  § I.—On the Relations existing between Oxygen and Yeast.The object of all science is a continuous reduction of the number of unexplained phenomena. It is observed, for instance, that fleshy fruits are not liable to fermentation so long as their epidermis remains uninjured. On the other hand, they ferment very readily when they are piled up in heaps, more or less open, and immersed in their saccharine juice. The mass becomes heated and swells; carbonic acid gas is disengaged, and the sugar disappears and is replaced by alcohol. Now, as to the question of the origin of these spontaneous phenomena, so remarkable in character as well as usefulness for man’s service, modern knowledge has taught us that fermentation is the consequence of a development of vegetable cells, the germs of which do not exist in the saccharine juices within fruits; that many varieties of these cellular plants exist, each giving rise to its own particular fermentation. The principal products of these various fermentations, although resembling each other in their nature, differ in their relative proportions and in the accessory substances that accompany them, a fact which alone is sufficient to account for wide differences in the quality and commercial value of alcoholic beverages. Now that the discovery of ferments and their living nature, and our knowledge of their origin, may have solved the mystery of the spontaneous appearance of fermentations in The least reflection will suffice to convince us that the alcoholic ferments must possess the faculty of vegetating and performing their functions out of contact with air. Let us consider, for instance, the method of vintage practised in the Jura. The bunches are laid at the foot of the vine in a large tub, and the grapes there stripped from them. When the grapes, some of which are uninjured, others bruised, and all moistened by the juice issuing from the latter, fill the tub—where they form what is commonly called the vintage—they are conveyed in barrels to large vessels fixed in cellars of a considerable depth. These vessels are not filled to more than three-quarters of their capacity. Fermentation soon takes place in them, and the carbonic acid gas finds escape through the bunghole, the diameter of which, in the case of the largest vessels, is not more than ten or twelve centimetres (about four inches). The wine is not drawn off before the end of two or three months. In this way it seems highly probable that the yeast which produces the wine under such conditions must have developed, to a great extent at least, out of contact with oxygen. No doubt oxygen is not entirely absent from the first; nay, its limited presence is even a necessity to the manifestation of the phenomena which follow. The grapes are stripped from the bunch in contact with air, and the must which drops from the wounded fruit takes a little of this gas Another equally exceptional characteristic of yeast and fermentation in general consists in the small proportion which the yeast that forms bears to the sugar that decomposes. In all other known beings the weight of nutritive matter assimilated corresponds with the weight of food used up, any difference that may exist being comparatively small. The life of yeast is entirely different. For a certain weight of yeast formed, we may have ten times, twenty times, a hundred times as much sugar, or even more decomposed, as we shall experimentally prove by-and-bye; that is to say, that whilst the The question before us is whether yeast is in reality an anaËrobian plant, and what quantities of sugar it may cause to ferment, under the various conditions under which we cause it to act. The following experiments were undertaken to solve this double problem:—We took a double-necked flask, of three litres (five pints) capacity, one of the tubes being curved and forming an escape for the gas; the other one, on the right hand side (Fig. 59), being furnished with a glass tap. We filled this flask with pure yeast-water, sweetened with 5 per cent. of sugar candy, the flask being so full that there was not the least trace of air remaining above the tap or in the escape tube; this artificial wort had, however, been itself aerated. The curved tube was plunged in a porcelain vessel full of mercury, resting on a firm support. In the small cylindrical funnel above the tap, the capacity of which was from 10 c.c. to 15 c.c. (about half a fluid ounce) we caused to ferment, at a temperature of 20° or 25° C. (about 75° F.), five or six cubic centimetres of the saccharine liquid, by means of a trace of yeast, which multiplied rapidly, causing fermentation, and forming a slight deposit of yeast at the bottom of the funnel above the tap. We then opened the tap, and some of the liquid in the funnel entered the flask, carrying with it the small deposit of yeast, Fig. 59. Fig. 59. Fig. 60. Fig. 60. Some exact experiments conducted by M. Raulin in our laboratory have established the fact that saccharine worts, like water, soon become saturated when shaken briskly with an Fig. 61. Fig. 61. The following is a description of two of these comparative fermentations and the results they gave. The fermentable liquid was composed of yeast-water sweetened with 5 per cent. of sugar-candy; the ferment employed was saccharomyces pastorianus. The impregnation took place on January 20th. The flasks were placed in an oven at 25° C. (77° F.). Flask A, without air. January 21st.—Fermentation commenced; a little frothy liquid issued from the escape-tube and covered the mercury. The following days, fermentation was active. Examining the yeast mixed with the froth that was expelled into the mercury by the evolution of carbonic acid gas, we found that it was very fine, young, and actively budding. February 3rd.—Fermentation still continued, showing itself by a number of little bubbles rising from the bottom of the liquid, which had settled bright. The yeast was at the bottom in the form of a deposit. February 7th.—Fermentation still continued, but very languidly. February 9th.—A very languid fermentation still went on, discernible in little bubbles rising from the bottom of the flask. Flask B, with air. January 21st.—A sensible development of yeast. The following days, fermentation was active, and there was an abundant froth on the surface of the liquid. February 1st.—All symptoms of fermentation had ceased. As the fermentation in A would have continued for a long time, being so very languid, and as that in B had been finished for several days, we brought to a close our two experiments on As might have been expected, the liquid in the flask B did not contain the least trace of sugar; that in the flask A still contained some, as was evident from the non-completion of fermentation, but not more than 4·6 grammes (71 grains). Now, as each flask originally contained 3 litres of liquid, holding in solution 5 per cent. of sugar, it follows that 150 grammes (2,310 grains) of sugar had fermented in the flask B, and 145·4 grammes (2,239·2 grains) in the flask A. The weights of yeast after drying at 100° C. (212° F.) were—
The proportions were 1 of yeast to 76 of fermented sugar in the first case, and 1 of yeast to 89 of fermented sugar in the second. From these facts the following consequences may be deduced: 1. The fermentable liquid (flask B), which since it had been in contact with air, necessarily held air in solution, although not to the point of saturation, inasmuch as it had been once boiled to free it from all foreign germs, furnished a weight of yeast sensibly greater than that yielded by the liquid which contained no air at all (flask A), or, at least, which could only have contained an exceedingly minute quantity. 