FLASK A, WITHOUT AIR.

January 21st.--Fermentation commenced; a little frothy liquid issued from the escape tube and covered the mercury.

The following days, fermentation was active. Examining the yeast mixed with the froth that was expelled into the mercury by the evolution of carbonic acid gas, we find that it was very fine, young, and actively budding.

February 3rd.--Fermentation still continued, showing itself by a number of little bubbles rising from the bottom of the liquid, which had settled bright. The yeast was at the bottom in the form of a deposit.

February 7th.--Fermentation still continued, but very languidly.

February 9th.--A very languid fermentation still went on, discernible in little bubbles rising from the bottom of the flask.

FLASK B, WITH AIR.

January 21st.--A sensible development of yeast.

The following days, fermentation was active, and there was an abundant froth on the surface of the liquid.

February 1st.--All symptoms of fermentation had ceased.

As the fermentation in A would have continued a long time, being so very languid, and as that in B had been finished for several days, we brought to a close our two experiments on February 9th.

To do this we poured off the liquids in A and B, collecting the yeasts on tared filters. Filtration was an easy matter, more especially in the case of A. Examining the yeasts under the microscope, immediately after decantation, we found that both of them remained very pure. The yeast in A was in little cl.u.s.ters, the globules of which were collected together, and appeared by their well-defined borders to be ready for an easy revival in contact with air.

As might have been expected, the liquid in flask B did not contain the least trace of sugar; that in the flask A still contained some, as was evident from the non-completion of fermentation, but not more than 4.6 grammes (71 grains). Now, as each flask originally contained three litres of liquid holding in solution 5 per cent of sugar, it follows that 150 grammes (2,310 grains) of sugar had fermented in the flask B, and 145.4 grammes (2,239.2 grains) in the flask A. The weights of yeast after drying at 100 degrees C. (212 degrees F.) were--

For the flask B, with air. ... ..1,970 grammes (30.4 grains). For the flask A, without air ... 1,368 grammes [Footnote: This appears to be a misprint for 1.638 grammes=25.3 grains.--D. C. R.].

The proportions were 1 of yeast to 76 of fermented sugar in the first case, and 1 of yeast to 89 of fermented sugar in the second.

From these facts the following consequences may be deduced:

1. The fermentable liquid (flask B), which since it had been in contact with air, necessarily held air in solution, although not to the point of saturation, inasmuch as it had been once boiled to free it from all foreign germs, furnished a weight of yeast sensibly greater than that yielded by the liquid which contained no air at all (flask A) or, at least, which could only have contained an exceedingly minute quant.i.ty.

2. This same slightly aerated fermentable liquid fermented much more rapidly than the other. In eight or ten days it contained no more sugar; while the other, after twenty days, still contained an appreciable quant.i.ty.

Is this last fact to be explained by the greater quant.i.ty of yeast formed in B? By no means. At first, when the air has access to the liquid, much yeast is formed and little sugar disappears, as we shall prove immediately; nevertheless the yeast formed in contact with the air is more active than the other. Fermentation is correlative first to the development of the globules, and then to the continued life of those globules once formed. The more oxygen these last globules have at their disposal during their formation, the more vigorous, transparent, and turgescent, and, as a consequence of this last quality, the more active they are in decomposing sugar. We shall hereafter revert to these facts.

3. In the airless flask the proportion of yeast to sugar was 1/59; it was only 1/79 in the flask which had air at first.

The proportion that the weight of yeast bears to the weight of the sugar is, therefore, variable, and this variation depends, to a certain extent, upon the presence of air and the possibility of oxygen being absorbed by the yeast. We shall presently show that yeast possesses the power of absorbing that gas and emitting carbonic acid, like ordinary fungi, that even oxygen may be reckoned amongst the number of food-stuffs that may be a.s.similated by this plant, and that this fixation of oxygen in yeast, as well as the oxidations resulting from it, have the most marked effect on the life of yeast, on the multiplication of its cells, and on their activity as ferments acting upon sugar, whether immediately or afterwards, apart from supplies of oxygen or air.

In the preceding experiment, conducted without the presence of air, there is one circ.u.mstance particularly worthy of notice.

