Part 8

The new memoirs published on the subject agreed in rejecting the hypothesis of any influence whatever of organisation or of life in the process of fermentation. Books, memoirs, dogmatic teaching, all were favourable to the theoretic ideas of Liebig. If a few rare observers indicated the presence in certain fermentations of living organisms, this presence was, in their opinion, a purely accidental fact, which, instead of favouring the phenomenon of fermentation, was injurious to it.

From his first investigation on lactic fermentation Pasteur was led to take an entirely different view of the matter. In this fermentation he recognised the presence and the action of a living organism, which was the ferment, just as yeast was the ferment of alcoholic fermentation. The lactic ferment was formed of cells, or rather of little rods nipped at their centres, extremely small, being hardly the thousandth part of a millimeter in diameter. It reproduced itself by fission--that is to say, the little rod divided itself at its middle and formed two shorter rods, which became elongated, nipped, in their turn, at their centres, each giving rise, as before, to two rods. Each of these, again, soon divided itself into two, and so on. Why had not this been observed prior to Pasteur? For the simple reason that chemists had never observed the production of lactic fermentation except in complex substances. They mixed chalk with their milk for the purpose of preserving the neutrality of the fermenting medium. They employed substances such as caseine, gluten, animal membranes, all of which, when examined by the microscope, exhibited a multitude of mineral or organic granules, with which the lactic ferment was confounded. Thus the first care of Pasteur, with the view of proving the presence of the ferment and its life, was to replace the cheesy matter and all its congeners by a soluble, nitrogenous body, which would permit of the microscopic examination of all the living cellular products.

In a memoir presented to the Academy of Sciences in 1857 Pasteur stated that there were 'cases where it is possible to recognise in lactic fermentation, as practised by chemists and manufacturers, above the deposit of chalk and the nitrogenous matter, a grey substance which forms a zone on the surface of the deposit. Its examination by the microscope hardly permits of its being distinguished from the disintegrated caseum or gluten which has served to start the fermentation. So that nothing indicates that it is a special kind of matter which had its birth during the fermentation. It is this, nevertheless, which plays the principal part.'

To isolate this substance and to prepare it in a state of purity, Pasteur boiled a little yeast with from fifteen to twenty times its weight of water. He then carefully filtered the liquid, dissolved in it about fifty grammes of sugar to the litre, and added to it some chalk. Taking then, by means of a drawn-out tube, from a good ordinary lactic fermentation a trace of the grey matter of which we have just spoken, he placed it as the seed of the ferment in the limpid saccharine solution. By the next day a lively and regular fermentation had set in, the liquid becoming turbid and the chalk disappearing, and one could distinguish a deposit which progressed continually as the chalk dissolved. This deposit was the lactic ferment.

Pasteur reproduced this experiment by substituting for the water of the yeast a clear decoction of nitrogenous plastic substances. The ferment invariably presented the same aspect and the same multiplication. These results, however, did not yet satisfy Pasteur. He desired more rigour in a subject of such theoretic importance. Might not the partisans of Liebig's theory argue, if not without subtlety yet with a semblance of justice, that the fermentation was not due to the formation and progressive growth of this feeble nitrogenous globular deposit, but rather to the nitrogenous matter dissolved during the decoction of the yeast used in the composition of the liquor? Up to a certain point it might be maintained that the dissolved matters which had been in contact with the oxygen of the air had been thrown into molecular motion, that this motion had been communicated to the fermentable matter, and that the deposit of the pretended organised ferment was but an accident--one of the physical changes or one of the precipitates so frequently observed in the modifications of albuminoid matters. In the observation of Cagniard-Latour and of Schwann as to the life of the yeast, Liebig saw nothing more. 'One cannot deny,' said he, 'the organisation of the yeast or its multiplication by budding, but these living cells are always associated with other dead cells in process of molecular alteration. It is these molecular motions which communicate themselves to the molecules of the sugar, break them up, and cause them to ferment.'

The arguments of Liebig derived great strength from the belief which was shared by all chemists that the cells of yeast perish during fermentation and form lactate of ammonia. On examining this assertion, Pasteur found that not only was there no ammonia formed during alcoholic fermentation, but that even if ammonia were added it disappeared, entering into the formation of new yeast cells. Was not this a proof of the potency of the organised ferment?

