N2 Production Rates Research Articles

Structure, Dynamics, and Reactivity for Light Alkane Oxidation of Fe(II) Sites Situated in the Nodes of a Metal-Organic Framework.

Metal organic frameworks (MOFs), with their crystalline, porous structures, can be synthesized to incorporate a wide range of catalytically active metals in tailored surroundings. These materials have potential as catalysts for conversion of light alkanes, feedstocks available in large quantities from shale gas that are changing the economics of manufacturing commodity chemicals. Mononuclear high-spin (S = 2) Fe(II) sites situated in the nodes of the MOF MIL-100(Fe) convert propane via dehydrogenation, hydroxylation, and overoxidation pathways in reactions with the atomic oxidant N2O. Pair distribution function analysis, N2 adsorption isotherms, X-ray diffraction patterns, and infrared and Raman spectra confirm the single-phase crystallinity and stability of MIL-100(Fe) under reaction conditions (523 K in vacuo, 378-408 K C3H8 + N2O). Density functional theory (DFT) calculations illustrate a reaction mechanism for the formation of 2-propanol, propylene, and 1-propanol involving the oxidation of Fe(II) to Fe(III) via a high-spin Fe(IV)═O intermediate. The speciation of Fe(II) and Fe(III) in the nodes and their dynamic interchange was characterized by in situ X-ray absorption spectroscopy and ex situ Mössbauer spectroscopy. The catalytic relevance of Fe(II) sites and the number of such sites were determined using in situ chemical titrations with NO. N2 and C3H6 production rates were found to be first-order in N2O partial pressure and zero-order in C3H8 partial pressure, consistent with DFT calculations that predict the reaction of Fe(II) with N2O to be rate determining. DFT calculations using a broken symmetry method show that Fe-trimer nodes affecting reaction contain antiferromagnetically coupled iron species, andhighlight the importance of stabilizing high-spin (S = 2) Fe(II) species for effecting alkane oxidation at low temperatures (<408 K).

Rates and Pathways of N2 Production in a Persistently Anoxic Fjord: Saanich Inlet, British Columbia

Marine oxygen minimum zones (OMZs) support 30-50% of global fixed-nitrogen (N) loss but comprise only 7% of total ocean volume. This N-loss is driven by canonical denitrification and anaerobic ammonium oxidation (anammox), and the distribution and activity of these two processes vary greatly in space and time. Factors that regulate N-loss processes are complex, including organic matter availability, oxygen concentrations, and NO2- and NH4+ concentrations. While both denitrification and anammox produce N2, the overall geochemical outcome of these processes are different, as incomplete denitrification, for example, produces N2O, which is a potent greenhouse gas. Rates of anammox and denitrification and more detailed ecophysiological knowledge of the microorganisms catalyzing these processes are needed to develop more robust models of N-loss in OMZs. To this end, we conducted monthly incubations with 15N-labeled N during anoxic conditions and deep-water renewal cycle in Saanich Inlet, British Columbia, a persistently anoxic fjord. Both denitrification and anammox operated throughout the low oxygen water column with depth integrated rates of anammox and denitrification ranging from 0.150.03 to 3.40.3 and 0.020.006 to 142 mmol N2 m-2 d-1, respectively. Most N2 production in Saanich Inlet was driven by denitrification, with high rates developing in response to enhanced substrate supply from deep water renewal. Dynamics in rates of denitrification were linked to shifts in microbial community composition. Notably, periods of intense denitrification were accompanied by blooms in an Arcobacter population against a background community dominated by SUP05 and Marinimicrobia. Rates of N2 production through denitrification and anammox, and their dynamics, were then explored through flux-balance modeling with higher rates of denitrification linked to the physiology of substrate uptake. Overall, both denitrification and anammox operated throughout the year, contributing to an annual N-loss of 2 x 10-3 Tg N2 yr-1, 37% of which we attribute to anammox and 63% to complete denitrification. Extrapolating these rates from Saanich Inlet to all similar coastal inlets in BC (2478 km2), we estimate that these inlets contribute 0.1 % to global pelagic N-loss.

