臺灣博碩士論文加值系統

石油組成成分中含有許多會危害自然環境的碳氫化合物，其中的芳香族碳氫化合物是最不易被分解的一類，若其廣泛分布在自然環境中，將會對生物以及人類造成莫大的毒害。因此，能有效快速分解此類碳氫化合物的生物復育已被認為是一種必要的趨勢。本研究利用分子生物之方法，建立本土性油污染分解菌群之16S rRNA基因選殖資料庫，可提供引子與探針設計及實行生物復育之應用。本研究共對2油污染場址(LY與FD)進行分子選殖，建立16S rRNA基因選殖資料庫。與先前研究所建立之YK污染場址選殖資料庫共同分析，結果共獲得47個OTUs，其中已被報導過與油污分解相關之OTUs共有16個，分別屬於Pseudomonas、Burkholderia、Azoarcus、Xanthomonas、Bacillus、Comamonas、Nocardioides、Sphingomona與Alcaligenes菌屬，其中Pseudomonas所佔比例為21.6%，為此三場址中數量最多之油污分解菌群。
此外，本研究自行修飾設計Pseudomonas菌屬16S rRNA基因專一性引子對PsF1與PsF3-PsR3。PsF1與真細菌16S rDNA廣泛性引子1392r配合使用可放大出一段約1100 bps之產物，可應用於以變性梯度凝膠電泳 (DGGE) 偵測Pseudomonas菌群結構。以semi-nested PCR-DGGE法偵測生物復育過程中Pseudomonas菌群結構之變化，結果顯示添加之營養鹽可能造成適應此環境且具有分解能力之Pseudomonas菌群大量生長，使指紋圖譜上出現特別顯著之條帶。然控制組與添加營養鹽之實驗組指紋圖譜有相同變化趨勢，皆於第24天之樣本中可發現最多量之條帶。PsF3-PsR3亦為Pseudomonas菌屬16S rRNA基因專一性引子對，可應用於即時定量PCR (Real-time PCR)之研究。本研究以Real-time PCR偵測復育場址於不同天數樣本中Pseudomonas 16S rRNA基因之數量，結果顯示各組別間Pseudomonas數量消長有相同之趨勢，皆於第14天到達最大數量之後遞減，至第24天到達最低。此外，針對控制組樣本所得數據加以分析，結果顯示各種生物復育方法對土壤中Pseudomonas族群含量皆造成一定影響，然油污降解能力在各組間則無明顯差異，顯示此場址原生菌群以有良好之油污分解能力。因此，在未來生物復育應用上，預先評估污染場址中原生菌群之油污分解能力為一不可或缺之指標，對於生物復育之時程與成本皆有很大之影響，具有相當之價值。

Petroleum hydrocarbons are the most widespread contaminants in the environment. Several of petroleum hydrocarbons are known mutagens or carcinogens for human and other organisms. They are difficult to be degraded in the natural environment. Bioremediation is considered a useful method to degrade the petroleum hydrocarbons using the oil-degrading bacteria. In this study, cultivation-independent methods were used to study the microbial communities in petroleum contaminated sites. Phylogenetic analysis of bacterial 16S rRNA gene clone libraries from the LY、FD and YK petroleum contaminated soil showed that 16 of 47 OTUs (operational taxonomic units) were oil-degrading bacteria which belong to Pseudomonas、Burkholderia、Azoarcus、Xanthomonas、Bacillus、Comamonas、Nocardioides、Sphingomonas and Alcaligenes. Proteobacteria was the dominant group (66%) and Pseudomonas was the most abundant genus (21.6%) in these 3 clone libraries.
In KH bioremediation site, there are 5 different treatments including bioaugmentation and biostimulation. Dynamic changes of bacterial communities in KH petroleum-contaminated site during bioremediation were monitored by PCR-DGGE, which used bacteria universal 16S rRNA gene primers. In the DGGE fingerprinting of KH site, the band at the same position with Pseudomonas markers was found in all samples. Our data indicate that Pseudomonas spp. may be important petroleum hydrocarbon-degradation bacteria in the KH site.
The changes of population size of Pseudomonas in KH site during bioremediation were monitored by Real-time PCR. In all 5 different bioremediation-treatments, the population size of Pseudomonas reached maximum in the 14th day (6.80*104 – 2.04*105copy numbers/ng DNA) and decreased to minimum in the 24th day (7.20*103 – 1.52*104copy numbers/ng DNA). The TPH-d (total petroleum hydrocarbons-diesel) degrading profile of KH site showed that the TPH-d in soil was decreased to 50-60% in the 14th day and was almost degraded in 24th day. The results show that the Pseudomonas population size was affected by bioremediation treatment, but the TPH-d degrading rate of different bioremediation group were the same in KH site. The addition of bacteria or biosurfantant did not improve degradation of the TPH-d in KH site. Our findings indicate that the oil-degrading ability of original bacteria in contaminated site should be considered as an important factor for future bioremediation, and monitoring the variation of main oil-degrading bacteria can help us to understand the efficiency of bioremediation process, and enhance bioremediation.

