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 partof a polluted estuary in northwest Europe was quantified usingtwo independent approaches. High-resolution N₂O microprofileswere recorded in sediment cores to which acetylene was addedto the overlying water and injected laterally into the sediment.The vertical distribution of the rate of denitrification supportedby nitrate uptake from the overlying water was then derivedfrom the time series N₂O concentration profiles. The rates obtainedfor the core incubations were compared to the rates predictedby a forward reactive transport model, which included rate expressionfor denitrification calibrated with potential rate measurementsobtained in flowthrough reactors containing undisturbed, 1-cm-thicksediment slices. The two approaches yielded comparable rateprofiles, with a near-surface, 2- to 3-mm narrow zone of denitrificationand maximum in situ rates on the order of 200 to 300 nmol cm^–3h^–1. The maximum in situ rates were about twofold lowerthan the maximum potential rate for the 0- to 1-cm depth intervalof the sediment, indicating that in situ denitrification wasnitrate limited. The experimentally and model-derived ratesof denitrification implied that there was nitrate uptake bythe sediment at a rate that was on the order of 50 (±10) nmol cm^–2 h^–1, which agreed well with directnitrate flux measurements for core incubations. Reactive transportmodel calculations showed that benthic uptake of nitrate atthe site is particularly sensitive to the nitrate concentrationin the overlying water and the maximum potential rate of denitrificationin the sediment.

INTRODUCTION

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INTRODUCTION

Anthropogenic inputs of nitrogen are a major cause of eutrophicationin aquatic environments (39). Denitrification, the bacterialdissimilatory reduction of nitrate to gaseous products, counteractseutrophication by removing inorganic nitrogen and releasingit to the atmosphere and by decomposing organic matter (34).The emissions resulting from denitrification, however, includenitrous oxide, a powerful greenhouse gas (8). Because of itsenvironmental importance, denitrification has been the subjectof numerous studies (for reviews, see references 12, 16, and34). Oxygen and nitrate concentrations, temperature, pH, andthe availability of suitable electron donor substrates (mainlyorganic carbon compounds) are considered the key factors controllingthe occurrence and rate of denitrification (6, 22, 38).

In estuarine and coastal environments, the most intense denitrificationoften occurs in sediments, where reduced solute transport ratesenable the establishment of anaerobic conditions. A number ofmethods have been developed to quantify denitrification andrelated nitrogen transformations in sediments; these methodsinclude, among others, benthic flux measurement (17), isotopepairing techniques (32), and microprofiling using nitrous oxide(31) or nitrate microsensors (9, 20). The available data showthat there are large spatial and temporal variations in denitrificationrates in nearshore sediments. Ideally, kinetic models of denitrificationshould account for these variations.

In this study, we combined potential denitrification rate measurements,N₂O microprofiling in acetylene block core incubations, andreactive transport modeling to quantify the depth distributionof the rate of denitrification in a sediment from the freshwaterpart of a macrotidal estuary. By combining the various approaches,we were able to verify the predictive capability of the kineticdescription of denitrification incorporated into the reactivetransport model and to assess the sensitivity of benthic nitrateremoval to changes in key model parameters.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Site description.
Sediment cores were collected in August 2001 and August 2002at an unvegetated intertidal site in the freshwater part ofthe Scheldt Estuary near Appels, Belgium. The Scheldt Riverhas a total length of 355 km and flows through France, Belgium,and The Netherlands. The average river discharge is on the orderof 120 m³ s^–1, while the tidal amplitude in the estuaryranges from 2 to 5 m (3). The Scheldt Estuary is heavily pollutedby municipal and industrial discharge and intensive livestockfarming. The large input of organic matter sustains high heterotrophicactivity in the upper freshwater part of the estuary, whichcan result in a pronounced oxygen depletion zone extending overa distance of 30 to 70 km (42). Intertidal sediments are theprimary site of benthic carbon mineralization in the ScheldtEstuary (36). A detailed description of the site is providedelsewhere (13). All cores used in the experiments describedhere were collected close to each other (<1 m).

Core incubations.
Sediment was sampled in August 2002 at low tide using Perspex(acrylic glass) cores with a diameter of 7.6 cm and length of35 cm. To determine the benthic NO₃^– flux, two cores containingabout 20 cm of sediment collected in August 2002 were incubatedfor 22 h with river water from the site. The overlying waterwas gently stirred and sparged with air to avoid developmentof anoxia. The overlying water of the cores was sampled periodicallyand analyzed to determine the NO₃^– content. The time-averagednitrate fluxes into the sediment were calculated from the concentrationdifferences measured in the overlying water between the startand end of the incubations, taking into account the known areaof the sediment and volume of overlying water.

