EXCHANGE THE BACTERIA OF NEBRASKA SOIL WITH SPECIAL REFERENCE TO THE FIXATION OF NITROGEN, AMMONIFICATION DENITRIFICATION IN NON-PROTEIN MEDIA, INCLUDING OBSERVATIONS ON THE REDUCTION OF NITRATES BY SOIL BACTERIA IN GENERAL BY JOHN J. PUTNAM OF THE UNIVERSITY SUBMITTED TO THE GRADUATE COLLEGE OF THE UNIVERSITY OF NEBRASKA IN CANDIDACY FOR THE DEGREE DOCTOR OF PHILOSOPHY FEBRUARY 20, 1913 1913 THE WOODRUFF PRESS LINCOLN, NEB. THE BACTERIA OF NEBRASKA SOIL WITH SPECIAL REFERENCE TO THE FIXATION OF NITROGEN, AMMONIFICATION DENITRIFICATION IN NON-PROTEIN MEDIA, INCLUDING OBSERVATIONS ON THE REDUCTION OF NITRATES BY SOIL BACTERIA IN GENERAL BY JOHN J. PUTNAM SUBMITTED TO THE GRADUATE COLLEGE OF THE UNIVERSITY OF NEBRASKA IN CANDIDACY FOR THE DEGREE DOCTOR OF PHILOSOPHY FEBRUARY 20. 1913 1913 THE WOODRUFF PRESS LINCOLN, NEB. THE BACTERIA OF NEBRASKA SOIL WITH SPECIAL REFERENCE TO THE FIXATION OF NITROGEN, AMMONIFICATION, DENITRIFICATION IN NON-PROTEIN MEDIA INCLUDING OBSERVATIONS ON THE REDUCTION OF NITRATES BY SOIL BACTERIA IN GENERAL HISTORICAL This work was undertaken with the idea of ascertaining if possible, some of the many chemical changes taking place through the action of bacteria indigenous to Nebraska soil. The fixation of nitrogen was first observed by M. Berthelot in 1885. He subsequently was able to prove that this pheno- menon is not brought about exclusively by a purely chemical process, but is due to the activity of micro-organisms. The discovery of an anaerobic organism by S. Winogradsky in 1893, Clostridium pasteurianum, which he found fixed from 2.5 to 3 mg of nitrogen per gram of dextrose consumed, marked the first advance along this important line. Recent observers have added a few organisms to the list, Beyerinck, Lohnis and Lipman having labored successfully in this field. In 1887 Schlossing and Muntz hazarded the opinion that the formation of nitrate within the soil is due to the vital activity of soil bacteria, and in a subsequent communication these two workers detailed some of the conditions requisite for the inception and course of nitrification. Much opposition developed from the advocates of the chemical theory. A re- examination of the comprehensive work of H. Plath by Lon- dalt, who undertook the task in consequence of an objection raised by B. Frank, led to a complete confirmation of Plath's discoveries in all particulars. It was thus ascertained in 1888, by the exclusion method, that in the oxidation process now under our notice the role of oxygen-carrier is played by living organisms, and that nitrification consequently is a physiological process. The discovery and closer investigation of these un- known organisms was shortly afterwards effected by S. Wino- gradsky, who isolated them in pure culture. Of great im- portance is the fact determined by Winogradsky that the numerous species of the group of nitrifying bacteria may be classified into two sharply divided sub-groups: Nitroso- bacteria and Nitro-bacteria. The nitroso-bacteria oxidize ammonia to nitrous acid, while the nitro-bacteria lack the faculty of attacking ammonia, but perform the important task of converting nitrous acid into nitric acid. We are indebted to E. Marchel for proving that the faculty of eliminating ammonia from albuminoids is common to many fungi. The potency of the different species was found by him to vary, the largest quantity being produced by Bacillus mycoides. The first researches along the line of denitrification were undertaken by Jules Reiset in 1854 and 1855. He asserted that free nitrogen was always evolved during the decomposi- tion of manure. Denitrification in arable soil was first noticed by Gappelsroder in 1862, and was long regarded as a purely chemical process. The first reference to the agency of bacteria in this decomposition was made by E. Mensel in 1875, and the earliest pure cultures of such organisms were obtained by U. Gay on and G. Dupetit in 1882. In succeeding years a large number of species, all capable of reducing nitrates, was made known. In 1888, P. Frankland was able to associate with the group in question 17 out of 32 species, and R. Warington 16 out of 25 species examined. A. Maassen, in 1902, found that out of 109 species, 85 were able to perform this function. But few of the organisms which have been observed to reduce nitrates to nitrites in pure culture, are able to continue the reduction to the liberation of free nitrogen. For an inquiry into the various functions performed by soil bacteria in general and with reference to the factors concerned in the fixation of nitrogen by azotobacter, ammonification, reduc- tion of nitrates and denitrification, in particular,seventy samples of soil were taken. These soils comprise perhaps all of the various types within our borders, with the possible exception of the alkali tracts which are interspersed over the western half of our state. Locations were made not with special reference to any favored locality or type of soil, but rather that the samples should be fairly representative of the whole state. These samples were taken in tubes which were constructed especially for our purpose, and are of steel bicycle tubing, eight inches long and one inch in diameter. Whenever possible, the earth was removed in a crust of an inch or less in thickness, and the tube forced into the ground beneath, to a depth of four or five inches. About one inch of dirt was removed from the tube with a sterile knife, and the cotton plug readjusted. On being returned to the laboratory the samples were transferred to sterile four ounce salt-mouth bottles, thoroughly mixed, and soil taken therefrom as desired. Experience has abund- antly proven that the whole process of sampling, transfer, etc., can be performed with such exactness that no contamination takes place. The following list shows the character of each of the soils investigated : 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Fine sand loam Fine sand loam Fine sand loam Fine sand loam Fine sand loam Fine sand loam Fine sand loam Fine sand loam Fine sand loam Fine sand loam Fine sand loam Fine sand loam Fine sand loam Fine sand loam Fine sand loam Silt loam Silt loam Silt loam Fine sand loam Fine sand loam Silt loam Silt loam Silt loam Silt loam 25 Silt loam 49 26 Silt loam 50 27 Loam 51 28 Loam 52 29 Loam 53 30 Loam 54 31 Fine sand loam 55 32 Loam 56 33 Loam 57 34 Loam 58 35 Loam 59 36 Loam 60 37 Loam 61 38 Loam 62 39 Loam 63 40 Silt loam 64 41 Loam 65 42 Loam 66 43 Loam 67 44 Silt loam 68 45 Fine sand loam 69 46 Fine sand loam 70 47 Silt loam 48 Muck Muck Loam Loam Silt loam Loam Clay loam Silt loam Silt loam Calcareus clay Fine sand loam Silt loam Fine sand loam Fine sand loam Gravel Fine sand loam Fine sand loam Fine sand loam Fine sand loam Fine sand loam Calcareus loam Fine sand loam Fine sand loam No more samples were taken at one time than could be handled with promptness, therefore necessitating several trips over the state. In order to approximate the number of bacteria within the soil, the seventy samples were plated both on nutrient agar, and Ashby's medium to which was added fifteen grams of agar- agar per liter. Nutrient medium recommended by Ashby for fixation of nitrogen by azotobacter: Mannite 20.00 grams Di-potassium phosphate 0.20 gram Magnesium sulphate 0.20 gram Sodium chloride 0.20 gram Calcium sulphate 0.10 gram Calcium carbonate 5.00 grams Distilled water 1000.00 grams A Kjeldahl determination disclosed the fact that good agar-agar contained 0.16% of nitrogen, or a little below the average of good soil. Therefore, each plate of the Ashby medium contained approximately .25 mg of nitrogen, or about .0025%, which is in the neighborhood of one one- hundredth the nitrogen content of a good loam. A little nitrogen must inevitably be carried over in the process of dilution, etc. The plates were counted after five days incubation at room temperature, which prevailed at about 33°C. The figures in the adjoining table represent the count per gram of soil dried at 100-1 10°C, to constant weight. NUMBER OF BACTERIA PER GRAM OF SOIL DRIED TO CONSTANT WEIGHT AT 100-110°C TABLE I Soil No. Nutrient agar Ashby's medium Moisture Per cent 1 693,000 1,155,000 13.44 2 94,900 940,000 5.23 3 3,301,000 3,056,000 18.22 4 1,673,000 3,705,000 16.33 5 1,793,000 2,577,000 10.77 6 210,000 1,053,000 5.09 7 288,000 2,138,000 6.41 8 381,000 448,000 10.83 9.. 1,353,000 1,466.000 11.37 TABLE I — Continued Soil No. Nutrient agar Ashby's medium Moisture Per cent 10 2,407,000 150,000 16.91 11 224,000 693,000 6.27 12 .... 153,000 54,800 8.91 13 15,300 404,000 13.50 14 75,800 162,000 7.73 15 289,000 133,000 13.55 16 34,000 10,600 5.95 17 42,900 219,000 44.45 18 790,000 11,900 16.56 19 50,600 562,000 11.09 20 115,000 399,000 4.98 21 1,530,000 3,178,000 15.05 22 737,000 1,989,000 9.15 23 474,000 830,000 15.68 24 946,000 2,601,000 15.44 25 165,000 1,650,000 9.54 26 82,000 671,000 15.11 27 65,000 412,000 7.96 28 293,000 1,084,000 11.44 29 215,000 420,000 7.15 30 135,000 135,200 9.80 31 59,000 462,000 15.59 32 40,700 184,000 14.23 33 639,000 575,000 6.15 34 713,000 594,000 7.56 35 36 409,000 445,000 301,000 434,000 7.19 7.95 37 .. 574,000 1,768,000 9.55 38 1,373,000 2,146,000 6.84 39 784,000 753,000 5.44 40. .. 389,000 811,000 5.11 41 716,000 791,000 6.49 42 236,000 381,000 6.97 43 1,795,000 2,218,000 5.32 44. 731,000 1,801,000 11.19 45 637,000 1,880,000 428 46 416,000 420,000 3.89 47... 873,000 484,000 7.15 48 1,886,000 551,000 31.08 49 1,965,000 573,000 38.95 50 . 335,000 1,300,000 7.71 51.. 364,000 1,287,000 6.79 52.. 74.000 391.000 5.43 8 TABLE I — Concluded Soil No. Nutrient Agar Ashby's Medium Moisture Per cent 53 254,000 637,000 5.91 54 255,000 210,000 9 79 55 1,602,000 554,000 16.98 56 1,006,000 524,000 8 56 57 11,000 4,500 9.70 58 . . 818,000 549,000 7.16 59 506,000 60,000 5.21 60 56,500 9,200 9.88 61 4,224 000 833 000 1241 62 10 000 4.48 63 468,000 60,300 3 90 64 947,000 904,000 7.12 65 158,000 70,000 4.30 66 134,000 17,000 11.98 67 40,000 30,700 .84 68 . . . 10,008,000 512,000 16.07 69 380,000 83,600 7.93 70.. 