exploding the pasteurian legend
TRANSCRIPT
Stereoisomerism
Pasteur entered the École normale supérieure, the famousteachers’ college in Paris, in 1843, aged 20, and in August1847 submitted theses for his doctorate in chemistry andphysics. In the physics thesis, following the views of thechemist Auguste Laurent who had worked for severalmonths in 1846–1847 in the same laboratory, Pasteurconcluded that substances of a similar crystalline formhad similar optical activity1. This conclusion prepares usfor his well known discovery of the d and l forms oftartaric acid. However, the evidence of his notebookssuggests that Pasteur’s greater concern at the time waswith crystal structure and waters of crystallization, aninterest that also arose from his contact with Laurent.
In March 1848, Pasteur presented a Memoir2 to theAcademy of Sciences in which he claimed that a group ofeight different salts of tartaric acid could be crystallizedtogether in any stoichiometry, a result that he recognizedas remarkable because the various tartrates belonged totwo crystallographic systems. (This work, undertaken withtwo students, subsequently proved to be unrepeatable.)
A particular problem arose over sodium ammoniumtartrate with eight waters of crystallization (four per tar-trate molecule) as sodium ammonium paratartrate, whichthe German chemist Eilhard Mitscherlich had found to beisomorphic with common tartrate, had been shown by theMontpellier chemist Charles Gerhardt to contain only twowaters of crystallization. In trying to resolve this anom-aly, Pasteur began to look for microscopic differences inthe crystal structures of common sodium ammoniumtartrate and the corresponding paratartrate. Mitscherlich,as Pasteur was aware, had also been baffled by the seem-ingly bizarre observation that, whereas common tartratewas optically active, paratartrate was not. It was from
these considerations and studies that Pasteur made theremarkable and unexpected observation that, althoughthe hemihedral crystal structure of paratartrate was thesame as for the common salt, many of the crystals were,in fact, mirror images of each other (Figure 2a,b)3. Manualseparation of the mirror image forms of the paratartratesfrom each other and their dissolution in water gavesolutions with equal and opposite optical activities. Thus,the puzzling lack of optical activity seen by Mitscherlichfor paratartrate was now explicable.
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Exploding the Pasteurian legend*Keith Manchester
In 1971, on the death of Louis Pasteur’s grandson, Pasteur Vallery-Radot, the collection of Pasteur’spersonal papers and notebooks, which had mostly been donated to the Bibliothèque nationale in Paris,became more accessible to scholars. Louis Pasteur (Figure 1) was one of the world’s greatest scientists,but since his death in 1895 his memory has been revered to an extent that almost borders on idolism. Oneconsequence of the improved access to Pasteur’s notebooks and correspondence was the publication in1995 of Gerald Geison’s book The Private Science of Louis Pasteur1, in which Geison compares what hebelieves to have been the more realistic sequence of steps by which Pasteur reached his unquestionablyfamous discoveries with the widely publicized Pasteurian legends that often read more like film scenarios.This article attempts to trace the stages by which Pasteur came to some of his celebrated conclusions inthe earlier years of his career.
Keith Manchester
Is at the Dept of Biochemistry, University of the Witwatersrand,Johannesburg, Wits 2050, South Africa.e-mail: [email protected]
Figure 1 Pasteur in 1857, aged 34, when Dean of the Faculty of Sciences in Lille (reproduced, with permission,from Ref. 1).
*This review was first published in Trends in BiochemicalSciences (2001) Vol. 26, pp. 632–636. We thank the authorsand TiBS for allowing us to reproduce it here. If citing the article, please refer to the original source.
It is at this point that Pasteur is supposed to have ex-claimed4 ‘Tout est trouvé!’ (‘All is discovered!’). But wasit? Certainly, the anomaly of optical activities observedby Mitscherlich could now be explained, but not theapparent co-crystallization of sodium ammonium tartratewith eight waters of crystallization and of the corre-sponding paratartrate with two, for it was surely incon-ceivable that the mixture of mirror image crystals wouldhave different waters of crystallization from those foundin common sodium ammonium tartrate. This must haveremained an enigma to Pasteur, for not until 1886 was itshown5 that only at a temperature below 27°C does sodiumammonium paratartrate crystallize in the hemihedral formthat Pasteur observed (Figure 2a,b), and that above 27°Cthe crystal structure is homohedral (Figure 2c), as Pasteurhad found for all the other paratartrates he had examined.Then, and only then, might Pasteur have said ‘Tout estvraiment trouvé!’ (‘Everything is truly discovered!’).
