Anammox bacteria and the anaerobic oxidation of ammonium in the oxygen minimum zone off northern Chile

ARTICLE IN PRESS Deep-Sea Research II 56 (2009) 1021–1031

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Anammox bacteria and the anaerobic oxidation of ammonium in the oxygen minimum zone off northern Chile Alexander Gala´n a,b, Vero´nica Molina b, Bo Thamdrup c, Dagmar Woebken d, Gaute Lavik d, Marcel M.M. Kuypers d, Osvaldo Ulloa b, a

´n, Casilla 160-C, Chile Programa de Doctorado en Oceanografı´a, Departamento de Oceanografı´a, Universidad de Concepcio ´fica en el Pacı´fico Sur-Oriental (FONDAP-COPAS), Universidad de Concepcio ´n Oceanogra ´n, Cabina 7-PROFC, Departamento de Oceanografı´a and Centro de Investigacio ´n, Chile Casilla 160-C, Concepcio c Nordic Center for Earth Evolution, Institute of Biology, University of Southern Denmark, DK-5230 Odense, Denmark d Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, D-28359 Bremen, Germany b

a r t i c l e in fo

abstract

Article history: Accepted 25 September 2008 Available online 5 November 2008

Anammox is the anaerobic oxidation of ammonium by nitrite or nitrate to yield N2. This process, along with conventional denitrification, contributes to nitrogen loss in oxygen-deficient systems. Anammox is performed by a special group of bacteria belonging to the Planctomycetes phylum. However, information about the distribution, activity, and controlling factors of these anammox bacteria is still limited. Herein, we examine the phylogenetic diversity, vertical distribution, and activity of anammox bacteria in the coastal upwelling region and oxygen minimum zone off northern Chile. The phylogeny of anammox bacteria was studied using primers designed to specifically target 16S rRNA genes from Planctomycetes in samples taken during a cruise in 2004. Anammox bacteria-like sequences affiliated with Candidatus ‘‘Scalindua spp.’’ dominated the 16S rRNA gene clone library. However, 62% of the sequences subgrouped separately within this cluster and together with a single sequence retrieved from the suboxic zone of the freshwater Lake Tanganyika. The vertical distribution and activity of anammox bacteria were explored through CARD-FISH (fluorescence in situ hybridization with catalyzed reporter deposition) and 15N labeling incubations, respectively, at two different open-ocean stations during a second cruise in 2005. Anammox bacterial CARD-FISH counts (up to 3000 cells ml1) and activity (up to 5.75 nmol N2 L1 d1) were only detected at the station subjected directly to the upwelling influence. Anammox cell abundance and activity were highest at 50 m depth, which is the upper part of the OMZ. In this layer, a high abundance of cyanobacteria and a marked nitrogen deficit were also observed. Thus, our results show the presence of a new subcluster within the marine anammox phylogeny and indicate high vertical variability in the abundance and activity of anammox bacteria that could be related to an intensification of carbon and nitrogen cycling in the upper part of the OMZ. & 2008 Elsevier Ltd. All rights reserved.

Keywords: Anammox Denitrification Oxygen minimum zone Eastern tropical South Pacific

1. Introduction The eastern tropical Pacific and the Arabian Sea are regions of intense upwelling and high productivity, with low ventilation rates and a high biological demand for oxygen at intermediate depths. These conditions allow the development of permanent oxygen minimum zones (OMZs; Deuser, 1975; Kamykowski and Zentara, 1990; Helly and Levin, 2004), which are thought to be responsible for 30–50% of the total nitrogen loss from the ocean, with heterotrophic denitrification being the commonly considered nitrogen loss mechanism (Gruber and Sarmiento, 1997; Codispoti et al., 2001). However, since the discovery of

 Corresponding author. Tel.: +56 41 220 3585; fax: +56 41 223 9900.

E-mail address: [email protected] (O. Ulloa). 0967-0645/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.dsr2.2008.09.016

anammox (Mulder et al., 1995; van de Graaf et al., 1995), several reports have partially attributed the fixed nitrogen loss to this process (Dalsgaard et al., 2003; Kuypers, et al., 2003, 2005). Recently, 15N-incubations were used to demonstrate that in the OMZs off northern Chile and Peru and in the shelf waters off Namibia, nitrogen was principally removed by the anammox process, whereas heterotrophic denitrification was generally not detected (Kuypers et al., 2005; Thamdrup et al., 2006; Hamersley et al., 2007). Considering these results and the widespread presence of anammox bacteria in diverse marine environments, i.e., anoxic basins, sediments, and even sea ice (Dalsgaard and Thamdrup, 2002; Dalsgaard et al., 2003; Kuypers et al., 2003; Trimmer et al., 2003; Risgaard-Petersen et al., 2004; Rysgaard and Glud, 2004), it appears that anammox contributes substantially (up to 50%) to nitrogen removal from the ocean (Devol, 2003).

