THE "DELFT SCHOOL" AND THE RISE OF GENERAL MICROBIOLOGY' C. B. vAN NIEL Hopkins Marine StationofStanford University, Pacific Grove, Calif. Antonievan Leeuwenhoekcertainly started somethingwhenhe began making his lenses and examining anything he could lay his hands on! His observations led, about 300 years ago, to the discovery of the "little animals", now known under the names of protozoa and bacteria. And thus Leeuwenhoek, the Delft draper and scientist, became the "Father of protozoology and bacteriology," asDobell (1)hassoaptlycalledhim.Thereisnodoubtthatthescienceofgeneral microbiology began in Delft. Itwas an exciting beginning. The animalcules were found almost everywhere, and they appeared to represent an astonishing array of sizes andshapes. Practi- cally any kind of material revealed their presencesa wonder to behold, a pleasuretowatch. AndLeeuwenhoek madethemostofhisdiscovery:witnessthe enormous number of letters which he sent to the Royal Society of London vividly describing his observations with many pertinent reflections upon their significance. Yet we do not ordinarily think of Leeuwenhoek as the founder of a "Delft School", or, for that matter, of any school. He was a solitary worker, and occa- sionally even reluctant to disclose to others the methods he employed. In con- sequence we are, even today, confined to speculation when it comes to deciding whether ornotLeeuwenhoekmighthavehituponawayofexamining specimens by using some sort of dark-field illumination, a possibility that was suggested byDobell (1, p. 331-2). This suggestion hasbeenmore fully discussed by Cohen (2) and Kingma Boltjes (3). Now, Leeuwenhoek did not start a "school," and so his methodology was handed down to posterity only insofar as his letters divulged. There were no pupilswhomightafterwardshaverevealed secretswhichthemasterhad decided not to publish. But his discoveries were so spectacular and so unexpected that theycouldnotfailtofiretheimaginationofothers,equallyimbuedwithcuriosity, that driving force of scientific endeavor. Hence his observations were, in the course oftime, repeatedandextended in otherplaces, andknowledge concerning the microbes gradually accumulated, until today there is available an amount of information so vast that it would be impossible for a single individual to be conversant with more than a small part of it. This is an unfortunate although inevitable result of expanding scientific activity: the interested individual must needs make a choice as to what shall occupy his mind and hands. Thus there is a real danger that he may become involved in minutiae; a narrow specialist who "knows more and more about less 1Based on the A. J. Kluyver Lecture delivered before the Society of American Bac- teriologists. Cincinnati, Ohio, May 19, 1949. 161 162 C. B. VAN NIEL [VOL. 13 and less." However, an increase in factual information, the only solid basis for scientific accomplishment, also brings with it the desire for organizing and inte- grating the details. If this is satisfactorily accomplished the isolated data can be connected together into a framework composed of general principles. And the latter mark the culminating advances of science. InwhatfollowsIshalltrytosketchthegradualdevelopmentofsomeprinciples in the realm of general microbiology; to show how these are associated with a "DelftSchool";andtoindicatehowtheyhavecontributedtotherapidlygrowing interest in thisfield. Obviously, Leeuwenhoek's discovery of the existence of the "little animals" raised problems concerning their origin, their activities, and the significance of the latter. Leeuwenhoek himself expressed opinions on these questions that were essentially identical with those which, two centuries later, became the established scientific views. During the intervening years the issues were, how- ever, ardentlydebatedonthebasis of seeminglyconflictingexperimentalresults, and these experiments have added greatlyto our store of knowledge. I shall not dwell upon the fascinating controversy about the spontaneous generation of the microbes versus their origin from preexisting ones. The battle, earlier fought over the origin of larger organisms, and quite recently again over that of viruses, ultimately led to an acknowledged victory of the proponents of the idea of biogenesis. In the meantime the discrepancies in the outcome of many crucial experiments gradually led to the development of an adequate methodology for the study of microorganisms. Most of the techniques now so confidently used represent modifications and refinements of methods that had once produced results interpreted in favor of spontaneous generation. Thus can the mistakes made in scientific investigations be turned to advantage, for