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Sy | es ; \" ‘Te i Pag ; | ” 1 . 1 : 5) | / | Sa \ ' i) ian) ‘i pris i ALBERT JAN KLUYVER HIS LIFE AND WORK RACE ak ERR ak SEE As i eet ere oe pa ALBERT JAN KLUYVER HIS LIFE AND WORK BIOGRAPHICAL MEMORANDA SELECTED PAPERS BIBLIOGRAPHY AND ADDENDA ie es] NORTH-HOLLAND PUBLISHING COMPANY, AMSTERDAM INTERSCIENCE PUBLISHERS INCG., NEW YORK No part of this book may be reproduced in any form by print, photoprint, microfilm or any other means without written permission from the publisher Sole Distributors for U.S.A.: Interscience Publishers Inc., New York Printed in the Netherlands by W. D. Meinema Ltd. — Delft EDITORS: A. F. KAMP, J. W. M. LA RIVIERE AND W. VERHOEVEN PREAMBLE Tuts book has been conceived in consultation with the children of the late Professor Kluyver. Colleagues, pupils, collaborators, and other associates from well-nigh every period of his life have un- stintingly responded to requests for contributions to the biographical section. The result aims at bearing witness to a sense of solidarity and reverence which is also manifested, as replica in effigy, by the bust sculptured by Professor Wenckebach through the initiative of Kluy- vers pupils. This bust was installed in the new Laboratory of Microbiology when the latter was being occupied; at that time, too, the plaque of Beijerinck was moved thence from the now abandoned old laboratory at the Nieuwelaan where the two scientists had con- ducted their illustrious studies for so many decennia. A strong impetus to the publication of the ‘Selected Papers’ was the desire to render comprehensible to future generations the influ- ence that Kluyver’s intellectual and human traits have exerted on science and on his associates in the widest sense of the word. Referring to the Apology for more specific details, a brief account of the composition of this book is here presented. Initially the possibility was considered of reprinting all of Kluyver’s publications. Closer scrutiny made it necessary to abandon such a plan. In part this was motivated by the fact that many of the con- cepts enunciated by Kluyver have long since been firmly incorporated into microbiological science. Others, representing notable stages in the development of this discipline at the time of their publication, have lost some of their significance in the course of years on account of later improvements in methodology and understanding. The scales were turned by the decision to let Kluyver’s work and personality speak to as large an audience as possible; and this inevitably implied a selection. Had this been confined to a choice from among the strictly original scientific papers, the resulting collection would have given too one- sided a picture of Kluyver’s range of activities. Consequently a num- VII PREAMBLE ber of review articles and the text of some lectures have been included; for particularly in these lectures he displayed that harmonious mastery over form and content, that highly developed linguistic and vividly stylistic ability that were so characteristically his own. The essence of this style reveals itself not only when he wrote in his mother tongue, but it is equally apparent from his publications in English, French, and German. At least one paper in each of these four languages has been incorporated here, while several papers, hitherto available only in the Dutch language, have been inserted in English translations. In this manner the collection may offer some novelties to interested peo- ple in foreign countries. It stands to reason that ample space has been reserved for an essay on Kluyver as a scientist, in which an attempt has been made to assess the totality of his contributions. The biographical section was written by several authors, each one covering a particular period of his life. For its composition contribu- tions were received from a former secondary school classmate who has written about this phase; from a colleague whom Kluyver befriended when they both were first-year students at the University, and who has dealt with the years prior to Kluyver’s assumption of the pro- fessorate; and from all those who were consecutively associated with him as senior staff members during the years of his professorship, and have covered this period. The biographical section closes with two orations spoken at the cremation ceremonies at Westerveld, viz., the one delivered by the Rector Magnificus of the Technological University in the name of organizations and persons within the confines of the University, and the testimonial of Kluyver’s closest friend, especially rewritten for publication in this book. The bibliography, finally, lists all of Kluyver’s publications. In addi- tion it contains a record of the Doctor’s dissertations and papers issued from his laboratory that do not bear his name. Apparently the Director did not have a rigid rule for determining whether or not his name should appear on a publication. Even so, the work done by his pupils was often concerned with the elaboration and testing of ideas that he supplied; and in spite of a consummate respect for the individuality of his coworkers he was wont to instill into every paper from his institute a characteristic and highly personal flavour. VIII PREAMBLE Although the entire book is a memorial to Kluyver, it seemed war- ranted to include a single ‘In Memoriam’. The choice fell on the one that appeared in the ‘Nieuwe Rotterdamse Courant’ ef May 15, 1956, written under the immediate influence of the realization of the loss that had been suffered. The present volume contains moreover a list of the numerous grateful appreciations written by Dutch and foreign scientists. Thus goes out into the world, and for the last time, a creation of A. J. Kluyver that represents an impressive part of his efforts; yet for the first time it has not been supervised by his own meticulous care. The compilers have ever been conscious of this regretted circumstance, and they have recognised how greatly it has added to their responsi- bility. May the reader, even of a future generation, glean from this volume some of the benefits that the lustre of Kluyver’s mind and personality so generously bestowed upon all who knew him. IX CONTENTS PREAMBLE vu CONTENTS x ILLUSTRATIONS opposite page 48 PART ONE BIOGRAPHICAL MEMORANDA FROM YOUTH TO PROFESSOR 3 1888-1905 The early years 3 1905-1916 Student and assistant 5 1916-1921 The tropical period 9 KLUYVER AS PROFESSOR; CHRONICLES OF THE LABORATORY 14 1922 Preparation 14 1923-1927 Inspiration 18 1928-1938 Consolidation 25 1939-1945 War and occupat.on 34 1945-1956 Recovery; the final years 38 KLUYVER AS SEEN BY HIS PUPILS 49 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY 68 Introduction 68 The prologue 79 The emergence of a programme 80 The unfolding of the programme 84 XI CONTENTS Further developments: phosphorylation and the nature of assimilation The nature of biocatalysis Comparative biochemistry Industrial microbiology and the submerged culture method for the study of mould metabolism The classification of micro-organisms Variability of micro-organisms Epilogue TWO FUNERAL ORATIONS O. Bottema A. van Rossem PART TWO SELECTED PAPERS MICROBIOLOGY AND INDUSTRY Inaugural address delivered in the Dutch language on the occasion of his accession to the Chair of Microbiology at the ‘Technological Uni- versity of Delft on January 18, 1922, under the title ‘Microbiologie en Industrie’. Translation of: ‘Microbiologie en Industrie’. Rede uitgesproken bij de aanvaarding van het ambt van Hoogleeraar in de Algemeene en Toegepaste Microbiologie aan de Technische Hoogeschool te Delft op Woensdag 18 Januari 1922. Delft, 1922. UNITY AND DIVERSITY IN THE METABOLISM OF MICRO-ORGANISMS Address delivered in the Dutch language at the general meeting of the Nederlandsche Chemische Vereeniging on April 24, 1924, under the title: ‘Eenheid en verscheidenheid in de stofwisseling der microben’. Translation of: ‘Eenheid en verscheidenheid in de stofwisseling der microben’. Chemisch Weekblad 21, 266, 1924. DIE EINHEIT IN DER BIOCHEMIE (Mit H. F. L. Donker) 1. Einleitung — 2. Die Zuriickfiihrung der Dissimilationsprozesse auf katalytische Wasserstoffiibertragung, falls Zucker als Substrate XII gI 98 114 130 138 146 152 157 157 159 186 201 CONTENTS fungieren — 3. Der Chemismus der katalytischen Wasserstoffiiber- tragung — 4. Anwendung der gegebenen Vorstellung des Chemismus der katalytischen Wasserstofftibertragung auf die Zuckerdissimila- tionsprozesse — 5. Anwendung der Wasserstofftibertragungstheorie auf sonstige Dissimilationsprozesse — 6. Die Assimilationsvorgange im Lichte der Wasserstoffiibertragungstheorie — 7. Uberblick iiber die aufgestellte Theorie der katalytischen Wasserstofftibertragung als Kern des biochemischen Geschehens — 8. Schlussbetrachtung. Chemie der Zelle und Gewebe 13, 134, 1926. This article is in various respects an extension of the views of the authors reached early in 1925, the essence of which has been published in the following preliminary communications in English: Proceedings Royal Academy of Sciences, Amsterdam 28, 297 and 605, 1925. QUELQUES REMARQUES SUR LA CLASSIFICATION DES LEVURES 268 Conférence faite a l’Assemblée générale de Union Internationale des Sciences biologiques a Bruxelles, le 13 Juillet 1931. Annales de Z ymologie Série IT, 50, 48, 1931. PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA (With C. B. van Niel) 282 1. Motives for a renewed discussion of the subject — 2. Principles in bacterial classification — 3. Critical examination of recent contri- butions to the classification of bacteria — 4. Outline of a rational sys- tem for bacterial classification on the basis of our present knowledge — Appendix: List of genera included in the tables and their diagnosis. Kentralblatt fiir Bakteriologie, Parasitenkunde und Infektionskrankheiten. Abt. II, 94, 369, 1936. LIFE’S FRINGES 329 Address delivered in the Dutch language at the XXVIth Nederlandsch Natuur- en Geneeskundig Congres on April 1, 1937 under the title “s Levens nevels’; The Dutch title contains a retrograde. Translation of: *’s Levens nevels’. Handelingen XXVJe Nederlandsch Natuur- en Geneeskundig Congres, 82, 1937. DIE KOHLENSAURE IM STOFFWECHSEL DER LEBEWESEN 349 Vortrag gehalten im Festsaal der Finnischen Technischen Hochschule in Helsinki den 14 Marz 1939. Suomen Kemistilehti 12, 81, 1939. XIII CONTENTS THREE DECADES PROGRESS IN MICROBIOLOGY 369 Lecture held on May 8, 1946, for ‘Danmarks Naturvidenskabelige Samfund’ and the ‘Biologisk Selskab’ after the ‘Emil Chr. Hansen Medal’ had been conferred on him. Antonie van Leeuwenhoek 13, 1, 1947. Naturens Verden 30, 161, 1946. HOMO MILITANS 3093 Address delivered in the Dutch language at the general meeting of the Hollandse Maatschappij der Wetenschappen on May 22, 1948, under the title ‘Homo militans’. Translation of: ‘Homo militans’. Haarlemse Voordrachten VIII. Haarlem 1949. HOMO MILITANS 416 Voordracht gehouden in de Algemene Vergadering van de Hollandse Maatschappij der Wetenschappen op 22 Mei 1948. Haarlemse Voordrachten VIII. Haarlem 1949. MICROBIAL METABOLISM AND ITS INDUSTRIAL IMPLICATIONS 424 Read before the inaugural meeting of the Microbiology Group of the Society of Chemical Industry in the Royal Institution, on March 7, 1951. Chemistry and Industry p. 136, 1952. AN ASPECT OF THE PROMOTION OF SCIENCE 473 Address delivered at the goth annual meeting of the National Academy of Sciences, in Washington, D.C., April 28, 1953. Kluyver represented the Royal Netherlands Academy of Sciences on this occasion and deliv- ered this address as guest of honour at the annual dinner. News Report, National Academy of Sciences-National Research Council, U.S.A., 3> 33> 1953: SOME ASPECTS OF NITRATE REDUCTION 483 Introduction — General considerations on true dissimilatory nitrate reduction — Fate of the hydrogen acceptor — Fate of the hydrogen donator — Molecular hydrogen as hydrogen donator — The influence of free oxygen on nitrate reduction. Lecture delivered at the 6th Congress for Microbiology held at Rome in September 1953, as part of the Symposium on Microbial Metabolism. Symposium Metabolismo Microbico, Istituto Superiore di Sanita. Roma, 1953. Da gils CONTENTS MICROBE AND LIFE Address delivered in the Dutch language at the meeting of ‘De ver- enigde afdelingen der Koninklijkke Nederlandse Akademie van Weten- schappen’ on April 4, 1955, under the title ‘Microbe en Leven’. Translation of: ‘Microbe en Leven’. Jaarboek der Koninklijke Nederlandse Akademie van Wetenschappen, Amsterdam, 1954-1955, P. 225. PART THREE BIBLIOGRAPHY AND ADDENDA PAPERS BY: A. J. KLUYVER DOCTOR’S THESES PREPARED UNDER KLUYVER’S DIRECTION PAPERS FROM THE LABORATORY FOR MICROBIOLOGY AT DELFT, PUBLISHED BY KLUYVER’S COLLABORATORS 1922-1956 PAPERS OF THE BIOPHYSICAL RESEARCH GROUP UTRECHT— DELFT STAFF MEMBERS OF THE LABORATORY FOR MICROBIOLOGY 1922-1956 HONOURS OF A. J. KLUYVER ARTICLES DEDICATED TO A, J. KLUYVER AND HIS WORK OBITUARY ARTICLES ‘IN MEMORIAM A. J. KLUYVER’ Published in the ‘Nieuwe Rotterdamse Courant’ of May 15, 1956. APOLOGY XV 593 349 aes 909 560 565 PARA ONE BIOGRAPHICAL MEMORANDA FROM YOUTH TO PROFESSOR THE EARLY YEARS 1888-1905 ALBERT JAN was the second child and only son sprung from the mar- riage of Jan Cornelis Kluyver and Marie Honigh. His father, born May 2, 1860, in Koog aan de Zaan, came from a family of merchants who for many generations had been living in the Zaan district, a region in the vicinity of Amsterdam. The family name is associated with ships; a ‘kluiver’ is a jib. Jan Cornelis and his somewhat elder brother, Albert, both were outstanding students at the but recently established secondary school in Zaandam, and the first members of the family to continue their formal education after graduating from this school. Albert chose the study of Letters at Leiden University, became editor of the Netherlands’ Dictionary, and afterwards professor at the University of Groningen. Jan Cornelis attended the Polytechnical Col- lege in Delft where he received the engineering degree, and at the age of 32 became professor of mathematics in Leiden. Thus the studies of these two brothers have notably enriched Dutch scholarship. The first position occupied by Jan Cornelis was that of instructor in mathematics at the Governmental High School in Breda (1883). Dur- ing his first year there he married Marie Honigh, likewise from the Zaan district, and who at the time was teaching in Haarlem (born September 27, 1859, in Zaandijk). In Breda a daughter was born to them, and a few years later, on Sunday, June 3, 1888, the son, Albert Jan. In 1890 the family moved to Amsterdam, where Kluyver had been appointed to the faculty of the Municipal High School at the Weteringschans. Thence he was called to the professorate at Leiden University, in 1892. Both in Amsterdam and in Leiden the family was augmented by the birth of a daughter. In 1896 the Uni- versity of Groningen conferred upon Jan Cornelis Kluyver an honor- ary doctorate. In Leiden the family first occupied a none too cheerful house in the Rembrandtstraat, though it commanded from the rear a magnificent BIOGRAPHICAL MEMORANDA view of the Witte Singel, a local canal. Later they moved to an archi- tecturally interesting, old-fashioned house on the Heerengracht. Finally, to the delight of father Kluyver, the family acquired a very modern, sunny, and colourful mansion at the Hoge Rindijk; it was from here that the children gradually dispersed. Kluyver died in Leiden, at the age of 72, on December 3, 1932, four years to a day after his wife. He was an exceptionally thorough mathematician, imbued with the contributions of the great mathe- maticians of the past, from whose works he often borrowed illustrative examples. When lecturing he was wont to cover blackboard after blackboard with beautifully regular letters and signs, without ever wavering. He was a stately, imposing figure, with a wittily ironic gleam in his grey eyes that caused many students rather to fear him. He was a splendid speaker and the address on ‘The Steady Evolution of Mathematics’ which, as Rector Magnificus, he delivered on Febru- ary 8, 1910, in the main auditorium of Leiden University, remains in my opinion the most beautiful paper on mathematics for laymen. It was after the Easter holidays in 1904 that I first set eyes on Albert Jan. He occupied a front row seat in the fourth grade classroom of the Leiden High School; a slender, blond boy, but recently graduated to ‘long pants’. Invariably he knew everything, and never failed a quiz; but he was in no way a braggart, rather did he appear timid and bashful. Not till after the summer holidays, when we had frankly expressed our delight at meeting again, did we begin regularly to study together for the final examinations. Mostly we worked in his room which we had virtually converted into a laboratory for practicing chemistry. Nearly every evening was concluded by a brief visit to the living room. Like all living-rooms in those days, this was a simply furnished apartment, with a rectangular table in the centre over which hung the lamp; there were no low little tables or cosy nooks. During the winter Albert Jan did his homework at that table in the family circle; his powers of concentration made him immune to distraction. On rare occasions, when upstairs we got stuck in a mathematical problem, we mustered up enough courage to ask the professor how it should be tackled. Usually he was engaged in reading a book or newspaper, and, almost without looking up or listening to us, he would then sketch 4 FROM YOUTH TO PROFESSOR the main elements of the solution in the margin of the paper, while making no bones about showing that he thought us far from bright. The presence of Mrs. Kluyver created in that room an atmosphere of order and kindliness that has remained with me to this day, half a century later. The final examinations became a great success. In those days they were not handled in and by the school, but administered by a large board which, in 1905, met in the city of Gouda. At the time this place could be reached from Leiden only by a combined railway and horse- drawn tramcar voyage. The trip to, and the sojourn in Gouda were a very special event for the three of us, the third one being C. Punt, who later became known as Kluyver’s partner in the famous tennis double. Outwardly Albert Jan was the image of his father, distinguished- looking, though a little less formal; and from his father he probably inherited also his uncommonly sharp intellect. But the charm that he radiated and that affected every one he met; his capacity for ingen- uously paying close attention to every human being; these he owed to his mother. And that was not the smaller part of the gift of grace that had been granted him in the form of his excellent parents. W. E.v. W. STUDENT AND ASSISTANT 1905-1916 In September, 1905, Kluyver registered as a first-year chemistry stu- dent at the Technological University in Delft. Our first meeting oc- curred during the hazing days, in the apartment of P. J. van Voorst Vader, an older student, living at Oude Delft 128a. J. P. Valkema Blouw, another older student, was at the piano singing Speenhoff dit- ties which were much in vogue at the time. A few freshmen, among them Kluyver and I, had been herded under the table; here, in some- what tenebrous circumstances and crouching position, began our acquaintance that was to lead to a life-long friendship. From 1905 till 1907 Kluyver resided in Delft; I noticed compar- atively little of him. After his sophomore year he moved to Leiden where for some years he roomed in the ‘Pancras House’, Hooglandse Kerkgracht 21, together with three students at Leiden University who studied chemistry, medicine, and theology, respectively. During those years Kluyver, though a member of the Delft student BIOGRAPHICAL MEMORANDA fraternity, did not care much for that city. To be sure, he had decided to become a chemical engineer; but his heart was set on Leiden from where he commuted to Delft. Here he participated but little in the activities of the student community; and, after the day’s work was done, he always returned to Leiden where, apart from rooming with his student friends, he also took part in community affairs. Having grown up in that city he knew many local families, and was a member of the lawn-tennis club ‘Ready’ and of other social clubs. In 1909 he passed, ‘with distinction’, the examinations that admitted him to candidacy for the degree of Chem. E. During his final year he studied mostly under Dr. J. Boeseken, Professor of Organic Chem- istry, and in June, 1910, he received the Chem. E. degree, again ‘with distinction’. Meanwhile he had taken several courses under Dr. G. van Iterson Jr., Professor of Microscopical Anatomy, who had offered him, even before the final examinations, a position as assistant in his institute.* It was during this period that a number of chemistry students, Kluyver among them, founded the ‘Natural Sciences Dispute’ which later became the nucleus of the Delft Section of the Netherlands’ Chemical Society. At its monthly meetings one of the members dis- cussed a subject in the natural sciences. Kluyver chose as his topic the chemical structure of chlorophyll. This lecture was published in 1911 in the ‘Pharmaceutisch Weekblad’ under the title, “Che chemistry of chlorophyll in the light of recent investigations’. It was Kluyver’s first publication, and he was not a little proud of it. But at the same time he realized even then the relativity of all things. The reprint he pre- sented to me had been inscribed in his fine, sharp script with the following quotation from Otto Ernst’s “Semper der Jiingling’, a widely read novel at that time: ‘Zwar blieb seine Wissenschaft einigermassen an der Oberflache; er sprach allerlei vom Chlorophyll, aber was es fiir eine Bedeutung hatte wusste er eigentlich selbst nicht’. During his student years Kluyver showed himself to be possessed of great mental ability, strong interests, and an extreme sense of duty. But it must not be concluded that he did nothing but study; in Leiden he cultivated that social intercourse with friends of both sexes which he needed so much. Here, especially during vacations, he indulged in * At the Technological University this position carried various major responsibil- ities, and was generally occupied only by persons with an advanced degree. 6 FROM YOUTH TO PROFESSOR sports; hockey in winter, and tennis during the remainder of the year. In tennis he excelled, and in those days he was one of the strongest players in Holland. The crowning glory came when, around 1910, he was a member of the Dutch team that was to play against a Belgian one, and won both singles matches. Soon thereafter he put his racket away, however, so that he could devote himself exclusively to his studies! From 1910-1916 Kluyver was assistant at the Laboratory for Micro- scopical Anatomy, later renamed Laboratory for Technical Botany. It was a period during which not many industrial positions were open. Moreover, Kluyver wanted to spend at least some years in pure re- search; and in Van Iterson’s institute he had a splendid opportunity to acquaint himself with various aspects of botany and eventually to master the field. The first two years offered scant opportunity for in- vestigations of his own. He assisted with everything pertaining to the teaching functions of the laboratory, such as the preparation of lecture demonstrations and of the laboratory courses. This involved a great deal of work, particularly because the institute’s staff could boast only one technician and a single gardener. Furthermore, he assisted in the laboratory courses, and partly supervised the work of the students specializing in the field. And when, in the spring of 1911, Van Iterson travelled to the Dutch East Indies as a delegate of the Netherlands’ Government to the International Fibre Congress in Surabaja, Kluy- ver was left in complete charge of the laboratory for several months. During the summer vacation of 1911 he spent some months working under Prof. Dr. Hans Molisch at the Plant-Physiological Institute of the University of Vienna. This led to a paper entitled ‘Observations on the effect of ultraviolet radiation on higher plants’ [1gr1]. He rel- ished the beauties of Vienna, and returned home via the Dolomites. It was logical to expect that Kluyver would attempt to prepare a Doctor’s dissertation. In the spring of 1g12 he began an investigation of biochemical sugar determinations, with the aim of elaborating this subject and, if possible, using the results for a thesis. For more than two years Kluyver threw himself into the problem with all his energy and endurance. Meanwhile he had moved back to Delft so that he could spend all his time on this study. This was particularly necessary because he regularly had to work evenings in the laboratory, and BIOGRAPHICAL MEMORANDA often the observations had to be extended till deep into the night. The preliminary results of his investigations were reported at the 14th ‘Netherlands’ Congress of Natural Sciences and Medicine’, in Delft, L913: On May 15, 1914, Albert Jan Kluyver received the degree of Doctor of Technical Science ‘with distinction’ on the basis of a sterling disser- tation, ‘Biochemical Sugar Determinations’, accompanied by no less than 27 propositions. It was just in time; on August 1 of that year the Dutch army was mobilized following the beginning of the first world war. Kluyver, too, was inducted into the army as buck private in the 4th Infantry Reg- iment in Leiden. He was fortunate in that his regiment was stationed there during the winter of 1914, so that he could spend his evenings with family and friends. His military career was not spectacular; he did not get promoted beyond the rank of corporal. But this was a blessing because it meant that, owing to Van Iterson’s urgent remon- strances that Kluyver could not be spared at the institute, he could get a discharge in May, 1915. Thus Kluyver returned to Delft and his assistantship. He was now 27 years old, and it became time to think of a future career. Van Iterson realized even then that this young man with his brilliant intellect would be capable of accomplishing great things. He considered it important that before settling down to a position in Holland, Kluyver should acquaint himself with life in the Dutch East Indies, or, more generally, in the tropics. The opportunity offered it- self rather unexpectedly; at the Department of Agriculture, Industry, and Commerce in Buitenzorg a post had been created for a consultant whose duty it would be to promote the native industries. Dr. H. J. Lovink, Director of Agriculture in the above-mentioned Department, consulted Van Iterson, and subsequently interviewed Kluyver for this position. The latter accepted, partly on Van Iterson’s advice. Meanwhile another and most fortunate change had taken place in Kluyver’s life. He had become engaged to Ejya van Lutsenburg Maas, candidate for the Chem. E. degree, who was a student at the Labor- atory for Technical Botany. This event undoubtedly influenced Kluyver’s decision to accept the consultantship in Buitenzorg; now he knew that he need not feel lonely there. On July 29, 1916, Albert Jan Kluyver and Helena Johanna van 8 FROM YOUTH TO PROFESSOR Lutsenburg Maas were married in The Hague. Following a brief honeymoon in Holland — it was war time — they embarked early in August for a much longer voyage. During those war years the trip East was not so simple and took a very long time. England forced the Dutch steamers to sail around Scotland, and to dock at both Kirkwall and Falmouth for a careful scrutiny of passengers and cargo. Subse- quently the ships sailed, via the Canary Islands and Capetown, around Africa. This provided an opportunity to visit his brother-in-law, Pijper, who was then a physician in South Africa, and later became professor of Bacteriology. The trip took more than two months, and not until October ro did the Kluyvers land in ‘Tandjong Priok. After a brief sojourn in Batavia they continued on to Buitenzorg, the resi- dence of the new industrial consultant. THE TROPICAL PERIOD 1916-1921 Kluyver spent three wonderful years (1916-1919) in Buitenzorg. His numerous letters written during that time give a clear picture of this period that was of such importance to him. He learned much from his contacts with various functionaries, both in his department and in other institutions; he became familiar with the bureaucratic and political atmosphere in Buitenzorg which, to be sure, disappointed him in many respects, but nevertheless increased his worldly wisdom. He was glad not to have missed this experience. He was disappointed with his field of activities, because he found neither a concrete plan, nor guidance as to definite lines of action. There was uncertainty and conflict. Initially, the Netherlands’ Govern- ment had intended that the consultant-to-be should devote himself to the study, promotion, and development of native industries. This was opposed by those who wanted to involve the consultant in the promo- tion of European enterprises. Throughout the years of Kluyver’s civil service (1916-1919) it thus remained in doubt for whom he should work, and the young consultant received too little support from his superiors in his difficulties. Furthermore, such plans as the creation of an independent department of industry, with an adequate laboratory in Bandung, did not materialize; the responsible officials had neg- lected to place the necessary funds on the 191g budget. 2) BIOGRAPHICAL MEMORANDA Was it surprising that, with the approaching termination of his first term of office, Kluyver was doubtful of his future? An attractive pros- pect in the Indies was the above-mentioned separate department in Bandung, perhaps under his own direction. But it appeared that he had not been forgotten in Holland. The Colonial Institute in Amster- dam offered him the directorship of a department for scientific research that was soon to be created and would be charged with the study of problems in colonial technology. In conjunction with this position, Van Iterson wanted to attach him also to the Technological University in Delft as extra-ordinary professor of phytochemistry. And when Van Iterson cabled him that the Government had agreed to the creation of such a chair, and that the Delft faculty concurred with Van Iter- son’s nomination of its future occupant, Kluyver accepted the position at the Colonial Institute. His first task was to make a survey of the coconut-fibre and copra- yarn industry in Ceylon, and on the Malabar Coast, with a view to determining whether such industries could be developed in Java. However, just when Kluyver’s European future seemed assured, but before starting his work with the Colonial Institute, an entirely differ- ent possibility opened up. This was a position with the Oil Manufac- turing Company ‘Insulinde’. Up to that time vegetable oils had been produced by purely empirical methods. The new director, M. H. Damme, Mech. E., recognized nevertheless that, were the industry to remain flourishing, scientific research would be imperative. Repeated- ly he urged Kluyver to join this concern as chemical adviser, in which position he would become director of a new, well-equipped laboratory in Bandung, and under exceptionally favourable financial conditions. The offer was so enticing that Kluyver finally accepted, after the Colo- nial Institute had been found willing to cancel the previous arrange- ment, though with the stipulation that the survey of the copra-fibre industry would still be undertaken. During the Buitenzorg episode the Kluyvers had made friends with several families, such as the Ruttens; Dr. L. Rutten was a geologist in the employ of the B.P.M., and later became professor in Utrecht. Others were Dr. Otto de Vries, Director of the Central Rubber Re- search Station, and subsequently extra-ordinary professor at the Med- ical University in Batavia; and H. J. Hellendoorn, Chem. E., also associated with the Rubber Research Station. IO FROM YOUTH TO PROFESSOR Kluyver terminated his work at the Department of Agriculture, Industry, and Commerce, and moved from his residence in Buitenzorg, Tjikeumeuh 32, where the couple had spent three happy years. Mrs. Kluyver traveled with their year-and-a-half old daughter to Holland, there to await the birth of her second child. For the study of the copra-fibre industry Kluyver, accompanied by Raden Mas Iso Reksohadiprodjo, Agric. E., trained in Wageningen, and an instructor in agriculture in Java, embarked early in December, 1919, on the 8.8. ‘Nawab’. They first went to Ceylon, thence to the Malabar Coast, via Madras to Calicut, and later to Cochin (Travan- core). Via Medan they returned to Java for consultation with the appropriate authorities and a discussion of the prospects for a copra industry. In May, 1920, Kluyver went to Holland in order to deliver a preliminary oral report at the Colonial Institute. The printed report of this enterprise appeared in 1923 as Communication No. XX of the Colonial Institute under the title, ‘Copra-fibre and Copra-yarn In- dustry’, a book of 300 pages, illustrated with beautiful photographs taken by Kluyver himself, and supplemented with numerous tables, maps, and appendices. It must be considered as the standard work on this difficult subject. Not till November, 1920, did Kluyver return to Java with his wife and children — in Holland a son had been added. He installed himself in Bandung, where the family enjoyed the friendship of Dr. J. Clay, pro- fessor at the local ‘Technological University, and his wife. His first task was to get the scientific laboratory of ‘Insulinde’ under way. But the high-flown expectations were soon dashed, for in 1920-1921 a serious economic crisis also developed in the Netherlands’ East Indies, and ‘Insulinde’ did not escape the consequences. Kluyver now realized that his position would soon come to an end; in June he and many other employees were told that they would be laid off in October, 1921. At this point Kluyver faced two alternatives: either to re-establish contacts with the Department of Agriculture, Industry, and Commerce in Buitenzorg, or to hope for a professorship at the Bandung Tech- nological University where a Division of Chemical Technology was being created; Kluyver had already been interviewed for this position. At this point something utterly unexpected happened: he received a cablegram from the Department of Chemical Technology of the Tech- It BIOGRAPHICAL MEMORANDA nological University at Delft, asking whether he would consent to being placed, as first choice, on the list of candidates for the chair of general and applied microbiology that would become vacant at the end of the academic year 1920-1921 owing to the retirement of M. W. Beyerinck who had reached the 70 years’ age limit. Kluyver was greatly astonished; he had expected that Prof. Dr. N. L. Sohngen, then at the Agricultural University in Wageningen, would be appointed as Beiyerinck’s successor, and never for a moment thought of himself in that position. With the image of his father before him, he soon realized the attraction of devoting the remainder of his life to science and teaching; but at the same time he recognized the great responsibility he would take upon himself. After careful delibera- tion he cabled his acceptance. Kluyver took leave of Java and, with his family, boarded the S.S. ‘Patria’ at T'andjong Priok on October 26, 1921, reaching Rotterdam at the end of November. Thus ended the ‘Indian adventure’, as he used to call it. Those who have read the foregoing account may perhaps be inclined to look upon this period as the story of two more or less unsuccessful ventures; but Kluyver himself was also keenly aware of another aspect. To be sure, there had been disappointments with the activities during these years, but these were counterbalanced by an enormous gain. The ‘adventure’ had taken him to Java and Sumatra, to parts of Ceylon and of India. He had become acquainted with peoples and conditions in the Orient. ‘This had vastly increased his range of vision, his insight into persons and circumstances, and left a body of expe- rience on which he could draw during a lifetime in Delft. In his inaugural address of January 18, 1922, he expressed this in his remarks to the student body in the following striking words: ‘...fate has taken me to far-away countries, and did not Goethe, in his ““Wahlverwandtschaften”’, say: ‘Die Gesinnungen andern sich ge- wiss in einem Lande, wo Elefanten und ‘Tiger zuhause sind’’? It is true that I encountered neither elephants nor tigers in nature; but the emancipating influence exerted by living in a foreign environment has not in the least worn off.’ Meanwhile the family had moved into the official residence at the Nieuwelaan, initially referred to as ‘Beijerinck’s house’. Although the view from the front of the house onto the street was somewhat 12 FROM YOUTH TO PROFESSOR dreary, the view from the rear, on the canal and beyond, amply com- pensated for this. Even more so did the splendid garden extending on both sides of the house; the many exotic trees and plants that Beije- rinck had introduced elicited also Kluyver’s interest and admiration, especially in later years. AsOy div. 13 KLUYVER AS PROFESSOR CHRONICLES OF THE LABORATORY PREPARATION 1922 WHEN, in December, 1921, Kluyver first set foot in his laboratory as professor, it was still permeated with the spirit of his great predecessor. ‘Beyerinck’, said Kluyver in a 1927 speech, ‘was the image of unbridled devotion, nay, utter submission, to research and science which imme- diately spoke to the minds of the students.’ But, apart from respect, that scientist also aroused in his pupils, at least initially, a feeling of awe. When he approached, all discussion stopped. What would be the first thing he would harp on: some minor detail that displeased him about an investigation that was obviously advancing rather slug- gishly; or, more trivial though just as frequently, any faint indication of a generally abhorred sloppiness, or the equally detested smell of lingering cigarette smoke? The attitude was coupled with a somewhat uncouth manner of speech; the tone was often uncomfortably direct. However, his successor would soon realize how long and how strongly that influence could make itself felt, even after the departure of this curious personage. Kluyver found himself facing an immense task. The fact that he had been chosen to occupy this world-renowned chair was generally re- garded as the result ofa willingness to make a credit appointment. He was considered as rather a layman in the field of microbiology. In the in- stitute of Van Iterson, Beijerinck’s most brilliant pupil, he had con- centrated on the study of technical botany, and for his doctor’s degree he had written an eminent thesis on quantitative sugar determinations by means of yeast fermentations. Although we may take it for granted that in the meantime Van Iterson had acquainted him with the most significant properties of the bacteria, his training and experience in microbiology had certainly not been broad. it KLUYVER AS PROFESSOR; CHRONICLES OF THE LABORATORY The personality and reputation of the new professor were known in the laboratory, and had frequently been discussed; the expectations were not very high. Kluyver sensed a mood of scientific snobbery in the assistants he had inherited from Beijerinck. They were still under the influence of the spontaneous, fickle, and — in spite of his having been born of a patrician family — rather unpolished Beijerinck; now they were confronted with the restrained, sensitive, utterly courteous successor. In the beginning these contrasts were keenly felt; they were to be- come even more pronounced. Kluyver, perhaps subconsciously, assumed that it would be one of his first tasks to transform the late-nineteenth century spirit of the institute into a modern one. The incident difficulties were increased because, shortly after his inauguration, a considerable number of students applied for space in his laboratory. During the post-war years Beijerinck had drawn very few disciples; but this had not worried him in the least. Thus the assistants at the time had come to consider the laboratory rather as a typical research institute, where, for all practical purposes, they should not be bothered with the education of students. As will appear from what follows, this laboratory of the Delft University, whose fame gradually extended beyond the city walls, the country’s boundaries, and eventually the oceans, became more and more a world centre of microbiology. No matter how long the duration of the study period had been, or however cursory the visit, it became a ‘must’ for any self-respecting microbiologist to have been there. But first of all the internal organization and working methods had to be remodeled. The copying press made place for the typewriter; one card index file after another was introduced. A house telephone was installed; a ban was declared against ringing, let alone shouting, for the technicians. A solemn quietude came to prevail. All of this commanded but little admiration among the staff, especially because the changes did not stop at arrangements instituted by the predecessor. In this re- spect it was irritating to see that Beijerinck’s holy-of-holies, the library, was desecrated by being in part converted into a classroom. Numer- ous purely botanical journal files unexpectedly wound up at the sec- ond-hand dealer’s. The garden, with all its exotic plants assembled by Beijerinck, received — or so it was believed — insufficient care and at- tention. 5) BIOGRAPHICAL MEMORANDA Gone — although, as later appeared, only temporarily — were the former heuristic discussions at the microscope in which the surprise element, so typical of Beijerinck’s attitude, required a perpetual alert- ness. One missed the instructive walks through the garden, peripatetic quiz sessions during which the hearers were kept on their toes even more because the open air seemed conducive to greater divagations, and thus to further provocative and unsuspected questions. In the lectures one missed Beiyerinck’s rhetoric and brilliance. The entire picture was different. ‘The new professor was seldom seen behind his microscope. Whenever he was not occupied with the students, he could always be found in his study, surrounded by piles of books. This pattern of behaviour can be understood if it is realized that Kluyver did not want to get singed by working on problems he had not yet recognized or, in any case, had not yet mastered. During the evening hours he studied superhumanly. He prepared himself; it is probable that he considered this difficult phase as an incubation period, full of tension, and requiring enormous efforts. As every one discovered, the reorganization progressed. The assist- ants saw their erstwhile unlimited opportunity for research curtailed; there were orders to be sent out; the books from the once so magnifi- cent library had to be sorted and assessed as to actual current value, reorganized according to a new system, spread out over a number of rooms to which the students had free access. The pupils increased in number and became ever more of a burden; they took up a hitherto unknown amount of time and care. Susceptivity to innovations which might prove to be just as good as, perhaps even better than the old order, had long ago been weakened by humdrum; and this prevented the assistants from recognizing how ingenious a man the new professor was. Nor did they see that he con- sidered the time ripe for doing the spadework that would lead to the development of the fundamentals of the ‘unity in biochemistry’, that gigantic edifice that slowly rose up amidst the venerable temples erected by Beiyerinck. Beijerinck had established himself in his country home at Gorssel. Although he had left Delft for good, and so definitively that he re- mained unwilling ever again to set foot in it, even on the occasion when Delft solemnly celebrated his golden doctor’s jubilee, his influ- 16 KLUYVER AS PROFESSOR; CHRONICLES OF THE LABORATORY ence nevertheless remained evident. He ordered his senior assistant, Den Dooren de Jong, to conduct various experiments, so that the latter found himself confronted with an insoluble dilemma. On the one hand he experienced the desire to maintain, also by regular visits to Gorssel, a cherished contact that had grown with the passage of time; on the other hand there were the daily activities in the Delft laboratory, in an atmosphere of sweeping renovation. And a choice had to be made. Disappointed, he left the once so beloved laboratory in the course of 1923, with the firm intention, following a higher exam- ple, never again to cross its threshold. At the leave-taking, Kluyver’s honesty and warm heart made him express regret that his associate had fared so badly during this first year, and declared that he owed him a debt of honour which he hoped to redeem sometime in the fu- ture. Den Dooren de Jong likes to tell of the royal manner in which this so-called obligation was subsequently redeemed; how much he owed Kluyver in connexion with the preparation of his thesis, and later with his investigations on bacteriophages which he conducted in Rotterdam; how the personal relationship developed into a strong tie of friendship; and how, to his great joy, his home could offer Kluyver a temporary but welcome and safe refuge during the second world war. Kluyver, too, visited his predecessor now and then in Gorssel. After such visits he returned not only mentally worn out, but occasionally even disturbed. That he had to digest various quaint, albeit undoubt- edly clever, remarks on the most diverse subjects could be tolerated. But he also became aware of insurmountable barriers, as on the occa- sion when Beierinck, in reply to an inquiry about a member of the staff, flatly pontificated: ‘X is an honest man, but he lies!’ A mind as rational as Kluyver’s could not stomach this. Consequently Kluyver could not be blind to the peculiarities of him whom he was wont to call ‘my great predecessor’; nevertheless he held him in the highest esteem, and never neglected to make this clear to his pupils. The lecture in which he introduced Beijerinck’s elective culture methods under the classical and characteristic title: “The mar- vels of a gram of garden soil’, usually contained a passage in which Kluyver underscored the significance of the elective culture method as follows: ‘Curators of culture collections often receive requests for cultures of 17 BIOGRAPHICAL MEMORANDA bacteria that can undoubtedly be found on the shoes of the petitioners. It is clear that such microbiologists are more in need of a thorough training in their discipline than of a pure culture of the bacterium they ask for.’ In Kluyver’s scientific approach the ‘red thread’ of Beijerinck’s work remained clearly discernable. In later years an unexpected ob- servation often elicited the remark: ‘Let’s see what Beierinck has to say about it’, and it usually turned out that the latter had already recor- ded the phenomenon. Kluyver’s deep appreciation of Beijerinck’s work will be apparent to anyone who peruses his contribution to the exten- sive Beijerinck biography [1940] under the title, ‘Beijerinck, the Micro- biologist’. A final indication of the considerable change that was accomplished during the single year in the guidance of the work and in the atmos- phere of the laboratory has its spatial origin outside the institute, though it resulted in the development of a strong inner solidarity. It was the influence of the professor’s domicile, an influence that initially manifested itself but timidly. This house, that Beyerinck had occupied with his sisters, was built under the same roof as the laboratory, and could be reached via the study. It now grew into the beloved home, where the Kluyver family more and more shared the cares of the laboratory, and whose hospital- ity for associates and visitors was equalled only by the readiness to listen and the open-mindedness that every one encountered in his contacts with the professor. INSPIRATION sh nome Sty) The biographer of these early years has a comparatively easy task in so far as his thoughts scarcely need to wander outside the walls of the institute at the Nieuwelaan. For the events that became the founda- tion for the later years all took place inside it. The laboratory and adjoining living quarters dated back to 1895, when the chair for Beijerinck was created. Built in the neo-gothic style of the period, with tall, narrow windows, it had been expanded, in 1911, by the addition of a wing that was, to be sure, better lighted, 18 KLUYVER AS PROFESSOR; CHRONICLES OF THE LABORATORY but whose soberness harmonized poorly with the other part. It had been intended as a temporary structure; but, like so many temporary things, it was predestined to last for a long time; it was not to be aban- doned till after Kluyver’s death. Already the laboratory had been changed from the domain of an imposing scientific Olympian into the more adequate workshop of a teacher whose repute as a man of charming courtesy gradually spread among the Delft students. Nonetheless, it is a striking indication of Kluyver’s inspiring personality that it was not his individual attrac- tiveness but the promising tenor of his inaugural address that brought the first of his students - Van Niel — to the door of his study the day after. This example was soon imitated. Thus, in the course of 1923, the laboratory gradually became occupied; at the end of the year some- body was at work in every usable space, from the ground floor to the attic. The inhabitants enjoyed the atmosphere of a laboratory where there was no longer any pressure of a requisite number of experiments, in anticipation of a final examination; where there was no crowding for space, as in other Delft laboratories; but where they entered into a small community that gradually became closer, and where each found work to do commensurate with his ambition and ability. Just the same, no matter how much this atmosphere of peace and devotion was respected by all, the spark of inspiration was still lacking. Subconsciously this was sensed in the laboratory where the reverbera- tion of Beijerinck’s discoveries could still be heard. But with his depar- ture an era had definitively come to an end; new, epoch-making finds of the kind that Beijerinck had made, could scarcely be expected any more. A few years later Kluyver himself was to formulate this situation by saying that the discovery of a truly novel type of bacteria would cause no less a sensation than that which the ‘Loch-Ness monster’ threatened to do at that time. Yet it was obvious that a science as young as microbiology stood only at the threshold of its development. Would it be vouchsafed Delft to contribute as much to its expansion as it had done to the foundations? Unquestionably this expectation was alive in the laboratory, but for the time being the activities had to be restricted to making a general survey of what had already been accomplished. It may have been the unexpected crystals in a glucose-calcium car- a BIOGRAPHICAL MEMORANDA bonate-agar plate, that De Leeuw had streaked with a suspension from an enrichment culture of vinegar bacteria, that initiated the further developments. But let us not ascribe too great a role to chance, of which Pasteur had said: ‘In the realm of observation chance favours only the prepared mind’. And here was a scientifically prepared mind that already knew the questions on which were based the gradually ripening answers; a mind, therefore, that was capable of recognizing even in minutiis those features that could promote the ripening. In this sense Acetobacter suboxydans contributed its share to a process that, once started, rapidly advanced and kept the whole laboratory under tension. In daytime little was spoken about these developments outside the assistants’ lab; at most a casual remark was dropped during the early morning hours, generally spiced by Kluyver’s predilection for exag- geration, such as: ‘As of last night, microbiology has once more under- gone a change of face’. The day was devoted to the students and cur- rent matters. In the evening and during a large part of the night the professor and his associate, H. J. L. Donker, ensconced themselves in the study, surrounded by books, and endeavoured to distill true unity out of the diversity of facts and apparently contradictory data. The students, with the exception of a few initiates, at first understood little more than that it must surely be an inspiring labour that called for so much exertion and yet left so few traces of fatigue behind. As the concepts took on an increasingly definite form, their signif- icance slowly began to penetrate to the students. The expectations grew more and more intense; until one morning the newly acquired insight found expression in the terse, and in its overstatement equally characteristic, phrase: ‘From elephant to butyric acid bacterium — it is all the same!’ The Unity in Biochemistry had been discovered. Can anything evoke a greater enthusiasm among the workers in a laboratory than a discovery of this sort, whose scientific importance is so evident? Everyone knew the enthusiasm that Pasteur had manag- ed to awaken in his pupils; everyone was admiringly aware of Beije- rinck’s pioneering work, particularly because of Kluyver’s example. Now all doubt as to whether the work of the ‘great predecessor’ could be continued had been dissipated; once again a new field had been opened to investigation. What did it matter if at first the extent was overrated? The vast horizon supplied the one element that had thus far been lacking: ‘Inspiration’. 20 KLUYVER AS PROFESSOR; CHRONICLES OF THE LABORATORY What delight it was to work now! A string of candidates for the doc- tor’s degree provided one brick after another towards the completion of the construction of the ‘unity’. All of this called for much and pains- taking analytical work; the number of fermentation balances assem- bled during those years is legion. The laboratory was always open, even to those who, because of their position, were not entitled to a key. For there was, after all, the pathway through the door of the director’s home, up the stairs, and through the study. Here Kluyver would be found at work, either alone or with one of his pupils. When, the work done, he descended to the laboratory in order to silence his disquietude about the primitive, gas-heated incubators, as he was wont to do every night, he would invariably find a number of his pupils still at work, distilling, analysing, hunting for fermentation products. How keenly he would then catch up with the latest developments! In the middle of the night he would await, with bated breath, the results of an experi- ment in that building that had already become known as the ‘light palace of the Nieuwelaan’. And if the results signified the happy conclu- sion of a particular investigation, the session was sometimes concluded with a spontaneous rendering of the national anthem. Although the attention was focussed largely on the biochemical ac- tivities of the micro-organisms, a profound interest was nevertheless maintained in morphological and developmental problems. It was during this period that the yeast, Sporobolomyces, was discovered; and the antics of Bacillus funicularius, curling itself up in strands, were watched with delight by the observers who, stretched out on a sofa, could thus make their observations through an inverted microscope. This was also the period during which a great intimacy developed between the professor and his closest associates. This always remained controlled because of the respect that his pupils felt for him, and which made it easy for them to accommodate to his courteous style. A con- tributory factor to this intimacy was the close vicinity of the home; the child’s voice that, around 6 p.m., could be heard through the corridor, summoning father to dinner; the encounters with Mrs. Kluyver who so often spent the evening in the study, cleaning up, ordering, writing, seeking an urgent consultation. And the professor also won the hearts of those pupils who did not belong to this more intimate circle. How amiably he could point out a mistake made by a beginner, correcting without discouraging. The 21 BIOGRAPHICAL MEMORANDA more advanced group was frequently astonished by the clarity of a verdict that could lay bare the fundamentals of a problem in a terse formulation. Striking, too, was his modesty. When he proclaimed: ‘The previous century — the century of Lavoisier — was the century of oxygen; this is the century of hydrogen’, it was not said in self-aggran- dizement, but out of respect for the work of scientists such as Warburg and Wieland. During a discussion with Séhngen about the merits of Beijerinck and Winogradsky, Kluyver once remarked: ‘It is such a pity that only our small group of microbiologists can understand what great scientists they were; what do others really know about them?’ Indubitably something of the realization that the founders of micro- biology did not always get their due can be gleaned from one of Kluy- ver’s last publications: “The Microbe’s Contribution to Biology’, writ- ten in collaboration with Van Niel. The opportunity openly to attest to his admiration for these founders was always welcomed by Kluyver. Already during the first year of his professorship he was in a position to sketch Pasteur’s merits in a public address, on the occasion of the commemoration of the cente- nary of Pasteur’s birthday. There we find the trenchant statement that shows how profoundly Kluyver recognized the connexion be- tween the minutest details and a general concept: ‘If nowadays bacte- riologists all over the world depend on the seemingly so flimsy cotton plugs; if, in their laboratories we can observe the intermittant glow of the inoculating needles; must we not then conclude that these attest to an expression of faith in the fundamental investigations of Pasteur?’ The significance of Beijerinck’s work was the subject of a lecture delivered by Kluyver on June 14, 1927, the day on which, fifty years earlier, Beijerinck had received the degree of Doctor of Science at Leiden University. On this occasion, too, a bronze plaque bearing the effigy of its first director was unveiled in the Laboratory for Micro- biology. Afterwards Kluyver travelled with a deputation to Gorssel to congratulate Beijerinck personally. In the place where the plaque was installed had formerly hung a ‘No Smoking’ sign; this now disappeared. Respect for the ‘great’ pre- decessor’ had caused Kluyver to retain it for more than five years in its original position. But it had been hard on Kluyver, who was an inveterate smoker. And whatever support the professor might count on from his associates, here they completely failed him. A prohibition 22 KLUYVER AS PROFESSOR; CHRONICLES OF THE LABORATORY that was no longer heeded after midnight had already lost its force. It lost more and more terrain; after 10 p.m.; after 6 p.m., when the younger students had gone; till finally the sign was removed and no obstacles remained in the way of enjoyment of ‘poison in one of its worst forms’, as Kluyver used to call it. During these years there were but few foreigners who came to the laboratory, and these only for cursory visits. Undoubtedly it was the old reputation of the institute that attracted them. A special impres- sion was created by the visit of Prof. and Mrs. Lindner on their return to Germany from a sojourn in Mexico where Lindner had studied the manufacture of pulque. The encounter with the great fermentation expert pleased Kluyver, the microbiologist, while in his function as host he was perhaps as much flattered by the real interest of his guests for the spirituous products of domestic origin that he offered them. No longer within the confines of the laboratory, though still within the borders of the city, Kluyver’s first contact with industry mate- rialized; during these years began his collaboration with ‘The Nether- lands’ Yeast and Alcohol Manufacturing Co., Ltd. This contact had a deeper origin than geographical proximity; it signified the re-establish- ment of historic connexions between applied and theoretical micro- biology. Van Marken, the founder of the yeast factory, was in many ways a progressive. As early as 1885 he recognized the desirability of scien- tific investigations of those microbiological processes that lie at the root of the manufacture of yeast; and to this end he succeeded in at- taching Beijerinck to his industry for the ten year period, 1885-1895. Unquestionably Beijerinck’s scientific acumen will have benefited the industry through important though indirect contributions. But he definitely was not the sort of person who would unreservedly devote himself to specifically industrial problems, and after his appointment as professor at the Polytechnical College, which later became the Technological University, his contacts with the industry practically ceased. Viewed from a technical-industrial angle, it is therefore all the more eratifying that, exactly at a time when technical developments were suf- ficiently far advanced to initiate the stormy growth ofa microbiological industry, Kluyver appeared as the man who was so eminently suited to resume the connexion, especially because the studies on the quan- BIOGRAPHICAL MEMORANDA titative aspects of the various fermentation processes had inevitably led him to consider the possibility of their industrial application. Consequently a more or less casual encounter with F. G. Waller, Chem. E., at that time a graduate student and later successor to his father as director of the yeast factory, was all that was needed to open negotiations. They led to many years of fruitful cooperation, which became formally established with Kluyver’s appointment as adviser in 1928. The first new fermentation products, manufactured on an industrial scale in 1928, were butanediol and acetylmethylcarbinol. Soon afterwards the latter was used for the production of diacetyl by chem- ical means. Some years later the manufacture of butanol and acetone was taken in hand. The development, and afterwards the supervision, of adequate procedures called for much technological-scientific work. As a consequence several of Kluyver’s pupils found excellent positions at the yeast factory. Kluyver himself paid regular weekly visits to the plant where he conferred with them on the problems they encoun- tered. Especially the butanol fermentation provided material for many discussions and extensive laboratory experimentation. Kluyver’s mastery of the literature, both in this particular field and in the most general sense of the word, was exceptionally stimulating, and great was the influence he wielded on the expansion of research work in the entire factory. In later years Kluyver also established connexions with many other industries that had to struggle with microbiological problems. When informed of their difficulties, Kluyver usually let one of his students conduct experiments on what, in the light of his experience, seemed to be the essential elements of the situation. Most common were the instances, such as food spoilage, anaerobic corrosion, obstruction, etc., in which the industry concerned was plagued by unwanted microbial activities. Naturally, the typical microbiological industries themselves also profited from his knowledge from time to time. Thus the useful- ness of microbiological research became more and more appreciated by industry in general, so that gradually an increasing number of Kluyver’s pupils went to work in breweries, the dairy industry, sugar factories, water and sewage purification plants, food preservation in- dustries, and even at the Royal Dutch Shell laboratory in Amsterdam, where Kluyver later became microbiological adviser. 24 KLUYVER AS PROFESSOR; CHRONICLES OF THE LABORATORY While these years of inspiration had yielded many important scien- tific successes, they also produced another fruit that filled Kluyver with great joy. This was his election to the Royal Netherlands’ Academy of Sciences in 1926; and for six years he could attend its monthly meetings in the company of his father. In this august body he estab- lished contacts with exponents of the diverse branches of the natural sciences for which the wide interests of his synthesizing mind hankered. And he deemed it an honour to communicate the results of investiga- tions conducted in his institute at the Academy meetings. At a riper age he would be called upon to serve the Academy as its highest functionary. CONSOLIDATION 1928-1938 The years that followed were happy ones. The work in his institute progressed steadily; more and more frequently Kluyver’s attention was solicited elsewhere; and the scientific world took increasing notice of his accomplishments, proofs of which became abundant. But, how- ever much his field of activity expanded, the Delft laboratory remained the nucleus. Kluyver keenly realized that the passion for work engendered in his pupils would inevitably lead to increased demands for a well-equipped institute. He had succeeded in bringing about some important im- provements. The gas-lit incubators had gradually been replaced by new, electric thermostats; a compressed-air line was installed through- out the laboratory. The latter greatly facilitated aeration experiments with the well-known Kluyver flasks [Kluyver, Donker, and Visser *t Hooft, 1925], which up to that time had been used in conjunction with water suction pumps. The new facilities considerably simplified the study of microbial oxidation processes. The kitchen, always an impor- tant part of a microbiological laboratory, was expanded. These changes had been accomplished in spite of a government subsidy which, by present standards, must be deemed very scanty in- deed. In fact, it was barely sufficient to defray the running expenses; very little was left for the purchase of new instruments. Hence the material aid of the Rockefeller Foundation, which made it possible to buy various much needed pieces of equipment was most welcome. Nevertheless most things had to be done with home-made apparatus, 25 BIOGRAPHICAL MEMORANDA so that on many an occasion great ingenuity was required to achieve good results with the primitive equipment. Although the limited sub- sidy was sometimes a source of annoyance, it nevertheless affected the general atmosphere but little; for deep down in his heart Kluyver found it rather sporting to reach a satisfactory solution with the avail- able funds. With almost fatherly pride he could show visitors what his collaborators had been able to construct with the simplest components. The division of labour between the members of the staff, now aug- mented by a second assistant, had not been changed after the first year. Problems of organization, as well as the care of the culture collection, were the primary responsibility of the ‘conservator’, or associate, who also assisted Kluyver in supervising the work of the students in the laboratory. The assistants were principally charged with the extensive preparatory work that was needed to provide the abundant demon- stration material displayed in the lecture courses, and with the prep- arations for the laboratory course that Beijerinck had originated and that had been greatly expanded by Kluyver. This course was intended to initiate those future chemical technologists who were interested in microbiology in the fundamentals of that science during a few after- noons and evenings per week over a six week period. Candidates for the doctor’s degree and special guest workers, some of them from abroad, were of course guided by Kluyver himself. It should be men- tioned, however, that this system was not rigorously adhered to; and mutual exchange of ideas and experiences was always warmly en- couraged. Besides the above-mentioned activities, the conservator and the as- sistants were expected to undertake a scientific investigation that would be suitable for a doctor’s thesis. Moreover, the discussions in connexion with the subjects treated in the lecture courses frequently occasioned the projection of special experiments that had to be con- ducted in addition to the other work. It was not always easy for the staff members to divide their attention properly over the various activities. Kluyver, too, found the combi- nation of research work, instruction, and organizational chores any- thing but ideal for his staff. As he sometimes remarked, he hardly dared ask one of his collaborators to have ‘his name included among the janitors in the university roster’. In Kluyver’s opinion the administrative and technical personnel 26 KLUYVER AS PROFESSOR ; CHRONICLES OF THE LABORATORY also constituted an important part of the laboratory team, indispens- able for the proper conduct of the experimental work. And they, in turn, were just as appreciative of Kluyver’s humane and often fatherly guidance as was the scientific staff. Thus, despite the inevitable turn-over inherent in a university insti- tution, a community had grown up that was deeply attached to the Delft laboratory. But no one manifested a greater attachment than he who was the center of this community. The rapidity with which Kluyver had made himself familiar with the problems of microbiology, and the mastery he had exhibited, had not failed to make a deep impression in scientific circles. Small wonder, then, that other universities cast covetous glances in the direction of the Delft scientist whenever there was a vacancy in some allied field. As early as 1926 Kluyver received an offer to occupy a chair for medical biochemistry that was being created at Leiden University. For a long time he weighed the alternatives: Holland’s most ancient university had formerly fascinated him, and a close contact with many medical colleagues would undoubtedly have been advantageous to his work; but he also realized that he would miss in Leiden all that he had built up in Delft. Thus he decided in favour of Delft. At about the same time he also declined a call of a different sort, viz., the director- ship of the Department of Agriculture in the Netherlands’ East Indies. Some compensation for his loyalty to the Technological University was that students from other Dutch universities now came to join the Delft students in his laboratory. This had been made possible by a new provision whereby certain departments in these universities had agreed to accept microbiology as taught in Delft in fulfilment of the requirements for a minor subject. Soon thereafter the first two Leiden biologists arrived: two girl students who had decided to complete their studies under Kluyver, and who soon found themselves completely at home in the new milieu. Later they were succeeded by many others. Kluyver was always greatly appreciative of this cooperation, especial- ly from Leiden University, where he also served on examining com- mittees; the most important single reason is probably that he thus found the scientific scope and atmosphere of his laboratory materially invigorated. During the ‘thirties’ Kluyver was once more faced with a similar dilemma as that which had confronted him a decade earlier. After the 27 BIOGRAPHICAL MEMORANDA death of Sdhngen, in 1934, he was sounded out about occupying the chair for microbiology in Wageningen. The biologist in him must have carefully balanced the attraction of being able to live in the most beau- tiful part of his native country against what the vicinity of Delft had to offer; Beijerinck had characterized the environs of Delft as a ‘botan- ical desert’. But once more loyalty to the Delft chair triumphed, prob- ably augmented by the recognition of the ties with the past that Van Leeuwenhoek and Beijerinck had woven between Delft and micro- biology. This supposition is therefore the more tempting because this past must have appeared to him more clearly than ever before, as a result of the active part he had taken in the publication of Beierinck’s Collected Works, completed in 1940 with the appearance of the final, biographical volume; and in the planning of a complete critical edi- tion by the Royal Netherlands’ Academy of Sciences of all the letters of Van Leeuwenhoek, begun in 1932 on the occasion of the tricenten- nial of Leeuwenhoek’s birth. Kluyver witnessed the publication of the first four volumes of this work. A final opportunity to change his scientific domicile came after the second world war, from Rutgers University, New Brunswick, U.S.A. Kluyver toyed with the idea of joining in the general trend to migrate to America; but nobody in his immediate environment really believed that it would ever develop into more than an idle contemplation. He had become too thoroughly entrenched in the affairs of his laboratory, of Delft, and of its Technological University. As could have been anti- cipated, the final decision was again a rejection. The work in the laboratory, conducted under the banner of the ‘unity’, progressed steadily. Sometimes, as in the case of redox potential meas- urements which, it was hoped, would clarify biochemical processes, the important results that were initially expected did not materialize. At other times the work assumed a significance that could not possibly have been foreseen at the time. A striking example is the development, by Kluyver and Perquin, of a method for the submerged cultivation of moulds. This was intended to provide physiologically uniform cell material for the study of the oxidative metabolism of these organisms. Kluyver was very pleased with this elegant procedure, which opened up the possibility of carefully controlled experimentation in an entirely new field. But he was far from suspecting that less than twenty years 28 KLUYVER AS PROFESSOR ; CHRONICLES OF THE LABORATORY later this very principle would be applied on an enormous scale in the new factories that sprang up like mushrooms all over the world. The stream of dissertations and publications drew more and more attention, now also abroad. The result was that not only graduate students from Dutch universities chose to complete their studies under Kluyver, but foreign guest workers also came to his laboratory. Soriano arrived from Argentina, in order to learn the methodology of fermentation research; Frateur, from Belgium, made an extensive study of the acetic acid bacteria; some other chemists from the same country sojourned for a time in Delft. Barker there succeeded in iso- lating methane-producing bacteria in pure culture, and discovered Clostridium kluyvert. He was the first of a string of guests from the U.S.A., and was followed by Starkey, Johnson, and Clifton. From Israel came Volcani; from Scandinavia, Hartelius and Wikén, who became Kluy- ver’s successor. The interest that Kluyver’s work had created abroad was apparent not only from the fact that so many foreigners came to work in his institute, but also from the invitations he received to visit universities in other lands in order personally to present his ideas. ‘The invitation to deliver a series of lectures during the spring semester of 1932 at Iowa State College in Ames was, for the laboratory community, the most momentous. He remained in the U.S. from April till the end of August, and, despite a crowded program, he still found time to keep his associates in Delft informed of his experiences. Kluyver could report that the Delft investigations were greatly appreciated, especially in Ames. The following excerpt from one of his letters illustrates to what extent the publications from his institute had come to influence the workers at Iowa State College: “The students here can be divided into those who can read Dutch, and the smaller category who cannot. Yesterday I met somebody who was pondering Scheffer’s mysterious fermentation balances, and who told me that he would rather read Dutch than German’. He, in turn, had great respect for American accomplishments, such as those of Buswell in Urbana: ‘With this technique he has demon- strated that the queerest compounds can be quantitatively converted into carbon dioxide and methane. Phenylacetic- and hydrocinnamic acids are instantly devoured, benzene nucleus and all! Let Mr. de Graaf imitate that as soon as possible; we are shamefully behind!’ 29 BIOGRAPHICAL MEMORANDA And about Madison: ‘My lecture on metabolism and redox poten- tial did not seem to be much appreciated; I think that they found this rather ‘“‘old hat’’. Besides, my imperfect English improvisation in an enormous hall — 200 in the audience, 1,000 seats! — will probably have contributed to the lukewarm reception.’ From another letter: “The danger here is that they appear to know the Delft theses even better than I; I had forgotten quite a bit from the older ones. Here they work a lot with Dutch-English dictionaries, and some masterpieces are available in English translation — a precious possession that is not accessible to everybody... For the rest, “A prophet is not without honour, save in his own country”’’, and for my American fame it would have been much better if I had remained be- hind the scenes. Here and there I could notice disappointment with my unassuming appearance, already on first acquaintance’, as he wrote to his friend, Van Rossem. At the time of Kluyver’s first American visit the depression was still very much in evidence there. Discussing the economy drive, then also raging in Delft, Kluyver wrote, for example: ‘In the U.S. the situation is perhaps even worse because there are so many privately endowed universities. In San Francisco they have recently buried ‘“‘John Depres- sion”, oh simple minds! More important is the fact that the price of hogs has risen, and that this has caused an upswing on the stock ex- change. May this reversal in trend be permanent!’ As appears from his letters, Kluyver was greatly impressed by the vigour of the Americans, particularly in the area of scientific investiga- tion. Presumably the respect was mutual; for before his return to Delft Iowa State College conferred upon him the honorary D. Sc. degree. During his sojourn in the U.S. heavy demands were made upon him; his program was always loaded. Here follows a summary of a ‘few days’ activities, literally quoted from his letter of June 4, 1932: ‘Lecture from 11 to 12; immediately by car to Rockford, a trip that lasted till to p.m.; early Tuesday on to Milwaukee, visited the acti- vated sludge plant; that evening a lecture before the local division of the American Chemical Society; early Wednesday on to Madison; visited many laboratories during the day, and had lunch and dinner with the “greats”? (Overton and Steenbock); evening lecture before the Wisconsin Chapter of the Amer. Chem. Soc.; Thursday, early breakfast, back by car to Ames, return 6 p.m., seminar at 7.30; Fri- 30 KLUYVER AS PROFESSOR; CHRONICLES OF THE LABORATORY day, lecture from 11 till 12, off by car to Minneapolis at noon, arrival at 7 p.m.; that evening reception by the members of the Bacteriology Department; Saturday a.m., visits to laboratories (Gortner’s, among others), at noon off by car to Hackensack at Birch Lake, in Northern Minnesota — land of the 1,000 lakes, each one of the dimensions of our Loosdrecht lakes — to the cabin of Dean Buchanan; Sunday and Mon- day spent with the Dean on his lake, fishing (!), canoeing (!), alter- nating with cooking and dish-washing; Monday evening, 7 p.m., by car to Minneapolis, stopped for an hour and a half with Kolthoff, con- tinued on to Ames, arrival Tuesday at 6 p.m.; at 7.30 my seminar; next morning lecture at 11. All in all, a rather formidable itinerary. Travelled 1600 miles, or 2500 km, in an automobile in a single week!’ In his own country Kluyver’s counsel in scientific matters was sought with increasing frequency. First love made him accept with alacrity an advisory function with the Governmental Fibre Institute. The tie lasted for many years, and was not severed when this institute became part of the ‘Central Organization for Applied Natural Scientific Re- search’ (T.N.O.) in the Netherlands. This is not surprising, for Kluy- ver had long ago recognized the importance of the sort of investiga- tions that were carried out there. And soon after T.N.O. had been established, Kluyver became a member of the board of trustees of the Central Organization; from 1932 on he served uninterruptedly in this function, and was a member of many of its committees. On January 1, 1935, began a collaboration that was to require much of Kluyver’s attention. The Rockefeller Foundation had made a grant to Dr. L. S. Ornstein, Professor of Physics at the University of Utrecht, for the establishment of a group that would be concerned with investigations of problems in the area of biophysics. Ornstein associated himself with Kluyver, and under their combined guidance the Biophysical Group Utrecht—Delft came into being. From the mem- ories of E. C. Wassink*, for many years an associate of this group, the following picture of Kluyver’s activity in it emerges: * The following account is a slightly adapted version of an article of Dr. Wassink, kindly submitted by him to the Editorial Committee. The work of the Biophysical Group Utrecht-Delft has been reviewed by Wassink in: Advances in Enzymol. 17, 119, 1951; and by Spruit and Spruit-van der Burg in: The luminescence of biological systems, edited by F. H. Johnson. Washington, 1955. p. 99. 31 BIOGRAPHICAL MEMORANDA ‘At the start the group concentrated on a study of bacterial lumi- nescence and photosynthesis, in a manner that would guarantee the closest possible collaboration of physicists and biologists. It was housed in the Physical Laboratory of Utrecht University, which, in 1938, was expanded by the addition of six new rooms for the group, made_pos- sible by a generous new subsidy from the Rockefeller Foundation. Although Ornstein supervised the daily activities, the younger mem- bers did not hesitate to communicate with Kluyver whenever they had obtained significant results, either by writing to him, or by visiting him in Delft, especially on Saturday afternoons. The most important con- tact between the group and its Delft leader was, however, maintained through Kluyver’s periodic visits to Utrecht, which occupied an entire day. These visits were always stimulating, strenuous, courteous, and friendly. The association of the two leaders, each a world-renowned authority in his own field, was particularly suitable to inculcate into the younger workers an appreciation of what is required of scientific work that is worthy of being judged by international standards. ‘On such days the experimental work was usually suspended, and the group assembled in Ornstein’s room for extensive discussions, while enjoying coffee and tobacco. The discussions were interrupted only for luncheon, in a nearby restaurant that was favoured by Ornstein. Kluyver used jokingly to regret the interruptions, and on one such occasion he discoursed, in science-fiction fashion, on the beneficent effects of food tablets that would immediately restore one’s capacity for work for several hours, and could be carried in goodly quantity in a waistcoat pocket. We may assume that Kluyver, despite everything, found it quite appropriate that the deprivations of war made it nec- essary to replace these luncheons by a simple meal, consisting of a few slices of bread and a tea- or coffee substitute, eaten in the laboratory. ‘At the start the two leaders had agreed that their names should not ordinarily appear on scientific publications by the group. Only once did they deviate from this principle. Because the publications were primarily biological, Kluyver took an active part in their preparation. He spent many weekends with the authors, whose experience in this respect was identical with that of Kluyver’s pupils. ‘Ornstein died in 1941; his end was undoubtedly accelerated by the cruel measures of the occupation. Under the difficult circumstances of wartime, Kluyver directed the program single-handedly until 32 KLUYVER AS PROFESSOR; CHRONICLES OF THE LABORATORY Milatz was appointed as Ornstein’s successor. Milatz resembled Orn- stein in many respects, and contributed a generous dose of optimism and enthusiasm which was much appreciated and helped to cheer up Kluyver during his visits. Thus the work was continued, even though the Rockefeller grant had ceased, until the critical situation during the winter of 1944 caused it to stop almost entirely. ‘After the end of the war the experiments were soon resumed. But the frequency of Kluyver’s visits to Utrecht gradually diminished, espe- cially during his last years. Milatz too found less and less time for the group because he was involved in the establishment of nuclear-physical studies. ‘In general, the close collaboration of a physicist and a biologist, en- visaged as an ideal during the early period, receded into the back- ground. ‘About the time of Kluyver’s death, Milatz left the physical labora- tory, so that the group was robbed of both its leaders. Till now they have not been replaced, and probably will not be; the present senior workers continue the work independently. One reason for this situation is surely that it would be difficult to find another team of leaders that could arouse the sort of scientific fervour that Kluyver and Ornstein engendered.’ Kluyver’s capacity for work and intellectual ability were devoted not only to science and technology. It was particularly characteristic of him that requests to serve the community in functions of minor im- portance were never refused. Thus, for many years, he served the city of his domicile as a member of its sanitary commission, whose task it was to advise the city council in matters of hygiene. Such work did not always give him satisfaction; he was, for example, considerably dis- turbed when, against the commission’s advice which he fully sup- ported, a cemetery was expanded in an area where, thirty years later, the new buildings of the Technological University were erected. Nevertheless, such experiences did not prevent him from taking upon himself other duties, such as the function, first of trustee, later of presi- dent of the board of trustees, of one of the local secondary schools. As his age advanced, his associates, and no doubt his children too, often advised him to give up such time-consuming activities; but he could not be moved to do so. He was probably convinced that in a ee BIOGRAPHICAL MEMORANDA democratic community everybody should take part in matters of the common weal to the full extent of his ability. This, by the way, was also Mrs. Kluyver’s stand; she too had always found time for social work in addition to caring for a growing and developing family. When in 1955 Kluyver, at his own request, was retired as president of the board of trustees of the secondary school, the Mayor of Delft presented him with the honorary silver emblem of the city; this was also intended as a posthumous appreciation of the considerable social contributions of his wife who had recently died. With this homage the city council gave a striking tribute to the civic-mindedness of both. Thus, at the end of the ‘thirties’, Kluyver led a life of harmony, rich by virtue of a close contact with much that fascinated him in science and society, both here and elsewhere. His fame had been established, and his career had reached a summit. On May 25, 1939, the occasion of Kluyver’s quarter-century doc- torate, “Chemisch Weekblad’ published a special issue. His teacher, Van Iterson; his colleague, Van Niel; and his pupil, Kingma Boltjes, each from his own vantage point, contributed essays that served to create an impressive balance of all that Kluyver had given during these twenty-five years to science and to his pupils. Yet, rather than presaging a continued assent, this commemoration turned out to be a turning point. The war rumble could already be heard distinctly; the ‘good old days’ were at an end. WAR AND OCCUPATION 1939-195 Pupils from the ’twenties and early ’thirties remember that Kluyver’s vocabulary contained such favorite slogans as ‘Never say die’, ‘Keep smiling’, ‘Some day we'll know’; but during the last years before the outbreak of the war another one came to the fore, the distinctly pes- simistic “This is a great and terrible world’. The unmistakable threat of what was to come depressed Kluyver perhaps more than others; but as long as it was still possible, the work was continued in the race against time. Kluyver was about to embark for a trip to New York, where he was to participate in the third Inter- national Congress for Microbiology, when the threat of war interfered ; oe KLUYVER AS PROFESSOR ; CHRONICLES OF THE LABORATORY the news reports were so ominous that he felt obliged to cancel his journey. On August 27, 1939, he wrote to Van Niel: ‘What hurts most is that now the plans for our reunion have come to naught. Believe me that the three months of vainly spent efforts in preparing for the congress and the tour that was to follow — on Thurs- day 240 lantern slides had been carefully packed in my suitcase — do not grieve me as much as the lost opportunity to see and talk with you again. The congress itself and the many lectures in numerous places that had been scheduled for the subsequent tour of the U.S. have never appealed to me; in any case, the superabundance of contacts would have rather got me down. Qui trop embrasse, . .. ‘Above all else I realize, however, that personal regret about the course of events sinks into nothingness in view of the tortures to which millions of people are subjected at this moment.’ On May 10, 1940, the Germans invaded Holland; as soon as the immediate turmoil of war had subsided, Kluyver called a meeting of the laboratory staff in order to consider the new situation. He ex- pressed his deep-seated conviction that in the long run right would eventually prevail, and he urged everyone to resume his work. ‘The meeting was concluded with the national anthem. Now began a period during which the laboratory population stead- ily declined; numerous necessities began to run out; and the contacts with foreign countries were broken off more and more. On the other hand, there also occurred a distinct change in Kluy- ver’s mental attitude towards his work. He was, after all, not the kind of scientist to whom his occupation means everything and who could work in total isolation, divorced from any and all ties with social events. His speculative mind, instead of concentrating on the work, strayed increasingly towards the experience of foreign occupation and war violence, alarming and laming by their brutal and chaotic irra- tionality. The news bulletins broadcast by the allies were faithfully taken in. The creative scientific work no longer progressed in a mood of playfulness, and often had to yield to activities concerned with the practical requirements of the moment. During the war days the laboratory and the home were a natural refuge for personnel, staff, and neighbours who were received with great hospitality and could seek protection in an improvised hiding trench. After the bombing of Rotterdam the laboratory was charged 39 BIOGRAPHICAL MEMORANDA with the bacteriological control of the drinking water which was supplied by that city, while Mrs. Kluyver had an important share in locating shelter for the refugees who had come from there. The laboratory garden was used for the cultivation of potatoes, peas, beans, and, not to be forgotten, tobacco. The scarcity of the latter product hit Kluyver particularly hard, and it must have been a great satisfaction to him that he could at least apply his knowledge of the tobacco fermentation in a directly useful manner. Agar could, of course, no longer be obtained from abroad. This served as a natural impetus to examine more closely the potentialities of silica gel as a substitute. Furthermore the bacteriological manufac- ture of lactate, used as an additive to fodder, was taken in hand in view of a threatening depletion. But in addition Kluyver still managed to devote his attention to dissertations and publications that were in preparation. During this time the sixth volume of Beierinck’s ‘Collected Works’ was completed, as also the section for the Dutch “Textbook of General Botany’ which he wrote in collaboration with Wassink, and a few papers that were pub- lished in ‘Antonie van Leeuwenhoek’, in English. He also contributed much to the writing of the second volume of the classification of the non-ascosporogenous yeasts. The cares weighed too heavily, however; no longer could a preoc- cupation with such activities bring solace. It was only through his iron sense of duty that he could bring himself to perform them, and even so the tempo was greatly reduced during these years. The device of Wil- liam the Silent, ‘Point n’est besoin d’espérer pour entreprendre, ni de réussir pour persévérer’, which he had many a time held up before his pupils, now had to serve as an inspiration to himself as well. Kluyver’s loyalty towards his country was equalled by that which he experienced towards what were then the Dutch East Indies. During his early years as a scientist he had been so profoundly impressed by this country that the Japanese occupation of this realm shocked him greatly. And his attachment to the Technological University, where he had been a student and later a professor for so many years, caused him to suffer as a personal attack the measures of the occupation forces against the Dutch universities. The dismissal of Jewish professors by the Nazis, later followed by the deportation of students who had re- fused to declare their loyalty to the occupant, almost completely lamed 36 KLUYVER AS PROFESSOR; CHRONICLES OF THE LABORATORY any instruction. He was often consulted about the difficult decisions that the Technological University had to make during these years, and his voice carried great authority. The role of leader in the resistance against the occupation was in- compatible with Kluyver’s character; but he was often a trusted ad- viser to those who fought the German attacks on students and uni- versities. He was deeply moved by the fate of hundreds of students who had been deported to Germany as forced labourers, and he established a bureau that endeavoured to remain in touch with each one of them. By amassing information he succeeded in greatly improving the defi- cient contacts with parents, and he acquired a fully documented knowledge of the regrettable conditions under which work was being done in many camps and industrial plants. This caused a strong pro- test to be launched with the German authorities; alas, it was of no avail. In many instances, only too frequently of a tragic nature, Kluyver could lend moral support. The yeast factory, too, experienced hitherto unknown difficulties. Kluyver learned about them when, in his capacity of adviser, he paid his weekly visits to that community which consisted so largely of his own pupils. But these visits were bright spots in his existence as well; occasions on which he could share with his former coworkers such simple pleasures as the daily cup of soup that was served in the plant in those times of severe food rationing. And it filled him with pride that during the last years of the war, when rumours about penicillin had begun to penetrate from allied sources, a research team at the yeast factory had managed to prepare batches of this ‘wonder drug’ of such purity that they aroused Sir Alexander Fleming’s unstinted admiration. Anxiety about his own family added to all the other worries of these troubled times, and Kluyver’s vitality and health were progressively undermined as a result of the food scarcity. Now and then he was forced to go into hiding or, with great bodily exertion, to provide food for his family. Delft was situated in the area known as ‘Fortress Hol- land’, where, during the hunger winter of 1944-1945, the horrors of war ran rampant. Nevertheless, weakened and emaciated as he was, he succeeded, with the aid of his faithful technician, Veenhoff, in maintaining the valuable pure culture collection almost fully intact, and this under circumstances that necessitated the improvisation of af BIOGRAPHICAL MEMORANDA even the barest essentials. By that time the work in the laboratory had obviously come to a complete standstill. RECOVERY ; THE FINAL YEARS 1945-1956 The liberation, dramatically ushered in by the famed allied food droppings, brought a welcome relaxation. With joy and gratitude Kluyver realized that his family and laboratory had come through the struggles unharmed. But he could not be blind to the total collapse of the fatherland, and to the immense problems that cried out for a solu- tion. More particularly, he realized that a vast deficit had developed in Europe in the field of scientific endeavour. ‘Beiyerinck has made Dutch microbiology great; but now we have been left utterly behind’ ; this is how he sized up the situation. And this view was strengthened when he could gradually acquaint himself in some detail with what had been accomplished outside the occupied territory. One of the first contacts he established with the outside world was with Sir Jack Drummond, who had arrived in the Netherlands with a food team. Soon afterwards news began to flow in from America, largely in the form of reprints collected for him by his friends during the war years. He was deeply moved by the fact that in the U.S.A. a ‘Delft Library Fund’ had been created through which the laboratory could acquire numerous books and journals. Perhaps the most striking token of sympathy came from Mr. Ben May, in Alabama, who pro- vided funds for the purchase of a Beckman spectrophotometer. In his own way, Kluyver honoured the donor by attaching a sign with the inscription ‘Ben May Institute’ to the door of the little room where the instrument was installed. With the aid of his coworkers, enormous quantities of reprints were sorted in the large class room. This operation progressed slowly, for Kluyver could not resist the temptation to browse around in, and comment on, this literature, and it was a valuable experience for his pupils. Avidly and admiringly Kluyver let the new knowledge sink in; the lectures were immediately brought up to date; and students and assistants were put to work, repeating various experiments in order to familiarize themselves with the newly developed techniques. Kluyver reacted to this sudden gush of new information in a man- 38 KLUYVER AS PROFESSOR ; CHRONICLES OF THE LABORATORY ner similar to that which he adopted when he had come to Delft as Beijerinck’s successor. Once again he chose not to follow the road of narrowing and restricting his attention to some specialized topics; once again he set himself the task of assimilating the new knowledge in its entirety, frequently consolidating his critical conclusions in papers in which the recently opened fields were reviewed. Already at a relatively early date, in 1949, Kluyver found an oppor- tunity personally to re-establish some of the broken contacts when he made a trip to the U.S.A. on behalf of the yeast factory. Building on the experience gathered during the war in the manufacture of penicil- lin, this concern had greatly expanded its activities in the pharmaceu- tical area, a development which had led to the appointment of a group of medical advisers, and Kluyver naturally became president of this advisory board. Meanwhile an entirely new methodology for the study of microbial morphology had begun to take shape in Delft. Just before the war, electron-optical developments abroad had led to the appearance on the market of the first electron-microscope. Surmising its great poten- ualities for biology, Kluyver and Mr. F. G. Waller of the yeast fac- tory joined forces and obtained the necessary material aid to enable Professor Dorgelo from the Department of Technological Physics to push his investigations in this field. During the war one of the students in that laboratory succeeded in constructing clandestinely an electron microscope that was regarded as the best in existence; and soon after- wards this instrument was in use every hour of the day, for the exam- ination of diverse objects, including many biological preparations. Be- fore long it became feasible to set aside a portion of the available time for microbiological studies, and eventually the microbiological labor- atory acquired its own electron microscope. Thus Kluyver’s strategic outlook had promoted, and in a particularly satisfying manner, a development that resulted in important pioneering work in the field of microbial morphology. As is evident from the bibliography and the list of dissertations, the scientific investigations in Kluyver’s institute were rapidly resumed. The study of inorganic hydrogen acceptors was expanded by further work on the reduction of carbonate and nitrate. Moreover, the results of studies on cellulose decomposition in the rumen of cows attracted 29 BIOGRAPHICAL MEMORANDA particular attention; these led Kluyver to remark that the rumen of herbivores should be considered as a large fermentation vat. As formerly, pupils came from Leiden and Utrecht, as well as from abroad. Fahraeus, Nickerson, De Ley, Erkema, Cantino, Van der Walt, Kistner, Pontieri, and Battley conducted experiments, each ona diffe- rent subject. Besides, a change in the curriculum of the Technological University now required students who wished to specialize in a biological area to spend an additional six weeks in Kluyver’s laboratory, during which they were to undertake a study of a special problem. Frequently the associate had to marshall all his resourcefulness in order to provide adequate space for the many applicants. A further handicap was the fact that in the post-war years it was extremely difficult to obtain various laboratory utensils. About 1950 Kluyver decided to have his staff — now increased by the addition of an assistant-in-chief, whose title was later changed to that of scientific officer — conduct the labor- atory courses. Furthermore, after the war a full-time assistant was appointed to take charge of the culture collection. Kluyver continued to pay close attention to the maintenance of an adequate instru- mentarium; and the laboratory acquired, for example, the equipment needed for tracer studies. But soon there was not enough space left to install additional apparatus. On January 18, 1947, Kluyver had occupied the chair for micro- biology for a period of 25 years. The ‘Netherlands’ Society for Micro- biology’ issued on that occasion a special Jubilee Volume (Antonie van Leeuwenhoek, Vol. 12) with numerous contributions by friends and admirers from all over the world. It took much gentle persuasion before Kluyver finally agreed to a small celebration in commemoration of the event; but the Saturday evening on which this took place was for him a truly happy occasion. Nearly all his collaborators of the past 25 years were present. The high light was indubitably the speech which the celebrant himself delivered. It took nearly two hours; he left out nothing and nobody, and recalled numerous details which clearly revealed that the element of personal interest generally domin- ated the contacts he maintained with his former pupils. Since the pre-war years Kluyver had undeniably imeem The ‘eager young tiger’ had gradually developed into one of the ‘grey eminences’ of science, upon whom many honours were bestowed, and who was more than ever called upon to deliver public addresses and 40 KLUYVER AS PROFESSOR; CHRONICLES OF THE LABORATORY to undertake assignments of longer duration. The world around him, too, had become a very different one; and in the wake of the upheavals many new problems had arisen that demanded solutions. It was evi- dent that these should be formulated by persons who were acutely aware of the changed conditions and of the events that had caused them. Above all, the war had emphatically underscored for him that science can have only relative significance, and represents but one of the many factors in the composite that governs all human endeavour. He felt that his position and gifts compelled him to accept responsibility for more than the mere pursuit of his special field of science. Perhaps he realized this obligation all the more strongly because it was one of the attractive features of his chair that he could devote his activities almost entirely to the education of only a small group of truly interested pupils. Thus he was privileged in comparison to many of his colleagues who had to spend most of their time and efforts in the ele- mentary training of large numbers of students. Consciously he now increased the sphere of his activities in the wide area of his interests that had already begun to unfold before the war. He continued to guide the scientific work in his institute; yet he also bore witness to the outside world of all he had gained in knowledge and insight during his rich life. His initial pessimism, which occasion- ally expressed itself in extremely sombre moods, and was caused by the recognition that he had fallen behind in his science, further ag- gravated by the great needs of the country, gradually dispersed; even- tually it was superseded by the constructive interest he took in the great problems that were in store. When the need for remodeling the system of higher education was felt in the Netherlands, Kluyver was appointed to the State Commis- sion that was charged with recommending measures that should be taken to achieve the desired goals. In 1947 he became Secretary, and a year later Rector Magnificus of the Technological University. The authority and respect he com- manded in the academic senate, and particularly among the students, were unrivaled. He knew how to win over the younger generation to his aims, and felt completely at home among the students. He was hos- pitality personified when officers of student organizations called on him, and during the existing housing shortage he set a personal example by making an apartment available in his home. 41 BIOGRAPHICAL MEMORANDA Obviously this aspect of his rectorate gave him much satisfaction, for ‘What can be more beautiful than to live in the hearts of the younger generation?’, as he wrote to a friend shortly before his death. He had also been elected to membership in the Council of the Royal Institute of Engineers. In this, as in many of his other official functions, he was frequently confronted with various problems that had their or- igin in marked changes in the social environment. These changes had made higher education accessible to large numbers of young people from non-intellectual milieus. At the same time, the country’s economy called for an intensive industrialization for which, in turn, many uni- versity graduates were needed. The task devolving upon the Technol- ogical University, of educating 5,000 instead of 2,000 students without lowering standards or abandoning tried traditions, was in fact an im- possible one. But Kluyver threw himself into the problem with his ac- customed thoroughness, and amassed facts and ideas from every side. He frequently referred to these problems in discussions with his asso- ciates and students in the laboratory. Although this caused a momen- tary interruption of the flow of the scientific work proper, it contrib- uted to the participation of his pupils in the real problems of the mo- ment. It was a great support for him that his wife had developed a similarly directed interest in the existing social problems. From 1947 till 1954 Kluyver was President of the Natural Sciences Section of the Royal Netherlands’ Academy of Sciences. His feeling for history led to the restoration of the proud headquarters of the Academy, the “Trippenhuis’, during his presidency. Moreover, there were problems of reorientation conditioned by the times, and Kluy- ver was too much attached to the Academy to hesitate in placing all his ability and energy at its disposal. With dash and dignity he re- presented Dutch science, compelling respect for his thorough know- ledge. An example of the manner in which he acquitted himself of this task may be found in this book, where the after-dinner speech, ‘An Aspect of the Promotion of Science’, delivered at the annual meeting of the National Academy of Sciences in Washington in 1953, has been reprinted. It was the second time that a foreign sister insti- tution, on this occasion the Netherlands’ Academy, had been hon- oured by an official invitation to attend such a meeting. Kluyver had already served the Academy in numerous committees, among others in the Leeuwenhoek Committee. During his presidency 42 KLUYVER AS PROFESSOR; CHRONICLES OF THE LABORATORY he was appointed, as Academy delegate, to the chairmanship of the ‘Commission Kluyver’, which was to prepare recommendations to the Government concerning atomic energy investigations in the Nether- lands. His forward-looking attitude and proficiency as a mediator were to set the tune in this commission. When the ‘Netherlands’ Re- actor Centre’ was established, he was made a member of its executive council. Shortly afterwards, on invitation by the Government, an Academy committee under his leadership studied the dangers to life connected with the detonation of atomic weapons. Although it is cer- tain that Kluyver owed these appointments to his great authority and diplomatic skill, it is worthy of note that in his 1922 inaugural address, reprinted in this volume, he had already anticipated that man would some day be able to exploit the enormous intra-atomic energy. In post-war years, as mentioned earlier, Kluyver was often invited to deliver special lectures. Once he had accepted such an invitation, he considered it beneath himself to live on past glory, and felt obli- gated to contribute the best that his experience could offer, always adapting the topic and presentation to his audience. This involved an extremely thorough and time-consuming preparation, which extended over weeks and used to continue till the very last moment, although it was always completed in time. A glance at the bibliography and the list of honours will suffice to indicate the number and variety of this sort of lectures, which often involved fatiguing journeys as well. These and the previously mentioned activities must be superim- posed on the regular daily schedule which, in these days, bore a truly kaleidoscopic character in its own right. Problems of instruction in the Department of Chemical Technology, committee activities in his own field, preparation for congresses and other scientific meetings, weekly and monthly visits to industrial concerns for which he acted as adviser, incidental requests for advice on technological or scientific matters, and naturally, the direction of the work in his laboratory, the lec- tures, and the examinations, these represent the most important cat- egories into which his work might be classified. In addition there were separate and special occasions that required his attention, such as the voyage to Trondheim in 1954, where he took part in the promotion of the Norwegian microbiologist, H. Larsen, who had conducted his work for the doctor’s degree under Van Niel. Another such example was the selection of Kluyver as promoter of 45 BIOGRAPHICAL MEMORANDA H.R.H. Prince Bernhard of the Netherlands when the latter received an honorary doctorate in 1951. This homage on the part of the Tech- nological University, to which Kluyver felt so deeply attached, must have been very dear to him, just as was another mark of distinction, which the Government evinced when it donated an authentic Leeu- wenhoek microscope to the Technological University on the occasion of its semi-centennial. It is not surprising that Kluyver often began a conference with a plaintive ‘Life is complicated!’ Nor is it astonishing that now and then he felt that his pupils did not receive their due, which made him limit the time for his own rest to the barest minimum. In this way he kept a firm grasp on the activities of his students and staff. And, although the personal contacts with the students may have appeared somewhat scanty in comparison with the situation in the thirties, it nevertheless remains true that they were both more numerous and more intensive than those which the students were apt to experience elsewhere in the university. Besides, the previously mentioned tendency to make his pupils partners in the manifold problems that occupied him added further to their unique educational opportunities. It goes almost without saying that, under the circumstances sketch- ed above, Kluyver’s amazing physical endurance could not stand up against the demands of his mental activities. For a number of years prior to his death he had been suffering from angina pectoris, but nothing could induce him to spare himself. He continued to smoke in- cessantly, albeit that he occasionally shifted to Egyptian cigarettes and limited himself to only one cigar per day. His perseverance was mag- nificently evident in 1953 when, during a rough crossing on his way to London, he and his chair tumbled down a couple of iron stair- cases on board ship. He suffered serious injuries, from which he did not recover till many weeks afterwards. Nonetheless, on the day after his arrival, and covered with bandages, he delivered the Leeuwen- hoek Lecture before the Royal Society. A member of the audience has described this feat as ‘one of the most heroic performances’ he had ever witnessed. | That in spite of all this he could keep up this extremely busy life for so long was largely owing to the care with which his wife sur- rounded him. When she took ill a shadow fell over Kluyver and the laboratory, for she too had occupied an important place there. During 44 KLUYVER AS PROFESSOR; CHRONICLES OF THE LABORATORY the post-war years it was she who saw to it that the ever-changing ranks in the personnel were regularly kept filled. She also played an active part in attempts to solve the housing difficulties of the staff. In fact, she was consulted whenever human problems arose in the labor- atory, and discussions between the professor and his staff about such problems always ended in conferences with the ‘unsalaried chief of personnel’ of the institute. Thus, when she died in 1952, it was also a severe loss to the laboratory. It hardly needs commentary that the next few years were particularly difficult for Kluyver; during this time his children proved a great support to him. But the most important consequence, the terrifying spectre of the lonely future that awaited him upon his retirement, was something of which nobody could re- lieve him; and there was irony in the circumstance that, just at that time, the plans for the new laboratory with adjoining living quarters began to assume a definite form. He had long looked forward to this development, and now he would scarcely be in a position to enjoy it any more. At the outbreak of the war, ‘the institute that had been promised me as early as 1924’ had not progressed much beyond the stage of a rough draft. When, after the liberation, funds were made available for newly equipping the Technological University, Kluyver hoped that now his new institute would soon come into being, so that he would still be in a position to use it for at least six or eight years before hand- ing it over, completely equipped and functioning, to his successor. With enthusiasm and thoroughness he had helped in the drafting of the plans; he had consulted architect friends and foreign acquaint- ances; he had gathered documents and designs of other laboratories and during his travels he had collected every pertinent datum of in- formation. But now it was a problem of financing, then again the city’s planning commission, that caused postponement or required modification of the plans. What had once been a joy gradually be- came a lingering burden, and with the passing of the years it finally was only a sense of duty towards the chair for microbiology itself that could still act as a stimulus. In addition, the impending departure from the old and familiar surroundings at the Nieuwelaan seriously troubled Kluyver, so that, when at long last it became evident that even under the most favourable conditions he would profit from the new institute for no more than a couple of years, he sometimes could 1) BIOGRAPHICAL MEMORANDA not resist the temptation to complain, during meetings with the build- ing committee, that ‘the last paper of a scientist is the description of his new laboratory’, although this did not diminish the gratitude with which Kluyver greeted the plans. The first pile was driven into the ground in 1955, and in the end the new institute, built according to Kluyver’s specifications, was not ready for occupancy, as a precious heritage, until 1958. In 1954 he returned enthusiastically from his extensive tour of the U.S.A. where, jointly with Van Niel, he had delivered the Prather lectures at Harvard University. The common European impression, that the supposed supremacy of scientific research in America need not be taken seriously, he denied emphatically. He was full of admi- ration for the American achievements in his own field, and searched for the secrets that could explain the success of those research insti- tutions. Now that the authorities had made available the bricks for the new building, Kluyver occupied himself with the question how, in the light of his recent experiences, it could be appropriately staffed with brains. The problem of equipping the new institute with modern apparatus too had occupied him during his travels, and the purchas- ing plan he formulated in 1955 greatly benefited by the information he had gathered. Van Niel’s visit to Europe, during which he spent several lengthy so- journs in the Delft laboratory, acted as a strong and refreshing stim- ulus. There was ample time and opportunity for extensive discussions, not only between the master and his former pupil, but also between Van Niel and the coworkers in the laboratory. The return of the asso- ciate, Verhoeven, who had spent a year in the U.S.A. as a Fellow of the Rockefeller Foundation, contributed further to the anticipation of modernization and of the fresh start that Kluyver would initiate in the new institute. The tragic element that scarcely came out into the open was, ob- viously, that both Kluyver and Van Niel prepared a new future for Delft microbiology in which neither of them would play a part. It had penetrated to only very few of those who daily surrounded Kluy- ver that only mental energy kept him going. But pupil and master realized that this was to be their last meeting, and the farewell in the spring of 1956 was difficult for both. 46 KLUYVER AS PROFESSOR ; CHRONICLES OF THE LABORATORY Kluyver did not fool himself as to his state of health, and occasion- ally intimated that he reckoned with the possibility of not personally participating in the developments of the near future. When, in rapid succession, the fathers of two members of the personnel died from old- age complaints, he expressed the hope that he himself would pass away amidst his work, and in full possession of his powers. On Saturday, May 12, he attended the annual meeting of the ‘Holland Association of Sciences’ in Haarlem; the next day he was occupied with matters concerning the future organization of the labor- atory, working as usual till late at night. During the first hours of May 14 the end came. Especially during the last year Kluyver had frequently been occupied with problems of life and death. In his lecture, ‘Microbe and Life’, which he used to call his ‘swan song’, he had expounded his ideas on the origin of life. While working on another lecture, which he deliv- ered on May g, 1956, before the Delft student organization ‘Vrije Studie’, he indicated to his associates in the course of several discus- sions that he would again touch upon his views on the problems of life. It was evident that the preparation for this lecture weighed heav- ily on his mind. But when he began his discourse in the old library of the student union which held so many memories of his own student days, he instantly captured his youthful audience, and it was touching to experience how his devotion succeeded in transmitting, seemingly in the form of a dialogue, the ripened conclusions of his life-long searchings to those whose careers still lay before them. Two days later he reported, as was his wont, to his scientific staff in the laboratory; at that time he also mentioned that his heart had trou- bled him considerably during the lecture, and that he had even tried, unsuccessfully, to consult his physician the next day. This was all the more remarkable because he never talked about his state of health, and immediately sidestepped any remark launched by others in this direction. Although he knew that dinner was waiting, he continued the discussion, which presently returned to the subject of the lecture. After having talked about the evolution of the micro-organisms, he turned, as if inevitably, to the influence of environment on man and, in support of a remark, he reiterated the example used in one of his earlier lectures, showing that Darwin and Lincoln, born on the self- 4] BIOGRAPHICAL MEMORANDA same day, owed their eminence in part to their environments; had they been exchanged right after birth, the world would have suffered the loss of a great biologist and a renowned statesman. It was impos- sible to escape the impression that he indulged in this scientific dis- course in order to express what was occupying his mind, and that he was actually applying his evaluation of the comparative significance of genetic heritage and the vicissitudes of life to himself. It was already well past 7 p.m. when his associate accompanied him to his house through the long corridor and the room where the yeast collection was stored. “Tomorrow I won ’t be here; [ll see you again on Monday’... 48 1. The young orofessor; 1926. 2. Kluyver amidst his colleagues of the Chemistry Division; 193 \e Meat’ Is gon dus Yekion to 4 ‘opi Fs Ne medisu. , and OF Sten Sp pee 05 hae & Generel f Hof gin fur the alin phase tes by (ole Anti Manin "yn a fk aed | Ma thn [Ba ly tn ‘nf LS we 4 oon Eyoddnsc deh / Mat Xho falls Fig tised ee re od catiat Lo Ke irtoun ite YELLrn , wblitie 4 tie a yell Cuff Atlin prespih, Chorin a nea 4 Proed . Tore ce ital inched in it We vind Ge pls hich paps A fa l ¢be Six. The pigrtat 4 again romorel farm ie ohn fa ‘7a hs by ichact? , mn He | Hh ielefa thal: «WL AA Deh sabe Ay ea) a Hin Sra mm Ma wht nitidim Ne, Bilin $e 0) erbr, “va : negt ord. Aiden rane Am @ bom Y OK heder. S4td need 4 vipat by lrg Dp, inh abl nif + Con he Ca, ie) Hs mth. Me eLinefo: Intl sotuben 4 Ki Us : _/ 8 , si ( A a¢ Tat ey ee a Pa, jm tr Liaida , Sinte 3 Hoe hae OL é slip) “a | : Aut hn pom Nylclh, Chelag. f ? ; Pare Ae “ i ogy | (Thi obloius) Letel. hy a fh tol Care He : Wiens ake las Wa ong Be a tub Sohigen / Vishll, Yulptate oS Pacehtman cmentaded b — Me Mincjrm 2 i by Pasa? » Craprration of te tas hbo 2 ofan abs a Fol rt Phist | feral Times ‘mr Te Jie ee usually racnpstabliaaS | in tethanct | (937 / tl ars Adoned 2 bmstat ritbhes pout. Yt som Become tnidtut Hat He predicts rBlsintd acendog To Mis Atal procedure ddpved trarkd ue tated Thie sus af tren percep hl jum He rakten prom munca bf fe ls fe Kee cubtune bac tA ie lest War thro mans fest be ae alels | Nore oven , Me, / oT yl ke rly Lorrobaled ‘wih A de ts ie wir, poat ae koe ees fire shee. To proveh ae F pgitel tins hy whieh Saree te, wiht 2 i i ma, Sas aur ee eS sich sig fel y pi date abhal: nA ee which Showed rape ud b Aight 0 ded Crepil mais drtle vhiel tu Porting showed 0 Sintbing Tae °C a) wulhes 4 fu raiplbrunloel af 22s? C. Hote, theld be cr ko t, tHin Cubes Ae thorrag a hjpead flake aS a D i Hosa mite pont Was sehainas Isa Niele Whe ts ue orechesin a Het aF bed Bo, aS forest Cin Inte feo od, Ca, f. fred f, He Hiylose | 56. ) I¢ i, act. 72, 40t \ manuscript page. J. Bz 3. 3. 13: iboratory; 1¢ c A commemoration at the | The laboratory in winter. 4. 5 ABORATORIUM « MIKROBIGLOGIE 6. Orla-Jensen visiting the laboratory; 1929. 7. Kluyver and a student; 1937- 8. An excursion; 1938. g. Kluyver and Van Niel; 1955; Promoter Kluyver at the promotion ceremony of Prince Bernhard of the Netherland: Prince Bernhard, Dr. Techn. Sci. h.c., and Kluyver after the promotion; 1¢ Kluyver as Rector Magnificus at the Student Union; 1948. 13. 14. Kluyver with two of his sisters; Kluyver as a youth; 1898. | } 1908. On the roof of the Student Union, Sociéteit Phoenix; 1903. Kluyver with his fiancée; 1916. Kluyver and Raden Mas _ Iso Reksohadiprodjo; 1920. The young Kluyver family in the tropics; 1919. é Holidays with his familyJin the Biesbos; 1933. Three generations of the Kluyver family; 1930. Another three generations of the Kluyver family; 1950. 22. Kluyver in his study; 1955. KLUYVER AS SEEN BY HIS PUPILS ‘He is great who is what he is from nature, and who never reminds us of others. But he must be related to us, and our life receive from him some promise of explanation. I cannot tell what I would know; but I have observed there are persons who, in their character and actions, answer questions which [ have not skill to put.’ (R. W. Emerson) To students at the Technological University in Delft the Laboratory of Microbiology used to be known as a quaint old building at the Nieuwelaan, bordering on the canal and adjoining the premises of the student’s Rowing Club; and with most of them knowledge went no further. To the chemistry students it was also one of the laboratories where they might choose to spend their last year before graduating with the Chemical Engineer’s degree. ‘The number of these latter who elect microbiology as their field of specialization has always been rel- atively small, however; students who are interested in things biolog- ical are naturally more apt to enroll in other universities where biol- ogy, instead of being merely an adjunct to the chemistry department, has its own rightful place in the curriculum. The Delft students who have qualified as candidates for the Chem. E. degree and enter the microbiological institute have had a rigorous training in all branches of chemistry, in physics, and in mathematics; but they have no more than a faint inkling of the properties of animate matter. There can be no doubt that their choice has usually been in- fluenced by a more or less conscious dissatisfaction with the inflex- ibility of the behaviour of molecules on the one hand, and on the other by the intriguing opportunity to study a new subject that will expose them to the alluring mysteries of life itself. It is precisely to this frame of mind that the mentality of Kluyver and the atmosphere of his institute were most receptive. Kluyver him- self had probably passed through a similar development during his studies at the Delft university; as a candidate for the Chem. E. degree he had majored in microscopical anatomy, and later he had continued 49 BIOGRAPHICAL MEMORANDA to prepare a doctor’s thesis on a biological subject under the guidance of Van Iterson. Following his appointment to the chair of microbiol- ogy, he had been compelled to master this field by himself, and this, too, must have tended to a certain feeling of kinship with his students. During his student days in Delft he had maintained a close contact with friends at the University of Leiden, which had stimulated his in- terest in biology and widened the scope of his knowledge. Nevertheless, he was proud to be an alumnus of Delft, and thus eminently suited to initiate his students in a field for which their previous training had hardly prepared them. Thus the microbiology novice was at once received into a congenial atmosphere that had been created by the Director and pervaded the entire laboratory. Its most striking feature was the spirit of complete freedom; there were no rules and regulations; it was taken for granted that everybody was enthusiastic, did his best, and behaved as a mature individual, showing responsibility towards the books, the stores, the equipment, and other workers. There were no formal restrictions; in principle everything was and should be possible; if there were obsta- cles, every effort was made to remove them. The only limiting factors that were recognized were the laws of nature, illness being grudgingly included among the latter. The lab was never really closed. The work was carried out in a true holiday spirit, and the Director’s response to a question about vacations, put by a visiting scientist, was as simple and natural as it was exact: ‘My dear Sir, here every day is a holiday’. The building itself, once described by a visiting journalist as a cross between a hunting lodge and a sanatorium, was designed throughout on functional principles to the point of barrenness; every nook and cranny was exploited and adapted to some special use; and the frequent changes, dictated by the needs of the moment, endowed it with a spirit of intensive and efficient internal life. This, together with the odd architecture, managed to create a romantic impression which was further enhanced by the appearance of the eminently prac- tical library rooms, and which reached its climax in the study of the professor, the ‘Olympos’, the centre of all things around which’ the laboratory and the house had been built. That large room had three windows, all facing south with a fine view of the canal. The remaining wall space had been used for the storage of books, reprints, and all kinds of documents. There were also 50 KLUYVER AS SEEN BY HIS PUPILS many photographs of friends, pupils, and colleagues, as well as curios and mementos, such as a replica of a Leeuwenhoek microscope, a stuff- ed kingfisher, and an Iowa State College pennant that often aroused the curiosity of visitors. The room had two doors, one leading to the house, the other to the laboratory. It contained the desk and chair that had belonged to Beyerinck, and it was said that during the first several years Kluyver could hardly overcome his awe, and did not dare use them. A weird assortment of four easy chairs around a small table in a corner of the room, and a huge table with six beautiful, antique, straight-backed chairs around it completed the furniture. It was in the chair at the head of this table that Kluyver did most of his work; when his collaborators were in the study, they installed them- selves to his left and right. Visitors were accommodated in the ‘cosy nook’, offered cigars and cigarettes, and interrogated shrewdly, yet so courteously, that they hardly noticed how much they revealed. Every interview was afterwards recorded on a separate sheet of note paper in a few highly pregnant words or sentences. All the drawers bulged with papers, especially in later years. Kluy- ver received innumerable reports and reprints, and because most of them contained something of interest to him, he could hardly ever bring himself to discard a single one. They were therefore stored in his ‘bar of science’, a specially constructed set of shelves, from which he could always extract the most diverse information pertinent to a particular discussion. The over-all impression of the room was neither one of beauty or academic, awe-inspiring austerity, nor of bohemian carelessness. Its quality was in harmony with its occupant; each object, by its nature and place, possessed a vital and graphic significance. ‘There was noth- ing to hide, and hence it revealed, honestly and unpretentiously, what its owner did and how he did it. It was a workshop that never failed to bring the visitor under its spell, radiating adventure and ac- complishment. No wonder that many of the students felt an instantaneous affinity when visiting the director in his room. This did not occur frequently, however; most discussions with the students took place at the work- bench in the laboratory or in the library, the domains of the students and of experiment. The formal guidance they received was negligible, and consisted mostly in the tacitly implied conclusions that could be 5! BIOGRAPHICAL MEMORANDA drawn from the discussion. Explicit advice or instructions were not given, for Kluyver knew that such direct help does not exert a lasting influence. He strongly felt that the initiative should come from the student; that a solution found by one’s own efforts is of immeasurably greater value than one given by another person. The discussions with the students were informal; seated on one of the low stools Kluyver examined their cultures with a small magnifying glass or under their microscopes, patiently letting himself be informed about recent devel- opments, putting in a question here and there to get a detail straight, and asking for suggestions as if he were the pupil. The next morning the student might find on his desk some reprints that Kluyver consid- ered pertinent; but it happened more often that during the conver- sation professor and student would migrate to the library where, as the search for information went on, pyramids of books and journals would soon accumulate on the tables. In the course of these explora- tions, usually attended by one of the assistants, the facts and argu- ments for and against certain interpretations were clearly developed, and thereafter the student was left to his own devices. Kluyver always groped for the essential aspects of a situation, and, like Beijerinck, in- sisted that an experiment should be simple; simplicity was to him also the best criterion of the merit of an interpretation or theory. Kluyver never used his authority and great experience to drive his pupils in a particular direction, though he tacitly assumed that, as rational beings, they would set their further course by the outcome of these discussions. He delighted in every spark of original thought or experiment. If the students deviated from the approach he had hinted at, he made sure that they were aware of the warning beacons, and then let them pro- ceed at their own peril. Even in cases of extreme stubbornness his exasperation was evident only in private conversations with his asso- ciates, and he refused to interfere. ‘We must have patience. Mr. X has to work out his own salvation; we cannot hand it to him on a platter’. The curriculum requirements included specified minimum periods of laboratory work, satisfactorily completed. Kluyver did not like to be the sole judge of what was ‘satisfactory’, and argued that the main point at issue in determining whether the student had done enough was the latter’s own opinion; ‘his soul had to be satisfied’. He saw no point in keeping students any longer than they themselves considered necessary; those who wanted to stay on were, of course, always wel- 52 KLUYVER AS SEEN BY HIS PUPILS come to do so. They ought to be wise enough to recognize their task, and otherwise to ask for guidance. They were expected to do as much as lay within their capacity. A natural corollary of this attitude towards the student was that those who preferred working under concrete orders in a definite sched- ule had difficulties in adjusting themselves. Some of them never did, and eventually left, disappointed. Kluyver was fully cognizant of this, but it did not make him spoil his game. He was tolerant of defects in scientific education and even in character, provided some enthusiasm and a positive will were evident; but these he neither could nor would supply if his own example failed to engender them. He accepted the fact that, after all, not every student can be sufficiently interested in microbiology to make him devote his major efforts to its pursuit, and he did not respect such persons any the less for it. In this manner Kluyver established an ideal relationship with his students in a very short time. It is hardly surprising that, once they had started to work in his laboratory, they more often than not felt as if they had landed in heaven. Their expectations were realized more completely than they could have dared to hope; for, with the intuition of youth, most of them quickly recognized the greatness of their teach- er. This made it easy for them to acquire a satisfactory knowledge of the elements of microbiological science. Usually the students had al- ready taken Kluyver’s introductory laboratory course in general mi- crobiology in which were included numerous enrichment cultures and pure culture isolations. Moreover, they could attend two series of lec- ture courses, one in general microbiology, the other dealing with one of four special topics that alternated from year to year. In his lectures Kluyver used an inductive approach; from a gradually accumulating body of experimental facts fundamental concepts were evolved. Every lecture was illustrated with an abundance of demonstration material. The greatest care was lavished on the first few lectures of the general microbiology course; for it was here that the sceptical young chemist was first formally initiated into the mysteries of living organisms. Here, the demonstrations included the scrupulously repeated classical experiments of Leeuwenhoek and Pasteur; and their significance, coupled with their simplicity and surprisingly primitive equipment, produced an imposing, nay, even solemn, and somewhat romantic effect. 53 BIOGRAPHICAL MEMORANDA A candidate for the Chem. E. degree was required to submit a de- tailed report of the research he had conducted during his final year, and this report had to be approved by the major professor. Kluyver discussed these reports with his own students punctiliously, from be- ginning to end, sometimes spending more than a full day on a single one. A few days later, professor and candidate met again for the offi- cial examination ceremony where, in the presence of the entire faculty of the chemical technology department, the candidate was subjected to an interrogation of some ten minutes’ duration by his major pro- fessor. On the whole, this examination was a mere formality; but it was characteristic of Kluyver that he always succeeded in making a sporting event of it by subtly creating the impression that the candi- date had to fight for a passing grade. The result was invariably grat- ifying; the ceremony gained in gravity, and afterwards the student could look back upon the performance with far more satisfaction than if he had been convinced that it was something of a farce. The degree earned, the new Chem. E. had to look for a job. Kluy- ver realized that a microbiological education limited to a single year did not suffice to make anyone a full-fledged microbiologist, and he did his utmost to help those who wanted to extend their training and experience by continuing to work in his laboratory. On the other hand, since Kluyver was regularly consulted by various industries, he often acted as an intermediary when job opportunities presented themselves. For the students it was but natural to seek the advice of the master when confronted with such problems; but they never ob- tained a direct answer. Kluyver would survey the available positions and possible careers, interspersed with questions about the inclinations of the person in question. The latter would afterwards go home with his ideas sorted out and his mind already half-way made up. Kluyver seldom went so far as to invite a promising student to stay on as an assistant; he clearly separated the interests of his students from those of the institute; and, even if they declared their preference for contin- uing to do academic work, he first called attention to various possibil- ities here and abroad, only at the end mentioning that a position might perhaps be found in his own laboratory. Then he would dwell in detail on the drawbacks of such a solution, and only hint at the positive aspects. But if the young graduate, after such ‘fair warning’, still insisted on his preference for an assistantship in the Delft institute, 54 KLUYVER AS SEEN BY HIS PUPILS Kluyver saw to it that this signified the start of a greatly intensified education. The first duty of the new assistant was usually the preparation of the demonstration material for the lecture courses. He could lean on an old, hand-written manual, compiled and amended by successive generations of assistants, for planning the execution of the experiments which were required for a particular lecture and which had been out- lined during the special weekly conferences with the professor. Never- theless, the task made heavy demands on the time and capacities of the young assistant. Every week new experiments were needed and even though, aided by the information contained in the manual, he managed to get them started with a minimum of delay, some of them were not immediately successful. The limited time available, with the deadline determined by the time of the lecture, did not always permit of repetitions, so that at the critical moment the demonstration mate- rial might expose more or less serious gaps, a permanent source of worry for the assistant. It was Kluyver’s custom to inspect the results just before the lecture, accompanied by the anxious assistant; he nev- er complained about the bad experiments but singled out for praise those that had come out well; and he could make the assistant feel that the whole success of the lecture actually depended on the latter’s contribution. One may, of course, question the efficacy of demonstra- tions as far as the education of an audience is concerned, and Kluyver himself certainly did not overestimate it. His main reason for perpet- uating the practice was its immense value in terms of the education of the assistant who had a unique opportunity to become familiar with a wide variety of organisms and broaden his practical experience un- der expert guidance. Another important reason was that these exper- iments led to the accumulation of valuable information, covering a wide range of subjects; occasionally they cast doubt on current views, and then yielded problems for more detailed studies afterwards. In addition to the above task the assistant usually conducted experi- ments on a subject of his own choosing, which had been agreed upon during extensive deliberations in which both the assistant and the pro- fessor voiced their ideas. It is not surprising that the great freedom Kluyver allowed his students implied that often a great diversity of problems was being simultaneously investigated in his laboratory. Al- though this widened the horizon, it may have impeded the rapid de- Do BIOGRAPHICAL MEMORANDA velopment of any one specific topic. Again it is clear that Kluyver did not close his eyes to this aspect of his policy; but he consciously did not encourage teamwork during the formative years. He preferred the cultivation of the single individual, who, thrown back on his own resources, could thus engage in a study for which he had evinced en- thusiasm from the start. The same type of guidance was received during the work for the thesis; here too the burden of initiative rested with the candidate, the professor acting the part of the obstinate pupil who had to be con- vinced. When a manuscript had finally been prepared, it was scruti- nized in the most meticulous fashion; every sentence, every word, was weighed and criticized with unassailable reasoning, leading to revisions in text and in the arrangement of data. An excellent impression of this process may be gained from the following description of Kingma Boltjes, which applies to the humblest publication and the most elab- orate thesis alike. ‘During lengthy evening sessions, often extending till deep into the night, the document is critically discussed. Subsequently, Kluyver literally makes an armed attack on the manuscript, while the author, according to his disposition, resists to a greater or lesser extent. But the end result is invariably the same. With a large pair of scissors the product is ruthlessly, though in a carefully considered manner, cut into pieces. The parts are moved about, and much has to be rewritten in order properly to fit the original pieces together into the new pat- tern. Undeniably, the construction thus becomes more logical, and particularly the readability is immensely improved. An attentive reader will readily recognize the publications prepared under Kluy- ver’s guidance by virtue of the numerous distinctive phrases they con- tain; Kluyver himself refers to them as “‘Leitfossilien” or graptolites. During this undertaking no trouble is too great for him; quietly, un- perturbedly, unaffected by time limits, the entire manuscript, down to the last comma, is examined so to speak under a magnifying glass, with the justification that ‘‘Genius is the infinite capacity for taking pains”. When at last the proofs arrive, Kluyver actively participates in their correction, and he thinks nothing of assisting with them till late in the night. Astounding in this connexion is Kluyver’s indefat- igableness. Whereas, by the time of the finish, the writer of a disser- tation is visibly worn out, Kluyver remains cool and collected to the 56 KLUYVER AS SEEN BY HIS PUPILS very end. Only if a number of theses are written in quick succession is it possible that he may show some signs of fatigue; but these he overcomes in an amazingly short time.’ With advancing seniority the assistants gradually became more in- volved in the organizational work of the laboratory and acted as aides to the director in many different spheres. ‘Thus they had an opportun- ity to observe, behind the scenes as it were, how he worked and han- dled people. Kluyver’s working days always started with a perusal of the mail. All ‘easy letters’ were disposed of right away, in later years the answer being dictated to his secretary. Some letters might fall into the cate- gory of ‘procurement of labour’, and these were put on one of the piles on the table, according to urgency and interest. Proofs were im- mediately corrected and returned. The receipt of reprints and other publications was generally acknowledged in a personal note that al- ways contained some cheering and pleasing remarks, in tune with the personality of the addressee. It was hard for Kluyver to use the same text twice, and he detested printed or mimeographed forms. He was always ready to help whenever his assistance was solicited. On the other hand, he was disinclined to beg favours from others, to ‘make a bow’, as he called it, unless it was absolutely necessary. ‘Vhen his letter made it clear that a favour was being asked, and that there was no obligation whatever to fulfil his request, for this was the way in which he himself liked to be treated; as he had learned, it minimized the chances of getting into trouble. No one could ever claim the right to the cooperation of others, and one should be duly grateful if it was granted. All in all, his attitude was reminiscent of the nobleman’s courtesy. He often referred letters to one of his associates for an opinion or to have some details checked before answering them. Now and then such letters indicated the author’s incompetence, or a distressing kind of reasoning. But Kluyver never allowed censorious or deprecatory re- marks to be made on such occasions; disposing of the clumsy aspects of the letter in some poignant statements, he would proceed to explain the material difficulties under which the writer worked, and empha- size his attainments in other respects. Sometimes inquiries were made about details of investigations still in progress, by people working on the same subject. In such cases a BIOGRAPHICAL MEMORANDA Kluyver’s first reaction was always to cite the memorable words of Cornelis Tromp, the Dutch admiral who had proposed to send pow- der and ammunition to his Spanish adversary in order to insure that the battle might go on without an insuperable handicap on one side. Or, to cheer up the associate in question, he would cite the words of Kipling: “They copied all they could follow, but they could not copy my mind, and I left them swearing and stealing a year and a half behind.’ But soon he would face the interests of his own pupils, and the end result was usually a satisfactory compromise. It was a delight to be in attendance when Kluyver was writing a letter or, for that matter, to witness the process of a well-considered argument taking shape in any manuscript from his hand. He used a vocabulary and style that were unique, and had developed, in Dutch as well as in other languages, a distinctive Kluyverian idiom which was apparent also in his speech. It was based on the premise that the meaning or effect aimed at should be inescapably clear, and expressed in phrases of irreproachable grammatical correctness. At the start a captatio benevolentiae, a bow to the reader, was needed to catch his at- tention; at the end a climax to drive the point home. Within this framework Kluyver took advantage of every opportunity to insert his typical, flourishing expressions, usually overstatements, the fruits of quick wit and rich imagination, designed to amplify his intentions with sudden gushes of surprising perspicacity. Not infrequently he spiced his speech and letters with veiled and highly subtle criticisms that were not always immediately grasped, and thus had the effect of a time bomb, producing an unexpected shock when later their true significance began to dawn upon the recipient. Instead of criticizing people to their faces, with the prospect of jeopardizing pleasant rela- tionships, he only supplied the pieces of a jigsaw puzzle from which the listener could afterwards reconstruct the true state of affairs in strict privacy; this eliminated the need for referring to it on later occasions. Praise and encouragement, too, were often wrapped up in the same way, and for the same reason. He liked to tease a little those who could stand it, including an audience or his readers, in order to dispel even the semblance of pomposity. Thus he evoked reactions that could not have been elicited as easily by other means. It has been noticed that many of his pupils unconsciously picked up some of the ways of their master, and could thus be unambiguously identified as such. 58 KLUYVER AS SEEN BY HIS PUPILS He took great pains with his texts; his mastery of languages was im- pressive. Although he had never learned Greek or Latin, nobody would have suspected this. Among foreign modern languages he seem- ed to have a preference for English; and armed with Roget’s “The- saurus’ and the Oxford and Ten Bruggencate’s Dutch-English diction- aries, he liked to hunt for the exact word. He had a predilection for words that appealed to him as expressive, and an aversion to weak ones, such as ‘interesting’ which, he used to say, ‘ought to be used only by one’s aunt in reporting on a lecture she had not understood’. Van Niel was a match for him in linguistic matters, and their collab- oration on the text of the Prather lectures was a delight to both. Although Kluyver was not the ivory-tower type of scientist, he al- lowed his social contacts and family life to usurp as little of his time as was reasonably possible. His wife, bearing the brunt of leading the household and of educating the five children, nobly and unselfishly shielded him from most cares. Kluyver did not like to bother with matters that seemed to him trivial, including the greater part of his personal affairs; and when his decision on some household transaction could not be dispensed with, Mrs. Kluyver frequently had to come to the study and compete for his attention with his collaborators. If, on such an occasion, Kluyver’s interest had been kindled and he started to analyse the matter in the same expansive manner in which he ap- proached a scientific problem, she was quick to pierce his eloquence in her businesslike, matter-of-fact way with some such ejaculation as ‘Don’t show off, Ab!’, thus saving her time and his; and Kluyver gra- ciously conceded the common sense of this attitude. Her vigour, cor- diality, and efficiency enabled her to cope easily with meals or garden parties for any number of guests who often turned up quite unexpect- edly. We may be sure that Kluyver did not take this essential support for granted or that he accepted it as his due; he was slightly apologet- ic about it, with an ever fresh sense of gratefulness. Thus Kluyver could devote virtually all his time and energy to his various tasks. The day was divided into three parts, starting at 9 a.m., 2 p.m., and 8 p.m., respectively, each one terminating at rather in- definite times. Only in later years did he allow himself a short break during the morning when, at 10.30, superb black coffee, much cov- eted by his associates, was served in the study. Kluyver adhered to his schedule with great punctuality, though never demonstratively so. He oo BIOGRAPHICAL MEMORANDA really felt unhappy and guilty if he did not work in the evening, per- haps out of a sense of duty left over from the early years when he had to spend all his time studying in order properly to prepare himself for teaching a science that was new to him. No doubt this attitude was intensified by the feeling that he had incurred the obligation to strive towards attaining a level of excellence such as Beijerinck had also reached by working incessantly. Kluyver had no ear for music, though he listened occasionally to an opera on the radio, gaily humming along. Apart from his interest in tennis and major sports events, he had no ‘worldly hobbies’ to which he could turn for relaxation. His only ruling passion was for work, ‘bound by neither time nor eternity’, and aided by his enormous reservoir of ° physical strength and power of recuperation. He never pampered him- self; and, although appreciative of the good things of this earth, he would not go out of his way to acquire them, and generally adhered to an austere regime. It was only a few months before his death that he confessed to having taken the first after-dinner nap in his life; ‘the colloquium had been so tedious and complicated’ was his apology. He was quite susceptible to the charms of travel. His visit to Rome in connexion with the 6th International Congress for Microbiology had filled him with such admiration and love for the Eternal City that he allowed himself the uncommon luxury of going to the cinema to see ‘Roman Holiday’; a strong additional motive was the fact that the leading actress was of Dutch descent. Nonetheless, it would never oc- cur to him to travel for its own sake. If he went on a short vacation at all, he usually stayed in Holland, most often in the country near Koot- wijk, where there was ample opportunity for walks in the woods, hunting mushrooms and picking blueberries with the children. From the small things in life — a beautiful morning, a subtle joke shared, the first drawings of his granddaughter, Eja, which he proudly displayed in his study — he derived a keen and genuine pleasure. What was the end to which Kluyver dedicated practically his entire life? It is certain that the pursuit of microbiology cannot be the only answer. Of necessity, during the first decade of his professorship this had to be his major occupation; but as soon as his accomplishments permitted, he branched out and attempted to relate his field to oth- ers, giving scope to his wide interests and desire for integration. Con- 60 KLUYVER AS SEEN BY HIS PUPILS sequently his membership in the Royal Netherlands’ Academy of Sciences, where each month he could meet with the outstanding re- presentatives of many branches of the natural sciences, was to him far more than an honour: it provided him with the means of satisfying his real need for a better comprehension of nature and of man. ‘Though he may not have been conversant with the methodological details in other fields, he nevertheless kept abreast of developments in biology, physics, astronomy, geology, and medicine. The extensive knowledge so acquired is reflected in many of his publications, and strikingly ap- parent from the obituary notices of Academy members which he wrote during his tenure of the presidency of this body. In addition he was a member of a whole spectrum of learned societies, committees, and editorial boards, thus encompassing a much broader field than microbiology proper. Although he was a scientist first and foremost, he did not shirk other duties resulting from his professorship, and he did not confine himself to those connected with his laboratory, faculty, or even university. He was deeply interested in the functional aspects of higher education in the Netherlands, and on many occasions, e.g. during his rectorate, his special gifts were naturally put to good use. Apart from contributing to the advancement, organization, and dis- semination of science, he felt it his duty and prerogative as a scientist to keep his finger on the pulse of the development of mankind in the broadest sense. He wanted to be informed about current events in the world at large, and to gain a reasonable interpretation of their mean- ing. To this end he read widely and voraciously, and subscribed to numerous Dutch and foreign journals, including the ‘New York Her- ald Tribune’, ‘Time Magazine’, ‘Biology and Human Affairs’, and ‘Impact of Science on Humanity’. Foreign policy fascinated him, and was often discussed with his associates. He had files of newspaper clip- pings on many matters of general interest, such as the Salk vaccine, and did not consider it beneath his dignity to peruse scientific reports written for the layman, particularly because these often suggested nov- el ways of presentation. Such an interest is comprehensible because he himself had a strong and efficacious inclination towards colourful dis- play, adapted to the audience of the moment, as is evident from many of the slides and charts he had designed. Against this background it is understandable that the second world 61 BIOGRAPHICAL MEMORANDA war and occupation of his country had a terrific impact on Kluyver; in a situation where logic and good intentions were superseded by brute force and malice he could not feel at ease. The real and potential disasters of the war took full possession of his keen mind and sensitive heart; they displaced many things that had previously occupied the forefront of his attention. Kluyver’s attitude towards science and human affairs that was so characteristic of his later years must have been conditioned during this time by the need for a reproportioning and reappraisal of values, opinions, and persons. The war intensified his preoccupation with both the larger issues and the individual. He had even fewer illusions, was more humble and warm-hearted, but at the same time more powerfully outspoken after the experience. Especially during the post-war years Kluyver displayed the qualities of a modern universal scientist who remained in close touch with the outside world. This exacted demands that are colossal in comparison with those required of the medieval scholar who had ample opportun- ity to pursue his own researches and at the same time to give his pupils extensive and profound instruction by merely letting them look over his shoulder. The social position and responsibility of the scientist, the tendency towards exponential development of research, and the dra- matic expansion of man’s technological resources, these were matters to which Kluyver devoted much of his time and energy, and with which he also confronted his collaborators. To the end he kept on learning, and he continued to teach his beloved specialty, microbiol- ogy, in a manner that emphasized more and more its place in the scheme of science and the implications of science for mankind. Free from the narrowness and irresponsibility with which the scientific pro- fession is so often stigmatized nowadays, he restored to it dignity and humanity, and set an example for contemporary and future scientists. We cannot say that Kluyver was compelled by an absorbing passion for truth, any more than that he was primarily motivated by the urge to promote the welfare of mankind. He worked because he liked it, and he was ever grateful for the opportunity given him. In later years he even felt apologetic about the position he occupied, because he thereby prevented a younger man from enjoying and exploiting its wealth of possibilities. He may have thought of his own activities as 62 KLUYVER AS SEEN BY HIS PUPILS pleasant and acceptable occupations, befitting the gentleman he was. Had he chosen to do so, he might have excelled in any one of a num- ber of fields quite remote from microbiology. His self-respect made it impossible for him to set any standards save the highest, the only challenge worthy of his endowments. ‘There was no point and no fun in doing something if it were not done as well as possible. Once engaged in a task, he never backed out; his pride al- lowed of no defeat. And in this manner he cultivated also in others a strong sense of responsibility and loyalty. His keen and critical intellect, sustained by reasonableness and a sense of proportion, made him the type of ‘classical scientist’ whom he characterized in his Washington speech. But there was more. Perhaps the gypsy vein, said to run in his family, expressed itself in the undeni- ably adventurous and romantic traits of his character, and most of all in his intuition and infinitely sensitive receptiveness to human emotion. These elements, unified into a harmonious bipolarity, conferred upon him an uncanny perceptivity through which he experienced reality in all its complexities. In both directions his vision extended all the way to the horizon, and the ominous comprehension of endless possi- bilities could not but produce an attitude of wariness and uncertainty. The need to make decisions was to him one of the crude necessities of life, and a decision once made always remained tentative and subject to an ‘agonising reappraisal’. But living with insecurity did not depress him, nor did it make him cynical. He had accepted it as inevitable, and learned to master vulnerability without becoming immune to it, by adopting a charming formality of manner that nonetheless made it easy to approach him. Through his courage and strength of mind he could keep his balance and act in practical life without having to distort the labyrinth of doubts into a more reassuring though false pattern of ‘truths’. He had no interest in seeking objective perfection — perhaps he was simply bored by it — but rather strove towards finding the best possi- ble solution for a given situation in which the pertinent factors, human as well as others, had been taken into account. His own work, conclu- sions, and decisions bore in his mind a predominantly conditional and experimental character, particularly because they had been fram- ed in a mood bordering on playfulness; to others they may have ap- peared as the last word and unassailable truths. 63 BIOGRAPHICAL MEMORANDA Every scientist knows, or should know, moments of doubt towards his own achievements. It is clear that Kluyver, who perpetually lived with doubt, so to speak, had become an expert in handling those sets of incomplete and obscure data that Nature is wont grudgingly to grant the biologist. He realized as few others that in science it is nec- essary to focus attention on some particular aspect, abstracted from its surroundings. He was a master in the art of emphasizing the cru- cial ones amongst a multitude of facts, and of distinguishing between scientific risks and reasonable interpretations. No wonder that many scientists, who instinctively turn to their neighbours for recognition and assurance, looked to him for appreciation of their work and res- toration of their peace of mind. To them he was a yardstick of achieve- ment; for science is but a frail network of concepts, kept intact by the courageous few who can substitute guiding principles for certitude. And Kluyver was one of them. Of course, his science and profession were constant victims of Kluy- ver’s pragmatic scrutiny, and his sense of humour did not stop at his own person. His self-respect could not be founded on the awe in which laymen may hold the scientist, nor on the esteem of his col- leagues, nor even on the recognition of participating in the improve- ment of the conditions for man’s existence. He was not deluded by the facile fallacy of mistaking results for aims. A clue to Kluyver’s at- titude may be found in a passage of the speech he delivered at a com- memoration ceremony for alumni of the Delft university who had per- ished during the war. Here he groped for the motives that had induc- ed these young people willingly to risk their life in the resistance movement. Vigorously denying that love for their country could have been their main incentive, he ventured to suggest that they had so acted because ‘C’était plus fort que moi’. Similarly, in his lecture enti- tled ‘Homo militans’ he called attention to the simple words, ‘Par honnéteté’, in which Camus formulates the answer to a comparable question in his book, ‘La Peste’. | The essence of Kluyver’s personality was revealed in his relations to people. He had a wonderful propensity for spurring others on to high- er attainments; by emotionally stimulating and coolly criticizing them, he carefully prepared the conditions under which they could develop to the best of their potentialities. He directed by soliciting ad- 64 KLUYVER AS SEEN BY HIS PUPILS vice; he taught by asking questions; he armed himself by refraining from fighting. With just that little more patience than the average, he could manoeuvre himself into the strongest position, and usually man- aged to accomplish what he wanted. He hardly ever worked in the laboratory with his own hands; he used people as well as his pen as his instruments. Indubitably these great talents could have assured Kluyver of great- er and more spectacular successes had he cared to exercise them to these ends. We can only conclude that he had diverted his ambitions from the common path and held uncommon personal views as to what is significant. Turning to Kluyver’s attainments, we may pass over the more ob- vious ones in the field of science, and concentrate on the less tangible effects of his labours. The profound influence he exerted on those around him has been stressed even in articles on Kluyver as a scientist. It was indeed a great and intense experience to be exposed to the ra- diance of his personality. He had acquired the rare ability to transfer effectively the fruits of his experience to other people, a quality per- haps greater than wisdom. He had known the sting of ambition, the tensions of failure and success, of fear and inferiority; but he had come through unscathed, soaring above them with a smile. He had related his knowledge of the ‘infinitely small’ to life as a whole and to man’s place in it. His teaching and research had grown from an end into a means; and by communicating his wisdom to his associates so that it always filled their individual needs, he made a lasting contribution to their education. With the rich expressiveness of his face and subtle speech he could uncover unsuspected potentialities in his pupils, and convey enduring aid and comfort. In this way he also transmitted moral codes that were almost unconsciously adopted by recognition. This powerful influence of Kluyver on his fellowmen has been touchingly described by Senez, who, after Kluyver’s death, wrote: ‘Jai fait a Delft, il y a déja plusieurs années, un trop court séjour, dont je garde un souvenir trés vif et trés précieux. Votre Maitre, Monsieur le Professeur Kluyver, m’y avait recu avec cette simplicité charmante qui était la sienne et les quelques jours passés auprés de lui ont trés profondément orienté ma carriére scientifique. Son rayonnement était immense et il existe dans le monde entier des chercheurs qui se con- 65 BIOGRAPHICAL MEMORANDA sidérent comme ses éléves. Permettez moi de me ranger dans leur nombre et de vous dire combien je suis attaché a sa mémoire.’ Another manifestation of Kluyver’s concern for individuals, char- acteristically cloaked in practical terms, may be found in his after- dinner speech in Washington on ‘An Aspect of the Promotion of Science’. Here he made an eloquent plea for the ‘romantic scientist’, the genius who has difficulties in adjusting himself to society. On the ground that ‘we cannot afford to let scientific geniuses perish’, he ad- vocated the establishment of institutions where these men could work under conditions attuned to their particular needs. Those who have known Kluyver will readily infer that this plea was not based on the stated motive alone, and that he was also, perhaps even chiefly, moved by a direct solicitude for these geniuses themselves. Practicing more than he preached, he extended these ideas to the humblest of his pupils, providing for them an environment where they could thrive and develop to the best of their abilities. This concern for others dominated all his actions, harmoniously im- buing them with added significance in purpose and effect. The re- sponse must have enriched his life, and may perhaps have compen- sated for the utter loneliness that his position inexorably engendered. In any group, whatever its function, he saw furthest, and thus irre- vocably became its focal point. It was not for him to be comforted; he could not afford to let responsibility slip away or to indulge in the easy escape into anger. His perception of the infinite potentialities of life had made him rec- ognize the human mind as the acme of evolutionary development. Hence he believed that man’s particular role in future evolution should rest upon the proper exercise of his spiritual powers. Only a few days before his death he attested to this conviction by quoting a fragment of the words of the Dutch poet, A. Roland Holst, engraved upon the National War Memorial in Amsterdam that had recently been unveiled: ‘Never, from ore to eagle, was any creature free beneath the sun, nor was the sun, nor were the stars. But spirit broke law, and in that breach uplifted man’. Thus, above all else, Kluyver was a man who moved in life’s true 66 KLUYVER AS SEEN BY HIS PUPILS domain, to which science is subordinated, and where success is not measured. And by doing so, he has imparted imperishable gifts to all those who had the great good fortune to cross his path. That he was revered by his pupils and associates is evident; and the reason may be summarized by another quotation from Emerson: ‘This is the key to the power of the greatest men — their spirit diffuses itself’. F.W. Mla R. 67 KLUYVER’?S CONTRIBUTIONS HO MICROBIOLOGY AND BIOCHEMISTRY ‘When Coleridge tried to define beauty, he returned always to one deep thought; beauty, he said, is ‘‘unity in variety’. Science is nothing else than the search to discover unity in the wild variety of nature — or more exactly, in the variety of our experience.’ (JF. Bronowski). INTRODUCTION Tue profound and inspiring analysis of the meaning of science from which the above passage has been quoted would have been readily subscribed to by Kluyver, whose scientific contributions offer a notable example of a perpetual search in that very sense. How successful this search has been is strikingly apparent from a comparison of the sta- tus of biochemical understanding in 1922, when Kluyver entered upon his career as a microbiologist, with that of to-day. In the second of a series of lectures delivered at Harvard University in 1954, Kluyver, after having reviewed the current ideas on biochem- ical reaction mechanisms, could state with full justification: ‘In concluding I should like to give my opinion that, mainly owing to its impressive metabolic diversity, the microbe has made a major contribution to our general insight into the essence of metabolism. There can be no doubt that studies on microbial metabolism have directly fertilized similar studies on animal metabolism in many ways’ [Kluyver and Van Niel, 1956, p. 72]. Nor can there be any doubt that the chief impetus to this develop- ment in our understanding of the essence of metabolism has been the enunciation of the concepts of the ‘unity in biochemistry’ and ‘comparative biochemistry’, both of them based upon the principle of hydrogen transfer as the common and fundamental feature of all metabolic processes. And it is hardly necessary to mention that these are the most far-reaching among the many contributions that science owes to A. J. Kluyver, even though his pertinent publications are but 68 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY rarely cited any longer. This, I believe, should be ascribed to the fact that they have been so completely assimilated that it has become superfluous to reiterate the basis on which present-day biochemistry is founded, just as for over a century chemists have stopped referring to Lavoisier and Dalton except in papers dealing with the history of their science. When, in 1922, Kluyver officially assumed his duties as Professor of general and applied microbiology at the Technological University of Delft, Holland, the knowledge of the chemical activities of micro- organisms was virtually restricted to an awareness of a large number of more or less specific transformations that can be brought about by the diverse and numerous representative types. Except in a few insti- tutions the study of biochemistry itself appeared to consist in little more than the development and application of methods for the analysis of urine and blood. Within ten years Kluyver succeeded in welding to- gether a vast amount of detailed information into a coordinated picture, whose strong and simple outlines encompassed the totality of the chemi- cal manifestations of all living organisms, and whose structure brought into strong relief the dynamic aspects of these processes. In another ten years the direction of biochemical research throughout the world had been guided into the paths mapped out by Kluyver. And the spec- tacular successes scored in enlarging and intensifying biochemical un- derstanding through investigations with appropriate micro-organisms, an approach repeatedly practiced and advocated by Kluyver, had con- vinced an increasing number of biochemists of the potentialities of such studies, with the logical result that the post-1940 biochemical literature has become predominantly occupied by publications on va- rious aspects of microbiological chemistry. These transformations at- test to the great influence exerted by Kluyver’s contributions; verily, it is difficult to overestimate their importance! It is the purpose of this essay to trace the main stages of Kluyver’s scientific development, and to indicate the wide area over which his fertile and philosophical mind was allowed to range during the 34 years of his professorship. It may then become evident that the many honours he received, and the great esteem in which he was held were richly deserved. But it must not be believed that his scientific em- inence alone is responsible for the deep attachment that he engen- dered in his friends, collaborators, and pupils alike. For he also pos- 69 BIOGRAPHICAL MEMORANDA sessed many captivating and inspiring traits that were not directly associated with his scientific endeavour. These, however, fall outside the scope of this paper; nevertheless, those who have had the privi- lege of knowing the Master personally will not lightly forget how much they have benefited from their every encounter with him, and will often be reminded of the treasures they have been permitted to add to their experience. THE PROLOGUE In his inaugural address, reprinted in this volume, the newly appoint- ed professor defended the thesis that some of the chemical activities of micro-organisms might be used to advantage on a commercial scale in order to supply the ever increasing need of an industrialized society for various raw materials. In this manner he could justify the inclu- sion of microbiology in the curriculum of the Technological Univer- sity, and hence his own position. In accordance with precedent this public lecture also embodied some plans for a special research pro- gramme. But it must be admitted that these were extremely vague. This is not difficult to understand; at that time Kluyver was by no means an accomplished or experienced microbiologist. During his ten- ure of the position as assistant to Professor G. van Iterson Jr., from 1910 till 1916, he had, however, evinced a number of attributes that had made his appointment to the chair which Beijerinck had been compelled to vacate on account of having reached the age limit im- posed by law, a sound ‘calculated risk’. Some of these characteristics can be appreciated from an examination of Kluyver’s [1914] major publication prior to that date, the thesis on ‘Biochemical Sugar Deter- minations’ which had earned him the degree of D. Sc. ‘with distinction’, the only manner in which the Technological University, on granting a degree, can express its recognition of superior merit. With the aid of a simple apparatus, somewhat modified after a model originally designed by Van Iterson, he had demonstrated the feasibility of accurately determining the amount of carbon dioxide produced by yeasts during anaerobic incubation with sugar solutions. And, because yeasts can be differentiated in part on the basis of the particular sugars they can ferment, it seemed possible to use a number of judiciously selected yeast types for the quantitative determination of any one of a variety of sugars in mixtures of these substances. 70 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY An extensive study of the properties of a large number of yeast strains led to the selection of a limited group exhibiting the most de- sirable patterns of sugar fermentation. It included two new isolates, described in the thesis as Torula monosa and T. dattila, n.sp.; like the other members of the small collection both had been found to ferment rapidly and completely glucose, fructose, and mannose. But T. monosa appeared to be incapable of fermenting any other sugar, whereas 7. dattila could also ferment sucrose. The additional strains were chosen because they could ferment one or more of the following sugars be- sides the above-mentioned ones: galactose, maltose, lactose, melibiose, and raffinose. For each of these strains the quantitative relationships between car- bon dioxide production and amount of fermentable sugar were next determined; thus it was established that there existed a linear propor- tionality between these quantities over a wide range of carbohydrate concentrations, and that the absolute value of the relationship showed slight but consistent differences between the individual cultures. In this manner the requisite calibration data were obtained; once these were available it was possible to compute the quantity of fermented sugar from the amount of carbon dioxide evolved. Consequently the quantity of glucose, fructose, and mannose combined could be esti- mated from the amount of carbon dioxide produced by T. monosa; sucrose from the extra carbon dioxide formed by TY. dattila; maltose from that evolved by Saccharomyces cerevisiae; and other sugars in like manner from the results of fermentations with other yeasts. During this investigation it was further established that the rafh- nose-fermenting yeasts can be divided into two categories; one group produces from the trisaccharide an amount of gas that corresponds closely with two molecules of carbon dioxide per molecule of raffinose, whereas the other liberates three times as much. The members of the former category, in contradistinction to those of the latter, were shown to be incapable of fermenting melibiose. Even closely related yeast types, such as bottom- and top-yeast, both classified as 8. cerevisiae, can be readily distinguished on this basis; only the former ferments raf- finose completely. The simplicity of the method, coupled with its great specificity, made it eminently suitable for the purpose of quantitative determina- tions of individual sugars in mixtures. Kluyver specifically studied a yl BIOGRAPHICAL MEMORANDA number of applications; he showed, for example, that it can be used to distinguish sharply between commercial jams and preserves pre- pared with the addition of sucrose, and of potato- or corn-sugar, re- spectively. This rests on the fact that the latter materials, produced by an enzymatic hydrolysis of starch, contain a high and reasonably uniform percentage of maltose, so that from the quantities of carbon dioxide produced by TY. monosa (sucrose and maltose negative), T. dat- tala (sucrose positive, maltose negative), and SS. cerevisiae (sucrose and maltose positive), respectively, the amounts of hexoses, sucrose, and maltose can be computed. A similar application was later developed by Den Dooren de Jong for the determination of lactose in bread; this permitted a ready detection of falsification of ‘premium bread’ which, according to the Dutch pure food laws, must be prepared with the addition of a specified minimum amount of milk to the dough. The constant lactose content of milk, together with the inability of baker’s yeast to ferment this sugar, imply that the quan- tity of lactose in ‘premium bread’ is rigorously determined by the amount of milk added. The simple and specific estimation of lactose by the fermentation method thus provides an accurate check. It may be mentioned that in his thesis Kluyver had already hinted at the ap- plication of lactose-fermenting yeasts for the analysis of milk products. Kluyver used his method also for the determination of the specific sugars present in glucosides; in leaves after periods of photosynthesis or storage in darkness; in germinating seeds and various other plant products; and in urine and blood. During these studies two unusual features of one particular yeast were discovered. The established pro- duction of carbon dioxide from pyruvic acid by yeasts led Kluyver to investigate the possibility that other acids, especially those generally encountered in fruits, might also yield carbon dioxide under the in- fluence of certain yeasts. In these experiments it was found that Schizosaccharomyces pombe does, in fact, decompose malic acid under anaerobic conditions, provided a fermentable sugar is simultaneously present. The fermentation of malic acid can then be expressed by the equation: | C,H,O; > 2 CO,+-C,H,OH In experiments on the fermentations in sugar-containing urine samples by various yeasts the observation was made that an initial carbon di- oxide production was occasionally superseded by the eventually com- 72 = oO .CTLCLCT. KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY plete disappearance of the gas. Pertinent tests revealed that this phe- nomenon, again encountered only with Sch. pombe, was associated with the development of an alkaline reaction in the fermented samples. ‘The alkali production could then be traced to the hydrolysis of urea by the yeast. Thus it was ascertained that this species is unique among yeasts in that it can ferment malic acid and possesses a strong urease activ- ity as well. The numerous experiments carried out in preparation for the develop- ment of the method of biochemical sugar determinations had revealed several regularities in the fermentation patterns of different yeasts. These can be summarized as follows: 1. Under anaerobic conditions yeasts can ferment only certain hexoses and oligosaccharides; pentoses, for example, are not fermented. 2. A yeast that can ferment any sugar is always capable of fermenting glucose, fructose, and mannose. 3. Asucrose-fermenting yeast can also ferment raffinose, and vice versa. 4. A maltose-fermenting yeast does not ferment lactose, nor can a lac- tose-fermenting yeast ferment maltose. But although these regularities had consistently turned up in the ex- tensive series of tests, they did not appear to have general validity. There were a number of reports in the literature indicating a contrary behaviour of certain yeast species. None of these reports was, however, based on studies carried out with the Van Iterson—Kluyver apparatus; and in his preliminary investigations Kluyver had become acutely aware of the shortcomings — as well as of the merits — of different me- thods for establishing the fermentative properties of yeasts. In order to be in a sound position to evaluate the contradictory reports, Kluyver therefore secured strains of all the yeasts for which a behaviour had been claimed different from what he had observed, and these cultures were subjected to a careful scrutiny, using a variety of methods. The results will be discussed in some detail because this will provide an opportunity to call attention to certain aspects of Kluyver’s scientific attitude. The relationship between the fermentation of glucose, fructose and mannose by yeasts had been previously noticed. To Kluyver the situa- tion seemed readily explicable in view of the closely similar stereo- chemical configuration of these sugars and the ease with which they 15 BIOGRAPHICAL MEMORANDA can be interconverted through their common enol form. Nevertheless, the reports indicating that some glucose-fermenting yeasts did not fer- ment mannose could not be dismissed offhand; and if such claims could be corroborated it would, of course, have become possible to determine mannose separately by appropriate fermentation tests.* This provided an additional incentive for a thorough re-examination of the yeasts for which a differential behaviour towards glucose and mannose had been observed. The results showed that the previous claims could not be upheld; all these strains fermented glucose as well as mannose. But the new experiments indicated why negative results had sometimes been recorded; this was usually the case when the fer- mentation rate was low, and the tests were conducted in an apparatus in which the inoculated sugar solution was in contact with air. Owing to the relatively high solubility of carbon dioxide in aqueous solutions, coupled with a rapid diffusion of this gas into the atmosphere, an ac- cumulation of sufficient magnitude to cause its appearance in the form of a detectible gas phase might thus be prevented. A corollary of this situation is that the amount of yeast used as inoculum and its rate of growth, hence of fermentation, in the medium supplied may yield spurious results unless a completely closed system is used. Since the Van Iterson—Kluyver apparatus represents such a system, with mercury as the movable barrier, carbon dioxide cannot escape, and will appear as a gas phase as soon as the aqueous solution has become saturated. Under atmospheric pressure this happens when about 1 ml of carbon dioxide has been formed per ml of solution; this requires the fermentation of approximately 4 mg of sugar. If the concentration of the fermentable sugar is above 0.4 per cent, the result ofa fermentation test can never be obscured by a low fermentation rate; eventually gas production will become observable. The test can be made even more sensitive by examining the result under reduced pressure. The empirical rule concerning the fermentation of sucrose and raf- finose also seemed to be challenged by the results of other investiga- tors. But in this case Kluyver’s attitude was perhaps more critical. Raffinose, as a galactose-glucose-fructose trisaccharide, possesses a sucrose configuration, which made the regularity he had observed with his own collection of yeasts readily intelligible if not downright predictable. Abandoning what thus appeared like a logical inference * See also p. 109. 