本研究目的為探討1,2-二氯乙烷(1,2-dichloroethane, 1,2-DCA)污染現地場址加強式生物整治法之成效與可行性，於台灣南部某一受1,2-DCA污染的場址中注入易溶於水的基質做為微生物之碳源與營養鹽，並於過程中監測地下水環境參數以及現地微生物相之變化。實驗中針對地下水質進行總有機碳(TOC)、氧化還原電位(ORP)、硫酸鹽濃度等偵測，以即時定量PCR (realtime-PCR)偵測地下水環境中已知1,2-DCA分解菌屬Dehalococcoides spp. 及Desulfitobacterium spp. 之數量變化，同時以聚合酶連鎖反應(PCR)方式放大現地微生物之16S rRNA基因片段後，利用變性膠體電泳(DGGE)及定序技術進行現場菌相分析，以了解現場微生物相之組成。實驗結果顯示，在注入C-mix基質4.5個月後，TOC含量上升，現場ORP及硫酸鹽濃度反應地下水環境已呈現厭氧還原狀態，因此較不耐氧的Dehalococcoides spp. 之數量開始增加，其數量介於104-106 cells/L，較耐氧的Desulfitobacterium spp. 數量則無明顯增加趨勢，其數量介於106-107之間，且現場之污染物濃度則隨分解菌數上升而下降。現地菌相分析實驗發現，基質注入後，因TOC含量上升，地下水井中微生物相豐富度也隨之上升，於整治末期達到豐富度之最大值，並且各井優勢菌種數量均有增加。菌相鑑定結果發現，場址中1,2-DCA降解相關菌種數量豐富，並且於基質注入後，其數量開始增加，並逐漸成為較優勢的菌種。鑑定出的脫氯、氯化物分解菌有：Acidovorax sp.，Dechlorosoma sp. ，Dehalobacter sp.，Dehalococcoides sp.，Hydrogenophaga sp.，Nitrosospira sp.，Pseudomonas sp.，Rhodoferax sp. 等菌種。脫硝、鐵還原、硫還原等菌則有：Comamonas sp.，Denitratisoma sp.，Desulfobulbus sp.，Ferrovum sp.，Gallionella sp.，Nitrobacter sp. 等菌種。另外值得注意的是，在整治末期從場址中鑑定出了Methylibium sp. ，Methylobacillus sp. ，Methylobacter sp. ，Methylomicrobium sp. ，Methylomonas sp. ，Methylophilus sp. ，Methylosarcina sp. ，Methylosoma sp. ，Methyloversatilis sp. ，Methylovorus sp. ，Methylovulum sp.等11種甲烷利用菌，它們可減少整治過程中甲烷因生物復育累積於場址中造成施工危險，或者逸散造成溫室效應。經由本研究可以得知所注入基質的確有改變現場地下水環境之效果，並且可促進1,2-DCA分解菌生長、微生物相豐富度相及降解相關功能菌種增加，對整治達到一定效果，因此加強式生物整治在污染場址實行現地生物復育是可行的，在未來之大規模生物復育上具有重要參考價值。

The aim of this study was to access the efficacy of an enhanced in situ bioremediation technology at a 1,2-dichloroethane (1,2-DCA) polluted site in southern Taiwan. A water-soluble substrate was injected into the groundwater to provide carbon sources for microbial growth. After substrate injection, increased total organic carbon (TOC) concentrations and microbial populations including Dehalococcoides spp. and Desulfitobacterium spp. were observed in the groundwater. Microbial diversity was analyzed using denaturing gradient gel electrophoresis (DGGE) and 16S rDNA sequencing to identify the bacterial strains. The results showed that after 4.5 months of substrate injection, the reduction-oxidation potential (ORP) changed from aerobic to anaerobic conditions. The less oxygen-tolerable 1,2-DCA degrading bacteria Dehalococcoides spp. started to accumulate in groundwater. However, the more oxygen-tolerable Desulfitobacterium spp. didn’t show a prominent change, although the ORP was suitable for Desulfitobacterium spp. to carry out reductive dechlorination. The DGGE results indicate that with the injected carbon sources and mineral nutrients, both the groundwater microbial diversity and the amount of dominant bacteria were increased. The 16S rDNA sequencing demonstrated that the amount and diversity of 1,2-DCA degradation-related bacteria also increased with the injection of substrate. Six groups of 1,2-DCA degradation related reactions were found: dechlorination, chlorinated-compound degradation, denitrification, iron-reduction, sulfate-reduction and methane-utilizing. Four species that can directly degrade 1,2-DCA were found: Dehalobacter sp., Dehalococcoides sp., Nitrosospira sp. and Pseudomonas sp. Moreover, 11 methane-utilizing bacterial species were also discovered. The presence of these methane-utilizing bacteria not only might assist the process of denitrification and sulfate-reduction, but also could diminish the emission of the greenhouse gas. The results of this study confirmed that the addition of substrates could affect the groundwater oxidation-reduction state and enhance the bioremediation at the 1,2-DCA-contaminated site. Thus, enhanced in situ bioremediation is a feasible technology for site remediation.

論文審定書 i
誌謝 ii
摘要 iii
ABSTRACT V
壹、前言 1
1.1 研究背景 1
1.1.1 1,2-二氯乙烷背景 1
1.1.2 含氯有機化合物在環境中特性 2
1.1.3 1,2-DCA在環境中特性 2
1.1.4 1,2-二氯乙烷毒性及物化特性 3
1.2 含氯脂肪族碳氫化合物分解 5
1.2 1,2-二氯乙烷生物分解 7
1.2.1 好氧生物分解 7
1.2.2 厭氧生物分解 8
1.2.3 已知1,2-DCA厭氧分解菌 10
1.3 受污染地下水之現地整治技術 11
1.3.1 地下水循環井 12
1.3.2 生物復育法 14
1.4 分子生物學監測技術 15
1.4.1 16s rDNA 15
1.4.2 變性梯度膠體電泳 15
1.4.3 即時定量PCR (realtime-PCR) 16
1.5 研究目的 17
貳、材料與方法 18
2.1 污染場址基質注入 18
2.2 地下水樣品微生物DNA萃取 19
2.3 聚合酶連鎖反應 21
2.4 變性梯度膠體電泳 22
2.5 MIXED DNA CLONING 及定序 23
2.6 NCBI比對序列 24
2.7 REALTIME-PCR 25
2.8 環境中樣品已知降解菌數量 27
參、結果 28
3.1 地下水水質參數與菌數監測 28
3.1.1 場址中TOC含量變化 28
3.1.2 場址中ORP及硫酸鹽濃度變化 29
3.1.3 場址中已知厭氧分解菌種數量變化 30
3.1.4 場址中污染物及降解產物濃度變化 33
3.2 微生物菌相鑑定與分析 34
3.2.1 場址中微生物相變化趨勢 34
3.2.2 場址中微生物組成及其代謝特性 35
肆、結論 41
伍、建議 44
引用文獻 45
圖表 65

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