they lead to the recognition of unexpected and unpredictable sources of error, and so permit the eventual elimination of the latter. But the apparent defeat of the doctrine of spontaneous generation left un- solved the fundamental problem of the origin of life. In recent years new ideas have been expressed, notably by Haldane (4), Oparin (5), and Horowitz (6) which have a strong scientific appeal because they suggest a way out of what otherwise would be an impasse. Whether these concepts can soon be made the basis for a renewed experimental attack cannot now be decided; the answer must be left to future studies. So much for the first problem. The question of the activities of the "little animals," too, was contemplated by Leeuwenhoek, and once again he reached a conclusion that was not to become part of our scientific outlook until two cen- turies later. I do not here refer to the concept that microorganisms can play a role as causative agents of disease, but to the far broader one concerning their function in the cycle of matter. It should be realized that the former activity represents nomore than a veryminor aspect of this general phenomenon. The important part played by microorganisms in transforming organic and inorganic substances on earth with the result that these may be used over and over again to sustain life of other organisms was first clearly expressed by Fer- 1949] "DELFT SCHOOL" AND MICROBIOLOGY 163 dinand Cohn (7) in 1872. In thus making possible the continuation of the great experiment ofevolutionthe "little animals" occasionally perform their task in a manner that clashes with the desires of man who, through ignorance and greed, has a propensity for eliminating various natural resources from participation in the natural cycle of matter, and often appears to regard the earth with all that is on it as his own private property. This has led to an unwarranted emphasis onsuchresentedactivitiesofthemicrobesaswouldinterferewithman'shoarding instinct, even to the point of making him lose sight of the fundamental signifi- cance of an uninterrupted continuation of the cycle. Those who have learned to viewlife inawidersensecanbuthopethat, througheducation, abetter compre- hension may gradually be reached, and the hoarding instinct be curbed-if there is still time. Our knowledge of the diverse types of microbes responsible for the specific major transformations of matter has advanced greatly since Cohn's pronounce- ment. The most important contributions to this problem we owe to M. W. Beijerinck (8), the second of the great Delft microbiologists. By introducing the principle of enrichment cultures he opened the way for a rational approach to microbial ecology. Although some of Beijerinck's specific discoveries are fairly wellknowntomostmicrobiologists, thefundamentalideasthatledtothemhave been appreciated far too little. This, I believe, isdueto the fact that Beijerinck, who could have written a treatise on enrichment cultures that would not have failedtoexertaprofound influence, neversomuchaspublishedapaperinwhich the principle was clearly formulated and its potentialities developed. When, in 1905, Beijerinck was awarded the Leeuwenhoek medal by the Koninlijke Akademie van Wetenschappen in Amsterdam, F.A.F.C. Went noted the above mentioned deficiency in hispresentation address withthe following words: "Thereisinyourpublications suchawealth oforiginalconcepts and of special approaches, often buried in a couple of sentences, that such a treatise would surely be anticipated with the utmost interest. It would then also become clear how many of the current ideas in microbiology we really owe to you; this is far morethan is apparent to thosewho merelyhavetaken superficial notice of your publications" (9). ItwasonlyonthisoccasionthatBeijerinckstatedhisobjectivesandapproach. I translatefromhisacceptance speech: "I am happy to note that the way in which I approach microbiology has the approval of the best judges. This approach can be concisely stated as the study of microbial ecology, i.e., of the relation between environmental conditions and the special forms of life corresponding to them. It is my conviction that, in our present state of understanding, this is the most necessary and fruitful direction to guide us in organizing our knowledge of that part of nature which dealswith the lowest limits of the organic world, and which constantly keeps before our mind the profound problem of the origin of life itself. Therefore it is a great satisfactiontomethattheAcademyapparentlywishestohonortheexperimenter who exploits this field. "In an experimental sense the ecological approach to microbiology consists of 164 C. B. VAN NIEL [VOL. 13 two complementary