Combining the 15N and acetylene inhibition techniques to examine the effect of acetylene of denitrification

Using acetylene to inhibit the reduction of N2O to N2 is the simplest and most commonly used technique to quantify denitrification. The acetylene treatment may not always inhibit N2O-reductase completely when soils are wet, low in NO3- or with a high C-to-NO3- ratio. Acetylene blocks nitrification as well as N2O-reductase, so if nitrification is the source of NO2- or NO3- for denitrification, then the denitrification rate would be underestimated by the C2H2 inhibition technique. Acetylene can also be metabolised in soil and so enhance denitrification. We show how the 15N method for measuring denitrification can be combined with the C2H2 inhibition technique to check the effect of acetylene on N2O-reductase, respiration and nitrification. No method exists, however, for the analysis of 15N in the N2 and N2O of headspace samples which also contain 10% v/v C2H2. We describe a technique to remove C2H2 from headspace samples with alkaline KMnO4 prior to analysis by isotope-ratio mass spectrometry. The C2H2 removal procedure had no effect on the determination of N2O concentration, or on the determination of the 15N content of N2O and N2. Then using data for fluxes of N2O, N2, (N2O + N2), CO2 and the size and 15N content of the soil NO2- pool for a soil incubation experiment, we show how the effects of C2H2 can be quantitatively assessed. The data also allowed the amount of denitrification, as estimated by the C2H2 inhibition technique, to be compared with that measured using the 15N technique. For the soil conditions during this incubation, 10% v/v C2H2 completely blocked N2O-reductase, slightly inhibited respiration and completely blocked nitrification. The average total flux of N gases as measured by 15N (4.3 nmol N g-1 h-1) was not significantly different (P > 0.05) from the average flux measured by the C2H2 inhibition method (3.9 nmol N g-1 h-1). Including C2H2 as a treatment in the 15N method provides a direct way of confirming that some of the assumptions in the C2H2 inhibition technique are valid.