本研究是針對烏山頂泥火山特殊地質中的土壤，做微生物多樣性的分析與研究；另一方面，透過培養的方式，分離可能存在泥火山泥
漿中的甲烷利用菌。其中主要利用的技術有Polymerase chain reaction (PCR)與Denaturing gradient gel electrophoresis (DGGE)，並以微生物的16S rRNA gene為標的來研究泥火山周圍的微生物菌相分佈與多樣性。最後透過PCR-DGGE所得到的DNA條帶加以定序，進一步與16S rRNA gene資料庫比對鑑定出各菌種。由目前所得到的結果顯示，烏山頂泥火山周圍各不同距離土樣，利用DGGE將DNA分離形成條帶後，再經過Quantity One軟體相似度分析，可以得知距
離火山口泥漿越遠的土壤，其菌相與火山口泥漿菌相差異越大。而主要存在火山口泥漿中菌種有Brevundimonas terrae、Ralstonia solanacearum、Ralstonia taiwanensis、Pseudonocardiaceae bacterium、Ochrobactrum anthropi)、Burkholderia phytofirmans、Stenotrophmonas sp.、Methylobacterium sp.、Cryseobacterium sp.、Sphingobacterium sp.、Stenotrophomonas sp. MG-3、Stenotrophomonas sp. EP01等，這些菌種都屬於高耐鹽性質與高pH值的種類。另外，Ochrobactrum anthropi與Burkholderia phytofirmans則是只有在火山口泥漿與5公尺土樣中發現； Ralstonia mannitolilytica、Ralstonia pickettii、Amycolatopsis rugosa、Maricaulis sp.則是只在距離火山口10公尺的土樣中發現，可能是10公尺土樣剛好位於泥漿與植被交界點而存在有特殊的菌種。最後在甲烷利用菌培養方面，已經成功培養出Methylobacterium organophilum與Ochrobactrum anthropi二株可以利用甲烷生長的菌種，並針對其生化反應進行測試，未來希望能夠探討此2株菌在生物復育上的應用，如分解石化污染物的能力等。

This research is focus on the analysis of microbial diversity and existance of methanotrophic microbial strains in Wu-Shan-Ding mud volcano areas. The microbial distribution and diversity in soil surrounding the mud volcano were analyzed by polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). The 16S rDNA sequences from PCR-DGGE bands were used to identify the bacterial strains with NCBI gene bank data. The results showed that the microbial diversity in the fresh erupted mud sample varied with other soil samples. The degrees of diversity were propotion to the distance away from the mud volcano. The main bacterial species found in the mud sample are Brevundimonas terrae, Ralstonia solanacearum, Ralstonia taiwanensis, Pseudonocardiaceae bacterium, Ochrobactrum anthropi, Burkholderia phytofirmans, Stenotrophmonas sp., Methylobacterium sp., Cryseobacterium sp., Sphingobacterium sp., Stenotrophomonas sp. MG-3, and Stenotrophomonas sp. EP01. According to documents, all of the above strains are tolerant to high salt and alkaline. The Ochrobactrum anthropi and Burkholderia phytofirmans were found only in the fresh erupted mud sample and the 5-meter soil sample, while Ralstonia mannitolilytica, Ralstonia pickettii, Amycolatopsis rugosa, and Maricaulis sp. were found only in 10-meter soil sample. In other word, all of the soil samples we examed in this study have their own specified bacterial strains. Two methanotrophic microganisms, Methylobacterium organophilum and Ochrobactrum anthropi were also successfully isolated in pure cultures. The biochemical characteristics of these two strains had been studied. These two strains show a potential might be able to use in the bioremediation of petroleum pollution.

中文摘要 II
Abstract IV
第一章 前言 1
1.微生物多樣性的重要性 1
2.泥火山的定義 2
3.台灣泥火山的分佈 2
4.烏山頂泥火山 3
5.烏山頂泥火山泥漿的成份 4
5.甲烷利用菌 5
6.甲烷利用菌在生物科技上的應用 6
7.分子生物在細菌多樣性分析上的重要性 6
8.16S rDNA在微生物上在多樣性研究上的應用 8
9.DGGE技術在土壤微生物分析的應用 9
第二章 實驗目的與流程 11
1.研究目的： 11
2.各採樣點簡述： 11
第三章 材料與方法 16
1.土壤樣品的處裡： 16
3. PCR放大16S rDNA： 17
4.PCR產物純化與濃縮： 20
5.DNA Ligation反應： 21
6.Competent cell製作： 22
7.Transformation： 23
8.質體DNA萃取： 23
9.DGGE電泳分離各16S DNA PCR產物： 25
10.DNA膠體圖譜之分析： 27
11. DNA定序與比對： 28
12.泥漿中甲烷利用菌培養與分離 29
13.菌種特性探討： 30
第四章 結果與討論： 31
1.16S rRNA gene PCR放大結果： 31
2.DGGE分析圖譜以及Quantity One相似度分析： 31
3.DGGE條帶序列比對結果： 33
4.火山口泥漿中菌種探討： 35
5.甲烷利用菌的培養與分離： 41
6.甲烷利用菌的基本生化測試： 42
7. Methylobacterium organophilum與Ochrobactrum anthropi在未來生物復育上的應用： 44
第五章 結論 47
參考文獻： 50
圖表 62
表格 84
附錄：培養基配方 91
附錄：定序之16s rDNA序列 (V6-V8 region) 92

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