ALCOHOLIC FERMENTATION 2nd Edition, 1914 by Arthur Harden

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ALCOHOLIC FERMENTATION

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HEAD OF THE BIOCHEMICAL DEPARTMENT, LISTER INSTITUTE, CHELSEA

SECOND EDITION

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PREFACE.

The following chapters are based on courses of lectures delivered at the London University and the Royal Institution during 1909–1910. In them an account is given of the work done on alcoholic fermentation since Buchner's epoch-making discovery of zymase, only in so far as it appears to throw light on the nature of that phenomenon. Many interesting subjects, therefore, have perforce been left untouched, among them the problem of the formation of zymase in the cell, and the vexed question of the relation of alcoholic fermentation to the metabolic processes of the higher plants and animals.

My thanks are due to the Council of the Royal Society, and to the Publishers of the "Journal of Physiology" for permission to make use of blocks (Figs. 2, 4 and 7) which have appeared in their publications.

A. H.

PREFACE TO THE SECOND EDITION.

In the New Edition no change has been made in the scope of the work. The rapid progress of the subject has, however, rendered necessary many additions to the text and a considerable increase in the bibliography.

A. H.

May,1914.

CONTENTS.

CHAPTER I. HISTORICAL INTRODUCTION.

[p001]

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.

CHAPTER II. ZYMASE AND ITS PROPERTIES.

Discovery of Zymase.

[p018]

The history of Buchner's discovery is of great interest [Gruber, [1908]; Hahn, [1908]]. As early as 1893 Hans and Eduard Buchner found that the cells of even the smallest micro-organism could be broken by being ground with sand [Buchner, E. and H., and Hahn, [1903], p. 20], and in 1896 the same process was applied by these two investigators to yeast, with the object of obtaining a preparation for therapeutic purposes. Difficulties arose in the separation of the cell contents from the ground-up mixture of cell membranes, unbroken cells, and sand, but these were overcome by carrying out the suggestion of Martin Hahn (at that time assistant to Hans Buchner) that kieselguhr should be added and the liquid squeezed out by means of a hydraulic press [Buchner, E. and H., and Hahn, [1903], p. 58]. The yeast-juice thus obtained was, in the first instance, employed for animal experiments, but underwent change very rapidly. The ordinary antiseptics were found to be unsuitable, and hence sugar was added as a preservative, and it was the marked action of the juice upon this added cane sugar that drew Eduard Buchner's attention to the fact that fermentation was proceeding in the absence of yeast-cells.

As in the case of so many discoveries, the new phenomenon was brought to light, apparently by chance, as the result of an investigation directed to quite other ends, but fortunately fell under the eye of an observer possessed of the genius which enabled him to realise its importance and give to it the true interpretation.

In his first papers [[1897, 1], [2]; [1898]], Buchner established the following facts: (1) yeast-juice free from cells is capable of producing the alcoholic fermentation of glucose, fructose, cane sugar, and maltose; (2) the fermenting power of the juice is neither destroyed by the addition of chloroform, benzene, or sodium arsenite [Hans Buchner, [1897]], by filtration through a Berkefeld filter, by evaporation to dryness at 30° to 35°, nor by precipitation with alcohol; (3) the fermenting power is completely destroyed when the liquid is heated to 50°. [p019]

From these facts he drew the conclusion "that the production of alcoholic fermentation does not require so complicated an apparatus as the yeast cell, and that the fermentative power of yeast-juice is due to the presence of a dissolved substance". To this active substance he gave the name of zymase.

Buchner's discovery was not received without some hesitation. A number of investigators prepared yeast-juice, but failed to obtain an active product [Will, [1897]; Delbrück, [1897]; Martin and Chapman, [1898]; Reynolds Green, [1897]; Lintner, [1899]]. A more accurate knowledge of the necessary conditions and of the properties of yeast-juice, however, led to more successful results [Will, [1898]; Reynolds Green, [1898]; Lange, [1898]], and it was soon established that, given suitable yeast, an active preparation could be readily procured by Buchner's method. Criticism was then directed to the effect of the admitted presence of a certain number of micro-organisms in yeast-juice [Stavenhagen, [1897]], but Buchner [Buchner and Rapp, [1897]] was able to show by experiments in the presence of antiseptics and with juice filtered through a Chamberland candle that the fermentation was not due to living organisms of any kind.

The most weighty criticism of Buchner's conclusion consisted in an attempt to show that the properties of yeast-juice might be due to the presence, suspended in it, of fragments of living protoplasm, which, although severed from their original surroundings in the cell, might retain for some time the power of producing alcoholic fermentation. This, it will be seen, was an endeavour to extend Nägeli's theory to include in it the newly discovered fact.

In favour of this view were adduced the similarity between the effects of many antiseptics on living yeast and on the juice, the ephemeral nature of the fermenting agent present in the juice, the effect of dilution with water, and the phenomenon of autofermentation which is exhibited by the juice in the absence of added sugar [Abeles, [1898]; v. Kupffer, [1897]; v. Voit, [1897]; Wehmer, [1898]; Neumeister, [1897]; Macfadyen, Morris, and Rowland, [1900]; Bokorny, [1906]; Fischer, [1903]; Beijerinck, [1897], [1900]; Wroblewski, [1899], [1901]].

A brief general description of the actual properties of yeast-juice and of the phenomena of fermentation by its means is sufficient to show the great improbability of this view.

The juice prepared by Buchner's method forms a somewhat viscous opalescent brownish-yellow liquid, which is usually faintly acid in reaction [compare Ahrens, [1900]] and almost optically inactive. It has a specific gravity of 1·03 to 1·06, contains 8·5 to 14 per cent. [p020] of dissolved solids, and leaves an ash amounting to 1·4 to 2 per cent. About 0·7 to 1·7 per cent. of nitrogen is present, nearly all in the form of protein, which coagulates to a thick white mass when the juice is heated.

A powerful digestive enzyme of the type of trypsin is also present, so that when the juice is preserved its albumin undergoes digestion at a rate which depends on the temperature [Hahn, [1898]; Geret and Hahn, [1898, 1], [2]; [1900]; Buchner, E. and H., and Hahn, [1903], pp. 287–340], and is converted into a mixture of bases and amino-acids. After about six days at 37°, or 10 to 14 days at the ordinary temperature, the digestion is so complete that no coagulation occurs when the juice is boiled. As this proteoclastic enzyme, like the alcoholic enzyme, cannot be extracted from the living cells, it is termed yeast endotrypsin or endotryptase. Fresh yeast-juice produces a slow fermentation of sugar, which lasts for forty-eight to ninety-six hours at 25° to 30°, about a week at the ordinary temperature, and then ceases, owing, not to exhaustion of the sugar, but to the disappearance of the fermenting agent. When the juice is preserved or incubated in the absence of a fermentable sugar this disappearance occurs considerably sooner, so that even after standing for a single day at room temperature, or two days at 0°, no fermentation may occur when sugar is added. The reason for this behaviour has not been definitely ascertained. As will be seen later on (p. [64]) the phenomenon is a complex one, but the disappearance of the enzyme was originally ascribed by Buchner to the digestive action upon it of the endotrypsin of the juice [[1897, 2]], and no better explanation has yet been found. Confirmation of this view is afforded by the fact that the addition of a tryptic enzyme of animal origin greatly hastens the disappearance of the alcoholic enzyme [Buchner, E. and H., and Hahn, [1903], p. 126], and that some substances which hinder the tryptic action favour fermentation [Harden, [1903]]. The amount of fermentation produced is almost unaffected by the presence of such antiseptics as chloroform or toluene, although some others, such as arsenites and fluorides, decrease it when added in comparatively high concentrations, and it is only slightly diminished by dilution with three or four volumes of sugar solution, somewhat more considerably by dilution with water. When it is filtered through a Chamberland filter the first portions of the filtrate are capable of bringing about fermentation, but the fermenting power diminishes in the succeeding portions and finally disappears. The juice can be spun in a centrifugal machine without being in any way altered, and no separation into more or less active layers takes place under these conditions. [p021]

The amorphous powder obtained by drying the precipitate produced when the juice is added to a mixture of alcohol and ether is also capable of producing fermentation, and the process of precipitation may be repeated without seriously diminishing the fermenting power of the product.

These facts clearly show that the various phenomena adduced by the supporters of the theory of protoplasmic fragments are quite consistent with the presence of a dissolved enzyme as the active agent of the juice, and at the same time that the properties demanded of the living fragments of protoplasm to which fermentation is ascribed are such as cannot be reconciled with our knowledge of living matter. If living protoplasm is the cause of alcoholic fermentation by yeast-juice, a new conception of life will be necessary; the properties of the postulated fragments of protoplasm must be so different from those which the protoplasm of the living cell possesses as to deprive the theory of all real value [Buchner, [1900, 2]; Buchner, E. and H., and Hahn, [1903], p. 33].

Further and very convincing evidence against the protoplasm theory is afforded by the behaviour of yeast towards various desiccating agents. When yeast is dried at the ordinary temperature it retains its vitality for a considerable period. If, however, the dried yeast be heated for six hours at 100° it loses the power of growth and reproduction but still retains that of fermenting sugar, and when ground with sand, kieselguhr and 10 per cent. glycerol solution yields an active juice [Buchner, [1897, 2]; [1900, 1]]. Preparations (known as zymin) obtained by treating yeast with a mixture of alcohol and ether [Albert, [1900], [1901, 1]], or with acetone and ether [Albert, Buchner, and Rapp, [1902]], show precisely similar properties (p. [38]). The proof in this case has been carried a step further, for the active juice obtained by grinding such acetone-yeast, when precipitated with alcohol and ether, yields an amorphous powder, still capable of fermenting sugar.