1,850,000 103,000 8.13 In forty of the samples the number of bacteria which developed visible colonies on the non-proteid medium were in excess, and in many instances in great excess, of those on the nutrient agar. It will be observed that in the remaining thirty samples the number of colonies in the Ashby medium very closely approximate those on the nutrient agar, and in but few in- stances were the colonies on nutrient agar in great preponder- ance. As to what is to be inferred from this it is difficult to con- jecture; nor can we conclude that we have here two distinct flora. One striking feature which invites attention to the Ashby plate, is the variety of pigments observable. Especially characteristic are the blue, violet, pink, purple, red and brown colors which develop after several days. Cladothrix dichotoma, which will be considered later, is one of the most common soil organisms which thrive on a lim- ited nitrogen supply. It is a matter of common knowledge that organisms growing under adverse conditions, or rather in 9 \ an environment other than the optimum, lose at least some of their distinguishing characteristics. Many of these organ- isms, when transferred to nutrient agar slants, grow vigorously without pigment production. The azotobacter develop on the Ashby medium in great profusion, however, the differentiation of the nitrogen-fixing bacteria is not sufficiently established to render an enumera- tion of them possible. THE FIXATION OF FREE NITROGEN BY BACTERIA The relation of bacteria to nitrogen is perhaps the most important problem which presents itself to the agriculturist; the reason being that while the nitrogen forms a very large proportion of the constituents necessary to the building up of plant tissue, it is present in the soil in a very limited quantity, and consequently constant cropping would tend toward ex- hausting the supply. The fixation of free nitrogen by bacteria is consummated in two widely different ways, commonly designated as the sym- biotic and non-symbiotic relation. Symbiosis involves a favor- able influence of one species upon another. Many observers contend that this symbiotic relation is detrimental to the host. The symbiotic relation existing between the leguminoceae and certain bacteria enables the former to absorb free nitrogen from the air and elaborate it into nitrogenous compounds. This metamorphosis takes place within the leguminous nodules, the earliest description of which was given by Malpighi in 1687, and this observer referred to them as galls, i. e., diseased excresences, an opinion also shared by later writers. Treviranus, in 1853, was the first to regard these nodules as normal growths, and thirteen years later they were studied by Woronin, who made the subsequently important observa- tion, that the formation contains entirely closed cells filled with bacteria. Beyerinck, in 1888, indubitably established the fungoid nature of these bacteria by isolating them from the nodules, and cultivating them further in artificial media. Some ex- hibited certain slight but undeniable differences which were not so extensive as to make their discoverer feel justified in 10 classifying the organisms as separate species. Beyerinck proposed the name Bacillus radicicola, for these nodule pro- ducing bacteria; as to whether there are more than one species, authorities are still undetermined. Hereby is evolved a rational system for the continuous addition of nitrogen to the soil, an increase which can not only be enjoyed and ap- propriated by the leguminous plants, but likewise by succeed- ing vegetable growth. The non-symbiotic fixation of nitrogen possesses the im- portant feature of having more universal application. The following aerobic species are the most vigorous nitrogen fixing organisms hitherto discovered: A. agilis, A. chroococcum, A. vinelandi, A. beyerincki, A. vitreum, and A. woodstowni. Of these A. chroococcum is in all probability the most common in our soil. I have isolated this organism from many parts of the state. STUDIES ON IMPURE CULTURES In order to determine the relative nitrogen-fixing power of our soils, the aforementioned samples were inoculated into Ash- by's medium, and the folio wing process and technique followed: One hundred cubic centimeters of Ashby's medium was meas- ured into 250 ccErlenmeyer flasks, and sterilized: these flasks, therefore, each contained two grams of mannite; the medium occupying about three-fourths of an inch in depth in the bottom of the flask, there remained above the surface an abundant air space. These flasks were each inoculated with one gram of soil, and incubated at room temperature, which prevailed at about 33°C, for twenty-one days. At the end of this period the entire contents of these flasks were transferred to the Kjeldahl apparatus and the total nitrogen content determined by the Kjeldahl method, the ammonia being distilled over into tenth-normal sulphuric acid and titrated back with tenth-normal sodium hydroxide, using congo red as indicator. The original nitrogen content of the soil was determined by the Kjeldahl method, using ten grams of sample. Each operation during the investigation was carefully checked in order to reduce the possible error to the limit of experimental manipulation. Samples number 48 and 49 were 11 the only typical muck soils available. It will be observed that these show a fixation of 4.38 and 5.02 mg respectively. One mg of nitrogen at 0°C and 760 mm pressure represents ap- proximately 0.80 cubic centimeters. The per cent of nitrogen determined for these soils dried to constant weight, at 100- 110°C, were .4331 and .5481, which is very greatly in excess of any of the remaining soils. The average of sixteen soils of which the nitrogen content ranged uniformly above .2024 per cent, with a limit of .5481 was 4.91 mg, while the average of sixteen soils which fixed more than 4.91 mg and which had a nitrogen content of uniformly less than .2024 per cent, was 6.72 mg. Soil number 1 which had a nitrogen content of .0926 per cent, fixed 10.74 mg which was the highest value of any. It is reasonable, therefore, to conclude that a soil which contains much above .1000 per cent of nitrogen, other things being favorable, may equal or surpass any other soil in nitrogen fixing possibilities. Probably the number of azotobacter present in the soil determined the speed of the reaction. Azotobacter chroococcum was found to be universally distributed over the state. Many of the cultures which evinced strong nitrogen-fixing properties were covered with an imperfect floating membrane of brownish color shading off to almost black. A. chroococcum was definitely isolated from an alfalfa field at a depth of three feet, to which particular reference will be made later. Fungus growth developed on the surface of the medium in some instances. It is significant that in those overrun with molds and similar vegetation, the liquid frequently exhibited decided colors, usually yellow, though in one instance pink. The following table shows the amount of nitrogen fixed in milligrams and the percentage of moisture and nitrogen in each of the seventy samples of soil investigated: 12 FIXATION OF NITROGEN ASHBY'S MEDIUM MANNITE TABLE II Soil No. Moisture Per cent N Fixed in Mg Per cent N in Sample 1 13.44 2 5.23 3 18.22 4 16.33 5 10.77 6 5.09 7 6.41 8 10.83 9 11.37 10 16.91 11 6.27 12 8.91 13 13.50 14 7.73 15 13.55 16 5.95 17 4.45 18 16.56 19 11.09 20 4.98 21 15.05 22 9.15 23 15.69 24 15.44 25 9.54 26 15.11 27 7.96 28 11.44 29 7.15 30 9.80 31 15.59 32 14.23 33 6.15 34 7.56 35 7.18 36 7.95 37.. 9.55 10.74 3.70 6.05 4.98 3.37 3.04 9.76 7.22 6.94 5.60 4.50 4.97 0.12 4.17 4.08 0.19 2.58 5.57 5.19 2.70 6.32 5.50 4.56 7.30 4.56 3.66 0.59 4.59 4.63 0.61 0.63 0.29 2.83 6.18 4.16 3.95 5.14 .0926 .1981 .2507 .1724 .1104 .1093 .1609 .1987 .1807 .2067 .1537 .1528 .1487 .1493 .2335 .1503 .1418 .1549 .1441 .1431 .2176 .2092 .2507 .2351 .2254 .2024 .2110 .2063 .2156 .1731 .1725 .1686 .1241 .2590 .1462 .1730 .1949 13 TABLE II — Continued Soil No. Moisture Per cent N Fixed in Mg Per cent N in Sample 38 6.84 39 5.44 40 5.11 41 6.49 42 6.97 43 5.32 44 11.19 45 4.28 46 3.89 47 7.15 48 31.08 49 38.95 50 7.71 51 6.79 52 5.43 53 5.81 54 9.79 55 16.98 56 8.56 57 9.70 58 7.16 59 5.21 60 '. ... 9.88 61 12.41 62 4.48 63 3.90 64 7.12 65 4.30 66 11.98 67 .84 68 16.07 69 7.93 70.. 8.13 4.26 2.74 5.77 5.39 2.21 8.57 4.23 7.72 7.66 3.41 4.38 5.02 4.33 4.93 0.15 2.63 0.29 5.65 4.07 0.00 4.06 0.13 0.00 6.84 0.00 1.36 0.00 0.57 0.00 0.24 3.87 2.73 5.17 .1683 .1720 .1667 .1852 .1477 .1745 .1800 .1153 .1028 .1274 .4331 .5481 .1574 .1876 .1381 .1371 .0472 .2031 .1016 .0172 .0722 .0875 .0176 .1831 .0051 .0425 .0911 .1006 .0347 .0308 .1604 .0598 .0892 14 THE AVAILABILITY OF VARIOUS COMPOUNDS EFFECTING NITROGEN FIXATION It having been previously established that carbohydrates were essential for the maximum efficiency of nitrogen fixation, many sugars have been studied in these investigations. In this connection I have employed the following: mannite, maltose, lactose, saccharose, dextrose, galactose, levulose, arabinose, dulcite, sorbit, raffinose, rhamnose, mannose, ery- thrite, xylose, quercit, glycerine, dextrin, inulin, calcium lactate, and calcium butyrate. These compounds were the best obtainable, mostly Kahlbaum's product, and were ac- curately assayed for nitrogen. Ten soils which showed good fixation on mannite were selected for this purpose: numbers 1, 2, 7, 10, 24, 34, 41, 43, 47 and 61. These soils were inoculated into Ashby's medium under conditions similar to those followed in the previous experiment, with the one exception that the mannite was replaced by a special sugar or other compound. While it would have been highly desirable to have the data for all the sugars on the ten samples, the prohibitive price on many rendered this quite impossible. An inspection of the table shows the ten highest averages as follows: Sorbit 8.32 mg Dulcite 6.21 mg Mannite 7.17 mg Arabinose 6.14 mg Maltose 6.34 mg Dextrose 5.32 mg Mannose 6.32 mg Galactose 5.08 mg Levulose 6.28 mg Rhamnose 4.92 mg The position held by sorbit is probably only possible because of the remarkable soil number 7. Of the disaccharides, maltose gave the best results, lactose second, and saccharose third. An impure sample of maltose which we had in the labora- tory, and which contained 15 mg of nitrogen per two grams of sugar, fixed an average on soils 2, 10, and 41 of 1.02 mg, while the same soils on pure maltose corrected for a very small per- centage of nitrogen, fixed an average of 4.97 mg. This may be accepted as additional testimony that the presence of nitro- genous compounds in considerable amounts is not conducive to high fixation. Mannose, the aldehyde of the alcohol man- 15 nite, might be expected to approach the latter in fixation, but this did not prove to be the case, yet it differed from maltose only in the second place of decimals. Erythrite fixed an average, on soils 1 and 43 in twenty-one days, of 0.18 mg, while on soils 10, 24, 34 and 41, in fifty-four days, an average of 5.59 mg was fixed. The slow fermentation of this sugar renders it useless for laboratory purposes. Dextrin and inulin gave comparable results which were inconsiderable. Probably twenty-one days is insufficient to develop the maxi- mum efficiency of these polysaccharides. Glycerine in soils 1 and 43 fixed an average of 1.13 mg in twenty-one days. Soils 7, 10, 24, 34, 41, 47, and 61, fixed an average of 6.64 mg in thirty-nine days. Soil number 1 fixed 3. 