To assess pore water transport, three additional cores wereincubated with 200 ml NaBr-amended river water from the site.The initial bromide concentrations in the overlying water afterNaBr addition were on the order of 10 mmol liter^–1. Thecores were incubated for 36 h. At the end of the incubation,one core was sectioned into 0.5-cm depth intervals, and thetwo other cores were sectioned into 1-cm intervals. Pore waterwas separated from the sediment slices by centrifugation (30min at 1,000 x g) and filtration (0.45-µm-pore-size Whatmanfilters) and stored at –20°C until it was analyzedto determine the bromide content. Overlying water samples werecollected at the beginning and end of the incubations and analyzedto determine the Br^– concentrations.

Flowthrough reactors.
Undisturbed, 1-cm-thick sediment slices were collected in August2002 with a specially designed hand-held corer. Potential nitratereduction rates were determined with slices corresponding todepth intervals of 0 to 1, 1 to 2, and 2 to 3 cm using flowthroughreactors (FTRs). The corer, coring technique, FTR design, andpotential rate determinations are described in detail elsewhere(18, 25). The reactors were supplied with deaerated inflow solutionscontaining variable nitrate concentrations (2.5, 5, 7.5, and10 mmol liter^–1; four reactors per depth interval) ata flow rate of 4 ml h^–1. Steady-state nitrate reductionrates (R_exp) (in nmol cm^–3 h^–1) were calculatedafter the nitrate concentration in the outflow reached a constantvalue, as described elsewhere (18). The nitrate reduction rateswere fitted to the Michaelis-Menten rate expression:

Formula 1 (1)
where K_m is the nitrate half-saturationconstant and R_max is the maximum nitrate reduction rate. Theaverage nitrate concentration in the reactor ([NO₃^–])was estimated to be the midpoint between the inflow and outflowconcentrations. The half-saturation constant was assigned avalue of 250 µmol liter^–1, the average value derivedfrom a large set of FTR experiments with estuarine sediments,which included additional sampling locations, sampling times,and depth intervals (18).

Microprofiling.
Perspex (acrylic glass) cores with a diameter of 4.2 cm anda length of 15 cm were collected at low tide in August 2001and August 2002. The cores contained about 10 cm of sedimentand were incubated at 25°C with river water from the site.The overlying water was gently mixed and sparged with air. Microprofilingwas started within 4 h after the cores were collected. Oxygenmicroprofiles were determined using a miniaturized Clark-typeoxygen sensor with an internal reference and a guard cathode(11, 30). Nitrous oxide profiles were determined with a miniaturizedClark-type nitrous oxide sensor with an internal reference anda guard cathode equipped with an oxygen front guard, makingit insensitive to oxygen (Unisense, Århus, Denmark). Microprofilesof nitrate were determined only in August 2002, using a LiquidIon eXchange-type (LIX) electrode (see reference 9) with a calomelreference electrode (Radiometer Copenhagen) and calibrated atthe in situ temperature (25°C) and salinity (0 ${per thousand}$ ). Lineartwo-point calibration of the oxygen and nitrous oxide microsensorswas performed with river water from the sampling site. The microelectrodeswere mounted on a micromanipulator driven by a computer-controlledmotor, making it possible to simultaneously measure verticaldistributions of oxygen, nitrate, and nitrous oxide.

Acetylene block experiments.
Conversion of N₂O to N₂ and nitrification were inhibited insediment cores collected in August 2001 and August 2002 by lateralinjection of acetylene-saturated water through silicone-filledholes positioned vertically along the core liner. The holeswere positioned every 0.5 cm from the sediment surface to asediment depth of 5 cm. The injections resulted in a C₂H₂ porewater concentration of 10% (vol/vol). Acetylene-saturated riverwater from the site was added to the core to obtain a finalC₂H₂ concentration of 10% (vol/vol) in the overlying water.After addition of C₂H₂, N₂O profiles were recorded with a depthresolution of 250 µm until 2 cm below the sediment surface.Two to eight profiles were recorded sequentially, approximatelyevery 15 min. Ambient oxygen, nitrous oxide, and nitrate (August2002 cores only) profiles were determined for sediment coreswithout C₂H₂ addition to determine background N₂O values andcheck for matrix effects on the sensor readings.

