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Volume 81, 1953
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The Effect of Ammonium Sulphate on the Growth of Dhar Yeast Cultures Containing Sucrose as Source of Carbon.

[Communicated by Dr. J. T. Salmon and read by title before the Wellington Branch, July 22, 1953; reccived by the Editor, July 22, 1953]

Summary

Loss of nitiogen in Dhar Yeast cultures increases with the increase of ammonium sulphate in the culture, within the limits studied in this paper. This change in the nitrogen metabolism of yeast is independent of its growth. Thus there is very little difference in the yield of yeast, calculated on the basis of sugar consumed, at different C/N ratios Although a larger amount of yeast in grams is produced at C/N 10 as compared to that at C/N 40, the consumption of sugar at C/N 40 is much less than what it is at C/N 10 so that the percentage of yeast yield, which is calculated on the basis of sugar consumed is nearly the same in both the cases.

Introduction

The data concerning the various factors which affect the growth of microorganisms is accumulating fast but the subtler aspects of nutrition, in which a nutrient may have a little or no effect on the extent of growth but exert a profound effect on metabolic processes such as the formation of specific enzymes, spores, capsular material, pigments, toxins and others, have so far not received the systematic investigation which they merit. More information how such changes influence nutrition is necessary for a better understanding of microbial ecology (1).

There is a great loss of nitrogen in the form of molecular nitrogen in the soil (2). This loss is chicfly due to physico-chemical reasons. Bacterial activity also accounts for some of this loss Plant leaves show loss of molecular nitrogen at a certain stage of their development (3). This loss is mainly due to enzymic activity Very little is known regarding this aspect of nitrogen metabolism in the case of micro-organisms and no work has been done to indicate whether certain factors do affect this phase of nitrogen metabolism.

In the case of Dhar Yeast* (4), Carbon Nitrogen ratio is one factor which affects the metabolic processes Within a certain limit it does not affect the growth of yeast but shows a great influence on the Nitrogen metabolism of the organism In this paper an account of such an effect is given.

The influence of C/N ratio on the loss of nitrogen has been very well studied in the case of soils. I have studied this loss of nitrogen in Dhar Yeast cultures, using sucrose and ammonium sulphate as sources of carbon and nitrogen respectively. I wanted to investigate what influence the C/N ratio of yeast culturc has on the nitrogen metabolism of this organism.

[Footnote] * This Yeast was isolated in the Allahabad University Laboratories from the toddy of Allahabad. This is a new variety of yeast which is found to give maximum amount of yeast and produce minimum amount of alcohol as compared to the other varieties found here.

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Experimental

I prepared several cultures each containing 0.10 g. of calcium carbonate, 0.10 g. of sodium chloride, 0.10 g. of potassium sulphate and 0.10 g. of disodium hydrogen phosphate. These salts were digested in dilute hydrochloric acid and to the clear solution 10 g. of sucrose and ammonium sulphate, equal to the amount mentioned against each culture in the tables, were added. Total volume of each culture was made to 400 cc. and pH of the culture was adjusted at 4; five 750 cc. flat-bottom flasks were used for holding the above cultures. All the chemicals used in preparing these cultures were of A.R. quality.

The flasks were cotton plugged and sterilised by heating in an autoclave for 30 minutes at 15lbs. pressure. After cooling, each culture was seeded with a trace of an activated sample of Dhar Yeast. After 30 days these cultues were analysed for their nitrogen, yeast, alcohol, acid and sugar contents. Kjeldahl and Gunning method was used for the estimation of nitrogen in the cultures and yeast cells.

The temperature variation during the period of fermentation was between 23.4 to 27.5 C.

Observations

Results obtained by the analysis of the above cultures are shown in the following tables.

