臺灣博碩士論文加值系統

本研究之目的係針對受MTBE與BTEX污染之人工模擬實驗系統進行生物復育測試，評估進行生物刺激及生物強化後對基質之分解效率，並利用統計學之回應曲面法(RSM)建立各基質最佳去除效率之氮源與溶氧的操作條件，同時獲得回應曲面之模型。此外，並採用分子生物技術中之聚合酶鏈鎖反應(PCR)與單股DNA構形多型性(SSCP)分析技術，以鑑別混合菌群之生物相結構變化情形，並建立生物復育槽之去除效率與其菌群結構間之關係。
研究結果顯示：(1)生物復育槽處理MTBE與BTEX之能力依序為ethylbenzene ＞p-xylene ＞toluene ＞benzene ＞MTBE；(2)經生物刺激及生物強化後之微生物於去除MTBE與BTEX之能力上，可使效率增加10~30 %左右，顯示氮源與溶氧量之重要性及所加入之純菌株確實對生物分解有極大之助益；(3)利用回應曲面法所獲致之結果如下：在不同氮源與溶氧量對Benzene受「生物刺激」之去除效率中，若將氮源量與溶氧量分別控制在65 mg/L與15.6 mg/L時，可使Benzene之去除效率提高至63.8 %；在不同氮源與溶氧量對MTBE+BTEX受「生物刺激」之去除效率中，若將氮源量與溶氧量分別控制在61 mg/L與15 mg/L時，可使 MTBE與BTEX之去除效率提高至62 %；在不同氮源與溶氧量對MTBE受「生物刺激＋生物強化」之平均去除效率中，若將氮源量與溶氧量分別控制在63 mg/L與14 mg/L時，可使MTBE之去除效率提高至46 %；在不同氮源量與溶氧量對MTBE+BTEX受「生物刺激＋生物強化」之平均去除效率中，若將氮源量與溶氧量分別控制在60 mg/L與15 mg/L，可使MTBE+BTEX之去除效率提高至73.8 %；溶氧對各基質去除效率的影響較氮源的影響顯著，因此整體而言溶氧的變化將較直接影響各基質的去除效率；(4)於進行不同氮源與溶氧之試程中，隨著MTBE與BTEX去除效率增加率之高低，可將微生物之菌群分為不同之族群；(5)由SSCP指紋圖譜中所呈現之主要bands (6、11)，應可代表生物復育槽內之主要去除MTBE及BTEX之優勢菌群。

The objectives of the research are to investigate the bioremediation capability in an MTBE- and BTEX-contaminated bioreactor, and to evaluate substrate removal efficiencies under biostimulation and bioaugmentation conditions. In addition, a relationship between removal efficiencies and concentrations of nitrogen source and oxygen will be determined by using response surface analysis. Exploring microbial community changes under various environmental conditions (concentrations of nitrogen source and oxygen) by using SSCP profiles of PCR-amplified 16S rDNA genes and linking the changes with simultaneously diminishing substrate concentration in the microcosms are the goals of this research.
Results of the research show that: (1) the removals for MTBE and BTEX decrease in the order of ethylbenzene, p-xylene, toluene, benzene, and MTBE; (2) additional 10–30 % removals for both MTBE and BTEX were observed via. biostimulation and bioaugmentation, thereby indicating the significant contributions of nitrogen source, oxygen and the substrates-degrading pure cultures; (3) the Benzene removal increased to 63.8 % via. biostimulation with the concentrations of nitrogen source and oxygen at 65 mg/L and 15.6 mg/L, respectively. The MTBE and BTEX removals increased to 62 % via. biostimulation with the concentrations of nitrogen source and oxygen at 61 mg/L and 15 mg/L, respectively. The MTBE removal increased to 46 % via. biostimulation and bioaugmentation with the concentrations of nitrogen source and oxygen at 63 mg/L and 14 mg/L, respectively. The MTBE and BTEX removals increased to 73.8 % via. biostimulation and bioaugmentation with the concentrations of nitrogen source and oxygen at 60 mg/L and 15 mg/L, respectively. Dissolved oxygen plays more significant role while compared to nitrogen source from the response surface analysis; (4) the microbial consortium were divided into different groups, by examining the increasing rates for MTBE and BTEX removal efficiencies; and (5) Bands-6 and -11 are the dominant species in degrading MTBE and BTEX, by comparing population profiles with SSCP pure culture patterns.

