ORIGINAL PAPERS ON DRENCHING.

The paper which one of us had the honour of reading before this Section on December 11, 1890,161 dealt chiefly with methods of bacteriological research, but especially in connection with the fermentation of bran as applied in the manufacture of light leathers. The object of the present paper is to give an account of further research into the nature of the fermentation and its products, the former communication being very incomplete. In further investigating the matter, we endeavoured—

1. To obtain a complete knowledge of the products of the actual fermentation as it takes place in practice.

2. To discover in what way the ferment acts, both on the materials fermenting and on the skins.

3. To examine in the same way the products of a pure cultivation of the bacterium causing the fermentation.

1. The Products of the Actual Fermentation.—These may be divided into three groups:—(1) gases; (2) volatile bodies; (3) non-volatile bodies. It was stated in the former paper that the ferment produced an inflammable gas along with considerable quantities of CO₂, H₂S, etc. The inflammable gas was thought by analogy from the researches of Tappeiner162 to be methane; it has, however, proved to be pure hydrogen. The absence of hydro-carbons was shown by the following method. Some of the gases were collected, the CO₂ and SH₂ removed by absorption with KOH, and the remaining gases exploded in a eudiometer tube with oxygen. The gases which remained after explosion did not diminish in volume after standing over KOH solution, showing absence of the paraffins and olefines.

For the purpose of analysis, about 1 1/2 litres of the gas were collected at a time in a large flask, fitted with a caoutchouc stopper and a funnel having an area of 28 sq. in. This was inverted, filled with the drench liquid, over the vat. The gas when collected was transferred immediately in the vat to a glass-stoppered bottle, sealed with a small quantity of the fermenting liquid and examined at once. Nine analyses have been made, most of them in duplicate. The following table, p.248, gives the results of three of these duplicate analyses, which have been performed by Hempel’s method, the hydrogen being estimated by combustion in air over heated palladinised asbestos.

We find that the gases given off during the fermentation are practically the same, with or without skins.

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 dulcitol163 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, 15,670c.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 16,200c.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 18·07grm.

These salts were treated with 200c.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. 219c.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; 75c.c. was taken and saponified with 80c.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. 71c.c. N/1 HCl was then added, and the apparatus connected with a condenser in the usual way. The distillate was acid. 800c.c. was taken off, forming fraction 1. The remainder of the acid required to neutralise the sodium hydrate was added, and another 800c.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.

The solution of the barium salts of fraction 2 was concentrated by evaporation, and to a portion excess of H₂SO₄ was added, the BaSO₄ filtered off, and the filtrate carefully neutralised with ammonia and a portion added to a solution of neutral ferric chloride. It was coloured dark red, and a finely-divided precipitate of basic acetate of iron came down on boiling. A second portion of the concentrated solution was heated with sulphuric acid and gave off a strong smell of acetic acid. To a third portion silver nitrate was added, and a slight excess of acetic acid; a little silver was deposited in the form of a film, showing a trace of formic acid.

Having thus shown the presence of butyric, acetic, and formic acids in the drench, we proceeded to ascertain the quantities formed in a normal fermentation of bran without skins. For this purpose a drench was made in a clean vessel with 10 litres of distilled water and 200grm. of bran mashed at a temperature of 38°C., and, after cooling to 33°, inoculated with bacteria from an actual drench which was fermenting vigorously; this was kept in the drench house so that the fermentation and general conditions might be exactly similar. In 48 hours all gases had ceased to be evolved, and the true fermentation was at an end. The liquid had an acid, not unpleasant smell, and was acid to litmus paper. The bran was strained off through muslin, washed with a little water, and well squeezed; the liquid measured 10 litres. Three litres of this were taken for the separation and estimation of the volatile acids, the remainder being set aside for examination and estimation of the non-volatile acids, etc. Two of the three litres were placed in a distilling flask with 5grm. of pure CaCO₃, and distilled down to one litre. The distillate was alkaline to litmus, and had a peculiar fishy smell; the remaining litre of drench was added, and the liquid taken down until the distillate ceased to be alkaline and only bad a faint smell. The alkaline distillate gave a yellow precipitate with Nessler’s solution, as well as the following reactions:—

