Chapter V
. I attached the name of _aërobian ferment_. The beer, after being transferred to a bottle that had been washed with hot water, was kept for several months in the heat of summer, without exhibiting the slightest trace of deterioration.
[Illustration: Fig. 76.]
[Illustration: Fig. 77.]
The essential conditions of the preceding experiment can readily be realized on the large scale. For this purpose we may employ the apparatus in the above sketch (Figs. 76 and 77) constructed of tin or tinned copper. As appears from the sketch, this consists of a cylindrical tub resting on a support, and closed at the top by a cover, whose lower edge fits into a gutter containing water. The wort prepared in the copper is led into the cylinder, a process which does not materially lower its temperature. Now we know that wort in breweries which has been cooled in contact with the air, and so got charged with disease-germs, will, nevertheless, recover its faculty of keeping for any length of time in pure air, if we again raise its temperature to 80° C. (176° F.) or even 70° or 75° C. (158°, 167° F.) Having filled the tub with the hot wort and put on the lid, we then connect, by means of a caoutchouc tube _c d_, the metal tube _a c_ (which opens into one of the tubulures projecting above the lid) with the system of tubes _d e_, _f g_, of which _d e_ is fixed to the cylinder; _e f_ is a caoutchouc junction connecting _e_ with the bent glass tube _g_. We then dash over the apparatus, lid, tubulures, and their corks a quantity of boiling water. This collects in the gutter in which the lid rests, and any excess overflows into a second gutter outside the first, where, however, it cannot remain, but passes away by means of a ring of small holes between the base of the outer trough _i i_ and the cylinder. The overflow is collected in a third trough at the bottom, whence it can be removed by a pipe M. T is a bent thermometer to indicate the temperature of the wort; its bulb is protected by an inlet socket _d d_, pierced with holes; _r_ is a stopcock for discharging the water in the gutter, which serves as a hydraulic junction between the cylinder and its lid; R, V, are stopcocks, or openings for the discharge of the liquid in the cylinder and its deposit. The next process is to cool the vessel, which may be done either by leaving it to itself, or by introducing a current of cold water through the tubulure E, soldered on to the lid. This tubulure is of the form of an inverted funnel, and is pierced at the bottom with a close row of holes, through which the cold water issues in a sheet over the surface of the cover. In whichever way the cooling is effected, the external air continues all the time to enter the vessel beneath the lid by way of the long, narrow passage _g f e d c a_, and must necessarily get purified by depositing in its course all fungoid-germs, just as happened in the case of the two-necked flask of air experiments. This, however, may be still further secured by introducing a small plug of cotton wool, or asbestos, into the end of the tube _g_.
The experiments which we have carried out with this apparatus have proved that, by adopting such an arrangement, beer, a liquid peculiarly liable to change, may be kept as long as we wish, for weeks or months, in contact with air, since the tube _g_ is open, without evincing the least symptom of disease. It matters little whether the leaves and strobiles of the hops are introduced with the hot wort or strained off; the result is the same. On the other hand, a leak in the apparatus from which the wort gets mixed with ordinary water from outside during cooling, will speedily effect a change in the wort and cause it to swarm with vibrios, or butyric ferment, lactic ferment, and other germs of disease, whilst its taste will be rendered extremely nauseous. It can only be through one’s own fault, that is, from want of skill in carrying out the operation, that any change can be brought about by the water in the gutter not being kept out of the fermenting vessel. That water may even become putrid without the organisms contained in it being able to reach the wort in the fermenting vessel. The apparatus may be of any size whatever; we have worked with vessels containing 12 hectolitres with as much ease and certainty as when we used an apparatus of 1 hectolitre (22 gallons).
It is easy to carry out the process of cooling in the presence of carbonic acid gas if we fit a bent tube, similar to _a c d e f g_, to the second tubulure D. Through this tube, or its companion, the gas can be passed as it issues from an apparatus in which it is generated, or from a gasometer filled with it, or from a vessel of beer undergoing fermentation.
However, there is no necessity that the cooling should take place in the fermenting vessel. It may be effected separately, in vessels of greater or less depth, in spiral coils surrounded with cold water, or in any kind of refrigerator, provided always that the conditions of purity are satisfied, and that the flow of the cooled wort takes place under the same conditions. Jets of steam, which are already extensively used for the cleansing of pipes in breweries, may be employed here with great advantage.
The pitching may be effected in various ways. A two-necked flask of a capacity of from 200 to 300 cc. (about 7 to 10 fl. ozs.), in which not more than 100 cc. (3-½ fl. ozs.) of wort has been fermented, will be sufficient for an apparatus of 1 hectolitre (22 gallons), although the flask may not contain more than 1 or 2 decigrammes (1-½ to 3 grains) of yeast. In the manufacture of beer, as at present conducted, the employment of so minute a quantity of yeast would lead to most disastrous results. The fermentation would unfailingly become lactic and butyric, since the foreign germs with which commercial worts and yeasts are always contaminated would have ample time to develop during the first twenty-four or forty-eight hours, whilst the small quantity of yeast used in the pitching could scarcely do more than begin to develop during that time. It is simply with the object of avoiding these secondary fermentations that the brewer uses large quantities of yeast for pitching.
After the wort and yeast have been _pulled up_,[163] a process which every practical brewer adopts after pitching, every part of the liquid is occupied by a multitude of yeast-cells, which seize upon the oxygen in solution, germinate with activity, turn to their own account the food-supplies most easily assimilated, and prevent the growth of the germs of disease-ferments. In the new process which we are now explaining, things happen quite differently. Our wort is pure, and our yeast is pure, and if only a single cell of yeast were introduced into the wort, the vital activity of this would be sufficient to bring about alcoholic fermentation, and to transform the wort into beer, without our having the least reason to apprehend the simultaneous development of any other organisms whatsoever. In short, the new process enables us to pitch with as small a quantity of yeast as we like. It is, nevertheless, inexpedient to employ too minute a quantity, since by doing so we should retard the commencement of fermentation.
[Illustration: Fig. 78.]
[Illustration: Fig. 79.]
In the case of an apparatus of 5 hectolitres (110 gallons) or double that capacity, the pitching may be accomplished by means of flasks holding from 4 to 9 litres (from 7 to 10 or 11 pints), (Fig. 79), or copper cans, tinned inside, holding from 10 to 15 litres (2-1/4 to 3-1/4 gallons), and provided at the upper conical end with glass tubes (Fig. 78). The vessel must be half or two-thirds filled with wort. For this purpose it will be well always to employ wort that has been preserved in bottles by Appert’s process. We must use a stopper provided with tubes, as represented in Fig. 79: _a b_ is a glass stopper which closes the india-rubber tube _b c_; _m n p_ is a fine glass tube, or, better still, made of copper.
The tap R being closed, a long india-rubber tube is attached to the extremity of the curved tube, and the flask is completely immersed in a hot-water bath; the india-rubber tube projects from the bath and keeps the interior of the vessel in communication with the external atmosphere. If the tube _m n p_ is of copper, we may avail ourselves of its flexibility and bend it upwards, so as to place its open extremity outside the bath. The water in the bath is then gradually raised to a temperature of 100° C. (212° F.), at which it is kept for a quarter or half an hour. In the case of copper cans, it is more convenient to place them over a gas-heater. They may be treated in the same manner as the flasks with curved necks. Vessels prepared in this manner may remain in a laboratory, or in any part of a brewery, for an indefinite time, without the wort in them undergoing the least change. It gradually darkens in colour through a direct oxidation of a purely chemical nature, but no tendency to disease will manifest itself.
Some days before we require to pitch an apparatus of several hectolitres, we impregnate one of these flasks or cans.[164] For this purpose we pass the flame of a spirit lamp over the tubes _c b a_ and _m n p_, to destroy the particles of dust that might pass inside at the moment when the stopper _a b_ is taken out, and then by means of a long, straight glass tube we take some of the liquid from a flask or vessel containing pure beer in a state of fermentation, and let a few drops of this, with the yeast that it holds in suspension, fall into the flask or can; the stopper _a b_ is once more passed through the flame and then replaced; generally in the course of one or two days the yeast develops in the flask sufficiently for the fermentation to show itself. We may shorten the operation still further by emptying into the can the contents of one of those double-necked flasks. To do this, we have simply to attach the straight tube of the flask to the india-rubber _b c_, and pour the liquid in. In a similar manner we introduce, through one of the tubulures surmounting the lid of the fermenting apparatus, the contents of the flasks or cans, either whilst they are still in
## active fermentation, or after fermentation is over. For this purpose,
the tap R is connected by means of an india-rubber tube (Fig. 79), with a tube passing through a cork fixed in one of the tubulures of the large apparatus. All this may be done in considerably less time than we have taken to describe it; and the operation may be performed accurately and safely by any one who has witnessed it a few times, even though he may not be skilled in chemical manipulations, especially if he takes care to bear in mind the very simple principles which we have explained.
Since certain parts of the apparatus—the outer opening of the tap, or the india-rubber tubing, for example—may contract particles of dust from the air, those parts, before being used, must be boiled in water, or washed with boiling water, or passed through the flame of a spirit lamp, to destroy the germs mixed with the particles of dust that settle upon them.
