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研究生： 謝崇良
Hsieh, Chung-Liang
論文名稱： 苗栗地區陸地深部打鹿砂岩地層岩石中菌種之分離與鑑定
The Isolation and Identification of the Indigenous Bacteria from Deep Subsurface Rocks of Talu Sandstone in the Miaoli Area
指導教授： 簡錦樹
Jean, Jiin-Shuh
學位類別： 碩士
Master
系所名稱： 理學院 - 地球科學系
Department of Earth Sciences
論文出版年： 2003
畢業學年度： 91
語文別： 中文
論文頁數： 68
中文關鍵詞： 鐵還原菌、深層菌、Bacillus、細菌分離及鑑定
外文關鍵詞： Ferric-reducing bacteria, Bacillus, the deep subsurface bacteria, bacterial isolation and identification.
相關次數： 點閱：194  下載：2 
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    • 深層菌的研究大部分都是利用鑽井之便來採集深層的地層水或是岩樣，其環境包含了含有石油的岩層及其地層水、湖泊的沉積岩、及地下噴出地表的熱泉水等都可能有深層菌的存在，而本研究僅以苗栗地區深部地層含石油的打鹿砂岩作為探討之對象。
    本研究的樣品為苗栗地區陸地油井所採得的深部岩層之岩屑(即打鹿砂岩)及其鑽泥，其深度皆在三千公尺以上。利用發酵培養基(TYG medium)和鐵還原培養基(MSA medium)來優厚培養岩屑中及鑽泥中的各類菌種，並進行菌株的分離，透過DNA萃取、聚合酶連鎖反應(PCR)、膠電泳、DNA定序及親緣關係樹等分子生物技術將菌種鑑定。同時亦進行菌種的生存溫度及鹽度範圍之測試，以了解菌群生存的適宜範圍，用以推測菌群的可能來源，並以電子顯微鏡觀察細菌型態。
    本研究實驗顯示所培養出來的菌屬可利用發酵或鐵還原反應獲得能量而生長，並且由鐵還原菌培養基的沉降試驗與鐵還原菌的優厚培養結果會產生可被磁鐵吸引的黑色物質，可確定培養基中紅棕色的Fe(Ⅲ)-oxyhydroxide轉變成黑色物質並非是單純的化學變化；由菌種鑑定及親緣關係樹得到30H1L菌株與Bacillus pumilus 與Bacillus sp. PB10有97﹪的核苷酸相似度，所以推定此分離菌為細菌Bacillus屬的一種。

    The study of deep subsurface bacteria is mainly from the deep subsurface rocks or geological formation water while drilling a well. The environments where the deep subsurface bacteria may be present include oil-bearing rocks, geological formation water, lacustrine deposits and sedimentary rock, hot spring, etc. However, only oil-bearing rocks in the Miaoli area were taken for this study.
    The samples of this study taken from the 3385-meter-deep subsurface rocks (i.e. Talu Sandstone) and drilling muds were collected in a certain drilling well in the Miaoli area. The indigenous bacteria from these rocks and muds are isolated and enriched with the MSA medium for the ferric-reducing bacteria and the TYG medium for the fermentative bacteria. The bacteria were identified followed by using the molecular biotechnology such as DNA extraction and purification, PCR (polymerase chain reaction), gel electrophoresis, gene sequencing and phylogenetic tree. Meanwhile, the laboratory tests for the ranges of temperature and salinity that the indigenous bacteria were adapted to the environment were performed. This is to understand the most optimal ranges for bacterial growth and to speculate the possible source of the indigenous bacteria. The electron microscopes were used to observe the morphology of bacteria.
