Abstract: Denitrification process is the main pathway for gaseous nitrogen loss in ecosystems, but it is difficult to quantify the rate of denitrification. The ¹⁵N natural abundance method is proved to be effective for quantifying denitrification rates at the ecosystem scale. However, this method requires an understanding of the nitrogen and oxygen isotope fractionation factors during denitrification. Currently, studies on the isotope fractionation factors during denitrification mainly focused on forest and cropland soils, and only few studies have been conducted in grassland soils. Here, we determine microbial nitrate consumption rates of four typical grassland soils from different regions in China (Daxinganling, Duolun, Eerguna, and Gangcha) under anaerobic condition. The nitrogen and oxygen isotope effects (ε) and their ratios (Δδ¹⁸O:Δδ¹⁵N) were then fitted using the Rayleigh fractionation model. Our results showed that the ¹⁵ε of grassland soils ranged from 21.6‰ to 32.0‰ (27.1‰±2.1‰), and the ¹⁸ε ranged from 10.4‰ to 15.7‰ (12.9‰±1.1‰). There was a certain difference in ¹⁵ε among different soils, which could be attributed to the differences in physical and chemical properties of different soil types and composition of denitrifying microbial communities. In addition, different calculation methods, initial nitrate concentration, nitrate dissimilatory reduction reaction, and possible nitrification in open environments may also affect the calculation of ¹⁵ε. Mean annual temperature and mean annual precipitation were important factors affecting ¹⁸ε value. The Δδ¹⁸O:Δδ¹⁵N ratios of grassland soils ranged from 0.38 to 0.49 (0.46±0.03), which were lower than previous studies on terrestrial and aquatic ecosystems and microbial pure cultures. The potential reason for the difference in Δδ¹⁸O:Δδ¹⁵N might be the re-oxidation of nitrite and oxygen isotope exchange with water. In conclusion, there were certain differences in fractionation effects among different grassland ecosystems. Thus, variables such as climate and ecosystem type should be comprehensively considered in quantifying denitrification rates.

Key words: denitrification, stable isotope technique, grassland, nitrogen and oxygen isotope fractionation effect, Δδ¹⁸O:Δδ¹⁵N