Limnol. Oceanogr., 54(2), 2009, 577–589 2009, by the American Society of Limnology and Oceanography, Inc. E Production of nitrogen gas via anammox and denitrification in intact sediment cores along a continental shelf to slope transect in the North Atlantic Mark Trimmer and Joanna Claire Nicholls School of Biological and Chemical Sciences, Queen Mary University of London, London E1 4NS, United Kingdom Abstract We measured the production of N2 gas from anammox and denitrification simultaneously in intact sediment cores at six sites along a transect of the continental shelf (50 m) and deeper slope (2000 m) in the North Atlantic. Maximum rates of total N2 production were measured on the shelf and were largely due to denitrification, with anammox contributing, on average, 33% of this production. On the continental slope, the production of N2 gas decreased but the proportion due to anammox reached a maximum of 65%. This change in both amount and dominant pathway of N2 production could be explained largely by the concentration of organic carbon at each site. With increasing carbon the total production of N2 increased rapidly while the response of anammox was not significant. On the continental slope, total N2 production fell below 2 mmol N m22 h21 and anammox was strongly related (r 5 0.95) to denitrification but the relative magnitude of anammox to denitrification (1.65 : 1) suggested that anammox could not be fuelled by NO { 2 from denitrification alone. On the shelf, however, where total N2 production was predominantly greater than 2 mmol N m22 h21, no relationship between anammox and denitrification was found and anammox remained constant at 1.4 mmol N m22 h21. Despite the constancy and greater availability of NO { 3 and lower temperatures on the continental slope, the significance of anammox to the total production of N2 appears primarily controlled by the overall rate of N2 production. Nitrogen (N) is generally considered to be limiting for the assimilation of carbon by primary production in the marine environment and the balance between N fixation and its removal via ‘denitrification’ is key to the regulation of primary production (Nixon et al. 1996; Falkowski 1997; Karl et al. 2002). Trends of increasing CO2 in the atmosphere are predicted to contribute to global warming and climate change, the effects of which appear to alter the balance between N fixation and N loss in coastal systems (Fulweiler et al. 2007) which, as such, necessitate a more comprehensive understanding of the removal of N in aquatic environments. Denitrification was thought to be the major pathway of N removal in the environment, but the discovery of anaerobic ammonium oxidation (anammox) in the laboratory in 1995 (Mulder et al. 1995) and the subsequent quantification of its potential contribution to N2 production in aquatic sediments, has altered the way in which we view the nitrogen cycle in such systems (Dalsgaard et al. 2005). Anammox is the formation of N2 gas from the oxidation of ammonium coupled to the reduction of NO { 2 at a 1 : 1 ratio under anaerobic conditions and is mainly thought to be an autotrophic metabolism (Van de Graaf et al. 1995; Güven et al. 2005). The biogeochemical significance of anammox is that it enables an anaerobic aquatic without the classic system to lose some of its NH z 4 coupling of aerobic nitrification to denitrification and it certainly helps explain the absence of NH z 4 in the anoxic parts of the world’s ocean (Devol 2003). The potential for anammox has now been reported for sediments from estuaries (Trimmer et al. 2003; RisgaardPetersen et al. 2004; Meyer et al. 2005) and coastal seas (Dalsgaard and Thamdrup 2002; Thamdrup and Dalsgaard 2002; Rysgaard et al. 2004) and, in addition, several anoxic bodies of water (Dalsgaard et al. 2003; Kuypers et al. 2003, 2005). The contribution of anammox to N2 production has been found to be very high in some sediments, accounting for up to 80% of N removal (Engström et al. 2005). The precise regulation of anammox in sediments is, as yet, unclear, though the current dogma is that denitrification decreases relative to anammox with increasing water depth offshore because of the associated decrease in availability of organic carbon needed to drive sediment mineralization and, thus, denitrification (Thamdrup and Dalsgaard 2002; Dalsgaard et al. 2005; Engström et al. 2005). In addition, anammox may be favored in colder regions and–or those { systems where NO { 3 and its reduction to NO 2 are both high and consistent (Rysgaard et al. 2004; Meyer et al. 2005; Trimmer et al. 2005). Despite this progress the majority of studies on anammox and denitrification in sediment are potentials based on anaerobic slurry incubations. Even before the advent of anammox, very few direct measurements of ‘denitrification’ or N2 production had been made in sediments beyond the estuarine or immediate coastal zone and our knowledge of N2 production in sediments in deeper (.100 m) offshore waters is scant (Devol 1991; Nowicki et al. 1997; and review by Steingruber et al. 2001). The majority of measurements of ‘denitrification’ in whole sediment cores over the past 25 yr have been made using either the isotope pairing technique (IPT; Nielsen 1992) or acetylene blockage (Sørensen 1978). Acknowledgments We thank the crew and technicians on board the R.R.S. Charles Darwin and R.V. Pelagia and also I. Sanders, A. Voak, C. Rooks, and A. Jaeschke for help with sampling and analysis at sea. We also thank the anonymous reviewers and the Associate Editor for their helpful suggestions and comments to improve this manuscript. This research has been supported by a Natural Environment Research Council grant (NER/A/S/2003/ 003) to M. T. 577 6.8 5.9 9.7 5.5 8.5 3.7 7.3 6.3 7.7 6.5 7.1 7.0 7.1 7.0 0.727 217 221 244 135 96 40 182 371 466 321 365 393 367 381 0.001 0.39 0.36 0.26 0.13 0.09 0.04 0.25 0.55 0.68 1.02 1.07 1.00 0.94 0.88 0.000 0.74 0.75 0.56 0.53 0.49 0.42 0.61 0.71 0.71 0.85 0.90 0.81 0.81 0.80 0.024 mud mud mud sand and mud mud and fine sand 1.41 1.49 1.69 1.77 1.77 1.64 1.63 1.53 1.52 1.23 1.31 1.29 1.28 1.36 0.021 mud and clay 20.4 20.8 15.8 16.8 9.8 11.4 15.4 6.1 1.6 5.4 6.0 1.1 4.7 4.0 0.001 50 100 100 05.79 05.92 32.00 35.56 34.84 34.16 6 6 5 5 5 5 13.15 13.08 52.00 52.98 07.22 07.15 51 51 53 53 54 54 Mean p 6 autumn spring autumn spring autumn spring 5 3 2 Mean 4 500 1000 3.9 3.9 9.5 9.6 10.6 10.4 7.5 10.6 8.6 11.7 8.3 14.4 7.5 10.2 0.183 2000 51.22 51.16 42.31 42.38 42.38 42.18 9 9 9 9 9 9 03.61 03.73 10.96 10.74 16.11 14.74 Autumn Spring Autumn Spring Autumn Spring 1 Latitude 48 48 48 48 48 48 Organic C (mmol cm23) Organic C (% dry wt) Porosity Sediment type Bulk density (g cm23) Ambient NO { 3 (mmol L21) Cruise Sampling methods and sites—The study sites (Fig. 1; Table 1) represent a depth transect from the North Atlantic continental slope (2000 m, Site 1), to Dundrum Bay in the Site Methods Bottom water temp. (uC) Because neither of these key methods is suitable for measuring production of N2 by sediments where