Additional sediment cores collected in August 2001 and August2002 were supplied with both nitrate and acetylene. One milliliterof a 10-mmol liter^–1 KNO₃ solution saturated with C₂H₂was injected laterally into each hole along the core liner,producing pore water NO₃^– concentrations of $~$ 1.7 mmol liter^–1.Nitrate was also added to the overlying, acetylene-saturatedwater to a final concentration of $~$ 2 mmol liter^–1 (comparedto ambient concentrations on the order of 200 µmol liter^–1).Time series microprofiles of N₂O were recorded as describedabove.

Analytical methods.
Nitrate, nitrite, and ammonium concentrations were determinedcolorimetrically with a Nutrient Autoanalyzer 3 (Bran and Luebbe),using standard procedures. Bromide concentrations were determinedby ion chromatography (IC Waters, Milford MA). Porosity wasdetermined from the weight loss of a core section having a knownvolume after oven drying at 105°C. Sediment organic C andtotal N contents were determined with a Carlo Erba CN analyzer.Inorganic C was removed before the analysis by shaking the samplewith 1 mol liter^–1 HCl twice (12 h and 4 h). Grain sizeanalyses of the bulk sediments were carried out with a MalvernInstruments Mastersizer S. The most probable numbers of denitrifyingorganisms were determined as described elsewhere (18). Mostof the sediment properties reported below were determined usingsediment sampled in August 2002; the exceptions were grain sizeand organic C and total N concentrations, which were determinedusing sediment collected in May 2002.

Reactive transport modeling.
Model calculations were carried out with the BiogeochemicalReaction Network Simulator, a flexible modeling environmentfor reactive transport calculations (1, 28; http://www.geo.uu.nl/ $~$ rtm/index).The time-dependent depth profiles of the concentrations of NO₃^–and N₂O plus the denitrification rate were described by thenon-steady-state mass conservation equation for a pore watersolute species (2):

Formula 2 (2)
wherex is the depth in the sediment, t is time, C is the concentrationof the solute, C₀ is the solute concentration at the sediment-waterinterface (SWI), D_s is the in situ molecular diffusion coefficientof the solute corrected for tortuosity (5), ${alpha}$ is the nonlocalexchange coefficient, and R is the net rate of production ofthe solute. At 25°C, 0 ${per thousand}$ salinity, and a porosity of 0.80,the D_s values were 2.10 x 10^–5 and 1.92 x 10^–5 cm²s^–1 for nitrous oxide and nitrate, respectively (5, 33).

The denitrification rate (R_den) was represented via a Michaelis-Menten-typeequation (37, 40), taking into account limitation by NO₃^–and inhibition by O₂:

Formula 3 (3)
wherethe default values of K_m and the O₂ inhibition constant () were set at 250 µmol liter^–1(18) and 5 µmol liter^–1 (23), respectively. R_maxwas initially assigned the value derived from the nitrate consumptionrate measurements in the flowthrough reactor experiments andthen optimized using the time-dependent N₂O profiles. Note thatR_max and K_m are apparent kinetic parameters that depend on factorssuch as organic matter availability and community composition.

The NO₃^– and N₂O concentrations measured for the overlyingwater were used as upper boundary conditions, while zero-concentrationgradients were used as lower boundaries. For the transient calculations,the measured ambient NO₃^– and N₂O profiles were used asthe initial conditions. Pore water O₂ profiles depend on a multitudeof reactions (41) and hence were not explicitly computed. Rather,the measured O₂ profiles were used in order to calculate theinhibition term in equation 3. The validity of this approachwas confirmed by the time series O₂ microprofiles, which showedthat there were no significant changes in the O₂ concentrationsover the duration of the incubations (see below).

The depth distributions of N₂O production rates during the acetyleneblock experiments were estimated from the time series N₂O microprofilesby solving equation 2 for the rate, R(x,t). The second-orderspatial derivative in the diffusion term was approximated bythe time-averaged change in slope of consecutively measuredconcentration profiles, whereas the partial derivative of concentrationwith respect to time was approximated as follows:

Formula 4 (4)
For simplicity, nonlocal exchangewas ignored (i.e., ${alpha}$ = 0).

