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

  • Tim Kalvelage ,

    tkalvela@mpi-bremen.de

    Affiliation Department of Biogeochemistry, Max Planck Institute for Marine Microbiology, Bremen, Germany

  • Marlene M. Jensen,

    Current address: Department of Environmental Engineering, Technical University of Denmark, Kongens Lyngby, Denmark

    Affiliation Department of Biogeochemistry, Max Planck Institute for Marine Microbiology, Bremen, Germany

  • Sergio Contreras,

    Current address: Large Lakes Observatory, University of Minnesota Duluth, Duluth, Minnesota, United States of America

    Affiliation Department of Biogeochemistry, Max Planck Institute for Marine Microbiology, Bremen, Germany

  • Niels Peter Revsbech,

    Affiliation Department of Biological Sciences, University of Aarhus, Aarhus C, Denmark

  • Phyllis Lam,

    Affiliation Department of Biogeochemistry, Max Planck Institute for Marine Microbiology, Bremen, Germany

  • Marcel Günter,

    Affiliation Department of Biogeochemistry, Max Planck Institute for Marine Microbiology, Bremen, Germany

  • Julie LaRoche,

    Affiliation Department of Marine Biogeochemistry, Leibniz Institute of Marine Sciences (IFM-GEOMAR), Kiel, Germany

  • Gaute Lavik,

    Affiliation Department of Biogeochemistry, Max Planck Institute for Marine Microbiology, Bremen, Germany

  • Marcel M. M. Kuypers

    Affiliation Department of Biogeochemistry, Max Planck Institute for Marine Microbiology, Bremen, Germany

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
PLOS
x

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.

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.

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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.

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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.

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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.

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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

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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).

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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.

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