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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2001, p. 3771–3778 0099-2240/01/$04.00⫹0 DOI: 10.1128/AEM.67.9.3771–3778.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Vol. 67, No. 9

MINIREVIEW Measurement of Denitrification in Sediments with the 15 N Isotope Pairing Technique ´ GA ¨ CHTER, SANDRA MARTINA STEINGRUBER,* JANA FRIEDRICH,† RENE AND BERNHARD WEHRLI Swiss Federal Institute for Environmental Science and Technology (EAWAG), Limnological Research Center, 6047 Kastanienbaum, Switzerland similation. Direct measurement of N2 production has the advantage to include coupled nitrification-denitrification but requires a very sensitive analysis because of the small N2 production compared to the N2 background. The mass-balance approach on larger systems such as whole lakes may lead to considerable errors due to a combination of the errors of each term in the mass-balance. The 15N IPT has the advantage that denitrification of both NO3⫺ diffusing from the overlaying water and NO3⫺ from nitrification within the sediment can be quantified. The purpose of this study is to briefly describe the principles of the 15N IPT, to review the different applications of the 15N IPT in sediments, to point out the advantages and the limitations of the method, and to assess the main research topics, which benefit from different applications of the method. The review covers publications between 1992 and 2000.

Due to increasing nitrogen concentrations in surface waters (6) and eutrophication of coastal waters (14, 26, 51), the quantification of denitrification rates in sediments of lakes, rivers, and estuaries has gained importance. Different methods for measuring denitrification have been developed: the acetylene inhibition technique (41), measurement of nitrate disappearance (2), calculation of nitrate fluxes into the sediment from pore water profiles (20), the 15N nitrate dilution method (16), direct measurement of N2 production (40), the nitrogen massbalance approach (1), and the 15N isotope pairing technique (IPT) (23). All of these methods have advantages but also potential problems and limitations (38, 39). The acetylene inhibition technique is a very simple method. It is widely used, and its sensitivity has been improved by N2 measurements based on N2/Ar ratios. However, many different studies have documented that the acetylene method systematically underestimates denitrification (20). Several causes may be responsible for artifacts: (i) inhibition of coupled nitrification-denitrification by acetylene (19), (ii) incomplete blockage of N2O reductase at low nitrate concentrations (3), (iii) incomplete blockage of N2O reductase when sulfide is present (3), (iv) diffusion of N2O toward deeper sediment layers and reduction to N2 (39), and (v) catalytic oxidation of NO into NO2 (4). The disappearance of nitrate can overestimate denitrification because nitrate may not only be denitrified but also reduced to ammonia (16, 20) or assimilated (36). On the other hand, it may also underestimate denitrification because it does not consider coupled nitrification-denitrification. The calculation of nitrate fluxes into the sediment from pore water profiles has the same problems as described for the nitrate disappearance method. In addition, the resulting fluxes across the sediment-water interface and the diffusive boundary layer often underestimate denitrification, because of insufficient vertical resolution of the profiles and because only diffusive and not turbulent transport is considered when the fluxes are calculated (11, 21). The 15N nitrate dilution method with subsequent measurement of 15N nitrate disappearance and 15N ammonia production quantifies denitrification and nitrification but still neglects coupled nitrification-denitrification and as-

PRINCIPLES OF THE

15

N IPT

Denitrification in the sediment can occur at the expense of NO3⫺ from the water column (DW) or of NO3⫺ produced within the sediment by nitrification (Dn). These two pathways can be analyzed by the 15N IPT, which was developed by Nielsen (23). The method relies on stable isotope tracers. The natural abundance of nitrogen isotopes is 99.64% of 14N and 0.36% of 15N. 15 NO3⫺ tracer is added to the sediment overlying water. This 15NO3⫺ mixes with the 14NO3⫺ present in the water column and in the upper sediment layer. Denitrification of this nitrate mixture (Dtot) produces N2 molecules with possible molecular masses of 28, 29, and 30 according to the isotopic signature of the tracer mixture. From the production rate of 29N2 (r29) and 30N2 (r30) it is possible to calculate denitrification of 15NO3⫺ (D15) as follows: D15⫽r29 ⫹ 2 䡠 r30

(1)

For the calculation of the denitrification rate D14 of unlabeled NO3⫺, Nielsen (23) derived the relation:

14

* Corresponding author. Mailing address: Dipartimento del Territorio, Divisione dell’Ambiente, Sezione Della Protezione dell’Aria e dell’Acqua, Via Salvioni 2a, 6500 Bellinzona, Switzerland. Phone: 4191-814-38-35. Fax: 41 (0) 91 814 44 33. E-mail: S. Steingruber@ticino .com.

