other: “Fermentation is the transference of the condition of motion of the molecules, atomic groups and atoms of the various compounds constituting the living plasma, to the fermenting material, in consequence of which equilibrium in the molecules of the latter is destroyed, the result being their disintegration.” He agreed with Pasteur that the presence of living cells is essential to the transformation of sugar into alcohol, but dissented from the view that the process occurs within the cell. This investigator held that the decomposition of the sugar molecules takes place outside the cell wall. In 1894 and 1895, Fischer, in a remarkable series of papers on the influence of molecular structure upon the action of the enzyme, showed that various species of yeast behave very differently towards solutions of sugars. For example, some species hydrolyse cane sugar and maltose, and then carry on fermentation at the expense of the simple sugars (hexoses) so formed. Saccharomyces Marxianus will not hydrolyse maltose, but it does attack cane sugar and ferment the products of hydrolysis. Fischer next suggested that enzymes can only hydrolyse those sugars which possess a molecular structure in harmony with their own, or to use his ingenious analogy, “the one may be said to fit into the other as a key fits into a lock.” The preference exhibited by yeast cells for sugar molecules is shared by mould fungi and soluble enzymes in their fermentative actions. Thus, Pasteur showed that Penicillium glaucum, when grown in an aqueous solution of ammonium racemate, decomposed the dextro-tartrate, leaving the laevo-tartrate, and the solution which was originally inactive to polarized light became dextro-rotatory. Fischer found that the enzyme “invertase,” which is present in yeast, attacks methyl-d-glucoside but not methyl-l-glucoside.
In 1897 Buchner submitted yeast to great pressure, and isolated a nitrogenous substance, enzymic in character, which he termed “zymase.” This body is being continually formed in the yeast cell, and decomposes the sugar which has diffused into the cell. The freshly-expressed yeast juice causes concentrated solutions of cane sugar, glucose, laevulose and maltose to ferment with the production of alcohol and carbon dioxide, but not milk-sugar and mannose. In this respect the plasma behaves in a similar manner towards the sugars as does the living yeast cell. Pasteur found that, when cane sugar was fermented by yeast, 49.4% of carbonic acid and 51.1% of alcohol were produced; with expressed yeast juice cane sugar yields 47% of carbonic acid and 47.7% of alcohol. According to Buchner the fermentative activity of yeast-cell juice is not due to the presence of living yeast cells, or to the action of living yeast protoplasm, but it is caused by a soluble enzyme. A. Macfadyen, G. H. Morris and S. Rowland, in repeating Buchner’s experiments, found that zymase possessed properties differing from all other enzymes, thus: dilution with twice its volume of water practically destroys the fermentative power of the yeast juice. These investigators considered that differences of this nature cannot be explained by the theory that it is a soluble enzyme, which brings about the alcoholic fermentation of sugar. The remarkable discoveries of Fischer and Buchner to a great extent confirm Traube’s views, and reconcile Liebig’s and Pasteur’s theories. Although the action of zymase may be regarded as mechanical, the enzyme cannot be produced by any other than living protoplasm.
Pasteur’s important researches mark an epoch in the technical aspect of fermentation. His investigations on vinegar-making revolutionized that industry, and he showed how, instead of waiting two or three months for the elaboration of the process, the vinegar could be made in eight or ten days by exposing the vats containing the mixture of wine and vinegar to a temperature of 20° to 25° C., and sowing with a small quantity of the acetic organism. To the study of the life-history of the butyric and acetic organisms we owe the terms “anaërobic” and “aërobic.” His researches from 1860 and onwards on the then vexed question of spontaneous generation proved that, in all cases where spontaneous generation appeared to have taken place, some defect or other was in the experiment. Although the direct object of Pasteur was to prove a negative, yet it was on these experiments that sterilization as known to us was developed. It is only necessary to bear in mind the great part played by sterilization in the laboratory, and pasteurization on the fermentation industries and in the preservation of food materials. Pasteur first formulated the idea that bacteria are responsible for the diseases of fermented liquids; the corollary of this was a demand for pure yeast. He recommended that yeast should be purified by cultivating it in a solution of sugar containing tartaric acid, or, in wort containing a small quantity of phenol. It was not recognized that many of the diseases of fermented liquids are occasioned by foreign yeasts; moreover, this process, as was shown later by Hansen, favours the development of foreign yeasts at the expense of the good yeast.
About this time Hansen, who had long been engaged in researches on the biology of the fungi of fermentation, demonstrated that yeast free from bacteria could nevertheless occasion diseases in beer. This discovery was of great importance to the zymo-technical industries, for it showed that bacteria are not the only undesirable organisms which may occur in yeast. Hansen set himself the task of studying the properties of the varieties of yeast, and to do this he had to cultivate each variety in a pure state. Having found that some of the commonest diseases of beer, such as yeast turbidity and the objectionable changes in flavour, were caused not by bacteria but by certain species of yeast, and, further, that different species of good brewery yeast would produce beers of different character, Hansen argued that the pitching yeast should consist only of a single species—namely, that best suited to the brewery in question. These views met with considerable opposition, but in 1890 Professor E. Duclaux stated that the yeast question as regards low fermentation has been solved by Hansen’s investigations. He emphasized the opinion that yeast derived from one cell was of no good for top fermentation, and advocated Pasteur’s method of purification. But in the course of time, notwithstanding many criticisms and objections, the reform spread from bottom fermentation to top fermentation breweries on the continent and in America. In the United Kingdom the employment of brewery yeasts selected from a single cell has not come into general use; it may probably be accounted for in a great measure by conservatism and the wrong application of Hansen’s theories.
Pure Cultivation of Yeasts.—The methods which were first adopted by Hansen for obtaining pure cultures of yeast were similar in principle to one devised by J. Lister for isolating a pure culture of lactic acid bacterium. Lister determined the number of bacteria present in a drop of the liquid under examination by counting, and then diluted this with a sufficient quantity of sterilized water so that each drop of the mixture should contain, on an average, less than one bacterium. A number of flasks containing a nutrient medium were each inoculated with one drop of this mixture; it was found that some remained sterile, and Lister assumed that the remaining flasks each contained a pure culture. This method did not give very certain results, for it could not be guaranteed that the growth in the inoculated flask was necessarily derived from a single bacterium. Hansen counted the number of yeast cells suspended in a drop of liquid diluted with sterilized water. A volume of the diluted yeast was introduced into flasks containing sterilized wort, the degree of dilution being such that only a small proportion of the flasks became infected. The flasks were then well shaken, and the yeast cell or cells settled to the bottom, and gave rise to a separate yeast speck. Only those cultures which contained a single yeast speck were assumed to be pure cultivations. By this method several races of Saccharomycetes and brewery yeasts were isolated and described.
The next important advance was the substitution of solid for liquid media; due originally to Schroter. R. Koch subsequently improved the method. He introduced bacteria into liquid sterile nutrient gelatin. After being well shaken, the liquid was poured into a sterile glass Petrie dish and covered with a moist and sterile bell-jar. It was assumed that each separate speck contained a pure culture. Hansen pointed out that this
