This was contained in a narrow-necked litre flask fitted with a rubber stopper, and narrow delivery tube dipping under mercury, and sterilised with all the usual precautions. The fermentation began on the second day, reached its height from the 6th–8th day, and continued for 39 days, when gas ceased to come off. The examination of the gases will be described later on. When the fermentation was over, the liquid was brought to boiling temperature. It was then examined for the volatile acids in exactly the same manner as we described in our previous paper.

140 c.c. normal HCl was added and distillation commenced; the distillate was acid. The distillation was continued until the distillate ceased to be acid, forming fraction I. Three more fractions were now distilled off using respectively 10, 20, and 17 c.c. N₁HCl.

The fractions were boiled with excess of BaCO₃ filtered, the BaCO₃ washed with hot water, the filtrate evaporated to dryness, and the barium salts dried at 130° C. till the weight was constant.177 The salts were then decomposed with strong H₂SO₄, ignited, and the barium sulphate weighed. The following is a tabulated statement of the results:—

Calculating fraction I. as a mixture of barium acetate and butyrate, and fractions II., III., and IV. as mixtures of barium acetate and formate,178 we get:—

Calculating the barium salts into their respective acids we get:—

The total volatile acids produced amounting to 1·8651 grm.

The residual liquid containing the non-volatile acids was submitted to the test for lactic acid previously used,179 and it was found to be present.

The method employed for estimating lactic acid in our previous communication proving somewhat difficult, we endeavoured to improve it by extracting the concentrated solution of the non-volatile acids on prepared pumice stone with ether in a paper thimble contained in a Soxhlet fat-extraction apparatus. After repeated trials we found that this method did not give accurate results. The solution was therefore titrated with  1/10 N sodium hydrate, using glazed litmus paper to determine the point of neutralisation. The acidity found corresponded to 2·438 grm. of lactic acid per 1000 c.c. of the fermented liquid.

We have done several other fermentations with this organism and find the same acids produced and the same gases evolved, the results just given being fully confirmed. At the same time the amount of the acids produced and their proportions vary, that is to say, the quantity of acid from a given fermentation cannot be predicted with absolute accuracy, although the conditions under which we carried out the experiments were made as like as possible.

We give the total acids from four fermentations to show the amount of variation. I. is a symbiotic fermentation caused by organisms α and β; the remainder are fermentations by α alone.

Comparison of Acids from Fermentation II. and III.180

Fermentation III., 2000 c.c.

Calculation of Barium Salts as Barium Butyrate, Acetate, and Formate.

The Gases.—In dealing with the gases evolved, we first compare those given off in the fermentation of glucose with that of bran under exactly similar conditions. The fermentation was conducted in open vessels as before described,181 and the gases were collected and examined in the same way.

Mean of three Analyses.

The composition of the gases is thus almost exactly similar, and, we think, fully proves our previous conclusions as to the change of the starch of the bran into glucoses by means of an unorganised ferment (cerealin).

In the closed fermentations we had previously collected only small quantities of gas over mercury, owing to the difficulty of continuously collecting large quantities which came off during the night.

In the fermentation of September 16, 1894, we collected the whole of the gas given off, taking samples every day over mercury, the gas coming off at night being collected over warm water. Of course this method does not give the total amount of gas evolved with absolute accuracy, but the exact composition of the gases was known from day to day, and the amount of CO₂ absorbed by the water could be calculated with moderate accuracy.

The fermentation was conducted in a narrow-necked litre flask fitted with a narrow delivery tube dipping under mercury, and sterilised with all the usual precautions. The temperature was maintained at 25°–30°, gas was evolved for 39 days, when it ceased to come off, the total amount collected being 3435 c.c. One-half of this quantity, however, came off in seven days. About 300 c.c. of CO₂ was absorbed by the water during the whole period. The diagram (Fig. 33) shows the manner of evolution of the gases, the ordinates representing volume of gas and the abscissæ lapse of time after inoculation. The following table shows the composition of the gas at different stages of the fermentation. (The fermentation (II.) is the one of which the chemical analysis has been previously given, page 273):—

Composition of Gases evolved in Fermentation of 1000 c.c.
Glucose with pure Ferment. September 16, 1894.

