REPORT

on an overseas mission in 1900.

Professor of the Imperial Tomsk University

V. V. Sapozhnikov.

On the initiative of the Tomsk Technological Institute, I was assigned for the autumn semester of 1900 to travel abroad for the practical study of modern methods of zymotechnology, and chiefly of methods of pure cultures. On the advice of the Director of the Institute, E. L. Zubashev, I chose the institutes of Dr. Jørgensen in Copenhagen and Prof. Wortmann in Geisenheim. In view of the limited duration of the mission, I did not set myself the goal of undertaking any special work, but limited my task specifically to a thorough acquaintance with the methods already developed. Therefore, concentrating my chief attention on the two institutes mentioned, and devoting to them the greater part of the time that was at my disposal, I only incidentally inspected several laboratories in Paris, Berlin, Munich, Nancy, and others. The present article is a report on my activities during the overseas mission and, chiefly, a description of the two stations mentioned.

I. Dr. Jørgensen’s Zymotechnical Laboratory in Copenhagen.

In Copenhagen there are two zymotechnical laboratories; one is attached to the enormous Carlsberg brewery, under the direction of Dr. Hansen; the other is directed by Dr. Jørgensen. The first is one of the most extensive and well-endowed institutions of this kind, equipped with the newest apparatus for special work in zymotechnology; but this laboratory does not accept beginners, giving place only to specialists who already possess the necessary methods for independent investigations; consequently there are very few trainees here. Jørgensen’s laboratory pursues a dual aim and consists of two substantially different departments. In the technical department

pure yeast cultures (chiefly brewing yeasts) are prepared for sale and for distribution to various factories. In the instructional department, brewers (and, in general, all those interested) are trained in methods of investigating lower organisms and preparing pure cultures. In summer one can find here a motley crowd of young people, up to 18 in number, accommodated in three small rooms. But the cramped premises are considerably redeemed by a well-devised program of studies, adapted to the various needs of the practical brewer, insofar as yeast is concerned. For people wholly inexperienced in microscopic investigations, the length of the course is set at 2½ months; but for those familiar with the microscope the course can, without particular detriment, be shortened to 1½ months. The fee in favor of the laboratory is 100 kroner per month and 10 kr. for a microscope, if the trainee does not have one of his own.

Under the immediate supervision of the head of the instructional department and of the assistant (in 1900 these were Dr. Holm and Mr. Brickfeldt), the trainees complete the following program *).

The work begins with acquaintance with the most common molds (Oidium, Penicillium, Aspergillus, Mucor, Thamnidium, Botrytis, Dematium, Cladosporium, and others). The aim of these investigations is to learn how to distinguish fungi which not infrequently contaminate yeast and may cause undesirable changes in fermentation products. Usually three kinds of cultures are set up: in beer wort in a Freudenreich flask, in a moist chamber, and on gelatin with wort, for macroscopic observation of the developing colonies. All work of transferring cultures is carried out in a special glass cabinet into which the necessary instruments are placed. The inside of the cabinet is sterilized with a solution of sublimate (1 in 500). By slightly raising the front wall of the cabinet and putting one’s hands inside, one can manipulate the cultures quite conveniently. Glass rods and other auxiliary instruments are sterilized in the flame of a gas burner.

Having become acquainted with the development of the vegetative cells and the separation of conidia in the forms listed, they proceed to fungi which, in the form of their cells, stand closer to yeasts and, to the inexperienced eye, may be confused with the latter; such are Monilia, Chalara, Mycoderma, Torula, and Saccharamyces apiculatus. After them come the true species of Saccharamyces, first the wild ones (cerevisiae, pastorianus, elipsoideus, fragilis, Ludwigii, membranifaciens, anomalus, exiguus, and others), and then also races of cultured yeasts (distilling yeasts, top- and bottom-fermenting brewing yeasts, species of wine yeasts).

*) Explanations in the laboratory can be given in Danish, Swedish, German, and English; and Dr. Jorgensen also understands French.

