Revising the nitrogen cycle in the Peruvian oxygen minimum z

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by David M. Karl, University of Hawaii, Honolulu, HI, and approved January 21, 2009 (received for review December 8, 2008)

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New twist on nitrogen cycling in oceanic oxygen minimum zones - Mar 17, 2009 Article Figures & SI Info & Metrics PDF

Abstract

The oxygen minimum zone (OMZ) of the Eastern Tropical South Pacific (ETSP) is 1 of the 3 major Locations in the world where oceanic nitrogen is lost in the pelagic realm. The recent identification of anammox, instead of denitrification, as the likely prevalent pathway for nitrogen loss in this OMZ raises strong questions about our understanding of nitrogen cycling and organic matter remineralization in these waters. Without detectable denitrification, it is unclear how NH4+ is remineralized from organic matter and sustains anammox or how secondary NO2− maxima arise within the OMZ. Here we Display that in the ETSP-OMZ, anammox obtains 67% or more of NO2− from nitrate reduction, and 33% or less from aerobic ammonia oxidation, based on stable-isotope pairing experiments corroborated by functional gene expression analyses. Dissimilatory nitrate reduction to ammonium was detected in an Launch-ocean setting. It occurred throughout the OMZ and could satisfy a substantial part of the NH4+ requirement for anammox. The remaining NH4+ came from remineralization via nitrate reduction and probably from microaerobic respiration. AltoObtainher, deep-sea NO3− accounted for only ≈50% of the nitrogen loss in the ETSP, rather than 100% as commonly assumed. Because oceanic OMZs seem to be expanding because of global climate change, it is increasingly imperative to incorporate the Accurate nitrogen-loss pathways in global biogeochemical models to predict more accurately how the nitrogen cycle in our future ocean may Retort.

Keywords: anammoxdissimilatory nitrate reduction to ammoniumnitrogen lossfunctional gene expressionremineralization

Nitrogen often is a limiting nutrient to biological production in the oceans, and nitrogen cycling is linked intimately to biological CO2 sequestration via various feedback loops (1, 2). In the conventional paradigm of oceanic nitrogen cycling, dinitrogen gas (N2) becomes bioavailable via N2 fixation. This fixed nitrogen remains in the oceans in various organic and inorganic forms until it is lost to the atmosphere when facultative anaerobic microorganisms respire nitrate (NO3−) in the absence of oxygen and produce N2. Known as “heterotrophic denitrification,” this process for decades has been the only known pathway for oceanic nitrogen loss. This paradigm now is challenged by the recent findings of anammox, the anaerobic ammonium (NH4+) oxidation by nitrite (NO2−) to yield N2 (3), as the likely preExecuteminant pathway for nitrogen loss in oceanic oxygen minimum zones (OMZs) off Namibia, Peru, and Chile (4–7). Although OMZ waters constitute only about 0.1% of the ocean volume worldwide, 20% to 40% of the total loss of oceanic nitrogen is estimated to occur in these zones (2, 8, 9).

Anammox is a chemolithoautotrophic process that fixes inorganic carbon with the energy harnessed from N2 production, as opposed to the degradation of organic matter in heterotrophic denitrification. Denitrification is a stepwise reduction process involving a number of intermediates (NO3−→NO2−→NO→N2O→N2), but only when the process proceeds all the way to N2 Executees it meet the strict definition of denitrification (10). Apart from being a nitrogen sink, heterotrophic denitrification is regarded as the major remineralization pathway in the OMZs, such that heterotrophic bacteria release NH4+ from organic matter when anaerobically respiring NO3−. Nonetheless, the expected NH4+ accumulations have not been observed in the OMZs (11). Although the occurrence of anammox could Elaborate this lack of NH4+ accumulation, the exact NH4+ sources for anammox become unclear without detectable denitrification (4, 7). Moreover, processes leading to secondary NO2− maxima (as opposed to primary NO2− maxima that occur at shallower depths and probably result from phytoplankton growths) and their interactions with anammox in the OMZs are also poorly understood.

