Part 9

Thirty-two years later, and after Pasteur had accumulated, during more than twenty years, proof upon proof that the theory of Liebig would not stand examination, a physician of Paris, M. Bouillaud, asked, with the insistent voice of a querulous octogenarian: 'Let M. Pasteur then tell us here, in presence of the Académie de Médecine, what are the ferments of the ferments.'

Before replying to this argument, which Liebig and M. Bouillaud believed to be irrefutable, Pasteur, wishing to mark all the phases of the phenomena, expounded in a short preamble the part played by atmospheric oxygen in the destruction of animal and vegetable matters after death. It is easy to understand, indeed, that fermentation and putrefaction only represent the first phase of the return to the atmosphere and to the soil of all that has lived. Fermentations and putrefactions give rise to substances which are still very complex, although they represent the products of decomposition of fermentable matters. When sugar ferments, a large proportion of it becomes gas; but alongside of the carbonic acid gas which is formed, and which is, indeed, a partial return of the sugar to the atmosphere, new substances, such as alcohol, succinic acid, glycerine, and materials of yeast, are produced. When the flesh of animals putrifies, certain products of decomposition, also very complex, are formed with the vapour of water and the other gases of putrefaction. Where, then, does nature find the agents of destruction of these secondary products?

The great fact of the destruction of animal and vegetable matters is accomplished by slow combustion, through the appropriation of atmospheric oxygen. Here, again, one must banish from science the preconceived views which assumed that the oxygen seized directly on the organic matter after death, and that this matter was consumed by purely chemical processes. It is life that presides over this work of death.

If fermentation and putrefaction are principally the work of microscopic anaérobies, living without free oxygen, the slow combustion is found very largely, if not exclusively, to depend upon a class of infinitely small aérobies. It is these last which have the property of consuming the oxygen of the air. It is these lower organisms which are the powerful agents in the return to the atmosphere of all which has lived. Mildew, mould, bacteria, which we have already noticed, monads, two thousand of which would go to make up a millimeter, all these microscopic organisms are charged with the great work of re-establishing the equilibrium of life by giving back to it all that it has formed.

To demonstrate the important part played everywhere by these microscopic organisms, Pasteur made two experiments. He first introduced into vessels air deprived of all dust. This process we shall have occasion to examine in all its details, in connection with the researches on spontaneous generation. In these vessels, containing pure air, were placed the water of yeast with sugar dissolved in it, milk, sawdust--all of which had been deprived by heat of the germs of the lower organisms. The vessels and their contents were then subjected to a temperature of twenty-five to thirty-five degrees Centigrade. In a series of parallel experiments, made under the same conditions and at the same temperature, Pasteur took no steps to prevent the germination of the little seeds of mould suspended in the air, or associated with the substances contained in the vessels, neither did he avoid other infinitely small germs of the class aérobies.

After some time the air of all the vessels of the two series was submitted to analysis, when, behold, a very interesting fact! In the vessels where life had been withdrawn from the organic matters--that is to say, where there were no germs--the air still contained a large proportion of oxygen. In the vessels, on the contrary, where the microscopic organisms had been allowed to develop, the oxygen was totally absent, having been replaced by carbonic acid gas. And, further, for this absorption and total consumption of the oxygen gas a few days had sufficed; while in the vessels without microscopic life there remained, after several years, a considerable quantity of oxygen in a free state, so weak is the proportion of oxygen that the organic matters consume directly and chemically when the infinitely small organisms are absent.

But can these microscopic organisms, after having decomposed or burnt up all these secondary products, be in their turn decomposed?

How, cried M. Bouillaud, repeating his question, can they be destroyed or decomposed? How can their materials, which are of the same order as those of all the living creatures of the earth, be gasified and caused to return to the atmosphere? After having been charged with the transformation of others, whose business will it be to transform them?