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Anammox bacteria are chemolithoautotrophic microorganisms that oxidize ammonium with nitrite (or nitrate) as the electron acceptor to obtain energy to fix CO2, thereby producing dinitrogen gas (Jetten et al., 2001). These metabolically versatile bacteria are, for example, capable of oxidizing short chain fatty acids with nitrate (Gu¨ven et al., 2005), co-oxidizing propionate and ammonium in the presence of nitrite and nitrate (Kartal et al., 2007a), and performing dissimilatory nitrate reduction to ammonium (Kartal et al., 2007b). In addition, anammox bacteria were recently shown to be able to tolerate higher O2 concentrations than originally established by Strous et al. (1997), being metabolically active at oxygen concentrations of up to 13 mmol O2 L1 (Jensen et al., 2008). Altogether, these results have important ecological and biogeochemical implications, since they extend the metabolic and environmental spectra of these bacteria. Anammox bacteria have an internal ribosome-free compartment, or anammoxosome, where the catabolism of anammox is assumed to take place. This physical feature results in a ringshaped fluorescence in situ hybridization (FISH) signal, which aids in cell recognition (Strous et al., 1999). The anammox process is carried out by bacteria belonging to a deep-branching monophyletic group within the Planctomycetes phylum, and can thus be studied through 16S rRNA gene surveys (e.g., Schmid et al., 2005, 2007). In fact, several 16S rRNA gene studies have been carried out in very diverse marine and estuarine environments (Bowman and McCuaig, 2003; Kuypers et al., 2003, 2005; Risgaard-Petersen et al., 2004; Tal et al., 2005; Kirkpatrick et al., 2006; Penton et al., 2006; Hamersley et al., 2007). Independent of the environment studied, 16S rRNA anammox sequences reveal low diversity and a close relationship to Candidatus ‘‘Scalindua sorokinii’’, the first tentative species identified in the water column of the Black Sea (Kuypers et al., 2003). One of the most intense OMZs is found in the eastern tropical South Pacific (ETSP; Morales et al., 1999), especially off northern Chile, a center of persistent coastal upwelling, where the continental shelf is narrow and oxygen-depleted waters can reach up into the photic zone, albeit with significant temporal variability in their depth distribution (Morales et al., 1999; Ulloa et al., 2001). We explored anammox bacteria richness in the Chilean OMZ for the first time through a phylogenetic analysis of the 16S rRNA genes. Also, we evaluated the anammox bacteria distribution and activity under two contrasting hydrographic conditions of this upwelling region through CARD-FISH and isotope-pairing techniques and explored its potential relationship with environmental variables.

2. Experimental procedures 2.1. Study area and water sampling The sampling sites are situated in a center of persistent coastal upwelling at the southern part of the ETSP OMZ (off Iquique 201S). The data were collected during two cruises: PRODEPLOY (July 2004; R/V Carlos Porter) and ZOMEI (September 2005; R/V Vidal Gormaz). In addition, bacterioplankton abundances were determined for the DINAMO cruise in order to compare our results with the anammox activities previously measured in the study area (for details about the DINAMO cruise, see Thamdrup et al., 2006). Vertical casts for hydrographic data were obtained using a rosette system equipped with a CTD (conductivity–temperature–depth) outfitted with previously calibrated oxygen (Seabird 23B Electronics, Bellevue, USA; accuracy at saturation levels under 2%: 2 mmol O2 L1) and fluorescence sensors (SeaBird Electronics, Bellevue, USA) and 12 8-L Niskin bottles. The samples for nutrient analyses were filtered and frozen until

laboratory analysis. Samples (2 ml) to estimate bacteria and cyanobacteria numbers were fixed with fresh glutaraldehyde (0.1% final concentration), incubated at room temperature for 10 min and stored at 80 1C until flow cytometry analysis. For CARD-FISH, water sample profiles were fixed with paraformaldehyde (final concentration 1%) and stored at 4 1C for at least 4 h. Thereafter, aliquots of 25, 50, and 100 ml of water from each depth were filtered through 0.2-mm polycarbonate filters (Millipore, 45 mm diameter), supported by filters of cellulose nitrate (0.45 mm), and stored frozen at 20 1C until further analysis. In addition, water samples for 15N-incubations were retrieved in a 30-L GO-FLO bottle from depths of 50 and 60 m for the ZOMEI cruise only. In general, during all water collections, N2 was injected into the headspace of the bottles (Niskin, GO-FLO) to avoid oxygenation of the samples from the suboxic depths. Finally, seawater (10 L) for DNA extraction was collected at 2, 10, 20, 40, 50, 60, 80, 100, 200, 300, and 400 m depths, pre-filtered through 3-mm pore-size polycarbonate filters, and filtered onto 0.2-mm pore-size membrane filters (Gelman, diameter 47 mm) with a gentle vacuum. The filters were immediately soaked in 1 ml of autoclaved DNA buffer (50 mM Tris-HCl (pH 9.0), 0.75 M sucrose, and 400 mM NaCl) and stored in liquid nitrogen until DNA extraction. 2.2. Nutrient analyses Ammonium was measured fluorometrically by using the orthophthaldialdehyde (OPA) method (Holmes et al., 1999). NO 2 was determined spectrophotometrically by manual colorimetric analyses (Bendschneider and Robinson, 1952). NO 3 was measured as NO 2 after reduction by a copper–cadmium column (Grasshoff and Koroleff, 1983). 2.3.

