含氯有機溶劑為土壤及地下水中常見之重質非水相溶液(dense non-aqueous phase liquids, DNAPL)污染物，而三氯乙烯(trichloroethylene, TCE)則為最具代表性之含氯有機物，且被環保署列為21種國內建議優先調查之揮發性有機污染物。由於DNAPL污染場址之整治是屬於長期性的工作，因此生物整治技術是較為經濟可行的整治方式。惟DNAPL之生物降解需長期注入主要基質，但基質之注入將造成阻塞及地下水酸化問題，造成基質傳輸效果不佳並影響地下水質，進而操作維護難度增加等問題。因此，本研究之目的為發展一種具緩衝pH能力之緩衝膠體基質(buffered colloidal substrate, BCS)，可緩釋基質及緩衝地下水之pH，以加強地下水中DNAPL之生物降解。研究中進行了BCS的合成及酸化試驗，最後在應用於微生物批次試驗中，觀察其TCE生物降解效率及酸化控制能力，並以分子生物技術進行菌相分析及菌種鑑定，了解在此環境下之優勢菌種。研究結果顯示，本研究所合成之BCS其放置一天後乳化安定性可達100%，放置96天後並無分層之現象且穩定性佳；酸化試驗BCS稀釋60倍監測45天，水中總有機碳所剩殘餘量最多約24%可繼續作為微生物所需之碳源，而pH值約8.7有利於還原脫氯之重要菌群生長；微生物批次試驗之BCS組別，其TCE降解率為85%以上，且反應到最後pH值可維持在約8.8，而菌相分析的結果也顯示此組別菌相豐富度最高，且由微生物定量分析的結果得知，第0天Dehalococcoides spp.(DHC)菌數約為4.53 × 105 gene copies/g，第145天DHC菌數約為6.11 × 104 gene copies/g，顯示DHC菌量並沒有銳減，故在此環境下適合脫氯菌群生長。因此綜合上述的結果，本研究所開發之BCS可作為透水性反應牆(permeable reactive barrier, PRB)，能有效吸附於土壤中，以提高土壤中有機質含量，以長期釋放碳源提供給微生物利用及釋放OH-中和酸化現象，進而加強還原脫氯之生物復育，達到污染物降解之目標。此研究符合綠色整治之目標，不會造成環境危害，以達永續經營。

Soil and groundwater at many existing and former industrial areas and disposal sites are contaminated by halogenated organic compounds that were released into the environment. When they are released into the subsurface, they tend to adsorb onto the soils and cause the appearance of DNAPL (dense-non-aqueous phase liquid) pool. The trichloroethylene (TCE) has been shown on induces hepatocellular carcinogens in mice and is a human carcinogen. Thus, TCE was used as the target compound in this study. As contaminated groundwater moves through the emplaced reactive zones, the contaminants are removed, and uncontaminated groundwater emerges from the downgradient side of the reactive zones. Application of in situ anaerobic bioremediation is a feasible technology to remediate DNAPL site. However, enhanced in situ bioremediation requires the injection of primary substrates, which would cause the acidification and odor problems of the subsurface environment. This would deteriorate the groundwater quality and cause the increase in maintenance cost. The objective of this proposed study is to develop buffered colloid substrate (BCS). The BCS can be applied in the saturated zone, which can release substrate from the colloid to enhance the reductive dechlorination of DNAPL in the saturated zone. Furthermore, the buffered colloid has the capability for pH control and prevents the decrease in pH value in groundwater. In this study, batch experiments were operated to test the feasibility of using BCS as the slow-released substrate and acidification control material. Several experimental conditions included the concentrations of contaminants and substrates, shacking speed, percentage of each component, acidification test, and molecular biotechnology. Results show BCS emulsion stability can reach 100% after place one day and no stratification for 96 days. Addition of BCS diluted 60-fold monitoring 45 days, the remaining residual amount of total organic carbon in water most about 24% can continue to serve as a carbon source of needed microorganisms, and a pH of about 8.7 dechlorination of important beneficial bacteria growth. Results from the microcosm study indicate that the addition of BCS would enhance the biodegradation rate of TCE under anaerobic conditions. Approximately 85% of TCE could be removed when BCS was added in the system. Increase in dehalococoides (DHC) population was observed after the addition of BCS. Results will be useful in designing a field-scale system to enhance the in situ bioremediation of chlorinated-solvent contaminated groundwater.

摘要 ii
Abstract iii
目錄 v
圖目錄 viii
表目錄 x
第一章 前言 1
1.1 研究緣起 1
1.2 研究目的 3
第二章 文獻回顧 4
2.1 土壤及地下水受含氯脂肪族碳氫化物污染之概況 4
2.1.1 含氯脂肪族碳氫化物之污染概況 4
2.1.2 三氯乙烯之特性及對人體之危害 7
2.1.3 三氯乙烯之傳輸機制 11
2.2 土壤與地下水污染整治技術之種類及應用趨勢 13
2.2.1 地下水之現地生物整治技術 16
2.2.2透水性反應牆 18
2.3 三氯乙烯之生物反應機制 22
2.3.1 三氯乙烯之好氧生物處理 22
2.3.2 三氯乙烯之厭氧生物處理 24
2.4 厭氧生物整治技術之挑戰 28
2.4.1 地下水pH對生物整治之影響 30
2.4.2 發展緩衝膠體基質 33
2.5 分子生物技術之應用 35
2.5.1 16S rDNA 在細菌演化分類與鑑定上的獨特性及應用 37
2.5.2 降解含氯脂肪族之菌種與基因 39
第三章 實驗設備與方法 41
3.1 研究流程 41
3.2 實驗設計 43
3.2.1 前導試驗 43
3.2.2 批次試驗 47
3.3 實驗材料與設備 48
3.3.1 實驗試藥 48
3.3.2 實驗儀器與設備 48
3.3.3 供試之土壤及地下水來源 49
3.3.4 供試之乳化型基質 50
3.4 實驗分析方法 50
3.4.1 水質分析 50
3.4.2 固體物成分分析 52
3.5 分子生物技術 53
3.5.1 土壤中微生物之DNA萃取 54
3.5.2 聚合酶鏈鎖反應 (Polymerase Chain Reaction, PCR) 54
3.5.3 DNA純化 56
3.5.4 即時定量PCR(real-time PCR，qPCR) 56
3.5.5 變性梯度膠電泳(denaturing gradient gel electrophoresis, DGGE) 57
3.5.6 DNA定序比對 58
第四章 結果與討論 59
4.1 緩衝膠體基質(BCS)之合成 59
4.1.1 乳化試驗 59
4.1.2 穩定性試驗 62
4.2 緩衝膠體基質(BCS)之可利用性 65
4.2.1 酸化試驗 65
4.3 厭氧微生物批次試驗 69
4.3.1 自然降解組 (N) 70
4.3.2 乳化型釋碳基質組 (E) 74
4.3.3 緩衝膠體基質組 (B) 79
4.3.4 綜合討論 83
4.4 分子生物技術 89
4.4.1 批次實驗之微生物菌相分析 (DGGE) 90
4.4.2 批次實驗之微生物菌量分析 (qPCR) 92
4.4.3 批次實驗之微生物功能基因分析 (qPCR) 94
第五章 結論與建議 96
5.1 結論 96
5.2 建議 98
參考文獻 99

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