The contamination of commercial 15N2 gas stocks with 15N-labeled nitrate and ammonium and consequences for nitrogen fixation measurements

Abstract

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

Figure 1. (a) δ¹⁵N_NO3+NO2 (log scale) of nitrate solutions (10–300 µmol L⁻¹) following equilibration with 0.1 mL ¹⁵N₂ gas from lecture bottles procured from three distributors.

Solutions were 40 mL for Sigma-Aldrich and Campro Scientific equilibrations, and 100 mL for Cambridge Isotopes equilibrations. The solid line corresponds to the δ¹⁵N_NO3 of the control solutions for Sigma-Aldrich and Cambridge Isotopes experiments (δ¹⁵N_NO3 = 23.5±0.5‰); the dashed line corresponds to controls for Campro Scientific experiments (δ¹⁵N_NO3 = 14.15±0.1‰). Paired symbols identify replicate experimental treatments. (b) Corresponding apparent δ¹⁸O_NO3+NO2 of the experimental nitrate solutions. The solid line corresponds to the δ¹⁸O_NO3 of control solutions for the Sigma-Aldrich and Cambridge Isotope experiments (δ¹⁸O_NO3 = 18.9±0.3‰); the dashed line corresponds to controls for Campro Scientific experiments (25.4±0.3‰).

Figure 2. (a) δ¹⁵N_NO3+NO2 (log scale) of higher sensitivity equilibrations of 10 µmol L⁻¹ nitrate solutions (10 mL) with 2 mL of ¹⁵N₂ gas from a Cambridge Isotopes or a Sigma-Aldrich bottle.

(b) Corresponding apparent δ¹⁸O_NO3 (log scale) of higher sensitivity equilibrations of the two stocks. n = the number of experimental replicates.

Figure 3. (a) δ¹⁵N_NH4 (log scale) of 5 µmol L⁻¹ ammonium solutions after equilibration with 0.1 mL ¹⁵N₂ gas from respective Sigma-Aldrich and Cambridge Isotopes lecture bottles vs. control solutions.

Sigma-Aldrich treatments utilized 40 mL ammonium solutions, whereas Cambridge Isotopes treatments utilized 100 mL ammonium solutions. (b) δ¹⁵N_NH4 of higher sensitivity equilibrations of 5 µmol L⁻¹ ammonium solutions (10 mL) with 2.0 mL ¹⁵N₂ gas from Cambridge Isotopes lecture bottles vs. control solutions. n = the number of experimental replicates.

Figure 4. The δ¹⁵N of particulate nitrogen (δ¹⁵N_PN) of D. tertiolecta harvested in stationary phase following growth in media containing sodium nitrate (and no ammonium) and equilibrated with ¹⁵N₂ gas from Sigma Aldrich or Cambridge Isotopes.

n = the number of experimental replicates.