D14 ⫽ D15 䡠 3771

r29 2䡠r30

(2)

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The total denitrification rate in the sediment is, of course: Dtot ⫽ D14 ⫹ D15 15

(3)



The denitrification rate of NO3 (D15) allows us to calculate denitrification of the 14N/15N nitrate mixture diffusing from the water column into the sediment (Dtot W ): Dwtot ⫽

D15 ε

(4)

where ε represents the isotopic nitrate enrichment during the incubation. It can normally be expressed as: ε⫽

[NO3⫺]a ⫺ [NO3⫺]b [NO3⫺]a

(5)

where the brackets indicate concentrations and the subscripts a and b refer to after and before the 15NO3⫺ tracer addition, respectively. Finally, coupled nitrification-denitrification within the sediment (Dn) can be estimated by the difference: tot Dn ⫽ Dtot ⫺ DW

(6)

tot W

If we assume that D follows a linear increase with higher tracer concentrations, this rate can now be extrapolated back to tracer-free conditions in order to obtain the natural denitrification rate DW with nitrate diffusing from the water column (15): tot 䡠 (1 ⫺ ε) DW ⫽ DW

(7)

All of the parameters mentioned here are depicted schematically in Fig. 1. Note that to simplify the figure we neglected that nitrogen is naturally enriched with a small amount of 15N. In the special case wherein there is initially no nitrate in the water column, the added 15NO3⫺ is not diluted. Therefore, Dtot W corresponds to D15, DW is zero, and Dn equals D14. The reader is referred to the methodological studies (15, 23) for more details about the derivation of these equations. APPLICATION OF THE

15

N IPT TECHNIQUE

The 15N IPT technique has basically been applied in four different ways: (i) in batch-mode assays (5, 7, 9, 10, 12, 13, 15, 17, 18, 19, 20, 23, 24, 25, 27, 28, 29, 30, 34, 35, 42, 43, 44, 45, 46, 47, 49, 50, 52, 53), (ii) in benthic flux chambers (20, 24), (iii) in enclosures (34), and (iv) in flow through systems (32, 33, 36, 37). A wide range of aquatic systems have been investigated thus far. The experimental conditions, and the resulting denitrification rates are summarized in Table 1. Batch-mode assays. The batch-mode assay (Fig. 2a) developed by Nielsen (23) has been applied to quantify denitrification in sediments of streams (23, 30), lakes (20, 34, 42, 45, 47, 52), estuaries (5, 7, 9, 13, 17, 25, 27, 28, 29, 35), coastal waters (12, 15, 24, 43, 44, 46, 49, 50, 53), shelf sediments (19), shallow reservoirs (10), and mangrove forests (18). In all studies, undisturbed 3- to 15-cm-long sediment cores (internal diameter, 2.6 to 20.0 cm) overlaid with 5 to 40 cm of water were sampled. In most studies the cores were submersed uncapped into a tank containing 10 to 70 liters of bottom water. In some studies the bottom water was replaced with artificial water (23, 45, 47, 52).

FIG. 1. Schematic representation of the transformation rates durtot ing a 15NO3⫺ tracer experiment. Dw is the total denitrification of nitrate from the water column. Dw is the denitrification of nitrate from the water column without tracer addition. Coupled nitrification-denitrification is given as Dn. Dtot refers to the total denitrification rate during the tracer experiment. The specific denitrification rates of 15N and 14N nitrate are given as D15 and D14, respectively, and the production rates of N2 with masses of 28, 29, and 30 are represented by r28, r29, and r30, respectively.

The water overlying the sediment was stirred with a magnetic stirrer (mostly 2 to 10 cm above the sediment) coupled to an external rotating magnet. Lohse et al. (19) switched the momentum of these magnets from clockwise to anticlockwise rotation every 5 s to minimize the pressure gradient at the sediment water interface. Jensen et al. (15) circulated the tank water with a pump. The water temperature was held constant at in situ conditions. In most of the studies, 15NO3⫺ was added after 0 to 24 h of preincubation to the tank to final concentrations of 5 to 333 ␮M. The final nitrate composition ranged between 5 and 100% of 15NO3⫺. After tracer addition, the water in the reservoir was allowed to equilibrate with sediment pore water for 10 to 30 min, sometimes by stirring the water manually with a rod. In order to obtain a near-steady-state efflux of labeled dinitrogen, Kristensen et al. (18) let the system equilibrate for 12 to 14 h. Afterward, the cores were capped with rubber stoppers. Capping the cores represents a reliable starting point for the accumulation of labeled N2. The cores were sometimes incubated in the dark to simulate in situ conditions (13, 15, 18, 20, 27, 34, 42, 45, 46, 47, 49), with a parallel daylight setup to study diurnal variation of denitrification (5, 7, 25, 30, 35, 44, 53) or by alternating light and darkness (10). During the incubation lasting from 0.4 to 24 h (depending on the season and on the station), the O2 concentration did not decrease to below 80 to 70% of saturation. To maintain O2 concentration at near in situ levels, Nielsen and Glud (24) purged the water with a N2-air mixture. The nitrate concentration in the water tank was normally