2. This same slightly aerated fermentable liquid fermented much more rapidly than the other. In eight or ten days it contained no more sugar; while the other, after twenty days, still contained an appreciable quantity. Is this last fact to be explained by the greater quantity of 3. In the airless flask the proportion of yeast to sugar was 1/89; it was only 1/76 in the flask which had air at first. The proportion that the weight of yeast formed bears to the weight of the sugar is, therefore, variable, and this variation depends, to a certain extent, upon the presence of air and the possibility of oxygen being absorbed by the yeast. We shall presently show that yeast possesses the power of absorbing that gas and emitting carbonic acid, like ordinary fungi, that even oxygen may be reckoned amongst the number of food-stuffs that may be assimilated by this plant, and that this fixation of oxygen in yeast, as well as the oxidations resulting from it, have the most marked effect on the life of yeast, on the multiplication of its cells, and on their activity as ferments acting upon sugar, whether immediately or afterwards, apart from supplies of oxygen or air. In the preceding experiment, conducted without the presence of air, there is one circumstance particularly worthy of notice. This experiment succeeds, that is to say, the yeast sown in the medium deprived of oxygen develops, only when this yeast is in a state of great vigour. We have already explained the meaning of this last expression. But we wish now to call attention to a very evident fact in connection with this point. We impregnate a fermentable liquid; yeast develops and fermentation appears. This lasts for several days and then ceases. Let us suppose that, from the day when fermentation If we compare under the microscope the appearance and character of the successive quantities of yeast taken, we shall see plainly that the structure of the cells undergoes a progressive change. The first sample which we take, quite at the beginning of the original fermentation, generally gives us cells rather larger than those later on, and possessing a remarkable tenderness. Their walls are extremely thin, the consistency and softness of their protoplasm is akin to fluidity, and their granular contents appear in the form of scarcely visible spots. The borders of the cells soon become more marked, a proof that their walls undergo a thickening; their protoplasm also becomes denser, and the granulations more distinct. Cells of the same organ, in the states of infancy and old age, should not differ more than the cells of which we are speaking, taken This being so it is evident, we repeat, that, to multiply in a fermentable medium, quite out of contact with oxygen, the cells of yeast must be extremely young, full of life and health, and still under the influence of the vital activity which they owe to the free oxygen which has served to form them, and which they have perhaps stored up for a time. When older, they reproduce themselves with much difficulty when deprived of air, and gradually become more languid; and if they do multiply, it is in strange and monstrous forms. A little older still, they remain absolutely inert in a medium deprived of free oxygen. This is not because they are dead; for in general they may be revived in a marvellous manner in the same liquid if it has been first aerated before they are sown. It would not surprise us to learn that at this point certain preconceived ideas suggest themselves to the mind of an attentive reader on the subject of the causes that may serve to account for such strange phenomena in the life of these beings which our ignorance hides under the expressions of youth and age; this, however, is a subject that we cannot pause to consider here. At this point we must observe—for it is a matter of great importance—that, in the operations of the brewer there is always a time when the yeasts are in this state of vigorous youth of which we have been speaking, acquired under the influence of free oxygen, since all the worts and all the yeasts of commerce are necessarily manipulated in contact with air, and so impregnated From the same circumstances it is clear that the brewer’s fermentations may, speaking quite strictly, last for an indefinite time, in consequence of the unceasing supply of fresh wort, and from the fact, moreover, that the exterior air is constantly being introduced during the work, and that the air contained in the fresh worts keeps up the vital activity of the yeast, as the act of breathing keeps up the vigour and life of cells in all living beings. If the air could not renew itself in any way, the vital activity which the cells originally received, under its influence, would become more and more exhausted, and the fermentation eventually come to an end. We may recount one of the results obtained in other experiments similar to the last, in which, however, we employed yeast which was still older than that used for our experiment with flask A (Fig. 60), and moreover took still greater precautions to prevent the presence of air. Instead of leaving the flask, as well as the dish, to cool slowly, after having expelled all air by boiling, we permitted the liquid in the dish to continue boiling whilst the flask was being cooled by artificial means; the end of the escape tube was then taken out of the still boiling dish and plunged into the mercury trough. In impregnating the liquid, instead of employing the contents of the small cylindrical funnel whilst still in a state of fermentation, we waited until this was finished. Under these conditions, fermentation was still going on in our flask, after a lapse of three months. We stopped it and found that 0·255 gramme (3·9 grains) of yeast had been formed, and that 45 grammes (693 grains) of sugar had fermented, the ratio between the weights of yeast and sugar being thus 0·255/45 = 1/176. In this experiment the yeast developed In such experiments we encounter another difficulty. If the yeast sown in the non-aerated fermentable liquid is in the least degree impure, especially if we use sweetened yeast-water, we may be sure that alcoholic fermentation will soon cease, if, indeed, it ever commences, and that accessory fermentations will go on. The vibrios of butyric fermentation, for instance, will propagate with remarkable facility under these circumstances. Clearly then, the purity of the yeast at the moment of impregnation, and the purity of the liquid in the funnel, are conditions indispensable to success. To secure the latter of these conditions, we close the funnel, as shown in Fig. 60, by means of a cork pierced with two holes, through one of which a short tube passes, to which a short length of india-rubber tubing provided with a glass stopper is attached; through the other hole a thin curved tube is passed. Thus fitted, the funnel can answer the same purposes as our double-necked flasks. A few cubic centimetres of sweetened yeast-water are then put in it and boiled, so that the steam may destroy any germs adhering to the sides. When cold the liquid is impregnated by means of a trace of pure yeast, introduced through the glass-stoppered tube. If these precautions are neglected it is scarcely possible to secure a successful fermentation in our flasks, because the yeast sown is immediately held in check by a development of anaËrobian vibrios. For greater security, we may add to the fermentable liquid, at the moment when it is prepared, a very small quantity of tartaric acid, which will prevent the development of butyric vibrios. Fig. 62. Fig. 62. The variation of the ratio between the weight of the yeast and that of the sugar decomposed by it now claims special attention. Side by side with the experiments which we have just described, we conducted a third lot by means of the flask C
Taking into consideration the volume of the flask, this shows a minimum of 50 c.c. (3·05 cub. in.) of oxygen to have been Fig. 63. Fig. 63. The next experiment was to increase the proportion of oxygen to a still greater extent, by rendering the diffusion of gas a more easy matter than it is in a flask, the air in which is in a state of perfect quiescence. Such a state of matters hinders the supply of oxygen, inasmuch as the carbonic acid, as soon as it is liberated, at once forms an immovable layer on the surface of the liquid, and so separates off the oxygen. To effect the purpose of our present experiment, we used flat basins having glass bottoms and low sides, also of glass, in which the depth of the liquid is not more than a few millimetres (less than 1/4-inch) (Fig. 63). The following is one of our experiments so conducted:—On April 16th, 1860, we sowed a trace of beer yeast (“high” yeast) in 200 c.c. (7 fl. oz.) of a saccharine liquid containing 1·720 grammes (26·2 grains) of sugar-candy. From April 18th our yeast was in good condition and well developed. We collected it, after having added to the liquid a few drops of concentrated sulphuric acid, with the object of checking the fermentation to a great extent, and facilitating filtration. The sugar remaining in the filtered liquid, determined by Fehling’s Formula 1. Formula 1. which is considerably higher than the preceding ones. We may still further increase this ratio by making our estimation as soon as possible after the impregnation, or the addition of the ferment. It will be readily understood why yeast, which is composed of cells that bud and subsequently detach themselves from one another, soon forms a deposit at the bottom of the vessels. In consequence of this habit of growth, the cells constantly covering each other prevents the lower layers from having access to the oxygen held in solution in the liquid, which is absorbed by the upper ones. Hence, those which are covered and deprived of this gas act on the sugar without deriving any vital benefit from the oxygen—a circumstance which must tend to diminish the ratio of which we are speaking. Once more repeating the preceding experiment, but stopping it as soon as we think that the weight of yeast formed may