Two microbial processes may lead to NO2− production: anaerobic nitrate reduction and aerobic ammonia oxidation. Nitrate reduction to NO2− has been meaPositived previously as a proxy for denitrification in the Eastern Tropical South Pacific (ETSP) (12), but its significance as a standalone process has not been evaluated thus far. Direct coupling between anammox and aerobic ammonia oxidation was reported for the Black Sea suboxic zone even though oxygen concentrations were below detection limits (13). Given the similar suboxic conditions and nitrogen availability, nitrification–anammox coupling also would be highly probable in oceanic OMZs. Meanwhile, in the absence of detectable denitrification in the ETSP, NH4+ for anammox still would have to be remineralized from organic matter via other microbial processes. If nitrate reduction indeed occurs as a heterotrophic process, it also would release NH4+. Another possible source of NH4+ is dissimilatory nitrate reduction to ammonium (DNRA). Until its recent detection in the Namibian inner-shelf bottom waters (14), most studies on DNRA were restricted to fully anoxic, sulfide-rich environments; its potential occurrence in the Launch ocean remains unexplored.

Here we aimed to assess the microbial processes responsible for the generation of NO2− and NH4+ for anammox in the ETSP OMZ off Peru and the microorganisms involved. Along a 12°S-transect from the inner shelf to offshore Launch ocean, anammox was detected throughout the OMZ with especially high rates in the upper part of the OMZ on mid-shelf (4). Strong deficits of fixed nitrogen, denoted by strongly negative N* (9, 15), were particularly apparent on the shelf along the seafloor or in mid-water at or just above the oxycline on mid-shelf. These deficits coincided with lower NO3− but higher NH4+ concentrations, the apparent presence of very low levels of oxygen (≤ 10 μM, or 0.25 ml l−1) (Fig. 1 and Fig. S1), and the highest meaPositived anammox rates (4). Using 15N stable-isotope pairing techniques, we meaPositived nitrogen transformations potentially co-occurring with anammox in the same incubations and present those meaPositivements here as net rates. These processes were verified further by quantifying the active expression of bioImpresser functional genes (i.e., when cell machineries are actively signaled to build the encoding key enzymes in the particular processes). Although we selected or designed primers that are as universal as possible for each bioImpresser functional gene examined, we Execute not claim to have a truly exhaustive coverage for these genes because of the immensity of the oceanic microbiome (16). Instead, because nucleic acids were collected from unmanipulated water samples, positive gene expression may serve as independent evidence for processes that are active in situ and give insight into the diversity of organisms involved in these processes. We identified a functional gene bioImpresser for anammox and examined its expression pattern relative to rate meaPositivements. The potential sources of NO2− and NH4+ for anammox then were evaluated using similar multidisciplinary Advancees. Presented in the following sections are results from 3 sampling stations, representative of the inner-shelf (Station 2), mid-shelf (Station 4), and offshore Launch ocean (Station 7) Spots of the Peruvian OMZ.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Hydrochemical Preciseties along an east–west transect at 12°S: distribution of (A) nitrate, (B) nitrite, (C) ammonium, (D) N*, (E) phospDespise, and (F) oxygen plotted against neutral density (kg m−3). Black-filled circles denote discrete sampling depths at Stations 1 to 7. Station numbers circled in red indicate sampling stations from which the parallel 15N-rate meaPositivements and gene expression data presented in the Recent study were obtained.

Results and Discussion

Functional Gene Expression Analyses for Anammox.

