| During the microbial production of organic acid, the rate of cell growth and organic acid production were gradually decreased with the decrease of pH value in the fermentation broth caused by organic acid accumulation. In order to further increase the titer, yield and productivity of organic acid, a potential way is to eliminate or alleviate the acid stress caused by the aim organic acid accumulation. In this thesis, a mutant strain RT-6, which could tolerate pH 1.91, was screened by adaptive evolution. Then, the mechanisms of the low-pH resistance of mutant RT-6 were carefully elucidated through comparing the physiological characteristics of mutant RT-6 and parent strain Torulopsis glabrata CCTCC M202019 under different pH conditions. Based on the elucidation, an optimum strategy was proposed to further increase the pyruvate production. The main results were described as follows:1. The reasons of pyruvate accumulation decrease the efficiency of pyruvate formation may be origin from:(1) lowing the medium pH; (2) bringing toxicity to cell growth. Then through adaptive evolution by transferring the parent strain at low pH continuously, we obtained a mutant strain RT-6 after 2160 adaptive generations. Dry cell weight, pyruvate titer and the end-point fermentation medium pH were determined in shake-flask fermentation medium at the initial pH 5.5 under nonneutralizing conditions for achieving better quantification of the mutant strain for acid tolerance and the results proved to be the enhancing ability of acid tolerance obtained by RT-6. Also, the strain RT-6 demonstrated the improvement of acid tolerance for other organic acids, such as propanoic acid, citric acid, lactic acid and acetic acid. Fermentation characteristics demonstrated that the strain RT-6 performed better cell growth and pyruvate production. At the end of the fermentation, the cell growth were increased by 21.5%(pH 5.5) and 61.8%(pH 4.7), respectively; the concentration of pyruvate were increased by 15.5%(pH 5.5) and 51.8%(pH 4.7), respectively.2. With the aim of elucidating the physiological characteristics of a low pH-tolerant strain Torulopsis glabrata RT-6, the intracellular pH, ATP level, the membrane bound H+-ATPase activity, the membrane fatty acid composition and the intracellular polyphosphate content of the parent strain CCTCC M202019 and the mutant strain RT-6 were carefully determined and detailed compared under different pH conditions. Compared to that of the parent strain, the intracellular pH was maintained higher level of the strain RT-6, which decreased from 7.11 (pH 5.5) to the 5.37 (pH 4.0), compared to the parent strain 7.03 (pH 5.5) to 5.08 (pH 4.0); The ATP content, the membrane bound H+-ATPase activity and the intracellular polyphosphate content of the mutant strain RT-6 were increased by 11.7%,13.6 %,3.5%(pH 5.5) and 61%,38.6%,30.8%(pH 4.0), respectively; Furthermore, the mutant strain RT-6 exhibited higher content of the unsaturated fatty acids and higher membrane fluidity. 3.Based on the above results, the nutrient and environmental conditions for efficient pyruvate production were optimized by single factor experiment, the optimum conditions were listed as follows (g/L):urea 7, sodium acetate 3, KH2PO43. With the optimum conditions, the cell growth, yield, productivity were increased by 23.1%,24.6%,29.6%, respectively. When B1 concentration in the culture media decrased from 0.02 mg/L to 0.012 mg/L, the cell growth decreased by 28.7% but the pyruvate titer increased by 19.4%. |
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