be determined by the balance (we find that this may be done twenty-four hours after impregnation with an inappreciable quantity of yeast) in this case the ratio between the weights of yeast and sugar is Formula 2. Formula 2. This is the highest ratio that we have been able to obtain. Under these conditions the fermentation of sugar is extremely languid: the ratio obtained is very nearly the same that ordinary fungoid growths would give. The carbonic acid evolved is principally formed by the decompositions which result from the assimilation of atmospheric oxygen. The yeast, therefore, lives and performs its functions after the manner of ordinary fungi: so far it is no longer a ferment, so to say; moreover, we might expect to find it cease to be a ferment at all if we We may here be permitted to make a digression. In his work on fermentations, which M. SchÜtzenberger has recently published, the author criticises the deductions that we have drawn from the preceding experiments, and combats the explanation which we have given of the phenomena of fermentation. M. SchÜtzenberger has failed to notice that the power of a ferment is independent of the time during which it performs its functions. We placed a trace of yeast in one litre of saccharine wort; it propagated, and all the sugar was decomposed. Now, whether the chemical action involved in this decomposition of sugar had required for its completion one day, or one month, or one year, such a factor was of no more importance in this matter than the mechanical labour required to raise a ton of materials from the ground to the top of a house would be affected by the M. SchÜtzenberger is astonished that fermentation can take place in the presence of free oxygen, if, as we suppose, the decomposition of the sugar is the consequence of the nutrition of the yeast, at the expense of the combined oxygen, which yields itself to the ferment. At all events, he argues, fermentation ought to be slower in the presence of free oxygen. But why should it be slower? We have proved that in the presence of oxygen the vital activity of the cells increases, so that, as far as rapidity of action is concerned, its power cannot be diminished. It might, nevertheless, be weakened as a ferment, and this is precisely what happens. Free oxygen imparts to the yeast an increased vital activity, but at the same time impairs rapidly its power as yeast—qu yeast, inasmuch as under this condition it approaches the state in which it can carry Fig. 64. Fig. 64. In presence of abundant air-supply, yeast vegetates with extraordinary activity. We see this in the weight of new yeast, comparatively large, that may be formed in the course In passing it is of interest to note how promptly the preceding results were turned to good account practically. In well-managed distilleries, the custom of aerating the wort and the juices, to render them more adapted to fermentation, has been introduced. The molasses, mixed with water, is permitted to run in thin threads through the air at the moment when the yeast is added. Manufactories have been erected, in which the manufacture of yeast is almost exclusively carried on. The saccharine worts, after the addition of yeast, are left to themselves, in contact with air, in shallow vats of large superficial area, realizing thus on an immense scale the conditions of the experiments which we undertook in 1861, and which we have already described in determining the rapid and easy multiplication of yeast in contact with air. The next experiment attempted was to determine the volume of oxygen absorbed by a known quantity of yeast, the yeast living in contact with air, and under such conditions that the absorption of air was comparatively easy and abundant. Fig. 65. Fig. 65. With this object we repeated the experiment that we performed with the large-bottomed flask (Fig. 62), employing a vessel shaped like Fig. B. (Fig. 65), which is, in point of We employed 60 c.c. (about 2 fluid ounces) of yeast-water, sweetened with 2 per cent. of sugar and impregnated with a trace of yeast. After having subjected our vessel to a temperature of 25° C. (77° F.) in an oven for fifteen hours, the drawn-out point was brought under an inverted jar filled with mercury and the point broken off. A portion of the gas escaped and was collected in the jar. For 25 c.c. of this gas we found, after absorption by potash, 20·6, and after absorption by pyrogallic acid, 17·3. Taking into account the volume which remained free in the flask, which held 315 c.c., there was a total absorption of 14·5 c.c. (0·88 cub. in.) of oxygen. It follows that in the production of 35 milligrammes (0·524 grain) of yeast there was an absorption of 14 or 15 c.c. (about 7/8 cubic inch) of oxygen, even supposing that the yeast was formed entirely under the influence of that gas: this is equivalent to not less than 414 c.c. for 1 gramme of yeast (or about 33 cubic inches for every 20 grains). Let us now return to the first experiment described in this paragraph (page 238), in which a flask of three litres capacity was filled with fermentable liquid, which, when caused to ferment, yielded 2·25 grammes of yeast, under circumstances where it could not obtain a greater supply of free oxygen than 16·5 c.c. (about one cubic inch). According to what we have just stated, if this 2·25 grammes (34 grains) of yeast had not been able to live without oxygen, in other words, if the original cells had been unable to multiply otherwise than by absorbing free oxygen, the amount of that gas required could not have been less than 2·25 × 414 c.c., that is, 931·5 c.c. (56·85 cubic inches). The greater part of the 2·25 grammes, therefore, had evidently been produced as the growth of an anaËrobian plant. Ordinary fungi likewise require large quantities of oxygen for their development, as we may easily prove by cultivating any mould in a closed vessel full of air, and then taking the weight of plant formed and measuring the volume of oxygen absorbed. To do this, we take a flask of the shape shown in Fig. 66, capable of holding about 300 c.c. (10-½ fluid ounces), and containing a liquid adapted to the life of moulds. We boil this liquid and seal the drawn-out point, after the steam has expelled the air wholly or in part; we then open the flask in a garden or in a room. Should a fungus-spore enter the flask, as will invariably be the case in a certain number of flasks out of several used in the experiment, except under special circumstances, it will develop there and gradually absorb all the oxygen contained in the air of the flask. Measuring the Fig. 66. Another equally striking proof of the truth of this theory is the fact, demonstrated in Chapter IV., that the ordinary moulds assume the character of a ferment when compelled to live without air, or with quantities of air too scant to permit of their organs having around them as much of that element as is necessary for their life as aËrobian plants. Ferments, therefore, only possess in a higher degree a character which belongs to many common moulds, if not to all, and which they share, probably, more or less, with all living cells, namely the power of living either an aËrobian or anaËrobian life, according to the conditions under which they are placed. It may be readily understood how, in their state of aËrobian life, the alcoholic ferments have failed to attract attention. Those ferments are only cultivated out of contact with air, at the bottom of liquids which soon become saturated with carbonic acid gas. Air is only present in the earlier developments of their germs, and without attracting the attention of the operator, whilst in their state of anaËrobian growth The possibility of living without oxygen, in the case of ordinary moulds, is connected with certain morphological modifications which are more marked in proportion as this faculty is itself more developed. These changes in the vegetative forms are scarcely perceptible in the case of penicillium and mycoderma vini, but they are very evident in the case of aspergillus, consisting of a marked tendency on the part of the submerged mycelial filaments to increase in diameter, and to develop cross partitions at short intervals, so that they sometimes bear a resemblance to chains of conidia. In mucor, again, they are very marked, the inflated filaments which, closely interwoven, present chains of cells which fall off and bud, gradually producing a mass of cells. If we consider the matter carefully, we shall see that yeast presents the same characteristics. For instance, what can more closely resemble the mucor of Plates V. and VI. than the saccharomyces of Figs. 33 and 37? Have we not in each case ramified chains of elongated cells or joints, more or less narrowed in the middle, and shorter segments or cells dropping off at the constrictions, and proceeding to bud in the liquid on their own account? Moreover, the