臺灣博碩士論文加值系統

中文摘要
2,4,6-三硝基甲苯(TNT)是一種芳香烴的化合物，在軍事和工業上被廣泛的使用，在過去這幾年間已經造成土壤與地下水的污染，生物復育是整治TNT汙染重要的方法。在本研究中將蚯蚓馴化在受2,4,6-三硝基甲苯(TNT)汙染的土壤，從蚯蚓的腸道分離出Citrobacter youngae strain E4，在搖瓶培養或土壤泥漿相中，探討有或沒有添加外源性氮源和碳源的情形下，微生物對TNT降解的能力。結果顯示，C. youngae strain E4在沒有外源性的胺基氮源和碳源的補充時無法降解TNT。但若同時添加胺基氮源和碳源能有效提升其降解TNT的能力，且TNT降解的能力也會隨添加量增加。因此C. youngae Strain E4降解TNT的能力為共代謝現象，也就是說C. youngae Strain E4為了要利用外源性的胺基氮源，使TNT亦被降解。同時發現補充外源性的營養能促進TNT的降解，也會增加細菌生長的數量並提高降解TNT的硝基還原酶，其中包含NemA、NfsA和NfsB的表現量，所以胺基氮源應該與誘導氮代謝的酵素有關。此外C. youngae strain E4應用到土壤泥漿相降解TNT汙染土，雖然TNT汙染土的濃度比較高但只要氮或碳源存在，則TNT降解的效率就會明顯的提高。本研究已發展出對生態環境友善且低成本的生物復育方法，當土壤受到汙染時可藉由生物刺激的方式並協同生物強化以去除土壤中TNT的汙染。  

Abstract
2,4,6-Trinitrotoluene (TNT), a nitroaromatic compound, widely used in military and industrial sites frequently caused soil and groundwater contamination in past decades. Bioremediation is a currently important method to remove TNT contamination. In this study, the earthworm Metaphire posthuma was acclimated in forest soil spiked with 2,4,6-trinitrotoluene (TNT), an earthworm enteric bacterium, Citrobacter youngae strain E4, was isolated to assess the capacity of microbial degradation of TNT in culture medium or in a soil slurry with/without exogenous nitrogen and carbon sources. As a consequence, C. youngae strain E4 was incapable of transforming TNT without a nitrogen or carbon amendment; however, the addition of amino-nitrogen and carbon amendments dramatically enhanced the efficacy of TNT degradation in a dose-dependent manner. Thus, metabolizing amino-nitrogen and carbon sources with simultaneous TNT degradation was verified as a process of cometabolism. The biostimulation, in terms of nutrient amendment on TNT degradation, was mediated by not only an increase in the cell number but also the up-regulation of nitroreductases, including NemA, NfsA and NfsB, which are involved in the reduction of TNT. The exogenous carbon source likely provided an adequate energy source for cell growth, and the nitrogen amendment might be responsible for the induction of enzymes relevant to nitrogen metabolism. Moreover, when C. youngae strain E4 was applied to a TNT-contaminated soil slurry in the presence of nitrogen and carbon amendments, TNT degradation was effectively enhanced, albeit with a higher concentration of TNT in the contaminated soil. In this study, we demonstrated a promising low-cost and ecofriendly bioremediation method to decontaminate TNT pollutants from contaminated soil through biostimulation in concert with bioaugmentation. 

目錄
致謝 I
中文摘要 III
英文摘要 IV
目錄 V
表目錄 VIII
圖目錄 VIII
附錄 X
第一章 文獻回顧 1
1.1 2,4,6-三硝基甲苯的基本特性 1
1.2 國內外TNT的相關法規 1
1.3 TNT的毒性與為害性 2
1.4 生物復育應用在TNT降解之技術 3
1.4.1 植生復育法 3
1.4.2 泥漿相生物降解 4
1.4.3 生物強化法 5
1.4.4 生物刺激法 6
1.4.5 微生物降解 7

第二章 實驗材料及方法 9
2.1 試驗的動物 9
2.2 土壤樣品來源 9
2.3 試驗用土壤配置 9
2.4 蚯蚓生物毒性測試 10
2.5 蚯蚓腸道菌株篩選 10
2.6 分子生物實驗技術 11
2.6.1 革蘭氏染色法 11
2.6.2 16SrDNA菌種鑑定和定序 11
2.7 總菌落數測定 15
2.8 TNT汙染土DNA萃取 16
2.9 C. youngae strain E4 RNA extraction 17
2.10 土壤泥漿相生物降解實驗分析方法 19
2.11 高效能液相層析儀分析條件/方法 20
2.12 TNT檢量線的製備 21
2.13 以蚯蚓腸道菌探討TNT降解能力之實驗設計 21
2.14 補充外源性氮源促進生物刺激降解TNT 21
2.14.1 以氯化氨 (NH4Cl) 替代氮源之實驗設計 22
2.14.2 以胺基氮源 (Peptone & Yeast Extract) 替代氮源之
實驗計 23
2.15 補充碳源促進生物刺激降解TNT 23
2.16 泥漿相生物降解TNT汙染土之實驗設計 24
2.17 反轉錄聚合酶連鎖反應 (RT-PCR) 24
2.18 生物急毒性試驗方法 25
2.19 實驗用的培養基配方 26
2.20 研究方法與步驟 28

