The problem of alcoholic fermentation, of the origin and nature of that mysterious and apparently spontaneous change which converted the insipid juice of the grape into stimulating wine, seems to have exerted a fascination over the minds of natural philosophers from the very earliest times. No date can be assigned to the first observation of the phenomena of the process. History finds man in the possession of alcoholic liquors, and in the earliest chemical writings we find fermentation, as a familiar natural process, invoked to explain and illustrate the changes with which the science of those early days was concerned. Throughout the period of alchemy fermentation plays an important part; it is, in fact, scarcely too much to say that the language of the alchemists and many of their ideas were founded on the phenomena of fermentation. The subtle change in properties permeating the whole mass of material, the frothing of the fermenting liquid, rendering evident the vigour of the action, seemed to them the very emblems of the mysterious process by which the long sought for philosopher's stone was to convert the baser metals into gold. As chemical science emerged from the mists of alchemy, definite ideas about the nature of alcoholic fermentation and of putrefaction began to be formed. Fermentation was distinguished from other chemical changes in which gases were evolved, such as the action of acids on alkali carbonates (Sylvius de le BoË, 1659); the gas evolved was examined and termed gas vinorum, and was distinguished from the alcohol with which it had at first been confused (van Helmont, 1648); afterwards it was found that like the gas from potashes it was soluble in water (Wren, 1664). The gaseous product of fermentation and putrefaction was identified by MacBride, in 1764, with the fixed air of Black, whilst Cavendish in 1766 showed that fixed air alone was evolved in alcoholic fermentation and that a mixture of this with inflammable air was produced by putrefaction. In the meantime it had been recognised that only sweet liquors could be fermented ("Ubi notandum, nihil fermentare quod non sit dulce," Becher, 1682), and finally Cavendish [p002] [1776] determined the proportion of fixed air obtainable from sugar by fermentation and found it to be 57 per cent. It gradually became recognised that fermentation might yield either spirituous or acid liquors, whilst putrefaction was thought to be an action of the same kind as fermentation, differing mainly in the character of the products (Becher).

As regards the nature of the process very confused ideas at first prevailed, but in the time of the phlogistic chemists a definite theory of fermentation was proposed, first by Willis (1659) and afterwards by Stahl [1697], the fundamental idea of which survived the overthrow of the phlogistic system by Lavoisier and formed the foundation of the views of Liebig. To explain the spontaneous origin of fermentation and its propagation from one liquid to another, they supposed that the process consisted in a violent internal motion of the particles of the fermenting substance, set up by an aqueous liquid, whereby the combination of the essential constituents of this material was loosened and new particles formed, some of which were thrust out of the liquid (the carbon dioxide) and others retained in it (the alcohol).

Stahl specifically states that a body in such a state of internal disquietude can very readily communicate the disturbance to another, which is itself at rest but is capable of undergoing a similar change, so that a putrefying or fermenting liquid can set another liquid in putrefaction or fermentation.

Taking account of the gradual accumulation of fact and theory we find at the time of Lavoisier, from which the modern aspect of the problem dates, that Stahl's theoretical views were generally accepted. Alcoholic fermentation was known to require the presence of sugar and was thought to lead to the production of carbon dioxide, acetic acid, and alcohol.

The composition of organic compounds was at that time not understood, and it was Lavoisier who established the fact that they consisted of carbon, hydrogen, and oxygen, and who made systematic analyses of the substances concerned in fermentation (1784–1789). Lavoisier [1789] applied the results of these analyses to the study of alcoholic fermentation, and by employing the principle which he regarded as the foundation of experimental chemistry, "that there is the same quantity of matter before and after the operation," he drew up an equation between the quantities of carbon, hydrogen, and oxygen in the original sugar and in the resulting substances, alcohol, carbon dioxide, and acetic acid, showing that the products contained the whole matter of the sugar, and thus for the first time giving a clear view of the chemical [p003] change which occurs in fermentation. The conclusion to which he came was, we now know, very nearly accurate, but the research must be regarded as one of those remarkable instances in which the genius of the investigator triumphs over experimental deficiencies, for the analytical numbers employed contained grave errors, and it was only by a fortunate compensation of these that a result so near the truth was attained.

Lavoisier's equation or balance sheet was as follows:—

The true composition of the sugar used was carbon 40·4, hydrogen 6·1, oxygen 49·4.

