Chapter V: The Alcoholic Ferments (2)

The fact which should claim all our attention, we repeat, is, that this exhausted, shrivelled-up, aged-looking yeast preserves its faculty of germination for several years; that, moreover, this faculty may be aroused by placing it in aerated nutritive media, in which case it will exhibit all the peculiarities which, under similar conditions, characterize some of the germ-cells found on the surface of our sweet domestic fruits. In other words, this yeast, instead of multiplying, as it always does in the course of several growths in saccharine musts, in the form of cells which detach themselves readily as soon as they have nearly attained the form and size of the mother-cells, begins to shoot out into such beautiful forms as those of _dematium pullulans_, producing like that ferment long, well-grown, branching filaments, as well as plump and frequently pyriform cells, as represented in Plate X.

The following figures (33 to 37) and descriptions of the observations to which they relate will furnish fresh proofs of our assertions. In these figures we see _saccharomyces pastorianus_, which has been exhausted in sweetened water or in yeast-water, undergo revival in saccharine musts, give rise to elongated, branching, pear-shaped forms, such as belong to the original ferments of fruits, and afterwards assume the most minute forms that we find in fermentations progressing or completed.

Let us examine Fig. 33. The history of this growth is as follows:—

Some spontaneous yeast which, after repeated cultivation, had acquired the aspect represented in Plate XI.—which aspect the _saccharomyces pastorianus_ generally assumes under these circumstances—was exhausted in sweetened water, and subsequently revived in must at 10° C. to 11° C. (51° F.). At this temperature germination was not very marked before the end of eight days; at a temperature of 20° C. (68° F.), it only took three days, under similar conditions. The sketch includes but one of the long branches from which the ferment cells and the budding joints took rise, but there were a great number more. Some of the forms represented in the figure bear a striking resemblance, it appears to us, to some of those of _dematium_, in Plate IX.; and even we may trace out the several peculiarities of form which distinguish the figures in the latter plate.

The next figure (34) represents the earliest forms of germination of another specimen of _saccharomyces pastorianus_ in wort, after it had been exhausted by four successive growths in sweetened water. We here see the large ferment-form which appears at the commencement of fermentation, in acid fruits, such as cherries and gooseberries (Plate IX.), associated with smaller forms, which follow it and emanate from it, in proportion as the process of budding is repeated. The field was covered with this minute form, and we had to search about considerably before we could find any of the large cells and the long, branching, jointed filaments which we have sketched. The reason of this was, that these large, extended filaments only appear at the beginning, when there is still an abundance of air, giving place, after repeated budding, to minute cells or short filaments, the ever-increasing number of which soon hides the others from sight.

Fig. 35 represents _saccharomyces pastorianus_ again, as it appears after having been exhausted by two years preservation in yeast-water, in contact with pure air. Strange to say, it has lost its elongated appearance, and would appear to have originated from a round ferment. The cells are much exhausted, and most of them seem to have a double border; their interior is very granular and of a yellowish colour. One might readily take the specimen to be a dead old ferment, which, however, it by no means is.

Fig. 36 represents the germination of this ferment, which had previously been revived in a flask of wort, at the temperature of the air, in May, 1875. The following are the details of our observation:—

We sowed a trace of the exhausted yeast (Fig. 35) in a flask of wort on May 16th. The sketch (Fig. 36) was made on May 19th, but on the 18th there was a sensible revival. It will be seen how much the little ferment had developed in the course of three days from the time when the process commenced. If we had waited a few days longer before taking our sample, we should probably have had difficulty in finding any cells or filaments of the large ferment form, as there would have been so few of them in comparison with the others.

In the above figure we should remark the chain of large cells and long-jointed processes, _a_, _b_, _c_, _d_ : _d_ is one of the cells that we sowed; it has become transparent, and its contents, which are slightly granular, have lost their brownish tint; _c_ is a large cell which sprang from the preceding one; its outline is clear, and it is full of fine, yellowish granules, which present a perfect resemblance to the large ferment-cells of fruits, proceeding from the germ-cells on the surface of those fruits, when it begins to appear in sweet juices; _b_ is a long filament, sprung from the preceding cell; and, last of all, _a_ is a joint and its bud, in which the border is not yet very clearly defined; it has scarcely any granules, and is finer than the others, belonging, in short, to the small ferment form represented in Plate XI. Here, then, we see the transition of the large ferment to the small, on the same branch, after two generations from the germination of the germ-cell _d_. This observation corroborates the opinion maintained by us, that in Figs. 33, 34, 36, as in Plate X., we have not a mixture of two ferments, the one consisting of large, elongated filaments, the other of small cells, but one and the same ferment, the differences in the form and size of which depend on particular conditions. The smallest ferment-form very soon becomes the only one visible, and it preserves its peculiar appearance in successive growths from inability to return to the full, elongated, filamentous forms before undergoing a prolonged exhaustion. The ferment of _mucor_ would probably afford similar indications: it would be very interesting to find out.