This experiment succeeds, that is to say, the yeast sown in the medium deprived of oxygen develops, only when this yeast is in a state of great vigour. We have already explained the meaning of this last expression. But we wish now to call attention to a very evident fact in connection with this point. We impregnate a fermentable liquid; yeast develops and fermentation appears. This lasts for several days and then ceases. Let us suppose that, from the day when fermentation first appears in the production of a minute froth, which gradually increases until it whitens the surface of the liquid, we take, every twenty-four hours, or at longer intervals, a trace of the yeast deposited on the bottom of the vessel and use it for starting fresh fermentations.

Conducting these fermentations all under precisely the same conditions of temperature, character and volume of liquid, let us continue this for a prolonged time, even after the original fermentation is finished. We shall have no difficulty in seeing that the first signs of action in each of our series of second fermentations appear always later and later in proportion to the length of time that has elapsed from the commencement of the original fermentation. In other words, the time necessary for the development of the germs and the production of that amount of yeast sufficient to cause the first appearance of fermentation varies with the state of the impregnating cells, and is longer in proportion as the cells are further removed from the period of their formation. It is essential, in experiments of this kind, that the quant.i.ties of yeast successively taken should be as nearly as possible equal in weight or volume, since, celeris paribus, fermentations manifest themselves more quickly the larger the quant.i.ty of yeast employed in impregnation.

If we compare under the microscope the appearance and character of the successive quant.i.ties of yeast taken, we shall see plainly that the structure of the cells undergoes a progressive change.

The first sample which we take, quite at the beginning of the original fermentation, generally gives us cells rather larger than those later on, and possessing a remarkable tenderness.

Their walls are exceedingly thin, the consistency and softness of their protoplasm is akin to fluidity, and their granular contents appear in the form of scarcely visible spots. The borders of the cells soon become more marked, a proof that their walls undergo a thickening; their protoplasm also becomes denser, and the granulations more distinct. Cells of the same organ, in the states of infancy and old age, should not differ more than the cells of which we are speaking, taken in their extreme states.

The progressive changes in the cells, after they have acquired their normal form and volume, clearly demonstrate the existence of a chemical work of a remarkable intensity, during which their weight increases, although in volume they undergo no sensible change, a fact that we have often characterized as "the continued life of cells already formed." We may call this work a process of maturation on the part of the cells, almost the same that we see going on in the case of adult beings in general, which continue to live for a long time, even after they have become incapable of reproduction, and long after their volume has become permanently fixed.

This being so, it is evident, we repeat, that, to multiply in a fermentable medium, quite out of contact with oxygen, the cells of yeast must be extremely young, full of life and health, and still under the influence of the vital activity which they owe to the free oxygen which has served to form them, and which they have perhaps stored up for a time. When older, they reproduce themselves with much difficulty when deprived of air, and gradually become more languid; and if they do multiply, it is in strange and monstrous forms. A little older still, they remain absolutely inert in a medium deprived of free oxygen. This is not because they are dead; for in general they may be revived in a marvellous manner in the same liquid if it has been first aerated before they are sown. It would not surprise us to learn that at this point certain preconceived ideas suggest themselves to the mind of an attentive reader on the subject of the causes that may serve to account for such strange phenomena in the life of these beings which our ignorance hides under the expressions of YOUTH and AGE; this, however, is a subject which we cannot pause to consider here.

At this point we must observe--for it is a matter of great importance--that in the operations of the brewer there is always a time when the yeasts are in this state of vigorous youth of which we have been speaking, acquired under the influence of free oxygen, since all the worts and the yeasts of commerce are necessarily manipulated in contact with air, and so impregnated more or less with oxygen. The yeast immediately seizes upon this gas and acquires a state of freshness and activity, which permits it to live afterwards out of contact with air, and to act as a ferment. Thus, in ordinary brewery practice, we find the yeast already formed in abundance even before the earliest external signs of fermentation have made their appearance. In this first phase of its existence, yeast lives chiefly like an ordinary fungus.

From the same circ.u.mstances it is clear that the brewer's fermentations may, speaking quite strictly, last for an indefinite time, in consequence of the unceasing supply of fresh wort, and from the fact, moreover, that the exterior air is constantly being introduced during the work, and that the air contained in the fresh worts keeps up the vital activity of the yeast, as the act of breathing keeps up the vigour and life of cells in all living beings. If the air could not renew itself in any way, the vital activity which the cells originally received, under its influence, would become more and more exhausted, and the fermentation eventually come to an end.

