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.

[Illustration: Fig. 41.]

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.

[Illustration: Fig. 42.]

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.

[Illustration: Fig. 43.]

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.

[Illustration: Fig. 44.]

[Illustration: Fig. 45.]

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.

[Illustration: Fig. 46.]

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.

What conclusion are we to draw from these facts? Apparently that “high” yeast is modified by heat in an acid and alcoholic medium, giving rise to caseous ferment. On the other hand, it might be conceived that the “high” yeasts on which we experimented were not pure, but contained, in a state of intermixture, some caseous ferment, and that by the application of a temperature of 50° C. (122° F.) to our alcoholic medium, the high ferment was all killed and the caseous ferment alone survived. It is a remarkable fact that this latter hypothesis, improbable as it seems, inasmuch as the microscope revealed no intermixture of ferments, seems, nevertheless, to be a true one. As a matter of fact, if we subject to a temperature of 50° C. for one hour in the medium in which it acts, not the “high” yeast of commerce but “high” yeast that is _absolutely pure_, this will perish utterly, and the wort after cooling may remain for years in an oven without either undergoing fermentation or developing any growth whatever of “high” ferment or “caseous” ferment.

On the other hand, if we impregnate this same alcoholic liquid with some of the caseous ferment and then heat the vessel to 50° C. for one hour, the caseous ferment will go on reproducing itself after the liquid has cooled down.[106]

It seems, therefore, impossible to admit that caseous ferment results from a modification of “high” ferment, and we are led to believe that in the preceding experiments it must have been the progeny of cells of caseous ferment present in the “high” yeasts of commerce, which cells, probably in consequence of their scarcity, the microscope was unable to reveal, but which, nevertheless, did exist, and went on reproducing themselves alone after the heating.

[Illustration: Fig. 47.]

This conclusion is supported by the following fact, which also tends to prove that in the case of the “high” English _pale ales_, caseous ferment plays a most important part. In the medium already described, we sowed the deposit from a bottle of good English pale ale. After having been heated the yeast went on growing, and we obtained the very beautiful specimen of caseous ferment represented in Fig. 47. The two dark globules are dead cells which had been killed. Two minute segments of lactic ferment are also visible in the sketch—the yeast which we sowed was, of course, impure—and their presence proves, we may observe, by the way, that lactic ferment also can withstand a temperature of 50° C. (122° F.) in the medium which we here employed. The yeast as sowed is represented in Fig. 48; it reminds us forcibly of certain forms of the caseous ferment. Amongst the globules, which for the most part were transparent and very young, there were some which appeared aged and of a yellowish colour and granular. These latter probably belonged to the yeast of manufacture. Their shape distinguishes them from “high” yeast, properly so called, as on the other hand it causes them to appear more like cells of a recent growth to which, there is no doubt, beer, after it is put in bottle, owes its effervescence and head. These various circumstances incline us to believe that the caseous ferment forms part of certain commercial yeasts, especially those used in the celebrated breweries of Bass and Allsopp, at Burton-on-Trent, in the manufacture of pale ale. Caseous yeast is, moreover, a “high” ferment, that is to say, it rises to the surface.

[Illustration: Fig. 48.]

§ V.—On a New Race of Alcoholic Ferments: Aërobian Ferments.

Mention has already been made of certain researches which we undertook with the object of ascertaining whether _mycoderma vini_, or efflorescence of wine, and _mycoderma cerevisiæ_, or efflorescence of beer, which grow equally well in all fermented liquids, have the power of becoming transformed into actual alcoholic ferment. The result of those researches was stated to be that these mycodermata do not become transformed into ferment, properly so called, and that whenever any such transformation has been supposed to have taken place, the ferment produced was derived from germs introduced by the air or by the utensils employed. What we did ascertain of the ferment-producing power of _mycoderma vini_, was merely that this plant, when submerged, is capable of causing sugar to ferment, in consequence of a certain continuous life possible to its cells, apart from the oxidations resulting from the presence of free oxygen, but without any generation of new cells taking place.