phaseswhich giveriseto an endless number of experiments. On the one hand it leads to investigating the conditions for the development of organisms that have for some reason or other, perhaps fortuitously, come to our attention; on the other hand to the discovery of living organisms that appear under predetermined conditions, either because they alone can develop, or because they arethemorefit andwin out over their competitors. Especially this latter method, in reality nothing but the broadest application of the elective culturemethod, is fruitful and truly scientific, and it is no exaggeration to claim that the rapid and surprising advances in general microbiology are due to this methodology. Nevertheless, andthisin spiteofthefact that Leeuwenhoek, more than two hundred years ago, already used this aspect of micro-ecology in some ofhisstudies, andthatPasteurwasenabledtomakemost ofhisgreatdiscoveries becausehewasguided bythe sameprinciple, thenumber of conscious exponents has so far remained very small. And I feel that I certainly may be reckoned among them because of the enthusiasm that is in me to contribute to the grand task that can here be accomplished" (10). That is all. Andwhowould bother to read these sentences, representing half a page of a printed speech, and written, like Leeuwenhoek's letters, in the Dutch language, some45 years ago? Beijerincknever got around to writing thetreatise Went had suggested, probably because he was more interested in doing experi- ments, and so the number of workers who consciously applied Beijerinck's principles remained small, limited, in fact, to thosewho had the good fortune of experiencing his influence, directly or indirectly. It is true that in 1907 Stock- hausen (11) published a number of essays on microbial ecology ("Enrichment cultures after Beijerinck") in the "Wochenschrift fMr Brauerei," also issued in bookform. But thirtyyearsafter its publication thefirst edition of thebook was still far from exhausted, and many microbiologists have probably never heard of it. Nonetheless, the fundamental significance of Beijerinck's work is slowly becoming recognized in wider circles, and the application of enrichment culture practices is spreading. Even suchup-to-date studies as those concerned with the search for antibiotics, with the attempts to culture various algae and protozoa, an endeavor so successfully pursued by Pringsheim (12), and with the selection of specific nutritional types of microbes; all such studies are now generally carried outwiththeconscious or unconscious inclusion of Beijerinck'sprinciples. Furthermore, ifonethinksaboutthereasonsforthereadyavailabilityofcultures of nearly all kinds of microorganisms (yeasts, algae, actinomycetes, sulfur and hydrogenbacteria,speciesofAcetobacter,Azotobacter,Aerobacter, Spirillum, Myco- bacterium, Propionibacterium, or Clostridium, lactic acid bacteria, bacteria de- composing cellulose, agar, or urea, denitrifying and sulfate-reducing bacteria, methane-producing,luminous, orphotosynthetic bacteria) itbecomesabundantly evident that these reasons arenot to be found primarily in the existence of pure culture collections, however useful a purpose they may serve, but chiefly in the simple methodology, based on Beijerinck's enrichment culture procedures, by which these organisms can regularly be procured. 19491 "DELFT SCHOOL" AND MICROBIOLOGY 165 Familiarity with the results that can be achieved by means of enrichment cultures also leads to the conclusion that the distribution of diverse sorts of microbes is ubiquitous. For example, the cellulose decomposing Cytophaga and Sporocytophaga species, the nitrogen fixing azotobacters and Clostridium pas- teurianum, or the hydrogen oxidizing bacteria that are known today can be isolatedfromsoil,mud,orwatersamplesinArgentina,Holland,Japan,Australia, Russia, or the U. S. with equal facility. And the pure cultures of one kind ob- tained in different placesgenerally do not show anymore differences among one another than do a variety of strains isolated in one and the same locality. A similarpictureispresentedbythemicrobesfoundinthose "natural" enrichment cultures encountered in different parts of our globe, such as in hot springs, brine pools and salt beds, sulfur or iron springs. A careful comparison shows that, where the environmental conditions are closely comparable, the same types of organisms appear. The significance of these facts for determinative bacteriology isthatweneednotthinkintermsoflocalmicrofloras and -faunas. Buttheyalso carry another, less obvious implication. There is now a large number of bacteria, yeasts, algae, and protozoa, inci- dentally observed under ill-defined conditions, that have been named and