Journal of Experimental Marine Biology and Ecology 359 (2008) 85–91

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Journal of Experimental Marine Biology and Ecology

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Denitrification, nitrate turnover, and aerobic respiration by benthic foraminiferans inthe oxygen minimum zone off Chile

Signe Høgslund a, Niels Peter Revsbech a, Tomas Cedhagen b, Lars Peter Nielsen a,⁎, Victor Ariel Gallardo c

a Department of Biological Sciences, University of Aarhus, Ny Munkegade Building 1540, DK-8000 Aarhus C, Denmarkb Department of Biological Sciences, University of Aarhus, Finlandsgade, 14 DK-8200, Aarhus N, Denmarkc Department of Oceanography and COPAS Center, University of Concepción, P.O. Box 160-C, Concepción, Chile

a r t i c l e i n f o

⁎ Corresponding author. Dept. of Biological ScienceAarhus, bld. 1540, DK-8000 Aarhus C, Denmark. Tel.: +2722.

E-mail address: biolpn@biology.au.dk (L.P. Nielsen).

0022-0981/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.jembe.2008.02.015

a b s t r a c t

Article history:Received 14 December 2007Received in revised form 7 February 2008Accepted 8 February 2008

Population density, nitrate turnover, and oxygen respiration of benthic foraminiferans were investigated inthe oxygen minimum zone (OMZ) off the Chilean coast. Live foraminiferans were found predominantly in theupper 3 mm of the sediment, and the nitrate accumulating species Nonionella cf. stella and Stainforthia sp.dominated with a combined standing stock of 2.0×106 Rose Bengal stained specimens m−2. The rate ofdenitrification in cells of N. cf. stella analyzedwith nitrous oxide microsensors during acetylene inhibitionwas84±33 pmol C individual−1 d−1. Multiplied with the standing stock of N. cf. stella and Stainforthia sp. thisyielded a minimum benthic denitrification rate of 173 µmol N m−2 d−1 by foraminiferans. Foraminiferaldenitrification, which seemed to account for almost all benthic denitrification at the investigated site will beoverlooked bymost conventional methods measuring benthic denitrification. Compared to the denitrificationrates, the potential rates of nitrate accumulation and oxygen respiration by N. cf. stella were an order ofmagnitude higher (864 pmol N individual−1 d−1 and 760±87 pmol C individual−1 d−1, respectively), whichseems an adaptation to the infrequent availability of nitrate and oxygen in the sediment surface.

© 2008 Elsevier B.V. All rights reserved.

Keywords:Aerobic respirationDenitrificationForaminiferaNitrate turnoverOxygen minimum zoneThioploca

1. Introduction

Since its discovery, the complete reduction of nitrate to dinitrogenhas been ascribed to the prokaryotic domains of Bacteria and Archaea(Shapleigh, 2006). Recently, however, denitrifying capacity has alsobeen found among foraminiferans, demonstrating that also eukar-yotes may directly remove nitrogen from the sea (Risgaard–Petersenet al., 2006).

Foraminiferans (Rhizaria: Foraminifera) (Adl et al., 2005) thrive inlow-oxygen environments and living specimens can be found manycentimeters down in the sediment where they tolerate both sulfideand absence of oxygen (Bernhard et al., 2003; Bernhard, 1993;Bernhard, 1992; Duchemin et al., 2005). High foraminiferan densitiesare found in the heart of oxygen minimum zones (OMZs) whereforaminiferans are often the most abundant benthic meiofauna(Bernhard et al., 2000; Gooday et al., 2000; Levin et al., 2002) playingan important role in mineralization (Woulds et al., 2007).

