, 320 Isonchus, 349 Isopoda in soil, 342 Isopods, 354, 355, 357 Julus, 355 Jumping beetles, 361 June bugs in soil, 361 K Karleria, 1\1 Kirchnerella, 233 Koch's postulates as applied to study of soil bacteria, 6 Kraussia, bacteria in, 105 Lacrymaria, 335 Lactarius, 278 Lactic acid, formation of, by bacteria, 178, 410, 412, 414, 440 Lamellibranchiata, 359 Larvae of insects in soil, 359 Lathyrus, nodule bacteria of, 134, 135, 136 Law of chance in counting bacteria, 24 Lead plant, nodule bacteria of, 137 Leaf glands, bacteria in, 140 Leaves, composition of, 428 decomposition of, 684 nodule formation in, 139 Lecithine, decomposition of, 485, 650 Lecythium, 332 Legume inoculation, 819 Leguminosae, mycorrhiza formation by, 274, 275 Lembus, 335 Lens, nodule bacteria of, 135, 136 Lepidoptera, 356, 358 Leptomyxa, 331 Leptothrix, 93, 287, 288, 402 ochracea, 93, 95, 100 Irichogenes, 93 Lespedeza, nodule bacteria of, 136 Leucine as a source of energy, 411 Levulans, decomposition of, 461 synthesis of, by bacteria, 383 Life cycles, of bacteria, 57, 117, 130 of protozoa, 324 Lignins, and their decomposition, 298, 429, 455-460 as a source of humus in soil, 693 Ligno-celluloses, 429, 455 Lignoceric acid in soil, 671, 672 Lima bean, nodule bacteria of, 136 Limax amoebae, 330 Lime, influence of, on biological ac- tivities, 530, 714, 743, 789 requirement of soil, as determined by growth of Azotobacter, 581 Limicolae in soil, 342, 353 Limonite, deposition of, 235 Linyphia, 355 Lionotus, 335 Lipoids in the microbial cell, 382 Lithobius, 355 Lithosphere, composition of, 624 Loliurn temulentum, mycorrhiza for- mation by, 272 Loxophyllum, 335 Lumbricus, 352 Lupinus, composition of, 428, 596 nodule bacteria of, 130, 134, 135, 137 Luzerne, composition of, 428 Lyngbya, 226 Lysine in soil, 479 M Macro-actinomyces, 309 Macrobiotus, 354 Macrosporium, 256, 807 Magnesium carbonate-gypsum block, 71 Magnesium, influence of, on biological activities in soil, 576, 789 transformation of, in soil, 664-665 Malate-glycerin agar, 291, 306 INDEX OF SUBJECTS 883 Mallomonas, 334 Manganese, as a source of energy, 59 salts, oxidation of, 93 stimulating action of, 665 transformation of, in soil, 665 Mannite decomposing capacity of soil, 729 medium, 112, 113 Manure, as a carrier of bacteria, 772-773 cellulose decomposing bacteria in, 434 composting of, 675 decomposition of, 444, 682 fertilizing action of, 773 influence of, on biological activities in soil, 771 influence of, on cellulose decompo- sition, 727, 728 influence of, on numbers of actino- myces in soil, 41 nitrogen transformation in the rot- ting of, 498, 686 numbers of bacteria in, 29-31 Marmots, in soil, 342 Marsh soils, occurrence of algae in, 224 Mastigamoeba, 333 Masligella, 333 Mastigophora, 312, 313, 333 Mathematical interpretation of results of plate counts of numbers of microorganisms, 24-27 Meat extract-peptone agar, 14 Media, for counting bacteria, 13-16 for counting fungi, 19 for cultivation of soil actinomyces, 291, 306 for cultivation of soil algae, 221 for cultivation of soil fungi, 241-243 for cultivation of soil protozoa, 317 for differentiation of actinomyces, 306 for isolation of algae, 218 for isolation of cellulose decomposing bacteria, 191, 195, 196, 198 for isolation of denitrifying bacteria, 185 for isolation of iron bacteria, 95 Media, for isolation of mycorrhiza fungi, 279 for isolation of nitrate-forming bac- teria, 74-75 for isolation of nitrite-forming bac- teria, 64-72 for isolation of nitrogen-fixing bac- teria, 107, 112 for isolation of nodule bacteria, 127 for isolation of urea bacteria, 207 for isolation of uric acid bacteria, 213 for isolation of sulfur oxidizing bac- teria, 84-90 Medicago, nodule bacteria of, 130, 134, 135, 136 Melanconiales, 257 Melanin, 698 Melanconium, 257 Melanospora, 763, 808 Melilotus, nodule bacteria of, 130, 135, 136 Melolontha, 356 Melosira, 227 Menoidium, 334 Mercaptans, formation of, by bacteria, 178, 614 Mermitbidae, 350 Merulius, 243, 257, 460 Mesophilic bacteria, 775 Mesotaenium, 229 Metabolism as a whole, 367 Metals, heavy, influence of, on nitrate formation, 534 Melapides, 336 Methane bacteria, 59, 96-98, 406 Methane formation, by bacteria, 177, 437 by cellulose decomposing bacteria, 193 in the decomposition of manure, 445 Methanomonas methanica, 96, 100, 406 Methods, of counting protozoa in the soil, 45 of cultivation of soil fungi, 241 of demonstration of mycorrhiza formation, 279 of demonstration of occurrence and abundance of fungi in the soil, 239 884 INDEX OF SUBJECTS Methods, of determination of ammonia in soil, 