Similar experiments made in 1871 by Marie von Manasseïn [[1872], [1897]], in which yeast was ground for six to fifteen hours with powdered rock crystal, yielded products which fermented sugar, but they contained unbroken yeast cells, so that the results obtained could not be considered decisive [Buchner and Rapp, [1898, 1]], although Frau von Manasseïn herself drew from them and from others in which sugar solution was treated with heated yeast, but not under aseptic conditions, the conclusion that living yeast cells were not necessary for fermentation.
Quite unsuccessful were also the attempts made to accomplish the separation of fermentation from the living cell by Adolf Mayer [[1879], p. 66], and, as we learn from Roux, by Pasteur himself, grinding, freezing, and plasmolysing the cells, having in his hands proved alike in vain. Extraction by glycerol or water, a method by which many enzymes can be obtained in solution, gave no better results [Nägeli and Loew, [1878]], and the enzyme theory of alcoholic fermentation appeared quite unjustified by experiment.
Having convinced himself of this, Nägeli [[1879]] suggested a new explanation of the facts based on molecular-physical grounds. According to this view, which unites in itself some of the conceptions of Liebig, Pasteur, and Traube, fermentation is the transference of a state of motion from the molecules, atomic groups, and atoms of the compounds constituting the living plasma of the cell to the fermentable material, whereby the equilibrium existing in the molecules of the latter is disturbed and decomposition ensues [[1879], p. 29]. [p016]
This somewhat complex idea, whilst including, as did Liebig's theory, Stahl's fundamental conception of a transmission of a state of motion, satisfies Pasteur's contention that fermentation cannot occur without life, and at the same time explains the specific action of different organisms by differences in the constitution of their cell contents. The really essential part of Nägeli's theory consisted in the limitation of the power of transference of molecular motion to the living plasma, by which the failure of all attempts to separate the power of fermentation from the living cell was explained. This was the special phenomenon which required explanation; to account for this the theory was devised, and when this was experimentally disproved, the theory lost all significance.
For nearly twenty years no further progress was made, and then in 1897 the question which had aroused so much discussion and conjecture, and had given rise to so much experimental work, was finally answered by Eduard Buchner, who succeeded in preparing from yeast a liquid which, in the complete absence of cells, was capable of effecting the resolution of sugar into carbon dioxide and alcohol [[1897, 1]].
In the light of this discovery the contribution to the truth made by each of the great protagonists in the prolonged discussion on the problem of alcoholic fermentation can be discerned with some degree of clearness. Liebig's main contention that fermentation was essentially a chemical act was correct, although his explanation of the nature of this act was inaccurate. Pasteur, in so far as he considered the act of fermentation as indissolubly connected with the life of the organism, was shown to be in error, but the function of the organism has only been restricted by a single stage, the active enzyme of alcoholic fermentation has so far only been observed as the product of the living cell. Nearest of all to the truth was Traube, who in 1858 enunciated the theorem, which was only proved for alcoholic fermentation in 1897, that all fermentations produced by living organisms are due to ferments secreted by the cells.
Buchner's discovery of zymase has introduced a new experimental method by means of which the problem of alcoholic fermentation can be attacked, and the result has been that since 1897 a considerable amount of information has been gained with regard to the nature and conditions of action of the enzymes of the yeast cell. It has been found that the machinery of fermentation is much more complex than had been surmised. The enzyme zymase, which is essential for fermentation, cannot of itself bring about the alcoholic fermentation of sugar, but is dependent on the presence of a second substance, termed, for [p017] want of a more reasonable name, the co-enzyme. The chemical nature and function of this mysterious coadjutor are still unknown, but as it withstands the temperature of boiling water and is dialysable, it is probably more simple in constitution than the enzyme. This, however, is not all; for the decomposition of sugar a phosphate is also indispensable. It appears that in yeast-juice, and therefore also most probably in the yeast cell, the phosphorus present takes an active part in fermentation and goes through a remarkable cycle of changes. The breakdown of sugar into alcohol and carbon dioxide is accompanied by the formation of a complex hexosephosphate, and the phosphate is split off from this compound and thus again rendered available for action by means of a special enzyme, termed hexosephosphatase. In addition to this complex of ferments, the cell also possesses special enzymes by which the zymase and the co-enzyme can be destroyed, and, further, at least one substance, known as an anti-enzyme, which directly checks this destructive action. It seems probable, moreover, that the decomposition of the sugar molecule takes place in stages, although much doubt yet exists as to the nature of these.
At the present moment the subject remains one of the most interesting in the whole field of biological chemistry, the limited degree of insight which has already been gained into the marvellous complexity of the cell lending additional zest to the attempt to penetrate the darkness which shrouds the still hidden mysteries.