HCl was added to the distillate in slight excess and the liquid evaporated to a small bulk; to a portion chloroform and alcoholic potash were added, and the liquid heated; no smell of isocyanides was given off; the body is, therefore, not a primary amine. Phosphomolybdic acid gives no precipitate, therefore the body is not an alkaloid. From the above tests and its characteristic smell, we conclude that the body is trimethylamine. The platinum salt was formed by evaporating the above concentrated solution of trimethylammonium hydrochloride with excess of platinum chloride, a precipitate of the platinum salt insoluble in alcohol being formed. There was not, however, a sufficient quantity from the three litres to ascertain the molecular weight.

Proceeding with the estimation of the volatile acids, 100c.c. N HCl being required to completely neutralise the CaCO₃ used, 50c.c. were first added and the distillation continued; the distillate was only very faintly acid. Four fractions were now distilled off, using respectively 10, 10, 10, and 20c.c. N HCl.165

Fraction 1 was boiled with excess of barium carbonate, filtered, the BaCO₃ washed with hot water, and the filtrate evaporated to dryness, and the Ba salts dried at 130°C. till the weight was constant; the salts were then decomposed with strong sulphuric acid, ignited, and the barium sulphate weighed.

Fractions 2, 3, and 4 were treated in an exactly similar way, the barium salts obtained and the weight and percentage of barium sulphate being shown in the following table:—

Calculating the fractions 1 and 2 as mixtures of barium butyrate and acetate, and fractions 3 and 4 as mixtures of barium acetate and formate, we may summarise the results thus:—

or as free acids:—

Proceeding now to (3) non-volatile bodies, we find these to consist of (1) acids, and (2) soluble carbohydrates. For examination, the remaining 4 litres of experimental drench were evaporated down to 1 litre and filtered; the residue (consisting of starchy matter) was well washed and the washings added to the filtrate, the whole placed in a large distilling-flask and distilled with continued addition of distilled water until the distillate was no longer acid (this required the addition and distillation of 4 litres of distilled water). A further deposit of solid matter rendering boiling dangerous, the liquid was again filtered and the residue washed free from acid. This residue was found to consist of nitrogenous organic matter and calcium phosphate, together with a trace of calcium oxalate, both being derived from the bran.

The clear liquid containing the non-volatile acids and other bodies was further concentrated and made up to 500c.c.; it was dark brown in colour and was very acid to litmus; owing to a trace of flocculent matter, it was again filtered.

The presence of lactic acid was shown in the following manner: 10c.c. of the liquid were placed in a small distilling-flask along with 2c.c. strong H₂SO₄ and about 0·5grm. potassium chromate in a little water. This was distilled and the vapours received in a test-tube surrounded by cold water; on adding magenta solution, decolorised by SO₂, to the liquid in the test-tube, a red colour was produced by the aldehyde formed from the lactic acid; aldehyde was also recognised by its smell. We find this an exceedingly delicate test for lactic acid, and as far as we know it is quite new in this form.

For 10c.c. of liquid to be examined, we find 2c.c. strong H₂SO₄ and 1grm. of potassium chromate to be the best proportions. Formic, acetic, propionic, butyric, valerianic, succinic, malic, tartaric, and citric acids do not give the reaction.

The liquid was now tested for succinic and malic acids. 25c.c. was taken and decolorised by treatment with pure animal charcoal for half an hour; the liquid was then filtered, the charcoal washed free from acid, and the filtrate concentrated; ammonia was added in slight excess, the precipitate of calcium phosphate filtered off. To the filtrate CaCl₂ was added in slight excess to remove the remainder of phosphates, the liquid filtered, and the filtrate cautiously neutralised with HCl; the addition of neutral ferric chloride to a portion gave no precipitate, showing the absence of succinic acid. To the remainder an equal volume of absolute alcohol was added and the liquid boiled; there was no precipitate. Twice the volume of absolute alcohol was then added and gave no precipitate. On the addition of four times the volume of absolute alcohol, a slight white precipitate came down, which evidently consisted of dextrin, from the manner in which it settled round the sides of the tube, and from its insolubility on adding HCl. We therefore conclude that there is no malic acid present, and that the only non-volatile acid produced is lactic acid.