Lavoisier expressed no view as to the agency by which fermentation was brought about, but came to a very definite and characteristic conclusion as to the chemical nature of the change. The sugar, which he regarded in harmony with his general views as an oxide, was split into two parts, one of which was oxidised at the expense of the other to form carbonic acid, whilst the other was deoxygenised in favour of the former to produce the combustible substance alcohol, "so that if it were possible to recombine these two substances, alcohol and carbonic acid, sugar would result".

From this point commences the modern study of the problem. Provided by the genius of Lavoisier with the assurance that the hitherto mysterious process of fermentation was to be ranked along with familiar chemical changes, and that it proceeded in harmony with the same quantitative laws as these simpler reactions, chemists were stimulated in their desire to penetrate further into the mysteries of the phenomenon, and the importance and interest of the problem attracted many workers.

So important indeed did the matter appear to Lavoisier's countrymen that in the year 8 of the French Republic (1800) a prize—consisting of a gold medal, the value of which, expressed in terms of the newly introduced metric system, was that of one kilogram of gold—was offered by the Institute for the best answer to the question: "What are the characteristics by which animal and vegetable substances which act as ferments can be distinguished from those which they are capable of fermenting?" [p004]

This valuable prize was again offered in 1802 but was never awarded, as the fund from which it was to be drawn was sequestrated from the Institute in 1804. The first response to this stimulating offer was an important memoir by citizen Thenard [1803], which provided many of the facts upon which Liebig subsequently based his views. Thenard combats the prevailing idea, first expressed by Fabroni (1787–1799), that fermentation is caused by the action of gluten derived from grain on starch and sugar, but is himself uncertain as to the actual nature of the ferment. He points out that all fermenting liquids deposit a material resembling brewer's yeast, and he shows that this contains nitrogen, much of which is evolved as ammonia on distillation. His most important result is, however, that when yeast is used to ferment pure sugar, it undergoes a gradual change and is finally left as a white mass, much reduced in weight, which contains no nitrogen and is without action on sugar. Thenard, moreover, it is interesting to note, differs from Lavoisier, inasmuch as he ascribes the origin of some of the carbonic acid to the carbon of the ferment, an opinion which was still held in various degrees by many investigators (see Seguin, quoted by Thenard).

Thenard's memoir was followed by a communication of fundamental importance from Gay-Lussac [1810]. A process for preserving food had been introduced by Appert, which consisted in placing the material in bottles, closing these very carefully and exposing them to the temperature of boiling water for some time. Gay-Lussac was struck by the fact that when such a bottle was opened fermentation or putrefaction set in rapidly. Analysis of the air left in such a sealed bottle showed that all the oxygen had been absorbed, and these facts led to the view that fermentation was set up by the action of oxygen on the fermentable material. Experiment appeared to confirm this in the most striking way. A bottle of preserved grape-juice was opened over mercury and part of its contents passed through the mercury into a bell-jar containing air, the remainder into a similar vessel free from air. In the presence of air fermentation set in at once, in the absence of air no fermentation whatever occurred. This connection between fermentation and the presence of air was established by numerous experiments and appeared incontestable. Fermentation, it was found, could be checked by boiling even after the addition of oxygen, and hence food could be preserved in free contact with the air, provided only that it was raised to the temperature of boiling water at short intervals of time. Gay-Lussac's opinion was that the ferment was formed by the action of the oxygen on the [p005] liquid, and that the product of this action was altered by heat and rendered incapable of producing fermentation, as was also brewer's yeast, which, however, he regarded, on account of its insolubility, as different from the soluble ferment which initiated the change in the limpid grape-juice. Colin, on the other hand [1825], recognised that alcoholic fermentation by whatever substance it was started, resulted in the formation of an insoluble deposit more active than the original substance, and he suggested that this deposit might possibly in every case be of the same nature.

So far no suspicion appears to have arisen in the minds of those who had occupied themselves with the study of fermentation that this change differed in any essential manner from many other reactions familiar to chemists. The origin and properties of the ferment were indeed remarkable and involved in obscurity, but the uncertainty regarding this substance was no greater than that surrounding many, if not all, compounds of animal and vegetable origin. Although, however, the purely chemical view as to the nature of yeast was generally recognised and adopted, isolated observations were not wanting which tended to show that yeast might be something more than a mere chemical reagent. As early as 1680 in letters to the Royal Society Leeuwenhoek described the microscopic appearance of yeast of various origins as that of small, round, or oval particles, but no further progress seems to have been made in this direction for nearly a century and a half, when we find that DesmaziÈres [1826] examined the film formed on beer, figured the elongated cells of which it was composed, and described it under the name of Mycoderma CerevisiÆ. He, however, regarded it rather as of animal than of vegetable origin, and does not appear to have connected the presence of these cells with the process of fermentation.

