Effect of Phosphate on the Total Fermentation Produced by Yeast-Juice.

The addition of a phosphate to yeast-juice not only produces the effect already described, but also enables a given volume of yeast-juice to effect a larger total fermentation, even after allowance is made for the carbon dioxide equivalent to the quantity of phosphate added. The increase in the case of ordinary yeast-juice has been found to amount to from 10 to 150 per cent. of the original total fermentation [p055] produced by the juice in the absence of added phosphate. The numbers contained in columns 1 and 2 of the table on p. [56] illustrate this effect, the ratio of the total in the presence of phosphate to that obtained in its absence being given, as well as that of the total in presence of phosphate less the equivalent of the phosphate added, to the original fermentation. The cause of this increase in the total fermentation is probably to be sought mainly in a protective action of the excess of hexosephosphate on the various enzymes, for, as has been stated above, the rate of fermentation after the termination of the initial period, is practically the same as in the absence of added phosphate (see p. [43]).

Now it follows from equation (1) (p. [51]) that in the total absence of phosphate no fermentation should occur, and the experimental realisation of this result would afford very strong evidence in favour of this interpretation of the phenomenon.

Hitherto, however, it has not been found possible to free the materials employed completely from phosphorus compounds which yield phosphates by enzymic hydrolysis during the experiment, but it has been found that when the phosphate contents are reduced to as low a limit as possible, the amount of sugar fermented becomes correspondingly small, and, further, that in these circumstances the addition of a small amount of phosphate or hexosephosphate produces a relatively large increase in the fermenting power of the enzyme.

When the total phosphorus present is thus largely reduced, the increase produced by the addition of a small amount of phosphate may amount to as much as eighty-eight times the original, in addition to the quantity equivalent to the phosphate, whilst the actual total evolved, including this equivalent, may be as much as twenty times the original fermentation. This result must be regarded as strong evidence in favour of the view that phosphates are indispensable for alcoholic fermentation.

The results indicated above were experimentally obtained in three different ways and are exhibited in the following table. In the first place (cols. 3 and 4), advantage was taken of the fact that the residues obtained by filtering yeast-juice through a Martin gelatin filter (p. [59]) are sometimes found to be almost free from mineral phosphates, whilst they still contain a small amount of co-enzyme. The experiment then consists in comparing the fermentation produced by such a residue poor in phosphate with that observed when a small amount of phosphate is added. The second method (col. 5) consisted in carrying out two parallel fermentations by means of a residue rendered inactive by filtration [p056] and a solution of co-enzyme free from phosphate and hexosephosphate (p. [67]) [Harden and Young, [1910, 2]].

The third method (col. 6) consisted in washing zymin with water, to remove soluble phosphates, and then adding to it a solution of co-enzyme containing only a small amount of phosphate, and ascertaining the effect of the addition of a small known amount of hexosephosphate upon the fermentation produced by this mixture [Harden and Young, [1911, 1]].

1
c.c.		2
c.c.		3
c.c.		4
c.c.		5
c.c		6
c.c.
Gas evolved in absence of added phosphate369 220 1·4 1·220·3 1·5
In the presence of added phosphate629 629 25·826·892·3132·7
Increase due to phosphate260 409 24·425·672·0131·2
Carbonic acid equivalent to phosphate63 61 16·916·816·8—
Increase after initial period197 348 7·58·855·2—
Ratio of totals1·72·918·421·34·588
Ratio of increase after initial period to original fermentation0·51·65·37·32·7—