The following is one of the most curious of the forms presented by _saccharomyces pastorianus_, occurring after exhaustion in a sweet mineral liquid. The ferment, taken from a closed vat, in which it had been used for beer, was sown in the mineral liquid on July 4th, 1873. The following days the ferment developed feebly, but perceptibly, and gradually increased in bulk. The flask was left to itself in an oven at 25° C. (77° F.) until December 3rd, when we ascertained that all the sugar had fermented. We then sowed a trace of the deposit, which had become abundant, in a flask of pure wort. On December 4th there was no perceptible change. On December 5th, however, fermentation was in active progress; a large quantity of froth covered the surface of the liquid, and a considerable deposit of ferment had already taken place at the bottom. We made a microscopical examination of this deposit, a sketch of which we append (Fig. 37). The dark, double-bordered cells are those which were sown but did not rejuvenesce. We may notice in different places several of these same cells, recognizable by their granular contents, which they are beginning to lose, to make room for germinating cells and joints, often numerous. For instance, in the group at the bottom of our figure one of the cells is in course of rejuvenescence and germination, and has given rise to no fewer than six cells, filaments, or groups of filaments. In different fields of our microscope we met with a crowd of branches, more or less ramified, and chains of cells, of greater or less length, of which we have sketched a few. In proportion as the budding of these branches is repeated, the cells and joints become more readily disunited, grow small, and assume the appearance of _saccharomyces pastorianus_ in ordinary growths, almost as represented in Plate XI. At first, when the old, exhausted cells begin to germinate, their appearance rather resembles that of _dematium pullulans_, as seen in the germination of many of the corpuscles on the surface of clusters of grapes or fruits, or their woody parts, some specimens of which are to be found in Plate IX.

We may briefly summarize the leading facts demonstrated in the above paragraph. We have seen that there are different alcoholic ferments. In the fermentation of natural saccharine juices, which, especially when acid, so readily undergo a decided alcoholic fermentation, the ferments originate in certain germ-cells, which are spread in the form of minute spherical bodies of a yellow or brown colour, isolated or in groups, over the exterior surface of the epidermis of the plant, and which are gifted with an extraordinary power of budding with ease and rapidity in fermentable liquids. The presence of atmospheric oxygen is indispensable to the germination of these germ-cells, a fact which explains Gay-Lussac’s observation that atmospheric oxygen is necessary for the commencement of spontaneous fermentation in must.[97] Of these various ferments one deserves special mention—namely, the variety termed _saccharomyces pastorianus_. As is the case with all ferments, when we gather it from the deposits produced in must that has been fermented by its action, it is composed entirely of oval or spherical cells or of short joints. When again placed in a similar must it buds, like all the ordinary ferments, and the buds detach themselves from the joints or mother-cells as soon as they have attained the size of these latter, from which time in the new deposit is reproduced the original ferment-form from which it sprung, and so on. Under certain conditions of exhaustion, however, which may be easily obtained, and which we have already accurately described, the cells undergo an absolute change as regards their capabilities of budding and germinating. Each cell, modified in its structure by the conditions we have mentioned, shows a tendency to shoot out all around its surface, with astonishing rapidity, into a multitude of buds, from many of which spring branching chains, covered in parts, and more especially at the internodes, with cells and jointed filaments, which fall off and bud in their turn, soon to present the forms of the yeast deposit. In this way _saccharomyces pastorianus_ seems to afford a kind of bond of union between the race of ferments on the one hand, and certain kinds of ordinary fungoid growths on the other. Of these latter the plant which De Bary has named _dematium_, and which is generally found on the surface of leaves or dead wood, more especially, however, on the wood of the vine at the end of autumn, the time of the vintage, presents a striking example.

There seems every reason to believe that at this period of the year one or more of the varieties of _dematium_ furnish cells of yeast, or even that the ordinary aërobian varieties of _dematium_ produce at a certain stage of their vegetation, in addition to aërobian cells and torulæ, other cells and torulæ which are anaërobian, that is, alcoholic ferments.

In this manner we arrive at the confirmation of an idea entertained by most authors who have studied yeast closely—namely, that it must be an organ detached from some more complex vegetable form. We may also add that in the case of _saccharomyces_ the chains of filaments, both tubular and fusiform, and septate cells more or less pyriform originating in them, when attentively observed, remind us forcibly of the filamentous chains and spore-balls, or conidia of _mucor racemosus_ when submerged, so that one might suppose that the spore-ferment of our _dematium_ is itself an organ detached from some still more complex vegetable form, in the same way that conidia-ferment of _mucor racemosus_ belongs to that more complex fungoid growth.

In the following passage De Bary uses, for the first time, the words _dematium pullulans_ (Hofmeister, vol. ii. p. 182, 1866). The German naturalist begins by citing the opinions of Bail, Berkeley, and H. Hoffmann, the first of whom maintains that _mucor mucedo_ becomes transformed into the yeast of beer, the second that yeast is a peculiar state of _penicillium_, and the third that it may be generated by fungi of very different nature, and especially by _penicillium glaucum_ and _mucor mucedo_. He goes on to say: “I have taken great pains to repeat the experiments of Bail, Berkeley, and H. Hoffmann, but I have never been able to confirm the results which they have stated, either in the case of growths in microscopic cells or in experiments performed in test-tubes with the purest possible substances—specially prepared solutions or must of wine and spores of penicillium, _mucor mucedo_, _botrytis cinerea_, &c.” On this point M. De Bary arrives at exactly the same results which we communicated to the _Société Philomathique_ and the _Société Chimique_ of Paris, as already given in Chap. IV. § 4, p. 128, note.

