乳化型釋碳基質(emulsified carbon-releasing substrate，ECS)為一種常被利用在生物復育污染場地時所添加到土壤中的營養基質，其經由微生物的代謝後可以促進微生物生長，並且增強微生物之生物降解功能。本研究對象為國內某一三氯乙烯污染場址，其內依地下水流方向鑿有五口井，從上游至下游依序編號為P64、P48、TW11、P22、P24，其中P22為ECS灌注井，其餘為採樣監測井。污染場址中之菌相監測係以16S rRNA基因為對象，利用聚合酶連鎖反應變性梯度膠體電泳(PCR-DGGE)方式進行菌相結構之分析。另外也利用即時定量PCR(real-time PCR)的方式偵測Dehalococcoides 菌屬及及還原脫氯基因(RDase)TceA、VcrA、BvcA之表現量，以評估污染物降解之生物活性。菌相分析結果顯示P22之菌相比其他口井之菌相豐富，這是由於加入ECS後促進微生物增長之故。而從real-time PCR對Dehalococcoides菌屬定量分析以及現場參數中的pH值高低，可以發現當pH值下降時，Dehalococcoides菌屬在環境中的數量也下降，但並不會影響Dehalococcoides菌屬的生長。從還原氧化電位(ORP)之檢測發現，污染場址一直保持在還原厭氧的狀態，有利於含氯有機物在污染場址中進行還原脫氯的反應。此外，亦自污染場址地下水中篩選出具有產氫氣能力的菌種Clostridium butyricum，可在三氯乙烯在還原脫氯的過程中，提供氫氣作為電子之供應者。由厭氧批次實驗的結果可以發現第28天加菌組之產氫基因(hydA)達到最高量1.7×1011 gene copies/L，三氯乙烯的濃度也持續下降，以此可以推測C. butyricum能穩定的提供氫氣，幫助Dehalococcoides菌屬進行還原脫氯反應。

The emulsified carbon-releasing substrate (ECS) is commonly used in bioremediation that can promote the microbial growth and enhance biodegradation process. The remediation object of this study is a local trichloroethylene (TCE) contaminated site. Based on the direction of the groundwater flow, there are five deep wells were dug in the remediation site, i.e. P64, P48, TW11, P22, and P24 respectively. P22 is the injection well and the others are sampling and monitoring wells. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique was used to monitor the microbial diversity during the remediation process. The real-time PCR was also used to detect the quantity of Dehalococcoides species as well as 3 dehalogenase genes (TceA、VcrA、BvcA) to evaluate the dechlorination activity. Results show that well P22 contained the most microbial diversity than all other wells because the injected ECS encouraged the microbial growth. The qPCR studies revealed that the content of Dehalococcoides species was related to pH value, the lower the pH value the less Dehalococcoides species present. However, the pH value did not overly affect the growth of Dehalococcoides species. The oxidation-reduction potential(ORP) indicated that the contaminated site was anaerobical and could stimulate the reductive dechlorination process. A hydrogen production Clostridium butyricum bacterial strain was isolated from the groundwater. The hydrogen produced by this strain can be used as an electron donor for TCE dechlorination. The expression of hydA gene of the anaerobic batch culture reached the highest amount (ie. 1.7× 1011 gene copies / L) at 28 day. The steady decreasing of TCE concentrations suggested that C. butyricum could provide hydrogen continually to assist the anaerobic dechlorination reaction.

論文審定書 i
誌謝 ii
摘要 iii
ABSTRACT iv
目錄 v
壹、前言 1
1.1 研究背景 1
1.2 三氯乙烯 2
1.3 氯乙烯 3
1.4 乳化型釋碳基質 4
1.5 含氯有機物之生物分解 4
1.5.1含氯有機物在好氧環境中的降解 4
1.5.2含氯有機物在厭氧環境中的降解 5
1.6 生物復育 7
1.7 微生態系統(Microcosm)研究 8
1.8 Clostridium butyricum以及hydA gene 8
1.9 分子生物學監測技術 9
1.9.1 16S rDNA 9
1.9.2聚合酶連鎖反應變性梯度膠體電泳 10
1.9.3即時定量PCR (real-time PCR) 11
1.10 研究目的 12
貳、材料與方法 14
2.1 藥品、培養基、儀器 14
2.2 污染場址及地理位置 14
2.3 現場水質參數監測 15
2.4 污染地下水樣品之genomic DNA萃取及純化 15
2.4.1 genomic DNA萃取 15
2.4.2 DNA純化 16
2.5 聚合酶連鎖反應-變性梯度膠體電泳(PCR-DGGE) 17
2.5.1聚合酶連鎖反應 17
2.5.2變性梯度膠體電泳 18
2.6 Cloning 19
2.6.1 Ligation 19
2.6.2 Transformation 19
2.6.3 NCBI nucleotide BLAST 20
2.7 即時聚合酶鏈鎖反應(Real-time PCR) 21
2.7.1標準曲線 21
2.7.2 Real-time PCR 22
2.8 篩選厭氧菌 23
固態培養基篩選 23
2.9 微生物系統厭氧批次實驗 24
2.9.1氣相層析儀/電子捕捉偵測器(GC/ECD) 24
2.9.2三氯乙烯生物降解實驗 24
參、結果與討論 26
3.1 地下水水質現場參數及菌數監測 26
3.1.1場址之總有機碳(TOC)含量變化 26
3.1.2場址之硫化氫(H2S)及甲烷(CH4)濃度變化 27
3.1.3場址中水溫及ORP之數值變化 29
3.1.4場址之pH值與厭氧分解菌數值變化 30
3.1.5 場址之含氯揮發性有機物(VOC)及還原脫氯基因數值變化 31
3.1.6厭氧分解菌Dehalococcoides菌屬及還原脫氯基因(RDase)數值變化 34
3.1.7微生物菌相分析 34
3.2 Clostridium butyricum厭氧批次實驗 35
3.2.1厭氧批次實驗之三氯乙烯(TCE)數值、pH值及產氫基因(hydA)變化 35
3.2.2 Dehalococcoides菌屬數值變化 37
肆、結論 39
伍、建議 42
參考文獻 43
圖附錄 51
表附錄 73

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