許多關於海洋微生物群落的研究顯示，微生物的群落組成會隨著環境的變化而改變。其中經由海底泥火山的噴發而不斷湧入海水中的甲烷、二氧化碳和氮氣 等，這些氣體對環境中微生物的影響不容小覷。但是對於海底泥火山之微生物組成與其餘海洋微生物群落的不同之處目前還尚未明瞭。透過次世代高通量測序技術的引入，推動了微生物群菌相分析領域的發展，並在傳統培養和基因方法的基礎上被廣泛應用。在本研究中，我們探討了位於台灣西南部海底泥火山附近的微生物群落組成，並將其與同一地區泥火山外的微生物群落進行比較。利用次世代高通量測序技術，分析了海底泥火山及非海底泥火山區域的海底底泥以及海水表層、中層和底層之海水樣品。結果顯示：在海水樣本中2個採樣點的表、中層優勢菌種相同，但是底層之優勢菌種不同。在表層及中層海水樣品中，優勢菌種分別為Alphaproteobacteria及Gammaproteobacteria；在底層海水樣品裡，海底泥火山區域則是以OD1_ABY1為主，而非泥火山區域以Alphaproteobacteria為主。其中OD1_ABY1為生長在厭氧環境下之菌種。另外在底泥樣本中，2個採樣點的優勢菌種不相同，且菌種種類的數量也相去甚遠。總結來說，海底泥火山微生物的菌相組成與同一地區的非火山地區有很大不同。因為海底泥火山的微生物群落的組成會受其獨特環境的影響，造成兩個地點的底泥樣品和底層海水優勢種的差異，在中表層海水樣品中則看不到此差異。隨著海底泥火山的噴發，甲烷、二氧化碳和氮氣等氣體的補充以及氧氣的缺乏，可能可以解釋這一劇烈的變化。

Large pool of study in marine microbial community has shown that the microbial composition within the community changes with environmental change. Submarine Mud Volcano, with methane, carbon dioxide, and nitrogen constantly erupted into the seawater, represents a unique and extreme environment for microorganisms to reside. However, it is not fully understood how the microbial composition is different than the rest of marine microbial community. The introduction of Next Generation High-throughput Sequencing advanced the field of metadata analysis in microbial community and since then has been widely used in addition to traditional culture and genotyping methods. In the present study, we examined the composition of microbial community near submarine mud volcano located in southwest Taiwan and compared it with the microbial community outside the volcano in the same region.Using next generation high-throughput sequencing, we compared samples collected from sediment, upper-level, mid-level, and bottom-level seawater. The results show the dominant strains are Alphaproteobacteria and Gammaproteobacteria in these sampling points of the surface and the middle seawater, but the bottom of the dominant strains is OD1_ABY1 in mud volcanic region. Also, the dominant strains is Alphaproteobacteria in general seabed region. In addition, the OD1_ABY1 is a bacterium that grow in an anaerobic environment. The methane from the volcanic eruption of the seafloor may cause the environment of some microorganism to be unsuitable. Moreover, the dominant strains of two points of the sediment sample are not the same, and the number of species is far from the same.In conclusion, the microbial composition from the submarine mud volcano is drastically different from the non-volcano area from the same region. The unique environment of submarine mud volcano manipulates the composition of microbial community which causes the difference in dominant species at sediment and bottom-level seawater but not mid- or upper-level seawater. Lack of oxygen along with the increased methane, carbon dioxide, and nitrogen may explain this drastic change.

