S. V. Lebedev

Professor of the Tomsk Technological Institute of EMPEROR NICHOLAS II.

CONTINUOUS

ALCOHOLIC FERMENTATION

(An experimental study).

Tomsk.
Partnership “Printing House of S. P. Yakovlev,” Makarovsky Lane, own building No. 4.
1915.

Preface.

In many branches of modern technology there are certain procedures and methods of work that have a place not because they are at present the most correct and expedient, but chiefly because contemporary practice in such industries has inherited them from previous generations, often from the time of hoary antiquity.

Thus, these are a kind of survivals, whose persistence in most cases is determined by the fact that, over time, these methods of work did not remain unchanged, but were to some extent improved, making use, for their development, of the advances attained in the study of those phenomena and forces of nature on whose application they are based.

Such survivals, despite their undoubted erroneousness from the standpoint of present-day possibilities, nevertheless often contain much that is valuable, put into them by the collective labor of a long succession of generations following one another.

In the respect indicated, in the worst conditions are found those industries whose origin dates back to epochs of the lower stages of human development, when man had to resolve, by the most primitive means, many varied and complex questions determined by the striving to secure his existence. From these strivings, in the course of time, arose the oldest branches of modern technology, among which also belongs the group of industries of alcoholic fermentation. Despite the extreme complexity of the processes reproduced here, connected with definite manifestations of life, the practice of these industries was able very early to develop its own procedures and methods of work, and moreover so successfully that they have survived to the most recent time.

Major scientific investigations and discoveries of the past century in the field of the study of the phenomena of fermentation had a very significant effect also on the practice of fermentation industries. However, many of the old methods of work not only were preserved, but even at the present time are held very firmly. An example of this is...

— II —

An example of this may be the method of periodic fermentation, which, both formerly and now, has proved to be the only form in which the process of alcoholic fermentation has everywhere been carried out up to the present day.

Whether this periodicity is a logical consequence of the conditions and tasks of fermentation-production technology, or whether it is merely an accident and a survival—this is a question which, as such, until quite recently remained entirely untouched. Meanwhile, it may be regarded as a generally known fact that any production gains considerably in economy of labor and in many other respects if the processes composing it proceed continuously. In view of this, it seems of interest: first, to establish the possibility of placing the fermentation process under such conditions as would allow it to be reproduced continuously and, at the same time, for an indefinitely long time; and, secondly, to ascertain how these conditions are reflected in the course of the fermentation process, in the activity and properties of the yeast, and also in the quantity and quality of the resulting fermentation product.

Obtaining appropriate answers to the questions enumerated was the aim of the present investigation.

S. Lebedev.

Tomsk,

1 June 1914.

CHAPTER I.

Periodic Fermentation and Its Particular Features

Although it is one of the most characteristic features of the products of the technology of alcoholic fermentation, ethyl alcohol is nevertheless only one of the many essential constituent parts of these products; therefore the reaction by which alcohol is formed by no means embraces all the complex and varied processes caused by the interaction of the yeast and the fermenting substrate. In view of this, in fermentation one must take into account many factors capable of influencing the yeast, the fermenting solution, and their interaction with one another. The planned regulation of the influence of all such factors constitutes the problem of controlling fermentation.

But the influence of each factor is determined by the conditions under which it manifests itself, and these conditions in fermentation are to a considerable extent governed by the chosen method of fermentation.

The simplest old method, still widely used up to the present time, and at the same time the starting point for all existing methods, is the method of periodic fermentation in vats, casks, bottles, and other fermentation vessels adopted in practice.

Characteristic of this method are: 1) the periodicity of operation; 2) confinement to one place, in the sense that fermentation from beginning to end is conducted in one and the same vessel; 3) the joint presence of the entire mass of yeast and fermenting substrate throughout the whole time of fermentation; 4) the multiplication of the introduced yeast and, consequently, the unavoidable expenditure of substance of the fermenting substrate on the formation of yeast; 5) the relatively rapid variability of the conditions under which the yeast works in the various stages of fermentation. A clear idea of the course of the fermentation process under the indicated method of fermentation can be given by the curve of Drawing No. 1, constructed according to the average hourly fermentation of sugar over separate periods of fermentation.

The experiment for constructing the curve of Drawing No. 1 was as follows. Into two flasks, each with a capacity of 1/2 liter, 300 cubic centimeters of previously sterilized settled [[unclear: continuation cut off]] were measured...

… beer-brewing wort. Both flasks, with the wort poured into them and closed with cotton plugs, were sterilized twice a day for one hour in flowing steam. Then into one of them, with the tip of a platinum wire, a minimal amount of a pure culture of top-fermentation yeast, race XII, was inoculated. After inoculation the cotton plug of the flask was replaced by a wooden one, equipped with a fermentation lock containing strong sulfuric acid. The flask thus fitted out was shaken slightly, weighed to an accuracy of 0.01 g, and placed in a thermostat at a temperature of \(17.5^\circ\text{C}\).

Subsequently, the weighing of the flask was repeated at definite intervals of time, ranging from 4 to 24 hours depending on the intensity of fermentation. From the loss in weight the amounts of carbon dioxide evolved during the separate periods of fermentation were determined; and on this basis the corresponding amounts of maltose fermented during this time were found. For this purpose the carbon dioxide was converted into sugar according to the equation:

\[ \mathrm{C}_{12}\mathrm{H}_{22}\mathrm{O}_{11} + \mathrm{H}_{2}\mathrm{O} = 4\,\mathrm{C}_{2}\mathrm{H}_{5}(\mathrm{OH}) + 4\,\mathrm{CO}_{2}. \]

Dividing the amounts of maltose fermented during the separate periods of fermentation by the duration of each of them, expressed in hours, and converting these quantities into percentages of the total amount of fermentable extract, we obtain, as a result, the values of the mean hourly fermentability for the separate periods of fermentation. These values are taken as the ordinates of the fermentation curve in drawing No. 1, the abscissas of which are the corresponding times from the start of fermentation, expressed in hours; the moment of yeast inoculation is considered the beginning of fermentation.

The determination of the amount of extract in the fermented sample of the original sweet wort was carried out not in it directly, but in a sample identical with it. For this purpose, in the latter the weight of the wort and the percentage content of extract in it were determined, the latter being established on the basis of a pycnometric measurement of the specific gravity of the filtered wort of this sample.

The fermentation diagram constructed on the basis of what was found and presented in drawing No. 1 clearly indicates that, with this method of fermentation, the fermentation process proceeds very unevenly. Initially, for a relatively long time, fermentation is not at all apparent. During this period yeast accumulates, and only when a sufficient quantity of it has formed does the fermentation process begin to manifest itself.

Average hourly fermentation of sugar, in weight percent of the extract of the initial wort, over separate periods of fermentation.

Curve of average hourly rates of sugar fermentation, in weight percent of the extract of the initial wort; \(t^\circ\) is maintained throughout the entire fermentation at \(11.5^\circ\text{C}\).

Average hourly sugar fermentation in separate periods of fermentation.

Horizontal axis: hours.

Drawing No. 1.

After this, under the influence of the rapid increase in the mass of working yeasts, over a comparatively short interval of time, the intensity of fermentation rises swiftly, reaches a maximum, and quickly falls to a very insignificant value, at whose level it then remains for a long time, slowly moving toward a minimum. The causes of the abrupt transition observed after the highest tension of the force of fermentation lie in the cessation of yeast reproduction and in the gradual disappearance of the fermentable substance, with which is simultaneously connected the continuing accumulation in the fermenting liquid of fermentation products that retard and weaken the work of the yeasts.

In view of the fact that, at each separate moment, the composition, properties, and temperature of the fermenting liquid change under the influence of the advancing process of fermentation, the yeasts of each of the subsequent generations must differ somewhat in their composition, and consequently also in their properties, from the preceding generations. As a result of this, the entire mass of yeasts formed toward the end of fermentation is not something entirely homogeneous and equally resistant in relation to the unfavorable effects of the various factors whose influence is manifested especially strongly toward the end of fermentation. Because of this, separate fractions of the working yeasts begin at different times gradually to weaken, ultimately becoming completely inactive in the sense of alcoholic fermentation.

The fully proven proposition concerning the great capacity of microorganisms, to a certain degree, to adapt themselves to unfavorable conditions may in the present case hold only on a very limited scale, owing to the short duration of the entire fermentation process and to the comparatively rapid change of conditions associated with the alteration of the fermentable medium.

For this reason, a certain part of the mass of yeasts found in the fermenting liquid, not being sufficiently adapted to the conditions of one or another period of fermentation, prove unable to manifest all their activity, which they might have displayed had they, over a longer period of time, adapted themselves to the conditions of the given medium.

As a result, the fermentative power of many yeast cells is weakened. With a low production of alcohol, and consequently of carbonic acid as well, the force carrying the cell upward is thereby insufficient, as a consequence of which the yeast cell settles to the bottom of the fermentation vessel, thus depriving itself of one of the important vital means, namely—move-

movement in the fermenting liquid. Under these conditions, having used up the nutrients of the surrounding medium within the sphere of its action, the yeast cell very soon begins to suffer from a lack of nourishment and from the unfavorable influence of the products of its own vital activity surrounding it. In the end, such a weakened yeast cell not only fails to assist; on the contrary, it impedes the proper course of fermentation, since it begins to enrich the fermenting liquid not so much with ethyl alcohol as with products undesirable for the yeasts themselves, products of the process of their self-digestion.

Thus, in the periodic method of fermentation under consideration, there occurs a gradual, fractionated settling and, in part, dying-off of the yeasts, which thereby become, to a certain extent, inactive.

According to Delbrück¹), during the after-fermentation period there first settle weak yeasts, and then, after a certain interval of time, even the strongest yeasts, which include: first, yeasts with a large reserve of zymase and with little peptonizing capacity; second, yeasts with the thinnest cell membrane—these are young cells of the last generations; and, finally, third, the strongest yeasts, richest in enzymes.

The successive separation of different fractions of yeast can be observed especially clearly in bottom-fermentation brewing, where this phenomenon has practical value and decisive importance for maintaining the purity of fermentation. As is known, from the yeast sediment that forms at the bottom of an exhausted brewing fermentation vat, only yeast of a definite quality and, consequently, of the corresponding generations—located only at a certain height of the settled yeast layer—is taken for further work. All the foregoing leads to the conclusion that fermentation in each individual period is not determined by the activity of the entire mass of available yeasts, but is maintained chiefly by the activity only of certain generations of yeasts, those most adapted to the environmental conditions of the given period of fermentation. Toward the end of the last period, relatively very few active cells remain freely floating in the fermenting liquid. The remaining mass of yeast settles to the bottom, whereby a considerable part of it proves—

¹) “Handb. d. Spiritusfabrikation” Maercker-Delbrück, 8th ed., p. 549.

proves either useless and even harmful during further fermentation for the reasons already indicated. In view of this, the removal from the fermenting liquid of this kind of yeast at a certain period appears expedient, and in other cases even necessary. This occurs when, in the finished product of fermentation, one must take into account not only the quantity of alcohol contained in it, but also the quality and quantity of all the substances present in it in a dissolved state besides alcohol. All these substances are here very important factors in the assessment of the quality of the finished product, since they determine to a considerable degree such decisive aspects of it as taste and aroma. Brewing and winemaking may serve as examples of such a case.

The freeing of the fermenting liquid from a certain portion of yeast may also, in other cases, have the purpose of interrupting the fermentation process at a definite stage of it, beyond which it is undesirable to carry the fermentation. The liberation of the fermenting liquid from the yeast that has settled out can be carried out either by decanting the fermented substrate from the yeast sediment, or by carefully drawing off the latter from the bottom of the fermenting vessel together with a small quantity of liquid carried along with it. The first method of separating yeast is used in ordinary periodic fermentation in brewing in vats and casks, and also in the Grünwald method and in the American method of fermentation; the second method takes place in the fermentation method proposed for brewing by Nathan.

In order to give an idea of how the fermentation process proceeds in each of the named methods, and how they differ in this respect both from one another and from the simplest method of periodic fermentation, the course of which is represented by the diagram of drawing No. 1, we shall pause for a brief characterization of each of them.

In brewing, both bottom and top fermentation are most commonly carried out by a method of fermentation that falls into two independently proceeding stages. The first of them, connected with work in open vats, proceeds relatively quickly and at a temperature higher in comparison with that which occurs in the second stage of fermentation. By the time the desired degree of fermentation is reached, usually corresponding to the fermentation of the greater part of the sugar to be converted into alcohol and carbon dioxide, the main mass of the yeast formed settles to the bottom of the fermenting vessel.

The wort standing above the sediment is carefully poured off from the latter and, together with a relatively small portion of the floating, strongest yeasts vigorously active during the given period of fermentation, is transferred to another vessel. After this, fermentation continues with a certain lowering of the temperature, at first under normal pressure and then under somewhat elevated pressure, caused by the formation of carbon dioxide in the fermenting liquid and by the tight closing of the fermenting vessel for a certain time during this period.

Toward the end of the second stage of fermentation, all insoluble matter, including the yeasts, settles out. The finished product is carefully poured off from the sediment, which is discarded.

For each new portion of wort to be fermented, the yeast is usually taken from the sediment formed in the preceding portion during the first stage of fermentation. For this purpose a definite fraction is selected, corresponding to the middle layer of the yeast sediment. The rest is discarded. The quantity of yeast introduced is such that, for each cell added, from 2 to 6 new ones are formed during fermentation.

In the Grünwald method¹, the entire fermentation process is divided into 4 stages, each of which proceeds in a separate vessel. In this method the yeast, before being pitched, is mixed at 12.5° R. with five percent of the wort being fermented at the same time, and is pitched at 10° R. The first stage of fermentation ends with the appearance of high kräusen, at which point the temperature gradually falls to 8° R. Then the wort is pumped into the next fermenting vessel, where the second stage proceeds, accompanied by a further lowering of the temperature to 6° R.

The settling of the yeast corresponds to the beginning of the third period, upon the onset of which the fermenting liquid is again transferred to a new, already third, vessel. The fourth stage corresponds to the final maturation of the beer and proceeds in casks in the usual manner.

The American method² of fermentation is characterized by the fact that in it fermentation is divided into several periods, each of which, as in the Grünwald method, takes place in a special fermenting vessel, whose capacities differ.

In this method the fermenting room is a multi-story building. On the uppermost of them, usually the fifth, are loc—

cooling apparatuses for the hot wort are located. On the fourth floor are vessels for adding yeast to the wort, after which fermentation here continues for 1–2 days.

From here the wort is directed into the actual fermentation vats, located on the third floor, where the main fermentation takes place. After the latter is completed, the fermenting liquid is transferred to vessels located one floor lower. Here, with a comparatively prolonged holding, the final fermentation and clarification take place, in most cases with the aid of chips. From here the beer, after carbonation, goes on sale.

Nathan’s method of fermentation¹ has as its aim the production of finished beer by bottom fermentation within 8–10 days. The means that make it possible to confine oneself to such a short period are: the use of rarefaction and periodic stirring during fermentation, carried out at 10–13° C. Fermentation takes place exclusively in a single closed vessel, which here has the form of a specially designed apparatus. The latter is provided with a water surface cooler, an agitator, devices for introducing yeast, for passing gases through the liquid and above it, and certain other devices that make it possible to observe and control the course of the fermentation process.

At any moment the fermentation apparatus can be connected with a vacuum, producing rarefaction in it. Operation by this method proceeds as follows: the hot wort comes directly from the mashing department into the fermentation apparatus; here it is cooled under the combined action of the surface cooler, the agitator, and the suction of air over the surface of the cooled wort. When the temperature of the wort has been lowered to 50–60° C., the air is replaced by carbon dioxide. This replacement is intended to eliminate the possibility of excessive multiplication of the yeast, associated with an excessively high degree of fermentation.

After cooling to 10° C., yeast is pitched into the wort by sucking into the apparatus the appropriate quantity of thick yeast, which is then thoroughly stirred with the wort for an hour in order to distribute it uniformly in the liquid. As soon as the increase in pressure, observed on the manometer, indicates the formation of carbon dioxide in the apparatus, the latter is connected with the vacuum, under the constant action

through which the entire fermentation process passes. The stirrer operates periodically. The initially very intensive multiplication of the yeast ceases very quickly.

The settling of the yeast, thanks to the periodically repeated stirring, begins at once and proceeds rapidly. In this process the yeast settling on the conical bottom of the fermenting vessel is removed by suction as it accumulates. By the 6th–7th day of fermentation, almost complete removal of the main mass of yeast is achieved. After this, carbon dioxide begins to be blown through the fermenting liquid. From the top of the apparatus it is drawn off, passed through purifying devices, and again conducted through the wort fermenting in the apparatus. This operation of repeatedly blowing carbon dioxide through the fermented but still unconditioned beer has the purpose of removing from it the bouquet of unripe young beer.

Next, the temperature of the contents in the apparatus is lowered to \(2^\circ\) C., and carbon dioxide is introduced there under a certain pressure to saturate the beer contained in the apparatus, after which it becomes the finished product.

Fermentation under reduced pressure, partly applied in Nathan’s method, is used on a wider scale in the method proposed earlier, in which fermentation under reduced pressure constituted its principal characteristic feature. This is the brewing method called Vakuumgärung¹.

The purpose of the method is to shorten the duration of fermentation, chiefly the period of secondary fermentation.

Thus, the period of main fermentation was carried out in the usual manner in open vats, whereas the secondary fermentation proceeded already in closed metal vessels, enamelled on the inside, with a conical bottom and equipped with pressure gauges, thermometers, vacuum gauges, etc. Secondary fermentation was conducted in this method under a constant reduced pressure of about 50 centimeters of mercury and was completed in 8–10 days. After this, the reduced pressure was eliminated and the beer was left in the same vessel for 3–4 days for the settling of suspended substances, and then saturation with carbon dioxide was carried out.

All the above-described methods of brewing proposed in beer production are characterized by the fact that the entire fermentation process is thereby divided into separate periods, carried out independently. This circumstance is of substantial importance.

If fermentation is conducted from beginning to end in one and the same vessel, with the fermenting substrate and the entire mass of yeast that has formed present together and continuously, then in this case, for each of the fermentation periods, one has to choose not the conditions most suitable for that period, but somewhat different ones—namely those under which the interests of the other periods, most distinct in their requirements from the given one, suffer the least. The reason for this is clear: it lies in the difference between the conditions, and consequently the requirements, of the separate stages of fermentation, and in the impossibility, when fermentation is carried out in only one vessel, of sharply separating the individual periods of fermentation from one another.

By the requirements of one or another period, from the point of view of the fermentation process, one must understand the totality of all conditions that favor the work of the yeast. But the mass of yeast present in the fermenting liquid is not something immutable and homogeneous. It contains within itself vigorous, energetically working cells, as well as weak ones that are dying off, and, finally, dead cells.

The conditions most favorable for fermentation will therefore be those that correspond to the best work of the strongest fraction of a given period. But at the same time it is quite possible that these “best conditions” for the strongest fraction of yeast of that period may simultaneously prove to be the “worst” for the weak ones, as a result of which the latter will perish definitively, having quickly been deprived of life under such conditions. The latter circumstance leads to the fact that these dead and dying yeasts, under the influence of factors both external and inherent in themselves, begin under these conditions to decompose and quickly enrich the fermenting wort with the products of their breakdown. This circumstance is especially undesirable where the quality of the finished product is determined to a considerable extent by its taste and aroma. Therefore, the removal of weak and dying yeasts during fermentation will always take place, to a greater or lesser degree, in all methods of fermentation in brewing. In this respect they all differ from the simplest method of fermentation, in which the fermentation process proceeds exclusively in a single vessel, without separation during fermentation of the yeast that settles out. This simplest method of fermentation is adopted in distilling and, of course, in yeast production. Distilling, as compared with brewing, has a different attitude toward the indicated

the question of separating yeast during fermentation is determined by the following.

