History of Science South African Journal of Science 103, September/October 2007 377 Louis Pasteur, fermentation, and a rival K.L. Manchester* Accusations of plagiarism, probably unjustified, concerning two eminent scientists over the first demonstration of fermentation by living organisms, still persist after a century and a half ONE HUNDRED AND FIFTY YEARS AGO, IN grains, did not fit with this rule, and this August 1857, Louis Pasteur gave a lecture discovery had led him to a special interest to the Société des Sciences de Lille entitled in the amyl alcohols. Nevertheless, and a ‘Lactate fermentation’, published soon little confusingly, the paper presented in after as a Mémoire.1 The title may sound Lille dealt instead with lactic fermenta- unexciting, but this historic paper marked tion,1 Pasteur declaring that: ‘I intend to Pasteur ’s first statement of a germ theory establish that, just as there is an alcoholic and a specific role for microorganisms in ferment, the yeast of beer, which is found metabolic processes. Pasteur was then everywhere that sugar is decomposed Fig. 1. Pasteur in 1857 when dean of the Faculty of 34 and professor of chemistry and dean into alcohol and carbonic acid, so also Sciences in Lille. [Musée Pasteur, Paris] of the new Faculté des Sciences in Lille there is a particular ferment, a lactic yeast, (Fig. 1), in the heart of the sugar beet always present when sugar becomes lactic both leucine and isoleucine exist in dextro growing region of northern France. acid.’ And this is precisely what he found. (+) and laevo (–) rotatory forms, their Pasteur ’s appointment was in part in- Critics have pondered over the years equivalent degradation to optically inac- tended to give help to industrialists in whether Pasteur formed his views on the tive isoamyl acohol and (optically) active Lille, but the opening sentence of his basis of the evidence or decided what the amyl alcohol results in loss of the asym- paper makes clear that he was led to result must be and proceeded to prove it! metry of a carbon atom in isoamyl alcohol, consider fermentations following his re- A prevailing view promoted by Justus which is retained in active amyl alcohol searches on the amyl alcohols and their von Liebig, up to this time, was that fer- (Fig. 3). To Pasteur’s considerable distress, remarkable crystallographic properties.2 mentation represented a form of decom- the two alcohols, as their barium His studies on fermentations led to a pro- position, perhaps initiated by yeasts sulphamylates, had exactly the same crys- tracted dispute with a contemporary, but proceeding essentially as a result of tal form.2 Antoine Béchamp, over the priority of his instabilities arising in molecules in fermen- findings which, remarkably, is main- tation liquors. On this view, amyl alcohol Fermentation correlative with life tained to this day and with which the possessed optical activity because it re- Yeast, when incubated with sugar latter part of this article deals. tained some of the properties of the alone, gradually disintegrates. Pasteur fermenting sugars. Pasteur considered recognized this event as one of the most Pasteur’s work on fermentation that amyl alcohol was too dissimilar to important points in Liebig’s theory of It will be recalled that, in 1846, as a sugars for this to be so (in which he was fermentation.4 If fermentation, Liebig student at the École Normale Supérieure correct), and that its optical activity derived argued, is a consequence of the develop- in Paris, Pasteur had had the remarkable from the living organism producing it (in ment and multiplication of cells, as others perspicacity and good fortune to notice which he was wrong). Subsequent work claimed, incubations containing sugar that the sodium ammonium salt of showed that the two amyl alcohols of alone should not produce alcohol, since paratartaric acid, a form of tartaric acid fermentation constitute breakdown prod- such a medium lacks the other essential that was indistinguishable chemically ucts of the amino acids leucine and iso- conditions for cell growth and division. from the commonly occurring tartaric leucine, present in nitrogenous materials Nevertheless, alcohol is produced under acid except that solutions were not optically in the fermentation media. Although these conditions. active, crystallized to give mixtures of two Pasteur showed that it was as a result of mirror image enantiomorphic forms the growth of yeast cells, which can feed (Fig. 2). These crystal forms, if carefully off the remnants of dead cells, that fermen- separated by hand and then dissolved in tation occurs.4 Thus, he reached the cele- water, gave optically active solutions of brated conclusion that ‘the breakdown of opposite rotations.3 Given the optical sugar into alcohol and carbonic acid is an activity of many organic compounds of action correlating with a vital phenome- natural origin, Pasteur had deduced non’, that is, fermentation is a property of that asymmetry (Pasteur used the term living cells. Pasteur was also able