The Preparation of Yeast-Juice.

Buchner's process for the preparation of active yeast-juice is characterised by extreme simplicity. The yeast employed, which should be fresh brewery yeast, is washed two or three times by being suspended in a large amount of water and allowed to settle in deep vessels. It is then collected on a filter cloth, wrapped in a press cloth, and submitted to a pressure of about 50 kilos, per sq. cm. for five minutes. The resulting friable mass contains about 70 per cent. of water and is free from adhering wort. The washed yeast is then [p022] mixed with an equal weight of silver sand and 0·2 to 0·3 parts of kieselguhr, care being taken that this is free from acid. The correct amount of kieselguhr to be added can only be ascertained by experience, and varies with different samples of yeast. The dry powder thus obtained is brought in portions of 300 to 400 grams into a large porcelain mortar and ground by hand by means of a porcelain pestle fastened to a long iron rod which passes through a ring fixed in the wall (Fig. 1). The mortar used by Buchner has a diameter of 40 cm. and the pestle and rod together weigh 8 kilos.

Fig. 1.

As the grinding proceeds the light-coloured powder gradually darkens and becomes brown, and the mass becomes moist and adheres to the pestle, until finally, after two to three minutes' grinding, it takes the consistency of dough, at which stage the process is stopped. The mass is next enveloped in a press cloth and submitted to a pressure of 90 kilos, per sq. cm. in a hydraulic hand press, the pressure being very gradually raised in order to avoid rupture of the cloth. The cloth required for 1000 grams of yeast measures 60 by 75 cm. and is previously soaked in water and then submitted to a pressure of 50 kilos, per sq. cm., retaining about 35 to 40 c.c. of water.

The juice runs from the press on to a folded filter paper, to remove kieselguhr and yeast cells, and passes into a vessel standing in ice water.

The yield of juice obtained by Buchner in an operation of this kind from 1 kilo. of yeast amounts to 320 to 460 c.c. It may be increased by re-grinding the press cake and again submitting it to pressure, and then amounts on the average to 450 to 500 c.c.

Since the cell membranes constitute about 20 per cent. of the weight of the dry yeast, this yield corresponds to more than 60 per cent. of the total cell contents of the yeast. It has been computed by Will [quoted by Buchner, E. and H., and Hahn, [1903], p. 66] that [p023] only about 20 per cent. of the cells are left unaltered by one grinding and pressing, and only 4 per cent. after a repetition of the process, at least 57 per cent. of the cells being actually ruptured by the double process, and the remainder to some extent altered. It seems probable from these figures that a certain amount of the juice may be derived from the unbroken cells, and Will expressly states that many unbroken cells have lost their vacuoles.

Fig. 2.

If the yeast be submitted to a process of regeneration, which consists in exposure to a well-aerated solution of sugar and mineral salts until fermentation is complete, the juice subsequently obtained [p024] is more active than that yielded by the original yeast [Albert, [1899, 1]].

A modified method of grinding yeast was introduced by Macfadyen, Morris, and Rowland [[1900]], who placed a mixture of yeast and sand in a jacketed and cooled vessel, in which a spindle carrying brass flanges was rapidly rotated [Rowland, [1901]]. One kilo. of yeast ground in this way for 3·5 hours yielded 350 c.c. of juice.

This grinding process was at first adopted by Harden and Young in their experiments but was afterwards abandoned in favour of Buchner's hand-grinding process, as it was found liable to yield juices of low fermenting power, probably on account of inefficient cooling during the grinding process. A slight modification of Buchner's process has, however, been introduced, the hand-ground mass being mixed with a further quantity of kieselguhr until a nearly dry powder is formed, and the mass packed between two layers of chain cloth in steel filter plates and pressed out in a hydraulic press at about 2 tons to the square inch (300 kilos. per sq. cm.). The press and plates are shown in section in Fig. 2. It has also been found convenient to remove yeast cells and kieselguhr from the freshly pressed juice by centrifugalisation instead of by filtration through paper, and to wash the yeast before grinding by means of a filter-press.

Working with English top yeasts Harden and Young have found the yield of juice extremely variable, the general rule being that the amount of juice obtainable from freshly skimmed yeast is smaller than that yielded by the same yeast after standing for a day or two after being skimmed. The yield for 1000 grams of pressed brewer's yeast varies from 150 to 375 c.c., and is on the average about 250 c.c.

Very fresh yeast occasionally presents the peculiar phenomenon that scarcely any juice can be expressed from the ground mass, although the latter does not differ in appearance or consistency from a mass which gives a good yield.

Extraction of Zymase from Unground Yeast.

1. Maceration of Dried Yeast.

A valuable addition to the methods of obtaining an active solution of zymase was made in 1911 by Lebedeff [[1911, 2]; [1912, 2]; see also [1911, 3], [7], and [1912, 1]]. This investigator had been in the habit of grinding dried yeast with water for preparing samples of yeast-juice of uniform character and observed that when the dried yeast was digested with sugar solution and the mixture heated, coagulation [p025] took place throughout the whole liquid, the proteins of the yeast having passed out of the cells. Further examination revealed the interesting fact that dried yeast readily yielded an active extract when macerated in water for some time. The quality of the resulting "maceration extract" depends on a considerable number of factors, the chief of which are: (1) the temperature of drying of the yeast; (2) the temperature of maceration; (3) the duration of maceration; and (4) the nature of the yeast, as well as, of course, the amount of water added in maceration.

In general the yeast should be dried at 25°–30° and then macerated with 3 parts of water for 2 hours at 35°.

The temperature of maceration may as a rule be varied, without detriment to the product provided that the time of maceration is also suitably altered; thus with dried Munich yeast, maceration for 4·5 hours at 25° is about as effective as 2 hours at 35°, whereas treatment for a shorter time at 25° or a longer time at 35° produces in general a less efficacious extract. Yeast dried at a lower temperature than 25° tends to yield an extract poor in co-enzyme (p. [59]) and hence of low fermenting power, this being especially marked at air temperature.

The subsequent treatment of the yeast during maceration may, however, be of great influence in such cases. Thus a yeast dried at 15° gave by maceration at 25° for 4·5 hours a weak extract (yielding with excess of sugar 0·33g. CO₂), whereas when macerated at 35° for 2 hours it yielded a normal extract (1·36g. CO₂).

The nature of the yeast is of paramount importance. Thus while Munich (bottom) yeast usually gives a good result, a top yeast from a Paris brewery was found to yield extracts containing neither zymase nor its co-enzyme in whatever way the preparation was conducted. The existence of such yeasts is of great interest, and it was probably due to the unfortunate selection of such a yeast for his experiments that Pasteur was unable to prepare active fermenting extracts and therefore failed to anticipate Buchner by more than 30 years (see p. 15). The English top yeasts as a rule give poor results [see Dixon and Atkins, [1913]] and sometimes yield totally inactive maceration extract. It is not understood why the enzyme passes out of the cell during the process of maceration and the whole method gives rise to a number of extremely interesting problems.

Method.—A suitable yeast is washed by decantation, filtered through a cloth, lightly pressed by means of a hand press, and then passed through a sieve of 5mm. mesh, spread out in a layer 1–1·5cm. thick and left at 25°–35° for two days. Fifty grams of the dried yeast is [p026] thoroughly and carefully mixed with 150 c.c. of water in a basin by means of a spatula and the whole digested for two hours at 35°. The mass often froths considerably. It is then filtered through ordinary folded filter paper, preferably in two portions, and collected in a vessel cooled by ice. The separation may also be effected by centrifuging or pressing out the mass, and the maceration may be conveniently conducted in a flask immersed in the water of a thermostat. It is not advisable to macerate more than 50 grams in one operation. Under these conditions 25–30 c.c. of extract are obtained after 20 minutes' filtration, 70–80 c.c. in twelve hours. Dried Munich yeast can be bought from Messrs. Schroder of Munich and serves as a convenient source of the extract.[1]

[1] The material supplied is occasionally found to yield an inactive extract and every sample should be tested.

This extract closely resembles in properties the juice obtained by grinding the same yeast, but it is usually more active and contains more inorganic phosphate (see p. [46]).

2. Other Methods.

Attempts to prepare active extracts from undried yeast in an analogous manner have so far not been very successful. Thus Rinckleben [[1911]] found that plasmolysis by glycerol (8 per cent.) or sodium phosphate (5 per cent.) sometimes yielded an active juice and sometimes a juice which contained enzyme but no co-enzyme, but more often an inactive juice incapable of activation (p. [64]) [see also Kayser, [1911]].

Giglioli [[1911]] by the addition of chloroform also obtained an active liquid. It appears in fact as though almost any method of plasmolysing the yeast cell may yield a certain proportion of zymase in the exudate.

An ingenious process has been devised by Dixon and Atkins [[1913]] who applied the method of freezing in liquid air which they had found efficacious for obtaining the sap from various plant organs. They thus succeeded in obtaining from yeast, derived from Guinness' brewery in Dublin, liquids capable of fermenting sugar and of about the same efficacy as the maceration extracts prepared by Lebedeff's method from the same yeast. The results were, however, in both cases very low, the maximum total production of CO₂ by 25 c.c. of liquid from excess of sugar being 32·5 c.c. (air temperature) or about 0·06g. Munich yeast on the other hand yields, either by maceration or grinding, a liquid giving as much as 1·5–2g. of CO₂ per 25 c.c., whilst [p027] English yeast-juice prepared by grinding often gives as much as 0·5–0·7g. of CO₂.

