Studies On The Fermentation Medium Optimization And Process Of Saccharopolyspora Spinosa

Spinosad, a kind of novel microbial green insecticide, is a secondary metabolite of aerobic fermentation by Saccharopolyspora spinosa. Because of its broad spectrum insecticidal activity as well as high-efficient, low-residue rate and no pollution to environment, spinosad has become the most promising bio-pesticides.Fermentation medium optimization is a commonly used method to improve the objective production in the fermentation process. In the study, we used mixture design to optimize the fermentation medium of spinosad by Saccharopolyspora spinosa. First of all, we used Plackett-Burman design to investigate the influence of glucose, soluble starch, sucrose, cotton seed flour, calcium carbonate, soybean flour, dextrin and corn steep liquor on the production of spinosad. Experiment results showed that glucose, cotton seed flour, corn steep liquor and soybean flour had a significant effect on the spinosad production. Then we set up the upper and lower limits of the four factors, and used D-mixture design to optimize the four factors. Regression model on Spinosad production was established, and then we got a second-order polynomial experimential model which expressed the good relationships of variables and response. The optimum fermentation medium for spinosad production were as follows:Gucose59g/L, cotton seed flour28g/L, corn steep liquor6.5g/L, soybean flour6.5g/L. The yield of spinosad was249.9mg/L in a20L bioreactor using the optimized medium, and was15.16%higher than the original medium.Precursor plays an important role in the fermentation process, an appropriate precursor can effectively improve the fermentation titer. First, we used single-factor experiment to study six possible precursors. Experiment results showed that sodium acetate and normal propyl alcohol had an inhibitory effect on the production of spinosad. Lysine, glutamic acid, fish meal peptone and n-butyl alcohol had a promotive effect on the production. Then we used orthogonal experiments to optimize the four promotive precursors. Experiment data showed that the primary and secondary relationship between the four precursors for spinosad production were as follows:fish meal peptone>lysine>glutamic acid> n-butyl alcohol, and the best components were glutamic acid(1.5g/L), lysine(2.0g/L), fish meal peptone(2.5g/L), n-butyl alcohol(0.4ml/L).The yield of spinosad was248.3mg/L with the medium, and14.95%higher than the original medium (216mg/L).We studied the extraction process of spinosad. When using solvent extraction method to extract the antibiotics, emulsifying phenomenon was serious, the emulsion layer contained a lot of pigment, which influenced the purity of the sample. In order to optimize the the extraction process and the purity of the spinosad, we separately collected the emulsion layer and extract phase. Then we filtrated the sample with0.22μm organic membrane to remove the impurities. The purity of the spinosad getting from extract phase was97.6%and the yield was85.71%. The purity of the spinosad getting from emulsion layer was61.5%and the yield was72.7%.The fermentation period of spinosad is12-14days, the production is low, and the cost is expensive, so the fermentation process need to be improved. This paper studied the optimization process for fermentation medium, the influence of precursor, and the fermentation extraction process. It includes several levels ranging from shake flask fermentation to the Bioreactor and the downstream processing process. The results provide some reference for spinosad fermentation, and have some value of applications.