Memoir on the alcoholic fermentation

1860 Louis Pasteur

Pasteur, L. 1860. Memoire sur la fermentation alcoölique. Annales de Chimie et de Physique, Vol. 58, 3rd Series, pages 323-426.

BY ALCOHOLIC FERMENTATION, I REFER to that fermentation which sugar undergoes under the influence of the ferment which is called beer yeast. It is the fermentation which is the source of alcohol in wine and all alcoholic beverages....
Any hesitation to apply the. words alcoholic fermentation and to realize their true meaning seems to me impossible, since they have been applied by Lavoisier, Gay-Lussac and Thenard to the fermentation of sugar by beer yeast....
In the first part of my memoir I will study the changes which occur in sugar during the alcoholic fermentation, and in the second part I will consider especially the ferment, its nature and the transformations which it undergoes.
[The first part is concerned with the products formed in the fermentation, such as alcohol, carbon dioxide, glycerol, and succinic acid, and the stoichiometric relationships to the sugar.]



I. Historical summary of the state of the science of beer yeast and its 
    modifications during alcoholic fermentation

In 1680 Leeuwenhoeck studied beer yeast under the microscope and found very small spherical or oval globules, but the chemical nature of this substance was unknown to him. Fabroni identified the yeast with gluten. This was some progress. It gave an indication that yeast might be an organic product. M. Thenard published a memoir in which he said: All natural sugary juices, in the process of spontaneous fermentation, deposit a substance which resembles beer yeast and which has the power of fermenting pure sugar. This yeast is animal in nature, since it is nitrogenous and yields ammonia upon distillation....
In his observations published in 1835 and 1837, M. Cagniard de Latour introduced a new idea. Before his time, yeast had been regarded as a vegetable product, produced in situ, which precipitated out in the presence of a fermentable sugar. M. Cagniard de Latour recognised "that the yeast was a mass of globules which reproduced by budding, and were not merely a simple chemical or organic substance." He concluded that "it is very probable that the production of carbon dioxide and the decomposition of sugar and its conversion into alcohol are effects of the growth of the yeast."
This opinion immediately found a powerful opponent in M. Liebig.
In his eyes, the ferment is an extremely unstable substance which decomposes itself and which causes fermentation as a result of the decomposition which it itself undergoes, during which it communicates this perturbation and disassimilation to the fermentable material. He expresses himself thus: "The experiments which we have revealed demonstrate the existence of a new cause which brings about decomposition and synthesis. This cause is nothing else than the movement which a body in the process of decomposition communicates to other substances in which the elements are held together very weakly. . . . Beer yeast, and in general all animal and vegetable materials undergoing putrefaction, communicate to other substances the state of decomposition in which they find themselves...."
M. Liebig has developed his opinions throughout the majority of his works with such a persistence and conviction that they have gradually triumphed. Today they are accepted generally in Germany and France. His ideas have been applied to other fermentations, such as the lactic, by MM. Fremy and Bautron.
As far as I can see, the reason that the ideas of M. Liebig have become gradually established amongst chemists is the following. There have been discovered during the last twenty years a large number of phenomena which have been placed in the same group with alcoholic fermentation and in which it has been impossible to recognise the existence of particular lower plants, but in which there was a substance which was undergoing decomposition. For example, if one places a sugar solution containing chalk with a nitrogenous material from some animal source, like casein, gluten, fibrin, gelatin, rennet, an animal membrane . . . one sees that the sugar gradually becomes converted into lactic acid. But although these animal materials are very diverse, the effect on the sugar is always the same. There is only one thing which appears to be similar in these nitrogenous materials; this is their gradual decomposition. A correlation is thus demonstrated between the transformation of sugar into lactic acid and an instability of the animal substance, a tendency to decompose.
 The work of M. Colin on alcoholic fermentation had already shown in 1825 that the analogous facts existed for it. He showed that these animal materials of diverse origin are able to induce the decomposition of sugar into
alcohol and carbon dioxide. 
Meanwhile a remarkable circumstance ought to have aroused the attention of, and cautioned, those who were concerned with alcoholic fermentation. Indeed, after the publication of the observations of M. Cagniard de Latour, M. Turpin, who had been in charge of the proceedings of  the Academy, studied, at the request of M. Thenard, the deposits which form in the alcoholic fermentation of sugar in the presence of egg white, and found that they consisted exclusively of globules of the beer yeast.
 Since one of the materials used by  M. Colin, albumin, did not cause an alcoholic fermentation while allowing for the appearance of yeast, it may be assumed that all the other nitrogenous substances behaved similarly, and because of their diversity would prove nothing more concerning M. Liebig's theory.
 But I hasten to add that nothing of the like would exist in the case of the very diverse and very numerous lactic fermentations. All of the workers agree that here there is only a chemical alteration of the animal substance. The facts concerning this fermentation and many other phenomena of the same order had therefore a decisive influence on the theory.
The idea of M. Cagniard de Latour, which at first had a certain amount of acceptance, was gradually abandoned. Many people did not contest the idea that beer yeast was organised, but it was believed to be partly destroyed by the fermentation, as had been stated by M. Thenard, and in common with all of the other nitrogenous materials acting as ferments, it was in this way that it acted on sugar. Such is the thought of M. Liebig.
Berzelius did not agree with the ideas of M. Liebig, while rejecting those of Cagniard de Latour and Schwann. For him fermentation was an action through contact. He did not believe at the same time in the existence of a living organism in the yeast. "It is only a chemical product which precipitates in the fermentation and which takes the ordinary form of a non-crystalline precipitate, even inorganic, of small balls which group themselves one after the other and form chains." Elsewhere he explains himself thusly: "It is clear that when the organised bodies decompose in the water, and when the dissolved materials precipitate, the latter should assume a form, and as they do not assume regular geometrical forms, the result must be other forms, depending on the nature of the bodies which influence them.... It is thus quite natural that they imitate the forms of the simplest organisms of plant life. Nevertheless form alone does not constitute life."
The chemical composition of yeast as published by M. Payen shows the following figures: Nitrogenous material, 62.73%, cellulose integument, 29.37%, fat, 2.10%, minerals, 5.80%. Elemental analysis by Schlossberger for ale yeast shows: Carbon, 50.05%, Hydrogen, 6.52%, Nitrogen, 31.59%, Oxygen, 11.84%. An analysis of the yeast ash by Mitscherlisch gives the following figures: phosphoric acid, 41.8%, potassium, 39.8%, soda ash, none, magnesium phosphate, 16.8%, calcium phosphate, 2.3%, proportion of ash in total, 7.65%.