58 mg in thirty-nine days and soil 43 fixed 4. 74 mg in the same time, a gain in the first instance of 2.61 mg and in the second of 2.45 mg in eighteen days. The slow fermentation of gly- cerine relegates it to the class with erythrite. In the work on mannite solutions one is struck with the great variety of odors, but perhaps the most characteristic is that of butyric acid. This led me to conclude that butyric acid or oxybutric was either one of the splitting products of mannite, or that according to an early discovery, two mole- cules of lactic acid were changed to one of butyric acid, giving off two molecules of carbon dioxide and two molecules of hy- drogen. After adding calcium butyrate to Ashby's medium it was inoculated with soils 1, 10 and 43, these yielded an aver- age fixation of 0.15 mg. The butyrate therefore seemed not available for carbon supply. In place of calcium butyrate, calcium lactate was next introduced using three grams to the flask, an equivalent of 2.45 grams calculated as free lactic acid. The average fixation for ten soils was 3.01 mg. The figures on this compound do not show the uniformity of the others, although soils 2, 7, 47 and 61 did remarkably well. The odor of butyric acid was not so pronounced as had been expected, but some cultures showed unmistakable evidence of its presence. In the fermentation of mannite considerable ethyl alcohol is split off. An analysis of the total acidity revealed approx- imately 30% acetic acid and 70% butyric acid. The following table shows the amount of nitrogen fixed when grown in a medium containing the compounds listed: 16 THE AVAILABILITY OF VARIOUS COMPOUNDS FOR NITROGEN FIXATION TABLE III " Soils 1 2 7 10 24 34 41 43 47 61 Maltose. . . . 5.24 4.06 11.21 4.92 4.53 5.70 5.93 4.40 5.79 11.61 Lactose. . . . 3.68 2.48 5.36 4.47 4.94 4.21 2.24 7.60 7.62 Saccharose. . 3.98 3.57 3.44 5.48 3.75 3.26 3.45 2.98 3.65 5.79 Mannite. . . . 10.74 3.70 9.76 5.60 7.30 6.18 5.39 8.57 7.66 6.84 Mannose. . . 11.10 3.50 10.90 4.57 3.35 5.94 4.91 5.58 10.31 3.01 Dextrose. . . 4.85 3.62 5.86 4.75 3.36 4.77 4.41 4.32 12.02 Levulose 4.84 4.55 9.82 6.62 5.00 4.80 6.94 5.98 6.64 7.69 Galactose. . . 5.87 3.17 4.78 4.42 5.35 4.36 3.74 7.48 6.56 Raffinose . . . 5.57 3.39 5.71 4.47 4.03 6.44 5.54 4.45 4.53 Rhamnose. . 5.06 4.66 3.94 5.60 5.61 4.43 5.15 Arabinose. . . 6.41 6.80 5.52 5.02 5.76 7.30 6.23 Dulcite 3.28 14.35 5.55 4.69 5.34 5.32 5.00 Erythrite . . . 0.23 7.83 4.02 8.07 2.45 0.13 Dextrin 3.70 1.88 3.52 3.64 2.02 Inulin 3.68 2.28 2.02 2.86 4.05 Sorbite 8.13 13.27 3.57 Xylose 6.93 3.84 3.42 Quercit 3.29 Glycerine . . . 3.58 3.37 6.50 6.90 6.53 7.90 4.74 6.14 9.18 Calcium Lactate 1.24 3.39 3.90 2.13 0.88 2.97 0.93 2.55 4.53 7.59 STUDIES ON AMMONIFICATION IN MIXED CULTURE The original 70 samples were used in connection with this experiment. The medium consisted of a solution of ten grams of Witte's peptone per liter of distilled water. One hundred cubic centimeters of this solution were measured into flasks of 500 cc capacity, sterilized, and inoculated with one gram of soil. After seven days incubation at 33° C, the contents of these Erlenmeyer flasks were transferred to the Kjeldahl apparatus, ten grams of magnesium oxide added, and the am- monia distilled over into semi-normal hydrochloric acid, and titrated back with semi-normal ammonium hydroxide, using congo red as indicator. In order to ascertain the percentage of nitrogen in the peptone, a composite sample was taken from the thirteen stock bottles, intimately mixed, and run by 17 the Kjeldahl method in triplicate. This composite sample which assayed 15.567% nitrogen was used in all ammonifica- tion experiments. Each flask therefore contained 155.67 mg of nitrogen. It will be observed that in soils number 49 and 61, over 80% of the nitrogen was evolved as ammonia. The muck soil 49 being a little below the loam. A survey of the table indicates that those soils which were especially active in fixing nitrogen also converted into ammonia more than 70% of the available nitrogen. AMMONIFICATION * IMPURE CULTURES TABLE IV Soil No. Nitrogen Evolved as Ammonia inMg Per cent of Nitrogen Evolved as Ammonia 1 . 11726 75 32 2 105 77 67 be 1 •s 21 TABLE VII Soil No. Mannite KNO3 Day on which Nitrate was Found to be Reduced 5 2 grams 1 gram 5th 10 2 grams 1 gram 5th 19 2 grams 1 gram 5th 22 2 grams 1 gram 5th 37 2 grams 1 gram 5th 61 2 grams 1 gram 5th 5 4 grams 1 gram 4th 10 6 grams 200 mg 4th 19 4 grams 200 mg 4th 22 4 grams 100 mg 4th 31 4 grams 500 mg 4th 37 6 grams 200 mg 4th 61. . 6 grams 200 mg 4th 61 4 grams 250 mg 4th Dextrose 37 2 grams 1 gram 5th 49 2 grams 1 gram 5th 61 2 grams 1 gram 5th The development within this medium was especially rapid, the evolution of gas beginning after the second day and continu- ing with increased vigor for some time. In the flasks which contained the gi eater amounts of carbohydrate the evidence of powerful reduction was most pronounced, the surface being rapidly overspread with fusarium and other fungi, which were not apparent on those with lower sugar content. The evolu- tion of gas was so violent in some cases as to force the felt- like growth from the surface, high above the liquid, as il- lustrated in the accompanying figure. The evidence here presented indicates that the reduction of nitrates is carried on with great vigor in the presence of considerable quantities of carbonaceous material. No appreciable difference could be detected in favor of either mannite or dextrose. 22 REDUCTION OF NITRATES CONTINUED IMPURE CULTURES: The medium used in this experiment was the 1% peptone solution to which was added one gram of potassium nitrate per liter. This solution was distributed in 150 cc. Erlenmeyer flasks in amounts of 50 cc each, and after inoculation with one gram of soil was incubated at 33°C for the length of time and with the results recorded below in table No. VIII. TABLE VIII Soil No. Time Per cent of Nitrite 1 30 hours 37 50 2 30 hours 44.00 3 30 hours 18 75 4 30 hours 31.25 5 30 hours 31.25 6 30 hours 25.00 7 30 hours 40.00 8 30 hours 15.00 9 30 hours 31.25 10 24 hours 50.00 11 24 hours 35.00 12 24 hours 37.50 13. . . 24 hours 31.30 14 24 hours 37.50 15 24 hours 50.00 16 24 hours 37.50 17 12 hours 50.00 18 24 hours 37.50 19 24 hours 43.80 20 24 hours 50.00 21 24 hours 21.88 22 24 hours 15.00 23 24 hours 21.88 24 24 hours 18.75 25 24 hours 18.75 26 24 hours 18.75 27 24 hours 15.25 28 24 hours 21.88 29 24 hours 37.50 30 24 hours 15.25 31 24 hours 18.75 32.. 24 hours 18.75 23 TABLE VIII— Continued Soil No. Time Per cent of Nitrite 33 30 hours 1875 34 30 hours 18 75 35 30 hours 22 00 36 30 hours 2500 37 30 hours 22 50 38 30 hours 1875 39 30 hours 18 75 40 30 hours 18.75 41.... 30 hours 22 50 42 30 hours 21.87 43 30 hours 1563 44 30 hours 21 87 45 36 hours 43.75 46 36 hours 4375 47 36 hours 31 25 48 36 hours 8.75 49 . .. 36 hours 33 30 50 36 hours 22 50 51 36 hours 27 50 52 36 hours 33 30 53 36 hours .45 54 36 hours 4375 55 24 hours 43 75 56 24 hours 46.25 57 24 hours 43 75 58 24 hours 4625 59 24 hours 43.74 60 24 hours 320 61 24 hours 4625 62 24 hours 43.75 63 24 hours 46.25 64 24 hours 43 75 65 24 hours 43.75 66 24 hours 31.25 67 24 hours 43 75 68 24 hours 43.75 69 24 hours 43.75 70 24 hours 43.75 The following solutions were used for determining the am- ount of nitrite present: 24 I a-Naphthylamine 1.00 gram Distilled water 100.00 grams II Sulphanilic acid 50 gram Dilute acetic acid 150.00 cc These solutions were kept in separate glass stoppered bottles. In the performance of the operation 5 cc of the culture medium were transferred to a Nesslerizing tube by means of a pipette, about 25 cc of distilled water added, and 1 cc of each of the above solutions introduced. The solution was brought up to the 50 or 100 cc mark with distilled water. The quan- titative estimation of the nitrite was determined by the col- orimic method. Every sample of soil without exception con- tained bacteria which reduced nitrates to nitrites. The odors emanating from these cultures and from the ammonfication experiments were exceedingly offensive. To determine the possibility of reduction of nitrates in soil infusion, 100 cc of distilled water was measured into each of nine 250 cc Erlen- meyer flasks; to each flask was added 100 mg of potassium ni- trate free from nitrite. These flasks were inoculated with soils 5, 47, 49, 51, 54, 55, 57, 60 and 61 respectively. They were incubated at 33°C for seven days. Tests were then made for nitrites and all without exception were found to be negative. No reduction is therefore probable except in the presence of considerable available nitrogen. STUDIES ON THE REDUCTION OF NITRITES IMPURE CULTURES: One hundred cubic centimeters of Ashby's medium without the carbohydrate, were measured into 250 cc Erlenmeyer flasks. To each flask was added vary- ing amounts of mannite, dextrose and potassium salts as in- dicated. These flasks were inoculated with one gram of the different soils and incubated at 28°C for the length of time and with the results recorded below in table No. IX. 25 TABLE IX Soil No. Mannite KNO2 Day on which Nitrite was Found to be Reduced 10 2 grams 1 gram 11 19 2 grams 1 gram 11 22 2 grams 1 gram 11 61 2 grams 1 gram 11 Soil No. Dextrose KN02 37 2 grams 1 gram 15 49 2 grams 1 gram 15 61 2 grams 1 gram 15 61.. 