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N-labeling incubations and analysis

N incubations were performed as described in Thamdrup et al. (2006). Briefly, 250 ml of water from specific depths were purged with helium for 15 min, immediately after adding 15Nlabeled and unlabeled nitrogen compounds. The final concentrations by tracer for each treatment were: 5 mM 15NH+4, 10 mM 15NO 3, 15 14 NO NH+4. The water was 5 mM 15NO 2 , and 5 mM 2 +5 mM dispensed into 12.6-ml glass vials (Exetainers, Labco, UK) and incubated at the in situ temperature (13 1C) for up to 48 h. Triplicate samples were taken after 0, 6, 15, 24, and 48 h by withdrawing 5 ml of water while replacing it with helium. Zinc chloride was injected to stop biological activity and vials were stored upside down for N2 analysis; 5 ml of water were withdrawn  + and used for analysis of NO 2 , NO3 , and NH4. The nitrogen isotopic composition of N2 (15N14N:14N14N and 15N15N:14N14N ratios) was determined by gas chromatography isotope-ratio-monitoring mass spectrometry (GC-irm-MS) using air as the standard. The excess 15N14N and 15N15N concentrations over time were calculated from the linear regression slopes as described previously (Thamdrup and Dalsgaard, 2002). In order to establish the error associated with the rate measurements, the replicates for one incubation experiment (15NH+4) were processed and found to be in the same range as previously reported and using the same GC-irm-MS (Kuypers et al., 2005; Hamersley et al., 2007). The stoichiometry of anammox for N2 production combines 1:1 atoms from NH+4+NO 2 (van de Graaf et al., 1995; Jetten et al., 2001). In the 15NH+4+14NOx incubations, the production of only 15 14 N N (29N2) rather than 15N15N (30N2) is a clear evidence of anammox activity. However, N2 must accumulate linearly without a lag phase, indicating that no intermediates (such as NO 2 or 15 NOx NO 3 ) are involved in the reaction. In the incubations with and 14NH+4 as the only sources of nitrogen, anammox and

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denitrification can be measured simultaneously, where 29N2 production is indicative of anammox and 30N2 production is indicative of denitrification. Thus the ratio of anammox to total N2 production can be calculated as the ratio of 29N2 to 29N2+30N2 (Thamdrup and Dalsgaard, 2002). 2.4. Cytometry and CARD-FISH Bacterioplankton (bacteria+archaea) and cyanobacteria (discriminated on the basis of their scatter signal, an index of cell size, and natural fluorescence) were counted using a Becton Dickinson, FACSCalibur (CA, USA) flow cytometer (flow rate: 30–40 ml min1; 410,000 events counted). The bacterioplankton abundance was measured using yellow-green fluorescence after nucleic acid staining with SYBR-I Green (530 nm). Forward scatter, side scatter, orange fluorescence from phycoerythrin (585 nm), and red fluorescence from chlorophyll (4650 nm) were measured after laser excitation (488 nm) for estimates of cyanobacterial abundance (Marie et al., 2000). A p5% error was associated with the picoplankton abundance determined using flow cytometry (D. Marie, unpublished data). Due to the high abundance of cells with autofluorescence (Fig. 4) in the particulate organic matter (mainly cyanobacteria, as confirmed by flow cytometry) (Fig. 2G–I), it was impossible to distinguish the anammox signal after a monolabeled FISH stain. Consequently, specific probes were used to detect and count anammox bacteria by CARD-FISH, following the protocol described by Pernthaler et al. (2002) with modifications by Woebken et al. (2007). The probes were BS820 (50 -TAATTCCCTCTACTTAGTGCCC-30 ) (Kuypers et al., 2003) and BS820C (50 -TAATCCCCTCTACTTAGTGCCC-30 ) (Hamersley et al., 2007) in addition to the unlabelled probe Amx820 used as a competitor (50 -AAAACCCCTCTACTTAGTGCCC-30 ; Schmid et al., 2000). The relative abundance of anammox cells was calculated in triplicate after enumerating the total picoplankton by 4,6-diamidino-2-phenylindole (DAPI) staining (Porter and Feig, 1980) under an epifluorescence microscope (Zeiss Axioplan 2) equipped with a 100-W Hg lamp and filter sets for DAPI, Cy3, and FLUOS. A comparison between DAPI and flow-cytometer estimates on data obtained during this study (linear regression) agreed well (R240.8) with the bacterioplankton counts (data not shown).

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2.6. Polymerase chain reaction Polymerase chain reaction (PCR) amplifications from total environmental DNA were performed using the forward primer Pla46 (Neef et al., 1998) and the universal reverse primer 1492 (Crump et al., 1999). The amplifications followed the previously described procedure by Schmid et al. (2000) with the following modifications. Reactions were carried out in 25 ml of 1X PCR buffer (50 mM KCl, 10 mM Tris-HCl, and 1% Triton X-100) containing 1.5 mM of MgCl2, 200 mM of each deoxynucleoside triphosphate, 0.2 mM of each primer, 1 U of Taq DNA polymerase, and 1 ml of template DNA (10 ng). Thermal cycling was carried out with an initial denaturation step of 5 min at 94 1C, followed by 35 cycles of denaturation at 94 1C for 1 min, annealing at 56 1C for 1.5 min, and elongation at 72 1C for 2 min, with no final elongation step. Negative and positive controls were carried out in parallel to each reaction. Amplification products were evaluated (presence, size) by agarose (1%) gel using standard electrophoresis procedures.