目錄
第一章 前言 1
1.1. 台灣西南海域 1
1.2. 海洋泥火山與天然氣水合物 2
1.3. 甲烷利用菌 3
1.4. 海洋細菌 4
1.5. 微生物多樣性 5
1.6. 16S rRNA基因之應用與特性 6
1.7. PCR-DGGE技術 8
1.7.1.聚合酶連鎖反應(Polymerase Chain Reaction, PCR) 8
1.7.2.變性梯度凝膠電泳(Denaturing Gradient Gel Electrophoresis, DGGE) 8
1.7.3.利用16S rRNA基因配合PCR-DGGE技術進行菌相分析 10
1.8. 次世代定序(Next Generation Sequencing, NGS) 10
1.8.1.Illumina定序原理 11
1.9. Alpha diversity 12
1.10.Beta diversity 12
1.11.研究目的 13
第二章 實驗材料方法 14
2.1 西南海域採樣 15
2.2 海水樣本水體分析 16
2.2.1生化需氧量(Biochemical oxygen demand，BOD)檢測 16
2.2.2化學需氧量(Chemical oxygen demand，COD)檢測 16
2.2.3氨氮(Ammonia)檢測 17
2.2.4硝酸鹽(Nitrate)檢測 17
2.2.5磷酸鹽(Phosphate)檢測 18
2.2.6矽酸鹽(Silicate)檢測 18
2.3 可培養菌種培養方法 18
2.3.1可培養西南海域海水細菌培養 18
2.3.2可培養西南海域底泥細菌培養 18
2.3.3甲烷利用菌培養 19
2.4 細菌total genomic DNA萃取 19
2.4.1.未經培養西南海域海水DNA萃取前處理 19
2.4.2.未經培養西南海域底泥DNA萃取前處理 20
2.4.3.未經培養西南海域海水/底泥細菌DNA萃取 20
2.4.4.可培養西南海域海水/底泥/甲烷細菌DNA萃取 20
2.4.5.瓊脂膠體電泳(Agarose gel electrophoresisi)檢視DNA 21
2.5 Polymerase chain reaction聚合酶鏈鎖反應 22
2.6 Denaturing gradient gel electrophoresis 23
2.6.140-60%變性梯度膠體製作 23
2.6.2進行梯度變性電泳 25
2.6.3SYBR green 染色 25
2.7 生化測試 25
2.8 次世代定序(Next Generation Sequencing, NGS) 27
2.9 定序、序列比對 28
第三章 結果與討論 29
3.1 西南海域底泥及海水樣本分析 29
3.2 西南海域可培養菌相 30
3.2.1底泥可培養菌相分布及分析 30
3.2.2海水可培養菌相分布及分析 31
3.2.3底泥可培養菌株生化測試暨文獻比對 34
3.2.4海水可培養菌株生化測試暨文獻比對 34
3.3 西南海域未經培養菌相 35
3.3.1底泥未經培養菌相分布及分析 35
3.3.2海水未經培養菌相分布及分析 38
3.4 次世代定序Alpha-diversity分析 40
3.5 菌相比較 41
3.5.1底泥可培養與未經培養間菌相比較 41
3.5.2採樣點1未經培養底泥各樣本間比較 41
3.5.3採樣點2未經培養底泥各樣本間比較 42
3.5.4採樣點1與採樣點2未經培養底泥間比較 42
3.5.5海水可培養與未經培養間菌相比較 43
3.5.6採樣點1未經培養海水各樣本間比較 44
3.5.7採樣點2未經培養海水各樣本間比較 44
3.5.8採樣點1與採樣點2未經培養海水間比較 45
3.5.9利用QIIME比對Greengene database之統計 46
3.6 甲烷利用菌篩選結果 47
第四章 結論 49
參考文獻 52
表 62
圖 89

Annarita P, Ilaria F, Ida R, Alessia G, Licia L and Barbara N (2017) Microbial Diversity in Extreme Marine Habitats and Their Biomolecules. Microorganisms Rev. 5020025:

Beth NO, Jason BS, Nina JK and Katrina JE (2011) Microbial Ecology of the Dark Ocean above, at, and below the Seafloor. Microbiol Mol Biol Rev.75(2):361–422.