less oxygen there is present, the more marked is the tendency to the formation of these budding cells, which isolate themselves and soon drop off. Who could ever imagine, in examining the ferment of mucor represented in Plate VI., that its first germ was the ordinary mucor that is found everywhere, with fine filaments, straight or ramified according to the variety, which send up aerial hyphae, terminating in little round heads bearing spores. So was it that in the ferment of Plate XI. we could scarcely recognize the ramified filaments of Figs. 33 and 37. It is a great presumption in favour of the truth of theoretical ideas when the results of experiments undertaken on the strength of those ideas are confirmed by various facts more recently added to science, and when those ideas force themselves “M. Pasteur gives the results of his researches on the fermentation of sugar and the development of yeast-cells, according as that fermentation takes place apart from the influence of free oxygen or in contact with that gas. His experiments, however, have nothing in common with those of Gay-Lussac, which were performed with the juice of grapes, crushed under conditions where they would not be affected by air, and then brought in contact with oxygen. “Yeast, when perfectly developed, is able to bud and grow in a saccharine and albuminous liquid, in the complete absence of oxygen or air. In this case but little yeast is formed, and a comparatively large quantity of sugar disappears—sixty or eighty parts for one of yeast formed. Under these conditions fermentation is very sluggish. “If the experiment is made in contact with the air, and with a great surface of liquid, fermentation is rapid. For the same quantity of sugar decomposed much more yeast is formed. The air with which the liquid is in contact is absorbed by the yeast. The yeast develops very actively, but its fermentative character tends to disappear under these conditions; we find, in fact, that for one part of yeast formed, not more than from four to ten parts of sugar are transformed. The fermentative “It seems, therefore, natural to admit that when yeast functions as a ferment by living apart from the influence of air, it derives oxygen from the sugar, and that this is the origin of its fermentative character. “M. Pasteur explains the fact of the immense activity at the commencement of fermentations by the influence of the oxygen of the air held in solution in the liquids, at the time when the action commences. The author has found, moreover, that the yeast of beer sown in an albuminous liquid, such as yeast-water, still multiplies, even when there is not a trace of sugar in the liquid, provided always that atmospheric oxygen is present in large quantities. When deprived of air, under these conditions, yeast does not germinate at all. The same experiments may be repeated with albuminous liquid, mixed with a solution of non-fermentable sugar, such as ordinary crystallized milk-sugar. The results are precisely the same. “Yeast formed thus in the absence of sugar does not change its nature; it is still capable of causing sugar to ferment, if brought to bear upon that substance apart from air. It must be remarked, however, that the development of yeast is effected with great difficulty when it has not a fermentable substance for its food. In short, the yeast of beer acts in exactly the same manner as an ordinary plant, and the analogy would be complete if ordinary plants had such an affinity for oxygen as permitted them to breathe by appropriating this element from unstable compounds, in which case, according to M. Pasteur, they would appear as ferments for those substances. “M. Pasteur declares that he hopes to be able to realize this result, that is to say, to discover the conditions under which certain inferior plants may live apart from air in the presence of sugar, causing that substance to ferment as the yeast of beer would do.” This summary and the preconceived views that it set forth 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. 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. 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. 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 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. “To speak here of alcoholic fermentation alone,” 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 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 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. 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 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 § 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 “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 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 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 § IV.—Fermentation of Dextro-Tartrate of Lime. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Pure, crystallized, neutral tartrate of lime | 100 grammes. |
| 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.
Fig. 67.
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
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.
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
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.
Fig. 68.
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.
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
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
| 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.] | |
Fig. 69.
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
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.
Before passing on we may remark that in this ready absorption of oxygen by bacteria we have a means of depriving fermentable liquids of every trace of that gas with a facility and success equal or even greater than by the method of preliminary boiling. Such a solution as we have described, if kept at summer heat, without any previous boiling, becomes turbid in the course of twenty-four hours from a spontaneous development of bacteria; and it is easy to prove that they absorb all the oxygen held in solution.
We may also call attention to another striking experiment, well suited to show the effect of differences in the composition of the medium upon the propagation of microscopic beings. The fermentation which we last described commenced on March 27th and continued until May 10th; that to which we are now to refer, however, was completed in four days, the liquid employed being similar in composition and quantity to that employed in the former experiment. On April 23rd, 1875, we filled a flask of the same shape as that represented in Fig. 69, and of similar capacity, viz., 6 litres, with a liquid composed as described at page 293. This liquid had been previously left to itself for five days in large open flasks, in consequence of which it had developed an abundant growth of bacteria. On the fifth day a few bubbles, rising from the bottom of the vessels, at long intervals, betokened the commencement of butyric fermentation, a fact, moreover, confirmed by the microscope, in the appearance of the vibrios of this fermentation in specimens of the liquid taken from the bottom of the vessels, the middle of its mass, and even in the layer on the surface that was swarming with bacteria. We transferred the liquid so prepared to the 6-litre flask arranged over the mercury. By evening a tolerably active fermentation had
Before we go any further, let us devote some attention to the vibrios of the preceding fermentations.
On May 27th, 1862, we completely filled a flask, capable of holding 2·780 litres (about five pints), with the solution of lactate and phosphates.
Fig. 70.
One of the best methods that can be employed for the microscopical examination of these vibrios, quite out of contact with air, is the following:—After butyric fermentation has been going on for several days in a flask, A (Fig. 71), we connect this flask by an india-rubber tube with one of the flattened bulbs previously described, page 156 (Fig. 31), which we then place on the stage of the microscope (Fig. 71). When we wish to make an observation we close, under the mercury, at the point b, the end of the drawn-out and bent delivery-tube. The continued evolution of gas soon exerts such a pressure within the flask, that when we open the tap r, the liquid is
Fig. 71.
Fig. 72.
Fig. 73.
On June 28th, fermentation was quite finished; there was no longer any trace of gas, nor any lactate in solution. All the infusoria were lying motionless at the bottom of the flask. The liquid clarified by degrees, and in the course of a few days became quite bright. Here we may inquire, were these motionless infusoria, which from complete exhaustion of the lactate, the source of the carbonaceous part of their food, were now lying inert at the bottom of the fermenting vessel—were they dead beyond power of revival?
