土壤被汽油、柴油及燃料油等油品(petroleum hydrocarbons)所污染是一個愈趨普遍且嚴重的問題。油污染之主要來源包括地下儲油槽(underground storage tanks, UST)漏油、油管破裂、及地面油品意外洩漏等。由於油品中之主要組成均可被生物分解去除，因此生物分解作用是油污染場址中造成污染物降解之最主要機制。因此，以現地自然或加強式(in situ natural or enhanced bioremediation)的生物分解法處理油污染之土壤是最經濟可行的方式。有時若因污染物在土壤中之濃度較高或整治時程等問題，亦可採用整合型復育技術或是整治列車(treatment train)之概念作為油污染土壤之整治概念，即應用界面活性劑之沖排或化學氧化技術為第一階段之整治方法，再以生物整治法進行第二階段之處理，已符合經濟及整治成效。
監測現地微生物族群及數量之變化和優勢菌群(dominant bacteria)之消長是評估生物分解及整治成效的有效方法之一。而分子生物之監測技術即是一種可行之應用方式。本研究之目的包括三個主要部份：(1)建立16S rDNA之分析技術，並以此分析技術評估微生物菌相在油污染場址之變化，(2)評估添加營養鹽對土壤中微生物菌相及優勢菌群之影響，及(3)評估以界面活性劑Simple GreenTM (SG)或類芬頓(Fenton like)氧化技術為油污染土壤整治之第一階段之整治方法時，SG及氧化劑對土壤中微生物菌相及優勢菌群之影響。本研究利用PCR (polymerase chain reaction, 聚合酶連鎖反應)與DGGE (denaturing gradient gel electrophoresis, 變性梯度膠泳)技術作為主要分析方法，探討不同環境及整治條件下微生物之菌相呈現，透過微生物16S rDNA (deoxyribonucleic acid, 去氧核糖核酸)基因序列的比對方式，針對上述樣品進而探討出完整的微生物消長情形。
在油污染場址監測樣品部份，屬於污染濃度程度較低的土壤菌相相似度有97%，屬於污染濃度程度較高的相似度則有84%，兩不同分類間相似度僅48%，而本樣品中定序(sequence)後的優勢菌種為Pseudomonas sp與Herbaspirillum sp.。除得知現地污染程度不同造成菌相有所差異外，此兩種菌群為可直接或間接降解石油碳氫化合物之微生物。
添加營養鹽以促進生物處理，與過氧化氫作Fenton-like法對石油碳氫化合物行氧化作用，就背景土壤與添加營養鹽兩樣品，其菌相相似度僅達70％，從有無添加過氧化氫則分為兩族群，此兩族群相似度僅有53％。若是在添加過氧化氫狀況下，有無添增營養鹽的相似度則僅達54%，最後添加過氧化氫此族群與背景土壤的菌相比較，則其相似度僅有28.62％。使用界面活性劑作沖排以增加降解效率部份，不同濃度(0.1 wt% , 0.01 wt%)造成的菌相差異其相似度達97%，與背景土讓之相似度則為85%。其定序而得之優勢菌種為Pseudomonas sp及Shewanella sp.。過氧化氫對於現地微生物會有相當程度影響，使之菌相有相當幅度變動。界面活性劑則因其為非離子型生物可分解之特質，對背景微生物較無影響。

關鍵字：PCR、DGGE、石油碳氫化合物

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目錄
頁次
摘要 I
謝誌 II
目錄 III
圖目錄 V
表目錄 VI
第一章 前言 1
1.1 研究緣起 1
1.2 研究目的 1
第二章 文獻回顧 2
2.1 石油碳氫化合物 2
2.2 自然衰減 3
2.3 PCR-DGGE分子生物技術的應用 4
2.4 不同環境條件下菌相呈現 6
第三章 材料與方法 15
3.1 樣品來源敘述 15
3.1.1 石油碳氫化合物污染場址監測 15
3.1.2 整治列車概念處理燃料油 17
3.2 Genomic DNA萃取 19
3.3 Genomic DNA瓊脂膠體電泳檢視及濃度測定 20
3.4 PCR 21
3.5 PCR產物純化與濃縮 23
3.6 DGGE 24
3.7 SYBR green I 染色與膠體拍攝與分析統計 25
3.8 基因定序及親緣關係樹 25
第四章 結果與討論 26
4.1 石油碳氫化合物污染場址監測 26
4.1.1 場址樣品genomic DNA萃取與聚合酶連鎖反應 26
4.1.2 場址樣品變性梯度膠體電泳結果 28
4.1.3 場址樣品菌相相似度分析 30
4.1.4 場址樣品基因定序及親緣關係樹 32
4.2整治列車概念處理燃料油 36
4.2.1 樣品genomic DNA萃取與聚合酶連鎖反應 36
4.2.2 整治列車各樣品變性梯度膠體電泳結果 39
4.2.3 整治列車各樣品菌相相似度分析 40
4.2.4 整治列車各樣品中界面活性劑組基因定序及親緣關係樹 45
第五章 結論與建議 49
5.1 結論 49
5.2 建議 49
參考文獻 51
附錄 樣品定序序列