Anammox and Denitrification in the Intertidal Sediment of the Hypereutrophic Yatsu Tidal Flat, Japan

An increase in available nitrogen loading in intertidal ecosystems causes eutrophication and macroalgae blooms. Denitrification and anaerobic ammonium oxidation (anammox) lead to the removal of bioavailable nitrogen, but few studies have examined this in intertidal sediments. The sediment anammox and denitrification rates in September 2015 and November 2016 were measured using a 15N tracer technique at two sites, with and without macroalgae, in the hypereutrophic Yatsu tidal flat, eastern Japan. At both sites, the rate of N2 production via anammox was consistently low compared with that via denitrification, accounting for < 7% of the total N2 production. In a fed-batch incubation experiment, the anammox rate increased in the surface sediment after 3 months. However, the contribution of anammox to nitrogen removal did not exceed that of denitrification, suggesting that denitrification is the major pathway for conversion of inorganic nitrogen to N2, and that anammox plays a limited role in nitrogen removal in the Yatsu tidal flat. Denitrification activity measured from August 2012 to January 2017 using the acetylene block method was higher in the sediment with macroalgae than that without. Multiple linear regression analysis revealed that denitrification in the sediment with macroalgae was limited by the nitrogen substrate, likely due to competition with macroalgae for nitrogen. Temperature and H2S production under macroalgae cover might also affect denitrification. In comparison, the organic carbon content was a key factor regulating heterotrophic denitrification in the sediment without macroalgae. These findings suggest that the occurrence of macroalgae changes the progress of denitrification in intertidal ecosystems.

Nitrous Oxide Production in Response to Oxygen in a Solar Greenhouse Vegetable Soil

To explore the sources of peak nitrous oxide (N2O) flushes in solar greenhouse vegetable field, an experiment was conducted with two conventional vegetable soils under different initial volume fractions of oxygen (O2) (0%, 1%, 3%, 5%, and 10%). A robotized incubation system was employed to analyze the gas kinetics[O2, N2O, nitric oxide (NO), nitrogen (N2), and carbon dioxide (CO2)] every 6 or 8 h and calculate the N2O/(NO+N2O+N2) index. Sodium chlorate (NaClO3) was used to inhibit the oxidation of NO2- to further explore the relationship between N2O and nitrite (NO2-). A parallel off-line incubation in triplicates was conducted under similar conditions to measure the dynamic changes in inorganic nitrogen content[ammonia (NH4+), nitrate (NO3-), and NO2-]. The results showed that N2O production under anaerobic condition was significantly higher than that under aerobic condition. The peak value of N2O in the soil collected from a straw-added plot (DIS) was significantly higher than that in the soil from non-straw added plot (DI) (P<0.01) when the volume fraction of oxygen was ≤ 1%. Oxygen can directly affect N2O production by delaying or inhibiting N2O reduction, with significant increase in N2O production rate under oxygen-depleted condition. However, the N2production rate decreased significantly with increase in initial oxygen volume fraction (P<0.01). When the initial volume fraction of oxygen was between 1% and 5%, a continuous accumulation of NO2- was observed during the incubation period, resulting in the significantly higher N2O/(NO+N2O+N2) index than that in either anaerobic or 10% of oxygen treatments. Furthermore, a linear correlation was observed between NO2- and N2O at 5% and 10% of oxygen with the addition of NaClO3 (R2 ≥ 0.85). Incomplete denitrification and nitrifier denitrification from NO2- induction co-occurred in the range of 1% and 5% volume fractions of oxygen, significantly increasing the soil N2O production and N2O/(NO+N2O+N2) index. In addition, N2O production under anaerobic condition was significantly higher than that under aerobic condition (P<0.01).

