We may here add a few words on the non-transformation of yeast into

_penicillium glaucum_.

If at any time during fermentation we pour off the fermenting liquid,

the deposit of yeast remaining in the vessel may continue there, in

contact with air, without our ever being able to discover the least

formation of _penicillium glaucum_ in it. We may keep a current of pure

air constantly passing through the flask; the experiment will give the

same result. Nevertheless, this is a medium peculiarly adapted to the

development of this mould, inasmuch as if we introduce merely a few

spores of _penicillium_, an abundant vegetation of that growth will

afterwards appear on the deposit. The descriptions of Messrs. Turpin,

Hoffmann, and Trécul have, therefore, been based on one of these

illusions which we meet with so frequently in microscopical

observations. When we laid these facts before the Academy,[159] M.

Trécul professed his inability to comprehend them:[160]

“According to M. Pasteur,” he said, “the yeast of beer is _anaërobian_,

that is to say, it lives in a liquid deprived of free oxygen; and to

become _mycoderma_ or _penicillium_ it is above all things necessary

that it should be placed in air, since, without this, as the name

signifies, an aërobian being cannot exist. To bring about the

transformation of the yeast of beer into _mycoderma cerevisiæ_ or into

_penicillium glaucum_, we must accept the conditions under which these

two forms are obtained. If M. Pasteur will persist in keeping his yeast

in media which are incompatible with the desired modification, it is

clear that the results which he obtains must be always negative.”

Contrary to this perfectly gratuitous assertion of M. Trécul’s, we do

not keep our yeast in media which are calculated to prevent its

transformation into _penicillium_. As we have just seen, the principal

aim and object of our experiment was to bring this minute plant into

contact with air, and under conditions that would allow the

_penicillium_ to develop with perfect freedom. We conducted our

experiments exactly as Turpin and Hoffmann conducted theirs, and exactly

as they stipulate that such experiments should be conducted—with the one

sole difference, indispensable to the correctness of our observations,

that we carefully guarded ourselves against those causes of error which

they did not take the least trouble to avoid. It is possible to produce

a ready entrance and escape of pure air in the case of the double-necked

flasks which we have so often employed in the course of this work,

without having recourse to the continuous passage of a current of air.

Having made a file-mark on the thin curved neck at a distance of two or

three centimetres (an inch) from the flask, we must cut round the neck

at this point with a glazier’s diamond, and then remove it, taking care

to cover the opening immediately with a sheet of paper which has been

passed through the flame, and which we must fasten with a thread round

the part of the neck still left. In this manner we may increase or

prolong the fructification of fungoid growths, or the life of aërobian

ferments in our flasks.

What we have said of _penicillium glaucum_ will apply equally to

_mycoderma cerevisiæ_. Notwithstanding what Turpin and Trécul may assert

to the contrary, yeast, in contact with air as it was under the

conditions of the experiment just described, will not yield _mycoderma

vini_ or _mycoderma cerevisiæ_ any more than it will _penicillium_.

The experiments described in the preceding paragraphs on the increase of

organized ferments in mineral media of the composition described, are of

great physiological interest. Amongst other results, they show that all

the proteic matter of ferments may be produced by the vital activity of

the cells, which, apart altogether from the influence of light or free

oxygen (unless, indeed, we are dealing with aërobian moulds which

require free oxygen), have the power of developing a chemical activity

between carbo-hydrates, ammoniacal salts, phosphates and sulphates of

potassium and magnesium. It may be admitted with truth that a similar

effect obtains in the case of the higher plants, so that in the existing

state of science we fail to conceive what serious reason can be urged

against our considering this effect as general. It would be perfectly

logical to extend the results of which we are speaking to all plants,

and to believe that the proteic matter of vegetables, and perhaps of

animals also, is formed exclusively by the activity of the cells

operating upon the ammoniacal and other mineral salts of the sap or

plasma of the blood, and the carbo-hydrates, the formation of which, in

the case of the higher plants, requires only the concurrence of the

chemical impulse of green light.

Viewed in this manner, the formation of the proteic substances would be

independent of the great act of reduction of carbonic acid gas under the

influence of light. These substances would not be built up from the

elements of water, ammonia, and carbonic acid gas, after the

decomposition of this last; they would be formed where they are found in

the cells themselves, by some process of union between the

carbo-hydrates imported by the sap, and the phosphates of potassium and

magnesium and salts of ammonia. Lastly, in vegetable growth, by means of

a carbo-hydrate and a mineral medium, since the carbo-hydrate is capable

of many variations, and it would be difficult to understand how it could

be split up into its elements before serving to constitute the proteic

substances, we may hope to obtain as many distinct proteic substances,

and even cellulose substances, as there are carbo-hydrates. We have

commenced certain studies in this direction.

If solar radiation is indispensable to the decomposition of carbonic

acid and the building up of the primary substances in the case of

higher vegetable life, it is still possible that certain inferior

organisms may do without it and nevertheless yield the most complex

substances, fatty or carbo-hydrate, such as cellulose, various organic

acids, and proteic matter; not, however, by borrowing their carbon

from the carbonic acid which is saturated with oxygen, but from other

matters still capable of acquiring oxygen, and so of yielding heat in

the process, such as alcohol and acetic acid, for example, to cite

merely carbon compounds most removed from organization. As these last

compounds, and a host of others equally adapted to serve as the

carbonaceous food of _mycoderms_ and the mucedines, may be produced

synthetically by means of carbon and the vapour of water, after the

methods that science owes to Berthelot, it follows that, in the case

of certain inferior beings, life would be possible even if it should

be that the solar light was extinguished.[161]

Footnote 111:

It has been remarked in practice that fermentation is facilitated by

leaving the grapes on the bunches. The reason of this has not yet been

discovered. Still we have no doubt that it may be attributed,

principally, to the fact that the interstices between the grapes, and

the spaces which the bunch leaves throughout, considerably increase

the volume of air placed at the service of the germs of ferment.

Footnote 112:

[NaHSO_{2}, now called _Sodium hyposulphite_. See p. 355,

footnote.—D.C.R.]

Footnote 113:

[This appears to be a misprint for 1·638 grammes = 25·3

grains.—D.C.R.]

Footnote 114:

[200 c.c. of liquid were used, which, as containing 5 per cent., had

in solution 10 grammes of sugar.—D. C. R.]

Footnote 115:

[International Science Series, vol. xx., pp. 179-182. London, 1876.—D.

C. R.]

Footnote 116:

Page 182, English edition.

Footnote 117:

This figure is on a scale of 300 diameters, most of the figures in

this work being of 400 diameters.

Footnote 118:

[It may be useful for the non-scientific reader to put it thus:—that

the 25 c.c. which escaped, being a fair sample of the whole gas in the

flask, and containing (1) 25 - 20·6 = 4·4 c.c., absorbed by potash and

therefore due to carbonic acid, and (2) 20·6 - 17·3 = 3·3 c.c.,

absorbed by pyrogallate, and therefore due to oxygen, and the

remaining 17·3 c.c. being nitrogen, the whole gas in the flask, which

has a capacity of 315 c.c., will contain oxygen in the above

proportion, and therefore its amount may be determined, provided we

know the total gas in the flask before opening. On the other hand, we

know that air normally contains, approximately, 1/5th its volume of

oxygen, the rest being nitrogen, so that, by ascertaining the

diminution of the proportion in the flask, we can find how many cubic

centimetres have been absorbed by the yeast. The author, however, has

not given all the _data_ necessary for accurate calculation.—D.C.R.]

Footnote 119:

This number is probably too small; it is scarcely possible that the

increase of weight in the yeast, even under the exceptional conditions

of the experiment described, was not to some extent at least due to

oxidation apart from free oxygen, inasmuch as some of the cells were

covered by others. The increased weight of the yeast is always due to

the action of two distinct modes of vital energy—activity, namely, in

presence and activity in absence of air. We might endeavour to shorten

the duration of the experiment still further, in which case we would

still more assimilate the life of the yeast to that of ordinary

moulds.

Footnote 120:

In these experiments, in which the moulds remain for a long time in

contact with a saccharine wort out of contact with oxygen—the oxygen

being promptly absorbed by the vital action of the plant (see our

_Mémoire sur les Générations dites Spontanées_, p. 54, note)—there is

no doubt that an appreciable quantity of alcohol is formed because the

plant does not immediately lose its vital activity, after the

absorption of oxygen.

A 300-c.c. (10-oz.) flask, containing 100 c.c. of must, after the air

in it had been expelled by boiling, was opened and immediately

re-closed, on August 15th, 1873. A fungoid growth—a unique one, of

greenish-grey colour—developed from spontaneous impregnation, and

decolorized the liquid, which originally was of a yellowish-brown.

Some large crystals, sparkling like diamonds, of neutral tartrate of

lime, were precipitated. About a year afterwards, long after the death

of the plant, we examined this liquid. It contained 0·3 gramme (4·6

grains) of alcohol, and 0·053 gramme (0·8 grain) of vegetable matter,

dried at 100° C. (212° F.). We ascertained that the spores of the

fungus were dead at the moment when the flask was opened. When sown,

they did not develop in the least degree.

Footnote 121:

We find in M. Raulin’s Note, already quoted, that “the minimum ratio

between the weight of sugar and the weight of organized matter, that

is, the weight of fungoid growth which it helps to form, may be

expressed 10/3·2 = 3·1.” JULES RAULIN, _Études chimiques sur la

végétation. Recherches sur le développement d’une mucédinée dans un

milieu artificiel_, p. 192, Paris, 1870. We have seen, in the case of

yeast, that this ratio may be as low as 4/1.

Footnote 122:

We shall show, some day, that the processes of oxidation due to growth

of fungi cause, in certain decompositions, liberation of ammonia to a

considerable extent, and that by regulating their action we might

cause them to extract the nitrogen from a host of organic _débris_, as

also, by checking the production of such organisms, we might

considerably increase the proportion of nitrates in the artificial

nitrogenous substances. By cultivating various moulds on the surface

of damp bread in a current of air, we have obtained an abundance of

ammonia, derived from the decomposition of the albuminoids effected by

the fungoid life. The decomposition of asparagus, and several other

animal or vegetable substances, has given similar results.

Footnote 123:

To determine the absence of cells of ferment in fruits that have been

immersed in carbonic acid gas, we must first of all carefully raise

the pellicle of the fruit, taking care that the subjacent parenchyma

does not touch the surface of the pellicle, since the organized

corpuscles existing on the exterior of the fruit might introduce an

error into our microscopical observations. Experiments on grapes have

given us an explanation of a fact generally known, the cause of which,

however, had hitherto escaped our knowledge. We all know that the

taste and aroma of the vintage, that is, of the grapes stripped from

the bunches and thrown into tubs, where they get soaked in the juice

that issues from wounded specimens, are very different from the taste

and aroma of an uninjured bunch. Now grapes that have been immersed in

an atmosphere of carbonic acid gas have exactly the flavour and smell

of the vintage; the reason is that, in the vintage tub, the grapes are

immediately surrounded by an atmosphere of carbonic acid gas, and

undergo, in consequence, the fermentation peculiar to grapes that have

been plunged in this gas. These facts deserve to be studied from a

practical point of view. It would be interesting, for example, to

learn what difference there would be in the quality of two wines, the

grapes of which, in the one case, had been perfectly crushed, so as to

cause as great a separation of the cells of the parenchyma as

possible; in the other case, left, for the most part, whole, as in the

case in the ordinary vintage. The first wine would be deprived of

those fixed and fragrant principles produced by the fermentation of

which we have just spoken, when the grapes are immersed in carbonic

acid gas. By such a comparison as that which we suggest, we should be

able to form an _à priori_ judgment on the merits of the new system,

which has not been carefully studied, although already widely adopted,

of milled, cylindrical crushers, for pressing the vintage.

Footnote 124:

We have sometimes found small quantities of alcohol in fruits and

other vegetable organs, surrounded with ordinary air, but always in

small proportion, and in a manner which suggested its accidental

character. It is easy to understand how, in the thickness of certain

fruits, certain parts of those fruits might be deprived of air, under

which circumstance they would have been acting under conditions

similar to those under which fruits act when wholly immersed in

carbonic acid gas. Moreover it would be useful to determine whether

alcohol is not a normal product of vegetation.

