Stable isotope approaches

Stable isotope techniques have a crucial role to play in the attribution of N2O emissions to different microbial processes. This may be done using estimation techniques (variations in natural abundance, site preference) or quantification techniques (using isotopic enrichment), which employ the 15N and 18O signatures of N2O determined by isotope ratio mass spectrometry. Here we give an outline of these approaches, but guide the reader to Baggs (2008) for theoretical details behind each of these approaches.

Natural abundance approaches rely on the biological fractionation against 15N and 18O. Fractionation during nitrification is generally higher than for denitrification, meaning that N2O produced during nitrification is more depleted (more negative 8) in 15N and 18O relative to substrates than that produced during denitrification (Wahlen and Yoshinari, 1985; Yoshida, 1988). Due to the different oxygen sources for NH3 oxidation to hydroxylamine (soil air, 818O 23.5 per mil (i.e. parts per thousand)) and oxidation of NH2OH to NO2- (soil water, 818O 10 per mil) the 818O in N2O from nitrifier denitrification should be intermediate in value (Sutka et al, 2006). The fractionation during nitrate ammonification has yet to be determined. Fractionation during ammonia oxidation has been shown to differ amongst the nitrifying bacteria. The differences in 815N in part arise because reduction of N2O to N2 enriches the remaining N2O in 15N (Webster and Hopkins, 1996; Barford et al, 1999). This approach has been applied to the determination of the microbial source of N2O in a range of ecosystems and controlled environment experiments, and is most advantageous in natural or unfertilized systems (e.g. Webster and Hopkins, 1996; Wrage et al, 2004; Perez et al, 2006).

Enrichment approaches have been developed aimed at quantifying the individual sources of N2O in situ. To date, these have mostly focused on distinguishing between nitrification and denitrification following application of 15N-labelled fertilizer. Application of 15N-NH4+ and/or 15N-NO3- to soil and attribution of the 15N-N2O fluxes to nitrification or denitrification depending on the 15N source applied negates the need for C2H2 inhibition (Baggs et al, 2003; Bateman and Baggs, 2005; Mathieu et al, 2006). For example, Baggs et al (2003) used this 15N-enrichment approach to verify that increased N2O emission under elevated atmospheric CO2 at the Swiss FACE experiment was mainly due to increased denitrification, with greater below-ground C allocation stimulating both denitrifier-N2O and -N2 production (Figure 2.3). Unfortunately, this approach is unable to distinguish denitrification from nitrate ammonification or nitrifier denitrification. A combined 15N-, 18O-enrichment approach has been proposed by Wrage et al (2005), involving application of 18O-labelled water to determine N2O production during nitrifier denitrification. However, quantification of nitrate ammonification N2O by the enrichment approach remains elusive.

Consideration of the isotopomer site preference of 15N in N2O has been applied to determine the microbial source of N2O in terrestrial systems (Yamulki et al, 2001; Bol et al, 2003; Well et al, 2006). N2O is a linear molecule, N-N-O, and the 14N/15N ratios of the central and outer N atoms can naturally vary. Site preference (SP) is termed as the difference in 815N between the central and outer N atoms in N2O, with different microbial processes and functional groups thought to exhibit distinct 15N-SPs (Bol et al, 2003; Sutka et al, 2003, 2004). Sutka et al (2006) demonstrated in cultures the potential for SP to distinguish between ammonia oxidation and nitrifier denitrification.

Figure 2.3 Total denitrifier 15N-(N2O + N2) production and the ratio of 15N-N2to 15N-N2O following application of 11.2g N m-2 (1 atom per cent excess 15N) to Lolium perenne swards under ambient (36Pa; empty symbols) and elevated (60Pa; solid symbols)

atmospheric CO2

Figure 2.3 Total denitrifier 15N-(N2O + N2) production and the ratio of 15N-N2to 15N-N2O following application of 11.2g N m-2 (1 atom per cent excess 15N) to Lolium perenne swards under ambient (36Pa; empty symbols) and elevated (60Pa; solid symbols)

atmospheric CO2

Source: Baggs et al (2003)

However, similarities between conventional denitrification and nitrifier denitrification mean that this approach alone will be unable to distinguish between these processes, and denitrification may need to first be quantified with a 15N-enrichment approach. This SP approach would seem to be insufficient to distinguish between all N2O sources on its own, but it provides a powerful means to partition between all processes when used in combination with other approaches.

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