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...

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.

Rapid Commun. Mass Spectrom. 2003; 17: 738-745.The calibration and calculation of the isotopomer composition of our internal N 2 O standard that was originally published has been revised. Because (Tables 1-4) do not change our conclusion that there is a significant difference in site preference of N 2 O formed during hydroxylamine oxidation by methanotrophic nitrification and by nitrification and nitrite reduction during nitrifierdenitrification.

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1. We estimated uptake of stream water dissolved organic carbon (DOC) through a wholestream addition of a 13 C-DOC tracer coupled with laboratory measurements of bioavailability of the tracer and stream water DOC. 2. The tracer, a leachate of 13 C-labelled tree tissues, was added to the head waters of White Clay Creek, Pennsylvania, U.S.A., over a 2-h period and followed 1.27 km downstream to generate mass transfer coefficients for DOC lability classes within the tracer. 3. From the longitudinal 13 C uptake curve, we resolved labile and semi-labile DOC classes within the 13 C-DOC tracer comprising 82% and 18% of the tracer respectively. 4. Plug-flow laboratory bioreactors colonized and maintained with stream water were used to determine the concentration of stream water DOC fractions that had a similar lability to the labile and semi-labile classes within the tracer and we assumed that stream water DOC and tracer DOC with comparable lability fractions in the bioreactors behaved similarly in the stream, i.e. they had the same mass transfer coefficients. 5. A small fraction (8.6%) of the stream water DOC was labile, travelling 238 m downstream before being taken up. The remaining bioavailable stream water DOC was semi-labile and transported 4.5 km downstream before being taken up. These uptake lengths suggest that the labile DOC is an energy source within a stream reach, while the semi-labile DOC is exported out of the reach to larger rivers and the downstream estuary, where it may provide energy for marine microbial communities or simply be exported to the oceans.