| Polycyclic aromatic hydrocarbons(PAHs)are persistent organic pollutants with a stable chemical structure and high toxicity.They are mainly released from the mining,refining,and transportation of fossil fuels.Determining practical and effective methods for PAH removal is essential for ensuring environmental protection.The microbial degradation of PAHs is a green and efficient method to remove these pollutants from the environment.As a high-molecular-weight PAH,it is difficult for microorganisms to directly use pyrene;however,adding a co-metabolism matrix can significantly enhance the removal of pyrene by microorganisms.In this study,using pyrene as the target pollutant,two pyrene-degrading bacteria were obtained from oil-contaminated soil in Shengli Oilfield through enrichment and cultivation.Molecular biology methods were used to identify bacterial strains,and study their physiological and biochemical characteristics,pyrene degradation rate,and co-metabolism properties.Through metabolite,enzyme activity,and whole genome sequencing analyses,the degradation pathway and co-metabolism mechanism of pyrene by degrading bacteria were discussed.The relevant research results are as follows:(1)Two pyrene-degrading bacteria were isolated from oil-contaminated soil in Shengli Oilfield.Here,16 S r DNA sequencing showed that the selected strains were Pseudomonas stutzeri M4 and Alterrothrobacter strain N1.The study of physiological and biochemical characteristics showed that the optimum growth temperature of the two strains was 35 °C and the optimum p H was 7.5.The degradation rates of strains M4 and N1 were 42.3% and 40.5%,respectively.(2)The pyrene metabolites in the two strains were explored separately.The intermediate metabolites of pyrene degradation were determined by gas chromatography-mass spectrometry(GC-MS)and liquid chromatography-mass spectrometry(LC-MS).Upstream metabolites,such as 1-hydroxypyrene and 1,2-dihydroxynaphthalene,as well as downstream metabolites,such as salicylic acid and phthalic acid,were detected,indicating that the two strains may degrade pyrene through a variety of metabolic pathways.(3)The 30-day degradation rate of pyrene was determined using saturated hydrocarbons,aromatic hydrocarbons,and crude oil separated from crude oil as cometabolism substrates.Compared to the control group without any co-metabolic matrix,the addition of a co-metabolic matrix could enhance pyrene degradation to varying degrees.Among them,strains M4 and N1 had the highest degradation rate using crude oil as the co-metabolism substrate,at 61.2% and 75.9%,respectively,and the cometabolism performance of strain N1 was stronger than that of M4.(4)The whole genome of strains M4 and N1 was sequenced.Gene annotation was based on homologous protein(GOG),gene ontology(GO),and Kyoto Encyclopedia of Genes and Genomes(KEGG).The results showed that both strains had salicylaldehyde dehydrogenase genes,protocatechuic acid 3,4-dioxygenase genes,naphthalene 1,2-dioxygenase genes,and aldol dehydrogenase genes.Strain N1 was annotated by RAST and the salicylic acid hydroxylase,gentisic acid 1,2-dioxygenase,catechol 1,2-dioxygenase,and ?-adipate ketohydrolase genes were obtained.Combined with a metabolite analysis,it was determined that pyrene degradation by M4 proceeds via the salicylic acid and phthalic acid pathways,while that by strain N1 proceeds via the salicylic acid,phthalic acid,and gentisic acid pathways.(5)Based on co-metabolism and metabolites,the enzyme activities of key metabolites in the process of pyrene metabolism were studied,and the main pathways for the degradation of pyrene were determined.The experimental results show that adding different co-metabolic substrates can significantly promote the activity of degrading enzymes in different degradation pathways.When crude oil is used as a cometabolism substrate,strain M4 mainly follows the salicylic acid pathway to achieve pyrene degradation,while strain N1 mainly follows the phthalic acid pathway to achieve pyrene degradation. |
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