Oxygen minimum zones cover more than one million km2 ofseafloor (Helly and Levin, 2004) and as much as 30–50% of the globalmarine nitrogen loss may occur in OMZs (Codispoti et al., 2001;Gruber and Sarmiento, 1997). Recent studies have shown that theanammox process converting nitrite plus ammonium into dinitrogen

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might be more important than denitrification in removing nitrogen inthe water column of OMZs (Kuypers et al., 2005). Denitrification byforaminiferans like Nonionella cf. stella and Stainforthia sp., both ofwhich inhabit OMZs (Risgaard–Petersen et al., 2006), could be anotheroverlooked factor in the OMZ nitrogen cycle. Nonionella stella is awidely distributed and dominant species in OMZ areas, constitutingup to 83% of the foraminiferan population in the Santa Barbara Basin(Bernhard and Reimers, 1991). Important nitrate competitors in thesediment of OMZs are the large filamentous sulfur bacteria of thegenus Thioploca which also accumulates nitrate in large quantities(Fossing et al., 1995). However, their activity does not contribute to theloss of fixed nitrogen because they reduce nitrate to ammonium andnot dinitrogen (Otte et al., 1999).

The present investigation was conducted with foraminiferans fromthe OMZ along the Chilean continental margin during the australsummerof 2006. TheOMZhere is formedwhennutrient-rich equatorialsubsurfacewater is brought to the surface by seasonal upwelling, whichinduces a high level of primary production (Ahumada et al., 1983). Thedecomposition of the sinking organic matter causes oxygen depletionand thus OMZ formation between 40–400mwater depth in the easternboundary Humboldt Current System off Chile and Peru (Morales et al.,1999).

Here we provide a first estimate of the foraminiferal contributionto benthic denitrification by measuring individual cell rates andpopulation densities. Rates of nitrate accumulation, aerobic respira-tion, and vertical distribution in the sediment are also measured inorder to evaluate the capability of the foraminiferans to compete for

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electron acceptors at the sea floor. We also evaluate the extent towhich foraminiferal denitrification has been, or could be, included inother measures of benthic denitrification.

2. Materials and methods

2.1. Sampling and site description

Intact sediment and bottom water was sampled in cores (9.5 cminner diameter) with a multicorer from the R/V Kay Kay in January2006 at Sta. 18 (Neira et al., 2001) on the continental shelf offConcepción Bay, central Chile (36°30′08′′S; 73°07′07′′W; 88m depth).The cores were stored at in situ temperature and transported withinthe same day to the laboratory.

The sampling site is characterized by intense spring-summerupwelling causing high nitrate and low oxygen concentrations abovethe shelf (Ahumada et al., 1983). At the time of sampling, theseconditions had probably persisted since the last major El Niño episode(1997–1998), and the sediment biota was dominated by Thioploca.

2.2. Biomass profiles

Within 6 hours after sampling, a sediment core was cut in 3 mmslices to 12 mm depth plus a 6 mm slice to 18 mm depth. Sodiumborate-buffered formalin was added to each slice to a finalconcentration of 2.4% for short term preservation of foraminiferansuntil staining and counting. Rose Bengal dissolved in tap water (0.1%)was added to each slice, and left over night (Walton, 1952). Aftergentle mixing subsamples, corresponding to 2.66 cm3 of sedimentfrom each slice were taken by a samplesplitter (Elmgren, 1973). To getsmall species, gentle sieving through a 50 µm screen was conductedbefore counting and sorting of tapwater-diluted samples under adissection microscope (40 times magnification). Only brightly stainedspecimens were counted.

2.3. Sample maintenance

Subsampled sediment cores (diameter 50 mm) were mounted to aclosed flume system consisting of a 30 cm long and 1 cm high flumechannel and a circulation pump. The top of the core fitted into a hole inthe middle of the flume channel, and the sediment was pushed upuntil the surface was in level with the flume bottom. Sediment coreand recycled anoxic bottom water was kept at in situ temperature.Nitrate was frequently added to maintain in situ concentration ofabout 10 µmol L−1 unless otherwise noted. The flume was made ofglass allowing observations with a dissection microscope from theside or the top, and a slit in the lid allowed for specimen collectionwith brush or pipette and insertion of microsensors.

2.4. Microsensors and determination of concentration profiles

Microsensors for oxygen, sulfide, NOx−, nitrous oxide, and pH were

used to analyze chemical profiles in a sediment core in the flume andin foraminiferan respiration experiments as described in detail below.

