Microbial N2O consumption in and above marine N2O production hotspots

The ocean is a net source of N2O, a potent greenhouse gas and ozone-depleting agent. However, the removal of N2O via microbial N2O consumption is poorly constrained and rate measurements have been restricted to anoxic waters. Here we expand N2O consumption measurements from anoxic zones to the sharp oxygen gradient above them, and experimentally determine kinetic parameters in both oxic and anoxic seawater for the first time. We find that the substrate affinity, O2 tolerance, and community composition of N2O-consuming microbes in oxic waters differ from those in the underlying anoxic layers. Kinetic parameters determined here are used to model in situ N2O production and consumption rates. Estimated in situ rates differ from measured rates, confirming the necessity to consider kinetics when predicting N2O cycling. Microbes from the oxic layer consume N2O under anoxic conditions at a much faster rate than microbes from anoxic zones. These experimental results are in keeping with model results which indicate that N2O consumption likely takes place above the oxygen deficient zone (ODZ). Thus, the dynamic layer with steep O2 and N2O gradients right above the ODZ is a previously ignored potential gatekeeper of N2O and should be accounted for in the marine N2O budget.


Introduction
Nitrous oxide (N 2 O) is not only a greenhouse gas with about 300 times greater radiative forcing per mole than carbon dioxide, it is also the dominant ozone-depleting agent emitted in the 21st century [1]. The N 2 O concentration in the atmosphere is increasing [2], and the rate of N 2 O emission is accelerating [3]. From 2007 to 2016, the ocean contributed 20% of global N 2 O emissions, and 35% of the natural sources on average [4]. N 2 O cycling in the ocean thus has the potential to exacerbate climate change, as well as being affected by associated chemical changes, such as ocean acidification [5]. The most intense sources and sinks of N 2 O in the ocean occur in oxygen minimum zones (OMZs) [6], which are marine regions characterized by a sharp O 2 gradient (oxycline) overlying an oxygen deficient zone (ODZ) where O 2 concentration is below the detection limit of a switchable trace oxygen (STOX) sensor (10 nM) [7]. There are multiple biological sources of N 2 O [8][9][10], but there is only one major biological sink [11] (Fig. S1): the reduction of N 2 O to N 2 by N 2 O-consuming microbes using the nitrous oxide reductase enzyme (N 2 OR). The possibility of N 2 O fixation has been suggested [12], but its mechanism is yet to be determined. N 2 O consumption in oxic waters, including the oxic layer of OMZs, has been ignored because this process was assumed to be part of the complete denitrification pathway (reduction of nitrate to N 2 gas) and to be restricted to suboxic/anoxic environments (such as ODZs) [13]. However, the oxic surface layer and the oxycline of OMZs above the ODZ could be of vital importance in regulating N 2 O emissions if N 2 O consumption occurs there. N 2 O concentration in the oxycline or the anoxic ODZ of OMZs can be ≥10-fold higher than atmospheric saturation at the air-sea interface [11]. If not consumed in situ, this excess N 2 O could diffuse through the oxycline and the surface layer or be upwelled into the surface where it can exchange with the atmosphere.
The functional marker of the operon encoding N 2 OR, nosZ, has been used as a proxy for the presence of N 2 Oconsuming microbes. Recent detection of nosZ genes and transcripts in oxic seawater [14,15] implies the potential for N 2 O consumption there. The presence of genes and transcripts, however, does not guarantee the successful translation or activity of the enzyme. Direct rate measurements are required to determine whether this microbial potential actually results in N 2 O consumption. Here, the abundance and community composition of N 2 O-consuming microbes were determined by qPCR and microarray, respectively. N 2 O-consuming microbes that contain only nosZ (i.e., none of the other genes in the complete denitrification pathway) are of particular interest, because their activity results in net N 2 O consumption. Based on the analysis of 652 draft or complete microbial genomes with one or more dentification genes, nosZ-only microbes are overrepresented among these isolates from the ocean compared to other ecosystems [16]. N 2 O consumption rates were also measured under a matrix of controlled N 2 O and O 2 concentrations in~3000 samples collected from oxic and anoxic depths in the Eastern Tropical North Pacific (ETNP) OMZ, one of the three major oceanic OMZs. O 2 tolerance and substrate kinetics of N 2 O consumption were determined and used to estimate in situ N 2 O consumption and production rates, which reflect the in situ conditions more accurately than directly using measured rates from incubation experiments without correcting for substrate additions.

