Substantial oxygen consumption by aerobic nitrite oxidation in oceanic oxygen minimum zones

Nitrite oxidation rates in the ETNP

We sampled six stations in the ETNP OMZ with DO concentrations <20 µM at depth (Figs. 1 and 2). Three stations extending out from the coast of Mexico (Stations 1–3) were AMZ stations with distinct SCMs and accumulations of nitrite in SNMs indicative of anaerobic N cycling (Fig. 2). We expected that these would be hotspots of nitrite oxidation given oxygen supply via photosynthesis in the SCM overlapping with nitrite supply via nitrate reduction. Across all stations and depths, we observed the highest nitrite oxidation rates at the AMZ stations (Fig. 2C, G, K). Station 1 is located nearest the coast and had a shallow OMZ at the time of our sampling, with 20 µM DO at 28 m depth and nitrite >1 µM at 100 m (Fig. 2A). Chlorophyll concentrations were also high in the upper water column (up to 5 mg m⁻³ at 20 m), with an SCM spanning 70–125 m (Fig. 2B). Nitrite oxidation displayed a local maximum at the base of the EZ at Station 1 (20–30 m), and then increased to higher levels (>100 nmol L⁻¹ day⁻¹; Fig. 2C). This increase at 100 and 125 m corresponded with the overlap between the bottom of the SCM and the top of the SNM. Nitrite oxidation rates then reached higher values at 150 m within the SNM at Station 1. Stations 2 and 3 displayed similar nitrite oxidation rate profiles to each other, including elevated rates in the SCM (Fig. 2G, K). Nitrite oxidation rates were similar in magnitude, and peak values at the base of the EZ and in the OMZ were also similar (69–96 nmol L⁻¹ day⁻¹). Depth patterns tracked oceanographic differences across the three AMZ stations, as the depth of all features increased moving offshore from Stations 1 to 2 to 3. For example, the SCM extended from 105 to 155 m at Station 2, while nitrite concentrations began to increase below 100 m; nitrite oxidation rates were elevated at 140 m and declined slightly with increasing depth (Fig. 2E–G). At Station 3, the SCM (120–180 m) and SNM (>140 m) depths were deeper, and nitrite oxidation rates increased from 100 to 200 m (Fig. 2I–K).

In contrast to these three AMZ stations (Stations 1–3), rate profiles at Stations 4–6 showed peaks at the base of the EZ followed by decreases with depth and lacked a pronounced rate increase within the OMZ (Supplementary Fig. 1). Parallel measurements of ammonia oxidation rates also showed this type of pattern at all stations (Supplementary Fig. 1). Subsurface maxima in ammonia oxidation tracked variations in the EZ across all six stations, but rates were not elevated in OMZ/AMZ waters—again contrasting with nitrite oxidation rate profiles at the AMZ stations. These data accord with earlier work in OMZs showing contrasting ammonia and nitrite oxidation rate profiles, and particularly high rates of nitrite oxidation in OMZ waters^{6,7,8,29,30,31}.

Initial DO concentrations for these measurements closely matched in situ values above the SCM (where DO concentrations are higher), and starting DO ranged from 260–1500 nM for measurements in and below the SCM. These DO concentrations are generally lower than those used for previous nitrite oxidation rate measurements in OMZs^6,9, but similar to work examining the oxygen affinity of nitrite oxidation²² and overall oxygen consumption^16,19. Elevated nitrite oxidation in the limited number of samples (n = 5) collected below the SCM (>125 m at Station 1, >155 m at Station 2, and >180 m at Station 3)—where little to no DO is typically available—should be considered potential rates and could have a number of possible explanations discussed below. Within the SCM, our data support the idea that nitrite oxidation contributes to ‘cryptic’ oxygen cycling¹⁵—i.e., that DO produced via oxygenic photosynthesis is rapidly consumed.

Oxygen consumption via nitrite oxidation

We determined the contribution of nitrite oxidation to overall oxygen consumption via parallel measurements of OCRs using in situ optical sensor spots—which are noninvasive, provide nearly identical results as other low-level measurement approaches³², are the only effective means of achieving substantial replication, and for which sensitivity increases as DO decreases^32,33. Decreases in DO were measured in both nitrite and ammonia oxidation rate sample bottles, as well as in three additional replicates, to leverage statistical power for increased sensitivity to low-level DO consumption (see “Methods”). Water column OCR profiles at all stations showed exponential declines with depth and decreasing DO concentrations (Fig. 2D, H, L and Supplementary Fig. 1). Rates were highest in the upper water column and declined sharply within the upper portion of the OMZ above the SCM. The majority of measurements within the SCM—where DO may be produced via photosynthesis—were 100 s of nmol L⁻¹ day⁻¹, with an overall range of 160–1380 nmol L⁻¹ day⁻¹. Below the SCM, DO would be available more rarely (e.g., ref. ¹⁶), and OCR measurements represent potential rates should oxygen be supplied; OCR ranged from 120 to 390 nmol L⁻¹ day⁻¹. OCR also tracked variations in DO across stations, with progressively steeper declines in OCR with depth from Station 6 to Station 1.

