The intramolecular distribution of nitrogen isotopes in N 2 O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N 2 O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N 2 O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 ؎ 1.2‰, 32.5 ؎ 0.6‰, and 35.6 ؎ 1.4‰ for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N 2 O from ammonia oxidation by N. europaea (31.4 ؎ 4.2‰) was similar to that produced during hydroxylamine oxidation (33.5 ؎ 1.2‰) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 ؎ 1.7‰), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N 2 O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (؊0.6 ؎ 1.9‰ and ؊0.5 ؎ 1.9‰, respectively) were similar to those during nitrate reduction (؊0.5 ؎ 1.9‰ and ؊0.5 ؎ 0.6‰, respectively), indicating no influence of either substrate on site preference. Site preferences of ϳ33‰ and ϳ0‰ are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N 2 O.Over the past several decades, anthropogenic activity, primarily agriculture, has doubled the annual input of biologically reactive nitrogen into the environment (14). This surplus of reactive nitrogen has stimulated natural microbial activity, the largest source of the greenhouse gas nitrous oxide (N 2 O) (17, 26). Ammonia-and methane-oxidizing organisms produce N 2 O during the oxidation of hydroxylamine (NH 2 OH) to nitrite (NO 2 Ϫ ). Ammonia-oxidizing bacteria also reduce NO 2 Ϫ to N 2 O and N 2 under anoxic conditions by a process termed nitrifier denitrification (12,22,23). Nitrous oxide can also be produced and consumed by heterotrophic denitrifying organisms. In this case, N 2 O is produced and consumed by the stepwise reduction of nitrate (NO 3 Ϫ ) to N 2 (33). The relative importance of nitrification and denitrification in N 2 O production has proven difficult to determine. Previous attempts to differentiate nitrification-and denitrification-mediated N 2 O production in soils using stable isotope approaches (4, 20, 30, 31, 32) relied on the observation that the fractionation factor associated with N 2 O production by denitrifiers (2) is substantially less than that associated with nitrification (34). The assumption was that N 2 O with a high ␦ 15 N value is indicative of denitri...

[1] Site preference (SP), the difference in d15 N between the central and outer nitrogen atoms in N 2 O, is a powerful approach for apportioning fluxes of N 2 O from soils to nitrification and denitrification (Sutka et al., 2006). A critical aspect of the use of SP data to apportion sources of N 2 O to nitrification and denitrification is the need to evaluate data for isotope shifts that may have occurred during N 2 O reduction in soils prior to its escape to the atmosphere. We present data on the isotopologue effects during reduction of N 2 O during anaerobic incubation of soils and pure cultures of denitrifying bacteria. N a with increased reduction. Consequently, a deviation from the linear mixing relationship between soil-derived and atmospheric N 2 O is an indication of extensive reduction. On the basis of our characterization of isotopic fractionation during N 2 O reduction, we show that the rate of reduction would have to be substantially greater than 10% of that of production to impact SP estimates of N 2 O from denitrification by more than a few percent. Nonetheless, reduction results in a small, but potentially important, increase in SP away from values proposed for bacterial denitrification (0%) toward those associated with production from nitrification (33%) (Sutka et al., 2006). On this basis, estimates of the proportion of N 2 O derived from denitrification obtained from SP values are underestimates and therefore conservative.Citation:

Identifying the importance of fungi to nitrous oxide (N2O) production requires a non-intrusive method for differentiating between fungal and bacterial N2O production such as natural abundance stable isotopes. We compare the isotopologue composition of N2O produced during nitrite reduction by the fungal denitrifiers Fusarium oxysporum and Cylindrocarpon tonkinense with published data for N2O production during bacterial nitrification and denitrification. The fractionation factors for bulk nitrogen isotope values for fungal denitrification were in the range -74.7 to -6.6 per thousand. There was an inverse relationship between the absolute value of the fractionation factors and the reaction rate constant. We interpret this in terms of variation in the relative importance of the rate constants for diffusion and enzymatic reduction in controlling the net isotope effect for N2O production during fungal denitrification. Over the course of nitrite reduction, the delta(18)O values for N2O remained constant and did not exhibit a relationship with the concentration characteristic of an isotope effect. This probably reflects isotopic exchange with water. Similar to the delta(18)O data, the site preference (SP; the difference in delta(15)N between the central and outer N atoms in N2O) was unrelated to concentration during nitrite reduction and, therefore, has the potential to act as a conservative tracer of production from fungal denitrification. The SP values of N2O produced by F. oxysporum and C. tonkinense were 37.1 +/- 2.5 per thousand and 36.9 +/- 2.8 per thousand, respectively. These SP values are similar to those obtained in pure culture studies of bacterial nitrification but quite distinct from SP values for bacterial denitrification. The large magnitude of the bulk nitrogen isotope fractionation and the delta(18)O values associated with fungal denitrification are distinct from bacterial production pathways; thus multiple isotopologue data holds much promise for resolving bacterial and fungal production. Our work further provides insight into the role that fungal and bacterial nitric oxide reductases have in determining site preference during N2O production.

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The relative importance of individual microbial pathways in nitrous oxide (N(2)O) production is not well known. The intramolecular distribution of (15)N in N(2)O provides a basis for distinguishing biological pathways. Concentrated cell suspensions of Methylococcus capsulatus Bath and Nitrosomonas europaea were used to investigate the site preference of N(2)O by microbial processes during nitrification. The average site preference of N(2)O formed during hydroxylamine oxidation by M. capsulatus Bath (5.5 +/- 3.5 per thousand) and N. europaea (-2.3 +/- 1.9 per thousand) and nitrite reduction by N. europaea (-8.3 +/- 3.6 per thousand) differed significantly (ANOVA, f((2,35)) = 247.9, p = 0). These results demonstrate that the mechanisms for hydroxylamine oxidation are distinct in M. capsulatus Bath and N. europaea. The average delta(18)O-N(2)O values of N(2)O formed during hydroxylamine oxidation for M. capsulatus Bath (53.1 +/- 2.9 per thousand) and N. europaea (-23.4 +/- 7.2 per thousand) and nitrite reduction by N. europaea (4.6 +/- 1.4 per thousand) were significantly different (ANOVA, f((2,35)) = 279.98, p = 0). Although the nitrogen isotope value of the substrate, hydroxylamine, was similar in both cultures, the observed fractionation (delta(15)N) associated with N(2)O production via hydroxylamine oxidation by M. capsulatus Bath and N. europaea (-2.3 and 26.0 per thousand, respectively) provided evidence that differences in isotopic fractionation were associated with these two organisms. The site preferences in this study are the first measured values for isolated microbial processes. The differences in site preference are significant and indicate that isotopomers provide a basis for apportioning biological processes producing N(2)O.