Enrichment And Degradation Efficiency Of Benzo[a]-pyrene Bacteria For Anaerobic Degradation

Polycyclic Aromatic Hydrocarobons（PAHs）are a class of persistent pollutants with strong toxicity,stable structure and widespread existence in natural environment.As a typical representative of high-molecular weight polycyclic aromatic hydrocarobons,benzo[a]pyrene（Ba P）is difficult to be degraded by microorganisms due to its complex chemical structure and strong toxicity.At present,there are many reports on aerobic benzo[a]pyrene degrading microorganisms,but the researches on benzo[a]pyrene degrading microorganisms under anaerobic conditions are still insufficient,and their degradation characteristics and degradation paths are not clear.Therefore,it is necessary to investigate the microbial composition and characteristics of anaerobic degradation of benzo[a]pyrene.In this study,benzo[a]pyrene was used as the only carbon source to acclimate and enrich benzo[a]pyrene anaerobic degrading bacteria from different anaerobic environments,and the dominant consortium with the best degradation effect was obtained.Based on the obtained dominant consortium,the influence of external environmental factors on the degradation ability of the consortium was investigated to determine the degradation performance of benzo[a]pyrene degrading consortium.The intermediate metabolites of benzo[a]pyrene degrading were analyzed by the Gas Chromatograohy-Mass Spectrometry to clarify the microbial degradation pathway of benzo[a]pyrene under anaerobic conditions.Finally,biostimulation,bioenhancement and natural attenuation remediation were carried out on the contaminated anaerobic sludge,and the change rule of key factors in the degradation process was paid attention to.Metagenomic sequencing technology was used to analyze the changing trend of microorganisms,so as to provide a theoretical basis for microbial remediation technology of polycyclic aromatic hydrocarobons pollution.The main research results are as follows:（1）9 anaerobic degradation consortium were successfully screened from the anaerobic sludge,the combined anaerobic sludge with sludge-straw,the combined anaerobic sludge with coking wastewater,among which the YQ-Ba P consortium cultured from the combined anaerobic sludge system with sludge-straw had the best degradation effect.The degradation rate of 30 mg/L benzo[a]pyrene stabilized at38.69±6.24%within 14 days.94.49%of the bacteria in the YQ-Ba P consortium belongs to bacterium phylum Proteobacteria,dominabt bacteria（Pseudomonas and Methyloversatilis）and discover new possibilities for polycyclic aromatic hydrocarobons metabolism between generic Quisquiliibacterium and Intrasporangium.YQ-Ba P consortium can degrade phenanthrene and pyrene in addition to benzo[a]pyrene.（2）YQ-Ba P consortium had a wide tolerance range to p H,temperature and substrate concentration,and the degradation effect of benzo[a]pyrene was best in neutral environment and range of 35℃～40℃.When benzo[a]pyrene concentration was 20 mg/L,the degradation rate reached 46.89%after 14 days of culture.The addition of bicarbonate as an electron acceptor increased the degradation rate of benzo[a]pyrene,which was 6.05%higher than that of the control group on day 14,while the sulfate group did not improve the degradation effect of benzo[a]pyrene.Fe³⁺and Cu²⁺promoted the degradation of benzo[a]pyrene,while Cd²⁺,Zn²⁺and Pb²⁺inhibited it.The intermediate metabolites in the process of degradation of benzo[a]pyrene by YQ-Ba P consortium were identified by the Gas Chromatograohy-Mass Spectrometry and the following degradation pathways were proposed:Benzo[a]pyrene fractured benzene ring to form phenanthrene or anthracene through initial reduction reaction,and phenanthrene was further degraded into naphthalene.The latter then follows the degradation pathway of naphthalene.Anthracene forms 9,10-anthraquinone and 9,10-dihydroanthracene in a series of reactions.（3）The degradation rate of polycyclic aromatic hydrocarobons in anaerobic sludge in the biostimulation group was the highest,reaching 39.50%after 28 days of culture,and the degradation rate of polycyclic aromatic hydrocarobons in the bioenhancement group was 13.46%higher than that in the natural decay group at the early stage of culture（day 7）.The addition of bacterial community had obvious degradation effect on5-ring polycyclic aromatic hydrocarobons in anaerobic sludge.There was no difference in p H and ORP among all groups,and all groups produced methane.The biofortification group had the highest yield（13.17 m L）.The addition of bicarbonate increased the population richness,but the microbial diversity of the three groups of biofortification,biostimulation and natural decay decreased with the increase of culture time.Chloroflexi became Proteobacteria after the addition of bacteria community.Statistically different bacteria genera were detected among the three groups,among which the natural attenuation group had the least amount of different bacteria.