| Spinosad is a new kind of high-efficiency biological pesticide, which is widely used and has enormous market demand. Spinosad is a secondary metabolite of Saccharopolyspora spinosa (S. spinosa) by aerobic fermentation, however, many problems exist during the fermentation process, among which improvement of spinosad yield, reduction of energy consumption and saving of spinosad costs are hot spots in research. Taking Saccharopolyspora spinosa ATCC49460(S. spinosaATCC49460) as a starting strain, through an analysis of key points of spinosad synthesis pathway, corresponding fermentation process control and optimization studies was carried out, in order to improve spinosad production and reduce energy consumption and save spinosad costs. The main research results are as follows:1. The study found that promoting the synthesis of PKS can increase spinosad production significantly by adding exogenous fatty acids, and promoting the substitution of methyl on rhamnose and forosamine can increase spinosad yield remarkably by adding exogenous S-adenosine methionine (SAM). Through further optimization, the control method and the fermentation process were established as follows:(1) The optimal way and type to add exogenous fatty acid was investigated. Comparing several common cheap exogenous fatty acids (soybean oil, rapeseed oil, corn oil, sunflower oil, peanut oil) effect, it’s found that rapeseed oil had the best effect, and then the rapeseed oil adding time and dose were optimized. When adding10g/L rapeseed oil in96h of the fermentation process, spinosad yield increased by4.0times.(2) A method for continuous supply of cheap SAM with co-culture saccharomyces cerevisiae was developed. At96h of the fermentation, inoculating7%(v/v) saccharomyces cerevisiae in fermentation process with continuous supply of the cheap SAM, could make spinosad yield increased by6.3times.2. Further investigation on relation among the variation of dissolved oxygen, cell growth, product synthesis, energy metabolism and key enzymes genes expression level was carried out to find the mechanism of demand of dissolved oxygen in different stages between strain growth and product synthesis process, and finally the four-stage dissolved oxygen regulation stratigy was established, i.e.,0-24h with dissolved oxygen of40%,24-96h50%,96-168h25%,168-240h25%. Compared to constant dissolved oxygen level of50%,40%,30%and25%, the yield of spinosad increased by652.1%,326.1%,546.8%and781.4%, respectively.3. A low-cost high-efficiency technology for spinosad production with non-grain fermentation was developed. Firstly, by analyzing the effect of cotton seed powder and yeast powder as the nitrogen source on the spinosad fermentation, under the premise of not affecting the spinosad yield, the cheap cottonseed powder partly replaced yeast powder, yeast powder consumption reduced by50%; Secondly, the effect of four non-grain carbon source alternatives on spinosad fermentation was studied, cheap DW significantly improved the spinosad production than glucose was found. Further optimization with response surface, the full non-grain substitution of the fermentation medium was realized, significantly reducing the costs of spinosad fermentation.4. A non-grain spinosad fermentation and co-production diosgenin was established. By strain breeding, strain S.spinosa-gm5-171can efficiently use DZW containing saponins, and the strain growths and synthesizes spinosad as intracellular products using DZW. At the end of the fermentation, the separation of the bacteria and fermented liquid containing saponins was conducted. Saponins can further converted into saponin, and bacteria can be obtained for spinosad production. The technology is to ensure more grain yield and the yield of saponin from sterilization, at the same time, abandon the traditional acid hydrolysis extraction process. The wastewater quantity is significantly reduced, BOD decreased from15000mg/L to450mg/L... |
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