A history of research on yeasts 2: Louis Pasteur and his contemporaries, 1850-1880
2000; Wiley; Volume: 16; Issue: 8 Linguagem: Inglês
10.1002/1097-0061(20000615)16
ISSN1097-0061
Autores Tópico(s)Yeasts and Rust Fungi Studies
Resumo… 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 contribution to biology, based on meticulous and ingenious experiments. He pointed out that, if the catalytic theory of Berzelius and von Liebig were valid, then during fermentation the 'ferment' would give up nothing and take nothing from the fermentable material.m By weighing the ingredients before and after fermentation, he showed that, quite to the contrary, during this process yeast takes something from the sugar. He concluded: 'The breakdown of sugar into alcohol and carbonic acid is associated with a vital process … in which the sugar takes a direct part in providing part of the material of the yeast globules'.n And in 1858 he reported the disappearance of ammonia when yeast both grew and also fermented glucose, in a medium containing ammonium tartrate. His explanation was as follows: … the ammonia is transformed into complex albuminoido material which enters the yeast at the same time as the phosphates give the new [yeast] globules their principal minerals. As for carbon, it is evidently supplied by the sugar.p In 1860, J. H. van den Broek, professor at the Military School of Utrecht, published research on the fermentation of grape juice, attributing fermentation to the growth of yeast cells77. In the same year, Pasteur affirmed the rôle of yeast in alcoholic fermentation49. The first part of this paper of 103 pages deals with the changes that occur in sugar brought about by alcoholic fermentation. The second part considers especially the 'ferment', its nature and the transformations it undergoes. He subjected fermentations to extensive quantitative analyses, and confirmed and greatly extended the observations he had described in his paper45 of 1857. Contrary to von Liebig's assumption78, only 95% of the products of fermenting invert sugar proved to be ethanol and carbon dioxide: the other 5% included glycerol, succinic acid and 'cellulose'.q Pasteur wrote '… we see that the yeast takes something from the sugar …'r Hence, alcoholic fermentation is a physiological process: The chemical changes of fermentation are associated with a vital activity, beginning and ending with the latter. I believe that alcoholic fermentation never occurs without either the simultaneous organization, development and multiplication of cells or the continued life of cells already formed. All the results in this paper seem to me completely in opposition to the opinions of Liebig and Berzelius … Now … in what does the chemical act of decomposing the sugar consist; and what is its precise cause? I confess that I simply do not know.s In addition, Pasteur produced a crop of yeast in a chemically defined medium of sugar, ammonium tartrate, and inorganic phosphate.t Nothing was present that could be putrefied by oxygen and extend its instability to the sugar, as von Liebig and his colleagues had stated78. In this way, Pasteur finally refuted von Liebig's assertion that yeast originates from the action of oxygen on the nitrogenous matter of fermentable liquid. Pasteur made strenuous efforts to establish an equation for alcoholic fermentation, as had Lavoisier some 70 years earlier4. He tried a number of methods, of which, as he wrote, '… the simplest of all succeeded …'. He believed it to be the only practical one. The sugar was made to ferment in a calibrated vessel, initially full of mercury, in which the substances needed were introduced successively. Unfortunately it was necessary to use only a small amount of sugar, because of the difficulty of managing mercury in large vessels. Those used had a volume of 350–450 cm3, neck included. Pasteur gives the details of one experiment: A flask with a long graduated neck was inverted over the mercury. Initially, I placed in that flask 1.440 g of sugar candy;u then 0.3 g of washed fresh yeast, as a little ball of firm paste. Finally I introduced into the flask 8.980 g of water at 15°C, and incubated it at 25 to 33°C.v In the same paper, Pasteur acknowledged the, then, unsolved problem of distinguishing between what we now know is enzymic action from fermentation as a physiological activity of whole, intact cells. He wrote: Should we say that yeast feeds on sugar and excretes alcohol and carbonic acid? Or, should we say, on the contrary, that yeast … produces a substance such as pepsin, which acts on the sugar and is soon exhausted, for no such substance is to be found in fermented liquids? I have nothing to say on the subject of these hypotheses. I neither accept them nor dismiss them and always wish not to go beyond the facts. And the facts tell me only that all true fermentations are associated with physiological phenomena.y The long-standing confusion between enzymic action and fermentation was at this time given particular emphasis by Pasteur's controversy with Berthelot, one