Whilst engaged in these researches, we were pursuing others in relation to the converse of the proposition just discussed, that is to say, respecting the possibility of ferment becoming transformed into _mycoderma vini_ or _mycoderma cerevisiae_. Our experiments in connection with this subject chiefly consisted in various endeavours by way of exhausting the yeast and subsequent revival of its growth. This exhaustion was effected by growing the yeast in excess of sweetened water, and at other times in unsweetened yeast-water, our efforts being directed to deprive it of all power of fermenting. We afterwards caused it to develop afresh in highly aerated, nutritious liquids, in order that we might see how it reproduced itself, and if its new form were that of a mycoderma. The yeast after having lost its power as a ferment, and being no longer able to act in pure sweetened water, nevertheless reproduced itself when placed in fermentable media, holding in solution materials adapted to its nutrition; yet we never succeeded in obtaining any organism besides the ferment, and, indeed, the identical variety of ferment on which we had operated. In no case was _mycoderma vini_ or _cerevisiæ_ produced, and we concluded that we were justified in stating that whenever the _mycoderma vini_ appeared on the surface of a fermented or fermentable liquid, its germ must have been introduced by the surrounding air, or have previously existed in the liquid, and that the reason why this germ multiplied so abundantly was because the liquid in question had been peculiarly adapted to the vitality of the plant.

In a laboratory where alcoholic fermentations are studied, these germs of _mycoderma vini_ exist in great abundance on the surfaces of different objects. This fact admits of easy proof; we have merely to open in such a laboratory some flasks containing yeast-water deprived of air, or yeast-water sweetened, or any natural saccharine medium, or any fermented liquid, which till the moment when our flasks were closed had been kept boiling (Chap. IV.); it would be a very rare thing, indeed, if _mycoderma vini_ did not develop in most of these flasks after the air was readmitted, especially if, shortly before this operation, the dust lying on the surface of the tables or floor of the laboratory had been stirred up by dusting or sweeping.

This series of experiments, the salient points of which we have just given, conducted with a view to ascertain whether yeast could be transformed into mycoderma, has led the way to certain results of special interest, results which concern all alcoholic ferments, and which in all probability will be found in the long run to apply to all aërobian ferments.

It being necessary for the conduct of our experiments to preserve our yeast in a state of purity for an indefinite period, often for a great length of time, in contact with pure air, we discovered that yeast was possessed of extraordinary vitality, and that it rarely perished completely throughout, inasmuch as we could almost invariably cause it to revive by bringing it into contact with fresh, fermentable liquid. This revival of the yeast—and it is to this point that we are most anxious to direct the attention of our readers—is effected from two distinct sources:—

1. By those cells of yeast which have not perished.

2. By cells of new formation.

We may give an example to explain this more clearly. In one of our two-necked flasks we cause some pure wort to ferment by employing yeast also in a state of purity. Fermentation completed, we leave the liquid to itself, not touching the flask again. The fermented liquor covers a deposit of yeast, apparently inert, and no trace of _mycoderma vini_ makes its appearance on the surface of the liquid. Let us suppose that we go on daily for a considerable time introducing a little of the yeast from this flask to a different flask of wort: the fresh flasks will begin to ferment. The only appreciable difference which these successive flasks will present, their impregnation having been effected at intervals of twenty-four hours, will be that, _ceteris paribus_, fermentation in them will be more and more slow in making its appearance. This difference, as we have already explained, will be due to the fact that the yeast in the first flask will, in the course of time, undergo, in each of its cells, a process which we cannot better describe than as a progressive _senescence_. The cells gradually become filled with amorphous granulations, their interior becomes yellow, and the protoplasm collects, either at the centre or near the borders; in short, the vitality of the yeast becomes feeble. When, however, it is taken out of the liquid in which it has fermented and introduced into a fresh saccharine wort, it gradually resumes its transparency, and then begins to germinate. These effects are the less rapidly brought about the longer the cells remain exhausting themselves in the first fermented liquid. They might be left in that liquid for such a length of time that they would eventually perish, a fact which would manifest itself in their absolute sterility and quiescence when sown in a fresh medium. In general, however, matters are not carried far enough for this to take place, and the yeast, preserved in a state of purity in its fermented liquid, retains the capacity of revival, which may then go on indefinitely. As a matter of observation, the cells of yeast, after causing the liquid to ferment, instead of remaining inactive, and so by living at their own expense gradually passing into a state of exhaustion, begin to bud again; at least this is true of many of them. Multiplying afresh in the fermented medium, under the influence of the air, they form a kind of mycodermic film on the liquid surface, or a ring round the sides of the flask, on a level with the liquid. This development might often be mistaken for _mycoderma vini_ or _cerevisiæ_; in reality there is not a single cell of _mycoderma_ formed. If we sow a trace of the new growth in a saccharine medium it will behave exactly as yeast would, budding and multiplying, and setting up fermentation in the liquid. And thus, in spite of its mycodermic aspect, this growth is nothing but yeast, since it gives rise to true alcoholic fermentation; but it is a kind of yeast which, under the foregoing conditions, lives after the manner of fungoid growths, absorbing the oxygen of the air and emitting carbonic acid gas. It appears on the surface of all fermented liquids, especially those which, like beer, contain carbohydrates, and its quantity is the greater, and its action the more rapid, in proportion as it has more perfect access to the air. We have termed this yeast _aërobian ferment_ or _fungoid ferment_.