de- scribed on the basis of certain more or less easily ascertainable properties. Whether such characteristics, mostly determined by the application of stereo- typedandarbitrarymethods, bearanydirectrelationtothosethatareimportant in connection with thenatural occurrence and survival of the organisms isoften doubtful. Idonotmean that itistotallyuselesstoknowthat onetypeofphoto- synthetic bacteria can liquefy gelatin or grow in glucose media while others do not, or that certain fluorescent pseudomonads, in contrast to others, can utilize arabinose or produce acid from raffinose. But knowledge of such properties is entirely inadequate to tell us anything concerning the normal activities of the organisms in question. If the latter were better understood, it would become possible to approach the problems of determinative bacteriology and classifica- tion in amorerational manner, and to eliminate much of the present confusion. For this purpose further studies with enrichment cultures are imperative. Beijerinck's great objective is still far from completed. It is necessary that con- ditions be more accurately controlled and specified, and that attention be paid to the effect on the outcome of enrichment culturesdueto suchvariables as the reactionofthemedium, thetemperature ofincubation, theconcentrations ofthe various nutrient and non-nutrient ingredients, thepresence orabsence ofmicro- nutrients and growth factors, etc. It is very probable that by means of such refinements an ever increasing number of microorganismswill becomeaccessible to isolation by enrichment culture techniques, and in thismannerwe shalllearn more about the normal activities of the organisms encountered than by con- tinued studies of pure cultures with standard methods. Of course, it must be admitted that such efforts may only slowly make it possible to recognize the naturalenvironmentofnumerousorganismsthathavebeenisolatedaccidentally, and whose properties are now most imperfectly known. When, in 1921, Beijerinck retired, a "Delft School" had been launched. The 166 C. B. VAN MEL [voL. 13 vast knowledge of themaster had in part been transmitted to his students, and some of them continued the traditions in other Dutch institutions. Also outside theNetherlandshismethodofapproachwasspreading;menlikeIssatchenkoand Krainsky, Melin, Gran, Krzemienievski, Kaserer, Stockhausen, and Stoklasa, who had worked in his laboratory, had gone back to their own countries and served as new nuclei abroad. And yet, when we think of a "Delft School" it is certainly not only these men who come to mind. Perhaps not even, in the first place, Beijerinck, but rather his successor, Albert Jan Kluyver, Corresponding Member of our Society of American Bacteriologists, the third of the great trio of Delft microbiologists, and the scientist in whose honor today's lecture is named. By developing the concept of comparative biochemistry Kluyver laid the foundation for an approach to biochemical problems that has proved to be oneofthemost fruitful ofour era. Ithasbrought order into a situation thatwas almostchaotic,andhasbecometheguidingprincipleforthestudyofthechemical activities of any and all living organisms. Two years after his inauguration Kluyver (13) madea survey of theprocesses known to occur in nature under the influence of microorganisms. It was a be- wildering picture that emerged. Not only did it show the endless variety of sub- stances, inorganic aswellasorganic, that can bedecomposed bybacteria, molds, yeasts, etc.; it also illustrated the enormous diversity of substances that could arise during these decompositions. Now, awareness of diversity, a prerequisite for scientific pursuit, engenders the desire to discover unifying principles. Thus theproblem arose:what common denominators can befound in thismultiplicity of microbial activities? The only one apparent in 1923 was the broadest possible generalization of Lavoisier's concept of biological oxidations as the source of energy for the main- tenance of life. Pasteur had extended this idea by recognizing that fermenta- tions, i.e. biological processes going on in the absence of air, are also energy yielding reactions. And Winogradsky, now some 60 years ago, had discovered organisms that could fulfill their energy requirements by oxidizing inorganic compounds. When computations of energy relations showed Kluyver that the multitude of known decompositions by microorganisms all proceeded with the liberation of energy it was, therefore, clear that Lavoisier's principle in this extended formcouldbeinvoked. But this general answer did not satisfy Kluyver. It begged the question of a mechanism. Afterall, thesedecompositions could also be considered aschemical