    The experimental results of the study showed that the viable bacterial strains could obtain energy and grow by utilizing the reaction products of the fermentative and ferric-reducing bacteria. The sedimentation tests and the enrichment of bacterial culture from the ferric-reducing medium showed that there exist magnetic dark materials. The transformation of the reddish-brown Fe(Ⅲ)-oxyhydroxide in the ferric-reducing medium accounts for resulting from the bacterial reaction rather than from the chemical reaction. The isolated and identified strain 30H1L has 97﹪ similarities in nucleotide to Bacillus pumilus and Bacillus sp. PB10. It can be defined that strain 30H1L belongs to genus Bacillus.

    摘要......................................................I Abstract..................................................II 致謝......................................................IV 目錄......................................................VI 表目錄....................................................X 圖目錄....................................................XI 第一章 緒 論.............................................1 1.1 研究動機與目的.......................................1 1.2 文獻回顧.............................................3 1.3 研究區域地質概況.....................................6 第二章 研究方法..........................................12 2.1 研究流程............................................12 2.2 岩樣取得及處置......................................14 2.2.1 岩樣的取得......................................14 2.2.2 樣品的製作......................................14 2.2.2.1 岩樣的切割與研磨............................14 2.2.2.2 菌體的脫附..................................14 2.3 細菌培養............................................15 2.3.1 培養基(medium)的製作............................15 2.3.1.1 鐵還原菌之MSA培養基........................15 2.3.1.2 發酵菌之TYG培養基..........................17 2.3.2 岩屑植入........................................17 2.3.2.1 鐵還原菌培養................................17 2.3.2.2 發酵菌培養..................................19 2.4 細菌純化與單一菌株..................................19 2.5 DNA萃取及純化 ..................................... 19 2.6 菌種的分子生物鑑定..................................21 2.6.1 Polymerase Chain Reaction (PCR)................21 2.6.2 DNA定序、比對及親源關係樹..................... 21 2.7 革蘭氏染色(Gram stain)..............................23 2.8 顯微鏡觀察前之備製過程..............................23 2.8.1 掃描式電子顯微鏡(SEM)..........................23 2.8.2 穿透式電子顯微鏡(TEM)..........................24 2.9 沉降試驗............................................25 2.10 菌體之生理試驗.....................................26 第三章 實驗結果..........................................27 3.1 鐵還原菌之MSA培養基培養............................27 3.1.1 優厚培養(enrichment culture)....................27 3.1.2 沉降試驗........................................29 3.1.3 生理試驗........................................31 3.2 發酵菌之TYG培養基培養..............................32 3.2.1 優厚培養(enrichment culture)....................32 3.2.2 膠電泳試驗(gel electrophoresis).................33 3.2.3 基因定序........................................33 3.2.4 資料比對及菌種鑑定..............................33 3.3 光學及電子顯微鏡觀察................................ 