Chapter IV

., that the ordinary moulds assume the character of a ferment when compelled to live without air, or with quantities of air too scant to permit of their organs having around them as much of that element as is necessary for their life as aërobian plants. Ferments, therefore, only possess in a higher degree a character which belongs to many common moulds, if not to all, and which they share, probably, more or less, with all living cells, namely the power of living either an aërobian or anaërobian life, according to the conditions under which they are placed.

It may be readily understood how, in their state of aërobian life, the alcoholic ferments have failed to attract attention. Those ferments are only cultivated out of contact with air, at the bottom of liquids which soon become saturated with carbonic acid gas. Air is only present in the earlier developments of their germs, and without attracting the attention of the operator, whilst in their state of anaërobian growth their life and action are of prolonged duration. We must have recourse to special experimental apparatus to enable us to demonstrate the mode of life of alcoholic ferments under the influence of free oxygen; it is their state of existence apart from air, in the depths of liquids that attracts all our attention. The results of their action are, however, marvellous, if we regard the products resulting from them, in the important industries of which they are the life and soul. In the case of ordinary moulds, the opposite holds good. What we want to use special experimental apparatus for with them is to enable us to demonstrate the possibility of their continuing to live for a time out of contact with air, and all our attention, in their case, is attracted by the facility with which they develop under the influence of oxygen. Thus the decomposition of saccharine liquids, which is the consequence of the life of fungi without air, is scarcely perceptible, and so is of no practical importance. Their aerial life, on the other hand, in which they respire and accomplish their process of oxidation under the influence of free oxygen, is a normal phenomenon, and one of prolonged duration which cannot fail to strike the least thoughtful of observers. We are convinced that a day will come when moulds will be utilized in certain industrial operations, on account of their power of destroying organic matter. The conversion of alcohol into vinegar in the process of acetification, and the production of gallic acid by the action of fungi on wet gall-nuts, are already connected with this kind of phenomena.[122] On this last subject, the important work of M. Van Tieghem (_Annales Scientifiques de l’École Normale_, vol. vi.) may be consulted.

The possibility of living without oxygen, in the case of ordinary moulds, is connected with certain morphological modifications which are more marked in proportion as this faculty is itself more developed. These changes in the vegetative forms are scarcely perceptible in the case of _penicillium_ and _mycoderma vini_, but they are very evident in the case of _aspergillus_, consisting of a marked tendency on the part of the submerged mycelial filaments to increase in diameter, and to develop cross partitions at short intervals, so that they sometimes bear a resemblance to chains of conidia. In _mucor_, again, they are very marked, the inflated filaments which, closely interwoven, present chains of cells which fall off and bud, gradually producing a mass of cells. If we consider the matter carefully, we shall see that yeast presents the same characteristics. For instance, what can more closely resemble the mucor of Plates V. and VI. than the _saccharomyces_ of Figs. 33 and 37? Have we not in each case ramified chains of elongated cells or joints, more or less narrowed in the middle, and shorter segments or cells dropping off at the constrictions, and proceeding to bud in the liquid on their own account? Moreover, the less oxygen there is present, the more marked is the tendency to the formation of these budding cells, which isolate themselves and soon drop off. Who could ever imagine, in examining the ferment of _mucor_ represented in Plate VI., that its first germ was the ordinary _mucor_ that is found everywhere, with fine filaments, straight or ramified according to the variety, which send up aerial _hyphae_, terminating in little round heads bearing spores. So was it that in the ferment of Plate XI. we could scarcely recognize the ramified filaments of Figs. 33 and 37.

