含氯有機溶劑污染場址之調查與整治有較高之難度，而污染源亦不易界定，而三氯乙烯(Trichloroethylene, TCE)由於工業上普遍被使用，具有脂溶性、高滲透及高揮發性等特性，一旦洩漏至地下水後則會造成大範圍的環境污染，且可能會透過飲用水等多種管道，將對人民的身體健康造成危害。近年來生物復育整治技術(biodegradation technology)被視為是一種較具發展性之現地整治技術，其原理為利用微生物分解土壤或地下水中之污染物，對於場址破壞性較小，是一種自然對環境無害的綠色整治技術。若只仰賴現地土壤地下水之原生微生物進行自然降解，其整治成本較不符合經濟效益，因此研究常會使用添加基質來增加其碳源濃度，透過提高碳源刺激微生物菌群生長進而加強生物復育整治成效。在添加基質過後，經過長時間發酵等作用，可能會導致水質酸化而降低污染整治成效，反而增加整治操作成本。因此，本研究選擇以溶解性較高之糖蜜搭配乳化植物油，製作乳化糖蜜基質(Emulsified Molasses substrate, EMS)，並且搭配碳酸鈉(Na2CO3)及碳酸氫鈉(NaHCO3)做為pH緩衝溶液，發展一種具有pH緩衝能力之基質，改善其水質易酸化之問題，進而提高整體生物復育整治成效。本研究先針對EMS進行基本特性分析，接著與市售乳化型基質(Emulsified Oil substrate, EOS®)搭配緩衝溶液進行前導實驗，觀察其pH緩衝能力與總有機碳(Total organic carbon, TOC)之消耗情形，並以不同稀釋濃度之緩衝溶液進行實驗，評估其緩衝能力。研究接續進行厭氧微生物批次實驗評估其降解污染物之能力，再以管柱流通性實驗模擬藥劑在土壤含水層中之傳輸情形，並模擬現地地下水水流之上中下游持續傳輸流動後的各項水質參數及污染物降解情形，最後批次及管柱實驗結果皆以分子生物技術分析菌相豐富程度，尋找對污染降解具有貢獻之菌種，以利後續現地實場應用。本研究之EMS經48小時穩定性實驗測試安定性皆為良好，且利用動態光散射儀(Dynamic Light Scattering, DLS)觀察油滴粒徑平均粒徑為0.427 μm。由基質及緩衝溶液前導實驗結果得知EMS之TOC消耗率約為70%，且搭配100 mM緩衝溶液最能有效控制水質酸化情形。由批次實驗結果得知EMS之TCE降解效率較佳，在90天實驗監測結果，TCE去除率可達98%，且副產物順-1,2-二氯乙烯[cis-1,2-Dichloroethene, cis-1,2-DCE(<0.7 mg/L)]及1,1-二氯乙烯[1,1- Dichloroethene, 1,1-DCE(<0.07 mg/L)]皆降解至低於第二類地下水污染管制標準，pH值亦控制在7-8之間，可有效控制水質酸化。變性梯度膠體電泳(Denaturing Gradient Gel Electrophoresis, DGGE)結果得知EMS組別之菌相豐富程度較佳。即時定量分析realtime-PCR(q-PCR)定量分析結果，第0天之Dehalococcoides spp.(DHC)菌數約為2.33E+02 gene copies/g，第90天DHC菌數約為1.78E+04 gene copies/g，其結果顯示添加本研究之EMS並搭配緩衝溶液可使環境維持中性且DHC菌量大幅生長。管柱實驗結果得知其管柱2生物反應區之TCE污染濃度降至降解至0.01 mg/L且監測到乙烯(ethane)生成，其濃度約為0.012-0.059 mg/L，而基質流至管柱3亦能持續利用進行生物復育，使目標污染物TCE及其副產物濃度皆能降解至低於第二類地下水污染管制標準。菌相分析結果得知基質流經管柱2及管柱3在實驗中後期菌相趨勢相較於自然降解組別皆呈現菌相增加之趨勢。綜合上述結果可知，本研究研發之EMS搭配緩衝溶液，能夠有效控制pH值且穩定持續提供碳源供微生物利用，且糖蜜所具有高溶解性佳及易於發酵之特性，亦能提升發酵產氫效率並增加其還原脫氯之整治效率。

