三硝基甲苯 (trinitrotoluene；TNT) 在軍事或民間用途上，均為廣泛使用的二級炸藥 (secondary explosive)。在許多環境中可以見到三硝基甲苯的污染，包括火藥製造與儲存廠、試驗以及訓練場所等。目前在TNT污染環境整治上，生物復育法是屬於對環境最友善的技術。本研究首先利用變性梯度膠體電泳 (denaturing gradient gel electrophoresis, DGGE) 對現地微生物菌相進行分析，並針對各菌種之16S DNA進行定序與比對，發現現地污染場址中即含有具有降解TNT能力之微生物。故利用TNT當作唯一氮源，針對TNT污染場址現地的原生菌進行篩選，共篩選出Achromobacter sp. 和Klebsiella sp. 2株可利用TNT為唯一氮源而生長的微生物，並進一步探討其在降解TNT污染土壤生物復育之可行性。實驗主要係採用好氧微生態系統 (aerobic microcosm) 方式，將2株菌種混合以不同批次實驗分別進行 (basal salt medium, BSM)及 (nitrogen free glucose salt medium, NFG) 兩種不同葡萄糖濃度培養基培養之降解TNT效率評估並利用HPLC監測最後TNT降解之產物。結果顯示在含葡萄糖濃度0.009% 之BSM培養基中好氧微生態系統進行第9天後，TNT降解率達到20%。而在含0.8%葡萄糖濃度之NFG培養基中微生態系統在第2天即可將TNT降解完畢，並可在HPLC偵測中發現4-amino-2,6-dinitrotoluene (4-A-2,6-DNT) 及 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT)等代謝副產物。

The 2,4,6-trinitrotoluene (TNT) is a secondary explosive widely used in military and civil purposes all over the world. Residual TNT had been found in contaminated soil and groundwater mainly releasing from manufacturing or demilitarization facilities. Bioremediation of using microbial degradation is a very promising technique to treat environmental pollution problems. In this study, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the microbial communities of a TNT polluted site. Two TNT degrading bacteria, Achromobacter sp. and Klebsiella sp. were isolated from the soils that could use TNT as the sole nitrogen source for their growth. Aerobic microcosm studies were conducted using these two bacteria to assess the effectiveness of removal TNT from contaminated soil. Both basal salt medium (BSM) and nitrogen free glucose (NFG) medium mixed with TNT were added into the experimented microcosms. Results show that approximately 20% of TNT was removed in microcosms with BSM medium after 9 days of incubation. Complete TNT removal was observed in microcosms using NFG as the medium. Moreover, The TNT degradation by-products, 4-amino-2,6-dinitrotoluene (4-A-2,6-DNT) and 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT) were detected in the microcosms.

論文審定書....................................................................................................i
誌謝...............................................................................................................ii
摘要..............................................................................................................iii
Abstract………………………………………………………………...….iv
目錄..............................................................................................................vi
圖目錄..........................................................................................................ix
表目錄...........................................................................................................x
第一章 前言……………………………………………………………….1
1.1 三硝基甲苯之特性............................................................................1
1.2 三硝基甲苯之健康危害....................................................................4
1.3 三硝基甲苯汙染物之處理方法........................................................7
1.3.1 物理處理法.................................................................................7
1.3.2 化學處理法.................................................................................8
1.3.3 生物處理法.................................................................................8
1.4 三硝基甲苯生物性分解研究..........................................................12
1.4.1 好氧降解...................................................................................12
1.4.2 厭氧降解...................................................................................14
1.5 分子生物技術檢測..........................................................................15
1.5.1 16S rDNA應用..........................................................................15
1.5.2 DGGE於菌相上之分析............................................................16
1.6 微生態系統(microcosm)研究.........................................................16
1.7 研究目的............................................................................................17
第二章 材料與方法...................................................................................18
2.1 三硝基甲苯污染場址菌相評估......................................................18
2.1.1 三硝基甲苯污染土壤之微生物DNA萃取............................18
2.1.2 聚合酶連鎖反應.......................................................................19
2.1.3 DGGE………………………………………………......……20
2.1.4 定序...........................................................................................21
2.1.4.1 agarose切膠及純化…………………………………….21
2.1.4.2 Ligation…………………………………………………22
2.1.4.3 Transformation………………………………………….22
2.1.4.4 Clone菌株確認及篩選…………………………………23
2.1.4.5 定序鑑定比對…………………………………………..23
2.2 三硝基污染土壤之菌株篩選..........................................................24
2.2.1 不同三硝基甲苯濃度培養基配置...........................................24
2.2.2不同三硝基甲苯濃度篩選出的菌種鑑定................................24
2.3微生態系統( microcosm)批次降解實驗..........................................25
2.3.1微生態系統培養基配置............................................................26
2.3.2分析方法:高效液相層析儀(High-Performance Liquid
Chromatography，HPLC) 偵測.........................................................27
2.3.3微生態系統( microcosm)菌數變化…………………………...28
2.4微生態系統( microcosm) DGGE菌相監測……………..…..…….29
第三章 結果與討論...................................................................................30
3.1 三硝基甲苯污染場址菌相分析......................................................30
3.1.1 場址中微生物菌相分析……………………………………...30
3.1.2場址中微生物組成……………………………………………31
3.2三硝基甲苯污染土壤之菌株篩選...................................................32
3.3微生態系統( microcosm)批次降解實驗..........................................32
3.3.1 BSM微生態組…………………………………………….......32
3.3.1.1 BSM微生態組-代謝產物……………………………….34
3.3.1.2 BSM微生態組-DGGE菌相…………………………….35
3.3.2 NFG 微生態組………………………………………………..36
3.3.2.1 NFG 微生態組-代謝產物……………………………....37
第四章 結論與建議...................................................................................38
參考文獻.....................................................................................................41
圖表.............................................................................................................50
附件……………………………………………………………………….64

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