2.7. Cloning and sequencing Anammox-specific PCR products were cloned by using a pGEM-T Easy Vector Systems and the JM109 high efficiency competent cells provided with the cloning kit (PROMEGA, Madison, USA). Inserts of the correct size were evaluated by PCR reactions using M13 PUC f–r vector-specific primers (Messing, 1983). Positive products were sequenced using M13 PUC f–r primers in the ABI 3730XL (ABI, Applied Biosystems) genetic analyzer by MACROGEN Inc. (Korea). To obtain high-quality sequences with a full overlap, the clones were sequenced with the following internal primers: BS820Gf (50 -GGGCACTAAGTAGAGG-30 ), BS820G (50 -CCTCTACTTAGTGCCC-30 ) (modified from Kuypers et al., 2003), and Brod541Gf (50 -GAGCACGTAGGT GGGTTTG-30 ) (modified from Penton et al., 2006). Consensus sequences were constructed by the overlap of sequences, obtained from M13 and BS820 forward and reversed and Brod541 forward using SeqMan (DNASTAR-Lasergene v6) software, after correcting the alignments by visual inspection. Chimera checks were done using Bellerophon (http://35.8.164.52/cgis/chimera.cgi?su=SSU) and CHECK-CHIMERA from the Ribosomal Database Project II (http://rdp.cme.msu.edu/).

2.5. DNA extraction 2.8. Phylogenetic analyses DNA extraction was modified from West and Scanlan (1999), including a physical breakage step according to Stevens et al. (2005) as follows: small pieces of filters were immersed in 3 ml of lysis solution (45 mM glucose, 23 mM Tris (pH 8.0), and 59 mM EDTA) with lysozyme (1 mg ml1). Samples were incubated at 37 1C for 1 h, frozen at 20 1C for 15 min, and thawed at 50 1C. These were then amended with 50 ml of proteinase K (10 mg ml1) and 350 ml of 10% sodium dodecyl sulfate and incubated at 37 1C for 30 min and at 55 1C for 10 min. The DNA was extracted by adding a volume of water-saturated phenol–chloroform–isoamyl alcohol (25:24:1) and centrifuged at 4000 rpm (5 min). The aqueous phase was removed and chloroform–isoamyl alcohol (24:1) was added again and the sample was centrifuged at 4000 rpm (5 min). The DNA was precipitated with 0.4 volume of sodium acetate (7.5 M) and 2 volume of cold ethanol (95%). Samples were incubated at 80 1C (20 min) and centrifuged at 13,000 rpm (10 min). Finally, the DNA pellet was cleaned with cold ethanol (70%), centrifuged at 13,000 rpm (10 min), and dried at room temperature (15 min). The DNA was resuspended in 50 ml of TE (50 mM Tris-HCl (pH 8.0), 10 mM EDTA) and stored at 80 1C. The DNA quality was checked in agarose gel.

Phylogenetic analyses were performed with the Phylip software package (Felsenstein, 1993), available from http://evolution.genetics.washington.edu/phylip/software.html, and the MEGA version 3.0 software package (Kumar et al., 2004), available from http://www.megasoftware.net/. The phylogenetic analyses were based on maximum likelihood (ML) and distance matrix methods (neighbor joining, NJ). Topologies were statistically evaluated by bootstrapping (100  resampling). The different phylogenetic analyses were compared visually and a consensus tree (Ludwig et al., 1998) was created based on the ML method. Multifurcations were generated when inconsistencies were found between ML and NJ results, following Ludwig et al. (1998). The consensus tree was drawn using the program Bosque (Ramı´rez-Flandes and Ulloa, 2008).

2.9. Nucleotide sequence accession numbers Sequences are published in GenBank under accession numbers EU039834–EU039866.

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3. Results The abundance, diversity, and activity of anammox bacteria were investigated using samples from two cruises to the OMZ off Iquique, northern Chile. During the PRODEPLOY cruise (July 2004), a profile of water samples was retrieved for DNA extraction; this was used for a qualitative analysis through PCR amplifications (Fig. 2J). After positive amplifications at several depths, one depth was chosen (50 m) to make a Plantomycetales-specific 16S rRNA gene clone library and to carry out a phylogenetic analysis of the anammox community. In September 2005, we revisited the study area (ZOMEI cruise) to evaluate the anammox process using stable isotopic techniques and to quantify the concentration of anammox bacteria in the water column using CARD-FISH at two sampling stations.

3.1. Hydrography The PRODEPLOY station (Pro2; 701170 W, 201170 S) was inside an upwelling zone at 15 km off the coast. The ZOMEI cruise covered one northern station (EW4; 701380 W, 201010 S) with upwelling influence and one southern station (EW9; 701330 W, 211000 S) outside the upwelling influence; both were 50 km off the coast (Fig. 1). Except for the bathymetric differences (Fig. 1), the biogeochemical variables in the water column were very similar between the stations for both cruises (Fig. 2). Station Pro2 showed a sharp oxycline with O2 concentrations dropping vertically from 275 mM in the mixed layer (10 m) to 12.0 mM at the upper OMZ boundary (50 m water depth). The O2 distribution at the ZOMEI stations was similar to that of Pro2, but the O2 dropped from 230 (nearly 100% saturation) to 6.0 mM (nearly 2% saturation) between the mixed layer (10–20 m) and the upper OMZ boundary (50 m). In both cruises, the vertical O2 distribution below the oxycline remained nearly constant (under the detection limit, o2 mM O2) until 400 m, the base of the suboxic zone (Fig. 2A–C). In general, the vertical NO 2 distribution showed a broad maximum within the OMZ, with concentrations up to 10 mM at

