Direct Measurements of Denitrification Gas (N₂, N₂O, NO) and CO₂ Emissions Using the Gas-Flow-Soil-Core Technique with Helium Environment Incubation

Denitrification is the key process of nitrogen transformation to close the global nitrogen cycle. Quantification of microbial denitrification rate and its ratios of products is the key step for obtaining insights into nitrogen cycling processes, though it is very difficult. To enable precise quantification of the rates of the entire process, as well as individual products, a system of gas-flow-soil-core technique with helium-environment incubation was recently established and a three-period incubation method was set up by the Karlsruhe Institute of Technology and the Institute of Atmospheric Physics, Chinese Academy of Sciences. Using this system, dynamic emission rates of dinitrogen (N₂), nitrous oxide (N₂O), nitric oxide (NO) and carbon dioxide (CO₂), which are the gaseous products of microbial denitrification, can be simultaneously and directly measured. In this study, we conducted the first soil incubation experiment to test the reliability of the measurements by this system in association with the newly proposed incubation method. Our experiment included two levels of initial soil nitrate (NO₃^-) content, which were around 10 and 100 mgN kg^-1d.s. (dry soil), respectively (hereinafter referred to as 10N and 100N). For either nitrate level, sufficient dissolved organic carbon (DOC) was initially supplied (glucose was added to establish an initial DOC content of around 300 mgC kg^-1d.s.). The incubated fresh soil was a silty clay loam of the northern China. It was sampled from a typical cropland rotationally cultivated with a double cropping system of summer maize and winter wheat and a single cropping system of cotton. Our results show that the microbial denitrification rate was significantly higher for the 100N than 10N treatments (p<0.01), and N₂ was the main product in both treatments (with mass fractions of 77% and 75%, respectively). The molar ratios were 1.2 (10N) to 1.5 (100N) for NO/N₂O and were 0.19 (both treatments) for N₂O/N₂. The measurements of individual denitrification gases recovered 81%-87% of disappeared nitrate during incubation. The direct dynamic detection of individual denitrification gases and the measurements of DOC, ammonium, nitrate, microbial biomass carbon, and microbial biomass nitrogen at the beginning and end of incubation gave mass balance rates of 92%-95% for nitrogen. These results suggest that the tested system, in association with the proposed incubation method, could precisely determine the dynamical rate of microbial denitrification. The molar ratios of NO/N₂O given by our data were greater than 1. This differs from previous knowledge of much lower ratios yielded by denitrifiers. This difference implicates that the NO/N₂O ratio of 1 may not be used as the threshold to indicate nitrification or denitrification processes.