to refute ‘dissymmetry’) was a feature of the chem- Liebig’s claim by showing that yeast istry of life and that this would correlate grows and ferments sugar in medium with hemihedral crystals. Optically active devoid of albuminoid (proteinaceous) amyl alcohol recovered in distillates from material, although containing ammonia alcoholic fermentations, particularly of and salts.5 *School of Molecular and Cell Biology, University of the But having made one clear and impor- Witwatersrand, Private Bag 3, WITS 2050, South Africa. Fig. 2. Hemihedral crystals of (+)– and (–)-sodium E-mail: firstname.lastname@example.org ammonium tartrate. Adapted from ref. 22. tant advance, Pasteur now risked pushing 378 South African Journal of Science 103, September/October 2007 History of Science with coloured indicators. He was surprised to see no effect of zinc chloride, since it showed an acid reaction. In this paper, Béchamp makes no suggestion that the appearance of the mould was a significant factor in the occurrence of inversion. Whether the thought had passed through his mind we do not know, which is unfor- tunate because it is this uncertainty which constitutes the basis of the controversy, which began to erupt around this time, Fig. 3. Structures of leucine and isoleucine, and the related amyl alcohols derived from them during fermentations. Another name for the amyl alcohols is fusel oil, an acrid, oily liquid occurring in insufficiently over priority between Béchamp and distilled alcoholic liquors. Pasteur as to who first showed the pro- duction of ferments by living organisms. himself into a potentially dubious position, turns into one of ‘subtle physiology’. In a subsequent paper,8 however, pub- this time in relation to vitalism, that is (Presumably the same could be said of lished at the beginning of 1858, Béchamp that certain metabolic properties of living Pasteur, where his academic study of pointed out that experiments he had con- matter cannot be observed outside the cell stereoisomerism led to fermentation.) ducted since 1855 forced him to modify and that only those fermentations carried Béchamp was anxious to establish whether his earlier conclusions. He now believed out by cells were to be regarded as the partial inversiona (hydrolysis) to glu- that cold water alone does not invert ‘proper’ fermentations. There was clear cose and fructose of cane sugar, dissolved sucrose, but that the reaction, when it evidence of soluble ferments (that is, in water and left to stand in stoppered occurs, is the result of a ‘true fermenta- enzymes) that operated outside cells, two bottles at room temperature for up to nine tion’. He concluded that moulds do not of the best-known examples being the months, was the result of an action of develop in the absence of air and, in their actions of diastase on starch and invertase water alone or had some other cause. absence, inversion does not occur; but if on sucrose. Thus it was not the ferment His first published work on this topic,7 simple solutions of sucrose are in contact that was living, but the cells that produced in 1855, was to show that, if to the sucrose with air, then moulds develop and inver- it, a point established unequivocally only solution he added 25% calcium or zinc sion occurs in proportion to the develop- with Buchner’s production, 40 years later chloride, no inversion was observed. In ment of the mould. With these findings, in 1897, two years after Pasteur’s death, of the solution without salts some mould Béchamp also regarded himself as the a juice from yeast capable of ethanolic appeared within a month, but in subse- first to show the action of ferments in fermentation.6 Pasteur was aware of such quent months it did not increase in extent, sugar solutions in the absence of proteina- a possibility but was never able experi- though inversion was then taking place. ceous material, that is, the ferment had to mentally to show it, and we know that in At this date it was known that acids could arise within the living cell. Buchner’s case the discovery was largely bring about inversion of sucrose. Béchamp a matter of luck. concluded from this experiment that the Controversy between Béchamp and acidity of salts was not comparable to the Pasteur in the academies Antoine Béchamp acidity of an acid, yet water acted on Things began quietly. At a meeting of While Pasteur was busy with his fermen- the sucrose by virtue of its acid nature, the Sociétés des Savantes in 1862, Pasteur, tations, another chemist was also actively although not showing an acid reaction in the presence of Béchamp, claimed studying the possible influence of living precedence for showing the appearance organisms to bring about fermentation. of living organisms in a medium devoid of Pierre Jacques Antoine Béchamp was albuminoid matter. The meeting report9 born the son of a miller in 1816 (Pasteur, reads: the son of a tanner, in 1822). Initially studying pharmacy in Strasbourg and M. Béchamp quoted some experiments, in teaching in various of the faculties in the which the