Comparing Denitrification Estimates for a Texas Estuary by Using Acetylene Inhibition and Membrane Inlet Mass Spectrometry

Characterizing denitrification rates in aquatic ecosystems is essential to understanding how systems may respond to increased nutrient loading. Thus, it is important to ensure the precision and accuracy of the methods employed for measuring denitrification rates. The acetylene (C₂H₂) inhibition method is a simple technique for estimating denitrification. However, potential problems, such as inhibition of nitrification and incomplete inhibition of nitrous oxide reduction, may influence rate estimates. Recently, membrane inlet mass spectrometry (MIMS) has been used to measure denitrification in aquatic systems. Comparable results were obtained with MIMS and C₂H₂ inhibition methods when chloramphenicol was added to C₂H₂ inhibition assay mixtures to inhibit new synthesis of denitrifying enzymes. Dissolved-oxygen profiles indicated that surface layers of sediment cores subjected to the MIMS flowthrough incubation remained oxic whereas cores incubated using the C₂H₂ inhibition methods did not. Analysis of the microbial assemblages before and after incubations indicated significant changes in the sediment surface populations during the long flowthrough incubation for MIMS analysis but not during the shorter incubation used for the C₂H₂ inhibition method. However, bacterial community changes were also small in MIMS cores at the oxygen transition zone where denitrification occurs. The C₂H₂ inhibition method with chloramphenicol addition, conducted over short incubation intervals, provides a cost-effective method for estimating denitrification, and rate estimates are comparable to those obtained by the MIMS method.     

Simultaneous measurement of denitrification and nitrogen fixation using isotope pairing with membrane inlet mass spectrometry analysis

A method for estimating denitrification and nitrogen fixation simultaneously in coastal sediments was developed. An isotope-pairing technique was applied to dissolved gas measurements with a membrane inlet mass spectrometer (MIMS). The relative fluxes of three N(2) gas species ((28)N(2), (29)N(2), and (30)N(2)) were monitored during incubation experiments after the addition of (15)NO(3)(-). Formulas were developed to estimate the production (denitrification) and consumption (N(2) fixation) of N(2) gas from the fluxes of the different isotopic forms of N(2). Proportions of the three isotopic forms produced from (15)NO(3)(-) and (14)NO(3)(-) agreed with expectations in a sediment slurry incubation experiment designed to optimize conditions for denitrification. Nitrogen fixation rates from an algal mat measured with intact sediment cores ranged from 32 to 390 microg-atoms of N m(-2) h(-1). They were enhanced by light and organic matter enrichment. In this environment of high nitrogen fixation, low N(2) production rates due to denitrification could be separated from high N(2) consumption rates due to nitrogen fixation. Denitrification and nitrogen fixation rates were estimated in April 2000 on sediments from a Texas sea grass bed (Laguna Madre). Denitrification rates (average, 20 microg-atoms of N m(-2) h(-1)) were lower than nitrogen fixation rates (average, 60 microg-atoms of N m(-2) h(-1)). The developed method benefits from simple and accurate dissolved-gas measurement by the MIMS system. By adding the N(2) isotope capability, it was possible to do isotope-pairing experiments with the MIMS system.     

Simultaneous Measurement of Denitrification and Nitrogen Fixation Using Isotope Pairing with Membrane Inlet Mass Spectrometry Analysis

