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年度	99	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	國立高雄師範大學	申請系所	生物科技系(所)
專案主持人	陳士賢	職等/職稱	教授
專案中文名稱	應用生物反應槽進行石化污染物之生物復育研究
中文關鍵字	生物復育；石油碳氫化合物；生物反應槽
專案英文名稱	Bioremediation of petroleum hydrocarbon contaminants by bioreactor
英文關鍵字	Bioremediation；Petroleum hydrocarbon；Bioreactor
執行金額
執行期間	2010/12/29 至 2011/12/28
計畫中文摘要	甲基第三丁基醚(Methyl tert-butyl ether, MTBE)於 1990 年代開始廣泛使用， 作為提高汽油辛烷值的含氧添加劑，由於具可水溶性及高揮發性；因此在地下水 層與地面水間具有高移動性，且可藉由揮發作用污染到地表土壤與底泥。 本研究主要探討利用來自石化污染場址中所分離的菌種(Pseudomonas sp. NKNU01, Bacillus sp. NKNU01, Klebsiella sp. NKNU01, Enterobacter sp. NKNU01, Enterobacter sp. NKNU02)，進行 MTBE 生物降解實驗，並找出最具有分解 MTBE 潛力菌種及其降解途徑。分別利用批次試驗求得單一菌種及混合菌種將 MTBE 當作碳源之降解能力、共代謝物質對微生物降解 MTBE 情形的影響。使用生物 反應槽利用批次試驗所得實驗條件進行提高 MTBE 濃度降解試驗。分解途徑以 蛋白質體技術進行鑑定，針對二維電泳蛋白質表現量差異兩倍以上蛋白質點，再 利用基質輔助雷射脫附-飛行時間質譜法(MALDI-TOF MS)分析與 PMFs(peptide mass fingerprints)資料庫比對鑑定。 在批次實驗中，混合菌加入共代謝物質降解效果為最佳，可降解 MTBE 約 達 64%，在單一菌種則是 Bacillus sp. NKNU01 為最佳，可降解 MTBE 約 21%； 共代謝物質挑選，屬正戊烷(Pentane)具有最佳效果。另外，不添加共代謝物質試 驗中，發現 Enterobacter sp. NKNU02 降解 MTBE 效率約達 29%較添加共代謝物 質時效率佳，且屬單一菌種試驗中具有最佳降解 MTBE 潛力菌種。 生物反應槽試驗結果顯示 Enterobacter sp. NKNU02 可於不添加共代謝物質 之實驗分解 MTBE 約達 56%；另一方面加入共代謝物試驗中，Bacillus sp. NKNU01 與 Klebsiella sp. NKNU01 降解 MTBE 皆約達 22%，與批次實驗相較之 下皆有顯著差異，故推測利用生物反應槽可促進生物降解效率提升。 結果顯示 Enterobacter sp. NKNU02 為具有最佳分解 MTBE 潛力菌種，蛋白 質 鑑 定 其 代 謝 蛋 白 包 含 alcohol dehydrogenase 、 phospho- glyceromutase 、 transaldolase 與 isocitrate dehydrogenase；另外利用 GC/MS 進行定性分析，推估 出具可能性之代謝產物，如 acetic acid、2-propenoic acid 與 2-propanol。實驗發 現 Enterobacter sp. NKNU02 進行 MTBE 分解時，不經過第三丁醛(tert-butyl formate, TBF)轉化，而直接氧化生成第三丁醇(tert-butyl alcohol, TBA)進而生成2-Propanol 與 Lactate 再進入 TCA cycle，完成 MTBE 代謝作用。 降解實驗添加 BTEX 以模擬汽油發生洩漏，結果發現 Enterobacter sp. NKNU02 分解 MTBE 會受 BTEX 所抑制，使降解率約減為 16%。然而，BTEX 亦會被 Enterobacter sp. NKNU02 分解，甲苯被分解效果為最佳約有 36%，苯約 有 32%，故此菌種對其他汽油污染物亦具有降解效用。本研究結果可應用提升汽 油污染場址之生物復育可能性。
計畫英文摘要	Methyl tert-butyl ether (MTBE) has been the most commonly used high octane additive to gasoline since 1990. The compound is partially water soluble and moderately volatile; thus, it is highly mobile in both groundwater and surface water and can be volatilized to contaminate the vadose zone, surface soils, and sediments. However, biological treatment of MTBE-contaminated groundwater appears to be the most economical, energy efficient, and environmentally sound approach. The objective of this reach was to investigate the biodegradation potential of MTBE by the microorganisms specified (Pseudomonas sp., Bacillus sp., Klebsiella sp., Enterobacter sp.) at a petroleum contaminated site. It was intended to evaluate the pure culture with the best ability of biodegradability of MTBE and to evaluate the biodegradation pathway for these microorganisms. MTBE can be metabolized by bacteria either as a primary carbon source, or cometabolized when bacterial growth requires another substrates at the batch experiments involving either mixed or pure cultures. The bioreactor with conditions obtained from the batch experiment was applied to enhance degradation of MTBE. The metabolic pathways and cellular responses of these microorganisms during growth on MTBE were studied through proteomics approach. Protein spots of interest were identified through database searching according to peptide mass fingerprints (PMFs) obtained using matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The pentane was used as the cometabolic chemical to enhance MTBE degradation. On the other hand, the pure culture (Enterobacter sp. NKNU02) shown the best degradation potential about 29 % of MTBE without adding pentane. Enterobacter sp. NKNU02 could degrade about 56 % of MTBE without adding pentane. Bacillus sp. NKNU01 and Klebsiella sp. NKNU01 could degrade about 22 % of MTBE with adding pentane. Comparing with the batch experiments, bioreactor could enhance MTBE degradation significantly. Four metabolic enzymes may involve alcohol dehydrogenase, phosphorglyceromutase, transaldolase, and isocitrate dehydrogenase. Assessing the potential products of MTBE degradation by gas chromatograph/mass spectrometer(GC/MS) involved acetic acid, 2-propenoic acid, and 2-propanol. Enterobacter sp. NKNU02 followed the pathway in the initial steps of MTBE degradation, without converting MTBE to tert-butyl formate, which was directly hydrolysed to tert-butyl alcohol, and then transformed 2-propanol and lactate to the TCA cycle (tricarboxylic acid cycle). MTBE and BTEX (benzene, toluene, ethylbenzene, and xylenes) can coexist in gasoline-contaminated groundwater, and MTBE-degradability of Enterobacter sp. NKNU02 could reduce about 16% of MTBE but inhibited by BTEX. However, it could also degrade BTEX including 36% of toluene and 32% of benzene. The effectiveness of bioremediation of MTBE will be assessed for potential field-scale application.