No direct comparison with the juice prepared by grinding was made by Dixon and Atkins, but it may be concluded from their results that the best method of obtaining an active preparation from the top yeasts used in this country is that of grinding. Maceration, freezing and plasmolysis alike yield poor results. With Munich yeast on the other hand the maceration process yields excellent results, whilst the liquid air process has not so far been tried.

Practical Methods for the Estimation of the Fermenting Power of Yeast-Juice.

In order to estimate the amount of carbon dioxide evolved in a given time and the total amount evolved by the action of yeast-juice on sugar, Buchner adopted an extremely simple method, which consisted in carrying out the fermentation in an Erlenmeyer flask provided with a small wash-bottle, which contained sulphuric acid and was closed by a Bunsen valve, and ascertaining the loss of weight during the experiment. Corrections are necessary for the carbon dioxide present in the original juice and retained in the liquid at the close of the experiment and for that present in the air space of the apparatus, but it was found that for most purposes these could be neglected. In cases in which greater accuracy was desired, the carbon dioxide was displaced by air before the weighings were made. A typical experiment of this kind, without displacement of carbon dioxide, is the following:—

The total weight of carbon dioxide evolved under these conditions is termed the fermenting power of the juice (Buchner).

A more accurate method [Macfadyen, Morris, and Rowland, [1900]] consists in passing the carbon dioxide into caustic soda solution and estimating it by titration. The yeast-juice, sugar, and antiseptic are placed in an Erlenmeyer flask provided with a straight glass tube, through which air can be passed over the surface of the liquid, and a conducting tube leading into a second flask which contains 50 c.c. of 10 per cent. caustic soda solution and is connected with the air by a guard tube containing soda lime. The juice can be freed from carbon dioxide by agitation in a current of air before the flask is connected to [p028] that containing the caustic soda solution, and at the end of the period of incubation air is passed through the apparatus, the liquid being boiled out if great accuracy is required. The absorption flask is then disconnected and the amount of absorbed carbon dioxide estimated by titration. This is carried out by making up the contents of the flask to 200 c.c., taking out an aliquot portion, rendering this exactly neutral to phenophthalein by the addition first of normal and finally of decinormal acid, adding methyl orange and titrating with decinormal acid to exact neutrality. Each c.c. of decinormal acid used in this last titration represents 0·0044 gram of carbon dioxide in the quantity of solution titrated.

Fig. 3.

These methods are only suitable for observations at considerable intervals of time. For the continuous observation of the course of fermentation Harden, Thompson and Young [[1910]] connect the fermentation flask with a Schiff's azotometer filled with mercury and measure the volume of gas evolved, the liquid having been previously saturated with carbon dioxide (Fig. 3). The level of the mercury in the reservoir is kept constant by a syphon overflow, as shown in the figure, or, according to a modification introduced by S. G. Paine, by a specially constructed bottle provided with two tubulures near the bottom. This ensures that no change in the pressure in the flask occurs, and the volume of gas observed is reduced to normal pressure by means of a table. Before making a reading it is necessary to shake the fermenting mixture thoroughly, as the albuminous liquid very readily becomes greatly supersaturated with carbon dioxide, so much so in fact that very little gas is evolved in the intervals between the shakings. The exact procedure in making an observation consists in shaking the flask [p029] thoroughly, replacing in the thermostat, allowing to remain for one minute, and then reading the level of the mercury in the azotometer. After the required time, say five minutes, has elapsed from the time at which the flask was first shaken, it is again removed from the bath, shaken as before, replaced, allowed to remain for one minute and the reading then taken. In this way readings can be conveniently made at intervals of three or five minutes or even less, and much more detailed information obtained about the course of the reaction than is possible by means of observations made at intervals of several hours.

Another form of volumetric apparatus, designed by Walton [[1904]], has been used by Lebedeff [[1909]].

An apparatus on a different principle has been designed by Slator [[1906]] for use with living yeast, but is equally applicable to yeast-juice, and a very similar form has been more recently employed by Iwanoff [[1909, 2]]. In this apparatus the change of pressure produced by the evolution of carbon dioxide is measured at constant volume, and comparative rates of evolution can be obtained with considerable accuracy, although the method has the disadvantage that the absolute volume of gas evolved is not measured. The apparatus consists of a bottle or flask connected with a mercury manometer. The fermenting mixture is placed in the bottle along with glass beads to facilitate agitation, the pressure is reduced to a small amount by the water-pump, and the rise of pressure is then observed at intervals, this being proportional to the volume of gas produced. As in the preceding case, the liquid must be well shaken before a reading is made.

The Alcoholic Fermentation of the Sugars by Yeast-Juice.

Yeast-juice brings about a slow fermentation of those sugars which are fermented by the yeast from which it is prepared as well as of dextrin, and of starch and glycogen, which are not fermented by living yeast.

(a) Relation to Fermentation by living Yeast.

Both in rate of fermentation and in the total fermentation produced, yeast-juice stands far behind the equivalent amount of living yeast. Taking 25 c.c. of yeast-juice to be equivalent to at least 36 grams of pressed yeast containing 70 per cent. of moisture, it is found that whereas the yeast-juice (from English top yeast) gives with glucose a maximum rate of fermentation of about 3 c.c. in five minutes, the living yeast ferments the sugar at the rate of about 126 c.c. in the same time, or [p030] about forty times as quickly. The total carbon dioxide obtainable from the yeast-juice, moreover, corresponds to the fermentation of only 2 to 3 grams of sugar, whilst the living yeast will readily ferment a much larger quantity, although the exact limit in this respect has not been accurately determined. The reasons for this great difference in behaviour will be discussed later on, after the various factors concerned in fermentation have been considered (p. [123]).

(b) Relation of Alcohol to Carbon Dioxide.

In all cases of fermentation by yeast-juice and zymin, the relative amounts of carbon dioxide and alcohol produced are substantially in the ratio of the molecular weights of the compounds, that is as 44: 46, so that for 1 part of carbon dioxide 1·04 of alcohol are formed. This has been shown for the juice and zymin from bottom yeasts by Buchner [Buchner, E. and H., and Hahn, [1903], pp. 210, 211], who obtained the ratios 1·01, 0·98, 1·01, and 0·99 from experiments in which from 8 to 15 grams of alcohol were produced. Similar numbers, 0·90, 1·12, 0·95, 0·91 and 0·92, have been obtained for the juice from top yeasts by Harden and Young [[1904]], who worked with much smaller quantities. The variable results obtained with juice from top yeast by Macfadyen, Morris and Rowland [[1900]], have not been confirmed.

(c) Relation of Carbon Dioxide and Alcohol Produced to the Amount of Sugar Fermented.

The construction of a balance-sheet between the sugar fermented and the products formed is of special interest in the case of alcoholic fermentation by yeast-juice, because, there being no cell growth as in the case of living yeast, an opportunity appears to be afforded of ascertaining whether the whole of the sugar is converted into alcohol and carbon dioxide, or whether some fraction of the sugar passes into any of the well-known subsidiary products of alcoholic fermentation by yeast, such as glycerol, fusel oil, or succinic acid. Unfortunately the question cannot be settled in this way. When the loss of sugar during the fermentation is estimated directly, it is usually found to be considerably greater than the sum of the alcohol and carbon dioxide produced from it. This fact was first observed by Macfadyen, Morris and Rowland [[1900]], and was then confirmed by Buchner [Buchner, E. and H., and Hahn, [1903], p. 212], in one instance, the excess of sugar lost over products being in this case about 15 per cent. of the total sugar which had disappeared. The matter was then more thoroughly investigated by Harden and Young [[1904]]. [p031]

The conditions under which the experiment must be carried out are not very favourable to the attainment of extreme accuracy. Yeast-juice contains glycogen and a diastatic enzyme which converts this into dextrins and finally into sugar. This process goes on throughout fermentation, tending to increase the sugar present and to make the apparent loss of sugar less than the sum of the products. In spite of this it was found that a certain amount of sugar invariably disappeared without being accounted for as alcohol or carbon dioxide, and this whether the fermentation lasted sixty or a hundred and eight hours, and independently of the dilution of the juice. This disappearing sugar amounted in some cases to 44 per cent. of the total loss of sugar, and on the average of twenty-five experiments was 38 per cent. Further information was sought by converting all the sugar-yielding constituents of the juice into sugar by hydrolysis before and after the fermentation. This process revealed the fact that when the glucose equivalent of the juice before and after fermentation was determined after hydrolysis with three times normal acid for three hours (and a correction made for the loss of reducing power experienced by glucose itself when submitted to this treatment), the difference was almost exactly equal to the alcohol and carbon dioxide produced. In other words, accompanying fermentation, a change proceeds by which sugar is converted into a less reducing substance, reconvertible into sugar by hydrolysis with acids. Similar results were subsequently obtained by Buchner and Meisenheimer [[1906]], who employed 1·5 normal acid and observed a small nett loss of sugar. Still more recently Lebedeff [[1909], [1910], see also [1913, 2]] has carried out similar estimations with the same result. It is doubtful whether the experiments which have so far been made on this point are sufficiently accurate to decide with certainty whether or not the loss of sugar is exactly equal to the sum of the carbon dioxide and alcohol produced. It has been shown by Buchner and Meisenheimer [[1906]] that glycerol is a constant product of alcoholic fermentation by yeast-juice (p. [95]), and no other source for this than the sugar has yet been found, so that it is not improbable that a small amount of sugar is converted into non-carbohydrate substances other than carbon dioxide and alcohol.