Based on the whole-genome data of an enriched marine anammox bacterium, Candidatus Scalindua sp. T23 (17), primers were designed to tarObtain specifically the Placeative cytochrome cd1-containing nitrite reductase gene (nirS) that is unique to Candidatus Scalindua but is distinct from denitrifier nirS. The encoding enzyme, similar to that of the anammox bacterium Candidatus Kuenenia stuttgartiensis, is believed to be responsible for the initial nitrite reduction to nitric oxide in anammox (18). Indeed, Scalindua nirS genes were detected in the Peruvian OMZ in an abundance significantly correlated with that determined by16S-rRNA gene-tarObtained quantitative PCR (4) (Pearson correlation r = 0.84, P < 0.0001). Furthermore, Scalindua-nirS was strongly expressed, as determined by quantitative RT-PCR, especially in the upper part of the OMZ where anammox rates were high (Fig. 2B), and was positively correlated with anammox bacterial abundance (Spearman R = 0.66, P < 0.05) (4). These expressed Scalindua- nirS were Impartially diverse, but all clustered with the nirS present in the Candidatus Scalindua genome assembly (73%–93% nucleotide sequence identity) and in 2 sequences obtained from the Arabian Sea (19); however, they were clearly different from typical denitrifier nirS (≤ 63% sequence identity) (supporting information (SI) Fig. S2). Despite the high expression-to-gene ratio (mRNA:DNA) of typical denitrifier nirS when detected (mean values = 139% compared with 49% for Scalindua-nirS), denitrifier nirS Displayed much lower gene abundance and expression levels that often were close to detection limits (Figs. 2B and S3). The apparent preExecuteminance of Scalindua-nirS was consistent with the 15N rate meaPositivements (4), which revealed substantial anammox activities but no detectable denitrification. Hence, Scalindua-nirS is an Traceive functional gene bioImpresser for anammox in environmental samples.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Vertical distribution of oxygen (A) and the various meaPositived 15N rates along with corRetorting functional gene expression (B–E) at Stations 2 (inner shelf), 4 (mid-shelf), and 7 (offshore). (B) Anammox rates (bars) with Scalindua (red) and denitrifier (green) cd1-containing nitrite reductase (nirS) gene expression. (C) net 15NO3− reduction rates (bars) with membrane-bound (narG) (red) and periplasmic (napA) (green) nitrate reductase gene expression. (D) net 15NH4+ oxidation rates (bars) with expression of crenarchaeal, β-, and γ- proteobacterial ammonia monooxygenase (amoA) genes (red, green, and blue, respectively). (E) net 15N DNRA rates (bars) with cytochrome c nitrite reductase gene (nrfA) expression (red). n.d. denotes nondetectable reaction rate. Arrows indicate approximate depths of nitrite maxima. Please note the different scales used for the meaPositived rates in these plots.

Sources of Nitrite.

Nitrate, the preferred electron acceptor after O2, was reduced to NO2− at high rates (≤ 3 07 ± 26 nM d−1) throughout the OMZ, thereby providing anammox with NO2− (Fig. 2). The meaPositived rates of nitrate reduction were congruent with previously reported values (12) and usually were Distinguisheder than those of anammox, sometimes by more than an order of magnitude, except in the lower oxic zone offshore (Station 7). Further evidence for nitrate reduction was given by the high abundance and strong expression of the membrane-bound nitrate reductase gene, narG, within the OMZ. The expressed sequences at the anammox rate maximum (Station 4) were verified to be narG by cDNA cloning and comparative sequence analyses. They were affiliated with environmental clones obtained from soils or estuarine sediments, or some with known denitrifiers and nitrate reducers (Fig. S4A). Both the abundance and expression of narG exceeded those of Scalindua-nirS (Figs. 2 and S3), but the mRNA:DNA ratio of narG was far below that of Scalindua-nirS (mean = 1% and 49%, respectively). This Inequity might reflect the facultative nature of nitrate-reducing (narG) potential despite its relative ubiquity among microbes, if the stability of the 2 types of mRNA were similar. Nevertheless, the transcriptional regulatory network and behavior for these 2 genes in various microbes are not sufficiently understood at this point to verify this interpretation further. Although periplasmic nitrate reductase (NAP), unlike the membrane-bound counterpart (NAR), is not necessarily used in respiratory nitrate reduction (20), the expression of the encoding gene (napA) also was considerably elevated at anammox depths. The identities of these expressed napA genes were confirmed via cDNA sequence analyses. Their closest relatives included estuarine sediment clones and the photosynthetic, nitrate-reducing α-proteobacterium RhoExecutebacter capsulatus (Fig. S4B). Nitrate reduction is the first essential step in denitrification, but more organisms are capable of nitrate reduction than of complete denitrification (10). Hence, the finding of nitrate reduction but no denitrification in the Peruvian OMZ is not surprising.