We may recount one of the results obtained in other experiments similar to the last, in which, however, we employed yeast which was still older than that used for our experiment with flask A (Fig. 2), and moreover took still greater precautions to prevent the presence of air. Instead of leaving the flask, as well as the dish, to cool slowly, after having expelled all air by boiling, we permitted the liquid in the dish to continue boiling whilst the flask was being cooled by artificial means; the end of the escape tube was then taken out of the still boiling dish and plunged into the mercury trough. In impregnating the liquid, instead of employing the contents of the small cylindrical funnel whilst still in a state of fermentation, we waited until this was finished. Under these conditions, fermentation was still going on in our flask, after a lapse of three months. We stopped it and found that 0.255 gramme (3.9 grains) of yeast had been formed, and that 45 grammes (693 grains) of sugar had fermented, the ratio between the weights of yeast and sugar being thus 0.255 divided by 45 = 1 divided by 176. In this experiment the yeast developed with much difficulty, by reason of the conditions to which it had been subjected. In appearance the cells varied much, some were to be found large, elongated, and of tubular aspect, some seemed very old and were extremely granular, whilst others were more transparent. All of them might be considered abnormal cells.

In such experiments we encounter another difficulty. If the yeast sown in the non-aerated fermentable liquid is in the least degree impure, especially if we use sweetened yeast-water, we may be sure that alcoholic fermentation will soon cease, if, indeed, it ever commences, and that accessory fermentations will go on. The vibrios of butyric fermentation, for instance, will propagate with remarkable facility under these circ.u.mstances. Clearly then, the purity of the yeast at the moment of impregnation, and the purity of the liquid in the funnel, are conditions indispensable to success.

To secure the latter of these conditions, we close the funnel, as shown in FIG. 2, by means of a cork pierced with two holes, through one of which a short tube pa.s.ses, to which a short length of india-rubber tubing provided with a gla.s.s stopper is attached; through the other hole a thin curved tube is pa.s.sed. Thus fitted, the funnel can answer the same purposes as our double-necked flasks. A few cubic centimetres of sweetened yeast-water are put in it and boiled, so that the steam may destroy any germs adhering to the sides; and when cold the liquid is impregnated by means of a trace of pure yeast, introduced through the gla.s.s- stoppered tube. If these precautions are neglected, it is scarcely possible to secure a successful fermentation in our flasks, because the yeast sown is immediately held in check by a development of anaerobian vibrios. For greater security, we may add to the fermentable liquid, at the moment when it is prepared, a very small quant.i.ty of tartaric acid, which will prevent the development of butyric vibrios.

[Ill.u.s.tration with caption: Fig. 4.]

The variation of the ratio between the weight of the yeast and that of the sugar decomposed by it now claims special attention.

Side by side with the experiments which we have just described, we conducted a third lot by means of the flask C (Fig. 4), holding 4.7 litres (8 1/2 pints), and fitted up like the usual two-necked flasks, with the object of freeing the fermentable liquid from foreign germs, by boiling it to begin with, so that we might carry on our work under conditions of purity. The volume of yeast-water (containing 5 per cent. of sugar) was only 200 cc.

(7 fl. oz.), and consequently, taking into account the capacity of the flask, It formed but a very thin layer at the bottom. On the day after impregnation the deposit of yeast was already considerable, and forty-eight hours afterwards the fermentation was completed. On the third day we collected the yeast after having a.n.a.lyzed the gas contained in the flask. This a.n.a.lysis was easily accomplished by placing the flask in a hot-water bath, whilst the end of the curved tube was plunged under a cylinder of mercury. The gas contained 41.4 per cent. of carbonic acid, and, after the absorption, the remaining air contained:--

Oxygen . ........................... ... 19.7

Nitrogen . ........................... . 80.3

100.0

Taking into consideration the volume of this flask, this shows a minimum of 50 cc. (3.05 cub. in.) of oxygen to have been absorbed by the yeast. The liquid contained no more sugar, and the weight of the yeast, dried at a temperature of 100 degrees C (212 degrees F.), was 0.44 grammes. The ratio between the weights of yeast and sugar is 0.44/10=1/22.7 [Footnote: 200 cc. of liquid were used, which, as containing 3 per cent., had in solution 10 grammes of sugar.--D.C.R.]. On this occasion, where we had increased the quant.i.ty of oxygen held in solution, so as to yield itself for a.s.similation at the beginning and during the earlier developments of the yeast, we found instead of the previous ratio of 1/76 that of 1/23.