A ferment which has finished its work, replied Pasteur, and which for want of aliment cannot continue it, becomes in its turn an accumulation, so to speak, of dead organic matters. Such, for example, would be an accumulation of yeast exposed to the air. Leave this mass to itself in summer temperature, and you will see appear in the interior of the mass anaérobic vibrios and the putrefactions associated with their life when protected from contact with the air. At the same time, on the surface of the entire mass--that is to say, that which finds itself in immediate contact with the oxygen of the air--the germs of bacteria, the seeds of mould will grow, and, by fixing the oxygen, determine the slow combustions which gasify the mass. The ferments of ferments are simply ferments. As long as the aérobic ferments of the surface have at their disposal free oxygen, they will multiply and continue their work of destruction. The anaérobic vibrios perish for want of new matter to decompose, and they form, in their turn, a mass of organic matter which, by and by, becomes the prey of aérobies. The portion of the aérobies which has lived becomes the prey either of new aérobies of different species, or of individuals of their own species, so that from putrefaction to putrefaction, and from combustion to combustion, the organic mass with which we started finds itself reduced to an assemblage of anaérobic and aérobic germs--of those same germs which were mixed up in the original primitive organic substances.

Though a collection of germs becomes again in its turn a collection of organic matter, subject to the double action of the phenomena of putrefaction and of combustion, there need be no anxiety as to their ultimate destruction; in the final analysis they represent life under its eternal form, for life is the germ, and the germ is life.

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Thus in the destruction of that which has lived, all reduces itself to the simultaneous action of these three great natural phenomena--fermentation, putrefaction, and slow combustion. A living organism dies--animal, or plant, or the remains of one or the other. It is exposed to the contact of the air. To the life which has quitted it succeeds life under other forms. In the superficial parts, which the air can reach, the germs of the infinitely small aérobies hatch and multiply themselves. The carbon, the hydrogen, and the nitrogen of the organic matters are transformed by the oxygen of the air, and under the influence of the life of these aérobies, into carbonic acid, vapour of water, and ammonia gas. As long as organic matter and air are present, these combustions will continue. While these superficial combustions are going on, fermentation and putrefaction are doing their work in the interior of the mass by the developed germs of the anaérobies, which not only do not require oxygen for their life, but which oxygen actually kills. Little by little, at length, by this work of fermentation and slow combustion, the phenomenon is accomplished. Whether in the free atmosphere, or under the earth, which is always more or less impregnated with air, all animal and vegetable matters end by disappearing. To arrest these phenomena an extremely low temperature is required. It is thus that in the ice of the Polar regions antediluvian elephants have been found perfectly intact. The microscopic organisms could not live in so cold a temperature. These facts still further strengthen all the new ideas as to the important part performed by these infinitely small organisms, which are, in fact, the masters of the world. If we could suppress their work, which is always going on, the surface of the globe, encumbered with organic matters, would soon become uninhabitable.

FOOTNOTE:

ACETIC FERMENTATION.

THE MANUFACTURE OF VINEGAR.

Soon afterwards Pasteur came upon a most curious illustration of the 'fixation' of atmospheric oxygen by a microscopic organism--the transformation of wine into vinegar. As its name indicates, vinegar is nothing else than wine turned sour. Everybody has remarked that wine, left to itself, in circumstances which occur daily, is frequently transformed into vinegar. This is noticed more particularly when bottles, having been uncorked, are left in a half-empty condition. Sometimes, however, wine turns sour even in corked bottles. In this case we may be sure that the bottles have been standing upright, and that corks more or less defective have permitted the air to penetrate into the wine. The presence of air, in fact, is indispensable to the chemical act of transforming wine into vinegar. How does this air intervene? And what is the little microscopic creature which, in conjunction with the air, becomes the agent of this fermentation?

In a celebrated lecture given at Orleans at the request of the manufacturers of vinegar in that town, Pasteur, after having stated the two foregoing scientific questions, proceeded to examine the difference between wine and vinegar. What takes place in the fermentation of the juice of the grape which yields the wine? The sugar of this juice disappears, giving place to carbonic acid gas, which is exhaled during fermentation, and to alcohol, which remains in the fermented liquid. Formerly, chemists gave the name of 'spirit' to all volatile matters which could be collected from distillation. Now, when we distil wine and condense the vapour in a worm surrounded by cold water, we collect the spirit of wine at the extremity of the worm--this, when the water with which it is mixed during distillation is withdrawn from it, we designate by the name of alcohol. Vinegar contains no alcohol. When distilled it yields water and a spirit. But this spirit is acid, with a very pungent odour, and not inflammable like spirit of wine. Separated from the water which had accompanied it during the distillation, this spirit takes the name of acetic acid. This is the form in which it is used in smelling bottles--in those bottles of English salts the vapour of which is so penetrating.