N2 production through denitrification and anammox across the continental margin (shelf–slope–rise) of the Ulleung Basin, East Sea

AbstractExperimental determinations of nitrogen cycling in deep‐sea sediments are strongly underrepresented in the databases. To investigate the total N2 production rates and relative contribution of denitrification and anaerobic ammonium oxidation (anammox) to benthic fixed‐N removal processes, we conducted 15N isotope‐labeling incubation experiments in whole cores and slurries at nine stations across the continental margin from the shelf (&lt; 200 m) and into the deep (&gt; 2000 m) Ulleung Basin (UB) in the East Sea. The total N2 production rates (anammox plus denitrification) in the center of the UB (8.4 ± 0.2 μmol N m−2 h−1) were high compared to most other deep‐sea sediments at similar water depths. Denitrification rates decreased from the shelf (7.6 ± 0.6 μmol N m−2 h−1) to the basin (3.2 ± 0.4 μmol N m−2 h−1), in proportion to benthic oxygen consumption, whereas anammox rates remained relatively constant or even increased slightly (1.3–4.1 μmol N m−2 h−1). The contribution of anammox to the total N2 production (ra) increased with increasing water depth from the shelf (ca. 17%) to the basin (ca. 56%). The enhanced ra in the center of the UB was associated with an increased availability of nitrite for anammox, which was likely a result of the competitive suppression of denitrification by manganese reduction under MnO2‐rich conditions. Our results emphasize the importance of anammox as a sink for reactive nitrogen in deep‐sea sediments and contribute toward a mechanistic understanding of the factors controlling benthic reactive nitrogen loss in the ocean.

Spatial Patterns of Groundwater Biogeochemical Reactivity in an Intertidal Beach Aquifer

AbstractBeach aquifers host a dynamic and reactive mixing zone between fresh and saline groundwater of contrasting origin and composition. Seawater, driven up the beachface by waves and tides, infiltrates into the aquifer and meets the seaward‐discharging fresh groundwater, creating and maintaining a reactive intertidal circulation cell. Within the cell, land‐derived nutrients delivered by fresh groundwater are transformed or attenuated. We investigated this process by collecting pore water samples from multilevel wells along a shore‐perpendicular transect on a beach near Cape Henlopen, Delaware, and analyzing solute and particulate concentrations. Pore water incubation experiments were conducted to determine rates of oxygen consumption and nitrogen gas production. A numerical model was employed to support field and laboratory interpretations. Results showed that chemically sensitive parameters such as pH and ORP diverged from salinity distribution patterns, indicating biogeochemical reactivity within the circulation cell. The highest respiration rates were found in the landward freshwater‐saltwater mixing zone, supported by high dissolved inorganic carbon. Chlorophyll a, a proxy for phytoplankton, and particulate carbon did not co‐occur with the highest respiration rates but were heterogeneously distributed in deeper and hypoxic areas of the cell. The highest rates of N2 production were also found in the mixing zone coinciding with elevated O2 consumption rates but closer to the lower discharge point. Model results were consistent with these observations, showing heightened denitrification in the mixing zone. The results of this work emphasize the relationship between the physical flow processes of the circulation cell and its biogeochemical reactivity and highlight the environmental significance of sandy beaches.

Ampicillin Mineralization by Denitrifying Process: Kinetic and Metabolic Effects.

The impact of the antibiotic ampicillin (AMP) on the metabolic and kinetics of denitrification process as well as the sludge ability for oxidizing it was evaluated in batch assays. Denitrifying reference assays with acetate-C and nitrate-N (C/N ratio of 1.1) were conducted for establishing the metabolic and kinetic performance of the denitrifying sludge. Assays amended with 10mg AMP-CL-1 were also performed. In reference assays, acetate and nitrate consumption efficiencies of 100% with a total conversion to HCO3- and N2 were achieved within 1.5h. When 10mg AMP-CL-1 was added, total and simultaneous consumption of nitrate-N, acetate-C, and AMP-C was achieved within 12h. The substrates were completely reduced to N2 and oxidized to HCO3-, respectively. No nitrite-N was registered at the end of culture. AMP caused a reversible inhibitory effect on specific nitrate and acetate consumption and N2 production rates. Complete consumption and mineralization of AMP associated to nitrate reduction to N2 were achieved. This work provides the first evidences on the metabolic and kinetic performance of a denitrifying sludge exposed to AMP. These results might be considered for proposing useful wastewater treatments where β-Lactam antibiotics can be present.

