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研究生:李世軒
研究生(外文):Shih-shuan Li
論文名稱:台灣綠島地熱區新穎耐熱性Exiguobacteriumsp.之研究
論文名稱(外文):Studies on Novel ThermotolerantExiguobacterium sp. Isolated from Geothermal Areas of Lutao, Taiwan
指導教授:呂誌翼蔡珊珊蔡珊珊引用關係
指導教授(外文):Jyh-yih LeuSan-san Tsay
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生命科學系碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:91
中文關鍵詞:耐熱菌菌種鑑定綠島微小桿菌
外文關鍵詞:Exiguobacteriumthermotolerantgreen island
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台灣擁有許多地熱資源,而這些高溫環境中蘊含著豐富的嗜熱性微生物。本研究自台東縣綠島鄉柚子湖地區的高溫海水水樣中,分離純化出耐熱性細菌。根據 16S rDNA gene sequence 定序比對結果篩選出 4 株序列比對較接近的菌株 NTU-1329、NTU-1330、NTU-1331 及 NTU-1337 進行本實驗。
本研究菌株在modified Thermus mediume 固體培養基上培養兩天後,呈現直徑 2-3 mm 的橘黃色、凸面具有光澤的菌落。經分析皆為格蘭氏陽性菌,有周鞭毛具移動性,不產孢子的好氧菌。在對數成長期呈現桿狀,穩定期時會變成球狀,細菌長約 1.16-1.45 μm 寬約 0.65-0.82 μm。最適生長條件為 30 oC,pH 7-11;在生化分析方面,4 株菌皆具有 alkaline phosphatase、esterase、leucine arylamidase 、acid phosphatase、α-glucosidase 、β-glucosidase、catalase、oxidase等酵素活性以及具有skim milk與starch水解的能力。在碳源利用上,主要可利用citrate 、D-mannose、D-xylose、L-glutamate、malate、pyruvic acid、L-proline、L-alaline、L-ornithine。脂肪酸組成主要為C16:0、isoC16:0、aiC17:0 以及 C18:0。
由16S rDNA結果分析顯示此 4 菌株與Exiguobacterium aestuarii 的相似度在 97.9 ~ 98.7 % 之間,在DNA G+C組成上為60.3 ~ 62.9 mol %。
根據形態、生理生化以及遺傳特性分析的結果發現,本研究的 4 株菌株與比對最相近的菌株E. aestuarii確實有差異,在酵素活性測試部分,最相近菌種 E. aestuarii 無 oxidase 與 starch 水解能力,本研究菌株皆有活性。本研究菌株 DNA G+C 組成皆大於 60 % 以上,而 Exiguobacterium 菌屬中目前無發現高於 60 % 的菌種。本研究菌株生長溫度最高可達 50 oC,為目前此屬中最高溫,而耐鹽度最高可達 15 %。因此推測本研究的四株菌株可能成為Exiguobacterium菌屬中的新菌種。
There are many geothermal areas which include variable microorganisms in Taiwan. The study is aimed to isolate the thermophiles from the sample of warm sea water located on Lyudao Township, Taitung County. According to the results of 16S rDNA sequences analyses, four isolates, NTU-1329, NTU-1330, NTU1331, and NTU-1337 were selected for further investigation.
Colonies are 2-3 mm in diameter after incubation for 2 days on modified Thermus agar at 40 oC and yellow-orange convex with entire margin and glossy. Cells are Gram positive, non-spore forming, motile with peritrichous flagella. Cells are short rods (0.73 μm x 1.23 μm) in the early stationary phase. Growth occurs at 30 oC and 50 oC, with optimal growth at 30 oC. The optimal pH for growth is pH 7-11 and growth does not occur in the presence of 16 % NaCl. All strains hydrolyze skim milk and starch. They show alkaline phosphatase, esterase, leucine arylamidase, acid phosphatase, α-glucosidase, β-glucosidase, catalase and oxidase activity. All strains utilize citrate, D-mannose, D-xylose, L-glutamate, malate, pyruvic acid, L-proline, L-alaline and L-ornithine. Major fatty acids are C16:0, isoC16:0, C18:0 and anteisoC17:0 .
