Effect of the Addition of Phosphate to a Fermenting Mixture of Yeast-Juice and Sugar.
When a suitable quantity[2] of a soluble phosphate is added to a fermenting mixture of glucose, fructose, or mannose with yeast-juice, the rate of fermentation rapidly rises, sometimes increasing as much as twenty-fold, continues at this high value for a certain period and then falls again to a value approximately equal to, but generally [p043] somewhat higher than, that which it originally had. Careful experiments have shown that during this period of enhanced fermentation the amounts of carbon dioxide and alcohol produced exceed those which would have been formed in the absence of added phosphate by a quantity exactly equivalent to the phosphate added in the ratio CO2 or C2H6O:R′2HPO4 [Harden and Young, [1906, 1]].
[2] The effect of an excess of phosphate is discussed later on, p. [71].
This result is of fundamental importance, and the evidence on which it rests deserves some consideration. Quantitative experiments on this subject require certain preliminary precautions. The acid phosphates are too acid to permit of any extended fermentation and the phosphates of the formula R′2HPO4 absorb a considerable volume of carbon dioxide with production of a bicarbonate, according to the reaction:—
R2HPO4 + H2CO3 ⇌ RHCO3 + RH2PO4.
The method which has been adopted, therefore, is to employ either a secondary phosphate saturated with carbon dioxide at the temperature of the experiment, or a mixture of five molecular proportions of the secondary phosphate with one molecular proportion of a primary phosphate, in which the amount of bicarbonate formed is negligible. In the former case it is necessary to ascertain whether any of the carbon dioxide evolved is derived from the bicarbonate by the action of acid originally present or produced in the yeast-juice or by a disturbance of the original equilibrium owing to the chemical change which occurs. This is done by acidifying duplicate samples with hydrochloric acid before and after the fermentation and measuring the gas evolved in each case. Any necessary correction can then be made. The calculation of the extra amount of carbon dioxide evolved from yeast-juice containing sugar when a phosphate is added involves an estimation of the amount which would have been evolved in the absence of added phosphate, and this is a matter of some difficulty. Since the final steady rate of fermentation attained is often slightly different from the initial rate, the practice has been adopted of ascertaining this final rate and then calculating the total evolution corresponding to it for the whole period from the time of the addition of the phosphate to the end of the observations. This amount deducted from the observed total leaves the extra amount of carbon dioxide formed, and it is this quantity which is equivalent to the phosphate added. Alcohol is simultaneously produced in the normal ratio. The justification for this method of calculation will be found later (p. [54]).
The following table, containing the results of experiments with [p044] glucose, fructose, and mannose, indicates very clearly the nature of the method of calculation and also of the agreement between observation and theory.
Three quantities of 25 c.c. of yeast-juice + 5 c.c. of a solution containing 1 gram of the sugar to be examined (a large excess) were incubated with toluene at 25° for one hour, in order to remove all free phosphate, and to each were then added 5 c.c. of a solution of sodium phosphate corresponding to 0·1632 gram of Mg2P2O7 and equivalent to 32·6 c.c. of carbon dioxide at N.T.P. The rates of fermentation were then observed until they had passed through the period of acceleration and had fallen and attained a steady value, the gases being measured moist at 19·3° and 760·15 mm.
| Glucose. | Mannose. | Fructose. | |
|---|---|---|---|
| Maximum rate attained, c.cs. per five minutes | 9·6 | 7 | 11·3 |
| Final rate of fermentation | 1·1 | 0·96 | 1·08 |
| Total carbon dioxide produced by fermentation in fifty-five minutes after addition of phosphate | 49·7 | 47·8 | 47·6 |
| Correction for evolution in absence of phosphate in fifty-five minutes | 12·1 | 10·6 | 11·9 |
| Extra carbon dioxide equivalent to phosphate | 37·6 | 37·2 | 35·7 |
| Extra carbon dioxide equivalent to phosphate at N.T.P. | 34·4 | 34 | 32·6 |
These numbers agree well with the value calculated from the phosphate added, viz. 32·6 [Harden and Young, [1909]].
Another experiment is illustrated graphically in Fig. 4, in which the volume of carbon dioxide evolved is plotted against time. The determination was in this case made by adding 25 c.c. of an aqueous solution containing 5 grams of glucose to one quantity of 25 c.c. of yeast-juice (curve A) and 5 c.c. of 0·3 molar solution of the mixed primary and secondary sodium phosphates, and 20 c.c. of a solution containing 5 grams of glucose to a second equal quantity of yeast-juice (curve B). Curve A shows the normal course of fermentation of yeast-juice with glucose. There is a slight preliminary acceleration during the first twenty minutes, due to free phosphate in the juice, and the rate then becomes steady at about 1·4 c.c. in five minutes. During this preliminary acceleration 10 c.c. of extra carbon dioxide are evolved, this number being obtained graphically by continuing the line of steady rate back to the axis of zero time. Curve B shows the effect of the added phosphate. The rate rises to about 9·5 c.c. in five minutes, i.e. to more than six times the normal rate, and then gradually falls until after an hour it is again steady and almost exactly equal to 1·4 c.c. per five minutes. Continuing the line of steady rate back to the axis of zero [p045] time it is found that the extra amount of carbon dioxide is 48 c.c. Subtracting from this the 10 c.c. shown in curve A as due to the juice alone, a difference of 38 c.c. is obtained due to the added phosphate. The amount calculated from the phosphate added in this case is, at atmospheric temperature and pressure, 38·9 c.c.
