[Agr, Biol. Chem. Vol. 30, No. 9, p. 925 `930, 1966] Biochemical Studies on the Mineral Components in Sake Yeast Part V. The Relationship of the Mineral Composition of Yeast to Fermentation By Tsuyoshi FUJITANI Niihanza Technical College, Niihama, Ehime-ken Received March 17, 1966 The critical concentrations of minerals in a growing medium for maximum fermentation of yeast were as follows: P, 1mmol/l; Mg,0.2 mmol/l; and K, 1 `2mmol/l. These values are lower than those for the saturation of the cells with each mineral. The order of the concentration for maximum fermentation (K>P>Mg) is in agreement with that for yeast growth. Only a small amount of mineral salt was required to increase the fermentative activity. Very small increase of fermentative activity was observed when the starved yeast was enriched with corresponding minerals by incubating cells with the mineral salt and glucose. In 1953, Rothstein and Demis1) demon strated that the fermentative activity of yeast cells is independent of the intracellular potas sium content but is completely dependent on the extracellular concentration of potassium. Afterward, Takemura2,3) stated that the potas sium concentration of a medium in which yeast has been grown is a factor affecting the potassium content of the cells and is the greatest factor controlling fermentative activ ity. This result is only one quantitative in vestigation concerning the effect of potas sium on the fermentative activity of sake yeast. However, there is scarcely any information on the effect of the other minerals on fermenta tion. I In the previous papers,4,5) the author 1 studied the relation between the mineral com position of sake yeast and its growth, and clari fied that the basal content of phosphorus is a factor controlling the growth, and the 1) A. Rothstein and C. Demis, Arch. Biochem. Biophys., 44, 18 (1953). 2) S. Takemura, J. Agr. Chem. Soc. Japan, 36, 569 (1962). 3) S. Takemura, ibid., 36, 717 (1962). 4) T. Fujitani, This Journal, 30, 558 (1966).5) T. Fujitani, ibid., 30, 568 (1966). growth-controlling mechanism of magnesium is identical with the case of phosphorus; and that the mechanism of potassium is different from that of phosphorus or magnesium. The present paper deals with the effect of each mineral on the fermentative activity of sake yeast and three elements are compared in terms of fermentation-controlling mecha nism. Anaerobic and aerobic carbon dioxide production of sake yeast cultured in media containing each mineral of various concentra tions was measured. The relationship of the mineral composition of the cells to the fermen tative activity and the one of the stimulation of fermentation by the mineral salt added to fermenting fluid to the concentration of the salt have been established. EXPERIMENTAL Test Organism. Sake yeast Kyokai No. 6 was used throughout the work. Culture and Preparation of Yeast Suspension. Culture was accomplished in a synthetic medium, in which the concentration of one of the minerals was varied and the other constituents were prepared ac-
926 Tsuyoshi FUJITANI cording to the prescription of the basal medium under the condition described in the previous reports.4,5) Having been grown in a test medium for three days, the yeast cells were centrifuged and washed with redistilled water three times. The washed cells were suspended in water at the concentration of 10 mg/ml. When the enrichment of the cells having been starved for one of mineral components is necessary, the cells were incubated in the medium containing glucose, lactic acid-sodium lactate buffer solution and the salt of corresponding mineral at 30 Ž overnight. For salt, ammonium biphosphate, magnesium sulfate and potassium sulfate were used. Analytical Methods. The analytical methods were the same as those described in the previous papers.6,7) Measurement of Fermentative and Respiratory Activities. Rates of fermentation and respiration were determined by the standard Warburg procedure. The amount of yeast cells used in Warburg experi ments was between 5 and 10 tng, with 3.2 ml final fluid volume in each flask. All suspensions were in redistilled water, with only glucose, as substrate, plus salt of each mineral. The salt was the same as the one used for the enrichment of starved cells. Sub strate concentration was 0.1 mol/l. The fluid was ad justed at ph 4.35 by an addition of 1 1111 of N/10 tris- (hydroxymethyl) aminomethane-succinic acid buffer solution. The temperature was 30 Ž in every experi ment. Carbon dioxide production was measured both in air and in purified nitrogen gas. RESULTS AND DISCUSSION Oxygen Uptake and Carbon Dioxide Production of Yeast Cells. Oxygen uptake and carbon dioxide production of the yeast cells grown in the basal medium were measured in air and in purified nitrogen gas, and the results are shown in Fig. 1. From the results obtained by the measurement in air, yeast cells apparently take up oxygen slowly and the rate of carbon di oxide production is almost equal to the one of oxygen uptake before an addition of glucose. This fact shows that endogenous respiration of sake yeast cultured statically is very low. After an addition of glucose, the rate of carbon 6) T. Fujitani, This Journal, 26, 762 (1962). 7) T. Fujitani, ibid., 29, 471 (1965). FIG. 1. Oxygen Uptake and Carbon Dioxide Produc tion of the Yeast Grown in the Basal Medium dioxide production increased dramatically, but a little increase of oxygen uptake was observed. It follows that almost all of substrate was decomposed through the anaerobic pathway. The result of the measurement in purified nitrogen gas shows that endogenous carbon dioxide production is negligible and that oxy gen in air inhibits fermentation. Accordingly, all the following measurements of carbon di oxide production were accomplished in puri fied nitrogen : CO2 production in the presence of glucose : O2 uptake in the presence of glucose œ : CO2 production in the absence of glucose : O2 uptake in the absence of glucose Air Purified nitrogen gas gas. Fermentative Activity of the Yeast Cells Starved for One of Essential Minerals. Fig.2 shows the carbon dioxide production of the yeast cells grown in a medium devoid of one of essential minerals. Carbon dioxide produc tion of phosphorus-starved cells decreased to