Upon this long period during which yeast was regarded merely as a chemical compound there followed, as has so frequently occurred in similar cases, a sudden outburst of discovery. No less than three observers hit almost simultaneously upon the secret of fermentation and declared that yeast was a living organism.

First among these in strict order of time was Cagniard-Latour [1838], who made a number of communications to the Academy and to the SociÉtÉ Philomatique in 1835–6, the contents of which were collected in a paper presented to the Academy of Sciences on 12 June, 1837, and published in 1838. The observations upon which this memoir was based were almost exclusively microscopical. Yeast was recognised as consisting of spherical particles, which were capable of [p006] reproduction by budding but incapable of motion, and it was therefore regarded as a living organism probably belonging to the vegetable kingdom. Alcoholic fermentation was observed to depend on the presence of living yeast cells, and was attributed to some effect of their vegetative life (quelque effet de leur vÉgÉtation). It was also noticed that yeast was not deprived of its fermenting power by exposure to the temperature of solid carbonic acid, a sample of which was supplied to Cagniard-Latour by Thilorier, who had only recently prepared it for the first time.

Theodor Schwann [1837], whose researches were quite independent of those of Cagniard-Latour, approached the problem from an entirely different point of view. During the year 1836 Franz Schulze [1836] published a research on the subject of spontaneous generation, in which he proved that when a solution containing animal or vegetable matter was boiled, no putrefaction set in provided that all air which was allowed to have access to the liquid was previously passed through strong sulphuric acid. Schwann performed a very similar experiment by which he showed that this same result, the absence of putrefaction, was attained by heating all air which came into contact with the boiled liquid. Wishing to show that other processes in which air took part were not affected by the air being heated, he made experiments with fermenting liquids and found, contrary to his expectation, that a liquid capable of undergoing vinous fermentation and containing yeast did not undergo this change after it had been boiled, provided that, as in the case of his previous experiments, only air which had been heated was allowed to come into contact with it.

Schwann's experiments on the prevention of putrefaction were unexceptionable and quite decisive. The analogous experiments dealing with alcoholic fermentation were not quite so satisfactory. Yeast was added to a solution of cane sugar, the flask containing the mixture placed in boiling water for ten minutes, and then inverted over mercury. About one-third of the liquid was then displaced by air and the flasks corked and kept inverted at air temperature. In two flasks the air introduced was ordinary atmospheric air, and in these flasks fermentation set in after about four to six weeks. Into the other two flasks air which had been heated was led, and in these no fermentation occurred. As described, the experiment is quite satisfactory, but Schwann found on repetition that the results were irregular. Sometimes all the flasks showed fermentation, sometimes none of them. This was correctly ascribed to the experimental difficulties, but none [p007] the less served as a point of attack for hostile and damaging criticism at the hands of Berzelius (p. 8).

The origin of putrefaction was definitely attributed by Schwann to the presence of living germs in the air, and the similarity of the result obtained with yeast suggested the idea that alcoholic fermentation was also brought about by a living organism, a conception which was at once confirmed by a microscopical examination of a fermenting liquid. The phenomena observed under the microscope were similar to those noted by Cagniard-Latour, and in accordance with these observations alcoholic fermentation was attributed to the development of a living organism, the fermentative function of which was found to be destroyed by potassium arsenite and not by extract of Nux vomica, so that the organism was regarded rather as of vegetable than of animal nature. This plant received the name of "Zuckerpilz" or sugar fungus (which has been perpetuated in the generic term Saccharomyces). Alcoholic fermentation was explained as "the decomposition brought about by this sugar fungus removing from the sugar and a nitrogenous substance the materials necessary for its growth and nourishment, whilst the remaining elements of these compounds, which were not taken up by the plant, combined chiefly to form alcohol".