RESULTS

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RESULTS

Sediment and pore water characteristics.
The uppermost centimeters of sediment at Appels had high porositiesand organic carbon contents on the order of 1.5 to 3% (wt/wt)(Table 1). At both sampling times, August 2001 and August 2002,O₂ penetrated approximately 2 to 3 mm below the SWI (Fig. 1).High NO₃^– concentrations were found in the river waterat the site (around 300 µmol liter^–1 and 200 µmolliter^–1 in August 2001 and August 2002, respectively).The ambient N₂O concentrations were always below the detectionlimit of the N₂O microsensor ( $~$ 1 µmol liter^–1 N₂O).The most probable numbers showed that microorganisms capableof denitrification were present at high densities ( $~$ 10⁸ cellscm^–3 wet sediment) at all depths investigated (0 to 3cm).

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TABLE 1. Sediment properties

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FIG. 1. Bromide (A) and oxygen (B) pore water profiles in sediment cores collected in August 2002 at Appels. Note the difference in the vertical scales. The three sets of symbols (measured data) and lines (model calculated) for bromide correspond to three replicate core incubations; the circles, diamonds, and squares correspond to the dotted, dashed, and solid lines, respectively. The calculated bromide profiles assume that there was no enhanced pore water mixing ( ${alpha}$ = 0). The dotted line in panel B corresponds to a model-predicted steady-state O₂ profile assuming that there was no enhanced mixing, whereas the solid line includes nonlocal pore water transport in the top 0. 75 mm with an ${alpha}$ value of 55 h^–1. See the text for details.

Enhanced pore water transport.
After 36 h, bromide penetrated to a depth of about 5 cm, andthere was good reproducibility for the three replicate cores(Fig. 1A). To quantify pore water mixing in addition to moleculardiffusion, the profiles were fitted to solutions of equation2, taking into account the depletion of Br^– in the overlyingwater with time. Except for the topmost 1 cm of sediment, simulationsassuming molecular diffusion only were in good agreement withthe measurements, implying that there was no deep ( $≥$ 1-cm) porewater irrigation. On a vertical scale smaller than that resolvedby the bromide measurements, however, a mixed zone in the uppermost0.75 mm (x_irr) was identified based on the distinct kink inthe O₂ microprofiles at that depth (Fig. 1B). An average valuefor the nonlocal exchange coefficient of 55 h^–1 was constrainedby fitting the O₂ microprofiles with equation 1, assuming asteady state and a Michaelis-Menten kinetic formulation forthe net rate of O₂ consumption. The affinity constant for O₂was set at 10 µmol liter^–1 (41), and the maximumO₂ consumption rate was estimated from 2 ${phi}$ D_s(C₀ – C_L)/L²,where the O₂ penetration depth (L) corresponds to the depthat which the O₂ concentration drops to 1 µmol liter^–1(i.e., C_L = 1 µmol liter^–1) and ${phi}$ is the porosity.A satisfactory match between measured and predicted O₂ profileswas obtained with ${alpha}$ values in the range from 10 to 100 h^–1(Fig. 1B).

The very shallow mixed zone is unlikely to be due to the activityof benthic infauna, which at Appels is dominated by oligochaeteworms (35). We believe that this zone reflects an experimentalartifact caused by the stirring of the overlying water in thecores. Air sparging and stirring of the overlying water werecarried out to avoid progressive chemical stratification andultimate anoxia of the waters just above the sediment. Thiswould have interfered with the establishment of a steady-statedistribution of denitrification activity, hence compromisingthe interpretation of time series N₂O microprofiles. Enhancedpore water mixing in the upper 0.75 mm of the sediment was accountedfor in the reactive-transport calculations by using the same ${alpha}$ values for all solute species (55 h^–1).

FTR experiments.
The maximum potential nitrate consumption rate obtained in theFTR experiments decreased from 585 nmol cm^–3 h^–1in the first centimeter of the sediment (0 to 1 cm) to 320 and225 nmol cm^–3 h^–1 in the two deeper intervals (1to 2 and 2 to 3 cm, respectively). Because of the high ratesof nitrate reduction, relatively high inflow NO₃^– concentrationswere needed in the FTR experiments in order to recover measurableNO₃^– concentrations in the outflow. As a result of thehigh average nitrate concentrations in the reactors, the measuredrates approached the maximum values, and the estimated R_maxvalues were not very sensitive to the value assigned to K_m.In additional acetylene block FTR experiments with Appels sediment,80 to 100% of the NO₃^– consumed was recovered as N₂O (18),implying that under the experimental conditions used, denitrificationto N₂ is the dominant pathway of nitrate reduction.

