The Contamination of Commercial (15)N(2) Gas Stocks with (15)N–Labeled Nitrate and Ammonium and Consequences for Nitrogen Fixation Measurements

We report on the contamination of commercial 15-nitrogen ((15)N) N(2) gas stocks with (15)N-enriched ammonium, nitrate and/or nitrite, and nitrous oxide. (15)N(2) gas is used to estimate N(2) fixation rates from incubations of environmental samples by monitoring the incorporation of isotopically labeled (15)N(2) into organic matter. However, the microbial assimilation of bioavailable (15)N-labeled N(2) gas contaminants, nitrate, nitrite, and ammonium, is liable to lead to the inflation or false detection of N(2) fixation rates. (15)N(2) gas procured from three major suppliers was analyzed for the presence of these (15)N-contaminants. Substantial concentrations of (15)N-contaminants were detected in four Sigma-Aldrich (15)N(2) lecture bottles from two discrete batch syntheses. Per mole of (15)N(2) gas, 34 to 1900 µmoles of (15)N-ammonium, 1.8 to 420 µmoles of (15)N-nitrate/nitrite, and ≥21 µmoles of (15)N-nitrous oxide were detected. One (15)N(2) lecture bottle from Campro Scientific contained ≥11 µmoles of (15)N-nitrous oxide per mole of (15)N(2) gas, and no detected (15)N-nitrate/nitrite at the given experimental (15)N(2) tracer dilutions. Two Cambridge Isotopes lecture bottles from discrete batch syntheses contained ≥0.81 µmoles( 15)N-nitrous oxide per mole (15)N(2), and trace concentrations of (15)N-ammonium and( 15)N-nitrate/nitrite. (15)N(2) gas equilibrated cultures of the green algae Dunaliella tertiolecta confirmed that the (15)N-contaminants are assimilable. A finite-differencing model parameterized using oceanic field conditions typical of N(2) fixation assays suggests that the degree of detected (15)N-ammonium contamination could yield inferred N(2) fixation rates ranging from undetectable, <0.01 nmoles N L(−1 )d(−1), to 530 nmoles N L(−1 )d(−1), contingent on experimental conditions. These rates are comparable to, or greater than, N(2) fixation rates commonly detected in field assays. These results indicate that past reports of N(2) fixation should be interpreted with caution, and demonstrate that the purity of commercial (15)N(2) gas must be ensured prior to use in future N(2) fixation rate determinations.