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MINIREVIEW TABLE 1. Denitrification values measured with the

Method

Batch-mode assay

Reference

Cabrita and Brotas (5) Christensen et al. (7) Dong et al. (9) Eriksson and Weisner (10) Friedrich et al. (unpublished) Glud et al. (12) Gran and Pitka¨nen (13) Jensen et al. (15) Krause-Jensen et al. (17) Kristensen et al. (18) Lohse et al. (19) Mengis et al. (20) Nielsen (23)

Water system

14

NO3⫺ concn (␮M)

15

N IPTa

15

Svensson et al. (46) Svensson and Leonardson (47) Tuominen et al. (49) Tuominen et al. (50) van Luijn et al. (52) Welsh et al. (53) Benthic flux chamber

Mengis et al. (20) Nielsen and Glud (25)

Deep lake Coastal water

213 0/8

62 35/50

Water enclosure

Risgaard-Petersen et al. (32)

Shallow lake

⬍1

100/30

Flow through system

Risgaard-Petersen et al. (32) Risgaard-Petersen et al. (33)

Shallow Shallow Estuary Shallow Estuary Shallow

Pind et al. (30) Risgaard-Petersen et al. (34) Rysgaard et al. (35) Steingruber (42) Sundba¨ck and Miles (43) Sundba¨ck et al. (44) Svensson (45)

Rysgaard et al. (36) Rysgaard et al. (37)

lake lake lake lake

0–71 0–27 3–1,171 ⬃333 24–110 9–12 9–14 0–70

13–60 50/100 333 3–36 70 100 30–60

0.6–0.7 ⬍1

30 61–83

0 0 0/8 0–300 ⬍10–1,200

56 19 55–76 50–100 ⫹50–100

14 15 40–520 0–325 0–1100 78 ⬍1–30 0.5 0.5 5–135 57 3–13 0 2–22

2 14 14 14 14

D values (mmol m⫺2 day⫺1)

NO3⫺ concn (␮M)

Estuary Estuary Estuary Shallow reservoir Shallow lake Coastal water Estuary Coastal water Estuary Mangrove forest Coastal water Deep lake River River Coastal water Estuary Estuary Estuary Estuary Estuary River Shallow lake Estuary Shallow lake Coastal water Coastal water Shallow lake Shallow lake Coastal water Shallow lake Coastal water Coastal water Shallow lake Coastal water

Nielsen and Glud (24) Nielsen et al. (25) Ogilvie et al. (27) Pelegrı´ and Blackburn (28) Pelegrı´ et al. (29)

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Dw

Dn

0.0–4.9 0.0–3.8 0–120

0–0.8 0.0–0.8 0–30

1.0–4.9 0.02–0.06

0.0–0.5 0.14–0.58

0.0 0.0 0.005–0.020 3.6/3.9 0 0 0.0/0.2

0.5–0.7 0.01–0.05 0.3 0 0.5 0.5 0.3

0.0–8.0

0.0–8.9

58 134 20–260 50–100 20–250 6

0.1–1.0 0.1–0.8 0.0–15.6

0.2–0.4 0.2–0.4 0.0–4.1

0.0–1.4 6.8–7.2

0.03–0.4 0.3–2.7

60 37 309

0.0–1.0

208 100 40–120 30

198 93 93 93 93

0.0–5.1 0.9–12.8ⴱⴱ 0.0–0.3

0.0–1.9 0.0–1.1 0.0–1.1 0.0–1.9 0.0–0.8ⴱⴱ 0.0–0.4

Dtot

0–5.6 0.0–4.0 0–150 0.4/0.5ⴱ 0.3–5.4 0.16–0.64 0.0–1.3 0.0–1.5 0.5–0.7 0.01–0.05 0.3 3.6/3.9 0.5 0.5 0.3/0.5 0.1–1.6 0.1–11.0 0.2–1.0ⴱⴱⴱ 0.3–1.4 0.3–1.2 0–18.0 0.1–2.4 0.05–1.80 5.0–7.4 0.0–1.4 0.0–2.9 0.5–2.6ⴱⴱ 3.0–9.3ⴱⴱ 0.0–7.0

0.4–3.4 0.02–0.10

0.1–0.7 0.0–0.3 0.4–3.4 0.05–0.14

4.3 0/0.01

0 0.3/0.4

4.3 0.3/0.5

0

1.2/3.6

1.2/3.6

0 ⬍0.05

0 0.4–2.5 1.6–4.8 3.6 4.8 7.6–0.6

3.6 0.1–0.5 0.4–0.8 0.1–0.6 0.8–1.0 0–2

3.6 0.9–2.6 2.1–5.2 3.7–4.2 5.6–5.8 7.6–2.6

a 15

N IPT was done, in some instances, with the addition of macrophytes (ⴱ), chironomids (ⴱⴱ), or Nereis sp. (ⴱⴱⴱ). A plus sign (⫹) indicates the increase in nitrate concentration due to tracer addition.

