Craig Tobias

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.

Co-Occurring Anammox, Denitrification, and Codenitrification in Agricultural Soils

ABSTRACT Anammox and denitrification mediated by bacteria are known to be the major microbial processes converting fixed N to N2 gas in various ecosystems. Codenitrification and denitrification by fungi are additional pathways producing N2 in soils. However, fungal codenitrification and denitrification have not been well investigated in agricultural soils. To evaluate bacterial and fungal processes contributing to N2 production, molecular and 15N isotope analyses were conducted with soil samples collected at six different agricultural fields in the United States. Denitrifying and anammox bacterial abundances were measured based on quantitative PCR (qPCR) of nitrous oxide reductase (nosZ) and hydrazine oxidase (hzo) genes, respectively, while the internal transcribed spacer (ITS) of Fusarium oxysporum was quantified to estimate the abundance of codenitrifying and denitrifying fungi. 15N tracer incubation experiments with 15NO3 − or 15NH4 + addition were conducted to measure the N2 production rates from anammox, denitrification, and codenitrification. Soil incubation experiments with antibiotic treatments were also used to differentiate between fungal and bacterial N2 production rates in soil samples. Denitrifying bacteria were found to be the most abundant, followed by F. oxysporum based on the qPCR assays. The potential denitrification rates by bacteria and fungi ranged from 4.118 to 42.121 nmol N2-N g−1 day−1, while the combined potential rates of anammox and codenitrification ranged from 2.796 to 147.711 nmol N2-N g−1 day−1. Soil incubation experiments with antibiotics indicated that fungal codenitrification was the primary process contributing to N2 production in the North Carolina soil. This study clearly demonstrates the importance of fungal processes in the agricultural N cycle.

Linking DNRA community structure and activity in a shallow lagoonal estuarine system.

Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are two nitrate respiration pathways in the microbial nitrogen cycle. Diversity and abundance of denitrifying bacteria have been extensively examined in various ecosystems. However, studies on DNRA bacterial diversity are limited, and the linkage between the structure and activity of DNRA communities has yet to be discovered. We examined the composition, diversity, abundance, and activities of DNRA communities at five sites along a salinity gradient in the New River Estuary, North Carolina, USA, a shallow temporal/lagoonal estuarine system. Sediment slurry incubation experiments with 15N-nitrate were conducted to measure potential DNRA rates, while the abundance of DNRA communities was calculated using quantitative PCR of nrfA genes encoding cytochrome C nitrite reductase, commonly found in DNRA bacteria. A pyrosequencing method targeting nrfA genes was developed using an Ion Torrent sequencer to examine the diversity and composition of DNRA communities within the estuarine sediment community. We found higher levels of nrfA gene abundance and DNRA activities in sediments with higher percent organic content. Pyrosequencing analysis of nrfA genes revealed spatial variation of DNRA communities along the salinity gradient of the New River Estuary. Percent abundance of dominant populations was found to have significant influence on overall activities of DNRA communities. Abundance of dominant DNRA bacteria and organic carbon availability are important regulators of DNRA activities in the eutrophic New River Estuary.

Role of Anaerobic Ammonium Oxidation (Anammox) in Nitrogen Removal from a Freshwater Aquifer

Anaerobic ammonium oxidation (anammox) couples the oxidation of ammonium with the reduction of nitrite, producing N2. The presence and activity of anammox bacteria in groundwater were investigated at multiple locations in an aquifer variably affected by a large, wastewater-derived contaminant plume. Anammox bacteria were detected at all locations tested using 16S rRNA gene sequencing and quantification of hydrazine oxidoreductase (hzo) gene transcripts. Anammox and denitrification activities were quantified by in situ (15)NO2(-) tracer tests along anoxic flow paths in areas of varying ammonium, nitrate, and organic carbon abundances. Rates of denitrification and anammox were determined by quantifying changes in (28)N2, (29)N2, (30)N2, (15)NO3(-), (15)NO2(-), and (15)NH4(+) with groundwater travel time. Anammox was present and active in all areas tested, including where ammonium and dissolved organic carbon concentrations were low, but decreased in proportion to denitrification when acetate was added to increase available electron supply. Anammox contributed 39-90% of potential N2 production in this aquifer, with rates on the order of 10 nmol N2-N L(-1) day(-1). Although rates of both anammox and denitrification during the tracer tests were low, they were sufficient to reduce inorganic nitrogen concentrations substantially during the overall groundwater residence times in the aquifer. These results demonstrate that anammox activity in groundwater can rival that of denitrification and may need to be considered when assessing nitrogen mass transport and permanent loss of fixed nitrogen in aquifers.

