Study On The Fermentation And Antimicrobial Substances Isolation And Purification Of Bacillus Marimus B-9987

Plant disease caused by bacterias and fungi are often difficult to control, because of the pathogens gene diversity and variability, pant diverisity and lack of effective pesticides.Bacillus marinus B-9987, a promising microbial pesticide, had a great antagonism effect on a wide range of plant pathogens, such as Ralstonia solanacearum, Erwinia carotovora and Botrytis cinerea. It has a promising prospect for controlling such plant disease. B. marinus B-9987has been researched for many years. The cell concentration at the end of fermentation was only1.5×109CFU/mL during the5L and50L fermentation process, and several bioactive substance has been separated for the fermentation broth, because of their instability, low in concentration and high separation cost, they have no business value.lt is important to establish a practical fermentation process and find a definite bioactive substance of B-9987.The culture medium of B-9987was preliminary optimized by orthogonal test (factors such as carbon sources, nitrogen sources, and inorganic salts). Cell concentration fluctuating during using the preliminary optimized medium fermentation process in the5L fermenter. Extracellular toxic substances, defoamer toxicity and restricted substrate consuming affected the cell culture and resulted cell concentration fluctuating during the fermentation process. Among them the A concentration (restricted substrate) played a major role. Adjusting the concentration of A can relieve the cell concentration fluctuating.The seed culture process was investigated in flask fermentation. The optimized medium was verified in5L fermenter and successfully scaled up into50L, which can serve as a practical fermentation process. The cell concentration and spore rate at the end of fermentation were5×109CFU/mL and above90%respectively. The cell concentration increased more than3times by the optimization.The fermentation broth which spore rate is more than90%were used for separation by testing the antifungal activity of fermentation broth at different times The separation of bioactive substances of B-9987broth was done as follow:First,1mol/L HCl was added into the B-9987broth to adjust pH to2.0in order to precipitating the sediments which contained bioactive substances; Second, the sediments were extracted with solvent A and further separated by silica gel column chromatography; At last, the bioactive components were analyzed by HPLC. Three main compounds were obtained and the structure of them needed further research.