II. The nitrogen of the yeast is never transformed into ammonia during the alcoholic fermentation. Instead of ammonia formation, a slight amount of it disappears

On 18 January, 1858, I placed 100 grams of sugar in a litre of water which contained in it the soluble substances from the beer yeast. To this I added a trace of the globules of fresh yeast. An analysis on a portion of this revealed that it contained 0.038 grams of ammonia per litre. On 5 February, the fermentation was ended. An analysis for ammonia revealed 0.020 grams per litre, or less ammonia than at the beginning.
On the 30th of April I repeated this experiment with 100 grams of sugar, but this time I used a very small amount of ordinary yeast, so that the fermentation could last for a longer time. I added only 1.037 grams of yeast (weight of material dried at 100¡). On the 30th of August the fermentation was still proceeding. A tube which lead out of the flask was always immersed in the liquid. The liquid was analysed on the 27th of November. All of the liquid contained only 0.0008 (grams of ammonia, and it is possible that it contained no ammonia and this minimum value was an error in the assay....
The consistency of the results and the data from many other experiments seem to leave no doubt of the principal fact. Not even the minimum quantity of nitrogen is formed during the alcoholic fermentation at the expense of the yeast. But these results show that there is a disappearance of ammonia from the original liquid. In order to study this phenomenon, I added ammonia from the original liquid. In this experiment I added 100 grams of sugar, 10 grams of washed yeast cake, and 0.200 grams of ammonium tartrate levorotatory, containing 0.0185 grams of ammonia. The fermentation lasted a long time. When the sugar was all gone, the liquid was assayed. It contained 0.0015 grams of ammonia.
I recovered unchanged all of the levorotatory tartaric acid free in the liquid. Therefore almost all of the ammonia added as tartrate had disappeared, as well as that which existed in the 10 grams of yeast....
In summary, we see that instead of the formation of ammonia during the alcoholic fermentation, that which is added disappears, especially in the case where there is insufficient albuminous [proteinaceous] material present because only a small amount of beer yeast was added. The studies in the following paragraphs still show us that the ammonia which disappears enters into the constitution of the yeast in the state of albuminous material.