It may easily be understood how this kind of production has escaped notice up to the present time. The conditions of our experiment were, in many respects, novel; a saccharine liquid had never before been caused to ferment by means of pure yeast, absolutely free from foreign germs; a fermented liquid had not previously been exposed to contact with pure air for an indefinite time. On the other hand, all ordinary fermented liquids, when left to themselves in contact with air, are a ready prey to _mycoderma vini_ or _aceti_ at their surface, and then give rise to true fungoid growths. The appearance of these organisms, which always takes place soon, has thus constantly concealed or prevented the development of the true aërobian ferments. In repeating the experiment described any alcoholic ferment may be used, and each one will be found to produce its own peculiar fungoid form of ferment. Another point worthy of notice is that these aërobian ferments, when they put forth buds in the act of fermentation, reproduce the forms of the original ferment, at least apparently so. In this respect they cannot be distinguished, notwithstanding the fact, surprising as it seems, that the two kinds of ferments are not identical. If we operate on a “low” yeast its aërobian ferment will differ physiologically from the ferment from which it sprung, presenting various special peculiarities which are not to be found in the original “low” yeast. In most of our experiments we have found the new aërobian ferment to be similar in its action to “high” yeast, rising to the surface, and producing a beer which possesses a greater fragrance than beer brewed with the identical “low” yeast from which it was derived. Lastly, the properties of an aërobious ferment are not peculiar to first growth, but are hereditary; by repeating the growth of the first aërobian ferment we do not cause them to disappear, we find them again in succeeding generations.

Notwithstanding these facts, it would be difficult to discover any very appreciable differences between the forms of the cells of any particular yeast and those of its aërobian ferment in course of development. So true is this, that the aërobian ferment of _saccharomyces pastorianus_ might even be caused to put on the forms of _dematium pullulans_, which we have had occasion to observe specially characterize this ferment after the cells have been subjected to a prolonged process of senescence.[107] This is evident from the following example, which will once again show the remarkable extent to which the forms of a particular organism may be varied by changes in composition of the nutritive medium:—

On August 6th, 1873, we took some of the ferment _saccharomyces pastorianus_ from a flask of wort that had undergone fermentation, and sowed a scarcely perceptible quantity of it in another flask containing a saline medium, composed as follows:—

Water containing about 10 per cent. of 150 c.c. (5-1/4 fl. sugar-candy oz.)

Ash of yeast 0·5 gramme (8 grs.)

Ammonic bitartrate 0·2 “ (3 grs.)

Ammonic sulphate 0·2 ” (3 grs.)

