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ALBERT JAN KLUYVER
HIS LIFE AND WORK

ALBERT JAN KLUYVER

HIS LIFE AND WORK

BIOGRAPHICAL MEMORANDA

SELECTED PAPERS
BIBLIOGRAPHY AND ADDENDA

q&?(

1959

NORTH-HOLLAND PUBLISHING COMPANY. AMSTERDAM

INTERSCIENCE PUBLISHERS INC.. 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

This 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-
ver's 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-

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.

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 Gourant' of 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.

CONTENTS

PREAMBLE VII

CONTENTS XI

ILLUSTRATIONS opposite page 48

PART ONE
BIOGRAPHICAL MEMORANDA

FROM YOUTH TO PROFESSOR 3

1 888- 1 905 The early years 3

1 905-1 9 16 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

1 928- 1 938 Consolidation 25

1939-1945 War and occupation 34

1 945- 1 956 Recovery; the final years 3^

KLUYVER AS SEEN BY HIS PUPILS 49

KLUYVER S CONTRIBUTIONS TO MICROBIOLOGY AND
BIOCHEMISTRY

Introduction
The prologue

r„",

70

The emergence of a programme 80

The unfolding of the programme 84

\i

CONTENTS

Further developments: phosphorylation and the nature of

assimilation 91

The nature of biocatalysis 98

Comparative biochemistry 114
Industrial microbiology and the submerged culture method for the

study of mould metabolism 130

The classification of micro-organisms 138

Variability of micro-organisms 146

Epilogue 152

TWO FUNERAL ORATIONS I 57

O. Bottema 157

A. van Rossem 159

PART TWO
SELECTED PAPERS

MICROBIOLOGY AND INDUSTRY 165

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 'Microbiologic en
Industrie'.

Translation of: ' Alicrobiologie en Industrie' . Rede uitgesproken bij de aanvaarding
van het ambt van Hoogleeraar in de Algemeene en Toegepaste Alicrobiologie aan de
Technische Hoogeschool te Delft op Woensdag 18 Januari 1922. Delft, 1922.

UNITY AND DIVERSITY IN THE METABOLISM OF

MICRO-ORGANISMS 186

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 verscheidenlrid in de stofwisseling der microben'.
Chemisch Weekblad 2 1 , 266, 1924.

die einheit in der biochemie (Mil H. J. L. Donker) 21 I

1. Einleitung - 2. Die Zuruckfiihrung der Dissimilationsprozesse
auf katalytische Wasserstoffiibertragung, falls Zucker als Substrate

CONTENTS

fungieren - 3. Der Chemismus der katalytischen Wasserstoffuber-
tragung - 4. Anwendung der gegebenen Vorstellung des Chemismus
der katalytischen Wasserstoffubertragung auf die Zuckerdissimila-
tionsprozesse - 5. Anwendung der Wasserstoffubertragungstheorie
auf sonstige Dissimilationsprozesse - 6. Die Assimilationsvorgange
im Lichte der Wasserstoffubertragungstheorie - 7. Uberblick fiber
die aufgestellte Theorie der katalytischen Wasserstoffubertragung
als Kern des biochemischen Geschehens - 8. Schlussbetrachtung.
Chemie der J^elle 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

Conference faite a l'Assemblee generale de l'Union Internationale des
Sciences biologiques a Bruxelles, le 13 Juillet 1 93 1 .
Annales de J^ymologie Serie II, 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.
Zentralblatt Jiir Bakteriologie, Parasitenkunde und Infektionskrankheiten.
Abt. II, 94, 369, 1936.

life's frlnges 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 XX] le Nederlandsch Natuur- en Geneeskundig Congres, 82, 1937.

DIE KOHLENSAURE IM STOFFWECHSEL DER LEBEWESEN 349

Yortrag gehalten im Festsaal der Finnischen Technischen Hochschule in
Helsinki den 14 Marz 1939.

Suomen Kemistilehti 12, 81, 1939.

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, /, 1947.
Naturens Verden 30, 161, 1946.