III. Production of yeast in a medium composed of sugar, ammonium salt, and phosphates

The experiments which follow will show all the power of organisation of yeast and put to an end all discussions on its nature:
In a solution of pure candy sugar containing 10 grams, I put the ash of I gram of yeast, 0.100 grams of ammonium tartrate (dextrorotatory) and the amount of fresh beer yeast which would fit on the head of a pin, which had been washed and which contained 80% water. Very remarkably, the globules which were added under these conditions developed, multiplied and the sugar fermented, at the same time that the minerals slowly dissolved and the ammonia disappeared. In other words, the ammonia was transformed into albuminous materials of a complex nature which entered into the composition of the yeast, at the same time that the phosphates were taken into the new globules produced. The carbon of the yeast is evidently furnished by the sugar.
In a similar mixture, the vessel was filled up to the neck and was well stoppered, and a gas tube was immersed in the liquid. After 24 to 36 hours, the liquid began to show signs that a fermentation had begun by the production of microscopic bubbles the which indicated that the liquid was already saturated with carbon dioxide . . .
The following day the liquid became progressively agitated, and because of the production of gas, foam filled the neck of the flask. A deposit gradually covered the bottom of the vessel. A drop of the deposit was examined under the microscope and showed an extensive development of yeast. The yeast looked very young, with swollen globules which were transparent, with out granules, and amongst them could be distinguished the r very easily each globule of the small quantity of yeast which had been originally used as seed. These globules have a thick envelope and stand out in a black circle. They are yellowish and filled with granules. But the way in which they are many times surrounded by young globules, indicates quite clearly that they have produced these globules which form the head of the chain.
 It is in the first days after inoculation that it is possible to make these interesting observations. At night, using gaslight for illumination, it is possible to distinguish the old globules  from the many more young ones, in the same way that one can distinguish  a black ball amongst many white balls.
Gradually the differences disappear and the new globules that form lose all appearance that they are in chains. One no longer sees buds. The globules are now very granular in the manner of adult or spent beer yeast.
 Nevertheless, the fermentation using ammonium as a source of nitrogen never becomes as active as when an albuminous material is used as a nitrogen source, such as that from grapes, or beet juice, or the soluble part of ordinary beer yeast. If one seeds into sugar water containing a little albuminous material some fresh yeast, process proceeds in general exactly like that described above, but the fermentation is perceptibly more active. For example, instead of the first appearance of bubbles of carbon dioxide after 36 to 48 hours, they have already appeared after 12 to 24 hours. In addition, the amount of yeast formed and deposited in the sane time is greater. But I repeat, in all respects is the process the same, except that it is more vigorous, and the products formed are exactly the same . . . 
It can be stated with certainty that the ammonium salt is indispensable for the fermentation. When yeast is seeded into a sugar solution containing yeast ash but no ammonium salt, there is hardly any sign of fermentation. Occasionally there is a fraction of a cubic centimetre of gas, hut this may be due to the ammonium content of the distilled water or the small amount of albuminous material carried over with the inoculum.
The necessity of sugar as a source of carbon for the yeast globules has been sufficiently proven that it requires no further experiments. Therefore, all that is necessary to bring about the phenomenon of fermentation are these things: sugar, nitrogenous substance, minerals....

