綠島沿岸熱泉內 Marinobacter spp. 及 Hydrogenophilus sp. 新穎嗜熱菌之研究

Studies on Novel Thermophilic Marinobacter spp. and Hydrogenophilus sp. Isolated from Coastal Hot Springs in Lutao, Taiwan

陳泰晏(Tai-Yen Chen)

指導教授 ： 蔡珊珊

國立臺灣大學/生命科學院/植物科學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.02257

摘要

本研究中從綠島朝日溫泉以及柚子湖地熱區域，分離篩選出六菌株，分別為NTU-445、NTU-1311、NTU-1438、NTU-1645、NTU-1689以及NTU-1701，經過初步之16S rDNA序列比對，可將這些菌株歸屬於 Marinobacter 以及 Hydrogenophilus 兩屬。第一群為NTU-1645、NTU-1689以及NTU-1701，與 Marinobacter lutaoensis BCRC 17087T 之16S rDNA相似度介於96.5 % ~ 97.2 % 之間，最適生長溫度為50 oC，為皆具單鞭毛，不產生孢子之革蘭氏陰性桿菌，經由外觀形態、生理特性以及遺傳特性等相關實驗，可發現分離菌株與 M. lutaoensis BCRC 17087T 除了16S rDNA序列之外，還有若干之差異性，包括了分離菌株可耐受之鹽度 (NaCl) 可高達15 %，且移動性較明顯，另外Citrate、D-cellulobiose、D-galactose、D-mannose、D-melibiose、D-raffinose、D-sobiotol、D-trehalose、D-xylose、glycerol、maltose 、myo-inostiol、L-arginine、L-ornithine、L-valine、Sodium Benzoate、n-butanol等等碳源之利用也有著明顯之差異，而脂肪酸之組成也有所差異，再加上DNA-DNA 雜合反應結果顯示，分離菌株與標準菌株之相似度皆小於 70 %，因此初步判定分離菌株NTU-1645、NTU-1689以及NTU-1701為 Marinobacter屬之新種菌株。
第二群則包括了NTU-445、NTU-1311以及NTU-1438，與Hydrogenophilus thermoluteolus NBRC 14978T 之16S rDNA序列相似度介於96.6 ~ 97.3 % 之間，最適生長溫度為50 oC，為具單鞭毛，不產生孢子之革蘭氏陰性桿菌，經過相關測試，發現分離菌株與不耐鹽的H. thermoluteolus NBRC 14978T 有明顯的差異，分離菌株具有鞭毛，且菌落呈現棕色，可於5 % 之NaCl以及pH 9之條件下生長，此外分離菌株有明顯之解脂酶活 (Lipase) 活性，另外D-fructose、D-galactose、D-melibiose、L-ramnose、L-proline、L-arginine及L-Alanine等碳源利用也有著顯著差異，且分離菌株也多了Alkaline phosphatase、Acid phosphatase、Naphthol-AS-BI-phosphohydrolase等酵素活性，而脂肪酸之組成也有所差異，且對於抗生素 Streptomycin 之抗性亦有所差異，加上DNA-DNA 雜合反應結果顯示，分離菌株與標準菌株之相似度皆小於 70 %，依照以上之實驗結果可初步判定，分離菌株NTU-445、NTU-1311以及NTU-1438為 Hydrogenophilus屬之新種菌株。

關鍵字

嗜熱菌 ； 綠島

並列摘要

In this study, six strains of thermophilic halotolerant bacteria were isolated from geothermal areas in Lutao, Taiwan. They were NTU-445, NTU-1311, NTU-1438, NTU-1645, NTU-1689 and NTU-1701. After amplifying and sequencing the 16S rDNA of those strains , the sequences were compared on NCBI database. According to the 16S rDNA sequences, it is found that the strains might be classified into two groups : Genus Marinobacter and Genus Hydrogenophilus.
Group I including isolated strains NTU-1645, NTU-1689 and NTU-1701, showed 96.5 % ~ 97.2 % 16S rDNA sequences similarity to Marinobacter lutaoensis BCRC 17087T, and 99.5 % ~ 99.7 % to each other. The optimal growth temperature of isolates was 50 oC. They were motile with single polar flagellum, non-spore forming gram-negative rods. The swarming motility of isolated strains is also more obvious than the type strain. The isolated strains could growth in medium containing 15 % NaCl, and utilitze citrate, D-cellulobiose, D-galactose, D-mannose, D-melibiose, D-raffinose, D-sobiotol, D-trehalose, D-xylose, glycerol, maltose, myo-inostiol, L-arginine, L-ornithine, L-valine as well as n-butanol as sole carbon source, while the type strain could not. Base on the DNA-DNA hybridization (lower than 70 %), the divergence in 16S rDNA (higher than 3 %), and some morphological, physiological, biochemical properties. The isolated strains could be considered as new species of Genus Marinobacter.
Group II including isolated strains NTU-455, NTU-1311 and NTU-1438 showed 96.6 % ~ 97.3 % 16S rDNA sequences similarity to Hydrogenophilus thermoluteolus NBRC 14978T, and 99.1 % ~ 99.7 % to each other. The optimal growth temperature of strains was 50 oC. They were non-spore forming gram-negative rods. The isolated strains were motile with single polar flagellum, and the colonies of isolated strains were brown color. Those morphological properties were different from type strain. The isolated strains could grow in the medium containing 5 % NaCl and pH 9, and utilize D-fructose, D-galactose, D-melibiose, L-ramnose, L-proline, L-arginine and L-alanine as sole carbon source, but type strain can not. The isolated strains also produced the following enzymes: Alkaline phosphatase, Acid phosphatase and Naphthol-AS-BI-phosphohydrolase, and were Streptomycin-resistant. According to the DNA-DNA hybridization (lower than 70 %), the divergence in 16S rDNA (higher than 3 %), and above results, the isolated strains could be considered as new species of Genus Hydrogenophilus.

並列關鍵字

thermophilic ； halotolrant ； hot springs ； lutao ； marinobacter ； hydrogenophilus

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