Julie Granger

Removal of nitrite with sulfamic acid for nitrate N and O isotope analysis with the denitrifier method

In environmental water samples that contain both nitrate (NO3-) and nitrite (NO2-), isotopic analysis of nitrate alone by all currently available methods requires pretreatment to remove nitrite. Sulfamic acid addition, used previously for this purpose (Wu JP, Calvert SE, Wong CS. Deep-Sea Research Part I - Oceanographic Research Papers 1997; 44: 287), is shown here to be compatible with the denitrifier method for both N and O isotope analysis of nitrate. Sulfamic acid at a pH of approximately 1.7 reduces nitrite to N2. Samples are then neutralized with base prior to isotope analysis, to alleviate the buffering demands of the bacterial media and as a precaution to prevent modification of nitrate during storage with the residual sulfamic acid at low pH. Under appropriate reaction conditions, nitrite is completely removed within minutes. Sulfamic acid treatment does not compromise the completeness of the conversion of nitrate into N2O or the precision and accuracy of N and O isotope measurements by the denitrifier method. Nitrite concentrations upwards of 7 times the ambient nitrate can be removed without affecting the isotope composition of nitrate. The method is applied to analyses of the coupled N and O isotopes of nitrate and nitrite in waters of the Mexican Margin, to illustrate its efficacy and utility when employed either in the field upon sample collection or in the lab after months of frozen sample storage.

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 (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.

Eukaryotic Assimilatory Nitrate Reductase Fractionates N and O Isotopes with a Ratio near Unity

In order to (i) establish the biological systematics necessary to interpret nitrogen (N) and oxygen (O) isotope ratios of nitrate ((15)N/(14)N and (18)O/(16)O) in the environment and (ii) investigate the potential for isotopes to elucidate the mechanism of a key N cycle enzyme, we measured the nitrate N and O isotope effects ((15)ε and (18)ε) for nitrate reduction by two assimilatory eukaryotic nitrate reductase (eukNR) enzymes. The (15)ε for purified extracts of NADPH eukNR from the fungus Aspergillus niger and the (15)ε for NADH eukNR from cell homogenates of the marine diatom Thalassiosira weissflogii were indistinguishable, yielding a mean (15)ε for the enzyme of 26.6 ± 0.2‰. Both forms of eukNR imparted near equivalent fractionation on N and O isotopes. The increase in (18)O/(16)O versus the increase in (15)N/(14)N (relative to their natural abundances) was 0.96 ± 0.01 for NADPH eukNR and 1.09 ± 0.03 for NADH eukNR. These results are the first reliable measurements of the coupled N and O isotope effects for any form of eukNR. They support the prevailing view that intracellular reduction by eukNR is the dominant step in isotope fractionation during nitrate assimilation and that it drives the (18)ε:(15)ε ≈ 1 observed in phytoplankton cultures, suggesting that this O-to-N isotope signature will apply broadly in the environment. Our measured (15)ε and (18)ε may represent the intrinsic isotope effects for eukNR-mediated N-O bond rupture, a potential constraint on the nature of the enzymes transition state.

Nitrogen losses in anoxic marine sediments driven by Thioploca-anammox bacterial consortia

Ninety per cent of marine organic matter burial occurs in continental margin sediments, where a substantial fraction of organic carbon escapes oxidation and enters long-term geologic storage within sedimentary rocks. In such environments, microbial metabolism is limited by the diffusive supply of electron acceptors. One strategy to optimize energy yields in a resource-limited habitat is symbiotic metabolite exchange among microbial associations. Thermodynamic and geochemical considerations indicate that microbial co-metabolisms are likely to play a critical part in sedimentary organic carbon cycling. Yet only one association, between methanotrophic archaea and sulphate-reducing bacteria, has been demonstrated in marine sediments in situ, and little is known of the role of microbial symbiotic interactions in other sedimentary biogeochemical cycles. Here we report in situ molecular and incubation-based evidence for a novel symbiotic consortium between two chemolithotrophic bacteria—anaerobic ammonium-oxidizing (anammox) bacteria and the nitrate-sequestering sulphur-oxidizing Thioploca species—in anoxic sediments of the Soledad basin at the Mexican Pacific margin. A mass balance of benthic solute fluxes and the corresponding nitrogen isotope composition of nitrate and ammonium fluxes indicate that anammox bacteria rely on Thioploca species for the supply of metabolic substrates and account for about 57 ± 21 per cent of the total benthic N2 production. We show that Thioploca–anammox symbiosis intensifies benthic fixed nitrogen losses in anoxic sediments, bypassing diffusion-imposed limitations by efficiently coupling the carbon, nitrogen and sulphur cycles.