measured before and after addition of the tracer in order to calculate the 15N enrichment ε. The production of 29N2 and 30 N2 was determined in two ways: (i) by following the production of 29N2 and 30N2 over time (time series experiment [15, 19, 23, 24, 34, 35, 52]) and (ii) by determining the concentration of 29 N2 and 30N2 at the beginning and the end of the incubation (endpoint experiment [5, 7, 9, 10, 12, 13, 18, 25, 27, 29, 30, 44, 45, 46, 47, 49, 53]). The first approach sacrifices cores at time intervals, whereas the second approach sacrifices all the cores at the same time. In fact, in both experiments the incubation was stopped by mixing the whole sediment or the upper centimeters of the sediment with the overlaying water to a slurry. To stop bacterial activity, a ZnCl2 solution was added to the cores before mixing. The slurry samples for 29N2 and 30N2 analysis were stored in gastight containers (exetainers), con-

taining some drops of a ZnCl2 solution. Entrapment of air was carefully avoided. Friedrich et al. (unpublished data) and Steingruber (42) stored the samples in evacuated glass vials containing some drops of a ZnCl2 solution, which were only half filled to permit equilibration of 29N2 and 30N2 with the gas phase. In contrast, Kristensen et al. (18) needed to sample only the water phase because the long preincubation time after tracer addition (12 to 14 h) after tracer addition led to a steady-state efflux of labeled dinitrogen. Before mixing the water column with the sediment, Lohse et al. (19), Nielsen and Glud (24), Friedrich et al. (submitted for publication), and Steingruber (42) sampled also the water column to determine the amount of 29N2 and 30N2 trapped in the sediment. They found that ⬎75, 80, ⬎73, and ⬎80%, respectively, of 29N2 and 30N2 was trapped in the sediment.

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FIG. 2. Set up of the application of the 15N IPT. The gray area corresponds to the sediment, the dotted area indicates the water without tracer, and the hatched area shows the water with tracer addition. (a) Batch-mode assay. The figure represents a water reservoir (labeled 1) with the incubation container filled with water (labeled 2), with three cylinders containing sediment, water, and a magnetic stirrer closed with rubber stoppers (black). (b) Flux chamber. The figure represents a lander lowered at the sediment surface and attached to a buoy; the flux chamber (labeled 1) is inserted in the sediment with two empty and two filled sampling syringes (labeled 2) and the magnetic stirrer. (c) Water enclosure. The figure represents the cylinder (labeled 1) pushed into the sediment and the plastic bag (labeled 2), which is open toward the sediment and toward the water surface. (d) Flow through system. The figure represents a water reservoir (labeled 1), with the incubation chamber (labeled 2) flowed through by a water flux and containing sediment, water, and a magnetic stirrer.

The time-series and the endpoint experiments have both advantages and drawbacks. The former permits one to control whether production of 29N2 and 30N2 occurs linearly with time. The disadvantage is that a single denitrification rate is based on a number of sediment cores, which may differ in quality due to heterogeneity of the sediments. The endpoint experiment avoids this problem because, for each incubated core, a specific denitrification rate is determined. However, no information is obtained about the linearity of the 29N2 and 30N2 production. Friedrich et al. (submitted for publication) and Steingruber (42) combined the two sampling methods to avoid the main disadvantages. They calculated the production of 29N2 and 30 N2 in the same way as described in the endpoint experiment, obtaining a denitrification rate for each core. In addition, they tested the linearity of the production by sampling the water phase of the cores with a syringe during the incubation at different time intervals. During the experiment the sampled water volumes were automatically replaced by tank water. Of course, such a linearity test in the overlying water rests on the assumption of a close coupling between the water column and the processes in the anerobic sediment horizons. The procedure will therefore be of limited value in sediments with a thick oxic layer.