transformation of cane sugar into grape sugar, brought about under the university, in 1854 he succeeded Pasteur influence of air, is always accompanied by (who had moved to Lille) as professor of moulds. These experiments agree with the chemistry. In 1856 he was appointed results obtained by M. Pasteur, who has- professor of medical chemistry and phar- tened to acknowledge that the fact put macy in the faculty of medicine of the forward by M. Béchamp is one of the most University of Montpellier (Fig. 4), where rigid exactness. he worked for 20 years. Béchamp believed The Académie des Sciences was an that a question of pure chemistry often important venue for airing and develop- a All naturally occurring sugars rotate the plane of polarized ing views and a place to put forward light to the right [dextrorotatory; a plus (+) rotation] or to the novel ideas. In 1864, Béchamp felt moved left [laevorotatory; a minus (–) rotation]. Polarimetry, a pro- to present a Mémoire 1 0 in which he cedure dating from the early years of the 19th century, was once one of the easiest ways of following a reaction such as suggested that only soluble ferments (like the hydrolysis of sucrose. Sucrose has a specific rotation of invertase, to which he gave the name +67°, glucose of +53° and fructose –92°. An equimolar zymase) were constant in their actions. mixture of glucose and fructose is therefore laevorotatory, whereas that of sucrose is dextrorotatory. Invertase thus The organized ferments (Pasteur ’s ‘prop- catalyses a reaction that results in the inversion of the plane erly called’ fermentations) generated of polarization from being to the right to being to the left. An variable amounts of products, according old name for fructose is invert sugar or laevulose, and for Fig. 4. Béchamp when professor of medical chemistry glucose, dextrose (or grape sugar), as opposed to cane and pharmacy at the University of Montpellier to circumstances, because they reflected sugar for sucrose. 1857–1875. From ref. 16. the nutritional activities of cells which History of Science South African Journal of Science 103, September/October 2007 379 consume organic materials, breaking accuracy of his experimental methods what he considered the impropriety of them down and converting them into from the aspersions cast upon them by Pasteur’s behaviour and the error of his simpler forms. It often requires several Pasteur.’ views of disease, which he described as successive fermentations (in modern ‘the greatest scientific silliness of the age’, terms, several enzymes) to produce the Études sur la Bière versus Les consumed him for the rest of his long total effect. For Béchamp, alcoholic fermen- Microzymas13 life.19 tation and the fermentations by organized In his book Les Microzymas, published in ferments are not ‘properly called’ fermen- 1883, Béchamp describes how Pasteur, in The microzymes tations—they are simply manifestations 1876 in his Études sur la Bière,15 ‘in cold The microzymes are a form of life that of nutrition, a remarkably prescient idea. blood’ tried to demolish him once and for Béchamp, over a period of 30 years, be- In 1872 we again find Béchamp telling the all. Pasteur wrote: lieved that he had discovered, beginning, Academy11 that he believed he was the as he points out in his book of this name,12 first to point out that organized ferments The first note of Béchamp on the inver- sion of sucrose is in 1855.7 There is no with his experiments carried out in the can develop in media in the absence of mention there of the influence of moulds, 1850s on the influence on moulds on the proteinaceous material and that fermen- the second where he states this influence hydrolysis of sucrose. In the book he tries tation is essentially an act of nutrition is of 4 January 1858,8 after my work on to bring together all the relevant data that which includes excretion. lactic fermentation, published 30 No- brought him to the belief that the micro- Conflict erupted yet again at the inter- vember 18571 where I establish for the zymes are at the basis of all life and death. national medical congress held in London first time that the lactic ferment is an or- ganized living being, that albuminoid When examining solutions in which the in 1881. Describing a session concerning hydrolysis of sucrose was taking place, the role of bacteria in disease,12 Béchamp, materials do nothing in the cause of fer- mentation, after also my first work on al- Béchamp observed extremely small micro- now from Lille, wrote: coholic fermentation published on 21 scopic forms, similar to those seen in fer- M. Pasteur began to lecture and sud- December 1857.4 What is certain, one is at mentations. In his paper of 1857 ,17 denly, in my presence, before I had said a pains to point out, is that Béchamp, who he designates them as little bodies and thus word, he condemned me in a general since 1855 has not suggested the action of he came to regard the molecular granula- anathema towards all aspects of hetero- moulds on sugar, although he had noted tions of