Buckley MR. (2004). The global genome question：Microbes are the key to understanding evolution and ecology. AmericanAcademy of Microbiology, Washington, DC,

Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Peña AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D,Koenig JE, Ley RE, Lozupone CA, McDonald DI, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J and Knight R (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 7:335–336

Chiu JK, Tseng WH and Liu CS, (2006) Distribution of gassy sediments andmud volcanoes offshore southwestern Taiwan. Terr. Atmos. Ocean. Sci. 17(4):703-722

Colby J, Dalton J and Whittenbury R (1979) Biological and biochemical aspects of microbial growth on C1 compounds. Ann. Rev. Microbiol. 33:481-517

Dimitrov LI (2002) Mud volcanoes—the most important pathway for degassing deeply buried sediments. Earth-Science Rev. 59:49–76.
Dimitrov LI (2003) Mud volcanoes—a significant source of atmospheric methane. Geo-Marine Letters. 23:155–161

Dukki H, Ilnam K, Ho KH, Hyun CK, Ok-Sun K, Bang YL, Jang-Cheon C, Hor-Gil H and Yoo KL (2014)Bacterial Communities of Surface Mixed Layer in the Pacific Sector of the Western Arctic Ocean during Sea-Ice Melting. PLOS ONE. 9:e86887

Geng T, XuYang Y, Hong L, XiaoHong X, Lars B and XiuQing Z (2010) Sequencing bias comparison of different protocols of MicroRNA library construction. BMC Biotechnology. 10:64

Guillem S and Shinichi S (2017) Marine microbial diversity. Current Biology. 27:489-494

Guillermo J, Mercedes U, Ana C, Aránzazu LL, Anicet RB, Javier T, Peter K, Anne BK, Daniel R, Juan FM, Francisco MC and Ramon RM (2013) Description of Bacillus toyonensis sp. nov., a novel species of the Bacillus cereusgroup, and pairwise genome comparisons of the species of the groupby means of ANI calculations. Systematic and Applied Microbiology. 36:383-391

Hanson RS and Hanson TE (1996) Methanotrophic bacteria. Microbiol. Rev. 60:439-471

Hawksworth DL and Mound LA (1991) Biodiversity of Microorganisms and Invertebrates : Its Role in Sustainable Agriculture. CAB International. P.17-30

Hugenholtz P and Huber T (2003) Chimeric 16S rDNA sequences of diverse origin A82are accumulating in the public databases. International Journal of Systematic and Evolutionary Microbiology. 53: 289–293

Inagaki F, Suzuki M, Takai K, Oida H, Sakamoto T, Aoki K, Nealson KH and Horikoshi K (2006) Microbial communities associated with geological horizons in coastal subseafloor sediments from the sea of okhotsk. Appl. Environ. Microbiol. 69:7224-7235.

Jannasch HW and Mottl MJ (1985) Geomicrobiology of deep-sea hydrothermal vents. Science. 229: 717–725.

Jan S, Wang J, Chern CS and Chao SY (2002) Seasonal variation of the circulation in the Taiwan Strait. Journal of Marine Systems. 35: 249-268

Lee Chen YI (1992) Summer phytoplankton community structure in the Kuroshio Current-related upwelling Northeast of Taiwan. Terrestrial,Atmospheric and Oceanic Sciences. 3: 305-320

Levin LA (2005) Ecology of cold seep sediments: interactions of fauna withflow chemistry and microbes. Oceanography and Marine Biology Rev. 43: 1-46

Lin CC, Lin ATS, Liu CS, Chen GY, Liao WZ and Schnurle P (2009) Geological controls on BSR occurrences in the incipient arc-continent collision zone off southwest Taiwan. Marine and Petroleum Geology. 26:1118-1131

Liu CS, Schnurle P, Wang YS, Chung SH, Chen SC and Hsiuan TH (2006) Distribution and characters of gas hydrate offshore of southwestern Taiwan.Terrestrial, Atmospheric and Oceanic Sciences. 17: 615-644

Lozupone C and Knight R (2005) UniFrac: a new phylogenetic method for comparing microbial communities. Appl Environ Microbiol. 71(12):8228-8235

Lozupone C, Lladser ME, Knights D, Stombaugh J and Knight R (2011) UniFrac: an effective distance metric for microbial community comparison. International Society for Microbial Ecology. 5(2):169

Lozupone CA, Hamady M, Kelley ST and Knight R (2007) Quantitative and qualitative β diversity measures lead to different insights into factors that structure microbial communities. Appl Environ Microbiol. 73(5):1576-1585

Lugwig W, Strunk O, Klugbauer S, Klugbauer N, Weizenegger M, Neumaier J,Bachleitner M, and Schleifer KH (1998). Bacterial phylogeny based on comparative equence analysis. Electrophoresis. 19:554-568.