Tormented, however, by the idea that, notwithstanding all these facts, the reasonings of Liebig might still find some credit, Pasteur worked earnestly to discover new facts capable of demonstrating that Liebig's theory was absolutely false. He made two crucial experiments, the one relating to the yeast of beer, or of alcohol, and the other relating to the lactic ferment. He introduced into a pure solution of sugar a small quantity of crystallisable salt of ammonia, then some phosphates of potash and magnesia, and he sowed in this medium an imponderable quantity, if we may so express it, of fresh cells of yeast. The cells thus sown multiplied, and the sugar fermented. In other words, the phosphorus, the potassium, the magnesium of the mineral salts, united to form the substances which compose the ferment. By this experiment, so simple and yet so demonstrative, the power of the organisation of the ferment was once for all established. The contact theory of Berzelius had no longer any meaning, since it was evident that the fermentable matter here furnished to the ferment one of its essential elements, namely, carbon. Liebig's theory of communicated molecular motion, originating in a nitrogenous albuminoid substance, had no better claim, since such substances had been discarded. The whole process took place between the sugar and a ferment germ which owed its life and development to nutritive matters, the most important of which was the fermentable substance. Fermentation, in short, was simply a phenomenon of nutrition. The ferment augmented in weight, feeding upon the sugar, and its vitality was such that it contrived to build up the complex materials of its own organisation by means of sugar and purely mineral elements.

In a second experiment, Pasteur demonstrated that, notwithstanding their smallness and the possibility of confounding them with the amorphous granules of caseine and gluten, the little particles of lactic ferment were indeed alive, and that they, and they only, were the cause of lactic fermentation. He mixed with some water, sweetened with sugar, a small quantity of a salt of ammonia, some alkaline and earthy phosphates, and some pure carbonate of lime obtained by precipitation. At the end of twenty-four hours the liquid began to get turbid and to give off gas. The fermentation continued for some days. The ammonia disappeared, leaving a deposit of phosphates and calcareous salt. Some lactate of lime was formed, and at the same time one could notice the deposition of the little lactic ferment. The germs of the lactic ferment had, in this case, been derived from particles of dust adhering to the substances themselves, of which the mixtures were made, or to the vessels used, or from the surrounding air. The chapter on spontaneous generation will render this clear.

It suffices here to state that the results of this second experiment were absolutely conclusive, and that the theories of contact force or of communicated motion, which up to that time had reigned in science, were completely overthrown.

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.'

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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.

III.

But there is still another class of chemical phenomena where the life without air of microscopic organisms is fully shown. Pasteur proved that in the special fermentation which bears the name of putrefaction the primum movens of the putrefaction resides in microscopic vibrios of absolutely the same order as those which compose the butyric ferment. The fermentation of sugar, of mannite, of gums, of lactate of lime, by the butyric vibrio, so closely resembles the phenomena of putrefaction, that one might call these fermentations the putrefaction of sugar and of the other products.

If it has been thought right to call the fermentation of animal matters putrefaction, it is because at the moment of the decomposition of fibrine, of albumen, of blood, of gelatine, of the substance of the tendons, &c., the sulphur, and even the phosphorus, which enter into their composition give rise to putrid odours, due to the evil-smelling gases of sulphur and phosphorus.

The phenomena of putrefaction being then simply fermentations, differing only in regard to the chemical composition of the fermenting matters, Liebig naturally included them in his general theory of the decomposition of organic matters after death. At a period long antecedent to Pasteur's labours it had been established that there existed in putrefying matters fungi or microscopic animalculæ, and the idea had taken shape that these creatures might have an influence in the phenomena. The proofs were wanting, but the notion of a possible relation remained. We may read in his 'Lessons on Chemistry' with what disdain Liebig mentioned these hypothetical opinions.

'Those who pretend to explain the putrefaction of animal substances by the presence of animalculæ,' he wrote, 'reason very much like a child who would explain the rapidity of the Rhine by attributing it to the violent motions imparted to it in the direction of Bingen by the numerous wheels of the mills of Mayence. Is it possible to consider plants and animals as the causes of the destruction of other organisms when their own elements are condemned to undergo the same decompositions as the creatures which have preceded them? If the fungus is the cause of the destruction of the oak, if the microscopic animalcula is the cause of the putrefaction of the dead elephant, I would ask in my turn what is the cause which determines the putrefaction of the fungus or of the microscopic animalcula when life is withdrawn from these two organisms?'