HOMO MILITANS 393

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 90th 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, !953-

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 Sanitd. Roma, 1953.
p. 71.

CONTENTS

MICROBE AND LIFE 503

Address delivered in the Dutch language at the meeting of 'De ver-
enigde afdelingen der Koninklijke 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,
rgj^^Sj^ P- 225.

PART THREE

BIBLIOGRAPHY AND ADDENDA

PAPERS BY A. J. KLUYVER 527

DOCTOR'S THESES PREPARED UNDER KLUYVER's DIRECTION 537

PAPERS FROM THE LABORATORY FOR MICROBIOLOGY AT DELFT,
PUBLISHED BY KLUYVER's COLLABORATORS I922-I956 540

PAPERS OF THE BIOPHYSICAL RESEARCH GROUP UTRECHT-
DELFT 548

STAFF MEMBERS OF THE LABORATORY FOR MICROBIOLOGY
I922-I956 554

HONOURS OF A. J. KLUYVER 556

ARTICLES DEDICATED TO A. J. KLUYVER AND HIS WORK 558

OBITUARY ARTICLES 559

'IN MEMORIAM A. J. KLUYVER' 560

Published in the 'Nieuwe Rotterdamse Courant' of May 15, 1956.

APOLOGY 565

PART ONE

BIOGRAPHICAL MEMORANDA

FROM YOUTH TO PROFESSOR

THE EARLY YEARS

i 888-1 905

Albert Jan was the second child and only son sprung from the mar-
riage of Jan Cornells Kluyver and Marie Honigh. His father, born
May 2, i860, 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 Cornells 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 Cornells 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 Cornells 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 Cornells 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 Rijndijk; 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, 1 9 10, 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

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
I 905-1916

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 2 1 , 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, 19 10, 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 191 1
in the 'Pharmaceutisch Weekblad' under the title, 'The 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 Jungling', a widely
read novel at that time: 'Zwar blieb seine Wissenschaft einigermassen
an der Oberflache; er sprach allerlei vom Chlorophyll, aber was es
fur 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.

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 191 1, 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 191 1 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' [191 1]. 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 191 2 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

7

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,

On May 15, 19 14, 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 191 4, 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, 191 5. 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 Eja 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, 1 91 6, Albert Jan Kluyver and Helena Johanna van

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 10 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

1 91 6-1 92 1

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 ol
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 19 19 budget.

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
beforf starting his work with the Colonial Institute, an entirely differ-
ent possibility opened up. This was a position with the Oil Manufac-
turing Company Tnsulinde'. 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.

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,
1 919, on the S.S. '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 Tnsulinde' under way. But the
high-flown expectations were soon dashed, for in 1 920-1 921 a serious
economic crisis also developed in the Netherlands' East Indies, and
Tnsulinde' 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-

1 1

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 1920-192 1 owing to the retirement of M. W.
Beijerinck 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 Beijerinck'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 Tandjong 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

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 thatBeije-
rinck had introduced elicited also Kluyver's interest and admiration,
especially in later years.

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.
'Beijerinck', 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 of a 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.

•I

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.

i">

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 Beijerinck'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 Beijerinck.

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 Beijerinck, 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

'7

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 Beijerinck 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 Beijerinck 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

1923-1927

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 191 1, 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-

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'.

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 funicular ius, 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 Sohngen 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

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
gratifying that, exactly at a time when technical developments were suf-
ficiently far advanced to initiate the stormy growth of a microbiological
industry, Kluyver appeared as the man who was so eminently suited
to resume the connexion, especially because the studies on the quan-

23

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

- 1 ?

BIOGRAPHICAL MEMORANDA

death of Sohngen, 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 Beijerinck'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 kluyveri. 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 Wiken, 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!'

_>.,

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 10 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 1 1 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 1 1 . 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 Utrccht-Dclft 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. //. 119.
1 951; 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

S3

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

I 939- I 945

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 ;

34

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

33

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 Beijerinck'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'esperer pour entreprendre, ni de
reussir pour perseverer', 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

37

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

i 945-1 956

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. 'Beijerinck 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-
tialities 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

39

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 on a 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 changed. 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.

