三氯乙烯(trichloroethylene, TCE)是一種具有潛在致癌性之揮發性有機化合物。由於三氯乙烯的水溶性低且密度大於水，故當三氯乙烯釋放到含水層後，會下沉、擴散到較低水力傳導區域並緩慢的溶解到地下水，進而形成重質非水相液體之污染源區域。Dehalococcoides (Dhc屬)是目前所知能夠在厭氧及中性pH之環境條件下，將含氯有機物還原脫氯至無毒乙烯之菌種，惟Dhc屬之還原性脫鹵素酶有多種的基因(pceA、tceA、bvcA、vcrA)，分別催化不同的生化反應，這些脫鹵素酶是藉由氫氣(H2)的氧化作用獲取能量。當Dhc屬還原脫氯含氯有機物時，硫酸鹽還原菌(sulfate-reducing bacteria, SRB)之存在會抑制Dhc屬之生長且會造成氯乙烯及順式二氯乙烯(cis-dichloroethylene, cis-DCE)之累積問題。本研究添加檸檬酸鐵[Fe (III)]及鉬酸鈉[Mo (VI)]兩種SRB抑制劑之結果顯示，在添加不同的SRB抑制劑及不同濃度進行厭氧生物還原脫氯試驗，含氯有機物降解所產生的副產物(氯乙烯)有降低，而乙烯則有上升之趨勢。接著探討較佳的SRB抑制劑種類及較適當的添加量，並以分子生物技術對批次實驗之Dhc屬、產氫菌及SRB或其他菌群變化進行評估。在分別添加Fe (III)及Mo (VI)抑制劑所測得的SRB菌量顯示，添加Fe(III) 0.6 mM及Mo(VI) 0.5 mM抑制劑時，可有效抑制SRB生長及有最佳之三氯乙烯降解效果，能減少SRB對基質與電子的消耗，增進Dhc屬之還原脫氯作用，改善副產物cis-DCE及氯乙烯之累積。本實驗成果有助於發展一套迅速有效之強化生物復育整治之綠色工法，供相關污染場址整治之應用。

Trichloroethylene (TCE) is a potentially carcinogenic volatile organic compound (VOC). Because TCE (with the density of greater than water) is low in water solubility, it will sink and diffuse into the lower hydraulic conduction zone and, then, will be slowly dissolved into the groundwater when releasing into the aquifer. Thus, TCE will form a dense non-aqueous phase liquid (DNAPL) in the pollution source area.It is known that Dehalococcoides (Dhc) are capable of dechlorinating TCE to non-toxic ethylene under anaerobic and neutral pH environments. Nonetheless, the genus Dhc (i.e., belongs to class Dehalococcoidia) with multiple reductive dehalogenase genes (pceA, tceA, bvcA and vcrA) can catalyze different biochemical reactions. The reductive dehalogenase genes obtain its energy via the oxidation of H2. During the anaerobic reductive dechlorination of chlorine-containing organic compounds, the existence of sulfate-reducing bacteria (SRB) can inhibit the growth of the genus Dhc and, thereby, cause the accumulation of vinyl chloride and cis-dichloroethylene (cis-DCE).In the present study, two chemicals (ferric citrate and sodium molybdate) for inhibiting the growth of SRB were used. The results showed that, during the dechlorination of chlorine-containing organic compounds, the concentration of vinyl chloride declined whereas the concentration non-toxic ethylene increased.Moreover, batch experiments were conducted to determine a better SRB inhibitor and an adequate dosing concentration of the inhibitor. Additionally, molecular biotechnology was also used to evaluate the prevalence of occurrence among the genus Dhc, hydrogen-producing bacteria and SRB.The addition of Fe(III) 0.6 mM and Mo(VI) 0.5 mM could effectively inhibit SRB. However, complete inhibition of SRB would not be beneficial to Dhc growth because SRB can provide trace growth elements, enhance Dhc for complete reduction and dechlorination. In this study, the addition of SRB inhibitors can reduce the consumption of matrix and electrons by SRB, and increase the content of Dhc, thereby reducing the accumulation of cis-DCE and VC, and achieving the effect of complete reduction and dechlorination.Results from this study will be helpful in developing a green remedial methods to enhance the reductive dechlorination of TCE and the developed techniques can be applied at other TCE-spill sites.

