Studies On The Batch Fermentation Kinetics And Extraction Process Of Spinosad

Spinosad (Spinosyns), a kind of novel microbial broad spectrum insecticide, is secondary metabolites of aerobic fermentation by naturally occurring antinomycetes Saccharopolyspora spinosa. Spinosad is effective on a wide variety of insects of crops. Attributes of spinosyns include high levels of activity against economical important insects, short half-life and large margins of safety for mammals, birds, fishes, et al.Fermentation kinetic model is important for optimizing control and up-scaling of fermentation process. In this study, firstly, we determined the biomass concentration using diphenylamine colorimetry methodology, and the patterns of metabolism of Saccharopolyspora spinosa SP06081 in a 20L bioreactor were studied. We proposed the kinetic models for biomass growth, spinosad production and substrates consumption in batch fermentation based on Logistic equation and structured model. According to the data of experiment, the parameter values of kinetic models were obtained by numeric integration coupled with Nelder-Mead simplex search method using MATLAB 7.0 software. The results showed that the diphenylamine colorimetry method was qualified for the determination of biomass in medium containing insoluble components and the experimental data was in good accordance with calculation values of kinetic models, the RE was 5.02%,7.37%,3.49%, respectively. The Logistic equation based kinetic models could simulate the batch fermentation process in 20 L bioreactors correctly.In order to enhance the fermentation productivity of spinosad, the kinetics property of fermentation process in 28℃-32℃were studied, the effects of operational temperature on cell metabolism, specifical growth speed and specifial spinosad formation speed were discussed in detail. Higher temperature was favorable for cell growth and decreased stage, and lower temperature was suitable for spinosad production, the final spinosad titer reached 408 mg/L at 28℃, which was 20% and 165% higher than 30℃,32℃, respectively. Maintained higner temperature in fast growth stage to improve cell propagation, and then reduced temperature to keep hign speed of spinosad formation, also prolongate the producing period, the final spinosad titer reached 185 mg/L, productivity was 0.77 mg/L·h, which was not enhanced, and this strategy needs further research.Thirdly, using extraction-antiextraction process, the influences of solvent, time, pH, temperature on soaking, extraction, antiextraction and crystallization were studied in order to establish the method of extracting spinosad from fermentation broth of Saccharopolyspora spinosa, and also, we determined the optimal time, pH, temperature for extraction, antiextraction and crystallization. The optimal conditions for spinosad separation from broth were:methanol extraction (1:1) for 4 hours, concentrated and extraction in 30℃, O/W=1, initial pH 7.5 for 5 min using ethyl acetate as the extracting solvent. Then antiextraction was carried out in 20℃, O/W=1 for 1 min using 0.2mol/L tartaric acid solution (pH=2.03). The pH of antiextraction phase was adjusted to 9.0 to spinosad crystallization. With the optimum conditions, the whole extraction process was practiced in small scale, the total yield was 71% and the purity of row products reached 61.3%. In this study, pH and temperature have significant effects on the yield of extraction and antiextraction. Because of low yield of single stage antiextraction process, total yield was not very high, so further research is very necessary to improve such process.