Breeding And Optimizing Fermentation Technology For The Production Of Thuringiensin By Bacillus Thuringiensis 032

Thuringiensin is a nucleotide analog produced by a number of strains of Bacillus thuringiensis belong to different serovars. It has a broad-spectrum of effectivity against many insects, nematodes and mites. Because its toxicity is much lower than a lot of chemical pesticide's, thuringiensin has a promising prospect.Taking Bacillus thuringiensis 032 which was screened out from 103 strains preserved in our laboratory as the original strain, a new strain named 032UDU with the steady capability of overproducing thuringiensin was obtained after UV and DES mutation. The yield of thuringiensin produced by 032UDU was 20% higher than that of the original strain.Based on the single-factor experiments, central composite experiment and response surface methodology were applied to optimize fermentation medium. The optimum composition of the fermentation medium was: 22.8 g/L glucose, 32.5 g/L nitrogen source A, 3.0 g/L soybean meal, 3.0 g/L nitrogen source B, 2.0 g/L CaCO₃, 2.0 g/L KH₂PO₄, 0.1 g/L MgSO₄. 7H₂O and 0.02 g/L MnSO₄. H₂O. Then the single-factor experiments were utilized to optimize the fermentation condition. And the optimized fermentation condition was determined as following, pH of medium was adjusted to 7.5 before sterilization and 20 mL broth was incubated at 30℃ for 24 h in 500 mL Erlenmeyer flasks after inoculating 3% seed of 12 h.Then the influence on the yield of thuringiensin with feeding different concentrations of glucose at different time was discussed. The yield of thuringiensin was improved with not only the increasing amount of biomass when glucose was feed at the exponential prophase but also the productivity of thuringiensin of the cell enhanced with glucose fed at the exponential anaphase. The output of thuringiensin was increased 40% compared with the batch fermentation when feeding 15 g/L glucose in two times at the exponential phase.The method of constant-speed feed could relieve the restraint caused by high concentration of glucose, and the amount of biomass and yield of thuringiensin were increased 73% and 75% respectively compared with that of batch-wise feed. But the good result was only owed to the increase of biomass. The concentration of glucose could be becoming to the need of cell when the feed speed was controlled by pH. The yield of thuringiensin and Y_P/X were increased 79% and 31 % respectively compared with that of batch fermentation. The feeding mode was conducted in cloudy medium and the yield ofthuringiensin was increased 83% compared with that of batch fermentation. The final yield of thuringiensin is 5.18 g/L.