of the most powerful members of the French scientific establishment14 and Professor of Organic Chemistry at the École Supérieure de Pharmacie. Relevant evidence came partly from the work of a lesser-known scientist, Moritz Traube, who eventually abandoned full-time research to take over the family wine merchant's business in Racibórz in the Prussian province of Silesia (now in Poland). Traube had been a pupil of von Liebig39 and in 1858 he had described clearly how all fermentations were caused by ferments which were substances produced by living organisms73. Then, in 1860, the year of Pasteur's big paper on alcoholic fermentation, Berthelot published a lucid and interesting account of his work on the inversion of sucrose (cane sugar) by beer yeast10. In 1842, Mitscherlich36 had found yeast extract could convert cane sugar into a laevo-rotary sugar which, in 1847, Dubrunfaut16 then showed to be a mixture of glucose and fructose (sucre de fruits). In his paper of 1860, Berthelot decribes the isolation of invertasez (ferment glucosique) by alcohol precipitation and disputes the views of Pasteur, who had said: …I think that the formation of grape sugaraa is due simply to the constant production of succinic acid, that this is only an incidental phenomenon and that it is never necessary that cane sugar must first become grape sugar to undergo fermentation … I do not think that yeast cells have any particular ability for transforming cane sugar into grape sugar. But succinic acid is a constant product of alcoholic fermentation, and the sugar must undergo in its presence the change that it undergoes generally owing to the action of acids.bb Berthelot's experiments showed that, on the contrary, succinic acid hardly inverted sucrose at all in conditions identical with those that held during fermentation; and furthermore, that inversion could occur in an alkaline medium. He dissolved 200 g of sugar candy (sucre candi) in water to a final volume of 1000 cm3. To 500 cm3 of this solution (A), he added 0.8 g of succinic acid and, to the other, 500 cm3 (B), 10 g of pressed beer yeast. After 16 h at 15–20°C, solution B was in full fermentation: it reduced cupropotassium tartrate and showed a big change in optical rotation. Solution A, on the other hand, gave barely perceptible reduction. A further solution (C) was the same as B, except that it also contained 10 g NaHCO3; there was then slow fermentation and the solution then gave a positive Fehling's reaction. From his results (Table 1) Berthelot concluded: 'It is not to succinic acid that one must attribute the inversion which follows the yeast's action … These facts prove that beer yeast inverts cane sugar by its own action and independently of the acidity of the solution.'cc 19 In further experiments, Berthelot mixed pressed yeast with twice its weight of water, then filtered the mixture and obtained a solution containing 1.5% of dissolved solids. This (presumably cell-free) filtrate rapidly inverted sucrose in the presence of 0.24 M NaHCO3. He wrote: 'The yeast extract thus contains a particular ferment, soluble in water and capable of changing cane sugar into invert sugar'.dd Furthermore, he found this ferment to be still active after redissolving and reprecipitating with alcohol. He had succeeded in isolating invertase from brewer's yeast. But he took his conclusions even further: One knows that the researches of Cagniard Latour, and especially those of Pasteur, have established that beer yeast consists of a mycodermic plant. From the new experiments that I am going to report, I have shown that the plant does not act on sugar physiologically, but simply by the ferments it secretes, in the same way as germinated barley secretes diastase, almonds secrete emulsin, the pancreas of an animal secretes pancreatin, and the stomach of the same animal secretes pepsin.ee Pasteur's immediate response to this attack was semantically adroit, but in part rather disingenuous: One can see … from Monsieur Berthelot's own words, that he calls substances soluble in water and capable of inverting sugar 'ferment'. Now everyone knows that many substances have this property, for example all the acids … When, however, we are concerned with cane sugar and beer yeast, I call only that which ferments the sugar 'ferment', that is, that which produces alcohol, carbonic acid, etc. As to inversion, I have not concerned myself with it. With respect to what causes it, I have only raised a doubt in passing in a note where I summarize three years of observations on alcoholic fermentation. Consequently, the contradiction that Monsieur Berthelot believes he has found, between my statements and the true facts, hold only because of the wider definition he gives for the word 'ferment', whereas I have always applied it only to substances that produce true fermentations.ff In 1861, Pasteur published his most famous work on yeast, in which he describes the contrasting effects of aerobic and anaerobic conditions on the fermentation of sugar51, 52. Once again, he made an observation