- 學位論文
香山濕地的非發酵性兼性厭氧細菌之分離與特性探討
Isolation and Characterization of Nonfermentative Facultative Anaerobic Bacteria from Shiangshan Wetland
摘要
海岸濕地是水陸交界的過渡地帶，當濕地被水浸潤時，微生物代謝時快速消耗氧氣，形成微氧甚至是無氧狀態。非發酵性兼性厭氧細菌除了可以有氧生活外，在溼地厭氧環境下可還原許多無機物質例如硝酸 (NO3-)、鐵 (Fe3+)、錳 (Mn4+)硫酸 (SO42-)，扮演溼地裡無機物循環重要的角色之一。 本研究於2007與2008年間於香山濕地客雅水資源回收中心施工區周邊及海山罟附近的海埔地採樣，樣本經序列稀釋後，注入PYG (Peptone-Yeast-Glucose）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）估得非發酵兼性厭氧細菌之生菌數約3.4×104∼2.4×106 cells /ml。 利用PYG (peptone-yeast-glucose)培養基在厭氧條件下篩選出不產酸的細菌，之後使用連續劃線法 (streak plate method)在有氧狀態下進行純化、保存、與繼代培養，總共分離出45株分離株，有31株均會行脫氮生活，且脫氮細菌生菌數 (約為3×104∼9.2×106 cells/ml)與非發酵兼性厭氧細菌之生菌數相當，推論本研究之非發酵性兼性嫌氣厭氧細菌之生態棲位以脫氮為主。 將分離株之16S rRNA gene以內切限制酶RsaΙ、AluΙ、DdeΙ進行限制片段多型性分析 (restriction fragment length polymorphism)，根據其圖譜總共可分為12種基因型。12種基因型代表株經16S rDNA定序，與NCBI blast資料庫之已發表菌株序列比對，可歸類為Marinobacter、Shewanella、Pseudoalteromonas、Marinobacterium、Lysinibacillus、Idiomarina、Halomonas、Marinomonas這八個菌屬，以Marinobacter為主。其中，比較第IV群代表株C19與相似菌種的16S rDNA序列與生理生化特性，初步研判為新種。
並列摘要
Coastal wetland is a transitional zone of land margin and water bodies. When wetland was inundated, respiring microorganisms quickly consume oxygen that is dissolved in the water or trapped in the soil. This causes suboxic or even anaerobic conditions. Nonfermentative facultative anaerobic bacteria can use either oxygen or other terminal electron acceptors such as nitrate (NO3-), oxidized iron (Fe3+), manganese (Mn4+) and sulfate (SO42-) and then cause nitrogen cycle, iron cycle, manganese cycle, and even sulfur cycle. They play an important role in mineral cycles. The samples were obtained from the coastal ecosystem near Koya Water Resource Recycling Center and Haishangu during years 2007 and 2008. After serial dilution, the samples were injected into PYG (Peptone-Yeast-Glucose) broth medium, and then incubated under anaerobic condition. The abundance of nonfermentative facultative anaerobic bacteria were evaluated by the method of most-probable-number (MPN) in PYG broth, and the MPN values were ranging from 3.4×104∼2.4×106 cells /ml. Forty-five nonfermentative isolates are obtained from PYG (Peptone-Yeast-Glucose) broth medium. We use streak plate method to get pure culture and then stored pure isolates in PY stab. There are thirty-one denitrifying isolates. In addition, it suggested most of nonfermentative facultative anaerobes are denitrifying bacteria according to the counting values of denitrifiers (3×104∼9.2×106 cells/ml) and nonfermentative facultative anaerobes (3.4×104∼2.4×106 cells /ml) in this study. Forty-five 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 eight genera, among them Marinobacter is the major, according to phylogenetic analysis of 16S rDNA sequences of representative isolates. The phenotypic properties of C19 are more similar to Pseudoalteromonas haloplanktis than to the most similar species. However, the C19 shows low 16S rDNA identity of 97.8 % with Pseudoalteromonas haloplanktis. It is necessary to study futhur to tell whether C19 is a novel species or not.