Oxygen microsensors (Revsbech, 1989) with 10 µm tips and detec-tion limitb0.5 µmol L−1were two-point calibrated in air-saturatedwaterand anoxic alkaline ascorbate. Hydrogen sulfide microsensors (Kühlet al., 1998) had tips of 20 µm, a detection limit b1 µmol L−1, andwere calibrated in relevant concentrations of Na2S in anoxic MES(2-morpholinoethanesulfonic acid) buffer at pH 6.7. Microsensorsfor pH measurements made according to Revsbech and Jørgensen(1986) had a 30-µm wide and 240 µm long sensing glass tip, anaccuracy of b0.05 pH units, and were calibrated in standard buffersof pH 4, 7, and 10. Total dissolved sulfide (H2Stot) was calculatedusing the relation between pH, total dissolved sulfide and H2S.Biosensors for NOx

− (Larsen et al., 1997) had tip diameters of 70 µm

and were two-point in situ calibrated in natural seawater with10 µmol L−1 nitrate and deep, nitrate-free sediment layers. Linearityand stirring insensitivity of the response was verified by calibrationin a beaker containing a 35‰ NaCl solution. The detection limit andsensitivity of the sensor was b1 µmol L−1. Nitrous oxide micro-sensors (Andersen et al., 2001) were made without an oxygen trapto improve the sensitivity and had tip diameters of 50–60 µm. Theywere calibrated in dilutions of nitrous oxide saturated water andhad a resolution and detection limit of b1 µmol L−1 nitrous oxide.

2.5. Nitrate content and accumulation

Intracellular nitrate+nitrite was analyzed by the method ofBraman and Hendrix (1989) where nitrate+nitrite are reduced tonitric oxide and detected by chemiluminescense. The setup wasoptimized for detecting small amounts of nitrate and direct manualsample insertion. The 0.6 mL reaction mixture of 0.1 mol L−1 VCl3 and2 mol L−1 HCl was held at 80 °C in a U-shaped glass tube and contin-uously bubbled with a carrier flow of 40 mL dinitrogen min−1 drivenby the nitric oxide detector (Eco Physics CLD 86, Eco Physics, DeurntenSwitzerland). The front end of the reaction tube was flushed withdinitrogen, and excess gas was vented out through a 2 mm wideopening allowing direct insertion of samples in the reaction liquidwithout air contamination or pressure changes.

Individual foraminiferans were picked and cleaned by a brush innitrate free artificial seawater under a dissection microscope, andspecies identity was determined before insertion in the reactor. Here,the hot acid mixture caused disintegration of the foraminiferans,release of nitrate and conversion to nitric oxide within a few seconds.Calibration was carried out by injecting 4 to 9 µL nitrate standardsprepared from analytical grade NaNO3 and pure demineralized water.The detection limit was 2 pmol nitrate corresponding to a 4 µL sampleof 0.5 µmol L−1. As a test for contamination and any possible artifacts,samples of 2 mm3 sediment without foraminiferans or Thioplocafilaments were analyzed following the same protocol.

Foraminiferal nitrate accumulation rates in the sediment weremeasured in the flume system. After a period of approximately24 hours without nitrate in the water, nitrate was added andmaintained at a concentration of 5–20 µmol L−1. Individuals of N. cf.stella were picked from the surface sediment at various times duringthe next two days and internal nitrate pools were analyzed.

2.6. Foraminiferal respiration rates

Foraminiferal oxygen and nitrate respiration rates were calculatedfrom linear steady state gradients of oxygen and nitrous oxide inmicrotubes with 3–5 individuals. Acetylene inhibits nitrous oxidereductase and thereby makes nitrous oxide the end product ofdenitrification (Smith et al., 1978). The steady state diffusion fluxes inthe tubes corresponding to the respiration rates were calculated byFick's first law of diffusion, J=-D ⁎ dC/dx, where J is the flux, dC/dx isthe measured concentration gradient, and D is the free diffusioncoefficient of oxygen and nitrous oxide at 12.3 °C and 34‰ salinity(1.58×10−5 cm2 s−1 (gas tables at Unisense, www.unisense.com/Default.aspx?ID=117). The total productions or consumptions werecalculated as the product of the flux and the cross section areaof the microtube (0.196 mm2). The production of nitrous oxidewas multiplied by 2 to account for both nitrogen atoms in theproduct.