The acidity was first determined by titrating 10c.c. of the solution with N/10 sodium hydrate, using glazed litmus paper to determine the point of neutralisation. The 10c.c. required 6·66c.c. N/10 NaOH, equivalent to 0·666 of N lactic acid = 0·7481grm. per litre of original drench.

In order to separate the lactic acid from the colouring matter and other bodies (dextrin and soluble starch) present, 100c.c. of the concentrated liquid was decolorised with 10grm. of animal charcoal, the mixture filtered and the charcoal well washed, the filtrate evaporated to dryness, and 15 drops of N/10 H₂SO₄ added to decompose any salts of lactic acid present; this was now extracted with ether, the ethereal extract of lactic acid placed in a distilling-flask, the ether distilled off, and the residue boiled with distilled water and pure calcium carbonate. The small amount of calcium sulphate present was removed by boiling the filtrate from the CaCO₃ with barium carbonate, and filtering. The filtered liquid now consisted of a solution of calcium lactate; it was evaporated to dryness in a platinum dish, and the residue dried at 110°C.; it was then washed first with ether, then absolute alcohol.166

The insoluble residue was dried at 110°C. till the weight was constant.

The total weight of calcium salts obtained was 0·7661grm. (from 4/5 litre of drench), of which 0·5493grm. yielded on ignition 0·1398grm. CaO = 25·45 per cent. Theory requires for calcium lactate (C₃H₅O₃)₂Ca = 25·69 per cent. CaO.

0·7661grm. calcium lactate in the quantity used = 0·9576grm. per litre = 0·7907grm. lactic acid per litre. The difference between this and the preceding amount of lactic acid found by titration, viz. 0·0426grm., is probably accounted for by the presence of a small quantity of salts of lactic acid in the drench.

The second part of the subject which we proposed to consider was “In what way does the ferment act on the bran and on the skins?” The average composition of bran is shown in the following table:—

It will be seen, as stated in the former communication,167 that the starch must be the principal body acted upon; but the cellulose is also an important constituent, and before going further it was necessary to ascertain if it took part in the fermentation. For this purpose some pure cellulose was prepared from cotton wool in the usual way, and small portions placed in tubes containing yeast-water168 as a nutrient material. These were sterilised by steaming; two tubes were inoculated from a pure cultivation of the bacteria obtained in 1889, two were inoculated from an actual drench, and three left uninoculated; all of them were placed on the incubator at a temperature of 30°–33°. On the second day the inoculated tubes were cloudy, but no gas was given off, nor was any acid formed; in 10 days the cellulose had not disappeared, nor on examination with the microscope could any action be detected. The experiment was repeated with peptone as a nutrient medium, but with the same result. The conclusion is that the bacterium does not attack the cellulose, which thus takes no part in the fermentation. The starch and nitrogenous bodies of the bran are therefore the only bodies acted upon by the bacteria in this fermentation.

From the fact that bran drenches ferment in the same way when mashed at all temperatures from 20°C. to 40°C., and that in all cases the starch is decomposed, it was supposed that the ferment was capable of attacking the starch in its undissolved condition. To ascertain if this were so, it was necessary to use pure cultivations in the laboratory.

The usual methods employed had thrown no light on this part of the subject, as in order to sterilise the solutions they had been repeatedly boiled, and were thus not comparable with the fermentation as it takes place in the works.

In order to get rid of this difficulty, the starch was sterilised in a dry condition in the hot-air oven, by heating for several hours on successive days to 110°C.

