三氯乙烯(trichloroethylene, TCE)為地下水體中常見的污染物，TCE之生物降解須長期注入主要基質，過去研究基質之注入往往造成地下水酸化，進而使脫氯菌的生長受到抑制。聚麩胺酸(gamma poly-glutamic acid, γ-PGA)是一種經由生化合成之高分子聚合物，具保濕、無毒、金屬螯合及生物可分解性等特性。本研究以γ-PGA做為微生物生長之基質進行管柱實驗以及現地模場試驗，評估加強TCE厭氧還原脫氯之成效，研究結合分子生物技術變性梯度膠體電泳(denaturing gradient gel electrophoresis, DGGE)及次世代定序(next generation sequencing, NGS)觀察菌相及菌種之變化，再以即時定量分析(real-time polymerase chain reaction, qPCR)觀察脫氯菌(Dehalococcoides sp., Dhc)量變化趨勢。γ-PGA基本特性分析結果顯示γ-PGA粒徑大小有效分佈到粗細顆粒間(包括黏土到砂粒)。管柱實驗結果得知，添加γ-PGA於管柱中做為碳源時，反應期間並未監測到出流水pH酸化問題維持中性環境，因γ-PGA含有胺基，胺基溶出後和水形成鹼性物質氨可中和微生物釋出有機酸，管柱出流水之TCE濃度經50天反應後由1.6降至0.009 mg/L，降解效率高達99%。由DGGE菌相分析γ-PGA確實可以增加環境中的菌相豐富度，以qPCR分析Dhc菌量，其初始菌量約4.6×103 gene copies/g soil，於第90天菌量達到3.1×106 gene copies/g soil。現地模場試驗的結果顯示，注藥井TCE初始濃度為0.127 mg/L，灌注藥劑第75天後，TCE濃度改善至0.008 mg/L(降解效率為93.7%)；下游監測井（距離注藥井1 m）之TCE濃度5周內由0.149 mg/L改善至0.035 mg/L（降解率約為63.6％），亦發現地下水pH值未有酸化情形發生。NGS菌種鑑定結果得知，本場址含有有可能將VC降解之菌種、可以將含氯有機物脫氯的菌種、合成維他命B12營造適合脫氯菌生長環境的菌種，以及能產生氫氣加速脫氯菌進行還原脫氯作用的菌種，顯示本場址的菌種有效的降解TCE至乙烯。qPCR結果顯示，注藥井初始脫氯菌之菌量為1.0×103 gene copies/L，於第75天菌量增加至5.63×107 gene copies/L。本研究成果顯示：γ-PGA具備pH緩衝的效果，提供促進脫氯菌生長之碳源，並維持厭氧還原環境和菌相豐富度。模場數據顯示γ-PGA可有效促進現場TCE之生物降解，使γ-PGA之基質系統成為一種更具經濟效益及環境友善之綠色整治工法。

