ORIGINAL PAPERS ON BATING.
The reprints of these papers being no longer available, I have thought
it best to print them in the present volume exactly as published,
with the exception of the paper entitled “Fermentation in the Leather
Industry” from which only so much is reproduced as relates to puering
and bating.
I. PART OF PAPER ENTITLED “FERMENTATION IN THE LEATHER INDUSTRY.”
Read before the Nottingham Section of the Society of Chemical
Industry, February 14, 1894.
In the next process, that of “bating,” the bacteria play a more
important part, and may be put in our second category, viz. helpful to
the tanner, though, from the very nature of the material used, it forms
a nidus for various putrefactive ferments, and is therefore dangerous.
The materials usually employed are bird dung and dog dung; the former
of these is sharp and piercing in its action, while the latter has a
more softening action on the skin. In both bates, however, a rapid
solution of the gelatinous and albuminous hide substance (coriin)
takes place. The hide fibres, however, are not attacked until all the
nutrient material in the liquid has been consumed by the bacteria.
Eitner (Der Gerber, xv., 158) states that a sterile bate is without
action on the skins, and appears in that article to attribute the
entire working of the bate to the action of micro-organisms. He
seems to have based his conclusions on experiments with an old bate
sterilised with creolin, which he found had no action on the skins. If,
however, a fresh bate be made and boiled for half an hour, then allowed
to cool to 90°–95° F., it will be found to have a considerable action
on the skins, though not so rapid a one as the unboiled bate. The
boiling kills all organisms, and fresh ones have not time to develop
from spores still remaining before the experiment is at an end.
Lately, the action of unorganised digestive ferments in the dung has
been found to be considerable. Thus bating is an exceedingly complex
process. The action appears to be threefold:--
(i) A purely chemical action of the soluble salts present in the
bate on the lime in the skin.[113]
(ii) An action due to organised ferments.
(iii) An action due to unorganised ferments.
[113] _References to bating_:
Procter. Text-book of Tanning, 1885, 184.
Der Gerber, x. (1884), 197, Mistbeizen.
Der Gerber, xv. (1889), 267.
J. Anal, and Appl. Chem., 1893, vii. 87, 95.
J.S.C.I., xii. 530, Palmer and Sandford.
Salomon, W. J., Tech. Quarter, 1892, v. 81, etc.
J.S.C.I., xii. 774, ix. 27.
The precise amount of influence each of these actions has on the skin
is difficult to assign. The chemical action of the ammonia compounds
dissolves the lime remaining in the skin, but the simple removal of the
lime is not sufficient, as may be shown by removing it completely with
dilute hydrochloric acid or other similar means, and washing perfectly
free from acid in distilled water. When tanned, such a skin is hard and
brittle.
The organised ferments or bacteria, of which there are many species
in the bate, probably act on the skin by secreting soluble ferments,
which have the power of dissolving hide fibre. I have isolated in plate
cultivations several species which liquefy the gelatin.
If skins are allowed to lie in the bate, zoogloæ of these bacteria
collect in the folds and attack the fine “grain,” so that the leather
is covered with lines and markings, or “flaked.” Thus the operation
is a very critical one, requiring careful watching; under certain
atmospheric conditions and at a temperature of 35°–40° C. the skin,
if only left a short time too long, will completely melt away. The
more skin substance is dissolved in the liquid, the more rapid and
pronounced the bacterial action becomes.
So far as is at present known the unorganised ferments in the dung,
besides those which are secreted by the bacteria, are mostly digestive
ferments[114] --pepsin, pancreatin, and trypsin--certain quantities
of which pass out of the animal’s body in an unchanged condition. Of
these, _pepsin_ only acts in an acid solution, and, although fresh
bate liquor is faintly acid to litmus, it quickly becomes neutralised
by the lime in the skins, so that the action of this ferment can only
be limited in extent. _Pancreatin_ will act in a neutral solution, and
has, therefore, a considerable effect on the skin.
[114] Harris and Gow, Jour. of Physiol., xiii. 469. G. Tammian,
Zeitschr. f. Physiol. Chem., xv. 271. O. Loew., J. Prak. Chem., 37,
101. J.S.C.I., 1888, 224.
SOME DUNG ANALYSES.
--------------------------+--------------------+--------------------------
Hen Dung | Dog Dung | Guano
--------------------------+--------------------+--------------------------
Per Cent. | Per Cent. | Per Cent.
Moisture 60·88 | Ca 43·049 | Urea 5·00
Organic matter† 19·22 | Mg 0·087 | K_{2}SO_{4} 7·90
Phosphates 4·47 | K 0·302 | NaCl 5·00
CaCO_{3} and | Na 0·438 | (NH_{4})_{3}PO_{4} 5·50
CaSO_{4} 7·85 | Si 0·004 | (NH_{4})_{2}O 0·60
Alkaline salts 1·09 | PO_{4} 3·446 | SiO_{2} 2·25
Silica and sand 6·69 | CO_{2} 7·464 | Ca_{3}(PO_{4})_{2} 9·00
------ | Cl 0·037 | MgNH_{4}PO_{4} 15·25
100.00 | Fe and loss 0·008 | Ammon. urate 15·25
| Organic | Organic matter
| matter 14·152 | (17 per cent. N) 41·73
† Containing | H_{2}O 31·013 | Moisture 6·52
N = ammonia 0·74 | ------- | ------
| 100.000 | 100.00
--------------------------+--------------------+--------------------------
W. I. Macadam, J.S.C.I., 1888, 80. _Viollet_, Diction d’Analyses Chim.
The dog dung actually used in a tannery is from animals fed on a more
vegetable diet, the one given having an extremely high percentage of
lime, no doubt owing to the animal having eaten bones. A dung as brought
from the kennels was found to contain:--
Per Cent.