This sterile starch was mixed with sterilised water in tubes plugged with sterile cotton-wool. Eight tubes were taken, as follows:—

1. Sterile starch and water.

2. Sterile starch and water inoculated pure culture.

3. Sterile starch and yeast-water inoculated pure culture.

4. Sterile starch and asparagin inoculated pure culture.

5. Yeast-water alone inoculated pure culture.

6. Dextrin169 and yeast-water inoculated pure culture.

7. Soluble starch170 and yeast-water inoculated pure culture.

8. Starch mucilage and yeast-water inoculated pure culture.

These were allowed to stand on the incubator at 33°–35°C., and examined for acid each day by the method described in the previous communication for starch testing. They all remained neutral, although the bacteria developed in all but No. 1. These experiments were repeated several times, with the same result in every case. They show that this ferment is unable to act on starch either in its insoluble or soluble condition, alone or in the presence of nitrogenous bodies.

Now it has been known for a considerable period that bran contains an unorganised ferment called cerealin,171 which is capable of changing starch into dextrin and other carbohydrates; but the information to be obtained about it was very meagre, and as it appeared that this body might play an important part in the fermentation, we proceeded to prepare some pure cerealin and to ascertain its action on pure starch. The cerealin was prepared by taking a kilo of bran and extracting it with 2 litres of distilled water at 30°C; the extract was filtered clear and 2 litres of strong spirits of wine containing 90 per cent. alcohol was added, when a flocculent precipitate separated, which was washed on a filter with alcohol, dehydrated with absolute alcohol, and dried over H₂SO₄. The cerealin thus prepared is an amorphous substance not quite white, difficultly soluble in water, though we think this is due to its having been coagulated, and that it might be prepared in some other way which would show it to be more soluble than that which you now see.

To show its action on starch we took 10grm. of pure starch in 200c.c. water at 40°C., and placed equal quantities in two flasks; to No. 1 about 0·1grm. of the cerealin was added; No. 2 was left blank. These were kept at 40°C. for 10 hours, the clear liquid filtered off, and examined with Fehling’s solution. No. 1 reduced it strongly, showing that glucoses were present in considerable quantity. No. 2 had no effect whatever.

Addition of alcohol to No. 1 gave a white precipitate; this was thrown on to a filter, washed with alcohol, and dried at 100°, again dissolved in water, and inverted by boiling the solution with 1/20 part of strong sulphuric acid and then neutralising with sodium hydrate. The resulting solution reduced Fehling’s solution, showing that the body was dextrin. Strong bran infusion (which of course contains cerealin) acting on a thick starch mucilage, liquefies it, and forms glucoses and dextrin.

We have thus shown that the cerealin produces glucoses as well as dextrin, both from solid starch and from starch mucilage. This is most important, as it has been previously shown by one of us that the ferment attacks glucose very easily.

The drenches were now examined in order to ascertain the presence of glucose and dextrin; in the former communication it was stated that these were absent. We find, however, that by concentrating the liquid, that both are present in the early stages. Samples were taken one hour after mashing, and at 3, 6, 12, and 18 hours, while the drench was working.

These were evaporated to 1/5 bulk, filtered, and divided into two portions, one of which was examined with Fehling’s solution; to the other alcohol was added; the white precipitate after addition of alcohol was filtered off, washed with alcohol, dried at 100°C., redissolved in water, and boiled with sulphuric acid to invert it, then examined with Fehling’s solution.

The following results were obtained:—

In a bran infusion kept from fermenting by a little ether or chloroform, the formation of glucose and dextrin goes on continuously, the glucose increasing in quantity; the action is, however, much slower than in the case of diastase; at the end of 12 hours, at a temperature of 40°C., about half the starch is transformed.

It appears from this and a number of other experiments that glucoses and dextrin are formed by the cerealin, the former only being decomposed by the bacteria almost as fast as it is produced, for after three hours no glucose is found in the drenches.