74 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY merely on the basis of a blind acceptance of the uncorroborated claims of others, even if they were eminent authorities, was never a Kluyverian attitude. Although deeply imbued with the overriding significance of experimental data, he was prone to exhibit his pro- nounced critical ability best when such results seemed to vitiate what appeared to be a rational hypothesis on all other counts. And, just as in the above-mentioned cases, a critical re-examination of the proper- ties of yeast strains that had been reported to behave otherwise led to an unexceptional confirmation of Kluyver’s own findings. There was, however, no such basis for the belief that the fermen- tation of maltose and lactose would be incompatible. Thus the rule established could not lay claim to an expectation of general validity. But at the time the literature contained no contrary reports and it was in his own laboratory that the first examples of yeasts that can ferment both maltose and lactose were discovered many years later, when Kluyver and Custers [1940] investigated the yeasts associated with ‘lambic’, a special product of the art of the Belgian beer brewers. ‘To date these Brettanomyces species still represent the only yeasts known to possess this property, and the genus is defined partly on this basis. Finally the thesis contains information pertaining to two other aspects of yeast physiology, viz., the fermentation of galactose, and the problem of the relation between fermentation and assimilation of various sugars. It had long been known that yeasts, although intrinsically able to ferment galactose, sometimes do so only very slowly, or after pro- longed periods of incubation. Various hypotheses had been advanced to account for this phenomenon, and Kluyver conducted some crucial experiments that supported the view that an adaptation process was involved. It was found, for example, that a yeast suspension, prepared from a culture in a galactose-containing medium, fermented galactose about equally as fast as glucose. The most intriguing experiments concerned with this problem were those in which it was demonstrated that at 40°C, a temperature at which growth is precluded but glucose fermentation is not affected, yeasts do not acquire the ability to ferment galactose at all. This established the importance of some growth pro- cess for the development of the new property. Kluyver considered it likely that this would also be reflected in the new-formation of cells, and carried out some experiments to test this. An appropriate yeast 75 BIOGRAPHICAL MEMORANDA was cultivated in a galactose-free medium, and thereafter exposed to this sugar. The appearance of the ability to ferment galactose was determined, and an attempt was made to relate this to growth as determined by cell counts. The results of these experiments were, however, inconclusive; and it was not until 1936 that the careful studies of Stephenson and Yudkin [1936] convincingly showed that the adaptation to galactose can take place in the absence of cell multi- plication. Scientifically the most interesting part of the thesis is that which deals with the studies on fermentation and assimilation of certain sugars by yeasts. It casts a clear light on Kluyver’s unusual ability to perceive the difficulties that may arise out of an uncritical acceptance of experimental results that would appear to be at variance with well- established and rational ideas; to detect potential flaws in experimental procedures; to devise experiments for decisively testing the merits of alternative hypotheses; and to synthesize conflicting interpretations. In 1910 Rose had discovered that Endomyces magnusii, a yeast that readily ferments glucose, fails to grow in Hayduck’s medium* with this sugar as the principal carbon source, yet develops profusely if maltose, which is not fermented by this species, is substituted for the glucose. Shortly afterwards Lindner and Saito, as well as Kita, had found that a number of other yeast species behave similarly. From these results the conclusion had been drawn that glucose cannot, whereas maltose can be assimilated by such yeasts. This situation was anomalous enough to warrant a more detailed investigation. The above-mentioned observations seemed to violate some fundamental ideas on the degradation of oligosaccharides wa the constituent hexose units, and on the significance of fermentation as the ultimate energy source for yeast during anaerobic existence. Evidently Kluyver was reluctant to abandon these concepts, for which a considerable body of sound experimental evidence had been amassed in the course of time, and hoped that it might be possible to find a more satisfactory explanation for the aberrant results. . First of all he repeated and readily confirmed the previous experi- ments of Rose, Lindner and Saito, and Kita. He also found several yeasts among his own collection that exhibited essentially the same * Tapwater, 0.025 per cent MgSQ,, 0.5 per cent KH,PO,, 0.5 per cent asparagine, 5 per cent sugar. 76 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY behaviour. Whereas S. cerevisiae, isolated from the baker’s yeast mar- keted by the Netherlands’ Yeast and Alcohol Manufactory, produced a crop of about 60 mg dry weight per 50 ml of Hayduck’s medium with glucose, several other yeasts grew to the extent of only 1-8 mg, although they yielded crops of 20-70 mg in maltose media. But these results were not considered sufficient by Kluyver to accept Lindner’s interpretation based on a differential assimilation of glucose and mal- tose; he looked for and finally found an explanation that did not con- travene the prevailing and generally fruitful notions on sugar metab- olism. The starting point was Kluyver’s familiarity with the investiga- tions of Bertrand on the often startling effects of minute amounts of certain substances on the development of diverse micro-organisms. This led Kluyver to consider the possibility that impurities in the maltose, rather than the sugar itself, might have been responsible for the good growth of yeasts that failed to develop in glucose media. ‘The validity of this hypothesis was substantiated by experiments with carefully purified maltose; when this was used as a substrate it proved to be as ineffective as glucose for growth of the yeasts. During the recrystallization of the maltose from 80 per cent alcohol Kluyver noticed the appearance of a flocculent precipitate; thus it became likely that it might have been this material that was respon- sible for the profuse growth of various yeasts in the maltose-contain- ing media. Nitrogen determinations carried out with the original maltose, a Kahlbaum C.P. product, confirmed the presence of con- siderable amounts of impurities, indicating as much as 0.22 per cent ‘protein’. Kluyver assumed that this would have been introduced with the amylase, presumably used in the manufacture of the maltose, and that the ‘protein’ was required for the growth of certain yeasts in the asparagine medium. He keenly realized that this should not be inter- preted to mean that asparagine is inadequate as a general nitrogen source, and pointed out that ‘it is readily conceivable that asparagine is unsuitable for the synthesis of particular cellular constituents, such as nucleic acids, for which the nitrogenous impurity can be used... even though asparagine may suffice for the synthesis of various other nitrogenous cell materials’. In principle this interpretation is, of course, the one currently used to explain the need for specific growth factors by diverse organisms, and it is highly probable that the phenomenon Kluyver had here 77 BIOGRAPHICAL MEMORANDA investigated to some extent belongs in the realm of vitamin require- ments by yeasts, the impure maltose being the source of such sub- stances. That Kluyver did not further pursue the problem can easily be understood if it be remembered that the main object of the thesis was the development of a satisfactory method for quantitative sugar determinations. And, although the problem posed by the experiments of Rose, of Lindner and Saito, and of Kita had to be disposed of in order to provide a solid foundation for an interpretation of the sugar fermentation data, it was certainly not necessary for Kluyver to at- tempt a chemical characterization of the impurities that exerted so marked an effect on yeast growth; it was quite sufficient to show that maltose itself could not be assimilated under conditions where glucose is not. This much may be granted. But it does not account for a curious omission in rounding out this investigation, and it is interesting to speculate on the significance of this fact. Kluyver had demonstrated that recrystallized maltose is not assimilated, and that the original preparation contained an alcohol-precipitable impurity; he had as- cribed to this impurity the striking effect of maltose on yeast growth. Now it must be evident that a simple experiment could have provided direct support for this hypothesis; it would have consisted in testing the growth of yeasts that responded in the characteristic fashion to the impure maltose also in asparagine media with glucose, supplemented with the flocculent precipitate. There is, however, no indication in Kluyver’s publication that this logical experiment was ever perform- ed. Does this imply that he had overlooked so obvious and simple a check? Not necessarily; it is at least equally probable that the experi- ment was carried out but, contrary to expectation, had yielded neg- ative results. On the basis of present-day knowledge of yeast nutrition such a negative result could be predicted with a high degree of proba- bility, because it is almost certain that vitamins of the B-group were involved as growth factors for the yeasts under investigation. It is quite reasonable to suppose that these substances were present in the impure maltose. Evidence to this effect is also furnished by the fact that Kluyver failed to observe an improvement of yeast growth in Hayduck’s medium with glucose to which 0.1 per cent ‘Witte’ peptone had been added. Now the B vitamins would not have been found in the alcohol-precipitable fraction; hence this material, too, would have 78 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY been ineffective in promoting growth. And confronted with a nega- tive result Kluyver may well have felt that it would be advisable not to mention it in order not to introduce facts that could more easily detract from the main point than help to explain it. This would have been sound scientific strategy and prudence; and Kluyver often made use of it when experiments in his laboratory yielded results that ap- peared to embarrass the orderly development of a particular investiga- tion. The only experiments in which it was shown that the various yeasts can grow abundantly at the expense of glucose pertain to yeast extract media. These had also been found satisfactory by Rose and by Lindner and Saito; but these authors had accounted for the good growth of their yeasts in glucose-yeast extract solutions by invoking a putative presence of glycogen in these media. Kluyver pointed out that this explanation fails to account for the extremely poor develop- ment of such yeasts in yeast extract without glucose, and thus con- cluded that, in an otherwise appropriate medium glucose can be as- similated as well as fermented. It may here be mentioned that this early encounter with conflicting results depending on the use of pure or impure maltose preparations later aided Kluyver and Hoogerheide in dispelling an apparent anomaly in the behaviour of certain yeasts towards maltose under aerobic and anaerobic conditions, respectively. When Trautwein and Weigand had announced that $. marxianus and S. exiguus, although unable to ferment maltose, could nevertheless oxidize this sugar, Kluyver felt that this was incompatible with the views he had mean- while developed concerning the relation between fermentative and oxidative metabolism. Hence a re-investigation of the situation was undertaken in collaboration with Hoogerheide [1933a]. From this it appeared that the rate of oxygen utilization by these yeasts in maltose solutions was dependent on the sugar concentration at levels far exceeding those at which glucose shows a similar effect; that in 10 per cent maltose solutions the rate drops sharply after relatively short periods of incubation during which the maltose concentration could not have been lowered appreciably; and that a 10 per cent maltose solution previously freed from fermentable monosaccharides by anaer- obic incubation with a yeast that cannot ferment maltose no longer causes an oxygen consumption noticeably in excess of that observed in sugar-free suspensions of the two yeasts. 79 BIOGRAPHICAL MEMORANDA But it must also be remarked that it has subsequently been shown that some yeasts can actually grow at the expense of disaccharides only in the presence of air, whilst their development in the absence of oxygen is restricted to media containing a fermentable mono- saccharide. In fact, Kluyver and Custers [1940], during a reinves- tigation of the differential behaviour of yeasts towards disaccharides under aerobic and anaerobic conditions, established that Brettanomyces anomalus, Candida parakrusei, T. dattila, and T. utilis can grow with maltose as the sole carbohydrate only in the presence of air, and that some other yeast species exhibit a similar pattern with respect to sucrose and lactose. THE EMERGENCE OF A PROGRAMME Whatever importance we may attach to these studies on yeast physiol- ogy and their application, it must be realized that they constituted Kluyver’s only contribution to microbiology, and practically the en- tire extent of his microbiological experience up to the time of his succession to Beiyerinck’s chair. Hence it is understandable, as already remarked in the introduction, that in his inaugural address he could only hint at some general lines of investigation, and these quite vaguely outlined, that might be pursued in his institute. During the first several months of his directorship he was occupied exclusively with preparing a background of knowledge of the field that would at least permit him to expound the fundamentals of microbiology in his courses, and to guide in an intelligent manner the work of the students who came to work under his direction. During this preparatory phase he received much support from the two assistants he had inherited from Beiyerinck, L. E. den Dooren de Jong and H. J. L. Donker. In later years he was wont to acknowledge the debt of gratitude he owed them, especially the former, who had acquired a thorough training under Kluyver’s famous predecessor, and possessed a considerable and integrated knowledge of the micro-organisms. Needless to say, Kluyver was an apt pupil. . The first impetus to the development of a more specific programme was provided by the fortuitous isolation of an unusual vinegar bac- terium. The isolation of vinegar bacteria from beer that had become acid after exposure to the air was one of the early experiments by 80 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY which new students were initiated in the mysteries of microbiological techniques. The morphological and physiological properties of the isolated pure cultures were then further investigated, which generally permitted their identification as members of the Acetobacter rancens group in the sense of Beijerinck. But it so happened that in 1923 F. J. G. de Leeuw, after streaking such a beer culture on a malt ex- tract-calcium carbonate-agar plate, obtained results strikingly different from those commonly encountered. The developing colonies caused an initial dissolution of the carbonate, due to acid production, that was far more pronounced than what was generally observed with cul- tures of A. rancens. Furthermore, the cleared areas gradually became riddled with well-developed and large crystals, in such abundance that it was relatively easy to obtain them in sufficient quantity for chemical analysis. The crystals were found to be composed of a slight- ly water-soluble calcium salt, readily soluble in dilute acid, from which solutions the original calcium salt could be reprecipitated by neutralization. This provided a simple and effective method for the purification of the material. The pure substance reduced Fehling’s solution in the cold, and was eventually identified as the calcium salt of 5-ketogluconic acid, a product of bacterial glucose oxidation that had been discovered in 1887 by Boutroux. A detailed study of the physiological and biochemical properties of the newly isolated bacterium [Kluyver and De Leeuw, 1924] re- vealed it to be a typical vinegar bacterium. It was named A. suboxy- dans to emphasize its pronounced tendency to perform only incom- plete oxidations. Thus it was found to oxidize simple primary alcohols to fatty acids, secondary alcohols to ketones, poly-alcohols to their keto-derivatives, aldoses to aldonic acids, and, if the latter possessed the requisite configuration, beyond this stage to the corresponding keto-acids. Many of these properties are shared by A. xylinum, Bert- rand’s ‘sorbose bacterium’, but the latter, in contrast to A. suboxydans, usually proceeds to oxidize the products of incomplete oxidation, after these have started to accumulate, so that ultimately only carbon di- oxide and water are produced. On account of its inability to effect a further oxidation of acetic acid, ketoses, etc., A. suboxydans could thus be regarded as the ideal acetic acid- and ‘sorbose’ bacterium. It has frequently proved its use- fulness for the preparation of ketoses from the respective poly-alcohols, 81 BIOGRAPHICAL MEMORANDA a procedure that was patented by Kluyver [Kluyver and Visser ’t Hooft, 1931]. The efficacy of such processes is attested to by the fact that the commercial production of sorbose, an important intermediate stage in the commercial manufacture of ascorbic acid by the Reich- stein method, is universally accomplished with cultures of this or- ganism. But the discovery of A. suboxydans was even more important because of the consequences it entailed for the evolution of Kluyver’s bio- chemical outlook. First of all, it had become apparent that the phys- iological group of the vinegar bacteria comprizes a number of repre- sentatives that are distinguishable by the extent to which they can oxidize various substrates. And the possibility of arranging these rep- resentatives in order so that the metabolic end products of one type can still serve as oxidation substrates for the subsequent ones further suggested that generally the stepwise oxidation of substrates might be analyzed with the aid of such a series of strains which would permit the isolation of the consecutive oxidation products from cultures with pro- gressively increasing oxidative capacity. Thus came about the desire to study in a comparative manner the biochemical activities of va- rious groups of micro-organisms. That Kluyver anticipated a considerable clarification of our under- standing of metabolic processes from such studies is clearly evident from the lecture he delivered before the Netherlands’ Chemical So- ciety in 1924. The major part of this lecture on the ‘Unity and diversity in the metabolism of micro-organisms’ was devoted to a discussion of the diversity; it could hardly have been otherwise because so little could be said at that time about a possible unity. Nevertheless, this aspect was also broached, and two different approaches were reviewed in some detail, viz., the energetic and the chemical ones. It is un- necessary to summarize the contents of this lecture which is included in this volume; suffice it to say that it illustrates the primitive state of understanding of biochemical phenomena in general. But it may not be superfluous to emphasize Kluyver’s insistence on the unifying effect that energetic considerations could exert on our interpretation of bio- chemical processes, equating, for example, all known cases of the so- called dissimilatory reactions, fermentative as well as oxidative, inas- much as they represented the mechanisms whereby energy is made available for the synthetic or assimilatory, and hence energy-requiring 82 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY functions. The importance of this attitude is that it made Kluyver recognize, already at this time, the fundamental problem of the man- ner in which the energetic coupling is brought about. The problem was posed in the form of a question; are the chemists familiar with cases in which two reactions that do not have any components in common can be energetically linked? From time to time Kluyver returned to this problem, and, as will be shown later on, he soon found himself in a position where he could make a significant contribution to its solution. It also seems desirable to call attention to the fact that the im- portance he attributed in this lecture to the outcome of some exper- iments on the nutrition of A. suboxydans now seems rather misplaced. These experiments had shown that the new bacterium could grow only in media containing, besides an oxidizable substrate, complex nitrogen compounds, such as are found in peptone or yeast extract, with one notable exception: in the presence of mannitol growth was observed in an otherwise mineral medium with an inorganic am- monium salt as the nitrogen source. From these results Kluyver ten- tatively concluded that perhaps the decrease in free energy during the first stages of the oxidation of mannitol and glucose, respectively, might be sufficiently different to enable the bacteria to utilise am- monia nitrogen for the synthesis of protein only during mannitol oxi- dation. We now know that A. suboxydans requires para-amino benzoic- and pantothenic acid for growth, so that it seems reasonable to as- cribe the positive result of the culture in the mannitol-mineral medium to an inadvertent contamination with these substances. But in mineral media supplemented with the two growth factors growth also occurs if, for example, glucose or gluconic acid is supplied as the main carbon source. Hence the arguments about the interrelations between carbon and nitrogen nutrition cannot any longer be taken seriously. In spite of this criticism these very arguments are interesting because they show that Kluyver was already beginning to think in terms of what gradually evolved into the concept of ‘comparative bio- chemistry’. The parallel drawn between the limited oxidative capa- city of A. suboxydans with its requirement for complex nitrogenous com- pounds on the one hand, and diabetes as a metabolic disease on the other may have been wrong in detail; yet it embodies the principle that, in the late ’thirties, came into prominence when the nature of 83 BIOGRAPHICAL MEMORANDA growth factors and their function in specific biochemical reactions began to be understood. Furthermore, the use of the various Acetobacter types may not have aided significantly in elucidating the consecutive steps involved in biological oxidations, but it cannot be denied that the approach Kluyver had suggested in 1924 is fundamentally the same as that which, more than two decades later, was employed with such spectacu- lar success in the analysis of biochemical mechanisms, and particu- larly biosyntheses, with mutant strains of micro-organisms that could be graded in a sequence similar to the one Kluyver had used in ar- ranging the different types of vinegar bacteria. During this same period Donker [1924], in a biochemical investigation of the anaerobic metabolism of Bacillus polymyxa, a sporeforming bac- terium frequently found in jars of improperly home-sterilized vege- tables, recognized the sugar fermentation of this organism as strikingly resembling that of Aerobacter aerogenes, whose fermentative metabolism had been clarified by the studies of Harden, Walpole, and Norris. This outcome showed that metabolic processes of the same kind may be encountered among organisms of different morphological groups, and hence to the tantalizing notion that such instances may be enor- mously useful in developing new and badly needed guides for solving taxonomic problems. The possibility of considering B. polymyxa as the equivalent among sporeforming bacteria of the non-sporeforming Aerobacter species was the justification for proposing a new generic rank for the former, with the name Aerobacillus to indicate its metab- olic relationship. Together with the differentiation of the genus Acetobacter along bio- chemical lines, this development has undoubtedly been responsible for Kluyver’s important contributions to the field of the classification of micro-organisms, and to the gradual emergence of ideas that have decisively influenced the trends in this field. THE UNFOLDING OF THE PROGRAMME In the course of the next two years the above-mentioned studies with the acetic acid bacteria and Aerob. polymyxa led to a development that signified one of the truly great advances in biochemistry. The recog- 84 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY nition of the stepwise oxidation processes, supporting the notion of differences in degree of oxidative capacity, along with the discovery of identical modes of sugar fermentation among different groups of bac- teria, these were the starting points for the inception of a theory of metabolism that soon permitted a unified and gratifyingly simple interpretation of metabolic processes in general. Following Donker’s studies with Aerob. polymyxa, a beginning was made with a coordination of the then existing knowledge of microbial fermentations. Kluyver’s outspoken desire to “discover unity in the wild variety’ soon made him perceive that all these processes can be considered as composites of a small number of major reaction types, characteristically displayed by specific groups of microbes. In the first publication on this subject eight groups of organisms were so differen- tiated, and these were shown to comprise only five fermentation pat- terns, as indicated by the products formed. Now the possibility to arrange the acetic acid bacteria in order of ascending oxidative capacity was based on the fact that the metabolic end products of one type could be considered as intermediate products of the subsequent types because the latter can further oxidize these compounds. This raised the question whether it might not be possible to use a similar approach in attempting to systematize the extant knowledge of fermentation processes as well. An auspicious support for such a possibility was the recognition that in diverse fermentations acetaldehyde had been postulated on good grounds as a more or less constant and crucial intermediate product; in fact, Neuberg had expressed the idea that sugar fermen- tations by various micro-organisms follow very similar paths. A survey of what was known and surmised with respect to different fermenta- tions soon led Kluyver and Donker [1924a] to a synthesis that held out great promise for a unified interpretation. The extent of this syn- thesis can best be appreciated by considering the general pattern that emerged, and that was based on the assumption of not more than four specific reaction types, as follows: z. A primary reaction in which the hexose molecule is split into two triose moieties. 2. Transformation of the latter, either to lactic acid, or to formic acid and acetaldehyde. 3. Dehydrogenations of formic acid and acetaldehyde. 85 BIOGRAPHICAL MEMORANDA 4. Condensation of acetaldehyde to acetyl methyl carbinol, or, vza acetaldol, to butyric acid; and of acetic acid to aceto-acetic acid followed by a decarboxylation to acetone and carbon dioxide. The dehydrogenations mentioned under 3 were depicted as pro- ceeding under the influence of the protoplasm of the fermenting cells, yielding an oxidation product and ‘protoplasm-H,’. Realizing that the protoplasm would have to be continuously regenerated from its reduced state, two types of suitable mechanisms were envisaged, viz., a. regeneration through the elimination of molecular hydrogen, and b. by means of an interaction with a reducible compound, 27.e. an acceptor molecule. Seven different examples of reactions of the latter type were listed in which triose, lactic acid, acetaldehyde, acetyl methyl carbinol, bu- tyric acid, acetone, and fructose, respectively, were reduced to the corresponding products, glycerol, propionic acid, ethanol, 2,3-butyl- ene glycol, butanol, iso-propanol, and mannitol, each one encount- ered in some fermentation process. The possibility was considered that an additional dehydrogenation reaction, of triose to pyruvic acid, might have to be invoked. But this was not deemed necessary because the occurrence of pyruvic acid as an important intermediate product had not yet been satisfactorily estab- lished. Kluyver and Donker pointed out that the formation of acetal- dehyde and carbon dioxide could equally well be represented by the reaction sequences, (1) f C,H,O,-+ ‘protoplasm’ — C,;H,O,-+‘protoplasm-H,’, and \ C,H,O, > CH,CHO+CO,; or (2) f C,;H,O, ~ HCOOH-+CH,CHO, and \ HCOOH- ‘protoplasm’ —> CO,-+‘protoplasm-H,’ ; the net result of these being obviously identical. A preference for one or the other would consequently have to be based on special consider- ations. Nevertheless, between the writing and proofreading of the paper such arguments had apparently occurred to the authors be- cause in a footnote the remark has been inserted that a continuation of the study has made the earlier mentioned extension desirable. Various combinations of these four simple reaction types could thus be used to interpret all known fermentation processes as the result of specific differences in the fate of the three key intermediate products, 86 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY triose, pyruvic acid, and acetaldehyde. This not only revealed the fund- amental similarity and interrelations between the diverse fermenta- tions, but it further suggested that these reaction types might also ac- count for some peculiar phenomena, incidentally discovered during studies on fermentations under special conditions, but never before in- tegrated with the normal fermentation patterns. Notably the ‘phyto- chemical reductions’ belonged to this category. Neuberg had coined this term to designate collectively the reduction of such utterly un- related and ‘foreign’ substances as, e.g., sulphur, methylene blue, or nitrobenzene, yielding hydrogen sulphide, leuco-methylene blue, or aniline, when the former were added to a sugar solution undergoing fermentation by yeast. It now seemed reasonable to interpret such reductions as the result of alternative regeneration reactions of proto- plasm, the added substances competing with acetaldehyde which nor- mally is exclusively involved in the regeneration process. ‘This concept suggested the general possibility of deliberately steering the fate of a particular intermediate product into channels not ordinarily followed, by creating special conditions during a fermentation that would inter- fere with the conventional reactions. A direct experimental verifica- tion was provided by experiments that were based on the fact, shown by Neuberg and Reinfirth in 1923, that yeast produces acetyl methyl carbinol if acetaldehyde is added to a fermenting sugar solution. ‘The carbinol is not a normal product of alcoholic fermentation, presum- ably because in this process the sole regenerating reaction involves the quantitative reduction of aldehyde to ethanol. But this should imply that under conditions that bring into play additional regeneration reactions, such as the ‘phytochemical reductions’, the aldehyde should partly escape reduction; and this fraction should therefore undergo a condensation to carbinol, which in turn might be further reduced to 2,3-butylene glycol. Appropriate experiments with yeast-sugar mix- tures to which methylene blue or sulphur had been added yielded the expected results. Subsequently Kluyver, Donker, and Visser *t Hooft [1925] showed that aeration of a yeast suspension in a sugar solution also gave rise to carbinol production; this result was anticipated be- cause it seemed likely that a regeneration of protoplasm could also be accomplished by a reaction of ‘protoplasm-H,’ with oxygen. Similarly those representatives of the true lactic acid bacteria in the sense of Orla-Jensen that normally produce, in addition to lactic acid, 87 BIOGRAPHICAL MEMORANDA carbon dioxide, acetic acid, ethanol, and glycerol, and had therefore been designated as ‘heterofermentative’ by Kluyver and Donker to distinguish them from the ‘homofermentative’ members that yield only lactic acid, could be induced to form acetyl methyl carbinol by cultivating them in fructose solutions. Such a behaviour had been anticipated as a consequence of the fact that the heterofermentative lactic acid bacteria were known to produce mannitol in fructose-, but not in glucose media. This mannitol formation could be considered as a particular case of a phytochemical reduction, and its occurrence would again prevent an equivalent reduction of acetaldehyde with its consequent condensation to carbinol. Thus the sugar fermentations by different organisms had been inter- preted as the net result of a small number of interrelated step reactions. The dynamic nature of these processes had been emphasized by show- ing that the fermentation pattern is not fixed but can be modified as a result of changes in the environmental conditions, and support for at least some of the postulated events had been provided by ingeni- ously contrived experiments. But even in the first paper the analysis had already progressed well beyond this point. The impetus to a further extension had been the earlier mentioned investigation of the vinegar bacteria. Ever since 1913 Wieland had advocated the idea that the oxidation of ethanol to acetic acid by these organisms should be interpreted as a mechanism involving primarily the transfer of hydrogen atoms from the oxidation substrate, ethanol, and subsequently from its first oxidation product, acetaldehyde (in its hydrated form), to oxygen. The discovery of the various incomplete oxidations performed by A. suboxydans had con- vinced Kluyver that Wieland’s theory was equally applicable to these oxidations. Hence, when it appeared that some of the central reactions in fermentations could be regarded as dehydrogenations of inter- mediate products by protoplasm, it logically followed that Wieland’s concept could also be invoked to account for these processes, and that the subsequent regeneration reactions of the reduced to the oxidized form of protoplasm, too, were essentially similar in nature. ‘Thus, not only the oxidations characteristic of the acetic acid bacteria, and, by extrapolation, all other biological oxidations, but even the majority of the step reactions in fermentations could be interpreted as specific 88 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY instances of hydrogen transfer mechanisms; and even the condensa- tion reactions appeared amenable to the same interpretation. This general theme was developed in more detail in a later pub- lication by Kluyver and Donker in which an array of arguments was presented to show how this concept of hydrogen transfer as the funda- mental feature of all metabolic processes could lead to yet further simplification and unification. Here it was also pointed out that, examined from this viewpoint, the apparently fundamental difference between fermentative and oxidative metabolic processes disappeared, and that the latter should be regarded as special cases of regeneration of protoplasm from its hydrogenated state through the transfer of the excess hydrogen to molecular oxygen. And the next step resulted in a simple and appealing synthesis of the conflicting ideas on biological oxidations that had been developed by Wieland and by Warburg, respectively. As mentioned, the former had interpreted biological oxidations as representing primarily instances of activation of sub- strate-hydrogen by biological catalysts, and had shown that they may proceed in the absence of oxygen provided a suitable alternative hy- drogen acceptor be present. Conversely, Warburg had long empha- sized the drastic inhibitory effects of substances such as cyanide, car- bon monoxide, and hydrogen sulphide on biological oxidations, and defended the thesis that these effects were compatible with the inact- ivation of iron-containing catalysts whose mode of action was con- cerned with the activation of molecular oxygen. Kluyver and Donker now showed that the presumably incompatible theories of Wieland and Warburg were complementary, and, in fact, were both needed to explain the dual aspects of the mechanism of biological oxidations which should involve, at one end, an activation of substrate-hydrogen (Wieland’s postulate), and, at the other, activation of molecular oxy- gen (Warburg’s theory). Almost simultaneously, and quite independ- ently, von Szent Gyorgyi in studies with plants, and Fleisch in studies with animals, had advanced the same idea. Nevertheless, it may here be stressed that Kluyver and Donker had arrived at the most general formulation in demonstrating that the Wielandian principle can be used for the interpretation not only of biological oxidations, but of any and all metabolic phenomena, whereas the Warburg principle is applicable only in those cases where oxygen is the final oxidant. Once this universal synthesis had been achieved, the problem of 89 BIOGRAPHICAL MEMORANDA biocatalysis itself was tackled. Because the essence of every metabolic process appeared to be hydrogen activation, and because Kluyver was prone to search for the ultimate in simplification and generalization, he concluded that one single property might suffice to account for the observed differences in the metabolic behaviour of diverse organisms. The most obvious feature that fulfilled such a requirement was a spe- cific ‘affinity’ of the protoplasm for hydrogen. The idea was developed as follows. A catalysed reaction involves the straining of certain bonds in the molecules of the reacting sub- stances, a ‘dislocation’, as Boeseken had termed it. Now, differences in the affinity for hydrogen of the protoplasm of different organisms would cause different degrees of such dislocations, which could ex- plain the variety of reactions that substrates and intermediate products can undergo under the influence of diverse organisms. An attractive aspect of this concept was that it eliminated the need to postulate, as was commonly done though without adequate supporting evidence, the occurrence in different cell types of a large number of haphazard- ly distributed enzymes. It was further pointed out that the same de- gree of dislocation in a molecule containing both hydrogen and oxygen atoms can result from a specific activation either of hydrogen or of oxygen, and that a definite affinity for hydrogen is equivalent to a reciprocal one for oxygen. Thus a catalyst with a high affinity for hydrogen possesses ipso facto a low one for oxygen, and vice versa. ‘This also means that a substrate may be dislocated to a comparable extent by catalysts that possess either a high or a low affinity for hydrogen, so that the metabolic processes of characteristically aerobic organisms, representing a high affinity for hydrogen, may come to resemble those of the most anaerobic types, effecting an equivalent substrate activa- tion through their high affinity for oxygen. Indeed, this affinity might even be so great that molecular oxygen would become firmly bound to the catalyst, thereby rendering the latter inactive for further sub- strate activation. This corollary provided a plausible explanation for the deleterious effect of oxygen on obligatory anaerobes. It is superfluous to enter here into a detailed discussion of the appli- cation of the principles of hydrogen activation and transfer to a large number of particular metabolic processes; this can be found in the classical paper by Kluyver and Donker, ‘Die Einheit in der Bio- chemie’, that has been reprinted in this volume. go KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY FURTHER DEVELOPMENTS: PHOSPHORYLATION AND THE NATURE OF ASSIMILATION The successes scored in interpreting the vast diversity of metabolic processes in a simple and unified manner understandably induced a desire more rigorously to test the applicability of the principles to spe- cific cases. Among the first to be so investigated was the mechanism of alcoholic fermentation. In part this may have been owing to Kluy- ver’s earlier preoccupation with this fermentation; but in part it must also be ascribed to the recognition that an as yet mysterious and un- explained phenomenon had been observed in this fermentation. The concept of hydrogen transfer had already made it possible to integrate the carbinol condensation and the phytochemical reductions with the more normal reactions. And the one aspect that had so far remained outside the scope of speculations now promised to shed some further light on the introductory conversion of a fermentable hexose molecule into the postulated two triose molecules. For nearly twenty years it had been known that the fermentation of sugar by yeast press juice or maceration juice is accompanied by the intermediate formation of hexose phosphate esters, and on the basis of quantitative experiments Harden and Young had established a rela- tionship between the formation of carbon dioxide and alcohol on the one hand, and of hexose phosphate on the other, that could be ex- pressed by the equation: 2C,H,,0,+2H,PO, > CH,O,(PO,H,).+2H,0+2CO,+2C,H,0H However familiar we may be with the current interpretation and sig- nificance of this equation, it should be remembered that in the ’twen- ties it did no more than paraphrase an over-all result, and that, taken at face value, it might suggest that concomitantly with the esterifica- tion of one sugar molecule another one was broken down to carbon dioxide and alcohol; a rational interpretation had not been suggested. It is equally significant to realize that during the early *twenties great strides had been made in our understanding of the structure of sugars; Haworth, Irvine, and their collaborators had established the ring structure of hexoses. It was this aspect that caused Kluyver to look for a potential connexion between the act of phosphorylation and the subsequent rupture of a hexose molecule into two triose moieties gi BIOGRAPHICAL MEMORANDA which might furnish an intelligible interpretation of the Harden and Young equation. Following the same line of reasoning that had been so successful in making metabolic processes more easily comprehensible as a result of the dislocation of substrate molecules under the influence of catalysts with appropriate affinities for hydrogen, it soon occurred to him that the ring configuration of a sugar, representing by far the most stable one, might prevent an effective attack by a biological cat- alyst, and that a primary modification of the structural pattern might be a prerequisite for initiating a subsequent break of the molecule be- tween the third and fourth carbon atoms. In coupling this notion with the equally plausible one that Harden and Young’s ester, as a hexose diphosphate, would probably be formed as a result of two con- secutive processes, involving hexose monophosphate as a first product, the concept emerged that the formation of this monophosphate per se might be the means by which the structural modification was accom- plished. Due to the strongly polar nature of the phosphate group the first phosphorylation would cause a redistribution of various bond strengths, and by assuming that this esterification involved the hy- droxyl group at carbon atom five after opening of the ring, a case could be made out for the specific weakening of the central carbon- carbon bond, as indicated in the following diagram: i O=-POr Ua where the dotted lines represent weakened, the dash-dotted ones rein- forced bonds. As a consequence of the specific activation of hydrogen attached to carbon atom four, this hydrogen could then be transferred to carbon atom three, at the same time causing a rupture of the car- bon chain. This ingenious hypothesis implied, therefore, a reaction sequence of the kind, (1) C,H,,.0,+H,PO, > C,H,,O;PO,H,+H,O, and (2) CgH,,O;PO,H, — C3H;0,PO,H, + C3H,Os, in which a triose molecule is generated alongside a triose-phosphate molecule as a direct consequence of the primary esterification. Under 92 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY the dislocating influence of the protoplasm the triose would be rapidly converted into carbon dioxide and alcohol; the triose phosphate would first have to be de-esterified before it, too, could yield the final fer- mentation products. By further postulating that the rate of hydrolysis of the triose phos- phate by yeast juice is smaller than the rate of formation of hexose monophosphate, of its decomposition to triose and triose phosphate, and of the triose breakdown, a result was obtained that was in full agreement with experimental observations. In the first place it ex- plained why the sugar decomposition was inextricably linked with the formation of phosphate esters; the latter served in effect to prepare the sugar molecule for its eventual breakdown. Secondly, the rapid for- mation of triose phosphate and triose from the hexose monophosphate, and the equally rapid disappearance of the triose, served to account for the fact that, as Harden and Young had established, per molecule of esterified phosphate one molecule of carbon dioxide and ethanol each were generated. Thirdly, the low rate of hydrolysis of the triose phosphate would cause this product to accumulate, so that gradually an increasing amount of inorganic phosphate would become tied up; following its complete disappearance the rate of production of carbon dioxide and ethanol must needs become dependent on phosphate lib- eration and the simultaneous triose formation by the dephosphoryla- tion reaction. Fourthly, the accumulation of triose phosphate, accord- ing to the proposed mechanism of its formation possessing the struc- ture of 2-phosphoglyceraldehyde, would result in its condensation to hexose diphosphate in a manner completely analogous to that of the condensation of acetaldehyde to acetyl methyl carbinol which is ob- served under conditions of aldehyde accumulation. And finally, the hexose diphosphate so formed should be a keto-derivative, and the identical one regardless of whether the fermentable sugar were glucose, fructose, or mannose. That this was so had been established by Har- den and co-workers. Without performing a single experiment, the available information had thus been used to develop an hypothesis that, for the first time, had accomplished an interpretation in which the formation of phos- phate esters had been incorporated as a logical and indispensable ele- ment in the picture of alcoholic fermentation. It stands to reason that the same mechanism was proposed to explain the initial stages of sugar gD BIOGRAPHICAL MEMORANDA degradation by any and all organisms that did not obviously effect a direct oxidation of hexoses to hexonic acids. Soon after the publication of the paper by Kluyver and Struyk [1926] in which this interpretation was advanced, it was adopted by all the leading investigators of carbohydrate biochemistry. But six years later the hypothesis, then no longer in keeping with newly es- tablished facts, was abandoned, and hexose diphosphate once again assumed the place of prominence it had lost. After another six years Lipmann had begun to perceive the significance of phosphorylation as a means of preserving metabolic energy in the form of ‘high energy phosphate bonds’. And Meyerhof [1945, 1947] provided a new and more refined explanation of the Harden and Young equation, based upon the lack of a sufficiently high ATP-ase activity of the yeast juice, a result of the fact that in the crude extracts this enzyme is quite labile and that it is strongly adsorbed on the structural elements of the yeast cells, so that only a small proportion of the total ATP-ase content of intact yeast appears in the extracted juice. Meanwhile the applicability of the principles developed in the course of 1924—1926 was tested for many different types of fermen- tations, and it cannot be denied that they rendered excellent service in helping to account for the butyric acid and butanol-acetone fermen- tations, the fermentations of the coli-aerobacter group of bacteria, the propionic acid fermentation, and the anaerobic processes of denitri- fication and sulphate reduction. In fact, these ideas were soon recognized as being of quite funda- mental significance. In consequence Kluyver was invited to deliver a series of lectures before the University of London, and two years later at Iowa State College in the U.S.A. The London lectures, given in 1930, were published in book form by the University of London Press in 1931. They contained a simple and highly condensed ac- count of the principles underlying the ‘unity in biochemistry’. It is here that for the first time the term ‘comparative biochemistry’ was launched, with the remark that, though as yet little developed, ‘this line of study . .. may in future win the same significance for biochem- istry as “‘comparative anatomy” has already long ago attained for anatomy’. Here, too, one can find the four equations that represent the various 94 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY possible types of hydrogen transfer reactions in their most generalized form, as: (1) AH+B-—> A+BH; (2) AH-B—> A-BH; (3) AH-B—> A+BH; and (4) AH+B > A.BH And here, finally, Kluyver developed his matured version of the con- cept that there is no need to assume a fundamental difference between those aspects of metabolism that had been so carefully differentiated in the 1924 lecture, vz., catabolism and anabolism. To be sure, the nature of assimilatory processes had already been discussed by Kluyver and Donker in 1926, in Part VI of ‘Die Einheit in der Biochemie’, where it had been stated that the principle of hy- drogen transfer can adequately account also for the synthesis of the major cell constituents, the carbohydrates, fats, and proteins, from the substrate molecules. The same theme was treated more fully in the paper Kluyver [1930] contributed to the first volume of the newly established ‘Archiv ftir Mikrobiologie’, on the interrelations between fermentation, oxidation, and assimilation. Now no longer pre-occu- pied with the problem of how to conceive of an energetic coupling of assimilatory and dissimilatory processes that did not have one or more components in common, Kluyver here reasoned that dissimilation should be considered as the means whereby, through oxidation-re- duction reactions, molecular entities are produced that have a higher energetic potential than the substrate itself. The formation of acetal- dehyde during the degradation of sugar was used as an example; through aldol condensations, dehydrations, and reductions, this sub- stance could be converted into fatty acids in a manner fully compat- ible with mechanisms encountered in the spontaneously occurring catabolic reactions. Since the formation of glycerol from sugar, as, for example, in yeast fermentation, is also part of a typical catabolic process, the synthesis of fat from sugar could be understood as the result of a sequence of step reactions none of which involved prin- ciples different from those used in explaining the spontaneous, energy liberating catabolic conversions. It had therefore become superfluous, and even appeared ill-advised, to postulate that biosyntheses cannot pro- ceed without a special influx of energy; rather should one interpret the 95 BIOGRAPHICAL MEMORANDA relation between breakdown and synthesis by considering the former as the means whereby are supplied the energetically elevated building blocks from which the synthetic reactions can proceed spontaneously. If we compare this concept with present-day knowledge it will be clear that there has not been a fundamental change in outlook. To be sure, our understanding of the intimate details of biochemical mecha- nisms has been immensely increased and broadened. We no longer think in terms of acetaldehyde as a key intermediate product, for example. Nonetheless, the mere substitution of ‘acetyl-coenzyme A’ for this substance is all that is required to show that the latest ideas on the mechanism of fatty acid synthesis are virtually identical with those that Kluyver had suggested more than 25 years ago. If we fur- thermore remember that acetyl-coenzyme A formation is currently interpreted as the direct result of catabolic conversions, it is evident that we have achieved a much more detailed, but not a fundamentally different comprehension of the mechanism of synthetic processes. This concept of assimilation and dissimilation as inextricably linked aspects of what Kluyver called ‘metabolism one and indivisible’ could have been experimentally tested, for example by determining the amount of growth (assimilation) of particular organisms in media with various substrates, inclusive of those which, as intermediate products, were presumed to be used as the direct participants in the synthetic processes. But it was generally believed that the amount of cell mater- ial formed in simple media is very small compared to the quantity of substrate used, which made it unlikely that accurate experiments of this sort would be feasible. Another deterrent to such studies was the high volatility and toxicity of acetaldehyde, one of the most crucial amongst the intermediate products. This situation was changed when Barker [1936], in studies on the oxidation of small amounts of substrates by resting cell suspensions of Prototheca zopfii, found that the quantities of oxygen consumed and carbon dioxide produced were far short of those required for complete oxidation of the substrates, and interpreted these results, as well as earlier ones obtained by Cook and Stephenson [1928] in similar stud- ies with B. coli, to mean that the oxidation was accompanied by an assimilation of considerable magnitude, sometimes amounting to more than half of the substrate carbon. The significance of this phenomenon did not escape Kluyver, and soon thereafter his collaborator, Giesber- 96 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY ger [1936Th], conducted numerous comparable experiments with various Spirillum species. ‘The report of this investigation contains an interesting discussion in which the notion of a ‘chip respiration’ was launched. This term was introduced to convey the idea that the oxi- dative dissimilation of a substrate might represent mainly a whittling down of its molecules to fragments that could be used as appropriate building blocks for the synthesis of cell materials. The parts that were intrinsically unsuited for this purpose would thus be eliminated as ‘chips’, generally in the form of carbon dioxide. The experimental results were, however, not entirely consistent with this idea; some of the over-all equations, expressing the quantitative relations between the amounts of substrate used, oxygen consumed, carbon dioxide evolved, and ‘assimilation products’ formed, were difficult to inter- pret in this manner. The only conclusions that could safely be drawn from the data were that the oxidative degradation of an organic sub- strate is quite generally accompanied by a massive assimilatory activ- ity, and that, even in the absence of assimilable nitrogen compounds, when protein synthesis and hence cell multiplication is precluded, a variety of assimilation products may be formed. Shortly afterwards Clifton [1937], in Kluyver’s laboratory, extended these studies, and discovered that this assimilatory activity can be pre- vented if the resting cell suspensions are exposed to low concentrations of dinitrophenol or sodium azide, which do not appreciably inhibit the oxidation of the substrates. Evidently the connexion between de- gradation and synthesis is severed under the influence of these com- pounds. Not until more than a decade later Loomis and Lipmann [1948] found an explanation for this effect by showing that dinitro- phenol blocks the formation of adenosine triphosphate that normally results from the transfer of phosphate from phosphorylated inter- mediate products such as acetyl phosphate. In current terminology, and entirely in keeping with Kluyver’s concept of the nature of bio- synthetic processes, this can be paraphrased by saying that under these conditions the necessary raw materials for anabolic reactions are prevented from being formed during the oxidation of the substrates. oy BIOGRAPHICAL MEMORANDA THE NATURE OF BIOCATALYSIS The developments mentioned in the last few pages show how firmly our current notions of metabolic reactions are based on the general concepts that Kluyver had advanced. Whenever a particular branch of science has reached the stage where such a generalization has been achieved, further progress is apt to result primarily from attempts to comprehend in an increasing- ly refined manner the mechanism underlying those phenomena that have been recognized as the most basic ones. Ultimately this approach should permit an interpretation of the behaviour of inanimate as well as animate systems in terms of the properties of the elementary par- ticles of matter, or of whatever may eventually take their place as the most fundamental constituents of the universe. Thus, when Kluyver had enunciated the main principles of bio- chemistry, it was logical to anticipate that important new advances in this field would come from studies aimed at acquiring a more pro- found understanding of the nature and mechanism of biocatalysis. Amongst the possible approaches that could be envisaged in 1930, one was obviously directed at investigations of the biocatalysts themselves. How much has been accomplished in this respect, owing to twenty- five years of increasingly intensive enzymological research, need not be discussed here. But Kluyver was reluctant to follow this line, with the consequence that not until the last few years were enzyme-chemical studies conducted in his institute. And it is pertinent to our subject to examine into the reasons for this attitude. At the time when the principles of biochemistry were gradually being developed, knowledge of enzyme chemistry was virtually non- existent. To be sure, the hydrolysis of starch under the influence of saliva had been known for about a century, and subsequently the hydrolysis of other complex carbohydrates, of fats, and of proteins by tissue extracts had been established. The agencies responsible for these hydrolyses appeared to be catalysts produced and often ex- creted by living cells; even if not excreted they could usually be extracted with ease. They had been designated as ‘ferments’ or ‘en- zymes’; they seemed to exhibit a high degree of specificity; but their chemical nature and mode of action were completely unknown as late as 1920. The inactivation of enzymatic activity by exposure to tem- 98 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY peratures that caused protein denaturation had suggested that en- zymes might be proteins or substances associated with proteins, and during the early ’twenties Willstatter and collaborators had assembled a mass of information in support of this view. Finally, in 1926, Sum- ner had announced the isolation of a crystalline protein that pos- sessed the power to hydrolyze urea to ammonium carbonate, thereby establishing that urease, at least, was a typical protein. It is understandable that the existence of hydrolytic enzymes had led to the assumption that comparable biological catalysts would be involved in other types of reactions brought about by living organ- isms. And when Buchner had prepared, from ground-up yeast, a cell- free press juice that could still promote a characteristic alcoholic fer- mentation of sugar, this was hailed as proof positive for the enzyme theory of fermentation. Some early successes were booked in preparing from other organisms cell-free juices that caused the formation of lactic acid from sugar, or an oxidation of alcohol or glucose. But the extrac- tion of such enzymes invariably proved more difficult than that of the hydrolases; it required the use of large quantities of cells, and, be- cause these enzymes were not excreted, they had to be liberated by rupturing the cells. In consequence the composition of these enzyme extracts was exceedingly complex, approximating that of the cells themselves. Now this marked difference in the behaviour of enzymes involved in hydrolyses and in typical dissimilatory reactions, respect- ively, had already been commented on by Kluyver in his 1924 lecture on the ‘Unity and Diversity in the Metabolism of Micro-organisms’. Here he had stressed what appeared to him the significant fact that hydrolyses represent preparatory events whereby large, non-diffusible molecules are broken down to small and readily diffusible units, and that, from the point of view of cellular economy, they are devoid of energetic significance. In contrast, the typical dissimilation processes are primarily concerned with energy provision of the cells, which accounted for the fact that the causative enzymes had to be rigorously cell-bound. Kluyver was not averse to considering dissimilatory transformations as enzymatic; in fact, in a series of lectures delivered in Amsterdam in 1929 he had categorically stated: ‘Biochemistry in its entirety is enzymatic’. Why, then, did he show so little inclination to include enzymological studies in his programme? There were two different factors to which we can assign an important role. 99 BIOGRAPHICAL MEMORANDA The first was his general philosophical outlook which made him wary of the tendency to explain biochemical reactions in terms of events mediated by specific enzymes. As long as there was no prospect that the chemical nature of the postulated enzymes could be eluci- dated or even approximated, this practice seemed like futile para- phrasing without adding anything to our understanding of the pro- cesses themselves. Worse, this approach, which had already induced many biochemists to name a specific enzyme for every biochemical reaction that could be surmised to exist, might easily lead to a false sense of comprehension, and to a mental image of a living cell as the depository of a vast number of such catalysts, distributed without rhyme or reason; a bag of enzymes rather than a smoothly func- tioning unit. Alternatively, the development of general principles that could help to account for the multifarious activities of living organisms in a chemically intelligible manner had given rise to the concepts of hydrogen transfer and the unity in biochemistry, and eventually to the notion that a single property should suffice to ex- plain the many conversions a cell is capable of performing. This ap- proach was clearly antagonistic to the one based upon the assumption of a haphazard multiplicity. It had, moreover, been fruitful; it was dedicated to a search for still greater unification; and it could be further exploited. If finally we remember the difficulties inherent in procuring extracts of nonhydrolytic enzymes, we can readily ap- preciate Kluyver’s belief that enzyme research would not be parti- cularly rewarding. Secondly, this attitude appeared to be vindicated by the results of the investigation on the coenzyme of alcoholic fermentation that he carried out with the collaboration of Struyk [1928]. As already stated, the exceedingly complex nature of an extract such as zymase made it seem hopeless to attempt a chemical characterization of the specific catalyst. But Harden had discovered that a yeast juice could be de- prived of its fermenting capacity by dialysis or ultrafiltration, and that the residue could be restored to activity by the addition of the dial- ysate or filtrate. Thus the factor in the dialysate responsible for this behaviour was evidently a substance of relatively small molecular size; in addition, it had been found resistant to prolonged boiling. ‘These properties of the so called co-zymase indicated that it would be more amenable than zymase itself to chemical investigation, and some bio- 100 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY chemists, notably Meyerhof and von Euler, were already engaged in studying the nature of this material. Now, the theory of alcoholic fermentation that Kluyver and Struyk had developed called for the participation of two different kinds of catalysts, viz., a phosphorylating and an hydrogen activating agent. Besides these nothing else should be needed with the possible excep- tion of a substance that could serve as the initial hydrogen acceptor for the conversion of methyl glyoxal into pyruvic acid. According to the theory, acetaldehyde should be suitable for this purpose, and Meyerhof, in fact, had already shown that a dialysed yeast juice could sometimes be reactivated by the addition of trace amounts of this substance. Meyerhof had also found that the fermentation induced by a zymase preparation was frequently accelerated by small amounts of hexose diphosphate. This, too, could be explained within the frame- work of Kluyver and Struyk’s theory by assuming that the phosphor- ylation would proceed more effectively with the hexose ester than with inorganic phosphate. So far the experimental results did not present serious obstacles to a simple explanation of the co-zymase effect. But it had also been established that zymase preparations, after ultrafiltration and pro- longed washing, yielded an inactive residue that could no longer be reactivated by hexose phosphate and acetaldehyde, although boiled yeast extract or the concentrated dialysate did restore the activity. Thus the problem was raised how this presumably more genuine co- zymase effect could be explained. Kluyver believed that the answer would be found in earlier studies of Buchner, Haehn, and others, who had postulated that the decline in fermenting capacity of a yeast juice was due to a decomposition of the zymase itself by proteolytic enzymes present in the original juice. This hypothesis could easily be elaborated to explain the reactivation of a zymase residue; it was but necessary to assume that the boiled yeast extract could in some manner protect the zymase against proteolytic degeneration. Numerous preliminary experiments seemed to support this expla- nation. Nevertheless, the reactivation of a thoroughly washed ultra- filtration residue was always accomplished by the addition of materials that could not be guaranteed to have been entirely devoid of co- zymase. And a crucial experiment that could serve to abolish the notion that an additional and functionally mysterious factor was need- IOI BIOGRAPHICAL MEMORANDA ed should carry precisely such a guarantee. This consideration led to the successful experiments in which it was shown that a filtered zymase residue, no longer capable of fermenting sugar, even when supple- mented with hexose diphosphate and acetaldehyde, and hence co- zymase free by the most rigorous standards, could be reactivated by mixing it with a boiled aliquot of the same preparation. This result was interpreted to mean that the boiled extract, in which the proteases had been destroyed, contained protease-inhibiting materials. The al- ternative explanation that a low-molecular weight substance needed for fermentation, a true coenzyme as we now understand this term, could be generated by boiling co-zymase-free zymase, was not con- sidered; in view of the fact that nothing was yet known about the nature and function of coenzymes this is not surprising. The conclusion of this extensive investigation was, therefore, that the co-zymase effect could be attributed to one or more of the following factors: 7. an initial hydrogen acceptor function, such as exhibited by acetal- dehyde; 2. an activation of the phosphorylating principle by hexose diphos- phate; and 3. protection of zymase against proteolytic degeneration. Thus it appeared that the term co-zymase had actually been applied to three entirely different types of substances, with different modes of action. For this reason Kluyver and Struyk argued that it would be best to ‘banish the notion of a coenzyme altogether from the litera- ture, and to replace it by the much more rational concept that a cell- free fermentation requires, in addition to zymase and phosphatase, the presence of an initial acceptor, of an hexose phosphate ester, and of substances that can regulate the proteolysis of the zymase system. ... Although the function of the coenzyme had thus far remained entirely obscure, it is now possible to indicate the nature of its activity, and even to link it with phenomena whose importance for the cell-free fermentation had long been recognized. ... Further cogitations then lead to the idea that the indispensability of the proteolysis-regulating factors should be ascribed to the fact that, for its maintenance and for the normal execution of its functions, the living cell requires the har- monious cooperation of the principles it contains. If this harmony is disrupted, as happens during the isolation of the zymase, and 102 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY even more during the subsequent filtration and washing, the l- ving substance undergoes selfdestruction.’ [Kluyver and Struyk, 1928. p. 258]. It is not difficult to understand that this investigation decided Kluyver against undertaking any further studies in the field of coen- zymes. And, despite the great advances in our knowledge of bio- chemical reaction mechanisms resulting from the eventual isolation and chemical identification of various coenzymes — advances that Kluyver fully appreciated and readily acknowledged — he never quite lost his earlier dislike for such studies. Even as late as 1954, in the second of the Harvard lectures, he conceded this in the passage: ‘...it is tempting to say a few words about the significance of the discovery of a coenzyme being involved in a certain reaction step. [I have to confess that my first mental reaction on hearing of such a discovery is to lower the flag to half-mast’ (p. 47). An additional effect of this study on ‘the so-called coenzyme of al- coholic fermentation’ was to strengthen still more his belief that studies with enzyme extracts were not apt to contribute much to bio- chemical comprehension. Even if — and this was doubtful enough in the ’twenties — better techniques could be invented for the prepara- tion of cell-free extracts, it seemed to him improbable that studies with such extracts would contribute anything beyond what had al- ready been inferred from investigations with intact organisms. ‘The studies on the cozymase problem afforded an instructive example; it was the previously developed theory of alcoholic fermentation that had led to predict what the coenzyme should be and do, and this had been supported by experiment. Meanwhile, these same studies had failed to provide a deeper insight into the mechanism of the process, and rather emphasized the disadvantages inherent in working with extracts in which the harmonious cooperation of various principles had been deranged. On the other hand, the concept that the essence of biocatalysis was the activation and transfer of hydrogen suggested that a more fundamental approach could be devised which would lead to a sounder organization and understanding of the various manifestations of the chemical activities of living organisms. ‘This was developed in the last of the earlier mentioned London lectures. Here, Kluyver first argued that there is no ground for believing that some biochemical events might not be mediated by enzymes, 103 BIOGRAPHICAL MEMORANDA concluding that ‘it has become very difficult to deny the enzymatic nature of those dissimilation processes for which the experimental proof is still lacking. For instance, I doubt if anyone would seriously maintain that sugar breakdown by the yeast cell is an enzymatic pro- cess, whereas the analogous fermentation by B. coli has a non-enzyma- tic character’ (p. 94). And what was true for dissimilatory reactions should hold equally for assimilation processes; after all, the synthesis of a new carbon-to-carbon link is encountered also in typical catabolic reactions. Nevertheless, he perceived that this thesis might be less readily accepted, and proceeded to disarm his potential opponents: ‘But you will perhaps observe that whilst it is easy to ferment sugar with the aid of the enzymes of yeast, it has not as yet been found possible to synthesize fat from sugar with these enzymes, a conversion which 1s nevertheless of general occurrence in the living yeast cell. However, on second thoughts this is not to be wondered at. For I have only to remind you how the conversion of sugar into fat demands for its successful completion a harmonious succession of a special set of pri- mary reactions out of the many that are possible. And the perfect harmony which is the one condition for such a long chain of reactions is the exclusive prerogative of the living cell’ (p. 95). Next he considered the implications of the enzymatic interpretation of metabolism: ‘The question then arises whether we have to conclude from the foregoing that a living cell should be considered as an arsenal filled up with enzymes, which successively are brought into action. Such a supposition would only be justified if every chemical reaction brought about by the cell required its own specific catalyst. ‘A survey of modern enzymological literature shows that the doc- trine of the extreme specificity of enzymes is adopted by almost all the leading authorities in this field. ... All the same, it seems worth while to dwell for a moment on the problem whether this doctrine is actually well founded... We should then like to observe in the first place that a catalyst can only be expected to be non-specific, i.e. be able to promote various reactions, in so far as these reac- tions are of the same nature. Now, we have seen that the primary reactions to which biochemistry can be reduced satisfy this demand in a high degree; all these reactions are either of the hydrolytic or of the oxidoreduction type. So at first sight it seems quite conceiv- 104 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY able that the same enzyme can promote several different conversions’ (p- 95-96). He then pointed out that catalysis involves the formation of a loose compound between catalyst and substrate, resulting in an activation of the latter. Thus specificity might be conditioned either by config- urational patterns or by the degree of activation which depends on the electrostatic properties of the catalyst. Hydrolases would tend to exhibit the former kind of specificity, whereas the oxido-reductases, acting on relatively small molecules and causing reactions with pronounced energetic effects, should display specificity largely be- cause of differences in the intensity of their electric fields. Kluyver concluded with the statement: ‘Ignoring all this, the experimen- tal material gathered with hydrolases has led biochemists to extend unconsciously the doctrine of great specificity to the whole field of enzymes... For every biochemical oxido-reduction... the ex- istence of a special ‘‘ase’’ has been postulated. In this way biochem- ical youth is nowadays poisoned by the necessity for learning nu- merous names of the barbaric succinofumarase and quercito-pyro- gallase type. ‘However, such a procedure is only practicable as long as the num- ber of substrates which are liable to dehydrogenation under the in- fluence of one and the same specific cell is hmited. But when we think of the bewildering diversity of compounds which are able to act as dehydrogenation substrates for the cells of Pseudomonas putida, ... it will be generally agreed that here the doctrine of extreme spe- cificity becomes untenable. For it can scarcely be conceived that the cells of the bacterium in question contain as many dehydrases* as there are suitable oxidation substrates for these cells. And, more- over, we should be obliged to assume that these cells have at their disposal specific catalysts for substrates such as bromo-succinic acid and bromopropionic acid which do not occur in nature, and which are only made by the conscious operations of the organic chemist. ‘So in this case there is not the slightest doubt that one and the same catalyst is capable of acting upon different substrates and very probably even on a large number of these. Once accepting the presence of such dehydrogenating “‘master keys” in bacterial * Ed. note: meant is obviously dehydrogenases. 105 BIOGRAPHICAL MEMORANDA cells, there is no clear reason why one should not go farther and accept the supposition that in Pseudomonas putida there is only one single oxido-reduction promoting agent which acts on all the sub- strates... And the same conclusion holds good for all the primary oxido-reductions which together constitute the typical fermentation process ‘ot a ‘cell! “This does not imply, however, that no specificity at all exists in oxido-reduction promoting agents. On the contrary, we shall have to seek, in the differences of the electrostatic properties of the agents of different specific cells, the explanation why some of these cells dehy- drogenate sugars only, others hydrocarbons as well, still others meth- ylamine or nitrites. And we may cherish the hope that the time will come when a wellfounded quantitative theory of catalysis will lead to a sharp characterisation of the electrostatic properties of the different catalysts, and in doing so make it possible to predict the nutritional requirements of the corresponding cells’ (p. g8—99). There is much in this reasoning that is as appealing as it is sound. Ironically, the argument about a single dehydrogenating agent for a large number of substrates has gained the strongest possible support from the isolation of coenzymes I and II, their identification as di- and triphosphopyridine nucleotides, and the finding that these are the molecular species directly involved in the dehydrogenation of numerous substrates. Admittedly, enzymological studies have also established that these coenzymes function only in cooperation with specific protein apoenzymes, so that the assumption of a multiplicity of enzymes is still a necessary adjunct hypothesis. But this aspect has gradually lost some of its perplexing consequences; the discoveries pertaining to the apparently universal occurrence of common path- ways, of a small number of cyclic mechanisms, and of the phenomenon of induced enzyme synthesis have eliminated the need for assuming that a particular cell is invariably equipped with as many types of specific protein molecules as there are substrates it can utilize. In reading Kluyver’s papers of this period during which he for- mulated the general principles of metabolism, one may perhaps find the approach too theoretical and speculative. This was also the crit- icism of some of his contemporaries, one of whom complained that what was really needed was ‘more matter and less art’. Kluyver was fully aware of this; but his philosophical inclination always made 106 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY him search for concepts that could help to tie various known facts to- gether, and he was wont to defend this attitude by quoting the felici- tous phrase of A. V. Hill, ‘It is dangerous to speculate too far, but it is foolish not to speculate at all’. It cannot be denied that, if an hypothesis can be made to accom- modate apparently unrelated or even conflicting observations, the ten- dency to speculate is sometimes conducive to generating a feeling of confidence rather than to performing experiments, especially such as might throw a different light on a particular problem. A case in point is the earlier discussed investigation on co-zymase. Surely the mechanism of alcoholic fermentation proposed by Kluyver and Struyk, however much of an advance it represented over other theories current at the time, still left a great deal to be explained. And the general state of ignorance concerning the composition and mode of action of enzymes was no justification for the tacit assump- tion that there should be no need for the cooperation of small mole- cules with as yet unexplained functions. Be that as it may, it is impossible to determine at just what point speculation will become dangerous. Moreover, the danger can easily be exaggerated, and in any case it is less so to science than to the speculator, who may be entranced by a preconceived notion to the point of not recognizing alternative possibilities. But since scientists, like microbes, vary a great deal in their predilections, there will al- ways be some who are not so taken in, and, by following other ap- proaches, provide the information that is essential in checking and correcting assumptions that lack a sufficient experimental basis. For science is a curious amalgam of art and matter, and its most out- standing characteristic is that rival hypotheses are ultimately re- solved not by personal preference but by the force of impersonal facts; accumulated observational evidence is the only final arbiter. This does not mean, however, that the elements of art and imagi- nation can be dismissed as irrelevant in scientific pursuits; they are as essential as factual data because it is through their exercise that extant knowledge can be integrated and new data can be collected in a meaningful manner. Besides, the search for general relationships has the refreshing effect that it leads to interpreting isolated events as coordinated phenomena, and thus guards against getting bogged down in details. 107 BIOGRAPHICAL MEMORANDA Kluyver, who was a master in the art of ‘discovering unity in the wild variety of nature’, had meanwhile started the search for a ‘quan- titative theory of catalysis’ that ‘could lead to a sharp characterization of the electrostatic properties of different catalysts’, and ‘might make it possible to predict the nutritional requirements of the corresponding cells. It was hoped that a study of the redox potentials established in metabolizing cultures of various micro-organisms would provide a promising approach to this problem, the underlying idea being that the specific affinity of the cells for hydrogen would express itself more or less clearly in the quantitatively measurable potentials. In a publication on redox potentials from his laboratory |Elema, Kluyver, and Van Dalfsen, 1934] the nature of the general problem was introduced as follows: *...a closer study of the connexion between the redox potentials established in microbial cultures and the nature of the metabolic pro- cesses had long been part of the programme of our institute. This was a logical consequence of the attempt to reduce the totality of bio- chemical events to chains of catalytic oxido-reduction reactions. ‘In choosing specific processes for studies of this sort we were guided by the following considerations. Firstly, 1t was necessary to select a metabolic process that can occur under fully physiological conditions in a medium of very simple composition. Secondly, it seemed essential for the time being to neglect the aerobic metabolic processes because under the usual conditions of cultivation these lead to heterogeneous situations in the culture medium. The slow diffusion of oxygen into the deeper layers of a culture liquid, and the conse- quent uneven distribution of the cells in the cultures will surely result in differences in redox potentials at different distances from the sur- face. * The meaning of this latter phrase may easily be misunderstood by the younger microbiologists and biochemists. In 1930, when our knowledge of specific growth factors was as primitive as that of enzymes, Kluyver could not have used the term ‘nutritional requirements’ in its current sense. From the context, and from know- ledge of the evolution of his ideas, it is obvious that he had in mind the possibility of predicting which of the great variety of potentially oxidizable or fermentable substrates — inorganic substances, hydrocarbons, alcohols, polyalcohols, fatty and substituted acids, carbohydrates, aromatic compounds, amines, amino acids, etc. — a particular organism should be able to utilize. 108 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY ‘Amongst anaerobic metabolic processes it seemed that particularly denitrification should fulfill the above-mentioned requirements’ (Pp. 319)- A beginning was therefore made with a study of the redox poten- tials in cultures of denitrifying bacteria. The results clearly indicated that the measured potentials were indeed dependent on the nature of the metabolic process. During the first phase of the cultures, when only nitrate was present, a potential of around —100 mV was estab- lished; when nitrite had accumulated the potential rose to a level of —4o mV; thereafter it gradually dropped again, eventually and quite sharply to a new low level of about —260 mV, coinciding with the disappearance of the nitrite. At this stage the addition of nitrate or nitrite sufficed to restore the earlier established potentials. On account of the drastic changes in hydrogen ion concentration during the development of cultures of denitrifying bacteria, and their inevitable effect on the redox potential, the results obtained were not interpretable in terms of absolute values; nevertheless, they showed that the type of metabolic process was reflected in the measured potentials. In passing it may here be mentioned that these studies had also led Elema in Kluyver’s laboratory to discover the reversible two-step oxidation of reduced pyocyanine and chlororaphine, simultaneously and independently found by Michaelis in the U.S.A. Spurred on by these promising results, the programme was soon ex- panded by Kluyver and Hoogerheide [1934] to studies on the poten- tials established in suspensions of micro-organisms provoking a char- acteristic alcoholic fermentation. The organisms used included vari- ous yeast species as well as the bacterium, Pseudomonas lindnert, which Lindner had discovered as the causative agent of the alcoholic fer- mentation of agave juice from which the Mexicans prepare the alco- holic beverage known as ‘pulque’. This bacterium had been thor- oughly studied by Kluyver and Hoppenbrouwers [1931] who had shown that it can ferment sugar to equimolar amounts of carbon dioxide and alcohol, accompanied by small quantities of lactic acid, and that, in contradistinction to yeasts, it can ferment glucose and fructose, but not mannose, so that it can be used for the quantitative determination of mannose in sugar mixtures by means of the previously discussed fermentation method. 109 BIOGRAPHICAL MEMORANDA Under anaerobic conditions all of these organisms, in suspensions containing a fermentable sugar, caused the establishment of a redox potential of 80-100 mV. The concordant data therefore suggested that this is the potential characteristic for alcoholic fermentation. ‘To- gether with the marked difference in the potentials of cultures of denitrifying bacteria, this further strengthened the belief that the redox potential may serve to define a specific type of metabolism, a first and important step in the direction of a quantitative description of metabolic processes in electrochemical terms. But it soon appeared that in the case of alcoholic fermentation the measured potentials conflicted with observations made by the use of other techniques, for shortly afterwards Fromageot and Desnuelle [1935] showed that a fer- menting yeast suspension can reduce dyes, such as Nile blue, whose nor- mal potential is considerably lower than the potentials determined by Kluyver and Hoogerheide. This, of course, implied that the reducing capacity of such a suspension must be much greater than what could be surmised from the values established with the aid of noble metal electrodes. Once again theoretical considerations of the situation provided a solution of this problem. In a subsequent publication Kluyver and Hoogerheide [1936b] argued that the potentials they had determined must be ascribed to the presence around the electrodes of a redox system of unknown composition that had diffused out of the metab- olizing cells into the surrounding medium. At first the nature of this system, referred to as the ‘bio-indicator’, had been deemed unim- portant. But now it became clear that this was not necessarily so; after all, the electrode can only measure a potential that is deter- mined by the continuous interaction of the bio-indicator system with the electrode on the one hand, and with the intracellular catalysts on the other. The argument continues: ‘Now in the previous communications we had tacitly assumed that the bio-indicators secreted by the cells would be able to display their mediating function over the entire range of potentials concerned in the metabolic activity. On closer consideration this may, however, be doubted... . It is by no means evident why it should be a general pro- perty of living cells to excrete into the surrounding medium an unin- terrupted series ofredox systems with decreasing normal potentials. ‘Thus it is possible, nay, even probable that the oxido-reduction processes IIo KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY participating in metabolism may often cause lower potentials than those measured by means of the metal electrodes in the cell suspensions.’ In this manner the apparent contradiction in the results obtained by Kluyver and Hoogerheide and by Fromageot and Desnuelle, re- spectively, could readily be explained. It was but necessary to assume that the dyes could interact with intracellular catalysts, representing a redox system at an even lower potential, but not excreted into the medium, and therefore unable to exert an effect on the electrodes. Consequently the potentials established at the electrodes did not necessarily represent the potentials inside the cells resulting from the particular metabolic activity under investigation. At the same time, the observed reduction of Nile blue suggested to Kluyver and Hooger- heide a simple method whereby the potential in the interior of metab- olizing cells might become measurable with the aid of an external electrode. This consisted in the addition to the cell suspension of a readily diffusible redox dye of appropriate normal potential; it was felt that the dye would mediate the necessary contact between the intracellular catalysts and the electrodes. A series of measurements conducted in this manner showed that now the electrode potentials in fermenting yeast suspensions were indeed much lower than those previously determined, and fell in the range of —30 to —40 mV. It is true that some exceptions were noted; in suspensions containing certain dyes with normal potentials high enough to warrant the ex- pectation that they should at least be partially reduced — e.g. indigo di-, tri-, and tetra sulphonates — the measured potentials were once again found to be around 100 mV. This result could, however, be attributed to an inability of these substances to penetrate into the cells, an interpretation that was supported by tests showing that such indicators are not reduced at all. These experiments led Kluyver and Hoogerheide to introduce a general technique for the measurement of redox potentials free from the ambiguity resulting from a dependency on excreted redox sys- tems. ‘The technique involves measurements of electrode potentials in cell suspensions supplemented with a mixture of redox dyes covering a wide range of normal potentials, a ‘universal indicator mixture’. Appropriate experiments with the various yeasts and Ps. lindnert then revealed that under these conditions an electrode potential of —30 to —40 mV was established in suspensions of all of them. This was therefore PEs BIOGRAPHICAL MEMORANDA considered to be the potential characteristic of alcoholic fermentation. In similar experiments with a large variety of homofermentative lactic acid bacteria a redox potential of —160mV was found in every case, which thus appeared to define the specific potential of a lactic acid fermentation. Here, too, some anomalies had to be faced, such as the fact that some Streptococcus species, although typical lactic acid bacteria in all respects, cannot reduce methylene blue, whose normal potential is surely high enough to cause its complete reduction in an environment with a redox potential as low as —160 mV. This contention was obviously supported by the rapid and complete re- duction of methylene blue in cultures of other lactic acid bacteria. Furthermore, some species differ markedly from others in their ability to reduce litmus and indigocarmin, a finding that seemed incompat- ible with the establishment of identical potentials in cultures of all the lactic acid bacteria containing the ‘universal indicator mixture’. The experience gained in the studies on the redox potentials of micro-organisms carrying out an alcoholic fermentation led, how- ever, to a ready explanation of these anomalies. First of all, it was found that the electrode potentials of cultures of various lactic acid bacteria not containing the indicator mixture were by no means iden- tical, and might even differ by as much as 200-300 mV. This sug- gested that some species do, in fact, excrete redox systems with much lower potentials than others. Secondly, this differential behaviour also made it obvious that the latter types cannot reduce indigocarmin; this dye does not ordinarily penetrate into living cells, so that its re- duction can be accomplished only under the influence of excreted redox systems at a sufficiently low potential. And thirdly, the failure of some species to reduce methylene blue could be attributed to the fact that this substance is quite toxic to these bacteria; they cannot grow and metabolize in media with the usually employed concen- tration of this dye, which accounts for the fact that no reduction can be observed. Initially these studies on the redox potentials in suspensions of metabolizing micro-organisms may well have appeared to hold out promise for the subsequent development of a quantitative theory of biocatalysis, and they were extended to a number of other metabolic processes. Thus Kingma Boltjes [1935], while studying the nitrifying bacteria in Kluyver’s laboratory, determined the potentials in cul- 112 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY tures of these organisms, and Roelofsen [1934], in the course of his investigations of the metabolism of the purple sulphur bacteria, meas- ured the potentials in suspensions of a Chromatium species, both in darkness and when exposed to light, thereby revealing that illumina- tion caused an instantaneous shift of the potentials to a new level, some 100 mV more positive than that of the suspension in darkness. While these measurements were of a rather incidental nature, this cannot be said of the study of Cozic [1936], who determined the poten- tials of suspensions of acetic acid bacteria during the oxidation of alcohol. This investigation was conducted with six different Acetobacter species, including A. peroxydans and A. suboxydans, thus covering the entire range of representatives with different oxidative capacities. It was found that, regardless of the species used, the potential was the same in all cases. This, no doubt, must have made Kluyver realize that the measured potentials were determined exclusively by the par- ticular metabolic process taking place in the suspensions of the mi- crobes under investigation; and it is probable that this recognition eventually convinced him that redox potentials could not provide the requisite data to define the ‘affinity for hydrogen’ of different organ- isms, which had been the primary aim of these studies and was in- tended to serve as the basis for a quantitative theory of biocatalysis. This would account for the fact that after 1936 no further work on this subject appears to have been carried out in his laboratory. That the decision to abandon this project was a sound one has become clear from the results of a quarter century’s research on iso- lated enzyme systems. For, although current knowledge of the redox potentials of these entities has made it possible to assign to many of them a definite place in the sequence in which they participate in biochemical reactions, it is apparent that ‘affinity for hydrogen’ is certainly not the only factor that determines their activity. However much enzyme research may have contributed to our understanding of the detailed mechanism of biocatalysis, it has not yet led to the emergence of concepts that can explain the potentialities of various microbes to metabolize diverse types of substrates without invoking a considerable degree of arbitrariness in the distribution of specific en- zymes among the organisms. Whether this will ever be feasible can only be decided by future developments. 