The method of cooling the wort in contact with carbonic acid prevents access of oxygen to the latter up to the time of pitching, so that the development of the yeast takes place apart from the influence of oxygen. Now, we know that these conditions necessitate the employment of a very young yeast—a yeast that is in course of active germination, such as may be taken from an incipient preparatory fermentation. Nevertheless, even with this, the development of the yeast under such conditions is extremely slow, and the fermentation takes from fifteen to twenty-five days; whilst, under the same circumstances, but with an aerated wort, it would be finished in from eight to twelve days. This is a considerable drawback, but, perhaps, a still more serious inconvenience is that the beer takes much longer to clarify, and does so with greater difficulty than those beers which are made with aerated worts. At the same time, this is largely compensated by the superior quality of the beer, which is stronger and has greater fulness on the palate, whilst the aroma of the hops is preserved to an extent never found in beers brewed by the ordinary process. Besides this, the yeast deposited at the bottom of the fermenting vessel is much less active, and, being of an older type, is revived with greater difficulty than that which forms in aerated worts. This, which might be considered a disadvantage, if we had to employ the yeast afterwards for pitching, has the great advantage of giving a beer which, when racked, undergoes its secondary fermentation only slowly, and with difficulty.[165] A beer of this kind is better adapted than ordinary beer to stand a long journey without developing great pressure inside the casks, and, if bottled, it will contain very little deposit, and will not froth violently when uncorked. The reason is, that a yeast is the more active, the more ready to multiply rapidly, and to work vigorously the more highly aerated the wort was in which it was grown. On the other hand, a yeast formed apart from air readily gets exhausted, and may even perish in the liquid in which it ferments, when that is kept out of contact with air; in other words, the vital action of yeast is more restricted when it has not been subjected to the action of oxygen during its formation.
If a great depth of wort, the surface of which alone is in contact with atmospheric air, is left to cool down, it will act in almost exactly the same manner as that which is cooled under an atmosphere of carbonic acid gas, because the oxygen of the air is very slow in pervading wort that is undisturbed. The gas will be taken into solution by the upper layer only, whilst the bulk of the liquid will remain unaffected by it. In some experiments which we conducted in a vessel which contained wort to a depth of 70 centimetres (27·5 inches), and which was provided with a tap that enabled us to draw off some of the liquid every day, until we had reduced the depth to 35 centimetres, we found, at the end of eight days, that there was not a trace of oxygen in solution at the latter depth. It is even probable that, considering the slow diffusion of the oxygen, on the one hand, and the combination that may take place between it and certain components of the malt, on the other hand, it would take a long time for all the wort, if undisturbed and of a certain depth, to become saturated with oxygen. In the vessel represented in Figs. 76 and 77 there is a considerable depth of wort to cool down. Nevertheless, the mere fact of the possibility of an aeration from the surface, whilst the wort is cooling down in contact with pure air, is enough to account for a certain effect that is produced on the yeast, later on, for the more youthful appearance of the yeast of the deposit, compared with that which we find in the case of wort cooled in the presence of carbonic acid gas. The difference between the results is particularly striking if, in both cases, we follow up microscopically the development of the yeast during the first few days succeeding the pitching.
The influence of the air on fermentation is considerable. In the ordinary process of brewing, fermentations would be almost impossible, and in every case most defective, if the wort, before being run into the fermenting vessels, were not aerated by its passage over the “coolers,” where the aeration is more or less effective, according as the liquid is more or less shallow. Worts and yeasts being impure, that is containing the germs of foreign ferments, those germs would have time to germinate in the fermenting vessels during the delay that the want of aeration in the wort would cause in the development of the yeast. We are aware that several inventions have been proposed to do away with the coolers, and we feel convinced that the object has been to remedy irregularities in fermentation. Considering the facts which we have published[166] on the development of yeast in the presence of air, and its inactivity in non-aerated media, such inventions ought to be supplemented by some means of further aeration for the prevention of the mischief that they must otherwise cause. In the existing process of brewing, the employment of coolers is a necessity.
The influence of the air on the vital action of the yeast may be proved in ways innumerable. The following is an experiment which we have often carried out with surprising results. A fermentation is going on; we draw off the liquid as rapidly as we please, and pour it back again into the vessel immediately. Within an hour we find a marked increase in the fermentation, evidenced by the liberation of a greater quantity of carbonic acid gas. This experiment may be performed with especial ease if we use the fermenting apparatus that we have described, for, by fitting a gas measurer to the escape tube _a b c d e f g_, the number of litres produced before the drawing off of the liquid may be compared with those obtained after. The least physical change in the running of the fermenting liquid whilst it is being drawn off, modifies the effect in question; such as change in the diameter of the stream, the height from which it falls, its greater or less scattering in falling, all influence it. Again, as might be expected from such results, corresponding modifications take place in the cells of yeast which come under the influence of the air. They become firmer in aspect and outline, their plasma becomes fuller, assumes a younger and more transparent aspect, and the vacuoles disappear. The molecular granulations, too, are less apparent. At a certain focus they disappear; at another they reappear, not as black spots, however, but as brilliant points so small as to be scarcely perceptible. If germination has been suspended it is resumed; in short, everything tends to prove—and having the yeast actually under our eyes we cannot doubt the fact—that the life of the cells is more decided, and the work of nutrition more active after they have been brought into contact with the oxygen of the air, and have absorbed a greater or less quantity of that gas.
Under the ordinary conditions of brewing, the atmospheric air is present in very varying quantities, whether introduced by the wort which holds more or less in solution, or by diffusion over the surface of the vessels, so that the same cells of yeast live by turns without air and with air. At first they absorb all the oxygen held in solution, and multiply under the influence of this absorption. Afterwards, when the supply has been exhausted, and various assimilations have resulted from it, they are deprived of it. Their life continues apart from oxygen, and if the vessel were closed, fermentation would be accomplished under these conditions, although more slowly. The vessel being open, a small quantity of air diffuses continuously through the layer of carbonic acid gas on the surface, and supports the vitality of the cells.
It is interesting to observe that, in the working of breweries, there are several empirical practices the explanation of which is to be found wholly in the fact that the aeration of wort or beer exercises a great influence on fermentation. In many breweries we have seen the pitching performed in the following manner: the brewer, having mixed his yeast in many times its volume of wort, pours all the thick liquid from a height from one bucket into another, and from that back again into the first, and so on a great many times, until the two buckets are filled with the froth enclosing air. In certain London breweries we have seen a bucket suspended by a pulley over the fermenting tun, which is 3 or 4 metres (10 or 12 feet) in depth; this the brewer, by means of a cord, can lower into the tun and pull up again at will, giving it a kind of see-saw movement which agitates the surface of the liquid and aerates it. The use of the fermenting tun itself and the racking of the wort from that tun into casks have the effect of aerating the beer and the yeast, and imparting to the latter a greater vigour and activity.
The resumption of fermentation in cask, after the beer has been run out of the tuns in “low” fermentation breweries is, in our opinion, principally due to the aeration of the beer at the moment when it is racked. The brewer ought to bear in mind that, during racking, every detail is of importance; it makes a great difference whether when the beer is run into the casks it falls from a height or is conducted by a tube to the bottom of the casks, whether it passes directly into the casks, or is poured into them from buckets, and whether it runs in a stream of small or large diameter, since these different methods cause the introduction of corresponding different quantities of air into the beer.
We have devised a simple arrangement for bringing the fermenting liquid into contact with various proportions of atmospheric air. Appended is a sketch of this apparatus (Fig. 80). Instead of one tube serving alike for the entrance and escape of gas, there are two similar ones, each of which opens into one of the tubulures on the cover. Round the other end of one of the tubes is fitted a kind of muff or bag, composed of a cylindrical cage of metallic gauze, over which a layer of well-combed cotton wool is placed, the whole being covered with a muslin bag. The object of this arrangement is to act as an air-filter for retaining the
## particles of dust. When fermentation has commenced in the apparatus, we
have simply to press momentarily the india-rubber connection between the tube from the lid and the tube with the bag. This will at once cause a regular stream of carbonic acid to issue from the end of the uncovered tube, whilst the air will enter by the filtering tube to take its place; and this arrangement will be maintained throughout the whole course of the fermentation, even if we omit the precaution of increasing the power of the syphon by making the tube for the escape of this gas longer than the other one.[167]
[Illustration: Fig. 80.]
It will be readily understood how, whether by this last method, or by the diameter of the tubes, we may vary the conditions of this circulation of air in the apparatus, on the surface of the beer.
§ II.—Method of Estimating the Oxygen held in Solution in Wort.
