本研究為利用分子生物學技術來評估厭氧現地生物整治含氯有機污染物之成效。本研究於國內某含氯有機污染場址注入乳化型釋碳基質(emulsified carbon-releasing substrate, ECS)，以促使現地微生物生長，同時使地下水中釋碳基質經厭氧分解產生之氫氣來加強含氯有機物之脫氯作用。抽取污染地下水中細菌的DNA，並針對其中細菌16S rRNA基因的特定區段進行PCR分析，再利用變性梯度膠體電泳(denaturing gradient gel electrophoresis, DGGE)進行菌相分析。為了瞭解Dehalococcoides的脫氯作用過程，利用即時定量PCR(quantitative real-time PCR)來偵測現地環境中具有脫氯作用菌群及還原脫氯功能基因的消長變化。研究結果顯示，整治期間現場監測井溫度大致都維持在25-30℃之間。注入釋碳基質後，總有機碳(total organic carbon, TOC)濃度遠高於未添加ECS前的背景值。而TOC慢慢被微生物利用而形成緩慢且穩定下降趨勢，亦顯示ECS為緩釋性基質且可提供微生物生長所需的營養源。在第一次注入ECS後，所有注藥井的氧化還原電位(oxidation-reduction potential, ORP)及溶氧(dissolved oxygen, DO)皆有呈現下降的趨勢呈還原狀態。場址中主要之污染物三氯乙烯下降至分析儀器的偵測極限(< 1 μg/L)，且場址地下水樣中亦測得三氯乙烯之分解副產物(二氯乙烯及氯乙烯)。依還原脫鹵酶基因的TceA、VcrA及BvcA含量變化與含氯有機物汙染物濃度的變化結果得知，此結果符合微生物於厭氧環境還原脫氯降解含氯有機物的途徑。在定量分析方面，雖然Dehalococcoides菌量會受到pH影響，但是在整治過程中仍能維持一定的菌量，此現象可說明此類菌群在現地厭氧脫氯生物復育整治中扮演重要的角色。現地地下水微生物菌相鑑定與分析結果發現，脫氯相關菌種有51種，其中Acidovorax spp.、Alcaligenes spp.、Clostridium spp.、Dehalobacter spp.、Dehalococcoides spp.、Desulfuromonas spp.、Enterobacter spp.、Hydrogenophaga spp.、Methylosinus spp.、Pseudomonas spp.、Ralstonia spp.、Rhodococcus spp.、Stenotrophomonas spp.、Variovorax spp.及Wautersia spp.等菌種。而檢測出之厭氧生物整治相關菌種:(1)脫硝作用相關的菌種有54種;(2)四價錳及三價鐵還原相關作用的菌種有12種;(3)硫酸鹽還原相關作用的菌種有9種;(4)氫氣產生的相關的菌種有Clostridium spp.及Ruminococcus spp.等。這些功能菌種的存在能幫助環境趨向厭氧且可促進微生物進行還原脫氯反應。由於地下水環境之異質性，ECS灌注區域仍有好氧或缺氧環境，因此汙染場址中也鑑定出13種具有與利用甲烷相關的菌種。此類甲烷氧化菌利用甲烷作為碳源，而限制了大量甲烷釋放於大氣中。本研究結果得知，注入ECS後，對污染場址的整治達到一定效果，也證實加強式生物整治法在含氯有機汙染物污染場址實行是可行的方法。

