High-Throughput Screening And Mutation Breeding Of Biosurfactant Producing Bacterium In Petroleum Contaminated Soil

Biosurfactants are secondary metabolites produced by microorganisms in the fermentation process.Biosurfactants are chemical compounds composed of amphipathic molecules containing hydrophilic and hydrophobic moieties,which can reduce surface tension and interfacial tension.Biosurfactants have the advantages of high surface activity,low toxicity,degradation,better stability under extreme temperature,p H and salinity conditions,and lower critical micelle concentration(CMC)values.Therefore,biosurfactants become excellent substitutes for synthetic surfactants,and have important applications in environmental protection,petroleum industry,food industry,agricultural field,pharmaceutical industry and other field.However,the widespread application of biosurfactants also faces some problems,such as limited strains that can produce biosurfactants,difficulty in optimizing the production process,low yield and low efficiency of purification process.Therefore,it is necessary to screen more strains that can produce biosurfactant from the nature,and improve the yield of biosurfactants through fermentation optimization,mutagenesis breeding and genetic engineering breeding.In this study,single cell culture and microplate screening were used to screen potential biosurfactant producing bacteria in oil-contaminated soil and oil well produced fluid,and the screened strains were re-screened by oil ring test,crude oil emulsification test and surface tension measurement.Then,Atmospheric and Room Temperature Plasma(ARTP)mutagenesis was employed to screen the mutants of high biosurfactants yield.The mutagenesis mechanism was preliminarily explored by genome resequencing.The main research contents and results of this paper were as follows:(1)A total of 272 strains of potential biosurfactants producing bacteria were screened from oil contaminated soil samples and oil well produced fluid samples by single cell culture and microplate detection.They belong to 4 Phylum12 Class 5Order18 Families25 Genus38 Species.The phylogenetic tree was constructed by MEGA X software,and the results showed that the selected strains were dispersed in the phylogenetic tree with high diversity.The genetic distance between 16 S r RNA sequences of different strains was calculated by MEGA X software,and the 272 strains had 63 different sequences.By measuring the oil spreading,surface tension and crude oil emulsification,the selected strains were screened again.The results showed that among the 63 strains with different sequences,28 strains could reduce the surface tension of the medium to different degrees,6 strains could produce obvious oil discharge rings,and 26 strains could emulsification the crude oil to different degrees.(2)Pseudomonas sp.L01 and Bacillus sp.N110 were treated by ARTP mutagenesis.The mutagenesis parameters were 120 W power,10 SLM gas capacity,strain L01 and N110 were treated for 30 s and 60 s,respectively,and the fatality rate reached 94.5% and 91.2%.A mutant with high and stability yield was obtained by initial screening of blood agar plate,re-screening of shaker fermentation and stability test of passage.The yield of L01 mutant 8-135 was 2.7 times higher than that of wild-type strain,reaching 3.5 g/L.The yield of N110 mutant 7-56 was stable at about0.7 g/L,which was 37% higher than that of the wild type.By comparing the growth curve and yield curve of wild-type and mutant strain,the growth capacity and metabolic rate of mutant strain were higher than that of wild-type strain.The biosurfactants produced by strain L01 and strain N110 were identified as glycolipids and lipopeptides by TLC.It was found that the critical micelle concentration(CMC)of the mutant was lower than that of the wild type,indicating that biosurfactant produced by mutant strains had better ability to form and aggregate.(3)The mechanism of ARTP induced strain L01 and strain N110 was preliminarily investigated by genome resequencing.Compared with the wild-type strain,132 non-synonymous mutation sites and 6870 SV mutation sites were detected in the five mutant strains,involving 1310 genes.The rhl A gene mutation was detected in mutant strains 8-303,8-384 and 9-74,which is one of the genes directly related to rhamnoid synthesis.Other genes involved in KEGG secondary metabolic pathways include amino acid metabolism,carbohydrate metabolism,energy metabolism,lipid metabolism,glycan biosynthesis and metabolism,secondary metabolite biosynthesis,cell growth and death,transcription,replication and repair,and membrane transport,and so on.It was speculated that the increase of biosurfactant yield in strain L01 was not only related to rhl A gene,but also the mutation of other genes may indirectly lead to the increase of biosurfactant yield.Genome resequencing of strain N110 wild-type and mutant was performed.Five mutant strains contained 132 SV mutation sites,involving 27 genes.Genes directly related to lipopeptide synthesis(spf、srf)were not detected,suggesting that the increase of lipopeptide production in strain N110 might be related to other genes.In this study,a high-throughput biosurfactant producing strain screening method was used,which combined single-cell culture with optical distortion detection of microplate.Provide more microbial resources for microbial enhanced oil recovery(MEOR).Then the yield of biosurfactant producing bacteria was improved by ARTP mutation breeding,and the mechanism of ARTP mutagenesis of microbial strains was preliminarily explored.It provides the necessary foundation and basis for the follow-up study on improving the yield of biosurfactants.