Results and discussion
N 2 O consumption rates and N 2 O-consuming microbes in the OMZ Potential N 2 O consumption rates (hereafter "measured rates") were determined in March and April 2018 at three stations in the ETNP OMZ (Fig. 1a). Anoxic incubations were amended with standard additions of ( 15 N) 2 O tracer with a final concentration of 50 nM at stations PS1 (on the west margin of the OMZ), PS2 (the open ocean station) and PS3 (the coastal station). Measured N 2 O consumption rates varied from zero to 5.1 nM d −1 at different depths (Fig. 1c, f, i). Measured rates in oxygen deficient waters were on the same order of magnitude (a few nM d −1 ) as previously measured rates in the ETNP, but lower than rates at one coastal station in that study [6], indicating high variability of N 2 O cycling in the coastal regions as previously suggested [17]. Notably, significant N 2 O consumption rates were measured in these anoxic incubations, even in samples collected from the oxycline and the oxygenated surface ocean (in situ [O 2 ] up to 199 µM, Tables S1, S2), where N 2 O-consuming microbes were present and the nosZ gene was transcribed (Fig. 1d, g, j). In the upper water column, measured N 2 O consumption rates were highest in the upper oxycline, above the peak of in situ N 2 O concentrations at each station (Fig. 1b, e, h), and the rate maximum at station PS2 was detected in samples collected from 60 m where in situ O 2 was 173.9 µM (Tables S1, S2). The rates of N 2 O consumption measured in surface waters stripped of oxygen were similar to or larger than rates measured in the ODZ. The larger consumption rates in the oxic layer given the same N 2 O and O 2 concentrations as the ODZ layer might be due to more available dissolved organic matter at the shallower depths compared to the deeper ODZ layer [18] and/or the presence of different microbial communities at these depths. N 2 O-consuming microbes in the oxic surface water and oxycline were at least as abundant (DNA) and transcriptionally active (in terms of nosZ RNA abundance) as those in ODZs (Fig. 1d, g, j). Diverse archetypes of N 2 Oconsuming microbes were detected in the oxic water above the ODZ at three stations in the ETNP, one station in the Arabian Sea, and were previously detected at two stations in the Eastern Tropical South Pacific (ETSP) (Fig. 2a) using a nosZ microarray. The microarray is not quantitative, but it can detect diverse, low abundance microbes from environmental microbial assemblages. Even though the microarray cannot represent every nosZ variant, the probe set (which includes marine, salt marsh, and terrestrial representatives) allowed us to determine that the community composition of the ETSP N 2 O-consuming microbial assemblages differs from the other two OMZs (Fig. 2a). Within the ETNP, the community composition of nosZ microbes at the RNA level differed between oxic and ODZ waters (Fig. 2b). The detection of N 2 O consumption in samples from the oxycline and the oxygenated surface seawater of the ETNP OMZ, and the presence of N 2 O-consuming microbes in all three major OMZs (Fig. 2a), indicate that microbes in the oxic layer above ODZs have the capacity to consume N 2 O at least under anoxic incubation conditions.