It has also been shown [Harden and Young, [1913]] that the deficit of sugar is not due to the formation of hexosephosphate (p. [47]), which has a lower reduction than glucose, and that the solution from which the sugar (either glucose or fructose) has disappeared actually contains some substance of relatively high dextrorotation and of low reducing power. [p032]

However this may be, it may be considered as established that during alcoholic fermentation sugar is converted by an enzyme into some compound of less reducing power, which again yields sugar on hydrolysis with acids. The exact nature of this substance has not been ascertained, but it appears likely that the process is a synthetical one resulting in the formation of some polysaccharide, possibly intermediate between the hexoses and glycogen.

A similar phenomenon has been observed with living yeast by Euler and Johansson [[1912, 1]], and Euler and Berggren [[1912]], whose interpretation of the observation is discussed later on (p. [57]).

(d) Fermentation of Different Carbohydrates. Autofermentation.

Yeast-juice and zymin ferment all the sugars which are fermented by the yeast from which they are prepared, and, in addition, a number of colloidal substances which cannot pass through the membrane of the living yeast cell, but which are hydrolysed by enzymes in the juice and thus converted into simpler sugars capable of fermentation [Buchner and Rapp, [1898, 3]; [1899, 2]]. Of the simple sugars which have been examined, glucose, fructose, and mannose are freely fermented, l-arabinose not at all, whilst the case of galactose is doubtful. Galactose is, however, fermented by juice prepared from a yeast which has been "trained" to ferment galactose [Harden and Norris, [1910]]. As regards both the rate of fermentation and the total amount of carbon dioxide evolved from glucose and fructose by the action of a definite amount of yeast-juice, Buchner and Rapp obtained practically identical numbers. Harden and Young [[1909]], using juice from top yeast, found that fructose was slightly more rapidly fermented and gave a somewhat larger total than glucose, whilst mannose was initially fermented at almost the same rate as glucose, but gave a decidedly lower total, the following being the average result:—

Among the disaccharides, cane sugar and maltose are freely fermented, and the juice can be shown like living yeast to contain invertase and maltase. The extent of fermentation does not differ materially from that attained with glucose. Lactose is not fermented.

Of the higher sugars raffinose is fermented by juice from bottom yeast, but more slowly than cane sugar or maltose. No experiments seem to have been made with juice from top yeast. [p033]

As regards the fermentation of the higher carbohydrates, very little experimental work has been carried out. Buchner and Rapp found that the fermentation of starch paste was doubtful, but that soluble starch and commercial dextrin were fermented with some freedom. No special study has been made of the diastatic enzymes which bring about the hydrolysis of these substances.

The fermentation of glycogen by yeast-juice is of considerable interest, since it is known that the characteristic reserve carbohydrate of the yeast cell is glycogen [see Harden and Young, [1902], where the literature is cited], and moreover that in living yeast the intracellular fermentation of glycogen proceeds readily, whereas glycogen added to a solution in which yeast is suspended is not affected. Yeast-juice contains a diastatic enzyme which hydrolyses glycogen to a reducing and fermentable sugar, so that in a juice poor in zymase to which glycogen has been added, the amount of sugar is found to increase, the hydrolysis of the glycogen proceeding more quickly than the fermentation of the resulting sugar [Harden and Young, [1904]], but the course of this enzymic hydrolysis of glycogen by yeast-juice has not yet been studied. As a rule, it is found both with juices from top and bottom yeast that the evolution of carbon dioxide from glycogen proceeds less rapidly and reaches a lower total than from an equivalent amount of glucose.

Since nearly all samples of yeast contain glycogen, yeast-juice and also zymin usually contain this substance as well as the products of its hydrolysis. These provide a source of sugar which enters into alcoholic fermentation, so that a slow spontaneous production of carbon dioxide and alcohol proceeds when yeast-juice is preserved without any addition of sugar. The extent of this autofermentation varies considerably, as might be expected, with the nature of the yeast employed or the preparation of the material, but is generally confined within the limits of 0·06 to 0·5 gram of carbon dioxide for 25 c.c. of juice.

In juice from bottom yeast it amounts to about 5 to 10 per cent. of the total fermentation obtainable with glucose [Buchner, [1900, 2]], whereas in juice from top yeasts, which gives a smaller total fermentation with glucose, it may occasionally equal, or even exceed, the glucose fermentation, and frequently amounts to 30 to 50 per cent. of it. It is therefore generally advisable in studying the effect of yeast-juice on any particular substance to ascertain the extent of autofermentation by means of a parallel experiment.

The maceration extract of Lebedeff (p. [24]) is usually, but not invariably [Oppenheimer, [1914, 2]], free from glycogen, which is hydrolysed [p034] and fermented during the processes of drying and macerating, and therefore as a rule shows no appreciable autofermentation.

(e) Effect of Concentration of Sugar on the Total Amount of Fermentation.

The kinetics of fermentation by zymase will be considered later on (p. [120]), but the effect on the total fermentation of different concentrations of sugar, this substance being present throughout in considerable excess, may be advantageously discussed at this stage. The subject has been investigated by Buchner [Buchner, E. and H., and Hahn, [1903], pp. 150–8; Buchner and Rapp, [1897]] using cane sugar, and he has found both for yeast-juice and for dried yeast-juice dissolved in water that (a) the total amount of fermentation increases with the concentration of the sugar; (b) the initial rate of fermentation decreases with the concentration of the sugar. The following are the results of a typical experiment, 20 c.c. of yeast-juice being employed in presence of toluene at 22°:—

The results as to the total fermentations in experiments of this kind are liable to be vitiated by the circumstance that when a low initial concentration of sugar is employed, the supply of sugar may be so greatly exhausted before the close of the experiment as to cause a marked diminution in the rate of fermentation and hence an unduly low total. Even allowing, however, for any effect of this kind, the foregoing table clearly shows the increase in total fermentation and the decrease in initial rate accompanying the increase of sugar concentration from 10 to 40 per cent. Working with a greater range of concentrations (3·3–53·3 grm. per 100 c.c.) Lebedeff has obtained similar results with maceration extract [[1911, 4]], but has found that the total amount fermented diminishes after a certain optimum concentration (about 33·3 grm. per 100 c.c.) is reached.

A practical conclusion from these experiments is that a high [p035] concentration of sugar tends to preserve the enzyme in an active state for a longer time. Simultaneously it prevents the development of bacteria and yeast cells.

(f) Effect of Varying Concentration of Yeast-Juice.

This subject, which is of considerable importance with reference to the question of the protoplasmic or enzymic nature of the active agent in yeast-juice, has been examined in some detail by Buchner [Buchner, E. and H., and Hahn, [1903], pp. 158–65] and by Meisenheimer [[1903]] for juices from bottom yeast, by Harden and Young [[1904]] for those from top yeast, and by Lebedeff [[1911, 4]] for maceration extract, the results obtained being in substantial agreement.

Dilution of yeast-juice with sugar solution, so that the concentration of the sugar remains constant, produces a small progressive diminution in the total fermentation, which only becomes marked when more than 2 volumes are added, and this independently of the actual concentration of the sugar. Dilution with water produces a somewhat more decided diminution, which, however, does not exceed 50 per cent. of the total for the addition of 3 volumes of water. The effect on maceration extract is somewhat greater but of the same kind. The autofermentation of juice from top yeast is scarcely affected by dilution with 4 volumes of water.

[p036]

On the whole, therefore, yeast-juice may be said to be only slightly affected by dilution even with pure water, and the effect of the latter can in no way be regarded as comparable with the poisonous effect which it exerts on living protoplasm, as suggested by Macfadyen, Morris, and Rowland [[1900]].

(g) The Effect of Antiseptics on the Fermentation of Sugars by Yeast-Juice.

Buchner has paid special attention to the effect of antiseptics on the course of fermentation by yeast-juice [Buchner and Rapp, [1897]; [1898, 2], [3]; [1899, 1]; Buchner and Antoni, [1905, 1]; Buchner and Hoffmann, [1907]; Buchner, E. and H., and Hahn, [1903], pp. 169–205; see also Albert, [1899, 2]; Gromoff and Grigorieff, [1904]; Duchaček, [1909]] in order (1) to obtain evidence as to the possibility of the active agent in yeast-juice consisting of fragments of protoplasm and not of a soluble enzyme, and (2) also to provide a safe method of avoiding contamination, by the growth of bacteria or yeasts, of the liquids used which were often kept at 25° for several days. The results of these experiments are briefly summarised in the following table, in which the effect of each substance on the total fermentation produced is noted:—

[p037]

The general result of these experiments is to show that quantities of antiseptics which are sufficient to inhibit the characteristic action of living cells have only a slight effect on the fermentative activity of yeast-juice. A large excess of the antiseptic in many cases produces a very decided diminution or total destruction of the fermenting power, and accompanying this a precipitation of the constituents of the juice. The decided increase of activity produced by small quantities of chloral hydrate, and to a less marked extent by chloroform and a few other substances, is of considerable interest. It is ascribed by Duchaček to a selective action on the proteoclastic enzyme, but without satisfactory evidence.