Mutualism between ribbed mussels and cordgrass enhances salt marsh nitrogen removal

AbstractSalt marsh ecosystems have declined globally and are increasingly threatened by erosion, sea level rise, and urban development. These highly productive, physically demanding ecosystems are populated by core species groups that often have strong trophic interactions with implications for ecosystem function and service provision. Positive interactions occur between ribbed mussels (Geukensia demissa) and cordgrass (Spartina alterniflora). Mussels transfer particulate nitrogen from the water column to the marsh sediments, which stimulates cordgrass growth, and cordgrass provides predator and/or heat stress refuge for mussels. Here, we test mussel facilitation of two functions in salt marshes that relate to N removal: microbial denitrification and water filtration. Microcosm experiments revealed that the highest rates of N2 production and nitrification occurred when mussels were present with marsh vegetation, suggesting that mussels enhanced coupling of the nitrification–denitrification. Surveys spanning the York River Estuary, Chesapeake Bay, showed that the highest densities of mussels occurred in the first meter for all marsh types with mainstem fringing (1207 ± 265 mussels/m2) being the most densely populated. The mussel population was estimated to be ~197 million animals with a water filtration potential of 90–135 million L/hr. Erosion simulation models demonstrated that suitable marsh habitat for ribbed mussels along the York River Estuary would be reduced by 11.8% after 50 years. This reduction in mussel habitat resulted in a projected 15% reduction in ribbed mussel abundance and filtration capacity. Denitrification potential was reduced in conjunction with projected marsh loss (35,536 m2) by 205 g N/hr, a 16% reduction. Because of the predominant occurrence of ribbed mussels at the marsh seaward edge and because the highest proportional loss will occur for fringing marshes (20%), shoreline management practices that restore or create fringing marsh may help offset these projected losses.

N2 production rates limited by nitrite availability in the Bay of Bengal oxygen minimum zone

Nitrogen losses have not been observed in the Bay of Bengal, unlike in other ocean oxygen minimum zones. Chemical and molecular analyses reveal that trace levels of oxygen inhibit nitrate formation, largely preventing microbial N2 production.

Application of the isotope pairing technique in sediments where anammox, denitrification, and dissimilatory nitrate reduction to ammonium coexist

The 15N isotope pairing technique is widely used to quantify anammox, denitrification, and dissimilatory nitrate reduction to ammonium (DNRA) in sediments. However, the effects of DNRA on anammox and denitrification in slurry incubations and on genuine N2 production in intact core incubations have not been fully explored. We developed a mathematical model describing these effects of DNRA, and tested this model in field and computational studies. Our model calculations revealed that for slurry incubations the presence of DNRA tends to under -and overestimate the actual anammox and denitrification rates, respectively, when calculated according to Thamdrup and Dalsgaard (2002). We found this underestimate of anammox to be proportional to the 15 NH4+ mole fraction (FA), and the overestimate of denitrification to be related to both FA and the relative contribution of anammox to the total N2 production (ra). We propose three alternative procedures to better quantify anammox and denitrification rates in slurry incubations and two procedures for intact core incubations. Our model calculations also revealed that for intact core incubations, the presence of DNRA leads to an overestimate of the genuine N2 production rate when calculated according to Risgaard-Petersen et al. (2003). This overestimate depends on the ra and the mole fractions of 15 NH4+ and 15 NO3− in intact core incubations. The results of our field experiments and numerical modeling indicated that the overestimate of the genuine N2 production was less than 1% at two sites in the East China Sea.

Contribution of Anammox to Nitrogen Removal in Two Temperate Forest Soils.

