Chapter VI (4)

This theory dates back as far as 1843. In 1846 Messrs. Boutron and Fremy, in a Memoir on lactic fermentation, published in the _Annales de Chimie et de Physique_, strained the conclusions deducible from it to a most unjustifiable extent. They asserted that one and the same nitrogenous substance might undergo various modifications in contact with air, so as to become successively alcoholic, lactic, butyric, and other ferments. There is nothing more convenient than purely hypothetical theories, theories which are not the necessary consequences of facts; when fresh facts which cannot be reconciled with the original hypothesis are discovered, new hypotheses can be tacked on to the old ones. This is exactly what Liebig and Fremy have done, each in his turn, under the pressure of our studies, commenced in 1857. In 1864 Fremy devised the theory of _hemi-organism_, which meant nothing more than that he gave up Liebig’s theory of 1843, together with the additions which Boutron and he had made to it in 1846; in other words, he abandoned the idea of albuminous substances being ferments, to take up another idea, that albuminous substances, in contact with air, are peculiarly adapted to undergo organization into new beings—that is, the living ferments which we had discovered—and that the ferments of beer and of the grape have a common origin.

This theory of hemi-organism was word for word the antiquated opinion of Turpin, as may be readily seen by referring to Chapter IV., section III. of the present work. The public, especially a certain section of the public, did not go very deeply into an examination of the subject. It was the period when the doctrine of spontaneous generation was being discussed with much warmth. The new word hemi-organism, which was the only novelty in M. Fremy’s theory, deceived people. It was thought that M. Fremy had really discovered the solution of the question of the day. It is true that it was rather difficult to understand the process by which an albuminous substance could become all at once a living and budding cell. This difficulty was readily solved by M. Fremy, who declared that it was the result of some power that was not yet understood, the power of “organic impulse.”[155]

Liebig, who, as well as M. Fremy, was compelled to renounce his original opinions concerning the nature of ferments, devised the following obscure theory (Memoir by Liebig, 1870, already cited):—

“There seems to be no doubt as to the part which the vegetable organism plays in the phenomenon of fermentation. It is through it alone that an albuminous substance and sugar are enabled to unite and form this particular combination, this unstable form under which alone, as a component part of the mycoderm, they manifest an action on sugar. Should the mycoderm cease to grow, the bond which unites the constituent parts of the cellular contents is loosened, and it is through the motion produced therein that the cells of yeast bring about a disarrangement or separation of the elements of the sugar into other organic molecules.”

One might easily believe that the translator for the _Annales_ has made some mistake, so great is the obscurity of this passage.

Whether we take this new form of the theory or the old one, neither can be reconciled at all with the development of yeast and fermentation in a saccharine mineral medium, for in the latter experiment fermentation is correlative to the life of the ferment and to its nutrition, a constant change going on between the ferment and its food-matters, since all the carbon assimilated by the ferment is derived from sugar, its nitrogen from ammonia, and phosphorus from the phosphates in solution. And even all said, what purpose can be served by the gratuitous hypothesis of contact-action or communicated motion? The experiment of which we are speaking is thus a fundamental one; indeed, it is its possibility that constitutes the most effective point in the controversy. No doubt Liebig might say, “but it is the motion of life and of nutrition which constitutes your experiment, and this is the communicated motion that my theory requires.” Curiously enough, Liebig does endeavour, as a matter of fact, to say this, but he does so timidly and incidentally: “From a chemical point of view, which point of view I would not willingly abandon, a _vital action_ is a phenomenon of motion, and, in this double sense of _life_ M. Pasteur’s theory agrees with my own, and is not in contradiction with it (page 6).” This is true. Elsewhere Liebig says:—

“It is possible that the only correlation between the physiological act and the phenomenon of fermentation is the production, in the living cell, of the substance which, by some special property analogous to that by which emulsin exerts a decomposing action on salicin and amygdalin, may bring about the decomposition of sugar into other organic molecules; the physiological act, in this view, would be necessary for the production of this substance, but would have nothing else to do with the fermentation (page 10).” To this, again, we have no objection to raise.

Liebig, however, does not dwell upon these considerations, which he merely notices in passing, because he is well aware that, as far as the defence of his theory is concerned, they would be mere evasions. If he had insisted on them, or based his opposition solely upon them, our answer would have been simply this: “If you admit with us that fermentation is correlated with the life and nutrition of the ferment, we agree upon the principal point. So agreeing, let us examine, if you will, the actual cause of fermentation;—this is a second question, quite distinct from the first. Science is built up of successive solutions given to questions of ever-increasing subtlety, approaching nearer and nearer towards the very essence of phenomena. If we proceed to discuss together the question of how living, organized beings act in decomposing fermentable substances, we will be found to fall out once more on your hypothesis of communicated motion, since, according to our ideas, the actual cause of fermentation is to be sought, in most cases, in the fact of life without air, which is the characteristic of many ferments.”