Footnote 125:

In these studies on plants living immersed in carbonic acid gas, we

have come across a fact which corroborates those which we have already

given in reference to the facility with which lactic and viscous

ferments, and, generally speaking, those which we have termed the

disease-ferments of beer, develop when deprived of air, and which

shows, consequently, how very marked their aërobian character is. If

we immerse beetroots or turnips in carbonic acid gas, we produce

well-defined fermentations in those roots. Their whole surface readily

permits the escape of the highly acid liquids, and they become filled

with lactic, viscous, and other ferments. This shows us the great

danger which may result from the use of pits, in which the beetroots

are preserved, when the air is not renewed, and that the original

oxygen is expelled by the vital processes of fungi, or other

deoxidizing chemical actions. We have directed the attention of the

manufacturers of beetroot sugar to this point.

Footnote 126:

LECHARTIER and BELLAMY, _Comptes rendus de l’Académie des Sciences_,

vol. lxix., pp. 366 and 466, 1869.

Footnote 127:

Those gentlemen express themselves thus: “In a note presented to the

Academy in November, 1872, we published certain experiments which

showed that carbonic acid and alcohol may be produced in fruits kept

in a closed vessel, out of contact with atmospheric oxygen, without

our being able to discover alcoholic ferment in the interior of those

fruits.

“M. Pasteur, as a logical deduction from the principles which he has

established in connection with the theory of fermentation, considers

that _the formation of alcohol may be attributed to the fact that the

physical and chemical processes of life in the cells of fruit continue

under new conditions, in a manner similar to those of the cells of

ferment_. Experiments, continued during 1872, 1873, and 1874, on

different fruits, have furnished results all of which seem to us to

harmonize with this proposition, and to establish it on a firm basis

of proof.” _Comptes rendus_, t. lxxix., p. 949, 1874.

Footnote 128:

PASTEUR, _Faites nouveaux pour servir à la connaissance de la théorie

des fermentations proprement dites_. (_Comptes rendus de l’Académie

des Sciences_, t. lxxv., p. 784). See, in the same volume, the

discussion that followed; also, PASTEUR, _Note sur la production de

l’alcool par les fruits_, same volume, p. 1054, in which we recount

the observations anterior to our own, made by Messrs. Lechartier and

Bellamy in 1869.

Footnote 129:

_Comptes rendus_, meeting of January 15th, 1872.

Footnote 130:

As a matter of fact, M. Fremy applies his theory of hemi-organism, not

only to the alcoholic fermentation of grape juice, but to all other

fermentations. The following passage occurs in one of his Notes

(_Comptes rendus de l’Académie_, t. lxxv., p. 979, October 28th,

1872):

“_Experiments on Germinated Barley._—The object of these was to show

that, when barley, left to itself in sweetened water, produces in

succession alcoholic, lactic, butyric, and acetic fermentations, these

modifications are brought about by ferments which are produced inside

the grains themselves, and not by atmospheric germs. More than forty

different experiments were devoted to this part of my work.” Need we

add that this assertion is based on no substantial foundation? The

cells belonging to the grains of barley, or their albuminous contents,

never do produce cells of alcoholic ferment, or of lactic ferment, or

butyric vibrios. Whenever those ferments appear they may be traced to

germs of those organisms, diffused throughout the interior of the

grains, or adhering to their exterior surface, or existing in the

water employed, or on the sides of the vessels used. There are many

ways of demonstrating this, of which the following is one: since the

results of our experiments have shown that sweetened water,

phosphates, and chalk very readily give rise to lactic and butyric

fermentations, what reason is there for supposing that if we

substitute grains of barley for chalk, the lactic and butyric ferments

will spring from those grains, in consequence of a transformation of

their cells or albuminous substances? Surely, there is no ground for

maintaining that they are produced by hemi-organism, since a medium

composed of sugar, or chalk, or phosphates of ammonia, potash, or

magnesia contains no albuminous substances. This is an indirect but

irresistible argument against the hemi-organism theory.

Footnote 131:

PASTEUR, _Mémoire sur la fermentation alcoolique_, 1860; _Annales de

Chimie et de Physique_. The word _globules_ is here used for _cells_.

In our researches we have always endeavoured to prevent any confusion

of ideas. We stated at the beginning of our Memoir of 1860, that: “We

apply the term _alcoholic_ to that fermentation which sugar undergoes

under the influence of the ferment known as _beer yeast_.” This is the

fermentation which produces wine and all alcoholic beverages. This,

too, is regarded as the type for a host of similar phenomena,

designated, by general usage, under the generic name of

_fermentation_, and qualified by the name of one of the essential

products of the special phenomenon under observation. Bearing in mind

this fact in reference to the nomenclature that we have adopted, it

will be seen that the expression _alcoholic fermentation_ cannot be

applied to every phenomenon of fermentation in which alcohol is

produced, inasmuch as there may be a number of phenomena having this

character in common. If we had not at starting defined that particular

one amongst the number of very distinct phenomena, which, to the

exclusion of the others, should bear the name _alcoholic

fermentation_, we should inevitably have given rise to a confusion of

language that would soon pass from words to ideas, and tend to

introduce unnecessary complexity into researches which are already, in

themselves, sufficiently complex to necessitate the adoption of

scrupulous care to prevent their becoming still more involved. It

seems to us that any further doubt as to the meaning of the words

_alcoholic fermentation_, and the sense in which they are employed, is

impossible, inasmuch as Lavoisier, Gay-Lussac, and Thénard have

applied this term to the fermentation of sugar by means of beer yeast.

It would be both dangerous and unprofitable to discard the example set

by those illustrious masters, to whom we are indebted for our earliest

knowledge of this subject.

Footnote 132:

See, for example, the communications of MM. Colin and Poggiale, and

the discussion on them, in the _Bulletin de l’Académie de Médicine_,

March 2nd, 9th, and 30th, and February 16th and 23rd, 1875.

Footnote 133:

We have elsewhere determined the formation of minute quantities of

volatile acids in alcoholic fermentation. M. Béchamp, who studied

these, recognized several belonging to the series of fatty acids,

acetic acid, butyric acid, &c. “The presence of succinic acid is not

accidental, but constant; if we put aside volatile acids that form in

quantities which we may call infinitely small, we may say that

succinic acid is the only normal acid of alcoholic fermentation.”

PASTEUR, _Comptes rendus de l’Académie_, t. xlvii. p. 224, 1858.

Footnote 134:

Traube’s conceptions were governed by a theory of fermentation

entirely his own, a hypothetical one, as he admits, of which the

following is a brief summary: “We have no reason to doubt,” Traube

says, “that the protoplasm of vegetable cells is itself, or contains

within it, a chemical ferment which causes the alcoholic fermentation

of sugar; its efficacy seems closely connected with the presence of

the cell, inasmuch as, up to the present time, we have discovered no

means of isolating it from the cells with success. In the presence of

air, this ferment oxidizes sugar, by bringing oxygen to bear upon it;

in the absence of air it decomposes the sugar by taking away oxygen

from one group of atoms of the molecule of sugar and bringing it to

act upon other atoms; on the one hand yielding a product of alcohol by

reduction, on the other hand a product of carbonic acid by oxidation.”

Traube supposes that this chemical ferment exists in yeast and in all

sweet fruits, but only when the cells are intact, for he has proved

for himself that thoroughly crushed fruits give rise to no

fermentation whatever in carbonic acid gas. In this respect this

imaginary chemical ferment would differ entirely from those which we

call _soluble ferments_, since diastase, emulsine, &c., may be easily

isolated.

For a full account of the views of Brefeld and Traube, and the

discussion which they carried on on the subject of the results of our

experiments, our readers may consult the _Journal of the Chemical

Society of Berlin_, vii. p. 872. The numbers for September and

December, 1874, in the same volume, contain the replies of the two

authors.

Footnote 135:

See PASTEUR, _Comptes rendus de l’Académie des Sciences_, t. lvi. p.

416.

Footnote 136:

[Carbonic acid being considerably more soluble than other gases

possible under the circumstances.—ED.]

Footnote 137:

We had to avoid filling the small flask completely, for fear of

causing some of the liquid to pass on to the surface of the mercury in

the measuring tube. The liquid condensed by boiling forms pure water,

the solvent affinity of which for carbonic acid, at the temperature we

employ, is well known.

Footnote 138:

The following is a curious consequence of these numbers and of the

nature of the products of this fermentation. The carbonic acid

liberated being quite pure, especially when the liquid has been boiled

to expel all air from the flask, and capable of perfect solution, it

follows that, the volume of liquid being sufficient and the weight of

tartrate suitably chosen—we may set aside tartrate of lime in an

insoluble, crystalline powder, along with phosphates at the bottom of

a closed vessel full of water, and find soon afterwards in their place

carbonate of lime, and, in the liquid, soluble salts of lime, with a

mass of organic matter at the bottom, without any liberation of gas or

appearance of fermentation ever taking place, except as far as the

vital action and transformation in the tartrate are concerned. It is

easy to calculate that a vessel or flask of five litres (rather more

than a gallon) would be large enough for the accomplishment of this

remarkable and singularly quiet transformation, in the case of fifty

grammes (767 grains) of tartrate of lime.

Footnote 139:

We treated the whole deposit with dilute hydrochloric acid, which

dissolved the carbonate of lime, and the insoluble phosphates of

calcium and magnesium; afterwards filtering the liquid through a

weighed filter paper. Dried at 100° C. (212° F.), the weight of

organic matter thus obtained was 0·54 gramme (8·3 grains), which was

rather more than 1/200th of the weight of fermentable matter.

Footnote 140:

Should the solution of lactate of lime be turbid, it may be clarified

by filtration, after previously adding a small quantity of phosphate

of ammonia, which throws down phosphate of lime. It is only after this

process of clarification and filtration that the phosphates of the

formula are added. The solution soon becomes turbid, if left in

contact with air, in consequence of the spontaneous formation of

bacteria.

Footnote 141:

The naturalist Cohn, of Breslau, who published an excellent work on

bacteria in 1872, described, after Mayer, the composition of a liquid

peculiarly adapted to the propagation of these organisms, which it

would be well to compare for its utility in studies of this kind with

our solution of lactate and phosphates. The following is Cohn’s

formula:—

Distilled water 20 c.c. (0·7 fl. oz.)

Phosphate of 0·1 gramme (1·5 grains).

potassium

Sulphate of 0·1 gramme (1·5 grains).

magnesium

Tribasic phosphate 0·01 gramme (0·15 grain).

of lime

Tartrate of ammonia 0·2 gramme (3 grains).

This liquid, the author says, has a feeble acid reaction and forms a

perfectly clear solution.

Footnote 142:

On the rapid absorption of oxygen by bacteria, see also our _Mémoire_

of 1872, _sur les Générations dites Spontanées_, especially the note

on page 78.

Footnote 143:

In what way are we to account for so great a difference between the

two fermentations that we have just described? Probably, it was owing

to some modification effected in the medium by the previous life of

the bacteria, or to the special character of the vibrios used in

impregnation. Or, again, it might have been due to the action of the

air, which, under the conditions of our second experiment, was not

absolutely eliminated, since we took no precaution against its

introduction at the moment of filling our flask, and this would tend

to facilitate the multiplication of anaërobian vibrios, just as, under

similar conditions, would have been the case if we had been dealing

with a fermentation by ordinary yeast.

Footnote 144:

In this case the liquid was composed as follows:—a saturated solution

of lactate of lime, at a temperature of 25° C. (77° F.) was prepared,

containing for every 100 c.c. (3-½ fl. oz.) 25·65 grammes (394 grains)

of the lactate, C_{6}H_{5}O_{5}_Ca_O [_new notation_,

C_{6}H_{10}_Ca_O_{6}]. This solution was rendered very clear by the

addition of one gramme of phosphate of ammonia and subsequent

filtration. For a volume of 8 litres (14 pints) of this clear,

saturated solution, we used [1 gramme = 15·43 grains]:—

Phosphate of ammonia 2 grammes.

Phosphate of 1 grammes.

potassium

Phosphate of 1 grammes.

magnesium

Sulphate of ammonia 0·5 gramme.

Footnote 145:

[1 millimetre = 0·039 inch: hence the dimensions indicated will

be—length, from 0·00039 to 0·00117, or even 0·00176 in.; diameter,

from 0·000058 to 0·000078, rarely 0.000117 in.]