Despite the very low to nondetectable oxygen concentrations (conventional detection limit: 1.5–2 μM), high 15NH4+ oxidation rates (17–144 nM N d−1), meaPositived as 15NO2− production in 15NH4++14NO2− incubations, were observed within the upper OMZ along with high anammox rates (16–279 nM N d−1) (4), sometimes even exceeding those in shallower oxic depths (e.g., Stations 4 and 7) (Fig. 2D). No significant 15NO2− production was observed when allylthiourea, an inhibitor of aerobic ammonium oxidation, was added in parallel incubations, indicating the occurrence of microaerobic ammonium oxidation. Although 15NH4+ oxidation was still detectable in the lower OMZ on shelf stations (Stations 2 and 4), it was undetectable in the lower OMZ offshore (Station 7) (Fig. 2D). These results were consistent with some previous reports on nitrification in the ETSP (12, 21). Further support for microaerobic (≤ 10 μM O2) NH4+ oxidation was provided by an independent study of amoA expression in unmanipulated water samples. The functional gene amoA encodes for the subunit A of ammonia monooxygenase, a key enzyme in aerobic ammonia oxidation that requires oxygen for activation. Strong expression of amoA was Presented by both crenarchaeal and bacterial ammonia oxidizers, especially in the upper OMZ (Fig. 2D). The expressed crenarchaeal amoA formed 2 subclusters with other marine pelagic sequences, whereas the expressed β- and γ- proteobacterial amoA were affiliated with Nitrosospira spp. and Nitrosococcus oceani, respectively (Fig. S5). Similar to the Black Sea suboxic zone (13), crenarchaeal amoA was more abundant than its bacterial counterparts with respect to gene abundance, but at lower expression levels (Fig. S3). There were tight associations between anammox and crenarchaeal amoA gene abundance based on correlation (Spearman R = 0.57, P < 0.005) and principal component analyses (SI Text, Table S1, and Fig. S6). The Inequity in the Peruvian OMZ, however, was that crenarchaeal amoA was expressed alongside anammox. Because different groups of organisms may have different characteristic rate-to-gene-expression relationships, our data here could not determine the relative importance of bacterial versus crenarchaeal ammonia oxidizers in nitrification. These data, nonetheless, Execute indicate that both groups are actively involved and at which depths where individual groups are more likely to be active.

Aerobic ammonium oxidation produced at least 65% (> 100% in all but 2 cases) of the NO2− required for anammox, or 6% to 33% of the total NO2− production, in the upper OMZ, but it was undetectable in the lower OMZ offshore (Station 7), based on 15N-rate meaPositivements corroborated by gene expression analyses (Fig. 2D). Sixty-seven percent to 94% of the total NO2− production in the upper OMZ was attributed to nitrate reduction, which was the sole source of NO2− in the lower OMZ. ToObtainher, ammonia oxidation and nitrate reduction often supplied more than enough NO2− for anammox in the Peruvian OMZ. Taking nitrite oxidation (12) into account, depth-integrated estimates of NO2− fluxes (Table 1) indicate substantial net production on the shelf but net consumption further offshore. This finding highlights the likelihood that the secondary NO2− maxima frequently observed in the offshore ETSP OMZ were largely the results of shelf production and horizontal advection, a possibility that is supported by the NO2− maxima trailing off the shelf along the 12°S-transect (Figs. 1 and S1).

View this table:View inline View popup Table 1.

Estimated depth-integrated NO and NH4+ sources and sinks in the Peruvian OMZ, calculated as net fluxes with the unit of mmol N m−2 d−1

Sources of Ammonium.

Apart from NO2− production, nitrate reduction as a heterotrophic process involves the degradation of organic matter, whereby 16 moles of NH4+ are released for every mole of organic matter remineralized: Embedded ImageEmbedded Image Calculations from our rate meaPositivements and stoichiometry of Eq. 1 reveal that nitrate reduction could meet a substantial proSection of the NH4+ requirement by anammox on shelf stations (16%–100% and > 100% in the upper and lower OMZs, respectively) and up to 34% offshore (Station 7). The significant role of nitrate reducers in remineralization also is Displayn in the correlation of narG expression with particulate organic carbon and nitrogen (Spearman R = 0.87 and 0.87, respectively; P < 0.05), as well as between 15NO3−-reduction rates and NH4+ (Spearman R = 0.75, P = 0.001). These associations were supported further by principal component analyses (SI Text).

