中文摘要

本研究係利用polymerase chain reaction (PCR) 及denaturing gradient gel electrophoresis (DGGE) 兩項分子生物學技術，以微生物的16S rDNA為標的來探討在石油碳氫化合物污染之地下水中微生物族群的分布與生物多樣性。經由PCR-DGGE所獲得DNA序列，進一步與16S rDNA資料庫進行比對，鑑定出各菌種，建構微生物的親緣演化樹，來瞭解石油碳氫化合物污染地區地下水微生物相之組成及變化。本實驗以中國石油公司高雄煉油廠，中國石油公司橋頭油庫及中國石油化學工業開發股份有限公司高雄廠為研究場址，進行石油碳氫化合物污染地區地下水微生物相分析。高雄煉油廠與橋頭油庫兩個廠址是以自然衰減污染為主要整治方法，而中國石油化學高雄廠則是以空氣注入 (air sparging) 方式配合生物性降解進行污染場址之整治。由實驗結果得知，高雄煉油廠之低度污染區地下水中有最豐富的細菌群聚，無污染區域次之，而高度污染區域則最少。中國石油橋頭油庫中高度污染區域的細菌群聚較無污染區域少。中國石油化學高雄廠在整治過程中，高度、低度及無污染區域的地下水微生物菌相皆有明顯的改變，三處的微生物多樣性有隨整治時間增長而增加的趨勢。在序列定序與比對的結果中，三處皆可能含有降解石油碳氫化合物的微生物存在。高雄煉油廠的高度污染地下水樣本中為Methylobacterium, Xanthobacter, Xanthomonas與Pseudomonas菌屬菌種。橋頭油庫的高度污染地下水樣本中為Flavobacterium菌屬菌種。中國石油化學高雄廠的高度污染地下水樣本中為Nocardia, Pseudomonas, Rubrivivax, Methylobacterium, 與Candida菌屬菌種，在低度污染地下水樣本中為Candida菌屬菌種。本實驗亦證實，利用地下水來進行石油碳氫化合物污染地區的生物復育的污染整治評估，相較於土壤樣本更為便利，比較不受腐質酸等物質干擾反應等因素的影響，而且菌相分析所需時間更為縮短。

Abstract

This research used molecular biological techniques such as polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) to analyze the dynamic microbial community and biodiversity in the groundwater contaminated with petroleum-hydrocarbons. The 16S rDNA sequences from all water samples were compared with the sequences of relative bacteria in the Ribosomal Database Project Bank to construct a phylogenetic tree. The results allowed us to understand the composition of the microbial communities in the petroleum-hydrocarbon contaminated groundwater. In this study, groundwater samples taken from the Chinese Petroleum Corporation Kaohsiung Refinery (CPCKR), Chinese Petroleum Corporation at Ciaotou fuel Tank Farm (CPCCTF) and China Petrochemical Development Corporation at Kaohsiung Factory (CPDCKF) were analyzed. The contaminated sites at CPDCKR and CPCCTF are remeated by natural attenuation. While the CPDCKF site is remeated by an enhanced air sparging bioremediation. In CPDCKR, we found that the low polluted area contained the richest microbial community, followed by the non-polluted area, and the high polluted area. At the CPCCTF site, the microbial community in the non-polluted area was richer than the high-polluted area. Increased microbial populations and variation in microbial community have beenobserved in non-polluted, less polluted, and highly polluted areas. The microbial community showed a dynamic succession of complexity during the bioremediation process at the CPDCKF site. From the 16S rDNA sequence analysis, it is possible that all samples contained petroleum-hydrocarbon degrading bacteria. These petroleum-hydrocarbon degrading bacteria include Methylobacterium, Xanthobacter, Xanthomonas and Pseudomonas at CPCKR site, Flavobacterium at CPCCTF site, Nocardia, Pseudomonas, Rubrivivax, Methylobacterium, and Candida at CPDCKF site. This study also demonstrates that it is more economic and reliable of using molecular techuiques to analyze the groundwater. Thus, groundwater samples can be used to replace soil samples for future work.

目錄
中文摘要…………………………………………………………Ⅰ
英文摘要…………………………………………………………Ⅲ
第一章 前言………………………………………………………1
1. 石油碳氫化合物污染對環境的衝擊……………………………1
2. 石油碳氫化合物污染地下水整治方式…………………………3
3. 分子生物技術在污染整治上的重要性與應用…………………4
4. 利用PCR-DGGE技術應用於環境微生物菌群結構的探討………6
5. 16S rDNA在細菌演化分類與鑑定上的獨特性及應用…………9
6. 分子生物技術應用於環境微生物研究的難題…………………11
7. 石油碳氫化合物降解微生物簡介………………………………12
8. 石油碳氫化合物污染廠址簡介…………………………………14
8.1中國石油公司高雄煉油廠……………………………………15
8.2中國石油公司橋頭油庫………………………………………17
8.3中國石油化學工業開發股份有限公司高雄廠………………17
9. 研究目的……………………………………………………………19
第二章 驗材料與方法……………………………………………22
1. 地下水之採樣………………………………………………22
2. 地下水中石油碳氫化合物污染指標分析…………………23
3. 地下水樣總生菌數量計數…………………………………24
4. 微生物genomic DNA萃取…………………………………24
4.1 Genomic DNA瓊脂膠體電泳檢視………………………26
4.2 Genomic DNA濃度與純度測定…………………………27
5. 地下水微生物Genomic DNA的純化………………………28
6. 聚合酶連鎖反應…………………………………………30
6.1 引子 (primer)………………………………………33
6.2 PCR-16S rDNA片段瓊脂膠體電泳檢視………………35
7. PCR-16S rDNA片段純化與濃縮…………………………36
8. 變性梯度膠體電泳………………………………………37
8.1 變性梯度膠體30﹪∼70﹪製作………………………38
8.2 進行變性梯度膠體電泳………………………………39
8.3 SYBR greenⅠ螢光染色……………………………40
9. DGGE Fimgerprinting pattern 統計分析…………40
10. PCR-16S rDNA定序與序列比對……………………41
11. 親緣演化樹 (Phylogeny tree)……………………43
第三章 結果…………………………………………………44
1. 地下水基本水質測定結果……………………………45
2. 地下水石油碳氫化合物污染指標分析監測結果………46
3. 地下水總生菌數量計數結果……………………………48
4. 微生物genomic DNA萃取結果……………………………49
5. PCR抑制物的去除…………………………………………51
6. 16S rDNA聚合酶連鎖反應結果…………………………52
7. DGGE電泳圖譜分析與比對結果…………………………53
8. DGGE電泳圖譜Fimgerprinting pattern統計分析結果……55
9. 16S rDNA V3 region 序列比對與分析結果………………57
10. 親緣演化樹的建構結果……………………………………60
第四章 討論………………………………………………………64
1. 石油碳氫化合物污染地下水環境中微生物活動狀態探討…64
2. 石油碳氫化合物污染地下水總生菌數變化分析……………66
3. 石油碳氫化合物污染地下水微生物菌相分析探討…………69
4 地下水取代土壤作為評估生物整治成效之可行性.………74
5. 分子生物技術尚須克服的問題……………………………75
6. PCR-DGGE菌相分析於生物復育研究的運用……………77
第五章 結論………………………………………………………79
參考文獻……………………………………………………82
圖表………………………………………………………98
附錄…………………………………………………………149

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