The glass microtube (inner diameter 0.5 mm, height 7.5 mm) wasfixed to the inside of a 20mL glass test tubemounted in a glass coolingbath to allow for a clear view by a dissection microscope. Microtubeand test tube were filled with nitrate -free artificial seawater andforaminiferans were picked from the sediment, thoroughly cleanedwith a brush, and transferred to the microtube with a pipette. The tipsof the microsensors were first positioned in the microtube about

Fig. 1. Surface of sediment core maintained in the flume. Numerous foraminiferansare sitting at the sediment water interface, arrows point out two individuals. WhiteThioploca filaments are stretching out into the bottom water. Scale bar 500 μm.

Fig. 2. (A) Abundance of calcareous live (Rose Bengal stained) foraminiferans(individuals cm−3) in the sediment at Sta. 18. (B) pH and H2Stot sediment microprofiles,and (C) nitrate microprofile at the sediment-water interface. Bars show standard errorof means (n=3 for all profiles). Notice the different depth scales.

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100 μm above the foraminiferans and concentration gradients abovethe cells were recorded in 250 μm steps. Oxygen respiration rates anddenitrification were measured on 3 replicate batches of N. cf. stellacontaining 5, 3, and 3 specimens. After oxygen profiling, water in themicrotube was exchanged with nitrate- and oxygen-free artificialseawater containing 1 kPa acetylene and a thin suspension of bakersyeast to consume all oxygen (verified by oxygen microsensors).Similar nitrous oxide measurements were performed in control ex-periments with 6 foraminiferans without acetylene, and in a micro-tube with acetylene but without foraminiferans.

In two cases internal nitrate pools were determined after finishingthe respiration measurements. This was done by removing foramini-ferans from the microtube with a pipette and analyzing each indi-vidual as described for nitrate uptake experiments.

3. Results

At the time of sampling, bottom water temperature and salinitywas 11 °C and 34‰, respectively, and no oxygen was detectable(b1 μmol L−1 measured with an oxygen microsensor). The Thioplocabiomass was 2.6 – 3.9 mm3 cm−2 (Holmkvist, L. et al. pers. comm.) andfilaments protruded up to several centimeters out of the sediment(Fig. 1). A 0.5 cm high porous layer of recently settled material wasseen at the top of the sediment, under which a 1 cm black sedimentlayer was followed by gray sediment.

Live (Rose Bengal stained) calcareous foraminiferans were con-centrated in the top 3 mm of the sediment with 190 cells cm−3

declining to less than 5 cells cm−3 below 9 mm depth (Fig. 2A). Depthintegrated standing stock was 280 individuals cm−2 at the site,assuming that foraminiferans living below 18 mm depth did notcontribute significantly. The two species Nonionella cf. stella andStainforthia sp. made up 73% of the stained foraminiferans (for SEMillustrations see, Risgaard–Petersen et al. (2006)). Among theremaining calcareous foraminiferans species of Bolivinitidae consti-tuted 12% of the total. The nitrate accumulating N. cf. stella andStainforthia sp. amounted to 205 individuals cm−2.

Observations with dissection microscope on intact sediment coreskept in theflume revealednumerous live andmotile foraminifera on theporous sediment surface (Fig. 1). They tended to accumulate on top ofsediment aggregates, but not on the protruding Thioploca filaments,probably due to bacterial slime secretion. The sediment surfacewas alsodensely interwoven with a variety of smaller filamentous bacteriadescribed by Gallardo and Espinoza (2007). A pronounced peak of dis-solved sulfide was measured in the upper 15 mm of the sediment(Fig. 2B) indicating high sulfate reduction rates in association with thefresh organic material. Sulfide concentrations were below 100 µmol L−1

in the top 3 mmwhere the majority of the foraminifera resided. Nitratehardly penetrated the sediment surface and the steep gradient in the

diffusive boundary layer just above indicated intense nitrate consump-tion at the sediment-water interface (Fig. 2C).

