Engineer D. N. Klimovskii.

On the Question of the Mineral Nutrition of Yeasts in Bread Mashes in Yeast Production

At the present time, two directions are being indicated in yeast production: on the one hand—to increase the yield of yeast as much as possible, reducing the formation of alcohol to a minimum; and on the other hand, in the event that alcohol is distilled off, to find the best situation in which the yeast yields are sufficiently high and the distillation of the fermented mash is economically advantageous. In the first case the best results are achieved by diluting the fermenting wort and by intensified blowing-through of air. The greatest yields are obtained when the wort is diluted to 1–1.5 Bl. Further dilution does not increase the yield.¹) This phenomenon becomes understandable if one compares the chemical composition of wort extract and yeast, as has been done in Table 1.

Table 1.

Average content²) in 100 g.

From the data of Table 1 it clearly follows that, with complete utilization of the nitrogen and ash of the wort extract for the building of yeast cells, part of the sugar must remain unused; consequently, further dilution of the wort cannot increase the yields. If it is desired to use completely the nitrogen-free constituents of the wort for the formation of yeast, it is evidently necessary to add mineral salts containing chiefly nitrogen, phosphorus, and potassium.

In practice, mineral nutrition is used in factory practice chiefly as nitrogenous nutrition and, to a lesser extent, phosphoric nutrition. In such a case one proceeds from the chemical composition of the breads used—

¹) S. V. Lebedev and I. Ya. Gorenburg, Publ. Tomsk State Univ., 170 pp., 1927.
²) E. Prior. Chemie u. Physiologie d. Malzes u. d. Bieres, pp. 307, 335, 356. 1896.

during mashing, the composition of the yeast, and the expected yield. For various breads, coefficients are given for assimilable nitrogen and phosphorus. Thus Bobrik¹ gives the following figures for assimilable constituents of breads, in %:

In mashes with barley and rye, for yields of 12–15 lb. per pood of mashed materials, there is no need to add salts. At yields of 18–20 lb., phosphorus proves to be in excess; the deficiency is felt only in nitrogen, and therefore mineral nitrogen is added in the form of a solution of ammonium sulfate. Higher yields also require mineral phosphorus, which is added either in the form of an extract from superphosphate or in the form of salts of phosphoric acid.

The mineral nutrition of yeasts as practiced has been illuminated from the theoretical side in the works of various authors. From the literature at our disposal, the following conclusions may be drawn regarding the assimilation of nitrogen by yeasts in fermentation industries: 1) The assimilable nitrogen of the wort in repeated fermentation may reach 80%.² Usually nitrogen assimilation ranges from 30% in brewing production to 60–65% with aeration by air.³ ⁴ 2) Yeasts make readier use of ammoniacal nitrogen than of organic nitrogen.⁵ ⁶ ⁷ 3) When yeasts assimilate ammonia from ammonium sulfate, the acidity increases owing to the liberation of free sulfuric acid.⁸ ⁹ 4) During their development, yeasts not only take up but also give off their nitrogen, in amino form, into the fermenting medium.¹⁰ ¹¹

The following experiments of Wolly and Sherdel¹² show the change in yeast yields with a variable ratio of mineral to organic nitrogen. The authors indicate that the use of ammoniacal nitrogen as mineral nutrition is possible only in the presence of a sufficient quantity of organic nitrogen. Otherwise the yeasts lose their capacity to multiply and ferment. Thus, in fermentation with an extract from malt sprouts, they obtained as the highest yield 18.88 g of yeast per 1 l, at 2.5% sugar and 0.0178% nitrogen in the solution. A higher dosage increases the nitrogen content in the yeasts but does not increase the yields. Then the nitrogen of the malt sprouts was replaced by per-

¹ Bobrik. Food Industry, 1924, p. 31.
² Classen. Zeitschr. f. ang. Chemie 443; 1926.
³ Kolbach. Wochenschr. f. Brauerei 245; 1925.
⁴ Laer. cited in W. f. B. 31, 181; 1922.
⁵ Lafar. Handbuch d. Techn. Mykologie IV, 100; 1905–1907.
⁶ Classen. in Chemisches Centralblatt. (I) 4, 601; 1928.
⁷ Thomas. cited in W. f. B. 2, 11; 1922.
⁸ Bermann. W. f. B. 43, 267; 1925.
⁹ Henneberg. cited in Zeitschr. f. techn. Biologie. Bd. VII, 229; 1919.
¹⁰ Classen. Z. f. an. Ch. 443; 1926.
¹¹ Euler and Funk. W. f. B. 1, 9; 114; 1927.
¹² Cited in Z. f. techn. Biologie. Bd. VII, 114; 1919.

