| This paper mainly studied the whole process of screening productive strain by mutation, optimizing fermentation conditions and the pretreatment on L-glutamine fermentation broth. Through physical and chemical mutagenesis, a productive strain had been screened and its physiological and biochemical characters had been identified. Later, the fermentation conditions were optimized, including the optimization of culture conditions about first generation seeds, the optimization of shake flask fermentation conditions, the process improvements about shake flask fermentation, the optimization of the expanded culture conditions and metabolic regulation. Finally, pretreatment on L-glutamine fermentation broth was done. The main research contents and conclusions included:(1) Screening L-glutamine high productive strain. By using C.glutamicum GS-01 as original strain and adopting the combination of UV, EMS and NTG mutagenesis, a high productive L-glutamine strain, which is named as GS-16 and has a double resistance and a good genetic stability, had been selected. Later, gelatin liquefaction test, Milk litmus test, indole test, VP test, H2S qualification test, methyl red test, nitrate reduction test, starch liquefaction test, urease and catalase qualification test and auxotrophic identification test have been done and the physiological and biochemical properties of the strain have been determined. After the initial optimization, the most yield of L-glutamine reached up to 22.5 g/L.(2) Optimization of fermentation conditions. Through the research of the optimization of the first seed culture medium and culture conditions, it was concluded that the best composition for the first seed culture medium was: glucose concentration 3%～4%, corn syrup 4.5%, potassium dihydrogen phosphate 0.2%～0.3%, initial pH 7.2, the temperature 30℃, shaking speed 300 r/min, culture time 6 h-7 h. Optimization of fermentation conditions: glucose 14%, ammonium sulfate 7%, corn syrup 2%～4%, potassium dihydrogen phosphate 0.25%, MgSO4·7 H2O 5%～6%, MnSO4·H2O 10 mg/L, ZnSO4·H2O 1 mg/L, CuSO4·5 H2O 1 mg/L, initial pH 7.2, the temperature 30℃, shaking speed 300rmp/min. The fermentation process improvement: initial glucose concentration was 5%. Later,5.5% glucose was supplemented respectively in 12 h and 24 h. At the beginning of fermentation,1 g CaCO3 added to 30 mL medium. The initial ammonia concentration was 7%.When it droped to a low concentration in 20 h-24 h,5% ammonium sulfate was supplemented.(3) The initial concentration of glucose in expanding seed culture was 8%. Vitamin H was added 5μg/L. Dissolved oxygen was controlled at 20%～40%. The initial speed of the seed tank was 250r/min. The initial rate of air flow was 200 L/h. The amount of dissolved oxygen was increased by upgrading gradually increase speed firstly and then ventilation flow repeatedly. On the basis of the seed expansing culture, the wet cells was 70～80 g/L in 10 L fermenter. According to this standard about optimum amount of wet cells, pH was positioned to 5.5～6.5. Later, the glucose concentration was maintained at 4%～5%, pH was adjusted by ammonia, and dissolved oxygen amount was maintained at 15%～25%. Final fermentation yield was up to 92.7 g/L after 48h and the production increased 1.1 times compared with shake flask fermentation.(4) Optimization of flocculation conditions:chitosan 250 mg/L, concentration of chitosan and sodium alginate 1:3, pH 5, temperature 25℃, stirring intensity 80 r/min, flocculation time 15 min. Activated carbon (GH-15) for glutamine fermentation was obvious and the decolorization rate reached up to 75%. Ultrafiltration for removing protein was obvious and the removing protein rate was up to 60%～80%, and it played a positive role on decolorization. |
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