anammox and denitrification coexist, the precise roles and regulatory factors of the two pathways of N2 production in intact sediment are currently unknown. Risgaard-Petersen et al. (2003) proposed the revised-IPT (r-IPT), with the addi{ 15 tional parameter r14 (the ratio of 14NO { x : NO x in the sediment), to distinguish between N2 production (p14) via anammox and denitrification. Subsequently we developed a method for determining r14 based on the distribution of 15N in N2O and, arguably, produced the first measurements of N2 production via anammox and denitrification in whole sediment cores (Trimmer et al. 2006). The aim of this study was to use the r-IPT to measure anammox and denitrification in intact sediment cores at six sites along a depth transect, from 2000 m on the continental slope in the North Atlantic, to 50 m on the shelf in the Irish Sea to improve our understanding of anammox and denitrification in the marine environment. Depth Longitude (m) Fig. 1. Station positions for both the spring and autumn cruises. C : N (atom) Trimmer and Nicholls Table 1. Decimal coordinates for each site plus some water and sediment characteristics for both the autumn and spring cruises. Mean values and significance values for a t-test comparison between sites grouped on either the continental slope (1, 2 and 3) or the shelf (4, 5 and 6) are given. 578 Anammox in shelf sediments Irish Sea (50 m, Site 6), and were selected to give a broad distribution of ambient NO { 3 and organic carbon but were, on the whole, muddy sediments. Sampling took place aboard the R.R.S. Charles Darwin from 12 September 2005 to 02 October 2005 (referred to hereon as the autumn cruise) and the R.V. Pelagia from 13 March 2006 to 03 April 2006 (referred to hereon as the spring cruise). Sediment samples were collected using a Netherlands Institute for Sea Research cylindrical box corer (50 cm in diameter) and sub-sampled on deck as whole sediment cores with overlying water into Plexiglass cores (25 cm long 3 3.4 cm inside diameter [i.d.]), sealed at one end with a bung. Cores were equilibrated to atmospheric pressure and incubation temperature (8uC at all sites) in storage barrels full of aerated site water, collected with a rosette water sampler fitted with Seabird Electronics sensor package (salinity, depth, temperature, conductivity, oxygen, and fluorescence). Sediment characteristics—Vertical sub-samples (5 cm) of sediment were collected from the box cores using truncated syringes (5 mL), transferred to small plastic bottles (Bijou; Bibby Sterilin) and immediately frozen on board. Later, the samples were defrosted before weighing, then drying and reweighing to calculate porosity. The sediment samples were then homogenized using a mortar and pestle, and a sub-sample (50–100 mg) placed into a preweighed scintillation vial (5 mL) and treated with HCl (2 mL, 1 mol L21 organic-free grade) as per Hedges and Stern (1984). Carbon and nitrogen content were analyzed using an elemental analyzer coupled to a continuous flow isotope ratio mass spectrometer ([CF/IRMS] Thermo-Finnigan; Delta Matt Plus) calibrated against known quantities of urea. Rates of anammox, denitrification, and dissimilatory nitrate reduction to ammonium (DNRA) in whole sediment cores—At each site two core experiments were conducted; a time-series experiment, designed to assess whether the mixing of 15N and 14N at the zone of reduction had reached equilibrium during the experiment; and a concentration-series experiment, designed to show changes in the distribution of 15N in the N2O and N2 pools with changes in 15N concentration. In total, 32 cores were used in these experiments. Time-series core experiment—After the equilibration period (,12 h), 14 cores were removed from the storage barrels and placed in a single holding tank (Trimmer et al. 2006). The overlying water in each core was then enriched 21 (50 mL of 116.3 mmol L21 with 15NO { 3 to ,50 mmol L Na15NO3 [99.3 15N atom%] Sigma-Aldrich) and carefully stirred before being bubbled gently with air through individual pieces of silicon (2 mm i.d.) tubing connected to a distributor and pump. The rate of bubbling was sufficient to fully oxygenate the water throughout the preincubation period but not enough to disturb the sediment surface. The duration of the pre-incubation period was driven by an estimate of time taken by 15NO { 3 to reach the nitrate reduction zone based upon oxygen penetration profiles (M. Trimmer unpubl.) and the diffusivity of NO { 3 579 based on porosity and temperature as per Li and Gregory (1974). The estimations agreed well with the lack of a significant relationship between r14 and time at all sites (see Results). At T0, a water sample (5 mL) was collected from each core, filtered (0.2 mm Minisart PlusTM; Sartorius UK) and { kept in the refrigerator until analysis for NH z 4 , NO 2 , and { NO 3 concentrations by automated segmented wet chemistry later that day (see below). The cores were then sealed and incubated in the dark at 8uC, with gentle stirring (,60 revolutions per minute [rpm]) of the water column. At Tf (time final) a further sample of water (5 mL) was taken and treated as above before the core was slurrified by gently mixing the sediment and overlying water with a Plexiglas rod. A slurry sample (20 mL) was carefully drawn off in a large syringe, and allowed to overflow into a gas-tight vial (Exetainer, 12 mL; Labco), fixed using formaldehyde solution (100 mL, 38% w/v) and sealed for later 15N-N2 and 15N-N2O analysis. A further slurry sample (5 mL) was carefully transferred into a bijou bottle and frozen immediately on dry-ice for 15NH z 4 analysis later in the home laboratory. The time-series cores were sacrificed from the second hour of incubation, every hour, for 14 h. Concentration-series whole core experiment—Fourteen cores were pre-incubated (as above) with 8-mmol L21 15N concentraincrements of 15NO { 3 (approximate final tions of 8, 16, 24, 32, etc., mmol L21) for at least 24 h before T0. At T0, the cores were treated as above. The concentration-series cores were sacrificed, as above, after a 12-h incubation period. An additional four cores were left unamended as references to determine the background abundance of 15N gases; these were sacrificed immediately, as above, at the beginning of the experiment. Traditional confirmation of anammox and additional slurry experiments with formate—In line with other studies on anammox in sediments we used anaerobic sediment slurries to confirm the presence of the anammox metabolism (Dalsgaard and Thamdrup 2002; Trimmer et al. 2003; Risgaard-Petersen et al. 2004). Briefly, working in a portable anaerobic glove box (Belle Technologies), sediment (20 mL from the top 2–4 cm of sediment from a box core) was homogenized with degassed site water (14 mL) and distributed into serum bottles (37 mL) before sealing with butyl rubber stoppers and aluminum crimps. The slurries were mixed on rollers (Spiramix; Thermo-Finnigan) overnight at 8uC to remove any traces of remaining oxygen or NO { x