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Title: Alcoholic Fermentation

Author: Arthur Harden

Release date: February 23, 2014 [eBook #44985]

Language: English

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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.


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 R. H. A. P.
 F. G. H.




 MONOGRAPHS ON BIOCHEMISTRY

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 THE NATURE OF ENZYME ACTION. By W. M. BAYLISS,
 D.Sc., F.R.S. Third Edition. 5s. net.

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 F.R.C.S. 4s. net.

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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/

 LONGMANS, GREEN AND CO.
 39 PATERNOSTER ROW, LONDON
 FOURTH AVENUE & 30TH STREET, NEW YORK
 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]




 CHAPTER II.

 ZYMASE AND ITS PROPERTIES.


 DISCOVERY OF ZYMASE.

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

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

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

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

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

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

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

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

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

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

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

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

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


 THE PREPARATION OF YEAST-JUICE.

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

 [Illustration: FIG. 1. (Mortar and Pestle--Ed.)]

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

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

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

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

 [Illustration: FIG. 2. (Hydraulic Hand Press--Ed.)]

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

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

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

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

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


 EXTRACTION OF ZYMASE FROM UNGROUND YEAST.

 /1. Maceration of Dried Yeast./

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

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

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

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

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

/Method./--A suitable yeast is washed by decantation, filtered through
a cloth, lightly pressed by means of a hand press, and then passed
through a sieve of 5mm. mesh, spread out in a layer 1-1·5cm. thick
and left at 25°-35° for two days. Fifty grams of the dried yeast is
[Pg 026] thoroughly and carefully mixed with 150 c.c. of water in
a basin by means of a spatula and the whole digested for two hours
at 35°. The mass often froths considerably. It is then filtered
through ordinary folded filter paper, preferably in two portions,
and collected in a vessel cooled by ice. The separation may also be
effected by centrifuging or pressing out the mass, and the maceration
may be conveniently conducted in a flask immersed in the water of a
thermostat. It is not advisable to macerate more than 50 grams in one
operation. Under these conditions 25-30 c.c. of extract are obtained
after 20 minutes' filtration, 70-80 c.c. in twelve hours. Dried Munich
yeast can be bought from Messrs. Schroder of Munich and serves as a
convenient source of the extract.[1]

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

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


 /2. Other Methods./

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

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

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

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


 PRACTICAL METHODS FOR THE ESTIMATION OF THE FERMENTING POWER OF
 YEAST-JUICE.

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

 March 22, 1899, Berlin bottom yeast V. 20 c.c. juice + 8 grams cane
 sugar + 0·2 c.c. toluene as antiseptic at 16°. Grams of carbon dioxide
 after

 24     48     72     96 hours.
  0·40   0·64   0·99   1·11

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

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

 [Illustration: FIG. 3. (Schiff's Azotometer--Ed.)]

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

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

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


 THE ALCOHOLIC FERMENTATION OF THE SUGARS BY YEAST-JUICE.

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


 (/a/) /Relation to Fermentation by living Yeast./

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


 (/b/) /Relation of Alcohol to Carbon Dioxide./

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


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

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

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

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

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

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


 (/d/) /Fermentation of Different Carbohydrates. Autofermentation./

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

   Sugar.     Relative Rates.    Relative Totals.
  Glucose         1                  1
  Fructose        1·29               1·15
  Mannose         1·04               0·67

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

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

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

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

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

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

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


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

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

      Cane Sugar.     │       CO{2} in grams after
 ─────────┬───────────┼──────────┬──────────┬──────────
  Weight. │ Per cent. │ 6 hours. │24 hours. │ 96 hours.
 ─────────┼───────────┼──────────┼──────────┼──────────
  2·2     │ 10        │ 0·17     │ 0·50     │ 0·55
  3·52    │ 15        │ 0·14     │ 0·53     │ 0·64
  5       │ 20        │ 0·13     │ 0·54     │ 0·73
  6·66    │ 25        │ 0·13     │ 0·52     │ 0·80
  8·56    │ 30        │ 0·12     │ 0·46     │ 0·81
  10·76   │ 35        │ 0·12     │ 0·40     │ 0·82
  13·33   │ 40        │ 0·11     │ 0·36     │ 0·82
  10·76   │ 35        │ 0·12     │ 0·40     │ 0·82
  13·33   │ 40        │ 0·11     │ 0·36     │ 0·82

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

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


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

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

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

 ┌───────────────────────────────────────────────────────────────────┐
 │   Nature of   Per cent.   Volumes of    Volumes of      Total     │
 │   Juice       of Sugar      Sugar      Water Added.  Fermentation │
 │   (1,2,3)     Employed     Solution                     in g.     │
 │               by Weight     Added.                    of CO{2}.   │
 │ Bottom Yeast──────────────────────────────────────────────────────┤
 │      1           29            0            --          0·99      │
 │                                1            --          1·13      │
 │                                2            --          0·92      │
 │                                4            --          0·79      │
 │      2            9            0            --          0·43      │
 │                                1            --          0·60      │
 │                                2            --          0·53      │
 │                                4            --          0·41      │
 │      3            9            --           0           0·46      │
 │                                --           1           0·32      │
 │                                --           2           0·33      │
 │                                --           3           0·36      │
 │ Top Yeast─────────────────────────────────────────────────────────┤
 │      1            0            --           0           0·29      │
 │           (Autofermentation)   --           2           0·29      │
 │                                --           3           0·28      │
 │      2           29            0            --          0·31      │
 │                                1            --          0·34      │
 │                                2            --          0·31      │
 │                                4            --          0·35      │
 │                                6            --          0·28      │
 │      3            7·4          --           0           0·44      │
 │                                --           1           0·35      │
 │                                --           2           0·30      │
 │                                --           3           0·28      │
 └───────────────────────────────────────────────────────────────────┘

[p036]

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


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

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

 Substance.                              Effect on Total Fermentation.

 Concentrated solution of glycerol         Slight diminution
      "          "     "  sugar               "   increase
 Toluene (to saturation or excess)         Less than 10 per cent.
                                             diminution
 Chloroform  0·5 per cent.                 Slight increase
      "      0·8 per cent. (saturation)    No change
      "      Large excess (17 per cent.)   64 per cent. diminution
 Chloral hydrate 0·7 per cent.             Increase up to 27 per cent.
        "    3·5-5·4 per cent.             Completely destroyed
 Phenol          0·1 per cent.             No change
   "             0·5   "                   40 per cent. diminution
   "             1·2   "                   Completely destroyed
 Thymol          1     "                   Slight diminution
   "             5     "                   Marked     "
 Benzoic acid    0·1   "                    7  per cent. diminution
       "         0·25  "                   26     "         "
 Salicylic acid  0·1   "                   10     "         "
       "         0·27  "                   35     "         "
 Formaldehyde    0·12  "                   20     "         "
      "          0·24  "                   30-60  "         "
 Acetone         6     "                   20     "         "
    "           14     "                   80     "         "
 Alcohol         6     "                    0-20  "         "
    "           14     "                   75     "         "
 Sodium fluoride 0·5   "                   90     "         "
       "         2     "                   Almost completely destroyed
 Ammonium fluoride 0·55 per cent.          Completely destroyed
 Sodium azoimide, NaN{3},  0·36 per cent.  Slight diminution
        "            "     0·71  "         Marked     "
 Quinine hydrochloride        1   "        Slight increase
 Ozone 10·4-34·8 milligrams per 20 c.c.    Marked diminution
 Hydrocyanic acid 1·2 per cent.            Completely destroyed

[p037]

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

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


 PERMANENT PREPARATIONS CONTAINING ACTIVE ZYMASE.

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

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

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

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

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

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

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

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

 ───────────┬──────────────────────────────────────────
 Volumes of │           Total Gas Evolved in
 Sugar      │   1      2      3      4     22·5 hours.
 Solution   │
 ───────────┼──────────────────────────────────────────
  5 c.c.    │ 15·7   31·6   44·8   56·5   233·3
 10 "       │  2·2   10·5   23     31·8   202·3
 20 "       │  0·9    2·4   13·6   23·7   125·5
 40 "       │  1·4    1·7    2·3    2·9    56·3
 ───────────┴──────────────────────────────────────────

[p040]

This behaviour appears to be due to the removal of soluble matter
essential for fermentation from the cell, which is discussed later
on. It follows that when zymin is being tested for fermenting power,
a uniform method should be adopted, and all comparative tests should
be made with the same volumes of added sugar solution. Ground zymin
appears to begin to ferment somewhat more slowly than unground (2 grm.
to 12·4 c.c. of sugar solution in each case), but eventually produces
the same total volume of gas [Buchner and Antoni, 1905, 1]. [p041]




 CHAPTER III.

 THE FUNCTION OF PHOSPHATES IN ALCOHOLIC FERMENTATION.


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

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

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

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

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

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

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

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


 EFFECT OF THE ADDITION OF PHOSPHATE TO A FERMENTING MIXTURE OF
 YEAST-JUICE AND SUGAR.

When a suitable quantity[2] of a soluble phosphate is added to a
fermenting mixture of glucose, fructose, or mannose with yeast-juice,
the rate of fermentation rapidly rises, sometimes increasing as
much as twenty-fold, continues at this high value for a certain
period and then falls again to a value approximately equal to, but
generally [p043] somewhat higher than, that which it originally had.
Careful experiments have shown that during this period of enhanced
fermentation the amounts of carbon dioxide and alcohol produced exceed
those which would have been formed in the absence of added phosphate
by a quantity exactly equivalent to the phosphate added in the ratio
CO{2} or C{2}H{6}O:R′{2}HPO{4} [Harden and Young, 1906, 1].

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

This result is of fundamental importance, and the evidence on which it
rests deserves some consideration. Quantitative experiments on this
subject require certain preliminary precautions. The acid phosphates
are too acid to permit of any extended fermentation and the phosphates
of the formula R′{2}HPO{4} absorb a considerable volume of carbon
dioxide with production of a bicarbonate, according to the reaction:--

 R{2}HPO{4} + H{2}CO{3} ⇌ RHCO{3} + RH{2}PO{4}.

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

The following table, containing the results of experiments with
[Pg 044] glucose, fructose, and mannose, indicates very clearly the
nature of the method of calculation and also of the agreement between
observation and theory.

Three quantities of 25 c.c. of yeast-juice + 5 c.c. of a solution
containing 1 gram of the sugar to be examined (a large excess) were
incubated with toluene at 25° for one hour, in order to remove all
free phosphate, and to each were then added 5 c.c. of a solution of
sodium phosphate corresponding to 0·1632 gram of Mg{2}P{2}O{7} and
equivalent to 32·6 c.c. of carbon dioxide at N.T.P. The rates of
fermentation were then observed until they had passed through the
period of acceleration and had fallen and attained a steady value, the
gases being measured moist at 19·3° and 760·15 mm.

 ─────────────────────────────────────────────────┬─────────────────────
                                                  │ Glucose.
                                                  │      ┌──────────────
                                                  │      │ Mannose.
                                                  │      │    ┌─────────
                                                  │      │    │Fructose.
 ─────────────────────────────────────────────────┼──────┼────┴─────────
 Maximum rate attained, c.cs. per five minutes    │  9·6 │  7      11·3
 Final rate of fermentation                       │  1·1 │  0·96    1·08
 Total carbon dioxide produced by fermentation in │      │
    fifty-five minutes after addition of phosphate│ 49·7 │ 47·8    47·6
 Correction for evolution in absence of phosphate │      │
    in fifty-five minutes                         │ 12·1 │ 10·6    11·9
 Extra carbon dioxide equivalent to phosphate     │ 37·6 │ 37·2    35·7
   "     "       "        "       "    " at N.T.P.│ 34·4 │ 34      32·6
 ─────────────────────────────────────────────────┴──────┴──────────────

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

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

 [Illustration: FIG. 4 (Graph of Carbon Dioxide Evolution v Time--Ed.)]

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

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

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

 Maximum rate attained,
    c.c. per five minutes                   14·1

 Final rate of fermentation                  6·2

 Total evolved by fermentation in eighty
    minutes after addition of phosphate    131

 Correction for evolution in absence of
    phosphate in eighty minutes             99·2

 Extra carbon dioxide at 16° and 767·1 mm   31·8

   "     "       "     "   N.T.P            29·8


Considering the small proportional rise in rate and the long period of
accelerated fermentation, the agreement between the volume observed,
29·8 c.c., and that calculated from the phosphate, 32·2, is quite
satisfactory [Harden and Young, 1910, 1.] Precisely the same relations
hold for maceration extract, but in this case it must be remembered
that a large amount of free phosphate is present in the extract, as
much as 0·3129 grm. Mg{2}P{2}O{7} being obtained from 20 c.c. in one
preparation, so that the original extract had the concentration of a
0·14 molar solution of sodium phosphate. It is in fact not improbable
that the delay in the onset of fermentation sometimes observed with
maceration extract (see Lebedeff, 1912, 2; Neuberg and Rosenthal,
1913) may be due to the presence of phosphate in so great an excess
of the amount which can be rapidly esterified by the enzymes that the
rate of fermentation is at first greatly lowered (see p. 71). When
this phosphate is removed by incubation with glucose or fructose,
the subsequent addition of phosphate produces the characteristic
action and the extra carbon dioxide evolved is, as with other yeast
preparations, equivalent to the phosphate added. An actual estimation
carried out in this way gave 35 c.c. of CO{2} for an addition of
phosphate equivalent to 32·9 c.c. [Harden and Young, 1912]. [p047]

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

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

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

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


 NATURE OF THE PHOSPHO-ORGANIC COMPOUND FORMED BY YEAST-JUICE AND ZYMIN
 FROM THE HEXOSES AND PHOSPHATE.

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

Phosphates undergo this characteristic change when the sugar
undergoing fermentation is glucose, mannose, or fructose, and it
may be said at once that no distinction can be established between
the products formed from these various hexoses; they all appear
to be identical. The compound produced is, as already mentioned,
not precipitated by ammoniacal magnesium citrate mixture, nor by
uranium acetate solution. It can, however, be precipitated by copper
acetate (Iwanoff) and by lead acetate (Young). The preparation of the
pure lead salt from the liquid obtained by fermenting a sugar with
yeast-juice or zymin in presence of phosphate is commenced by boiling
and filtering the liquid. Magnesium nitrate solution and a small
quantity of caustic soda solution are then added to precipitate any
free phosphate, and the liquid well stirred and allowed to stand over
night. To the neutralised filtrate lead acetate is then added together
with sufficient caustic soda solution to maintain the reaction neutral
to litmus, until no further precipitate is formed. The liquid is then
filtered or, better, centrifugalised, and the precipitate repeatedly
washed with water until a portion of the clear filtrate gives no
reduction when boiled with Fehling's solution. It is essential that
this washing should be thorough as evidence has recently been obtained
of the formation under certain conditions of a hexosephosphate,
the lead salt of which is not so sparingly soluble as that of the
hexosediphosphate [Harden and Robison, 1914]. The lead precipitate
is then suspended in water, decomposed by a current of sulphuretted
hydrogen, the clear filtrate freed from sulphuretted hydrogen by a
current of air, and finally neutralised with caustic soda. The removal
of phosphate and conversion into lead salt are repeated twice, and the
resulting lead salt is then found to be free from nitrogen and to have
a composition represented by the formula C{6}H{10}O{4}(PO{4}Pb){2}.
Lebedeff carries out the preparation in a somewhat different manner.
The fermentation is effected by means of air-dried yeast (150 grams
to 1 litre of water, 210 grams cane-sugar and 105 grams of a mixture
of 2 parts Na{2}HPO{4} and 1 part NaH{2}PO{4}) and the liquid (about
700 c.c.) after boiling and filtering, is treated with an equal volume
of acetone. About 300 c.c. of a thick liquid is precipitated and
this is redissolved in water and precipitated by an equal volume of
acetone two or three times. The final liquid is then precipitated with
warm lead acetate solution and filtered and washed with dilute lead
acetate solution until the filtrate is clear and no longer reduces
Fehling's solution after removal of the lead [1910]. Euler and Fodor
[1911] on the other hand precipitate the free phosphate with magnesia
mixture and then add acetone, dissolve the syrup thus precipitated in
water and add copper [p049] acetate solution. A blue copper salt
is precipitated which is thoroughly washed with water and used for
the preparation of solutions of the acid. A solution of the free acid
can readily be prepared by the action of sulphuretted hydrogen on the
lead salt suspended in water. It forms a strongly acid liquid, which
requires exactly two equivalents of base for each atom of phosphorus
present to render it neutral to phenolphthalein. It decomposes when
evaporated, leaving a charred mass containing free phosphoric acid.
The acid is slightly optically active, and has [/a/{D}] = + 3·4°. A
number of amorphous salts have been prepared by precipitation from
a solution of the sodium salt, and of these the silver, barium, and
calcium salts have been analysed with results agreeing with the
general formula C{6}H{10}O{4}(PO{4}R′{2}){2}. The magnesium, calcium,
barium, and manganese salts, which are only sparingly soluble, are
all precipitated when their solutions are boiled but re-dissolve on
cooling, and this property can be utilised for their purification. The
alkali salts have only been obtained as viscid residues.

A difference of opinion exists as to the molecular weight and
constitution of this substance. Iwanoff [1909, 1] regards it as a
triosephosphoric acid, C{3}H{5}O{2}(PO{4}H{2}), basing this view
on the preparation of an osazone which melted at 142°, but when
recrystallised from benzene gave a product melting at 127°-8°,
which had the same appearance, melting-point, and nitrogen content
as the triosazone formed by the action of phenylhydrazine on the
oxidation products of glycerol. Neither Lebedeff [1909] nor Young
could obtain Iwanoff's osazone, and all attempts to reduce the acid
with formation of glycerol either by sodium amalgam or hydriodic acid
were unsuccessful (Young). There is therefore practically no serious
experimental evidence in favour of Iwanoff's view.

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

 C{6}H{5}NH·NH{2}·H{2}PO{4}·C{4}H{5}(OH){3}·
                                       C(N{2}HC{6}H{5})CH(N{2}HC{6}H{5})

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

 Na{2}PO{4}·C{4}H{5}(OH){3}·(CN{2}HC{6}H{5})·CH(N{2}HC{6}H{5})

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

 (C{6}H{5}NH·NH{2}·H{2}PO{4}){2}·C{6}H{7}(OH){3}·N{2}HC{6}H{5}.

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

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

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

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

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

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

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

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


 THE EQUATION OF ALCOHOLIC FERMENTATION.

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

 (1) 2C{6}H{12}O{6} + 2PO{4}HR{3} =
        2CO{2} + 2C{2}H{6}O + 2H{2}O + C{6}H{10}O{4}(PO{4}R{2}){2}.

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

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

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


 CYCLE OF CHANGES UNDERGONE BY PHOSPHATE IN ALCOHOLIC FERMENTATION.

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

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

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

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

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

This last reaction may be represented by the equation

 (2) C{6}H{10}O{4}(PO{4}R{2}){2} + 2H{2}O =
                                       C{6}H{12}O{6} + 2PO{4}HR{2}.

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

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

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

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

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

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

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

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

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

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

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


 EFFECT OF PHOSPHATE ON THE TOTAL FERMENTATION PRODUCED BY YEAST-JUICE.

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

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

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

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

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

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

 ───────────────────────────┬──────┬──────┬──────┬──────┬──────┬───────
                            │   1  │   2  │   3  │  4   │   5  │   6
 ───────────────────────────┼──────┼──────┼──────┼──────┼──────┼───────
                            │ c.c. │ c.c. │ c.c. │ c.c. │  c.c │  c.c.
                            │      │      │      │      │      │
  Gas evolved in absence of │      │      │      │      │      │
    added phosphate         │ 369  │ 220  │  1·4 │  1·2 │ 20·3 │   1·5
  In the presence of        │ 629  │ 629  │ 25·8 │ 26·8 │ 92·3 │ 132·7
 ───────────────────────────┼──────┼──────┼──────┼──────┼──────┼───────
  Increase due to phosphate │ 260  │ 409  │ 24·4 │ 25·6 │ 72·0 │ 131·2
  Carbonic acid equivalent  │      │      │      │      │      │
    to phosphate            │  63  │  61  │ 16·9 │ 16·8 │ 16·8 │  --
 ───────────────────────────┼──────┼──────┼──────┼──────┼──────┼───────
  Increase after initial    │      │      │      │      │      │
     period                 │ 197  │ 348  │  7·5 │  8·8 │ 55·2 │  --
 ───────────────────────────┼──────┼──────┼──────┼──────┼──────┼───────
  Ratio of totals           │ 1·7  │ 2·9  │ 18·4 │ 21·3 │  4·5 │  88
  Ratio of increase after   │      │      │      │      │      │
    initial period to       │      │      │      │      │      │
    original fermentation   │ 0·5  │ 1·6  │  5·3 │  7·3 │  2·7 │  --
 ───────────────────────────┴──────┴──────┴──────┴──────┴──────┴───────


 PRODUCTION OF A FERMENTABLE SUGAR FROM HEXOSEPHOSPHATE BY THE ACTION OF
 AN ENZYME CONTAINED IN YEAST-JUICE.

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

When, however, a solution of a hexosephosphate is exposed to the
action of either yeast-juice or zymin, entirely or partially freed
from co-enzyme, this sugar, being no longer fermented, accumulates and
can be examined. It has thus been found [Harden and Young, 1910, 2]
that a sugar is in fact produced in this way which can be fermented
by living yeast and exhibits the reactions of fructose, although the
presence of other hexoses is not excluded. The products of the enzymic
hydrolysis of the hexosephosphates therefore appear to be the same as,
or similar to, those formed by the action of acids [Young, 1909].

A further consequence of these facts is that a hexosephosphate will
yield carbon dioxide and alcohol when it is added to yeast-juice or
zymin, and this has also been found to be the case [Harden and Young,
1910, 2; Iwanoff, 1909, 1]. [p057]


 MECHANISM OF THE FORMATION OF HEXOSEDIPHOSPHORIC ACID.

On this subject little is yet known, but a number of extremely
interesting results, the interpretation of which is still doubtful,
have been obtained by Euler and his colleagues. Euler has obtained
a yeast [Yeast H of the St. Erik's brewery in Stockholm] which
differs from Munich yeast in several respects. A maceration extract
prepared from the yeast dried at 40° in a vacuum produces no effect
on a glucose solution containing phosphate. If, however, the glucose
solution be previously partially fermented with living yeast and
then boiled and filtered, the addition of the extract prepared from
Yeast H brings about the esterification of phosphoric acid without
any accompanying evolution of carbon dioxide [Euler and Ohlsén, 1911,
1912].

Euler interprets this as follows: (/a/) Glucose itself is not directly
esterified, but must first undergo some preliminary change, which is
brought about by the action of living yeast. No proof of the existence
of a new modification of glucose in this solution has however been
advanced, other than its behaviour to extract of Yeast H, so that
Euler's conclusion cannot be unreservedly accepted. It is moreover
possible and even more probable that some thermostable catalytic
substance (perhaps a co-enzyme) passes from the yeast into the
glucose solution and enables the yeast extract to attack the glucose
and phosphoric acid. A very small degree of esterification was also
produced when an extract having no action on glucose and phosphate
was added to glucose which had been treated with 2 per cent. caustic
soda for forty hours, but the nature of the compound formed was not
ascertained [Euler and Johansson, 1912, 4]. (/b/) The esterification
of phosphoric acid without the evolution of carbon dioxide implies
that the enzyme by which this process is effected is distinct from
that which causes the actual decomposition of the sugar. Euler goes
further than this and regards the enzyme as a purely synthetic one,
giving it the name of hexosephosphatese to distinguish it from the
hexosephosphatase which hydrolyses the hexosephosphate.

The evidence on which this conclusion is based cannot be regarded as
satisfactory, inasmuch as it consists in the observation that /in
presence of sugar/ yeast extract does not hydrolyse the phosphoric
ester. This, however, could not be expected since hydrolysis and
synthesis under these conditions would ultimately proceed at equal
rates.

In any case the adoption of this nomenclature is inconsistent with the
conception of an enzyme as a catalyst and is therefore inadvisable
until the reaction has been much more thoroughly studied. [p058]

It may further be pointed out that no proof has yet been advanced that
the phosphoric ester produced without evolution of carbon dioxide is
identical with hexosediphosphoric acid produced with evolution of
carbon dioxide. It is by no means improbable that it represents some
intermediate stage in the production of the latter (see p. 117).

Euler's other results on this subject may be briefly summarised as
follows:--

(1) In presence of excess of sugar the esterification of the
phosphoric acid proceeds by a monomolecular reaction and is most rapid
in faintly alkaline reaction [Euler and Kullberg, 1911, 3].

(2) When yeast extract has been heated for 30 minutes to 40° it
effects the esterification of phosphoric acid at a much greater
rate than the unheated extract (2-10 times). Heating at 50° for 30
minutes however completely inactivates the extract. The cause of the
activation is as yet unknown. The temperature coefficient for the
unheated extract (17·5°-30°) is 1·4-1·5 for 10° rise of temperature
[Euler and Ohlsén, 1911].

(3) Yeasts which in the dried state all produce rapid esterification
of phosphoric acid, yield extracts of very unequal powers in this
respect [Euler, 1912, 1]. [p059]




 CHAPTER IV.

 THE CO-ENZYME OF YEAST-JUICE.


In the previous chapter reference was made to the fact that the
addition of boiled yeast-juice greatly increases the amounts of carbon
dioxide and alcohol formed from sugar by the action of a given volume
of yeast-juice.

When the boiled juice is dialysed the substance or substances to which
this effect is due pass into the dialysate, the residue being quite
inactive. In order to ascertain the effect on the process of alcoholic
fermentation of the complete removal of these unknown substances
from yeast-juice itself, dialysis experiments were instituted with
fresh yeast-juice, capable of bringing about an active production of
carbon dioxide and alcohol from sugar. It was already known from the
experiments of Buchner and Rapp [1898, 1] that dialysis in parchment
paper for seventeen hours at 0° against water or physiological salt
solution only produced a diminution of about 20 per cent. in the total
amount of fermentation obtainable, and in view of the less permanent
character of the juice from top yeasts a more rapid method of dialysis
was sought. This was found in the process of filtration under pressure
through a film of gelatin, supported in the pores of a Chamberland
filter candle, which had been introduced by Martin [1896].

In this way it was found possible to divide the juice into a residue
and a filtrate, each of which was itself incapable of setting up the
alcoholic fermentation of glucose, whereas, when they were reunited,
the mixture produced almost as active a fermentation as the original
juice [Harden and Young, 1905, 1; 1906, 2].

The apparatus employed for this purpose consists of a brass tube
provided with a flange in which the gelatinised candle is held by
a compressed india-rubber ring, and is shown in section in Fig. 5.
Two such apparatus are used, each capable of holding about 70 c.c.
of the liquid to be filtered. The tubes, after being filled with the
yeast-juice, are connected by means of a screw joint with a cylinder
of compressed air and the filtration carried out under a pressure of
50 atmospheres, [p060] the arrangement employed being shown in Fig.
6. In the earlier experiments 25 to 50 c.c. of yeast-juice were placed
in each tube and the filtration continued until no more liquid passed
through. The residue was then washed several times /in situ/ by adding
water and forcing it through the candle. The time occupied in this
process varied from six to twelve hours with different preparations of
yeast-juice. The candle was then removed from the brass casing and the
thick, brown-coloured residue scraped off, dissolved in water, and at
once examined. It was subsequently found to be possible to dry this
residue /in vacuo/ over sulphuric acid without seriously altering the
fermenting power, and this led to a slight modification of the method,
which is now conducted as follows. Two quantities of 50 c.c. each of
yeast-juice are filtered, without washing, and the residues spread
on watch-glasses and dried /in vacuo/. Two fresh quantities of 50
c.c. are then filtered through the same candles and the residues also
dried. The 200 c.c. of juice treated in this way give a dry residue
of 17 to 24 grams. The residue is then dissolved in 100 c.c. of water
and filtered in quantities of 50 c.c. through two fresh gelatinised
candles and the residue again dried. A considerable diminution in
weight occurs, partly owing to incomplete removal from the candle
and brass casing, and the final residue only amounts to about 9 to
12 grams. Occasionally it is necessary to repeat the processes of
dissolving in water, filtering, and drying, but a considerable loss
both of material and fermenting power attends each such operation.

 [Illustration FIG. 5. (Filter Apparatus, Tube/Flange Detail--Ed.)]

The sticky residue dries up very rapidly /in vacuo/ to a brittle,
scaly mass, which is converted by grinding into a light yellow powder.

The filtrate was invariably found to be quite devoid of fermenting
power, none of the enzyme passing through the gelatin.

 [Illustration: FIG. 6. (Filter Apparatus--Ed.)]

/Properties of the Filtered and Washed Residue./--The residue
prepared as described above consists mainly of the protein, glycogen,
and dextrins of the yeast-juice, and is almost free from mineral
phosphates, but contains a certain amount of combined phosphorus. It
also contains the enzymes of the juice, including the proteoclastic
enzyme, and the hexosephosphatase (p. 54). Its solution in water is
usually quite inactive to glucose or fructose, but in some cases
produces a small and evanescent fermentation. When the original
filtrate or a corresponding [p061] quantity of the filtrate from
boiled fresh yeast-juice is added, the mixture ferments glucose or
fructose quite readily. The following table shows the quantitative
relations observed, the sugar being in all cases present in excess:--

 ──────────────────────┬────────┬──────────┬────────┬────────┬──────────
                       │        │ Filtrate │ Boiled │ Water  │  CO{2}
 No.    Material.      │ Volume.│  added.  │ Juice  │ added. │ evolved.
                       │        │          │ added. │        │
                       │        │   c.c.   │  c.c.  │  c.c.  │
 ──────────────────────┼────────┼──────────┼────────┼────────┼──────────
  1   Undried and      │        │          │        │        │
     unwashed residue  │ 15 c.c.│    0     │   0    │  15    │ 0     g.
                       │ 15 "   │   15     │   0    │   0    │ 0·035 "
  2    "        "      │ 15 "   │    0     │   0    │  15    │ 0·024 "
                       │ 15 "   │    0     │  15    │   0    │ 0·282 "
  3  Undried and washed│        │          │        │        │
      residue          │ 25 "   │    0     │   0    │   0    │  0·4 c.c.
                       │ 25 "   │    0     │  25    │   0    │268    "
  4     "       "      │ 20 "   │    0     │   0    │   0    │  8·3  "
                       │ 20 "   │   20     │   0    │   0    │ 90·3  "
  5   Washed and dried │        │          │        │        │
      residue          │ 1 gram │          │        │        │
                       │   in   │          │        │        │
                       │ 15 c.c.│    0     │   0    │   0    │  0    "
                       │    "   │    0     │  12    │   0    │108    "
  6       "   "        │ 1 gram │          │        │        │
                       │   in   │          │        │        │
                       │ 25 c.c.│    0     │   0    │   0    │  0    "
                       │    "   │    0     │  25    │   0    │364    "
 ──────────────────────┴────────┴──────────┴────────┴────────┴──────────

[p062]

These experiments lead to the conclusion that the fermentation of
glucose and fructose by yeast-juice is dependent upon the presence,
not only of the enzyme, but also of another substance which is
dialysable and thermostable.

Precisely similar results were subsequently obtained by Buchner and
Antoni [1905, 2] by the dialysis of yeast-juice. One hundred c.c. of
juice were dialysed for twenty-four hours at 0° against 1300 c.c. of
distilled water, and the dialysate was then evaporated at 40° to 50°
to 20 c.c. The fermenting power of 20 c.c. of the dialysed juice was
then determined with the following additions:--

 (1) 20 c.c. of dialysed juice + 10 c.c. of water gave
        0·02 gram CO{2}.
 (2) 20 c.c. of dialysed juice + 10 c.c. of evaporated dialysate gave
        0·52 gram CO{2}.
 (3) 20 c.c. of dialysed juice + 10 c.c. of boiled juice gave
        0·89 gram CO{2}.

It was shown in the previous chapter that phosphates are essential to
fermentation, and hence it becomes necessary to inquire whether the
effect of dialysis is simply to remove these. Experiment shows that
this is not the case. Soluble phosphates do not confer the power of
producing fermentation on the inactive residue obtained by filtration.
Moreover, when yeast-juice is digested for some time before being
boiled, it is found, as will be subsequently described, that the
boiled autolysed juice is quite incapable of setting up fermentation
in the inactive residue, although free phosphates are abundantly
present [Harden and Young, 1906, 2].

