Optimization Of Biocontrol Agent B99-2 Fermentation Process And The Research Of Its WP, Microcapsule

Bacillus subtilis is dominant bacteria in ecological environment of soil and plant, which was isolated from natural soil by our lab and finally identified as Bacillus subtilis. B99-2 has a broad-spectrum resistance on Fusarium oxysporum, Rhizoctonia solani, Cladosporium fulvum, Botrytis cinerea, Colletotrichum orbiculare, Ralstonia solanacearum etc.In order to put biocontrol agent B99-2 into use effectively, the culture medium of B99-2 was optimized. Corn flour and soybean meal were screened from 12 kinds of common carbon sources and 10 kinds of common nitrogen sources, which would improve the bacterial biomass and shorten the sporulation time. We also found that CaCO3 was effective for sporulation, while KNO3 could greatly improve the biomass. The fermentation conditions were optimized by Response Surface Methodology. The final composition was:corn flour 24.24 g/L, KNO3 6.99g/L, soybean meal 26.09 g/L, MgSO4 5.00 g/L, MnSO4 18mg/L, KH2PO4 0.3 g/L, K2HPO4 2.0 g/L and CaCO3 4.2 g/L. Under these conditions, the biomass of B99-2 could reach 7.23×109cfu/mL, which was increased by 22.54% to 5.90×109cfu/mL. The 150 L fermenter experiment was carried out by optimized parameters. The ferment biomass could come up to 2.05×1010cfu/mL and the sporulation time was advanced by 18 h compared to flask fermentation.Secondly, WP component of Bacillus subtilis, such as carriers (filler), wetting agent, suspending agent, stabilizer, UV protection agents etc, were screened. The optimized composition was:diatomite 10%, suspending agent PVA 7.2%, wetting agent D4254.8%, stabilizer CMC-Na 2% and protectant CMC 0.1%. Every index could match the state standard.The effective and cheap wall materials of microcapsule were screened and the spray drying parameters were optimized. The final conditions of B99-2 microcapsule making were: bacterial solution content 3%, wall material content 20%, multiple emulsion adjuvant HLB 13, emulsifier 0.4%, emulsification speed 600 r/min., wind temperature 170℃and solution flow 400 mL/h. Through this way, the biomass of B99-2 microcapsule agent could reach 4.05×10 cfu/g and also had long-time storage and good anti-UV ability.