Acetylene block core incubations.
In sediment cores in which C₂H₂ was added to the overlying water,as well as injected laterally into the sediment, increasingpore water concentrations of N₂O occurred as a function of time.A selected number of N₂O depth profiles from a time series experimentcarried out in August 2002 are shown in Fig. 2A. The profilesexhibited a distinct maximum at a depth of around 3 mm, withmaximum concentrations of $~$ 35 µmol liter^–1 measuredafter 95 min. The maximum N₂O concentrations occurred belowthe depth where O₂ penetrated (Fig. 2). Oxygen and NO₃^–microprofiles determined alongside the N₂O profiles showed littlechange during the total time of the incubations (Fig. 2B). Aswas observed for the ambient O₂ microprofiles (Fig. 1B), thenitrate and O₂ profiles recorded during the C₂H₂ block experimentsshowed that there was a pronounced change in gradient at a depthof around 0.75 mm (Fig. 2B).

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FIG. 2. Examples of microprofiles of N₂O (A), NO₃^– (B), and O₂ (inset) in an acetylene block core incubation experiment carried out in August 2002. The profiles were recorded 30, 59, and 95 min after addition of C₂H₂ to the overlying water and the pore waters via lateral injection ports.

The rate profiles calculated at different times during the acetyleneblock experiments did not differ significantly over the timescale of the incubations (Fig. 3). The results showed that therewas a narrow, 2- to 3-mm-thick layer of denitrification, locatedbetween the depths of penetration of O₂ and NO₃^– (Fig.3). The denitrification activity peaked at depths of around3 mm in both 2001 and 2002, and the maximum rates were in therange from 200 to 400 nmol N cm^–3 h^–1.

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FIG. 3. N₂O production rates for a number of selected time intervals in the same acetylene block core incubation experiment as in Fig. 2. Each rate profile was derived using two successive N₂O profiles (measurement times are indicated), assuming that there was no enhanced pore water transport ( ${alpha}$ = 0). The shading corresponds to the uppermost sediment layer which exhibits evidence of enhanced pore water transport. See the text for a complete discussion.

The negative rates in the uppermost millimeters were due tothe additional removal of N₂O by enhanced pore water transport(see above). When the negative rates were ignored, the integratedN₂O production rates in the sediment were on the order of 60± 12 nmol N cm^–2 h^–1 for August 2001 and50 ± 8 nmol N cm^–2 h^–1 for August 2002. Notethat because acetylene also blocks nitrification, these integratedN₂O production rates should have reflected only denitrificationsupported by NO₃^– uptake from the overlying water.

Addition of NO₃^– to the overlying water and sediment porewaters resulted in N₂O concentrations that were more than 1order of magnitude higher than the values observed in the acetyleneblock incubations with no added NO₃^– (compare Fig. 2Aand 4). The maximum concentrations also occurred deeper in thecores, indicating that NO₃^– added below the ambient nitratepenetration depth was actively used in denitrification. Thisobservation agreed with the observed presence of denitrifyingmicroorganisms several centimeters below the SWI, as well asthe high potential nitrate reduction rates measured with theflowthrough reactors at depths below 1 cm.

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FIG. 4. Examples of N₂O profiles recorded during a core incubation experiment (August 2002) after simultaneous addition of C₂H₂ and NO₃^– to the overlying water and pore water. See the text for details.

Nitrate fluxes.
The benthic NO₃^– uptake flux, obtained from the measuredloss of nitrate from the overlying water in the unamended coreincubations in August 2002, was 45 ± 14 nmol N cm^–2h^–1. Diffusive nitrate fluxes were also estimated fromthe linear portions of the depth profiles for NO₃^– concentrationsbelow the zone of enhanced transport, according to the followingequation:

Formula 5 (5)
wherethe concentration gradient was evaluated below 0.75 mm. Forthe near-steady-state nitrate profiles measured during the acetyleneblock experiments with no added nitrate in August 2002 (Fig.2B), equation 5 yielded downward nitrate fluxes into the zoneof denitrification of 60 ± 15 nmol N cm^–2 h^–1.The nitrate fluxes were thus the same order of magnitude asthe integrated N₂O production rate in the August 2002 acetyleneblock core incubations with no added nitrate.