目錄

封面內頁
簽名頁
博碩士論文暨電子檔案上網授權書 iii
中文摘要 iv
英文摘要 vi
誌謝 viii
目錄 ix
圖目錄 xiii
表目錄 xvii
第一章 緒論 1
1.1前言 1
1.2研究目的 3
第二章 文獻回顧 5
2.1 MTBE與BTEX之簡介及其物化特性 5
2.2 生物刺激對生物整治之影響 10
2.2.1 溶氧 10
2.2.2 氮源 13
2.3生物強化對生物整治之影響 14
2.4地下水生物復育之相關研究 16
2.5 MTBE與BTEX之生物分解特性 18
2.6總生菌數與分子生物之關係 21
2.7 分子生物技術於環境微生物菌群結構分析之相關研究 25
2.7.1 分子生物在污染整治上之重要性與應用 25
2.7.2 聚合酶鏈鎖反應原理 26
2.7.3 單股DNA構型多行性分析 26
2.7.4 16S rRNA基因放大與定序 28
2.7.5 微生物種類辨識方法 29
2.7.6 分子生物技術於環境微生物之應用 31
2.8 回應曲面法之應用 39
2.8.1回應曲面法之原理 39
2.8.2中心混層設計 41
第三章 材料與方法 42
3.1研究材料與儀器設備 44
3.1.1菌種來源 44
3.1.2藥品種類 44
3.1.3分子生物之儀器設備 48
3.1.4其他相關使用材料 48
3.1.5儀器設備 48
3.2研究方法與步驟 51
3.2.1 MTBE與BTEX批次降解能力評估 51
3.2.2生物反應器之組裝 52
3.2.3生物復育槽之去除效率評估方法與步驟 54
3.2.3-1生物復育槽之去除效率評估 54
3.2.3-2生物復育槽之檢量線 55
3.2.3-3 菌量之分析方法 55
3.2.4生物復育槽中MTBE與BTEX之分解實驗 56
3.3分子生物技術建立 63
3.3.1微生物之總體DNA萃取方法 64
3.3.2聚合酶連鎖反應 64
3.3.3單股DNA構形多型性分析步驟 66
3.3.4 微生物種類辨識 68
3.3.5菌群相似度與群叢分析 69
第四章 結果與討論 70
4.1 批次實驗 70
4.1.1不同氮源量之批次實驗結果 70
4.1.2不同溶氧量之批次實驗結果 72
4.1.3比較氮源與溶氧對MTBE與BTEX降解之影響 74
4.2生物刺激與生物強化之分解效率提升成效 76
4.2.1回應曲面模型之分析 82
4.2.2 MTBE與BTEX受生物刺激(無生物強化)之回應曲面分析 84
4.2.3 MTBE與BTEX受生物刺激(有生物強化)之回應曲面分析 95
4.2.4 MTBE與BTEX受生物刺激與生物刺激(有生物強化)之回應曲面分析 107
4.3氮源與溶氧對生物復育槽內菌群結構之變化影響 117
4.3.1 生物刺激之影響 117
4.3.2 生物強化之影響 118
4.4菌種定序比對分析 124
第五章 結論與建議 131
5.1結論 131
5.2建議 132
參考文獻 134
附錄A 生物復育槽之基質濃度檢量線 154
附錄B 菌量檢量線 158

