7.-Vitality of Yeast in Nutrient Solutions and in the Dry State.

Hansen's researches have likewise furnished explanations of the vitality of saccharomycetes in nutrient solutions. The results were the following: The best preserving liquid for these fungi is, as stated in Section II., a 10 per cent. solution of saccharose. In the course of more than twenty years' observations, and in experiments with forty-four species and varieties, the only ones which died in this solution were Sacch. Ludwigii, Carlsberg bottom yeast No. 2, and its asporogenous variety, in several cases, however, only after some years, and, indeed, only some of the growths; as regards all the other species and varieties no dying off was observed, although the majority of them had been sixteen to seventeen years and several of them more than twenty years in the sugar solution. The behaviour of saccharomycetes is essentially different in wort, and here great irregularities prevail. In one case the same species died frequently in five months, in another it still lived after twelve years; usually death occurred early. In preservation in water the extent of the seeding is of great moment, ie., whether it is a small or a large one; in the first case the species were dead after one and a half to two years, in the latter they were still living after ten years. The stronger cells, in fact, live at the cost of the weaker ones. The resisting power of yeast towards drying varies very much also; this depends mainly on whether the single cell is subjected to the drying or the cells lie together in large quantity and thus form a thick layer. Hansen drew attention to this in 1885. Later on he made experiments on the resistance of the isolated cell to drying by dipping a piece of platinum wire into a yeast mass, placing the wire in an empty Freudenreich flask and shaking the latter in such a way that the small quantity of yeast on the wire was spread over the bottom and walls of the flask. He thus

obtained as thin a layer of yeast as possible. It then resulted that under these circumstances the saccharomycetes remain alive only for a short time. Some species died in less than five days. Sacch. Marxianus held out longest, being alive for three months, also Sacch. anomalus and Sacch. membranæ faciens, which lived eighty and sixty-five days réspectively. These facts refer to the vegetative cells. Under the same circumstances spores lived at least five months.

In addition, Hansen made numerous experiments relating to the drying of yeast cells in other ways, viz., when placed in layers on filter paper and when put on cotton wool in small flasks with a wadding plug (Section II., pp. 62 and 117). In the first case, as a rule, the vegetative cells died in the course of a year; under the same circumstances the spores lived for one to two years longer. On the cotton wool, the species tested lived for more than a year, some even more than three years; under these conditions they formed spores, and it is probable that this had something to do with their longer period of life. Experiments in practice on the preservation of brewery yeast by drying have been described in Section II., p. 118.

8.-Disease Yeasts and Mixed Fermentations.

Diseases caused by Fermentation Products.—We have seen from the above that yeasts occur which are the cause of diseases in beer. As an example of such a yeast, Sacch. Pastorianus I. may be mentioned, which, according to Hansen's experiments (1882), produces a bitter taste and disagreeable smell in bottom fermentation beer. If the quantity of this yeast amounts to that of the stock yeast, the disease is very noticeable; even when it only amounts to the disease is still appreciable. It may, at the same time, have an undesirable action on the clarification, and cause turbidity. Will has isolated two similar disease yeasts

22

in Bavarian bottom fermentation breweries; the one gives the beer a sharp, bitter after-taste and produces strong turbidity, the other causes a somewhat sweet disagreeable aromatic taste, and a bitter astringent after-taste, as well as turbidity.

Diseases caused by Turbidity.-Several wild yeast species produce in beer the disease called yeast turbidity by multiplying rapidly during storage, particularly in bottles, the beer becoming filled with cells, the specific gravity of which is such that they remain suspended. This was demonstrated by Hansen in his experiments with Sacch. Pastorianus III. and Sacch. ellipsoideus II. (1883). The same holds for these disease yeasts as for the foregoing, namely, that they must be present at the beginning of the primary fermentation in order to be able to cause the disease. An infection, therefore, which takes place only at the end of the primary fermentation, while bringing the beer into the storage cellar, is without effect. If it here amounts to of the latter and the beer is also casked with an extract of 7.5 per cent. Balling, the storage being interrupted after two and a third months, the disease will make its appearance. However, this will not happen if the quantity of extract is reduced to 67 per cent. Balling, and the beer is stored for at least three months.