Fig. 4.
After the expiration of seventy minutes from the commencement of the experiment, a second addition is made of an equal amount of phosphate. The whole phenomenon then recurs, as shown in curve C, the maximum rate being slightly lower than before, about 6 c.c. per five minutes, and the rate again becoming finally steady at 1·4 c.c. as before. The extra amount of carbon dioxide evolved in this second period obtained graphically as in the former case, is 107–68 = 39 c.c.
It may be noted that in this case the observations after each addition last fifty to seventy minutes, so that an error of 0·1 c.c. per five minutes in the estimated final rate would make an error of 1 to 1·4 c.c. in the extra amount of carbon dioxide, i.e. about 3 to 4 per cent. of the total, and this is approximately the limit of accuracy of the method. [p046] The results are more precise when the yeast-juice employed is an active one, since, when the fermenting power of the juice is low, the initial period of accelerated fermentation is unduly prolonged and the calculation of the extra amount of carbon dioxide is rendered uncertain.
Zymin (p. [38]) yields precisely similar results to yeast-juice, but in this case the rate of fermentation is not so largely increased. This has the effect that the extra amount of carbon dioxide cannot be quite so accurately estimated for zymin, because a slight error in the determination of the final rate of fermentation has a greater influence on the result. The equivalence between the extra amount of carbon dioxide evolved and the phosphate added is, however, unmistakable, as is shown by the following results of an experiment with zymin, in which 6 grams of zymin (Schroder) + 3 grams of fructose (Schering) + 25 c.c. of water were incubated at 25° in presence of toluene until a steady rate had been attained. Five c.c. of a solution of sodium phosphate equivalent to 32·2 c.c. carbon dioxide at N.T.P. were then added.
| Maximum rate attained, c.c. per five minutes | 14·1 |
| Final rate of fermentation | 6·2 |
| Total evolved by fermentation in eighty minutes after addition of phosphate | 131 |
| Correction for evolution in absence of phosphate in eighty minutes | 99·2 |
| Extra carbon dioxide at 16° and 767·1 mm | 31·8 |
| Extra carbon dioxide at N.T.P | 29·8 |
Considering the small proportional rise in rate and the long period of accelerated fermentation, the agreement between the volume observed, 29·8 c.c., and that calculated from the phosphate, 32·2, is quite satisfactory [Harden and Young, [1910, 1].] Precisely the same relations hold for maceration extract, but in this case it must be remembered that a large amount of free phosphate is present in the extract, as much as 0·3129 grm. Mg2P2O7 being obtained from 20 c.c. in one preparation, so that the original extract had the concentration of a 0·14 molar solution of sodium phosphate. It is in fact not improbable that the delay in the onset of fermentation sometimes observed with maceration extract (see Lebedeff, 1912, 2; Neuberg and Rosenthal, 1913) may be due to the presence of phosphate in so great an excess of the amount which can be rapidly esterified by the enzymes that the rate of fermentation is at first greatly lowered (see p. [71]). When this phosphate is removed by incubation with glucose or fructose, the subsequent addition of phosphate produces the characteristic action and the extra carbon dioxide evolved is, as with other yeast preparations, equivalent to the phosphate added. An actual estimation carried out in this way gave 35 c.c. of CO2 for an addition of phosphate equivalent to 32·9 c.c. [Harden and Young, [1912]]. [p047]
Within the limits imposed by the experimental conditions, then, the fact is well established that the addition of a soluble phosphate to a fermenting mixture of a hexose with yeast-juice, maceration extract, dried yeast, or zymin causes the production of an equivalent amount of carbon dioxide and alcohol.
This fact indicates that a definite chemical reaction occurs in which sugar and phosphate are concerned, and this conclusion is confirmed when the fate of the added phosphate is investigated. If an experiment, such as one of those described above, be interrupted as soon as the rate of fermentation has again become normal, and the liquid be boiled and filtered, it is found that nearly the whole of the phosphorus present passes into the filtrate, but that only a small proportion of this exists as mineral phosphate, whilst the remainder, including that added in the form of a soluble phosphate, is no longer precipitable by magnesium citrate mixture [Harden and Young, [1905, 2]].
A similar observation was made at a later date by Iwanoff [[1907]], who had previously observed [[1905]] that living yeast, like many other vegetable organisms, converted mineral phosphates into organic derivatives. Iwanoff employed zymin and hefanol (p. [38]) instead of yeast-juice, and found that phosphates were thereby rendered non-precipitable by uranium acetate solution, but did not observe the accelerated fermentation caused by their addition.
The foregoing conclusions have been strikingly confirmed by experiments with maceration extract carried out by Euler and Johansson [[1913]], in which both the carbon dioxide evolved and the phosphate rendered non-precipitable by magnesia were determined at intervals. When dried yeast is employed as the fermenting agent, the amount of phosphate esterified in the earlier stages is greater than would be expected, but ultimately becomes exactly equivalent to the carbon dioxide evolved.