The aim of distillery production is alcohol alone, i.e. the most complete possible conversion into it of all the sugar present in the fermenting mash. Taste and aroma, which are connected with the composition of the by-products of fermentation, in this case have absolutely no significance, since the substances that produce these properties, like everything else present in the fermented mash, are separated at the outset from the finished product of fermentation, which is rectified spirit. In view of all this, in distilling there is absolutely no place for the separation, during fermentation, of weak yeasts that gradually settle out.

At first glance such an operation might even appear to contradict the very basic aim of fermentation in distilling—namely, obtaining the greatest quantity of alcohol. Indeed, if the by-products of fermentation, produced more intensively by dying and weak yeasts, have absolutely no negative significance for the aims of distilling, since they do not enter into the finished product, then in that case yeasts gradually leaving the sphere of action, though weakening, may still be regarded as useful workers. True, their productivity is very small, but they nevertheless provide a certain contribution to the overall work. Apparently it is this kind of view that underlies the method of fermentation now adopted in distillery production.

However, the correctness of such a view is far from being decided by the considerations just cited. In the present case what is very essential is, first, the relation of the active yeast fraction, vigorous in the sense of alcohol productivity, to the products produced by weakened and dying yeasts, and, secondly, the relation of the latter to the ethyl alcohol that has formed. If yeasts leaving the sphere of action diminish the useful work of the active mass of yeast, or cause a loss of alcohol, for example by burning it in considerable quantity, then, given a certain scale of these processes, the removal from the fermenting mash of settling yeasts will be unquestionably necessary for distilling as well. In the opposite case, under these conditions, an indifferent attitude toward a certain part of the yeasts present in the fermenting liquid—such as exists at present in distilling—would be a mistake. Thus, there arises

the question, still almost entirely untouched, of the relation of the spent yeast in the fermenting liquid to the alcohol contained in it and to the useful work of the active fraction of the mass of yeast formed during fermentation. Closely connected with this is the need to clarify how many times during fermentation, and at what stages of it, the undesirable portion of the yeast should be separated, and how this may affect the course of fermentation, as well as the quality and quantity of the finished product. The latter already concerns the question of losses of fermentable substance in alcoholic fermentation. By loss or waste is meant the expenditure of fermentable substance on those phenomena and processes connected with fermentation which do not directly lead to the transformation of the initial material into the final products of fermentation. By the latter are meant: extract remaining unfermented, the alcohol formed, and the carbon dioxide released during fermentation, taken in a definite quantitative ratio to the alcohol formed, determined by the sugar present and corresponding to it according to the fermentation equation. In the fermentation of, for example, brewer’s or distiller’s wort, the quantitative ratio of alcohol and CO₂ is determined by the equation for the fermentation of maltose:

\[ \mathrm{C}_{12}\mathrm{H}_{22}\mathrm{O}_{11} + \mathrm{H}_{2}\mathrm{O} = 4\mathrm{C}_{2}\mathrm{H}_{5}(\mathrm{OH}) + 4\mathrm{CO}_{2}. \]

In winemaking, where the fermentable sugar is chiefly glucose, the fermentation equation must be taken in the following form:

\[ \mathrm{C}_{6}\mathrm{H}_{12}\mathrm{O}_{6} = 2\mathrm{C}_{2}\mathrm{H}_{5}(\mathrm{OH}) + 2\mathrm{CO}_{2}. \]

The phenomenon of loss during fermentation is due, to a certain extent, to the known expenditure of fermentable substance on the construction of the bodies of newly formed yeasts and on their maintenance. The fermentation diagram (see drawing No. 1, p. 3) with minimal inoculations indicates that under these conditions a considerable period, in comparison with the duration of the entire fermentation process, is occupied by the accumulation of the yeast mass.

The practice of individual branches of the technology of alcoholic fermentation substantially shortens the duration of the period of yeast accumulation, by taking for inoculation into the fermenting wort not a minimal quantity of them, but considerable masses, ranging approximately from \(1/10\) to \(1/4\) of the entire quantity of yeast that can form by the end of fermentation in the given volume of fermenting wort. In this case, for each subsequent trial the yeast is taken from the preceding one, thereby establishing—

a successive connection is established between the individual samples of the fermenting wort. The addition of a considerable quantity of so-called “mother” yeast is also significant in the sense of reducing the amount of loss, which will be the smaller the more yeast is taken for pitching.

But with the periodicity of the fermentation operation, when the latter is conducted not in the entire mass of liquid intended for fermentation, but in separate, comparatively small quantities of it, treated independently, the expenditure on yeast cannot be completely eliminated even by such quantities of mother yeast as would be such that all the yeast obtained at the end of fermentation in the preceding portion of wort would be introduced wholly into the next portion, equal to it in size and intended for fermentation. The reason for this lies in the fact that, in periodic operation, the yeast in each separate sample of fermenting wort passes through three essentially different stages: it accumulates, develops, and dies off. At the same time, during the interval from the beginning to the end of fermentation, the mass of yeast formed undergoes certain quantitative changes, having its minimum and maximum. The latter does not correspond to the final stage of fermentation, but to some intermediate one. This explains why, in the method of periodic fermentation under factory conditions, even in the very best case, some multiplication of the mother yeast is inevitable, together with a certain loss, associated with it, of the extract of the fermenting wort.

In the practical conditions of individual production facilities employing the technique of alcoholic fermentation, because of many accompanying circumstances and special requirements, the mother yeast, as was indicated, usually amounts to from \(1/10\) to \(1/2\) of its entire quantity obtained by the end of fermentation. Under such conditions the expenditure on yeast already represents a quantity of such magnitude that even the practical side of production may be interested in its fluctuations. A reduction of the expenditure on yeast may stand in a certain relation to an increase in the yield of the finished product.

Thus, the expenditure on yeast is one of the characteristic features of periodic fermentation; the study of it may make it possible to approach more closely a critical assessment of this universally widespread method of fermentation, and therefore this aspect of the phenomenon under consideration should be examined somewhat more closely and in greater detail.

CHAPTER II.

The Consumption and Fluctuations of Its Magnitude in Periodic Fermentation.

The first investigation of alcoholic fermentation from the quantitative side was undertaken by the creator of all modern chemistry—Lavoisier, who came to the conclusion that the essence of this process is, to a considerable extent, exhausted by the splitting of sugar into ethyl alcohol and carbon dioxide.¹) Further investigations in this direction led to the chemical equation of alcoholic fermentation, which was first given by Gay-Lussac.²) With corrections to the chemical formulas of sugar and alcohol, which had initially been taken erroneously, the fermentation equation assumed the following form.³)

\[ \mathrm{C}_{6}\mathrm{H}_{12}\mathrm{O}_{6} = 2\,\mathrm{CO}_{2} + 2\,\mathrm{C}_{2}\mathrm{H}_{5}(\mathrm{OH}). \]

This equality indicates that at that time alcoholic fermentation was regarded as a process in which: first, the fermentable sugar passes completely into alcohol and carbon dioxide, and, second, these fermentation products are always formed in a constant quantitative ratio. But already the experiments of Dubrunfaut⁴) showed that it is practically impossible to attain such yields of fermentation products as theoretically should have been obtained according to Gay-Lussac’s equation. Later, Pasteur’s extensive investigations⁵) definitely established that part of the fermentable sugar is constantly expended on the construction of the substance of the newly arising yeasts and on the formation of by-products of fermentation, some of which Pasteur determined qualitatively and quantitatively.⁶) Elion⁷) proved that

¹) Ann. de chim. et de phys. (3 sér.) 1860, vol. 58, p. 325.
²) Ibid., p. 328.
³) “Die Gärungschemie,” Ad. Mayer. 1906, p. 24.
⁴) Compt. rend. de l’Ac. 1856, vol. 42, p. 945.
⁵) “Mémoire sur la fermentation alcoolique,” Pasteur, Ann. de chim. et de phys. (3 sér.) 1860, p. 323.
⁶) “Études sur le vin,” Pasteur, p. 214.
⁷) “Handb. d. Techn. Myk.,” Lafur IV, p. 373.

part of the fermentable sugar is assimilated by the yeast, and therefore is not converted into the final products of fermentation. Thus it turned out that, during fermentation, sugar is not completely converted into alcohol and carbon dioxide, as was interpreted by the Gay-Lussac equation. Judging from the experiments of Pasteur¹, Jodlbauer², Kosutany³, and others, in the best case only about 95% of all the fermentable sugar passes into alcohol and carbon dioxide. As regards the existence of a constant quantitative ratio between the alcohol and carbon dioxide formed during fermentation, as was assumed from the Gay-Lussac equation, it turned out that this, too, does not always actually occur.

Yeast, under certain conditions, may need oxygen, like higher organisms, whereby carbon dioxide is formed without the corresponding formation of alcohol. Thus Giltay and Aberson⁴ found that during fermentation with vigorous blowing-through of air, only 75% of the fermentable sugar decomposes in the proportions determined by the fermentation equation, whereas without blowing-through, 90% decomposes in this way. Laer⁵ showed that a certain excess of carbon dioxide obtained during fermentation with blowing-through is the result of yeast respiration. Buchner and Rapp⁶ established that the amount of carbon dioxide obtained exclusively at the expense of yeast respiration by the combustion of sugar may, under certain conditions, reach \(1/7\), reckoned on the total amount of fermented sugar. According to the experiments of Jodlbauer⁷, the quantitative ratio between the alcohol and carbon dioxide formed during fermentation is to a certain degree determined by the age of the yeast used for fermentation; moreover, older cultures form relatively less carbon dioxide than cultures of vigorous young yeast, while the data of Lindet and Marsais⁸ indicate a dependence of this same ratio on the phase of fermentation. It appears that at the beginning of the fermentation process the formation of alcohol proceeds relatively more vigorously than at its end.

Another of the causes producing a disturbance of constancy in the ratio of alcohol and carbon dioxide formed during fermentation is also the vital activity of the yeast after the disappearance of all the sugar in the fermenting liquid. According to the experiments of Kayser and Demolon¹), oxidative processes then take place, among other things, causing the formation of carbon dioxide without the corresponding accumulation of alcohol according to the fermentation equation, since in this case the carbon dioxide arises not through the work of zymase, but mainly as a result of the activity of oxidases, the presence of which in yeast has been demonstrated by Tolomei²), Grüss³), Henneberg⁴), Issajew⁵), and others. In Effront’s⁶) opinion, under certain conditions carbon dioxide can be formed in very considerable quantities not only from carbohydrates, but also from proteins.

All that has been set forth above leads to the conclusion that, of the fermentable sugar, only one part is converted into alcohol and carbon dioxide, while another passes into yeast substance as well as into by-products of fermentation; finally, a certain quantity of sugar undergoes transformations leading to the formation of carbon dioxide without a corresponding accumulation of alcohol. Pasteur assumed that, of 100 parts by weight of fermented sugar, 1.0–1.5% is expended on the formation of yeast⁷), and 4.5–6.0% of it goes to by-products⁸), of which 2.5–3.6 parts by weight of glycerin, 0.5–0.7% of succinic acid, and a certain quantity of other substances present in the yeast and in the fermented substrate are obtained.

Thus, according to Pasteur, glycerin and succinic acid—these, as he supposed, constant companions of alcoholic fermentation—are allegedly the chief cause of the loss of sugar to by-products of fermentation. Moreover, the magnitude of this loss, in Pasteur’s opinion, should always fluctuate within the limits given by him. Meanwhile, it was later established that the quantity of by-products may deviate considerably from the limits indicated by Pasteur, which is found

¹) Wochenschr. f. Brauer. 1909, pp. 446–447.
²) “Die Enzyme,” Green—Windisch, 1901, p. 309.
³) Wochenschr. f. Brauer. 1901, p. 310.
⁴) Zeitschr. f. Spir.—Ind. 1904, p. 96.
⁵) Zeitschr. f. physiol. Chem. 1904, p. 132.
⁶) Wochenschr. f. Brauer. 1905, p. 444.
⁷) Ann. de chim. et de phys. 1860 (3 Sér.) 58-m, p. 344.
⁸) Ibid., p. 362.

depending on such factors as: the temperature and rate of fermentation, its phase, the individuality of the sugar being fermented, the race of yeast, and much else.

All of the above, in connection with fluctuations in the quantities of glycerin and succinic acid obtained, led Müller-Thurgau and Wortmann¹ to a view that treats the named products of fermentation as having no direct relation to the sugar being fermented. Therefore the source at whose expense they arise may be not only sugar, but also other substances. In Delbrück’s opinion,² for example, glycerin is formed through the action of yeast lipase at the expense of the transformation of fats. Boussingault³ obtained it from yeast when the latter was left under water at a temperature of 40–41° C. This made it possible to assume the formation of glycerin at the expense of yeast lecithin, discovered in them by Hoppe-Seyler.⁴ In the opinion of Blumenthal and Grüss,⁵ the initial substance for succinic acid, along with carbohydrates, may also be proteins, or rather the products of their decomposition. These same protein-derived products, according to F. Ehrlich,⁶ are the material at whose expense higher alcohols also arise, constituting the principal part of the fusel oil of raw spirits obtained in distillery production. But the chief expenditure of protein decomposition products in alcoholic fermentation is connected with the formation of yeast.

From all that has been set forth above it is clear that the fermentable sugar is expended not only on the formation of ethyl alcohol and carbonic acid, but is also consumed for by-products, in the formation of which certain other substances present in the solution of the fermenting substrate also take part.

While recognizing the presence of yeast during fermentation, Lavoisier⁷ nevertheless considered them to have no significance in the quantitative ratios of alcohol and carbonic acid formed from sugar.

Gay-Lussac was evidently of the same view, judging by the fermentation equation he gave. However Pasteur,⁸ basing himself

in experiments, arrived at different conclusions, considering that the formation of yeast causes a certain consumption of the fermented sugar, the numerical value of which he determined as 1.0–1.5% of the amount of the latter.

In view of the practical impossibility, both with respect to yeast and with respect to certain other by-products of alcoholic fermentation, of strictly separating the consumption of sugar from the consumption of the other constituent parts of the fermentable substrate, it appeared more convenient to connect, by a definite numerical dependence, the entire sum of dissolved substances of the fermentable liquid with the sum of all the products of fermentation.

This was, to a certain extent, done by Balling¹. According to his investigations, 100 parts by weight of fermented extract of brewer’s wort give: 48.391 parts by weight of alcohol, 46.286 of carbon dioxide, and 5.323 of yeast, reckoning the latter as the dry substance contained in it. In grape must, from 100 parts by weight of fermented extract there are obtained 49.977 parts by weight of alcohol, 47.803 of carbon dioxide, and 2.219 of yeast.

Thus, in fermentation under brewing conditions with malt wort, dry yeast substance is obtained in the amount of 10.9 parts by weight of the alcohol formed, whereas in the fermentation of grape must—4.49.

On the basis of the preceding considerations, Balling² gives, instead of Gay-Lussac’s fermentation equation, his own equation, namely

\[ E = e + A + K + H, \]

where the following designations are adopted:

\(E\)—the fermented extract of the original wort;

\(e\)—the extract remaining after fermentation;

\(A\)—the weight amount of alcohol formed;

\(K\)—the weight amount of carbon dioxide, obtained in a constant ratio to the alcohol formed, equal to \(1 : 0.9565\);

\(H\)—the dry substance of the yeast.

Balling’s equation assumes between the alcohol and carbon dioxide formed during fermentation the same ratio as was adopted by Gay-Lussac, but in addition introduces still—

a known expenditure on yeast. In this it is assumed that the alcohol obtained, the carbon dioxide, taken in the indicated ratio to the former, the yeast formed, and the unfermented extract that remains—all this, in sum, must be equal to the extract being fermented.

From a comparison of the equations of Gay-Lussac and Balling it is evident that the latter treats the process of fermentation somewhat more broadly than the former; however, it too is not capable of encompassing the entire totality of phenomena that occur in the process of alcoholic fermentation. One of the reasons for this lies in the incorrectness of Balling’s accepted proposition concerning the constancy of the quantitative ratio of the alcohol and carbon dioxide obtained during fermentation and supposedly formed, at the expense of decomposition and transition into these products, from the fermentable sugar.

Meanwhile, as has already been indicated, this in reality does not occur, since a certain amount of carbon dioxide can be obtained without the formation of alcohol, and moreover at the expense of a certain part not only of the sugar but also of the non-sugar of the fermentable substrate.

In addition, in alcoholic fermentation, as is known, other so-called secondary products of fermentation also occur, some of which, under certain conditions, may be formed even in considerable quantities; examples of these may be glycerin, succinic acid, and higher alcohols. These circumstances, however, are not at all taken into account by Balling’s equation, which, by virtue of this, lacks—judging by its formulation—one or several terms of the equation corresponding to the indicated transformations of the fermentable extract. All these quantities ought to have entered into the equation as components of that part of it in which the products of fermentation obtained are summed.

With regard to yeast, Balling considered it possible to adopt a quite definite and constant value of the expenditure in relation both to the fermentable extract and to the alcohol formed. However, as we have seen, Balling adopted different numerical values of these quantities in brewing and in winemaking.

Thus, Balling, judging from his equation of fermentation, regarded the expenditure on yeast as a quantity whose absolute value may vary depending on the conditions of fermentation, but which, under given conditions, throughout the entire ...

the fermentation process always remains proportional to the quantity of alcohol formed. As to the dependence of the magnitude of the consumption for yeast on many very significant factors, such as: the quantity of yeast pitched, the temperature of fermentation, the concentration of the fermenting substrate, etc., Balling’s equation says nothing. It is quite possible that this was not even included among Balling’s aims when he formulated his equation of fermentation, since the latter was adapted to fairly definite conditions, such as are set in individual cases by one or another industry, e.g., winemaking or brewing, whereby a certain constancy is ensured in the conditions of fermentation and in the final results to which it leads. However, when Balling’s data are checked, certain discrepancies are encountered in them, both with regard to the magnitude itself and to the constancy of the consumption for yeast, even within the limits of only one particular industry. For example, in bottom fermentation in brewing, according to Schönfeld, it turns out that in one series of experiments the consumption for yeast was equal to 5.8%¹), and in another—6.8%²) by weight of all the alcohol formed. Both of the cited figures differ from one another and, moreover, both deviate considerably from the corresponding magnitude given by Balling for these conditions and equal to 10.9% by weight of the alcohol obtained during fermentation. The latter thus proves to be almost twice as large as the corresponding values of Schönfeld.

Judging from the data cited, the quantitative ratios of alcohol, carbon dioxide, and yeast obtained during fermentation, as accepted by Schönfeld³), are much closer to the actual state of affairs than those proposed by Balling. But this is limited only to the conditions of bottom fermentation; when passing from them to top fermentation, already in brewing one has to observe a deviation from Schönfeld’s ratios in the direction of those proposed by Balling⁴).

The difference between top and bottom fermentation consists in the temperature, in the duration of fermentation, and in the degree of multiplication of the yeast. All these circumstances are closely connected with that quantity of fermented extract, by which the consumption for yeast is usually understood.

¹) Wochenschr. f. Brauer. 1910, p. 59.
²) Wochenschr. f. Brauer. 1911, p. 212.
³) Wochenschr. f. Brauer. 1910, p. 59.
⁴) Wochenschr. f. Brauer. 1910, p. 223.