Footnote 146:

The carbonaceous supply, as we remarked, had failed them, and to this

failure the absence of vital action, nutrition, and multiplication was

attributable. The liquid, however, contained butyrate of lime, a salt

possessing properties similar to those of the lactate. Why could not

this salt equally well support the life of the vibrios? The

explanation of the difficulty seems to us to lie simply in the fact

that lactic acid produces heat by its decomposition, whilst butyric

acid does not, and the vibrios seem to require heat daring the

chemical process of their nutrition.

Footnote 147:

To do this, it is sufficient first to fill the curved ends of the

stop-cocked tubes of the flasks, as well as the india-rubber tube _c

c_, which connects them, with boiling water that contains no air.

Footnote 148:

We find this fact, which we published as long ago as 1863, confirmed

in a work of H. Hoffmann’s published in 1869, under the title _Mémoire

sur les bactéries_, which has appeared in French (_Annales des

Sciences naturelles_, 5th series, vol. xi.). On this subject we may

cite an observation that has not yet been published. Aërobian bacteria

lose all power of movement when suddenly plunged into carbonic acid

gas; they recover it, however, as if they had only been suffering from

anæsthesia, as soon as they are brought into the air again.

Footnote 149:

These doubts might easily be removed by putting the matter to the test

of direct experiment.

Footnote 150:

ROBIN, _Sur la nature des fermentations_, &c. (_Journal de l’Anatomie

et de la Physiologie_, July and August, 1875, p. 386).

Footnote 151:

LIEBIG, _Sur la fermentation et la source de la force musculaire_

(_Annales de Chimie et de Physique_, 4th series, t. xxiii. p. 5,

1870.)

Footnote 152:

It is important that we should here remark that, in the fermentation

of pure solution of sugar by means of yeast, the oxygen originally

dissolved in the water, as well as that appropriated by the globules

of yeast in their contact with air, has a considerable effect on the

activity of fermentation. As a matter of fact, if we pass a strong

current of carbonic acid through the sugared water and the water in

which the yeast has been treated, the fermentation will be rendered

extremely sluggish, and the few new cells of yeast which form will

assume strange and abnormal aspects. Indeed this might have been

expected, for we have seen that yeast, when somewhat old, is incapable

of development or of causing fermentation, even in a fermentable

medium containing all the nutritive principles of yeast, if the liquid

has been deprived of air; much more should we expect this to be the

case in pure sugared water, likewise deprived of air.

Footnote 153:

DOEBEREINER, _Journal de Chimie de Schweigger_, vol. xii. p. 129, and

_Journal de Pharmacie_, vol. i. p. 342.

MITSCHERLICH, _Monatsberichte d. Kön. Preuss. Akad. d. Wissen. zu

Berlin_, and _Rapports annuels de Berzelius_, Paris, 1843, 3rd year.

On the occasion of a communication on the inversion of cane sugar, by

H. Rose, published in 1840, M. Mitscherlich observed: “The inversion

of cane sugar in alcoholic fermentation is not due to the globules of

yeast, but to a soluble matter in the water with which they mix. The

liquid obtained by straining off the ferment on a filter paper,

possesses the property of converting cane sugar into uncrystallizable

sugar.”

BERTHELOT, _Comptes rendus de l’Académie_. Meeting of May 28th, 1860.

M. Berthelot confirms the preceding experiment of Mitscherlich, and

proves, moreover, that the soluble matter of which that author speaks

may be precipitated with alcohol without losing its invertive power.

M. Béchamp has applied Mitscherlich’s observation, concerning the

soluble fermentative part of yeast, to fungoid growths, and has made

the interesting discovery that fungoid growths, like yeast, yield to

water a substance that inverts sugar. When the production of fungoid

growths is prevented by means of an antiseptic the inversion of sugar

does not take place.

We may here say a few words respecting M. Béchamp’s claim to priority

of discovery. It is a well-known fact that we were the first to

demonstrate that living ferments might be completely developed, if

their germs were placed in pure water, together with sugar, ammonia,

and phosphates. Relying on this established fact, that moulds are

capable of development in sweetened water, in which, according to M.

Béchamp, they invert the sugar, our author asserts that he has proved

that, “living organized ferments may originate in media which contain

no albuminous substances.” (See _Comptes rendus_, vol. lxxv. p. 1519.)

To be logical, M. Béchamp might say that he has proved that certain

moulds originate in pure sweetened water, without nitrogen or

phosphates or other mineral elements, for such a deduction might very

well be drawn from his work, in which we do not find the least

expression of astonishment at the possibility of moulds developing in

pure water, containing nothing but sugar without other mineral or

organic principles.

M. Béchamp’s first Note on the inversion of sugar was published in

1855. In it we find nothing relating to the influence of moulds. His

second, in which that influence is noticed, was published in January,

1858, that is, subsequently to our work on lactic fermentation, which

appeared in November, 1857. In that work we established, for the first

time, that the lactic ferment is a living organized being, that

albuminous substances have no share in the production of fermentation,

and that they only serve as the food of the ferment. M. Béchamp’s Note

was even subsequent to our first work on alcoholic fermentation, which

appeared on December 21st, 1857. It is since the appearance of these

two works of ours that the preponderating influence of the life of

microscopic organisms, in the phenomena of fermentation, has been

better understood. Immediately after their appearance M. Béchamp, who,

from 1855, had made no observation on the action of fungoid growths on

sugar, although he had remarked their presence, modified his former

conclusions. (_Comptes rendus_, January 4th, 1858.)

Footnote 154:

“There are two classes of ferments; the first, of which the yeast of

beer may be taken as the type, perpetuate and renew themselves if they

can find in the liquid in which they produce fermentation food enough

for their wants; the second, of which diastase is the type, always

sacrifice themselves in the exercise of their activity.” (DUMAS,

_Comptes rendus de l’Académie_, t. lxxv. p. 277, 1872.)

Footnote 155:

FREMY, _Comptes rendus de l’Académie_, vol. lviii. p. 1065, 1864.

Footnote 156:

See our Memoir of 1860 (_Annales de Chimie et de Physique_, vol.

lviii.) p. 61, and following, and especially pp. 69 and 70, where the

details of the experiment will be found.

Footnote 157:

PASTEUR, _Comptes rendus de l’Académie des Sciences_, vol. lxxiii. p.

1419, 1871.

Footnote 158:

In his Memoir of 1870, Liebig has made a remarkable admission: “My

late friend Pelouze,” he says, “had communicated to me, nine years

ago, certain results of M. Pasteur’s researches on fermentation. I

told him that, just then, I was not disposed to alter my opinion on

the cause of fermentation, and that if it were possible by means of

ammonia to produce or multiply the yeast in fermenting liquors,

industry would soon avail itself of the fact, and that I would wait to

see if it did so; up to the present time, however, there has not been

the least change in the manufacture of yeast.” We do not know what M.

Pelouze’s reply was; but it is not difficult to conceive so sagacious

an observer remarking to his illustrious friend, that the possibility

of deriving pecuniary advantage from the wide application of a new

scientific fact had never been regarded as the criterion of the

exactness of that fact. We could prove, moreover, by the undoubted

testimony of very distinguished practical men, notably by that of M.

Pezeyre, director of distilleries, that upon this point also Liebig

was mistaken.

Footnote 159:

PASTEUR, _Comptes rendus de l’Académie_, vol. lxxviii. pp. 213-216.

Footnote 160:

TRÉCUL, _Comptes rendus de l’Académie_, vol. lxxviii. pp. 217, 218.

Footnote 161:

See on this subject the verbal observations which we addressed to the

Academy of Sciences, at its meetings of April 10th and 24th, 1876.

CHAPTER VII.

New Process for the Manufacture of Beer.

The principles established in the course of this work implicitly involve

the conditions of a new process of manufacture, the essential feature of

which would consist in the production of a beer of excellent keeping

qualities, we might even say a beer that could not undergo alteration.

It will not be difficult now to make ourselves clear on the point.

We have shown in the first place, that the changes which take place in

the ferment, the wort, and the beer itself, are due to the presence of

microscopic organisms of an entirely different character to that of the

ferment-cells properly so called, which organisms, by simultaneously

giving rise, in the course of their multiplication in the wort, ferment,

or beer, to other products, make the materials difficult to keep or

effect their deterioration. Again, we have seen that these

change-producing organisms, the ferments of disease, never arise

spontaneously in the wort or beer, but, whenever they make their

appearance in these fluids, have been imported from without, either in

company with the yeast, or from accession of atmospheric dust, or from

contact with the vessels, or from the materials themselves which the

brewer uses in his manufacture. Moreover, we know that these

disease-ferments, or their germs, are destroyed when the wort has its

temperature raised to the boiling-point. And, following up the

inferences from such facts, we have seen that wort exposed to pure air,

after having been heated to boiling, remains absolutely free of any sort

of fermentation.

Inasmuch then as the disease-germs of wort and beer are destroyed in the

copper in which the wort is boiled, and as, by employing a perfectly

pure ferment, we guard against the admission of any foreign ferment of

an evil character, we have it in our power to prepare a beer which shall

be incapable of undergoing any pernicious fermentation whatsoever. This

we shall have effected provided we can take the wort as run off from the

coppers, cool and manipulate it out of contact with ordinary air or in

contact with pure air, charge it with a pure yeast, and, lastly, store

the beer when the fermentation is complete in vessels thoroughly

purified from disease-ferments.[162]

§ I.—Preliminary Experiments.

We may readily satisfy ourselves as to the truth of these inferences.

The following is one of the earliest experiments which I devised with a

view to establish their certainty. Into a flask with a straight neck of

about a litre (1-3/4 pints) capacity, a quantity of wort from a brewery

was introduced and there raised to boiling, and whilst the vapour still

issued from the neck of the flask, connection was made with a two-necked

flask in which the cultivation of pure yeast had been carried on. The

cork and glass tube used for this purpose had previously been treated

with boiling water.

[Illustration: Fig. 75.]

When the wort had cooled down in the flask and matters were arranged as

represented in Fig. 75, I raised the two-necked flask so as to cause a

little of the liquid and yeast to flow into the wort. Thereupon

fermentation was set up, and the resulting carbonic acid gas made its

escape by the drawn-out end of the doubled-necked flask. The entire

arrangement with its supporting stand remained in this connection for

eighteen months, sometimes on a stove, sometimes in the laboratory,

exposed to all the variations of external temperature. At the end of

that time I tasted the beer in the flask; it was perfectly sound, and

the ferment, submitted to the microscope, showed not the slightest trace

of any foreign ferments: and, doubtless, the experiment might have been

protracted over any number of years with the same result.

The only change that occurs in course of time is the appearance in the

neck of the flask at the surface of the beer of a deposit of small

prominences resembling a crystallization, but which really consists of

those forms of ferment to which in Chapter V. I attached the name of

_aërobian ferment_. The beer, after being transferred to a bottle that

had been washed with hot water, was kept for several months in the heat

of summer, without exhibiting the slightest trace of deterioration.

[Illustration: Fig. 76.]

[Illustration: Fig. 77.]

The essential conditions of the preceding experiment can readily be

realized on the large scale. For this purpose we may employ the

apparatus in the above sketch (Figs. 76 and 77) constructed of tin or

tinned copper. As appears from the sketch, this consists of a

cylindrical tub resting on a support, and closed at the top by a cover,

whose lower edge fits into a gutter containing water. The wort prepared

in the copper is led into the cylinder, a process which does not

materially lower its temperature. Now we know that wort in breweries

which has been cooled in contact with the air, and so got charged with

disease-germs, will, nevertheless, recover its faculty of keeping for

any length of time in pure air, if we again raise its temperature to 80°

C. (176° F.) or even 70° or 75° C. (158°, 167° F.) Having filled the tub

with the hot wort and put on the lid, we then connect, by means of a

caoutchouc tube _c d_, the metal tube _a c_ (which opens into one of the

tubulures projecting above the lid) with the system of tubes _d e_, _f

g_, of which _d e_ is fixed to the cylinder; _e f_ is a caoutchouc

junction connecting _e_ with the bent glass tube _g_. We then dash over

the apparatus, lid, tubulures, and their corks a quantity of boiling

water. This collects in the gutter in which the lid rests, and any

excess overflows into a second gutter outside the first, where, however,

it cannot remain, but passes away by means of a ring of small holes

between the base of the outer trough _i i_ and the cylinder. The

overflow is collected in a third trough at the bottom, whence it can be

removed by a pipe M. T is a bent thermometer to indicate the temperature

of the wort; its bulb is protected by an inlet socket _d d_, pierced

with holes; _r_ is a stopcock for discharging the water in the gutter,

which serves as a hydraulic junction between the cylinder and its lid;

R, V, are stopcocks, or openings for the discharge of the liquid in the

cylinder and its deposit. The next process is to cool the vessel, which

may be done either by leaving it to itself, or by introducing a current

of cold water through the tubulure E, soldered on to the lid. This

tubulure is of the form of an inverted funnel, and is pierced at the

bottom with a close row of holes, through which the cold water issues in

a sheet over the surface of the cover. In whichever way the cooling is

effected, the external air continues all the time to enter the vessel

beneath the lid by way of the long, narrow passage _g f e d c a_, and

must necessarily get purified by depositing in its course all

fungoid-germs, just as happened in the case of the two-necked flask of

air experiments. This, however, may be still further secured by

introducing a small plug of cotton wool, or asbestos, into the end of

the tube _g_.

