1,2-二氯乙烷(1,2-dichloroethane, 1,2-DCA)為工業上常用之含氯有機物，對生物具有毒性。由於其屬於重質非水相液體，容易在地表下造成累積，造成整治上的困難。本研究係利用好氧及厭氧微生態系統批次實驗的方式，加入可激發微生物生長之不同基質，以促進現地微生物之代謝反應，達到降解1,2-二氯乙烷的效果。研究結果顯示好氧批次實驗中，自然衰減組(A1)與好氧污泥組(A3)在第7天的污染物分解效率超過90%，基質添加組(A2)在第14天時分解效率超過95%。而厭氧批次實驗中，除了自然衰減組(B1)外，其他組別在第10天時，皆能有效分解1,2-二氯乙烷至50%以下。厭氧污泥組(B3)甚至在第五天的污染物分解率就已達到80%。DGGE菌相分析的結果可以看出隨著實驗的進行，菌相產生變化，其中基質添加組(A2, B2)可能因砂糖促進不同的菌群生長，造成菌相改變最大。菌種鑑定的結果顯示各批次實驗中均出現可分解1,2-二氯乙烷之菌群，如Klebsiella、Pseudomonas、Rhodoferax及Xanthobactor，以及其他降解相關的菌群等。即時定量PCR結果則發現，Dehalococcoides spp.菌數變化符合厭氧基質添加組(B2)的1,2-二氯乙烷濃度變化，推測可能是本實驗中主要降解1,2-二氯乙烷的菌群。而Desulfitobacterium spp.菌數亦符合好氧基質添加組(A2)與厭氧批次實驗所有組別(B1, B2, B3, B4)的1,2-二氯乙烷濃度趨勢，推測Desulfitobacterium spp.可能是這些組別中主要降解1,2-二氯乙烷的菌群。

1,2-dichloroethane (1,2-DCA) is a popular industrial chlorinated organic chemical. Because 1,2-DCA is a dense non-aqueous phase liquid and easily accumulated in deep soil and water, it is difficult to be removed from the contaminated sites. In this study, aerobic and anaerobic microcosm batch experiments were performed to evaluate the feasibility of biodegradation of 1,2-DCA by adding different growth substrates. The aerobic microcosm results show that approximately 90% of 1,2-DCA removal was observed in the natural degradation group (A1) and the aerobic sludge addition group (A3) after 7 days of incubation. Up to 95% of 1,2-DCA removal could be observed in the substrate supplement group in after 14 days of incubation. In the anaerobic microcosm studies, 50% of 1,2-DCA removal could be obtained in all groups after 10 days except for the natural degradation group (B1). Moreover, the degradation efficiency for the anaerobic sludge group (B3) reached 80% of 1,2-DCA removal in 5 days. The DGGE profiles show that the microbial diversity varied with time and the sugar supplement groups (A2, B2) exhibited the most microbial diversity. Bacterial clones results revealed that the 1,2-DCA biodegradable microbial strains were presented in the microcosms, such as Klebsiella, Pseudomonas, Rhodoferax and Xanthobactor. The real-time PCR results indicated that the Dehalococcoides spp. was the major bacterium that was responsible for the degradation of 1,2-DCA in the anaerobic substrate supplement group (B2). Desulfitobacterium spp. could be the dominant 1,2-DCA degrading bacterium for the aerobic substrate supplement group (A2) and all of the anaerobic groups (B1, B2, B3, B4).

目錄
論文審定書 i
誌謝 ii
摘要 iii
Abstract v
目錄 vii
圖目錄 x
表目錄 xi
(一) 前言 1
1.1 1,2-二氯乙烷之物化特性 1
1.2 1,2-二氯乙烷之人體危害 3
1.3 國內1,2-二氯乙烷之污染案例 5
1.4 重質非水相液體之整治技術 6
1.5 加強式生物復育 7
1.6 1,2-二氯乙烷之生物分解 8
1.6.1 好氧生物分解 8
1.6.2 厭氧生物分解 11
1.7 微生態系統研究 (Microcosm study) 12
1.8 分子生物技術檢測環境微生物 13
1.8.1 16 S rDNA之偵測 14
1.8.2 變性梯度膠體電泳(Denaturing Gradient Gel Electrophoresis, DGGE) 15
1.8.3 菌種鑑定 16
1.8.4 即時定量PCR (Real-time PCR) 17
1.9 研究目的 18
(二) 研究方法 19
2.1 微生態系統好氧批次生物分解實驗 19
2.2 微生態系統厭氧批次生物分解實驗 21
2.3 污染物分析 24
2.4 利用分子生物技術分析批次實驗中之優勢菌群 25
2.4.1 微生物DNA的萃取及純化 25
2.4.2 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 26
2.4.3 變性梯度膠體電泳(Denaturing Gradient Gel Electrophoresis, DGGE) 28
2.4.4 SYBR greenⅠ螢光染色 29
2.4.5 DNA定序與NCBI資料庫比對序列 29
2.4.6 即時定量PCR (Real-time PCR) 30
(三) 結果與討論 32
3.1 微生態系統批次實驗 32
3.1.1 好氧批次實驗 32
3.1.2 厭氧批次實驗 34
3.2 微生態系統批次實驗菌相分析與鑑定 37
3.2.1 好氧批次實驗 37
3.2.2 厭氧批次實驗 39
3.3 即時定量PCR偵測Dehalococcoides spp.與Desulfitobacterium spp.數量 41
3.3.1 好氧批次實驗 42
3.3.2 厭氧批次實驗 46
(四) 結論 52
(五) 建議 54
(六) 參考文獻 55
(七) 附圖 64
(八) 附表 74

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