497 of determination of numbers of microorganisms in the soil, 6 of isolation of anaerobic bacteria from soil, 164 of isolation of soil algae, 218 of studying actinomyces, 291 of studying animal population, 342 of studying cellulose decomposition, 443 of studying soil microorganisms, 6 Metopus, 336 Mice in soil, 342 Micro-actinomyces, 309 Microaerophile bacteria, 160 Microbial activities in the soil, proof of, 5-6 Microbial cell, chemical composition of, 377 Microbiological condition of soil, meth- ods of determination, 709 Micrococcus, 58, 118, 153, 208 cytophagus, 195 melanocyclus, 195 nitrosus, 73 ochraceus luteus, 762 paraffinae, 204 pyogenes, 182, 208, 209, 212 selenicus, 91 ureae, 208, 209, 212, 762 ureae liquefaciens, 208 Microcoleus, 220, 226 Microcystis, 226 Microgromia, 332 Micromonospora, 295 Microorganisms, and partial steriliza- tion of soil, 763-766 role of, in humus formation, 693 Microscopic counts compared with plate counts, 27 methods for study of soil bacteria, 7-11 methods for study of soil fungi, 240 study of actinomyces, 291 Microsiphonales, 287 Microspira aestuarii, 189 agar-liquefaciens, 199 Microspira desulfuricans, 188, 611, 612 Microspora, 228 Milk, growth of actinomyces on, 298 Milnesium, 354 Mineral composition, of microbial cells, 3S0, 625 of plants, 429 Mineral requirements, of algae, 231 of Azotobacter, 571 of fungi, 242, 260 Mineral transformation in soil, 614, 644-668 Mineralization of organic matter, 522 Minerals, role of, in bacterial metab- olism, 368, 667-668 Mites in soil, 342 Mixed and pure cultures in the study of soil organisms, 641 Modification of nitrate-forming bac- teria by culture, 76 Modification of nodule bacteria, 134 Moisture, influence of, on bacterial numbers in soil, 34 influence of, on biological activities in soil, 621-624, 781-784 influence of, on nitrogen-fixation, 5S3 influence of, on plant parasites, 812 Moisture film, 633 Molds (see Fungi) Moles in soil, 342 Molluscs in soil, 342, 357, 359 Monas, 82, 334 Monilia, 3, 238, 252, 265, 779 sitophila, 495 Moniliaceae, 267 Moniliopsis, 278 Monocilia, 228 Monohystera, 346, 347, 350 Mononchus, 346, 348, 349, 350, 818 Monosiga, 333 Monosaccharides, decomposition of, by microorganisms, 466 Monosporium, 254, 260, 265 Moraria, 355 Morphological characters of bacteria, 57 Morphology, of anaerobic nitrogen fixing bacteria, 108 of Azotobacter, 117 INDEX OF SUBJECTS 885 Morphology, of Bad. radicicola, 130 of nitrite-forming bacteria, 72 Mortierellaceae, 249 Mosaic virus in soil, 806 Moss protonema in soil, 215, 225 Mougeotia, 229 Mucedinaceae, 252, 289 Mucilages, 460 Mucor, 3, 23S, 243, 244, 248, 258, 259, 278, 444, 459 amylomyces, 379 glomerula, 260, 637 hiemalis, 495 pusillus, 244 ramannianus, 275 stolonifer, 266 Mucoraceae, 247, 248, 249, 267 Mucorales, 239, 241, 243, 244, 247, 260, 266, 268, 275 Mucorineae, 3, 246 Mucuna utilis, nodule bacteria of, 136 Mycelial filaments in the soil, 239 Myceliphlhora, 252 Mycelium of actinomyces, 287, 293 Mycelium radicis atrovirens, 278, 279 Mycetophilidae, 358 Mycobacteria, 59, 158, 204, 205, 286, 465 Mycobacterium album, 465 lacticola, 97 paraffinae, 465 phlei, 97, 782 rubiacearum, 140 rubrum, 465 Mycoderma, 155 Mycocriny, 694 Mycodextrans, 460 Mycogalactans, 460 Mycogone, 255, 264, 265 Mycomycetes, 277, 278 Mycorrhiza formation, 270, 803 fungi, 236, 270-284 role of, in plant nutrition, 280 Myriapods in soil, 342, 355, 357, 361, 362 Myrica, nodule formation by, 105, 138, 139 Myxobacteria, 59, 159 Myxomycetes, 236 Myxophyceae (see Cyanophyceae) N Ndegleria gruberi, 325, 330, 331 Naphthalene, decomposition of, 466 Nassula, 335 Navicula, 224, 227 Nebela, 332 Nematodes, in soil, 3, 341, 345-351, 357, 358, 804 destruction of, in partial steriliza- tion, 763 methods of determination, 343 temperature of control, 815 N ematogonium, 254 Nemathelminthes in soil, 341 Neocosmopora, 763 Nessler's reagent, 67 Nitrate "fermentation," 183, 185 formation as index of nitrogen availability, 539 formation as influenced by plant residues, 706 formation in soil and in solution, 536 -forming bacteria, 59, 62, 6S, 74, 151 -reducing bacteria, 174, 180 reduction in nature, 103, 552 reduction and cellulose decomposi- tion, 200 Nitrates, assimilation of, 269, 544 as sources of oxygen, 420, 545 influence of, on cellulose decomposi- tion, 728 influence of, on nitrate-forming bac- teria, 394 influence of, on nitrogen fixation, 133, 575 reduction of, and energy utilization, 420, 546 reduction of, by actinomyces, 29S, 302 reduction of, by Azotobacter, 576 reduction of, to ammonia, 182 reduction