IV. Study on the relationship between the yeast and the sugar

We now arrive at a very delicate point in these researches. I would like to speak of the relationship which exists between the sugar and the yeast.
It will hardly be a question of the relationship between the atoms, but rather the more intimate relationships, the physiological connections.
I would like to indicate first several details concerning the structure of the beer yeast globules.
There can be no doubt that the globules form small vesicles with elastic walls, full of a liquid with which is associated a soft material which is more or less granular and vacuolar which is situated directly within the wall. This vesicle gradually reaches the centre as the globule ages.
The wall of the cell is elastic. Indeed, when a sample of water containing young globules is allowed to dry on a microscope slide resting on the microscope stage, the contraction of the sample which occurs as the result of the introduction of air presses the globules together and one can see them become deformed and become more or less polyhedric.
The contents of the globules, especially the central contents, are liquid. This is proven by the presence in most of the adult globules of one or more granules which exhibit movement similar to Brownian movement. It would be very difficult to say if it is true Brownian movement. The cause of Brownian movement, which is probably purely physical, is so little understood, that it cannot be said whether it can exert an effect on the free granules in the centre of the globule right through the wall of the yeast. The budding of the globules constitutes an important discovery of M. Cagniard de Latour. It can be represented, after M. Mitscherlich, as the change from Figure 3 to Figure 4, in which it can be seen that the new globule begins as a simple bulge. I have confirmed these observations of M. Mitscherlich. I have seen this process quite clearly many times. Soon the small bud, while remaining attached, appears to have its own wall, and is itself a true globule. The movements of the liquid only detach it when it has reached the size of the mother globule. Therefore its attachment is close and firm.
Is the bud borne, as many people have felt, by an effect of contact, of a pressure between the internal wall of one of the granules of the globule? I have seen nothing which might confirm this opinion, and I believe it to he inexact.
On one hand, the transparent globules, those without apparent granules, are always those globules which are budding, while on the other hand, the development of granules appears to occur only in older globules. It appears that the older the globule, the less active it is, and the less able to bud.
I do not believe the statement of M. Mitscherlich, already advanced by Cagniard de Latour and Turpin, that the globules of yeast are able to burst frequently and empty their granules which then spread through the liquid like seeds which then enlarge and become ordinary globules of yeast.
I can state that I have never observed this phenomenon in the course of three years of careful and frequent study on beer yeast, studies which were carried out under a wide variety of conditions of development. The fact that the volume of the globules of yeast during their action on sugar is quite uniform argues against this theory. Those globules which are smaller in size are not free, but attached to the larger globules in the form of buds. It is clear that if the yeast did reproduce by granules which u ere liberated into the medium by large globules, then one would find all sizes of globules amongst those which are free....
Sugar never undergoes alcoholic fermentation without the presence of living globules of yeast. Reciprocally, globules of yeast are never formed without the presence of sugar or a carbohydrate material or without the fermentation of this material. Any statements which are contrary to this principle have been derived from incomplete or inexact experiments.
All of the chemical work on alcoholic fermentation indicates that it can be accomplished through two separate circumstances, depending on whether the yeast is added to a solution of pure sugar, or whether the sugar solution is mixed with albuminous material. In the first case, it is said, the ferment acts, but it does not reproduce. In the second case, it acts, but it does reproduce. This second case is what occurs during the manufacture of beer.
M. Liebig has said: "If the fermentation is a consequence of the development and reproduction of the globules, this could not cause the fermentation in pure sugar solutions since this solution lacks the conditions necessary for the maintenance of vital activity. This solution does not contain the nitrogenous material necessary for the production of the nitrogen substances of the globules. In this case the globules cause the fermentation, not because they continue to develop, but because of the metamorphosis of their internal nitrogen which decomposes into ammonia and other products. That is to say, because of a chemical decomposition which is completely the opposite of an organic action."
The facts which I have reported are obviously in opposition to these views, and I am certain that whether the yeast is mixed in a pure sugar solution or in a sugar solution containing albuminous material, the phenomena are in many ways similar. In both cases the yeast is organised and multiplies. Only in the first case, when the fermentation is ended, all of the globules, young and old, are deprived of soluble nitrogenous material. The nitrogenous nutrients are fixed in an insoluble state in the new globules that have been formed. The aggregate of these globules does not therefore have an action on the pure sugar water. There are only enough nitrogenous nutrients for the globules which may still be young enough to act and to multiply. On the contrary-, in the case of fermentation in the presence of an albuminous material there are plenty of globules which are exhausted, but the majority of new globules are filled with nitrogenous material and minerals and with the aid of these nutrients are quite able to act when introduced into a new sugar solution....