113 BIOGRAPHICAL MEMORANDA COMPARATIVE BIOCHEMISTRY The concept of the ‘unity in biochemistry’ evolved from the idea that all metabolic processes can be interpreted as sequences of step reac- tions, each one representing an intra- or intermolecular transfer of hydrogen under the influence of cellular catalysts. During its develop- ment several types of sugar fermentations had been analyzed in some detail, and it had been found that all of these could be explained as the end result of an identical primary conversion, leading through hexose monophosphate to methyl glyoxal, with modifications arising only in the fate of the latter compound (See, e.g., Kluyver’s review [1935]). It has already been mentioned that, by changing the environmental conditions during the fermentation, the intermediate products derived from methyl glyoxal could be diverted into channels that were more or less abnormal for the particular organism; the formation of acetyl methyl carbinol by yeast and lactic acid bacteria in media with added hydrogen acceptors is a good case in point. Later Kluyver and Molt [1939] showed that B. coli can also be induced to form this substance in trace amounts. Of even greater significance was the application of the theory to the fermentations of substances other than the common hexoses. An ex- cellent example is furnished by the studies of Braak [1928Th] in Kluy- ver’s laboratory on the fermentation of glycerol; they established that the anaerobic decomposition of the polyalcohol by members of the coli-aerogenes group of bacteria may follow one of two distinct pat- terns. The first yields products that qualitatively and quantitatively resemble those of a sugar fermentation by the same organism; this type of fermentation is characteristic for strains that can decompose glycerol only in the presence of an additional hydrogen acceptor with which glycerol can first be converted into a triose, the latter being the genuine fermentable substrate. The second is encountered in those cases where glycerol can be fermented in the absence of an additional acceptor; these fermentations are characterized by the appearance of trimethylene glycol (1,3-propane diol) as a fermentation end product in an amount roughly equal to one-half of the glycerol fermented, the other half being recoverable in the form of products typical of a normal sugar fermentation. Such results could easily be understood by as- suming an initial conversion of glycerol in which one molecule of the 114 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY substrate is oxidized with the concomitant reduction of another, ac- cording to the equation: 2C,H,O, > C;H,O;+C,H,0,+H,O An essentially similar situation was encountered some years later when Barker [1936b, 1937a], during his sojourn in the Delft institute, stud- ied the fermentation of malic, fumaric, and tartaric acids by bacteria of the coli-aerogenes group, and of glutamic acid by anaerobic spore- forming bacteria. Here, too, comparable initial conversions, such as 2C,H,O; > C,H,O;+C,H,O,+H,O could be invoked to explain the accumulation of succinic acid, and of other products by a further degradation of oxaloacetic acid through pyruvic acid. The fermentation of the methyl pentose, rhamnose, by B. rham- nosifermentans provides a good example of the ready applicability of the theory to another aberrant type of metabolism. Kluyver and Schnellen [1937] found that in this fermentation 1,2-propane diol is formed in an amount approximately corresponding to one mole per mole of rhamnose fermented. Apart from an indication in an earlier report that it may be produced in small quantities during the commer- cial manufacture of glycerol by fermentation of sugar in the presence of sulphite, this was the first, and so far the only record of the appear- ance of the asymmetrical propane diol as a major metabolic end prod- uct. Its formation was explained by postulating a rupture of rham- nose to lactic and glyceric aldehydes, the former acting as the virtu- ally exclusive hydrogen acceptor for those reactions by which the glyceraldehyde is further degraded. The formation of succinic acid, which was found in considerable quantities in this fermentation, was, as in the previous studies of Braak [1g28Th] and Scheffer [1928Th] on fermentations by members of the coli-aerogenes group, attributed to a cleavage of hexose into a two- and a four-carbon fragment; the hexose was assumed to be generated by a condensation of two mole- cules of glyceraldehyde. A somewhat more detailed discussion of this aspect will be found in a later section of this chapter. Not less fruitful than in the above-mentioned cases was the applica- tion of the theory to those metabolic phenomena that can best be 115 BIOGRAPHICAL MEMORANDA described as anaerobic oxidations. These are processes in which a sub- strate can be oxidized under strictly anaerobic conditions with the simultaneous reduction of certain mineral constituents of the medium. Specific examples are the processes known as nitrate, nitrite, and sul- phate reduction. Formerly these had been interpreted as reactions in which the reducible component of the medium supplied the oxygen necessary for the oxidation of the substrate; now they could more properly be regarded as examples of substrate dehydrogenations with nitrate, nitrite, or sulphate acting as the specific hydrogen acceptors. Thus the oxidation of a particular substrate, H,A, could be envisaged to take place with the participation of various acceptors, and the several possibilities expressed by the over-all reaction equations: 2H,A+ O, — 2A+2H,0; 4H,A+HNO; > 4A+3H,0+H;N; 3H,A+HNO, — 3A+2H,0+H,N; 4H,A+H,SO, — 4A+4H,O+H,S; obviously all of these represent special instances of the general equa- tion: H,A+B— A+H,B These comparisons were particularly significant because they eventu- ally suggested an explanation for the general mechanism of the meth- ane fermentation which Kluyver, in his London lectures, had char- acterized as ‘the ultimo ratio in the domain of oxido-reduction. ... It is obvious that the extreme form which can be conceived for such a process will be, that part of the carbon atoms present in the substrate attain their highest reduction stage, i.e. methane, another part of these carbon atoms their highest stage of oxidation, i.e. carbon dioxide’ (p. 75). At that time Kluyver had to admit, however, that the ‘mech- anism of this type of fermentation is as yet quite unknown’ (p. 76). But a comparative-biochemical consideration of the methane fer- mentation as another anaerobic oxidation process later suggested that, in analogy with the nitrate, nitrite, and sulphate reduction, the meth- ane fermentation could be interpreted as a case of substrate oxidation coupled with the reduction of carbon dioxide, a ‘carbonate reduction’ : 4H,A+ CO, — 4A+2H,0+CH, This hypothesis received strong support from Barker’s [1936a] dis- Ge KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY covery, in Kluyver’s laboratory, of a methane fermentation in which ethanol, the only organic ingredient of the medium, was oxidized quantitatively to acetic acid, while simultaneously methane was formed, and an equivalent amount of carbon dioxide disappeared: 2CH,CH,OH-+ CO, + 2CH,COOH+CH, Later the origin of methane from carbon dioxide was established quite unambiguously by Barker and collaborators [1940] in experi- ments demonstrating that the methane formed by a pure culture of the causative bacteria during the oxidation of ethanol in the presence of CO, was, indeed, “CH,. The rationale of Barker’s use of ethanol as the oxidation substrate for the study of methane production had its origin in yet another application of the comparative biochemical approach. It was based on the fact that various micro-organisms oxidize this substance only as far as acetic acid; this applies, for instance, to the strictly aerobic acetic acid bacteria, notably A. suboxydans, as well as to particular representatives of the strictly anaerobic sulphate reducing bacteria, as had been shown by Kluyver’s collaborator, Baars [1930Th], in his extensive studies of this group of organisms. The advantage of such an incomplete oxidation is, of course, that the initial substrate can still be recognized in the oxidation product, which justifies the in- ference that the substrate carbon cannot be implicated in the forma- tion of methane. A comparable incomplete oxidation was later used by Kluyver and Schnellen [Kluyver, 1939a; Schnellen, 1947Th] to provide additional evidence for the origin of methane from carbon dioxide, before Barker had carried out his experiments with the radio-active carbon isotope. They succeeded in inducing a methane fermentation with isopropanol as the oxidizable substrate, and showed that the latter was quantitatively oxidized to acetone, while simultaneously carbon dioxide disappeared in an amount equivalent to that of the methane generated, according to the equation: 4CH,CHOHCH,-+ CO, > 4CH,COCH,+ CH,+2H,O During their studies on the methane fermentation Kluyver and Schnellen [1947] also discovered two anaerobic bacterial species that can produce methane in an environment that contains carbon mon- oxide as the only oxidizable substrate [Schnellen, 1947Th]. Appro- fig BIOGRAPHICAL MEMORANDA priate experiments led to the conclusion that the conversion of carbon monoxide by these organisms, Methanosarcina barkert and Methano- bacterium formicicum, proceeds in two stages, v2z., an initial conversion of carbon monoxide and water to carbon dioxide and hydrogen, fol- lowed by a reduction of carbon dioxide with the hydrogen liberated in the first reaction; the fermentation can thus be expressed by the equations: 4CO +4H,O — 4CO,+ 4H, CO,+4H, -— CH,+2H,O 4CO +2H,0 — 3C0,+ CH, Ms. barkeri could accomplish this conversion in an atmosphere of pure carbon monoxide; Mb. formicicum appeared somewhat sensitive to this gas, tolerating it in concentrations up to about 12 per cent. While investigating the methane fermentation in ethanol-calctum carbonate media, Barker [1937a] had also observed the formation of higher fatty acids, especially butyric and caproic acids, in those crude cultures that contained, besides the methane producing organisms, an anaerobic sporeforming bacterium. This organism was subsequent- ly isolated in pure culture and described as Clostridium kluyveri; this became the starting point for the highly important studies of Barker and Stadtman on the mechanism of fatty acid synthesis. In the meantime it had also become apparent from studies in other laboratories that carbon dioxide could no longer be considered as a mere end product of the metabolic activities of non-photosynthetic or- ganisms acting on organic substrates. For simultaneously with the demonstration that the methane fermentation represents a process of carbon dioxide reduction, three other metabolic reactions had become known that involve carbon dioxide as one of the participating molec- ular species. These were: 1. The synthesis of formic acid from carbon dioxide and hydrogen under the influence of B. coli, which Woods had shown to catalyze the reversible reaction, HCOOH = CO,+H, This must have come as a severe shock to Kluyver who, some ten years earlier, had strenuously maintained that such a formation of formic acid, postulated by De Graaff, was too improbable to be taken seriously. 118 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY 2. The formation of acetic acid from carbon dioxide and hydrogen by Clostridium aceticum, which Wieringa [1936, 1940] had discov- ered during a study of the utilization of hydrogen gas by micro- organisms under anaerobic conditions. 3. The assimilation of carbon dioxide during the fermentation of elycerol by propionic acid bacteria, observed by Wood and Werk- man. This observation led to the recognition that many cell types can catalyze such a process in which carbon dioxide is condensed with a three-carbon compound, resulting in the formation of four- carbon dicarboxylic acids; this has become the basis on which the production of succinic acid in various fermentations is best explained. This, too, must have come as a complete surprise to Kluyver who until then had defended the thesis that the formation of succinic acid during the anaerobic decomposition of sugars and related com- pounds could only be attributed to an initial cleavage of hexose into a two- and a four-carbon fragment, a possibility first suggested by Virtanen. The reasoning followed by Kluyver is of sufficient general interest to warrant a brief discussion. Kluyver started from the premise that there were only two alternative mechanisms that could account for the formation of succinic acid; in addition to the one suggested by Virtanen and mentioned above, there was the reaction proposed by Thunberg and Wieland which involved a condensation of two molecules of acetic acid with the simultaneous elimination of two hydrogen atoms: HOOC.CH,-++H,C-COOH — HOOC. CH,:CH,:COOH-+-2H This mode of formation had, however, been ruled out by the following argument. If the primary degradation of a hexose molecule invariably yields two triose moieties, then all products with two carbon atoms and their derivatives should be accompanied by an equimolar amount of one-carbon products which, in fermentations, are generally rep- resented by formic acid and carbon dioxide. Now, the results of quantitative analyses of fermented sugar solutions invariably showed that, whenever succinic acid was encountered among the products, there was a marked deviation from the required ratio of two- and one-carbon products in favour of the former. This was considered sufficient to dismiss the Thunberg-Wieland mechanism as a possible source of the dicarboxylic acid. 119 BIOGRAPHICAL MEMORANDA Furthermore, these analytical data indicated that part of the two- carbon products must have originated by a different mode of sugar de- composition, which would not imply the formation of an equivalent amount of one-carbon products; and this was conveniently provided by the postulated degradation to two-and four-carbon fragments, the latter being the immediate source of the succinic acid. And because the quantitative relations between the amounts of succinic acid, two- carbon compounds, and one-carbon products closely agreed with those predicted on the basis of an assumed simultaneous occurrence of both a symmetrical and an asymmetrical cleavage of the sugar, this was consequently considered as conclusive evidence in favour of the mechanism proposed by Virtanen. It will be obvious that this kind of reasoning is pertinent only as long as the one-carbon fragments produced in the course of the fermentation do not undergo subsequent changes by which they are converted into substances with more carbon atoms. The above- mentioned investigations of Wood and Werkman on the actual dis- appearance of carbon dioxide during a fermentation suffice, however, to render the above argument invalid. However great the jolt may have been, Kluyver quickly rallied to the new situation. In a lecture delivered in Helsinki in 1939 he re- viewed the recent developments concerning the participation of car- bon dioxide in the metabolism of heterotrophic micro-organisms and their implications; it was on this occasion that he ventured the remark: ‘Under these circumstances we may well ask .. . whether we should not make ourselves familiar with the idea that even the cells of certain animal organs can assimilate carbon dioxide. ‘If at present a lecturer were to proclaim that the cattle in the pasture, nay, even the members of his audience assimilate carbon dioxide, he may expect to be showered with energetic protests. Never- theless, the temptation to do so is certainly not easy to resist!’ (p. 86). In this lecture Kluyver also discussed certain experimental results of his collaborator, Hes, who had shown that suspensions of various micro-organisms failed to metabolize in a rigorously carbon dioxide- free environment. In trying to account for this fact Kluyver considered a number of possibilities without, however, reaching a satisfactory conclusion. This is not surprising; it was not until the role of the di- carboxylic acids in oxidative metabolism, and particularly in the tri- E20 KLUYVER’S CONTRIBUTIONS TO MIGROBIOLOGY AND BIOCHEMISTRY carboxylic acid cycle, had been clarified that a rational interpretation could be advanced. Meanwhile Kluyver’s theory of metabolism had also opened the way to a re-interpretation of the mechanism of photosynthesis. In ‘Die Einheit in der Biochemie’ this was foreshadowed in the brief discussion of the properties of the purple sulphur bacteria. Here it was pointed out that the earlier attempts of Winogradsky, Engelmann, Molisch, and others to account for the behaviour of these organisms suffered from the fact that they had attributed the importance of light for the development of the purple bacteria to the production of oxygen by a decomposition of carbonic acid with the aid of absorbed radiant energy, even though ‘no one has yet succeeded in demonstrating oxygen production under the most diverse conditions’. But once the dehydrogenation theory of biochemical reactions is accepted, this fundamental difficulty disappears because ‘it is then no longer im- perative to assume that the acceptor for the dehydrogenation of hy- drogen sulphide or sulphur must needs be oxygen; it would be entire- ly possible for some other acceptor to play this role. It is merely nec- essary to retain the principle that the hydrogenated acceptor com- pound can transfer the hydrogen to carbon dioxide with the aid of radiant energy’ (p. 175). The work begun in Kluyver’s laboratory on the metabolism of the purple bacteria soon led to a generalized formulation* of photosyn- thesis as a light-dependent transfer of hydrogen to carbon dioxide with a concomitant dehydrogenation of an appropriate oxidizable sub- stance, H,A, according to the overall equation: light CO,+-2H,A — CH,O+H,0+2A, an exact counterpart of Kluyver’s equation expressing the metabolic processes of non-photosynthetic organisms. This formulation made it clear that oxygen evolution in photosynthesis will be observable only if H,O is the ultimate hydrogen donor for the reduction of carbon dioxide; in all other cases a different dehydrogenation product, ‘A’, should make its appearance. Afterwards Muller [1933Th], on the basis of experiments concluded in Kluyver’s laboratory, established that organic substances may serve as hydrogen donors in purple bac- * Ed. note: C. B. van Niel. Arch. Mikrobiol. 3, 1, 1931. 121 BIOGRAPHICAL MEMORANDA teria photosynthesis, and a year later Roelofsen [1934, 1935Th], in the same institute, made the important discovery that molecular hy- drogen can also fulfil this function. Kluyver’s continued interest in this subject has made itself felt in the many and important contribu- tions to photosynthesis published by the ‘Biophysical Group’ in Utrecht, largely supported by the Rockefeller Foundation, and of which Kluy- ver was one of the two directors. This same group also significantly advanced our knowledge of bioluminescence. Although several of the publications on this phe- nomenon deal with its physical aspects, some were clearly inspired by the comparative biochemical approach; these may here be men- tioned in particular. In a series of measurements on the relation between oxygen con- sumption and intensity of luminescence of suspensions of luminous bacteria under diverse conditions, it was convincingly established that specific inhibitors of respiratory activity could reduce the rate of oxygen utilization by a large factor without appreciably diminishing the amount of radiant energy emitted. This result led to the notion that bioluminescence, although characteristically an oxidative pro- cess, should be interpreted as an incidental side reaction of the normal respiratory mechanism. It also became the basis of a simple modifi- cation of Beijerinck’s classical method for demonstrating the depend- ence of bioluminescence on the presence of oxygen, with which this point can be illustrated in a striking and elegant manner. ‘The exper- iment of Beijerinck is carried out as follows: a glass tube is filled al- most completely with a suspension of luminous bacteria, whereupon the open end of the tube is closed off. After standing for some time the suspension no longer emits light except at the very boundary between the liquid column and the gas phase, because the oxygen initially present in the suspension has been completely consumed. If now one turns the tube upside-down, the air passes through the liquid, which causes the entire suspension temporarily to become luminescent once more; the duration of this spectacle depends on the amount of oxygen that has gone into solution as the air bubble passes through the suspension, and on the rate of its consumption, which latter is nor- mally determined by the density of the suspension. The experiment can be repeated many times, and will only fail if all the oxygen in the gas phase has been used up. 122 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY In the above-mentioned modification this experiment is conducted with two such tubes. When it has been ascertained that after turning the tubes upside-down the luminescence persists in both tubes for approximately the same length of time, a small amount of cyanide is added to one of the tubes, and after the suspensions have once more become non-luminous, both tubes are simultaneously inverted. The immediately observable intensity of the light emission is not per- ceptibly different in the two tubes; but in the tube with cyanide the luminescence lasts very much longer, demonstrating that the oxygen supply, through inhibition of the respiration, disappears at a greatly reduced rate. Another important contribution of this group was the unequivocal demonstration with the aid of luminous bacteria that, contrary to what had been claimed by Keilin and Hartree, oxygen is not required for the decomposition of hydrogen peroxide by catalase. In a gas-tight apparatus, in which the absence of even trace amounts of oxygen could be guaranteed by virtue of the fact that a suspension of lumi- nous bacteria failed to emit light, catalase and hydrogen peroxide were mixed. This resulted in the instantaneous reappearance of lumi- nescence, signifying that oxygen was being produced, and hence that the enzymatic decomposition of the peroxide does not depend on the presence of free oxygen. Afterwards Keilin and Hartree found that their original contention was based on a misinterpretation of their experimental results, and that these were caused by the inactivation of the catalase by toxic nitrogen-oxygen compounds that were present in the gas they had used to render their apparatus oxygen-free. In the preface to his lectures at Harvard University on the anatomy of science, G. N. Lewis, one of the great philosopher-scientists of this century, stated that ‘the strength of science lies in its naiveté’. This also holds for Kluyver’s biochemical speculations and generalizations, and he was himself fully aware of it, as is shown by his use of Lewis’ phrase as the motto for the preface to his own London lectures. The attribute of strength of these biochemical concepts is amply evident from the fact that, in reducing the whole of biochemistry to a very small num- ber of reaction patterns, a great simplification was achieved which also permitted inferences with respect to some hitherto enigmatic bio- chemical processes that thereby appeared in a fundamentally new 123 BIOGRAPHICAL MEMORANDA light, and thus contributed to their eventual solution. Furthermore, the comparative biochemical approach often indicated an immediate answer to some less baffling problems. A good example is furnished by the identification of a pigment produced by Pseudomonas aureofa- ciens, discovered in Kluyver’s laboratory, as phenazine-a-carboxylic acid. It is significant that Kluyver [1956], in his posthumous publi- cation on the subject, introduced the experimental part in the follow- ing words: ‘It is well known that the genus Pseudomonas comprises several pig- ment-producing species, and that in all cases in which the constitution of these pigments has been established they have been found to be phenazine derivatives. ... (Thus it seemed) worthwhile to look out for further pigment-producing species within the genus Pseudomonas in order to check if still other phenazine derivatives occur as natural products. ‘The opportunity presented itself in 1936 when Mr. Bouman, working in this laboratory, incidentally came across a bacterium which was easily identified as a Pseudomonas species, and which attracted atten- tion by an ample formation of yellow crystals in its colonies’ (p. 406). But the element of naiveté was not lacking either; it cannot be denied that Kluyver sometimes found it hard to resist the temptation to promulgate an explanation of certain phenomena even in the ab- sence of an adequate body of factual information to support it; and the tendency to be satisfied with such an explanation if only it sup- ported the concept of comparative biochemistry and the ‘unitary theory’ may occasionally have delayed more searching studies. This aspect is illustrated by the studies on yeast metabolism that were intended to show that certain observations made elsewhere, al- though at first sight contradicting Kluyver’s theory, could nevertheless be reconciled with it. The investigation of the co-zymase problem discussed earlier was a case in point; here another example may be mentioned. After having invoked an identical series of primary reactions for the transformation of sugar into two triose moieties for the explanation of all known sugar fermentations, it was, to use a favourite phrase of his, ‘tempting to postulate’ that the same conversions would also be in- volved in the initiation of the oxidative degradation of sugars by many different organisms. This ‘unitary theory’ was threatened when Lunds- gaard published his studies on the effect of mono iodoacetic acid on 124 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY the fermentative and oxidative metabolism of yeast in which it had been shown that fermentation can be completely suppressed by con- centrations of the poison that had no appreciable effect on the oxi- dative metabolism. Kluyver and Hoogerheide [1933b], after having corroborated Lunds- gaard’s experiments and determined the exact concentration of iodo- acetic acid that produced the differential effect under the conditions of their experiments, then found that even the slightest raise in con- centration of the inhibitor immediately affected the respiratory activ- ity as well. This result was used to defend the thesis that the situation was fully compatible with the unitary theory. The argument was developed on the basis of the following assumptions: zr. Methyl glyoxal is the immediate substrate for both fermentation and oxidation; 2. oxidation takes precedence over fermentation, but the rate of the former is limited by the capacity of the oxygen-activ- ating catalyst; 3. in the absence of iodoacetic acid the rate of methyl glyoxal formation greatly exceeds that of oxygen activation, so that under these conditions a considerable surplus fermentation will be observable; and 4. the principal effect of iodoacetic acid is that it diminishes the rate of formation of methyl glyoxal. From these assumptions it logically followed that an inhibition of the oxidative sugar metabolism of yeast by iodoacetic acid can be expected only if its concentration has become sufficiently high to cause the rate of methyl glyoxal formation to drop to the point where it is less than the rate of oxygen activation; at lower concentrations the poison will merely cause a decrease in the rate of fermentation. This explanation is certainly not without merit. But the above as- sumptions also imply that, at the concentration where iodoacetic acid just begins to inhibit the respiratory metabolism, methyl glyoxal must still be generated at a commensurate rate, so that at this concen- tration a comparable rate of fermentation should still be observable under anaerobic conditions. This is contrary to fact. Kluyver and Hoogerheide were well aware of this difficulty, and explained the disturbing fact by invoking the auxiliary hypothesis that, in addition to inactivating the methyl glyoxal-producing enzyme system, iodo- acetic acid also causes an oxidation of reduced glutathione which latter substance would play a vital role in the fermentation reactions proper. Similarly, the early studies by Kluyver and co-workers on the oxi- 125 BIOGRAPHICAL MEMORANDA dation of disaccharides by yeasts that cannot ferment these sugars, were, as has been mentioned earlier, largely attempts to negate the occurrence of such phenomena which were contrary to the expecta- tions based on the unitary theory. Only at a later stage, when experi- ments in his own laboratory had incontrovertibly shown the reality of such occurrences, was the situation investigated in somewhat greater detail. In the publication with Custers [1940] on the suitability of disaccharides as respiration and assimilation substrates for glucose fer- menting yeasts that do not ferment these sugars the problem is intro- duced in a manner calculated to emphasize the difference with which it had been treated by others who had recorded similar observations: ‘It is true that these authors do not offer any special comments to their results’ (p. 123), suggesting that they may have been unaware of the fundamental problem that was raised by such experimental findings. When they had established that certain yeasts can oxidize a number of disaccharides which they cannot ferment, Kluyver and Custers next attempted to detect the presence in such yeasts of enzymes that can hydrolyze these sugars to their constituent hexose units. The positive results of these experiments indicated that the inability of the yeasts to ferment the disaccharides could not be attributed to the ab- sence of appropriate hydrolases per se. This made it unnecessary to ascribe the oxidation of such sugars to the operation of a mechanism that did not involve a preliminary hydrolysis, sometimes postulated and designated as a ‘direct’ oxidation of disaccharides. Since the hy- drolases were present, the only way to explain the non-fermentability of the disaccharides seemed to call for the assumption that ‘under anaerobic conditions these hydrolases are inactivated either complete- ly, or at least to such an extent that the fermentability of the disac- charide is not detected by the relatively insensitive routine methods for the determination of this property’ (p. 159). Two possible reasons for the inactivity of the hydrolytic enzymes under anaerobic conditions were briefly discussed. The first one im- plicated a differential permeability of the disaccharides in the pres- ence and absence of oxygen. After reviewing the results obtained by various investigators in studies on cell permeability under different conditions it became clear that in some cases permeability may be increased, in others decreased by anaerobiosis. This situation led to the following argument: 126 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY ‘We must, therefore, conclude that the question of the influence of oxygen on the permeability of yeast cells has not yet definitely been settled. ‘Nevertheless, it is difficult to conceive that anaerobiosis should decrease the permeability of the yeast cells for the disaccharides under consideration. For these cells show a normal Pasteur-effect with glu- cose or well-fermentable disaccharides as fermentable substrates, prov- ing that by withdrawal of oxygen the permeability for these sugars either remains unchanged, or even — according to Dixon’s conception — is considerably increased. Now, it seems extremely improbable that the cells behave quite differently towards supposedly unfermentable disaccharides than towards the other sugars mentioned. ‘On rejecting the permeability hypothesis we have to accept the second possibility, vzz., that the inactivation is due to a reversible change of the catalyst itself. Taking into account that withdrawal of oxygen inevitably leads to an increase in the state of reduction in the interior of the cell, it is only logical to assume that the said change is connected with a reduction process. ‘Until now no evidence for the idea that carbohydrases are in- activated by reducing agents is available’ (p. 160). This statement is difficult to interpret. Does it mean that, by using this negative phraseology, the authors tried to evade another problem that could here be raised? For it is obvious that this explanation does not answer the question why these same disaccharides can readily be fermented by other yeasts under equally reducing conditions, so that it could legitimately have been said that there was proof positive to the effect that the hydrolases are not inactivated simply as a result of the absence of oxygen. Moreover, Kluyver and Custers had ob- tained from autolyzed yeast suspensions preparations exhibiting hydro- lytic activity, and it should have been easy to test the effect of an- aerobiosis and of reducing substances on the activity of the hydrolases. Nevertheless, the publication contains no evidence that such experi- ments were either carried out or even contemplated. These remarks suggest that, once a reasonably satisfactory explanation had been found, the problem was considered settled. It is, of course, possible that the experiments had actually been made, but, contrary to expec- tation, had yielded results that refuted the contention. A somewhat similar situation has previously been discussed in connexion with 127 BIOGRAPHICAL MEMORANDA Kluyver’s early investigation of the assimilation of disaccharides in which it had been found that maltose could act as a satisfactory nu- trient by virtue of the impurities it contained. But in that case the predicament was a fundamentally different one because the fact that purified maltose failed to induce growth could only mean that other substances were responsible for the effects and it was not necessary to identify them. In the present case the question was whether or not anaerobiosis could inhibit the activity of hydrolases of particular yeasts. Hence a negative result would have invalidated the inter- pretation proposed by Kluyver and Custers, and the withholding of pertinent experimental results would not have been sound scientific strategy. Therefore it seems more reasonable to conclude that the experiments were not performed. This same publication also contains some remarks indicative of Kluyver’s dissident attitude towards the concept of a ‘direct’ oxidation of disaccharides. It was claimed, for example, that ‘it is also quite difficult to conceive that a disaccharide will undergo oxidation with- out preceding hydrolysis. ... Such a “‘direct” oxidation . . . is further quite incompatible with the unitarian theory of respiration and fer- mentation’ (p. 122). Again the implication seems to be that the ‘uni- tarian theory’ must be considered as the most satisfactory guide in for- mulating interpretations of biochemical phenomena. But in science a theory, no matter how attractive, must yield to facts, and in later years Kluyver had to admit the existence of such direct oxidations. In fact, with De Ley and Rijven [1951] he extended the earlier observations of Stodola and Lockwood on the oxidation of maltose and lactose to malto- and lactobionic acids. In a way this was not too revolutionary a discovery, for these oxidations are carried out by members of the Pseudomonas group with a strictly oxidative metabolism, and it was al- ready known that at least some species of this group also oxidize glucose directly to gluconic acid. In this, as in other respects, these microbes resemble the acetic acid bacteria, and for the latter an ini- tial non-oxidative conversion of sugar to triose had never been as- sumed. | Strict adherence to the unitary theory of fermentation and oxida- tion had to be abandoned, however, when soon afterwards it was established that the typically fermentative metabolism of some bac- teria also proceeds via a primary oxidation of hexose sugars. By the 128 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY use of isotopically labeled glucose Gunsalus and co-workers, Gest and Lampen, and Gibbs and De Moss demonstrated that the distribution of the labeled carbon atoms of the substrate among the fermentation products arising under the influence of heterofermentative lactic acid bacteria and <_ymomonas mobilis (Ps. lindnert) is entirely incompatible with the functioning of a mechanism of sugar decomposition whereby the hexose is first broken down to two triose molecules. Present information strongly supports the concept that the reaction sequence consists in an initial oxidation of the substrate to gluconic, and next to keto-gluconic acid; a decarboxylation of the latter; and finally a cleavage of the resulting pentose to a two- and three-carbon fragment. Meanwhile De Ley had shown that the potentially fermentative Aerobacter species can also carry out such a direct oxidation of glucose, while Horecker and co-workers discovered a cyclic pathway for the oxidation of glucose via gluconic acid and pentose that now appears to be of quite general occurrence in many different kinds of cells. Needless to say, these developments have indicated that the old ‘uni- tarian theory’ can at best be applied to a very much more restricted number of cases than was at first believed. In his second Harvard lecture Kluyver discussed in detail some of these newer findings, and introduced the startling change in outlook with the remark: ‘Fortunately, however, this age has also its “‘scep- tical biologists” ’ (p. 38), concluding that review with the statement: ‘Meanwhile we have learned that “there are more paths between heaven and earth” than until recently had been dreamt of in the phi- losophy of the comparative biochemist’ (p. 45). This sentence represents an admission of the inadequacy of the older concepts. Indubitably it has become evident that the small number of reaction patterns initially envisaged by Kluyver as constituting the whole of biochemistry must be greatly expanded if we are to com- prehend the various biochemical processes in a manner more in keep- ing with mounting experience, and it seems a foregone conclusion that a continued study of biochemical mechanisms will reveal a number of as yet unknown types. Kluyver had begun his search for unity in the wild variety of bio- chemical manifestations in 1923. In a single decade this search had produced a theory that has inspired a whole generation and guided its efforts into productive channels. Is it to be wondered at that Kluy- 129 BIOGRAPHICAL MEMORANDA ver, having experienced the powerful influence of these unsophistic- ated concepts, did not always succeed in immediately freeing himself of the admittedly somewhat naive notion that they would suffice to account for all the facts? | But it is also understandable that lately the diversity has once again come to occupy the centre of attention. This has had the salutary effect of greatly expanding our factual knowledge; and, just as the multiplicity of elementary particles now accepted by physicists must sooner or later engender the desire to discover new unifying principles, so it is to be expected that in biochemistry, too, the ever increasing number of recognized reaction patterns is apt eventually to lead to the emergence of concepts from which an even more profound unity will become apparent. In the Harvard lectures Kluyver alluded to such a development in the following passage: ‘Thus we are led to the conclusion that the most fundamental char- acter of the living state is ... a continuous and directed movement of electrons. «4, ‘Such reflections suggest the possibility of gradually achieving an even greater simplification and unification of our views on the mech- anism of metabolism than can presently be envisaged’ (p. 71-72). This testifies to a thorough appreciation of the nature of science. INDUSTRIAL MICROBIOLOGY AND THE SUBMERGED CULTURE METHOD FOR THE STUDY OF MOULD METABOLISM In his inaugural address Kluyver had emphasized the potential use- fulness of micro-organisms for the large-scale production of certain types of raw materials needed by the organic-chemical industry. A strong argument in its favour was the consideration that this would retard the frightening rate at which the deposits of fossil fuels were being depleted, because the microbiological processes of greatest im- portance in this respect are based upon the decomposition of agricul- tural materials, often even available as waste products. Kluyver re- mained keenly aware of the dangerous tendency of mankind to exploit the limited and irreplaceable natural resources of our world without paying heed to the obvious consequences. This is evident from the remarks he made in the lecture he delivered before the ‘Holland’s Society of Sciences’ under the title ‘Homo militans’: 130 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY ‘There is but one enemy of Homo sapiens, and probably the most frightening of all, whom I shall deliberately disregard; this is his brother, Homo ignorans. Whosoever wishes to become deeply disturbed by the manner in which, through ignorance and thoughtlessness, man himself is engaged in undermining his terrestrial existence, should read the recent book by Fairfield Osborne, “‘Our Plundered Planet’’. In this treatise, dedicated “‘to all who care about tomorrow’’, the famed President of the New York Zoological Society demonstrates how man, in consequence of the alarmingly rapid populaticn increase, has resorted more and more to a rapacious exploitation, and thus has gradually become a geological force that even now is rapidly changing the once flourishing appearance of our planet into something akin to the desultory landscape of the moon’. It was, therefore, not so much his belief that a Professor of Micro- biology at a Technological University should concern himself prima- rily with the industrial application of micro-organisms on account of his position, but rather his penetrating insight into the problems of an industrial civilization, that caused him to pay close attention to the potentialities of the microbial world as purveyors of useful ingredients for industry. In connexion with the discussion of the discovery of A, suboxydans it has already been mentioned that he had appre- ciated the usefulness of the mildly and specific oxidative properties of this organism for the production of various keto-compounds, and consequently had patented this application. The same kind of fore- sight had been displayed in the case of the microbiological formation of 2,3-butane diol and acetyl methyl carbinol during the fermentation of carbohydrates under the influence of Aerobacter and Aerobacillus cul- tures. Kluyver did not fail to realize that the glycol might become an important starting material for the manufacture, by a simple dehydra- tion process, of butadiene, which in turn is readily convertible into polyenes with rubber-like properties. The extensive studies carried out during the second world war, especially in the laboratories of the Canadian Research Council, have fully substantiated Kluyver’s ex- pectations, and it seems therefore all the more regrettable that the synthetic rubber industry has so exclusively concentrated its efforts on the production of butadiene from petroleum products, thereby causing an additional drain on the fossil fuels rather than making use of carbohydrates for the manufacture of butadiene. 131 BIOGRAPHICAL MEMORANDA Furthermore, the butane diol fermentation could also be modified so as to yield acetyl methyl carbinol instead of the glycol as a major end product. Scheffer, in Kluyver’s laboratory, had shown that this can be accomplished by a proper aeration of Aerobacter cultures in sugar media; this apparently prevents the reduction of the primarily formed carbinol to a large extent, so that under these conditions it can accumulate in significant amounts during the fermentation. Now, acetyl methyl carbinol had been noted as a regular component of wine vinegar by Visser *t Hooft [1925a, 1925b, 1925Th] in Kluyver’s institute; and he had also shown that its concentration in vinegar can be used as an excellent index of the quality of this condiment. Even more important was the fact that, in 1927, Kluyver and co-workers had identified the substance principally responsible for the character- istic and delicate flavour of high-grade butter as diacetyl, which also appears to be an important flavouring ingredient of many another food product [Kluyver, Van Niel and Derx, 1g29a, 1929b]. The ready convertibility of acetyl methyl carbinol into diacetyl, either by means of a purely chemical oxidation or, even better, with the aid of A. suboxy- dans, thus emphasized the potential usefulness of the butylene glycol fermentation for several different purposes. When, after the first decade of Kluyver’s directorship of the Delft laboratory, the most important microbial sugar fermentations had been surveyed, he could begin to pay special attention to the metab- olic activities of various moulds, also briefly mentioned in the inau- gural address. These often lead to the production of organic acids, notably gluconic, fumaric, and citric acids, although the fundamental mechanism whereby these substances are formed were not clearly understood. It could thus be expected that a better comprehension of this type of metabolism might permit the industrial microbiologist to use conditions which improved the yield of these particular com- pounds, or which might be conducive to making the production of some other substances, sometimes formed in very small quantity, economically feasible. Apart from this the insight gained into metab- olic reactions generally might be considerably increased by such a study of oxidative processes. From the beginning it was evident that a commercially useful aspect of mould metabolism is that which is characterized by an in- complete oxidation of the substrate. Furthermore, the large number 132 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY of previous investigations in this field had clearly indicated that the nature of the metabolic conversion is often strikingly dependent on the conditions under which the organisms were grown. This led to the fundamental problem in how far it would be possible to differentiate between the influence of environmental conditions obtaining during growth and affecting mainly the potentialities of the cells, and of those existing during the later stages of a culture and causing a particular cell type to produce either one or another metabolic product. Early experiments on mould metabolism had generally been con- ducted with growing cultures in stationary liquid media. Here the environmental conditions are subject to continuous changes, owing to the assimilation of various ingredients of the culture medium and to the accumulation of metabolic products. This had made a reasonably critical evaluation and interpretation of the results of such experiments well-nigh impossible. Besides, with such cultures it could not be as- certained whether a particular product was derived directly from the substrate provided, or might have originated by a very different, round-about route. The reason for this is that growth implies the for- mation of new cell materials, including reserve products, so that the latter, rather than the substrate itself, might be the direct precursors of the metabolic products in question. The formation of kojic acid by the mould, Aspergillus flavus, is a case in point. It is easy to envisage its formation from glucose; the closely similar constitution of the two substances suggests that the conversion would consist in a partial oxidation and dehydration of the sugar. But the same mould had also been reported to produce kojic acid when grown at the expense of various other substrates, such as pentoses, erythritol, and glycerol; and this is rather difficult to understand as the result of a direct de- gradation because their constitution does not at all resemble that of the product. It seemed therefore much more reasonable to assume that in these cases the substrates are first converted into reserve materials that subsequently can yield glucose, and that the kojic acid is actually formed only from this substance. In order to test this hypothesis Kluyver and Perquin [1933b] pre- pared a culture of Asp. flavus in the manner described in more detail below, and exposed aliquots to solutions of glucose, fructose, galactose, arabinose, xylose, mannitol, erythritol, and glycerol. Analysis of the 133 BIOGRAPHICAL MEMORANDA cultures showed that, except in the glucose solution, kojic acid was not produced, thus supporting the contention that the positive results obtained by others with growing cultures were due to substrate trans- formations via glucose, and involving growth of the mould. It should, however, be mentioned that this experiment does not provide con- clusive evidence in favour of the proposed interpretation; it is at least possible that mycelia grown in the presence of other substrates might have yielded different results. But the main point of this discussion is to illustrate the fundamental difficulties inherent in the use of growing cultures for the study of mould metabolism. It was therefore a distinct improvement when the method of using pre-grown mycelium was introduced. This could first be freed of the initial medium by washing, and then tested for its effect on specific substrates under much better controlled conditions. Nevertheless, even this technique had been found far from satisfactory ; this follows clearly from the fact that it is virtually impossible to du- plicate the results of such experiments. It was the keen recognition of the complex situation encoun- tered in cultures of filamentous fungi that led Kluyver and Perquin [1933a] to develop a new methodology for the study of mould metabolism; this was published in 1933 under the name of ‘agi- tated cultures’. The arguments for abandoning the stationary cul- tures, even those with preformed mycelial mats, have been set forth so clearly, and are so convincing that they are here quoted in some detail. Having raised the question what factors are responsible for the oftentimes incomplete oxidations performed by moulds, Kluyver and Perquin concluded: ‘It is clear that a priori two different reasons could be held respon- sible for this behaviour. Firstly, it is conceivable that the cells are characterized by a specific low oxidative capacity. This is true, for example, in the case of A. suboxydans, whose oxidative activity towards certain substrates is restricted to a single dehydrogenation, even under widely different external conditions. But secondly, it has long been known that in many cases an incomplete oxidation is determined by outside influences, so that cells fully capable of performing a complete substrate oxidation may, under special circumstances, cause the ac- cumulation of incomplete oxidation products. For such materials 134 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY > Duclaux has introduced the suggestive term, “‘products of suffering”’ (p. 69).* Now, the experiments of Molliard had shown that the mould, Asp. niger, which can oxidize sugar without causing the accumulation of any acid whatever, may be induced to form considerable quantities of gluconic, citric, or oxalic acid by growing it in a culture medium with drastically reduced nitrogen, phosphate, or potassium content. ‘Consequently’, continued Kluyver and Perquin, ‘it was obvious that for this mould at least the industrially important acid production is not a physiological, but rather a pathological process. Viewed in this light the inadequacy of the methods hitherto used in the study of mould metabolism hardly needs commentary. It must be evident how utterly heterogeneous are the conditions under which the individual cells exist in a mycelial pad growing on the surface of a liquid culture medium. Whereas the cells in the upper layers are bathed in the oxygen of the atmosphere, they must depend for their supply of nutrients on the dif- fusion of the latter through the mycelium, and only that fraction which is not absorbed by the cells lower down will reach them. Conversely, the latter type of cells swim in a substrate-rich environment, but have access to very limited amounts of oxygen because this gas is largely consumed in the upper layers. Between these two extremes all possible intermediate stages exist. It must be realized that part of the cells may be so poorly nourished that they become considerably weakened, and may even undergo autolysis. ‘In connexion with results such as those of Molliard’s this implies that the metabolism of the individual cells in the mycelium will ex- hibit pronounced mutual differences, and that the customary anal- ysis of the over-all processes in such a liquid culture can only provide information concerning the combined results of the diverse metabolic processes that proceed under the influence of the various cell types. ‘If one further keeps in mind that the structure of the mycelial mat * After the discovery of A. suboxydans with its intrinsically low oxidative capac- ity, the transient accumulation of incomplete oxidation products in liquid cultures of A. xylinum was attributed by Visser ’*t Hooft [1925] to the poor oxygen provision underneath the thick pellicle which is the characteristic growth form of this organ- ism. This notion is supported by the fact that on glucose-calcium carbonate-agar plates A. xylinum does not even produce detectible amounts of acid. Kluyver used to refer to this bacterium as a ‘physically conditioned sorbose bacterium’, in con- trast to the ‘ideal sorbose bacterium’ which is, of course, A. suboxydans. P30 BIOGRAPHICAL MEMORANDA — either adhering closely to the surface of the medium, or forming more or less pronounced and raised folds — can often be greatly modified by factors that are difficult to control, then it is no longer surprising that different results are so often obtained in duplicate cultures under pre- sumably identical conditions. But even if such cultures were to show satisfactory agreement it is still impossible to specify sharply the con- ditions responsible for a particular direction of the metabolic pro- cesses. If, for example, a particular situation leads to the formation of large amounts of citric acid, it is impossible to decide which of the cells in the mycelium, developing under heterogeneous conditions, have been responsible for the acid production. And even if it were possible to determine which cells have produced the acid, and to specify the optimum conditions for this process, the result would still be of only limited significance, because it would be erroneous to con- clude that any cell of the organism would display the metabolic activ- ities characteristic of the acid production under these same optimum conditions. One can find many examples in the literature showing that the metabolism of mould cells is greatly dependent on the conditions under which the cells were grown, and we shall presently add further proof for this statement. Now it is obvious that these conditions are quite heterogeneous for the cells developing in a mycelial mat on a liquid medium. In summary we may therefore conclude that one has to reckon with the presence in such a mat of cells of heteroge- neous origin, exposed to heterogeneous conditions. It will then be clear that one can obtain at best a very restricted picture of the metabolic activities of moulds with the aid of the usual stationary cultures. Consequently a deeper penetration into the problems of this metab- olism is possible only if it is investigated with cell material of homoge- neous composition, studied under homogeneous conditions’ (p. 70). This, of course, raised the question how such cell material of homo- geneous composition could be procured. At the outset it seemed pos- sible that this might be accomplished by growing the mould so that it would develop submerged in ‘a culture liquid rather than on top of it. This would necessitate aeration of the culture in order to supply the cells with an adequate amount of oxygen. After testing a number of different arrangements the most satisfactory results were obtained by inoculating a liquid medium with mould spores, and placing the culture on a shaking apparatus, thus maintaining the contents in a 136 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY state of continuous and violent agitation. ‘This completely prevented pellicle formation and at the same time assured a plentiful oxygen supply. In such cultures the mould developed in the form of small balls, of uniform size, composed of tangled threads of cells that had originated under conditions as nearly homogeneous as possible. ‘The balls of mycelium could be conveniently handled; a simple filtration through ordinary filter paper sufficed to collect and wash them free of adhering medium. Thereafter uniform suspensions could be pre- pared from the residue on the filter paper, permitting experiments to be conducted with a rigorous control of factors, varied one at a time. Application of this technique to problems of kojic, gluconic, and citric acid formation showed that the results were reproducible to a remarkable degree. This also led to a better specification of the op- timum conditions for the production of these substances, although it contributed little to advancing our understanding of the mechanism whereby they are generated. The significance of this new methodology is difficult to overestimate. Foster, in his book ‘Chemical Activities of Fungi’, has paid due credit to it in the passage: ‘From much of the foregoing it is evident that for the most effective studies on mold physiology and biochemistry, none of the methods already described are completely adequate. Submerged growth cul- tures theoretically and practically afford the closest approach to the ideal method of studying mold metabolism. The principles involved in the technique, and the implications possible for the interpretation of physiological studies with fungi under different conditions were first clearly enunciated in 1933 in a classic paper... by Professor A. J. Kluyver and his student and collaborator, L. H.C. Perquin. This and subsequent papers by these authors represent the first attempt to study mold metabolism systematically under strictly controlled con- ditions, and to elucidate some fundamental principles obtained thereby’ (p. 51). Those who are familiar with current practices in the industrial production of penicillin, streptomycin, and other substances formed by filamentous microbes will realize how completely this method has been adopted in microbiological practice. But that this method was devised some twenty five years ago in Kluyver’s laboratory, and on purely theoretical grounds, is insufficiently appreciated. 137 BIOGRAPHICAL MEMORANDA THE CLASSIFICATION OF MICRO-ORGANISMS In the inaugural address Kluyver had also stressed the need for an intensive study of micro-organisms in various directions as a prelimin- ary to developing a more satisfactory system of classification than those in use at the time. ‘It is well known’, he had asserted, ‘that even to-day there exists an exasperating confusion in the area of the clas- sification of the Schizomycetes’. Although the confusion was all too evident from the mere fact that several conflicting and uncoordinated systems were in use side by side, it is doubtful whether Kluyver could have indicated in what respects these systems were defective, or how they could be improved. For it must be realized that a good system- atist must above all be thoroughly familiar with the material to be classified, and this requires an extensive first-hand knowledge and ex- perience such as Kluyver certainly did not yet possess. But through the isolation of numerous pure cultures of diverse micro-organisms and the study of their characteristics in his laboratory this deficiency was gradually repaired. And together with his amazing memory and acute sense of order, the experience gained soon helped him to acquire the background necessary for making constructive proposals. The chief reason for the confused state of bacterial classification was that bacteriologists with divergent aims and fields of interest had not been able to agree on what properties should constitute acceptable criteria for the segregation of taxonomic categories. As is only natural, each one had brought his own experience and preferences to bear on the problem. But these were often so different that several independent approaches had been advocated, each one supported by one or more specialists, and leading to several almost ‘private’ systems. Differentiation on the basis of morphology had long ago been used by Ferdinand Cohn as a means of classifying the bacteria known to him in a few ‘form genera’. In due course some additional morpholog- ical features, such as flagellation, spore formation, and staining prop- erties, had permitted a much needed expansion of the number of these entities. Nevertheless, it soon became clear that the genera com- prised types whose further differentiation was desirable, and this could be accomplished only on the basis of non-morphological criteria. Hence ecology, pathogenicity, and specific biochemical and serol- ogical properties had been introduced as additional aids in distin- 138 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY guishing pure cultures of bacteria one from another. Usually, how- ever, the identification of a culture did not seem to require the deter- mination of more than a few of such characteristics, and this frequently caused all others to be neglected. As a consequence one and the same organism might be recognized by one specialist on account of its ap- pearance and mode of growth in various culture media, by another because of its effects on experimental animals, by yet others on the basis of the occurrence of particular chemical changes observed in certain environments. The result was that many bacterial cultures were only partially characterized, and not always by means of the same criteria, so that a single type might even be described under a variety of names. As long as the total number of known bacterial types was relatively small, the few morphologically characterized groups, the ‘form gen- era’ established in the early days of bacteriological research, sufficed as a primary basis for descriptive and nomenclatorial purposes. How- ever, the rapid increase in knowledge eventually led to the practice of combining groups of bacteria into genera that were no longer de- marcated primarily or exclusively with reference to morphological features. Especially Winogradsky and Beierinck introduced many ‘genera’ of this sort; Nitrosomonas, Nitrobacter, Acetobacter, Azotobacter, Aerobacter, Photobacterium, Thiobacillus, and Lactobacillus may be men- tioned as examples. Now, the great advantage of such genera is that they effectively speed up identification of a newly isolated strain; any bacterium that lives in an alcohol-containing medium and produces acetic acid therein immediately becomes recognizable as an Aceto- bacter species; a luminous bacterium is, ipso facto, a Photobacterium; or a non-sporeforming nitrogen fixing bacterium an Azotobacter. But the practice of originating such genera also had some distinct disadvan- tages. For example, neither Winogradsky nor Beijerinck had ever taken the trouble to explain his reasons for proposing such genera, probably because they had considered them to be self-evident. As a consequence the undesirable implications of the procedure became clear only at a later date. Was it sound practice to include physiological properties amongst the diagnostic features of a genus? As early as 1872 Cohn had dis- cussed this problem in connexion with the differentiation of species within his form genera, and warned of the dangers inherent in this 139 BIOGRAPHICAL MEMORANDA practice. His negative attitude was based on a simple and convincing argument. Consider, he had said, two almond trees, one producing sweet, the other one bitter almonds. Obviously these two specimens exhibit a physiological difference that is as striking as it is constant. Nevertheless, no botanist would ever be induced to regard these trees as different species. And what guarantee was there that physiol- ogical differences between two types of bacteria were any more sig- nificant than those exhibited by the two almond trees? Now if, for these reasons, it appeared ill-advised to use physiological charact- eristics for the differentiation of species, how much more forcibly would the argument apply to Winogradsky’s and Beijerinck’s genera! Furthermore, one might legitimately ask whether such differences were constant. Also in this respect the dangers were certainly not imaginary; it need but be pointed out that Beijerinck himself had shown how easily a strain of luminous bacteria may permanently lose its ability to emit light; this happens, for example, if it is cultivated at moderately high temperatures, where growth is not adversely affected. This implies that such a modified strain would henceforth have to be identified as a member of a different genus! Despite this criticism many of the above-mentioned genera have been perpetuated, largely because they proved to be so eminently useful. No doubt this argues strongly for the thesis that Winogradsky and Beijerinck both had a highly developed ‘taxonomic intuition’. Later studies have shown that many of their genera represent assem- blages of organisms with a considerable number of common, more or less basic properties. And spurred on by the example set by the two great general microbiologists, the tendency to employ physiological characteristics in bacterial systematics gradually spread. In view of the paucity of morphological properties and the obvious need for more refined supplementary means of differentiation, this was inevitable. The most elaborate and consistent use of generic names for groups of bacteria with similar physiological properties was made by Orla- Jensen in his attempt to construct a system of bacterial classification that was purported to reflect the phylogenetic relationships of these microbes. He began by recognizing two morphologically distinct lines, represented by the orders Cephalotrichinae and Peritrichinae, comprising the bacteria with polar and peritrichous flagella, respectively; non- motile bacteria were, somewhat arbitrarily, assigned to one or the 140 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY other of these groups. The subdivision of the two orders into genera was based primarily on physiological criteria, and the genera, in- cluding many new ones, were therefore largely physiologically defined. The new names proposed were designed to express directly the main morphological and physiological attributes of their members. Although the approach had much to recommend it, the increased use of physiological properties also led to developments that may be deemed less satisfactory. The medically oriented bacteriologists now felt justified, and I believe rightly so, in regarding pathogenicity as a differential character on a par with certain biochemical ones. Eventu- ally this led to the creation of special genera for disease-producing bacteria, and these sometimes overlapped with genera of non-patho- genic organisms. For the development of a consistent system of clas- sification it therefore became necessary to assess the relative values of several unrelated physiological properties, and, in the absence of acknowledged standards, it usually proved impossible to achieve con- cordance amongst specialists of different persuasion. Another unfor- tunate result of the application of physiological properties for the crea- tion of taxa of higher order was that the extremely limited compre- hension of what constitutes the fundamental characteristics often led to the use of what later could be regarded as very minor qualities. Now, as mentioned earlier, the discovery of Acetobacter suboxydans, and the comparison of its properties with those of other acetic acid bacteria, provided the opening wedge for Kluyver’s entrance in the field of bacterial taxonomy. The very appearance of cultures of this organism on sugar-calcium carbonate-agar plates left no doubt that it represented a hitherto unknown type, and the biochemical studies had shown that it could be characterized by its low oxidative capac- ity. Because the other representatives of the acetic acid bacteria also seemed to exhibit differences in this respect, it had become possible to arrange all of them in order of their oxidizing ability, and to assign to those with clearly recognizable differences the status of a species or species group. This approach could also be applied readily to some strains of acetic acid bacteria that Visser ’t Hooft had isolated from ditch water, through elective cultures in an acidic mineral medium with alcohol as organic substrate. These strains differed from all other Acetobacter cultures in being catalase negative and able to oxidize molecular hydrogen. At the time this latter feature suggested that they 141 BIOGRAPHICAL MEMORANDA possessed an exceptionally high affinity for hydrogen, which was equiv- alent to having a high oxidative capacity, a supposition that was fur- ther supported by the fact that they did not produce acid from glucose, apparently causing a complete oxidation of this substrate without the temporary accumulation of incomplete oxidation products. These or- ganisms, for obvious reasons, became known as Acetobacter peroxydans. The studies on the acetic acid bacteria had therefore shown that biochemical properties could be advantageously used for the sub- division of a genus already partially characterized on the basis of physiological criteria. Hence it is understandable that, when the fundamental principles of biochemistry had begun to emerge, Kluy- ver expected that they would be even more efficaceous for the clas- sification of micro-organisms in a still larger framework, promising that ultimately such a classification might become based on numer- ically determinable differences in the affinity of the protoplasm of various microbes for hydrogen. Pending the establishment of appro- priate methods for the quantitative evaluation of this property, one could begin by using in its place the general metabolic patterns of the organisms which could be ascertained by relatively simple analytical procedures. This meant that biochemical characteristics far more fundamental than, for example, the ability to ferment one or more particular sugars, would come to serve as the basis for the creation of physiologically homogeneous entities, comparable in many respects with Cohn’s ‘form genera’. It remained to develop a scheme in which the old form genera and the new physiological groups could be combined, so that satisfactorily circumscribed genera would ensue. That morphological and physiological features could be conveniently merged had already been shown by Donker’s analysis of the fermentation pattern of Bac. polymyxa which had indicated the striking similarity in physiological respect of this morphologically typical sporeformer and the non- sporeforming Aerobacter species. By proposing the new genus, Aero- bacillus, for the former, the feasibility of this kind of approach had been sufficiently demonstrated. . Meanwhile the study of the propionic acid bacteria in Kluyver’s laboratory had shown that these organisms represent a remarkably homogeneous group with respect to both their general morphological and biochemical properties. This justified the resurrection of Orla- 142 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY Jensen’s previously proposed genus, Propionibacterium. But this in- vestigation had also indicated a close morphological resemblance be- tween these organisms and several other types of bacteria, such as the lactic acid-, the coryne-, and the mycobacteria. All these organisms can be characterized as non-sporeforming, Gram-positive, and per- manently immotile bacteria. This in turn had suggested the possibility of establishing, in addition to the two major morphological groups recognized by Orla-Jensen, a third one for these immotile organisms. These developments resulted, in 1936, in a fairly elaborate attempt to formulate the basic ideas on the classification of bacteria that had gradually taken shape. The timing was shrewd; at the International Congress for Microbiology scheduled to meet in London later during that year, the International Committee for Nomenclature and Classi- fication of which Kluyver was a member would thus be in a position to take cognizance of the proposals, elaborated if necessary by Kluyver himself. Because the paper has been reprinted in this volume it is not nec- essary to discuss it in detail; suffice it to say that it was felt to represent a first step in organizing the Eubacteria in a manner somewhat com- parable to that in which Mendelejeff had arranged the chemical el- ements, and with the same advantage of indicating the potential existence of groups of bacteria with combined morphological and physiological properties not as yet encountered; if discovered, these could immediately be incorporated in the system in their appropriate places. The recent discovery of polarly flagellated bacteria with a fermentation pattern like that of the coli-group of organisms, and for which Asai et al. [1956] have aptly proposed the generic name, Alwy- vera, testifies to the usefulness of the approach. So do several generic names, created since 1936, and based on the same principles, such as, for example, Methanobactertum and Butyribacterium. The simple guidelines employed in 1936 were adequate to achieve a reasonably satisfactory subdivision of those bacteria that are charac- terized by an obligatorily or facultatively fermentative metabolism, be- cause it is easy to distinguish between the diverse and specific fermen- tation patterns. But a subdivision of bacteria with a strictly oxidative type of metabolism could not then be accomplished by a comparable approach; hence the corresponding genera were less satisfactorily defined. Much has meanwhile been learned about patterns of oxi- dative metabolism which suggests that an imaginative use of this in- ta5 BIOGRAPHICAL MEMORANDA formation may permit the establishment of subgroups more nearly equivalent to those based on particular anaerobic processes. Although the outline of bacterial classification presented in 1936, like any and all other systems that have thus far been proposed, is subject to the criticism that it does not rest on a truly phylogenetic basis [Van Niel 1955], its rational features have had a beneficial influence on systematic bacteriology generally, and many of the genera there delin- eated have gradually been incorporated in the current systems. Besides the main outline mentioned above, the contributions to bacterial classification of Kluyver and his associates comprise many publications on problems of a more limited scope. The monographic treatments of the genera Acetobacter [Visser *t Hooft, 1925Th; Fra- teur, 1950], Protaminobacter [Den Dooren de Jong, 1926Th, 1927], Propionibacterium [Van Niel, 1928Th], Pediococcus [ Mees, 1934Th], and Spirillum [Giesberger, 1936Th]; of the group of sulphate reducing bac- teria [Baars, 1930Th], brine bacteria [Hof, 1935Th], and methane producing bacteria [Schnellen, 1947Th], all these will long remain amongst the indispensable references to these groups. Giesberger later extended his studies of the spirilla to the reddish-brown members of the genus Rhodospirillum; Kingma Boltjes [1934Th, 1936] made a careful investigation of the nitrifying bacteria during which he rediscovered Hyphomicrobium vulgare, whose strange life cycle, including the multi- plication by budding, was described in detail; Mayer [1938Th] and Perquin [1939] made notable contributions to our knowledge of the dextran-producing Betabacterium vermiforme and Streptobacterium dex- tranicum, respectively; and Kaars Sijpesteyn [1948Th, 1949, 1951] to that of the cellulose-decomposing bacteria in rumen, leading to the establishment of the new genus, Ruminococcus. Here may also be men- tioned the discovery of Pseudomonas beyerinckit | Kluyver and Hof, 1939; Kluyver, Hof and Boezaardt, 1939], characterized by the production of a purple pigment only if the organism is grown in the presence of inositol, which is oxidized to tetra hydroxyquinone, whose calcium and magnesium complexes are responsible for the colour of the cul- tures; of Ps. aureofaciens [Kluyver, 1956]; and of Hydrogenomonas car- boxydovorans [Kistner, 1953, 1954] which can live at the expense of the oxidation of carbon monoxide. The comparative studies of the aerobic nitrogen fixing bacteria of the Azotobacter [Kluyver and Van Reenen, 1933; Kluyver and Van den Bout, 1936] and Bezyerinckia 144 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY [Kluyver and Becking, 1955] groups, and of the nitrate-reducing sporeforming bacteria [Verhoeven, 1950, 1952Th] contain valuable information relating bacterial taxonomy to ecology; to this category may be added Elazari-Volcani’s [1939] investigation of Ps. indigofera. Kluyver’s contributions to classification are not restricted to the bac- terial kingdom, however. Through his early studies with yeasts he maintained a profound interest in this group of organisms. He had in- herited Beijerinck’s culture collection which included many yeasts, and soon after assuming his duties as Professor of Microbiology Kluy- ver arranged with Professor Johanna Westerdijk, Director of the re- nowned ‘Central Bureau of Fungus Cultures’ * in Baarn, Holland, to have the yeasts represented in the Baarn collection transferred to the Delft institute. Thus was established what is probably the world’s most outstanding collection of yeast pure cultures. It has served as the basis for a series of monographs on the yeasts [Stelling-Dekker, 1931 Th; Lodder, 1934Th; Diddens and Lodder, 1942; Lodder and Kreger-van Rij, 1952] comprising the best so far published in this field. ‘The prin- ciples guiding the development of an up-to-date system of yeast classifi- cation, adopted in these treatises, can be found in Kluyver’s publication of 1931 which has also been included in this volume. Before the first of these monographs had appeared, Kluyver’s ex- tensive knowledge of microbiological literature and his keen scient- ific insight had permitted him to recognize the curious phenomenon of mirror-image formation by certain pink yeasts when cultivated on solid media in an inverted position as associated with a mechanism of spore discharge that is characteristically encountered among the basi- diomycetes. This suggested that, contrary to the universally held opinion, not all yeasts should be considered as primitive or reduced ascomycetes, but rather that the basidiomycetes also embrace a fully comparable series of progressively more complex fungi whose simplest representatives are the typical yeasts for which the genus Sporobolomyces was created [Kluyver and Van Niel, 1925, 1927]. Similarly, Kluyver’s wide experience and microbiological acuity were responsible for the recognition that organisms described by certain specialists as new yeast species or even genera, were, in reality, * A note on the history of the Central Bureau of Fungus Cultures has been published by K. B. Raper in Mycologia 49, 884 [1957]. hao BIOGRAPHICAL MEMORANDA members of the group of aerobic sporeforming bacteria; this applies, for instance, to the species, Schizosaccharomyces hominis, [Dorrepaal, 1930], and to the genus, Schzzotorulopsis [Verkaik, 1931]. Besides these contributions should be noted the monographs dealing with the yeast genus, Brettanomyces |[Custers, 1940Th], and with the species, Candida pulcherrima [van der Walt, 1952Th]. The latter con- tains the fascinating observations on the pigments produced by this yeast in the presence of various heavy metals. This study eventually led Kluyver and co-workers [1953] to the isolation and characteri- zation of pulcherrimin, surely one of the most interesting chelating agents known to-day. VARIABILITY OF MICRO-ORGANISMS From the preceding section it will be clear that Kluyver knew micro- organisms as more than entities performing special biochemical trans- formations, which implies that he was also familiar with that bane of the systematist that can conveniently be described by the collective term, ‘variability’. Variability expresses itself in physiological as well as morphological respects. Both of these have been studied in Kluyver’s institute, and some of the contributions to this aspect of microbial behaviour will here be briefly reviewed. In 1925 Elion [1925] had there discovered a bacterial sulphate re- duction at temperatures as high as 65°C; the causative agent had been isolated and described as Vibrio thermodesulfuricans; it was the third spe- cies of sulphate-reducing bacteria, the previously known ones being the mesophilic freshwater V. desulfuricans and the marine JV. aestuari. The isolation of V. thermodesulfuricans, unable to grow at temperatures be- low 30°C, presented a curious problem, however. The cultures had been started with an inoculum of mud taken from a ditch below a heavy layer of ice, and it seemed most improbable that even during warm seasons the temperature of the mud in this ditch would ever reach the minimum temperature required for growth of the new species. This raised the question how its apparently regular occurrence in this environment could be explained. Some years later this situation led Kluyver and Baars to the idea that V. thermodesulfuricans might not be an autonomous species, but 146 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY rather a modified form of the mesophilic V. desulfuricans. By using large inocula of a pure culture of the latter, and incubating transfers at increasingly elevated temperatures, it proved indeed possible to effect a change in the properties of the organism which appeared to involve a gradual shift in the entire temperature range within which the strain could develop. In like manner the reverse was also accom- plished, and a thermophilic culture changed into a mesophilic one. These experimentally induced modifications strongly supported the view that V. desulfuricans and V. thermodesulfuricans represented more or less stabilized races of one and the same organism rather than dis- tinct and separate species. This further led to experiments designed to determine whether V. desulfuricans and V. aestuartt might be similarly related. Hitherto these species had been distinguished solely because the former can develop normally in media with o—1.5 per cent sodium chloride, but is inhibited by higher concentrations, and unable to grow with more than 2.5 per cent, whereas the latter grows equally rapidly in media with 1-3 per cent, and not at all with less than 0.5 per cent salt. By transferring cultures to media with different salt content it was found that the range of salt concentrations at which un- inhibited growth can occur is just as little fixed as is the temperature range, and that these two types can also be gradually interconverted. Consequently it was proposed to recognize only V. desulfuricans as a taxonomically sound species; the others were graphically designated as ‘physiological artefacts’ [Kluyver and Baars, 1932; Baars, 1930Th]. The thermophilic nature of Elion’s isolate could be more easily understood when Starkey [1938], working in Kluyver’s laboratory, dis- covered that sulphate reduction at elevated temperatures is caused by a sporeforming bacterium. At the time this was considered to be ident- ical with the ‘adapted’ mesophilic cultures of Baars, and designated as Sporovibrio. But recent studies have shown that the experimental results of Kluyver and Baars are not reproducible. Eventually this situation has led Campbell et al [1957] to identify Starkey’s Sporovibrio as Clostridium nigrificans, a sporeforming anaerobe that had long been known as the causative agent of hydrogen sulphide production in cer- tain canned products. This conclusion has lately been corroborated in Kluyver’s laboratory as well as by Starkey. It therefore seems probable that the earlier results must be attributed to the use of impure cultures. Interconversion of freshwater and marine strains of Desulfovibrio 147 BIOGRAPHICAL MEMORANDA desulfuricans has also been reinvestigated elsewhere, and it has been found that strains of varied origin display unpredictable differences in behaviour in this respect. Some strains appear to be readily adapt- able, others not at all [Littlewood and Postgate, 1957]. While thus the variability of some physiological properties of the sulphate reducing bacteria appears to be less general or pronounced than was claimed in 1930, the studies of Kluyver and Manten [1942] with a culture of an hydrogen-oxidizing bacterium, identified as Hydro- genomonas flava, provided an example of another sort of physiological variability. By means of manometric experiments it was established that suspensions of this organism grown in mineral media at the expense of hydrogen oxidation, can oxidize hydrogen gas as well as organic substrates, and that such oxidations can even proceed simul- taneously; whereas cultures grown in media with’ organic compounds in the absence of hydrogen completely failed to oxidize the latter. This indicated that ‘obviously the formation of the special hydrogen oxi- dizing system only takes place when the bacteria are grown under autotrophic conditions’. Such a behaviour is reminiscent of that dis- _ played by certain yeasts with respect to their ability to ferment galac- tose, and, as mentioned earlier, Kluyver was fully cognizant of its significance. The experiments with H. flava revealed furthermore that, after prolonged cultivation on organic media in the absence of gaseous hydrogen, its ability to grow autotrophically was irretrievably lost. A fully satisfactory explanation of this phenomenon, previously also ob- served by Grohmann, has not yet been proposed. This investigation of H. flava, together with renewed studies in Kluyver’s institute on bacterial denitrification, eventually led to a search for naturally occurring micro-organisms that can oxidize hy- drogen with nitrate as the sole oxidant. The existence of such organ- isms was probable on theoretical grounds, but had never been con- clusively demonstrated. By means of appropriate elective cultures the presence of such specialists in soil samples was readily ascertained, although it appeared that the bacteria that can carry out this con- version can grow in an anaerobic environment with hydrogen and nitrate only if a small amount of yeast extract is present in the medium. A careful examination of such cultures showed that they contained only one predominant bacterial type which closely resembled the 148 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY denitrifying bacterium that Beijerinck had previously and rather cur- sorily described under the name Micrococcus denitrificans; the latter was not known to be a potential hydrogen bacterium, however. Just as H. flava, the new isolates could oxidize hydrogen as well as organic compounds, and hydrogen was not oxidized by cultures grown in or- ganic media. Furthermore, these strains could use either oxygen or nitrate as oxidant, although nitrate reduction was exhibited only by cultures grown anaerobically in the presence of nitrate, a behaviour that is also characteristic of other denitrifying bacteria. Once these facts had been established, it was possible to test the suspicion that the new isolates were indeed identical with M. denitrificans, and this was confirmed when it was found that Beijerinck’s original culture of that organism, maintained for many years in the Delft culture collec- tion on routine organic media, could still be induced to form a nitrate- reducing as well as a hydrogen-oxidizing enzyme system when cultiv- ated under the requisite conditions. In contrast to H. flava, M. denitri- ficans evidently retains its potentiality to live as a hydrogen bacterium even after prolonged growth on organic media. In his Harvard lectures Kluyver referred to these studies as exam- ples of microbial variability due to enzymatic adaptation, or, to use the current term, to induced enzyme formation, and stated: ‘...it seems warranted to conclude that M/. denitrificans offers a striking example of life’s flexibility. It can live as a heterotrophe, de- pending on the oxidation of some organic substrate, not only with free oxygen, but also with nitrate as a substitute for the latter; next it turns out to be a fully autotrophic organism able to thrive on the Knallgas system; and finally it emerges as a chemometatrophic organism dis- playing the seemingly exceptional quality of thriving on the system molecular hydrogen-nitrate. But it has been clearly shown that the ability to use hydrogen as donor and nitrate as acceptor depends on the presence of special enzymes which are produced only in response to well-defined conditions during growth. ‘How strongly the potentiality to produce these enzymes is fixed in the genetic apparatus of the organism is demonstrated in a most con- vincing way by the behaviour of Beijerinck’s original culture, which, maintained for forty years — that is, for thousands of generations — on peptone agar, on being transferred to an environment where mole- cular hydrogen is the only energy source available, answers nature’s pes, BIOGRAPHICAL MEMORANDA challenge by the brave device: “Here I am, I can also act differ- ently"? \(p. 107). But the microbial world also offers many examples of a kind of var- iability that is not primarily induced or controlled by environmental conditions. A fairly common sort is what eventually became known as ‘dissociation’; it manifests itself through the appearance of different types of colonies on one and the same culture plate, streaked with a suspension of a pure culture of some micro-organism. Special terms and abbreviations, such as ‘smooth’ (‘S’), ‘rough’ (‘R’), ‘mucoid’ (‘M’), etc., had been introduced to characterize various colony types. The reality of this phenomenon had been established beyond question, but a generally accepted interpretation was lacking even as late as the middle ’thirties. As early as 1900, and again in 1912, long before the name ‘disso- ciation’ had been introduced, Beijerinck had described several cases of variability of this kind, and claimed that they represented mutations in the sense of Hugo de Vries, regularly occurring in cultures of micro- organisms. This concept had, however, been sharply contested, the opponents arguing that mutations can be observed only in organisms that reproduce sexually. Such a mode of reproduction was unknown amongst the bacteria, which were not even considered to possess a nucleus. Consequently Beijerinck’s interpretation of microbial varia- bility had fallen into disrepute. Another theory to account for the dissociation phenomenon and which had gained prominence during the nineteen-thirties, had been developed by Hadley. It was based on the assumption that bacterial cultures regularly display ontogenetic variations, and that different ‘cyclostages’ are recognizable by the specific colony types correspond- ing to them, the ‘rough’ phase representing the culminating, or ‘repro- ductively mature’ stage through which every species would have to pass in due course. Nevertheless, this concept smacked too much of the ‘life cycle’ theories of Lohnis and Enderlein, formulated in order to account for morphological variations which others, with good rea- sons, had attributed to the use of impure cultures; and Hadley’s spe- culations were not regarded very sympathetically by most of the leading microbiologists. It is clear, therefore, that the situation was confused and that further experiments were needed to resolve it. 150 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY The opportunity to carry out such an investigation in Kluyver’s laboratory presented itself when Mayer [1938Th] there encountered a typical case of dissociation during his studies on the microbiology of the ‘Tibi grain’. This material, like the ‘ginger beer plant’ in Ene- land, was used in some countries for the preparation of mildly alco- holic, effervescent beverages from sugar solutions flavoured with var- ious fruits. Mayer showed that these grains, which multiply during the fermentation, are composed of a yeast, isolated in pure culture and identified as Saccharomyces intermedius, and of a rod-shaped hetero- fermentative lactic acid bacterium characterized by its ability to pro- duce large capsules of dextran in sucrose media. ‘This bacterium was also isolated in pure culture and named Betabactertum vermiforme; it represented the first instance of a rod-shaped, dextran-forming lactic acid bacterium. Mayer had moreover accomplished the synthesis of typical “Tibi grains’ from the pure cultures of the two components. Starting with fresh ‘Tibi grains’ from fermenting solutions, Mayer had frequently observed that the lactic acid bacteria colonies develop- ing on sucrose-gelatin plates were all of one kind. But during the iso- lation of Betab. vermiforme it had also been found that different kinds of colonies appeared on glucose-agar plates streaked with suspensions prepared from single, well-isolated colonies of uniform appearance that had grown on solid media. In addition to the more numerous ‘rough’ colonies, representing the initial type, irregular flat ones were encountered, and it was shown that every suspension of a ‘rough’ colony invariably gave rise to a certain proportion of colonies of the flat type. It is important to note that this behaviour was observed with cultures on glucose-containing media; here capsule formation never occurs, and this precludes the possibility that contaminating bacteria would be regularly entrapped in the stiff mucus. By the side of the flat, irregular colonies, smooth elevated ones were also found occa- sionally. Associated with the changes in colonial structure was a loss in ability to produce dextran from sucrose; this applied to both the flat and the smooth types. A meritoriously critical evaluation of the sequence of events and of many other aspects of the observations finally led to the conviction that the observed ‘dissociation’ could best be explained as the result of mutations, even though at the time this interpretation was prob- ably the least favoured by microbiologists. The reasoning that led to 151 BIOGRAPHICAL MEMORANDA this conclusion appears modern even to-day, and it is striking that Mayer carried out some experiments in order to test the possibil- ity that the formation of dextran might be induced in the strains that had lost the capacity to produce capsules in sucrose media, by in- cubating the latter with killed cells of dextran-producing strains, 2.