In the course of the following days the ferment began to develop, although with difficulty, the fermentation revealing itself by collections of bubbles appearing here and there on the surface of the liquid. We left the flask undisturbed till the 25th of November following. On that day we found a very white deposit of ferment covering the yeast-ash that had not been taken into solution, and a ring of aërobian ferment on a level with the surface of liquid; all the sugar had disappeared; the liquid contained 5·2 per cent. of alcohol, by volume, at a temperature of 15° C. (59° F.); and, lastly, in consequence of the purity of the materials employed, there was no trace of the formation of fungoid growths, whether of _mycoderma vini_ or of _mycoderma cerevisiæ_, on the surface of the liquid, or of vibrios or lactic-ferment below the surface.

Thus then we see—and several other examples throughout this work confirm the fact—that saccharine liquids holding mineral salts in solution are as capable of complete fermentation as any media of natural composition. It is true that ferment develops slowly and with difficulty in them, and at times takes on rather curious forms, but, nevertheless, it does develop in the media and carry on a fermentation in which not the minutest particle of sugar is left undecomposed. This is true, at least, in the case of _saccharomyces pastorianus_, but there are other ferments which in such media are checked in their multiplication and in their continued action on sugar. One condition indispensable to the accomplishment of fermentation in such a sweetened mineral medium, by means of _saccharomyces pastorianus_, is the absolute purity of the materials and of the ferment. It is necessary that the life and physiological action of the latter should be in no way interfered with by the presence of other microscopic organisms. We shall have occasion to revert to this important detail in connection with our growths.

[Illustration: Fig. 49.]

Fig. 49 represents the ferment as it appeared when examined on August 11th, 1873. We can no longer recognize in it any _saccharomyces pastorianus_. The general appearance is spherical, and there are a number of clusters of budding cells which remind one at first sight of the mode of germination of brewers’ “high” yeast. At _a_, _a_, _a_, we see globules from which irregular abortive filaments have sprung, a proof of difficult germination. No such monstrosities could ever have occurred if we had used beer-wort or must as our nutritive medium.

On November 25th we made another examination and sketch of the ferment, the appearance of which did not differ materially from that given above. The general appearance was the same, consisting mostly of globules joined together in clusters of two or three or more. No separation, such as occurs in the case of ferment formed in natural worts, had taken place. The ferment, moreover, was very irregular, and comprised cells of all sizes. We sowed some of it in a flask of pure wort. On November 26th there was no apparent development: on November 27th, however, not more than forty-eight hours after impregnation, there was a considerable deposit of white ferment at the bottom of the liquid, and fermentation was so active that the surface of the liquid was covered with an abundant froth. This shows us the wonderful vitality and recuperative power possessed by germs which, left to themselves for about four months, revived so readily. It proves too that the reviving influences took effect on some aërobian ferment. From the mode of life of this latter being similar to that of a surface fungoid growth, it does not become exhausted as the cells of ordinary ferment do. Now the cells which, sown on August 6th, had become exhausted by prolonged stay in the mineral liquid, and were almost inert, would have required several days for their revival; but in the experiment described the revival was rapid, and this rapidity proves, as we have said, that the revival must have taken place in cells of aërobian ferment.

[Illustration: Fig. 50.]

Taking some fresh yeast from the bottom of the liquid we examined and made a sketch of it (Fig. 50). The field was filled with round and oval cells, jointed and ramified filaments, budding and multiplying in the most remarkable manner, reminding us of the germination of the cells of yeast exhausted in sweetened water, and also of the germination in the form of _dematium pullulans_ of certain germ-cells which are spread over the surface of sweet, domestic fruits. We could never grow tired, as we wrote it in our original notes, of sketching this beautiful plant, which establishes very clearly a transition between one of the best defined cellular ferments, viz., _saccharomyces pastorianus_, and certain forms of very common fungoid growths, those of _dematium_, and even of the most common mould, _mucor mucedo_ or _racemosus_, when it vegetates beneath the surface of a liquid and acts as a ferment.[108] We have here, as in these cases, filamentous chains branching into other similar chains, composed of more or less elongated cells, which at length fall off and germinate exactly as the conidia-bearing _hyphae_ of _mucor_ do.

The aërobian ferment of “high” yeast, in whatever medium we cultivated it, presented no peculiarity, as far as its forms were concerned. It was composed of cells of spherical shape, like ordinary “high” yeast, and germinated in the same way as the latter.