Let us briefly see what Liebig thinks of the experiment in which fermentation is produced by the impregnation of a saccharine mineral medium, a result so greatly at variance with his mode of viewing the question.[156] After deep consideration he pronounces this experiment to be inexact, and the result ill-founded. Liebig, however, was not one to reject a fact without grave reasons for his doing so, or with the sole object of evading a troublesome discussion. “I have repeated this experiment,” he says, “a great number of times, with the greatest possible care, and have obtained the same results as M. Pasteur, excepting as regards the formation and increase of the ferment.” It was, however, the formation and increase of the ferment that constituted the point of the experiment. Our discussion was, therefore, distinctly limited to this: Liebig denied that the ferment was capable of development in a saccharine mineral medium, whilst we asserted that this development did actually take place, and was comparatively easy to prove. In 1871 we replied to M. Liebig before the Paris Academy of Sciences in a Note, in which we offered to prepare in a mineral medium, in the presence of a commission to be chosen for the purpose, as great a weight of ferment as Liebig could reasonably demand.[157] We were bolder than we should, perhaps, have been in 1860; the reason was that our knowledge of the subject had been strengthened by ten years of renewed research. Liebig did not accept our proposal, nor did he even reply to our Note. Up to the time of his death, which took place on April 18th, 1873, he wrote nothing more on the subject.[158]

When we published, in 1860, the details of the experiment in question, we pointed out at some length the difficulties of conducting it successfully, and the possible causes of failure. We called attention particularly to the fact that saccharine mineral media are much more suited for the nutrition of bacteria, lactic ferment, and other lowly forms, than they are to that of yeast, and in consequence readily become filled with various organisms from the spontaneous growth of germs derived from the particles of dust floating in the atmosphere. The reason why we do not observe the growth of alcoholic ferments, especially at the commencement of the experiments, is because of the unsuitableness of those media for the life of yeast. The latter may, nevertheless, form in them subsequent to this development of other organized forms, by reason of the modification produced in the original mineral medium by the albuminous matters that they introduce into it. It is interesting to peruse, in our Memoir of 1860, certain facts of the same kind relating to fermentation by means of albumens—that of the blood, for example, from which, we may mention incidentally, we were led to infer the existence of several distinct albumens in the serum, a conclusion which, since then, has been confirmed by various observers, notably by M. Béchamp. Now, in his experiments on fermentation in sweetened water, with yeast-ash and a salt of ammonia, there is no doubt that Liebig had failed to avoid those difficulties which are entailed by the spontaneous growth of other organisms than yeast. Moreover, it is possible that, to have established the certainty of this result, Liebig should have had recourse to a closer microscopical observation than from certain passages in his Memoir he seems to have adopted. We have little doubt that his pupils could tell us that Liebig did not even employ that instrument without which any exact study of fermentation is not merely difficult but well-nigh impossible. We ourselves, for the reasons mentioned, did not obtain a simple alcoholic fermentation any more than Liebig did. In that particular experiment, the details of which we gave in our Memoir of 1860, we obtained lactic and alcoholic fermentation together; an appreciable quantity of lactic acid formed and arrested the propagation of the lactic and alcoholic ferments, so that more than half of the sugar remained in the liquid without fermenting. This, however, in no way detracted from the correctness of the conclusion which we deduced from the experiment, and from other similar ones; it might even be said that, from a general and philosophical point of view—which is the only one of interest here—the result was doubly satisfactory, inasmuch as we demonstrated that mineral media were adapted to the simultaneous development of several organized ferments, instead of only one. The fortuitous association of different ferments could not invalidate the conclusion that all the nitrogen of the cells of the alcoholic and lactic ferments was derived from the nitrogen in the ammoniacal salts, and that all the carbon of those ferments was taken from the sugar, since, in the medium employed in our experiment, the sugar was the only substance that contained carbon. Liebig carefully abstained from noticing this fact, which would have been fatal to the very groundwork of his criticisms, and thought that he was keeping up the appearance of a grave contradiction by arguing that we had never obtained a simple alcoholic fermentation. It would be unprofitable to dwell longer upon the subject of the difficulties which the propagation of yeast in a saccharine mineral medium formerly presented. As a matter of fact, the progress of our studies has imparted to the question an aspect very different from that which it formerly wore; it was this circumstance which emboldened us to offer, in our reply to Liebig before the Academy of Sciences in 1871, to prepare, in a saccharine mineral medium, in the presence of a commission to be appointed by our opponent, any quantity of ferment that he might require, and to effect the fermentation of any weight of sugar whatsoever.

