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年度	102	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	申請系所	生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	開發生物復育技術整治受三硝基甲苯污染之土壤-實驗模場研究
中文關鍵字	三硝基甲苯、生物復育、本土微生物
專案英文名稱	Process development on bioremediation of trinitrotoluene contaminated soils by indigenous microorganisms by lab pilot-scale study
英文關鍵字	Trinitrotoluene; bioremediation; Indigenous microorganisms
執行金額
執行期間	2012/12/10 至 2013/12/9
計畫中文摘要	土壤及地下水(以下簡稱土水)被火炸藥類物質所污染是一個長久以來被嚴重 忽視的問題。由於部分火炸藥類物質[例如三硝基甲苯(trinitrotoluene (TNT)，環三 亞甲基三硝胺(hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)，環四亞甲基四硝胺 (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) (HMX)]已確定或可能具致癌性，因 此許多歐美國家均已訂出此類火炸藥類物質在土水之管制標準，並積極進行火炸 藥類物質污染場址之整治。環保署目前亦已著手研訂土水之火炸藥類物質管制標 準，並將於近期公告施行。屆時火炸藥類物質之土水污染場址均需提出污染改善 或整治策略。目前國內針對火炸藥水體與土壤污染的處理技術主要還是使用傳統 的直接焚燒法，在高溫下燃燒，達到氧化分解有毒物質的目的。但是其價格昂貴 且其燃燒後的二次污染物將對生態及人體健康產生影響。因此，發展經濟有效且 環境友善之火炸藥類物質污染土水整治技術有其必要性。在綠色整治(green remediation)之原則下，污染場址之調查與整治規劃必須有著和傳統不同之思維。 在整治技術方面，應優先採用生物處理為主之整治系統，即儘可能減少採用物化 處理為主之整治系統。本計畫之主要目的即是針對火炸藥類物質污染場址中最具 代表性之 TNT 為目標污染物，探討本土分離之微生物，應用於 TNT 之生物復育 之可行性評估，以達到綠色整治之目的。 本研究延續上一期的成果，對篩選分離之本土 TNT 分解微生物進行完整之分 析探討，主要將著重於前一期所篩選之本土菌株對 TNT 降解效率的評估，同時也 將前期所建立之 DGGE 監測環境微生物多樣性之平台應用於實驗室模場規模 TNT 污染土壤生物復育研究過程中，微生物族群狀態之觀測。研究執行內容與成果包 括(1)本土分離菌株對 TNT 分解之探討；(2)環境樣本之 TNT 標準方法檢測平台技 術之建立：利用 USEPA method 8330A 與環保署公佈之「火炸藥物質檢測方法－高 效液相層析儀/紫外光偵測器法」(NIEA M804.00B)之檢測方法，建立 TNT 污染土 壤中殘餘之 TNT 濃度檢測之標準流程； (3) 使用高效液相層析儀建立檢測一些TNT 代謝的可能中間產物的方法，包括苯、甲苯與二甲苯的檢測；(4) 使用醱酵技 術大量培養本土 TNT 分解菌株；(5) 使用實驗室批次規模進行本土菌株對三硝基 甲苯污染之生物復育評估以及(6) 使用變性梯度凝膠電泳(DGGE)直接分析土壤樣 本中細菌種類，探討 TNT 污染土壤在降解過程中微生物相之變化。這些研究初步 成果將有助於我們未來規劃可應用於三硝基甲苯污染場址的原址生物復育之條件 與使用之菌株。 初步成果顯示可降解 TNT 本土菌株的多樣性，亦分離純化並收 藏數株可應用於 TNT 降解之菌種。研究結果亦建議生物刺激法搭配原生菌株可達 成 TNT 之生物降解。
計畫英文摘要	Soil and groundwater contaminated by munitions compounds has been a crucial issue in environmental protection. These compounds including trinitrotoluene (TNT), hexahydro-1,3,5- trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro -1,3,5,7-tetrazocine (HMX) are reported as highly toxic and carcinogenic. Therefore, control and remediation of contamination of nitroaromatic compounds including TNT are very critical issues in environmental protection in Europe and United States. Control and regulation standard of these munitions compounds have also been reviewed by Taiwan Environmental Protection Administration and will be announced recently. Proposal for the remediation of the contaminated sites will then be required. Compared to physical or chemical processes, biological remediation (microbial bioremediation and phytoremediation) are more economy and environmental friendly approaches and also more acceptable by general publics. In this study, we aim to develop bioremediation approaches for TNT by microbial degradation with indigenous microorganisms isolated from TNT contaminated soil and evaluate the feasibility of bioremediation for TNT pollution using these microorganisms. In the present study, we continued to characterize the indigenous microbial isolates and evaluated their ability in TNT biodegradation. Micorbial diversity (microbial population) of TNT contaminated soil was also examined by Denaturing Gradient Gel Electrophoresis (DGGE). The main goals and the results obtained from this project included (1) Evaluation of TNT biodegradation and biotransformation by indigenous isolates; (2) Establish the method for detection of TNT residues in the soil using High performance Liquid Chromatography (HPLC) according to USEPA method 8330A and NIEA M804.00B by EPA, Taiwan; (3) Development and evaluation of the methods for detection of intermediates produced by microbial metabolism of TNT as well as benzene, toluene and xylene by HPLC; (4)Cultivation of TNT-degrading bacterium using batch culture technique by Lab scale fermenter; (5) Development and evaluation of bioremediation of trinitrotoluene contaminated soils by indigenous microorganisms with lab-scale model; (6) Examination of microbial diversity during bioremediation of TNT contaminated soil in lab by DGGE. The preliminary results obtained from this study will help us to evaluate and propose schemes for in situ bioremediation of TNT contaminated site in the fuure. Preliminary results indicated the diversity of TNT-degrading microorganisms. We have isolated and collected several strains of indigenous TNT-degrading microorganisms in our culture collection. The results also suggest that combination of biostimulation and bioaugmentation is a feasible approach for development of bioremediation of TNT pollution.