A method for estimating denitrification and nitrogen fixation simultaneously in coastal sediments was developed. An isotope-pairing technique was applied to dissolved gas measurements with a membrane inlet mass spectrometer (MIMS). The relative fluxes of three N₂ gas species (²⁸N₂, ²⁹N₂, and ³⁰N₂) were monitored during incubation experiments after the addition of ¹⁵NO₃⁻. Formulas were developed to estimate the production (denitrification) and consumption (N₂ fixation) of N₂ gas from the fluxes of the different isotopic forms of N₂. Proportions of the three isotopic forms produced from ¹⁵NO₃⁻ and ¹⁴NO₃⁻ agreed with expectations in a sediment slurry incubation experiment designed to optimize conditions for denitrification. Nitrogen fixation rates from an algal mat measured with intact sediment cores ranged from 32 to 390 μg-atoms of N m⁻² h⁻¹. They were enhanced by light and organic matter enrichment. In this environment of high nitrogen fixation, low N₂ production rates due to denitrification could be separated from high N₂ consumption rates due to nitrogen fixation. Denitrification and nitrogen fixation rates were estimated in April 2000 on sediments from a Texas sea grass bed (Laguna Madre). Denitrification rates (average, 20 μg-atoms of N m⁻² h⁻¹) were lower than nitrogen fixation rates (average, 60 μg-atoms of N m⁻² h⁻¹). The developed method benefits from simple and accurate dissolved-gas measurement by the MIMS system. By adding the N₂ isotope capability, it was possible to do isotope-pairing experiments with the MIMS system.     

Vertical distribution of denitrification in an estuarine sediment: integrating sediment flowthrough reactor experiments and microprofiling via reactive transport modeling

Denitrifying activity in a sediment from the freshwater part of a polluted estuary in northwest Europe was quantified using two independent approaches. High-resolution N(2)O microprofiles were recorded in sediment cores to which acetylene was added to the overlying water and injected laterally into the sediment. The vertical distribution of the rate of denitrification supported by nitrate uptake from the overlying water was then derived from the time series N(2)O concentration profiles. The rates obtained for the core incubations were compared to the rates predicted by a forward reactive transport model, which included rate expression for denitrification calibrated with potential rate measurements obtained in flowthrough reactors containing undisturbed, 1-cm-thick sediment slices. The two approaches yielded comparable rate profiles, with a near-surface, 2- to 3-mm narrow zone of denitrification and maximum in situ rates on the order of 200 to 300 nmol cm(-3) h(-1). The maximum in situ rates were about twofold lower than the maximum potential rate for the 0- to 1-cm depth interval of the sediment, indicating that in situ denitrification was nitrate limited. The experimentally and model-derived rates of denitrification implied that there was nitrate uptake by the sediment at a rate that was on the order of 50 (+/- 10) nmol cm(-2) h(-1), which agreed well with direct nitrate flux measurements for core incubations. Reactive transport model calculations showed that benthic uptake of nitrate at the site is particularly sensitive to the nitrate concentration in the overlying water and the maximum potential rate of denitrification in the sediment.     

Denitrification in coastal ecosystems: methods, environmental controls, and ecosystem level controls, a review

In this review of sediment denitrification in estuaries and coastal ecosystems, we examine current denitrification measurement methodologies and the dominant biogeochemical controls on denitrification rates in coastal sediments. Integrated estimates of denitrification in coastal ecosystems are confounded by methodological difficulties, a lack of systematic understanding of the effects of changing environmental conditions, and inadequate attention to spatial and temporal variability to provide both seasonal and annual rates. Recent improvements in measurement techniques involving ¹⁵ N techniques and direct N₂ concentration changes appear to provide realistic rates of sediment denitrification. Controlling factors in coastal systems include concentrations of water column NO ₃ ^– , overall rates of sediment carbon metabolism, overlying water oxygen concentrations, the depth of oxygen penetration, and the presence/absence of aquatic vegetation and macrofauna. In systems experiencing environmental change, either degradation or improvement, the importance of denitrification can change. With the eutrophication of the Chesapeake Bay, the overall rates of denitrification relative to N loading terms have decreased, with factors such as loss of benthic habitat via anoxia and loss of submerged aquatic vegetation driving such effects.     