The filtration residue is never obtained quite free from combined
phosphorus, but the production from this of the phosphate necessary
for fermentation to proceed, may be so slow as to render the test for
co-enzyme uncertain, owing to the absence of sufficient phosphate.
When a filtration residue is being tested it is therefore necessary
to secure the presence of sufficient phosphate to enable the
characteristic reaction to proceed, and at the same time to avoid
adding phosphate in too great concentration, as this may, in the
presence of only small amounts of enzyme or co-enzyme, inhibit the
fermentation (p. 71). The proof that a filtration residue or dialysed
juice is quite free from co-enzyme is therefore a somewhat complicated
matter, and not only involves the experimental demonstration that
the material will not ferment sugar, but also that this power is not
imparted to it by the addition of a small concentration of phosphate.
As it has been found (p. 73) that the fermentation of fructose is
less affected than that of glucose by the presence of excess of
phosphate, the practical method of examining a filtration residue
for co-enzyme is to test its action on a solution of fructose (1)
alone and (2) in presence of a small concentration of phosphate. If
the residue produces no action [p063] in either case, but produces
fermentation when a solution of co-enzyme is added in the presence of
the same concentration of phosphate as was previously employed, it may
be concluded that this sample was free from co-enzyme but contained
enzyme; such an experiment also affords a definite proof that the
co-enzyme does not consist of phosphate.

This dialysable, thermostable substance, without which alcoholic
fermentation cannot proceed, has been provisionally termed the
co-ferment or co-enzyme of alcoholic fermentation. This expression
was first introduced by Bertrand [1897], to denote substances of
this kind, and he applied it in two instances--to the calcium salt
which he considered was necessary for the action of pectase on
pecten substances, and to the manganese which he supposed to be
essential for the activity of laccase. Without inquiring whether these
substances are precisely comparable in function with that contained
in yeast-juice, the term may be very well applied to signify the
substance of unknown constitution without the co-operation of which
the thermolabile enzyme of yeast-juice is unable to set up the process
of alcoholic fermentation. The active agent of yeast-juice consisting
of both enzyme and co-enzyme may be conveniently spoken of as the
fermenting complex, and this term will occasionally be employed in the
sequel.

The co-enzyme is present alike in the filtrates from fresh yeast-juice
and from boiled yeast-juice, and is also contained in the liquids
obtained by boiling yeast with water and by washing zymin or dried
yeast with water.

Practically the only chemical property of the co-enzyme, other than
that of rendering possible the process of alcoholic fermentation,
which has so far been observed, is that it is capable of being
decomposed, probably by hydrolysis, by a variety of reagents,
prominent among which is yeast-juice. This was observed by Harden and
Young in the course of their attempts to prepare a completely inactive
residue by filtration. In many cases a residue was obtained which
still possessed a very limited power of fermentation, only a small
amount of carbon dioxide being formed and the action ceasing entirely
after the expiration of a short period; on the subsequent addition
of boiled juice, however, a very considerable evolution of carbon
dioxide was produced. This was interpreted to mean that the residue
in question contained an ample supply of enzyme but only a small
proportion of co-enzyme, and that the latter was rapidly destroyed,
so that the fermentation soon ceased. The boiled juice then added
provided a further proportion of co-enzyme by the aid of which the
surplus enzyme was [p064] enabled to carry on the fermentation. This
view was confirmed by adding to a solution of a completely inactive
filtration residue and glucose successive small quantities of boiled
juice and observing the volumes of carbon dioxide evolved after each
such addition. Thus in one case successive additions of volumes of
3 c.c. of boiled juice produced evolutions of 8·2, 6, and 6 c.c. of
carbon dioxide. In another case two successive additions of 15 c.c.
of boiled juice produced evolutions of 54 and 41·2 c.c. On the other
hand, the enzyme itself also gradually disappears from yeast-juice
when the latter is incubated either alone or with sugar (p. 20).

The cessation of fermentation in any particular mixture of enzyme and
co-enzyme may, therefore, be due to the disappearance of either of
these factors from the liquid. If the amount of co-enzyme present be
relatively small it is the first to disappear, and fermentation can
then only be renewed by the addition of a further quantity, whilst the
addition of more enzyme produces no effect. If, on the other hand,
the amount of co-enzyme be relatively large, the inverse is true; the
enzyme is the first to disappear, and fermentation can only be renewed
by the addition of more enzyme, a further quantity of co-enzyme
producing no effect. It has, moreover, been found that the co-enzyme,
like the enzyme, disappears more rapidly in the absence of glucose
than in its presence, incubation at 25° for two days being as a rule
sufficient to remove all the co-enzyme from yeast-juice from top
yeasts in the absence of sugar, whilst in the presence of fermentable
sugar co-enzyme may still be detected at the end of four days.

In all the experiments carried out by Harden and Young with juice
from English top yeast it was found that when a mixture of the juice
with glucose was incubated until fermentation had ceased, the further
addition of co-enzyme in the form of boiled juice did not cause any
renewal of the action; in other words, the whole of the enzyme had
disappeared.

On the other hand, Buchner and Klatte [1908], working with juice and
zymin prepared from bottom yeast, observed the extremely interesting
fact that after the cessation of fermentation the addition of an equal
volume of boiled juice caused a renewed decomposition of sugar, and
that the processes of incubation until no further evolution of gas
occurred and re-excitation of fermentation by the boiled juice could
be repeated as many as six times. Thus in one experiment the duration
of the fermentation was extended from three to a total of twenty-four
days, and the total gas evolved from 0·73 gram to 2·19 grams. The
phenomenon has been found to be common to yeast from Munich and
[p065] from Berlin as well as to zymin and maceration extract, and
it was further observed that the boiled juice from one yeast could
regenerate the juice from another, although the quantitative relations
were different.

In these samples of yeast-juice, therefore, there is present a
natural condition of affairs precisely similar to that obtaining in
the artificial mixtures of inactive filtration residue and co-enzyme
solution made by Harden and Young. The balance of quantities is such
that the co-enzyme disappears before the enzyme, leaving a certain
amount of enzyme capable of exercising its usual function as soon as
sufficient co-enzyme is added. This establishes an interesting point
of contrast with the juice prepared from top yeast in England, in
which the enzyme does not outlast the co-enzyme [Harden and Young,
1907]. The difference may be due to some variation in the relative
proportions of enzyme and co-enzyme or of the enzymes to which
the disappearance of each of these is presumptively due, or to a
combination of these two causes. It was, however, found, even in the
juice from bottom yeast, that incubation for three days at 22° without
the addition of sugar caused the disappearance of the enzyme as well
as of the co-enzyme, and left a residue alike incapable of being
regenerated by the addition of co-enzyme or of restoring the power of
producing fermentation to an inactive mixture containing enzyme and
sugar.

If the fermenting power of the juice is to be preserved by repeated
regeneration for a long period, it is absolutely necessary to add the
co-enzyme solution each time as soon as fermentation has ceased, since
the enzyme in the absence of this addition rapidly disappears, even in
the presence of sugar.

This result is probably to be explained, at all events in the main, by
the presence in the co-enzyme solution of the antiprotease to which
reference has already been made [Buchner and Haehn, 1910, 2]. This
agent, the constitution of which is still unknown, protects proteins
in general from the action of digestive enzymes, and on the assumption
that the alcoholic enzyme of yeast-juice belongs to the class of
proteins, may be supposed to lessen the rate at which this enzyme is
destroyed by the endotryptase of the juice. This antiprotease is, like
the co-enzyme (p. 68), destroyed by lipase but is more stable than the
co-enzyme towards hydrolytic agents, and can be obtained free from
co-enzyme by boiling the solution for some hours alone or by heating
with dilute sulphuric acid. Such a solution possesses no regenerative
power, but still retains its power of protecting proteins against
digestion and of preserving the fermenting power of yeast-juice.
[p066]

It must, however, be remembered that the addition of a phosphate alone
may greatly prolong the period of fermentation of yeast-juice (p.
55), and sugar is well known to exert a similar action. It appears,
therefore, that the existence of the enzyme is prolonged not only
by the presence of the antiprotease but also by that of sugar and
hexosephosphate, into which phosphate passes in presence of sugar.
Similar effects are exerted on the co-enzyme by sugar and probably
also by hexosephosphate.

The fermenting complex, therefore, in the presence of these
substances, either separately or together, falls off more slowly in
activity and is present for a longer time, and for both of these
reasons produces an increased amount of fermentation. It seems
probable also that the hexosephosphatase is similarly affected,
so that the supply of free phosphate is at the same time better
maintained, and the rate of fermentation for this reason decreases
more slowly than would otherwise be the case.

It is in this way that an explanation may be found of the remarkable
increase in total fermentation, which is produced by the addition to
yeast-juice and sugar of boiled yeast-juice, containing free phosphate
(which passes into hexosephosphate) as well as co-enzyme, of boiled
autolysed yeast-juice, containing free phosphate but no co-enzyme, or
of phosphate solution alone.

In no case is the original rate of fermentation greatly increased
after the initial acceleration has disappeared, but in every case
the total fermentation is considerably augmented, and this is no
doubt mainly to be attributed, as just explained, to the diminished
rate of decomposition of the fermenting complex and probably of the
hexosephosphatase.

Although both enzyme and co-enzyme are completely precipitated from
yeast-juice, as already described (p. 38), by 10 volumes of acetone,
the co-enzyme is less easily precipitated than the enzyme, and a
certain degree of separation can therefore be attained by fractional
precipitation [Buchner and Duchaček, 1909]. The enzyme cannot,
however, be completely freed from co-enzyme in this manner, and the
process is attended by a very considerable loss of enzyme. This is
probably due to the fact that only small quantities of acetone can
be added (1·5 to 3 volumes), in order to avoid precipitation of
co-enzyme, and that the precipitates thus formed contain a large
proportion of water, a condition which appears to be fatal to the
preservation of the enzyme.

It is, however, not quite certain whether it is the zymase or the
hexosephosphatase which is destroyed in these cases, as no attempt
[Pg 067] was made to distinguish between them. In any case the
precipitates obtained by fractional treatment with acetone, even when
reunited, produce a much smaller fermentation than the original juice
or the powder prepared by bringing it into 10 volumes of acetone.

Attempts to isolate the co-enzyme from boiled yeast-juice have also
been hitherto unsuccessful. It has, however, been found possible to
remove a considerable amount of material from the solution without
affecting the co-enzyme. When 1 volume of alcohol is added to boiled
yeast-juice, a bulky precipitate, consisting largely of carbohydrates,
is produced, and the filtrate from this is found to contain the
co-enzyme and can be freed from alcohol by evaporation. Further
precipitation with alcohol has not led to useful results.

When a solution which has been treated in this way is precipitated
with lead acetate and kept neutral to litmus, the free phosphate
and hexosephosphate are thrown down and the co-enzyme remains in
solution. The filtrate can be freed from lead by means of sulphuretted
hydrogen and neutralised, and then forms a solution of co-enzyme free
from phosphate and hexosephosphate but still containing combined
phosphorus. More complete purification than this has not yet been
accomplished. Occasionally the precipitate of lead salts retains some
of the co-enzyme, apparently by adsorption, but usually the greater
part remains in the solution (Harden and Young).

The co-enzyme is partially removed from yeast-juice by means of a
colloidal solution of ferric hydroxide (Resenscheck). A precipitate
is thus obtained which contains phosphorus and resembles boiled
yeast-juice in its regenerative action on yeast-juice rendered
inactive by fermentation. It has not, however, so far been found
possible to isolate any definite compound from this precipitate. There
are also indications that when yeast-juice, either fresh or boiled,
is electrolysed, the co-enzyme tends to accumulate at the cathode
[Resenscheck, 1908, 1, 2].

Buchner and Klatte [1908] made use of yeast-juice rendered free from
co-enzyme by incubation with sugar solution to examine the nature
of the agent by which the co-enzyme is destroyed. This agent is
certainly an enzyme, since boiled yeast-juice can be preserved with
unimpaired powers for a considerable length of time, and suspicion
fell naturally, in the first instance, on the endotryptase of the
yeast cell. Direct experiment showed, however, that yeast-juice,
which, when fresh, rapidly destroyed the co-enzyme of boiled juice,
lost this power on preservation, but retained its proteoclastic
properties without diminution, so that the tryptic enzyme could not be
the one concerned. The direct action of commercial trypsin on boiled
yeast-juice also yielded [p068] a negative result, although this
cannot strictly be regarded as an indication of the effect of the
specific proteoclastic enzymes of yeast-juice. On the other hand, it
was found that when boiled juice was treated for some time with an
emulsion containing the lipase of castor oil seeds, the co-enzyme was
completely destroyed. This is a result of great importance, inasmuch
as it probably indicates that the co-enzyme is chemically allied to
the class of substances hydrolysable by lipase, i.e. to the fats and
other esters.

Further, observations by Buchner and Haehn [1909] have shown that
digestion with potassium carbonate solution containing 2·5 grams per
100 c.c. also brings about the destruction of the co-enzyme, and that
this is also slowly accomplished by the repeated boiling of the juice.
The co-enzyme is also destroyed both by acid and alkaline hydrolysis,
and when the solution is evaporated to dryness and the residue ignited.

Beyond this general indication nothing is known of the chemical
nature of the co-enzyme. The intimate relation of phosphoric acid
to the process of fermentation renders it not impossible that the
co-enzyme may contain this group, but there is no definite evidence
for such a belief. Purely negative results have been obtained
with all the substances of known composition which have yet been
tested, among these being soluble phosphates, hexosephosphates and
a number of oxidisable and reducible substances, such as quinol,
p-phenylenediamine, methylene blue, peptone beef broth, etc. (Harden
and Young; Harden and Norris [1914]; see also Euler and Bäckström
[1912]), glycero-phosphates (Buchner and Klatte).

The precise function of the co-enzyme is even more obscure than
its chemical nature. The system of reacting substances consisting
of fermentable material, enzyme and co-enzyme, bears, however, an
obvious superficial resemblance to many of the systems required for
the accomplishment of chemical changes in the animal or vegetable
organism. Such a triad of substances is, for example, requisite for
the process by which the red blood corpuscles of an animal are broken
up by the serum of a different animal into the blood of which the red
corpuscles of the first animal have been injected. This effect is only
produced when two substances are present, the amboceptor or immune
body and the complement. The analogy may be carried to a further
stage since the amboceptor is, like the co-enzyme, more thermostable
than the complement, which therefore corresponds with the enzyme.
Immune serum can, in fact, be freed from complement by being heated at
57-60° for half an hour, whereas the amboceptor is unaffected by this
treatment. On the other hand, the complement and amboceptor do not
[p069] appear to act like enzymes but rather like ordinary chemical
reagents, remaining in combination even after the blood corpuscle has
been broken up, whereas the enzyme and co-enzyme of yeast-juice are
again liberated when the reaction between sugar and phosphate has been
completed. [Pg 070]




 CHAPTER V.

 ACTION OF SOME INHIBITING AND ACCELERATING AGENTS ON THE ENZYMES OF
 YEAST-JUICE.


One of the most interesting and at the same time most difficult
problems concerning enzyme action in general is the nature of the
inhibiting or accelerating effect produced by many substances upon
the rate or total result of the chemical process set up in presence
of the enzyme. Inhibition, it is usually supposed, involves either
the decomposition of the enzyme, in which case it is irreversible,
its removal from the sphere of action by some change in its mode of
solution, or the formation of an inactive or less active compound
between the enzyme and the inhibiting agent. This compound it may
sometimes be possible to decompose, with the result that the activity
of the enzyme is restored. A striking example of this, to which
allusion has already been made, is the effect of hydrocyanic acid on
alcoholic fermentation (p. 37).

Acceleration of enzyme action can in some cases be ascribed to the
fact that the accelerating substance possesses an assignable chemical
function in the reaction, so that an increase in the concentration of
this substance causes an increase in the rate of the reaction. As we
have seen in Chapter III, this is the explanation of the accelerating
effect of phosphates on fermentation by yeast-juice. In many other
cases, however, no such chemical function can be traced, as, for
example, in the effect of neutral salts on the hydrolytic action of
invertase, or the effect of the addition of the co-enzyme to zymase,
and it is necessary to fall back on some assumption, such as that the
accelerating agent acts by increasing the effective concentration of
the enzyme or by combining either with the enzyme or the substrate,
forming a compound which undergoes the reaction more readily.

The interest in the following examples of inhibition and acceleration
of fermentation by yeast-juice lies not only in their relation to
these general problems but also, and perhaps chiefly, in their
bearing on the specific problem of the nature and mode of action of
the various agents concerned in the production of alcohol and carbon
dioxide from sugar in the yeast-cell. [p071]


 I. INFLUENCE OF CONCENTRATION OF PHOSPHATE ON THE COURSE OF
 FERMENTATION.

Prominent among these instances of inhibition and acceleration are
the phenomena attendant on the addition of excess of phosphate to
yeast-juice.

When a phosphate is added to a fermenting mixture of a sugar and
yeast-juice, the effect varies with the concentration of the phosphate
and the sugar and with the particular specimen of yeast-juice
employed. With low concentrations of phosphate in presence of excess
of glucose the acceleration produced is so transient that no accurate
measurements of rate can be made. As soon as the amount of phosphate
added is sufficiently large, it is found that the rate of evolution of
carbon dioxide very rapidly increases from five to ten times, and then
quickly falls approximately to its original value.

As the concentration of phosphate is still further increased, it is
first observed that the maximum velocity, which is still attained
almost immediately after the addition of the phosphate, is maintained
for a certain period before the fall commences, and then, as the
increase in concentration of phosphate proceeds, that the maximum is
only gradually attained after the addition, the period required for
this increasing with the concentration of the phosphate. Moreover,
with still higher concentrations, the maximum rate attained is less
than that reached with lower concentrations, and further, the rate
falls off more slowly. The concentration of phosphate which produces
the highest rate, which may be termed the optimum concentration,
varies very considerably with different specimens of yeast-juice
[Harden and Young, 1908, 1].

All these points are illustrated by the accompanying curves (Fig. 7)
which show the rate of evolution per five minutes plotted against
the time for four solutions in which the initial concentrations of
phosphate were (A) 0·033, (B) 0·067, (C) 0·1, and (D) 0·133 molar,
the volumes of 0·3 molar phosphate being 5, 10, 15, and 20 c.c. in
each case added to 25 c.c. of yeast-juice, and made up to 45 c.c, each
solution containing 4·5 grams of glucose. The time of addition is
taken as zero, the rate before addition being constant, as shown in
the curves.

It will be observed that 5 and 10 c.c. (A and B) give the same
maximum, whilst 15 c.c. (C) produce a much lower maximum, and 20 c.c.
(D) a still lower one, the rate at which the velocity diminishes
after the attainment of the maximum being correspondingly slow in
these last two cases. By calculating the amount of phosphate which
has disappeared as such from the amount of carbon dioxide evolved,
[p072] it is found that the maximum does not occur at the same
concentration of free phosphate in each case.

 [Illustration: FIG. 7. (Graph CO{2} rate vs time--Ed.)]

These results suggest that the phosphate is capable of forming two
or more different unstable associations with the fermenting complex.
One of these, formed with low concentrations of the phosphate, has
the composition most favourable for the decomposition of sugar,
whilst the others, formed with higher concentrations of phosphate,
contain more of the latter, probably associated in such a way with
the fermenting complex as to render the latter partially or wholly
incapable of effecting the decomposition of the sugar molecule. As the
fermentation proceeds slowly in the presence of excess of phosphate,
the concentration of the latter is reduced by conversion into
hexosephosphate, and a re-distribution of phosphate occurs, resulting
in the gradual change of the less active into the more active
association of phosphate with fermenting complex, and a consequent
rise in the rate of fermentation.

In those cases in which the maximum rate corresponding to the optimum
concentration of phosphate is never attained, some secondary cause may
be supposed to intervene, such as a permanent change in a portion of
the fermenting complex, accumulation of the products of the reaction,
etc.

It is also possible as suggested by Buchner for the analogous case of
arsenite (p. 78) that the addition of increasing amounts of phosphate
causes a progressive but reversible change in the mode of dispersion
[p073] of the colloidal enzyme, and that this has the secondary
effect of altering the rate of fermentation. No decisive evidence is
as yet available upon the subject.

The results obtained by Euler and Johansson [1913] to which reference
has already been made indicate that in presence of a moderate excess
of phosphate esterification is more rapid than production of carbon
dioxide. No explanation of this phenomenon has yet been given, but it
might obviously be due either to the production of some phosphorus
compound which subsequently takes part in the production both of
hexosediphosphate and of carbon dioxide, or, less probably, to the
entire independence of the two changes--esterification of phosphate
and production of carbon dioxide--which might then be differently
affected by the presence of excess of phosphate and therefore take
place at different rates.


 II. REACTION OF FRUCTOSE WITH PHOSPHATES IN PRESENCE OF YEAST-JUICE.

Although, as has been pointed out (p. 42), glucose, mannose, and
fructose all react with phosphate in a similar manner in presence of
yeast-juice, there are nevertheless certain quantitative differences
between the behaviour of glucose and mannose on the one hand, and
fructose on the other, which appear to be of considerable importance.
Fructose differs from the other two fermentable hexoses in two
particulars: (1) the optimum concentration of phosphate is much
greater; (2) the maximum rate of fermentation attainable is much
higher [Harden and Young, 1908, 2; 1909].

These points are clearly illustrated by the following results, which
all refer to 10 c.c. of yeast-juice, and show that the optimum
concentration of phosphate for the fermentation of fructose is
from 1·5 to 10 times that of glucose, and that the maximum rate of
fermentation for fructose in presence of phosphate is 2 to 6 times
that of glucose.

 ───────┬─────────┬──────────────────────┬──────────────────────
  Sugar │ Total   │   Optimum Volume of  │ Maximum Rate in Cubic
    in  │ Volume. │ 0·6 Molar Phosphate  │ Centimetres of CO{2}
  Grams.│         │        in c.c.       │   per Five Minutes
        │         │ Glucose. │ Fructose. │ Glucose. │ Fructose.
 ───────┼─────────┼──────────┼───────────┼──────────┼───────────
    2   │   35    │     2    │    5      │   7·5    │    32·2
    4       50          1        10          5·4         28·4
    1·6     23          2         5          8           17
    1       25          1·75      5          5·2         25·9
    2       25          5         7·5       16·2         31·2
    2       20          2         3·5        7·9         22·6
    2       22·5        0·75      2          3·4         22·2
 ───────────────────────────────────────────────────────────────

[p074]

It is interesting to note that the two high rates, 32·2 and 31·2 c.c.
per five minutes, are equal to about half the rate obtainable with
an amount of living yeast corresponding to 10 c.c. of yeast-juice,
assuming that about 16 to 20 grams of yeast are required to yield this
volume of juice, and that this amount of yeast would give about 56
to 70 c.c. of carbon dioxide per five minutes at 25°, which has been
found experimentally to be about the rate obtainable with the top
yeast employed for these experiments.


 III. EFFECT OF THE ADDITION OF FRUCTOSE ON THE FERMENTATION OF GLUCOSE
 OR MANNOSE IN PRESENCE OF A LARGE EXCESS OF PHOSPHATE.

When the maximum rate of fermentation of glucose or mannose by
yeast-juice in presence of phosphate is greatly lowered by the
addition of a large excess of phosphate, the addition of a relatively
small amount of fructose (as little as 2·5 per cent. of the weight of
the glucose) causes rapid fermentation to occur. This induced activity
is not due solely to the selective fermentation of the added fructose,
since the amount of gas evolved may be greatly in excess of that
obtainable from the quantity added.

Another way of expressing the same thing is to say that the optimum
concentration of phosphate (p. 71) is greatly raised when 2·5 per
cent. of fructose is added to glucose, and that consequently the
rate of fermentation rises. The effect is extremely striking, since
a mixture of glucose and yeast-juice fermenting in the presence of a
large excess of phosphate at the rate of less than 1 c.c. of carbon
dioxide in five minutes may be made to ferment at six to eight times
this rate by the addition of only 0·05 gram of fructose (2·5 per cent.
of the glucose present), and to continue until the total gas evolved
is at least five to six times as great as that obtainable from the
added fructose, the concentration of the phosphate being the whole
time at such a height as would limit the fermentation of glucose alone
to its original value.

The effect is not produced when the concentration of the phosphate is
so high that the rate of fermentation of fructose is itself greatly
lowered.

This remarkable inductive effect is specific to fructose and is not
produced when glucose is added to mannose or fructose, or by mannose
when added to glucose or fructose, under the proper conditions of
concentration of phosphate in each case.

This interesting property of fructose, taken in connection with the
[p075] facts that this sugar in presence of phosphate is much more
rapidly fermented than glucose or mannose, and that the optimum
concentration of phosphate for fructose is much higher than for
glucose or mannose, appears to indicate that fructose when added to
yeast-juice does not merely act as a substance to be fermented, but in
addition, bears some specific relation to the fermenting complex.

All the phenomena observed are, indeed, consistent with the
supposition that fructose actually forms a permanent part of the
fermenting complex, and that, when the concentration of this sugar in
the yeast-juice is increased, a greater quantity of the complex is
formed. As the result of this increase in the concentration of the
active catalytic agent, the yeast-juice would be capable of bringing
about the reaction with sugar in presence of phosphate at a higher
rate, and at the same time the optimum concentration of phosphate
would become greater, exactly as is observed. The question whether,
as suggested above, fructose actually forms part of the fermenting
complex, and the further questions, whether, if so, it is an essential
constituent, or whether it can be replaced by glucose or mannose with
formation of a less active complex, remain at present undecided, and
cannot profitably be more fully discussed until further information is
available.

It must, moreover, be remembered that different samples of yeast-juice
vary to a considerable extent in their relative behaviour to glucose
and fructose, so that the phenomena under discussion may be expected
to vary with the nature and past history of the yeast employed.


 IV. EFFECT OF ARSENATES ON THE FERMENTATION OF SUGARS BY YEAST-JUICE
 AND ZYMIN.

The close analogy which exists between the chemical functions of
phosphorus and arsenic lends some interest to the examination of the
action of sodium arsenate upon a mixture of yeast-juice and sugar, and
experiments reveal the fact that arsenates produce a very considerable
acceleration in the rate of fermentation of such a mixture [Harden
and Young, 1906, 3; 1911, 1]. The phenomena observed, however, differ
markedly from those which accompany the action of phosphate.

The acceleration produced is of the same order of magnitude as that
obtained with phosphate, but it is maintained without alteration
for a considerable period, so that there is no equivalence between
the amount of arsenate added and the extra amount of fermentation
effected. Further, no organic arsenic compound corresponding in
composition with the hexosephosphates appears to be formed.

Increase of concentration of arsenate produces a rapid inhibition of
[p076] fermentation, probably due to some secondary effect on the
fermenting complex, possibly to be interpreted as the formation of
compounds incapable of combining with sugar and hence unable to carry
on the process of fermentation. An optimum concentration of arsenate
therefore exists just as of phosphate, at which the maximum rate is
observed, and this optimum concentration and the corresponding rate
vary with different samples of juice and are less for glucose than
for fructose. The rate of fermentation by zymin is relatively less
increased than that by yeast-juice.

Owing to the fact that the rate is permanently maintained the addition
of a suitable amount of arsenate increases the total fermentation
produced to a much greater extent than phosphate.

The nature of these effects may be gathered from the result of a few
typical experiments. In one case the rate of fermentation of glucose
by yeast-juice was raised by the presence of 0·03 molar arsenate from
2 to 23 c.c. per five minutes, and the total evolved in ninety-five
minutes from 51 to 459 c.c. The accelerating effect on 20 c.c. of
juice, of as little as 0·005 c.c. of 0·3 molar arsenate, containing
0·11 mgrm. of arsenic, can be distinctly observed, but the maximum
effect is usually produced by about 1 to 3 c.c., the concentration
being therefore 0·015 to 0·045 molar. Greater concentrations than
this produce a less degree of acceleration accompanied by a shorter
duration of fermentation, as shown by the following numbers which
refer to 20 c.c. of yeast-juice in a total volume of 40 c.c.
containing 10 per cent. of glucose:--

 ─────────────┬──────────────────┬────────────────
   C.cs. of   │     Molar        │    Maximum
   0·3 Molar  │ Concentration of │    Rate of
   Arsenate   │    Arsenate.     │ Fermentation.
   in 40 c.c. │                  │
 ─────────────┴──────────────────┴────────────────
     0             0                    3·5
     0·005         0·0000375            6·3
     0·01          0·000075             8
     0·02          0·00015             14·2
     0·04          0·0003              19·9
     0·1           0·00075             29·7
     0·2           0·0015              35
 ─────────────────────────────────────────────────
     0·5           0·00375             34·9
     1·0           0·0075              29·5
     2·0           0·015               23·2
     5·0           0·0375              14·5
    10·0           0·075                8·7
    15·0           0·1125               5·3
    20             0·15                 3·2
 ─────────────────────────────────────────────────

The contrast between glucose and fructose in their relations to [p077]
arsenate are well exhibited in the following table, in which the
rates of fermentation produced by arsenate in presence of excess of
glucose and fructose respectively are given:--

 ────────────────────────────────────────────────────
                                       Rate.
   Concentration of Arsenate. ───────────────────────
                                Glucose.   Fructose.
 ────────────────────────────────────────────────────
   0·0075 molar                   12·1       26·6
   0·0225 (opt. for glucose)      13·4       --
   0·0525 (opt. for fructose)     --         45·8
   0·1125                         5·1        39
 ────────────────────────────────────────────────────

Here the optimum concentration for fructose is more than twice that
for glucose, whilst the maximum rate of fermentation obtainable with
fructose is between three and four times the maximum given by glucose.


 V. EFFECT OF ARSENITES ON THE FERMENTATION PRODUCED BY YEAST-JUICE.

Effects somewhat similar to those produced by arsenates were observed
by Buchner [Buchner and Rapp, 1897; 1898, 1, 2, 3; 1899, 2; Buchner,
E. and H., and Hahn, 1903, pp. 184-205] when potassium arsenite was
added to yeast-juice. This substance, the action of which on yeast had
been adduced by Schwann as a proof of the vegetable nature of this
organism, was employed by Buchner on account of its poisonous effect
on vegetable cells as an antiseptic and as a means of testing for the
protoplasmic nature of the agent present in yeast-juice. Its effect on
the fermentation was, however, found to be irregular, and at the same
time it did not act as an efficient antiseptic in the concentrations
which could be employed. Even 2 per cent. of arsenious oxide, added as
the potassium salt, had in many cases a decided effect in diminishing
the total fermentation obtained with cane sugar, and this effect
increased with the concentration. A number of irregularities were also
observed which cannot here be discussed. It was further found that in
some cases 2 per cent. of arsenious oxide inhibited the fermentation
of glucose but not of saccharose, or of a mixture of glucose and
fructose, whilst its effect on fructose alone was of an intermediate
character.

The important observation was also made by Buchner that the
addition of a suitable quantity of arsenite as a rule caused a
greatly increased fermentation during the first sixteen hours even
in experiments in which the total fermentation was diminished. By
examining the effect of arsenite on fermentation in a similar manner
to that of arsenate, Harden and Young [1911, 1] have found that a
close analogy exists [Pg 078] between the effects and modes of
action of these substances, but that arsenite produces a much smaller
acceleration than arsenate. An optimum concentration of arsenite
exists, just as in the case of arsenate, which produces a maximum
rate of fermentation. Further increase in concentration leads to
inhibition, and in no case is there any indication of the production
of an exactly equivalent amount of fermentation as in the case of
phosphate. In various experiments with dialysed, evaporated, and
diluted yeast-juice in which 2 per cent. of arsenious oxide was found
by Buchner to inhibit fermentation, it is probable that, owing to the
small amount of fermenting complex left, this amount of arsenious
oxide was considerably in excess of the optimum concentration,
although Buchner ascribes the effect to the removal of some of the
protective colloids of the juice, owing to the prolonged treatment to
which it had been subjected.

The extent of the action of arsenite appears from the following
results. In one case a rate of 1·7 c.c. was increased to 7 c.c. by
0·06 molar arsenite. In another experiment it was found that the
optimum concentration was 0·04 molar arsenite, the addition of which
increased the rate three-fold. As in the case of arsenate the optimum
concentration and the corresponding maximum rate of fermentation are
considerably greater for fructose than for glucose. The relative
rates produced by the addition of equivalent amounts of arsenate and
arsenite (1 c.c. of 0·3 molar solution in each case to 20 c.c. of
yeast-juice) were 27·5 and 3·1, the original rate of the juice being
1·7. In general the optimum concentration of arsenite is considerably
greater than that of arsenate.

The inhibiting effects of higher concentrations of arsenite and
arsenate also present close analogies, but this most interesting
aspect of the question has not yet been sufficiently examined to repay
detailed discussion. Buchner [Buchner, E. and H., and Hahn, 1903,
pp. 199-205] has suggested that the inhibition is due primarily to
some change in the colloidal condition of the enzyme and has shown
that certain colloidal substances appear to protect it, as does also
sugar. The possibility is also present that inactive combinations of
some sort are formed between the fermenting complex and the inhibiting
agent, in the manner suggested to account for the inhibiting effect of
excess of phosphate (p. 72). It seems most probable that the effect is
a complex one, in which many factors participate.


 NATURE OF THE ACCELERATION PRODUCED BY ARSENATE AND ARSENITE.