KÜtzing's memoir, the third of the trio [1837], also dates from 1837, and his opinions, like those of Cagniard-Latour, are founded on microscopical observations. He recognises yeast as a vegetable organism and accurately describes its appearance. Alcoholic fermentation depends on the formation of yeast, which is produced when the necessary elements and the proper conditions are present and then propagates itself. The action on the liquid thus increases and the constituents not required to form the organism combine to form unorganised substances, the carbonic acid and alcohol. "It is obvious," says KÜtzing, in a passage which roused the sarcasm of Berzelius, "that chemists must now strike yeast off the roll of chemical compounds, since it is not a compound but an organised body, an organism."

These three papers, which were published almost simultaneously, were received at first with incredulity. Berzelius, at that time the arbiter and dictator of the chemical world, reviewed them all in his "Jahresbericht" for 1839 [1839] with impartial scorn. The microscopical evidence was denied all value, and yeast was no more to be regarded as an organism than was a precipitate of alumina. Schwann's experiment (p. 6) was criticised on the ground that the fermenting power of the added yeast had been only partially destroyed in the [p008] flasks in which fermentation ensued, completely in those which remained unchanged, the admission of heated or unheated air being indifferent, a criticism to some extent justified by Schwann's statement, already quoted, of the uncertain result of the experiment.

Berzelius himself regarded fermentation as being brought about by the yeast by virtue of that catalytic force, which he had supposed to intervene in so many reactions, both between substances of mineral and of animal and vegetable origin [1836], and which enabled "bodies, by their mere presence, and not by their affinity, to arouse affinities ordinarily quiescent at the temperature of the experiment, so that the elements of a compound body arrange themselves in some different way, by which a greater degree of electro-chemical neutralisation is attained".

To the scorn of Berzelius was soon added the sarcasm of WÖhler and Liebig [1839, 1839]. Stimulated in part by the publications of the three authors already mentioned, and in part by the report of Turpin [1838], who at the request of the Academy of Sciences had satisfied himself by observation of the accuracy of Cagniard-Latour's conclusions, WÖhler prepared an elaborate skit on the subject, which he sent to Liebig, to whom it appealed so strongly that he added some touches of his own and published it in the "Annalen," following immediately upon a translation of Turpin's paper. Yeast was here described with a considerable degree of anatomical realism as consisting of eggs which developed into minute animals, shaped like a distilling apparatus, by which the sugar was taken in as food and digested into carbonic acid and alcohol, which were separately excreted, the whole process being easily followed under the miscroscope.

Close upon this pleasantry followed a serious and important communication from Liebig [1839], in which the nature of fermentation, putrefaction, and decay was exhaustively discussed. Liebig did not admit that these phenomena were caused by living organisms, nor did he attribute them like Berzelius to the catalytic action of a substance which itself survived the reaction unchanged. As regards alcoholic fermentation, Liebig's chief arguments may be briefly summarised. As the result of alcoholic fermentation, the whole of the carbon of the sugar reappears in the alcohol and carbon dioxide formed. This change is brought about by a body termed the ferment, which is formed as the result of a change set up by the access of air to plant juices containing sugar, and which contains all the nitrogen of the nitrogenous constituents of the juice. This ferment is a substance remarkably susceptible of change, which undergoes an uninterrupted and progressive metamorphosis, of [p009] the nature of putrefaction or decay, and produces the fermentation of the sugar as a consequence of the transformation which it is itself undergoing.

The decomposition of the sugar is therefore due to a condition of instability transferred to it from the unstable and changing ferment, and only continues so long as the decomposition of the ferment proceeds. This communication of instability from one substance undergoing chemical change to another is the basis of Liebig's conception, and is illustrated by a number of chemical analogies, one of which will suffice to explain his meaning. Platinum is itself incapable of decomposing nitric acid and dissolving in it; silver, on the other hand, possesses this power. When platinum is alloyed with silver, the whole mass dissolves in nitric acid, the power possessed by the silver being transferred to the platinum. In like manner the condition of active decomposition of the ferment is transferred to the sugar, which by itself is quite stable. The central idea is that of Stahl (p. 2) which was thus reintroduced into scientific thought.