In the formation of vinegar in contact with air the alcohol disappears, and is replaced by acetic acid. The air has thus given up something to the wine. Atmospheric air every one knows to be a mixture of nitrogen and oxygen, the nitrogen in the proportion of four-fifths of the total volume, and the oxygen of one-fifth. Well, in the transformation of wine into vinegar the nitrogen remains inactive. It is the oxygen alone which enters into combination with the alcohol. You ask for the proof of this? Take a bottle of wine turned sour, a bottle which at the same time is stopped hermetically; if the oxygen of the air contained in the bottle has combined with the alcohol, then, instead of air, there will be nothing in the bottle but nitrogen gas. Turn the bottle upside down and open it in a basin of water. The water of the basin will rush into the bottle to fill the partial vacuum created by the disappearance of the oxygen. The volume of water which enters the bottle is precisely equal to a fifth part of the total original volume of the air which the bottle contained at the time when it was closed. Moreover, it is easy to show that the gas which remains in the bottle has the properties of nitrogen gas. A lighted match is extinguished in it as if plunged into water, and a bird dies immediately in it of asphyxia.

If we confine our knowledge to what has gone before, it would seem that alcohol diluted with water and exposed to the air ought to furnish acetic acid. It is not so, however. Pure water alcoholised to the degree of ordinary wines may remain for whole years in contact with the air, without the least acetification. In this difference between natural wine and pure water alcoholised, and exposed to contact with air, we touch upon a vital point in the phenomena of fermentation. The celebrated theory of Liebig, which Pasteur was destined to overthrow, might be thus summed up:--If pure alcoholised water cannot become sour in contact with air, as is the case with wine, it is because the pure alcoholised water lacks the albuminoid substance which exists in the wine in a state of chemical alteration, and which is a ferment capable of causing the oxygen of the air to combine with the alcohol. And the proof, according to Liebig, that things act rigorously thus is, that if you add to the mixture of water and alcohol a little flour, or a little meat-juice, or even a minute quantity of any vegetable juice, the acetic fermentation arises, as if by compulsion. In other words, by the addition of a small quantity of any nitrogenised substance in process of alteration, you cause the union of the oxygen of the air with the alcohol.

There is doubtless always in the wine, when it turns sour, a necessary intermediary, producing the fixation of the oxygen of the air; since in no circumstances can pure alcohol, diluted to any degree whatever with pure water, transform itself into vinegar. But this necessary intermediary is not, as the German theory would have it, a dead albuminoid substance; it is a plant, and of all plants one of the simplest and most minute, which has been known from time immemorial under the name of flower of vinegar. This little fungus is invariably present on the surface of a wine which is being transformed into vinegar. Liebig was not ignorant of this, but he regarded it as a simple coincidence. Do we not know, said he, that whenever an infusion of organic matter is exposed to the air it becomes covered with a cryptogamic vegetation, or is invaded by a crowd of animalculæ? Is not vinegar a vegetable infusion? Vinegar affords a refuge to the flower of vinegar, just as it gives refuge to what are called the little eels of vinegar.

We can appreciate here the uncertainties of pure observation. The great art--and no one practised it better than Pasteur--consists in instituting decisive experiments which leave no room for an inexact interpretation of facts. These decisive proofs of the true part played by the little microscopic fungus, by this flower of vinegar, this mycoderma aceti, are thus formulated by Pasteur. It is but another example of the method which he used in alcoholic, lactic, and tartaric fermentations. The theories of Berzelius, of Mitscherlich, and of Liebig were destined again to receive the rudest shocks by the demonstration of these rigorous facts.

Let us place a little wine in a bottle, then hermetically seal it, and leave it to itself. In these conditions the wine becomes sour. But if we take the precaution of putting the bottle into hot water, so that the wine and the air in the bottle may be heated for some instants to a temperature of 60° Centigrade, and if, after cooling, we leave the bottle to itself, the wine in these conditions will never become transformed into vinegar. The heating, however, must have left intact the albuminoid or nitrogenous substances contained in the wine. These, then, cannot constitute the ferment of the vinegar. Can it be maintained that by heating the wine to 60° we have altered the albuminoid matter, which is, on this account, no longer able to act as a ferment, or, in other words, no longer able to determine the union of the oxygen of the air with the alcohol? This hypothesis falls to pieces before the following experiment. Open the bottle, blow into it with bellows, so that the once heated wine shall come into contact with ordinary air, and the acetification of the wine will take place.