In explanation of the remarkable accelerating action of arsenates
and arsenites two obvious possibilities present themselves. In
the [p079] first place the arsenic compound may actually replace
phosphate in the reaction characteristic of alcoholic fermentation,
the resulting arsenic analogue of the hexosephosphate being so
unstable that it undergoes immediate hydrolysis, and is therefore
only present in extremely small concentration at any period of the
fermentation and cannot be isolated. In the second place it is
possible that the arsenic compound may accelerate the action of the
hexosephosphatase of the juice, and thus by increasing the rate of
circulation of the phosphate produce the permanent rise of rate. With
this effect may possibly be associated a direct acceleration of the
action of the fermenting complex.

The experimental decision between these alternative explanations is
rendered possible by the use of a mixture of enzyme and co-enzyme free
from phosphate and hexosephosphate. As has already been described (p.
55) a mixture of boiled yeast-juice, which has been treated with lead
acetate, glucose or fructose, and washed zymin can be prepared which
scarcely undergoes any fermentation unless phosphate be added. If now
arsenates or arsenites can replace phosphate, they should be capable
of setting up fermentation in such a mixture. Experiment shows that
they do not possess this power. For fermentation to proceed phosphate
must be present and it cannot be replaced either by arsenate or
arsenite [Harden and Young, 1911, 1].

The effect of these salts on the action of the hexosephosphatase can
also be ascertained by a modification of the foregoing experiment.
If a hexosephosphate be made the sole source of phosphate in such
a mixture as that described above, in which it must be remembered
abundance of sugar is present, the rate at which fermentation can
proceed will be controlled by the rate at which the hexosephosphate
is decomposed with formation of phosphate. Experiment shows that in
the presence of added arsenate or arsenite the rate of fermentation
is largely increased, so that the effect of these salts must be to
increase the rate of liberation of phosphate, or in other words, to
accelerate the hydrolytic action of the hexosephosphatase.

This conclusion is even more strikingly confirmed by a comparison
of the direct action of yeast-juice on hexosephosphate in presence
and in absence of arsenate, as measured by the actual production of
free phosphate. In a particular experiment this gave rise to 0·0707
gram of Mg{2}P{2}O{7} in the absence of arsenate and 0·6136 gram of
Mg{2}P{2}O{7} in the presence of arsenate.

The results obtained with arsenite are throughout very similar to
those given by arsenate, but are not quite so striking. It may
therefore be affirmed with some confidence that the chief action
of arsenates [p080] and arsenites in accelerating the rate of
fermentation of sugars by yeast-juice or zymin, consists in an
acceleration of the rate at which phosphate is produced from the
hexosephosphate by the action of the hexosephosphatase.

It has further been found that arsenates, and to a less
degree arsenites, also produce an acceleration of the rate of
autofermentation of yeast-juice and of the rate at which glycogen is
fermented. This turns out to be due in all probability to an increase
in the activity of the glycogenase by the action of which the sugar
is supplied which is the direct subject of fermentation. Thus in one
case an initial rate of fermentation of glycogen of 1·9 c.c. per five
minutes was increased by 0·05 molar arsenate to 9·7 and the amount
of carbon dioxide evolved in two hours from 38 to 158 c.c. Even this
enhanced production of glucose from glycogen, however, is not nearly
sufficient for the complete utilisation of the phosphate also being
liberated by the action on the hexosephosphatase, for the addition of
an excess of sugar produces a much higher rate, in this case 36 c.c.
per five minutes. The effect of arsenate on the rate of action of the
glycogenase seems therefore to be much smaller than on that of the
hexosephosphatase.

No other substances have yet been found which share these interesting
properties with arsenates and arsenites, and no advance has been made
towards an understanding of the mechanism of the accelerating action
of these salts on the specific enzymes which are affected by them.
[p081]




 CHAPTER VI.

 CARBOXYLASE.


An observation of remarkable interest, which promises to throw
light on several important features of the biochemistry of yeast,
was made in 1911, and has since then formed the subject of detailed
investigation by Neuberg and a number of co-workers.

It was found that yeast had the power of rapidly decomposing a
large number of hydroxy-and keto-acids [Neuberg and Hildesheimer,
1911; Neuberg and Tir, 1911; see also Karczag, 1912, 1, 2]. The
most important among these are pyruvic acid, CH{3}·CO·COOH, and
a considerable number of other aliphatic a-keto-acids which are
decomposed with evolution of carbon dioxide and formation of the
corresponding aldehyde:--

 R·CO·COOH = R·CHO + CO{2}.

The reaction is produced by all races of brewer's yeast which have
been tried, as well as by active yeast preparations and extracts and
by wine yeasts [Neuberg and Karczag, 1911, 4; Neuberg and Kerb, 1912,
2]. The phenomenon can readily be exhibited as a lecture experiment by
shaking up 2 g. of pressed yeast with 12 c.c. of 1 per cent. pyruvic
acid, placing the mixture in a Schrötter's fermentation tube, closing
the open limb by means of a rubber stopper carrying a long glass
tube and plunging the whole in water of 38-40°. Comparison tubes of
yeast and water and yeast and 1 per cent. glucose may be started at
the same time, and it is then seen that glucose and pyruvic acid are
fermented at approximately the same rate [Neuberg and Karczag, 1911,
1]. If English top yeast be used it is well to take 0·5 per cent.
pyruvic acid solution and to saturate the liquids with carbon dioxide
before commencing the experiment. The production of acetaldehyde can
be readily demonstrated by distilling the mixture at the close of
fermentation and testing for the aldehyde either by Rimini's reaction
(a blue coloration with diethylamine and sodium nitroprusside) or by
means of p-nitrophenylhydrazine which precipitates the hydrazone,
melting at 128·5° [Neuberg and Karczag, 1911, 2, 3]. [p082]

As the result of quantitative experiments it has been shown that 80
per cent. of the theoretical amount of acetaldehyde can be recovered.
The salts of the acids are also attacked, the carbonate of the metal,
which may be strongly alkaline, being formed. Thus taking the case
of pyruvic acid, the salts are decomposed according to the following
equation:--

 2CH{3}·CO·COOK + H{2}O = 2CH{3}·CHO + K{2}CO{3} + CO{2}.

Under these conditions a considerable portion of the aldehyde
undergoes condensation to aldol [Neuberg, 1912]:--

 2CH{3}·CHO = CH{3}·CH(OH)·CH{2}·CHO.

This change appears to be due entirely to the alkali and not to an
enzyme since the aldol obtained yields inactive β-hydroxybutyric
acid on oxidation [Neuberg and Karczag, 1911, 3; Neuberg, 1912]. The
various preparations derived from yeast which are capable of producing
alcoholic fermentation also effect the decomposition of pyruvic acid
in the same manner as living yeast. They are, however, more sensitive
to the acidity of the pyruvic acid, and it is therefore advisable to
employ a salt of the acid in presence of excess of a weak acid, such
as boric or arsenious acid, which decomposes the carbonate formed but
has no inhibiting action on the enzyme [Harden, 1913; Neuberg and
Rosenthal, 1913].

As already mentioned the action is exerted on α-ketonic acids as
a class and proceeds with great readiness with oxalacetic acid,
COOH·CH{2}·CO·COOH, all the three forms of which are decomposed, with
α-ketoglutaric acid, and with α-ketobutyric acid. Hydroxypyruvic
acid CH{2}(OH)·CO·COOH is slowly decomposed yielding glycolaldehyde,
CH{2}(OH)·CHO, and this condenses to a sugar [Neuberg and Kerb,
1912, 3; 1913, 1]. Positive results have also been obtained with
diketobutyric, phenylpyruvic, p-hydroxyphenylpyruvic, phenylglyoxylic
and acetonedicarboxylic acids [Neuberg and Karczag, 1911, 5].


 RELATION OF CARBOXYLASE TO ALCOHOLIC FERMENTATION.

With regard to the relation of carboxylase to the process of alcoholic
fermentation, nothing definite is yet known. As Neuberg points out
[see Neuberg and Kerb, 1913, 1] the universal presence of the enzyme
in yeasts capable of producing alcoholic fermentation, and the
extreme readiness with which the fermentation of pyruvic acid takes
place create a [p083] strong presumption that the decomposition of
pyruvic acid actually forms a stage in the process of the alcoholic
fermentation of the sugars. On the other hand Ehrlich's alcoholic
fermentation of the amino-acids (p. 87) provides another function
for carboxylase--that of decomposing the α-ketonic acids produced
by the deaminisation of the amino-acids. It must be remembered in
this connection that carboxylase is not specific in its action, but
catalyses the decomposition not only of pyruvic acid but also of
a large number of other α-ketonic acids, including many of those
which correspond to the amino-acids of proteins and are doubtless
formed in the characteristic decomposition of these amino-acids by
yeast. Carboxylase undoubtedly effects one stage in the production of
alcohols from amino-acids, whether it is also the agent by which one
stage in the alcoholic fermentation of sugar is brought about still
remains to be proved.

A comparison of the conditions of action of carboxylase and zymase
has revealed several interesting points of difference. Neuberg and
Rosenthal [1913] have observed that the fermentation of pyruvic
acid by maceration extract commences much more rapidly than that
of glucose and interpret this to mean that in the fermentation of
glucose a long preliminary process occurs before sufficient pyruvic
acid has been produced to yield a perceptible amount of carbon
dioxide. The long delay (3 hours) which they sometimes observed in
the action of maceration juice on glucose is however by no means
invariable (see p. 46), but in any case indicates that the sugar
fermentation can be affected by conditions which are without influence
on the pyruvic fermentation. A similar conclusion is to be drawn
from the fact that the pyruvic acid fermentation is less affected
by antiseptics than the glucose fermentation [Neuberg and Karczag,
1911, 4; Neuberg and Rosenthal, 1913], chloroform sufficient to stop
the glucose fermentation brought about by yeast or dried yeast being
usually without effect on the fermentation of the pyruvates either
alone or in presence of boric or arsenious acid. A more important
difference is that carboxylase decomposes pyruvic acid in the absence
of the co-enzyme which is necessary for the fermentation of glucose
[Harden, 1913; Neuberg and Rosenthal, 1913]. This can be demonstrated
experimentally by washing dried yeast or zymin with water (see p.
63) until it is no longer capable of decomposing glucose (Harden),
or by allowing maceration extract to autolyse or dialyse until it is
free from co-enzyme (Neuberg and Rosenthal). The zymase of maceration
extract is moreover inactivated in 10 minutes at 50-51°, whereas after
this treatment the carboxylase is still active. [p084]

The only conclusion that can be legitimately drawn from these highly
interesting facts is that if the decomposition of pyruvic acid
actually be a stage in the alcoholic fermentation of glucose the
soluble co-enzyme is required for some change precedent to this,
so that in its absence the production of pyruvic acid cannot be
effected. [p085]




 CHAPTER VII.

 THE BY-PRODUCTS OF ALCOHOLIC FERMENTATION.


When pure yeast is allowed to develop in a solution of sugar
containing a suitable nitrogenous diet and the proper mineral
salts, the liquid at the close of the fermentation contains not
only alcohol and some carbon dioxide but also a considerable number
of other substances, some arising from the carbonaceous and others
from the nitrogenous metabolism of the cell. Prominent among the
non-nitrogenous substances which are thus found in fermented sugar
solutions are fusel oil, succinic acid, glycerol, acetic acid,
aldehyde, formic acid, esters, and traces of many other aldehydes and
acids. In addition to these substances which are found in the liquid,
there are also the carbonaceous constituents of the newly formed cells
of the organism, comprising the material of the cell walls, yeast gum,
glycogen, complex organic phosphates, as well as other substances.

The attention of chemists has been directed to these compounds since
Pasteur first emphasised their importance as essential products of
the alcoholic fermentation of sugar, and his example was generally
followed in attributing their origin to the sugar.

The study of cell-free fermentation by means of yeast-juice or zymin
has, however, revealed the facts that certain of these substances are
not formed in the absence of living cells, and that their origin is to
be sought in the metabolic processes which accompany the life of the
cell. Their source, moreover, has been traced not to the sugar but to
the amino-acids, formed by the hydrolysis of the proteins, which occur
in all such liquids as beer wort, grape juice, etc., which are usually
submitted to alcoholic fermentation. This has so far been proved with
certainty for the fusel oil and succinic acid, and rendered highly
probable for all the various aldehydes and acids of which traces have
been detected.


 FUSEL OIL.

All forms of alcohol prepared by fermentation contain a fraction
of high boiling-point, which is termed fusel oil, and amounts to
about [p086] 0·1 to 0·7 per cent. of the crude spirit obtained
by distillation. This material is not an individual substance, but
consists of a mixture of very varied compounds, all occurring in small
amount relatively to the ethyl alcohol from which they have been
separated. The chief constituents of the mixture are the two amyl
alcohols, isoamyl alcohol,

 (CH{3}){2}·CH·CH{2}·CH{2}·OH,

and /d/-amyl alcohol,

 CH{3}·CH(C{2}H{5})·CH{2}·OH,

which contains an asymmetric carbon atom and is optically active. In
addition to these, much smaller amounts of propyl alcohol and isobutyl
alcohol are present, together with traces of fatty acids, aldehydes,
and other substances.

The origin of these purely non-nitrogenous compounds was usually
sought in the sugar of the liquid fermented, from which they were
thought to be formed by the yeast itself or by the agency of bacteria
[Emmerling, 1904, 1905; Pringsheim, 1905, 1907, 1908, 1909], whilst
others traced their formation to the direct reduction of fatty acids.
Felix Ehrlich has, however, conclusively shown in a series of masterly
researches that the alcohols, and probably also the aldehydes,
contained in fusel oil are in reality derived from the amino-acids
which are formed by the hydrolysis of the proteins.

The close relationship between the composition of leucine,

 (CH{3}){2}·CH·CH{2}·CH(NH{2})·COOH,

and isoamyl alcohol,

 (CH{3}){2}·CH·CH{2}·CH{2}·OH,

had previously led to the surmise that a genetic relation might exist
between these substances, but the idea had not been experimentally
confirmed. In 1903 Ehrlich discovered [1903; 1904, 1, 2; 1907, 2;
1908; Ehrlich and Wendel, 1908, 2] that proteins also yield on
hydrolysis an isomeride of leucine known as isoleucine, which has the
constitution

 CH{3}·CH(C{2}H{5})·CH(NH{2})·COOH,

and therefore stands to /d/-amyl alcohol,

 CH{3}·CH(C{2}H{5})·CH{2}·OH,

in precisely the same relation as leucine to isoamyl alcohol. This
suggestive fact at once directed his attention to the problem of the
origin of the amyl alcohols in alcoholic fermentation. Using a pure
culture of yeast, and thus excluding the participation of bacteria in
the change, he found that leucine readily yielded isoamyl alcohol,
and isoleucine /d/-amyl alcohol when these amino-acids were added in
the pure state [p087] to a solution of sugar and treated with a
considerable proportion of yeast [1905; 1906, 2, 3; 1907, 1, 3]. The
chemical reactions involved are simple ones and are represented by the
following equations:--

 (1) (CH{3}){2}·CH·CH{2}·CH(NH{2})·COOH + H{2}O
             Leucine
                         = (CH{3}){2}CH·CH{2}CH{2}·OH + CO{2} + NH{3}
                                  Isoamyl alcohol

 (2) CH{3}·CH(C{2}H{5})·CH(NH{2})·COOH + H{2}O
            Isoleucine
                        = CH{3}·CH(C{2}H{5})·CH{2}·OH + CO{2} + NH{3}
                               /d/-Amyl alcohol

The experiments by which these important changes were demonstrated
were of a very simple and convincing character [Ehrlich, 1907, 1]. Two
hundred grams of sugar and 3 to 10 grams of the nitrogenous substance
to be examined were dissolved in 2 to 2·5 litres of tap water in
a 3 to 4 litre flask, the liquid was sterilised by being boiled
for several hours, and after cooling 40 to 60 grams of fresh yeast
were added and the flask allowed to stand at room temperature until
the whole of the sugar had been decomposed by fermentation. In the
earlier experiments the amyl alcohols were isolated and identified by
conversion into the corresponding valerianic acids, but as a rule the
fusel oil as a whole was quantitatively estimated in the filtrate by
the Röse-Herzfeld method [Lunge, 1905, p. 571].

The following are typical results. (1) An experiment carried out as
above without any addition of leucine gave 97·32 grams of alcohol
containing 0·40 per cent. of fusel oil. (2) When 6 grams of synthetic,
optically inactive leucine were added, 97·26 grams of alcohol were
obtained, containing 2·11 per cent. of fusel oil, which was also
optically inactive; 2·5 grams of leucine were recovered, so that 87
per cent. of the theoretical yield of isoamyl alcohol was obtained
from the 3·5 grams of leucine decomposed. (3) In the presence of 2·5
grams of /d/-isoleucine (prepared from molasses residues), 200 grams
of sugar gave 93·99 grams of alcohol, containing 1·44 per cent. of
fusel oil, which was lævo-rotatory. This corresponds with 80 per cent.
of the theoretical yield of /d/-amyl alcohol from the isoleucine added.

This change, which Ehrlich has termed the alcoholic fermentation
of the amino-acids, although brought about by living yeast,
does not appear to occur at all when zymin [Ehrlich, 1906, 4;
Pringsheim, 1906] or yeast-juice [Buchner and Meisenheimer, 1906]
is substituted for the intact organism, nor is it effected even
by living yeast in the absence of a fermentable sugar [Ehrlich,
1907, 1]. The reaction appears indeed to be intimately connected
with the nitrogenous metabolism of the cell, and the whole of the
ammonia produced is at once assimilated and does not appear in the
fermented liquid. Other amino-acids [p088] undergo a corresponding
change, and the reaction appears to be a general one. Thus tyrosine,
OH·C{6}H{4}·CH{2}·CH(NH{2})·COOH, yields p-hydroxyphenylethyl alcohol,
or tyrosol [Ehrlich, 1911, 1; Ehrlich and Pistschimucka, 1912, 2],
OH·C{6}H{4}·CH{2}·CH{2}OH, a substance of intensely bitter taste,
which was first prepared in this way and is probably one of the
most important factors in determining the flavour of beers, etc.
Phenylalanine, C{6}H{5}·CH{2}·CH(NH{2})·COOH, in a similar way yields
phenylethyl alcohol, C{6}H{5}·CH{2}·CH{2}OH, one of the constituents
of oil of roses, whilst tryptophane,

     C{6}H{4}
    ╱    ╲
   ╱      ╲
 HN       C·CH{2}·CH(NH{2})·COOH,
   ╲    ╱╱
    ╲  ╱╱
     CH

yields tryptophol,

     C{6}H{4}
    ╱    ╲
   ╱      ╲
 HN       C·CH{2}·CH{2}OH,
   ╲    ╱╱
    ╲  ╱╱
     CH

which was also first prepared in this way [Ehrlich, 1912] and has a
very faintly bitter, somewhat biting taste.

The extent to which the amino-acids of a medium in which yeast is
producing fermentation are decomposed in this sense depends on the
amount of the available nitrogen and on the form in which it is
present. Thus the addition of ammonium carbonate to a mixture of yeast
and sugar was found to lower the production of fusel oil from 0·7
to 0·33 per cent. of the alcohol produced. The addition of leucine
alone raised the percentage from 0·7 to 2·78, but the addition of
both leucine and ammonium carbonate resulted in the formation of only
0·78 per cent. of fusel oil, The production of fusel oil therefore
and the character of the constituents of the fusel oil alike depend
on the composition of the medium in which fermentation occurs. This
affords a ready explanation of the fact that molasses, which contains
almost equal amounts of leucine and isoleucine, yields a fusel oil
also containing approximately equal amounts of isoamyl alcohol and
/d/-amyl alcohol [Marckwald, 1902], whilst corn and potatoes, in which
leucine preponderates over isoleucine, yield fusel oils containing a
relatively large amount of the inactive alcohol. The subject is, in
fact, one of great interest to the technologist, for as Ehrlich points
out "the great variety of the bouquets of wine and aromas of brandy,
cognac, arrak, rum, etc., may be very simply referred to the manifold
variety of the proteins of the raw materials (grapes, corn, rice,
sugar cane, etc.) from which they are derived".

Yeast can also form fusel oil at the expense of its own protein, but
this only occurs to any considerable extent when the external [p089]
supply of nitrogen is insufficient. Under these circumstances the
amino-acids formed by autolysis may be decomposed and their nitrogen
employed over again for the construction of the protein of the cell.

The yield is also influenced by the condition of the yeast employed
with regard to nitrogen, a yeast poor in nitrogen being more
efficacious in decomposing amino-acids than one which is already well
supplied with nitrogenous materials. The nature of the carbonaceous
nutriment and finally the species of yeast are also of great
importance [see Ehrlich, 1911, 2; Ehrlich and Jacobsen, 1911].

A very important characteristic of the action of yeast on the
amino-acids is that the two stereo-isomerides of these optically
active compounds are fermented at different rates. When inactive,
racemic leucine is treated with yeast and sugar, the naturally
occurring component, the /l/-leucine, is more rapidly attacked, so
that if the experiment be interrupted at the proper moment the other
component, the /d/-leucine, alone is present and may be isolated in
the pure state. In an actual experiment 3·8 grams of this component
were obtained in the pure state from 10 grams of /dl/-leucine
[Ehrlich, 1906, 1], so that the whole of the /l/-leucine (5 grams)
had been decomposed but only 1·2 grams of the /d/-leucine. This
mode of action has been found to be characteristic of the alcoholic
fermentation of the amino-acids by yeast. In all the instances
so far observed, both components of the inactive amino-acid are
attacked, but usually the naturally occurring isomeride is the more
rapidly decomposed, although in the case of β-aminobutyric acid both
components disappear at the same rate [Ehrlich and Wendel, 1908, 1].
This reaction therefore must be classed along with the action of
moulds on hydroxy-acids [McKenzie and Harden, 1903], and the action
of lipase on inactive esters [Dakin, 1903, 1905], in which both
isomerides are attacked but at unequal rates, and differs sharply from
the action of yeast itself on sugars [Fischer and Thierfelder, 1894],
and of emulsin, maltase, etc., which only act on one isomeride and
leave the other entirely untouched [see Bayliss, 1914, pp. 55, 77,
117].


 SUCCINIC ACID.

The origin of the succinic acid formed in fermentation has also
been traced by Ehrlich [1909] to the alcoholic fermentation of the
amino-acids. It was shown by Buchner and by Kunz [1906] that succinic
acid like fusel oil is not formed during fermentation by yeast-juice
or zymin, and, in the light of Ehrlich's work on fusel oil, several
[p090] modes of formation appeared possible for this substance
[Ehrlich, 1906, 3]. The dibasic amino-acids might, for example,
undergo simple reduction, the NH{2} group being removed as ammonia
and replaced by hydrogen. Aspartic acid would thus pass into succinic
acid:--

 COOH·CH{2}·CH(NH{2})·COOH + 2H =
                           COOH·CH{2}·CH{2}·COOH + NH{3}.

This change can be effected in the laboratory only by heating
with hydriodic acid. Biologically it has been observed [E. and H.
Salkowski, 1879] when aspartic acid is submitted to the action of
putrefactive bacteria, and almost quantitatively when /Bacillus coli
communis/ is cultivated in a mixture of aspartic acid and glucose
[Harden, 1901]. In this case a well-defined source of hydrogen exists
in the glucose, which when acted on by this bacillus yields a large
volume of gaseous hydrogen, which is not evolved in the presence of
aspartic acid. Some such source is also available in the case of
yeast, although it cannot be chemically defined, for this organism is
known to produce many reducing actions, which are usually ascribed to
the presence of reducing ferments or reductases in the cell.

A similar action would convert glutamic acid,

 COOH·CH{2}·CH{2}·CH(NH){2}·COOH,

into glutaric acid,

 COOH·CH{2}·CH{2}·CH{2}·COOH,

which also is found among the products of fermentation, whilst the
monamino-acids would pass into the simple fatty acids.

On submitting these ideas to the test of experiment, however, Erhlich
found that the addition of aspartic acid did not in any way increase
the yield of succinic acid, and that of all the amino-acids which were
tried only glutamic acid, COOH·CH{2}·CH{2}·CH(NH{2})·COOH, produced a
definite increase in the amount of this substance. Further experiments
showed that glutamic acid was actually the source of the succinic
acid, the relations being quite similar to those which exist for the
production of fusel oil.

Succinic acid is formed whenever sugar is fermented by yeast, even in
the absence of added nitrogenous matter, and amounts to 0·2 to 0·6 per
cent. of the weight of the sugar decomposed, its origin in this case
being the glutamic acid formed by the autolysis of the yeast protein.
When some other source of nitrogen is present, such as asparagine or
an ammonium salt, the amount falls to 0·05 to 0·1. If glutamic acid be
added it rises to about 1 to 1·5 per cent. but falls again to about
0·05 to 0·1 when other sources of nitrogen, such as asparagine or
ammonium salts, are simultaneously available, either in the presence
or [p091] absence of added glutamic acid. As in the case of fusel
oil, the production does not occur in the absence of sugar, and is not
effected by yeast-juice or zymin.

The chemical reaction involved in the production of succinic acid
differs to some extent from that by which fusel oil is formed,
inasmuch as an oxidation is involved:--

 COOH·CH{2}·CH·CH(NH{2})·COOH + 2O =
                               COOH·CH{2}·CH{2}·COOH + NH{3} + CO{2}.

From analogy with the production of amyl alcohol from leucine, glutamic
acid would be expected to yield γ-hydroxybutyric acid:--

 COOH·CH{2}·CH{2}·CH(NH{2})·COOH + H{2}O =
                           NH{3} + CO{2} + COOH·CH{2}·CH{2}·CH{2}·OH.

As a matter of fact this substance cannot be detected among the
products of fermentation, but succinic acid as already explained is
formed. This acid might, however, possibly be formed by the oxidation
of the γ-hydroxybutyric acid:--

 COOH·CH{2}·CH{2}·CH{2}·OH + 2O = COOH·CH{2}·CH{2}·COOH + H{2}O,

although this change is on biological grounds improbable.

The conversion of the group ─CH(NH{2})─ into the terminal CH{2}·OH
in fusel oil, or COOH in succinic acid, may possibly be effected in
several different ways, the most probable of which are the following:--

I. Direct elimination of carbon dioxide, followed by hydrolysis of the
resulting amine:--

 (1) R·CH(NH{2})·COOH = R·CH{2}·NH{2}+ CO{2}.

 (2) R·CH{2}·NH{2} + H{2}O = R·CH{2}·OH + NH{3}.

The reaction (1) is actually effected by many bacteria and has been
employed for the preparation of bases from amino-acids [cf. Barger,
1914, p. 7], although there is no direct evidence that it can be
brought about by yeast. On the other hand reaction (2) has actually
been observed with some yeasts. Thus it has been found [Ehrlich and
Pistschimuka, 1912, 1] that many "wild" yeasts produce this change
with great readiness in presence of sugar, glycerol or ethyl alcohol
as sources of carbon and grow well in media in which amines, such
as p-hydroxyphenylethylamine or iso-amylamine, form the only source
of nitrogen. /Willia anomala/ (Hansen), a yeast which forms surface
growths, succeeds admirably under these conditions, whereas culture
yeasts are much less active in this way, although they produce a
certain amount of change. It is therefore possible that this mode of
decomposition plays some part in the production of fusel oil, but in
the case of culture yeasts it is entirely subordinated to the mode
next to be discussed. [p092]

II. Oxidative removal of the ─NH{2} group with formation of an
α-ketonic acid:--

 (1) R·CH(NH{2})·COOH + O = R·CO·COOH + NH{3}

followed by the decomposition of the ketonic acid into carbon dioxide
and an aldehyde and the subsequent reduction or oxidation of the
aldehyde:--

 (2) R·CO·COOH = R·CHO + CO{2}.

 (3) (/a/) R·CHO + 2H = R·CH{2}OH.
     (/b/) R·CHO + O = R·COOH.

The evidence for the occurrence of reaction (1) is supplied by the
experiments of Neubauer and Fromherz [1911]. Having previously
found that amino-acids undergo a change of this kind in the animal
body, Neubauer investigated their behaviour towards yeast. Taking
/dl/-phenylaminoacetic acid, C{6}H{5}·CH(NH{2})·COOH, it was found
that the changes produced were essentially the same as in the animal
body. The /l/-component of the acid was partly acetylated and partly
unchanged, whereas the /d/-component of the acid yielded benzyl
alcohol, C{6}H{5}·CH{2}·OH, phenylglyoxylic acid, C{6}H{5}·CO·COOH,
and the hydroxy-acid C{6}H{5}·CH(OH)·COOH. Since however this
hydroxy-acid was produced in the /l/-form it probably arose by the
asymmetric reduction of phenylglyoxylic acid, a reaction which can
be effected by yeast as was also found to be the case in the animal
body [see Dakin, 1912, pp. 52, 78]. Moreover it was shown that
when the effects of yeast on a ketonic acid and the corresponding
hydroxy-acid were compared, the alcohol was formed in much better
yield from the ketonic acid (70 per cent.) than from the hydroxy-acid
(3-4 per cent.), the actual example being the production of
tyrosol (p-hydroxyphenylethyl alcohol), OH·C{6}H{4}·CH{2}·CH{2}OH,
from p-hydroxyphenylpyruvic acid, OH·C{6}H{4}·CH{2}·CO·COOH,
and p-hydroxyphenyl-lactic acid, OH·C{6}H{4}·CH{2}·CH(OH)·COOH
respectively.

Neubauer by these experiments established two extremely important
points. 1. That the amino-acids actually yield the corresponding
α-ketonic acids when treated with yeast and sugar solution.
2. That the a-ketonic acids under similar conditions give the
alcohol containing one carbon atom less in good yield, whereas the
corresponding hydroxy-acids only give an extremely small amount of
these alcohols.

It is therefore probable that at an early stage in the decomposition
of the amino-acids by yeast a ketonic acid is produced, which then
undergoes further change.

The source of the oxygen required for this reaction and the mechanism
of oxidation have not yet been definitely ascertained. It is possible
[p093]

                                              OH
                                             ╱
 that hydrated imino-acids of the type R·C─COOH are first formed
                                             ╲
                                              NH{2}

[Knoop, 1910], but these have not as yet been isolated.

The spontaneous production of ketonic aldehydes from amino-acids and
from hydroxy-acids in aqueous solution, which has been demonstrated by
Dakin and Dudley [1913], points however to the possibility that the
ketonic acid may be a secondary product derived from the corresponding
ketonic aldehyde [see also Dakin, 1908; Neuberg, 1908, 1909]. This
itself may either arise directly from the amino-acid or from a
previously formed hydroxy-acid, the latter alternative being, however,
improbable in view of the small yield of alcohol obtained from
hydroxy-acids by the action of yeast in the experiments of Neubauer
and Fromherz.

 R·CH(NH{2})·COOH → R·CH(OH)·COOH

 R·CH(NH{2})·COOH ⇌ R·CO·CHO

 R·CO·CHO ⇌ R·CH(OH)·COOH

 2R·CO·CHO + O{2} → 2R·CO·COOH

(2) Whatever be the exact mode by which the ketonic acid is formed,
it appears most probable that a compound of this nature forms the
starting-point for the next stage in the production of the alcohols.
The researches of Neuberg, which have already been discussed on p.
81, have revealed a mechanism in yeast--the enzyme carboxylase--by
which these α-ketonic acids are rapidly broken up into an aldehyde and
carbon dioxide:

 R·CO·COOH = R·CHO + CO{2}

and it can scarcely be doubted that this is the actual course of the
reaction.

(3) The final conversion of the aldehyde into the corresponding
alcohol is also a change which it has been proved can be effected
by yeast [Neuberg and Rosenthal, 1913] probably by the aid of the
reductase which is one of the weapons in its armoury of enzymes.

Yeast is capable of producing many vigorous reducing actions and
rapidly reduces methylene blue and sodium selenite. It is in all
probability due to a reaction of this kind that the iso-amylaldehyde
and isovaleraldehyde were reduced to the alcohols in Neuberg and
Steenbock's experiments [1913, 1914], and that considerable quantities
of ethyl alcohol are formed in the sugar free fermentation of pyruvic
acid [Neuberg and Kerb, 1913, 1] (see later p. 110 for a discussion of
this question).

A further possibility exists that in some cases the aldehyde may
[p094] be simultaneously oxidised and reduced or the molecule of one
aldehyde reduced and that of another oxidised with production of the
corresponding acid and alcohol by an "aldehydo-mutase," similar to
that which has been observed by Parnas [1910] in many animal tissues.
Finally the aldehyde may simply be converted into the corresponding
acid by oxidation as appears to take place in the formation of
succinic acid.

The intermediate production of an aldehyde would thus be consistent
both with the production of alcohols and acids from amino-acids.

Fusel oil would be formed by the reduction of the aldehydes arising
from the simple monobasic amino-acids, succinic acid would be produced
by oxidation of the aldehyde derived from the dibasic glutamic acid.

In favour of this view is to be adduced the fact that aldehydes such
as isobutyraldehyde and valeraldehyde have been found in crude spirit,
whilst acetaldehyde is a regular product of alcoholic fermentation
[see Ashdown and Hewitt, 1910]. Benzaldehyde, moreover, has been
actually detected as a product of the alcoholic fermentation of
phenylaminoacetic acid, C{6}H{5}·CH(NH{2})·COOH [Ehrlich, 1907, 1].
Further, the aldehydes so produced would readily pass by oxidation
into the corresponding fatty acids, small quantities of which are
invariably produced in fermentation.

