Endogenous alcoholic fermentation in yeast

Abstract

During fermentation of glucose by yeast in a state of divisional rest, 2/3 parts of the glucose absorbed are degraded producing equimolecular quantities of CO2 and ethanol (10, 16). Determination of the increase in dry weight of the cells showed that the remaining third was assimilated, probably under the form of glucogen. In spite of a certain intracellular content of glucogen yeast is not capable of producing an appreciable spontaneous endogenous alcoholic fermentation (23, 24, 26, 27, 28, 33).

Aerobically yeast also assimilates part of the glucogen used, probably in a proportion greater than during fermentation (18, 30, 31. 33). In spite of this, various authors have shown that glucogen respiration takes place, only with great lentitude (24, 29).

It has been supposed that the glucose-6-phosphate glucose-l-phosphate process would be found in vivo very displaced towards glucose-l-phosphate, although it has been shown that the action is completely reversible in vitro.

Rothstein and Berke (23, 24) have found that the addition of adequate quantities of dinitrophenol DNP or sodium azide induces the anaerobic production of considerable amounts of carbon dioxide in yeast cells without exterior substrate. In the present work, the author shows how, without the intervention of Chemical efectors, it is also possible to induce a considerable endogenous alcoholic fermentation in yeast.

In the experiments fresh baker’s yeast washed twice in distilled water was used.

For determination of the quantity of yeast, measurements of sediment constant at 1.800 r. p. m. were done. In fig. 1 and in Table I are found the equivalents for dry and damp weight.

For the absorption measurements 'of glucose, fundamentally the procedure employed by Rothstein and Larrabee (20) was followed. The period of emptying of reserves is brought about in distilled water and with three or four hours aeration. The pH of the suspensions is adjusted with HC1 0.1 N to 3.S immediately before absorption. After 30 minutes absorption, centrifuging is done and glucose in the supernatant liquid is deterinined photocolorimetrically with the Folin-Wu (9, 11, 17) reactive.

Production of CO3 is measured by the standard technique of Warburg (2, 32). Anaerobic conditions are obtained by replacing air by previously purified nitrogen.

Table III contains the results of various experiments in which on the one hand the slight anaerobic endogenous metabolism of yeast is shown 9 gr. of fresh yeast were dispersed in a 5 % solution of glucose to a total volume of 100 mi The suspension was maintained at 30° C. and a strong current of air was passed for an hour. Aflerwards, the suspension was centrifuged and the sediment washed twice in distilled water and put at pH 3.5. The suspension of yeast previously submitted to this treatment produces in anaerobic conditions a much greater amount of CO3 than without incubation (Table III).

Endogenous metabolism can also be increased if the cells are incubated anaerobically (Table III). But the increase is greater when incubation is carried out with aeration (Tables III, IV and V). For incubations of short duration, the influence of oxygen seems to be more considerable (Tables III and IV).

Incubation time has a great influence in the anaerobic production of CO3, but 1/4 of an hour is sufficient to induce a significant increase (Tables III and IV).

With time, the production of CO3 by cells previously incubated, disminishes (Table V).

As shown by Rothstein (20, 22), for a cellular concentration of 0.03 mi./mi. small quantities of uranyl nitrate strongly inhibit the utilization of exterior glucose (Table VI). For the same amount of cells the uranyl ion does not appreciably inhibit anaerobic production of CO3 by the incubated cells at least up to a concentration of 10”3 M (Table VII). A concentration of lO"2 M of sodium fluoride strongly inhibits approximately in the same degree utilization of exterior glucose and anaerobic production of CO3 in a suspension in distilled water of yeast cells previously incubated (Tables VIII and IX).

The fact that the production of CO3 induced by incubation is not sensitive to uranyl and increases with incubation time (Table IV), shows its endogenous origin.

Production of CO3 is connected with previous assimilation of glucose in the incubation process. It is inhibited by NaF in the same proportion as the exogenous fermentation. These facts show that the CO3 produced really originates from an endogenous alcoholic fermentation.

Without previous incubation, endogenous fermentation is very weak (Table IV). However, this does not seem to mean that the cells are at an extraordinarily low level of glucidic reserves. Rothstein and, Larrabee (23) showed that even in yeast cells starved in distilled water the DNP and azide are capable of producing appreciable endogenous fermentation. In agreement with the authors quoted, we think that a certain interior level of glucogen exists wich is not normally fermentable.

It seems probable that a saturation level exists of the assimilation capacity of glucose which can be influenced by experimental conditions. During incubation, an exceptionally high level of interior glucogen is probably reached.

According to the influence of oxygen in incubation on subsequent endogenous alcoholic fermentation (Tables III and IV), it can be suggested that the saturation level of glucogen within the cells should be reached earlier through aerobic incubation. This being so, the relation between oxidative and anoxic assimilation would be very much influenced by the duration of the «experiments. This would perhaps explain some discrepancies between the authors in the percentages of glucose assimilation by yeast (18, 33).