Practical Methods for the Estimation of the Fermenting Power of Yeast-Juice.
In order to estimate the amount of carbon dioxide evolved in a given time and the total amount evolved by the action of yeast-juice on sugar, Buchner adopted an extremely simple method, which consisted in carrying out the fermentation in an Erlenmeyer flask provided with a small wash-bottle, which contained sulphuric acid and was closed by a Bunsen valve, and ascertaining the loss of weight during the experiment. Corrections are necessary for the carbon dioxide present in the original juice and retained in the liquid at the close of the experiment and for that present in the air space of the apparatus, but it was found that for most purposes these could be neglected. In cases in which greater accuracy was desired, the carbon dioxide was displaced by air before the weighings were made. A typical experiment of this kind, without displacement of carbon dioxide, is the following:—
| March 22, 1899, Berlin bottom yeast V. 20 c.c. juice + 8 grams cane sugar + 0·2 c.c. toluene as antiseptic at 16°. Grams of carbon dioxide after | ||||
|---|---|---|---|---|
| 24 | 48 | 72 | 96 hours. | |
| 0·40 | 0·64 | 0·99 | 1·11 | |
The total weight of carbon dioxide evolved under these conditions is termed the fermenting power of the juice (Buchner).
A more accurate method [Macfadyen, Morris, and Rowland, [1900]] consists in passing the carbon dioxide into caustic soda solution and estimating it by titration. The yeast-juice, sugar, and antiseptic are placed in an Erlenmeyer flask provided with a straight glass tube, through which air can be passed over the surface of the liquid, and a conducting tube leading into a second flask which contains 50 c.c. of 10 per cent. caustic soda solution and is connected with the air by a guard tube containing soda lime. The juice can be freed from carbon dioxide by agitation in a current of air before the flask is connected to [p028] that containing the caustic soda solution, and at the end of the period of incubation air is passed through the apparatus, the liquid being boiled out if great accuracy is required. The absorption flask is then disconnected and the amount of absorbed carbon dioxide estimated by titration. This is carried out by making up the contents of the flask to 200 c.c., taking out an aliquot portion, rendering this exactly neutral to phenophthalein by the addition first of normal and finally of decinormal acid, adding methyl orange and titrating with decinormal acid to exact neutrality. Each c.c. of decinormal acid used in this last titration represents 0·0044 gram of carbon dioxide in the quantity of solution titrated.
Fig. 3.
These methods are only suitable for observations at considerable intervals of time. For the continuous observation of the course of fermentation Harden, Thompson and Young [[1910]] connect the fermentation flask with a Schiff's azotometer filled with mercury and measure the volume of gas evolved, the liquid having been previously saturated with carbon dioxide (Fig. 3). The level of the mercury in the reservoir is kept constant by a syphon overflow, as shown in the figure, or, according to a modification introduced by S. G. Paine, by a specially constructed bottle provided with two tubulures near the bottom. This ensures that no change in the pressure in the flask occurs, and the volume of gas observed is reduced to normal pressure by means of a table. Before making a reading it is necessary to shake the fermenting mixture thoroughly, as the albuminous liquid very readily becomes greatly supersaturated with carbon dioxide, so much so in fact that very little gas is evolved in the intervals between the shakings. The exact procedure in making an observation consists in shaking the flask [p029] thoroughly, replacing in the thermostat, allowing to remain for one minute, and then reading the level of the mercury in the azotometer. After the required time, say five minutes, has elapsed from the time at which the flask was first shaken, it is again removed from the bath, shaken as before, replaced, allowed to remain for one minute and the reading then taken. In this way readings can be conveniently made at intervals of three or five minutes or even less, and much more detailed information obtained about the course of the reaction than is possible by means of observations made at intervals of several hours.
Another form of volumetric apparatus, designed by Walton [[1904]], has been used by Lebedeff [[1909]].
An apparatus on a different principle has been designed by Slator [[1906]] for use with living yeast, but is equally applicable to yeast-juice, and a very similar form has been more recently employed by Iwanoff [[1909, 2]]. In this apparatus the change of pressure produced by the evolution of carbon dioxide is measured at constant volume, and comparative rates of evolution can be obtained with considerable accuracy, although the method has the disadvantage that the absolute volume of gas evolved is not measured. The apparatus consists of a bottle or flask connected with a mercury manometer. The fermenting mixture is placed in the bottle along with glass beads to facilitate agitation, the pressure is reduced to a small amount by the water-pump, and the rise of pressure is then observed at intervals, this being proportional to the volume of gas produced. As in the preceding case, the liquid must be well shaken before a reading is made.