...ordinary phosphoric-acid ammonium. Under the same conditions, the highest yield, 19.28 g, was obtained with 40% ammoniacal nitrogen and 60% organic nitrogen; with 50% ammoniacal and 40% organic nitrogen the yield fell to 17.7 g; with 90% ammoniacal and 10% organic nitrogen—to 10 g, and with 100% ammoniacal—to 10 g. In these experiments the highest yield is given with 40% organic nitrogen.

With regard to the influence of phosphorus on the multiplication of yeast and, chiefly, the quantitative ratios between the phosphorus content in the fermenting medium and the yields of yeast, there are few indications in the literature. Berman and Kuhl¹ indicate that the phosphoric acid added, calculated for the expected yeast yields according to the formula

\[ x \cdot \frac{\% \text{ content of } P_{2}O_{5} \text{ in superphosphate}}{100} = a \cdot \frac{\% \text{ content of } P_{2}O_{5} \text{ in yeast}}{100}, \]

where

\(x\)—kg of superphosphate, \(a\)—yield of yeast in kg, is insufficient. Some excess is necessary. This excess must not be very great, so as not to produce undesirable properties in the yeast, even if the fermentation does not show an abnormal appearance.

Malkov² gives a favorable concentration of \(P_{2}O_{5}\) for the multiplication of yeast. According to his experiments, a concentration of \(P_{2}O_{5}\)—0.07%—is the most favorable. An increase or decrease of this concentration lowers the yield.

The question of the mineral nutrition of yeast, connected with increasing yields, finds its reflection in practice. The series of excerpts from the German patent literature cited below indicates the ways in which foreign technology has approached the solution of this question³.

D. D. P. 303222. The mash was prepared from sugar and mineral salts. Nutrient salts were used in an amount corresponding to half of the sugar employed. When sulfuric-acid ammonium was used as the source of nitrogen, a rapid assimilation of the mineral salts entering the yeast took place, with an extremely strong increase in acidity (liberated sulfuric acid), which was neutralized by the addition of alkalis or alkaline salts.

D. R. P. 304242. For the mineral nutrition of yeast, free or bound alkalis or their carbonates were used in the form of a continuous inflow of solution. During yeast growth, a weakly alkaline reaction is maintained in the mash. In this way special neutralization of the mash during fermentation is unnecessary.

D. R. P. 304211. First of all, as much mother yeast is introduced as can be obtained from the prepared quantity of liquid.

D. R. P. 300663. Sugar and purely mineral salts are used. It is shown here that yeast multiplies in strongly diluted solutions of sand or raffinade when at least 15 parts by weight of salts are used per 100 parts of sugar. The processing is carried out at con-

concentration is at most 2%. The resulting yeast has normal properties and color.

D. R. P. 300623. Work with a highly diluted mash is begun in the usual way, and the mash of the same higher concentration is added to the diluted mash slowly and continuously. Owing to the continuing decomposition of the nutrients by the yeast, only such a concentration of the mash is used in which the nutrients and the metabolic products formed are present in an amount that is well and rapidly assimilated by the yeast.

D. R. P. 304243. Into a solution containing seed yeast, mineral salts are first introduced, from which the yeast liberates acids, so that the alkalis or alkaline salts flowing in are bound by the free acids. Ammonia is added to the nutrient substrate, which gradually flows to the yeast, and it combines with the liberated acids.

The above-mentioned patents are based on the following principles: 1) the composition of the mash does not correspond to the chemical composition of the yeast; namely: when the extract of the mash is used completely for building yeast cells, there are not enough nitrogenous and mineral constituents of the mash, and therefore mineral salts are added; 2) in order to reduce the harmful influence of metabolic products, highly diluted media are used¹; 3) the yeast is maintained in a state as if of hunger with respect to nutrients; for this purpose, a large quantity of yeast is charged at once, and the inflow of mash is carried out slowly and continuously.