of, to atmospheric nitrogen 183, 548 reduction of, to nitrites, ISO source of, in soil, 523 transformation of, by microor- ganisms, 543 886 INDEX OF SUBJECTS Nitrification, 62, 527, 637, 836 as influenced by carbon-nitrogen ratio of soil, 509 methods of studying, 715 Nitrifying bacteria, numbers of, in soil, 37, 38, 77 isolation of, from soil, 63, 68 limiting and optimum reaction of, 77 occurrence of, in soil, 67, 77 Nitrifying capacity of soil as index of soil fertility, 713,715-717 Nitrite formation, from inorganic and organic substances, 527 in the reduction of nitrates, 298 Nitrite-forming bacteria, 59, 62, 151 Nitrite oxidation, mechanism of, 526 Nitrite utilization by actinomyces, 298-299 Nitrobacter, 74, 75, 79, 100, 400, 637 growth of, on organic media, 76 Nitrogen compounds, assimilation of, by algae, 234 assimilation of, by bacteria, 368 as sources of energy for autotrophic bacteria, 62, 389 decomposition of, in presence of non- nitrogenous, 505 decomposition of, in organic matter, 705 in leachings, as influenced by plant residues, 706 in manure, 498 influence of, on plant growth, 502 non-protein nitrogen, decomposition of, 485 utilization of by bacteria and fungi, 502 Nitrogen fixation, and respiration, 566 as influenced by partial steriliza- tion of soil, 757 by algae, 105, 231 by algae, in symbiosis with Azoto- bacter, 120, 232 by anaerobic bacteria, 109, 179 by higher plants, 106 by mycorrhiza fungi, 281 by nodule bacteria, 103, 122-140 by soil fungi, 105, 207 Nitrogen fixation, chemistry of pro- cess, 572 chemical fixation in soil, 103 in nature, 103 in soil, 560 influence of available nitrogen on, 575 influence of organic matter on, 579 non-symbiotic, 104, 111, 558 non-symbiotic, importance of in soil, 585-588 symbiotic, 106, 122, 588 Nitrogen-fixing bacteria, 5, 27 aerobic, 59, 106, 112, 558 (see also Azotobacter) anaerobic, 59, 106, 107, 166, 175, 558 (see also Bac. amylobacter and Clostridium) numbers of, in soil, 37, 38 symbiotic, 59 Nitrogen-fixing capacity of soil, 729-734 Nitrogen gas, formation of in decom- position of organic matter, 183 formation of, in nitrate reduction, 183, 184, 550 losses of, in soil, 554 Nitrogen, oxides of, formation by bac- teria, 178, 184, 549 transformation by microorganisms, 504 transformation in the rotting of manure, 498 transformation in the decomposition of organic matter in the soil, 500 Nitrogen utilization, by actinomyces, 298 by algae, 231 by fungi, 267 Nitrogenous substances, decomposi- tion of, by fungi, 266 Nilromicrobium, 77 Nitrosococcus, 72, 73, 74, 79, 100 Nitrosomonads, 8 Nitrosomonas, 72, 79, 100, 400, 637 europea, 72 javanensis, 73, 95 Nitschia, 224, 227 Nocardia, 287, 288, 402 INDEX OF SUBJECTS 887 Noctuidae, 361 Nodularia, 224, 225, 226 Nodule bacteria, groups of, 136 nitrogen fixation by, 122-140, 595 Nodule formation, by actinomyces, 310 by leguminous plants, 124, 128 by non-leguminous plants, 138 in leaves of plants, 105 Non-leguminous plants, inoculation of with nodule bacteria, 828 Non-spore forming bacteria, 4, 10, 60, 141, 150, 153, 209 Non-symbiotic nitrogen fixation, chem- istry of, 572 Nostoc, 220, 224, 225, 227 Nostococcaceae, 217, 223, 226 N uclearia, 331 Nucleic acids, 474, 479, 651 Nucleobacter, 651 Nucleo-proteins, decomposition of, 651 Numbers of actinomyces in the soil, 39^2 of anaerobic bacteria in soil, 38 of anaerobic nitrogen fixing bacteria in soil, 110 of bacteria at different depths of soil, 34-36 of bacteria in different seasons of year, 31-34 of bacteria in manure, 29 of microorganisms in the soil, 3-50 of microorganisms in soil as an index of soil fertility, 711 of nitrifying bacteria in soil, 38 of nodule bacteria in soil, 129 of fungi in the soil, 42-44 of physiological groups of bacteria, 36 of protozoa in the soil, 45-50 Nutrition of protozoa, 315 O Obligate anaerobic bacteria, 160-163 parasitism, 804 relation between mycorrhiza and host plant, 283 Occurrence of heterotrophic bacteria in soil, 145, 153 Occurrence, of microorganisms in the soil, 3-5 of trophic and encysted protozoa in soil, 325, 329 Odor production by actinomyces, 303 Oedocephalum, 253 Oedothorax, 355 Oidium, 3, 252, 287, 288, 289, 441, 775 Oicomonas, 318, 330, 334 Oligochaeta, 342, 351, 353, 357, 358 Oligonitrophilic bacteria, 104 Oligotricha, 336 Omnivagous nematodes, 346 Onobrychis saliva, nodule bacteria of, 135 Onychodromus, 336 Oomycetes, 247, 275 Oospora, 252, 287, 288, 289 Orange-liquefying bacteria in soil, 27 Orcheomyces, 276 Orchidaceae, mycorrhiza formation by, 271,276,277,283 Organic acids, decomposition of, 468 Organic matter, decomposition of, by actinomyces. 