土壤及地下水被火炸藥類物質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三硝基甲苯 (trinitrotoluene (TNT))已確定或可能具致癌性。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，探討自高雄市汙染TNT土壤分離出Citrobacter sp.，然後將此菌應用於TNT之生物復育之可行性評估，以達到綠色整治之目的。進一步利用系統生物學方法 (功能性基因體和蛋白質體法)，尋找相關重要降解基因/蛋白質和生物反應性基因/蛋白質，決定此菌降解TNT分子機制，可提供未來如何強化此菌對TNT降解重要策略參考。故擬訂三年計劃如下: 第一年建立Citrobacter sp.降解TNT之最佳培養條件、最大容忍濃度、環境因子對降解之影響及好/氧條件下代謝副產物之種類。最後，以生物反應器建立模擬汙染TNT模場，評估Citrobacter sp.降解TNT之能力。接著第二年，針對Citrobacter sp.結合RNA-seq方法 ，研討Citrobacter sp.全基因體定序及在菌種經TNT處理前後，進行其功能性基因體分析所有有表現量差異之基因 (> or = 2 or <2, p<0.05)，利用生物資訊分析者這些表現量有差異基因，可推測菌種降解TNT之可能分子機制。除此之外，RNA-seq結果對於相關降解基因或相關重要生物反應性基因，具有表現量差異基因均以既時定量 PCR 驗證這些基因表現量是否符合RNA-seq結果。第三年進行Citrobacter sp.降解TNT前後之蛋白質體之系統性分析，再結合第二年所獲得功能性基因體數據比較分析後，找出主要作用TNT 之酵素。將此酵素以分子生物法大量表現此酵素，然後純化此酵素。進一步探討主要降解酵素之最適反應酸鹼值與溫度、酵素動力、基質專一性等多種特性;並整合酵素固定化技術以開發具有高處理效率之TNT生物技術整治方法， 本計劃整合微生物學，基因體學，蛋白質體學和生物資訊學應用在生物復育研究，此研究獲得數據，未來可擬定如何加強菌種生物復育能力策略參考，以及針對在汙染環境中許多變數 (如pH，溫度等)，可調控相關降解或重要生物反應性基因或蛋白質表現量，使菌種生長和降解能力表現最佳化。 再者，本計劃所使用分子生物方法特點具有高效率和全面性，可檢測所有基因和蛋白質表現。且基因體和蛋白質體同時 ;Soil and groundwater contaminated by munitions compounds has been a crucial issue in environmental protection. Trinitrotoluene (TNT) has been classified as carcinogens. The aim of this proposal is to evaluate the potential of Citrobacter sp. applied to biodegrade TNT-contaminated soil/water. A total of consecutive three years will be focused on bioremediation as follows. First year, we will establish the optimal conditions for bioremediation by Citrobacter sp., including the maximal tolerance of TNT toxicity by this bacterium, the byproducts of TNT bioconversion, and the application of bioreactor containing contaminated soil/water mixed with Citrobacter sp. Second year, the whole genome of Citrobacter sp.will be established as the reference template due to its lacking genome. Using Citrobacter sp. as the mapping reference, we compared the transcriptome responses in between Citrobacter sp.with and without TNT, which deduce the molecular mechanisms of TNT biodegradation by Citrobacter sp. Additionally, some differential genes related to TNT bioremediation/resistance will be validated by qRT-PCR. Third year, we will perform the proteomic analysis for Citrobacter sp. to biodegrade TNT or not. The combined analysis of proteomic and transcriptome will enable us to find out the key enzyme for bioremediation. After the purification of the enzyme, we determine its specificity, activity at the defined pH, and kinetic studies. Finally, the immobilized enzyme will be evaluated for its potency in enhancing TNT bioremediation.