Mardis ER (2008) Next-generation DNA sequencing methods. Annual Review of Genomics and Human Genetics. 9:387-402

Mark JC, Alan C and Nathalie DH (2010) Surface area and the seabed area, volume, depth, slope, and topographic variation for the world's seas, oceans, and countries. Environ. Sci. Technol. 44:8821–8828

Mark JC and Chhaya C (2017) Marine Biodiversity, Biogeography,Deep-Sea Gradients, and Conservation.Current Biology. 27:511–527

Maria GP, Vasilios L, Euripides GS and Konstantinos AK (2010) Prokaryotic community structure and diversity in the sediments of an active submarine mud volcano (Kazan mud volcano, East Mediterranean Sea). FEMS Microbiol Ecol.429–444.

Masayo O, Akira H, Takeshi K and Tateo F (1994) Photobacterium histaminum sp. nov., a Histamine-Producing Marine Bacterium. International Journal of Systematic Bacteriology. 44: 631-636

Moitinho SL, Bayer K, Cannistraci CV, Giles EC, Ryu T, Seridi L, Ravasi T and Hentschel U (2013) Specificity and transcriptional activity of microbiota associated with low and high microbial abundance sponges from the Red Sea. Molecular Ecology. 23: 1348–1363

Muyzer G, Waal EC de, and Uitterlinden AG. (1993). Profiling of complex microbial populatio ns by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59: 695-700.

Muyzer G and Ramsing NB (1995) Molecular methods to study the organization of microbial communities. Wat. Sci. Teeh. 32:1-9.

Muyzer G, Teske A, Wirsen CO and Jannasch HW (1995) Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments. Arch Microbiol. 164: 165–172.

Muyzer G and Smalla K (1998) Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek. 73:127-141.

Nubel U, Ferran GP, Kuhi M and Muyzer G (1999) Quantifying microbial diversity: morphotypes, 16S rRNA genes, and carotenoids of oxygenic phototrophs in microbial mats. Appl. Environ. Microbiol. 65: 422-430.

Oren A (2008) Microbial life at high salt concentrations: phylogenetic and metabolic diversity. Saline Systems Rev. 4:2

Palys T, Nakamura LK and Cohan FM (1997) Discovery and classification of ecological diversity in the bacteria world: the role of DNA sequence data. Int. J. Syst.Bacteriol. 47:1445-1156.

Qiliang L, Yang L and Zongze S (2014) Bacillus xiamenensis sp. nov., isolated from intestinal tract contents of a flathead mullet (Mugil cephalus). Antonie van Leeuwenhoek.105:99–107

Robert AH and Steven JM (2008) The new paradigm of flow cell sequencing. Genome Research. 18:839–846

Satyanarayana T, Raghukumar C and Shivaji S (2005) Extremophilic microbes: diversity and perspectives. Current Science. 89: 78–90

Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ and Weber CF (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol. 75:7537–7541

Shih TT (1967) A survey of active mud volcano in Taiwan and a study of their types and the character of the mud. Petroleum Geology of Taiwan.5:259-311.

Simon C, Wiezer A, Strittmatter AW and Daniel R (2009) Phylogenetic diversity and metabolic potential revealed in a glacier ice metagenome. Applied and Environmental Microbiology. 75:7519–7526

Simon C and Daniel R (2011) Metagenomic analyses: past and future trends. Applied and Environmental Microbiology. 77: 1153–1161.

Sogin ML, Morrison HG, Huber JA, Mark Welch D, Huse SM, Neal PR, Arrieta JM and Herndl GJ (2006) Microbial diversity in the deep sea and the underexplored "rare biosphere. Proc Natl Acad Sci U S A. 103:12115-12120.