In a pure sugar solution the decomposition of the ferment soon comes to an end and fermentation then ceases. In beer wort or vegetable juices, on the other hand, more ferment is continually formed in the manner already described from the nitrogenous constituents of the juice, and hence the sugar is completely fermented away and unexhausted ferment left behind. Liebig's views were reiterated in his celebrated "Chemische Briefe," and became the generally accepted doctrine of chemists. There seems little doubt that both Berzelius and Liebig in their scornful rejection of the results of Cagniard-Latour, Schwann and KÜtzing, were influenced, perhaps almost unconsciously, by a desire to avoid seeing an important chemical change relegated to the domain of that vital force from beneath the sway of which a large part of organic chemistry had just been rescued by WÖhler's brilliant synthetical production of urea and by the less recognised synthesis of alcohol by Hennell (see on this point Ahrens [1902]). A strong body of evidence, however, gradually accumulated in favour of the vegetable nature of yeast, so that it may be said that by 1848 a powerful minority adhered to the views of Cagniard-Latour, Schwann, and KÜtzing [see Schrohe, 1904, p. 218, and compare Buchner, 1904]. Among these must be included Berzelius [1848], who had so forcibly repudiated the idea only ten years before, whereas Liebig in the 1851 edition of his letters does not mention the fact that yeast is a living organism (Letter XV).

The recognition of the vegetable nature of yeast, however, by no [p010] means disproved Liebig's view of the nature of the change by which sugar was converted into carbon dioxide and alcohol, as was carefully pointed out by Schlossberger [1844] in a research on the nature of yeast, carried out in Liebig's laboratory but without decisive results.

Mitscherlich was also convinced of the vegetable character of yeast, and showed [1841] that when yeast was placed in a glass tube closed by parchment and plunged into sugar solution, the sugar entered the glass tube and was there fermented, but was not fermented outside the tube. He regarded this as a proof that fermentation only occurred at the surface of the yeast cells, and explained the process by contact action in the sense of the catalytic action of Berzelius, rather than by Liebig's transference of molecular instability. Similar results were obtained with an animal membrane by Helmholtz [1843], who also expressed his conviction that yeast was a vegetable organism.

In 1854 SchrÖder and von Dusch [1854, 1859, 1861] strongly reinforced the evidence in favour of this view by succeeding in preventing the putrefaction and fermentation of many boiled organic liquids by the simple process of filtering all air which had access to them through cotton-wool. These experiments, which were continued until 1861, led to the conclusion that the spontaneous alcoholic fermentation of liquids was due to living germs carried by the air, and that when the air was passed through the cotton-wool these germs were held back.

At the middle of the nineteenth century opinions with regard to alcoholic fermentation, notwithstanding all that had been done, were still divided. On the one hand Liebig's theory of fermentation was widely held and taught. Gerhardt, for example, as late as 1856 in the article on fermentation in his treatise on organic chemistry [1856], gives entire support to Liebig's views, and his treatment of the matter affords an interesting glimpse of the arguments which were then held to be decisive. The grounds on which he rejects the conclusions of Schwann and the other investigators who shared the belief in the vegetable nature of yeast are that, although in some cases animal and vegetable matter and infusions can be preserved from change by the methods described by these authors, in others they cannot, a striking case being that of milk, which even after being boiled becomes sour even in filtered air, and this without showing any trace of living organisms. The action of heat, sulphuric acid, and filtration on the air is to remove, or destroy, not living organisms but particles of decomposing matter, that is to say, ferments which would add their activity to that of the oxygen of the air. Moreover, many ferments, as for example diastase, act without [p011] producing any insoluble deposit whatever which can be regarded as an organism.

"Evidemment," he concludes, "la thÉorie de M. Liebig explique seule tous les phÉnomÈnes de la maniÈre la plus complÈte et la plus logique; c'est À elle que tous les bons esprits ne peuvent manquer de se rallier."

On the other hand it was held by many to have been shown that Liebig's view of the origin of yeast by the action of the air on a vegetable infusion was erroneous, and that fermentation only arose when the air transferred to the liquid an active agent which could be removed from it by sulphuric acid (Schulze), by heat (Schwann), and by cotton-wool (SchrÖder and von Dusch). Accompanying alcoholic fermentation there was a development of a living organism, the yeast, and fermentation was believed, without any very strict proof, to be a phenomenon due to the life and vegetation of this organism. This doctrine seems indeed [Schrohe, 1904] to have been widely taught in Germany from 1840–56, and to have established itself in the practice of the fermentation industries.