4i

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

43

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

45

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 9, 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-

47

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; I'll see you again
on Monday' . . .

48

1. The young professor; 1926.

2. Kluyver amidst his colleagues of the Chemistry Division; 1935.

J.pku X Hid a ISahFtxI 'j Mu. bif*i3t fin. 'dj. 3u }>*&' - *-* y
■ x & U&i i$ , U #<*/ had*- '+** tester h *i Aj±^iWJ %. iduu^

* I Sf Co Hi »*£.:Ua~. .

- ' j ^ ( ""-,; -if .jlX. %M A •* fa* <-j{ -h fin >~ , "fl 3 - hi)" - 5

ttiii ... 7* hi-', t I* M ~ - /" U*» k ft *-tft m fy *Lj

""7/? j ~~S • hi it ' r '

DS*

< ; fe^|

dhnojiv* Sv&trf* fchao )yuul h^^udi tu SL.L3k<tvJ, Se&. (

fjlplidi <uJ Ifart4lfatfjn> CKduti&i ^foyt4L /& cHle\tfn*.

/4 <Vi*j/«& 4lffc*i fl <»ffA.t W^i.* |Jw*-/".

V^ Jw>. ^ iw'^.f &tf ^/ ^Xt^ ir4i^ A«e^^ t iLr

tit* Ha U^ h%th ***.{*$ Mjp *,&}( * W rj Hi cJlfcu h£tUa )

Siht^ii d" lofC aJ hdtktb I Hit *cuflh*tt*r J 2*S* C\ H^^tt/U
tyMRMtWi* Ik u. JtMx tncL<U», '« kit a? <iul kvj&J

jiSk.d*

3. A manuscript page. J. Bact. 72, 406, 1956.

4. The laboratory in winter.

5. A commemoration at the laboratory; 1933.

h^ l - vv- '"H^.-ifk '^Lj^^MflEI

arms-wm^i *

WW^^M. K

1 ^m ^ .
1 ' J|| •' ^

P 1 | * J

■

Ff •* ^^

Vf-

6. Orla-Jensen visiting the

laboratory; 1929.

1

7. Kluyver and a student;

1

1937-

8. An excursion; 1938.

9. Kluyver and Van Niel;

1955-

i V3

^T

a

?

-■n

1

?

1

1

1

l

v.*L fk/.yf

8

.^^

io. Promoter Kluyver at the promotion ceremony of Prince Bernhard of the Netherlands; 1951

11. Prince Bernhard, Dr. Techn. Sci. h.c, and Kluyver after the promotion; 1951.

12. Kluyver as Rector Magnificus at the Student Union; 1948.

h

13. Kluyver as a youth; 1898.

14. Kluyver with two of his sisters;
1908.

On the roof of the Student
Union, Societeit Phoenix; 1903.

16. Kluyver with his fiancee; 1916.

17. Kluyver and Raden Mas Iso
Reksohadiprodjo; 1920.

The young Kluyver family in the
tropics; 1919.

Holidays with his family^in the
Biesbos; 1933.

20. Three generations of the Kluyver
family; 1930.

2 1 . Another three generations of the
Kluyver family; 1950.

5.W*JL

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 I 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 Beijerinck, 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

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-

55

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. Then
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

57

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, Airs. 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

59

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'etait plus fort que moi'. Similarly, in his lecture enti-
tled 'Homo militans' he called attention to the simple words, 'Par
honnetete', 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:
'J'ai fait a Delft, il y a deja plusieurs annees, un trop court sejour, dont
je garde un souvenir tres vif et tres precieux. Votre Maitre, Monsieur
le Professeur Kluyver, m'y avait recu avec cette simplicite charmante
qui etait la sienne et les quelques jours passes aupres de lui ont tres
profondement oriente ma carriere scientifique. Son rayonnement etait
immense et il existe dans le monde entier des chercheurs qui se con-

65

BIOGRAPHICAL MEMORANDA

siderent comme ses eleves. Permettez moi de me ranger dans leur
nombre et de vous dire combien je suis attache a sa memoire.'