Benthic flux chambers. Application of the 15N IPT to benthic chambers (Fig. 2b) is based on the same principles as the batch-mode assay. However, addition of 15NO3⫺ and sampling occur in situ at the sediment surface and not in a waterfilled container. Nielsen and Glud (24) used this technique to study coastal sediments, and Mengis et al. (20) adapted it to study deep lake sediments. The flux chambers contained one to two chambers, covering a sediment area between 400 and 900 cm2 and enclosing 3 to 12 liters of water. 15NO3⫺ was added from a syringe to the sediment overlaying water reaching a concentration of 35 and 50 ␮M (24) and 119 ␮M (20). During the experiment the water volume was continuously mixed with a magnetic stirrer, keeping the diffusive boundary between 0.4 and 1 mm. Oxygen was measured with sensors, and water samples were taken at regular time intervals. Nielsen and Glud (24) incubated samples for only 3 h, ensuring that oxygen was not depleted by more than 25%. Risgaard-Petersen et al. (33) and Rysgaard et al. (37) showed that the concentrations of the N species did not change linearly with time if the O2 concentration decreased by more than 20%. Mengis et al. (20) incubated samples for 60 h and, although oxygen was depleted by 75%, NO3⫺, NH4⫹, and N2 still changed linearly with time. After incubation the flux chambers were retrieved on deck.

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Water samples for determination of 29N2 and 30N2 were filled in exetainers (12.4 ml) containing 250 ␮l of ZnCl2 (50% [wt/ wt]) without entrapping gas bubbles and closed gastight (24). To extract 28N2, 29N2, and 30N2 entrapped in the sediment Nielsen and Glud (24) took sediment cores from the chamber, mixed the sediment and the overlaying water gently, and sampled the mixture as described above. Mengis et al. (20) did not sample the slurry and calculated the denitrification from the increase of labeled dinitrogen in the sampled water volumes. Mengis et al. (20) also included the isotopic composition of NO3⫺ and NH4⫹ in their analysis in order to obtain a mass balance of 15N. Water enclosures. Similar to the flux chambers, enclosures (Fig. 2c) are setups permitting in situ measurements. However, enclosures integrate over larger spatial scales. Mixing of the water is promoted by wave forces and not by a magnetic stirrer. The enclosure of Risgaard-Petersen et al. (34) consisted of a plastic bag that was open on two sides, fixed to a metal cylinder (inner diameter, 150 cm; height, 120 cm), and pushed into the sediment of a shallow lake. 15 NO3⫺ was added to the water to final concentrations of 30 and 100 ␮M. In order to estimate the outgassing of N2, Ar was added as a second tracer to the enclosure. During an incubation of 24 h, the water column and the sediment were sampled with glass tubes. Samples of the sediment and the water column were obtained with a Plexiglas tube and mixed after a ZnCl2 solution was added to stop bacterial activity. Samples were preserved with ZnCl2 and stored in exetainers until analyses. Flow through systems. In contrast to the applications of the 15 N IPT discussed thus far, flowthrough systems (Fig. 2d) are operated at steady-state conditions (32, 36). Sediment cores were incubated in gastight glass chambers connected to a thermostated continuous flowthrough system. Artificial freshwater or seawater was supplied via the inflow. The flow rate ranged from 17 to 300 ml h⫺1. Nitrate concentrations of the inflows ranged from 50 to 200 ␮M, with about 13% to 99% as 15NO3⫺. Risgaard-Petersen et al. (32, 33) and Rysgaard et al. (36, 37) adjusted the oxygen and the N2 concentration in the inflowing water with a gas-mixing system. The water in the chamber was gently mixed by a magnetic stirrer not disturbing the sediment. Preincubation for reaching steady-state conditions lasted between 7 and 14 days, and the incubation itself lasted between 12 and 32 days. During the incubation inlet and effluent were sampled periodically. In the study of Rysgaard et al. (36), the outflowing water passed through a 50-ml flask that was heated to 75°C to strip the gases. Gas samples for analyses of isotopic composition of N2 were taken with a syringe through a butyl stopper. Oxygen concentrations were measured with a microsensor (36, 37). As mentioned above, the application of the 15N IPT in a flowthrough system has the advantage of operating at steadystate conditions. For this reason the flowthrough system is suitable for studying such different parallel processes as assimilation, nitrification, and mineralization. ANALYSES AND CALCULATIONS Continuous-flow isotope ratio mass spectrometers are typically used to perform analyses for the IPT. The sample prep-

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aration of such a modern setup is much simpler than for conventional dual-inlet mass spectrometers. To analyze the isotopic composition of N2 in water samples, an He headspace is introduced into the exetainers and, after vigorous shaking for a few minutes, more than 98% of the N2 is found in the gas phase. The gas phase is then injected into a gas chromatograph in line with a triple-collector mass spectrometer to obtain the isotopic composition of N2. The concentrations of 29N2 and 30N2 in the water or slurry is calculated in the following way: [29N2] ⫽

[29N2] 28 䡠 [ N 2] [28N2]