By the latter, according to Balling’s equation, and consequently also according to Schönfeld, one must understand the difference between the amount of fermented extract and the sum of the remaining extract, the alcohol formed, and the corresponding amount of carbonic acid, taken in the ratios of the equation

\[ \mathrm{C_6H_{12}O_6 = 2CO_2 + 2C_2H_5(OH).} \]

With such a formulation, the expenditure for yeast naturally includes that which in reality has nothing in common with it, namely: the consumption of fermentable substance for the formation of by-products of fermentation; for carbon dioxide arising without the corresponding formation of alcohol; and, finally, this also includes loss due to evaporation of alcohol, if fermentation is carried out in open vats. The magnitude of the latter is highly dependent on temperature, on the duration of fermentation, on the concentration of the fermenting wort, and, finally, on the character and thickness of the layer of foam covering the surface of the fermenting liquid. Under the conditions of bottom fermentation in brewing, all these circumstances are such that evaporation of alcohol occurs to a very small extent and has almost no appreciable influence on the quantitative ratio of the fermented extract to the resulting fermentation products. Whereas in top-fermentation brewing, and still more in distilling, conditions are such that evaporation of alcohol already causes appreciable losses here.

According to Durin, mash with an alcohol content of 6–7% lost, with the escaping carbon dioxide and through evaporation, up to 1.5% of the total amount of alcohol.

According to Riess¹), mashes with an alcohol content reaching 10% lost only 1.12% of all the alcohol in carbon dioxide, and still more from surface evaporation. According to Foth²), the loss of alcohol, calculated on the volume of the mash, is 0.2% in a closed vat; in an open one, with air moving in the room and at 20°R, 0.5%; and at 27°R, 0.95% alcohol.

Thus, evaporation of alcohol and its removal by carbon dioxide can very appreciably alter the magnitude of what is often understood, not quite correctly, as the expenditure for yeast. As regards its actual numerical value, it is, of course,

¹) “Handb. d. Spiritusfabrik.” 8 Aufl. p. 678.
²) Ibid., p. 679.

stands in direct relation to the possibility of yeast reproduction under the given fermentation conditions. The latter are very unequal, depending on the totality of everything that determines the course of the fermentation process. Consequently, for example, in bottom-fermentation brewing the quantity of pitched yeast increases by 2.5–3.5 times, while in top-fermentation brewing by 4–8¹) times. The difference in this respect is still sharper when brewing is compared with distillery production, where for one part by weight of yeast pitched into the mash tub, by the end of fermentation 12–16²) parts by weight of yeast are obtained. Approximately the same ratios of the magnitude of the pitching and the yield, namely 8–12, are also found in the air-yeast industry. Consequently, the expenditure on yeast in distilling must be considerably higher than in brewing.

Speaking of expenditure on yeast under strictly defined fermentation conditions in one or another industry, it should also be borne in mind that the magnitude of the yield, or the reproductive capacity of the yeast, is in a certain dependence on the quantity of mother yeast introduced and intended for reproduction. According to practical data, Balcke³) found that, with an increase in the quantity of mother yeast pitched, the quantity of yeast obtained after fermentation also increases to a considerable extent. The reason for this lies in the increase in the number of producing mother cells, so that, taking 6 of them instead of 4, for example, and assuming that each of them gives 3 new ones during fermentation, we obtain that, with an increase in the quantity of mother yeast by 3, the increase in yield reaches \((3 \times 6) - (3 \times 4) = 6\). This circumstance has practical significance in yeast production. However, such a dependence exists only within certain narrow limits of very definite conditions; beyond these limits, an entirely opposite dependence has to be observed.

Reichard and Riel⁴), in factory experiments in brewing, found that, when pitching 8.2, 12.5, and 19.0 Kil. respectively into 25 hectoliters of wort, by the end of fermentation they obtained such quantities—

¹) Wochenschr. f. Brauer. 1911, p. 223.

²) Die Gärungsführung. Delbrück u. Hayduck, 1911, p. 21.

³) Wochenschr. f. Brauer. 1884, p. 385. Cited in the same journal for 1911, p. 233.

⁴) Zeitschr. f. d. ges. Brauwes. 1897, p. 28. Cited in Wochenschr. f. Brauer. 1911, p. 232.

of yeast: 35, 40, and 50 kil., i.e., when the quantity of pitching yeast is increased by 10.8 kil., the yield rises by 15 kil., and, consequently, per 1 kil. of pitching yeast in the three indicated cases there are obtained respectively 4.3, 3.1, and 2.7 kil. of yeast. Similar practical data are also given by Thausing¹ concerning fermentation in brewing, wherein it is shown that 1 liter of pitching yeast, after the completion of the main fermentation, gives the following quantities of pure yeast, expressed in liters, in Table I.

Table I.

Pitching yeast added per 1 hectoliter of wort.	Obtained liters of yeast per 1 hectoliter of wort at breweries: A	Obtained liters of yeast per 1 hectoliter of wort at breweries: B	Obtained liters of yeast per 1 hectoliter of wort at breweries: C	Obtained liters of yeast per 1 hectoliter of wort at breweries: D
1/2 liter . . . . .	4.90	5.96	5.21	4.48
1/3 liter . . . . .	3.32	3.90	3.90	3.00
2/3 liter . . . . .	2.49	2.75	2.68	2.30

Schönfeld’s experiments² lead to the conclusion that, when the amount of yeast inoculum is changed in the ratio 4.1:8.9, the reproductive capacity of a unit of pitching yeast changes in the ratio 2.5:6.0. According to A. Brown,³ when 0.93, 3.75, and 7.44 yeast cells are inoculated per 1 volume of wort, the yield of one pitching cell proves different in all three cases considered and is respectively equal to 26.2, 6.9, and 2.6 cells formed by the end of fermentation.

An even more sharply marked phenomenon appears in the experiments of F. Hayduck and G. Anders,⁴ presented in Table II.

Table II.

Experiment	Inoculation of yeast per 1 liter of wort	Number of cells per unit volume (0.0005 m.m.) At the beginning of fermentation $p$	Number of cells per unit volume (0.0005 m.m.) At the end of fermentation $k$	Productivity of one cell of mother yeast $\dfrac{(p-k)}{k}$
A	0.5 gram	1.1	55	49
A	1 —	2.2	58	25
A	2 —	4.4	60	13
A	4 —	8.8	68	7
A	8 —	17.6	77	4
A	12 —	26.4	94	2.9
A	20 —	44.0	96	1.2
B	10 grams	22	50	1.5
B	20 —	44	61	0.4
B	40 —	88	104	0.2
B	70 —	154	198	0.2
B	100 —	220	230	0.05

Comparing the ratio of the quantities of yeast produced by a unit of inoculum with the ratio of the quantities of yeast inoculated into one and the same volume of fermenting substrate, we obtain the following weight ratios of yeast:

	Inoculated	Obtained
according to Schönfeld . . .	1.0 : 2.3	2.1 : 1.0
“ Brown . . .	1.0 : 8.1	10.1 : 1.0
“ Hayduck . . .	1.0 : 10.0 1.0 : 40.0	30.0 : 1.0 40.8 : 1.0

Thus, it turns out that the reproductive capacity of yeast, other conditions being equal, is the greater the smaller the inoculum, and conversely; and therefore, much or little constant yeast ...

per unit volume of wort, their number toward the end of fermentation in the one case and in the other, other conditions being equal, will be approximately the same. Thus, apparently, such maximum inoculations are possible in which the multiplication of the inoculated yeast is almost completely excluded.

Very close to this is the last case in Hayduck’s experiments, when for 220 yeast cells sown before fermentation, only 10 new cells are obtained after it, i.e., when, owing to the almost complete exclusion of yeast multiplication, the expenditure on them during fermentation becomes very close to zero.

From the foregoing it follows that the expenditure on yeast, apart from other conditions, is determined by the magnitude of the inoculation of mother yeast. By virtue of this, in individual plants for the technology of alcoholic fermentation, which use unequal quantities of mother yeast, the expenditure on yeast must be different. If, nevertheless, certain norms are encountered there concerning the magnitude of the expenditure on yeast, connected by a constant numerical ratio with the quantity of fermented extract or with the alcohol obtained, then the reason for this is quite clear: such norms can be applicable only within very narrow limits of strictly defined fermentation conditions of one or another plant, outside of which they prove to have no place at all.

Still more than the magnitude of the inoculation, the access of air to the fermenting liquid affects the yeast’s capacity for multiplication, and, consequently, also the expenditure during fermentation. Some idea of the magnitude of this influence may be given by a comparison of the quantities of yeast obtained in brewing and in yeast production by the method associated with blowing air through. Delbrück and Hayduck¹ cite the following data, taken from factory practice: in brewing, the greatest amount of pressed yeast that can be obtained by weight of the mash material is about 6%, whereas in air-yeast production, with prolonged and vigorous blowing of air through the fermenting wort, the yeast yield may reach 40% and even more by weight of the mash material. Thus, in the latter case almost 7 times more yeast is obtained than in the former. Of course, a direct comparison

of the figures adduced, and the possibility of any definite conclusions on their basis concerning the influence of blowing air on the growth of yeast does not appear possible. In the present case one must bear in mind the unequal conditions of fermentation in the one production and in the other, both in the sense of temperature, concentration, and composition of the fermenting mash, and also with respect to the capacity for multiplication of those races of yeast that are used in brewing and in yeast production. In the latter, races are employed that possess a high capacity for multiplication, whereas bottom-fermentation brewer’s yeasts, by their nature, prove to be comparatively weakly multiplying. This circumstance, in connection with the low fermentation temperatures adopted in brewing, determines the tendency of brewer’s yeasts to enrich themselves with proteins to a considerably greater extent than is the case with the top-fermentation yeasts of distilling and yeast production. According to Delbrück ¹), the protein content in yeasts obtained in brewing is about 15%; in yeast production, however, about 10%. Taking into account the yields of yeast indicated earlier, one may see that per 100 Kil. of material mashed, the amount of nitrogen assimilated by the yeast in brewing production corresponds to \(0.15 \times 6 = 0.9\) kilograms of proteins, whereas in yeast production it is \(0.10 \times 40 = 4.0\) kilograms. Thus, the blowing of air, together with all the conditions of fermentation in yeast production, which differ markedly from the conditions of fermentation in brewing, leads to an increase by 4–5 times in the amount of nitrogen assimilated by the yeast.

The data cited, owing to the difference in conditions, are comparable only to a certain degree, and therefore can give only a very approximate idea of the magnitude of the influence of blowing on the reproductive capacity of yeast of one and the same race and under identical conditions. More definite conclusions can be obtained only in experiments directly comparing fermentation under completely identical conditions with blowing of air and without it.

Such experiments were carried out by Pedersen ²). Determining successively, in different periods of fermentation, the amounts of fermented extract and the weight of the yeast obtained, he arrived at the results given in Table III.

¹) “Die Gärungsführung” Delbrück and Hayduck, p. 9.
²) “Die Gärungschemie” Ad. Mayer, pp. 160–162.

Table III.

Duration of fermentation in days	Amount of fermented extract in grams per 1 liter of wort. With aeration.	Amount of fermented extract in grams per 1 liter of wort. Without aeration.	Amount of yeast formed in grams per 1 liter of wort. With aeration.	Amount of yeast formed in grams per 1 liter of wort. Without aeration.	One weight part of yeast in a unit of time ferments sugar. In the presence of oxygen.	One weight part of yeast in a unit of time ferments sugar. Without access of oxygen.
2	2.8	2.6	0.55	0.74	0.8	0.6
3	7.0	5.4	2.02	0.91	0.6	0.9
5	27.8	23.4	3.16	2.10	1.2	1.4
6	34.4	31.8	3.07	2.29	1.3	1.6
7	42.2	40.2	3.15	2.42	1.4	1.7
8	54.2	41.2	4.11	1.79	1.3	1.9
10	60.0	52.5	4.10	2.14	1.2	1.7
12	69.0	68.8	4.85	2.46	0.9	1.5

In calculating the fermentative power of one weight part of yeast over 24 hours, from the amount of fermented extract there is subtracted that part of it which is spent on the formation of yeast, and to the amount of the latter there is added 0.84 gr. of yeast initially taken for fermentation per each liter of wort.

The results of Pedersen’s experiments lead to the conclusion that, with aeration, the fermentative power of yeast in some cases decreases by 40 percent; its capacity for reproduction, however, increases approximately twofold. According to Delbrück¹, aeration, to a certain degree, can increase the yield of yeast threefold in comparison with what is obtained without an influx of air and under otherwise equal conditions.

In the question of losses during fermentation, which is closely connected with the amount of yeast formed, it is necessary to take account of the composition of the latter. The basis of the substance of the yeast cell is—

plasma, characterized by the fact that it continuously undergoes various transformations, being subject to constant destruction and new formation. The products of decomposition are in part utilized again, being expended in the construction of the substance of the plasma; in part, however, they are excreted by it, and in this latter case either pass beyond the limits of the cellular organism, as a product of its vital activity that is unnecessary and even harmful to the working organism, or else are retained to a certain degree by the organism. Finally, the plasma of the yeast cell has the ability to absorb and temporarily accumulate in its mass very considerable quantities of certain substances, which under certain conditions may again disappear, undergoing decomposition in the process of developing the energy necessary for maintaining the vital processes of the yeast cell. Thus, the plasma of the yeast cell is the center of a very active activity: in it the material necessary for the growth of the yeast is assimilated; here too it may accumulate as a nutritive reserve which, under certain conditions, is utilized and disappears, passing beyond the limits of the organism. All this, closely connected with the vital activity of the yeast organism, indicates that, in addition to reproduction, there are also causes by virtue of which the mass of working yeast may change quantitatively. Thus, the loss during fermentation must depend not only on the number of newly formed yeast cells, but also on the composition of the yeast already present.

Like any organism, yeast contains a considerable quantity of water, fluctuating around 70% by weight of pressed yeast. The dry matter of yeast consists of proteins, carbohydrates, fats, mineral substances, and certain other chemical compounds, the relative amount of which is very small, while their chemical nature and significance in the life of yeast have not yet been sufficiently elucidated.

In the literature there are data concerning the chemical composition of yeast, some of which concern the elemental composition, while others relate to the content of certain chemical compounds that are important from the point of view of the processes of vital activity of the yeast organism, of which they form part as such. Below is given the chemical composition of yeast, as reported by various authors¹:

Table IV.

Author	Yeasts	C.	H.	N.	O.	S.
Schlosberg	Top-fermenting	49.9	6.6	12.1	31.4	—
"	Bottom-fermenting	48.0	6.5	9.8	35.7	—
Mitscherlich	Top-fermenting	47.0	6.6	10.0	—	0.6
Hessenland	Bottom-fermenting	49.3	8.2	10.5	—	—
Wagner	Top-fermenting	49.8	6.8	9.2	—	—
"	Bottom-fermenting	44.4	6.0	9.2	—	—

Nägeli and Löw ¹) found, per 100 parts by weight of yeast:

Component	Content
Cellulose and vegetable mucilage	37.0%
Ordinary albumin	36.0 "
Protein substances, readily decomposable	9.0 "
Peptone, precipitated by acetic-acid lead	2.0 "
Fat	5.0 "
Ash	7.0 "
Extractive substances	4.0 "

The ash content in the dry matter of yeast ranges from 2.5% according to Schlosberg and Wagner, to 7.5–8.8% according to Mitscherlich and Bull ²); but it may reach still more considerable values; for example, Hirt ³) gives an analysis of yeast with an ash content of 9.94%:

¹) “Chemie und Physiologie des Malzes und des Bieres.” Prior. 1896, p. 355.
²) “Die Gärungschemie.” Ad. Mayer. 1906, p. 112.
³) Wochenschr. f. Brauer. 1911, p. 177.

Table V.

	Yeast I	Yeast II	Refers to
Glycogen	12.67%	38.56%	dry substance
Protein	59.55%	46.56%	dry substance
Ash	9.94%	6.81%	of the yeast.
\(P_2O_5\)	5.62%	3.51%	of the yeast.
\(P_2O_5\)	56.54%	52.70%	ash.
Specific gravity	1.0997	1.1138	pressed yeast.
Specific gravity	1.494	1.532	dry substance.

With regard to the composition of yeast ash, the following data are available:¹)

Table VI.

Authors.	Mitscherlich.	Mitscherlich.	Bull.	Belahoubek.	Bechamp.
Yeasts	Top.	Bottom.	Top from Weissbier.	Pressed yeast.	Beer yeast.
Phosphoric acid	53.9%	59.4%	54.7%	51.1%	54–58%
Potash	39.8%	28.3%	35.2%	38.7%	29–31%
Soda	—	—	0.5%	1.8%	0.8–1.9%
Magnesia	6.0%	8.1%	4.1%	4.2%	4–7%
Lime	1.0%	4.3%	4.5%	2.0%	1.6–2.5%
Silicic acid	Traces	—	—	1.6%	Traces
Iron oxide	—	—	0.6%	0.1%	0.8–7.3%
Sulfuric acid	—	—	—	0.6%	5–6%
Chlorine	—	—	0.1%	0.03%	—

¹) “Die Gärungschemie.” Ad. Mayer. 1906, p. 112.

The entire mass of the substance of the yeast cell is subject to fluctuations in composition, not excluding its active basis, which is the proteins. The quantity of the latter, calculated on the dry substance of the yeast, varies within very wide limits, from 31.25 to 75%, which corresponds to a change in the nitrogen content from 5 to 12%¹.

The numerical material presented gives a sufficient idea of the diversity of yeasts that they may exhibit in respect of their chemical composition. The causes of this lie, first, in the individual peculiarity of separate races of yeast; second, in the composition of the fermenting substrates; and, finally, in the fact that the chemical composition of yeasts, like that of every other living organism, is not something constant and unchanging, but fluctuates within very broad limits depending on the totality of all those factors to whose influence the given organism is subjected in the separate periods and stages of its development. These qualitative changes in the yeasts may also be reflected in the quantitative side, in which case three possibilities arise: the weight of yeast either decreases, or increases, or, finally, remains unchanged. Reproduction in the present case must, of course, be regarded as a transient phenomenon, all the more so since later, during the whole process of fermentation carried out in the ordinary way, it appears, for many reasons, as a relatively short period. Delbrück², for example, considers reproduction to be completed by the beginning of the principal fermentation.

As to the possibility, during fermentation, of a decrease in the substance of the yeasts, Thenard³ had already expressed himself; but this was established quite definitely by experiment for the first time only by Pasteur⁴. Thus, according to his experiments it turned out, for example, that 0.424 gram of sugar and 10 g of yeast, the latter calculated on dry substance, gave during fermentation 0.6 g of alcohol and 300 cubic centimeters of carbonic acid, instead of the 110 cubic centimeters which should have been evolved in this case. Since the carbonic acid formed could only in part have arisen at the expense of the sugar taken for the experiment, the remaining quantity of carbonic acid obtained must therefore be attributed to the transformed substance of the yeasts themselves. The explanation of this—

Pasteur explained the phenomenon itself by proceeding from the fact that yeast cells contain a certain substance which can be converted into fermentable sugar, transformed by the action of the yeast into alcohol and carbon dioxide. And indeed Pasteur obtained, by boiling yeast with dilute acid, fermentable sugar in an amount of about 20% of the dry matter of the yeast. He mistakenly attributed the formation of alcohol to the cellulose entering into the cell walls of the yeast. The work of Salkowski¹ and Cremer² established that the sugar fermented in this case, namely d-glucose, is obtained not through the transformation of the cellulose of the yeast wall, but as the result of the hydrolysis of yeast glycogen. On the other hand, during fermentation there is observed, as it were, a disappearance of sugar, as was established by Jodlbauer,³ who used the fermentation method for the quantitative determination of sugar. It turned out that part of the fermentable sugar seemed to vanish, not passing into alcohol and carbon dioxide, but disappearing from the fermenting substrate.

Subsequently the possibility of this phenomenon was also established in cell-free fermentation with the aid of pressed yeast juice; this can be explained⁴ by the presence in the yeast and in its pressed juice of a synthesizing enzyme that causes reversion and the formation of glycogen, or of some other derivatives,⁵ at the expense of the fermentable sugar. A loss of sugar of this kind, as Euler and Berggren⁶ indicate, during the fermentation of glucose by living yeast may reach 5–15% of its initial content in the fermenting liquid, if the sugar ferments only halfway.

