Isolation and characterization of denitrifying bacteria within a seagrass bed (Zostera japonica) in Siangshan Wetland

• Main Authors: Cheng-Wei Lo, 羅政為
• Other Authors: 謝文陽
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/15041432018971852651

碩士 === 國立臺灣大學 === 海洋研究所 === 104 === 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.