(8)

where brackets indicate the concentrations and [29N2] 䡠 [28N2]⫺1 is the ratio obtained with mass spectrometry. [28N2] is calculated with Henry’s law: KH(T) ⫽

p N2 [ N 2] 28

(9)

where KH(T) is the temperature-dependent Henry constant and pN2 is the partial pressure of dinitrogen in the atmosphere (0.78 atm). Calculation of the production rates of 29N2 (r29) and 30N2 (r30) depends on the experimental setup. For the time-series incubations of the batch-mode assay, production rates r29 and r30 are obtained from a relation like the following (here shown for r29): r29 ⫽

m29 䡠 (Vw ⫹ ␾Vs) A

(10)

where m29 is the slope of the linear regression line of [29N2] plotted against time, A is the surface of the incubated sediment, Vw is the incubated water volume, Vs is the volume of the sediment (both in liters), and ␾ is the sediment porosity. For the endpoint incubation in the batch-mode assay, r29 and r30 are calculated as follows (here shown for r29): r29 ⫽

[29N2]f ⫺ [29N2]i 䡠 (Vw ⫹ ␾Vs) A䡠t

(11)

where [29N2]f and [29N2]i represent the final and initial concentrations of 29N2 and t is the time interval. Initial concentrations can be determined by analyzing the water phase before capping the cores or by scarifying a reference core at the beginning of the experiment and measuring the concentrations in the water-sediment slurry. The production rates of labeled dinitrogen in the flux chambers should be determined using the formula of the endpoint experiment (equation 11), where [29N2]f is the concentration of 29 N2 in the water-sediment slurry of the core subsampled at the end of the experiment. For calculating r29 and r30 in the enclosure, equation 10 can be used. In this case, when [29N2] and [30N2] are plotted against time to obtain m29 and m30, not only the amounts of 29N2 and 30 N2 measured in the slurry should be considered but also the amount lost to the atmosphere. The method used to estimate the gas loss is described elsewhere (34). The production rates r29 and r30 in the flowthrough system are calculated as follows (here shown for r29):

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r29 ⫽

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[29N2]out ⫺ [29N2]in 䡠F A

(12)

where [29N2]out and [29N2]in are the concentrations of 29N2 and N2 in the outlet and in the inlet, respectively. F is the water flow through the chamber. The calculated production rates of 29N2 and 30N2 (r29 and r30) can then be inserted in equations 1 to 6 to calculate the denitrification of NO3⫺ from the water column and coupled nitrification-denitrification. 30

DISCUSSION Assumptions of the 15N IPT. The 15N IPT is based on four assumptions (23, 35), which are briefly discussed below. (i) Added 15NO3ⴚ does not interfere with denitrification of in situ NO3ⴚ. This requirement can be tested by adding different amounts of 15NO3⫺ to the sediment overlaying water (23). The rates obtained from such experiments (Dw and Dn, Fig. 1) should remain independent of the concentration of the added tracer. In several studies (15, 19, 24, 29, 35, 45), 15NO3⫺ was added from a minimum of 10 ␮M to a maximum of 400 ␮M without modifying the in situ denitrification rate. However, this makes it not superfluous to test the interference of the addition of 15NO3⫺ before every experiment, because the optimal tracer range depends not only on the nitrate concentration but also on the heterogeneity, stability, and activity of the sediment and the incubation process. (ii) Denitrification of nitrate from the water column (Dwtot) should increase linearly with the nitrate concentration. The calculation of Dw (denitrification of in situ 14NO3⫺) from Dwtot (denitrification of 14NO3⫺ plus added 15NO3⫺) depends on this assumption. This assumption can also be tested by adding different amounts of the 15N tracer. At elevated nitrate concentrations, denitrification may no longer depend linearly on the concentration of nitrate. At high nitrate concentrations saturation effects may occur due to limited supply of electron donors or Michaelis-Menten-type saturation. In addition, the nitrate penetration depth will increase and cause a nonlinear response of denitrification rates to the concentration in the overlying water. (iii) Labeling of in situ NO3ⴚ with 15NO3ⴚ in the water column and in the sediment must be homogeneous. Heterogeneity of the sediment and bioturbation can affect the ratio between nitrification and NO3⫺ flux at different spots in the sediment. As a result, dinitrogen is produced preferentially as isotope pairs 28N2 and 30N2. Higher r30 and r28 rates compared to r29 cause an underestimation of D14 and D15 (see equations 1 and 2). However, Nielsen (23) reports that at higher concentrations of 15NO3⫺ more of the denitrified 14NO3⫺ will be measured as 29N2. As a result, the possible miscalculation of 28 N2 production will be of less significance compared to situations with lower concentrations of 15NO3⫺. Nielsen suggests that the 15NO3⫺ concentrations are high enough when D14 becomes independent of the 15NO3⫺ concentrations. However, van Luijn et al. (52) argue that in the case of anaerobic microsites that may exist in the aerobic layer, nitrifying and denitrifying bacteria may be ideally positioned, allowing a tightly coupled nitrification-denitrification. Accordingly, around these microsites the condition of