[Illustration: Fig. 51.]

Fig. 51 represents the revival of this aërobian ferment. We recognize here the branched mode of budding and spherical contour characteristic of “high” yeast proper. Nor does the aërobian ferment of “low” yeast present any special peculiarities, in forms, dimensions, and mode of growth closely resembling the “low” yeast from which it is derived. At the commencement of its restoration, however, if this is performed in sweetened water, the cells in the groups are larger than those which are subsequently developed.

[Illustration: Fig. 52.]

Fig. 52 represents the aërobian ferment of yeast used in “low”-fermentation breweries, examined forty-eight hours after pitching. We find that groups resembling that at a are of very rare occurrence. They are to be seen only at the very beginning, generally only for the first few hours of the renewed activity. Very soon, however, they develop cells which are of the size of the oval cells budding at _b_.

[Illustration: Fig. 53.]

Fig. 53 represents the aërobian caseous yeast which forms rather rapidly, in thick, greasy-looking pellicles, on the surface of liquids which have been fermented by means of caseous ferment. The larger form of cells, _a_ and _b_, is not often met with.

[Illustration: Fig. 54.]

On May 27th, 1875, we sowed, in a flask of wort, a trace of a pellicle of this kind, which had formed on the surface of a flask in which fermentation had been set up by means of caseous yeast in May of the preceding year. On May 30th fermentation began to reveal its presence by a voluminous froth, and the newly-formed yeast had reached the bottom of the flask. A small quantity was taken out by a capillary glass tube, and a sketch of the ferment made; this is given in Fig. 54. Amongst the cells which occupy the field there are groups of some of larger size. These are not distinct forms mixed with the others, but simply another illustration of the fact that old cells in course of revival, especially when they have been exhausted in sweetened water, as we have just observed of the aërobian ferment of “low” yeast, commence with forms of larger diameter or more elongated than the ordinary forms peculiar to the ferment which at a later stage are developed from them. We have seen how marked and exaggerated this feature was in the case of _saccharomyces pastorianus_.

Let us again call attention to the forms of aërobian ferment furnished by the yeast which we have already described under the name of _new “high” yeast_. Fig. 55 represents this aërobian ferment, as taken on November 27th, 1873, from a pellicle of rather greasy and moist appearance, on the surface of a flask of fermented beer-wort which had been impregnated on July 21st, 1872. It might readily be mistaken for ordinary “high” yeast, yet no two ferments can be more distinct.

[Illustration: Fig. 55.]

[Illustration: Fig. 56.]

On November 27th, 1873, we sowed a trace of this ferment in a flask of wort. From the 29th, with a continuous temperature of 25° C. (77° F.), a considerable deposit of yeast began to form, and the froth of fermentation covered the whole surface of the liquid. We took a little of this deposit for examination; it is represented in Fig. 56. The field is occupied with oval cells of great uniformity. We recognize the aspect of the original yeast (Fig. 43). Here and there, indeed, we come across some cells of larger size, such as those at _a_ and _b_, which is another illustration of the remark that we have just made respecting the forms which revived exhausted cells take on at the commencement of a new germination.

The physical aspect of the several aërobian ferments is in general so characteristic that we are often able by simple inspection to distinguish between them as they occur on the surface of liquids. _Saccharomyces pastorianus_ in its aërobian state forms a crown of cells round the sides of the vessel at the surface of the liquid, which crown is broken up by the least agitation of the liquid; its vitality continues for years.

The aërobian ferment of “high” yeast appears in the form of small isolated teats on the surface of the fermented liquid. It develops rather sluggishly, and has no great vitality.

The aërobian form of “low” yeast develops as a somewhat fragile layer, the least agitation precipitating it to the bottom of the vessel in a cloud of very small irregular flocks, that do not diffuse through the liquid as they fall. With free access to air it retains life for a long time.

The aërobian ferment of caseous yeast forms a continuous greasy-looking pellicle, gradually thickening, which breaks up into fragments when shaken. With a supply of air it lives very long, and the pellicle gradually increases in thickness.