The reader who has attentively studied the facts which we have placed before him cannot, in our opinion, entertain the least doubt on the subject of the possible multiplication of the vibrios of a fermentation of lactate of lime out of contact with atmospheric oxygen. If fresh proofs of this important proposition were necessary, they might be found in the following observations, from which it may be inferred that atmospheric oxygen is capable of suddenly checking a fermentation produced by butyric vibrios, and rendering them absolutely motionless, so that it cannot be necessary to enable them to live. On May 7th, 1862, we placed in the oven a flask holding 2·580 litres (4-½ pints), and filled with the solution of
There is a most simple method of observing the deadly effect of atmospheric air upon vibrios. We have seen in the microscopical examination made by means of the apparatus represented in Fig. 71, how remarkable were the movements of the vibrios when absolutely deprived of air, and how easy it was to discern them. We will repeat this observation, and at the same time make a comparative study of the same liquid, under the microscope, in the ordinary way, that is to say, by placing a drop of the liquid on an object-glass, and covering it with a thin glass slip, a method which must necessarily bring the drop into contact with air, if only for a moment. It is surprising what a remarkable difference is observed immediately between the movements of the vibrios in the bulb and of those under the glass. In the case of the latter we generally see all movement at once cease near the edges of the glass, where the drop of liquid is in direct contact with the air; the movements continue for a longer or shorter time about the centre, in proportion as the air is more or less intercepted by the vibrios at the circumference of the liquid. It does not require much skill in experiments of this kind to enable one to see plainly that immediately after the glass has been placed on the drop, which has been affected all over by atmospheric air, the whole of the vibrios seem to languish and to manifest symptoms of illness—we can think of no better expression to explain what we see taking place—and that they gradually recover their activity about the centre, in proportion as they find themselves in a
Some most curious facts are to be found in connection with an observation, the correlative and inverse of the foregoing, on the ordinary aËrobian bacteria. If we examine below the microscope a drop of liquid full of these organisms under a coverslip, we very soon observe a cessation of motion in all the bacteria which lie in the central portion of the liquid, where the oxygen rapidly disappears to supply the necessities of the bacteria existing there; whilst, on the other hand, near the edges of the cover-glass the movements are very active, in consequence of the constant supply of air. In spite of the speedy death of the bacteria beneath the centre of the glass, we see life prolonged there if by chance a bubble of air has been enclosed. All round this bubble a vast number of bacteria collect in a thick, moving circle, but as soon as all the oxygen of the bubble has been absorbed they fall apparently lifeless, and are scattered by the movement of the liquid.
We may here be permitted to add, as a purely historical matter, that it was these two observations just described, made successively one day in 1861, on vibrios and bacteria, that first suggested to us the idea of the possibility of life without air, and caused us to think that the vibrios which we met so frequently in our lactic fermentations must be the true butyric ferment.
We may pause a moment to consider an interesting question in reference to the two characters under which vibrios appear in butyric fermentations. What is the reason that some vibrios exhibit refractive corpuscles, generally of a lenticular form,
In a fermentation of glycerine in a mineral medium—the glycerine was fermenting under the influence of butyric vibrios—after we had determined the, we may say, exclusive presence
Another observation which still more closely accords with this hypothesis is given in our work on the silkworm disease (vol. i., page 256). We there demonstrate that, when we place in water some of the dust formed of desiccated vibrios, containing a host of these refractive corpuscles, in the course of a very few hours large vibrios appear, well-developed rods fully grown, in which the brilliant points are absent; whilst in the water no process of development from smaller vibrios is to be discerned, a fact which seems to show that the former had issued fully grown from the refractive corpuscles, just as we see colpoda issue with their adult aspect from the dust of their cysts. This observation, we may remark, furnishes one of the best proofs that can be adduced against the spontaneous generation of vibrios or bacteria, since it is probable that the same observation applies to bacteria. It is true that we cannot say of mere points of dust, examined under the microscope, that one particular germ belongs to vibrio, another to bacterium; but how is it possible to doubt that the vibrios issue, as we see them, from an ovum of some kind, a cyst, or germ, of determinate character, when, after having placed some of these indeterminate motes of dust into clean water, we suddenly see, after an interval of not more than one or two hours, an adult vibrio crossing the field of the microscope, without our having been able to detect any intermediate state between its birth and adolescence?
It is a question whether differences in the aspect and nature of vibrios, which depend upon their more or less advanced age, or are occasioned by the influence of certain conditions of the
Fig. 74.
From a consideration of all the facts detailed in this paragraph we can have no hesitation in concluding that, on the one hand, in cases of butyric fermentation, the vibrios which abound in them and constitute their ferment, live without air or free oxygen; and that, on the other hand, the presence of gaseous oxygen operates prejudicially against the movements and activity of those vibrios. But now does it follow that the
We are compelled here to admit that vibrios frequently abound in liquids exposed to the air, and that they appropriate the atmospheric oxygen, and could not withstand a sudden removal from its influence. Must we, then, believe that such vibrios are absolutely different from those of butyric fermentations?
We must not forget, however, that aËrobian torulÆ and anaËrobian ferments present an example of organisms apparently identical, in which, however, we have not yet been able to discover any ties of a common origin. Hence we were forced to regard them as distinct species; and so it is possible that there may likewise be aËrobian and anaËrobian vibrios without any transformation of the one into the other.
The question has been raised whether vibrios, especially those which we have shown to be the ferment of butyric and many other fermentations, are, in their nature, animal or vegetable. M. Ch. Robin attaches great importance to the solution of this question, of which he speaks as follows
We are unable to see the matter in the same light as our learned colleague does; to our thinking, we should be labouring under a great delusion were we to suppose “that it is quite as serious an omission not to determine the animal or vegetable nature of a ferment as it would be to confound nitrogen with hydrogen, or urea with stearine.” The importance of the solutions of disputed questions often depends upon the point of view from which these are regarded. As far as the result of our labours is concerned, we devoted our attention to these two questions exclusively:—1. Is the ferment, in every fermentation properly so called, an organized being? 2. Can this organized being live without air? Now, what bearing can the
M. Robin, however, would have no difficulty in determining the limits of the two kingdoms. According to him, “every variety of cellulose is, we may say, insoluble in ammonia, as also are the reproductive elements of plants, whether male or female. Whatever phase of evolution the elements which reproduce a new individual may have reached, treatment with this reagent, either cold or raised to boiling, leaves them absolutely intact under the eyes of the observer, except that their contents, from being partially dissolved, become more transparent. Every vegetable, whether microscopic or not, every mycelium, and every spore thus preserves in its entirety its special characteristics of form, volume, and structural arrangements; whilst in the case of microscopic animals, or the ova and microscopic embryos of different members of the animal kingdom, the very opposite is the case.”
