本研究主旨為探討以加強式生物整治法進行三氯乙烯污染場址之整治成效及監測。於現地的三氯乙烯污染場址，添加微生物生長所需的碳源、營養鹽，以促使微生物生長進而增強三氯乙烯之降解。實驗中針對A、B兩個三氯乙烯污染場址的地下水樣檢測微生物的菌相變化，方法為針對細菌16S rRNA基因的特定區段進行PCR，再利用變性梯度膠體電泳(DGGE)進行菌相分析，並將DNA條帶進行定序與比對，以確認其所相對應的微生物。此外，亦以Dehalococcoides spp.的16S rRNA基因設計專一性的引子，利用即時定量PCR來偵測現地環境中此菌群的趨勢變化。研究結果顯示，加入乳化型釋氫基質後，營養源被分解產生適合厭氧還原脫氯作用發生的環境。檢測 volatile organic compounds，包括trichloroethene (TCE)，1,1-dichloroethene (1,1-DCE) 和 vinyl chloride (VC) 的測值發現，場址A與B均有下降，表示有還原脫氯的反應在持續進行中。菌相分析的結果則觀察到，注入乳化型釋氫基質後微生物豐富度有增加，並且下游監測井的菌相較注入井豐富。而菌種鑑定的結果則發現，A與B兩場址均出現可降解三氯乙烯相關之菌種，例如：Acidovorax spp.、Burkholderiales及Pseudomonas sp.，以及其他β-proteobacterium、Comamonadaceae、Iron-reducing bacterium、Hydrogenophilaceae、Variovorax spp.、Clostridium spp.、Geobacter spp.、Rhodoferax ferrireducens、Dehalospirillum multivorans、Dehalococcoides spp.等。Dehalococcoides spp.為厭氧生物整治三氯乙烯污染場址之指標性生物，因此本研究偵測兩個場址於基質注入前後Dehalococcoides spp.的含量變化以解釋厭氧生物整治的成效。結果顯示，A場址的菌量介於4.47×103-8.26×104 CFU/ L，B場址的菌量則介於4.60×102-9.31×107 CFU/ L。基質注入之後，兩個場址中此菌群的含量都有增加，證明添加乳化型釋氫基質可有效地刺激Dehalococcoides菌群的增生，促使現地環境中三氯乙烯降解。總而言之，本研究依據不同時間點的菌相分析及Dehalococcoides菌群含量變化之結果，可提供強而有力的證據來評估現地厭氧生物整治法之成效。

The goal of this study was to use molecular biology techniques to access and monitor the efficacy of bioremediation on a trichloroethene (TCE) polluted site. We added emulsified hydrogen releasing materials to stimulate onsite microbial growth and the biodegradation of TCE. This process was known as enhanced bioremediation. In this study, there were two bioremediation sites had been treated anaerobically. Groundwater samples were taken periodically for microbial analysis. Denaturing gradient gel electrophoresis (DGGE) was used to evaluate the variations in microbial community structures during the in situ groundwater remediation. The DGGE DNA bandings were sequenced to determine the 16S rRNA gene sequences and identify the dominate bacterial species. In addition, we used Dehalococcoides spp. 16S rRNA genes as the targets to do real-time PCR. Results show that the emulsified hydrogen releasing materials could enhance anaerobic reductive dechlorination. After addition of emulsified hydrogen releasing materials, we found that the volatile organic compounds concentrations (i.e., TCE, 1, 1-DCE and VC) were decreased. In microbial analysis, the diversities of the microbial community were increased after nutrient supplement. According to the DNA sequencing results, there were 31 bacterial species had been found that related to TCE degradation (i.e., Acidovorax sp., Burkholderiales, Pseudomonas sp., β-proteobacterium, Comamonadaceae, Iron-reducing bacterium, Hydrogenophilaceae, Clostridium sp., Geobacter sp., Rhodoferax ferrireducens, Dehalospirillum multivorans and Dehalococcoides spp.). Dehalococcoides spp. can be used as a biomarker to evaluate the efficacy of anaerobic bioremediation on a TCE contaminated site. Therefore, we quantified Dehalococcoides populations to explain the capacity of bioremediation after addition of emulsified hydrogen releasing materials to groundwater. Results reveal that Dehalococcoides cell numbers of site A were 4.47×103-8.26×104 CFU/liter, site B were 4.60×102-9.31×107 CFU/liter. This data indicated that the addition of emulsified substrate would increase the growth of total Dehalococcoides population under anaerobic conditions. Overall, results from this study demonstrated that the microbial analysis and quantities of Dehalococcoides at different time points can provide useful information to proceed with bioremediation methods.

論文審定書 .............................................................................i
謝誌 ........................................................................................ii
中文摘要 .............................................................................. iii
Abstract ..................................................................................v
壹 前言....................................................................................1
1.1 研究緣起..........................................................................1
1.2 三氯乙烯（Trichloroethylene, TCE）之物化特性與人體之危害.............................................................................3
1.3 含氯有機物整治方法......................................................6
1.3.1 現地化學氧化技術（in situ chemical oxidation, ISCO）...................................................................................6
1.3.2 透水性反應牆 （Permeable reactive wall）..........8
1.3.2.1 釋氧基質...................................................................9
1.3.2.2 釋氫基質...................................................................9
1.4 微生物降解含氯有機物................................................11
1.4.1 好氧環境中含氯有機物的降解................................12
1.4.1.1 好氧氧化作用（直接代謝）.................................13
1.4.1.2 好氧氧化作用（共代謝）.....................................13
1.4.2厭氧環境中含氯有機物的降解.................................14
1.4.2.1 厭氧還原脫氯反應（直接代謝）.........................16
1.4.2.2 厭氧還原脫氯反應（共代謝）.............................17
1.5 環境檢測現地污染場址................................................17
1.6分子生物學之監測技術.................................................18
1.6.1變性梯度膠體電泳.....................................................19
1.6.2菌種鑑定.....................................................................20
1.6.3 即時定量PCR（quantitative PCR，qPCR）.....21
1.7 研究目的........................................................................23
貳 材料與方法.....................................................................24
2.1研究場址及添加基質整治之介紹.................................24
2.1.1 場址A監測井設置與採樣時間..................................24
2.1.2 場址B監測井設置與採樣時間..................................26
2.2 現地地下水質參數監測................................................28
2.3 地下水環境微生物DNA萃取.......................................29
2.4 聚合酶連鎖反應............................................................30
2.5 變性梯度膠體電泳........................................................31
2.6 以clone library進行定序..............................................32
2.7 NCBI比對序列..............................................................33
2.8 即時定量 PCR..............................................................34
2.8.1 配置PCR反應液........................................................34
2.8.2 建立標準曲線............................................................35
2.8.3 環境中樣品Dehalococcoides spp. 數量..............36
參 結果與討論.....................................................................37
3.1 地下水質監測分析........................................................37
3.1.1 現地DO，pH以及ORP數值變化............................37
3.1.2 TCE降解趨勢............................................................39
3.1.3 現地水質數值變化 ....................................................41
3.2 三氯乙烯污染場址之菌相分析與鑑定........................43
3.3 利用即時定量PCR偵測現地Dehalococcoides spp.之變化趨勢..........................................................................47
肆 結論.................................................................................52
伍 建議.................................................................................55
參考文獻..............................................................................56
圖表......................................................................................70

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