Mineral matter 4·679
Organic " 9·731
Water " 85·590
------
100·000
I find by experiments with the purified ferments that their action is
very slow compared with the bate itself. Two portions of the same
skin were taken: one of them was treated with a 1 per cent. solution
of pepsin acidified with 0·2 per cent. of hydrochloric acid, the
other in a bate liquor of dog’s dung; both at a temperature of 40° C.
(104° F.). At the end of one hour, the skin in the pepsin solution was
considerably “fallen,” but that in the manure solution was bated nearly
away, i.e. the greater part of it was dissolved.
A 1 per cent. solution of pancreatin (Merck’s) was found to act far
more rapidly than pepsin. At 40° C. in a neutral solution the skin
fell rapidly, and the action continued even in the cold. In this
experiment it was found that in 15 hours the liquid was swarming with
minute bacteria. In order to guard against the influence of bacteria
a similar experiment, at the suggestion of Mr. H. R. Procter, of the
Yorkshire College, Leeds, was tried with the addition to the pancreatic
solution of 1·5 per cent. of chloroform; this prevents the development
of bacteria, while it does not interfere with the action of the
pancreatin. The skin was reduced as before, but in neither case had it
the peculiar touch of a “puered” skin, nor were the characteristics of
the leather the same.
II. NOTES ON THE CONSTITUTION AND MODE OF ACTION OF THE DUNG BATE
IN LEATHER MANUFACTURE. Read before the Nottingham section of the
Society of Chemical Industry, Oct. 26, 1898.
In a paper read before the Society of Chemical Industry (J.S.C.I.,
1894, 219), the author gave an outline of the bating process as far
as he had then investigated it. Further researches have shown that,
although the views then put forward must be somewhat modified, in the
main they are correct.
In 1895–96 a series of experiments was undertaken with a view of
ascertaining how much of the reducing action of the bate was due to:
(1) the chemical constituents; (2) the bacteria; (3) the digestive
ferments and enzymes.
The puer used was dog dung obtained freshly every week from hunting
kennels. It contained on an average: water, 85 per cent.; organic
matter, 10 per cent.; mineral matter, 5 per cent.; about 3 per cent. of
the organic is soluble and about 1 per cent. of the mineral matter.
According to Gamgee (Phys. Chem. 2), a dog fed on flesh diet excretes
from 27 to 40 grm. of fæces in 24 hours, of which 12·9 grm. are solids.
On a bread diet the amount of fæces is much more.
An ultimate analysis gave the following results:--
---------------+------------+----------
| Bread Diet | Meat Diet
---------------+------------+----------
C | 47·39 | 43·44
H | 6·59 | 6·47
N | 2·92 | 6·50
O | 36·08 | 13·58
Mineral matter | 7·02 | 30·01
---------------+------------+----------
These figures alone are sufficient to show what variable quantity is
the puer employed in a tannery.
The mineral matter is composed as follows:--
--------------------+----------------------+-------+------
Soluble in water {|NaCl and Na_{2}SO_{4} | 1·37} | 4·00
{|Sodium phosphate | 2·65} |
| | |
{|Earthy phosphates |80·37} |
Insoluble in water {|Ferric phosphate | 2·09} |94·93
{|CaSO_{4} | 4·52} |
{|Silicic acid | 7·94} |
--------------------+----------------------+-------+------
The ammonia compounds combine with the lime remaining in the skin, but
the constitution of these compounds was unknown.
The puer was examined for ammonia by Schlösing’s method, 50 c.c. being
mixed with 50 c.c. milk of lime placed under an air-tight bell-glass,
together with an open vessel containing a measured quantity of standard
acid, for 36 hours. No NH_{3} was given off.[115]
[115] This is not invariably the case: other samples of puer examined
have given off NH_{3} when similarly treated, but only in small
quantity. A similar instance has been observed by Rideal and Orchard
in the liquefaction and decomposition of gelatin by B. fluorescens
liquefaciens (The Analyst, October 1897), where the quantity of
ammonia produced was insignificant, amounting even after 16 days’
incubation to only 0·168 grm. of N per 100 c.c., corresponding to
0·204 grm. of ammonia.
The mixture of dung and Ca(OH)_{2} was filtered and a perfectly clear
filtrate of a golden-yellow colour obtained. This contained free
amines, calcium salts of volatile and non-volatile acids.
It was distilled until the distillate was no longer alkaline, and made
up to 500 c.c. 50 c.c. required for neutralisation 5·7 c.c. of (N/100)
H_{2}SO_{4}, equivalent to 0·1938 grm. of NH_{3} per litre of original
puer, being the amines set free by Ca(OH)_{2} in cold solution.
A further 50 c.c. was distilled with NaOH, the amines given off
neutralised 8 c.c. of (N/1) HCl, equivalent to 2·72 grm. of NH_{3} per
litre. The neutralised liquid was evaporated to dryness; the residue,
consisting of amine hydrochlorates, amounted 0·27 grm. A qualitative
test showed both primary and secondary amines to be present.
