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.
"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 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 cc. (rather more than 5 fluid ounces) of yeast— water, containing 2 1/2 per cent, of milk-sugar. The solution was prepared in one of our double-necked flasks, with the necessary precautions to secure the 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 November 15th, pouring some wort on the deposit of the plant, which remained in the flask. In less than five hours from the time we placed it in the oven, the plant 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 cause 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 tinder 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 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 should like to 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 gas, in consequence of a renewed action starting in the interior cells of the grapes, in such a way that these cells would assume the functions 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. [Footnote: 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 miscroscopical 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 the 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 into 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 once 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 a priori judgment on the merits of the new system, which had not been carefully studied, although already widely adopted, of milled, cylindrical crushers, for pressing the vintage.]
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 Ecole 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:[Footnote: 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 east to understand how, in the thickness of certain fruits, certain parts of those fruits might be deprived of air, under which circumstances they would have been acting under conditions similar to those under which fruits act when wholly immersed in the carbonic acid gas. Moreover, it would be useful to determine whether alcohol is not a normal product of vegatation.]—On July 21, 1872, we placed twenty-four of these plums under a glass bell, which we immediately filled with carbonic acid gas. The plums had been gathered on the previous day. By the side of the bell 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 bell, 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 Berard 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 than these facts could we have 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 bell, one of which is in contact with the objects, are touched alternately. It also makes itself evident in the formation of 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. [Footnote: In these studies of plants living immersed in carbonic acid gas, we have come across a fact which corroborated 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 or beer, develop when deprived of air, and which shows, consequently, how very marked their aerobian character is. If we immerse beet-roots 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 beet-roots 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 nave directed the attention of the manufacturers of beet-root sugar to this point.]
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 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 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 Ecole Normale Superieure, and his coadjutor, M. Bellamy. [Footnote: Lechartier and Bellamy, Comptes rendus de l'Academie des Sciences, vol. lxix., pp., 366 and 466, 1869.] In 1821, in a very remarkable work, especially when we consider the period when it appeared, Berard 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 Berard 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 fore-shadowed 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. [Footnote: 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 principle 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 PRECESSES 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.] 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. [Footnote: PASTEUR, Faites nouveaux pour servir a la connaissance de la theorie des fermentations proprement dites. (Comptes rendus de l'Academie 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.] M. Fremy, in particular, was desirous of finding in these 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, quoted word for word in the preceding section, furnish the most conclusive evidence in favor 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 that yeast does. Such is the case with the fungi already studied; such, too, is the case with the fruits employed in the experiments of Messrs. Lechartier and Bellamy, and in our own experiments, the results of which not only confirm those obtained by these gentlemen, but even extend them, in so far as we have shown that fruits, when surrounded with carbonic acid gas immediately produce alcohol. When surrounded with air, they live in their aerobian state and we have no fermentation; immersed immediately afterwards in carbonic acid gas, they now assume their anaerobian state, and at once begin to act upon the sugar in the manner of ferments, and emit heat. As for seeing in these facts anything like a confirmation of the theory of hemi-organism, imagined by M. Fremy, the idea of such a thing is absurd. The following, for instance, is the theory of the fermentation of the vintage, according to M. Fremy. [Footnote: Comptes rendus, meeting of January 15th, 1872.]
"To speak here of alcoholic fermentation alone," our author says, "I hold that in the production of wine it is the juice of the fruit itself that, in contact with air, produces grains of ferment, by the transformation of the albuminous matter; Pasteur, on the other hand, maintains that the fermentation is produced by germs existing outside of the grapes." [Footnote: 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'Academie, 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 the exterior surface, or existing in the water employed, or on the side 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 and 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.]
Now what bearing on this purely imaginary theory can the fact have, that a whole fruit, immersed in carbonic acid gas, immediately produces alcohol and carbonic acid? In the preceding passage which we have borrowed from M. Fremy, an indispensable condition of the transformation of the albuminous matter is the contact with air and the crushing of the grapes. Here, however, we are dealing with UNINJURED FRUITS IN CONTACT WITH CARBONIC ACID GAS. Our theory, on the other hand, which, we may repeat, we have advocated since 1861, maintains that all cells become fermentative when their vital action is protracted in the absence of air, which are precisely the conditions that hold in the experiments on fruits immersed in carbonic acid gas. The vital energy is not immediately suspended in their cells, and the latter are deprived of air. Consequently, fermentation must result. Moreover, we may add, if we destroy the fruit, or crush it before immersing it in the gas, it no longer produces alcohol or fermentation of any kind, a circumstance that may be attributed to the fact of the destruction of vital action in the crushed fruit. On the other hand, in what way ought this crushing to affect the hypothesis of hemi-organism? The crushed fruit ought to act quite as well, or even better than that which is uncrushed. In short, nothing can be more directly opposed to the theory of the mode of manifestation of that hidden force to which the name of hemi-organism has been given, than the discovery of the production of these phenomena of fermentation in fruits surrounded with carbonic acid gas; whilst the theory, which sees in fermentation a consequence of vital energy in absence of air, finds in these facts the strictest confirmation of an express prediction, which from the first formed an integral part of its statement.
We should not be justified in devoting further time to opinions which are not supported by any serious experiment. Abroad, as well as in France, the theory of the transformation of albuminous substances into organized ferments had been advocated long before it had been taken up by M. Fremy. It no longer commands the slightest credit, nor do any observers of note any longer give it the least attention; it might even be said that it has become a subject of ridicule.