In 1857 commenced the classical researches of Pasteur which finally decided the question as to the origin and functions of yeast and led him to the conclusion that "alcoholic fermentation is an act correlated with the life and organisation of the yeast cells, not with the death or putrefaction of the cells, any more than it is a phenomenon of contact, in which case the transformation of sugar would be accomplished in presence of the ferment without yielding up to it or taking from it anything" [1860]. It is impossible here to enter in detail into Pasteur's experiments on this subject, or indeed to do more than indicate the general lines of his investigation. His starting-point was the lactic acid fermentation.

The organism to which this change was due had hitherto escaped detection, and as we have seen the spontaneous lactic fermentation of milk was one of the phenomena adduced by Gerhardt (p. 10) in favour of Liebig's views. Pasteur [1857] discovered the lactic acid producing organism and convinced himself that it was in fact a living organism and the active cause of the production of lactic acid. One of the chief buttresses of Liebig's theory was thus removed, and Pasteur next proceeded to apply the same method and reasoning to alcoholic fermentation. Liebig's theory of the origin of yeast by the action of the oxygen of the air on the nitrogenous matter of the fermentable liquid was conclusively and strikingly disproved by the brilliant device of producing a crop of yeast in a liquid medium containing only comparatively [p012] simple substances of known composition—sugar, ammonium tartrate and mineral phosphate. Here there was obviously present in the original medium no matter which could be put into a state of putrefaction by contact with oxygen and extend its instability to the sugar. Any such material must first be formed by the vital processes of the yeast. In the next place Pasteur showed by careful analyses and estimations that, whenever fermentation occurred, growth and multiplication of yeast accompanied the phenomenon. The sugar, he proved, was not completely decomposed into carbon dioxide and alcohol, as had been assumed by Liebig (p. 8). A balance-sheet of materials and products was constructed which showed that the alcohol and carbon dioxide formed amounted only to about 95 per cent. of the invert sugar fermented, the difference being made up by glycerol, succinic acid, cellulose, and other substances [1860, p. 347]. In every case of fermentation, even when a paste of yeast was added to a solution of pure cane sugar in water, the yeast was found by quantitative measurements to have taken something from the sugar. This "something" was indeterminate in character, but, including the whole of the extractives which had passed from the yeast cells into the surrounding liquid, it amounted to as much as 1·63 per cent. of the weight of the sugar fermented [1860, p. 344].

Pasteur was therefore led to consider fermentation as a physiological process accompanying the life of the yeast. His conclusions were couched in unmistakable words: "The chemical act of fermentation is essentially a phenomenon correlative with a vital act, commencing and ceasing with the latter. I am of opinion that alcoholic fermentation never occurs without simultaneous organisation, development, multiplication of cells, or the continued life of cells already formed. The results expressed in this memoir seem to me to be completely opposed to the opinions of Liebig and Berzelius. If I am asked in what consists the chemical act whereby the sugar is decomposed and what is its real cause, I reply that I am completely ignorant of it.

"Ought we to say that the yeast feeds on sugar and excretes alcohol and carbonic acid? Or should we rather maintain that yeast in its development produces some substance of the nature of a pepsin, which acts upon the sugar and then disappears, for no such substance is found in fermented liquids? I have nothing to reply to these hypotheses. I neither admit them nor reject them, and wish only to restrain myself from going beyond the facts. And the facts tell me simply that all true fermentations are correlative with physiological phenomena."

Liebig felt to the full the weight of Pasteur's criticisms; his reply [p013] was long delayed [1870], and, according to his biographer, Volhard [1909], caused him much anxiety. In it he admits the vegetable nature of yeast, but does not regard Pasteur's conclusion as in any way a solution of the problem of the nature of alcoholic fermentation. Pasteur's "physiological act" is for Liebig the very phenomenon which requires explanation, and which he still maintains can be explained by his original theory of communicated instability. On some of Pasteur's results, notably the very important one of the cultivation of yeast in a synthetic medium, he casts grave doubt, whilst he explains the production of glycerol and succinic acid as due to independent reactions. The phenomenon of fermentation is still for him one which accompanies the decomposition of the constituents of the cell, rather than their building up by vegetative growth. "When the fungus ceases to grow, the bond which holds together the constituents of the cell contents is relaxed, and it is the motion which is thus set up in them which is the means by which the yeast cells are enabled to bring about a displacement or decomposition of the elements of sugar or other organic molecules." Pasteur replied in a brief and unanswerable note [1872]. All his attention was concentrated on the one question of the production of yeast in a synthetic medium, which he recognised as fundamental. The validity of this experiment he emphatically reaffirmed, and finally undertook, from materials supplied by Liebig himself, to produce as much yeast as could be reasonably desired. This challenge was never taken up, and this communication formed the last word of the controversy. Pasteur had at this time firmly established his thesis, no fermentation without life, both for alcoholic fermentation and for those other fermentations which are produced by bacteria, and had put upon a sound and permanent basis the conclusions drawn by Schulze, Cagniard-Latour, Schwann, and KÜtzing from their early experiments. It became generally recognised that putrefaction and other fermentative changes were due to specific organisms, which produced them in the exercise of their vital functions.