43 3.3.1 光學顯微鏡......................................43 3.3.2 掃描式電子顯微鏡(SEM)...........................43 3.3.3 穿透式電子顯微鏡(TEM)...........................46 第四章 討論..............................................49 4.1 樣品污染控制........................................49 4.2 沉降試驗之意義......................................49 4.3 鐵還原菌在固態培養基培養之問題......................51 4.4 細菌之生理特性......................................52 4.5 細菌生存環境及棲地特性..............................52 第五章 結論與建議........................................55 5.1 結論................................................55 5.2 建議................................................56 參考文獻..................................................58 附錄1 打鹿砂岩中分離出菌株之核苷酸序列................... 62 附錄1a:30H1L-1菌株.....................................62 附錄1b:30H1L-2菌株.....................................63 附錄1c:30H1L-3菌株.....................................64 附錄1d:30H1L-4菌株.....................................65 附錄1e:30H1L-5菌株.....................................66 附錄2 Bacillus pumilus strain KL-052之16S rRNA部分核苷酸 序列(資料來源：NCBI資料庫)......................... 67 自述......................................................68 表2-1 鐵還原菌之MSA medium組成成分(per liter)............16 表2-2 發酵菌之TYG medium組成成分(per liter)..............18 表2-3 發酵菌之TYG medium中的Solution M 組成成分(per liter).................................18 表2-4 PCR實驗所使用之引子(Bennasar et al.,1988) ..........22 表2-5 PCR混和液(mixture)組成成分 .........................22 表3-1 鐵還原菌之MSA培養基優厚培養樣品....................27 表3-2 鐵還原菌之MSA培養基優厚培養結果....................28 表3-3 鹽度生長範圍之結果(起始時間5/14) ...................32 表3-4 溫度生長範圍之結果(起始時間5/14) ...................32 表3-5 發酵菌之TYG培養基優厚培養結果......................34 表3-6 30H1L-1菌株與B.pumilus strain KL-052比對之結果， 核苷酸相似度為95﹪..................................37 表3-7 30H1L-2菌株與B.pumilus strain KL-052比對之結果， 核苷酸相似度為97﹪..................................38 表3-8 30H1L-3菌株與B.pumilus strain KL-052比對之結果， 核苷酸相似度為93﹪..................................39 表3-9 30H1L-5菌株與B.pumilus strain KL-052比對之結果， 核苷酸相似度為97﹪..................................40 表3-10 所選擇的17株菌種與30H1L菌株的核苷酸相似度的比較..41 圖1-1 2500倍的SEM鑽泥電顯圖 .............................2 圖1-2 15000倍的SEM鑽泥電顯圖。為圖1-1之局部放大，可 見直徑約0.6μm以孢子方式存在之球狀菌體 ............2 圖1-3 本研究試驗鑽探井之地質剖面簡圖(資料來源：中國石 油公司台灣油礦探勘總處鑽井隊，1999) ................8 圖1-4 台灣西北部油氣田分布圖(取自林麗華及郭政隆，1998)....9 圖1-5 全球性海水變化曲線(中新世至現今)(取自Haq et al., 1987)，以及台灣地區中新統地層對比(楊耿明及丁信修 ，1997)........................................... 10 圖2-1 研究流程圖.........................................13 圖3-1 不同試管培養基在30日內之沉降速率變化.............. 30 圖3-2 左邊試管為鐵還原菌之MSA培養基，其固態物質無法被磁 鐵所吸起；右邊試管則為培養後產生能被磁鐵所吸引之黑 色生成物 ..........................................31 圖3-3 膠電泳結果圖，膠電泳實驗顯示五個菌株(30H1L-1、 30H1L-2、30H1L-3、30H1L-4、30H1L-5)的band皆分 布於接近1500bp的位置............................. 35 圖3-4 30H1L菌株與其他菌株(如表3-10)間之親緣關係。30H1L 菌株與Bacillus pumilus A586最為相近，可確定30H1L 菌株為Bacillus屬。其橫條數字0.01代表每100個鹼基 有一個相異 ........................................42 圖3-5 在1000倍光學顯微鏡下可見30℃厭氧性發酵菌之TYG 培養基所培養出之藍紫色革蘭氏陽性球菌 ..............44 圖3-6 在1000倍光學顯微鏡下可見30℃喜氧性發酵菌之TYG 培養基所培養出之粉紫色革蘭氏陰性桿菌 .............. 44 圖3-7 在11000倍之SEM觀察下可見許多長約0.46μm的似菌體 鑲嵌在鐵還原菌之MSA培養基固態物中…...............45 圖3-8 在15000倍之SEM觀察下可見一長約3.67μm、寬約0.67 μm的桿狀似菌體，與似菌體接觸的乃是鐵還原菌之MSA 培養基中的固態物...................................45 圖3-9 在30000倍之TEM觀察為喜氧性發酵菌之TYG培養基所得 的30H1L革蘭氏陰性菌之TEM照片。菌體長約為3.33μm 、寬約0.83μm，其中箭頭所指乃陰性菌獨有的外膜.....47 圖3-10 在20000倍之TEM觀察30H1L岩樣經厭氧性發酵菌之TYG 培養基所得之革蘭氏陽性球菌之TEM照片，菌體直徑約為 0.75μm ...........................................48

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