Oxygen Sensitivity of Anammox and Coupled N-Cycle Processes in Oxygen Minimum Zones

Tim Kalvelage; Marlene M. Jensen; Sergio Contreras; Niels Peter Revsbech; Phyllis Lam; Marcel Günter; Julie LaRoche; Gaute Lavik; Marcel M. M. Kuypers

doi:10.1371/journal.pone.0029299

Abstract

Nutrient measurements indicate that 30–50% of the total nitrogen (N) loss in the ocean occurs in oxygen minimum zones (OMZs). This pelagic N-removal takes place within only ∼0.1% of the ocean volume, hence moderate variations in the extent of OMZs due to global warming may have a large impact on the global N-cycle. We examined the effect of oxygen (O₂) on anammox, NH₃ oxidation and NO₃⁻ reduction in ¹⁵N-labeling experiments with varying O₂ concentrations (0–25 µmol L⁻¹) in the Namibian and Peruvian OMZs. Our results show that O₂ is a major controlling factor for anammox activity in OMZ waters. Based on our O₂ assays we estimate the upper limit for anammox to be ∼20 µmol L⁻¹. In contrast, NH₃ oxidation to NO₂⁻ and NO₃⁻ reduction to NO₂⁻ as the main NH₄⁺ and NO₂⁻ sources for anammox were only moderately affected by changing O₂ concentrations. Intriguingly, aerobic NH₃ oxidation was active at non-detectable concentrations of O₂, while anaerobic NO₃⁻ reduction was fully active up to at least 25 µmol L⁻¹ O₂. Hence, aerobic and anaerobic N-cycle pathways in OMZs can co-occur over a larger range of O₂ concentrations than previously assumed. The zone where N-loss can occur is primarily controlled by the O₂-sensitivity of anammox itself, and not by any effects of O₂ on the tightly coupled pathways of aerobic NH₃ oxidation and NO₃⁻ reduction. With anammox bacteria in the marine environment being active at O₂ levels ∼20 times higher than those known to inhibit their cultured counterparts, the oceanic volume potentially acting as a N-sink increases tenfold. The predicted expansion of OMZs may enlarge this volume even further. Our study provides the first robust estimates of O₂ sensitivities for processes directly and indirectly connected with N-loss. These are essential to assess the effects of ocean de-oxygenation on oceanic N-cycling.

Citation: Kalvelage T, Jensen MM, Contreras S, Revsbech NP, Lam P, Günter M, et al. (2011) Oxygen Sensitivity of Anammox and Coupled N-Cycle Processes in Oxygen Minimum Zones. PLoS ONE 6(12): e29299. https://doi.org/10.1371/journal.pone.0029299

Editor: Jack Anthony Gilbert, Argonne National Laboratory, United States of America

Received: August 31, 2011; Accepted: November 23, 2011; Published: December 28, 2011

Copyright: © 2011 Kalvelage et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The study was funded by the Max-Planck-Gesellschaft (www.mpg.de), Deutsche Forschungsgemeinschaft (DFG) (www.dfg.de), the DFG-funded Sonderforschungsbereich 754 “Climate-Biogeochemistry Interactions in the Tropical Ocean” (www.sfb754.de), the European Research Council, grant 267233 (www.erc.europa.eu) and the Danish Council for Independent Research: Natural Sciences, grant 10-083140 (http://en.fi.dk/councils-commissions/the-danish-council-for-independent-research). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Oxygen (O₂) is one of the key regulatory factors of major biogeochemical cycles in the marine environment [1]. The distribution of dissolved O₂ in the world's oceans is regulated by gas exchange between surface waters and the lower atmosphere, advective processes within the ocean, as well as the biological processes of photosynthesis and respiration. Oxygen, entering the ocean interior mainly at high latitudes, is distributed throughout the global ocean via thermohaline circulation. In the ocean's sunlit surface layer, phytoplankton produces O₂ and fixes carbon dioxide (CO₂) in to biomass. Near the base of the euphotic zone, concentrations of O₂ are generally at their lowest as photosynthesis diminishes or ceases altogether while the repiration of sinking organic matter by heterotrophic micro-organisms consumes O₂ at maximal rates.

Subsurface regions of severely reduced O₂ concentrations (O₂≤5 µmol L⁻¹), the so-called oxygen minimum zones (OMZs), are found along the eastern boundaries of the ocean basins in the subtropics and tropics (e.g. off California, Namibia, Peru/Chile) and in the Arabian Sea. Typically in these regions, wind-driven circulation results in the upwelling of nutrient-rich deep waters, fueling high primary production in the euphotic zone. The high surface productivity results in high export of organic matter and thus strong respiration in subsurface waters. Combined with the poor ventilation of these water masses [2], [3], this leads to permanently O₂-depleted to anoxic conditions at mid-depths [4]–[6].

Although OMZs (if defined by O₂≤5 µmol L⁻¹) account for only ∼0.1% of the global ocean volume [7], they play a key role in controlling the oceans' nutrient inventory as 30–50% of the oceanic nitrogen (N) loss is estimated to occur therein [7], [8]. The recharge of such N-deficient waters from these regions back to adjacent surface waters limits primary production and thus carbon (C) sequestration in large parts of the tropical oceans. N-loss as primarily the formation of gaseous dinitrogen (N₂) can occur via two pathways: (1) heterotrophic denitrification, the reduction of nitrate (NO₃⁻) to gaseous dinitrogen (N₂) via a sequence of intermediates (NO₃⁻→NO₂⁻→NO→N₂O→N₂) and (2) anammox, the anaerobic oxidation of ammonium (NH₄⁺) with nitrite (NO₂⁻) to N₂. In the OMZs of Namibia and Peru/Chile, on which the current study focuses, anammox has been identified as the major N-loss pathway based on ¹⁵N-labeling experiments, whereas heterotrophic denitrification was often not detectable or only measured sporadically [9]–[11].

In the course of global climate change and increasing anthropogenic pressures on the marine environment, coastal and open ocean OMZs have been expanding and intensifying in the last decades [12], [13]. A continuing decline in dissolved O₂ due to reduced O₂ solubility and enhanced stratification [14], as well as coastal and open ocean eutrophication [15], [16], is expected. De-oxygenation will have the greatest effect on water masses already deficient in O₂ as these are often at or near the thresholds for anaerobic processes such as anammox or denitrification. Deutsch et al. [17] calculated that a reduction of the mean upper ocean O₂ content by only 1% would mean a doubling of water masses with O₂≤5 µmol L⁻¹, thus significantly enlarging the ocean volume potentially affected by N-loss.

However, the sensitivities of anammox and denitrification to changes in dissolved O₂ and their upper O₂ limits in the marine environment are largely unknown. N-loss attributed to denitrification has been reported to occur at up to 20 µmol L⁻¹ of O₂ [18]. Nonetheless, direct measurements of denitrification under controlled exposure to low O₂ concentrations in OMZs are lacking. Active anammox bacteria have been found to be abundant at O₂ concentrations up to 9 and 20 µmol L⁻¹ in the Namibian and Peruvian upwelling systems, respectively [9], [10], and it has been suggested that marine snow aggregates could provide suitable anoxic micro-niches at ambient O₂ concentrations up to 25 µmol L⁻¹ [19], [20]. Off Peru/Chile the measured anammox rates were often the highest at the base of the oxycline and in the upper OMZ [10], [11], [21], likely associated with intensified remineralization of organic matter in these water layers. This further indicates that, unlike their cultured counterparts, which are inhibited at O₂ concentrations as low as 1 µmol L⁻¹ [22], marine anammox bacteria can tolerate O₂ concentrations higher than the upper O₂ limit (5 µmol L⁻¹) often used to restrict anaerobic processes in biogeochemical models [23]. Recently, Jensen et al. [24] investigated the O₂ sensitivity of anammox in the near-anoxic zone of the Black Sea water column and showed that anammox bacteria remained active up to ∼9 µmol L⁻¹ of O₂. Still unknown is whether this relatively high O₂ tolerance is widespread amongst anammox bacteria in the major OMZs of the world's oceans.

Although anammox is an autotrophic process, it relies on other N-cycling processes for the required reactive substrates NO₂⁻ and NH₄⁺, e.g. NH₃ oxidation to NO₂⁻ and heterotrophic nitrate (NO₃⁻) reduction to NO₂. The co-occurrence of these aerobic and anaerobic processes together with anammox requires them to be adapted to a certain overlapping range of O₂ concentrations. Thus far, it remains unclear whether or not processes coupled to anammox can proceed in the same range of O₂ as assumed for anammox (0–20 µmol L⁻¹), or if they show different O₂ sensitivities that might hence restrict N-loss to a narrower O₂ regime. Under anoxic conditions, NO₃⁻ is the next thermodynamically favored electron acceptor, which can be used by a variety of micro-organisms to oxidize organic matter [25]. In OMZ waters, secondary NO₂⁻ maxima are often interpreted as active NO₃⁻ reduction [26], [27]. The formation of NO₂⁻ from NO₃⁻ is the first step in both denitrification and dissimilatory nitrate reduction to ammonium (DNRA), but it can also be considered as a stand-alone process, as more micro-organisms are known capable of reducing NO₃⁻ to NO₂⁻ than to N₂ or NH₄⁺ [25], [28]. Heterotrophic NO₃⁻ reduction to NO₂⁻ has been measured at high rates in the Peruvian OMZ [29], [30], and has been estimated to account for approximately two thirds of the NO₂⁻ required for anammox in this region [30]. At the same time, NO₃⁻ reduction also provides an important source of NH₄⁺ released from oxidized organic matter [30], [31]. Lipschultz et al. [29] investigated the effect of varying O₂ concentrations on NO₃⁻ reduction to NO₂⁻ in the Peruvian OMZ. They observed that NO₃⁻ reduction rates doubled under anoxic conditions (N₂ atmosphere) compared to in situ conditions (2.5 µmol L⁻¹ of O₂), while rates decreased by ∼75% at 20 µmol L⁻¹ of O₂.

When O₂ is present, NO₂⁻ can be produced aerobically by NH₃ oxidizing bacteria and archaea in the first step in nitrification. Rates of NH₃ oxidation are generally highest near the upper OMZ boundaries [32], [33]. In the Peruvian OMZ, this is also where anammox bacteria are most active [10]. These bacteria are partly fueled by NH₃ oxidation in this zone [30]. A similarly tight coupling between anammox and NH₃ oxidation was shown earlier for the Black Sea [34]. The occurrence of NH₃ oxidizers is, however, not restricted to the upper OMZ. They have been found active at non-detectable concentrations of O₂ (<1–2 µmol L⁻¹) in the core of OMZs [30], [33], [35] and are thus obviously well adapted to near-anoxic O₂ conditions. When Lipschultz et al. [29] investigated the O₂ sensitivity of NH₃ oxidation in the Peruvian OMZ, the inferred de-oxygenation of the samples only caused a ∼50% decrease in activity relative to ambient O₂ (2.5 µmol L⁻¹), whereas no stimulation was achieved by an increase to ∼20 µmol L⁻¹ of O₂.