An attempt has also been made to prove that we have contradicted ourselves, inasmuch as in 1860 we published our opinion that alcoholic fermentation can never occur without a simultaneous occurrence of organization, development, and multiplication of globules; or continued life, carried on from globules already formed. [Footnote: PASTEUR, Memoire 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 of 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 Thenard 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 these illustrious masters, to whom we are indebted for our earliest knowledge of this subject.] Nothing, however, can be truer than that opinion, and at the present moment, after fifteen years of study devoted to the subject since the publication to which we have referred, we need no longer say, "we think," but instead, "we affirm," that it is correct. It is, as a matter of fact, to alcoholic fermentation, properly so called, that the charge to which we have referred relates—to that fermentation which yields, besides alcohol, carbonic acid, succinic acid, glycerine, volatile acids, and other products. This fermentation undoubtedly requires the presence of yeast—cells under the conditions that we have named. Those who have contradicted us have fallen into the error of supposing that the fermentation of fruits is an ordinary alcoholic fermentation, identical with that produced by beer yeast, and that, consequently, the cells of that yeast must, according to own theory, be always present. There is not the least authority for such a supposition. When we come to exact quantitative estimations—and these are to be found in the figures supplied by Messrs. Lechartier and Bellamy—it will be seen that the proportions of alcohol and carbonic acid gas produced in the fermentation of fruits differ widely from those that we find in alcoholic fermentations properly so called, as must necessarily be the case since in the former the fermentaction is effected by the cells of a fruit, but in the latter by cells of ordinary alcoholic ferment. Indeed we have a strong conviction that each fruit would be found to give rise to special action, the chemical equation of which would be different from that in the case of other fruits. As for the circumstance that the cells of these fruits cause fermentation without multiplying, this comes under the kind of activity which we have already distinguished by the expression CONTINUOUS LIFE IN CELLS ALREADY FORMED.
We will conclude this section with a few remarks on the subject of equations of fermentations, which have been suggested to us principally in attempts to explain the results derived from the fermentation of fruits immersed in carbonic acid gas.
Originally, when fermentations were put amongst the class of decompositions by contact-action, it seemed probable, and, in fact, was believed, that every fermentation has its own well- defined equation which never varied. In the present day, on the contrary, it must be borne in mind that the equation of a fermentation varies essentially with the conditions under which that fermentation is accomplished, and that a statement of this equation is a problem no less complicated than that in the case of the nutrition of a living being. To every fermentation may be assigned an equation in a general sort of way, an equation, however, which, in numerous points of detail, is liable to the thousand variations connected with the phenomena of life. Moreover, there will be as many distinct fermentations brought about by one ferment as there are fermentable substances capable of supplying the carbon element of the food of that same ferment, in the same way that the equation of the nutrition of an animal will vary with the nature of the food which it consumes. As regards fermentation producing alcohol, which may be effected by several different ferments, there will be as in the case of a given sugar, as many general equations as there are ferments, whether they be ferment-cells properly so called, or cells of the organs of living beings functioning as ferments. In the same way the equation of nutrition varies in the case of different animals nourished on the same food. And it is from the same reason that ordinary wort produces such a variety of beers when treated with the numerous alcoholic ferments which we have described. These remarks are applicable to all ferments alike; for instance, butyric ferment is capable of producing a host of distinct fermentations, in consequence of its ability to derive the carbonaceous part of its food from very different substances, from sugar, or lactic acid, or glycerine, or mannite, and many others.
When we say that every fermentation has its own peculiar ferment, it must be understood that we are speaking of the fermentation considered as a whole, including all the accessory products. We do not mean to imply that the ferment in question is not capable of acting on some other fermentable substance and giving rise to fermentation of a very different kind. Moreover, it is quite erroneous to suppose that the presence of a single one of the products of a fermentation implies the co-existence of a particular ferment. If, for example, we find alcohol among the products of a fermentation, or even alcohol and carbonic acid gas together, this does not prove that the ferment must be an alcoholic ferment, belonging to alcoholic fermentations, in the strict sense of the term. Nor, again, does the mere presence of lactic acid necessarily imply the presence of lactic ferment. As a matter of fact, different fermentations may give rise to one or even several identical products. We could not say with certainty, from a purely chemical point of view, that we were dealing, for example, with an alcoholic fermentation properly so called, and that the yeast of beer must be present in it, if we had not first determined the presence of all the numerous products of that particular fermentation under conditions similar to those under which the fermentation in question had occurred. In works on fermentation the reader will often find those confusions against which we are now attempting to guard him. It is precisely in consequence of not having had their attention drawn to such observations that some have imagined that the fermentation in fruits immersed in carbonic acid gas is in contradiction to the assertion which we originally made in our Memoir on alcoholic fermentation published in 1860, the exact words of which we may here repeat:—"The chemical phenomena of fermentation are related essentially to a vital activity, beginning and ending with the latter; we believe that alcoholic fermentation never occurs"—we were discussing the question of ordinary alcoholic fermentation produced by the yeast of beer—"without the simultaneous occurrence of organization, development, and multiplication of globules, or continued life, carried on by means of the globules already formed. The general results of the present Memoir seem to us to be it direct opposition to the opinions of MM. Liebig and Berzelius." These conclusions, we repeat, are as true now as they ever were, and are as applicable to the fermentation of fruits, of which nothing was known in 1860, as they are to the fermentation produced by the means of yeast. Only, in the case of fruits, it is the cells of the parenchyma that function as ferment, by a continuation of their activity in carbonic acid gas whilst in the other case the ferment consists of cells of yeast.