Thus, the fermentable sugar is consumed not only for the construction of the substance of new cells arising during reproduction, but also goes toward the accumulation, in the body of already existing old cells, of reserve nutritive substances in the form, for example, of glycogen. It turns out in this connection that, for the construction of yeast substance, it is necessary, for example according to Pasteur, only

about 1%, and according to Gyltay and Aberson¹ 3.8% by weight of the entire quantity of sugar processed. For the accumulation of a nutritive carbohydrate reserve, judging by the quantity of glycogen in the yeast, considerable quantities of sugar are required. According to the data of Pavy and Bywaters², fresh commercial yeast contains about 5% of it by weight, which amounts to 25% of the dry substance. When it is left for 2–3 hours in sugar solutions, depending on the concentration of the latter, the quantity of glycogen in the yeast doubles or triples; moreover, the glycogen content may rise even to 16% by weight of the yeast, which amounts to about 75% by weight of its dry substance. Laurent³ found in one case a glycogen content equal to 32.6% of the dry substance of the yeast. Krampf and Hirt⁴ give—12.67%; 13.27%; 38.56% and 39.90%. Laurent regards glycogen as a constant constituent part of the yeast cell, usually occurring in it in rather considerable quantities, whereas Henneberg⁵ proved the possibility of cases in which yeast has no glycogen at all, or contains it in the most insignificant quantities. The presence, appearance, and disappearance of glycogen in yeast, apart from the composition of the fermenting substrate, are determined by many causes. Thus Henneberg⁶ showed that the ability of yeast to accumulate a greater or smaller quantity of glycogen depends on its race. Especially rich in glycogen are, for example, top-fermenting brewery yeasts—race B, and distillery yeasts—race II. High temperature, strong aeration⁷, and a considerable concentration of wort favor the formation of glycogen. Fermenting cells show glycogen as long as there is a sufficient quantity of sugar in the fermenting wort. With the disappearance of the latter in the fermenting wort, the glycogen content in the yeast also decreases. At a high fermentation temperature glycogen is lost more rapidly than at a low one, which is why yeast in distilling loses glycogen considerably earlier than in brewing, where

fermentation is conducted at a lower temperature. In yeasts rich in glycogen and spread in a thin layer in the air, the glycogen disappears at a temperature of 22° C after 3 hours, and at 8° C after 48 hours. Remaining under water without contact with air, the yeast retains glycogen still longer. In weak wort, with vigorous aeration, glycogen is very rapidly lost.

The appearance of glycogen in yeast, as well as its disappearance, without representing anything strictly definite, depends on many factors and above all on temperature¹. Thus, the glycogen content in yeast under certain conditions, if the temperature is not high, 24 hours after the beginning of fermentation proves still to be very small, whereas at a high temperature and with the other conditions the same as in the preceding case, in the same time the yeast manages to accumulate a very large quantity of glycogen. Its greatest content at low fermentation temperatures in brewing is reached only on the 7th–8th day; in fermentation at high temperatures, e.g. in distilling, the maximum occurs already on the second day. In wort with a concentration of 7° Ball., glycogen begins to form after 1¾ hours, whereas at a concentration of 26° Ball. only after 24 hours; moreover, the maximum in the first case is reached after 24 hours, and in the second after 5 days.

From what has been presented concerning glycogen, whose content in yeast can fluctuate within very wide limits, it is clear that this alone can reduce or increase severalfold the expenditure on yeast, which must therefore depend to a considerable extent on the stage of fermentation, since in the separate stages of fermentation the glycogen content in yeast changes very sharply.

During fermentation, not only glycogen is subject to known fluctuations in quantitative terms, but also other constituent parts of the body of the yeast cell, not excluding the actual basis of the latter—namely, the protein substances entering into its composition. The process of nitrogen assimilation and its fate after uptake by the yeast naturally very early began to interest many investigators of the phenomena of fermentation.

Already Pasteur² pointed out that, under certain conditions of fermentation, yeast, while losing in its weight, may become noticeably poorer

nitrogen, the content of which, under certain conditions, decreases from 9.77% to 5.50%.

Bechamp and Schutzenberger¹) were the first to express the view that, in the phenomenon indicated, two independently proceeding processes must be distinguished, one of which leads to the conversion into alcohol and carbon dioxide of the reserve carbohydrates of the yeast cell, chiefly glycogen, whereas the other causes the decomposition of the protein substance of the yeast.

The correctness of this view was subsequently confirmed by a whole series of later investigations, carried out chiefly by Salkowsky and Wiel²). According to the work of Hahn³) and others, the active agent of this decomposition is the proteolytic enzyme of yeast—endotryptase—which causes the formation of products of decomposition of the protein molecule.

Thus, during fermentation, in certain cases there may occur a decrease in the protein substances of yeast, associated with a diminution of the expenditure on yeast, whereas in other cases the opposite phenomenon is possible.

The experiments of Wysmann⁴) give a certain idea of the successive changes in the nitrogen content of yeast during fermentation. He found that, upon introducing yeast into the fermenting liquid, a rapid increase in the nitrogen content of the yeast is observed, which, apparently, is due to the accumulation in the yeast cell of nitrogen-containing nutrients before the period of vigorous reproduction. In the further course of fermentation, a certain decrease in the nitrogen content is found. The latter was initially 7.09%, calculated on the dry substance of the yeast; after one hour it rose to 9.90%, then after 2 hours it became 9.60%, after 3 hours—9.55%, and, finally, after ten hours it decreased to 6.40%.

Delbrück⁵) believes that during the period of reproduction, ending by the beginning of the main fermentation, the yeast no longer has the possibility of increasing the percentage content of nitrogen, since the absorbed nitrogen is given over to the newly formed young cells. When reproduction is finished, however, the nitrogen absorbed goes to

¹) “Handb. d. Techn. Mykol.” Lafar IV p. 439.
²) “Die Zymasegärung” p. 291.
³) “Handb. d. Techn. Mykol.” Lafar IV p. 439.
⁴) Ibid., vol. I, p. 243.
⁵) “Ueber d. Eiw. im. Hefeleb.” F. Hayduck, p. 53.

nutrition only for those cells which assimilate it; as a result, the nitrogen content in the yeast increases. Delbrück regards this phenomenon as the fattening of the yeast, and accordingly denotes it by the word “Mästen.” This phenomenon occurs, for example, in brewing, when yeast, having been repeatedly and successively cultivated in media rich in assimilable nitrogen-containing compounds, very considerably increases its percentage content of proteins, while becoming, in Delbrück’s opinion¹, very sluggish in terms of reproduction, but energetic with respect to sugar fermentation.

The reverse case is also possible, when, owing to a deficiency of the corresponding nutrition in the surrounding medium, the yeast is forced to use the nitrogen of its own body, which leads to a lowering of its content in the yeast substance.

Thus, by artificially inducing—e.g., by raising the temperature or introducing aeration—an intensified multiplication of yeast without a corresponding increase in nutrition by nitrogen-containing substances, it is possible to cause nitrogen depletion in the yeast. Such experiments were carried out by M. Hayduck and led to the result that, after the end of fermentation associated with intensified multiplication owing to an elevated temperature, the yeast lost nitrogen to a certain extent, lowering its content, for example, from 9.888% to 9.262%, from 10.047% to 9.329%, from 9.456 to 9.288%.

According to Henneberg’s experiments², yeast rich in proteins, with a protein quantity above 53%, in most cases either has no glycogen at all, or contains very little of it; in the opposite case, the quantity of glycogen may reach very high limits.

Thus, glycogen and proteins are constituent parts of the yeast substance which, to a certain extent, mutually exclude one another.

All that has been set forth above indicates that the loss obtained during fermentation depends chiefly on the following factors: 1) on the composition and concentration of the fermenting substrate; 2) on the fermentation temperature; 3) on the race of yeast; 4) on the amount of pitching; 5) on the degree of fermentation which marks the end of fermentation in the given production; 6) on whether aeration takes place during fermentation or not, and also on

many other conditions in connection with which fermentation proceeds. The influence of these factors in individual branches of alcoholic fermentation production is highly unequal, as a result of which the fermentation losses in them will not only differ, but in other cases may not even be comparable. The reasons for this, moreover, lie in the influence on the magnitude of the loss during fermentation of a number of concomitant circumstances that have nothing in common with the process of alcoholic fermentation in the strict sense of the word, such as, for example, mechanical losses of the fermentable extract, side fermentations, evaporation and volatilization of alcohol, etc. All this creates such a sharp difference in the magnitude of the practical norms of fermentation losses in individual fermentation industries that in any case it cannot be explained solely by the unequal conditions of the fermentation process itself in the strict sense of the word; thus, for example, according to the data of Kibu ^1), in air-yeast production with a fully equipped plant, the loss during fermentation amounts to about 14% by weight of the material consumed. In good distillery production it is, according to Delbrück ^2), equal to 7.5% by weight of the sugar taken for fermentation.

In brewing, however, judging from the data of the specialized literature, the loss during fermentation is taken to be equal to 3–4½% ^3) by weight of the fermentable extract, although in practice it is often taken as still greater, whereas according to Schönfeld’s study ^4) it is in reality considerably lower and lies at about 2%. Thus, the practical norms of losses during fermentation in individual technical industries can in any case not be used as a scale for comparison with fermentation losses under other conditions. Such a scale could be furnished by data from special investigations concerning the magnitude of the fermentation loss and its fluctuations in connection with definite conditions of fermentation, in the sense of the composition, concentration of the wort, temperature and degree of fermentation and, finally, the race of yeast.

As regards the influence of the degree of fermentation on the magnitude of the loss during fermentation, the question has scarcely been touched upon at all. Yet it is of interest in many respects. In view of this, in the present case, in order to clarify the dependence

1) Chem. Zeitung 1911, p. 435.
2) “Hand. d. Spiritusfabr.” Maercker-Delbrück, 8th ed., p. 681.
3) Wochenschr. f. Brauer, 1909, p. 437.
4) Ibid., 1905, pp. 407–9.

the magnitude of the loss of extract undergoing fermentation on the degree of fermentation, the corresponding experiments were set up, the description and results of which are given below.

The wort intended for these experiments is placed in a Karlsberg copper flask with a capacity of 15 liters and brought in a sand bath to boiling, which is maintained for one hour. After this, the opening through which steam escaped from the flask during boiling is closed with a sterile cotton filter. The boiling is repeated twice after 2 days. Then the wort is left at room temperature so that the insoluble substances separated during boiling may settle. After 5–7 days the wort is carefully drawn off from the settled sediment through the lower opening of the flask, filtered through a dense paper filter, and dispensed with a pipette in portions of 100 cubic centimeters into glass flasks each having a capacity of 500 cubic centimeters. Before being filled with wort, these flasks are closed with cotton stoppers and sterilized by heating in a drying oven at 160–170° C for one hour. Two series of flasks prepared in this way are taken. Into the flasks of one series, 200 cubic centimeters are poured; into the flasks of the other series—100 cubic centimeters of the same wort and 100 cubic centimeters of water. Owing to this, in the second series of flasks the wort has a concentration twice as low as in the first. After filling, each of the flasks is again immediately closed with a cotton stopper, and then all of them are simultaneously sterilized with flowing steam in a Koch sterilizer. One day after the final sterilization of the wort, all the flasks, except two of them in each series, are inoculated, by means of an eye of platinum wire, with a pure culture of a definite race of yeast. After this the cotton stoppers of the flasks are replaced by ordinary stoppers provided with fermentation locks containing strong sulfuric acid. The fermentation flasks equipped in the manner indicated are shaken slightly, weighed to an accuracy of 0.01 gram, and placed for fermentation in a thermostat at the corresponding temperature. All inoculations were carried out in an inoculation cabinet previously sterilized by wiping it with a wall sponge moistened with a solution of mercuric chloride of concentration 0.001%. The stoppers with fermentation locks, before the fermentation flasks were closed with them, were flamed in the flame of a Bunsen burner.

The yeast inoculated into the fermentation flasks was first refreshed by two-day fermentation in beer wort at 25° C in Freudenreich flasks. Of such flasks, one

simultaneously inoculated with one and the same yeast culture; for each experiment two were taken—one for each row of flasks. In this way all the fermentation flasks were simultaneously inoculated with one and the same yeast culture, taken for each row from a special Freudenreich flask assigned to it.

The progress of fermentation was observed by the loss in weight of the flasks. Accordingly, after certain intervals necessary for attaining the desired degree of fermentation, the corresponding flasks were successively opened and their contents analyzed.

By shaking, the wort was to a considerable extent freed from the carbon dioxide contained in it and was then transferred quantitatively, by repeated rinsing of the fermentation flask with water, into a weighed volumetric flask. The capacity of the latter, with undiluted original wort, was equal to one liter, and with diluted wort—to \(1/2\) liter.

After bringing the contents of the volumetric flask up to the mark with water and weighing it to an accuracy of 0.01 gram, after thorough shaking the liquid is filtered with the aid of a large glass funnel, tightly covered during filtration with a glass plate in order to eliminate the possibility of evaporation.

Filtration was carried out through a dry, dense paper filter. In the filtrate, the content of alcohol, of extract remaining unfermented, and of nitrogen is determined quantitatively—the latter by the Kjeldahl method.

To determine the extract, about 150 grams of the indicated filtrate are weighed into a glass dish to an accuracy of 0.01 g. On a water bath this sample is evaporated to \(1/3\) of its initial volume, cooled, and brought up with water exactly to the initial weight. After this, the specific gravity of the resulting liquid is determined pycnometrically; on the basis of this, from Balling’s tables ^1) the percentage content of extract contained in the filtrate of the solution of the investigated sample of fermented wort is found. Multiplying the percentage content of extract thus found by the weight of the whole sample, after it has been transferred into the volumetric flask and diluted by adding water to the mark, we obtain, with some approximation, the total amount of extract remaining unfermented.

To determine the alcohol content, 100 grams of filtrate are weighed, to an accuracy of 0.01 gram, into a distillation flask of 500 cubic centimeters; 50 cubic centimeters of water are added, after

^1) “Das chemische Laboratorium des Brauers” W. Windisch, 1907, p. 40.

of it is distilled off with a dephlegmator into a 100 cu. cent. receiver. The receiver is a calibrated volumetric flask of 100 cu. cent.; after filling with the alcoholic distillate to the mark, its contents are topped up with water until the weight of the entire liquid becomes exactly 100.00 g.

In the liquid thus obtained the specific gravity is determined pycnometrically, and from Hehner’s table¹ the weight percentage of alcohol is found, referring directly to the filtrate of the solution of the fermented sample under investigation.

All determinations of specific gravity were made with pycnometers of 50 cu. cent. capacity. The total alcohol content in the sample was obtained by multiplying the alcohol percentage found in the sample by its weight, after it had been transferred to a volumetric flask and diluted by adding water to the definite volume of this flask. The product of these quantities gives the amount of alcohol formed during fermentation.

In the brewing wort fermented in the experiments under consideration, alcohol is formed chiefly at the expense of maltose, and therefore the equation determining the quantitative relation between sugar and the alcohol formed from its fermentation may be taken as follows:

\[ C_{12}H_{22}O_{11} + H_{2}O = 4 C_{2}H_{5}(OH) + 4 CO_{2}. \]

It follows from this that, in order to obtain one part by weight of alcohol, 1.859 parts by weight of maltose must be fermented. On this basis, from the amount of alcohol found in each of the samples under investigation, one can calculate how much maltose had fermented. Adding this latter quantity to the amount of unfermented extract remaining and subtracting the sum from the amount of extract contained in the sweet wort of each fermenting sample, we obtain the loss during fermentation in each individual case.

• To determine the total amount of extract in the original wort contained in each fermentation flask of a given series, two of them, left without yeast inoculation, were used.

The determination of extract in the sweet wort is carried out under exactly the same conditions as in the fermented wort; that is, all the sweet wort of the fermentation flask is quantitatively transferred into the corresponding calibrated volumetric flask. The capacity of the latter is 1 liter for undiluted wort, and 1/₂ liter for diluted wort.

¹ “Die Untersuchung landw. und gewerblich wichtiger Stoffe,” J. König, 3rd ed., 1906, p. 1031.

Then the volumetric flask is filled with water up to the mark, weighed, shaken, and its contents filtered through a dense dry paper filter. The specific gravity of the filtrate is determined pycnometrically, on the basis of which, in the manner already indicated, the quantity of extract contained in the original fermenting wort of each individual fermentation flask of the given series is calculated.

In addition to the extract in the filtrate of the solution of the sweet fermenting wort, the nitrogen content was also determined by the Kjeldahl method; for each determination, 50 cubic centimeters of the solution were taken. All determinations were carried out in parallel samples.

A certain inaccuracy of the indicated method of calculating the loss of extract during fermentation is due to the fact that in the present case the entire sample is taken as a solution, whereas in it, besides the liquid, there is also a certain quantity of insoluble substances, e.g. yeast, coagulated proteins, etc. For this reason the weight of the solution to which, in the preceding calculations, we refer the percentage content of extract and alcohol found by analysis should in reality be somewhat less than that adopted. The numerical value of this reduction depends on the volume occupied by the yeast and by other insoluble substances separated during fermentation.

From the experimental data of Schönfeld¹) it follows that in brewing, toward the end of fermentation in the vats, the quantity of yeast and of all insoluble precipitates formed during fermentation, reckoned as thick yeast, corresponds to 1.72 percent by volume of the fermenting liquid; and since the volumes of the fermenting and fermented liquids are very close to one another, and according to Kosutany²) are even equal to one another, the same percentages may also be referred to the fermented wort. But in the “thick yeast” counted by Schönfeld, the actual content of yeast represents only half the volume, since the rest is occupied by liquid, and therefore the volume of yeast as such amounts in this case to only 0.85 percent by volume of the fermented wort. Under the conditions of the experiments considered, the fermented wort, when transferred to the volumetric flask, is diluted fivefold; therefore the volume occupied by the yeast in this case amounts only to

\[ \left(\frac{0.85}{5}\right)=0.17\% \]

of the volume of the entire sample solution. This is also the cause of that

1) Wochenschr. f. Brauer. 1910, p. 58.
2) Die landwirtschaftl. Versuchs-Station. 1897, vol. XLIX, p. 182.

a small error that occurs in this case when the entire weight of the diluted sample transferred to the volumetric flask is taken as the solution to which the percentage content of extract and alcohol, found by analysis in the filtrate of each investigated sample of fermenting wort, refers. The obtained numerical value of the decrease in the volume of the solution of the fermenting liquid is connected with the particular conditions of fermentation and with a definite stage of it, to which Schönfeld’s data apply. Thus, the numerical value of the volume correction cannot be something constant, if only because the quantity of yeast, and consequently its volume, changes in different periods of fermentation. For this reason, for each separate case the indicated correction must be established separately by calculating the volume occupied by the yeast present in the fermenting wort of the given sample.

Under the conditions of the experiments considered, this volume may be determined with a certain approximation on the assumption that the entire loss is caused by the passage of the fermentable substance only into the yeast, i.e., that the numerical value of the loss expresses exclusively the quantity of dry matter of the yeast that has formed.

The content of dry matter in pressed yeast, freed from the excess liquid present, for example, in thick yeast, amounts, according to many authors, to about 30% of its weight.

The specific gravity of yeast, according to Schönfeld and Rommel¹), and also Kusserow²), Hirt³), and others, may on the average be taken as equal to 1.10.