These OCR results are similar to the limited previous measurements that have been conducted in OMZ regions, with some key differences. In particular, they are consistent with previous measurements of rapid DO consumption in the SCM, with OCR rates ranging from 482 to 1520 nmol-O₂ L⁻¹ day⁻¹ in the ETSP, and from 55 to 418 nmol-O₂  L⁻¹ day⁻¹ in the ETNP¹⁵. Earlier OCR measurements conducted in the ETNP near Stations 1 and 3 (across a wide range of DO values) likewise ranged from 420 to 828 nmol L⁻¹ day⁻¹ in the SCM near Station 1, and from 101 to 269 nmol L⁻¹ day⁻¹ in the SCM near Station 3 (ref. ¹⁶). Above the SCM, previous OCR measurements in the ETNP spanned 2260 to 662 nmol L⁻¹ day⁻¹ from the EZ to the edge of the OMZ; these values are lower than our measurements at 44 and 67 m depth at Station 2, but in line with our remaining measurements above the SCM. OCR reached 1610 nmol L⁻¹ day⁻¹ in the SCM in Namibian shelf waters and 200–400 nmol L⁻¹ day⁻¹ in the SCM off Peru¹⁸. Kalvelage et al.¹⁸ furthermore observed sharp decreases with depth in the ETSP, with rates declining from >1000 nmol L⁻¹ day⁻¹ above the SCM.

This pattern of declining OCR with increasing depth and decreasing DO was also evident in our dataset and contrasted with that of nitrite oxidation rates, which were notably elevated in the SCM at the AMZ stations (Fig. 2). We directly compared nitrite oxidation rates with OCR, assuming that each mole of nitrite is oxidized using ½ mole of O₂ (ref. ⁵). We found that nitrite oxidation systematically increased as a proportion of overall OCR at lower DO levels (Fig. 3A, B). Nitrite oxidation was responsible for up to 69% of OCR at Station 1, although most values were closer to 10–40% at Stations 2 and 3 (Fig. 3A, B). In contrast, ammonia oxidation contributed <5% of oxygen consumption in the OMZ (Supplementary Fig. 1). Overall, these data demonstrate consistent patterns in the contribution of nitrite oxidation to OCR, as the proportion of DO consumed by nitrite oxidation increased at progressively lower DO concentrations across multiple stations.

Effects of oxygen manipulation experiments on nitrite oxidation and OCR

We corroborated these observations by experimentally manipulating DO concentrations to measure the response of OCR, as well as ¹⁵NO₂⁻ nitrite oxidation rates, to changing DO (Fig. 4). To examine responses of different assemblages in different depth regions, we conducted experiments on the edge of the OMZ at Stations 2–4, in the SCM at Stations 2 and 3, and in the SNM at Station 3 and an additional sampling Station 3.5 (Table 1). (We could not conduct additional experiments at Stations 1 and 2 owing to a hurricane in the region at the time of sampling in 2018 and added Station 3.5 as a result.) Oxygen manipulations were designed to quantify OCR and nitrite oxidation across the full spectrum of DO concentrations that occur from the OMZ edge to its core—rather than solely probing their lower limits—in order to constrain rate responses to changing DO within OMZs.

Oxygen manipulation experiments were consistent with water column profiles and provide additional evidence that nitrite oxidation can be a substantial oxygen sink. In all experiments, we found that OCR declined with experimentally decreased DO (Fig. 4). These declines in OCR were particularly steep below 1–2 µM DO across different experiments. For the SCM and SNM experiments, DO concentrations >1 µM are obviously higher than expected to occur in situ (Table 1), but these concentrations were included for comparison across experiments and with earlier studies^9,15; as a result, OCR measurements for SCM and SNM samples conducted at DO concentrations >100 s of nM represent potential rates. For OMZ edge samples, OCR values in the µM range were higher than observed in profiles—most likely due to the effects of bubbling¹⁹, which could physically break down the organic matter present in higher concentrations at these depths (Table 1). Throughout all experiments, rate magnitudes in the 100 s of nM DO concentration range (11–820 nmol L⁻¹ day⁻¹) were similar to profile measurements (Fig. 2), as well as to previous measurements in OMZs^15,16,18,19 (see above).

