不同类型土壤反硝化作用分馏系数的研究

Denitrification is a major process contributing to the removal of nitrogen (N), through nitrate (NO3-), from ecosystems, but its rate is difficult to quantify. The natural abundance of isotopes can be used to identify the occurrence of denitrification and has recently been used to quantify denitrification rates at the ecosystem level. However, the technique requires an understanding of the isotopic enrichment factor associated with denitrification. To date, despite numerous studies of the N isotope effects performed using pure cultures of heterotrophic denitrifying bacteria, groundwater and sediments, only few studies have investigated soils of terrestrial ecosystem (forest, grassland and agricultural soils). In the proposed project, soils collected from forest, grassland and agriculture ecosystems in different climate zones in China will be incubated under anaerobic lab conditions to investigate the isotope fractionation of N and O of NO3- during denitrification by native soil microbial communities, and the relationship between N and O isotopes (△δ18O:△δ15N). In addition, combined with biological inhibitors, real-time fluorescence quantitative PCR, high-throughput sequencing and microbial pure culture, the N isotope effects of denitrifying fungi and bacteria can be also determined. This study aims to reveal the differences of the isotope fractionation of nitrate during denitrification in different types of terrestrial ecosystem in different climate zones, as well as its controlling mechanisms. The measured N isotope effects would be further applied to estimate the denitrification rates of global terrestrial ecosystems. In the context of global changes, these results are useful to understand and predict climate change and the response of terrestrial ecosystem to N deposition.

反硝化过程是生态系统气态氮损失的主要途径，然而由于传统方法的诸多限制，生态系统尺度上的反硝化速率一直难以量化。硝酸盐的15N/14N比值被用于量化生态系统尺度上的反硝化速率，但是利用该方法需要考虑反硝化过程中的同位素分馏效应。然而，目前对于陆地生态系统（森林、草地和农业生态系统）土壤反硝化作用分馏系数的研究有限。因此，本研究拟选取我国不同气候区、不同类型土壤为研究对象，通过室内厌氧培养实验，结合生物抑制剂和分子生物学技术，测定不同类型土壤微生物群落反硝化作用N和O同位素分馏效应以及其比值（△δ18O:△δ15N），并区分真菌和细菌反硝化作用N和O同位素分馏效应的差异。本研究结果将揭示不同气候区、不同生态系统类型土壤反硝化作用同位素分馏效应的差异及其控制因子，并进一步量化陆地生态系统反硝化作用速率，在全球变化的背景下，对于理解和预测氮沉降对陆地生态系统影响以及全球气候变化有着十分重要的意义。

本项目以我国不同气候区不同生态系统（森林、农田和草地）的15个土壤为研究对象，利用室内厌氧培养实验测定土壤微生物群落反硝化作用N 和O 同位素分馏效应及其比值（∆δ18O: ∆δ15N），旨在揭示不同生态系统类型土壤反硝化作用同位素分馏效应的差异及其控制因子，从而提高量化陆地生态系统反硝化作用速率的精度。研究发现，森林、农田和草地生态系统的15ε分别为28.2 ± 2.6‰、31.5 ± 3.8‰和26.8 ± 1.9‰，18ε分别为8.9 ± 1.3‰、9.3 ±1.0‰和12.7 ± 1.2‰，∆δ18O: ∆δ15N分别为0.30 ± 0.04、0.32 ± 0.06和0.46 ± 0.03。不同生态系统之间的15ε和∆δ18O: ∆δ15N没有显著差异。本研究中15ε均值（29.1 ± 1.8‰）高于以往研究中土壤15ε均值（23.3 ± 1.4‰），而∆δ18O: ∆δ15N均值（0.35 ± 0.03‰）则低于以往研究结果（0.62 ± 0.08‰）。我们推测土壤理化性质（pH、C/N等）会显著影响土壤中的微生物特征（酶活性、基因表达、NO2-与水的氧原子交换等），进而显著影响15ε和∆δ18O: ∆δ15N的测定值。不同计算公式得出的15ε也存在显著差异。另外，陆地土壤15ε显著高于其它培养环境（淡水、海洋等），这可能是由于水体环境中硝化作用对15ε的稀释以及不同环境中真菌和细菌的丰度差异所导致。我们的结果表明不同气候区不同生态系统类型15ε存在一定差异，因此在评估陆地生态系统反硝化损失时需要综合考虑气候和生态系统等变量，从而更准确的量化陆地生态系统气态氮损失。