Estimating the effects of seawater intrusion on an estuarine nitrogen cycle by comparative network analysis

Nitrogen (N) removal from estuaries is driven in part by sedimentary microbial processes. The processes of denitrification and anaerobic ammonium oxidation (anammox) remove N from estuaries by producing di-nitrogen gas, and each can be coupled to N recycling pathways such as nitrification and dissimilatory nitrate reduction to ammonium (DNRA). Environmental conditions in estuaries influence sedimentary N cycling processes; therefore, seawater intrusion may affect the coupling of N cycling processes in the freshwater portions of estuaries. This study investigated the potential effects of seawater intrusion on these process couplings through a comparative modeling approach. We applied environ analysis, a form of ecological network analysis, to two N cycling mass-balance network models constructed at freshwater (oligohaline) and saltwater (polyhaline) sites in the Cape Fear River Estuary, North Carolina. We used a space-for-time substitution to predict the effects of seawater intrusion on the sedimentary N cycle. Further, we conducted an uncertainty analysis using linear inverse modeling to evaluate the effects of parameterization uncertainty on model results. Nitrification coupled to both denitrification and anammox was 2.5 times greater in the oligohaline model, while DNRA coupled to anammox was 2.7 times greater in the polyhaline model. However, the total amount of N2 gas produced relative to the nitrogen inputs to each network was 4.7% and 4.6% at the oligohaline and polyhaline sites, respectively. These findings suggest that changes in water chemistry from seawater intrusion may favor direct over coupled nitrogen removal, but may not substantially change the N removal capacity of the sedimentary microbial processes.

Molecular and stable isotope methods to detect and measure anaerobic ammonium oxidation (anammox) in aquatic ecosystems.

Numerous microbial processes transform nitrogen (N) but three anaerobic respiratory pathways remove fixed N from the environment: denitrification (nitrate conversion to N(2)), anaerobic ammonium oxidation (anammox; ammonium plus nitrite conversion to N(2)), and nitrite dependent methane oxidation (nitrite conversion to N(2)). Nitrification becomes a part of N removal processes as a supplier of nitrite (NO(2)(-)) and nitrate (NO(3)(-)) to anammox and denitrifying bacteria in anoxic water and sediments. It is important to detect and measure anammox and denitrification to understand biogeochemical N cycle and to estimate N removal potential in aquatic ecosystems. Denitrification has been extensively studied in many ecosystems to examine diversity and spatial and temporal dynamics of denitrifying communities as well as to understand its importance in regional and global N cycles. Nitrite dependent methane oxidation was recently discovered as a new pathway of removing fixed N and just started to examine its importance in different ecosystems. Anammox has undergone limited examination, although the number of studies is continuously increasing. There are many questions remaining in order to understand the factors controlling activities and community structures of anammox bacteria in different ecosystems. This chapter reviews both molecular and stable isotope methods to detect and measure anammox in anoxic sediments and water.

Nitrogen reduction pathways in estuarine sediments: Influences of organic carbon and sulfide

Potential rates of sediment denitrification, anaerobic ammonium oxidation (anammox), and dissimilatory nitrate reduction to ammonium (DNRA) were mapped across the entire Niantic River Estuary, CT, USA, at 100–200 m scale resolution consisting of 60 stations. On the estuary scale, denitrification accounted for ~ 90% of the nitrogen reduction, followed by DNRA and anammox. However, the relative importance of these reactions to each other was not evenly distributed through the estuary. A Nitrogen Retention Index (NIRI) was calculated from the rate data (DNRA/(denitrification + anammox)) as a metric to assess the relative amounts of reactive nitrogen being recycled versus retained in the sediments following reduction. The distribution of rates and accompanying sediment geochemical analytes suggested variable controls on specific reactions, and on the NIRI, depending on position in the estuary and that these controls were linked to organic carbon abundance, organic carbon source, and pore water sulfide concentration. The relationship between NIRI and organic carbon abundance was dependent on organic carbon source. Sulfide proved the single best predictor of NIRI, accounting for 44% of its observed variance throughout the whole estuary. We suggest that as a single metric, sulfide may have utility as a proxy for gauging the distribution of denitrification, anammox, and DNRA.