中文摘要 ........................................................... I
英文摘要 ........................................................... II
致謝 .............................................................. III
目錄 .............................................................. IV
表目錄 ............................................................ VII
圖目錄 ............................................................ VIII
第一章 前言 ...................................................... 1
1-1 研究緣起 .................................................. 1
1-2 研究目的 .................................................. 3
1-3 研究架構 .................................................. 4
第二章 文獻回顧 .................................................. 5
2-1 石油碳氫化合物的污染 ................................... 5
2-2 石油碳氫化合物在環境中的分解 ........................... 6
2-2-1 分解石油碳氫化合物的微生物 ................................ 6
2-3 分子生物技術應用於環境微生物生態之研究 ................. 7
2-3-1 聚合酵素鏈鎖反應 .......................................... 9
2-3-2 變性梯度凝膠電泳 .......................................... 10
2-3-3 限制酵素片段長度多型性分析法 .............................. 11
2-3-4 末端螢光標定限制酵素片段長度多型性分析法 .................. 12
2-3-5 螢光原位雜合法 ............................................ 13
2-3-6 單股構型多態性法 .......................................... 15
2-3-7 即時定量聚合酵素連鎖反應 .................................. 16
第三章 材料與方法 ................................................ 18
3-1 油污染土壤樣本來源 ........................................ 18
3-2 DNA 的萃取方法 ............................................ 18
3-2-1 Miller-modified method (MMB) .............................. 18
3-2-2 MoBio kit (Stults et al., 2001) ........................... 19
3-3 分析方法 .................................................. 20
3-3-1 聚合酵素鏈鎖反應(Polymerase chain reaction，PCR) .......... 20
3-3-2 瓊脂膠體電泳 .............................................. 22
3-3-3 16S rRNA基因分子選殖 (Cloning) ............................ 22
3-3-4 變性梯度凝膠電泳 .......................................... 23
3-3-5 變性梯度凝膠電泳上之DNA片段萃取 ........................... 23
3-3-6 限制酵素片段長度多型性分析法(Restriction Fragment Length Polymorphism(RFLP) ............................................................. 23
3-3-7 定序 ...................................................... 26
3-3-8 親緣關係分析 .............................................. 26
3-4 假單胞菌屬專一性引子之設計與PCR條件之建立 ................. 26
3-4-1 假單胞菌屬專一性引子PsF1之設計與PCR條件之建立 ............. 26
3-4-2 假單胞菌屬專一性Semi-nested PCR-DGGE偵測方法之建立 ........ 27
3-4-3 假單胞菌屬專一性引子對PsF3-PsR3之設計與PCR反應條件之最佳化 27
3-4-4 假單胞菌屬專一性引子對PsF3-PsR3之設計與PCR反應條件之最佳化 28
3-4-5 假單胞菌屬專一性即時定量PCR反應標準曲線之建立 ............. 28
3-5 主要儀器 .................................................. 29
第四章 結果與討論 ................................................ 30
4-1 利用分子生物技術分析油污土壤的菌群結構 .................... 30
4-1-1 LY油污土壤之選殖分析 ...................................... 30
4-1-2 FD油污土壤之選殖分析 ...................................... 32
4-1-3 油污染場址選殖資料庫之分析 ................................ 38
4-2 利用分子生物方法監測復育過程中油污土壤的菌群結構變化 ...... 44
4-2-1 以DGGE監測生物復育過程中土壤菌群結構之變化 ................ 44
4-3 以Semi-nested PCR-DGGE法快速檢測環境中油污染分解菌群-Pseudomonas菌屬 ................................................................. 47
4-3-1 Pseudomonas菌屬DGGE biomarker之建立 ....................... 47
4-3-2 以semi-nested PCR-DGGE法生物監測油污染土壤中假單胞菌屬菌群結構 ................................................................. 49
4-3-3 利用semi-nested PCR-DGGE法監測生物復育過程土壤中Pseudomonas菌屬菌群結構之變化 ........................................................... 52
4-4 土壤樣本中Pseudomonas菌群之定量分析 ....................... 55
4-4-1 Pseudomonas菌屬專一性引子之設計與Real-time PCR條件之建立 .. 55
4-4-2 以Real-time PCR偵測生物復育過程中Pseuomonas菌屬含量之變化 . 55
第五章 結論與建議 ................................................ 59
第六章 參考文獻 .................................................. 61
自述 ............................................................... 75

表目錄
Table 3-1 Primers used in this study ............................... 21
Table 3-2 Procedure of cloning .................................... 24
Table 3-3 Procedure of DGGE analysis ............................... 25
Table 4-1 Phylogenetic affiliations of the 16S rDNA clone library of the LY oil-contaminated soil sample ....................................... 35
Table 4-2 Phylogenetic affiliations of the 16S rDNA clone library of the FD oil-contaminated soil sample ....................................... 37
Table 4-3 Oil degrading bacterium in FD、LY、YK clone library ...... 43
Table 4-4 Phylogenetic affiliations of the DGGE cut band clone library50


圖目錄
Fig 1-1 Schematic representation the steps for studying of petroleum-contaminated soil bacterial community by culture-independent approaches ......................................................... 4
Fig 3-1. Semi-nested PCR-DGGE methods for monitoring Pseudomonas community structure .......................................................... 27
Fig 4-1. Phylogenetic dendrogram of 16S rDNA bacterial sequence from the LY oil-contaminated soil .............................................. 34
Fig 4-2. Phylogenetic dendrogram of 16S rDNA bacterial sequence from the FD oil-contaminated soil .............................................. 36
Fig 4-3. Phylogenetic dendrogram of 16S rDNA bacterial sequence from the LY、FD and YK oil-contaminated soil .................................... 42
Fig 4-4. DGGE fingerprinting and the illustration of the change of microbial community through week 0 to week 6 in KH bioremediation site. NE group46
Fig 4-5. Test of semi-nested PCR-DGGE method to detect Pseudomonas populations ......................................................... 48
Fig 4-6. DGGE fingerprinting and illustration of Pseudomonas populations in FD oil contaminated soil ............................................... 51
Fig 4-7. DGGE fingerprinting and the illustration of the dynamic change of Pseudomonas community through week 0 to week 6 in KH bioremediation site................................................................. 54
Fig 4-8. Variation of Pseudomonas 16S rRNA genes copy numbers during bioremediation ...................................................... 58

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