4 errams 1 crram 18 Soils number 37, 49, and 61 in dextrose gave on the eleventh day very appreciable reaction for nitrite; not until the fifteenth day did this totally disappear. An inspection of this table conveys the idea at once that the nitrite disappears from the mannite-containing medium more rapidly than from the dextrose. THE REDUCTION OF NITRITES IN PEPTONE SOLUTION IMPURE CULTURES: Fifty cubic centimeters of a medium containing ten grams of peptone, together with one gram of potassium nitrite per liter of distilled water, were measured into 150 cc Erlenmeyer flasks and sterilized. These flasks were each inoculated with one gram of soil and incubated at 33°C. After periods of time as indicated in the table number X, the solutions were tested for the presence of nitrites. On sterilizing peptone-nitrite solution in the Arnold, the liquid assumes a more decided yellow color than the peptone-nitrate solution. A quantitative determination shows that a portion of the nitrite has combined with the peptone, therefore the results are invariably low. All of the soils contained organisms which rapidly reduced nitrites to free nitrogen. The rapidity 26 of this reaction is very marked. After twenty-four hours the surface of the liquid is covered with foam, and at the end of two days very little nitrite remains. Those cultures in which the evolution of gas was most pronounced, evolved disagreeable odors; while those which developed but slight activity were comparatively odorless. But few species of bacteria reduce nitrites to free nitrogen in straight peptone media. The fluorescens group are of special importance in producing this change. Representatives of this class were isolated from many of these soils and were probably indigenous to all. TABLE X Soil No. Time Per cent of Nitrite 1 2 days 28.00 2 2 days 17.60 3 , 2 days 0.00 4 2 days 14.00 5 2 days 8.00 6 2 days 10.00 7 ... 2 days 14.00 8 2 days 0.00 9 . . 2 days 0.00 10.. 2 days 12.00 11 . 2 days 32.00 12 2 days 14.00 13 2 days 50.00 14 2 days 12.00 15 2 days 56.00 16 2 days 24.00 17 2 days 28.00 18 2 days 4.00 19 2 days 56.00 20 2 days 12.00 21.. . 5 days 2.40 22 5 days 0.00 23 5 days 0.00 24 5 days 0.00 25 5 days 5.60 26. . 5 days 0.00 27 5 days 0.00 28 5 days 0.00 29.. 5 days 0.00 27 TABLE X — Continued Soil No. Time Per cent of Nitrite 30 5 days 0 00 31 5 days 0 00 32 5 days 0 00 33 6 days 0 00 34 6 days 0 00 35 6 days 000 36 6 days 0 00 37 6 days 0.00 38 6 days ooo 39 6 days o oo 40 6 days 0 00 41 6 davs 0 00 42 6 days 0.00 43 6 davs 000 44 ....... 6 days 0 00 45 3 days 0.00 46 3 days 0 00 47 6 days 0 00 48 6 days 0.00 49 6 days 24 00 50 6 days 0.00 51 6 davs 12 00 52 6 days 0.00 53 6 days 0.00 54 ... . 4 days 0.00 55 4 days 0 00 56 4 days 0.00 57 4 days 44 00 58 4 days 0 00 59 4 days 0.00 60 4 days 50 00 61 4 days 0 00 62 4 days 70.00 63 4 davs 0.00 64 4 days 000 65 4 days 0 00 66 4 davs 63 00 67 4 days 000 68 4 days 0 00 69 4 days 0.00 70 4 days 0.00 28 BACTERIA CONTENT OF THE SUBSOIL An investigation was begun at the instigation of Hon. George Coupland, Regent of the University of Nebraska, to determine the lowest depth of subsoil in which micro-organisms might be found. In order to facilitate the sampling it was necessary that the subsoil, to the total depth projected, should be exposed. Therefore a hole, which was approximately four feet in diameter and twenty-one feet deep, was dug in an alfalfa field on the farm of Mr. Coupland. Twenty-one samples were taken at intervals of one foot along this per- pendicular line. Four surface samples were also taken, east, west, north and south of the excavation, at a distance of ten feet. These samples represent two and four inch depths. All samples were plated both on nutrient agar and on Ashby's medium. Table number XI, shows no striking variation to the sixth foot, except that the fourth level is abnormally high. The oscillation thence to the thirteenth level is neither sur- prising nor unprecedented, but the great preponderance on the thirteenth level is unaccountable. No visible stratum of impervious earth was observed. Alfalfa roots penetrated to the lowest depth. While the number of bacteria on the thir- teenth level was very great, yet, the flora was little diversified. Cladothrix dichotoma being the principal representative. Undoubted azotobacter were isolated from the third level. Of the different species isolated from the above samples, five fermented lactose bouillon with gas production. Fifty- eight per cent reduced nitrates to nitrites. Excavation showing method of sampling. 29 NUMBER OF BACTERIA PER GRAM OF SUBSOIL DRIED TO CONSTANT WEIGHT AT 100— 110°C TABLE XI Depth Nutrient Agar Ashby's Medium 2 inches . 2,500.000 610.000 4 inches 660 000 458,000 1 foot 290,000 417,000 2 feet 282,000 250,000 3 feet 169 000 185 000 4 feet 277,000 210,000 5 feet 156,000 114,000 6 feet 66,000 47,000 7 feet 11,000 2,000 8 feet 7.400 7,100 9 feet 700 300 10 feet 1,200 1,000 11 feet 4700 2,700 12 feet 1,200 2,600 13 feet 26,500 116,000 14 feet 50 000 15 feet 000 000 16 feet 000 000 18 feet 000 000 19 feet 000 000 20 feet 000 000 THE FATE OF UREA IN THE SOIL IMPURE CULTURES: To asertain the changes which take place when urea is added to nitrogenous media, 100 cc of the 1% peptone solution were measured into 250 cc Erlenmeyer flasks and sterilized. To each flask was added one gram of urea. They were then inoculated with soils 1, 10, 13, 25, 27, 28, 34, 35, 45, 49, 61 and 68. After seven days incubation at 33°C, each flask gave off strong odor of ammonia. An inquiry into the presence of carbonate was then instituted with positive result. I therefore conclude that the organisms which transform urea to ammonium carbonate in the presence of abundant available nitrogen supply, are universally distri- buted within our soil. To ascertain the trend of the reaction 30 in nitrogen-poor media, flasks were filled with Ashby's medium as before, one gram of urea introduced, and inoculated as in the nitrogenous medium. After forty eight hours the surface was covered with gas bubbles and on the fourth day a strong odor of ammonia was evolved from each flask. It therefore appears that in the presence of an abundant nitrogen supply urea is converted into ammonium carbonate, and that this process is not impeded by the presence of carbohydrate in great excess, but is rather promoted, even though the nitrogen content be very small. Several flasks of Ashby's medium were inoculated with soils and thio-urea introduced in place of urea. The growth in these flasks was much less pronounced than in those con- taining urea. Evidently ammonium sulphite was not formed. Hippuric acid is split, in nitrogenous media, into benzoic acid and amino-acetic acid. Several flasks of Ashby's medium were inoculated with soil and one gram of hippuric acid in- troduced. After a few days the surface was overgrown with molds, later a vigorous evolution of carbon dioxide was per- ceptible, the overlying growth being forced high in the flask. In a second experiment the hippuric acid was neutralized with sodium hydroxide before being transferred to the Ashby's medium. The splitting of the hippuric acid molecule into benzoic acid and amino-acetic acid, and the subsequent union of the benzoic acid and calcium carbonate to form calcium benzoate, necessitates the liberation of considerable quanti- ties of carbon dioxide. Amino-acetic acid (Glycocoll) is reduced by soil bacteria to ammonia and acetic acid, this re- duction is consummated both in nitrogenous and non-nitro- genous media. THE REDUCTION OF NITRATES TO NITRITES The breaking down of organic compounds by bacterial agency, falls under two categories; simple cleavage, and partial elementary disintegration of the proteid and carbohydrate molecule. In the first category we are concerned with the simple splitting off of groups from the original relatively com- plex molecule. Among the cleavage products may be men- tioned alcohols, esters, mercaptans, amino-acids, phenol, 31 skatol, indol, acids, glycols, etc. The formation of nascent hydrogen by the action of destructive organisms on car- bohydrate and proteid compounds may be best illustrated by a careful study of the products obtained by the destructive distillation of coal, wood and other products of animal and vegetable origin. In the destructive distillation of coal we get as products: 02, H2, N2, S2, Cx, H20, NH3, H2S, CH4, CO, C02, C2H2, C2H4, C6H6, CS2, etc. All of these products are obtained in small or large amounts depending on the com- position of the coal, character of heating, etc. Can these products be explained in any other way than that the complex proteid molecules undergo in this process of destructive dis- tillation, complete disintegration into their constitutent elements: C, 0, H, N, S.? These elements must exist mo- mentarily in the active or nascent state. Because of their great chemical affinity these active elements then combine with each other to form inactive molecules which are free to pass off from the sphere of action. In the formation of these simple molecules some of the atoms have combined with different atoms, while some have combined with other atoms of the same kind. As a result of the first method we get: H20, NH3, H2S, CO, C02, C2H4, CH4, etc. As a result of the second method we get: Cx (coke or soot), N2, H2, 02, S2, etc. For the reduction of nitrates in pure culture, a medium consisting of peptone 1% and potassium nitrate 1% was em- ployed. 