Under these conditions, the volume occupied by the yeast is determined by formula (I); the weight of the liquid of the sample solution corresponding to this volume is given by formula (II), and the corrections to the amounts found of the extract remaining in the sample and of the fermented maltose are obtained from formulas (III) and (IV).

\[ \frac{100 \times t}{S_z \times U_g} \qquad \text{(I);} \qquad \frac{100 \times t}{S_g \times U_g} \times U_p \qquad \text{(II)} \]

\[ \frac{100 \times t}{S_g \times U_g} \times U_p \times e \qquad \text{(III);} \qquad \frac{100 \times t}{S_g \times U_g} \times U_p \times 1{,}859 \times a \qquad \text{(IV),} \]

¹) Wochenschr. f. Brauer. 1906, p. 523.
²) Wochenschr. f. Brauer. 1906, p. 527.
³) Wochenschr. f. Brauer. 1906, p. 436.

where the following notation has been adopted:

$t$—the loss of extract in grams, found by the preceding calculations, which did not take into account the yeast and the volume occupied by it;

$S_g$—the weight percentage content of dry matter in the yeast, taken as equal to 30%;

$U_g$—the specific gravity of the yeast, equal to 1.10;

$U_p$—the specific gravity of the filtrate of the solution of the fermented-wort sample directly;

$a$—the weight percentage of alcohol in the filtrate of the solution of the fermented-wort sample;

$e$—the weight percentage of the extract remaining unfermented in the filtrate of the solution of the same fermented-wort sample;

1.859—the coefficient for converting alcohol into fermented sugar.

The numerical values of the corrections obtained from formulas (III) and (IV) represent the amounts of extract and fermented sugar remaining after fermentation which are attributable to the solution of fermented wort taken up in the volume of yeast of the given sample. Therefore, the corresponding amounts of both fermented maltose and remaining extract, found by calculations in which the entire weight of the fermented-wort solution was treated as liquid—despite the fact that this weight also included the yeast present there—must be reduced by the amounts of these corrections.

Under the indicated conditions of dilution of each fermented-wort sample before its analysis, the corrections for the volume occupied by the yeast amount in total, as the calculations showed, to about 10% of the value of the loss obtained when the yeast present in the solution is not taken into account.

With direct determination of the quantity of yeast, the described method of analysis, with the indicated method of calculating the loss and with corrections for the yeast, could yield accurate results. But when no direct determination of the quantity of yeast is made, the magnitude of the loss during fermentation is obtained with a certain approximation. The degree of the latter is determined by the magnitude of the difference between the total loss and the actual loss due to the yeast. By completely ignoring the yeast in the calculation, we make an error in determining the loss of about 10% of its magnitude.

TABLE VII.

Experiment No.	Series No.	Sample No.	Extract, grams	Nitrogen, grams	Concentration of the initial wort, °Ball.	Yeast race	Fermentation temperature, °C	Fermentation duration, days	Fermentation duration, hours
1	—	—	36.1998	0.2667	16.91	XII, upper ferm.	28.5—29.0°
1	A	1	36.1998	0.2667	16.91	XII, upper ferm.	28.5—29.0°	1	5.5
1	A	2	36.1998	0.2667	16.91	XII, upper ferm.	28.5—29.0°	1	11.0
1	A	3	36.1998	0.2667	16.91	XII, upper ferm.	28.5—29.0°	1	14.5
1	A	4	36.1998	0.2667	16.91	XII, upper ferm.	28.5—29.0°	1	20.5
1	A	5	36.1998	0.2667	16.91	XII, upper ferm.	28.5—29.0°	2	7.0
1	A	6	36.1998	0.2667	16.91	XII, upper ferm.	28.5—29.0°	2	13.5
1	A	7	36.1998	0.2667	16.91	XII, upper ferm.	28.5—29.0°	3	0.5
1	A	8	36.1998	0.2667	16.91	XII, upper ferm.	28.5—29.0°	3	10.0
1	—	—	17.7971	0.1315	8.61	XII, upper ferm.	28.5—29.0°
1	B	1	17.7971	0.1315	8.61	XII, upper ferm.	28.5—29.0°	1	5.5
1	B	2	17.7971	0.1315	8.61	XII, upper ferm.	28.5—29.0°	1	9.0
1	B	3	17.7971	0.1315	8.61	XII, upper ferm.	28.5—29.0°	1	11.5
1	B	4	17.7971	0.1315	8.61	XII, upper ferm.	28.5—29.0°	1	13.0
1	B	5	17.7971	0.1315	8.61	XII, upper ferm.	28.5—29.0°	1	20.5
1	B	6	17.7971	0.1315	8.61	XII, upper ferm.	28.5—29.0°	2	13.5
1	B	7	17.7971	0.1315	8.61	XII, upper ferm.	28.5—29.0°	3	0.5
1	B	8	17.7971	0.1315	8.61	XII, upper ferm.	28.5—29.0°	4	6.0

Maltose, g	Extract remaining, g	Fermented extract, g	Degree of fermentation according to remaining extract, %	Loss of extract during fermentation, g	Loss of extract during fermentation, % of content in the original mash	Loss of extract during fermentation, % of fermented extract	Loss of nitrogen, g	Loss of nitrogen, % of its content in the original wort
1.9085	32.2436	3.9562	10.93%	2.0477	5.66%	51.76%	0.0594	22.27%
6.7559	27.1466	9.0532	25.01%	2.2973	6.35%	25.37%	0.0628	23.55%
11.0872	21.8246	14.3752	39.71%	3.2880	9.06%	22.87%	—	—
12.5949	20.1841	16.0157	44.24%	3.4208	9.45%	21.36%	0.0662	24.82%
16.6872	16.6920	19.5078	53.89%	2.8206	7.79%	14.46%	0.0696	26.10%
19.6749	14.6923	21.5075	59.41%	1.8376	5.08%	8.52%	0.0700	26.25%
21.8915	12.5658	23.6340	65.28%	1.7425	4.81%	7.37%	0.0772	28.95%
23.1773	11.5346	24.6652	68.32%	1.4879	4.11%	6.03%	0.0693	25.98%
2.4458	14.4407	3.3564	18.86%	0.9106	5.12%	27.13%	0.0338	25.71%
4.2100	12.3900	5.4071	30.38%	1.1971	6.73%	22.14%	—	—
6.7872	9.4556	8.3415	46.87%	1.5563	8.74%	18.66%	0.0471	35.82%
7.8585	8.4042	9.3929	52.78%	1.5344	8.62%	16.33%	—	—
9.2539	7.0956	10.7015	60.13%	1.4476	8.13%	13.50%	0.0540	41.06%
10.9909	5.6063	12.1908	68.50%	1.1999	6.74%	9.84%	0.0539	40.99%
11.2361	5.4735	12.3236	69.24%	1.0875	6.11%	8.82%	0.0539	40.99%
11.5753	5.2650	12.5321	70.42%	0.9568	5.37%	7.63%	0.0468	35.59%

TABLE VIII.

Experiment no.	Series no.	Sample no.	Content in the initial wort: extract, grams	Content in the initial wort: nitrogen, grams	Concentration of the initial wort in Balling degrees	Yeast strain	Temperature, °C	Duration of fermentation: days	Duration of fermentation: hours
2	—	—	39.7200	0.2667	18.45	XII v. br.	17.5°	4	18.0
”	A	1	”	”	”	”	”
”	”	2	”	”	”	”	”	5	6.0
”	”	3	”	”	”	”	”	5	18.0
”	”	4	”	”	”	”	”	6	18.0
”	”	5	”	”	”	”	”	7	2.0
”	”	6	”	”	”	”	”	7	19.0
”	”	7	”	”	”	”	”	8	21.0
2	—	—	20.0070	0.1330	9.66	XII v. br.	17.5°
”	B	1	”	”	”	”	”	3	18.0
”	”	2	”	”	”	”	”	4	8.0
”	”	3	”	”	”	”	”	4	18.0
”	”	4	”	”	”	”	”	5	13.0
”	”	5	”	”	”	”	”	6	21.0
”	”	6	”	”	”	”	”	9	2.0

Total content for each sample, in grams: Maltose fermented	Total content for each sample, in grams: Residual extract	Total content for each sample, in grams: Fermented extract	Degree of fermentation by residual extract	Loss of extract during fermentation: In grams	Loss of extract during fermentation: As % of its content in the original wort	Loss of extract during fermentation: As % of fermented extract	Loss of nitrogen: In grams	Loss of nitrogen: As % of its content in the original wort
4.8187	33.0676	6.6524	16.75%	1.8337	4.62%	27.56%	0.0293	10.99%
9.6849	27.6203	12.0987	30.46%	2.4148	6.08%	19.96%	0.0348	13.05%
12.6082	24.5508	15.1692	38.19%	2.5610	6.45%	16.88%	0.0349	13.09%
16.5826	20.7221	18.9979	47.83%	2.4153	6.08%	12.71%	0.0348	13.05%
18.5142	19.5361	20.1839	50.80%	1.6697	4.20%	8.27%	0.0420	15.75%
20.8637	17.5129	22.2071	55.91%	1.3434	3.38%	6.05%	0.0562	21.07%
23.1626	15.2551	24.4649	61.59%	1.3023	3.28%	5.32%	0.0554	20.77%
2.9243	15.6797	4.3273	21.63%	1.4030	7.01%	32.42%	—	—
6.3104	12.2877	7.7193	38.58%	1.4089	7.04%	18.25%	0.0275	20.68%
8.3060	10.5384	9.4686	47.33%	1.1626	5.81%	12.28%	0.0344	25.86%
10.4419	8.6133	11.3937	56.95%	0.9518	4.75%	8.35%	0.0416	31.28%
11.7033	7.3612	12.6458	63.21%	0.9425	4.71%	7.45%	0.0459	34.44%
12.1599	7.0163	12.9907	64.93%	0.8308	4.15%	6.39%	0.0485	36.47%

TABLE IX

Exp. no.	Series	Sample	Extract in original wort of each sample, g	Nitrogen, g	Concentration of original wort, °Ball.	Yeast race	Temperature, °C	Days	Hours
3	—	—	38.6118	0.2876	17.98	XII top ferm.	8–10°
″	A	1	″	″	″	″	″	7	4.0
″	″	2	″	″	″	″	″	7	19.0
″	″	3	″	″	″	″	″	8	19.0
″	″	4	″	″	″	″	″	10	6.0
″	″	5	″	″	″	″	″	11	8.0
″	″	6	″	″	″	″	″	12	13.0
″	″	7	″	″	″	″	″	20	8.0
3	—	—	19.2863	0.1444	9.29	XII top ferm.	8–10°
″	B	1	″	″	″	″	″	7	4.0
″	″	2	″	″	″	″	″	7	19.0
″	″	3	″	″	″	″	″	8	19.0
″	″	4	″	″	″	″	″	10	20.0
″	″	5	″	″	″	″	″	11	22.0
″	″	6	″	″	″	″	″	13	3.0
4	—	—	38.6118	0.2876	17.98	D bottom ferm.	8–10°
″	A	1	″	″	″	″	″	6	21.0
″	″	2	″	″	″	″	″	7	9.0
″	″	3	″	″	″	″	″	8	11.0
″	″	4	″	″	″	″	″	9	11.0
″	″	5	″	″	″	″	″	11	3.0
″	″	6	″	″	″	″	″	12	21.0

Maltose	Fermented	Extract remaining	Extract fermented	Degree of fermentation by remaining extract	Loss of extract during fermentation, in grams, max.	Loss of extract during fermentation, as % of its original dry matter	Loss of extract during fermentation, as % of fermented extract	Loss of nitrogen, in grams, max.	Loss of nitrogen, as % of its original dry matter
3.9582	33.9659	4.6459	12.03%	0.6877	1.78%	14.80%	0.0752	26.16%
7.8941	29.8260	8.7858	22.76%	0.8917	2.31%	10.15%	0.0805	27.93%
13.6533	23.6751	14.9367	38.68%	1.2834	3.34%	8.59%	0.0875	30.43%
17.7292	19.2749	19.3369	50.08%	1.6077	4.16%	8.31%	0.0913	31.76%
20.8671	16.0356	22.5762	61.06%	1.7091	4.43%	7.57%	0.0946	32.88%
23.0053	14.5423	24.0895	62.34%	1.0642	2.91%	4.42%	0.0946	32.90%
24.1190	13.5266	25.0752	64.94%	0.9562	2.61%	3.81%	0.0909	31.61%
1.9458	16.9738	2.3125	11.99%	0.3667	1.90%	15.86%	0.0371	25.66%
4.4025	14.1082	5.1781	26.84%	0.7756	4.02%	14.98%	0.0405	28.03%
8.3300	9.8177	9.4686	49.09%	1.1386	5.90%	12.02%	0.0474	32.86%
10.5399	7.6470	11.8393	60.35%	1.1004	5.71%	9.45%	0.0476	32.97%
11.3479	7.2495	12.0368	62.41%	0.6889	3.57%	5.72%	0.0475	32.90%
12.0205	6.7714	12.5119	64.87%	0.4914	2.55%	3.93%	0.0475	32.90%
9.9241	27.2092	11.4026	29.53%	1.4785	3.82%	12.96%	0.0784	27.25%
11.7729	21.8722	13.7396	35.58%	1.9667	5.09%	14.31%	0.0827	28.75%
15.6482	20.8722	17.7396	45.94%	2.0914	5.42%	11.78%	0.0864	30.05%
18.6590	18.5686	20.0432	51.91%	1.3842	3.58%	6.91%	0.0866	30.12%
20.9660	16.3302	22.2816	57.71%	1.3156	3.41%	5.90%	0.0865	30.07%
23.3149	14.2472	24.3616	63.10%	1.0427	2.72%	4.30%	0.0865	30.07%

If, however, we take the total loss wholly as the loss to yeast, then the inaccuracy of the determination will already be governed only by the difference between the indicated losses. But this difference is, of course, smaller than the total loss, and therefore the error caused by it must be as many times smaller than the error of the preceding determination as the said difference is smaller than the total loss; that is, in any case, the maximum inaccuracy of such a determination will thereby be less than 10% of the true value of the total loss.

The numerical magnitude of this error for each separate case could be established from the quantitative ratio of the loss to yeast and the loss to other gaseous and liquid by-products of alcoholic fermentation. In short, it is established by the quantitative ratio of the “determinate” and “indeterminate loss” in fermentation. Here by the concept of “determinate loss” in the present case is meant the loss to yeast, whereas all the remaining losses are covered by the concept of “indeterminate loss.”

From a comparison of the magnitudes of the total losses of extract and of nitrogen losses, expressed in grams and given in Tables VII, VIII, and IX, one may form a certain idea of the quantitative ratio of determinate and indeterminate losses in the individual cases of fermentation.

The indeterminate loss is a magnitude that increases as the degree of fermentation rises. Moreover, the increase is both absolute and relative. The former is because, toward the end of fermentation, a maximum of by-products of fermentation is obtained; the latter because the determinate loss, conditioned by the formation of yeast substance, becomes smaller toward the end of fermentation owing to a certain decrease in the mass of yeast.

A high content of proteins and glycogen in a certain measure exclude one another. Moreover, the higher percentage of proteins in the yeast corresponds to the end of fermentation. Let us take the sample with the highest degree of fermentation from the series under consideration and assume in it the maximum content of proteins in the yeast, for example, 70% by weight. This assumes the conditions under which the indeterminate loss will be the highest, i.e., when the difference between the weight of the total loss and the weight of the dry matter of the yeast formed will be the greatest. The indicated difference in the sample with the highest degree of fermentation will, at the same time, correspond to the greatest indeterminate loss among those possible for the given series of fermenting samples. In all the preced—

in the best-fermented samples of the given series, with a lower degree of fermentation, the numerical values of the undetermined losses will be either equal to or lower than those in the last sample. Taking the maximum undetermined loss of the last sample as applying to all the other samples of the given series, we compare the ratio of the magnitudes of the determined and undetermined losses at different degrees of fermentation. As for the maximum undetermined loss of a given series of fermenting samples, it is found in the following way. For this purpose the magnitude of the loss of nitrogen in the sample with the highest degree of fermentation is converted, by multiplication by 6.25, into the corresponding quantity of proteins. Then, taking their content in the yeast as equal to 70% by weight of dry matter, we find, from this, the weight of the latter, which represents the determined loss in the given sample. Subtracting this value from the total loss of the same sample, we find the maximum undetermined loss for the given series of samples of mash, identical in the conditions of fermentation but different in the degree of fermentation attained in them.

The determined losses of all the other samples with a lower degree of fermentation are obtained by subtracting the maximum undetermined loss found above from the total loss of each individual fermenting sample of the given series. The determined losses of the various samples of fermenting mash, calculated in the indicated manner, in one or another series of them, are compared in Table X.

From the data of this table it is evident that the losses due to yeast, found by calculation, vary within the limits of from 38 to 94% of the magnitude of the total loss, which corresponds to fluctuations of the undetermined loss within the limits of from 62 to 6 of the same percent.

But, as was indicated earlier, with complete disregard of the yeast formed, an error of about 10% of the magnitude of the latter is made in the results of the calculations leading to the magnitude of the total loss. If, however, the total loss is attributed entirely to the yeast, then the inaccuracy of the calculation is determined only by the magnitude of the undetermined loss, and therefore the resulting error in calculating the magnitude of the total loss must fluctuate within the limits from \(0.10 \times 0.62\) to \(0.10 \times 0.06\), i.e., within the limits from 6.2 to 0.6% of the true magnitude of the total loss obtained during the fermentation of one or another individual sample. These limits are in reality even narrower, since the undetermined losses for each sample of one series or another, taken in the indicated calculations, are somewhat larger than they actually are; this is a consequence of

Table X.

Experiment no.	Row	Sample no.	Degree of fermentation	Loss (in grams), total	Loss (in grams), undetermined	Loss (in grams), determined	Determined loss in % of total loss	Fermentation conditions
1	A	1	10.93%	2.0477	0.8692	1.1785	57.5%	28.5—29.5°C. 16.91° Ball. XII race in br.
1	A	2	25.01 "	2.2973	" ↑ "	1.4281	62.2 "	28.5—29.5°C. 16.91° Ball. XII race in br.
1	A	3	39.71 "	3.2880	" | "	2.4188	73.6 "	28.5—29.5°C. 16.91° Ball. XII race in br.
1	A	4	44.24 "	3.4208	" | "	2.5506	74.6 "	28.5—29.5°C. 16.91° Ball. XII race in br.
1	A	5	53.89 "	2.8206	" | "	1.9514	69.2 "	28.5—29.5°C. 16.91° Ball. XII race in br.
1	A	6	59.41 "	1.8326	" | "	0.9634	52.6 "	28.5—29.5°C. 16.91° Ball. XII race in br.
1	A	7	65.28 "	1.7425	" ⇵ "	0.8733	50.1 "	28.5—29.5°C. 16.91° Ball. XII race in br.
1	A	8	68.32 "	1.4879	0.8692	0.6187	41.5 "	28.5—29.5°C. 16.91° Ball. XII race in br.
1	B	1	18.86%	0.9106	0.4179	0.4927	54.1%	28.5—29.5°C. 8.61° Ball. C. XII race in br.
1	B	2	30.38 "	1.1971	" ↑ "	0.7792	65.1 "	28.5—29.5°C. 8.61° Ball. C. XII race in br.
1	B	3	46.87 "	1.5563	" | "	1.1384	73.1 "	28.5—29.5°C. 8.61° Ball. C. XII race in br.
1	B	4	52.78 "	1.5344	" | "	1.1165	72.7 "	28.5—29.5°C. 8.61° Ball. C. XII race in br.
1	B	5	60.13 "	1.4476	" | "	1.0297	71.1 "	28.5—29.5°C. 8.61° Ball. C. XII race in br.
1	B	6	68.50 "	1.1999	" | "	0.7820	65.2 "	28.5—29.5°C. 8.61° Ball. C. XII race in br.
1	B	7	69.24 "	1.0875	" ⇵ "	0.6696	61.6 "	28.5—29.5°C. 8.61° Ball. C. XII race in br.
1	B	8	70.42 "	0.9568	0.4179	0.5389	56.3 "	28.5—29.5°C. 8.61° Ball. C. XII race in br.
2	A	1	16.75%	1.8337%	0.8077	1.0260	55.9%	17.5° C. 18.45° Ball. XII race in br.
2	A	2	30.46 "	2.4148 "	" ↑ "	1.6071	66.6 "	17.5° C. 18.45° Ball. XII race in br.
2	A	3	38.19 "	2.5610 "	" | "	1.7533	68.5 "	17.5° C. 18.45° Ball. XII race in br.
2	A	4	47.83 "	2.4153 "	" | "	1.6076	66.6 "	17.5° C. 18.45° Ball. XII race in br.
2	A	5	50.80 "	1.6697 "	" | "	0.8620	51.6 "	17.5° C. 18.45° Ball. XII race in br.
2	A	6	55.91 "	1.3434 "	" ⇵ "	0.5357	39.9 "	17.5° C. 18.45° Ball. XII race in br.
2	A	7	61.59 "	1.3023 "	0.8077	0.4946	38.0 "	17.5° C. 18.45° Ball. XII race in br.