¢., by using the approach that several years later gave rise to the spectac- ular experiments of Avery and associates on the transformation of pneumococci under the influence of desoxyribonucleic acids from dif- ferent types of these organisms. That the attitude which yielded the final conclusion was based on a careful appraisal of all the known facts, and was anything but dog- matic is evident from the formulation of the verdict: ‘The old idea of Beijerinck’s that bacterial variation is the result of gene mutations possesses a degree of probability that is unmatched by any other current interpretation.’ (p. 182). As is well known, this concept came into prominence during the next decade, and it is now universally accepted as the most satisfactory explanation of many forms of microbial variability. The term “dissoci- ation’ has been relegated to limbo, largely because of Braun’s [1947] masterly analysis of that phenomenon on the basis of the mutational concept. Braun approached this problem by studying the kinetic aspects of the gradual increase of mutant types in bacterial cultures, which he could attribute to a strong selective influence of the pro- gressively changing medium on the growth of the original organism and of the different variants. In this connexion it is interesting to recall Mayer’s observation that cultures of Betab. vermiforme, grown in yeast extract-sucrose solutions, show a much lower incidence of mutants than do comparable cultures in glucose solutions or on solid media. This, of course, suggests a similar selective effect, and it accounts for the occurrence of but one colony type of lactic acid bacteria on plates prepared from fresh “Tibi grains’. EPILOGUE In 1939 the ‘Delft School’, by which here is meant the many asso- ciates and students of Albert Jan Kluyver, celebrated the twenty-fifth anniversary of the date on which he had received his Doctor’s diploma. On this occasion the Dutch chemical weekly, ‘Chemisch Weekblad’, 152 KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY published a special issue containing various contributions in which Kluyver’s numerous accomplishments were reviewed. The paper deal- ing with his eminence as a microbiologist and biochemist ends with the following passage: “The law will permit him to continue his activities as Professor at the Technological University for nineteen more years. It is to be hoped that he may have the opportunity to devote himself during this ime span without interruption to investigations in the area he has chosen as his special field of endeavour, and in which his achievements have already been so numerous. In that event it may assuredly be expected that many more significant contributions from his laboratory will en- rich our knowledge, and that many students as well as established in- vestigators will experience the beneficent influence of his personality and institute.’ (p. 319). Unfortunately, this wish has not been fulfilled. There have been long and painful interruptions, and his death, two years before the scheduled date of his retirement, has shortened the expected duration of his association with the famed microbiological institute in Delft still more. In the end it turned out that less than half of the remaining nineteen years were actually available for his scientific pursuits. Exactly a year after the above-mentioned celebration, Holland was invaded, and for many years suffered under the German occupation. During this period the work in the microbiological laboratory came to a virtual standstill. This explains, for example, why the studies on the methane fermentation, in part already performed prior to the 1939 Helsinki lecture in which Kluyver had mentioned some of the early results, were not completed and published until 1947. And when the war was ended, conditions for the resumption of scientific activities were anything but favourable. Apart from the lack of necessary equip- ment there were other causes that hampered the development of his programme. In other countries, especially in the U.S.A., scientific in- vestigations had been continued, albeit at a reduced rate and with a pronounced emphasis on application to the war effort. The vast quantity of papers published during this period was not accessible to Kluyver until after the liberation, and when he realized how much had been accomplished, he despaired of ever being able to catch up with the advances made in the interim during which he had been entirely shut off from contacts with science and scientists. 6 Po BIOGRAPHICAL MEMORANDA Although physically much weakened as a result of the severe depri- vations suffered, and mentally shaken by the horrors experienced during the war years, Kluyver set himself the task to assimilate as rapidly as possible the accumulated knowledge, particularly in those fields that most interested him. This led to a number of important re- views, such as those he prepared on the use of isotopically labeled com- pounds for the study of biochemical reaction mechanisms [1947<, -b]. It also made him aware of the great strides that had been made in the field of enzyme chemistry. Although, as has previously been indicateds Kluyver had not been overly sanguine concerning the contribution, that could be expected from this approach, the laboratory had ac- quired, as early as 1938, a Booth-Green mill for the preparation of enzyme extracts from bacteria, and this instrument had been used in attempts to obtain cell-free extracts of luminous bacteria that still possessed the capacity to emit light. Unfortunately, the results had not been encouraging. Later, Tuynenburg Muys [1949] constructed a simple and effective apparatus for disrupting microbial cells by grinding. But it was not until very recently that la Riviére carried out the first successful experi- ments with bacterial enzyme preparations in Kluyver’s institute [1956]. At about the same time a beginning was there made with studies on carbon dioxide assimilation by potentially autotrophic bacteria with the aid of labeled carbon dioxide. During the early years of his directorship Kluyver spent practically all his time in the laboratory, encouraging the students and guiding their efforts by daily discussions in which the status of their work was reviewed in the most minute detail, leading to suggestions for new experiments and approaches. These sessions also served to acquaint the students with pertinent information which Kluyver provided from his own vast store of factual knowledge, and to point out poten- tial relationships of experimental results with what at first sight often appeared to be unrelated subjects. In this manner the students gradu- ally became imbued with an increasingly profound appreciation of the true meaning of scientific research, while the close and intensive cooperation between the professor and his pupils led to the develop- ment of an enthusiastic and stimulating atmosphere such as is rarely encountered in a scientific laboratory. Noes KLUYVER’S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY But in later years a considerable proportion of Kluyver’s time was occupied by numerous official functions, many of which required the preparation of special reports and lectures. ‘Thus, in spite of the fact that he rigorously adhered to his arduous schedule of work, from 9 a.m. till midnight and after, not excluding Sundays, the time he could spend on the supervision and guidance of the work in his institute was severely curtailed. A compensating feature of Kluyver’s activities during these later years is, however, that they resulted in the pub- lication of many lectures of great general importance, and one can- not but be grateful for the painstaking efforts and profound thought that went into their preparation. Some of these lectures are re- printed in this volume; they show an increasing preoccupation with the fundamental problems that humanity is facing, and wisdom in the formulation of specific solutions. ‘Thus they are apt to contribute to the development of those attitudes that are so desperately needed in order that we may eventually witness the fulfilment of the ardent wish that Kluyver expressed in the final sentence of the lecture he delivered before the combined sections of the ‘Koninklike Nederlandse Akade- mie van Wetenschappen’ under the title ‘Microbe and Life’, a lecture to which he was wont to refer as his ‘swan song’: ‘Heavy is the responsibility that rests on mankind; may we succeed in finding the right way’. BIOGRAPHICAL MEMORANDA REFERENCES References to articles of which Kluyver was author or co-author are found in the ‘Bibliography’ (p. 527); references marked with Th are included in the list of Doc- tor’s dissertations prepared in Kluyver’s laboratory (p. 537), and publications by his students and associates have been assembled in a separate list (p. 540). References to other papers are arranged alphabetically below. ASAI, T., OKUMUTA, S. and TSUMODA, T. 1956. Proc. Japan. Acad. 32, 488. BARKER, H. A. 1936. J. Cellular. Gomp. Physiol. 8, 231. BARKER, H.A., RUBEN, S.and KAMEN, M. D. 1940. Proc. Nat. Acad. Sci. U.S. 26, 426. BRAUN, W. 1947. Bact. Rev. 17, 75. CAMPBELL, L. L., FRANK, H. A. and HALL, E. R. 1957. J. Bact. 73, 516. COOK, R. P. and STEPHENSON, M. 1928. Biochem. J. 22, 1368. FROMAGEOT, CL. and DESNUELLE, P. 1935. Biochem. Z. 279, 34. LITTLEWOOD, D. and POSTGATE, J. 1957. J. Gen. Microbiol. 17, 378. LOOMIS, W. F. and LIPMANN, F. 1948. J. Biol. Ghem. 173, 807. MEYERHOF, O. 1945. J. Biol. Ghem. 157, 105. MEYERHOF, O. 1947. Antonie van Leeuwenhoek 12, 140. NIEL, C. B. VAN 1955. In: A century of Progress in the Natural Sciences, 1853-1953. San Francisco. STEPHENSON, M. and YUDKIN, J. 1936. Biochem. J. 30, 506. WIERINGA, K. T. 1936. Antonie van Leeuwenhoek 3, 263. WIERINGA, K. T. 1940. Antonie van Leeuwenhoek 6, 251. TWO FUNERAL ORATIONS Out of the incomprehensible fullness of his being, Albert Jan Kluyver has given so generously and in so many directions that a multitude of people, here and elsewhere, must have cherished the impression that he belonged to them; and they all must now feel dejected by a deep sense of sadness and loss. He belonged to the world of his science which has bestowed on him its most coveted honours. He belonged to this country which he elevated and to which he brought fame whenever he represented it abroad. He belonged to the city of Delft which he, the world-renowned scientist, loved and served as a good and simple citizen. He belonged to the Delft University, whose brilliant pupil he once had been, and where for so many years he learned, taught, and worked in that unique scientific position that one is tempted to call a ‘Regal Chair’. Above all, he belonged to his family, to his children and their mother who preceded him. If, on the occasion of this last farewell, a few words are spoken on behalf of the entire university, this is done in clear recognition of the fact that within its confines, too, his activities and solicitude were so diverse that many will experience the desire to bear witness to their esteem and sorrow. In the first place we think of those who were his collaborators in the secluded community of the quarters that were his home and his workshop; who surrounded him with their daily cares; and on whom the thought that they will never see him again must leave an impression of unutterable emptiness. We think of the aca- demic youth, the students to whom he was so deeply devoted; to whom his anxiety and vigilant compassion went out during the dark years; and whom, only a few days ago, he addressed on the science of invisible life. I may speak, too, in the name of the Board of Trustees and the Department of Chemical Technology who but recently, im- pelled by their admiring respect, had jointly decided upon exceptional provisions for the chair of microbiology, just because it was Kluyver’s. And I also speak on behalf of the Academic Senate, simply to say that from its midst has been taken he who was incontestably the foremost. 7 BIOGRAPHICAL MEMORANDA In our memory he stands in his accustomed place in the council chamber, holding forth in his characteristic attitude, advising and warning and offering a solution; and we knew that it was right to accept his council and to pursue the path that he laid out. On special occasions we would ask Kluyver to be our leader, and we felt secure and protected. To him we could go with our troubles, and we were met with endless patience for our questions, and received attention and understanding, consolation and strength. Truly, towards him whom we have lost, all words seem powerless. His profundity and versatility silence us. But we have always known that he was not just an exceptional scientist, but first and foremost a great and complete human being, and that his talents were alloyed with a noble steadfastness of character, and a tender, deep-seated sensitivity. About inessentials he could talk playfully, even flippantly; but he never compromised when faced with fundamentals. In him there was room for the significant side by side with the fleeting, and without endangering the unity. He who worked at the farthest borders was at the same time attentive to the daily chores. And another combination of opposites: his mind that was so supple, full of esprit, and open to the unknown, liked to preserve an old but meaningful form. In his style of life the dignity of the magistrate could harmoniously be blended with the utmost simplicity. Order, justice, and balance conferred upon him the invulnerability on which one could build and trust. The word ‘classical’ comes to mind; and another, ‘self-possession’ in its multiple connotations. This rich life has now come to an end. In a spirit of reverence and affection, deeply moved and in sorrow, the University commemorates him, and is grateful for what it has been vouchsafed in the person of Kluyver. O. Bottema TWO FUNERAL ORATIONS On this occasion I wish to speak in the capacity of a friend — I may even say an intimate friend — of the deceased. We were bound by a friendship that, begun in September, 1905, when we first registered as students in Delft, has now come to an end. When, after having spent some years in the tropics, Kluyver re- turned to Delft to occupy Beijerinck’s chair in 1921, that friendship continued without interruption. Both of us were married; both of us reared five children who also became mutual friends. Never has there been a single note of discord; small wonder, then, that the friendship was firm, and considered as something self-evident. Thus I have been in a position to observe Kluyver throughout the 35 years during which he developed into an ornament of the Technol- ogical University and a scientist of international repute. It is the man Kluyver whom I want to bring back to your memory in these mo- ments. It is amply recognized that Kluyver gradually built up a school of microbiologists. What is a school? It implies that a group belonging to a younger generation conduct experiments and publish their results under the guidance of a mentor, and that they all bear the imprint of some fundamental idea of the master. How could the master do this? Firstly because his fertile mind could initiate profound concepts; but also because he cooperated with his assistants and students; because he knew how to inspire them with ardour for their science. This, only a great man can do. One will never see a school develop if the teacher does not possess great human qualities. Kluyver has lived and toiled for his students. That was particularly evident when they took their degree under him and were writing their theses. It often happened that, despite pressure from his family, Kluyver did not take — or postponed taking — a vacation because he stayed behind to work with the pupil; occasionally he might take the student along. And then Kluyver would concentrate on that work with his pupil for entire weekends, and not infrequently have his pupils stay on as guests. And in this manner much was accomplished. Every publication was permeated with the fundamental concepts of the master. And every pupil departed with gratitude towards his teacher; every one had gained much, not only in scientific knowledge, but also — some more, others less — worldly wisdom which they could carry as valuable lug- r59 BIOGRAPHICAL MEMORANDA gage for life; for Kluyver was not only a very gifted man with an ex- ceptional intelligence and insatiable curiosity, he was also a wise man. What is wisdom? ‘Wisdom is a native gift of intuition, ripened and given application by experience, for understanding the nature of things, certainly of living things, most certainly of the human heart’. These are the words of T.S. Eliot, spoken when he received the Hanseatic Goethe prize for 1954. But I can also illustrate, by some eminently practical examples, what Kluyver’s wisdom implied. He never rebuked; he never told any one off, as so many of us are apt to do. He never became irate; during those fifty years I have never seen him angry. He was a wise man. This he was also to his students and pupils. His wisdom towards them included also the recognition that not all persons are alike, that not every one was as he; and he not merely tolerated, but actually encouraged different personalities. There was but one human trait he disliked: pretentiousness, would-be learning, hypocrisy, insincerity. Is it surprising that he was venerated by his pupils, and that, when he had been professor for 25 years and declined a grand celebration, his pupils gave him a dinner party at which they toasted him so ‘pro- fusely and so touchingly? Kluyver was also a man of style. Has not Buffon said that ‘Car le style, c’est de PFhomme méme’? This was a most essential element of his character. He was a man of decorum, and he despised vulgarity. His feeling for style was also expressed in his brilliant lectures on his specialty and related matters. Yet one should not think that it came to him easily, by some sort of intuition. It was achieved through hard work, by revising, by pondering every single word. In his conversation, too, he was original and singular. His diction was intensely personal, sometimes spiced with expressions that had their origin in the district where his father and mother had been born, and where his grandfather had owned a mill. He liked slogans, which he replaced by new ones every few years. During the *thirties, when he was exceptionally busy, I remember that the favorite one was: “This is a great and terrible world’. But in his later years he had become more resigned, and was wont to say: “Well, that’s how it is’. His conversation was brilliant in another respect. In a few words he could outline a situation, either in science or in politics. He often 160 TWO FUNERAL ORATIONS explained to me some microbiological problem, fully clarifying the subject in a few terse sentences. In spite of the fact that many honours were conferred upon him — honorary doctorates, the Hansen- and Copley medals, election to many foreign scientific societies — Kluyver remained a modest man. He never prided himself on these distinctions; he always made it ap- pear as if he owed them to some accident or chance. When he had been awarded the Emil Christian Hansen medal in Copenhagen and had returned to Delft, his students and associates celebrated the occasion by presenting him with an enormous cake bearing the inscription, ‘Tam only a simple man’. It was one of his own, characteristic slogans. Kluyver seldom spoke of things to come; rarely mentioned the fact that, at the end of 1956, he would have to give up his beloved home, and a year later his old laboratory. It was as if he himself doubted that he would witness these events. And now this beautiful life has suddenly been cut off. Former pupils, spread out over the entire world, will share these moments with us. They will gratefully remember their master. Every one of them has taken along something he had received from him; in difficult moments they will all be inclined to ask: ‘What would Kluyver have said?’ Nothing is ever lost in this world. Kluyver is no more; but the seed he has sown on the fields of science and in our hearts has long since germinated, and those who have known him well will retain the memory of an eminent scientist, but above all of a complete human being with a big heart. And if we can do this, how much more strongly will these memories remain alive in his children who have known him in other ways, and who will be grateful that they had so excellent a father. The relation with his children I cannot better express than in the words in which he characterized his own education, and which are taken from his inaugural lecture in which he addressed his parents thus: ‘The education you gave me was characterized by acts of love rather than by verbose theory’. This friend of half a century, who actually never knew any rest, — may he now rest in peace. A. v. Rossem 161 PART TWO SELECTED PAPERS MICROBIOLOGY AND INDUSTRY “This enables the chemist to regard micro-organisms as Co-practitioners of his craft, and the chemical achievements of these humble agents have continued to excite his admiration since they were revealed by Pasteur.’ (‘The Laboratory of the Living Organism’, Presidential Address delivered by Dr. M. O. Forster, F. R.S. to Section B of the British Association for the Advancement of Science, at Edinburgh on September 1, 1921.) INSTRUCTION at a present-day Technological University has grad- ually expanded into a grand symphony of pure and applied science. Only the exceptionally practiced ear can adequately value the role of the individual instruments, 7.e., the various branches of these sci- ences, separately. Besides, scientists will but rarely be provided with an opportunity to perform as soloists before an audience that is not composed exclusively of professional experts. The newly appointed professor who, in accordance with tradition, embarks on his career with a public address faces a task that calls for an appraisal of two aspects. This is so because on the one hand the listener may be in- clined to attach to this address a special significance in that it will permit him to evaluate the speaker’s ideas concerning the manner in which the latter intends to carry out the duties of his new office. On the other hand, the introductory remark implies that this lecture is also important from the point of view of his specialty because it cannot fail to influence the position that will be assigned to his branch in the composite. The latter consideration weighs more heavily on me than personal responsibility now that, after a lapse of nearly a quarter century, a spokesman for microbiology may once again request the attention of the assembled representatives of technological higher edu- cation. And this is all the more so because of the fact that it is my privilege to speak in the same city where Antony van Leeuwenhoek, the father of microbiology, used to live and work. As a subdivision of biology, microbiology is perhaps in greater need of an occasional opportunity to make itself heard than any other sub- ject taught at this university. The reason is that at first sight there 165 SELECTED PAPERS appears to be a chasm between the science of life and modern engi- neering sciences that precludes any possibility of co-ordination. Never- theless, it is my firm conviction that a closer consideration will reveal the situation to be otherwise, and that, side by side with the other fields of study, microbiology may lay claim to a profound interest on the part of all those who are concerned about the education of the prospective chemical engineer. Hence my aim to-day will be to make you partisans of this view, and for this purpose I shall discuss the rdéle that microbes play in human economy. Human society depends nowadays, among many other things, on a large number of organic substances for its multifarious needs. A gigan- tic organic-chemical industry, this term to be understood in its broadest sense, performs the requisite conversions of organic compounds. First of all I want to examine the question whether in the present day and age microbes may still fulfil a function in this industrial transfor- mation process. Obviously, there is every reason for posing this question. For, despite the fact that in the beginning of the previous century the synthesis of the manifold organic substances was considered to be the exclusive prerogative of living organisms, there has gradually developed a strong reaction against this notion. A professor of organic chemistry is not likely to pass up the opport- unity to refer in his inaugural address to the discovery that was made on February 22, 1828, and to do so with consummate satisfaction. And rightly so; for on that memorable day the barriers which until then had sharply divided the products of animate from those of inan- imate matter were shattered in consequence of the accomplishment of the celebrated chemist, Friedrich Wohler, who had succeeded in converting ammonium cyanate into urea. An endless field for study, inaccessible till that moment, was thereby opened up for the chemist. And [I need not belabour the point that chemistry and chemical industry have nobly acquitted themselves in this respect, and scored prodigious successes. The number of organic substances that the chem- ist has learned to prepare in his laboratory runs into the hundreds of thousands, and the application of these discoveries has caused sweep- ing changes in the world’s enterprises. 166 MICROBIOLOGY AND INDUSTRY For the time being the organic-chemical industry still remained dependent for its raw materials on the fossil and extant vegetable kingdom. The conversion of carbon dioxide of the atmosphere into organic matter, accomplished in the leaves of green plants under the influence of solar energy, remains a problem that the chemical engi- neer has so far failed to solve, at least in an economically practicable manner. Now it is in particular in connexion with the utilisation of the fos- sil raw materials, coal, bitumen, lignite, and petroleum, that the organic-chemical industry can boast of its greatest triumphs. Even though in many places the sugar-, oil-, and starch industries have been enormously expanded, one should not lose sight of the fact that in all these cases the plants, e.g. the sugar cane, coconut, or potato, are the true manufacturers of the desired raw materials; it is merely the isolation therefrom of the ingredients most valuable to man that is carried out in the factories. The world’s reservoirs of fossil raw materials are, however, far from inexhaustible; on the contrary, sundry investigations concur in leading to the conclusion that several of these products will be depleted in the not too distant future. Their ever increasing exploitation makes one fear for the worst in this respect. In a study of the energy provision of the U.S.A. Steinmetz has calculated that since 1870 the coal produc- tion has increased on the average by 6.35 per cent annually. From 10 million tons in 1852 it has risen to 100 million tons in 1882; in 1920 it has probably exceeded tooo million tons; and, if the increase con- tinues at the same rate, it should amount to 10,000 million tons by 1958. Similar considerations generally apply to other countries, except for the fact that the world war has caused temporary perturbations. Although Svante Arrhenius recently estimated that the known coal deposits in the world would be sufficient for 1500 years on the basis of current consumption data, the Steinmetz figures indicate that the time at which coal rationing will have to be imposed in certain parts of the world is apt to be reached considerably earlier, in fact, in the foresee- able future. The prospects for the petroleum industry are even more dire. Au- thorities whose judgement carries weight do not consider it improb- able that within a few decades the maximum rate of petroleum pro- duction will be reached. Equally pessimistic views have frequently 167 SELECTED PAPERS been expressed in connexion with the problem of the world’s energy provision. Nevertheless, there is always the bright prospect that be- fore a state of emergency will have arisen, human cunning will have succeeded in fixing solar energy directly, 7.e., without the aid of the plant world, in a form that is useful to society. Even the possibility that some day man will be in a position to exploit the enormous intra- atomic energy cannot any longer be relegated merely to the realm of fiction, as indicated by scientists of the stature of Richardson and Rutherford. But the exhaustion of fossil fuels is important not only from the point of view of energetics. I have already mentioned that they supply a significant fraction of the raw materials needed by the organic- chemical industry. The gradual depletion and the resulting higher price of the fossil raw materials will consequently entail that the chem- ical industry will gravitate more and more towards procuring its start- ing materials immediately from the present-day plant world.* Thus the intensification of agriculture, and its development along lines of modern industry, will in the future become more and more the order of the day. This will mean that the chemical industry will be provided primarily with a few staple agricultural products whose value derives largely from their content of the three major groups of substances, the carbohydrates, oils, and proteins. And then it are par- ticularly the carbohydrates — the sugars, starches, and cell-wall constitu- ents — that will become prominent as raw materials. Now the specific advantages of microbiological mechanisms are strikingly apparent in the transformations of carbohydrates into numerous other products. With an ease that elicits the organic chem- ist’s envy various microbes can split the glucose molecule, each one yielding its own more or less specific products. ‘Thus the common yeast, Saccharomyces cerevistae, furnishes alcohol and carbon dioxide; Lactobacillus fermentum and related types produce significant quantities of lactic acid; Fernbach’s Bacillus macerans manufactures butanol and * The fact that we can currently observe diversions in the opposite direction, for example in the manufacture of alcohol from coal via calcium carbide, acetylene, and acetaldehyde, or in the production of synthetic fats from paraffins by way of their oxidation to fatty acids, does not detract from the validity of the general argument. By increasing the utilization of the fossil raw materials such developments can only hasten their depletion. 168 MICROBIOLOGY AND INDUSTRY acetone; the recently isolated Bacillus acetoethylicus yields acetone and ethanol; Beijerinck’s Granulobacter saccharobutyricum makes butyric acid; Duclaux’s Tyrothrix tenuis, dihydroxyacetone. Various moulds of the genus Citromyces may give rise to the production of appreciable quan- tities of citric acid from glucose; Wehmer’s Aspergillus fumaricus con- verts it into fumaric acid with very good yields. It would not be dif- ficult to augment these examples with many others, but I fear that this would become tiresome. The above survey may, however, suffice to make you realise that a sub- stance such as glucose, easily procurable by purely chemical or bioche- mical methods from a staple product such as starch, can be converted into diverse products with the aid of micro-organisms. And it seems to me that these are exactly the compounds that are of prime importance to the organic chemical industry as building blocks for various syntheses. I believe that no organic chemist is likely to contradict me when I say that the purely chemical manufacture ofthe above mentioned substances from glucose will cause him far greater difficulties than one is apt to encounter in the corresponding biochemical transformations. In his opening address of the Congrés de la chimie industrielle, ‘L’avenir de la chimie organique’, none other than Sir William Pope has quite recently emphasized the considerable difference between organic chemistry, both of the past and present, and the conversions that proceed in living organisms. On the one hand Pope notes the high temperatures and the powerful, though rather unspecific chemical reagents that are still indispensable to the chemist; on the other hand the extremely selective mechanisms of the living cell, functioning at ordinary temperatures. It is true that Pope points out that the meth- odology of modern organic chemistry shows an increasing tendency to approximate that of biochemistry, since the application of catalysts, and the consequent use of colloidal reaction media, are practiced more and more by the organic chemist. But this does not alter the fact that the distance that will have to be traversed before the organic chemist possesses a set of catalysts that would enable him to perform the conversions summarized above without the aid of microbes still appears very great. This raises the question whether biochemical conversions, proceed- ing under the influence of micro-organisms, are currently of impor- tance for the organic-chemical industry. 169 SELECTED PAPERS Let us first of all consider the alcoholic fermentation of sugars. The enormous economic significance of biochemical alcohol manufacture, so universally practiced by primitive and by the most highly civilized societies alike, does not need extensive illustration. Simmonds esti- mates the average annual production by the seven most important producing countries over the period rg09-1913 as more than 436 million Imperial gallons of 100 per cent alcohol. If one realizes that the practice of single-cell culture methods has convincingly shown that potentially one single yeast cell, of which around 2.10!° make up one gram of compressed yeast, suffices for the production of so vast a quantity of alcohol, this will provide a striking demonstration of the macro-achievement of a micro-organism. The above-mentioned estimate of 436 million gallons includes, however, 310 million gallons that have served for human consump- tion. One might therefore be inclined to think that an extension of pro- hibition over the rest of the world, following the example set by the U.S.A., might cause irreparable damage to this industry. But if such an extension were to be introduced gradually, such consequences need not be feared on account of the general situation that I have already indicated, viz., that the potential exhaustion of fossil fuels cries out for substitutes derived from the present-day plant kingdom. As mentioned earlier, one cannot sustain high expectations with respect to future petroleum production. In even stronger measure does this apply to the production of petrol; as is known, this occurs in reasonable amounts in some crudes only. In view ofthe undreamt-of expansion of automobile traffic it will be evident that serious concern is felt in var- ious circles in connexion with the possibility that the petrol production might not keep pace with the increase of motor vehicle traffic. For a considerable time a search for petrol substitutes has already been in- stituted, and universally the conviction has been gained that industrial alcohol offers the best prospects. In 1918 the British Government therefore appointed an ‘Alcohol Motor Fuel Committee’ that was charged with making a study of an eventual large increase of the al- cohol industry, and at the same time of the difficulties inherent in the use of alcohol in petrol combustion engines. Aided by national com- mittees in different parts of the Empire, this committee has issued a report in which it calls attention to the particular suitability of the tropical and subtropical regions for an increased carbohydrate pro- 170 MICROBIOLOGY AND INDUSTRY duction with conjunct manufacture of alcohol. The drawbacks initial- ly attaching to the use of alcohol in petrol combustion engines appear to have been conquered satisfactorily both in South Africa and in Australia. Hence the future of the biochemical alcohol manufacture seems to be assured. While discussing this industry I cannot forego mentioning specific- ally the successful application, in France and a few other countries, of the ‘amylo-process’, because this supplies one of the outstanding ex- amples of the imaginative use of micro-organisms as aids in chemical technology. It also demonstrates that the primitive procedures of Oriental peoples often contain the germ of a methodology that can effectively be used in competition with Western processes. It has long been known that from olden times the Chinese have possessed the secret of preparing alcoholic beverages from rice. For this purpose they use a material that has commonly been designated by European investigators as ‘Chinese yeast’, and that was first stud- ied in 1885 by Calmette in Indo-China.* According to a formula ob- tained by Calmette, the preparation of ‘Chinese yeast’ requires no less than 46 ingredients, most of which are parts of plants with special flavouring qualities. The active principle, however, consists of the microbes that are always present in these ingredients, and among which a mould, probably belonging to the genus Mucor, is particularly predominant. This mould has been obtained in pure culture; it is characterized by a very strong diastatic power, t.e., the ability to con- vert starch to sugars. Calmette then attempted to use the diastatic properties of this and of related moulds also in Western European alcohol industry, for here, too, starch-rich raw materials are converted into sugar by diastatic enzymes, as, for example, in breweries, where one generally depends on the diastase found in malt, z.e., germinated barley. Now the applica- tion of mould diastases seems to offer a number of advantages, such as the fact that the hydrolysis of starch yields virtually no non-fermen- table dextrins. After many reverses Calmette’s collaborators have final- ly succeeded in sufficiently eliminating the practical difficulties, so that a few years ago Delamar estimated that 6 million hectolitres of alcohol were produced by the amylo-process. In due course the initial- * A very similar product is generally available in the Dutch East Indies under the name ‘ragi’; it was investigated by Went and Prinsen Geerligs. ig SELECTED PAPERS ly employed moulds have been replaced by others that are even more satisfactory; and currently only those known as Rhizopus Delamar and Mucor Boulard find application. Still dissatisfied with the results, Effront and Boidin have aimed for still higher goals. ‘They had found that in both the malt- and the amylo- process powerful proteases are operative in addition to the amylases. This causes the major part of the proteins present in the raw material to be converted into simple, water-soluble nitrogen compounds that are lost in the course of further operations. Moreover, even the use of mould diastase requires a rather costly pretreatment of the starch- rich material with steam under pressure. These investigators there- fore attempted to prepare a potent diastase that can act on native starch, and that leaves the proteins largely intact. In this manner the nitrogenous material can be recovered after the alcohol has been dis- tilled off, and used as fodder. They ultimately succeeded in finding the desired biochemical catalyst in a specially adapted culture of Bacillus mesentericus. If properly prepared, one unit weight of such a culture is capable of hydrolysing 1,000 unit weights of starch. Following the treatment with Bac. mesentericus the raw material is subjected to a brief action by Rh. Delamar in order to produce exactly the minimum amount of soluble nitrogen compounds needed for a good development of the yeast during the third stage of the process. Thus one sees how in Effront’s modification of the amylo-process the entire conversion of starch into alcohol is accomplished by the successive operations of three different micro-organisms. Proceeding now to a consideration of some other biochemical con- versions, I may state that for the technical manufacture of lactic acid the fermentation method possesses a virtually undisputed monopoly. Chapman estimates that in 1909 Germany produced no less than 1500 tons of lactic acid for export, over and above the amount used inside the country. Also in Holland the commercial manufacture of lactic acid has lately been introduced. Most intriguing is furthermore the history of the biochemical man- ufacture of acetone. During the years of the great rubber boom chem- ists in many countries tried to find a method for the manufacture of synthetic rubber. The British chemist, Perkin, devised a procedure in which butanol was used as the starting material. In conjunction with this process the French bacteriologist, Fernbach, developed a method 172 MICROBIOLOGY AND INDUSTRY for the production of butanol and acetone from potato starch by fermentation with a bacterium he isolated, and which is probably identical with Bac. macerans studied in 1905 by Schardinger. On ac- count of the gradual changes in the rubber market the manufacture of synthetic rubber never got started. But in 1915, when a great de- mand for acetone developed in England for the manufacture of ex- plosives (‘cordite’), Fernbach’s pupil, Weizmann, succeeded in adapt- ing Fernbach’s procedure to the large-scale production of acetone.* From England this process was exported to Canada where the ‘British Acetones Ltd.’ built a large factory in Toronto for the new process. In 1918 this factory contained no less than 22 functioning fermenters, each one with a capacity of 30,000 gallons. From Canada the process was introduced into the U.S.A.; meanwhile, Fernbach had succeeded in developing his process on a technical scale in France. During peace-time the commercial success of this process obviously depends on the possibility of finding a ready market for the butanol which is produced in an amount twice as great as that of the acetone. Butanol had not previously been used in chemical industry; various possibilities for a potential utilisation of this substance have already been suggested, but as yet it is not possible to express a final verdict concerning their feasibility. Meanwhile Northrop, Ashe and Morgan had isolated their Bac. acetoethylicum which produces acetone accompanied by ethanol instead of butanol. Furthermore, subsequent investigations showed that this bacterium is not particular as far as its nutrition is concerned; it pro- duces the same substances from pentoses in quite satisfactory yields. This opens up a means for economically disposing of the corn cobs, now representing a waste product which, in the U.S.A., amounts to 20 million tons annually. Whatever else may develop from these industries, it may suffice to state that estimates show that, at the time of the armistice, 5,000 tons of acetone and twice that amount of butanol had been produced in the U.S.A. alone, and this owing to the activity of a microbe that twenty years ago had probably never been considered worthy of hu- man attention. In this connexion I may point out that the thousands * Whether Weizmann used the initial isolate of Fernbach is not quite certain. In one of his later patents he recommends the use of Granulobacter pectinovorum, discovered by Beyerinck and van Delden as a causative agent of flax retting. 173 SELECTED PAPERS of cultures employed in these industrial fermentations may well have originated from one bacterial cell whose dimensions, in comparison with those of the earlier mentioned yeast cell, are very small indeed. If we further realize that in a relatively short time that single, minute cell has given rise to the construction of many imposing factories in various parts of the world, then it is unavoidable not to be impressed by the enormous powers that reside in even the smallest living entities. Then, too, we cannot escape the notion that, in view of the thousands of microbes that so far have only superficially been investigated, and of the unknown numbers of microbes that occur on earth but have never yet been observed by man, a rational exploration and exploita- tion of the powers of the microbial world may still bring many benefi- cial results to mankind. This possibility is strikingly illustrated by some procedures worked out in Germany during the war emergency. I refer to the microbiolog- ical production of fat and protein. It had long been known that under certain conditions various micro-organisms accumulate fats and oils as intracellular reserve products. Encouraged by the great demand for fat during the war, Lindner made a careful study of this phenomenon, hoping thereby to accomplish a technically feasible procedure for the conversion of carbohydrates into fats. In a sample of some material collected from the sap flow of a birch tree and which his pupil Schret- tenfeger had sent Lindner from the Eastern front, the latter discov- ered an organism, Endomyces vernalis, that appeared eminently suitable for such a fat synthesis. It was found possible to obtain good yields of this organism with a fat content of 60 per cent, calculated on a dry- weight basis, by growing it in sugar media with a small amount of ammonium sulphate as nitrogen source. A comparable situation is encountered in the microbiological pro- tein synthesis, also evolved in the ‘Institut fiir Garungsgewerbe’ in Berlin. Starting from the consideration that, after elimination of the bitter ingredients introduced by the hops, the brewery yeast repre- sents an eminently suitable, protein-rich fodder, Lindner further investigated the possibility of manufacturing a protein-rich yeast pro- duct from readily available inorganic nitrogen sources, primarily am- monium sulphate, reasoning that, at least during periods of severe food scarcity, such a process might be economically important. As a result of this investigation many factories in Germany actually pro- 174 MICROBIOLOGY AND INDUSTRY duced a ‘mineral yeast’ during the war, at which time it acquired im- portance as food for both man and animal. For this purpose a yeast was used that is closely related to the common alcohol yeast but that, when cultivated in shallow layers of liquid exposed to air, displays virtually no fermentative activity so that under these conditions a strictly aerobic respiration is accompanied by a copious multiplication. On Chapman’s [1921] authority, who spoke in this connexion of ‘true Teutonic enthousiasm’, I may state that it was this process that tempted Hayduck to the pronouncement that not until man is in a position to convert his evening newspaper so rapidly into sugar that the protein produced therefrom can be consumed the next morning at breakfast will one of the greatest problems of this century have been solved! It is also worth mentioning that the reverse of the last-mentioned process, vz., a microbiological conversion of the organic nitrogen compounds present in waste materials into inorganic nitrogen com- pounds is the basis of a native British Indian industry that is current- ly still most important in that country. It may surprise many of you to learn that before the war British India, despite a far from negligible native consumption, exported on the average 15,000 tons of salpetre annually, while during the war this figure reached approximately 50,000 tons. All of this nitrate had been produced by the natives in a primitive manner, by making use of the powerful bacterial nitrification process that, in the tropics, can proceed under very favourable condi- tions. The preceding remarks have opened up many unsuspected prospects for applied microbiology; nevertheless, the existing possibilities have by no means been exhausted. Thus far I have discussed micro-organ- isms as biochemical catalysts more or less on a par with the catalysts of pure chemistry. But this implies a serious undervaluation that calls for a redress. Another aspect is evidently revealed by the power of organisms to reproduce, which consequently means that, in contrast to what happens to purely chemical catalysts, an enhanced production of biocatalysts occurs during microbial conversions as a result of cell multiplication. In addition I must emphasize the great adaptiveness that appears to be inherent in living organisms. In this connexion I think of the surprising modifications in the metabolic properties of a cell under the influence of chemical changes in the medium in which 175 SELECTED PAPERS it lives. Striking examples of this sort have become known, especially during recent years, and they do not suffer from a lack of technological significance. A first instance may be derived from an investigation of the Ameri- can microbiologist, Currie. Whereas oxalic acid, in addition to carbon dioxide, had been found as a significant by-product of the carbo- hydrate metabolism of the intensively studied mould, Aspergillus niger, Currie succeeded in demonstrating that the oxidation of carbohydrates by this mould proceeds in consecutive stages, and that citric acid is one of the earliest intermediate products. Currie further proved that, by an appropriate choice of environmental conditions, the metabolism of Asp. niger can be so guided that citric acid production is enhanced, while the further oxidation of this product to oxalic acid and carbon dioxide is inhibited. Thus he was able to increase the citric acid pro- duction from sugar to 50 per cent, whereas under the conditions com- monly employed never more than trace amounts of this substance are found. Here is another example. Pasteur already knew that during a nor- mal alcoholic fermentation a small quantity of glycerol, ordinarily about 2-3 per cent of the fermented sugar, is formed in addition to the major products, alcohol and carbon dioxide. When during the war Germany suffered from a serious shortage of glycerol, owing to the deficit of fats, various investigators raised the question whether it might not be possible under special conditions to induce the yeast to an increased glycerol production. Probably fully independently, this problem was simultaneously solved by Connstein and Liidecke, and by Neuberg and collaborators in Germany, by Schweizer in Switzer- land, and, later, by Eoff and co-workers in the U.S.A. All four groups found that the yield of glycerol can be increased to 25~30 per cent of the sugar by adding appreciable amounts of sodium sulphite to the normal, sugar-containing media. In Germany this process has been used on a large scale. What makes this discovery particularly interesting is the fact that Neuberg observed that besides glycerol only very little alcohol was formed, while a large amount of acetaldehyde, equivalent to that of glycerol, was recovered. I cannot here enter into a discussion of the enormous significance that this discovery has exerted on our inter- pretation of the chemical conversions that take place during a normal 176 MICROBIOLOGY AND INDUSTRY alcoholic fermentation. I shall only emphasize that a microbe, used as long as man remembers for the manufacture of alcoholic liquors, and unquestionably studied more thoroughly than any other microbe, after half a century of intensive microbiological research suddenly ap- peared capable of performing hitherto unimagined chemical conver- sions. If such thoroughly studied organisms as the ubiquitous yeast and Asp. niger can occasion such surprises even to-day, the thou- sands and thousands of microbes, thus far studied only cursorily if at all, may yet offer limitless possibilities for biochemical transformations that may also be important from an industrial point of view. But at the same time we realize that the inflexible concept of microbes as catalysts capable of performing but one specific conversion implies a serious underestimate of the capacities of these organisms, and we must bow before the impressive potentialities of life. Now one might justifiably object that, from a commercial point of view, many of the above-mentioned processes have not been a lasting success, and have not or only barely been able to weather the economic post-war competition. To this I can only reply that I have chosen these examples because they seemed to involve some ideas of general significance. It should, however, not be concluded that they represent the only examples of an industrial application of micro-organisms. Had I wished to restrict myself to long established and renowned industries in which microbes play a leading réle, I might have discussed the man- ufacture of wine, beer, and vinegar; the dairy industry; the retting of fibres; the production of drinking water; the practice of sewage dis- posal; etc. I might then also have dwelt on a few flourishing primitive industries of the Orient, such as the ‘ontjom’- and ‘tempé’-industry in Java, an industry that may well await its Calmette in order to gain significance for the tropical oil industry which is now conducted on a Western pattern.* But I might rightfully be convicted of pronounced one-sidedness if I tried to advocate the misapprehension that at all times microbes are the benefactors of the industrialist. Far from it; there are numerous cases in which the microbes interfere in a most undesirable manner in the production process. * Owing to Went’s investigations much has been learned about the microbiol- ogical process of ‘ontjom’ and ‘tempé’ production; nevertheless, a rational indus- trial application of these findings has not as yet been attempted. 177 SELECTED PAPERS It is well known that the oleomargarine industry, which during the war years underwent a rapid expansion especially in England, was seriously threatened by the poor keeping qualities of its product. Oleo- margarine appeared to be particularly vulnerable to the deterioration known as rancidity. Owing to the outstanding studies of Jacobsen we now know that rancidity, both of vegetable fats and, as Orla-Jensen had proved, of dairy butter, is caused by micro-organisms. It will therefore be necessary to attach great value to various means by which the product can be protected against contamination during the successive stages in its production. If we may believe a recent study of Stokoe it will even be necessary to pay the closest possible attention to such measures already during the production of the plant oils that are used as the starting material in the oleomargarine industry. During the war large quantities of sugar, produced in tropical coun- tries, could not immediately be shipped, and thus had to be temporar- ily stored there. In this case, too, deterioration was soon observed. Kopeloff and collaborators in the U.S.A., and Amons in Java, could unequivocally demonstrate that the deterioration was again the result of microbial activities. In some cases the source of the contamination could be located in the factories, and measures could thus be devised to restrict the deleterious effects to a minimum. Browne estimates that these microbes caused the Cuban sugar harvest of 1916 alone to suffer a loss in value of $ 1,500,000. Let me finally remind you of the industries concerned with the preservation of foodstuffs, which owe their existence to the ubiquit- ousness of spoilage-provoking microbes. Guided by intelligent pure food laws, the large American canning industries have increasingly been forced to adopt practices that are based on the results of scientific investigations dealing with the destruction of micro-organisms as a function of particular conditions. This certainly is an example worthy of being emulated! Whereas microbiology thus serves industry firstly by providing leads for the proper management of biochemical transformations, and sec- ondly by developing adequate techniques for a rational elimination of unwanted micro-organisms, there is yet a third area in which it can unfold its powers to the benefit of industry. In this connexion I have in mind the indispensability of scientific investigations for the proper 178 MICROBIOLOGY AND INDUSTRY management of every industry. I may here refer, for example, to the studies of Pfeiffer showing that the rate at which various kinds of wood are decomposed under the influence of methane producing bac- teria may provide important indications as to the durability of these materials in actual practice. Biochemical sugar determinations can certainly be of importance in the scientific control of the fermentation industries, in particular if new raw materials are being used; a good example is furnished by the gradually developing alcohol manufacture from sawdust. And microbiological studies can be of importance even for the more remote petroleum industry. With the aid of micro-organisms that can decompose the hydrocarbons of the paraffin series, and that had pre- viously been studied by Sohngen, ‘Tausz and Peter recently accom- plished the isolation of naphthenes, not attacked by these bacteria, from mixtures of naphthenes with paraffin hydrocarbons. This represents a separation that offered well-nigh insurmountable difficulties when at- tempted by purely chemical methods, even in Engler’s laboratory. It would not be difficult to cite many more instances, but I must already have taxed your attention unduly. The famous biologist, Huxley, calculated at one time that the econ- omic advantages that France reaped from Pasteur’s fundamental microbiological discoveries were sufficient to pay, during a period of only twenty years, for the entire war debts incurred in the war of 1870—1871, debts that were then considered stupendous. Is it too bold to propose that, as a consequence of the above discussion, we may expect that during the next few decennia microbiological science may significantly contribute to an alleviation of the economic consequences of the world war? Before closing I must, however, dispose of a misapprehension that may have arisen out of the previous remarks. I have used the time at my disposal largely to give you an idea of the great importance of microbiological applications in industry. But I should not like to leave you with the impression that the study of general or theoretical microbiology, which I shall also have to teach, appeals less strongly to me; nothing could be farther from the truth. When Leibniz once urged Van Leeuwenhoek to see to it that his method for grinding microscope lenses would be transmitted to later generations, Van Leeuwenhoek replied: 179 SELECTED PAPERS ‘To train young people to grind lenses, and to found a sort of school for this purpose, I can’t see there’d be much use: because many students at Leyden have already been fired by my discoveries and my lens-grinding, and three lens-grinders have gone there in consequence ; to whom the students have repaired, to learn how to grind lenses. But what’s come of it? Nothing, as far as I know: because most students go there to make money out of science, or to get a reputation in the learned world. But in lens-grinding, and discovering things hidden from our sight, these count for nought. And I ’m satisfied too that not one man in a thousand is capable of such study, because it needs much time, and spending much money; and you must always keep on think- ing about these things, if you are to get any results. And over and above all, most men are not curious to know: nay, some even make no bones about saying: What does it matter whether we know this or not?’ * Even though, after a lapse of 250 years, our opinions as to the econ- omic importance of the microbial world may differ from those held by Van Leeuwenhoek, I should not wish to be counted among those to whom knowledge is important only if in a material sense one ‘can get something out of it’. I, too, am not insensitive to the fascination which the study of nature and the struggle to penetrate into its tenaci- ously guarded secrets holds for the investigator. Moreover, I keenly realize that the study of general microbiology is a primary prerequisite also for future progress in the realm of microbiological applications, and this at the same time justifies the inclusion of this purely scientific subject in the curriculum of a technological university. In a noteworthy paper Rahn [1921] has recently made a plea for theoretical bacteriology which he characterizes as the foster-child among the sciences. He points out that, in contradistinction to most of the latter, in Germany bacteriology has been able to develop only as an accessory, as the handmaiden principally of medicine and agri- cultural science, from the very start. Not a single German university can boast of the inclusion of an institute for theoretical bacteriology ; nowhere does bacteriology find a refuge where, in peace and quiet, it may be studied for its own sake. It is true that bacteriology, as a subdivision of botany, can right- * Ed. note: This passage has been copied from Clifford Dobell’s [1932] superb translation of Van Leeuwenhoek’s reply (p. 325). 180 MICROBIOLOGY AND INDUSTRY fully engage the interest of those who hold positions in the universities’ botany schools, and several amongst these botanists have made mer- itorious contributions to microbiological science. Nevertheless, if we survey the extent of the plant kingdom, and recognise how relatively modest a place the microbes occupy therein, we realize that the pro- fessor of botany can and should devote no more than a small fraction of his time and efforts to bacteriology. But the present status of bac- teriological science imperatively calls for a large number of investig- ators who can dedicate their entire life to studies in this field. I shall mention but a single example in support of this claim. It is well known that even to-day there exists an exasperating confusion in the area of the systematics of the Schizomycetes. In this respect the students of bacteriology have even been compared to insulars who, each one sitting tight on his own little island, are wont to hurl minor verities at each other across oceans of misunderstanding. Surely this picture is hardly painted too darkly! In consequence of the minute dimensions of the bacteria, their morphological characteristics cannot have the same significance in bacterial systematics as they do in the taxonomy of the higher plants. Hence physiological and ecological properties must occupy a far more prominent place in bacterial taxonomy, and, together with morpho- logical criteria, they must be wielded into an encompassing and har- monious whole. Thus it follows that an intensive investigation of the multifarious bacteria, extended in many different directions, is re- quired to escape from the present impasse. Meanwhile, the Society of American Bacteriologists has taken the first steps in the right direction [Winslow e¢ al. 1920]. It has appointed a committee on the classification of bacteria which has evolved a framework that may be called successful in many respects, and that consequently deserves the whole-hearted attention of bacteriologists from all over the world. It would be most desirable if some degree of international agreement could soon be reached concerning this matter. Whereas Rahn, too, calls attention to the example of the U.S.A. as one of the few countries where bacteriology is studied for its own sake, and exhorts his compatriots to follow this lead, he has at the same time made a statement in which we may rejoice. I quote: ‘Die Profes- sur Beijerinck’s in Delft (Holland) entspricht ebenfalls den Bediirf- nissen der theoretischen Bakteriologie.’ And also outside Germany 181 SELECTED PAPERS voices are raised complaining about the neglect of microbiology as a science. Let me cite Nicolle, the Director of the Pasteur Institute in Tunesia, who after his visit to Paris recently wrote [Lichtenberger, 1921]: ‘Les études microbiologiques se meurent. Le pays qui a produit Pasteur, Duclaux, Laveran, Roux, pour ne pas citer que les plus illus- tres et qui a recueilli Metchnikoff, laisse, sans en témoigner nul souci, périr une science qui lui a valu jusqu’ a présent une belle part de sa gloire’. If I contemplate this state of affairs, and particularly if I recall Rahn’s sober tribute to my great predecessor’s work in the field of theoretical bacteriology, then it is inevitable that I close my address by returning to my starting point. Once more shall I express my profound feeling of responsibility towards microbiology, not merely as a subject to be taught at this university, but also as a science. I shall endeavour to serve it according to the best of my modest abilities. Members of the Board of Trustees, It is for me a reason of deep-felt gratitude that you have been willing to propose my name to the Government for the chair of microbiology ; me who resided at so great a distance from my mother-country, and who must perforce have been a more or less mythical personality to you. To me this is proof that in your choice you have also been guided by considerations of potentialities, which implies confidence in my future efforts and ability. It will be my serious endeavour not to shame this confidence. Moreover, I want to express the hope that you will not deny me your support. Personally I do not doubt that you also represent that spirit of progressiveness and that keen insight that were exhibited by the educational authorities when, in 1895, they decided to include bac- teriology as a subject in the curriculum of the Polytechnical School, a farsightedness which, as I have previously indicated, elicits the envy of other countries even to-day. What I have said may contribute to keeping alive the notion that, amongst the various sciences taught at this university, general and applied microbiology deserves to occupy an important place, at present, and perhaps even more so in future. Eighly esteemed Beyerinck, Amongst the congratulations that I received as a result of my appoint- 182 MICROBIOLOGY AND INDUSTRY ment none was more valuable to me than your brief announcement that it caused you satisfaction that I had been selected as your suc- cessor. Not yet a year ago authorities whose opinion is of significance also to you have honoured you, ‘anerkannten Meister der mikrobiologi- schen Wissenschaft’, as Lindner said, and have attempted to do hom- age to your merits in connexion with the exemplary and dedicated manner in which you have served this science. A feeble reiteration of such praise from my side cannot be agreeable to you. But here I will proclaim my deep recognition that the occupancy of this chair implies also that at least part of that which your genius and unremitting en- deavour has created is now entrusted to my care. It would be fruitless for me to attempt to maintain the lustre that your institute has acquired by virtue of your work. But to contribute to making this golden quarter century of Dutch microbiology some- thing that will continue to live in the minds of a new generation, that is within my power. Professors of the Technological University, On entering upon my duties I wish to assure my colleagues at the Technological University that I greatly appreciate being included in their community. I hope that on occasion I may be permitted to appeal to you for assistance. This applies particularly to the members of the Department of Chemical Technology, amongst whom I may greet many of my former teachers. Although the isolation of micro-organ- isms may be an important aspect of my future endeavour, isolation of microbiology will never be its aim. I am far too much aware of the fact that our separate fields interpenetrate than that I would not at- tach great importance to a close co-operation. Allow me to benefit from your wide knowledge and mature experience. Not in the last place do I think in this connexion of you, dear Boeseken. My remarks may have suggested a certain competitiveness between purely chemical and biochemical procedures in industry; but this does not imply a lack of realisation that biochemical problems can be solved only with the aid of organic chemistry. Although some- thing may have been added to our previous relationship of teacher and pupil, I wish nevertheless that in many respects this relationship may be perpetuated. 183 SELECTED PAPERS Dear Van Iterson, That I, who have been privileged to be your long-time assistant, have now been called upon to succeed him whose assistant you yourself have been for many years, reminds me of the atavistic phenomena that are so characteristic of certain higher plants, to use another biological analogy. To me this is proof of the close relationship that exists be- tween the subjects we are to teach. It seems to me that our teaching will profit if at all times we inculcate a recognition of this relationship into the future chemical engineers. It is for this reason, too, that I here want to express the ardent hope that you may be found willing to grant me your continued support, tutelage, and friendship which you have so lavishly accorded me hitherto. Be convinced that I shall always remember that it was you who showed me the way to inde- pendent research. During the nearly six years that have elapsed since I left your laboratory I have realized how much more you have im- parted to me for life. My parents, My presence here is certainly in no small measure the result of your devoted cares. I feel fortunate that I may publicly thank you for this. The education you gave me was characterized by acts of love rather than by verbose theory. For that very reason I shall not expand this fervent testimony. Ladies and gentlemen, students of the Technological University, Even though so little time has gone by since the moment when I left this university, I cannot claim that we are mutually acquainted. It 1s probable that all of us will have changed too much during that brief span. On the one hand, the war has also caused upheavals in the student community whose significance I cannot yet fathom. On the other hand, fate has taken me to far-away countries, and did not Goethe, in his ‘Wahlverwandtschaften’, say that ‘Die Gesinnungen andern sich ge- wiss in einem Lande, wo Elefante und Tiger zuhause sind’? | It is true that I encountered neither elephants nor tigers in a free state; but the emancipating influence exerted by life in a foreign en- vironment has not worn off. But, although we may not yet be able to meet as old acquaintances, I should like to express the hope that we may soon have reached that stage. 184. MICROBIOLOGY AND INDUSTRY I wish to address a special message particularly to the prospective chemical engineers. My presence in Delft will probably remain un- noticed by many of you. But to those who, impelled perhaps by an inborn love of living nature, should decide to include in their program the study of microbiology I want to say that, whenever possible, I shall ever try to aid and guide you. I have already briefly alluded to my sojourn in the beautiful tropical Netherlands. In that environment I have acquired many good friends who will be gratefully remembered. Apart from that, however, I must stress another point. Whoever has been so favoured as to spend, as it was my lot, a number of years in that enchanted state of symbiosis be- tween Europeans and Orientals, recognizes at the time of departure that he has incurred a mental debt of honour. I consider it my duty emphatically to acknowledge this. May it be vouchsafed me to redeem part of this debt by awakening in some of my future pupils an interest in the growing and fermenting communities of the Far East, an in- terest that at some time will express itself in deeds. And I shall ex- perience a feeling of exultance if their pursuits in that part of the world were to give them a degree of satisfaction such as I was privileged to taste, and if their efforts were to accrue to the benefit of that com- munity. REFERENCES CHAPMAN, A. CH. 1921. J. Roy. Soc. Arts rrg, 618. DOBELL, C. 1932. Antony van Leeuwenhoek and his ‘Little Animals’. Amsterdam. LICHTENBERGER, A. 1921. Revue des deux Mondes, Nov. 15th. RAHN, O. 1921. Naturwissenschaften 9, 734. WINSLOW, E. A., BROADHURST, J., BUCHANAN, R. E., KRUMWIEDE, CH., ROGERS, L. A. and SMITH, G. H. 1920, J. Bact. 5, 191. UNITY AND DIVERSITY IN THE METABOLISM OF MICRO-ORGANISMS ‘My daughter Alice is a student in High School. One of the prescribed courses is General Science. The section on Bacteria left her with a vague impression of a world teeming with deadly germs awaiting an opportunity to infect mankind. It seems probable that this malignant conception of bacteria is very generally held. ‘In reality civilization owes much to the microbe.’ (From the Preface of A. I. Kendall, ‘Civilisation and the Microbe’. Boston, 1923 ) WHEN, some time ago, I received the esteemed invitation to deliver a lecture on some biochemical subject before the Netherlands’ Chem- ical Society, I gladly accepted because a true microbiologist may never pass up the opportunity to contribute to the vindication of the smallest living organisms. For there is an altogether too prevalent notion that the microbes, as irreconcilable enemies of man, plant, and animal, deserve attention only in order to make it possible the better to combat them. However understandable such a concept may be in view of the beneficial effects resulting from the brilliant discoveries of the rdle of microbes in numerous diseases, it nevertheless does not detract from the fact that it represents an extremely warped picture of reality. But I shall not use the time at my disposal to show you how, in a microbe-less world, the conditions for human life on earth would soon no longer be realized, so that man possesses at least as many friends as enemies in the domain of the microbes. Taking advantage of the fact that I am addressing a chemically trained audience, I shall rather limit myself to an attempt at striking a sensitive chord by dis- cussing the chemical potentialities of micro-organisms. I trust that a sober review of these capacities may suffice to make you regard these smallest living beings with a little more sympathy in the future. The most specific characteristic of living matter resides in its metab- olic properties. It is an empirical fact that the maintenance of life re- quires a continuous supply of special chemical substances which, in 186 UNITY AND DIVERSITY IN THE METABOLISM OF MICRO-ORGANISMS the living cell, undergo transformations that lead to their partial excretion in altered form. You are sufficiently familiar with the notion that this applies to the higher organisms; the idea that it is equally true for the microbes follows immediately from the consideration that in so many cases the presence of microbes is forced upon us by the very fact that we observe chemical changes. When milk turns sour, when sugar- or protein- containing solutions start to ferment, it is first of all metabolic activ- ities that draw our attention, and, after half a century of microbiol- ogical research, will lead to the inference that microbes are present. As the title of to-day’s lecture indicates, it is my intention to dis- cuss the unity and diversity in microbial metabolism. But I shall take the liberty of changing the sequence and first to dwell upon the diver- sity. Later on I shall then try to indicate some aspects of the unity by which this diversity is tied together. In doing so I shall also follow in the main the historical development of microbiology. When, owing to Pasteur’s pioneer investigations, the idea had be- come established that fermentation, putrefaction, and mineralization were none other than metabolic processes of microscopically small organisms, microbiologists obviously considered it their first duty to make a survey of the multitude of different causative agents. Chemically the diversity manifested itself in two ways. It was ob- served on the one hand that one and the same medium might yield a variety of metabolic products; on the other that different microbes differ greatly in their requirements for particular chemical substances. Let me first illustrate the former aspect with some examples. If I inoculate a series of flasks, each containing a sterile solution of 5 per cent glucose in yeast extract, with pure cultures of a film-forming yeast (Mycoderma cerevisiae), and with some common moulds, such as Aspergillus niger and Citromyces glaber, respectively, a chemical analysis of the cultures at the end of their development will yield different results. Confining the investigation to a determination of the fate of the consumed sugar, it will be found that the film yeast has oxidized it to carbon dioxide and water; hence the over-all metabolism resem- bles that of animals. In contrast we shall find that Asp. niger and C. glaber have produced sizable amounts of oxalic and citric acid, respec- tively, in addition to carbon dioxide. Consequently this simple exper- iment reveals metabolic differences although these are not as yet very 187 SELECTED PAPERS pronounced. Much more spectacular is the result of a larger exper- iment in which flasks with the same medium are inoculated with pure cultures of Saccharomyces cerevisiae (baker’s yeast), Lactobacillus delbriickit (the so-called ‘domesticated’ lactic acid bacterium), Lactobacillus fer- mentum (a ‘wild’ lactic acid bacterium), Bacterium colt, Bacterium aerogenes, Bacterium typhosum, Granulobacter saccharobutyricum, and Granulobacter buty- licum, and incubated under exclusion of air. After some time it will be evident that the yeast has converted the sugar largely into ethanol and carbon dioxide; L. delbriickit into lactic acid; L. fermentum into lactic and acetic acids, ethanol and carbon dioxide; B. coli into lactic, acetic, and succinic acids, carbon dioxide, and hydrogen; B. aerogenes into the same products with, in addition, 2,3-butylene glycol; B. typhosum into formic, acetic, and lactic acids, and ethanol; G. saccharobutyricum into butyric and acetic acids, carbon dioxide, and hydrogen; G. buty- licum into butanol, acetone, carbon dioxide, and hydrogen. Thus a remarkable diversity emerges. Nevertheless, this diversity in products obtainable from a single substrate is almost negligible in comparison with the differences that various microbes exhibit with respect to their nutrient requirements. For their studies on micro-organisms the early microbiologists de- pended more or less on the fortuitous appearance of special types. But gradually they became aware of correlations between the initial com- position of the medium and the microbes therein encountered. ‘To mention just one example: it soon became clear that true yeasts are found only in sugar media. After the introduction of pure culture methods had permitted a closer investigation of metabolic activities, it became increasingly evident that substances which represent excel- lent foodstuffs for one type of microbe may be less so, if not entirely unsuitable, for others. Once this had been recognized, the idea ob- viously occurred that the investigator possessed a powerful tool for encouraging the development of certain microbes at the expense of others present in the inoculum. Although ultimately this method of elective cultures harks back to Pasteur and Raulin, it is in the hands of Beijerinck and Winogradsky that it has indubitably produced the richest harvest. Well-nigh overwhelming is the metabolic diversity revealed by the consistent application of their ‘enrichment cultures’ ; some examples may illustrate this. Based on Berthelot’s studies, showing that the increase in bound 188 UNITY AND DIVERSITY IN THE METABOLISM OF MICRO-ORGANISMS nitrogen of fallow soils was unquestionably the result of a biological process, Winogradsky [1902] succeeded in isolating one of the caus- ative agents, Clostridium pasteurianum, by means of the elective culture method in 1893. It turned out to be an organism that can grow only in the absence of air. The following remarkable experiment throws a clear light on its extra-ordinary metabolism.* A solution containing glucose and all other elements necessary for growth with the excep- tion of nitrogen is inoculated with spores of this bacterium. The cul- ture is placed in a container which is thereupon evacuated in order to remove the toxic oxygen. Even after prolonged incubation one does not observe any changes. At this point gaseous nitrogen, freed from the last traces of oxygen and of nitrogenous compounds, is ad- mitted. After 24 hours a vigorous fermentation ensues, and in a few days the sugar has completely disappeared, the bacteria have grown profusely, and nitrogen has been fixed. ‘This demonstrates the remark- able phenomenon of a living organism that is roused to life from a latent state by the inert gaseous nitrogen! Closely related to this curious organism is the bacterium, Azoto- bacter chroococcum, that was discovered a few years later by Beijerinck and Van Delden. It, too, fixes nitrogen, and to an even greater extent. But in contrast to C. pasteurianum it can do so only if an ample supply of oxygen ensures a rapid oxidation of organic substrates.** Is it surprising that the activities of these organisms have elicited the envy of men like Haber? Yet other specialists among bacteria demand our attention. How curious a group of organisms are not the urea bacteria that more or less intensively convert urea into ammonium carbonate! Special men- tion deserves Urobacillus pasteurtt which under suitable conditions com- * In spite of the fact that the assimilation of gaseous nitrogen by Cl. pasteurianum has long been established beyond any doubt, it is, as far as I am aware, only recently that the elegant demonstration has been devised (by H. J. L. Donker, Assistant at the Laboratory of Microbiology of the Technological University) which illustrates the sharp contrast between the harmful influence of oxygen and the salutary effect of nitrogen gas. For the benefit of those who might wish to repeat this experiment, it must be mentioned that the addition of a small amount of humate to the culture medium is indispensable for its success. This does not affect the sense of the experi- ment. ** For additional details of the organisms studied by Beijerinck, reference may be made to his ‘Collected Works’, published in 1922. 189 SELECTED PAPERS pletely hydrolyses a 10 per cent urea solution and thus can tolerate with impunity the alkaline reaction of a 16 per cent ammonium car- bonate solution. I may not omit an organism such as Bacillus oligocarbophilus, for which Beijerinck and Van Delden showed that it can derive its nutrients from the traces of organic matter that never seem to be lacking in laboratory air.* Also the group of bacteria that feed on hydrocarbon vapours, studied by Sdhngen [1903], may not be neglected. But even this diversity is still limited in one respect. The carbon, essential for the growth of the organisms so far mentioned, must be supplied in the form of an organic substance, though the nature of the latter may vary enormously. This implies that in the end these organisms derive their food from other organisms, hence their des- ignation as heterotrophs. Nevertheless, it is almost forty years ago since Winogradsky ex- pressed the idea that even in this regard the diversity of microbial metabolism is not limited. To be sure, it had long ago been estab- lished that chlorophyll-containing microbes could build up their cel- lular constituents from carbon dioxide and other minerals, by virtue of the absorbed solar energy. But that colourless microbes, without the aid of radiant energy, could do so too is a concept so daring that even to-day it is surprising that a human mind has ventured to propose it. A genius like Winogradsky [1887] did not hesitate, however, to conclude, as early as 1887, from his simple and ingenious experiments that the colourless Beggiatoa alba, frequently encountered in sulphur spring waters, can grow in darkness in a strictly mineral medium. The organic cellular constituents would be synthesized from carbon diox- ide, a process made possible because the necessary energy would be obtained from the oxidation of hydrogen sulphide, first to sulphur, and next to sulphate. In addition to Beggiatoa this would also apply to a large group of bacteria, partly colourless, partly purple, that exhibit the common characteristic of depositing free sulphur in their cells. Subsequent experiments have amply confirmed the correctness of Winogradsky’s concept. Consciously executed elective cultures have shown that, in addition to the relatively large organisms that deposit * Later investigators are inclined primarily to implicate traces of carbon monox- ide, a gas that is extremely toxic for higher animals, but can certainly be used as the sole carbon source by B. oligocarbophilus. See: Centr. Bakt. II, 57, 309, 1922. 1gO UNITY AND DIVERSITY IN THE METABOLISM OF MICRO-ORGANISMS colloidal sulphur in the form of droplets and as a reserve material in their cells, and which had long been known to naturalists, there exists another large group of small bacteria that, though producing sulphur extra-cellularly when grown in sulphide media, yet must be reckoned as belonging to the physiological group of sulphur bacteria on account of their metabolic behaviour which is similar to that of Beggvatoa. These bacteria, discovered by Nathansohn, but studied more particul- arly by Beijerinck, Lieske [1912], and Jacobsen [1912, 1914], have recently been much discussed in America, and permit me to give you another striking example of the chemical proficiency of the microbes. In a series of papers the American microbiologist, Waksman [1922], has reported on the astounding properties of an organism, designated as Thiobacillus thiooxidans, that can also oxidize sulphur powder to sul- phuric acid, and get along with carbon dioxide as the sole carbon source. It differs from Thiobacillus thioparus, earlier described by Beijerinck and by Jacobsen, especially by its insensitivity to acid which is so pro- nounced that in certain media the pH reaches 0.6. Nay, J. G. Lipman, the well-known editor-in-chief of ‘Soil Science’, has communicated in a recent paper that 7. thiooxidans can still effect a perceptible sul- phur oxidation in solutions containing 5 per cent sulphuric acid. It should not surprise you that this powerful ability to produce sulphuric acid has been exploited in different directions in the U.S.A. I shall mention but one example because, in a sense, it implies an attack on one of the branches of chemical industry. Lipman and Waksman have already carried out experiments on a rather extensive scale to test the possibility of circumventing the commercial manufac- ture of superphosphate by fertilizing the soil with a mixture of natural phosphate and free sulphur, and thus causing the localized formation of sulphuric acid, hence also of superphosphate, through the activity of T. thiooxidans. In certain soils this treatment appears to have given fully satisfactory results. As far as an impressive chemical performance is concerned the last- mentioned bacterium is still exceeded, however, by Beijerinck’s 7. denitrificans which can grow in the complete absence of oxygen and gaseous carbon dioxide in a medium containing sulphur, potassium nitrate, chalk, and a small amount of phosphate; thus it should be able to produce organic matter from these compounds in deeper soil layers. IgI SELECTED PAPERS I could display before you several other specialists which resemble the sulphur bacteria in being able to grow in purely mineral media. Time limitations prevent me from dwelling on them; I shall merely mention some other examples, such as the bacteria that oxidize nitrite to nitrate, or ammonia to nitrite, and for which glucose is almost as toxic as is sublimate for other organisms. Let me finally also refer to the various types of organisms for which mixtures of hydrogen and oxygen, methane and oxygen, and hydrogen and nitrous oxide con- stitute nutrients. If we consider all these data it is well-nigh impossible not to be impressed by the enormous diversity in microbial metabolism. If we further survey the trends in microbiology during the past few decennia it is hard to avoid the conclusion that the attempts to demonstrate this diversity have largely dominated the investigations. It was not so much the microbes themselves that were the starting point for the studies; rather was the mere suspicion that certain chemical trans- formations might occur in nature a sufficient impetus to formulate the hypothesis that there would be microbes to accelerate them. And the correctness of the hypothesis was substantiated in nearly every case by those who had learned to utilize the elective culture method. The very consideration that the cycle of matter on earth is closed, and that consequently the naturally occurring hydrocarbons must eventually be converted into carbon dioxide and water, led Sdhngen to the dis- covery and isolation of an important group of bacteria, members of the genus Mycobacterium, that can use these substances as carbon source. I can cite an even more striking instance. The studies of the above- mentioned autotrophic bacteria, of which the sulphur-, nitrite-, and ammonia-oxidizing bacteria are the best-known examples, uncon- sciously led Nathansohn to the bold hypothesis that still other reac- tions, proceeding very slowly at ordinary temperatures, might serve as a basis for microbial metabolism even if the oxidizable compound is not known to occur in nature. Thus he could ascertain the fact that the exclusively man-made thiosulphate can serve as the major nutri- ent for particular types of sulphur bacteria. These examples may suffice to show how gradually the investiga- tions became subservient to what might be called a ‘microbiological imperialism’. It became a contest to open up new, seemingly inacces- 192 UNITY AND DIVERSITY IN THE METABOLISM OF MICRO-ORGANISMS sible areas for the microbes. Barely had one specialist among micro- organisms been discovered when another even more spectacular one was announced; the microbiological theatre resembled one grand naturalistic vaudeville show. However great may be our admiration for those who, through their intuition and ingenious experimentation, have guided this flight of general microbiological discoveries, and however indispensable has been this thorough exploration of the microbial world for the contin- ued development of microbiology, in the long run this approach could not persistently be satisfying. Even while the main current of microbiological research was concentrated on the diversity, a field of study gradually developed in which a search for unity in the diversity became apparent. It would be unjust to depict this trend as an entire- ly new and recent development. Far from it: many classical figures in microbiological science, and its founder, Pasteur, in the very first place, must receive honourable mention in this connexion. Neverthe- less, one may safely say that the attempt to view microbial metabolism in the light of the outcome of general physiological research has been deliberately initiated by the German physiologist and hygienist Rub- ner, only at the beginning of the twentieth century. And we may 1m- mediately add that thus far this approach has found very little re- sponse. This regrettable fact can be readily explained by the circum- stance that microbiology has hitherto developed primarily as an ap- plied science. By far the majority of medical, technological, and agri- cultural microbiologists have studied the rdle of microbes in the im- portant and eminently practical problems they had to face. And of the relatively very small number of investigators who did have the oppor- tunity and desire to study microbes for their own sake, the majority was under the spell of the diversity; quite understandably so in view of the fascination of this type of work. If now I am going to embark on the attempt to give you a glimpse of the unity that can be discovered in microbial metabolism, I do so with the full knowledge that it is but a meagre account I have to offer. That nevertheless I have ventured to solicit your attention for this topic finds its explanation in the fact that I shall thus have the opportunity to make you realize that this still presents an immense field of endeavour. It seems to me that the solution of the problems in 193 SELECTED PAPERS this area is indispensable to elevate microbiology from its status as a largely descriptive branch of science to a higher plane, and equally so to the application of microbiology to many situations. And I am firmly convinced that only a close cooperation between microbiol- ogists and accomplished chemists can lead to advances in this respect. Let me then first of all show you how the unity in the divergent metabolic processes of the microbes finds expression in the fact that we recognise in them the same general trends that have come to light as a result of investigations of the metabolism of higher organisms. With- out discussing this aspect in detail, the following remarks may serve to illustrate this point. Studies of the metabolism of higher organisms have unequivocally shown that one can always distinguish two types of processes. Part of the foodstuffs is converted into cell materials, the latter being used either for growth or for replacement of degenerated cellular constituents. Another fraction of the food appears to owe its significance largely to the therein accumulated chemical energy which is unleashed by the living cell, thus enabling it to carry out energy-requiring functions; this part is ultimately degraded, generally producing heat. ‘These two processes are differentiated as assimilation and dissimilation.