Wortmann has recently drawn attention to the fact that turbidity of a particular kind may make its appearance in wine, by the cell wall of dead cells dissolving, and the contents of the latter being distributed.

Diseases similar to the above have also been observed in top fermentation breweries (de Bavay, Frew).

Competitive Relations.-When two or more species are present together in a nutrient solution they have, as a rule, an injurious action on one another as regards multiplication, a state of competition arising among them.

Hansen was the first to make experiments in this direc

tion. In his paper of 1881 he describes the competitive relations in beer wort between Sacch. apiculatus and brewery bottom yeasts. It resulted that when the same number of cells of both species was seeded in the same flask, the multiplication of both was smaller than in the corresponding flasks in which the same number of cells of each species had been seeded separately. The increase of Sacch. apiculatus was considerably lessened, but the amount of alcohol formed was the same both in the flask with the mixed seeding, and in that which contained Sacch. cerevisia only. In another series of experiments, carried out in a similar, manner, but with the difference that the seeding of Sacch. apiculatus contained twice the number of cells in that of Sacch. cerevisiae, the result was the same as regards the mutual retarding action which the two species exercised on multiplication, but here somewhat less alcohol was formed in the flask with the two species than in that containing Sacch. cerevisia alone. The increase of Sacch. cerevisiae was also restrained to a greater extent than in the first series of experiments. The experiments were carried out at various temperatures, and with different species of brewery bottom yeast. The main result was that Sacch. apiculatus, as the weaker probably at the close of the primary fermentation, is checked in the struggle with Sacch. cerevisia, but that it can also exercise a restraining action on the increase of its stronger rival and on the alcohol production of the latter. When each species was in a separate flask Sacch, apiculatus increased more rapidly than Sacch. cerevisie; with equally numerous seedings the proportion was 3: 1.

A very remarkable result was obtained by Hansen in his experiments in practice with mixed fermentations of brewery yeast species, viz., Carlsberg bottom yeasts No. 1 and No. 2. The chief result was that the pitching yeast gives a beer of less stability when it consists of a mixture

of two brewery yeast species than when it consists of one of the species only. In these mixtures the species present in smallest proportion acted as a disease yeast, making the beer less stable. The experiments showed that this happened not only when the two species were mixed in the proportion 9:1, but even when the proportion was 19:1. The disease then appeared only when the storage of the beer was interrupted after 1 to 13 month; after three months' storage only a faint indication of it was noticeable.

In his experiments on mixed seeding, Vuylsteke found that when a mixture of Sacch. cerevisia I. and Sacch. Pastorianus I. was seeded in wort, the number of cells of the former species per unit of volume increased from the first to the second day from 1 to 481 and 5·18 respectively; the number of cells of Sacch. Pastorianus I. rose from 1 to 13.3 and 12-2 respectively. From this the increase of Sacch. Pastorianus I. had been

133

=

2.76 and

4.81

12.2 5.18

times greater than that of Sacch. cerevisia I.

2:35

When a

mixture of Sacch. cerevisiae I. and Sacch. Pastorianus III. was taken for pitching, the cells of Sacch. cerevisiæ I. increased in the proportion 1 to 5:02 and 1 to 4.62 in the first twenty-four hours, and the cells of Sacch. Pastorianus III. in the proportion 1 to 3:57 and 1 to 3:01. The increase of the cells of Sacch. Pastorianus III. compared with the cell increase of Sacch. cerevisia I. was thus 5 0.71 and 302 0.65.

5902

=

4.62

G. Syrée, in a research on the competitive struggle between the culture yeast Frohberg and Sacch. Pastorianus III., has recently obtained the following results: He employed, as culture substratum in his experiments, yeast water with 10 per cent. of saccharose added. At 25 C. the Frohberg yeast when by itself exhibited an energy of multiplication of 1110 (after four days) and a multiplying power of 1659 (after four weeks); at a temperature of 5° to 6° C. the numbers were respectively 383 and 474.