transformations, and since the beginning of the 19th century much construc- tive thought had gone into making chemical reactions intelligible on the basis of the atomic and molecular theories. That it might ultimately be possible to bring the comprehension of biochemical transformations up to the same level was, consequently, a reasonable expectation. Besides, progress had been made in this direction. The researches of Neuberg onalcoholicfermentationbyyeastshadachievedaninterpretation ofthisprocess as the net result of a series of consecutive step reactions, each one chemically conceivable and simple innature. Wieland hadtackled the problem oftheoxida- 19491 "DELFT SCHOOL" AND MICROBIOLOGY 167 tion of alcohol to acetic acid by acetic acid bacteria and contended that this oxidation should be considered as composed of two stages, viz., the oxidation of alcohol to aldehyde, and of the latter-in the form of ahydrate-to acetic acid. Boththeseoxidations appearedtobereactionsinwhichtwohydrogenatomsare eliminated from a substrate molecule and transferred to any one of a number of hydrogen acceptors, suchas 02, quinone, methyleneblue, etc. Fromtheseresults Wieland had drawn the conclusion that all biological oxidations could be inter- preted as primarily composed of series of dehydrogenations, with 02 acting as the normal, but not the only possible H-acceptor. Harden and his coworkers, especially Grey, had made a good beginning with the resolution of the coli and aerobacter fermentations; and Fred and Peterson, as well as Speakman, of the butanol-acetone fermentation. It would take too long to review the developments that led Kluyver, in a few years, to the masterly syntheses represented by the two major publications: "The unity in biochemistry" (14), and "Thechemical activities of micro6rgan- isms" (15). It is in thelatter treatise thattheterm "comparative biochemistry" was first used, and Kluyver envisaged for it an influence which could benefit biochemistry in a manner similar to that in which the concept of "comparative anatomy" had helped to bring order into the mass of isolated anatomical ob- servations. Kluyver's keen and critical mind recognized the potential significance of the ideas that Neuberg, Wieland, Warburg, Harden, and afew othershad advanced to account for more or less specific biochemical events. Soon it became evident to him that those concepts could be welded together into a very few general principles, applicable to all biochemical phenomena. The most basic of these generalizations is the extension of the ideas of Neuberg and of Wieland to their ultimatelimits. Thus, anybiochemical process, whether oxidation, fermentation, or synthetic reaction, was considered as a chain of step reactions, each one of which represented a simple mechanism in which hydrogen is transferred from one molecule, the H-donor, to another, the H-acceptor. The only apparent ex- ception to this principle was exhibited in the metabolism of complex molecules, composed of a number of simple entities, for example the polysaccharides (com- plexesof simplesugars), proteins (complexesofaminoacids), andfats(complexes of fatty acids and glycerol). Such complexes would first be converted to their constituentunitsbyhydrolytic cleavages, withtheproductssubsequentlyunder- going the various hydrogen-transfer reactions. In this manner the existence of the many hydrolytic enzymes-glucosidases, proteinases, lipases, etc.-could be fitted into the general picture. Many of the known facts concerning diverse metabolic processes could be readily incorporated into this concept. In the course of the following years numerous additional cases were investigated in his laboratory, and the outcome of this activity did much to strengthen the evidence for the soundness of the postulates. Italso indicated thattheinitial stagesinthebiochemicaltransforma- tionsofaspecific substancewereverysimilar, ifnotidentical,nomatterwhatthe final result proved to be. For example, the evidence strongly suggested the 168 C. B. VAN NIEL [VOL. 13 probability that in practically all instances of sugar decomposition the carbohy- dratewouldfirst bedegraded to three-carbonmoieties; thedifferences intheend productsreflecteddifferences inthefateoftheseuniversal intermediateproducts. It is important to realize that the arguments applied to a great diversity of processes, such as the alcoholic and lactic acid fermentations, the "mixed acid" fermentation characteristic of the coli group, the butane-diol fermentation of Aerobacter and Aerobacillus species, the propionic acid fermentation, the butyric acid and thebutanol fermentations, the fermentations in which acetone and iso- propanol are produced. Also, many of the oxidative degradations