of fundamental importance. He put 100 cm3 of sugar solution into a 250 cm3 flask and boiled the solution to remove the oxygen (Figure 2). After cooling, he introduced a very small amount of beer yeast and placed the drawn out neck of the flask under mercury (Figure 3). The yeast grew only a little and the sugar fermented: 60–80 parts of sugar were consumed for one part of yeast formed. Pasteur's apparatus for sterilizing and removing oxygen from a sugar solution in flask A. After cooling, the end of the curved tube was placed under mercury, as in Figure 3 (Études sur la Bière, Figure 60) A double-necked flask, with one neck drawn out and placed under mercury (Études sur la Bière, Figure 59) If, however, in a similar experiment contact with the air is allowed over a large surface area … much more yeast is produced for the same quantity of sugar consumed. The air loses oxygen as a result of its absorption by the yeast. The yeast grows vigorously in these conditions, but its capacity to ferment tends to disappear. For one part of yeast formed, only 4 to 10 parts of sugar are transformed. The yeast nevertheless retains its capacity to cause fermentation. Indeed it appears greatly increased if it is again cultured with sugar in the absence of free oxygen.gg Hence Pasteur had found the growth yield per gram of sugar consumed to be up to 20 times greater aerobically than anaerobically. Writing on bacterial putrefaction55 in 1863, Pasteur introduced the terms 'aerobic' and 'anaerobic' disarmingly: 'I propose with all kinds of misgivings these new words aerobic and anaerobic, to indicate the existence of two classes [of microbe] … those which survive only in the presence of free oxygen gas, and those which can multiply without contact with free oxygen'.hh In a recent critical paper on this aspect of Pasteur's work with yeasts, Lagunas comments:33 … the enormous difference in growth yield, observed by Pasteur, between aerobic and anaerobic cultures cannot be ascribed to the energetic benefit of the respiration of sugars but is better explained by the ability of aerobic yeast to utilize fermentation products [presumably ethanol] and the inability of anaerobic yeast to grow after a few generations. Both D-glucose and D-fructose are now well known to repress the aerobic catabolism of Saccharomyces cerevisiae, even in fully aerobic conditions18. Pasteur, however, was working with sucrose; but, since sucrose hydrolysis by invertase occurs outside the plasma-membrane of Saccharomyces cerevisiae, it is glucose and fructose, the products of sucrose hydrolysis, that are transported into the cells; hence respiration is repressed also when sucrose is the carbon source27, presumably by the products of sucrose hydrolysis. These findings suggest that it would be useful to compare the behaviour of wild-type Saccharomyces cerevisiae with that of mutants with no invertase in which, as Zimmermann and his colleagues have described30, sucrose is hydrolysed by cytosolic α-glucosidase. Pasteur was fully aware, not only of widely different kinds of microbe, but also of different sorts of yeast. These were beginning to be described from many sources, such as beer, vines, cheese, rotten wood, trees and truffles; and human urine, intestines, mouth, skin and hair (Table 2). Pasteur's work on beer and wine yeasts gives some account of different yeasts, although he was never much interested in taxonomy. As late as 1876, in his Études sur la Bière, he wrote: 5 35 22 12 62 4 69 13 5 7 3 80 8 64 63 67 John Quekett's drawing of yeast cells, published in 1852 (ref.62, p. 18) Drawings of cells of yeast‒like fungi by A. C. J. Corda, published in 1854 (ref.13, p. 5). Corda was the first mycologist to indicate cell sizes consistently and published technical illustrations of the microscopes he used2 I have never given specific names to these different yeasts, any more than to the other microscopic organisms that I have had occasion to study. This is not so much from indifference towards nomenclature, as because I have been exclusively preoccupied with the physiological functions of these little beings, and have therefore always been afraid of attaching too much importance to exterior characters. Many a time I have found that forms different in appearance often belong to the same species and that similar forms can hide profound differences.ii Pasteur's Études sur le Vin56, first published in 1866, was largely concerned with diseases of wine and how to prevent them. He was particularly interested in the wines of the Arbois area, in the Jura, where his father was a tanner and where Pasteur himself had been at school. There he studied the celebrated vin jaune of Château-Châlons, a long-keeping, very alcoholic wine, rather like an unfortified fino sherry. Like such sherry, the vins jaunes are left on ullage for several years66, 68. That is, the cask is not filled up at each racking (the transfer of wine off its lees from one cask to another). The surface of the wine in the cask is therefore exposed to air and develops a thick, white