With anammox as well as NO₃⁻ reduction being apparently tolerant to relatively high O₂ and NH₃ oxidation being apparently able to cope with severe O₂ depletion, an expansion of OMZs might indeed drive larger water masses to greater N-deficits. This would potentially exacerbate N-limitation of primary production in large parts of the ocean and thus affect the oceans' capacity to attenuate the rising atmospheric CO₂. However, at present no study has systematically investigated the O₂ sensitivities of anammox and concurrent N-cycling processes in oceanic OMZs, and thus the future nutrient balance in these regions remains speculative at best.

In this paper, we present results for the Namibian and Peru/Chile upwelling systems, two of the most productive regions in the worlds' oceans associated with massive N-loss, where we explored the effect of O₂ on anammox, NH₃ oxidation and NO₃⁻ reduction throughout the OMZ.

Materials and Methods

Ethics Statement

The necessary permissions were obtained from the governments of Namibia and Peru to carry out research in their waters.

Water sampling and nutrient analyses

Samples were taken on two cruises to the OMZs off Namibia (M76/2) and Peru (M77/3), where upwelling persists year-round, onboard R/V Meteor in May/June 2008 and December/January 2008/2009, respectively (Fig. 1). A pump-CTD system was used to collect water samples just below the oxycline, through the core of the OMZ, down to ∼375 m depth off the coast of Peru. The pump CTD system was equipped with a conventional amperometric O₂ micro-sensor to obtain vertical profiles of dissolved O₂. In addition, the recently developed STOX (Switchable Trace amount OXygen) sensor [6], which allows high-accuracy O₂ measurements in near-anoxic environments (detection limit: 50–100 nmol L⁻¹ during our deployments), was deployed. At least five measuring cycles after ≥10 min sensor equilibration at a given sampling depth were used to calculate O₂ concentrations. Water samples were taken with a depth resolution of 1–2 m for nutrient analyses. NH₄⁺ was measured fluorometrically [36] and NO₂⁻ was analyzed spectrophotometrically [37] on board. Water samples for NO₃⁻ and PO₄³⁻ were stored frozen until spectrophotometric determination [37] with an autoanalyzer (TRAACS 800, Bran & Lubbe) in a shore-based laboratory. Detection limits for NH₄⁺, NO₂⁻, NO₃⁻ and PO₄³⁻ were 10, 10, 100 and 100 nmol L⁻¹, respectively. N-deficits were calculated from the measured fixed inorganic N- and PO₄³⁻ concentrations as N* (in µmol L⁻¹) following Gruber and Sarmiento [8]: N* = [NH₄⁺]+[NO₂⁻]+[NO₃⁻]−16×[PO₄³⁻]+2.9 µmol kg⁻¹×density in kg L⁻¹.

Download:

Figure 1. Locations of the sampled stations and distribution of dissolved O₂.

Maps show sampling locations on the A) Namibian shelf and in the B) OMZ off Peru during cruises M76-2 and M77-3, respectively. Water samples were collected by pump-CTD (max. sampling depth: ∼375 m). The oxygen sensitivities of anammox and coupled N-cycling processes were investigated at sampling stations indicated by numbers (red circles). Vertical distributions of dissolved O₂ are plotted along blue lines.

https://doi.org/10.1371/journal.pone.0029299.g001

¹⁵N labeling experiments

Incubation experiments were carried out at two shallow shelf stations off Namibia (St. 206 and 252) and four stations off Peru (St. 36, 44, 54 and 63), ranging from coastal to open ocean settings (Fig. 1 and Table 1). Based on O₂ profiles, three to six depths per station were chosen for a standard series of ¹⁵N-labeling experiments. The experimental procedure for ¹⁵N-labeling experiments has been described in detail previously [9], [31], [38]. Briefly, N-loss by either anammox or heterotrophic denitrification was measured as the production of ¹⁵N-labeled N₂ in ¹⁵NH₄⁺ (+¹⁴NO₂⁻), ¹⁵NO₂⁻ (+¹⁴NH₄⁺) and ¹⁵NO₃⁻ (+¹⁴NO₂⁻) (isotopes: Campro scientific) time-series incubations carried out in 12-ml Exetainers (Labco, UK). At each time interval (about 0, 6, 12, 24 and 48 h) production in one replicate Exetainer was terminated by the addition of saturated mercuric chloride to stop biological activity. The N-isotopic composition of N₂ gas produced in these experiments was determined by GC/IRMS (Fisons VG Optima). Afterwards, rates of NH₃ oxidation to NO₂⁻ and those of NO₃⁻ reduction to NO₂⁻ were determined in the same samples as net ¹⁵NO₂⁻ production in ¹⁵NH₄⁺+¹⁴NO₂⁻ and ¹⁵NO₃⁻+¹⁴NO₂⁻ incubations respectively. The N-isotopic composition of NO₂⁻ was determined by GC/IRMS after conversion to either nitrous oxide (N₂O) by sodium azide [39], or to N₂ by sulfamic acid [40], [41]. Rates were calculated from the slope of linear regression of ¹⁵N-production as a function of time. Only significant and linear production of ¹⁵N-species without an initial lag-phase was considered (t-tests, p<0.05; R²>0.8). The net production rates presented here have been corrected for the mole fractions of ¹⁵N in the original substrate pools but not for isotope dilution due to any other concurrent N-consumption or production processes in the course of the incubation.

Download:

Table 1. Concentrations of O₂, NH₄⁺, NO₂⁻ and N-conversion rates in ¹⁵N-labeling experiments in the OMZs off Namibia and Peru.

https://doi.org/10.1371/journal.pone.0029299.t001

Oxygen sensitivity experiments

In order to determine the effect of varying O₂ concentrations on N-cycle processes, one to two depths per station were sampled for additional O₂ sensitivity experiments. Samples were taken from the upper OMZ, where aerobic and anaerobic N-cycle processes have been shown to co-occur [30], except one sample taken deeper in the core of the Peruvian OMZ (St. 36). Samples were obtained in 250-mL serum bottles and purged with helium (He) for approximately 15 min to remove any initial O₂ and to lower the N₂ background in order to enhance the detection limit of ²⁹N₂ and ³⁰N₂ [38]. As a small sample volume was lost during He-purging, the bottles were then refilled with a second He-purged sample from the same depth to avoid headspace. Afterwards, air-saturated water from the same depth was added to the serum bottles in exchange for part of the de-oxygenated water to adjust samples to the desired O₂ concentration. At St. 206 and 252 (Namibian OMZ) three samples each were adjusted to ∼3.5, 7.5 and 12 µmol L⁻¹ of O₂, whereas at St. 36, 44, 54 and 63 (Peruvian OMZ) the experimental setup was extended and five samples each were adjusted to ∼1.5, 3, 6, 12, and 24 µmol L⁻¹ of O₂. One sample, to which no air-saturated water was added, served as an anoxic control at all stations. After additions of either ¹⁵NH₄⁺+¹⁴NO₂⁻, ¹⁵NO₂⁻ (+¹⁴NH₄⁺) or ¹⁵NO₃⁻+¹⁴NO₂⁻, samples were transferred into replicate vials (Exetainers, Labco) for time-series incubations. Except for the incubations with only ¹⁵NO₂⁻, ¹⁴N-species were added to all experiments to exclude substrate limitation, which would otherwise complicate the interpretation of any O₂ effects on the processes of interest. Moreover, keeping the ¹⁴N-pool of the product of a certain reaction well above the expected concentrations produced from the added ¹⁵N-substrate could minimize any further conversion of the newly formed ¹⁵N-products by co-occurring processes. The rate measurements for the various processes were carried out as described above. To exclude formation of ²⁹N₂ due to coupled nitrification-denitrification in incubations amended with ¹⁵NH₄⁺ we added allylthiourea (ATU; final concentration 84 µmol L⁻¹) to an additional sample of the highest O₂ treatment (∼11.5 µmol L⁻¹) at St. 206 and 252. ATU is a specific inhibitor of aerobic NH₃ oxidation [42]–[44] and does not affect anammox activity shown at least in sediments [45]. Two sets of incubations were performed in parallel at St. 206 and 252 and one sample per time-point was sacrificed to measure dissolved O₂. For the remaining stations, O₂ concentrations were determined only for the initial time-point in each ¹⁵N-incubation experiment. We used a custom-built, fast-responding O₂ micro-sensor (Clark-type; MPI Bremen) for most measurements (detection limit: ∼0.5 µmol L⁻¹ of O₂), except at St. 206 where a STOX sensor was used for selected samples.

Data analysis

We applied least-squares fitting to each set of samples of the O₂ sensitivity experiments using Excel's solver function [46].

Results

Hydrochemistry in the Namibian OMZ

The water column was poorly stratified over the Namibian shelf at St. 206 and 252 during the time of sampling, as indicated by a weak density gradient, along with the vertical profiles of dissolved O₂ and inorganic N-species (Fig. 2A). At both stations O₂ declined gradually with depth, from ∼200 µmol L⁻¹ in the surface waters to less than 10 µmol L⁻¹ at ∼80 m. STOX measurements at the incubation depths revealed O₂ concentrations as low as 0.60±0.11 µmol L⁻¹ at St. 206. In the central OMZ at St. 252 (Table 1), the sensor was at its detection limit (100 nmol L⁻¹ of O₂ during M76-2). Ammonium concentrations were typically in the range of 1–3 µmol L⁻¹ in the oxic zone (<80 m) and decreased to 0.1–0.5 µmol L⁻¹ at the base of the oxycline (Fig. 2B). Towards the sediment-water interface NH₄⁺ concentrations increased up to 4.5 (St. 206) and 2.5 µmol L⁻¹ (St. 252). Nitrite concentrations were fairly constant in the upper ∼100 m (0.1–0.5 µmol L⁻¹) and increased to ∼2 and ∼4 µmol L⁻¹ in the bottom waters at St. 206 and 252, respectively. The increase in both NO₂⁻ and NH₄⁺ in the lower OMZ was accompanied by a sharp decrease in NO₃⁻ concentrations, with minimum concentrations of ∼12 µmol L⁻¹ in the lowest sampling depths at both stations.

Download:

Figure 2. Physicochemical zonation and N-conversion rates at selected stations.

