The Isolation Of Polycyclic Aromatic Hydrocarbons Degrading Bacteria And The Study On Polycyclic Aromatic Hydrocarbons Degradation Pathways

Polycyclic aromatic hydrocarbons(PAHs)widely existing in petroleum pollutants,characteriged by hydrophobicity and stability,are one of the key pollutants in environmental science and engineering research.The activated sludge in the oil production wastewater treatment system was taken as the research object.Through the identification of intermediate products and metagenomic sequencing analysis,the degradation mechanism of three typical PAHs of naphthalene,phenanthrene and anthracene by petroleum hydrocarbon degrading bacteria was studied.Metagenome sequencing was used to analyze the changes of microbial community structure and dominant bacteria in activated sludge in SBR tanks.The bacteria in the activated sludge tank belonged to 76 phylums and 861 genera.The predominant bacteria in the three samples of WZ1,WZ2 and WZ3 were mainly from 5 phylums,ie.Proteobacteria,Actinobacteria,Chloroflexi,Planctomycetes,and Cyanobacteria.The relative abundances of Proteobacteria and Actinobacteria were 49.41% and 12.13% respectively,which played a major role in the SBR activated sludge system.From the perspective of the genus,the dominant bacteria of WZ1,WZ2,and WZ3 were mainly Mycobacterium,Ardenticatena,Azospirillum,Skermanella,Inquilinus,Thialkalivibrio and Leptolyngbya,etc.12 genera.Among the three samples treated with PAHs,bacteria belonged to 32 phylums and 628 genera.The dominant bacteria belonged to Proteobacteria,Bacteroidetes and Actinobacteria.The dominant genera in the P1 and X3 samples were Halomonas,Marinobacter,Vitellibacter,Nitratireductor,Altibacter and Pseudomonas.The average relative abundance of Halomonas in the P1 and X3 was 32.12%,which was the first dominant genus in the P1 and X3 samples.Seven salt-tolerant(30‰)bacteria were isolated and named Y2,Y3,YA,YB,NY1,NY2 and NY3.It was identified that Y2 was Alcanivorax,Y3 was Altibacter,NY1 was Idiomarina,and YA,YB,NY2,and NY3 were all Halomonas.Seven strains were used for TOC degradation test.After 3 days of cultivation at 35?,pH 7.0,and salinity of 30‰,the TOC degradation rates of Y2,NY1,and NY2 were 93.02%,93.29%,and 92.76%,respectively.Seven strains were used for PAHs degradation test.After 7 days of cultivation at 35?,pH 7.0,salinity 30‰,PAHs concentration of 100 mg/L,the degradation rates of naphthalene by Y2,NY2 and NY3 were 88.02% and 93.76% and 87.26%.The degradation rates of anthracene by NY1,NY2 and NY3 were 83.22%,81.45% and 88.13% respectively.The degradation rates of phenanthrene by NY2 and NY3 were 75.86% and 83.90%,respectively.PAHs degradation test was carried out using consortium WZ-7.The degradation effect was better than that of the original strain treatment at 35?,pH 7.0,salinity 30‰ and PAHs concentration of 100 mg/L for 7 days.The degradation rates of naphthalene,anthracene and phenanthrene were 97.08%,90.34% and 87.04%,respectively.Gas chromatography-mass spectrometry(GC-MS)was used to analyze the intermediate products of WZ-7 degrading three PAHs.Four intermediate products were detected during the degradation of naphthalene,including 1,2-dihydroxynaphthalene,2-carboxybenzaldehyde,phthalic acid and protocatechuic acid.Five intermediate products were detected during the degradation of anthracene,including phthalic acid,protocatechuic acid,6,7-benzocoumarin,1,2-dihydroxyanthracene and 3-hydroxy-2-naphthoic acid.Four intermediate products were detected during the degradation of phenanthrene,including 1-hydroxy-2-naphthoic acid,1,2-dihydroxynaphthalene,7,8-benzocoumarin and phthalic acid.According to the KEGG functional annotation,19 genes encoding PAHs degrading enzymes were annotated in the WZ-7.Genes related to phenanthrene degradation are phdE,phd F,phdG,and pcaL,genes related to anthracene degradation were nahAc and phdI,and genes related to naphthalene degradation were nahAb,catA and pcaL.Based on the intermediate metabolites detected by GC-MS and the identified potential degradation genes,the degradation pathways of three PAHs by WZ-7 were determined.Naphthalene was first dihydroxylated in C1 and C2 carbon sites,1,2-dihydroxyl naphthalene was decomposed into phthalic acid by ring-opening cleavage at ortho position.Anthracene was first dihydroxylated in C1 and C2 carbon sites,1,2-dihydroxyanthracene was cleaved at the meta position and converted to 3-hydroxy-2 naphthalic acid,and then decarboxylated to phthalic acid.Phenanthrene was dihydroxylated at C3 and C4 carbon sites,3,4-dihydroxyl phenanthrene was cleaved at the meta position to form 1-hydroxy-2-naphthalic acid which was then decarboxylated to 1,2-dihydroxynaphthalene.1,2-Dihydroxynaphthalene cleaved at the meta position and converted to phthalic acid.The three PAHs were eventually degraded to phthalic acid,which was converted to protocatechuic acid by the action of dioxygenase and decarboxylase.Protocatechuic acid was decomposed into pyruvate under the action of dioxygenase,and finally entered the tricarboxylic acid cycle to complete degradation.