Vertical Distribution of Denitrification in an Estuarine Sediment: Integrating Sediment Flowthrough Reactor Experiments and Microprofiling via Reactive Transport Modeling

Denitrifying activity in a sediment from the freshwater part of a polluted estuary in northwest Europe was quantified using two independent approaches. High-resolution N₂O microprofiles were recorded in sediment cores to which acetylene was added to the overlying water and injected laterally into the sediment. The vertical distribution of the rate of denitrification supported by nitrate uptake from the overlying water was then derived from the time series N₂O concentration profiles. The rates obtained for the core incubations were compared to the rates predicted by a forward reactive transport model, which included rate expression for denitrification calibrated with potential rate measurements obtained in flowthrough reactors containing undisturbed, 1-cm-thick sediment slices. The two approaches yielded comparable rate profiles, with a near-surface, 2- to 3-mm narrow zone of denitrification and maximum in situ rates on the order of 200 to 300 nmol cm⁻³ h⁻¹. The maximum in situ rates were about twofold lower than the maximum potential rate for the 0- to 1-cm depth interval of the sediment, indicating that in situ denitrification was nitrate limited. The experimentally and model-derived rates of denitrification implied that there was nitrate uptake by the sediment at a rate that was on the order of 50 (± 10) nmol cm⁻² h⁻¹, which agreed well with direct nitrate flux measurements for core incubations. Reactive transport model calculations showed that benthic uptake of nitrate at the site is particularly sensitive to the nitrate concentration in the overlying water and the maximum potential rate of denitrification in the sediment.     

Denitrification measurements in aquatic sediments: A comparison of three methods

Measurements of denitrification using the acetylene inhibition,¹⁵N isotope tracer, and N₂ flux methods were carried out concurrently using sediment cores from Vilhelmsborg sø, Denmark, in an attempt to clarify some of the limitations of each technique. Three experimental treatments of overlying water were used: control, nitrate enriched, and ammonia enriched water. The N₂ flux and¹⁵N tracer experiments showed high rates of coupled nitrification/denitrification in the sediments. The acetylene inhibition method did not capture any coupled nitrification/denitrification. This could be explained by acetylene inhibition of nitrification. A combined¹⁵N tracer/acetylene inhibition experiment demonstrated that acetylene inhibition of N₂O reduction was incomplete and the method, therefore, only measured approximately 50% of the denitrification due to nitrate from the overlying water. Similar rates of denitrification due to nitrate in the overlying water were measured by the N₂ flux method and the acetylene inhibition method, after correcting for the 50% efficiency of acetylene inhibition. Rates of denitrification due to nitrate from the overlying water measured by the¹⁵N tracer method, however, were only approximately 35% or less of those measured by the acetylene inhibition or N₂ flux methods.     

Modeling denitrification in aquatic sediments

Sediment denitrification is a major pathway of fixed nitrogen loss from aquatic systems. Due to technical difficulties in measuring this process and its spatial and temporal variability, estimates of local, regional and global denitrification have to rely on a combination of measurements and models. Here we review approaches to describing denitrification in aquatic sediments, ranging from mechanistic diagenetic models to empirical parameterizations of nitrogen fluxes across the sediment-water interface. We also present a compilation of denitrification measurements and ancillary data for different aquatic systems, ranging from freshwater to marine. Based on this data compilation we reevaluate published parameterizations of denitrification. We recommend that future models of denitrification use (1) a combination of mechanistic diagenetic models and measurements where bottom-waters are temporally hypoxic or anoxic, and (2) the much simpler correlations between denitrification and sediment oxygen consumption for oxic bottom waters. For our data set, inclusion of bottom water oxygen and nitrate concentrations in a multivariate regression did not improve the statistical fit.     

Denitrification Rates in a Marine Sediment as Measured by the Acetylene Inhibition Technique

A method has been developed for measurement of denitrification activity in sediments by application of the acetylene inhibition technique. Acetylene-saturated water was injected, at close intervals, into sediment cores which were then incubated for a few hours at the in situ temperature. Frozen segments of the cores were assayed for accumulation of N₂O by a combined gas extraction and detection system. The segments were thawed under a stream of helium from which N₂O (and other gases) was collected in a liquid N₂ trap, and the quantity of N₂O was measured by gas chromatography. The maximum rate of denitrification in a coastal marine sediment was 35 nmol of N per cm³ of sediment per day at 2.5°C, and the rate of denitrification for the total sediment was 0.99 nmol of N per m² per day.     

