Study On The Strategies For Enhancing Process Performance Of Pyruvate Fermentation By Torulopsis Glabrata CCTCC M202019

In order to get a high yield, high concentration and high productivity of fine chemicals production, the physiological and process performance of the microorganisms must be investigated to improve the efficiency of the aim metabolites production. This thesis chose Torulopsis glabrata CCTCC M202019, a multi-vitamin auxotrophic and over-production pyruvate yeast, as a model system, to develop the novel and feasible strategies to enhance pyruvate production. In this thesis, the effects of environmental and nutritional conditions on the pyruvate production were carefully investigated. The main results of this thesis were described as following:1 It was found that the cease of cell growth and pyruvate production was due to the deficiency of the limited substrate (vitamin) and the cell specific growth rate (μ) was determined by the vitamin availability in the fermentation broth. High cell concentration (24.8 g/L) was achieved at higherμset with low pyruvate yield (0.48 g/g), while the pyruvate concentration (81 g/L) was high at lowerμset with low cell concentration (17.6 g/L). Based on the analysis of cell physiological states at different values ofμset, a novel and simpleμset controlling strategy, in which the value ofμset was shifted from 0.05 to 0.02 at 34 h and then to 0.01 h-1 at 64 h, was proposed through a vitamin assigning strategy. By using this strategy, a high pyruvate concentration (91.2 g/L) and yield (0.75 g/g) were achieved, higher 34.5% and 22.9% than the corresponding values of the optimal batch fermentation, respectively.2 The batch cultivation processes of T. glabrata CCTCC M202019 for pyruvate production under diverse initial glucose concentrations were investigated. It was concluded that high concentration, high yield and high productivity of both yeast cell and pyruvate could not be consolidated in a batch process. High glucose concentration could inhibit cell growth and pyruvate production, while low glucose concentration couldn't provide sufficient substrate for pyruvate production. With the aim of enhancing the pyruvate production by T. glabrata, fed-batch culture should be investigated in detail. The effects of different cultivation modes such as batch feeding, constant-rate feeding and constant glucose concentration feeding on pyruvate production were also studied. As a whole, fed-batch culture with low glucose feeding rate and 20-30 g/L glucose controlled in the fermentation broth was the most suitable approach to attain not only high concentrations but also high yields and high productivities of yeast cell and pyruvate in the cultivation of T. glabrata CCTCC M202019. Furthermore, based on kinetic model analysis, a four-stage glucose-control strategy was proposed. A high concentration (83.1 g/L), yield (0.621 g/g) and productivity (1.00 g/(L h)) of pyruvate were achieved by applying this strategy, which were higher 21.3%, 21.6% and 29.9% than the corresponding value of batch fermentation.3 The effect of temperature, varied from 26 oC to 34 oC, on production of pyruvate was investigated. A modified Logistic equation and Luedeking-Piret equation were successfully applied to estimate kinetics of cell growth and pyruvate production. The effect of temperature on the estimated kinetic parameters was further studied by regression analysis. Based on kinetic parameters analysis, a temperature-shifting strategy was proposed, in which, at 0-8 h, culture was performed at 34 oC to obtain a high specific cell growth rate, and at 9-42 h, the temperature was decreased step by step from 34 oC to 27 oC to keep a high pyruvate production rate. After 42 h, the temperature was maintained at 27 oC to weaken inhibition of pyruvate and offer constant production capacity of pyruvate in the anaphase of fermentation. A high concentration (80.4 g/L), yield (0.70 g/g) and productivity (1.32 g/(L h)) of pyruvate were achieved by applying this strategy, which were 12.9%,6.9% and 32.8% higher than batch fermentation with constant temperature 30 oC, respectively. Meanwhile, the distribution of metabolic flux was redirected from by-product accumulation to pyruvate production and the final glycerol andα-KG concentrations were only 2.10 g/L and 1.57 g/L, which were 59.2% and 44.7% of the corresponding values of 30 oC respectively.