The action of these amine hydrochlorides was tried upon skin, pieces of
limed sheep grain previously washed in water being used. The solution
was maintained at 35°C. In the first experiment 0·27 grm. was dissolved
in 100 c.c. of water. In two hours the skin was considerably reduced,
but had not the touch of a “puered” skin. It tanned well and was a good
colour, showing that the lime had been removed, but the leather was not
sufficiently soft.
With a strength of 1 grm. per 100 c.c. the action was hastened, the
same result being obtained in 1-1/4 hours, but the coriin of the skin
was not dissolved. A comparison piece of the same skin in dung was
bated too much.
With a view of comparing the action of analogous bodies, skin was
treated in a solution of anilin hydrochloride, 1 grm. per 100 c.c. for
1-1/4 hours at 35° C. The solution was acid, lime was removed, and the
skin felt very similar to that in the previous experiment. Coriin was
not dissolved, and the skin did not “come down.”
Of the mineral constituents mentioned above, only the chlorides[116]
have a reducing action on skin, the other compounds being inert, e.g.
silicates, or merely supply food for the bacteria.
[116] Procter, Text Book of Tanning, p. 85.
For a general outline of the decompositions taking place in dung,
see Dr. Herfeld’s paper (J.S.C.I., May 1895). We need only consider
the action of the chief organic compounds, as the amount of chlorine
is too small (in one case 0·053 per cent. on the dry dung) to be of
importance. The principal compounds are:--
_Organic Acids._--Formic, acetic, butyric, valerianic, lactic, malic,
tartaric, citric, and glyceric.
_Amido Compounds._--Leucin, tyrosin, glutamic acid, glutamine,
asparagin, glycocol.
_Bases_, consisting of amines, skatol, and indol, and varying amounts
of ammonia, according to the age of the dung.
The organic acids exist partly as salts of the alkaline earths, partly
combined with amines. A distillation of “puer,” after acidifying with
H_{2}SO_{4}, gave 2·2 grm. per litre of volatile acids reckoned as
acetic. No HCl came over. On decomposing the sodium salts of the acids
with H_{2}SO_{4}, the smell of butyric acid predominated. I have also
shown the presence of lactic acid in the dung, but have been unable to
determine the amount.
_Action of Amido Compounds._--A mixture of glycocol and leucin[117] was
prepared by boiling 8 grm. of gelatin in 400 c.c. of water, acidified
with 1 c.c. of HCl for two hours with inverted condenser. The acid was
then neutralised with ammonia. The solution had a very considerable
reducing action on skin at 35° C. in 90 minutes, but not sufficient for
practical purposes. It appears to dissolve a little coriin in a similar
way to dilute acids. It was found on further investigation that the
action was largely due to the NH_{4}Cl present.
[117] According to the text books; but for later researches see Paal
and Schilling, Chem. Zeit. 1895, 1487; also J.S.C.I., 1898, 589.
_Action of Dilute Acids._--Although the bate in practice is almost
always alkaline, yet a fresh bate is _acid_, and it may be as well,
before proceeding, to consider briefly the action of weak and dilute
acids on skin.
The fibres of the skin have only a limited capacity for holding acids,
and soon begin to swell abnormally and partially dissolve. Reimer[118]
has found that the material may be reprecipitated by lime water. It
forms a fibrous mass, which has not the sticky feel of gelatin, but is
at once converted into that body by boiling.
[118] H. K. Procter, “Text Book of Tanning,” 1885, p. 18.
The author has examined the action of dilute H_{2}SO_{4} on sheep
skins, and this may be considered typical of the action of acids on
skin generally.
When puered and drenched sheep skin is treated for 20 minutes with a
very dilute solution of H_{2}SO_{4} (1/280), it swells up considerably
and becomes soft and semi-transparent, The fibres of the skin, which
under normal conditions have a diameter of 4µ–6µ become 20µ or more; on
staining with picro-carmin there are seen to be certain fibres which
are unaffected or only slightly affected by the acid. These are the
elastic fibres[119] and the capillary blood vessels.
[119] See Procter, “Text Book of Tanning,” pp. 8 and 21.
On filtering a solution of the above strength in which skin in excess
has been treated for 20 minutes, and evaporating 100 c.c. to dryness
in a platinum dish, a residue of 0·4992 grm. was obtained. A second
experiment gave 0·496 grm. of which 61 per cent. was organic matter.
By Kjeldahling another portion of the residue, the N in 0·12 grm.
amounted to 0·0122 grm. equivalent to 0·073 grm. of skin substance;
i.e. 60 per cent. of the soluble matter is skin. It is evident that
the matter in solution is a compound of skin substance and H_{2}SO_{4}
together with a little soluble mineral matter contained in the skin.
The amount of substance dissolved depends upon the condition and
previous treatment of the skin.
In the presence of NaCl, which prevents the swelling of the fibres,
puered sheep grains take up 7·0 c.c. of (N/1) H_{2}SO_{4} from
a solution containing 17 c.c. of (N/1) H_{2}SO_{4} (0·833 grm.
H_{2}SO_{4}) and 7·8 grm. of NaCl per 100 c.c. About 300 grm. wet skin
per litre were used. The skin substance dissolved by the acid in the
time named does not appear to be more than that dissolved by the weak
acids of a drench, although an equivalent weight of H_{2}SO_{4} has a
much more powerful swelling action on skin than these acids.