Potential rates of nitrification and denitrification in an oligotrophic freshwater sediment system

Potential rates of nitrification and denitrification were measured in an oligotrophic sediment system. Nitrification potential was estimated using the CO oxidation technique, and potential denitrification was measured by the acetylene blockage technique. The sediments demonstrated both nitrifying and denitrifying activity. E_h, O₂, and organic C profiles showed two distinct types of sediment. One type was low in organic C, had high O₂ and E_h, and had rates of denitrification 1,000 times lower than the other which had high organic C, low O₂, and low E_h. Potential nitrification and denitrification rates were negatively correlated with E_h. This suggests that environmental heterogeneity in denitrifier and nitrifier populations in oligotrophic sediment systems may be assessed using E_h before sampling protocols for nitrification or denitrification rates are established. There was no correlation between denitrification and nitrification rates or between either of these processes and NH₄ ⁺ or NO₃ ^– concentrations. The maximum rate of denitrification was 0.969 nmole N cm^–3 hour^–1, and the maximum rate of nitrification was 23.6 nmole cm^–3 hour^–1, suggesting nitrification does not limit denitrification in these oligotrophic sediments. Some sediment cores had mean concentrations of 6.0 mg O₂/liter and still showed both nitrification and denitrification activity.     

Acetylene inhibition technique underestimates in situ denitrification rates in intact cores of freshwater sediment

Abstract Denitrification in intact sediment cores was measured by the acetylene inhibition technique and compared with the nitrate flux between water and sediment. Less than half of the nitrate-N consumed by the sediment could be recovered as nitrous oxide-N. The low recovery rate of nitrous oxide from intact sediment cores indicated losses of nitrous oxide by diffusion down to nitrate-free sediment layers, where reduction of nitrous oxide may take place. In sediment slurries 100% of nitrate-N could be recovered as nitrous oxide-N as long as the nitrate concentration in the liquid phase was above 10 μM. Nitrous oxide added to nitrate-free sediment slurries was reduced regardless of whether acetylene was present or not. Therefore denitrification may be significantly underestimated by this method.     

Perfusion method for assaying microbial activities in sediments: applicability to studies of n(2) fixation by c(2)h(2) reduction

A perfusion method for assaying nitrogenase activity (acetylene reduction) in marine sediments was developed. The method was used to assay sediment cores from Spartina alterniflora (salt marsh), Zostera marina (sea grass), and Thalassia testudinum (sea grass) communities, and the results were compared with those of conventional sealed-flask assays. Rates of ethylene production increased progressively with time in the perfusion assays, reaching plateau values of 2 to 3 nmol . g of dry sediment . h by 10 to 20 h. Depletion of interstitial NH(4) was implicated in this stimulation of nitrogenase activity. Initial acetylene reduction rates determined by the perfusion assay of cores from the Spartina community ranged from 0.15 to 0.60 nmol of C(2)H(4) . g of dry sediment . h. These rates were similar to those for sediments assayed in sealed flasks without seawater when determined over linear periods of C(2)H(4) production. Initial values obtained by using the perfusion method were 0.66 nmol of C(2)H(4) . g of dry sediment . h for sediments from Zostera communities and 0.70 nmol of C(2)H(4) . g of dry sediment . h for sediments from Thalassia communities. In all cases, rates determined by simultaneous slurry assays were lower than those determined by the perfusion method.     