Pasteur subsequently [1875] came to the conclusion that fermentation was the result of life without oxygen, the cells being able, in the absence of free oxygen, to avail themselves of the energy liberated by the decomposition of substances containing combined oxygen. This view, which did not involve any alteration of Pasteur's original thesis but was an attempt to explain the physiological origin and function of fermentation, gave rise to a prolonged controversy, which cannot be further discussed in these pages. [p014]

Nevertheless, Liebig's desire to penetrate more deeply into the nature of the process of fermentation remained in many minds, and numerous endeavours were made to obtain further insight into the problem. In spite of an entire lack of direct experimental proof, the conception that alcoholic fermentation was due to the chemical action of some substance elaborated by the cell and not directly to the vital processes of the cell as a whole found strenuous supporters even among those who were convinced of the vegetable character of yeast. As early as 1833 diastase, discovered still earlier by Kirchhoff and Dubrunfaut, had been extracted by means of water from germinating barley and precipitated by alcohol as a white powder, the solution of which was capable of converting starch into sugar, but lost this power when heated [Payen and Persoz, 1833]. Basing his ideas in part upon the behaviour of this substance, Moritz Traube [1858] enunciated in the clearest possible manner the theory that all fermentations produced by living organisms are caused by ferments, which are definite chemical substances produced in the cells of the organism. He regarded these substances as being closely related to the proteins and considered that their function was to transfer the oxygen and hydrogen of water to different parts of the molecule of the fermentable substance and thus bring about that apparent intramolecular oxidation and reduction which is so characteristic of fermentative change and had arrested the attention of Lavoisier and, long after him, of Liebig.

Traube's main thesis, that fermentation is caused by definite ferments or enzymes, attracted much attention, and received fresh support from the separation of invertase in 1860 from an extract of yeast by Berthelot, and from the advocacy and authority of this great countryman of Pasteur, who definitely expressed his opinion that insoluble ferments existed which could not be separated from the tissues of the organism, and further, that the organism could not itself be regarded as the ferment, but only as the producer of the ferment [1857, 1860]. Hoppe-Seyler [1876] also supported the enzyme theory of fermentation, but differed in some respects from Traube as to the exact function of the ferment [see Traube, 1877; Hoppe-Seyler, 1877].

Direct experimental evidence was, however, still wanting, and Pasteur's reiterated assertion [1875] that all fermentation phenomena were manifestations of the life of the organism remained uncontroverted by experience.

Numerous and repeated direct experimental attacks had been made [p015] from time to time upon the problem of the existence of a fermentation enzyme, but all had yielded negative or unreliable results.

As early as 1846 a bold attempt had been made by LÜdersdorff [1846] to ascertain whether fermentation was or was not bound up with the life of the yeast by grinding yeast and examining the ground mass. A single gram of yeast was thoroughly ground, the process lasting for an hour, and the product was tested with sugar solution. Not a single bubble of gas was evolved. A similar result was obtained in a repetition of the experiment by Schmidt in Liebig's laboratory [1847], the grinding being continued in this case for six hours, but the natural conclusion that living yeast was essential for fermentation was not accepted, on the ground that during the lengthy process of trituration in contact with air the yeast had become altered and now no longer possessed the power of producing alcoholic fermentation, but instead had acquired that of changing sugar into lactic acid [see Gerhardt, 1856, p. 545].

Similar experiments made in 1871 by Marie von ManasseÏn [1872, 1897], in which yeast was ground for six to fifteen hours with powdered rock crystal, yielded products which fermented sugar, but they contained unbroken yeast cells, so that the results obtained could not be considered decisive [Buchner and Rapp, 1898, 1], although Frau von ManasseÏn herself drew from them and from others in which sugar solution was treated with heated yeast, but not under aseptic conditions, the conclusion that living yeast cells were not necessary for fermentation.

