The organism to which this change was due had hitherto escaped detection, and as we have seen the spontaneous lactic fermentation of milk was one of the phenomena adduced by Gerhardt (p. [10]) in favour of Liebig's views. Pasteur [[1857]] discovered the lactic acid producing organism and convinced himself that it was in fact a living organism and the active cause of the production of lactic acid. One of the chief buttresses of Liebig's theory was thus removed, and Pasteur next proceeded to apply the same method and reasoning to alcoholic fermentation. Liebig's theory of the origin of yeast by the action of the oxygen of the air on the nitrogenous matter of the fermentable liquid was conclusively and strikingly disproved by the brilliant device of producing a crop of yeast in a liquid medium containing only comparatively [p012] simple substances of known composition—sugar, ammonium tartrate and mineral phosphate. Here there was obviously present in the original medium no matter which could be put into a state of putrefaction by contact with oxygen and extend its instability to the sugar. Any such material must first be formed by the vital processes of the yeast. In the next place Pasteur showed by careful analyses and estimations that, whenever fermentation occurred, growth and multiplication of yeast accompanied the phenomenon. The sugar, he proved, was not completely decomposed into carbon dioxide and alcohol, as had been assumed by Liebig (p. [8]). A balance-sheet of materials and products was constructed which showed that the alcohol and carbon dioxide formed amounted only to about 95 per cent. of the invert sugar fermented, the difference being made up by glycerol, succinic acid, cellulose, and other substances [[1860], p. 347]. In every case of fermentation, even when a paste of yeast was added to a solution of pure cane sugar in water, the yeast was found by quantitative measurements to have taken something from the sugar. This "something" was indeterminate in character, but, including the whole of the extractives which had passed from the yeast cells into the surrounding liquid, it amounted to as much as 1·63 per cent. of the weight of the sugar fermented [[1860], p. 344].
Pasteur was therefore led to consider fermentation as a physiological process accompanying the life of the yeast. His conclusions were couched in unmistakable words: "The chemical act of fermentation is essentially a phenomenon correlative with a vital act, commencing and ceasing with the latter. I am of opinion that alcoholic fermentation never occurs without simultaneous organisation, development, multiplication of cells, or the continued life of cells already formed. The results expressed in this memoir seem to me to be completely opposed to the opinions of Liebig and Berzelius. If I am asked in what consists the chemical act whereby the sugar is decomposed and what is its real cause, I reply that I am completely ignorant of it.
"Ought we to say that the yeast feeds on sugar and excretes alcohol and carbonic acid? Or should we rather maintain that yeast in its development produces some substance of the nature of a pepsin, which acts upon the sugar and then disappears, for no such substance is found in fermented liquids? I have nothing to reply to these hypotheses. I neither admit them nor reject them, and wish only to restrain myself from going beyond the facts. And the facts tell me simply that all true fermentations are correlative with physiological phenomena."
Liebig felt to the full the weight of Pasteur's criticisms; his reply [p013] was long delayed [[1870]], and, according to his biographer, Volhard [[1909]], caused him much anxiety. In it he admits the vegetable nature of yeast, but does not regard Pasteur's conclusion as in any way a solution of the problem of the nature of alcoholic fermentation. Pasteur's "physiological act" is for Liebig the very phenomenon which requires explanation, and which he still maintains can be explained by his original theory of communicated instability. On some of Pasteur's results, notably the very important one of the cultivation of yeast in a synthetic medium, he casts grave doubt, whilst he explains the production of glycerol and succinic acid as due to independent reactions. The phenomenon of fermentation is still for him one which accompanies the decomposition of the constituents of the cell, rather than their building up by vegetative growth. "When the fungus ceases to grow, the bond which holds together the constituents of the cell contents is relaxed, and it is the motion which is thus set up in them which is the means by which the yeast cells are enabled to bring about a displacement or decomposition of the elements of sugar or other organic molecules." Pasteur replied in a brief and unanswerable note [[1872]]. All his attention was concentrated on the one question of the production of yeast in a synthetic medium, which he recognised as fundamental. The validity of this experiment he emphatically reaffirmed, and finally undertook, from materials supplied by Liebig himself, to produce as much yeast as could be reasonably desired. This challenge was never taken up, and this communication formed the last word of the controversy. Pasteur had at this time firmly established his thesis, no fermentation without life, both for alcoholic fermentation and for those other fermentations which are produced by bacteria, and had put upon a sound and permanent basis the conclusions drawn by Schulze, Cagniard-Latour, Schwann, and Kützing from their early experiments. It became generally recognised that putrefaction and other fermentative changes were due to specific organisms, which produced them in the exercise of their vital functions.