This view of the nature of the alcoholic fermentation of the
amino-acids is undoubtedly to be preferred to that previously
suggested by Ehrlich [1906, 3] according to which a hydroxy-acid is
first formed and then either directly decomposed into an alcohol and
carbon dioxide or into an aldehyde and formic acid, the aldehyde being
reduced and the formic acid destroyed (see p. 115).

 R·CH(NH{2})·COOH → R·CH(OH)·COOH

 R·CH(OH)·COOH → R·CH{2}OH + CO{2}  or  R·CHO + H·CO{2}H

 R·CHO → R·CH{2}OH

The most probable course of the decomposition by which isoamyl
alcohol and succinic acid are produced from leucine and glutamic acid
respectively is therefore the following:--

                            (/a/) /Isoamyl Alcohol./
 (1) (CH{3}){2}·CH·CH{2}·CH(NH{2})·COOH  (3) (CH{3}){2}CHCH{2}·CHO+CO{2}
                   Leucine                      Isovaleraldehyde

 (2) (CH{3}){2}·CH·CH{2}·CO·COOH         (4) (CH{3}){2}·CH·CH{2}·CH{2}OH
       α-Ketoisovalerianic acid                 Isoamyl alcohol

                             (/b/) /Succinic Acid./
 (1) COOH·CH{2}·CH{2}·CH(NH{2})·COOH     (3) COOH·CH{2}CH{2}·CHO + CO{2}
               Glutamic acid                  Succinic semialdehyde

 (2) COOH·CH{2}·CH{2}·CO·COOH            (4) COOH·CH{2}·CH{2}·COOH
       α-Keto-glutaric acid                   Succinic acid

[p095]


 GLYCEROL.

Of the three chief by-products of alcoholic fermentation, only
glycerol remains at present referable directly to the sugar. This
substance, as shown by the careful experiments of Buchner and
Meisenheimer [1906], is formed by the action both of yeast-juice and
zymin to the extent of 3·8 per cent. of the sugar decomposed, and
no other source for its production has so far been experimentally
demonstrated. If it be true that during the decomposition of sugar
into alcohol and carbon dioxide, substances containing three carbon
atoms are formed as intermediate compounds (see p. 100), it is obvious
that these might by reduction be converted into glycerol which would
thus be a true by-product of the alcoholic fermentation of sugar. [See
Oppenheimer, 1914, 2.] It has, however, been suggested that it may in
reality be a product of decomposition of lipoid substances or of the
nuclein of the cell (Ehrlich).

The effect of Ehrlich's work has been clearly to distinguish the
chemical changes involved in the production of fusel oil and succinic
acid from those concerned in the decomposition of sugar into alcohol
and carbon dioxide, and to bring to light a most important series of
reactions by means of which the yeast-cell is able to supply itself
with nitrogen, one of the indispensable conditions of life. [p096]




 CHAPTER VIII.

 THE CHEMICAL CHANGES INVOLVED IN FERMENTATION.


It has long been the opinion of chemists that the remarkable and
almost quantitative conversion of sugar into alcohol and carbon
dioxide during the process of fermentation is most probably the result
of a series of reactions, during which various intermediate products
are momentarily formed and then used up in the succeeding stage of the
process. No very good ground can be adduced for this belief except the
contrast between the chemical complexity of the sugar molecule and
the comparative simplicity of the constitution of the products. Many
attempts have, however, been made to obtain evidence of such a series
of reactions, and numerous suggestions have been made of probable
directions in which such changes might proceed. In making these
suggestions, investigators have been guided mainly by the changes
which are produced in the hexoses by reagents of known composition.
The fermentable hexoses, glucose, fructose, mannose, and galactose,
appear to be relatively stable in the presence of dilute acids at the
ordinary temperature, and are only slowly decomposed at 100°, more
rapidly by concentrated acids, with formation of ketonic acids, such
as levulinic acid, and of coloured substances of complex and unknown
constitution.

In the presence of alkalis, on the other hand, the sugar molecule is
extremely susceptible of change. In the first place, as was discovered
by Lobry de Bruyn [1895; Bruyn and Ekenstein, 1895; 1896; 1897, 1, 2,
3, 4], each of the three hexoses, glucose, fructose, and mannose is
converted by dilute alkalis into an optically almost inactive mixture
containing all three, and probably ultimately of the same composition
whichever hexose is employed as the starting-point.

This interesting phenomenon is most simply explained on the assumption
that in the aqueous solution of any one of these hexoses, along with
the molecules of the hexose itself, there exists a small proportion
of those of an enolic form which is common to all the three hexoses,
as illustrated by the following formulæ, the aldehyde formulæ [p097]
being employed instead of the γ-oxide formulæ for the sake of
simplicity:--

   CHO          CHO          CH{2}(OH)     CH(OH)
   │            │            │             ║
  HCOH        HOCH           CO            COH
   │            │            │             │
 HOCH         HOCH         HOCH          HOCH
   │            │           │              │
  HCOH         HCOH         HCOH          HCOH
   │            │            │             │
  HCOH         HCOH         HCOH          HCOH
   │            │            │             │
   CH{2}(OH)    CH{2}(OH)    CH{2}(OH)     CH{2}(OH)
  Glucose.     Mannose.     Fructose.     Enolic form.

This enolic form is capable of giving rise to all three hexoses,
and the change by which the enolic form is produced and converted
into an equilibrium mixture of the three corresponding hexoses is
catalytically accelerated by alkalis, or rather by hydroxyl ions.
In neutral solution the change is so slow that it has never been
experimentally observed; in the presence of decinormal caustic
soda solution at 70° the conversion is complete in three hours.
Precisely similar effects are produced with galactose, which yields
an equilibrium mixture containing talose and tagatose, sugars which
appear not to be fermentable.

The continued action even of dilute alkaline solutions carries the
change much further and brings about a complex decomposition which
is much more rapidly effected by more concentrated alkalis and at
higher temperatures. This change has been the subject of very numerous
investigations [for an account of these see E. v. Lippmann, 1904,
pp. 328, 713, 835], but for the present purpose the results recently
obtained by Meisenheimer [1908] may be quoted as typical. Using
normal solutions of caustic soda and concentrations of from 2 to 5
grams of hexose per 100 c.c., it was found that at air temperature in
27 to 139 days from 30 to 54 per cent. of the hexose was converted
into inactive lactic acid, C{3}H{6}O{3}, from 0·5 to 2 per cent.
into formic acid, CH{2}O{2}, and about 40 per cent. into a complex
mixture of hydroxy-acids, containing six and four carbon atoms in the
molecule. Usually only about 74 to 90 per cent. of the sugar which had
disappeared was accounted for, but in one case the products amounted
to 97 per cent. of the sugar. About 1 per cent. of the sugar was
probably converted into alcohol and carbon dioxide. No glycollic acid,
oxalic acid, glycol, or glycerol was produced.

The fact that alcohol is actually formed by the action of alkalis on
sugar was established by Buchner and Meisenheimer [1905], who obtained
small quantities of alcohol (1·8 to 2·8 grams from 3 kilos. of cane
sugar) by acting on cane sugar with boiling concentrated caustic
soda [p098] solution. It is evident that under these conditions an
extremely complex series of reactions occurs, but the formation of
alcohol and carbon dioxide and of a large proportion of lactic acid
deserves more particular attention.

The direct formation of alcohol from sugar by the action of alkalis
appears first to have been observed by Duclaux [1886], who exposed a
solution of glucose and caustic potash to sunlight and obtained both
alcohol and carbon dioxide. As much as 2·6 per cent. of the sugar was
converted into alcohol in a similar experiment made by Buchner and
Meisenheimer [1904]. When the weaker alkalis, lime water or baryta
water, were employed instead of caustic potash, however, no alcohol
was formed, but 50 per cent. of the sugar was converted into inactive
lactic acid [Duclaux, 1893, 1896]. Duclaux therefore regarded the
alcohol and carbon dioxide as secondary products of the action of a
comparatively strong alkali on preformed lactic acid. Ethyl alcohol
can, in fact, be produced from lactic acid both by the action of
bacteria [Fitz, 1880] and of moulds [Mazé, 1902], and also by chemical
means. Thus Duclaux [1886] found that calcium lactate solution exposed
to sunlight underwent decomposition, yielding alcohol and calcium
carbonate and acetate, whilst Hanriot [1885, 1886], by heating calcium
lactate with slaked lime obtained a considerable quantity of a liquid
which he regarded as ethyl alcohol, but which was shown by Buchner and
Meisenheimer [1905] to be a mixture of ethyl alcohol with isopropyl
alcohol.

It appears, therefore, that inactive lactic acid can be quite readily
obtained in large proportion from the sugars by the action of alkalis,
whilst alcohol can only be prepared in comparatively small amount and
probably only as a secondary product of the decomposition of lactic
acid.

The study of the action of alkalis on sugar has, however, yielded
still further information as regards the mechanism of the reaction
by which lactic acid is formed. A considerable body of evidence has
accumulated, tending to show that some intermediate product of the
nature of an aldehyde or ketone containing three carbon atoms is first
formed.

Thus Pinkus [1898] and subsequently Nef [1904, 1907], by acting on
glucose with alkali in presence of phenylhydrazine obtained the
osazone of methylglyoxal, CH{3}·CO·CHO. This osazone may be formed
either from methylglyoxal itself, from acetol, CH{3}·CO·CH{2}·OH, or
from lactic aldehyde, CH{3}·CH(OH)·CHO [Wohl, 1908]. Methylglyoxal
itself may also be regarded as a secondary [p099] product derived
from glyceraldehyde, CH{2}(OH)·CH(OH)·CHO, or dihydroxyacetone,
CH{2}(OH)·CO·CH{2}(OH), by a process of intramolecular dehydration, so
that the osazone might also be derived indirectly from either of these
compounds [see also Neuberg and Oertel, 1913]. Methylglyoxal itself
readily passes into lactic acid when it is treated with alkalis, a
molecule of water being taken up:--

 CH{3}·CO·CHO + H{2}O = CH{3}·CH(OH)·COOH.

Further evidence in the same direction is afforded by the interesting
discovery of Windaus and Knoop [1905], that glucose is converted by
ammonia in presence of zinc hydroxide into methyliminoazole,

 CH{3}·C·NH
       ║ ╲
       ║  CH,
       ║ ╱╱
       CH·N

a substance which is a derivative of methylglyoxal.

The idea suggested by Pinkus that acetol is the first product of the
action of alkalis on sugar has been rendered very improbable by the
experiments of Nef, and the prevailing view (Nef, Windaus and Knoop,
Buchner and Meisenheimer) is that the first product is glyceraldehyde,
which then passes into methylglyoxal, and finally into lactic acid:--

 (1) C{6}H{12}O{6} = 2CH{2}(OH)·CH(OH)·CHO.

 (2) CH{2}(OH)·CH(OH)·CHO = CH{3}·CO·CHO + H{2}O.

 (3) CH{3}·CO·CHO + H{2}O = CH{3}·CH(OH)·COOH.

All these changes may occur at ordinary temperatures in the presence
of a catalyst, and in so far resemble the processes of fermentation by
yeasts and bacteria.

The first attempt to suggest a scheme of chemical reactions by which
the changes brought about by living organisms might be effected
was made in 1870 by Baeyer [1870], who pointed out that these
decompositions might be produced by the successive removal and
re-addition of the elements of water. The result of this would be to
cause an accumulation of oxygen atoms towards the centre of the chain
of six carbon atoms, which, in accordance with general experience,
would render the chain more easily broken. Baeyer formulated the
changes characteristic of the alcoholic and lactic fermentations as
follows, the intermediate stages being derived from the hydrated
aldehyde formula of glucose by the successive removal and addition of
the elements of water: [p100]

    I.         II.       III.         IV.       V.

 CH{2}·OH   CH{2}...OH    CH{3}       CH{3}     CH{3}
 │          │             │           │         │
 CH·OH      COH..H        CH·OH       CH(OH)    CH{2}
 │          │             │           │        ╱
 CH·OH      C..OH..H      C(OH){2}    CO      O
 │          │             │          ╱         ╲
 CH·OH      COH...H       C(OH){2}  O           CO
 │          │             │          ╲         ╱
 CH·OH      COH...H       C(OH){2}    CO      O
 │          │             │           │        ╲
 CH(OH){2}  CH...(OH){2}  CH{3}       CH(OH)    CO
                                      │        ╱
                                      CH{3}   O
                                               ╲
                                                CH{2}
                                                │
                                                CH{3}

The immediate precursor of alcohol and carbon dioxide is here seen to
be the anhydride of ethoxycarboxylic acid (V), whilst that of lactic
acid is lactic anhydride (IV). (Baeyer does not appear, as recently
stated by Meisenheimer [1907, p. 8], Wohl [1907, 2], and Buchner
and Meisenheimer [1909] to have suggested that lactic acid was an
intermediate product in alcoholic fermentation, but rather to have
represented independently the course of the two different kinds of
fermentation, the alcoholic and the lactic.)

It was subsequently pointed out by Buchner and Meisenheimer [1904]
that Baeyer's principle of oxygen accumulation might be applied
in a different way, so that a ketonic acid would be produced, the
decomposition of which, in a manner analogous to that of acetoacetic
acid, would lead to the formation of two molecules of lactic acid,
from which the final products alcohol and carbon dioxide might be
directly derived, as shown in the following formulæ:--

 CHO           COOH      COOH      CO{2}
 ·             ·         ·         ────────
 CH(OH)        CH(OH)    CH(OH)    CH{2}·OH
 ·             ·         ·         ·
 CH(OH)        CH{2}     CH{3}     CH{3}
 ·             ·         ──────    ────────
 CH(OH)        CO        COOH      CO{2}
 ·             ·         ·         ────────
 CH(OH)        CH(OH)    CH(OH)    CH{2}·OH
 ·             ·         ·         ·
 CH{2}(OH)     CH{3}     CH{3}     CH{3}

A scheme based on somewhat different principles has been propounded
by Wohl [Lippmann, 1904, p. 1891], and has been accepted by Buchner
and Meisenheimer [1905] as more probable than that quoted above. Wohl
and Oesterlin [1901] were able to trace experimentally the various
stages of the conversion of tartaric acid (I) into oxalacetic acid
(III), which can be carried out by reactions taking place at the
ordinary temperature, and they found that the first stage consisted in
the removal of the elements of water leaving an unsaturated hydroxy
derivative (II) which in the second stage underwent intramolecular
change into the corresponding keto-compound (III): [p101]

     COOH              COOH     COOH
     ·                 ·        ·
     CH(OH) minus H  = C(OH) ⇌  CO
     ·            ·    ║        ·
     CH(OH)       OH   CH       CH{2}
     ·                 ·        ·
     COOH              COOH     COOH

       I.               II.      III.
 Tartaric acid.                Oxalacetic acid.

This change differs in principle from that assumed by Baeyer,
inasmuch as the second stage is not effected by the re-addition of
water, but by the keto-enol transformation, which is now usually
ascribed to the migration of the hydrogen atom, although the same
result can theoretically be arrived at by the addition and removal
of the elements of water. The analogy of this process to what might
be supposed to occur in the conversion of sugar into carbon dioxide
and alcohol was pointed out by Wohl and Oesterlin, and subsequently
Wohl developed a theoretical scheme of reactions by which the process
of alcoholic fermentation could be represented. In the first place
the elements of water are removed from the α and β carbon atoms of
glucose (I) and the resulting enol (II) undergoes conversion into
the corresponding ketone (III), which has the constitution of a
condensation product of methylglyoxal and glyceraldehyde, and hence
is readily resolved by hydrolysis into these compounds (IV). The
glyceraldehyde passes by a similar series of changes (V, VI) into
methylglyoxal, and this is then converted by addition of water into
lactic acid (VII), a reaction which is common to all ketoaldehydes
of this kind. Finally, the lactic acid is split up into alcohol and
carbon dioxide (VIII):--

          CHO                CHO              CHO
          │                  │                │
          CH(OH)       H     C(OH)            CO
          │      minus ·     ║                │
          CH(OH)       OH    CH           ⇌   CH{2}
          │                  │                │
          CH(OH)             CH(OH)           CH(OH)
          │                  │                │
          CH(OH)             CH(OH)           CH(OH)
          │                  │                │
          CH{2}(OH)          CH{2}(OH)        CH{2}(OH)

            I.                  II.             III.
          Glucose.

 Methylglyoxal
 CHO                                         COOH    CO{2}
 │                                           │       ------
 CO                   + H{2}O                CH(OH)  CH{2}OH
 │                                           │       │
 CH{3}                                       CH{3}   CH{3}
 ───────────────────────────────────────────────────────────
 CHO                 CHO     CHO          COOH    CO{2}
 │            H      │       │            │       ------
 CH(OH) minus ·      C(OH) ⇌ CO + H{2}O   CH(OH)  CH{2}OH
 │            OH     ║       │            │       │
 CH{2}(OH)           CH{2}   CH{3}        CH{3}   CH{3}

   IV.               V.         VI.        VII.     VIII.

 Glyceraldehyde.             Methyl-      Lactic   Alcohol
                            glyoxal.      acid.     and
                                                    carbon
                                                    dioxide.

[p102]

This scheme agrees well with the current ideas as to the formation
of lactic acid from glucose under the influence of alkalis (p.
99). It postulates the formation as intermediate products of no
less than three compounds containing a chain of three carbon
atoms--glyceraldehyde, methylglyoxal, and lactic acid.


 THE LACTIC ACID THEORY OF ALCOHOLIC FERMENTATION.

A practical interest was given to these various schemes by the fact
that Buchner and Meisenheimer adduced experimental evidence in favour
of the view that lactic acid is an intermediate product in the
formation of alcohol and carbon dioxide from sugar by fermentation
[1904, 1905, 1906, 1909].

These observers proved by a series of very careful analyses that
yeast-juice frequently, but not invariably, contains small quantities
of lactic acid, not exceeding 0·2 per cent. When yeast-juice is
incubated alone or with sugar the amount of lactic acid may either
increase or decrease. Moreover, lactic acid added to the juice is
sometimes diminished and sometimes increased in quantity. On the whole
it appears that the addition of a considerable quantity of sugar or of
some lactic acid favours the disappearance of lactic acid. Juices of
low fermenting power produce a diminution in the lactic acid present,
those of high fermenting power an increase.

In all cases the amounts of lactic acid either produced or destroyed
are very small in relation to the volume of the yeast-juice employed.

Throughout the whole series of experiments the greatest increase
amounted to 0·47 per cent. on the juice employed, and the greatest
decrease to 0·3 per cent. [See also Oppenheimer, 1914, 1.] Buchner and
Meisenheimer at one time regarded these facts as strong evidence that
lactic acid is an intermediate product of alcoholic fermentation. It
was thought probable that the production of alcohol and carbon dioxide
from sugar occurred in at least two stages and under the influence of
two distinct enzymes. The first stage consisted in the conversion of
sugar into lactic acid, and for the enzyme which brought about this
decomposition was reserved the name zymase or yeast-zymase. The lactic
acid was then broken down into alcohol and carbon dioxide by the
second enzyme, lactacidase.

This theory, which is quite in harmony with the current ideas as to
the mode of decomposition of sugars by alkalis, and is also consistent
with Wohl's scheme of reactions, is open to adverse criticism from
several points of view. In the first place, it is noticeable that the
total amount of lactic acid used up by the juice is extremely small,
even [p103] in the most favourable cases, relatively to the amount of
the juice [Harden, 1905], and it may be added to the sugar-fermenting
power of the juice. Moreover, as pointed out by Buchner and
Meisenheimer themselves [1909], no proof is afforded that the lactic
acid which disappears is converted into alcohol and carbon dioxide. It
is not even certain, although doubtless probable, that the lactic acid
which occurs or is produced in the juice is really derived from sugar.

The most weighty criticism of the theory is that of Slator [1906,
1907; 1908, 1, 2], which is based on the consideration that if lactic
acid be an intermediate product of alcoholic fermentation the reaction
by which it is fermented must proceed at least as rapidly as that by
which it is formed, in order to prevent accumulation of lactic acid.
The fermentation of lactic acid by yeast should therefore proceed at
least as rapidly as that of glucose. So far is that from being the
case that it has been experimentally demonstrated that lactic acid is
not fermented at all by living yeast. This conclusion was rendered
extremely probable by Slator, who showed that lactic acid, even in
concentrations insufficient to prevent the fermentation of glucose, is
not fermented to any considerable extent. The final proof that lactic
acid is neither formed nor fermented by pure yeast has been brought
by Buchner and Meisenheimer in a series of very careful quantitative
experiments carried out with a pure yeast and with strict precautions
against bacterial contamination [1909, 1910].

At first sight this fact appears decisive against the validity of
the lactic acid theory, and it is recognised as such by Buchner and
Meisenheimer. Wohl has, however, suggested that the non-fermentability
of lactic acid by yeast is not really conclusive [1907, 1; see also
Franzen and Steppuhn, 1912, 1]. The production of lactic acid from
glucose is attended by the evolution of a considerable amount of
heat (22 cal.), and it is possible that at the moment of production
the molecule of the acid is in a condition of activity corresponding
with a much higher temperature than the average temperature of the
fermenting liquid. Under these circumstances the molecule would be
much more susceptible of chemical change than at a later period when
temperature equilibrium had been attained. It has, however, been
pointed out by Tafel [1907], that such a decomposition of the lactic
acid would occur at the very instant of formation of the molecule,
so that no ground remains even on this view for assuming the actual
existence of lactic acid as a definite intermediate product. It has
also been suggested by Luther [1907] that an unknown isomeride of
lactic acid is formed as an intermediate product and fermented, and
that traces of lactic [p104] acid are formed by a secondary reaction
from this, but no satisfactory evidence for this view is forthcoming.
There still remains a doubt as to whether the living yeast-cell is
permeable to lactic acid, a fact which would of course afford a very
simple explanation of the non-fermentability of the acid. Apart from
this, however, it is difficult, in face of the evidence just quoted,
to believe that lactic acid is in reality an intermediate product in
alcoholic fermentation.


 METHYLGLYOXAL, DIHYDROXYACETONE AND GLYCERALDEHYDE.

As regards the fermentability by yeast of compounds containing three
carbon atoms, which may possibly appear as intermediate products
in the transformation of sugar into carbon dioxide and alcohol,
many experiments have been carried out, with somewhat uncertain
results. Care has to be taken that the substance to be tested is
not added in such quantity as to inhibit the fermenting power of
the yeast or yeast-juice, and further that the conditions are such
that the substance in question, often of a very unstable nature, is
not converted by some chemical change into a different fermentable
compound. It is also possible that the substance to be tested may
accelerate the rate of autofermentation in a similar manner to
arsenates (pp. 80, 126) and many other substances. These are all
points which have not up to the present received sufficient attention.
In the case of living yeast the further question arises of the
permeability of the cell.

Methylglyoxal, CH{3}·CO·CHO, has been tested by Mayer [1907] and
Wohl [1907, 2] with yeast, and by Buchner and Meisenheimer both with
acetone-yeast [1906] and yeast-juice [1910], in every case with
negative results, but it may be noted that the concentration employed
in the last mentioned of these experiments was such as considerably to
diminish the autofermentation of the juice.

Glyceraldehyde, CH{2}(OH)·CH(OH)·CHO, was also tested with yeast
with negative results by Wohl [1898] and by Emmerling [1899], who
employed a number of different yeasts. The same negative result
attended the experiments of Piloty [1897] and Emmerling [1899] with
pure dihydroxyacetone. Fischer and Tafel [1888, 1889], however, had
previously found that glycerose, a mixture of glyceraldehyde and
dihydroxyacetone prepared by the oxidation of glycerol, was readily
fermented by yeast, agreeing in this respect with the still older
observations of Van Deen and of Grimaux. The reason for this diversity
of result has not been definitely ascertained, but it has been
supposed by Emmerling to lie in the formation of some fermentable
sugar from [p105] glycerose when the latter is subjected to too high
a temperature during its preparation.

On the other hand, Bertrand [1904] succeeded in fermenting pure
dihydroxyacetone by treating a solution of 1 gram in 30 c.c. of liquid
with a small quantity of yeast for ten days at 30°, the best result
being a fermentation of 25 per cent. of the substance taken. Moreover,
Boysen-Jensen [1908, 1910, 1914] states that he has also observed both
the formation from glucose and the fermentation of this substance by
living yeast, but the amounts of alcohol and carbon dioxide produced
were so minute and the evidence for the production of dihydroxyacetone
so inconclusive that the experiments cannot be regarded as in any
way decisive [see Chick, 1912; Euler and Fodor, 1911; Karauschanoff,
1911; Buchner and Meisenheimer, 1912]. A careful investigation by
Buchner [1910] and Buchner and Meisenheimer [1910] has led them to
the conclusion that both glyceraldehyde and dihydroxyacetone are
fermentable. Glyceraldehyde exerts a powerful inhibiting action both
on yeast and yeast-juice, and was only found to give rise to a very
limited amount of carbon dioxide, quantities of 0·15 to 0·025 gram
being treated with 1 gram of yeast or 5 c.c. of yeast-juice and a
production of 4 to 12 c.c. of carbon dioxide being attained.

When 0·1 gram of dihydroxyacetone in 5 c.c. of water was brought in
contact with 1 gram of living yeast, about half was fermented, 17 c.c.
of carbon dioxide (at 20° and 600 mm.) being evolved in excess of the
autofermentation of the yeast (13 c.c.). A much greater effect was
obtained by the aid of yeast-juice, and the remarkable observation
was made that whilst yeast-juice alone produced comparatively little
action a mixture of yeast-juice and boiled yeast-juice was much
more effective, quantities of 20 to 50 c.c. of yeast-juice mixed
with an equal volume of boiled juice, which in some experiments was
concentrated, yielding with 0·4, 1, and 2 grams of dihydroxyacetone
almost the theoretical amount of carbon dioxide and alcohol in
excess of that evolved in the absence of this substance. It was
further observed that the fermentation of this substance commenced
much more slowly than that of glucose. No explanation of either of
these facts has at present been offered. The conclusion drawn from
their experiments by Buchner and Meisenheimer that dihydroxyacetone
is readily fermentable, was confirmed by Lebedeff [1911, 1], who
further made the important observation that during the fermentation
of dihydroxyacetone the same hexosephosphoric acid is produced as is
formed during the fermentation of the hexoses. Lebedeff accordingly
propounded a scheme of alcoholic fermentation according to which the
hexose [p106] was first converted into two molecules of triose,
the latter being first esterified to triosephosphoric acid and
then condensed to hexosediphosphoric acid, which then underwent
fermentation, after being hydrolysed to phosphoric acid, and some
unidentified substance, probably an unstable modification of a hexose,
much more readily attacked by an appropriate enzyme than the original
glucose or fructose [1911, 1, pp. 2941-2].

The idea that the sugar is first converted into triose and this into
triosemonophosphoric acid had been previously suggested by Iwanoff
who postulated the agency of a special enzyme termed /synthease/
[1909, 1], and supposed that this triosemonophosphoric acid was then
directly fermented to alcohol, carbon dioxide and phosphoric acid.
According both to Iwanoff and Lebedeff the phosphoric ester is an
intermediate product and its decomposition provides this sole source
of carbon dioxide and alcohol. This is quite inconsistent with the
facts recounted above (Chap. III), which prove that the formation
of the hexosephosphate is /accompanied/ by an amount of alcoholic
fermentation exactly equivalent to the quantity of hexosephosphate
produced, and that the rate of fermentation rapidly falls as soon as
the free phosphate has disappeared, in spite of the fact that at that
moment the concentration of the hexosephosphate is at its highest,
whereas according to Iwanoff's theory it is precisely under these
conditions that the maximum rate of fermentation should be maintained.

It has also been shown that the arguments adduced by Iwanoff in favour
of the existence of his synthease are not valid [Harden and Young,
1910, 1].

The fermentation of dihydroxyacetone was moreover proved by Harden
and Young [1912] to be effected by yeast-juice and maceration extract
at a much slower rate than that of the sugars, in spite of the fact
that the addition of dihydroxyacetone did not inhibit the sugar
fermentation. The same thing has been shown for living yeast by Slator
[1912] in agreement with the earlier results of Buchner [1910] and
Buchner and Meisenheimer [1910].

The logical conclusion from Lebedeff's experiments would appear
rather to be that dihydroxyacetone is slowly condensed to a hexose
and that this is then fermented in the normal manner [Harden and
Young, 1912; Buchner and Meisenheimer, 1912; Kostytscheff, 1912,
2]. Buchner and Meisenheimer, however, regard this as improbable on
the ground that dihydroxyacetone, being symmetric in constitution,
would yield an inactive hexose of which only at most 50 per cent.
would be fermentable. Against this it may be urged, however, [p107]
that enzymic condensation of dihydroxyacetone might very probably
occur asymmetrically yielding an active and completely fermentable
hexose. Buchner and Meisenheimer, however, still support the view
that dihydroxyacetone forms an intermediate stage in the fermentation
of glucose and adduce as confirmatory evidence of the probability of
such a change the observation of Fernbach [1910] that this compound is
produced from glucose by a bacillus, Tyrothrix tenuis, which effects
the change both when living and after treatment with acetone.

The balance of evidence, however, appears to be in favour of the
opinion that dihydroxyacetone does not fulfil the conditions laid down
by Slator (see p. 103) as essential for an intermediate product in the
process of fermentation [see also Löb, 1910].

Lebedeff subsequently [1912, 4; Lebedeff and Griaznoff, 1912]
extended his experiments to glyceraldehyde and modified his theory
very considerably. Using maceration extract it was found in general
agreement with the results of Buchner and Meisenheimer (p. 105) that
20 c.c. of juice were capable of producing about half the theoretical
amount of carbon dioxide from 0·2 gram of glyceraldehyde, whereas 0·4
gram caused coagulation of the extract and a diminished evolution
of carbon dioxide. The addition of phosphate diminished rather than
increased the fermentation. Even in the most favourable concentration
however (0·2 gram per 20 c.c.) the glyceraldehyde is fermented much
more slowly than dihydroxyacetone or saccharose, as is shown by the
following figures:--

 ─────────────────┬────────────────────────────┬─────────────────────
 20 c.c. Extract  │     CO{2} in grams in      │  Duration    Total
   + 0·2 gram.    │    successive periods of   │ of fermen-  CO{2}.
                  │6 hours.│18 hours.│24 hours.│  tation.
 ─────────────────┼────────┴─────────┴─────────┼───────────────────
 Cane sugar       │ 0·050     0·000     0·000  │      6       0·05
 Dihydroxyacetone │ 0·042     0·000     0·000  │      6       0·042
 Glyceraldehyde   │ 0·008     0·022     0·005  │     48       0·035
 ─────────────────┴────────────────────────────┴───────────────────

Further, during an experiment in which 0·129 gram of CO{2} was
evolved in 22·5 hours from 0·9 gram of glyceraldehyde in presence of
phosphate, no change in free phosphate was observed, whereas in a
similar experiment with glucose a loss of about 0·2 gram of P{2}O{5}
would have occurred. Hence the fermentation takes place without
formation of hexosediphosphate. This was confirmed by the fact that
the osazone of hexosephosphoric acid was readily isolated from the
products of fermentation of dihydroxyacetone (0·259 gram of CO{2}
having been evolved in twenty hours) but could not be obtained from
those of glyceraldehyde (0·138 gram CO{2} in twenty hours). [p108]

This result is extremely interesting, although it is not impossible
that the rate of fermentation of the glyceraldehyde is so slow that
any phosphoric ester produced is hydrolysed as rapidly as it is formed.

Lebedeff regards the experiments as proof that phosphate takes no part
in the fermentation of glyceraldehyde and bases on this conclusion and
his other work the following theory of alcoholic fermentation.

1. The sugar is split up into equimolecular proportions of
glyceraldehyde and dihydroxyacetone:--

 (a) C{6}H{12}O{6} = C{3}H{6}O{3} + C{3}H{6}O{3}.

2. The dihydroxyacetone then passes through the stages previously
postulated (p. 106).

 (b) 4 C{3}H{6}O{3} + 4 R{2}HPO{4} = 4 C{3}H{5}O{2}PO{4}R{2} + 4 H{2}O.

 (c) 4 C{3}H{5}O{2}PO{4}R{2} = 2 C{6}H{10}O{4}(R{2}PO{4}){2}.

 (d) 2 C{6}H{10}O{4}(R{2}PO{4}){2} + 4 H{2}O =
                                     2 C{6}H{12}O{6} + 4 R{2}HPO{4}.

After which the hexose, C{6}H{12}O{6} re-enters the cycle at (a).