10 cc of this medium were introduced into each tube and sterilized. These tubes were inoculated with the various organisms and incubated at 33°C for ten days. At the expiration of this time they were tested for the presence of nitrates and nitrites. The presence of nitrate was deter- mined by the addition of a 1% solution of diphenylamine in pure concentrated sulphuric acid. The nitrite was deter- mined according to the method used in the previous work on impure cultures. Those cultures which failed to reduce nitrates to nitrites within ten days were duplicated and the time extended to thirty days for a final reading. The organisms in the following list were obtained from the celebrated Krai collection, Vienna Austria, Prof. Kraus Cura- tor; and from the American Museum of Natural History, 32 New York, Prof. C. E. A. Winslow Curator. No attempt was made to determine whether they were true to name, the only precaution being that they were in pure culture. It is quite improbable that any considerable collection of species would be assembled without some repetition under different names. Not all the organisms listed are strictly soil bacteria, several of the intestinal group being purposely included. A few have no connection with soil fertility whatever. CATALOG OF ORGANISMS 1. Bacterium acetosum [Henneberg] 2. Bacterium lactis aerogenes [Escherich] 3. Bacillus brassicae acidae 4. Micrococcus agilis [Ali-Cohen] 5. Bacillus acidi lactici [Hueppe] 6. Micrococcus albidus 7. Bacillus amylovorus 8. Bacillus anthracis 9. Bacillus pseudo-anthracis 10. Bacillus anthracoides 11. Bacterium annulatum A 12. Bacterium annulatum B 13. Bacillus aquatile 14. Bacillus arborescens [Frankland] 15. Bacillus argentinensis [Kayser] 16. Micrococcus ascoformans 17. Bacillus asterosporus 18. Bacterium aurantiacus 19. Sarcina aurantiaca 20. Bacillus Baccarinii [Macchiati] 21. Bacterium beticolum 22. Micrococcus brunneus 23. Bacillus budapestinensis [Ajtay] 24. Bacillus butyricus [Hueppe] 25. Bacillus candicans 26. Micrococcus candicans [Flugge] 27. Monila Candida 28. Bacillus campestris 29. Rhodobacillus capsulatus 33 30. Bacillus cereus [Frankland] 31. Bacillus cereulens 32. Micrococcus cereus 33. Micrococcus carneus 34. Micrococcus cinnabareus 35. Bacillus cloacae [Jordan] 36. Micrococcus citreus 37. Bacillus constrictus 38. Micrococcus concentricus 39. Bacillus coli commune [Kruse] 40. Bacillus coli-anaerogenes 41. Bacillus carotovorus [Jones] 42. Bacillus cyanogenes [Hueppe] 43. Bacillus cylindrosporus [Burchard] 44. Bacillus creusus 45. Bacillus cyaneus 46. Bacterium crysogloia 47. Bacillus denitrificans 48. Bacillus dendroides 49. Pseudomonas destructans 50. Bacillus disciformans 51. Bacillus enteritidis [Gaertner] 52. Bacillus esterigenes [Krai] 53. Bacillus esterigenes A 54. Bacillus esterigenes D 55. Bacterium lactis erythrogenes [Grotenfeldt] 56. Bacillus ethacinicus 57. Bacillus ethaceto succinicus 58. Bacillus ferruginous 59. Bacillus faecalis alcaligenes [Petruschky] 60. Sarcina flava 61. Micrococcus flavus [Flugge] 62. Bacillus flavidus 63. Bacterium filiforme [Henrici] 64. Bacterium filifaciens [H. Jensen] 65. Bacillus Fitzianus 66. Bacillus fluorescens liquefaciens [Flugge] 67. Bacillus fluorescens non liquefaciens 68. Bacillus fluorescens tenuis 69. Bacillus Frostii 34 70. Bacillus fuchsinus [Balkhout] 71. Sarcina gasformans 72. Bacillus graviolens [A. Meyer et Gottheil] 73. Bacterium aquatile griseum 74. Micrococcus grossus [Henrici] 75. Bacterium Hartlebi [H. Jensen] 76. Bacillus havaniensis 77. Bacillus herbicoli aureus 78. Bacillus helvolus [Zimmermann] 79. Bacillus hoagii 80. Bacillus hyponitrous 81. Bacillus immobile 82. Bacillus indicus 83. Bacillus indigoferus [Voges] 84. Bacillus irritans 85. Bacillus ivilans 86. Bacillus jasminocyaneus 87. Bacillus juglandis 88. Bacillus kiliensis 89. Bacillus lactis 90. Bacillus lactorubefaciens [Gruber] 91. Bacillus lateritia 92. Bacillus levans 93. Bacillus lactis amari liquefaciens [Freudenreich] 94. Bacillus liodermos 95. Bacillus limosus 96. Sarcina liquefaciens [Frankland] 97. Bacillus liquefaciens 98. Bacillus lactis niger 99. Bacillus liquefaciens niger 100. Bacillus loxosus [Burchard] 101. Bacterium aquatile gasformans non liquefaciens 102. Micrococcus luteus 103. Sarcina lutea 104. Streptococcus luteus liquefaciens 105. Bacillus maidis 106. Bacillus melonis [Winslow] 107. Bacillus mesentericus fuscus 108. Bacillus mesentericus niger 109. Bacillus mesentericus ruber 35 110. Bacillus mesentericus vulgatus 111. Bacillus megatherium [De Bary] 112. Bacillus miniaceus [Zimmermann] 113. Bacillus proteus mirabilis 114. Sarcina mobilis 115. Moellers grass bacillus, Mist. 116. Bacterium muris [E. Klein] 117. Bacillus mycoides [Flugge] 118. Bacillus nanus 119. Bacillus ochraceus [Zimmermann] 120. Bacillus oleraceae 121. Bacillus olfactorius 122. Oidium lactis 123. Bacillus oleae [Schiff-Giorgini] 124. Cladothrix odorifera 125. Cladothrix dichotoma. 126. Bacillus oxalatus 127. Bacterium para-coli gasformans anindolicum [Kayser] 128. Bacillus parvus 129. Rhodobacillus palustis 130. Bacillus Petasites [A. Meyer et Gottheil] 131. Bacterium Petroselini [Bur chard] 132. Bacillus prodigiosus [Flugge] 133. Bacillus lactis proteolyticus [Rullman] 134. Bacillus plicatus 135. Bacterium phytophtorum 136. Bacillus proteus 137. Bacillus pumilis [A. Meyer et Gottheil] 138. Bacillus punctatus 139. Bacillus fluorescens putidus [Flugge] 140. Bacillus phosphorescens 141. Pseudomonas pyocyanea 142. Bacterium radiatum [Kayser] 143. Pseudomonas radicicola, clover 144. Bacillus ramosus non liquefaciens 145. Bacillus rosaceus 146. Micrococcus roseus [Eisenberg] 147. Bacillus of ropy milk 148. Micrococcus rhodochrous 149. Bacillus brunneus mycoides roseus 36 150. Bacillus capsulatus roseus 151. Bacillus ruber 152. Micrococcus ruber 153. Bacillus subtilis var ruber 154. Bacillus ruber Plymouth 155. Bacillus rubidus 156. Spirillum rubrum 157. Bacterium rugosum [Henrici] 158. Bacillus ruber of Kiel 159. Spirillum rugula 160. Bacterium rubilum 161. Bacillus ruminatus 162. Bacillus rutilus 163. Bacillus rutilensis 164. Spirillum serpens 165. Bacillus silvaticus [Arthur Meyer et Neide] 166. Bacillus simplex [A. Meyer et Gottheil] 167. Vibrio saprophilus 168. Micrococcus sordidus 169. Bacillus luteus sporogenes [Wood, Smith et Baker] 170. Bacterium der sorbose 171. Bacillus solanisaprus 172. Bacillus sphaericus [Arthur, Meyer et Neide] 173. Staphlococcus cereus aureus 174. Staphlococcus pyogenes citreus 175. Staphlococcus pyogenes albus 176. Staphlococcus pyogenes aureus 177. Bacillus ochraceus subflavus 178. Bacterium subflavum 179. Micrococcus sulfur 180. Bacillus subtilis [Ehrenberg] 181. Bacterium Stutzeri [H . Jensen] 182. Bacillus synxanthus [Cohn] 183. Bacterium tremellioides [Schottelius] 184. Bacillus tumefaciens 185. Bacillus tumescens 186. Bacillus typhosus [Eberth] 187. Bacillus para- typhosus 188. Sarcina ventriculi [Goodsir] 189. Bacillus violaceus [Jordan] 37 190. Azotobacter vinelandii [Lipman] 191. Micrococcus viticulosus 192. Bacillus proteus viridis 193. Bacillus aquatile villos 194. Bacillus vivax 195. Spirillum volutans 196. Bacillus proteus vulgaris [Hauser] 197. Bacterium xanthochlorum 198. Bacillus xylinum 199. Bacillus proteus Zenkeri [Hauser] 200. Bacillus Zopfii 201. Boden I. [Tsiklinsky-Sudpolar expedition 1903-5] 1. Bacterium acetosum: good growth; vigorous evolution of gas on addition of acid; nitrate content considerably diminished; Strong formation of nitrite. 2. Bacterium lactis aerogenes: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 3. Bacillus brassicae acidae: good growth; moderate evolution of gas on addition of acid; nitrate content considerably diminished; Weak formation of nitrite. 4. Micrococcus agilis: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 5. Bacillus acidi lactici: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 6. Micrococcus albidus: moderate growth; slight evolution of gas on addition of acid; nitrate content not greatly diminished; Weak formation of nitrite. 7. Bacillus amylovorus: good growth; strong evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 8. Bacillus anthracis: good growth; vigorous evolution of gas on ad- dition of acid; nitrate content greatly diminished; Strong formation on nitrite. 9. Bacillus pseudo-anthracis: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 10. Bacillus anthracoides: good growth; moderate evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 38 11. Bacterium annulatum A: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 12. Bacterium annulatum B: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 13. Bacillus aquatile: good growth; vigorous evolution of gas on addi- tion of acid; nitrate content greatly diminished; Strong formation of nitrite. 14. Bacillus arborescens: good growth; vigorous evolution of gas addi- tion of acid; nitrate content greatly diminished; Strong formation of nitrite. 15. Bacillus argentinensis: good growth; moderate evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 16. Micrococcus ascoformans: fair growth; slight evolution of gas on addition of acid; nitrate content not greatly diminished; Weak formation of nitrite. 17. Bacillus asterosporus: scant growth; slight evolution of gas on addition of acid; nitrate content not greatly diminished; Weak formation of nitrite. 18. Bacterium aurantiacus: good growth; no evolution of gas on addi- tion of acid; nitrate content unchanged; No formation of nitrite. 19. Sarcina aurantiaca: scant growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 20. Bacillus Baccarinii: fair growth; slight evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 21. Bacterium beticolum: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly reduced; Moderate formation of nitrite. 22. Micrococcus brunneus: moderate growth; slight evolution of gas on addition of acid; nitrate content not greatly diminished; Weak formation of nitrite. 23. Bacillus budapestiensis: good growth; no evolution of gas on addi- tion of acid; nitrate content very slightly diminished; Very weak formation of nitrite. 24. Bacillus butyricus: good growth; no evolution of gas on addition of acid; nitrate content unchanged' No formation of nitrite. 25. Bacillus candicans: moderate growth; no evolution of gas on addi- tion of acid; nitrate content unchanged; No formation of nitrite. 26. Micrococcus candicans: moderate growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 39 27. Monila Candida: fair growth; slight evolution of gas on addition of acid; nitrate content not greatly diminished; Weak formation of nitrite. 