The Contamination of Commercial 15N2 Gas Stocks with 15N-Labeled Nitrate and Ammonium and Consequences for Nitrogen Fixation Measurements

We report on the contamination of commercial 15-nitrogen (N-15) N-2 gas stocks with N-15-enriched ammonium, nitrate and/or nitrite, and nitrous oxide. N-15(2) gas is used to estimate N-2 fixation rates from incubations of environmental samples by monitoring the incorporation of isotopically labeled N-15(2) into organic matter. However, the microbial assimilation of bioavailable N-15-labeled N-2 gas contaminants, nitrate, nitrite, and ammonium, is liable to lead to the inflation or false detection of N-2 fixation rates. N-15(2) gas procured from three major suppliers was analyzed for the presence of these N-15-contaminants. Substantial concentrations of N-15-contaminants were detected in four Sigma-Aldrich N-15(2) lecture bottles from two discrete batch syntheses. Per mole of N-15(2) gas, 34 to 1900 mmoles of N-15-ammonium, 1.8 to 420 mmoles of (15)Nnitrate/nitrite, and >= 21 mmoles of N-15-nitrous oxide were detected. One N-15(2) lecture bottle from Campro Scientific contained >= 11 mmoles of N-15-nitrous oxide per mole of N-15(2) gas, and no detected N-15-nitrate/nitrite at the given experimental N-15(2) tracer dilutions. Two Cambridge Isotopes lecture bottles from discrete batch syntheses contained >= 0.81 mu moles N-15-nitrous oxide per mole (15)N2, and trace concentrations of N-15-ammonium and N-15-nitrate/nitrite. N-15(2) gas equilibrated cultures of the green algae Dunaliella tertiolecta confirmed that the N-15-contaminants are assimilable. A finite-differencing model parameterized using oceanic field conditions typical of N-2 fixation assays suggests that the degree of detected N-15-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 N-15(2) gas must be ensured prior to use in future N-2 fixation rate determinations.