The experiments which we have carried out with this apparatus have

proved that, by adopting such an arrangement, beer, a liquid peculiarly

liable to change, may be kept as long as we wish, for weeks or months,

in contact with air, since the tube _g_ is open, without evincing the

least symptom of disease. It matters little whether the leaves and

strobiles of the hops are introduced with the hot wort or strained off;

the result is the same. On the other hand, a leak in the apparatus from

which the wort gets mixed with ordinary water from outside during

cooling, will speedily effect a change in the wort and cause it to swarm

with vibrios, or butyric ferment, lactic ferment, and other germs of

disease, whilst its taste will be rendered extremely nauseous. It can

only be through one’s own fault, that is, from want of skill in carrying

out the operation, that any change can be brought about by the water in

the gutter not being kept out of the fermenting vessel. That water may

even become putrid without the organisms contained in it being able to

reach the wort in the fermenting vessel. The apparatus may be of any

size whatever; we have worked with vessels containing 12 hectolitres

with as much ease and certainty as when we used an apparatus of 1

hectolitre (22 gallons).

It is easy to carry out the process of cooling in the presence of

carbonic acid gas if we fit a bent tube, similar to _a c d e f g_, to

the second tubulure D. Through this tube, or its companion, the gas can

be passed as it issues from an apparatus in which it is generated, or

from a gasometer filled with it, or from a vessel of beer undergoing

fermentation.

However, there is no necessity that the cooling should take place in the

fermenting vessel. It may be effected separately, in vessels of greater

or less depth, in spiral coils surrounded with cold water, or in any

kind of refrigerator, provided always that the conditions of purity are

satisfied, and that the flow of the cooled wort takes place under the

same conditions. Jets of steam, which are already extensively used for

the cleansing of pipes in breweries, may be employed here with great

advantage.

The pitching may be effected in various ways. A two-necked flask of a

capacity of from 200 to 300 cc. (about 7 to 10 fl. ozs.), in which not

more than 100 cc. (3-½ fl. ozs.) of wort has been fermented, will be

sufficient for an apparatus of 1 hectolitre (22 gallons), although the

flask may not contain more than 1 or 2 decigrammes (1-½ to 3 grains) of

yeast. In the manufacture of beer, as at present conducted, the

employment of so minute a quantity of yeast would lead to most

disastrous results. The fermentation would unfailingly become lactic and

butyric, since the foreign germs with which commercial worts and yeasts

are always contaminated would have ample time to develop during the

first twenty-four or forty-eight hours, whilst the small quantity of

yeast used in the pitching could scarcely do more than begin to develop

during that time. It is simply with the object of avoiding these

secondary fermentations that the brewer uses large quantities of yeast

for pitching.

After the wort and yeast have been _pulled up_,[163] a process which

every practical brewer adopts after pitching, every part of the liquid

is occupied by a multitude of yeast-cells, which seize upon the oxygen

in solution, germinate with activity, turn to their own account the

food-supplies most easily assimilated, and prevent the growth of the

germs of disease-ferments. In the new process which we are now

explaining, things happen quite differently. Our wort is pure, and our

yeast is pure, and if only a single cell of yeast were introduced into

the wort, the vital activity of this would be sufficient to bring about

alcoholic fermentation, and to transform the wort into beer, without our

having the least reason to apprehend the simultaneous development of any

other organisms whatsoever. In short, the new process enables us to

pitch with as small a quantity of yeast as we like. It is, nevertheless,

inexpedient to employ too minute a quantity, since by doing so we should

retard the commencement of fermentation.

[Illustration: Fig. 78.]

[Illustration: Fig. 79.]

In the case of an apparatus of 5 hectolitres (110 gallons) or double

that capacity, the pitching may be accomplished by means of flasks

holding from 4 to 9 litres (from 7 to 10 or 11 pints), (Fig. 79), or

copper cans, tinned inside, holding from 10 to 15 litres (2-1/4 to 3-1/4

gallons), and provided at the upper conical end with glass tubes (Fig.

78). The vessel must be half or two-thirds filled with wort. For this

purpose it will be well always to employ wort that has been preserved in

bottles by Appert’s process. We must use a stopper provided with tubes,

as represented in Fig. 79: _a b_ is a glass stopper which closes the

india-rubber tube _b c_; _m n p_ is a fine glass tube, or, better still,

made of copper.

The tap R being closed, a long india-rubber tube is attached to the

extremity of the curved tube, and the flask is completely immersed in a

hot-water bath; the india-rubber tube projects from the bath and keeps

the interior of the vessel in communication with the external

atmosphere. If the tube _m n p_ is of copper, we may avail ourselves of

its flexibility and bend it upwards, so as to place its open extremity

outside the bath. The water in the bath is then gradually raised to a

temperature of 100° C. (212° F.), at which it is kept for a quarter or

half an hour. In the case of copper cans, it is more convenient to place

them over a gas-heater. They may be treated in the same manner as the

flasks with curved necks. Vessels prepared in this manner may remain in

a laboratory, or in any part of a brewery, for an indefinite time,

without the wort in them undergoing the least change. It gradually

darkens in colour through a direct oxidation of a purely chemical

nature, but no tendency to disease will manifest itself.

Some days before we require to pitch an apparatus of several

hectolitres, we impregnate one of these flasks or cans.[164] For this

purpose we pass the flame of a spirit lamp over the tubes _c b a_ and _m

n p_, to destroy the particles of dust that might pass inside at the

moment when the stopper _a b_ is taken out, and then by means of a long,

straight glass tube we take some of the liquid from a flask or vessel

containing pure beer in a state of fermentation, and let a few drops of

this, with the yeast that it holds in suspension, fall into the flask or

can; the stopper _a b_ is once more passed through the flame and then

replaced; generally in the course of one or two days the yeast develops

in the flask sufficiently for the fermentation to show itself. We may

shorten the operation still further by emptying into the can the

contents of one of those double-necked flasks. To do this, we have

simply to attach the straight tube of the flask to the india-rubber _b

c_, and pour the liquid in. In a similar manner we introduce, through

one of the tubulures surmounting the lid of the fermenting apparatus,

the contents of the flasks or cans, either whilst they are still in

active fermentation, or after fermentation is over. For this purpose,

the tap R is connected by means of an india-rubber tube (Fig. 79), with

a tube passing through a cork fixed in one of the tubulures of the large

apparatus. All this may be done in considerably less time than we have

taken to describe it; and the operation may be performed accurately and

safely by any one who has witnessed it a few times, even though he may

not be skilled in chemical manipulations, especially if he takes care to

bear in mind the very simple principles which we have explained.

Since certain parts of the apparatus—the outer opening of the tap, or

the india-rubber tubing, for example—may contract particles of dust from

the air, those parts, before being used, must be boiled in water, or

washed with boiling water, or passed through the flame of a spirit lamp,

to destroy the germs mixed with the particles of dust that settle upon

them.

The method of cooling the wort in contact with carbonic acid prevents

access of oxygen to the latter up to the time of pitching, so that the

development of the yeast takes place apart from the influence of oxygen.

Now, we know that these conditions necessitate the employment of a very

young yeast—a yeast that is in course of active germination, such as may

be taken from an incipient preparatory fermentation. Nevertheless, even

with this, the development of the yeast under such conditions is

extremely slow, and the fermentation takes from fifteen to twenty-five

days; whilst, under the same circumstances, but with an aerated wort, it

would be finished in from eight to twelve days. This is a considerable

drawback, but, perhaps, a still more serious inconvenience is that the

beer takes much longer to clarify, and does so with greater difficulty

than those beers which are made with aerated worts. At the same time,

this is largely compensated by the superior quality of the beer, which

is stronger and has greater fulness on the palate, whilst the aroma of

the hops is preserved to an extent never found in beers brewed by the

ordinary process. Besides this, the yeast deposited at the bottom of the

fermenting vessel is much less active, and, being of an older type, is

revived with greater difficulty than that which forms in aerated worts.

This, which might be considered a disadvantage, if we had to employ the

yeast afterwards for pitching, has the great advantage of giving a beer

which, when racked, undergoes its secondary fermentation only slowly,

and with difficulty.[165] A beer of this kind is better adapted than

ordinary beer to stand a long journey without developing great pressure

inside the casks, and, if bottled, it will contain very little deposit,

and will not froth violently when uncorked. The reason is, that a yeast

is the more active, the more ready to multiply rapidly, and to work

vigorously the more highly aerated the wort was in which it was grown.

On the other hand, a yeast formed apart from air readily gets exhausted,

and may even perish in the liquid in which it ferments, when that is

kept out of contact with air; in other words, the vital action of yeast

is more restricted when it has not been subjected to the action of

oxygen during its formation.

If a great depth of wort, the surface of which alone is in contact with

atmospheric air, is left to cool down, it will act in almost exactly the

same manner as that which is cooled under an atmosphere of carbonic acid

gas, because the oxygen of the air is very slow in pervading wort that

is undisturbed. The gas will be taken into solution by the upper layer

only, whilst the bulk of the liquid will remain unaffected by it. In

some experiments which we conducted in a vessel which contained wort to

a depth of 70 centimetres (27·5 inches), and which was provided with a

tap that enabled us to draw off some of the liquid every day, until we

had reduced the depth to 35 centimetres, we found, at the end of eight

days, that there was not a trace of oxygen in solution at the latter

depth. It is even probable that, considering the slow diffusion of the

oxygen, on the one hand, and the combination that may take place between

it and certain components of the malt, on the other hand, it would take

a long time for all the wort, if undisturbed and of a certain depth, to

become saturated with oxygen. In the vessel represented in Figs. 76 and

77 there is a considerable depth of wort to cool down. Nevertheless, the

mere fact of the possibility of an aeration from the surface, whilst the

wort is cooling down in contact with pure air, is enough to account for

a certain effect that is produced on the yeast, later on, for the more

youthful appearance of the yeast of the deposit, compared with that

which we find in the case of wort cooled in the presence of carbonic

acid gas. The difference between the results is particularly striking

if, in both cases, we follow up microscopically the development of the

yeast during the first few days succeeding the pitching.

The influence of the air on fermentation is considerable. In the

ordinary process of brewing, fermentations would be almost impossible,

and in every case most defective, if the wort, before being run into the

fermenting vessels, were not aerated by its passage over the “coolers,”

where the aeration is more or less effective, according as the liquid is

more or less shallow. Worts and yeasts being impure, that is containing

the germs of foreign ferments, those germs would have time to germinate

in the fermenting vessels during the delay that the want of aeration in

the wort would cause in the development of the yeast. We are aware that

several inventions have been proposed to do away with the coolers, and

we feel convinced that the object has been to remedy irregularities in

fermentation. Considering the facts which we have published[166] on the

development of yeast in the presence of air, and its inactivity in

non-aerated media, such inventions ought to be supplemented by some

means of further aeration for the prevention of the mischief that they

must otherwise cause. In the existing process of brewing, the employment

of coolers is a necessity.