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Table 1—Concentrations of ammonium sulphate and sucrose in the beginning and the concentration of sugars after fermentation.
S.N. 1. 2. 3. 4.
g. of (NH4)2SO4 in the culture in the beginning. 0.4714 0.9428 1.4142 1.8856
g. of sucrose added in the culture in the beginning. 10.00 10.00 10.00 10.00
C/N ratio of the culture in the beginning. 40.00 20.00 13.33 10.00
g. of sugar left in the culture after fermentation. 3.86 0.69 0.29 0.00
g. of reducing sugar left in the culture. 3.86 0.69 0.29 0.00
g. of non-reducing sugar left. 0.00 0.00 0.00 0.00
Percentage of sugar consumed calculated on the basis of original sugar. 61.41 93.10 97.10 100.00

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Table 2—Amounts of acid, alcohol and yeast produced during fermentation.
Acid.
S.N. 1. 2. 3. 4.
g. equ. of volatile acid formed in the culture. 0.0102 0.0134 0 0182 0 0215
g equ. of nonvolatile acid formed. 0.4616 0.7819 0.8040 0 8665
g. equ. of total acid formed. 0.4718 0.7953 0.8222 0.8880
Percentage of acid formation calculated on the basis of sugar used. 7.81 8.58 8.64 8.88

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Alcohol.
g. of ethyl alcohol formed in the culture. 0.40 0 52 0.60 0.84
Percentage of alcohol formation calculated on the basis of sugar used. 0.61 5 55 6.10 8.40

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Yeast.
g of yeast produced during fermentation. 1.3748 2 2538 2.2456 2.3192
Percentage of yeast yield calculated on the basis of sugar used. 22 31 24 16 23.04 23.19
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Table 3—Nitrogen present in the culture before fermentation, after fermentation, the amount of nitrogen in yeast cells and the loss of nitrogen during yeast growth.
S.N. 1. 2. 3. 4.
Percentage of N in the culture in the beginning. 0.025 0.050 0.075 0.100
g. of nitrogen in the culture before fermentation. 0.1000 0.2000 0.3000 0.4000
g. of nitrogen left in the culture after fermentation. 0.0063 0.0288 0.1156 0.2219
g. of nitrogen used up during yeast growth. 0.0937 0.1712 0.1844 0.1781
g. of ntirogen taken up by the yeast cells. 0.0756 0.1235 0.1235 0.1275
g. of yeasts formed by consumption of 1 g. of nitrogen. 14.63 13.13 12.18 13.02
g. of nitrogen lost during yeast growth. 0.0182 0.0573 0.0608 0.0505
Percentage of loss of N calculated on the basis of N consumed during fermentation. 19.40 33.48 32.99 28.48
Discussion
1.

The percentage of alcohol formation shows remarkable incerase with increased concentration of sugar in the yeast cultures, whereas the yield of yeast shows little differences except that at high concentration of ammonium sulphate in the culture, yeast yield shows a decreasing trend.

2.

The percentage loss of nitrogen, calculated on the basis of total nitrogen consumed during fermentation, increases with the increase of ammonium sulphate in the culture, except at such concentrations at which the C/N is 10, at which it is slightly less than that of C/N 13.33.

3.

Grams of dry yeast produced by consuming 1 g. of nitrogen is between 12 to 14 at all concentrations in consideration.

4.

Consumption of sugar during the period of yeast growth increases with the decrease of Carbon-Nitrogen ratio. It is minimum at C/N 40 and maximum at 10.

Acknowledgement

I am very much indebted to Prof. N. R. Dhar for his valuable advice on this work.

References

1. Stokes, J. L., 1952. Nutrition of Micro-organisms. Ann. Rev. Microbiol., Vol. 6.

2. Dhar, N. R., and Pant, M. C., 1949. J. Indian Chemical Society, 26, 227–30.

3. Vickery, H. B., Pucher, G. W., Wakeman, A. J., and Leavenworth, C. S., 1946. Conn. Agr. Expt. Sta. Bull., 496, 1–93.

4. Dhar, N. R., and Krishna Bahadur, 1950. Pro. Nat. Acad. Sci., India, Vol. 19, Part II, 55–59.