covering of yeast, fleur du vin, probably Saccharomyces cerevisiae (compare the flor of sherry, see ref.21), underneath which the wine is generally quite limpid. Figure 6 shows Pasteur's illustration of yeast cells from a similar growth on a wine surface. Cells of Mycoderma vini (probably Saccharomyces cerevisiae) from the surface of a wine (Études sur le Vin, 2nd edn, Figure 2) Études sur la Bière57, which appeared 10 years later, similarly dealt with alcoholic fermentation and the diseases of beer. It also compares brewing with wine-making. Indeed, this work describes some elegant experiments on yeasts associated with wine grapes, probably carried out in the autumn of 1872. In these experiments, forty 250–300 cm3 flasks (Figure 7) were filled with filtered and boiled must (unfermented grape juice). Wine grapes were washed with a few cm3 of water, which, when examined under a microscope, were found to contain microbes. (Pasteur refers to a similar observation already made with gooseberries by H. Hoffmann28). Ten flasks were left without additions. A few drops of the grape-washings were put in each of a second group of ten. To a third lot were added boiled grape-washings. Finally, a drop of grape juice, taken from the inside of an uninjured grape, was introduced into each of a last set of flasks. To do this, the tube of each flask was bent and drawn out to a fine point (Figure 8), which was then closed in a flame. The point was broken off inside the grape and a drop of juice drawn into the flask by the reduced pressure therein. The broken point was then sealed in a flame. After incubation, the only samples of must to ferment were those in flasks with unboiled washings. The kind of flask used for experiments on the juice in grapes (Études sur la Bière, Figure 8) A. One neck of flask drawn to a fine point (a). B. Fine point of the flask thrust into grape (Études sur la Bière, Figures 9A and B) The importance of these findings comes in part from the work of Edmond Fremy, who worked in Paris as a professor at the École Polytechnique and then at the Muséum d'Histoire Naturelle39. According to him, 'albuminous material' (protein) from inside grapes is transformed into yeast grains by a vital force. Pasteur's experiments showed the yeast which ferments the grapes to come from the exterior and not the inside of the grapes. And Pasteur pointed out that Liebig's theory, on the transformation of albuminous substances into ferments after oxidation, was also disproved. The term 'vital force' or Lebenskraft varied in meaning, sometimes with mystical overtones. It was often used to refer to processes in living organisms that could not be carried out by chemists, so rejection of the concept was important for the development of the scientific study of those processes. In 1862, Pasteur discussed further the question of where wine yeasts came from54. He described how yeasts could be found in different fruit juices of high acidity. If the juices were less acid, bacteria would grow too. This he reported for clear, filtered grape juice; he enlarged on these findings in Études sur la Bière (ref.57, pp. 148–149). To examine these yeasts under the microscope, Pasteur used a glass tube blown out into a flat bulb (Figure 9) and placed on the microscope stage (Figure 10) These bulbs were made by the famous Bonn glass-blower, Heinrich Geissler. Pasteur's illustration (Figure 11) shows two kinds of yeast. The first, which began the fermentation, was the small, apically budding, lemon-shaped, Saccharomyces apiculatus (now named Hanseniaspora uvarum). The second, which continued the fermentation, was a larger yeast with round cells (probably Saccharomyces cerevisiae), which Pasteur called either Saccharomyces pastorianus or Saccharomyces ellipsoideus. These names had been given by Max Reess in his book67, published in 1870, on fungi capable of alcoholic fermentation. Reess described various new yeast species (Table 2), illustrated with good drawings, which also include clear depictions of asci with ascospores. He discussed the possibility of ascospore formation being a sexual process, but considered that there was insufficient evidence for this idea. Pasteur's account of the succession of yeasts in making a wine, is consistent with more recent surveys20 of wine that has been made without adding cultured yeast. German-made glass bulb, used by Pasteur to examine yeast cells with a microscope. A tube is blown out into a flat bulb, the sides of which in the centre are sufficiently close together to contain only a thin layer of liquid (Études sur la Bière, Figure 31) The glass bulb shown in Figure 9 is here in situ on the microscope stage (Études sur la Bière, Figure 71) Pasteur's drawings of the cells of two kinds of yeast found in fermenting grape must: he called the small lemon-shaped yeast Saccharomyces apiculatus and the larger round-celled yeast Saccharomyces pastorianus or Saccharomyces ellipsoideus (Études sur la Bière, Figure 27) By the 1870s, even von Lie
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