【摘要】：目前我國正大力推廣再生水的應用，保障再生水的水質安全是關鍵問題。病原菌在消毒壓力下會進入活性但不可培養（VBNC）狀態，無法用培養法檢測出，但仍保留原有的毒力因子和致病性，可在消毒壓力解除后復活繁殖而導致再生水回用的微生物健康風險。本研究針對VBNC病原菌復活風險，建立了逆轉錄活性和細胞膜完整性的檢測方法體系，深入分析再生水系統中VBNC病原菌的存在去除特性和復活特性，探究VBNC病原菌復活的關鍵影響因子；應用焦磷酸測序技術，研究病原菌的群落結構變化；評估再生水市政雜用的微生物健康風險。在此基礎上提出控制VBNC病原菌復活的方法。 本研究結果表明，進水中可培養大腸桿菌、糞腸球菌、沙門氏菌、分枝桿菌的濃度水平為3~5LogCFU/100mL。經過深度處理工藝，出水中處于VBNC狀態的大腸桿菌、糞腸球菌、沙門氏菌濃度水平為1.5~3.8Log CE/100mL，占各自總活性菌含量的40~100%；分枝桿菌對消毒劑的抗性較強，出水中VBNC狀態分枝桿菌濃度為1Log CE/100mL，占總活性菌的20%。膜處理工藝對可培養菌與活性菌的去除量相近約為1Log。消毒工藝對培養菌的去除量可達2~3Log，對活性菌的去除量1Log，對分枝桿菌作用不顯著。 再生水貯存輸送過程中，處于VBNC狀態的病原菌大量復活，四種細菌的復活率達0.3~2.9Log。余氯與微生物濃度水平存在相關性。有機物和營養元素會促進微生物在管網中的復活，但其變化與微生物的復活不存在顯著相關性。 采用454焦磷酸測序分析再生水生產、貯存和輸送過程中群落結構的變化，結果表明經過消毒工藝、貯存和輸送，微生物群落結構發生顯著變化。管網末端，Proteobacteria是主要的門類，包含大量病原菌Burkholderiales。采用典范對應分析（CCA），考察理化因子對生物群落的影響，發現TN、TP、NO3-N和TOC是最重要的影響因素。 對再生水用作市政雜用的微生物健康風險進行評價，，得出年健康風險值與病原菌濃度水平的相關曲線。再生水出廠中，VBNC病原菌含有的潛在風險值比可培養病原菌高2個數量級。經貯存輸送后，該風險值成為顯性風險值，影響再生水回用的微生物健康風險。
[Abstract]:At present, our country is popularizing the application of reclaimed water. It is a key problem to ensure the safety of reclaimed water quality. The pathogen can enter into the active but unculturable (VBNC) state under disinfection pressure, which can not be detected by culture method, but it still retains the original virulence factor and pathogenicity. Microbial health risks that can be reactivated to reproduce after disinfection pressure is released, leading to reuse of reclaimed water. In this study, the reverse transcription activity and cell membrane integrity of VBNC pathogens were analyzed by reverse transcriptase assay, and the removal and reactivation characteristics of VBNC pathogens were analyzed. To explore the key factors influencing the revival of VBNC pathogens; to study the community structure changes of pathogens by pyrosequencing technology; and to evaluate the microbial health risk of municipal mixed use of reclaimed water. On this basis, the method of controlling the resurrection of VBNC pathogens was put forward. The results showed that the concentration of Escherichia coli, Enterococcus faecalis, Salmonella and Mycobacterium in the influent was 3 5 LogCFU / 100 mL. After advanced treatment, the concentration levels of Escherichia coli, Enterococcus faecalis and Salmonella in VBNC in the effluent were 1.5 ~ 3.8Log CE / 100 mL, accounting for 40,100 of the total active bacteria, and the resistance of Mycobacterium to disinfectants was stronger. The concentration of Mycobacterium in VBNC state was 1 Log CE / 100 mL, accounting for 20% of the total active bacteria. The removal of culturable bacteria and active bacteria by membrane treatment process was similar to that of 1Log. The removal amount of culture bacteria was 2 ~ 3Log. the removal amount of active bacteria was 1Log. the effect on Mycobacterium was not significant. During the storage and transportation of reclaimed water, the pathogenic bacteria in the state of VBNC were resurrected in large numbers, and the resurrection rate of the four bacteria reached 0.3n2.9Log. Residual chlorine was correlated with microbial concentration. Organic matter and nutrient elements can promote the resurrection of microorganisms in the pipe network, but there is no significant correlation between the changes and the resurrection of microorganisms. 454 pyrosequencing was used to analyze the changes of community structure during the production, storage and transportation of reclaimed water. The results showed that the microbial community structure changed significantly after disinfection, storage and transportation. Proteobacteria at the end of pipe network is the main category, including a large number of pathogenic bacteria Burkholderia. Canonical correspondence analysis (CCA) was used to investigate the effects of physicochemical factors on biota. It was found that TNT TPNO3-N and TOC were the most important factors. The microorganism health risk of reclaimed water used as municipal miscellaneous was evaluated and the correlation curve between annual health risk value and pathogen concentration level was obtained. The potential risk value of VBNC pathogens in reclaimed water was two orders of magnitude higher than that of culturable pathogens. After storage and transportation, the risk value becomes dominant risk value, which affects the microbial health risk of reclaimed water reuse.
【學位授予單位】：清華大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R123;X824

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