Reactive transport modeling.
A baseline simulation of the August 2002 acetylene block experimentwas carried out using the maximum potential nitrate reductionrate measured for the 0- to 1-cm sediment slice (R_max = 585nmol cm^–3 h^–1), the default nitrate half-saturationconstant (K_m = 250 µM), and the default oxygen inhibitionconstant ( Formula 5 = 5 µmol liter^–1), plus the mixing parameters derived from theO₂ microprofiles (x_irr = 0.075 cm, ${alpha}$ = 55 h^–1). Sensitivityanalyses were then performed by multiplying or dividing thekinetic parameter values (R_max, K_m, and ) by 2. The results are summarized in Table 2,which also shows the model-predicted effect of the overlyingwater NO₃^– concentration on pore water N₂O productionin the acetylene block core incubation experiment.

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TABLE 2. Model sensitivity analyses: effects of the NO₃^– concentration in the overlying water and kinetic parameters (R_max, K_m, and Formula 5 ) on pore water N₂O buildup during simulated acetylene block core incubations^a

Additional forward reactive transport simulations were carriedout to match the measured N₂O concentration buildup in the sediment,by fixing either K_m or R_max to the values derived from FTR experiments.When K_m was set at 250 µmol liter^–1, R_max had tobe adjusted to 950 nmol cm^–3 h^–1 in order to reproducethe time series N₂O profiles (Fig. 5A). With this set of parameters,the calculated NO₃^– concentration profile closely resembledthe measured profile (Fig. 5B). As observed, most denitrifyingactivity was predicted to be restricted to the 1.5- to 4-mmdepth interval, although the modeled rates tended to be somewhatlower than the N₂O production rates estimated from the timeseries N₂O profiles (Fig. 6A). There was also a slight offsetof about 0.5 mm between the calculated and observed depths atwhich the maximum N₂O concentration and production rate werereached.

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FIG. 5. Measured and model-predicted N₂O profiles (A) and measured and predicted NO₃^– profiles (B) after 59 min of incubation in an acetylene block core incubation experiment (August 2002) (see Fig. 2 and 3). The dashed lines are the modeled depth distributions obtained using a K_m value of 250 µmol liter^–1 and an R_max value of 585 nmol cm^–3 h^–1; the solid lines indicate modeled depth distributions obtained using a K_m value of 250 µmol liter^–1 and an R_max value of 950 nmol cm^–3 h^–1. In both simulations, the existence of a thin layer of enhanced pore water transport (x_irr = 0.75 mm) is assumed, with a constant exchange coefficient ( ${alpha}$ ) of 55 h^–1. The inset shows the N₂O accumulation after 1, 2, 3, and 4 h. See the text for details.

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FIG. 6. In situ denitrification rates supported by nitrate uptake from the overlying water (A), maximum potential denitrification rates measured in flowthrough sediment reactors (B), and most probable numbers (MPN) of denitrifiers (C) in the top 3 cm of the sediment at Appels (August 2002). The solid line in panel A indicates the model-predicted in situ denitrification rate distribution in an acetylene block core incubation, obtained using a K_m value of 250 µmol liter^–1, an R_max value of 950 nmol cm^–3 h^–1, an ${alpha}$ value of 55 h^–1, and an x_irr value of 0.75 mm. The rectangles in panel A indicate the denitrification rates derived from the time series N₂O microprofiles (negative rates at the top were excluded for clarity).

An equally good fit to the observed values was obtained whenwe fixed R_max at 585 nmol cm^–3 h^–1 and reduced K_mto 100 µmol liter^–1 (results not shown). With bothsets of K_m and R_max values, the depth-integrated steady-staterate of N₂O production calculated with the reactive transportmodel for August 2002 was 37 nmol N cm^–2 h^–1. Irrespectiveof the actual K_m and R_max values used, the reactive transportsimulations predicted that the NO₃^– concentration profilesshould reach steady state shortly after C₂H₂ injection, whilethe buildup of N₂O in the pore waters should continue for severalhours (Fig. 5A, inset). These predictions were consistent withthe observations.

DISCUSSION

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DISCUSSION

Rate distribution and nitrate limitation.
The results of the acetylene block experiments performed withcores with no added nitrate indicate that shortly (<15 min)after injection of C₂H₂, the depth profiles for pore water O₂and NO₃^– reach steady state (Fig. 2B). The N₂O productionrates derived from the time series N₂O microprofiles also implythat there was little variation in the depth distributions ofdenitrification activity during the experiments (Fig. 3).

Production of N₂O in the acetylene block experiments startedat depths where pore water O₂ concentrations dropped below 50µmol liter^–1 and ended when pore water NO₃^–was exhausted (Fig. 2B and 3). As a result, denitrificationwas restricted to a sediment layer that was about 3 mm thick.The presence of such thin zones of denitrification has beenreported previously for stream sediments (6), lake sediments(14, 15, 19), and estuarine sediments (4, 20).

