甲基第三丁基醚(methyl tert-butyl ether, MTBE)為一含氧之化學物質，其使用始於1978年，主要用於取代鉛做為增進辛烷值之汽油添加劑，並藉以改善燃燒效率，達到改善空氣品質之目的。MTBE由於具有價格低廉、運送方便及製造、混合容易等優點，目前已是最常使用之汽油添加劑。此外，由於大量使用及不當處置之故，MTBE亦為目前地下水中常見污染物之一。MTBE已被證實對動物具有致癌性，美國環境保護署並將其歸類為疑似對人體具致癌性之物質，且建議其在飲用水中之管制值應介於或低於20-40 μg/L間。在台灣，台灣環境保護署亦已將MTBE列管為第四類毒性化學物質。

自然衰減、自然生物復育及加強式生物復育等整治技術由於具有經濟性及環境友善性，目前已是受到矚目的整治選項。一般而言，受污染場址中往往存在可降解污染物之現地微生物，這些微生物對場址中污染物之移除扮演著極為重要之角色。雖然早期的研究結果顯示MTBE的生物降解性並不顯著，但是近年來許多實驗室及現場的研究結果皆顯示，MTBE可在好氧及厭氧的條件下被微生物降解。此外，目前一些可得的MTBE自然衰減成功的案例及證據，亦使得自然衰減成為一可行的整治策略。然而，現地環境狀況及營養條件不佳時，往往會造成生物降解速率變低，進而影響整治之成效。因此評估現地微生物在不同場址條件下的對污染物的降解能力及分離、鑑定污染場址中的現地優勢微生物，以獲得適當的整治參數是極為重要的。此外，自然生物降解及其他非生物機制對污染團移除及控制的貢獻度亦應進行評估，以獲得充足的資訊，提供未來場址整治技術選擇時之參考。

本研究主要分為兩個主題，在第一主題中我們以微生物批次實驗(microcosm study)評估某MTBE污染場址中(場址A)，現地微生物對MTBE的生物降解性，並利用微生物鑑定技術-變性梯度膠體電泳(denaturing gradient gel electrophoresis, DGGE)鑑定場址中降解MTBE之優勢菌種。在本研究的第二主題中，我們選擇了兩個受MTBE污染的場址(場址A及場址B)進行詳細的場址調查，以評估場址中MTBE之自然衰減潛能。此外，我們亦利用自然衰減模式BIOSCREEN評估自然衰減機制對於MTBE污染團控制之貢獻度。本研究主要目的包括下列各項：
(1)評估現地微生物在不同氧化還原條件下對MTBE之降解能力。
(2)確認可降解MTBE之優勢菌種，提供未來整治時之應用。
(3)評估以自然衰減控制MTBE污染團之可行性。
(4)以自然衰減模式BIOSCREEN評估自然生物降解在自然衰減中之貢獻度。

第一主題中微生物批次實驗結果顯示，MTBE可在好氧且不添加其他碳源之條件下被含水層土壤中的微生物降解。實驗期間亦偵測到MTBE生物降解副產物第三丁基醇(tert-butyl alcohol, TBA)之生成，而TBA在實驗結束時亦可被完全去除。好氧組實驗結果顯示，氧氣應為場址A中MTBE生物降解之主要限制因子。因此若能提供場址A充足的氧氣，場址中之MTBE即可藉由自然生物降解機制移除。以清洗後土壤菌液為微生物來源之組別的實驗結果顯示，現地微生物可利用MTBE為單一碳源及能源，其生物降解速率為0.597 mg/g cells/h (0.194 nmole/mg cells/h)。厭氧組實驗結果顯示，MTBE在各種不同厭氧條件下並無法被現地微生物降解。研究結果顯示，好氧生物降解為場址中優勢的生物降解機制，因此好氧生物復育應是較為適合此場址的整治選項。DGGE分析之結果顯示，好氧MTBE降解菌Pseudomonas sp.及Xanthomonas sp.可能存在於場址A中。雖然微生物批次實驗結果顯示，MTBE無法在厭氧條件下被降解，但微生物鑑定實驗結果卻發現厭氧MTBE降解菌可能存在於場址中。此外，厭氧批次實驗中電子接受者(如硝酸鹽及三價鐵)的消耗及苯、甲苯、乙苯、二甲苯、三甲苯的降解，皆顯示此場址中厭氧微生物的活性極佳。

第二主題中現場調查結果顯示，MTBE的自然衰減確實發生於場址A及場址B，場址B中MTBE的污染團可藉由自然衰減機制有效的被控制，但場址A中MTBE的污染團已移動超出場址邊界。調查結果顯示，場址A及場址B中MTBE一階自然衰減速率分別為0.0021及0.0048 1/day。根據BIOSCREEN模擬結果顯示，自然生物降解機制可移除場址A中78%及場址B中59%之MTBE，因此自然生物降解對MTBE污染團的控制具有極大的貢獻。此外，稀釋及延散作用應為場址下游區域MTBE衰減之主要機制。現場調查結果顯示，縱使場址中具有高微生物活性，有時亦無法有效控制污染團。本主題之研究結果顯示，自然衰減或可做為受MTBE污染場址的整治選項，但需在下列二個前提下進行：(1)詳細的場址調查；及(2)自然衰減的發生及效率已被確認。

