(c) Relation of Carbon Dioxide and Alcohol Produced to the Amount of Sugar Fermented.
The construction of a balance-sheet between the sugar fermented and the products formed is of special interest in the case of alcoholic fermentation by yeast-juice, because, there being no cell growth as in the case of living yeast, an opportunity appears to be afforded of ascertaining whether the whole of the sugar is converted into alcohol and carbon dioxide, or whether some fraction of the sugar passes into any of the well-known subsidiary products of alcoholic fermentation by yeast, such as glycerol, fusel oil, or succinic acid. Unfortunately the question cannot be settled in this way. When the loss of sugar during the fermentation is estimated directly, it is usually found to be considerably greater than the sum of the alcohol and carbon dioxide produced from it. This fact was first observed by Macfadyen, Morris and Rowland [[1900]], and was then confirmed by Buchner [Buchner, E. and H., and Hahn, [1903], p. 212], in one instance, the excess of sugar lost over products being in this case about 15 per cent. of the total sugar which had disappeared. The matter was then more thoroughly investigated by Harden and Young [[1904]]. [p031]
The conditions under which the experiment must be carried out are not very favourable to the attainment of extreme accuracy. Yeast-juice contains glycogen and a diastatic enzyme which converts this into dextrins and finally into sugar. This process goes on throughout fermentation, tending to increase the sugar present and to make the apparent loss of sugar less than the sum of the products. In spite of this it was found that a certain amount of sugar invariably disappeared without being accounted for as alcohol or carbon dioxide, and this whether the fermentation lasted sixty or a hundred and eight hours, and independently of the dilution of the juice. This disappearing sugar amounted in some cases to 44 per cent. of the total loss of sugar, and on the average of twenty-five experiments was 38 per cent. Further information was sought by converting all the sugar-yielding constituents of the juice into sugar by hydrolysis before and after the fermentation. This process revealed the fact that when the glucose equivalent of the juice before and after fermentation was determined after hydrolysis with three times normal acid for three hours (and a correction made for the loss of reducing power experienced by glucose itself when submitted to this treatment), the difference was almost exactly equal to the alcohol and carbon dioxide produced. In other words, accompanying fermentation, a change proceeds by which sugar is converted into a less reducing substance, reconvertible into sugar by hydrolysis with acids. Similar results were subsequently obtained by Buchner and Meisenheimer [[1906]], who employed 1·5 normal acid and observed a small nett loss of sugar. Still more recently Lebedeff [[1909], [1910], see also [1913, 2]] has carried out similar estimations with the same result. It is doubtful whether the experiments which have so far been made on this point are sufficiently accurate to decide with certainty whether or not the loss of sugar is exactly equal to the sum of the carbon dioxide and alcohol produced. It has been shown by Buchner and Meisenheimer [[1906]] that glycerol is a constant product of alcoholic fermentation by yeast-juice (p. [95]), and no other source for this than the sugar has yet been found, so that it is not improbable that a small amount of sugar is converted into non-carbohydrate substances other than carbon dioxide and alcohol.
It has also been shown [Harden and Young, [1913]] that the deficit of sugar is not due to the formation of hexosephosphate (p. [47]), which has a lower reduction than glucose, and that the solution from which the sugar (either glucose or fructose) has disappeared actually contains some substance of relatively high dextrorotation and of low reducing power. [p032]
However this may be, it may be considered as established that during alcoholic fermentation sugar is converted by an enzyme into some compound of less reducing power, which again yields sugar on hydrolysis with acids. The exact nature of this substance has not been ascertained, but it appears likely that the process is a synthetical one resulting in the formation of some polysaccharide, possibly intermediate between the hexoses and glycogen.
A similar phenomenon has been observed with living yeast by Euler and Johansson [[1912, 1]], and Euler and Berggren [[1912]], whose interpretation of the observation is discussed later on (p. [57]).
(d) Fermentation of Different Carbohydrates. Autofermentation.
Yeast-juice and zymin ferment all the sugars which are fermented by the yeast from which they are prepared, and, in addition, a number of colloidal substances which cannot pass through the membrane of the living yeast cell, but which are hydrolysed by enzymes in the juice and thus converted into simpler sugars capable of fermentation [Buchner and Rapp, [1898, 3]; [1899, 2]]. Of the simple sugars which have been examined, glucose, fructose, and mannose are freely fermented, l-arabinose not at all, whilst the case of galactose is doubtful. Galactose is, however, fermented by juice prepared from a yeast which has been "trained" to ferment galactose [Harden and Norris, [1910]]. As regards both the rate of fermentation and the total amount of carbon dioxide evolved from glucose and fructose by the action of a definite amount of yeast-juice, Buchner and Rapp obtained practically identical numbers. Harden and Young [[1909]], using juice from top yeast, found that fructose was slightly more rapidly fermented and gave a somewhat larger total than glucose, whilst mannose was initially fermented at almost the same rate as glucose, but gave a decidedly lower total, the following being the average result:—
| Sugar. | Relative Rates. | Relative Totals. |
|---|---|---|
| Glucose | 1 | 1 |
| Fructose | 1·29 | 1·15 |
| Mannose | 1·04 | 0·67 |
Among the disaccharides, cane sugar and maltose are freely fermented, and the juice can be shown like living yeast to contain invertase and maltase. The extent of fermentation does not differ materially from that attained with glucose. Lactose is not fermented.