Inhibitors such as nitrapyrin and chlorate, and biocides such as cyclohexamide (De Boer and Kowalchuk, 2001) have been applied to soil to distinguish the relative contributions of denitrification and heterotrophic and autotrophic nitrification to N2O emissions. Acetylene (C2H2) gas at 10kPa was first recognized as an inhibitor of the N2O reductase involved in denitrification (Yoshinari and Knowles, 1976), then the inhibition of nitrification at lower concentrations (10Pa or 0.01 per cent v/v) was noted (Hynes and Knowles, 1978). The suppression by C2H2 of N2O reduction to N2 by denitrifying bacteria is due to non-competitive inhibition of the enzyme N2O reductase, and requires high partial pressures (10kPa) to have an effect (Webster and Hopkins, 1996); however, recovery is rapid after C2H2 removal.
These actions of C2H2 have led to the established procedure of applying 10Pa C2H2 to soil to inhibit nitrification, and 10kPa C2H2 to inhibit both nitrification and N2O reduction in denitrification (Klemedtsson et al, 1988). Many studies have used this C2H2 inhibition methodology to demonstrate the simultaneous production of N2O by denitrification and nitrification (e.g. Maag and Vinther, 1996; Webster and Hopkins, 1996), and to show how they are differently affected by soil water content and soil texture. For example, the proportion of N2O produced by nitrification has been found to increase with soil water content (Maag and Vinther, 1996), and nitrification has been identified as the predominant source of N2O from dry soils and denitrification as the predominant source above 100 per cent soil water-holding capacity (Webster and Hopkins, 1996). Both the initiation of inhibition and recovery (Kester et al, 1996) are dependent on the diffusion capacity of C2H2 within the soil, which is related to the soil texture and water content. However, there are well-recognized limitations of the acetylene inhibition technique, which have led to the development and adoption of alternative stable isotope approaches. The limitations with C2H2 include an underestimation of denitrification by preventing the supply of nitrifier-NO3~, C2H2 itself can be decomposed and used as a substrate for denitrification if C is limiting, it inhibits nitrate ammonification, the extra pair of electrons that would have been used to reduce N2O to N2 can increase the reduction of NO3~ in denitrification, and its diffusion into fine-textured or saturated soil can be slow (Groffman et al, 2006).
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