Optical activity, crystalline hemihedry and life
As a consequence of his findings with sodium ammoniumparatartrate, Pasteur attempted to establish a new scien-tific law, namely a general correlation between opticalactivity and crystalline hemihedry. Though this ‘law’eventually proved to be untenable, the struggles led himinexorably to another series of correlations, namely be-tween optical activity and the activity of living cells, andlater between living cells and fermentation.
In 1850, the chemist Victor Dessaignes demonstrated thataspartate could be produced in the laboratory from fumarate6
(Figure 3) – this was, to Pasteur, an unwelcome observationbecause fumarate was optically inactivewhereas natural aspartate was active. In fact,Laurent had pointed out in the early 1840show some organic alkaloids (e.g. nicotine)were optically active in the natural state, butwould possibly be inactive in their syntheticor artificially prepared forms7. Pasteur, to hisgreat satisfaction8, showed that Dessaignes’saspartate was indeed optically inactive.
However, Pasteur refused to concede thatthe lack of optical activity arose because aracemic mixture of aspartates was produced(which was the case). Instead, he preferredto believe, presumably because both formsof the currently known racemate of tartratewere optically active and of natural origin,in the existence of different species identi-cal in chemical, but different in optical,properties – one produced chemically andthe other naturally. In this he was curiouslyrecreating Mitscherlich’s dilemma. In fact,in 1853, in experiments seeking to resolvethe components of paratartrate by heatingwith cinchonine, Pasteur did discoveranother form of tartaric acid, namely theméso or ‘untwisted’ form9 which, as we nowknow, is optically inactive as a result of in-ternal compensation (Figure 4a). But thereis no méso form of aspartate.
Thus, each discovery led to an equally inconsistent newphenomenon; the young Pasteur seemed to be a genius in making important findings on the basis of false startingpremises. Because of Pasteur’s later fame and thepopularity of portraits from his later years, we tendautomatically to see him as a patriarchal figure. At thetime of the work just described, Pasteur was in his late20s. It is impressive that he should have espoused suchbold hypotheses based on slender evidence. One cannotbut admire the clarity of his writings and the rigour of hisexperimentation.
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(a) (c)(b)
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Figure 2 (a) and (b) Hemihedral crystals of (+) and (–) sodium ammonium tartrate, respectively, and (c) holohedral crystal of sodium ammonium paratartratesuch as is obtained with crystallization above 27°C. The mineralogist and crystallographer René Juste Haüy referred to the full symmetrical development ofcrystal faces as holohedral; if half of any form were missing this was referred to ashemihedral. Forms (a) and (b) possess four waters of crystallization per tartratemolecule; form (c) possesses only one water per tartrate molecule. Reproduced,with permission, from Ref. 28.
C
C
COOH
COOH
H
H
C
C
COOH
COOH
H
H
NH2
H
C
C
COOH
COOH
H2N
H
H
H
C
C
COOH
COOH
H
H
H
H
(a) (b) (c) (d)
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Figure 3 Structures of (a) fumaric, (d) succinic, and (b) and (c) aspartic acids. Formation of aspartic acidfrom either fumaric or succinic acid results in a racemic mixture of the D- and L-forms of aspartic acid.
C
C
COOH
COOH
H
OH
C
C
COOH
COOH
H
H
OH
HO
C
C
COOH
COOH
HO
OH
H
H
C
C
COOH
COOH
HO
H
H
H
(a) (b) (c) (d)
Ti BS
OH
H
Figure 4 Stereochemical configurations of tartaric and malic acids: (a) mesotartaric acid, (b) L-(+)tartaric acid, (c) D-(–)tartaric acid and (d) L-(–)-malic acid.
The unequivocal synthesis of paratartrate from succin-ate was performed by William Perkin and Francis Duppain 1860 (Ref. 10), thus showing that a racemic mixture ofoptically active species can be made by chemical meansfrom an optically inactive precursor. This was acceptedby Pasteur.