Trichloroethene(TCE) is a type of recalcitrant contaminant commonly found in groundwater. Provide carbon sources for a long period of time for TCE-bioremediation is necessary . Related research indicates that acidification problems often be accompanied with injecting substrate and that problems inhibits biological dechlorination mechanisms. The gamma poly-glutamic acid (γ-PGA) is a biopolymer synthesized by biochemical processes. In addition, its characteristics of moisture resistance, no toxicity, and chelating ability with metals. In this study, column experiments and field scale test were performed to evaluate the feasibility of using γ-PGA as a primary substrate to enhance the dechlorinating process of TCE degradation under anaerobic conditions. The predominant bacterial species and change of bacterial diversity during TCE bioremediation were using different molecular biology techniques to analyses, it were including denaturing-gradient gel electrophoresis (DGGE), next generation sequencing (NGS), and real-time PCR (qPCR). Among them, qPCR observed the change trend of Dehalococcoides(Dhc). Result of particle size showed that the γ-PGA could distribution well in soil particles. The results of column experiments showed that when γ-PGA were useded as a carbon source in the column, the acidity of the outflow water was not monitored during the reaction to maintain the neutral environment. Because γ-PGA contains an amine function group, and the amine group was dissolved after add system, and the formation of alkaline substance ammonia in water neutralizes the release of organic acid by the microorganism. The TCE concentration of the outflow of the column is reduced from 1.6 to 0.009 mg/L after 50 days of reaction, and the degradation efficiency is as high as 99%. The DGGE results show that γ-PGA could increase the abundance of bacteria in the environment. The data of qPCR for Dhc showed that the initial bacterial amount is about 4.6×103gene copies/g soil, and the amount of bacteria reaches 3.1×106gene copies/g soil on the 90th day. The results of field scale test showed that the initial concentration of TCE in the injection well was 0.127 mg/L. After the 75th day of the infusion, the TCE concentration was decreased to 0.008 mg/L (degradation efficiency was 93.7%). The TCE concentration of the downstream monitoring well (1 m from the injection well) were decreased from 0.149 mg/L to 0.035 mg/L within 5 weeks (degradation rate was about 63.6%). It was also found that the pH of the groundwater did not acidify. According to the identification results of NGS strains, the site contains strains that can completely reduce dechlorination, strains that can dechlorinate iii chlorinated aliphatic hydrocarbons, synthetic coenzymefactor B12 to create strains suitable for the growth environment of dechlorination bacteria, and produce hydrogen that coluld accelerate the reduce dechlorination of dechlorination bacteria effectively degrade TCE to ethylene.qPCR results show that the amount of the initial Dhc in the injection well was 1.0×103gene copies/L, and the amount of bacteria increased to 5.63×107gene copies/L on the 75th day. The results show that γ-PGA has a pH buffering effect, provides a carbon source that promotes the growth of dechlorination bacteria. The γ-PGA also can maintains an anaerobic reduction environment and bacterial abundance. The result of field scale test data showed that γ-PGA can effectively promote the biodegradation of TCE in situ, making the matrix system of γ-PGA a more economical and environmentally friendly green remediation method.

論文審定書 i
公開授權書 ii
誌謝 iii
摘要 iv
Abstract v
圖目錄 ix
表目錄 xi
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 含氯有機物來源與特性 4
2.2 地下水污染物處理技術之種類 7
2.2.1 物化處理 7
2.2.2 生物整治 8
2.3 TCE生物降解反應機制 12
2.3.1 好氧共代謝作用 13
2.3.2 厭氧還原脫氯作用 14
2.4 不同基質於土壤地下水之應用性 17
2.4.1 乳化型釋碳基質 17
2.4.2 乳化零價鐵 18
2.3.3 糖蜜 19
2.4.4 γ-PGA 20
2.5 環境微生物以及分子生物技術之應用 24
2.5.1 微生物多樣性之檢測方法 25
2.5.2 以即時定量PCR監測脫氯菌種與基因 28
2.5.3 以次世代定序監測菌群變化 29
第三章 實驗設備與方法 31
3.1 研究架構 31
3.2 實驗藥品與器材 33
3.2.1 實驗藥品 33
3.2.2 實驗器材 34
3.3 實驗設計 35
3.3.1 γ-PGA基本特性分析 35
3.3.2 管柱實驗 37
3.3.3 現地模場 39
3.4 採樣與樣品分析方法 41
3.4.1 水質分析方法 41
3.5 菌相分析 42
3.5.1 DNA萃取 42
3.5.2 聚合酶連鎖反應 43
3.5.3 變性梯度膠體電泳 44
3.5.4 即時定量分析 45
第四章 結果與討論 48
4.1 特性分析 48
4.1.1 黏度試驗 48
4.1.2 粒徑分布與界達電位 49
4.1.3 元素分析 50
4.1.4 均質試驗 50
4.1.5 流通試驗 51
4.2 管柱實驗 52
4.2.1 水質參數變化 52
4.2.2 化學參數變化 57
4.2.3 污染物變化趨勢 64
4.2.2 菌相變化分析 68
4.3 現地模場 74
4.3.1 水質變化趨勢 75
4.3.2 化學參數變化 80
4.3.3 污染物變化趨勢 84
4.3.3 菌相變化分析 88
4.4 應用NGS評估模場菌種 89
4.5 基質成本效益評估 96
第五章 結論與建議 97
5.1 結論 97
5.2 建議 99
第六章 參考文獻 100

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