We have thus shown that the acids and gases are produced from the starch contained in the bran, the starch being first changed into glucoses by the action of an unorganised ferment or ferments; and that the glucoses are decomposed by a specific organism, the nitrogenous material in the bran serving for its nutriment; that the action is the same with or without skins although there appears to be a little more H₂S gas given off from drenches containing skins, than from those containing none.

The ferment has no direct action on the skins. This may be shown by taking a piece of limed skin, in which a considerable portion of the lime exists as carbonate, and submitting it to the action of the ferment; in this case the action goes on much longer than in the drenches, being complete in about 15 days, but the skin may be left in the resulting liquid for three months without undergoing further change than solution of the lime, provided that suitable means be taken to exclude moulds, which, by destroying the organic salts and acids produced, enable putrefactive fermentation to begin.

It has been thought that the bran itself exercised some peculiar action on the skin, and possibly this may be so to a slight extent,172 as the sweet bran drench is occasionally used on the continent, but if skins are placed in a mixture of bran and water (in the proportions for drenching) which is prevented from working by the addition of a minute quantity of HgCl₂ 1/10000, such a drench has no action on them, and when tanned they are harsh and hard, similar experiments have been made on a smaller scale, using ether and chloroform to prevent fermentation, with the same results.

In order to show whether the acids alone were the cause of the action on the skins, an artificial drench was made up of the following composition—

0·5grm. per 1000 glacial acetic acid.
1·0 " " " lactic acid sp. gr. 1·210.

In this skins were worked intermittently for 1 1/2 –2 hours, and it was found that in this time they were in a similar condition to skins which had been in a drench from 12–16 hours. They were afterwards tanned, and found to be good leather, and in every way equal to similar skins which had been “drenched.” A number of experiments have been tried with sulphuric and hydrochloric acids in order to ascertain if these had a similar action, but the results have not been satisfactory.

With regard to the third portion of the research, viz. the products of a pure cultivation of the bacteria, we have obtained a good number of results; but as the description of the experiments is of a greater length than we anticipated, and as there is still some work to do in verifying them, we are obliged to leave this portion for another paper, which we hope to have ready by the next session.

In conclusion, we may summarise the results obtained up to the present in the fermentation of bran by the organism we have used; remarking that there may be other organisms capable of fermenting bran in a somewhat similar manner.

1. It has been shown that the fermentation investigated is due to a specific organism, of which we find no account, and which, pending further experiments, we have therefore provisionally named Bacterium furfuris.

2. That the starch and nitrogenous bodies in the bran, alone take part in the fermentation, the starch being first transformed into glucoses and dextrin by the action of an unorganised ferment or ferments; the glucoses and nitrogenous bodies only, being decomposed by the bacteria, with the formation of formic, acetic, butyric, and lactic acids, and the simultaneous evolution of hydrogen, carbon dioxide, nitrogen, and a small quantity of sulphuretted hydrogen. The following table shows the quantities found in an experimental drench per 1000c.c.

We find in actual work that the quantity of acid produced varies from 1 to 3grm. per litre.

3. That if these acids are applied to the skins in the same proportions as they occur in the drench, the action on them is the same, and much quicker than an ordinary drench.

4. That the gas therefore, has no action on the skins per se, with the exception of floating and distending them, and so enabling them better to take up the acids.

We are indebted to Mr. H.R. Procter, of the Yorkshire College, Leeds, and to Dr. Percy F. Frankland for valuable suggestions in carrying out some of the work.

Isolation of Pure Culture.—In our previous communication on bran fermentation as applied in the manufacture of light leathers,173 we gave an account of the actual fermentation and its products, together with the mode of action on the bran and on the skins for which this fermentation is used, reserving to the present paper an account of the products of a pure cultivation of the bacteria causing the fermentation.

The cultivation used in the first experiments for this purpose was one isolated in 1889, and used in the cellulose and starch experiments described in the above-mentioned paper.

