環保專案成果報告查詢系統

專案計畫編號	EPA-99-GA103-03-A236-1
經費年度	099
計畫經費	994 千元
專案開始日期	2010/12/29
專案結束日期	2011/12/28
專案主持人	陳士賢
主辦單位	土污基管會
承辦人	尤衍翔
執行單位	國立高雄師範大學
專案分類	土壤及地下水
中文關鍵字	甲基第三丁基醚、生物降解、生物反應槽、共代謝作用、正戊烷
英文關鍵字	Methyl tert-butyl ether, biodegradation, bioreactor, cometabolism, pentane
協同主持人	陳師慶
共同主持人
計畫聯絡人	陳士賢
計畫聯絡電話	07-7172930#7312
計畫聯絡信箱	cschen@nknu.edu.tw

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。本研究結果可應用於提昇汽油污染場址之生物復育成效。
中文關鍵字	甲基第三丁基醚、生物降解、生物反應槽、共代謝作用、正戊烷

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。本研究結果可應用於提昇汽油污染場址之生物復育成效。

中文關鍵字

甲基第三丁基醚、生物降解、生物反應槽、共代謝作用、正戊烷

類型	檔名	檔案大小	說明	最新版公開日期
期末報告	期末報告.pdf	2MB		2012/2/1 上午 12:00:00

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, phosphor- glyceromutase, 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.
英文關鍵字	Methyl tert-butyl ether, biodegradation, bioreactor, cometabolism, pentane

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, phosphor- glyceromutase, 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.

英文關鍵字

Methyl tert-butyl ether, biodegradation, bioreactor, cometabolism, pentane