Influence of Algal Community Structure on Denitrification Rates in Periphyton Cultivated on Artificial Substrata

We conducted a field survey of periphyton cultivated on benthic mesh installations in freshwater aquatic systems, including two constructed wetlands and a pond, and also studied periphyton grown on a benthic mesh in laboratory mesocosms. The objectives of this study were to (1) determine if periphyton cultivated on benthic mesh denitrifies at higher rates than the underlying sediments and (2) determine if denitrification rates within periphyton vary with characteristics such as algal and bacterial community structure and biomass. We measured denitrification potential rates of field and laboratory periphyton by the acetylene inhibition method. We characterized algal community composition by algal identification and bacterial community composition by terminal restriction fragment length polymorphisms. Periphyton collected on benthic mesh from our field sites denitrified at significantly higher rates than the underlying sediments, regardless of sampling site or season. Results from both our field survey and laboratory studies indicated a significant, positive correlation between diatom presence and denitrification rate. In our laboratory studies, we found that periphyton with the highest diatom abundance showed the highest denitrification rates as well as a distinct bacterial community composition. These results suggest a synergistic relationship between diatoms and denitrifying bacteria that warrants further study.     

Annual Pattern of Denitrification and Nitrate Ammonification in Estuarine Sediment

The seasonal variation and depth distribution of the capacity for denitrification and dissimilatory NO₃⁻ reduction to NH₄⁺ (NO₃⁻ ammonification) were studied in the upper 4 cm of the sediment of Norsminde Fjord estuary, Denmark. A combination of C₂H₂ inhibition and ¹⁵N isotope techniques was used in intact sediment cores in short-term incubations (maximum, 4 h). The denitrification capacity exhibited two maxima, one in the spring and one in the fall, whereas the capacity for NO₃⁻ ammonification was maximal in the late summer, when sediments were progressively reduced. The denitrification capacity was always highest in the uppermost 1 cm of the sediment and declined with depth. The NO₃⁻ ammonification was usually higher with depth, but the maximum activity in late summer was observed within the upper 1 cm. The capacity for NO₃⁻ incorporation into organic material was investigated on two occasions in intact sediment cores and accounted for less than 5% of the total NO₃⁻ reduction. Denitrification accounted for between 13 and 51% of the total NO₃⁻ reduction, and NH₄⁺ production accounted for between 4 and 21%, depending on initial rates during the time courses. Changes of the rates during the incubation were observed in the late summer, which reflected synthesis of denitrifying enzymes. This time lag was eliminated in experiments with mixed sediment because of preincubation with NO₃⁻ and alterations of the near-environmental conditions. The initial rates obtained in intact sediment cores therefore reflect the preexisting enzyme content of the sediment.     

Effect of organic loading on nitrification and denitrification in a marine sediment microcosm

Abstract The effects of organic additions on nitrification and dentrification were examined in sediment microcosms. The organic material, heat killed yeast, had a C/N ratio of 7.5 and was added to sieved, homogenized sediments. Four treatments were compared: no addition (control), 30 g dry weight (dw) m⁻² mixed throughout the 10 cm sediment column (30M), 100 g dw m⁻² mixed throughout sediments (100M), and 100 g dw m⁻² mixed into top 1 cm (100S). After the microcosms had been established for 7–11 days, depth of O₂ penetration, sediment-water fluxes and nitrification rates were measured. Nitrification rates were measured using three different techniques: N-serve and acetylene inhibition in intact cores, and nitrification potentials in slurris. Increased organic additions decreased O₂ penetration from 2.7 to 0.2 mm while increasing both O₂ consumption, from 30 to 70 mmol O₂ m⁻² d⁻¹, and NO₃⁻ flux into sediments. Nitrification rates in intact cores were similar for the two methods. Highest rates occurred in the 30M treatment, while the lowest rate was measured in the 100S treatment. Total denitrification rates (estimated from nitrification and nitrate fluxes) increased with increased organic addition, because of the high concentrations of NO₃⁻ (40 μM) in the overlaying water. The ratio of nitrification: denitrification was used as an indication of the importance of nitrification as the NO₃⁻ supply for denitrificaion. This ratio decreased from 1.55 to 0.05 iwth increase organic addition.     