Sun SC and Liu CS (1993) Mud diapirs and submarine channel deposits in offshore Kaohsiung-Hengchun, southwest Taiwan. Petrol. Geol. Taiwan 28:1-14.

Svetlana ND, Claudia K and Peter FD (2005) Methylocella Species Are Facultatively Methanotrophic. J. Microbiol. 13:4665-4670

Tatsuhiko H, Tomohiro T, Akira I, Yuki M, Hideaki M, Juichiro A, Kei O and Fumio I (2017) Atribacteria from the Subseafloor Sedimentary Biosphere Disperse to the Hydrosphere through Submarine Mud Volcanoes. Frontiers in Microbiology. 8:1135.

Trotsenko YA and Khmelenina VN (2002) Biology of extremophilic and extremotolerant methanotrophs. Arch Microbiol. 177:123-131.

Watanabe K, Teramoto M, Futamata H and Harayma S (1998) Molecular detection, isolation, and physiological characterization of functionally dominant phenol-degrading bacteria. Appl. Environ. Microbiol. 64:4396-4402.

Whitman WB, Coleman DC and Wiebe WJ (1998) Prokaryotes: the unseen majority. Proc. Natl. Acad. Sci. USA. 95: 6578-6583.

Whittenbury R, Phillips KS, Wilkinson JF (1970) Enrichment, Isolation and Some Properties of Me thane-utilizing Bacteria. J.Gen.Microbiol. 61:205-218

Woese CR. (1987) Bacterial evolution. Microbiol. Rev. 51:221-271.

Yu, HS and Wen YH (1992) Physiographic characteristics of the continental margin off southwestern Taiwan. J. Geol. Soc. China 36:337-351.

Yuan R, Shao-yi C, Hai-yan Y and Liu-jie D (2015) Lysinibacillus cresolivorans sp. nov., an m-cresol-degrading bacterium isolated from coking wastewater treatment aerobic sludge. Int J Syst Evol Microbiol. 65:4250–4255

Zhang Y, Maignien L, Zhao X, Wang F and Boon N (2011) Enrichment of a microbial community performing anaerobic oxidation of methane in a continuous high-pressure bioreactor. BMC Microbiology. 11:137

邱協棟，陳松春，許樹坤，蔡慶輝，王詠絢，劉家瑄。(2015)。臺灣西南海域永安及好景海脊區之近表層天然氣水合物賦存潛能。鑛冶 59/2，p17-p32。

邵廣昭。(2009)。系統分類學的演進。中央研究院生物多樣性研究中心

郭倍甄。(2007)。廢水廠污泥及米糠複合基質之能源化可行性研究。國立台中教育大學環境教育研究所碩士論文。

陳太山，石文卿。(2013)。臺灣西南部的泥火山分佈與油氣探勘潛能。鑛冶 57/3，p65-p74。

陳松春，許樹坤，王詠絢，蔡慶輝。(2012)。臺灣西南海域高屏斜坡上活躍的泥火山群與可能的天然氣水合物賦存。鑛冶 56/3，p73-p88。

陳金泉，傅子琅，李法西。(1982)。關於閩南-台灣淺灘漁場上升流的研究。台灣海峽 1:5-13

張阡肇。(2005)。台灣泥火山沈積物之特性、來源與西南部石灰岩體之隱示。臺灣大學地質科學研究所學位論文

曾威豪，劉家瑄。(2007)。台灣西南海域的泥火山。科學發展，p18-p25。

趙榮台。(2004)。生物多樣性。(www.tmue.edu.tw/~envir2/bitdiv/2004715/07.pdf)

趙鴻椿。(2003)。台灣地區泥火山氣體成分分析及其對全球甲烷來源的可能影響。國立成功大學地球科學研究所碩士論文。

駱守凡。(2008)。海洋次生重晶石在台灣西南海域泥火山沉積物之分佈。國立成功大學碩士論文。

盧鴻復，吳朝榮，李忠潘。(2007)。臺灣東北海域湧升流之時空變化。第29屆海洋工程研討會論文集，國立成功大學。pp. 613-618