A piece of puered and drenched sheep skin was carefully washed in
distilled water and then dried _in vacuo_ until it ceased to lose
weight; the absolute dry weight was 4·2670 grm. The skin was soaked
down in distilled water and again drenched for 30 hours in a clean bran
drench, developing approximately 0·8 grm. lactic acid, and 0·5 grm.
acetic acid per litre (see this Journal, May 1893). It was again washed
and dried _in vacuo_. It weighed 4·12 grm., a loss of 0·14 grm. = 3·44
per cent.
The action, therefore, of the acids is a reducing one, inasmuch as skin
is dissolved, but as the fibres hold a certain amount of acid, the skin
appears to be plumped. Where the acid is a mineral one the skin tans
plump, but with a brittle and inelastic fibre, weak organic acids make
a plump, soft leather with a moderately elastic fibre, giving to the
skin a somewhat india-rubber feel. We thus see that weak acids alone do
not produce a similar result to a dung bate, which causes the skin to
fall and produces a flat soft leather with a stretch in it, which will
not spring back.
I have also tried the action of the following Na and NH_{3} salts upon
skin:--
_Sodium lactate_, C_{3}H_{5}O_{3}Na, prepared by neutralising 2 grm.
lactic acid with sodium carbonate in 1000 c.c. water. The solution
was used under the same conditions as the other bating experiments,
viz. at 37° C. the reducing action was nil, but on prolonging the
digestion, the medium was found to be very suitable for the development
of putrefactive organisms, and, as a consequence, the skin was attacked
and partly peptonised.
_Ammonium lactate_, prepared in a similar way, had an almost identical
action on the skin, but was more effective in removing the last traces
of lime.
_Ammonium butyrate_, prepared as above, removed lime, but instead of a
reducing action it appeared to plump the skin slightly.
_Action of Bile._
Amongst other compounds in the dung which might have some action on
skin were the bile salts and bile colouring matters. (For a full
description of these, see Gamgee, Phys. Chem. ii.)
Experiments were made with ox bile, 25 c.c. being diluted with 250 c.c.
of water. Pieces of sheep grain previously washed in water were
digested in this solution at 37° C. for 1–4 hours. The bile had no
reducing action on the skin and in fact hardened it, at the same time
staining it a dirty yellowish brown, the colour differing from that of
the colouring matter of dung (hydrobilirubin). Even after continued
standing there was little or no development of bacteria, the bile
appearing to have an antiseptic action.
_Action of Bacteria._
Having ascertained the action of the chief chemical constituents of the
dung, that of the bacteria was next examined. In the first experiment,
eliminating all but bacterial action, a tube of nutrient gelatin was
inoculated from an active bate. In two days the gelatin had liquefied
along the needle track.
The culture was now transferred to the following solution:--
Gelatin 4 grm.
Dextrose 4 "
K_{2}HPO_{4} 1·0 "
MgSO_{4} 0·2 "
NaCl 0·4 "
Water 2000 c.c.
The growth in this was fairly rapid and the smell very slight at the
end of a week. A piece of washed sheep grain worked in this culture
at 37° C. for four hours was considerably reduced, the grain was
not attacked, the action differing in this respect from a prolonged
puering. This mixed culture was grown in various other nutrient media,
gelatin, gelatin and mineral salts, broth made from sheep and calf
fleshings, etc., with practically the same results. In no case did the
bating action equal that of dung, and was insufficient for practical
purposes.
A cursory bacteriological examination of dung will show that the number
of species of bacteria it contains is very large, but on making a
plate cultivation from a sufficiently dilute infusion of fresh dog
dung, most of the colonies developing appear to belong to four or five
species. The following is a list of all the organisms which, so far as
my knowledge goes, have been isolated from different kinds of dung:--
LIST OF KNOWN BACTERIA IN DUNG.
{These organisms have
1. _Bacillus fluorescens putridus_ {been formerly de-
2. " " _liquefaciens_ {scribed as Bacterium
3. _Proteus vulgaris_. {termo by various
4. " _mirabilis_. {authors.
5. _Bacillus subtilis_. }J. T. Wood, J.S.C.I.,
6. _Micrococcus fulvus_. } 1890, 28.
7. _Bacterium ureæ_. (Cohn.)}
8. _Bacillus saprogenes, I., II., III_. _Vide_ Herfeld, J.S.C.I.,
May 1895.
9. _Bacillus butyricus_.
10. _B. putrificus coli_.
11. _B. pyocyaneus_.
12. _B. janthinus_.
13. _B. coprogenes fœtidus_.
14. _B. pyogenes fœtidus_.
15. _B. prodigiosus_.
16. _B. zenkeri_.
17. _B. magnus_.
18. _B. spinosus_.
19. _B. liquefaciens_.
20. _B. coli_.
21. _B. duclauxii = B. ureæ_.
22. _Micrococcus ureæ_.
23. " " _liquefaciens_.
24. _Proteus sulphureus_.
25. _Bacterium sulphureum_.
26. _Bacillus amylobacter_.
27. _Clostridium butyricum_.
28. _B. acidi paralactici_.
29. _Bacillus I_. }Isolated from horse manure by
30. " _II_. }S. A. Severin, Centr.-Bl. f. Bakt. ii.
31. " _III. } [1], 97.
32. _Spirillum serpens_. }
33. " _tenue_. }
34. " _undula_. }Isolated by Kutscher, Zeit. f.
35. " _volutans_.} Hyg. xx. 45.