before (80 mL of addition, through the septa, of 15N labeled NH z 4 120 mmol L21 15NH4Cl [99.4 15N atom%] Sigma-Aldrich) and 1 4 NO { (final concentrations 500 mmol L 2 1 and 3 100 mmol L21, respectively). The slurries were returned to the rollers overnight before taking a sample of the headspace (1 mL) and transferring it to a water filled gas-tight vial (12 mL; Exetainer). The samples were then stored upside-down before direct analysis of the headspace as described below. At Sites 2 (continental slope, 1000 m) and 6 (shelf, 50 m) additional slurry experiments were designed to show the potential effect of carbon supplements on the relative contributions of anammox and denitrification to the 580 Trimmer and Nicholls production of N2. Briefly, at each site, 64 sediment slurries were created as above with four left unamended as references. The remaining serum bottles were injected with 14NH z (final concentration 500 mmol L21) and then 4 divided into the four treatments: 15NO { + formate; 3 15NO { + formate; 15NO { ; and 15NO { . Half of the 2 3 2 experimental bottles (30) were injected with sodium formate (final concentration 500 mmol L21); 15 of these were then injected with increasing aliquots of 15NO { 3 (final concentrations 1–15 mmol L21), and the remaining 15 injected with increasing aliquots of 15NO { 2 (final concentrations 1–15 mmol L21). The other 30 serum bottles were treated in the same way but without the addition of formate and after incubation overnight the serum bottles were treated as above. The amounts of 15N gas per unit of slurry and the relative contribution of anammox and denitrification to the production of N2 were calculated according to Dalsgaard and Thamdrup (2002). 15N-N and 15N-N2O gas analyses—On return to the home laboratory, a headspace (1 mL analytical grade He) was introduced into the gas-tight vials from the core experiments using a two-way valve and a syringe. The vials were then shaken vigorously and stored upright at 22uC to allow the gases to equilibrate between the water phase and headspace. For N2 analysis, samples of the headspace (40 mL) for both the core and slurry experiments were then injected using an auto-sampler into an elemental analyzer (which merely served as an interface), but bypassing the reduction and oxidation columns so that 15N labeled N2O would not be reduced to 15N-N2. Gases were separated on the elemental analyzer’s gas chromatograph (GC) column prior to passing to the CF/IRMS. Calibration was performed with N2 in helium over air-equilibrated water at 22uC and the mass charge ratios for mass charge ratios (m/z) 28, m/z 29, and m/z 30 nitrogen (28N2, 29N2, and 30N2) measured. For N2O isotope ratio analysis, samples of the headspace were sub-sampled (10–100 mL depending on the concentration of N2O in the headspace predetermined on a GC fitted with a m-electron capture detector) using a gastight syringe (Vici Precision Sampling) into an air filled gastight vial. The entire content of the gas-tight vial was then swept, using a two-way needle and analytical grade He, to a trace gas preconcentrator (Cryo-Focusing; PreCon, Thermo-Finnigan), where the gases are dried and scrubbed of most of the CO2 before being cryo-focused twice in liquid N2 and final separation of N2O from CO2 on a PoraPLOT Q capillary column. The sample then passes to the CF/IRMS via an interface (ConFlo III Interface, Thermo-Finnigan) and the mass charge ratios for m/z 44, m/z 45, and m/z 46 (44N2O, 45N2O, and 46N2O) measured. Calibration was performed with known amounts of N2O (98 mL L21; Scientific and Technical Gases) over the range 0.41–13.25 nmol N2O (S 44N2O, 45N2O, and 46N2O) and was linear between 0.8 pmol and 99 pmol for 45N2O and 46N O. Here, for clarity, we refer to 15N labeled N O as 2 2 45N O and 46N O and not 29N 16O and 30N 16O. 2 2 2 2 2 15NH z analysis for estimation of net DNRA—The frozen slurry samples from the time-series incubations were 4 defrosted and immediately mixed with KCl (6 mL of 2 mol L21 KCl). The samples were then mixed on rollers (as above) in the dark at 4uC for half an hour, before centrifuging. The supernatant was then sub-sampled into two gas-tight vials (3 mL Exetainer; 1 mL in each), before being bubbled with oxygen-free nitrogen (British Oxygen Company) for 10 min to remove any 15N gases dissolved in solution. A further sample of the supernatant was frozen for later measurement of NH z 4 concentration by wet chemistry (see nutrient analyses below). The 15NH z 4 content of the sample was then quantified using the hypobromite oxidation method of Rysgaard and Risgaard-Petersen (1997). The lids of both of the gas-tight vials were removed, and to one of the vials a blocked hypodermic needle was inserted and 50 mL of prepared hypobromite solution added to the cap of the needle. The other vial (reference sample) contained only the sample liquid. The lids were then replaced and sodium hydroxide (50 mL of 12 mol L21) was injected through the septa into the sample liquid of all the vials. The samples were then agitated gently and left for at least 24 h before analysis of 15N-N gas in the headspace by mass spectrometry as 2 described above. The 15N labeling in the sediment due to 15NH z was determined by subtracting the signal of the 4 reference sample from the signal of the sample with the added hypobromite, generating a proportion of 15N in the sample. This was then multiplied by the concentration of NH z 4 determined from the nutrient sample (as described below), taking into account the dilution by KCl. Ambient rates of net DNRA based on the reduction of 14NO { 3 were calculated according to DNRA ~ r14 | 15 NHz 4 ð1Þ Where r14 is calculated from the distribution of 15N in the N2O from each core (see below) and 15NH z 4 is the rate of 15NH z production (mmol m22 h21). 4 { Nutrient analyses—Concentrations of NO { 3 , NO 2 , and z NH 4 were measured using a segmented flow auto-analyzer (Skalar) and standard colorimetric techniques. The limit of and NO { was detection and precision for NO { 3 2 21 6 5%. 0.2 mmol L21 6 1%, and for NH z 0.5 mmol L 4 Calculation of anammox and denitrification—The revised isotope pairing technique (r-IPT) was used to estimate the genuine total production of N2 (p14 N2 as N from both anammox and denitrification) using the theory and methods described in Risgaard-Petersen et al. (2003) and Trimmer et al. (2006). The r-IPT estimates total N production as follows: r-IPT p14 ~ 2r14 | p29 N2 z p30 N2 | ð1 { r14 Þ ð2Þ and anammox (p14 anammox) as follows: p14 anammox ~ 2r14 | p29 N2 { 2 | r14 | p30 N2 ð3Þ Denitrification is then taken as the difference between Eq. 2 and Eq. 3. p29N2 and p30N2 are the production of 29N2 and 30N after 15NO { amendment and r 2 14 is the ratio between 3 Anammox in shelf sediments Table 2. Pearson correlation analysis between water depth, water (temperature and NO { 3 ), and sedimentary organic matter for all six sites across the shelf and continental slope. Top value in each row is the correlation coefficient (R), the bottom value the significance (p), n 5 12. Depth Temperature (uC) NO { 3 (mmol L21) R p 20.744 0.006 0.940 0.000 Organic C Organic C (% dry wt) (mmol cm23) 20.545 0.067 20.480 0.114 14NO { { and 15NO { 3 in the NO 3 reduction zone. In Eqs. 2 30 and 3 2 and p N2 are directly quantifiable using mass spectrometry, and r14 can be calculated from the distribution of 15N in the N2O pool, according to Eq. 4 (Trimmer et al. 2006). 