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, CH3·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, CH2(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]].