In studying yeasts, in addition to observations on the development of vegetative cells, the conditions of spore formation are determined; for this purpose the culture in beer wort, after the excess liquid has been poured off, is transferred to a gypsum plate placed in a Petri dish. The rate of formation of spores forms the basis of the analytical table for determining the race of yeasts, namely:

Note. Beer yeasts of bottom fermentation form spores after 5–7 days of culture on a gypsum plate.

Besides yeasts and mold fungi, the program of the trainees includes acquaintance with certain species of bacteria that are of importance in technology, such as Bacterium aceti, Pasteurianum, Kutzingianum, species of Sarcina, Pediococcus, and others.

When the trainee has become sufficiently acquainted with the organisms listed, he is given simple analyses containing 2–3 species, which he must identify. In all, up to 50 analyses are given, and, if time permits, even more.

An important part of the practical work in Jørgensen’s laboratory is acquaintance with the methods of pure cultures of yeasts.

Pure cultures constitute a necessary condition of rational fermentation technology; without them one can sometimes still manage in winemaking, but for proper brewing they are obligatory. Pasteur was the first to demonstrate the important significance of the purity of yeast culture in his classic work Étude sur la bière; investigating diseases of wine and beer, he found their cause in the presence in the products of fermentation of foreign organisms, namely bacteria; but only half of the conditions of the disease had been revealed by him; a very important circumstance escaped him, namely that some races of wild

yeasts also exert a very unfavorable influence on the quality of the product; such are the species apiculatus, pastorianus, and others. The honor of this discovery belongs to the Danish scientist Hansen. Consequently, by a pure culture of yeasts in the modern sense one must understand a culture of a definite race, without admixture not only of bacteria and molds, but also of yeasts of some other race. Only when yeasts of a definite race, suited to the given particular case, are put into operation can the technician be assured of the good quality of the product produced by them.

The basic requirement in preparing pure cultures is to obtain progeny of yeasts from a single, individual cell. This requirement is fully met by Hansen’s most commonly used method, known as the dilution method) (Verdünnungsmethode). They begin by subjecting the given culture to fractionation; for this purpose, with a platinum loop, they take a drop of the culture and transfer it onto a cover glass on which a grid of 25 divisions has been engraved and, placing it in a moist chamber, count the number of yeast cells in one drop. After this, one or several drops of the same culture are diluted with sterilized water to such an extent that in the diluted culture there is one cell in every other drop of water. For a more regular distribution of the cells in the liquid, the latter is shaken for ten minutes. The shaken liquid is distributed by means of a pipette, one drop at a time, into Freudenreich flasks with sterilized wort*) (up to 60). The flasks are left undisturbed for 48 hours, to allow colonies of yeasts visible to the naked eye to develop. On careful inspection it is easy to notice that white specks of colonies have appeared on the bottom of the flasks; in some flasks one each, in others two each (rarely more); there are also some that have remained uninfected. Selecting precisely those flasks in which one colony has developed, and multiplying the cultures by transferring them into large vessels with sterilized wort, we obtain pure cultures by fractionation.

However, this method is still far from perfect; here one can speak of the purity of the culture only with greater or lesser probability, because even in the case when we find one colony in a flask, it is difficult to vouch that it developed from one cell and not from two.

A further improvement in the method of pure cultures was the use of solid substrates, where the cells are fixed in a definite place, which makes it possible to observe the development of one and the same cell and its growth into a colony over the course of several days.

) This method was first applied by Lister to bacteria of lactic fermentation.
*) After 4–5 drops, the pipettes must be changed.

The method proposed by Schröter and improved by R. Koch (Plattenkultur) was perfected and applied by Hansen to pure cultures of yeasts. For this purpose one may make use of a culture already purified by the fractionation method and subject it to final purification. A drop of the culture, diluted with sterilized water, is transferred into a melted mixture of gelatin with wort; the latter is gently shaken (vigorous shaking must be avoided, so as not to cause abundant formation of air bubbles in the gelatin). A drop of gelatin with the culture is carefully spread with the blunt end of a glass rod over a cover glass bearing a grid of 16 divisions. For convenience in counting and marking, numbers are engraved within each division*). The cover glass is placed in a moist chamber; the solidified drop of gelatin is examined under the microscope, and on a ruled sheet of paper all the cells located in the various divisions of the grid are marked.

