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標題: | 東沙島潟湖沉積物中硝酸還原/脫氮細菌的分離和特性探討 Isolation and characterization of nitrate reducing/denitrifying bacteria from the lagoon sediment of Dongsha Island |
作者: | Hao-Ting Hsia 夏浩庭 |
指導教授: | 謝文陽(Wung-Yang Sheih) |
關鍵字: | 東沙島,潟湖,脫氮作用,硝酸還原, Dongsha Island,lagoon,denitrification,nitrate-reducing, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 東沙環礁國家公園為台灣第七座國家公園,範圍包括東沙島以及其外圍圓形環礁,基於生態保育理由,目前未開放一般民眾登島,島上環境因而幾乎沒有人為干擾,能忠實呈現原始樣貌。東沙島內潟湖為多種海草分布地,此潟湖環境中氮元素循環跟存在此間沉積物中細菌密切相關。本研究首先針對東沙島潟湖海草床沉積物中與氮循環脫氮過程有關的硝酸還原細菌和脫氮細菌進行菌株分離,以及分離株生理、生化特性的初步探討。樣本稀釋液接種到內含有倒置 Durham tube 之 polypeptone-yeast extract-nitrate (PYN) broth 試管中以MPN法估算菌數,之後藉由平板四區連續畫線方式純化分離出 64 株細菌。針對分離株進行革蘭氏反應、發酵產酸、酵素活性、以及不同生長能力等測試以及16S rRNA基因序列分析,最後依序列結果進行菌株分群並挑選各群代表株進行硝酸還原以及脫氮基因檢測 。MPN計數的部分,硝酸還原菌數介在1.1 × 107至2.4 × 109 cells/g wet wt.之間,而脫氮菌數則介在3.0 × 101 至 4.3 × 107 cells/g wet wt.之間,同時發現樣點間有無海草生長會影響估算菌數。依照片段16S rRNA 基因序列定序的結果,64株分離株屬於Pseudoalteromonas, Vibrio, Marinobacter, Marinobacterium, Shewanella, Neptunomonas, Idiomarina, Alcanivorax以及 Halomonas等 9屬菌種,依此結果分群並挑選23株菌株為分群代表株,再次確認其脫氮能力並進行脫氮基因相關實驗以及硝酸還原化學試劑檢測。結果顯示東沙島潟湖沉積物中,脫氮細菌以Marinobacter屬的細菌為主,而硝酸還原細菌則是以Vibrio屬的細菌為主要菌群,且此結果與前人研究比較可發現不論樣點位於台灣本島或離島,或是陸源性氮源的有無,其主要脫氮菌群皆為Marinobacter屬的細菌。藉由本研究結果可初步了解並記錄東沙島潟湖中參與氮元素循環中硝酸還原和脫氮過程的細菌種類,未來可對其脫氮效率做進一步研究或作為該處微生物研究之基礎。 The Dongsha Atoll National Park is the seventh national park in Taiwan. So far, the atoll remains close for tourism, and thus the environment is still in relatively pristine state. Two sand ridges extend to the west of the island embracing a small lagoon which connect to the main lagoon of the atoll. This lagoon is an important habitat for sea grasses and sedimentary bacteria closely related to the nitrogen cycle. In this study, we isolated the nitrate-reducing or denitrifying bacteria sampled from Dongsha small lagoon. Preliminary biochemical and physiological characterizations of the isolates were performed. We used most-probable-number (MPN) to estimate the abundance of denitrifying and nitrate reducing bacteria. After serial dilutions, samples were inoculated in polypeptone-yeast extract-nitrate (PYN) broth medium. After incubation, the MPN value of nitrate-reducing bacteria was between 1.1 × 107 to 2.4 × 109 cells/g wet wt. and the value of denitrifying bacteria was between 3.0 × 101 to 4.3 × 107 cells/g wet wt. According to the phylogenetic analysis of the partial 16S rRNA gene, sixty-four isolates belong to nine genera, including Pseudoalteromonas, Vibrio, Marinobacter, Marinobacterium, Shewanella, Neptunomonas, Idiomarina, Alcanivorax and Halomonas. We also used specific primer pairs in conserved regions of nitrite reductase gene (nirS and nirK) to determine the nir gene type of denitrifying or nitrate reducing isolates with PCR. The result showed that Marinobacter and Vibrio were the major genera of bacteria which participate the denitrification in the lagoon of Dongsha Island. Compared with previous studies, whether there were contenetal nitrogen source or not, bacteria of Marinobacter played key role in denitrification. This study provides important information about bacteria which involve in nitrogen cycle in Dongsha lagoon and can be an useful basic knowledge for microbial research in future. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67799 |
DOI: | 10.6342/NTU201701956 |
全文授權: | 有償授權 |
顯示於系所單位: | 海洋研究所 |
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