appeared to proceed by the same initial stages. It would be foolish to insist that the principles of comparative biochemistry would not have been developed if it had not been for Kluyver's penetrating ap- proach, just as it would be foolish to contend that microorganisms would not have been discovered if Antonie van Leeuwenhoek had not done so. In the late twenties there were others who were beginning to think along similar lines, and thereconciliation ofWieland'sandofWarburg'sideasonthenatureofbiological oxidations was proposed almost simultaneously by Kluyver and Donker, Szent- Gyorgi, and Fleisch, in three entirely independent publications. Nevertheless, the familiarity with the vast diversity of the conditions under which life can exist and manifest itself, especially in the world of microorganisms, made avail- able for Kluyver's scientific contemplation an immensely greater range of pat- terns than that presented by the higher plants and animals. And the result was the enunciation of the most far-reaching generalization. The attempts at interpreting various biochemical phenomena in greater detail ledKluyverandhiscollaboratorstopostulateanumberofspecific stepreactions, leading to a small group of common intermediate products. Itwas clearly recog- nized that some substrates or intermediates could undergo more than one par- ticular conversion. The extent to which each of the possible transformations occurswould, ofcourse, dependonthenatureoftheorganisms, i.e., itsenzymatic composition. But even for the same organism the result is usually not fixed be- cause environmental conditions, such astemperature, concentration of substrate or intermediate products, reaction of the medium, the presence or absence of special hydrogen donors or acceptors, could readily influence the magnitude of thedifferentconversions. Itis, therefore, impossibletopredicttheexactoutcome of a biochemical process in terms of the precise quantities in which each of the endproductswillbeformed. Thefrequently observedfluctuationsinthisrespect need not be disturbing, however; they become readilyunderstandable asthere- sult ofacomplicated interplay betweenthevariouspotentiallypossiblereactions inwhich theintermediate products canparticipate. Whenviewed inthismanner a biochemical reaction becomes more clearly a dynamic event, to be represented by series of steps with variations in several directions rather than by a single chemical equation with fixed quantitative relations between the end products. Many of the step reactions and intermediate products postulated by Kluyver sometwentyyearsagoappearoutmodedto-day.Surely'nobiochemistwouldnow seriously consider methyl glyoxal, for example, in the central position which 1949] "DELFr SCHOOL") AND MICROBIOLOGY 169 Kluyverassignedtoit inhisLondon Lectures (15). Muchhasbeen accomplished in the intervening years through the brilliant work of many scientists. The chemical nature of several intermediate products has been established with in- creasing precision; theinteractions and conversions ofthese compounds can now be represented by reaction chains far more elaborate than was once deemed possible. In large part this astounding penetration into details of biochemical mechanismshasresultedfromtheisolationofspecific enzymeswithwhichpartial conversions can be investigated under rigorously defined conditions. And much of this work has been done with microorganisms; those who have attended the symposium on thefirstday of ourmeetingswill realizethis. Furthermore,newprincipleshavebeenintroduced. Among-themostimportant onesmust bementioned Michaelis' theory (16) of the single-electron shifts; Lip- mann's concept (17) of the high-energy phosphate bond and its significance for the preservation and storage of energy; and the ideas concerning the transfer of whole blocks of atoms, as in trans-aminations, trans-methylations, trans- acetylations, trans-glucosidations andtrans-phosphorylations. It hasbeen aphe- nomenal development. But, although these advances have shown the need for modifying the earlier postulated details, they have also served to substantiate the validity of Kluyver's main thesis regarding the fundamental unity in bio- chemistry. The basic similarity in thebiochemical behavior of so many different organisms is now generally admitted. It is emphasized by the occurrence of the same amino acids, vitamins, enzymes, etc., in all forms of life, and by the par- ticipation of a number of identical intermediate products in practically all meta- bolic activities. The recognition of this unity is Kluyver's great contribution; it is also the starting point of "comparative biochemistry". Predicated upon the fact that a particular substance, whether