Fermentation Optimization And Purification Of Surfactin Produced By Bacillus Amyloliquefaciens

Surfactin,as a green biosurfactant,has broad application prospects in various fields,such as agriculture,food,pharmaceutics,daily chemicals,oil recovery and bioremediation.Surfactin is mainly produced by fermentation,however,its commercialization is restricted by low biosynthesis efficiency,severe foaming,and the high cost of purification.Therefore,this study optimized the fermentation of Bacillus amyloliquefaciens and the process of purification,which is helpful to promote the industrialization of surfactin.In this study,the wild-type strain of B.amyloliquefaciens MT45 was used as the research object.In flask,the surfactin yield was increased by adding nanomaterials,and the molecular mechanism of this high-production was revealed by electron microscopy and transcriptomic analysis.In 7 L bioreactor,the optimal fermentation strategy based on foam fractionation was explored for controlling the foaming and increasing surfactin yield.Finally,this study provided a process of separation and purification,which was simple and suitable for industrialization.The purified surfactin was used for qualitative analysis.The main results of this study are as follows:（1）The secretion of surfactin is an important factor affecting the surfactin yield.This study analyzed the effect of seven nanomaterials on the secretion and surfactin yield.The results showed that Fe nanoparticles significantly increased the surfactin yield from 4.93 g?L^-1 to 7.15g?L^-1 with an optimal addition amount of 5 g?L^-1.Moreover,electron microscopy and transcriptomic analysis were used to to reveal the mechanism of this high-production,which could be summarized for three reasons:（a）After adding Fe nanoparticles,the cell membranes were damaged,and the expressions of genes related to the electron transport chain were down-regulated.However,the expressions of genes related to oxidoreductase activity,sucrose transport,and surfactin secretion were up-regulated.It indicated that Fe nanoparticles induced membranes leakage of B.amyloliquefaciens MT45 through physical damage and oxidative stress,and the cells could also trigger the antioxidative defense system.As a result,Fe nanoparticles increased the permeability of membranes,resulting the sucrose ingestion and surfactin secretion more effective.（b）The genes encoding glycerolipid synthase and fatty acid synthesis were up-regulated,indicating that cells might repair the damaged membranes by enhancing the fatty acid synthesis,which could provide more precursors for surfactin.（c）The genes involved in the modules of glycolysis and TCA cycle,amino acid metabolism,and modularly enzymatic synthesis of surfactin were up-regulated,which might divert more energy and primary substrates for surfactin synthesis.（2）In order to solve the problem of severe foaming during the fermentation in bioreactor,the strategies of controlling foaming were studied in 7 L bioreactor.After adding organic silicon and soybean oil antifoams,the results showed that organic silicon was very effective for defoaming,but the surfactin yield was as low as 1.42 g?L^-1.Addtion of soybean oil antifoam could increase the surfactin yield to 1.96 g?L^-1,but the defoaming effect was poor.However,the foam fractionation not only solved the problem of foaming,but also increased the surfactin yield to 2.39 g?L^-1.Based on the foam fractionation,the fermentation conditions in bioreactor were optimized.After fermentation for 60 h,the surfactin yield increased to 6.04 g?L^-1 by controlling pH at 7,DO≧20%,and feeding with a constant rate of 1.39 mL?min^-1 for 30 h.The working volume was stabilized at 3.5 L.Furthermore,the surfactin yield increased to 9.18 g?L^-1 by adding Fe nanoparticles,which was 6.46 times higher than using organic silicon antifoam.It was the highest yield in the reported wild-type strains.This study provided a basis for the industrial production of surfactin.（3）In order to solve the problems of complex operations in current purification process and promote the industrialization of surfactin,this study provided a simple purification process with high recovery rate and purity of surfactin,and it was suitable for industrialization.The purification process was as follows:The organic solvent was added into supernatant for removing protein.Then,the organic solvent was recovered by rotary evaporator.Finally,the purified surfactin were obtained by acid precipitation and pickling for removing small molecular impurities.Among them,the optimal method of removing protein was adding ethanol to broth(V_broth:V_ethanol=1:1),and the pH was 6～7.As a result,the rate of protein removal was85.08%,the recovery yield of surfactin was 99.71%,and the purity was 6.18%.The optimal method for removing small molecule impurities was that adjust pH to 2 and pickle twice by H₂SO₄ aqueous solution（pH=2）.The obtained surfactin was used for the qualitative analysis,the results showed that the purified surfactin contained C₁₃-C₁₇ surfactin.Among them,the C₁₅-surfactin had the highest abundance,accounting for 34%.In this study,a simple and efficient method was provided for the separation and purification of surfactin,and it was more suitable for industrial application.The recovery yield of surfactin was 96.28%,and the purity was94.22%.