理學院: 海洋研究所指導教授: 謝文陽羅政為Lo, Cheng-WeiCheng-WeiLo2017-03-032018-06-282017-03-032018-06-282015http://ntur.lib.ntu.edu.tw//handle/246246/274071脫氮作用為氮循環中的一個重要環節，具有脫氮能力的細菌在自然界的脫氮作用中扮演著非常重要的角色，脫氮細菌的種類繁多，其中有許多種類在沿海河口生態系中能夠大量的移除含氮汙染物。本研究針對生長在台灣香山濕地河口的日本鰻草，其根圈沉積物中脫氮細菌菌相進行研究。使用最大可能計數法(most-probable-number counts) 和 PYN ( polypepton-yeast-nitrate) 液體培養基估算日本鰻草根圈沉積物中的脫氮菌數。估得脫氮細菌在根圈沉積物中的菌數介7.5 x 103至1.5 x 105cells/g wet wt. 間。並且由沉積物樣本中分離得到65株脫氮菌分離株。 65株脫氮菌分離株進行各種生理生化特性檢測後發現，所有分離株皆屬於革蘭氏陰性菌，其中33株具鈉鹽需求性，應屬於海洋原生種細菌。65株分離株進行16S rRNA基因定序，分析定序結果後，脫氮菌分離株分別屬於9個菌屬，其中以 Marinobacter 和 Shewanella 兩菌屬為主要組成。根據16S rRNA序列比對結果，分離株A41與最相近標準株 Halomonas mongliensis的序列相似度為97.6%，以及B10-1與最相近標準株Pseudoalteromonas shioyasakiensis 的序列相似度為98.3% ，皆明顯低於可區分細菌種與種間的閾值 (98.65%)，分離株A41及 B10-1的生理生化特性也與其相關標準株有明顯不同。在多元分類探討的前提下，初步研判分離株A41及B10-1有可能為新種的脫氮細菌。在脫氮基因研究方面，利用亞硝酸根還原酶 (nitrite reductase) 基因 (nirK 和 nirS) 中的保守區域引子對，透過聚合酶連鎖反應 ( polymerase chain reaction) 分析亞硝酸根還原酶基因序列的多樣性。在本實驗中，歸類為Escherchia, Shewanella, Photobacterium, Pseudoalteromona和 Marinobacter分群之細菌可檢測出nirS基因，可成功定序的分離株之nirS 基因序列經分析後，與NCBI資料庫中最相近的基因序列相似度都在95 % 以上。屬Vibrio和Ochrobactrum 分群之細菌則具有nirK基因，可成功定序的分離株之nirK 基因序列經分析後，與NCBI資料庫中最相近的可培養菌株基因序列相似度則低於84%。Denitrification is a microbially catalyzed respiration process and plays an important role in nitrogen cycle. Microorganisms that transfer redox equivalents from the oxidation of a carbon source to an N oxide under anaerobic conditions are known as denitrifers. Denitrfying bacteria are impotant dentrifiers in global nitrogen cycle and they are known to be phylogenetically diverse. Many species of dentrifying bacteria can remove organic pollutant in costal regions or estuaries efficiently . This study aims to isolate and characterize the denitrifying bacteria from a Zostera japonica dominated seagrass bed in Siangshan Wetland. We use the method of most-probable-number (MPN) to estimate the abundance of denitrifying bacteria. After serial dilution, the sediment samples were inoculated into PYN (polypepton-yeast-nitrate) broth medium, and then incubated under anaerobic condition at 25oC. After the incubation, the MPN values were ranged from 7.5 x 103 to 1.5 x 105cells/g wet wt. Sixty-five denitrifying bacteria were isolated from all sample . According to their biochemical and physiological characteristics, all isolates are Gram negative and thirty isolates required sodium ion for growth. All isolates were divided into nine groups, according to the phylogenetic analysis of the 16S rRNA gene . These isolates belong to nine genera, among them Marinobacter and Shewanella were the major group. A41 and B10-1 shared low similarity of 16S rRNA sequence with the known species. However, the phenotypic properties between A41 and its phylogenetic related strains were different. After comparison, B10-1 also had the same result. According to polyphasic data gained in this study , these two denitrifying isolates might be classified as two novel denitrifying species by furthur researches. Besides phenotypic properties characterization and phylogenetic analysis of denitrifying bacteria, this study also used specific primer pairs in conserved regions of nitrite reductase gene (nirK and nirS) to determine the nir gene type of denitrifying isolates with PCR. Some isolates belong to Escherchia, Shewanella, Photobacterium, Pseudoalteromona and Marinobacter had nirS amplicons, and two isolates belong to Vibrio and Ochrobactrum had nirK gene. NirS amplicons belong to Shewanella and Escherichia were sequenced and both showed more than 95% identity to nirS gene in NCBI database. NirK amplicon belonged to Ochrobactrum was sequenced and show less than 84% identity to nirK gene of culturable bacteria in NCBI database.3512963 bytesapplication/pdf論文公開時間: 2015/12/1論文使用權限: 同意有償授權(權利金給回饋學校)香山濕地日本鰻草脫氮細菌沉積物根圈WetlandZostera japonicadenitrificationseagrassbeddenitrifying bacteriarhizosphere[SDGs]SDG15thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/274071/1/ntu-104-R02241210-1.pdf