The influence of the air on the vital action of the yeast may be proved

in ways innumerable. The following is an experiment which we have often

carried out with surprising results. A fermentation is going on; we draw

off the liquid as rapidly as we please, and pour it back again into the

vessel immediately. Within an hour we find a marked increase in the

fermentation, evidenced by the liberation of a greater quantity of

carbonic acid gas. This experiment may be performed with especial ease

if we use the fermenting apparatus that we have described, for, by

fitting a gas measurer to the escape tube _a b c d e f g_, the number of

litres produced before the drawing off of the liquid may be compared

with those obtained after. The least physical change in the running of

the fermenting liquid whilst it is being drawn off, modifies the effect

in question; such as change in the diameter of the stream, the height

from which it falls, its greater or less scattering in falling, all

influence it. Again, as might be expected from such results,

corresponding modifications take place in the cells of yeast which come

under the influence of the air. They become firmer in aspect and

outline, their plasma becomes fuller, assumes a younger and more

transparent aspect, and the vacuoles disappear. The molecular

granulations, too, are less apparent. At a certain focus they disappear;

at another they reappear, not as black spots, however, but as brilliant

points so small as to be scarcely perceptible. If germination has been

suspended it is resumed; in short, everything tends to prove—and having

the yeast actually under our eyes we cannot doubt the fact—that the life

of the cells is more decided, and the work of nutrition more active

after they have been brought into contact with the oxygen of the air,

and have absorbed a greater or less quantity of that gas.

Under the ordinary conditions of brewing, the atmospheric air is present

in very varying quantities, whether introduced by the wort which holds

more or less in solution, or by diffusion over the surface of the

vessels, so that the same cells of yeast live by turns without air and

with air. At first they absorb all the oxygen held in solution, and

multiply under the influence of this absorption. Afterwards, when the

supply has been exhausted, and various assimilations have resulted from

it, they are deprived of it. Their life continues apart from oxygen, and

if the vessel were closed, fermentation would be accomplished under

these conditions, although more slowly. The vessel being open, a small

quantity of air diffuses continuously through the layer of carbonic acid

gas on the surface, and supports the vitality of the cells.

It is interesting to observe that, in the working of breweries, there

are several empirical practices the explanation of which is to be found

wholly in the fact that the aeration of wort or beer exercises a great

influence on fermentation. In many breweries we have seen the pitching

performed in the following manner: the brewer, having mixed his yeast in

many times its volume of wort, pours all the thick liquid from a height

from one bucket into another, and from that back again into the first,

and so on a great many times, until the two buckets are filled with the

froth enclosing air. In certain London breweries we have seen a bucket

suspended by a pulley over the fermenting tun, which is 3 or 4 metres

(10 or 12 feet) in depth; this the brewer, by means of a cord, can lower

into the tun and pull up again at will, giving it a kind of see-saw

movement which agitates the surface of the liquid and aerates it. The

use of the fermenting tun itself and the racking of the wort from that

tun into casks have the effect of aerating the beer and the yeast, and

imparting to the latter a greater vigour and activity.

The resumption of fermentation in cask, after the beer has been run out

of the tuns in “low” fermentation breweries is, in our opinion,

principally due to the aeration of the beer at the moment when it is

racked. The brewer ought to bear in mind that, during racking, every

detail is of importance; it makes a great difference whether when the

beer is run into the casks it falls from a height or is conducted by a

tube to the bottom of the casks, whether it passes directly into the

casks, or is poured into them from buckets, and whether it runs in a

stream of small or large diameter, since these different methods cause

the introduction of corresponding different quantities of air into the

beer.

We have devised a simple arrangement for bringing the fermenting liquid

into contact with various proportions of atmospheric air. Appended is a

sketch of this apparatus (Fig. 80). Instead of one tube serving alike

for the entrance and escape of gas, there are two similar ones, each of

which opens into one of the tubulures on the cover. Round the other end

of one of the tubes is fitted a kind of muff or bag, composed of a

cylindrical cage of metallic gauze, over which a layer of well-combed

cotton wool is placed, the whole being covered with a muslin bag. The

object of this arrangement is to act as an air-filter for retaining the

particles of dust. When fermentation has commenced in the apparatus, we

have simply to press momentarily the india-rubber connection between the

tube from the lid and the tube with the bag. This will at once cause a

regular stream of carbonic acid to issue from the end of the uncovered

tube, whilst the air will enter by the filtering tube to take its place;

and this arrangement will be maintained throughout the whole course of

the fermentation, even if we omit the precaution of increasing the power

of the syphon by making the tube for the escape of this gas longer than

the other one.[167]

[Illustration: Fig. 80.]

It will be readily understood how, whether by this last method, or by

the diameter of the tubes, we may vary the conditions of this

circulation of air in the apparatus, on the surface of the beer.

§ II.—Method of Estimating the Oxygen held in Solution in Wort.

The use of carbonic acid gas and the cooling of the wort, in contact

with that gas or in contact with very limited quantities of pure air,

are by no means necessary to the application of the new process. There

is only one thing that is absolutely essential—which is, the _purity_ of

the gases in the presence of which the wort is cooled and treated. If,

therefore, it is well to aerate our wort, either before or during

fermentation, this may be done, on the sole condition that the air

employed does not introduce any germs of disease that are likely to

develop in the beer during fermentation or afterwards. The question of

aerating the wort is not, however, so simple a matter as it seems at

first sight. A very simple observation will show that wort cannot be

safely oxygenated by exposure, without precaution, to the air, even

leaving out of account the germs of disease which that air may contain.

It is easy to show that finished wort has a decided flavour and aroma of

hops, as well as a sweet taste, and that it leaves a certain pleasant,

bitter after-taste on the palate. When we taste it in this condition we

cannot help thinking that a liquor of the kind, after fermentation,

ought to constitute a very valuable beverage, as wholesome as it is

pleasant. Now all this pleasant and refreshing sensation that the wort

leaves on the palate, which is due as much to the aroma as to the

bitterness of the hop, disappears absolutely, we may say, if the wort is

left exposed to contact with air for a sufficient time, and that whether

the air be warm or cold. We may easily perform the experiment in one of

our two-necked flasks, in which we can preserve the wort, in contact

with pure air, without any fear of change. The oxygen of the air enters

into combination with the substances that the hop introduces into the

wort, and the wort, in consequence of this oxidation, gradually becomes

transformed into a saccharine decoction, without odour, in which even

the bitter flavour is destroyed or hidden. In other words, the wort

grows weak and flat, in just the same way that beer and wine do, as well

as all the various natural or artificial worts which serve to produce

them. Thus it is evident that considerable care is necessary in

subjecting wort and beer, whether in course of manufacture or finished,

to the action of atmospheric air. If, therefore, it is a good thing to

supply wort with oxygen, as we have already pointed out, in order to

facilitate the fermentation and nourish the yeast, it is, on the other

hand, important that the quantity supplied to it should not be too

great, otherwise we may injure the quality of the beer, and particularly

its fulness on the palate, that is its apparent strength, which has very

little to do with the proportion of alcohol in it. The strength of a

beer is intimately connected with those substances introduced by the

hops into the wort and thus into the beer, to which we previously

alluded, and of which too little is known; their properties and the

palatableness resulting from them are very readily affected by the

oxygen of the air.[168]

We have, therefore, to ascertain the measure in which air occurs during

the process of brewing, and whether, in the actual process, there may

not be too great a proportion of active oxygen present. The study of

this subject requires that we should know what quantities of oxygen may

be held in solution in the wort or absorbed by direct combination.

Fortunately this has been rendered a comparatively easy matter by a

rapid method of estimating the oxygen held in solution in liquids of

various kinds, devised by M. Schützenberger in 1872. As soon as this

method was made known, we requested M. Raulin, who was attached to our

laboratory as assistant-director, to apply it to the determination of

oxygen in wort. This he did with his accustomed skill, devising certain

alterations of details which rendered the method at the same time surer

and more expeditious.

The principal feature in M. Schützenberger’s process consists in the

employment of a salt, the properties of which that chemist was the first

to recognize; he has named it _hydrosulphite of soda_, and it is

obtained by the action of zinc filings on a solution of bisulphite of

soda, out of contact with air.

Hydrosulphite of soda S^2O^2,NaO,HO, which is isomeric with hyposulphite

of soda, only differs from the bisulphite by two equivalents of

oxygen.[169] When brought into contact with free oxygen, it absorbs that

gas instantaneously and becomes converted into bisulphite; similarly

when mixed with water, it immediately absorbs the oxygen held in

solution. Again there are colouring matters, such as M. Coupier’s

soluble aniline blue, that are instantaneously decolourized by

hydrosulphite of soda, whilst they resist the action of the bisulphite.

If, taking care to avoid the access of air, we add hydrosulphite of soda

to a certain volume of water—a litre, for example—that has been deprived

of air and faintly coloured with Coupier’s blue, we shall see that a few

drops will be sufficient to effect the decoloration. If, on the

contrary, the water is aerated, the decoloration will not be effected

before a sufficient quantity of the hydrosulphite has been added to

absorb the oxygen in solution, and the volume of the reagent required is

in proportion to the quantity of oxygen in solution in the water. To

render the process sensitive, we must dilute the hydrosulphite to such

an extent that 10 c.c., for example, may correspond very nearly with 1

c.c. of oxygen. If the reagent would keep we should only have to

determine directly, once for all, the volume of oxygen that a known

volume of the liquid could absorb; but, in consequence of its extreme

liability to change through contact with air, it is necessary to titrate

the liquid every time before using it. This is easily done in the

following manner:—

According to the observations of Messrs. Schützenberger and Lalande, the

hydrosulphite decolourizes an ammoniacal solution of sulphate of copper,

reducing the copper to a lower state of oxidation; the sulphite and

bisulphite having no action as long as there is an excess of ammonia. We

prepare a strongly ammoniacal solution of sulphate of copper, containing

such a quantity of copper that 10 c.c. of the liquid will correspond, as

far as action on the hydrosulphite is concerned, with 1 c.c. of oxygen.

Calculation by equivalents gives us the correct value verified by direct

experiment.[170]

The object of the modification which M. Raulin has introduced, is to

avoid the loss of time thus occasioned by the changes which take place

in the titrated liquids by long keeping, as well as certain errors which

may arise from the acidity of the wort. On this latter point M.

Schützenberger has remarked that the quantities of hydrosulphite of soda

corresponding with one and the same volume of oxygen vary with the

acidity of the liquid operated upon, a phenomenon which that skilful

chemist explains by the formation of oxygenated water, of varying

stability in media of different acidity.

Instead of determining the strength of the titrated solution of

hydrosulphite before each operation, we take the solution as it happens

to be, and determine its strength by causing it to act on a known volume

of pure water saturated with oxygen at a certain temperature. The tables

of solubility of oxygen in water give the exact volume of oxygen on

which the measured volume of hydrosulphite used has acted. According to

Bunsen, about one minute’s brisk shaking in a closed bottle, with excess

of air, will be sufficient to effect the maximum saturation of the water

at the temperature at which we operate.

For experiments on wort we require:—

1. A 2-litre (3-½ pints) flask, A, containing _saturated_ hydrosulphite

of soda,[171] of such strength that 2·5 c.c. will be sufficient to

absorb almost all the oxygen in 50 c.c. of water saturated with air at

the ordinary temperature (that is, 1 volume of hyposulphite must equal

20 volumes of water).

2. A 2-litre flask, B, containing a solution of indigo-carmine, 50 c.c.

of which will be decolourized by about 20 c.c. of the hydrosulphite.

This solution contains about 20 grammes (30·7 grains) of commercial

indigo-carmine per litre (1·76 pints).

3. An apparatus, C, for the production of hydrogen.

4. An experimental apparatus composed of a burette, D, graduated in

tenths of a cubic centimetre, and a three-necked Wollf’s bottle, E.

5. A flask, F, holding about 100 c.c. provided with a straight tube

divided into tenths of a cubic centimetre, and containing a solution of

ammonia of such strength that about ten drops of it will neutralize the

acidity in 50 c.c. (1·76 fl. oz.) of wort.

To perform the operation we shake about 150 c.c. (5·3 fl. oz.) of

distilled water, at the existing temperature, in a 1-litre flask for a

minute or so; this saturates it with air, and we must at the same time

note the temperature. To be extremely precise, we should note also the

barometrical pressure.

[Illustration: Fig. 81.]

Into the bottle E we introduce about 50 c.c. of the indigo solution, and

200 c.c. of water at about 60° C. (140° F.), and fill the tube _e_ to

the point _b_ with water saturated with air; we then expel the air from

the bottle E by a current of hydrogen. The blue colour of the liquid in

the bottle is then very carefully brought to a yellow tint, by running

in, drop by drop, the hydrosulphite with which the burette D is filled.

We next pour 50 c.c. of distilled water saturated with air into the

funnel _a_, and pass it into the flask; the blue colour reappears. We

must then bring back the colour to exactly the same tint of yellow. Let

_n_ represent the number of divisions on the burette denoting the volume

of hydrosulphite employed for this purpose.