3 p29N r14 ~ p45 N2 O 2 | p46 N2 O ð4Þ The respective contributions of p14 supported by either NO { 3 from the overlying water (p14w) or that coupled to nitrification in the sediment (p14n) as per Nielsen (1992) were calculated according to Risgaard-Petersen et al. (2003): r14 w r14 ð5Þ p14 n ~ p14 { p14 w ð6Þ p14 w ~ p14 | 15NO { in the where r14w is the ratio of 14NO { 3 to 3 overlying water determined by the difference in concentration of NO { 3 between the reference and experimental cores. The two parameters p14w and p14n were further allocated to anammox and denitrification as Aw, An, Dw, and Dn, by substitution into Eqs. 5 and 6, respectively. The term r14 can be converted into a more familiar parameter, which is useful when interpreting these calculations (i.e., q). The term q is the proportion of 15N in the produced N gas pool and is directly related to r14: q~ 1 r14 z 1 ð7Þ Strictly this definition should only be used to describe the proportion of 15N in the produced gases if they were generated from random isotope pairing (Hauck et al. 1958). Here we use it to describe the proportion of 15N for both 15N gas species (i.e., q’ N and q N O) and look for 2 2 deviations from 1 : 1 to indicate the presence of anammox (Trimmer et al. 2006). Hence, if anammox is present, by definition, q’ N2 cannot reflect random isotope pairing because the total N2 produced will contain 14N from both { the NH z 4 and NO 3 pools. Results Site characteristics—The coordinates for each station along with some characteristics for the bottom water and sediment are reported in Table 1 and a correlation analysis 581 between some of the measured variables and depth is presented in Table 2. The temperature of the bottom water was negatively correlated with depth, decreasing from 14.4uC on the shelf at 50 m to 3.9uC at 2000 m. In addition, the temperature was more consistent at Sites 1, 2, and 3 on the continental slope, changing by ,2% between the two seasons, but it changed by .18% at Sites 4, 5, and 6 on the shelf (48% at Site 6). Counter to this, bottom water NO { 3 was positively correlated with depth, peaking at 20.8 mmol L21 at 2000 m and was, overall, higher on the continental slope compared to on the shelf. The majority of the sites had muddy sediment with porosity .0.7 but it was lower at Site 2 (0.53, 0.56) and particularly low at the partly sand and mud Site 3 (0.42, 0.49). The organic carbon content of the sediment, expressed as either a percentage of dry weight or as a concentration, was, on average, greater on the shelf compared to on the continental slope but there was no overall simple relationship with water depth (Tables 1 and 2). Detecting anammox using differences in the 15 N gas pools—Here we use r14 calculated from the distribution of 15 N in the N 2 O as a proxy for the ratio of 14NO { : 15NO { (r ) in the NO { reduction zone and, 14 x x 3 ideally, this ratio should be constant throughout the incubation. The values for r14 calculated from the 15NN2O (i.e., ‘true’ r14) and 15N-N2 (i.e., potentially ‘false’ r14: anammox contributes more 14N and increases r14 in the N2 pool) in the time-series experiments were essentially constant with time. Examples of the maximum and minimum differences in r14 for N2O and N2 from Site 1 (continental slope) and Site 4 (shelf), respectively, are given in Fig. 2A and B. Clearly there was a marked difference in the two derivations of r14 at Site 1, whereas they were more similar at Site 4. Additionally, the relationship between r14 as a function of 15NO { 3 concentration in the overlying water showed r14 decreasing, predictably, with increasing 15NO { at all sites on both cruises and again examples for 3 Sites 1 and 4 are given in Fig. 2C and D. Again there was clear separation between r14 for the N2O compared to that for the N2 at Site 1 but not so much at Site 4. To visualize more clearly the effect of anammox on the distribution of 15N in the two respective gas pools r14 (as a function of 15NO { 3 ) was converted into the term q. Regression analysis of q’ N2 vs. q N2O showed a significant linear relationship between the two distributions throughout the study (for example, Fig. 2E and F) at all sites (p , 0.05). Further, the intercept was not significantly different from zero at any site (p . 0.05), that is, had no 15NO { 3 been added there would indeed be no 15N in either the N2 or N2O gas pools. Deviations from a slope of 1 : 1 indicate significant anammox activity (Trimmer et al. 2006) and all of the slopes of q’ N2 vs. q N2O were significantly different to 1 : 1 (t-test, p , 0.05) at all sites; though no significant production of 15N gases could be measured at the more sandy Site 3 in the spring. Total N2 production, anammox, and denitrification in whole sediment cores—Our estimates of the rates of N2 582 Trimmer and Nicholls Fig. 2. Selected examples showing the maximum and minimum differences between r14 derived from 15N2 and 15N2O as a function of time for (A) Site 1 (continental slope) and (B) Site 4 (shelf), and as a function of 15NO { 3 addition to the overlying water for (C) Site 1 and (D) Site 4. (E) and (F) are q’ N2 vs. q N2O plots derived from the r14 data at Sites 1 and 4, respectively. The solid line represents a first-order linear regression through the data, the dashed line represents a slope of 1 : 1 (i.e., where q’ N2 5 qN2O), which was taken as no significant anammox activity. Regression coefficients (r2 and b1 [i.e., slope], p , 0.05 in all cases) are included to illustrate the difference between the measured slope and 1 : 1 and the respective differences between the two sites. All data from the spring cruise. Anammox in shelf sediments 583 Fig. 3. Production of 14N2 gas (as N) in whole sediment cores as a function of water depth at all sites on both cruises: (A) total production (anammox plus denitrification) and broken down into (B) anammox, and (C) total denitrification. Open and closed symbols indicate spring and autumn cruises, respectively, with circles and squares indicating the continental slope and shelf sites, respectively. Numbers indicate sites. Strength of the relationship between depth and each respective process on the continental slope is also indicated (r; points are means 6 SE, n 5 14). (D) Total production of N2 and that by anammox as a function of the concentration of organic carbon in the sediment. Data are mean values for both cruises at each site, except Site 3 where there was no spring data. production by either anammox or denitrification are based on the mean values from the concentration-series experiments, where n 5 14 in each case. Comparisons across the two