根據微生物批次實驗及現場調查結果顯示，此二場址中的MTBE可在好氧及厭氧的條件下被現地微生物族群降解。此外，以自然衰減做為受MTBE污染場址的整治選項，應為一可行之方式。本研究之成果有助於確認適當的生物復育條件，並設計一經濟有效的生物復育系統如監測式自然衰減或現地或現場的MTBE生物復育系統，以提供受污染場址整治之用。

Methyl tert-butyl ether (MTBE) has been used as a gasoline additive to improve the combustion efficiency and to replace lead since 1978. It is the most commonly used oxygenate now due to its low cost, convenience of transfer, and ease of blending and production. MTBE has become a prevalent groundwater contaminant because it is widely used and it has been disposed inappropriately. MTBE has been demonstrated an animal carcinogen. The US Environmental Protection Agency (US EPA) has temporarily classified MTBE as a possible human carcinogen and has set its advisory level for drinking water at 20-40 µg/L based on taste and odor concerns. The Taiwan Environmental Protection Administration (TEPA) also classifies it as the Class IV toxic chemical substances.

Currently, natural attenuation (NA) as well as natural bioremediation or enhanced bioremediation are attractive remediation options for contaminated sites due to their economic benefit and environmental friendly. In general, in situ microorganisms at the contaminated site play a very important role in site restoration. Although early studies suggested that the biodegradability of MTBE was not significant, recent laboratory and field reports reveal that MTBE can be biodegraded under aerobic and anaerobic conditions. In addition, evidences and some successful cases of MTBE attenuation have been reported that make natural attenuation a considerable remedial strategy. However, the biodegrading rate might decrease if the nutritional and physiological requirements are not met. Thus, it is important to assess the biodegradability of natural microorganisms under various site conditions to obtain optimal remedial conditions. Contributions of intrinsic biodegradation and other abiotic mechanisms to the removal and control of contaminants should also be evaluated to provide sufficient information for remedial option determination. Moreover, isolation and identification of the dominant native microorganisms will be helpful to following remediation tasks.

In the first part of this study, microcosm study and microbial identification technologies (denaturing gradient gel electrophoresis, DGGE) were applied to assess the biodegradability of MTBE by indigenous microbial consortia and to identify the dominant microorganisms at a MTBE-contaminated site (Site A). In the second part of this study, thorough field investigations were performed to evaluate the occurrence of natural attenuation of MTBE at two MTBE-contaminated sites (Site A and Site B). In addition, a natural attenuation model, BIOSCREEN, was performed to assess the effectiveness of natural attenuation on MTBE containment. The main objectives of this study contained the following:
(1)Evaluate MTBE biodegradability under different redox conditions by the indigenous microorganisms.
(2)Determine the dominant native microorganisms in MTBE biodegradation for further application.
(3)Assess the feasibility of using natural attenuation to control the MTBE plume.
(4)Evaluate the contributions of intrinsic biodegradation patterns on natural attenuation processes by BIOSCREEN.

Results from the microcosm study reveal that MTBE could be biodegraded by aquifer sediments without the addition of extra carbon sources under aerobic conditions. The production of tert-butyl alcohol (TBA), a degradation byproduct of MTBE, was detected. Complete removal of TBA was also observed by the end of the experiment. Results from aerobic microcosms study indicate that oxygen might be the major limiting factor of MTBE biodegradation at Site A. Thus, MTBE at this site could be removed via natural biodegradation processes with the supplement of sufficient oxygen. Microcosm study with extracted supernatant of aquifer sediments as the inocula show that the indigenous microorganisms were capable of using MTBE as the sole carbon and energy source. The calculated MTBE degradation rate was 0.597 mg/g cells/h or 0.194 nmole/mg cells/h. No MTBE removal was observed under various anaerobic conditions. Results suggest that aerobic biodegradation was the dominant degradation process and aerobic bioremediation might be a more appropriate option for the site remediation. According to the results of DGGE analysis, aerobic MTBE-biodegrading bacteria, Pseudomonas sp. and Xanthomonas sp., might exist at this site. Although results of microcosm study show that MTBE could not be degraded under anaerobic conditions, the microbial identification indicates that some novel anaerobic microbes, which could degraded MTBE, might be present at this site. In addition, anaerobic microbes caused the consumption of electron acceptors (e.g., nitrate, ferric iron) and removal of benzene, toluene, ethylbenzene, xylenes (BTEX), 1,2,4-trimethyl benzene (1,2,4-TMB), and 1,3,5-trimethyl benzene (1,3,5-TMB) (TMBs) in the anaerobic microcosms. These results also indicate that the potential of anaerobes activities was high at Site A.

