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年度	100	專案性質	實驗性質
專案類別	研究專案	研究主題	整治
申請機構	元智大學生	申請系所	物科技與工程研究所
專案主持人	簡志青	職等/職稱	教授
專案中文名稱	三硝基甲苯污染土壤菌株種類之探討與具降解三硝基甲苯潛力菌株之篩選與開發
中文關鍵字	三硝基甲苯、環境生物技術、微生物降解
專案英文名稱
英文關鍵字	Trinitrotoluene, Environmental biotechnology, Microbial biodegradation
執行金額
執行期間	2011/11/26 至 2012/11/25
計畫中文摘要	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，另外戰爭時期遭強烈轟炸的遺址亦可 能有大量三硝基甲苯的殘留，還有工礦爆破以及戰後火藥處理等都是環境遭受三 硝基甲苯污染的來源。遭受三硝基甲苯污染環境包括土壤與水體都會對生物以及 人體的健康造成極大的影響。因此，如何有效的對受污染的環境進行整治亦是一 個在土壤與地下水的環境保護重要的課題。 自然衰減 (Natural attenuation)無法對遭三硝基甲苯污染的環境達到整治的效 果。化學處理法中，利用高級氧化程式 (advanced oxidation processes (AOPs))對含 有不易降解之有機化合物的污染，乃是一具相當潛力的整治技術。在處理三硝基 甲苯污染的方面，則有臭氧氧化法、半導體光催化法、超音波高級氧化法、過氧 化氫氧化(Fenton 法、H2O2 + UV, photo-Fenton 與 Fenton-like 程式)、光觸媒法等。 然而對環境污染進行整治，利用生物復育法仍是屬於對環境最友善的技術。三硝 基甲苯的生物整治技術中，常用的方法有微生物降解(Microbial biodegradation)與植 生復育 (Phytoremediation) 等方法。本研究計畫主旨便是開發利用污染場址中的本 土微生物 (indigenous strains)來進行三硝基甲苯污染土壤處理。 本研究以台灣南部某一處遭受三硝基甲苯污染的場址的土壤為樣本來源，進 行微生物種類之探討，並從該場址之土壤中分離純化具降解三硝基甲苯之本土微 生物，作為後續進行現地整治的生物復育可行性之探討。 本年度的主要研究成 果，包括(1)建立使用變性梯度凝膠電泳(DGGE)直接分析土壤樣本中細菌種類的研究平臺，包括直接萃取土壤樣本總DNA 技術，以聚合酶連鎖反應放大細菌16S rRNA 基因之程式，以及變性梯度凝膠電泳的製備與結果分析；(2) 利用富集培養 方法，從含有三硝基甲苯污染的土壤中篩選與分離培養具降解三硝基甲苯之本土 環境微生物，包括利用三硝基甲苯為生長之唯一氮源之菌株與利用三硝基甲苯為 唯一碳源與氮源之細菌。同時也開始篩選可降解三硝基甲苯之真菌類微生物；(3) 針對篩選出之菌株，進行純化培養與菌株之初步鑑定，並與不經培養方式所獲得 之微生物種類做一比較；(4) 初步完成挑選之優勢菌株的特性分析，包括在實驗室 中培養基的生長情形，使用高效液相層析儀分析培養基中三硝基甲苯的殘留量以 初步評估篩選之本土菌株降解三硝基甲苯之能力。
計畫英文摘要	Trinitrotoluene (TNT) is a secondary explosive widely used both for military and civil purposes all over the world. Residual TNT could be detected as an environmental pollutant both in soil and groundwater due to the release of TNT to the environment from manufacturing or demilitarization facilities and also after munitions firing/detonation or leakage from explosive remnants of war. The presence of TNT in the soil and groundwater has been associated with adverse impacts on biological activities and human communities. Therefore, Control and remediation of TNT-containing products in soil and groundwater are critical issues in environmental protection. Regarding to the approaches for the remediation of TNT contamination, natural attenuation appears not effective enough. Chemical processes for TNT removal such as advanced oxidation processes (AOPs) appears to be one of the most promising methods for the treatment of soils and waters contaminated with non-easily biodegradable organic compound including nitroaromatic compounds such as TNT. Some AOPs techniques concerning the degradation of TNT include processes based on ozone; processes based on hydrogen peroxide (H2O2 + UV, Fenton, photo-Fenton and Fenton-like processes); photocatalysis etc. However, bioremediation is the most environmental friendly strategy for the treatment of environmental contamination. Biodegradation/biotransformation of TNT can be approached by microbial bioremediation and phytoremediation. Among them, bioremediation of TNT by microbial degradation is a very promising technique. The aim of this project is therefore to establish the platform for the study and monitoring the microbial diversity in the soil and wastewater contaminated with TNT. In this study, we evaluated microbial diversity of a soil sample obtained from southern Taiwan which was contaminated with high concentration of TNT. Indigenous microorganisms which can degrade/utilize TNT were also isolated and characterized and their potential for using in bioremediation of TNT also evaluated. The main goals of this study include (1) Establish platform for studying microbial diversity of TNT-contaminated soil by culture independent techniques (i.e. 16S rRNA gene analysis and DGGE); (2) Isolation of TNT-degrading microorganisms from TNT contaminated soil by enrichment culture techniques; (3) Cultivation and identification of the isolated strains and (4) Characterization of TNT-degrading isolates and evaluate TNT degradation efficient by these microorganisms.