A history of research on yeasts 2: Louis Pasteur and his contemporaries, 1850-1880

… alcoholic fermentation is a process correlated with the life and organization of yeast cells, not with the death or putrefaction of the cells. Nor is it a phenomenon of contact, for in that case the transformation of the sugar would occur in the presence of the ferment without giving anything to it or taking anything from ita (Pasteur, 1860). Introduction…755 Pasteur's passage from chemistry to microbiology…756 Pasteur on alcoholic fermentation…758 Problem of distinguishing between activities ofwhole organisms and of enzymes…760 Quantitative differences between aerobic and anaerobic sugar utilization…762 Yeasts of wine and beer…763 References…769 The first article of this series4 concerned the early nineteenth century, when yeast was first seen to be a living organism. Yeasts and fermentation then began to be studied by biologists as well as by chemists. This, the second article, is on the period 1850–1880, when yeasts became widely recognized as microbes. Different kinds of yeast were described, and their physiology began to be studied. At this time, those influenced by the earlier chemical approaches of Berzelius and von Liebig were in conflict with the newer biologists who followed Schwann. The chemists interpreted changes produced by microbes in terms of catalysis. Hence they helped to found enzymology. The biologists, in contrast, made advances in microbiology, especially microbial physiology. The protagonists were the two French scientists, Louis Pasteur (1822–1895) (Figure 1) and Pierre Eugène Marcellin Berthelot (1827–1907). Louis Pasteur in 1857, professeur et doyen de la Faculté des sciences de Lille. © Institut Pasteur (Archive Photographique Musée Pasteur) Pasteur began as an outstanding research chemist and became one of the most distinguished microbiologists of all time. A master of experimental research, both academic and applied, he is described as an exceedingly serious man, totally obsessed with his scientific work, humourless, politically conservative, royalist and a Catholic by convention. He publicized his researches brilliantly but was sensitive to and highly intolerant of adverse criticism. The most interesting of the many biographies of Pasteur include those of Vallery-Radot76 (his son-in-law), Duclaux17, Dubos15 and Geison25. Berthelot, the son of a medical man who heroically tended the sick in the slums of Paris, became a leading chemist who made major contributions to synthetic organic chemistry39. Although brought up a Catholic, Berthelot became a sceptic, even rather anti-clerical, and a republican. He wrote in simpler French than Pasteur and, as another contrast, Geison25 draws attention to a letter from Pasteur's wife to their daughter, written on a wedding anniversary: ‘Your father, very busy as always, says little to me, sleeps little, and gets up at dawn—in a word, continues the life that I began with him thirty-five years ago today.’ (ref.75, p. 418). Berthelot was more affectionate. When his wife was dying, she asked her children: ‘Qu'arrivera-t-il de mon mari quand je n'y serai plus?’. His response to them was: ‘Je sens que je ne survivrai pas à votre mère’. Indeed, he died an hour after her. A state honour without precedent permitted them to be buried together in the Panthéon39. Pasteur began work on the fermentation of sugar by yeast in the late 1850s. By then, many research workers and practical men in the brewing industry were coming to accept the conclusions reached by Theodor Schwann and others, 20 years earlier, that yeast was a living organism. Accordingly, biologists were beginning to take over the study of fermentation from chemists (see ref.4). The biological nature of yeast and fermentation was, however, still a matter of confusion and controversy. Some influential scientists still held that fermentation and putrefaction were not caused by living microbes. This was despite the results of many experiments, notably those of Schröder and von Dusch70, who had found that filtering air through cotton-wool prevented both putrefaction and fermentation in boiled organic liquids. Charles Frédéric Gerhardt, Professor of Chemistry and Pharmacy at Strasbourg, was one who still held von Liebig's views: he stated that milk sours after boiling and even in filtered air, and that it then contains no living organisms.b Moreover, even as late as 1870, some eminent scientists, including von Liebig,c could not distinguish clearly between microbial fermentation and what is now known to be enzymic activity (e.g. ref.6).d Between 1855 and 1875, however, Pasteur established, unequivocally, (a) the rôle of yeast in alcoholic fermentation, (b) fermentation as a physiological phenomenon, (c) differences between the aerobic and anaerobic utilization of sugar by yeasts. By the age of 25, Pasteur had already reported the connexion between enantiomorphism and optical activity40, 41, 53. In 1848 he was appointed Professor of Chemistry at the University of Strasbourg; and in 1856 he was awarded the Rumford Medal of the Royal Society of London for his crystallographic research81. Pasteur gives a clear account of why he nevertheless changed from chemistry to microbiology. His researches on the optical activity of organic compounds such as tartrates, asparagine and malic acid had led him to believe that all organic compounds with optical activity were formed