The most favorable conditions are when there is one cell in each square of the grid, although a larger number of cells may also be present, provided only that they do not lie too close to one another. In order to notice all the cells lying in the field of the microscope, a certain skill and great attentiveness are required; otherwise one may miss many cells, which will reveal themselves on the next day, when they begin to develop into colonies.

After several days (2–3) in the moist chamber, small colonies develop on the gelatin in the form of white specks visible to the naked eye. Now, at least with respect to some of them, one may be fully assured that they have developed from a single cell. Next, some of the marked colonies should be inoculated into liquid wort; for this purpose the cover glass is carefully removed from the chamber without turning it over, the chosen colony is touched with the end of a copper wire clamped in forceps, and it is dropped into a small flask with wort. The resulting sediments of the pure culture are examined under the microscope and are transferred for propagation first into small and then into large Pasteur flasks. Such is Hansen’s most perfected and widespread method of pure cultures.

Alongside this method one should mention another, less troublesome but also less exact method: the so-called drop method (Tropfenmethode) of the Berlin professor Lindner. Lindner’s method consists in taking a culture diluted with water on an ordinary steel writing pen and applying it to a cover glass in the form of small droplets (16–20), at some distance one

) See Jörgensen, Die Mikroorganismen d. Gärungsindustrie*. 1898.

from another. In this case such a degree of dilution is necessary that in each droplet there should be one cell—at any rate, only such droplets can then be admitted to the work. The cover glass is placed in a moist chamber, and the developing colonies are checked for some time under the microscope. After 2–3 days the droplet, together with the colony, may be transferred by means of a piece of filter paper into a flask with wort, etc. In addition to the speed and convenience of manipulation, this method also has the advantage that, from the form and character of the freely developing colonies, it is already possible to conclude whether we are dealing with one or several species of yeast. But the method is not free of a substantial drawback, namely: the droplets of water with the developing colonies increase at the expense of the water vapor of the moist chamber, often merge together, and in that case it is difficult to vouch that the cells have not crossed over from one droplet into another. However, contamination of the culture can be avoided by completely discarding those droplets which have merged with one another or have come too close to one another.

The course of practical work at Jørgensen’s institute usually ends with experiments in the fermentation of beer wort on pure cultures of yeasts prepared by the practical student himself (top-fermenting, bottom-fermenting, and apiculatus yeasts), for a comparative evaluation of the taste and bouquet of the beer obtained. Alongside this, the alcohol content in various products is determined with the aid of an ebullioscope.

As I have already mentioned above, the technical department of Jørgensen’s institute prepares yeasts for dispatch, and not only beer yeasts, but also distillery yeasts and yeasts for the fermentation of fruit and berry juice. For this purpose the culture is propagated in Pasteur and Carlsberg flasks, and from the latter, after the excess liquid has been poured off, is transferred into metal vessels of up to 2 liters capacity with distilled water. Well closed with a metal tap, the vessels with yeasts are sent over considerable distances and remain in good condition for several weeks (5–6).

In small quantities the culture can be transported in an even more compact and portable form, namely in a small test tube on cotton wool (everything, of course, sterilized). Two or three drops of culture, transferred onto cotton wool, gradually dry out and can be preserved in this form for several months; cultures prepared by me in this way in September were revived after arrival in Tomsk—in January, and all proved to be fully viable. Such stability of cultures in the dry state can be explained, in my opinion, by the fact that, with slow drying, spores form in the cells, which better resist

final loss of water than vegetative cells. In the same way I subsequently stored wine yeasts as well, and with equal success; I did not lose a single culture. Ten cultures of wine yeasts (duplicates), stocked in November, I transferred into grape must in July of the following year; and only two of them failed to develop—the rest proved to be fully viable. Finally, one culture of Menes’s Hungarian yeasts was renewed after a year and proved quite viable and active. In grape must, cultures can remain alive from 2–3 months to half a year, and perhaps longer. Finally, for still more prolonged preservation of cultures in laboratories, Hansen recommends using a 10% sucrose solution. Such cultures need be renewed only after a year has elapsed.