There should be nothing very surprising in the fact that fermentation can originate in fruits and form alcohol without the presence of yeast, if the fermentation of fruits were not confounded completely with alcoholic fermentation yielding the same products and in the same proportions. It is through the misuse of words that the fermentation of fruits has been termed alcoholic, in a way which has misled many persons. [Footnote: See, for example, the communications of MM. Colin and Poggiale, and the discussion on them. In the Bulletin de l'Academie de Medecine, March 2d, 9th, and 30th, and February 16th and 23rd, 1875.] In this fermentation, neither alcohol nor carbonic acid gas exists in those proportions in which they are found in fermentation produced by yeast; and, although we may determine in it the presence of succinic acid, glycerine, and a small quantity of volatile acids [Footnote: We have elsewhere determined the formation of minute quantities of volatile acids in alcoholic fermentation. M. Bechamp, 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' Academie, t. xlvii., P. 224, 1858] the relative proportions of these substances will be different from what they are in the case of alcoholic fermentation.
III. REPLY TO CERTAIN CRITICAL OBSERVATIONS OF THE GERMAN NATURALISTS, OSCAR BREFELD AND MORITZ TRAUBE.
The essential point of the theory of fermentation which we have been concerned in proving in the preceding paragraphs may be briefly put in the statement that ferments properly so called constitute a class of beings possessing the faculty of living out of contact with free oxygen; or, more concisely still, we may say that fermentation is a result of life without air.
If our affirmation were inexact, if ferment cells did require for their growth or for their increase in number or weight, as all other vegetable cells do, the presence of oxygen, whether gaseous or held in solution in liquids, this new theory would lose all value, its very raison d'etre would be gone, at least as far as the most important part of fermentations is concerned. This is precisely what M. Oscar Brefeld has endeavoured to prove in a Memoir read to the Physico-Medical Society of Wurzburg on July 26th, 1873, in which, although we have ample evidence of the great experimental skill of its author, he has nevertheless, in our opinion, arrived at conclusions entirely opposed to fact.
"From the experiments which I have just described," he says, "it follows, in the most indisputable manner, that A FERMENT CANNOT INCREASE WITHOUT FREE OXYGEN. Pasteur's supposition that a ferment, unlike all other living organisms, can live and increase at the expense of oxygen held in combination, is, consequently, altogether wanting in any solid basis of experimental proof. Moreover, since, according to the theory of Pasteur, it is precisely this faculty of living and increasing at the expense of the oxygen held in combination that constitutes the phenomenon of fermentation, it follows that the whole theory, commanding though it does such general assent, is shown to be untenable; it is simply inaccurate."
The experiments to which Dr. Brefeld alludes, consisted in keeping under continued study with the microscope, in a room specially prepared for the purpose, one or more cells of ferment in wort in an atmosphere of carbonic acid gas free from the least traces of free oxygen. We have, however, recognized the fact that the increase of a ferment out of contact with air is only possible in the case of a very young specimen; but our author employed brewer's yeast taken after fermentation, and to this fact we may attribute the non-success of his growths. Dr. Brefeld, without knowing it, operated on yeast in one of the states in which it requires gaseous oxygen to enable it to germinate again. A perusal of what we have previously written on the subject of the revival of yeast according to its age will show how widely the time required for such revival may vary in different cases. What may be perfectly true of the state of a yeast to-day may not be so to-morrow, since yeast is continually undergoing modifications. We have already shown the energy and activity with which a ferment can vegetate in the presence of free oxygen, and we have pointed out the great extent to which a very small quantity; of oxygen held in solution in fermenting liquids can operate at the beginning of fermentation. It is this oxygen that produces revival in the cells of the ferment and enables them to resume the faculty of germinating and continuing their life, and of multiplying when deprived of air.
In our opinion, a simple reflection should have guarded Dr. Brefeld against the interpretation which he has attached to his observations. If a cell of ferment cannot bud or increase without absorbing oxygen, either free or held in solution in the liquid, the ratio between the weight of the ferment formed during fermentation and that of oxygen used up must be constant. We had, however, clearly established, as far back as 1861, the fact that this ratio is extremely variable, a fact, moreover, which is placed beyond doubt by the experiments described in the preceding section. Though but small quantities of oxygen are absorbed, a considerable weight of ferment may be generated; whilst if the ferment has abundance of oxygen at its disposal, it will absorb much, and the weight of yeast formed will be still greater. The ratio between the weight of ferment formed and that of sugar decomposed may pass through all stages within certain very wide limits, the variations depending on the greater or less absorption of free oxygen. And in this fact, we believe, lies one of the most essential supports of the theory which we advocate. In denouncing the impossibility, as he considered it, of a ferment living without air or oxygen, and so acting in defiance of that law which governs all living beings, animal or vegetable, Dr. Brefeld ought also to have borne in mind the fact which we have pointed out, that alcoholic yeast is not the only organized ferment which lives in an anaerobian state. It is really a small matter that one more ferment should be placed in a list of exceptions to the generality of living beings, for whom there is a rigid law in their vital economy which requires for continued life a continuous respiration, a continuous supply of free oxygen. Why, for instance, has Dr. Brefeld omitted the facts bearing on the life of the vibrios of butyric fermentation? Doubtless he thought we were equally mistaken in these: a few actual experiments would have put him right.