by living organisms. He wrote: … never, in any circumstances, is an optically active compound produced by a non-living body, while almost all the substances elaborated by nature in vegetable organisms are asymmetrical, in the manner of tartaric acid …e Pasteur's first communication on microbial activity concerned lactic acid fermentation and was made on 3 August 1857 to the Société des Sciences de Lille. He began by explaining his interest in fermentation: After devoting, up to now, all my efforts in trying to discover the relations which exist between the chemical, optical and crystallographic properties of certain substances, with the aim of explaining their molecular constitution, it will perhaps seem astonishing for me to begin on physiological chemistry, so apparently remote from my first work. The two are nevertheless closely tied together.f Pasteur went on to clarify this relationship. In 1855, he had shown one of the amyl alcohols formed during the fermentation of beet juice to be optically active43. Although derived from sugar, which is also optically active, the difference in structure between the sugar and the alcohol was too great for the asymmetrical arrangement of the atoms to have been retained. This observation persuaded Pasteur that it would be of special interest to study how the ‘ferment’ produces these two alcohols.g But, as often happens … my work … has changed from its original direction, so that the results that I am publishing today appear incongruous with my earlier studies… Ultimately, I hope to be able to show the connexion between the phenomena of fermentation and the molecular asymmetry characteristic of substances of organic origin.h He commented further: … I have discovered a mode of fermentation of tartaric acid, which occurs very easily with ordinary dextro-tartaric acid and very badly or not at all with laevo-tartaric acid. Now, something remarkable, … when one subjects paratartaric acid, formed by the combination, molecule to molecule, of two tartaric acids, dextro and laevo [i.e. the racemic mixture], to this same mode of fermentation, the paratartaric acid is separated into the dextro acid which ferments and the laevo acid which remains intact …i Pasteur had laid the foundation for the concept of stereospecificity. In the first 15 years of the nineteenth century, during the Napoleonic wars, the British blockade had prevented the French from importing cane sugar from the West Indies or south-east Asia. Sugar beet was therefore grown widely in northern France and many sugar-beet factories were built1, 34, 72, and by 1854, when Pasteur was appointed Professor of Chemistry at Lille, the fermentation of beet sugar for producing alcohol (needed mainly for industrial use) had become a major industry in the Lille region15. At the time of his appointment, the Ministre de l'Instruction Publique wrote to the Rector of the university: ‘… in order to produce useful and far-reaching results, whilst keeping up with scientific theories, Monsieur Pasteur … must nevertheless adapt the numerous applications to the genuine needs of the country …’j Accordingly, in 1856, when a local producer of alcohol from beet sugar, a Monsieur Bigo, had serious failures of fermentation, he consulted Pasteur. Bigo's son, who studied with Pasteur, wrote: Pasteur had seen under the microscope that the globules were round when the fermentation was sound, that they were lengthening when the deterioration began and that they were all fully lengthened when the fermentation became lactic. This very simple method enabled us to watch the process and to avoid the failures of fermentation that had formerly been often experienced …k In his work on lactic acid fermentation, Pasteur44, 46, 48 established four general requirements for such research: (a) for the fermentation to be studied, optimum conditions must be found; (b) the simplest possible substances must be used; (c) the organisms that appear during the fermentation must be examined with a microscope and their appearance shown to be constant; (d) a minute trace of the presumptive cause must be able to produce the characteristic fermentation. These principles may be likened to the famous ‘postulates’ propounded by Koch31 in 1878.l The École Normale in Paris was founded in 1794 and was one of the two grandes écoles throughout the nineteenth century14. From 1857, Pasteur was director of scientific studies at the École and greatly strengthened its scientific work. In 1857, he published his first paper on alcoholic fermentation45. Like so much of his research, as well as its practical importance, it made a fundamental to on and He the of Berzelius and von Liebig were then during fermentation the ‘ferment’ would up and take from the By the and after fermentation, he to the during this process yeast something from the He of sugar into alcohol and acid is with a process … in which the sugar a in of the of the yeast in he reported the of when yeast both and also in a His was as … the is into which the yeast at the same time as the the globules their for it is by the In at the of published research on the fermentation of fermentation to the of yeast In the same Pasteur the rôle of yeast in alcoholic The first of this paper of with the changes that occur in sugar brought by alcoholic fermentation. The second especially the its nature and the it He to and and greatly the he had described in his of to von Liebig's