圖目錄

圖2.8-1中心混層設計之星點及中心點補充實驗圖 41
圖3-1 整體研究架構流程圖 43
圖3.2-1模擬現地地下水環境之生物反應系統 53
圖3.2-2 生物復育槽之氧氣進流濃度監測曲線 58
圖3.2-3中心混層設計圖 61
圖3.3-1分子生物技術分析流程圖 63
圖3.3-2 SSCP流程圖 67
圖4.1-1 不同氮源濃度下MTBE與BTEX之降解效率圖 71
圖4.1-2 不同溶氧濃度下MTBE與BTEX之降解效率圖 73
圖4.1-3不同溶氧與氮源濃度下混合菌對MTBE與BTEX之去除效率關係 75
圖4.2-1(a)~(c)為不同溶氧與氮源量試程下之去除效率圖 78
圖4.2-2 不同氮源與溶氧之試程對生物刺激、生物強化與空白實驗組之比較圖 81
圖4.2-3不同溶氧與氮源試程下之總生菌數長條圖 82
圖4.2-4 不同氮源與溶氧量對Benzene受生物刺激(無生物強化)去除效率之回應曲面圖 90
圖4.2-5不同氮源與溶氧量對Benzene受生物刺激(無生物強化)去除效率之等去除效率線圖 90
圖4.2-6不同氮源與溶氧量對Toluene受生物刺激(無生物強化)去除效率之回應曲面圖 91
圖4.2-7 不同氮源與溶氧量對Toluene受生物刺激(無生物強化)去除效率之等去除效率線圖 91
圖4.2-8 不同氮源與溶氧量對Ethylbenzene受生物刺激(無生物強化) 去除效率之回應曲面圖 92
圖4.2-9不同氮源與溶氧量對Ethylbenzene受生物刺激(無生物強化)去除效率之等去除效率線圖 92
圖4.2-10不同氮源與溶氧量對p-xylene受生物刺激(無生物強化)去除效率之回應曲面圖 93
圖4.2-11不同氮源與溶氧量對p-xylene受生物刺激(無生物強化)去除效率之等去除效率線圖 93
圖4.2-12不同氮源與溶氧量對MTBE+BTEX受生物刺激(無生物強化)去除效率之回應曲面圖 94
圖4.2-13不同氮源與溶氧量對MTBE+BTEX受生物刺激(無生物強化)去除效率之等去除效率線圖 94
圖4.2-14不同氮源與溶氧量對MTBE受生物刺激(有生物強化)去除效率之回應曲面圖 101
圖4.2-15不同氮源與溶氧量對MTBE受生物刺激(有生物強化)去除效率之等去除效率線圖 101
圖4.2-16不同氮源與溶氧量對Benzene受生物刺激(有生物強化)去除效率之回應曲面圖 102
圖4.2-17 不同氮源與溶氧量對Benzene受生物刺激(有生物強化)去除效率之等去除效率線圖 102
圖4.2-18 不同氮源與溶氧量對Toluene受生物刺激(有生物強化)去除效率之回應曲面圖 103
圖4.2-19 不同氮源與溶氧量對Toluene受生物刺激(有生物強化)去除效率之等去除效率線圖 103
圖4.2-20 不同氮源與溶氧量對Ethylbenzene受生物刺激(有生物強 化)去除效率之回應曲面圖 104
圖4.2-21不同氮源與溶氧量對Ethylbenzene受生物刺激(有生物強化)去除效率之等去除效率線圖 104
圖4.2-22 不同氮源與溶氧量對p-xylene受生物刺激(有生物強化)去除效率之回應曲面圖 105
圖4.2-23 不同氮源與溶氧量對p-xylene受生物刺激(有生物強化)去除效率之等去除效率線圖 105
圖4.2-24 不同氮源與溶氧量對MTBE+BTEX受生物刺激(有生物強化)去除效率之回應曲面圖 106
圖4.2-25 不同氮源與溶氧量對MTBE+BTEX受生物刺激(有生物強化)去除效率之等去除效率線圖 106
圖4.2-26 不同氮源與溶氧量對MTBE受生物刺激與生物強化下去除效率之回應曲面圖 113
圖4.2-27 不同氮源與溶氧量對MTBE受生物刺激與生物強化下去除效率之等去除效率線圖 113
圖4.2-28 不同氮源與溶氧量對Benzene受生物刺激與生物強化下去除效率之回應曲面圖 114
圖4.2-29 不同氮源與溶氧量對Benzene受生物刺激與生物強化下去除效率之等去除效率線圖 114
圖4.2-30 不同氮源與溶氧量對Toluene受生物刺激與生物強化下去除效率之回應曲面圖 115
圖4.2-31 不同氮源與溶氧量對Toluene受生物刺激與生物強化下去除效率之等去除效率線圖 115
圖4.2-32 不同氮源與溶氧量對MTBE+BTEX受生物刺激與生物強 化下去除效率之回應曲面圖 116
圖4.2-33 不同氮源與溶氧量對MTBE+BTEX受生物刺激與生物強化下去除效率之等去除效率線圖 116
圖4.3-1 不同氮源與溶氧試程下經生物刺激後菌群結構之相對相似度與群叢關係及SSCP指紋圖譜。(a)以PCR-SSCP分佈之指紋圖譜；(b)菌群間相對相似度及群叢關係圖 121
圖4.3-2 不同氮源與溶氧試程下經生物刺激與生物強化後菌群結構之相對相似度及群叢關係與SSCP指紋圖譜。(a)以PCR-SSCP分佈之指紋圖譜；(b)菌群間相對相似度及群叢關係圖 122