M. De Bary goes on to say: “In researches of this kind it is difficult to eliminate two sources of error. On the one hand, it is beyond doubt that cells of ferment are actually scattered over everything, and that, consequently, they may easily get into the experimental liquid along with the spores that we sow, and so occasion mistakes. On the other hand, there are a great many fungi which develop budding processes similar to yeast, but incapable of producing fermentation, which yet in some cases spring directly from spores as well as from mycelium, especially we may instance _exoascus_. This last observation is especially applicable to the extraordinarily numerous variety of fungi which rank under the Dematiei and Sphaeriacei, and which I shall term, for convenience of naming, _dematium pullulans_.”[98]

We shall conclude this paragraph with a remark that has doubtless presented itself to the minds of our readers, which is, that it would be impossible to carry out the experiments we have described if we could not make sure of dealing with pure ferments, or, at least, with mixtures the components of which are sufficiently well known for us to assign to each the effect produced by it in the total phenomena observed. It would be extremely difficult to continue growths of yeast-deposit in sweetened water or in a moist atmosphere if the little plant were mixed with spores of other fungoid growths, a variety of ferment-forms, and germs of bacteria, vibrios, or infusoria in general. All these foreign organisms would tend to develop just in proportion as the conditions of the media were more or less favourable to their growth, and, in a very few days, our flasks would be filled with swarms of beings which, in most cases, would entirely conceal the facts relating to those forms, the separate study of which it was our object to follow out. We shall have occasion, therefore, to examine, in a subsequent paragraph, the preparation of ferments in a state of purity. At present we may state that yeast, which in its ordinary condition is a mass of cells so liable to change that its preservation in a moist state is impossible, manifesting in the course of a few days during the winter, and in twenty-four hours during the heat of summer, all the signs of incipient putrefaction, thereby losing its distinctive characteristics, is nevertheless capable, when pure, of enduring the highest atmospheric temperatures for whole years without showing the least signs of putrid change or contamination with any other microscopic organisms, and without the cells losing their power of reproduction. In the presence of facts like these, the theory of spontaneous generation must seem chimerical. The hypothesis of the possible transformation of yeast into _penicillium glaucum_, bacteria, and vibrios, or conversely, which the theories of Turpin, H. Hoffmann, Berkeley, Trécul, Hallier, and Béchamp involve, is equally refuted by these facts.

§ II.—On “Spontaneous” Ferment.

The expression _spontaneous ferment_ may be applied to any ferment that appears in a fermentable liquid without having been purposely sown in it. In this respect the ferments mentioned in the preceding paragraph, those of all saccharine juices of fruit which ferment when left to themselves—the ferments of wine, for example—are spontaneous. The term, however, is not altogether appropriate, because, after all, the process is the same as if an actual sowing had been made, since, as we have shown, it is absolutely necessary for the juice to come into contact with the surfaces of the fruit, so that the ferment may be mixed with it, and so produce subsequent fermentation. Therefore, although we may apply the term _spontaneous ferment_ to the ferments of fruits, we intend that expression to apply in this paragraph solely to those ferments that are generated in a saccharine liquid, in which, by previous boiling, we have destroyed all ferment germs, and which, nevertheless, enters into fermentation after being exposed in free contact with air. In such a case it is entirely from the particles of dust floating in the air that the ferment germs that appear in the liquid are derived. Such are typical _spontaneous_ fermentations, and it is of the ferment so obtained that we are about to speak.

In the course of the researches which we undertook in order to ascertain whether _mycoderma vini_, or vinous efflorescence, became transformed, in the case of beer, into actual alcoholic ferment—researches which were the more protracted and varied in consequence of their leading to the condemnation as erroneous, on the faith of new and more precise experiments, such as those given in Chap. IV. § 2, of that transformation, in which we had for long believed—we had occasion to observe several spontaneous fermentations of this kind in various saccharine liquids. We then proceeded to describe our method of conducting the experiments. Having brought about the development of a film of _mycoderma vini_ or _cerevisiæ_ on the surface of a liquid, fermented or not, we submerged that film in wort, which we afterwards put into long-necked flasks, in which alcoholic fermentation generally took place in the course of a few days. This fermentation in no way resulted from the transformation of the cells constituting the efflorescence into ferment. The mycodermic film merely acted as a receptacle of true ferment germs, wafted thither with the particles of dust floating in the air of the laboratory, which germs developed in the liquid into actual alcoholic ferments amongst the cells of the submerged mycoderma. By conducting experiments in this manner we brought about several spontaneous fermentations, the germs of which could have been introduced by nothing but the particles of dust in the air. These fermentations, which we were obliged to follow very carefully with the microscope from the time when they first manifested themselves, on account of the transformation that we were seeking, which transformation we thought might possibly be that of the cells of _mycoderma vini_ into cells of ferment, generally gave us during the first days of fermentation the large, elongated, branchy ferment represented in Plate X., which was succeeded by the small ferment represented in Plate XI.[99]