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3.2. Cytometry and CARD-FISH The bacterioplankton abundance had a multimodal distribution during both cruises (Fig. 2G–I), with a maximum in the top 20-m layer, a secondary peak just below the oxycline (upper OMZ boundary; 50 m) associated with an SFM at stations Pro2 and EW4, and a broad maximum within the OMZ. The bacterial abundance was higher (430%) for EW4 than for the station outside the upwelling influence (EW9). The cyanobacteria distribution and abundance also showed differences between both cruises (Fig. 2G–I). The differences in cyanobacteria abundance between the stations were more conspicuous than for bacteria, being 95% higher at EW4 than at EW9. The presence of anammox bacteria was restricted to the suboxic zone and to the station directly influenced by coastal upwelling (EW4). An anammox density maximum of 3000 cells ml1 (0.45% of the total number of DAPI-stained cells) was found in the upper part of the OMZ (50 m depth), where it coincided with the SFM. The anammox distribution showed a vertical gradient to the OMZ core (Fig. 2K) with cell numbers decreasing to 450 cells ml1 (0.17% of total number of DAPIstained cells) at 300 m. Representative photomicrographs of hybridizations with anammox-specific probes and autofluorescence cells are shown in Fig. 4.

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100 m and similar levels at 150–200 m. A NO 3 maximum (410 mM) was associated with the upper OMZ boundary. Ammonium concentrations were low and remained almost constant throughout the suboxic zone (from the detection limit of 0.01–0.03 mM), presenting maxima of 0.8 and 1.8 mM at oxic depths (20 and 30 m) for the PRODEPLOY and ZOMEI stations, respectively. The deficit in dissolved inorganic nitrogen (DIN), expressed as N* (Gruber and Sarmiento, 1997), showed strong DIN removal within the OMZ during both cruises. At the Pro2 station, N* peaked at 25.5 mM at 40 m, whereas at the ZOMEI stations, the maxima were 20.8 mM at 75 m (EW4) and 16.7 mM at 40 m (EW9) (Fig. 2D–F). The vertical distribution of physical and chemical parameters in the upper OMZ can be divided into three layers (Fig. 3; see also Farı´as et al., 2007). In the first layer (oxycline), located between the base of the mixed layer and the upper OMZ boundary (25.6osto26.0 kg m3), reduction of the high NO 3 pool begins, whereas NO 2 remains low and O2 falls from 250 to 10 mM. The second layer or transition zone is the upper OMZ boundary (26.1osto26.2 kg m3). This thin layer is characterized by the beginning of the accumulation of NO 2 , a marked nitrogen deficit, and sometimes by the presence of a secondary fluorescence maximum (SFM). The third layer is the OMZ core (st426.2 kg m3), where the O2 concentration remains almost constant (O2 o10 mM). In this layer, NO 3 continues to decrease, whereas a large NO 2 accumulation is observed. Anammox bacteria, when detected by CARD-FISH, were found exclusively in the two deepest layers, but their cell abundances and activity were highest in the second layer (i.e., just below the upper oxycline).

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Fig. 1. Study area and location of sampling stations in the oxygen minimum zone (OMZ) off northern Chile.

N-labeling anoxic incubations were performed to estimate anammox activity in the suboxic zone during the ZOMEI cruise with water taken from two depths at station EW4 (50 and 60 m) and from one depth (50 m) at EW9. Anaerobic ammonium oxidation was only detected at 50 m at station EW4 in anoxic 15 14 + NO incubations with 15NH+4, 15NO 3 , and 2 + NH4 but not  15 with NO2 , as indicated by a significant production of 14N15N (the slope of the linear regression significantly differed from 0,

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Fig. 2. Vertical distribution of physical and chemical variables (mean7SD), abundance of bacterioplankton and anammox cells (mean7SD), and depths for PCR screening. (A–C) Fluorescence (green diamonds), temperature (red line), oxygen (dotted blue line), and salinity (black line); (D–F) nitrite (green triangles), nitrate (blue squares), ammonium (red diamonds), and N* (dotted line); (G–I) abundance of bacterioplankton by flow cytometry (blue circles) and cyanobacteria (Prochlorococcus: red squares; Synechococcus: green diamonds); (J–L) abundance of total picoplankton, DAPI-stained (blue circles), anammox bacteria (green diamonds), and sampling depths at which PCR screening was carried out (black stars) and the Plantomycetales-specific 16S rRNA gene clone library was constructed (red star) for the PRODEPLOY cruise (Pro2) (A, D, G, J), and at the northern (EW4) (B, E, H, K) and southern stations (EW9) (C, F, I, L) of the ZOMEI cruise. Note axes have different scales.

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Fig. 3. Schematic model for the distribution of anammox cells (red line) at the upper OMZ boundary based on the distribution of physical and chemical parameters.

p o0.05) (Fig. 5). In contrast, the production of 14N15N at 60 m was not statistically significant, and no detectable 15N15N production occurred in any of the 48-h 15N incubations for either station. The absence of 15N15N production and the linear accumulation of 14 15 N N without an observable lag phase (which means that labels were transformed directly to N2 without extracellular intermediates) indicate that anammox was the active process. However, we cannot exclude the possibility of shorter lag-phases of minutes to a few hours. The anammox rates ranged from 2.1170.1 nmol N2 L1 d1 (15NH+4 incubation; detection limit of 0.5 nmol L1 d1) + 14 to 5.75 nmol N2 L1 d1 (15NO 2 + NH4 incubation; detection limit 1 1 of 0.5 nmol L d ), considering all different tracers. On the other hand, no significant changes were observed in the concentration of nitrite, nitrate, or ammonium over the incubation time (data not shown). This is consistent with the fact that the anammox rates obtained in this study would not lead to detectable changes in the ammonium or nitrite concentrations.