uniform labeling is still not fulfilled, even when the 15NO3⫺ concentration increases, so that coupled nitrification-denitrification is still underestimated. Middelburg et al. (22) demonstrated in a modeling study that the condition of homogeneous labeling is fulfilled only when nitrification and denitrification occur in distinct separate zones. Small-scale heterogeneity supporting denitrification within the aerobic layer interfere with this condition and may underestimate the denitrification rate. A final resolution over the controversy of the role of microsites is likely to come from studies combining microsensor or tomographic work to characterize sediment heterogeneity. Nielsen et al. (23) and Welsh et al. (53) also suggested an underestimation of coupled nitrification-denitrification when the sediments are colonized by macrophytes. The reason is that coupled nitrification-denitrification can occur close to the roots deep in the sediment remote from the diffusion zone of the 15N tracer. (iv) A stable NO3ⴚ concentration gradient across the sediment water interface must be established shortly after 15NO3ⴚ addition. This is important, because otherwise 15NO3⫺ will not be immediately available for denitrification, leading to an underestimation of the initial denitrification rate. A too-short incubation time results in an increasing production rate of 29N2 and 30N2. If 15NO3⫺ is initially well mixed in the water column, the time to establish a stable gradient depends on the oxygen penetration depth. It determines the diffusion length of nitrate molecules to reach denitrifying bacteria. The time to reach a steady-state gradient depends on microbial activity: it will be longer in oligotrophic than in eutrophic systems and longer in winter than in summer. According to Nielsen (23), the equilibration time was 8 min at an oxygen penetration depth of 1 mm. Dalsgaard (8) developed a model that predicts the equilibration time as a function of the oxygen penetration depth. Due to their long preincubation times, the flowthrough systems exhibit the most stable nitrate gradients. To meet all four conditions, D14 must be independent of the tracer concentration and D15 must increase linearly with increasing 15NO3⫺ concentrations. Furthermore, measuring the production of labeled N2 in a time series allows us to verify a constant denitrifying activity. Whether the fulfillment of these tests also excludes the underestimation of denitrification in the case of coupled nitrification-denitrification within microsites is still a matter of controversy. Comparison of different applications and methods. Lohse et al. (19) and Svensson (45) compared the acetylene inhibition technique with the 15N IPT in the batch-mode assay. The 15N IPT yielded denitrification rates two times (19) and three to eight times (45) higher than the acetylene inhibition technique because acetylene inhibits also nitrification-denitrification, which was the main denitrification mechanism. Similar observations were reported by Seitzinger et al. (39). Van Luijn et al. (52) observed that denitrification rates measured with the 15N IPT were also smaller than those estimated with the N2 flux method. In contrast, Risgaard-Petersen et al. (32) observed good agreement of the two methods in the same flowthrough system. These authors concluded that the bad agreement of the two methods described by van Luijn et al. (52) was due to the longer preincubation time necessary for the N2 flux method, which caused an accumulation of nitrate and ammonia, leading to an overestimation of denitrification. However, results from

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the N2 flux experiment of van Luijn et al. (52) agreed well with the nitrogen mass-balance of the studied lake, while the results obtained from the 15N IPT in the batch-mode assay resulted in lower denitrification rates. These authors proposed an underestimation by the IPT due to the presence of coupled nitrification-denitrification in microsites within the aerobic layer. In contrast, Nielsen et al. (25) found a good agreement between the nitrogen mass-balance of an estuary and results from the 15 N IPT in the batch-mode assay. Similar results were reported in two deep lakes (20). In the study of Steingruber (42), denitrification rates from the batch-mode assay were higher than the results from a mass-balance calculation of a shallow lake. It was proposed that the sampled bottom water for the incubation experiment may not have been representative for the whole lake (differences in nitrate concentrations). Comparison of the 15N IPT in the batch-mode assay and in an enclosure experiment in a shallow lake yielded denitrification rates 6 to 26 times higher in the enclosure (34). In the same lake the annual average denitrification estimated from whole lake mass-balance calculations was about three times higher than the rates estimated with the 15N IPT in the batch-mode assay. These authors concluded that the batch-mode assay underestimated in situ denitrification rates in shallow lakes due to lower turbulent mixing and thus transport of nitrate into the sediment. Mengis et al. (20) and Nielsen and Glud (24) compared the application of the 15N IPT to the use of a benthic chamber with the batch-mode assay. The two methods yielded similar results which also corresponded well to the results from the mass-balance calculations. In summary, the 15N IPT applied in different experimental designs compared to other methods to measure denitrification yielded sometimes contradictory results. However, it seems that the application of the 15N IPT to the batch-mode assay, the flowthrough system, and probably also the flux chamber may give correct results for deep lakes or estuaries, while it may underestimate denitrification rates in shallow waters due to suboptimal turbulent mixing in the setups. Advantages and limitations of different applications of the IPT. Three major limitations of the IPT deserve further attention independent of the mode of application. (i) So far no technique provides satisfactory simulation of the turbulent mixing conditions in shallow lakes. In the future, 15NO3⫺ tracer could be added to whole ponds in order to incubate the tracer under the most realistic conditions. (ii) It is still not clear whether an underestimation of denitrification in the case of coupled nitrification-denitrification in microniches of oxic zones can be excluded, when D14 is proved to be independent of the amount of added tracer. More research in order to clarify this issue is needed. (iii) The anaerobic oxidation of ammonium into dinitrogen with nitrate serving as an electron acceptor (anammox reaction), which has been recently reported to occur in sea sediments (T. Dalsgaard, Abstr. ASLO Aquat. Sci. Meet. 2001), can also limit the application of the IPT. Ogilvie et al. (27) suggested that in the presence of the anammox reaction, coupled nitrification-denitrification may be overestimated, while the total denitrification result would be correct. Further research is needed regarding this issue. The 15N IPT applied to the batch-mode assay is a straightforward, quick, and reliable method to determine denitrification rates. A major problem arises when denitrification occurs