Here let me describe one of these experiments. In the beginning of March, 1872, we grew some _mycoderma vini_, obtained from wine, on some wort contained in a shallow basin. On March 6th we submerged the efflorescence and put it all together, liquid and film, into a long-necked flask. On March 9th we detected incipient fermentation, and on March 12th we took a sketch of the yeast of the deposit, as given in Plate XII. This is the large and long branching, more or less pear-shaped form, which occurs at the beginning of fermentation in the sweet and acid musts of our domestic fruits. On March 16th we made another sketch of the deposit, in which the proportion of cells, in the form of elongated segments and filaments, reminded us, in some measure, of the filamentous mycelium of typical fungoid growths much diminished. In this case, however, the majority of cells were oval, round, and in short segments. On this day, March 16th, we added some fresh wort to that which had fermented, with the object of prolonging the duration of fermentation and increasing the proportion of yeast. On March 19th we made a fresh sketch, which it is not necessary either to reproduce; suffice it to say, that the yeast was now considerably more regular and uniform in appearance.

Spontaneous ferment, therefore, very often occurs in this large ferment-form, which, by repeated developments in the act of fermentation, becomes reduced by degrees after successive generations to the ferment which, following Dr. Rees, we have named _saccharomyces pastorianus_, a polymorphous ferment which must be studied closely that it may not be confounded with others, inasmuch as it is so universally diffused that we very seldom fail to find it in any ferment which has been exposed in contact with ordinary air, at least, we may repeat, in a laboratory devoted to researches on fermentation. We have found the same thing occur in a brewery, being there mixed with the ferments used in brewing.

There are, no doubt, several varieties of this _saccharomyces_. We sometimes find amongst the spontaneous ferments which repeated growths have brought to a more or less uniform state, the forms represented in Plate XI., but the cells and segments much smaller. Amongst others, Dr. Rees has distinguished a _saccharomyces exiguus_.

Fig. 38 represents another spontaneous ferment, which appeared in a boiled saccharine wort, which entered into fermentation after being exposed to the air of the laboratory.

The sketch was made directly after the fermentation had commenced. Probably this is simply one of the earlier forms assumed by the _saccharomyces_, or by one of its varieties. It will be seen that the alcoholic ferment is associated with another little filiform ferment, probably the lactic. The spontaneous ferments are almost always impure, a circumstance that may be readily understood if we bear in mind the results described in Chapters III. and IV.

§ III.—On “High” and “Low” Ferments.

The ferments mentioned in the preceding paragraphs do not belong, properly speaking, to industrial products; that is to say, in actual practice there are no operations in which the ferments of fruits and spontaneous ferments are employed for the purpose. It is quite true that these ferments are the cause of the fermentations from which wine, cider, gin, rum, gentiana, mead, &c., are derived, but these fermentations are spontaneous, they take place without the intervention of man, and without man’s directing their production, or taking any notice of the agent which starts them.

In the manufacture of beer, on the other hand, the practice is quite different. We may say that the wort is never left to ferment spontaneously, the fermentation being invariably produced by the addition of yeast formed on the spot in a preceding operation, or procured from some other working brewery, which, again, had at some time been supplied from a third brewery, which itself had derived it from another, and so on, as far back as the oldest brewery that can be imagined. A brewer never prepares his own yeast. We have already had occasion to remark that the interchange of yeasts amongst breweries is a time-honoured custom, which has been observed in all countries at all periods, as far back as we can trace the history of brewing. The yeasts which in the present day produce beer in the brewery of Tourtel, near Nancy, in that of Grüber, at Strasburg, that of Dreher, at Vienna, and others, came originally from breweries, where and when it would be hard to say. In the case of the first working brewery, the yeast was, no doubt, derived from some spontaneous fermentation, which took place in an infusion of barley that had been left to itself, or, from some natural spontaneous ferment, and nothing could be easier than to realize this fact again. In the brewing industry there are two distinct modes of fermentation:—“high” fermentation and “low” fermentation, some of the distinctive characteristics of which we have pointed out in Chapter I. It may be questioned whether the spontaneous yeast employed in the first brewery, or that which a wort left to itself in the present day would yield, would be of the “high” or “low” type. It may be concluded from what we have said on the subject of spontaneous fermentations in wort, that wort, left to itself, would furnish ferments more or less resembling those of wine. We have never obtained in spontaneous fermentations of wort either a distinctly “high” ferment, or a distinctly “low” one, properly so called; nor, further, have we ever obtained either one of these distinct kinds, with its industrial characteristics, in experiments on the ferments of fruits. What, then, was the origin of the “high” and “low” ferments now used by brewers? What was the nature of their original germs? These are questions which we are unable to answer, but we are very much inclined to think that we have here another example of the modifications which plants as well as races of animals undergo, and which become hereditary in the course of prolonged domestication. We know nothing of corn in its wild state, we cannot tell what its first grain was like. We know nothing of the silk-worm in its original state, and we are ignorant of the characters of the race that furnished the first egg.

These reflections may seem to favour the supposition that there is a real difference between “high” yeast and “low” yeast, and that both of these differ from spontaneous ferments and the ferments of domestic fruits. These are propositions demanding most careful consideration, for it is generally admitted that these ferments become intermixed, that their morphological differences are merely a question of medium, and that the transition of one to the other is a simple matter. The following facts seem to contradict such statements.