309 decomposition of, by mycorrhiza fungi, 282 decomposition of, in manure, 498 decomposition of, in soil, 500, 510, 673-681 influence of, on activities of micro- organisms, 699 influence of, on autotrophic bacteria, 61 influence of, on nitrification, 531 influence of, on nitrite and nitrate- forming bacteria, 395, 528 influence of, on nitrogen fixation, 579 influence of, on oxidation processes, 523, 540 influence of, on soil population, 768 nature of, 669 role of, in soil processes, 625 transformation of, in soil, 669-707 utilization of, by algae, 231 Organic media for the cultivation of algae, 222 nitrogen, nitrification of, 715 INDEX OF SUBJECTS Oribatidae, 355 Ornithopus, nodule bacteria of, 134, 135, 137 Orobus tuberosus, mycorrhiza formation by, 274 Orthoptera, 358 Oscillatoria, 220, 224, 226 Oscillatoriaceae, 223, 226 Osmotic pressure in the growth of microorganisms, 370 Oxalic acid, formation by micro- organisms, 212, 410 Oxidase, 521 Oxidation processes in the soil, 520-541, 717-720 Oxidation-reduction phenomena, 163, 385, 520 Oxidative deaminization, 483 Oxidizing power of soil, 736 Oxygen absorption, by soil organisms, 163 from atmosphere, 170 Oxygen, influence of, on the growth of anaerobic bacteria, 169 limit for the growth of bacteria, 161 removal in the cultivation of anaer- obic bacteria, 169 requirement of actinomyce3, 299 requirement of protozoa, 314 supply of, in the growth of algae, 234 tension, in the growth of bacteria, 160, 170 tension, influence of, on nitrate formation, 535 Oxylricha, 336 Ozonium, 257, SOS, 813 Pachybasium, 254 Palmitic acid, 411 Pamphagus, 332 Pandorina, 334 Pantostomatinae, 333 Paraffinbacterium, 204 Paraffin oil, decomposition of, by bac- teria, 204, 465 Paraffin, oxidation of, 97, 204 Paraffinic acid in soil, 671 Paramoecium, 314, 317, 318, 319, 335, 637 Parasitic fungi in soil, 709 nematodes in soil, 348 Parasitism among soil microorganisms, 803, 804 of mycorrhiza, 283 Partial sterilization of soil, 743-749 Partridge pea, nodule bacteria of, 136 Pasture lands, insect fauna in, 356 Pavetta, nodule formation in leaves of, 105, 106, 139, 804 Peanut, nodule bacteria of, 136 Peat, bacteria in, 785 Peat formation, 695 Pectins, 428, 446, 460 Pectin-decomposing bacteria in soil, 38, 175, 203 Pectinobacter amylophilum, 203 Pectins, utilization of, by fungi, 242 Pecto-celluloses, 429 Pellia epiphylla, mycorrhiza formation by, 277, 804 Penicillium, 3, 238, 244, 251, 253, 258, 259, 260, 265, 275, 418, 443, 444, 454, 465, 642, 803 brevicaule, 303, 556, 652 chrysogenum, 495 expansum, 244, 382, 779 glaucum, 380, 382, 417, 652 intricatum, 495 italicum, 261, 637 luteum, 270, 604 variabile, 261 Penium, 229 Pennatae, 227 Pentosans, decomposition of, by micro- organisms, 446 determination of, 453 occurrence of, 452 Peptone-decomposing bacteria in soil, 37 Peranema, 334 Perhydridase, 521 Peritricha, 336 Peronosporales, 247 Peroxidase, 521 Petalomonas, 334 INDEX OF SUBJECTS Petroleum, oxidation of, by bacteria, 97, 204 Phacus, 334 Phaeophyceae, 230 Phalansterium, 333 Phaseolus, nodule bacteria of, 134, 135, 136, 137 Phenol, oxidation of, by bacteria, 205, 465 Philodina, 351 Phoma, 270, 276, 277, 281, 804, 807 Phormidium, 224, 225, 226 Phosphate-sulfur composts, 614 Phosphates, solubilization of, as a result of direct bacterial action, 614, 652 solubilization of, as a result of inter- action with acids formed by microorganisms, 656-660 influence of, on nitrogen-fixation, 577 reduction of, in soil, 556 Phosphatides, synthesis of, by micro- organisms, 382 Phosphorus compounds in soil, 625, 649 content of bacteria, 650, 651 organic in soil, decomposition of, 650-652 requirements of soil, as indicated by growth of Azotobacter, 577, 730, 731 transformation in soil, 650-660 Photosynthetic utilization of energy, 4 Phycomycetes, 247, 265, 275, 277 Phyllomitus, 333 Phyllomonas, 333 Phryganella, 332 Phycobacteria, 59 Phycocyanin, 215 Phycomycetes, 3, 259 Physiological activities as a basis of bacterial classification, 57 Physiological groups of bacteria, num- bers of, in soil, 36 Physiology of anaerobic bacteria, 110, 176 Physomonas, 334 Phytin, decomposition of, 651 Phytoflagellata, 315, 335 Phytomonadinae, 334 Phytophthora, 247, 763, 807 Phytosterol in soil, 671 Picoline carboxylic acid in soil, 671 Pigment formation by microorganisms, 303, 439 Pilobolaceae, 249 Pine needles, composition of, 428 Pinnularia, 227 Piptocephalis, 249 Pisum, nodule bacteria of, 130, 134, 135, 136 Placocysta, 332 Plagiotricha, 336 Planarians, 345 