regions are based on the means for each set of three sites from both cruises (e.g., Sites 1, 2, and 3 on the continental slope, n 5 6). Total production of N2 gas (anammox plus denitrification) ranged from 0.31 mmol N m22 h21 to 6.84 mmol N m22 h21 and was greater in the autumn compared to the spring at all sites (Fig. 3A). Overall, production was significantly greater on the shelf (Sites 4, 5, and 6, mean 5 4.82 6 0.83 SE, n 5 6) compared to on the continental slope (1, 2, and 3 mean 5 1.02 6 0.36 SE, n 5 5, p 5 0.004) and increased significantly (r 5 0.9, p , 0.05) with depth on the continental slope. Across the six sites total N2 production was positively correlated with the organic carbon content of the sediment, when expressed as either a percentage (org C% dry wt: r 5 0.678, p 5 0.022, n 5 11) or as a concentration (mmol cm23: r 5 0.698, p 5 0.017). The averages for the two cruises for total N2 production as a function of the concentration of organic carbon are given in Fig. 3D; note the site numbers and how this was not simply a function of depth. Production of N2 gas from anammox ranged from 0.11 mmol N m22 h21 to 2.51 mmol N m22 h21 and increased significantly (r 5 0.9, p , 0.05) with depth on the continental slope but, on average, there was no significant difference in anammox between the shelf and the continental slope (1.38 6 0.24 SE to 0.61 6 0.26 SE, n 5 6 and 5 respectively; Fig. 3B). In addition, compared to differences in total N2 production, anammox was relatively constant between autumn and spring at the majority of sites (Fig. 3A and B). Anammox was not significantly 584 Trimmer and Nicholls related to the concentration of organic carbon in the sediment (r 5 0.508, p 5 0.111, n 5 11), though the relationship held for the carbon content of the sediment as a percentage (r 5 0.754, p 5 0.007, n 5 11). Regardless of the expression of sediment carbon content, the important point is the steepness of the relationship for total N2 production relative to that for anammox (Fig. 3D). The production of N2 gas from denitrification was 0.20– 5.83 mmol N m22 h21 across all sites (Fig. 3C) and was significantly greater on the shelf compared to on the continental slope (3.44 6 0.76 SE to 0.42 6 0.11 SE, respectively; p 5 0.01). There was a marked increase in denitrification between the two seasons, especially on the shelf. Denitrification tended to decrease with depth (r 5 20.608, p 5 0.047) but followed a similar pattern as for total N2 production as a function of the concentration of organic carbon (r 5 0.676, p 5 0.022; i.e., denitrification is the difference between the two sets of data in Fig. 3D). Although there was a positive relationship (r 5 0.73, p 5 0.011, n 5 11) between depth and the contribution from anammox (ra) to the total production of N2 gas across the six sites, it was much stronger for the continental slope on its own (r 5 0.96; Fig. 4A). In addition, the contribution from anammox to the production of N2 was markedly different between the spring and autumn on the shelf but more consistent on the continental slope. Regardless of depth, greater contributions from anammox to the total production of N2 gas were associated with lower rates of total production; though the data are suggestive of an optimum for the significance of anammox at around 2 mmol N m22 h21 of total N2 production (Fig. 4B). Total production of N2 gas was dominated by pw on the continental slope (mean for Sites 1–3, 68%) and pn on the shelf (mean for Sites 4–6, 81%). DNRA—Evidence for DNRA (i.e., the presence of 4 in the slurrified whole core sample), was only found at Sites 4 and 6 (both on the shelf). The data were noisy but the enrichment in terms of excess signal for 29N2 over total signal (sum of 28N2 and 29N2) was significant. For example, at Site 6 we measured a mean signal for excess 29N2 over total of 6.07 3 1024 6 SE 1.9 3 1024 and at Site 4, 4.9 3 1024 6 SE 1.29 3 1024, which gave respective t-values of 3.8 and 3.2 (t-critical 5 1.771, n 5 14). Whereas at Site 5, for example, we had 5.4 3 1026 6 SE 1.1 3 1025 and a t-value of only 0.5 and the same was also true for Sites 1, 2 and 3. Our best estimate for the rates 22 h21 and of net 14NH z 4 production were 0.001 mmol N m 0.005 mmol N m22 h21 for Sites 4 and 6, respectively. 15NH z Traditional confirmation of anammox and additional slurry experiments with formate—In line with other studies on anammox in sediments, we confirmed its presence at all six sites with measurable production of 29N2 gas from 15NH z and 14NO { in anaerobic slurries (data not shown; 3 4 one-tailed t-test against zero production, p , 0.05). With the additional slurry experiments there was no difference in { the gas yield between NO { 3 and NO 2 and the data presented in Fig. 5 are the mean values for the two as NO { x . The addition of formate had no significant effect on Fig. 4. Contribution of anammox to total N2 production (%) in whole sediment cores as a function of (A) depth (same symbols as Fig. 3, panel A), and (B) rate of total N2 production (points are means, n 5 14). the yield of gas from denitrification at either Site 2 or Site 6 (Fig. 5A and B) but it caused a significant suppression in the yield of gas by anammox at both sites (Fig. 5C and D; ANCOVA, p , 0.001). Discussion Application of the revised isotope pairing technique (rIPT)—We have used a revised version of a widely applied technique (IPT; Nielsen 1992) and it is worthwhile explaining its application and the validity of our data (rIPT; Risgaard-Petersen et al. 2003; Trimmer et al. 2006). Anammox interferes with the original IPT and causes an overestimate of N2 production, but it can be accounted for { 15 if the ratio of 14NO { 3 : NO 3 (r14) in the nitrate reduction zone is known. Assuming that denitrification makes N2O and N2, while anammox makes only N2, we can use the Anammox in shelf sediments 585 Fig. 5. Additional sediment slurry experiments with the yield of N2 gas from denitrification as a function of amended 15NO { x concentration at (A) Site 2 (continental slope) and (B) Site 6 (shelf) and the yield of N2 gas from anammox at (C) Site 2 and (D) Site 6, with and without the addition of formate, on the spring cruise only. The separation between the yield of N2 gas from anammox for both with and without formate was highly significant at both sites (p , 0.001). ratio of 15N to 14N in the N2O pool as a proxy for r14 (Trimmer et al. 2006). In the presence of anammox the proportion of 15N in the N2 pool (q’) will be diluted relative to the N2O pool (q) and q’ , q. As an example we can use the data from slurries in Fig. 5 to estimate the potential contribution of anammox to the production of N2 (ra) at each site and use this to predict the offset in q’ relative to q (as in Fig. 2E and F) in our intact core experiments according to (Trimmer et al. 2006): ra ~ 2{2| 2{ q0 q q0 q ð8Þ With an ra from the slurries of 0.28 and 0.12 for Sites 2 and 6 respectively, we would predict an average ratio for q’ : q of 0.85 : 1 and 0.95 : 1 for the intact cores