Nitrate uptake capacity of foraminiferans at the surface of thesediment core, on which the chemical profiling was performed, wasstudied by following the nitrate content of individual N. cf. stella afternitrate starvation. Individuals picked at the end of a period ofapproximately 24 hours with no nitrate in the circulating flumewater showed an average (±SEM) nitrate content of 58±11 pmol (n=6)(Fig. 3). Four hours after nitrate addition, the internal nitrate contenthad increased to an average (±SEM) of 201±49 pmol (n=10), yielding

Fig. 3. Nitrate content in individual N. cf. stella picked from the sediment surface during2 days following nitrate addition to the water after 1 day without nitrate.

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an initial nitrate uptake rate of minimum 864 pmol individual−1 d−1.This corresponds to an internal concentration rate of 7 mmol L−1 h−1

based on an average volume ofN. cf. stella of 5.2×10−3 mm3 (Risgaard–Petersen et al., 2006). In the following hours the uptake rate declinedand an average steady state content of 242±31 pmol (n=29) wasattained.

Oxygen and nitrate respiration were measured on the sameindividuals using batches of the dominant species, N. cf. stella, inmicroprofiling experiments. The rates of oxygen consumption were660, 934, and 687 pmol O2 individual−1 d−1 for the experimentsshown in Fig. 4A, B, and C respectively. Average (±SEM.) oxygenrespiration rate for the 3 replicates was thus 760±87 pmol O2

individual−1 d−1. Nitrous oxide production showed denitrificationrates of 151, 62 and 39 pmol N individual−1 d−1 in A, B, and C, res-pectively (Fig. 4), and the average (±SEM) denitrification rate for the 3replicates was 84±33 pmol N individual−1 d−1. Nitrous oxideproduction was not detectable (concentrations b0.4 µmol N L−1) incontrol experiments without acetylene, which shows that theforaminifera conducted complete denitrification to dinitrogen. Thestoichiometry of organic carbon oxidation associated with eitheroxygen respiration or nitrate reduction to nitrous oxide is 1:1, and therates above therefore directly translates into carbon oxidation rates.

The average internal nitrate contents of the specimens at the end ofthe experiment shown in Fig. 4B and Cwas 172±70pmol nitrate (n=6).

4. Discussion

4.1. Foraminiferan fauna

The total density of calcareous foraminiferanswasonly 10–50%of theobserved densities in the Peruvian (Levin et al., 2002) and other OMZareas (summarized by Gooday et al., 2000). Typically, species richness isvery low in oxygen poor areas with two or three species constituting asmuch as 80% of the fauna (Gooday et al., 2000; Sen Gupta andMachain–Castillo, 1993). This was also the case at the sampled site where the

Fig. 4. Steady state oxygen and nitrous oxide profiles in oxic and anoxic microtubes w

nitrate accumulating species N. cf. stella and Stainforthia sp. dominatedthe species composition (Fig. 2A). Preliminary results suggests thatsome, if not all other species at the site also rely on nitrate accumulation.

Nonionella stella is known to dominate the species composition atsome locations in the Santa Barbara Basin constituting up to 83% of thebenthic foraminiferan fauna (Bernhard and Reimers, 1991) and arealso reported from other low-oxygen settings (Diz et al., 2006).Stainforthia species are highly tolerant to anoxia (Alve, 2003) and theBuliminacea, to which Stainforthia sp. belongs, are found in severallow-oxygen settings (Gooday et al., 2000; Sen Gupta and Machain–Castillo 1993). It is therefore reasonable to assume that dominance ofnitrate accumulating species is a wide-spread phenomenon in OMZenvironments.