3.4. Sequence and phylogenetic analyses The screening of the 16S rRNA gene clone sequences against the NCBI database showed that 48% of the clones (n ¼ 80)

matched uncultured sequences related to anammox bacteria, mainly Candidatus ‘‘Scalindua sorokinii’’ (95–97% identity). Most of the non-anammox bacteria-like sequences were related to uncultured Planctomycetes and g-Proteobacteria (data not shown). The comparison of anammox bacteria-like high quality sequences (1458 bp) from the study area indicated the presence of two operational taxonomic units (OTUs) that were about 3% different (41 nucleotide substitutions). The phylogenetic relationships of the anammox bacteria-like 16S rRNA genes from the study area are presented in a consensus tree (Fig. 6). Candidatus ‘‘Scalindua spp.’’ sequences were clearly separated from the rest of the anammox candidate genera (Anammoxoglobus, Brocadia, Jettenia, Kuenenia) in both of the phylogenetic approaches used: NJ and ML with bootstrap values of X99%. All the anammox bacteria-like sequences retrieved from the study area fell within the Candidatus ‘‘Scalindua spp.’’ cluster. A large fraction of these sequences (62%) grouped in a cluster together with a single sequence derived from the suboxic zone of Lake Tanganyika (DQ444400). This subcluster was supported by bootstrap values (X76%) with all the phylogenetic methods used (see Fig. 6). The rest of the sequences (38%) were more diversified and distributed within the Candidatus ‘‘Scalindua’’ cluster (see Fig. 6).

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Fig. 4. Epifluorescence micrographs stained with CARD-FISH. Hybridization with the anammox-specific probe mix BS-820 and BS-820-C. The left panel shows autofluorescent cells in red (arrows). In the middle panel, anammox bacteria are shown in green (circles). The right panel shows combined micrographs; in blue: DNAcontaining cells after stain with DAPI; in red: autofluorescent cells; in green: anammox cells stained with fluorescein-labeled tyramide. Note that turquoise cells are anammox bacteria stained by both dyes.

4. Discussion

et al., 2007). Thus, further studies on the diversity and genomics of natural anammox communities are required.

4.1. Anammox bacteria community structure in the ETSP-OMZ The 16S rRNA-gene analyses revealed that anammox bacteria-like sequences represent an important proportion (48%) of our Planctomycetes-specific clone library constructed with DNA obtained from the upper part of the OMZ. Members of the anammox group were also retrieved from the OMZ in the study area in a general diversity study of bacterial 16S rRNA genes (Stevens and Ulloa, 2008), indicating that they are a characteristic component of the total OMZ bacterial community. All the retrieved anammox bacteria-like sequences fell within the Candidatus ‘‘Scalindua’’ cluster, as in many other marine systems (e.g., Schmid et al., 2007). However, apart from finding sequences closely related to the widespread marine uncultured Candidatus ‘‘Scalindua spp.’’ cluster, we found a distinct subcluster sharing only 96% similarity with Candidatus ‘‘Scalindua brodae’’ (95.8–96.3%) and Candidatus ‘‘Scalindua sorokinii’’ (95.7–96.3%). An unexpected result was that members of this more distantly related anammox clade formed a cluster together with a single sequence obtained from the shallowest part of the suboxic zone of the freshwater lake Tanganyika (100–110 m; Schubert et al., 2006), with which they were highly similar (98.5–98.8%). The presence of this ‘‘novel subcluster’’ suggests that the upper boundary of the OMZ off northern Chile holds a more diverse anammox community than that reported so far for other aquatic systems. These results have physiological implications, since small differences in the 16S rRNA gene sequences (o3%) might be linked to great variations at the genomic level, as demonstrated, for example, for different strains of the cyanobacterium Prochlorococcus (Dufresne et al., 2003; Rocap et al., 2003; Kettler

4.2. Anammox bacterial activity in the ETSP-OMZ The anaerobic incubations of water samples using 15NH+4 showed a linear production of 14N15N (Fig. 5A) in samples from the upper part of the OMZ (50 m). This linearity indicates that anammox bacteria were active from time 0, suggesting that they are also active in situ. The absence of 15N15N production indicates 14 NO that 15NH+4 was oxidized with 14NO 2 and/or 3 and not through another non-nitrogenous oxidant agent (Murray et al., 1995), consistent with the anammox pathway. In addition, the apparent lack of a lag phase excludes the possibilities that the 15N in N2 comes from a possible coupling between anammox and nitrifier–denitrifier organisms. Such coupling would require that 15 NH+4 oxidation) be mixed with the the 15NO 2 (originated from  huge NO2 ambient pool; therefore, the lag phase would be many days. The dominance of 14N15N over 15N15N production in the + 15 14 NO anaerobic incubations with 15NO 3 and 2 + NH4 supported the anammox activity (Fig. 5B and C, respectively), which ranged from 2.11 to 5.75 nmol N2 L1 d1. 15NH+4 and 15NO 3 incubations resulted in similar anammox rates (2.11 and 2.41 nmol N2 L1 d1, respectively). The anammox rates obtained in this study are in the lower range of those reported for the upwelling area of the eastern South Pacific, that is 4.8–16.8 nmol L1 d1 at the same location (Thamdrup et al., 2006) and 1.5–384 nmol L1 d1 off Peru (Hamersley et al., 2007), as well as for coastal marine areas like Golfo Dulce and the shelf waters of the Benguela upwelling system and Lake Tanganyika (9.6–480 nmol L1 d1) (Dalsgaard et al., 2003; Kuypers et al., 2005; Schubert et al., 2006).

