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標題: | 東沙島潟湖沉積物中固氮細菌及氮清除細菌的分離和特性研究 Isolation and characterization of nitrogen-fixing/nitrogen-scavenging bacteria from a lagoon in Dongsha Island |
作者: | Yen-ting Chen 陳彥婷 |
指導教授: | 謝文陽 |
關鍵字: | 沉積物,平板計數法,東沙,固氮菌,16S rRNA基因, Dongsha Island,lagoon,seagrass-bed sediment,nitrogen-fixing bacteria,nitrogen-scavenging bacteria,16S rRNA gene, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 固氮作用是氮循環中一個重要環節,自然界可利用氮源大多經由具有固氮能力細菌行生化作用,固氮細菌的種類多元,並普遍存在海洋各種環境中。
本研究針對生長在台灣西南海域中東沙島小潟湖的海草床以及非海草床沉積土中固氮細菌以及氮源清除細菌進行了分離和分類探討。 使用NFG (nitrogen-free glucose) 液體培養基在厭氧條件下增菌培養,配合並利用最大可能計數法 (most-probable-number counts) ,估得潟湖沉積物中固氮細菌/氮源清除菌在不同海草床沉積物的菌數介於 2.8×〖10〗^3至 4.6×〖10〗^5 cells/g wet wt.,在非海草床沉積物的菌數介於 1.0×〖10〗^1至 7.0×〖10〗^3 cells/g wet wt.。 從培養液明顯變混濁試管共分離出64株固氮/氮源清除細菌,進行其各種生理生化特性檢測後發現,分離株大部分為革蘭氏陰性菌,只有一株為革蘭氏陽性。利用16S rRNA 基因列比對進行親源分析,可將64株分離株分為17個屬層級菌群,其中Vibrio為最大組成菌屬。利用固氮酶(nitrogenase)基因(nifH) 中保守區域引子對,透過聚合酶反應 (polymerase chain reaction) 分析固氮酶基因序列多樣性,從歸類為 Vibrio、Shewanella、Nesiotobacter以及Nitratireductor等屬之 15 株細菌檢測出nifH基因,其中以Vibrio占最多 (12株)。依照16S rRNA 基因序列比對結果,共有5株含有固氮基因分離株代表株與最相近標準株序列相似度低於區分種間閾值(98.7%),為潛在新種固氮菌,其中代表株A3以及 B3-1 與最相近標準株序列相似度低於可區分細菌屬間的閾值(95.0 %),再比對相關生理、生化特性後,初步研判具有發表為新屬潛力。 Nitrogen fixation is an essential step in nitrogen cycle since atmospheric N2 is inaccessible to most organisms, therefore it is the main process that N2 gas is transformed into ammonia. N2 fixation is exclusively carried out by specialized bacteria which are called diazotrophs. Diazotrophs exist in a diverse marine environment. The aim of the study was to isolate and characterize the nitrogen-fixing/nitrogen-scavenging bacteria from a lagoon in Dongsha Island. The abundance of the nitrogen-fixing/nitrogen-scavenging bacteria in seagrass-bed and non-seagrass-bed sediments were enumerated by the most-probable-number (MPN) method and incubated in NFG (nitrogen-free glucose) media under anaerobic condition. The values of MPN ranged between 2.8×〖10〗^3and 4.6×〖10〗^5 cells/g wet wt. in seagrass-bed sediments and between 1.0×〖10〗^1 and 7.0×〖10〗^3 in non-seagrass-bed sediments. Sixty-four nitrogen-fixing/nitrogen-scavenging bacteria isolates were isolated. Based on their physiological and biochemical characteristics, most isolates were Gram negative, only one isolate was Gram positive. According to phylogenetic analysis of the 16S rRNA gene, all isolates were divided into 17 groups based on genus level. Majority of them were the genus Vibrio consisted the majority. The primer pairs in conserved regions of nitrogenase gene (nifH) were used to determine the nitrogen-fixing isolates with PCR. Fifteen isolates belonging to Vibrio, Shewanella, Nitratireductor and Nesiotobacter genus had the nifH gene, and most of them were from the genus of Vibrio. Five strains containing amplified nifH gene shared low similarity of 16S rRNA sequences with the known species, which might be classified as novel nitrogen-fixing species. Among them, strain A3 and B3-1 might be proposed two species in a novel genus. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67375 |
DOI: | 10.6342/NTU201702499 |
全文授權: | 有償授權 |
顯示於系所單位: | 海洋研究所 |
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