Anaerobic ammonium oxidation with nitrite reduction to dinitrogen (termed anammox) has been reported to be an important process for removing fixed nitrogen (N) in marine ecosystems and in some agricultural and wetland soils. However, its importance in upland forest soils has never been quantified. In this study, we evaluated the occurrence of anammox activity in two temperate forest soils collected from northeastern China. With (15)N-labeled NO3 (-) incubation, we found that the combined potential of the N2 production rates of anammox and codenitrification ranged from 0.01 ± 0.01 to 1.2 ± 0.18 nmol N per gram of soil per hour, contributing 0.5% to 14.4% of the total N2 production along the soil profile. Denitrification was the main pathway of N2 production and accounted for 85.6% to 99.5% of the total N2 production. Further labeling experiments with (15)NH4 (+) and (15)NO2 (-) indicated that codenitrification was present in the mixed forest soil. Codenitrification and anammox accounted for 2% to 12% and 1% to 7% of the total N2 production, respectively. Two anammox species, "Candidatus Brocadia fulgida" and "Candidatus Jettenia asiatica," were detected in this study but in very low abundance (as indicated by the hzsB gene). Our results demonstrated that the anammox process occurs in forest soils, but the contribution to N2 loss might be low in these ecosystems. More research is necessary to determine the activities of different N2 releasing pathways in different forest soils. In this study, we examined the anammox activity in temperate upland forest soils using the (15)N isotope technique. We found that the anammox process contributed little to the N2 production rate in the studied forest soil. Two anammox organisms, "Candidatus Brocadia fulgida" and "Candidatus Jettenia asiatica," were detected. In addition, we found that codenitrification was another N2 production pathway in forest soils. Our results could contribute to the understanding of soil gaseous N losses and microbial controls in forest soils.

Rates of N2 production and diversity and abundance of functional genes associated with denitrification and anaerobic ammonium oxidation in the sediment of the Amundsen Sea Polynya, Antarctica

A combination of molecular microbiological analyses and metabolic rate measurements was conducted to elucidate the diversity and abundance of denitrifying and anaerobic ammonium oxidation (anammox) bacteria and the nitrogen gas (N2) production rates in sediment underlying the highly productive polynya (Stns. 10 and 17) and the sea-ice zone on the outer shelf (Stn. 83) of the Amundsen Sea, Antarctica. Despite the high water column productivity, the N2 production rates by denitrification (0.04–0.31nmolNcm−3sed.h−1) and anammox (0.13–0.26nmolNcm−3sed.h−1) were lower than those measured in other polar regions. In contrast, gene copy number (106–107copiescm−3 of nirS and nosZ genes targeting denitirifiers and 105–107copiescm−3 of 16S rRNA genes related to anammox bacteria) of the two bacterial groups at Stn. 17 was similar compared to those of other organic-rich environments. The majority of the nirS sequences were affiliated with Gammaproteobacteria (54% and 61% of the total nirS gene at Stns. 17 and 83, respectively), which were closely related to Pseudomonas aeruginosa. Most nosZ sequences (92% and 72% of the total nosZ genes at Stns. 17 and 83, respectively) were related to the Alphaproteobacteria, which were closely related to Ruegeria pomeroyi and Roseobacter denitrificans. Most (98%) of the sequences related to anammox bacteria were affiliated with Candidatus Scalindua at Stn. 17. Consequently, despite the low metabolic activity, the abundance and composition of most denitrifying and anammox bacteria detected from the ASP were similar to those reported from a variety of marine environments. Our results further imply that increased labile organic matter production resulting from a shift of the phytoplankton community from Phaeocystis to diatoms in response to rapid melting of sea ice stimulates metabolic activities of the denitrifying and anammox bacteria, thereby enhancing the N removal process in the ASP.

