Study On Optimization Of Fermentation Conditions And The Biocontrol Effect Of Bacterium B579

Bacillus subtilis is a beneficial soil bacterium with strong environmental adaptability and resistance, as it can produce heat and stress tolerance spores. It also can antagonize a variety of pathogens; also, it is safety to human. Thus, it is commonly used for biological control of crop diseases.In this thesis Bacillus subtilis B579, which was screened in our previous research, was studied. The seed culture condition in the flask was determined. The result showed that BYP was the better seed medium, and the optimum conditions for seed preparation were as follows:seed age14h, inoculum volume7%. The optimum pH for fermentation is7.0.In this study feeding strategys were optimized. The result showed that glucose feedback feeding strategy was the best one, and glucose concentration should be controlled at3.0g/L-6.0g/L. during the fermentation of B. subtilis B579. The final cell concentration was9.5×109CFU/mL, which represented a1.9-fold increase when compared with those of unoptimized condition. In order to obtain higher spore concentration, the sporulation condition was optimized using LB medium. Two-step control strategy was used, in the first phase (0-10h) to improve cell growth, and in the second phase (10h-30h) to improve spore formation. Four factors, initial concentration of glucose, fermentation pH, the second phase temperature and shaking speed, were optimized using the methods of single factor test and orthogonal experiment. The initial concentration of glucose showed a significant effect on sporulation (P<0.05). The optimal condition for the sporulation of B. subtilis B579was as follows:initial concentration of glucose5.0g/L, fermentation pH7.0, the temperature for the first phase37℃, and the shaking speed for the first phase180r/min, the temperature for the second phase40℃, and the shaking speed for the second phase200r/min. Under this conditions, after30h cultivation, the spore concentration and sporulation ratio were9.4×108CFU/mL and91.0%, respectively, which represent a6.7-fold and2.4-fold increase compared with those unoptimized.According to preliminary results, the fermentation of B579were conducted in a7L scale fermenter, with an inoculum size of7%(v/v) for a final volume of5L. Cultivation temperature and pH were maintained constant at37℃and7.0respectively. After30h fermentation the final cell concentration was1.1×1010CFU/mL. Feed-batch fermentation were conducted under the following condition:temperature37℃, pH7.0, DO10%, then supplemented glucose to the fermenter during the fermentation, and kept the glucose concentration at3.0-6.0g/L, after35h fermentation the final cell concentration was3.9×1010CFU/mL, which represented a3.8-fold increase compared with those unoptimized. Applying the pre-optimised spore formation condition in the fermenter, at the end of fermentation, it made a spore concentration of2.8×1010CFU/mL and spore efficiency75%.The biocontrol agent was made by the technology of spray drying. Analysis of the inhibitory effect of it was carried out. Pot trial studies under greenhouse conditions were carried out; the result showed that biocontrol bacterium B579was able to reduce disease incidence by73.6%it also can significantly promote the growth of the cucumber. It increased budding rate, seedling height, fresh weight and vigor index by15%,40%,30%and49%, respectively.