It is a great presumption in favour of the truth of theoretical ideas when the results of experiments undertaken on the strength of those ideas are confirmed by various facts more recently added to science, and when those ideas force themselves more and more on our minds, in spite of a _primâ facie_ improbability. This is exactly the character of those ideas which we have just expounded. We propounded them in 1861, and not only have they remained unshaken since, but they have served to foreshadow new facts, so that it is much easier to defend them in the present day than it was to do so fifteen years ago. We first called attention to them in various notes, which we read before the Chemical Society of Paris, notably at its meetings of April 12th and June 28th, 1861, and in papers in the _Comptes rendus de l’Académie des Sciences_. It may be of some interest to quote here, in its entirety, our communication of June 28th, 1861, entitled, “Influences of Oxygen on the Development of Yeast and on Alcoholic Fermentation,” which we extract from the _Bulletin de la Société Chimique de Paris_:—

“M. Pasteur gives the results of his researches on the fermentation of sugar and the development of yeast-cells, according as that fermentation takes place apart from the influence of free oxygen or in contact with that gas. His experiments, however, have nothing in common with those of Gay-Lussac, which were performed with the juice of grapes, crushed under conditions where they would not be affected by air, and then brought in contact with oxygen.

“Yeast, when perfectly developed, is able to bud and grow in a saccharine and albuminous liquid, in the complete absence of oxygen or air. In this case but little yeast is formed, and a comparatively large quantity of sugar disappears—sixty or eighty parts for one of yeast formed. Under these conditions fermentation is very sluggish.

“If the experiment is made in contact with the air, and with a great surface of liquid, fermentation is rapid. For the same quantity of sugar decomposed much more yeast is formed. The air with which the liquid is in contact is absorbed by the yeast. The yeast develops very actively, but its fermentative character tends to disappear under these conditions; we find, in fact, that for one part of yeast formed, not more than from four to ten parts of sugar are transformed. The fermentative character of this yeast, nevertheless, continues, and produces even increased effects, if it is made to act on sugar apart from the influence of free oxygen.

“It seems, therefore, natural to admit that when yeast functions as a ferment by living apart from the influence of air, it derives oxygen from the sugar, and that this is the origin of its fermentative character.

“M. Pasteur explains the fact of the immense activity at the commencement of fermentations by the influence of the oxygen of the air held in solution in the liquids, at the time when the action commences. The author has found, moreover, that the yeast of beer sown in an albuminous liquid, such as yeast-water, still multiplies, even when there is not a trace of sugar in the liquid, provided always that atmospheric oxygen is present in large quantities. When deprived of air, under these conditions, yeast does not germinate at all. The same experiments may be repeated with albuminous liquid, mixed with a solution of non-fermentable sugar, such as ordinary crystallized milk-sugar. The results are precisely the same.

“Yeast formed thus in the absence of sugar does not change its nature; it is still capable of causing sugar to ferment, if brought to bear upon that substance apart from air. It must be remarked, however, that the development of yeast is effected with great difficulty when it has not a fermentable substance for its food. In short, the yeast of beer acts in exactly the same manner as an ordinary plant, and the analogy would be complete if ordinary plants had such an affinity for oxygen as permitted them to breathe by appropriating this element from unstable compounds, in which case, according to M. Pasteur, they would appear as ferments for those substances.

“M. Pasteur declares that he hopes to be able to realize this result, that is to say, to discover the conditions under which certain inferior plants may live apart from air in the presence of sugar, causing that substance to ferment as the yeast of beer would do.”

This summary and the preconceived views that it set forth have lost nothing of their exactness; on the contrary, time has strengthened them. The surmises of the last two paragraphs have received a valuable confirmation from recent observations made by Messrs. Lechartier and Bellamy, as well as by ourselves, an account of which we must put before our readers. It is necessary, however, before touching upon this curious feature in connection with fermentations to insist on the accuracy of a passage in the preceding summary, the statement, namely, that yeast could multiply in an albuminous liquid, in which it found a non-fermentable sugar, milk-sugar for example. The following is an experiment on this point:—On August 15th, 1875, we sowed a trace of yeast in 150 c.c. (rather more than 5 fluid ounces) of yeast-water, containing 2-½ per cent. of milk-sugar. The solution was prepared in one of our double-necked flasks, with the necessary precautions to secure absence of germs, and the yeast sown was itself perfectly pure. Three months afterwards, November 15th, 1875, we examined the liquid for alcohol; it contained only the smallest trace; as for the yeast, which had sensibly developed, collected and dried on a filter paper, it weighed 0·050 gramme (0·76 grain). In this case we have the yeast multiplying without giving rise to the least fermentation, like a fungoid growth, absorbing oxygen, and evolving carbonic acid, and there is no doubt that the cessation of its development in this experiment was due to the progressive deprivation of oxygen that occurred. As soon as the gaseous mixture in the flask consisted entirely of carbonic acid and nitrogen, the vitality of the yeast was dependent on, and in proportion to, the quantity of air which entered the flask in consequence of variations of temperature. The question now arose, was this yeast, which had developed wholly as an ordinary fungus, still capable of manifesting the character of a ferment? To settle this point we had taken the precaution, on August 15th, 1875, of preparing another flask, exactly similar to the preceding one in every respect, and which gave results identical with those described. We decanted this on November 15th, pouring some wort on the deposit of the plant, which remained in the flask. In less than five hours from the time when we placed it in the oven, the plant had started fermentation in the wort, as we could see by the bubbles of gas rising to form patches on the surface of the liquid. We may add that yeast in the medium which we have been discussing will not develop at all without air.

