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ALCOHOLIC FERMENTATION
2ND EDITION, 1914.
By ARTHUR HARDEN.
MONOGRAPHS ON BIOCHEMISTRY
EDITED BY
R. H. A. PLIMMER, D.Sc.
AND
F. G. HOPKINS, M.A., M.B., D.Sc., F.R.S.
GENERAL PREFACE.
The subject of Physiological Chemistry, or Biochemistry, is enlarging
its borders to such an extent at the present time that no single
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deal with it as a whole, so as to give both a general and a detailed
account of its present position. It is, moreover, difficult, in the
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a science by means of new editions, and such volumes are therefore
issued when much of their contents has become obsolete.
For this reason, an attempt is being made to place this branch
of science in a more accessible position by issuing a series of
monographs upon the various chapters of the subject, each independent
of and yet dependent upon the others, so that from time to time, as
new material and the demand therefor necessitate, a new edition of
each monograph can be issued without reissuing the whole series. In
this way, both the expenses of publication and the expense to the
purchaser will be diminished, and by a moderate outlay it will be
possible to obtain a full account of any particular subject as nearly
current as possible.
The editors of these monographs have kept two objects in view:
firstly, that each author should be himself working at the subject
with which he deals; and, secondly, that a /Bibliography/, as complete
as possible, should be included, in order to avoid cross references,
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proceed from physiological chemistry to what may be now more properly
termed chemical physiology. This will depend upon the success which
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may be of interest at the time.
R. H. A. P.
F. G. H.
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LONGMANS, GREEN AND CO.,
LONDON, NEW YORK, BOMBAY, CALCUTTA, AND MADRAS.
ALCOHOLIC FERMENTATION
BY
ARTHUR HARDEN, PH.D., D.SC., F.R.S.
PROFESSOR OF BIOCHEMISTRY, LONDON UNIVERSITY
HEAD OF THE BIOCHEMICAL DEPARTMENT, LISTER INSTITUTE, CHELSEA
[Illustration: sailing ship in crown-like frame]
/SECOND EDITION/
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BOMBAY, CALCUTTA, AND MADRAS
1914
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 PAGE
I. HISTORICAL INTRODUCTION 1
II. ZYMASE AND ITS PROPERTIES 18
III. THE FUNCTION OF PHOSPHATES IN ALCOHOLIC FERMENTATION 41
IV. THE CO-ENZYME OF YEAST-JUICE 59
V. ACTION OF SOME INHIBITING AND ACCELERATING AGENTS ON
THE ENZYMES OF YEAST-JUICE 70
VI. CARBOXYLASE 81
VII. THE BY-PRODUCTS OF ALCOHOLIC FERMENTATION 85
VIII. THE CHEMICAL CHANGES INVOLVED IN FERMENTATION 96
IX. THE MECHANISM OF FERMENTATION 119
BIBLIOGRAPHY 136
INDEX 155
[p001]
CHAPTER I.
HISTORICAL INTRODUCTION.
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:--
Carbon. Hydrogen. Oxygen.
95·9 pounds of sugar (cane sugar) consist 26·8 7·7 61·4
of These yield:--
57·7 pounds of alcohol containing 16·7 9·6 31·4
35·3 " carbon dioxide containing 9·9 -- 25·4
2·5 " acetic acid containing 0·6 0·2 1·7
----- ---- -----
Total contained in products 27·2 9·8 58·5
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]. 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. [p018]