圖目錄
頁次
圖3.1 場區全圖及監測井分布圖 16
圖4.1 樣品CT-3之gradient PCR測試結果 26
圖4.2 場址樣品PCR試驗結果 27
圖4.3 場址樣品PCR產物純化試驗結果 28
圖4.4 場址樣品DGGE試驗結果 29
圖4.5 場址樣品DGGE光帶篩選比對結果 30
圖4.6 場址樣品相似度分析結果 31
圖4.7 場址樣品DGGE切取光帶之PCR試驗結果 33
圖4.8 場址樣品DGGE切取光帶濃度稀釋之PCR試驗結果 33
圖4.9 場址樣品親緣關係樹 35
圖4.10 整治列車各樣品PCR結果 36
圖4.11 樣品B1之gradient PCR測試結果 37
圖4.12 整治列車各樣品調整後PCR產物純化結果 38
圖4.13 加強式生物處理樣品變性梯度膠體電泳結果 39
圖4.14 整治列車各樣品中營養鹽與過氧化氫組之光帶篩選結果 41
圖4.15 整治列車各樣中營養鹽與過氧化氫組相似度分析結果 42
圖4.16 整治列車各樣品中界面活性劑組之光帶篩選結果 43
圖4.17 整治列車各樣品中界面活性劑組相似度分析結果 44
圖4.18 加強式生物處理樣品DGGE光帶膠體之PCR試驗結果 45
圖4.19 將加強式生物處理樣品親緣關係樹 48

表目錄
頁次
表2.1 石油碳氫化合物管制標準 2
表2.2 相關文獻整理 8
表3.1 整治列車處理各樣品成份 18
表3.2 PCR反應中各詳細參數 22
表3.3 DGGE膠體配置細目 24
表4.2 場址樣品定序相關結果 34
表4.3 加強式生物處理樣品定序結果 47

1. Altschul, S. F., Gish, W., Miller, W., Myers, E, W., Lipman, D, J., 1990. Basic local alignment search tool, Journal Molecular Biology, 215, 403-410.
2. Amann, R. I., Ludwig, W., Schliefer, K. H., 1995. Phylogenetic identification and in situdetection of individual microbial cells without cultivation, Microbiology Reviews, 22, 143-169.
3. Bano, N., Hollibaugh, J. T., 2002. Phylogenetic Composition of Bacterioplankt on Assemblages from the Arctic Ocean, Applied and Environmental Microbiology, 68, 505-518.
4. Barbara, T., Wonerow, K., Paschke, K., 1999. DGGE is more sensitive for the detection of somatic point mutations than direct sequencing, Biotechniques, 27, 266-268.
5. Benoit, J. M., Gilmour, C. C., Heyes, A., Mason, R. P., Miller, C. L., 2003. Geochemical and biological controls over methylmercury production and degradation in aquatic ecosystems, ASC Symposium Series, 835, 262–297.
6. Borden, R. C., Gomez, C. A., Becker, M. T., 1995. Geochemical indicators of intrinsic bioremediation, Ground Water, 33, 180-189.
7. Chandler, D. P., Jarrell, A. E., Roden, E. R., Golova, J., Chernov, B., Schipma, M. J., Peacock, A. D., Long, P. E., 2006. Suspension array analysis of 16S rRNA from Fe- and SO42- reducing bacteria in uranium-contaminated sediments undergoing bioremediation, Applied and Environmental Microbiology, 72, 4276-4287.
8. Deeb, R. A., Scow, K. M., Lisa, A. C., 2000. Aerobic MTBE biodegradation: an examination of past studies, current challenges and future research directions, Biodegradation, 11, 171-186.
9. Duarte, G. F., Rosado, A. S., Seldin, L., Keijzer-Wolters, A. C., van Elsas, J. D., 1998. Extraction of ribosomal RNA and genomic DNA from soil for studying the diversity of the indigenous bacteria community, Journal of Microbiological Methods, 32, 21-29.
10. Elbeltagy, A., Nishioka, K., Sato, T., Suzuki, H., Ye, B., Hamada, T., Isawa, T., Mitsui, H., Minamisawa, K., 2001. Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species, Applied and Environmental Microbiology, 67, 5285-5293.
11. Felske, A., Engelen, B., Nübel, U., Backhaus, H., 1996. Direct ribosomeisolation from soil to extract bacterial rRNA for community analysis, Applied and Environmental Microbiology, 62, 4162–4167.


