Isolation Of Efficient Petroleum Hydrocarbon Degrading Bacteria And Bioaugmentation Of Petroleum Contaminated Soil Remediation

Microbial degradation is the main function of petroleum hydrocarbon pollution removal in natural environment.The metabolic pathway of petroleum hydrocarbon degradation by single bacteria in pure culture has been extensively studied,but the interaction mechanism between microorganisms and metabolism of petroleum hydrocarbons by bacterial community remain to be further studied and explored.Therefore,in this thesis,the enrichment culture of indigenous degrading bacteria was obtained from petroleum-contaminated soil of Dagang oilfield to reveal the mechanism of different microorganisms in the process of petroleum hydrocarbon degradation.Isolation and recombination of degradation bacteria were used to construct efficient petroleum degradation bacterial community to explore the synergistic remediation effect of bacteria community soil and provided theoretical and experimental basis for microbial remediation and restoration of petroleum contaminated soil.In this study,petroleum contaminated soil from Dagang oilfield in Tianjin was collected,and petroleum hydrocarbon pollutants were used as the only carbon source to enrich and domesticate the indigenous degrading bacteria community,and finally a efficient petroleum degradation bacterial community with dynamic and stable bacterial structure was formed.Through degradation experiments and 16S r RNA gene sequencing found that the degradation rate of petroleum hydrocarbon at 1%（m/v）concentration reached 79.3%in three days,and the degradation half-life was 0.94d by the second-order degradation kinetics curve of degradation process.The degradation experiments showed that the degradation process of petroleum hydrocarbons was divided into three stages（Stage1,2 and 3）and cluster analysis and principal coordinate analysis found that the samples in these three stages were one group respectively,indicating that the community structure of degrading bacteria was significantly affected by the concentration of petroleum hydrocarbons.LEf Se method was used to analyze the bacterial community of different species in three different periods,and it was found that Stage1 had the most significant difference species,including Pseudoxanthomonas,Variovorax,Bordetella,Stage2 had the fewest significant differences,such as Aggregatibacter and the species with significant differences in Stage3 included Rhodococcus,Mycobacterium,etc.Combined with the changes in community species composition,it was found that the abundance of Rhodococcus and other bacteria increased during the degradation of petroleum hydrocarbons,indicating that these bacteria played a very important role in the degradation of petroleum hydrocarbons.Through high-throughput sequencing and PICRUSt function prediction,according to the literatures and KEGG database for enzymes and functional genes needed in different degradation stages of petroleum hydrocarbons and the contribution of different microorganisms to these enzymes,Rhodococcus had a high effect on the upstream oxygenation process and the downstream dehydrogenation metabolic process of alkane degradation.Mycobacterium,Acinetobacter and Pseudomonas also contributed to upstream metabolic process of alkane degradation.Other Variovorax,Achromobacter,Bordetella and Achromobacter were mainly involved in the downstream degradation metabolism of alkanes,and the alkane metabolic network of the bacterial community was initially mapped.Rhodococcus in indigenous degraded bacteria was screened and purified,and it was found that Rhodococcus indeed had a good degradation effect of petroleum hydrocarbons,indicating that the bioinformatics analysis method could identify target strains for the screening of degraded bacteria.Pure culture of microorganisms is the basis for further study on the function of microbial community.In this study,two strains of efficient petroleum hydrocarbon degrading bacteria were screened from indigenous degrading bacteria.Strain A2-1 was identified as Acinetobacter junii and the strain A17 was Rhodococcus qingshengii.Both strains had the best growth condition at 30℃,p H 9.0,and were tolerant to 4%salinity.Under the optimum conditions,the degradation rates of petroleum hydrocarbons of strains A17 and A2-1 could reach 65.6%and 67.8%,respectively,after incubation for 3days.According to the second-order degradation kinetics,the degradation half-lives were 34.25h and 37.27h,respectively.They could degrade alkanes with different carbon chains(C₈～C₃₅)and had good degradation ability to long chain alkanes(C₂₅～C₃₅).The genome sizes of strains A17 and A2-1 were 6.71Mbp and 3.25Mbp,respectively.They had 18 and 3 functional genes related to alkane degradation,respectively,including long-chain alkane degradation genes Lad A and Alm A and the metabolic pathway of alkanes begins with terminal and sub-terminal hydroxylation of alkanes.Strains A17 and A2-1 were combined to form a two-bacteria system for degrading petroleum hydrocarbons,and the mechanism of its co-degradation of petroleum hydrocarbons was investigated.It was found that different inoculation ratios in the two-bacteria system had no significant effect on the degradation effect of petroleum hydrocarbons,and had high cell viability and degradation effect under alkaline condition（p H9.0）.The degradation rate of petroleum hydrocarbons reached 71.3%within 3 days,and the half-life of petroleum hydrocarbons degradation was 28.56h under second-order reaction kinetics.Mix indigenous degrading bacteria and two-bacteria system builded co-cultivation microbial community,we find the alkanes,branched alkanes and long chain alkane component degradation effect has significantly improved,and high biomass,cell activity,the content of extracellular protein and polysaccharide than other treatment group have significantly improved the representative occur between microbial synergy to produce more degradation enzyme and biological surface active agent,It enhances the transport and degradation effect of petroleum hydrocarbon pollutants.Biomass,cell activity,extracellular protein and polysaccharide were also significantly increased compared with other treatment groups,indicating that the synergistic action of microorganisms produced more degradation enzymes and biosurfactants,which enhanced the transport and degradation effect of petroleum hydrocarbon pollutants.In addition,the abundance of strain A17 and A2-1was quantified during the degradation process.It was found that strain A17 grew rapidly in the early stage,consistent with the growth of the whole bacterial community,and the abundance of strain A2-1 increased rapidly in the late stage of degradation,suggesting that strain A17 dominated the entire petroleum hydrocarbon degradation process and provided carbon source and energy for the growth of co-culture bacteria,strain A2-1played a role in the degradation of long-chain hydrocarbons in the later stage.High-throughput analysis showed that the addition of two-bacteria system did not cause the diversity change of the bacterial community and could cooperate and reduce the competitive inhibition with indigenous microorganisms,this result contribute to improve the long-term degradation effect of pollutants.In the bioreremediation experiment of petroleum hydrocarbon contaminated soil（39.1 mg/g）,the two-bacteria system could remove 71.8%petroleum hydrocarbon in the soil in 50 days,and can significantly reduce the biological toxicity of soil.Partial correlation analysis showed that petroleum hydrocarbon degradation rate had a significant positive correlation with electrical conductivity and enzyme activity,but a significant negative correlation with p H.Soil microbial community diversity analysis showed that adding degrading bacteria could increase the diversity,uniformity and stability of soil microbial community,and the colonization of bacterial strains A2-1 and A17 in soil enhanced the continuous restoration effect of petroleum hydrocarbons.Collectively,various biomarkers including microbial abundance,genes,patterns,andphenotypes were identified for completion assessment of the bioremediation in a historically polluted farmland-oilfield mixed area.These biomarkers included but were not limited to the abundance of petroleum hydrocarbon degraders（as microbial markers）,the genes of sulfur respiration,energy generation,and phenanthrene degradation（as genetic markers）,the F:B ratio and the organization of functions of microbiomes（as systematic markers）,and biofilm-forming and oxygen utilization（as phenotypic markers）.Therefore,we propose that predicted functional diversity along with microbial community dynamic can be applied as a powerful tool for probing bioremediation completion and microbial restoration in petroleum contaminated soil.