O 2 tolerance of N 2 O-consuming microbes
The O 2 tolerance of N 2 O-consuming microbes was determined by incubating seawater under a range of O 2 conditions and measuring the O 2 concentration corresponding to each rate using Pyroscience optical O 2 sensors. N 2 O consumption rates were highest when O 2 was lowest in almost all incubations (Fig. 3). The highest rates were measured in anoxic incubations with samples from the oxic seawater (Fig. 3b, c and Table S3), and surprisingly, these rates were much higher than rates measured in samples collected from anoxic depths at the same station ( Fig. 3d-g). These results suggest that N 2 O-consuming microbes found in the oxic layer have the potential to metabolize N 2 O more rapidly than anaerobic organisms when conditions become anoxic ( Fig. 1d, g, j). At stations PS1 and PS3, the higher potential of N 2 O consumption also corresponds to higher nosZ gene copy numbers in the oxic layer (Fig. 1d, j). N 2 O consumption rates in samples from oxic seawater, but not the ODZ, decreased sharply with increasing O 2 , indicating that N 2 O-consuming microbes from oxic seawater (Fig. 3a-c) were more sensitive to O 2 than those from anoxic seawater ( Fig. 3d-g). Although N 2 O consumption in samples from the oxic layer did not occur at high O 2 concentrations, it started rapidly (≤1 day) after transitioning from oxic in situ conditions to anoxic incubation conditions (Fig. S2). The speed of this response might be due to the growth of N 2 Oconsuming microbes, fast enzyme (N 2 OR) translation, or a response by already translated N 2 OR in the oxic seawater prior to sampling.
While our rate measurements suggest that N 2 OR is not active under oxic conditions, molecular data obtained here (Fig. 1d, g, j) and previously [15] show that both nosZ RNA and N 2 OR can be synthesized under oxic conditions. This phenomenon is seen in other environments as well, for example, an obligate aerobe from soil requires O 2 to initiate  [37] and red dashed lines indicate the N 2 O concentration at equilibrium with the atmosphere [38]. c, f, i Black circles with black bars indicate measured N 2 O consumption rates with standard errors (also shown in Table S2). Error bars are standard errors of linear regression slope calculated from 15 point time-course incubations (three replicates at 5 time points). Blue circles indicate estimated in situ rates, which will be discussed in the "Estimated in situ N 2 O consumption and production rates" section. d, g, j Filled black circles indicate copy number of nosZ DNA in samples collected from the same casts from which rates were measured, filled gray circles indicate nosZ DNA from other casts at the same stations sampled within the same week, and red circles indicate copy numbers of nosZ RNA from the same casts. None of the RNA error bars overlapped with the zero line. Error bars are standard deviation (n = 3). Error bars are not shown if smaller than the symbol.
nosZ expression, and can use the N 2 OR enzyme to consume N 2 O to survive temporary anoxia [19]. Another microbial culture continually makes N 2 OR and stores the enzymes inside their cells under oxic conditions, which was proposed as a "bet-hedging" strategy by this facultative anaerobe to allow for a rapid transition into anoxic environments [20]. Regardless of the mechanism of the rapid response, our results indicate that microbes from oxic seawater have the genetic potential to consume N 2 O, that the consumption was not limited by organic matter supply (in situ limitation by organic matter would prevent the observed increase in rate with increasing N 2 O concentration, Fig. 4a-c) and that they could consume N 2 O under anoxic conditions. These anoxic conditions could occur at a small scale in otherwise oxic water; for example they could be associated with phytoplankton colonies [14] and other particles [21,22], especially in the productive and dynamic oxyclines of the OMZ with strong O 2 and N 2 O gradients at shallow depths.

Substrate affinity of N 2 O-consuming microbes
In incubations without O 2 , measured K m values (halfsaturation constants of the Michaelis-Menten curve) ( Fig. 4) for N 2 O consumption were in excess of in situ N 2 O concentrations at every station and depth (Fig. 1b, e, h), indicating that the in situ N 2 O concentrations were too low to saturate the N 2 O consumption rate. Notably, the potential maximum rate of N 2 O consumption in the oxic layer, upon removal of O 2 , was much higher than that in the ODZ at the same station (Fig. 4), and the substrate affinities of N 2 Oconsuming microbes were distinct between oxic and anoxic depths (Fig. 4). Consistently, the community composition of N 2 O-consuming microbes at the RNA level was also different between oxic layers and anoxic ODZs of the ETNP (Fig. 2b). These results indicate that different kinetics parameters should be applied to the estimate of the anoxic N 2 O sink and the newly discovered potential N 2 O sink in the oxic layer. The difference in substrate affinities is likely due to diverse N 2 O-consuming microbes occupying different niches, because their affinities for N 2 O can vary by two orders of magnitude as shown in pure cultures [23]. This difference might be obscured when microbes in different layers are mixed up by physical processes such as upwelling and eddies.