Stations are plotted for cruises M76-2 and M77-3 to the OMZs off A) Namibia and B) Peru, respectively. Water depths were 111 m at St. 252 and 2845 m at St. 36. N* was calculated from the fixed inorganic N- and PO₄³⁻ concentrations (data not shown). Anammox rates were determined in ¹⁵NH₄⁺ (St. 206) and ¹⁵NO₂⁺+¹⁴NH₄⁺ incubations (St. 36). All rates are net rates corrected for the percentage of ¹⁵N in the pool of the respective N-species. Error bars for rates are standard errors calculated from linear regression.

https://doi.org/10.1371/journal.pone.0029299.g002

Hydrochemistry in the Peruvian OMZ

The stations sampled in the Peruvian OMZ were located on the shelf (St. 62), shelf edge (St. 44) and in the open ocean (St. 36 and 54). Similar to the Namibian shelf stations, the shallowest site (St. 62) was characterized by low density gradients and a gradual decline in O₂ between ∼20 and 50 m. In contrast, the water column was highly stratified further offshore. Strong pycnoclines, centered around 65, 30 and 55 m at St. 44, 54 and 36, respectively, and a steep oxycline indicated oxygenated surface waters and OMZ were well separated (Figure 2A). Oxygen decreased from ∼250 µmol L⁻¹ in the surface to less than 10 µmol L⁻¹ at 66 (St. 44), 35 (St. 54) and 75 m (St. 36). A local O₂ maximum (10 to 25 µmol L⁻¹) was found between 90 and 100 m at St. 36, likely due to some lateral advection of more oxygenated water. At all four stations, STOX measurements at the incubation depths revealed traces of O₂ in the central OMZ at best; mostly here O₂ concentrations remained below the detection limit of the STOX sensor (∼50 nmol L⁻¹ of O₂ during M77-3). Ammonium concentrations were low and typically 0.05 to 0.1 µmol L⁻¹ throughout the OMZ as well as in the surface layer (Fig. 2B). On the shelf, concentrations of NH₄⁺ were slightly elevated at the base of the oxycline (up to ∼0.4 µmol L⁻¹ at St. 62). At the open-ocean stations (St. 54 and 36) NH₄⁺ maxima of ∼2 µmol L⁻¹ were measured at 20 and 35 m, which coincided with NO₂⁻ maxima (up to 1 µmol L⁻¹). In general, NO₂⁻ concentrations in the surface waters remained below 0.5 µmol L⁻¹, whereas NO₂⁻ accumulated to over 5 µmol L⁻¹ in the core of the OMZ at all stations. Nitrate concentrations were as low as ∼1 µmol L⁻¹ on the shelf (St. 62). Further off-shore less pronounced NO₃⁻ concentration minima were detected (∼12 at St. 44 and ∼20 µmol L⁻¹ at St. 54 and 36).

N-cycling in the Namibian and Peruvian OMZs

Distribution of anammox activity.

Over the Namibian shelf a strong increase in the N-deficit was observed below the oxycline. Minimum values for N* (down to −19 µmol L⁻¹) were found in the central OMZ, suggesting N-loss therein. We measured ¹⁵N¹⁴N formation in all of our ¹⁵NH₄⁺ (+¹⁴NO₂⁻) and ¹⁵NO₂⁻-incubations at the three depths sampled per station (Table 1). Corrected for the labeling percentage (i.e. the mole fraction of ¹⁵N in the respective N-substrate pool), rates were comparable in ¹⁵NH₄⁺ and ¹⁵NO₂⁻ experiments. As no increase in ¹⁵N¹⁵N was detectable in either ¹⁵NO₂⁻ or ¹⁵NO₃⁻ incubations, the formation of ¹⁵N-labeled N₂ was attributed to anammox activity and not denitrification. At both stations, anammox rates and N-loss inferred from N* increased with depth (Fig. 2C). Rates ranged from 13 to 43 nmol N L⁻¹ d⁻¹ at the base of the oxycline to 144 to 496 nmol N L⁻¹ d⁻¹ in the central OMZ and were generally higher at St. 252.

In the OMZ off Peru, the N-deficit was strongest over the shelf (N* = −33 µmol L⁻¹; St. 62) and less pronounced towards the open ocean (N* = 10 µmol L⁻¹; St. 54), indicating the highest N-loss likely occurred near the coast. Six depths per station were sampled and ¹⁵N¹⁴N formation in ¹⁵NH₄⁺+¹⁴NO₂⁻ and ¹⁵NO₂⁻+¹⁴NH₄⁺ was measured in 22 out of 24 incubation depths (Table 1). No formation of ¹⁵N-labeled N₂ was detectable at 150 and 337 m at St. 36. As for the Namibian OMZ, whenever N₂ formation occurred all of the ¹⁵N-labeled N₂ produced was recovered as ²⁹N₂ and there was no detectable increase in ¹⁵N¹⁵N over time detected in either ¹⁵NO₂⁻ or ¹⁵NO₃⁻ incubations. Thus, anammox was the only detectable active N₂-producing pathway, while there was no clear evidence for denitrification activity at the time of our sampling. In general, high anammox activity corresponded with more negative N*, i.e. a more pronounced N-deficit (Fig. 2C). Over the Peruvian shelf, anammox rates (25 to 108 nmol N L⁻¹ d⁻¹; St. 62) were comparable to those measured over the Namibian shelf (St. 206). Further offshore in the Peruvian OMZ, rates dropped to as low as one tenth of those measured near the coast (2.2 to 9.4 nmol N L⁻¹ d⁻¹; St. 54).

Distribution of nitrate reduction to nitrite activity.

Nitrate reduction was measured as ¹⁵NO₂⁻ production in all ¹⁵NO₃⁻+¹⁴NO₂⁻ incubations carried out in the OMZ overlying the Namibian shelf. Nitrate reduction occurred uniformly over the three sampled depths, at rates around 100 and 360 nmol N L⁻¹ d⁻¹ at St. 206 and 252, respectively (Table 1).

Off Peru, NO₃⁻ reduction could be detected in 21 out of 23 ¹⁵NO₃⁻+¹⁴NO₂⁻ incubation experiments. The vertical distribution of NO₃⁻ reducing activity was slightly variable and high NO₃⁻ reduction rates did not always coincide with a noticeable accumulation of NO₂⁻. Similar to anammox activity, maximum rates of NO₃⁻ reduction were generally detected over the shelf (up to 215 nmol N L⁻¹ d⁻¹) and decreased towards the open ocean (up to 48 nmol N L⁻¹ d⁻¹).

Distribution of ammonia oxidation activity.

Ammonia oxidation, measured as ¹⁵NO₂⁻ production in ¹⁵NH₄⁺+(¹⁴NO₂⁻) incubation experiments, was detected at all incubation depths (Table 1). At St. 206 ¹⁵N-labeling experiments were carried out under anoxic conditions, whereas samples were incubated at in situ O₂ (<1 µmol L⁻¹) at St. 252. Rates increased with depth at St. 206 (from 29 to 84 nmol N L⁻¹ d⁻¹) but remained rather constant at St. 252 (∼100 nmol N L⁻¹ d⁻¹).

Off Peru, NH₃ oxidation to NO₂⁻ was determined in ¹⁵NH₄⁺+¹⁴NO₂⁻ incubations under anoxic conditions (St. 44 and 54) or at in situ O₂ levels (St. 36 and 62). Maximum NH₃ oxidation rates ranged between 15 and 47 nmol N L⁻¹ d⁻¹. There was no obvious trend in nitrifying activity between coastal and open-ocean stations. Ammonia oxidation was generally confined to the upper OMZ, where O₂ was still measurable. However, despite an apparent lack of O₂ in situ (i.e. O₂ concentrations were below detection) shipboard experiments revealed NH₃ oxidation activity also at St. 54 at 75 m as well as in the central OMZ at St. 62 (1.7 to 5.0 nmol N L⁻¹ d⁻¹).

Oxygen sensitivity of anammox and coupled N-cycle processes

Oxygen sensitivity of anammox.

Anammox activity, as indicated by ¹⁵N¹⁴N production from ¹⁵NH₄⁺ and ¹⁵NO₂⁻, was measurable in all O₂ manipulation experiments without lag phase at the Namibian shelf stations (Table 2). Oxygen concentration and N₂ formation showed a significant negative correlation for the incubations with ¹⁵NH₄⁺ as well as ¹⁵NO₂⁻ at St. 206 and the one with ¹⁵NH₄⁺ at St. 252 (Pearson r = −0.95 to −0.99, P<0.05). Similar responses to increased O₂ were observed for the incubations amended with ¹⁵NH₄⁺ and ¹⁵NO₂⁻ at both stations. Activity decreased with increasing O₂ and was, on average, ∼85%, ∼70% and ∼50% of the anoxic control at ∼3.7, ∼8.1 and ∼11.3 µmol L⁻¹ of oxygen, respectively (Fig. 3A). Over the course of the incubation (0–48 h) O₂ concentrations in the ¹⁵N-labeling experiments did not vary significantly (±0.44 µmol L⁻¹ on average). No substantial difference in ¹⁵N¹⁴N production was observed between ¹⁵NH₄⁺-labeled incubations with and without ATU. This indicates that anammox rather than coupled nitrification-denitrification was the process responsible for the production of ¹⁵N-labeled N₂ at 11–12 µmol L⁻¹ of dissolved O₂.

Download:

Figure 3. N-cycle processes in Namibian and Peruvian OMZ waters with respect to dissolved oxygen.