~ œ Biochemical Studies on the Mineral Compo nents in Sake Yeast. Part V 927 FIG. 2. Carbon Dioxide Production of the Yeast : Normal Starved for One of Essential Minerals : Phosphorus-starved (once), phosphorus content is 0.06mmol/g œ : Phosphorus-starved (twice) 0.05mmol/g, phosphorus content is : Magnesium-starved, magnesium content is 0.02mmol/g : Potassium-starved, potassium content is 0.004mmol/g about one half the rate of the normal cells In this case, the phosphorus content of the cells had been fallen to 0.06mmol/g (th( basal content) as was evident in the previous report.4) On the other hand, the fermenta tive activity of magnesium-starved and potas sium-starved cells fell down to a very low rate of carbon dioxide production characteristically. When phosphorus-starved cells were in cubated in a phosphate-free medium again, phosphorus content decreased a little and the carbon dioxide production decreased to about one half the rate of the cells having been starved for phosphorus once, but did not fall down to such a low level as that of magnesium - starved or potassium-starved cells. This fact shows that the starvation of phosphorus em ployed is unsufficient to decrease the fermenta tive activity of the cells. It is, therefore, as sumed that phosphorus-starvation is charac teristically different from magnesium - or potassium-starvation on the physiological meaning. Relation between the Content of Each Essential Mineral of Yeast Cells and their Fermentative Activity. In Fig. 3, the phosphorus content of the cells and the rate of carbon dioxide pro duction are plotted against the initial concen tration of phosphate of a medium in which the cells have been grown. When phosphate con centration of a growing medium is low, phos phorus content of the cells is low and the carbon dioxide production is also low. Al though the cells are not saturated with phos phorus, the carbon dioxide production reaches to the maximum at 1mmol of phosphate in a liter of a growing medium. When the phosphate concentration is 5mmol/l, the phosphorus content reaches to the saturated content, but the rate of carbon dioxide pro duction is lower than the rate at 1mmol/l. The above results were obtained in a fer menting fluid free from phosphate. When phosphate salt is added to the fluid at the concentration of 10mmol/l, the curve shows FIG. 3. Phosphorus Content of Yeast Cells and their Fermentative Activity : CO2 production in a phosphate-free fluid : CO2 production in a fluid containing phosphate at the concentration of 10mmol/1 ~ : Phosphorus content of the cells
œ 928 Tsuyoshi FUJITANI in various concentrations was measured in the same way. Fig. 4 clearly shows that the cells FIG. 4. Magnesium Content of Yeast Cells and their Fermentative Activity production in a magnesium-free fluid : CO2 : CO2 production in a fluid containing magnesium at the concentration of 5mmol/l ~ Magnesium of the : content cells grown in a medium containing magnesium at the concentration of 0.2mmol/l have the max imum fermentative activity both in the pres ence and absence of magnesium in fermenting fluid and that the acceleration of fermentation by an addition of magnesium to the fluid is smaller than the case of phosphorus. However, the magnesium content of the cells is less than the saturated content when the maximum fer mentative activity is obtained in a medium in which concentration is 0.2mmol/l. Fig. 5 illustrates the relation between the potassium concentration of a growing medium and the fermentative activity of the cells. The potassium content of the cells is also illustrated in this figure. Apparently the optimum con centration of potassium in a growing medium for fermentation is 1 `2 mmol/l regardless of the presence of potassium in fermenting fluid. When the concentration is 1 `2mmol/l, the FIG. 5. Potassium Content of Yeast Cells and their Fermentative Activity production in a potassium-free fluid œ: CO2 : COE production in a fluid containing potassium at the concentration of 10mmol/ ~ Potassium of the : content cells a different feature. When the phosphate con centration of a growing medium is low, the carbon dioxide production increases greatly. Although one gram of the obtained cells con tains merely 0.1mmol of phosphorus, the carbon dioxide production reaches to the max imum at 0.2 `0.5mmol of phosphate in a liter of a growing medium. The increase of carbon dioxide production of the cells, grown in a medium containing a large amount of phosphate, is small. Carbon dioxide-producing ability of the cells grown in media containing magnesium potassium content of the cells is not quite saturated like the cases of phosphorus and magnesium. From Figs. 3, 4 and 5, the order of the concentrations of minerals in a growing medi um for the cells to show maximum fermenta tive activity is K>P>Mg, and this order is in agreement with that of the critical concen tration for yeast growth, sugar consumption and ethanol formation which was demon strated in the previous report.8) Transfer of Minerals into Yeast Cells and the Change of Fermentative Activity. When the corresponding mineral salt was added to the fermenting fluid, the fermentative activity of the cells having been starved for one of min erals increased greatly. On this increase of the activity, the possible causes for increment in activity are (1) the mineral salt added to the fluid penetrated into the cells and the content of the corresponding mineral in the 8) T. Fujitani, This Journal, 29, 477 (1965).