Significant K m for N 2 O consumption could be determined for the oxic layer at station PS2 and the oxic-anoxic interface at station PS3 (Fig. 4b, e). The lack of Michaelis-Menten kinetics in samples from anoxic ODZs (Fig. 4f, g) implies that factors (e.g., organic matter) other than the added substrate were limiting N 2 O consumption. K m for the oxic layer at station PS3 was likely larger than that of station PS2, because the rate was not saturated even at the maximum N 2 O concentration (Fig. 4c). As for station PS1, the decreasing rate at the 4th datapoint suggests a mixture of N 2 O-consuming microbes with different substrate affinities (Fig. 4a). Although not significant, K m was 110 (±230) nM when only including the first four datapoints. When excluding the 4th datapoint, K m was 1354 (±653) nM for the oxic layer at station PS1. Larger K m values in samples from the oxic layer indicate that microbes there have lower affinity for N 2 O.
Notably, nosZ archetypes closely related to A. dehalogenans were among the top five most abundant archetypes at almost all examined depths from the ETNP (Tables S4,  S5), ETSP [15], and Arabian Sea (Table S6), implying the importance of A. dehalogenans-like N 2 O-consuming microbes in both oxic layers and ODZs of all major OMZs. Furthermore, the K m (1.3 μM) of Anaeromyxobacter dehalogenans determined in cultures [23] was within the range of the K m values determined in this study (2.8 μM in the oxic layer and 0.3 μM at the top of the ODZ, Fig. 4). Different overall community composition, but similar affiliation of the most abundant archetypes, implies that the difference in community composition between the oxic and ODZ assemblages in the ETNP (Fig. 2b) and the difference between the ETSP and the other two OMZs (Fig. 2a) results AS (Arabian Sea)). Arabian Sea samples include two samples from the oxic layer above the ODZ and two inside the ODZ at station 1 (19N, 66E) collected on a previous cruise [34], and ETSP samples include four samples from oxic layers and four inside ODZs. ETSP data were obtained from a previous study [15]. b nosZ data from the ETNP only. from a diversity of low abundance microbes rather than a few abundant clades. The microarray probes cannot identify exact species, but can differentiate among archetypes representing unknown microbes, such as the A. dehalogenans-like N 2 O-consuming types that were detected in most of these samples. The vital role of A. dehalogenanslike microbes and other microbes in the same clade has been demonstrated in soils [24,25]. Because A. dehalogenans possesses nosZ but no other denitrification genes [26], A. dehalogenans-like microbes may decouple N 2 O consumption from its production, resulting in a net N 2 O sink at depths where they dominate N 2 O-consuming assemblages.
In addition to anoxic incubations, we also examined N 2 O consumption kinetics in oxic incubations. Consistent with the low O 2 tolerance of N 2 O-consuming microbes, especially in the oxic layers (Fig. 3), the kinetics of N 2 O consumption could not be determined under most incubations with O 2 additions because N 2 O consumption rates were not detected (Fig. 4). Only samples from the oxic-anoxic interface at station PS3 showed Michaelis-Menten kinetics in the oxic incubation (Fig. 4e), likely because the O 2 addition in this incubation (4.5 μM, Table S3) was less than all the other oxic incubations (8.1-342.0 μM, Table S3) and N 2 O-consuming microbes in ODZs had higher tolerance to increasing O 2 concentration than those from the oxic layer (Fig. 3e) [10,28]. In addition, unlike the presence of nosZ in the oxic layers, the group of denitrifiers represented by nitrite reductase genes (nirK and nirS) were very rare in the oxic layers [29]. These observations suggest that denitrification can be carried out in a

Estimated in situ N 2 O consumption and production rates
Rates of biogeochemical processes inferred from incubation experiments can be biased away from in situ values due to the dependence of rates on substrate concentrations and other environmental factors (e.g., O 2 concentration), which often differ between in situ and incubation conditions. However, the new quantitative information on N 2 O consumption kinetics and effects of environmental factors like O 2 derived here can be used to estimate in situ rates.