“High” Ferment.—Fig. 39 represents some “high” yeast taken from a deposit after fermentation, and Fig. 40 the same yeast in course of propagation in some aerated wort. In comparing “high” yeast with other alcoholic ferments at the same stage of development, there are three points which are especially striking: the diameter of its cells is relatively large, their general aspect is rounder, and when they are undergoing propagation their mode of budding produces a markedly ramified appearance, so that the cells always occur in clusters and branches. Fig. 40 gives a very exact idea of these characters. To investigate satisfactorily the branching habit of growth peculiar to this ferment we should examine it during the first few hours of its propagation, when, under the influence of the oxygen dissolved in the fermentable liquid, its vital activity is greatest. Later on, often on the day following the sowing, the groups become disconnected, and at the end of the fermentation the cells have quite separated from each other, not more than 2 or 3 per cent. remaining united, and even these in groups of not more than two cells together. This is represented in Fig. 39.

To give an idea of the rapidity with which this ferment multiplies, we may state that our sketch (Fig. 40) was made under the following conditions:—On April 28th, 1874, we caused a flask of wort to ferment by means of a trace of “high” yeast. On the morning of the next day, that is fourteen hours afterwards, an appreciable deposit of yeast had formed, and some frothy patches appeared on the surface of the liquid, showing that fermentation had set in. On May 1st we decanted the beer, substituting for it water sweetened with 10 per cent. of sugar. On May 2nd we decanted the sweetened water, and substituted a fresh quantity containing the same percentage of sugar. On May 3rd, at mid-day, we took some of the fermenting liquid from this flask and put it into a flask of wort; five hours after the introduction of the ferment we made the sketch in question. The field is covered with branching clusters, the groups being sketched exactly as they occurred in the field. Their activity was due to the condition of the ferment, and to the perfect fitness of the nutritive medium for its vegetation. In sweetened water the budding of the cells was considerably less active; no branching groups of cells are to be found. Budding, nevertheless, occurs to a considerable extent, but it is limited to one bud, or two at the most, to each cell. Fermentation in pure sweetened water is mostly correlative with the duration of vital activity in the globules already formed.

Let us next suffer our yeast to exhaust itself by keeping it in a great excess of sweetened water for a very long time; we shall then be able to observe its process of revival, and see if we can find any facts analogous to those presented by _saccharomyces pastorianus_ (Chapter V. § 1).

With this object in view, on May 6th, 1874, we impregnated two fresh flasks of sweetened water with some of the contents of the before-mentioned flask, which we had refilled with sweetened water on May 2nd. On May 13th we decanted the liquid, which was still very sweet, from one of these two fresh flasks, which could hardly be said to have fermented at all—the quantity of yeast in them being so small—and replaced it with some wort. Strange to say, on the morning of the 14th we found an appreciable growth of yeast, and a froth of carbonic acid gas on the surface of the liquid. The yeast therefore was not dead, although its fermentative powers had been exhausted. There was, however, no remarkable feature in connection with its revival, nor did we find the slightest trace of any of the elongated ferment-form. What we got was simply the ramified groups of “high” yeast again, in round cells, but nothing more.

Fearing that our yeast might not have remained for a sufficient time in the sweetened water for exhaustion, we set aside, for a whole year, the other flask which we had prepared on May 6th. On May 16th, 1875, we decanted the sweetened liquid and replaced it with wort. This time, however, there was no revival of the yeast; it had perished. Fortunately, we had also saved the flask of yeast and sweetened water which was prepared on May 2nd, 1874, as already mentioned, and in this case, as will be seen, the vitality of the yeast had not been extinguished, doubtless, in consequence of the formation of what we shall presently designate by the name of _aërobian ferment_. On May 16th, 1875, we decanted the liquid from this last flask, and replaced it with wort. On the next day the surface of the wort was covered with a thin froth, indicating the commencement of fermentation. The microscope revealed nothing extraordinary, or indicative of the fermentation of any special ferment. To assure ourselves that our ferment had remained “high,” we sowed some of it in a fresh flask of wort on May 19th, and then, seven hours after impregnation, submitting it to examination, we could find nothing but ramified groups in fine condition, without a single elongated cell, indeed, it would have been impossible to find a more beautiful specimen of “high” yeast, or one of a more decided character.

It would seem, therefore, that “high” yeast cannot, under any circumstances, assume the form and character of the ferment _saccharomyces pastorianus_, or of other known ferments. We are justified, therefore, in regarding it as a distinct species of ferment, an opinion which is supported by other circumstances.

1. In equal quantities of saccharine wort a considerably greater growth of “high” yeast is obtained than of other yeasts. We need no very rigorous proofs to convince ourselves of this fact: for by simply causing equal volumes of the same wort to ferment, the one being pitched with _saccharomyces pastorianus_, for example, the other with “high” yeast, we shall obtain a perceptibly greater volume of “high” yeast than of the other, in certain cases even five or six times as much.

2. “High” yeast is of a tougher texture than the others, separating, when the fermented liquor and its deposit is shaken up, into lumps which refuse to disappear; whereas _saccharomyces pastorianus_ diffuses through the whole liquid with the greatest ease.

3. “High” yeast produces a special beer, with a peculiar flavour, well known to consumers, but little esteemed at the present day. Hence the gradual displacement of breweries worked on the old “high” fermentation system by others in which “low” yeast (of which more anon) alone is employed.