36. _Vibrio I_. }
37. " _II_. }
38. " _III_. }
39. _Spirillum_ from pig dung. Smith. Centr.-Bl. f. Bakt.
xvi. [1], 124.
40. _Streptococcus liquefaciens coli_. Gamgee. Phys.
Chem. ii.
41. _Bacillus mycoides_.[120]
[120] Since the above was written, Severin (Centr.-Bl. iii. [2],
628) has described seven other species of bacteria, isolated from
horse dung; for the description and property of these the original
memoir must be consulted. Popp and Becker, German Patent 86,335, 28,
describe fourteen species which they isolated from dog and pigeon
dung, of which three have considerable bating action on skins, and
which form the subject of the patent. Some of these are probably
identical with organisms in the above list.
There are besides many unnamed species of bacteria in dung, among which
are the two following isolated by me from puer--
(_a_) _Plate Cultures._--Small yellowish colonies, slightly
fluorescent, liquefy the gelatin slowly. Rods resembling B. subtilis
when at rest, but moving with a rapid undulatory motion. Cultures in
nutrient gelatin have a considerable reducing action on skin.
(_b_) _Plate Cultures._--Bluish colonies liquefying gelatin rapidly,
micro-bacteria in pairs. Not so marked an action on skin as (_a_). The
organism resembles Proteus vulgaris, but swarming islets not observed.
Of the species mentioned above, a pure culture of B. subtilis in meat
broth, maintained for one week at 35° C., had _no_ reducing action on
skin. B. fluorescens liquefaciens had a moderate reducing action.
The species developing on the plates vary according to the age of the
dung. In horse manure, according to Severin,[121] bacilli predominate
in the early stages. At the end of two to three weeks micro-bacteria,
cocci and diplococci make their appearance, while the bacillar form
becomes scarce. In three months cocci predominate, forming zoogloæ;
strepto- and staphylo-cocci and spirilli were seldom found. Yeasts and
sarcina were never observed. From observations extending over several
years it is evident that a similar cycle occurs in dog dung. When quite
fresh it contains comparatively few bacteria; then certain groups of
species take possession, causing decompositions in the dung; these in
their turn give place to other species, which decompose the products
formed by the first, so that no one species produces or can produce the
complex chemical and physiological changes which take place, or the
bodies necessary for the bating of skin, as has been supposed by some
observers.
[121] Centr.-Bl. ii. [1], 97.
None of the experiments, either with mixed or pure cultures of
bacteria, had a perfect reducing action on skin, although the action
was considerably greater than that of the chemical solutions. It was
found, however, that by adding a small quantity of the amines above
mentioned to the bacterial cultures, the reducing action became nearly
as rapid and effective as with dung itself; and it was, therefore,
clear that the principal bating effect was due to a combination of two
things, viz. an active growth of bacteria in the presence of amine
compounds.
It was now necessary to ascertain the mode of action of the bacteria on
the skins.
I have previously shown[122] the action of digestive ferments or
enzymes to be notable in the case of pancreatin. A careful study of the
behaviour of the digestive enzymes in the animal body shows, however,
pretty conclusively, that they are all destroyed before the fæcal
matter is discharged,[123] and, therefore, the enzymes in the dung are
not from this source.
[122] J.S.C.I., 1894, March 31, 218–221.
[123] See Gamgee, Phys. Chem. 2.
An attempt was made to filter the dung diluted with water through a
Berkefeld filter, and thus obtain a clear filtrate containing all the
enzymes and free from bacteria; it was found impossible to filter the
liquid in this way.
The method of Claudio Fermi[124] was also tried, but a clear filtrate
could not be obtained.
[124] Ann. de Micrograph, ii. [6], March 20, 1896.
The author has succeeded in obtaining the enzymes best in the following
manner:--About 150 c.c. of the above-mentioned “puer” was well
mixed with an equal quantity of glycerin and allowed to stand for
seven days; it was then capable of being filtered on a filter pump,
although very slowly, and yielded a clear filtrate of a deep golden
brown colour; the filtrate was poured in a thin stream into a tall
vessel containing about 1500 c.c. of 98 per cent. alcohol. A copious
flocculent precipitate of the albuminous matters and enzymes was thrown
down, the alcohol was filtered off and the precipitate washed on
the filter with absolute alcohol, and dried over sulphuric acid. The
resulting amorphous body was of light brown colour, and became darker
when exposed to the air. The amount obtained from 150 c.c. of dung was
0·55 grm. (3·66 grm. per litre). The body consists of a mixture[125]
of all the enzymes existing in the dung along with other albuminous
bodies. It has a very slight diastatic action on starch. 0·5 grm. in
100 c.c. of water at 35° C. had a very considerable reducing action
on skin. A further experiment with 0·5 grm. of amine hydrochlorides,
0·5 grm. of enzymes and 100 c.c. of water, at 35° C. brought down
a piece of limed sheep grain in 30 minutes exactly like puer; the
reaction of the solution at the commencement of the experiment was
faintly alkaline; at the end of the experiment it was considerably
alkaline.
[125] I did not consider it necessary to purify the enzymes, the
object being to examine the action of the bacterial products apart
from the living organisms.
It was noted that the fæcal odour of the glycerin solution disappeared
on standing for two or three weeks, and the solution smelt strongly of
ethyl butyrate, the enzymes being still present.