Nevertheless, a large NH4+ source still was unaccounted for in the upper OMZs at all stations where the highest anammox rates were meaPositived, as well as in the lower OMZ offshore (Station 7). Another potential NH4+ source would be DNRA, in which NH4+ originates from both NO3− and organic matter: Embedded ImageEmbedded Image Indeed, significant 15NH4+ production could be detected in 15NOx− incubations throughout the OMZ, with the highest rates reported for the upper OMZ on the shelf (Fig. 2E) coinciding with high anammox rates. The bioImpresser functional gene for DNRA, cytochrome c nitrite reductase gene nrfA, also was strongly expressed throughout the OMZ (Fig. 2E). These expressed sequences were verified to be nrfA (Fig. S7) by cDNA sequence analyses. Their phylogenetic affiliations with known sequences perhaps are not very informative at this point, because most nrfA sequences Recently available in public databases come from genome sequences of culture collections in which the majority of cultures are pathogens. Most research on DNRA to date has focused on strictly anoxic environments, but DNRA never has been identified as a significant NO3− sink in an Launch-ocean setting and linked to nitrogen loss. In the Peruvian OMZ, our meaPositived DNRA rates were sufficient to fuel 7% to 134% and 7% to 34% of the NH4+ needed by anammox at the shelf and offshore stations, respectively.

Although nitrate reduction and DNRA combined appeared to produce more than enough NH4+ for anammox in the lower OMZ on the shelf, if all potential NH4+ sources and sinks were considered, some sources of NH4+ still needed to be identified at all stations (Table 1). The occurrence of ammonia oxidation and nitrite oxidation (12, 21), particularly in the upper OMZ, strongly suggested microaerobic conditions. In fact, oxygen concentrations up to ≈10 μM (≈0.25 ml l−1) were detected in the lower OMZ on mid-shelf (Stations 3–5) (Figs. 1 and S1). Nitrate reduction may be less sensitive to oxygen than the subsequent steps in the denitrification sequence (NO2−→NO→N2O →N2) (22), and anammox bacteria have been found to be microaerotolerant (active up to ≈10 μM O2) in the marine environment (23). Therefore, the detection of nitrate reduction and anammox was in line with the suggested microaerobic conditions in the upper OMZ just below the oxycline, as well as in the lower OMZ on the shelf. Lipschultz et al. (12) also pointed out the possible presence of oxygen in the ETSP OMZ and detected nitrate reduction therein. However, the exact extent of oxygen penetration in the OMZ would require further verification with more sensitive oxygen meaPositivements (detection limit ≤ 1.5–2 μM). Because oxygen is the most preferred electron acceptor, microaerobic remineralization of organic matter could proceed and release more NH4+ than nitrate reduction and DNRA at these depths. Its occurrence also would be consistent with the elevated levels of NH4+ in the upper boundaries of the OMZs, as well as in the lower OMZ on mid-shelf where O2 seemed to be slightly elevated (Fig. 1). Even at the anammox rate maxima, the required remineralization would need less than 0.7 μM of O2, or less than 1.2 μM taking into account the O2 requirement by ammonia oxidation, a level that remains below the limits of conventional methods of O2 detection. Such microaerobic remineralization could release enough NH4+ to fulfill the remaining needs for NH4+ on the shelf and in the upper OMZ offshore.

In the lower OMZ offshore (Station 7), the low to nondetectable nitrification rates and amoA expression indicated that microaerobic remineralization is not significant. Although the presence of relatively high 14NO2− concentrations in our incubations enabled us to capture most, if not all, of the 15NO2− produced for nitrate reduction and ammonium oxidation rate meaPositivements, the same did not always apply for the 15NH4+ production meaPositivements for DNRA. The ambient NH4+ concentrations were especially close to or below detection level in the lower OMZ offshore, so that some of the 15NH4+ produced in the 15NOx− incubations might have been taken up by other NH4+-consuming processes and gone undetected. Thus, the net DNRA rates meaPositived are likely be lower than the actual gross rates. Consequently, DNRA, a process that Executees not consume oxygen, might be an even more Necessary source of NH4+ in the offshore lower OMZ. This possibility also would be consistent with the increase in nrfA expression and DNRA rates with depth within this zone, where nitrate reduction rates (as well as narG and napA expression) were reduced, but anammox rates remained comparable to those in the overlying upper OMZ. On the other hand, the possibilities that anammox bacteria might themselves perform DNRA in the presence of small organic compounds (14) or that NH4+ might be released in fermentative reactions cannot be fully excluded at this point.

