High-throughputscreening Of Mutants Of Surfactin High-producing Bacillus Subtilis,fermentation Optimization And Purification

Surfactin,one of the most powerful biosurfactants,is a lipopeptide-type biosurfactant generated by Bacillus subtilis.The chemical structure of surfactin is composed of seven amino acids bonded to the carboxyl and hydroxy groups on long chain fatty acids(C13–C15).Surfactin has a great value in environmental management and medical treatment.However,the high cost of surfactin production and its low yields impose restrictions on its range of commercial applications.In this study,surfactin high yield strains were constructed by ARTP mutation and high throughput(High-Throughput)screening and then fermentated in 5 L fermentation for surfactin production.Eventually,we obtained purified surfactin by preparative liquid chromatographic.Following are details about the major steps of this study:(1)We used ARTP(atmospheric pressure and room temperature plasma)to obtain rapid mutagenesis of Bacillus subtilis E7 in atmospheric and room temperature.The initial strain was cultured for9 hours and then mutagenesis for 220 s in ARTP.The results showed that there were some differences in the phenotype of the mutants.The initial screening of the mutant strains was carried out on the basis of the differences of phenotype.With the help of the initial screening,combined with the classical blood plate method(diametr of hemolytic circle),the strain with high yield surfactin can be rapidly screened.(2)To improve the screening fiux,the 24 shaken microtiter plates(24 Microflask)were used to carry out the fermentation experiments instead of common triangle flask.Surfactin was used to detect the change of color after the reaction of surfactin with CPC-BTB reagent by Microplate reader,and the mutants with relatively higher yield could be screened out quickly.The results of HPLC test showed that the activity of surfactin was increased from 0.12 g/L to 0.8 g/L,and the yield was increased by 7 times.A combination of appearance screening,diametr of hemolytic circle,cultured in Microflask,multifunctional enzyme standard detector,shake flask test and high performance liquid chromatography(HPLC)methods enhanced the screening of flux and speeded up the screening process.We successfully constructed a high-throughput screening system for mutant strain with high yield of surfactin.(3)The results showed that the direct fermentation of fermented broth was serious and the utilization rate of nutrientsand the yield of surfactin was low.The addition of soymilk to defoamer can not inhibit the production of foam and make it difficult for later extraction.Finally,the 5 L fermenter level ofsurfactin production was collected by foam collection and reflux,and the defoaming tower was added on the basis of the model,which further enhanced the effectiveness of the reflux.The combination of ventilation and agitation speed showed that it would lead to severe blistering and required dissolved oxygen could not guarantee the normal growth and metabolism of cells,which was not conducive to surfactin fermentation production.After comparison with the ventilation 1 vvm,stirring speed 300 rpm under the conditions of surfactin obtained the highest yield,2.392 g/L.(4)The supernatant of the fermentation broth was adsorbed and eluted using macroporous resin to obtain a lipopeptide biosurfactant crude product.The concentrate of surfactin is 58.9%.The crude product was detected by LC/MS,indicating that the crude product was mainly composed of bacillomycin D,fengycin,iturin and surfactin.The results showed that the mass ratio of the product was 1008,1022,1036,1050,which was surfactin(A B C D)by using the preparation liquid phase to collect surfactin.