Soil and groundwater at many existing and former industrial areas and disposal sites are contaminated by halogenated organic compounds that were released into the environment. When they are released into the subsurface, they tend to adsorb onto the soils and cause the appearance of DNAPL (dense-non-aqueous phase liquid) pool. In this study, TCE will be used as the target compound for the feasibility and pilot-scale studies. The cost-effective approach for the remediation of the TCE-contaminated aquifers is the application anaerobic reductive dechlorination for TCE biodegradation. Enhanced in situ bioremediation requires the injection of primary substrates. In this study, the emulsified molasses substrate (EMS) containing pH buffering chemicals (Na2CO3 and NaHCO3) was prepared as the substrate with the capability of pH control. Batch experiments were conducted to evaluate the effectiveness of total organic carbon (TOC) release and pH control for EMS and Emulsified Oil substrate (EOS®). Anaerobic microcosm study was performed to evaluate the feasibility of using EMS as the primary substrate for the enhancement of TCE dechlorination. A column experiment was performed following the microcosm study to assess the transport and distribution phenomena of EMS in aquifers. The variations in microbial diversity and dominant bacteria during the biodegradation process were conducted using a series of molecular biology techniques including DNA extraction, polymerase chain reaction (PCR) amplification, denaturing gradient gel electrophoresis (DGGE), and quantitative PCR (qPCR or real-time PCR) analyses. The globule diameter of the EMS was around 0.427 μm analyzed by the Dynamic light scattering (DLS) method. Results show that EMS with 100 mM of buffering solution could effective control pH and maintain a neutral condition with a TOC consumption of 70%. Results from the microcosm study show that up to 98% of TCE could be removed during the 90-day operational period. The concentrations of TCE dechlorination byproducts [e.g., cis-1,2-dichloroethene (cis-1,2-DCE), 1,1-dichloroethene (1,1-DCE)] were also dropped to below the groundwater standard (<0.7 mg/L). The pH value was maintained in the range from 7 to 8 during the microcosm experiment. Results from the DGGE results show that the microcosms with EMS had a higher microbial diversity. The population of Dehalococcoides spp. (DHC) increased from 2.33E+02 gene copies/g on day 0 to 1.78E+04 gene copies/g on day 90. This indicates that the EMS addition resulted in the increase in DHC population probably due to the optimal pH condition for the growth of DHC. Results from the column study show that the TCE concentrations dropped to below 0.01 mg/L in the column effluents with the ethene concentrations of 0.012 to 0.059 mg/L. Moreover, the DGGE results also show that an increased microbial diversity was observed in soils collected from the third column (the last column of the three-column system). Results from this study indicate that EMS is a substrate with a buffering capability that can be applied to enhance TCE removal through the anaerobic dechlorination mechanism. The enhanced in situ anaerobic bioremediation is a promising technology to remediate TCE-contaminated groundwater.

摘要 i
目錄 vi
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 2
第二章 文獻回顧 3
2.1含氯脂肪族碳氫化合物 3
2.1.1含氯脂肪族碳氫化合物污染概況 3
2.2三氯乙烯之特性 4
2.2.1三氯乙烯之物理化學特性及毒性危害 4
2.2.2三氯乙烯之傳輸路徑 5
2.3地下水污染整治技術 10
2.3.1物理及化學整治技術 10
2.3.2生物復育整治技術 11
2.4三氯乙烯之生物降解 12
2.4.1三氯乙烯之好氧生物降解 12
2.4.2三氯乙烯之厭氧生物降解 13
2.5基質與緩衝溶液之特性介紹 15
2.5.1糖蜜之基本特性 15
2.5.2厭氧微生物暗發酵產氫代謝途徑 17
2.5.3油品介紹與特性 18
2.5.4緩衝溶液特性介紹 21
2.6 氫氣濃度效應 23
2.7分子生物技術之應用 24
2.7.1 16SrDNA之獨特性及應用 24
2.7.2降解含氯脂肪族之菌種與基因 25
2.7.3聚合酶鏈鎖反應(Polymerase Chain Reaction, PCR) 26
2.7.4瓊酯膠體電泳(Agarose Gel Electrophoresis) 27
2.7.5變性梯度膠體電泳(Denaturing gradient gel electrophoresis, DGGE) 28
第三章 實驗設備與方法 29
3.1研究流程 29
3.2實驗材料與設備 32
3.2.1實驗材料 32
3.2.2實驗儀器與設備 32
3.3實驗設計 33
3.3.1 基質前導實驗 33
3.3.2不同緩衝溶液緩衝能力實驗 33
3.3.3厭氧微生物批次降解實驗 37
3.3.4管柱流通性實驗 37
3.3.5管柱實驗 38
3.4實驗分析方法 39
3.5分子生物分析 39
3.5.1土壤微生物DNA萃取 39
3.5.2聚合酶鏈鎖反應(Polymerase Chain Reaction, PCR) 40
3.5.3變性梯度膠體電泳(Denaturing Gradient Gel Electrophoresis，DGGE) 41
3.5.4 Mixed DNA cloning及定序 42
3.5.5即時定量PCR(realtime-PCR) 42
3.6成分分析 45
3.6.1界達電位分析 45
3.6.2動態光散射儀分析 45
3.7水質分析 45
第四章 結果與討論 46
4.1EMS配製及特性分析 46
4.1.1添加植物油對於糖蜜乳化之影響 47
4.1.2EMS之最佳合成實驗 48
4.1.3EMS基本性質 50
4.1.4 EMS安定性實驗 51
4.2 EMS之生物可利用性評估 52
4.2.1 EMS與緩衝溶液之前導實驗 52
4.2.2不同緩衝溶液濃度之緩衝能力試驗 55
4.3厭氧微生物批次實驗 57
4.3.1控制滅菌組(KC) 57
4.3.2自然降解組(LC) 60
4.3.3乳化型糖蜜基質組(EMS) 63
4.3.4市售乳化型基質組(EOS®) 68
4.4氫氣濃度批次實驗 71
4.5厭氧微生物批次實驗菌相分析 73
4.5.1 DGGE菌相分析 73
4.5.2即時定量分析 realtime-PCR (q-PCR) 75
4.6厭氧微生物管柱實驗 78
4.6.1 管柱流通性實驗 79
4.6.2管柱水質及總有機碳 82
4.6.3管柱總鐵、亞鐵、硫酸鹽、硫化物 86
4.6.4管柱污染物與甲烷變化趨勢 90
4.7管柱實驗菌相分析 94
4.7.1 DGGE菌相分析 94
第五章 結論與建議 96
5.1結論 96
5.2建議 97
參考文獻 98

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