These remarks on the criticisms of Dr. Brefeld are also applicable to certain observations of M. Moritz Traube's, although, as regards the principal object of Dr. Brefeld's attack, we are indebted to M. Traube for our defence. This gentleman maintained the exactness of our results before the Chemical Society of Berlin, proving by fresh experiments that yeast is able to live and multiply without the intervention of oxygen. "My researches," he said, "confirm in an indisputable manner M. Pasteur's assertion that the multiplication of yeast can take place in media which contain no trace of free oxygen. ... M. Brefeld's assertion to the contrary is erroneous." But immediately afterwards M. Traube adds: "Have we here a confirmation of Pasteur's theory? By no means. The results of my experiments demonstrate on the contrary that this theory has no true foundation." What were these results? Whilst proving that yeast could live without air, M. Traube, as we ourselves did, found that it had great difficulty in living under these conditions; indeed he never succeeded in obtaining more than the first stages of true fermentation. This was doubtless for the two following reasons: first, in consequence of the accidental production of secondary and diseased fermentations which frequently prevent the propagation of alcoholic ferment; and, secondly, in consequence of the original exhausted condition of the yeast employed. As long ago as 1861, we pointed out the slowness and difficulty of the vital action of yeast when deprived of air; and a little way back, in the preceding section, we have called attention to certain fermentations that cannot be completed under such conditions without going into the causes of these peculiarities. M. Traube expresses himself thus: "Pasteur's conclusion, that yeast in the absence of air is able to derive the oxygen necessary for its development from sugar, is erroneous; its increase is arrested even when the greater part of the sugar still remains undecomposed. IT IS IN A MIXTURE OF ALBUMINOUS SUBSTANCES THAT YEAST, WHEN DEPRIVED OF AIR, FINDS THE MATERIALS FOR ITS DEVELOPMENT." This last assertion of M. Traube's is entirely disproved by those fermentation experiments in which, after suppressing the presence of albuminous substances, the action, nevertheless, went on in a purely inorganic medium, out of contact with air, a fact, of which we shall give irrefutable proofs. [Footnote: Traube's conceptions are 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 gas 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.]
IV. FERMENTATION OF DEXTRO-TARTRATE OF LIME.
[Footnote: See PASTEUR, Comptes rendus de l'Academie des Sciences, t. lvi., p. 416.]
Tartrate of lime, in spite of its insolubility in waters is capable of complete fermentation in a mineral medium.
If we put some pure tartrate of lime, in the form of a granulated, crystalline powder, into pure water, together with some sulphate of ammonia and phosphates of potassium and magnesium, in very small proportions, a spontaneous fermentation will take place in the deposit in the course of a few days, although no germs of ferment have been added. A living, organized ferment, of the vibrionic type, filiform, with tortuous motions, and often of immense length, forms spontaneously by the development of some germs derived in some way from the inevitable particles of dust floating in the air or resting on the surface of the vessels or material which we employ. The germs of the vibrios concerned in putrefaction are diffused around us on every side, and, in all probability, it is one or more of these germs that develop in the medium in question. In this way they effect the decomposition of the tartrate, from which they must necessarily obtain the carbon of their food without which they cannot exist, while the nitrogen is furnished by the ammonia of the ammoniacal salt, the mineral principles by the phosphate of potassium and magnesium, and the sulphur by the sulphate of ammonia. How strange to see organization, life, and motion originating under such conditions! Stranger still to think that this organization, life, and motion are effected without the participation of free oxygen. Once the germ gets a primary impulse on its living career by access of oxygen, it goes on reproducing indefinitely, absolutely without atmospheric air. Here then we have a fact which it is important to establish beyond the possibility of doubt, that we may prove that yeast is not the only organized ferment able to live and multiply when out of the influence of free oxygen.
Into a flask, like that represented in FIG. 9, of 2.5 litres (about four pints) in capacity, we put:
Pure, crystallized, neutral tartrate of lime. .. 100 grammes Phosphate of ammonia. ... . ... . .. ... . ... 1 grammes Phosphate of magnesium. ... . ... . ... . ... .. 1 grammes Phosphate of potassium. ... . ... . ... . .. 0.5 grammes Sulphate of ammonia. ... . ... . ... . ... .. 0.5 grammes (1 gramme = 15.43 grains)
To this we added pure distilled water, so as entirely to fill the flask.
In order to expel all the air dissolved in the water and adhering to the solid substances, we first placed our flask in a bath of chloride of calcium in a large cylindrical white iron pot set over a flame. The exit tube of the flask was plunged in a test tube of Bohemian glass three-quarters full of distilled water, and also heated by a flame. We boiled the liquids in the flask and test-tube for a sufficient time to expel all the air contained in them. We then withdrew the heat from under the test- tube, and immediately afterwards covered the water which it contained with a layer of oil and then permitted the whole apparatus to cool down.