In reviewing these ferments we may naturally ask ourselves the question whether the “high” ferments of which we have spoken—the industrial one concerned in the “high” fermentation of breweries, and the other which we have termed _new “high” ferment_—are not aërobian ferments of “low” yeasts. We are inclined to think that the ferment which in the preceding paragraph we termed _new “high” ferment_, may, perhaps, be the aërobian form of the “low” yeast employed by Alsatian and German brewers. We have studied this new “high” ferment side by side with the aërobian ferment of “low” yeast, and the result we have arrived at is, that in appearance and mode of germination, as well as in the flavour and quality of the beers which they produce, they greatly resemble one another. In the last respect, however, we cannot say that the identity is quite absolute, and hence it is with some doubt that we suggest the possible identity of the two ferments. As regards the ordinary “high” yeast of breweries, it may well be supposed, both from its power of rising to the surface during fermentation and from the peculiar smell and flavour of its beer, that we have in it the aërobian ferment of some “low” yeast, as to the identity of which, however, we can say nothing, having no knowledge as to where it is to be found; or, indeed, any certainty that such a yeast actually exists.

In writing these lines an idea suggests itself which might be profitably made the subject of serious experimental study. What would be the peculiar properties of the aërobious ferment-form of an aërobian yeast? Certain facts incline us to believe that these forms differ from each other just as a “low” yeast differs from its aërobian ferment. If this were actually the case it would be very interesting to compare the peculiar properties of an indefinite series of aërobian ferments, all derived from a common origin. We find recorded in our laboratory notes that a certain aërobian ferment of the second generation produced a beer different from that produced by the same ferment of the first generation, being possessed of a fragrance so marked that, on entering our laboratory, in which only a few litres of this beer were fermenting, we were at once struck by the powerful odour which it emitted.

§ VI.—The Purification of Commercial Yeasts.

We have already stated that the researches detailed in the preceding chapter require for their successful prosecution that the ferments on which we experiment should be absolutely free from germs of other organisms, and we have shown how impossible it would be, if this condition were not complied with, to follow for weeks or months, sometimes even years, the changes which occur in a yeast maintained in contact with air, either in sweetened water or in a liquid which has fermented under its influence. Equally necessary is it that the saccharine worts employed should also be exempt from these impurities, as well as the air, which is being constantly renewed at the surface of the liquids. These last conditions may be realized by the adoption of our double-necked flasks, with which a laboratory for research of this kind should be furnished, always ready for use, filled with the different kinds of liquids that may be required.

In general, the inconveniences resulting from the impurity of a yeast employed do not immediately manifest themselves, in consequence of the enormous preponderance of the true yeast, which, in comparison with the foreign germs that contaminate it, may be so great that microscopical examination fails to reveal even the presence of these latter. Again, it is a well-known fact that the abundance of one growth in a limited medium operates to the prejudice of a less abundant one, inasmuch as the first consumes the nutritive materials at the expense of the second, and more particularly the needful amount of oxygen. It follows, that when a saccharine liquid is impregnated with commercial yeast, nothing but yeast may be detected for a time, and one is led to believe in the purity of the subsequent growth. This, however, supposes that the external conditions, as well as those of the medium of growth, are equally adapted to the life of the yeast and that of those organisms present as impurities; for if these conditions rather favoured the nutrition of the latter, we should be sure to find their proper developments appearing at an early stage. For example, when the growth of yeast becomes sluggish, we have invariably the development of such after-growths. The principal germs, having exhausted the saccharine liquid which has fermented under their influence and is no longer adapted for their growth, cease to develop, and have their place taken by ferments of disease, spores of moulds, mycodermata, &c., the growth of which proceeds more or less rapidly, in proportion as the character of the liquid and the surrounding temperature are more or less suited to their growth.

