含氯乙烯類化合物為國內外土壤與地下水中常見的污染物質，而厭氧脫鹵程序為目前重要的生物復育法之一。於此程序中，Dehalococcoides屬在還原四氯乙烯(PCE)至無害的乙烯(ETH)中扮演關鍵性的角色，而還原脫鹵酵素(RDase)的分析則有助於進一步地了解氯乙烯類化合物的降解路徑。此外，參與脫氯反應的其他的功能性微生物與微生物社會能提供建立基質降解網絡的完整性資訊。本研究目的為利用次世代定序法(NGS)來解析位於台灣四個污染場址中的微生物社群組成，與利用以DNA為生物標記的SybrGreen-qPCR法定量在執行生物復育約1年的兩個場址中、不同時期的8種功能性微生物與4種RDase基因。首先，NGS結果顯示完整性與含有機鹵化物(OHRB)降解之微生物社群與污染場址特性具有高度的相關性。
在受四氯乙烯污染的場址一中，添加EcoClean藥劑有助於增殖或活化功能性微生物的增長與基因表現。Desulfitobacterium和Dehalococcoides屬分別負責PCE降解和轉換三氯乙烯(TCE)至氯乙烯(VC)。 統計分析結果顯示，溶氧(DO)與總有機碳(TOC)和氯烯類化合物中氯分子數的分解具有高度的相關性。 而在另一個受三氯乙烯(TCE)污染的場址六中，添加SL-90SRW藥劑能誘發tceA和bvcA基因的表現來進行順-1,2-二氯乙烯(cis-1,2-DCE)的降解。於此系統中，Dehalococcoides屬在生物復育後期被觀察到取代原生的Desulfitobacterium屬。
在生物復育潛能方面，當現地污染場址中Dehalococcoides屬具有3.2510-3 fmole/cell-day的降解速率時能達到50%的生物復育可能性。相對於在經兩年優勢培養的系統中，Dehalococcoides屬具有76%的去除能力於5個月的測試期中降解10 μM的三氯乙烯濃度。進一步探討本研究所檢測的所有參數，分析66個有效數據的結果顯示，溶氧為發生還原脫氯反應的關鍵因子，且Clostridia綱為活化OHRB家族中RDase表現的重要能量供應者。 而共發生分析的結果再次證明Dehalococcoides屬中tceA 和 vcrA基因為最重要的RDase酵素來催化存在於台灣各場址中氯烯類物種的轉換。
最後，本研究所發展的專家系統，結合現地場址背景參數與微生物資訊，在生物復育效率的評估方面提供一個嶄新的視野，期待未來能幫助現地操作者制定有用的整治策略來去除環境中殘留的有毒化合物。

Chlorinated ethenes are an important class of contaminants present in the soil and groundwater sites. Bioremediation with anaerobic dehalogenation is a promising method for chloroethenes degradation. Genus Dehalococcoides plays a critical role in reduction of tetrachloroethene (PCE) to harmless ethene (ETH). Furthermore, several reductive dehalogenases (RDase) extracted from genus Dehalococcoides may provide the important message for chloroethenes degradation pathway. In addition, other functional microbes and microbial community involved in the dechlorination give the comprehensive information for establishment of substrate degrading network.
This study aimed to clarify the composition of comprehensive communities in four contaminated sites distributed in Taiwan using next-generation sequencing (NGS) analysis. In addition, this study also used SybrGreen-qPCR method with DNA biomarker to quantify the population and abundance of 8 functional microbes and 4 RDase genes in two contaminated sites during about 1 year and different stages of bioremediation. NGS results showed that the distribution of comprehensive and OHRB-containing communities displayed high correlation with the properties of contaminated sites.
In the case of site I contaminated with PCE, addition of EcoClean benefited to enlarge or activate the growth of functional microbes and gene expression. Genera Desulfitobacterium and Dehalococcoides were responsible for transformation of PCE to trichloroethylene (TCE) and TCE to vinyl chloride (VC), respectively. Statistical analyses showed that both dissolved oxygen (DO) and total organic carbon (TOC) had high correlation with the decomposition of chloroethenes. In another case of site VI contaminated with TCE, addition of SL-90SRW induced the expression of tceA and bvcA genes, resulting in further dechlorination of cis-1-2-DCE. Moreover, genus Dehalococcoides replaced genus Desulfitobacterium in the site after latter period of bioremediation.
For bioremediation potential, 50% probability was attained when Dehalococcoides population performed the degradation rate of 3.2510-3 fmole/cell-day in the field site. To fit the result of 2-year enriched culture, genus Dehalococcoides had the capability of 76% removal probability to degrade TCE concentration of 10 μM within 5 months. To further investigate the occurrence frequency of all indicators determined in this study, 66 effective data showed that DO was the key factor for occurrence of reductive dechlorination and class Clostridia was the important donor for activation of RDase expression in the OHRB family. Results of co-occurrence analysis proved again that both of tceA and vcrA genes in genus Dehalococcoides were the most important RDase to catalyze the transformation of chlorinated species in Taiwan.
Finally, the expert system developed in this study provided a new insight in the evaluation of bioremediation efficiency, in combination of background geochemical parameters and microbial information. It may serve as a useful tool for the managers and engineers to determine the remediation strategies and plans for the clean-up of chloroethene-contaminated sites.

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