* Doubtless the dissimilatory process is the more essential charact- eristic of life. Whereas other typical vital phenomena, such as growth, reproduction, internal or external motion, may frequently be lacking, dissimilation is absent only in some stages of latent life, resulting, for example, from desiccation. Consequently it had been established that in the case of higher or- ganisms the perpetuation of life is tied to a continuous conversion of chemical into other forms of energy, and Rubner demonstrated that the first law of thermodynamics applies to the energy transformations in the animal body, while Atwater showed this also for man. As far as the nature of the energy-providing reactions is concerned, Lavoisier had already pointed to the significance of the respiratory process, 2.e., the slow combustion taking place in living organisms, * Perhaps the terms ‘Bau-’ and ‘Betriebsstoffwechsel’, customarily used in the German literature, make for a clearer distinction. For a further consideration of these problems see, e.g., M. Rubner, ‘Kraft und Stoff im Haushalte der Natur’ (1909), and the chapter by C. Oppenheimer, ‘Energetik der lebenden Substanz’, in his ‘Handb. d. Biochemie d. Menschen u.d. Tiere’, 2nd ed., Vol. II, p. 223, 1923. pols: UNITY AND DIVERSITY IN THE METABOLISM OF MICRO-ORGANISMS which reveals itself through their requirement for oxygen. The valid- ity of this concept was emphasized when Rubner experimentally es- tablished his principle of ‘isodynamic substitution’. He showed that, up to a point, proteins, fats, and carbohydrates can replace one another, in a weight ratio that is inversely proportional to the heat of combustion of these substances, in the adult animal for the mainte- nance of the same vital functions; in other words that, within certain limits, equality in chemical energy corresponds to equality in food value. As early as 1885 Rubner pointed out that in all probability it would be possible that also in the metabolism of micro-organisms a conver- sion could be designated which derives iis significance for the organism entirely from the resultant energy liberation. Now Pasteur, who, in 1860, had discovered the first instance of organisms that can multiply in the complete absence of oxygen, had intuitively recognized the con- nexion between the absence of respiration in these organisms and the fermentation process that characterizes their mode of lie. Meanwhile the mutual substitution of respiration and fermentation was initially considered largely from a material angle, so that the term fermentation was used to indicate a respiration with bound oxy- gen. The need to consider this substitution primarily on the basis of energetics was first formulated with sufficient clarity by Rubner as a logical consequence of his attempt to interpret the metabolism of any and all living creatures from a single point of view. But not until 1902 did he begin his series of micro-calorimetric measurements of the metabolism of various microbes which corroborated the validity of these hypotheses. Since that time it has been satisfactorily established that the metabolic activities of every microbe comprise the processes whereby new cell material is synthesized, as well as dissimilatory pro- cesses characterized by the fact that chemical energy is utilized by the living cell for its energy-requiring functions, and finally appears as heat. A number of the most important dissimilatory processes encountered among various groups of micro-organisms is summarized in ‘Table I.* * In computing the majority of the caloric effects listed in this Table, I have been privileged to profit by the authoritative advice of my colleague, P. E. Verkade, to whom, here too, I want to express my sincere gratitude. It may be mentioned that an attempt has been made to calculate, as accurately as possible, the differences between the heats of combustion of the aqueous solutions of the substrates and of the metabolic products. 195 SELECTED PAPERS TABLE I. MICROBIAL DISSIMILATORY PROCESSES * A. Oxydative processes Examples of organisms: 1. C,H,.O,+60, = 6CO,+6H,0O+676 cal. various fungi 2. C,H;OH+ 30, = 2CO,+3H,0+325'/,cal. mycodermic yeasts (‘Kahmhefen’) 3. CH,NH,COOH- 11/,0, = 2CO,+H,O+ +NH,+ 142 cal. many aerobic bacteria 4. C,H,0H+O, = CH;COOH+H,0O+ 1161/, cal. acetic acid bacteria 5. CH,+20, = CO,+2H,0-+210 cal. methane oxidizing bacteria 6. H,+1/,.0..= H,O+68?/, cal. hydrogen oxidizing bacteria 4, NH,+11/,0, = HNO,+-H,O+79 cal. nitrite forming bacteria 8. KNO,+1/,0, = KNO,+ 22 cal. nitrate forming bacteria g. H,S+14/,0, = H,O+S-+67 cal. sulphur bacteria 10. S+11/,0,+H,O = H,SO,+ 141 cal. sulphur bacteria B. Fermentative processes 11. C,H,,O, = 2C,H,0H-+2C0,+ 25 cal. yeasts 12. C,H,,O, => 2C;H,0,+-28 cal. lactic acid bacteria 13. C,.H,.O, = C,H,0,+2C0,+2H,+15 cal. butyric acid bacteria 14. C,H,.O,+1/.H,O = CH,CGHOHCOOH+ +CO,+H,+1/,GH,COOH+ +1/,C,H;0H-+8 cal. coliform bacteria 15. (CH,COO),Ca+H,O = CaCO,+CO,+ +2CH,-+ 19 cal. methane bacteria 16. CO(NH,),+2H,O = (NH,),CO;+6 cal. urea splitting bacteria 17. C,H,OH-+2.4KNO, = 1.2N,+ +1.2K,CO,+0.8C0,+ 3H,O-+ 293 cal. denitrifying bacteria 18. C,H,OH-+11/,CaSO, = 11/,H,S+ + 11/,CaCO,+1/,CGO,.+ 11/,H,O+14 cal. sulphate reducing bacteria * This Table makes no attempt whatever at completeness. In particular, thelist of oxidative dissimilation processes with organic substrates can be expanded indef- initely. As for the fermentative dissimilation processes, the most important types known today are represented, with the exception of the anaerobic decomposition of protein breakdown products. These have been omitted because it is very difficult to denote them by means of simple equations. See, however, Arch. f. Hyg., 66, 209, 1908. 196 UNITY AND DIVERSITY IN THE METABOLISM OF MICRO-ORGANISMS The first ten represent oxidative dissimilation reactions of which the upper three occur also in higher animals, the others being typical dissimilatory processes of the earlier mentioned microbes. ‘The dissim- ilations listed under the heading ‘fermentative’ are examples of trans- formations that satisfy the energetic requirements of organisms that live temporarily or permanently in the absence of oxygen. These fermentative processes, understandably, are characterized by a considerably smaller caloric effect, and the correctness of the ener- getic approach to these transformations is reflected in the fact that per unit weight of the causative organism comparatively much more food is used, and much greater amounts of metabolic products are formed during fermentative existence. Particularly clearly is this phenomenon revealed by organisms which, depending on circumstances, derive their energy either from an oxidative or from a fermentative dissim- ilation. This is true, for example, for yeast; and Pasteur had already established that the sugar consumption per unit weight is considerably greater during anaerobic than during aerobic cultivation; in the latter case part of the sugar is also oxidized. Now it cannot be doubted that the energetic interpretation of metab- olism can also be used for the clarification and systematization in other directions. In the first place it forces the investigator to develop a clearer picture than is customary of the metabolism of the organism studied. To mention an example: it is usually stated without further specification that the extensively investigated B. coli can grow in meat extract broth both with and without sugar, and that it is a facultative anaerobe, implying that it can develop both in the presence and in the absence of oxygen. Many a microbiologist does not realize, however, that the dissimilation of this bacterium consists in an oxidative degra- dation of proteinaceous split products on the one hand, but in a fer- mentative decomposition of carbohydrates or sugar alcohols on the other, so that the presence of the last-mentioned substances is a pre- requisite for anaerobic growth. This is in contrast to bacteria of the Proteus-group which are usually similarly characterized in the litera- ture, although they appear to possess the property of obtaining energy from a fermentation of protein degradation products so that they can lead an anaerobic existence even in the absence of carbohydrates. While this example indicates that the vague designation of an organ- ism as a facultative anaerobe should be replaced by a rational con- 197 SELECTED PAPERS sideration of potential dissimilation processes in order to permit a proper insight into the conditions needed for development, the follow- ing example may further illustrate this. The organisms that can be assembled in the group of genuine lactic acid bacteria are often designated as facultatively anaerobic. In a sense this is permissible because the majority can indeed develop in the presence as well as in the absence of air. It would, however, be a glaring error if one were to infer from this fact that these organisms, like the above-mentioned facultative anaerobes, can display both an oxidative and a fermentative metabolism. On the contrary, even in the presence of air the lactic acid bacteria do not utilize oxygen in their metabolism. They are therefore purely fermentative organisms; carbohydrates or sugar alcohols are essential for their growth; and they differ from other strictly fermentative microbes only in the fact that free oxygen does not completely inhibit their development. In this connexion it is not without significance to point out that the absence of an oxidative metabolism among the lactic acid bacte- ria is undoubtedly related to a property that Beierinck in 1893 estab- lished for all lactic acid bacteria, viz., the absence of the enzyme cat- alase which decomposes hydrogen peroxide into water and oxygen, and which had been found in all cells of higher plants and animals, and until then also in all microbes that had been tested. Later Orla- Jensen pointed out that the exclusively anaerobic butyric acid bacte- ria also lack catalase, and we have established the same situation in the case of anaerobic protein-decomposing bacteria. The widely ac- cepted, though still hotly disputed [Dakin 1922] hypothesis that in all aerobic organisms, the higher as well as the lower, catalase plays a role in the transfer of oxygen to the oxidizable substrate is undoubted- ly supported by these facts. Conversely it seems likely that a study of the occurrence of catalase in a newly isolated microbe can be fruitfully employed to determine the presence of an oxidative metabolism. The great significance of the establishment of the nature of a mi- crobe’s dissimilation processes, these primarily characterizing its’ me- tabolism, also resides in the fact that the natural relationships of micro-organisms are unquestionably expressed in their metabolism.* * But — as Orla-Jensen too points out correctly — it does not follow that identical metabolic processes may not also be encountered among phylogenetically independ- ent evolutionary lines. 198 UNITY AND DIVERSITY IN THE METABOLISM OF MICRO-ORGANISMS This idea has been used in an eminent manner by Orla-Jensen [1909 | in his sketch of a general system of classification of bacteria. By as- signing every microbe to one of the natural groups, whose delineations become progressively clearer, it will frequently be possible to predict its properties on the basis of its relationships, and it will also become feasible to indicate more rational culture conditions for it. For example, one frequently hears complaints concerning the difficulty of cultiva- ting numerous streptococci; this must in the first place be ascribed to the fact that the investigators fail to realize that they are working with organisms belonging to the group of genuine lactic acid bacteria. Table II indicates the distribution of dissimilatory processes among some important groups of microbes. It shows that, as a rule, oxidative processes do not exhibit any great specificity as far as the nature of the oxidizable substrate is concerned. Not infrequently the oxidizing ca- pacity extends to nitrogen-containing compounds, to carbohydrates and sugar alcohols, and to organic acids, although in the series, acetic acid bacteria, moulds, aerobic sporeformers, and the Pseudomonas group,* a decreasing tendency towards carbohydrate oxidation and an increasing one towards the oxidation of nitrogen-containing com- pounds may be detected. In contrast, the fermentative processes are generally more dependent on a specific substrate, even though in some groups several potentialities may co-exist. When further considering the meaning of the energetic aspects of metabolism we are led to still other notions. That man or a higher animal requires energy is a notion that we immediately seem to rec- ognize as befitting. The maintenance of the body temperature, the performance of internal and external work, these are inconceivable without energy supply. But if one raises the question what function is served by the continuous flow of energy which, according to experi- ence, is requisite for the perpetuation of life even of a non-motile mi- crobe, maintained at constant temperature, and not displaying any internal movements, there appear to be only two rational answers. On the one hand we may assume that the energy conversion is simply a necessary condition for the living substance as such; on the other hand it is tempting to postulate that there exists a close connexion between * Although the organisms assembled in this group exhibit physiological similarities, they do not form a ‘natural group’; see the previous footnote. 199 TABLE II. DISSIMILATORY PROCESSES OF SOME IMPORTANT GROUPS OF MICRO-ORGANISMS Oxidative dissimilatory processes Fermentative dissimilatory processes autotrophic bacteria acetic acid bacteria yeasts fungi : : 8 lactic acid bacteria coliform bacteria anaerobic cellulose decomposing aerobic " er bacteria sporeforming eee of the bacteria roteus group butyric acid bacteria urea splitting anaerobin bacteria protein fermenting bacteria Pseudomonas Micrococcus s : ee: methane bacteria Sarcina denitrifying Spirillum bacteria Vibrio Mycobacterium P sulfate reducing bacteria b-——S Inorganic compounds organic substances containing nitrogen salts of organic acid AUAAUTANIT carbohydrates and sugaralcohols UNITY AND DIVERSITY IN THE METABOLISM OF MICRO-ORGANISMS this incessant utilization of chemical energy and the other side of metabolism, viz., the assimilation, or the production of new cell con- stituents. In contemplating this possibility the problem arises whether such a situation should not be apparent if one were to succeed in reducing the extent of assimilation by a microbe to a minimum, so that the visual new-formation of cell material, ¢.e. multiplication, is excluded. We then encounter the problem whether it is possible to maintain microbial cells alive by the supply of a food ration that will just prevent multiplication. Rubner [1913] has made an attempt at experimentally testing this possibility with yeast, and had concluded that it does not exist. One may, however, raise serious objections against the tech- nique he used, so that we can only state that in the realm of microbes this fundamental problem is still unsolved. But even in those cases where visual growth fails to take place we must always count with the occurrence of a process of replacement of worn-out cell materials, so that it remains possible to regard the function of dissimilation as one serving assimilation. On the other hand it is patently true that at least part of the dissimilatory energy is indispensable for assimilation whenever we can ascertain that the latter is accompanied by an obvious increase in energy. A good exam- ple of such a situation is provided by the autotrophic bacteria which produce organic matter, such as bacterial protein, from carbon diox- ide. In the case of such organisms, sometimes improperly designated as chemosynthetic, the microbiologists have consequently been wont to stress the energy-providing reactions. If it is remembered, however, that experience has shown how, for example, the sulphur bacteria must oxidise 32 g of sulphur to sul- phuric acid in order to fix 1 g¢ of carbon in the form of bacterial cells (dissimilation process 10, Table I), it is immediately apparent that the autotrophic bacteria, too, retain only a small fraction of the dissimilatory energy in the form of assimilation products. This point is still more obvious in the case of microbes that depend on organic nutrients. Rubner’s calorimetric measurements have shown convinc- ingly that the autolysis of yeast cells, like most enzymatic hydrolyses, proceeds with a very small heat production. Conversely it must there- fore be true that the formation of yeast cells from the products of auto- lysis implies an equally small energy fixation. Nevertheless, the energy 201 SELECTED PAPERS utilization of the dissimilation that accompanies the reconstitution of yeast in an advanced state of autolysis by the sudden addition of sugar is quite considerable. On this basis one would again be inclined to conclude that the formation of cell constituents represents a process that occurs with a very low energy efficiency, in other words, that the energy derived from some chemical transformation can co-operate in a second reaction only if concomitantly a considerable part of the energy is dissipated as heat. If now we try to formulate this problem more succinctly, it must be stated that the metabolism of a living cell, which presumably pro- ceeds isothermically, shows us a reaction that leads to an increase in free energy, v2z., the assimilation, which obviously is conceivable only if concomitantly a second reaction occurs which proceeds with a larger loss of free energy, so that the free energy of the entire system decreases considerably. Hence I should like to ask the physical chem- ists amongst my audience whether they know of similar cases of cou- pled reactions in inanimate systems, and in how far they would have fundamental objections to accepting the occurrence of such energetic coupling. Euler and Af Ugglas [1911] have posed this question as early as 1911, though perhaps in a less pregnant form. They believe that such energetic interplay between two materially independent reactions 1s conceivable if both reactions are accelerated by one and the same catalyst. They elucidate this by referring to displacements in equilib- ria that must neccessarily happen if the catalyst forms compounds with the two substrates as well as with the reaction products. Without here entering into a detailed evaluation, it follows from the necessity to assume a coupling of dissimilation and assimilation that it is entirely inconceivable that the energy-yielding dissimilatory process would occur under the influence of a catalyst that can be divorced from the remainder of the living cell. Such a concept, advocated rather thought- lessly by chemists after Buchner’s discovery of zymase, can only be defended by those who are blind to the biological significance of fer- mentation as the dissimilatory process of the yeast, and which conse- quently has to be considered in connexion with the assimilation. That the dissimilatory reactions, in contrast to the preparatory conversions of foodstuffs, occupy a special position in the cell is clearly reflected by the fact that the latter occur under the influence of en- 202 UNITY AND DIVERSITY IN THE METABOLISM OF MICRO-ORGANISMS zymes that frequently can be quantitatively separated from the cells, whereas the former have tenaciously resisted the efforts to isolate the causative enzymes. It is true that Buchner has succeeded in extracting a very small fraction of the fermenting capacity of some specific yeasts from the cells, but various arguments can be adduced in favour of the statement that in those instances the prominent dissimilation catalysed by the living protoplasm is accompanied by a weak sugar fermentation proceeding under the influence of a zymase function that can be isolated in small amounts. This concept is strengthened by the rather ridiculously low yield that Buchner and his collabora- tors, Gaunt and Meisenheimer [1906], obtained in their attempts to isolate a lactozymase and an alcoholoxidase from lactic and acetic acid bacteria, respectively. In spite of this, they interpret their results as supporting their enzymatic theory of metabolism. Similarly, the old controversy as to whether the urea decomposition by urea bacte- ria does or does not proceed under the influence of a soluble enzyme, urease, thus appears in a new light. From the genuine urea bacteria, for which the decomposition of urea indubitably is also a dissimilatory process, a separation of urease is practically impossible, as Beijerinck has already shown. In other organisms the splitting of urea appears virtually to have lost its energetic significance, in view of the occur- rence of other dissimilatory processes. Thus it becomes understandable that Jacoby [1917, 1923] succeeded in isolating a soluble urease from Proteus bacteria; this he would certainly not have accomplished had he used Urobacillus pasteurii.* I do not have the opportunity to dwell much longer on the con- cepts that arise out of the energetic considerations of metabolism. That it would undoubtedly be extremely rewarding to submit the entire problem of microbial metabolism to a renewed study in con- nexion with the second law of thermodynamics may here be hinted at. The great significance of this problem for general physiology has been pointed out by Zwaardemaker [1906]. Application of these principles to the relatively simple situation in microbes has hardly been begun thus far; nevertheless, it should almost inevitably be pro- ductive of significant results. * The ready solubility of soybean urease is undoubtedly closely linked to the fact that the decomposition of urea is energetically insignificant for the cells of higher plants because their mode of life is predominantly oxidative. 203 SELECTED PAPERS It is extremely tempting, for instance, to try to establish a relation between the quantitative values of the dissimilation reactions of the various microbes and their characteristic assimilatory processes. Should it not be possible that in this way one might learn to under- stand why the butyric acid fermentation as dissimilatory process enables the butyric acid bacteria to build up their proteins from am- monia nitrogen, whereas the lactic acid fermentation does not permit the lactic acid bacteria to accomplish this, though they can use pep- tones for this purpose? It is evident that the caloric effects of the dissim- ilation processes do not provide an adequate yardstick in this respect, but that it is necessary to determine the free energy decrease of these processes. It may be pointed out by the way that for this reason one may easily conceive of the existence of organisms whose dissimilatory reactions represent spontaneously occurring endothermic processes, causing them, in contrast to all known organisms, not to produce heat but rather to absorb this from their surroundings. Baron and Polanyi [1913] have pointed out that in the case of re- actions that proceed with a small caloric effect the decrease in free energy by no means agrees with the caloric effect, whereas in oxida- tive processes the discrepancy between the two is not very great. If this be so, and if we must also conclude that the dissimilatory energy conversions are used for assimilation, then we might expect that for one and the same organism, or for closely related types, an identical dissimilatory energy conversion should express itself in identical assim- ilatory effects. The opportunity to check this is offered by the am- monia- and nitrite-oxidizing bacteria, organisms with very similar metabolic properties but which derive their energy from dissimilations with different caloric effects. Now it is no doubt interesting that, while the caloric effects of these reactions are 79 and 22, the ratios of nitrogen oxidized to carbon assimilated by the two organisms are 35/1 and 135/1, respectively. This, therefore, agrees with the expectation that they would be inversely proportional to the caloric effects. I have, however, lingered too long in discussing the energetic unity in metabolism. It would please me if you had gained the im- pression that the energetic approach may already open up some at- tractive vistas, but that a closer investigation has hardly been begun thus far. It is not only in energetic respect that we may discern a unity in 204 UNITY AND DIVERSITY IN THE METABOLISM OF MICRO-ORGANISMS the metabolism of micro-organisms. Also in material respect there exists a much greater unity than was assumed not so long ago. This has been shown by recent studies; again, the circumstances do not permit me to document this statement extensively. But it may be pointed out that the investigations of Neuberg and collaborators have made it very likely that acetaldehyde occurs as an intermediate pro- duct in the fermentations provoked by yeast [cf. Fuchs 1922], coli bacteria [Neuberg and Nord 1g1g], butyric acid bacteria [Neuberg and Arinstein 1921], and cellulose decomposers [Neuberg and Cohn 1923]. Furthermore, this intermediate product arises in butyric acid fermentations both of substrates with 6 and with 3 carbon atoms per molecule, which at least renders the formation of butyric acid with its 4 carbon atoms from compounds of the C; type somewhat more intelligible. The occurrence of pyruvic acid as an intermediate pro- duct in diverse fermentations is equally suitable to demonstrate the unity in material respect of at first sight very different processes. Even stronger does the material unity in metabolic reactions ap- pear if now we consider the microbes that are characterized by a typically oxidative dissimilation. It is evident from Table II that this manner of fulfilling the energetic requirements, characteristic of all higher plants and animals, is also encountered amongst rather diver- gent groups of microbes. In contrast to the relatively extensive chem- ical investigations of fermentative processes, these aerobic decompo- sitions have not yet been much studied, however. The explanation for this situation lies at hand; in general, aerobic organisms utilize the energy of the proffered foodstuffs to the maximum extent, that is to say that the oxidation is carried as far as possible, and the food is oxidized to carbon dioxide and water, accompanied by ammonia in the case of the oxidation of nitrogenous substrates. Nevertheless, exceptions to this rule have long been known. When acetic acid bacteria had been discovered, it was immediately appar- ent that they provided an instance of organisms characterized by an incomplete oxidative metabolism. This manifested itself not merely in the incomplete oxidation of alcohol to acetic acid, but also of glucose to gluconic acid. Among other groups of aerobic organisms, too, some specialists gradually appeared. Thus the mould, Aspergillus niger, ac- quired some reputation as the causative agent of what has unfortun- ately been called an oxalic acid fermentation; it was found that if 205 SELECTED PAPERS the organism is grown on sugar-rich media, considerable quantities of oxalic acid were formed. A number of other moulds, closely related to the well-known Penicillium glaucum, appeared to produce citric acid under similar conditions; this has been considered a sufficient reason for collecting these specialists in a separate genus, Citromyces. Moreover, as early as 1886 the French chemist, Boutroux [1886], had discovered a conversion of glucose, in the presence of calcium carbon- ate, into calcium keto-gluconate under the influence of an aerobic bacterium that was not described. And around 1900 appeared the publications of Bertrand [1904] on the biochemistry of the so-called sorbose bacterium (Acetobacter xylinum) which rightly caused a sensa- tion also amongst organic chemists. Although from the start it was obvious that this bacterium should be classed with the acetic acid bacteria, its metabolism showed nonetheless important deviations from that of the bacteria used in the manufacture of vinegar. This appeared, for instance, from the fact that it produced large amounts of sorbose when cultivated in the presence of sorbitol; mannitol yielded fructose; and glycerol dihydroxyacetone. All of these represent conversions that the ordinary acetic acid bacteria did not effect. Hence a number of specialists had gradually begun to stand out amidst the at first sight metabolically quite uniform group of aerobic microbes. And the discoverers of these organisms did not tire of sing- ing their praise. Now it is certainly most interesting that recent investigations have shown that the performances of all these apparently so diverse special- ists may be correlated. Firstly, the experiments of Currie [1917], and particularly those of Molliard [1920, 1922], showed that by changing the culture conditions it is possible at will to cause A. niger to convert an important fraction of the sugar into gluconic, or citric, or oxalic acid. Consequently one could make this organism, hitherto known only as an oxalic acid specialist, produce substances that until that time had been known only as specific products of acetic acid bacteria, and of Citromyces species, respectively. This very fact made it quite likely that the above-mentioned compounds are none other than inter- mediate products of the oxidative degradation of glucose to carbon dioxide and water. The difference between the specialists would then merely consist in the fact that one organism would stop at an earlier intermediate stage. 206 UNITY AND DIVERSITY IN THE METABOLISM OF MICRO-ORGANISMS Perhaps it may appear to you that the interpretation has always lain at hand that metabolic products such as citric, oxalic, and obviously gluconic acids are normal metabolic products in the oxidation of glucose. This notion had not been generally accepted, however. ‘This appears from the fact that Butkewitsch [1922, 1923] who, during the past several years, has carried out extensive experimental investigations on citric and oxalic acid formation by moulds, has independently reached the same conclusion only quite recently. Experiments carried out in the Delft laboratory during the last year on the metabolism of various acetic acid bacteria have also led to the same concept of a stepwise oxidative degradation. To begin with, De Leeuw probably recovered the bacterium used by Boutroux, and long since lost; it turned out to be an acetic acid bacterium which we have called Acetobacter suboxydans. This organism also shows a close relationship to Bertrand’s sorbose bacterium, although it clearly differs from the latter in some respects. The investigations have shown that A. suboxydans can carry out the same mild oxidations of different sugar alcohols as have al- ready been mentioned in connexion with the sorbose bacterium. But it also appeared that A. suboxydans possesses a still weaker oxidative capacity than A. xylinum, as evidenced, for example, by the fact that the former oxidizes calcium gluconate only to ketogluconate, while the latter can also oxidize the gluconate to carbonate. Besides, A. sub- oxydans, in contrast to A. xylinum, cannot be induced to oxidize sub- stances like dihydroxyacetone and potassium ketogluconate. ‘Thus the latter bacterium appears capable of further oxidizing its characteristic metabolic products under suitable conditions. And this shows up the intermediate nature of these incomplete oxidations. Still more clearly does the correctness of these concepts follow from the fact that we [Kluyver and De Leeuw 1924] have succeeded in inducing acetic acid bacteria that under ordinary culture conditions completely oxidize substrates like glycerol and mannitol to bring about incomplete oxidations by the use of methods similar to those employed by Molliard in his studies on Asp. niger. A few words may here be devoted to a discussion of these methods through which oxidations may be halted at various intermediate stages. Molliard paid attention primarily to the quantitative regulation of the amounts of N, P, and K in the culture medium. In connexion with earlier observations of Beijerinck and Hoyer, we made use of the fact that the 207 SELECTED PAPERS nature of the nitrogenous foodstuffs often determines whether a par- ticular substrate, hence also a particular intermediate product, will or will not be further oxidized. This also reveals a very striking relation between dissimilation and assimilation, and perhaps may open a way for penetrating more deeply into these problems. After all, it is quite remarkable that A. suboxydans can grow perfectly well in a mineral medium containing a few per cent mannitol if nitrogen is supplied in the form of an ammonium salt. It is thus certain that this bacterium can use ammonia nitrogen for the synthesis of its proteins. But it appears also that this organism does not grow in the same medium if glucose is substituted for mannitol, whereas, in the presence of pep- tone, glucose is very rapidly oxidized, and the bacteria then multiply profusely. Glucose is therefore fully adequate as an energy-providing substrate. Thus we see how two substances, each one utilizable in its own function, can furnish an inadequate combination as food. Might it perhaps be possible to ascribe this unexpected phenomenon to a difference in the decrease in free energy of the first stages of the oxida- tion of mannitol and glucose, respectively, a difference that might cause ammonia nitrogen to be used for the synthesis of bacterial protein in the one, but not in the other case? We don ’t know; but we can learn from this example that the occurrence of a biological oxidation does not depend only on the nature and condition of the living cells and the presence of an oxidizable substrate, but that it is conditioned by very subtle modifications in the composition of the medium. A further consideration of these problems inevitably reminds us of those other phenomena of incomplete and insufficient biological oxi- dations that are characteristic of the pathological deviations in the metabolism of man and animals known as diabetes. Is it a tenuous comparison if we say that we cure A. suboxydans of diabetes if, by the addition of a small amount of peptone, we resuscitate the cells of this organism, suspended in a nutrient medium that contains glucose and ammonia nitrogen, and which, in view of the outcome of the experi- ment with mannitol, should constitute a complete medium? And in studying the beneficial but as yet rather mysterious insulin effect, is there not reason to pay attention to the possibility that under the influence of this hormone chemically readily detectable substances may be excreted into the blood stream whose presence suffices to 208 UNITY AND DIVERSITY IN THE METABOLISM OF MICRO-ORGANISMS induce the oxidative degradation of the sugar by the body cells?* I gladly leave the answer to these questions to those more competent; but I believe that it was not unwarranted to pose them here, partic- ularly because there is an ever-increasing amount of evidence in favour of a fundamental unity in the mechanism of biological oxida- tions that extends to everything that lives aerobically; a final example may suffice to illustrate this. It has long been known that rancid coconut oil contains, among other substances, various ketones, such as methyl nonyl ketone, methyl heptyl ketone, etc. Dr. Derx has recently called my attention to in- vestigations by Stokoe [1922] and by himself, demonstrating that these substances originate under the influence of moulds which apparently produce them by an incomplete oxidation of the fatty acids formed from the oil. Now it is certainly most striking that these microbes effect an incomplete oxidation of, ¢.g., lauric acid with the formation of methyl nonyl ketone, in complete agreement with Knoop’s theory of fatty acid oxidation by higher organisms, according to which the biological oxidation of fatty acids is initiated at the 6-carbon atom, thereby producing f-keto acids from which ketones are formed by decarboxylation, a type of conversion that, in the body of the diabet- ic, causes the production of compounds such as acetoacetic acid and acetone. And many other arguments could be advanced in support of the thesis that the mechanism of oxidative dissimilatory processes re- veals a high degree of unity. It seems to be a practicable task to determine the successive inter- mediate oxidation stages of physiologically important compounds such as glucose, glycerol, etc., with numerous micro-organisms that exhibit a low oxidative capacity. At the same time one may attempt to ascertain in a quantitative manner the ease with which the various steps are accomplished. It is hardly doubtful that such investigations will increase our comprehension of the nature of microbial metabolism. But by virtue of the above-mentioned unity it will also be eminently * Even if one had to assume that the oxidation of glucose proceeds by the round- about way of glycogen, lactacidogen, and lactic acid — as has been proved for the transformations during muscle contraction by the investigations of Embden, Meyer- hof, Laquer, and others — similar considerations would nevertheless apply to the later oxidative phases of this process. See the reviews by F. Laquer, ‘Insulin’, Natur- wiss., 12, 89, 1924, and by O. Meyerhof, ‘Die Energieumwandlungen im Muskel’, Naturwiss., 12, 181, 1924. 209 SELECTED PAPERS useful for our understanding of the metabolic activities of higher organisms which are much less amenable to experimentation. How- ever this may be, I hope that my discussion may have convinced you that a study of microbial metabolism offers many an intriguing prob- lem to the chemist, particularly in view of both the existing diversity and the manifest unity. REFERENCES BARON, J. and POLANYI, M. 1913. Biochem. Z. 53, I. BERTRAND, G. 1904. Ann. Chim. Phys. 8e série 3, 18. BOUTROUX, L. 1886. Compt. rend. 102, 924. BUCHNER, E., GAUNT, R. and MEISENHEIMER, J. 1906. Ann. 349, 125, 140. BUTKEWITSCH, WL. 1922. Biochem. Z. 137, 327, 338. BUTKEWITSCH, WL. 1923. Biochem. Z. 142, 195. CURRIE, J. N. 1917. J. Biol. Ghem. 37, 15. DAKIN, H. D. 1922. Oxidations and Reductions in the Animal Body. London p. 19. EULER, H. VON and UGGLAS, B. AF IgII. Z. allgem. Physiol. 12, 374. FUCHS, W. 1922. Der gegenwartige Stand des Garungsproblems. Stuttgart. JACOBSEN, H. GC. 1912. Folia Microbiologica 1, 487. JACOBSEN, H. Cc. 1914. Folia Microbiologica 3, 155. JACOBY, M. 1917. Biochem. Z. 8&4, 355. JACOBY, M. 1923. Biochem. Z. ro, 168. KLUYVER, A. J. and LEEUW, F. J. G. DE 1924. Tijdschr. Vergelijk. Geneesk. ro, 170. LIESKE, R. 1912. Sitzber. Heidelberg. Akad. Wiss. Abt. B, no. 6. MOLLIARD, M. 1920. Compt. rend. soc. biol. 72, 479. MOLLIARD, M. 1922. Compt. rend. soc. biol. 87, 967. MOLLIARD, M. 1922. Compt. rend. 174, 881. NEUBERG, C. and NORD, F. F. 1919. Biochem. Z. 96, 133. NEUBERG, G. and ARINSTEIN, B. 1921. Biochem. Z. 117, 269. NEUBERG, C. and COHN, R. 1923. Biochem. Z. 139, 527. ORLA-JENSEN, S. 1909. Centr. Bakt. Parasitenk. Abt. II, 22, 305. RUBNER, M. 1913. Die Ernahrungsphysiologie der Hefezelle bei alkoholischer Ga- rung. Leipzig. SOHNGEN, N. L. 1913. Centr. Bakt. Parasitenk. Abt. IT, 37, 595. STOKOE, W.N. 1922. J. Soc. Chem. Ind. London 4o, 76. WAKSMAN, S. A. 1922. Soil Sci. 13, 329. WAKSMAN, S. A. and JOFFE, J. 8. 1922. J. Bact. 7, 231, 239, 605, 609; J. Biol. Ghem. 59, 35+ WINOGRADSKY, S. 1887. Botan. Ztg. 45, 489, 513, 529, 545, 569, 585, 606. WINOGRADSKY, S. 1902. Centr. Bakt. Parasitenk. Abt. II, 9, 43. ZWAARDEMAKER, H. 1906. Ergeb. Physiol. 5, II, 108. DIE EINHEIT IN DER BIOCHEMIE (MIT H. J. L. DONKER) I. EINLEITUNG Die Biochemie hatte sich bis vor kurzem in gewissem Sinne der Haupt- sache nach als deskriptive Wissenschaft entwickelt. Die verschieden- sten Organismen wurden auf ihre chemischen Leistungen untersucht, und ein ungeheures experimentelles Material hat sich allma&hlich an- gehauft. Wenn man versucht dieses zu iiberblicken, ist man vor allem erstaunt tiber die ausserordentliche Verschiedenheit der chemischen Umsetzungen, welche von der lebenden Zelle bewirkt werden k6nnen. Allerdings hat man allmahlich gelernt, in den biochemischen Vor- gangen zwei Arten zu unterscheiden. Immer deutlicher hat sich ge- zeigt, dass die grosse Gruppe der hydrolytischen Spaltungen und Ver- esterungen sich scharf von den anderen Stoffwechselvorgangen ab- trennen lasst. In Ubereinstimmung hiermit hat C. Oppenheimer in der neulich erschienenen Auflage seines grossen Werkes ‘Die Fer- mente’!* eine Trennung vorgenommen zwischen den die hydrolyti- schen Vorgange katalysierenden Enzymen: den Hydrolasen und den ibrigen ‘eigentlichen’ Stoffwechselfermenten, wofiir er gemeinsam mit C. Neuberg? den Klassifikationsnamen ‘Desmolasen’ in Vorschlag ge- bracht hat. Oppenheimer bemerkt in diesem Zusammenhange: ‘ wahrend jene (die hydrolytischen Fermente) nur einfache Spaltungs- vorgange an den sekundaren Bindungen des Kohlenstoffes mit O oder N katalysieren, die ohne nennenswerte Abnahme der freien Energie verlaufen, beschleunigen diese Fermente Prozesse, welche die ent- scheidend wichtigen Kohlenstoffverbindungen voneinander trennen, die ‘‘Bindungen lésen’’, die Desmolyse im Gegensatz zur Hydrolyse be- wirken. Wesentlich dabei ist auch der Unterschied, dass diese Vor- gange in ihrer Gesamtheit unter Abgabe von freier Energie verlaufen. Die Desmolasen sind also die eigentlichen Stoffwechselfermente; sie befordern die entscheidenden Vorgiange, bei denen die Zelle sich die * Footnotes and references have been assembled in the original form on pp. 262—267. 211 SELECTED PAPERS chemische Energie der ihr zugefitihrten Nahrstoffe oder auch der eigenen Leibessubstanz nutzbar macht, sie in andere Energieformen, vor allem in Warme und mechanische Leistung umsetzt. Fiir diese Vorgange sind die Wirkungen der Hydrolasen nur die allerdings un- umgdngliche Vorbereitung’.® Wir zitieren hier diese Ausfithrungen Oppenheimers nur, um von vornherein klarzustellen, dass man zweifellos berechtigt ist, die hy- drolytischen Vorgange von den eigentlichen Stoffwechselprozessen ab- zutrennen. In dieser Abhandlung werden namlich die biochemischen Hydrolysen grésstenteils unberiicksichtigt bleiben; die Aufmerksam- keit soll nur auf die ‘eigentlichen’ Stoffwechselprozesse gerichtet werden. Wenn man also die biochemischen Hydrolysen ausser Betracht lasst, wird man jedoch vom physiologischen Standpunkte aus in den restierenden Stoffwechselvorgangen immer noch zwei Arten unter- scheiden kénnen, namlich die sogenannten dissimilatorischen und die assimilatorischen Prozesse.4 Sogar eine oberflachliche Betrachtung des Gesamtstoffwechsels lehrt, dass ein Teil der Nahrung in Produkte tibergefithrt wird, welche von der Zelle entweder ‘sofort oder doch bald ausgeschieden werden (Dissimilation), wahrend ein anderer Teil der Nahrung als mehr oder weniger wichtige Zellsubstanz zuriick- gehalten wird (Assimilation). Beide Prozesse sind jedoch aufs innigste miteinander verkniipft, wie wir im 6. Teil naher auseinandersetzen werden. Es sind die dissimilatorischen Stoffwechselvorgange, die gewohn- lich mit den Begriffen ‘Atmung’ und ‘Garung’ angedeutet werden, welche wir an erster Stelle naher betrachten wollen. Dann werden wir auch den assimilatorischen Vorgangen unsere Aufmerksamkeit widmen. Weil es erwiinscht ist, den gemeinsamen Charakter von Atmung und Garung fortwahrend klar vor Augen zu haben, haben wir es fiir angebracht gehalten, die Begriffe ‘oxydative Dissimilation’ und ‘fer- mentative Dissimilation’ einzufiihren. Wir sind uns bewusst, dass wir uns damit in Gegensatz stellen zu der wblichen Nomenklatur. Be- dauerlicherweise werden auch in neueren Arbeiten typische aerobe Atmungsprozesse, die also nur unter Aufnahme des freien Sauerstoffs vor sich gehen, noch 6fters mit dem Namen Garung (‘Fermentation’ in franzosischer und englischer Sprache) belegt.> Wie unerwiinscht die- 212 DIE EINHEIT IN DER BIOCHEMIE ser Verwirrung stiftende Brauch ist, folgt ohne weiteres aus der Tat- sache, dass die Stoffwechselforschung in neuester Zeit allgemein wie- der die Richtigkeit des Kerns des alten Pasteurschen Satzes ‘La fer- mentation est la vie sans air’ anerkennt.* Man sollte daher die Be- eriffe ‘Garung’, ‘fermentation’, ‘fermentativ’ reservieren fir diejenigen Dissimilationsprozesse, welche im Gegensatz zu den Atmungsprozes- sen ohne Mitwirkung des freien Sauerstoffs vor sich gehen.’ Wahrend nun die Gleichwertigkeit von Atmung und Garung in physiologischer Hinsicht schon zur allgemeinen Anerkennung gelangt ist, hat man in den allerletzten Jahren auch angefangen darauf hinzu- weisen, dass diesen, beim ersten Blick in rein chemischer Hinsicht so verschiedenen Prozessen, doch ebenfalls eine weitgehende chemisch iibereinstimmende Grundlage eigen ist. -Seit langem hat man schon ausser Zweifel gestellt, dass der Sauer- stoff in den weitaus meisten Fallen bei den aeroben Atmungsprozessen erst in das Spiel der Umsetzungen eingreift, nachdem das Atmungs- substrat eine anaerobe Umsetzung durchgemacht hat. Mit dieser Er- kenntnis war der Gegensatz im Chemismus der Atmungs- und Ga- rungsprozesse schon in bedeutender Weise aufgehoben. Immerhin blieb dieser Gegensatz noch in auffallender Weise bestehen, weil die letzte Phase des Atmungsprozesses als Verbrennungsreaktion einen grossen Unterschied im Vergleich mit den Teilprozessen des Garungs- vorganges aufzuweisen schien. In neuester Zeit ist aber auch diese Liicke in der einheitlichen Be- trachtung des Chemismus von Atmung und Gdrung auf glicklichste Weise beseitigt worden. Als wichtigster Beitrag in dieser Hinsicht ist zweifellos die von Hein- rich Wieland entwickelte Theorie des Chemismus der aeroben At- mungsprozesse zu betrachten. Wahrend es sich eriibrigt an dieser Stelle darauf naher einzugehen, mége nur kurz angedeutet werden, dass durch Wieland ein tiberwaltigendes Material zusammengebracht wurde zur Erhartung seiner Auffassung — fiir welche Palladin’, Bredig’® u.a. auch schon friither, jedoch mit wenigerem Nachdruck, eingetreten waren —, dass das Wesen der biochemischen Oxydationskatalyse an erster Stelle auf eine Aktivierung von Wasserstoffatomen im Oxydati- onssubstrat (eventuell nach vorhergehender Hydratierung) zuriick- zufiihren sei, wahrend die Rolle des Sauerstoffs zuriickgedrangt wurde, indem derselbe nur als Akzeptor dieses aktivierten Wasserstoffs Be- 213 SELECTED PAPERS deutung habe. Diese letzte Ansicht wurde dadurch plausibel gemacht, dass auch bei vielen biologischen Oxydationen der Sauerstoff durch andere gut reduzierbare Substanzen, also durch andere ‘Wasserstoff- akzeptoren’, ersetzt werden konnte. Die Wielandsche Theorie des Chemismus der aeroben Atmungs- prozesse hat schwer um Anerkennung ringen miissen. Dies ist dadurch zu erklaren, dass sie in einem Zeitpunkt zur vollen Entwicklung ge- kommen war, als die Ergebnisse der bewundernswerten Untersuchun- gen von O. Warburg iiber die Rolle des Eisens bei der biochemischen Oxydationskatalyse die Aufmerksamkeit der Biochemiker auf sich lenkten. Wenn auch auf die Warburgschen Auffassungen hier nicht naher eingegangen werden wird, wollen wir hier nur feststellen, dass unabhangig von Fleisch, von v. Szent-Gyérgyi und auch von uns darauf hingewiesen wurde, welche grossen Vorteile eine Vereinigung der Wielandschen und der Warburgschen Ansichten tiber den Che- mismus der Atmungsvorgange bietet. Dafiir braucht man nur anzu- nehmen, dass der gasfOrmige Sauerstoff an sich ungeeignet ist, als Akzeptor des nach Wieland aktivierten Wasserstoffes zu dienen, und dass der Sauerstoff — im Gegensatz zu den andern Wasserstoffakzep- toren — in Ubereinstimmung mit den Ansichten Warburgs sich nur mit dem aktivierten Wasserstoff vereinigen kann, wenn er vorher von einer in der Zelle anwesenden Eisenkomponente in eine hoherwertige Eisen-Sauerstoffverbindung tbergeftihrt worden ist. Auch Oppenheimer hat sich neulich in einer mustergiltigen kriti- schen Betrachtung des vorhandenen experimentellen Materials zu- gunsten dieser Auffassung ausgesprochen. Das wichtigste Ergebnis dieser ‘einheitlichen Deutung’ — wie Oppen- heimer sie nennt — des Chemismus der Atmungsprozesse ist nun zweifellos, dass sie die Oxydationsvorgange in engsten Zusammen- hang bringt mit bestimmten Teilprozessen der Gdrungsvorgange. Diese hat man allmahlich immer mehr auf Teilreaktionen zurtickge- fihrt, bei denen bestimmten Zwischenprodukten der Garung Wasser- stoff entzogen wurde, welcher alsdann auf ein anderes Zwischen- produkt iibertragen wurde. Das bekannteste, von Neuberg immer wieder in den Vordergrund geriickte Beispiel, ist die Cannizzaro-Um- lagerung oder ‘Dismutation’ der Aldehyde, bei der die eine Halfte der Aldehydmenge zu Saure dehydriert, die andere Halfte zu Alkohol re- duziert wird. Doch auch auf andere gekuppelte Dehydrierungs- und 214 DIE EINHEIT IN DER BIOCHEMIE Hydrierungsvorgange im anaeroben Abbau der Dissimilationssub- strate ist schon mehrmals die Aufmerksamkeit gelenkt worden. Wir konnen aus den obigen Betrachtungen schliessen, dass die grosse Bedeutung der Oxydoreduktion — wie wir die gekuppelte Dehydrierung und Hydrierung kurz bezeichnen kénnen — fiir die Erklarung des Chemismus der Dissimilationsvorgange immer deutlicher in den Vor- dergrund tritt. Die obenstehenden Ausfithrungen beabsichtigen nur eine kurze Ubersicht von dem heutigen Stande unserer Kenntnisse des Chemis- mus der Dissimilationsvorgange zu geben. Fiir die Dokumentierung dieser Betrachtungsweise kann in erster Linie auf die schon erwahnte rezente Zusammenfassung von Oppenheimer verwiesen werden. Das an jener Stelle zusammengebrachte Material beschrankt sich jedoch der Hauptsache nach auf die aeroben Atmungsprozesse ; von den Garungsprozessen wird fast nur die alkoholische Garung beriicksichtigt. Das der mikrobiologischen Stoffwechselforschung entnommene Mate- rial, das uns in unserer vorlaufigen Mitteilung selbstandig zu ahniichen Schliissen gefiihrt hat, ist ausser Betracht geblieben, obwohl unsere diesbeziigliche Abhandlung beilaufig erwahnt worden ist.'* Wir glauben daher, dass es niitzlich sein wird, unsere Betrachtungen hier kurz wiederzugeben, um so mehr als wir dabei zu Vorstellungen und Schliissen gelangt sind, die nach unserer Meinung von grosserer Tragweite sind. Wahrend namlich Oppenheimer in Ubereinstimmung mit der iiblichen Auffassung zwar den Oxydoreduktionsvorgangen eine her- vorragende Stellung bei den Dissimilationsprozessen zuschreibt, be- merkt er doch ausdriicklich: ‘Andererseits umfasst aber der Begriff Oxydoreduktasen auch wieder nicht das gesammte, chemisch und bio- logisch zusammengehorige Gebiet, da andere wichtige Teilfermente nicht dazugehoren, wie die Karboxylase u.a.’.'* Dagegen haben wir schon vor einem Jahre darauf hingewiesen, dass alle Teilprozesse der Dissimilation — soweit nicht Veresterungen und Hy- drolysen eingreifen' — als Oxydoreduktionsprozesse zu betrachten sind. Wenn auch diese Aussprache an und fiir sich bedeutungslos schei- nen kann, so befahigt sie doch zu Schliissen, die geeignet sind, in hohem Grade Ordnung in das Chaos der biochemischen Erscheinun- gen zu bringen. Dies mége in einigen Ziigen angedeutet werden. Angesichts der Tatsache, dass man in sehr vielen Fallen mit gutem 215 SELECTED PAPERS Erfolge versucht hat, die von der lebenden Zelle hervorgerufenen biochemischen Umsetzungen auch mit den aus der Zelle abgetrenn- ten Enzymen zu bewirken, hat sich die Vorstellung allgemein einge- biirgert, dass eine Zelle ein Magazin ist, das mit den verschiedensten Werkzeugen gefiillt ist. Jedes dieser Werkzeuge befahigt die Zelle zu einer speziellen chemischen Reaktion. In welcher Weise diese Werk- zeuge iiber die verschiedenen Zellen verteilt sind, entzieht sich dabei jeder theoretischen Uberlegung. Es scheint, dass jede artspezifische Zelle durch das Vorkommen einer bestimmten, aber willkiirlichen, Enzymkombination gekennzeichnet ist. Wie weit diese Auffassung, wenn man auch die hydrolytischen En- zyme in Betracht zieht, berechtigt ist, werden wir hier dahingestellt lassen. Beschranken wir uns aber auf die eigentlichen Dissimilations- vorgange, dann folgt aus dem Zuriickfiihren aller Teilprozesse auf Oxydoreduktionen die Moglichkeit, den ganzen Vorgang der Dissi- milation fiir eine Zelle einem und demselben Agens zuzuschreiben. Die Unterschiede, welche die Dissimilationsvorgange der verschieden- artigen Zellen aufweisen, sollten bei dieser Auffassung ihre Ursache finden in einer quantitativen Abstufung einer und derselben Eigen- schaft des oxydoreduzierenden Agens. Indem wir nun diese Eigen- schaft auf Grund der neueren Auffassungen tiber die chemische Kata- lyse naher zu prazisieren versuchten, ergab sich die Moglichkeit, dabei einen Zusammenhang mit ganz andern Eigenschaften der lebenden Zellen durchaus plausibel zu machen. Wir hoffen dies durch die folgenden Ausfiihrungen naher zu er- lautern und den Wert unserer Betrachtungsweise wenigstens als Ar- beitshypothese begriinden zu kénnen. 2. DIE ZURUCKFUHRUNG DER DISSIMILATIONSPROZESSE AUF KATALYTISCHE WASSERSTOFFUBERTRAGUNG, FALLS ZUCKER ALS SUBSTRATE FUNGIEREN Die Veranlassung zu unseren Betrachtungen war der Wunsch, die ausserst verschiedenen Umsetzungen, welchen Zuckerarten bei den fermentativen Dissimilationsvorgangen der unterschiedenen Mikroben- arten unterliegen, von einem gemeinschaftlichen Gesichtspunkte aus zu tberblicken. In unserer ersten, oben zitierten Mitteilung haben wir naher begriindet, wie es méglich ist, die zur fermentativen Zucker- 216 DIE EINHEIT IN DER BIOCHEMIE dissimilation befahigten Mikrobenarten in einer sehr beschrankten Zahl natiirlicher Gruppen unterzubringen. Die Vernachlassigung dieses Gesichtspunktes ist einer der Griinde, weshalb unsere Kenntnis tiber die Art des mikrobiellen Zuckerabbaus bis heute noch so mangelhaft ist. Ein zweiter Grund fiir diese Situation liegt darin, dass man bis vor kurzem wenig Erfolg mit den Versuchen gehabt hat, sich eine nahere Vorstellung von dem Chemismus der fiir die verschiedenen Gruppen charakteristischen Art des Zuckerabbaus zu machen. Anfanglich hat man versucht, die von einem Organismus bewirkte Umsetzung durch eine einzelne chemische Gleichung wiederzugeben. Als Bei- spiel sei nur die vor langer Zeit von Harden’ fir die Glukosegarung durch Bacterium coli vorgeschlagene Gleichung angefihrt: 2C,H,,0,+H,O > 2C,H,O;+C,H,O,+C,H,O+2CO,+2H, Eine derartig einfache zusammenfassende Formel kann jedoch nicht befriedigen, schon deshalb nicht, weil die Quantitaten der entstan- denen Garungsprodukte je nach den dusseren Umstanden in hohem Grade wechseln. Dieser Umstand hat einige Forscher dazu gebracht, eine Erklarungs- weise zu geben, wobei angenommen wurde, dass jedes der gefundenen Garungsprodukte sein Entstehen einer besonderen, unabhangig vor sich gehenden Umbildung des Zuckers verdankt.!® In den Fallen, bei denen wahrend der Garung Kohlensaéure und Wasserstoff gebildet wird, ist es selbstverstandlich leicht, fiir jedes Garungsprodukt eine einfache Gleichung niederzuschreiben, wobei dieses Produkt unter Freiwerden der beiden genannten Gase aus dem Zuckermolekiil her- geleitet wird. Dagegen springt sofort ins Auge, zu welchen unhaltbaren Konse- quenzen eine derartige Auffassung fiir diejenigen Garungsprozesse fiihrt, bei welchen kein freier Wasserstoff entsteht. So nimmi z.B. Baumgartel!?, um auf diese Weise das Entstehen von Produkten wie Glyzerin bezw. Mannit bei der Milchséuregarung zu erklaren, seine Zuflucht zu Gleichungen wie: 7C,H,,0,+6H,O > 12C,;H,O,+6CO, Glukose Glyzerin Fruktose Mannit 217 SELECTED PAPERS Zwischen diesen beiden Extremen steht die Auffassungsweise, nach welcher bei den meisten Garungsprozessen eine Anzahl mehr oder weniger unabhangiger Umsetzungen eintreten. Die gebildeten Ga- rungsprodukte werden unter dieser Voraussetzung teilweise gleich ge- richteten, teilweise verschieden gerichteten Umsetzungen ihr Ent- stehen verdanken. Eines der wenigen ausgearbeiteten Beispiele hierfiir findet man im Studium von Grey!§ iiber die Glukosevergaérung durch Bacterium colt. Hierbei wird die Bildung der Milchséure unabhangig von der Ent- stehung der tibrigen Produkte gedacht, die aus dem Glukosemolekil gemiass folgender Gleichung entstehen: C,H,,0,+H,O > 2CO,+2H,+C,H,OH+CH,COOH Obgleich diese letzte Auffassung zweifellos die richtige ist, hat man sie bis jetzt noch wenig zur Erklarung des Chemismus der verschiedenen Garungsprozesse benitzt. Eine neue Epoche im Studium der Garungsprozesse wurde einge- leitet von den grundlegenden Untersuchungen von Neuberg!® und seinen Mitarbeitern iiber die alkoholische Garung. Bei diesen Unter- suchungen wurde u.m. der Nachweis erbracht, dass der bei genannter Garung auftretende Alkohol in letzter Instanz durch Reduktion von Azetaldehyd gebildet wurde.” Spater folgte eine Reihe von Unter- suchungen tber verschiedene, durch andere Mikroben bewirkte fer- mentative Zuckerdissimilationsprozesse, woraus sich ergab, dass aller Wahrscheinlichkeit nach auch hierbei Azetaldehyd als Zwischen- produkt auftritt. Ausser bei der alkoholischen Garung, wiesen sie dies nach fur die Zuckervergaérung durch B. coli communis, B. lactis aerogenes, B. dysen- teriae, ‘“Gasbrandbazillus’, Bac. butylicus Fitz und Bac. butyricus Fitz, wo- bei sie ebenfalls Azetaldehyd mit Hilfe von Natrium- oder Kalzium- sulfit bezw. Dimedon ‘abfangen’ konnten.”? Auch Peterson und Fred konnten auf dieselbe Weise Azetaldehyd- bildung fiir ein dem B. coli nahestehendes Bakterium, fiir die sporen- bildende Art Bac. acetoethylicum und fiir ein Milchséurebakterium, nam- lich Lactobacillus pentoaceticus nachweisen.?? Durch De Graaff und Le Févre wurde dieser Nachweis auch fiir weitere Vertreter der Koli- Typhusgruppe (B. typhosum, B. paratyphosum A und B) erbracht.”* Auf diese Beobachtungen — welche noch durch viele andere, auf die 218 DIE EINHEIT IN DER BIOCHEMIE hier nicht eingegangen werden kann, gestiitzt werden — griindet Neu- berg die tiberaus wichtige Ansicht, dass bei den verschiedenen Zucker- garungen zunachst tberall die gleichen Spaltungen auftreten, und dass die beobachteten Unterschiede in den Garungsendprodukten nur auf Unterschiede in den sekunddren Umwandlungen der primaren Spaltungsprodukte zuriickzufiihren sind. Obgleich diese Erkenntnis zweifellos einen gewaltigen Fortschritt bedeutet, geniigt sie nicht, um ohne weiteres eine klare Einsicht in das Spiel der Umsetzungen zu geben und die Entstehung der sehr zahl- reichen Garungsprodukte zu erklaren. Wahrend bei der alkoholischen Garung, bei der die Produkte des Zuckerabbaus sich praktisch auf Athylalkohol, Kohlensaure und ein wenig Glyzerin beschranken, ein wichtiger Teil des Vorganges klargelegt worden ist, waren die Umset- zungen des Zuckermolekiils bei den bakteriellen Garungen noch groésstenteils im Dunkeln geblieben. Im folgenden wollen wir auseinandersetzen, wie die Vorgange in allen Fallen vollstdndig auf eine Kette von Oxydoreduktionen, oder wie wir es lieber ausdriicken méchten, auf eine katalytische Uber- tragung von Wasserstoff zuriickzufiihren sind. Wahrend wir uns in diesem Teil bis jetzt auf eine Betrachtung der fermentativen Zuckerdissimilationsprozesse beschrankt haben, wollen wir gemass der Einleitung jetzt auch die oxydativen Zuckerdissimila- tionsprozesse in Betracht ziehen. Denn hierdurch wird deutlich wer- den, wie die Wielandsche Auffassung der aeroben Atmungsprozesse als Dehydrierungsvorgange einen tiberaus gliicklichen Anschluss die- ser Prozesse an die Garungsprozesse erméglicht und eben diese ‘Tat- sache ein wichtiges Argument zugunsten dieser Auffassung liefert. Wie gesagt, werden wir uns an dieser Stelle auf die Betrachtung eines einzelnen Dissimilationssubstrates, namlich der Glukose, be- schranken.24 Dabei wird klar ans Licht kommen, dass, wie verschie- denartig die Umsetzungen in den verschiedenen Organismengruppen auch sein mogen, sie alle vollstandig auf Oxydoreduktionswirkungen zurtickzufithren sind. Wir sind uns dabei bewusst, dass eine vollstandige Dokumentierung der von uns in den einzelnen Fallen angenommenen Reaktionen des Zuckerabbaus nicht gegeben wird. Doch sprechen daftir in den meis- ten Fallen wichtige Argumente, auf die wir aber leider mit Hinsicht auf den Rahmen dieser Abhandlung nicht ausfiihrlich eingehen konnen. 219 SELECTED PAPERS Wir werden unsere Ausfiihrungen mit einer Betrachtung der Or- ganismen mit ausgesprochen oxydativem Charakter anfangen und allmahlich immer mehr fermentativ geneigte Organismen folgen lassen, um schliesslich mit den véllig anaeroben Organismen zu enden. I. Organismen mit ausgesprochen oxydativem Charakter Beispiele hierfiir sind die Essigsaurebakterien und zahlreiche Schim- melpilze. Wie langst bekannt, wird Glukose von allen Essigsdurebakterien und zahlreichen Schimmelpilzen an erster Stelle zu Glukonsaure oxy- diert. Nach der Wielandschen Auffassung beruht dieser Vorgang auf einer Dehydrierung des Glukosemolekiils, wobei aber vorher an der Aldehydgruppe eine Hydratierung stattgefunden hat. Diese Dehydrierung findet aber in diesen Fallen nur statt, wenn gleichzeitig ein kraftiger Wasserstoffakzeptor anwesend ist, also: C,H,,.0,- H,O > C,H,,0,+Akzeptor-Wasserstoff Normalerweise ist dieser Akzeptor der — gemass der Warburgschen Ausfiihrungen zunachst an organisch gebundenem Eisen aktivierte — Sauerstoff, welcher dabei schliesslich in Wasser tibergeftihrt wird. Doch konnte Wieland zeigen, dass fiir die Oxydation von Athylalko- hol der Sauerstoff auch von andern kraftigen Akzeptoren wie Methy- lenblau ersetzt werden kann. Wir haben diesen Befund auch fur die Glukoseoxydation feststellen konnen. Die gebildete Glukonsaure unterliegt dann unter dazu giinstigen Be- dingungen einer ganzen Reihe weiterer Dehydrierungen, ebenfalls mit Sauerstoff als Akzeptor, wobei in vielen Fallen wahrscheinlich Zitro- nensdiure, Fumarsaure und Oxalsaure als Zwischenprodukte auf- treten, welche schliesslich zu den Endprodukten Kohlensaure und Wasser fihren.?° Bei allen weiter unten zu besprechenden Umsetzungen findet eine direkte Dehydrierung des Glukosemolekiils nicht statt. Zahlreiche Belege sind dafiir vorhanden, dass der Zucker in den weitaus meisten Fallen zuerst in Verbindungen vom C,-Typus tibergefiihrt wird. Uber die Art der dabei auftretenden Reaktionen sind wir noch ungeniigend 220 DIE EINHEIT IN DER BIOCHEMIE unterrichtet. Doch ist die Hypothese, dass diese Umsetzung in allen Fallen tiber den Glyzerinaldehyd zu dem Methylglyoxal(hydrat) fihrt, wie Neuberg dies fiir die alkoholische Garung der Zucker angenommen hat, durchaus wahrscheinlich. Dabei ist aber zu be- riicksichtigen, dass bei dieser Spaltung des Glukosemolekiils die von Harden und Young?® bei der alkoholischen Garung entdeckte biochemische Phosphorylierung eine ausschlaggebende Rolle spielt. Angesichts der Tatsache, dass eine ahnliche Phosphorylierung des Zuckers von Embden und Laquer?? auch bei dem Kohlenhydratstoff- wechsel im Muskelgewebe festgestellt worden ist und Virtanen sie auch fiir die Zuckerdissimilation durch wahre Milchsaéurebakterien?® und durch Propionsaurebakterien®® ausser Zweifel gestellt hat, ist es sehr wahrscheinlich, dass die Zuckerspaltung immer unter Beteili- gung der anorganischen Phosphate vor sich gehen wird. In einer neulich erschienenen Abhandlung hat einer von uns (K1.) gemeinsam mit A. P. Struyk*! dargetan, dass viele wichtige Argu- mente dafiir sprechen, dass der Verlauf der primaren Zuckerspaltung wie folgt vor sich geht: C,H,,0,+ PO,R,H — C,H,,0;(PO,R,) +H,O C,Hy,O;(PO,R,) mag C3;H,O3+ C3H;O,(PO Rg») C,H,;O,(PO,R,)-+H,O — C,H,O;+PO,R,H In diesen Gleichungen steht fiir den Fall, dass Glukose gespalten wird, C3;H,O, fiir Glyzerinaldehyd. Wie eine nahere Betrachtung des Chemismus des eigentlichen Spaltungsvorganges des Glukose-mono- phosphorsaureesters uns gelehrt hat, ist dieser Vorgang zurtickzu- fiihren auf eine intramolekulare Dehydrierung und Hydrierung, wo- bei das an das vierte C-Atom gebundene Wasserstoffatom auf das dritte C-Atom ubertragen wird, unter gleichzeitiger Sprengung der Bindung zwischen diesen beiden C-Atomen. Der gebildete Glyzerinaldehyd unterliegt nun weiteren Umlage- rungen, welche ebenfalls auf eine intramolekulare Dehydrierung und Hydrierung zuriickzufiithren sind, und wobei schliesslich Methyl- glyoxalhydrat resultiert. Die wahrscheinlichste Vorstellung dieser Umlagerung ist wohl die hierunter folgende: 221 SELECTED PAPERS CH,OH-CHOH-CHO -> CH,OH-CHOH.- oon —> CH,-CHOH- cn —> CH,-CHOH.: ei —> CH,-COH:- of" — ped ere ib een OH Lo) ee OH ae CH, CC Pan CH,-CO- Oa OH OH San In diesem Schema ist das vom wirksamen Agens aktivierte Wasser- stoffatom als H angegeben, wie dies auch in den weiteren Ausfth- rungen immer geschehen wird. Fiir die weitere Erklarung wird nur Gebrauch gemacht von der durchaus berechtigten Annahme, dass die genannten Verbindungen alle Neigung haben, in wasseriger L6- sung Gleichgewichte zu bilden mit isomeren Verbindungen, welche durch Hydratierung, Ringschluss (Wasserabspaltung) und Ringoff- nung (ebenfalls Hydratierung) aus ihnen entstehen. Das entscheidende Moment dabei ist nur die vom wirksamen Agens katalysierte Uber- tragung des Wasserstoffatoms vom zweiten auf das dritte C-Atom. Wir sind nun der Ansicht, dass alle bei den weiter zu besprechenden Arten des Zuckerabbaus angetroffenen Verbindungen aus dem Me- thylglyoxalhydrat durch eine Reihe von Oxydoreduktionen gebildet werden. Um bei der Behandlung der einzelnen Organismengruppen Wiederholungen zu vermeiden, haben wir alle in Betracht kommenden Reaktionen in das folgende Schema untergebracht. Dabei sind die vom wirksamen Agens im Sinne Wielands aktivierten Wasserstoff- atome durch Fettdruck hervorgehoben worden. Die betreffenden Re- aktionen sind je nach ihrem Charakter in Gruppen vereinigt, welche wohl keiner Erlaéuterung bediirfen. I. Umlagerungen des Methylglyoxalhydrats ZOH LOH a. CH,-CO- GOH —+ CH,:-C= O- oe eee CH,-CHOH-COOH NG Milchsaure b. CH,-CO- COH —> CH,:-C = O-C—O—H — CH;:CHO + HCOOH Na Agemideted Ameisensaure Nu POH 2 DIE EINHEIT IN DER BIOCHEMIE /OH yee CH,:CO-COH — CH,-C = Oe —H — CH,-CO-COOH -> H Hens ehe tenure >: H H Akzeptor J — CH;:CO-C*-O—H — CH,-CHO + CO, Apecidaid II. Kondensationsreaktionen DE. CH,-COH + >C-CH; _> CH,-COH + Sar © Ny OO _> CH,-CO-CHOH.CH, Azetylmethylkarbinol Sac DC: CHs > yo 4H cH,-c¢ + HSc-CHO > CH,-C Ca O KH 0” 1 WSc.CHO > Nu | HZ was -> CH,-CHOH-CH,-CHO > CH,-CHOH-CH,-CAOH —> NOH —> CH,-CHOH-CH, ces H — CH,-CH,-CH,-COOH \OH Buttersaure OH O Hv CH,C’ +HSC-COOH>CH,-c@ +H C-COOH> \oH’ H” \OH H/ /OH +CH,-Cé -CH,-COOH -> CH,-CO-CH,:COOH —> OH ZO ++ CH,;-CO-CH,-C + GHCO7CH.-— CO, \O—H Azeton Dehydrierungsreaktionen HCOOH — HCOOH — CO, + 2H wil Sa CH,;-CHO — CH,-C—OH + CH,C--O—H — CH,;COOH + 2H NOH SOV Essigsaure CH,-CO.- cZOu > CH,:-CO.- oan CH,-CO-COOH+ 2H \OH NOH (= Reaktion Ic) Brenztraubensaure usw. 223 SELECTED PAPERS IV. Hydrierungsreaktionen a. 2H — H, gasformiger Wasserstoff b. O+2H— H,O aktivierter Sauerstoff G CH,OH-CHOH-CHO-+2H — CH,OH-CHOH-CH,OH Glyzerin d. CH,- CHOH-COOH-+2H -> CH,-CH,- COOH Propionsaure e. CH,-CHO+2H > CH,-CH,OH Athylalkohol ee CH,-CO-CHOH- CH,-++2H -> CH,- CHOH-CHOH-CH, 2,3-Butylenglykol g. CH,-CH,-CH,-COOH+4H — CH,-CH,-CH,-CH,OH+H,O n-Butylalkohol h. C,H,,.0O,+2H — C,H,,0, Fruktose Mannit de uSW. Hierzu ist nur noch zu bemerken, dass die Dehydrierungsreaktionen immer gekoppelt mit der einen oder anderen Hydrierungsreaktion verlaufen, wenn man jedenfalls — wie wir es getan haben — die even- tuell stattfindende Entwickelung gasformigen Wasserstoffs auch als eine Hydrierung (des atomaren Wasserstoffs) betrachten will. Wir wollen nun kurz darauf hinweisen, dass man die in den jetzt folgenden Gruppen auftretenden Formen des Zuckerabbaues nahezu vollstandig auf bestimmte Kombinationen der im Schema aufgenom- menen Reaktionen zuriickfiihren kann. IT. Organismen mit obligat aerobem Charakter, aber mit etwas geringerem Oxy- dationsvermégen als die Gruppe I Hierzu geh6ren die Zellen der héheren Pflanzen und Tiere (wenigstens die des Muskelgewebes), wie auch viele aeroben Bakterien. Beim Muskelgewebe der héheren Tiere wird das gebildete Methyl- glyoxalhydrat unter anaeroben Bedingungen in Milchsdure umge- lagert (Reaktion Ia). Diese Saure wird dann unter aeroben Bedingun- gen dehydriert, wobei der Sauerstoff als Akzeptor auftritt (Reaktion IVb). Normalerweise findet aller Wahrscheinlichkeit nach bei der Ruheatmung schon vor dem Auftreten der Milchsaéure eine Dehydrie- rung von Methylglyoxalhydrat mit Sauerstoff als Akzeptor statt, wo- bei Brenztraubensaure entsteht, welche Saure dann einer intramole- 224, DIE EINHEIT IN DER BIOCHEMIE kularen Dehydrierung und Hydrierung zu Azetaldehyd und Kohlen- sdure unterliegt (Reaktion Ic). Der Azetaldehyd wird dann weiter dehydriert bis zu Kohlensaure und Wasser, wobei ebenfalls Sauerstoff als Akzeptor auftritt (Reaktion IVA). Bei den hodheren Pflanzen findet unter anaeroben Bedingungen eine Umlagerung statt, wie sie bei den Alkoholhefen angetroffen wird (vergleiche die nachstfolgende Gruppe). Unter normalen Bedingun- gen findet man dieselben Verhaltnisse wie fiir die Ruheatmung des Muskelgewebes angegeben. Bemerkt sei nur noch, dass in dieser Gruppe die unter anaeroben Bedingungen verlaufenden Umsetzungen nicht geniigen, um das Energiebediirfnis der Zelle zu befriedigen. Insofern die Organismen der nachfolgenden Gruppen III, IV und V in der Lage sind, den Zucker unter Sauerstoffzutritt zu dissimilieren, wird der (aktivierte) Sauerstoff in die Garungsvorgange eingreifen, in- dem er an die Stelle der Garungsakzeptoren tritt. Gerade wie bei der Gruppe II wird dieser Eingriff in letzter Instanz zu der Bildung von Kohlensaure und Wasser fithren. In den folgenden Abschnitten wird jedoch nur die fermentative Zuckerdissimilation betrachtet werden. III, Organismen, welche zur alkoholischen Vergarung des Kuckers befahigt sind Hierzu gehéren einzelne Schimmelpilze wie Mucor racemosus und vor allem die Alkoholhefen. Der Zuckerabbau verlauft hier gemass der bekannten Anschauung Neubergs mittels der Reaktionen Ic und IVe. Da aber der fir die erste Phase der Reaktion Ic benGtigte Akzeptor erst bei der zweiten Phase dieser Reaktion gebildet wird, ist es klar, dass die alkoholische Garung einen einleitenden Akzeptor benotigt. Unter Umstanden wird vielleicht der Glyzerinaldehyd diese Rolle spielen und dabei nach Reaktion [Vc in Glyzerin iibergefiihrt werden. Es ist aber nicht un- wahrscheinlich, dass in vielen Fallen der freie Sauerstoff der Luft seine Akzeptorwirkung entfalten wird (Reaktion IV)). IV. Die fakultativ anaeroben zur fermentativen Kuckerdissimilation befahigten sporenbildenden Bakterien Man kann in dieser Gruppe zwei Untergruppen unterscheiden: a. Die Untergruppe von Bac. acetoethylicum Northrop 225, SELECTED PAPERS Hierzu gehort auch die bekannte Art Bac. macerans Schardinger. Der fer- mentative Zuckerabbau ist hier eingehend von verschiedenen ameri- kanischen Forschern studiert. Die Garungsprodukte waren: Athyl- alkohol, Azeton, Wasserstoff, Kohlensaure, Essigsdure, Ameisensdure und Milchsaure.*? Die sich hier abspielenden Reaktionen sind vor allem,die Reakti- onen Id und in geringem Masse auch Ia. Die Garungsprodukte Athyl- alkohol, Essigsdure und Azeton entstehen zweifellos durch die Reak- tionen [Ve, IIId und IIId+- IIc aus dem Azetaldehyd. b. Die Untergruppe von Bac. polymyxa Beijerinck Hierzu gehort auch Bac. asterosporus Arthur Meyer. Diese Untergruppe ist eingehend von dem einen von uns (D.) untersucht.*% Hier treten 2,3-Butylenglykol, Athylalkohol, Kohlensaure und Wasserstoff als Hauptprodukte auf. Auch hier entstehen der Azet- aldehyd und die Kohlensaure hauptsachlich nach Reaktion Id und IIIa. Das 2,3-Butylenglykol entsteht zweifellos nach den Reaktionen Ila und IVf. V. Die Bakterien der Kolt- Typhus-Gruppe Die Vorgange bei der fermentativen Zuckerdissimilation der Bakte- rien dieser Gruppe sind vor allem von Harden* und von Grey*® auf- geklart worden. Weitere Angaben sind bei Neuberg und Nord und bei De Graaff und Le Feévre anzutreffen. Es empfiehlt sich, hier drei Untergruppen zu unterscheiden: a. Untergruppe von B. typhosum Die ersten Reaktionen sind zweifellos Ia und Id. Der bei der letzten Reaktion gebildete Azetaldehyd wird gemass den Reaktionen IIId und IVe in Essigsaure und Athylalkohol iibergefithrt. b. Untergruppe von B. coli Angesichts der Tatsache, dass Greys ‘selected strains’ of B. colt, bei denen das Vermoégen zur Gasbildung verloren gegangen war, einen Stoffwechsel hatten, der grosse Ahnlichkeit mit dem von B. typhosum zeigte und somit einen merkbaren Ertrag an Ameisensaure aufwies, ist wohl anzunehmen, dass hier neben Reaktion Ia auch Reaktion Id auftritt. Die Kohlenséure und der Wasserstoff werden dabei gemass Reaktion IIIa gebildet. Der Azetaldehyd wird wiederum gemass den Reaktionen IIIb und IVe in Essigsaéure und Athylalkohol tbergefiihrt. 226 DIE EINHEIT IN DER BIOCHEMIE Teilweise wird auch Glyzerinaldehyd als Akzeptor auftreten und in Glyzerin tibergefiithrt (Reaktion [Vc). Wahrend wir friiher ange- nommen haben, dass die Bernsteinsdure einer Dehydrierung von Essigsdure entstammte, ist es angesichts der rezenten Untersuchung Virtanens iiber die Entstehung von Bernsteinsaéure bei der Propion- sduregdrung (s.d.) und im Zusammenhang mit den quantitativen Er- gebnissen Greys wahrscheinlicher, dass die Bernsteinsaure auch hier einer nebenbei vor sich gehenden Spaltung des nicht-phosphorylierten Zuckers in eine C,- und eine C,-Verbindung seinen Ursprung ver- dankt. c. Untergruppe von B. aerogenes Die Situation ist hier im grossen und ganzen dieselbe wie in Unter- gruppe b. Nur wird der Azetaldehyd hier nicht ausschliesslich nach den Reaktionen IIIb und [Ve umgesetzt, doch gemass der Reaktion IIa in Azetylmethylkarbinol itbergefiihrt, das dann unter Mitwirkung des aus Reaktion IIIa stammenden Wasserstoffs nach Reaktion IVfin 2,3-Butylenglykol umgewandelt wird. VI. Die Gruppe der wahren Milchsdurebakterien Hierbei lassen sich wieder zwei Untergruppen unterscheiden. a. Die Untergruppe der heterofermentativen Milchsaurebakterien Der erste, der tiber die Zuckerdissimilation in dieser Untergruppe ein- gehende Versuche anstellte, war Jan Smit**, der mit Lactobacillus fer- mentum arbeitete. In neuester Zeit sind ausfiihrliche Untersuchungen mit einer sehr verwandten Bakterienart Lactobacillus pentoaceticus ge- macht von Fred, Peterson und Mitarbeitern.*? Die Ergebnisse stimmen bei beiden Bakterienarten in der Auf- fassung iiberein, dass neben Reaktion Ia auch Reaktion Id verlauft. Der nach der Reaktion IIIa gebildete Wasserstoff wird dabei quanti- tativ fiir die Hydrierungsreaktionen [Vc und IVe benutzt. Wird Fruktose an Stelle von Glukose dissimiliert, dann tritt die Hydrierungsreaktion IV’ in den Vordergrund. b. Die Untergruppe der homofermentativen Milchsaurebakterien Zu dieser Untergruppe gehéren Bakterien wie Lactobacillus delbriickt, Lactobacillus bulgaricus u.a., welche bekanntlich aus Zucker nur Milch- sdure (Reaktion Ia) bilden. 227 SELECTED PAPERS VII. Die Gruppe der Propionsdurebakterten Uber die Zuckerdissimilation in dieser Gruppe sind in den letzten Jahren eingehende Untersuchungen von Virtanen**® ver6ffentlicht worden. Dieser Forscher fand als Produkte der Zuckervergaérung Propionsaure, Essigsaure, Bernsteinsdure und Kohlensdure. Es unter- liegt wohl keinem Zweifel, dass das Methylglyoxalhydrat in bedeu- tender Menge in Milchsaure umgelagert wird gemiass Reaktion Ia. Virtanen halt es nun fiir wahrscheinlich, dass diese Umlagerung quantitativ verlauft und dass die weiteren Produkte aus Milchsaure entstehen, indem ein Teil dieser Saure unter Kohlensdureabspaltung und gleichzeitiger Oxydation in Essigsaure tibergeht. Der dieser Dehy- drierung entstammende Wasserstoff wiirde auf den andern Teil der Milchsaure tibertragen werden, wobei diese Saure in Propionsaure iibergefiihrt wird. Es ist jedoch nicht ausgeschlossen, dass ein ‘Teil des Methylglyoxalhydrats gar nicht in Milchsaure, sondern nach unserer Reaktion I/in Ameisensaure und Azetaldehyd tibergefiihrt wird, welche beide Korper dann dehydriert werden (Reaktionen [IIa und IIId), wobei ebenfalls Milchsaéure als Akzeptor auftritt und in Propionsaure umgesetzt wird (Reaktion IVd). Die gebildete Bernsteinsdure wird aller Wahrscheinlichkeit nach auf anderem Wege, namlich aus dem nicht phosphorylierten Zucker gebildet.*® VIIT. Die Gruppe der Buttersdure- und Butylalkoholbakterten Uber den in den letzten 15 Jahren fiir die technische Bereitung von Butylalkohol und Azeton herangezogenen Gdarungsprozess, wobei Starke der Hauptsache nach in Butylalkohol, Azeton, Kohlensaure und Wasserstoff umgesetzt wird, sind von englischer, kanadischer und amerikanischer Seite mehrere Publikationen erschienen.*” Einer von uns (D.) hat weiter tiber den Vorgang der Zuckerdissimilation auch fiir andere Vertreter dieser Gruppe ausgebreitete Versuche ange- stellt. Wir kénnen hier wieder zwei Untergruppen unterscheiden: a. Untergruppe der Buttersdurebakterien In dieser Untergruppe findet zuerst wieder eine Unnlasenae des Methylglyoxalhydrats statt in Ameisensaure und Azetaldehyd gemass unserer Reaktion Ib. Die Ameisensaéure wird praktisch vollstandig zu Kohlensaure und gasformigem Wasserstoff dehydriert (Reaktionen IIIa und IVa). Der Azetaldehyd unterliegt dann teilweise einer Um- 228 DIE EINHEIT IN DER BIOCHEMIE lagerung, wobei der Wasserstoff von einem Molekiil auf ein zweites, unter gleichzeitiger Kuppelung dieser Molekiile zu Aldol, iibertragen wird. Das gebildete Aldol unterliegt weiter einer intramolekularen Dehydrierung und Hydrierung, wobei es in Buttersaure tibergeftihrt wird (Reaktion IIb). Ein anderer Teil des Azetaldehyds wird in Essigsdure und gasformigen Wasserstoff umgewandelt (Reaktionen IIIé und IVa). b. Untergruppe der Butylalkoholbakterien Wahrend in der erstgenannten Untergruppe alle Aufnahme von Wasserstoff durch die anderen Garprodukte unterbleibt, findet die- selbe in der jetzt zu besprechenden Untergruppe wohl statt. Dabei wird ein Teil des den Dehydrierungen entstammenden Wasserstoffs auf die Buttersaure ibertragen, wobei diese in n-Butylalkohol tiber- gefiihrt wird (Reaktion IVg). Ausserdem findet aber eine Ubertra- gegung von Wasserstoff von einem Molekiil Essigsaure auf ein zweites Molekiil, unter gleichzeitiger Kuppelung beider Molekiile zu Azet- essigsdure statt, die dann einer intramolekularen Dehydrierung und Hydrierung unterliegt, woraus Azeton und Kohlensdure resultieren (Reaktion IIc). Der Unterschied im Verhalten beider Untergruppen wird sehr wahrscheinlich dadurch bedingt, dass die in der zweiten Untergruppe dazugekommenen Reaktionen nur bei einer bestimmten Saurekon- zentration mit messbarer Geschwindigkeit vor sich gehen. Die Bak- terien der ersten Untergruppe sind nun saureempfindlicher als die Butylalkoholbakterien und sterben schon, bevor der ‘kritische’ Saure- grad erreicht worden ist. 3. DER CHEMISMUS DER KATALYTISCHEN WASSERSTOFFUBERTRAGUNG Wenn wir das in den vorangehenden Teilen Behandelte iiberblicken, so sehen wir, dass es modglich ist, die verschiedenartigsten dissimilato- rischen Umwandlungen des Zuckermolekiils vollstandig auf eine Kette katalytischer Ubertragungen von Wasserstoff zuriickzufiithren. Dabei kann diese Ubertragung unserer Ansicht nach sowohl intermolekularer als auch intramolekularer Natur sein. Obwohl die intermolekulare Uber- tragung als gekoppelter Dehydrierungs- und Hydrierungsvorgang von verschiedenen Forschern mehrfach zur Erklarung bestimmter Stoff- 229 SELECTED PAPERS wechselvorgange herangezogen war, stand eine konsequente An- wendung dieses Erklarungsprinzips in der vergleichenden Biochemie noch aus. Ausserdem weicht unsere Anschauung von der anderer Forscher dadurch in prinzipieller Weise ab, dass wir — wie schon in unserer vorlaufigen Mitteilung bekannt gegeben — auf die Moglich- keit hingewiesen haben, auch die unter Kohlenstoffverkniipfung vor sich gehenden Kondensationsreaktionen und Umlagerungsreaktionen als eine katalytische Ubertragung von Wasserstoff aufzufassen und somit die Moéglichkeit zu schaffen, alle Dissimilationsteilprozesse von einem gemeinschaftlichen Gesichtspunkte aus zu iiberblicken.*? Die Berechtigung dieser Anschauung wird am deutlichsten hervor- treten, wenn wir jetzt den Chemismus der katalytischen Wasserstoff- iibertragung einer naheren Analyse unterwerfen. Dabei diirfte vorausgeschickt werden, dass ein Vorgang, wobei unter dem Einflusse eines anorganischen Katalysators Wasserstoff aus einer organischen Substanz gelést und auf eine zweite organische Sub- stanz tbertragen wird, auch in der reinen Chemie durchaus nicht un- bekannt ist. Wir verweisen in diesem Zusammenhange nur auf die friiher zitierte Abhandlung von Bredig und Sommer und auf eine Arbeit von Armstrong und Hilditch*?, deren Titel “The Transference of Hydrogen from Saturated to Unsaturated Organic Compounds in the Liquid State in the Presence of Metallic Nickel’ schon fiir sich spricht. Das Vermégen der lebenden Substanz zur Bewirkung einer derartigen Katalyse steht also gar nicht ohne Analogon in der leblosen Natur da. Weitere Beispiele dafiir sind iibrigens in den von Wieland studierten Fallen der Oxydationskatalyse zu finden. So konnte dieser Forscher zeigen, dass die z.B. von den Essigbakterien bewirkte Ubertragung von Wasserstoff aus Alkohol und Azetaldehyd (-hydrat) auf Sauer- stoff oder Methylenblau auch mit Hilfe von Palladiumschwarz als Katalysator vor sich geht. Wie kénnen wir uns jetzt den Vorgang dieser Ubertragungskatalyse naher vorstellen ? Hierzu miissen wir zuriickgreifen auf die Anschau- ungen der Chemiker iiber die rein chemische Katalyse. In dieser Hin- sicht sind nun die von Boéeseken** und Prins*® entwickelten Anschau- ungen von der allergréssten Bedeutung.*® In Ubereinstimmung mit den von diesen beiden Forschern ent- wickelten Betrachtungen geht beim ersten Teil der Katalyse, d.h. bei 230 DIE EINHEIT IN DER BIOCHEMIE der Dehydrierung des Substrates durch das Protoplasma der kataly- sierenden Zelle ein Einfluss auf das Substrat aus, wobei diese Substanz eine Anderung erfahrt, welche durch Béeseken als Dislokation be- zeichnet wird. Diese Anderung ist logischerweise eine Funktion der chemischen Affinitaten des Katalysators zu dem Dehydrierungssub- strat oder besser gesagt, zu in diesem Substrate vorhandenen Atomen. Indem ein Teil der Affinitaten dieser Atome von den Affinitaten des Katalysators beschlagnahmt wird, tritt in erster Phase eine Lockerung der Bindungen zwischen einzelnen Atomen im Molekiul auf. Es liegt nun auf der Hand, dass wir in dieser Beziehung an erster Stelle an die Affinitat des Katalysators zu den Wasserstoff- und Sauer- stoffatomen im Substrate denken miissen. Dabei muss von vornherein festgestellt werden, dass die Affinitaten einer Substanz zu Wasserstoff und Sauerstoff keine unabhangigen Eigenschaften sind, sondern derart zusammenhangen, dass zu einer grossen Affinitat zu Sauerstoff immer eine kleine Afhinitat zu Wasserstoff gehort und umgekehrt, gerade wie dies sich auch im Verhalten der Metalle gegeniiber Sauerstoff und Wasserstoff zeigt. a. Uberwiegende Affintat des Katalysators zu Wasserstoff Dass eine Affinitat des die Dehydrierung einleitenden Agens zu Was- serstoff zu einer Aktivierung von einem oder mehreren im Substrate anwesenden Wasserstoffatomen fiihren kann, mége auf folgende Weise erlautert werden. Die Sattigung eines Teiles der Affinitat zu Wasser- stoff von dem an und fiir sich ungesattigten Katalysator wird zur Folge haben, dass der Wasserstoff mit einer geringeren Kraft an den Substratrest gebunden bleibt. Hierdurch wird in diesem Rest ein Zustand von ‘Ungesattigtheit’ eintreten. Dieser Zustand wird in- zwischen bewirken, dass die freigewordene Bindungskraft sich sonst irgendwo im Molekiil zu sattigen versucht. Je nachdem diese Neigung befriedigt wird, oder je nachdem mehr oder weniger ausgesprochene Atomverschiebungen in dem Molekiil stattfinden, wird der Wasser- stoff mit einer geringeren Kraft an den Molekiilrest gebunden bleiben, d.h. der Wasserstoff wird aktiviert. Im extremsten Fall wird dieser Zu- stand zu einem Ubergang des Molekiilrestes in eine in sich selbst ge- sattigte Verbindung fiihren, wobei dann die Bindung zwischen dem Wasserstoffatom und dem Molekiilrest vollstandig gelést wird. In diesem Fall wiirden aber die Wasserstoffatome am Katalysator ge- 231 SELECTED PAPERS bunden bleiben, was jedoch bedeuten wiirde, dass die Reaktion kei- nen Fortgang nimmt. Dabei kann sich dann aber der Fall einstellen, dass die Bindungsneigung zweier Wasserstoffatome die Affinitat des Katalysators tiberwindet, und die Reaktion unter Freiwerden gas- formigen Wasserstoffs fortschreitet.*” Letzter Fall der Dehydrierung wird jedoch nur ausnahmsweise ein- treten. In den meisten Fallen wird der Katalysator die Bindung des Wasserstoffs am Substratrest nur lockern. In diese Situation wird nur dann eine Anderung eintreten, wenn noch eine dritte Verbindung — der sogenannte Wasserstoffakzeptor — hinzukommt, welche ebenfalls freie Affinitat zum Wasserstoff besitzt. In diesem Falle wird der Was- serstoff auf letztgenannte Verbindung iibergehen kénnen, wobei im Substratrest Affinitatsverschiebungen eintreten, welche zu einem neu- en in sich gesattigten Zustande fihren. Der Katalysator steht dann wieder fiir einen neuen Angriff zur Verfiigung, worauf das Spiel der Bindungskrafte sich wiederholt. Ob die geschilderte Wasserstoffiibertragung wirklich stattfindet, wird nun von drei Faktoren bedingt werden: a. der Kraft, womit das zu aktivierende Wasserstoffatom im Substrat gebunden ist, 5. der Bin- dungsneigung des Katalysators zu Wasserstoff, c. der Bindungsneigung des Akzeptors zu Wasserstoff. Wenn wir diesen letzten Faktor einen Augenblick konstant halten, indem wir einen Vergleich machen in Anwesenheit desselben Akzeptors bei der Dehydrierung zweier Sub- strate, worin die zu aktivierenden Wasserstoffatome mit verschiedener Kraft gebunden sind, dann leuchtet es sofort ein, dass das Substrat, worin der Wasserstoff am kraftigsten gebunden ist, auch einen Kata- lysator mit grésserer Affinitat zu Wasserstoff fordert, um eine passende Aktivierung des Wasserstoffs zu bewirken, als das andere Substrat. Dabei ist jedoch zu bemerken, dass eine passende Wasserstoffaktivie- rung ebenso wenig erreicht wird, wenn die betreffende Affinitat des Katalysators zu gross ist, denn dann wird das Wasserstoffatom eben zu kraftig an den Katalysator gebunden bleiben. In diesem Falle schiesst, wie Prins in diesem Zusammenhang bemerkt, der Kata- lysator, vom Standpunkt der Katalyse betrachtet, itiber das Ziel hinaus. Die optimale Wasserstoftaktivierung wird nun in den verschiedenen Substraten, je nachdem das Wasserstoffatom, welches aktiviert wird, fester im Substrat gebunden ist, geringer sein, so dass dann fur das 232 DIE EINHEIT IN DER BIOCHEMIE Gelingen der Katalyse auch ein kraftiger Wasserstoffakzeptor not- wendig ist. Das Prinzip obiger Anschauung, wonach die Aktivierung des Was- serstoffs eine Folge ist von der Bindungsneigung des Katalysators zu Wasserstoff, wird auch von Wieland*® zur Erklarung seiner rein che- mischen mit Hilfe von Platin oder Palladium bewirkten Katalysen herangezogen. Inzwischen werden wir sehen, dass die Wasserstoffaktivierung ebenfalls eine Folge der freien Affinitat des Katalysators zu Sauer- stoff sein kann. b. Uberwiegende Affinitat des Katalysators zu Sauerstoff Dass eine iiberwiegende Affinitat des Katalysators zu Sauerstoff eben- falls zu einer Wasserstoffaktivierung ftihren kann, moége durch Folgen- des erlautert werden. Betrachten wir zuerst die Dehydrierungssub- strate, worin das in einer Hydroxylgruppe vorhandene Wasserstoff- atom aktiviert wird. In diesem Falle wird man auf Grund analoger Erwagungen, wie oben auseinandergesetzt, annehmen diirfen, dass eine Sattigung eines Teiles der Bindungsneigung des Sauerstoffatomes der Hydroxylgruppe von dem an sich ungesattigten Katalysator statt- findet, wodurch das Wasserstoffatom mit geringerer Kraft an das Sauerstoffatom gebunden bleibt, d.h. das Wasserstoffatom wird akti- viert. Die Anwesenheit eines geniigend wasserstoffbegierigen Akzep- tors wird dann in bestimmten Fallen geniigen, um eine Ubertragung des Wasserstoffs auf den Akzeptor stattfinden zu lassen. Das Los- reissen des Wasserstoffs wird aber die Ursache sein, dass im Substrat- rest Affinitatsverschiebungen eintreten, wobei dieser in einen neuen gesattigten Zustand iibergeht. Dabei wird die Bindung des Sauer- stoffatomes mit dem Katalysator wieder gelést, worauf dieser fiir einen neuen katalytischen Eingriff zur Verfiigung steht. In den Fallen, worin in sauerstoffhaltigen Substraten kein Wasser- stoff direkt an den Sauerstoff gebunden ist, wird unter Umstanden ebenfalls eine Wasserstoffaktivierung eintreten k6nnen. Um dies ein- zusehen, braucht man nur zu realisieren, dass die Beschlagnahme eines Teils der Bindungsneigung des Sauerstoffs durch den Katalysa- tor zur Folge haben wird, dass die Bindung des Sauerstoffatomes am Molekiilrest geschwacht wird. Das Atom des Restes, woran das Sauer- stoffatom gebunden ist, wird deshalb ebenfalls ungesattigt werden und 235 SELECTED PAPERS daher versuchen, anderswo im Molekiil Affinitaten zu beschlagnah- men. Die Folge dieses Vorganges wird eine Lockerung der Bindungen von Wasserstoffatomen, d.h. eine Wasserstoffaktivierung sein k6nnen.*® Wir legen grossen Wert darauf, hier festzustellen, dass eine ahn- liche Erklarung der Wasserstoffaktivierung durch eine Affinitat des Katalysators zu Sauerstoff bereits von Prins fiir die von ihm studierten Reduktionsprozesse gegeben wird. Aus obigen Auseinandersetzungen folgt nun, dass wenigstens bei den sauerstoffhaltigen Dehydrierungssubstraten die Wasserstoffaktivierung sowohl bewirkt werden kann durch die Bindungsneigung des Kataly- sators zu Wasserstoff als durch diejenige zu Sauerstoff. Da jedoch eine grosse Affinitat des Katalysators zu Wasserstoff immer eine kleine Affinitat zu Sauerstoff bedeutet und umgekehrt, wird in der Regel nur einer dieser beiden Faktoren praktische Bedeutung haben.*! Zu diesem allen miissen wir nur noch hinzufiigen, dass in bestimm- ten Fallen der Katalysator ebenfalls Affinitaten von Wasserstoff- oder Sauerstoffatomen in dem als Akzeptor auftretenden Molekiil beschlag- nahmen wird, was sich unter Umstanden in einer Verstarkung der akzeptierenden Wirkung dussern wird. Dabei ist zu beachten, dass dadurch bisweilen einander entgegenarbeitende ‘T'endenzen entstehen, indem zwar eine gréssere Wasserstoffafhinitat des Protoplasmas auch bei optimaler Aktivierung des Wasserstoffs einen kraftigeren Akzeptor fordert, aber gleichzeitig diese Affinitat auch eine kraftigere Aktivie- rung eines an sich wenig kraftigen Akzeptors bedingen kann. 4. ANWENDUNG DER GEGEBENEN VORSTELLUNG DES CHEMISMUS DER KATALYTISCHEN WASSERSTOFFUBERTRAGUNG AUF DIE ZUCKERDISSIMILATIONSPROZESSE Wir wollen jetzt naher betrachten, inwieweit die im vorigen Teil ge- gebene Hypothese iiber den Chemismus der katalytischen Wasserstofl- iibertragung sich eignet, um angewendet werden zu konnen bei der Erklarung der im 2. Teil behandelten Dissimilationsprozesse, wobei Zucker als Substrate fungieren. Wir sahen, dass bei unseren Ausfiihrungen die fiir die katalytische Wasserstoffiibertragung unentbehrliche Wasserstoffaktivierung — so- wohl die Folge einer Affinitat des Katalysators zu Wasserstoff als die 234 DIE EINHEIT IN DER BIOCHEMIE einer Affinitat zu Sauerstoff sein kann. Wir wollen uns jetzt die Frage vorlegen, ob es Griinde gibt anzunehmen, dass diese Vor- stellung auch bei den oben genannten biochemischen Katalysen zutrifft. Hierbei ist das die Katalyse bewirkende Agens das Protoplasma*? der betreffenden Zelle. Wenn wir uns jetzt fragen, ob irgendwelche Anzeichen vorhanden sind, dass das Protoplasma der verschiedenen Zellen freie Affinitat zu Sauerstoff oder zu Wasserstoff hat, dann stossen wir sofort auf die ausserordentliche Sensibilitat der obligat anaeroben Organismen dem freien Sauerstoff gegeniiber. Wenn man bedenkt, dass ausserst geringe Quantitaten Sauerstoff den Tod dieser Organismen in sehr kurzer Zeit herbeifthren, dann ist man selbst- verstandlich gezwungen, hierbei an eine chemische Wirkung dieser Substanz zu denken. Es liegt dann auf der Hand, an eine Bindung des Sauerstoffs durch das die Dissimilation bewirkende Agens, d.h. durch das Protoplasma, zu denken, wodurch der fiir das Leben unentbehr- liche Dissimilationsprozess ausgeschaltet wird. Hier ist also die An- nahme einer Affinitat des Protoplasmas zu Sauerstoff durchaus moti- viert, und die Wasserstoffiibertragung bei den obligat anaeroben Organismen lasst sich ungezwungen dieser Affinitat zuschreiben. Unter den von uns besprochenen Gruppen gilt dies fiir die Gruppe der Buttersaéure- und Butylalkoholbakterien, deren Dissimilation also eine Folge der ausgesprochenen Affinitat des Protoplasmas dieser Bakterien zu Sauerstoff ist. Aber diese Sachlage trifft auch fiir die Gruppen der Propionsaurebakterien und der wahren Milchsaure- bakterien zu, deren Vertreter zwar in gewohnlichem Sinne nicht obligat anaerob sind, sich jedoch nicht nur am ippigsten bei Sauer- stoffausschluss vermehren, sondern in vielen Fallen sogar ausge- sprochen luftscheu sind. Dass diese Bakterien anscheinend dem freien Sauerstoff gegeniiber resistent sind, findet seine Erklarung einmal darin, dass die Affinitat des Protoplasmas zu Sauerstoff hier geringer ist als bei den obligat anaeroben Organismen, dann in dem Umstand, dass die Sauerstoffempfindlichkeit durchweg beurteilt wird nach Ver- suchen bei gleichzeitiger Anwesenheit guter Dissimilationssubstrate. In diesem Falle wird nicht das freie Protoplasma dem Angriffe des Sauerstoffs ausgesetzt sein, sondern das Protoplasma wird jetzt ge- schiitzt von den aktivierten Wasserstoffatomen des an ihn locker ge- bundenen Substrates. Diese Atome werden dann vom Sauerstoff ak- 235 SELECTED PAPERS zeptiert, wodurch die Gefahr abgelenkt wird und der Dissimilations- vorgang einfach teilweise in andere Bahnen gefiihrt wird. Diese Vorstellung gibt gleichzeitig eine Erklarung fiir die von Ber- thelot®’ festgestellte, aber nicht aufgeklarte Tatsache, dass eine Zu- gabe von brenztraubensauren Salzen die Kultur von sogar obligat anaeroben Organismen in offenen an der Luft stehenden Gefassen ermoglicht. Eben weil diese Salze oder der daraus allmahlich gebildete Azetaldehyd leicht zu aktivierenden Wasserstoff enthalt, wird der von den Anaerobiern kraftig aktivierte Wasserstoff das Protoplasma vor dem Sauerstoff der Luft schiitzen. Fiir alle weiteren von uns betrachteten Gruppen liegt nun kein Grund vor, an eine direkte Bindung des Sauerstoffs an das Protoplasma zu denken. Im Gegenteil weist der Umstand, dass bei allen diesen Or- ganismen das Protoplasma zu einer katalytischen Zerlegung von Was- serstoffsuperoxyd unter Sauerstoffentwicklung imstande ist, darauf hin, dass das wasserstoffiibertragende Agens keine ausgesprochene freie Affinitat zu Sauerstoff besitzt. Es liegt nun auf der Hand anzunehmen, dass bei diesen Gruppen von aeroben oder fakultativ aeroben Organismen die freie Affinitat des Protoplasmas zu Wasserstoff der Faktor ist, welche hier die kata- lytische Wasserstoffiibertragung bewirkt. Hierfiir spricht an erster Stelle die Tatsache, dass die Essigbakte- rien die katalytische Dehydrierung von Alkohol zu Azetaldehyd und von dieser Substanz zu Essigsdure bewirken, was auch mit Hilfe von Palladiumschwarz vorgenommen werden kann. Und fiir die Erkla- rung dieser letzten Katalyse zieht Wieland, wie schon bemerkt, eben- falls die freie Afinitat des Palladiums zu Wasserstoff heran. Deutlicher noch spricht die Berechtigung der Annahme einer freien Affinitat des Protoplasmas zu Wasserstoff bei den aeroben Organismen aus der Tatsache, dass man in der letzten Zeit festgestellt hat, dass es zahlreiche Bakterien gibt, welche imstande sind, gasf6rmigen Wasser- stoff zu verbrennen.