The goal of this study is using molecular biology techniques to assess the effectiveness of anaerobic in situ bioremediation of chlorinated organic contaminants. In this study, a chlorinated organic contaminated site was injected with emulsified carbon-releasing substrate (ECS) to promote in situ microbial growth, as well as to release hydrogen generated by anaerobic decomposition of the ECS to enhance dechlorination of chlorinated organic compounds. DNAs were extracted from groundwater samples to analyze the microbial communities by using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). In order to understand the role of Dehalococcoides species in the dechlorination process, quantitative real-time PCR was used to detect the presence and shift of these bacteria and associated dehalogenase genes. The results showed all temperatures of the monitoring wells were generally maintained at 25-30℃ during this study. After ECS was injected, the total organic carbon (TOC) raised and its concentration was much higher than the background. The TOC was consumed gradually by microorganisms, indicated that ECS was a sustained-release matrix and could provide a source of nutrients for microbial growth. At the beginning of ECS injection, oxidation-reduction potential and dissolved oxygen of all injection wells slowly decreased to reduced state that could enhance the following anaerobic bioremediation. The trichloroethylene decreased below the detection limit (<1 μg/L). The trichlorethylene-degradation byproducts, dichloroethenes and vinyl chloride, were found in groundwater samples. The expression of reductive dehalogenase genes (TceA, VcrA and BvcA) were consistent with the concentration of chlorinated organic contaminants indicated that reductive dechlorination process was developed in this anaerobic environment. In quantitative analysis, the amount of Dehalococcoides species was affected by pH, but still maintained a constant number during the remediation processes, demonstrated that Dehalococcoides species played an important role in anaerobic dechlorination. Fifty-one dechlorination bacterial species were identified from the groundwater, including Acidovorax spp., Alcaligenes spp., Clostridium spp., Dehalobacter spp., Dehalococcoides spp., Desulfuromonas spp., Enterobacter spp., Hydrogenophaga spp., Methylosinus spp., Pseudomonas spp., Ralstonia spp., Rhodococcus spp., Stenotrophomonas spp., Variovorax spp., Wautersia spp. and so on. Other anaerobic bioremediation related bacteria species found including: (1) 54 denitrifying associated species; (2) 12 manganese(IV) reduction and iron(III) reduction species; (3) 9 sulfate-reducing species; (4) Clostridium spp. and Ruminococcus spp. that could generate hydrogen. All these bacteria could create an anaerobic environment and promote the reductive dechlorination. Due to the diversity of groundwater environment, the ECS injection area still possesses both aerobic and anoxic regions. Thirteen bacterial methanotrophs were recognized from the groundwater. These methanotrophic bacteria consumed methane as their carbon source and cut down the release of methane into the atmosphere. In conclusion, the injection of ECS is an effective and feasible bioremediation way to treat chlorinated organic contaminated sites.

審定書 i
誌謝 ii
中文摘要 iii
Abstract v
壹、 前言 1
1.1 研究緣起 1
1.2 三氯乙烯之性質及管制標準 2
1.3 含氯有機物整治方法 3
1.3.1 乳化型釋碳基質(emulsified carbon-releasing substrate, ECS) 4
1.4 微生物分解含氯有機物的反應機制 4
1.4.1 含氯有機物的好氧分解 4
1.4.2 含氯有機物的厭氧分解 5
1.5 現地生物復育機制 6
1.6 環境監測 7
1.7 分子生物學監測技術 7
1.7.1 16S rDNA 7
1.7.2 PCR-DGGE 8
1.7.3 Real-time PCR (qPCR) 9
1.8 研究目的 10
貳、 材料與方法 12
2.1 研究場址及整治方法 12
2.2 水質參數監測 13
2.3 樣品微生物的genomic DNA萃取 13
2.4 以PCR-DGGE進行菌相分析 14
2.4.1 聚合酶連鎖反應(PCR) 14
2.4.2 變性梯度膠體電泳(DGGE)分析 14
2.5 菌種分析與鑑定 16
2.5.1 聚合酶連鎖反應(PCR) 16
2.5.2 回收PCR產物 16
2.5.3 Ligation 17
2.5.4 Transformation 17
2.5.5 篩選及確認藍白菌落 17
2.5.6 定序比對分析 18
2.6 即時聚合酶鏈鎖反應(Real-time PCR) 18
2.6.1 基因片段篩選 18
2.6.2 建立標準曲線 18
2.6.3 配置反應液 18
2.6.4 環境中樣品數量 19
參、 結果與討論 20
3.1 水質參數、Dehalococcoides菌屬及不同還原脫鹵酶基因的監測分析 20
3.1.1 水溫變化 20
3.1.2 TOC含量變化 20
3.1.3 DO及ORP數值變化 21
3.1.4 硫化氫(H2S)及甲烷(CH4)濃度變化 22
3.1.5 pH與Dehalococcoides(Dhc)菌屬數值變化 22
3.1.6 含氯有機物濃度及不同還原脫鹵酶(RDase)基因數量變化 23
3.1.7 Dhc菌屬與RDase基因數量變化 25
3.2 微生物菌相鑑定與分析 26
3.2.1 微生物菌相分析 26
3.2.2 微生物菌相鑑定 26
肆、 結論 30
伍、 參考文獻 33
圖附錄 62
表附錄 81

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