at the two sites. We actually measured a ratio for q’ : q of 0.71 : 1 and 0.83 : 1, which gives higher estimates for ra in the cores of 0.45 and 0.29, respectively. This increase in ra in cores relative to slurries is consistent with our initial measurements in fjord and estuarine sediments (Trimmer et al. 2006) and can be explained by the anammox bacteria having to compete for any NO { x with far more of the facultative anaerobes in a slurry, relative to that in an intact sediment core. Our measurements agree with our predictions for the offset in q’ : q across a range of anammox activity and we have also demonstrated that r14 is constant with time (i.e., { 14 that the 15NO { 3 and NO 3 are uniformly mixed), which is the same criterion required of the original IPT (Nielsen 1992). In addition, taking all of the data together for the six sites revealed no significant dependency between our estimates of anammox (p14A) and our additions of 15NO { (r2 5 0.092). Taken individually, however, and at 3 Site 5 only, the estimate of p14A did decrease significantly 21 and 110 mmol L21 at with 15NO { 3 between 20 mmol L 21 (r2 5 0.44, 20.009 mmol N m22 h21 per 15NO { 3 mmol L 586 Trimmer and Nicholls p 5 0.019) but removal of the first two concentrations of 15NO { (22 and 29 mmol L21) removed this dependency, 3 which may have been due to inadequate mixing at these lower concentrations. Overall we are confident that the revised-IPT provides a robust estimate of genuine N2 production by both anammox and denitrification. Existing knowledge on N2 gas production—The discovery of anammox has altered our fundamental understanding of the N cycle in the marine environment and beyond (Dalsgaard et al. 2005). The data presented here, are to our knowledge, the first simultaneous measurements of anammox and denitrification in intact sediment cores from along a continental shelf to slope transect. Rysgaard et al. (2004) did report areal rates for anammox and denitrification measured in intact cores of arctic sediment (to 100 m), but their approach relied on an indirect correction for anammox derived from slurries, rather than being measured directly in cores as reported here. Even before anammox, data for N2 production in coastal and more offshore sediments were scarce. Data reported for coastal seas using the N2 flux method give a range for production of 10–141 mmol N m22 h21 but represent an integral for production by both anammox and denitrification (Devol 1991; Devol et al. 1997; Nowicki et al. 1997). Alternatively, estimates of N2 gas production made using the IPT of Nielsen (1992) typically range from 0 mmol N m22 h21 to 300 mmol N m22 h21 (Steingruber et al. 2001) but, besides being for shallow coastal waters, are likely to have been overestimated if significant anammox was unaccounted for (Risgaard-Petersen et al. 2003; Trimmer et al. 2006). Comparison in the current study between N2 production calculated with either the IPT or r-IPT showed a significant overestimation using the original IPT of between 18% and 188% (paired t-test, p , 0.001) at all but Sites 2 and 3 during the autumn cruise. Regulation of N2 gas production—Along our gradient the total production of N2 gas was positively related to the concentration of organic carbon in the sediment (Fig. 3D), which is consistent with that measured in transects from estuaries and coastal regions to deeper waters in both sediment cores and slurries (Nowicki et al. 1997; Dalsgaard and Thamdrup 2002). In contrast, the relationship between anammox and the concentration of organic carbon was not significant, which suggested other factors maybe key in regulating anammox activity (Fig. 3D). The increasing separation between each line is a measure of the increasing contribution of denitrification to N2 production and, whereas denitrification was able to exploit the increase in available carbon, anammox could not (Dalsgaard et al. 2005). Despite the likely greater availability of NH z 4 to potentially supply anammox as organic carbon increased, the increased demand for electron acceptors (NO { 3 and { NO { 2 ) may have limited the availability of NO 2 for anammox (Dalsgaard et al. 2005; Trimmer et al. 2005). On the shelf, the majority of N2 production was coupled to nitrification in the sediment (pn 5 An + Dn), as would be expected away from riverine influences (Seitzinger 1988) and, in contrast, on the continental slope, fuelled by NO { 3 Fig. 6. Anammox as a function of denitrification for all sites on both cruises. Filled symbols are data from Sites 1, 2, and 3 on the continental slope (Site 1 is at the top) and the open symbols are data from Sites 4, 5, and 6 on the shelf. A first-order regression has been fitted through the data for the continental slope sites, forced through the origin because the intercept was not significantly different to zero and the regression coefficients r2 and b1 (i.e., slope [p 5 0.03]) are included. from the overlying water column (pw 5 Aw + Dw), in agreement with previous measurements of N2 flux at 630 m (Devol 1991) and the respective difference in bottom water NO { across the two regions. Rysgaard et al. (2004) 3 reported a strong positive correlation between water column NO { 3 and potential rates of anammox but, overall, we found no such relationship here (r 5 20.43, p . 0.05). Maximal anammox activity, measured at Site 5, was actually sustained by nitrification in the sediment (An), rather than NO { 3 in the overlying water (Aw), and such a correlation would not be expected. Overall, our trend agrees very well with that reported formerly in the literature, that anammox can be a highly significant, if not the major pathway of N2 production, in less reactive sediments (Thamdrup and Dalsgaard 2002; Rysgaard et al. 2004; Engström et al. 2005). Coupling anammox to a supply of NO { 2 —Three common reduction have been suggested to supply pathways of NO { 3 { NO { to anammox, either directly as NO to NO { 2 3 2 { through NO 3 respiration (Nicholls et al. 2007), or indirectly via leakage of NO { 2 as part of the denitrification { (NO { R NO R NO R N2O R N2; Dalsgaard and 3 2 Thamdrup 2002; Trimmer et al. 2003; 2005) or DNRA z { processes (NO { 3 R NO 2 R NH 4 ). { If the source of NO 2 for anammox was denitrification, the two could be coupled, as has been suggested previously (Rysgaard et al. 2004). Plotting anammox as a function of denitrification revealed a very strong positive correlation between the two processes at Sites 1, 2, and 3 on the continental slope but not at Sites 4, 5, and 6 on the shelf (Fig. 6). This alone suggested different mechanisms of Anammox in shelf sediments regulation of anammox across the regions, although there was overlap between Site 5 and the continental slope when denitrification was ,1 mmol N m22 h21. In this analysis, which considers the dataset as a whole, the ratio of anammox to denitrification on the continental slope was 1.65 : 1 (b1 in Fig. 6), which is equivalent to 62% of N2 production coming from anammox, relative to a mean of 52% by site (Fig. 