3. The fermentation of the glyceraldehyde occurs according to the
scheme developed by Kostytscheff (p. 109), pyruvic acid being formed
along with hydrogen and then decomposed into carbon dioxide and
acetaldehyde, which is reduced by the hydrogen. Lebedeff, however,
suggests [1914, 1, 2] that glyceric acid is first formed (1) and then
converted by an enzyme, which he terms /dehydratase/ into pyruvic acid
(2):--

 (1) CH{2}(OH)·CH(OH)·CHO + H{2}O → CH{2}(OH)·CH(OH)·CH(OH){2}
     CH{2}(OH)·CH(OH)·CH(OH){2} → CH{2}(OH)·CH(OH)·COOH + 2H

 (2) CH{2}(OH)·CH(OH)·COOH = CH{3}·CO·COOH + H{2}O.

The experimental basis for this idea is the fact that glyceric acid is
fermented by dried yeast and maceration juice [compare Neuberg and Tir,
1911].

This scheme has the merit of recognising the fact that the carbon
dioxide does not wholly arise from the products of decomposition
of hexosephosphate, nor from its direct fermentation. The function
assigned to the phosphate is that of removing dihydroxyacetone and
thus preventing it from inhibiting further conversion of hexose into
triose, according to the reversible reaction

 C{6}H{12}O{6} ⇌ 2 C{3}H{6}O{3}.

This however appears to be quite inadequate, since, on the one hand,
the fermentation of glucose proceeds quite freely in presence of as
much as 5 grams per 100 c.c. of dihydroxyacetone [Harden and Young,
1912], and on the other hand alcoholic fermentation appears not to
proceed at all in the absence of phosphate (see p. 55). This forms
the chief objection to the theory in its present form. The slow rate
at which [p109] glyceraldehyde is fermented also affords an argument
against the validity of Lebedeff's view, but this may possibly be
accounted for to some extent by the fact that glyceraldehyde is a
strong inhibiting agent so that it might be more rapidly fermented if
added in a more dilute condition.

The unfermented glyceraldehyde cannot be recovered from the solution
and nothing is known as to its fate except that it readily gives rise
both to lactic acid and glycerol [Oppenheimer, 1914, 1, 2]. Evidently
the reaction between glyceraldehyde and yeast-juice is by no means a
simple one.


 THE PYRUVIC ACID THEORY.

The third stage of Lebedeff's theory postulates the intermediate
formation of pyruvic acid. This idea immediately suggested itself
when it became known that yeast was capable of rapidly decomposing
/a/-ketonic acids with evolution of carbon dioxide [see Neubauer and
Fromherz, 1911, p. 350; Neuberg and Kerb, 1912, 4; Kostytscheff, 1912,
2].

This scheme has been differently elaborated by different workers.
According to Kostytscheff it involves (1) the production of pyruvic
acid from the hexoses, a process accompanied by loss of hydrogen;
(2) the decomposition of pyruvic acid into acetaldehyde and carbon
dioxide; and (3) the reduction of the acetaldehyde to ethyl alcohol.

 (1) C{6}H{12}O{6} = 2CH{3}·CO·COOH + 4[H].

 (2) 2CH{3}·CO·COOH = 2CH{3}·CHO + 2CO{2}.

 (3) 2CH{3}·CHO + 4H = 2CH{3}·CH{2}·OH.

1. As regards the production of pyruvic acid from the hexoses by
yeast, the only direct evidence is afforded by the experiments of
Fernbach and Schoen [1913] who have obtained a calcium salt having
the qualitative properties of a pyruvate by carrying out alcoholic
fermentation by yeast in presence of calcium carbonate, but have
not yet definitely settled either the identity of the acid or its
origin from sugar. Pyruvic acid is, however, very closely related to
several substances which are intimately connected both chemically and
biochemically with the hexoses. Thus lactic acid is its reduction
product,

 CH{3}·CO·COOH  → CH{3}·CH(OH)·COOH,
                + 2H

glyceraldehyde can readily be converted into it by oxidation to
glyceric acid followed by abstraction of water (Erlenmeyer), [p110]

 CH{2}(OH)·CH(OH)·CHO → CH{2}(OH)·CH(OH)·COOH → CH{3}·CO·COOH,
                      + O                      −H{2}O

and finally methylglyoxal CH{3}·CO·CHO is its aldehyde.

2. The decomposition of pyruvic acid into acetaldehyde and carbon
dioxide has already been fully discussed (Chapter VI). The
universality of the enzyme carboxylase in yeasts and the rapidity of
its action on pyruvic acid form the strongest evidence at present
available in favour of the pyruvic acid theory. Given the pyruvic
acid, there is no doubt that yeast is provided with a mechanism
capable of decomposing it at the same rate as an equivalent amount of
sugar.

3. The final step postulated by the pyruvic acid theory is the
quantitative reduction to ethyl alcohol of the acetaldehyde formed
from the pyruvic acid.

The idea that acetaldehyde is an intermediate product in the various
fermentations of sugar has frequently been entertained [Magnus Levy,
1902; Leathes, 1906; Buchner and Meisenheimer, 1908; Harden and
Norris, D., 1912] although no very definite experimental foundation
exists for the belief. It is, however, a well-known fact that traces
of acetaldehyde are invariably formed during alcoholic fermentation
[see Ashdown and Hewitt, 1910], and this is of course consistent with
the occurrence of acetaldehyde as an intermediate product. Important
evidence as to the specific capability of yeast to reduce acetaldehyde
to alcohol has been obtained by several workers. Thus Kostytscheff
[1912, 3; Kostytscheff and Hübbenet, 1913] found that pressed yeast,
dried yeast and zymin all reduced acetaldehyde to alcohol, 50 grams
of yeast in 10 hours producing from 660 mg. of aldehyde 265 mg. of
alcohol in excess of the amount produced by autofermentation in
absence of added aldehyde. Maceration extract was found to reduce both
in absence and in presence of sugar, whereas Lebedeff and Griaznoff
[1912] obtained no reduction in presence of sugar, and observed
that the power of reduction was lost by the extract on digestion,
a circumstance which suggests the co-operation of a co-enzyme in
the process. Neuberg and Kerb [1912, 4; 1913, 1] have also been
able to show by large scale experiments that alcohol is produced in
considerable quantity by the fermentation of pyruvic acid by living
yeast in absence of sugar and that the yield is increased by the
presence of glycerol. When treated with 22 kilos, of yeast, 1 kilo,
of pyruvic acid yielded 241 grams of alcohol in excess of that given
by the yeast alone, whilst in presence of glycerol the amount was
360 grams, the amount theoretically obtainable being 523 grams. The
function of the glycerol is not understood but is probably that of
lessening the rate of destruction of the yeast enzymes. [p111]

That yeast possesses powerful reducing properties has long been
known and many investigations have been made as to the relation of
these properties to the process of alcoholic fermentation. Thus
Hahn (Buchner, E. and H., and Hahn, 1903, p. 343) found that the
power of reducing methylene blue was possessed both by yeast and
zymin and on the whole ran parallel to the fermenting power in the
process of alcoholic fermentation. The intervention of a reducing
enzyme was suggested by Grüss [1904, 1908, 1, 2] and was supported by
Palladin [1908]. The latter observed that zymin which reduces sodium
selenite and methylene blue in absence of sugar almost ceases to do
so in presence of a fermentable sugar, and concluded that the great
diminution of reduction during fermentation was due to the fact that
the reducing enzyme was largely combined with a different substrate
arising from the sugar, the reduction of which was necessary for
alcoholic fermentation. Grüss, however, found that with living yeast
the reduction is greatly increased in presence of a fermentable sugar,
while Harden and Norris, R. V. [1914] confirmed the observation of
Grüss, but found that the reducing power of zymin is not seriously
affected by the presence of a fermentable sugar in concentration less
then 20 grams per 100 c.c., whilst its fermenting power for glucose is
inhibited by 1 per cent. sodium selenite. Hence Palladin's conclusion
cannot be regarded as proved.

Interesting attempts have been made by Kostytscheff and later by Lvoff
to obtain evidence of the participation of a reductase in alcoholic
fermentation by adding some substance which would be capable either
of taking up hydrogen and thus preventing the reduction of the
acetaldehyde or of converting the aldehyde into some compound less
liable to reduction.

Kostytscheff [1912, 1; 1913, 1, 2; 1914; Kostytscheff and Hübbenet,
1913; Kostytscheff and Scheloumoff, 1913; Kostytscheff and Brilliant,
1913] has examined the effect of the addition of zinc chloride, chosen
with the idea that it might polymerise the aldehyde and thus remove it
from the sphere of action. As pointed out by Neuberg and Kerb [1912,
1] this action is not very probable, and it was subsequently found
[Kostytscheff and Scheloumoff, 1913] that the effect of added zinc
salts was more probably specifically due to the zinc ion. Fermentation
of sugar by dried yeast still proceeds when 0·6 gram of ZnCl{2} is
added to 10 grams of the yeast and 50 c.c. of water, whereas it ceases
in the presence of 1·2 gram of ZnCl{2}. Even the addition of 0·075
gram however greatly diminishes the rate of fermentation and the
total amount of sugar decomposed. The most noteworthy effect is that
the production of acetaldehyde is increased both in autofermentation
and [p112] in sugar fermentation. The course of the reaction is
further modified in the sense that the percentage of sugar used
up which can be accounted for in the products decreases, in other
words the "disappearing sugar" (p. 31) increases. In long continued
fermentations moreover and particularly with high concentrations
of zinc chloride less alcohol is produced than is equivalent to
the carbon dioxide evolved. The interpretation of these results is
difficult. Kostytscheff takes them to mean (1) that the zinc salt
modifies one stage of the reaction so that a higher concentration of
intermediate products is obtained, and (2) that the carbon dioxide and
alcohol must be produced at different stages or their ratio, in the
absence of secondary changes, would be unalterable.

Alternative interpretations are, however, by no means excluded. Thus
Neuberg and Kerb [1912, 1; 1913, 2] do not regard it as conclusively
proved that the aldehyde really arises from the sugar since they
have observed its production in maceration extract free from
autofermentation. The method used by Kostytscheff for the separation
of alcohol and aldehyde (treatment with bisulphite) has also proved
unsatisfactory in their hands and the results obtained as to the
reduction of acetaldehyde by yeast, etc., are not accepted. They also
consider that in any case the small amounts produced (less than 0·2
per cent. of the sugar used) would not afford convincing evidence
that the aldehyde is an intermediate product, although it must be
admitted that no large accumulation of an intermediate product
could be reasonably expected. It may also be pointed out that the
increase in "disappearing sugar" may be simply due to the fact that
in the controls the whole of the sugar was fermented, so that any
polysaccharide formed at an earlier stage would have been hydrolysed
and fermented, whereas in the presence of zinc chloride excess of
sugar was present throughout the whole experiment.

Lvoff [1913, 1, 2, 3] has made quantitative experiments on the effect
of methylene blue both on the sugar fermentation and autofermentation
of dried yeast and maceration extract. In presence of sugar the
methylene blue causes a decrease in the extent of fermentation, the
difference during the time required for reduction of the methylene
blue being represented by an amount of glucose equimolecular to
the latter. In the absence of sugar on the other hand an excess of
carbon dioxide equimolecular to the methylene blue is evolved but
no corresponding increase in the alcohol production occurs. The
effect of methylene blue is evidently complex and it is impossible
at present to say whether Lvoff's contention is correct that the
methylene blue actually [p113] interferes with the fermentation by
taking up hydrogen (2 atoms per molecule of glucose) destined for
the subsequent reduction of some intermediate product or whether the
effect is one of general depression of the fermenting power which
would be presumably proportional to the concentration of methylene
blue and inversely proportional to that of the fermenting complex
[see Harden and Norris, R. V., 1914]. In any case it will be noticed
that Lvoff s interpretation of the results is at variance with the
requirements of Kostytscheff's theory (p. 109) according to which 4
atoms of hydrogen should be given off by a molecule of glucose.

Kostytscheff [1913, 2; Kostytscheff and Scheloumoff, 1913] has also
observed a depression of the extent of fermentation by methylene blue
without any serious alteration in the ratio of CO{2} to alcohol,
although an increase occurs in the production of acetaldehyde.

On the whole it cannot be said that the evidence gathered from
experiments on the reduction of acetaldehyde and methylene blue is
very convincing. All that is established beyond doubt seems to be that
yeast possesses a reducing mechanism for many aldehydes [see also
in this connection Lintner and Luers, 1913; Lintner and von Liebig,
1911; as well as Neuberg and Steenbock, 1913, 1914] and colouring
matters. This mechanism appears to be capable of activity in the
absence of sugar and it is to be supposed that in accordance with the
views of Bach [1913] the necessary hydrogen is derived from water
and that some acceptor for the oxygen simultaneously liberated is
also present. There seems however at the moment to be no sufficient
reason to suppose that this mode of reduction is in any way altered
by the presence of sugar and until the production of intermediate
products equivalent to the amount of substance reduced is actually
demonstrated, the conclusions of these workers may be regarded as not
fully justified.

Neuberg and Kerb [1913, 2] themselves tentatively propose a
complicated scheme possessing some novel features according to which
methylglyoxal is the starting-point for the later stages of the change.

(/a/) A small portion of this is converted by a reaction which may be
variously interpreted as a Cannizzaro transformation or a reductase
reaction into glycerol and pyruvic acid.

 CH{2}:C(OH)·CHO + H{2}O  H{2}     CH{2}(OH)·CH(OH)·CH{2}(OH)
                          │                glycerol
                    +     │    =              +
   CH{2}:C(OH)·CHO        O          CH{2}:C(OH)·COOH
                                        Pyruvic acid

(/b/) The pyruvic acid is then decomposed by carboxylase yielding
aldehyde and carbon dioxide (equation 2, p. 109). [p114]

(/c/) The aldehyde and a molecule of glyoxal then undergo a Cannizzaro
reaction and yield alcohol and pyruvic acid,

 CH{3}·CO·CHO     O        CH{3}·CO·COOH
               +  │     =         +
 CH{3}·CHO        H{2}     CH{3}·CH{2}(OH)

and the latter then undergoes reaction (/b/).

A small amount of glycerol is thus necessarily formed, as is actually
found to be the case.

The experimental foundation for stages (/a/) and (/c/) will be awaited
with great interest, as well as the proof that methylglyoxal is
readily fermentable (see p. 104).


 THE FORMIC ACID THEORY.

An interesting interpretation of the phenomena of fermentation was
attempted by Schade [1906] based upon the conception that glucose
under the influence of catalytic agents readily decomposes into
acetaldehyde and formic acid. It was subsequently found that the
experimental evidence upon which this conclusion was founded had
been wrongly interpreted [Buchner, Meisenheimer, and Schade, 1906;
Schade, 1907], but Schade has succeeded in devising an interesting
series of reactions by means of which alcohol and carbon dioxide can
be obtained from sugar by the successive action of various catalysts.
The following are the stages of this series: (1) Glucose, fructose,
and mannose are converted by alkalis into lactic acid along with other
products. (2) Lactic acid when heated with dilute sulphuric acid
yields a mixture of acetaldehyde and formic acid:--

 CH{3}·CH(OH)·COOH = CH{3}·CHO + H·COOH.

(3) It has long been known that formic acid is catalysed by metallic
rhodium at the ordinary temperature into hydrogen and carbon dioxide,
and Schade has found that when a mixture of acetaldehyde and formic
acid is submitted to the action of rhodium the acetaldehyde is reduced
to alcohol at the expense of the hydrogen and the carbon dioxide is
evolved:--

 CH{3}·CHO + H·COOH = CH{3}·CH{2}(OH) + CO{2}.

Schade suggests [1908] that the fermentation of sugar may proceed by a
similar series of reactions catalysed by enzymes, the acetaldehyde and
formic acid being derived not from the relatively stable lactic acid
but more probably from a labile substance capable of undergoing change
either into lactic acid or into aldehyde and formic acid.

It will be noticed that this theory resembles the pyruvic acid [p115]
theory in postulating the immediate formation of acetaldehyde but
differs from it by supposing that the reduction is effected at the
expense of formic acid produced at the same time.

The acetaldehyde question has already been discussed. In view of the
fact that formic acid is a regular product of the action of many
bacteria on glucose [see Harden, 1901], Schade's theory of alcoholic
fermentation may be said to be a possible interpretation of the facts.
Formic acid is known to be present in small amounts in fermented sugar
solutions and the actual behaviour of yeast towards this substance
has been investigated in some detail by Franzen and Steppuhn [1911;
1912, 1, 2], who have obtained results strongly reminiscent of those
obtained with lactic acid by Buchner and Meisenheimer (p. 102).
Many yeasts when grown in presence of sodium formate decompose a
certain proportion of it, whereas in absence of formate they actually
produce a small amount of formic acid--the absolute quantities being
usually of the order of 0·0005 gram molecule (0·023 gram) per 100
c.c. of medium in 4 to 5 days. Only in the case of /S. validus/ did
the consumption of formic acid in 5 days reach 0·0017 gram molecule
(0·08 gram). Somewhat similar but rather smaller results were given
by yeast-juice, a small consumption of formic acid being usually
observed. The possibility thus exists that formic acid may be an
intermediate product of alcoholic fermentation and Franzen argues
strongly in favour of this view.

Direct experiment, on the other hand, shows that yeast-juice cannot
ferment a mixture of acetaldehyde and formic acid, even when these
are gradually produced in molecular proportions in the liquid by
the slow hydrolysis of a compound of the two, ethylideneoxyformate,
OHC·O·CH(CH{3})·O·CH(CH{3})·O·CHO, this method being adopted to avoid
the inhibiting effect of free acetaldehyde and formic acid [Buchner
and Meisenheimer, 1910]. Nor is the reduction of acetaldehyde assisted
by the presence of formate [Neuberg and Kerb, 1912, 4; Kostytscheff
and Hübbenet, 1912].

A modified form of Schade's theory has been suggested by Ashdown
and Hewitt [1910], who have found that when brewer's yeast is
cultivated in presence of sodium formate the yield of aldehyde,
as a rule, becomes less. They regard the aldehyde as derived from
alanine, CH{3}·CH(NH{2})·COOH, one of the amino-acids formed from the
proteins by hydrolysis, which is known to be attacked by yeast in the
characteristic manner (p. 87), forming alcohol, carbon dioxide, and
ammonia. Fermentation is supposed to proceed in such a way that the
sugar is first decomposed into two smaller molecules, C{3}H{6}O{3}
[p116] (equation i), and that these react with formamide to produce
alanine and formic acid (ii). The alanine then enters into reaction
with formic acid, producing alcohol, carbon dioxide, and formamide
(iii):--

 (i)   C{6}H{12}O{6} = 2C{3}H{6}O{3}.

 (ii)  C{3}H{6}O{3} + H·CO·NH{2} = CH{3}·CH(NH{2})·COOH + H·COOH.

 (iii) CH{3}·CH(NH{2})·COOH + H·COOH =
                               CH{3}·CH{2}·OH + CO{2} + H·CO·NH{2}.

According to this scheme all the sugar fermented passes through the
form of alanine, and the formic acid acts along with the enzyme
as catalyst, passing into formamide in reaction (iii) and being
regenerated in (ii). The alanine is in the first place derived from
the hydrolysis of proteins, or possibly by the reaction of the
C{3}H{6}O{3} group with one of the higher amino-acids:--

 C{3}H{6}O{3} + C{n}H{2n+1}·CH(NH{2})·COOH =
                     C{n}H{2n+1}·CH{2}·OH + CO{2} + CH(NH{2})·COOH.

There is as little positive evidence for this course of events as for
that postulated by Schade, and the theory suffers from the additional
disability that the chemical reactions involved have not been realised
in the laboratory. Direct experiments with yeast-juice, moreover,
show that a mixture of alanine with formic acid or a formate is not
fermented, whilst neither the added mixture nor formamide seriously
effects the action of the juice on glucose.


 OTHER THEORIES.

Among other suggestions may be mentioned that of Kohl [1909] who
asserts that sodium lactate is readily fermented, whilst Kusseroff
[1910] holds the view that the glucose is first reduced to sorbitol
and the latter fermented, in spite of the fact that sorbitol itself in
the free state is not fermented by yeast.

The rapid appearance and disappearance of glycogen in the yeast cell
at various stages of fermentation [see Pavy and Bywaters, 1907; Wager
and Peniston, 1910] has led to the suggestion [Grüss, 1904; Kohl,
1907] that this substance is of great importance in fermentation, and
represents a stage through which all the sugar must pass before being
fermented. The fact that the formation of glycogen has been observed
in yeast-juice by Cremer [1899], and that complex carbohydrates are
also undoubtedly formed (p. 31), are consistent with this theory. The
low rate of autofermentation of living yeast, which is only a few per
cent. of the rate of sugar fermentation, renders this supposition
very improbable (Slator), as does the fact that the fermentation of
glycogen by yeast-juice is usually slower than that of glucose [see
also Euler, 1914].

An entirely different explanation of the chemical changes attendant on
alcoholic fermentation has been suggested by [p117] Löb [1906; 1908,
1, 2; 1909, 1, 2, 3, 4; 1910; Löb and Pulvermacher, 1909], founded on
the idea that the various decompositions of the sugar molecule both
by chemical and biological agents are to be explained by a reversal
of the synthesis of sugar from formaldehyde. As the sugar molecule
can be built up by the condensation of formaldehyde, so it tends to
break down again into this substance, and the products observed in any
particular case are formed either by partial depolymerisation in this
sense or by partial re-synthesis following on depolymerisation.

Löb has adduced many striking facts in favour of this view, and has
shown that very dilute alkalis produce no lactic acid but formaldehyde
and a pentose as primary products. These substances represent
the first stage of depolymerisation and are also formed by the
electrolysis of glucose.

Löb has himself been unable to detect definite intermediate products
of fermentation by adding reagents, such as aniline, ammonia, and
phloroglucinol, which would combine with such substances and prevent
their further decomposition [1906].

The occurrence of traces of formaldehyde as a product of alcoholic
fermentation by yeast-juice [Lebedeff, 1908] is at least consistent
with this theory, but no decisive evidence has so far been obtained
either for or against it.

In all the foregoing attempts to indicate the probable stages in the
production of alcohol and carbon dioxide from sugar, a single molecule
of the sugar forms the starting-point. The facts recounted in Chapter
III as to the function of phosphates in alcoholic fermentation, which
are summed up in the equation:--

 2C{6}H{12}O{6} + 2R{2}HPO{4} =
        2CO{2} + 2C{2}H{6}O + 2H{2}O + C{6}H{10}O{4}(PO{4}R{2}){2},

render it in the highest degree probable that two molecules of the
sugar are concerned. The most reasonable interpretation of this
equation appears to be that in the presence of phosphate and of the
complicated machinery of enzyme and co-enzyme two molecules of the
hexose, or possibly of the enolic form, are each decomposed primarily
into two groups.

Of the four groups thus produced, two go to form alcohol and carbon
dioxide and the other two are synthesised to a new chain of six carbon
atoms, which forms the carbohydrate residue of the hexosephosphate.
The introduction of the phosphoric acid groups may possibly occur
before the rupture of the original molecules, and may even be the
determining factor of this rupture, or again this introduction may
take place during or after the formation of the new carbon [p118]
chain. Sufficient information is not yet available for the exact
formulation of a scheme for this reaction. Such a scheme, it may be
noted, would not necessarily be inconsistent with the views of Wohl
and of Buchner as to the way in which the carbon chain of a hexose is
broken in the process of fermentation, but would interpret differently
the subsequent changes which are undergone by the simpler groups
which are the result of this rupture. The reaction might thus proceed
without the formation of definite intermediate products, whilst
opportunity would be afforded for the production of a small quantity
of by-products such as formaldehyde, glycerol, lactic acid, acetic
acid, etc., by secondary reactions.

A symmetrical scheme can readily be constructed for such a change, but
much further information is required before any decisive conclusion
can be drawn as to the precise course of the reaction which actually
occurs in alcoholic fermentation. [p119]




 CHAPTER IX.

 THE MECHANISM OF FERMENTATION.


The analysis of the process of alcoholic fermentation by yeast-juice
and other preparations from yeast which has been carried out in the
preceding chapters has shown that the phenomenon is one of a very
complex character. The principal substances directly concerned in the
change appear to be the enzyme and co-enzyme of the juice, a second
enzyme, hexosephosphatase, and, in addition, sugar, phosphate, and
the hexosephosphate formed from these. During autofermentation two
other factors are involved, the complex carbohydrates of the juice,
including glycogen and dextrins, and the diastatic ferment by which
these are converted into fermentable sugars. It is also possible that
the supply of free phosphate is partially provided by the action of
proteoclastic ferments on phosphoproteins. Under special circumstances
the rate at which fermentation proceeds may be controlled by the
available amount of any one of these numerous substances.

When the juice from well-washed yeast is incubated, the phenomenon
of autofermentation is observed. The juice contains an abundant
supply of enzyme, co-enzyme, and phosphate or hexosephosphate, and
in this case the controlling factor is usually the supply of sugar,
which is conditioned by the concentration of the diastatic enzyme or
of the complex carbohydrates as the case may be. When this is the
case the measured rate of fermentation is the rate at which sugar is
being produced in the juice, this being the slowest of the various
reactions which are proceeding under these circumstances. If sugar be
now added, an entirely different state of affairs is set up. As soon
as any accumulated phosphate has been converted into hexosephosphate,
the normal rate of fermentation which is usually higher than that
of autofermentation is attained, and, provided that excess of sugar
be present, fermentation continues for a considerable period at a
slowly diminishing rate and finally ceases. During the first part of
this fermentation the rate is controlled entirely by the supply of
free phosphate, and this depends mainly on the concentration of the
hexosephosphatase and of the hexosephosphate, and only in a secondary
degree on the decomposition [p120] of other phosphorus compounds by
other enzymes and on the concentration of the sugar. The amount of
hexosephosphate in yeast-juice is usually such that an increase in
its concentration does not greatly affect the rate of fermentation,
and hence the measured rate during this period represents the rate
at which hexosephosphate is being decomposed, and this in its turn
depends on the concentration of hexosephosphatase, which is therefore
the controlling factor. As fermentation proceeds, the concentration
of both enzyme and co-enzyme steadily diminishes, as already
explained, probably owing to the action of other enzymes, so that at
an advanced stage of the fermentation, the controlling factor may
be the concentration of either of these, or the product of the two
concentrations (see p. 122). The hexosephosphatase appears invariably
to outlast the enzyme and co-enzyme. The condition at any moment
could be determined experimentally if it were possible to add enzyme,
co-enzyme and hexosephosphatase at will and so ascertain which of
these produced an acceleration of the rate.

Unfortunately this can at present be only very imperfectly
accomplished, owing to the impossibility of separating these
substances from each other and from accompanying matter which
interferes with the interpretation of the result.

A third condition can also be established by adding to the fermenting
mixture of the juice and sugar a solution of phosphate. The supply
of phosphate is now almost independent of the action of the
hexosephosphatase, and the measured rate represents the rate at which
reaction (1), p. 51, can occur between sugar and phosphate in the
presence of the fermenting complex consisting of enzyme and co-enzyme.
This change is controlled, so long as sugar and phosphate are present
in the proper amounts, by the concentration of the fermenting complex
or possibly of either the enzyme or the co-enzyme. If only a single
addition of a small quantity of phosphate be made, the rate falls as
soon as the whole of this has been converted into hexosephosphate and
the reaction then passes into the stage just considered, in which the
rate is controlled by the production of free phosphate.

Although these varying reactions have not yet been exhaustively
studied from the kinetic point of view, owing to the experimental
difficulties to which allusion has already been made, investigations
have nevertheless been carried out on the effect of the variation of
concentration of yeast-juice and zymin as a whole, as well as of the
carbohydrate. Herzog [1902, 1904] has made experiments of this kind
with zymin, and Euler [1905] with yeast-juice, whilst many of the
results [p121] obtained by Buchner and by Harden and Young are also
available.

The actual observations made by these authors show that the initial
velocity of fermentation is almost independent of the concentration
of sugar within certain limits, but decreases slowly as the
concentration increases. When the velocity constant is calculated on
the assumption that the reaction is monomolecular [see Bayliss, 1914,
Chap. VI], approximate constancy is found for the first period of the
fermentation. This method of dealing with the results is, however,
as pointed out by Slator, misleading, the apparent agreement with
the law of monomolecular reactions being probably due to the gradual
destruction of the fermenting complex.

Experiments with low concentrations of sugar are difficult to
interpret, the influence of the hydrolysis of glycogen and of
dextrins on the one hand, and the synthesis of sugar to more complex
carbohydrates on the other (p. 31), having a relatively great effect
on the concentration of the sugar. Unpublished experiments (Harden
and Young) indicate, however, that the velocity of fermentation
remains approximately constant, until a certain very low limit of
sugar concentration is reached, and then falls rapidly. The fall in
rate, however, only continues over a small interval of concentration,
after which the velocity again becomes approximately constant and
equal to the rate of autofermentation. During this last phase, as
already indicated, the velocity is generally controlled by the rate
of production of sugar and no longer by that of phosphate, this
substance being now present in excess. In other words, the rate of
fermentation of sugar by yeast-juice and zymin is not proportional
to the concentration of the sugar present as required by the law
of mass, but, after a certain low limit of sugar concentration, is
independent of this and is actually slightly decreased by increase in
the concentration of the sugar.

The relations here are very similar to those shown to exist by Duclaux
[1899] and Adrian Brown [1902] for the action of invertase on cane
sugar and are probably to be explained in the manner suggested by
the latter. According to this investigator, the enzyme unites with
the fermentable material, or as it is now termed, the substrate or
zymolyte, forming a compound which only slowly decomposes so that it
remains in existence for a perceptible interval of time. The rate of
fermentation depends on the rate of decomposition of this compound
and hence varies with its concentration. This conception leads to
the result that the rate of fermentation will increase with the
concentration of the substrate up to a certain limit and will then
remain [p122] constant, unless interfered with by secondary actions.
This limit of concentration is that at which there is just sufficient
of the material in question present to combine with practically
the whole of the enzyme, so that no further increase in its amount
can cause a corresponding increase in the quantity of its compound
with the enzyme or in the rate of fermentation which depends on the
concentration of that compound.

The curve relating the rate of action of such an enzyme with the
concentration of the zymolyte therefore consists of two portions, one
in which the rate at any moment is proportional to the concentration
of the zymolyte, according to the well-known law of the action
of mass, and a second in which the rate at any moment is almost
independent of that concentration, approximately equal amounts being
decomposed in equal times whatever the concentration of the substrate.

The results of the experiments with yeast-juice therefore indicate
that what is being measured is a typical enzyme action, but afford
no information as to which of the many possible actions is the
controlling one, a fact which must be ascertained for each particular
case in the manner indicated above.

Clowes [1909], using washed zymin free from fermenting power
and adding various volumes of boiled yeast extract, found that
the velocity of reaction was proportional to the product of the
concentrations of zymin and yeast extract up to a certain optimum
concentration. He interprets these concentrations as representing
the concentrations of zymase and co-enzyme, but they also represent
the concentrations of hexosephosphatase (present in the zymin) and
phosphate (present in the yeast extract), so that at least four
factors were being altered instead of only two.

It has already been mentioned that Euler and Kullberg [1911, 3] found
the conversion of phosphate into hexosephosphate in presence of excess
of glucose to proceed according to a monomolecular reaction (p. 58).

The rate of fermentation is diminished by dilution of the yeast-juice,
but less rapidly than the concentration of the juice. Herzog found
that when the relation between concentration of enzyme and the
velocity constant of the reaction is expressed by the formula
K{1}/K{2} = (C{1}/C{2})^{/n/} where K{1} and K{2} are the velocity
constants corresponding with the enzyme concentrations C{1} and
C{2}, the value for /n/ is 2 for zymin, whilst Euler working with
yeast-juice obtained values varying from 1·29 to 1·67 and decreasing
as K increased.

The temperature coefficient of fermentation by zymin was found [p123]
by Herzog to be K{24·5°}/K{14·5°} = 2·88, which agrees well with the
value found by Slator for yeast-cells (p. 129).

When we endeavour to apply the results of the investigations of the
fermentation of sugar by yeast-juice, zymin, etc., to the process
which goes on in the living cell, considerable difficulties present
themselves. A scheme of fermentation in the living cell can, however,
easily be imagined, which is in harmony with these results. According
to the most simple form of this ideal scheme, the sugar which has
diffused into the cell unites with the fermenting complex and
undergoes the characteristic reaction with phosphate, already present
in the cell, yielding carbon dioxide, alcohol, and hexosephosphate.
The latter is then decomposed, just as it is in yeast-juice, but more
rapidly, and the liberated phosphate again enters into reaction,
partly with the sugar formed from the hexosephosphate and partly
with fresh sugar supplied from outside the cell. The main difference
between fermentation by yeast-juice and by the living cell would then
consist in the rate of decomposition of the hexosephosphate, for it
has been shown that yeast-juice in presence of sufficient phosphate
can ferment sugar at a rate of the same order of magnitude (from 30 to
50 per cent.) as that attained by living yeast.

The difference between the two therefore would appear to lie not so
much in their content of fermenting complex as in their very different
capacity for liberating phosphate from hexosephosphate and thus
supplying the necessary conditions for fermentation.