Hydrocyanic acid, even in dilute solution, completely suspends the fermenting power of the juice, without, however, producing any permanent change in the fermenting complex, as is shown by the fact that when the hydrocyanic acid is removed by a current of air, the juice regains its fermenting power. In this respect hydrocyanic acid behaves precisely as with many other enzymes and with colloidal platinum [Bredig, [1901]]. Sodium arsenite is a pronounced protoplasmic poison, which rapidly destroys the power of growth and reproduction in living cells, and was therefore applied to yeast-juice to differentiate between protoplasmic and enzymic action. It was, however, found that the action of this substance was complicated by some unknown factor and very irregular results were obtained [Buchner, E. and H., and Hahn, [1903], pp. 193 ff.]. These phenomena appear to be of the same order as those produced by the addition of arsenates to yeast-juice [Harden and Young, [1906, 3]], and will be discussed along with the latter (p. [77]).

Permanent Preparations Containing Active Zymase.

A considerable number of preparations have been obtained in the dry state which retain some proportion of the fermenting power of yeast or yeast-juice.

Starting with yeast-juice, it is possible to arrive at this result either by evaporation or precipitation. When the juice is very rapidly evaporated to a syrup at 20° to 25° and then further dried at 35°, either in the air or in a vacuum and finally exposed over sulphuric acid in a vacuum desiccator, a dry brittle mass is obtained which is soluble in water and retains practically the whole of the fermenting power of the juice. The success of the preparation depends on the nature of the yeast from which the juice is derived, Berlin yeasts V and S yielding much less satisfactory results than Munich yeast. The powder when [p038] thoroughly dry is found to retain its properties almost unimpaired for at least a year, and can be heated to 85° for eight hours without undergoing any serious loss of fermenting power [Buchner and Rapp, [1898, 4]; [1901]; Buchner, E. and H., and Hahn, [1903], pp. 132–9].

Active powders can also be obtained by precipitating yeast-juice with alcohol, alcohol and ether, or acetone. The preparation is best effected by bringing the juice into 10 volumes of acetone, centrifuging at once and as rapidly as possible, washing, first with acetone and then with ether, and finally drying over sulphuric acid. The white powder thus obtained is not completely soluble in water but is almost entirely dissolved by aqueous glycerol (2·5 to 20 per cent.), forming a solution which has practically the same fermenting power as the original juice. The precipitation can be repeated without any serious loss of fermenting power. Prolonged contact of the precipitate with the supernatant liquid, especially when alcohol or alcohol and ether are used, causes a rapid loss of the characteristic property [Albert and Buchner, [1900, 1], [2]; Buchner, E. and H., and Hahn, [1903], pp. 228–246; Buchner and Duchaček, [1909]].

Dry preparations capable of fermenting sugar can also be readily obtained from yeast without any preliminary rupture of the cells. Heat alone (yielding a product known as hefanol) or treatment with dehydrating agents may be used for this purpose, and a brief allusion has already been made (p. [21]) to the different varieties of permanent yeast (Dauerhefe) obtainable in these ways. The most important of these products are the dried Munich yeast (Lebedeff, see p. 25), and the material known as zymin, which is now made under patent rights for medicinal purposes by Schroder of Munich. The latter has proved of value in the investigation of the production of zymase in the yeast cell [Buchner and Spitta, [1902]], and of many other problems concerned with alcoholic fermentation. In order to prepare it 500 grams of finely divided pressed brewer's yeast, containing about 70 per cent. of water, are brought into 3 litres of acetone, stirred for ten minutes, and filtered and drained at the pump. The mass is then well mixed with 1 litre of acetone for two minutes and again filtered and drained. The residue is roughly powdered, well kneaded with 250 c.c. of ether for three minutes, filtered, drained, and spread on filter paper or porous plates. After standing for an hour in the air it is dried at 45° for twenty-four hours. About 150 grams of an almost white powder containing only 5·5 to 6·5 per cent. of water are obtained. This is quite incapable of growth or reproduction but produces a very considerable amount of alcoholic fermentation, far greater indeed than a corresponding [p039] quantity of yeast-juice. Two grams of the powder corresponding to 6 grams of yeast and about 3·5 to 4 c.c. of yeast-juice, are capable of fermenting about 2 grams of sugar, whereas the 4 c.c. of yeast-juice would on the average only ferment from one-quarter to one-sixth of this amount of sugar. The rate produced by this amount of zymin is about one-eighth of that given by the corresponding amount of living yeast [Albert, [1900]; Albert, Buchner, and Rapp, [1902]]. The proteoclastic ferment is still present in zymin, which undergoes autolysis in presence of water in a similar manner to yeast-juice [Albert, [1901, 2]].

As already mentioned an active juice can be prepared by grinding acetone-yeast with water, sand, and kieselguhr, and this process presents the advantage that samples of yeast-juice of approximately constant composition can be prepared at intervals from successive portions of a uniform supply of acetone-yeast.

Preparations of acetone-yeast, made from yeast freed from glycogen by exposure in a thin layer to the air for three or four hours at 35° to 45°, or eight hours at the ordinary temperature [Buchner and Mitscherlich, [1904]], show practically no autofermentation and may be used analytically for the estimation of fermentable sugars.

All the foregoing preparations exhibit the same general properties as yeast-juice, as regards their behaviour towards the various sugars, antiseptics, etc.

When zymin is mixed with sugar solution without being previously ground, it exhibits a peculiarity which is of some practical interest. The time which elapses before the normal rate of fermentation is attained and the total fermentation obtainable vary with the amount of sugar solution added, the time increasing and the total diminishing as the quantity of this increases. This phenomenon appears to have been noticed by Trommsdorff [[1902]], and a single experiment of Buchner shows the influence of the same conditions [Buchner, E. and H., and Hahn, [1903], p. 265, Nos. 700–1]. Harden and Young have found that when 2 grams of zymin are mixed with varying quantities of 10 per cent. sugar solution the following results are obtained:—

[p040]

This behaviour appears to be due to the removal of soluble matter essential for fermentation from the cell, which is discussed later on. It follows that when zymin is being tested for fermenting power, a uniform method should be adopted, and all comparative tests should be made with the same volumes of added sugar solution. Ground zymin appears to begin to ferment somewhat more slowly than unground (2 grm. to 12·4 c.c. of sugar solution in each case), but eventually produces the same total volume of gas [Buchner and Antoni, [1905, 1]].

CHAPTER III. THE FUNCTION OF PHOSPHATES IN ALCOHOLIC FERMENTATION.

[p041]

In the course of some preliminary experiments (commenced by the late Allan Macfadyen, but subsequently abandoned) on the production of anti-ferments by the injection of yeast-juice into animals, the serum of the treated animals was tested for the presence of such antibodies both for the alcoholic and proteoclastic enzymes of yeast-juice, and it was then observed that the serum of normal and of treated animals alike greatly diminished the autolysis of yeast-juice.

As the explanation of the comparatively rapid disappearance of the fermenting power from yeast-juice had been sought, as already mentioned (p. [20]), in the hydrolytic action of the tryptic enzyme which always accompanies zymase, the experiment was made of carrying out the fermentation in the presence of serum, with the result that about 60 to 80 per cent. more sugar was fermented than in the absence of the serum [Harden, [1903]].

This fact was the starting-point of a series of attempts to obtain a similar effect by different means, in the course of which a boiled and filtered solution of autolysed yeast-juice was used, in the hope that the products formed by the action of the tryptic enzyme on the proteins of the juice would, in accordance with the general rule, prove to be an effective inhibitant of that enzyme. This solution was, in fact, found to produce a very marked increase in the total fermentation effected by yeast-juice, the addition of a volume of boiled juice equal to that of the yeast-juice doubling the amount of carbon dioxide evolved [Harden and Young, [1905, 1]]. This effect was found to be common to the filtrates from boiled fresh yeast-juice and from boiled autolysed yeast-juice, and was ultimately traced in the main, not to the antitryptic effect which had been surmised, but to two independent factors, either of which was capable in some degree of bringing about the observed result.

Boiled yeast-juice was indeed found to possess a decided anti-autolytic effect, as determined by a comparison of the amounts of nitrogen rendered non-precipitable by tannic acid in yeast-juice alone [p042] and in a mixture of yeast-juice and boiled juice on preservation [Harden, [1905]]. The anti-autolytic effect, however, appeared to vary independently of the effect on the fermentation, and the conclusion was drawn, as stated above, that the increase in the alcoholic fermentation was not directly dependent on the decrease in the action of the proteoclastic enzyme but was due to some independent cause. The property possessed by boiled yeast-juice of diminishing the autolysis of yeast-juice has now been carefully examined by Buchner and Haehn [[1910, 2]] and ascribed by them to a soluble antiprotease (p. [65]).

The two factors to which the increase in fermentation produced by the addition of boiled juice were ultimately traced were (1) the presence of phosphates in the liquid, and (2) the existence in boiled fresh yeast-juice of a co-ferment or co-enzyme, the presence of which is indispensable for fermentation [Harden and Young, [1905, 1], [2]].

The former of these factors will be here discussed and the co-enzyme will form the subject of the following chapter.

The general fact that sodium phosphate increases the total fermentation produced by a given volume of yeast juice was observed on several occasions by Wroblewski [[1901]] and also by Buchner [Buchner, E. and H., and Hahn, [1903], pp. 141–2], who ascribed the action of this salt to its alkalinity, comparing it in this respect with potassium carbonate and remarking that the increase in both cases took place chiefly in the first twenty hours of fermentation. The increased amount of fermentation following the addition of boiled yeast-juice was also noted by Buchner and Rapp [[1899, 2], No. 265, p. 2093] in a single experiment.