摘要 i
Abstract ii
誌謝 iv
目錄 v
圖目錄 viii
表目錄 xi
第一章 前言 1
1.1研究源起 1
1.2研究目的 2
第二章 文獻回顧 3
2.1 含氯碳氫化合物污染概況 3
2.2 土壤地下水整治技術 6
2.3 三氯乙烯之特性 9
2.4 三氯乙烯之生物反應機制 13
2.4.1 三氯乙烯之好氧生物降解 13
2.4.2 三氯乙烯之厭氧生物降解 16
2.5 基質之特性介紹 18
2.5.1 聚麩胺酸(γ-PGA)之基本特性 18
2.5.2 γ-PGA在環境上之應用 19
2.5.3不同基質於土壤之地下水之應用性 20
2.6 Dehalococcoides之特性介紹 21
2.7 硫酸還原菌 26
2.7.1硫酸還原菌(sulfate-reducing bacteria,SRB)概況 26
2.7.2硫酸還原菌與產氫菌及脫氯菌之相互關係 30
2.8抑制硫酸鹽介紹 33
2.8.1抑制硫酸鹽之機制介紹 33
2.8.2抑制硫酸鹽之藥劑 35
2.9分子生物在地下水生物復育之應用 37
2.9.1降解含氯乙烯類之菌種與基因 38
第三章 實驗設備與方法 40
3.1 研究流程 40
3.2.1 實驗材料 42
3.2.2 實驗設備 43
3.2.3配置污染地下水及馴養污泥 43
3.2.4硫酸還原菌馴養 44
3.2.5脫氯球菌馴養 45
3.2.6聚麩酸胺 47
3.3實驗設計 47
3.3.1產氫菌前導最佳配比試驗 47
3.3.2 厭氧微生物批次實驗 47
3.4實驗分析項目 48
3.4.1 水質分析 48
3.5分子生物分析 50
3.5.1微生物DNA萃取 50
3.5.2 DNA純化 51
3.5.3即時定量PCR (realtime-PCR，qPCR) 52
第四章 結果與討論 56
4.1 產氫菌最佳化試驗 56
4.1.1 不同比例產氫菌之TCE濃度變化及副產物變化 56
4.1.2 不同比例產氫菌之微生物功能基因分析 (qPCR) 59
4.1.3 不同比例產氫菌之硫酸鹽(SO42-)及硫化物(HS-)濃度變化 62
4.1.4 不同比例產氫菌之pH、DO、ORP及TOC之數值變化 64
4.2 添加硫酸鹽組別 67
4.2.1 添加硫酸鹽組別之TCE濃度變化及副產物變化 67
4.2.2 添加硫酸鹽組別之微生物功能基因分析 (qPCR) 68
4.2.3 添加硫酸鹽組別之硫酸鹽(SO42-)及硫化物(HS-)濃度變化 70
4.2.4 添加硫酸鹽組別之pH、DO、ORP及TOC之數值變化 72
4.3添加SRB抑制劑(Fe3+)批次實驗組別 74
4.3.1 添加SRB抑制劑(Fe3+)之TCE濃度變化及副產物變化 74
4.3.2 添加SRB抑制劑(Fe3+)之微生物功能基因分析(qPCR) 77
4.3.3 添加SRB抑制劑(Fe3+)之硫酸鹽(SO42-)及硫化物(HS-)濃度變化 79
4.3.4 添加SRB抑制劑(Fe3+)之pH、DO、ORP及TOC之數值變化 81
4.4 添加SRB抑制劑(MoO42-)批次實驗組別 84
4.4.1 添加SRB抑制劑(MoO42-)之TCE濃度變化及副產物變化 84
4.4.2 添加SRB抑制劑(MoO42-)之微生物功能基因分析(qPCR) 86
4.4.3 添加SRB抑制劑(MoO42-)之硫酸鹽(SO42-)及硫化物(HS-)濃度變化 88
4.4.4 添加SRB抑制劑(MoO42-)之pH、DO、ORP及TOC之數值變化 90
4.5 各組別最佳配比之微生物功能基因分析(qPCR)綜合比較 92
第五章 結論與建議 96
5.1 結論 96
5.2 建議 97
參考文獻 98

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