We should be glad to learn that the employment of a drop of ammonia would enable us to pronounce an opinion, with this degree of confidence, on the nature of the lowest microscopic beings; but is M. Robin absolutely correct in his assumptions? That gentleman himself remarks that spermatozoa, which belong to animal organisms, are insoluble in ammonia, the effect of which is merely to make them paler. If a difference of action in certain reagents, in ammonia, for example, were sufficient to determine the limits of the animal and vegetable kingdoms, might we not argue that there must
In like manner the difficulty which M. Robin has raised in objecting to the employment of the word germ, when we cannot specify whether the nature of that germ is animal or vegetable, is in many respects an unnecessary one. In all the questions which we have discussed, whether we were speaking of fermentation or spontaneous generation, the word germ has been used in the sense of origin of living organism. If Liebig, for example, said of an albuminous substance that it gave birth to ferment, could we contradict him more plainly than by replying: “No; ferment is an organized being, the germ of which
In our Memoir of 1862, on so-called spontaneous generations, would it not have been an entire mistake to have attempted to assign specific names to the microscopic organisms which we met with in the course of our observations? Not only would we have met with extreme difficulty in the attempt, arising from the state of extreme confusion which even in the present day exists in the classification and nomenclature of these microscopic organisms, but we should have been forced to sacrifice clearness in our work besides; at all events, we should have wandered from our principal object, which was the determination of the presence or absence of life in general, and had nothing to do with the manifestation of a particular kind of life in this or that species, animal or vegetable. Thus we have systematically employed the vaguest nomenclature, such as mucors, torulÆ, bacteria, and vibrios. There was nothing arbitrary in our doing this, whereas there is much that is arbitrary in adopting a definite system of nomenclature, and applying it to organisms but imperfectly known, the differences or resemblances between which are only recognizable through certain characteristics, the true signification of which is obscure. Take, for example, the extensive array of widely different systems that have been invented during the last few years for the species of the genera bacterium and vibrio in the works of Cohn, H. Hoffmann, Hallier, and Billroth. The confusion which prevails here is very great, although we do not of course by any means place these different works on the same footing as regards their respective merits.
M. Robin is, however, right in recognizing the impossibility of maintaining in the present day, as he formerly did, “that fermentation is an exterior phenomenon, going on outside cryptogamic cells, a phenomenon of contact. It is probably,” he adds, “an interior and molecular action at work in the inmost recesses of the substance of each cell.” From the day when we first
We have devoted the greater part of this chapter to the establishing with all possible exactness the extremely important physiological fact of life without air, and its correlation to the phenomena of fermentations properly so called—that is to say, of those which are due to the presence of microscopic cellular organisms. This is the chief basis of the new theory that we propose for the explanation of these phenomena. The details into which we have entered were indispensable on account of the novelty of the subject no less than on account of the necessity we were under of combating the criticisms of the two German naturalists, Drs. Oscar Brefeld and Traube, whose works had cast some doubts on the correctness of the facts upon which we had based the preceding propositions. We have much pleasure in adding that at the very moment when we were revising the proofs of this chapter, we received from M. Brefeld an essay, dated from Berlin, January, 1876, in which, after describing his later experimental researches, he owns with praiseworthy frankness that Dr. Traube and he were both of them mistaken. Life without air is now a proposition which he accepts as perfectly demonstrated. He has witnessed it in the case of mucor racemosus, and has also verified it in the case of yeast. “If,” he says, “after the results of my previous researches, which I
To bring one’s self to believe in a truth that has just dawned upon one is the first step towards progress; to persuade others is the second. There is a third step, less useful perhaps, but highly gratifying nevertheless, which is, to convince one’s opponents.
We, therefore, have experienced great satisfaction in learning that we have won over to our ideas an observer of singular ability, on a subject which is of the utmost importance to the physiology of cells.
§ VI.—Reply to the Critical Observations of Liebig, Published in 1870.
In the Memoir which we published, in 1860, on alcoholic fermentation, and in several subsequent works, we were led to a different conclusion on the causes of this very remarkable phenomenon from that which Liebig had adopted. The opinions of Mitscherlich and Berzelius had ceased to be tenable in the presence of the new facts which we had brought to light. From
“I had admitted,” he says, “that the resolution of fermentable matter into compounds of a simpler kind must be traced to some process of decomposition taking place in the ferment, and that the action of this same ferment on the fermentable matter must continue or cease according to the prolongation or cessation of the alteration produced in the ferment. The molecular change in the sugar would, consequently, be brought about by the destruction or modification of one or more of the component parts of the ferment, and could only take place through the contact of the two substances. M. Pasteur regards fermentation in the following light:—The chemical action of fermentation is essentially a phenomenon correlative with a vital action, beginning and ending with it. He believes that alcoholic fermentation can never occur without the simultaneous occurrence of organization, development, and multiplication of globules, or continuous life, carried on from globules already formed. But the idea that the decomposition of sugar during fermentation is due to the development of the cellules of the ferment, is in contradiction with the fact that the ferment is able to bring about the fermentation of a pure solution of sugar. The greater part of the ferment is composed of a substance that is rich in nitrogen and contains sulphur. It contains, moreover, an appreciable quantity of phosphates, hence it is difficult to conceive how, in the absence of these elements in a pure solution
Notwithstanding Liebig’s belief to the contrary, the idea that the decomposition of sugar during fermentation is intimately connected with a development of the cellules of the ferment, or a prolongation of the life of cellules already formed, is in no way opposed to the fact that the ferment is capable of bringing about the fermentation of a pure solution of sugar. It is manifest to any one who has studied such fermentation with the microscope, even in those cases where the sweetened water has been absolutely pure, that ferment-cells do multiply, the reason being that the cells carry with them all the food-supplies necessary for the life of the ferment. They may be observed budding, at least many of them, and there can be no doubt that those which do not bud still continue to live; life has other ways of manifesting itself besides development and cell-proliferation.
If we refer to the figures on page 81 of our Memoir of 1860, Experiments D, E, F, G, H, I, we shall see that the weight of yeast, in the case of the fermentation of a pure solution of sugar, undergoes a considerable increase, even without taking into account the fact that the sugared water gains from the yeast certain soluble parts, since, in the experiments just mentioned, the weights of solid yeast, washed and dried at 100° C. (212° F.), are much greater than those of the raw yeast employed, dried at the same temperature.
In these experiments we employed the following weights of yeast, expressed in grammes (1 gramme = 15·43 grains)—
| 2·313 |
| 2·626 |
| 1·198 |
| 0·699 |
| 0·326 |
| 0·476 |
which became after fermentation, we repeat, without taking
| grammes. | grains. | ||
| 2·486 | [Increase | 0·173 = | 2·65 |
| 2·963 | “ | 0·337 = | 5·16 |
| 1·700 | ” | 0·502 = | 7·7 |
| 0·712 | “ | 0·013 = | 0·2 |
| 0·335 | ” | 0·009 = | 0·14 |
| 0·590 | “ | 0·114 = | 1·75 |
Have we not in this marked increase in weight a proof of life, or, to adopt an expression which may be preferred, a proof of a profound chemical work of nutrition and assimilation?
We may cite on this subject one of our earlier experiments, which is to be found in the Comptes rendus de l’AcadÉmie for the year 1857, and which clearly shows the great influence exerted on fermentation by the soluble portion that the sugared water takes up from the globules of ferment:—
“We take two equal quantities of fresh yeast that have been washed very freely. One of these we cause to ferment in water containing nothing but sugar, and, after removing from the other all its soluble particles—by boiling it in an excess of water and then filtering it to separate the globules—we add to the filtered liquid as much sugar as was used in the first case along with a mere trace of fresh yeast, insufficient, as far as its weight is concerned, to affect the results of our experiment. The globules which we have sown bud, the liquid becomes turbid, a deposit of yeast gradually forms, and, side by side with these appearances, the decomposition of the sugar is effected, and in the course of a few hours manifests itself clearly. These results are such as we might have anticipated. The following fact, however, is of importance. In effecting by these means the organization into globules of the soluble part of the yeast that we used in the second case, we find that a considerable quantity of sugar is decomposed. The following are the results of our experiment: 5 grammes of yeast caused the fermentation of 12·9
How is it possible to maintain that, in the fermentation of water containing nothing but sugar, the soluble portion of the yeast does not act, either in the production of new globules or the perfection of old ones, when we see, in the preceding experiment, that after this nitrogenous and mineral portion has been removed by boiling, it immediately serves for the production of new globules, which, under the influence of the sowing of a mere trace of globules, causes the fermentation of much sugar?
