| Poly γ-glutamate (γ-PGA) is an unusual anionic, water-soluble and high viscous polypeptide in which glutamate is polymerized via y-amide linkage. For its excellent characters such as high efficient water-absorption, completely biodegradable and non-toxic to human, γ-PGA has attracted a variety of interest in the past decade and proved to be a safe and environmental friendly material that has potential applications in a wide range of industry and agriculture such as food, cosmetics, medicine and manure. γ-PGA and its derivatives can be used as thickener, humectant, drug carrier, heavy metal absorber, feed additives etc. This natural biopolymer is worth researching and developing to a commercial material.In the present study, the aim was to investigate the production of γ-PGA in liquid fermentation and solid state fermentation and achieve a series of optimal process parameters. A newly isolated strain B. subtilis CCTCC202048 was employed in this study. The factors considered critical to γ-PGA production in fermentation were studied and optimized in this thesis paper. A novel method of metabolic flux analysis was adopted to study the strain's metabolic character, which can provide many rules or clues for reconstructing metabolic pathway or optimizing fermentation process parameter to get a higher production.The production of γ-PGA and biomass were greatly influenced by the composition of medium and culture conditions according to the results attained in flask liquid fermentation experiments, which were conducted to investigate the optimal process parameter for γ-PGA production. The highest γ-PGA yield was achieved in the conditions that describe as following (g/L): Glutamate 20.0, Glucose 80.0, Citric acid 6.0, CSL 1.0, Glycerol 10.0 NH4C1 7.0, K2HPO4 3H2O 0.5, MgSO4 7H2O 0.5, FeCl3-6H2O 0.04, CaCl2 2H2O 0.5, MnSO4 H2O 0.052, initial pH 7.5, temperature 37℃, culture time 60 h, volume level 20 mL (250 mL flask), seed age 10 h, inoculum level 1 %. This yield was 2.14 times more than the one before being optimized.The solid state fermentation (SSF) was carried out for the production of y-PGA. The maximum γ-PGA production (83.61 g /kg of dry substrate) was obtained in the mixed substrates of soybean cake powder and wheat bran (11:9 w/w) supplemented with glutamate (40.14 g/kg), citric acid (18.50 g/kg), NH4NO3 (20.05 g/kg) and mineral salts (MgSO4 7H2O 0.5 g/kg, CaCl2 2H2O 0.5 g/kg, FeCl3 6H2O 0.04 g/kg and MnSO4H2O 0.104 g/kg), with initial moisture content as 65%, at 40°C incubated for 42 h. This yield was four times more than the one before being optimized. It has been the first report on optimization of γ-PGA production in SSF so far.MFA was employed to analyze the metabolic flux distribution of this γ-PGA productive strain. These experiments were conducted in batch fermentation and fed-batch fermentation using 10 L fermentor. According to the results of MFA in B. subtilis CCTCC202048, it was deduced that most of glutamate monomers which composed γ-PGA were derived from extra-cell glutamate, oxygen supply was short for biomass and γ-PGA synthesis when culture became viscous during later logarithmic phase, and extra-glutamate inhibit the pathway from citric acid to a-ketoglutarate in TCA cycle. Then the metabolic flux distribution was optimized by Fluxanalyzer procedure-Fluxoptimization. An ideal metabolic network was shown for y-PGA production. In comparison of the difference between ideal network to actual one, some helpful hints of y-PGA production optimization for next step were attained.
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