of the of sugar to be and the acid and Pasteur wrote ‘… that the yeast something from the sugar alcoholic fermentation is a physiological The chemical changes of fermentation are with a beginning and with the I believe that alcoholic fermentation occurs without the and of or the life of already the results in this paper seem to me in to the of Liebig and Berzelius … … in what the chemical of the sugar and what is its I that I not In Pasteur produced a of yeast in a of sugar, and was that could be by and its to the sugar, as von Liebig and his had In this Pasteur von Liebig's that yeast from the of on the matter of Pasteur made efforts to an for alcoholic fermentation, as had some years He a of of as he ‘… the simplest of all He it to be the practical The sugar was made to ferment in a of in which the substances were it was to a of sugar, of the of in had a of Pasteur gives the of one A with a was over the I in that of sugar then of as a little of I into the of at and it at to In the same Pasteur then, of distinguishing between what now is enzymic from fermentation as a physiological activity of intact cells. He wrote: that yeast on sugar and alcohol and on the that yeast … produces a such as which on the sugar and is for no such is to be found in I have to on the of these I accept them them and not to the the me that all are with physiological The confusion between enzymic and fermentation was at this time by Pasteur's with Berthelot, one of the most of the French scientific and Professor of Chemistry at the École de from the work of a who research to take over the wine in in the of in had been a of von and in he had described clearly how all were caused by ferments which were substances produced by living in the of Pasteur's paper on alcoholic fermentation, Berthelot published a and interesting account of his work on the of by In had found yeast could cane sugar into a sugar in then to be a of and de In his paper of Berthelot the of by alcohol and the of Pasteur, who had that the of is to the of acid, that this is an phenomenon and that it is that cane sugar must first become sugar to fermentation … I not that yeast have any for cane sugar into acid is a of alcoholic fermentation, and the sugar must in its presence the that it to the of on the acid at all in conditions with those that held during and that could occur in an He of sugar in to a of of this he of acid and, to the of After at was in fermentation: it and a in optical on the A was the same as that it also was then fermentation and the then a From his results 1) Berthelot is not to acid that one must the which the … These that yeast cane sugar by its and of the of the In experiments, Berthelot yeast with its of then filtered the and a of This in the presence of He wrote: yeast contains a in and of cane sugar into he found this ferment to be still active after and with He had in from he his conclusions even further: that the researches of and especially those of Pasteur, have established that yeast of a From the that I am to I have shown that the not on sugar but by the ferments it in the same as the of an and the of the same Pasteur's response to this was but in rather … from Monsieur that he substances in and of sugar that many substances have this for all the … however, are concerned with cane sugar and I that which ferments the sugar that that which produces acid, to I have not concerned with to what I have a in in a I years of on alcoholic fermentation. the that Monsieur Berthelot he has between my and the of the he gives for the I have it to substances that produce In Pasteur published his most famous work on in which he the of aerobic and anaerobic conditions on the fermentation of he made an observation of fundamental He of sugar into a and boiled the to the (Figure After he a very of yeast and the of the under (Figure The yeast a little and the sugar of sugar were for one of yeast Pasteur's for and from a sugar in After the of the was under as in 3 la A with one and under la however, in a with the air is over a … much more yeast is produced for the same of sugar The air as a of its by the The yeast in these but its to ferment to one of yeast to of sugar are The yeast nevertheless its to cause fermentation. it greatly it is with sugar in the of Hence Pasteur had found the of sugar to be up to 20 than on in Pasteur the terms and with all kinds of these aerobic and to the of two … those which in the presence of and those which without with In a paper on this of Pasteur's work with … the difference in by Pasteur, between aerobic and anaerobic be to the of the of but is by the of aerobic yeast to fermentation and the of anaerobic yeast to after a and are now well known to the aerobic of even in fully aerobic Pasteur, however, was with by occurs the of it is and the of that are into the is also when is the by the of These that it would be useful to the of with that of with no in as and his have is by Pasteur was fully not of widely kinds of but also of of These were beginning to be described from many such as and and and Pasteur's work on and wine yeasts gives some account of he was much in late as in his la he wrote: 3 of yeast cells, published in p. of of by published in p. was the first to and published of the he I have to these any more than to the organisms that I have had to This is not so much from as I have been with the physiological of these little and have therefore been of too