淡水環境中新穎氨氧化古菌Nitrososphaera sp.之分離及其特性鑑定之研究

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Title:	淡水環境中新穎氨氧化古菌Nitrososphaera sp.之分離及其特性鑑定之研究
Other Titles:	Isolation and characterization of novel ammonia-oxidizing archaea Nitrososphaera sp. from freshwater environment
Authors:	蕭百淳 Hsiao, Pai-Chun
Contributors:	黃啟裕 Huang, Chi-Yu 環境科學與工程學系
Keywords:	氨氧化古菌;氨氧化;硝化作用;Nitrososphaera amoA gene;AOA;ammonia oxidation;nitrification;Nitrososphaera
Date:	2014
Issue Date:	2015-04-15T03:04:12Z (UTC)
Abstract:	硝化是全球氮循環中不可或缺的部分，而氨氧化則是硝化作用之速率限制步驟。氨氧化早期被認為是氨氧化細菌（ammonia oxidizing bacteria, AOB）所主導，直到近年來由於分子生物技術的進步，許多研究結果顯示，氨氧化古菌（ammonia oxidizing archaea, AOA）在各種環境中廣泛存在，對環境中的氨氧化作用有著重要的貢獻，因此對氨氧化古菌也有越來越多的關注和探討。本研究從淡水魚缸中採集底泥樣本進行氨氧化古菌之富集培養，利用添加抗生素、固態培養基培養、過濾培養以及連續稀釋等方法純化，獲得一株穩定之氨氧化古菌，暫命名為strain THU。經分析strain THU的16S rDNA序列，與已成功培養之氨氧化古菌Nitrososphaera gargensis相似度為97%。strain THU細胞形態呈現球桿狀，具有鞭毛構造以及移動性，大小直徑約為0.35~0.5 ?m，長度約為0.75~1.8 ?m。其生長環境為中性pH，氧氣為生長時所需之必要物質;培養後期培養液不會混濁，呈現透明狀。該菌株生長溫度範圍為25~55℃，最適生長溫度為45℃。以硫酸銨當作基質利用，其可生長氨氮濃度範圍為0.05~2 mM，最適生長基質濃度為0.05 mM。鹽濃度可生長範圍為0~0.1%，最適生長鹽濃度為0.1%。震盪培養對該菌株氨氧化有輕微抑制效果，光照則會完全抑制，然而，添加有機酸（甲酸鈉、乙酸鈉及丙酮酸鈉）對於培養菌株時，並無明顯刺激生長及氨氧化之作用，其中甲酸鈉反而會有些微抑制。根據本研究結果顯示，Strain THU與目前已知之氨氧化古菌特性不同，可推斷strain THU為Nitrososphaera屬之一株新穎氨氧化古菌，因此，暫時將本研究所分離之氨氧化古菌命名為”Candidatus Nitrososphaera taiwanensis”。 Ammonia oxidation is critical to nitrification and is often thought to be driven only by ammonia-oxidizing bacteria (AOB). However, recent metagenomic studies have revealed the existence of unique ammonia monooxygenase subunit (amoA) genes derived from ammonia-oxidizing archaea (AOA). Discovery of ammonia-oxidizing archaea involving in ammonia oxidation has dramatically changed the perception of the diversity of microbes involved in nitrification. AOA are thought to have advantages over AOB in extreme environments, such as extreme temperature, salinity and pH. Therefore, AOA play more important role than AOB on ammonia oxidation in harsh environments. In this study a novel aerobic, moderately thermophilic ammonia oxidizing archaea was enriched from freshwater aquarium. Phylogenetic analysis of 16S rDNA sequence and amoA gene sequence of the strain showed that the isolate was closely related to Nitrososphaera viennensis and Nitrososphaera gargensis, there for the isolate was tentatively named strain THU. Cells of strain THU were coccobacillus, sized of 0.35~0.5 ?m in diameter and 0.75~1.8 ?m in length. Cells were motile with monotrichous flagellum. Growth of the isolate occurred in the range of temperature 25~55℃, salinity 0~0.1% and ammonia concentration 0.05~2 mM. Optimal growth conditions was found at 45℃, pH 7.01, salinity at 0.1% and ammonia concentration at 0.05 mM. Ammonia oxidation by the isolate was slightly inhibited by shaking and completely inhibited by white-light. Addition of organic substrates (formate, acetate and pyruvate) did not stimulate the growth and ammonia oxidation. There results suggested that strain THU differed from currently known AOA and was proposed to designate the novel strain as “Candidatus Nitrososphaera taiwanesis”
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