Planobacillus nitrofigens, 121 Planococcus, 58 Planostreptococcus, 58 Planosarcina, 58, 208 ureae, 208 Plant composition at different stages of growth, 673 diseases caused by actinomyces, 809 diseases caused by bacteria found in the soil, 806 diseases caused by fungi found in the soil, 806-809 growth and nitrogenous decomposi- tion products, 501 growth and saprophytic soil organ- isms, 801 growth, influence of, on microbiologi- cal activities in soil, 705, 792-800 infection as influenced by soil en- vironment, 810 infection, as influenced by soil reaction, 813 nutrition, role of mycorrhiza in, 282 parasitic nematodes, 804-805 residues, decomposition of, by fungi, 266 residues, influence of, upon plant growth, 516 secretions, 643, 692 Plants, non-leguminous, nitrogen-fixa- tion by, 106 forming mycorrhiza, 273-275 890 INDEX OF SUBJECTS Plasmodiophora brassicae, 236, 803, 812, 814, 816 Plasmodium formation, 236 Plasmoptic mycorrhiza, 277 Plastic equivalent, 417 Plate counts compared with micro- scopic counts, 27 Plate method, for determining numbers of soil bacteria, 13 for isolation of bacteria, 55 Plates, preparation of, 21 Plathelminthes, 341, 345 Plectoascineae, 251 Plectonema, 225, 227 Plectridium, 173, 178, 204 peclinovorum, 173 Pleclus, 346, 349, 350 Pleurococcus, 228, 234 Pleuronema, 335 Pleurotaenium, 229 Pleurotricha, 336 Podocarpus, nodule formation by, 138, 139 Poisons, influence of, on actinomyces, 302 Poisson series, 26 Polydesmus, 355 Polypodiaceae, mycorrhiza formation by, .273 Polypseudopodius, 334 Polytoma, 334 Porrho?nma, 355 Potassium, available, 662 in soil, 625 influence of, on nitrogen fixation, 577 transformation of, in soil, 615, 660-662 Potato as reducing agent, 171 Potato scab, 301, 616 Potato wart, 236 Potatoes, symbiosis with mycorrhiza fungi, 274 Primitive activities of microor- ganisms, 645 Prismatolaimus, 350 Probable error in bacterial counts, 26 Proleptomonas, 333 Proof of microbial activities in soil, 5 Prorhynchus, 345 Prorodon, 335 Protein content of natural organic materials, 470 decomposition of, 299, 475, 480, 679 hydrolysis, 473 physical and chemical properties of, 470 synthesis by Azotobacter, 570 synthesis, in soil, 479 Protein decomposing bacteria, num- bers of, in soil, 38, 175 Protein-forming bacteria, 183 Proteins of microorganisms, decom- position of, 268 Proteolytic bacteria, 154, 166 Proteomyxa, 331 Protoasci, 250 Protococcus, 234 Proteus vulgaris (see Bad. vulgar e) Protoderma, 228 Protomastiginae, 333 Protosiphon, 228, 233 Protozoa, 311-340 autotrophic forms, 315, 316 classification of soil protozoa, 313, 329 cysts of, in soil, 45 heterotrophic forms, 315 importance of, in soil, 336 influence of, on nitrogen fixation, 338 isolation of pure cultures, 320 life history of, 324 media for cultivation of, 317 methods of determination of num- bers, 45 morphology of, 311-314 numbers of, in the soil, 45-50, 327 occurrence of trophic forms and cysts in soil, 3, 325, 329 physiology of, 314 reproduction of, 312 staining of, 323 Protozoan theory of soil fertility, 757 Prowazekia, 333 Pseudococcus, 362 Pseudochlamys, 332 Pseudodifflugia, 332 INDEX OF SUBJECTS 891 Pseudomonas, 58 caudalus (see Bad. caudatum) fluorescens (see Bad. fluorescens) radicicola (see Bad. radicicola) Pseudomycorrhiza, 278 Psilocybe, 257 Psychrophilic bacteria, 775 Pulmonata, 357 Punctiform bacteria in soil, 151, 153 Pure culture study, 53, 641 Purification of anaerobic bacteria, 165 Purple bacteria, 79, 83 Putrefaction, 175, 477 Pyrenomycetes, 275 Pyrenomycetineae, 251 Pyrocatechin, action of, on bacteria, 165, 167 oxidation of, by bacteria, 205 Pyrogallic acid, use of, in the isolation of anaerobic bacteria, 170 Pyruvic acid, 414, 439, 469, 574, 681 Pythiacystis, 808 Pythium, 238, 247, 807, 813, 815, 816 Quadrula, 332 Q R Radiation, influence of, on actino- myces, 299 Radiobacter group of bacteria, 59, 106,112,212,582 Raisin agar, 18 Raphidiophrys, 331 Raphidium, 228 Ray fungi, 288 (see Actinomyces) Reaction, influence of, on actinomyces, 301, 636, 637 influence of, on anaerobic nitrogen- fixing bacteria, 111, 637 influence of, on denitrification, 551 influence of, on growth of anaerobic bacteria, 176 influence of, on growth of fungi, 244, 262, 637 influence of, on hydrogen bacteria, 102, 637 , Reaction, influence of, on nitrate formation, 528, 637 influence of, on nitrate-reducing bacteria, 186 influence of, on nitrogen-fixation, 581, 595 influence of, on nodule bacteria, 133, 637 influence of, on plant pathogenic fungi, 813 influence of, on potato scab, 809 influence of, on protozoa, 314, 