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]