Gases

A

B

C

CO2 and H2S

21·9

25·2

42·4

O2

1·0

2·1 

3·6

H2

53·1 

46·7

28·2

N2

24·0

26·0

25·8

A is from a vat containing no skins, 1–2 days.
B from a vat containing skins, 2–3 days.
C from a vat containing skins, 3–4 days.

The H2S is present only in small quantities from 1–2 per cent. Its presence was shown by aspirating the gases dissolved in 1 litre of drench through a dilute solution of lead acetate containing a few drops of acetic acid. The gases were liberated by heating the liquid, and at the same time aspirating air through it. The H2S is present both in the gases evolved from drenches which do not contain skins, and from those which do, though to a slightly greater extent in the latter. The amount of CO2 given off increases as the fermentation proceeds; the oxygen also increases, the nitrogen remaining practically constant. We consider that some of the nitrogen given off is that dissolved in the water, the oxygen being partly used up by the ferment in its earlier stages; the remainder of the nitrogen is probably produced from the decomposition of the nitrogenous bodies contained in the bran. It may be noted here that the bran fermentation under ordinary circumstances ceases on the fourth day, and sometimes earlier.

Frankland and Frew, in a paper on a pure fermentation of mannitol and dulcitol[163] have shown the hydrogen and carbon di-oxide given off were produced by the decomposition of formic acid, the ferment producing formic acid and the latter immediately splitting up into an equal number of molecules of carbonic anhydride and hydrogen. We have every reason to believe, from experiments which will be included in the third section of the paper, that the source of the H and CO2 is the same in the fermentation we are considering.

2. Volatile Bodies.—These may be divided into (1) acids, and (2) amines. We had previously shown the absence of aldehyde by the rosaniline reaction, and of alcohol by Lieben’s iodoform test.

In the first experiment to determine the acids, 15,670 c.c. of the liquid from a normally fermenting vat was taken when the fermentation was at its height; this was submitted to distillation, and to the last portions distilled water added and 16,200 c.c. distilled over; the distillate was neutralised with sodium carbonate, and the whole was then evaporated in a porcelain dish, the residue dried first at 100° C., then over strong sulphuric acid, the weight of the sodium salts of the volatile acids thus obtained being 18·07 grm.

These salts were treated with 200 c.c. absolute alcohol and 20 c.c, strong H2SO4; heat was evolved, and there was a strong smell of ethyl acetate and butyrate.[164] The mixture was allowed to stand for 24 hours and then distilled on the oil-bath, the temperature for a long time remaining at 81° C., finally rising to 96° C. 219 c.c. of distillate was obtained, and to this a saturated solution of common salt was added, but the esters of the volatile acids did not separate out. The whole was again redistilled with the same result.

Failing in this way to separate the acids in the form of their esters, the mixture of esters and alcohol was examined qualitatively; 75 c.c. was taken and saponified with 80 c.c. N/1 NaOH in a distilling flask, with inverted condenser, for half an hour, until all the fragrant smell of the esters had disappeared. 71 c.c. N/1 HCl was then added, and the apparatus connected with a condenser in the usual way. The distillate was acid. 800 c.c. was taken off, forming fraction 1. The remainder of the acid required to neutralise the sodium hydrate was added, and another 800 c.c. distilled off, forming fraction 2. Fraction 1 smelt strongly of butyric acid, fraction 2 of acetic acid. The fractions were then each boiled for half an hour with excess of barium carbonate; this was filtered off and washed, the filtrate evaporated to dryness, and dried at 130°.

A portion of barium salt of fraction 1 was heated with alcohol and sulphuric acid and gave the characteristic pineapple smell of ethyl butyrate.