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 Hoist, 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.

J. W. M. la R.

67

KLUYVER'S CONTRIBUTIONS TO
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.'

(J. Bronowski).

INTRODUCTION

The 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 1 954, 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 1 9 16, 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 [19 14] 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 T.
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 T. 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 raffi-
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 these 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 S. 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

7i

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 T. monosa (sucrose and maltose negative), T. dat-
tila (sucrose positive, maltose negative), and S. 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 4 H 6 5 -> 2 C0 2 +C 2 H 5 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

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:
/. 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.
j. A sucrose-fermenting yeast can also ferment rafhnose, 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

73

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 i 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 of a 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
'Iambic', 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 19 10 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 via
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 P er cent MgS0 4 , 0.5 per cent KH 2 P0 4 , 0.5 per cent asparagine,
5 per cent sugar.

76

KLUYVER'S CONTRIBUTION'S 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 o. i 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 S. 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 Beijerinck'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 Beijerinck, 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, 1 931]. 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 evoked 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:
/. 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, via
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 5 were depicted as pro-
ceeding under the influence of the protoplasm of the fermenting cells,
yielding an oxidation product and 'protoplasm-H 2 '. 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, i.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,

. , / C 3 H 6 3 + 'protoplasm' -> C 3 H 4 3 -}-'protoplasm-H 2 ', a nd

^ M C 3 H 4 3 -> CH 3 CHO + C0 2 ; or

/ C 3 H 6 3 -> HCOOH+CH 3 CHO, and

I HCOOH+ 'protoplasm' -> C0 2 +'protoplasm-H 2 ';

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,
bv 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 Reinfurth 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 2 ' 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 ofacetaldehyde 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

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.

90

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:

2 C 6 H 12 6 -2H 3 P0 4 -> G 6 H 10 O 4 (PO 4 H 2 ) 2 +2H 2 O+2GO 2 +2C 2 H 5 OH

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

9 1

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 :

H

! li
C 6 ^c 5 ^C 4 c 3 ^c 2 C 1 ,

! i I!

O— :P0 4 = OH

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,

( i ) G 6 H 12 6 + H 3 P0 4 -> C 6 H n 5 P0 4 H 2 + H 2 0, and

(2) CeHnC^PC^Ha -> C 3 H 5 2 P0 4 H 2 + G 3 H 6 3 ,

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

93

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 1 924-1 926 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
^S ? were published in book form by the University of London
Press in 193 1. 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
fine 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:

(i) AH+B->A-fBH;

(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, viz., 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 fur 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 [1936TI1], 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.

')7

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 tern-

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.

')')

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-

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-

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 :

i. an initial hydrogen acceptor function, such as exhibited by acetal-
dehyde ;
2. an activation of the phosphorylating principle by hexose diphos-
phate; and
j. 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 li-
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 is
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 limited. 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.

IO5

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 of 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. 98-99).

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 fight 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, it 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'

(P-3!9)-

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 — ioo mV was estab-
lished; when nitrite had accumulated the potential rose to a level of
— 40 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 lindneri, 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 [193 1] 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 of redox systems with decreasing normal potentials. Thus
it is possible, nay, even probable that the oxido-reduction processes

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. lindneri 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

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 — 1 60 mV 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-

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.

13

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 [1928Tb] 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 3 H 8 3 -» C 3 H 6 3 +C 3 H 8 2 +H 2

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

2 C 4 H 6 5 -> C 4 H 4 5 +C 4 H 6 4 +H 2

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 [1928Tb] and Scheffer [1928TI1]
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

"5

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 2 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 2 A+ 2 -> 2A+ 2 H 2 0;
4 H 2 A+HN0 3 -> 4A+3H 2 0+H 3 N;
3 H 2 A+HN0 2 -> 3A+ 2 H 2 0+H 3 N;
4 H 2 A+H 2 S0 4 -> 4 A+ 4 H 2 0+H 2 S;

obviously all of these represent special instances of the general equa-
tion:

H 2 A+B-^ A+H 2 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' :

4 H 2 A+C0 2 -> 4 A+2H 2 0+GH 4

This hypothesis received strong support from Barker's [1936a] dis-

116

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:

2 CH 3 CH 2 OH+C0 2 -> 2CH 3 COOH+CH 4

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 n C0 2 was, indeed, n CH 4 .