We repeat this last operation immediately, taking 50 c.c. of the wort,

the oxygen of which we wish to determine, having first introduced into

the funnel _a_ a sufficient number of drops of the ammoniacal solution

to neutralize the acidity of the wort. Let _n´_ represent the number of

divisions of hydrosulphite employed to restore the yellow tint in the

case of the wort.

We once more perform the experiment with 50 c.c. of saturated water; let

_n´´_ be the number found.[172]

The ratio which the quantity of oxygen held in solution in the wort

bears to the quantity of oxygen contained in the same volume of water

saturated with air, at the temperature _t_, and under the pressure H,

will be

[Illustration: Formula 3: _n´_/((_n_ + _n´´_)/2).]

it will be sufficient in most cases to bear in mind this ratio.

When we want to deduce the absolute quantity of oxygen held in solution

in a volume V of the wort, we have merely to multiply this ratio by the

quantity of oxygen contained in the same volume of water saturated with

air, at the temperature _t_ and under the pressure H, a very simple

problem if we know the coefficients of the solubility of oxygen in water

at different temperatures. These coefficients are given for ordinary

temperatures in the following table, which was compiled by Bunsen. We

have restricted the numbers to three places of decimals:—

Temperatures. Coefficients.

0° C. (32° F.) 0·040

1° C. (33·8° F.) 0·040

2° C. (35·6° F.) 0·039

3° C. (37·4° F.) 0·038

4° C. (39·2° F.) 0·037

5° C. (41·0° F.) 0 036

6° C. (42·8° F.) 0·035

7° C. (44·6° F.) 0·035

8° C. (46·4° F.) 0·034

9° C. (48·2° F.) 0·033

10° C. (50·0° F.) 0·033

11° C. (51·8° F.) 0·032

12° C. (53·6° F.) 0 031

13° C. (55 4° F.) 0·031

14° C. (57·2° F.) 0·030

15° C. (59·0° F.) 0·030

16° C. (60·8° F.) 0·029

17° C. (62·4° F.) 0·029

18° C. (64·4° F.) 0·029

19° C. (66·2° F.) 0·028

20° C. (68·0° F.) 0·028

The primary condition which enables us to rely on the exactness of this

method is the fact which we have mentioned above, that a liquid if

shaken up with air for one minute will become perfectly saturated with

oxygen. Substantially this is the case. In estimating the oxygen in

different parts of a liquid treated thus, we have invariably obtained

the same figures to within about 1/50th.

It is true that the variable quantity of the oxygen held in solution in

the liquid contained in the part of the tube _eb_, as well as the oxygen

absorbed during the treatment of the liquid in contact with air,

constitute causes of error. Experience, however, proves that these

causes of error are insignificant, as long as we have to deal with a

liquid the aeration of which is not very far removed from the point of

saturation, and whose solubility-coefficient for oxygen is not widely

different from that of water for the same gas. Under such conditions we

have always found a constant ratio, to within about 1/40th between the

same liquid and air-saturated distilled water, placed under the same

circumstances.

If, on the other hand, we have to deal with a liquid which holds but a

minute quantity of oxygen in solution, the causes of error mentioned may

very seriously affect our results, and it will be absolutely necessary

to avoid them. The liquid experimented on must be treated out of contact

with air, by aspirating it directly from the vessel that contains it

into the pipette H, which is graduated for 50 c.c., and causing it to

pass thence into the flask E, by substituting the pipette for the funnel

_a_. Finally, before arranging the pipette, we cause a small quantity of

the liquid in the flask, which has been previously brought to the exact

yellow tint, to pass, by pressure, through the tube _eb_, so as to avoid

the cause of error that is likely to result from the air held in

solution in the liquid of that tube.

The liquid, the oxygen of which has to be determined, may also be passed

directly from the vessel containing it into the flask E; the rest of the

operation being performed as already described.

It was by this method that the oxygen held in saturate solution in wort

was determined. The following are the principal results obtained by M.

Raulin:—

1. At different pressures the ratio between the quantities of oxygen

held in solution in water and in wort is, all other conditions being

similar, constant. This ratio has been found equal to 1·20 in the case

of wort and water saturated with air at the ordinary pressure, and 1·24

in the case of wort and water saturated with pure oxygen.

2. The ratio between the coefficient of the solubility of oxygen in

water and that of its solubility in wort is very nearly constant at

different temperatures, increasing, however, slightly as the temperature

diminishes.

This ratio has been found to be—

Temperatures.

26° C. (78·8° F.) 1·20

19·5° C. (67·1° F.) 1·25

4° C. (39·2° F.) 1·37

Another wort gave the following results:—

Temperatures.

9° C. (48·2° F.) 1·15

21° C. (69·8° F.) 1·10

25° C. (77·0° F.) 1·07

3. The ratio between the quantities of oxygen held in solution in water

and those held in solution in wort increases with the concentration of

the wort. By evaporating the same wort to different degrees of

concentration, and afterwards saturating it with air, at the same

temperature, we obtained the following figures for the ratio in

question:—

Weak wort 1·06

The same evaporated to half 1·15

“ ” “ 2/5 1·27

” “ ” 3/10 1·45

“ ” “ 1/6 1·96

4. Worts of different origin, but of the same density and temperature,

when saturated with oxygen, always contain very nearly the same quantity

of that gas.

Two portions of the same wort, shaken up with air, one being hot the

other cold, then left to themselves for some time, and afterwards

saturated with air, at the same temperature, gave the figures 1·22 for

the ratio between the oxygen in the water and that in the wort.

Different worts of the same density, saturated at a temperature of 15°

C. (59° F.), gave the following ratios:—

Wort kept in a bottle with air for 19 1·140

months

Wort recently prepared 1·142

Wort kept in a bottle without air for 20 1·142

months, aerated for 18 days

Wort evaporated to dryness and made up 1·126

with water

5. The solubility of oxygen in wort differs very little from the

solubility of oxygen in sweetened water of the same density.

An experiment was made with a solution of sugar on the one hand, and

with wort more or less diluted with water on the other hand, at the same

temperature of 11° C. (51·8° F.). The following figures were obtained

for the ratios of solubility:—

Solution Wort.

of Sugar.

Marking 17·9° 1·278 1·27

Balling[173]

“ 14·0° ” 1·190 1·15

“ 7·0° ” 1·092 1·06

6. From the preceding results it is easy to deduce a general formula

which shall give the coefficient of solubility of oxygen in any wort,

marking B° by _Balling_, and at temperature _t_°.

From the figures of (2) it follows that above and below the temperature

of 15° C. (59° F.), the ratio which the coefficient of solubility of

oxygen in water bears to that of the solubility of the same gas in wort

varies about 0·006 for each degree of the thermometer. From the figures

of (3) it follows that the same ratio varies about 0·002 for each degree

of _Balling_ above and below the 15th degree on the instrument.

By taking _c_ for the coefficient of solubility of oxygen in water at

_t_° C., and _c´_ for that of oxygen in wort also at _t_° C., and having

a density B, by _Balling_ at 15° C.; and taking X for the ratio

_c_/_c´_, at 15° C. and 15° _Balling_, we shall have

_c_/_c´_ = X + (B - 15) 0·022 - (_t_ - 15) 0·006.

By carefully ascertaining the ratio _c_/_c´_ for different worts, and

adopting the preceding formula, we have found for X a mean value of

1·16.

The definitive formula, therefore, is:

(1) _c_/_c´_ = 1·16 + (B-15) 0·022 - (_t_ - 15) 0·006,

or again,

(2) _c_/_c´_ = 0·86 - (B - 15) 0·016 + (_t_ - 15) 0·004.

The coefficient _c_ of the solubility of oxygen in water will be found

in the table given a few pages back.

§ III.—On the Quantity of Oxygen existing in a state of Solution in

Brewers’ Worts.[174]

The wort, when it comes from the copper in which it is boiled with the

hops, remains exposed upon the coolers for a time, the length of which

varies according to circumstances, the most important of which is the

exterior temperature. The average time is from seven to eight hours,

during which the volume of the wort diminishes, whilst its density

increases; at the same time, it deposits its proteinaceous matters and

absorbs oxygen from the air, either by way of solution or of

combination.

In the present paragraph we shall confine ourselves to the uncombined

oxygen held in a state of solution in wort, recognizable by the change

of colour produced by its action on white indigo.

The use of the coolers enables the brewer to obtain his wort in two

distinct states of limpidity—filtered wort and unfiltered wort. At the

same time there is a further difference between these worts, namely, in

the quantity of oxygen held in solution. The unfiltered wort comes

direct from the coolers; the wort to be filtered, mixed with a part of

the deposit, is run into a special vessel, from which it is distributed

over the filtering surfaces, which are generally of felt; filtered

bright, it is then received in a reservoir, from which it is distributed

amongst special fermenting vessels. Falling through the air in a thin

stream of drops, it must necessarily have become charged with a greater

quantity of oxygen than ordinary wort. In good breweries it is put apart

by itself to ferment, and the yeast which it yields is firmer and

deposits more easily than that of unfiltered wort. As for the

fermentation, it is, under similar conditions, quicker by a day or a day

and a half than in the case of ordinary wort. The difference in the

quantity of oxygen held in solution in the two kinds of wort is greater

in proportion as the external temperature is lower; in winter it may be

twice as great as in summer. The reason is that in summer a boiling wort

does not obtain a minimum temperature of 20° C. (68° F.), on the best

coolers, in less than six or seven hours. After leaving the coolers it

is passed over a refrigerator. In winter it attains that temperature in

about three hours, or less, which then goes on sinking on the coolers.

During the last two or three hours which are employed in bringing the

temperature still lower, as also during the running off, the wort

absorbs an appreciable quantity of oxygen. In other words, wort in

winter remains for a longer time at low temperatures, in free contact

with air.

Another circumstance unites with this exposure upon the coolers to

increase the aeration of the wort; the wort is run into the fermenting

tuns through pipes of large sectional area, more or less bent, and

carries with it by suction considerable quantities of air, which, from

the continual agitation, gets well mixed with it. The effect of this

mixing in the pipes is to considerably increase the proportion of air in

solution in the wort, especially in winter, when the temperature of the

wort is lower; and from the figures given below we may, although it is

very variable, put the average increase at a quarter of the whole

amount. The calculation has been made by comparing the quantities of air

held in solution in two samples of the same wort, one of which was taken

from the coolers at the moment of “turning out,”[175] and the other from

the fermenting vessel after it was filled.

Let us call the ratio between the quantity of oxygen held in solution by

a wort, and that which the same wort would hold in solution if saturated

at the same temperature, the _degree of saturation_ of that wort at the

temperature _t_.

The determination of degrees of saturation is reduced to a comparison of

the number of divisions of hydrosulphite _n_ which satisfies the wort in

the first case, with the number _n´_ corresponding with the same wort

saturated at the same temperature. The ratio _n_/_n´_ gives the degree

of saturation at the temperature _t_.

In experiments made with a wort at 14·5° _Balling_ as mean density, we

found the following results:—

In summer, in the case of worts reduced to the temperature of 5° C. (41°

F.) by a refrigerator, the degrees of saturation may be set down as—

For unfiltered worts 0·500

For filtered worts 0·800

In winter, in the case of some worts which were racked at a temperature

of from 3° to 4° C. (37·4° to 39·2° F.), without the use of a

refrigerator, we found the saturation complete in both worts. In the

case of a very low external temperature, however (-10° C., 14° F.), we

have failed to determine the saturation in an unfiltered wort. As

regards the mean winter figures, in the case of worts racked at a

temperature of 5° C. (41° F.), they may be fixed at these:—

For unfiltered wort 0·850

For filtered wort 0·950

In autumn and spring we find the mean figures to be intermediate between

those given above:—

For unfiltered wort 0·500 to 0·850

For filtered wort 0·800 to 0·950

From these ratios it is easy to find the quantity of oxygen contained in

brewers’ worts, if we also refer to Bunsen’s Tables and the formula (2)

given in the preceding section. At the temperature of 5° C. (41° F.), at

which the above worts were “gathered,”[176] and not taking into account

the very small correction that should be made for the difference of half

a degree on Balling, we find, by this formula, as the ratio of the

coefficients of the solubility of oxygen in saturated wort and in water—

_c´_/_c_ = 0·82

Now, at the temperature of 5° C., the quantity of oxygen held in

solution in 1 litre of water is, according to Bunsen, 0·036 litre, at

the atmospheric pressure, and therefore at the pressure of 1/5th

atmosphere, which is that of the oxygen in atmospheric air, it will be—

0·036/5 litre = 7·2 c.c.—[that is, 2 cubic inches per gallon.]