[Ill.u.s.tration with caption: Fig. 5]

The next experiment was to increase the proportion of oxygen to a still greater extent, by rendering the diffusion of gas a more easy matter than in a flask, the air in which is in a state of perfect quiescence. Such a state of matters hinders the supply of oxygen, inasmuch as the carbonic acid, as soon as it is liberated, at once forms an immovable layer on the surface of the liquid, and so separates off the oxygen. To effect the purpose of our present experiment, we used flat basins having gla.s.s bottoms and low sides, also of gla.s.s, in which the depth of the liquid is not more than a few millimetres (less than 1/4 inch) (Fig. 5). The following is one of our experiments so conducted:--On April 16th, 1860, we sowed a trace of beer yeast ("high" yeast) in 200 cc. (7 fl. oz.) of a saccharine liquid containing 1.720 grammes (26.2 grains) of sugar-candy. From April 18th our yeast was in good condition and well developed. We collected it, after having added to the liquid a few drops of concentrated sulphuric acid, with the object of checking the fermentation to a great extent, and facilitating filtration. The sugar remaining in the filtered liquid, determined by Fehling's solution, showed that 1.04 grammes (16 grains) of sugar had disappeared. The weight of the yeast, dried at 100 degrees C. (212 degrees F.), was 0.127 gramme (2 grains), which gives us the ratio between the weight of the yeast and that of the fermented sugar 0.123/1.04=1/8.1, which is considerably higher than the preceding ones.

We may still further increase this ratio by making our estimation as soon as possible after the impregnation, or the addition of the ferment. It will be readily understood why yeast, which is composed of cells that bud and subsequently detach themselves from one another, soon forms a deposit at the bottom of the vessels. In consequence of this habit of growth, the cells constantly covering each other prevents the lower layers from having access to the oxygen held in solution in the liquid, which is absorbed by the upper ones. Hence, these which are covered and deprived of this gas act on the sugar without deriving any vital benefit from the oxygen--a circ.u.mstance which must tend to diminish the ratio of which we are speaking. Once more repeating the preceding experiment, but stopping it as soon as we think that the weight of yeast formed may be determined by the balance (we find that this may be done twenty-four hours after impregnation with an inappreciable quant.i.ty of yeast), in this case the ratio between the weights of yeast and sugar is gr/024 yeast/0 gr. 09 sugar=1/4. This is the highest ratio we have been able to obtain.

Under these conditions the fermentation of sugar is extremely languid: the ratio obtained is very nearly the same that ordinary fungoid growths would give. The carbonic acid evolved is princ.i.p.ally formed by the decompositions which result from the a.s.similation of atmospheric oxygen. The yeast, therefore, lives and performs its functions after the manner of ordinary fungi: so far it is no longer a ferment, so to say; moreover, we might expect to find it to cease to be a ferment at all if we could only surround each cell separately with all the air that it required. This is what the preceding phenomena teach us; we shall have occasion to compare them later on with others which relate to the vital action exercised on yeast by the sugar of milk.

We may here be permitted to make a digression.

In his work on fermentations, which M. Schutzenberger has recently published, the author criticises the deductions that we have drawn from the preceding experiments, and combats the explanation which we have given of the phenomena of fermentation.

[Footnote: International Science Series, vol. xx, pp. 179-182.

London, 1876.--D. C. R.] It is an easy matter to show the weak point of M. Schutzenberger's reasoning. We determined the power of the ferment by the relation of the weight of sugar decomposed to the weight of the yeast produced. M. Schutzenberger a.s.serts that in doing this we lay down a doubtful hypothesis, and he thinks that this power, which he terms FERMENTATIVE ENERGY, may be estimated more correctly by the quant.i.ty of sugar decomposed by the unit-weight of yeast in unit-time; moreover, since our experiments show that yeast is very vigorous when it has a sufficient supply of oxygen, and that, in such a case, it can decompose much sugar in a little time, M. Schutzenberger concludes that it must then have great power as a ferment, even greater than when it performs its functions without the aid of air, since under this condition it decomposes sugar very slowly.