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 [1930Tb], 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, 1947Tb] 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 3 CHOHCH 3 +C0 2 -> 4CH 3 COCH 3 +CH 4 +2H 2

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, 1947TI1]. Appro-

117

BIOGRAPHICAL MEMORANDA

priate experiments led to the conclusion that the conversion of carbon
monoxide by these organisms, Methanosarcina barken and Methano-
bacterium formicicum, proceeds in two stages, viz-, 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: 4 CO + 4 H 2 ^ 4 C0 2+4 H 2

C0 2 + 4 H 2 -> CH 4 +2H 2
4 CO +2H 2 -> 3C0 2 + CH 4

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-calcium
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 :
/. 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^C0 2 +H 2

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
glycerol 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 3 + H 3 C • COOH -> HOOC • CH 2 • 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.

"9

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-

KLUYVER S CONTRIBUTION'S TO MICROBIOLOGY 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 2 A, according to the overall equation:

light
C0 2 +2H 2 A -> CH 2 0+H 2 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 2 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 [1933Tb], 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.

BIOGRAPHICAL MEMORANDA

teria photosynthesis, and a year later Roelofsen [1934, 1935TI1], 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 naivete'. 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 aureqfa-
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 naivete 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 :
/. 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-

[25

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, viz., 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. lindneri) 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 appeals
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. . . .

'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 population 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.

'3

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, 1925Tb] 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, 1929a, 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
w r ell-nigh impossible. Besides, with such cultures it could not be as-
certained whether a particular product w r as 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 r 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 w r hen 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- 6 9 )-*

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.

J 35

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 nitration
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 Colin 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 Beijerinck 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

[OGRAPHICAL 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

1'

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, Kluy-
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, Methanobacterium 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-

143

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, 1925TI1; Fra-
teur, 1950], Protaminobacter [Den Dooren de Jong, 1926TI1, 1927],
Propionibacterium [Van Niel, 1928TI1], Pediococcus [Mees, 1934Tb], and
Spirillum [Giesberger, 1936Tb] ; of the group of sulphate reducing bac-
teria [Baars, 1930Tb], brine bacteria [Hof, 1935Tb], and methane
producing bacteria [Schnellen, 1947Tb], 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 Rho do spirillum; Kingma Boltjes [1934Tb, 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 [1938Tb] and
Perquin [1939] made notable contributions to our knowledge of the
dextran-producing Betabacterium vermiforme and Streptobacterium dex-
tranicum, respectively; and Kaars Sijpesteyn [1948Tb, 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 beijerinckii [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, 1 956] ; 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 Beijerinckia

144

KLUYVER S CONTRIBUTIONS TO MICROBIOLOGY AND BIOCHEMISTRY

[Kluyver and Becking, 1955] groups, and of the nitrate-reducing
sporeforming bacteria [Verhoeven, 1950, 1952Tb] contain valuable
information relating bacterial taxonomy to ecology; to this category
may be added Elazari-Volcani's [1939] investigation of Ps. indigo/era.

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, 1934Tb; 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 1 93 1 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].

145

SIOGRAPIIICAL MEMORANDA

members of the group of aerobic sporeforming bacteria; this applies,
for instance, to the species, Schizosaccharomyces hominis, [Dorrepaal,
1930], and to the genus, Schizotorulopsis [Verkaik, 193 1].

Besides these contributions should be noted the monographs dealing
with the yeast genus, Brettanomyces [Custers, 1940Tb], and with the
species, Candida pulcherrima [van der Walt, 1952Tb]. 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 thermode sulfuric ans ; it was the third spe-
cies of sulphate-reducing bacteria, the previously known ones being the
mesophilic freshwater V. desulfuricans and the marine V. aestuarii. The
isolation of V. thermo desulfuricans, 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. thermo desulfuricans 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. aestuarii might be similarly
related. Hitherto these species had been distinguished solely because
the former can develop normally in media with 0-1.5 P er 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, 1930Tb].