The majority of the foraminiferans were found in the surfacehorizon (0–3 mm) which is in close proximity to the bottom-waternitrate source (Fig. 2A and C). Distance to the nitrate source couldrestrict the depth to which these small (∼0.2 mm) organisms are ableto migrate, but other parameters, such as sulfide, may also limit depthoccurrence, since the foraminiferan fauna was found above the zonewith a 230 µmol L−1sulfide peak at 10 mm depth (Fig. 2B).

4.2. Turnover of nitrate and oxygen respiration in foraminiferans

The denitrification activity of N. cf. stella has not yet been traced to aspecific organelle. Diatom chloroplasts occur inN. stella and assimilativenitrate reductase from preserved diatom chloroplasts has been iden-tified and suggested to play a role in foraminiferal nitrogen metabolism(Grzymski et al., 2002). Assimilative pathways, however, are energyconsuming and never lead to dinitrogen production, and are there-fore unlikely to be involved in the dissimilative nitrate reduction offoraminifera. Careful examination of another denitrifying foraminiferan,Globobuliminia pseudospinescens, excluded that denitrification wasassociated with endosymbiontic bacteria (Risgaard–Petersen et al.,2006).

Several observations confirmed that the investigated species werenot affected by the incubation in the flume or in the microtube. Intactsediment cores incubated for weeks in the flume system retained theappearance they had when retrieved from the sea, except that theprotrusion of Thioploca filaments became less pronounced. Cellsretained the internal nitrate storage during collection and rinsingwithbrushes and pipettes. If left over night in dishes, they gatheredsediment particles indicating active pseudopodial network. Whenincubated in microtubes the foraminifera showed constant oxygenand nitrate respiration rates for several hours with no signs of stress.

All analyzed foraminiferal cells contained nitrate and respired at aconstant rate, suggesting that their anaerobic metabolismwas neitherelectron acceptor nor electron donor limited at this time. The obtainedaverage denitrification rate of 84±33 pmol N individual−1 d−1 for N. cf.stella (Risgaard–Petersen et al., 2006) was therefore likely to reflect aconstant respiration in balance with growth and activity of the cells.

ith N. cf. stella lying in the bottom. (A) 5 individuals, and (B) and (C) 3 individuals.

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Despite the normal absence of oxygen in the bottomwater and theinability to store oxygen, N. cf. stella maintained a capacity to respirewith this more potent electron acceptor at a rate of 760±87 pmoloxygen individual−1 d−1, which is about 10 times higher than thedenitrification rate (Fig. 4). Compared to respiration rates measured in5 genera of estuarine foraminiferans of similar size (Hannah et al.,1994), the rates obtained here are two orders of magnitude lower,suggesting that N. cf. stella has a relatively low level of activity.

The minimum potential nitrate uptake rate of 864 pmol Nindividual−1 d−1 was 10 times higher than the continuous con-sumption rate of 84±33 pmol N individual−1 d−1, and the steady-statenitrate storage of about 230 pmol N individual−1 (Fig. 3) wassufficient to feed denitrification for 2.5 days. Nonionella cf. stella thusappeared well adapted to infrequent availability of nitrate in theenvironment. The microsensor profiles showed that nitrate was onlyavailable at the very surface of the sediment (Fig. 2C) where manyforaminiferans were observed to gather (Fig. 1). Other studies havealso shown a linkage between certain foraminiferan species and thenitrate reduction zone which in these cases occur within thesediment (Duchemin et al., 2005, Licari et al., 2003, Jorissen et al.,1998). We suggest that this linkage may not only be due to a par-ticular food source and predation pressure, but also to the availabilityof nitrate as an electron acceptor. Nitrate should therefore be consid-ered a main parameter, in line with oxygen and organic matter,controlling benthic foraminiferan vertical distribution and generalcommunity structure.

Many foraminiferans were observed down to 9 mm depth (Fig. 2A)and there are several, still speculative, ways by which they aquirenitrate. They could transport nitrate down in the sediment by ver-tical migrations like the coexisting bacteria Thioploca (Huettel et al.,1996) and as suggested for the foraminiferan G. pseudospinescens(Risgaard–Petersen et al., 2006). With the ability to transport materialin the pseudopodial network (Cedhagen and Frimanson, 2002) itmight also be possible that the foraminerans use pseudopodia to takeup nitrate at the sediment surface or to even extend the networkfurther out to compete with Thioploca filaments traversing thediffusive boundary layer (Zopfi et al., 2001). A third option at thissite could be parasitizing on the abundant Thioploca filamentscontaining up to 500 mmol L−1 nitrate (Fossing et al., 1995).