In conclusion, I visited the huge “Ni-Caresberg” brewery in Copenhagen and a small brewery in Helsingborg on the Swedish shore, where a new method of fermentation under reduced pressure has been introduced (Vacuumbottich), which is usually maintained at about half an atmosphere. The principal advantage of this method consists in shortening the time required for fermentation. The process is usually completed in 8 days.

2. The Station for Pure Yeast Culture in Geisenheim

Hansen’s method, which had produced such fruitful results in winemaking and brewing, did not remain without influence in the field of rational viticulture as well. In Germany, one of the first—the plant-physiological laboratory at the school of horticulture, fruit-growing, and viticulture in Geisenheim *)—set itself the task of carrying out the method of pure cultures as applied to the fermentation of grape juice.

But, with its modest premises and means, the scientific laboratory could not successfully satisfy the broad and urgent demands of practice, and therefore in 1894, in connection with the scientific laboratory, there was established here the “Station for the Culture of Pure Yeasts of the German Society of Winegrowers” **), with a subsidy from the Prussian Ministry of Agriculture, Domains, and Forests. The station is at present under the direction of the director of the plant-physiological laboratory, Professor Wortmann, and has almost merged with it into a single institution. The choice of site for the station proved to be as fortunate as possible: it lies in the center of the very rich winegrowing district of the Rheingau and therefore can maintain the closest possible contact with winegrowers and practitioners.

) Kgl. Lehranstalt für Obst, Wein—und Gartenbau zu Geisenheim am Rhein.
*) Hefe—Reinzuchtstation.

The tasks of the station are quite broad: on the one hand, purely scientific investigations are carried out here into the phenomena of fermentation and the work of various races of pure yeasts, as well as of other organisms whose participation must be taken into account in the fermentation of grape juice; on the other hand, the results obtained here are immediately transferred into the practice of winemaking, in order to give winemakers receiving yeasts from the station a full guarantee that the yeasts issued from here are not only pure, but that the very choice of the race has been made with reference to the given technical conditions and is based on the laboratory’s scientific investigations*). The station does not confine itself merely to issuing yeasts according to the winemakers’ requirements, but is in constant communication with the latter, receiving from them various reports from the practice of winemaking and subjecting them to new scientific elaboration, or offering this or that advice and instruction. How great the station’s connection with the population is may be judged from the fact that in 1898 it received and satisfied up to 1,500 different inquiries. In the hottest period of winemaking, i.e. in October and November, the station daily receives requests for pure yeast cultures, samples of young or unsuccessful wines for investigation, this or that inquiry, and so forth; so that two assistants and two secretaries are overwhelmed with work. For this reason the station, apart from pupils of the schools, is reluctant to accept outside trainees, limiting their number to one or two at a time**).

Following Prof. Wortmann’s instruction, my work in Geisenheim coincided with the time of the grape harvest, so that a sufficient quantity of material was always at hand. Being familiar with the morphology of yeasts from my work in Copenhagen, here I paid primary attention to the physiological side of fermentation. I wished to carry out a series of elementary experiments in which, in miniature, one could observe the principal moments of fermentation and become tangibly convinced of the significance of various factors, such as the use of pure cultures in comparison with spontaneous fermentation, the influence of the relative sugar content in the must, its acidity, and so on. Since the experiments had a purely programmatic character and their results present nothing essentially new, I report on them only in the briefest outline.