Because of inhibition by O₂ in the uppermost 1 to 2 mm and,more importantly, because of the lack of NO₃^– at depthsof >5 mm, only a small fraction of the denitrifying capacityof the sediment was utilized. This is shown in Fig. 6, wherepotential and in situ rates of denitrification are compared(Fig. 6A and B). It is also clearly evident from the instantaneousresponse of N₂O production to nitrate addition to the pore waters(Fig. 4) and is consistent with the presence of denitrifierswell below the depth of NO₃^– penetration (Fig. 6C).

The denitrification rates inferred from the acetylene blockcore incubations are lower than the maximum potential rate (R_max)measured for the 0- to 1-cm depth interval in the FTR experimentsby a factor of about 2 (Fig. 6). Furthermore, the NO₃^–concentrations in the overlying water during the two samplingtimes fall in the range of nitrate half-saturation concentrations(K_m) derived from FTR experiments with estuarine sediments (200to 600 µmol liter^–1) (18). Within the zone of maximumN₂O production (2 to 4 mm), the NO₃^– concentrations arewell below the K_m.

Taken together, the results indicate that denitrification inthe sediment is limited by nitrate. Consequently, the residentdenitrifying community rapidly adjusts its level of activityto increased nitrate availability, as shown by the results ofthe NO₃^– addition core incubations (Fig. 4). The excessdenitrifying capacity also implies that there is efficient utilizationby the denitrifying community of nitrate produced by nitrification.In the mid-Scheldt Estuary downstream from Appels, up to 85%of total denitrification in intertidal sediments may be coupledto nitrification (21). The ability to rapidly respond to changesin the NO₃^– concentration should be a useful adaptationin intertidal estuarine sediments where the pore water compositionand redox state can vary substantially on short time scalesdue to tidal flooding and irrigation by benthic infauna.

Microprofiling versus FTR experiments.
Acetylene block and FTR experiments yield different, but complementary,kinetic information. The high-resolution denitrification ratedistributions inferred from the N₂O microprofiles provide aprecise determination of the in situ reaction zone (Fig. 3).In contrast, FTR experiments are designed to measure potentialrates and parameterize predictive rate equations. Furthermore,the spatial resolutions of the two approaches are quite different,as the FTRs integrate the potential denitrifying activity over1-cm depth intervals. However, as illustrated here, the differencesbetween the two experimental approaches can be bridged throughreactive transport modeling.

With the R_max value obtained in the 0- to 1-cm FTR experimentand the baseline K_m value, the model underpredicts the buildupof N₂O; the calculated maximum N₂O pore water concentrationsare about one-third lower than the observed values (Table 2).Nonetheless, the forward reactive transport model calculationsare able to forecast the general features of the acetylene blockcore incubation experiments. In particular, the model predictsthat production of N₂O is restricted mainly to a narrow 2- to3-mm zone. In addition, the model-calculated depth distributionof the N₂O production rate rapidly reaches steady state, whilethe pore water N₂O concentrations keep increasing for severalhours.

Better agreement of the magnitudes of the concentrations andproduction rates of N₂O is obtained with the model by increasingR_max or decreasing K_m (Fig. 5). The significant decrease inthe potential denitrification rate with depth (Table 1 and Fig.6B) suggests that the average R_max for the 0- to 1-cm depthinterval (585 nmol cm^–3 h^–1) represents a lowerlimit for R_max in the zone of denitrification (0.1 to 0.5 cm).Furthermore, matching the N₂O profiles with an R_max value of585 nmol cm^–3 h^–1 requires a K_m value (100 µM)that is at the extreme low end of the range obtained in a largerset of FTR experiments with Appels sediment (200 to 600 µM)(18). Therefore, by combining the various experimental and model-derivedconstraints, our best estimates for the ranges of the kineticparameters in equation 3 are as follows: for K_m, 100 to 500µM; and for R_max, 585 to 950 nmol cm^–3 h^–1.Note that varying Formula 5 by a factor of 2 has only a minor effect on the predicted N₂O concentrationsand production rate (Table 2).

The K_m values derived from FTR experiments with natural estuarinesediments (18) are higher than the value determined for a pureculture of a nitrate-reducing bacterium, Paracoccus denitrificans( $~$ 10 µM) (26). They are, however, in line with the typicallyhigher whole-sediment K_m values obtained in slurry or core incubationexperiments (10, 17, 24, 27). The great variability of whole-sedimentK_m values among different studies, which range from 10 to 600µmol liter^–1, may reflect the great diversity ofbenthic communities of denitrifiers, which contain organismsexhibiting a wide range of nitrate affinities.