The advantages of nitrate storage should be compared with theenergetic cost of accumulating nitrate against a concentrationgradient. The minimum energy required to concentrate a solutex-fold up from ambient concentrations is: log10(x) ×5.7 kJ mol−1

(Harold,1986). The internal nitrate pool inN. cf. stella is concentrated bya factor 3,000 as compared to the surrounding environment (Risgaard–Petersen et al., 2006). This yields an energy cost of 19.8 kJ mol−1 nitrate.If we assume a free energy yield of -566 kJ mol−1 nitrate fromdenitrification (recalculated after Jørgensen, 2000) and subtract 66%lost in ATP formation (recalculated after Jørgensen, 2000 and Spanninget al., 2007), then only about 10% of the energy gained from respirationis used for nitrate accumulation.

The life strategy based on nitrate accumulation and respiration isso far reported for the prokaryotes Thioploca that live side by sidewith the foraminiferans in the OMZ (Fossing et al., 1995), for themore cosmopolitan vacuolated Beggiatoa in oxygen poor sediments(Mussmann et al., 2003), and for the giant unicellular Thiomargarita inthe Namibian OMZ (Schulz et al., 1999). These organisms dominate thebenthic biota in every case and a picture of nitrate accumulation as ageneral solution to successful survival in organic- or sulfide-rich andoxygen-poor environments emerges.

4.3. Denitrification and dissimilative nitrate reduction to ammonium inOMZ sediments

Denitrification performed by foraminiferans and dissimilativenitrate reduction to ammonium carried out by Thioploca have very

different implications for the marine environment, since the firstprocess removes nitrogenwhile the other retains nitrogen as an easilyavailable nutrient. Studies show that reduction of nitrate to ammo-nium dominates benthic nitrate reduction when Thioploca is present(Molina et al., 2004; Zopfi et al., 2001). By their ability to stretch theirlong filaments above the general diffusive boundary layer Thioplocaobtain a very efficient nitrate uptake and apparently oust prokaryoticdenitrification. Very little nitrate thus reaches the sediment surface atlocations with abundant Thioploca biomass (Fig. 2C) (Zopfi et al.,2001). This implies that the foraminiferal mediated denitrificationmay constitute a significant fraction of the benthic dinitrogenproduction in the OMZ.

Denitrification by Stainforthia sp. was not measured directly, butbased on the similar depth distribution (Fig. 2A) and nitrate content(Risgaard–Petersen et al., 2006), we assume that they denitrified likeN. cf. stella and with the same average individual rate. Multiplying thecombined abundance of the nitrate accumulating Stainforthia sp. andN. cf. stella with the average denitrification rate, we estimate a totalforaminiferal mediated denitrification of 173 µmol N d−1 m−2. Thisestimated rate is considered conservative since it is likely to findmoredenitrifying species among the remaining 27% of the foraminifera atthe site. The estimate corresponds to the denitrification rate of250 µmol N d−1 m−2 measured in January 1999 in the Bay ofConcepción (Graco et al., 2001). Higher denitrification rates up to2900 µmol N d−1 m−2 have, however, also been measured in the bay(Farias et al., 2004). These rates are still small compared to the nitrateuptake of more than 20000 µmol N d−1 m−2 reported from the samearea, which contains dense populations of Thioploca (Huettel et al.,1996). Thioploca populations are diminished by several orders ofmagnitude during El Niño events, and they seem to take years torecover (Schulz et al., 2000). Foraminiferans may flourish in theseperiods with low Thioploca biomass, thereby causing a shift fromammonium to dinitrogen as the major end product of nitratereduction. Complementary spatial distribution of foraminiferans andThioploca has actually been observed in the OMZ off Pakistan, whereforaminiferal densities increased by a factor of 4 by moving only 1.5 mfrom the inside to the outside of a sulfur bacterial mat, with largely thesame foraminiferan species at both locations (Erbacher and Nelskamp,2006). Variations in benthic communities may thus be the majordeterminant for nitrogen recycling or removal in OMZ sediments.

