| Glutathione, as a tripeptide of glutamate, cysteine and glycine, is the most abundant non-protein thiol compound present in living organisms. It is used as a pharmaceutical compound and can be used in food additives, cosmetic industries and farming industries. There are many ways to produce glutathione, but microbial fermentation is considered as the most promising approach in the industrial production of glutathione.In this study, Candida utilis SZU 07-01 which could accumulate glutathione intracellularly was selected for glutathione production. In order to achieve high concentration, high yield and high productivity, initially, the effect of diverse nitrogen sources and C/N ratios on cell growth and glutathione production by C. utilis SZU 07-01 in shaking flasks was studied. Then, batch fermentations and fed-batch fermentations under constant/exponential feeding strategies were studied in 5 L fermenter. On this basis, we developed a polynomial feeding strategy, which was applied to realize the high-cell-density fermentation of glutathione. To further enhance the total production of glutathione, amino acids adding strategy was discussed. The main results were showed as follows:(1) Effect of diverse nitrogen sources and C/N ratios on glutathione production was studied in shaking flasks. It was found that, urea was beneficial to cell growth while (NH4)2SO4 was favorable to glutathione production, and an optimal C/N ratio of 8.3 mol/mol and 5.6 mol/mol existed for (NH4)2SO4 and urea, respectively.(2) The utilization of nitrogen source was analysed, and when it was 30%, the glutathione concentration was the highest no matter the yeast was cultivated under sole or mixed nitrogen sources. Batch fermentation of glutathione using the sole nitrogen source was conducted under the optimal C/N ratios, and it was indicated that urea was more favorable for cell growth and glutathione production than (NH4)2SO4. Then the reason for the increase of dry cell weight and glutathione production by urea was quantitatively described in detail by the comparison of parameters obtained from fermentation kinetic models.(3) A series of batch glutathione fermentation were carried out and the optimal initial glucose concentration was found to be about 26.0 g/L. Then fed-batch fermentations under constant/exponential feeding strategies were used to enhance glutathione production. Based on results above, a polynomial glucose feeding strategy was developed under which both the cell and glutathione productivity were improved. Further more, fed-batch fermentation under this strategy with a total glucose concentration of 200 g/L was successfully performed, the dry cell weight and glutathione concentration reached 91.20 g/L and 825.0 mg/L, respectively.(4) Effect of L-methionine on cell growth and glutathione production of C. utilis SZU 07-01 was studied in shaking flasks. It was showed that, L-methionine was beneficial for both cell growth and glutathione production. When the concentration of L-methionine added up to 60 mmol/L, both the glutathione concentration and the intracellular glutathione content were the highest. Since intracellular glutathione content is another important factor in the high-cell-density fermentation of glutathione, a single-shot addition of L-cysteine was used to increase glutathione production at 27 h as the glucose feeding stopped. Results showed that as L-cystine concentration reached 30 mmol/L, glutathione concentration reached the maximum of 1104.3 mg/L and the intracellular glutathione content was 2.10%.(5) Finally, L-methionine with concentration of 60 mmol/L was added at the batch phase, then, a single-shot addition of L-cysteine with concentration of 30 mmol/L was also added after the glucose feeding stopped to further enhance the glutathione production. Using this amino adding strategy, the final glutathione yield was further enhanced to 1247.1 mg/L, the intracellular content reached 2.41%, and the glutathione productivity reached 26.00 mg/(L·h).
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