Observations made at intervals of a few minutes instead of twenty hours have, however, revealed the fact that phosphates play a part of fundamental importance in alcoholic fermentation and that their presence is absolutely essential for the production of the phenomenon.

Effect of the Addition of Phosphate to a Fermenting Mixture of Yeast-Juice and Sugar.

When a suitable quantity[2] of a soluble phosphate is added to a fermenting mixture of glucose, fructose, or mannose with yeast-juice, the rate of fermentation rapidly rises, sometimes increasing as much as twenty-fold, continues at this high value for a certain period and then falls again to a value approximately equal to, but generally [p043] somewhat higher than, that which it originally had. Careful experiments have shown that during this period of enhanced fermentation the amounts of carbon dioxide and alcohol produced exceed those which would have been formed in the absence of added phosphate by a quantity exactly equivalent to the phosphate added in the ratio CO₂ or C₂H₆O:R′₂HPO₄ [Harden and Young, [1906, 1]].

[2] The effect of an excess of phosphate is discussed later on, p. [71].

This result is of fundamental importance, and the evidence on which it rests deserves some consideration. Quantitative experiments on this subject require certain preliminary precautions. The acid phosphates are too acid to permit of any extended fermentation and the phosphates of the formula R′₂HPO₄ absorb a considerable volume of carbon dioxide with production of a bicarbonate, according to the reaction:—

R₂HPO₄ + H₂CO₃ ⇌ RHCO₃ + RH₂PO₄.

The method which has been adopted, therefore, is to employ either a secondary phosphate saturated with carbon dioxide at the temperature of the experiment, or a mixture of five molecular proportions of the secondary phosphate with one molecular proportion of a primary phosphate, in which the amount of bicarbonate formed is negligible. In the former case it is necessary to ascertain whether any of the carbon dioxide evolved is derived from the bicarbonate by the action of acid originally present or produced in the yeast-juice or by a disturbance of the original equilibrium owing to the chemical change which occurs. This is done by acidifying duplicate samples with hydrochloric acid before and after the fermentation and measuring the gas evolved in each case. Any necessary correction can then be made. The calculation of the extra amount of carbon dioxide evolved from yeast-juice containing sugar when a phosphate is added involves an estimation of the amount which would have been evolved in the absence of added phosphate, and this is a matter of some difficulty. Since the final steady rate of fermentation attained is often slightly different from the initial rate, the practice has been adopted of ascertaining this final rate and then calculating the total evolution corresponding to it for the whole period from the time of the addition of the phosphate to the end of the observations. This amount deducted from the observed total leaves the extra amount of carbon dioxide formed, and it is this quantity which is equivalent to the phosphate added. Alcohol is simultaneously produced in the normal ratio. The justification for this method of calculation will be found later (p. [54]).

The following table, containing the results of experiments with [p044] glucose, fructose, and mannose, indicates very clearly the nature of the method of calculation and also of the agreement between observation and theory.

Three quantities of 25 c.c. of yeast-juice + 5 c.c. of a solution containing 1 gram of the sugar to be examined (a large excess) were incubated with toluene at 25° for one hour, in order to remove all free phosphate, and to each were then added 5 c.c. of a solution of sodium phosphate corresponding to 0·1632 gram of Mg₂P₂O₇ and equivalent to 32·6 c.c. of carbon dioxide at N.T.P. The rates of fermentation were then observed until they had passed through the period of acceleration and had fallen and attained a steady value, the gases being measured moist at 19·3° and 760·15 mm.

These numbers agree well with the value calculated from the phosphate added, viz. 32·6 [Harden and Young, [1909]].

Another experiment is illustrated graphically in Fig. 4, in which the volume of carbon dioxide evolved is plotted against time. The determination was in this case made by adding 25 c.c. of an aqueous solution containing 5 grams of glucose to one quantity of 25 c.c. of yeast-juice (curve A) and 5 c.c. of 0·3 molar solution of the mixed primary and secondary sodium phosphates, and 20 c.c. of a solution containing 5 grams of glucose to a second equal quantity of yeast-juice (curve B). Curve A shows the normal course of fermentation of yeast-juice with glucose. There is a slight preliminary acceleration during the first twenty minutes, due to free phosphate in the juice, and the rate then becomes steady at about 1·4 c.c. in five minutes. During this preliminary acceleration 10 c.c. of extra carbon dioxide are evolved, this number being obtained graphically by continuing the line of steady rate back to the axis of zero time. Curve B shows the effect of the added phosphate. The rate rises to about 9·5 c.c. in five minutes, i.e. to more than six times the normal rate, and then gradually falls until after an hour it is again steady and almost exactly equal to 1·4 c.c. per five minutes. Continuing the line of steady rate back to the axis of zero [p045] time it is found that the extra amount of carbon dioxide is 48 c.c. Subtracting from this the 10 c.c. shown in curve A as due to the juice alone, a difference of 38 c.c. is obtained due to the added phosphate. The amount calculated from the phosphate added in this case is, at atmospheric temperature and pressure, 38·9 c.c.

Fig. 4.

After the expiration of seventy minutes from the commencement of the experiment, a second addition is made of an equal amount of phosphate. The whole phenomenon then recurs, as shown in curve C, the maximum rate being slightly lower than before, about 6 c.c. per five minutes, and the rate again becoming finally steady at 1·4 c.c. as before. The extra amount of carbon dioxide evolved in this second period obtained graphically as in the former case, is 107–68 = 39 c.c.

It may be noted that in this case the observations after each addition last fifty to seventy minutes, so that an error of 0·1 c.c. per five minutes in the estimated final rate would make an error of 1 to 1·4 c.c. in the extra amount of carbon dioxide, i.e. about 3 to 4 per cent. of the total, and this is approximately the limit of accuracy of the method. [p046] The results are more precise when the yeast-juice employed is an active one, since, when the fermenting power of the juice is low, the initial period of accelerated fermentation is unduly prolonged and the calculation of the extra amount of carbon dioxide is rendered uncertain.

Zymin (p. [38]) yields precisely similar results to yeast-juice, but in this case the rate of fermentation is not so largely increased. This has the effect that the extra amount of carbon dioxide cannot be quite so accurately estimated for zymin, because a slight error in the determination of the final rate of fermentation has a greater influence on the result. The equivalence between the extra amount of carbon dioxide evolved and the phosphate added is, however, unmistakable, as is shown by the following results of an experiment with zymin, in which 6 grams of zymin (Schroder) + 3 grams of fructose (Schering) + 25 c.c. of water were incubated at 25° in presence of toluene until a steady rate had been attained. Five c.c. of a solution of sodium phosphate equivalent to 32·2 c.c. carbon dioxide at N.T.P. were then added.

Considering the small proportional rise in rate and the long period of accelerated fermentation, the agreement between the volume observed, 29·8 c.c., and that calculated from the phosphate, 32·2, is quite satisfactory [Harden and Young, [1910, 1].] Precisely the same relations hold for maceration extract, but in this case it must be remembered that a large amount of free phosphate is present in the extract, as much as 0·3129 grm. Mg₂P₂O₇ being obtained from 20 c.c. in one preparation, so that the original extract had the concentration of a 0·14 molar solution of sodium phosphate. It is in fact not improbable that the delay in the onset of fermentation sometimes observed with maceration extract (see Lebedeff, 1912, 2; Neuberg and Rosenthal, 1913) may be due to the presence of phosphate in so great an excess of the amount which can be rapidly esterified by the enzymes that the rate of fermentation is at first greatly lowered (see p. [71]). When this phosphate is removed by incubation with glucose or fructose, the subsequent addition of phosphate produces the characteristic action and the extra carbon dioxide evolved is, as with other yeast preparations, equivalent to the phosphate added. An actual estimation carried out in this way gave 35 c.c. of CO₂ for an addition of phosphate equivalent to 32·9 c.c. [Harden and Young, [1912]]. [p047]

Within the limits imposed by the experimental conditions, then, the fact is well established that the addition of a soluble phosphate to a fermenting mixture of a hexose with yeast-juice, maceration extract, dried yeast, or zymin causes the production of an equivalent amount of carbon dioxide and alcohol.

This fact indicates that a definite chemical reaction occurs in which sugar and phosphate are concerned, and this conclusion is confirmed when the fate of the added phosphate is investigated. If an experiment, such as one of those described above, be interrupted as soon as the rate of fermentation has again become normal, and the liquid be boiled and filtered, it is found that nearly the whole of the phosphorus present passes into the filtrate, but that only a small proportion of this exists as mineral phosphate, whilst the remainder, including that added in the form of a soluble phosphate, is no longer precipitable by magnesium citrate mixture [Harden and Young, [1905, 2]].

A similar observation was made at a later date by Iwanoff [[1907]], who had previously observed [[1905]] that living yeast, like many other vegetable organisms, converted mineral phosphates into organic derivatives. Iwanoff employed zymin and hefanol (p. [38]) instead of yeast-juice, and found that phosphates were thereby rendered non-precipitable by uranium acetate solution, but did not observe the accelerated fermentation caused by their addition.

The foregoing conclusions have been strikingly confirmed by experiments with maceration extract carried out by Euler and Johansson [[1913]], in which both the carbon dioxide evolved and the phosphate rendered non-precipitable by magnesia were determined at intervals. When dried yeast is employed as the fermenting agent, the amount of phosphate esterified in the earlier stages is greater than would be expected, but ultimately becomes exactly equivalent to the carbon dioxide evolved.

Nature of the Phospho-organic Compound formed by Yeast-Juice and Zymin from the Hexoses and Phosphate.