The material for my experiments was pear, apple, and chiefly grape must, supplied in abundance by the station. In all cases the must was examined for sugar and acid content, and the wine obtained, in addition, for alcohol content. Sugar was determined by titration,

*) Bericht der Kgl. Lehranstalt. 1899. p. 77.
**) Prof. Wortmann earnestly asked me to inform, on occasion, my compatriots who intend to come to Geisenheim for practical training that it is necessary to notify him of their arrival at least a month in advance; otherwise one may find oneself without a place in the laboratory.

with Fehling’s solution, acids—by titration with a \(1/10\)-normal solution of caustic potash, and alcohol—pycnometrically. The experiments were carried out in glass vessels of 1–2 liters’ capacity with glass “fermentation locks,” so that the energy of fermentation could easily be determined from the loss of weight. In parallel with these experiments I set up others, in smaller dimensions, preserving the same conditions, especially for microscopic observations, in order to remain constantly informed of events in the plant world inhabiting the must.

In the first series of experiments (6) I observed the fermentation of apple and pear juice with a pure culture of yeast and without it; moreover, the apple must was taken either natural or sugared. The differences in the process of fermentation with a pure culture and without it amount, in general, to the following.

I. With a pure culture, fermentation becomes evident after 24 hours and at the end of the second day reaches its maximum, after which the fermentation curve falls rapidly, and the main fermentation ends in 7 days; thereafter the evolution of carbon dioxide gas becomes barely noticeable. The total loss in weight over 18 days is greater than in the case of spontaneous fermentation.

II. Spontaneous fermentation becomes noticeable only toward the end of the second day, and the maximum (less intense) is shifted to the 3rd–5th day; the decline of the fermentation curve is not so rapid, and appreciable evolution of carbon dioxide gas can still be observed on the 10th day.

The amount of alcohol formed in cases I and II is approximately the same; sugaring in both cases, of course, increases the alcohol content. More acids are formed in case I, which should be reflected in the greater stability of the product obtained from fermentation with pure cultures of yeast.

The bouquet of the wine obtained with a pure culture is noticeably better, even for a layman.

In the control vessels intended for microscopic examination, one can observe a great difference, especially noticeable in the first days of fermentation. In must fermenting spontaneously, S. apiculatus, species of Torula, Dematium, Mucor, and other fungi, introduced into the must from the skin of the berries, develop abundantly, and only on the 3rd or 4th day do the true yeasts gain the upper hand over all of them. In a vessel with a pure culture, the true noble yeasts immediately take possession of the field and do not allow extraneous organisms to develop. In both cases the fermentation ends with true yeasts, because the especially widespread and harmful apiculatus cannot tolerate an alcohol content above 3%, but even its brief activity leaves its imprint on the quality of the product.

In the second series of experiments I determined the influence of various races of yeasts on the quality of wine.

For the experiments I took freshly pressed grape juice from this year’s harvest (Rissling); sugar content 25.5%. Acid content—14.25‰. The must was poured into 4 vessels, 1.5 liters each.

I was left to ferment spontaneously.

II was inoculated with a pure culture of the yeast Piesport.

III ” ” ” ” Gau Bickelheim Goldberg

IV ” ” ” ” Steinberg.

Fermentation was observed for 18 days, but during this period it had not yet ended and the wine had not begun to clear; nevertheless, samples of the wines were taken.

The curves of fermentation energy generally coincided. The maximum evolution of carbon dioxide gas occurred for II, III, and IV on the third day, for I—on the fourth. The highest rise was represented by the curve of the IIIrd vessel (Gau Bickelheim Goldberg), somewhat lower the curve of the IInd vessel (Piesport), still lower IV—(Steinberg), and finally the least of all for I (Spontan). The fall of the curves stands in inverse relation to the height of their rise. The temperature during fermentation fluctuated within the limits of 12–19° C.

The bouquets of the four wines were distinctly different, and with pure cultures considerably higher.

The composition of the wines is expressed by the following table.

In the third series of experiments I observed fermentation with different sugar contents in the juice.

The material for the experiments was sterilized grape juice with a sugar content of 12.8%. The first bottle contained 6.4% sugar; to the next five, such an amount of cane sugar was added as to obtain juice with sugar contents of 10, 20, 30, 40, and 50%. The acid content was the same in all vessels—14.2 pro mille. All 6 vessels were inoculated with a pure culture of one and the same race of yeast and left to ferment for 13 days.