Experiment No.	Series No.	Sample No.	Degree of fermentation	Consumption (in grams): total	Consumption (in grams): undetermined	Consumption (in grams): determined	Determined consumption as % of total consumption	Fermentation conditions
2	B	1	21.63%	1.4030	0.3978	1.0052	71.6%	17.5° C. 9.66° Ball. XII race in br.
2	B	2	38.58 „	1.4089	„ ↑ „	1.0111	71.8 „	17.5° C. 9.66° Ball. XII race in br.
2	B	3	47.33 „	1.1626	„ | „	0.7648	65.9 „	17.5° C. 9.66° Ball. XII race in br.
2	B	4	56.95 „	0.9518	„ | „	0.5540	58.2 „	17.5° C. 9.66° Ball. XII race in br.
2	B	5	63.21 „	0.9425	„ [[unclear: mark]] „	0.5447	57.8 „	17.5° C. 9.66° Ball. XII race in br.
2	B	6	64.93 „	0.8308	0.3978	0.4330	52.1 „	17.5° C. 9.66° Ball. XII race in br.
3	A	1	12.03%	0.6877	0.1446	0.5431	79.0%	8—10° C. 17.98° Ball. XII race in br.
3	A	2	22.76 „	0.8917	„ ↑ „	0.7471	83.8 „	8—10° C. 17.98° Ball. XII race in br.
3	A	3	38.68 „	1.2834	„ | „	1.1388	88.7 „	8—10° C. 17.98° Ball. XII race in br.
3	A	4	50.08 „	1.6077	„ | „	1.5631	97.2 „	8—10° C. 17.98° Ball. XII race in br.
3	A	5	61.06 „	1.7091	„ | „	1.5645	91.5 „	8—10° C. 17.98° Ball. XII race in br.
3	A	6	62.34 „	1.0642	„ [[unclear: mark]] „	0.9196	84.9 „	8—10° C. 17.98° Ball. XII race in br.
3	A	7	64.94 „	0.9562	0.1446	0.8116	84.8 „	8—10° C. 17.98° Ball. XII race in br.
3	B	1	11.99%	0.3667	0.0673	0.2994	81.6%	8—10° C. 9.29° Ball. XII race in br.
3	B	2	26.84 „	0.7756	„ ↑ „	0.7083	91.3 „	8—10° C. 9.29° Ball. XII race in br.
3	B	3	49.09 „	1.1386	„ | „	1.0713	94.1 „	8—10° C. 9.29° Ball. XII race in br.
3	B	4	60.35 „	1.1004	„ | „	1.0331	93.9 „	8—10° C. 9.29° Ball. XII race in br.
3	B	5	62.41 „	0.6889	„ [[unclear: mark]] „	0.6216	90.2 „	8—10° C. 9.29° Ball. XII race in br.
3	B	6	64.87 „	0.4914	0.0673	0.4241	86.3 „	8—10° C. 9.29° Ball. XII race in br.
4	A	1	29.53%	1.4785	0.2774	1.2011	81.2%	8—10° C. 17.98° Ball. D. race in br.
4	A	2	35.58 „	1.9667	„ ↑ „	1.6893	85.9 „	8—10° C. 17.98° Ball. D. race in br.
4	A	3	45.94 „	2.0914	„ | „	1.8140	86.7 „	8—10° C. 17.98° Ball. D. race in br.
4	A	4	51.91 „	1.3842	„ | „	1.1068	80.0 „	8—10° C. 17.98° Ball. D. race in br.
4	A	5	57.71 „	1.3156	„ [[unclear: mark]] „	1.0382	78.9 „	8—10° C. 17.98° Ball. D. race in br.
4	A	6	53.10 „	1.0497	0.2774	0.7723	73.6 „	8—10° C. 17.98° Ball. D. race in br.

because, to each of the fermented samples, the smallest indeterminate loss of that series has been assigned. Meanwhile, at medium, and still more at low, degrees of fermentation, the indeterminate loss may be not only considerably less than the maximum, but in some cases may even be equal to zero.

Examining, from Tables VII, VIII, and IX, the losses of extract during fermentation, one can see that the latter are not something constant in magnitude, but are determined numerically by the totality of all the conditions under which fermentation is conducted; in connection with this, fluctuations in the numerical magnitude of the losses may occur within very wide limits. Judging from the results obtained in the experiments performed, the concentration of the fermenting wort and the temperature of fermentation are very important factors here. An increase in concentration, all other conditions being equal, increases the absolute numerical value of the losses. Thus, for example, the weight of the final loss in series “A” of experiment No. 3 is almost twice as great as the weight of the corresponding loss in series “B” of the same experiment, owing to the increase in the concentration of the fermenting wort. In the remaining experiments this phenomenon is also observed, though not to the same degree.

At the same time it is not difficult to see that the loss increases disproportionately to the rise in concentration of the fermenting wort. Consequently, it turns out that the loss, expressed as a percentage of the fermenting extract, in most cases relatively increases as the concentration decreases.

An increase in temperature, by promoting the multiplication of yeast, has the effect of increasing the absolute numerical value of the loss, as follows from a comparison of the final losses of experiment No. 3 in its series “A” and “B” with the corresponding losses in the same series of experiments No. 1 and No. 2. But, in addition to the intensified multiplication of yeast, the increase in losses at high temperatures is apparently also explained by the extraordinary growth of indeterminate losses that occurs here. In this regard, an indication is given by comparing the magnitudes of the determinate losses as percentages of the total losses, presented in Table X, from which it is evident that at 29° C the determinate loss is equal to, or even less than, the indeterminate loss, whereas at low temperatures it amounts to a value 6–7 times greater than the latter. This circumstance explains, to a considerable extent, the sharp difference in the magnitude of normal losses obtained during fermentation and accepted in the practice of distilling and brewing.

The influence of high fermentation temperatures on the increase in loss is a fact known also from practical data of factory distilling; thus, for example, Heinzelmann and Hesse¹ long ago pointed out that, by maintaining a lower temperature during the main fermentation and the period of after-fermentation, one can obtain a comparatively increased yield of alcohol.

Not being constant under changing conditions, the loss of extract during fermentation, even when those conditions remain unchanged, proves to be a quantity whose numerical value varies greatly at the different stages of fermentation. At the same time, what matters is not only the process of formation of new organisms, the number of which ceases to increase after a certain stage of the fermentation process, but also the ability of these organisms to absorb certain substances, temporarily accumulating them in their body as nutrient reserves. And this causes significant changes in the mass of yeast at different degrees of fermentation, which in turn is closely connected with fluctuations in the numerical value of the total loss, which, judging from the data of Tables VII, VIII, and IX, undergoes considerable changes in the course of fermentation.

Thus, when comparing losses in individual cases of fermentation, it is absolutely necessary to take equal, or at least mutually close, degrees of fermentation; otherwise, incomparable quantities will be compared.

If the loss during fermentation can vary considerably in its absolute magnitude, then these fluctuations are manifested still more when the loss is expressed as a percentage of the fermented substance. In this case the magnitude of the fermentation loss changes, decreasing many times from the beginning to the end of fermentation. The reason for this is that, in periodic fermentation under the usual conditions of factory fermentation, and still more in the laboratory with minimal inoculations by means of an ear of platinum wire, at the beginning of fermentation what proceeds chiefly is the growth and accumulation of the yeast mass, whereas subsequently only fermentation takes place, ending with the fermentation of the nutrient reserves of the yeast body itself. A graphic representation of the decline in the magnitude of the loss during fermentation, expressed as a percentage of the fermented extract, is given, for example, by the diagram in drawing No. 2, relating to experiment No. (1—A). In this drawing, the degrees of fermentation are plotted on the abscissa axis—

Chart No. 2.

Vertical axis: in % of fermented extract
Horizontal axis: Degree of fermentation

Chart No. 3.

Vertical axis: in % of fermented extract
Horizontal axis: Degree of fermentation

...and on the ordinate axis—the total loss of fermentation, expressed as percentages of the fermented extract. The changes in the magnitude of the same loss, but expressed as percentages of the extract being fermented, have an entirely different character; this is presented by the diagram in the following drawing No. 3.

The question now arises as to the relation between the data obtained, concerning the magnitude and fluctuations of fermentation losses under the various conditions of the laboratory-fermentation experiments considered, and those losses which may be observed under the conditions of factory practice in the individual branches of the technique of alcoholic fermentation.

Of course, a direct comparison and any definite conclusions from a straightforward juxtaposition of the one and the other do not appear possible. This is indicated by the fact that fermentation in a factory setting is closely connected with many processes and phenomena that affect the magnitude of the loss incurred, but have nothing in common with fermentation in the proper sense of the word. Moreover, in the experiments considered, with minimal inoculations, the entire mass of yeast formed arises at the expense of the fermentable extract, whereas under factory fermentation conditions a considerable part of it is always introduced into the fermentable substrate immediately before fermentation. This reduces the loss on yeast, and by so much the more, the greater the quantity of yeast introduced. But if the data obtained cannot serve directly as a measure of the correctness of the magnitude of the losses occurring in the individual branches of fermentation technique, then in any case they indicate with sufficient definiteness by what factors, and to what extent, the magnitude of the loss obtained in one case or another is conditioned, as well as the possible fluctuations of that loss depending on the conditions that determine the phenomenon of fermentation as such. Thus, when the fermentation process is reproduced with brewer’s wort identical to that which was taken for the experiments considered, and when fermentation is conducted with minimal inoculations of the same pure cultures of yeasts under conditions eliminating the possibility of losses, infection, etc., then in such cases the losses obtained earlier, and likewise the degree of fluctuation in their magnitude, may already serve as a scale applicable to the losses obtained here. On the basis of all that has been set forth above concerning the losses occurring in periodic fermentation, the following conclusions may be drawn:

1) The loss of fermentable extract is unavoidable, but its magnitude varies depending on the totality of all the conditions that determine the course of fermentation.

2) The principal factors determining the magnitude of the loss are the composition of the fermentable substrate, its concentration, and the fermentation temperature.

3) Under quite definite and constant fermentation conditions, the loss varies with the degree of fermentation.

4) The total loss of extract during fermentation consists of the “definite loss” to yeast and the “indefinite loss” to liquid and gaseous by-products of fermentation.

5) The quantitative ratio of definite to indefinite losses is not constant when the fermentation temperature changes; an increase in the latter changes the indicated ratio in the direction of increasing the indefinite losses.

All that has been set forth, and the conclusions drawn, lead to the view that all the proposed formulas determining the magnitude of the loss during fermentation, proceeding from the quantitative ratios of the fermentable, fermented, and remaining extract and of the fermentation products obtained, still lack a very great deal in order to encompass the phenomenon of fermentation in its entirety; and therefore the numerical magnitudes of the losses to which such formulas lead may, in individual cases, deviate very considerably from reality. Consequently, when it is necessary to judge the correctness of the magnitude of the loss under one or another set of fermentation conditions, special experiments must be carried out to determine these losses and their fluctuations for the given fermentation conditions.

This, incidentally, was one of the chief tasks of the experiments analyzed in this chapter.

CHAPTER III.

The Conditions of Continuous Fermentation and Its Inevitable Connection with a Continuously Operating Fermentation Apparatus.

In all branches of the technology of alcoholic fermentation, the entire mass of fermenting wort is usually divided into separate portions that are small in comparison with the total fermenting mass, each of which undergoes fermentation independently. Therefore the fermentation vessels are alternately filled with fermenting liquid and emptied of it, and thus the work of fermentation is carried out periodically.

In each separate portion the process of fermentation proceeds analogously to what is shown by the diagram in drawing No. 1, i.e., it takes place under approximately the same conditions that correspond to the indicated case of fermentation. Some negative aspects of the latter method of fermentation, as adopted in the practice of individual industries, are to a certain extent mitigated, but the general character of the course of the fermentation process is thereby not altered in the least. Its principal features are determined by the fact that the fermentation of each separate portion of wort requires the addition of a certain quantity of yeast, which multiplies, reaches the maximum of its development, and thereafter begins to die off.

The consequence of this is, first, an inevitable loss of substance to yeast; second, a great unevenness in the course of fermentation, since a significant part of its duration is connected, under these conditions, on the one hand with development and, on the other hand, with the gradual decline of the active mass of working yeast.

The practice of individual industries to a certain extent shortens the period of fermentation development, which is achieved by the use of relatively large quantities of pitching yeast. There have also been attempts to shorten the duration of the latter period, when the activity of the yeast proves to be greatly weakened. An example of this may be fermentation under reduced pressure.

But all this, like much else, can only mitigate, but not eliminate, the fundamental shortcomings of all methods in general that are connected with the periodicity of fermentation work. Under these conditions all the wort being processed is fermented in separate, very small portions in comparison with the total productivity of the plant. This entails great inconvenience in the sense of the need to carry out repeated emptying, cleaning of the fermentation vats, and preparing them for the next fermentation.

At the same time it is necessary to carry out the removal of the yeast, its purification, and pitching into the wort to be fermented. In short, under these conditions a very great deal of manual labor is required, connected with the assembly and disassembly of each individual fermentation vessel.

This feature entails a great inconvenience, which is especially strongly felt where manual labor is costly.

For this reason, in America, for example, a method of fermentation was proposed for brewing, and found some application, which to a considerable extent eliminates the inconvenience indicated. This method, called the “method of continuous fermentation” according to the Forward system, developed by Schalk,^1) and applied at Ruppert’s brewery in New York. With this method,^2) fermentation is carried out in closed metallic horizontal cylindrical vessels, 87 feet long and with a capacity of 1700 hectoliters each. Several such vessels are required for continuous operation. At the beginning of operation, cooled sterile wort at fermentation temperature is poured into the first of them, with the addition to it of twice the amount of yeast in comparison with that required under normal conditions.

By blowing sterile air, conducted through a pipe laid on the bottom of the fermentation vessel, the yeast is distributed uniformly in the liquid. The blowing is repeated on the second and third day of fermentation. Twenty-four hours after filling, at the time when signs of activity appear, the first vessel is connected by a pipeline with the second, whereby the fermenting wort is divided equally between them. Then each of them is topped up with fresh sterile wort. After this the first fermentation vessel is separated from the second and remains at rest for independent and final fermentation. At this point a valve is opened, located at one quarter of the height of the fer-

^1) Jahrb. d. Versuch u. Lehranst. f. Brauerei in Berlin 1908, p. 584.

^2) Illustr. Brauerei Lexikon. 1910, p. 538.

fermentation vessel from above. Through this valve, during fermentation, from the first vessel into the second, together with the foam being formed, passes a portion of the yeast cells carried at that time to the surface of the fermenting wort.

After two days the second vessel is connected with a third, still empty one, in order to pour into the latter half of the contents of the second vessel, and then the whole operation is carried out as was done with the first vessel. All this is completed by separating the second fermentation vessel for independent fermentation. Thereafter, in the same order, the process proceeds successively: pouring from each vessel into the next, separating the preceding one, and allowing it to finish fermenting independently. When the time comes to pour from the last vessel, the first is already empty, cleaned, and prepared to receive half the quantity of wort that has fermented in the last vessel.

Then, in the subsequent work, all the operations are performed successively in the order already indicated.

From the description given of this method it is evident that, with the indicated mode of conducting fermentation, in essence each fermentation vessel works independently, and that in each of them the fermentation process proceeds independently of the course of fermentation in the other vessels.

Each two adjacent vessels are connected only for the short interval of time required for pouring half of the wort into the next vessel from the preceding one, where it has already fermented for one day. On the last day of fermentation in each fermentation vessel, air is passed over the surface of the fermenting wort, which promotes the settling of the yeast. For convenience in emptying and cleaning the fermentation vessels, they are arranged with an inclination of 8 inches over their 87-foot length.

The advantages of this method amount to the cleanliness of fermentation, the rapidity of fermentation owing to the large pitching of yeast, and the elimination of operations connected with selecting, washing, and pitching yeast. The very large size of the fermentation vessels in this system, compared with the usual capacity of brewery fermentation vats, considerably reduces the size of the fermentation room, as well as the manual labor connected with emptying, filling, and cleaning the fermentation vats. At the same time, thanks to the metal enameled vessels, the troublesome work of tarring and paraffining the fermentation vessels is also eliminated.

A similar method of fermentation, also called discontinuous, was proposed for distilling as well, but it found application only in a few distilleries producing alcohol from beet molasses and from beets.

This method was tested by Lüder¹ as applied to potato distilling, in connection with purification fermentation according to the method given by Henneberg² for eliminating possible infection under these conditions.

The essence of this method, and its advantages over ordinary periodic fermentation, consists in the fact that it eliminates the work of preparing yeast for each mash to be fermented; instead, the yeast is taken directly at the beginning of the period of main fermentation, together with the wort being fermented. In Lüder’s experiments, one half of the capacity of the fermentation vat was drawn off.

After part of the fermenting mash has been drawn off, both the first drawn-off vat and the second, newly filled vat are topped up with mash that has not yet fermented. After twenty-four hours, one third of the contents of the second fermentation vat is transferred to the next one, after which both vats, as in the preceding case, are filled up with fresh mash. The work then proceeds in this order.

The chief advantage of this method, compared with the ordinary one, consists in eliminating the special operation of preparing yeast and in considerably shortening the duration of fermentation, since under these conditions the period of saccharification is almost absent.

The elimination of infection, which is unavoidable in open fermentation vessels, is achieved, when such infection appears, by adding to the withdrawn third of fermenting wort, intended for the next mash, a considerable quantity of some antiseptic—for example, sulfuric acid—which is allowed to act for a short interval at comparatively high concentrations. In this way the fermenting medium is cleansed of harmful microorganisms and weak yeasts, which perish under these conditions. As a result, only the strongest generation of yeast remains, whose activity subsequently initiates fermentation. When the desired degree of purification has been reached, the vat is topped up with sweet mash, whereby the concentration of the antiseptic—

decreases, reaching a normal value. In his experiments, Lülder conducted the purification fermentation in such a way that, when infection appeared, he added sulfuric acid to the withdrawn third of the fermenting vessel up to an acidity of \(2.85^\circ\), and allowed the acid to act at this concentration for two hours. After this, the vat was filled up with a sweet mash.

The results obtained in this way, according to Lülder, were good. The same conclusion was reached by trials of this method under factory-practice conditions¹, where the purification fermentation made it possible for months to conduct the work in the indicated order. However, cases are possible in which the infection reaches such proportions that combating it creates considerable difficulties, and this already leads to very great inconveniences. The latter circumstance compels practice to regard the indicated method of fermentation with great caution, and explains why it has not found wide dissemination.

Both of the methods of fermentation described, proposed for brewing and for distillery production and called “methods of continuous fermentation,” are in essence completely identical. What is characteristic of both is that each separately fermented batch includes a considerable portion of yeast taken directly from the preceding batch, together with the fermenting mash at the beginning of the period of main fermentation. Thus, a certain successive connection is established among all the separately fermented batches. The latter, in a somewhat different form, also takes place under ordinary fermentation conditions both in brewing and in distilling.