These enzymes were prepared from dung, and it now remained to prepare
them in the laboratory by the action of bacteria alone. For this
purpose 200 c.c. of a mixed culture of dung bacteria in the solution
above described, seven days old, was mixed with 200 c.c. of dilute
alcohol (alcohol 65, water 100) and well shaken. Gelatin and albuminoid
bodies are by this means precipitated. The liquid was filtered and
poured into eight times its volume of 98 per cent. alcohol. The
precipitate which came down was washed with absolute alcohol and dried
in the usual way. The enzymes thus obtained were redissolved in water
and the former experiment with skin repeated with this solution. The
same effect was produced, showing conclusively that it is the enzymes
produced by the bacteria, acting in conjunction with the amines, which
bate the skin. It would seem that the special action of the enzymes is
aided by the presence of amine compounds, in addition to the chemical
action which these latter have upon the skin. The action is shown to
be interdependent, i.e., bacterial action alone is insufficient, and
chemical action alone is insufficient, the true bating action being a
combination of the two.
In conclusion the author has pleasure in acknowledging the valuable
assistance of Mr. H. S. Shrewsbury in carrying out some of the
experiments.
III. FURTHER NOTES ON THE ACTION OF THE DUNG BATE.[126]
[126] Reprinted from the Journal of the Society of Chemical Industry,
November 30, 1899, No. 11, vol. xviii.
The present notes are a continuation of those published in 1898. It has
been impossible for me to follow up the subject except in a desultory
manner, but I think it desirable to put further results, however
meagre, before you with the object of throwing more light on the
complex process of bating hides and skins.
Since the previous notes were written, Dr. Th. Körner[127] has called
attention to the physics of the process, a matter which I had not
previously mentioned. He explains part of the action by differences
of osmotic pressure. The ammonium salts and salts of organic bases
contained in the dung, possess a smaller osmotic pressure than the
solution of calcium hydrate contained in the skins. The ammonium ion
endeavours to unite with the hydroxyl ion of the calcium hydrate to
form undissociated ammonium hydroxide. In consequence of the osmotic
pressure and the electrostatic attraction of the oppositely charged
ions, the calcium hydrate solution is withdrawn from the skin and the
latter falls.
[127] 10 Jahresber. d. Deutsch. Gerberschule zu Freiberg, 1898–99:
Beiträge zur Kentniss der wissenschaftlichen Grundlagen der Gerberei,
p. 32.
_Experiments with Bacteria on a Large Scale._
I have shown[128] that simple cultures of bacteria in certain nutrient
media, in the absence of amines and other bodies, have an imperfect
bating action on skin. This was the case, whether the cultures were
pure or mixed, but the mixed cultures have a better action than
the pure cultures of any of the organisms I have hitherto used.
Bacteriological processes in nature are usually carried on by a
mixture of species of bacteria; still there are fermentations like the
spontaneous souring of milk, the formation of nitrites from nitrates,
and the ammoniacal fermentation of urea, which may be considered
as natural pure cultures. These are examples of what I may call the
selective influence of the nutrient medium. The nutrient medium used
in my former experiments was a solution of gelatin and mineral salts,
such as is used in general bacteriological work, modified in various
ways. On careful consideration, this did not appear to be a medium at
all corresponding to the natural bate. The albuminoids in the process
of digestion are peptonised, and dung contains only those bodies which
the animal is incapable of assimilating. It is from these bodies that
the necessary enzymes are produced by bacteria. Of the numerous trials
with pure cultures of different organisms from dung, I found that
most of those having the property of secreting bating enzymes, were
non-liquefying bacteria. Popp and Becker have also pointed out that
peptonising bacteria do not exert a favourable reducing action on skin.
It is impracticable to grow these non-peptonising organisms on a large
scale in a solid medium, and it occurred to me that gelatin, previously
peptonised by chemical means, would be a favourable medium. Experiments
with hydrochloric and sulphuric acids gave hopeful results. The acid
was neutralised with ammonia after the chemical action was complete,
and the bacteria cultivated in this solution.
[128] J.S.C.I., 1898, p. 1011, see previous paper, p. 152.
A better result was obtained by digesting 10 grm. gelatin, 5 grm.
lactic acid (anhydrous by calculation), 100 c.c. of water, in a closed
vessel on the water bath for three hours. A slight black precipitate of
melanoidic acid (humic acid) is formed. The liquid is of a clear brown
colour, and contains a large number of nitrogenous bodies resulting
from the breaking down of the gelatin molecule. It has been impossible
for me to ascertain its exact composition, but I have partially
examined it with the following results:--
1. A small quantity of the liquid poured into absolute alcohol, gives
a white precipitate which re-dissolves on the addition of about 30 per
cent. water. This shows absence of gelatin.
2. On saturating with ammonium sulphate, a brownish precipitate is
thrown down which is perfectly soluble in cold water. It consists of
gelatose lactates which are evidently formed in an analogous manner,
to the gelatose hydrochlorides prepared by C. Paal.[129] The gelatoses
correspond to the protoses formed during the digestion of albumin. In
one sample 1·3 grm. gelatose lactates per 100 c.c. was found.
[129] Ber. 25, 1202; also Allen, “Comm. Org. Analysis,” iv., 466. F.
Marpmann, Centralblatt f. Bakt. 2, 5, 67.
3. The filtrate from (2) was dialysed against running water for
15 hours to get rid of the ammonium sulphate. The resulting
gelatin-peptones, or gelatones, amounted to 9·1 grm. per 100 c.c. In
other words, the heating under pressure had transformed 87 per cent. of
the original gelatin into true peptones.
