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]].
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 + 0·2 gram. | CO2 in grams in successive periods of | Duration of fer- ment- ation | Total CO2 | ||
|---|---|---|---|---|---|
| 6 hours. | 18 hours. | 24 hours. | |||
| Cane sugar | 0·050 | 0·000 | 0·000 | 6 | 0·05 |
| Dihydroxy- acetone | 0·042 | 0·000 | 0·000 | 6 | 0·042 |
| Glycer- aldehyde | 0·008 | 0·022 | 0·005 | 48 | 0·035 |
Further, during an experiment in which 0·129 gram of CO2 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 P2O5 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 CO2 having been evolved in twenty hours) but could not be obtained from those of glyceraldehyde (0·138 gram CO2 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) C6H12O6 = C3H6O3 + C3H6O3.
2. The dihydroxyacetone then passes through the stages previously postulated (p. [106]).
(b) 4 C3H6O3 + 4 R2HPO4 = 4 C3H5O2PO4R2 + 4 H2O.
(c) 4 C3H5O2PO4R2 = 2 C6H10O4(R2PO4)2.
(d) 2 C6H10O4(R2PO4)2 + 4 H2O = 2 C6H12O6 + 4 R2HPO4.
After which the hexose, C6H12O6 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) CH2(OH)·CH(OH)·CHO + H2O → CH2(OH)·CH(OH)·CH(OH)2
→ CH2(OH)·CH(OH)·COOH + 2 H
(2) CH2(OH)·CH(OH)·COOH = CH3·CO·COOH + H2O.
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
C6H12O6 ⇌ 2 C3H6O3.
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.