The use of carbonic acid gas and the cooling of the wort, in contact with that gas or in contact with very limited quantities of pure air, are by no means necessary to the application of the new process. There is only one thing that is absolutely essential—which is, the _purity_ of the gases in the presence of which the wort is cooled and treated. If, therefore, it is well to aerate our wort, either before or during fermentation, this may be done, on the sole condition that the air employed does not introduce any germs of disease that are likely to develop in the beer during fermentation or afterwards. The question of aerating the wort is not, however, so simple a matter as it seems at first sight. A very simple observation will show that wort cannot be safely oxygenated by exposure, without precaution, to the air, even leaving out of account the germs of disease which that air may contain. It is easy to show that finished wort has a decided flavour and aroma of hops, as well as a sweet taste, and that it leaves a certain pleasant, bitter after-taste on the palate. When we taste it in this condition we cannot help thinking that a liquor of the kind, after fermentation, ought to constitute a very valuable beverage, as wholesome as it is pleasant. Now all this pleasant and refreshing sensation that the wort leaves on the palate, which is due as much to the aroma as to the bitterness of the hop, disappears absolutely, we may say, if the wort is left exposed to contact with air for a sufficient time, and that whether the air be warm or cold. We may easily perform the experiment in one of our two-necked flasks, in which we can preserve the wort, in contact with pure air, without any fear of change. The oxygen of the air enters into combination with the substances that the hop introduces into the wort, and the wort, in consequence of this oxidation, gradually becomes transformed into a saccharine decoction, without odour, in which even the bitter flavour is destroyed or hidden. In other words, the wort grows weak and flat, in just the same way that beer and wine do, as well as all the various natural or artificial worts which serve to produce them. Thus it is evident that considerable care is necessary in subjecting wort and beer, whether in course of manufacture or finished, to the action of atmospheric air. If, therefore, it is a good thing to supply wort with oxygen, as we have already pointed out, in order to facilitate the fermentation and nourish the yeast, it is, on the other hand, important that the quantity supplied to it should not be too great, otherwise we may injure the quality of the beer, and particularly its fulness on the palate, that is its apparent strength, which has very little to do with the proportion of alcohol in it. The strength of a beer is intimately connected with those substances introduced by the hops into the wort and thus into the beer, to which we previously alluded, and of which too little is known; their properties and the palatableness resulting from them are very readily affected by the oxygen of the air.[168]
We have, therefore, to ascertain the measure in which air occurs during the process of brewing, and whether, in the actual process, there may not be too great a proportion of active oxygen present. The study of this subject requires that we should know what quantities of oxygen may be held in solution in the wort or absorbed by direct combination. Fortunately this has been rendered a comparatively easy matter by a rapid method of estimating the oxygen held in solution in liquids of various kinds, devised by M. Schützenberger in 1872. As soon as this method was made known, we requested M. Raulin, who was attached to our laboratory as assistant-director, to apply it to the determination of oxygen in wort. This he did with his accustomed skill, devising certain alterations of details which rendered the method at the same time surer and more expeditious.
The principal feature in M. Schützenberger’s process consists in the employment of a salt, the properties of which that chemist was the first to recognize; he has named it _hydrosulphite of soda_, and it is obtained by the action of zinc filings on a solution of bisulphite of soda, out of contact with air.
Hydrosulphite of soda S^2O^2,NaO,HO, which is isomeric with hyposulphite of soda, only differs from the bisulphite by two equivalents of oxygen.[169] When brought into contact with free oxygen, it absorbs that gas instantaneously and becomes converted into bisulphite; similarly when mixed with water, it immediately absorbs the oxygen held in solution. Again there are colouring matters, such as M. Coupier’s soluble aniline blue, that are instantaneously decolourized by hydrosulphite of soda, whilst they resist the action of the bisulphite. If, taking care to avoid the access of air, we add hydrosulphite of soda to a certain volume of water—a litre, for example—that has been deprived of air and faintly coloured with Coupier’s blue, we shall see that a few drops will be sufficient to effect the decoloration. If, on the contrary, the water is aerated, the decoloration will not be effected before a sufficient quantity of the hydrosulphite has been added to absorb the oxygen in solution, and the volume of the reagent required is in proportion to the quantity of oxygen in solution in the water. To render the process sensitive, we must dilute the hydrosulphite to such an extent that 10 c.c., for example, may correspond very nearly with 1 c.c. of oxygen. If the reagent would keep we should only have to determine directly, once for all, the volume of oxygen that a known volume of the liquid could absorb; but, in consequence of its extreme liability to change through contact with air, it is necessary to titrate the liquid every time before using it. This is easily done in the following manner:—
According to the observations of Messrs. Schützenberger and Lalande, the hydrosulphite decolourizes an ammoniacal solution of sulphate of copper, reducing the copper to a lower state of oxidation; the sulphite and bisulphite having no action as long as there is an excess of ammonia. We prepare a strongly ammoniacal solution of sulphate of copper, containing such a quantity of copper that 10 c.c. of the liquid will correspond, as far as action on the hydrosulphite is concerned, with 1 c.c. of oxygen. Calculation by equivalents gives us the correct value verified by direct experiment.[170]
The object of the modification which M. Raulin has introduced, is to avoid the loss of time thus occasioned by the changes which take place in the titrated liquids by long keeping, as well as certain errors which may arise from the acidity of the wort. On this latter point M. Schützenberger has remarked that the quantities of hydrosulphite of soda corresponding with one and the same volume of oxygen vary with the acidity of the liquid operated upon, a phenomenon which that skilful chemist explains by the formation of oxygenated water, of varying stability in media of different acidity.
Instead of determining the strength of the titrated solution of hydrosulphite before each operation, we take the solution as it happens to be, and determine its strength by causing it to act on a known volume of pure water saturated with oxygen at a certain temperature. The tables of solubility of oxygen in water give the exact volume of oxygen on which the measured volume of hydrosulphite used has acted. According to Bunsen, about one minute’s brisk shaking in a closed bottle, with excess of air, will be sufficient to effect the maximum saturation of the water at the temperature at which we operate.
For experiments on wort we require:—
1. A 2-litre (3-½ pints) flask, A, containing _saturated_ hydrosulphite of soda,[171] of such strength that 2·5 c.c. will be sufficient to absorb almost all the oxygen in 50 c.c. of water saturated with air at the ordinary temperature (that is, 1 volume of hyposulphite must equal 20 volumes of water).
2. A 2-litre flask, B, containing a solution of indigo-carmine, 50 c.c. of which will be decolourized by about 20 c.c. of the hydrosulphite. This solution contains about 20 grammes (30·7 grains) of commercial indigo-carmine per litre (1·76 pints).
3. An apparatus, C, for the production of hydrogen.
4. An experimental apparatus composed of a burette, D, graduated in tenths of a cubic centimetre, and a three-necked Wollf’s bottle, E.
5. A flask, F, holding about 100 c.c. provided with a straight tube divided into tenths of a cubic centimetre, and containing a solution of ammonia of such strength that about ten drops of it will neutralize the acidity in 50 c.c. (1·76 fl. oz.) of wort.
To perform the operation we shake about 150 c.c. (5·3 fl. oz.) of distilled water, at the existing temperature, in a 1-litre flask for a minute or so; this saturates it with air, and we must at the same time note the temperature. To be extremely precise, we should note also the barometrical pressure.
[Illustration: Fig. 81.]
Into the bottle E we introduce about 50 c.c. of the indigo solution, and 200 c.c. of water at about 60° C. (140° F.), and fill the tube _e_ to the point _b_ with water saturated with air; we then expel the air from the bottle E by a current of hydrogen. The blue colour of the liquid in the bottle is then very carefully brought to a yellow tint, by running in, drop by drop, the hydrosulphite with which the burette D is filled.
We next pour 50 c.c. of distilled water saturated with air into the funnel _a_, and pass it into the flask; the blue colour reappears. We must then bring back the colour to exactly the same tint of yellow. Let _n_ represent the number of divisions on the burette denoting the volume of hydrosulphite employed for this purpose.
We repeat this last operation immediately, taking 50 c.c. of the wort, the oxygen of which we wish to determine, having first introduced into the funnel _a_ a sufficient number of drops of the ammoniacal solution to neutralize the acidity of the wort. Let _n´_ represent the number of divisions of hydrosulphite employed to restore the yellow tint in the case of the wort.
We once more perform the experiment with 50 c.c. of saturated water; let _n´´_ be the number found.[172]
The ratio which the quantity of oxygen held in solution in the wort bears to the quantity of oxygen contained in the same volume of water saturated with air, at the temperature _t_, and under the pressure H, will be
[Illustration: Formula 3: _n´_/((_n_ + _n´´_)/2).]
it will be sufficient in most cases to bear in mind this ratio.
When we want to deduce the absolute quantity of oxygen held in solution in a volume V of the wort, we have merely to multiply this ratio by the quantity of oxygen contained in the same volume of water saturated with air, at the temperature _t_ and under the pressure H, a very simple problem if we know the coefficients of the solubility of oxygen in water at different temperatures. These coefficients are given for ordinary temperatures in the following table, which was compiled by Bunsen. We have restricted the numbers to three places of decimals:—
Temperatures. Coefficients. 0° C. (32° F.) 0·040 1° C. (33·8° F.) 0·040 2° C. (35·6° F.) 0·039 3° C. (37·4° F.) 0·038 4° C. (39·2° F.) 0·037 5° C. (41·0° F.) 0 036 6° C. (42·8° F.) 0·035 7° C. (44·6° F.) 0·035 8° C. (46·4° F.) 0·034 9° C. (48·2° F.) 0·033 10° C. (50·0° F.) 0·033 11° C. (51·8° F.) 0·032 12° C. (53·6° F.) 0 031 13° C. (55 4° F.) 0·031 14° C. (57·2° F.) 0·030 15° C. (59·0° F.) 0·030 16° C. (60·8° F.) 0·029 17° C. (62·4° F.) 0·029 18° C. (64·4° F.) 0·029 19° C. (66·2° F.) 0·028 20° C. (68·0° F.) 0·028
The primary condition which enables us to rely on the exactness of this method is the fact which we have mentioned above, that a liquid if shaken up with air for one minute will become perfectly saturated with oxygen. Substantially this is the case. In estimating the oxygen in different parts of a liquid treated thus, we have invariably obtained the same figures to within about 1/50th.
It is true that the variable quantity of the oxygen held in solution in the liquid contained in the part of the tube _eb_, as well as the oxygen absorbed during the treatment of the liquid in contact with air, constitute causes of error. Experience, however, proves that these causes of error are insignificant, as long as we have to deal with a liquid the aeration of which is not very far removed from the point of saturation, and whose solubility-coefficient for oxygen is not widely different from that of water for the same gas. Under such conditions we have always found a constant ratio, to within about 1/40th between the same liquid and air-saturated distilled water, placed under the same circumstances.