Isotopic overprinting of nitrification on denitrification as a ubiquitous and unifying feature of environmental nitrogen cycling

Significance Stable isotopes of nitrate have long provided a tool for tracking environmental sources and biological transformations. However, divergent interpretations of fundamental nitrate isotope systematics exist among disciplinary divisions. In an effort to transcend disciplinary boundaries of terrestrial and marine biogeochemistry, we use a quantitative model for coupled nitrogen and oxygen isotopes of nitrate founded on benchmarks established from microbial cultures, to reconcile decades of nitrate isotopic measurements in freshwater and seawater and move toward a unified understanding of cycling processes and isotope systematics. Our findings indicate that denitrification operates within the pervasive context of nitrite reoxidation mechanisms, specifically highlighting the relative importance of nitrification in marine denitrifying systems and anammox in groundwater aquifers. Natural abundance nitrogen and oxygen isotopes of nitrate (δ15NNO3 and δ18ONO3) provide an important tool for evaluating sources and transformations of natural and contaminant nitrate (NO3−) in the environment. Nevertheless, conventional interpretations of NO3− isotope distributions appear at odds with patterns emerging from studies of nitrifying and denitrifying bacterial cultures. To resolve this conundrum, we present results from a numerical model of NO3− isotope dynamics, demonstrating that deviations in δ18ONO3 vs. δ15NNO3 from a trajectory of 1 expected for denitrification are explained by isotopic over-printing from coincident NO3− production by nitrification and/or anammox. The analysis highlights two driving parameters: (i) the δ18O of ambient water and (ii) the relative flux of NO3− production under net denitrifying conditions, whether catalyzed aerobically or anaerobically. In agreement with existing analyses, dual isotopic trajectories >1, characteristic of marine denitrifying systems, arise predominantly under elevated rates of NO2− reoxidation relative to NO3− reduction (>50%) and in association with the elevated δ18O of seawater. This result specifically implicates aerobic nitrification as the dominant NO3− producing term in marine denitrifying systems, as stoichiometric constraints indicate anammox-based NO3− production cannot account for trajectories >1. In contrast, trajectories <1 comprise the majority of model solutions, with those representative of aquifer conditions requiring lower NO2− reoxidation fluxes (<15%) and the influence of the lower δ18O of freshwater. Accordingly, we suggest that widely observed δ18ONO3 vs. δ15NNO3 trends in freshwater systems (<1) must result from concurrent NO3− production by anammox in anoxic aquifers, a process that has been largely overlooked.

Coupled nitrate N and O stable isotope fractionation by a natural marine plankton consortium

The stable nitrogen (N) and oxygen (O) isotope ratios (15N/14N and 18O/16O, respectively) of nitrate (NO3-) were measured during incubations of freshly collected seawater to investigate the effect of light intensity on the isotope fractionation associated with nitrate assimilation and possible co-occurring regeneration and nitrification by in situ plankton communities. Surface seawater was collected off the coast of Vancouver, Canada, in late fall and in late summer and was incubated under different laboratory light conditions for 10 and 30 days, respectively. In the late summer experiments, parallel incubations were supplemented with 15NH4+ and H218O tracers to monitor co-occurring nitrification. Differences in irradiance in the fall incubations resulted in reduced nitrate consumption at low light, but had no distinguishable impact on the N-isotope isotope effect (15e) associated with NO3- assimilation, which ranged between 5 and 8‰. The late-summer community incubations, in contrast, showed reduced growth rates at low light and more elevated 15e of 11.9 ± 0.4‰, compared to 8.4 ± 0.3‰ at high-light conditions. The seasonal differences could reflect physiological adaptations of the fall plankton community to reduced irradiance, such that their incubation at low light did not elicit the increase in proportional cellular nitrate efflux required to raise the isotope effect. In both the fall and summer incubations, the ratio of the coincident rises in the δ15N and δ18O of NO3- was comparable to previous monoculture phytoplankton experiments, with a ∆δ18O:∆δ15N of ~1, regardless of light level. A decoupling of ∆δ18O:∆δ15N is expected if nitrification occurs concomitantly with nitrate assimilation. The lack of such decoupling is best explained by the absence of significant nitrification in any of our study’s treatments, an interpretation supported by our inability to identify any tracer 15N and 18O uptake into the NO3- pool in the late-summer community incubations.