And, consequently, in the case of saturated wort, it will be—

7·2 c.c. × 0·82 = 5·904 c.c.—[that is, 1·62 cub. inches per gall.]

Multiplying this last number of c.c. by the different _degrees of

saturation_ found, we shall obtain the volumes of oxygen held in

solution in 1 litre of different worts:—

Summer worts {Unfiltered 0·500 × 5·904 c.c. = 2·952 c.c.

{Filtered 0·800 × 5·904 “ = 4·723 ”

Winter worts {Unfiltered 0·850 × 5·904 “ = 5·018 ”

{Filtered 0·950 × 5·904 “ = 5·609 ”

It is important to notice that we are here dealing with wort taken from

the fermenting vessel just before it was pitched; that is to say, when

the quantity of oxygen held in solution was as large as the treatment to

which it had been subjected allowed of its being. The mode of taking it

for examination is as follows:—A burette, H (Fig. 81), is plunged into

the fermenting vessel, the temperature of which at the time is

ascertained very exactly, the upper part of the burette being fitted

with an india-rubber tube, _a b_, longer than itself. The liquid is then

sucked up the tube, and soon completely fills the apparatus and runs out

at _b_ (Fig. 82). By lowering the tube the whole arrangement thus forms

a syphon, and enables us to let the wort that we are experimenting on

flow for some minutes; when every trace of air has been thus expelled,

the lower tap is closed and the liquid is introduced into

Schützenberger’s apparatus.

[Illustration: Fig. 82.]

As for the saturated wort, the value of which in oxygen serves to

determine one of the elements of the degree of saturation, it is readily

obtained by introducing a volume of from 100 c.c. to 150 c.c. of wort

into a 2-litre or 3-litre flask, and shaking it briskly so as to

saturate it with air; it is then poured into a settling-glass, to

separate it from the great quantity of froth formed in the shaking, and

then, by means of a graduated pipette, 50 c.c. is taken for examination.

We have spoken of the influence that oxygen has on the activity of

yeast, on its development and, consequently, on the progress of

fermentation. Moreover, we know, from experiments already mentioned,

which we communicated to the Academy and the Chemical Society in 1861,

that the rapid development of yeast in contact with air is in reciprocal

relation to the disappearance of the oxygen from the air. Knowing the

conditions of the aeration of wort from the moment when it arrives on

the coolers until the moment when, in the fermenting tun, it is about to

be pitched, it would be interesting to ascertain what happens to the

oxygen dissolved in the wort at the moment of pitching, how yeast is

affected when suddenly brought into contact with that oxygen; what part,

in short, that gas plays in fermentation.

Let us therefore follow up, hour by hour, the degree of saturation after

pitching, in Tourtel’s brewery. On November 4th, 1875, some wort at 14°

Balling was pumped on to the coolers at 7 p.m., and at 4 a.m. went down

to a 32-hectolitre (700 gallons) tun, its temperature then being 6° C.

(42·8° F.) The pitching, in which about 100 grammes (3·2 oz. troy) of

pressed yeast was used per hectolitre (22 gallons), took place at 5 a.m.

The following is the curve of the degrees of saturation of the oxygen,

as drawn by Messrs. Calmettes and Grenet.

[Illustration: Fig. 83.]

The abscissæ represent the time expressed in hours, and the ordinates

give the degrees of saturation of the wort with oxygen. It will be seen

that about twelve hours after the pitching, and at a temperature of 6°

C., all the oxygen had disappeared, absorbed by the yeast. We shall find

that wort by itself, unassociated with yeast, would also have combined

with oxygen; but in the course of twelve hours, at 6° C., this

combination would have been scarcely appreciable in absence of yeast. It

follows, therefore, that the oxygen in solution is taken up by the

yeast, under the conditions of which we are speaking. This has been

proved directly by an experiment. A double quantity of yeast was

employed for a tun similar to the preceding one, and it was found that

the oxygen in solution disappeared completely in less than half the time

that it took to disappear in the first case.[177] It is very important

to notice that in our 32-hectolitre tun, at the moment when we

determined the complete disappearance of the oxygen in solution, the

cells of yeast had assumed a younger and fuller appearance than they had

at first; but they had not multiplied at all up to that time, nor were

there even any buds then visible on them. The oxygen, therefore, must be

stored up somehow in the cells, taken up by their oxidizable matters to

be brought into work subsequently, or to act as a _primum movens_ of

life and nutrition, spreading its influence over several successive

generations of cells.

§ IV.—On the Combination of Oxygen with Wort.

The atmospheric oxygen is not merely taken into solution by wort; it

also combines with it, as a very simple experiment will suffice to show.

If we place in a tinned iron vessel some boiling wort, separated from

the hops in the copper, and cool it suddenly by plunging it into iced

water, and after having cooled it down in this manner to 15° or 20° C.

(59° or 68° F.), saturate it with oxygen, by shaking it briskly in a

large flask, and then completely fill a vessel with it and close it up

for twelve hours, we shall find at the end of that time, if we test it

with the hydrosulphite of soda, as we have described in § II., that it

does not contain a trace of free oxygen. The whole of the gas which was

originally held in solution will have entered into combination, that is

to say, the liquid, first coloured blue with the indigo-carmine, and

then brought to a yellow tint by means of the hydrosulphite of soda,

will not regain its original blue colour through the action of this

wort. The following experiments were undertaken with the object of

studying this property of wort, and in order that we might form some

idea of its importance, and of the total quantity of oxygen that wort

can absorb under certain special circumstances. The experiments were

performed in our own laboratory on wort from Tourtel’s brewery, which M.

Calmettes had forwarded to us in bottles prepared in the brewery at

Tantonville, in the following manner: Each bottle was filled with

boiling wort taken from the copper and closed with a bored cork, through

which the neck of a funnel passed; the funnel also was filled with the

wort, and the whole preserved from contact with air by a layer of oil.

The next day the bottles were corked full by the help of a bottling

needle,[178] previously heated, with perfect corks that had been passed

through the flame. The bottles arrived in Paris in very good condition,

quite full of the liquid up to the corks. They were left undisturbed for

one or two days at the same temperature as that to which they had been

exposed during the corking and the journey. The object of this was to

afford time for a deposit of the wort to form at the bottom of each

bottle. As a matter of fact, we know that wort boiling in the copper is

charged with proteinaceous matters and other floating and insoluble

substances. The wort above the deposit was turbid and opaline; it was in

this state when we used it for our experiments. It may be taken for

granted, without risk of appreciable error, that the wort had been

absolutely deprived of oxygen in solution, inasmuch as it had been

bottled when boiling, and had cooled down out of contact with air. As

for the quantity of oxygen that it might have held in combination, this

must have been insignificant, although there must have been some, since

the wort had been exposed to the air in the copper; the oxygen in

combination, however, could have had no appreciable influence on the

results which we obtained. Let us call this wort _boiled wort_.

_First Experiment._—Into a straight-necked flask we introduced a certain

measured quantity of this wort by means of a syphon, taking care that

the syphon should only act on the opaque wort, and should not reach the

deposit at the bottom of the bottle. We then drew out the neck to a fine

tube in the flame and boiled the wort; and during ebullition we sealed

the end of the fine tube. After it had cooled, we arranged that pure air

should enter the flask. To do this we made a file mark near the fine

closed point of the flask, and connected the point by a piece of

india-rubber tubing with a glass tube containing a column of asbestos,

which we heated. We then broke off the point of the flask inside the

india-rubber tube, so that the air entered the flask after being

filtered through the asbestos. We removed the india-rubber tube and

sealed up once more the fine end of the neck at the point where we had

broken it off. Finally, to aerate the wort to saturation, we shook the

flask briskly for some minutes, and then placed it in a hot-water bath,

where we left it for about a quarter of an hour. We afterwards removed

it to an oven at 25° (77° F.). We repeated the same operation next day

and the four succeeding days.

The wort, which at first was scarcely coloured, gradually assumed a

reddish-brown tint, and deposited an amorphous matter, but without

brightening. It became clear, however, when filtered, which was not the

case with the turbid, opaline wort in the bottles when they arrived.

The following is an analysis of the air in the flask, made immediately

after a renewed and vigorous shaking, the object of which was to

saturate the wort with air before analyzing the supernatant air:—

November 29th.

Temperature at which the flask was refilled with air 4° C.

(29·2° F.)

Atmospheric pressure 751 mm. (29·6 ins.)

Total volume of flask 333 c.c. (20·32 cub. in.)

Volume occupied by the wort 120 “ ( 7·32 ” )

December 8th.

Volume of gas analyzed 27·6 c.c. (1·68 cub. ins.)

After treatment with potash 27·4 c.c. (1·67 “ )

” “ pyrogallol 22·4 ” (1·36 “ )

Oxygen 5·0 c.c. (0·305 cub. in.)

Composition of the gas:— Per cent.

Oxygen 18·25

Nitrogen 81·57

The formula which we deduced above (§ II.) allows us to conclude that at

the temperature of 8° C. (46·4° F.), which was the temperature at which

the wort was saturated before the analysis given above, the quantity of

oxygen in solution in the 120 c.c. (4·2 fl. oz.) of wort was 0·84 c.c.

(0·051 cub. in.).

At the moment when the flask was closed, the total volume of oxygen,

calculated to zero and 760 mm. (30 in.) pressure, was 44·73 c.c. (2·729

cub. in.).

At the moment when the analysis was finished, the volume of oxygen was

calculated to the same conditions of temperature and pressure, 38·86

c.c. (2·355 cub. in.); 5·87 c.c. (0·374 cub. in.) has, therefore,

disappeared. Now, as there is 0·84 c.c. (0·051 cub. in.) in solution,

there has, consequently, been an absorption, by combination with 120

c.c. of wort, of 5·03 c.c. (0·32 cub. in.) of oxygen, or 41·7 c.c. per

litre (11·6 cub. ins. per gallon).

_Second Experiment._—In a similar experiment, in which, however, the

flask was kept for five days at a rigorously constant temperature of 55°

C. (131° F.), day and night, and in which the supernatant air was not

shaken up with the wort, we found—

Volume of gas analyzed 28·5

After treatment with potash 28·3

“ ” “ pyrogallol 23·0

Oxygen 5·3

Composition of the gas:— Per cent.

Oxygen 18·6

Nitrogen 81·4

Total oxygen at first 29·40

“ ” remaining 26·04

“ ” that has disappeared 3·36

“ ” in solution 0·54

“ ” in combination 2·82

Or per litre, 35·2 c.c. (9·8 cub. ins. per gallon).

The colour of the wort in this experiment had become sensibly similar to

that of the wort in the preceding experiment.

_Third Experiment._—In another experiment we left the flask, for the

same length of time again, after it had been refilled with air and

reclosed, at a temperature which varied between 2° and 4°C. (35·6° and

39·2° F.). In this case we found—

Volume of air analyzed 27·8

After the action of potash 27·8

After pyrogallic acid 22·3

Oxygen 5·5

Composition of the gas:— Per cent.

Oxygen 19·7

Nitrogen 80·3

Total oxygen at first 29·40 c.c.

“ ” remaining 27·58

“ ” that has disappeared 1·82

“ ” in solution 0·44

“ ” in combination 1·38

Or per litre, 17·20 c.c. (4·8 cub. ins. per gallon).

In this last experiment the wort was scarcely darker in colour. Its

colour, compared with that of wort cooled on the coolers in the brewery,

was slightly darker; but the difference, although it existed, was

scarcely appreciable. We shall revert to this fact, which is of

importance, presently.

_Fourth Experiment._—The following series of experiments were undertaken

to enable us to form some idea of the rapidity with which oxygen is

absorbed by wort.

We employed three flasks. A, B, C, of the following capacities:—

A = 234

B = 214

C = 203

into which we introduced the following quantities of wort (boiled wort,

without air):—

Into A 96 c.c.

“ B 84 ”

“ C 84 ”

The necks of the flasks were then drawn out and sealed in a flame, the

liquid being at a temperature of 5° C. (41° F.). The flasks were then

placed in a hot-water bath and kept at 100° C. (212° F.) for a quarter

of an hour. The flask A was repeatedly shaken during cooling, as also

was the flask B, this being omitted in the case of the flask C.