A simple calculation based on the phosphorus content of living yeast
[Buchner and Haehn, 1910, 2] shows that the whole of this phosphate
must pass through the stage of hexosephosphate every five or six
minutes in order to maintain the normal rate of fermentation, whereas
in an average sample of yeast-juice the cycle, calculated in the same
way, would last nearly two hours.

Wherein this difference resides is a difficult question, which cannot
at present be answered with certainty.

In the first place it must be remembered that a very great
acceleration of the action of the hexosephosphatase is produced
by arsenates (p. 79), and this suggests the possibility that some
substance possessing a similar accelerating power is present in
the yeast-cell and is lost or destroyed in the various processes
involved in rendering the yeast susceptible to phosphate. The great
variety of these processes--extraction of yeast-juice by grinding and
pressing, drying and macerating, heating, treating with acetone and
with toluene--renders this somewhat improbable, and so far no such
substance has been detected. [p124]

A comparison of living yeast, zymin, and yeast-juice shows that these
are situated on an ascending scale with respect to their response to
phosphate. Taking fructose as the substrate in each case, yeast does
not respond to phosphate at all (Slator), the rate of fermentation
by zymin is approximately doubled (p. 46), and that by yeast-juice
increased ten to forty times, whilst the maximum rates are in each
case of the same order of magnitude. Euler and Kullberg, however,
have observed an acceleration of about 25 per cent. in the rate
of fermentation of yeast in presence of a 2 per cent. solution of
monosodium phosphate, NaH{2}PO{4} [1911, 1, 2].

The high rate of fermentation by living yeast and its lack of response
to phosphate may possibly be explained by supposing that the balance
of enzymes in the living cell is such that the supply of phosphate
is maintained at the optimum, and the rate of fermentation cannot
therefore be increased by a further supply.

A further difference lies in the fact that yeast-juice and zymin
respond to phosphate more strongly in presence of fructose than of
glucose, whereas yeast ferments both sugars at the same rate (p. 131),
and this property has been shown to be connected with the specific
relations of fructose to the fermenting complex. It seems possible
that these differences are associated with the gradual passage from
the complete living cell of yeast, through the dead and partially
disorganised cell of zymin to yeast-juice in which the last trace of
cellular organisation has disappeared and the contents of the cell are
uniformly diffused throughout the liquid. Living yeast is, moreover,
not only unaffected by phosphate but only decomposes hexosephosphate
extremely slowly (Iwanoff).

Some light is thrown on these interesting problems by the effect of
antiseptics on fermentation by yeast-cells and by yeast-juice. The
action of toluene has hitherto been most completely studied, and
this substance is an extremely suitable one for the purpose since it
has practically no action whatever on fermentation by yeast-juice.
The experiments of Buchner have, in fact, shown that the normal rate
of fermentation and the total fermentation produced, are almost
unaffected by the presence of toluene even in the proportion of 1 c.c.
to 20 c.c. of yeast-juice. What then is the effect of toluene on the
living yeast-cell? When toluene in large excess is agitated with a
fermenting mixture of yeast and sugar, the rate of fermentation falls
rapidly at first and then more slowly until a relatively constant rate
is attained which gradually decreases in a similar manner to the rate
of fermentation by yeast-juice. Thus at air temperature (16°) 10 grams
of [p125] yeast suspended in 50 c.c. of 6 per cent. glucose solution
gave the following results when agitated with toluene:--

 ───────────────────────┬───────────────┬───────┬──────────
   Time after Addition  │ C.c. of CO{2} │ Time. │ C.c. per
    of Toluene, Minutes │   per Minute. │       │ Minute
 ───────────────────────┴───────────────┴───────┴──────────
             0                 4·6         6        1·6
             1                 4           8        1·2
             2                 3·3        12        0·85
             3                 2·6        24        0·8
             4                 2          32        0·5
             5                 1·8                constant
 ──────────────────────────────────────────────────────────

Simultaneously with this, the yeast acquires the property of
decomposing and fermenting hexosephosphate and of responding to the
addition of phosphate. This last property is only acquired to a small
degree in this way but it becomes much more strongly developed if
the pressed yeast be washed with toluene on the filter pump. Thus 10
grams of yeast after this treatment fermented fructose at 1·2 c.c. per
three minutes; after the addition of phosphate (5 c.c. of 0·6 molar
phosphate) the rate rose to 6·9 and then gradually fell in the typical
manner [Harden, 1910; see also Euler and Johansson, 1912, 3].

The current explanation of the great decrease in rate of fermentation
which attends the action of toluene and other antiseptics on living
yeast, and also follows upon the disintegration of the cell, appears
to be that in living yeast the high rate of fermentation is maintained
by the continued production of relatively large fresh supplies
of fermenting complex, and that when the power of producing this
catalytic agent is destroyed by the poison, the rate of fermentation
falls to a low value, corresponding to the store of zymase still
present in the cell (cf. Buchner, E. and H., and Hahn, 1903, pp. 176,
180).

This explanation implies that the rate of fermentation after the
action of the toluene represents the amount of fermenting complex
present, a supposition which has been shown (p. 53) to be highly
improbable. It further necessitates, as also pointed out independently
by Euler and Ugglas [1911], a rapid destruction of the fermenting
complex both in the process of fermentation and by the action of the
antiseptic, as otherwise the store of zymase remaining in the dead
cell would be practically the same as that contained in the living
cell at the moment when it was subjected to the antiseptic, and this
store would therefore suffice to carry out fermentation at the same
rate in the dead as in the living cell. No such rapid destruction,
however, occurs in yeast-juice, as judged by the rate of fermentation,
which falls off [p126] slowly and to about the same extent in the
presence or absence of toluene. Moreover, as shown above, it is
highly probable that the actual amount of fermenting complex in
yeast-juice is a large fraction of that present at any moment in
the cell, and is capable under suitable conditions of producing
fermentation at a rate comparable with that of the living cell.

This last criticism also applies to the view expressed by Euler
[Euler and Ugglas, 1911; Euler and Kullberg, 1911, 1, 2] that in the
living cell the zymase is partly free and partly combined with the
protoplasm; when the vital activity of the cell is interfered with,
the combined portion of the zymase is thrown out of action and only
that which was free remains active.

The suggestion made by Rubner [1913] that the action of yeast on sugar
is in reality chiefly a vital act, but that a small proportion of
the change is due to enzyme action, is similar in its consequences
to that of Euler and may be met by the same arguments. Buchner and
Skraup [1914] have moreover shown that the effects of sodium chloride
and toluene on the fermenting power of yeast which were observed by
Rubner, can be explained in other ways.

Some other explanation must therefore be sought for this phenomenon.
Great significance must be attached in this connection to the relation
noted above between the degree of disintegration and disorganisation
of the cell and the fall in the normal rate of fermentation. It seems
not impossible that fermentation may be associated in the living cell
with some special structure, or carried on in some special portion
of the cell, perhaps the nuclear vacuole described by Janssens and
Leblanc [1898], Wager [1898, 1911; Wager and Peniston, 1910] and
others which undergoes remarkable changes both during fermentation
and autofermentation [Harden and Rowland, 1901]. The disorganisation
of the cell might lead to many modifications of the conditions,
among others to the dilution of the various catalytic agents by
diffusion throughout the whole volume of the cell. As a matter of
observation the dilution of yeast-juice leads to a considerable
diminution of the rate of fermentation of sugar, and it is possible
that this is one of the chief factors concerned. That phenomena
of this kind may be involved is shown by the remarkable effect of
toluene on the autofermentation of yeast. Whereas the fermentation
of sugar is greatly diminished by the action of toluene, the rate of
autofermentation, which is carried on at the expense of the glycogen
of the cell, is greatly increased. In a typical case, for example, the
autofermentation of 10 grams of yeast suspended in 20 c.c. of water
amounted to 28 c.c. in 4·8 hours [p127] at 25°, whereas the same
amount of yeast in presence of 2 c.c. of toluene gave 97·6 c.c. in
the same time.

Many salts produce a similar effect on English top yeasts (in which
the autofermentation is large) [Harden and Paine, 1912], whereas
Neuberg and Karczag in Berlin [1911, 2] were unable to observe this
phenomenon.

A necessary preliminary of the fermentation of glycogen is its
conversion by a diastatic enzyme into a fermentable sugar, and it
is probable that the effect of the disorganisation of the cell by
toluene is that this enzyme finds more ready access to the glycogen,
which is stored in the plasma of the cell. No such acceleration
of autofermentation is effected by the addition of toluene to
yeast-juice, and hence the result is not due to an acceleration of the
action of the diastatic enzyme on the glycogen.

This effect of toluene is similar in character to the action of
anæsthetics on the leaves of many plants containing glucosides and
enzymes, whereby an immediate decomposition of the glucoside is
initiated [see H. E. and E. F. Armstrong, 1910].

Although as indicated above Euler's theory cannot apply to zymase
itself, if applied to the hexosephosphatase it would afford a
consistent explanation of the facts. According to this modified view
it would be the hexosephosphatase of yeast which existed largely in
the combined form, so that in extracts, in dried yeast and in presence
of toluene only the small fraction which was free would remain active.
The zymase on the other hand would have to be regarded as existing to
a large extent in the free state so that it would pass into extracts
comparatively unimpaired in amount and capable under proper conditions
(i.e. when supplied with sufficient phosphate) of bringing about a
very vigorous fermentation. The theory of combined and free enzymes is
undoubtedly of considerable value, although it cannot be considered as
fully established.


 FERMENTATION BY LIVING YEAST.

Much important information as to the nature of the processes involved
in fermentation has been acquired by the direct experimental study of
the action of living yeast on different sugars.

This phenomenon has formed the subject of several investigations
from the kinetic point of view, and its general features may now be
regarded as well established.

The difficulty, which must as far as possible be avoided in
quantitative experiments of this sort with living yeast, is the
alteration [p128] in the amount or properties of the yeast, due to
growth or to some change in the cells. This has been obviated in
the work of Slator [1906] by determining in every case the initial
rate of fermentation, so that the process only continues for a very
short period, during which any change in the amount or constitution
of the yeast is negligible. The method has the further advantage that
interference of the products of the reaction is to a large extent
avoided. The pressure apparatus already described (p. 29) was employed
by Slator, the rate of production of carbon dioxide being measured by
the increase of pressure in the experimental vessel.


 INFLUENCE OF CONCENTRATION OF DEXTROSE ON THE RATE OF FERMENTATION.

With regard to this important factor it is found that the action
of living yeast follows the same law as that of most enzymes (p.
121); within certain wide limits the rate of fermentation is almost
independent of the concentration of the sugar. This conclusion has
been drawn by many previous investigators from their experiments
[Dumas, 1874; Tammann, 1889; Adrian Brown, 1892; O'Sullivan, 1898,
1899] and is implicitly contained in the results of Aberson [1903],
although he himself regarded the reaction as monomolecular.

 [Illustration: FIG. 8. (Graph Fermentation Velocity vs Conc.
 Dextrose--Ed.)]

Slator, working with a suspension of ten to twelve yeast-cells per
1/4000 cubic millimetre at 30°, obtained the results which are
embodied in the curve (Fig. 8).

This shows that, for the amount of yeast in question, the rate of
fermentation is almost constant for concentrations of glucose between
[p129] 1 and 10 grams per 100 c.c., but gradually decreases as the
concentration increases. Below 1 gram per 100 c.c. the rate decreases
very rapidly with the concentration.

It follows from this, in the light of what has already been said (p.
121), that the action of living yeast on sugar follows the same course
as a typical enzyme reaction, although in this case, as in that of
yeast-juice, no information is given as to the exact nature of this
reaction.


 INFLUENCE OF THE CONCENTRATION OF YEAST.

It appears to be well established that, when changes in the quantity
and constitution of the yeast employed are eliminated, the rate of
fermentation is exactly proportional to the number of the yeast-cells
present (Aberson, Slator). This result might be anticipated, as
pointed out by Slator, from the fact that the fermentation takes place
within the cell, each cell acting as an independent individual.

The diffusion of sugar into the yeast-cell which necessarily precedes
the act of fermentation has been shown by Slator and Sand [1910] to
occur at such a rate that the supply of sugar is always in excess of
the amount which can be fermented by the cell.


 TEMPERATURE COEFFICIENT OF ALCOHOLIC FERMENTATION BY YEAST.

The temperature coefficient of fermentation by living yeast has been
carefully determined by Slator by measurements of the initial rates at
a series of temperatures from 5° to 40° C. The coefficient is found to
be of the same order as that for many chemical reactions, but to vary
considerably with the temperature, a rise in temperature corresponding
with a diminution in the coefficient. The following values were
obtained for glucose; they are independent of the concentration
of yeast and glucose, the class of yeast, and presence or absence
of nutrient salts, and remain the same when inhibiting agents are
present. Almost precisely the same ratios are obtained for fructose
and mannose:--

 t.     V{/t/ + 5}/V{/t./}     V{/t/ + 10}/V{/t./}
  5          2·65                   5·6
 10          2·11                   3·8
 15          1·80                   2·8
 20          1·57                   2·25
 25          1·43                   1·95
 30          1·35                   1·6
 35          1·20

Aberson's result, K{/t/ + 10}/K{/t/} = 2·72, which represents the
mean coefficient for 10° between 12° and 33°, agrees well with this.
[p130]


 ACTION OF ACCELERATING AGENTS ON LIVING YEAST.

Slator [1908, 1] was unable to find any agent which greatly
accelerated the rate of fermentation of living yeast. Small
concentrations of various inhibiting agents which are often supposed
to act in this way were quite ineffective, and phosphates, which
produce such a striking change in yeast-juice, were almost without
action (cp. p. 124).

Euler and Bäckström [1912], however, have made the important
observation that sodium hexosephosphate causes a considerable
acceleration although it is itself neither fermented nor hydrolysed
under these conditions. The extent of this is evident from the
following numbers:--

 ────────────────────────────────────────────────────
       20 c.c. of 20 per cent. glucose solution.
    0·25 g. yeast [Yeast H of St. Erik's brewery].
 ────────────────────┬───────────────────────────────
   Without addition. │ + 0·5 g. Na hexosephosphate.
 ────────┬───────────┼───────────┬───────────────────
   Time. │  CO{2}.   │    Time.  │     CO{2}.
    Min. │           │     Min.  │
 ────────┼───────────┴───────────┴───────────────────
     46  │   10·5           37            8
     76  │   17·5           73           19
    197  │   45            188           52·5
    347  │   74·5          321          123
    488  │   95            450          193·5
 ────────────────────────────────────────────────────

The observation has been confirmed with English top yeast (Harden and
Young, unpublished experiments), but no explanation of the phenomenon
is at present forthcoming.

Euler has also found [Euler and Cassel, 1913; Euler and Berggren,
1912] that yeast extract, sodium nucleinate and ammonium formate also
increase the rate of fermentation of glucose by yeast, but these
results have been criticised by Harden and Young [1913] on the ground
that the possibility of growth of the yeast during the experiment has
not been excluded.


 FERMENTATION OF DIFFERENT SUGARS BY YEAST.

Many valuable ideas as to the nature of fermentation have been
obtained by a consideration of the phenomena presented by the action
of yeast on the different hexoses. Of these only glucose, fructose,
mannose, and galactose are susceptible of alcoholic fermentation by
yeast, the stereoisomeric hexoses prepared in the laboratory being
unfermentable, as are also the pentoses, tetroses, and the alcohols
corresponding to all the sugars. The yeast-cell is therefore much more
limited in its power of producing fermentation than such an organism
as, for example, /Bacillus coli communis/, which attacks substances
as [p131] diverse as arabinose, glucose, glycerol and mannitol, and
yields with all of them products of the same chemical character,
although in varying proportions.

A careful examination of a number of different genera and species
of the Saccharomycetaceæ and allied organisms by E. F. Armstrong
[1905] has shown that all yeasts which ferment glucose also ferment
fructose and mannose. Armstrong grew his yeasts in a nutrient solution
containing the sugar to be investigated, and his experiments are
open to the criticism that the organisms were hereby afforded an
opportunity for becoming acclimatised to the sugar. His results,
therefore, only demonstrate the fact that the organisms in question
when cultivated in presence of the sugars examined brought about
their fermentation, and do not exclude the possibility that the same
organism when grown in presence of a different sugar might not be
capable of fermenting the one to which it had in the other type of
experiment become acclimatised.

This has actually been shown to be the case for galactose by Slator
[1908, 1], and it is possible that this circumstance explains the
negative results obtained by Lindner [1905] with /S. exiguus/ and
/Schizosaccharomyces Pombe/ upon mannose, a sugar which, according to
Armstrong, is fermented by both these organisms.

The same problem has been attacked quantitatively by Slator, who has
shown that living yeast of various species and genera ferments glucose
and fructose at approximately the same rate. Moreover, when the yeast
is acted upon by various inhibiting agents, such as heat, iodine,
alcohol, or alkalis, the crippled yeast also ferments glucose and
fructose at the same rate.

With mannose the relations are somewhat different. The relative rate
of fermentation of mannose and glucose by yeast is dependent on the
variety of the yeast and the treatment which it has received. Fresh
samples of yeast ferment mannose more quickly than glucose, but by
older samples the glucose is the more rapidly decomposed. This is
especially the case with yeast, the activity of which has been partly
destroyed by heat, the relative fermenting power to mannose being
sometimes reduced by this treatment from 120 per cent. of that of
glucose to only 12 per cent. (Slator).

A further difference consists in the fact that with certain yeasts the
rate of fermentation of glucose is somewhat increased by monosodium
phosphate whilst that of mannose is unaffected [Euler and Lundeqvist,
1911].

Mixtures of glucose and fructose are fermented by yeast at the
[p132] same rate as either the glucose or the fructose contained in
the mixture would be alone. When, however, mannose and glucose are
fermented simultaneously interference between the reactions takes
place, and this is especially evident when the yeast has comparatively
little action on mannose. The following are the results obtained by
Slator:--

 ┌───────────────────────┬─────────────────────────────────────────┐
 │                       │               Relative Rates.           │
 │         Yeast.        ├─────────────┬─────────────┬─────────────┤
 │                       │2·5 per cent.│2·5 per cent.│2·5 per cent.│
 │                       │             │             │  Glucose +  │
 │                       │   Glucose.  │   Mannose.  │2·5 per cent.│
 │                       │             │             │  Mannose.   │
 └───────────────────────┴─────────────┴─────────────┴─────────────┘
   S. Thermantitonum           100           105           92

   Brewery yeast,
   53 per cent. activity
   destroyed by heat           100            21           33

   Brewery yeast,
   60 per cent. activity
   destroyed by heat           100            12           42
 ──────────────────────────────────────────────────────────────────

The case of galactose merits special attention. Previous
investigations [see Lippmann, 1904, p. 734] have shown that the
fermentation of galactose by yeast differs greatly from that of the
other hexoses. The subject has been re-investigated by E. F. Armstrong
[1905], and by Slator [1908, 1]. Armstrong carried out his experiments
in the manner already described (p. 131), and found that some yeasts
had, and others had not, the power of fermenting galactose, although
all were capable of fermenting glucose, fructose, and mannose.

Slator made quantitative experiments on the same subject. He was able
to confirm the statement which had previously been made, that certain
yeasts which have the property of fermenting galactose possess it only
after the yeast has become acclimatised by culture in presence of the
sugar. This was shown for brewery yeast and for the species mentioned
below. This phenomenon is one of great interest and is strictly
analogous to the adaptation of bacteria which has now been quite
conclusively established [Neisser, 1906].

 ┌────────────────────┬────────────────────┬───────────────────────┐
 │                    │                    │   Relative Rates.     │
 │     Yeast.         │ Mode of Culture.   ├──────────┬────────────┤
 │                    │    Grown in:       │ Glucose. │ Galactose. │
 └────────────────────┴────────────────────┴──────────┴────────────┘
   S. Carlsbergensis    wort                   100         <1
          "             hydrolysed lactose     100      86, 83, 85,
          "                 "         "                 25, 46, 51,
          "                 "         "                 69, 54, 155
   S. Cerevisiæ         wort                   100          <1
          "             hydrolysed lactose     100      21, 26, 29
   S. Thermantitonum.   wort                   100         <1
          "             hydrolysed lactose     100      77, 53, 35
   S. Ludwigii          wort                   100         <1
          "             hydrolysed lactose     100         <1
 ───────────────────────────────────────────────────────────────────

[p133]

It will be seen that in one case the rate of fermentation of galactose
was considerably greater than that of glucose. /S. Ludwigii/ did not
respond to the cultivation in hydrolysed lactose, but, as Slator
points out, it is quite possible that repeated cultivation in this
medium might effect the change, and this would be strictly analogous
to the results obtained with bacteria. Slator's results have been
confirmed by Harden and Norris, R. V. [1910], and by Euler and
Johansson [1912, 2] who have made an exceedingly interesting study of
the progress of the adaptation. As in the case of mannose the rates
of fermentation of glucose and galactose are differently affected by
agents such as heat and alcohol; moreover, the rate of fermentation of
mixtures of dextrose and galactose is in no case either the sum or the
mean of the rates obtained with the separate sugars. The temperature
coefficient of the fermentation of galactose also differs slightly
from that of the other hexoses.

 ┌───────────────────┬────────────────────────────────────┐
 │                   │          Relative Rates.           │
 │      Yeast.       ├──────────┬────────────┬────────────┤
 │                   │ Glucose. │ Galactose. │ Glucose +  │
 │                   │          │            │ Galactose. │
 └───────────────────┴──────────┴────────────┴────────────┘
   S. Cerevisiæ           100           0          103
        "                 100          34          103
   S. Carlsbergensis      100         155          119
   S. Thermantitonum      100          76          124
 ──────────────────────────────────────────────────────────

Assuming that his conclusion that all yeasts which ferment glucose
also ferment fructose and mannose is correct, Armstrong has drawn
attention to the fact that these three hexoses are also related by the
possession of a common enolic form (p. 97) and has suggested that this
enolic form is the substance actually fermented to carbon dioxide and
alcohol [1904].

The idea that such an intermediate form is the direct subject of
fermentation has much to recommend it. In the first place it is
almost certain, as already pointed out, that the sugars in aqueous
solution do exist, although to a very small extent, in this enolic
form. The slow rate at which equilibrium is established in aqueous
solution, however, must be taken as definite evidence that under
these circumstances the enolic form is only produced very slowly
[compare Lowry, 1903]. This has been used by Slator [1908, 1] as an
argument against the probability of the preliminary conversion of
the sugars into the enolic form before fermentation. It appears,
however, quite possible that under the influence of the fermenting
complex of the yeast-cell, or of special enzymes, this change might
occur much more rapidly, [p134] and at different rates with the
different sugars. This reaction might in fact control the observed
rate of fermentation. This conception affords a simple explanation of
the different rates of fermentation of mannose and glucose, and also
of galactose, the enolic form of which is quite different, by yeast
under different circumstances, but does not explain the uniformity
of rate observed by Slator for glucose and fructose nor the results
with mixtures of sugars. The direct fermentation of a common enolic
form is also consistent with the fact that the same hexosephosphate is
produced from all three hexoses.

Slator himself prefers the view that the first stage of fermentation
consists in the rapid combination of the sugar with the enzyme,
producing a compound, which then breaks up at a rate which determines
the observed rate of fermentation. This rate will of course vary
with the nature of the compound, so that if two sugars form the
same compound they will be fermented at the same rate; if they form
different compounds, different rates may result. Slator supposes
that glucose and fructose form the same compound with the enzyme.
This, however, appears to involve an intramolecular change of the
same order as the production of the enolic form, and moreover is not
absolutely essential, as it is probably sufficient to suppose that
the two compounds derived from glucose and fructose are very similar,
although possibly not absolutely identical. Mannose and galactose, on
the other hand, form stereoisomeric compounds, and the capacity of the
fermenting complex to form these compounds may be affected by various
agents to a different extent from its capacity for combining with
glucose or fructose.

A third theory has also been suggested to explain these phenomena,
according to which the various sugars are fermented by different
enzymes [see Slator, 1908, 1]. The uniformity of the result obtained
with glucose and fructose suggests that these two sugars are fermented
by the same enzyme (glucozymase), mannose and galactose by different
ones (mannozymase and galactozymase). This would afford a simple
explanation of the different rates of fermentation for different
sugars and of different degrees of sensitiveness towards reagents.

If, however, a separate and independent mechanism were present for
each sugar, the rate of fermentation of mixtures should be the sum of
the rates for the constituents. This, as shown above, is not found to
be the case, and it is therefore necessary to suppose, either that
one sugar influences the fermentation of another in some unknown way,
or that only a part of the mechanism of fermentation is specific
for the particular sugar. Thus the enzyme may be specific and, the
co-enzyme [p135] non-specific, so that only a certain maximum rate
is attainable, or again, the supply of free phosphate may be the
controlling factor.

In the prevailing state of ignorance as to the exact function of
the co-enzyme and of the conditions upon which the velocity of
fermentation in the cell depends, it is at present impossible to
decide between these various theories, but they all offer points of
attack which justify the hope that much further information can be
obtained by experimental inquiry.

It will be seen from the foregoing that Buchner's discovery of zymase
has opened a chapter in the history of alcoholic fermentation which
is yet far from being completed. In every direction fresh problems
present themselves, and it cannot be doubted that as in the past, the
investigation of the action of the yeast-cell will still prove to
be of fundamental importance for our knowledge of the mode in which
chemical change is brought about by living organisms. [p136]




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     Ber., 3, 63-75.

 BARGER, G. (1914), /The simpler natural bases/ (this
 series)                                                        90

 BAYLISS, W. M. (1914), /The nature of enzyme action/      89, 121
     (Longmans, Green & Co., London.)

 BEIJERINCK, M. W. (1897), /Weitere Beobachtungen über
 die Octosporushefe/                                            19
     Centr. Bakt. Par., Abt. II., 3, 449-454.

 BEIJERINCK, M. W. (1900), /Ueber Chinonbildung durch
 Streptothrix chromogena und Lebensweise dieses Mikroben/       19
     Centr. Bakt. Par., Abt. II., 6, 2-12.

 BERTHELOT, M. (1857), /Sur la fermentation alcoolique/         14
     Compt. rend., 44, 702-706.

 BERTHELOT, M. (1860), /Sur la fermentation glucosique du
 sucre de canne/                                                14
     Compt. rend., 50, 980-984.

 BERTRAND, G. (1897), /Sur l'intervention du manganèse
 dans les oxydations provoquées par la laccase/                 63
     Compt. rend., 124, 1032-1035.

 BERTRAND, G. (1904), /Étude Biochimique de la Bacterie du
 Sorbose/                                                      105
     Ann. Chim. Phys., (8), 3, 181-288.

 BERZELIUS, JACOB (1836), /Einige Ideen über eine bei der
 Bildung organischer Verbindungen in der lebenden Natur
 wirksame, aber bisher nicht bemerkte Kraft/                     8
     Berz. Jahresbericht, 15, 237-245.

 BERZELIUS, JACOB (1839)                                         7
     Berz. Jahresbericht, 18, 400-403.

 BERZELIUS, JACOB (1848)                                         9
     Berz. Jahresbericht, 27, 500.

 BOKORNY, TH. (1906), /Ueber die Trennung von Leben und
 Gärkraft in der Hefe/                                          19
     Pflüger's Arch., 114, 535-544.

 BOYSEN-JENSEN, P. (1908), /Die Zersetzung des Zuckers
 während des Respirationsprocesses/. (Vorläufige Mitteilung)   105
     Ber. deut. bot. Ges., 26a, 666-667.

 BOYSEN-JENSEN, P. (1910), /Sukkersönderdelingen under
 Respirationsprocessen hos höjere Planter/                     105
     Dissertation, Köbenhavn. (H. H. Hagerup.)

 BOYSEN-JENSEN, P. (1914), /Die Zersetzung des Zuckers bei
 der alkoholischen Gärung/                                     105
     Biochem. Zeitsch., 58, 451-466.

 BREDIG, GEORG (1901), /Anorganische Fermente/                  37
     (Engelmann, Leipzig), 68.

 BROWN, ADRIAN, J. (1892), /Influence of oxygen and
 concentration on alcoholic fermentation/                      128
     J. Chem. Soc., 61, 369-385.

 BROWN, ADRIAN, J. (1902), /Enzyme action/                     121
     J. Chem. Soc., 81, 373-388.

 BRUYN, C. A. LOBRY DE (1895), /Action des alcalis sur les
 hydrates de carbone, I./ (Expériences provisoires)             96
     Rec. trav. chim., 14, 156-165.

 BRUYN, C. A. LOBRY DE, and W. A. VAN EKENSTEIN (1895),
 /Action des alcalis sur les sucres, II. Transformation
 réciproque des uns dans les autres des sucres glucose,
 fructose et mannose/                                           96
     Rec. trav. chim., 14, 203-216.

 BRUYN, C. A. LOBRY DE, and W. A. VAN EKENSTEIN (1896),
 /Action des alcalis sur les sucres, III. Transformations
 des sucres sous l'influence de l'hydroxyde de plomb/           96
     Rec. trav. chim., 15, 92-96.

 BRUYN, C. A. LOBRY DE, and W. A. VAN EKENSTEIN (1897, 1),
 /Action des alcalis sur les sucres, IV. Remarques générales/   96
     Rec. trav. chim., 16, 257-261.

 BRUYN, C. A. LOBRY DE, and W. A. VAN EKENSTEIN (1897, 2),
 /Action des alcalis sur les sucres, V. Transformation de la
 galactose, les tagatoses et la galtose/                        96
     Rec. trav. chim., 16, 262-273.

 BRUYN, C. A. LOBRY DE, and W. A. VAN EKENSTEIN (1897, 3),
 /Action des alcalis sur les sucres, VI. La glutose et la
 pseudo-fructose/                                               96
     Rec. trav. chim., 16, 274-281.

 BRUYN, C. A. LOBRY DE, and W. A. VAN EKENSTEIN (1897, 4),
 /Action de l'eau bouillante sur la fructose/                   96
     Rec. trav. chim., 16, 282-283.

 BUCHNER, EDUARD (1897, 1), /Alkoholische Gärung ohne
 Hefezellen./ [Vorläuf. Mitt.]                              16, 18
     Ber., 30, 117-124.

 BUCHNER, EDUARD (1897, 2), /Alkoholische Gärung ohne
 Hefezellen./ Zweite Mitt.                              18, 20, 21
     Ber., 30, 1110-1113.

 BUCHNER, EDUARD (1898), /Ueber zellenfreie Gärung/             18
     Ber., 31, 568-574.

 BUCHNER, EDUARD (1900, 1), /Zymase aus getöteter Hefe/         21
     Ber., 33, 3307-3310.

 BUCHNER, EDUARD (1900, 2), /Bemerkungen zur Arbeit
 von A. Macfadyen, G. H. Morris and S. Rowland "Ueber
 ausgepresstes Hefezellplasma (Buchner's Zymase)"https://www.gutenberg.org/          21, 33
     Ber., 33, 3311-3315.

 BUCHNER, EDUARD (1904), /Zur Geschichte der
 Gärungstheorien/                                                9
     Wochensch. Brauerei, 21, 507-510.

 BUCHNER, EDUARD (1910), /Sur la fermentation
 alcoolique du Sucre/                                     105, 106
     Bull. Soc. Chim.

 BUCHNER, EDUARD, und WILHELM ANTONI (1905, 1), /Weitere
 Versuche über die zellfreie Gärung/                        36, 40
     Zeitsch. physiol. Chem., 44, 206-228.

 BUCHNER, EDUARD, und WILHELM ANTONI (1905, 2),
 /Existiert ein Coenzym für die Zymase?/                        62
     Zeitsch. physiol. Chem., 46, 136-154.

 BUCHNER, EDUARD, HANS BUCHNER, und MARTIN HAHN (1903),
 /Die Zymasegärung. Untersuchungen über den Inhalt der
 Hefezellen und die biologische Seite des Gärungsproblems/ 18, 20,
          21, 22, 30, 34, 35, 36, 37, 38, 39, 42, 77, 78, 111, 125
     [R. Oldenbourg, München, pp. viii, 416.]

 BUCHNER, EDUARD, und FRANZ DUCHAČEK (1909), /Ueber
 fraktionierte Fällung des Hefepresssaftes/                 38, 66
     Biochem. Zeitsch., 15, 221-253.

 BUCHNER, EDUARD, und HUGO HAEHN (1909), /Ueber das
 Spiel der Enzyme im Hefepresssaft/                         62, 68
  Biochem. Zeitsch., 19, 191-218.

 BUCHNER, EDUARD, und HUGO HAEHN (1910, 1), /Ueber eine
 Antiprotease im Hefepresssaft/                                 42
     Biochem. Zeitsch., 26, 171-198.

 BUCHNER, EDUARD, und HUGO HAEHN (1910, 2), /Studien über
 den Phosphorgehalt der Hefe und einiger Hefepräparate/    65, 123
     Biochem. Zeitsch., 27, 418-426.

 BUCHNER, EDUARD, und ROBERT HOFFMANN (1907), /Einige
 Versuche mit Hefepresssaft/                                    36
     Biochem. Zeitsch., 4, 215-234.