Here, too, we have an explanation of the rapid change that occurs in brewers’ yeast when left to itself after fermentation. In such a mass of cells, kept apart from any food-supply, and only with difficulty able to keep themselves in life by consuming their own soluble contents, we have an excellent field for the development of foreign germs. In this way we may have a rapid putrefaction in yeast, to which there will be a correspondingly rapid growth of organisms in the liquid, where they find, as well as in the yeast-cells, appropriate nourishment. Nothing could better confirm this view of the matter than the array of facts, by way of antithesis, already described, in which we have seen a pure yeast remain for an indefinite time in contact with pure air, without undergoing any putrefaction, or manifesting other changes than those which result from the combustions peculiar to living cells when left to support themselves, in a moist state, in contact with oxygen.

In the process of brewing, as soon as fermentation is finished, or rather, as soon as certain physical effects are produced, for instance, when the beer falls bright, or, as the French say technically, when the yeast breaks up,[109] the beer is racked; subsequently the yeast, which is left in a plastic layer at the bottom of the vessels, is collected, washed, and kept under water in a cool place, to be used again in the course of twenty-four or forty-eight hours. Brewers never care to keep their yeast for a longer time before using it, especially in summer. We can understand how this practice prevents the foreign germs which are mixed with the yeast from living and reproducing; but although the conditions of brewing, as far as the treatment of the yeast is concerned, may, in a certain measure, prevent the development of these germs of disease, nevertheless they are there, and from their extreme minuteness, pass into the beer in greater or less number, however bright it may have been rendered by racking. There they only await conditions favourable to their existence to enable them to develop, and to affect more or less injuriously the qualities of that delicate beverage.

On December 15th, 1872, we bought nine samples of beer in different large cafés in Paris, which had all come from the best breweries of Strasburg, Nancy, Vienna, and Burton. After leaving them for twenty-four hours, we decanted all our samples, and then sowed a drop of the deposit of each in flasks of pure wort. On January 2nd, 1873, we examined the ferments formed in these worts, which had been kept in an oven at a temperature of 20° C. (68° F.), and also tasted the beers produced; they all had an abominable taste, and each contained diseased ferments.

At the same time, by way of comparison, we impregnated other flasks of wort with pure ferments. None of the beers of this series acquired a bad taste or produced foreign ferments; they only became flat.

When we review the operations of the brewer’s art, we are surprised by the comparative perfection to which that art has been brought by the laborious experience of years, and the more so when we consider that, as regards the question of the diseases of beer, the brewer has never been guided by any such rigorous principles as those which we have explained in this work. We have already given proofs of this in our first chapter.

The beer is racked and separated from its yeast before fermentation has entirely ceased. The principal reason for this is that it is necessary that the beer, after being run into cask, should work again and undergo a secondary fermentation, in order that it may not be invaded by the parasites, of which we have already spoken, as would not fail to be the case if the beer were suffered to remain in a state of perfect quiescence. Not only is the beer racked before it has attained its limit of attenuation, but in addition to this, and also with the view of checking the development of parasites, it is placed in cellars sensibly cooler than the temperature of fermentation, low as that is in the case of “low” beers: the temperature of the cellars being not higher than 2° or 3° C. (36° F.).

Unfortunately, the requirements of trade prevent our complying with these exigencies to the end. When the beer is sold it is conveyed away, no matter what the season may be, and deposited in the retailer’s cellar, for a longer or shorter time, according to the variations of consumption. On a warm day beer will be in great demand; the next day, if rain or cold have come on, the demand will be very limited, since beer is, in our climate at least, a drink for hot weather. From causes of this nature, the beer may have to remain a long time in the cellars of the retailers or consumers. By way of precaution, indeed, it is put into very small casks, which permit of a frequent renewal of the supply, and is conveyed to distances by express trains, and during the night; it is even sent away in wagons provided with a kind of double case, the outer jacket being filled with ice, which keeps the air surrounding the casks constantly cold. Such are some of the troublesome measures taken to obviate the danger that we have pointed out. They operate very injuriously in restricting the trade and raising the price of beer. It is a matter of extreme importance, then, that our produce should be better removed from the action of those microscopic enemies which beer contains; in other words, that this beverage should have less cause to fear circumstances favourable to the development of the germs of impurity with which it is always contaminated, as a natural consequence of the methods of manufacture at present adopted. The question of alteration in the flavour of beer should be regarded from another point of view which merits equal attention. We have seen that there are different kinds of beer, each of which corresponds to a special ferment from which it derives its flavour and aroma, and, in a word, everything which gives it a value in the eyes of the consumer. It very often happens, especially in badly-managed breweries, and more particularly in those in which several beers are manufactured, that the yeast is a mixture of different ferments. The evil effects of such a mixture are experienced in the course of manufacture, and still more so in the beer after manufacture. Brewers in good “low” fermentation breweries, who brew what is called _stock_ beer, during the winter months, for consumption in summer, up to August and September, are very anxious to prevent the development of a _vinous_ flavour in beers of this kind. According to our observations, this vinous flavour seems to be principally due to an intermixture with the pitching yeast of _saccharomyces pastorianus_ or its varieties, one of the peculiarities of which ferment is that in the course of time it imparts a decided vinous flavour to beer. If this ferment were not present amongst the yeast-cells—and here we are speaking of an absolute, so to say, mathematically absolute absence—the beer produced would gradually grow old in the store cellars, without ever acquiring any vinous flavour, properly so called.