Use of the [(14)C]leucine incorporation technique to measure bacterial production in river sediments and the epiphyton.

Bacterial production is a key parameter for the understanding of carbon cycling in aquatic ecosystems, yet it remains difficult to measure in many aquatic habitats. We therefore tested the applicability of the [(14)C]leucine incorporation technique for the measurement of bulk bacterial production in various habitats of a lowland river ecosystem. To evaluate the method, we determined (i) extraction efficiencies of bacterial protein from the sediments, (ii) substrate saturation of leucine in sediments, the biofilms on aquatic plants (epiphyton), and the pelagic zone, (iii) bacterial activities at different leucine concentrations, (iv) specificity of leucine uptake by bacteria, and (v) the effect of the incubation technique (perfused-core incubation versus slurry incubation) on leucine incorporation into protein. Bacterial protein was best extracted from sediments and precipitated by hot trichloroacetic acid treatment following ultrasonication. For epiphyton, an alkaline-extraction procedure was most efficient. Leucine incorporation saturation occurred at 1 microM in epiphyton and 100 nM in the pelagic zone. Saturation curves in sediments were difficult to model but showed the first level of leucine saturation at 50 microM. Increased uptake at higher leucine concentrations could be partly attributed to eukaryotes. Addition of micromolar concentrations of leucine did not enhance bacterial electron transport activity or DNA replication activity. Similar rates of leucine incorporation into protein calculated for whole sediment cores were observed after slurry and perfused-core incubations, but the rates exhibited strong vertical gradients after the core incubation. We conclude that the leucine incorporation method can measure bacterial production in a wide range of aquatic habitats, including fluvial sediments, if substrate saturation and isotope dilution are determined.     

Direct flux and 15N tracer methods for measuring denitrification in forest soils

Estimates of denitrification are one of the key uncertainties in the terrestrial nitrogen (N) cycle, primarily because reliable measurements of this highly variable process—especially the production of its terminal product (N₂)—are difficult to obtain. We evaluated the ability of gas-flow soil core and ¹⁵N tracer methods to provide reliable estimates of denitrification in forest soils. Our objectives were to: (1) describe and present typical results from new gas-flow soil core and in situ ¹⁵N tracer methods for measuring denitrification, (2) discuss factors that affect the relevance of these methods to actual in situ denitrification, and (3) compare denitrification estimates produced by the two methods for a series of sites in a northern hardwood forest ecosystem. Both methods were able to measure accumulations of N₂ over relatively short (2–5 h) incubations of either unamended or tracer-amended intact soils. Denitrification rates measured by the direct flux soil core method were very sensitive to incubation oxygen (O₂) concentration and decreased with increased O₂ levels. Denitrification rates measured by the in situ ¹⁵N tracer method were very sensitive to the ¹⁵N content of the nitrate (NO₃ ^?) pool undergoing denitrification, which limits the applicability of this method for quantifying denitrification in N-poor ecosystems. While its ability to provide accurate estimates of denitrification was limited, the ¹⁵N tracer method provided estimates of the short-term abiotic and biotic transformations of atmospheric N deposition to gas. Furthermore, results suggest that denitrification is higher and that N₂O:N₂ ratios are lower (<0.02) than previously thought in the northern hardwood forest and that short-term abiotic and biotic transformations of atmospheric N deposition to gas are significant in this ecosystem.     

Denitrification in Marine Sediment: Measurement of Capacity and Estimate of In Situ Rate

The acetylene block technique was used to assay denitrification in undisturbed sediment cores of an intertidal mud flat. Nitrogen loss due to this process was estimated at 1 to 41 kg of N/hectare (ha) per year. Anaerobic nitrate-saturated slurry of the same sediment had a denitrification capacity of 1,026 kg of N/ha per year. The acetylene block technique failed at low nitrate concentrations, so that denitrification at average in situ nitrate concentrations could not be determined. Denitrification followed zero-order kinetics at nitrate concentrations high enough to allow successful blockage of N₂O reduction. Thus, an estimate of in situ rates based on kinetic parameters and in situ nitrate concentrations was impossible. No denitrification was observed in a slurry of the top 1.5 cm under aerated conditions and nitrate saturation. In undisturbed sediment, significant denitrification occurred in few discrete sites within a matrix of undetectable or low activity. Despite numerous errors contributing to the uncertainty of the estimate of in situ rates, the result obtained by this method was considered more valuable than the determination of denitrification capacities. Methods which include severe changes of physical and chemical parameters may frequently lead to overestimates of denitrification rates.     