A) Anammox measured as ¹⁵N¹⁴N production in ¹⁵NO₂⁻ (+¹⁴NH₄⁺) and ¹⁵NH₄⁺+¹⁴NO₂⁻ incubations. B) NO₃⁻ reduction measured as ¹⁵NO₂⁻ production in ¹⁵NO₃⁻+¹⁴NO₂⁻ incubations. C) NH₃ oxidation measured as ¹⁵NO₂⁻ production in ¹⁵NH₄⁺+¹⁴NO₂⁻ incubations. N-conversion rates are given as percentages of the highest rate observed ( = 100%) for the different O₂ treatments at each incubation depth. Adjusted O₂ concentrations were verified by micro-sensor measurements. Parentheses in figure legend indicate the corresponding sampling depths at each station shown. Station numbers with double digits and triple digits represent the Peruvian and Namibian stations, respectively. Shelf and open ocean stations are represented by red and blue symbols, respectively. The O₂ sensitivity assays indicate an upper O₂ limit for N-loss due to anammox of ∼20 µmol L⁻¹ (grey shading).

https://doi.org/10.1371/journal.pone.0029299.g003

Download:

Table 2. Rates of NH₃ oxidation, NO₃⁻ reduction and anammox measured at varying concentrations of dissolved O₂.

https://doi.org/10.1371/journal.pone.0029299.t002

In the OMZ off Peru, ¹⁵N¹⁴N production rates in ¹⁵NH₄⁺ and ¹⁵NO₂⁻ incubations decreased with increasing O₂ concentrations in all O₂ manipulation experiments. However, substantial differences in the O₂ sensitivity of anammox were found between stations. Over the Peruvian shelf, adjusted O₂ levels and N₂ production were linearly and negatively correlated up to 14.4 µmol L⁻¹ O₂ at St. 44 (Pearson r = −0.99, P<0.05) and 10.9 µmol L⁻¹ at St. 62 (Pearson r = −0.96, P<0.05). No rates were detectable beyond ∼20 µmol L⁻¹ of O₂. At the open-ocean stations in the Peruvian OMZ, anammox activity appeared to be more sensitive to the added O₂ (Fig. 3A). At St. 36, ∼30% activity of the anoxic control experiment remained detectable when O₂ was increased from the in situ ∼1.2 µmol L⁻¹ (measured by STOX) to 5.5 µmol L⁻¹ of O₂ in the 120 m sample. In comparison, anammox was fully inhibited at 2.8 µmol L⁻¹ of O₂ already in the 180 m sample, where O₂ was not detectable by the STOX sensor in situ. A similarly strong O₂ response was seen at St. 54, where rates dropped to zero at 4.0 µmol L⁻¹ of O₂ in the 75 m incubation experiment.

Oxygen sensitivity of nitrate reduction to nitrite.

Nitrate reduction rates in the O₂ sensitivity assay carried out for the Namibian OMZ waters, decreased with increasing O₂ concentrations (Table 2). The incubation experiments at St. 206 revealed a stronger negative response to elevated O₂ levels than those performed at St. 252. Activity at St. 206 was reduced to ∼30% of the anoxic control in the highest O₂ treatment (7.3 µmol L⁻¹), whereas a doubling of the O₂ concentration (14.7 µmol L⁻¹) led to a decrease in NO₃⁻ reduction rates to ∼60% of the control experiment at St. 252 (Fig. 3B).

In the Peruvian OMZ, production of ¹⁵NO₂⁻ from ¹⁵NO₃⁻ was never fully inhibited by O₂, not even in the highest O₂ treatments (∼25 µmol L⁻¹ of O₂). Nevertheless, NO₃⁻ reduction rates showed marked differences in their sensitivity towards elevated O₂ levels between and within our experimental stations. For example at St. 36, NO₃⁻ reduction activity in the upper OMZ sample (120 m) at St. 36 did not vary significantly among the various O₂ treatments (1.4 to 27.1 µmol L⁻¹ of O₂), while activity decreased to ∼10% of the control experiment in samples taken deeper (180 m) in the OMZ when adjusted to 25.5 µmol L⁻¹ of O₂ (Figure 3B).

Oxygen sensitivity of ammonia oxidation.

Rates of NH₃ oxidation to NO₂⁻ showed no significant difference over the range of the applied O₂ concentrations (∼1–12 µmol L⁻¹) in the Namibian OMZ samples (Table 2). Activity varied by a maximum of ∼15% among the different O₂ treatments but without any systematic trends (Fig. 3C).

Similar to the observations for the Namibian shelf, ¹⁵NO₂⁻ production in the ¹⁵NH₄⁺ experiments conducted for the Peruvian shelf (St. 44) and at open-ocean (St. 54) stations showed no marked differences among the different O₂ treatments (∼1–25 µmol L⁻¹). Only the control experiment (0.8 µmol L⁻¹ O₂) at St. 54 suggested a slightly lower NH₃ oxidation rate (−35%) compared to the higher O₂ treatments (Fig. 3C).

Discussion

Oxygen sensitivity of anammox in OMZ waters

In the investigated samples from both the Namibian and Peruvian OMZ, the only N₂-forming pathway detected by ¹⁵N-labeling experiments was anammox. This confirms the results from earlier studies, which detected N-loss due to anammox but not denitrification in these regions [9]–[11]. The highest anammox rates (on the order of 500 nmol N L⁻¹ d⁻¹) were measured in the Namibian shelf waters. Off Peru, rates declined from ∼50 nmol N L⁻¹ d⁻¹ over the shelf to <10 nmol N L⁻¹ d⁻¹ at the open ocean sites. This may be explained by differences in surface productivity between the two upwelling systems [47] as well as between Peruvian coastal and open-ocean waters, since organic matter transport ultimately fuels all processes delivering NH₄⁺ and NO₂⁻ for the anammox reaction [30], [31]. Anammox often showed the highest rates in the upper OMZ, as seen in previous studies [10], [11], [21] probably in response to the high NH₄⁺ release from the enhanced remineralization of particulate organic matter at the base of the oxycline, below which all three activities decreased with depth. There were exceptions, however, particularly at depths close to the seafloor on the shelf, where exceptionally high rates were likely supported by NH₄⁺ diffusing out of the sediment [9], [48], [49] (S. Sommer, pers. comm.).

In the O₂ tolerance assays, N-loss due to anammox was in fact detectable at O₂ levels significantly higher (up to ∼15 µmol L⁻¹) than that generally used to define OMZs (<5 µmol L⁻¹ of O₂). Anammox activity in samples taken at the shallow sites appeared the least affected by increasing O₂. The rates therein remained measurable even at adjusted O₂ concentrations of 10 to 15 µmol L⁻¹. These are almost twice as high as the anammox O₂-tolerance level previously determined in the Black Sea suboxic zone [24]. In comparison, anammox activity appeared increasingly sensitive to O₂ towards the open ocean and deeper in the OMZ, where rates were not detectable above 2.8 to 5.5 µmol L⁻¹ of O₂ (St. 36 and 54). Based on the observed negative linear correlation between the measured rates and adjusted O₂ levels, the upper O₂ limit for anammox to proceed in the OMZs is estimated to be ∼20 µmol L⁻¹ (Table 3 & Fig. 3).

Download:

Table 3. Overview of the response of NH₃ oxidation, NO₃⁻ reduction and anammox to changes in dissolved O₂.

https://doi.org/10.1371/journal.pone.0029299.t003

The apparently higher O₂ tolerance at the shelf stations may be explained by an adaptation of anammox bacteria to fluctuations in dissolved O₂ due to the presence of a less stable oxycline at the upper boundary of the OMZ. Vertical mixing is usually enhanced in coastal upwelling regions. This was indicated by a weak density gradients and a gradual O₂ decline over the Namibian shelf, where the level of dissolved O₂ are known to be variable [50]. In the open-ocean off Peru, ventilation of the OMZ from above is hindered due to strong stratification [51]. The dissolved O₂ content is perhaps most stable within the core of the OMZ, where the highest O₂ sensitivity of anammox was measured in our current study (180 m at St. 36). With O₂ concentrations consistently below 1–2 µmol L⁻¹, anammox bacteria thriving therein are unlikely to have adapted to higher O₂ levels compared to their counterparts in more dynamic environments.

Alternatively, marine snow particles have been speculated to provide “anoxic” micro-environments in which O₂ is sufficiently depleted to favor N-loss at ambient O₂ levels <25 µmol L⁻¹ [9], [20], while some anammox bacteria have been shown to be potentially particle-associated in the Namibian OMZ [20]. Hence, higher abundance of particles in coastal waters than further offshore or in the core of the OMZ might also explain the apparently higher O₂ tolerance by anammox bacteria near the coast.

Oxygen sensitivity of nitrate reduction in OMZ waters

The reduction of NO₃⁻ to NO₂⁻, was detected at high rates at the shallow shelf stations both off Namibia and Peru (∼100 to 360 nmol L⁻¹ d⁻¹) and decreased with increasing distance from the coast in the Peruvian OMZ (∼10 to 50 nmol L⁻¹ d⁻¹ at St. 36). The rates measured off Peru are consistent with earlier results from ¹⁵N-labeling experiments in the same region [29], [30] and a similar rate distribution was recently reported for the Arabian Sea OMZ [52], [53].

Reduction of NO₃⁻ to NO₂⁻ showed a high degree of variability in O₂ sensitivity amongst stations. No effect of increasing O₂ on NO₃⁻ reduction was observed in the 120 m incubations at St. 36. At the remaining stations, the correlation between activity and adjusted O₂ concentrations was non-linear and could be best described by an exponential function, as determined by least-squares fitting (Table 3 & Fig. 3b). Our results from two shelf stations in the Namibian (St. 252) and Peruvian (St. 62) OMZs further confirmed earlier observations by Lipschultz et al. [29] that NO₃⁻ reduction was only moderately affected by increasing O₂. About 50% of NO₃⁻ reduction activity remained when O₂ was adjusted to ∼14 to 17 µmol L⁻¹ in our above-mentioned samples (Table 3). More pronounced sensitivity to O₂ was detected at St. 206 on the Namibian shelf and at 180 m at St. 36 off Peru, where rates were reduced by ∼50% relative to the control already at ∼4 µmol L⁻¹ of O₂.

The observation, that in general NO₃⁻ reduction activity was only moderately affected by increasing concentrations of O₂ may at first seem at odds with the fact that NO₃⁻ respiration is generally considered an anaerobic process. However, it has been reported from experiments with cultures and environmental samples that complete or partial denitrification can take place under aerobic conditions [54]–[56]. Moreover, the different enzymes involved in the step-wise reduction on NO₃⁻ to N₂ during denitrification, differ in their O₂ sensitivity. In various bacterial strains the NO₂⁻ and nitrous oxide (N₂O) reductase appear to be most sensitive with respect to O₂, whereas the NO₃⁻ reductase is the most O₂-tolerant enzyme [57]–[59]. This O₂ tolerance could explain the observation that even the highest O₂ additions did not lead to a full inhibition of NO₃⁻ reduction in the samples taken from the Namibian and Peruvian OMZ waters. However, the detected variability in terms of O₂ sensitivity among the different incubation experiments and the lack of any response at 120 m at St. 36 remains puzzling. One possible explanation might be the high phylogenetic diversity and thus variable physiology of the NO₃⁻ reducers inhabiting the OMZ waters [30], [60].

Oxygen sensitivity of ammonia oxidation in OMZ waters

Ammonia oxidizing activity seemed widespread throughout the OMZ overlying the Namibian shelf, as indicated by high NO₂⁻ production rates. Off Peru, nitrifying activity peaked at the base of the oxycline, where the highest NH₄⁺ release due to remineralization of sinking organic matter can be expected. Though O₂ was not always detectable in situ, NH₃ oxidation rates could be detected at these upper OMZ depths, consistent with previous studies [30], [33], [35].