表目錄

表2.1-1目前公告之地下水污染管制標準 8
表2.1-2 MTBE與BTEX之物化特性 9
表2.5-1 MTBE與BTEX生物分解之可行性相關研究 22
表2.7-1傳統方法對環境中微生物之培養可能率 30
表2.7-2分子生物應用技術之相關文獻 33
表3.1-1碳源藥品清單 45
表3.1-2模擬地下水之營養鹽配比 45
表3.1-3寡核苷酸引子種類 46
表3.1-4 PCR與SSCP相關藥品清單 46
表3.1-5分子生物技術之相關藥品種類 47
表3.1-6分子生物技術之儀器設備清單 49
表3.1-7本研究分子生物技術其它使用相關材料清單 50
表3.2-1實驗設計組數表 60
表3.2-2三階層二變數中心混層實驗設計反應參數實驗值範圍 61
表3.3-1 PCR升溫程式 65
表3.3-2 PCR藥品與比例 65
表3.3-3 SSCP膠片配比 67
表3.3-4 SSCP變性劑(Loading dye)成分與配比 68
表4.2-1為各試程中進行生物刺激及生物刺激與生物強化後效率增加率 81
表4.2-2各基質(MTBE與BTEX)受「生物刺激(無生物強化)」之去除效率 87
表4.2-3各基質回應曲面之變異分析表：生物刺激(無生物強化)(1/2) 87
表4.2-3各基質回應曲面之變異分析表：生物刺激(無生物強化)(2/2) 88
表4.2-4各基質最佳回應曲面模型之迴歸分析表：生物刺激(無生物強化) 89
表4.2-5各基質(MTBE與BTEX)受「生物刺激(有生物強化)」之去除效率 98
表 4.2-6 各基質回應曲面之變異分析表：生物刺激(受生物強化) 99
表4.2-7各基質最佳回應曲面模型之迴歸分析表：生物刺激(受生物強化) 100
表4-2-8 各基質(MTBE與BTEX)受「生物刺激+生物刺激(有生物強化)」之去除效率 109
表4.2-9 各基質回應曲面之變異分析表：「生物刺激+生物刺激(有生物強化)」之平均(1/2) 110
表4.2-9 各基質回應曲面之變異分析表：「生物刺激+生物刺激(有生物強化)」之平均(2/2) 111
表4.2-10 各基質最佳回應曲面模型之迴歸分析表：「生物刺激+生物刺激(受生物強化)」平均 112
表4.3-1 生物刺激之指紋圖譜條件對照表 121
表4.3-2 生物強化之指紋圖譜條件對照表 123
表4.4-1 MTBE菌(Pseudomonas aeruginosa YAMT521)與Pseudomonas aeruginosa ATCC 27853序列比對結果 126
表4.4-2 Benzene菌(Ralstonia sp. YABE411)與Ralstonia sp. P-10序列比對結果 127
表4.4-3 Toluene菌(Pseudomonas sp. YATO411)與Pseudomonas sp. PH1序列比對結果 128
表4.4-4 Ethylbenzen菌(Pseudomonas putida YABE411)與Pseudomonas putida序列比對結果(1/2) 129
表4.4-4 Ethylbenzene菌(Pseudomonas putida YABE411)與Pseudomonas putida序列比對結果(2/2) 130

