Isolation, Identification, Biocontrol Potential And Fermentation Systematic Optimization Of Brevibacillus Laterosporus AMCC100017

To lay the foundation for the further development and utilization of biocontrol strains with good colonization ability, bacterial strain with particular biocontrol effect was isolated, screened and identified from soil selectively. In this study, various kinds of soil samples contained farmland, orchard, vegetable plot were collected from Shandong Tai’an. After water-curing treatment10min at80℃, with Fusarium moniliforme and Fusarium oxysporum as target pathogens, antagonistic experiment was used to screen antagonistic bacteria and evaluate its inhibitory ability. Lastly, five spore-forming bacterium with good biocontrol effect were screened. The best strain was classified and identified by morphological characteristics, physiological biochemical characteristics, sequencing of16S rDNA and analysis of biological sequences similarity comparing in GenBank. The strain was identified Brevibacillus laterosporus, the preservation number was AMCC100017. Protease activity was measured by Folin-Ciocalteu method; cellulase activity, chitinase activity, pectinase activity were measured by spectrophotometry. Results showed that protease activity was higher, which reached to1687.2U/mL after fermented6d. Antimicrobial spectrum experiment indicated that the strain has strong antagonistic effect against various plant pathogenic fungus, especially Fusarium and Rhizoctonia.Brevibacillus laterosporus can produce various active substances with widely effective application potential of antimicrobial, nematicide and immune adjustment. In this study, the screened strain Brevibacillus laterosporus AMCC100017is a good biocontrol strain with great development and utilization potential for fungal diseases of crops especially wheat sharp eyespot, fruit tree diseases especially apple replantation obstacle and biological control of nematodes. It is the bottle-neck of industrial practice to obtain the adequate amounts of both bacteria and spores. Liquid fermentation experiment of Brevibacillus laterosporus strain AMCC100017was carried out in this study. Bacteria amount and spore formation amount were systematically optimized by single factor experiment, Plackett-Burman (PB) design and response surface methodology (RSM). The result showed that the optimal carbon source, nitrogen source and inorganic salt were corn flour, fish peptone and MgSO4. The optimal fermentation conditions were temperature35℃, initial pH7.5, volume40mL/250mL, inoculation quantity2%, bacteria fermentation time24h, fermentation time of spore formation48h, respectively. Temperature, volume and inoculation quantity were tested to be significant factors that affected the bacteria amount using the PB design, besides, carbon source, temperature and fermentation time were significant factors affected the spore amount. The optimized conditions by response surface analysis were temperature34℃, volume39mL/250mL, inoculation quantity2%, corn flour59g/L, spore formation fermentation time48h. The highest bacteria amount was6.2×109CFU/mL and the spore amount was2.5×109CFU/mL in the validation experiments.Amplification culture step by step is necessary in the development process of microbial products from laboratory to industrial production. On the basis of shake flask fermentation test of Brevibacillus laterosporus AMCC100017, pilot tests on5L and20L fermenters were conducted, respectively. Results showed that when the fermenter parameters set to rotating speed250r/min and ventilation volume4m/(m min), fermentation effect is the best, the highest number of living bacterium reached2.5×109CFU/mL.