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.

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To provide mechanistic constraints to interpret nitrogen (N) and oxygen (O) isotope ratios of nitrate ( NO3−), 15N/14N and 18O/16O, in the environment, we measured the enzymatic NO3− N and O isotope effects (15ε and 18ε) during its reduction by NO3− reductase enzymes, including (1) a prokaryotic respiratory NO3− reductase, Nar, from the heterotrophic denitrifier Paracoccus denitrificans, (2) eukaryotic assimilatory NO3− reductases, eukNR, from Pichia angusta and from Arabidopsis thaliana, and (3) a prokaryotic periplasmic NO3− reductase, Nap, from the photoheterotroph Rhodobacter sphaeroides. Enzymatic Nar and eukNR assays with artificial viologen electron donors yielded identical 18ε and 15ε of ∼28‰, regardless of [ NO3−] or assay temperature, suggesting analogous kinetic mechanisms with viologen reductants. Nar assays fuelled with the physiological reductant hydroquinone (HQ) also yielded 18ε ≈ 15ε, but variable amplitudes from 21‰ to 33.0‰ in association with [ NO3−], suggesting analogous substrate sensitivity in vivo. Nap assays fuelled by viologen revealed 18ε:15ε of 0.50, where 18ε ≈ 19‰ and 15ε ≈ 38‰, indicating a distinct catalytic mechanism than Nar and eukNR. Nap isotope effects measured in vivo showed a similar 18ε:15ε of 0.57, but reduced 18ε ≈ 11‰ and 15ε ≈ 19‰. Together, the results confirm identical enzymatic 18ε and 15ε during NO3− assimilation and denitrification, reinforcing the reliability of this benchmark to identify NO3− consumption in the environment. However, the amplitude of enzymatic isotope effects is apt to vary in vivo. The distinctive signature of Nap is of interest for deciphering catalytic mechanisms but may be negligible in most environments given its physiological role.

Hydroxyl radical (•OH) is produced in soils from oxidation of reduced iron (Fe(II)) by dissolved oxygen (O2) and can oxidize dissolved organic carbon (DOC) to carbon dioxide (CO2). Understanding the role of •OH on CO2 production in soils requires knowing whether Fe(II) production or O2 supply to soils limits •OH production. To test the relative importance of Fe(II) production versus O2 supply, we measured changes in Fe(II) and O2 and in situ •OH production during simulated precipitation events and during common, waterlogged conditions in mesocosms from two landscape ages and the two dominant vegetation types of the Arctic. The balance of Fe(II) production and consumption controlled •OH production during precipitation events that supplied O2 to the soils. During static, waterlogged conditions, •OH production was controlled by O2 supply because Fe(II) production was higher than its consumption (oxidation) by O2. An average precipitation event (4 mm) resulted in 200 µmol •OH m−2 per day produced compared to 60 µmol •OH m−2 per day produced during waterlogged conditions. These findings suggest that the oxidation of DOC to CO2 by •OH in arctic soils, a process potentially as important as microbial respiration of DOC in arctic surface waters, will depend on the patterns and amounts of rainfall that oxygenate the soil.

Taxon-specific nitrogen (N) isotope data from the summertime Sargasso Sea have previously suggested reliance of prokaryotic phytoplankton on regenerated N but a greater importance of nitrate assimilation for eukaryotic phytoplankton. To investigate this further, particles collected in the summer from , 100 m at the Bermuda Atlantic Time-series Study site were incubated in particle-free, unamended surface seawater (from 4 m and 30 m) with measured ambient nitrate and ammonium concentrations. Preincubation and postincubation particles were sorted using flow cytometry, and the d 15 N of separated prokaryotic and eukaryotic phytoplankton was analyzed. In July 2009 and 2010, nitrate was undetectable throughout the euphotic zone (the upper , 100 m). The d 15 N of prokaryotic biomass was initially low and remained low upon incubation, with one exception out of six. Preincubation eukaryotic d 15 N was higher than or similar to prokaryotic d 15 N and decreased or remained low after incubation. These data confirm the expectation that, in nitrate-deplete water, all phytoplankton shift toward a low d 15 N characteristic of recycled N assimilation. In June 2010, the euphotic zone nitrate concentration was anomalously high (, 1 mmol L 21 ), and the d 15 N of all populations varied as a function of depth, with highest d 15 N in the surface where nitrate was lowest. After incubation in surface water, the d 15 N of all sorted phytoplankton increased, suggesting nitrate assimilation by all groups, including Prochlorococcus, the nitrate assimilatory capabilities of which have been debated. This study supports the use of the d 15 N of sorted phytoplankton as an indicator of the dominant N source fueling their growth.