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]