Quite unsuccessful were also the attempts made to accomplish the separation of fermentation from the living cell by Adolf Mayer [1879, p. 66], and, as we learn from Roux, by Pasteur himself, grinding, freezing, and plasmolysing the cells, having in his hands proved alike in vain. Extraction by glycerol or water, a method by which many enzymes can be obtained in solution, gave no better results [NÄgeli and Loew, 1878], and the enzyme theory of alcoholic fermentation appeared quite unjustified by experiment.

Having convinced himself of this, NÄgeli [1879] suggested a new explanation of the facts based on molecular-physical grounds. According to this view, which unites in itself some of the conceptions of Liebig, Pasteur, and Traube, fermentation is the transference of a state of motion from the molecules, atomic groups, and atoms of the compounds constituting the living plasma of the cell to the fermentable material, whereby the equilibrium existing in the molecules of the latter is disturbed and decomposition ensues [1879, p. 29]. [p016]

This somewhat complex idea, whilst including, as did Liebig's theory, Stahl's fundamental conception of a transmission of a state of motion, satisfies Pasteur's contention that fermentation cannot occur without life, and at the same time explains the specific action of different organisms by differences in the constitution of their cell contents. The really essential part of NÄgeli's theory consisted in the limitation of the power of transference of molecular motion to the living plasma, by which the failure of all attempts to separate the power of fermentation from the living cell was explained. This was the special phenomenon which required explanation; to account for this the theory was devised, and when this was experimentally disproved, the theory lost all significance.

For nearly twenty years no further progress was made, and then in 1897 the question which had aroused so much discussion and conjecture, and had given rise to so much experimental work, was finally answered by Eduard Buchner, who succeeded in preparing from yeast a liquid which, in the complete absence of cells, was capable of effecting the resolution of sugar into carbon dioxide and alcohol [1897, 1].

In the light of this discovery the contribution to the truth made by each of the great protagonists in the prolonged discussion on the problem of alcoholic fermentation can be discerned with some degree of clearness. Liebig's main contention that fermentation was essentially a chemical act was correct, although his explanation of the nature of this act was inaccurate. Pasteur, in so far as he considered the act of fermentation as indissolubly connected with the life of the organism, was shown to be in error, but the function of the organism has only been restricted by a single stage, the active enzyme of alcoholic fermentation has so far only been observed as the product of the living cell. Nearest of all to the truth was Traube, who in 1858 enunciated the theorem, which was only proved for alcoholic fermentation in 1897, that all fermentations produced by living organisms are due to ferments secreted by the cells.

Buchner's discovery of zymase has introduced a new experimental method by means of which the problem of alcoholic fermentation can be attacked, and the result has been that since 1897 a considerable amount of information has been gained with regard to the nature and conditions of action of the enzymes of the yeast cell. It has been found that the machinery of fermentation is much more complex than had been surmised. The enzyme zymase, which is essential for fermentation, cannot of itself bring about the alcoholic fermentation of sugar, but is dependent on the presence of a second substance, termed, for [p017] want of a more reasonable name, the co-enzyme. The chemical nature and function of this mysterious coadjutor are still unknown, but as it withstands the temperature of boiling water and is dialysable, it is probably more simple in constitution than the enzyme. This, however, is not all; for the decomposition of sugar a phosphate is also indispensable. It appears that in yeast-juice, and therefore also most probably in the yeast cell, the phosphorus present takes an active part in fermentation and goes through a remarkable cycle of changes. The breakdown of sugar into alcohol and carbon dioxide is accompanied by the formation of a complex hexosephosphate, and the phosphate is split off from this compound and thus again rendered available for action by means of a special enzyme, termed hexosephosphatase. In addition to this complex of ferments, the cell also possesses special enzymes by which the zymase and the co-enzyme can be destroyed, and, further, at least one substance, known as an anti-enzyme, which directly checks this destructive action. It seems probable, moreover, that the decomposition of the sugar molecule takes place in stages, although much doubt yet exists as to the nature of these.

At the present moment the subject remains one of the most interesting in the whole field of biological chemistry, the limited degree of insight which has already been gained into the marvellous complexity of the cell lending additional zest to the attempt to penetrate the darkness which shrouds the still hidden mysteries.