Removal rates of dissolved munitions compounds in seawater.

The historical exposure of coastal marine systems to munitions compounds is of significant concern due to the global distribution of impacted sites and known toxicological effects of nitroaromatics. In order to identify specific coastal regions where persistence of these chemicals should be of concern, it is necessary to experimentally observe their behavior under a variety of realistic oceanographic conditions. Here, we conduct a mesocosm scale pulse addition experiment to document the behavior of two commonly used explosives, 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in simulated marine systems containing water and sediments collected from Long Island Sound, CT. The addition of sediments and sediment grain-size had a major influence on the loss rates of all compounds detected. RDX and reduced TNT products were removed from seawater only in the presence of sediment, and TNT degraded significantly faster in the presence of sediment. Both compounds were removed from the system faster with decreasing grain-size. Based on these findings and a thorough review of the literature, we hypothesize that in addition to bacterial abundance and nutrient availability, TNT removal rates in coastal marine waters may be controlled by sorption and rapid surface-mediated bacterial transformation, while RDX removal rates are controlled by diffusion into sedimentary anoxic regions and subsequent anaerobic bacterial breakdown. A comparison of published removal rates of RDX and TNT highlights the extreme variability in measured degradation rates and identifies physicochemical variables that covary with the breakdown of these munitions compounds.

Chemical formation of hybrid di-nitrogen calls fungal codenitrification into question.

Removal of excess nitrogen (N) can best be achieved through denitrification processes that transform N in water and terrestrial ecosystems to di-nitrogen (N2) gas. The greenhouse gas nitrous oxide (N2O) is considered an intermediate or end-product in denitrification pathways. Both abiotic and biotic denitrification processes use a single N source to form N2O. However, N2 can be formed from two distinct N sources (known as hybrid N2) through biologically mediated processes of anammox and codenitrification. We questioned if hybrid N2 produced during fungal incubation at neutral pH could be attributed to abiotic nitrosation and if N2O was consumed during N2 formation. Experiments with gas chromatography indicated N2 was formed in the presence of live and dead fungi and in the absence of fungi, while N2O steadily increased. We used isotope pairing techniques and confirmed abiotic production of hybrid N2 under both anoxic and 20% O2 atmosphere conditions. Our findings question the assumptions that (1) N2O is an intermediate required for N2 formation, (2) production of N2 and N2O requires anaerobiosis, and (3) hybrid N2 is evidence of codenitrification and/or anammox. The N cycle framework should include abiotic production of N2.

Mineralization of RDX-Derived Nitrogen to N2 via Denitrification in Coastal Marine Sediments

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a common constituent of military explosives. Despite RDX contamination at numerous U.S. military facilities and its mobility to aquatic systems, the fate of RDX in marine systems remains largely unknown. Here, we provide RDX mineralization pathways and rates in seawater and sediments, highlighting for the first time the importance of the denitrification pathway in determining the fate of RDX-derived N. (15)N nitro group labeled RDX ((15)N-[RDX], 50 atom %) was spiked into a mesocosm simulating shallow marine conditions of coastal Long Island Sound, and the (15)N enrichment of N2 (δ(15)N2) was monitored via gas bench isotope ratio mass spectrometry (GB-IRMS) for 21 days. The (15)N tracer data were used to model RDX mineralization within the context of the broader coastal marine N cycle using a multicompartment time-stepping model. Estimates of RDX mineralization rates based on the production and gas transfer of (15)N2O and (15)N2 ranged from 0.8 to 10.3 μmol d(-1). After 22 days, 11% of the added RDX had undergone mineralization, and 29% of the total removed RDX-N was identified as N2. These results demonstrate the important consideration of sediment microbial communities in management strategies addressing cleanup of contaminated coastal sites by military explosives.