| This paper optimize the DHA fermentation process, enhance supply of NADPH by adding malic acid at the oil accumulation stage in Schizochytrium sp as object, and improve DHA production yield and rate. For the purpose, the work carried out in four aspects as follows.(1) Malic acid, the activator of malic enzymatic activity was introduced to the fermentation system at fast lipid accumulation stage to enhance NADPH supply. DHA product increased from 3.75 to 5.51 g/L by the addition of 4 g/L malic acid at 24h of the fermentation. DHA content of total fatty acids also showed an apparent increase from 25 to 40%,which laid a solid foundation for the further optimization of next phase.(2) The mechanism of inducing effect of malic acid by adjusting adding way and quantity from the perspective of fermentation reactor filling material flow process was also discussed:To ensure optimal malic acid supply at the early stage and supplementation with appropriate dose during the process of fermentation could obtain better inducing effect. Constant concentration flow way of malic acid is explorated. The one-time addition of 4g/L malic acid at 24h of fermentation time, which ensure early fermentation moderate amounts of malic acid supply. And then then flowing MA with the speed of 0.038g/L·h-1 during the process of fermentation and malic acid concentration maintained the 4 g/L, which ensure the release of malic acid induction at the larger degree..Finally, compared with malic acid one-time addition, the fungus biomass, oil content, DHA production rate, and yield have further improved at constant concentration of flow mailic acid fermentation. high cell density (114.27g/L), high lipid content (93.82g/L), high DHA percentage (38.72%) and high DHA productivity (36.33g/L) were achieved by using this strategy, which were more than malic acid one-time addition increase by 5.90%、8.70%、8.37% and38.64% respectively.(3) The effect of different malic acid addition ways on fermentation process from analysis of two closely related to lipid metabolism key enzyme(malic enzyme and glucose 6-phosphate dehydrogenase). The change trend of two enzyme activities, biomass growth, oil and DHA production were explorated. I Enzyme activity peak value in advance and enzyme activity reduction of ME and G6PDH at fast lipid accumulation stage could result in the shortage of NADPH supply and thus reduce the production of DHA by analyzing the changes of cell growth, lipid accumulation, fatty acid composition and the key enzymes for acetyl-CoA and NADPH synthesis during the fermentation stages; In this part, Constant concentration adding mode of malic acid was proved to be more useful in improving the activity of ME and delaying enzymatic activity peak arrival time which then strengthen the supplies of NADPH during oil accumulation stage.(4) The thesis derived the dynamics model for the fermentation production of DHA from Schizochytrium sp. The models for growth kinetics, product generation and the substrate consumption rate of Schizochytrium sp. were derived by the use of empirical models based on the experimental data via the non-linear fitting of the Origin software. Upon examination, the fission in the fermentation process of vibro calculation value and experimental value is very close. The correlation index of model is above 0.98, suggesting the DHA dynamics model better reflects the fission vibrio batch fermentation process. It was proved that the fitted values and the experimental values were very close, the model relevant parameters were above 0.98. indicating that the mathematical model could well reflect the growth of Schizochytrium sp. in batch fermentation process. |
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