*4 Das wichtigste Ergebnis der neueren Arbeiten ist zweifellos, dass diese biokatalytische Umsetzung des Knallgases nicht, wie man bis vor kurzem annahm, eine merkwiirdige Eigenschaft weniger ganz be- stimmter Bakterienarten ist,°> sondern dass Bakterien aus ganz ver- schiedenen Gruppen und zwar solchen, die auch zu ganz anderen Dis- similationsprozessen imstande sind, unter Umstanden auch den gas- 236 DIE EINHEIT IN DER BIOCHEMIE formigen Wasserstoff verarbeiten. In diesem Zusammenhang muss be- merkt werden, dass Visser ’t Hooft neulich in diesem Laboratorium nachweisen konnte, dass es auch typische Essigbakterien gibt, die zu dieser Katalyse befahigt sind.*® Es leuchtet sofort ein, welche wichtige Stiitze die Erklarung der Dissimilationsprozesse auf Grund des Wasserstoffaktivierungsprinzips hierdurch findet. Denn es geht hieraus hervor, dass die Wasserstofl- oxydation durchaus den andern von diesen Organismen bewirkten Oxydationen gleichzustellen ist. Uberlegt man dazu, dass fiir die Knallgaskatalyse mit Hilfe von Pailadium sehr viel zugunsten der Auffassung spricht, dass dieser Katalysator die Spaltung des moleku- laren Wasserstoffs durch seine freie Affinitat zu elementarem Wasser- stoff bewirkt, dann scheint es durchaus berechtigt, dieses Erklarungs- prinzip auch bei den sonstigen Dissimilationsprozessen heranzuziehen. Dass eben nicht alle mit Wasserstoffaffinitat ausgeriisteten Zellen zu einer Aktivierung und einer darauf anschliessenden Ubertragung der Atome gasformigen Wasserstoffs befahigt sind, ist eine logische Konse- quenz der gegebenen Theorie der Katalyse. Denn auch im Wasser- stoffmolekiil sind die Atome mit einer bestimmten Kraft gebunden und diese Atome werden nur von einem Katalysator mit einer ganz bestimmten Bindungsneigung fiir Wasserstoff geniigend aktiviert. So- wohl ein Uber- wie ein Untermass wird wieder schadlich sein fiir die Katalyse, die unter Umstanden dadurch ganz fortbleiben kann. Die obigen Ausfithrungen werden geniigen, um die Annahme zu berechtigen, dass die Dissimilationsprozesse in den ersten sechs der im 2. Teil behandelten Gruppen eine direkte Folge sind von der Affini- tat des Protoplasmas der Zellen zu Wasserstoff. Und zwar liegt es auf der Hand vorauszusetzen, dass diese Affinitat bei der ersten Gruppe am grossten ist, um von diesen ausgesprochen aeroben Organismen ausgehend immer niedriger zu werden, bis schliesslich bei den Milch- siurebakterien ein Uberwiegen der Sauerstoffaffinitat eintritt. Es bedarf wohl kaum naherer Auseinandersetzung, dass wir in den Unterschieden der Groésse der Wasserstoffaffinitat bei den verschie- denen Gruppen die Ursache sehen, dass die Zuckerdissimilation bei diesen Gruppen so verschiedene Wege einschlagt. Wenn man diese Unterschiede voraussetzt, ist es eine logische Konsequenz der im 3. Teil entwickelten Auffassung iiber den Chemismus der katalytischen Wasserstoffiibertragung, dass eine bestimmte Wasserstoffaffinitat des 23/ SELECTED PAPERS Protoplasmas geeignet ist, eine optimale Wasserstoffaktivierung zu bewirken bei Wasserstoffatomen, die mit einer bestimmten Kraft im Substrat gebunden sind. Eine groéssere oder eine kleinere Wasserstoff- affinitat des Protoplasmas wird zur Folge haben, dass die in Betracht kommenden Wasserstoffatome entweder gar nicht oder in ungenii- gender Weise aktiviert werden, so dass damit die Katalyse nicht oder jedenfalls mit viel geringerer Geschwindigkeit stattfindet. Bei den betrachteten wasserstoffreichen Substraten wird aber eine solche gréssere oder kleinere Wasserstoffaffhinitat in vielen Fallen eben eine optimale Aktivierung bewirken im Gegensatz zu den zuerst betrach- teten Wasserstoffatomen, d.h. die katalytische Wasserstoffiibertragung wird einen anderen Weg einschlagen. In unserer Auffassung ist also die grosse Verschiedenheit, die der Zuckerabbau in den verschiedenen Gruppen aufweist, in erster Linie auf eine quantitative Anderung einer einzigen Eigenschaft des Proto- plasmas zurickzufiihren. Ganz in Ubereinstimmung mit unserer Ansicht, dass bei den aero- ben Gruppen, bei denen die Wasserstoffaffinitat am gréssten ist und infolgedessen die optimale Wasserstoffaktivierung stets kleiner als die optimale Wasserstoffaktivierung der Gruppen mit geringerer Wasser- stoffaffinitat, steht die Tatsache, dass bei den ersten Gruppen stets sehr kraftige Wasserstoffakzeptoren — normalerweise der nach War- burg aktivierte Luftsauerstoff oder auch Methylenblau — zum Gelin- gen der Katalyse notwendig sind. Fur die Organismen mit geringerer Wasserstoffaffinitat geniigen immer schwachere Akzeptoren fiir die Wasserstoffiibertragung. Hierdurch wird auch erklarlich, dass bei den mehr aeroben Organismen niemals gasformiger Wasserstoff in den Stoffwechsel auftritt. Gegen die oben entwickelte Auffassung, dass jede bestimmte Was- serstoffaffinitat vor allem ganz bestimmte Katalysen bewirkt, konnte man nun anfihren, dass es bestimmte Teilreaktionen gibt, welche bei vielen Gruppen auftreten, wie z. B. die Milchsaéurebildung bei Grup- pe II bis IX. Dazu muss bemerkt werden, dass es sich bei diesen Reaktionen vor allem um intramolekulare Umlagerungen und Konden- sationsreaktionen handelt. Bei einer naheren Betrachtung kann es nun gar nicht wundern, dass in diesen Fallen die Katalyse in hohem Grade unabhangig ist von der Wasserstoffaffinitat des Protoplasmas; denn hierbei wird eine kraftigere Bindung des Wasserstoffatomes vom 236 DIE EINHEIT IN DER BIOCHEMIE Protoplasma notwendigerweise zur Folge haben, dass auch der be- treffende Akzeptor — bei der intramolekularen Umlagerung der Mole- kulrest — bei den Kondensationen ein zweites Substratmolekiil, mehr ungesattigt wird und also kraftigere Akzeptorwirkung entfalten wird. Es ist nicht méglich, hier fiir alle in unserem Schema aufgenomme- nen Katalysen eine nahere Betrachtung anzustellen, wie eine spezielle Katalyse unter dem Einflusse der Wasserstofl- bezw. der Sauerstoff- affinitat des Protoplasmas vor sich geht. Als Beispiel wollen wir nur die verschiedenartigen Umwandlungen des Azetaldehyds, der in allen Gruppen als Zwischenprodukt beim Zuckerabbau*’ auftritt, einer naheren Betrachtung unterwerfen. Bekanntlich hat der Azetaldehyd folgende Strukturformel: Inzwischen gibt diese Formel ungenitigende Auskunft tiber die prazise Affinitatsverteilung im Molekiil. Die grosse Neigung des Azetalde- hyds zu Anlagerungen an die CO-Gruppe, lasst erkennen, dass die unten folgende Vorstellung zweifellos die Natur des Azetaldehyds besser wiedergibt. Dabei sind schwachere Bindungen als eine normale Valenz mit .--, starkere Bindungen mit == wiedergegeben. In wel- chem Masse die Schwachung oder die Verstarkung der verschiedenen Bindungen auftritt, lasst sich nicht sagen. Nur ist anzunehmen, dass die drei H-Atome in der CH,-Gruppe nicht mit gleicher Kraft gebun- den sind, und dass eine Beschlagnahme der Affinitat dieser Kohlen- stoffatome sich vor allem in einer Lockerung des am schwachsten ge- bundenen H-Atomes aussern wird. Daher konnen wir uns den Azetal- dehyd wie folgt denken: pcan’ Das ungesattigte Sauerstoffatom wird nun bewirken, dass das Azet- aldehyd in verdiinnter wasseriger Losung zum Teil ein Molekiil Was- ser addiert. Die Anlagerung der OH-Gruppe an das Kohlenstoffatom und des Wasserstoffatomes an das Sauerstoffatom wird jedoch nur locker sein. Dieses letzte Atom wird immer noch mehr als eine Valenz 239 SELECTED PAPERS des Kohlenstoffatomes in Anspruch nehmen, wodurch die sonstigen Bindungen dieses Kohlenstoffatomes gelockert werden. Folglich k6n- nen wir das auftretende Gleichgewicht wiedergeben als: H H aS : ee Oo x HO HC oe CO] a A B Bei allen weiteren Betrachtungen muss also mit dem Vorhandensein dieser beiden Reaktionsformen des Azetaldehyds gerechnet werden. Eine kraftige Wasserstoffaffinitat, wie die der Essigbakterien z.B., hat offenbar zur Folge, dass die beiden pradisponierten (mit x ange- deuteten) H-Atome der Form B in passender Weise aktiviert werden, um eine Ubertragung auf einen kraftigen Wasserstoffakzeptor zu er- moglichen, bei Anwesenheit des (nach Warburg aktivierten) Sauer- stoffs auf diesen. Dabei wird dann neben Wasser Essigsdure gebildet. Fehlt aber dieser Wasserstoffakzeptor, dann wird auch die A-Form des Azetaldehyds als Akzeptor geniigen, wie Neuberg und Windisch*® neulich gezeigt haben, und die Folge wird eine Cannizzaro-Umlage- rung oder Dismutation des Azetaldehyds sein, wobei gleiche Teile Athylalkohol und Essigsaure gebildet werden. Bei wahrscheinlich noch kraftigerer Wasserstoffaffinitat des Proto- plasmas wird ausser den aktiven H-Atomen der Form B auch das pradisponierte H-Atom der Form A geniigend aktiviert werden, um zur Katalyse geeignet zu sein. Diese Aktivierung bedeutet aber zu gleicher Zeit eine kraftige Verstarkung der ‘Akzeptorwirkung’ des O- Atomes in Form A. Das Resultat ist eine katalytische Ubertragung des aktivierten Wasserstoffatomes eines Molekiils A auf das Sauerstoff- atom eines zweiten Molekiils A. Die dadurch ungesattigt gewordenen C-Atome lagern sich aneinander, und eine Aldolkondensation findet Stati." Bei den Gruppen mit weniger kraftiger Wasserstoffaffinitat tritt neben der Dehydrierung von Form B und Ubertragung der betref- fenden Wasserstoffatome auf Sauerstoff oder Azetaldehyd (Dismuta- tion) noch eine andere Katalyse ein. Indem die Wasserstoffaffinitat offenbar nicht ausreicht, um beide pradisponierten Wasserstoffatome 240 DIE EINHEIT IN DER BIOCHEMIE der Form B optimal zu aktivieren, beschrankt die Aktivierung sich hier fast nur auf das an das C-Atom gebundene H-Atom. In diesem Fall wird nur dieses eine H-Atom auf das O-Atom der Form A iiber- tragen und die beiden dadurch ungesattigt gewordenen C-Atome (respektive der Form A und der Form B) lagern sich aneinander: es findet eine Kondensation des Azetaldehyds zu Azetylmethylkarbi- nol (Azetoin) statt. Hierdurch wird begreiflich, dass zwei zweifellos so nah verwandte Bakterienarten, wie B. coli und B. aerogenes augen- scheinlich so verschiedene Zucker-Dissimilationsprozesse aufweisen. Jetzt ist es aber klar, dass die fiir das letztere Bakterium charakteris- tische Azetylmethylkarbinol-kondensation eben nur eine unvollstandig gelungene, fiir B. coli typische Dismutation des Azetaldehyds bedeutet. Ferner hat man bei all diesem zu beachten, dass zwar eine be- stimmte Wasserstoffaffinitat des Protoplasmas eine ganz bestimmte Wasserstofftibertragung vorzugsweise katalysiert, dass man aber dar- aus nicht schliessen darf, dass das Protoplasma nicht auch andere Wasserstofftibertragungen katalysieren kann, wenn die bevorzugte Reaktion aus irgend einem Grunde nicht eintreten kann. Wahrend z. B. bei der alkoholischen Garung der Azetaldehyd (in der A-Form) normalerweise ganz als Akzeptor fiir den im Methyl- glyoxalhydrat aktivierten Wasserstoff in Anspruch genommen wird, tritt bei kraftiger Aeration der (aktivierte) Luftsauerstoff als Akzeptor fiir den genannten Wasserstoff auf, so dass der Azetaldehyd von dem Protoplasma der Hefe in andere Richtungen verarbeitet werden kann. Einerseits wird der Azetaldehyd zu Kohlensaure und Wasser dehydriert, andererseits wird er zu Azetylmethylkarbinol kondensiert. Ausserdem wird jedoch unter den genannten Bedingungen der Luftsauerstoff aller Wahrscheinlichkeit nach schon vorangehend an der Azetalde- hydbildung in das Spiel der Reaktionen eingreifen. In diesen Ande- rungen des normalen Garungsvorganges unter dem Einflusse des Sauerstoffs liegt eben das Geheimnis der Lufthefefabrikation. Auch durch Zuftigung anderer geeigneter Wasserstoffakzeptoren zu einer garenden Zuckerlésung kann man dafiir sorgen, dass die Akzeptor- funktion des Azetaldehyds teilweise ausgeschaltet wird.* Eine geringe Sauerstoffaffinitat des Protoplasmas wird nun eben- falls eine Aktivierung der pradisponierten H-Atome in den beiden Formen des Azetaldehyds zur Folge haben, so dass auch hier noch Dehydrierung der Form Bund Ubertragung auf gleichzeitig vorhandene 241 SELECTED PAPERS Wasserstoffakzeptoren unter Essigsdurebildung vorkommt. Bei einer erésseren Sauerstoffaffinitat wird offenbar ein Freikommen von den Wasserstoffatomen aus Form B unter Bildung gasformigen Wasserstoffs sehr gefordert, wie dies von uns fiir die Buttersdurebakterien durchaus wahrscheinlich gemacht worden ist.®? Bei Form A wird die Sauerstoff- affinitat ebenfalls eine kraftige Aktivierung des pradisponierten Was- serstoffatomes bewirken, da aber hier der eventuell entstehende Mole- kiilrest nicht existenzfahig ist, kann eine Ubertragung dieses Wasser- stoffatomes nur auf ein zweites Molekiil unter gleichzeitiger Anlage- rung der betreffenden Kohlenstoffatome, also unter Aldolkondensa- tion stattfinden. Diese Reaktion ist dann der Ausgangspunkt fiir die bei den betreffenden Organismen so vorherrschende Buttersadure- (und Butylalkohol-) bildung. 5. ANWENDUNG DER WASSERSTOFFUBERTRAGUNGSTHEORIE AUF SONSTIGE DISSIMILATIONSPROZESSE Im vorigen Teil haben wir die verschiedensten Zucker-Dissimilations- prozesse zu erklaren versucht durch eine einzige Eigenschaft des Pro- toplasmas, die von Fall zu Fall quantitativ verschieden ist. Es drangt sich nun die Frage auf, ob eine ahnliche Vorstellung auch fir die spe- ziell in der Mikrobenwelt deutlich ans Licht tretenden Dissimilations- prozesse anderer Substrate zutrifft. Wir sind der Ansicht, dass dies ebenfalls méglich ist. Betrachten wir hierzu zuerst die weiteren oxydativen Dissimilationsprozesse. Es leuch- tet sofort ein, dass wir bei allen diesen Prozessen die vereinigte Wie- land-Warburesche Anschauung (Oppenheimers ‘Einheitliche Deu- tung’) ungehindert anwenden kénnen. Die Frage ist nur, ob es auch hierbei geniigt, nur eine einzelne Eigenschaft des Protoplasmas zur Erklarung des Verhaltens der verschiedenen Organismen zu den ver- schiedenen Dissimilationssubstraten heranzuziehen. Beim ersten Anblick scheint die ausgesprochene Spezifizitat der verschiedenen Organismen sich einer solchen Auffassung zu wider- setzen. Zwar koénnte man z. B. das ausgesprochene Vermégen ver- schiedener Mykobakterien® zur Dehydrierung der von den meisten Organismen nicht angegriffenen Kohlenwasserstoffe (wie Benzin, Erd- 6l u. dgl.) dadurch erklaren, dass in diesen Substraten mit kraftig ge- bundenen Wasserstoffatomen nur Wasserstoffaktivierung auftritt, 242 DIE EINHEIT IN DER BIOCHEMIE wenn das Protoplasma, wie dies dann bei den betreffenden Myko- bakterien der Fall sein wiirde, eine sehr grosse Wasserstoffaffinitat besitzt. Doch begegnet man bei einer solchen Auffassung in anderen Fallen Schwierigkeiten. Es ist z. B. bekannt, dass verschiedene Bakterien der Pseudomonas- Gruppe vor allem angepasst sind an eine oxydative Verarbeitung von Produkten der Eiweisshydrolyse. Doch sind dieselben auch imstande, anstatt dieser Produkte Salze von organischen Sauren als Dissimila- tionssubstrat zu benutzen. Dieser letzten Eigenschaft begegnet man aber ebenfalls in ausgesprochenem Masse bei den verschiedensten Essigsaure- bakterien,®* weshalb man auf eine gleiche oder nahezu gleiche Wasser- stoffaffinitat bei diesen beiden Bakteriengruppen schliessen muss. Damit scheint jedoch die ‘Tatsache, dass die Pseudomonas-Arten mit Zucker und Alkoholen, die Acetobacter-Arten mit Peptonen als Dissimilations- substrat schlecht oder gar nicht auskommen, unvereinbar. Wir sind jedoch der Ansicht, dass dieser Widerspruch nur schein- bar ist. Unsere im 3. Teil gegebene Anschauungsweise des Chemis- mus der katalytischen Wasserstofftibertragung ist namlich in einer Hinsicht absichtlich unvollstandig gewesen. Wir haben uns namlich darauf beschrankt, eine Vorstellung zu geben, nach welcher die Wasserstoffaffinitat 1m Substrat, die Wasserstoffaffinitat des Kataly- sators und die Bindungsneigung des Akzeptors zu Wasserstoff — die drei Faktoren, welche uber das Zustandekommen der Katalyse ent- scheiden — unveranderliche Gréssen sind. In Wirklichkeit wird dies aber nicht zutreffen; denn die physische Chemie lehrt, dass diese Eigenschaften, die in den Oxydoreduktionspotentialen der genannten Substanzen ihren quantitativen Ausdruck finden, in hohem Grade abhangig sind von der herrschenden Wasserstofhonenkonzentration.®® Nun brauchen wir nur noch daran zu denken, dass einerseits jede oxydative Verarbeitung von hydrolysierten Eiweissspaltprodukten eine Produktion von Ammoniak zur Folge hat, und die Dissimilation sich also in einem alkalischen Medium abspielt, dass andererseits die oxydative Verarbeitung von Zucker und Alkoholen immer mit inter- mediarer Produktion von Saéuren zusammengeht, um einzusehen, dass die nahezu beim Neutralpunkte vor sich gehende Verarbeitung der organischen Salze durch beide Bakteriengruppen gar nicht einzu- schliessen braucht, dass beide sich auch gegenttber den weitergenann- ten Substraten ahnlich verhalten werden. is SELECTED PAPERS Wenn wir dies alles beriicksichtigen, so steht vorlaufig der Annahme nichts im Wege, dass der ganze Unterschied im Verhalten der ein- zelnen Organismen den verschiedenen Dissimilationssubstraten gegen- iiber seine Ursache darin findet, dass das Protoplasma eine Wasser- stoff- oder Sauerstoffaffinitat besitzt, die fiir eine jede Art in einem charakteristischen Zusammenhange mit der Wasserstofhionenkonzen- tration steht und sowohl bei zu niedriger wie bei zu hoher Konzen- tration dieser Ionen auf Null herabsinkt. Dabei sind die Grenzen des zulassigen Wasserstoffionengebietes ftir jede Art verschieden. Diese Auffassung findet, wie man einsehen wird, eine Parallele in dem Zu- sammenhang zwischen Enzymwirkung und Wasserstofhionenkonzen- tration, der in den klassischen Arbeiten von Sorensen, Michaelis u.a. festgestellt worden ist. In Ubereinstimmung mit dieser Ansicht steht nun die Tatsache, dass eine Anderung der Wasserstoffionenkonzentration innerhalb des fiir einen Organismus zulassigen Gebietes zur Folge hat, dass be- stimmte Wasserstoffiibertragungskatalysen ausfallen und andere der- artige Katalysen dafiir an deren Stelle treten. Das schdnste Beispiel dafiir ist die bekannte von Neuberg entdeckte sogenannte dritte Ver- garungsform des Zuckers bei der alkoholischen Garung. Wahrend die Alkoholhefe normalerweise den Wasserstoff des Methylglyoxalhydrats auf Azetaldehyd tibertragt, tritt in einem alkalischen Medium Glyze- rinaldehyd als Akzeptor in den Vordergrund, wobei dann der Azetal- dehyd ebenfalls teilweise zu Essigsaure dehydriert wird und der daraus entstammende Wasserstoff auf ein zweites Molekiil Aldehyd tber- tragen wird, unter Bildung von Athylalkohol. Wir haben weiter feststellen konnen, dass es gelingt, die Zucker- vergarung durch B. coli durch Pufferung des Mediums derartig zu verandern, dass der Azetaldehyd nicht, wie es normalerweise stets der Fall ist, in Athylalkohol und Essigsaure, sondern teilweise in Aze- tylmethylkarbinol (also wie bei B. aerogenes) iibergefithrt wird. Als drittes Beispiel mége angefiihrt werden, dass Kostytschew und Afanassjewu gezeigt haben, dass Schimmelpilze, wie Penicillium. glau- cum, die unter anaeroben Bedingungen normalerweise aus Zucker keine Spur Athylalkohol bilden, hierzu sehr merkbar befahigt werden, wenn man den Versuch in schwach alkalischer Lésung vornimmt.*®* Diese Beispiele sind noch durch viele andere zu vermehren, doch wird das obige geniigen, um unsere Anschauungsweise zu rechtfertigen. 244 DIE EINHEIT IN DER BIOCHEMIE Wir wollen nun noch mit einigen Beispielen zeigen, wie fruchtbar die allgemeine, katalytische Wasserstoffibertragungstheorie in ihrer Anwendung auf die verschiedensten Dissimilationsprozesse ist. I. Der Nitrifikationsvorgang Bekanntlich steht es seit den klassischen Untersuchungen Winograds- kys — die in denjenigen Meyerhofs®’ einen wichtigen Abschluss gefun- den haben — fest, dass der Nitrifikationsvorgang, d. h. der Ubergang der Ammonsalze in Nitrate, in zwei Stufen verlauft. Dabei wird jede dieser beiden Stufen, d. h. die Oxydation der Ammonsalze zu Nitriten und die Oxydation der Nitriten zu Nitraten von einer spezifischen Bak- terienart bewirkt. Aus den genannten Untersuchungen geht klar hervor, dass die beiden Bakterienarten aus den von ihnen bewirkten anorganischen Oxyda- tionsvorgangen ihre Energie nehmen und dass dieselben die Dissimi- lationsprozesse dieser Organismen darstellen. Meistens findet man diese Vorgange wie folgt wiedergegeben. Fiir das Nitritbakterium: NH,+30 — HNO,+H,O-+ 79 cal. und fiir das Nitratbakterium: HNO,+0 — HNO,-+ 21,6 cal. Konnen wir nun die katalytische Wasserstoffiibertragungstheorie auch auf diese Prozesse anwenden ? Wie wir sehen werden, ist dies durchaus méglich und wir erreichen damit den Vorteil, dass die enge Verwandtschaft dieser Vorginge mit den normalen Atmungsprozes- sen deutlich hervortritt. Es ist nun wahrscheinlich, dass der Dissimilationsprozess der Nitrit- bakterien sich folgenderweise abspielen wird: OH ns | OH Hydroxylamin ‘H H 2 O La / ae ZL —iH+0 — W\. +H,O > NA OH+0 > Nx Nagi te NS Oe INK O:H Untersalpetrige Saure OH Salpetrige Saure 245 SELECTED PAPERS Der Dissimilationsprozess der Nitratbakterien konnte dann wie folgt wiedergegeben werden: OH O J O n€ +H,0 > NNX~ +H, Xo ‘SO Kaliumnitrat Kaliumnuitrit 248 DIE EINHEIT IN DER BIOCHEMIE yes O ag | +2H,O n< + w| SS OK Kaliumnitrit Untersalpetrigsaures Kalium Dabei enstammt der fiir die Hydrierung beniitzte Wasserstoff der gleichzeitig anwesenden organischen Substanz, die nebenbei auch einer intramolekularen Dehydrierung (und Hydrierung) unterliegen kann, welche zu einer Kohlensaurebildung Anlass gibt. Die Kohlen- sdure wird aus dem untersalpetrigsauren Kalium die Saure in Frei- heit setzen, die bekanntlich in verdiinnter wasseriger Lésung leicht das Anhydrid N,O (Lachgas) abspaltet. ~Das Anhydrid ist aber, wie auch aus den direkten Versuchen von Beijerinck und Minkman’ hervorgeht, ebenfalls noch imstande als Wasserstoffakzeptor aufzutreten, wobei es in freien Stickstoff wber- geftihrt wird: N. N o+2H —> ||| +H,O N N Die gegebene Auffassung des Denitrifikationsprozesses erklart also in deutlicher Weise die Entstehung von Nitriten und N,O als Zwischen- produkte der Denitrifikation, wie dies von vielen Forschern, insbeson- dere auch von den beiden genannten, nachgewiesen worden ist. Dass verschiedene Bakterienarten die Denitrifikation bis zu verschiedenen Stufen bewirken, ist eine logische Konsequenz, denn die Nitrate, Nitrite und das Stickstoffoxydul sind als Wasserstoffakzeptoren nicht eleichwertig. Eine Wasserstoffaffinitaét, wie das Protoplasma einer be- stimmten Bakterienart sie aufweist, kann gerade geeignet sein flr die Wasserstoffiibertragung aus einem speziellen Substrat auf Nitrate, nicht aber auf Nitrite oder auf Stickstoffoxydul. Ein grosser Vorteil der gegebenen Anschauung besteht weiter darin, dass die ebenfalls von Beijerinck und Minkman festgestellte merk- wirdige bakterielle Verarbeitung eines Gemisches von gasf6rmigem Wasserstoff und Stickstoffoxydul gemass folgender Gleichung: N,O++-H, = N,-+H,O ganz in den Rahmen der iiblichen Denitrifikationsprozesse hineinpasst. 249 SELECTED PAPERS Ubrigens ist die besprochene Gruppe noch interessant in Hinsicht auf eine Bemerkung Wielands, folgendermassen formuliert: ‘Die extremste Forderung der Dehydrierungstheorie, den Sauerstoff bei funktionellen Vorgangen der Zelle durch einen anderen Wasserstoff- akzeptor zu ersetzen, hat sich bis jetzt nicht erfiillen lassen’.”4 Die denitrifizierenden Bakterien sind nun ein schénes Beispiel dafiir, dass es aerobe Organismen gibt, bei denen sich der Luftsauerstoff vollstandig durch einen anderen Wasserstoffakzeptor ersetzen lasst, ohne dass der normale Entwicklungsgang dieser Organismen davon beeintrachtigt wird. IV. Dissimilationsprozesse der Bakterien der Proteus-Gruppe Bekanntlich sind die Bakterien der Proteus-Gruppe einerseits denen der Coli-aerogenes-Gruppe nahe verwandt, indem bei beiden der anaerobe Zuckerabbau wesentlich in derselben Richtung verlauft. Anderseits unterscheidet die Proteus-Gruppe sich von der zweitgenannten dadurch, dass die Vertreter der ersteren unter anaeroben Bedingungen ebenfalls zu einer Verarbeitung von hydrolytischen Eiweissspaltprodukten im- stande sind (Faulnis). Der Chemismus dieser Vorgange ist noch grossten- teils in Dunkel gehiillt. Durch Nawiasky’? ist jedoch dieser Vorgang fiir den Fall des Asparagins als Substrat nahezu vollstandig aufgeklart. Die von ihm aufgestellten Umlagerungen sind nun darauf zuriick- zufiihren, dass ein Teil der aus Asparagin in erster Instanz durch Hy- drolyse gebildeten Asparaginsaure vollstandig bis zu Wasser, Kohlen- siure und Ammoniak dehydriert wird, wobei ein anderer Teil des Asparagins als Wasserstoffakzeptor auftritt und dabei unter Ammo- niakabspaltung zuerst zu Bernsteinsdure und teilweise weiter bis zu Essigsaure hydriert wird. Der Unterschied zwischen den Dissimilationsprozessen der Koli- bakterien und denen der Proteusbakterien ist demnach darauf zurick- zufiithren, dass die Wasserstoffaffinitat der letzteren noch beim pH>7 die angegebene Wasserstoffiibertragung erméglicht. Die vier gegebenen Beispiele liessen sich noch durch zahlreiche andere vermehren”*, doch glauben wir, dass sie geniigen werden, um zu zeigen, auf welche mannigfache Art durch die katalytische Wasser- stoffiibertragungstheorie der Einblick in die Dissimilationsvorgange vergrossert werden kann. 250 DIE EINHEIT IN DER BIOCHEMIE 6. DIE ASSIMILATIONSVORGANGE IM LICHTE DER WASSERSTOFFUBERTRAGUNGSTHEORIE Wie schon in der Einleitung bemerkt wurde, lassen sich im Gesamt- stoffwechsel vom physiologischen Standpunkte aus zweierlei Typen von Vorgangen unterscheiden: assimilatorische und dissimiatorische Prozesse. Hierbei sollen als dissimilatorische Vorgange diejenigen be- zeichnet werden, bei denen ein Teil der dargebotenen Nahrung in solche Produkte tibergefiihrt wird, die von der Zelle entweder sofort oder doch bald ausgeschieden werden und dann in der Regel fiir sie keinen Wert mehr haben.7? Diesen gegeniiber stehen die assimilato- rischen Vorgange, also diejenigen chemischen Prozesse, welche die Nahrung in mehr oder weniger wichtige, doch normalerweise auf- tretende Zellsubstanzen wherfthren. Wahrend die Dissimilationsprozesse im Vorhergehenden geniigend beriicksichtigt sind, wollen wir jetzt die Frage kurz streifen, in wiefern auch die Bildung der Zellsubstanzen restlos — abgesehen von den hy- drolytischen und esterifizierenden Vorgangen — auf eine Kette von katalytischen Wasserstoffiibertragungen zurtickgefithrt werden kann. Dabei ist zuerst zu beriicksichtigen, dass, wie die Erfahrung lehrt, die assimilatorischen Vorgange, die am deutlichsten bei der Zellver- mehrung hervortreten, niemals stattfinden, ohne dass sich gleich- zeitig dissimilatorische Vorgange abspielen. Dieser Zusammenhang erklart auch, dass bestimmte Assimilationsprodukte auf einem ener- getisch héheren Plan stehen als die verarbeitete Nahrung. Dies ist eben nur moglich, weil die freie Energie bei sonstigen Vorgangen eine grossere Abnahme gefunden hat, sodass auch die freie Energie des ganzen Systems abgenommen hat. Der Vorgang der katalytischen Wasserstoffiibertragung ist nun durchaus geeignet, eine nahere Vorstellung zu geben, wie dieser che- mische Potentialhub einzelner Komponenten des ganzen Systems vor sich gehen kann. Denn es ist eben charakteristisch ftir diesen Vorgang, dass bei diesem Prozess der Wasserstoffakzeptor energetisch gehoben wird, dem dann gegeniiber steht, dass das Substrat bei der Dehydrie- rung einen grésseren Verlust an freier Energie erfahrt. In diesem Zu- sammenhange sei bemerkt, dass auch Wieland betont, dass nur solche Reaktionen im Spiel der Hydrierungen und Dehydrierungen ein- treten, bei denen der Energiegewinn bei der Hydrierung grosser ist als 251 SELECTED PAPERS die Aufwendung zur Dehydrierung. Man kann diesen Satz auch so formulieren: ‘dass nur solche Reaktionen eintreten, bei denen die Zu- nahme der freien Energie, welche der Akzeptor bei der Hydrierung erfahrt, kleiner ist als die Abnahme der freien Energie, welche das Substrat bei der Dehydrierung erfahrt’. Aus dieser Auseinandersetzung folgt einerseits, dass der chemische Potentialhub einzelner Reaktionsprodukte durchaus nicht auf assimi- latorische Vorgange beschrankt ist, sondern dass dieser vielmehr auch bei dissimilatorischen Vorgangen Ofters auftritt. Daraus konnen wir schliessen, dass in physikochemischer Hinsicht durchaus kein Unter- schied zwischen beiden 'Typen von chemischen Prozessen existiert, so- dass von vornherein sich nichts dagegen widersetzt, auch die Ent- stehung der typischen Assimilationsprodukte auf eine Kette kataly- tischer Wasserstoffiibertragungen zuriickzufiihren.”® Im folgenden werden wir uns darauf beschraénken, das Problem fiir die wichtigsten der verschiedenen Substanzen, welchen man in den Zellen begegnet, namlich die Kohlenhydrate, Fette und Eiweissstoffe, kurz zu betrachten. Was zuerst die Kohlenhydrate betrifft, denen man als Reservesub- stanz und auch als Geriistsubstanz haufig begegnet, so kann folgendes bemerkt werden: soweit die zu verarbeitende Nahrung schon Kohlen- hydrate enthalt, kann die Bildung der komplexen Kohlenhydrate ein- fach durch Kondensation der einfachsten Bausteine, namlich der Zucker, stattfinden. Ausgeschlossen ist es dabei jedoch nicht, dass sich die Sache nicht so einfach verhalt, und dass die Synthese ihren Aus- gangspunkt nimmt bei irgendwelchen Zucker-Abbauprodukten der Dissimilationsprozesse. Dabei kann man sich denken, dass irgendein dehydriertes Substrat unter Umstanden wieder als Wasserstoffakzep- tor fungiert bei irgend einer Dehydrierung, welche mit grésserer Ab- nahme an freier Energie verlauft als die Dehydrierung des urspriing- lichen Substrates. Dieselbe Situation findet man dann zuriick in den Fallen, worin die Nahrung nur Substanzen enthalt, welche in energetischer Hin- sicht minderwertiger sind als die zu synthetisierenden Kohlenhydrate, wie dies z. B. zutrifft fiir die Riickverwandlung von Milchsaure in Glykogen in den Muskelzellen bei der aeroben Phase der Muskel- arbeit. Auch hier brauchen offenbar nur die bei der Bildung der Milch- sdure eingetretenen Dehydrierungen und Hydrierungen riickgangig 252 DIE EINHEIT IN DER BIOCHEMIE gemacht zu werden, was durch die gleichzeitig stattfindende Ver- brennung eines Teiles der Milchsaure energetisch erméglicht wird. Zwar wollen wir hier offen gestehen, dass es nicht ohne weiteres ver- standlich ist, wie sich dies Riickgingigmachen bei den zntra-moleku- laren Dehydrierungen und Hydrierungen abspielt, aber immerhin kann man sich denken, dass hier die entwertete Atomgruppe zuerst energetisch gehoben wird, indem sie als Akzeptor bei einer ‘Dissimi- lationsdehydrierung’ auftritt und dann die zweite, urspriinglich ener- getisch gehobene Atomgruppe in einem neuen Dehydrierungsvorgang wieder degradiert wird. Auch in den Fallen, worin die Kohlenhydratsynthese aus Kohlen- sdure stattfindet, unterlieget es kaum einem Zweifel, dass hierbei an erster Stelle eine Reduktion dieser Saure tiber Ameisensdure zu For- maldehyd stattfindet. Wir werden hierbei die photochemische Kohlen- sdureassimilation durch die griinen Pflanzen ausser Betracht lassen,*® obgleich der enge Zusammenhang dieses Vorganges mit der Kohlen- sdureassimilation durch die autotrophen Bakterien unverkennbar ist. Es ist nun zweifellos kein Zufall, dass wir das Vermogen zur Kohlen- sdureassimilation unter den Bakterien nur bei denjenigen Gruppen antreffen, die zu der schwierigen Dehydrierung anorganischer Sub- strate imstande sind. In unserem Vorstellungskreis sind diese Bak- terien — vor allem die nitrifizierenden- und Schwefelbakterien — dazu befahigt, weil die grosse freie Affinitat ihres Protoplasmas zu Wasser- stoff es erméglicht, die Wasserstoffatome in Substraten, worin dieselben fest gebunden sind, zu aktivieren. Die Kohlensaure (in ihrer Hydrat- form) wird aber von der exzeptionell grossen Affinitat des Protoplas- mas zu Wasserstoff aktiviert werden, wodurch sie befahigt wird als Akzeptor aufzutreten. Was an zweiter Stelle die Fette betrifft, so k6nnen wir uns dariiber ganz kurz fassen, weil diese Frage eine weitgehende Losung gefun- den hat durch die Untersuchungen von Haehn und Kinttof*. Ohne naher darauf einzugehen, wollen wir nur konstatieren, dass die Vor- stellung viel Wahrscheinliches hat, dass die Fettsynthese bei dem Azet- aldehyd ihren Ausgangspunkt nimmt und aus dieser Substanz durch abwechselnde Kondensation (d. h. intermolekulare Wasserstofftiber- tragung) und Hydrierung allmahlich in die héheren Fettsduren um- gewandelt wird. Der Wasserstoff — und damit die bendtigte Energie — fiir diese Hydrierung wird dabei den Dehydrierungen der gleich- 250 SELECTED PAPERS zeitig verlaufenden Dissimilation eines anderen Teiles des Nahrsub- strates entstammen. Schliesslich moéchten wir noch einige Aufmerksamkeit der Synthese des Eiweisses schenken. Durch die rezenie Arbeit von Knoop und Oesterlin*! ist dieser Vorgang, jedenfalls was die Synthese der Amino- sduren betrifft, klargestellt. Die genannten Forscher konnten schéne experimentelle Nachweise dafiir bringen, dass diese Synthese der Hauptsache nach den umgekehrten Weg einschlagt, welcher bei der oxydativen Dissimilation der Aminosauren befolgt wird. Diese oxydative Verarbeitung der Aminosauren ist eine der ver- breitetsten Dissimilationsprozesse. Wahrscheinlich spielen sich dabei die folgenden Reaktionen ab: A R-CHNH,-COOH+0 > R-C = NH-COOH+H,0O _/ OH B R-CG = NH-COOH+H,O>R-G¢___ -COOH NH, OH _/OH os R-C¢_ -GOOH+H,O-+R-C€__ .COOH+NH, NH, OH OH P) Roce -COOH > R-C = 0-COOH+H,0 NOH Die gebildete Ketosaéure wird dann weiter in der bekannten Weise dehydriert, wobei als erstes Zwischenprodukt zweifellos der Aldehyd LO entstehen wird (Streckersche Reaktion). Auch dieser Dissimilationsprozess ist also auf eine Kombination von Hy- drolysen und katalytischen Wasserstofftibertragungen zuriickzufiihren. Die wichtigen Ergebnisse von Knoop und Oéesterlin machen es nun ausserst wahrscheinlich, dass die Synthese der Aminosauren umge- kehrt ihren Ausgangspunkt nimmt bei den Ketoséuren und Ammo- niak, welches System dann durch eine bei der Dissimilation statt- findende Dehydrierung (mit grésserem Potentialfall als die der Dehy- drierung von Aminosaure zu Iminosaure) zu Aminosaure hydriert wird. Auf die iiberaus grosse Wahrscheinlichkeit, dass weiter die Orga- nismen, welche zur Eiweisssynthese aus atmospharischem Stickstoffim- 254 DIE EINHEIT IN DER BIOCHEMIE stande sind, als erste Stufe den freien Stickstoff zu Ammoniak hy- drieren mit Hilfe des dem Dissimilationssubstrat entstammenden aktivierten Wasserstoffs, ist schon von Wieland hingewiesen worden (‘biologische Habersynthese’). Dabei ist es jedoch nicht ausgeschlos- sen, dass diese Hydrierung nur stattfindet, wenn der Stickstoff zuvor ebenfalls aktiviert wird, genau wie dies beim Sauerstoff mit Hilfe der Warburgschen Eisenverbindung immer geschieht. Der eine von uns (D.) hat die grosse Bedeutung eines Zusatzes kolloidaler Eisenver- bindungen ftr die Entwicklung der anaeroben stickstoffbindenden Bakterien (Clostridium pasteurtanum Winogradsky) mit Sicherheit fest- stellen k6nnen, und auch fiir die wichtigsten aeroben stickstoffbinden- den Bakterien (Azotobacter chroococcum Beijerinck) ist ahnliches schon friiher von Krzemieniewski und von Prazmowski nachgewiesen. Obgleich die hier gegebene Ubersicht ausserst unvollstandig ist und verschiedene Punkte zweifellos weiterer Aufklarung bediirfen, glau- ben wir doch, dass sie geniigen wird den Eindruck zu erwecken, dass das Prinzip der katalytischen Wasserstoffiibertragung geeignet ist, auch die assimilatorischen Vorgange in ihren Hauptziigen verstand- lich zu machen. Die gegebene Vorstellung vom Wesen der assimilatorischen Vor- gange erklart auch den unverkennbaren Zusammenhang zwischen der Natur der Dissimilationsprozesse der verschiedenen Organismen- gruppen und der Fahigkeit dieser Organismen zu assimilatorischen Synthesen. Dies zeigt sich z. B. in der Tatsache, dass die wahren Milchsaurebakterien, welche durch einen Mangel von sowohl kraftiger Wasserstoff- als Sauerstoffafhinitat gekennzeichnet sind, nicht imstande sind, mit anorganischen Stickstoffverbindungen oder mit Verbindun- gen wie Harnstoff auszukommen. Auch das ganze vielumstrittene Biosproblem asst sich mit grosser Wahrscheinlichkeit auf denselben Umstand zurickfihren. 7. UBERBLICK UBER DIE AUFGESTELLTE THEORIE DER KATALYTISCHEN WASSERSTOFFUBERTRAGUNG ALS KERN DES BIOCHEMISCHEN GESCHEHENS Die in den vorhergehenden Teilen niedergelegten Ausfiihrungen kann man kurz zusammenfassen als einen Versuch, die durch Wieland fiir die oxydativen Dissimilationsprozesse ausgearbeitete Dehydrierungs- 2005. SELECTED PAPERS theorie konsequent auch auf die fermentativen Dissimilationsprozesse zu tibertragen. Obgleich die Wielandsche Theorie friiher schon mehr- mals fiir bestimmte Teilreaktionen in den fermentativen Dissimilations- prozessen zur Erklarung herangezogen worden war, stand eine kon- sequente Durchfiihrung dieses Prinzips auf alle — oder jedenfalls auf die wichtigsten — Dissimilationsprozesse noch aus. Der gemachte Versuch scheint uns in hohem Grade erfolgreich, in- dem man, sich zuerst beschrankend auf die Prozesse, wobei Zucker (Glukose) als Dissimilationssubstrat fungiert, klar erblicken kann, wie sich die verschiedensten bekannten Dissimilationsprodukte mit Hilfe einer Kette von katalytischen Wasserstoffiibertragungsreaktionen von dem Substrat ableiten. Die von Wieland verteidigte Auffassung der Wasserstoffaktivierung, die fiir die aeroben Dissimilationsprozesse mehr oder weniger eine wissenschaftliche Abstraktion ist, sieht man allmahlich bei den mehr anaeroben Organismen zur Wirklichkeit werden, indem hier grosse Quantitaten gasfOrmigen Wasserstoffs aus dem Giarungsgefass entweichen. Sonnenklar tritt weiter der allmah- liche Ubergang des aeroben Lebens in das anaerobe an den Tag. Es tritt nicht nur bei der obigen Betrachtung die volle Berechtigung des beriihmten Pasteurschen Satzes ‘La fermentation est la vie sans air’ sehr deutlich hervor, sondern die Ahnlichkeit im Wesen der At- mung und Giarung wird so vollkommen, dass man mit gleichem Rechte die Atmung als eine von dem — durch Warburgs Eisenverbin- dung aktivierten — Sauerstoff in andere Bahnen gelenkte Garung auffassen kann.8? Die nur nebensiachliche Bedeutung des Sauerstoffs tritt besonders schén bei den denitrifizierenden Bakterien hervor, bei denen wir den Fall realisiert finden, dass der Sauerstoff sich ohne irgend einen nachteiligen Einfluss auf den normalen Entwicklungs- gang durch andere Akzeptoren (Nitrate) ersetzen lasst. Neben der gegebenen Erweiterung der Wielandschen Theorie in quantitativer Hinsicht haben wir die Theorie der Oxydoreduktion auch in prinzipieller Weise erweitert, indem wir darauf hinwiesen, dass es méglich ist, auch diejenigen Teilreaktionen, welche auf einer einfachen Umlagerung bestimmter Substanzen beruhen, als kataly- tische Wasserstoffiibertragungsprozesse intramolekularer Art zu_be- trachten. Dasselbe gilt ebenfalls fiir die im Stoffwechsel nicht seltenen Kondensationsreaktionen. Dadurch war die Méglichkeit gegeben, alle von einer Zelle be- 256 DIE EINHEIT IN DER BIOCHEMIE wirkten Teilprozesse der Dissimilation — abgesehen von den hydrolyti- schen und esterifizierenden Vorgangen, welche aber grosstenteils nur vorbereitender Natur sind — der Wirkung eines einzelnen Agens zu- zuschreiben. Es braucht kaum betont zu werden, wie die Vorstellung der biochemischen Arbeitsweise der lebenden Zelle hierdurch vereinfacht wird. Bis jetzt ist es fast allgemein iiblich, jede der in dem Dissimilations- prozess sichergestellten Teilreaktionen der Wirkung eines spezifischen Enzyms zuzuschreiben. Diese Enzyme haben dann je nach der Natur der von ihnen bewirkten Reaktionen Namen bekommen als: Kata- lase, Reduktase, Zymase, Laktozymase, Alkoholoxydase, Karboxy- lase, Karboligase, Methylglyoxalase, Aldehydmutase, Schardingers Enzym u. s. w. Auf Grund des Vermégens einer Zelle, Bernsteinsdure zu Fumar- saure zu dehydrieren, wurde z. B. auf die Anwesenheit einer Sukzino- fumarase geschlossen. So lange man seine Aufmerksamkeit nur auf die normalerweise vorkommenden Stoffwechselvorgange lenkte, war eine derartige Handlungsweise noch durchfiihrbar; seitdem aber die mo- derne Forschung jeden Tag deutlicher zeigt, dass die Zellen auch gegentiber ihnen in der Natur nie oder dusserst selten gebotenen Sub- stanzen kraftige Wirkungen entfalten, fiihrt diese Auffassung ad ab- surdum. Man kann sich doch unméglich vorstellen, dass, um nur ein Beispiel zu geben, die von Beijerinck beschriebenen Bakterien, welche Querzit in Pyrogallol tberftihren, dies bewirken dank der Anwesen- heit einer Querzitopyrogallolase, welche sie fiir den seltenen Fall, dass sie Querzit begegnen, bereit halten! Obgleich man in beschranktem Masse schon gefiihlt hatte, dass die Wirkung der genannten Enzyme nicht so streng spezifisch aufgefasst werden konnte und man z. B. der Karboxylase eine allgemeine Wir- kung den e-Ketoséuren gegeniiber zuschrieb, ist es nicht schwierig, an einem Beispiel zu demonstrieren, welche Befreiung die von uns angegebene weitgehende Unifikation der wirksamen Agentia mit sich bringt. Durch Neuberg und Windisch®* ist in einer schOnen experimen- tellen Untersuchung gezeigt worden, dass Essigbakterien beim Ab- schluss freien Sauerstoffs Aldehyde weitgehend umlagern in gleiche Teile der betreffenden Alkohole und Saéuren (z. B. Azetaldehyd in Athylalkoho] und Essigsaure). Die genannten Forscher schliessen 27 SELECTED PAPERS daraus auf die Anwesenheit eines die Dismutation des Aldehyds be- wirkenden Enzyms in diesen Bakterien, wogegen wenig einzuwenden scheint. Dies fiihrt sie jedoch zu dem Schluss, dass ein Essigbakterium, welches unter normalem Sauerstoffzutritt Alkohol oxydiert und dabei zweifellos als erstes Oxydationsprodukt Azetaldehyd bildet, diesen Aldehyd auch unter aeroben Verhaltnissen mit Hilfe des genannten Enzyms nur zur Halfte in Essigsaure tberfiihren und die zweite Halfte in Alkohol riickverwandeln wird, um denselben wieder in der ange- gebenen Weise zu verarbeiten. So entsteht Neubergs ‘Pilgerschrittschema’ fiir die normale durch Essigbakterien bewirkte Oxydation von Alkohol zu Essigsaure: Athylalkohol Athylalkohol Athylalkohol aa es f Y | U Azetaldehyd Azetaldehyd usw. as. YU \ Essigsaure Essigsaure Diese an sich wenig wahrscheinliche Vorstellung begriinden sie weiter durch einen einzigen Versuch, bei dem mit einem Trockenpraparat der Bakterien auch bei einem offenbar sehr mangelhaften Luftzutritt ein Teil des verarbeiteten Azetaldehyds als Alkohol wiedergefunden werden konnte. Die Notwendigkeit zur Annahme eines derartig komplizierten Vor- ganges fallt sofort weg, wenn wir die Dismutierung des Aldehyds unter anaeroben Bedingungen der Wirkung des allgemeinen Wasserstoff tiber- tragenden Agens zuschreiben, welches bei Abwesenheit eines anderen Akzeptors den der Dehydrierung eines Molekiils Azetaldehyd ent- stammenden Wasserstoff auf ein zweites Molekiil iibertragt. Wir sehen dann sofort ein, dass bei tadelloser Liiftung der aus Athylalkohol primar gebildete Azetaldehyd normalerweise ebenfalls einer Dehydrierung unterliegen wird, aber dass in diesem Falle der aktivierte Sauerstoff, nicht ein zweites Molekiil Aldehyd, als Akzeptor fiir diesen Wasser- stoff auftreten wird. Eine Riickbildung von Alkohol wird dabei héch- stens von ungeniigend beliifteten Zellen bewirkt werden. Man wird gegen unsere Auffassung der einheitlichen Natur der wirksamen Agentia den Einwand erheben, dass demnach alle Zellen zu allen diesen Umsetzungen imstande sein miissten. Dagegen wollen 258 DIE EINHEIT IN DER BIOCHEMIE wir zunachst bemerken, dass die moderne Forschung auch immer deutlicher lehrt, dass die verschiedensten Zellen zu iiberraschend vielen Umsetzungen fahig sind. Es eriibrigt sich wohl, hierfiir Beispiele zu geben, da sie in grésster Menge zu Gebote stehen. Dass jedoch das Agens verschieden gearteter Zellen nicht dasselbe ist und bestimmte Falle katalytischer Wasserstoffiibertragung merk- lich bevorzugt, andere nur langsam oder mit praktisch unmerklicher Geschwindigkeit fordert, findet seine Ursache in einer quantitativen Abstimmung des Katalysators. Im Anschluss an friihere Beirachtungen uber den Chemismus der Katalyse bei rein chemischen Prozessen haben wir nun die nach- folgende Hypothese iiber die Ursache der katalytischen Wasserstoff- ubertragung aufgestellt. Die katalytische Wirkung des Agens — wel- ches wir vorlaufig einfach mit dem Protoplasma der Zelle identifiziert haben — sollte bedingt sein durch die freie Affinitat des Protoplasmas zu Wassersioff oder zu Sauerstoff. Diese Affinitat wiirde fiir eine jede artspezifische Zelle eine bestimmte Grésse haben und eben diese Grosse, welche fiir bestimmte Katalysen optimal sein wiirde, wiirde die Richtung der Dissimilation bestimmen. Die verschiedenen Zellen wirden sich je nach dem Masse ihrer Aerobie und Anaerobie gewisser- massen in eine Reihe ordnen lassen mit abnehmender Wasserstoff- affinitat, welche bei den Anaerobiern in eine zunehmende Sauerstoff- affinitat umschlagt. Diese Hypothese wird gestiitzt durch die Tat- sache, dass sie einen Zusammenhang griindet mit anderen hervor- tretenden Eigenschaften der betrachteten Zellen, vor allem mit der Sauerstoffempfindlichkeit der anaeroben Organismen und mit dem Vermogen der Verarbeitung gasf6rmigen Wasserstoffs durch viele ausgesprochen aerobe Organismen. Auch die Empfindlichkeit der am kraftigsten oxydierenden Organismen, der nitrifizierenden Bak- terien, gegeniiber den fiir die meisten tibrigen Zellen am besten assimi- lierbaren organischen Substanzen, lasst sich in diesem Rahmen be- trachten. Die gegebene Hypothese musste jedoch noch eine sehr plausible Erweiterung erfahren, indem angenommen werden musste, dass die genannte Wasserstoffaffinitat der Zelle keine absolut konstante Grosse ist, sondern dass dieselbe innerhalb des Gebietes der fiir die Zelle zu- lassigen Wasserstoffionenkonzentrationen mit dieser Konzentration schwankt. Die unendliche Verschiedenheit des biochemischen Ver- 259 SELECTED PAPERS haltens der verschiedenen artspezifischen Zellen ware dabei zuriick- zufiihren auf eine Verschiedenheit einer einzelnen Eigenschaft des Protoplasmas — der Wasserstoff- bezw. der Sauerstoffaffinitat — in ihrer Abhangigkeit von den, fiir jede Art typischen, zulassigen Wasser- stoffionenkonzentrationen. Die Tatsache, dass auch die Dehydrie- rungsneigung der Substrate und Hydrierungsneigung der Akzeptoren bekanntlich eine Funktion der Wasserstoffonenkonzentrationen sind, traet wesentlich dazu bei, die dussere Verschiedenheit des bioche- mischen Geschehens trotz seiner inneren Einheit zu vermehren. Wenn man daneben beriicksichtigt, dass die oxydative oder fermen- tative Dissimilation von Zuckerarten immer zur intermediaren oder definitiven Bildung von Sauren, die Dissimilation der hydrolytischen Eiweissspaltprodukte immer zur Bildung von Ammoniak fihrt, dann kommt man zu der Ansicht, die Anpassung der verschiedenen Zellen an diese wichtigsten Substratgruppen darin zu begriinden, dass das Protoplasma dieser Zellen bei den dazu geh6renden Wasserstoffionen- konzentrationen eine passende Wasserstoffaffinitat erworben hat. Schliesslich haben wir gemeint andeuten zu konnen, dass nicht nur die dissimilatorischen, sondern auch die assimilatorischen Vorgange wesentlich auf katalytische Wasserstofftibertragung zuriickgefthrt werden konnen. 8. SCHLUSSBETRACHTUNG Wir mochten diese Arbeit nicht abschliessen, ohne klar auszusprechen, dass wir uns vollbewusst sind, dass hier nur ein sehr unvollendeter Versuch gemacht worden ist, eine gewisse Ordnung in die biochemi- schen Vorgange zu bringen. Der Umfang des betrachteten Materials liess es nicht zu, die Einzelheiten immer geniigend zu begriinden. Wir sind also darauf vorbereitet, dass der kritische Leser an vielen Stellen Einwande erheben wird. Doch sind wir tberzeugt, dass die Anwen- dung der gegebenen Betrachtungen als Arbeitshypothese in vielen Spezialfallen Vorteile abwerfen wird. Immerhin sind wir vollig iiberzeugt, dass es notwendig sein wird, die angewendeten Begriffe auf eine festere, physikochemische Basis zu stellen. Dabei unterliegt es kaum einem Zweifel, dass die von uns be- nutzte freie Affinitat des Protoplasmas zu Wasserstoff oder zu Sauer- stoff ihren quantitativen Ausdruck finden wird in dem Oxydoreduk- 260 DIE EINHEIT IN DER BIOCHEMIE tionspotential des Protoplasmas. Angesichts der Tatsache, dass in jiingster Zeit ein erfolgreicher Versuch gemacht worden ist, dieses Potential fiir das Protoplasma einer lebenden Zelle zu bestimmen,*4 offmet sich die Moglichkeit, zahlenmassig zu entscheiden, ob eine Zelle imstande sein wird, eine bestimmte gekuppelte Dehydrierung und Hydrierung zu bewirken. Wenn ausserdem einmal die Oxydo- reduktionspotentiale fiir die Systeme Substrat-dehydriertes Substrat und Akzeptor-hydrierter Akzeptor in geniigenden Fallen festgestellt worden sind, wird man auch dazu kommen k6nnen vorauszusagen, ob ein bestimmtes einfaches Substanzsystem fiir die betreffende Zelle eine passende Nahrung bieten wird oder nicht. In diesem Zusammenhang wird auch das nahere Studium der an- organischen wasserstoffiibertragenden Katalysatoren zweifellos wich- tige Anhaltspunkte liefern k6nnen. Die Wielandschen Versuche leh- ren, dass das Palladium in seinen oxydoreduktiven Eigenschaften dem Protoplasma vieler aeroben Bakterien (u.m. Essigbakterien) sehr nahe steht. Obgleich eine typische Vergérung des Zuckers mit Hilfe anorga- nischer Katalysatoren wohl ausgeschlossen sein wird, da bei den bio- logischen Vergarungen die Bildung und Spaltung der Phosphorsaure- ester unentbehrlich scheint, werden die rein oxydoreduktiven Teil- prozesse sich zweifellos alle mit anorganischen Katalysatoren repro- duzieren lassen. Dies ist ja auch heute schon weitgehend der Fall. Wie dies auch sein mége, fest steht wohl, dass die mit Riicksicht auf die aeroben Atmungsprozesse gemachte Aussage Thunbergs: “dass der Wasserstoff als das elementare, gemeinsame Brennmaterial der Zellen zu betrachten ist’ in seiner Tendenz noch viel zu beschrankt ist, und dass in den kommenden Jahrzehnten der Wasserstoff in seinen verschiedenen Aktivierungsstadien im Mittelpunkte der ganzen bio- chemischen Forschung stehen wird. 261 OO Nm Il. 12. 13. 14. 15. 16. 17, 18. 19. SELECTED PAPERS NOTEN UND LITERATUR C. OPPENHEIMER, Die Fermente und ihre Wirkungen, 5 Aufl., Leipzig 1926. Cc. NEUBERG und C. OPPENHEIMER, Biochem. Z. 166, 450, 1925. a.a.O., 5.1213. In Ubereinstimmung hiermit ist die in deutscher Sprache mehr iibliche Unter- scheidune in Betriebsstoffwechsel und Baustoffwechsel. So z.B.: J. H. QUASTEL, Biochem. J. 78, 365, 1925; C. NEUBERG und F. WIN- pDiscH, Biochem. Z. 166, 454, 1925. Vergleiche z.B.: 0. MEYERHOF, Naturwissenschaften 7, 253, 1919; Ders., Ber. deutsch. chem. Ges. 58, 991, 1925; 8S. KOSTYTSCHEW, Z.f. physiol. Chem. 177, 141, 1920; M. SCHOEN, Bull. de l’Inst. Pasteur 24, 1, 1926. — Eine nahere experi- mentelle Begriindung lieferten F. HAYDUCK und H. HAEHN, Biochem. Z. 128, 586, 1922. ; Dass man heute das Adjektiv ‘fermentativ’ noch 6fters gebraucht als Synonym fiir ‘enzymatisch’, bringt keine Anderung in unsere Anschauung. Dass man eben dazu iibergegangen ist, die Begriffe ‘Enzym’ und ‘enzymatisch’ einzu- fihren, beweist schon, dass man gefiihlt hat, dass die alteren Begriffe ‘Ferment’ und ‘fermentativ’ in genannter Hinsicht verwirrend wirken. Es eriibrigt sich wohl, hier die betreffende umfangreiche Literatur vollstandig anzufithren. Es mége nur verwiesen werden auf die Ubersicht in Oppenheimer, ‘Die Fermente’, und weiter auf die folgenden zusammenfassenden Darstellungen von WIELAND selbst: ‘Ergebnisse der Physiologie’ 20, 477, 1922; Ber. deutsch. chem. Ges. 55, 639, 1922 und auf das von WIELAND bearbeitete Kapitel in Oppenheimers ‘Handbuch der Biochemie’, 2. Aufl., Bd. IT, S. 252, 1923. W. PALLADIN, Biochem. Z. 18, 151, 1909; Id. 27, 442, 1910; Id. 35, 1, 1911; Id. 49, 381, 1913; Id. 60, 171, 1914. G. BREDIG und F. SOMMER, Z. f. physik. Chem. 70, 34, 1910; G. BREDIG, Ber. deutsch. chem. Ges. 47, 546, 1914. Fir die Warburgschen Ansichten vergleiche man seine diesbeziiglichen Ab- handlungen in Biochem. Z. 119, 1921 bis heute. Herr Professor Oppenheimer war so freundlich zu berichten, dass er unsere Separata erst empfangen hatte, als die betreffenden Abschnitte schon im Druck fertig standen. OPPENHEIMER, a.a.O., S. 1214. Wie bei der biochemischen Phosphorylierung vorangehend an den Zucker- zerfall; vergl. A.J. KLUYVER und A. P. STRUYK, Versl. Kon. Akad. v. Wet. Amsterdam 35, 177, 1926. A. HARDEN, J. Chem. Soc. 79, 612, 1901. Beispiele hierfiir findet man u.a. bei A. Kirow, Untersuchungen zur Butter- sduregarung, Ref. Centr. f. Bakt. Parasitenk. II. Abt., 37, 534, 1912. T. BAUMGARTEL, Grundriss der theoretischen Bakteriologie, Berlin 1924. E. C. GREY, Proc. Royal Soc. Ser. B. 87, 472, 1914; siehe auch E. C. GREY, Ibid. go, 92, 1919. Es eriibrigt sich hier wohl, eine vollstandige Literaturangabe dieser allgemein 262 20. 215 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. $2. DIE EINHEIT IN DER BIOCHEMIE bekannten Abhandlungen zu geben. Bis 1923 findet man sie in tibersichtlicher Weise wiedergegeben bei A. Harden, Alcoholic Fermentation, 3rd Ed., Lon- don 1923. Fir diese Ansicht war auch friiher Kostytschew eingetreten. Vergleiche S. KOSTYTSCHEW, Z. f. physiol. Ch2m. 79, 130, 1912; Ibid. 79, 359, 1912. Cc. NEUBERG und F.F.NORD, Biochem. Z. 96, 133, 1919; C. NEUBERG, F. F. NORD und E. WOLFF, Ibid. rr2, 144, 1920; C. NEUBERG und B. ARINSTEIN, Ibid. 117, 269, 1921; H. KUMAGAWA, Ibid. 137, 157, 1922. Ein Vorlaufer ist E. c. GREY, der 1913 schon die Bildung von Azetaldehyd bei der von B. coli bewirk- ten Garung nachwies (Biochem. J. 7, 359, 1913). W. H. PETERSON and E. B. FRED, J. Biol. Chem. 44, 29, 1920. W. C. DE GRAAFF und A. J. LE FEVRE, Biochem. Z. 155, 313, 1925. Ausnahmsweise wird auch Fruktose als Substrat beriicksichtigt werden. Vergleiche hierzu die folgenden Arbeiten iber den Chemismus der oxydativen Verarbeitung von Zucker durch Schimmelpilze: J. N. CURRIE, J. Biol. Chem. 31, 15, 1917. M. MOLLIARD, C.R. Soc. Biol. 82, 719, 1919; Ibid. 83, 479, 1920: Ibid. 87, 967, 19223; Ibid. 90, 13995, 1924; C. R. Acad. d..Sci. 174, 881, 1922. W. BUTKEWITSCH, Biochem. Z. 129, 445, 1922; Ibid. 129, 455, 1922; Ibid. 129, 464, 1922; Ibid. 137, 327, 1922; Ibid. 131, 338, 1922; Ibid 132, 556, 1922; Ibid. 136, 224, 1923; Ibid. rg2, 195, 1923; Ibid. 154, 177, 1924. Idem, Jahrb. f. wis- sensch. Botanik 64, 637, 1925. R. FALCK und VAN BEYMA THOE KINGMA, Ber. deutsch. chem. Ges. 57, 915, 1924. R. FALCK und s. w. KAPUR, Ibid. 57, 920, 1924. K. BERNHAUER, Biochem. Z. 153, 517, 1924. C. WEHMER, Ber. deutsch. chem. Ges. 58, 2616, 1925. R. SCHREYER, Ber. deutsch. chem. Ges. 58, 2647, 1925. Fiir den weiteren Abbau des Zuckers uber das Glukonsaurestadium hinaus durch Essigbakterien verweisen wir nach: A.J. KLUYVER und F. J. G. DE LEEUW, Die deutsche Essigindustrie 29, 175, 1925; und F. VISSER ’T HOOFT, Biochemi- sche onderzoekingen over het geslacht Acetobacter, Diss. Delft 1925. A. HARDEN and wW. J. YOUNG, Proc. Royal Soc. Ser. B. 77, 405, 1906. G. EMBDEN und F. LAQUER, Z. physiol. Chem. 93, 94, 1914; Ibid. 98, 181, 1917; Ibid. 173, 1, 1921. A. I. VIRTANEN, Z. physiol. Chem. 138, 136, 1924. A. I. VIRTANEN, Comment. physico-mathematicae Societas Scientiarum Fen- nica II, Nr. 20, 1925. In seiner zuletzt zitierten Abhandlung hat Virtanen jedoch wahrscheinlich ge- macht, dass in den Fallen, in denen die normalerweise stattfindende biochemi- sche Phosphorylierung durch Mangel and Ko-Enzym nicht eintreten kann, bei den Propionsaurebakterien auch eine direkte Spaltung des Glukosemolekiils in Verbindungen der C,- und der C,-Reihe auftritt. Diese Spaltung sollte nach Virtanen auch im normalen Stoffwechsel der genannten Bakterien als Neben- prozess stattfinden. A. J. KLUYVER und A. P. STRUYK, a.a.O. Fur die Dokumentierung der oben- gegebenen Auffassung mége auf diese Abhandlung verwiesen werden. J. H. NORTHROP, L. H. ASHE and R. R. MORGAN, J. Ind. Eng. Chem. 11, 723, 263 33- 34- 35: 36. 37: 38. 59: 40. 41. 42. 43- 44. 45: 46. 47: 48. SELECTED PAPERS 1919. J. H. NORTHROP, L. H. ASHE and K. J. SENIOR, J. Biol. Chem. 39, 1, 1919. C. F. ARZBERGER, W. H. PETERSON and E. B. FRED, J. Biol. Chem. 44, 465, 1920. W. H. PETERSON, E. B. FRED and J. H. VERHULST, J. Ind. Eng. Chem. 13, 757, 1921. E. B. FRED, W. H. PETERSON and J. A. ANDERSON, Ibid. 15, 126, 1923. Vorlaufige Publikation in: Tijdschr. v. Vergl. Geneesk. 1924. Ausftihrliche Publikation erscheint demnachst. A. HARDEN, J. Chem. Soc. 79, 612, 1901. A. HARDEN and D. NORRIS, Proc. Royal Soc. Ser. B. 84, 492, 1912. E. C. GREY, Proc. Royal Soc. Ser. B. 87, 461, 1914; Ibid. 87, 472, 1914; Ibid. go, 75, 1919; Ibid. go, 92, 1919; Ibid. 97, 294, 1920. JAN sMIT, Bacteriologische en chemische onderzoekingen over de melkzuur- gisting, Diss. Amsterdam 1913. W. H. PETERSON and E. B. FRED, J. Biol. Chem. 47, 431, 1920; Ibid. 42, 273, 1920; Ibid. 42, 175, 19203; Ibid. 44, 29, 1920; Ibid. 48, 385, 1921; Ibid. 53, 111, 1922; Ibid. 64, 643, 1925. A. I. VIRTANEN. Societas Scientiarum Fennica, Comment. Physico-Mathemati- cae 1, Nr. 36; 1923; Ibid. 2, Nr. 20; 1925. Siehe auch: J. M. SHERMAN, J. Bact. 6, 379, 1921. A. I. VIRTANEN, a.a.O. Ausser verschiedenen technisch wichtigen Publikationen sind hier zu erwahnen H. B. SPEAKMAN, J. Biol. Chem. 47, 319, 1920; Ibid. 43, 401, 1920; Ibid. 58, 395, 1923; H. B. SPEAKMAN and J. F. PHILIPS, J. Bact. g, 183, 1924; J. REILLY, W. J. HICKINBOTTOM, FR. R. HENLEY and A. GC. THAYSEN, Biochem. J. 14, 229, 1920; W. H. PETERSON, E.B. FRED and L.G. SCHMIDT, J. Biol. Chem. 60, 627, 1924. Ausfiihrliche Publikation erscheint demnachst. Nur die hydrolytischen Vorgange und Esterifikationen fallen — wie schon mehr- mals bemerkt — aus diesem Schema. Bei der Zuckerdissimilation begegnen uns nur die Bildung der Hexosemonophosphorsaureester und die damit zusammen- hangende Spaltung der ‘Triosemonophosphorsaureester. E. F. ARMSTRONG and T. P. HILDITCH, Proc. Royal Soc. Ser. A. 96, 322, 1920. Vergl. auch N. ZELINSKY und N. GLINKA, Ber. 44, 2305, IQII. Eine Ubersicht von Béesekens ‘Dislokationstheorie’ findet man in: Rec. trav. chim. 39, 623, 1920; und Proc. Royal Acad. Amsterdam 25, 210, 1922. Die Grundlagen der fiir unsere Betrachtungen ebenfalls sehr wichtigen Anschau- ungen von Prins findet man in seiner Dissertation ‘Bijdrage tot de kennis der katalyse’, Delft 1912. Speziell diirfte auch Erwahnung finden seine Abhandlung iiber den Zusammenhang zwischen Katalyse und Affinitat in Chem. Weekblad 14, 63, 1917. Fir die heterogene Katalyse im allgemeinen vergleiche man die rezente Zu- sammenfassung von E. F. ARMSTRONG und T. B. HILDITGH, Chem. Ind. 44, 701, 1925 und auch die Arbeit von H. s. TAYLOR, J. phys. Chem. 28, 897, 1925. Es moége sogleich bemerkt werden, dass dieser Fall wahrscheinlich vorliegt bei der Dehydrierung von Ameisensaure von Bakterien der Koligruppe. WIELAND, a.a.O. 264 49. 50. 51. 52. 53: 54- OT: 58. 60. hit DIE EINHEIT IN DER BIOCHEMIE Vergleiche hierzu den nachsten Teil. Siehe hierfiir: H. J. PRINS, Rec. trav. chim. 42, 473, 1923. Einen analogen Fall findet man bei der Verseifung verschiedener Ester, die sowohl von Wasserstoffionen wie von Hydroxylionen beschleunigt wird. In saurem Medium ist jedoch nur die Wirkung der Wasserstoffionen, in alkali- schem Medium nur die Wirkung der Hydroxylionen von praktischer Be- deutung. Die Frage, ob man hierbei an das Protoplasma selbst oder an irgend ein den Wasserstoff iibertragendes Enzym zu denken hat, ist in diesem Zusammenhang ganz bedeutungslos. Die wichtigen Versuche von Warburg, Meyerhof, Rubner u.a., aus welchen hervorgeht, dass Atmung und Garung in hohem Grade an die Struktur der Zelle gebunden sind, berechtigen uns ganz allgemein von Proto- plasma zu sprechen. Bull. Soc. Chim. Biol. 6, 326, 1924. Vergl. die neueren Arbeiten von w. RUHLAND, Ber. deutsch. bot. Ges. go, 180, 1922 und G. GROHMANN, Centralbl. Bakt. Parasitenk. II Abt. 67, 256, 1924. So werden in der systematik der ‘Society of American Bacteriologists’ die wasserstoffoxydierenden Bakterien noch in ein Genus: Hydrogenomonas ver- eint. Vergl. BERGEY’s Manual of Determinative Bacteriology, Baltimore 1923. F. VISSER ’T HOOFT, Biochemische onderzoekingen over het geslacht Acetobac- ter. Diss. Delft 1925. Bei der ersten Gruppe ist dies durch die Untersuchung von CLARA COHEN, Bio- chem. Z. 112, 139, 1920 und von Cc. NEUBERG und F. F. NORD, Biochem. Z. 96, 158, 1919 ebenfalls nachgewiesen. Fir die sonstigen Gruppen ist die betreffende Literatur schon angefuhrt. Cc. NEUBERG und F. WINDISCH, Biochem. Z. 166, 454, 1925. Nach den obigen Ausfithrungen braucht es wohl keiner Erlauterung, dass wir nicht mit der Auf- fassung der Autoren dieser interessanten Arbeit einverstanden sind, dass diese Dismutation des Azetaldehyds auch bei Anwesenheit anderer kraftiger Wasser- stoffakzeptoren, wie Luftsauerstoff, also bei der normalen Essigsaurebildung, stattfinden wird. Wir kommen hierauf im 7. Teil zuriick. Dieser Vorgang liegt zweifellos vor bei den kraftig aeroben, fettbildenden Mikro- organismen. Vergl. hierzu: H. HAEHN und w. KINTTOF. Chem. Zell. Gew. 12, 157, 1925. Bedingung fir das Eintreten einer kraftigen Aldolkondensation, und damit auch fur die Fettbildung, ist offenbar, dass der Azetaldehyd jedenfalls teilweise durch kraftige Liiftung vor einer Akzeptorwirkung geschiitzt wird. Dies steht in vollkommener Ubereinstimmung mit den experimentellen Ergebnissen von Lindner, Haehn und Kinttof. Diese ‘karboligatische’ Wirkung der Hefe gegeniiber zugefiigtem Azetaldehyd ist zuerst von C. NEUBERG und E. REINFURTH, Biochem. Z. 143, 553, 1923; nachgewiesen worden. Fir die Dokumentierung obiger Anschauung vergleiche man: A. J. KLUYVER, H. J. L. DONKER und F, VISSER ’T HOOFT, Biochem. Z. 167, 361, 1925. 265 62. 63. 64. 65. 66. 67: 68. 78. oe 80. 81. 82. SELECTED PAPERS Nach unver6ffentlichten Untersuchungen des einen von uns (D.) N. L. SOHNGEN, Centralbl. Bakt. Parasitenk. II Abt. 37, 595, 1913. Vergl. z.B.: F. VISSER ’T HOOFT, Diss. a.a.O. Man siehe z.B.: W. MANSFIELD CLARK, The Determination of Hydrogen Ions, Baltimore 1923, Chapter XVI. Vergl. hierfiir: s. KostyTscHEW, Die Pflanzenatmung, Berlin 1924, S. 89. O. MEYERHOF, Pfliigers Archiv. f. d. ges. Physiol. 164, 353, 1916; ebenda, 165, 229, 1916; ebenda, 166, 240, 1917. Ganz vollstandig korrekt ist die gegebene Erklarung nicht, da vor allem die Vermehrungs- und weniger die Dissimilationsvorgange bei diesen Bakterien durch die Zufiigung organischer Substanzen verhindert werden. Dies bedeutet, dass eben die Kohlensaure, welche nur nach Aktivierung durch das Protoplasma als Akzeptor auftreten kann, von der fest an das Protoplasma gebundener orga- nischer Substanz verhindert wird, sich an das Protoplasma anzulagern. Ver- gleiche hierfiir den nachsten Teil. W. BAVENDAMM, Die farblosen und roten Schwefelbakterien. Jena 1924. L. G. M. BAAS BECKING, Annals of Bot. 39, 613, 1925. Diese Frage konnte hier nur kurz angedeutet werden. Die weitere wichtige Literatur auf diesem Gebiete — besonders die sch6nen Arbeiten Buders — miissen hier unberiicksichtigt bleiben. Oder Schwefel. Vergleiche Beijerincks: Thiobacillus denitrificans. M. W. BEIJERINCK und D. GC. J. MINKMAN, Centralbl. Bakt. Parasitenk. IT Abt. 25, 30, IQIO. H. WIELAND in: Oppenheimers Handbuch der Biochemie, 2. Aufl. Jena 1923, Bd. 2, S. 265. P. NAWIASKY, Archiv. f. Hyg. 66, 209, 1908. Fir die sehr verbreitete oxydative Dissimilation der Aminosduren siehe man den nachsten Teil. . Jedoch gibt es auch hier Ausnahmen. So scheiden die Butylalkoholbakterien in der ersten Garungsphase z.b. Buttersdure in das umgebende Medium ab. Nachdem sich diese Saure hierin angehauft hat wird sie in der zweiten Garungs- phase wieder in den Stoffwechsel bezogen und in Butylalkohol iibergefiihrt. Vergleiche die im 2. Teil bei der genannten Gruppe angefiihrte Literatur. Dies hindert nicht, dass die Dissimilationsprozesse gegeniiber den Assimilations- prozessen sowohl in materieller wie in energetischer Hinsicht meistens bedeu- tend vorherrschen, was vor allem auf Konzentrationsverhaltnisse der in der Zelle anwesenden Substanzen zuriickzufihren ist. Vergleiche hierzu: T. THUNBERG, Svensk. Kemisk Tidskr. 145, 1923. Zitiert nach Wochenschr. f. Brauerei 47, 33, 1924. G. KLEIN und 0. WERNER, Biochem. Z. 168, 361, 1926. H. HAEHN und w. KINTTOF, Chem. Zell. Gew. 12, 115, 1925. F. KNOOP und HUBT. OESTERLIN, Z. physiol. Chem. 148, 294, 1925. Absichtlich ist hier der in den letzten Jahren von O. Meyerhof verkiindigte An- tagonismus zwischen Atmung und Garung ausser Betracht gelassen. Dass die Muskelzelle nach Wiederherstellung der zeitweise unterbrochenen Aerobiose 266 DIE EINHEIT IN DER BIOCHEMIE die angehaufte Milchsaure teilweise in Glykogen riickverwandelt, ist nur ein sehr auf der Hand liegender Spezialfall der Bildung eines typischen Assimilations- produktes. Auch die Garung ist zur Bildung derartiger Assimilationsprodukte durchaus befahigt und ein prinzipieller Gegensatz zwischen Atmung und Garung ist hierin nicht zu sehen. Wir hoffen auf diese Frage spater zuriickzu- kommen. 83. a.a.O. 84. J. NEEDHAM and D, NEEDHAM, Proc. Royal Soc. Ser. B. 98, 259, 1295. 267 QUELQUES REMARQUES SUR LA CLASSIFICATION DES LEVURES Cr n’est que pour vous donner quelqwidée du travail du ‘Cen- traalbureau voor Schimmelcultures’, institution subventionnée par la Section de Botanique de notre union, que j’ose attirer votre attention sur quelques remarques inévitablement superficielles concernant la classification des levures. L’apparition récente d’une publication assez volumineuse de la plume de Mme Stelling-Dekker [1931] sur les levu- res sporogénes semble justifier un bref apercu de ce qui a été fait dans le secteur des levures du bureau pendant les derniéres années. Pour ceux qui ne connaissent pas le catalogue du ‘Centraalbureau’, il faut observer que la collection du bureau, grace a une coopération internationale remarquable, comprend un trés grand nombre de cul- tures pures de levures. C’est bien ce fait qui nous a décidé entreprise trés audacieuse d’entamer une révision critique de la classification des levures. Car il sera superflu d’insister sur le fait que la classifica- tion de ce groupe de microorganismes, qui par son importance pratique a attiré attention de tant de personnes d’une éducation scientifique limitée, se trouve encore dans un état plus ou moins chaotique. Tout d’abord, il me semble indiqué de m/arréter un instant a la question ‘qu’est-ce qu’une levure’? Le mot vulgaire ‘levure’ ne laisse aucun doute sur son origine: depuis un temps immémorial on a donné le nom ‘levure’ au principe qui faisait lever les substances exposées a une fermentation alcoolique. C’est au génie de Pasteur que nous de- vons la connaissance de la nature biologique de ce principe et les re- cherches ultérieures ont bientét montré la pluralité des microbes qui sont responsables du processus de la fermentation alcoolique. En outre une étude plus approfondie prouvait bientdt existence de champig- nons étroitement reliés a la levure de la vinification au point de vue morphologique et qui pourtant étaient dépourvus de la propriété de faire fermenter les sucres. C’est ainsi qu’on accepte aujourd’ hui l’exis- tence d’un groupe étendu de champignons auxquels le nom vulgaire ‘levure’ est applicable. 268 QUELQUES REMARQUES SUR LA CLASSIFICATION DES LEVURES Conformément a cet exposé M. Guilliermond, autorité éminente sur ce groupe de microbes, a donné la définition suivante: ‘On entend par levure, au sens botanique, tout champignon unicellulaire quelles que soient ses proprictés biochimiques, de forme ovale ou sphérique, qui se multiplie par bourgeonnement’. Toutefois, il me faut observer que cette définition ne peut nous satis- faire complétement, car nous devons a M. Lindner, de Berlin, et a feu mon prédécesseur Beijerinck la découverte de plusieurs espéces de champignons qui ont tous les caractéres des levures mentionnées jus- qu’ici, avec la seule exception qu’ils ne se multiplient pas par bourge- onnement, mais par cloisonnement transversal. A premiére vue on pourrait croire qu il sufhrait de modifier simple- ment la définition donnée par Guilliermond en ajoutant les mots: ‘ou par cloisonnement transversal’ pour ladapter a la découverte des Schizosaccharomyceétes. Mais en y réfléchissant, c’est clair que ce n’est pas ainsi, car l’addition des mots ‘ou par cloisonnement transversal’ comporterait que la grande majorité des bactéries satisferait a la défi- nition de Guilliermond. J espére vous démontrer tout a ’heure que c’est justement cette difficulté de bien délimiter le groupe de levures qui est responsable de beaucoup de méprises. D’abord, il me semble désirable d’essayer par une autre voie d’arri- ver a une définition scientifique de la notion ‘levure’. La seule procé- dure praticable me parait, de partir des propriétés d’une espéce de levure incontestable et de répondre alors a la question, quels organis- mes montrent des affinités suffisamment étroites pour les incorporer a lespéce initiale dans le méme groupe naturel. C’est tout indiqué de choisir comme espéce initiale ’organisme qui provoque la fermentation spontanée du jus de raisins. Des centaines de recherches nous ont appris que cette expérience méne presque tou- jours au développement prépondérant d’une espéce de levure connue depuis longtemps sous le nom scientifique de ‘Saccharomyces ellipsoideus . Que savons nous maintenant des propriétés de cette levure? D’a- bord on peut observer que c’est un organisme qui se conforme bien a la définition donnée par Guilliermond. Mais il est évident que cette formule ne suffit pas pour établir la place qu’il faut lui assigner dans la classification des champignons. I] est donc d’une extréme importan- ce que nous devons a Reess la découverte de l’existence d’un vrai cycle 269 SELECTED PAPERS évolutif de la levure de vin. C’est Reess qui a observé pour la premiére fois que cette levure ne se multiplie pas seulement par bourgeonne- ment, mais que dans certaines conditions la levure de vin donne lieu a la formation d’un sporange, dont les spores a leur tour sont capables de se transformer de nouveau en cellules levures typiques. Les belles recherches morphologiques et cytologiques de Guillier- mond montrent indubitablement que ces sporanges sont parfaitement les homologues de l’asque des Ascomycetes, en d’autres termes, que la levure de vin appartient a cette classe de champignons. C’est ainsi que la question de la délimitation du groupe des levures s'est réduite a cette question: quels champignons appartenant a la classe des Ascomycetes sont suffsamment étroitement reliés a la levure de vin pour les rassembler dans un seul groupe naturel, celui des levures? Ce sont encore les études admirables de Guilliermond qui nous ont montré le chemin pour répondre a cette derniére question. I] résulte de ces recherches que plusieurs espéces du genre Endomyces ne different de certaines espéces de levures typiques que par le fait qu’elles mon- trent aussi par la forme levure une forme mycélienne typique, ou plutét que les formes levures ne sont que des conidies qui se multiplient par bourgeonnement. A leur tour ces espéces d’Endomyces montrent une relation étroite avec le champignon décrit par Mlle Stoppel sous le nom d’Eremascus fertilis dont la multiplication végétative cependant s'est restreinte a la forme mycélienne. Mais il y a plus. On connait aussi d’autres espéces d’Endomyces qui sont également reliées a lEremascus fertilis mais qui se distinguent des espéces mentionnées d’abord par le fait qu’on n’y rencontre jamais la formation de conidies. Au contraire ces derniéres espéces sont carac- térisées par le fait que leur mycélium se divise souvent en particules, en d’autres termes, il y a formation d’oidies. Il est évident que ce sont ces espéces dont on peut dériver en accentuant cette propriété ce qui méne a la réduction totale de la forme mycélienne, les espéces de le- vure, dont j’ai parlé plus haut, a savoir celles qui se multiplient par cloisonnement transversal. Ce dernier résultat nous permit de définir le groupe naturel des levures comme un sous-groupe d’ Ascomycetes, caractérisé d’un coté par la prépondérance plus ou moins absolue de l’existence unicellulaire et de l’autre par sa relation évidente avec 1’ Eremascus fertilis. 270 QUELQUES REMARQUES SUR LA CLASSIFICATION DES LEVURES Il faut ajouter qu’on connait d’ailleurs de nombreuses espéces qui dans leurs propriétés montrent une concordance frappante avec les levures ainsi définies, mais qui se séparent jusqu’a présent par le simple fait qu’elles sont incapables de produire des ascospores. II est évident qu’il convient de ranger ces derniéres levures parmi les funge imperfecti de situation douteuse dans le systéme naturel. Ici je ne m’occuperai plus de ces levures imparfaites. Vous vous demanderez peut-étre pourquoi j’insiste si fort a préciser la notion ‘levure’. Je réponds a cette question que l’expérience nous montre que ces idées — quoiqu’elles datent déja de plus de vingt ans — ne sont pas généralement répandues méme parmi les travailleurs dans ce domaine de la mycologie. Aussi n’est-il pas rare qu’on décrive comme étant de nouvelles espéces de levures des microbes qui en réa- lité sont trés éloignés d’elles. Dans les derniéres années le Centraal- bureau a eu deux fois l’occasion de fixer l’attention sur de telles er- reurs. D’abord il s’agissait d’un organisme deécrit par le Docteur Bene- dek, de Leipzig, sous le nom de Schizosaccharomyces hominis. La décou- verte de cette espéce semblait intéressante entre autre parce que ce serait le premier exemple d’un Schizosaccharomyceéte habitant les régions tempérées. Toutefois une étude faite par Mlle Dorrepaal [1930] au laboratoire du Centraalbureau apprit bientot que Porganisme de Benedek avait bien une ressemblance morphologique superficielle avec les Schizosaccharomycétes, mais qwil n’y avait aucun doute que cet organisme n’appartient point aux Ascomycetes. Au contraire, il était évident qu’en réalité le Schizosaccharomyces hominis était une bac- térie banale voisine du Bacillus megatherium de Bary. Le second cas, encore plus récent, est parfaitement analogue. Seule- ment ici il ne traitait pas exclusivement de la création erronée d’une nouvelle espéce, mais méme d’un nouveau genre de levures. Ici je fais allusion a la création du genre Schizotorulopsis par Ciferri, savant d’ail- leurs bien connu par d’autres études approfondies sur les levures aspo- rogenes. La recherche faite par Mlle Verkaik au laboratoire du Cen- traalbureau sur la seule espéce du nouveau genre: Schizotorulopsis al- fonsecai Ciferri établit aussi avec certitude que cet organisme n’était qu’une espéce de bactérie, remarquable par ses grandes dimensions sur certains terrains de culture. Ici la preuve de la nature bactérienne de Porganisme est spécialement victorieuse, parce que Mlle Verkaik [1931] réussit a démontrer que lorganisme est pourvu de motilité, si on le 271 SELECTED PAPERS cultive dans un milieu approprié. Conformément a cette observation la présence de fouets pouvait étre prouvee. Jai insisté sur ces deux exemples, parce qu’ils montrent combien il est important d’avoir des notions claires sur la délimitation du groupe des levures, car il faut noter que le Schizosaccharomyces hominis autant que le Schizotorulopsis alfonsecat se conforment trés bien a la définition courante donnée par Guilliermond, du moins quand on y apporte Pamendement inévitable sur la multiplication par cloisonnement trans- versal. La tache que Mme Stelling s’était posée, était donc un examen cri- tique de toutes les cultures de levures sporogénes présentes dans la collection du Centraalbureau. Le nombre des espéces représentées, ré- pandues sur 19 genres, se montait a 159 ou 73°, du nombre des espé- ces, dont la description pouvait étre trouvée aprés une enquéte minu- tieuse de la littérature. Le but des recherches de Mme Stelling était double: d’abord il fallait examiner si les propriétés des cultures se conformaient aux des- criptions originales données par les auteurs. Mais en vue de la témé- rité avec laquelle un grand nombre de ces auteurs ont proposé de nouvelles espéces pour les levures isolées par eux il était aussi indispen- sable de se rendre compte si l’existence d’une telle espéce était suffi- samment justifiée. Le tableau peut donner quelque idée des grandes lignes de la classi- fication que Mme Stelling a cru bon d’accepter pour le moment. Vous y retrouverez les pensées qui ont conduit M. Guilliermond dans ses considérations sur la phylogénie des levures. Sans me perdre dans les détails un bref exposé de la planche me semble étre utile. D’abord ensemble des levures sporogénes est réunie en une seule famille, celle des Endomycetaceae. Une séparation nette entre celle-ci et une seconde, a laquelle les levures dans un sens plus limité apparte- naient: la famille des Saccharomycetaceae, nous a paru impraticable. Néanmoins, il convient de distinguer dans la famille des Endomyce- taceae quatre sous-familles. D’abord celle des Eremascoideae avec le seul genre Evremascus. Cette sous-famille est caractérisée — comme nous Pavons déja observé — par l’absence d’autres formes végétatives que la forme mycélienne. Dans I’Evemascus donc il n’y a pas de conidies- levures et pas d’oidies. La seconde sous-famille est celle des Endomy- 272 QUELQUES REMARQUES SUR LA CLASSIFICATION DES LEVURES coideae. La, on trouve deux genres qui se distinguent par le fait que dans le premier: Endomyces on trouve a coté de la forme mycélienne la forme oidie, tandis que dans le genre Schizosaccharomyces ce n’est que la forme oidie qui subsiste. La troisiéme sous-famille nous montre une situation analogue. Ici on trouve encore un genre dans lequel la forme mycélienne subsiste: c’est le nouveau genre Endomycopsis. Mme Stelling s’est décidée de réunir a ce genre toutes les espéces d’Endomyces qui en contraste avec l’espéce typique de ce genre, a savoir Endomyces decipiens. Ce sont la les espéces qui sont indiquées par Guilliermond comme étant les an- cétres des vraies levures bourgeonnantes. Quant a ce groupe, qui comprend la majorité des espéces de levures décrites jusqu’a ce jour, il convient de distinguer encore deux sous- groupes. Tandis que pour la plupart des levures chaque partie de la membrane de la levure peut figurer comme point de départ pour la formation des bourgeons, quelques genres sont caractérisés par le fait que le bourgeonnement des cellules ovales s’accomplit exclusivement aux poles. Cette particularité donne a ces levures un aspect typique auquel il faut aussi attribuer quelque valeur au point de vue de la classification. J’abuserais trop de votre indulgence en faisant la revue de tous les genres des tribus des Saccharomyceteae et des Nadsonteae. Je me bornerai donc a faire encore quelques remarques générales sur les caractéres qui se prétent a la délimitation des genres et des espéces. Vous aurez observé que pour les grandes lignes de la classification les proprictés morphologiques externes: forme mycélienne, forme oidie, forme levure et type de bourgeonnement, nous ont sufi. Cependant il va sans dire que tous les genres ainsi délimités ont un caractére commun: celui de produire des ascospores. Aussi est-il facile a com- prendre que les particularités du mode de formation des asques auront aussi une grande importance au point de vue de la classification. Pour ceci il faut distinguer entre la formation d’asques par partheé- nogénése et entre la formation d’asque précédée d’une copulation de deux cellules-levures. En outre pour les genres Torulaspora et Schwan- niomyces il est typique que les cellules qui se transforment en asques essairent de copuler au moyen d’un ou plusieurs tubes sans y jamais par- venir. Dans le cas d’une copulation précédente il convient de différen- cier encore entre la copulation isogamique et hétérogamique. 273 PTR, saoAWOTUURMYIgG o Of 0) “Oe saoAmodreqaq (a) G9 Booge — & C) es “pax pynussue yp] COLA) 79D @® “paxo “S*S RIYII VLA © 0) erytd CH i- ae 5 © | : Ua] “pO “pO snoseIptgv0r) vIuOSpeN vaodseynio J, 2 S Q9¢ 2. VE SOO § BCS A “Md & y esis f (3) oy, Cew © *PAXO “UO “PAxg “ULI viodsojeulaN viodsetuasurpy saohuoreyroesosA7Z sooAulorvyooesoOZztyIg a a S 6 ©) ce, Ee @ / /) ys § aT *s's sgOAUIOIRYIILG ws? y “pax vyfosodsouoyy sapooAWloreYyIILG saoAOIvYyIIVG sooAUopuy snoseulosy avotuOspeN ava}a0AUIOIeYIIVG svasdooAuiopuy avaproiodsoiewia Ny IvaIpIogAWIOILYIILG aeaprooAwopuy ICIPTOIS CUI S$ | | aeooea0AUIOpuy QUELQUES REMARQUES SUR LA CLASSIFICATION DES LEVURES Un cas spécial de la copulation hétérogamique est encore rencontré dans les levures du genre Nadsonia. Ici on trouve d’abord copulation entre une cellule-mére et une cellule-fille. Aprés l’accomplissement de la fusion l’oeuf forme au pole opposé au gaméte male un bourgeon dans lequel émigre son contenu et se transforme en un asque aun seul ascospore. Enfin la forme et aspect des ascospores — présence d’un globule oléagineux, a paroi lisse ou verruqueuse — présentent aussi des carac- teres utiles pour la différenciation des genres. Toutefois tous ces caractéres de nature morphologique ne suffisent pas dans tous les cas pour la délimitation des genres. On connait depuis longtemps des espéces qui ne montrent pas de différences essentielles au point de vue morphologique avec la levure de vin et d’autres espé- ces volsines, mais qui pourtant s’en distinguent nettement par leur conduite dans les milieux de culture liquides. Tandis que la plupart des espéces de levures cultivées dans une solution sucrée sont caracté- risées par un développement diffus, qui se termine par la formation d’un dépot, les especes mentionnées plus haut végétent sur une telle solution en formant un voile mycodermique. I] est évident que cette divergence est le symptéme d’une différence marquée dans le métabolisme des deux groupes. Apparemment les levures a formation de voile ont une tendance beaucoup plus accentuée a entrainer l’oxygéne libre dans leur métabolisme. Si lon considére que l'étude des diverses espéces de levure est étroite- ment hée a celle des transformations biochimiques provoquées par ces organismes, il n’y a pas lieu de s’étonner du fait que les caractéres physiologiques ont toujours eu une grande importance dans la classi- fication des levures. De méme, il me parait aussi qu’une telle procédure est entiérement justifiable. Puisque le métabolisme n’est qu’une réaction chimique du protoplasme vivant sur les substances nutritives présentes dans le mi- lieu de culture, il convient de considérer les différences en métabolisme comme désignant des différences en propriétés de la partie la plus im- portante de la cellule vivante: le protoplasme. I] est donc clair qu’on ne peut pas négliger dans la classification la grande difference qui existe entre les levures d’une dissimilation fer- mentative et celles d’une dissimilation préférablement oxydative qui se manifeste bien souvent dans la formation des voiles. 275 SELECTED PAPERS C’est ainsi que Mme Stelling, en accord d’ailleurs avec les auteurs précédents, a assuré la délimitation des genres Pichia et Hansenula avec les autres genres de la tribu des Saccharomyceteae. Enfin je dois faire la remarque que Mme Stelling s’est décidée de reunir en une quatriéme sous-famille, a savoir celle des Nematosporoi- deae, les trois genres, souvent considérés comme des levures douteuses: Monosporella, Nematospora et Coccidiascus. Jusqu’a ce jour aucune espéce de Monosporella et de Coccidiascus n’a jamais été cultivée. Ces genres étaient donc inaccessibles a un nouvel examen. Quant a Nematospora les recherches récentes de Guilliermond donnent Ja preuve définitive de la nature ascomycete des espéces de ce genre. En outre, les descrip- tions de la formation d’asque et de la forme aiguille ou fuseau des ascospores dans Monosporella et Coccidiascus semblent bien justifier la reunion de ces deux genres avec Nematospora en un seul sous-groupe des Endomycetaceae. Jusqu’ici je ne vous ai parlé que de la délimitation des genres. Ici la contribution de Mme Stelling se borne principalement a avoir donné des diagnoses plus achevées des genres déja créés par les tra- vailleurs antérieurs. ‘Toutefois le travail du Centraalbureau s’est dirigé principalement vers Pexamen critique de la validité des nombreuses espéces qui ont été décrites jusquici. Pour se faire une idée des difficultés quw’il fallait surmonter a ce sujet, il faut savoir que la plupart des mycologistes qui isolaient une levure dans des conditions un peu anormales ont cru avoir le droit de décrire cette levure comme ¢tant une nouvelle espéce. Bien souvent tout effort a manqué pour comparer la culture isolée avec des espéces déja exis- tantes. I] faut avouer que la description insuffisante et presque toujours in- cidentelle de la plupart des espeéces rendait une telle comparaison extrémement difficile sinon impossible. La situation était devenue peu a peu si chaotique que méme les auteurs les plus compétents étaient incapables de donner un apergu des différentes espéces réunies en un genre quelconque. I] fallait donc une recherche expérimentale comparative des diver- ses espéces en faisant usage de méthodes plus ou moins standardisées pour éliminer la confusion existante. La circonstance favorable que 72% des espéces décrites — et pour plusieurs des genres ce nombre était encore beaucoup plus élevé — 276 QUELQUES REMARQUES SUR LA CLASSIFICATION DES LEVURES étaient représentées dans la collection du Centraalbureau, a permis a Mme Stelling d’entamer cette recherche. Ce travail, dans lequel elle a été assistée par les demoiselles Dorre- paal et Verkaik n’a pas été en vain, ce qui résulte du fait que Mme Stelling donne dans sa monographie des arguments acceptables pour réduire le nombre des genres de 18 a 15 et le nombre des espéces étu- diées de 159 a 85. En outre, elle a signalé dans sa monographie plusieurs exemples qui démontrent la nécessité de transférer une espéce d’un genre dans un autre. Il en resulte que Mme Stelling peut donner pour chaque genre une clef dichotomique qui permet la détermination des diverses espéces du genre. Dés maintenant il est possible de donner une réponse plus exacte a la question s’il y a lieu d’identifier une levure nouvellement isolée avec une des espéces étudiées. Dans le cas opposé, on peut attendre que les diagnoses données par Mme Stelling faciliteront une délimi- tation exacte des nouvelles espéces. On peut méme espérer que le travail exécuté stimulera la découverte de nouvelles espéces aux ca- ractéres intéressants; dans l'état actuel tout mycologiste hésite a ajouter une nouvelle espéce au grand nombre, que personne ne savait distinguer avec certitude. D’autre part — et voici ce qui est encore plus important — l’ordre atteint aura un effet préventif contre la créa- tion de nouvelles espéces qui en réalité n’ont aucun droit d’existence. Qu’il me soit permis de faire encore quelques remarques sur les caractéres acceptés par Mme Stelling pour la délimitation des espéces. I] n’est pas étonnant qu’il faille donner encore plus d’attention aux propriétés biochimiques, car il ne faut jamais oublier que le rdéle des diverses levures dans la nature, ainsi que leur application industrielle, est déterminé en premiere ligne par le métabolisme de ces organismes. I] va sans dire qu’a cet égard le type de dissimilation, soit oxydative, soit fermentative, est d’une importance primaire. Mais ce probleme une fois résolu, if faut tenir compte du fait que les diverses levures montrent de grandes variations dans leur pouvoir d’attaquer les divers sucres. Le mycologiste qui voudrait classer une levure aura donc a répondre a la question générale: l’organisme posséde-t-il un pouvoir fermentatif et dans l’affirmative quels des sucres sont fermentescibles. Voila une question trés facilement posée, mais on se trouve bien embarrassé d’y répondre. 277 SELECTED PAPERS Pourtant ce point est trés important et il faut avouer que la non- chalance avec laquelle cette question a été traitée par la grande ma- jorité des savants est responsable en partie de la confusion existante. A ce point de vue, il est désirable d’examiner les méthodes en usage pour la détermination de la fermentescibilité d’un sucre. Toutes ces méthodes sont fondées sur le méme principe: on ense- mence une solution de sucre avec la levure et constate s’il y a évolu- tion de lacide carbonique. Toutefois en réfléchissant un peu sur ce critérium son insuffisance saute bientot aux yeux. Car il est évident que non seulement la dis- similation fermentative, mais aussi la dissimilation oxydative (la respiration) méne a la production de lacide carbonique. Il n’y a qu’une seule différence: c’est que leffet énergétique de la combus- tion intégrale d’un sucre surpasse plusieurs fois celui de la fermenta- tion de ce sucre. Ainsi la quantité du sucre transformé dans le cas de fermentation est toujours beaucoup plus grande que dans le cas de respiration. I] en est de méme pour la quantité de lacide carbonique produite dans les deux cas. Dans les levures respirantes cette quantité est toujours si minime que Vacide carbonique se dissout entiérement dans le milieu aqueux et il n’y a pas d’évolution visible de gaz. Dans le cas, au contraire, ot: la levure fait fermenter le sucre, la vitesse de la production de Pacide carbonique est généralement si grande que Pacide carbonique échappe du milieu a létat gazeux. Néanmoins faut-il conclure de ces considérations, que le résultat dune telle expérience dépendra d’un coté de la vitesse avec laquelle Pacide carbonique est produit par la levure, et de l'autre de la vitesse avec laquelle Pacide carbonique peut échapper du milieu par simple diffusion. En vue du fait qu’on se sert d’appareils trés divers pour examen de la fermentescibilité des sucres, il n’est pas étonnant qu’on rencontre bien souvent des résultats contradictoires dans la littérature. La situation est encore aggravée par la circonstance que le déve- loppement récent de la physiologie nous montre que le pouvoir fermen- tatif d’une cellule est loin d’étre un caractére absolu. Au contraire on sait qu’aussi des organismes d’une dissimilation oxydative prononcée, comme plusieurs des moisissures les plus ordinaires, sont capables de provoquer une fermentation alcoolique sous des conditions spéciales. Parmi ces conditions, Pabsence de l’oxygéne libre est probablement la plus importante. 278 QUELQUES REMARQUES SUR LA CLASSIFICATION DES LEVURES A premiére vue, il parait bien facile de tenir compte de cette der- niére circonstance. Mais ici une autre difficulté se présente. L’expé- rience nous a appris que les levures sont incapables de se développer sous des conditions strictement anaérobies. Alors si on ensemence un milieu anaérobie il ne se produira presque pas de développement de la levure et la quantité d’acide carbonique produite sera trés limitée a cause du petit nombre des cellules qui provoquent la fermentation. Bien souvent cette quantité ne dépassera pas celle qui peut étre dis- soute dans le milieu et on conclura a tort une absence de pouvoir fermentatif. Si au contraire on ensemence la levure dans un milieu auquel l’oxygeéne libre a accés, la levure peut se développer, mais dans ces conditions la respiration peut supprimer toute fermentation. Pour- tant c’est bien possible que la méme levure démontrera nettement son pouvoir fermentatif, si on rassemble une grande quantité de cellules dans un volume relativement petit d’un milieu sucré. Un exposé de application des principes énoncée a l’examen critique des méthodes usuelles demanderait trop de votre patience; pour cela il faut se référer au livre de Mme Stelling. Toutefois les considérations données suffront pour démontrer qu’avec des levures qui ont une dissimilation fermentative peu pro- noncée on ne peut pas attribuer beaucoup de valeur au résultat des experiences de fermentation. I] y a heureusement un grand nombre d’espéces qui ont un pouvoir fermentatif tellement accentué que loxygéne a faible concentration ne peut point supprimer la fermentation. Dans ce cas-ci il est d’une extréme importance d’observer l’action dune levure vis-a-vis des différents sucres. Le résultat d’une telle re- cherche constitue alors un caractére spécifique d’une grande impor- tance. L’ensemble des résultats obtenus par Mme Stelling présente quel- que intérét, car il en résulte que la distribution du pouvoir fermen- tatif des diverses levures vis-a-vis des différents sucres n’est pas tout a fait capricieuse, comme la plupart des auteurs a ce sujet nous laissent croire. Au contraire quelques régularités a cet égard sur lesquelles j’ai déja fixé Pattention en 1914 ont été bien confirmées par les recherches trés étendues de Mme Stelling. D’abord une levure ne fait jamais fermenter un sucre si elle ne fait pas fermenter la glucose. Deuxitmement pour toute levure qui fait 279 SELECTED PAPERS fermenter la glucose, la fructose et la mannose sont aussi fermen- tescibles. La troisieme régle est que jamais une levure n’est capable de faire fermenter toutes les deux, la maltose et la lactose. Aucun des nombreux exemples rapportés dans la littérature qui sont en contradiction avec ces régles n’a résisté a une inspection scru- puleuse. En revenant a la question de la classification des espéces, il faut en- core observer qu’aussi la maniére dont les différentes levures se com- portent vis-a-vis des milieux nutritifs synthétiques a fourni des données importantes. Quant aux résultats concrets obtenus dans la révision des espéces je ne veux que citer quelques-uns a titre d’exemple. En 1925 M. Redaelli a décrit sous le nom ‘Saccharomyces cavernicul@’ une nouvelle espéce isolée par lui des poumons d’une personne at- teinte de la tuberculose. Une inspection minutieuse de la culture authentique nous a appris cependant que cet organisme était parfaite- ment identique avec le Saccharomyces fragilis de Jorgensen, une levure trés répandue et qu’on presque toujours peut isoler du petit-lait. Récemment M. Nishiwaki a décrit une nouvelle levure pour la- quelle il a créé le nouveau genre Zygosaccharomycodes. La justesse d’une telle proposition a été déja contestée par M. Guilliermond qui a as- signé a cette levure une place dans le genre Zygosaccharomyces. En bon- ne harmonie avec cette opinion nos recherches ont montré lidentité du Zygosaccharomycodes japonicus avec la Zygosaccharomyces mandshuricus, levure décrite par Saito, il y a déja quinze ans. On peut espérer que l’augmentation de la possibilité d’identifier une levure contribuera aussi a une extension de notre connaissance de la répartition des diverses espéces. Jusqu’ici opinion a été trés répandue qu’a l’exception de quelques espéces ubiquitaires, la répartition de la plupart des espéces est trés limitée. Le grand nombre de cas dans lesquels les noms spécifiques sont dérivés de noms géographiques font témoignage de cette maniére de voir. Toutefois nous avons déja rencontré plusieurs exemples qui s’oppo- sent a cette idée. Une levure, isolée du lait par nous et provisoirement décrite sous le nom Saccharomyces galactosus s’est montrée nettement identique a une levure isolée par Naganishi, 4 Dairen en Manschou- rie, il y a 14 ans et décrite par ce savant comme Saccharomyces dairensts. 