But in the so-called methods of “continuous fermentation” considered, the simplest and most convenient form of connection has been chosen, eliminating all intermediate operations associated with the comparatively prolonged period of propagation of the yeast introduced into each newly fermented batch, which causes a temporary slowing in the course of the fermentation process in each separate fermented batch.

This is not present in the described methods of continuous fermentation, when fermentation of each new batch begins directly—

...respectively, with the vigorous activity of a large quantity of inoculated yeast in the stage of the main fermentation.

However, the difference between the ordinary methods of fermentation and the proposed “continuous” ones completely disappears in those periods that follow the main fermentation. In both the one and the other case, the activity of the yeast, having rapidly fallen after the main fermentation to a certain level, then begins to decline very slowly and, finally, after a relatively long interval of time, ceases altogether. In order to establish more clearly the difference in the course of the fermentation process under the usual periodic methods of fermentation and under the indicated “continuous” ones, let us imagine the following.

All the wort subject to fermentation is divided into separate, identical samples. One half of them is fermented in the usual manner, the other by the indicated method of “continuous” fermentation. Let us assume, moreover, that in both cases the entire fermentation is carried out only in a single series of successive samples following one after another and having between them a succession link in the sense of the transfer of part of the yeast inoculated from each preceding sample into the one following it. The moment of sampling and transfer of the yeast is determined by the very method of fermentation. In the periodic method of fermentation this is done when fermentation in the preceding sample is completely finished; in the “continuous” method, at the beginning of the period of main fermentation.

As regards the quantity of inoculated yeast, let us take the extreme limits. In periodic fermentation we take almost negligible quantities of inoculated yeast, for example, corresponding to an inoculation made with the eye of a platinum wire or even with a drop of yeast culture for the whole sample. Under such conditions the whole mass of yeast formed toward the end of fermentation of an individual sample arises in that very sample during the first period of fermentation, as the result of multiplication of the inoculated yeast.

In the “continuous” method of fermentation the inoculation of yeast is taken to be maximal, in the form of half the quantity of the fermenting wort of the preceding sample, removed at the beginning of the main fermentation. Thus the mass of inoculated yeast in the latter case constitutes a very considerable quantity.

In order to trace the fermentation while observing all the above-mentioned conditions, corresponding experiments were set up with fermentation at a temperature of \(20.0^\circ\) C of beer sterile wort with a concentration of \(17.0^\circ\) Ball. The fermentation in this case was...

was carried out with samples of 400 cubic centimeters in tall cylindrical vessels 65 mm in diameter and 350 mm high, closed with rubber stoppers fitted with fermentation attachments containing strong sulfuric acid. In those vessels in which fermentation was conducted by the “continuous” method, the stoppers, besides the fermentation attachments, also had two bent glass tubes. One of them was short, the other long, not reaching the bottom of the vessel by only 2–3 centimeters. On the outer ends of both tubes were rubber tubes closed with glass rods. Drawing No. 4 shows such a fermentation vessel in assembled form.

Drawing No. 4.

In the “continuous” fermentation method, yeast is added only to the first vessel, in the amount of one drop of a culture previously prepared in Freudenreich’s flask.

All subsequent vessels of this series receive inoculum in the form of half the fermenting wort taken from the preceding fermenting sample at the beginning of the period of main fermentation.

In order to take off the fermenting wort, the outer ends of the long tubes of the vessel from which the fermenting wort is being withdrawn, and of the vessel into which it is desired to transfer this wort, are connected with rubber tubing. The latter vessel contains not 400 cubic centimeters, but only 200 cubic centimeters of sterile wort. When, with all the precautions necessary to eliminate possible infection of the wort, the vessels are connected with one another, air is drawn through cotton wool into the short tube of the first of them, and [the text continues]

air is blown through a sterile filter. Owing to this, half of the fermenting wort is displaced from the first vessel into the second vessel. After this, 200 cubic centimeters of fresh sterile sweet wort are introduced into the first vessel. This is done in the same way as was done when drawing off the fermenting wort. The exact determination of the quantities of liquid drawn off or added to each separate vessel is established by repeated weighing before the decanting or filling and after it.

Two days after the moment of drawing off the fermenting wort from the first vessel, in the same order the wort is drawn from the second into the third vessel, then, after another two days, from the third into the fourth, and so on. In all the fermentation vessels the course of fermentation is observed from the data of weighing, carried out with an accuracy of up to 0.01 gram. In this way, from the loss in weight caused by the removal of the carbonic acid formed, the quantity of fermented sugar is determined. Relating the latter to the time and expressing it in weight percentages of the fermentable extract contained in the 400 cubic centimeters of sweet wort falling to each separate fermentation vessel, we obtain certain quantities expressing the strength of fermentation at its various stages.

The results of these experiments are presented graphically in diagrams No. 5 and No. 6.

Representing graphically the course of fermentation proceeding successively in the fermenting samples of each row of them, we obtain, for periodic fermentation, the curve of diagram No. 5, and for the Forward-Schall method, the curve of diagram No. 6.

Considering the diagrams of drawing No. 6, one can see that in each subsequent vessel the intensity of fermentation in the principal period of fermentation increases, and very considerably. This circumstance appears to be mainly the result of the fact that, under the conditions of the experiment under consideration, the cooling of the fermenting liquid was only by air, through the walls of the fermentation vessel, which proved quite insufficient for maintaining the desired constant fermentation temperature in the fermenting liquid itself. At the same time, in each subsequent vessel, under the stated experimental conditions, the mass of inoculated and active yeast gradually increased; therefore the period of main fermentation, correspondingly, proceeded with a gradually increasing quantity of heat developed by the yeast. Owing to this, the fermentation temperature of this period in each subsequent vessel was higher than in the preceding one. This also ...

Average hourly fermentation of sugar, in % of the extract introduced into the wort.

Continuous fermentation.

Drawing No. 1.
2.25 liters.
Axis label: Hours.

Average hourly fermentation of sugar, in % of the extract introduced into the wort.

Continuous fermentation.

Drawing No. 2.
2.5 liters.
Axis label: Hours.

Average hourly fermentation of sugar, in % of the extract introduced into the wort.

Continuous fermentation.

Drawing No. 3.
[[unclear: volume in liters]].
Axis label: Hours.

was the cause of a corresponding increase in the intensity of fermentation with each subsequent transfer, which is clearly marked by the diagrams in Fig. No. 6. This phenomenon appears to be transient and, of course, undesirable for many quite understandable reasons. It can be eliminated very easily by strictly regulating the temperature of fermentation, which must be maintained and gradually varied in exactly the same way in all the fermentation vessels of a given series. Under this condition, the fermentation process should proceed in accordance with what is shown by the diagrams in Fig. No. 7.

A comparison of the diagrams in Fig. No. 5 and No. 6, and also No. 5 and No. 7, gives an idea both of the difference and of the similarity in the course of the fermentation process under the methods of periodic and Forward-Schalk fermentation.

In the first of these, the intensity of the reproduced fermentation process in a series of successively fermented samples now rises to a maximum, now falls to the complete cessation of fermentation. If one takes into account the relative duration of the periods of maximum and minimum work of the yeasts during the fermentation of each individual sample, it will turn out that the productivity of this and of all analogous methods of periodic fermentation, in the sense of utilization of time and of the fermentation vessel, will be very small.

The diagram in Fig. No. 5 clearly points to the causes of this. There are two such causes. One of them consists in the excessively long duration of the interval of time separating the period of principal fermentation of one sample from the corresponding period of the sample following it. The other cause lies in the periodicity of the action of the fermentation apparatus, the working time of which under these conditions is spent chiefly on the development and gradual extinction of the fermentation process reproduced in them. Desiring to make more expedient use of working time, Forward and Schalk devised the described method, in which the first of the causes of the low productivity of periodic fermentation is to a considerable extent eliminated. This is achieved by the fact that the period of principal fermentation of each subsequent fermenting sample begins almost immediately after the period of principal fermentation of the preceding sample. The latter proves possible because, in the method of fermentation under consideration, not only one, as in periodic fermentation, but several batches immediately following one another are at work simultaneously.

in the other vessel being mashed. In one of them fermentation is only just beginning, whereas in the others it is already coming to an end; and between these extreme samples there are always intermediate ones, in which the process is in the stage of principal fermentation. Owing to this, in a given series of successively mashed samples, the fermentation process in several of them will always, at each separate moment, be in the stage of principal fermentation. This circumstance gave grounds for calling the Forward-Schalk method a method of “continuous fermentation.”

But this continuity refers only to the phenomenon of the reproduction of the period of principal fermentation throughout the entire series of mashed samples, regarded as something whole; for this, however, there are in reality no grounds whatever. For when each individual sample of this series is mashed in the usual manner, in which each fermentation vessel nevertheless operates periodically, with all the features and disadvantages inherent in this method of mashing. Consequently, the second cause of the low degree of utilization of working time and of the operating apparatus, inherent in all methods of periodic mashing, is not eliminated by the Forward-Schalk method, but only partly mitigated. The latter is achieved in the present case only by shortening the duration of the period of development of the fermentation process, which is a consequence of the large inoculations of yeast present here.

As regards the negative aspects of the last period of mashing, connected with the gradual and slow dying down of the fermentation process that takes place in each individual mashed sample, in this respect the Forward-Schalk method and analogous methods in no way differ from the corresponding conditions of ordinary periodic mashing. In both cases the final period is equally prolonged and is associated with an unproductive expenditure of working time and with unproductive use of the fermentation apparatus.

Thus, in the Forward-Schalk method, despite the continuity in respect to the reproduction of the fermentation process in the form of its highest manifestation in the various samples of the mashed series of them, there nevertheless remains entirely the periodicity of the action of each fermentation apparatus, which here is each separately taken fermentation vessel.

Meanwhile, this circumstance is one of the most important causes that do not permit the maximum to be attained, both in terms of the utilization of time and in respect to—

...of using a fermenting apparatus. The latter is possible only when, first, continuity in the operation of each fermenting apparatus is ensured and, second, when it is possible constantly to maintain this operation of the apparatus at the level of its highest productivity. These conditions are not satisfied either by methods of periodic fermentation or by the so-called methods of “continuous fermentation” of the type of F. Foward-Schalk’s and analogous methods, each of which operates with a greater or lesser number of separate, independently but nevertheless periodically functioning fermenting apparatuses. In connection with this there is yet another negative aspect of all such methods, consisting in the impossibility, during separate periods of fermentation, of using the full productive power of the available mass of working yeast. This is due to the rapid change in the fermentation conditions over the separate periods, which the adaptability of the yeast does not have time to follow. As a result, weak, dying generations appear, which in turn exert a negative influence on the course of fermentation. Their presence in the fermenting liquid compels one to take them into account, and this to a significant degree constrains, in the sense of possibility, the creation of optimal conditions for the operation of the active fraction of the yeast in each period. The latter circumstance is to some extent eliminated by those fermentation methods in which the entire process is divided into separate periods carried out independently, each of them being preceded by the removal of the mass of undesirable yeast fractions.

But if the separated yeast proves unsuitable and inappropriate for the fermentation conditions of the subsequent period, then, in a situation corresponding to that period, it represents valuable workers. And therefore, by maintaining conditions that meet the requirements of the given yeast fraction, we preserve its working capacity, eliminate dying off, and, together with this, avoid the harmful influence of the products of the vital activity of perishing yeast and of the decomposition products of yeast that have been deprived of life on the active working fraction of them.

During the fermentation of a definite quantity of wort, or of another substrate, the fermentation process in a given sample proceeds continuously until all the fermentable sugar has disappeared. In parallel, the fermented medium changes; its composition and properties at each separate moment create for the work of the yeast a new, gradually changing environment. Thus—

therefore, to maintain definite conditions with respect to the composition and properties of the medium in which fermentation is taking place—proceeding according to the diagram of drawing No. 1—would be equivalent to a complete cessation of this process, which in any case does not constitute the desired solution of the given question.

Such might be the situation in which, from each separate fraction of yeast requiring the corresponding setting and medium, the other is continuously brought into the form of the fermentable substrate with the corresponding degree of fermentation and at the temperature required by the given generation of yeast; the products of fermentation, however, are continuously removed.

This creates the possibility of repeatedly using for purposes of fermentation the entire mass of yeast obtained, which can reduce the expenditure of fermentable substance on the formation of yeast in each new portion of the fermentable substrate and, moreover, reduce the duration of fermentation, since thereby the first, low-productivity period of yeast multiplication is completely eliminated.

The realization of the indicated conditions may lead to a new method of fermentation, permitting not only the continuous conduct of the fermentation operation, but also reproduction of the fermentation process itself in a continuously operating fermenting apparatus. Two cases are possible here. In the first of them, the yeast cells that form are retained in a certain space of the fermenting apparatus, through which the fermentable wort flows at a known rate. The magnitude of the rate is determined by the duration of contact between the yeast and the wort necessary in order, under the given conditions, to attain during this time the corresponding degree of fermentation. Such a formulation of the question embraces only one aspect of it, namely the repeated use of the yeast mass that has formed. The other, no less important aspect is not touched upon at all—namely, the division of the mass of working yeast into separate fractions and the placement of each of them under conditions appropriate as to the temperature and composition of the medium in which it remains. Thus, what is characteristic of this method of fermentation is the complete confinement of the entire mass of yeast to a definite space through which the fermentable substrate flows continuously. This method is used in the production of vinegar; the negative aspects of its application to alcoholic fermentation, however, follow definitely from what has been said earlier.

The second possible case of continuous fermentation in a continuously operating fermentation apparatus makes it possible to effect only a relative immobility of the yeast, which, in this case, like the mash, but only at a lower velocity, moves through a definite space constituting the fermentation apparatus. In practice, such a situation could be realized by forcing the fermenting mash to flow through a series of vessels connected with one another by narrow tubes. After the entire system has been filled, it is continuously supplied with fermentable wort, entering at a known rate into the initial vessel and leaving the system through the final one.

In this case the yeast present in the fermenting liquid is displaced not only under the influence of the liquid flowing through the vessels in the direction of motion of the latter, but also independently from below upward, and back again downward, by the fermentation it induces. Owing to this, the yeast is to a certain extent kept back from the movement of the liquid and therefore accumulates in the fermentation vessels, partly being retained in them. In addition, some portion of the yeast settles at the bottom of the fermentation vessels, which, of course, hinders its motion both in the vertical direction and in the direction of the flow of the fermenting liquid.

This settling is the result of, first, the process of fermentation itself, associated with a change in the physiological state of the yeast and in the physical properties of the fermenting liquid; and, second, of the immediate cause of yeast sedimentation, which takes place not at once, but gradually; moreover, in each separate stage corresponding to its degree of fermentation, an appropriate fraction of yeast is separated out. Owing to this, on the path of successively connected vessels of the apparatus, from each preceding one into the next there passes not the whole mass of yeast, but only a definite fraction, the one most adapted to the conditions of the fermenting medium in the given vessel. With continuous fermentation this medium in each separate vessel of the apparatus is constantly maintained by the flow of wort, and thus the yeast of each fraction remains for a prolonged time under identical conditions; as a result, owing to this, it can sufficiently adapt itself and develop the maximum of its productive powers under the given conditions. This is one of the important advantages of fermentation in a continuously operating apparatus of the second type indicated by us, something not present in fermentation apparatuses operating periodically. All this leads to the conclusion-

… that, under the conditions of continuous fermentation under consideration, in a continuously operating apparatus there may occur a fractionation of the yeasts, which are then distributed among the separate vessels of the apparatus according to their properties; at the same time, to each of these fractions there is continuously supplied a medium corresponding to its requirements.

The first possible and examined case of continuous fermentation, in which all the yeast is retained entirely within a definite space of the fermentation apparatus, possesses certain negative aspects inherent in batch fermentation. They are a consequence of the joint presence of the entire mass of active yeast in the substrate being fermented. This circumstance compels one to abandon such a form of fermentation; therefore, in the subsequent study of the conditions of continuous fermentation, the form chosen was that which corresponds to the second case discussed, tending only toward the relative immobility of the yeast as compared with the motion of the fermenting liquid.

CHAPTER IV.

Continuously Operating Fermentation Apparatuses of the S. V. Lebedev System

In studying the phenomenon of alcoholic fermentation under conditions of continuous fermentation, meeting the above-stated requirements, one must first of all confront the question of the appropriate fermentation apparatus. The scheme of such an apparatus was already outlined earlier; it follows logically from the stated conditions of fermentation and leads to such a fermentation apparatus as represents a series of communicating vessels. But, in addition to the requirements that determine the apparatus in its general features, established by the characteristic peculiarities of the selected method of fermentation, in designing it one must also take into account many details and incidental circumstances that have important practical significance. Failure to observe the latter may lead to the fact that, alongside the phenomenon being studied, chance occurrences may arise which, under certain conditions, will interfere with the observations, obscuring the phenomenon under study. Such a circumstance, with which in the present case one must above all reckon, is the possibility of infection. To eliminate the latter, the individual vessels of the fermentation apparatus must be closed and supplied with appropriate outlets for the carbon dioxide that is formed.

At the same time, the vessel to the apparatus must be placed under conditions that make it possible conveniently and precisely to regulate it, while completely eliminating the possibility of infection caused by the apparatus of the sterile fermentable substrate.

For each individual vessel of the fermentation apparatus, a shape is desirable that would ensure such a layer of liquid as would make it possible to draw off the fermenting mash from a level sufficiently remote both from the surface of the fermenting liquid and from the bottom of the fermentation vessel. This is important from the standpoint of the possibility of removing from the liquid being drawn off undesirable fractions of yeast, such as, for example, floating on the surface or settled yeast.

The indicated requirement is best fulfilled with a cylindrical shape with a diameter smaller than the height.

corresponding to the working capacity of the vessel. In view of this, in constructing the apparatus its separate vessels were given the form of tall cylinders.

In choosing the dimensions of the vessels of the fermentation apparatus, it was necessary to strive to reduce their working volume; this was dictated by the need to be economical in the consumption of the fermenting substrate. But it was also impossible to go beyond certain limits in this direction, since the contents of each separate fermentation vessel nevertheless had to be quite sufficient to make possible all the necessary tests both of the fermenting liquid from each vessel of the apparatus and of the yeast present in it. In addition, it was necessary to reckon with the need to be able to introduce into the apparatus relatively small quantities of liquid, quite uniformly, over a very long interval of time. A working capacity satisfying these requirements is a volume of 350–400 cubic centimeters. In view of the possibility, in some cases, of vigorous foaming, the total capacity of each fermentation vessel is increased approximately twofold in comparison with the working capacity selected for it.

Drawing No. 8.

For the possibility of observing the course of the fermentation process in the individual vessels and at different periods of operation of the fermentation apparatus, the vessels of the latter were initially made of glass. While affording great convenience with respect to the possibility of observing the external changing signs that characterize the fermentation process at its separate stages, the glass vessels, however, greatly hinder the work of sterilization, assembly, and preparation of the apparatus for fermentation. In view of this, subsequently, when the phenomenon had already been sufficiently grasped, the glass fermentation vessels of the apparatus were replaced by corresponding copper ones, tinned on the inside.

The form and dimensions of each separate fermentation vessel of the apparatus are presented in Drawing No. 8.

The diameter of the vessel is 65 mm, height 340 mm. Tube \(p\) is intended for the inflow of the fermenting liquid; for the discharge, however,

the last, after passing through the vessel, is the tube orst. In each vessel, taken separately, the degree of filling is determined by the height of the level of the upper part of the overflow elbow st of the discharge tube. In the same way, when the fermentation vessels are connected successively with one another, the moment of final filling of each of them is determined by the level of the liquid surface in the last vessel.