Method for Measuring Rates of NH(4) Turnover in Anoxic Marine Sediments, Using a N-NH(4) Dilution Technique

A method is described for the determination of the net and total rates of NH(4) production and NH(4) incorporation at different depths in an anoxic marine sediment. N-NH(4) was added to the sediment NH(4) pool, and the N content was assayed after 0, 2, and 5 days of incubation. The pool size changed during incubation; this change in pool size is incorporated into a model which predicts the dynamics of N-NH(4) dilution. A simple microdiffusion of NH(3) was followed by an emission spectrometry analysis of N content. This procedure avoided all problems of cross-contamination. The model was tested and rates were measured in four sediment cores, at seven different depths. The high correlation coefficients (mean, 0.96 for the 0- to 2-, 2- to 4-, 4- to 6-, and 6- to 8-cm sediment fractions) indicated that the model was correct and that the measured rates were valid. The immediate distribution of N-NH(4) between interstitial and exchangeable NH(4) pools indicated that it was the combined pool that was turning over. In the 0- to 2-cm fraction at 17 degrees C the net rate of NH(4) production was 274 (standard deviation, 31) nmol cm day, and the mean total rate of NH(4) production was 309 (standard deviation, 39) nmol cm day; both rates decreased to <1% of these values in the 12- to 14-cm fractions.     

Denitrification, dissimilatory reduction of nitrate to ammonium, and nitrification in a bioturbated estuarine sediment as measured with N and microsensor techniques

Nitrogen and oxygen transformations were studied in a bioturbated (reworked by animals) estuarine sediment (Norsminde Fjord, Denmark) by using a combination of N isotope (NO(3)), specific inhibitor (C(2)H(2)), and microsensor (N(2)O and O(2)) techniques in a continuous-flow core system. The estuarine water was NO(3) rich (125 to 600 muM), and NO(3) was consistently taken up by the sediment on the four occasions studied. Total NO(3) uptake (3.6 to 34.0 mmol of N m day) corresponded closely to N(2) production (denitrification) during the experimental steady state, which indicated that dissimilatory, as well as assimilatory, NO(3) reduction to NH(4) was insignificant. When C(2)H(2) was applied in the flow system, denitrification measured as N(2)O production was often less (58 to 100%) than the NO(3) uptake because of incomplete inhibition of N(2)O reduction. The NO(3) formed by nitrification and not immediately denitrified but released to the overlying water, uncoupled nitrification, was calculated both from NO(3) dilution and from changes in NO(3) uptake before and after C(2)H(2) addition. These two approaches gave similar results, with rates ranging between 0 and 8.1 mmol of N m day on the four occasions. Attempts to measure total nitrification activity by the difference between NH(4) fluxes before and after C(2)H(2) addition failed because of non-steady-state NH(4) fluxes. The vertical distribution of denitrification and oxygen consumption was studied by use of N(2)O and O(2) microelectrodes. The N(2)O profiles measured during the experimental steady state were often irregularly shaped, and the buildup of N(2)O after C(2)H(2) was added was much too fast to be described by a simple diffusion model. Only bioturbation by a dense population of infauna could explain these observations. This was corroborated by the relationship between diffusive and total fluxes, which showed that only 19 to 36 and 29 to 62% of the total O(2) uptake and denitrification, respectively, were due to diffusion-reaction processes at the regular sediment surface, excluding animal burrows.