280 QUELQUES REMARQUES SUR LA CLASSIFICATION DES LEVURES Un second exemple est fourni par une levure regue il y a quelques mois par le Centraalbureau de Ile de Célébes, aux Indes Néerlan- daises. Cette levure n’avait aucun caractére qui permit de la distin- guer du Saccharomyces mangini, levure rapportée par la mission Cheva- lier de Afrique Occidentale. Je vais conclure, j’ai déja trop exigé de votre patience. Je serai bien heureux si mon exposé vous a convaincu que le Centraalbureau est une institution qui mérite bien l’appui de notre Union. Mais sur- tout je vous serai extrémement reconnaissant si vous voulez bien apres votre retour chez vous, animer vos collégues-mycologistes de donner leur coopération précieuse aux travaux du Centraalbureau.* * Ed note: This paper was published from a manuscript used for oral delivery, and Professor Kluyver did not have an opportunity to smooth out certain passages before it appeared in print. He often expressed his regrets about this development. The Editors have made some minor alterations in the original version which, they believe, are in accordance with the intentions of the author. BIBLIOGRAPHIE DORREPAAL, GC. 1930. Zentr. Bakt. Parasitenk. Abt. II, 82, 11. STELLING-DEKKER, N. M. 1931. Die Hefesammlung des ‘Centraalbureau voor Schim- melcultures’, Erster Teil, Die Sporogenen Hefen. Verhandel. Koninkl. Akad. Wetenschap. Amsterdam XXVIII. VERKAIK, Cc. 1931. Zentr. Bakt. Parasitenk. Abt. II, 85, 153. 281 PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA (WITH CG. B. VAN NIEL) I. MOTIVES FOR A RENEWED DISCUSSION OF THE SUBJECT Art the present time only two systems of bacterial classification are in more general use. In Europe the system of Lehmann and Neumann [1926] is still prevalent, whilst in America many bacteriologists adhere to the system given in Bergey’s Manual of Determinative Bacteriology [1934]. Yet it is certain that several investigators have felt that these systems are unsatisfactory both from a practical and from a taxonomic standpoint. As a consequence several new or amended systems have been pro- posed which, however, have failed to draw the attention of the major- ity of bacteriologists. Amongst these there are a few contributions which well deserve a wider appreciation, because they are based upon what seems to the authors of the present essay a sound realization of the principles of scientific taxonomy. These studies often give evidence of a profound knowledge of the literature pertaining to the subject. Nevertheless, it is to be regretted that the most notable of the authors seem to be unaware of the existence of similarly directed efforts. Hence valuable suggestions offered by one author have not been considered by the kindred writers, although these might have materially aided in ensuring the success of the various attempts. Under these circumstances it seems profitable to expound again the cuiding principles of a rational taxonomy, to point out the deficiencies of the recently proposed systems, and finally to develop a classification which is largely built on the meritorious elements of many preceding studies and in which, on the other hand, fallacies inherent in many of these are avoided. 282 PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA 2. PRINCIPLES IN BACTERIAL CLASSIFICATION If one examines the various systems of bacteria proposed up till now, it becomes evident that many of these systems are almost entirely the outcome of purely utilitarian motives. Very often such artificial systems are ultimately impractical because as a rule newly discovered facts necessitate profound modifications or even the construction of new systems at short intervals. Naturally, the only truly scientific foundation of classification is to be found in an appreciation of the available facts from a phylogenetic point of view. Only in this way can the natural interrelationships of the various bacteria be properly understood. It has to be admitted at once that, inasmuch as the course of phylogeny will always remain un- known, the basis of a true phylogenetic system of classification will be very unstable indeed. On the other hand it cannot be denied that the studies in comparative morphology made by botanists and zoologisis have made phylogeny a reality. Under these circumstances it seems appropriate to accept the phylogenetic principle also in bacteriological classification. The question then arises in which characters phylogeny expresses itself. There is no doubt that in this respect morphology remains the first and most reliable guide. It is, however, a commonplace in system- atic bacteriological literature to bewail the scantiness of suitable mor- phological data. This, in turn, is chiefly responsible for the unsatis- factory state of bacterial taxonomy. This situation has, already a long time ago, induced several authors to apply characters of a physiological nature in addition. The first steps in this direction were made timidly, but gradually the defenders of the good right of a physiological basis for taxonomy have become more and more numerous. It seems superfluous to dwell upon this evolution here. It may suffice to remark that nowadays the indispensability of physiological char- acters for the purpose of classification has been generally accepted, which is only natural because, after all, these physiological differences must be considered as expressions of variations in submicroscopical morphology. In this connexion the predominant problem is only how the macro- morphological and the micromorphological (physiological) characters 283 SELECTED PAPERS should be rated. On the one hand we may refer to Orla-Jensen’s [1909] classification which is an extreme example of the concept that phys- iological characters should determine the main lines of the system. In contrast herewith Prévot [1933] recently formulates one of his laws of bacteriological systematics as follows: ‘les caractéres physiologiques sont des caractéres spécifiques’, thus forbidding the use of physiological characters for the demarcation of units of higher systematic rank than the species. However, in another passage of his treatise Prévot obvious- ly takes exception to his law by allowing for the creation of genera on a physiological basis in such cases where otherwise the morphological genera would become overburdened. Personally we are of the opinion that Prévot rightly emphasizes the priority of morphological over physiological characters. Yet his restric- tion that the application of the latter should be confined to delimita- tion of species is of a quite arbitrary nature. In accepting the taxon- omic value of physiological characters it cannot be understood why they could not also be applied for the demarcation of higher system- atic units. In support of this view we wish to observe that such a procedure is not at all limited to bacteriological classification. A typical example is offered by algologists who do not hesitate to support the separation of the class of Heterocontae from that of the Jsocontae by such typical physiol- ogical features as the nature of the storage products, the proportion of the pigments present, and the chemical constituents of the cell wall [West and Fritsch, 1927]. There is, moreover, a second reason why it is only rational to use physiological characters not exclusively for differentiation of species but also for uniting them into higher groups. This reason is to be found in the fact, that the mere act of cultivating a bacterium, of thus gradually familiarizing oneself with the various aspects of the organism, leads to a more or less unconscious inclusion of the physiological char- acteristics in fixing its systematic position. In other words, no bacteriol- ogist can be satisfied with a classification which combines in one genus organisms which behaved quite dissimilarly in his preliminary work. To cite just one example: the microscopical appearance of some of the aerobic sporeforming bacteria and that of certain types of butyric acid bacteria may show a striking resemblance, yet the technique in hand- ling and the nutritional requirements of both groups are so obviously 284 PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA different that it will be distasteful to the majority of bacteriologists to incorporate these organisms into one and the same genus. One is thus led to the conclusion that among the physiological char- acteristics there are some which should be used in separating larger groups and the question is which ones may be deemed to be essential in this respect. Now it is clear that differences in nutritional require- ments are conditioned by differences in the metabolic activities of the organisms. Thus the problem resolves itself in grading the metabolic properties according to their intrinsic value. A lack of insight in the fundamentals of metabolism has thus far been the great stumbling- block for a rational application of physiological characteristics in tax- onomy and it also explains the horror with which many systematicians have witnessed their ever increasing use. In surveying the metabolism of the various bacteria one is struck by its great diversity which contrasts sharply with the relatively great uni- formity characteristic for the metabolism of higher plants and animals. An analysis of this situation bears out that, whereas the anabolic proc- esses are essentially similar in both cases the fundamental differences are to be found in katabolism. It is especially the many different ways in which bacteria succeed in meeting their energetic requirements which draw the attention. Besides the respiration process, as it 1s also found in higher organisms, we encounter numerous instances in which this process is substituted for by conversions in which free oxy- gen does not take any part. Moreover, all these processes are char- acterized as well by the diversity of their substrates as by the different ways in which these substrates are converted into the final products of katabolism. The fundamental nature of the energy providing processes justifies the view that they should be rated first amongst the physiological characters suitable for classification, the more so since these properties are typically reflected in the cultural behaviour of the organisms. If, in consequence of this, one is led to the creation of systematic groups on the basis of katabolic properties is not the principle of phylogeny as a taxonomic basis endangered? This needs not be the case provided this principle is duly subordinated to the requirements ensuing from a primarily morphological classification. For it does not seem excluded at all that in a single morphological group a physiolo- gical evolution is responsible for the differences in katabolism observed, 285 SELECTED PAPERS whereas parallel evolutionary processes may have taken place in various morphological units. One might object to this hypothesis, since the differences in kata- bolism seem to be of such an absolute nature as to exclude any evolu- tionary idea. However, recent development in the study of bacteria] metabolism seems well suited to modify this view. A closer analysis of the katabolic processes has revealed their fundamental unity and made it possible to connect the differences observed largely with differences of a quantitative nature in some special property of the living cell [Kluyver, 1931]. This quantitative gradation implies that also the physiological groups do not show any sharp lines of demarcation. Hence, the bound- aries imposed by the intuition of the investigators will always bear a more or less arbitrary character. The situation seems comparable with that existing in colour distinction. Although every one will accept the validity of such clearly distinguishable colours as red, green, blue, yet a glance at the spectrum is convincing evidence that also in this case boundaries are lacking. It is obvious that the resulting taxonomic difficulty will be felt most severely in the determination of the ultimate systematic unit, the species. The masterly way in which Benecke [1912], as early as 1912, has expressed himself on this matter justifies the quotation in extenso of the following passage. ‘Was sind nun die Arten, d.h. die niedrigsten von den eben aufge- fiuhrten systematischen Einheiten? ‘Die Antwort lautet: Das, was der Forscher, welcher die Art aufstellt, nach seinem ‘‘wissenschaftlichen Takt’? darunter zusammenfasst. An- ders kann die Frage offenbar darum nicht beantwortet werden, weil die Natur selbst keine Arten kennt, sondern nur Individuen mit ihrer Aszendenz und Deszendenz, also nur sog. “‘Linien’’, und solche Linien fasst eben der Systematiker zu Linienbiindeln zusammen, die er Arten nennt. Wie gross oder wie klein er sein Biindel schniiren will, das hangt von seiner wissenschaftlichen Auffassung ab, die von der eines anderen mehr oder minder abweichen kann. Freimachen von dieser subjektiven Umgrenzung der Arten wiirde sich der Systematiker dann, wenn er auf noch niedrigeren Einheiten als den Arten fussen wollte, eben jenen Linien. Er miisste dann alle Bakterien in Form von Einzel- kulturen ziichten, und zwar unter den denkbar verschiedensten Be- 286 PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA dingungen, unter denen sie tiberhaupt zu leben vermégen, wiirde sich all ihre Formen und Eigenschaften in Abhangigkeit von diesen Bedin- gungen merken und sie in die Diagnose der betreffenden Linie auf- nehmen. Alle die reinen Linien, die dann keine Unterschiede in der Diagnose aufweisen wiirden, miisste er zu systematischen Einheiten zusammenfassen, zu sog. elementaren Arten, und diese von willkiir- licher Umgrenzung wenigstens einigermassen freien Einheiten nach Gutdiinken zu hdheren Einheiten zusammenfassen. Tatsachlich ist das nicht modglich; das braucht nicht weiter begriindet zu werden, denn jene eben skizzierte Arbeit wurde kein Ende absehen lassen. So miissen denn ftir die praktischen Zwecke der Systematik, d.h. um eine Ubersicht iiber die Formen zu erméglichen, héhere systemaiische Ein- heiten gewahlt werden.’ Hence it follows that in an attempt to subdivide the organisms be- longing to one ‘natural’ group of bacteria into species one shall have to create as many species as there are organisms which differ in ‘suf- ficiently fundamental’ characters, regardless of the possible existence of intermediate types. It depends entirely upon the ‘scientific tact’ of the investigator which characters will be deemed worthy of the desig- nation ‘sufficiently fundamental’.* It is here the place to give a short survey of the characters which may be taken into consideration in this respect. a. Morphological characters It is self-evident that the shape of the cells is of outstanding importance for determining the place of a bacterium in any phylogenetic system. The same holds, of course, for the mode of reproduction and the oc- currence of special resistent stages such as endospores, gonidia etc. In addition to these characters the size and the structure of the cells may give valuable indications. With regard to structure it is especially the presence of organs for locomotion, flagella, and the way in which they are attached to the cell which has long been recognized as affording insight in mutual relationships. In this connexion it must, however, be remarked that experience has taught that in cases of immotility of the cells we have to discriminate between ‘incidental’ and ‘genuine’ immotility. For, as has * It goes without saying that similar difficulties will also be encountered in marking off the higher systematic units, though to a lesser extent. 287 SELECTED PAPERS been emphasized by Orla-Jensen, we sometimes meet with immotile species which unquestionably are closely related to motile ones, where- as, on the other hand, one is struck by the fact that in several large natural groups motility is completely lacking Finally one diagnostic character of wide application, wiz., the Gram stain, may also be considered as dependent on structural properties of the cell and the many instances of a close correlation of the outcome of the Gram test with several other characters leave no doubt as to its significance also for taxonomic purposes. b. Phystological characters The behaviour of the various bacteria in relation to temperature and osmotic pressure of the environment has often been used in the char- acterization of species. Recent investigations tend to throw doubt upon the soundness of this procedure, because of the wide range of adapta- tion which is manifested by most bacteria. As has already been set forth the physiological characters of prime importance are those which are connected with the metabolism of the organism. Although in this respect the significance of the katabolic properties of the cells has been rightly stressed, yet one should not lose sight of the fact that in some cases the occurrence of special prod- ucts of anabolism can also be made use of. A first division of katabolic activity has to be made according to the source of the energy required by the cell. Here we must distinguish be- tween the organisms capable of utilizing radiant energy and those which are dependent upon the chemical energy supplied by one or more con- stituents of the medium. In the latter case, which is the more common with bacteria, a further subdivision imposes itself according to the role of the free oxygen in katabolism. Firstly we find bacteria in which this gas is as indispensable as it is for higher plants and animals. But in addition to this category there exist organisms which can satisfy their energy requirements also in the absence of oxygen. Finally there is a third group of bacteria, which thrive only in its absence, or nearly complete absence. In each of these groups a further subdivision can be made both on the basis of the nature of the favoured katabolic substrates and on the basis of the mode of decomposition as evidenced by the nature of the final katabolic products. It may be added at once that this predilection 288 PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA for a special group of substrates — carbohydrates, protein decomposi- tion products, or organates — involves an adaptation to life in an envi- ronment of a special range of hydrogen ion concentration. It must be pointed out, however, that a judicious selection of the criteria on which the above mentioned subdivision should be founded, is most essential. In this connexion it cannot be sufficiently stressed that one should not confuse the physiological significance of the ability to derive energy from the desmolytic transformation of a special group of substrates with the ability to hydrolyze compounds of a complex nature, like proteins, polysaccharides, fats etc. For it must be clear that there exists a far more fundamental difference between an organism which splits glucose into lactic acid and a second organism which prod- uces out of the same substrate butyric and acetic acid, carbon dioxide and hydrogen, than between two lactic acid bacteria only one of which attacks maltose. In the latter case both organisms derive their energy from the same conversion of glucose into lactic acid, the hydrol- ysis of the maltose into glucose being only an introductory act devoid of any energetic significance. Differences in the hydrolytic capacity should never be applied for distinguishing systematic groups, they can merely be taken into account for the differentiation of species. The neglect of this point of view is undoubtedly responsible for the dislike with which morphologically inclined taxonomists view the application of physiological characters for the delimitation of system- atic units larger than species. Finally some remarks should be made regarding a physiological character of rather wide application in present day taxonomy, v7z., pathogenicity. This appears to be a character of very doubtful value. For the case is not rare that a pathogenic organism is so closely related to a non-pathogenic one that the two are undistinguishable except with the aid of infection experiments. The creation of separate genera on the basis of such a character is objectionable, because this implies that even the generic nature of an organism cannot be decided upon independently of a knowledge of its previous history. The same diffi- culty holds, albeit to a lesser extent, for a differentiation of species or the basis of pathogenicity.* * (Cf. particularly the valuable remarks made by O. Rahn, Zentralbl. Bakt. Para- sitenk. Abt. IT. 78, 1, 1929: 79, 321, 1929; and Stapp, C., Schizomycetes (Spalt- pilze oder Bakterien) in: Sorauer, Handb. d. Pflanzenkrankh. Bd. 2. 1928. S. 1-295. 289 SELECTED PAPERS In summarizing the above it is tempting to conclude that on the one hand phylogeny has led to the origin of various morphological groups, whilst, on the other hand, an evolution in metabolic properties has occurred which together are responsible for the almost unlimited diversity of bacterial species. Before attempting to apply these prin- ciples in the construction of a system, we will first critically examine the more recent contributions to the classification of bacteria. 3. CRITICAL EXAMINATION OF RECENT CONTRIBUTIONS TO THE CLASSIFICATION OF BACTERIA The large number of papers in which ideas on bacterial taxonomy are advanced makes an endeavour to give a moderately complete survey altogether impossible. ‘Therefore we shall confine ourselves to a con- sideration of the more strictly taxonomic publications. The system of Lehmann and Neumann [1926], adapted to modern needs since its inception in 1896, may still lay claim to a serious con- sideration. The outstanding feature, and at the same time the weak- ness of the system, is undoubtedly its simplicity. Although for the separation of the families of the Schizomycetales morphological char- acters are used exclusively, it would seem that a far better use could be made of such characters to express natural relationships. In this case the Desmobacteriaceae, which have nothing in common with the other five families, would have been set aside as a group equivalent to the orders of Schizomycetales and Actinomycetales. The same holds for the family of the Spzrochaetaceae. The remaining four families seem at first sight acceptable as units resulting from a gross subdivision. Yet it appears from a closer inspection of the accepted genera that their grouping violates the principle of natural relationship in many respects. Thus one finds in the large, physiologically very heterogeneous, genus Bacterium organisms with polar flagella which are much more closely related to the family Spirillaceae than to the greater part of the other Bacterium species. Furthermore, the unmistakable relationship be- tween the species of the genus Plocamobacterium with those of the Pro- actinomycetaceae is fully neglected, even to the extent of incorporating them in different orders. As regards the family of the Desmobactertaceae its extremely heterogeneous nature should be emphasized. Apart from the fact that Lehmann and Neumann apologetically incorporate 290 PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA in this family several large groups of bacteria not possessing the diag- nostic characters of the family (Thiobacillus species, purple sulphur bacteria, the coccoid iron bacteria described by Molisch, etc.) there is no doubt that the recognized genera: Beggiatoa, Leptothrix, Creno- thrix, Cladothrix and Thiothrix show neither affinities to the remaining genera of the Schizomycetales nor to one another. It is clearly evident that in proposing this family Lehmann and Neumann have more or less unconsciously used physiological char- acters. Obviously the main reason for uniting the organisms in question into one family is not so much their morphological similarity as the peculiarity of their metabolism. The most outstanding contribution of the system of the German authors and the one which has found general recognition is undoubt- edly the realization of the close affinities existing between the genuine Actinomycetes and the genera Corynebacterium and Mycobacterium. A revolution in the principles of bacterial taxonomy was brought about in the years 1908 and 1909 by the appearance of Orla-Jensen’s publications [1908, 1909]. For the first time the classification of the bacteria was based mainly on the physiological characters. The idea underlying the proposed system is a phylogenetic one. A genealogy is developed as a result of considerations concerning the succession of physiological types in the course of the evolution of life on earth. Migula’s influence is observable in the emphasis placed on the signi- ficance of the difference in flagellation. The mode of insertion of the locomotive organs is used for the separation of the bacteria into two orders: the Cephalotrichinae and the Peritrichinae. A place was assigned to the immotile bacteria in either one of these orders on the basis of their physiological relationship to motile species. Orla-Jensen [1921] has returned to the subject in a paper in which he makes another eloquent plea for his main theses. With the exception of a few minor amendments the system as proposed in 1908 is main- tained. The system has the great merit that it is built upon a sound evalua- tion of what is fundamental in the various physiological characters as manifested by the value attached to the energetically important katabolic properties. Another meritorious feature of Orla-Jensen’s system is the creation of several new genera for physiologically well defined groups, together with the introduction of a rational code of 2gI SELECTED PAPERS nomenclature for all genera, fully independently of the principle of priority. On the other hand, it is not surprising that the manifest neglect of the importance of morphological characters other than the flagellation has given rise to severe criticism. Without entering into a discussion of the numerous objections raised, it may suffice to give here a few examples in which in consequence of this the principles of natural relationship have been violated. The place of the immotile groups in the system is not satisfactory. Here physiological characters, like special nitrogen requirements, have been decisive in the position of the genera Streptococcus, Caseobactertum, and Propionibacterium amongst the Peritri- chinae, in the position of Mycomonas, Corynemonas and Actinomyces amongst the Cephalotrichinae. The reason for this is that Orla-Jensen has adopted the view that one and the same physiological evolution has occurred only once in phylogeny. The possibility is not considered that such an evolution may have taken place independently in various morphologically different groups. Morphologically the three first mentioned genera show such close affinities to the genera Mycobacterium and Corynebacterium, that the grouping of these five genera in the two different orders is inaccept- able. In addition the designation of the two last mentioned genera as Mycomonas and Corynemonas, thus suggesting a relationship between the representatives of these groups and the cephalotrichous genera, has its foundation exclusively in the oxidative character of their katabolism. Here again we encounter the idea that in determining the systematic position of a group metabolism dominates over morphology. Another instance of artificial grouping is offered by the creation of the genus Carboxydomonas for the permanently immotile Bacillus oligo- carbophilus discovered by Beijerinck and Van Delden. The systematic position given to this organism by Orla-Jensen — also expressed in its new generic name — is merely based on the idea, that there is a close resemblance between the metabolism of the organism in question and that of the representatives of the genera Hydrogenomonas and Methano- monas. Morphologically, however, the species of the latter genera are so typically related to the Pseudomonas species and so distinctly different from B. oligocarbophilus that Orla-Jensen’s classification has to be rejected. In spite of the criticisms given we want to emphasize that in our 292 PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA opinion Orla-Jensen’s contribution to systematics — the prominent place assigned to rightly evaluated physiological characters — marks a milestone in the development of bacterial taxonomy. In their entirety Orla-Jensen’s views have never been accepted. It is the merit of Buchanan to have advocated a system of classification in which a limited use has been made of Orla-Jensen’s ideas. In subdividing the order of the Eubacteriales Buchanan recognizes one family exclusively on the basis of physiological characteristics. The autotrophic nitrifying bacteria which Orla-Jensen considered as re- presentatives of the most primitive among micro-organisms were here united in the family Nitrobactertaceae. Buchanan’s system was more or less the foundation of that which soon afterwards evolved out of the work of a committee appointed by the Society of American Bacteriologists, of which Buchanan originally was a member. For a description of the development of the work of this committee we may refer to the data presented in Buchanan’s [1925] most valuable, exhaustive monograph on bacterial systematics. It may suffice to state that the results have ultimately been laid down in Bergey’s [1923, 1925, 1930, 1934] well-known Manual of Deter- minative Bacteriology. In the following discussion we will therefore limit ourselves to a criticism of the system as developed in the last edition of this book. As for the earlier editions we will only remark that in these — especially in the first one — the tentative character of the outlined system was duly emphasized in the preface. Gradually the attitude of the editing committee has, however, changed and has grown more and more self-confident, probably owing to the commercial success of the book. This should be regretted because the publication of this cooperative effort has led to an abundance of sound criticism which might have been usefully incorporated in later editions. By ignoring this criticism the benificial effect which might have resulted from the cooperative character of the work — the first formal coopera- tion in the history of bacterial taxonomy — has been more or less nulli- ja ra The final outcome can best be described as a compromise between the most divergent ideas which have been expressed in the course of time. Anyone who has had the opportunity to peruse the book will have been struck by the fact that morphological, physiological, nomen- clative, utilitarian, cultural and pathogenic properties have been used 293 SELECTED PAPERS in the building up of the system in the most arbitrary way. The result is a complete lack of homology in the various groups, as has already been emphasized by Prévot (l/.c.). In addition to this serious short- coming an utter disregard for the significance of mutual relationships between natural groups is apparent. In support of these statements the following instances may be cited. The first one of the five families of the order Eubacteriales, viz., the Nitro- bacteriaceae, is a curious conglomerate of organisms the majority of which have nothing in common save the fact that the average — usu- ally medically trained — bacteriologist is unfamiliar with them. Where- as Buchanan, in creating this family, clearly intended to separate the nitrifying bacteria with their remarkable autotrophic mode of life from those bacteria for which organic substances are a necessary pre- requisite for their development, this principle was violated when the Committee included in this family organisms which ‘may use in their metabolism’ also ‘simple carbon containing compounds’. Apart from the question what one has to understand by the restriction ‘simple’ it will suffice to remark that nearly all bacteria will answer this require- ment. Nor can such a ‘simple’ metabolism have been meant to be im- perative since any bacteriologist acquainted with genera like Aceto- bacter and Rhizobium will cultivate representatives of the genera in media containing an abundance of complex organic compounds! The next family, Coccaceae, shows a clear-cut example of lack of homology in its subdivision in genera. No argument whatever is ad- vanced for the sudden use of the character of pigment formation in the demarcation of the genus Rhodococcus. ‘This emphasis on the occur- rence of a red pigment in representatives of this genus, although noth- ing is known about a possible metabolic significance of the pigment, is most astounding, since the occurrence of a yellow or even an orange pigment amongst members of the genus Micrococcus does not seem to offer any ground for separating these from the non-pigmented species. Proceeding to the third family, Spirllaceae, we will only point out that here again we meet with an instance where the occurrence of a red pigment is not considered of sufficient importance to be used as a generic character, for Spirillum rubrum Esmarch is classified together with the colourless spirillae. Unfortunately in this case — and this in contrast to what has been remarked regarding the Rhodococcus species — there would have been every reason for separating Sp. rubrum from the 204 PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA other species, because here the pigment fulfils a fundamental metabolic function, Sp. rubrum being a typical purple bacterium. A documen- tation of this insight is in this case quite superfluous since this opinion is fully shared by Bergey et al. as is proved by the fact that we meet with the same organism again in an entirely different order (!) as Rhodospirillum rubrum (Esmarch) Molisch. The next family, Bactertaceae, offers no end of examples of heterogen- eity, arbitrariness, utilitarianism, and disregard for natural relation- ship. The subdivision of this family into its twelve tribes has obviously been dictated by the tendency to keep apart the bacteria of importance to the hygienist from all others. In the key to the tribes this is obtained by giving the attribute ‘pathogenic for animals’ to the representatives of the former group. This leads to the remarkable situation that in this group of ‘pathogens’ several utterly harmless organisms, like many Aerobacter and Alcaligenes species, are encountered. Apart from this lack in consistency, making the organisms in question fully in- determinable, no bacteriologist who isolates a bacterium from soil, water, dairy products etc. will ever be able to decide whether it belongs to the ‘would-be pathogens’ of Bergey et al.. Hence another group of organisms, like the Escherichia species, becomes in most cases also indeterminable. The attempt to reinforce the antithesis pathogenic— non-pathogenic by ascribing to the former group an optimum tem- perature of 37.5 °C, against one of 30 °C or less to the latter group, shows the same lack of consistency as regards its practical application. Numerous are the instances in which species with low temperature optima are found in the so-called pathogenic group, whereas the authors also do not hesitate to include bacteria with a temperature optimum of 37 °C or even higher (Serratia spec., Lactobacillus spec.) in the saprophytic group. Moreover in the species diagnosis several organisms of this group are reported to be pathogenic or having the intestinal canal as their normal habitat! If one now proceeds to the subdivision of the large group of sapro- phytes or plant parasites one is struck by the miscellany of characters which are used in the demarcation of the various tribes. One semi- morphological property, the outcome of the Gram-stain, is used; the other characteristics are all physiological and of a very dubious nature, such as the ability to grow either well or poorly on ordinary media, the ability to digest cellulose or not, etc. Attention should be drawn 295 SELECTED PAPERS furthermore to the great importance attached to the character of pigment production. Whilst in genera like Sportllum, Propronibacterium, Mycobacterium the production of yellow, orange, brown and red pig- ments is not considered to be of sufficient importance for making generic distinctions, here a special tribe is created for uniting the pigmented forms which are then divided into four genera on the basis of the colour. The occurrence of yellow pigments which in the genus Micrococcus is deemed inessential is here raised to a character of generic rank (Flavobacterium). Not all bacteriologists will realize, moreover, that for the correct determination of the common fluorescents he must reject the designation of the pigment as yellow but characterize it as green or blue-green. If he succeeds in avoiding this pitfall he will still be lost in case he is dealing with one of the numerous representatives of the genus Phytomonas which also produce a fluorescent pigment. For it is impossible to arrive at the last mentioned genus without this time having designated the same colour as pale yellow! The arbitrariness may have been sufficiently demonstrated by the foregoing examples. Yet it is worth-while to note two more instances which at the same time clearly show the lack of homology in the vari- ous systematic units. In the tribe Erwineae the two genera are distin- guished by the mode of insertion of the flagella; on the contrary the genera Flavobacterium, Chromobactertum and Achromobacter contain or- ganisms with polar as well as such with peritrichous flagella. The second instance is the arbitrary use of the character of the Gram-stain. It has been mentioned above that the main subdivision of the sapro- phytes is based on this property; on the other hand in the group of animal parasites we encounter Gram-positive and Gram-negative species within the same genus (cf. e.g. Alcaligenes, Bacterovdes). We shall not continue the detailed criticism of Bergey’s classification of the Eubacteriales but only make a few remarks regarding the re- maining orders, documenting our opinion that in these orders the same inconsistency, lack of homology etc. are to be found. The second order, Actinomycetales, owes its origin to the just evaluation by Lehmann and Neumann of the natural relationship between the Actinomycetes and the bacteria of the genera Mycobacterum and Corynebacterium. ‘The com- mon characteristics of these groups are morphological and comprise positive Gram-stain, the lack of ability to form true endospores and permanent immobility. These properties are indeed included in the 296 PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA diagnosis of the order as outlined in Bergey’s manual. It is therefore perplexing to find in the family Mycobacteriaceae Gram-negative as well as motile organisms. Any one isolating an organism answering the diagnostic requirements of the genera Mycoplana, Cellvibrio and Actino- bacillus would not hesitate a moment to include his organism in the order of the Eubacteriales. A similar example is furnished by the species Leptothrix hyalina which, although it is stated not to form a sheath, is incorporated in the order of the Chlamydobacteriales regardless of the fact that in the key to the orders the occurrence of a sheath is decisive for including the organism in the order in question. As for the next order, Thiobacteriales, we will only state that here, in contradistinction to what holds for the foregoing orders, morpho- logical characters are fully left out of consideration in delimiting the order. The essential metabolic function of the sulfur or (and) the photosynthetic activity with the aid of the bacteriopurpurin pigment complex serve to characterize the order. Considered from this point of view it is rather surprising that the genus Thiobacillus is not included in this group. With these comments on one of the most widely used systems the deplorable present state of bacterial classification is sufficiently illus- trated. During the course of the development of the American system a number of important studies on bacterial taxonomy appeared. One of the most outstanding contributions to a solution of the problem is undoubtedly the paper by Pringsheim [1923]. This publication con- tains several very sound general considerations on the principles of taxonomy with corresponding critical remarks on the previously pro- posed systems. We will confine ourselves to a brief review of the scheme as advocated by the author. Then we meet with a first subdivision of the bacteria into five orders, each one of which may be called homo- geneous and homologous. The principal feature of Pringsheim’s subdivision of the first order, Eubacteriales, is unquestionably the fact that the usual, somewhat primitive, classification on the basis of spheres, rods and spirals is not strictly maintained inasmuch as the Spirtllaceae also include the rod- shaped bacteria with polar flagella, 7.e. the genus Pseudomonas Migula. The natural affinities of the representatives of this genus to those of the 297 SELECTED PAPERS genus Vibrio, as already pointed out by Migula, is so unmistakable that this amendment must be considered a decided improvement. Following the order of the Eubacteriales we encounter the order of the Rhodobacteriales comprising the natural group of the photosynthetic purple bacteria in two suborders: the sulfur-containing and the sulfur- free species. The merit of the demarcation of this order as compared with that of the Thiobacteriales Buchanan is to be found in the fact that the colourless sulfur bacteria of the genera Beggtatoa, Thiothrix and Thioploca, which show so different natural affinities (colourless Cyano- phyceae as rightly suggested by Pringsheim), are not mixed up with the purple bacteria. Characteristic for Pringsheim’s order of the Myco- bacteriales is that it is again restricted to the homogeneous group formed by the genera Corynebacterium, Mycobacterium and Actinomyces. Signifi- cant for an appreciation of the clear insight of the author is his sug- gestion that the rod-shaped lactic acid bacteria might also belong to this order. The fifth order of the Desmobacteriales needs no comment since Pringsheim himself stresses its provisional nature. From all the systems of classification based mainly on the morphol- ogy of the organisms Pringsheim’s scheme is the most satisfactory one, and it compares especially favourably with Enderlein’s [1925] revolu- tionary attempt at classifying the bacteria according to principles of comparative morphology and ontogeny (‘cyclogeny’). This author rejects all previous attempts at classification because they are not based on a careful and thorough cytological study and from a theo- retical standpoint his plea for attaching predominant importance to cytological and ontogenetic characters is very convincing. However, as pointed out earlier in this paper, our knowledge of characters of this kind is necessarily extremely limited and Enderlein’s own contribu- tions in this field do not change this situation to any appreciable extent. For it has escaped the attention of this zoologist that by far the greater part of the cytological details reported by him are theoreti- cally undetectable since the dimensions of the structures described are below the limits of the resolving power of the microscope. This implies that most of the ‘life-cycles’ which are at the basis of Enderlein’s sys- tem are fully artificial.* However tempting the classification outlined may be at first sight (cf. e.g. the table on p. 236), its value must be * For sound ideas concerning the term ‘life-cycle’ the reader is referred to the note by Ch.-E. A. Winslow, Science. 87, 314, 1935. 298 PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA judged from its results. A system which unites in one genus Proteus vulgaris and Lactobacillus delbriickit, and assumes close relationships of these with Acetobacter, Rhizobium (all in one and the same subfamily of the Ezsenbergiinae) and with Sclerothrix (Mycobacterium tuberculosis) and Corynebacterium by placing all these genera in one of the 15 fam- ilies, condemns itself. Janke [1926], who in 1926 had already published a critical essay on the subject of bacterial systematics, later developed a system which 1s clearly influenced by Enderlein’s views [ Janke, 1929]. It seems super- fluous to give here a complete survey of Janke’s system. We will only remark that we encounter in this system many well-known groups — to which Janke assigns the rank of families — v2z., Coccaceae, Bacillaceae, Bacteriaceae, Corynebacteriaceae, Spirillaceae, Spirochaetaceae, Desmobacte- riaceae and Myxobacteriaceae. A discussion of the question whether the establishment of these units as families is more or less justified may be omitted here. Our remarks will be confined to the subdivision of the families. Janke’s critical attitude towards the use of physiological characters for classificatory purposes leads him to restrict his genera to those groups which can be identified with the aid of morphological characters only. This means that e.g. in the family of the Bacillaceae the common aerobic and anaerobic sporeforming bacteria are united in the one genus Bacillus thus rejecting the genus Clostridium of va- rious older systems. Moreover, many bacteriologists will be shocked to meet with the genus Azotobacter in this family. The Jjustifica- tion of this procedure is found in the statement: ‘Sporenbildende Stabchen gehdren zum Entwicklungskreis (LOohnis)’. The rather doubtful observations of Loéhnis have, however, never been corrob- orated. It is also worth mentioning that the external morphology which is responsible for the maintenance of a separate genus Azoto- bacter in Janke’s system is frequently nearly duplicated in species closely related to Bacillus megaterium [Dianowa and Woroschilowa, 1931] which is accepted by this author as a type species for a sub- group of the genus Bacillus. The trend of thought mentioned above also accounts for the occur- rence of only two genera, Bacterium and Fusiformis, in the family Bacte- riaceae. The weakness of Janke’s principles of classification is clearly evidenced by the heterogeneity of the genus Bacterium. The diversity of organisms collected by Enderlein in the subfamily of the Eisenbergiinae 299 SELECTED PAPERS — which has already been criticized above — is found here in one and the same division of the genus Bacterium. The 1o contributions by Rahn [Rahn, 1929] and collaborators [Rahn, Laubengeyer and Mansfield, 1929] to the classification of bac- teria will be mentioned here firstly for their very sound, meritorious criticisms of Bergey’s system. For these we refer to the original papers and we will only remark that it is most discouraging that since their publication two new editions of Bergey’s manual have appeared from which it is evident that the editing committee did not pay any heed to Rahn’s amply documented considerations. In addition to calling attention to various fallacies in Bergey’s system Rahn has also given a number of constructive suggestions, the most important of which will be briefly reviewed here. In the first place Rahn and Laubengeyer point out the close relationship between several of the Bacteroides spe- cies and the Lactobacilli and propose to abandon the illdefined genus by incorporating these species in the genus Lactobacillus, whilst group- ing the remaining quite dissimilar species with some other genera. Rahn and Mansfield then show the desirability and the feasibility of collecting all polar flagellates out of the family of the Bacteriaceae into a separate family Pseudomonadaceae as had already been proposed by Winslow et al. in 1917. Finally there is a plea by Rahn for a due recog- nition of the intimate relation between the Streptococci and the natural group of the rod-shaped lactic acid bacteria. Rahn goes so far as to advocate the removal of the tribe Streptococceae from the family of the Coccaceae and its inclusion in the Bacteriaceae. The most elaborate effort to revolutionize bacterial taxonomy in later years is undoubtedly the monograph published by Pribram [1933], an extension of the ideas expressed in an earlier paper [Pribram, 1929]. ‘The most radical principle introduced is the division of the bacteria into three subclasses which are based chiefly on ecological considera- tions. ‘hus the subclass Algobacteria is meant to comprise all the forms adapted to a life in water, as evidenced a.o. by the motility with the aid of polar flagella. The second subclass, Eubacterta, is made up of those bacteria whose habitat is the animal body or complex waste products of plant or animal origin. This habitat has led to forms which are either immotile or motile by means of peritrichous flagella and which are characterized by their ability to attack complex molecules. Finally the third subclass, Mycobacteria, is adapted to a life in soil and 300 PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA shows a distinct tendency to differentiation as manifested by the occurrence of spore formation, branching, etc. Now the foundation of a classification chiefly on ecologic principles is undoubtedly very dangerous, as is clearly demonstrated by the ob- vious impossibility to apply such a scheme to the classification of higher organisms, where a more scientific taxonomy can be achieved on the basis of characters which have an undisputably phylogenetic value. It is therefore not surprising that a closer inspection of Pribram’s system reveals its inadequacy. Thus, in order to defend the motility of a bacterium as a typical feature of an aquatic habitat Pribram has to resort to the enormity that the occurrence of peritrichous flagella amongst the representatives of the third group does not conflict with their supposed ‘sessile’ character, since here the flagella do not serve the purpose of locomotion but just act as organs to replenish the food supply.* Moreover we find amongst the Algobacteria numerous species which are immotile, as well as organisms like various Micrococcus and Sarcina species which according to their habitat belong to the second subclass. And how can the author justify the inclusion of the peritrichous genera Serratia (and Hillhousia?) in the first subclass? The same holds, al- though for different reasons, for the genera Myxococcus, Chondromyces and Polyangium. ‘The dissemination of a natural group like the Thiorhodaceae over three orders should not remain without protest and no investigator of this group of organisms will tolerate the plac- ing of Rhabdomonas (N.B. as type genus) between the genera Beggiatoa and Yhioploca and this merely on the basis of an alleged contractility of the cells. Our criticisms of the second subclass are based for the greater part on the extreme heterogeneity of its constituent units. As for the second family our objections are substantially the same as those which have been brought forward in connexion with our review of Enderlein’s subfamily of the Evsenbergiinae. We might remark in this connexion that all motile acetic acid bacteria (Ulvina spec.) which have been studied with respect to the mode of insertion of flagella have proved to be polar. In consequence their inclusion in the subclass Ewbacteria conflicts with the first mentioned character in the diagnosis of this * We have found the same critical remark in the attractive review of Pribram’s monograph by C, Stapp, Zentralbl. Bakt. Parasitenk. Abt. II, 8, 514, 1934. 301 SELECTED PAPERS group. The objections raised by Rahn with respect to the genus Bacte- roides apply also to the third family Bacterovdaceae. The attempt to unite in one subclass those bacteria which show a tendency towards modes of reproduction other than fission is not ob- jectionable in itself. However, the marked differences between the two orders makes it preferable to look upon these subgroups as terminal stages in the development of entirely different morphological groups and therefore, in our opinion, they should be ranged separately with their simpler ancestries. The advisability of the addition of several Gram-negative genera to the otherwise well characterized group of the Mycobacteriales seems also questionable. The preceding remarks will suffice to make it clear that, although Pribram’s genera are homogeneous and much better characterized than many of the genera adopted by Bergey et al., we cannot approve of the way in which they are arranged in larger units. Which factors can be made responsible for this difference in appreciation? ‘There can be no doubt that this is chiefly due to a different evaluation of the im- portance of various characters. Although Pribram pretends to attach a distinct value to the type of flagellation, yet the exceptions he allows in the consistent application of this diagnostic character are so numer- ous that already on this account a great deal of confusion results. Pribram’s neglect of the suggestion made by Orla-Jensen that one should discern between ‘incidental’ and ‘genuine’ immotility — as discussed earlier — is another disturbing factor. Finally it is the in- sufficient appreciation of the importance of the Gram-reaction which should be criticized. On the one hand Pribram fully recognizes the value of this character which even accounts for the creation of several new genera. On the other hand the author fails to realize the obvious mutual affinities between many of the Gram-positive and many of the Gram-negative genera respectively. Perhaps it is more correct to say that these mutual relationships do not fully escape the attention of the author, since in various places mention is made of the fact that certain species may be considered as connecting links between two often widely separated groups. Our principal grievance against Pribram’s classification is that such considerations have remained without any influence on the final form of his system. The last contribution to bacterial classification is the important paper by Prévot [1933] which has already been quoted. This study is 302 PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA especially attractive because the author starts with a clear formulation of the rules, principles and laws which, according to his insight, should govern bacterial systematics. Prévot’s first law states that the general morphology enables one to divide the bacterial kingdom in classes and these in orders. As classes Prévot recognizes the six orders previously established by Bu- chanan and a preliminary attempt is made at subdividing the first class (Eubacteriales) in five orders which apparently correspond to the five families of the American system. There is, however, the important difference that Prévot envisages the possibility of demarcating the Nitrobacteriaceae on the basis of an ellipsoidal cell form. Apart from the fact that our criticism of the said family as defined in the American system is not obviated by Prévot’s redefinition, it seems to us very doubtful whether a distinction between two groups of the fundamental significance of an order can be founded on the difference between an ellipsoidal and a cylindrical cell shape. The failure of such a procedure is obvious if it is remembered that some of the most typical represent- atives of the Bacteriaceae have originally been described as Micrococcus species (Micrococcus prodigiosus, etc.). The ambiguity of the said char- acter has, already a long time ago, led to abandoning Cohn’s differen- tiation of the genera Bactertum and Bacillus on the basis of the length of the rods. We can pass the second law in silence. As for the third law we wel- come the stress laid on the importance of the Gram-stain, especially since good arguments are given for the view that the outcome of this staining reaction depends on structural characters. The homogeneity of a family in this respect is required by Prévot. The fourth law formulates that more special morphological prop- erties (flagella, capsules, biometrical constants) have a generic value. Why the use of these features should be restricted to these smaller groups is not clearly stated, however. To us this limitation does not seem justified particularly with reference to the flagellation. The remaining laws do not invite further comments; we only refer to our opposition to the sixth law in which Prévot restricts the use of physiological characters to the delimitation of species. Prévot has confined himself to apply the above-mentioned principles to the elaboration of a detailed classification of the Coccaceae. ‘The out- standing feature of his system as compared with previous ones is the BeS SELECTED PAPERS early separation of the Gram-negative and the Gram-positive cocci, which ultimately leads to the creation of the new Gram-negative genus Veillonella which is the counterpart of the Gram-positive genus Micro- coccus. In consequence of this the final system has the great merit of a logical and consistent structure. It should, however, be questioned whether it is commendable to restrict the physiological characters to specific delimitation. We can agree with the author that ‘aerobic’ and ‘anaerobic’ mode of life as based solely on the sensitivity of the organ- ism towards free oxygen is a rather dangerous character. Yet it seems to us that the katabolic nature of the organism is a much more essen- tial property and should not have been left out of consideration in the demarcation of higher groups than species. For it can hardly be doubted that the relationship between aerobic Micrococcus species and aerobic Sarcina species is at least as close as that between the latter and the anaerobic Sarcina species such as Zymosarcina ventriculi (Goodsir) Smit [Smit, 1930]. 4. OUTLINE OF A RATIONAL SYSTEM FOR BACTERIAL CLASSIFICATION ON THE BASIS OF OUR PRESENT KNOWLEDGE The preceding pages have not only served the purpose of exposing the manifold weaknesses and inconsistencies of the more recent systems, they may also perform the function of illustrating the considerations to which the general principles laid down in Section 2 lead in con- crete instances. Therefore in the following attempt at sketching an outline of a rational bacterial system we shall also draw upon material which has already been used in the discussion of our attitude towards previous classifications. It may be recalled that in the foregoing we have had the opport- unity to point out that the assumption of both a morphological and a physiological evolution seems justified. A true reconstruction of the course of evolution is the ideal of every taxonomist. But, as has been rightly emphasized by Pringsheim, such a reconstruction is not feas- ible, firstly because the accepted relationships remain conjectural, and secondly because a number of the connecting links will be missing in the bacterial kingdom as it exists to-day. With Pringsheim we are of the opinion that ‘ein wirklich wissenschaftliches System, das also mit Kritik aufgestellt ware, gar nichts anderes tun konnte, als zu ver- 394 PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA suchen, alle gegenseitigen Beziehungen der Organismen, die auf ir- gendeine Verwandtschaft hindeuten, wiederzugeben’. The practical application of this line of thought obliges us to face the question whether in the construction of the system morphological or physiological characters should have priority in the expression of relationships between those systematic groups which are no longer morphologically and physiologically homogeneous. In our opinion the binomial nature of nomenclature accentuates the demand that genera be systematic units which are characterized as well by a more or less complete morphological homogeneity as by a fundamental agreement in metabolic properties. This means, of course, that for the distinction of species within a genus only characters of secondary importance, such as biomeiric constants, hydrolytic abilities, the occurrence of pigments not determining the metabolic type, etc. can be applied. Consequently the question raised above must be answered in order to make possible an arrangement of the genera in such a way as to satisfy best the natural relationships existing between them. We have decided upon the use of morphological criteria as main guiding principle in the creation of systematic units above the rank of genera. In doing so we are fully aware that this choice may appear arbitrary. The resulting system unintentionally suggests that a mor- phological evolution has been primary and that in the various stages of morphological development an independent, though sometimes parallel, physiological differentiation has occurred afterwards. Yet it does not seem excluded at all that in special cases the order of events has been the reverse and that in reality parallel morphological evolu- tions have taken place in two physiologically different groups. There is however, only a limited number of examples in which morphological differentiation in a clearly defined physiological group strongly suggests itself, whereas the instances are numerous that a typical katabolic process is found in groups morphologically so un- related that affinities on the basis of physiology seem fully incompatible with the evolutionary idea. Moreover, in the former case the range of morphological differentiation extends but over a small number of closely related morphological units, as is e.g. clearly shown by the groups of purple bacteria. Therefore in a mainly morphological system such physiologically related groups will remain together, whilst, on 305 SELECTED PAPERS the other hand, in a mainly physiological system morphologically related groups will be widely dispersed. Guided by these general considerations we shall first of all give a survey of the morphological units which can be distinguished in the bacterial kingdom and of the way in which they seem to be mutually related. This will be followed by the differentiation of the morphol- ogically homogeneous groups on the basis of katabolism. It seems acceptable that the diversity of bacterial forms is the out- come of various independent morphological evolutions which have had their starting-point in the simplest form both existent and con- ceivable: the sphere. The existing forms suggest that four such evolutionary lines have to be distinguished. a. In the first place we can observe that certain bacteria in which the spherical form is still fully maintained display a tendency to form complexes of a more or less regular appearance. Whereas little signif- icance can be attached to the diplococcus form since its occurrence is inevitable in the reproduction process of the coccus, the formation of chains of four or more cocci is typical for several species. Obviously in this case one direction is preferential in cell division, the causative polar- ity becoming manifest owing to the fact that the cells remain attached. In those spherical bacteria in which division takes place in two directions which meet at right angles complexes may originate which possess the typical form of tetrads or tetracocci. If finally the division occurs in three directions of space the outcome may be the formation of regular packets or sarcinae. It must be emphasized, however, that the mentioned morphological groups comprise several transitional forms so that their delimitation, although desirable, is subject to many difficulties. The occurrence of both Gram-positive and Gram-negative represent- atives among the coccoid forms indicates a micromorphological evolu- tion which, although its direction cannot be traced, justifies a differen- tiation according to this character. The highest developmental stage in the group of spherical organ- isms is in all probability displaid by the cocci able to form endospores, the existence of which has recently been firmly established by an in- vestigation of Gibson [1935]. b. Motility occurs only sporadically among the cocci, yet it in- 306 PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA dicates that also the flagellated rods find their origin in this primitive group, the more so since the two typical modes of flagellation are al- ready encountered. It seems only logical that the short Gram-negative polarly flagellated rods, as for instance characteristic for the species of the genus Pseudo- monas, owe their origin to a Gram-negative monoflagellated coccus.* The polarity which arises from the attachment of a single flagellum to the sphere may easily induce a polar deviation in the form of the bacterium. In an analogous way the type of motility characteristic for polarly flagellated organisms, viz., the rotation of the cell according to its longitudinal axis, may well be responsible for a gradual deforma- tion of the cell resulting in the comma or vibrio form. The numerous Micrococeaceae Be ft aK ‘ Mvycobacteriaceac transitional forms between this morphological type and the spirillum forms leave no doubt as to the origin of the latter group. c. The appearance of more than one flagellum distributed over the * ‘The same argumentation holds, of course, for the evolution of polarly flagellated Gram-positive rods (the newly created genus Listerella) in relation to a motile Gram- positive coccus. 307 SELECTED PAPERS surface of a coccus may well have been the starting-point for the devel- opment of motile rods with peritrichous flagella. As a result of this the representatives of the Gram-negative colon group (in its widest sense) and of the Gram-positive genus Aurthia would have arisen. It lies at hand that the endospore forming rods with peritrichous flagella rep- ‘resent a higher stage of development of these groups. d. The fourth line of morphological evolution of the spherical cells seems to lead via the streptococci to the short Gram-positive rods in the group of lactic acid bacteria and corynebacteria. ‘The further development of these universally immotile bacteria can have given rise to the mycobacteria which apparently form the connecting link with the simpler actinomycetes. The various stages of these four independent evolutionary trends are represented in the following diagram (Fig. 1, see p. 307). It appears appropriate to assign the rank of a family to each one of the four domains described and the names Micrococcaceae, Pseudo- monadaceae, Bacteriaceae and Mycobacteriaceae are indicated. The various morphological groups distinguished in each family will be designated as tribes. In accordance herewith the following tribes will be recognized : Family Tribes Micrococceae Streptococceae Micrococcaceae ; Sarcineae Sporosarcineae Pseudomonadeae Pseudomonadaceae Vibrioneae Spirilleae { Bacterieae \ Bacilleae | Corynebacterieae Bacteriaceae Mycobacteriaceae — i \ Mycobacterieae Next we have listed the known, well distinguishable types of kata- bolism and so classified the bacteria answering the morphological re- quirements of the tribe according to their characteristic type of kata- bolism. As has already been argued organisms which on the basis of this procedure are collected in one and the same group should together constitute a genus. 308 PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA In several instances this classification does not involve any difficulty, because the energetic requirements of many bacteria can be met by only one type of katabolic reaction. There are, however, also numerous cases in which it is clear that the organism can derive its energy from two or even more clearly distinct types of katabolism. This holds e.g. for the so-called facultatively anaerobic bacteria which in the presence of air are characterized by an oxidative katabolism (respiration), but which, under anaerobic conditions, depend upon some special type of fermentation. In those cases it is, of course, desirable to classify the organism in question according to its most characteristic type of kata- bolism, that is the type which permits the distinction from otherwise related organisms. This implies that for organisms capable of develop- ment under anaerobic conditions the katabolic process involved in this mode of life has been determinative, regardless of the question whether or not the organism also possesses a respiratory mechanism. If two different types of anaerobic katabolism, e.g. saccharolytic and proteolytic, are represented, the latter, as being the rarer, has been decisive. In this way the system of classification represented in table I has been obtained. The principles underlying this system would logically imply the creation of new generic names for all actually occurring combinations of fundamental morphological characters and special katabolic types. Experience shows, however, that in the majority of cases the natural groups obtained in this way coincide in all major points with various genera recognized in the systems now in use, at the same time demon- strating that our classificatory principles have, more or less unconsci- ously, already been applied by many of our predecessors. In all these cases we have, for practical reasons, maintained these current generic names, although naturally the generic diagnoses had to be amended more or less considerably. In a few instances we have rejected a current generic name, although its diagnosis was sufficiently suited to justify its use. This was done because the generic name in question might give rise to confusion as a result of current nomenclature. Thus we have dropped the names Thiobacillus and Rhodobacillus, since these names wrongly suggest that they cover sporeforming bacteria. On analogous grounds we have used the generic name Sulfospirillum instead of Thiospira. 309 TABLE I. THE GENERA TO BE DISTINGUISHED IN THE VARIOUS Cephalotrichous (and related immotile) rod-shaped bacteria Fam. Pseudomonadaceae ~~ SN = R g S S & = SS S n-s gg: ales aS eae oy BS o A 5 10 Patan eh © @ is del om EE ars S ‘x ne oe ae Photo-autotrophic: a. Bacteria with green pigment complex (Chlorobacteria) : ‘ - ~ - b. Bacteria with purple pigment complex. ( Thiorhodaceae) . : : , ‘ 5 Thiospirillum Chromatium Thiothece Photo-heterotrophic: a. Bacteria with brown pigment complex (Phaeobacteria) . : ; ‘ Phaeospirillum — Phaeomonas b. Bacteria with purple pigment complex (Athiorhodaceae) . : : ; : : ‘ Rhodospirillum Rhodovibrio Rhodomonas Chemo-autotrophic: a. Bacteria which oxidize inorganic sulfur compounds (Leucothiobacteria) —. , . | Sulfospirillum ~ Sulfomonas b. Bacteria which oxidize ferrous iron (and manganese) : ‘ ; ‘ : <- Te - Didymohelix — Sideromonas a) c. Bacteria which oxidize ammonia . : : - - Nitrosomonas d. Bacteria which oxidize nitrites ‘ ‘ ‘ -- - Nitrobacter Chemo-heterotrophic: I. Bacteria with obligatory oxidative Spirillum Vibrio Acetobacter katabolism Pseudomonas Rhizobium ; Azotobacter II. Fermentative: Listerella? a. ‘Mixed acid’ fermentation . - ~ - b. Symmetric dimethylglycol (2,3- “butylene- glycol) fermentation . : - - . Aeromonas ? c. Alcoholic fermentation —. : : : - - Zymomonas d. Butyric acid (butylalcohol and acetone) fermentation ; ‘ : : , ; - ~ - e. Protein fermentation . : , : : - - = f. Propionic acid fermentation . . . - — = g. Homofermentative lactic acid fermentation F : ‘ : : 3 = = = h. Heterofermentative lactic acid fermentation : : : : : ‘ - - a 7. Sulfate reduction i - Desulfovibrio = k. Methane fermentation (CO, -reduction) - - Methano- bacterium? TRIBES ON THE BASIS OF KATABOLIC PROPERTIES Spherical bacteria Fam. Micrococcaceae Permanently immotile rod-shaped bacteria Fam. Mycobacteriaceae Peritrichous (and related immotile) rod-shaped bacteria Fam. Bacteriaceae ' N nm n U = ae 2g a a bo 8 so Ee s — 3 Ee oS aa < S 82 8 x as aes ~ 8 Cos S = Paes en 2 co 8 hh som S = avs oS o) ie, 3 sos 3858 s S aaa ‘a8 o= ~ WS = Ss Gus 2S Ss Se 8 Gs gins of § Soo as cS 5 ASR g ashrcias mS wD Bos 2S 3g § <= a ® p(s flees ae oo SS oes - & oS con 4ZES ee ES as 5 ac 4 2 2 a a 50 Ss 69 em va r) oR 0) - co) -— Oo oy ae o a) a oO 2 ee Za 4 & 4 9 ze wo 2 Bites: £2 3 oe es woe cna gf aa 64 cea oe oe ae a ch Re oF Ae So = re a Chlerobium — = = = Est _ Thiopoly- Thiopedia coccus > Thio- - - = = == = Sarcina Rhodococcus _ — - = = = = Achro- = = = = = = = matium Siderocapsa - - — - ~ - - Nitrosococcus = — ~ = = = = Neisseria? Gaffkya Sporo- - Coryne- Myco- Kurthia Bacillus Micrococcus Sarcina sarcina bacterium bacterium | Alcali- genes ~ ~ - - — - Bacterium - - - - - - - Aerobacter Aero- bacillus = Zymo- = — - - - Zymo- sarcina bacillus — Butyri- — — — = = Clostri- sarcina dium Veillonella - - Peptostrep- | Fusiformis - - Peptoclos- Peptococcus tococcus ? tridium - — - — Propioni- — - = bacterium = Pedio- - Strepto- Strepto- Thermo- - - coccus coccus bacterium bacterium - — = Beta- Beta- = = = coccus bacterium Methano- Methano- - - = - — = coccus sarcina SELECTED PAPERS It is only self-evident that in a number of cases we have been obliged to establish new genera. The name Desulfovibrio for the sulfate reducing bacteria first described by Beijerinck and Van Delden needs no further comment. Zymomonas has been chosen as a suitable name for polarly flagellated bacteria causing alcoholic fermentation like Pseudomonas lindnert (Termobacterium mobile Lindner), Aeromonas for similar organisms which ferment sugars in a way probably closely related to the fermentation type characteristic of the genera Aerobacter and Aerobacillus. The genus Methanobacterium has been created for the methane producing rod-shaped bacteria described by Séhngen [1906]. Similarly Methanosarcina is a new generic name for the related sarcina, Methanococcus for the small spherical forms causing this type of fermen- tation [Groenewege, 1920]. Peptococcus and Peptostreptococcus represent the anaerobic proteolytic micrococci and streptococci respectively ; Butyrisarcina has been adopted as a designation for the butyric acid producing Zymosarcina maxima (Lindner) Smit and possibly allied forms. Finally the name Zymobacillus has been introduced for the faculta- tively anaerobic sporeforming bacteria of the type Bacillus macerans Schardinger. Provisionally, and with great reservation, we have added two genera, Phaeospirillum and Phaeomonas, for bacteria belonging to the group of organisms which Utermohl [1924] has designated as Phaeobacteria. These show very close affinities with the Athiorhodaceae, both morphol- ogically and physiologically. It is entirely possible that a careful study of these brown bacteria will reveal facts which would justify their inclusion in the corresponding genera of the purple bacteria, Rhodo- spirillum and Rhodomonas. In the composition of the table some difficulty was experienced, however, in view of the fact that for a few of the existing genera the katabolic properties are still insufficiently known. In some cases the relationships of such genera to organisms, whose position could be readily determined, were more or less evident, and therefore their place in the table has tentatively been indicated. This holds e.g. for the genera Aeromonas and Fustformis. In other cases even such indica- tions are lacking, and so a few genera well-known to the medical bac- teriologist could not yet be classified. This applies e.g. to Pasteurella, Dialister, Haemophilus, Listerella and to the species Corynebacterium diph- theriae which, owing to its indubitable fermenting capacity, cannot be 312 PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA grouped with the obligatory oxidative saprophytic corynebacteria. For analogous reasons, but now because of the scantiness of mor- phological data, the position of the genera N?trobacter, Didymohelix, Sideromonas, Kurthia, and Methanobacterium must be deemed privisional. The preliminary nature of the table manifests itself also by the circumstance that in a restricted number of cases it was considered desirable to maintain a number of genera which according to the principle adopted should have been amalgamated. Because we have felt that there possibly exist sufficiently essential differences between such genera as Azotobacter, Acetobacter, Kurthia, and the genera with which they are grouped, we have refrained from rashly abolishing them, although the morphological differentiation which is at the basis of our system does not suffice to keep them sepa- rated. In some instances a separation is already fully justified on the basis of the outcome of the Gram-stain. In this connexion we refer to the genera Kurthia-Alcaligenes, and Listerella-Pseudomonas, the species of the first-mentioned genus of each couple being Gram-positive, those of the second one Gram-negative. We may trust, moreover, that future investigations will provide the means for a more adequate generic differentiation in the cases under discussion. When we now proceed to an examination of the table as a whole, we must first of all realize that its two-dimensional nature evidently does not permit of a true representation of the natural relationships of the various genera. The postulated many-sided morphological evolution starting from the group of spherical organisms in reality asks for a grouping of the three families of rod-shaped organisms around that of the Micrococcaceae. Notwithstanding this shortcoming of the table it is obvious that in many of the physiological groups distinguished the distribution of the genera is far from random, as a glance at the upper half of the table shows convincingly. Apparently there is a close correlation between the phototrophic and chemo-autotrophic modes of life and morpholo- gy. In this domain it is therefore rather inessential whether a physiol- ogical or a morphological evolution is assumed to have been primary. The mutual relationships remain clearly expressed in either case. Another physiological group, viz., that of the true lactic acid bac- teria, also shows a definite correlation between physiological and morphological properties. es) SELECTED PAPERS A further striking feature of the table is undoubtedly the occurrence of an obligatory oxidative metabolism in nearly all morphological types. Although this is partly due to the fact that in the present state of our knowledge it is impossible to carry through a subdivision of the aerobic metabolism into different types in a way comparable with that which has been applied in the case of fermentative metabolism, yet the table suggests that in the group of the obligatory oxidative heterotrophic bacteria the main morphological evolution has occurred. In this line of thought the physiological evolution will have taken place in the different stages of this primary development. In this con- nexion it must be remembered that in several cases the fermentative metabolism is only a supplement to the oxidative mode of life. The assumption made would also account for the more or less haphazard distribution of the various katabolic types in the different morphologi- cal units. A few words remain to be said with regard to the distribution of the Gram-positive and Gram-negative genera in the system. As has been remarked before we attach sufficient importance to this character to require that a genus be homogeneous in this respect. Yet it appears that the system given does not result in a clear-cut separation of the Gram-positive and the Gram-negative genera. For the family of the Micrococcaceae this differentiation has already been remarked upon. In the family of the Pseudomonadaceae Gram-negative genera are strongly predominant, whereas on the contrary in the family of the Mycobac- teriaceae only Gram-positive organisms are encountered. Finally it does not seem excluded that in the family of the Bactertaceae a parallel devel- opment has taken place in both Gram-positive and Gram-negative forms. If the reported occurrence of Gram-negative Bacillus species should be confirmed a division of this genus will become inevitable which might lead to a clarification of the affinities in the family as a whole. It will not have escaped attention that in the foregoing most ‘bacte- ria’ which, already in previous systems, have been separated from the so-called Eubacteriales have been left out of consideration. This holds for the Spirochaetales, the Myxobacteriales, the Actinomycetales (p.p.) and the Chlamydobacteriales. The Thiobacteriales in the sense of Buchanan have been included with the exception of the genera Beggiatoa, Thio- thrix and Thioploca. The representatives of the latter genera show such oo PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA unmistakable morphological affinities with the Cyanophyceae that in our opinion they must be considered as colourless derivatives of the genera Oscillatoria, Phormidium and Schizothrix (see also Pringsheim l.c.). As for the Spirochaetales and the Myxobacterzales they form well-defined groups whose affinities with the Eubacteriales are at least doubtful. Therefore they may well be ignored here. On the other hand the re- lationship of the Actinomycetes to the Eubacteriales is very clear; as is generally accepted they represent stages of higher development of the Mycobacterieae. At the same time they form such a special and extensive group by themselves that their classification falls outside the scope of this study. The remaining order of the Chlamydobacteriales cannot so easily be disposed of. At first sight the representatives of genera like Leptothrix, Crenothrix and Sphaerotilus do not seem at all related to any of the Eu- bacteriales. Although we cannot yet express any definite opinion regard- ing the taxonomy of this group it appears probable that it is very heterogeneous and that it harbours organisms (Crenothrix!) which are closely related or belong to the Eumycetes, as well as organisms which, like Sphaerotilus natans, are related to sheath-free filamentous Eubacte- riales (Bacillus, Mycobacterium and Thermobacterium species). In this respect it is tempting to draw the attention on the one hand to such organisms as Leptothrix hyalina (Migula) Bergey et al. and Sphaerotilus paludosus Smit [Smit, 1934] which, although they are usually reckoned to belong to the Chlamydobacteriales, are reported to lack a sheath, and on the other hand to a bacterium, like Bacillus funicularius, which, al- though a true Bacillus species, forms a distinct sheath under special nutritional conditions [Kluyver and Van Niel, 1926]. Moreover, the latter phenomenon is also frequently encountered in the group of the true lactic acid bacteria [Orla-Jensen, 1919]. Table II gives a survey of the families, tribes and genera of the order Eubacteriales. After all that has already been said this table will not need any further comment. In concluding we wish to make the following remarks. As has been amply set forth in the discussion of the general prin- ciples of classification it is necessary both from a scientific and from a practical standpoint to aim at a system which is worthy of the design- ation ‘natural’. We fully realize that the result of our own classifica- tory attempt has only very imperfectly approximated this goal. on TABLE II. Thiospirillum Phaeospirillum Rhodospirillum Sulfospirillum Spirillum ieibe | Spirilleae Chromatium Tribe Rhodovibrio Vibrioneae ae wohelix brio Desulfovibrio —, Thiothece Phaeomonas Rhodomonas Sulfomonas Sideromonas Nitrosomonas Nitrobacter Acetobacter Pseudomonas Rhizobium Azotobacter Listerella Aeromonas XK ymomonas Tribe Pseudo- monadeae Methanobacterium SELECTED PAPERS Tribe Sarcineae Family Pseudo- monadaceae Tribe Micrococceae Tribe Streptococceae | | [ Thiopedia Thiosarcina Gaffkya Sarcina XK ymosarcina Butyrisarcina Pediococcus | Methanosarcina Family Muicro- coccaceae Chlorobium Thiopolycoccus Rhodococcus Achromatium Siderocapsa Nitrosococcus Neisseria Micrococcus Veillonella Peptococcus Methanococcus | Streptococcus Betacoccus Peptostreptococcus Y Actinomycetales THE FAMILIES, TRIBES, AND GENERA OF THE EUBACTERIALES Bacillus | Aerobacillus fire Kymobacillus . Clostridium Bacilleae Peptoclostridium Family Bacteriaceae Kurthia ; Alcaligenes Tribe Bacterium Bacterieae Aerobacter Corynebacterium Dib pele | Corynebacterieae Propionibacterium Fusiformis Family Myco- | bacteriaceae Tribe | Thermobacterium Mycobacterieae | Mycobacterium PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA On the other hand it seems to us that the system as outlined in this paper marks a definite progress as compared with previous ones. To justify this statement it will suffice to bring out two features. Firstly the system given is characterized by its simplicity as well as by its consistency. The principle that a genus in all cases is defined by the fundamental morphological and katabolic properties makes for units which are much more easily distinguishable than the majority of the genera in the previous systems. In addition the system offers the advantage of permitting the ready incorporation of organisms with still unknown combinations of morphological and physiological char- acters. Therefore it may lay claim to the epithet ‘rational’. But secondly it seems to us that to a certain extent the system, al- though artificial, also answers the requirements of a true natural system. For the natural relationships between the various bacteria which in the present state of our knowledge can be vaguely perceived find a definite expression in the aspect of the system. That this aspect is not final is certain. Future investigations will deepen our insight into the natural relationships of different bacterial groups and the system will have to be modified accordingly. Nevertheless it appears likely that the idea which is largely respon- sible for the outline, viz., the occurrence of both morphological and katabolic evolution in the bacterial kingdom, will reappear in future classifications, thus perhaps justifying the use of the word prospects in the title of this paper. In the meantime the system in its present imperfect shape may well serve the purpose of stimulating interest and research in this field. 317 SELECTED PAPERS APPENDIX LIST OF GENERA INCLUDED IN THE TABLES AND THEIR DIAGNOSIS* I. Tribe Sprrilleae 1. Thiospirillum Winogradsky, 1888. Spiral bacteria, motile by means of cephalotrichous flagella. No endospores formed. Photo-autotrophic, containing a red to purple pigment complex. Nor- mally reducing carbon dioxide with the simultaneous oxidation of H,S or other inorganic sulfur compounds. The type species is Thiospirillum sanguineum (Ehrenberg) Winogradsky. 2. Phaeospirillum nov. gen. Spiral bacteria, motile by means of cephalotrichous flagella. No endospores formed. Photo-heterotrophic, containing a brown pigment complex. The type species to be assigned in the near future. 3. Rhodospirillum Molisch, 1907. Spiral bacteria, motile by means of cephalotrichous flagella. No endospores formed. Photo-heterotrophic, containing a red to purple pigment complex. The type species is Rhodospirillum rubrum (Esmarch) Molisch. 4. Sulfospirillum nov. gen. Syn.: Thiospira Wislouch, 1914. Spiral bacteria, motile by means of cephalotrichous flagella. No endospores formed. Chemo-autotrophic, oxidizing H,S or other inorganic sulfur com- pounds. The type species is Sulfospirillum winogradskyi (Omelianski). 5. Spirillum Ehrenberg, 1830. Spiral bacteria, motile by means of cephalotrichous flagella. No endospores formed. Chemo-heterotrophic, oxidizing various organic compounds. Gram negative. The type species is Spirillum undula (Miller) Ehrenberg. * Although the diagnoses of those genera which have been retained from earlier systems of classification have nearly all been subject to more or less considerable amendments, we have indicated this only explicitly by the suffix ‘Emend.’ in those cases in which the amendment introduced involved the elimination of well established species from the genus. Secondly it has to be emphasized that the following generic diagnoses have pur- posely been kept broad, although they might, in most cases, well have been elaborat- ed on the basis of the available information concerning the representatives described until now. We have refrained from this in order to permit the future incorporation of species which deviate in minor respects from the collectivity now included in the genus, but which, nevertheless, show the morphological features of the tribe, and the katabolic activity deemed characteristic of the genus. 318 PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA II. Tribe Vibrioneae 1. Chromatium Perty, 1852. Slightly curved rods, motile by means of cephalotrichous flagella. No endo- spores formed. Photo-autotrophic, containing a red to purple pigment com- plex. Normally reducing carbon dioxide with the simultaneous oxidation of H.S or other inorganic sulfur compounds. The type species is Chromatium okenii Perty. 2. Rhodovibrio Molisch, 1907. Slightly curved rods, motile by means of cephalotrichous flagella. No endo- spores formed. Photo-heterotrophic, containing a red to purple pigment complex. The type species is Rhodovibrio parvus Molisch. 3. Didymohelix Griffith, 1853. Syn.: Gallionella Ehrenberg, 1838. Curved rods, motile (?). No endospores formed. Chemo-autotrophic, oxidizing ferrous iron. The ferric hydroxide is deposited in the form of a twisted band which carries the organism at the top. The type species is Didymohelix ferruginea (Ehrenberg) Griffith. 4. Vibrio Miller, 1773. Curved rods, motile by means of cephalotrichous flagella. Occasionally spiral forms are present. No endospores formed. Chemo-heterotrophic, oxidizing various organic compounds. Gram-negative. The type species is Vibrio comma (Schroeter) Bergey et al. 5. Desulfovibrio nov. gen. Curved rods, motile by means of cephalotrichous flagella. Occasionally spiral forms are present. No endospores formed. Chemo-heterotrophic, anaerobic, oxidize organic substances with the simultaneous reduction of sulfate to sulfide. The type species is Desulfovibrio desulfuricans (Beijerinck), Syn.: Spirillum desul- furicans Beijerinck. III. Tribe Pseuwdomonadeae 1. Thiothece Winogradsky, 1888*. Ellipsoidal to rod-shaped bacteria, motile by means of cephalotrichous flagella. No endospores formed. Photo-autotrophic, containing a red to purple pigment complex. Normally reducing carbon dioxide with the simultaneous oxidation of H,S or other inorganic sulfur compounds. The type species is Thiothece gelatinosa Winogradsky. * Tt seems highly doubtful whether the other genera created by Winogradsky for similarly shaped organisms (Thiocystis, Lamprocystis, Amoebobacter, Thiodictyon) are sufficiently different to be maintained. Also various of the purple sulfur bacte- ria which have been described as Chromatium species should be reckoned to Thiothece. og SELECTED PAPERS Phaeomonas nov. gen. Ellipsoidal to rod-shaped bacteria, motile by means of cephalotrichous flagella. No endospores formed. Photo-heterotrophic, containing a brown pigment complex. The type species is Phaeomonas varians (Ewart), Syn.: Streptococcus varians Ewart. Rhodomonas Orla-Jensen, 1909. Emend. (!).* Syn.: Rhodobacillus Molisch, 1907. Ellipsoidal to rod-shaped bacteria, motile by means of cephalotrichous flagella. No endospores formed. Photo-heterotrophic, containing a red to purple pig- ment complex. The type species is Rhodomonas palustris (Molisch), Syn.: Rhodobacillus palustris Molisch. Sulfomonas nov. gen. Syn.: Thiobacillus Beijerinck, 1904. Ellipsoidal to rod-shaped bacteria, motile by means of cephalotrichous flagella. No endospores formed. Chemo-autotrophic, oxidizing H,S or other inorganic sulfur compounds. The type species is Sulfomonas thiopara (Beijerinck), Syn.: Thiobacillus thioparus Beijerinck, Sideromonas Cholodny, 1922. Ellipsoidal to rod-shaped bacteria, immotile (?). No endospores formed. Chemo-autotrophic, oxidizing ferrous iron. ‘The type species is Sideromonas confervara Cholodny. Nitrosomonas Winogradsky, 1892. Ellipsoidal to rod-shaped bacteria, motile by means of cephalotrichous flagella. No endospores formed. Chemo-autotrophic, oxidizing ammonia to nitrite. The type species is Nitrosomonas europaea Winogradsky. Nitrobacter Winogradsky, 1892. Ellipsoidal to rod-shaped bacteria, either immotile or motile by means of cephalotrichous flagella. No endospores formed. Chemo-autotrophic, oxi- dizing nitrite to nitrate. The type species is Nitrobacter winogradskyi Buchanan. Acetobacter Beyerinck, 1900. Ellipsoidal to rod-shaped bacteria, either immotile or motile by means of cephalotrichous flagella. No endospores formed. Chemo-heterotrophic, oxi- dizing various organic compounds, with a marked tendency to form acids as products of incomplete oxidation. Adapted to life in acid media. The type species is Acetobacter pasteurianum (Hansen) Beijerinck. Pseudomonas Migula, 1894. Ellipsoidal to rod-shaped bacteria, either immotile or motile by means of * Tt has to be clearly understood that the diagnosis of this genus as given above is not at all in agreement with the diagnosis given by Orla-Jensen who uses the name as a synonym for Chromatium. Nevertheless, the name in question is adopted here to match the names of the related genera Rhodospirillum, Rhodovibrioand Rhodococcus. 320 10. Il. 12. 13. 14. 15. IV. PROSPECTS FOR A NATURAL SYSTEM OF CLASSIFICATION OF BACTERIA cephalotrichous flagella. No endospores formed. Chemo-heterotrophic, oxi- dizing various organic compounds. Gram-negative. Adapted to life in neutral to slightly alkaline media. The type species is Pseudomonas fluorescens (Fligge) Migula. Rhizobium Frank, 1889. Ellipsoidal to rod-shaped bacteria, motile by means of cephalotrichous flagella. Branched and swollen involution forms. No endospores formed. Chemo-hetero- trophic, oxidizing various organic compounds. The association of the bacteria and Leguminosae fixes atmospheric nitrogen (root-nodules!). The type species is Rhizobium radicicola (Beijerinck). Azotobacter Beijerinck, 1901. Ellipsoidal to rod-shaped bacteria, motile by means of cephalotrichous flagella. No endospores formed. Chemo-heterotrophic, oxidizing various organic sub- stances. Capable of fixing atmospheric nitrogen. The type species is Azotobacter chroococcum Beijerinck. Listerella Pirie, 1927. Short rod-shaped bacteria, motile by means of cephalotrichous flagella. No endospores formed. Chemo-heterotrophic, oxidizing various organic compounds. Gram-positive. The type species is Listerella monocytogenes (Murray et al.) Pirie. Aeromonas nov. gen. Short rod-shaped bacteria, motile by means of cephalotrichous flagella. No endospores formed. Chemo-heterotrophic, oxidizing various organic com- pounds, and capable of fermenting carbohydrates with the production of carbon dioxide and hydrogen, and 2,3-butylene glycol (?). Gram-negative. The type species is Aeromonas liquefaciens (Beijerinck), Syn.: Aerobacter liquefaciens Beijerinck,. A ymomonas nov. gen. Ellipsoidal to rod-shaped bacteria, motile by means of cephalotrichous flagella. No endospores formed. Chemo-heterotrophic, oxidizing various organic com- pounds, and capable of fermenting carbohydrates with the production of carbon dioxide, ethyl alcohol, and lactic acid. Gram-negative. The type species is