Role of nitrite and nitric oxide in the processes of nitrification and denitrification in soil: Results from 15N tracer experiments

Recent research has proven soil nitrite to be a key element in understanding N-gas production (NO, N2O, N2) in soils. NO is widely accepted to be an obligatory intermediate of N2O formation in the denitrification pathway. However, studies with native soils could not confirm NO as a N2O precursor, and field experiments mainly revealed ammonium nitrification as the source of NO. The hypothesis was constructed, that the limited diffusion of NO in soil is the reason for this contradiction. To test this diffusion limitation hypothesis and to verify nitrite and NO as free intermediates in native soils we conducted through-flow (He/O2 atmosphere) 15N tracer experiments using black earth soil in an experimental set up free of diffusion limitation. All of the three relevant inorganic N soil pools (ammonium, nitrite, nitrate) were 15N labelled in separate incubation experiments lasting 81 h based on the kinetic isotope method. During the experiments the partial pressure of O2 was decreased in four steps from 20% to about 0%. The net NO emission increased up to 3.7 μg N kg-1 h-1 with decreasing O2 partial pressure. Due to the special experimental set up with little to no obstructions of gas diffusion, only very low N2O emission could be observed. As expected the content of the substrates ammonium, nitrate and nitrite remained almost constant over the incubation time. The 15N abundance of nitrite revealed high turnover rates. The contribution of nitrification of ammonium to the total nitrite production was approx. 88% under strong aerobic soil conditions but quickly decreased to zero with declining O2 partial pressure. It is remarkable that already under the high partial pressure of 20% O2 12 % of nitrite is generated by nitrate denitrification, and under strict anaerobic conditions it increases to 100%. Nitrite is present in two separate endogenous pools at least, each one fed by the nitrification of ammonium or the denitrification of nitrate. The experiments clearly revealed that nitrite is almost 100% the direct precursor of NO formation under anaerobic as well as aerobic conditions. Emitted N2O only originated to about 100% from NO under strict anaerobic conditions (0-0.2% O2), providing evidence that NO is a free intermediate of N2O formation by denitrification. To the best of our knowledge this is the first time that NO has been detected in a native soil as a free intermediate product of N2O formation at denitrification. These results clearly verify the “diffusion limitation” hypothesis.