Chapter VI (2)

“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 a 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 c.c. (rather more than 5 fluid ounces) of yeast-water, containing 2-½ per cent. of milk-sugar. The solution was prepared in one of our double-necked flasks, with the necessary precautions to secure 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 on November 15th, pouring some wort on the deposit of the plant, which remained in the flask. In less than five hours from the time when we placed it in the oven, the plant had 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 causes 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 under 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 the 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 would 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, in consequence of a renewed action starting in the interior cells of the grapes, in such a way that these cells would assume the function 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.[123]

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 _École 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_[124]:—On July 31st, 1872, we placed twenty-four of these plums under a glass cylinder, which we immediately filled with carbonic acid gas. The plums had been gathered on the previous day. By the side of the cylinder 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 cylinder, 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 Bérard 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 could we have than these facts 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 cylinder, one of which is in contact with the objects, are touched alternately. It also makes itself evident in the formation of vapour, which condenses in 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.[125]

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, _i.e._ a 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 certain 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 _École Normale Supérieure_, and his coadjutor, M. Bellamy.[126] In 1821, in a very remarkable work, especially when we consider the period when it appeared, Bérard 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 Bérard 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 foreshadowed 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.[127] 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.[128] M. Fremy, in particular, was desirous of finding in those 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, which we have quoted word for word in the last paragraph, furnish the most conclusive evidence in favour 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 as yeast does. Such is the case with the fungi already studied in Chapter IV.; 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 aërobian state, and we have no ferment-action; immersed immediately afterwards in carbonic acid gas, they now assume their anaërobian 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.[129]

“To speak here of alcoholic fermentation alone,”[130] 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; M. Pasteur, on the other hand, maintains that the fermentation is produced by germs existing outside the skin of the grapes.”

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 experiment 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 was 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.[131] 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 our 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 ferment-action 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 a 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 paragraph with a few remarks on the subject of the 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 had 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, 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, and that they were present in those proportions, characteristic of that 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 globules already formed. The general results of the present Memoir seem to us to be in 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 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 vital activity in carbonic acid gas_, whilst in the other case the ferment consists of the 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 ordinary 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.[132] In this fermentation, neither alcohol nor carbonic acid gas exists in those proportions in which they are found in fermentations produced by yeast; and although we may determine in it the presence of succinic acid, glycerine, and a small quantity of volatile acids,[133] 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 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, 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’être_ 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 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 paragraph. 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 between 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 anaërobian 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 sure 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 paragraph, 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.[134]

§ IV.—Fermentation of Dextro-Tartrate of Lime.[135]

Tartrate of lime, in spite of its insolubility in water, 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 materials 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. 67, of 2·5 litres (about four pints) in capacity, we put:—

Pure, crystallized, neutral tartrate of 100 grammes.
lime

Phosphate of ammonia 1 gramme.

Phosphate of magnesium 1 gramme.

Phosphate of potassium 0·5 gramme.

Sulphate of ammonia 0·5 gramme.

(1 gramme=15·43 grains.)

To this we added pure distilled water, so as to entirely 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 fortnight. It might have remained for a century without ever manifesting the least sign of fermentation, the fermentation of the tartrate being a consequence of life, and life after the 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.c. (about 17 minims) of liquid and deposit from another flask, similar to the one we have 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 doubt 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 a 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 c.c. (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.[136]

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° C. and 28° C. (77° F. and 83° 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 a gauge-mark on the neck.[137] 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 2·135 litres, gave a total of 10·457 litres (638·2 cubic inches) at 20° and 760, which calculated to 0° C. and 760 mm. atmospheric pressure (32° F. and 30 inches) gave a weight of 19·70 grammes (302·2 grains) of carbonic acid.

Exactly half of the lime of 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.[138] 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.[139] 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 punctate, 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 at 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 greater 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 a 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[140] 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. 69, 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 was 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° C. and 30° C. (77° F. and 86° F.); then, after having refilled the small cylindrical tap-funnel with carbonic acid, we passed into it with all necessary precautions 10 c.c. (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 till 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.[141] 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.