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Fig. 6. Consensus phylogenetic tree based on the maximum likelihood method and X700 nucleotides of the 16S RNA gene related to the anammox bacteria sequences retrieved from the study area and representatives of this group. Sequences from this study are depicted in bold. Dots on nodes indicate bootstrap (100 resamplings) values higher than 70% obtained with both treeing methods (maximum likelihood, ML; neighbor joining, NJ). Nitrosospira multiformis (AY123807) was used as an outgroup. The scale bar represents the estimated percentage of the sequence divergence.

Fig. 5. Production of 15N-labeled N2 from incubations during the ZOMEI cruise at EW4, 50 m. (A) Incubations with 15NH+4. (B) Incubations with 15NO 3 . (C) 14 + Incubations with 15NO 2 + NH4. Lines are linear regressions.

The low rates obtained here, as compared to previous studies in the area, could be due to substrate availability for the anammox process (see Table 1). The studies by Thamdrup et al. (2006) and Hamersley et al. (2007) were done in the summer, whereas our study was carried out at the beginning of winter, when coastal upwelling is less intense and there is less phytoplankton biomass in surface waters (Yuras et al., 2005). Primary productivity is enhanced in summer by the greater fertilization of the euphotic zone with nutrient-rich upwelled waters (Daneri et al., 2000). On the other hand, the lack of detection of anammox cells and activity and the lower bacterioplankton abundance found at Station EW9 (see Fig. 2I and L), situated outside the upwelling influence, support the idea that differences in substrate availability—not only between seasons but also among sampling sites—could be influencing the anammox rates. A tight coupling between carbon

fixation and bacterial consumption (up to 96% of the primary production is utilized by bacteria) was reported off northern Chile (Troncoso et al., 2003). In addition, higher bacterioplankton abundances were found at the same stations where Thamdrup et al. (2006) measured higher anammox activities off Chile (Table 1). Considering that an important proportion of the substrates used by the anammox communities should ultimately come from the degradation of sinking organic matter, these results suggest a possible dependency of the anammox process on the substrate remineralized by the heterotrophic bacterioplankton community and potentially by other players in nitrogen regeneration like heterotrophic nanoplankton (Molina et al., 2005). Denitrification activity was not detected during our 15Nincubations, based on the absence of 15N15N production in 15 15 NO NO 3 and 2 incubations. These results support previous reports from the ETSP (Thamdrup et al., 2006; Hamersley et al., 2007). However, a diverse community of denitrifying bacteria has been found in the OMZ off northern Chile through the study of genes encoding for nitrite reductase (nirS) (Castro-Gonza´lez et al., 2005). In addition, denitrification seems to govern 92% of the nitrous oxide production and consumption (Castro-Gonza´lez and Farı´as, 2004; Farı´as et al., 2007). Thus, it is possible that incomplete denitrification was taking place during our experiments, with NO 2 or N2O as a final product and not N2.

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Table 1 Biogeochemical and microbiological characteristics at the stations and the depths of maximum anammox rates (50/60 m) in the OMZ off northern Chile.

Season OMZ upper boundarya (m) Surface chlorophyll-ab (satellite-based) Anammox ratesc +d NH4 NOd 3 NOd 2 3d PO4 d N* Bacterioplanktone Procholorococcuse Synechococcuse

ZOMEI cruise (Sta. EW 9; 24 September)

ZOMEI cruise (Sta. EW 4; 22 September)

DINAMO cruise (Sta. 1; 21 March)

DINAMO cruise (Sta. 2; 24 March)

DINAMO cruise (Sta. 1; 24 March)

Winter 39 0.6

Winter 39 0.6

Summer 30 1.00

Summer 30 1.00

Summer 30 1.00

0.00 0.01 8.51 4.40 2.01 14.25 1080 2.10 0.50

2.11 0.02 7.83 4.31 2.26 18.36 1286 68.64 30.94

4.80 0.034 16.82 6.53 2.56 12.72 832 22.58 5.65

11.28 0.05 19.12 4.83 2.45 10.70 1298 14.26 6.67

17.52 0.02 19.46 6.44 2.40 8.34 1629 6.88 10.42

a

Depth limit considered where dissolved oxygen declines below 10 mmol L1. In mg L1. c Rates to 15NH+4 incubations in nmol N2 L1 d1. d In mmol L1. e In 103 cells ml1. b