4. Lastly, one characteristic of “high” yeast, which it shares in common with some other ferments, although not with all, and which, from a practical point of view, deserves special mention, is that as fermentation proceeds the yeast rises to the surface of the liquid. Whilst the process of the manufacture of beer by this ferment is going on, the yeast is seen to work out of the bung-holes, flowing over in considerable quantity. The ferment named after the author, as well as “low” yeast, does not possess this property: it remains at the bottom of the vessels. When “high” fermentation takes place in vessels that are not filled, the ferment forms a thick layer, a kind of cap on the surface of the beer. This characteristic may be witnessed even in the fermentation of very small quantities of liquid. In our flasks, in which the volume of fermenting wort does not exceed 100 c.c. or 150 c.c. (about 4 or 5 fluid ounces), we may perceive, as the violence of fermentation subsides, and the head falls, the sides of the vessel covered to a height of from 1 cm. to 2 cm. (about 3/4-in.) above the surface of the liquid, with particles of yeasty matter, in little masses, or in a thin film, raised to that height by the head, and left behind when that fell.

“Low” Ferment.—Whilst high yeast performs its functions in the breweries in which it is used at somewhat high temperatures—namely, between 16° C. and 20° C. (60° F. to 68° F.)—“low” yeast is never employed at a higher temperature than 10° C. (50° F.), and it is even thought preferable that it should not be subjected to more than 6° C., 7° C., or 8° C. (43° F. to 46° F.). At these comparatively low temperatures “high” yeast would have no perceptible action, whereas it is at such temperatures that “low” yeast best performs its functions.

In our Memoir on alcoholic fermentation, published in 1860, in the _Annales de Chimie et de Physique_, the idea of the identity of the two yeasts was accepted; but we had at that time made no special observations of our own on the subject.

Upon closer investigation we are inclined to believe that the two yeasts are quite distinct. We might keep our “high” yeast at the lowest temperatures that it can bear, and repeat our growths under these conditions; or, on the other hand, we might subject the “low” yeast to temperatures higher than those at which it ordinarily grows, without ever succeeding in changing the first into the second or the second into the first, supposing, of course, that each of our yeasts was pure to begin with. If they were intermixed the change in the conditions of development would cause one or the other to preponderate, and incline us to believe that a transformation had really occurred.

It is true that brewers generally are of a different opinion. Most of them assert that “low” yeast cultivated at a high temperature becomes “high” yeast; and conversely, that “high” yeast becomes “low” by repeated growths at a low temperature. Many have told us that they have proved this. Nevertheless it is our belief that the success of such transformation has been but apparent, attributable in each case to the fact, as we have just stated, of their having operated on a mixture of the two yeasts.

Mitscherlich, and various authors after him, have asserted that “high” yeast propagates by budding, and “low” yeast, on the contrary, by spores, formed by the endogenous division of the protoplasm of the cells, and set free by the rupture of the cell-wall, which then, increasing in size, assume the character of ordinary cells. But we have never been able to confirm this.

Fig. 41 represents a field of low yeast, taken from the deposit in a vat after the fermentation of the beer was finished. The granular matter mixed with the cells is altogether amorphous although in many cases perfectly spherical. It is a product in no way related to this yeast (see Plate I., No. 7).[100] “High” yeast and all the ferments of beer have this kind of deposit associated with them. There is no doubt that confused observations as regards these minute bodies have been the cause of the error which we had to deal with in connection with a particular mode of reproduction of low yeast, as to which we have already fully expressed our views (Chap. V. § 1, p. 146).

Comparing Fig. 41 with Fig. 40 (p. 189), it may be seen that the general aspect of low yeast is distinguished, in its early stages, although in no very decided manner, from that of “high” yeast, by being slightly smaller and less round or spherical in its cells than the latter.[101] These differences, however, would escape an unpractised eye.

As to the case of “high” yeast, the deposits of “low” yeast after fermentation appear as scattered, isolated cells; we do not find more than two or three per cent. of united cells. Nevertheless the two yeasts present, as we shall see, quite marked differences in the character of their budding and multiplication.

On May 28th, 1875, we put a trace of pure, unicellular, “low” yeast, taken at the end of a fermentation, into a flask of wort. On May 29th, sixteen hours after impregnation, the temperature during the night having been 15° C. (59° F.), we made a sketch of the yeast before its development had become apparent to the naked eye. No perceptible development, that is to say, no visible deposit at the bottom of the liquid and formation of patches of froth on the surface, took place before May 30th. A mere glance at Fig. 42 will be sufficient to enable us to detect a considerable difference between it and Fig. 40, which represents the multiplication of the cells of “high” yeast. The cells of the “low” yeast are slightly smaller and rather more oval, as we have already had occasion to notice, and the budding processes are considerably less ramified, in consequence of which there is a comparative absence of globular clusters which are so striking a feature in the development of “high” yeast, when examined early enough. Moreover, if we cause our “low” yeast to age, by leaving it for a longer or shorter time in the beer which it has formed, or if we exhaust it in sweetened water by leaving it for whole months in a volume of sweetened water considerably larger than what it is capable of fermenting, and then proceed to revive it and cause it to propagate in an aerated saccharine wort capable of nourishing it, this yeast will resume its original aspect, as sketched and described. At most we shall observe certain minute differences in the size of the cells in successive growths. A very remarkable industrial characteristic of this yeast is the fact that it never rises to the surface, no matter at what temperature it may be working, whether between 6° C. and 8° C. or 15° C. and 20° C.;[102] in other words, it is not buoyed up by the carbonic acid gas when the fermentation is at its height. At the end of the fermentation, the surface of the liquid and the sides of the vessel above the level of the liquid are clean and not covered with the yeast, which remains altogether at the bottom of the fermented liquid. Moreover, the weight of new yeast which it yields is always less than that yielded by “high” yeast, for the same quantity of fermentable liquid, although greater than that which _saccharomyces pastorianus_ would give. Lastly, the beer possesses a flavour and delicacy which cause it to be held in higher esteem by consumers than beers produced by means of other ferments.[103]