All these propositions are expressed especially vividly and fully in Effront’s work “Synthesis of Proteins by Saccharomycetes”². Its most characteristic part is as follows: “Yeast in the dry state contains from 47 to 50% proteins and from 47 to 45% nitrogen-free organic substance, the composition of which very closely approaches sugar.

The need for sugar for the building of new cells fluctuates greatly, while the synthesis of complex organic substances proceeds by one or another process, which schematically could be expressed by the following formulas:

\[ \text{(A)}\quad 2\,\mathrm{C_6H_{12}O_6} + 4\,\mathrm{NH_3} \rightarrow (\mathrm{C_{12}H_{22}N_4O_4}) + 8\,\mathrm{H_2O} \]

\[ \text{(B)}\quad \left\{ \begin{aligned} 1.\;&3\,\mathrm{C_6H_{12}O_6} + 3\,\mathrm{O_2} \rightarrow 6\,\mathrm{CH_3{-}CH{=}O} + 6\,\mathrm{CO_2} + 6\,\mathrm{H_2O} \\ 2.\;&6\,\mathrm{CH_3{-}CH{=}O} + 3\,\mathrm{N} \rightarrow \mathrm{C_{12}H_{20}N_3O_4} + 2\,\mathrm{H_2O} \end{aligned} \right. \]

The yield of yeast and the liberated \(\mathrm{CO_2}\) could be studied by us in the course of synthesis:

Series I. Anaerobic fermentation. Medium: 1 kg of glucose, ammonium sulfate, and yeast ash in 12 l of water.

Tonkin yeast was inoculated and left at a temperature of 38.5° C. Fermentation continued for 11 hours, at a higher temperature of 37°.

Series II. Fermentation with very strong aeration. Medium: 3% glucose, phosphates, ammonium sulfate, and yeast ash. Before inoculation \(P_{\mathrm{H}}\) is brought to 3.8. Sugar and assimilable nitrogen are supplied in such a way that the liquid never contains more than the amount that the yeast can assimilate in 30 minutes. The duration of fermentation is 7–8 hours. The total amount of glucose introduced reached 180 g.

In the first series the amount of \(CO_2\) corresponds exactly to the amount of alcohol produced; on the other hand, for the formation of 100 g of yeast, 103.4 g of glucose were required.

These facts show that the synthesis of proteins in this case follows a formula that approaches formula (A). The yeast obtained contains 48% proteins and 43.9% nitrogen-free organic substances. According to formula (A), 1 g of protein requires 1.27 g of glucose. For 100 g of yeast, therefore, the following is required:

\[ (48 \times 1.27) + 43.9 = 104.8 \text{ g.} \]

Thus, in reality the yield of yeast (106.1 g) comes very close to scheme (A).

The yeast obtained in the second series contains 49.1% protein and 41.8% nitrogen-free substances. According to formula (B) (2 g of glucose per 1 g of protein), 100 g of glucose should have formed 71.4 g of yeast and 34 g of \(CO_2\). The actual yield was 50 g of yeast and 58 g of \(CO_2\). This yield corresponds, according to scheme (B), to 78.4 g of glucose and 26.4 g of \(CO_2\). Since in fact 58 g of \(CO_2\) were found, the excess arose at the expense of burned glucose.

Dividing this excess by 1.46, since 180 g of glucose, when completely burned, give 264 g of \(CO_2\), we find the amount of glucose decomposed:

\[ \frac{58 - 26.4}{1.46} = 21.4 \text{ g.} \]

In the present work the author carries out aerobic and anaerobic fermentation with one and the same race of yeast. In aerobic fermentation, the greater part of the sugar goes into the construction of plasma, and the energy necessary for the life of the yeast is supplied by a more economical process than alcoholic fermentation, namely by the burning of sugar to carbon dioxide. In connection with this, considerably more yeast is obtained in aerobic fermentation than in anaerobic fermentation. Considering the conditions of one and the other fermentation, it may be said that a number of factors—namely aeration and dilution of the medium, the constant concentration of the nutrient substrate, and a large amount of mother yeast—favor the aerobic life of yeast. It has already been indicated above that these propositions formed the basis of the methods used in industry. Thus, the question of increasing yields is reduced to the creation of conditions favorable to the aerobic life of yeast. Of such conditions for yeast production, the influence of wort concentration¹) and of the intensity of air blowing²) has been analyzed. In connection, however, with the question of mineral—

¹) S. V. Lebedev and I. K. Gorenburg. Izv. Tom. Gos. Un., 1927.
²) S. V. Lebedev and D. N. Klimovskii. Food Industry, 1927.

