Such is the large volume of oxygen necessary for the development of one gramme of yeast when the plant can assimilate this gas after the manner of an ordinary fungus.
Let us now return to the first experiment described in the paragraph on page 292 in which a flask of three litres capacity was filled with fermentable liquid, which, when caused to ferment, yielded 2.25 grammes of yeast, under circumstances where it could not obtain a greater supply of free oxygen than 16.5 cc. (about one cubic inch). According to what we have just stated, if this 2.25 grammes (34 grains) of yeast had not been able to live without oxygen, in other words, if the original cells had been unable to multiply otherwise than by absorbing free oxygen, the amount of that gas required could not have been less than 2.25 X 4l4 cc., that is, 931.5 cc. (56.85 cubic inches). The greater part of the 2.25 grammes, therefore, had evidently been produced as the growth of an anaerobian plant.
Ordinary fungi likewise require large quantities of oxygen for their development, as we may readily prove by cultivating any mould in a closed vessel full of air, and then taking the weight of plant formed and measuring the volume of oxygen absorbed. To do this, we take a flask of the shape shown in Fig. 8, capable of holding about 300 cc. (10 1/2 fluid ounces), and containing a liquid adapted to the life of moulds. We boil this liquid, and seal the drawn-out point after the steam has expelled the air wholly or in part; we then open the flask in a garden or in a room. Should a fungus-spore enter the flask, as will invariably be the case in a certain number of flasks out of several used in the experiment, except under special circumstances, it will develop there and gradually absorb all the oxygen contained in the air of the flask. Measuring the volume of this air, and weighing, after drying, the amount of plant formed, we find that for a certain quantity of oxygen absorbed we have a certain weight of mycelium, or of mycelium together with its organs of fructification. In an experiment of this kind, in which the plant was weighed a year after its development, we found for 0.008 gramme (0.123 gram) of MYCELIUM, dried at 100 degrees C. (212 degrees F.), an absorption that amounted to not less than 43 cc. (2.5 cubic inches) of oxygen at 25 degrees. These numbers, however, must vary sensibly with the nature of the mould employed, and also with the greater or less activity of its development, because the phenomena is complicated by the presence of accessory oxidations, such as we find in the case of mycoderma vini and aceti, to which cause the large absorption of oxygen in our last experiment may doubtless be attributed. [Footnote: 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 Memoire sur les Generations dites Spontanees, p. 54. note)—there is no doubt that an appreciable quantity of alcohol is formed because the plant does not immediately lose vital activity after the absorption of oxygen.
A 300 cc. (10-oz.) flask, containing 100 cc. of must, after the air in it had been expelled by boiling, was open and immediately re-closed on August 15th, 1873. A fungoid growth—a unique one, of greenish-grey colour—developed from spontaneous impregnation, and decolourized 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 degrees C. (212 degrees 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.]
The conclusions to be drawn from the whole of the preceding facts can scarcely admit of doubt. As for ourselves, we have no hesitation in finding them the foundation of the true theory of fermentation. In the experiments which we have described, fermentation by yeast, that is to say, by the type of ferments properly so called, is presented to us, in a word, as the direct consequence of the processes of nutrition, assimilation and life, when these are carried on without the agency of free oxygen. The heat required in the accomplishment of that work must necessarily have been borrowed from the decomposition of the fermentable matter, that is from the saccharine substance which, like other unstable substances, liberates heat in undergoing decomposition. Fermentation by means of yeast appears, therefore, to be essentially connected with the property possessed by this minute cellular plant of performing its respiratory functions, somehow or other, with oxygen existing combined in sugar. Its fermentative power—which power must not be confounded with the fermentative activity or the intensity of decomposition in a given time—varies considerably between two limits, fixed by the greatest and least possible access to free oxygen which the plant has in the process of nutrition. If we supply it with a sufficient quantity of free oxygen for the necessities of its life, nutrition, and respiratory combustions, in other words, if we cause it to live after the manner of a mould, properly so called, it ceases to be a ferment, that is, the ratio between the weight of the plant developed and that of the sugar decomposed, which forms its principal food, is similar in amount to that in the case of fungi. [Footnote: We find in M. Raulin's note 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 as 10/3.2=3.1." JULES RAULIN, Etudes chimiques sur la vegetation. Recherches sur le developpement d'une mucedinee dans un milieu artificiel, p. 192, Paris, 1870. We have seen in the case of yeast that this ratio may be as low as [Proofers note: unreadable symbol] On the other hand, if we deprive the yeast of air entirely, or cause it to develop in a saccharine medium deprived of free oxygen, it will multiply just as if air were present, although with less activity, and under these circumstances its fermentative character will be most marked; under these circumstances, moreover, we shall find the greatest disproportion, all other conditions being the same, between the weight of yeast formed and the weight of sugar decomposed. Lastly, if free oxygen occurs in varying quantities, the ferment-power of the yeast may pass through all the degrees comprehended between the two extreme limits of which we have just spoken. It seems to us that we could not have a better proof of the direct relation that fermentation bears to life, carried on in the absence of free oxygen, or with a quantity of that gas insufficient for all the acts of nutrition and assimilation.
Another equally striking proof of the truth of this theory is the fact previously demonstrated 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 aerobian 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 aerobian or anaerobian life, according to the conditions under which they are placed.
It may be readily understood how, in their state of aerobian life, the alcoholic ferments have failed to attract attention. These ferments are only cultivated out of contract 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 anaerobian 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 utilised in certain industrial operations, on account of their power in 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. [Footnote: 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 debris, as also, by checking the production of such organisms, we might considerably increase the proportion of nitrates in the artificial nitrogenous substances. By cultivating the 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 similar results.] On this last subject, the important work of M. Van Tieghem (Annales Scientifiques de l'Ecole 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. * * * *
It is a great presumption in favor 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 prima facie improbability. This is exactly the character of those ideas which we have just expounded. We pronounced 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 Comtes rendus de l'Academie 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 Societe Chimique de Paris:—
"M. Pasteur gives the result 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 into contact with oxygen.