Optimization For The Fermentation Technology Of Lipopeptides Producing Bacterium Bacillus Amyloliquefaciens ES-2-4 And Separation Of Lipopeptides

Strains of Bacillus can produce variety of antimicrobial lipopeptides that mainly include three families. They are Surfactin family, Iturin family and Fengycin family. Lipopeptides have great potential applications in biomedicine because of their antibacterial, antifungal, antivirus and antimycoplasma properties. Moreover lipopeptides was widely utilized in industries such as food, cosmetics, agriculture, enhanced oil recovery, and bioremediation of oil-contaminated sites. However, lipopeptides have not yet been employed extensively in industry because of the high production and recovery costs. Thus finding the method to reduce the costs of lipopeptides is very important. In this study, we used the method of selecting microorganisms capable of producing lipopeptides in high yields and optimizing fermentation and recovery system conditions to lower the costs of the production of lipopeptides. The results were described as follows:Bacillus amyloliquefaciens ES-2 was implantated with low-energy nitrogen ion beam to breed mutants of high yield of antimicrobial lipopeptides. The effects of different doses of nitrogen ion implantation on the survival and positive mutations rate was studied. The mutant strain designated as B. amyloliquefaciens ES-2-4 showing higher yield of antimicrobial lipopeptides was obtained. The concentration of the lipopeptides in fermentation liquid increased by 15.2% compared with the original strain. We also studied the fermentation process between the mutant and the original strain. It indicated that the mutant with shorter lag phase, longer stable phase and higher yield of the lipopeptides, which was suit for large-scale production. Plackett-Burman design was undertaken to evaluate the effect of the thirteen factors. In the following phase of the optimization process, Uniform design was applied to get the optimum composition of the crucial factors. By statistical analysis, the significant factors affecting the yield of the antimicrobial substance were determined as follows: glucose, L-monosodium glutamate, temperature and rotated speed. The optimal composition was 42 g/L glucose, 14 g/L L-monosodium glutamate, 27℃and 200r/min. The production of the antimicrobial substance was increased from 3.87 g/L to 5.41 g/L, which was 39.7% higher than that of the control. Furthermore, one of the artificial intelligence technologies named as genetic algorithm (GA) was also used for optimization. The optimal result was obtained after 50 generations, which was the same as the composition obtained by uniform design.A single-factor design experiment was employed to screen carbon source of fermentation medium for industrial application from four agricultural products (millet power, corn power, soybean meal, wheat bran). Uniform design was used to optimize the optimal composition of carbon source and nitrogen source. After optimization the yield of lipopeptides reached 6.876 g/L when millet power was 48.0 g/L and L-monosodium glutamate was 15.0 g/L.Foaming was applied for separating lipopeptides in the culture broth directly in our study. The effect of agitation rate and aeration rate on the concentration of lipopeptides in the foam separated from culture broth was studied. Uniform design was used to get the optimal composition of the two factors. The results showed that the concentration of lipopeptides in the foam was able to reach 18.609 g/L, and the recovery rate was up to 89% when agitation rate was 200r/min and aeration rate was 12 L/min, which was the optimal composition.