28. Bacillus campestris: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 29. Rhodobacillus capsulatus: moderate growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 30. Bacillus cereus: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 31. Bacillus cereulens: good growth; moderate evolution of gas on addition of acid; nitrate content appreciably diminished; Weak formation of nitrite. 32. Micrococcus cereus: moderate growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 33. Micrococcus carneus: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 34. Micrococcus cinnabareus: fair growth; slight evolution of gas on addition of acid; nitrate content not greatly diminished; Weak formation of nitrite. 35. Bacillus cloacae: good growth; vigorous evolution of gas on addi- tion of acid; nitrate content greatly diminished; Strong formation of nitrite. 36. Micrococcus citreus: fair growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 37. Bacillus constrictus: good growth; vigorous evolution of gas on addition of acid nitrate content greatly diminished; Strong formation of nitrite. 38. Micrococcus concentricus: fair growth; no evolution of gas on addi- tion of acid; nitrate content unchanged; No formation of nitrite. 39. Bacillus coli commune: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 40. Bacillus coli-anaerogenes: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 41. Bacillus carotovorus: fair growth; slight evolution of gas on addi- tion of acid; nitrate content not greatly diminished; Weak formation of nitrite. 42. Bacillus cyanogenes: good growth; slight evolution of gas on addi- tion of acid; nitrate content not greatly diminished; Weak formation of nitrite. 43. Bacillus cylindrosporus: fair growth; slight evolution of gas on addition of acid; nitrate content not greatly diminished; Weak formation of nitrite. 44. Bacillus creusus: good growth; moderate evolution of gas on addi- tion of acid; nitrate content considerably diminished; Moderate formation of nitrite. 45. Bacillus cyaneus: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 46. Bacterium crysogloia: moderate growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 47. Bacillus denitrificans: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 48. Bacillus dendroides: fair growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 49. Pseudomonas destructans: moderate growth; fair evolution of gas on addition of acid; nitrogen content slightly diminished; Weak formation of nitrite. 50. Bacillus disciformans: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 51. Bacillus enteritidis: good growth; vigorous evolution of gas on addi- tion of acid; nitrate content greatly diminished; Strong formation of nitrite. 52. Bacillus esterigenes: fair growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 53. Bacillus esterigenes A: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 54. Bacillus esterigenes D: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 55. Bacterium lactis erythrogenes: good growth; slight evolution of gas on addition of acid; nitrate content not greatly diminished; Weak formation of nitrite. 56. Bacillus ethacinicus: fair growth; moderate evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 57. Bacillus ethaceto succinicus: good growth; moderate evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 58. Bacillus ferruginous: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 41 59. Bacillus faecalis alcaligenes: good growth; slight evolution of gas on addition of acid; nitrate content not greatly diminished; Weak formation of nitrite. 60. Sarcina flava: fair growth; slight evolution of gas on addition of acid; nitrate content not greatly diminished; Weak formation of nitrite. 61. Micrococcus flavus: moderate growth; slight evolution of gas on addition of acid; nitrate content not greatly diminished; Weak formation of nitrite. 62. Bacillus flavidus: moderate growth; weak evolution of gas on addi- tion of acid; nitrate content not greatly diminished; Weak formation of nitrite. 63. Bacterium filiforme: good growth; slight evolution of gas on addi- tion of acid; nitrate content not greatly diminished; Weak formation of nitrite. 64. Bacterium filifaciens: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 65. Bacillus Fitzianus: good growth; slight evolution of gas on addition of acid; nitrate content not greatly diminished; Weak formation of nitrite. 66. Bacillus fluorescens liquefaciens: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 67. Bacillus fluorescens non-liquefaciens: good growth; vigorous evolu- tion of gas on addition of acid; nitrate content greatly dimin- ished; Strong formation of nitrite. 68. Bacillus fluorescens tenuis: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 69. Bacillus Frostii: good growth; slight evolution of gas on addition of acid; nitrate content not greatly diminished. Weak formation of nitrite. 70. Bacillus fuchsinus: moderate growth; no evolution of gas on ad- dition of acid; nitrate content unchanged; No formation of nitrite. 71. Sarcina gasformans: moderate growth; no evolution of gas on addition of acid; nitrite content unchanged; No formation of nitrite. 72. Bacillus graviolens: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 73. Bacterium aquatile griseum: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 74. Micrococcus grossus: moderate growth; slight evolution of gas on addition of acid; nitrate content not greatly diminished; Weak formation of nitrite. 42 75. Bacterium Hartlebi: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 76. Bacillus Havaniensis: good growth; moderate evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 77. Bacillus herbicoli aureus: moderate growth; slight evolution of gas on addition of acid; nitrate content diminished; Weak formation of nitrite. 78. Bacillus helvolus: good growth; moderate evolution of gas on ad- dition of acid; nitrate content diminished; Fair formation of nitrite. 79. Bacillus Hoagii: moderate growth; fair evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 80. Bacillus hyponitrous: scant growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 81. Bacillus immobile: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 82. Bacillus indicus: good growth; slight evolution of gas on addition of acid; nitrate content not greatly diminished; Weak formation of nitrite. 83. Bacillus indigoferus: good growth; moderate evolution of gas on addition of acid; nitrate content slightly diminished; Moderate formation of nitrite. 84. Bacillus irritans: good growth; moderate evolution of gas on ad- dition of acid; nitrate content slightly diminished; Weak formation of nitrite. 85. Bacillus ivilans: moderate growth; slight evolution of gas on ad- dition of acid; nitrate content not greatly diminished; Weak formation of nitrite. 86. Bacillus jasminocyaneus: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 87. Bacillus juglandis: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 88. Bacillus kiliensis: moderate growth; no evolution of gas on ad- dition of acid; nitrate content unchanged; No formation of nitrite. 89. Bacillus lactis: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 90. Bacillus lactorubefaciens: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 43 91. Bacillus lateritia: good growth; slight evolution of gas on addition of acid; nitrate content almost unchanged; Very weak formation of nitrite. 92. Bacillus levans: moderate growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 93. Bacillus lactis amari liquefaciens: good growth; slight evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 94. Bacillus liodermos: good growth; moderate evolution of gas on addition of acid; nitrate content considerably diminished; Moderate formation of nitrite. 95. Bacillus limosus: moderate growth; slight evolution of gas on addition of acid; nitrate content not materially changed; Weak formation of nitrite. 96. Sarcina liquefaciens: fair growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 97. Bacillus liquefaciens: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 98. Bacillus lactis niger: good growth; moderate evolution of gas on addition of acid; nitrate content not greatly diminished; Weak formation of nitrite. 99. Bacillus liquefaciens niger: good growth; moderate evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 100. Bacillus loxosus: good growth; vigorous evolution of gas on ad- dition of acid; nitrate content greatly diminished; Strong formation of nitrite. 101. Bacterium aquatile gasformans non-liquefaciens: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 102. Micrococcus luteus: fair growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 103. Sarcina lutea: moderate growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 104. Streptococcus luteus liquefaciens: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 105. Bacillus maidis: scant growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 44 106. Bacillus melonis: good growth; vigorous evolution of gas on ad- dition of acid; nitrate content greatly diminished; Strong formation of nitrite. 107. Bacillus mesentericus fuscus: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 108. Bacillus mesentericus niger: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 109. Bacillus mesentericus ruber: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 110. Bacillus mesentericus vulgatus: good growth; slight evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 111. Bacillus megatherium: good growth; no evolution of gas on addi- tion of acid; nitrate content unchanged; No formation of nitrite. 112. Bacillus miniaceus: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 113. Bacillus proteus mirabilis: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 114. Sarcina mobilis: fair growth; slight evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 115. Moeller's grass bacillus, Mist: good growth; moderate evolution of gas on addition of acid; nitrate content slightly diminished; Moderate formation of nitrite. 