4A). In contrast, the average contribution of anammox to N2 production on the shelf was 33%. Dalsgaard et al. (2003) presented an eloquent argument for a tight coupling between anammox and denitrification in the water column of the Golfo Dulce, based on the combined stoichiometry for the two reactions and a common Redfield ratio of 6.6 : 1 for the organic matter being mineralized. Indeed, they predicted that 29% of N2 production would be due to anammox which, on average, agreed very well with their measurements (19–35% mean ,27%). This is interesting because our similar value of 33%, on the shelf, suggested anammox could be fuelled z by NO { 2 and NH 4 coming from denitrification in this region, though the two were not related. On the shelf, conditions for denitrification were more favorable and total production of N2 maximal (Fig. 3C). The relationship on the continental slope holds up to ,2.5 mmol N m22 h21 (total N2 production) or the lower rate of denitrification measured at Sites 4 and 6. It may be that the rate of denitrification at Sites 4 and 6 was always { sufficient to ‘leak’ enough NO { 2 to avoid NO 2 limitation of anammox but that an alternative factor kept anammox constant. In contrast, this was not likely to be the case on the continental slope with a contribution to the production of N2 from anammox of up to 65%. Dalsgaard et al. (2003) argued that .29% of N2 coming from anammox could be due to the mineralization of organic matter with a lower C : N ratio (i.e., more N released per mole of carbon oxidized with NO { 3 ). Our measurements of C : N for the organic matter in the sediment do not necessarily support this, because the ratio tended to be higher (autumn) when the contribution of anammox to N2 production was greatest. Even if NH z 4 was in excess in the sediment, anammox would still require a source of NO { 2 which, at contributions of up to 65% from anammox to N2 production (Fig. 4), would require denitrification to be very inefficient and ‘lose’ 46% of its NO { 2 . Alternatively, such a tight coupling on the continental { slope may be via an ‘upstream’ supply of NO { 2 from NO 3 respiration, which fuelled both anammox and denitrification when total N2 production was low (i.e., ,2 mmol N m22 h21) and organic C ,220 mmol C cm23 sediment. This certainly helps explain the pattern in the contribution of anammox to N2 production at Sites 1, 2, and 3, because although the rates of anammox and denitrification were different across the two seasons, the difference was proportional and the contribution to N2 production via each source remained constant at each site respectively. Blaszczyk (1993) measured a significant accumulation of { NO { 2 (up to 70% of NO 3 reduction) from Paracoccus denitrificans growing on minimal medium (ethanol, acetate, { or methanol) with NO { 3 , but no accumulation of NO 2 587 with growth on nutrient broth. Hence, here, under similar carbon limitation, the first step of N removal stalls after { NO { 3 reduction and liberates NO 2 which, in turn, is taken by both anammox and denitrification. This continued because the availability of carbon increased with both anammox and denitrification increasing. Eventually conditions became more favorable for ‘complete’ denitrification (.2 mmol N m22 h21 total N2 production), NO { 2 was consumed and, despite the availability of NH z 4 in the sediment, anammox could not proceed without NO { 2 and became proportionally less significant. Given that anammox and denitrification appear to have similarly very 21 high affinities for NO { 2 , possibly as low as 0.1 mmol L (Dalsgaard and Thamdrup 2002), it is the availability of organic carbon to sustain heterotrophic respiration that enables denitrification to eventually win out over anammox for a common source of NO { 2 . DNRA and its effect on anammox—An additional factor to consider is the potential for DNRA found in the intact sediment cores at Sites 4 and 6, because the production of 15NH z from the reduction of 15NO { could potentially 3 4 enable anammox to generate 30N2, which would undermine the central assumptions of anammox 15N techniques (Thamdrup and Dalsgaard 2002; Trimmer et al. 2006; Kartal et al. 2007). The net (because some of the 15NH z 4 will be oxidized to NO { x or potentially directly to N2 via anammox) rates of DNRA were very low compared to the gross rates of anammox and denitrification, representing ,0.04% and 0.13% of N2 gas production at Sites 4 and 6, respectively. We can conclude from this that unless gross DNRA was directly linked to anammox as suggested by 15NH z Kartal et al. (2007; i.e., metabolism of 15NO { 3 to 4 and N2, coupled to the oxidation of simple organics), the z contribution of 15NH 4 to the anammox N2 gas pool would pool was be negligible, because the ambient 14NH z 4 relatively large (,30 mmol L21 data not shown). At Site 6, however, the slurry experiment showed that the addition of organic carbon suppressed the production of N2 by anammox, as it did also at Site 2, but without a concomitant rise in denitrification (Fig. 5). If there were 15NH z an entirely intracellular metabolism of 15NO { 3 to 4 and N2 then we may have expected an increase in the 15NH z ), which production of 30N2 (15NO { x pairing with 4 would have appeared as an increase in denitrification, but we did not (Fig. 5). The potential for DNRA does represent an alternative sink for NO { x in the sediments z and NH and an alternative source of NO { 2 4 for anammox but it appears of minor importance and does not appear to be directly part of the anammox metabolism itself. Maintenance of anammox—Several researchers have argued that anammox is more successful where environmental conditions are relatively constant and that denitrification is more flexible in response to environmental change (Risgaard-Petersen et al. 2004; Rysgaard et al. 2004; Dalsgaard et al. 2005). In addition, it has been suggested that both the availability and consistency in a supply of NO { 3 maybe essential for maintaining anammox (Rysgaard et al. 2004; Risgaard-Petersen et al. 2005; Trimmer et 588 Trimmer and Nicholls al. 2005) and anammox has been shown to be favored at lower temperatures relative to denitrification (Dalsgaard and Thamdrup 2002; Rysgaard et al. 2004). Certainly anammox was consistently more significant at Sites 1, 2, 3, 21 in the and 5 where NO { 3 never drops below 5 mmol L overlying bottom water and, at Sites 1, 2, and 3, temperatures were lower. Sites 1–3 are in deep upwelling water in the North Atlantic and Site 5 is in the gyre system of the western Irish Sea (Trimmer et al. 1999; Horsburgh et al. 2000). In contrast, Sites 4 and 6 are more variable 21 in the spring and and NO { 3 can reach ,1 mmol L summer (Gowen et al. 2000). This was further corroborated by the relative change in bottom-water NO { 3 between the two seasons, where the difference was greatest at Sites 4 and 6 (74% and 343%, respectively), but less marked (,16%) at Sites 1, 2, 3, and 5. Despite this, the dominant factor in the significance of anammox to the production of N2 along our transect was the overall metabolic rate of N2 production. References BLASZCZYK, M. 1993. Effect of medium composition on the denitrification of nitrate by Paracoccus denitrificans. Appl. Environ. Microbiol. 