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deep in the sediment, causing an extended equilibration time and, as a result, a changing production rate of dinitrogen. In such cases the flowthrough system will provide more accurate results. The flux chamber technique is more time-consuming. The quite complex benthic chambers are difficult to transport and to deploy (48). However, it allows measurements with minimal sediment disturbance under in situ conditions. The disadvantages of the 15N IPT in flux chambers are basically the same as for the batch-mode assay. In addition, the mixing of the tracer at the beginning of the experiment cannot be controlled manually. This usually causes a nonlinear production of labeled N2 during the startup phase. When most labeled dinitrogen remains in the sediment (24), denitrification has to be determined with an endpoint experiment. This implies that a sediment-water slurry has to be sampled on board. If degassing of the sediment due to decreased pressure and/or manipulation cannot be prevented, this might cause a problem. In addition, careful timing is essential for autonomous benthic landers (48), because the time period between the end of the incubation and the sampling of the slurry can be quite long. Application of the 15N IPT to a flowthrough system requires a relatively complicated technical setup. However, because steady-state conditions are maintained, this approach avoids problems of inhomogeneous mixing of the tracer. Comparison of input and output concentrations offers an additional way to determine denitrification rates. It also allows experimental variation of parameters such as light, concentrations of chemical species in the water, and temperature. Long preincubation and incubation times, which may change the microbial activity and concentration of chemical species in the sediment, are the major disadvantages of this method. In conclusion, the 15N IPT is, in spite of its limitations, a powerful technique for quantifying denitrification rates in aquatic systems if it is carefully applied and if the results are critically evaluated. ACKNOWLEDGMENTS We thank Antonin Mares and Christian Dinkel for their skilled field and laboratory work. In particular, we are indebted to Martin Mengis for measurements on the mass spectrometer and for suggestions regarding the 15N IPT. We are also grateful to the constructive comments of two anonymous reviewers. We acknowledge a grant of EAWAG to S.M.S. and financial support from the Swiss National Foundation for J.F. (project no. 31-54043.98). REFERENCES 1. Ahlgren, I. 1967. Limnological studies of Lake Norrviken, a eutrophicated Swedish lake. 1. Water chemistry and nutrient budget. Hydrologie 29:53–90. 2. Andersen, J. M. 1977. Rates of denitrification of undisturbed sediment from six lakes as a function of nitrate concentration, oxygen, and temperature. Arch. Hydrobiol. 80:147–159. 3. Binnerup, S. J., K. Jensen, N. P. Revsbech, M. H. Jensen, and J. Sørensen. 1992. Denitrification, dissimilatory reduction of nitrate to ammonium, and nitrification in a bioturbated estuarine sediment as measured with 15N and microsensor techniques. Appl. Environ. Microbiol. 58:303–313. 4. Bollmann, A., and R. Conrad. 1997. Acetylene blockage technique leads to underestimation of denitrification rates in oxic soils due to scavenging of intermediate nitric oxide. Soil Biol. Biochem. 29:1067–1077. 5. Cabrita, M. T., and V. Brotas. 2000. Seasonal variation in denitrification and dissolved nitrogen fluxes in intertidal sediments of the Tagus estuary, Portugal. Mar. Ecol. Prog. Ser. 202:51–65. 6. Carpenter, S. R., N. F. Caraco, D. L. Correll, R. W. Howarth, A. N. Sharpley, and V. H. Smith. 1998. Nonpoint pollution of surface waters with phosphorus and nitrogen. Ecol. Appl. 8:559–568. 7. Christensen, P. B., S. Rysgaard, N. P. Sloth, T. Dalsgaard, and S.

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