116. Bacterium muris: fair growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 117. Bacillus mycoides: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 118. Bacillus nanus: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 119. Bacillus ochraceus: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 120. Bacillus oleraceae: moderate growth; fair evolution of gas on ad- dition of acid; nitrate content slightly diminished; Weak formation of nitrite. 121. Bacillus olfactorius: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 45 122. Oidium lactis: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 123. Bacillus oleae: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 124. Cladothrix odorifera: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 125. Cladothrix dichotoma: good growth; no evolution of gas on ad- dition of acid; nitrate content unchanged; No formation of nitrite. 126. Bacillus oxalatus: good growth; strong evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 127. Bacterium paracoli gasformans anindolicum: good growth; moder- ate evolution of gas on addition of acid; nitrate content slightly diminished; Moderate formation of nitrite. 128. Bacillus parvus: good growth; vigorous evolution of gas on ad- dition of acid; nitrate content greatly diminished; Strong formation of nitrite. 129. Rhodobacillus palustis: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 130. Bacillus Petasites: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 131. Bacterium Petroselini: good growth; moderate evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 132. Bacillus prodigiosus: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 133. Bacillus lactis proteolyticus: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 134. Bacillus plicatus: moderate growth; slight evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 135. Bacterium phytophtorum : fair growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 136. Bacillus proteus: good growth; moderate evolution of gas on ad- dition of acid; nitrate content slightly diminished; Weak formation of nitrite. 137. Bacillus pumilus: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 46 138. Bacillus punctatus: good growth; vigorous evolution of gas on ad- dition of acid; nitrate content greatly diminished; Strong formation of nitrite. 139. Bacillus fluorescens putidus: moderate growth; no evolution of gas on addition of acid; nitrate content undiminished; No formation of nitrite. 140. Bacillus phosphorescens: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 141. Pseudomonas pyocyanea: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 142. Bacterium radiatum: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 143. Pseudomonas radicicola, clover: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 144. Bacillus ramosus non liquefaciens: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 145. Bacillus rosaceus: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 146. Micrococcus roseus: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 147. Bacillus of ropy milk: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 148. Micrococcus rhodochrous: moderate growth; slight evolution of gas on addition of acid; nitrate content not greatly diminished; Weak formation of nitrite. 149. Bacillus brunneus mycoides roseus: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 150. Bacillus capsulatus roseus: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 151. Bacillus ruber: moderate growth; fair evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 152. Micrococcus ruber: moderate growth; fair evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 153. Bacillus subtilis var ruber: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 47 154. Bacillus ruber Plymouth: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 155. Bacillus rubidus: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 156. Spirillum rubrum: moderate growth; no evolution of gas on ad- dition of acid; nitrate content unchanged; No formation of nitrite. 157. Bacterium rugosum: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 158. Bacillus ruber of Kiel: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 159. Spirillum Rugala: moderate growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 160. Bacterium rubilum: fair growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 161. Bacillus ruminatus: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 162. Bacillus rutilus: good growth; moderate evolution of gas on ad- dition of acid; nitrate content slightly diminished; Weak formation of nitrite. 163. Bacillus rutilensis: fair growth; moderate evolution of gas on addition of acid; nitrate content slighlty diminished; Weak formation of nitrite. 164. Spirillum serpens: moderate growth; no evolution of gas on the addition of acid; nitrate content unchanged; No formation of nitrite. 165. Bacillus silvaticus: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 166. Bacillus simplex: good growth; vigorous evolution of gas on ad- dition of acid; nitrate content greatly diminished ,v Strong formation of nitrite. 167. Vibrio saprophilus: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 168. Micrococcus sordidus: moderate growth; slight evolution of gas on addition of acid; nitrate content diminished; Weak formation of nitrite. 169. Bacillus luteus sporogenes: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 48 170. Bacterium der sorbose: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 171. Bacillus solanisparus: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 172. Bacillus sphaericus: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 173. Staphlococcus cereus aureus: good growth; moderate evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 174. Staphlococcus pyogenes citreus: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 175. Staphlococcus pyogenes albus: good growth; moderate evolution of gas on addition of acid; nitrate content slightly reduced; Weak formation of nitrite. 176. Staphlococcus pyogenes aureus: good growth; fair evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 177. Bacillus ochraceus subflavus: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong furmation of nitrite. 178. Bacterium subflavum: fair growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 179. Micrococcus sulfur: moderate growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 180. Bacillus subtilis: good growth; moderate evolution of gas on ad- dition of acid; nitrate content slightly diminished; Weak furmation of nitrite. 181. Bacterium Stutzeri: good growth; strong evolution of gas on ad- dition of acid; nitrate content greatly diminished; Strong formation of nitrite. 182. Bacillus synxanthus: good growth; moderate evolution of gas on addition of acid; nitrate content moderately diminished; Weak formation of nitrite. 183. Bacterium tremellioides: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 184. Bacillus tumefaciens: fair growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 185. Bacillus tumescens: good growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 49 186. Bacillus typhosus: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 187. Bacillus para-typhosus: moderate growth; slight evolution of gas on addition of acid; nitrate content not greatly diminished; Weak formation of nitrite. 188. Sarcina ventriculi: fair growth; moderate evolution of gas on addition of acid; nitrate content considerably diminished; Moderate formation of nitrite. 189. Bacillus violaceus: moderate growth; fair evolution of gas on .ad- dition of acid; nitrate content slightly diminished; Weak formation of nitrite. 190. Azotobacter vinelandi: good growth; moderate evolution of gas on addition of acid; nitrate content considerably diminished; Weak formation of nitrite. 191. Micrococcus viticulosus: moderate growth; slight evolution of gas on addition of acid; nitrate content not greatly diminished; Weak formation of nitrite. 192. Bacillus viridis: good growth; vigorous evolution of gas on ad- dition of acid; nitrate content greatly diminished; Strong formation of nitrite. 193. Bacillus aquatile villos: moderate growth; slight evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 194. Bacillus vivax: fair growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 195. Spirillum volutans: good growth; moderate evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 196. Bacillus proteus vulgaris: good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 197. Bacterium xanthochlorum: good growth; moderate evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 198. Bacillus xylinum: good growth; moderate evolution of gas on addition of acid; nitrate content slightly diminished; Weak formation of nitrite. 199. Bacillus proteus Zenkeri: good growth; moderate evolution of gas on addition of acid: nitrate content slightly diminished; Weak formation of nitrite. 200. Bacillus Zopfii: scant growth; no evolution of gas on addition of acid; nitrate content unchanged; No formation of nitrite. 201. Boden I (Tsiklinsky-Sudpolar expedition, 1903-5): good growth; vigorous evolution of gas on addition of acid; nitrate content greatly diminished; Strong formation of nitrite. 