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]