This vinous flavour develops more especially in English beers when these are kept. It is an easy matter to show that in English beers, after their manufacture, _saccharomyces pastorianus_ and the ferment which we have termed _caseous_, which also imparts a peculiar flavour, form almost exclusively, notwithstanding the fact that the yeast used in the manufacture of English beer is a ferment essentially distinct from _saccharomyces pastorianus_.

The secondary fermentation which takes place in “high” and “low” beers stored in cask after manufacture, is very often due to this same ferment, which may be recognized by elongated jointed cells, at times more or less ramified, as well as by the influence which it exercises upon the flavour of the beer.

We may add that the general result of our researches has convinced us that “high” yeast cannot transform itself, any more than “low” yeast can, into the ferment of which we are speaking, and that whenever a beer produced by means of “high” or “low” yeast develops a foreign ferment, this ferment must have existed in the original yeast in the form of germs, which, from their extreme scarcity, often fail to be detected by means of the microscope. The best proof that we can give of this is the fact that a beer produced by means of “high” or “low” yeast, if left to itself for months or years, will never contain in its deposit anything besides the yeast that was used in its manufacture, provided that that was pure to begin with. Now this can never be the case in dealing with actual commercial beers, no matter what they may be or in what brewery they may be produced. In all beers, in the course of time, in addition to diseased ferments, ferments essentially different from those used in their manufacture will appear, and notably _saccharomyces pastorianus_; this result must be attributed to the general impurity of commercial yeasts.

In certain cases the intermixture of ferments is to be feared almost as much as the presence of diseased ferments, when these latter have not developed to any great extent. We have often seen our fermentations invaded by ferments differing absolutely from those which we originally employed. The repetition of growths, and more particularly changes in the composition of our fermentable media, purposely made with the view of attaining certain results, often produce complications of this kind. For a long time we were unable to realize the true significance of the results of some of our experiments, in consequence of the facts which we have just explained, as well as those detailed in the preceding paragraph, having escaped our notice; indeed, our ignorance of those facts added greatly to the difficulty and length of our researches. Our labours from the commencement of this work to the date of its publication have extended over not less than five years, and no one can know better than ourselves with what advantage we might devote a still longer time to it; but, as Lavoisier says, one would never give anything to the world if he delayed doing so until he fully attained unto his ideal aims, which always seem more distant the more one increases one’s efforts in the attempt.

Our preceding observations show how extremely important it is to employ pure yeasts to obtain, on the one hand, well flavoured beers, whilst adhering to the processes at present existing in breweries, and on the other, beers of good keeping qualities, less liable to injury, less dependent on actual commercial requirements, capable, that is, of withstanding conditions favourable to the development of ferments prejudicial to the soundness of the produce, what we have named ferments of disease.

In the case of intermixture of alcoholic ferments, we may sometimes manage to effect their separation by taking advantage of their unequal vitalities in different media of cultivation. On December 17th, 1872, we made a powder of commercial Dutch yeast and plaster, as described in

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