Polymorphism Of Aromatic Dioxygenase Genes And Metagenomic Sequencing In Petroleum Contaminated Soils

Petroleum is the most important energy substance and chemical raw material in today's society.Inevitably,leakage occurs in all aspects of oil exploration,transportation,storage,and processing.The aromatic hydrocarbon compounds in various components of petroleum are very biologically toxic.It is particularly important to eliminate them from the environment.Biodegradable remediation technology is considered to be the most economical,safe,and effective means of all oil organic pollutant removal methods.When oil leaks into the ecological environment,it will have an impact on the environmental microbial community structure.Therefore,it is of theoretical significance to study the changes of microbial community structure,functional genes and metabolic pathways over time in the process of natural degradation of petroleum,and to screen organism-degrading strains of organisms,to search for functional genes that are characteristic targets of pollution,and to carry out bioremediation.In this paper,soils with different years of petroleum pollution are taken as the research object,and clone libraries are used to study the types of aromatic dioxygenase genes in different samples and their changes with time.16 S rRNA amplicon sequencing analysis of bacterial communities in petroleum contaminated soils and background soils.Metagenomic sequencing technology was used to study the effect of petroleum pollution on soil macro-genome changes,and to explore the law of petroleum pollution on soil microbial community structure and metagenomic changes over time.The following results were obtained through research:(1)The nid nested primer set obtained by the experimental screening was used to amplify the aromatic dioxygenase gene in oil-contaminated soil in the Zhundong Oilfield,and a clone library was constructed to obtain 5 types of aromatic dioxygenase genes:(1)Phthalate dioxygenase gene;(2)Aromatic oxygenase gene;(3)Naphthalene dioxygenase gene;(4)PAH ring-hydroxylating dioxygenase gene;(5)Benzoate dioxygenase gene.As the contamination time increased,the number of gene types in the sample increased,the abundance of the PAH ring-hydroxylating dioxygenase gene decreased,and the abundances of benzoate dioxygenase gene and naphthalene dioxygenase genes increased.(2)By 16 S rRNA amplicon sequencing analysis,Actinobacteria and Proteobacteria were the dominant phyla in the soil of the Zhundong oilfield.Through Non-Metric Multi-Dimensional Scaling,it was found that the soil microbial community structure in the soil after 1 year of petroleum pollution was similar to that in the background soil.With the increase of pollution time,the difference in the structure of the contaminated soil microbial flora increased,and the microbial community in the soil contaminated for 5 years formed a unique group.It is clearly different from the background sample,indicating that the contaminant remediation has not been completed,the contaminants have not been completely degraded,and petroleum-degrading bacteria have been more easily screened in the soil that has been contaminated for five years.(3)Metagenomic measurements showed that with the increase of petroleum pollution time,the number of species,the number of genes,and the number of metabolic pathways increased.Among the 14 petroleum organic pollutant degradation pathways obtained,except that the PAH degradation pathway abundance decreases as the pollution time increases,the abundance of metabolic-related genes in most low-molecular-weight aromatic hydrocarbon degradation pathways(such as the benzoic acid degradation pathway)increases with the contamination time,it shows that in the soil that has been polluted by oil for 5 years,the microbial communities and functional genes that can use aromatic hydrocarbons are extremely rich.RNA processing and modification function and cytoskeleton function gene abundance decreased with increasing contamination time,carbohydrate transport and metabolism function and extracellular structures function gene abundance increased with the increase of petroleum contamination time,and the abundance of the remaining 20 types of COG functional genes increased in varying degrees at 5 years of degradation.In this paper,the soils of different years of petroleum pollution are taken as the research object to study the regularity of the changes of aromatic dioxygenase genes,microbial community structure and metagenomics in contaminated soil over time.The results of the experiments all show that with the prolonged time of petroleum pollution,the diversity of microorganisms,the number of genes and the number of metabolic pathways have increased.And the microbial community in the contaminated soil will form a unique community structure as the substrate spectrum changes.Microbial degradation uses organic pollutants in petroleum as a carbon source material to reduce the poisoning effect of pollutants on itself.Degradation products obtained during the degradation process are used to synthesize carbohydrates necessary for their own growth and reproduction,and provide energy.