Our knowledge of the facts detailed in the preceding chapters concerning pure ferments and their manipulation in the presence of pure air, enables us to completely disregard those causes of embarrassment that result from the fortuitous occurrence of the germs of organisms, different in character from the ferments, introduced by the air or from the sides of vessels, or even by the ferment itself.

Let us once more take one of our double-necked flasks (Fig. 22, p. 110), which we will suppose is capable of containing three or four litres (six to eight pints).

Let us put into it the following:—

Pure distilled
water.

Sugar candy 200 grammes.

Bitartrate of 1·0 “
potassium

” “ ammonia 0·5 ”

Sulphate of ammonia 1·5 “

Ash of yeast 1·5 ”

[1 gramme = 15·43
grains.]

Let us boil the mixture, to destroy all germs of organisms that may exist in the air or liquid or on the sides of the flask, and then permit it to cool, after having placed, by way of extra precaution, a small quantity of asbestos in the end of the fine, curved tube. Let us next introduce a trace of ferment into the liquid, through the other neck, which, as we described, is terminated by a small piece of india-rubber tube closed with a glass stopper.

Here are the details of such an experiment:—

On December 9, 1873, we sowed some pure ferment—_saccharomyces pastorianus_. From December 11, that is, within so short a time as forty-eight hours after impregnation, we saw a multitude of extremely minute bubbles rising almost continuously from the bottom, indicating that at this point the fermentation had commenced. On the following days, several patches of froth appeared on the surface of the liquid. We left the flask undisturbed in the oven, at a temperature of 25° C. (77° F.). On April 24, 1874, we tested some of the liquid, obtained by means of the straight tube, to see if it still contained any sugar. We found that it contained less than two grammes, so that 198 grammes (4·2 oz. Troy) had already disappeared. Some time afterwards the fermentation came to an end; we carried on the experiment, nevertheless, until April 18, 1875.

There was no development of any organism absolutely foreign to the ferment, which was itself abundant, a circumstance that, added to the persistent vitality of the ferment, in spite of the unsuitableness of the medium for its nutrition, permitted the perfect completion of fermentation. There was not the minutest quantity of sugar remaining. The total weight of ferment, after washing and drying at 100° C. (212° F.), was 2·563 grammes (39·5 grains).

In experiments of this kind, in which the ferment has to be weighed, it is better not to use any yeast-ash that cannot be dissolved completely, so as to be capable of easy separation from the ferment formed. Raulin’s liquid, the composition of which we have already given (p. 89, footnote), may be used in such cases with success.

All the alcoholic ferments are not capable to the same extent of development by means of phosphates, ammoniacal salts, and sugar. There are some whose development is arrested a longer or shorter time before the transformation of all the sugar. In a series of comparative experiments, 200 grammes of sugar-candy being used in each case, we found that whilst _saccharomyces pastorianus_ effected a complete fermentation of the sugar, the caseous ferment did not decompose more than two-thirds, and the ferment which we have designated _new “high” ferment_ not more than one-fifth: and keeping the flasks for a longer time in the oven had no effect in increasing the proportions of sugar fermented in these two last cases.

We conducted a great number of fermentations in mineral media, in consequence of a circumstance which it may be interesting to mention here. A person who was working in our laboratory asserted that the success of our experiments depended upon the impurity of the sugar-candy which we employed, and that if this sugar had been pure—much purer than was the ordinary, white, commercial sugar-candy, which up to that time we had always used—the ferment could not have multiplied. The persistent objections of our friend, and our desire to convince him, caused us to repeat all our previous experiments on the subject, using sugar of great purity, which had been specially prepared for us, with the utmost care, by a skilful confectioner, Seugnot. The result only confirmed our former conclusions. Even this did not satisfy our obstinate friend, who went to the trouble of preparing some pure sugar for himself, in little crystals, by repeated crystallizations of carefully-selected commercial sugar-candy; he then repeated our experiments himself. This time all his doubts were overcome. It even happened that the fermentations with the perfectly pure sugar instead of being slow were very active, when compared with those which we had conducted with the commercial sugar-candy.

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.