寡营养淡水环境中氨氧化古菌及其氨氧化特性研究

Recently, ammonia-oxidizing archaea (AOA)was found as a main member of microbes in ammonia oxidation, and the number of it is higher than ammonia-oxidizing bacteria (AOB)in kinds of environments. In natural ecosystem, the microbial ecology of community distribution, abundance and evolution, ammonia oxidation, biochemical and biophysical characteristics, and genome bioinformation of AOA in freshwater still need more and precisely research.In this project, we will study the distribution of AOA/AOB in vertical and horizontal districts of oligotrophic freshwater to analyze the evolution of AOA/AOB, the response of AOA/AOB community stability and change to physical and chemical factors. Microbial ecology of AOA/AOB in oligotrophic freshwater will be revealed by the research of their distribution, abundance, community structure, and diversity. Stable isotope probing (SIP) will be used to labelled the nucleotide of N-element transformation microbes, and the ammonia-oxidizing ability of AOA/AOB in freshwater will be studied based on the SIP labelled DNA and RNA. After the analysis, the contribution of ammonia oxidation by AOA and AOB will be figured out clearly. Basing on the microbial ecology research, we will design special enrichment method to obtain freshwater type AOA stains. After isolating the AOA strans, we will characterize their biochemistry and biophysics including the response to dissolve oxygen and ammonia concentration, ammonia oxidation, even denitrifiaction kinetics and so on.AOA genome sequence will be obtained by high throughput and metagenomic sequencing techniques. Basing on the genome sequence, genome bioinformation of AOA including gene structure, ammonia oxidation enzyme system, metabiotic pathway, and energy regulation will be analyzed. In the meantime, the genome and functional genes of freshwater type AOA will be compared with the sequences of the known AOA in databases. This project will be helpful for the revelation of ammonia oxidation in freshwater, and the expansion of ecology, biochemistry and biophysic, and genome bioinformations of AOA in natural environemnt. So, the research project has important theory and application values for bioremediation of ammonium polluted environments.

近期研究发现氨氧化古菌(AOA)也是自然界中氨氧化作用的主要贡献者，且在很多环境中数量高于氨氧化细菌(AOB)，但它们在环境中的群落、丰度、进化等生态学信息、氨氧化特性、基因组生物信息仍较少。本项目以寡营养淡水环境为对象，深入研究其中AOA/AOB的垂直和水平分布、AOA/AOB的物种进化和群落结构对理化因子的响应，揭示它们在环境中的分子生态学；利用SIP核苷酸标记，研究环境中AOA/AOB的氨氧化活性，分析它们对氨氧化作用的贡献率；通过对淡水型AOA富集，分离到纯化的菌株，研究AOA氨氧化动力学、呼吸作用、反硝化等理化特性；通过高通量和宏基因组测序技术获得AOA的基因组序列，研究淡水型AOA的基因结构、氨氧化酶系、能量及代谢调控等生物信息，并与已知AOA进行比较。本课题的完成将有助于揭示淡水环境中氨氧化作用和拓展AOA的生态、理化和基因组信息，对环境生物修复具有重要的理论意义和应用价值。

氨氧化微生物催化的氨氧化反应在氮的生物地球化学循环中扮演了重要角色，对环境的生态修复和污染治理也有重要意义。目前大量的研究表明，自然界和人工生态系统中氨氧化古菌(AOA)的数量和所起的作用要大于氨氧化细菌(AOB)。为了揭示珠江淡水生态系统中AOA和AOB的氨氧化特性及对氨氧化作用的贡献，我们利用了克隆文库、高通量测序、宏基因组、宏转录组等分子生态学的方法，研究了它们在珠江环境中的分布、丰度和群落结构，结果表明：AOA主要分布于水层和沉积物层交汇处，而AOB主要分布于水体表面，AOA的amoA基因主要与Group1.1b的土壤来源的Candidatus Nitrososphaera有较高的同源性，而nirK基因与已知的AOA的nirk序列相似性较低；宏基因组和宏转录组揭示了珠江水体与沉积物中的微生物群落结构，根据序列统计分析，水体宏基因组中前五位的菌是Candidatus Chloracidobacterium thermophilum、Pseudanabaena biceps、Leptolyngbya boryana、Oscillatoria acuminata和Microcystis aeruginosa，沉积物宏基因组中前五位的菌是Dechloromonas aromatica、Candidatus Contendobacter odensis、Anaerolinea thermophila、Pyrococcus horikoshii和Candidatus Competibacter denitrificans；根据序列分析，宏基因组和宏转录组中amoA基因主要来源于Nitrosomonas，而检出的AOA的序列很少，因此，根据宏基因组中基因的丰度和宏转录组中基因的表达，我们认为珠江生态系统中AOB可能是主要的氨氧化作用推动者。为了挖掘更多的AOA基因信息并进一步阐明AOA的氨氧化特性，我们通过优化AOA富集和筛选的策略，期望从该淡水生态系统中得到AOA菌株或富集物；在不断尝试不同抗生素、碳源、温度等条件后使富集物中AOA的丰度达到了30%，并且随着富集的进行AOA的氨氧化活性也有了显著的提高。尽管当前我们未能达到项目的预期目标，但我们摸索出了一种新的AOA富集和筛选的方法，将在较短时间内得到丰度>90%的AOA富集物或者菌株，并继续完成项目原定的研究计划。

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