§ IV.—On the Existence and Production of Other Species of Ferment.

Our present knowledge of the alcoholic ferments embraces the following, without taking into account the ferment-form of _mucor_:—

The ferment named after the author, which is found associated with the ferments of the grape and other domestic fruits, and with spontaneous ferments in general.

The ferment of “high” beer.

The ferment of “low” beer.

To these must be added the ordinary ferment of wine, and that called _apiculatus_, although, indeed, these last are of little practical importance, since, in general, they soon become lost amongst others of greater vitality, in the spontaneous fermentation of fruits. These are not the only alcoholic ferments; a study of the germ-cells diffused over the surface of fruits, grains, and stalks of all vegetables in different countries, would doubtless lead to the discovery of many new ones. We are even inclined to believe that one ferment might give rise to a multitude of others. The investigations which we have undertaken in this direction are as yet not far advanced; we may, however, be allowed merely to state the principle which governs them. A ferment is a combination of cells, the individuals of which must differ more or less from each other. Each of these cells has certain generic and specific peculiarities which it shares with the neighbouring cells; but over and above this, certain peculiar characteristics which distinguish it, and which it is capable of transmitting to succeeding generations. If, therefore, we could manage with some species of ferment to isolate the different cells that compose it, and could cultivate each of these separately, we should obtain as many specimens of ferments, which would, probably, be distinct from one another, inasmuch as each of them would inherit the individual peculiarities of the cell from which it originated. Our endeavours are directed to realizing this result practically, by first thoroughly drying a ferment and reducing it to fine powder. We have seen (Chap. III. § 6) that this mode of experiment is practicable, that in a powder composed of yeast and plaster the ferment preserves its faculty of reproduction for a very long time. If we now drop a small quantity of this powder from a sufficient height, and then, at a certain distance below the cloud of dust so formed, open several flasks previously deprived of air and containing a fermentable liquid that has been boiled, immediately closing them all up again, under such circumstances it is conceivable that some of the cells of yeast diffused in the cloud of dust, and separated widely in the act of falling, will enter some of our flasks singly, and there develop an appreciable weight of ferment, all the cells of which will have sprung from the same mother-cell. We have proved that flasks may be easily impregnated under these conditions, and our preliminary observations, although incomplete, seem to favour the idea that numerous varieties of ferment are to be obtained by these means.

Spontaneous ferments, properly so called, of which we have already spoken, are, after all, the result of sowings of this kind. Originating in liquids which have been boiled, and then left to themselves in contact with the air in a place where cells or germs must have existed, these ferments must necessarily often spring from single germs or from a limited few, and this also would probably be a means of developing distinct varieties of ferments.

Without dwelling longer on the practical consequences likely to result from the ideas which we have just expressed, we shall proceed to describe two new alcoholic ferments, which differ widely from those already mentioned.

_New “High” Ferment._—We met with this ferment accidentally, under the following circumstances:—On February 12th, 1873, we had brewed in the laboratory about 2-½ hectolitres (rather over 50 gallons) of wort, 10 litres (about two gallons) of which were set aside to cool in a white-iron trough, and left during the night exposed to free contact with air in the underground part of the laboratory, where we have a small experimental brewery. Next day we put some of this latter into a bottle; the wort soon began to show evidence of change, various productions made their appearance on the surface of the liquid, and a deposit of yeast settled at the bottom. On May 23rd, perceiving bubbles of gas and a steady fermentation set up in the wort, which remained all the time corked up, and fearing that the bottle might burst by the increasing internal pressure, we drew the cork. A considerable liberation of gas at once took place, accompanied by a voluminous foam which half emptied the bottle. A microscopical examination of the deposit from the disturbed liquid led to the discovery of a very homogeneous yeast, associated with various other organisms; it was clearly a yeast which we had not hitherto met with amongst the spontaneous ferments which we had had occasion to study. Thinking that this might be a new species of ferment which would probably produce a beer that was also unknown, we set to work to purify it by cultivation in flasks of pure wort, during the months of May, June, and July. Our last growths, of August 4th, 1873, were preserved, in order that we might assure ourselves of the purity of the beer, and, consequently, of the ferment. On November 15th its purity was established. On that date we made some beer with this ferment, which had now been left to itself for several months in contact with pure air. The beer which we obtained resembled no known variety; consequently the ferment must itself have been a distinct one, differing from others, especially those which we have been considering in this chapter.