Optical activity of fermentation products
It seems that Pasteur’s increasing interest in fermentationafter his move from Strasbourg to Lille in 1855 had littleto do with the industrial interests of the region as the storyis usually told4 but, as he himself put it, arose from the‘inflexible’ logic of his research that took him from‘crystallography and molecular chemistry to the study offerments’11. The amyl alcohols that were known to ariseduring alcoholic fermentations, particularly when usinggrain as the source of sugar, had proved to be the rocks onwhich Pasteur’s concept of the correlation betweenoptical activity and hemihedrism finally foundered, as hewas unable to find any evidence of hemihedral facets incrystals of the optically active amylates (nor any con-vincing excuses for this as he had managed to provide forthe many other exceptions)12. Moreover, two forms ofamyl alcohol arose in fermentation, one optically active,the other not.
The optically active form of amyl alcohol posed afurther problem, namely the origin of its optical activity.The perceived wisdom, derived from the famous Germanchemist Justus von Liebig and others, was that it camefrom the optical activity of the sugar from which it wasformed during fermentation, a process that, at the time,was considered by the Liebig school to be essentially oneof decomposition not involving living organisms. How-ever, Pasteur thought that the structure of the amylalcohols, based at this time on empirical formulae andproperties, were too far removed from that of sugars toretain any vestige of the sugar’s optical activity. There-fore, the origin of the amyl alcohols and, in particular, anyasymmetry and hence optical activity, must have arisen asa result of the action of living cells, given the assumptionthat asymmetrical molecules were only to be found innatural products.
Fermentation must, therefore, be one of the activities of living cells, a view that had already been clearly
established independently by other in-vestigators such as Charles Cagniard-Latour, Theodor Schwann and FriedrichKützing 20 years previously13. Althoughtheir views were opposed by Liebig and theSwedish chemist Jacob Berzelius, thedifferent fermentation products must beexpected to result from the activity ofdifferent organisms. Thus, in his firstMemoir on fermentation to the Academy ofSciences in 1857 (Ref. 14), Pasteur boldlystates ‘I intend to establish that, just asthere is an alcoholic ferment, the yeast ofbeer, which is found everywhere wheresugar is decomposed into alcohol andcarbonic acid, so also there is a particular
ferment, a lactic yeast, always present when sugarbecomes lactic acid.’ And this is precisely what he found.
Ironically, as we now know, optically active amylalcohol arises during alcoholic fermentations not fromsugar but from isoleucine, itself asymmetric in its ali-phatic chain (Figure 5). Thus, the origin of opticallyactive amyl alcohol is as proposed by Liebig, except notfrom sugar. It is, however, produced by the yeast cell andnot by the albuminoids (proteinaceous components) ofthe fermentation medium. In addition, another amyl alco-hol is derived from leucine and does not possess anasymmetric carbon (Figure 5).
Fermentation correlative with life
Yeast, when incubated with sugar alone, gradually disinte-grates. Pasteur recognized this event as one of the mostimportant points in Liebig’s theory of fermentation15. Iffermentation, Liebig argued, is a consequence of the de-velopment and multiplication of cells, as others claimed,incubations containing sugar alone should not producealcohol as such a medium lacks the other essential con-ditions for cell growth and division. Nevertheless, alcoholis produced under these conditions.
Pasteur showed that fermentation occurs as a result ofthe growth of yeast cells that feed off the remnants ofdead cells15. Thus, he reached the celebrated conclusionthat ‘the breakdown of sugar into alcohol and carbonicacid is an action correlating with a vital phenomenon’;that is, fermentation is a property of living cells. Pasteurwas also able to refute Liebig’s position by showing thatyeast grows and ferments sugar in medium devoid ofalbuminoid material, but containing ammonia and salts16.