the histologists as being organized genesis.b I was waiting to speak, because I their presence, has now modified his former conclusions. and living ferments. was due to lecture after him. But soon I was obliged to go down from my place to Béchamp came up with a startling find- the front to sit opposite M. Pasteur The implications of this is that the change ing in 1866.20 It was a well-established because he had dared to say “that even if of Béchamp’s ideas, which took place procedure to add chalk, mainly powdered there were any points in my results, I had between his first paper in 18557 and the limestone, to lactic and other acid fermen- only incorporated his ideas and made note which appeared in January 1858,8 tations to maintain neutrality. But Béchamp them mine”. In short M. Pasteur had just occurred after he had heard of Pasteur ’s asks whether this is the only role of the claimed a priority of views and made an work as presented to the Academy in chalk, which, as well as consisting of the accusation of unprecedented plagiarism. November1 and December4 1857. To us, fossil remains of crustaceans, he finds still In an indignant voice I demanded of M. Pasteur to prove his assertion, since I familiar with long intervals between contains a whole generation of extremely would myself show him that the contrary submission of a manuscript and its even- small organisms, smaller than the yeasts. was true. M. Pasteur, refusing a public tual publication, this charge would seem Not only do they exist, but they are alive, discussion, left the session. improbable, but on occasion publication despite their extreme geological age. They could be very rapid. grow with a rare energy like ferments Béchamp goes on to say that The Times Béchamp’s answers in Les Microzymas12 [yeasts]. They are the most active ferments newspaper carried full details of the inci- to Pasteur ’s accusations are, first, outrage Béchamp has encountered and they dent. Actually, The Times report of 8 that Pasteur could make such suggestions, nourish themselves on very diverse organic August13 was more restrained and the and secondly that all his (Béchamp’s) new substances. summary of Pasteur ’s lecture (delivered ideas were contained in his memoir of To the organisms from lime (killed in French, as was Béchamp’s) was directed 1857. Unfortunately, the latter does not when heated to 300°C) Béchamp gives the towards criticism of the work of Charlton exist, but an apologist16 describes how this name Microzyma cretae, but microzymes Bastian over spontaneous generation. memoir is his paper in Annales de Chimie,17 (meaning ‘minute ferments’) are found However, Béchamp is reported to have which for unknown reasons appeared everywhere, including in soil. Béchamp’s ‘affirmed that the microzymas in chalk only in September 1858. Dates of submis- paper20 is logically presented, with se- [see below] did exist and that if Pasteur sion of manuscripts are not indicated in quential arguments, and the remarkable has not obtained such results it was the final publication. nature of his results is clear to him. Could because his experiments were badly It is interesting to note, in parenthesis, it be that Béchamp was genuinely observ- conducted. Béchamp held that the cause how both Béchamp and Pasteur started ing specific bacteria? Or was it a case of of disease and death lay in the animal their careers more as physical scientists artefacts in the microscopes of the time? itself.’ The report of the same session in the British Medical Journal 14 describes than biologists, but were gradually led Béchamp as ‘vindicating his claim to into biology through the study of aspects Promotion of Béchamp against priority in the discovery of the organisms of fermentation, then turned their atten- Pasteur [microzymes] which caused the fermen- tion to the diseases of man and of higher Béchamp’s views would by now have tation of milk. He also defended the animals. Béchamp, like Pasteur, also been long since forgotten had they not worked on the diseases of wine and of been espoused by anti-vivisectionists and b Heterogenesis could mean either the birth or organization silkworms, making significant findings protagonists of alternative medicine. of a living being otherwise than from a parent of the same which did not attract the publicity of Pasteur ’s use of experimental animals kind—a process called by Charlton Bastian arche- Pasteur’s studies, and again Béchamp felt aroused the wrath of the former, and the biosis—or the generation of animal or vegetable life of low organization from inorganic (i.e. non-living) matter—called he had reason to accuse Pasteur of plagia- desire of the latter to believe that disease by Thomas Huxley abiogenesis. rizing his work.18 Béchamp’s anger at originating from microorganisms is a 380 South African Journal of Science 103, September/October 2007 History of Science fiction, set them firmly against Pasteur in ing to microzymes. Airborne germs arise 5. Pasteur L. (1858). Nouveaux faits concernant l’histoire de la fermentation