Using these kinetic parameters most in situ N 2 O consumption rates were inferred to be zero in oxic layers (Fig. 1c, f, i) based on the high sensitivity of those microbes to O 2 (Fig. 3a-c). The exception was 90 m at station PS2 where the in situ O 2 concentration (4.4 µM, Table S1) was likely to be low enough to allow N 2 O consumption based on similar kinetics in anoxic incubations and incubations at PS3 with 4.5 µM O 2 (Fig. 4e). In situ N 2 O consumption rates in anoxic ODZs were simulated by the Michaelis-Menten equation using the K m value determined here (Fig. 4e) and the in situ N 2 O concentrations (Fig. 1b, e, h). Measured N 2 O consumption rates had maxima in the upper oxycline above the N 2 O concentration peaks at all three stations, but the maxima in estimated in situ rates at these stations occurred at or below the oxic-anoxic interface, and the highest rate at station PS2 (6.3 nM d −1 ) was at 850 m, the lower edge of the ODZ (Fig. 1c, f, i). The secondary peak of N 2 O consumption inside the ODZ at station PS2 was greatly reduced after corrections were made using in situ N 2 O concentrations due to low in situ N 2 O concentrations (Fig. 1e). The peak inside the ODZ at station PS1, however, was larger after correction because the in situ N 2 O concentration (~80 nM, Fig. 1b) was higher than that in incubations (50 nM). The persistently high N 2 O concentration in the ODZ core at station PS1 reflects the slow N 2 O removal by denitrification at the margin of the OMZ. The lack of a SNM, a typical feature for anoxic ODZs, at station PS1 (Fig. S3) is also consistent with its position at the oceanic edge of the OMZ. The difference between measured rates and kinetics-corrected in situ rates indicates the need for more information on the kinetics of N 2 O consumption under different environmental conditions and in different OMZ regions. In situ N 2 O production rates were modeled from estimated in situ N 2 O consumption rates, N 2 O concentrations, advection, and diffusion using a 1-D steady-state framework (Fig. 5), which reflects a weak lateral advection and upwelling scenario as in a previous study [6]. Production and consumption rates were mostly balanced but were decoupled at the sharp N 2 O concentration gradient at station PS3. The decoupling of the production and consumption was due to the strong N 2 O fluxes from physical processes (i.e., advection and diffusion) in the sharp N 2 O gradient coinciding with the sharp O 2 gradient. This decoupling was not reported previously because measurements of N 2 O consumption were all below the bottom of the upper oxycline [6]. Notably, the modeled production rates in the oxic layer at stations PS1 and PS3 (Fig. 5) with 199.0 and 89.9 µM in situ O 2 were negative considering advection, diffusion and zero estimated consumption rates (Table S1). Since the production rate cannot be negative, this analysis suggests that N 2 O consumption occurs at least sometimes at these depths to balance the N 2 O flux from physical processes (assuming steady state). Consistent with the model results, N 2 O consumption rates were detected when oxygen is above the 4.5 µM threshold especially at station PS3 (Fig. 3c, e, j). The significant rates under oxic conditions might imply more micro-anoxic sites (e.g., particulate organic matter) at the coastal station. Although we chose 4.5 µM as a conservative oxygen threshold here, different thresholds for N 2 O consumption need to be determined for different environmental conditions in future studies. Particles and other microsites might disintegrate during sampling and purging, so the in situ N 2 O consumption rates in the oxic layer were potentially underestimated by the incubation experiments.