4.4. Implications to measures of marine denitrification

The foraminiferan storage and transportation of nitrate in thesediment challenges our measurements of marine denitrification. Thecommonly used isotope pairing technique requires homogenousmixing of added 15NO3

− into the nitrate pool being denitrified(Groffman et al., 2006). This also applies to the internally storednitrate, and the incubation time therefore has to exceed the turnovertime of the internal nitrate pool, which is a couple of days (this study)or even weeks (Risgaard–Petersen et al., 2006). This may explainthe observations made by Farias et al. (2004), who investigatedthe seasonal benthic nitrogen turnover at the same site (Sta. 18) in1998–2000 when the population of Thioploca had not yet recoveredsince the 1997–98 El Niño event. They noticed that other processesthan those driven by Thioploca and other known nitrate reducingbacteriawere required to fully explain the large sediment nitrate poolsand the imbalances between nitrate fluxes, nitrification, and deni-trification rates. We suggest, that denitrifying foraminiferans were theunknown actors which accounted for most of the sediment nitratepool at that time, and which caused significant underestimation of thedenitrification rate by the applied short term isotope pairingtechnique. The foraminiferan population should have been about 10times larger than observed in the present study to account forthe imbalance. This is not unrealistic considering the minimal com-petition from Thioploca at that time and reports of such high

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foraminiferan population densities at other OMZ locations (Goodayet al., 2000; Levin et al., 2002).

The acetylene block technique worked well for measuringdenitrification rates of isolated cells in this study, but failed to detectany denitrification activity in intact sediment cores that we sampled atthis location at a time with similar benthic conditions and Thioplocaabundance (b40 µmol N m-2 d-1; N. P. Revsbech, unpubl. data, 1993).This is explained by the fact, that the acetylene block is inefficient insulfidic environments (Groffman et al., 2006).

Direct measurements of dinitrogen flux will probably be a betterchoice for determination of total benthic dinitrogen productionincluding the contribution from foraminiferans (Groffman et al,2006). Due to the deep occurrence of some denitrification activity,however, long incubation times of sediment cores may be necessary tore-establish a stable situation where the sediment-water dinitrogenflux matches the dinitrogen production rate. Alternatively, thedinitrogen flux could be measured in situ using benthic chambers.

A recent model study suggests that marine sediments beyond thecontinental shelf are much more important global nitrogen sinks thanpreviously assumed (Middelburg et al., 1996). The model is based onreaction-transport processes driven by organicmatter input and bottomwater concentrations of oxygen and nitrate. Active uptake and transportof nitrate by foraminiferansmeans that denitrificationmayaccount for alarger part of the anaerobic degradation than themodel predicts. To theextent that foraminiferans appear to be important denitrifiers incontinental slopes and abyssal sediments, the benthic nitrogen sinkmight therefore need to be adjusted further upwards. Areal denitrifica-tion estimates for the Peru-Chile OMZ based on stoichiometricapproaches and mass balances (Codispoti et al., 2001) should not needrevision, even if benthic denitrification appears not to be dominated byprokaryotes but by foraminiferans as this study indicates.

Acknowledgements

We thank B. B. Jørgensen for organizing and leading the 2006Thioploca–Chile Expedition and all the participants for help andsupport. J. G. Kuenen, J. L. Nielsen, and N. Risgaard–Petersen contrib-uted with suggestions and help in the laboratory. The staff of DichatoMarine Biological Station and the crew of R/V Kay Kay of theUniversity of Concepción are thanked for their help and hospitality.Anonymous reviewers are thanked for comments improving thepaper. This work was supported by the Max Planck Gesellschaft andthe Danish Natural Science Research Council grant 21–03–0500. [SS]

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