The fermentation curves characterize the process as follows:

I. Fermentation began on the 2nd day, reached a maximum on the 3rd; after remaining at the same height on the fourth day, on the 5th the curve fell steeply; consequently the main fermentation passed in 5 days. Clarification of the wine began on the 8th day.

II. The curve up to the 4th day coincides with the preceding ones, but then falls much more gradually, and the main fermentation is completed only on the 7th day.

III. The beginning of fermentation is as in I and II; the maximum is higher on the 4th day, and there is a very gradual fall of the curve to the end of the experiment.

IV. The beginning of fermentation is as in I, II, and III; the maximum is on the 4th day, and in general the curve is similar to III.

V. The beginning of fermentation is on the 3rd day; a gradual rise to the maximum on the 6th day and the same gradual fall to the end of the experiment.

VI. The beginning of fermentation is on the 5th day; a weak rise of the curve to the maximum on the 7th day and a weak decline to the end of the experiment.

The following table gives the principal moments of fermentation and the composition of the resulting wine:

It is evident from the table that the most favorable conditions for fermentation and for the yield of alcohol lie between 20 and 30% sugar, with 30% already lying beyond the optimum. In the practice of Rhine winemaking, the greatest sugar content in grape must was observed in 1893, a year famous for its wines. The Riesling variety yielded must with a sugar content above 35%. The wine fermented very slowly; in the first year the alcohol content reached barely 5%, and the slow fermentation continued into the second and third years, after which still not all the sugar had been consumed. The best wines of 1893 reached a price of up to 100 marks per bottle.

In the last series of experiments I studied the influence of the acidity of grape must on fermentation. Sterilized grape juice was distributed into 6 vessels of 400 cc each, and, with all other conditions kept equal, a gradually increasing acidity was established in the different vessels, from weakly alkaline to 50 pro mille.

Fermentation continued for 12 days, except in vessel I (weakly alkaline), where there was no fermentation at all. During this time all the sugar was consumed. The total loss in weight in all the vessels, except the second (neutral), was approximately the same (24–25 gr.); the second vessel lost only 14.3 gr.; at the same time the acidity of the neutral solution increased toward the end of fermentation (to 2.48 pro mille), which was noticeable from the change in the color of the liquid from dark brown to yellow. In the others the acidity remained approximately—

approximately the same as the initial value (6.4, 14.6, 25, 50).—The alcohol content was, in general, one and the same, namely about 6.9%; only with an acid content of 25 pro mille does it decrease to 6.6, and with 50 pro mille to 6.5.

To characterize the course of fermentation, the following may be noted. In the neutral must, fermentation begins weakly and reaches its maximum only on the 6th day, whereas the other vessels gave a steep rise of the curve with a maximum on the 4th day, and moreover an acid content of 25 pro mille was especially favorable and gave the greatest rise of the curve. Of some interest among the data presented, in my opinion, is the considerable formation of acids in the neutral must, by which the most favorable conditions for the vital activity of the yeasts were prepared. Alongside this it should be noted that, with the same alcohol yield as in the other vessels (about 6.9%), the loss in carbon dioxide was much smaller (14.3 as against 24–25). The deficiency of the evolved carbon dioxide is fairly closely covered by the other acids formed in the wine \((2.48 \times 4 = \text{about } 10)\).

The preparation of pure yeast cultures in the Geisenheim laboratory begins with the separation of the yeasts from aerobic bacteria and other organisms. For this purpose fermenting grape must is poured into an Erlenmeyer flask, which is placed bottom upward. Two glass tubes are passed through the stopper; one almost reaches the bottom of the flask and, emerging with its end above the liquid, serves for the admission of air, while the other barely projects above the stopper. When fermentation is finished, the yeasts settle on the stopper, and it is enough to open the second tube in order to obtain a drop of yeast free from bacteria. The further separation of the yeasts is carried out by means of culture on gelatin (Plattenkultur*).

