Alcoholic Fermentation / Second Edition, 1914

by Arthur Harden

Glycerol.

[p095]

Of the three chief by-products of alcoholic fermentation, only glycerol remains at present referable directly to the sugar. This substance, as shown by the careful experiments of Buchner and Meisenheimer [[1906]], is formed by the action both of yeast-juice and zymin to the extent of 3·8 per cent. of the sugar decomposed, and no other source for its production has so far been experimentally demonstrated. If it be true that during the decomposition of sugar into alcohol and carbon dioxide, substances containing three carbon atoms are formed as intermediate compounds (see p. [100]), it is obvious that these might by reduction be converted into glycerol which would thus be a true by-product of the alcoholic fermentation of sugar. [See Oppenheimer, [1914, 2].] It has, however, been suggested that it may in reality be a product of decomposition of lipoid substances or of the nuclein of the cell (Ehrlich).

The effect of Ehrlich's work has been clearly to distinguish the chemical changes involved in the production of fusel oil and succinic acid from those concerned in the decomposition of sugar into alcohol and carbon dioxide, and to bring to light a most important series of reactions by means of which the yeast-cell is able to supply itself with nitrogen, one of the indispensable conditions of life.

CHAPTER VIII. THE CHEMICAL CHANGES INVOLVED IN FERMENTATION.

[p096]

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:—

	C	HO		C	HO		C	H2(OH)		C	H(OH)
	│			│			│			║
H	C	OH	HO	C	H		C	O		C	OH
	│			│			│			│
HO	C	H	HO	C	H	HO	C	H	HO	C	H
	│			│			│			│
H	C	OH	H	C	OH	H	C	OH	H	C	OH
	│			│			│			│
H	C	OH	H	C	OH	H	C	OH	H	C	OH
	│			│			│			│
	C	H2(OH)		C	H2(OH)		C	H2(OH)		C	H2(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, C₃H₆O₃, from 0·5 to 2 per cent. into formic acid, CH₂O₂, 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, CH₃·CO·CHO. This osazone may be formed either from methylglyoxal itself, from acetol, CH₃·CO·CH₂·OH, or from lactic aldehyde, CH₃·CH(OH)·CHO [Wohl, [1908]]. Methylglyoxal itself may also be regarded as a secondary [p099] product derived from glyceraldehyde, CH₂(OH)·CH(OH)·CHO, or dihydroxyacetone, CH₂(OH)·CO·CH₂(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:—

CH₃·CO·CHO + H₂O = CH₃·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,

CH3·	C	·NH·	C	H
	║		║		,
H	C	────	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) C₆H₁₂O₆ = 2 CH₂(OH)·CH(OH)·CHO.
(2) CH₂(OH)·CH(OH)·CHO = CH₃·CO·CHO + H₂O.
(3) CH₃·CO·CHO + H₂O = CH₃·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.
C	H2·OH
│
C	H·OH
│
C	H·OH
│
C	H·OH
│
C	H·OH
│
C	H(OH)2

II.
C	H2 . . . OH
│
C	OH . . H
│
C	. . OH . . H
│
C	OH . . . H
│
C	OH . . . H
│
C	H . . . (OH)2

III.
C	H3
│
C	H . OH
│
C	(OH)2
│
C	(OH)2
│
C	(OH)2
│
C	H3

IV.
		C	H3
		│
		C	H(OH)
		│
		C	O
	╱
O
	╲
		C	O
		│
		C	H(OH)
		│
		C	H3

V.
		C	H3
		│
		C	H2
	╱
O
	╲
		C	O
	╱
O
	╲
		C	O
	╱
O
	╲
		C	H2
		│
		C	H3

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:—

C	HO
·
C	H(OH)
·
C	H(OH)
·
C	H(OH)
·
C	H(OH)
·
C	H2(OH)

C	OOH
·
C	H(OH)
·
C	H2
·
C	O
·
C	H(OH)
·
C	H3

C	OOH
·
C	H(OH)
·
C	H3
─	───
C	OOH
·
C	H(OH)
·
C	H3

C	O2
─	───
C	H2·OH
·
C	H3
─	───
C	O2
─	───
C	H2·OH
·
C	H3

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]

C	OOH				C	OOH		C	OOH
·					·			·
C	H(OH)		H		C	(OH)		C	O
·		−	·	=	║		⇌	·
C	H(OH)		OH		C	H		C	H3
·					·			·
C	OOH				C	OOH		C	OOH
I. Tartaric acid					II.			III. 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):—

C	HO			C	HO		C	HO
│				│			│
C	H(OH)			C	(OH)		C	O
│			H	║			│
C	H(OH)	−	OH	C	H	⇌	C	H2
│				│			│
C	H(OH)			C	H(OH)		C	H(OH)
│				│			│
C	H(OH)			C	H(OH)		C	H(OH)
│				│			│
C	H2(OH)			C	H2(OH)		C	H2(OH)
I. Glucose.				II.			III.

Methyl- glyoxal
C	HO							C	OOH	C	O2
│								│		─	───
C	O		+H2O					C	H(OH)	C	H2OH
│								│		│
C	H3							C	H3	C	H3
C	HO		C	HO		C	HO	C	OOH	C	O2
│			│			│		│		─	────
C	H(OH)	H	C	(OH)	⇌	C	O + H2O	C	H(OH)	C	H2OH
│		·	║			│		│		│
C	H2(OH)	− HO	C	H2		C	H3	C	H3	C	H3
IV. Glyceral- dehyde.			V.			VI. Methyl- glyoxal.		VII. Lactic acid.		VIII. Alcohol 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.