In short, he is disposed to draw from our observations the very opposite conclusion to that which we arrived at.

M, Schutzenberger has failed to notice that the power of a ferment is independent of the time during which it performs its functions. We placed a trace of yeast in one litre of saccharine wort; it propagated, and all the sugar was decomposed. Now, whether the chemical action involved in this decomposition of sugar had required for its completion one day, or one month, or one year, such a factor was of no more importance in this matter than the mechanical labour required to raise a ton of materials from the ground to the top of a house would be affected by the fact that it had taken twelve hours instead of one. The notion of time has nothing to do with the definition of work. M.

Schutzenberger has not perceived that in introducing the consideration of time into the definition of the power of a ferment, he must introduce at the same time, that of the vital activity of the cells which is independent of their character as a ferment. Apart from the consideration of the relation existing between the weight of fermentable substance decomposed and that of ferment produced, there is no occasion to speak of fermentations or of ferments. The phenomena of fermentation and of ferments have been placed apart from others, precisely because, in certain chemical actions, that ratio has been out of proportion; but the time that these phenomena require for their accomplishment has nothing to do with either their existence proper, or with their power. The cells of a ferment may, under some circ.u.mstances, require eight days for revival and propagation, whilst, under other conditions, only a few hours are necessary; so that, if we introduce the notion of time into our estimate of their power of decomposition, we may be led to conclude that in the first case that power was entirely wanting, and that in the second case it was considerable, although all the time we are dealing with the same organism--the identical ferment.

M. Schutzenberger is astonished that fermentation can take place in the presence of free oxygen, if, as we suppose, the decomposition of the sugar is the consequence of the nutrition of the yeast, at the expense of the combined oxygen, which yields itself to the ferment. At all events, he argues, fermentation ought to be slower in the presence of free oxygen. But why should it be slower? We have proved that in the presence of oxygen the vital activity of the cells increases, so that, as far as rapidity of action is concerned, its power cannot be diminished.

It might, nevertheless, be weakened as a ferment, and this is precisely what happens. Free oxygen imparts to the yeast a vital activity, but at the same time impairs its power as yeast--qua yeast, inasmuch as under this condition it approaches the state in which it can carry on its vital processes after the manner of an ordinary fungus; the mode of life, that is, in which the ratio between the weight of sugar decomposed and the weight of the new cells produced will be the same as holds generally among organisms which are not ferments. In short, varying our form of expression a little, we may conclude with perfect truth, from the sum total of observed facts, that the yeast which lives in the presence of oxygen and can a.s.similate as much of that gas as is necessary to its perfect nutrition, ceases absolutely to be a ferment at all. Nevertheless, yeast formed under these conditions and subsequently brought into the presence of sugar, OUT OF THE INFLUENCE OF AIR, would decompose more IN A GIVEN TIME than in any other of its states. The reason is that yeast which has formed in contact with air, having the maximum of free oxygen that it can a.s.similate is fresher and possessed of greater vital activity than that which has been formed without air or with an insufficiency of air. M. Schutzenberger would a.s.sociate this activity with the notion of time in estimating the power of the ferment; but he forgets to notice that yeast can only manifest this maximum of energy under a radical change of its life conditions; by having no more air at its disposal and breathing no more free oxygen. In other words, when its respiratory power becomes null, its fermentative power is at its greatest. M.

Schutzenberger a.s.serts exactly the opposite (p. 151 of his work-- Paris, 1875) [Footnote: Page 182, English edition], and so gratuitously places himself in opposition to facts.

In presence of abundant air supply, yeast vegetates with extraordinary activity. We see this in the weight of new yeast, comparatively large, that may be formed in the course of a few hours. The microscope still more clearly shows this activity in the rapidity of budding, and the fresh and active appearance of all the cells. Fig. 6 represents the yeast of our last experiment at the moment when we stopped the fermentation. Nothing has been taken from imagination, all the groups have been faithfully sketched as they were. [Footnote: This figure is on a scale of 300 diameters, most of the figures in this work being of 400 diameters].