The contents of flask A were submitted to analysis as soon as it was

quite cooled—that is to say, in about three hours. The analysis of

contents of B and C was delayed for about twenty-four hours. We took the

precaution of not commencing the analysis before we had shaken the

flasks for a few minutes, so that the wort in all of them might be

saturated at a fixed temperature, and thus enable us to ascertain the

exact quantity of oxygen in solution.

The analyses showed that the worts in the three flasks contained:—

Flask A, oxygen in combination, per litre 20 c.c.

“ B, ” “ ” 21·4 c.c.

“ C, ” “ ” 16·8 c.c.

Several facts may be deduced from these experiments: the shaking up of

the wort with air has a marked effect on the absorption; a very

appreciable absorption immediately follows the shaking up of the wort

when warm; whereas, in the case of cold wort that has remained

undisturbed, the absorption takes place slowly.

The results of the preceding experiments plainly show that the wort,

which is very hot when it comes on to the coolers, where it remains for

several hours, must absorb an appreciable quantity of oxygen by

combination; but these same experiments teach us nothing definite

concerning the volume of oxygen that is actually absorbed. We can only

gather from the remark which concludes the third experiment given above,

that the total quantity of oxygen absorbed by the wort in Tourtel’s

brewery, during the time that it remains on the coolers, must be less

than 17 c.c. per litre (4·7 cubic inches per gallon), inasmuch as the

coloration effected by combined oxygen in the proportion of 17 c.c. per

litre was considerably greater than that of the wort taken from the

backs in the brewery.

If we knew the curve of cooling on the Tourtonville coolers we might

easily, in experiments conducted in our laboratory, assimilate the

conditions of our experiments to those of the oxidation of the wort in

the brewery, by exposing wort in contact with air in closed flasks to

temperatures varying according to the indications of the curve in

question. For this purpose, we induced M. Calmettes to study the process

of cooling upon the coolers at Tantonville. In Fig. 84 the figures found

in one of that gentleman’s experiments are given.

The abscissæ represent the time expressed in hours; the ordinates, the

degrees of temperature. The exterior temperature was 0° C. (32° F.); the

atmosphere was calm. The wort was pumped on to the coolers at 5.20 p.m.,

its temperature then being 85° C. (185° F.), and the operation of

pumping lasted from 5.20 to 5.30 p.m. The first determination was made

at 5.30 p.m., and was repeated every ten minutes until 7.30 p.m. Between

7.30 and 8.30 p.m. it was repeated every twenty minutes; after that, it

was repeated every half-hour until 2 a.m., when the wort went down to

the fermenting vessels. The mean depth of the wort was 8·5 centimetres

(3·1 inches).

[Illustration: Fig. 84. Curve of cooling of the wort on the coolers

(December 18th, 1875).]

Having determined the rate of cooling in the brewery, we made the

following experiment: a known quantity of wort from the copper—deprived,

consequently, of oxygen—in the same condition as when it comes on the

coolers, was put into a graduated, cylindrical vessel, which was then

closed with an india-rubber cork, and placed immediately, without being

shaken, in a hot water bath at 85° C. (185° F.). Another vessel similar

to the preceding one, and having a thermometer passed through the cork,

and immersed in the wort, enabled us to observe the temperature. The

temperature was gradually reduced, in exact accordance with the data of

the preceding curve, until the water, in the course of eight hours and a

half, was brought down to 10° C. (50° F.). It is true, that we cannot

pretend to have realized all the conditions of the coolers, in this

manner, but we approached them very nearly; moreover, it was an

approximation rather than a rigorous determination that we desired to

obtain. We then collected over mercury the air which remained in the

flask, and analyzed it very carefully; at the same time, with

Schützenberger’s apparatus, we determined the oxygen held in solution in

the wort so treated. From the results thus obtained we easily found the

quantity of oxygen that had disappeared—that is, the oxygen which the

wort had acquired from the atmosphere of the flask, and which had

combined with the oxidizable matters of the wort.

The volume of the flask being 815 c.c., that of the wort 391 c.c., and

the depth of the liquid 8 cm., we found an absorption by combination of

9·49 c.c. of oxygen per litre of wort (2·63 cub. ins. per gallon).

Another flask treated in the same manner gave us similar results.

As the oxygen in solution has so great an influence on fermentation, it

is important that we should, likewise, know the effect produced by the

oxygen in combination. The following considerations and experiments may

throw some light on this subject:—

We have already remarked that natural saccharine worts oxidize, and

acquire colour in contact with air, and that this coloration disappears

when these worts are caused to ferment. This furnishes one presumption,

that the oxygen in combination disappears then, from, being abstracted

by the ferment. A similar phenomenon is observable in the case of wort.

After having acquired a marked dark shade by remaining in contact with

pure air, it loses this colour very appreciably during fermentation; and

if the wort does not quite regain the colour which it originally had

when it came from the copper, this circumstance is probably owing to the

fact that the quantity of oxygen in combination with the wort is larger

than that which is abstracted by the yeast. We have seen that yeast

absorbs oxygen, since, in the case of a saccharine wort, more or less

saturated with oxygen in solution, when fermentation commences, the

first effect of the ferment is to cause that oxygen to combine with its

own substance. We should, therefore, expect to find the oxygen in

combination, as well as that held in solution, in wort, abstracted by

the yeast and contributing to the activity of fermentation. As a matter

of fact, this is proved by direct experiments, for the fermentation of a

wort that has oxidized in contact with air, or of one from which all the

oxygen that was held in solution in it has disappeared by direct

combination, is much more easy, rapid, and complete than the

fermentation of the same wort when it contains no oxygen, whether free

or combined. These experiments were as follows: we boiled some _copper

wort_ in a large double-necked flask, like those shown in Fig. 73; all

the air being expelled, pure air was allowed to enter the flask; and

when the wort was cool it was saturated with this air, by being shaken

briskly for a quarter of an hour. The wort was then forced by a pressure

of air, applied to the extremity of the S-shaped tube, into smaller

flasks, similar to the preceding ones; these we filled completely, and

then plunged the end of their sinuous tubes under mercury. After waiting

for two or three days, a longer time than was required for the oxygen in

solution to enter into combination—a fact which we confirmed by means of

a similar flask, which served as a standard—we caused the wort, so

prepared, to ferment in the flasks, and side by side, for the sake of

comparison, some _copper wort_ that contained no air in solution or

combination.

In other experiments we operated on pure wort, saturated with oxygen in

combination, by being allowed to remain for one year in an open flask in

contact with pure air. This wort was deprived of air in solution by a

protracted boiling over mercury. It was then pitched, out of contact of

air, with an old yeast. The yeast underwent no development at all, a

proof that oxygen in combination cannot act like oxygen that is free, or

simply in solution, in effecting the revival of the yeast; nevertheless,

after the revival has been once started by means of a small quantity of

air, fermentation declares itself with much greater facility than in the

case of copper wort, placed under the same conditions, but deprived of

oxygen in combination.

§ V. On the Influence of Oxygen in Combination on the Clarification of

Wort.

Oxygen in combination has another effect which it is essentially

important to point out, for it concerns the clarification of beer. One

of the most valued properties of this beverage is its limpidity and

brilliancy. We know from the results of the fourth experiment in the

preceding paragraph that in the case of a wort shaken up when hot with

air, and examined as soon as cold, that is, after an interval of only

three hours, we find a notable volume of oxygen to have been absorbed by

combination; in the experiment to which we allude, this volume was not

less than 20 c.c. of oxygen per litre of wort. The shaking up of the

wort when cold with air saturated it with oxygen in solution, but the

quantity of oxygen which under these conditions entered into

combination, in the course of three hours, is insignificant, although

saturation by solution may be attained in the course of one minute’s

shaking. If two samples of the same wort are shaken up with air, one of

them being hot and the other cold, and both filtered after having been

left undisturbed for twenty-four hours, or even immediately after the

agitation, we cannot fail to be struck with the great difference that

they will present in point of brightness. The wort that was shaken up

hot will have more colour, and will be brilliant; the other will be

turbid, and will not become clear for five or six days, when left to

itself in contact with air and filtered again. This explains a fact that

may be easily verified in practice: Boiled wort, if cooled down

suddenly, or slowly but out of contact with air, or shaken up cold in

contact with air, is opaque when filtered; whilst the same wort, cooled

down on the coolers where it has taken a certain quantity of oxygen into

combination, generally passes through the filter very bright. The

intelligent brewer is uneasy when this is not the case, for it cannot be

denied that the easy clarification of wort has a favourable influence on

the easy clarification of beer.

It would, nevertheless, be a grave error to suppose that the

clarification of beer must necessarily follow that of wort, and we may

be permitted to make a digression here on the subject, to prove this

statement.

On February 3rd, 1874, we brewed 2 hectolitres (44 gallons) of beer. The

boiling wort, hops and all, was run into a vessel like that represented

in Fig. 80, but provided in addition with a false bottom, pierced with

holes and fixed at 1 centimetre (0·39 inch) above the true bottom of the

vessel; this was meant to retain the spent hops. The temperature of the

wort in the vessel after it was filled, February 3rd, 4 p.m., was 90° C.

(194° F.), that of the room was 10° C. (50° F.). We permitted the wort

to cool down gently, without running cold water over the vessel. The

wort indicated a density of 14° Balling.

The following temperatures were taken:—

Temp. of Temp. of

Wort. Room.

Feb. 4, 11 a.m. 38° C. (100·4° F.) 9° C. (48·2° F.)

7 p.m. 30° C. ( 86° F.) 9° C. (48·2° F.)

11.30 p.m. 26·3° C. ( 79·3° F.) 9° C. (48·2° F.)

Feb. 5, 9 a.m. 21° C. ( 69·8° F.) 8° C. (46·4° F.)

12 a.m. 19·75° C. ( 66·6° F.) 8° C. (46·4° F.)

4 p.m. 18° C. ( 64·4° F.) 8·5° C. (47·3° F.)

Feb. 6, 11 a.m. 14° C. ( 57·2° F.) 8° C. (46·4° F.)

Feb. 7, 2 p.m. 11° C. ( 51·8° F.) 7° C. (44·6° F.)

At the end of this time the wort drawn from the smaller tap half-way up

the vessel had already become very bright, although it was taken from

the bulk of the liquid above the deposit of hops.

On February 8th the temperature of the wort was 9·5° C. (49·1° F.), and

that of the room 5° C. (41° F.); the wort was again very bright. Taken

from the small tap and tested by Schützenberger’s process it gave no

evidence of free oxygen in solution, although its surface was in contact

with air. It continued absolutely pure, the arrangements of our vessel,

as we have already explained, allowing only such air to enter as was

first deprived of its disturbing germs.

Not till February 12th, after we had again determined the purity and

brilliant clearness of the wort, a brilliancy which we can compare with

nothing so well as Cognac, without the faintest trace of cloudiness, did

we set it to ferment in a vessel similar to that in which it had cooled,

but without the false bottom. In the process of transfer we effected its

aeration by causing it to fall on a small inverted tinned iron capsule

some 4 or 5 centimetres (1-½ to 2 inches) in diameter. By this

arrangement the wort took up air to the extent of rather more than a

third of its saturate capacity, that is to say, by spreading over the

capsule, and falling from it in a kind of sheet, it absorbed a volume of

oxygen more than a third of the total amount of oxygen which it was

capable of absorbing at the existing temperature; this was 12° C. (53·6°

F.) at the moment when the wort was drawn off. The pitching was

accomplished with a 6-litre flask containing about 4 litres (7·04 pints)

of beer that had been in “low” fermentation from February 3rd. The beer

was cleansed on February 24th, and had a density of 5-1/4° Balling. We

collected 2·345 kilos (75·39 oz. troy) of yeast, containing 56 per

cent., that is, 1·313 kilos (42·21 oz. troy) of pressed yeast,

containing 36·7 per cent. of yeast dried at 100° C. (212° F.), that is

482 grammes (15·49 oz. troy) for the brew, which would give 241 grammes

(7·748 oz. troy) of yeast formed per hectolitre (22 gallons).

The beer was turbid when drawn off, and the small glassful that we

removed did not brighten in twenty-four or even forty-eight hours. The

samples for some days previously had been in the same condition. The

yeast existed as a fine deposit without any straggling yeast about the

sides. The want of brightness was dependent rather on spurious colour

than on any actual turbidity. We may here remark that if in the

preceding experiment the wort had taken up oxygen into combination as

well as into solution at the time that it was aerated, the other

conditions being the same, the beer would have been bright and better.