substrate or intermediate product, can undergo only a limited number of immediate transformations, sometimes only a single one, these can be explored by investigating the fate of such compounds under the influence of different organisms. The results so far obtained have amply demonstrated thefruitfulness of this line of study. Agood example is furnished by the methane fermentation, a process in which various alcohols and fatty acids are decomposed to methane, generally accom- panied by the production of carbon dioxide. Now, the primary attack on those substratescannotreadilybeconceivedofasanythingbutastraightdehydrogena- tion. Hence an external hydrogen acceptor is required. Decompositions of the same substrates are known to occur in the presence of oxygen, nitrate, and sul- fate, andthese substances, actingashydrogen acceptors, arethus converted into H20, H3N, orH2S respectively. Thisled to the ideathatthemethane fermenta- tion represents a similar substrate oxidation with C02 as hydrogen acceptor, a postulateforwhichtheinvestigationsofBarker (19)havefurnishedexperimental evidence. Thedegradationofthehigherfattyacidsduringthemethanefermenta- tion has been shown by Mrs. Stadtman (personal communication) to follow exactly the path required by the Knoop-Dakin theory for this process in higher animals. Hence the methane fermentation no longer occupies a totally unique 170 C. B. VAN NIEL [VOL. 13 position. The details of the mechanism whereby carbon dioxide is reduced to methaneremain to be elucidated, and thesemight yield important resultsforan understanding ofthemechanism of photosynthesis. I realize that thismay seem a far-fetched conclusion. However, the following considerations, in exposingthetrend ofthoughtuponwhichthisdeductionrests, should make it appear reasonable. A comparison between the photosynthetic activities of green plants and of green and purple bacteria suggested, several years ago, that photosynthesis should be interpreted as a process of carbon di- oxide reduction with hydrogen obtained by a photochemical decomposition of water (20). This, inturn, impliesthat thereactionsmore immediately concerned with the assimilation and reduction of carbon dioxide must themselves be non- photochemical processes. It should consequently be possible to reach a better understanding oftheessentialfeatures ofthesereactionsbyacomparativestudy of all cases in which carbon dioxide is similarly involved. And those include not only the carbon dioxide assimilation by chemo-autotrophic microbes, but also the Wood and Werkman reaction (21), the formation of other di- and tricar- boxylic acids by carbon dioxide addition to various keto-compounds (22), the production of acetic acid from C02 and hydrogen by Clostridium aceticum (23), andthemethanefermentation. Suchacomparative studywouldmakeitpossible to discover the common denominators of all these processes, and therefore con- tribute to a more detailed picture of the photosynthetic reaction. Two decades ago Kluyver advocated the use of microorganisms for compara- tive biochemical studies. On several occasions he stressed the advantages they offered, both on account of the ease of handling them under controlled and re- producible conditions, andbecause of the enormous biochemical versatility en- counteredwithinthisgroup. Itisoftenpossibleto selectaspecific microorganism assingularlyappropriateforagivenproblembecauseitcarriesoutacertaintype of reaction to the exclusion of almost any other. But it is equally important to realize that one may find among these creatures the best examples of seemingly quitedifferent biochemical propertieswithrespecttotheconversionofaparticu- lar substrate. Both ofthese aspects areimportant for a comparative biochemical approach. If it be further remembered that by the application of Beijerinck's principle ofenrichment culturesmanyoftheorganismsaresoreadilyprocurable, it will be clear that the case for the microbes-and for the microbiologist-is pretty strong. This has obviously been recognized. During the past decade there has been a rapidly growing interest in comparative biochemistry as well as in micro6rgan- isms. It isno longerunusual to find a large fraction of thepages of physiological and biochemical journals occupied by publications dealing with the activities of fungi, protozoa, andbacteria. Even inthefield ofgeneticsthemold Neurospora, the yeasts, Escherichia coli, Paramecium, and bacteriophages are successfully competing with Oenothera, Zea mais, and Drosophila. Whennowadaysenzymereactionsarestudiedbymethodsrangingfromkinetic measurements (24, 25) to theuseofmashed cells, of dried cell preparations (26), of cultures supplied with sub-optimal amounts of growthfactors (27-30), of in-