Distinguishing nitrous oxide production from nitrification and denitrification on the basis of isotopomer abundances

The intramolecular distribution of nitrogen isotopes in N2O 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 N2O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N2O 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 parts per thousand, 32.5 +/- 0.6 parts per thousand, and 35.6 +/- 1.4 parts per thousand for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N2O from ammonia oxidation by N. europaea (31.4 +/- 4.2 parts per thousand) was similar to that produced during hydroxylamine oxidation (33.5 1.2 parts per thousand) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 +/- 1.7 parts per thousand), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N2O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (-0.6 +/- 1.9 parts per thousand and -0.5 +/- 1.9 parts per thousand, respectively) were similar to those during nitrate reduction (-0.5 +/- 1.9 parts per thousand and -0.5 +/- 0.6 parts per thousand, respectively), indicating no influence of either substrate on site preference. Site preferences of similar to 33 parts per thousand and similar to 0 parts per thousand are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N2O.