During the fermentation of the wort, foam is formed in the apparatus, and not only inside the vessels, but also in the transfer elbows of the discharge tubes, on the surface of the overflowing liquid. The presence of foam here, as experience has shown, greatly hampers the operation of the apparatus and may in some cases even completely disturb the regularity of the transfer of liquid from vessel to vessel, with which the normal course of operation of the entire fermentation apparatus is closely connected. It was therefore necessary to take measures to remove the level at which the liquid passes in the transfer tubes from the surface of the fermenting liquid, on which the foam accumulates. This is achieved by placing the level of the upper part of the lateral elbow of the discharge tube of the last fermentation vessel of the apparatus somewhat higher than in all the preceding vessels. The upper part of the tube orst opens into the gas space of the fermentation vessel, which serves for the removal of the carbon dioxide formed in the transfer tube; otherwise, over time, it would accumulate here and, by its presence, would disturb the regularity of the operation of the fermentation apparatus. The internal diameter both of the feed tubes and of the discharge tubes is about 6 mm. Each fermentation vessel is closed with a wooden stopper with a T-shaped glass tube for the removal of the carbon dioxide that forms. These tubes in the adjacent fermentation vessels are connected with one another by rubber. The free elbow of the T-shaped tube of the last fermentation vessel is connected by rubber either with a fermentation lock containing sulfuric acid, or with a three-way glass tube. One end of the latter is connected with the gas outlet of the receiver of the fermented wort, and the other with Drechsel bottles containing a small amount of water, through which the carbon dioxide formed in the apparatus passes. The feeding vessel is a somewhat modified copper, tinned on the inside, Carlsberg flask, equipped with a water gauge and consisting of a copper tinned vessel with a capacity of 10–15 liters, the shape of which is shown in drawing No. 9.

A caoutchouc tube is fitted onto the upper outlet tube of the feeding vessel; upon completion of sterilization of the liquid intended for fermentation, it is closed with a cotton biological filter. Onto the lower drain tube, located \(1\frac{1}{2}\) vershoks above the bottom of the feeding vessel, a thick-walled caoutchouc tube is fitted

Drawing No. 9.    Drawing No. 10.

with an internal diameter of 3–4 mm. During sterilization, or when the sterile wort is kept in the vessel, this outlet rubber tube is closed with a glass, fire-polished rod and, in addition, carries a strong screw clamp. When the feeding vessel is in operation and connected with a continuously operating fermentation apparatus, its outlet rubber tube is connected with the inlet tube of the transfer control funnel. The latter is placed between the outlet opening of the feeding vessel and the inlet tube of the first fermentation vessel of the apparatus. Drawing No. 10 gives an idea of the form and dimensions of the transfer control funnel.

The feed tube \(e\) has an external diameter of 8 mm; the outlet narrow aperture of its drawn-out end \(k\), however, is no more than 0.5 mm. This latter circumstance makes it possible to introduce the nutrient wort into the fermentation vessel in very small drops. The thick-walled rubber of the outlet tube of the feeding vessel, the screw clamp on it, and, finally, the very narrow opening of the outlet end of the feed tube of the control receiving funnel—all this makes it possible to vary the inflow of wort within the widest limits, setting it with great precision by the number of drops per minute to any hourly rate. Control of the strength of the wort flow is carried out simply and conveniently by counting drops per minute, which, with the glass transfer funnel, can be done at any moment without any difficulty. The carbon dioxide formed during fermentation in the transfer control funnel is removed through its tube \(d\), connected by rubber with one of the ends of the T-shaped outlet gas tube of the first fermentation vessel of the apparatus. The significance of the receiving control funnel is reduced to the following three important points: regulation of the inflow of wort, control of its magnitude, and elimination of the possibility of infection of the sterile liquid fed to the apparatus.

After passing through the fermentation apparatus, the fermented liquid from the outlet tube of its last vessel is directed through a rubber hose into a receiver. The latter is, depending on the purpose of the experiment, either a measuring cylinder closed with a cotton plug, or an ordinary glass bottle of suitable capacity. The receiving bottle is closed with a stopper through which pass two glass tubes, whose arrangement is shown in drawing No. 11.

One of them, \(st\), lowered further down, serves for the inflow of fermented wort and is connected by rubber with the drain tube of the last fermentation vessel of the apparatus. Tube \(m\) serves to remove from the receiver the carbon dioxide developing in it, which from there is directed through the previously mentioned three-way tube to Drechsel bottles with water.

The general view of the described fermentation apparatus, designated as type A, is shown in assembled form in drawing No. 12.

Drawing No. 12.

Labels in the drawing: Gas; Air; Water outlet; Water.

Continuously operating fermentation apparatus of the S. V. Lebedev system in assembled form.
(Type—A.)

In the last fermentation vessel of the apparatus, the upper part of the lateral branch of the outlet tube, denoted by the letter \(w\), as already indicated, is situated higher than in the other fermentation vessels. The individual parts of the apparatus are connected with one another by means of rubber tubes.

To maintain a definite fermentation temperature during the experiment, all the fermentation vessels are placed in a water bath, which is a long metal box, the side walls and most of the bottom of which, except for a small central area, are covered with insulating sheathing. The box has a false perforated bottom, placed 2 cm above the actual bottom. Between the two, along the length of the bath, runs a thin tube with a large number of openings, bent double in the horizontal plane. This tube serves for the inflow of air, by which, during the experiment, the water in the bath is continuously stirred. The temperature is maintained by a small flame of a gas burner connected to a thermoregulator placed in the water of the bath.

Under these conditions, during the experiment the temperature fluctuations were within about \(0.5\)—\(1.0^\circ\mathrm{C}\), and only at low fermentation temperatures, owing to the relatively sharp fluctuations in the fermentation room, did these fluctuations reach, in all over the entire fermentation period, two degrees.

The outlet tube of the last fermentation vessel of the apparatus passes through a rubber stopper fitting tightly into an opening in the wall of the bath at the corresponding height. Along the upper edges of the longitudinal walls of the bath, opposite one another in pairs, wire hooks are placed. By means of each pair of them, situated opposite one another, and also by means of a cord fastened at its ends to the hooks and encircling the neck of the fermentation vessel, each of the latter is drawn tightly to the false bottom of the bath.

This is necessary so that, when the bath is filled with water, the fermentation vessels do not float up, but, while retaining their proper position, at the same time remain under water for the known part of their height that is filled with the fermenting liquid. To observe the temperature and its fluctuations during the experiments, minimum and maximum thermometers are placed in the bath.

The preparation of the parts of the apparatus for its assembly consisted in the following. The feeding vessel is half-filled with water, heated, and boiled for one hour.

Then the rubber sleeve placed on the upper outlet metal tube of the vessel is closed with a cotton biological filter. After cooling, the water is drained from the vessel through the lower discharge opening, and it is filled with sterile wort, freed by settling from the insoluble substances that had precipitated during the preliminary sterilization. After the feeding vessel has been filled with wort, the latter is sterilized twice, at an interval of one day, by heating and boiling for one hour in a sand bath. After each boiling, the rubber sleeve on the upper outlet tube of the vessel is closed with a fresh cotton biological filter. The fermentation vessels and the receiving bottles for the fermented wort are tightly closed with well-fitted and lapped stoppers, with the corresponding outlet glass tubes inserted into them. All openings of the vessels and of the outlet tubes are closed with cotton plugs. The same is done with the openings of the transfer control funnel. After this, all these glass parts of the fermentation apparatus are placed in a cold dry sterilizer. The latter is gradually heated with a gas burner to 170° C., and at this temperature all its contents are left for one hour. After cooling, everything is removed from the sterilizer and is ready for assembly, which is carried out by connecting the separate parts of the apparatus with rubber tubes.

Before connecting the parts of the apparatus, the rubber tubing intended for this purpose is first sterilized for one hour with live steam. For this, a large glass flask is taken and closed with a rubber stopper into which an outlet glass elbow tube has been inserted; onto this tube is fitted the rubber tube of the appropriate diameter and sufficient length needed for the connection. The flask thus equipped is brought to boiling by heating on a strong gas burner. Then, for one hour, the steam issuing from the flask and passing through the rubber tube sterilizes it. After this, without ceasing the vigorous heating of the tube, pieces of the required length are cut from the latter with scissors; these are immediately used to connect to one another the separate tubes of the various parts of the fermentation apparatus. In doing so, the cotton plugs that closed the openings during sterilization are removed after flaming; the ends of the glass tubes and of the cut rubber are flamed, and, with all the necessary precautions and the requisite ...

with a rapid operation, the cotton plugs are replaced by sterilized rubber tubes connecting them.

In this way all the separate parts of the fermentation apparatus are connected with one another, except for the transfer control funnel. The latter is first of all connected to the feed vessel containing the wort intended for fermentation. This is done as follows. A screw clamp is tightly closed on the thick-walled rubber fitted over the outlet tube of the feed vessel, with the end of a glass rod inserted into the rubber placed beside it. The previously singed cotton plug closing the opening of the inlet tube of the transfer control funnel is removed; the end of the tube is flamed and, with the necessary precautions, is quickly inserted, instead of the glass rod, into the thick-walled discharge rubber of the feed vessel. After this, the lower end of the attached transfer control funnel is raised so that it assumes a vertical position with its upper end downward, and the whole is then located above the level of the discharge opening of the feed vessel. Maintaining this position, the inlet tube of the transfer funnel is filled with wort by cautiously opening the screw clamp. This must be done so that the wort in the tube reaches exactly to the outlet opening, without overflowing beyond it and at the same time displacing all the air filling the tube.

Complete removal of the latter from the inlet tube of the transfer funnel is an indispensable condition for making it possible to establish a uniform inflow of wort and to regulate it accurately thereafter.

Then the transfer control funnel is lowered. The feed vessel is placed in the position intended for it during the experiment, and only after this is the lower end of the transfer funnel flamed, freed from the cotton plug, and, in the order already indicated, connected by sterile rubber to the inlet opening of the first fermentation vessel.

After this the fermentation apparatus is ready for inoculation with yeast.

The yeast intended for the experiment is seeded by transfer and by preliminary two-day fermentation in a thermostat at 25° C. This fermentation is carried out in a Freudenreich flask containing 30 cubic centimeters of sterile beer wort, into which the yeast is inoculated with an eye of platinum wire. After two days of fermentation, the fermented wort, under the corresponding

the precautions, is poured off from the precipitate that has formed. The latter is shaken with the small quantity of liquid remaining. From this, with a small sterile pipette, a mass of yeast is taken, and 1–2 drops of this culture are introduced, with the necessary precautions, into the first fermentation vessel of the apparatus, namely into the vessel that is directly connected with the transfer control funnel.

The glass pipette by which the yeast is inoculated is sterilized before use. For this purpose its wide end is closed with a small piece of cotton wool introduced into the opening. The pipette itself is wrapped in paper. The resulting packet is tied with thread and sterilized for an hour at \(170^\circ\) C.

After the inoculation of the yeast, the sterilized rubber gas-outlet tubes of the transfer control funnel and of the first fermentation vessel of the apparatus are connected. This completes the assembly of the apparatus. Then the screw clamp on the rubber delivery tube of the feed vessel is immediately opened, and in this way the first fermentation vessel of the apparatus is filled with wort, after which the inflow of wort is stopped. The water bath is filled with water of the appropriate temperature, maintained during the experiment by means of a burner for thermostats connected to a gas thermoregulator immersed in the water of the bath of the fermentation apparatus. Temperature uniformity is also ensured by continuous stirring of the water, which is produced by passing air brought through the small openings of a tube laid along the bottom of the bath under its false bottom. Minimum and maximum thermometers are immersed in the bath, from whose readings a record is kept of the temperature and its fluctuations during the experiment. After the inoculation of the yeast and the filling of the first fermentation apparatus, fermentation begins. Initially it proceeds without an inflow of wort. When, however, the dark-colored discharges that have coagulated begin to be carried to the surface of the foam formed during fermentation, the screw clamp on the thick-walled rubber delivery tube of the feed vessel is opened, and in this way a continuous inflow of wort to the apparatus is established. The strength of the wort flow is determined by the number of drops per minute and is regulated by opening the screw clamp to a greater or lesser extent.

Thus the fermentation apparatus is put into operation, and thereafter its work proceeds continuously under constant conditions with respect to temperature and the strength of the wort flow.

When all the wort intended for the experiment has fermented, the experiment ends and the apparatus is taken apart. Its disassembly, depending on the aims pursued by the experiment, may be carried out in different ways. When it is desirable to trace which stages of fermentation correspond to the individual vessels of the apparatus, how the mass of yeast being formed is distributed in them, etc., then in this case, at the end of the experiment, each fermentation vessel of the apparatus must be removed from their common circuit, and its contents analyzed separately. The disassembly of the fermentation apparatus is conducted in accordance with this requirement.

When the experiment is finished, first of all the thick-walled rubber drain tube of the feeding vessel is closed by a screw clamp. The water is drained from the bath. In the middle of each rubber tube connecting the outlet tube of the preceding vessel with the inlet tube of the following fermentation vessel of the apparatus, two strong clamps are placed side by side, by which the connecting rubber tubes are tightly compressed. The clamps are arranged in the same way between the inlet control funnel and the first fermentation vessel, and also between the receiver and the last fermentation vessel.

After this, between each two adjacent clamps placed side by side, the rubber tubes are cut, and in this way the fermentation apparatus is divided into the separate elements composing it. Then the contents of each fermentation vessel are carefully decanted from the yeast sediment into a dry paper folded dense filter, Schleicher-Schüll factory No. 572½, placed in a glass funnel with ground edges. To remove the possibility of evaporation during filtration, the funnel is covered with glass. A dry glass flask serves as the receiver for filtration. In the filtered fermented wort, the contents of extract, alcohol, and, in some cases, nitrogen are determined by the methods already indicated, the last by Kjeldahl’s method.

When it is necessary to determine the amount of yeast formed, the following is done. The yeast sediment from the fermentation vessel remaining on the filter after decanting the fermented wort is, with repeated rinsing with water, transferred quantitatively into a tall glass cylinder having a tightly fitting stopper. Into the same cylinder, after the main mass of liquid has drained from the filter, the remnants of the last yeast retained by the filter are transferred. For this purpose the funnel with the filter is transferred onto the cylinder. At the end of the filter, with a thin glass rod, an opening is made—

the opening, through which the entire quantity of yeast remaining on the filter is washed quantitatively into the cylinder. The washing of the yeast was carried out by means of a very fine, strong jet of water, obtained by fitting a rubber hose onto a water tap, into which a glass tube with a very narrow drawn-out end is inserted. By opening the water tap to a greater or lesser extent one can obtain a jet of water of any desired force. Under these conditions, with the filters indicated, the washing of the yeast into the cylinder proceeds easily and quickly and requires only the most insignificant amount of water.

The cylinder with the yeast transferred into it is filled with cooled water, to which is added such a quantity of a normal solution of sulfuric acid as is needed so that in the cylinder a liquid is obtained with an acidity corresponding approximately to \(2^\circ\) Delbrück. The purpose of this is to eliminate the possibility of the development of infectious organisms, and also to suppress the growth and fermentation of the collected yeast. After this, the cylinder, filled up with water, is shaken vigorously and placed for 10–12 hours in a bath with melting ice. During this interval the yeast settles as a dense layer. The clarified liquid is carefully decanted, and the yeast is separated from the remaining liquid on a Nutsche apparatus; here it is immediately washed with cold water, the excess of which is removed by repeated squeezing between layers of filter paper and by pressing under a definite pressure. The quantity of yeast thus obtained is determined by weighing to an accuracy of 0.1 g. In addition, the nitrogen content in it is determined by the Kjeldahl method.

The fermented wort entering the receiver is subjected to the same treatment and analogous analysis. Its examination is carried out by separate samples as they accumulate, the receiver being changed at the same time. In the initial fermenting wort the extract content and nitrogen are determined by the Kjeldahl method.

Another method of dismantling the fermentation apparatus was used in those experiments whose purpose was to determine the balance of the fermentable substance and the magnitude of its loss during fermentation. Under these conditions, the entire feeding vessel is determined by weighing to an accuracy of 0.1 g before the experiment and after it, whereby the quantity of initial sweet wort subjected to fermentation is established.

At the end of the experiment, first, in the order already indicated, only the feeding vessel and the receiver of the fermented wort are separated.

After this, the contents of the undismantled fermentation apparatus are drained through the outlet tube and the rubber tube of the last fermentation vessel into a glass bottle weighed to an accuracy of 0.1 g. The draining is carried out by raising the end of the bath containing the fermentation vessels mounted in it where the first of them is placed. Owing to this, the liquid flows from vessel to vessel through the connecting tubes and is directed into the receiving bottle set up for collection. Next comes the washing of the apparatus; for this purpose the wash water is directed first into the first, then into the second and the following fermentation vessels. The washings are directed into the same bottle into which the fermented mash was drained. When the apparatus proves to be sufficiently washed, it is dismantled in the order indicated earlier, and each part of it is then washed separately with a thin, strong stream of water. All the washings are transferred, without loss, again into the receiving bottle, into which there also enters the fermented mash from the receivers and the water from Drechsel bottles. The order of dismantling and washing in this case is fully determined by the need to transfer the entire contents of the fermentation apparatus quantitatively into the receiving weighed bottle.

The difference in the weights of the latter before and after filling gives the weight of the solution of fermented mash together with the washings and the yeast. By reducing the obtained value by the weight of the yeast, the amount of which is determined, as indicated earlier, we obtain the weight of only the solution of fermented mash. Having determined by analysis the composition of the latter and knowing its weight, we obtain the weight quantities of the alcohol and extract contained in it. And since the amount of extract subjected to fermentation has also been determined, all this makes it possible to establish the magnitude of the loss of extract during fermentation. This latter is also the aim of the experiments in which the dismantling of the fermentation apparatus is carried out only in the manner described.

It is not difficult to see that a necessary condition for the success of experiments of this kind is the accuracy of the quantitative determination of the extract and alcohol in the fermented mash. Meanwhile, the indicated construction of the glass fermentation apparatus of type A, in connection with its dismantling, presents considerable difficulties in this respect as regards the possibility of the practical execution of such experiments. In view of this, for experiments whose aim is to ascertain the magnitude of the loss during fermentation, another continuously operating fermentation apparatus of type B was constructed.

Drawing No. 13.

Section along KK and LL

Section along AA′ BB′

Section along CD

Section along EF

Apparatus cover.

Continuously operating fermentation apparatus of the system of S. V. Lebedev.
(Type—B.)

The construction of this apparatus is shown in Drawing No. 13, and its arrangement during operation is visible from Drawing No. 14.

Drawing No. 14.

Continuously operating fermenting apparatus of the system of S. V. Lebedev, in assembled view.
(Type—B.)

In this apparatus of type—B, the changes concerned only the form, but not the essence of the matter, which remains the same as in the fermenting apparatus of type—A. The fermenting apparatus of type—B is a long, tall, and narrow rectangular closed box, made of tinned copper. This entire

1. Jahrb. d. Ver. d. Spir. Ind. 1911, pp. 266–267. ↩↩↩↩↩↩↩↩↩↩↩↩↩↩↩↩↩↩

2. “Gärungsbakt. Praktikum” Henneberg, pp. 240 and 244. ↩↩↩↩↩↩↩↩↩↩↩

3. “Handb. d. Techn. Myk.” Lafar IV. p. 96. ↩↩↩↩↩↩

4. Wochenschr. f. Brauer. 1911, p. 182. ↩↩↩↩↩↩

5. Ibid., 1910, p. 288. ↩↩↩↩

6. “Gärungsbakt. Praktikum” Henneberg 1909, p. 200. ↩↩↩↩

7. Wochenschr. f. Brauer. 1911, p. 182. ↩↩↩

8. Ann. de chim. et. de phys., 1860 (3e [[unclear: series/page obscured]]). ↩↩