Biochemical Studies on the Mineral Components in Sake Yeast. Part V 929 TABLE I. CHANGES IN FERMENTATIVE ACTIVITY AND IN MINERAL COMPOSITION OF YEAST STARVED FOR ONE OF ESSENTIAL MINERALS BY THE CULTURE IN A MEDIUM CONTAINING CORRESPONDING MINERAL SALT The yeast cells having been starved for one of essential minerals were incubated overnight at 30 Ž in a medium con taining glucose of 0.5mol/1, N/10 lactic acid-sodium lactate buffer solution and corresponding mineral salt. After the culture, the cells were centrifuged and washed with water. The content of each mineral and fermentative activity were measured. cells increased in quantity; (2) the starved cells required the corresponding mineral salt for fermentation. For the elucidation of this, the cells having been starved for each mineral were incubated overnight in a medium con taining glucose and the corresponding mineral salt and the fermentative activity of the cells were measured. Table I indicated that when phosphorus-starved cells were incubated in a medium containing glucose and phosphate salt, phosphate penetrated into the cells and phosphorus content of the cells increased greatly, but the increase of the fermentative activity was low. The fermentative activity was not influenced by the change of magne sium content induced by the incubation in a medium containing glucose and magnesium salt. When potassium-starved cells were in cubated in a potassium-rich medium, the potassium content increased and the fermen tative activity increased slightly. These facts shows that the fermentative ac tivity is mainly influenced by the constitution of the medium in which yeast has been grown and is not a function of the content of min erals in the cells. It is, furthermore, assumed that the increase of fermentative activity by an addition of the corresponding mineral salt to the fermenting fluid is not caused by the transfer of minerals into the cells. Change of Fermentative Activity by the Addi tion of Mineral Salt to Fermenting Fluid. The above result on the effect of mineral salt in a fluid was obtained from the experiment in which the salt was added at high concentration (P: 10mmol/l, Mg: 5mmol/l, K: 10mmol/l). However, no information was obtained on the concentration of these salts required for the maximum increase of the activity. Fig. 6 FIG. 6. Change of Fermentative Activity of Yeast Starved for One of Essential Minerals by the Addi tion of Corresponding Mineral Salt to Fermenting Fluid. : : Phosphorus-starved (once) œ : Phosphorus-starved (twice) Magnesium-starved : Potassium-starved
930 Tsuyoshi FUJITANI shows the change of carbon dioxide-producing ability by the concentration of mineral salt added to the fermenting fluid. From the result, it is determined that the concentration required for the maximum increase is as follows: Phosphorus, 0.1mmol/l; magnesium, 0.1 mmol/l and potassium, 1mmol/l. The fact that the maximum increase of carbon dioxide production occurred by an addition of the salts at these low concentration denies the transfer concept, because the increase of the content of each mineral must be very small even if the salt added at these low concentra tion is completely taken up into the cells. The data on the same experiment on the cells having been starved for phosphorus twice are also shown in Fig. 6. The critical concen tration of phosphate for the maximum in. crease on the twice starved cells is similar in value with the concentration on the once starved cells. From the results and discussion mentioned above, it is concluded that the main factor controlling fermentation is the mineral com position of a medium in which yeast has been grown and the mineral composition of fer menting fluid is the sub-factor. Acknowledgement. The author wishes to ex press his sincere thanks to Prof. T. Matsuura of Hiroshima University, not only for his criticism but also for his interest and en couragement. Thanks are also given to Mr. K. Tabuchi of this laboratory for his assistance.