In the O₂ sensitivity assays, NH₃ oxidation at most decreased slightly in the anoxic control (St. 54) when compared to the higher O₂ treatments. No stimulation at higher O₂ levels (20 to 25 µmol L⁻¹ of O₂) was achieved. A similar observation was made by Lipschultz et al. [29], though they detected a 50% reduction of activity in their assumedly anoxic control. Our results suggest a relatively high O₂ affinity of aerobic NH₃ oxidizers in both OMZs investigated. It has been shown that cultured bacterial NH₃ oxidizers, including marine nitrifiers, are, in principle, able to cope with very low O₂ concentrations down to at least ∼2 µmol L⁻¹ [61]–[63]. The only cultured marine aerobic ammonia oxidizing archaea investigated so far appears to have a limited capacity to survive under near anoxic conditions [64]. However, a higher O₂ affinity of archaeal NH₃ oxidizers in the environment is indicated by results from the Peruvian OMZ, which suggest that both bacterial and archaeal NH₃ oxidizers are active at undetectable in situ O₂ levels (<1.5–2 µmol L⁻¹) [30].

Based on our findings, the minimum O₂ concentration for NH₃ oxidizer to be active in OMZ waters is most likely in the nanomolar range. An adaptation of aerobic micro-organisms to extremely low O₂ has been shown in a recent study by Stolper et al. [65]. They demonstrated aerobic growth in a culture experiment at an O₂ concentration ≤3 nmol L⁻¹. Alternatively, when O₂ is scarce, NH₃ oxidizer may also grow anaerobically via the oxidation of NH₃ with gaseous nitrogen dioxide (NO₂) or tetraoxide (N₂O₄) [66]. However, as these compounds are rare in the marine environment, it is unlikely that this is of major ecological significance.

Implications for N-loss in the future ocean and our understanding of N-cycling in modern OMZs

In summary, the current study shows that O₂ is a major controlling factor for anammox activity in OMZ waters. Based on our O₂ assays we estimate the upper limit for anammox to be ∼20 µmol L⁻¹ O₂, which is significantly higher than previously shown for the Black Sea (Table 3 & Fig. 3). In contrast, NH₃ oxidation and NO₃⁻ reduction as the main NH₄⁺ and NO₂⁻ sources for anammox were little or only moderately affected by changing concentrations of dissolved O₂. Intriguingly, aerobic NH₃ oxidation was active at non-detectable O₂ concentrations, while NO₃⁻ reduction to NO₂⁻, which is generally considered to be an anaerobic process, was fully active up to at least 25 µmol L⁻¹ O₂. Hence, aerobic and anaerobic N-cycle pathways in OMZs can co-occur over a larger range of O₂ concentrations than previously assumed. The zone where N-loss can occur is primarily controlled by the O₂-senstivity of anammox and not by the O₂-senstivity of the tightly coupled aerobic NH₃ oxidation and anaerobic NO₃⁻ reduction.

Additionally, our results indicate that N-loss and other N-cycling processes within such O₂ regimes would be controlled by other environmental factors such as substrate availability. For instance, the (near) anoxic conditions in the core of the OMZ do not confer the highest NO₃⁻ reduction and anammox rates despite the ideal O₂ regime. Surface water productivity and therewith export of particulate organic matter into the OMZ might play an important role in controlling anammox activity. Sinking organic matter is the ultimate source of the required reactive substrates NO₂⁻ and NH₄⁺ for anammox and it may also provide suitable anoxic micro-environments for anammox bacteria in zones of higher ambient O₂ [9], [20].

The fact that anammox in the marine environment can proceed at O₂ levels ∼20 times higher than those known to inhibit enrichment cultures of anammox bacteria (∼1 µmol L⁻¹) [22] enlarges the global oceanic volume potentially affected by N-loss from the previously estimated 0.1% tenfold to ∼1% (O₂≤20 µmol L⁻¹) [67]. In addition, recent reports show that OMZs have been expanding and intensifying worldwide, particularly in the tropical Atlantic and Pacific [13]. Such expansions of the OMZs would mean an even greater increase in ocean volume potentially subject to active N-loss processes in the coming years. In other words, progressively more fixed inorganic N may be removed from the oceans, and larger areas in the subtropics and tropics might experience enhanced N-limitation due to the recharge of N-deficient waters back to the surface in the future. In the long run, negative feedbacks might also ensue from increasing N-loss and ocean warming. Less productive surface waters would export less organic matter to subsurface waters and lead to reduced O₂ consumption rates. The stronger stratification due to the warming of the upper ocean might also hamper upwelling of nutrient-rich water to the surface, therewith reducing export production and the respiration of O₂ in OMZs.

The relative significance of these positive and negative feedback mechanisms, or how they may counteract each other and eventually influence global oceanic nutrient budgets, would require further investigations complemented with realistic global biogeochemical modeling. To date, the models used to develop future scenarios of the global ocean nutrient balance have rarely taken into account coupling N-cycling processes, and certainly not their respective O₂ sensitivities.

In light of the above presented results, the simple switching from aerobic to anaerobic respiration at ∼5 µmol L⁻¹ of O₂ often implemented in models [23] appears not realistic. The current study provides the first robust estimates of O₂ sensitivities for processes directly and indirectly connected with N-loss. These factors are necessary for biogeochemical models to collectively and accurately assess the effects of ocean de-oxygenation on N-cycling in OMZs and neighboring water masses, and hence global oceanic N-balance.

Acknowledgments

We sincerely thank the cruise leaders Kay Emeis (M76/2) and Martin Frank (M77/3) as well as the crews of the cruises onboard R/V Meteor for their support at sea. We are grateful for the technical and analytical assistance of Gabriele Klockgether, Daniela Franzke, Inka Boosmann, Violeta Leon, Aurelien Paulmier, Moritz Holtappels, Andreas Ellrott, Volker Meyer, Philipp Hach and Michael Jünemann. We thank Gail Arnold and Rachel Foster for reading the early version of the manuscript and offering valuable comments to improve the article.

Author Contributions

Conceived and designed the experiments: MMJ JL GL MMMK. Performed the experiments: TK MMJ SC GL. Analyzed the data: TK MMJ MG PL. Contributed reagents/materials/analysis tools: NPR. Wrote the paper: TK PL GL MMMK.