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

K.LUYVER 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

H9

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 191 2, 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 [1938TI1] 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 Eng-
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 intermedins, 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 Betabacterium 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

!5!

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, i.e.,
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 time
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.

153

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 [1947a, -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 Riviere 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.

154

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 'Koninklijke 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'.

C. B. v. JV.

*55

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. 1 956. Proc. Japan. Acad. 32, 488.
barker, h. a. 1936. J. Cellular. Comp. Physiol. #, 231.

barker, h. a., ruben, s. and kamen, m. d. 1940. Proc. Nat. Acad. Sci. U.S. 26, 426.
braun, w. 1947. Bact. Rev. //, 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. 2jg, 34.

littlewood, d. and postgate, j. 1957.J. Gen. Microbiol. 77, 378.

loomis, w. f. and lipmann, f. 1948. J. Biol. Ghem. 773, 807.

meyerhof, o. 1945. J. Biol. Chem. 757, 105.

meyerhof, o. 1 947. 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. 1 936. Biochem. J. 30, 506.
wieringa, k. t. 1 936. Antonie van Leeuwenhoek 3, 263.
wieringa, k. t. 1940. Antonie van Leeuwenhoek 6, 251.

156

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.

J 57

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

I 5 8

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-

159

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 'Gar le
style, c'est de l'homme meme'? 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

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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,
'I am 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

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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. 0. Forster, F. R. S. to Section B of the British Association for the
Advancement of Science, at Edinburgh on September i, 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, i.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

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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 role
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.

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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 1000 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

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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, i.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 cerevisiae, 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.

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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 of the 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 Congres 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.

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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 1 909-1913 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 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 mzVro-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 of the 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-

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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, i.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, i.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.

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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

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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 19 15, 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
1 9 18 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 Beijerinck and van Delden as a causative agent of flax retting.

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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 Tnstitut fur 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-

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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, viz., 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

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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

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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 role, 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 'tempe'-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 'tempe' production; nevertheless, a rational indus-
trial application of these findings has not as yet been attempted.

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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 Anions 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

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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 — 1 87 1, 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 :

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'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).

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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 et 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

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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,
192 1] : 'Les etudes 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 temoigner nul souci,
perir une science qui lui a valu jusqu' a present 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, tne Y 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.

Highly esteemed Beijerinck,
Amongst the congratulations that I received as a result of my appoint-

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merit 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.

Xot 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.

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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 is
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.

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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. ig2I. J. Roy. SoC. Arts Tig, 6l8.

dobell, c. 1932. Antony van Leeuvvenhoek and his 'Little Animals'. Amsterdam.
lichtenberger, a. 1921. Revue des deux Mondes, Nov. 15th.
rahn, o. 1 92 1. Naturwissenschaften g, 734.

WINSLOW. E. A., BROADHURST. J., BUCHANAN, R. E., KRUMWIEDE, CH., ROGERS.

l. a. and smith, g. H. 1920, J. Bact. 5, igi.

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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 role 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

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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 delbriickii
(the so-called 'domesticated' lactic acid bacterium), Lactobacillus fer-
mentum (a 'wild' lactic acid bacterium), Bacterium coli, 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. delbriickii 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

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 pasteurii which under suitable conditions com-

* In spite of the fact that the assimilation of gaseous nitrogen by CI. 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.

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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 Sbhngen [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.

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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 Beggiatoa.
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 T. 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 T.
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.

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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 Sohngen 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 im-
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 role 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

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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, i.e., the slow combustion taking place in living organisms,

* Perhaps the terms 'Bau-' and 'BetriebsstoffwechseP, 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.

194

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 its significance for the organism
entirely from the resultant energy liberation. Now Pasteur, who, in
i860, 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 life.

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 L*

* 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.