Fig. 43 represents the rejuvenescence of this ferment. Comparing this figure with Fig. 42, we see that this ferment presents a considerable resemblance to “low” yeast in dimensions, method of budding, and oval shape; but the feature which distinguishes it essentially from “low” yeast is that it rises to the surface, like “high” yeast. Buoyed up by the gas during fermentation, it forms a layer of yeast on the surface of the fermenting liquid, where it remains after the head has fallen. Some of this head of yeast likewise adheres to the sides of the vessel above the level of the liquid.

In short, by the greater regularity of its forms and the uniform dimensions of its cells, this ferment is to be easily distinguished from _saccharomyces pastorianus_; its aspect, which is oval instead of spherical, and the ramified form of its chains of cells, which is less marked than in the case of “high” yeast, also prevent our confounding it with the latter ferment; in its rising character it differs absolutely from “low” yeast; lastly, it may be distinguished from all other ferments by the flavour of the beer that it produces.

The ferment which we discovered in this accidental way may be utilized. Indeed, we may ask, is it not to be found already in our beer? We are inclined to believe that it is. After the war of 1870, some Viennese traders established at Maisons-Alfort, near Paris, a manufactory of yeast for bakers. They saccharified by means of malt a mixture of the meals of rye, maize, and barley, which they then caused to ferment. One day we had occasion to study the yeast produced in this establishment, and although we did not submit it to a sufficient number of consecutive experiments to enable us to speak positively, we are under the impression that the yeast produced at Maisons-Alfort is a “high” one, differing from what may be properly termed the “high” yeast of breweries in which “high” fermentation is practised, but presenting a great resemblance to the “high” yeast of which we have been speaking. It would be interesting to confirm the opinion of their possible identity by fresh studies, and the best way of doing this would be to compare the qualities of beer which the two yeasts could produce.

_Caseous Ferment._—We give the title caseous for a reason that will presently appear, to a ferment which we came across also accidentally. We were trying different methods of purifying yeasts, and for this purpose had composed a liquid formed of:

Ordinary wort 150 c.c.[104]
Water saturated with bi-tartrate of potash 50 c.c.
Alcohol of 90° 25 c.c.

Quantities of this liquid were placed in several of our double-necked flasks, submitted to boiling, then, after cooling, impregnated with different ferments, and kept in a water-bath at 50° C. (122° F.) for one hour.

In operating under these conditions with brewers’ “high” yeast, say, for instance, with what is called Dutch yeast, a kind well known in distilleries, fermentation shows itself in the course of a few days, in spite of the increased temperature to which our liquid, which is hopped and slightly acid and alcoholic, has been subjected. The time required for the resumption of fermentation depends both upon the degree of temperature to which the yeast has been exposed and upon the duration of its exposure. These, however, are not the points upon which we now wish to dwell. It is of greater importance to notice that the new yeast has none of the characteristics of “high” ferment, of which Dutch yeast seems to be exclusively composed, if we do not take into account impurities which cannot be avoided in a commercial product of this nature. Other specimens of Dutch yeasts would give the same results.

Figs. 44 and 45 represent this new ferment magnified to the same extent as the other ferments have generally been, that is 400/1; it will readily be seen how different its form is from that of “high” yeast, how far it is from having the spherical aspect and mode of budding characteristic of that ferment. In Fig. 45 the ferment is represented in a mass; in Fig. 44 we see the ramified groups, the cells and segments of which form, after separation, the yeast of the deposit. It thus appears to be composed of jointed branches of greater or less length, which, at the junctions of the segments, put forth similar cells or segments of a round, oval, pyriform, cylindrical, or other shape; in all its characters recalling the description of _dematium_. Moreover, the cells and segments exhibit a greater sharpness of outline, as well as a more marked transparency and refractive power than are found in the majority of ferments; but the most curious physical characteristic of this ferment is its plasticity and elasticity, if we may use those terms. It can only be made to diffuse through water with great difficulty; when shaken up in it, it sinks to the bottom quickly as a clotted sediment, and the supernatant liquid appears scarcely at all charged with globules in suspension. Again, when placed on a microscope slide and compressed by the cover-glass, it returns to its original form on removal of the pressure. It is from these considerations that we have given to it the name of _caseous ferment_.[105] Lastly, this ferment produces a beer of a peculiar kind, which cannot be confounded with other kinds of beer known in the present day. We should add that it preserves its characteristics in repeated growths, and that we have never found it reproduce ordinary “high” yeast.

When caseous ferment is sown in a saccharine medium charged with mineral salts, its aspect, form, and mode of budding differ completely from what they are when the ferment exists in a natural medium, such as wort or other liquid adapted to the nutrition and life of ferments.

Fig. 46 represents this ferment in course of development, forty-eight hours after it had been sown in a saline medium (we employed Raulin’s fluid, substituting bi-tartrate for the nitrate of ammonia). It will be seen how different its aspect is from that of the preceding figures; it is still capable, however, of resuming the forms of the latter if cultivated afresh in natural saccharine worts.

“High” yeast from a “high” fermentation brewery in the Ardennes, after having been exposed to heat under the conditions given above, likewise produced caseous ferment, without a trace of “high” ferment, just as happened in the case of the Dutch yeast. All the “high” yeasts used in brewing seem to behave in the same manner.