During the wine-making season the station constantly maintains cultures of several of the most commonly used races in an active state. The yeasts are cultivated in thick-walled glasses into which up to 400 cc. of sterilized grape juice is poured; the glasses are placed in a thermostat, where the temperature is maintained at about 22° C. When a request is received, 5-day cultures are taken and transferred into an ordinary half-bottle, which has previously been washed with alcohol and an excess of must of the given culture. They are stoppered with an ordinary cork and covered with sealing wax. All manipulations are carried out directly in the room; sterilizing cabinets are not used at all, and, it is said, infection of the cultures has not been observed up to the present.

When the program of my work was completed, I had only to visit one of the famous Rhenish wine-cellars, also—

*) See R. Meissner. Anleitung z. mikrosk. Unters. und Reinzüchtung. Stuttgart 1901, p. 76.

Schloss Johannisberg, located 4–5 kilometers from Geisenheim, was chosen by me. The old castle, with its cellars, is situated on the summit of a hill, whose gently sloping southern sides are covered with vineyards of excellent vines. The manager of the estate, Mr. Henisch, very kindly showed us through the cellars, one of which is considered to have existed for as long as 1000 years. To my question as to what races of yeasts they work with, the answer, to my surprise, was that pure cultures are still not used here—not because this is regarded as useless, but simply out of fear that the introduction of pure cultures might give rise to talk of artificial methods, perhaps even of falsification of the wine, and might damage the firm’s established reputation. Thus, willy-nilly, giving way to inertia, they are obliged to adhere to the old irrational method. While inspecting the Johannisberg cellars I noticed one particular detail: namely, all the vaults and walls of the cellars were entirely covered with a felt-like tissue of fungus. Black shreds of this felt hung down on the chandeliers and covered them. The main mass of the mycelium had already died and dried up, and from the touch of a lit candle began to smolder like tinder. The winemaker treasured this natural covering of the cellar walls, attributing to it an important influence on the quality of the wine. But, of course, there can be no question here of a direct influence; spores of the fungus, if they got into the wine, could only spoil it. It is more likely that the mycelium, possessing great hygroscopicity, regulates the humidity of the air in the cellar and, by keeping it more or less constant, indirectly affects the wine.

For lack of time I did not manage to visit another cellar where I might have seen the use of pure cultures on a large scale.

In Paris I visited the zymotechnical department of the Pasteur Institute, directed by M. Fernbach, but found nothing here deserving of special attention. Perhaps this was due to the rebuilding of the laboratory and the establishment of the brewing school, but apart from old Koch cultures on gelatin and several propagators placed in a temporary shed, I found nothing. There was also an almost complete absence of trainees. Having visited, moreover, several wineries of Burgundy near Dijon, thanks to the kind assistance of Prof. Legras, I became convinced that here almost nothing is heard about the use of pure cultures. In general, judging by what I saw and heard, one may conclude that the introduction of the rational method into winemaking in France lags far behind Germany.

Nor did the small private laboratory of Jacquemin in Nancy, to which I made a special trip from Germany, give me anything. At the present time it is simply a small factory of yeasts (chiefly wine—

... where cultures are distributed to factories in large glass flasks.

In passing, I visited the laboratories of Weigmann in Kiel, Obri in Munich, Koch in Oppenheim, Müller-Thurgau in Wädensweil near Zurich, and Lindner in Berlin.

In conclusion, I intended to devote myself to the study of the more common and widespread diseases of cultivated plants; for this purpose I first planned to visit Prof. Frank’s laboratory in Berlin (Gesundheitsamt). But Frank died unexpectedly, his laboratory was left without a head, and I settled in the laboratory of Prof. Wittmack at the Higher Agricultural School. Making use partly of preparations that were in Wittmack’s laboratory, and partly of material from Frank’s laboratory, kindly supplied to me by Dr. Krüger, I focused chiefly on diseases of potatoes and beets, but in this area too there proved to be not especially much material.

