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研究生:胡太龢
研究生(外文):Tai-he Hu
論文名稱:石油碳氫化合物污染土壤之菌相監測
論文名稱(外文):Monitor of the Microbial Diversivity in Pertroleum-hydrocarbons Contaminated Soils
指導教授:高志明高志明引用關係
指導教授(外文):Jimmy C. M. Kao
學位類別:碩士
校院名稱:國立中山大學
系所名稱:環境工程研究所
學門:工程學門
學類:環境工程學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:65
中文關鍵詞:石油碳氫化合物
外文關鍵詞:PCRDGGE
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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
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
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