Biogeochemical implications.
The integrated N₂O production rates determined in the acetyleneblock core incubations, the direct nitrate flux determinations,and the reactive transport modeling results all point to thehigh capacity for nitrate removal by the intertidal estuarinesediments in the upper Scheldt Estuary (21). High rates of benthicdenitrification supported by nitrate uptake from the water column,similar to those at Appels (37 to 60 nmol N cm^–2 h^–1),have been reported for nitrate-rich streams ( $~$ 120 nmol N cm^–2h^–1) (7) and eutrophic estuaries in Denmark (40 to 140nmol N cm^–2 h^–1) (4, 20).

The experimentally calibrated reactive transport model providesfurther insight into the role of the kinetic parameters andthe overlying water chemistry in denitrification activity inthe sediments (Table 2). In particular, the benthic NO₃^–uptake and subsequent denitrification are very sensitive tothe NO₃^– concentration in the overlying water. The watercolumn NO₃^– concentrations in the freshwater part of theScheldt Estuary exhibit large seasonal and interannual variations,with values ranging from a few micromoles per liter to 400 µmolliter^–1. For this range of overlying water NO₃^–concentrations, the model-predicted depth-integrated denitrificationrates increase almost linearly with increasing NO₃^– concentrationsat the water-sediment interface; that is, benthic nitrate uptakein the upper Scheldt Estuary should directly scale to the watercolumn nitrate concentration.

For maximum potential denitrification rates up to about 1,000nmol cm^–3 h^–1, the model-calculated integrated rateof denitrification increases with increasing R_max (Table 2).At even greater values of R_max, however, the depth-integrateddenitrification rate asymptotically approaches a maximum value(results not shown), reflecting the increasing rate-limitingeffect of physical transport of nitrate from the water columnto the zone of denitrification. Thus, a benthic denitrifyingcommunity exhibiting a potential metabolic capacity much lowerthan that inferred here from the combined experimental and modelingresults (R_max range, 585 to 950 nmol cm^–3 h^–1) wouldunderutilize the high NO₃^– concentrations in the ScheldtRiver water, whereas a community with a much higher R_max wouldbe only marginally more efficient in removing NO₃^– fromthe water column.

Although estuarine sediments are recognized as a sink for reactivenitrogen (3, 34), the role of benthic denitrification is oftenignored in estuarine nitrogen models. For example, Regnier andSteefel (29) considered only water column denitrification intheir non-steady-state N model of the Scheldt Estuary. The averagevalues for the river discharge (30 m³ s^–1) and dissolvedinorganic N concentration (600 µmol liter^–1) yielda daily input of reactive dissolved inorganic nitrogen (NH₄⁺plus NO₃^–) to the upper Scheldt Estuary of around 22 tonsN during the summer months. Multiplying the benthic nitrateuptake obtained in this study ( $~$ 60 nmol cm^–2 h^–1)by the surface area of freshwater tidal sediments (12.2 x 10⁶m²) (P. Regnier, personal communication) yields a daily benthicuptake of about 2.5 tons of NO₃-N in the upper estuary alone.Although this is a very rough estimate, it indicates that sedimentsmay be responsible for the removal of a considerable fractionof the reactive inorganic N entering the estuary.

ACKNOWLEDGMENTS

We thank Pieter Kleingeld, Debby Los, and Rick Canavan for fieldand laboratory assistance, Gaby Eickert and Ines Schröderfor preparing the NO₃^– sensors, Jeffrey Abell and CarolineSlomp for comments on an earlier draft, and Parisa Jourabchiand Pierre Regnier for contributions to the modeling tools usedin this study. The constructive comments of three journal reviewershelped improve the manuscript. The Netherlands Institute ofEcology in Yerseke (NIOO-CEMO) kindly provided logistical support.

This study was supported by the Netherlands Organisation forScientific Research (NWO; Pioneer and Veni Programmes).

FOOTNOTES

* Corresponding author. Mailing address: Department of Earth Sciences-Geochemistry, Utrecht University, P.O. Box 80021, 3508 TA Utrecht, The Netherlands. Phone: 31 30 2536220. Fax: 31 30 2535302. E-mail: anniet{at}geo.uu.nl.

Published ahead of print on 27 October 2006.

REFERENCES

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