637 limiting and optimum for growth of Azotobader, 582, 637, 792 limiting and optimum for growth of Bad. radicicola, 594, 636, 637 limiting and optimum for growth of nitrifying bacteria, 66, 77, 637 limiting and optimum for growth of sulfur-oxidizing bacteria, 90, 91, 400, 637 Reaction, of media, 118 of soil, 633-638 of soil, as influenced by cellulose decomposition, 451 Reaction velocity, 372 Red clover, nodule bacteria of, 136 Reducing power of soil, 736 Reductase, 521 Reduction of nitrates to atmospheric nitrogen, 183 of nitrates to ammonia, 182 of nitrates to nitrites, 180 of sulfates by bacteria, 556, 610 Reduction processes in soil, 522, 542-557 Reductive deaminization, 483 Resin acids and esters in soil, 671 Respiration and nitrogen-fixation, 566 coefficient, 417 equivalent, 417 of nitrate-forming bacteria, 392 Rhabditis, 347, 349, 350 Rhabdolaimus, 350 Rhadocoelae, 345 Rhizobium, numbers of, in soil, 37, 39 leguminosarum, 126, 825 (see Bad. radicicola) 892 INDEX OF SUBJECTS Rhizobium leguminosum, 136 radicicolum, 136 Rhizoctonia, 257, 276, 277, 278, 279, 763, 807, 814, 815, solani, 257, 277, 803, 806, 807, 808, 812 Rhizomyxa, 278 Rhizopoda, 313, 331 Rhizopus, 3, 238, 247, 258, 259, 266, 454, 461 arrhizus, 247 nigricans, 247, 495, 803, 809 nodosus, 247 Rhizosphere, 155, 643 Rhodobacteria, 59 Rhodomonas, 334 Rhodophyceae, 230 Rhopalomyces, 253 Rhynchota, 358 River flukes, 345 Rivularia, 227 Rivulariaceae, 227 Robinia, nodule bacteria of, 137 Robertus, 355 Rocks, decomposition of, by micro- organisms, 645 Root-knot nematode, 348 Root-rots in soil, 807 Rose-bengal stain for soil bacteria, 9 Rosellinia necalrix, 808 Rotatoria (see Rotifers) Rotifers, occurrence of. in soil, 3, 342, 351 Roundworms in soil, 341 Rubiaceae, nodule formation in leaves of, 139 Russula, 257, 276, 278 Rusts, 763, 808 S Saccharolytic bacteria, 166, 172 Saccharomyces, 251 Saccharomycopsis, 251 Sainouron, 333 Salpingoeca, 333 Salt concentration, influence of, on biological activities in soil, 301, 787 Saltpeter beds, 62 Salts, influence of, on nitrification, 531 influence of, on nitrogen fixation, 576 Sampling of soil, 19 Saprolegniales, 247 Saprophytism among soil microor- ganisms, 803 Sarcina, 58, 154 flava, 154 lutea, 490, 491,507, 508 Sarcodina, 313, 331 Scarabaeidae, 361 Scarlet runner bean, nodule bacteria of, 137 Scenedesmus, 228 Schinzia leguminosarum, 126 Schizomycetes, 236 Schizophyceae (see Cyanophyceae) Schizophyllum, 355 Schweitzer's reagent, 431 Sclerotia, 257 Sclerotinia, 264, 763, 779, 807, 814, 815 Sclerotium, 257 rolfsii, 807, 808, 812 Scytonema, 224, 227 Scytonemaceae, 227 Seasons of the year, influence of, on biological activities, 40, 779-7S1 influence of, on numbers of bacteria, 31-34 Seed germination, role of fungi in, 280 Seeds of plants as carriers of bacteria, 106 Segmented worms, 342 Selective culture method, 54 destruction of microorganisms in partial sterilization, 761 media, use of, in separation of bac- teria, 166 media, use of, in feeding of protozoa, 317 Selenates, reduction of, 303, 556 Selenium, oxidation of, 91 Separation of anaerobic bacteria, 166 Septoria, 815 Seradella, nodule bacteria of, 137 Serological method of differentiation of nodule bacteria, 134, 137 Sick soils, 326, 709, 794, S08 INDEX OF SUBJECTS 893 Siderocapsa, 93 Sideromonas, 93 Silica gel media, 68, 166, 196, 220 plates for isolation of Azotobacter, 113 Silicates, decomposition of , by bacteria, 646 Single-cell cultures, isolation of, 55, 169 Single-spore cultures, 245 Sinigrin, 600 Slime molds, 236 Slime of bacteria, 460 Slow growing bacteria, 153 Smuts in soil, 763, 808 Sodium formate, action of, on bacteria, 165, 171 pyrogallate, use of, in the cultivation of anaerobic bacteria, 167, 170 sulfide as reducing agent, 171 sulfite as reducing agent, 171 sulphindigotate, action of, on bac- teria, 165 Soil, as a medium for growth of micro- organisms, 619 atmosphere, 638-040 colloidal condition of, 622, 626 composition of, 619, 620 energy transformation in, 425 environments, relation to plant in- fection, 810 extract agar, 15 extract gelatin, 17 fertility and microbiological ac- tivities, 708 gases, 626 humus, nature of, 669 inoculation, principles of, 817-833 inoculation, with non-symbiotic bac- teria, 105 oxidation processes in, 522 population, 642 reaction and microbiological activi- ties, 633-638 sampling, 19-21 solution, 631-633 sterilization, 641 temperature, 640 use of, for legume inoculation, 821 Soil, variability, 20 Soja (see Glycine) Solanaceae, mycorrhiza formation by, 275 Solid media for isolation of nitrite