The importance of these results can escape no one; they prove clearly that the fermentative character is not an invariable phenomenon of yeast-life, they show that yeast is a plant which does not differ from ordinary plants, and which manifests its fermentative power solely in consequence of particular conditions under which it is compelled to live. It may carry on its life as a ferment or not, and after having lived without manifesting the slightest symptom of fermentative character, it is quite ready to manifest that character when brought under suitable conditions. The fermentative property, therefore, is not a power peculiar to cells of a special nature. It is not a permanent character of a particular structure, like, for instance, the property of acidity or alkalinity. It is a peculiarity dependent on external circumstances and on the nutritive conditions of the organism.

§ II.—Fermentation in Saccharine Fruits Immersed in Carbonic Acid Gas.

The theory which we have, step by step, evolved, on the subject of the causes of the chemical phenomena of fermentation, may claim a character of simplicity and generality that is well worthy of attention. Fermentation is no longer one of those isolated and mysterious phenomena which do not admit of explanation. It is the consequence of a peculiar vital process of nutrition which occurs under certain conditions, differing from those which characterize the life of all ordinary beings, animal or vegetable, but by which the latter may be affected, more or less, in a way which brings them, to some extent. within the class of ferments, properly so called. We can even conceive that the fermentative character may belong to every organized form, to every animal or vegetable cell, on the sole condition that the chemico-vital acts of assimilation and excretion must be capable of taking place in that cell for a brief period, longer or shorter it may be, without the necessity for recourse to supplies of atmospheric oxygen; in other words, the cell must be able to derive its needful heat from the decomposition of some body which yields a surplus of heat in the process.

As a consequence of these conclusions it should be an easy matter to show, in the majority of living beings, the manifestation of the phenomena of fermentation; for there are, probably, none in which all chemical action entirely disappears, upon the sudden cessation of life. One day, when we were expressing these views in our laboratory, in the presence of M. Dumas, who seemed inclined to admit their truth, we added: “We would make a wager that if we were to plunge a bunch of grapes into carbonic acid gas, there would be immediately produced alcohol and carbonic acid, in consequence of a renewed action starting in the interior cells of the grapes, in such a way that these cells would assume the function of yeast-cells. We will make the experiment, and when you come to-morrow—it was our good fortune to have M. Dumas working in our laboratory at that time—we will give you an account of the result.” Our predictions were realized. We then endeavoured to find, in the presence of M. Dumas, who assisted us in our endeavour, cells of yeast in the grapes; but it was quite impossible to discover any.[123]

