環保專案成果報告查詢系統

專案計畫編號	EPA-97-U1G1-02-103
經費年度	097
計畫經費	1,929 千元
專案開始日期	2008/04/03
專案結束日期	2009/04/02
專案主持人	陳士賢
主辦單位	土污基管會
承辦人	王禎
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	石化污染, 生物復育, 甲基第三丁基醚, 多環芳香族碳氫化合物, 甲基第三丁基醚分解菌, 蚯蚓
英文關鍵字	Petroleum hydrocarbon contamination, bioremediation, methyl tert butyl ether(MTBE), polynuclear aromatic hydrocarbons(PAHs), MTBE-degrading bacteria, earthworm
協同主持人	陳師慶,梁世雄
共同主持人
計畫聯絡人	陳士賢
計畫聯絡電話	076051016
計畫聯絡信箱	cschen@nknucc.nknu.edu.tw

中文摘要	本計畫發展受石化產品污染之土壤及地下水場址生物復育技術，目標為完成石化污染場址土壤及地下水之菌種菌相分析，建立本土分解石化污染物之菌種資料庫，及應用蚯蚓於生物復育之可行性探討，及其分解石化污染物之效率研究，作為石化污染場址進行生物復育之參考依據。本研究利用denaturing gradient gel electrophoresis (DGGE)，以微生物的16S rDNA為標的，分析中國石油公司橋頭油庫具有分解石油碳氫化合物(petroleum hydrocarbons)的微生物，並用real-tme PCR分析與分解BTEX（苯、甲苯，二甲苯與乙苯）有關的六種基因(phe、rmo、nah、tod、tol和bph4)，探討污染場址之生物降解可能性。MTBE (methyl tertiary butyl ether)已知在石油污染物中是較難被微生物分解利用，本研究以培養方式自污染場址分離具分解MTBE的微生物。DGGE結果顯示其微生物菌相會隨著石油碳氫化合物的污染程度增加而減少，且地下水微生物菌相以β-Proteobacteria中Burkholderia菌屬及δ-Proteobacteria中Geobacteraceae菌科為可能分解石油碳氫化合物的微生物。從real-tme PCR分析結果顯示有遭受石油碳氫化合物污染的地下水，皆具有phe基因，可得知該地下水中的微生物可能具有分解BTEX的能力。在利用culture的方式分離具分解MTBE的微生物方面，分離並純化出五株菌株（Pseudomonas sp. NKNU01、Bacillus sp. NKNU01、Klebsiella sp. NKNU01、Enterobacter sp. NKNU01與Enterobacter sp. NKNU02），並將五株菌株個別與混合做MTBE降解試驗，結果顯示三星期內混合菌株降解率32％明顯高於單一菌株（Pseudomonas sp. NKNU01降解率16％、Bacillus sp. NKNU01降解率15％、Klebsiella sp. NKNU01降解率20％、Enterobacter sp. NKNU01降解率20％與Enterobacter sp. NKNU02降解率25％）。以蚯蚓發展石化污染土壤之環境復育技術部份，經耐受實驗結果選定Eisenia fetida(紅蚯蚓)和Perionyx excavatu(掘穴環爪蚓)此兩種蚯蚓作為復育多環狀芳香族碳氫化合物(polybuclear aromatic hydrocarbons, PAHs)實驗物種。比較兩種蚯蚓復育PAHs成效，並無明顯差異，實驗結果顯示柴油污染土壤的最佳復育天數為60天，可減少PAH濃度為100 mg/kg的柴油污染土壤中90%以上PAHs，比較120日復育期程後，掘穴環爪蚓在低及中柴油污染土壤中存活率較紅蚯蚓為高，但紅蚯蚓在高柴油污染土壤有較高存活率，顯示其繁殖後代使存活率回升。蚯蚓復育PAHs結果發現土壤若受柴油污染後，土壤所吸附之PAHs濃度將隨時間自然衰減，其衰減程度將視場址物化條件及是否有PAH分解菌等條件影響，但蚯蚓存在與運動行為，將有助於減低污染土壤之PAH濃度，因此可提高PAHs之生物降解。以本計畫實驗設計柴油高污染土壤為例，蚯蚓可在TPH濃度數萬至十萬(mg/kg)環境下協助PAHs減量，此點可作為設計蚯蚓對石化污染場址整治時決策判定的參考依據。因此若於柴油污染土壤，在蚯蚓可耐受的污染物濃度範圍，提供適合蚯蚓生存的因子(如濕度、堆肥)，添加蚯蚓有助提高復育之能力，並加速去除土壤中之污染物質PAHs，同時使用蚯蚓復育可考慮搭配其他生物復育方法，並作為國內為數眾多的石化污染場址進行現地或離地強化性生物復育工具是適切可行。本計畫探討微生物篩選及蚯蚓對石化污染物降解，透過篩選MTBE降解優勢菌種及蚯蚓對PAHs減量成效，判定石化污染場址中污染源減量，建立國立石化污染場址菌種與無脊椎動物資料庫，以促進國際資訊交換及與國內土壤及地下水整治業者知識分享，藉此整合資源並提供石化污染場址復育最佳決策依據。篩選出五種MTBE分解菌及評估適合PAH分解潛勢之蚯蚓，此五株菌株已申請存放在新竹財團法人食品工業發展研究所，未來可提供學界、產業界與土壤及地下水整治業者，應用於國內為數眾多之石化污染場址(如加油站、石化工業區、軍事儲槽設施)進行生物復育施作。
中文關鍵字	石化污染, 生物復育, 甲基第三丁基醚, 多環芳香族碳氫化合物, 甲基第三丁基醚分解菌, 蚯蚓