It may be presumed that the other and simpler nitrogenous bodies
formed, are the same as those produced by the treatment of gelatin
solutions by dilute mineral acids, and that the bases combine with the
free lactic acid. An excellent list of these and a very interesting
account of the protamines and hexones which go to form the complex
albumin molecule is given in a paper by Dr. A. Kossel.[130]
[130] Rev. Gen. des Sciences, 1899, p. 380.
The solution of gelatone lactates and free lactic acid prepared as
above, was neutralised with sodium carbonate and diluted to 1000 c.c.;
it was found to be a very good medium for the growth of the bating
organisms after the addition of a small quantity of potassium phosphate.
In looking round for suitable means of testing the action of both pure
and mixed cultures of bacteria on a practical scale, it occurred to me
that the Carlsberg vessels, as used by Hansen for the pure cultivation
of yeast,[131] would answer for the cultivation of bacteria also. I was
unable to obtain any information on the subject, and was surprised to
find that the apparatus is very little used in England. I procured two
of these vessels from Jensen of Copenhagen, and found they answered
very well. The bacteria were transferred from the test tube in which
the original inoculation had been made to a Pasteur flask of 250 c.c.
capacity containing the nutrient solution; when the growth in this
flask was sufficiently vigorous the Carlsberg vessel was inoculated
from it in the manner described by Hansen, and with all the usual
precautions.
[131] Jorgensen, the “Micro-organisms of Fermentation,” p. 20.
After three days at a temperature of 37° C. the whole of the 10
litres was used for inoculating 100 litres of the nutrient medium
above mentioned contained in a clean barrel standing in a room,
the temperature of which was maintained at 37° C. By using this
comparatively large volume of pure culture for pitching, if I may be
allowed to use a familiar brewing term, the large culture was kept
practically pure, although it could not be supplied with germ-free air,
as in the case of the Carlsberg vessel.
Using a nutrient medium containing 1 per cent. original gelatin, the
maximum bating effect was obtained with the majority of organisms in
three days.
In my previous notes I showed[132] that a mixture of digestive enzymes
and amine hydrochlorates was effective in bating skin. The liquid in
this case was free from bacteria. It was found, however, that the
action of the bate was hastened when an active growth of bacteria was
going on at the same time in the liquid, even though the quantity of
enzyme present was smaller. This is what one might infer from a careful
study of the process. It appears better for small quantities of enzymes
to be produced in the liquid as required, than to use larger quantities
of stale enzymes.
[132] J.S.C.I., 1898, p. 1012.
Mr. Loxley Meggitt was kind enough to concentrate for me about 170
litres of a culture of bating organisms. The concentration was
conducted _in vacuo_ at a temperature below 50° C., at which the
enzymes were certainly not injured, and it was found that when this
concentrated culture was diluted to the same strength as the original,
the action was considerably diminished. This diminution of activity was
due in part to a loss of volatile products in the evaporation, but more
especially to the absence of an active fermentation going on in the
liquid.
The nutrient medium above described, besides acting in a selective
manner on a mixture of bacteria, and providing them with food stuff,
contains amido compounds and other bodies, which are eminently
favourable to the proteolytic action of the enzymes, and therefore act
in the same way as the amine hydrochlorates which I used in former
experiments.
The action of the following bacteria grown in this medium has been
tried on skin. The pure cultures are lettered in continuation of
previous descriptions (see previous paper, p. 162).
1. Bacillus _c_, isolated from pigeon dung bate. Small, round,
bluish-white colonies, standing up slightly above the surface of the
gelatin, but not spreading out on the surface, non-liquefying pairs of
micro-bacteria. This was the principal organism found in a pigeon dung
bate as used for the bating of E.I. kips. Grown in nutrient broth or in
the special medium it had no reducing effect on skin.
2. Bacillus pyocyaneus from blue skin, i.e. skin in an early stage of
putrefaction. No action.
3. Mixed culture from fresh pigeon dung, collected in a sterile vessel.
This contained very few bacteria capable of developing in the special
medium. The action on skin was no more than that due to the chemical
compounds present.
4. Mixed culture from fresh dog dung. This contained remarkably few
species of bacteria which came to a good development in the special
medium. The principal organisms were a jointed bacillus with extremely
rapid vibrionic movement, and a small bacterium in pairs; the action on
skin was perceptible but slight.
5. Mixed culture from dog dung one month old, as used in the bate. The
action of this in a certain medium has already been described (see
p. 160). In the special medium it was distinctly better and almost
equal to that of the dung bate.
6. Mixed culture from fresh fæces gave a similar result to (3). A
microscopic examination showed pairs of small bacteria and micrococci,
but no bacillar forms.
7. Mixed culture from fresh horse dung had a moderate but distinct
action on skin, about equal to (4).
8. Bacillus _d_, isolated from wool infusion. Very slight action.
9. Bacillus _e_, isolated from wool infusion. Similar result to (8).
10. Mixed culture of bacilli _d_ and _e_ only. Very powerful bating
action, skin bated more rapidly than with dung.
These experiments tend to confirm the conclusion I previously arrived
at, viz. that no single species of bacteria produces the complex
chemical and physiological changes which take place in the dung, and
which result in the production of the bodies necessary for the proper
bating of skin. It is a well-known fact that dog dung requires keeping
for at least a month before it gives the best result. During this time
it undergoes a kind of fermentation and continues to improve up to two
months, after which it deteriorates. The rate of fermentation depends
upon the season.