The formation and properties of the compound produced from phosphates in the manner just described have been investigated by Harden and Young [[1905, 2]; [1908, 1]; [1909]; [1911, 2]], Young [[1909]; [1911]], Iwanoff [[1907]; [1909, 1]], Lebedeff [[1909]; [1910]; [1911, 5], [6]; [1912, 3]; [1913, 1]]; and Euler [[1912, 1]; Euler and Fodor, [1911]; Euler and Kullberg, [1911, 3]; Euler and Ohlsén, [1911]; [1912]; Euler and Johansson, [1912, 4]; Euler and Bäckström, [1912]], but its exact constitution cannot as yet be regarded as definitely known. [p048]

Phosphates undergo this characteristic change when the sugar undergoing fermentation is glucose, mannose, or fructose, and it may be said at once that no distinction can be established between the products formed from these various hexoses; they all appear to be identical. The compound produced is, as already mentioned, not precipitated by ammoniacal magnesium citrate mixture, nor by uranium acetate solution. It can, however, be precipitated by copper acetate (Iwanoff) and by lead acetate (Young). The preparation of the pure lead salt from the liquid obtained by fermenting a sugar with yeast-juice or zymin in presence of phosphate is commenced by boiling and filtering the liquid. Magnesium nitrate solution and a small quantity of caustic soda solution are then added to precipitate any free phosphate, and the liquid well stirred and allowed to stand over night. To the neutralised filtrate lead acetate is then added together with sufficient caustic soda solution to maintain the reaction neutral to litmus, until no further precipitate is formed. The liquid is then filtered or, better, centrifugalised, and the precipitate repeatedly washed with water until a portion of the clear filtrate gives no reduction when boiled with Fehling's solution. It is essential that this washing should be thorough as evidence has recently been obtained of the formation under certain conditions of a hexosephosphate, the lead salt of which is not so sparingly soluble as that of the hexosediphosphate [Harden and Robison, [1914]]. The lead precipitate is then suspended in water, decomposed by a current of sulphuretted hydrogen, the clear filtrate freed from sulphuretted hydrogen by a current of air, and finally neutralised with caustic soda. The removal of phosphate and conversion into lead salt are repeated twice, and the resulting lead salt is then found to be free from nitrogen and to have a composition represented by the formula C₆H₁₀O₄(PO₄Pb)₂. Lebedeff carries out the preparation in a somewhat different manner. The fermentation is effected by means of air-dried yeast (150 grams to 1 litre of water, 210 grams cane-sugar and 105 grams of a mixture of 2 parts Na₂HPO₄ and 1 part NaH₂PO₄) and the liquid (about 700 c.c.) after boiling and filtering, is treated with an equal volume of acetone. About 300 c.c. of a thick liquid is precipitated and this is redissolved in water and precipitated by an equal volume of acetone two or three times. The final liquid is then precipitated with warm lead acetate solution and filtered and washed with dilute lead acetate solution until the filtrate is clear and no longer reduces Fehling's solution after removal of the lead [[1910]]. Euler and Fodor [[1911]] on the other hand precipitate the free phosphate with magnesia mixture and then add acetone, dissolve the syrup thus precipitated in water and add copper [p049] acetate solution. A blue copper salt is precipitated which is thoroughly washed with water and used for the preparation of solutions of the acid. A solution of the free acid can readily be prepared by the action of sulphuretted hydrogen on the lead salt suspended in water. It forms a strongly acid liquid, which requires exactly two equivalents of base for each atom of phosphorus present to render it neutral to phenolphthalein. It decomposes when evaporated, leaving a charred mass containing free phosphoric acid. The acid is slightly optically active, and has [a_D] = + 3·4°. A number of amorphous salts have been prepared by precipitation from a solution of the sodium salt, and of these the silver, barium, and calcium salts have been analysed with results agreeing with the general formula C₆H₁₀O₄(PO₄R′₂)₂. The magnesium, calcium, barium, and manganese salts, which are only sparingly soluble, are all precipitated when their solutions are boiled but re-dissolve on cooling, and this property can be utilised for their purification. The alkali salts have only been obtained as viscid residues.

A difference of opinion exists as to the molecular weight and constitution of this substance. Iwanoff [[1909, 1]] regards it as a triosephosphoric acid, C₃H₅O₂(PO₄H₂), basing this view on the preparation of an osazone which melted at 142°, but when recrystallised from benzene gave a product melting at 127°–8°, which had the same appearance, melting-point, and nitrogen content as the triosazone formed by the action of phenylhydrazine on the oxidation products of glycerol. Neither Lebedeff [[1909]] nor Young could obtain Iwanoff's osazone, and all attempts to reduce the acid with formation of glycerol either by sodium amalgam or hydriodic acid were unsuccessful (Young). There is therefore practically no serious experimental evidence in favour of Iwanoff's view.

On the other hand, Harden and Young regard the acid as a diphosphoric ester of a hexose. This view is based on the fact that when the acid is boiled with water, or an acid, free phosphoric acid is produced along with a levo-rotatory solution containing fructose and possibly a small proportion of some other sugar or sugars. (Euler and Fodor however did not obtain a hexose in this way [[1911]].) The acid itself only reduces Fehling's solution after some hours in the cold, rapidly when boiled, whereas when its solution is first boiled, and then treated with Fehling's solution in the cold, the products of decomposition bring about reduction in a few minutes. The reduction brought about when the acid is boiled with Fehling's solution is considerably less (33 per cent.) than that produced by an equivalent amount of glucose. The behaviour of the compound towards phenylhydrazine is also in complete agreement [p050] with this view. Lebedeff found [[1909], [1910]] that the acid or its salts heated with phenylhydrazine in presence of acetic acid gave an insoluble compound which was ultimately found to be the phenylhydrazine salt of hexosemonophosphoric acid osazone

C₆H₅NH·NH₂·H₂PO₄·C₄H₅(OH)₃·C(N₂HC₆H₅)CH(N₂HC₆H₅)

[Lebedeff, [1910]; [1911, 6]; Young, [1911]]. After recrystallisation from alcohol this compound forms yellow needles, melting at 151°–152°. It is decomposed by caustic soda yielding a sodium salt

Na₂PO₄·C₄H₅(OH)₃·(CN₂HC₆H₅)·CH(N₂HC₆H₅)

and on boiling with caustic soda decomposes giving a hexosazone (free from phosphorus) which is probably glucosazone, and in addition glyoxalosazone, probably as the result of a secondary decomposition. Towards acids it is remarkably stable yielding with hydrochloric acid a hexosonephosphoric ester from which the original osazone can be regenerated (Lebedeff). Lebedeff at first [[1910]] argued from the formation of this osazone that the original hexosephosphate contained only one phosphoric acid group per molecule of hexose. It was however shown by Young [[1911]] and subsequently confirmed by Lebedeff [[1911, 6]] that one molecule of phosphoric acid is split off during the formation of the osazone, even in neutral solution. Moreover it has been found that in the cold hexosediphosphoric acid reacts with 3 molecules of phenylhydrazine forming the diphenylhydrazine salt of hexosediphosphoric acid phenylhydrazone

(C₆H₅NH·NH₂·H₂PO₄)₂·C₆H₇(OH)₃·N₂HC₆H₅.

This compound crystallises out when 1 volume of alcohol is added to a solution of 3 molecules of phenylhydrazine in one of the acid and forms colourless needles melting at 115°–117°. p-Bromophenylhydrazine yields an analogous compound melting at 127°–128°.

Precisely the same products are given with phenylhydrazine by the hexosephosphoric acid prepared from glucose, mannose, and fructose, proving that all these sugars yield the same hexosediphosphoric acid, a point of fundamental importance.

Direct measurements of the molecular weight of the acid by the freezing-point method, combined with the determination of the degree of dissociation by the rate of cane-sugar inversion, are indecisive, but indicate that the acid has a molecular weight considerably higher than that required for a triosephosphoric acid.

A similar uncertainty attaches to the determination of the molecular weight from the freezing-point depression and conductivity of the acid potassium salt [Euler and Fodor, [1911]]. Euler however concludes [p051] that both a hexosediphosphoric acid and a triosemonophosphoric acid are formed, but has not prepared any derivatives of the latter.

As regards the constitution of the hexosephosphoric ester several suggestions have been made by Young, but no decisive evidence at present exists. The identity of the products from glucose, mannose, and fructose may be explained by regarding the acid as a derivative of the enolic form common to these three sugars (p. [97]), or by supposing that portions of two sugar molecules may be concerned in its production. The formation and composition of the hydrazone and osazone are of great importance as they indicate that in all probability one of the phosphoric acid residues is united with the carbon atom adjacent to the carbonyl group of the hexose. They moreover render it certain that the original phosphoric ester is a hexosediphosphoric ester and not a triosemonophosphoric ester.

Hexosediphosphoric acid has not as yet been discovered in the animal body. The action of a number of enzymes upon it has been examined [Euler, [1912, 2]; Euler and Funke, [1912]; Harding, [1912]; Plimmer, [1913]] with the following results.

The lipase of castor oil seeds, a glycerol extract of the intestinal mucous membrane of the rabbit and pig, and an aqueous extract of bran have a slow hydrolytic action, whereas pepsin and trypsin are without effect. Feeding experiments with rabbits and dogs indicate that the ester is capable of hydrolysis in the animal body, a large proportion of the phosphorus being excreted as inorganic phosphate. The ester is also decomposed by Bacillus coli communis.