In short, Liebig is not justified in saying that the solution of pure sugar, caused to ferment by means of yeast, contains none of the elements needed for the growth of yeast, neither nitrogen, sulphur, nor phosphorus, and that, consequently, it should not be possible, by our theory, for the sugar to ferment. On the contrary, the solution does contain all these elements, as a consequence of the introduction and presence of the yeast.
Let us proceed with our examination of Liebig’s criticisms:—
“To this,” he goes on to say, “must be added the decomposing action which yeast exercises on a great number of substances,
This statement, with all due deference to the opinion of our illustrious critic, is by no means correct. Yeast has no action on malate of lime, or on other calcareous salts formed by vegetable acids. Liebig had previously, much to his own satisfaction, brought forward urea as being capable of transformation into carbonate of ammonia during alcoholic fermentation in contact with yeast. This has been proved by us to be erroneous. It is an error of the same kind that Liebig again brings forward here. In the fermentation of which he speaks (that of malate of lime), certain spontaneous ferments are produced, the germs of which are associated with the yeast, and develop in the mixture of yeast and malate. The yeast merely serves as a source of food for these new ferments without taking any direct part in the fermentations of which we are speaking. Our researches leave no doubt on this point, as is evident from the observations on the fermentations of tartrate of lime previously given.
It is true that there are circumstances under which yeast brings about modifications in different substances. Doebereiner and Mitscherlich, more especially, have shown that yeast imparts to water a soluble material, which liquefies cane-sugar and produces inversion in it by causing it to take up the elements of water, just as diastase behaves to starch or emulsin to amygdalin.
M. Berthelot also has shown that this substance may be isolated by precipitating it with alcohol, in the same way as diastase is precipitated from its solutions.
M. Paul Bert, in his remarkable studies on the influence of
We now come to Liebig’s principal objection, with which he concludes his ingenious argument, and to which no less than eight or nine pages of the Annales are devoted.
Our author takes up the question of the possibility of causing yeast to grow in sweetened water, to which a salt of ammonia and some yeast-ash have been added—a fact which is evidently incompatible with his theory that a ferment is always an albuminous substance on its way to decomposition. In this case the albuminous substance does not exist; we have only the mineral substances which will serve to produce it. We know that Liebig regarded yeast, and, generally speaking, any ferment whatever, as being a nitrogenous, albuminous substance which, in the same way as emulsin, for example, possesses the power of bringing about certain chemical decompositions. He connected fermentation with the easy decomposition of that albuminous substance, and imagined that the phenomenon occurred in the following manner:—“The albuminous substance on its way to decomposition possesses the power of communicating to certain other bodies that same state of mobility by which its own atoms are already affected; and through its contact with other bodies it imparts to them the power of decomposing or of entering into other combinations.” Here Liebig failed to perceive that the ferment, in its capacity of a living organism, had anything to do with the fermentation.
This theory dates back as far as 1843. In 1846 Messrs. Boutron and Fremy, in a Memoir on lactic fermentation, published in the Annales de Chimie et de Physique, strained the
This theory of hemi-organism was word for word the antiquated opinion of Turpin, as may be readily seen by referring to Chapter IV., section III. of the present work. The public, especially a certain section of the public, did not go very deeply into an examination of the subject. It was the period when the doctrine of spontaneous generation was being discussed with much warmth. The new word hemi-organism, which was the only novelty in M. Fremy’s theory, deceived people. It was thought that M. Fremy had really discovered the solution of the question of the day. It is true that it was rather difficult to understand the process by which an albuminous substance could become all at once a living and budding cell. This difficulty was readily solved by M. Fremy, who declared that it was the result of some power that was not yet understood, the power of “organic impulse.”
“There seems to be no doubt as to the part which the vegetable organism plays in the phenomenon of fermentation. It is through it alone that an albuminous substance and sugar are enabled to unite and form this particular combination, this unstable form under which alone, as a component part of the mycoderm, they manifest an action on sugar. Should the mycoderm cease to grow, the bond which unites the constituent parts of the cellular contents is loosened, and it is through the motion produced therein that the cells of yeast bring about a disarrangement or separation of the elements of the sugar into other organic molecules.”
One might easily believe that the translator for the Annales has made some mistake, so great is the obscurity of this passage.
Whether we take this new form of the theory or the old one, neither can be reconciled at all with the development of yeast and fermentation in a saccharine mineral medium, for in the latter experiment fermentation is correlative to the life of the ferment and to its nutrition, a constant change going on between the ferment and its food-matters, since all the carbon assimilated by the ferment is derived from sugar, its nitrogen from ammonia, and phosphorus from the phosphates in solution. And even all said, what purpose can be served by the gratuitous hypothesis of contact-action or communicated motion? The experiment of which we are speaking is thus a fundamental one; indeed, it is its possibility that constitutes the most effective point in the controversy. No doubt Liebig might say, “but it is the motion of life and of nutrition which constitutes your experiment, and this is the communicated motion that my theory requires.” Curiously enough, Liebig does endeavour, as a matter of fact, to say this, but he does so timidly and incidentally: “From a chemical point of view, which point of
“It is possible that the only correlation between the physiological act and the phenomenon of fermentation is the production, in the living cell, of the substance which, by some special property analogous to that by which emulsin exerts a decomposing action on salicin and amygdalin, may bring about the decomposition of sugar into other organic molecules; the physiological act, in this view, would be necessary for the production of this substance, but would have nothing else to do with the fermentation (page 10).” To this, again, we have no objection to raise.
Liebig, however, does not dwell upon these considerations, which he merely notices in passing, because he is well aware that, as far as the defence of his theory is concerned, they would be mere evasions. If he had insisted on them, or based his opposition solely upon them, our answer would have been simply this: “If you admit with us that fermentation is correlated with the life and nutrition of the ferment, we agree upon the principal point. So agreeing, let us examine, if you will, the actual cause of fermentation;—this is a second question, quite distinct from the first. Science is built up of successive solutions given to questions of ever-increasing subtlety, approaching nearer and nearer towards the very essence of phenomena. If we proceed to discuss together the question of how living, organized beings act in decomposing fermentable substances, we will be found to fall out once more on your hypothesis of communicated motion, since, according to our ideas, the actual cause of fermentation is to be sought, in most cases, in the fact of life without air, which is the characteristic of many ferments.”