In the mineral nutrition of yeasts in bread mashes, the factor of a constant concentration of salts acquires especially important significance. Mineral salts, in addition to replenishing the constituent parts lacking in the wort for the construction of yeast cells, may also influence the character of yeast life, depending on the manner in which they are supplied.

Thus Effron¹ showed that, if the quantity of nitrogenous nutrition is maintained at a definite concentration, the yields of yeast increase considerably. It may be assumed that a constant concentration of phosphorus, whose important significance in the construction of living matter is sufficiently well known,² may likewise influence the multiplication of yeast. Thus, in mineral nutrition, two aspects must be taken into account: the most favorable quantity of mineral salts and the method of supplying them.

In unpublished work carried out in the Laboratory of Nutrient Substances of the Siberian Technological Institute under the direction of Prof. S. V. Lebedev, the first part of the question was addressed. Mineral nutrition in the form of ammonium sulfate and an extract from superphosphate was supplied in various concentrations at the beginning of fermentation. From a series of experiments there was determined the concentration of salts that gave the greatest increase in yeast yields in comparison with yields from wort without the addition of salts.

The purpose of the present work is to trace the change in the principal constituent parts of the wort during fermentation and the influence of the constancy of their initial concentration on the yield of yeast. The initial concentration of wort salts was chosen, as an approximate one, in order to reveal the character of its influence on yields.

The experiments were carried out as follows: fermentation was conducted in a fermentation vessel constructed according to the type of apparatus for continuous alcoholic fermentation of the system of Prof. S. V. Lebedev.³ The fermentation vessel is a copper tinned cylinder, 10 cm in diameter and 70 cm high, closed at the top by a removable lid. In the lower part, up to a height of 20 cm, the cylinder is surrounded by a water bath, making it possible to thermostat the fermenting medium. In the upper part of the cylinder there is a branch pipe for the outlet of air, and in the lower part one for taking samples during fermentation. Air is supplied by a rotary air pump and distributed by means of a star-shaped bubbler placed inside the cylinder. The wort, in the quantity necessary for all the experiments, was taken from the yeast factory of Tomselprom, sterilized by boiling in a Karlsberg flask, from which it was taken in the quantity necessary for each experiment.

The composition of the initial wort was as follows: 12 Blg.; in 100 cc of wort there are contained: \(P\)—0.072 g, \(N\)—0.263 g, \(K_2O\)—0.108 g, maltose—11.45 g. The yeast was prepared in the laboratory from a pure culture of race XII, isolated from the stock yeast of the same factory. The yeast inoculum for each experiment was prepared separately by a special method, with aeration, from the main wort diluted twofold, and was checked for purity under the microscope. The inoculum yeast contained: \(N\)—2.31%, \(P\)—0.62. For the experiments, the main wort was diluted with tap wa—

was placed in the fermentation vessel in the required amount, where it was brought to the fermentation temperature of \(30^\circ\). After this, the seed yeast was added and aeration was begun. After the end of fermentation, the yeast was filtered off on a Büchner funnel through a paper filter, washed, and pressed on filter paper until the paper no longer became moistened. The yeast was then weighed with an accuracy of 0.1 g. The moisture content of such yeast reached 75%, with fluctuations within 1%. In the mash and in the yeast, phosphorus was determined, as needed, colorimetrically by Briggs with hydroquinone and sodium sulfite; nitrogen by Kjeldahl; ammonia nitrogen by distillation with magnesia under reduced pressure; maltose by Bertrand; and \(P_{\mathrm{H}}\) electrometrically with a quinhydrone electrode.

In the first experiments, the question of the change in nitrogen content in the medium during fermentation was investigated. The experimental conditions were as follows: 300 cc of the original wort was brought to two liters, which gave a wort of 2.35° Bl.; air—1000 l per hour; seed yeast—3.75 g. Duration of fermentation: 9 hours. During fermentation, samples of 55 cc were taken every hour. Nitrogen was determined in 50 cc of the filtered mash. The data of the experiment are presented in Fig. 1.