Denitrification Dominates Sediment Nitrogen Removal and Is Enhanced by Bottom-Water Hypoxia in the Northern Gulf of Mexico

Nutrient loads from the Mississippi River watershed are associated with seasonal development of bottom-water hypoxia in the northern Gulf of Mexico. Microbial nitrogen (N) transformations at the sediment-water interface are important in determining system productivity and the development and maintenance of hypoxia. Intact sediment cores were incubated in a continuous-flow system with stable isotope tracers to identify and quantify important N sources (e.g., N fixation), sinks (e.g., denitrification and anammox), and links (e.g., dissimilatory nitrate reduction to ammonium, DNRA). Microbial N sinks on the Louisiana-Texas continental shelf remove up to 68 % of the total N load from the Mississippi River watershed, and up to 29 % of this N removal (mean = 11.8 ± 1.7 %) may be due to anammox. The highest N2 production rates and ammonium effluxes were observed at low bottom-water oxygen concentrations, and sediments were a significant source of ammonium to the water column at all times. DNRA and heterotrophic N fixation were not consistent pathways for total sediment N fluxes, but their potential importance to N balance and productivity in the system warrants further study and inclusion in ecosystem models. Physical disturbance from passage of two hurricanes in 2008 resulted in lower N cycling rates and sediment oxygen consumption, with sediment processes migrating into the water column. Denitrification is the dominant N sink in the northern Gulf of Mexico and provides a valuable ecosystem service by mitigating N loads from the Mississippi River watershed, particularly during seasonal bottom-water hypoxia events.

Nitrous oxide emission from agricultural soils

Anthropogenic nitrogen loading, particularly fertiliser usage in agricultural soils is thought to be a potentially important source of nitrous oxide (N2O) emission, which can be controlled by properly managed fertiliser usage. In this research, laboratory scale experiments were conducted to evaluate N2O production and emission from agricultural soils under variable oxygen availability conditions. N2O was observed to be released from the reactors in the absence of oxygen as well as at low concentrations of oxygen. With the increase of oxygen concentration, N2O production decreased. In addition, N2O emission was found to coincide with corresponding nitrate (NO3−) depletion, indicating that denitrification was the dominating process that was responsible for N2O production. The depletion of NO3− was described by the sum of two exponential functions, i.e., NO3− reduction for N2O and NO3− reduction for N2. The simulation of the experimental results using above model demonstrated that N2 production dominated over N2O production in the absence of oxygen. Both N2O production rate and N2 production rate decreased with the increase of oxygen concentration. However, the decrease of N2 production rate was more pronounced than that of N2O production rate.

Influence of environmental factors on net N₂ and N₂O production in sediment of freshwater rivers.

Denitrification is an important N removal process in aquatic systems but is also implicated as a potential source of global N₂O emissions. However, the key factors controlling this process as well as N₂O emissions remain unclear. In this study, we identified the main factors that regulate the production of net N₂ and N₂O in sediments collected from rivers with a large amount of sewage input in the Taihu Lake region. Net N₂ and N₂O production were strongly associated with the addition of NO₃(-)-N and NH₄(+)-N. Specifically, NO₃(-)-N controlled net N₂ production following Michaelis-Menten kinetics. The maximum rate of net N₂ production (V max) was 116.3 μmol N2-N m(-2) h(-1), and the apparent half-saturation concentration (k m) was 0.65 mg N L(-1). N₂O to N₂ ratios increased from 0.18 ± 0.03 to 0.68 ± 0.16 with the addition of NO₃(-)-N, suggesting that increasing NO₃(-)-N concentrations favored the production of N₂O more than N₂. The addition of acetate enhanced net N₂ production and N₂O to N₂ ratios, but the ratios decreased by about 59.5% when acetate concentrations increased from 50 to 100 mg C L(-1), suggesting that the increase of N₂O to N₂ ratios had more to do with the net N₂ production rate rather than acetate addition in this experiment. The addition of Cl(-) did not affect the net N₂ production rates, but significantly enhanced N₂O to N₂ ratios (the ratios increased from 0.02 ± 0.00 to 0.10 ± 0.00), demonstrating that the high salinity effect might have a significant regional effect on N₂O production. Our results suggest that the presence of N-enriching sewage discharges appear to stimulate N removal but also increase N₂O to N₂ ratios.

