Thorn Sugar Zhongshengmycin Produce Bacteria Strain Selection And Optimization Of Fermentation Conditions

Spinosad, which was a novel microbial broad-spectrum insecticide, was secondary metabolites from the aerobic fermentation of a naturally occurring actinomycetes bacterium, Saccharopolyspora spinosad. Spinosad, which was composed of a mixture of two most active naturally occurring metabolites (spinosyns A and D), was effective on a wide variety of crops. Attributes of spinosad included: unique chemistry and mode of action, high levels of activity against economically important pests, a short half-life, degradation to natural building blocks, and large margins of safety for mammals, birds, fish and even most beneficial insects. In this dissertation, strain improvement, optimization of spinosad fermentation process and scale-up of fermentation processes in 10 L fermentor. were studied.According to the biosynthesis pathway and the metabolic regulation of spinosad, stram improvement was performed by mutation and rational screening to improve the productivity. Through treament by rational sceening and mutagens such as UV, DES, LiCl, r -ray and microwave etc, spinosad over-producing strain NS-1 was obtained. Productivity of strain NS-1 reached 97.83ug/mL, which was 254.71% higher than that of the original strain CT-20-I. Subculture test indicated that the hereditary character of strain NS-1 was stable.In this dissertation, the medium composition and the fermentation conditions for spinosad production were investigated. The optimal medium composition, which was obtained through single factor experiments and orthogonal design experiments, was asfbllows(g/L): glucose 0.6g, maltose 0.3g, cottonseed flour 0.3g, fish meal 0.05g, corn steep liquor 0.15g, NH4Cl 0.0lg, CoCl20.0003g, methyl oleate 0.1g, calcium carbonate 0.5g, precursor 0.3g (added at 72h for batch fermentation). The optimal fermentation conditions were as follows: initial pH valuer,of medium 7.0, the inoculum amount 10%, loading amount 30ml in 250ml shaking flask, culture temperature 30@ and after 96h at 28@. The productivity of the optimal fermentationcondition reached 128.21ug/mL, which was increased by 31.05% compare to that of the original fermentation condition.Based on the above experimental data, tests in 10L fermentor were also carried out. The fermentation kinetics of spinosad over-producing strain NS-1 was studied in this dissertation. Compared with shake-flask fermentation in 10L fermentor, a mase of foam were produced, which were controlled by adding an antifoam agent. Finally, the scale-up of spinosad fermentation from shaking flask to 10L fermentor was realized successfully, and productivity of spinosad high-producing strain NS-1 in 10L fermentor reached 66.21 ug/mL which was decreased by 62ug/mL compare to that of the original fermentation condition .