Optimization And Screening Of The Fermentation And Preparation Conditions For Saccharopolyspora Spinosa

Spinosad, a kind of novel microbial green insecticide, is a secondary metabolite of aerobic fermentation by soilactinomycete Saccharopolyspora spinosa. Attributes to its major biological active component, Spinosad has a powerful effect on a wide variety of pests, including Lepidoptera, Thysanoptera, Coleoptera, Diptera, Hymenoptera and has the features of broad spectrum, high insecticide vitality, short half-life and to the Environmental Friendly.Medium optimization is an important approach of improving yield by batch fermentation, but its effect is limited due to the needed nutrition is varied from time to time. Appropriate fed-batch fermentation is the basis of high production due to its flexibility and controllability. Using fed-batch fermentation to increase the production of spinosad is the aim of our research. In this study, we determinates the best initial glucose concentration, the optimum feeding glucose concentration and the best feeding strategy. The final fed-batch fermentation strategy for spinosad production is as follow:the initial glucose concentration is 60 g/L, and increase the glucose concentration by 12.5 g/L every two days. The final production of spinosad and biomass are increased by 15.7% and 19.7% respectively in a 20 L bioreactor fermentation system.Dissolved oxygen concentration (DO) is an important factor of fermentation process parameters. We studied the kinetics property of the batch fermentation of spinosad under different DO(40%～60%) and the effects of temperature on cell growth, substrate consumption and products formation. The result showed that a higher DO was prefered to accelerate the metabolism but to the disadvantage of production.A relative lower DO concentration was prefered to product formation while the extremely low DO may restrain the cell growth.A three-stage DO control strategy was used to shorten fermentation cycle and increase productivity in the meantime:0-72 h the DO was kept at 55%,72～168 h the DO was kept at 50%,168～240 h the DO was kept at 45%. Finally, the spinosad production acheived 394.48 mg/L, which increased by 18.8% than those of 50% DO.The spinosad production was further increased by combining the above-mentioned DO-control system and fed-batch fermentation process. Under the optimum condition, the final spinosad production reached 410 mg/L, which was higher than that of DO-uncontrolled, DO-controlled and DO-shift-controlled methods respectively.Further, in order to ameliorate the extraction process and improve the purity of product, based on the physicochemical characteristics of spinosad, The best methods for spinosad refinement are:dissolve the semi-finished products five times with acetone, gather the acetone and filter through the 0.22μm organic membrane. Then volatiliz the acetone to spinosad crystallization. The purity of spinosad reached 78.27%, and the total yield was 60.01%.