forming bacteria, 68 Solution method, 710 Solvent action of algae, 235 Sordaria, 251 Soy bean, nodule bacteria of, 137 Spathidium, 335 Specific differentiation of actinomyces, 290 Sphaeronema, 257 Sphaeropsidales, 257, 276 Sphenoderia, 332 Sphenomonas, 334 Spicaria, 254 Spirillaceae, 58 Spirillum, 58, 82, 83, 141 Spirochaeta, 58 cytophaga, 195, 196, 203, 368, 439, 770 Spiromonas, 334 Spitonema, 334 Spirophyllum, 93, 94, 95, 100, 402 Spirostomum, 336 Spondylomorum, 217 Spongomonas, 334 Spongospora subterranea, 808, 812, 815 Sporangiophorae, 247 Spore-forming bacteria, 10, 60, 141, 142, 143, 209 Spores of fungi, germination of, 246 Sporogenous hyphae of actinomyces, 294 Sporormia, 251 Sporozoa, 314 Sporotrichum, 3, 254, 264, 265 Spring vetch, nodule bacteria of, 136 Stachobotrys, 255, 264 Staining, of flagella, 132 of nitrite-forming bacteria, 74 of protozoa, 323 Standard loop for counting protozoa, 45 Staphyinidae, 358 Staphylococcus, 612, 631, 641 Starch agar, 39, 291 894 INDEX OF SUBJECTS Starch, as a source of energy, 561 decomposition of, 461 Starch-zinc iodide reagent, 67 Staurastrum, 229 Steam, as an agent of partial steriliza- tion, 746, 748 Stemphylium, 256, 258, 260, 265, 490 Sterilization of soil, 641 and solubilization of minerals, 655 Stichococcus, 224, 228, 233 Stichotricha, 336 Stigonema, 224, 227 Stigonemaceae, 227 Stilbaceae, 3, 256 Stimulation of bacteria by antiseptics, 756 Straw, bacteria in, 30 composition of, 428 decomposition in soil, 684, 704 influence of, upon the growth of plants, 516 Streptococcus, 58 pyogenes, 208 Streptothrix, 287, 288 Strombidium, 336 Strophostyles, nodule formation by, 135, 137 Stubble, composition of, 428 Slylonychia, 336 Sty s anus, 257 Sugar-beet nematode, 348 Sulfate-reducing bacteria, 174, 188 Sulfate reduction, 188-189, 556 Sulfates, as sources of oxygen for anaerobic bacteria, 420 reduction of, by bacteria, 610-612 reduction of, and energy utilization, 420 Sulfide bacteria, 79, 398 Sulfides, oxidation of, 604 Sulfur bacteria, 59, 78-91, 399 Sulfur, chemical oxidation of, 603, 604 energy utilization in the oxidation of, 397 liberation in decomposition of pro- teins, 602, 612 oxidation of, by autotrophic bac- teria, 605-609 Sulfur, oxidation of, by heterotrophic organisms, 604 oxidation of, in soil, 540, 602, 605 presence of, in plants and in soil, 601 reduction of, 610 sources of, in soil, 600 transformation of, by microorgan- isms, 600-616 use of, for control of soil-born dis- eases, 816, 832 Sulfur-phosphate composts, 88, 614 Sulfuric acid, formation of, by bac- teria, 90, 608 Summer bacteria, 32 Sweet pea, nodule bacteria of, 136 Symbiosis, between bacteria and legu- minous plants, 122-140, 804 between bacteria and non-legumi- nous plants, 105 Symbiotic action, between Azotobacter and other bacteria, 121, 560, 641 between Azotobacter and algae, 121, 232 in cellulose decomposition, 201 in mycorrhiza, 280 Symbiotic nitrogen fixation, chemistry of, 591 Symphyla, 355, 357 Symplastic stage in bacterial develop- ment, 57 Syncephalis, 249 Syncephalastrum, 249 Synchitrium, 236, 763, 808, 814 Synechoccus, 226 Synedra, 227 Synsporium, 255 Synthetic agar, 16 Systematic position of actinomyces, 287 Tachinidae, 356 Tap water gelatin, 16 Tardigrada, 342, 354 Tarsonemidae, 355 Tartaric acid as a source of energy, 411 Taurine, 614 Tellurates, reduction of, 303, 556 INDEX OF SUBJECTS 895 Temperature, influence of, on actino- myces, 299 influence of, on bacterial numbers, 32 influence of, on biological activities in soil, 774 influence of, on energy utilization, 419 influence of, on fungi, 244 influence of, on nitrate formation, 539, 776 influence of, on nitrogen fixation, 583 influence of, on oxidation processes in soil, 523 influence of, on period of incubation of plates, 23 influence of, on protozoa, 314, 320, 327 of soil and biological activities, 640 of soil and plant diseases, 811 Temperature, selective use of, in the separation of soil bacteria, 166 Teratocephalus, 350 Termites in soil, 339, 363 Terricolae, 342, 351 Testacea, 332 Testacella, 359 Testaceous rhizopods, 315 Tetmemorus, 229 Tetracoccus, 154 Tetramitus, 334 Thamnidium, 258 Thamnidiaceae, 249 Thecamoebae in soil, 48, 326, 328 Thermoascus, 418 Thermophilic actinomyces, 300 bacteria, 155-158, 774, 775 anaerobic bacteria, 166, 176 cellulose-decomposing bacteria, 157, 439-441 denitrifying bacteria, 157, 188 nitrogen-fixing bacteria, 157 occurrence of, bacteria, 156 Thielavia basicola, 808, 811 Thiobacillus, 78, 151, 605 denitrificans, 79, 86, 87, 100, 188, 398, 399, 400, 637 thiooxidans, 79, 89, 