References

1. Falkowski PG (2008) Earth's Biogeochemical Cycles. Science 320: 1034–1039.
- View Article
- Google Scholar
2. Wyrtki K (1962) Circulation and Water Masses in the Eastern Equatorial Pacific Ocean. Deep-Sea Research. pp. 11–13.
3. Karstensen J, Stramma L, Visbeck M (2008) Oxygen minimum zones in the eastern tropical Atlantic and Pacific oceans. Progress in Oceanography 77: 331–350.
- View Article
- Google Scholar
4. Kamykowski D, Zentara S (1990) Hypoxia in the world ocean as recorded in the historical data set. Deep-Sea Research 37(12): 1861–1874.
- View Article
- Google Scholar
5. Helly JJ, Levin LA (2004) Global distribution of naturally occurring marine hypoxia on continental margins. Deep-Sea Research 51: 1159–1168.
- View Article
- Google Scholar
6. Revsbech NP, Larsen LH, Gundersen J, Dalsgaard T, Ulloa O, et al. (2009) Determination of ultra-low oxygen concentrations in oxygen minimum zones by the STOX sensor. Limnology and Oceanography: Methods 7: 371–381.
- View Article
- Google Scholar
7. Codispoti LA, Brandes JA, Christensen JP, Devol AH, Naqvi SW, et al. (2001) The oceanic fixed nitrogen and nitrous oxide budgets: Moving targets as we enter the anthropocene? Scienta Marina 65: 85–105.
- View Article
- Google Scholar
8. Gruber N, Sarmiento JL (1997) Global Patterns of Marine Nitrogen Fixation and Denitrification. Global Biogeochemical Cycles 11: 235–266.
- View Article
- Google Scholar
9. Kuypers MM, Lavik G, Woebken D, Schmid M, Fuchs BM, et al. (2005) Massive nitrogen loss from the Benguela upwelling system through anaerobic ammonium oxidation. Proc Natl Acad Sci USA 102(18): 6478–6483.
- View Article
- Google Scholar
10. Hamersley MR, Lavik G, Woebken D, Rattray JE, Lam P, et al. (2007) Anaerobic ammonium oxidation in the Peruvian oxygen minimum zone. Limnology and Oceanography 52(3): 923–933.
- View Article
- Google Scholar
11. Thamdrup B, Dalsgaard T, Jensen MM, Ulloa O, Farias L, et al. (2006) Anaerobic ammonium oxidation in the oxygen-deficient waters off northern Chile. Limnology and Oceanography 51(5): 2145–2156.
- View Article
- Google Scholar
12. Naqvi SW, Jayakumar DA, Narvekar PV, Naik H, Sarma VV, et al. (2000) Increased marine production of N2O due to intensifying anoxia on the Indian continental shelf. Nature 408(6810): 346–349.
- View Article
- Google Scholar
13. Stramma L, Johnson GC, Sprintall J, Mohrholz V (2008) Expanding Oxygen-Minimum Zones in the Tropical Oceans. Science 320(5876): 655–658.
- View Article
- Google Scholar
14. Keeling RF, Körtzinger A, Gruber N (2009) Ocean Deoxygenation in a Warming World. Annual Review of Marine Science 463–493.
- View Article
- Google Scholar
15. Diaz RJ, Rosenberg R (2008) Spreading Dead Zones and Consequences for Marine Ecosystems. Science 321(5891): 926–929.
- View Article
- Google Scholar
16. Duce RA, LaRoche J, Altieri K, Arrigo KR, Baker AR, et al. (2008) Impacts of Atmospheric Anthropogenic Nitrogen on the Open Ocean. Science 320(5878): 893–897.
- View Article
- Google Scholar
17. Deutsch C, Brix H, Ito T, Frenzel H, Thompson L (2011) Climate-Forced Variability of Ocean Hypoxia. Science 33: 336–339.
- View Article
- Google Scholar
18. Smethie WM Jr (1987) Nutrient regeneration and denitrification in low oxygen fjords. Deep-Sea Research 34(5–6): 983–1006.
- View Article
- Google Scholar
19. Ploug H (2001) Small-scale oxygen fluxes and remineralization in sinking aggregates. Limnology and Oceanography 46(7): 1624–1631.
- View Article
- Google Scholar
20. Woebken D, Fuchs BA, Kuypers MA, Amann R (2007) Potential interactions of particle-associated anammox bacteria with bacterial and archaeal partners in the Namibian upwelling system. Applied and Environmental Microbiology 73(14): 4648–4657.
- View Article
- Google Scholar
21. Galán A, Molina V, Thamdrup B, Woebken D, Lavik G, et al. (2009) Anammox bacteria and the anaerobic oxidation of ammonium in the oxygen minimum zone off northern Chile. Deep-Sea Research Part II 56(16): 1021–1031.
- View Article
- Google Scholar
22. Strous M, Van Gerven E, Kuenen JG, Jetten M (1997) Effects of Aerobic and Microaerobic Conditions on Anaerobic Ammonium-Oxidizing (Anammox) Sludge. Applied and Environmental Microbiology 63(6): 2446–2448.
- View Article
- Google Scholar
23. Paulmier A, Kriest I, Oschlies A (2009) Stoichiometries of remineralisation and denitrification in global biogeochemical ocean models. Biogeosciences 6(1): 2539–2566.
- View Article
- Google Scholar
24. Jensen MM, Kuypers MM, Lavik G, Thamdrup B (2008) Rates and regulation of anaerobic ammonium oxidation and denitrification in the Black Sea. Limnology and Oceanography 53(1): 23–36.
- View Article
- Google Scholar
25. Zumft WG (1997) Cell Biology and Molecular Basis of Denitrification. Microbiology and Molecular Biology Reviews 61(4): 533–616.
- View Article
- Google Scholar
26. Cline JD, Richards FA (1972) Oxygen Deficient Conditions and Nitrate Reduction in the Eastern Tropical North Pacific Ocean. Limnology and Oceanography 17(6): 885–900.
- View Article
- Google Scholar
27. Codispoti LA, Packard TT (1980) Denitrification Rates in the Eastern Tropical South-Pacific. Journal of Marine Research 38(3): 453–477.
- View Article
- Google Scholar
28. Gonzales PJ, Correia C, Moura I, Brondino CD, Moura JJ (2006) Bacterial nitrate reductases: Molecular and biological aspects of nitrate reduction. Journal of Inorganic Biochemistry 100: 1015–1023.
- View Article
- Google Scholar
29. Lipschultz F, Wofsy SC, Ward BB, Codispoti LA, Friedrich G, et al. (1990) Bacterial transformations of inorganic nitrogen in the oxygen-deficient waters of the Eastern Tropical South Pacific Ocean. Deep-Sea Research 37(10): 1513–1541.
- View Article
- Google Scholar
30. Lam P, Lavik G, Jensen MM, van De Vossenberg J, Schmid M, et al. (2009) Revising the nitrogen cycle in the Peruvian oxygen minimum zone. Proc Natl Acad Sci USA 106(12): 4752–4757.
- View Article
- Google Scholar
31. Dalsgaard T, Canfield DE, Petersen J, Thamdrup B, Acuna-Gonzalez J (2003) N2 production by the anammox reaction in the anoxic water column of Golfo Dulce, Costa Rica. Nature 422(6932): 606–608.
- View Article
- Google Scholar
32. Ward BB, Zafiriou OC (1988) Nitrification and nitric oxide in the oxygen minimum of the eastern tropical North Pacific. Deep-Sea Research 35(7): 1127–1142.
- View Article
- Google Scholar
33. Ward BB, Glover HE, Lipschultz F (1989) Chemoautotrophic Activity and Nitrification in the Oxygen Minimum Zone off Peru. Deep-Sea Research 36(7): 1031–1051.
- View Article
- Google Scholar
34. Lam P, Jensen MM, Lavik G, McGinnis DF, Muller B, et al. (2007) Linking crenarchaeal and bacterial nitrification to anammox in the Black Sea. Proc Natl Acad Sci USA 104(17): 7104–7109.
- View Article
- Google Scholar
35. Molina V, Farías L (2009) Aerobic ammonium oxidation in the oxycline and oxygen minimum zone of the eastern tropical South Pacific off northern Chile (∼20°S). Deep-Sea Research Part II 56(16): 1032–1041.
- View Article
- Google Scholar
36. Holmes RM, Aminot A, Keroul R, Hooker BA, Peterson BJ (1999) A simple and precise method for measuring ammonium in marine and freshwater ecosystems. Canadian Journal of Fisheries and Aquatic Sciences 56: 1801–1808.
- View Article
- Google Scholar
37. Grasshoff K, Ehrhdardt K, Kremling K, Anderson LG (1999) Determination of nutrients. Methods of seawater analysis. Wiley. pp. 159–226.
38. Holtappels M, Lavik G, Jensen MM, Kuypers MM (2011) N-labeling experiments to dissect the contributions of heterotrophic denitrification and anammox to nitrogen removal in the OMZ waters of the ocean. Methods in Enzymology 486: 223–251.
- View Article
- Google Scholar
39. McIlvin MR, Altabet MA (2005) Chemical Conversion of Nitrate and Nitrite to Nitrous Oxide for Nitrogen and Oxygen Isotopic Analysis in Freshwater and Seawater. Analytical Chemistry 77(17): 5589–5595.
- View Article
- Google Scholar
40. Granger J, Sigman DM, Prokopenko MG, Lehmann MF, Tortell PD (2006) A method for nitrite removal in nitrate N and O isotope analyses. Limnology and Oceanography: Methods 4: 205–212.
- View Article
- Google Scholar
41. Füssel J, Lam P, Lavik G, Jensen MM, Holtappels M, et al. (2011) Nitrite oxidation in the Namibian oxygen minimum zone. ISME Journal. In press.
- View Article
- Google Scholar
42. Hall GH (1984) Measurement of Nitrification Rates in Lake Sediments: Comparison of the Nitrification Inhibitors Nitrapyrin and Allylthiourea. Microbial Ecology 10(1): 25–36.
- View Article
- Google Scholar
43. Hall GH, Jeffries C (1984) The Contribution of Nitrification in the Water Column and Profundal Sediments to the Total Oxygen Deficit of the Hypolimnion of a Mesotrophic Lake (Grasmere, English Lake District). Microbial Ecology 10(1): 37–46.
- View Article
- Google Scholar
44. Bedard C, Knowles R (1989) Physiology, Biochemistry, and Specific Inhibitors of CH4, NH4+, and CO Oxidation by Methanotrophs and Nitrifiers. Microbiological Reviews 53(1): 68–84.
- View Article
- Google Scholar
45. Jensen MM, Thamdrup B, Dalsgaard T (2007) Effects of Specific Inhibitors on Anammox and Denitrification in Marine Sediments. Applied and Environmental Microbiology 73(10): 3151–3158.
- View Article
- Google Scholar
46. Kemmer G, Keller S (2010) Nonlinear least-squares data fitting in Excel spreadsheets. Nature Protocols 5(2): 267–281.
- View Article
- Google Scholar
47. Carr M (2002) Estimation of potential productivity in Eastern Boundary Currents using remote sensing. Deep-Sea Research 49: 59–80.
- View Article
- Google Scholar
48. Lavik G, Stuhrmann T, Bruchert V, Van Der Plas A, Mohrholz V, et al. (2008) Detoxification of sulphidic African shelf waters by blooming chemolithotrophs. Nature 457: 581–584.
- View Article
- Google Scholar
49. Bohlen L, Dale AW, Sommer S, Mosch T, Hensen C, et al. (2011) Benthic nitrogen cycling traversing the Peruvian oxygen minimum zone. Geochimica et Cosmochimica Acta 75(20): 6094–6111.
- View Article
- Google Scholar
50. Chapman P, Shannon LV (1985) The Benguela ecosystem. Part II. Chemistry and related processes. Mar Biol Ann Rev 23: 183–251.
- View Article
- Google Scholar
51. Fuenzalida R, Schneider W, Garces-Vargas J, Bravo L, Lange C (2009) Vertical and horizontal extension of the oxygen minimum zone in the eastern South Pacific Ocean. Deep-Sea Research II 56: 992–1003.
- View Article
- Google Scholar
52. Nicholls JC, Davies CA, Trimmer M (2007) High-resolution profiles and nitrogen isotope tracing reveal a dominant source of nitrous oxide and multiple pathways of nitrogen gas formation in the central Arabian Sea. Limnology and Oceanography 52(1): 156–168.
- View Article
- Google Scholar
53. Lam P, Jensen MM, Kock A, Lettmann KA, Plancherel Y, et al. (2011) Origin and fate of the secondary nitrite maximum in the Arabian Sea. Biogeosciences 8: 1565–1577.
- View Article
- Google Scholar
54. Robertson LA, Kuenen JG (1984) Aerobic denitrification: a controversy revived. Archives of Microbiology 139(4): 351–354.
- View Article
- Google Scholar
55. Robertson LA, Dalsgaard T, Revsbech N, Kuenen JG (1995) Confirmation of ‘aerobic denitrification’ in batch cultures, using gas chromatography and 15N mass spectrometry. FEMS Microbiology Ecology 18(2): 113–119.
- View Article
- Google Scholar
56. Gao H, Schreiber F, Collins G, Jensen MM, Kostka JE, et al. (2009) Aerobic denitrification in permeable Wadden Sea sediments. ISME Journal 4(3): 417–426.
- View Article
- Google Scholar
57. Körner H, Zumft WG (1989) Expression of Denitrification Enzymes in Response to the Dissolved Oxygen Level and Respiratory Substrate in Continuous Culture of Pseudomonas stutzeri. Applied and Environmental Microbiology 55(7): 1670–1676.
- View Article
- Google Scholar
58. Coyne MS, Tiedje JM (1990) Induction of denitrifying enzymes in oxygen-limited Achromobacter cycloclastes continuous culture. FEMS Microbial Ecology 73: 263–270.
- View Article
- Google Scholar
59. McKenney DJ, Drury CF, Findlay WI, Mutus B, McDonnell T, et al. (1994) Kinetics of denitrification by Pseudomonas fluorescens: Oxygen Effects. Soil Biology & Biochemistry 26(7): 901–908.
- View Article
- Google Scholar
60. Hartsock A, Shapleigh JP (2011) Physiological Roles for Two Periplasmic Nitrate Reductases in Rhodobacter Sphaeroides 2.4.3 (ATCC 17025). Journal of Bacteriology 193(23): 6483–6489.
- View Article
- Google Scholar
61. Gundersen K (1966) The Growth and Respiration of Nitrosocystis oceanus at Different Partial Pressures of Oxygen. J gen Microbiol 42: 387–396.
- View Article
- Google Scholar
62. Carlucci AF, Mcnally PM (1969) Nitrification by marine bacteria in low concentrations of substrate and oxygen. Limnology and Oceanography 14(5): 736–739.
- View Article
- Google Scholar
63. Goreau TJ, Kaplan WA, Wofsy SC, McElroy MB, Valois FW, et al. (1980) Production of NO2- and N2O by Nitrifying Bacteria at Reduced Concentrations of Oxygen. Applied and Environmental Microbiology 40(3): 526–532.
- View Article
- Google Scholar
64. Martens-Habbena W, Berube PM, Urakawa H, de La Torre JR, Stahl DA (2009) Ammonia oxidation kinetics determine niche separation of nitrifying Archaea and Bacteria. Nature 461(7266): 976–979.
- View Article
- Google Scholar
65. Stolper DA, Revsbech NP, Canfield DE (2010) Aerobic growth at nanomolar oxygen concentrations. Proc Natl Acad Sci USA 107(44): 18755–18760.
- View Article
- Google Scholar
66. Schmidt I, Bock E (1997) Anaerobic ammonia oxidation with nitrogen dioxide by Nitrosomonas eutropha. Arch Microbiol 167: 106–111.
- View Article
- Google Scholar
67. Lam P, Kuypers MM (2011) Microbial Nitrogen Cycling Processes in Oxygen Minimum Zones. Annual Review of Marine Science 317–347.
- View Article
- Google Scholar

[ref1] 1. Falkowski PG (2008) Earth's Biogeochemical Cycles. Science 320: 1034–1039.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Wyrtki K (1962) Circulation and Water Masses in the Eastern Equatorial Pacific Ocean. Deep-Sea Research. pp. 11–13.

