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The use of microbial fermentation production process of an organic acid, the reaction rate of the fermentation process is often suppressed with the accumulation of organic acids and how to eliminate or mitigate this acid stress caused by the accumulation of organic acids from the target metabolite inhibition, is to obtain an organic acid fermentation process in high yield, high yield and high production intensity key. Therefore, this thesis intends to a massive accumulation of extracellular pyruvate smooth ball yeast (Torulopsis glabrata) quadruple vitamins (thiamine, biotin, pyridoxine and niacin) auxotrophic strain CCTCC M202019, for starting strain . The adaptive evolution screened a tolerance to pH 1.91 mutant strains RT-6 compared acid and fermentation characteristics, its acid resistance mechanism was discussed, and finally to further optimize the fermentation medium composition of the mutant, laid the foundation for the understanding of Torulopsis yeast acid stress tolerance mechanism and increase pyruvate production. The key findings are as follows: 1. Investigated the accumulation of pyruvate fermentation process, to inhibit fermentation mainly from two aspects: on the one hand, by reducing the medium pH; On the other hand by pyruvate toxicity. After adaptive evolution, continuous transfer at low pH strains obtained after 2160 generations of evolution screening a mutant strain RT-6, from bacteria concentration, pyruvate production and extracellular pH effects of mutant RT -6 acid resistance and found that the mutant RT-6 acid resistance has been markedly improved. The mutant RT-6 in other types of organic acids such as propionic acid, citric acid, lactic acid and acetic acid tolerance was also significantly improved. The fermentation results show that mutant strains RT-6 in normal conditions (pH 5.5) under acidic conditions (pH 4.7) final bacteria concentration increased 21.5% and 61.8%, respectively, compared with the original bacteria, pyruvate production increased by 15.5% and 51.8 %. 2 compares the mutant RT-6 with the original bacteria the intracellular pH in normal conditions and under acidic conditions, ATP, H-ATPase membrane fatty acids, polyphosphates physiological parameter difference. The results showed that: the mutants to maintain a high level of intracellular pH, reduced from 7.11 (pH 5.5) to 5.37 (pH 4.0), which decreased to 7.03 (pH 5.5) 5.08 (pH 4.0) from the original bacteria; mutant intracellular ATP H-ATPase enzyme activity and polyphosphate content respectively: 11.8%, 13.6%, 3.5% (pH 5.5) and 61.1%, 38.6%, 30.9% (pH 4.0); the mutant membrane fatty acid saturation enhancement film fluidity stronger. Mutant RT-fermentation medium optimized by single factor screening to determine the best nitrogen source for urea; pyruvate production as the goal, the composition of the fermentation medium level of each single factor concentration optimization, and ultimately determine urea, sodium acetate and potassium dihydrogen phosphate concentration followed 7g / L, 3g / L and 3 g / L. Validation to optimize the optimum fermentation medium tank, the bacteria concentration and pyruvate were increased by 23.1% and 24.6%, respectively, the pyruvate production strength increased by 29.6%. To reduce the high bacteria concentration, the more the flow of carbon flow in the direction of pyruvate, we optimize the concentration of vitamin B1, the optimal concentration of 0.012 mg / L, after the end of the fermentation bacteria concentration decreased by 28.7%, increase in the amount of pyruvate by 19.4%.
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