Let us briefly see what Liebig thinks of the experiment in which fermentation is produced by the impregnation of a saccharine mineral medium, a result so greatly at variance with
Our knowledge of the facts detailed in the preceding chapters concerning pure ferments and their manipulation in the presence of pure air, enables us to completely disregard those causes of embarrassment that result from the fortuitous occurrence of the germs of organisms, different in character from the ferments, introduced by the air or from the sides of vessels, or even by the ferment itself.
Let us once more take one of our double-necked flasks (Fig. 22, p. 110), which we will suppose is capable of containing three or four litres (six to eight pints).
Let us put into it the following:—
| Pure distilled water. | |
| Sugar candy | 200 grammes. |
| Bitartrate of potassium | 1·0 “ |
| ” “ ammonia | 0·5 ” |
| Sulphate of ammonia | 1·5 “ |
| Ash of yeast | 1·5 ” |
| [1 gramme = 15·43 grains.] |
Let us boil the mixture, to destroy all germs of organisms that may exist in the air or liquid or on the sides of the flask, and then permit it to cool, after having placed, by way of extra precaution, a small quantity of asbestos in the end of the fine, curved tube. Let us next introduce a trace of ferment into the liquid, through the other neck, which, as we described, is terminated by a small piece of india-rubber tube closed with a glass stopper.
Here are the details of such an experiment:—
On December 9, 1873, we sowed some pure ferment—saccharomyces pastorianus. From December 11, that is, within so short a time as forty-eight hours after impregnation, we saw a multitude of extremely minute bubbles rising almost continuously from the bottom, indicating that at this point the fermentation had commenced. On the following days, several
There was no development of any organism absolutely foreign to the ferment, which was itself abundant, a circumstance that, added to the persistent vitality of the ferment, in spite of the unsuitableness of the medium for its nutrition, permitted the perfect completion of fermentation. There was not the minutest quantity of sugar remaining. The total weight of ferment, after washing and drying at 100° C. (212° F.), was 2·563 grammes (39·5 grains).
In experiments of this kind, in which the ferment has to be weighed, it is better not to use any yeast-ash that cannot be dissolved completely, so as to be capable of easy separation from the ferment formed. Raulin’s liquid, the composition of which we have already given (p. 89, footnote), may be used in such cases with success.
All the alcoholic ferments are not capable to the same extent of development by means of phosphates, ammoniacal salts, and sugar. There are some whose development is arrested a longer or shorter time before the transformation of all the sugar. In a series of comparative experiments, 200 grammes of sugar-candy being used in each case, we found that whilst saccharomyces pastorianus effected a complete fermentation of the sugar, the caseous ferment did not decompose more than two-thirds, and the ferment which we have designated new “high” ferment not more than one-fifth: and keeping the flasks for a longer time in the oven had no effect in increasing the proportions of sugar fermented in these two last cases.
We conducted a great number of fermentations in mineral
We may here add a few words on the non-transformation of yeast into penicillium glaucum.
If at any time during fermentation we pour off the fermenting liquid, the deposit of yeast remaining in the vessel may continue there, in contact with air, without our ever being able to discover the least formation of penicillium glaucum in it. We may keep a current of pure air constantly passing through the flask; the experiment will give the same result. Nevertheless, this is a medium peculiarly adapted to the development of this mould, inasmuch as if we introduce merely a few spores of penicillium, an abundant vegetation of that growth will afterwards appear on the deposit. The descriptions of Messrs. Turpin, Hoffmann, and TrÉcul have, therefore, been based on one of these illusions which we meet with so frequently in microscopical observations.
“According to M. Pasteur,” he said, “the yeast of beer is anaËrobian, that is to say, it lives in a liquid deprived of free oxygen; and to become mycoderma or penicillium it is above all things necessary that it should be placed in air, since, without this, as the name signifies, an aËrobian being cannot exist. To bring about the transformation of the yeast of beer into mycoderma cerevisiÆ or into penicillium glaucum, we must accept the conditions under which these two forms are obtained. If M. Pasteur will persist in keeping his yeast in media which are incompatible with the desired modification, it is clear that the results which he obtains must be always negative.”
Contrary to this perfectly gratuitous assertion of M. TrÉcul’s, we do not keep our yeast in media which are calculated to prevent its transformation into penicillium. As we have just seen, the principal aim and object of our experiment was to bring this minute plant into contact with air, and under conditions that would allow the penicillium to develop with perfect freedom. We conducted our experiments exactly as Turpin and Hoffmann conducted theirs, and exactly as they stipulate that such experiments should be conducted—with the one sole difference, indispensable to the correctness of our observations, that we carefully guarded ourselves against those causes of error which they did not take the least trouble to avoid. It is possible to produce a ready entrance and escape of pure air in the case of the double-necked flasks which we have so often employed in the course of this work, without having recourse to the continuous passage of a current of air. Having made a file-mark on the thin curved neck at a distance of two or three centimetres (an inch) from the flask, we must cut round the neck at this point with a glazier’s diamond, and then remove it, taking care to cover the opening immediately with a sheet of paper
What we have said of penicillium glaucum will apply equally to mycoderma cerevisiÆ. Notwithstanding what Turpin and TrÉcul may assert to the contrary, yeast, in contact with air as it was under the conditions of the experiment just described, will not yield mycoderma vini or mycoderma cerevisiÆ any more than it will penicillium.
The experiments described in the preceding paragraphs on the increase of organized ferments in mineral media of the composition described, are of great physiological interest. Amongst other results, they show that all the proteic matter of ferments may be produced by the vital activity of the cells, which, apart altogether from the influence of light or free oxygen (unless, indeed, we are dealing with aËrobian moulds which require free oxygen), have the power of developing a chemical activity between carbo-hydrates, ammoniacal salts, phosphates and sulphates of potassium and magnesium. It may be admitted with truth that a similar effect obtains in the case of the higher plants, so that in the existing state of science we fail to conceive what serious reason can be urged against our considering this effect as general. It would be perfectly logical to extend the results of which we are speaking to all plants, and to believe that the proteic matter of vegetables, and perhaps of animals also, is formed exclusively by the activity of the cells operating upon the ammoniacal and other mineral salts of the sap or plasma of the blood, and the carbo-hydrates, the formation of which, in the case of the higher plants, requires only the concurrence of the chemical impulse of green light.
Viewed in this manner, the formation of the proteic substances would be independent of the great act of reduction of carbonic acid gas under the influence of light. These substances would not be built up from the elements of water, ammonia,
If solar radiation is indispensable to the decomposition of carbonic acid and the building up of the primary substances in the case of higher vegetable life, it is still possible that certain inferior organisms may do without it and nevertheless yield the most complex substances, fatty or carbo-hydrate, such as cellulose, various organic acids, and proteic matter; not, however, by borrowing their carbon from the carbonic acid which is saturated with oxygen, but from other matters still capable of acquiring oxygen, and so of yielding heat in the process, such as alcohol and acetic acid, for example, to cite merely carbon compounds most removed from organization. As these last compounds, and a host of others equally adapted to serve as the carbonaceous food of mycoderms and the mucedines, may be produced synthetically by means of carbon and the vapour of water, after the methods that science owes to Berthelot, it follows that, in the case of certain inferior beings, life would be possible even if it should be that the solar light was extinguished.