 BUCHNER, EDUARD, und F. KLATTE (1908), /Ueber das Koenzym
 des Hefepresssaftes/                                       64, 67
     Biochem. Zeitsch., 8, 520-557.

 BUCHNER, EDUARD, und JAKOB MEISENHEIMER (1904), /Die
 chemischen Vorgänge bei der alkoholischen Gärung/    98, 100, 102
     Ber., 37, 417-428.

 BUCHNER, EDUARD, und JAKOB MEISENHEIMER (1905), /Die
 chemischen Vorgänge bei der alkoholischen Gärung./ (Zweite
 Mitteilung)                                      97, 98, 100, 102
     Ber., 38, 620-630.

 BUCHNER, EDUARD, und JAKOB MEISENHEIMER (1906), /Die
 chemischen Vorgänge bei der alkoholischen Gärung./ (Dritte
 Mitteilung)                                  31, 87, 95, 102, 104
     Ber., 39, 3201-3218.

 BUCHNER, EDUARD, und JAKOB MEISENHEIMER (1908), /Ueber
 Buttersäuregärung/                                            110
     Ber., 41, 1410-1419.

 BUCHNER, EDUARD, und JAKOB MEISENHEIMER (1909), /Ueber
 die Rolle der Milchsäure bei der alkoholischen Gärung des
 Zuckers/                                            100, 102, 103
     Zeitsch. wiss. Landwirtschaft, 38, Ergänzungsband
     V., 265-288.

 BUCHNER, EDUARD, und JAKOB MEISENHEIMER (1910), /Die
 chemischen Vorgänge bei der alkoholischen Gärung./ (IV.
 Mitteilung)                               103, 104, 105, 106, 115
     Ber., 43, 1773-1795.

 BUCHNER, EDUARD, und JAKOB MEISENHEIMER (1912), /Die
 chemischen Vorgänge bei der alkoholischen Gärung/        105, 106
     Ber., 45, 1633-1643.

 BUCHNER, EDUARD, J. MEISENHEIMER, und H. SCHADE (1906),
 /Zur Vergärung des Zuckers ohne Enzyme/                       114
     Ber., 39, 4217-4231.

 BUCHNER, EDUARD, und SIGURD MITSCHERLICH (1904),
 /Herstellung glykogenarmer Hefe und deren Anwendung zum
 Zuckernachweis im Harn/                                        39
     Zeitsch. physiol. Chem., 42, 554-562.

 BUCHNER, EDUARD, und RUDOLF RAPP (1897), /Alkoholische
 Gärung ohne Hefezellen/                            19, 34, 36, 77
     Ber., 30, 2668-2678.

 BUCHNER, EDUARD, und RUDOLPH RAPP (1898, 1), /Alkoholische
 Gärung ohne Hefezellen/                                15, 59, 77
     Ber., 31, 209-217.

 BUCHNER, EDUARD, und RUDOLF RAPP (1898, 2),
 /Alkoholische Gärung ohne Hefezellen./ (5 Mitteilung)      36, 77
     Ber., 31, 1084-1090.

 BUCHNER, EDUARD, und RUDOLF RAPP (1898, 3),
 /Alkoholische Gärung ohne Hefezellen./ (6 Mitteilung)  32, 36, 77
     Ber., 31, 1090-1094.

 BUCHNER, EDUARD, und RUDOLF RAPP (1898, 4),
 /Alkoholische Gärung ohne Hefezellen./ (7 Mitteilung)          38
     Ber., 31, 1531-1533.

 BUCHNER, EDUARD, und RUDOLF RAPP (1899, 1),
 /Alkoholische Gärung ohne Hefezellen./ (8 Mitteilung)          36
     Ber., 32, 127-137.

 BUCHNER, EDUARD, und RUDOLF RAPP (1899, 2),
 /Alkoholische Gärung ohne Hefezellen./ (9 Mitteilung)  32, 42, 77
     Ber., 32, 2086-2094.

 BUCHNER, EDUARD, und RUDOLF RAPP (1901), /Alkoholische
 Gärung ohne Hefezellen./ (10 Mitteilung)                       38
     Ber., 34, 1523-1530.

 BUCHNER, EDUARD, und SIEGFRIED SKRAUP (1914), /Ist die
 Enzym-Theorie der Gärung einzuschränken?/                     127
     Ber., 1914, 47, 853-870.

 BUCHNER, EDUARD, and ALBERT SPITTA (1902), /Zymasebildung
 in der Hefe/                                                   38
     Ber., 35, 1703-1706.

 BUCHNER, HANS (1897), /Die Bedeutung der aktiven löslichen
 Zellprodukte für den Chemismus der Zelle/                      18
     München. med. Wochensch. (No. 12), 44, 300-302, and 322.


 CAGNIARD-LATOUR (1838), /Mémoire sur la Fermentation
 vineuse./ (Présenté à l'Académie des Sciences, le 12 juin,
 1837)                                                           5
     Ann. Chim. Phys., 68, 206-222.

 CAVENDISH, HENRY (1766), /Three papers containing
 experiments on factitious air. Part III. Containing
 experiments on the air, produced by fermentation and
 putrefaction./ (Read 13 Nov., 1766)                             2
     Phil. Trans., 175-184.

 CHICK, FRANCES (1912), /Die vermeintliche
 Dioxyacetonbildung während der alkoholischen Gärung und
 die Wirkung von Tierkohle und von Methylphenylhydrazin auf
 Dioxyaceton/                                                  105
     Biochem. Zeitsch., 40, 479-485.

 CLOWES, GEORGE H. A. (1909), /A critical study of the
 conditions under which zymase and its associated coenzyme
 bring about alcoholic fermentation/                           122
     Proc. Exper. Biol. and Medicine, 6, 44-46.

 COLIN (1825), /Mémoire sur la Fermentation du Sucre/. (Lu
 à l'Académie des Sciences, le 31st janvier, 1825)               5
     Ann. Chim. Phys., 28, 128-142.

 CREMER, M. (1899), /Ueber Glykogenbildung im Hefepresssaft/   116
     Ber., 32, 2062-2064.


 DAKIN, H. D. (1903), /The hydrolysis of ethyl mandelate by
 lipase/                                                        89
     Proc. Chem. Soc., 19, 161.

 DAKIN, H. D. (1905), /The fractional hydrolysis of
 optically inactive esters by lipase Part II./                  89
     J. Physiol., 32, 199-206.

 DAKIN, H. D. (1908), /The oxidation of leucin,
 α-amido-isovaleric acid and of α-amido-n-valeric acid with
 hydrogen peroxide/                                             93
     J. Biol. Chem., 4, 63-76.

 DAKIN, H. D. (1912), /Oxidations and reductions in the
 animal body/ (this series)                                     92

 DAKIN, H. D., and H. W. DUDLEY (1913), /The
 interconversion of α-amino-acids, α-hydroxyacids, and
 α-ketonic aldehydes. Part II./                                 93
     J. Biol. Chem., 15, 127-143.

 DELBRÜCK, MAX (1897), /Alkoholische Gärung ohne
 Hefezellen/                                                    19
     Wochensch. Brauerei, 14, 363-364.

 DESMAZIÈRES (1826)                                              5
     Annales des Sciences naturelles, 10, 42-67.

 DIXON, HENRY H., and W. R. G. ATKINS (1913), /The
 extraction of zymase by means of liquid air./ (Preliminary
 Note)                                                      25, 26
     Sci. Proc. Roy. Dublin Soc., 14, 1-8.

 DUCHAČEK, FRANZ (1909), /Einwirkung verschiedener
 Antiseptika auf die Enzyme des Hefepresssaftes/                36
     Biochem. Zeitsch., 18, 211-227.

 DUCLAUX, E. (1886), /Sur les transformations chimiques
 provoquées par la lumière solaire/                             98
     Compt. rend., 103, 881-882.

 DUCLAUX, E. (1893), /Sur les analogies entre les procès de
 fermentation et de combustion solaire/                         98
     Ann. Inst. Pasteur, 7, 751-754.

 DUCLAUX, E. (1896), /Études sur l'action solaire/.
 (Premier Mémoire)                                              98
     Ann. Inst. Pasteur, 10, 129-168.

 DUCLAUX, E. (1899)                                            121
     Mikrobiologie, 2, 142.

 DUMAS, JEAN BAPTISTE (1874), /Recherches sur la
 fermentation alcoolique/                                      128
     Ann. Chim. Phys., 3, 57-108.


 EHRLICH, FELIX (1903), /Ueber neue stickstoffhaltige
 Bestandteile der Zuckerabläufe/                                86
     Zeitsch. Verein. Rübenzucker-Ind., 809-829.

 EHRLICH, FELIX (1904, 1), /Ueber das natürliche Isomere des
 Leucins/                                                       86
     Ber., 37, 1809-1840.

 EHRLICH, FELIX (1904, 2), /Ueber den neuen optischaktiven
 Nichtzucker, das Isoleucin/                                    86
     Zeitsch. Verein. Rübenzucker-Ind., 775-803.

 EHRLICH, FELIX (1905), /Ueber die Entstehung des Fuselöles/    87
     Zeitsch. Verein. Rübenzucker-Ind., 539-567.

 EHRLICH, FELIX (1906, 1), /Ueber eine Methode zur Spaltung
 racemischer Aminosäuren mittels Hefe/                          89
     Biochem. Zeitsch., 1, 8-31; Zeitsch.
     Verein. Rübenzucker-Ind., 840-860.

 EHRLICH, FELIX (1906, 2), /Verfahren zur Gewinnung von
 Fuselölen und dessen Bestandteilen/                            87
     German Patent Kl. 120, Nr. 177174, vom. 1,
     4, 1905 (17 Nov., 1906).

 EHRLICH, FELIX (1906, 3), /Die chemischen Vorgänge bei der
 Hefegärung/                                            87, 90, 94
     Biochem. Zeitsch., 2, 52-80; Zeitsch.
     Verein. Rübenzucker-Ind., 1145-1168.

 EHRLICH, FELIX (1906, 4), /Zur Frage der Fuselölbildung
 der Hefe/                                                      87
     Ber., 39, 4072-4075.

 EHRLICH, FELIX (1907, 1), /Ueber die Bedingungen der
 Fuselölbildung und über ihren Zusammenhang mit dem
 Eiweissaufbau der Hefe/                                    87, 94
     Ber., 40, 1027-1047; Zeitsch. Verein.
     Rübenzucker-Ind., 1907, 461.

 EHRLICH, FELIX (1907, 2), /Ueber das natürliche Isomere
 des Leucins/                                                   86
     Ber., 40, 2538-2562.

 EHRLICH, FELIX (1907, 3), /Die Rolle des Eiweisses und der
 Eiweissabbauprodukte bei der Gärung/                           87
     Jahrb. d. Versuchs u. Lehranstalt f. Brauerei
     in Berlin, 10, 515-529.

 EHRLICH, FELIX (1908), /Ueber eine Synthese des Isoleucins/    86
     Ber., 41, 1453-1458; Zeitsch. Verein.
     deutsch. Zuckerind., 1908, 528-533.

 EHRLICH, FELIX (1909), /Ueber die Entstehung der
 Bernsteinsäure bei der alkoholischen Gärung/                   89
     Biochem. Zeitsch., 18, 391-423.

 EHRLICH, FELIX (1911, 1), /Ueber die Vergärung des
 Tyrosins zu p-Oxyphenylethyl alkohol. (Tyrosol)/               88
     Ber., 44, 139-147.

 EHRLICH, FELIX (1911, 2), /Ueber die Bildung des
 Plasmaeiweisses bei Hefen und Schimmelpilzen/                  89
     Biochem. Zeitsch., 36, 477-497.

 EHRLICH, FELIX (1912), /Ueber Tryptophol
 (β-Indolylæthylalkohol), ein neues Gärprodukt der Hefe aus
 Aminosäuren/                                                   88
     Ber., 45, 883-889.

 EHRLICH, FELIX, and K. A. JACOBSEN (1911), /Ueber die
 Umwandlung von Aminosäuren in Oxysäuren durch Schimmelpilze/   89
     Ber., 44, 888-897.

 EHRLICH, FELIX, and P. PISTSCHIMUKA (1912, 1),
 /Ueberfuhrung von Aminen in Alkohole durch Hefe und
 Schimmelpilze/                                                 91
     Ber., 45, 1006-1012.

 EHRLICH, FELIX, and P. PISTSCHIMUKA (1912, 2), /Synthesen
 des Tyrosols und seine Umwandlung in Hordenin/                 88
     Ber., 45, 2428-2437.

 EHRLICH, FELIX (mit A. WENDEL) (1908, 1), /Ueber die
 Spaltung racemischer Amidosäuren mittels Hefe, II./            89
     Biochem. Zeitsch., 8, 438-466.

 EHRLICH, FELIX, und ADOLPH WENDEL (1908, 2), /Zur Kenntnis
 der Leucinfraktion des Eiweisses/                              86
     Biochem. Zeitsch., 8, 399-437.

 EMMERLING, O. (1899), /Das Verhalten von Glycerinaldehyd
 und Dioxyaceton gegen Hefe/                                   104
     Ber., 32, 542-544.

 EMMERLING, O. (1904), /Ueber den Ursprung der Fuselöle/        86
     Ber., 37, 3535-3538.

 EMMERLING, O. (1905), /Ueber den Ursprung der Fuselöle/        86
     Ber., 38, 953-956.

 EULER, HANS (1905), /Chemische Dynamik der zellfreien
 Gärung/                                                       120
     Zeitsch. physiol. Chem., 44, 53-73.

 EULER, HANS (1912, 1), /Ueber die Wirkungsweise der
 Phosphatese/. (3 Mitteilung)                               47, 58
     Biochem. Zeitsch., 41, 215-223.

 EULER, HANS (1912, 2), /Verhalten der
 Kohlenhydratphosphorsäure-ester im Tierkörper. Nach
 Versuchen von E. Thorin und D. Johansson/                      51
     Zeitsch. physiol. Chem., 79, 375-397.

 EULER, HANS (1914), /Ueber die Rolle des Glykogens bei der
 Gärung durch lebende Hefe/                                    116
     Zeitsch. physiol. Chem., 89, 337-344.

 EULER, HANS, and BÄCKSTRÖM, HELMA (1912), /Zum Kenntnis
 der Hefegärung/. (2 Mitteilung)                       47, 68, 130
     Zeitsch. physiol. Chem., 77, 394-401.

 EULER, HANS, and TH. BERGGREN (1912), /Ueber die primäre
 Umwandlung der Hexosen bei der alkoholischen Gärung/      32, 130
     Zeitsch. Gärungsphysiol., 1, 203-218.

 EULER, HANS, and HENRY CASSEL (1913), /Ueber Katalysatoren
 der alkoholischen Gärung/. Vorläufige Mitteilung              130
     Zeitsch. physiol. Chem., 86, 122-129.

 EULER, HANS, and A. FODOR (1911), /Ueber ein
 Zwischenprodukt der alkoholischen Gärung/     47, 48, 49, 50, 105
     Biochem. Zeitsch., 36, 401-410.

 EULER, HANS, and YNGVE FUNKE (1912), /Ueber die Spaltung
 der Kohlenhydratphosphorsäure-ester/                           51
     Zeitsch. physiol. Chem., 77, 488-496.

 EULER, HANS, and DAVID JOHANSSON (1912, 1), /Umwandlung
 des Zuckers und Bildung der Kohlensäure bei der
 alkoholischen Gärung/                                          32
     Zeitsch. physiol. Chem., 76, 347-354.

 EULER, HANS, and DAVID JOHANSSON (1912, 2),
 /Untersuchungen über die chemische Zusammensetzung und
 Bildung der Enzyme. IV. Ueber die Anpassung einer Hefe an
 Galaktose/                                                    133
     Zeitsch. physiol. Chem., 78, 246-265.

 EULER, HANS, and DAVID JOHANNSON (1912, 3), /Ueber den
 Einfluss des Toluols auf die Zymase und auf die Phosphatese/  125
     Zeitsch. physiol. Chem., 80, 175-181.

 EULER, HANS, and DAVID JOHANSSON (1912, 4), /Versuche über
 die enzymatische Phosphatbindung/                          47, 57
     Zeitsch. physiol. Chem., 80, 205-211.

 EULER, HANS, and DAVID JOHANSSON (1913), /Ueber die
 Reaktionsphasen der alkoholischen Gärung/          47, 52, 54, 73
     Zeitsch. physiol. Chem., 85, 192-208.

 EULER, HANS, and SIXTEN KULLBERG (1911, 1),
 /Untersuchungen über die chemische Zusammensetzung und
 Bildung der Enzyme/                                      124, 126
     Zeitsch. physiol. Chem., 71, 14-30.

 EULER, HANS, and SIXTEN KULLBERG (1911, 2), /Ueber das
 Verhalten freier und an Protoplasma gebundener
 Hefenenzyme/                                             124, 126
     Zeitsch. physiol. Chem., 73, 85-100 and
     partly in Arkiv. Kem. Min. Geol., 4, No. 13, 1-11.

 EULER, HANS, and SIXTEN KULLBERG (1911, 3), /Ueber die
 Wirkungsweise der Phosphatese/                        47, 58, 122
     Zeitsch. physiol. Chem., 74, 15-28.

 EULER, HANS, and GUNNAR LUNDEQVIST (1911), /Zur Kenntnis
 der Hefegärung/                                               131
     Zeitsch. physiol. Chem., 72, 97-112.

 EULER, HANS, and HJALMAR OHLSÉN (1911), /Ueber den
 Einfluss der Temperatur auf die Wirkung der
 Phosphatese/                                           47, 57, 58
     Biochem. Zeitsch., 37, 313-320.

 EULER, HANS, and HJALMAR OHLSÉN (1912), /Ueber die
 Wirkungsweise der Phosphatese, II/                         47, 57
     Zeitsch. physiol. Chem., 76, 468-477.

 EULER, HANS, and BETH AF. UGGLAS (1911), /Untersuchungen
 über die chemische Zusammensetzung und Bildung der Enzyme./
 (2 Mitteilung)                                           125, 126
     Zeitsch. physiol. Chem., 70, 279-290.


 FERNBACH, A. (1910), /Sur la dégradation biologique des
 hydrates de carbone/                                          107
     Compt. rend., 151, 1004-1006.

 FERNBACH, A., and SCHOEN M. (1913), /L'acide pyruvique,
 produit de la vie de la levure/                               109
     Compt. rend., 157, 1478-1480.

 FISCHER, EMIL, und JULIUS TAFEL (1888), /Oxydation des
 Glycerins/                                                    104
     Ber., 21, 2634-2637.

 FISCHER, EMIL, und JULIUS TAFEL (1889), /Oxydation des
 Glycerins, II./                                               104
     Ber., 22, 106-110.

 FISCHER, EMIL, und HANS THIERFELDER (1894), /Verhalten der
 verschiedenen Zucker gegen reine Hefen/                        89
     Ber., 27, 2031-2037.

 FISCHER, HUGO (1903), /Ueber Enzymwirkung und Gärung/          19
     Centr. Bakt. Par., Abt. II., 10, 547-8.

 FITZ, ALB. (1880), /Ueber Spaltpilzgärungen/. (6
 Mitteilung)                                                    98
     Ber., 13, 1309-1312.

 FRANZEN, HARTWIG, and O. STEPPUHN (1911), /Ein Beitrag zur
 Kenntnis der alkoholischen Gärung/                            115
     Ber., 44, 2915-2919.

 FRANZEN, HARTWIG, and O. STEPPUHN (1912, 1),
 /Vergärung und Bildung der Ameisensäure durch Hefen/     103, 115
     Zeitsch. physiol. Chem., 77, 129-182.

 FRANZEN, HARTWIG, and O. STEPPUHN (1912, 2), /Berichtigung
 zu der Abhandlung: Ueber die Vergärung und Bildung der
 Ameisensäure durch Hefen/                                     115
     Zeitsch. physiol. Chem., 78, 164.


 GAY-LUSSAC, LOUIS JOSEPH (1810), /Extrait d'un Mémoire sur
 la Fermentation/. (Lu à l'Inst., 3 Dec, 1810)                   4
     Ann. Chim. Phys., 76, 245-259.

 GERET, L., und M. HAHN (1898, 1), /Zum Nachweis des im
 Hefepresssaft enthaltenen proteolytischen Enzyms/              20
     Ber., 31, 202-205.

 GERET, L., und M. HAHN (1898, 2), /Weitere Mitteilungen
 über das im Hefepresssaft enthaltene proteolytische Enzym/     20
     Ber., 31, 2335-2344.

 GERET, L., und M. HAHN (1900), /Ueber das Hefe-endotrypsin/    20
     Zeitsch. Biologie, 40, 117-172.

 GERHARDT, CHARLES (1856), /Traité de Chimie Organique/, 4,
 537-546                                                    10, 15

 GIGLIOLI, J. (1911), /Della probabile funzione degli olii
 essenziali e di altri prodotti volatili delle piante, quale
 causa di movemento dei succhi nei tessuli viventi/             26
     Atti. R. Accad. Lincei, 20, II., 349-361.

 GREEN, J. REYNOLDS (1897), /The supposed alcoholic enzyme
 in yeast/                                                      19
     Annals of Botany, 11, 555-562.

 GREEN, J. REYNOLDS (1898), /The alcohol-producing enzyme
 of yeast/                                                      19
     Annals of Botany, 12, 491-497.

 GROMOFF, T., und O. GRIGORIEFF (1904), /Die Arbeit der
 Zymase und der Endotryptase in den abgetöteten Hefezellen
 unter verschiedenen Verhältnissen/                             36
     Zeitsch. physiol. Chem., 42, 299-329.

 GRUBER, M. (1908), /Eduard Buchner/                            18
     München, med. Wochensch., 342.

 GRÜSS, J. (1904), /Untersuchungen über die Atmung und
 Atmungsenzyme der Hefe/                                       111
     Zeitsch. Ges. Brauwesen, 27, 689.

 GRÜSS, J. (1908, 1), /Ueber den Nachweis mittelst
 Chromogramm-Methode dass die Hydrogenase aktiv bei der
 Alkoholgärung beteiligt ist/                                  111
     Ber. deutsch, botan. Ges., 26A, 191-196.

 GRÜSS, J. (1908, 2), /Hydrogenase oder Reduktase?/            111
     Ber. deutsch, botan. Ges., 26A, 627-630,
     Abstract J. Inst. Brewing, 1909, 344.

 HAHN, MARTIN (1898), /Das proteolytische Enzym des
 Hefepresssaftes/                                               20
     Ber., 31, 200-201.

 HAHN, MARTIN (1908), /Zur Geschichte der Zymaseentdeckung/     18
     München. med. Wochensch., 515.

 HANRIOT, M. (1885, 1886), /Sur la décomposition pyrogénée
 des acides de la série grasse/                                 98
     Bull. Soc. Chim., 43, 417; 45, 79-80.

 HARDEN, ARTHUR (1901), /The chemical action of Bacillus
 coli communis and similar organisms on carbohydrates and
 allied compounds/                                         90, 115
     J. Chem. Soc., 79, 612-628.

 HARDEN, ARTHUR (1903), /Ueber alkoholische Gärung mit
 Hefepresssaft (Buchner's "Zymase") bei Gegenwart von
 Blutserum/                                                 20, 41
     Ber., 36, 715-716.

 HARDEN, ARTHUR (1905), /Zymase and alcoholic
 fermentation/                                             42, 103
     J. Inst. Brewing, 11, No. 1.

 HARDEN, ARTHUR (1910), /Recent researches on alcoholic
 fermentation/                                                 125
     J. Inst. Brewing, 16, 623-639.

 HARDEN, ARTHUR (1913), /The enzymes of washed zymin and
 dried yeast/ (Lebedeff). /I. Carboxylase/                  82, 83
     Biochem. J., 7, 214-217.

 HARDEN, ARTHUR, and DOROTHY NORRIS (1912), /The bacterial
 production of acetylmethylcarbinol and 2-3 butylene glycol
 from various substances/                                      110
     Proc. Roy. Soc., B., 84, 492-499.

 HARDEN, ARTHUR, and ROLAND V. NORRIS (1910), /The
 fermentation of galactose by yeast and yeast-juice./
 (Preliminary Communication)                               32, 133
     Proc. Roy. Soc., B., 82, 645-649.

 HARDEN, ARTHUR, and ROLAND V. NORRIS (1914), /The enzymes
 of washed zymin and dried yeast/ (Lebedeff). /II.
 Reductase/                                           68, 111, 113
     Biochem. J., 8, 100-106.

 HARDEN, ARTHUR, and S. G. PAINE (1912), /The action of
 dissolved substances on the autofermentation of yeast/        127
     Proc. Roy. Soc., B., 84, 448-459.

 HARDEN, ARTHUR, and ROBERT ROBISON (1914), /A new
 phosphoric ester obtained by the aid of yeast-juice./
 (Preliminary Note)                                             48
     Proc. Chem. Soc., 30, 16-17.

 HARDEN, ARTHUR, and SYDNEY ROWLAND (1901),
 /Autofermentation and liquefaction of pressed yeast/          126
     J. Chem. Soc., 79, 1227-1235.

 HARDEN, ARTHUR, J. THOMPSON, and W. J. YOUNG (1910),
 /Apparatus for the collection of gases evolved in
 fermentation/                                                  28
     Biochem. J., 5, 230-235.

 HARDEN, ARTHUR, and W. J. YOUNG (1902), /Glycogen from
 yeast/                                                         33
     J. Chem. Soc., 81, 1224-1233.

 HARDEN, ARTHUR, and W. J. YOUNG (1904), /Gärversuche mit
 Presssaft aus obergäriger Hefe/                        30, 33, 35
     Ber., 37, 1052-1070.

 HARDEN, ARTHUR, and W. J. YOUNG (1905, 1), /The alcoholic
 ferment of yeast-juice/                                41, 42, 59
     J. Physiol., 32. Proceedings of 12 November, 1904.

 HARDEN, ARTHUR, and W. J. YOUNG (1905, 2), /The
 influence of phosphates on the fermentation of glucose by
 yeast-juice./ (Preliminary communication, 1 June, 1905)    42, 47
     Proc. Chem. Soc., 21, 189-191.

 HARDEN, ARTHUR, and W. J. YOUNG (1906, 1), /The alcoholic
 ferment of yeast-juice/                                        43
     Proc. Roy. Soc., B., 77, 405-420.

 HARDEN, ARTHUR, and W. J. YOUNG (1906, 2), /The alcoholic
 ferment of yeast-juice. Part II. The co-ferment of
 yeast-juice/                                               59, 62
     Proc. Roy. Soc, B., 78, 369-375.

 HARDEN, ARTHUR, and W. J. YOUNG (1906, 3), /Influence
 of sodium arsenate on the fermentation of glucose by
 yeast-juice/. (Preliminary notice)                         37, 75
     Proc. Chem. Soc, 22, 283-284.

 HARDEN, ARTHUR, and W. J. YOUNG (1907)                         65
     Biochem. Centr., 6, 888.

 HARDEN, ARTHUR, and W. J. YOUNG (1908, 1), /The alcoholic
 ferment of yeast-juice. Part III. The function of
 phosphates in the fermentation of glucose by
 yeast-juice/                                           47, 53, 71
     Proc. Roy. Soc., B., 80, 299-311.

 HARDEN, ARTHUR, and W. J. YOUNG (1908, 2), /The
 fermentation of mannose and lævulose by yeast-juice/.
 (Preliminary note)                                             73
     Proc. Chem. Soc., 24, 115-117.

 HARDEN, ARTHUR, and W. J. YOUNG (1909), /The alcoholic
 ferment of yeast-juice. Part IV. The fermentation of
 glucose, mannose, and fructose by yeast-juice/     32, 44, 47, 73
     Proc. Roy. Soc., B., 81, 336-347.

 HARDEN, ARTHUR, and W. J. YOUNG (1910, 1), /The function
 of phosphates in alcoholic fermentation/                  46, 106
     Centr. Bakt. Par., Abt. II., 26, 178-184.

 HARDEN, ARTHUR, and W. J. YOUNG (1910, 2), /The alcoholic
 ferment of yeast-juice. Part V. The function of phosphates
 in alcoholic fermentation/                             44, 52, 56
     Proc. Roy. Soc., B., 82, 321-330.

 HARDEN, ARTHUR, and W. J. YOUNG (1911, 1), /The alcoholic
 ferment of yeast-juice. Part VI. The effect of arsenates
 and arsenites on the fermentation of the sugars by
 yeast-juice/                                       56, 75, 77, 79
     Proc. Roy. Soc., B., 83, 451-475.

 HARDEN, ARTHUR, and W. J. YOUNG (1911, 2), /Ueber die
 Zusammensetzung der durch Hefepresssaft gebildeten
 Hexosephosphorsäure I/.                                        47
     Biochem. Zeitsch., 32, 173-176.

 HARDEN, ARTHUR, and W. J. YOUNG (1912), /Der Mechanismus
 der alkoholischen Gärung/                            46, 106, 108
     Biochem. Zeitsch., 40, 458-478.

 HARDEN, ARTHUR, and W. J. YOUNG (1913), /The enzymatic
 formation of polysaccharides by yeast preparations/       31, 130
     Biochem. J., 7, 630-636.

 HARDING, VICTOR J. (1912), /The action of enzymes on
 hexosephosphate/                                               51
     Proc. Roy. Soc., B., 85, 418-422.

 HELMHOLTZ, HERMANN LUDWIG (1843), /Ueber das Wesen der
 Fäulnis und Gärung/                                            10
     Arch. Anat. Physiol. Joh. Müller, 5, 453-462.

 HERZOG, R. O. (1902), /Ueber alkoholische Gärung, I./         120
     Zeitsch. physiol. Chem., 37, 149-160.

 HERZOG, R. O. (1904), /Ueber die Geschwindigkeit
 enzymatischer Reaktionen/                                     120
     Zeitsch, physiol. Chem., 41, 416.

 HOPPE-SEYLER, F. (1876), /Ueber die Processe der
 Gärungen und ihre Beziehungen zum Leben der Organismen/        14
     Pflüger's Archiv, 12, 1-17.

 HOPPE-SEYLER, F. (1877), /Ueber Gärungen. Antwort auf
 einen Angriff des Herrn Moritz Traube/                         14
     Ber., 10, 693-695.


 IWANOFF, LEONID (1905), /Ueber Umwandlungen des Phosphors
 in der Pflanze/                                                47
     S. Travaux de la Soc. des Naturalistes de St.
     Petersburg, 34.

 IWANOFF, LEONID (1907), /Ueber die Synthese der
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     Zeitsch. physiol. Chem., 50, 281-288.

 IWANOFF, LEONID (1909, 1), /Ueber die Bildung der
 phospho-organischen Verbindung und ihre Rolle bei der
 Zymasegärung/                                    47, 49, 56, 106
     Centr. Bakt. Par., Abt. II., 24, 1-12.

 IWANOFF, LEONID (1909, 2), /Ueber einen neuen Apparat für
 Gärungsversuche/                                               29
     Centr. Bakt. Par., Abt. II., 24, 429-432.

 JANSSENS, A., and A. LEBLANC (1898), /Recherches
 cytologiques sous la cellule de levure/                       126
     La Cellule, 14, 203-241.


 KARAUSCHANOFF, S. (1911), /Zur Frage nach der Bedeutung
 des Dioxyacetons als eines intermediären Produktes der
 alkoholischen Gärung/                                         105
     Ber. deutsch, bot. Ges., 29, 322.

 KARCZAG, L. (1912, 1), /Ueber die Gärung der verschiedenen
 Weinsäuren/                                                    81
     Biochem. Zeitsch., 38, 516-518.

 KARCZAG, L. (1912, 2), /In welcher Weise wird die
 Weinsäure durch Hefe angegriffen?/                             81
     Biochem. Zeitsch., 43, 44-46.

 KAYSER, E. (1911), /Sur le suc de levure de bière/             26
   Compt. rend., 152, 1279-1280.

 KNOOP, F. (1910), /Ueber den physiologischen Abbau der
 Säuren und die Synthese einer Aminosäure im Tierkorper/        93
     Zeitsch. physiol. Chem., 67, 487-502.

 KOHL, F. G. (1907), /Ueber das Glykogen und einige
 Erscheinungen bei der Sporulation der Hefe/                   116
     Ber. deut. bot. Ges., 25, 74-85.

 KOHL, F. G. (1909), /Alkoholische Gärung/                     116
     Inaug. Diss. Leipzig Abstr. in Zeitsch.
     Brauereiwesen, 32, 406-7, and J. Inst.
     Brewing, 15, 710-711.

 KOSTYTSCHEFF, S. (1912, 1), /Bildung von Acetaldehyd bei
 der alkoholischen Zuckergärung/. (Vorläufige Mitteilung)      111
     Ber., 45, 1289-1293.

 KOSTYTSCHEFF, S. (1912, 2), /Ueber Alkoholgärung/. (1
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 alkoholischen Zuckergärung/                              106, 109
     Zeitsch. physiol. Chem., 79, 130-145.

