II.
The light shed by these experiments quickly extended its sphere; and Pasteur lost no time in discovering a new ferment, that of butyric acid. Having shown the absolute independence which exists between the ferment of butyric acid and the others, he found, contrary to the general belief, that the lactic ferment is incapable of giving rise to butyric acid, and that there exists a butyric fermentation having its own special ferment. This ferment consists of a species of vibrio. Little transparent cylindrical rods, rounded at their extremities, isolated or united in chains of two or three, or sometimes even more, form these vibrios. They move by gliding, the body straight, or bending and undulating. They reproduce themselves by fission, and to this mode of generation their frequent arrangement in the form of a chain is due.
Sometimes one of the little rods, with a train of others behind it, agitates itself in a lively manner as if to detach itself from the rest. Often, also, the little rod, after being broken off, holds on still to its chain by a mucous transparent thread.
These little infusoriæ may be sown like the yeast of beer or the lactic ferment. If the medium in which they are sown is suitable for their nourishment, they will multiply to infinity; but the character most essential to be observed is, that they may be sown in a liquid which contains only ammonia and crystallisable substances, together with the fermentable substances, sugar, lactic acid, gum, &c. The butyric fermentation manifests itself as these little organisms multiply. Their weight sensibly increases, though it is always minute in comparison with the quantity of butyric acid produced; this is found to be the case in all other fermentations.
This experiment no doubt resembles those made with the alcoholic and lactic ferments. But it is distinguished from them by one circumstance eminently worthy of attention. The butyric ferment, by its motions and by its mode of generation, furnishes the irrefutable proof of its organisation and of its life. This ferment, moreover, presented to Pasteur a new and unexpected peculiarity. The vibrios live and multiply without the smallest supply of air or of free oxygen. Not only, indeed, do they live without air, but the air destroys them and arrests the fermentation which they initiate. If a current of pure carbonic acid is made to pass into the liquid where they are multiplying, their life and reproduction do not appear to be at all affected by it. If, on the contrary, instead of the current of carbonic acid we employ one of atmospheric air for only one or two hours, the vibrios fall without movement to the bottom of the vessel, and the butyric fermentation which was dependent on their existence is immediately arrested.
Pasteur designated this new class of organisms by the name of anaérobies; that is to say, beings which can live without air. He reserves the designation aérobies for all the other microscopic beings which, like the larger animals, cannot live without free oxygen. 'It matters little,' added Pasteur, 'whether the progress of science makes of this vibrio a plant or an animal; it is a living organism, endowed with motion, which is a ferment and which lives without air.'
In meditating upon these facts, and upon the general character of fermentation, Pasteur soon found himself in a position to approach more nearly to the essential nature of these mysterious phenomena. In what way do microscopic organisms provoke the phenomena of fermentation?
The organism eats, if one may say so, one part of the fermentable matter. But how does this phenomenon of nutrition differ so much from that of higher beings? In general, for a given weight of nutritive matter which the animal takes in, it assimilates a quantity of the same order. In fermentation, on the contrary, the ferment, while nourishing itself with fermentable matter, decomposes a quantity great in comparison to its own individual weight. Again, the butyric ferment lives without free oxygen. Is there not, said Pasteur, a hidden relation between the property of being a ferment and the faculty of living without free oxygen? Are not vibrios which imperatively require for their nutrition and multiplication the presence of oxygen gas those which will never have the properties of ferments?
Pasteur then contrived a series of experiments with the view of placing in parallelism these two curious physiological facts: life without air and the characteristics of ferments.
We know how wine and beer are prepared. The must of grapes and the must of beer are placed in wooden vats, or in barrels of greater or less dimensions. Whether the fermentation proceeds from germs taken from the exterior surface of the grapes, or from a small quantity of ferment sown in the must under the form of yeast, as in the fermentation of beer, the life of the ferment, its multiplication, the augmentation of its weight, are so many vital actions which to a certainty cannot borrow from the free oxygen of the external air, or from that originally dissolved in the must, an appreciable quantity of this gas. All the life of the cells of the ferment which multiplies itself indefinitely appears then to take place apart from free oxygen gas. In certain breweries in England the fermenting vats have sometimes a capacity of several thousands of hectolitres; and the fermentation liberates pure carbonic acid, a gas much heavier than atmospheric air, which rests on the surface of the liquid in the vat in a layer thick enough to protect the liquid underneath from any contact with the external air. All this liquid mass, then, is inclosed between the wooden sides of the vat and a deep layer of heavy gas which contains no trace of free oxygen. In this liquid, nevertheless, the life of the cells of the ferment and the production of all its constituents go on for several days with extraordinary activity. Here certainly we have life without air, and the ferment character expresses itself in the enormous difference between the weight of the ferment formed and collected from the vats under the name of yeast, at the end of the operation, and the weight of the sugar which has fermented, transforming itself into alcohol, carbonic acid, and various other products.
Pasteur has studied experimentally that which takes place when, without otherwise changing the conditions of these phenomena, the arrangement is so modified as to permit the introduction of the free oxygen of the atmosphere. It sufficed for this purpose to provoke a fermentation of the must of beer, or the must of grapes, upon shallow glass dishes presenting a large surface, or in a flat-bottomed wooden trough with sides a few centimeters in height, instead of in deep vats as before. In these new conditions the fermentation manifests an activity even more extraordinary than it did in the deep vats. The life of the ferment is itself singularly enhanced, but the proportion of the weight of the decomposed sugar to that of the yeast formed is absolutely different in the two cases. While, for example, in the deep vats, a kilogram of ferment sometimes decomposes seventy, eighty, one hundred, or even one hundred and fifty kilograms of sugar, in the shallow troughs one kilogram of the ferment will be found to correspond to only five or six kilograms of decomposed sugar. These proportions between the weight of the sugar which ferments and the weight of the ferment produced, constitute the measure of what one might call the ferment's character—of that character which distinguishes its mode of life from that of all other existences, great or small, in which the weight of the organising matter and the assimilated alimentary matter are about equal. In other words, the more free oxygen the yeast ferment consumes, the less is its power as a ferment. Such is the case in the shallow troughs where the extended surface is exposed to the contact of the oxygen of the air. The more, on the contrary, the life of the ferment is carried on without the presence of free oxygen, the greater is its power of decomposing and of fermenting the saccharine matter. This is the case in deep casks. The intimate co-relation then between life without air and fermentation appears complete.
The unexpected light which these facts threw upon the cause of the phenomena of fermentation made a forcible impression upon all thinking minds. 'In these infinitely small organisms,' M. Dumas said one day to M. Pasteur before the Academy of Sciences, 'you have discovered a third kingdom—the kingdom to which those organisms belong which, with all the prerogatives of animal life, do not require air for their existence, and which find the heat that is necessary for them in the chemical decompositions which they set up around them.'
The work of Pasteur, demonstrating that fermentation was always dependent on the life of a microscopic organism, continued without interruption. One of the most remarkable of his researches is that which relates to the fermentation of the tartrate of lime. The demonstration of life and of fermentation without free oxygen is in this paper carried to the utmost limits of experimental rigour and precision.