50 THE FOLLOWING ORGANISMS REDUCED NITRATE TO NITRITE 1. Bacterium acetosum; 2. Bacterium lactis aerogenes; 3. Bacillus brassicae acidae; 5. Bacillus acidi lactici; 6. Mi- crococcus albidus; 7. Bacillus amylovorus; 8. Bacillus anth- racis; 10. Bacillus anthracoides; 11. Bacterium annulatumA; 12. Bacterium annulatum B; 13. Bacillus aquatile; 14. Bacil- lus arborescens; 15. Bacillus argentinensis; 16. Micrococcus ascoformans; 17. Bacillus asterosporus; 20. Bacillus baccar- inii; 21. Bacterium beticolum; 22. Micrococcus brunneus; 23. Bacillus budapestiensis; 27. Monila Candida; 30. Bacillus cereus; 31. Bacillus cereulens; 34. Micrococcus cinnabareus; 35. Bacillus cloacae; 37. Bacillus constrictus; 39. Bacillus coli commune; 40. Bacillus coli-anaerogenes; 41. Bacillus carotovorus; 42. Bacillus cyanogenes; 43. Bacillus cylin- drosporus; 44. Bacillus creusus; 47. Bacillus denitrificans; 49. Pseudomonas destructans; 50. Bacillus disciformans; 51. Bacillus enteritidis; 55. Bacterium lactis erythrogenes; 56. Bacillus ethacinicus; 57. Bacillus ethaceto succinus; 58. Bacillus ferruginous; 59. Bacillus faecalis alcaligenes; 60. Sarcina flava; 61. Micrococcus flavus; 62. Bacillus flavidus; 63. Bacterium filiforme; 64. Bacterium filifaciens; 65. Bacil- lus Fitzianus; 66. Bacillus fluorescens liquefaciens; 67. Bacillus fluorescens non liquefaciens; 69. Bacillus Frostii; 73. Bacillus aquatile griseum; 74. Micrococcus grossus; 75. Bacterium Hartlebi; 76. Bacillus Havaniensis; 77. Bacillus herbicoli aureus; 78. Bacillus helvolus; 79. Bacillus Hoagii; 81. Bacillus immobile; 82. Bacillus indicus; 83. Bacillus indigoferus; 84. Bacillus irritans; 85. Bacillus ivilans; 86. Bacillus jasminocyaneus; 89. Bacillus lactis; 90. Bacillus lactorubefaciens; 91. Bacillus lateritia; 93. Bacillus lactis amari liquefaciens; 94. Bacillus liodermos; 95. Bacillus limosus; 98. Bacillus lactis niger; 99. Bacillus liquefaciens niger; 100. Bacillus loxosus; 101. Bacterium aquatile gas- formans non liquefaciens; 106. Bacillus melonis; 107. Bacillus mesentericus fuscus; 108. Bacillus mesentericus niger; 109. Bacillus mesentericus ruber; 110. Bacillus mesentericus vulgatus; 112. Bacillus miniaceus; 114. Sarcina mobilis, 115. Moeller's grass bacillus, Mist.; 117. Bacillus mycoides; 51 118. Bacillus nanus; 120. Bacillus oleraceae; 121. Bacillus olfactorius; 123. Bacillus oleae; 126. Bacillus oxalatus; 127. Bacterium paracoli gasformans anindolicum; 128. Bacillus parvus; 129. Rhodobacillus palustis; 131. Bacterium Petro- selini; 132. Bacillus prodigiosus; 133. Bacillus lactis proteo- lyticus; 134. Bacillus plicatus; 136. Bacillus proteus; 138. Bacillus punctatus; 140. Bacillus phosphorescens; 141. Pseudomanaspyocyanea; 143. Pseudomonas radicicola clover; 146. Micrococcus roseus; 147. Bacillus of ropy milk; 148. Micrococcus rhodochrous; 149. Bacillus brunneus mycoides roseus; 150. Bacillus capsulatus roseus; 151. Bacillus ruber; 152. Micrococcus ruber; 153. Bacillus subtilis var ruber; 154. Bacillus ruber Plymouth; 157. Bacterium rugosum; 158. Bacillus ruber of Kiel; 162. Bacillus rutilus; 163. Bacillus rutilensis; 165. Bacillus silvaticus; 166. Bacillus simplex; 167. Vibrio saprophilus; 168. Micrococcus sordidus; 170. Bacterium der sorbose; 171. Bacillus solanisparus; 173. Staphlococcus cereus aureus; 175. Staphlococcus pyogenes albus; 176. Staphlococcus pyogenes aureus; 177. Bacillus ochraceus subflavus; 180. Bacillus subtilis; 181. Bacterium Stutzeri; 182. Bacillus synxanthus; 183. Bacterium tremel- lioides; 186. Bacillus typhosus; 187. Bacillus paratyphosus; 188. Sarcina ventriculi; 189. Bacillus violaceus; 190. Azoto- bacter vinelandii; 191. Micrococcus viticulosus; 192. Bacillus proteus viridis; 193. Bacillus aquatile villos; 195. Spirillum volutans; 196. Bacillus proteus vulgaris; 197. Bacterium xanthochlorum; 198. Bacillus xylinum; 199. Bacillus proteus Zenkeri; 201. Boden I. (Tsiklinsky Siidpolarexpedition 1903—5). THE FOLLOWING ORGANISMS DID NOT REDUCE NITRATE TO NITRITE 4. Micrococcus agilis; 9. Bacillus pseudo-anthracis; 18. Bacterium aurantiacus; 19. Sarcina aurantiaca; 24. Bacillus butyricus; 25. Bacillus candicans; 26. Micrococcus candicans; 28. Bacillus campestris; 29. Rhodobacillus capsulatus; 32. Micrococcus cereus; 33. Micrococcus carneus; 36. Micrococ- cus citreus; 38. Micrococcus concentricus; 45. Bacillus cyaneus; 46. Bacterium crysogloia; 48. Bacillus dendroides; 52 51. Bacillus esterigenes; 53. Bacillus esterigenes A; 54. Bacil- lus esterigenes D; 68. Bacillus fluorescens tenuis; 70. Bacillus fuchsinus; 71. Sarcina gasformans; 72. Bacillus graviolens; 80. Bacillus hyponitrous; 87. Bacillus juglandis; 88. Bacillus kiliensis; 92. Bacillus levans; 96. Sarcina liquefaciens; 97. Bacillus liquefaciens; 102. Micrococcus luteus; 103. Sarcina lutea; 104. Streptococcus luteus liquefaciens; 105. Bacillus maidis; 111. Bacillus megatherium; 113. Bacillus proteus mirabilis; 116. Bacterium muris; 119. Bacillus ochraceus; 122. Oidium lactis; 124. Cladothrix odorifera; 125. Cladothrix dichotoma; 130. Bacillus Petasites; 135. Bacterium phyto- phtorum; 137. Bacillus pumilis; 139. Bacillus fluorescens putidus; 142. Bacterium radiatum; 144. Bacillus ramosus non liquefaciens; 145. Bacillus rosaceus; 155. Bacillus rubidus; 156. Spirillum rubrum; 159. Spirillum Rugula; 160. Bac- terium rubilum; 161. Bacillus ruminatus; 164. Spirillum serpens; 169. Bacterium luteus sporogenes; 172. Bacillus sphaericus; 174. Staphlococcus pyogenes citreus; 178. Bac- terium subflavum; 179. Micrococcus sulfur; 184. Bacillus tumefaciens; 185. Bacillus tumescens; 194. Bacillus vivax; 200. Bacillus Zopfii. Of the 201 organisms under consideration 139, or 69.1%, reduced nitrate to nitrite, and 62, or 30.9%, did not effect this reduction. Those organisms which produce green pig- ment almost invariably reduce nitrate to free nitrogen. This reduction takes place very rapidly, after forty-eight hours no nitrite remains in solutions of small concentration. It is impossible to declare from the vigor of the growth of the organism respecting its ability to effect the reduction of nitrate to nitrite. However, those bacteria which failed to perform such reduction were commonly found among those whose growth was slow and at best feeble. Many organisms were inoculated into Giltay's medium in the hope that it would prove available for reduction experi- ments. This proved to be the case with soil inoculation, but in pure culture the slow growth of all and the refusal of many bacteria to develop in this synthetic medium, did not prove encouraging. Calcium glycerophosphate and calcium lac- tophosphate were also tried in this connection. These com- pounds proved to be unstable in solution and were difficult \ 53 to sterilize intact. Nothing was found superior to peptone although this medium in the absence of mineral salts and car- bonaceous material is far from the optimum requirement of most bacteria. DENITRIFICATION It must be evident that of all the organisms which have been studied, comparatively few reduce nitrite to free nitrogen in pure culture. Of the seventy soils under consideration, all of those which would be considered as suitable for crop pro- duction reduced nitrite to free nitrogen in a very short period of time. It must therefore be concluded that either the very few species of bacteria which effect such reduction are univer- sally distributed, or that those organisms which will not per- form this function in pure culture will work in symbiosis to effect this end. The latter phenomenon has been established with reference to a few organisms and will doubtless be extended to include a great variety. The operation of this function is not confined to nitrogenous media but is performed with equal vigor in Ashby's medium in which either glucose or man- nite are employed. The reduction seemingly taking place a little slower in the case of glucose. In simple soil infusion of considerable concentration I have been unable to detect the slightest evidence of reduction of nitrates or nitrites on addi- tion of these salts. That these reductions are effected wholly according to the nascent hydrogen theory seems improbable. By soil inoculation of the media I have been unable to reduce sulphates to hydrogen sulphide except in a very few instances, while with sewage sludge no difficulty is experienced. The phosphates also seem refractory. A selective action involving energy, nutrition, etc., may be concerned. But eight species of bacteria are commonly cited as re- ducing nitrites to free nitrogen in pure culture: Bacterium centropunctatum, (H. Jensen), Bacterium filifaciens (H. Jensen), Bacterium Hartlebi (H. Jensen), Bacterium nitro- vorum (H. Jensen), Pseudomonas pyocyanea (Migula), Bacil- lus denitrificans (H. Jensen) and probably two members of the Fluorescens group: Bacillus fluorescens and Bacillus fluorescens liquefaciens. Maassen observed that Bacillus praepollens broke down nitrates to free nitrogen only in sym- 54 biosis with other bacteria, and that nitrites were not reduced to free nitrogen except in harmony with organisms which re- duced nitrates to nitrites. He discovered that the following organisms, in cooperation with Bacillus praepollens, would effect this reduction: "B. acidi lactici, B. capsulatus, B. cre- moides, B. cuniculicida mobilis, B. diphtheria columbarum, B. enteritidis Gartneri, B. from lean meat, B. indigonaceus, B. mycoides, B. mesentericus ruber, B. mesentericus Flugge I, III and VII, B. mustelae septicus, B. miniaceus, B. pro- digiosus, B. pneumoniae, B. proteus mirabilis, B. proteus vulgaris, B. psitticosis, B. rhinoscleromatis, B. ruber of Kiel, B. ruber plymouth, B. ruber purpureus, B. suipestifer, B. Hog-cholera (Salmon), B. swine plague, B. typhi-abdominalis, B. typhi-murium, B. violaceus, B. coli commune I, II, III, IV, B. lactis aerogenes, B. phosphorescens, M. candicans, Staph- lococcus pyogenes albus, Staphlococcus pyogenes aureus, Sarcina flava II, Vibrio Blankenese, Vibrio Mottlau II, Vibrio tyrogenes Deneke." An attempt was made to isolate the organisms from the soil, which perform the function of reducing nitrates to free nitrogen. As far as possible all the organisms were isolated from soil number 9. None of these bacteria reduced nitrates to free nitrogen in pure culture. Of the fifteen different species thus isolated various combinations were made in the hope of discovering a symbiotic relation, but of the many combina- tions thus effected in no instance did I succeed in securing the desired result. After fishing the different colonies from a great number of plates, the agar in several was carefully rolled together with a sterile spatula and introduced into a medium prepared for the reduction of nitrates, those plates which con- tained the greater number of colonies invariably reduced the nitrate to free nitrogen, but in some of the plates on which were few colonies no reduction took place. I am under obligations to Dr. A. F. MacLeod, Assistant Professor of Physical Chemistry in Beloit College, Beloit, Wisconsin, for valuable suggestions and assistance; and like- wise to Dr. H. H. Waite, Professor of Bacteriology and Patho- logy in the University of Nebraska, for valuable suggestions and assistance. 28801*; MJ UNIVERSITY OF CALIFORNIA LIBRARY