Pasteur, having made one clear and important advance,now risked pushing himself in a potentially dubiousposition, this time in relation to vitalism; that is, thatcertain metabolic properties of living matter cannot beobserved outside the cell. There was clear evidence, atleast to other acute minds such as Marcelin Berthelot atthe Collège de France, of soluble ferments (i.e. enzymes)that operated outside cells, two of the best-known ex-amples being the actions of diastase on starch andinvertase on sucrose. Berthelot, in a concise and hard-hitting Memoir17, proceeded to demolish Pasteur’s claimthat the inversion of sucrose was a consequence of the
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CH3 . CH . CH2 . CHNH2 . COOH
Leucine
Isoamyl alcohol
CH3
CH3 . CH . CH2 . CH2OH
CH3
CH3 . CH2 . CH . CHNH2 . COOH
Isoleucine
Active amyl alcohol
CH3
CH3 . CH2 . CH . CH2OH
CH3
Figure 5 Structures of leucine and isoleucine and the related amyl alcohols derived from themduring fermentations. Another name for the amyl alcohols is fusel oil, an acrid oily liquid occurringin insufficiently distilled alcoholic liquors. Systematic names are 3-methyl-1-butanol (for isoamylalcohol) and 2-methyl-1-butanol (for active amyl alcohol).
acidity of succinic acid formed during fermentation, andwas not caused by the action of a ferment. Berthelot sug-gested that there was no fundamental difference betweenthe soluble and insoluble ferments – it was not theferment that was living, but the cell that produced it.
It is not obvious why Pasteur, no doubt stung by theattack but at the same time with a background of chem-istry rather than biology, was not willing to accept thisseemingly very sensible suggestion. He sought instead torebut Bethelot’s proposal with two rather lame remarks18
– first by saying that he was not very interested in solubleferments because similar actions were carried out bymany substances, and second by describing only thosefermentations carried out by cells as ‘proper’ fermen-tations. Thus, fermentation so defined had to be correla-tive with a vital phenomenon. It is hard to believe thatPasteur could not have been aware of the tautologicalnature of this claim. It was only 16 years later with thediscovery of urease in urine that Pasteur was reluctantlyforced to change his stance19.
It is fanciful to speculate whether, by acceptingBerthelot’s viewpoint, Pasteur might have become less ofa microbiologist and more of a biochemist! Cell-freefermentation might have been discovered several decadesearlier, although we know that Pasteur did make severalunsuccessful attempts to prepare cell-free preparationscapable of carrying out fermentation from yeast, and thatEduard Buchner’s discovery20 of cell-free fermentation,in 1897, two years after Pasteur’s death, was by chance.
Spontaneous generation
The concept of spontaneous generation, the study of whichnow occupied Pasteur’s attention, had a long and con-troversial history. Spontaneous generation, the evolutionof low forms of life from inanimate matter (e.g. moulds,worms and bacteria during putrefaction) and the trans-mutation of species had, at different times, been used bothin support of, and against belief in, a divine creator. Aroundthe 1860s, the prevailing conservative and religious at-mosphere of France was against spontaneous generation,and Pasteur himself was by temperament of a politicallyconservative, if not deeply religious, persuasion. In hisbook, Geison1 raises the interesting question of whether,in his studies of spontaneous generation, Pasteur waspossibly unconsciously or deliberately influenced byphilosophical, religious and political interests.
In 1864, Pasteur gave a famous lecture on spontaneousgeneration1, remarking that ‘neither religion, norphilosophy, nor atheism, nor materialism, nor spiritualismhas any place here… It is a question of fact, I haveapproached it without any preconceived ideas.’ However,such remarks should be treated with caution for as late as1884 Pasteur was describing how he had been driven tothe study of fermentation by ‘the almost inflexible logicof [my] studies’ and by his ‘preconceived ideas’ to whichhe clung tenaciously even in the presence of powerfulevidence against them1. Neither admission reflects ad-versely on Pasteur, but they should make us cautious ofaccepting at face value public statements made to impressdistinguished and fashionable audiences.
The concept of spontaneous generation, which Pasteursought to vanquish, might indeed seem absurd to thescientifically educated today, brought up from birth withthe germ theory of disease. However, there are plenty ofindividuals around for whom the germ theory of diseaseis anathema. More importantly, 150 years ago even thescientifically educated were still entitled to ask ‘Where isthe proof that spontaneous generation does not occur?’.The report of the International Medical Congress21 inLondon in 1881 shows Pasteur still battling 20 years afterthe Pouchet affair (see below) with educated individualswho continued to cling to outmoded ideas such that micro-organisms evolve into each other. Pasteur’s publi-cations22–24 confirming that all fermentations are dependenton introduced germs are as rigorous as always and do notreveal any unscientific approach.
