| The overuse,misuse and accumulation of antibiotics in the environment have created tremendous selection pressure,greatly accelerating the emergence and enrichment of antibiotic resistant bacteria(ARB)and inducing the diffusion and spread of antibiotic resistance genes(ARGs)in the environment.Since swage water is an important environmental reservoir of antibiotics and ARGs,municipal sewage treatment plant,as the main centralized treatment site of sewage,aims to ensure the biological safety of effluent.Disinfection is an important safeguard to ensure the biological safety of the treated effluent.Because of their respective advantages,chlorination and ultraviolet(UV)disinfection have become the two most commonly used disinfection methods.However,traditional methods of microbial culture and molecular biology cannot comprehensively profile the community structure and antibiotic resistome of living and dead bacteria,nor can they systematically and thoroughly reveal the variation characteristics of microbial structure and ARGs diversity and abundance under disinfection pressure.This thesis chose the effluent of a sewage treatment plant in Nanjing as the research object,and a gradient dose experiment of chlorination,UV and sequential UV/chlorination disinfection was set up under laboratory conditions to systematically compare the effects of different disinfection methods on microbial community and antibiotic resistome(ARGs and mobile genetic elements(MGEs))structure,in domestic sewage.In addition,since the antibiotic resistance of the surviving bacteria that are insensitive to disinfectants deserves greater concerns,this thesis studied the effects of chlorination and UV disinfection on the microbial community and ARGs of living bacteria using the technologies of propidium monoazide(PMA),multiple displacement amplification(MDA)and metatranscriptome.The potential hosts of ARGs were also identified by assembling metagenome and metatranscriptome.The contents and conclusions are as follows.(1)The variation patterns of the microbial structure before and after chlorination and UV disinfectionIn this thesis,gradient dose experiments of chlorination,UV and sequential UV/chlorination disinfection were set up,and Illumina Miseq high-throughput sequencing and metagenomic analysis combined with cluster analysis were used to explore the variation patterns of microbial structure in domestic sewage.The results showed that there was a threshold of disinfection dose for both chlorination and UV disinfection.Specificially,the threshold of chlorination and UV chlorination was 40 mg/L and 40 mJ/cm2,respectively.When the disinfection doses exceeded the threshold,the microbial structure no longer changed with the increase of disinfection dose.At this time,the change of microbial structure caused by chlorination was greater than that by UV disinfection.In addition,chlorination and UV disinfection showed different ability to remove different bacteria.Chlorination significantly reduced the relative abundance of Proteobacteria and Actinobacteria,while enriched Chloroflexi and Chlorobi.UV disinfection significantly reduced the relative abundance of Proteobacteria,while enriched Firmicutes,Bacteroidetes,A ctinobacteria,Cyanobacteria and Chloroflexi.Furthermore,sequential chlorination/UV disinfection effectively removed the bacteria that are insensitive to the single disinfectant.(2)The variation patterns of antibiotic resistome before and after chlorination and UV disinfectionIn this thesis,gradient dose experiments of chlorination,UV and sequential UV/chlorination disinfection were set up,and Illumina Hiseq high-throughput sequencing and metagenomic analysis were used to explore the change patterns of antibiotic resistome(ARGs and MGEs)in domestic sewage.Network analysis was also used to explore the correlation among microbial structure,ARGs and MGEs.The results showed that both chlorination and UV disinfection reduced the relative abundance of ARGs in domestic sewage,but the removal capacity was limited and there was a certain preference.From the pespective of the types of ARGs,chlorination removed all kinds of ARGs,but enriched macrolide-lincosamide-streptogramin B(MLSB)ARGs,while UV disinfection was not found capable of removing tetracyclines,multi-drug and sulfonamides resistance genes.From the pespective of the mechanisms of ARGs,chlorination effectively removed the ARGs with any resistance mechanisms,while UV disinfection only removed ARGs of drug enzymatic destruction and efflux pump.Furthurmore,the removal efficiency of combined disinfection on ARGs and MGEs was similar to that of chlorination,but greater than that of UV disinfection.(3)The variation patterns of the microbial structure and ARGs diversity of living bacteria before and after chlorination and UV disinfectionIn this thesis,the microbial structure and ARGs distribution patterns of living bacteria after chlorination were explored through Illumina Hiseq sequencing,metagenomic and metatranscriptomic analysis.The microbial structure and ARGs diversity of living bacteria after UV disinfection were explored through Illumina Hiseq sequencing and assembly of metagenomic and metatranscriptomic data.The results showed that after chlorination(0.5 mg/L),the microbial structure and ARGs distribution of living bacteria were significantly different from those of mixed living and dead bacteria,indicating that PMA could help reveal the real microbial structure.But there were significant differences between the results from DNA analysis and RNA analysis,in terms of the community structure,ARGs and MGEs abundance.UV disinfection(20 mJ/cm2)resulted in a slight decrease in the relative abundance of transcripts of ARGs,but a significant increase in the relative abundance of MLSB transcripts.From the perspective of living bacteria,chlorination disinfection posed a greater effect on microbial structure and antibiotic resistome composition in domestic sewage than UV disinfection. |
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