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.]
SECOND PART
CONCERNING THE TRANSFORMATLONS OF THE BEER YEAST
DURING ALCOHOLIC FERMENTATION
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. |