59: 3951–3953. DALSGAARD, T., D. E. CANFIELD, J. PETERSEN, B. THAMDRUP, AND J. ACUÑA-GONZÁLEZ. 2003. N2 production by the anammox reaction in the anoxic water column of Golfo Dulce, Costa Rica. Nature 422: 606–608. ———, AND B. THAMDRUP. 2002. Factors controlling anaerobic ammonium oxidation with nitrite in marine sediments. Appl. Environ. Microbiol. 68: 3802–3808. ———, ———, AND D. E. CANFIELD. 2005. Anaerobic ammonium oxidation (anammox) in the marine environment. Res. Microbial. 156: 457–464. DEVOL, A. H. 1991. Direct measurement of nitrogen gas fluxes from continental shelf sediments. Nature 349: 319–321. ———. 2003. Solution to a marine mystery. Nature 422: 575–576. ———, L. A. CODISPOTI, AND J. P. CHRISTENSEN. 1997. Summer and winter denitrification rates in western Arctic shelf sediments. Cont. Shelf Res. 17: 1029–1033. ENGSTRÖM, P., T. DALSGAARD, S. HULTH, AND R. C. ALLER. 2005. Anaerobic ammonium oxidation by nitrite (anammox): Implications for N2 production in coastal marine sediments. Geochim. Cosmochim. Acta 69: 2057–2065. FALKOWSKI, P. G. 1997. Evolution of the nitrogen cycle and its influence on the biological sequestration of CO2 in the ocean. Nature 387: 272–275. FULWEILER, R. W., S. W. NIXON, B. A. BUCKLEY, AND S. L. GRANGER. 2007. Reversal of the net dinitrogen gas flux in coastal marine sediments. Nature 448: 180–182. GOWEN, R. J., D. K. MILLS, M. TRIMMER, AND D. B. NEDWELL. 2000. Production and its fate in two coastal regions of the Irish Sea: The influence of anthropogenic nutrients. Mar. Ecol. Prog. Ser. 208: 51–64. GÜVEN, D., AND oTHERS. 2005. Propionate oxidation by and methanol inhibition of anaerobic ammonium-oxidizing bacteria. Appl. Environ. Microbiol. 71: 1066–1071. HAUCK, R. D., S. W. MELSTED, AND P. E. YANKWICH. 1958. Use of N-isotope distribution in nitrogen gas in the study of denitrification. Soil Sci. 86: 287–296. HEDGES, J. I., AND J. H. STERN. 1984. Carbon and nitrogen determination of carbonate-containing solids. Limnol. Oceanogr. 29: 657–663. HORSBURGH, K. J., A. E. HILL, J. BROWN, L. FERNAND, R. W. GARVINE, AND M. M. P. ANGELICO. 2000. Seasonal evolution of the cold pool gyre in the western Irish Sea. Prog. Oceanogr. 46: 1–58. KARL, D., AND oTHERS. 2002. Dinitrogen fixation in the world’s oceans. Biogeochem. 57/58: 47–98. KARTAL, B., M. M. M. KUYPERS, G. LAVIK, J. SCHALK, H. J. M. OP DEN CAMP, M. S. M. JETTEN, AND M. STROUS. 2007. Anammox bacteria disguised as denitrifiers: Nitrate reduction to dinitrogen gas via nitrite and ammonium. Environ. Microbiol. 9: 635–642. KUYPERS, M. M. M., AND oTHERS. 2005. Massive nitrogen loss from the Benguela upwelling system through anaerobic ammonium oxidation. Proc. Natl. Acad. Sci. 102: 6478–6483. ———, AND ———. 2003. Anaerobic ammonium oxidation by anammox bacteria in the Black Sea. Nature 422: 608–611. LI, Y. H., AND S. GREGORY. 1974. Diffusion of ions in sea water and in deep-sea sediments. Geochim. Cosmochim. Acta 38: 703–714. MEYER, R. L., N. RISGAARD-PETERSEN, AND D. E. ALLEN. 2005. Correlation between anammox activity and microscale distribution of nitrite in a subtropical mangrove sediment. Appl. Environ. Microbiol. 71: 6142–6149. MULDER, A., A. A. VAN DE GRAAF, L. A. ROBERTSON, AND J. G. KUENEN. 1995. Anaerobic ammonium oxidation discovered in a denitrifying fluidized bed reactor. FEMS Microbiol. Ecol. 16: 177–184. NICHOLLS, J. C., C. A. DAVIES, AND M. TRIMMER. 2007. High resolution profiles and nitrogen isotope tracing reveal and dominant source of nitrous oxide and multiple pathways of nitrogen gas production in the central Arabian Sea. Limnol. Oceanogr. 52: 156–168. NIELSEN, L. P. 1992. Denitrification in sediments determined from nitrogen isotope pairing. FEMS Microbiol. Ecol. 86: 357–362. NIXON, S. W., AND oTHERS. 1996. The fate of nitrogen and phosphorus at the land–sea margin of the North Atlantic Ocean. Biogeochem. 35: 141–180. NOWICKI, B. L., E. REQUINTINA, D. VAN KEUREN, AND J. R. KELLY. 1997. Nitrogen losses through sediment denitrification in Boston Harbor and Massachusetts Bay. Estuaries 20: 626–639. RISGAARD-PETERSEN, N., R. L. MEYER, AND N. P. REVSBECH. 2005. Denitrification and anaerobic ammonium oxidation in sediments: effects of microphytobenthos and NO32. Aquat. Microb. Ecol. 40: 67–76. RISGAARD-PETERSON, N., R. L. MEYER, M. SCHMID, M. S. M. JETTEN, A. ENRICH-PRAST, S. RYSGAARD, AND N. P. REVSBECH. 2004. Anaerobic ammonium oxidation in an estuarine sediment. Aquat. Microb. Ecol. 36: 293–304. ———, L. P. NIELSEN, S. RYSGAARD, T. DALSGAARD, AND R. L. MEYER. 2003. Application of the isotope pairing technique in sediments where anammox and denitrification coexist. Limnol. Oceanogr. Methods 1: 63–73. RYSGAARD, S., R. N. GLUD, N. RISGAARD-PETERSEN, AND T. DALSGAARD. 2004. Denitrification and anammox activity in Arctic marine sediments. Limnol. Oceanogr. 49: 1493–1502. ———, AND N. RISGAARD-PETERSEN. 1997. A sensitive method for determining nitrogen-15 isotope in urea. Mar. Biol. 128: 191–195. SEITZINGER, S. P. 1988. Denitrification in freshwater and coastal marine ecosystems: Ecological and geochemical significance. Limnol. Oceanogr. 33: 702–724. SØRENSEN, J. 1978. Denitrification rates in a marine sediment as measured by the acetylene inhibition technique. Appl. Environ. Microbiol. 36: 139–143. STEINGRUBER, S. M., J. FREIDRICH, R. GÄCHTER, AND B. WEHRLI. 2001. Measurements of denitrification in sediments with the 15N isotope pairing technique. Appl. Environ. Microbiol. 67: 3771–3778. Anammox in shelf sediments THAMDRUP, B., AND T. DALSGAARD. 2002. Production of N2 through anaerobic ammonium oxidation coupled to nitrate reduction in marine sediments. Appl. Environ. Microbiol. 68: 1312–1318. TRIMMER, M., R. J. GOWEN, B. M. STEWART, AND D. B. NEDWELL. 1999. The spring bloom and its impact on benthic mineralization rates in western Irish Sea sediments. Mar. Ecol. Prog. Ser. 185: 37–46. ———, J. C. NICHOLLS, AND B. DEFLANDRE. 2003. Anaerobic ammonium oxidation measured in sediments along the Thames Estuary, United Kingdom. Appl. Environ. Microbiol. 69: 6447–6454. ———, ———, N. MORLEY, C. A. DAVIES, AND J. ALDRIDGE. 2005. Biphasic behavior of anammox regulated by nitrate and nitrite in an estuarine sediment. Appl. Environ. Microbiol. 71: 1923–1930. 589 ———, N. R ISGAARD -P ETERSEN , J. C. N ICHOLLS , AND P. ENGSTRÖM. 2006. Direct measurement of anaerobic ammonium oxidation (anammox) and denitrification in intact sediment cores. Mar. Ecol. Prog. Ser. 326: 37–47. VAN DE GRAAF, A. A., A. MULDER, P. DE BRUIJN, M. S. M. JETTEN, L. A. ROBERTSON, AND J. G. KUENEN. 1995. Anaerobic oxidation of ammonium is a biologically mediated process. Appl. Environ. Microbiol. 61: 1246–1251. Edited by: Bo Thamdrup Received: 27 June 2008 Accepted: 18 October 2008 Amended: 13 November 2008
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