The 16S rDNA sequence similarities of NTU-1329, NTU-1330, NTU1331, and NTU-1337 to Exiguobacterium aestuarii are between 97.9 ~ 98.6 %. The DNA G+C contents of the isolates are between 60.3 ~ 62.9 %. On the basis of morphological, physiological and genetic data, there are differences between the four strains and E. aestuarii. It is found oxidase-negative and no hydrolysis of starch in E. aestuarii but not in four strains. The maximal growth temperature and pH of four strains are the highest in the genus of Exiguobacterium. As a result, the four strains might be a novel species in the genus of Exiguobacterium.
中文摘要 I
英文摘要 III
縮寫對照表 IV
第一章 前言 1
一、嗜熱性微生物 1
二、台灣與綠島的地熱環境 2
三、台灣嗜熱性微生物之研究 3
四、Exiguobacterium 4
五、細菌的分類 6
六、研究目的與架構 10
第二章 材料與方法 11
一、材料 11
(一) 菌種 11
(二) 藥品與酵素 11
(三) 培養基 11
(四) 儀器 12
二、方法 13
(一) 樣本採集 13
(二) 嗜熱菌之分離、純化與保存 13
1. 菌種之分離與純化 13
2. 菌種之保存 14
2.1 菌種短期與中期保存 14
2.2 菌種長期保存 14
(三) 菌種分析 15
1. 形態觀察 15
1.1 菌落型態 15
1.2 格蘭氏染色 15
1.2 內孢子染色 16
1.3 移動性測試 16
1.4 穿透式電子顯微鏡觀察 16
2.生理生化特性分析 16
2.1 溫度生長範圍測試 17
2.2 酸鹼度生長範圍測試 17
2.3 耐鹽度測試 17
2.4 單一碳源的利用能力 17
2.5 API-ZYM 酵素活性檢測 18
2.6 澱粉水解能力測試 18
2.7 蛋白酶活性測試 19
2.8 幾丁質水解測試 19
2.9. 幾丁聚醣水解測試 19
2.10 Tween 20 與 Tween 80 的水解測試 20
2.11 氧化酶活性測試 20
2.12 觸酶活性測試 20
2.13 脂肪酸組成分析 21
2.14 抗生素測試 21
3.菌種遺傳特性分析 22
3.1 DNA G+C 含量分析 22
3.2 16S rDNA 序列分析 23
第三章 結果 27
一、嗜熱菌之分離與純化 27
二、菌種鑑定 27
(一) 形態分析 27
1. 菌落形態 27
2. 格蘭氏染色 28
3. 內孢子染色 28
4. 移動性測試 28
5. 穿透式電子顯微鏡觀察 28
(二) 生理特性分析 29
1. 溫度生長範圍測試 29
2. 酸鹼度生長範圍測試 29
3. 耐鹽度測試 29
4. 單一碳源利用能力 30
5. API-ZYM 酵素活性檢測 31
6. 澱粉水解測試 31
7. 蛋白酶活性測試 31
8. 幾丁質水解測試 32
9. 幾丁聚醣水解測試 32
11. 氧化酶活性測試 32
12. 觸酶活性測試 33
13. 脂肪酸組成分析 33
14. 抗生素測試 34
(三) 遺傳分析 34
1. DNA G+C 含量分析 34
2. 16S rDNA 序列分析 34
第四章 討論 36
ㄧ、 Exiguobacterium菌種鑑定 36
二、綠島嗜熱菌之研究 38
三、嗜鹼嗜熱細菌的發展與應用 38
第五章 結論 40
參考文獻 41
圖表 51
附錄一、10X Castenholz basal salt solution 74
附錄二、API-ZYM 酵素套件呈色對照表 75
附錄三、抗生素測試對照表 76
附錄四、分離菌株 16S rDNA 序列 77
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