During my stay in Berlin I was able to attend a lecture by Prof. Buchner, where he expounded his work on the study of the yeast enzyme, which he called zymase; thanks to the kindness of his assistant, Dr. Albert, I became acquainted in the laboratory with the methods for obtaining zymase and received preparations of it for demonstration in Tomsk.

We have long known the initial substance for alcoholic fermentation (sugar), the final products (alcohol, carbon dioxide, and small quantities of glycerin and succinic acid), and the active cause—yeast cells; but until recent times the very mechanism, or, more precisely, the chemistry, of this process remained obscure—all the more so because it had not been possible, by laboratory means, to produce the act of fermentation without the participation of plants—yeasts.

Different scholars have viewed the mechanics of fermentation in two ways. Some, like Pasteur, regarded this mysterious process as a physiological process, as a surrogate of respiration, as life without the oxygen of the air; this view was partly confirmed by the phenomenon of the so-called intramolecular respiration, respiration without oxygen, of higher plants and even of certain animals, whereby alcohol is also formed in their tissues. Others equated the action of yeasts with enzymes (diastase, invertin, etc.) and saw in yeast cells merely an apparatus for preparing the enzyme that then produces the chemical act characteristic of fermentation. Strictly speaking, these two views cannot be opposed, because all physiological acts of the transformation of matter ultimately reduce to chemical transformations, but the direction of the chemical process may also be different.

One way or another, the investigations of recent years make much more probable the original supposition concerning the participation of an amorphous enzyme, manufactured by the yeast cell. It was precisely in this direction that Prof. Buchner worked and obtained, apparently, very eloquent results. He succeeded in obtaining from yeast an amorphous substance which, without the participation of living cells, carries out the same transformation that we have in the process of fermentation. He called this substance zymase.

The method of obtaining zymase consists, in general, in pressing out the juice from yeast cells and precipitating from it the zymase in the form of a powder.

Several pounds of pure brewer’s yeast are carefully washed free of admixtures of beer and are placed for the first time under a press in order to remove water. After this the mass of yeast is placed in a large porcelain mortar and is ground with a heavy pestle together with sand and tripoli, whereby the cells themselves are crushed and their contents are released. The ground mass of yeast, wrapped in a cloth, is again placed under the same press, resembling a press for squeezing grape juice, and is subjected to stronger pressure. The juice obtained may either be thickened directly by evaporation, or the zymase may be precipitated with alcohol in the form of an amorphous white powder.

A well-dried enzyme is preserved for months or even more than a year, retaining its fermentative properties. It is enough to take a 10–20% solution of cane or beet sugar and to mix in approximately an equal amount of zymase diluted in water; immediately a rapid evolution of carbon dioxide begins, the liquid foams as in ordinary fermentation, and alcohol gradually accumulates in it.

The experiment mentioned shows that zymase is not a chemical individual, not a single enzyme, but contains two, or several, enzymes, because cane sugar is first inverted into grape sugar (the first transformation), and this latter already decomposes into alcohol and carbon dioxide (the second transformation). In this process, conditioned by the presence of a chemically active substance, secondary products are also obtained in small quantities, such as glycerin and succinic acid. This circumstance makes it very probable that several enzymes are contained in zymase, and at the present time Buchner is striving to separate these enzymes; to what extent he succeeds in this, future investigations will show.

Despite the enormous interest of Buchner’s discoveries, I believe that they have more theoretical significance than practical, and that hardly ever will the enzyme supplant, in winemaking and brewing, the living cell. The principal enzyme of alcoholic fermentation is, evidently, one and the same in ...

in all races of yeasts, how varied are the products of their vital activity! The same grape, the same beer wort, yield different beverages depending on the race of yeast, with different alcohol content, unequal stability, with different bouquets, and so on. The causes of this difference are so elusive that they do not admit of even an approximate determination, and for a long time to come the complex chemistry of the yeast cell, not analyzed in detail, will remain, on the whole, the sole laboratory of good wine and beer.