If, on the other hand, we have to deal with a liquid which holds but a minute quantity of oxygen in solution, the causes of error mentioned may very seriously affect our results, and it will be absolutely necessary to avoid them. The liquid experimented on must be treated out of contact with air, by aspirating it directly from the vessel that contains it into the pipette H, which is graduated for 50 c.c., and causing it to pass thence into the flask E, by substituting the pipette for the funnel _a_. Finally, before arranging the pipette, we cause a small quantity of the liquid in the flask, which has been previously brought to the exact yellow tint, to pass, by pressure, through the tube _eb_, so as to avoid the cause of error that is likely to result from the air held in solution in the liquid of that tube.
The liquid, the oxygen of which has to be determined, may also be passed directly from the vessel containing it into the flask E; the rest of the operation being performed as already described.
It was by this method that the oxygen held in saturate solution in wort was determined. The following are the principal results obtained by M. Raulin:—
1. At different pressures the ratio between the quantities of oxygen held in solution in water and in wort is, all other conditions being similar, constant. This ratio has been found equal to 1·20 in the case of wort and water saturated with air at the ordinary pressure, and 1·24 in the case of wort and water saturated with pure oxygen.
2. The ratio between the coefficient of the solubility of oxygen in water and that of its solubility in wort is very nearly constant at different temperatures, increasing, however, slightly as the temperature diminishes.
This ratio has been found to be—
Temperatures. 26° C. (78·8° F.) 1·20 19·5° C. (67·1° F.) 1·25 4° C. (39·2° F.) 1·37
Another wort gave the following results:—
Temperatures. 9° C. (48·2° F.) 1·15 21° C. (69·8° F.) 1·10 25° C. (77·0° F.) 1·07
3. The ratio between the quantities of oxygen held in solution in water and those held in solution in wort increases with the concentration of the wort. By evaporating the same wort to different degrees of concentration, and afterwards saturating it with air, at the same temperature, we obtained the following figures for the ratio in question:—
Weak wort 1·06 The same evaporated to half 1·15 “ ” “ 2/5 1·27 ” “ ” 3/10 1·45 “ ” “ 1/6 1·96
4. Worts of different origin, but of the same density and temperature, when saturated with oxygen, always contain very nearly the same quantity of that gas.
Two portions of the same wort, shaken up with air, one being hot the other cold, then left to themselves for some time, and afterwards saturated with air, at the same temperature, gave the figures 1·22 for the ratio between the oxygen in the water and that in the wort.
Different worts of the same density, saturated at a temperature of 15° C. (59° F.), gave the following ratios:—
Wort kept in a bottle with air for 19 1·140 months
Wort recently prepared 1·142
Wort kept in a bottle without air for 20 1·142 months, aerated for 18 days
Wort evaporated to dryness and made up 1·126 with water
5. The solubility of oxygen in wort differs very little from the solubility of oxygen in sweetened water of the same density.
An experiment was made with a solution of sugar on the one hand, and with wort more or less diluted with water on the other hand, at the same temperature of 11° C. (51·8° F.). The following figures were obtained for the ratios of solubility:—
Solution Wort. of Sugar.
Marking 17·9° 1·278 1·27 Balling[173]
“ 14·0° ” 1·190 1·15
“ 7·0° ” 1·092 1·06
6. From the preceding results it is easy to deduce a general formula which shall give the coefficient of solubility of oxygen in any wort, marking B° by _Balling_, and at temperature _t_°.
From the figures of (2) it follows that above and below the temperature of 15° C. (59° F.), the ratio which the coefficient of solubility of oxygen in water bears to that of the solubility of the same gas in wort varies about 0·006 for each degree of the thermometer. From the figures of (3) it follows that the same ratio varies about 0·002 for each degree of _Balling_ above and below the 15th degree on the instrument.
By taking _c_ for the coefficient of solubility of oxygen in water at _t_° C., and _c´_ for that of oxygen in wort also at _t_° C., and having a density B, by _Balling_ at 15° C.; and taking X for the ratio _c_/_c´_, at 15° C. and 15° _Balling_, we shall have
_c_/_c´_ = X + (B - 15) 0·022 - (_t_ - 15) 0·006.
By carefully ascertaining the ratio _c_/_c´_ for different worts, and adopting the preceding formula, we have found for X a mean value of 1·16.
The definitive formula, therefore, is:
(1) _c_/_c´_ = 1·16 + (B-15) 0·022 - (_t_ - 15) 0·006,
or again,
(2) _c_/_c´_ = 0·86 - (B - 15) 0·016 + (_t_ - 15) 0·004.
The coefficient _c_ of the solubility of oxygen in water will be found in the table given a few pages back.
§ III.—On the Quantity of Oxygen existing in a state of Solution in Brewers’ Worts.[174]
The wort, when it comes from the copper in which it is boiled with the hops, remains exposed upon the coolers for a time, the length of which varies according to circumstances, the most important of which is the exterior temperature. The average time is from seven to eight hours, during which the volume of the wort diminishes, whilst its density increases; at the same time, it deposits its proteinaceous matters and absorbs oxygen from the air, either by way of solution or of combination.
In the present paragraph we shall confine ourselves to the uncombined oxygen held in a state of solution in wort, recognizable by the change of colour produced by its action on white indigo.
The use of the coolers enables the brewer to obtain his wort in two distinct states of limpidity—filtered wort and unfiltered wort. At the same time there is a further difference between these worts, namely, in the quantity of oxygen held in solution. The unfiltered wort comes direct from the coolers; the wort to be filtered, mixed with a part of the deposit, is run into a special vessel, from which it is distributed over the filtering surfaces, which are generally of felt; filtered bright, it is then received in a reservoir, from which it is distributed amongst special fermenting vessels. Falling through the air in a thin stream of drops, it must necessarily have become charged with a greater quantity of oxygen than ordinary wort. In good breweries it is put apart by itself to ferment, and the yeast which it yields is firmer and deposits more easily than that of unfiltered wort. As for the fermentation, it is, under similar conditions, quicker by a day or a day and a half than in the case of ordinary wort. The difference in the quantity of oxygen held in solution in the two kinds of wort is greater in proportion as the external temperature is lower; in winter it may be twice as great as in summer. The reason is that in summer a boiling wort does not obtain a minimum temperature of 20° C. (68° F.), on the best coolers, in less than six or seven hours. After leaving the coolers it is passed over a refrigerator. In winter it attains that temperature in about three hours, or less, which then goes on sinking on the coolers. During the last two or three hours which are employed in bringing the temperature still lower, as also during the running off, the wort absorbs an appreciable quantity of oxygen. In other words, wort in winter remains for a longer time at low temperatures, in free contact with air.
Another circumstance unites with this exposure upon the coolers to increase the aeration of the wort; the wort is run into the fermenting tuns through pipes of large sectional area, more or less bent, and carries with it by suction considerable quantities of air, which, from the continual agitation, gets well mixed with it. The effect of this mixing in the pipes is to considerably increase the proportion of air in solution in the wort, especially in winter, when the temperature of the wort is lower; and from the figures given below we may, although it is very variable, put the average increase at a quarter of the whole amount. The calculation has been made by comparing the quantities of air held in solution in two samples of the same wort, one of which was taken from the coolers at the moment of “turning out,”[175] and the other from the fermenting vessel after it was filled.
Let us call the ratio between the quantity of oxygen held in solution by a wort, and that which the same wort would hold in solution if saturated at the same temperature, the _degree of saturation_ of that wort at the temperature _t_.
The determination of degrees of saturation is reduced to a comparison of the number of divisions of hydrosulphite _n_ which satisfies the wort in the first case, with the number _n´_ corresponding with the same wort saturated at the same temperature. The ratio _n_/_n´_ gives the degree of saturation at the temperature _t_.
In experiments made with a wort at 14·5° _Balling_ as mean density, we found the following results:—
In summer, in the case of worts reduced to the temperature of 5° C. (41° F.) by a refrigerator, the degrees of saturation may be set down as—
For unfiltered worts 0·500 For filtered worts 0·800
In winter, in the case of some worts which were racked at a temperature of from 3° to 4° C. (37·4° to 39·2° F.), without the use of a refrigerator, we found the saturation complete in both worts. In the case of a very low external temperature, however (-10° C., 14° F.), we have failed to determine the saturation in an unfiltered wort. As regards the mean winter figures, in the case of worts racked at a temperature of 5° C. (41° F.), they may be fixed at these:—
For unfiltered wort 0·850 For filtered wort 0·950
In autumn and spring we find the mean figures to be intermediate between those given above:—
For unfiltered wort 0·500 to 0·850 For filtered wort 0·800 to 0·950
From these ratios it is easy to find the quantity of oxygen contained in brewers’ worts, if we also refer to Bunsen’s Tables and the formula (2) given in the preceding section. At the temperature of 5° C. (41° F.), at which the above worts were “gathered,”[176] and not taking into account the very small correction that should be made for the difference of half a degree on Balling, we find, by this formula, as the ratio of the coefficients of the solubility of oxygen in saturated wort and in water—
_c´_/_c_ = 0·82
Now, at the temperature of 5° C., the quantity of oxygen held in solution in 1 litre of water is, according to Bunsen, 0·036 litre, at the atmospheric pressure, and therefore at the pressure of 1/5th atmosphere, which is that of the oxygen in atmospheric air, it will be—
0·036/5 litre = 7·2 c.c.—[that is, 2 cubic inches per gallon.]
And, consequently, in the case of saturated wort, it will be—
7·2 c.c. × 0·82 = 5·904 c.c.—[that is, 1·62 cub. inches per gall.]