中文摘要

本計畫發展受石化產品污染之土壤及地下水場址生物復育技術，目標為完成石化污染場址土壤及地下水之菌種菌相分析，建立本土分解石化污染物之菌種資料庫，及應用蚯蚓於生物復育之可行性探討，及其分解石化污染物之效率研究，作為石化污染場址進行生物復育之參考依據。本研究利用denaturing gradient gel electrophoresis (DGGE)，以微生物的16S rDNA為標的，分析中國石油公司橋頭油庫具有分解石油碳氫化合物(petroleum hydrocarbons)的微生物，並用real-tme PCR分析與分解BTEX（苯、甲苯，二甲苯與乙苯）有關的六種基因(phe、rmo、nah、tod、tol和bph4)，探討污染場址之生物降解可能性。MTBE (methyl tertiary butyl ether)已知在石油污染物中是較難被微生物分解利用，本研究以培養方式自污染場址分離具分解MTBE的微生物。DGGE結果顯示其微生物菌相會隨著石油碳氫化合物的污染程度增加而減少，且地下水微生物菌相以β-Proteobacteria中Burkholderia菌屬及δ-Proteobacteria中Geobacteraceae菌科為可能分解石油碳氫化合物的微生物。從real-tme PCR分析結果顯示有遭受石油碳氫化合物污染的地下水，皆具有phe基因，可得知該地下水中的微生物可能具有分解BTEX的能力。在利用culture的方式分離具分解MTBE的微生物方面，分離並純化出五株菌株（Pseudomonas sp. NKNU01、Bacillus sp. NKNU01、Klebsiella sp. NKNU01、Enterobacter sp. NKNU01與Enterobacter sp. NKNU02），並將五株菌株個別與混合做MTBE降解試驗，結果顯示三星期內混合菌株降解率32％明顯高於單一菌株（Pseudomonas sp. NKNU01降解率16％、Bacillus sp. NKNU01降解率15％、Klebsiella sp. NKNU01降解率20％、Enterobacter sp. NKNU01降解率20％與Enterobacter sp. NKNU02降解率25％）。以蚯蚓發展石化污染土壤之環境復育技術部份，經耐受實驗結果選定Eisenia fetida(紅蚯蚓)和Perionyx excavatu(掘穴環爪蚓)此兩種蚯蚓作為復育多環狀芳香族碳氫化合物(polybuclear aromatic hydrocarbons, PAHs)實驗物種。比較兩種蚯蚓復育PAHs成效，並無明顯差異，實驗結果顯示柴油污染土壤的最佳復育天數為60天，可減少PAH濃度為100 mg/kg的柴油污染土壤中90%以上PAHs，比較120日復育期程後，掘穴環爪蚓在低及中柴油污染土壤中存活率較紅蚯蚓為高，但紅蚯蚓在高柴油污染土壤有較高存活率，顯示其繁殖後代使存活率回升。蚯蚓復育PAHs結果發現土壤若受柴油污染後，土壤所吸附之PAHs濃度將隨時間自然衰減，其衰減程度將視場址物化條件及是否有PAH分解菌等條件影響，但蚯蚓存在與運動行為，將有助於減低污染土壤之PAH濃度，因此可提高PAHs之生物降解。以本計畫實驗設計柴油高污染土壤為例，蚯蚓可在TPH濃度數萬至十萬(mg/kg)環境下協助PAHs減量，此點可作為設計蚯蚓對石化污染場址整治時決策判定的參考依據。因此若於柴油污染土壤，在蚯蚓可耐受的污染物濃度範圍，提供適合蚯蚓生存的因子(如濕度、堆肥)，添加蚯蚓有助提高復育之能力，並加速去除土壤中之污染物質PAHs，同時使用蚯蚓復育可考慮搭配其他生物復育方法，並作為國內為數眾多的石化污染場址進行現地或離地強化性生物復育工具是適切可行。本計畫探討微生物篩選及蚯蚓對石化污染物降解，透過篩選MTBE降解優勢菌種及蚯蚓對PAHs減量成效，判定石化污染場址中污染源減量，建立國立石化污染場址菌種與無脊椎動物資料庫，以促進國際資訊交換及與國內土壤及地下水整治業者知識分享，藉此整合資源並提供石化污染場址復育最佳決策依據。篩選出五種MTBE分解菌及評估適合PAH分解潛勢之蚯蚓，此五株菌株已申請存放在新竹財團法人食品工業發展研究所，未來可提供學界、產業界與土壤及地下水整治業者，應用於國內為數眾多之石化污染場址(如加油站、石化工業區、軍事儲槽設施)進行生物復育施作。