It is remarkable that the hexosephosphate is not fermented nor hydrolysed by living yeast, a fact observed by Iwanoff, Harden and Young, and Euler, although, according to the experiments of Paine [[1911]], the yeast cell is at all events partially permeable to the sodium salt.

The Equation of Alcoholic Fermentation.

An equation can readily be constructed for the reaction in which hexosephosphate is formed, the data available being the formula of the product and the relation between the phosphate added and the carbon dioxide and alcohol produced:—

(1) 2 C₆H₂O₆ + 2 PO₄HR₃  =
2 CO₂ + 2 C₂H₆O + 2 H₂O + C₆H₁₀O₄(PO₄R₂)₂.

According to this, two molecules of sugar are concerned in the change, the carbon dioxide and alcohol being equal in weight to one [p052] half of the sugar used, and the hexosephosphate and water representing the other half.

Additional confirmation of this equation is afforded by the determination of the ratio between sugar used and carbon dioxide evolved when a known weight of sugar together with an excess of phosphate is added to yeast-juice at 25°. The phenomena then observed are precisely similar to those which occur when a phosphate is added to a fermenting mixture of yeast-juice and excess of sugar as described above. The rate of fermentation rapidly rises and then gradually falls until a rate is attained approximately equal to that of the autofermentation of the juice in presence of phosphate. At this point it is found that the extra amount of carbon dioxide evolved, beyond that which would have been given off in the absence of added sugar, bears the ratio expressed in equation (1) to the sugar added [Harden and Young, [1910, 2]]. The results of four estimations made in this way were (a) 0·2 grams of glucose gave 26·5 and 27·9 c.c. of carbon dioxide at N.T.P.; (b) 0·2 grams of fructose gave 27·9 and 28·9 c.c. The carbon dioxide calculated from the sugar added in each of the four cases is 26·96 c.c.

It has also been shown by Euler and Johansson [[1913]] that in the fermentation of a mixture of equivalent amounts of phosphate and glucose, the whole of the glucose had disappeared when the whole of the phosphate had become esterified.

Cycle of Changes Undergone by Phosphate in Alcoholic Fermentation.

According to equation (1) the free phosphate present is used up in the reaction, and the question at once arises whether there is any source from which a constant supply of free phosphate can be elaborated in the juice, or whether some other change occurs which results in the formation of carbon dioxide and alcohol in the absence of free phosphate. The experimental evidence points in the direction of the former of these alternatives, but the question is a very difficult one to decide with absolute certainty.

When a mixture of a phosphate with yeast-juice and sugar is examined at intervals and the amount of free phosphate estimated, the following stages are observed:—

1. During the initial period of accelerated fermentation following the addition of the phosphate, the concentration of free phosphate rapidly diminishes.

2. At the close of this period, the amount of free phosphate [p053] present is very low, and, as long as active fermentation continues, no marked increase in it occurs.

3. As alcoholic fermentation slackens and finally ceases, a marked and rapid rise in the amount of free phosphate occurs at the expense of the hexosephosphate, which steadily diminishes in amount, and this change is brought about by an enzyme in the juice and ceases if the liquid be boiled.

This last reaction may be represented by the equation

(2) C₆H₁₀O₄(PO₄R₂)₂ + 2 H₂O = C₆H₁₂O₆ + 2 PO₄HR₂.

In the light of this equation, together with equation No. 1, given above, all these facts can be simply and easily understood.

The rapid diminution in the amount of free phosphate during stage 1 corresponds with the occurrence of reaction (1). During the whole period of fermentation the enzymic hydrolysis of the hexosephosphate is proceeding according to equation (2). Up to the end of stage 2 the phosphate thus produced enters into reaction, according to equation (1), with the sugar which is present in excess and is thus reconverted into hexosephosphate, so that as long as alcoholic fermentation is proceeding freely, no accumulation of free phosphate can occur.

As soon as alcoholic fermentation ceases, however, it is no longer possible for the phosphate to pass back into hexosephosphate, and hence it accumulates in the free state.

A similar hydrolysis of hexosephosphate and accumulation of phosphate occur when a solution of hexosephosphate is treated with yeast-juice which has been deprived of the power of fermentation by dialysis, or with zymin freed from co-enzyme by washing (p. [63]).

The actual rate of fermentation observed in any particular case in presence of excess of sugar, enzyme, and co-enzyme must on this view depend on the supply of phosphate which is available.

In presence of an adequate amount of phosphate, as well as of sugar, the highest rate attained represents the maximum velocity at which reaction (1) can proceed in that sample of yeast-juice or zymin, and this high rate is characteristic of the initial period of accelerated fermentation which follows the addition of a suitable quantity of phosphate. By the simple expedient of renewing the supply of phosphate as rapidly as it is converted into hexosephosphate, this high rate can be maintained for a considerable time [Harden and Young, [1908, 1]]. In this way, for example, an average rate of evolution of carbon dioxide of 15 c.c. in five minutes was maintained for an hour and a [p054] quarter, whereas the normal rate in the absence of added phosphate was 3 c.c.

As soon as all the free phosphate has entered into the reaction, however, the supply of phosphate depends in the main on the rate at which the resulting hexosephosphate is decomposed, and the rate of fermentation now attained is conditioned by the rate at which reaction (2) proceeds, and this evidently depends on the existing concentration of the hydrolytic enzyme, which may be provisionally termed hexosephosphatase.

The rates attained during the initial period of rapid fermentation and the subsequent period of slow fermentation are thus seen to represent the velocities of two entirely different chemical reactions.

These considerations also explain why it is the extra carbon dioxide evolved during the initial period, and not the total carbon dioxide, which is equivalent to the added phosphate. As the production of phosphate is proceeding throughout the whole period at a rate which is equivalent to the normal rate of fermentation, it is obviously necessary to deduct the corresponding amount of carbon dioxide from the total evolved in order to ascertain the amount equivalent to the added phosphate.

An explanation is also afforded of the fact that a considerable increase in the concentration of hexosephosphate does not materially increase the normal rate of fermentation. This is probably due to the circumstance that, in accordance with the general behaviour of enzymes in presence of excess of the fermentable substance, the hexosephosphatase hydrolyses approximately equal amounts of hexosephosphate in equal times whatever the concentration of the latter may be, above a certain limit.

According to the experiments of Euler and Johansson [[1913]] the hydrolytic activity of the hexosephosphatase is greatly diminished by the presence of toluene.

Effect of Phosphate on the Total Fermentation Produced by Yeast-Juice.

The addition of a phosphate to yeast-juice not only produces the effect already described, but also enables a given volume of yeast-juice to effect a larger total fermentation, even after allowance is made for the carbon dioxide equivalent to the quantity of phosphate added. The increase in the case of ordinary yeast-juice has been found to amount to from 10 to 150 per cent. of the original total fermentation [p055] produced by the juice in the absence of added phosphate. The numbers contained in columns 1 and 2 of the table on p. [56] illustrate this effect, the ratio of the total in the presence of phosphate to that obtained in its absence being given, as well as that of the total in presence of phosphate less the equivalent of the phosphate added, to the original fermentation. The cause of this increase in the total fermentation is probably to be sought mainly in a protective action of the excess of hexosephosphate on the various enzymes, for, as has been stated above, the rate of fermentation after the termination of the initial period, is practically the same as in the absence of added phosphate (see p. [43]).

Now it follows from equation (1) (p. [51]) that in the total absence of phosphate no fermentation should occur, and the experimental realisation of this result would afford very strong evidence in favour of this interpretation of the phenomenon.

Hitherto, however, it has not been found possible to free the materials employed completely from phosphorus compounds which yield phosphates by enzymic hydrolysis during the experiment, but it has been found that when the phosphate contents are reduced to as low a limit as possible, the amount of sugar fermented becomes correspondingly small, and, further, that in these circumstances the addition of a small amount of phosphate or hexosephosphate produces a relatively large increase in the fermenting power of the enzyme.

When the total phosphorus present is thus largely reduced, the increase produced by the addition of a small amount of phosphate may amount to as much as eighty-eight times the original, in addition to the quantity equivalent to the phosphate, whilst the actual total evolved, including this equivalent, may be as much as twenty times the original fermentation. This result must be regarded as strong evidence in favour of the view that phosphates are indispensable for alcoholic fermentation.

The results indicated above were experimentally obtained in three different ways and are exhibited in the following table. In the first place (cols. 3 and 4), advantage was taken of the fact that the residues obtained by filtering yeast-juice through a Martin gelatin filter (p. [59]) are sometimes found to be almost free from mineral phosphates, whilst they still contain a small amount of co-enzyme. The experiment then consists in comparing the fermentation produced by such a residue poor in phosphate with that observed when a small amount of phosphate is added. The second method (col. 5) consisted in carrying out two parallel fermentations by means of a residue rendered inactive by filtration [p056] and a solution of co-enzyme free from phosphate and hexosephosphate (p. [67]) [Harden and Young, [1910, 2]].

The third method (col. 6) consisted in washing zymin with water, to remove soluble phosphates, and then adding to it a solution of co-enzyme containing only a small amount of phosphate, and ascertaining the effect of the addition of a small known amount of hexosephosphate upon the fermentation produced by this mixture [Harden and Young, [1911, 1]].

Production of a Fermentable Sugar from Hexosephosphate by the Action of an Enzyme Contained in Yeast-Juice.

The sugar which, according to equation (2) accompanies the phosphate formed by the enzymic hydrolysis of hexosephosphate is under ordinary circumstances fermented by the alcoholic enzyme of the juice and thus escapes detection.