12. Fierer, N., Schimel, J. P., Holden, P. A., 2003. Variations in microbial community composition through two soil depth profiles, Soil Biology and Biochemistry, 35, 167-176.
13. Fiorenza, S., Rifai, H. S., 2003. Review of MTBE Biodegradation and Bioremediation, Bioremediation Journal, 7, 1-35.
14. Gabriela, F. D., Rosado, A. S., Seldin, L., de Araujo, W., van Elsas, J. D., 2001. Analysisof bacterial community structure in sulfurous-oil-containing soils and detection of species carrying dibenzothiophene desulfurization (dsz) gene, Applied and Environmental Microbiology, 67,1052-1062.
15. Garibsu, C., Alkorta, I., 1999. Utilization of genetically engineered microorganisms (GEMS) for bioremediation, Journal of Chemical Technology and Biotechnology, 74, 599–606.
16. Gyaneshwar, P., James, E. K., Reddy, P. M., Ladha, J. K., 2002. Herbaspirillum colonization increases growth and nitrogen accumulation in aluminum-tolerant rice varieties, New Phytologist, 154, 131–145.
17. Hall, B. G., 2001. Tutorial: Create a tree. In “Phylogenetic trees made easy: a how-to manual for molecular biologists” (B. G. Hall, Ed.), 19-29, Sinauer Associates, Sunderland.
18. Hamamura, N., Olson, S. H., Ward, D. M., Inskeep, W. P., 2005. Diversity and functional analysis of bacterial communities associated with natural hydrocarbon seeps in acidic soils at Rainbow Springs, Yellowstone National Park, Applied and Environmental Microbiology, 71, 5943-5950.
19. Hamamura, N., Olson, S. H., Ward, D. M., Inskeep, W. P., 2006. Microbial Population Dynamics Associated with Crude-Oil Biodegradation in Diverse Soils, Applied and Environmental Microbiology, 72, 6316-6324.
20. Hendrickx, B., Dejonghe, W., Boenne, W., Brennerova, M., Cernik, M., Lederer, T., Bucheli-Witschel, M., Bastiaens, L., Verstraete, W., Top, E. M., Diels, L., Springael, D., 2005. Dynamics of an oligotrophic bacterial aquifer community during contact with a groundwater plume contaminated with benzene, toluene, ethylbenzene, and xylenes: an in situ mesocosm study, Applied and Environmental Microbiology, 71, 3815-3825.
21. Holben, W. E., 1994. Isolation and purification of bacterial DNA from soil. In: Mickelson,S.H (Ed.), Methods of Soil Analysis, Part 2, Soil Society of America, Madison, WI, 727-751.
22. Kang, N., Lee, D. S., Yoon, J., 2002. Kinetic modeling of Fenton oxidation of phenol and monochlorophenols, Chemosphere, 47, 915-924.


23. Kao, C. M., Chen, S. C., Liu, J. K., and Wang, Y. S., 2001. Application of microbial enumeration technique to evaluate the occurrence of natural bioremediation, Water Research, 35, 1951-1960.
24. Kao, C. M., Prosser, J., 2001. Evaluation of natural attenuation rate at a gasoline spill site, Journal of Hazardous Materials, 82, 275-289.
25. Kerin, E. J., Gilmour, C. C., Roden, E., Suzuki, M. T., Coates, J. D., Mason, R. P., 2006. Mercury Methylation by Dissimilatory Iron-Reducing Bacteria, Applied and Environmental Microbiology, 72, 7919-7921.
26. Kleikemper, J., Pombo, S. A., Schroth, M. H., Sigler, W. V., Pesaro, M., Zeyer, J., 2005. Activity and diversity of methanogens in a petroleum hydrocarbon-contaminated aquifer, Applied and Environmental Microbiology, 71, 149-158.
27. Krsek, M., Wellington, E. M. H., 1999. Comparison of different methods for the isolation and purification of total community DNA from soil, Journal of Microbiological Methods, 39, 1–16.
28. Maidak, B. L., Larsen, N., McCaughey, M. J., Overbeek, R., Olsen, G. J., Fogel, K.,Blandy, J., Woese, C. R., 1994. The ribosomal database project, Nucleic Acid Research, 22, 3485-3487.
29. Muyzer, G., De Waal, E. C., Uitterlinden, A. G., 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes encoding for 16S rRNA, Applied and Environmental Microbiology, 59, 695-700.
30. Ni Chadhain, S. M., Norman, R. S., Pesce, K. V., Kukor, J. J., Zylstra, G. J., 2006. Microbial dioxygenase gene population shifts during polycyclic aromatic hydrocarbon biodegradation, Applied and Environmental Microbiology, 72, 4078-4087.
31. Prince, R.C., 2000. Biodegradation of Methyl tertiary-Butyl Ether (MTBE) and Other Fuel Oxygenares, Critical Reviews Microbiology, 26, 163-178.
32. Rios-Hernandez, L. A., Gieg, L. M., Suflita, J. M., 2003. Biodegradation of an alicyclic hydrocarbon by a sulfate-reducing enrichment from a gas condensate-contaminated aquifer, Applied and Environmental Microbiology, 69, 434-443.
33. Roling, W. F., Milner, M. G., Jones, D. M., Fratepietro, F., Swannell, R. P., Daniel, F., Head, I. M., 2004. Bacterial community dynamics and hydrocarbon degradation during a field-scale evaluation of bioremediation on a mudflat beach contaminated with buried oil, Applied and Environmental Microbiology, 70, 2603-2613.

