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.
Highlights
Nitrogen (N) is a major nutrient required universally by photosynthetic organisms
Ammonium and nitrate solutions were prepared with salts or with solutions obtained from different distributors: sodium nitrate (NaNO3: CAS 7631-99-4), potassium nitrate (KNO3: CAS 7757-79-1), and ammonium chloride (NH4Cl: CAS 12125-02-9) from Fisher Scientific (Pittsburgh, PA); analytical-grade potassium nitrate (CAS 7757-79-1) from Fluka Analytical and a gravimetric solution of ammonium chloride from SPEX CertiPrep (Metuchen, NJ)
This study reveals that some commercial 15N2 gas stocks contain contaminant 15N-labeled bioavailable nitrogen species, including nitrate/nitrite, ammonium and nitrous oxide
Summary
Nitrogen (N) is a major nutrient required universally by photosynthetic organisms. Its availability in the environment can directly affect the ecology and productivity of terrestrial and marine ecosystems, with important implications for the regional and global carbon cycles. The 15N2 tracer assay was originally developed when artificially 15N-enriched substrate N2 first became available [4]. This approach was superseded by the acetylene reduction technique, as the cost and availability of high precision isotope ratio measurements proved restrictive [3]. It is generally the preferred method to quantify N2 fixation rates in both terrestrial and aquatic environments [1], owing to its high sensitivity, and ability to provide qualitative and quantitative constraints on the translocation and the fate of biologically fixed N [8,9,10]
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