DO concentrations were also continuously monitored in a subset of experimental bottles, and DO consumption was consistently linear (see “Methods”). The few exceptions occurred in several experiments conducted at DO concentrations <235 nM; in these experiments, OCR declined as DO was consumed over time (Supplementary Fig. 2). These observations are consistent with Tiano et al.¹⁶ and allowed us to calculate the low-level DO affinity of the community (following ref. ¹⁶), in addition to overall values based on variations across all experimental incubations (following ref. ¹⁹; Supplementary Note 1). However, we note that both reflect mixed assemblages of microorganisms that use DO to oxidize a variety of substrates (Supplementary Note 1). These substrates can include nitrite, as well as different forms of organic matter, reduced sulfur compounds, and possibly methane³⁴. Low-level results were consistent with previous observations indicating high affinity for DO (K_m ranged from 53 to 127 nM DO), and, importantly, all data demonstrate that OCR decreases as DO decreases (Supplementary Note 1 and Table 1).

In line with earlier work^22,24, nitrite oxidation rates also declined in response to decreasing DO in most of our experiments (Fig. 4). However, nitrite oxidation rates were less sensitive to declining DO than OCR (Supplementary Note 2). Nitrite oxidation was therefore responsible for progressively higher proportions of overall OCR as DO was experimentally decreased (Fig. 3C). In fact, we found that 97% of OCR in the SCM at Station 2 could be explained by measured nitrite oxidation rates—indicating that nitrite oxidation alone can sustain all DO consumption at DO concentrations just below 393 nM. However, most values were closer to 10–20%, and this contribution was obviously variable across experiments: while our experiment in the SCM at Station 2 showed that nitrite oxidation can consume all available DO (Fig. 4B), the experiment with 20 µM [DO] water at Station 2 suggests that, even at low DO levels, nitrite oxidation would not consume all DO (Fig. 4A). This highlights the fact that nitrite oxidation is unlikely to be dominant on the edge of the OMZ due to higher DO and organic matter concentrations, along with lower nitrite concentrations, compared with the SCM (Figs. 2 and 3 and Table 1). Overall, patterns in experiments were notably similar to results from rate profiles: both showed increases in the contribution of nitrite oxidation to DO consumption below 2 µM DO, both followed significant power-law relationships (experimental r² = 0.40, P < 0.0005; profiles r² = 0.51, P < 0.0001), and both displayed similar values (Fig. 3B, C).

These data tie together multiple aspects of OMZ biogeochemistry into a coherent picture. Although nitrite oxidation rates decline as DO is experimentally decreased at individual depths—and so with particular assemblages of NOB—different depths/assemblages display different properties (Fig. 4 and Supplementary Note 2). More importantly, this decline is always less severe for nitrite oxidation than for overall OCR (Figs. 2–4). NOB are therefore effective at scavenging DO (Figs. 3 and 4; ref. ¹⁵), and the concentration range over which they become increasingly important (0.2–1 µM DO) matches model predictions involving a nitrogen–oxygen feedback loop in OMZs driven by oxygen depletion via nitrite oxidation¹⁷.

Isotopic, 16S rRNA, and metagenomic constraints on nitrite oxidation

To further verify our findings, we applied natural abundance stable isotope measurements, 16S rRNA sequencing, and metagenome sequencing to water samples and nucleic acid samples collected in parallel with nitrite oxidation rate measurements (Fig. 5). The dual N and O isotopic composition of dissolved nitrate (δ¹⁵N and δ¹⁸O, respectively; Supplementary Fig. 3) provides an effective constraint on nitrite oxidation under low DO conditions due to isotopic “overprinting” by nitrite oxidation^35,36. While the respiratory reduction of nitrate (denitrification) leads to equal 1:1 increases in δ¹⁵N and δ¹⁸O (refs. ^37,38), deviations in 1:1 N:O isotope behavior arise from a decoupling of the N and O systems. These deviations are widely interpreted as reflecting cryptic reoxidation of nitrite under low DO, where the reduction of nitrate to nitrite removes an O atom, and the subsequent reoxidation of nitrite to nitrate appends a new O atom derived from ambient water^39,40. Deviations from the 1:1 relationship are represented via Δ(15,18) values⁴¹, where more negative values represent larger departures from the 1:1 relationship.

At Stations 1–3, dual nitrate isotope values exhibited nonlinear trends (Fig. 5B and Supplementary Fig. 4) that are consistent with isotopic overprinting by nitrite oxidation^35,36. Δ(15,18) values were lowest (i.e., deviations were largest) at 50–150 m at Station 1, 100–140 m at Station 2, and 110–160 m at Station 3. The lower portions of these depth ranges overlapped with the upper portion of the SCM (75–12, 105–155, and 120–180 m, respectively) and tracked depth variations between stations. Peak deviations at Stations 1 and 2 were just above the SCM (75 and 100 m), while the peak at Station 3 corresponded closely with the SCM (140 m). Throughout these ETNP OMZ sites, our nitrate isotope data are consistent with previous observations and interpretations of rapid recycling between nitrate and nitrite^35,41,42, ultimately evidenced by isotopic overprinting of the nitrate reduction signal by nitrite oxidation.