But the master experiment is the following. We have seen that pure alcoholised water never turns sour unless some albuminoid matter is introduced into it. Pasteur saw that this albuminoid matter might be completely suppressed and replaced by saline crystallisable substances, alkaline and earthy phosphates, to which has been added a little phosphate of ammonia. In these conditions, especially if the alcoholised water be acidulated by small quantities of pure acetic acid, one actually sees the mycoderm developing, and the alcohol transforming itself into acetic acid. It is not possible to demonstrate in a more convincing manner that the albuminoid matters of the wine are not in this case the acetic ferment. These albuminoid matters, however, contribute to the acetic fermentation, but only as being an aliment to the mycoderma aceti, and notably a nitrogenous aliment. The true and only ferment of vinegar is the little fungus; it is the great agent of the phenomenon; it, indeed, accomplishes all.

Is there not a great charm in seeing an obscure subject clearly illuminated by facts well understood and well interpreted? If in a bottle containing wine and air and raised to a temperature of 50° or 60° the wine never turns sour, it is because the germs of the mycoderma aceti, which the wine and the air hold in suspension, are deprived of all vitality by the heat. Placed, however, in contact with ordinary air, this once-heated wine can turn sour; because, though the germs of the mycoderma aceti contained at first in the wine are killed, this is not the case with those derived from the surrounding air. Pure alcoholised water never turns sour, even in contact with ordinary air, and with whatever germs this air may carry, or that may be found in the dust of the vessels which receive it. The reason is that these germs cannot become fertile because of the absence of their indispensable food. Wine in bottles well filled and laid flat do not acetify; this is because the mycoderm cannot multiply for lack of oxygen. Without doubt the air constantly penetrates through the pores of the cork, but always in such feeble quantities that the colouring matters of the wine, and other more or less oxydisable constituents, take possession of it without leaving the smallest quantity for the germs of the mycoderm which are generally suspended in the wine. When the bottle is upright the conditions are quite altered. The desiccation of the cork renders it much more permeable to the air, and the germs of the mycoderm on the surface of the liquid, if any exist there, are enveloped by air.

Thus, to recapitulate in a few words the principles which have just been established; it is easy to see that the formation of vinegar is always preceded by the development, on the surface of the wine, of a little plant formed of strangulated particles, of an extreme tenuity, and the accumulation of which sometimes takes the form of a hardly visible veil, sometimes of a wrinkled film of very slight thickness, and greasy to the touch, because of the various fatty matters which the plant contains.

This cryptogam has the singular property of condensing considerable quantities of oxygen and of provoking the fixation of this gas upon the alcohol, which is thereby transformed into acetic acid. The little mycoderm is not less exacting than larger vegetables. It must have its appropriate aliments. Wine offers them in abundance: nitrogenous matters, the phosphates of magnesia and of potash. The mycoderm thrives, moreover, in warm climates. To cultivate it in temperate regions like ours it is well to warm artificially the places where it is cultivated. But if wine contains within itself all the elements necessary to the life of the little mycoderm, this life is further promoted by rendering the wine more acid through the addition of acetic acid.

What, then, can be more simple than to produce vinegar from wine--a manufacture which justly makes the reputation of the town of Orleans? Take some wine, and after having mixed with it one-fourth or one-third of its volume of vinegar already formed, sow on its surface the little plant which does the work of acetification. It is only necessary to skim off, by means of a wooden spatula, a little of the mycodermic film from a liquid covered with it, and to transfer it to the liquid to be acetified. The fatty matters which it contains render the wetting of it difficult. Thus, when we plunge into the liquid the spatula covered with the film, the latter detaches itself and spreads out over the surface instead of falling to the bottom. When we operate in summer, or in a room heated to 15° or 25° Centigrade in winter, in twenty-four or forty-eight hours at most, the mycoderm covers the whole liquid, so easy and rapid is its development. After some days all the wine has become vinegar.

On one occasion, in a discussion which he was holding at the Academy of Sciences, Pasteur, wishing to affirm the prodigious activity of the life and multiplication of this little organism, expressed himself thus:--