Next day we applied a finger to the open extremity of the exit- tube, which we then plunged in a vessel of mercury. In this particular experiment which we are describing, we permitted the flask to remain in this state for a fort-night. It might have remained there for a century without ever manifesting the least sign of fermentation, the fermentation of the tartrate being a consequence of life, and life after boiling no longer existed in the flask. When it was evident that the contents of the flask were perfectly inert, we impregnated them rapidly, as follows: all the liquid contained in the exit-tube was removed by means of a fine caoutchouc tube, and replaced by about 1 c. (about 17 minims) of liquid and deposit from another flask, similar to the one we have just described, but which had been fermenting spontaneously for twelve days; we lost no time in refilling completely the exit tube with water which had been first boiled and then cooled down in carbonic acid gas. This operation lasted only a few minutes. The exit-tube was again plunged under mercury. Subsequently the tube was not moved from under the mercury, and as it formed part of the flask, and there was neither cork nor india-rubber, any introduction of air was consequently impossible. The small quantity of air introduced during the impregnation was insignificant and it might even be shown that it injured rather than assisted the growth of the organisms, inasmuch as these consisted of adult individuals which had lived without air and might be liable to be damaged or even destroyed by it. Be this as it may, in a subsequent experiment we shall find the possibility removed of any aeration taking place in this way, however infinitesimal, so that no doubts may linger on this subject.
The following days the organisms multiplied, the deposit of tartrate gradually disappeared, and a sensible ferment action was manifest on the surface, and throughout the bulk of the liquid. The deposit seemed lifted up in places, and was covered with a layer of dark-grey colour, puffed up, and having an organic and gelatinous appearance. For several days, in spite of this action in the deposit, we detected no disengagement of gas, except when the flask was slightly shaken, in which case rather large bubbles adhering to the deposit rose, carrying with them some solid particles, which quickly fell back again, whilst the bubbles diminished in size as they rose, from being partially taken into solution, in consequence of the liquid not being saturated. The smallest bubbles had even time to dissolve completely before they could reach the surface of the liquid. In course of time the liquid was saturated, and the tartrate was gradually displaced by mammillated crusts, or clear, transparent crystals of carbonate of lime at the bottom and on the sides of the vessel.
The impregnation took place on February 10th, and on March 15th the liquid was nearly saturated. The bubbles then began to lodge in the bent part of the exit-tube, at the top of the flask. A glass measuring-tube containing mercury was now placed with its open end over the point of the exit-tube under the mercury in the trough, so that no bubble might escape. A steady evolution of gas went on from the 17th to the 18th, 17.4 cc. (1.06 cubic inches) having been collected. This was proved to be nearly absolutely pure carbonic acid, as indeed might have been suspected from the fact that the evolution did not begin before a distinct saturation of the liquid was observed. [Footnote: Carbonic add being considerably more soluble than other gases possible under the circumstances.—ED.]
The liquid, which was turbid on the day after its impregnation, had, in spite of the liberation of gas, again become so transparent that we could read our handwriting through the body of the flask. Notwithstanding this, there was still a very active operation going on in the deposit, but it was confined to that spot. Indeed, the swarming vibrios were bound to remain there, the tartrate of lime being still more insoluble in water saturated with carbonate of lime than it is in pure water. A supply of carbonaceous food, at all events, was absolutely wanting in the bulk of the liquid. Every day we continued to collect and analyze the total amount of gas disengaged. To the very last it was composed of pure carbonic acid gas. Only during the first few days did the absorption by the concentrated potash leave a very minute residue. By April 26th all liberation of gas had ceased, the last bubbles having risen in the course of April 23rd. The flask had been all the time in the oven, at a temperature between 25 degrees C. and 28 degrees C. (77 degrees F. and 83 degrees F.). The total volume of gas collected was 2.135 litres (130.2 cubic inches). To obtain the whole volume of gas formed we had to add to this what was held in the liquid in the state of acid carbonate of lime. To determine this we poured a portion of the liquid from the flask into another flask of similar shape, but smaller, up to the gaugemark on the neck. [Footnote: 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. This smaller flask had been previously filled with carbonic acid. The carbonic acid of the fermented liquid was then expelled by means of heat, and collected over mercury. In this way we found a volume of 8.322 litres (508 cubic inches) of gas in solution, which, added to the 2.135 litres, gave a total of 10.457 litres (638.2 cubic inches) at 20 degrees and 760 mm., which, calculated to 0 degrees, C. and 760 mm. atmospheric pressure (32 degrees F. and 30 inches) gave a weight of 19.700 grammes (302.2 grains) of carbonic acid.
Exactly half of the lime in the tartrate employed got used up in the soluble salts formed during fermentation; the other half was partly precipitated in the form of carbonate of lime, partly dissolved in the liquid by the carbonic acid. The soluble salts seemed to us to be a mixture or combination of 1 equivalent of metacetate of lime, with 2 equivalents of the acetate, for every 10 equivalents of carbonic acid produced, the whole corresponding to the fermentation of 3 equivalents of neutral tartrate of lime. [Footnote: 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, alone 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 50 grammes (767 grains) of tartrate of lime.]. This point, however, is worthy of being studied with greater care: the present statement of the nature of the products formed is given with all reserve. For our point, indeed, the matter is of little importance, since the equation of the fermentation does not concern us.