中文關鍵字

石化污染, 生物復育, 甲基第三丁基醚, 多環芳香族碳氫化合物, 甲基第三丁基醚分解菌, 蚯蚓

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-以微生物及蚯蚓發展石化污染土壤之環境復育技術.pdf	3MB	[期末報告]公開完整版	2009/8/1 上午 12:00:00

摘要	The objectives of this research are to: (1) screen and identify gasoline oxygenate (i.e., methyl tert butyl ether (MTBE)) degrading microorganisms from contaminated sites by denaturing gradient gel electrophoresis (DGGE), detect and quantify bacteria that are closely associated with degradation of MTBE by quantitative real-time polymerase chain reaction (PCR), (2) conduct feasibility study of using earthworm to remediate diesel contaminated soil and study effectiveness of ex-situ treatment of diesel contaminated soils at presence of earthworm for cleanup of diesel contaminated soils. The 16S rDNA-denaturing gradient gel electrophoresis (DGGE) was performed to identify the petroleum-hydrocarbons-degrading bacteria in the contaminated ground water. Realtme PCR was performed to provide biodegradation ability by analyzing six genes (phe, rmo, nah, tod, tol and bph4) connected with BTEX (benzene, toluene, ethylbenzene, xylenes) degradation. It has proved difficult to bacteria using MTBE as sole cabon and energy source, so a methyl tertiary butyl ether (MTBE)-metabolizing bacteria were cultured, and then identified with their separate 16S rDNA sequences. All of these sequences were blasted with NCBI database. The significance of this study is to enhance the efficacy of petroleum bioremediation with the establishment of databank of petroleum-hydrocarbons-degrading bacteria. Results for DGGE showed microbial community reduce as the pollution increase and Burkholderia and Geobacteraceae are the bacteria could degradate petroleum hydrocarbons. PHE gene was detected by realtme PCR in petroleum hydrocarbons-impacted wells could indicate the ability for biodegradation. Five bacteria (Pseudomonas sp. NKNU01, Bacillus sp. NKNU01, Klebsiella sp. NKNU01, Enterobacter sp. NKNU01 and Enterobacter sp. NKNU02) were cultured, and then proceeded with MTBE-metabolizing by single bacteria and mix culture. Results for MTBE-metabolizing show the degradation rate and degradation percent for mix culture (32%) was higher than single bacteria (Pseudomonas sp. NKNU01, 16%; Bacillus sp. NKNU01, 15%; Klebsiella sp. NKNU01, 20%; Enterobacter sp. NKNU01, 20% and Enterobacter sp. NKNU02, 25%) during three weeks. In order to assess applicability of using earthworms to degrade polynuclear aromatic hydrocarbons (PAHs), batch experiments were conducted. Two species of earthworm (Eisenia fetida and Perionyx excavatu) were adopted to degrade diesel-contaminated soils for exposure time of 120 days. The total PAHs concentration is around 25, 50, and 100 mg/kg in diesel contaminated soils, respectively. Removal efficiency of PAHs was evaluated after 120 day incubation. The results indicated that removal efficiency of PAHs was compatible among two species. After 60 day exposure, total PAH was reduced to less than 10% in soils of all contaminant levels. The results of control sets illustrated that PAHs may also be degraded by microorganisms in contaminated soils. Bioaccumulation of PAHs in earthworm tissues was not observed in this study. It is believed that microorganisms can assist the degradation of PAHs. Given the proper conditions such as proper humidy, nutrients, to allow earthworms to survive in the contaminated, earthworm can be used for bioremediation of contaminated soil. This study focused on tha potential options of bioremediation. Bacteria community in petroleum hydrocarbon contaminated site was observed and potential MTBE-metabolizing bacteria was evaluated. Applicability of earthworms for remediating PAHs was feasible, in particular, for enhanced bioremediation. As a result, bioremediation technologies will be recommended for cleanup of petroleum contaminated soils depending on their contamination levels, composition of contaminants, soil characteristics and climate.
英文關鍵字	Petroleum hydrocarbon contamination, bioremediation, methyl tert butyl ether(MTBE), polynuclear aromatic hydrocarbons(PAHs), MTBE-degrading bacteria, earthworm