Pasteur subsequently [[1875]] came to the conclusion that fermentation was the result of life without oxygen, the cells being able, in the absence of free oxygen, to avail themselves of the energy liberated by the decomposition of substances containing combined oxygen. This view, which did not involve any alteration of Pasteur's original thesis but was an attempt to explain the physiological origin and function of fermentation, gave rise to a prolonged controversy, which cannot be further discussed in these pages. [p014]
Nevertheless, Liebig's desire to penetrate more deeply into the nature of the process of fermentation remained in many minds, and numerous endeavours were made to obtain further insight into the problem. In spite of an entire lack of direct experimental proof, the conception that alcoholic fermentation was due to the chemical action of some substance elaborated by the cell and not directly to the vital processes of the cell as a whole found strenuous supporters even among those who were convinced of the vegetable character of yeast. As early as 1833 diastase, discovered still earlier by Kirchhoff and Dubrunfaut, had been extracted by means of water from germinating barley and precipitated by alcohol as a white powder, the solution of which was capable of converting starch into sugar, but lost this power when heated [Payen and Persoz, [1833]]. Basing his ideas in part upon the behaviour of this substance, Moritz Traube [[1858]] enunciated in the clearest possible manner the theory that all fermentations produced by living organisms are caused by ferments, which are definite chemical substances produced in the cells of the organism. He regarded these substances as being closely related to the proteins and considered that their function was to transfer the oxygen and hydrogen of water to different parts of the molecule of the fermentable substance and thus bring about that apparent intramolecular oxidation and reduction which is so characteristic of fermentative change and had arrested the attention of Lavoisier and, long after him, of Liebig.
Traube's main thesis, that fermentation is caused by definite ferments or enzymes, attracted much attention, and received fresh support from the separation of invertase in 1860 from an extract of yeast by Berthelot, and from the advocacy and authority of this great countryman of Pasteur, who definitely expressed his opinion that insoluble ferments existed which could not be separated from the tissues of the organism, and further, that the organism could not itself be regarded as the ferment, but only as the producer of the ferment [[1857], [1860]]. Hoppe-Seyler [[1876]] also supported the enzyme theory of fermentation, but differed in some respects from Traube as to the exact function of the ferment [see Traube, [1877]; Hoppe-Seyler, [1877]].
Direct experimental evidence was, however, still wanting, and Pasteur's reiterated assertion [[1875]] that all fermentation phenomena were manifestations of the life of the organism remained uncontroverted by experience.
Numerous and repeated direct experimental attacks had been made [p015] from time to time upon the problem of the existence of a fermentation enzyme, but all had yielded negative or unreliable results.
As early as 1846 a bold attempt had been made by Lüdersdorff [[1846]] to ascertain whether fermentation was or was not bound up with the life of the yeast by grinding yeast and examining the ground mass. A single gram of yeast was thoroughly ground, the process lasting for an hour, and the product was tested with sugar solution. Not a single bubble of gas was evolved. A similar result was obtained in a repetition of the experiment by Schmidt in Liebig's laboratory [[1847]], the grinding being continued in this case for six hours, but the natural conclusion that living yeast was essential for fermentation was not accepted, on the ground that during the lengthy process of trituration in contact with air the yeast had become altered and now no longer possessed the power of producing alcoholic fermentation, but instead had acquired that of changing sugar into lactic acid [see Gerhardt, [1856], p. 545].