Encouraged by this result, we undertook fresh experiments on grapes, on a melon, on oranges, on plums, and on rhubarb leaves, gathered in the garden of the _École Normale_, and, in every case, our substance when immersed in carbonic acid gas, gave rise to the production of alcohol and carbonic acid. We obtained the following surprising results from some _prunes de Monsieur_[124]:—On July 31st, 1872, we placed twenty-four of these plums under a glass cylinder, which we immediately filled with carbonic acid gas. The plums had been gathered on the previous day. By the side of the cylinder we placed other twenty-four plums, which were left there uncovered. Eight days afterwards, in the course of which time there had been a considerable evolution of carbonic acid from the cylinder, we withdrew the plums and compared them with those which had been left exposed to the air. The difference was striking, almost incredible. Whilst the plums which had been surrounded with air (the experiments of Bérard have long since taught us that, under this latter condition, fruits absorb oxygen from the air and emit carbonic acid gas in almost equal volume) had become very soft and watery and sweet, the plums taken from under the jar had remained very firm and hard, the flesh was by no means watery, but they had lost much sugar. Lastly, when submitted to distillation, after crushing, they yielded 6·5 grammes (99·7 grains) of alcohol, more than 1 per cent. of the total weight of the plums. What better proof could we have than these facts of the existence of a considerable chemical action in the interior of fruit, an action which derives the heat necessary for its manifestation from the decomposition of the sugar present in the cells? Moreover, and this circumstance is especially worthy of our attention, in all these experiments we found that there was a liberation of heat, of which the fruits and other organs were the seat, as soon as they were plunged in the carbonic acid gas. This heat is so considerable that it may at times be detected by the hand, if the two sides of the cylinder, one of which is in contact with the objects, are touched alternately. It also makes itself evident in the formation of vapour, which condenses in little drops on those parts of the bell which are less directly exposed to the influence of the heat resulting from the decomposition of the sugar of the cells.[125]

In short, fermentation is a very general phenomenon. It is life without air, or life without free oxygen, or, more generally still, it is the result of a chemical process accomplished on a fermentable substance, _i.e._ a substance capable of producing heat by its decomposition, in which process the entire heat used up is derived from a part of the heat that the decomposition of the fermentable substance sets free. The class of fermentations, properly so called, is, however, restricted by the small number of substances capable of decomposing with the production of heat, and at the same time of serving for the nourishment of lower forms of life, when deprived of the presence and action of air. This, again, is a consequence of our theory, which is well worthy of notice.

The facts that we have just mentioned in reference to the formation of alcohol and carbonic acid in the substance of ripe fruits, under certain special conditions, and apart from the action of ferment, are already known to science. They were discovered in 1869 by M. Lechartier, formerly a pupil in the _École Normale Supérieure_, and his coadjutor, M. Bellamy.[126] In 1821, in a very remarkable work, especially when we consider the period when it appeared, Bérard demonstrated several important propositions in connection with the maturation of fruits:—

I. All fruits, even those that are still green, and likewise even those that are exposed to the sun, absorb oxygen and set free an almost equal volume of carbonic acid gas. This is a condition of their proper ripening.

II. Ripe fruits placed in a limited atmosphere, after having absorbed all the oxygen and set free an almost equal volume of carbonic acid, continue to emit that gas in notable quantity, even when no bruise is to be seen—“as though by a kind of fermentation,” as Bérard actually observes—and lose their saccharine particles, a circumstance which causes the fruits to appear more acid, although the actual weight of their acid may undergo no augmentation whatever.

In this beautiful work, and in all subsequent ones of which the ripening of fruits has been the subject, two facts of great theoretical value have escaped the notice of the authors; these are the two facts which Messrs. Lechartier and Bellamy pointed out, for the first time, namely, the production of alcohol and the absence of cells of ferments. It is worthy of remark that these two facts, as we have shown above, were actually foreshadowed in the theory of fermentation that we advocated as far back as 1861, and we are happy to add that Messrs. Lechartier and Bellamy, who, at first, had prudently drawn no theoretical conclusions from their work, now entirely agree with the theory we have advanced.[127] Their mode of reasoning is very different from that of the savants with whom we discussed the subject before the Academy, on the occasion when the communication which we addressed to the Academy, in October, 1872, attracted attention once more to the remarkable observations of Messrs. Lechartier and Bellamy.[128] M. Fremy, in

## particular, was desirous of finding in those observations a confirmation

of his views on the subject of _hemi-organism_, and a condemnation of ours, notwithstanding the fact that the preceding explanations and, more

## particularly our Note of 1861, which we have quoted word for word in the

last paragraph, furnish the most conclusive evidence in favour of those ideas which we advocate. Indeed, as far back as 1861 we pointed out very clearly that if we could find plants able to live when deprived of air, in the presence of sugar, they would bring about a fermentation of that substance, in the same manner as yeast does. Such is the case with the fungi already studied in