After the completion of fermentation there was not a trace of tartrate of lime remaining at the bottom of the vessel: it had disappeared gradually as it got broken up into the different products of fermentation, and its place was taken by some crystallized carbonate of lime—the excess, namely, which had been unable to dissolve by the action of the carbonic acid. Associated, moreover, with this carbonate of lime there was a quantity of some kind of animal matter, which, under the microscope, appeared to be composed of masses of granules mixed with very fine filaments of varying lengths, studded with minute dots, and presenting all the characteristics of a nitrogenous organic substance. [Footnote: 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 degrees C. (212 degrees F.), the weight of the organic matter thus obtained was 0.54 gramme (8.3 grains), which was rather more than 1/200 of the weight of fermentable matter.] That this was really the ferment is evident enough from all that we have already said. To convince ourselves more thoroughly of the fact, and at the same time to enable us to observe the mode of activity of the organism, we instituted the following supplementary observation. Side by side with the experiment just described, we conducted a similar one, which we intermitted after the fermentation was somewhat advanced, and about half of the tartrate dissolved. Breaking off with a file the exit-tube at the point where the neck began to narrow off, we took some of the deposit from the bottom by means of a long straight piece of tubing, in order to bring it under microscopical examination. We found it to consist of a host of long filaments of extreme tenuity, their diameter being about 1/1000th of a millimetre (0.000039 in.); their length varied, in some cases being as much as 1/20th of a millimetre (0.0019 in.). A crowd of these long vibrios were to be seen creeping slowly along, with a sinuous movement, showing three, four, or even five flexures. The filaments that were at rest had the same aspect as these last, with the exception that they appeared punctuate, as though composed of a series of granules arranged in irregular order. No doubt these were vibrios in which vital action had ceased, exhausted specimens which we may compare with the old granular ferment of beer, whilst those in motion may be compared with young and vigorous yeast. The absence of movement in the former seems to prove that this view is correct. Both kinds showed a tendency to form clusters, the compactness of which impeded the movements of those which were in motion. Moreover, it was noticeable that the masses of these latter rested on tartrate not yet dissolved, whilst the granular clusters of the others rested directly on the glass, at the bottom of the flask, as if, having decomposed the tartrate, the only carbonaceous food at their disposal, they had then died on the spot where we captured them, from inability to escape, precisely in consequence of that state of entanglement which they combined to form, during the period of their active development. Besides these we observed vibrios of the same diameter, but of much smaller length, whirling round with great rapidity, and darting backwards and forwards; these were probably identical with the longer ones, and possessed greater freedom of movement, no doubt in consequence of their shortness. Not one of these vibrios could be found throughout the mass of the liquid.
We may remark that as there was a somewhat putrid odour from the deposit in which the vibrios swarmed, the action must have been one of reduction, and no doubt to this fact was due the greyish coloration of the deposit. We suppose that the substances employed, however pure, always contain some trace of iron, which becomes converted into the sulphide, the black colour of which would modify the originally white deposit of insoluble tartrate and phosphate.
But what is the nature of these vibrios? We have already said that we believe that they are nothing but the ordinary vibrios of putrefaction, reduced to a state of extreme tenuity by the special conditions of nutrition involved in the fermentable medium used; in a word, we think that the fermentation in question might be called putrefaction of tartrate of lime. It would be easy enough to determine this point by growing the vibrios of such fermentation in media adapted to the production of the ordinary forms of vibrio; but this is an experiment which we have not ourselves tried.
One word more on the subject of these curious beings. In a great many of them there appears to be something like a clear spot, a kind of bead, at one of their extremities. This is an illusion arising from the fact that the extremity of these vibrios is curved, hanging downwards, thus causing a greater refraction at that particular point, and leading us to think that the diameter is greater at that extremity. We may easily undeceive ourselves if we watch the movements of the vibrio, when we will readily recognize the bend, especially as it is brought into the vertical plane passing over the rest of the filament. In this way we will see the bright spot, THE HEAD, disappear, and then reappear.
The chief inference that it concerns us to draw from the preceding facts is one which cannot admit of doubt, and which we need not insist on any further—namely that vibrios, as met with in the fermentation of neutral tartrate of lime, are able to live and multiply when entirely deprived of air.
V.—ANOTHER EXAMPLE OF LIFE WITHOUT AIR—FERMENTATION OF LACTATE OF LIME
As another example of life without air, accompanied by fermentation properly so called, we may lastly cite the fermentation of lactate of lime in a mineral medium.
In the experiment described in the last paragraph, it will be remembered that the ferment liquid and the germs employed in its impregnation came in contact with air, although only for a very brief time. Now, notwithstanding that we possess exact observations which prove that the diffusion of oxygen and nitrogen in a liquid absolutely deprived of air, so far from taking place rapidly, is, on the contrary, a very slow process indeed; yet we were anxious to guard the experiment that we are about to describe from the slightest possible trace of oxygen at the moment of impregnation.
We employed a liquid prepared as follows: Into from 9 to 10 litres (somewhat over 2 gallons) of pure water the following salts [Footnote: 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.] were introduced successively, viz:
Pure lactate of lime. ... . ... . ... . ... . .. 225 grammes Phosphate of ammonia. ... . ... . ... . ... . .. 0.75 grammes Phosphate of potassium. ... . ... . ... . ... .. 0.4 grammes Sulphate of magnesium. ... . ... . ... . ... ... 0.4 grammes Sulphate of ammonia. ... . ... . ... . ... . ... 0.2 grammes (1 gramme = 15.43 grains.)