34. Sarah, J. M., Stephen, J. R.,Venosa, A. D., Davis, G. A., Chang, Y. j., White, D. C., 1999. Microbial population changes during bioremediation of an experimental oil spill, Applied and Environmental Microbiology, 65, 3566-3574.
35. Seghers, D., Wittebolle, L., Top, E. M., Verstraete, W., Siciliano, S. D., 2004. Impact of Agricultural Practices on the Zeamays L. Endophytic Community, Applied and Environmental Microbiology, 70, 1475-1482.
36. Smets, B. F., Pritchard, P. H., 2003. Elucidating the microbial component of natural attenuation, Current Opinion in Biotechnology, 14, 283-288.
37. Smith, A. E., Hristova, K., Wood, I., Mackay, D. M., Lory, E., Lorenzana, D., Scow, K. M., 2005. Comparison of biostimulation versus bioaugmentation with bacterial strain PM1 for treatment of groundwater contaminated with methyl tertiary butyl ether (MTBE), Environmental Health Perspectives, 113, 317-322.
38. Struchtemeyer, C. G., Elshahed, M. S., Duncan, K. E., McInerney, M. J., 2005. Evidence for aceticlastic methanogenesis in the presence of sulfate in a gas condensate-contaminated aquifer, Applied and Environmental Microbiology, 71, 5348-5353.
39. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., Higgins, D. G., 1997. The ClustalX-Windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools, Nucleic Acids Research, 25, 4876-4882.
40. Trumbore, S., 2000. Age of soil organic matter and soil respiration : radiocarbon constraints on belowground C dynamics, Ecological Applications, 10, 399–411.
41. Van Hamme, J. D., Singh, A., Ward, O. P., 2003. Recent advances in petroleum microbiology, Microbiology and Molecular Biology Reviews, 67, 503-49.
42. Vinas, M., Sabate, J., Espuny, M. J., Solanas, A. M., 2005. Bacterial community dynamics and polycyclic aromatic hydrocarbon degradation during bioremediation of heavily creosote-contaminated soil, Applied and Environmental Microbiology, 71, 7008-7018.
43. Wiedemeier, T. H., Rifai, H. S., Newell, C. J., Wilson, J. T., 1999. Natural Attenuation of Fuels and Chlorinated Solvents in the Subsurface, John Wiley & Sons, New York.
44. Zhang, Y., Miller, R. M., 1994. Effect of a Pseudomonas rhamonlipid biosurfactant on cell hydrophobicity and biodegradation of octadecane, Applied and Environmental Microbiology, 60, 2101-2106.
45. 方瑋寧，2002，MTBE好氧分解之可行性研究。國立中山大學環境工程研究所，碩士論文。
46. 行政院環保署，http://www.epa.gov.tw(網頁)
47. 行政院環保署毒管處，http://www.epa.gov.tw/J/toxic/index.html(網頁)
48. 張莉茹，2004，以自然衰減整治受石油碳氫化合物污染之地下水。國立中山大學環境工程研究所，碩士論文。
49. 陳鴻易，2003，以16S rDNA分析法監測石油污染土壤中微生物相之變化。國立中山大學生物科學系(研究所)，碩士論文。
50. 郭雅鈴，2005，應用監測式自然衰減法整治受石油碳氫化合物污染之地下水。國立中山大學環境工程研究所，碩士論文。
51. 梁書豪，2005，以Fenton-like氧化處理受燃料油污染之土壤。國立中山大學環境工程研究所，碩士論文。
52. 曾妤馨，2002，以葉綠體核酸序列推衍蹄蓋蕨類之親緣關係。國立中山大學生物科學系(研究所)，碩士論文。
53. 謝昶毅，2003，以PCR-DGGE 技術分析石油碳氫化合物污染地下水之微生物相。國立中山大學生物科學系(研究所)，碩士論文。