湖泊沉积物中氨氧化菌群落的海拔梯度格局及驱动机制研究

Nitrogen is a key element for the lake ecosystems. Ammonia oxidation is the rate limiting step of nitrification, which is an integral part of the global nitrogen cycle. Aerobic biological ammonia oxidation is accomplished by two groups of microorganisms, ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). The community structure of AOA and AOB could be affected by various environmental factors. Therefore, environmental factors varied along an elevational gradient may affect the AOA and AOB and subsequently the ammonia oxidation process. In this proposed study, the lakes at different elevations in the eastern part of Tibetan Plateau were chosen as the study sites. Field investigation will be firstly performed. The variations of the community composition, abundance and activity of AOA and AOB in lake sediments along the elevational gradient will be analyzed with multiple molecular biological techniques including barcoded pyrosequencing, terminal restriction fragment length polymorphism (T-RFLP), cloning and sequencing, real-time qPCR, and quantitative reverse transcription PCR (qRT-PCR). Multivariate statistical analysis would be used to investigate the relationships between ecological distribution of AOA and AOB and environmental factors. The in situ microcosms will be subsequently constructed within representative lakes at different elevations. The sediment transplant and nutrient addition experiments will be carried out. The community composition of AOA and AOB and nitrification rate will be measured to investigate their responses to the varied elevation and increased nutrient loading. Thereafter, microcosms will be constructed in laboratory to examine the effects of single environmental factor on the community and autotrophic metabolism of ammonia-oxidizing microorganisms in lake sediments at different elevations with the stable isotope probing technique. The obtained results would be helpful to elucidate the elevational distribution patterns of AOA and AOB in lake sediments, and reveal the driving mechanisms of variations in composition and function of ammonia-oxidizing microorganisms along an elevational gradient and provide further insights into the biogeochemical cycle of nitrogen in lake ecosystems.

氮是湖泊生态系统关键元素，微生物驱动的氨氧化过程是氮循环的关键步骤。氨氧化微生物易受多种环境因素影响，海拔梯度带来的环境改变可能会导致氨氧化菌群落产生空间分异。本研究拟选取我国青藏高原东部不同海拔高度湖泊为研究场所，首先进行野外调查，应用多种分子生物学技术分析湖泊沉积物中氨氧化菌群落结构、丰度和活性等沿海拔梯度的变化特征，分析其与环境因子的关系。然后选取不同海拔典型湖泊，构建野外模拟系统，进行沉积物移植及营养盐添加试验，将氨氧化菌群落组成与硝化速率关联，探讨氨氧化菌群落及其功能对海拔气候环境改变及营养盐负荷增加的响应。最后室内构建微宇宙，借助稳定性同位素核酸探针技术分析氨氧化菌群落生理生态，探讨单一环境因子变化对不同海拔湖泊沉积物中氨氧化菌群落的影响。预期成果将阐明沉积物中氨氧化菌群落沿海拔梯度的分布特征，揭示氨氧化菌群落及其功能沿海拔梯度变化的机理，有助于加深对湖泊氮循环过程的认识。

相对于对大型动、植物海拔分布格局的充分认识，人们对湖泊沉积物中微生物沿海拔梯度的分布规律则知之甚少，对氨氧化微生物群落沿海拔梯度变化方面的研究还非常缺乏。本课题选取我国青藏高原东部不同海拔高度（525-4490 m）湖泊，研究了沉积物中细菌群落以及氨氧化原核微生物群落沿着海拔梯度的分布规律，并探索形成此海拔格局的驱动因素。研究发现，湖泊沉积物中细菌群落结构和多样性海拔梯度格局明显。细菌群落α多样性随着海拔高度增加而单调下降，细菌群落β多样性随着海拔距离的增加而增加。谱系结构分析结果表明，环境过滤是影响细菌群落多样性海拔梯度格局的重要过程，随着海拔高度增加，环境过滤作用增强。对于所有沉积物样品而言，检测的因子中对细菌群落多样性影响相对贡献最大的是海拔，其次是pH。虽然海拔和化学因子是影响细菌群落结构及多样性的主要驱动因子，但是两者的影响作用有一定的差别。表层沉积物受到海拔和理化等多种因子显著影响，底层沉积物主要受到海拔因子的影响。对不同海拔湖泊沉积物中氨氧化微生物群落的研究发现，氨氧化细菌AOB的多样性随海拔增加减少，而氨氧化古菌AOA的多样性与海拔无相关关系。不同海拔沉积物中氨氧化微生物的丰度与海拔无相关关系，氨氧化古菌AOA的丰度与TP显著正相关，氨氧化细菌AOB的丰度与NO3-、NO2-、TN和TP正相关。不同海拔高度氨氧化微生物优势类群有一定差异。对不同海拔湖泊沉积物中反硝化微生物类群（nirK、nirS和nosZ）进行分析，结果显示，nirS基因类群的多样性高于nirK基因类群。相较于其他海拔处，在海拔4282m处三种反硝化微生物的多样性均是最低的。三种反硝化微生物的多样性和丰度随着海拔的增加没有明显的变化趋势。海拔、温度和pH是影响反硝化微生物群落最重要的环境因子。