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) C₆H₁₂O₆ = 2 CH₃·CO·COOH + 4[H].
(2) 2 CH₃·CO·COOH = 2 CH₃·CHO + 2 CO₂.
(3) 2 CH₃·CHO + 4 H = 2 CH₃·CH₂·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,

CH₃·CO·COOH + 2 H → CH₃·CH(OH)·COOH,

glyceraldehyde can readily be converted into it by oxidation to glyceric acid followed by abstraction of water (Erlenmeyer), [p110]

CH₂(OH)·CH(OH)·CHO + O → CH₂(OH)·CH(OH)·COOH
CH₂(OH)·CH(OH)·COOH − H₂O → CH₃·CO·COOH,

and finally methylglyoxal CH₃·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 ZnCl₂ 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 ZnCl₂. 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 CO₂ 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.

CH₂:C(OH)·CHO + H₂O	H₂ + │ = O	CH₂(OH)·CHOH·CH₂(OH) (glycerol) +
CH₂:C(OH)·CHO	H₂ + │ = O	CH₂:C(OH)·COOH (Pyruvic acid)

(b) The pyruvic acid is then decomposed by carboxylase yielding aldehyde and carbon dioxide (equation 2, p. [109]). [p114]

CH₃·CO·CHO	O	CH₃·CO·COOH
	+ │ =	+
CH₃·CHO	H₂	CH₃CH₂(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]).