much to a time I have found that in appearance often to the same and that Pasteur's first published in was concerned with of wine and how to He was in the of the in the his was a and Pasteur had been at he studied the of a very alcoholic rather an Like such the are on for the is not up at of wine its from one to The of the wine in the is therefore to air and a of the of which the wine is Pasteur's of yeast from a on a wine of from the of a wine la which years with alcoholic fermentation and the of also brewing with Indeed, this work some on yeasts with wine in the of In these experiments, (Figure were with filtered and boiled must were with a of when examined under a were found to microbes. to a observation already made with by were without A of the were in of a second of a were boiled a of from the of an was into of a of the of was and to a (Figure which was then in a The was the and a of juice into the by the The was then in a After the of must to ferment were those in with The of for on the juice in la of to a of the into la and The of these in from the work of who in Paris as a at the École and then at the to from is into yeast by a Pasteur's the yeast which ferments the to from the and not the of the Pasteur that Liebig's on the transformation of substances into ferments after was also The or in with was often to to in living organisms that could not be by so of the concept was for the of the scientific study of those In Pasteur the of wine yeasts He described how yeasts could be found in of the were acid, would This he reported for filtered he on these in la these yeasts under the Pasteur a into a (Figure and on the microscope (Figure These were made by the famous Pasteur's (Figure two kinds of The which began the fermentation, was the The which the fermentation, was a yeast with round which Pasteur or These had been by in his published in 1870, on of alcoholic fermentation. described yeast with which also include clear of with He the of a but that was for this Pasteur's account of the of yeasts in a is with more of wine that has been made without by Pasteur to yeast with a A is into a the of which in the are together to a of la The shown in is in on the microscope la Pasteur's of the of two kinds of yeast found in he the yeast and the yeast or la By the even von Liebig had a for he now yeasts as living of a and the of the yeast on the presence and of substances which become of the living he still the concept of fermentation as He to have had in the experimental he was for to distinguish between of the yeast and its utilization of He wrote: The that the of sugar during fermentation on the and of yeast is with the that the yeast produces fermentation in a of the yeast of substances in and it also contains a of and it has been to in the of these in the sugar fermentation, the of be From however, scientists still that alcoholic fermentation and putrefaction were not of microbial physiology. Pasteur, however, the to a of as a and on some by which were published by Berthelot after death in was one of the of and a of experimental his the concept of living microbes as the cause of fermentation, something had not or even with Pasteur This some with Berthelot and is clearly of great interest to on the of such as but the of yeasts or of alcoholic fermentation. Pasteur's however, made in the late that he still had with distinguishing between the activities of microbes and those of The passage was concerned with of the of alcohol from sugar in a … it is an to me that could believe that I would be by the of ferments in as or by the of alcohol from sugar, of living cells. … I not now the for the of these ferments or the of their in this of Pasteur had already his from that in in his with Berthelot the of in when the of to in he had by then He wrote: yeast of produces a ferment which cane sugar the years in this article, had been a in fermentation and the activities of or of both the in the of von Liebig and the with Pasteur. These changes in were on the scientific by experimental especially those of Pasteur and The in is well by the of published in which an of biological greatly from which he had held 20 years The ferments are as Liebig substances in the process of able to their chemical activity to substances that are but are chemical compounds to the not possible to all substances, a chemical and produce changes in compounds by of chemical by Pasteur) of fermentation as a of the activity of organisms is … The of is ferments are the of the most The confusion produced by the of the ‘ferment’ led in to for the who was a and a at his The have not general on the one it was that chemical and so could not be the was already to yeast and organisms … while on the it was that yeast could not be then all including man, would have to be so … to why the so much I have the to a and I the to some of the known substances, by many This is not to any but it that in yeast something occurs that this or that which is to to the The is however, to be to the of but is to that more organisms from which the and so be are not from the organisms as some would have us By alcoholic fermentation as a of the physiological activity of yeasts was not scientific to that time, the of enzymic separated from that of living cells, of the rôle of in in the nineteenth and into the century, this rôle become (see to and for much and and to and for and of the The of a research from the Royal Society is