[ref3] 3. Karstensen J, Stramma L, Visbeck M (2008) Oxygen minimum zones in the eastern tropical Atlantic and Pacific oceans. Progress in Oceanography 77: 331–350.
View Article
Google Scholar

[6] View Article

[7] Google Scholar

[ref4] 4. Kamykowski D, Zentara S (1990) Hypoxia in the world ocean as recorded in the historical data set. Deep-Sea Research 37(12): 1861–1874.
View Article
Google Scholar

[9] View Article

[10] Google Scholar

[ref5] 5. Helly JJ, Levin LA (2004) Global distribution of naturally occurring marine hypoxia on continental margins. Deep-Sea Research 51: 1159–1168.
View Article
Google Scholar

[12] View Article

[13] Google Scholar

[ref6] 6. Revsbech NP, Larsen LH, Gundersen J, Dalsgaard T, Ulloa O, et al. (2009) Determination of ultra-low oxygen concentrations in oxygen minimum zones by the STOX sensor. Limnology and Oceanography: Methods 7: 371–381.
View Article
Google Scholar

[15] View Article

[16] Google Scholar

[ref7] 7. Codispoti LA, Brandes JA, Christensen JP, Devol AH, Naqvi SW, et al. (2001) The oceanic fixed nitrogen and nitrous oxide budgets: Moving targets as we enter the anthropocene? Scienta Marina 65: 85–105.
View Article
Google Scholar

[18] View Article

[19] Google Scholar

[ref8] 8. Gruber N, Sarmiento JL (1997) Global Patterns of Marine Nitrogen Fixation and Denitrification. Global Biogeochemical Cycles 11: 235–266.
View Article
Google Scholar

[21] View Article

[22] Google Scholar

[ref9] 9. Kuypers MM, Lavik G, Woebken D, Schmid M, Fuchs BM, et al. (2005) Massive nitrogen loss from the Benguela upwelling system through anaerobic ammonium oxidation. Proc Natl Acad Sci USA 102(18): 6478–6483.
View Article
Google Scholar

[24] View Article

[25] Google Scholar

[ref10] 10. Hamersley MR, Lavik G, Woebken D, Rattray JE, Lam P, et al. (2007) Anaerobic ammonium oxidation in the Peruvian oxygen minimum zone. Limnology and Oceanography 52(3): 923–933.
View Article
Google Scholar

[27] View Article

[28] Google Scholar

[ref11] 11. Thamdrup B, Dalsgaard T, Jensen MM, Ulloa O, Farias L, et al. (2006) Anaerobic ammonium oxidation in the oxygen-deficient waters off northern Chile. Limnology and Oceanography 51(5): 2145–2156.
View Article
Google Scholar

[30] View Article

[31] Google Scholar

[ref12] 12. Naqvi SW, Jayakumar DA, Narvekar PV, Naik H, Sarma VV, et al. (2000) Increased marine production of N2O due to intensifying anoxia on the Indian continental shelf. Nature 408(6810): 346–349.
View Article
Google Scholar

[33] View Article

[34] Google Scholar

[ref13] 13. Stramma L, Johnson GC, Sprintall J, Mohrholz V (2008) Expanding Oxygen-Minimum Zones in the Tropical Oceans. Science 320(5876): 655–658.
View Article
Google Scholar

[36] View Article

[37] Google Scholar

[ref14] 14. Keeling RF, Körtzinger A, Gruber N (2009) Ocean Deoxygenation in a Warming World. Annual Review of Marine Science 463–493.
View Article
Google Scholar

[39] View Article

[40] Google Scholar

[ref15] 15. Diaz RJ, Rosenberg R (2008) Spreading Dead Zones and Consequences for Marine Ecosystems. Science 321(5891): 926–929.
View Article
Google Scholar

[42] View Article

[43] Google Scholar

[ref16] 16. Duce RA, LaRoche J, Altieri K, Arrigo KR, Baker AR, et al. (2008) Impacts of Atmospheric Anthropogenic Nitrogen on the Open Ocean. Science 320(5878): 893–897.
View Article
Google Scholar

[45] View Article

[46] Google Scholar

[ref17] 17. Deutsch C, Brix H, Ito T, Frenzel H, Thompson L (2011) Climate-Forced Variability of Ocean Hypoxia. Science 33: 336–339.
View Article
Google Scholar

[48] View Article

[49] Google Scholar

[ref18] 18. Smethie WM Jr (1987) Nutrient regeneration and denitrification in low oxygen fjords. Deep-Sea Research 34(5–6): 983–1006.
View Article
Google Scholar

[51] View Article

[52] Google Scholar

[ref19] 19. Ploug H (2001) Small-scale oxygen fluxes and remineralization in sinking aggregates. Limnology and Oceanography 46(7): 1624–1631.
View Article
Google Scholar

[54] View Article

[55] Google Scholar

[ref20] 20. Woebken D, Fuchs BA, Kuypers MA, Amann R (2007) Potential interactions of particle-associated anammox bacteria with bacterial and archaeal partners in the Namibian upwelling system. Applied and Environmental Microbiology 73(14): 4648–4657.
View Article
Google Scholar

[57] View Article

[58] Google Scholar

[ref21] 21. Galán A, Molina V, Thamdrup B, Woebken D, Lavik G, et al. (2009) Anammox bacteria and the anaerobic oxidation of ammonium in the oxygen minimum zone off northern Chile. Deep-Sea Research Part II 56(16): 1021–1031.
View Article
Google Scholar

[60] View Article

[61] Google Scholar

[ref22] 22. Strous M, Van Gerven E, Kuenen JG, Jetten M (1997) Effects of Aerobic and Microaerobic Conditions on Anaerobic Ammonium-Oxidizing (Anammox) Sludge. Applied and Environmental Microbiology 63(6): 2446–2448.
View Article
Google Scholar

[63] View Article

[64] Google Scholar

[ref23] 23. Paulmier A, Kriest I, Oschlies A (2009) Stoichiometries of remineralisation and denitrification in global biogeochemical ocean models. Biogeosciences 6(1): 2539–2566.
View Article
Google Scholar

[66] View Article

[67] Google Scholar

[ref24] 24. Jensen MM, Kuypers MM, Lavik G, Thamdrup B (2008) Rates and regulation of anaerobic ammonium oxidation and denitrification in the Black Sea. Limnology and Oceanography 53(1): 23–36.
View Article
Google Scholar

[69] View Article

[70] Google Scholar

[ref25] 25. Zumft WG (1997) Cell Biology and Molecular Basis of Denitrification. Microbiology and Molecular Biology Reviews 61(4): 533–616.
View Article
Google Scholar

[72] View Article

[73] Google Scholar

[ref26] 26. Cline JD, Richards FA (1972) Oxygen Deficient Conditions and Nitrate Reduction in the Eastern Tropical North Pacific Ocean. Limnology and Oceanography 17(6): 885–900.
View Article
Google Scholar

[75] View Article

[76] Google Scholar

[ref27] 27. Codispoti LA, Packard TT (1980) Denitrification Rates in the Eastern Tropical South-Pacific. Journal of Marine Research 38(3): 453–477.
View Article
Google Scholar

[78] View Article

[79] Google Scholar

[ref28] 28. Gonzales PJ, Correia C, Moura I, Brondino CD, Moura JJ (2006) Bacterial nitrate reductases: Molecular and biological aspects of nitrate reduction. Journal of Inorganic Biochemistry 100: 1015–1023.
View Article
Google Scholar

[81] View Article

[82] Google Scholar

[ref29] 29. Lipschultz F, Wofsy SC, Ward BB, Codispoti LA, Friedrich G, et al. (1990) Bacterial transformations of inorganic nitrogen in the oxygen-deficient waters of the Eastern Tropical South Pacific Ocean. Deep-Sea Research 37(10): 1513–1541.
View Article
Google Scholar

[84] View Article

[85] Google Scholar

[ref30] 30. Lam P, Lavik G, Jensen MM, van De Vossenberg J, Schmid M, et al. (2009) Revising the nitrogen cycle in the Peruvian oxygen minimum zone. Proc Natl Acad Sci USA 106(12): 4752–4757.
View Article
Google Scholar

[87] View Article

[88] Google Scholar

[ref31] 31. Dalsgaard T, Canfield DE, Petersen J, Thamdrup B, Acuna-Gonzalez J (2003) N2 production by the anammox reaction in the anoxic water column of Golfo Dulce, Costa Rica. Nature 422(6932): 606–608.
View Article
Google Scholar

[90] View Article

[91] Google Scholar

[ref32] 32. Ward BB, Zafiriou OC (1988) Nitrification and nitric oxide in the oxygen minimum of the eastern tropical North Pacific. Deep-Sea Research 35(7): 1127–1142.
View Article
Google Scholar

[93] View Article

[94] Google Scholar

[ref33] 33. Ward BB, Glover HE, Lipschultz F (1989) Chemoautotrophic Activity and Nitrification in the Oxygen Minimum Zone off Peru. Deep-Sea Research 36(7): 1031–1051.
View Article
Google Scholar

[96] View Article

[97] Google Scholar

[ref34] 34. Lam P, Jensen MM, Lavik G, McGinnis DF, Muller B, et al. (2007) Linking crenarchaeal and bacterial nitrification to anammox in the Black Sea. Proc Natl Acad Sci USA 104(17): 7104–7109.
View Article
Google Scholar

[99] View Article

[100] Google Scholar

[ref35] 35. Molina V, Farías L (2009) Aerobic ammonium oxidation in the oxycline and oxygen minimum zone of the eastern tropical South Pacific off northern Chile (∼20°S). Deep-Sea Research Part II 56(16): 1032–1041.
View Article
Google Scholar

[102] View Article

[103] Google Scholar

[ref36] 36. Holmes RM, Aminot A, Keroul R, Hooker BA, Peterson BJ (1999) A simple and precise method for measuring ammonium in marine and freshwater ecosystems. Canadian Journal of Fisheries and Aquatic Sciences 56: 1801–1808.
View Article
Google Scholar

[105] View Article

[106] Google Scholar

[ref37] 37. Grasshoff K, Ehrhdardt K, Kremling K, Anderson LG (1999) Determination of nutrients. Methods of seawater analysis. Wiley. pp. 159–226.