Based on the results from the field investigation, natural attenuation of MTBE was occurring at both sites. MTBE plume at Site B could be effectively controlled via natural attenuation processes. Nevertheless, MTBE plume at Site A has migrated to a farther downgradient area and passed the boundary of the site. Field investigation results indicate that the natural attenuation mechanisms of MTBE at both sites were occurring with the first-order attenuation rates of 0.0021 and 0.0048 1/day at Sites A and B, respectively. According to BIOSCREEN simulation, biodegradation was responsible for 78% and 59% of MTBE mass reduction at Sites A and B, respectively. The intrinsic biodegradation had significant contributions on the control of MTBE plumes. Moreover, the dilution and dispersion processes might be the major mechanisms for the attenuation of MTBE in the downgradient areas. However, results also reveal that intrinsic biological processes might still fail to contain the plume if the selected point of compliance is not appropriate. Results of this study suggest that natural attenuation might be feasible to be used as a remedial option for the remediation of MTBE-contaminated site on the premise that (1) detailed site characterization has been conducted, and (2) the occurrence and effectiveness of natural attenuation processes have been confirmed.

Based on the results from the field investigation and laboratory microcosm studies, MTBE could be biodegraded by natural microbial populations at the studied sites under both aerobic and anaerobic conditions and natural attenuation would be applied as a remedial option at MTBE-contaminated sites. Results from this study would be useful in determining the favorable bioremediation conditions and designing an efficient and cost-effective bioremediation system such as monitored natural attenuation (MNA) or in situ or on-site MTBE bioremediation system for field application.

謝誌 I
摘要 II
Abstract V
Table of Contents IX
List of Tables XII
List of Figures XIII
Chapter 1. Introduction 1
1.1 Background of the Study 2
1.2 Objectives 3
Chapter 2. Literature Review 5
2.1 Usage of MTBE 6
2.2 Contamination of MTBE 7
2.3 MTBE Toxicity and Health Effects 9
2.4 Biodegradability of MTBE 12
2.4.1 Aerobic Biodegradation 13
2.4.1.1 Metabolic Biodegradation of MTBE by Pure and Mixed Cultures 13
2.4.1.2 Cometabolic Biodegradation of MTBE by Pure and Mixed Cultures 15
2.4.1.3 Biodegradation of MTBE in the Environment 17
2.4.2 Anaerobic Biodegradation 21
2.4.3 Pathway of MTBE Biodegradation 23
2.5 Natural Attenuation 24

2.6 Application of PCR-DGGE to the Analysis of Microbial Communities 26
Chapter 3. Natural Biodegradation of MTBE under Different Environmental Conditions: Microcosm and Microbial Identification Studies 38
Abstract 39
3.1 Introduction 41
3.2 Site Description 43
3.3 Materials and Methods 44
3.3.1 Aquifer Sediments and Groundwater Collection 44
3.3.2 Microcosm Study 44
3.3.2.1 Aerobic Microcosm Study 45
3.3.2.2 Denitrifying Microcosm Study 46
3.3.2.3 Iron Reducing Microcosm Study 47
3.3.2.4 Methanogenic Microcosm Study 48
3.3.3 Analytical Methods 48
3.3.4 Microbial Identification 49
3.4 Results and Discussion 51
3.4.1 Aerobic Microcosm 51
3.4.1.1 Microcosms with Aquifer Sediments as the Inocula and Different Initial Oxygen Concentrations 51
3.4.1.2 Microcosms with Extracted Supernatant of Aquifer Sediments as the Inocula 52
3.4.1.3 Microcosms with Activated Sludge as the Inocula 53
3.4.1.4 Calculation of MTBE Biodegradation Rates in the Aerobic Microcosms 54
3.4.2 Denitrifying Microcosm 55
3.4.3 Iron Reducing Microcosm 57
3.4.4 Methanogenic Microcosm 57
3.4.5 Summary of Microcosm Study 58
3.4.6 Microbial Identification 59
3.5 Summary 61
Chapter 4. Natural Attenuation of MTBE at Two Petroleum- Hydrocarbon Spill Sites 89
Abstract 90
4.1 Introduction 91
4.2 Site Description 94
4.3 Materials and Methods 95
4.3.1 Field Investigation 95
4.3.2 Analytical Methods 95
4.3.3 BIOSCREEN 96
4.4 Results and Discussion 97
4.4.1 Field Investigation 97
4.4.2 BIOSCREEN 100
4.5 Summary 101
Chapter 5. Conclusions and Recommendations 115
5.1 Conclusions 116
5.2 Recommendations 120
References 122
作者簡歷及著作 140

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