This cultivation had not been obtained from a single colony from gelatin, and in order to make quite sure that the cultures used were pure, it was decided to make another attempt to isolate the bacillus by plate cultivation. Previous attempts to do this had failed, bate organisms and gelatin liquefying bacilli, developing in such numbers that the plates were spoiled before the organism, which caused the fermentation, had time to develop; beside which the organisms, as obtained direct from the drenches, grew with difficulty in the ordinary nutrient gelatin. A special gelatin was therefore prepared of the following composition:—

Plates of this gelatin in Petri dishes were prepared from the previously used supposed pure cultures which had been preserved in sealed tubes. These were found to be dead. A modification of the method previously described by one of us175 was adopted.

A solution of nutrient glucose was inoculated from a working drench, and as soon as the liquid was observed to become cloudy, a tube of the solid glucose gelatin was inoculated from it by plunging in a platinum needle. In two days the bacteria developed along the needle track. Fig.32 shows the appearance of the tube four days after inoculation, a bubble of gas being formed in the solid gelatin. On the following day, the tube was broken, and from the portion where gas was given off most vigorously other tubes of solid and liquid media were inoculated. Acid was quickly formed in the nutrient glucose solutions. In the gelatin tubes, the bacteria developed well in the depth. The now purified culture was passed through three more glucose gelatin tubes, each time also a glucose tube being inoculated. From the last of these tubes a very minute quantity was taken 12 hours after inoculation on the point of a platinum needle, and a streak culture made on glucose gelatin. In 24 hours a growth could be seen on the surface of the gelatin in the form of minute dots perfectly separated one from another.

Organisms causing Bran Fermentation.
Pure Cultures.

From one of these dots a tube was inoculated and from this several plate cultivations were made. The colonies which developed on these plates were of two kinds, the majority being round, yellowish and of small size, a smaller number spreading out on the surface of the gelatin and slightly iridescent. These surface expansion colonies when examined with a low power appear like a milky drop, with very fine granular contents, the whole surrounded by wavy lines which follow exactly the irregular contour of the expansion. The small round colonies growing in the depth occur in the proportion of about 3 to 1 of the surface expansion colonies. The microscopic appearance of the bacteria composing the two kinds of colonies, is almost exactly similar, they are extremely small and regular in size, 0·75µ ×0·5µ to 0·7µ ×1µ. When spread upon a slide, they are not readily miscible with water, and appear greasy. Both colonies inoculated into glucose tubes produced acid. The existence of these two organisms was confirmed in the following way:—A glucose tube was inoculated from a drench in active fermentation; as soon as the liquid became cloudy, a second tube was inoculated from it by means of a platinum needle; from this tube the fermentation was carried through two more tubes; a plate cultivation was made from the last tube 10 hours after inoculation. Again, the two kinds of colonies developed exactly similar in every respect to those obtained from the streak cultures.

It seems probable from these results, and also from a comparison of the fermentations made with the organisms from an actual drench,176 and from purified cultures with those from a single organism, which are described in the present paper, that the action in the drenches is a symbiotic one in which two or more organisms take part.

The Fermentations.—During the time occupied by the isolation of pure cultures of the bacteria, two fermentations were conducted with the supposed pure cultures. These fermentations (or rather the second of them, for the first was unfortunately lost through the breakage of a bottle) may prove of considerable interest as throwing some light on the symbiotic action of the two organisms.

The first fermentation with pure cultures of the bacillus a (B. furfuris) obtained from a single colony in glucose gelatin, was inoculated on September 16, 1894, the composition of the fermenting liquid being—

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.

140c.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 17c.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 1·8651grm.

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 2·438grm. of lactic acid per 1000c.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 a and ; the remainder are fermentations by a 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 3435c.c. One-half of this quantity, however, came off in seven days. About 300c.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, page273):—

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

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

In a second fermentation (III.) we endeavoured to ascertain the exact amount of CO₂ evolved, as in the previous fermentation this had not been done. It was therefore decided to absorb the CO₂ by means of potash.

The fermentation in this case was conducted in a narrow-necked flask of 2000c.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 1000c.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 2000c.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 a and , the relative amounts being very close, while in all the fermentations with a 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.

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