Rational Breeding Of Spinosad Producing Strain And Optimization Of Spinosad Fermentation Process

Spinosad,which was a novel microbial broad-spectrum insecticide, was secondary metabolites from the aerobic fermentation of a naturally occurring actinomycetes bacterium,Saccharopolyspora spinosa. Compare with normal insecticides,it has advantages of favorable mammalian and environmental profile.In this dissertation,the determination of spinosad by HPLC,the strain improvement by rational selection,Response Surface Methodology（RSM） was used to optimize fermentation medium and scale-up of fermentation processes in 5L fermentor were studied.Analyzing method of spinosad by HPLC was introduced.The result shows that the relative standard deviation,the coefficients of variation, the linear correlation and the average recovery were 1.044,0.415%,0. 99970,and 99.79%respectively.At room temperature,using methanol as leaching liquor,lixiviate the broth by the proportion of 1:4（broth/methanol,v/v）,processed with supersonic wave（100W） for 10min,the time of extraction was two hours and leaching liquor pH9.0.Based on the biosynthetic pathway and metabolic regulation of S. spinosa,a relational selection procedure was performed to obtain a higher spinosad-producing strain.After six UV mutations,the higher productive strain S.spinosa 6-18 was selected,which could grow well on the medium with corresponding concentration of rhamnose,acetate,propionate, erythromycin,2-deoxygen-D-glucose（2-DOG） and phosphate.The productivity reached 503.4mg/L,which was 2.2 times than that of the parent strain S.spinosa L-2.The optimization of the fermentation progress was studied. Response Surface Methodology（RSM） was used to optimize fermentation medium for spinosad production by S.spinosa H-2.The results showed that glucose 64.5g/L,maltose 20g/L,corn steep liquor 2g/L,bean oil 40g/L,proflo cottonseed flour 25g/L,soybean cake meal 2.4g/L,peptone 25g/L,and CaCO₃5g/L combined the most suitable media for spinosad production,which increased the productivity of S. spinosa H-2 from 278.1mg/L to 508.7mg/L.And the optimal cultural conditions of S.spinosa S-15 involved:the inoculation amount 10%, inoculation circle 48h,pH8.0 before sterilized,ferment for 7 days, loading amount 25mL in 250mL shaking flask,culture temperature 30℃and after 3 days at 28℃. The curve of the growth of S.spinosa,utilization of glucose,pH, dissolved oxygen（DO） and production of spinosad were studied by cultivated S.spinosa H-2,S.spinosa 6-18 in a 5L fermentation.The kinetics curve of fed batch fermentation about S.spinosa 6-18 was also studied.The productivity of S.spinosa 6-18 in a 5L fermentation was 636.7mg/L,which was 27%more than that in the shaking flasks.