FIG. No. 1.

It is evident from the curve that by the 6th hour the utilization of nitrogen almost ceases. The yeast stops multiplying, having used up the organic nitrogen present in the wort. At the fifth hour of fermentation, ammonium sulfate may be added, thereby making up the deficiency of assimilable nitrogen. In that case the yeast, having assimilable nitrogen, could continue to multiply. Table 2 presents the results of experiments carried out for this purpose.

Table 2.

The first experiment was the control, without addition of ammonia. In the second experiment, at the fifth hour, 2 g of \((\mathrm{NH}_4)_2\mathrm{SO}_4\) with a content of 20.9% N were added in order to bring the nitrogen content up to the initial value. The results of the experiment showed that in this case only about 50% of the ammoniacal nitrogen is assimilated from ammonium sulfate. Therefore, in the third experiment, 1 g of \((\mathrm{NH}_4)_2\mathrm{SO}_4\) was added. In this experiment the nitrogen of ammonium sulfate was used completely. The results of the experiments show that the addition of ammonium sulfate lowers the yeast yield, but increases the nitrogen content in the yeast up to a certain limit. At the fifth hour, judging from the nitrogen-utilization curve, approximately about 85% of the total amount of yeast is formed, and 85% of the control yield amounts to 25.5 g—the quantity of yeast approximately equal to the yields of the second and third experiments. Thus, from the moment ammonium sulfate is added, the yeast ceases to multiply, while at the same time using a certain amount of the supplied nitrogen under the given conditions.

Subsequently, an experiment was carried out in which the change in the content of the principal constituents of the wort—nitrogen, phosphorus, and maltose—was determined. The experimental conditions were as follows: 200 k.s. of initial wort were diluted to 2 liters; air, 800 l per hour; seed yeast, 5 g. For each sample, 60 k.s. were taken every hour. In the filtered mash, determinations were made of nitrogen, phosphorus, and maltose.

Table 3.

In 100 k.s. of mash, potassium was determined, and only traces of it were found. This shows that potassium is used almost completely. Therefore, in subsequent experiments, for neutralization of the wort during ...

...of fermentation, the normal potash solution was taken. The data are presented in Table 3 and, for clarity, are shown as curves in Fig. 2.

Fig. No. 2.

On examining the curves in Fig. 2, the rapid drop in the maltose content in the wort is striking. This drop is far from proportional to the fall in nitrogen and phosphorus, the disappearance of which from the fermenting medium characterizes the multiplication of the yeasts. On the basis of Effront’s work cited above, this fact may be explained by the circumstance that, under the given conditions, the yeasts live an anaerobic life, obtaining the energy necessary for their development through fermentation. Indeed, throughout the entire fermentation a distinct odor of alcohol was observed. In order to change the fermentation conditions in the direction favoring, according to the supposition stated above, the aerobic life of the yeasts, the following experiments were carried out with a constant initial concentration of phosphorus and nitrogen separately, and of both together. Nitrogen was added in the form of ammonium sulfate, phosphorus—in the form of an extract from superphosphate with a content of 1.8 mg in 1 kg of wort. The salts were pri-

were poured in drops from a burette placed on the lid of the fermentation vessel, according to the hourly requirement calculated on the basis of the data in Table 4. In all, 50 cc of extract was added, i.e. 90 mg P and 1.6 g \((\mathrm{NH}_4)_2\mathrm{SO}_4\), or 334 mg N. The inflow was completed in the sixth hour, and fermentation continued for 2 hours.

The results of the experiments are presented in Table 4.

Table 4

The first 4 experiments were carried out with seed yeast taken from the Tomskspirtprom yeast plant. The yeast contained about 2% mycoderma. The duration of fermentation was 8 h. The last 3 experiments were carried out with a pure culture of the same yeast maintained in the laboratory. The duration of fermentation was 7 h. Columns 5 and 6 give the P and N content both present in the wort and added over the entire course of the experiment.