The total quantity of CO₂ actually collected = 1563 c.c. = 3·090 grm.; the amount of CO₂ due to decomposition of the CaCO₃ by the acids produced was found to be 667 c.c. = (1·3189 grm). The vol. of hydrogen collected was 1086 c.c. = 0·973 grm.

The fermentation in this case was conducted in a narrow-necked flask of 2000 c.c. capacity, connected by means of a narrow glass tube with two potash bulbs containing strong caustic potash, and furnished with a delivery tube dipping under water; the whole apparatus stood upon an iron plate, and was maintained at a temperature of 25°–30° in the same manner as the previous fermentation. The gases were evolved for 21 days—a considerably shorter period than the 1000 c.c. fermentation; but resembling it in that one-half the gas was evolved in eight days. The diagram shows the curve as in the previous fermentation, which it resembles for the first 14 days, afterwards however stopping suddenly. When the fermentation was at an end the flask and contents were heated to boiling point, at the same time a current of air free from CO₂ was drawn through it, and the CO₂ given off being collected in potash bulbs as in the fermentation. Unfortunately the estimation of the CO₂ was rendered valueless owing to an accident.

The table shows the composition of the gases other than CO₂ evolved in this second fermentation.

Gases from Fermentation of 2000 c.c. (excluding CO₂)
Fermentation III.

The gas from days 18–21 was unfortunately mixed with air. On comparing the mean composition of gases other than CO₂ collected from both fermentations, we get the following result:—

If now the O and part of the N in the proportion to form air be taken away, the composition of the gases from the two fermentations is found to be almost exactly similar:—

The gases from a third fermentation were almost exactly similar in composition, but the total volume was not measured.

A remarkable fact in this fermentation is the evolution of free N, which seems to be rare, except in the case of putrefactive organisms, as in the vast number of fermentative decompositions due to bacteria, almost the only gases found are carbonic anhydride, hydrogen, H₂S, and marsh gas.

Gayon182 in 1875, in an investigation on the putrefaction of eggs, collected the gas given off from large ostrich eggs, and found in it 29 per cent. of nitrogen; he adds, however, that its presence may be due to the accumulation of a certain quantity of air in the air-bubble before putrefaction.

Béchamp183 found that yeast cells under suitable conditions, but sugar being withheld, produced pure nitrogen along with leucin, tyrosin, a soluble albuminous substance coagulable by heat, an enzyme, a peculiar gummy substance, phosphates and acetic acid, alcohol and CO₂. These are almost the only instances where observers of repute have been convinced of the evolution of free N by bacteria. We find since the above work was carried out that Immendorf184 has found certain bacteria in dung which form ammonium nitrate, and this body, as is known, splits up at a comparatively low temperature into nitrogen and water.

From the bacteriological as well as the chemical results, it is now evident that the fermentation as it takes place in practice is a symbiotic one in which two organisms play the most important part, and very probably cause the entire fermentation. This is shown by comparing the acids produced by the fermentation in the works with those produced by a mixture of the organisms α and β, the relative amounts being very close, while in all the fermentations with α alone a much less proportion of lactic acid is produced, as the following table shows:—

The acetic acid, as far as we can ascertain, is produced directly from dextrose without the previous production of alcohol, since the presence of the latter is not shown by its tests at any stage of the fermentation. We have also ascertained that the organism is without action on dilute solutions of alcohol, in yeast water, no acid being produced.

We are indebted to Mr. H. S. Shrewsbury for the analysis of some of the gases and volatile acids, and also for the preparation of the diagrams. In conclusion we may state that the investigation of this fermentation in the tannery has been the means of pointing the way to a still more complicated process, viz., “bating.” It may even be possible in the future to place these processes on somewhat the same footing as the accurately understood fermentations in the brewing industry although the difficulties in the way are much greater.