Multiplying this last number of c.c. by the different _degrees of saturation_ found, we shall obtain the volumes of oxygen held in solution in 1 litre of different worts:—
Summer worts {Unfiltered 0·500 × 5·904 c.c. = 2·952 c.c. {Filtered 0·800 × 5·904 “ = 4·723 ”
Winter worts {Unfiltered 0·850 × 5·904 “ = 5·018 ” {Filtered 0·950 × 5·904 “ = 5·609 ”
It is important to notice that we are here dealing with wort taken from the fermenting vessel just before it was pitched; that is to say, when the quantity of oxygen held in solution was as large as the treatment to which it had been subjected allowed of its being. The mode of taking it for examination is as follows:—A burette, H (Fig. 81), is plunged into the fermenting vessel, the temperature of which at the time is ascertained very exactly, the upper part of the burette being fitted with an india-rubber tube, _a b_, longer than itself. The liquid is then sucked up the tube, and soon completely fills the apparatus and runs out at _b_ (Fig. 82). By lowering the tube the whole arrangement thus forms a syphon, and enables us to let the wort that we are experimenting on flow for some minutes; when every trace of air has been thus expelled, the lower tap is closed and the liquid is introduced into Schützenberger’s apparatus.
[Illustration: Fig. 82.]
As for the saturated wort, the value of which in oxygen serves to determine one of the elements of the degree of saturation, it is readily obtained by introducing a volume of from 100 c.c. to 150 c.c. of wort into a 2-litre or 3-litre flask, and shaking it briskly so as to saturate it with air; it is then poured into a settling-glass, to separate it from the great quantity of froth formed in the shaking, and then, by means of a graduated pipette, 50 c.c. is taken for examination.
We have spoken of the influence that oxygen has on the activity of yeast, on its development and, consequently, on the progress of fermentation. Moreover, we know, from experiments already mentioned, which we communicated to the Academy and the Chemical Society in 1861, that the rapid development of yeast in contact with air is in reciprocal relation to the disappearance of the oxygen from the air. Knowing the conditions of the aeration of wort from the moment when it arrives on the coolers until the moment when, in the fermenting tun, it is about to be pitched, it would be interesting to ascertain what happens to the oxygen dissolved in the wort at the moment of pitching, how yeast is affected when suddenly brought into contact with that oxygen; what part, in short, that gas plays in fermentation.
Let us therefore follow up, hour by hour, the degree of saturation after pitching, in Tourtel’s brewery. On November 4th, 1875, some wort at 14° Balling was pumped on to the coolers at 7 p.m., and at 4 a.m. went down to a 32-hectolitre (700 gallons) tun, its temperature then being 6° C. (42·8° F.) The pitching, in which about 100 grammes (3·2 oz. troy) of pressed yeast was used per hectolitre (22 gallons), took place at 5 a.m. The following is the curve of the degrees of saturation of the oxygen, as drawn by Messrs. Calmettes and Grenet.
[Illustration: Fig. 83.]
The abscissæ represent the time expressed in hours, and the ordinates give the degrees of saturation of the wort with oxygen. It will be seen that about twelve hours after the pitching, and at a temperature of 6° C., all the oxygen had disappeared, absorbed by the yeast. We shall find that wort by itself, unassociated with yeast, would also have combined with oxygen; but in the course of twelve hours, at 6° C., this combination would have been scarcely appreciable in absence of yeast. It follows, therefore, that the oxygen in solution is taken up by the yeast, under the conditions of which we are speaking. This has been proved directly by an experiment. A double quantity of yeast was employed for a tun similar to the preceding one, and it was found that the oxygen in solution disappeared completely in less than half the time that it took to disappear in the first case.[177] It is very important to notice that in our 32-hectolitre tun, at the moment when we determined the complete disappearance of the oxygen in solution, the cells of yeast had assumed a younger and fuller appearance than they had at first; but they had not multiplied at all up to that time, nor were there even any buds then visible on them. The oxygen, therefore, must be stored up somehow in the cells, taken up by their oxidizable matters to be brought into work subsequently, or to act as a _primum movens_ of life and nutrition, spreading its influence over several successive generations of cells.
§ IV.—On the Combination of Oxygen with Wort.
The atmospheric oxygen is not merely taken into solution by wort; it also combines with it, as a very simple experiment will suffice to show. If we place in a tinned iron vessel some boiling wort, separated from the hops in the copper, and cool it suddenly by plunging it into iced water, and after having cooled it down in this manner to 15° or 20° C. (59° or 68° F.), saturate it with oxygen, by shaking it briskly in a large flask, and then completely fill a vessel with it and close it up for twelve hours, we shall find at the end of that time, if we test it with the hydrosulphite of soda, as we have described in § II., that it does not contain a trace of free oxygen. The whole of the gas which was originally held in solution will have entered into combination, that is to say, the liquid, first coloured blue with the indigo-carmine, and then brought to a yellow tint by means of the hydrosulphite of soda, will not regain its original blue colour through the action of this wort. The following experiments were undertaken with the object of studying this property of wort, and in order that we might form some idea of its importance, and of the total quantity of oxygen that wort can absorb under certain special circumstances. The experiments were performed in our own laboratory on wort from Tourtel’s brewery, which M. Calmettes had forwarded to us in bottles prepared in the brewery at Tantonville, in the following manner: Each bottle was filled with boiling wort taken from the copper and closed with a bored cork, through which the neck of a funnel passed; the funnel also was filled with the wort, and the whole preserved from contact with air by a layer of oil. The next day the bottles were corked full by the help of a bottling needle,[178] previously heated, with perfect corks that had been passed through the flame. The bottles arrived in Paris in very good condition, quite full of the liquid up to the corks. They were left undisturbed for one or two days at the same temperature as that to which they had been exposed during the corking and the journey. The object of this was to afford time for a deposit of the wort to form at the bottom of each bottle. As a matter of fact, we know that wort boiling in the copper is charged with proteinaceous matters and other floating and insoluble substances. The wort above the deposit was turbid and opaline; it was in this state when we used it for our experiments. It may be taken for granted, without risk of appreciable error, that the wort had been absolutely deprived of oxygen in solution, inasmuch as it had been bottled when boiling, and had cooled down out of contact with air. As for the quantity of oxygen that it might have held in combination, this must have been insignificant, although there must have been some, since the wort had been exposed to the air in the copper; the oxygen in combination, however, could have had no appreciable influence on the results which we obtained. Let us call this wort _boiled wort_.
_First Experiment._—Into a straight-necked flask we introduced a certain measured quantity of this wort by means of a syphon, taking care that the syphon should only act on the opaque wort, and should not reach the deposit at the bottom of the bottle. We then drew out the neck to a fine tube in the flame and boiled the wort; and during ebullition we sealed the end of the fine tube. After it had cooled, we arranged that pure air should enter the flask. To do this we made a file mark near the fine closed point of the flask, and connected the point by a piece of india-rubber tubing with a glass tube containing a column of asbestos, which we heated. We then broke off the point of the flask inside the india-rubber tube, so that the air entered the flask after being filtered through the asbestos. We removed the india-rubber tube and sealed up once more the fine end of the neck at the point where we had broken it off. Finally, to aerate the wort to saturation, we shook the flask briskly for some minutes, and then placed it in a hot-water bath, where we left it for about a quarter of an hour. We afterwards removed it to an oven at 25° (77° F.). We repeated the same operation next day and the four succeeding days.
The wort, which at first was scarcely coloured, gradually assumed a reddish-brown tint, and deposited an amorphous matter, but without brightening. It became clear, however, when filtered, which was not the case with the turbid, opaline wort in the bottles when they arrived.
The following is an analysis of the air in the flask, made immediately after a renewed and vigorous shaking, the object of which was to saturate the wort with air before analyzing the supernatant air:—
November 29th.
Temperature at which the flask was refilled with air 4° C. (29·2° F.)
Atmospheric pressure 751 mm. (29·6 ins.)
Total volume of flask 333 c.c. (20·32 cub. in.)
Volume occupied by the wort 120 “ ( 7·32 ” )
December 8th.
Volume of gas analyzed 27·6 c.c. (1·68 cub. ins.)
After treatment with potash 27·4 c.c. (1·67 “ )
” “ pyrogallol 22·4 ” (1·36 “ )
Oxygen 5·0 c.c. (0·305 cub. in.)
Composition of the gas:— Per cent.
Oxygen 18·25
Nitrogen 81·57
The formula which we deduced above (§ II.) allows us to conclude that at the temperature of 8° C. (46·4° F.), which was the temperature at which the wort was saturated before the analysis given above, the quantity of oxygen in solution in the 120 c.c. (4·2 fl. oz.) of wort was 0·84 c.c. (0·051 cub. in.).
At the moment when the flask was closed, the total volume of oxygen, calculated to zero and 760 mm. (30 in.) pressure, was 44·73 c.c. (2·729 cub. in.).
At the moment when the analysis was finished, the volume of oxygen was calculated to the same conditions of temperature and pressure, 38·86 c.c. (2·355 cub. in.); 5·87 c.c. (0·374 cub. in.) has, therefore, disappeared. Now, as there is 0·84 c.c. (0·051 cub. in.) in solution, there has, consequently, been an absorption, by combination with 120 c.c. of wort, of 5·03 c.c. (0·32 cub. in.) of oxygen, or 41·7 c.c. per litre (11·6 cub. ins. per gallon).