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TABLE I. MICROBIAL DISSIMILATORY PROCESSES*

A. Oxydative processes
i . C 6 H 12 6 +60 2 = 6C0 2 +6H 2 0+676 C al.
2. C 2 H 5 OH+ 3 2 = 2 C0 2 + 3 H 2 0+325 1 / 2 cal.

CH 2 NH 2 COOH+i 1 / 2 2 = 2C0 2 +H 2 0+

+NH 3 +i42cal.

C 2 H 5 OH+0 2 = CH 3 COOH+H 2 0+

1 16 1 / 2 cal.

CH 4 +20 2 = C0 2 +2H 2 0+2iocal.

H 2 +V 2 2 = HoO + GSVaCal.

NHg+iVaO, = HN0 2 +H 2 + 79 cal.

9-
10.

KNO a + 1 /2°2 = KNO3+22 cal.

H 2 S + V 2 2 = H0O + S + 67 cal.
S+iVaOa+Hp = H 2 S0 4 +i 4 i cal.

Examples of organisms :
various fungi
mycodermic yeasts
('Kahmhefen')

many aerobic bacteria

acetic acid bacteria
methane oxidizing bacteria
hydrogen oxidizing bacterid
nitrite forming bacteria
nitrate forming bacteria
sulphur bacteria
sulphur bacteria

B. Fermentative processes

1 1.
12.

13-
14.

i.>

C 6 H 12 O e - 2C 2 H 5 OH+2C0 2 +2 5 cal.

C 6 H 12 6 = 2C 3 H 6 3 +28 cal.

C 6 H 12 6 = C 4 H 8 2 +2CO ? +2H 2 +i5 cal.

C 6 H 12 6 + 1 / 2 H 2 = CH 3 CHOHCOOH+

+ CO a +H 2 + 1 /2CH 3 COOH+

+ 1 / 2 C 2 H 6 OH+8 cal.

(CH 3 COO) 2 Ca + H 2 = CaC0 3 +CO a +

+ 2CH 4 +igcal.

CO(NH 2 ) 2 +2H 2 = (NH 4 ) 2 C0 3 +6cal.

C 2 H 5 OH+2.4KN0 3 = i.2N 2 +

+ i.2K 2 C0 3 +o.8C0 2 +3H 2 + 293 cal.

G 2 H 5 OH+i 1 / 2 GaS0 4 = 1V2H2S+

+ i 1 / 2 CaG0 3 + 1 / 2 C0 2 + iV 2 H 2 0+ 14 cal.

yeasts

lactic acid bacteria

butyric acid bacteria

coliform bacteria

methane bacteria
urea splitting bacteria

denitrifying bacteria

sulphate reducing bacteria

* This Table makes no attempt whatever at completeness. In particular, the list 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.

[9 6

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-

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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 Beijerinck 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.

T 99

TABLE II. DISSIMILATORY PROCESSES OF SOME IMPORTANT
GROUPS OF MICRO-ORGANISMS

Oxidative dissimilatory processes

autotrophic
bacteria

i m j uuu i nuui

&&&&&a

aerobic

sporeforming

bacteria

Pseudomonas

Micrococcus

Sarcina

Spirillum

Vibrio

Mycobacterium?

Fermentative dissimilatory processes

yeasts

coliform bacteria

bacteria of the
Proteus group

urea splitting
bacteria

denitrifying
bacteria

lactic acid
bacteria

anaerobic
cellulose
decomposing
bacteria

butyric acid
bacteria

anaerobic
protein
fermenting
bacteria

methane bacteria

sulfate reducing
bacteria

inorganic compounds
carbohydrates and sugaralcohols

£v:*X*j organic substances containing nitrogen
5§§§§§§j salts of organic acid

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, i.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 [191 3] 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

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, viz., 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 [191 1] have posed this question as early as
191 1, though perhaps in a less pregnant form. They believe that such
energetic interplay between two materially independent reactions is
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 offer-
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-

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 [191 7, 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.

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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 [191 3] 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

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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 [Xeuberg and Nord 1919], 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 3 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

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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 [191 7], 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.

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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

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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

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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, e.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 ^-carbon atom,
thereby producing /5-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