4.3. Vertical distribution of anammox bacteria Maximum anammox bacteria cell counts were 3000 cells ml1, corresponding to 0.45% of all DAPI-stained cells (Fig. 2K). The abundance was in the lower range of previously reported values from the Black Sea and Benguela upwelling region (1900–22,000 cells ml1) (Kuypers et al., 2003, 2005). The maximum anammox cell abundance (3000 cells ml1) and the rates obtained for the 15NH+4 incubations (2.11 nmol L1 d1) gave a cell-specific anammox activity of 0.7 fmol NH+4 cell1 d1. In the OMZ off Peru, Hamersley et al. (2007) also found lower cellspecific potential anammox activity (0.4–2.4 fmol NH+4 cell1 d1) compared to that reported for the Benguela upwelling region (up to 4.5 fmol NH+4 cell1 d1) (Kuypers et al., 2005). The anammox cell distribution showed a conspicuous maximum associated with the upper OMZ boundary, just below the oxycline at Station EW4 (Fig. 2), coinciding with the anammox activity depth distribution reported previously at the same location by Thamdrup et al. (2006). These authors found that anammox rates peaked in the shallowest part of the OMZ (55/ 60 m), whereas its activity decreased steeply with depth. A similar anammox bacteria abundance and activity distribution were found off Peru (Hamersley et al., 2007). The vertical distribution of bacterioplankton mirrored that of the nitrite profile within the OMZ, unlike the vertical distribution of anammox bacteria, whose abundance declined steeply towards the OMZ core (Fig. 2). The nitrite concentrations at the depths where anammox bacteria were present were usually higher than the saturation concentrations of this substrate for the natural anammox activity (p3 mmol L1; e.g., Dalsgaard and Thamdrup, 2002), whereas oxygen concentrations were low. Even off Namibia and in the Black Sea, where nitrite concentrations are always far below 3 mmol L1, there is little evidence of nitrite limitation for the anammox process (Jensen, 2006). Therefore, nitrite and oxygen should not be the controlling factors for anammox in the OMZ. Considering that the anammox depth distribution (e.g., Thamdrup et al., 2006) is consistent with that of the settling of organic matter, the availability of ammonium then becomes the main candidate for controlling the abundance and activity of anammox bacteria. In agreement with these results, anammox rates were found to be ammonium-limited in Golfo Dulce, and local mineralization of organic nitrogen and ammonium

transformation by the anammox process were tightly coupled (Dalsgaard et al., 2003). The vertical distribution of the anammox bacteria and activity, therefore, appears to be mainly controlled by the source of ammonium, which is, in turn, dependent on the mineralization of organic nitrogen that falls from the upper levels or that is eventually produced at depth (see below).

4.4. Anammox bacteria at the upper boundary of the ETSP-OMZ The organic matter that maintains the microbial community in the upper part of the OMZ is mainly produced at the surface. However, it could also be produced locally if an autotrophic community is present, such as the cyanobacteria Synechococcus and Prochlorococcus, as evidenced through the SFM. The presence of cyanobacteria could be an additional source of organic matter within the shallowest part of the OMZ, if they indeed fix inorganic carbon and do not live heterotrophically, as suggested by Johnson et al. (1999) for the Arabian Sea. In fact, intense nitrogen and carbon recycling occurs in this layer, mainly related to nanoheterotrophs grazing on bacterioplankton, including cyanobacteria (e.g., Molina et al., 2005; Cuevas and Morales, 2006). The occurrence of the highest abundance and activity of anammox bacteria together with this potentially autotrophic cyanobacteria community is surprising because both communities should compete for the same substrates (e.g., ammonium, nitrite). Usually, environmental gradients or ecotones are characterized by high levels of diversity and are considered to be important zones in diversification and speciation (e.g., Smith et al., 2001). Indeed, a diverse metabolic bacterial community appears to inhabit the upper part of the OMZ. In this layer, denitrification contributes to the accumulation of N2O (Farı´as et al., 2007). Nitrification also contribute to N2O production (Castro-Gonza´lez and Farı´as, 2004; Farı´as et al., 2007) and to dark carbon fixation rates (Molina and Farı´as, 2009). On the other hand, Ward et al. (1989) found the highest abundance of nitrifying bacteria and highest activities on the same horizon in the Peruvian OMZ. In the suboxic zone of the Black Sea, Lam et al. (2007) detected an active expression of the ammonium monooxygenase subunit gene (amoA), a functional marker of the presence of aerobic ammonium oxidizers, suggesting a potential nitrification–anammox coupling via nitrite. However, both communities should also compete for

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ammonium, in which case the ecological advantage of this coupling is not so clear. Molina et al. (2007) also found the amoA gene on the same horizon where the anammox library was constructed (40 m, PRODEPLOY). Thus, these findings reflect the complexity of the microbial interactions in this transition layer. In summary, our results show marked vertical variability in the abundance and activity of anammox bacteria that seems to be related to the availability of ammonium. Anammox bacteria peak in the upper part of the OMZ off Chile and present higher microdiversity than that previously reported for marine environments. The presence of several microbial groups associated with this maximum suggests a tight interaction of the anammox process with other microbial processes occurring in this layer.

Acknowledgments The present article is partly based on the doctoral work of A.G., who was financially supported by the DAAD (German Academic Exchange Services) and the Millennium Scientific Initiative (Grant EBMA P04/007). V.M. was supported by a Marine Genomics postdoctoral fellowship (Grant PBCT RUE 004). We thank the captains and crews of the research vessels Carlos Porter and Vidal Gormaz. A.G. gratefully acknowledges B.B. Jo¨rgensen and R. Amann for their support during his visit to the MPI-MM. We also acknowledge G. Alarco´n for assisting on board and for the flow cytometry analysis, L. Belmar for her skillful technical assistance with the N2 incubations, W. Rojas for the map of the study area, and M. Varas for the nutrient analysis. This research was supported by the Chilean National Commission for Scientific and Technological Research through the FONDAP program (Grant 15010007) and by the Agouron Institute (Grant AI-MME1.05) to O.U.

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