_Second Experiment._—In a similar experiment, in which, however, the flask was kept for five days at a rigorously constant temperature of 55° C. (131° F.), day and night, and in which the supernatant air was not shaken up with the wort, we found—
Volume of gas analyzed 28·5
After treatment with potash 28·3
“ ” “ pyrogallol 23·0
Oxygen 5·3
Composition of the gas:— Per cent.
Oxygen 18·6
Nitrogen 81·4
Total oxygen at first 29·40
“ ” remaining 26·04
“ ” that has disappeared 3·36
“ ” in solution 0·54
“ ” in combination 2·82
Or per litre, 35·2 c.c. (9·8 cub. ins. per gallon).
The colour of the wort in this experiment had become sensibly similar to that of the wort in the preceding experiment.
_Third Experiment._—In another experiment we left the flask, for the same length of time again, after it had been refilled with air and reclosed, at a temperature which varied between 2° and 4°C. (35·6° and 39·2° F.). In this case we found—
Volume of air analyzed 27·8
After the action of potash 27·8
After pyrogallic acid 22·3
Oxygen 5·5
Composition of the gas:— Per cent.
Oxygen 19·7
Nitrogen 80·3
Total oxygen at first 29·40 c.c.
“ ” remaining 27·58
“ ” that has disappeared 1·82
“ ” in solution 0·44
“ ” in combination 1·38
Or per litre, 17·20 c.c. (4·8 cub. ins. per gallon).
In this last experiment the wort was scarcely darker in colour. Its colour, compared with that of wort cooled on the coolers in the brewery, was slightly darker; but the difference, although it existed, was scarcely appreciable. We shall revert to this fact, which is of importance, presently.
_Fourth Experiment._—The following series of experiments were undertaken to enable us to form some idea of the rapidity with which oxygen is absorbed by wort.
We employed three flasks. A, B, C, of the following capacities:—
A = 234
B = 214
C = 203
into which we introduced the following quantities of wort (boiled wort, without air):—
Into A 96 c.c.
“ B 84 ”
“ C 84 ”
The necks of the flasks were then drawn out and sealed in a flame, the liquid being at a temperature of 5° C. (41° F.). The flasks were then placed in a hot-water bath and kept at 100° C. (212° F.) for a quarter of an hour. The flask A was repeatedly shaken during cooling, as also was the flask B, this being omitted in the case of the flask C.
The contents of flask A were submitted to analysis as soon as it was quite cooled—that is to say, in about three hours. The analysis of contents of B and C was delayed for about twenty-four hours. We took the precaution of not commencing the analysis before we had shaken the flasks for a few minutes, so that the wort in all of them might be saturated at a fixed temperature, and thus enable us to ascertain the exact quantity of oxygen in solution.
The analyses showed that the worts in the three flasks contained:—
Flask A, oxygen in combination, per litre 20 c.c.
“ B, ” “ ” 21·4 c.c.
“ C, ” “ ” 16·8 c.c.
Several facts may be deduced from these experiments: the shaking up of the wort with air has a marked effect on the absorption; a very appreciable absorption immediately follows the shaking up of the wort when warm; whereas, in the case of cold wort that has remained undisturbed, the absorption takes place slowly.
The results of the preceding experiments plainly show that the wort, which is very hot when it comes on to the coolers, where it remains for several hours, must absorb an appreciable quantity of oxygen by combination; but these same experiments teach us nothing definite concerning the volume of oxygen that is actually absorbed. We can only gather from the remark which concludes the third experiment given above, that the total quantity of oxygen absorbed by the wort in Tourtel’s brewery, during the time that it remains on the coolers, must be less than 17 c.c. per litre (4·7 cubic inches per gallon), inasmuch as the coloration effected by combined oxygen in the proportion of 17 c.c. per litre was considerably greater than that of the wort taken from the backs in the brewery.
If we knew the curve of cooling on the Tourtonville coolers we might easily, in experiments conducted in our laboratory, assimilate the conditions of our experiments to those of the oxidation of the wort in the brewery, by exposing wort in contact with air in closed flasks to temperatures varying according to the indications of the curve in question. For this purpose, we induced M. Calmettes to study the process of cooling upon the coolers at Tantonville. In Fig. 84 the figures found in one of that gentleman’s experiments are given.
The abscissæ represent the time expressed in hours; the ordinates, the degrees of temperature. The exterior temperature was 0° C. (32° F.); the atmosphere was calm. The wort was pumped on to the coolers at 5.20 p.m., its temperature then being 85° C. (185° F.), and the operation of pumping lasted from 5.20 to 5.30 p.m. The first determination was made at 5.30 p.m., and was repeated every ten minutes until 7.30 p.m. Between 7.30 and 8.30 p.m. it was repeated every twenty minutes; after that, it was repeated every half-hour until 2 a.m., when the wort went down to the fermenting vessels. The mean depth of the wort was 8·5 centimetres (3·1 inches).
[Illustration: Fig. 84. Curve of cooling of the wort on the coolers (December 18th, 1875).]
Having determined the rate of cooling in the brewery, we made the following experiment: a known quantity of wort from the copper—deprived, consequently, of oxygen—in the same condition as when it comes on the coolers, was put into a graduated, cylindrical vessel, which was then closed with an india-rubber cork, and placed immediately, without being shaken, in a hot water bath at 85° C. (185° F.). Another vessel similar to the preceding one, and having a thermometer passed through the cork, and immersed in the wort, enabled us to observe the temperature. The temperature was gradually reduced, in exact accordance with the data of the preceding curve, until the water, in the course of eight hours and a half, was brought down to 10° C. (50° F.). It is true, that we cannot pretend to have realized all the conditions of the coolers, in this manner, but we approached them very nearly; moreover, it was an approximation rather than a rigorous determination that we desired to obtain. We then collected over mercury the air which remained in the flask, and analyzed it very carefully; at the same time, with Schützenberger’s apparatus, we determined the oxygen held in solution in the wort so treated. From the results thus obtained we easily found the quantity of oxygen that had disappeared—that is, the oxygen which the wort had acquired from the atmosphere of the flask, and which had combined with the oxidizable matters of the wort.
The volume of the flask being 815 c.c., that of the wort 391 c.c., and the depth of the liquid 8 cm., we found an absorption by combination of 9·49 c.c. of oxygen per litre of wort (2·63 cub. ins. per gallon). Another flask treated in the same manner gave us similar results.
As the oxygen in solution has so great an influence on fermentation, it is important that we should, likewise, know the effect produced by the oxygen in combination. The following considerations and experiments may throw some light on this subject:—
We have already remarked that natural saccharine worts oxidize, and acquire colour in contact with air, and that this coloration disappears when these worts are caused to ferment. This furnishes one presumption, that the oxygen in combination disappears then, from, being abstracted by the ferment. A similar phenomenon is observable in the case of wort. After having acquired a marked dark shade by remaining in contact with pure air, it loses this colour very appreciably during fermentation; and if the wort does not quite regain the colour which it originally had when it came from the copper, this circumstance is probably owing to the fact that the quantity of oxygen in combination with the wort is larger than that which is abstracted by the yeast. We have seen that yeast absorbs oxygen, since, in the case of a saccharine wort, more or less saturated with oxygen in solution, when fermentation commences, the first effect of the ferment is to cause that oxygen to combine with its own substance. We should, therefore, expect to find the oxygen in combination, as well as that held in solution, in wort, abstracted by the yeast and contributing to the activity of fermentation. As a matter of fact, this is proved by direct experiments, for the fermentation of a wort that has oxidized in contact with air, or of one from which all the oxygen that was held in solution in it has disappeared by direct combination, is much more easy, rapid, and complete than the fermentation of the same wort when it contains no oxygen, whether free or combined. These experiments were as follows: we boiled some _copper wort_ in a large double-necked flask, like those shown in Fig. 73; all the air being expelled, pure air was allowed to enter the flask; and when the wort was cool it was saturated with this air, by being shaken briskly for a quarter of an hour. The wort was then forced by a pressure of air, applied to the extremity of the S-shaped tube, into smaller flasks, similar to the preceding ones; these we filled completely, and then plunged the end of their sinuous tubes under mercury. After waiting for two or three days, a longer time than was required for the oxygen in solution to enter into combination—a fact which we confirmed by means of a similar flask, which served as a standard—we caused the wort, so prepared, to ferment in the flasks, and side by side, for the sake of comparison, some _copper wort_ that contained no air in solution or combination.
In other experiments we operated on pure wort, saturated with oxygen in combination, by being allowed to remain for one year in an open flask in contact with pure air. This wort was deprived of air in solution by a protracted boiling over mercury. It was then pitched, out of contact of air, with an old yeast. The yeast underwent no development at all, a proof that oxygen in combination cannot act like oxygen that is free, or simply in solution, in effecting the revival of the yeast; nevertheless, after the revival has been once started by means of a small quantity of air, fermentation declares itself with much greater facility than in the case of copper wort, placed under the same conditions, but deprived of oxygen in combination.
§ V. On the Influence of Oxygen in Combination on the Clarification of Wort.