Temperature response of denitrification and anammox reveals the adaptation of microbial communities to in situ temperatures in permeable marine sediments that span 50° in latitude

Abstract. Despite decades of research on the physiology and biochemistry of nitrate/nitrite-respiring microorganisms, little is known regarding their metabolic response to temperature, especially under in situ conditions. The temperature regulation of microbial communities that mediate anammox and denitrification was investigated in near shore permeable sediments at polar, temperate, and subtropical sites with annual mean temperatures ranging from −5 to 23 °C. Total N2 production rates were determined using the isotope pairing technique in intact core incubations under diffusive and simulated advection conditions and ranged from 2 to 359 μmol N m−2 d−1. For the majority of sites studied, N2 removal was 2–7 times more rapid under simulated advective flow conditions. Anammox comprised 6–14% of total N2 production at temperate and polar sites and was not detected at the subtropical site. Potential rates of denitrification and anammox were determined in anaerobic slurries in a temperature gradient block incubator across a temperature range of −1 °C to 42 °C. The highest optimum temperature (Topt) for denitrification was 36 °C and was observed in subtropical sediments, while the lowest Topt of 21 °C was observed at the polar site. Seasonal variation in the Topt was observed at the temperate site with values of 26 and 34 °C in winter and summer, respectively. The Topt values for anammox were 9 and 26 °C at the polar and temperate sites, respectively. The results demonstrate adaptation of denitrifying communities to in situ temperatures in permeable marine sediments across a wide range of temperatures, whereas marine anammox bacteria may be predominately psychrophilic to psychrotolerant. The adaptation of microbial communities to in situ temperatures suggests that the relationship between temperature and rates of N removal is highly dependent on community structure.

New high precision approach for measuring 15N–N2 gas fluxes from terrestrial ecosystems

Dinitrogen (N2) production from denitrification and anaerobic ammonium oxidation represents a loss of reactive nitrogen (N) from terrestrial and aquatic ecosystems to the atmosphere. The large 15N additions required to detect 15N2 production against the high atmospheric background precludes the use of the 15N tracer technique in natural terrestrial ecosystems. We present an isotope ratio mass spectrometry technique that dramatically improves the precision of 15N2 measurements. The approach uses gas chromatography to remove oxygen and gas purification techniques to remove water vapor and trace gases that can interfere with 15N2 analysis. The analytical precision for manual gas sample injection was 0.018‰ δ15N; this translates to a minimum detectable N2 flux of 0.12 ng-N g−1 h−1 over a 24 h incubation using our experimental parameters. We measured denitrification-derived N2 production rates of 0.67 ± 0.04 ng N g−1 dry soil h−1 following the addition of 0.1 μg 98 atom % 15N–NO3− g−1 dry soil to anaerobically-incubated soils; rates were significantly higher with the addition of 1.0 μg 98 atom % 15N–NO3− g−1 dry soil (p < 0.001, n = 5), averaging 1.3 ± 0.03 ng N g−1 dry soil h−1. The N2:N2O ratio at 24 h after 15N addition was 48 ± 12 and 5.4 ± 1.9 for the 0.1 μg 15N g−1 and 1.0 μg 15N g−1 treatments, respectively. The decrease in the N2:N2O with the addition of only 1 μg 15N g−1 underscores the need to use very low rates of 15N addition to accurately characterize denitrification dynamics. This analytical advance will allow us to better estimate the N2:N2O ratio of denitrification, constrain ecosystem N budgets, and explore the mechanisms of and controls on N2 production.