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林依蓉、劉文佐，(2001)，多氯聯苯厭氧馴養降解菌群微生物多樣性解析，碩士論文，國立中央大學生命科學研究所碩士論文。
林啟文、吳志鴻 、陳立軒、林虹君，(2003)，ETBE、MTBE及TAME之生物降解中間產物探討，第28屆廢水處理技術研討會。
林啟文、吳志鴻、陳政遠、鄭雅文、林虹君、陳立軒，(2003)，本土MTBE分解菌對甲基第三丁基醚之動力模式探討，第八屆生化工程研討會論文集。
林啟文、鄭雅文、陳信源、洪照先、羅伊翔，(2003)，MTBE分解菌之外加碳源及最適量化研究，第一屆土壤與地下水研討會。
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吳春生，(2002)，以生物曝氣法整治受地下儲槽洩漏之石化系有機污染物模場研究，碩士論文，國立中山大學環境工程研究所。
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陳信源、林啟文，(2003)，甲基第三丁基醚分解菌之分解能力與重金屬抑制效應研究，碩士論文，大葉大學環境工程研究所。
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陳谷汎，(2001)，以生物復育法整治2,4-二氯酚污染之地下水，碩士論文，國立中山大學環境工程研究所。
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張怡塘、李俊福、洪俊雄、李佳熹、張綺砡、楊子君、周希瓴、王灝頤、詹惠萍、葉榮美，(2003)，應用螢光原位雜交技術(FISH)偵測土壤與地下水中微生物族群結構，第一屆土壤與地下水研討會。
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曾怡禎、張權英，(2001)，利用分子生物方法分析微生物社會的結構，環境保護分子生物科技策略論壇會議資料集(III)。
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黃俊霖、劉文佐，(2001)，以分子生物技術探討厭氧生物產氫程序之菌群結構，碩士論文，國立中央大學環境工程研究所
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鄭幸雄、陳乃慈、簡杏純、邱瓊芳、吳坤龍，(2003)，污染場址馴化之微生物對柴油降解性之研究，第一屆土壤與地下水研討會。
鄭幸雄、吳坤龍、曾怡禎、莊蕙萍，(2003)，高溫厭氧流體化床處理PAN人造纖維廢水之微生物族群動態研究，第28屆廢水處理技術研討會。
鄭幸雄、白明德、趙禹杰，(2003b)，厭氧產氫菌分解高分子化合物及petone之產氫機制，第28屆廢水研討會論文集。
鄭幸雄、林秋裕、曾怡禎、李季眉、林信一、陳幸德，(2003c)，複合基質生物產氫機制及程序之應用整合研究-2002年，第28屆廢水研討會論文集。
簡青紅、曾怡禎、張權英、劉憶芬，(2003)，厭氧生物產氫反應槽的微生物社會結構，第28屆廢水處理技術研討會。
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謝昶毅，(2003)，以PCR-DGGE技術分析石油碳氫化合物污染地下水之微生物相，碩士論文，國立中山大學生物科學研究所。
潘結昌，(2000)，利用反應曲面法尋求以Aspergillus terreus生產lovastatin之培養基最適化研究，碩士論文，朝陽大學應用化學系。
蘇立群，(2005)，受無鉛汽油添加劑甲基第三丁基醚污染場址整治排氣及受污水之處理：活性污泥曝氣池法及生物滴濾塔法，碩士論文，國立中山大學環境工程研究所
蕭嘉瑢，(2004)，複合基質厭氧氫發酵生物程序操控之功能評估及分生檢測生態之研究，碩士論文，國立成功大學環境工程學系。
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