Oxygen in combination has another effect which it is essentially important to point out, for it concerns the clarification of beer. One of the most valued properties of this beverage is its limpidity and brilliancy. We know from the results of the fourth experiment in the preceding paragraph that in the case of a wort shaken up when hot with air, and examined as soon as cold, that is, after an interval of only three hours, we find a notable volume of oxygen to have been absorbed by combination; in the experiment to which we allude, this volume was not less than 20 c.c. of oxygen per litre of wort. The shaking up of the wort when cold with air saturated it with oxygen in solution, but the quantity of oxygen which under these conditions entered into combination, in the course of three hours, is insignificant, although saturation by solution may be attained in the course of one minute’s shaking. If two samples of the same wort are shaken up with air, one of them being hot and the other cold, and both filtered after having been left undisturbed for twenty-four hours, or even immediately after the agitation, we cannot fail to be struck with the great difference that they will present in point of brightness. The wort that was shaken up hot will have more colour, and will be brilliant; the other will be turbid, and will not become clear for five or six days, when left to itself in contact with air and filtered again. This explains a fact that may be easily verified in practice: Boiled wort, if cooled down suddenly, or slowly but out of contact with air, or shaken up cold in contact with air, is opaque when filtered; whilst the same wort, cooled down on the coolers where it has taken a certain quantity of oxygen into combination, generally passes through the filter very bright. The intelligent brewer is uneasy when this is not the case, for it cannot be denied that the easy clarification of wort has a favourable influence on the easy clarification of beer.
It would, nevertheless, be a grave error to suppose that the clarification of beer must necessarily follow that of wort, and we may be permitted to make a digression here on the subject, to prove this statement.
On February 3rd, 1874, we brewed 2 hectolitres (44 gallons) of beer. The boiling wort, hops and all, was run into a vessel like that represented in Fig. 80, but provided in addition with a false bottom, pierced with holes and fixed at 1 centimetre (0·39 inch) above the true bottom of the vessel; this was meant to retain the spent hops. The temperature of the wort in the vessel after it was filled, February 3rd, 4 p.m., was 90° C. (194° F.), that of the room was 10° C. (50° F.). We permitted the wort to cool down gently, without running cold water over the vessel. The wort indicated a density of 14° Balling.
The following temperatures were taken:—
Temp. of Temp. of Wort. Room.
Feb. 4, 11 a.m. 38° C. (100·4° F.) 9° C. (48·2° F.)
7 p.m. 30° C. ( 86° F.) 9° C. (48·2° F.)
11.30 p.m. 26·3° C. ( 79·3° F.) 9° C. (48·2° F.)
Feb. 5, 9 a.m. 21° C. ( 69·8° F.) 8° C. (46·4° F.)
12 a.m. 19·75° C. ( 66·6° F.) 8° C. (46·4° F.)
4 p.m. 18° C. ( 64·4° F.) 8·5° C. (47·3° F.)
Feb. 6, 11 a.m. 14° C. ( 57·2° F.) 8° C. (46·4° F.)
Feb. 7, 2 p.m. 11° C. ( 51·8° F.) 7° C. (44·6° F.)
At the end of this time the wort drawn from the smaller tap half-way up the vessel had already become very bright, although it was taken from the bulk of the liquid above the deposit of hops.
On February 8th the temperature of the wort was 9·5° C. (49·1° F.), and that of the room 5° C. (41° F.); the wort was again very bright. Taken from the small tap and tested by Schützenberger’s process it gave no evidence of free oxygen in solution, although its surface was in contact with air. It continued absolutely pure, the arrangements of our vessel, as we have already explained, allowing only such air to enter as was first deprived of its disturbing germs.
Not till February 12th, after we had again determined the purity and brilliant clearness of the wort, a brilliancy which we can compare with nothing so well as Cognac, without the faintest trace of cloudiness, did we set it to ferment in a vessel similar to that in which it had cooled, but without the false bottom. In the process of transfer we effected its aeration by causing it to fall on a small inverted tinned iron capsule some 4 or 5 centimetres (1-½ to 2 inches) in diameter. By this arrangement the wort took up air to the extent of rather more than a third of its saturate capacity, that is to say, by spreading over the capsule, and falling from it in a kind of sheet, it absorbed a volume of oxygen more than a third of the total amount of oxygen which it was capable of absorbing at the existing temperature; this was 12° C. (53·6° F.) at the moment when the wort was drawn off. The pitching was accomplished with a 6-litre flask containing about 4 litres (7·04 pints) of beer that had been in “low” fermentation from February 3rd. The beer was cleansed on February 24th, and had a density of 5-1/4° Balling. We collected 2·345 kilos (75·39 oz. troy) of yeast, containing 56 per cent., that is, 1·313 kilos (42·21 oz. troy) of pressed yeast, containing 36·7 per cent. of yeast dried at 100° C. (212° F.), that is 482 grammes (15·49 oz. troy) for the brew, which would give 241 grammes (7·748 oz. troy) of yeast formed per hectolitre (22 gallons).
The beer was turbid when drawn off, and the small glassful that we removed did not brighten in twenty-four or even forty-eight hours. The samples for some days previously had been in the same condition. The yeast existed as a fine deposit without any straggling yeast about the sides. The want of brightness was dependent rather on spurious colour than on any actual turbidity. We may here remark that if in the preceding experiment the wort had taken up oxygen into combination as well as into solution at the time that it was aerated, the other conditions being the same, the beer would have been bright and better.
It follows from this experiment that a wort may be _perfectly bright_ at the moment when it is pitched, yet fail to produce a beer which shall be bright when racked, or one that will brighten subsequently otherwise than with great difficulty. We may add that when we repeated this same experiment, cooling the wort, however, as rapidly as the conditions of our apparatus permitted, and employing iced water, the beer appeared very nearly bright when it was racked, and brightened pretty quickly in cask and in bottle. The total duration of cooling was not longer than two hours.
The question here arises what part does the oxygen combined with wort play in the clarification of the latter, or in the clarification of beer? Although it may be difficult to give a definite answer to this question, we must bear in mind that in cases where the beer brightens best, if we examine it under the microscope during fermentation, we see, besides the clusters of yeast-cells, floating amorphous particles, which are larger and more compact than those to which the turbidity of worts and muddy beers is due, a circumstance which should lead us to suppose that the oxygen in combination with the wort has the effect of modifying the nature of the amorphous deposit which is produced during the fermentation of the wort. During boiling, the hop yields to the wort a variety of resinous, odorous, and astringent substances, which, for the most part, are held in solution by the presence of sugar and dextrin. At the moment when, under the influence of the yeast, which is itself more or less oxidized, the sugar becomes transformed into alcohol and carbonic acid, a portion of the bitter and resinous matters of the hop becomes insoluble and remains in a state of suspension in the liquid. It is very probable that at this point it is when the combined oxygen assumes its function of modifying the physical structure of these insoluble particles, agglomerating them, so that they become more easily deposited.[179]
Moreover, oxidation tends to form a special precipitate in the wort, which precipitate contributes towards the collection and deposition of the very fine particles suspended in the wort, by a mechanical action, similar to that which we notice in fining operations. On the coolers an effect of this kind is produced. The wort in the copper contains insoluble matters which pass on to the coolers. Very bright when boiling, it grows turbid as it cools, and then contains two kinds of insoluble substances: 1. Substances insoluble alike in the hot and cold liquid, some of which even, as we have just seen, are formed under the influence of heat and air: all these substances precipitating rapidly to the bottom of the vessels. 2. Very fine particles insoluble in the cold, but soluble in the hot liquid, appearing as the wort cools down, and giving it a milky appearance. If the air does not come into play they remain in suspension for an indefinite time, so to say. Wort taken boiling from the copper and cooled down, therefore, forms a considerable deposit at the bottom of the bottles. Now, if we put this wort into bottles without filling them, putting into some only the milky wort from above the deposit, and into others the same wort along with some of the deposit, then raise it to 100° C. (212° F.), and before it has time to cool down shake it up with air a good many times, it will be readily seen that the wort in the bottles containing the deposit will brighten more rapidly and satisfactorily than those in the bottles without the deposit. The deposits which are insoluble in the copper have, therefore, an influence on the clarification. We must add, however, that this influence cannot be compared with that of direct oxidation.
The “turning out” of the wort and its stay upon the coolers to a certain extent exhibit the different conditions which take part in its clarification, inasmuch as the wort charged with its insoluble matters is run off very hot, and with more or less violence against the external air.
§ VI.—Application of the Principles of the New Process of Brewing with the Use of Limited Quantities of Air.
We have now an idea of the quantities of oxygen which occur, free or combined, in the actual processes of manufacture. We know, moreover, that an excess of air may be injurious, especially to the aroma of the beer, and to that quality which consumers prize so highly, which goes by the name of _bouche_. It must, therefore, be important to ascertain whether in existing processes the proportion of active oxygen may not be excessive.
The best practical means of determining this would consist in comparing the products of different processes with progressively increasing access of air, starting from none at all, as in the case of cooling in the presence of an atmosphere of carbonic acid gas. The following arrangement (Fig. 85) permits us to realize these conditions:—
[Illustration: Fig. 85.]
The wort brought to a temperature between 75° and 80° C. (167° and 176° F.) in the double-bottomed vessel C, passes by the tube _a b_ into a refrigerator, such as Baudelot’s, for example, but acting in an inverse manner to the ordinary mode of using Baudelot’s; that is to say, the wort is made to circulate inside the tubes, whilst the cold water plays on the outside.[180] The wort when cooled, its temperature being indicated by a thermometer _c_, passes down by the tube _c_DD to fill the fermenting vessel A. This vessel is made of tinned iron, or, better still, tinned copper, and has a cover provided with a man-hole and eye-hole; _m n_ one of the tubes for the circulation of air during fermentation; its connecting-tube is not represented, it would be behind the vessel.
At the point _d_ there is a pipe for admission of pure air; this is represented on a larger scale at T. The wort, as it runs through the large tube, carries with it air from outside, and this air is calcined on its way in by means of a flame which plays on the copper tube through which it passes. This arrangement supplies a third or more of the total quantity of oxygen that the wort is capable of acquiring by solution at the temperature at which we work.