[ref38] 38. Holtappels M, Lavik G, Jensen MM, Kuypers MM (2011) N-labeling experiments to dissect the contributions of heterotrophic denitrification and anammox to nitrogen removal in the OMZ waters of the ocean. Methods in Enzymology 486: 223–251.
View Article
Google Scholar

[109] View Article

[110] Google Scholar

[ref39] 39. McIlvin MR, Altabet MA (2005) Chemical Conversion of Nitrate and Nitrite to Nitrous Oxide for Nitrogen and Oxygen Isotopic Analysis in Freshwater and Seawater. Analytical Chemistry 77(17): 5589–5595.
View Article
Google Scholar

[112] View Article

[113] Google Scholar

[ref40] 40. Granger J, Sigman DM, Prokopenko MG, Lehmann MF, Tortell PD (2006) A method for nitrite removal in nitrate N and O isotope analyses. Limnology and Oceanography: Methods 4: 205–212.
View Article
Google Scholar

[115] View Article

[116] Google Scholar

[ref41] 41. Füssel J, Lam P, Lavik G, Jensen MM, Holtappels M, et al. (2011) Nitrite oxidation in the Namibian oxygen minimum zone. ISME Journal. In press.
View Article
Google Scholar

[118] View Article

[119] Google Scholar

[ref42] 42. Hall GH (1984) Measurement of Nitrification Rates in Lake Sediments: Comparison of the Nitrification Inhibitors Nitrapyrin and Allylthiourea. Microbial Ecology 10(1): 25–36.
View Article
Google Scholar

[121] View Article

[122] Google Scholar

[ref43] 43. Hall GH, Jeffries C (1984) The Contribution of Nitrification in the Water Column and Profundal Sediments to the Total Oxygen Deficit of the Hypolimnion of a Mesotrophic Lake (Grasmere, English Lake District). Microbial Ecology 10(1): 37–46.
View Article
Google Scholar

[124] View Article

[125] Google Scholar

[ref44] 44. Bedard C, Knowles R (1989) Physiology, Biochemistry, and Specific Inhibitors of CH4, NH4+, and CO Oxidation by Methanotrophs and Nitrifiers. Microbiological Reviews 53(1): 68–84.
View Article
Google Scholar

[127] View Article

[128] Google Scholar

[ref45] 45. Jensen MM, Thamdrup B, Dalsgaard T (2007) Effects of Specific Inhibitors on Anammox and Denitrification in Marine Sediments. Applied and Environmental Microbiology 73(10): 3151–3158.
View Article
Google Scholar

[130] View Article

[131] Google Scholar

[ref46] 46. Kemmer G, Keller S (2010) Nonlinear least-squares data fitting in Excel spreadsheets. Nature Protocols 5(2): 267–281.
View Article
Google Scholar

[133] View Article

[134] Google Scholar

[ref47] 47. Carr M (2002) Estimation of potential productivity in Eastern Boundary Currents using remote sensing. Deep-Sea Research 49: 59–80.
View Article
Google Scholar

[136] View Article

[137] Google Scholar

[ref48] 48. Lavik G, Stuhrmann T, Bruchert V, Van Der Plas A, Mohrholz V, et al. (2008) Detoxification of sulphidic African shelf waters by blooming chemolithotrophs. Nature 457: 581–584.
View Article
Google Scholar

[139] View Article

[140] Google Scholar

[ref49] 49. Bohlen L, Dale AW, Sommer S, Mosch T, Hensen C, et al. (2011) Benthic nitrogen cycling traversing the Peruvian oxygen minimum zone. Geochimica et Cosmochimica Acta 75(20): 6094–6111.
View Article
Google Scholar

[142] View Article

[143] Google Scholar

[ref50] 50. Chapman P, Shannon LV (1985) The Benguela ecosystem. Part II. Chemistry and related processes. Mar Biol Ann Rev 23: 183–251.
View Article
Google Scholar

[145] View Article

[146] Google Scholar

[ref51] 51. Fuenzalida R, Schneider W, Garces-Vargas J, Bravo L, Lange C (2009) Vertical and horizontal extension of the oxygen minimum zone in the eastern South Pacific Ocean. Deep-Sea Research II 56: 992–1003.
View Article
Google Scholar

[148] View Article

[149] Google Scholar

[ref52] 52. Nicholls JC, Davies CA, Trimmer M (2007) High-resolution profiles and nitrogen isotope tracing reveal a dominant source of nitrous oxide and multiple pathways of nitrogen gas formation in the central Arabian Sea. Limnology and Oceanography 52(1): 156–168.
View Article
Google Scholar

[151] View Article

[152] Google Scholar

[ref53] 53. Lam P, Jensen MM, Kock A, Lettmann KA, Plancherel Y, et al. (2011) Origin and fate of the secondary nitrite maximum in the Arabian Sea. Biogeosciences 8: 1565–1577.
View Article
Google Scholar

[154] View Article

[155] Google Scholar

[ref54] 54. Robertson LA, Kuenen JG (1984) Aerobic denitrification: a controversy revived. Archives of Microbiology 139(4): 351–354.
View Article
Google Scholar

[157] View Article

[158] Google Scholar

[ref55] 55. Robertson LA, Dalsgaard T, Revsbech N, Kuenen JG (1995) Confirmation of ‘aerobic denitrification’ in batch cultures, using gas chromatography and 15N mass spectrometry. FEMS Microbiology Ecology 18(2): 113–119.
View Article
Google Scholar

[160] View Article

[161] Google Scholar

[ref56] 56. Gao H, Schreiber F, Collins G, Jensen MM, Kostka JE, et al. (2009) Aerobic denitrification in permeable Wadden Sea sediments. ISME Journal 4(3): 417–426.
View Article
Google Scholar

[163] View Article

[164] Google Scholar

[ref57] 57. Körner H, Zumft WG (1989) Expression of Denitrification Enzymes in Response to the Dissolved Oxygen Level and Respiratory Substrate in Continuous Culture of Pseudomonas stutzeri. Applied and Environmental Microbiology 55(7): 1670–1676.
View Article
Google Scholar

[166] View Article

[167] Google Scholar

[ref58] 58. Coyne MS, Tiedje JM (1990) Induction of denitrifying enzymes in oxygen-limited Achromobacter cycloclastes continuous culture. FEMS Microbial Ecology 73: 263–270.
View Article
Google Scholar

[169] View Article

[170] Google Scholar

[ref59] 59. McKenney DJ, Drury CF, Findlay WI, Mutus B, McDonnell T, et al. (1994) Kinetics of denitrification by Pseudomonas fluorescens: Oxygen Effects. Soil Biology & Biochemistry 26(7): 901–908.
View Article
Google Scholar

[172] View Article

[173] Google Scholar

[ref60] 60. Hartsock A, Shapleigh JP (2011) Physiological Roles for Two Periplasmic Nitrate Reductases in Rhodobacter Sphaeroides 2.4.3 (ATCC 17025). Journal of Bacteriology 193(23): 6483–6489.
View Article
Google Scholar

[175] View Article

[176] Google Scholar

[ref61] 61. Gundersen K (1966) The Growth and Respiration of Nitrosocystis oceanus at Different Partial Pressures of Oxygen. J gen Microbiol 42: 387–396.
View Article
Google Scholar

[178] View Article

[179] Google Scholar

[ref62] 62. Carlucci AF, Mcnally PM (1969) Nitrification by marine bacteria in low concentrations of substrate and oxygen. Limnology and Oceanography 14(5): 736–739.
View Article
Google Scholar

[181] View Article

[182] Google Scholar

[ref63] 63. Goreau TJ, Kaplan WA, Wofsy SC, McElroy MB, Valois FW, et al. (1980) Production of NO2- and N2O by Nitrifying Bacteria at Reduced Concentrations of Oxygen. Applied and Environmental Microbiology 40(3): 526–532.
View Article
Google Scholar

[184] View Article

[185] Google Scholar

[ref64] 64. Martens-Habbena W, Berube PM, Urakawa H, de La Torre JR, Stahl DA (2009) Ammonia oxidation kinetics determine niche separation of nitrifying Archaea and Bacteria. Nature 461(7266): 976–979.
View Article
Google Scholar

[187] View Article

[188] Google Scholar

[ref65] 65. Stolper DA, Revsbech NP, Canfield DE (2010) Aerobic growth at nanomolar oxygen concentrations. Proc Natl Acad Sci USA 107(44): 18755–18760.
View Article
Google Scholar

[190] View Article

[191] Google Scholar

[ref66] 66. Schmidt I, Bock E (1997) Anaerobic ammonia oxidation with nitrogen dioxide by Nitrosomonas eutropha. Arch Microbiol 167: 106–111.
View Article
Google Scholar

[193] View Article

[194] Google Scholar

[ref67] 67. Lam P, Kuypers MM (2011) Microbial Nitrogen Cycling Processes in Oxygen Minimum Zones. Annual Review of Marine Science 317–347.
View Article
Google Scholar

[196] View Article

[197] Google Scholar

Figures

Abstract

Introduction

Materials and Methods

Ethics Statement

Water sampling and nutrient analyses

15N labeling experiments

Oxygen sensitivity experiments

Data analysis

Results

Hydrochemistry in the Namibian OMZ

Hydrochemistry in the Peruvian OMZ

N-cycling in the Namibian and Peruvian OMZs

Distribution of anammox activity.

Distribution of nitrate reduction to nitrite activity.

Distribution of ammonia oxidation activity.

Oxygen sensitivity of anammox and coupled N-cycle processes

Oxygen sensitivity of anammox.

Oxygen sensitivity of nitrate reduction to nitrite.

Oxygen sensitivity of ammonia oxidation.

Discussion

Oxygen sensitivity of anammox in OMZ waters

Oxygen sensitivity of nitrate reduction in OMZ waters

Oxygen sensitivity of ammonia oxidation in OMZ waters

Implications for N-loss in the future ocean and our understanding of N-cycling in modern OMZs

Acknowledgments

Author Contributions

References

¹⁵N labeling experiments