Diversity of denitrifying bacteria in the sediments from Shiangshan Wetland

脫氮細菌是自然界氮循環中脫氮作用的主導者，其中有許多種類能夠有效率地處理高氮量有機污染物，它們對環境之貢獻力備受學界關注。本研究於2006年至2007年間，在新竹香山濕地客雅水資源回收中心預定地及海山罟附近的海埔地，進行沉積物中脫氮細菌多樣性調查。採得樣本經序列稀釋後，注入PYN（polypepton-yeast-nitrate）液體培養基，進行厭氣增菌培養，並利用最可能計數法（most-probable-number counts）估得脫氮菌數約介於2.9×103至4.6×107 cells/g wet wt.。4株脫氮分離株之16S rDNA由三種限制內切酶（RsaI、AluI和DdeI）反應後，依據個別RFLP圖譜，可將所有菌株歸納為12種基因型；而以16S rDNA序列為基礎的親緣關係分析顯示，各基因型代表菌株分屬於7個不同菌屬，而多數歸類為Marinobacter和Halomonas兩屬。據16S rDNA序列比對結果，分離株N26與Oceanicola batsensis有最相近序列相似度（97.2%），Maritimibacter alkaliphilus（95.9%）次之，然而後者與N26的生理生化特性較為近似，但N26卻具有運動能力和兼性嫌氣的特性，迥異於此二種細菌。另ㄧ分離株N41的生理生化特性與最相近菌種Marinobacter sediminum（16S rDNA序列相似度97.3%）有較大的差異，特別是N41為兼性嫌氣性，與Marinobacter sediminum絕對好氣的特性不符。經由多元分類探討，初步研判N26和N41有可能為新種的海洋細菌。脫氮基因研究方面，利用亞硝酸根還原酶基因（包括nirS和nirK）中保守區域的引子對，配合聚合酶連鎖反應，調查各基因型分群之脫氮菌株具有的nir基因型式。在本實驗中，屬Marinobacter和Idiomarina分群之細菌檢測出nirS基因；屬Photobacterium分群之細菌則具有nirK基因，經序列分析後，與EMBL資料庫中最相近的胺基酸演繹序列為Shewanella loihica的NirK，相似度為78 %。其他包括Halomonas等六個基因型分群則未偵測到nir基因的存在。Denitrifying bacteria are predominant species in global nitrogen cycle. Since many species can treat organic pollutant containing high nitrogen efficiently, so their contribution to environment was gaining attention by the microbial ecologists. This study was aimed to investigate the diversity of denitrifying bacteria in the sediments from Shiangshan Wetland. The soil samples were obtained from the coastal ecosystem near Koya Water Resource Recycling Center and Haishangu during years 2006 and 2007. After serial dilution, the samples were injected into PYN (polypepton-yeast-nitrate) broth medium, and then incubated under anaerobic condition. The abundance of denitrifying bacteria were calculated by the method of most-probable-number (MPN) in PYN broth, and the MPN values were found ranged from 2.9×103 to 4.6×107 cells/g wet wt.ixty-four denitrifying isolates are divided into twelve groups, according to the restriction fragment length polymorphisms (RFLP) analysis with three endonucleases, RsaI, DdeI and AluI. These bacteria are belonging to seven genera, among them Marinobacter and Halomonas were the major, according to phylogenetic analysis of 16S rDNA sequences of representative isolates.26 and N41 shared low identities of 16S rDNA sequence with the known species. However, the phylogeneic relationship between N26 and Oceanicola batsensis was closer than other known species, but the phenotypic properties of N26 were similar to Maritimibacter alkaliphilus. Furthermore, N26 was motile and facultative anaerobic, which differed from Oceanicola batsensis and Maritimibacter alkaliphilus. Strain N41 compared with other known species showed the highest 16S rDNA sequence identity of 97.3% with Marinobacter sediminum R65T. However, N41 was facultative anaerobic but M. sediminum was aerobic. Polyphasic data accumulated in this study revealed that the two denitrifying isolates could be classified as two novel species. Besides diversity and phylogenetic analysis of denitrifying bacteria, this study also used 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 Marinobacter and Idiomarina genera had nirS gene, and one isolate (N20) belong to Photobacterium genus had nirK gene. This nirK gene was sequenced and shared 78% amino acid identity to NirK of Shewanella loihica. Other six genotypic groups including Halomonas could not detect either of the Nir gene with these primer pairs.誌謝 I文摘要 II文摘要 III一章 前言 1一節 脫氮細菌在海洋環境中的分布 2二節 脫氮細菌的分子研究方法 3三節 脫氮細菌和海洋環境間的相互關係 4四節 問題與未來展望 6五節 研究背景 7六節 研究動機 7二章 材料與方法 9一節 培養基 9二節 樣本採集 9三節 培養、計數與分離 9四節 脫氮能力之檢測 10五節 16S rDNA基因型分析 10六節 脫氮基因（nirS和nirK）之分析 14七節 分離株之形態及生理、生化、化學特性檢測 16三章 結果 22一節 計數 22二節 16S rDNA-RFLP分析 22三節 脫氮活性與脫氮基因之序列分析 23四節 各脫氮細菌16S rDNA-RFLP分群代表株之序列分析 23五節 脫氮分離株之生理生化特性分析 25四章 討論 27一節 計數 27二節 脫氮細菌多樣性之探討 29三節 親緣演化與脫氮特性關聯性之探討 31四節 脫氮作用與脫氮基因之探討 33五節 脫氮分離株N26和N41與相近菌種的比較 34五章 結論 36考文獻 37 46 52表 61錄 6