摘要

The objectives of this research are to: (1) screen and identify gasoline oxygenate (i.e., methyl tert butyl ether (MTBE)) degrading microorganisms from contaminated sites by denaturing gradient gel electrophoresis (DGGE), detect and quantify bacteria that are closely associated with degradation of MTBE by quantitative real-time polymerase chain reaction (PCR), (2) conduct feasibility study of using earthworm to remediate diesel contaminated soil and study effectiveness of ex-situ treatment of diesel contaminated soils at presence of earthworm for cleanup of diesel contaminated soils. The 16S rDNA-denaturing gradient gel electrophoresis (DGGE) was performed to identify the petroleum-hydrocarbons-degrading bacteria in the contaminated ground water. Realtme PCR was performed to provide biodegradation ability by analyzing six genes (phe, rmo, nah, tod, tol and bph4) connected with BTEX (benzene, toluene, ethylbenzene, xylenes) degradation. It has proved difficult to bacteria using MTBE as sole cabon and energy source, so a methyl tertiary butyl ether (MTBE)-metabolizing bacteria were cultured, and then identified with their separate 16S rDNA sequences. All of these sequences were blasted with NCBI database. The significance of this study is to enhance the efficacy of petroleum bioremediation with the establishment of databank of petroleum-hydrocarbons-degrading bacteria. Results for DGGE showed microbial community reduce as the pollution increase and Burkholderia and Geobacteraceae are the bacteria could degradate petroleum hydrocarbons. PHE gene was detected by realtme PCR in petroleum hydrocarbons-impacted wells could indicate the ability for biodegradation. Five bacteria (Pseudomonas sp. NKNU01, Bacillus sp. NKNU01, Klebsiella sp. NKNU01, Enterobacter sp. NKNU01 and Enterobacter sp. NKNU02) were cultured, and then proceeded with MTBE-metabolizing by single bacteria and mix culture. Results for MTBE-metabolizing show the degradation rate and degradation percent for mix culture (32%) was higher than single bacteria (Pseudomonas sp. NKNU01, 16%; Bacillus sp. NKNU01, 15%; Klebsiella sp. NKNU01, 20%; Enterobacter sp. NKNU01, 20% and Enterobacter sp. NKNU02, 25%) during three weeks. In order to assess applicability of using earthworms to degrade polynuclear aromatic hydrocarbons (PAHs), batch experiments were conducted. Two species of earthworm (Eisenia fetida and Perionyx excavatu) were adopted to degrade diesel-contaminated soils for exposure time of 120 days. The total PAHs concentration is around 25, 50, and 100 mg/kg in diesel contaminated soils, respectively. Removal efficiency of PAHs was evaluated after 120 day incubation. The results indicated that removal efficiency of PAHs was compatible among two species. After 60 day exposure, total PAH was reduced to less than 10% in soils of all contaminant levels. The results of control sets illustrated that PAHs may also be degraded by microorganisms in contaminated soils. Bioaccumulation of PAHs in earthworm tissues was not observed in this study. It is believed that microorganisms can assist the degradation of PAHs. Given the proper conditions such as proper humidy, nutrients, to allow earthworms to survive in the contaminated, earthworm can be used for bioremediation of contaminated soil. This study focused on tha potential options of bioremediation. Bacteria community in petroleum hydrocarbon contaminated site was observed and potential MTBE-metabolizing bacteria was evaluated. Applicability of earthworms for remediating PAHs was feasible, in particular, for enhanced bioremediation. As a result, bioremediation technologies will be recommended for cleanup of petroleum contaminated soils depending on their contamination levels, composition of contaminants, soil characteristics and climate.

英文關鍵字

Petroleum hydrocarbon contamination, bioremediation, methyl tert butyl ether(MTBE), polynuclear aromatic hydrocarbons(PAHs), MTBE-degrading bacteria, earthworm