V. In all alcoholic fermentations a portion of the sugar is fixed in the yeast as cellulose

I allowed to ferment 100 grams of sugar in 750 cubic centimetres of water with 2.626 grams (dry weight) of yeast. After 20 days, I recovered 2.96S grams (dry weight) of yeast. I boiled this with sulphuric acid (diluted 20 times) for 6 to 8 hours, and also a sample of yeast before fermentation. The weight of the fermented yeast was 1.707 grams (dry weight) and the weight of the unfermented yeast was 1.730 grams. The residue insoluble in sulphuric acid was collected on a tared filter and dried at 100°. The filtrate was saturated with barium carbonate to neutralise and then the amount of sugar was estimated both by Fehling's solution and by the amount of carbon dioxide released during fermentation. The values were calculated for the original weights of yeast (2.626 and 2.965 grams). It was found that 2.626 grams of yeast gave an insoluble nitrogen residue of 0.391 grams (14.8%) and a fermentable sugar value of 0.532 grams. The 2.965 gram sample gave an insoluble nitrogen residue of 0.634 grams (21.4¡/O) and 0.918 grams of fermentable sugar. These results show that ( I ) in the fermentation of 100 grams of sugar by 2.626 grams of yeast, it fixes into itself 0.4 grams of carbohydrate material transformable into fermentable sugar by sulphuric acid. (2) There is an increase in the amount of nitrogenous material insoluble in dilute sulphuric acid. This last result is a new proof that during fermentation, there is a fixation into an insoluble state of the albuminous materials of a soluble nature which are present within the active yeast globules.
It is still necessary to determine if the boiling with dilute sulphuric acid has dissolved all of the cellulose. I determined the amount of cellulose in the yeast by the method of M. Schlossberger.
These results indicate that boiling with dilute sulphuric acid has removed all of the cellulose. The amount of cellulose in the 2.626 grams of yeast corresponds to 0.532 grams or 20.2%. . . . The yeast collected after fermentation, 2.965 grams, had an amount of cellulose which corresponds to 0.91X grams of sugar, or 31 9%~ so that there was an increase of 11% in the amount of cellulose present in the yeast after fermentation.
This considerable increase in the weight of cellulose during the fermentation of sugar is another proof to add to all that I have presented, concerning the living state of the yeast during the alcoholic fermentation.

Comments by Thomas Brock
Pasteur's main contribution to the problem of alcoholic fermentation is the application of quantitative methods for determining what has happened to the various substances. By doing this, he was able to show that the yeast actually increased in weight, nitrogen, and carbon content during the fermentation process. This added considerable support to the argument that the yeast was really a living organism. Bur perhaps the most important contribution in this paper is that the yeast can actually increase extensively in weight and produce alcohol even in a liquid which lacks proteinaceous materials of a natural source. He obtained an active alcoholic fermentation in what we would today call a synthetic (or defined) medium, consisting merely of trace elements, ammonium salt and sugar. The problem became considerably clarified by this observation, since it could bc easily shown in such a defined medium that the fermentation always proceeded with the development of the yeast, and the increase in protein in the yeast was accompanied by a decrease in nitrogen of the medium. Pasteur's long and fairly precise paper, of which only a small portion is excerpted here, can be said to have ended the controversy regarding the nature of the alcoholic fermentation.