F represents the arrangement of the reversed funnel in which the tube _m n_ terminates. Its mouth is closed with cotton-wool held in place between two pieces of wire gauze, for the purpose of purifying the air that enters by it into the fermenting vessel during fermentation.
_v_ is an entrance tap for steam, by means of which the vessel and refrigerator are cleansed from all extraneous germs before each fermentation, and before the wort passes into the refrigerator.
When the fermenting vessel A is at work, we may start a fermentation in a second vessel in the following manner: opening a small tap situated at about a third of the height of the vessel, we pass a few litres of the fermenting beer into a can of tinned copper, previously purified by a current of steam, and filled with pure air. This can is then emptied into the fresh vessel, an operation of no difficulty, since we have merely to connect the tap of the can with the small tap of the vessel, and lastly, the vessel is filled with wort, which then mixes with the fermenting liquid. These various manipulations, it is evident, are performed under conditions of complete purity, without the slightest contact of the liquids either with the exterior air or with utensils contaminated by disturbing germs.[181]
It is seldom that an industry adopts at once in their entirety new practices which would necessitate a re-arrangement of plant, and the process of which we are speaking would require such re-arrangement, as far as the fermenting vessels and the method of cooling the wort are concerned. The new process would, however, be of great value if once introduced, simply for the manufacture of pure ferment and pure wort, or even for that of pure ferment alone. In other words, we might retain the ordinary methods employed in low fermentation, use the same method of cooling or the new one, the same fermenting vessels, and the process of fermentation at low temperatures; the yeast, however, would be prepared in a state of purity in the closed vessel which we have described, collected in those vessels, aerated, and then employed after the old-established custom; better still, the pitching might be performed with beer in the act of undergoing pure fermentation.
Above the fermenting-stage there might be arranged a room for the vessels used in the new process, from which the pure beer could be run for pitching purposes into the large tuns in the brewery below. It is true that beer prepared in this manner would not be perfectly pure, but from the results which have been obtained by working on this system, there is no doubt that it would possess keeping qualities far superior to those of beer made with ordinary yeast, even supposing that beer to have been treated with every possible precaution, and to be as pure as any produced in the best regulated breweries.
In the month of September, 1874, we conducted an experiment at Tantonville, in a closed vessel capable of holding 6 hectolitres (132 gallons). The deposit of yeast served to pitch an open vessel, the wort of which had, moreover, been cooled under conditions of purity. The cooling had been effected by means of the Baudelot refrigerator, represented in Fig. 85, the wort in the closed vessel having been similarly treated. For shortness sake, we may designate the closed vessel and its beer by the letter K, and use the letter M for the open vessel and its beer, and T for the corresponding beer of the brewery. The vessel K was pitched on September 4th, and racked on September 17th, the beer then showing a density of 5·5° Balling.
The beers K and M were sent to Paris at the same time as some barrels of the beer T, brewed by the ordinary process; and samples of these different beers, which arrived on October 22nd, were procured from five different cafés for purposes of examination.
The beer M did not suffer by comparison with the beer T. The similarity between the flavours of these two was so close as to puzzle even experienced judges. In both cases the beer was brilliantly clear. In two cafés the beer M was even preferred to T, being considered softer on the palate (_moelleuse_) and of more decided character (_corsée_) than T, a circumstance which may be explained by the fact that its wort had been less aerated.
The beer K, although very clear and bright, was considered inferior to M, but the sole reason of this was that at the date when it was tasted—November 3rd—it did not froth. As we have already remarked, a peculiarity of the beers made in closed vessels is that their secondary fermentation takes a longer time to develop. The yeast held in suspension in the beer, at the moment when it is drawn off, is, in the case of all beers, the yeast of a supplementary fermentation, if we may use that expression. In the ordinary process of brewing, this yeast, in consequence of the greater aeration of the wort at the commencement of fermentation, is more active, or, rather, more ready to revive and multiply than is that which develops in closed vessels. If the barrels of the K beer had been tapped on the 12th or 15th of November, instead of on the 3rd, it is probable that they would have contained as much carbonic acid gas as the beer M contained at the earlier date. This delay in the resumption of fermentation, which characterizes beer made in closed vessels, is an advantage, inasmuch as it facilitates the transmission of the beer to long distances, besides giving us the smallest deposits of yeast in cask or bottle, as we have already pointed out.
In comparing the keeping qualities of the beer M and the beer T (the latter being the brewery beer), we made the following observations:—[182]
On November 25th we began to detect in the brewery beer an unsound flavour; a large deposit, too, had formed; the beer had lost its brilliancy, and frothed enormously. The deposit swarmed with diseased ferments, especially those represented in Nos. 1 and 7 of Plate I. The beer M, on the contrary, was in brilliant condition, with an insignificant deposit, and an ordinary froth, if anything, rather small, and beautifully bright.
On December 3rd the beer M was still good, very clear, and in excellent preservation; it was considered by professional brewers as remarkably sound.
December 22nd, the same beer M was still very bright and good.
January 20th, the beer was still bright; for the first time, however, we detected in the deposit in the bottles, which was still small, the filaments of turned beer. This unsoundness was in its earliest stage. Now, comparing the relative unsoundness of the two beers, we see that M kept at least two months longer than the corresponding brewery beer. This example shows us that as far as the keeping powers and the quality of beer are concerned, the existing process would gain considerably by the employment of pure wort and pure ferment; and, indeed, it seems likely that the new process may be introduced into breweries with this object in view.
In the course of the summer of 1875 we made the following observations on the keeping qualities of a beer brewed on the new system, all the details of which had been rigorously carried out. The beer brewed at Tantonville during the months of June and July, at a temperature of 13° C. (55·4° F.), in 50-litre and 80-litre casks (11 and 18-gallon), had been sent by slow trains to Arbois (Jura), where we were staying for a time. The temperature of the wine cellars in which these barrels were stored was, on June 1st, 12·5° C. (54·5° F.); this rose gradually until September 1st, when it attained 18° C. (64·4° F.). In this cellar the brewery beer, brewed in the ordinary way, underwent change in the course of fifteen days or three weeks, whilst the beer brewed on the new system remained sound for several months. It is true that some of the barrels lost their frothiness, and that the beer in them underwent a peculiar vinous change, but these effects in no way depend on the conditions peculiar to the new process.
Comparing the beers K, M, T, of which we have been speaking, we see that, however useful the aeration and oxidation of the wort may be in quickening fermentation and facilitating clarification, yet it is by no means indispensable to the success of our operations that we should introduce into our worts large quantities of oxygen, whether by solution or combination. Beyond a certain limit—a limit which is undoubtedly overstepped in the existing process—oxygen is injurious to the palate characteristics and aroma of beer.
These comparisons have proved to us that the new process can be applied to wort aerated to the third of its saturate-capacity for oxygen, and pitched with a good “low” yeast, taken from the fermentation of a wort aerated in the same way, and that the beers thus obtained not only possess vastly superior keeping properties, but are equal in quality and superior in palate-fulness to beers brewed with the same wort on the existing system. We should be perfectly justified in forming this conclusion as to the _strength_[183] of the beer furnished by the new process, even if on tasting it we found that the new beer M was merely equal in strength to Tourtel’s beer brewed in the ordinary manner, since the wort in the new process, other conditions being the same, is weaker than the same wort treated in the usual way, from not having undergone that evaporation on the coolers which concentrates it. If we were to restore to the concentrated wort of ordinary brewing all the water lost by it through evaporation, the beer that we should obtain would be sensibly weakened.[184]
One thing, however, is that we must employ good varieties of “low” yeast. We have seen how the employment of certain forms of yeast renders the clarification of beers difficult, as well as extremely slow, and almost prevents their falling bright at the end of fermentation. These yeasts, moreover, frequently impart to beer a peculiar yeast-bitten flavour, which does not disappear even after a prolonged stay in cask. Even repeated growth of these yeasts, whether in closed or in open vessels, and no matter what quantity of air we may supply them with before fermentation, seems to have no effect in changing their character. The only thing we can do with these varieties of yeast is to get rid of them with all speed, and to replace them with others.
Notwithstanding the comparative success that has attended various trials of the new process on the commercial scale, that process has not yet been practically adopted: and here we must bear in mind that we have not to deal with any casual invention or mechanical improvement that could be introduced all at once into the working of a brewery; we are dealing with operations of considerable delicacy, which necessitate the adoption of a special plant to carry them out. Under such conditions time and labour are required to effect a change in the established processes of a great industry. This, however, cannot diminish the confidence that we have in the future of our process, and it is our hope that the same confidence will be shared in by all those who may give this work an attentive perusal.
Footnote 162:
M. Galland, a brewer in Maxéville, near Nancy, published with his name, in November, 1875, a pamphlet, which was reproduced in the brewing journals of that date, bearing the title, _It is said, “the air being impure, let us exclude it;” I say, “The air being impure, let us purify it.”_ These two aphorisms, together or apart, constitute the essential novelty of my researches on beer, and M. Galland is mistaken in attempting to appropriate the merit of the second alternative (see my note in the _Comptes rendus_ of the 17th November, 1873, and the text of the letters-patent obtained 13th March of that year). M. Galland has devised some arrangements for putting the latter of these two schemes into practice; but it is possible, of course, to effect this in a variety of ways. M. Velten, a brewer in Marseilles, had already accomplished this in his efforts to carry out practically the procedure advocated in the present work.
Footnote 163:
[Non-technically, stirred about.—ED.]
Footnote 164:
As stated in the paragraph on aërobian ferments, in