Breeding Of L- Ornithine High-yielding Strain And Optimizating Of The Fermentation Process

In order to improve the fermentation level of the L-ornithine, this paper focuses on the strain breeding for high producing L-ornithine and optimizating of the fermentation process. To improve the yield of L-ornithine, reduce the production cost, the main research contents and results are as follows:(1) Corynebacterium glutamicum S.L-18 as a starting strain was stepwise mutated with protoplast UV and Diethyl sulfate. One L-ornithine high-producing strain VO-26 (Arg-+NaFr) with genetic marker was screened through shake tube primary screening, shake flask compound screening, genetic marker test. This strain could produce 38.5g/L of L-ornithine under the fermentation condition without being optimized, as compared with the the original strain SL-1829.6 g/L, the production level of the L-ornithine has increased by 30.1%.(2) The best strain medium of strain VO-26 strains (g/L):glucose 10, yeast extract B 8, beef extract E 10, NaCl 3, agar powder 16. The Best strains preservation method:30% glycerol preservation method for L-ornithine strains can be used to save for six months. Lyophilization for L-ornithine high-producing strains can be used to save for a long period of time.(3) The best seed mediumand(g/L):sugar A 20, (NH4)2SO4 15, MgSO4-7 H2O 2.5, corn steep liquor 3, yeast extract A 15, K2HPO4 1, NaH2PO4 0.5, KH2PO4 0.5, CaCO3 15(food grade), pH 7.0.Through studying different fermentation conditions on the L-ornithine seed, the best seed culture conditions was optimized:Temperature 31?; pH 7.0; Liquid volume 50mL/500mL round bottom flask, Eight layer gauze sealing; Shaker speed 210r/min; Seed period 12 h.(4) The batch fermentation conditions of strain VO-26 in shake flask were studied, the fermentation medium was optimized by single factor optimization and orthogonal design experiment. The best fermentation mediumand(g/L):Glucose 140, (NH4)2SO460, Yeast extract C 15, organic nitrogen source B 5, KH2PO4 1, MgSO4-7H2O 2.5, K2HPO4 0.5, NaH2PO4 0.5, MnSO4-H2O 0.01, FeSO4-7H2O 0.01, CaCO3 18.Through studying different fermentation conditions on the L-ornithine fermentation, the best fermentation conditions was optimized:Fermentation temperature 31?; pH 6.8-7.0; Inoculated volume 10%; Liquid volume 20mL/500mL round bottom flask; Shaker speed 220r/min; Fermentation cycle 72 h.Under the optimum condition, the VO-26 strain could produce 49.89g/L of L-ornithine acid and its conversion rate (L-ornithine acid/glucose) could reach 35.92%,which increased 12.70% and 7.67% respectively related to the initial condition(44.27/L,33.36%). And the production volume of L-ornithine than the original strain SL-18 increased 68.55%.Through the mutation breeding and optimizating process of being fermented by L-ornithine producing strain, the strain VO-26 could produce L-ornithine 49.89g/L, and could reach the fermentation production requirements of L-ornithine enterprise.