臺灣博碩士論文加值系統

本實驗取自丹大溫泉、鹿角坑溫泉、四稜溫泉及烏來溫泉的樣區水中，經篩選分離、培養在 50 oC後，發現上述四個樣區的溫泉水中皆可篩選出 12 株相近粉紅色的細菌，將這些細菌的 16S rDNA 保守序列片段與 NCBI databases 資料庫比對。經過初步的基本測試後，挑選了編號NTU-400、NTU-402、NTU-1162、NTU-1290、NTU-1611五株菌株來做研究。根據形態、生理、生化實驗及16S rDNA序列結果分析可將這五株菌分為兩群，此兩群菌株彼此間相似度為92.5 % ~ 93.4 %。第一群包括NTU-400、NTU-402，此兩株菌為粉紅色菌落革蘭氏陰性球菌，與 Roseomonas gilardii ATCC49956T 16S rDNA相似度分別為 94.3 %及 94.2 %，且彼此之間的相似度為 99 %，DNA G＋C 含量為 68.6及 70.2 mol%；第二群包括實驗菌株 NTU-1162、NTU-1290、NTU-1611 此三株菌為粉橘色菌落革蘭氏陰性短桿菌，與 Roseomonas lacus TH-G33T 16S rDNA 親緣相似度分別為 96.8 % 及 96.7 % 及96.1 %，且彼此之間的相似度為 99.0 % ~ 99.7 % 之間，DNA G＋C 含量為 60.4 ~ 64.1 mol%。所有分離株皆有運動性，單一鞭毛，且最適生長條件為 30 ~ 50 oC，pH 5 ~ pH 12；在酵素的測試方面，皆有很強的氧化酶反應、觸酶活性alkaline phosphatase、esterase。脂肪酸組成多以 16：0、18：0、18：1 為主；在抗生素抗性的測試部份，所有的菌株對於抑制細胞壁合成之抗生素 vancomycin，抑制細胞膜功能之抗生素 polymyxin B、colistin 及抑制核酸合成之抗生素nalidixic acid有較好的抗性。藉由本實驗鑑定的結果建議：第一群實驗菌株 NTU-400 與 NTU-402 可分類為 alphaproteobacteria 的新菌屬；而第二群實驗菌株 NTU-1162、NTU-1290、NTU-1611 則分類為Roseomonas 屬中另一新菌種。

In this study, the samples of hot springs CL, WR, LGK, DD were serially diluted, spread on modified Thermus medium and further incubated at 50 oC. In total, 12 strains were obtained from 4 hot spring samples. The 16S rDNA sequences of 12 strains were determined, and then analyzed in NCBI database of 12 strains. Five strains NTU-400, NTU-402, NTU-1162, NTU-1290, and NTU-1611 were selected to be representatives for further research, which demonstrated with 93 % ~ 94 % similarity of 16S rDNA sequences to Roseomonas gilardii ATCC49956T. According to the 16S rDNA sequences and morphological, physiological, and biochemical experiments, the isolated strains can be clustered into two groups. The two groups showed 92.5 % ~ 93.4 % similarity of 16S rDNA sequences each other. Group I including experimental strains NTU-400 and NTU-402 showed 94.3 % and 94.2 % similarity to Roseomonas gilardii ATCC49956T respectively and 99.8 % each other. Both strains are Gram-negative, pink-pigmented, cocci and the DNA G＋C contents were 68.6 and 70.2 mol% . Group II including NTU-1162, NTU-1290 and NTU-1611 strains showed 96.8 %, 96.7 % and 96.1 % similarity of 16S rDNA sequences to Roseomonas lacus TH-G33T respectively, and 99.0 % ~ 99.7 % each other. The strains are gram negative, orange-pigmented, rods and the DNA G＋C contents are 60.4, 62.5 and 64.1 mol% . All the isolates have a single polar flagellum, the optimal growth temperature at 30 ~ 50 oC, PH 5 ~ 12 and maximal growth temperature at 55 oC. Oxidase, catalase, alkaline phosphatase and esterase are evidently positive. They are positive in reactions of β - hemolysis. The major compositions of cellular fatty acids are C16:0, C18:0 and C18:1. These isolates were resistant to the Vancomycin (30 μg), Polymyxin (300 U), Colistin (10 μg) and Nalidixic acid (30 μg). According to the morphological, physiological and genetic results, the group I can be classified as a new genus of alphaproteobacteria, while group II can be a new species of genus Roseomonas.

致謝
中文摘要 i
英文摘要 iii
表目錄 v
圖目錄 vi
第一章 前言
一、微生物 1
二、嗜熱性微生物 3
三、台灣的地熱環境 6
四、台灣嗜熱性微生物之研究 7
五、Roseomonas 菌屬 8
六、研究目的 10
第二章 材料與方法
一、材料
（一）實驗菌株 12
（二）藥品與酵素 12
（三）培養基 12
（四）儀器 13
二、方法
（一）樣本採集 15
（二）嗜熱菌之分離、純化與保存
1. 菌株之分離與純化 15
2. 菌株之保存 15
（三）菌種鑑定
1. 形態特性觀察 17
2. 生理特性分析 20
3. 遺傳分析 27
第三章 實驗結果
一、實驗菌株 33
二、 形態特性 33
三、 生理特性 35
四、 脂肪酸組成分析 40
五、 DNA G＋C 含量分析 41
六、 16S rDNA 序列分析 41
七、 16S rDNA 親緣演化分析 42

第四章 討論
一、 微生物的分類方法 43
二、 台灣本土新穎嗜熱性菌種之發現 45
第五章 總結 51
參考文獻 52
圖表 71
附錄一、 菌種鑑定技術及基本流程圖 96
附錄二、API ZYM 套組分析表 97
附錄三、 分離菌株之 16S rDNA 序列 98

陳懋彥 2002 台灣嗜熱性細菌之研究。國立台灣大學植物學研究所博士論文

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