On March 23rd, 1875, we filled a 6 litre (about 11 pints) flask, of the shape represented in FIG. 11, and placed it over a heater. Another flame was placed below a vessel containing the same liquid, into which the curved tube of the flask plunged. The liquids in the flask and in the basin were raised to boiling together, and kept in this condition for more than half-an-hour, so as to expel all the air held in solution. The liquid was several times forced out of the flask by the steam, and sucked back again; but the portion which re-entered the flask was always boiling. On the following day when the flask had cooled, we transferred the end of the delivery tube to a vessel full of mercury and placed the whole apparatus in an oven at a temperature varying between 25 degrees C. and 30 degrees C. (77 degrees F. and 86 degrees F.) then, after having refilled the small cylindrical tap-funnel with carbonic acid, we passed into it with all necessary precautions 10 cc. (0.35 fl. oz) of a liquid similar to that described, which had been already in active fermentation for several days out of contact with air and now swarmed with vibrios. We then turned the tap of the funnel, until only a small quantity of liquid was left, just enough to prevent the access of air. In this way the impregnation was accomplished without either the ferment-liquid or the ferment- germs having been brought in contact, even for the shortest space, with the external air. The fermentation, the occurrence of which at an earlier or later period depends for the most part on the condition of the impregnating germs, and the number introduced in the act, in this case began to manifest itself by the appearance of minute bubbles from March 29th. But not until April 9th did we observe bubbles of larger size rise to the surface. From that date onward they continued to come in increasing number, from certain points at the bottom of the flask, where a deposit of earthy phosphates existed; and at the same time the liquid, which for the first few days remained perfectly clear, began to grow turbid in consequence of the development of vibrios. It was on the same day that we first observed a deposit on the sides of carbonate of lime in crystals.
It is a matter of some interest to notice here that, in the mode of procedure adopted, everything combined to prevent the interference of air. A portion of the liquid expelled at the beginning of the experiment, partly because of the increased temperature in the oven and partly also by the force of the gas, as it began to be evolved from the fermentative action, reached the surface of the mercury, where, being the most suitable medium we know for the growth of bacteria, it speedily swarmed with these organisms. [Footnote: 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 cc. (0.7 fl. oz.) Phosphate of potassium. ... . ... ...0.1 gramme (1.5 grains) Sulphate of magnesium. ... . ... . 0.1 gramme (1.5 grains) Tribasic phosphate of lime. ... ... 0.01 gramme (0.15 grain) Tartrate of ammonia. ... . ... . ... 0.2 gramme (3 grains)
This liquid, the author says, has a feeble acid reaction and forms a perfectly clear solution.] In this way any passage of air, if such a thing were possible, between the mercury and the sides of the delivery-tube was altogether prevented, since the bacteria would consume every trace of oxygen which might be dissolved in the liquid lying on the surface of the mercury. Hence it is impossible to imagine that the slightest trace of oxygen could have got into the liquid in the flask.
Before passing on we may remark that in this ready absorption of oxygen by bacteria we have a means of depriving fermentable liquids of every trace of that gas with a facility and success equal or even greater than by the preliminary method of boiling. Such a solution as we have described, if kept at summer heat, without any previous boiling, becomes turbid in the course of twenty-four hours from a SPONTANEOUS development of bacteria; and it is easy to prove that they absorb all the oxygen held in solution. [Footnote: On the rapid absorption of oxygen by bacteria, see also our Memoire of 1872, sur les Generations dites Spontanees, especially the note on page 78.] If we completely fill a flask of a few litres capacity (about a gallon) (Fig. 9) with the liquid described, taking care to have the delivery-tube also filled, and its opening plunged under mercury, and, forty- eight hours afterwards by means of a chloride of calcium bath, expel from the liquid on the surface of the mercury all the gas which it holds in solution, this gas, when analyzed, will be found to be composed of a mixture of nitrogen and carbonic acid gas, WITHOUT THE LEAST TRACE OF OXYGEN. Here, then, we have an excellent means of depriving the fermentable liquid of air; we simply have completely to fill a flask with the liquid, and place it in the oven, merely avoiding any addition of butyric vibrios, before the lapse of two or three days. We may wait even longer; and then, if the liquid does become impregnated spontaneously with vibrio germs, the liquid, which at first was turbid from the presence of bacteria, will become bright again, since the bacteria, when deprived of life, or, at least, of the power of moving, after they have exhausted all the oxygen in solution, will fall inert to the bottom of the vessel. On several occasions we have determined this interesting fact, which tends to prove that the butyric vibrios cannot be regarded as another form of bacteria, inasmuch as, on the hypothesis of an original relation between the two productions, butyric fermentation ought in every case to follow the growth of bacteria.