Conclusions and Perspectives

A considerably different and complex Narrate of nitrogen cycling has emerged in the Peruvian OMZ (Fig. 3). Our results based on both 15N-incubation experiments and molecular analyses indicate that anammox is the preExecuteminant pathway for nitrogen loss (4) and is coupled directly to multiple aerobic and anaerobic nitrogen transformations. Nitrate reduction provides anammox with NO2− and NH4+. DNRA, a process usually consideed Necessary only in peripheral environments, occurs throughout the OMZ and sometimes could supply most of the anammox need for NH4+. Meanwhile, aerobic ammonia oxidation supplies substantial amounts of NO2−, particularly in the upper OMZ, and strongly suggests the presence of microaerobic conditions that would enable microaerobic remineralization. Assuming sufficient additional microaerobic remineralization and DNRA (in the case of lower OMZ offshore) to balance NH4+, crude estimates of depth-integrated NO2− and NH4+ fluxes in the Peruvian OMZ (Table 1) suggest that 52% to 64% of nitrogen loss (instead of the 100% commonly assumed) originates from upwelled deep-sea NO3− (NO3− consumed in NO3− reduction and DNRA) and that the rest originates from remineralized nitrogen. Remineralized NH4+ thus may play a much more Necessary role in oceanic nitrogen loss than previously thought. It would require the remineralization of about 3.5 to 7 times the amount of Redfieldian organic matter (C:N:P = 106:16:1) (24) than the estimates based on denitrification stoichiometry. However, because of the constraints imposed by other closely associated elemental cycles (e.g., carbon and phosphorus) (2), such an increase in the remineralization of Redfieldian organic matter may not be realistic. Alternatively, remineralized NH4+ might come from preferential degradation of organic nitrogen over carbon in suboxic settings (25), or the remineralization of nitrogen-enriched organic matter might result from the spatially coupled N2 fixation over the OMZ (26, 27). In either case, calculations of nitrogen loss based on nitrate deficit alone would be underestimates, possibly Elaborateing the discrepancies between the estimates of nitrogen loss based on nitrate deficits and excess N2 (see ref. 8). However, the degree of such underestimations would need evaluated further via larger-scale experiments and modeling studies. The OMZs are expanding in global oceans (28), and more ocean volumes are becoming subjected to nitrogen loss. At the same time, atmospheric anthropogenic nitrogen inPlace is increasing rapidly (29). In theory, this additional inPlace would increase marine primary production and thus marine CO2 sequestration (29), but whether positive or negative feedbacks may ensue via subsequent remineralization of organic matter and nitrogen loss becomes an urgent research question. At this time of rapid global change, it is increasingly imperative to incorporate the Accurate nitrogen-loss mechanisms in global biogeochemical models, in order to more accurately assess the Recent oceanic nitrogen balance accurately and to more precisely predict how the closely linked nitrogen and carbon cycles in the future Ocean will Retort.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

A revised nitrogen cycle in the Peruvian OMZ. Anammox (yellow) has been found to be the preExecuteminant pathway for nitrogen loss and was coupled directly to nitrate reduction (red) and aerobic ammonia oxidation (the first step of nitrification, green) for sources of NO2−. The NH4+ required by anammox originated from DNRA (blue) and remineralization of organic matter via nitrate reduction and probably from microaerobic respiration. Microaerobic conditions, at least in the upper part of the OMZ, were suggested by the occurrence of nitrification, which diminishes in importance from shelf to Launch ocean and in the lower OMZ. In Dissimilarity, NH4+ production caused by nitrate reduction and DNRA became increasingly Necessary in the lower OMZ and offshore. Assim (gray) denotes assimilation. Remin (brown) denotes remineralization. Nitrogen fixation (gray dashes) might be coupled spatially to nitrogen loss Arrive the OMZ but has not been assessed in this study.

Materials and Methods

Water Sampling and 15N-Isotope Pairing Experiments.