It is evident from these facts that the bacteria present in the dung
when it leaves the animal’s body do not produce the required enzymes
and chemical compounds, and that these are produced by bacteria which
obtain access from the air. It is also evident that the production of
these enzymes depends upon the composition of the nutrient medium,
since this exerts a selective influence on the species of bacteria
obtaining access to it. In the spontaneous souring of milk numerous
bacteria in the air have free access to the milk, yet the lactic
ferment is generally so pure that it may be and is used as a pure
culture on a large scale in the manufacture of lactic acid. Dog dung
is a favourable medium for the bacteria secreting the bating enzymes;
but if dextrose or other carbohydrate be added to fresh dung an acid
fermentation is set up which effectually prevents the development of
the bating organisms.
Reasoning along these lines I was led to try some cultures of air
bacteria in my special medium, and I found that a good source of such
organisms likely to have a useful effect was a sweating stove as used
for the depilation of skins. The hair roots are loosened by bacterial
action; the wool, when it slips, brings away with it the epidermis.
The root portions of the wool were cut off and digested in water at
35° C., the liquid strained off and used for making plate cultures
in the usual way. In making the attenuations for the plate cultures,
the fourth was found to be practically a pure culture of the organism
I have called bacillus _d_, or the sweating bacillus. It forms large
whitish colonies, spreading on the surface, with irregular contour. The
bacilli are very small, mostly in pairs, but sometimes joining together
in thread-like forms. (Fig. 21.)
Grown in the special medium it had little or no action on skin. At the
same time I found that a culture made from the original liquid, i.e.,
a mixed culture of the sweating organisms, had an exceedingly powerful
bating action; indeed, the skin was bated more rapidly than with dung.
All the experiments carried out so far tend to prove that mixed
cultures of suitable bacteria possessed the required action, whereas
pure cultures do not. A further examination of the infusion of wool
roots showed that the bacteria contained in it, consisted practically
of two species only. The first of these I have already described,
the second one, bacillus _e_ (Fig. 22), forms small brownish-yellow
boat-shaped colonies on gelatin plates, very similar to one of the dung
bacteria. They consist of plump cells, two or three times the size of
bacillus _d_, united in pairs and short chains and surrounded by a
capsule; the cells appear to vary considerably in size. Cultivated in
the same way as the others it has very little action on skin. It is
evident from these facts that the growth of the bacteria is a symbiotic
one; separately they exert little or no action, whereas used together
the action is most remarkable.
The author wishes to express his indebtedness to Mr. J. Golding,
F.I.C., of University College, Nottingham, who made the first pure
cultures of these “wool” bacteria.
_The Influence of Solid Matter in the Bate._
In making comparative tests of my artificial bates and dog dung, I
usually took the latter from the paddles in which it was diluted
ready for use, and maintained both solutions at exactly the same
temperature. Parts of the _same_ skin were then digested in each
solution, and the results noted after 1-1/2 hours. In order to make
them more strictly comparable I filtered the dung bate, so as to employ
the matter actually in solution when the bate was made up. It was found
that the filtered bate had far less action than when it was used in an
unfiltered condition. That this is not wholly due to the passing into
solution of some of the solid matter during the bating was shown by
adding an inert solid, viz., kaolin, to the filtered bate, when the
action was greatly hastened. On adding kaolin to the artificial bate
and keeping the liquid agitated, the same result was obtained. It seems
that the finely-divided solid matter acts as a carrier for the enzymes,
perhaps by a kind of mass action, each particle offering a surface
vastly greater than the molecules of dissolved solids. In the case of
“puer,” the organic insoluble matter is gradually brought into solution
by bacterial action as the bating proceeds, though I do not believe
this occurs to any great extent.
The following quantities of soluble and insoluble matter were found in
puer wheels in actual practice per 100 c.c.:--
-------------+-------+------+------+------
---- | 1 | 2 | 3 | 4
-------------+-------+------+------+------
Total solids | 10·20 | 8·63 | 8·64 | 3·26
Soluble | 6·00 | 4·57 | 6·19 | 2·14
Insoluble | 4·20 | 4·06 | 2·45 | 1·12
-------------+-------+------+------+------
1. Wheel in constant use for a week.
2. Wheel freshly made up.
3. The same wheel after one lot of skins.
4. From pigeon dung bate pit.
On comparing 2 and 3 it will be seen that apparently 1·61 grm. of
the insoluble matter has passed into solution. This is not entirely
the case, for part of the additional soluble matter is lime and hide
substance from the skins, while part of the insoluble at the same time
adheres to the skins and is not estimated.
In conclusion, it will be seen that it is now quite possible to
produce an artificial dung bate, or rather a bate having the essential
properties of a dung bate, by producing the required enzymes by
fermentation and then adding to them the amine compounds. Such a bate
I am able to show you here. The chief difficulty in its practical
application in the tannery is the question of cost. If this can be
overcome, and efforts are being made to overcome it, there is no doubt
that the tanners of the next generation will be supplied with a bate
which will do the same work as the dung bate, and which will have a
definite and uniform composition and properties, and a regular and
certain action. The tannery will become cleaner and healthier, and
what is perhaps of equal importance, the sewage effluents will be
comparatively harmless.