16S rRNA gene and transcript sequencing revealed a similar pattern to rate profiles, experiments, and isotopic data, while also enabling identification of NOB that are abundant and active in the ETNP OMZ. Based on DNA, Nitrospina 16S rRNA amplicon sequence variants (ASVs; ref. ⁴³) comprised up to 3.4–5.4% of all ASVs at Stations 1–3 (Fig. 5C), and relative abundances were consistent with biogeochemical data. Abundances and isotopic deviations were well-correlated (r² = 0.70–0.84, P < 0.05) at Stations 1 and 3, for instance, while abundances correlated with nitrite oxidation rates (r² = 0.20–0.46, P < 0.05) at Stations 2 and 3. Patterns observed for DNA samples were accentuated for RNA, with discrete peaks in 16S rRNA transcripts generally occurring at deeper depths (Fig. 5D). Nitrospina 16S rRNA peaked at 125 m at Station 1 (1.8% of all 16S rRNA transcripts; with an additional upper water column peak at 50 m), 140 m at Station 2 (3.2%), and particularly 140–180 m at Station 3 (1.6–7.1%). All of these depths lie within the SCM at each station. Although 20 Nitrospina ASVs were identified, three were dominant (each >1% of all ASVs, together constituting 72–100% of all Nitrospina 16S rRNA gene sequences and transcripts). One of these ASVs was found only in OMZ samples at Stations 1–3 and was not detected above the OMZ, nor at Stations 4 and 5 (Supplementary Fig. 5). This was also the lone ASV observed in the OMZ at Station 3, where Nitrospina were most active based on their comparatively high percentages of all 16S rRNA transcripts. In contrast, no Nitrococcus ASVs and only one low-abundance Nitrospira ASV were identified out of >11,000 ASVs and >3.5 million 16S sequences from 73 DNA and 73 RNA samples. In line with earlier work in the ETNP^7,26, other OMZs^6,44, and the variations in DO affinity observed across oxygen manipulation experiments (Fig. 4), these results indicate that particular Nitrospina ecotypes—and one ASV in particular—are significant for low-oxygen nitrite oxidation, while other ASVs may be more important at different depths and DO concentrations.

To validate the genetic potential for nitrite oxidation by Nitrospina under low DO concentrations, we sequenced metagenomes collected on the OMZ edge, at the SCM, and within the OMZ core at Stations 1, 2, and 3. Nitrite oxidoreductase (nxr) genes from Nitrospina were prevalent at all stations and depths, establishing the genomic potential for nitrite oxidation throughout the ETNP OMZ (Table 2). nxr and all Nitrospina genes were most abundant in the SCM, consistent with a range of omic data showing maximal Nitrospina gene or enzyme abundances in the upper portion of AMZs^2,7,15,26. In addition, multiple high-affinity cytochrome c oxidase genes from Nitrospina were present in all samples—indicating that Nitrospina is capable of consuming DO at the low concentrations found within the OMZ. These genes were more common in SCM and SNM metagenomes than at the OMZ edge, consistent with Nitrospina ecotype distributions, and with the lower DO concentrations found in the SCM and SNM (Table 2). Cytochrome bd-type oxidase genes from Nitrospina were also detected in all metagenomes except the OMZ edge sample at Station 1—although these lack quinol binding sites and may not function as canonical oxidases^26,45. Sun et al.²⁶ also suggested that chlorite dismutase genes may be relevant for anaerobic metabolism in Nitrospina, and these were present in all metagenomes (Supplementary Table 1). We also recovered formate dehydrogenase and nitrate reductase genes indicative of anaerobic metabolism in Nitrospina²⁵ (Supplementary Table 1). Both of these genes were absent from OMZ edge metagenomes at Stations 1 and 2, showing higher relative abundances in the SCM, and especially the SNM. Nitrospina-derived nitrate reductase genes were present at similar levels as nitrite oxidoreductase in the SNM, while less common in the SCM (Supplementary Table 1). Finally, Prochlorococcus genes were prevalent from the OMZ edge to core, and especially within the SCM, supporting the idea that oxygenic photosynthesis and cryptic oxygen cycling occur throughout the ETNP OMZ (Table 2). Overall, metagenomic data are consistent with experimental data and 16S data and indicate that Nitrospina is tightly tuned to DO concentrations in the ETNP.

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