90, 91, 92, 100, 399, 400, 401, 606, 609, 615, 637, 832 Thiobacillus thioparus, 79, 85, 86, 91, 92, 100, 398, 399, 606 Thiobacteria, 59 Thiobacteriales, 78 Thiobacterium, 83 Thionic acid bacteria, 79, 88, 398 Thiophysa volulans, 82 Thioploca, 80 Thiospirillum, 79, 82 Thiosulfate, chemical oxidation of, 603 oxidation of, by sulfur bacteria, 84, 188, 606 media for sulfur bacteria, 84 Thiothrix, 79, 80, 81, 100, 397, 398, 606 Thiovulum, 79, 82 Thread-forming bacteria, 79, 80 Thysanoptera, 358 Thysanura, 358 Tick trefoil, nodule bacteria of, 136 Ticks in soil, 342 Tilachlidiujn, 256 Tilletia tritici, 812 Tipula, 356 Tipulidae, 361 Tissues as reducing agents, 171 Toluol, as an agent of partial steriliza- tion of soil, 753, 755 influence of, on nitrate formation, 536 oxidation of, by bacteria, 97, 205 Tolypothrix, 227 Torula ammoniacale, 206, 251, 255 Toxin theory of partial sterilization of soil, 760 Trachelomonas, 334 Trailing wild bean, nodule bacteria of, 137 Tremaloda in soil, 341, 345 Tribonema, 229 Tri-calcium phosphate (see Phosphate) Trichocera, 358 Trichocladium, 264 Trichoderma, 3, 253, 258, 259, 260, 265, 443, 514, 642, 701, 817 koningi, 250, 259, 260, 267, 379, 495, 808 lignorum, 808 Tricholoma, 276, 278 Trichothecium, 255, 258, 459 INDEX OF SUBJECTS Trifolium, nodule bacteria of, 130, 134, 135, 136 Trigonella, nodule bacteria of, 135, 136 Trilobus, 350 Trimethylamine in soil, 479 Trinema, 332 Triphaena, 358 Tripyla, 348, 350, 358 Trochalworms in soil, 342 Trochelminthes in soil, 342 Trochilia, 335 Trochiscia, 224, 225, 228 Trombidiidae, 355 Trophic protozoa in the soil, 47, 326 Tuber formation and plant evolution, 273 Tuberculareaceae, 3, 257 Tumbler method, 687 Turbellaria in soil, 341, 345 Tylenchorhynchus, 348 Tylenchus, 348, 349, 350, 810, 818 Tylopharynx, 350 Tyroglyphidae, 355 Tyrosine, decomposition of, 484 U Ulmic acid, 697 Ulmin, 697 Ulothrix, 225, 228 Ultramicroscopic organisms in the soil, 5 Ur eolus, 334 Urea as a source of nitrogen, 299 Urea bacteria, 27, 60, 151, 202-211 anaerobic, 176, 210 classification of, 208 influence of oxygen tension on, 210 isolation of, 207 numbers of, in soil, 37, 38, 207 Urea, formation of, in the decomposi- tion of proteins, 299 formation of, in the decomposition of uric acid, 212, 486 decomposition of, 206, 486, 487 nitrate agar, 16 Uredinales, 257 Uric acid, 206, 211, 486, 487 bacteria, 60, 211 Urine, bacteria in, 30 Urobacillus, 208, 488 Urobacterium, 209 Urococcus, 208 Urocystis, 811 Uroleptus, 336 Uronema, 335 Urophlyctis alfalfae in soil, 808 Urosarcina, 208 Urostyla, 336 Urolricha, 335 Ustilaginales, 257 Utilization quotient, 416 Vacuum, use in the cultivation oi bacteria, 170 Vaginicola, 336 Vampyrella laterita, 331 Variability, of actinomyces, 304 of bacteria in soil, 20 Vaucheria, 228 Vegetable organic matter, composition of, 427 Velvet bean, nodule bacteria of, 136 Vertebrates in soil, 31, 342, 362 Verticillium, 254, 258, 260, 265, 278, 812 Vibrio cholerae, 806 denitrificans, 187 thermo-desulfuricans, 189, 611 Vibrion septique, occurrence of. in soils, 173 Vicia, nodule bacteria of, 130, 134, 135, 136 Vigna, nodule bacteria of, 135, 136 Vinca minor, mycorrhiza formation by, 277 Virulence of nodule bacteria, 588 Volcanothrix silicophila, 647 Vorlicella, 336 W Washed agar media, 70 Water blanks for counting bacteria, 19 Waxes, decomposition of, 463 Waxes in soil, 463, 671 Wheat nematode, 348 Wheel animalcules in soil, 342 INDEX OF SUBJECTS 897 White clover, nodule bacteria of, 136 White sweet clover, nodule bacteria of, 136 Wild indigo, nodule bacteria of, 136 Willia, 251 Wilts in soil, 807 Winter bacteria, 32 Wireworms, 361 Wood residues, influence of, upon plant growth, 518 Wood's clover, nodule bacteria of, 137 Worms in soil, 50, 341 X ■ Xanthine in soil, 479, 671 Xanthophyll in algae, 215 Xylol, oxidation of, by bacteria, 97, 205 Yeast, as a reducing agent, 171 in soil, 3, 11 Yeast, reduction of nitrates by, 181 Yellow sweet clover, nodule bacteria of, 137 Yellow trefoil, nodule bacteria of, 137 Yellows, as influenced by soil environ- ment, 811 Zigzag clover, nodule bacteria of, 136 Zinc, transformation of, in soil, 666 Zooglea formation by nitrite-forming bacteria, 73 Zygodesmus, 255 Zygogonium, 229 Zygomycetes, 3, 247 Zygorhynchus, 248, 258, 259, 275, 642 molleri, 260, 275, 379, 444 vuilleminii, 495 Zygospore, isolation of, 246 Zymotic efficiency, 710 ■ HIE ' ffl -"'■-•' is ' ■ ' ■" ■■ ■-,■',• $*&*!» isaBs m : iac&fx ■■ '-'■•. : ■.'.■" ■■ ■''""■■■■'■•.'■■■':- ..••■- ; ■.-■•■-'••■-■•■'-' ■' 1 ' ■-• '■■ ■• ■ ■■•'.;":■:.•■■■.■ 111111