東沙島潟湖兼性嫌氣性發酵細菌的分離及特性研究

Isolation and characterization of facultatively anaerobic, fermentative bacteria from lagoon of Dongsha Island

陳姿尹(Tzu-Yin Chen)

指導教授 ： 謝文陽

國立臺灣大學/理學院/海洋研究所/碩士(2017年)

https://doi.org/10.6342/NTU201702344

摘要

發酵作用是微生物於嫌氣情況下，分解有機物並得到能量的過程。葡萄糖是海洋環境中最豐富的有機物質之一，許多海洋細菌可以透過代謝海水中葡萄糖而參與碳元素循環。本研究目標在於東沙島潟湖具有發酵葡萄糖能力細菌之分離及特性探討，並分析不同種類沉積物細菌的親緣關係，且調查潟湖中兼性嫌氣性發酵細菌之生物多樣性。最大可能計數法 (most-probable-number counts)顯示，潟湖沉積物兼性嫌氣性發酵細菌數量介於2.4 x 105 - 2.4 x 107cells/g wet wt.之間，其中，海草碎屑、珊瑚碎屑、泰來草與圓葉水絲根圈沉積物的菌數最多。從樣本中共分離出42株兼性嫌氣性發酵細菌，皆為革蘭氏陰性、嗜鹽性，且可以生長於25 – 30℃、pH 6-9，與1 - 5%氯化鈉，大部份菌株具有過氧化氫酶與氧化酶活性。16S rRNA基因序列親緣關係分析顯示，這些細菌皆為Gammaproteobacteria綱，並可進一步分類為Halomonadaceae科的Cobetia屬、Oceanospirillaceae科的Neptunomonas屬，與Vibrionaceae科的Grimontia、Aliivibrio、Vibrio屬。其中，83.3% (35/42)的分離株被歸類為Vibrio細菌。菌株A4、A5與其最相似菌種之16S rRNA基因序列相似度僅92.0 %，因此被推測為新屬且新種的細菌。親緣關係分析顯示，這二株新屬細菌屬於Aeromonadaceae科，並暫時將之命名為Dongshaea marina。此外，菌株B1、H2與H5之16S rRNA基因序列相似度與它們最相近Vibrio標準菌株比較，皆低於97.3 %，這三株菌株未來亦可能被發表為Vibrio屬之新種細菌。綜合以上所述，本研究顯示，Vibrio為大部份潟湖沉積物樣本中可培養兼性嫌氣性發酵細菌的優勢菌屬。然而，在無植物生長的一般沉積物樣本發酵菌中，則以Aliivibrio與Neptunomonas為優勢。所有本研究分離之菌株，皆為東沙島潟湖沉積物中發酵作用的潛在參與者。

關鍵字

東沙島 ； 潟湖 ； 醣類 ； 發酵細菌 ； 16S rRNA基因

並列摘要

Fermentation is the process that microorganisms decompose organic materials and gain energy under anaerobic conditions. Glucose is one of the most abundant organic matters in the marine environment. Many of the marine bacteria may involve in carbon cycling through metabolizing glucose in the seawater. The objectives of this study were to isolate and characterize the glucose-fermenting bacteria from lagoon of Dongsha Island,to analyze the phylogenetic relationships of bacteria in different types of sediments and to investigate the biodiversity of facultatively anaerobic, fermentative bacteria in the lagoon. Most-probable-number (MPN) counts revealed that the facultatively anaerobic, fermentative bacteria in the lagoon sediments ranged from 2.4 x 105 to 2.4 x 107 cells/g wet wt. Higher counting values were obtained in samples from seagrass debris, coral rubble,and the sediments surrounding the rhizosphere of Thalassia hemprichii and Cymodocea rotundata. Forty-two strains of facultatively anaerobic, fermentative bacteria were isolated from the samples. All strains were Gram-negative, and halophilic. All of them grew in 25-30 ℃, pH 6-9, and 1-5 % NaCl. Majority of these strains showed catalase and oxidase activities. Phylogenetic analysis based on 16S rRNA gene sequences revealed that all isolates belonged to class Gammaproteobacteria, which could be further classified as members of the genusCobetia within family Halomonadaceae, the genusNeptunomonas within family Oceanospirillaceae, and thegenera Grimontia, Aliivibrio, andVibriowithin familyVibrionaceae.Among these isolates, 83.3 % (35/42)were classified to be Vibrio species. Strains A4 and A5might be proposed to represent a novel genus and species in the future since they had16S rRNA gene sequence similarity below 92.0 % compared with their most closely relatedspecies. Phylogenetic analysis revealed the potentially new genus bacteria belonged to family Aeromonadaceaeand were named Dongshaea marina tentatively. Additionally,strain B1, H2, and H5 had 16S rRNA gene sequence similarity below 97.3 % in comparison to the type strains of the closestVibrio species.These three strains also might be proposed to be novel Vibrio species in the future.In summary, this study showed that Vibrio was the dominant genus in culturable facultatively anaerobic, fermentative bacteria within most lagoon sediment samples. However, in bulk sediment sample, Aliivibrio and Neptunomonas were the dominant fermentative bacteria. All strains isolated in the study would bepotential participants in fermentation process in lagoon sediment of Dongsha Island.

並列關鍵字

Dongsha Island ； lagoon ； carbohydrate ； fermentative bacteria ； 16S rRNA gene

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