We may also call attention to another striking experiment, well suited to show the effect of differences in the composition of the medium upon the propagation of microscopic beings. The fermentation which we last described commenced on March 27th and continued until May 10th; that to which we are now to refer, however, was completed in four days, the liquid employed being similar in composition and quantity to that employed in the former experiment. On April 23, 1875, we filled a flask of the same shape as that represented in Fig. 11, and of similar capacity, viz., 6 litres, with a liquid composed as described at page 69. This liquid had been previously left to itself for five days in large open flasks, in consequence of which it had developed an abundant growth of bacteria. On the fifth day a few bubbles, rising from the bottom of the vessels, at long intervals, betokened the commencement of butyric fermentation, a fact, moreover, confirmed by the microscope, in the appearance of the vibrios of this fermentation in specimens of the liquid taken from the bottom of the vessels, the middle of its mass, and even in the layer on the surface that was swarming with bacteria. We transferred the liquid so prepared to the 6 litre flask arranged over the mercury. By evening a tolerably active fermentation had begun to manifest itself. On the 24th this fermentation was proceeding with astonishing rapidity, which continued during the 25th and 26th. During the evening of the 26th it slackened, and on the 27th all signs of fermentation had ceased. This was not, as might be supposed, a sudden stoppage due to some unknown cause; the fermentation was actually completed, for when we examined the fermented liquid on the 28th we could not find the smallest quantity of lactate of lime. If the needs of industry should ever require the production of large quantities of butyric acid, there would, beyond doubt, be found in the preceding fact valuable information in devising an easy method of preparing that product in abundance. [Footnote: 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 anaerobian vibrios, just as, under similar conditions, would have been the case if we had been dealing with a fermentation by ordinary yeast.]
Before we go any further, let us devote some attention to the vibrios of the preceding fermentations.
On May 27th, 1862, we completely filled a flask capable of holding 2.780 litres (about five pints) with the solution of lactate and phosphates. [Footnote: In this case the liquid was composed as follows: A saturated solution of lactate of lime, at a temperature of 25 degrees C. (77 degrees F.), was prepared, containing for every 1OO cc. (3 1/2 fl. oz.) 25.65 grammes (394 grains) of the lactate, C6 H5 O5 Ca O (NEW NOTATION, C6 H10 Ca O6) This solution was rendered very clear by the addition of 1 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 potassium. ... . ... . ... . ... 1 gramme Phosphate of magnesium. ... . ... . ... . ... 1 gramme Sulphate of ammonia. ... . ... . ... . ... 0.5 gramme]
We refrained from impregnating it with any germs. The liquid became turbid from a development of bacteria and then underwent butyric fermentation. By June 9th the fermentation had become sufficiently active to enable us to collect in the course of twenty-four hours, over mercury, as in all our experiments, about 100 cc. (about 6 cubic inches) of gas. By June 11th, judging from the volume of gas liberated in the course of twenty-four hours, the activity of the fermentation had doubled. We examined a drop of the turbid liquid. Here are the notes accompanying the sketch (Fig. 12) as they stand in our note-book: "A swarm of vibrios, so active in their movements that the eye has great difficulty in following them. They may be seen in pairs throughout the field, apparently making efforts to separate from each other. The connection would seem to be by some invisible, gelatinous thread, which yields so far to their efforts that they succeed in breaking away from actual contact, but yet are, for a while, so far restrained that the movements of one have a visible effect on those of the other. By and by, however, we see a complete separation effected, and each moves on its separate way with an activity greater than it ever had before."
One of the best methods that can be employed for the
microscopical examination of these vibrios, quite out of contact with air, is the following. After butyric fermentation has been going on for several days in a flask, (Fig. 13), we connect this flask by an india-rubber tube with one of the flattened bulbs previously described, which we then place on the stage of the microscope (Fig. 13). When we wish to make an observation we close, under the mercury, at the point B, the end of the drawn- out and bent delivery-tube. The continued evolution of gas soon exerts such a pressure within the flask, that when we open the tap R, the liquid is driven into the bulb LL, until it becomes quite full and the liquid flows over into the glass V. In this manner we may bring the vibrios under observation without their coming into contact with the least trace of air, and with as much success as if the bulb, which takes the place of an object glass, had been plunged into the very centre of the flask. The movements and fissiparous multiplication of the vibrios may thus be seen in all their beauty, and it is indeed a most interesting sight. The movements do not immediately cease when the temperature is suddenly lowered, even to a considerable extent, 15 degrees C. (59 degrees F.) for example; they are only slackened. Nevertheless, it is better to observe them at the temperatures most favourable to fermentation, even in the oven where the vessels employed in the experiment are kept at a temperature between 25 degrees C. and 30 degrees C. (77 degrees F. and 86 degrees F.).
We may now continue our account of the fermentation which we were studying when we made this last digression. On June 17th that fermentation produced three times as much gas as it did on June 11th, when the residue of hydrogen, after absorption by potash, was 72.6 per cent.; whilst on the 17th it was only 49.2 per cent. Let us again discuss the microscopic aspect of the turbid liquid at this stage. Appended is the sketch we made (Fig. 14) and our notes on it: "A most beautiful object: vibrios all in motion, advancing or undulating. They have grown considerably in bulk and length since the 11th; many of them are joined together in long sinuous chains, very mobile at the articulations, visibly less active and more wavering in proportion to the number that go to form the chain, of the length of the individuals." This description is applicable to the majority of the vibrios which occur in cylindrical rods and are homogeneous in aspect. There are others, of rare occurrence in chains, which have a clear corpuscle, that is to say, a portion more refractive than other parts of the segments, at one of their extremities. Sometimes the foremost segment has the corpuscle at one end, sometimes the other. The long segments of the commoner kind attain a length of from 10 to 30 and even 45 thousandths of a millimetre. Their diameter is from 1 1/2 to 2, very rarely 3, thousandths of a millimetre. [Footnote: 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.—D. C. R.]