The factory yeast gave increased yields in comparison with the laboratory yeast. This is explained by the circumstance that a culture kept in the laboratory had somewhat lost its viability, as is observed during storage in collections. The factory yeast also proved more sensitive to changes in the medium. It may be noted that a constant initial concentration of phosphorus increases the yield, whereas the same concentration of nitrogen lowers it. Thus in the second experiment, at a constant concentration, the yield was 101.2%; in the third, at a constant concentration of N, 87.3%; in the fourth experiment, 92.8%, where the harmful effect of nitrogen was compensated by the favorable effect of phosphorus. The amount of added mineral nitrogen in relation to the total nitrogen of the wort reaches 48.1%. In the above-cited work by Wolff and Scherdel it is indicated that the highest yield was obtained with 40% mineral and 60% organic nitrogen. With 50% mineral nitrogen, the yields decrease. The experimental data confirm this. In Wolff’s experiments, nitrogen was supplied at the beginning

fermentation in these experiments at the end and during the course of fermentation. It is evident that, whatever the time at which mineral nitrogen was introduced—at the beginning of fermentation, at the end of fermentation, or gradually throughout the whole period of fermentation—when its amount exceeds 50%, the multiplication of the yeasts is retarded.

In experiment No. 7, the amount of ammoniacal nitrogen remaining in the mash was taken into account. A total of 0.210 g was found. In the form of $(\mathrm{NH})_2\mathrm{SO}_4$, 0.334 g had been introduced. Thus, 0.124 g, or 37.1%, of mineral nitrogen was utilized. In all, 0.458 g of nitrogen was used by the yeasts. Consequently, 0.334 g was utilized from organic nitrogen, which gives a percentage utilization of organic nitrogen of 63.7%. In the preceding experiment, without the addition of ammonium sulfate, the degree of utilization was 63.5%. It is evident that the addition of ammonium sulfate does not reduce the degree of utilization of organic nitrogen. Ammoniacal nitrogen is absorbed in addition to what can be used by the yeasts from the wort. This situation somewhat contradicts the proposition stated earlier, that yeasts make readier use of ammoniacal nitrogen than of organic nitrogen. This circumstance, in connection with the harmful effect of mineral nitrogen on yeast multiplication, argues that the addition of mineral nitrogen should be carried out with great caution, especially since an increase in the nitrogen content of yeasts does not always guarantee their better baking capacity¹.

Experiments with a pure culture gave negative results with respect to increasing the yields. The yields in all cases proved to be the same. In comparison with factory yeasts, they proved more indifferent to the composition of the medium. Meyerhof² indicates that wild yeasts possess a higher respiratory capacity than cultured yeasts. It may be supposed that this property of the wild yeasts contained in factory yeasts could naturally have manifested itself in the present experiments, which were aimed precisely at intensifying aerobic life. In connection with this, the question must be raised of choosing a yeast race for experiments of this kind, whose purpose is to obtain the greatest possible yield. Evidently, not all yeast races possess the property of changing their respiratory power. Thus, in the above-cited experiments of Effront, Tonkin yeasts were used, with a readily changing respiratory function.

The data of experiments carried out with wort 1.2 WI with strong aeration permit the following conclusions to be drawn for these conditions:

1. The addition of nitrogenous nourishment at the moment of its disappearance in the fermenting medium lowers the yeast yield, but increases the nitrogen content in them.
2. A constant initial concentration of nitrogen either lowers the yield (factory yeasts) or has no effect.
3. A constant initial concentration of phosphorus either raises the yield (factory yeasts) or has no effect.

It also follows from these experiments that the aerobic life of yeasts can proceed only with a limited inflow of sugar. In such

In that case, each cell will use it without carrying the fermentation to completion, but in the form of intermediate products of fermentation. In the work cited above, Effront considers acetaldehyde to be such a substance. To clarify this circumstance, experiments must be carried out with highly diluted wort at a constant concentration not only of salts but also of sugar. Experiments in this direction are under way.

Laboratory of Nutrients
Siberian Technological Institute
Tomsk.

1. Malkov, Soviet Flour Milling and Bread Baking. 1929, p. 164. ↩↩↩↩

2. Biochemische Zeitschrift. 165, 43; 1925. ↩↩↩↩

3. S. V. Lebedev. Continuous Alcoholic Fermentation. Tomsk, 1915. ↩↩