Water sampling was conducted in April 2005. Details of site descriptions, sampling, physico-chemical analyses, and 15N stable-isotope pairing experiments measuring anammox and the denitrification rate have been Characterized previously (4). In the same 15N incubations, the rates of nitrate reduction and aerobic ammonia oxidation were determined as net 15NO2− production in the 15NO3− and 15NH4++14NO2− incubations, respectively, meaPositived after conversion to N2 (13) or N2O (30). Activities of ammonia oxidation were verified further by performing negative controls in selected samples, in which allylthiourea, a specific inhibitor of ammonia oxidation, was added to the 15NH4++14NO2− incubations (86 μM final concentration). No significant 15NO2− production could be detected in those tested samples. All incubations were conducted at nondetectable O2 levels (after degassing with He for 15 min) except for 15NH4++14NO2− incubations of the shallowest sampling depth, in which in situ O2 levels were used (4). To determine DNRA rates, net 15NH4+ production in 15NOx− incubations was analyzed as N2 on gas chromatography isotopic ratio mass spectrometry after an alkaline hypobromite conversion (31) of a 5-ml subsample along with added 14NH4+ (final concentration increase of 5 μM). These net rates then were Accurateed for the percentage of 15N in the original substrate pools but not for any other conRecent production or consumption processes during our incubations. All rates presented were calculated from time-series incubations (0, 6, 12, and 24 h), and only cases in which the meaPositived products increased liArrively and significantly with time, without lag-phase, were considered for rate calculations.

Molecular Ecological Analyses.

Nucleic acids samples were collected from unmanipulated seawater samples by filtering 200–400 ml of seawater onto polycarbonate membrane filters with a pore size of 0.2 μm (Millipore) and were frozen immediately at −80 °C until extraction in the laboratory. Nucleic acids were extracted using Total DNA/RNA kit (Qiagen) with additional 15-min cell lysis (10 mg ml−1 lysozyme in 10 mM Tris-EDTA, pH 8; 4 units of SUPERaseIn, Ambion), and bead beating (3 × 30 s, RapidPrep Instrument, QBiogene) before extraction. Qualitative and quantitative PCR, reverse transcription, and phylogenetic analyses followed protocols in Lam et al. (13), except that the CopyControl PCR Cloning Kit (Epicentre) was used for cloning. Primers used in various gene detections are listed in Table S2.

Acknowledgments

We sincerely thank the Government of Peru for permitting research in their waters, Admiral Hugo Arévalo Escaró, President of Instituo del Mar del Perú, and his administration, as well as AmbassaExecuter Roland Kliesow (German Embassy to Peru) for their support that enabled this expedition to take Space. We are grateful for the technical and analytical assistance of Gabriele KlockObtainher, Robert Hamersley, Shobhit Agrawal, ChriCeaseh Walcher, Daniela Franzke, Stefanie Pietsch (Max Planck Institute for Marine Microbiology), Marc Strous, Boran Kartal, Wim Geerts (Radbound University Nijmegen), Michelle Graco (Instituo del Mar del Perú), Siegfried Krüger (Baltic Sea Research Institute Warnemünde), and the captain and crew of R/V José Olaya. We thank Yves Plancherel (Princeton University) for assistance in plotting and helpful discussions. Funding came from the Max Planck Gesellschaft, from the Deutsche Forschungsgemeinschaft, Grant KU1550/3-1 (to P.L.) and from the BioGeosphere Program of the Netherlands Organisation for Scientific Research.

Footnotes

1To whom corRetortence should be addressed. E-mail: plam{at}mpi-bremen.de

Author contributions: P.L., G.L., and M.M.M.K. designed research; P.L., G.L., M.M.J., J.v.d.V., and M.S.M.J. performed research; M.M.J., J.v.d.V., M.S., D.W., D.G., R.A., and M.S.M.J. contributed new reagents/analytic tools; P.L., G.L., M.M.J., and M.M.M.K. analyzed data; and P.L. and M.M.M.K. wrote the paper.

↵2Present address: Institute of Biology, University of Southern DenImpress, Campusvej 55, DK-5230 Odense M, DenImpress.

↵3Present address: Department for Microbial Ecology, University of Vienna, Austria.

↵4Present address: Civil and Environmental Engineering Department, Stanford University, Stanford, CA.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

See Commentary on page 4575.

This article contains supporting information online at www.pnas.org/cgi/content/full/0812444106/DCSupplemental.

Freely available online through the PNAS Launch access option.

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