蒙古高原河流湿地厌氧氨氧化及反硝化菌群空间异质性与脱氮特征研究

Anammox and denitrification are two main pathways causing nitrogen loss in natural wetlands.However,whether anammox and denitrification in river wetland from Inner Mongolia steppe with grazing use can cause nitrogen loss is not yet clear. Xilin River grazing wetlands located in semi-arid region of Inner Mongolia Plateau would be selected as the main investigated area, at the same time,the sampling sites or plots would be designed according to grazing,resting and landscape spatial heterogeneity.Specifically,at landscape scale along the gradient from coarse alluvium in turbulent flow zone and fine alluvium from slow flow zone of the river-bed located in shallow waters, then to coarse or fine diluvium from river floodplain under grazing environment. At the same time,grazing plot and resting one would be took account under the same natural habitat.For key microbial process causing soil nitrogen loss,based specific region of the 16S rRNA gene,the community structure,biodiversity,abundance and their spatial distribution about anaerobic ammonium oxidizing microoganisms and denitrifying microoganisms will be deeply researched by 454 pyrosequencing technique.In order to quantitatively assess the contribution of anammox microoganisms and denitrifying microoganisms producing gaseous nitrogen(N2,N2O,NO),spatial variability and abundance of key functional genes(such as hao, hzo of anaerobic ammonium oxidizing microoganisms, nir, nor,nos of denitrifying microoganisms) will be analyzed by real-time fluorescence quantitative PCR.At the same time,anammox and denitrification activity, and their contribution to the emission of gaseous nitrogen(N2,N2O,NO) will be determined by 15N isotope tracer technique.Meanwhile,combined with gas chromatography technique after collecting the undisturbed soil column,the emission flux of nitrous oxide will be deeply determined. Certainly, in the view of wetland microbial functional diversity, the causes of high spatial heterogeneity about anaerobic ammonium oxidizing bacteria and denitrifying bacteria will be revealed, furthermore, the mechanism of which processes could cause nitrogen losses will be found out. Which can clarify population fluctuation of anaerobic ammonium oxidizing bacteria and denitrifying bacteria in the different habitat conditions, reveal their ecological adaptability and environmental response mechanism about the kind of microbial functional groups in the grazing wetland,in order to make sure the critical processes causing nitrogen losses by anammox bacteria or/and denitrifying bacteria. Finally,these results will be able to improve the underlying data about anaerobic ammonium oxidizing bacteria and denitrifying bacteria in the grazing wetland of the Mongolian Plateau,such as spacial distribution, diversity, function and dynamics. On the other hand,these can also provide a scientific basis in order to explore and solve environmental problems caused by overgrazing in river wetland of the Mongolian Plateau.

厌氧氨氧化和反硝化是天然湿地脱氮的重要途径，蒙古高原草原区锡林河湿地放牧利用引起的氮逸失问题尚不明晰。针对引起土壤氮逸失的关键微生物学过程，基于16S rRNA基因特征区域采用454测序技术分析湿地土壤中厌氧氨氧化菌和反硝化菌群落结构、多样性、丰度及其分布动态；利用定量PCR技术分析该类菌群关键功能基因（hao、hzo,nir、nor、nos）丰度及分布特征；利用原状土柱气相色谱法分析N2O的排放量及分布特征；利用15N示踪技术分析厌氧氨氧化及反硝化活性及其对N2的贡献率。厘清不同生境下关联的厌氧氨氧化和反硝化菌群相互间的消长规律及引起土壤氮逸失的主要途径，定量评估关键功能基因丰度和相应气体排放量的关系。以期从基质环境多样、微生物功能群及代谢活性的角度揭示放牧湿地氮逸失关键功能菌群的空间异质性成因及其对脱氮过程的生态调控机制。为探索解决蒙古高原河流湿地过度放牧引起的氮逸失问题提供科学依据。

反硝化和厌氧氨氧化细菌是天然湿地脱氮的重要驱动者。本项目利用分子生态技术对蒙古高原锡林河湿地不同环境梯度的厌氧氨氧化、反硝化细菌群落组成、分布、丰度及多样性进行研究，主要成果如下：【1】通过控制试验研究锡林河湿地N2O释放及反硝化菌群对氮添加的响应。发现：（1）氮添加显著提高了河流湿地N2O的释放量及反硝化菌群丰度，培养3天后120-300 kg N ha-1及培养6天后240-300 kg N ha-1剂量组N2O的释放量与空白组之间均存在显著性差异（p＜0.05），氮素剂量组180-300 kg N ha-1与空白组间16S rDNA和反硝化菌基因nosZ丰度存在显著（p＜0.05）和极显著性差异（p＜0.01），但与N2O释放量之间无显著相关性；（2）氮添加明显改变河流湿地nirS型反硝化菌群落结构，降低反硝化菌群多样性，变形菌门中 Sulfuritalea hydrogenivoran、Anaerolinea thermophila和Comamonadaceae I-Bh25-7等优势菌群对外源氮添加响应敏感。【2】锡林河湿地厌氧氨氧化细菌群落由4类已知菌群和1类未知菌群组成。其中（1）Ca.Brocadia-like和New cluster是最优势属；Ca.Kuenenia-like和Ca. Anammoxoglobus-like为次优势属；Ca.Jettenia-like只在低河漫滩湿地检测到；（2）Ca.Brocadia-like沿河流中心、河流边缘、低河漫滩、高河漫滩及放牧样点呈先降后升的分布趋势，而New cluster呈现先升后降分布趋势。（3） 锡林河湿地厌氧氨氧化细菌的丰度为1.11×104copies -2.73×105copies/kg土，放牧样点最高，5个样点间厌氧氨氧化细菌丰度存在显著性差异（p <0.05）。（4）CCA分析表明，Ca. Anammoxoglobus 和Ca.Brocadia是放牧样点最优势的厌氧氨氧化菌属，由TN、TOC、TP、NH4-N、NO3-N、含水率等环境因子正向驱动，由pH反向驱动；而Ca.Kuenenia、New Cluster、Ca.Jettenia主要分布在河流中心、河流边缘、低河漫滩、高河漫滩处，由pH正向驱动，由TN、TOC、TP、NH4-N、NO3-N、含水率等环境因子反向驱动。