alcoolique. Compt. favour of Béchamp’s ideas. from microzymes in dead plant and Rend. 47, 1011–1013. Following the death of Béchamp in animal life. Béchamp writes: ‘The micro- 6. Buchner E. (1897). Alcoholische Gährung ohne 1908, an event ignored in France, a Dr zyme is at the beginning and at the end of Hefezellen. Ber. Dt. Chem. Ges. 30, 117–124. Montague Leverson from Baltimore every cell organization. It is the funda- 7. Béchamp A. (1855). Note sur l’influence que l’eau pure et certaines dissolutions salines exercent sur persuaded a writer and sympathizer, mental anatomical element by which the le sucre de canne. Compt. Rend. 40, 436–438. Ethel Douglas Hume, to put together cellules, the tissues, the organism, the 8. Béchamp A. (1858). De l’influence que l’eau pur some notes he had collected as a book that whole of an organism are constituted ou chargée de diverse sels exerce à froid sur the was published first in 1923 under the title living.’12 sucre de canne. Compt. Rend. 46, 44–47. 9. Meeting report (1862). Revues des Sociétés des Béchamp or Pasteur? A Lost Chapter in the Savantes I, 81. History of Biology and pours scorn on Coda 10. Béchamp A. (1864). Sur la fermentation alcoo- Pasteur ’s efforts and accuses him of It seems likely that, in the 1850s and lique. Compt. Rend. 58, 601–605. plagiarism and fabrication.16 The book 1860s, Béchamp and Pasteur were inde- 11. Béchamp A. (1872). Seconde observation sur quelques communications récentes de M. Pasteur, was republished in 1932 and has gone pendently making similar discoveries—a notamment sur la théorie de la fermentation through numerous subsequent editions not unknown phenomenon in science. alcoolique. Compt. Rend. 75, 1519–1523. and modifications of title (more recently Accusations of plagiarism are therefore 12. Béchamp A. (1883). Les Microzymas: L’hétérogénie, being attributed to Douglas Hume!).c It is l’histogénie, la physiologie et la pathologie. Librairie probably not justified. Pasteur without J.-B. Baillière, Paris. of course increasingly true that much question was aggressive and intolerant of 13. Report of International Medical Congress (1881). human disease and suffering cannot be opposition and treated Béchamp shabbily, The Times (London), 8 August, p. 6. attributed to infection but stems from but Béchamp led himself into a theory of 14. Report of International Medical Congress (1881). changes within us, but in this the micro- Br. Med. J. 2, 547–548. such generality that was both its strength 15. Pasteur L. (1876). Études sur la Bière. Gautier-Villars, zymes do not have a place. and its weakness—it could be used to Paris. With a medical colleague, Alfred Estor, explain too much, but did not lend itself to 16. Hume E.D. (1923). Béchamp or Pasteur? A lost Béchamp observed granulations in cells experimental testing. In a France increas- chapter in the history of biology. Covici-McGee, (they mainly studied liver of different Chicago. ingly idolizing Pasteur and his memory, 17. Béchamp A. (1858). De l’influence que l’eau pure, species).21 In the physiological state these Béchamp was bound to become increas- ou chargée de divers sels, exerce, à froide, sur le granules, which they considered to be ingly ignored. Only Miss Hume16 and fol- sucre de canne. Ann. Chim. 3rd ser., 54, 28–42. microzymes, are spherical, but outside lowers, for their own reasons, have kept 18. Béchamp A. (1867). Lettre adressée à M. le the cell they develop into bead-like elon- Président, au sujet de la communication faite par his name alive. M. Pasteur le 29 avril dernier. Compt. Rend. 64, gated structures, eventually recognizable 1042–1043. as bacteria. From observations of bacteria 19. Béchamp A. (1903). Louis Pasteur: ses plagiats in blood, they conclude that bacteria, far 1. Pasteur L. (1857). Mémoire sur la fermentation chimicophysiologiques et medicaux. Chez l’auteur, appelée lactique. Compt. Rend. 45, 913–916. Paris. from being the cause of illness, are, to the 20. Béchamp A. (1866). Du rôle de la craie dans les 2. Pasteur L. (1856). Isomorphisme entre des corps contrary, the result. isomères, les uns actifs, les autres inactifs sur la fermentations butyrique et lactique, et des Thus, in death the microzymes become lumière polarisée. Compt. Rend. 43, 1259–1264. organismes actuellement vivants qu’elle contient. bacteria, eventually reducing the cells of Compt. Rend. 63, 451–455. 3. Pasteur L. (1848). Mémoire sur la relation qui peut higher organisms to dust, and then revert- exister entre la forme cristalline et la composition 21. Béchamp A. and Estor A. (1868). De l’origine et du chimique, et sur la cause de la polarisation développement des bactéries. Compt. Rend. 66, c The most recent edition I have been able to track down is rotatoire. Compt. Rend. 26, 535–538. 859–863. published by Health Research, in 2003 (ISBN-10 4. Pasteur L. (1857). Mémoire sur la fermentation 22. Findlay A. (1965). A Hundred Years of Chemistry, 3rd 0787311286).There are also items on the Web. alcoolique. Compt. Rend. 45, 1032–1036. edn, p. 47. Wiley, New York.