Effect of aquacultured oyster biodeposition on sediment N&lt;sub&gt;2 production in Chesapeake Bay

Suspension feeding bivalves have the potential to mitigate estuarine and coastal marine eutrophication by permanently removing nitrogen (N) from the system. We conducted an integrated field and laboratory examination of the effect of eastern oyster biodeposition on sedi- ment denitrification (DNF) and anammox (AMX) rates to quantify the N removal potential of oys- ter aquaculture using 2 commercial-scale sites in Chesapeake Bay, USA. Sediment N2 production rates were measured using 2 techniques, 15 N isotope tracer (n = 51) and N2:Ar (n = 30). Oyster biodeposit N-load rates explained 21% of variation in sediment N2 production (DNF and AMX). Oyster sediment N2 production rates ranged from 0.00 to 1.56 mmol N m �2 d �1 and were almost always lower than reference sediments. From laboratory-based biodeposit addition and field- based forced biodeposit accumulation experiments, we found ~2.50 mmol N m �2 d �1 to be the max- imum sediment N2 production capacity of these sediments, regardless of increasing organic N or labile organic carbon delivery rates. We found no evidence to support the contention that biode- position associated with oyster aquaculture significantly impacts annual N removal via sediment N2 production, i.e. stimulation or inhibition, above reference rates, but there was evidence that sediment NH4 + efflux rates were increased. We estimate the N removal rate via sediment N2 pro- duction at similar oyster cultivation sites (1750 m 2 ) with 5 × 10 5 oysters ranges from 0.49 to 12.60 kg N yr �1 , compared to 2.27 to 16.72 kg N yr �1 at reference sites. Thus, aqua cultured oyster biode- position did not have a ubiquitously enhancing effect on N removal rates via N2 production and is therefore unlikely to be effective as a policy initiative for eutrophication mitigation.

Inhibition of anaerobic ammonium oxidizing (anammox) enrichment cultures by substrates, metabolites and common wastewater constituents

Anaerobic ammonium oxidation (anammox) is an emerging technology for nitrogen removal that provides a more environmentally sustainable and cost effective alternative compared to conventional biological treatment methods. The objective of this study was to investigate the inhibitory impact of anammox substrates, metabolites and common wastewater constituents on the microbial activity of two different anammox enrichment cultures (suspended and granular), both dominated by bacteria from the genus Brocadia. Inhibition was evaluated in batch assays by comparing the N2 production rates in the absence or presence of each compound supplied in a range of concentrations. The optimal pH was 7.5 and 7.3 for the suspended and granular enrichment cultures, respectively. Among the substrates or products, ammonium and nitrate caused low to moderate inhibition, whereas nitrite caused almost complete inhibition at concentrations higher than 15mM. The intermediate, hydrazine, either stimulated or caused low inhibition of anammox activity up to 3mM. Of the common constituents in wastewater, hydrogen sulfide was the most severe inhibitor, with 50% inhibitory concentrations (IC50) as low as 0.03mM undissociated H2S. Dissolved O2 showed moderate inhibition (IC50=2.3–3.8mgL−1). In contrast, phosphate and salinity (NaCl) posed very low inhibition. The suspended- and granular anammox enrichment cultures had similar patterns of response to the various inhibitory stresses with the exception of phosphate. The findings of this study provide comprehensive insights on the tolerance of the anammox process to a wide variety of potential inhibiting compounds.

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

Anammox and denitrification mediated by bacteria are known to be the major microbial processes converting fixed N to N(2) gas in various ecosystems. Codenitrification and denitrification by fungi are additional pathways producing N(2) in soils. However, fungal codenitrification and denitrification have not been well investigated in agricultural soils. To evaluate bacterial and fungal processes contributing to N(2) production, molecular and (15)N 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. (15)N tracer incubation experiments with (15)NO(3)(-) or (15)NH(4)(+) addition were conducted to measure the N(2) production rates from anammox, denitrification, and codenitrification. Soil incubation experiments with antibiotic treatments were also used to differentiate between fungal and bacterial N(2) 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 N(2)-N g(-1) day(-1), while the combined potential rates of anammox and codenitrification ranged from 2.796 to 147.711 nmol N(2)-N g(-1) day(-1). Soil incubation experiments with antibiotics indicated that fungal codenitrification was the primary process contributing to N(2) production in the North Carolina soil. This study clearly demonstrates the importance of fungal processes in the agricultural N cycle.