 KOSTYTSCHEFF, S. (1912, 3), /Ueber Alkoholgärung/. (2
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 Acetaldehyd durch lebende und getötete Hefe. Von S.
 Kostytscheff und E. Hübbenet/                                 110
     Zeitsch. physiol. Chem., 79, 359-374.

 KOSTYTSCHEFF, S. (1913, 1), /Ueber den Mechanismus der
 alkoholischen Gärung/                                         111
     Ber., 46, 339.

 KOSTYTSCHEFF, S. (1913, 2), /Ueber Alkoholgärung. III. Die
 Bedingungen der Bildung von Acetaldehyd bei der Gärung von
 Dauerhefe/                                               111, 113
     Zeitsch. physiol. Chem., 83, 93-111.

 KOSTYTSCHEFF, S. (1914), /Ueber Alkoholgärung/ (6
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 lebenden Hefe/                                                111
     Zeitsch. physiol. Chem., 89, 367-372.

 KOSTYTSCHEFF, S., and E. HÜBBENET (1913), /Zur Frage der
 Reduktion von Acetaldehyd durch Hefesaft/                110, 115
     Zeitsch. physiol. Chem., 85, 408-411.

 KOSTYTSCHEFF, S., and W. BRILLIANT (1913), /Ueber
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 Gegenwart von Zinkchlorid/                                    111
     Zeitsch. physiol. Chem., 85, 507-516.

 KOSTYTSCHEFF, S., and A. SCHELOUMOFF (1913), /Ueber
 Alkoholgärung. IV. Ueber Zuckerspaltung durch Dauerhefe in
 Gegenwart von Zinkchlorid/                               111, 113
     Zeitsch. physiol. Chem., 85, 493-506.

 KUNZ, R. (1906), /Ist die bei der alkoholischen Hefegärung
 entstehende Bernsteinsäure als Spaltungsprodukt des Zuckers
 anzusehen?/                                                    89
     Zeitsch. Unters. Nahr. Genussmittel, 12, 641-645.

 KUPFFER, C. V. (1897)                                          19
     München, med. Wochensch., 44, 321-322.

 KUSSEROFF, R. (1910), /Eine neue Theorie der alkoholischen
 Gärung/                                                       116
     Centr. Bakt. Par., Abt. II., 26, 184-187.

 KÜTZING, FRIEDRICH (1837), /Mikroskopische Untersuchungen
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     J. pr. Chem., 11, 385-409.


 LANGE, H. (1898), /Beitrag zur alkoholischen Gärung ohne
 Hefezellen/                                                    19
     Wochensch. Brauerei, 15, 377-378.

 LAVOISIER, A. (1789), /Traité Élémentaire de Chymie/,
 Chap. XIII                                                      2

 LEATHES, J. B. (1906), /Problems in Animal Metabolism/        110
     London, 81-86.

 LEBEDEFF, ALEX. V. (1908), /Auftreten von Formaldehyd bei
 der zellfreien Gärung/                                        117
     Biochem. Zeitsch., 10, 454-457.

 LEBEDEFF, ALEX. V. (1909), /Versuche zur Aufklärung des
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     Biochem. Zeitsch., 20, 114-125.

 LEBEDEFF, ALEX. V. (1910), /Ueber
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     Biochem. Zeitsch., 28, 213-229.

 LEBEDEFF, ALEX. V. (1911, 1), /Ueber den Mechanismus der
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     Ber., 44, 2932-2942.

 LEBEDEFF, ALEX. V. (1911, 2), /Extraction de la zymase par
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     Compt. rend., 152, 49-51.

 LEBEDEFF, ALEX. V. (1911, 3), /Sur l'extraction de la
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     Compt. rend., 152, 1129.

 LEBEDEFF, ALEX. V. (1911, 4), /La zymase est-elle une
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     Ann. Inst. Past., 25, 682-694. (Bull.
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 der Phosphatese/                                               47
     Zeitsch. physiol. Chem., 75, 499-500.

 LEBEDEFF, ALEX. V. (1911, 6), /Ueber Hexosephos
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     Biochem. Zeitsch., 36, 248-260.

 LEBEDEFF, ALEX. V. (1911, 7), /Sur l'extraction de la
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     Bull. Soc. Chim., IV., 9, 744-750.

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     Chem. Zeit., 36, 365.

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     Ann. Inst. Past., 26, 8-37.

 LEBEDEFF, ALEX. V. (1912, 3), /Notiz über "Phosphatese"https://www.gutenberg.org/       47
     Biochem. Zeitsch., 39, 155-157.

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     Biochem. Zeitsch., 46, 483-489 (and Bull.
     Soc. Chim., IV., 11, 1039-1041).

 LEBEDEFF, ALEX. V. (1913, 1), /Ueber die Veresterung von
 Dioxyaceton mit Phosphaten/                                    47
     Zeitsch. physiol. Chem., 84, 305.

 LEBEDEFF, ALEX. V. (1913, 2), /Ueber den kinetischen
 Verlauf der alkoholischen Gärung/                              31
     Zeitsch. Gärungsphysiol., 2, 104-106.

 LEBEDEFF, ALEX. V. (1914, 1), /Ueber den Mechanismus
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 Polyoxy-carbonsäuren/                                         108
     Ber., 1914, 47, 660-672.

 LEBEDEFF, ALEX. V. (1914, 2), /Notij über zellenfreie
 Gärung der Poly-oxycarbonsäuren/                              108
   Ber., 1914, 47, 965-967.

 LEBEDEFF, ALEX. V., and N. GRIAZNOFF (1912), /Ueber den
 Mechanismus der alkoholischen Gärung/                    107, 110
     Ber., 45, 3256-3272.

 LIEBIG, JUSTUS V. (1839), /Ueber die Erscheinungen der
 Gärung, Fäulnis und Verwesung und ihre Ursachen/                8
     Annalen, 30, 250-287.

 LIEBIG, JUSTUS V. (1870), /Ueber die Gärung und die Quelle
 der Muskelkraft/                                              13
     Annalen, 153, 1-47; 137-228.

 LINDNER, PAUL (1905), /Mikroskopische Betriebskontrolle in
 den Gärungsgewerben/                                          131
     [Berlin, 4th edition], 234.

 LINTNER, C. J. (1899)                                          19
     Chem. Zeit, 23, 851.

 LINTNER, C. J., and H. J. V. LIEBIG (1911), /Ueber die
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 Gärung/                                                       113
     Zeitsch. physiol. Chem., 72, 449-454.

 LINTNER, C. J., and H. LÜERS (1913), /Ueber die Reduktion
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 Gärung/                                                       113
     Zeitsch. physiol. Chem., 88, 122-123.

 LIPPMANN, E. V. (1904), /Die Chemie der Zuckerarten/ 97, 100, 132
     Braunschweig.

 LÖB, WALTER (1906), /Zur chemischen Theorie der
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   Landwirtsch. Jahrb., 35, 541; Chem. Zeit.,
   42, 540; Zeitsch. Elektrochem., 1906,
   12, 282; 1907, 13, 311-516.

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     Biochem. Zeitsch., 12, 78-96.

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     Biochem. Zeitsch., 12, 466-472.

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     Biochem. Zeitsch., 17, 132-144.

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     Biochem. Zeitsch., 20, 516-522.

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     Biochem. Zeitsch., 22, 103-105.

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 Zuckersynthese (von Walter Löb und Georg Pulvermacher)/       117
     Biochem. Zeitsch., 23, 10-26.

 LÖB, WALTER (1910), /Zur geschichte der chemischen
 Gärungshypothesen/                                            107
     Biochem. Zeitsch., 29, 311-315.

 LÖB, WALTER, und GEORG PULVERMACHER (1909), /Zur Kenntnis
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 glycerins und des Glykols/                                    117
     Biochem. Zeitsch., 17, 343-355.

 LOWRY, T. MARTIN (1903), /Studies of dynamic isomerism. I.
 The mutarotation of glucose/                                  133
     J. Chem. Soc, 83, 1314-1323.

 LÜDERSDORFF, F. V. (1846), /Ueber die Natur der Hefe/          15
     Ann. Physik., 76, 408-411.

 LUNGE, G. (1905), /Chemisch-technische
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     (Berlin, 5te Aufgabe), 3, 571.

 LUTHER (1907)                                                 103
     Zeitsch. Elektrochem., 13, 517.

 LVOFF, SERGIUS (1913, 1), /Zymase und Reduktase in ihren
 gegenseitigen Beziehungen/                                    112
     Ber. deut. bot. Ges., 31, 141-147.

 LVOFF, SERGIUS (1913, 2), /Sur le rôle de la reductase
 dans la fermentation alcoolique/                              112
     Bull. Acad. Sci. St. Pétersbourg, 501-532.

 LVOFF, SERGIUS (1913, 3), /Hefegärung und Wasserstoff/         112
     Zeitsch. Gärungsphysiol., 3, 289-320.

 MACFADYEN, ALLAN, G. HARRIS MORRIS, and SYDNEY ROWLAND
 (1900), /Ueber ausgepresstes Hefezellplasma/ (/Buchner's
 "Zymase"https://www.gutenberg.org/)                                         19, 24, 27, 30
     Ber., 33, 2764-2790.

 MCKENZIE, ALEX., and ARTHUR HARDEN (1903), /The biological
 method for resolving inactive acids into their optically
 active components/                                             89
     J. Chem. Soc, 83, 424-438.

 MAGNUS-LEVY, A. (1902), /Ueber den Aufbau der hohen
 Fettsäuren aus Zucker/                                        110
     Arch. Anat. Physiol., 365-368.

 MANASSEÏN, MARIE V. (1872), /Mikroskopische
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     [Stuttgart], 128.

 MANASSEÏN, MARIE V. (1897), /Zur Frage von der
 alkoholischen Gärung ohne lebende Hefezellen/                  15
     Ber., 30, 3061-3062.

 MARCKWALD, W. (1902), /Ueber die Trennung der
 Amylalkohole des Fuselöles. III./                              88
     Ber., 35, 1595-1601.

 MARTIN, C. J. (1896), /A rapid method of separating
 colloids from crystalloids in solutions containing both/       59
     J. Physiol., 20, 364-371.

 MARTIN, C. J., and H. G. CHAPMAN (1898), /An endeavour to
 procure an alcoholic ferment from yeast-cells/                 19
     Proc. physiol. Soc., 11 June, ii.

 MAYER, ADOLF (1879), /Lehrbuch der Gärungschemie 3.
 Ausgabe/                                                       15
     Heidelberg, Carl Winters' Univ.-buchh.

 MAYER, P. (1907), /Zur Frage der Vergärbarkeit von
 Methylglyoxal/                                                104
     Biochem. Zeitsch., 2, 435-437.

 MAZÉ, P. (1902), /Recherches sur les modes d'utilisaion du
 carbone térnaire par les végétaux et les microbes/             98
     Ann. Inst. Pasteur, 16, 446; Compt.
     rend., 134, 241.

 MEISENHEIMER, JAKOB (1903), /Neue Versuche mit
 Hefepresssaft/                                                 35
     Zeitsch. physiol. Chem., 37, 518-526.

 MEISENHEIMER, JAKOB (1907), /Ueber die chemischen
 Vorgänge bei den als Enzymreaktionen erkannten Gärungen/      100
     Biochem. Centr., 6, 1-13.

 MEISENHEIMER, JAKOB (1908), /Ueber das Verhalten der
 Glukose, Fructose und Galaktose gegen verdünnte Natronlauge/   97
     Ber., 41, 1009-1019.

 MITSCHERLICH, EILHARD (1841), /Ueber die chemische
 Zersetzung und Verbindung mittels Contactsubstanzen/           10
     Monatsber. K. Akad. d. Wissensch. Berlin, 1841,
     392; Ann. Physik., 1842, 55, 209-229.


 NÄGELI, C. V. (1879), /Theorie der Gärung/                     15
     [München. R. Oldenbourg,] 156.

 NĀGELI, C., und O. LOEW (1878), /Ueber die chemische
 Zusammensetzung der Hefe/                                      15
     Annalen, 193, 322-348.

 NEF, J. U. (1904), /Dissoziationsvorgänge in der
 Glycolglycerinreihe/                                           98
     Annalen, 335, 247-333.

 NEF, J. U. (1907), /Dissoziationsvorgänge in der
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 Zuckerarten gegen die Fehling'sche Lösung sowie gegen
 andere Oxydationsmittel/                                       98
     Annalen, 357, 214-312.

 NEISSER, M. (1906), /Ein Fall von Mutation nach de Vries
 bei Bakterien und andere Demonstrationen/                     132
     Cent. Bakt. Par., Abt. I., Ref. 38, Append., 98-102.

 NEUBAUER, OTTO and KONRAD FROMHERZ (1911), /Ueber den
 Abbau der Aminosäuren bei der Hefegärung/                 92, 109
     Zeitsch. physiol. Chem., 70, 326-350.

 NEUBERG, CARL (1908), /Chemische Umwandlungen durch
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 Sonnenlichtes/                                                 93
     Biochem. Zeitsch., 13, 305-320.

 NEUBERG, CARL (1909), /Chemische Umwandlungen durch
 Strahlenarten/. II. Mitteilung. /Wirkungen des elektrischen
 Gleichstroms/                                                  93
     Biochem. Zeitsch., 17, 270-292.

 NEUBERG, CARL (1912), /Ueber zuckerfreie Hefegärungen,
 VII. Bildung von β-Oxybuttersäurealdehyd (Aldol) bei der
 Vergärung von Brenztraubensäure/                               82
     Biochem. Zeitsch., 43, 491-493.

 NEUBERG, CARL, and A. HILDESHEIMER (1911), /Ueber
 zuckerfreie Hefegärungen, I./                                  81
     Biochem. Zeitsch., 31, 170-176.

 NEUBERG, CARL, and L. KARCZAG (1911, 1), /Die Gärung der
 Brenztraubensäure und Oxalessigsäure als Vorlesungsversuch/    81
     Ber., 44, 2477-2479.

 NEUBERG, CARL, and L. KARCZAG (1911, 2), /Ueber
 zuckerfreie Hefegärungen, III./                           82, 127
     Biochem. Zeitsch., 36, 60-67.

 NEUBERG, CARL, and L. KARCZAG (1911, 3), /Ueber
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 der Hefe/                                                      82
     Biochem. Zeitsch., 36, 68-75.

 NEUBERG, CARL, and L. KARCZAG (1911, 4), /Ueber
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     Biochem. Zeitsch., 36, 76-81.

 NEUBERG, CARL, and L. KARCZAG (1911, 5), /Ueber
 zuckerfreie Hefegärungen, VI./                                 82
     Biochem. Zeitsch., 37, 170-176.

 NEUBERG, CARL, and JOHANNES KERB (1912, 1), /Ueber
 zuckerfreie Hefegärungen, VIII. Enstehung von Acetaldehyd
 bei der sog. Selbstgärung/                               111, 112
     Biochem. Zeitsch., 43, 494-499.

 NEUBERG, CARL, and JOHANNES KERB (1912, 2), /Ueber
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 durch Weinhefen/                                              81
     Biochem. Zeitsch., 47, 405-412.

 NEUBERG, CARL, and JOHANNES KERB (1912, 3),
 /Ueber zuckerfreie Hefegärungen, X. Die Gärung der
 α-Ketobuttersäure/                                             82
     Biochem. Zeitsch., 47, 413-420.

 NEUBERG, CARL, and JOHANNES KERB (1912, 4), /Entsteht bei
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     Zeitsch. Gärungphysiol., 1, 114-120.

 NEUBERG, CARL, and JOHANNES KERB (1913, 1), /Ueber
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 Hefegärung/                                           82, 93, 110
     Biochem. Zeitsch., 53, 406-419; Ber.,
     46, 2225-2228.

 NEUBERG, CARL, and JOHANNES KERB (1913, 2), /Ueber
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 Aldehydbildung bei der Gärung von Hexosen sowie bei der
 sog. Selbstgärung/                                       112, 113
     Biochem. Zeitsch., 58, 158-170.

 NEUBERG, CARL, and W. OERTEL (1913), /Studien über
 Methylglyoxalbildung/                                          99
     Biochem. Zeitsch., 55, 495-503.

 NEUBERG, CARL, and P. ROSENTHAL (1913), /Ueber zuckerfreie
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     Biochem. Zeitsch., 51, 128-142.

 NEUBERG, CARL, and H. STEENBOCK (1913), /Ueber die Bildung
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 Valeraldehyd in Amyl Alkohol/                             93, 113
     Biochem. Zeitsch., 52, 494-503.

 NEUBERG, CARL, and H. STEENBOCK (1914), /Ueber die Bildung
 höherer Alkohole aus Aldehyden durch Hefe, II./           93, 113
     Biochem. Zeitsch., 59, 188-192.

 NEUBERG, CARL, and L. TIR (1911), /Ueber zuckerfreie
 Hefegärungen, II./                                             81
     Biochem. Zeitsch., 32, 323-331.

 NEUMEISTER, R. (1897), /Bemerkungen zu Eduard Buchner's
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     Ber., 30, 2963-2966.

 OPPENHEIMER, MAX (1914, 1), /Ueber die Bildung von
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     Zeitsch. physiol. Chem., 89, 45-62.

 OPPENHEIMER, MAX (1914, 2), /Ueber die Bildung von
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     Zeitsch. physiol. Chem., 89, 63-77.

 O'SULLIVAN, JAMES (1898), /On the rate of alcoholic
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     J. Soc. Chem. Ind., 17, 559-560.

 O'SULLIVAN, JAMES (1899), /The hydrolytic and
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     J. Inst. Brewing (3), 5, 161-175.


 PARNAS, JAKOB (1910), /Ueber fermentative
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 durch Gewebesäfte/                                             94
     Biochem. Zeitsch., 28, 274-294.

 PAINE, SYDNEY, G. (1911), /The permeability of the yeast
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     Proc. Roy. Soc,, B., 84, 289-307.

 PALLADIN, W. (1908), /Beteiligung der Reduktase im
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     Zeitsch. physiol. Chem., 56, 81-88.

 PASTEUR, LOUIS (1857), /Mémoire sur la fermentation
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     Compt. rend., 45, 913-916.

 PASTEUR, LOUIS (1860), /Mémoire sur la fermentation
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     Ann. Chim. Phys., (3), 58, 323-426.

 PASTEUR, LOUIS (1872), /Note sur la mémoire de M.
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     Ann. Chim. Phys., (4), 25, 145-151.

 PASTEUR, LOUIS (1875), /Nouvelles observations sur la
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     Compt. rend., 80, 452-457.

 PAVY, F. W., and H. W. BYWATERS (1907), /On glycogen
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     J. Physiol., 36, 149-163.

 PAYEN, ANSELME et PERSOZ (1833), /Mémoire sur la
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     Ann. Chim. Phys., 53, 73-92.

 PILOTY, OSCAR (1897), /Ueber eine neue Totalsynthese des
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     Ber., 30, 3161-3169.

 PINKUS, GEORG (1898), /Ueber die Einwirkung von
 Benzhydrazid auf Glukose/                                      98
     Ber., 31, 31-37.

 PLIMMER, ROBERT HENRY ADERS (1913), /The metabolism of
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     Biochem. J., 7, 43-71.

 PRINGSHEIM, HANS H. (1905), /Zur Fuselölfrage/                 86
     Ber., 38, 486-487.

 PRINGSHEIM, HANS H. (1906), /Ueber die Bildung von Fuselöl
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     Ber., 39, 3713-3715.

 PRINGSHEIM, HANS H. (1907), /Ueber die Stickstoffernährung
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     Biochem. Zeitsch., 3, 121-286.

 PRINGSHEIM, HANS H. (1908), /Ueber die Unterdrückung der
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     Biochem. Zeitsch., 10, 490-497.

 PRINGSHEIM, HANS H. (1909), /Bemerkungen zur Mitwirkung
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     Biochem. Zeitsch., 16, 243-245.


 RESENSCHECK, FRIEDERICH (1908, 1), /Einwirkung des
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     Biochem. Zeitsch., 9, 255-263.

 RESENSCHECK, FRIEDERICH (1908, 2), /Einwirkung von
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     Biochem. Zeitsch., 15, 1-11.

 RINCKLEBEN, P. (1911), /Gewinnung von Zymase aus frischer
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     Chem. Zeit., 35, 1149-1150.

 ROWLAND, SYDNEY (1901), /A method of obtaining
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     J. Physiol., 27, 53-56.

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     Rubner's Archiv. Physiologie Suppl. Bd., 1-369.


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     Ber., 12, 648-655.

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     Zeitsch. physikal. Chem., 57, 1-46.

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     Zeitsch. physikal. Chem., 60, 510-512.

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     Biochem. Zeitsch., 7, 299-326.

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     Annalen, 51, 193-212.

 SCHMIDT, C. (1847), /Gärungsversuche/                          15
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 SCHROEDER, H. (1859, 1861), /Ueber Filtration der Luft in
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     Annalen, 109, 35-52; 117, 273-295.

 SCHROEDER, H., und TH. V. DUSCH (1854), /Ueber Filtration
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     Annalen, 89, 232-343.

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     Hefe, Gärung und Fäulnis, 208-229 [Parey, Berlin].

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     Ann. Physik., 39, 487-489.

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 SLATOR, ARTHUR (1908, 1), /Studies in Fermentation/. Part
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                                                131, 132, 133, 134
     J. Chem. Soc., 93, 217-242.

 SLATOR, ARTHUR (1908, 2), /Factors which influence
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     Chem. News, 98, 175, and Brit. Ass. Reports, 1908.

 SLATOR, ARTHUR (1912), /Ueber Dioxyaceton als
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     Ber., 45, 43-46.

 SLATOR, ARTHUR, and H. J. S. SAND (1910), /Studies
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     J. Chem. Soc, 922-927.

 STAHL, GEORG ERNST (1697), /Zymotechnia fundamentalis/          2
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 STAVENHAGEN, A. (1897), /Zur Kenntnis der
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     Ber., 30, 2422-2423 and 2963.


 TAFEL, JULIUS (1907), /Ueber Zwischenprodukte bei
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     Ber., 40, 3318-3321.

 TAMMANN, G. (1889), /Ueber die Wirkung der Fermente/          128
     Zeitsch. physikal. Chem., 3, 25-37.

 THENARD, LOUIS JACQUES (1803), /Mémoire sur la
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     Ann. Chim. Phys., 46, 294-320.

 TRAUBE, MORITZ (1858), /Theorie der Fermentwirkungen/          14
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     Centr. Bakt. Par., Abt. II., 8, 82.

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     Compt. rend., 7, 369-402.


 VOIT, CARL V. (1897)                                           19
     München, med. Wochensch., 44, 321.

 VOLHARD, JAKOB (1909), /Justus von Liebig/                     13
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 WAGER, HAROLD (1898), /The nucleus of the yeast plant/        126
     Annals of Botany, 12, 449.

 WAGER, HAROLD (1911), /The yeast cell/                        126
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     Zeitsch. physikal. Chem., 47, 185-222.

 WEHMER, C. (1898)                                              19
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 WILL, H. (1897), /Alkoholische Gärung ohne Hefezellen/         19
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     Ber., 31, 1796-1801.

 WOHL, A. (1907, 1), /Ueber Oxyfumar- und Oxymaleïnsäure/      103
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     Biochem. Zeitsch., 5, 45-65, and Zeitsch.
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 WROBLEWSKI, A. (1898), /Gärung ohne Hefezellen/                19
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     Centr. Physiol., 13, 284-297.

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     J. pr. Chem., (2), 64, 1-70.


 YOUNG, W. J. (1909), /The hexosephosphate formed by
 yeast-juice from hexose and phosphate/                     47, 56
     (Prel. Note, Proc. Chem. Soc., 1907, 65.) Proc.
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 YOUNG, W. J. (1911), /Ueber die Zusammensetzung der
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     Biochem. Zeitsch., 32, 178-188.




 INDEX.


 ACETALDEHYDE, as an intermediate product of alcoholic
      fermentation, 110.

 -- reduction of by yeast, 110.

 Acetone-yeast, 38.

 Alanine, as an intermediate product of alcoholic fermentation, 115.

 Alcohol, formation of, from sugar by alkalis, 97.

 Alcoholic fermentation, attempts to separate enzymes of, from
      yeast-cell, 15.

 -- -- by-products of, 85.

 -- -- equation of, 51.

 -- -- Gay-Lussac's theory of, 4.

 -- -- Iwanoff's theory of, 106.

 -- -- kinetics of, 120, 128.

 -- -- Lavoisier's views on, 3.

 -- -- Liebig's theory of, 8.

 -- -- Nägeli's theory of, 15.

 -- -- of the amino-acids, 87.

 -- -- -- -- theory of, 91.

 -- -- Pasteur's researches on, 11.

 -- -- Traube's enzyme theory of, 14.

 Alkalis, effect of, on hexoses, 96.

 Amino-acids, alcoholic fermentation of, 87.

 -- stereoisomerides of, fermented at different rates by yeast, 89.

 d-Amyl alcohol, formation of from isoleucine, 86.

 Antiprotease in yeast-juice, 42, 65.

 Antiseptics, action of, on yeast-juice, 19, 36.

 Arsenate, effect of, on fermentation by yeast-juice and zymin, 73.

 -- -- on autofermentation of yeast-juice, 80.

 -- nature of acceleration produced by, 78.

 Arsenite, effect of, on fermentation by yeast-juice, 77.

 -- -- on autofermentation of yeast-juice, 80.

 -- nature of acceleration produced by, 78.

 Autofermentation of yeast-juice, 33, 119.

 -- -- effect of arsenates and arsenites on, 80.


 BAEYER'S theory of fermentation, 99.

 Boiled yeast-juice, effect of, on fermentation by yeast-juice, 41.


 CARBOXYLASE, 81, 93.

 -- relation of to alcoholic fermentation, 83.

 Co-enzyme, effect of electric current on, 67.

 -- enzymic destruction of, 63.

 -- of yeast-juice, 59.

 -- precipitation of, by ferric hydroxide, 67.

 -- properties of, 63.

 -- removal of, from yeast-juice, 59.

 -- separation from phosphate and hexosephosphate, 67.

 Concentration of sugar, effect of, on fermentation by
      yeast-juice, 34,


 DAUERHEFE, 38.

 Diastatic enzyme of yeast-juice, 33.

 Dihydroxyacetone, fermentability of, 104.

 -- formation of, in fermentation, 105.

 Dried yeast (Lebedeff), 24, 38.


 ENDOTRYPTASE, 20.

 Enzyme action, laws of, 121.

 Enzymes, combined with protoplasm, 126.

 Equation of alcoholic fermentation, 51.


 FERMENTATION by yeast-juice, causes of cessation of, 64.

 Fermenting complex, 63.

 -- power of yeast-juice, estimation of, 27.

 Formaldehyde, production of in alcoholic fermentation, 117.

 Formic acid theory of fermentation, 114.

 Fructose, fermentation of, by yeast-juice, 32.

 -- -- in presence of phosphate, 73.

 -- relation of, to fermenting complex, 74.

 Fusel oil, formation of, from amino-acids, 85.


 GALACTOSE, fermentation of, by yeast, 131.

 -- fermentation of, by yeast-juice, 32.

 Glucose, fermentation of, by yeast-juice, 32.

 Glyceraldehyde, fermentability of, 104.

 Glyceric acid, fermentation of, 108.

 Glycerol, formation in fermentation, 95.

 Glycogen as an intermediate product of alcoholic fermentation, 116.

 -- fermentation of, by yeast-juice, 33.

 -- removal of, from yeast, 39.

 Grinding of yeast by hand, 22.

 -- -- -- mechanical, 23.

 Glutamic acid, decomposition of, by yeast, 90.


 HEFANOL, 38.

 Hexosediphosphoric acid phenylhydrazone, hydrazine salt of, 50.

 Hexosemonophosphoric acid osazone, hydrazine salt of, 50.

 Hexosephosphatase, 54.

 -- effect of arsenate and arsenite on action of, 79.

 Hexosephosphate, constitution of, 51.

 -- enzymic decomposition of, in yeast-juice, 56.

 -- -- hydrolysis of, 51.

 -- formation of, 48.

 -- hydrolysis of, by acids, 49.

 -- preparation of, 48.

 -- properties of, 49.

 -- theory of formation of, 57, 117,

 Hexoses, action of alkalis on, 96,


 ISOAMYL alcohol, formation from leucine, 87.

 Isoleucine, decomposition of, by yeast, 87.


 α-KETONIC acids, fermentation of, 81.


 LACTIC acid, destruction of, by yeast-juice, 102.

 -- -- formation from sugars by alkalis, 97.

 -- -- -- of, in yeast-juice, 102.

 -- -- non-fermentability of, by yeast, 103.

 -- -- theory of fermentation, 102.

 Leucine, decomposition of, by yeast, 87.


 MACERATION extract, preparation of, 25.

 Mannose, fermentation of, by yeast, 131.

 -- -- of by yeast-juice, 32.

 Methylglyoxal, conversion of, into lactic acid, 101.

 -- non-fermentability of, 104.

 -- as an intermediate product of alcoholic fermentation, 113.


 OXALACETIC acid, formation of, from tartaric acid, 101.


 PERMANENT yeast, 38.

 Phenylethyl alcohol, 88.

 Phosphate, changes of, in alcoholic fermentation, 47.

 -- effect of, on fermentation by yeast-juice, 42.

 -- -- -- -- -- by zymin, 46.

 -- -- -- -- -- of fructose, 73.

 -- -- of on total fermentation of yeast-juice, 54.

 -- influence on fermentation of concentration of, 71.

 -- inhibition by, 71.

 Phosphates, essential for alcoholic fermentation, 55.

 Proteoclastic enzyme of yeast, 20.

 Protoplasmic theory of activity of yeast-juice, 19.

 Pyruvic acid, fermentation of, 81.

 -- -- theory of fermentation, 109.


 RATE of fermentation, controlling factors of, 119.

 Reductase, intervention of, in alcoholic fermentation, 111.


 SERUM, effect of, on fermentation by yeast-juice, 41.

 Succinic acid, formation of, in fermentation, 89.

 -- -- formed from glutamic acid by yeast, 90.

 Synthetic enzyme in yeast-juice, 32.


 TEMPERATURE coefficient of fermentation by yeast, 129.

 -- -- -- -- by zymin, 122.

 -- -- -- esterification of phosphoric acid by yeast extract, 58.

 Tryptophol, 88.

 Tyrosol, 88.


 WOHL'S theory of fermentation, 101.


 YEAST, action of toluene on, 124.

 -- and yeast-juice, fermentation by, compared, 29, 124.

 -- discovery of the vegetable nature of, 5.

 -- fermentation by, 127.

 -- -- of different sugars by, 130.

 -- influence of concentration of dextrose on fermentation by, 128.

 -- -- -- -- of, on rate of fermentation, 129.

 -- -- of toluene on autofermentation of, 126.

 -- nature of the process of fermentation by, 123.

 -- temperature coefficient of fermentation by, 129.

 -- theories of fermentation by, 133.

 Yeast-juice and yeast, fermenting powers compared, 29, 124.

 -- co-enzyme of, 59.

 -- dialysis of, 59, 62.

 -- effect of arsenate on fermentation by, 75.

 -- -- of concentration of sugar on fermentation by, 34.

 -- -- of dilution on fermentation by, 35.

 -- -- of phosphate on total fermentation, produced by, 54.

 -- estimation of fermenting power of, 27.

 -- evaporation of, 37.

 -- filtration of through gelatin filter, 59.

 -- precipitation of, 38.

 -- preparation of, 21.

 -- properties of, 19.

 -- ratio of alcohol and carbon dioxide, produced by, 30.

 -- synthesis of complex carbohydrate by, 31.

 -- variation of rate of fermentation by, with concentration of
      sugar, 121.


 ZYMASE, Buchner's discovery of, 16.

 -- enzymic destruction of, 64.

 -- properties of, 18.

 -- regeneration of inactive, 64.

 -- separation from co-enzyme, 59.

 Zymin, 21, 38.

 -- fermentation by, 39.

 -- rate of fermentation by, 39.

 -- temperature coefficient of fermentation by, 122.


 ABERDEEN: THE UNIVERSITY PRESS




TRANSCRIBER'S ENDNOTE.

Full stops, middle dots "·", or even dots aligned with the top of the
font were variably used in the original as decimal points, and also
for denoting chemical bonding. These have been rendered as middle
dots herein.

Tables and formulas have been edited for clarity and readability,
while honoring the original form. In particular, some tables and
chemical equations were made more compact to control line length.

The reference to Colin's paper on page 5 has been changed from 1826
to 1825, to agree with the corresponding entry in the Bibliography.
The reference to Turpin's paper on page 8 was changed to 1838 from
1839, for the same reason.

On page 93, the complex reaction system has been revised into four
simple reactions. The unbalanced original equations have been
retained. Similarly on page 94, the original large diagram was
converted into four simple reactions.

The incorrect formula for the enol II. in the equation for glucose
dehydration near bottom of page 101 was corrected.

Equations (1) on page 108 showing production of glyceric acid from
glyceraldehyde have been simplified and clarified.







*** END OF THE PROJECT GUTENBERG EBOOK ALCOHOLIC FERMENTATION ***


    

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