第三章 結果 29
3.1 蚯蚓之毒性試驗 29
3.2 富集培養 (enrichment culuter) 篩選蚯蚓腸道菌降解TNT
29
3.3 菌種鑑定 29
3.4 以蚯蚓腸道菌測試對TNT的降解能力 31
3.5 評估細菌的接種量降解TNT的效能 32
3.6 補充外源性氮源進行生物刺激降解TNT 32
3.7 補充碳氮源進行生物刺激降解TNT 34
3.8 補充胺基氮源在生物刺激下對nitroreductase表現量的影響
34
3.9 以生物刺激和生物強化的方式共同進行降解TNT汙染土 35
3.10 泥漿相生物降解以補充碳源對TNT汙染土的降解 36
3.11 藉由C. youngae strain E4降解TNT並測試代謝物的生物急
毒性試驗 38

第四章 討論 40
4.1 分離M.posthuma的腸道菌以及TNT的生物毒性影響 40
4.2 泥漿相生物降解處理TNT的降解效能 41
4.3 以TNT做為唯一的氮源 (營養源) 42
4.4 補充外源性無機銨基氮源 (NH4Cl) 對TNT降解的影響 42
4.5 補充碳源對TNT降解的影響 44
4.6 代謝TNT與nitroreductase的關係 46
4.7 TNT代謝產物及生物毒性試驗 48

第五章 結論 50

參考文獻 51
 
表目錄
表1.1 TNT之物理及化學特性 87
表1.2 TNT危害毒性及曝露濃度資料 88
表3.1 以image J計算總細胞數在TNT汙染土之DNA的相對含量 89
表3.2 C. youngae strain E4處理TNT-NFG在好氧環境和厭氧環境中
有無添加胺基氮源之生物急毒性試驗 90
表3.3 C. youngae strain E4處裡培養基的成份說明表並後續進行生
物急毒性試驗 91

圖目錄
圖1.1 TNT化學結構 61
圖1.2 微生物降解TNT好氧環境中的代謝途徑 62
圖1.3 微生物降解TNT厭氧環境中的代謝途徑 63
圖2.1 虎班猛水蚤不同時期胚胎發育之成果 64
圖3.1 不同天數M. Posthuman在TNT土壤濃度 (100、125、150、175
和200 mg/kg) 14D-LC50的死亡率 65
圖3.2 分離菌株之革蘭氏染色圖 (Gram's stain) 66
圖3.3 E4 strain 16SrRNA PCR V6-V9片段膠圖 67
圖3.4 16SrDNA序列菌種親源關係圖 68
圖3.5 API 20E生化特性菌種鑑定 69
圖3.6 在相同的培養條件比較有或沒有添加碳源 (葡萄糖) 對TNT
的降解效能與細菌的生長關係 70
圖3.7 以固定碳源 (葡萄糖) 去除LB (Luria broth) 對TNT的降解
效能 71
圖3.8 評估C. youngae strain E4的接種量對TNT降解效率之影響
72
圖3.9 補充有機胺基氮源對C. youngae strain E4降解TNT與細菌
生長的關係 73
圖3.10 補充無機胺基氮源對C. youngae strain E4降解TNT與細菌
生長的影響 74
圖3.11 補充碳源(葡萄糖)對C. youngae strain E4降解TNT與細菌
生長的影響 75
圖3.12 NemA蛋白之序列比對 76
圖3.13 NemA RT-PCR primer的設計 77
圖3.14 NemA degenerate primer將PCR放大的DNA序列並使用
NCBI data base核對NemA (OYE family) 78
圖3.15 以RT-PCR偵測C. youngae E4的nitroreductase表現量
的差異 79
圖3.16 泥漿相生物降解TNT汙染土以補充有機胺基氮源和
C. youngae strain E4不同時間點的變化 80
圖3.17 偵測TNT的中間代謝產物 81
圖3.18 泥漿相生物降解TNT汙染土以補充碳源(糖蜜) C. youngae
strain E4在不同時間點的變化 82
圖3.19 偵測不同時間點 (24、48、72和168小時) 總細胞數的DNA量
83
圖3.20 TNT-NFG好氧培養之虎班猛水蚤生物急毒性試驗96hr-LC50 84
圖3.21 TNT-NFG厭氧培養有無添加胺基氮源之虎斑猛水蚤生物急毒性試
驗96hr-LC50 85
圖3.22 為TUa毒性簡易標示圖 86
 

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