A practice of the Academy of Sciences was to set upcommissions to adjudicate disputed matters of interest,sometimes by the award of a prize. This they did in 1859following the publication of Félix-Archimède Pouchet’smonumental treatise Hétérogenie, ou traité de lagénération spontanée in support of spontaneous gen-eration. Pouchet, a respected naturalist working in Rouen,suggested that new organisms arose from the effects of a mysterious ‘plastic force’ that could be found in plantand animal debris as well as in living organisms them-selves. He reported the spontaneous generation of micro-organisms in boiled hay infusions. Members of the twoappointed commissions were heavily biased againstPouchet’s views, even to the point of announcing de-cisions without examining the entries, and it seems thatnone of the other academics noticed how superficially thecommissioners carried out their charge1. None of this canbe held against Pasteur, but he does seem to have been on the winning side from the start. (The Chemistry sec-tion of the Academy awarded Pasteur the Jecker Prize in 1861 for his work on fermentation and praised him forhis most laudable activity and enlightened zeal. In 1862he received the Alhumbert Prize of the Academy for hismemoir on ‘Organised particles which exist in theatmosphere’.)
Why did Pasteur not attempt to repeat Pouchet’sexperiments showing the development of organisms insterilized hay infusions and, thus, apparently supportingspontaneous generation? Probably he did not do so be-cause he was fully convinced from his own work thatPouchet’s contradictory findings must have some trivialtechnical explanation. However, one can ask whether, hadPasteur been more on the defensive, that is, if theprevailing opinion in influential circles was more proPouchet, Pasteur would have felt himself obliged, despitehis own convictions, to repeat Pouchet’s experiments todemonstrate their error. As we now know, Pasteur wouldhave found himself in difficulties because the technicalproblem in Pouchet’s experiments was the presence ofheat-resistant spores whose role in Pouchet’s results wereonly discovered 14 years later25. Thus, one can argue thatin this instance, the prevailing climate of opinion didfavour acceptance of a point of view for which theevidence was not unequivocal.
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Conclusions
René Dubos, in his biography of Pasteur26, suggests thatPasteur carried with him to the grave the dream of hisscientific youth – the fantastic vision of developing tech-niques for the creation or the modification of life byintroducing asymmetrical forces into chemical reactions.Indeed, in the 1850s, Pasteur attempted some bizarreexperiments to try to induce plants to alter the chirality oftheir products. In this he was unsuccessful, but howexcited he would have been to read a recent paper inNature27 showing experimentally that magnetochiralanisotropy – an effect linking chirality and magnetism –can give rise to enantiomeric excess in a photochemicalreaction – an effect that could have played a role in theorigin of the homochirality of life.
Dubos suggests that Émile Duclaux, one of Pasteur’sbiographers and a former collaborator, was probably cor-rect in regarding the subsequent studies on spontaneousgeneration and fermentations as consequences ofPasteur’s early visions. Indeed, it is a striking fact thatPasteur devoted much of his later life to demonstratingthat nature operates as if it were impossible to achievewhat he himself had failed to do. Indeed, he proved thatall claims of life out of lifeless material were based onfaulty observation or unskilled experiments; that life alwayscomes from a ‘germ’, from life. He also demonstratedthat this ‘germ’ imparts on the new life that it creates adirectional force so intense that each living being, how-ever simple, possesses specific properties and functions.Each microbe, Pasteur ultimately showed, is the specificagent of a particular fermentation, of a particular disease.Just as he had failed in his attempts to create or modifylife, so he proved that others, who had claimed to besuccessful where he had failed, had been merely thevictims of illusion.
Pasteur was a scientist of extraordinary experimentalskill, endowed with an exceptional ability to attack con-troversial problems by selecting for critical examinationthe weak points in a theory he intended to disprove. Hewas also frank, stubborn, prodigiously self-confident andintensely serious. He displayed no restraint in controversyand was constitutionally incapable of suffering criticismin silence. But, perhaps these are some of the charac-teristics that stood him in good stead and enabled him to revolutionize our understanding in so many fields – so many that this article has only covered the first 20years of the 50 years of Pasteur’s intensely creative life.It is, nonetheless, instructive to understand in greater de-tail the manner of thinking that enabled Pasteur to reachhis great discoveries.
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