Implications for the oceanic N 2 O budget
The annual N 2 O emission rate of the ocean is 3-5 Tg-N yr −1 based on recent estimates [4,30]. Developing an understanding of what controls the major biological sink of N 2 O is vitally important to better constrain estimation of the highly uncertain [17] marine N 2 O budget. Using direct rate measurements, we demonstrated the ability of microbial assemblages in the oxic layer above N 2 O production hotspots to consume N 2 O under anoxic conditions and quantified the rate dependence on N 2 O and O 2 concentrations. The potential N 2 O consumption rate in oxic seawaters was at least two orders of magnitude faster than that in ODZs under favorable conditions (i.e., low O 2 and high N 2 O concentrations). Even though N 2 O is unlikely to be consumed when surface seawater is saturated with O 2 , N 2 O consumption started rapidly after switching to anoxic conditions. N 2 O consumption also occurred in the oxycline where O 2 concentration was low and N 2 O concentration was high. High N 2 O concentrations in the oxycline are attributed to production from both denitrification and nitrification, with dentification as the main source [10]. Assuming anoxia occurs transiently in the layer above the ODZ, the consumption rate (0.7-10.3 nM d −1 ) estimated from measured rates using in situ N 2 O concentrations (Table S1) are on the same order of magnitude as N 2 O production rates measured in the ETNP OMZ [10]. These rates suggest a potential gross N 2 O sink in the layer above the ODZ of all OMZs of 0.02-0.32 Tg-N yr −1 assuming a 10 cm transiently anoxic depth interval of this layer with a volume of 3.04 × 10 11 m 3 [31]. Although anoxia is unlikely to occur in such a large area at once, this potential gross sink of N 2 O is considerable relative to an annual oceanic N 2 O emission of 3-5 Tg-N yr −1 [4,30].
Anoxic conditions could occur in microsites such as particles [21] and phytoplankton aggregates [14] in the oxic layer. nosZ transcripts were found to be highly enriched in particle-associated fractions compared to free-living fractions in the ETNP OMZ [22]. Additionally, eddies, upwelling and other dynamic mixing events could lead to the shoaling of low oxygen, high N 2 O seawater, and N 2 Oconsuming microbes [32]. O 2 concentrations in the oxycline above the ODZ of the ETNP varied from nearly 100 µM to below detection within days or weeks based on both Argo float data (Fig. S4) and conductivity-temperature-depth (CTD) Seabird data (Fig. S5). Thus, microbes in the surface layer or subsurface microbes being brought to the surface layer could consume a portion of N 2 O before it escapes into the atmosphere when surface anoxia occurs.
The detection of viable N 2 O-consuming microbes in the upper oxyclines of all three OMZs implies a potential role for unconventional nosZ-containing microbes in regulating the N 2 O budget. The presence of these microbes in all three OMZs and their potential N 2 O sink raises the necessity of quantifying this potential in the other two OMZs. The substrate kinetics and biological information obtained in this study provide previously lacking parameters for the characterization of N 2 O consumption in marine N 2 O models. Applying the new O 2 threshold obtained here for N 2 O consumption (4.5 µM) to a mechanistic 1-D biogeochemistry model [6] produces N 2 O peaks and O 2 profiles similar to our measurements at the open ocean station when the O 2 threshold for N 2 O production via denitrification was also increased (to 20 µM [10, 28]) (Fig. S6). The O 2 thresholds for N 2 O production and consumption along the redox gradient of the ocean need further investigation, but the high O 2 tolerance of N 2 O production from denitrification in the modified model is consistent with previous experimental results showing the persistence of N 2 O production from NO 3 − at 7 and 23 µM O 2 in the ETNP [10] and ETSP [28], respectively. Further kinetics and molecular experiments are required to investigate the cooccurrence of microbes with different substrate kinetics in the same sample (Fig. 4a) and the spatial variation of microbial communities for both N 2 O consumption and production. The OMZs are not only the most intense N 2 O cycling regions [6,10], but also contribute to a large seasonal variation to the global N 2 O emissions [30]. Thus, the findings here will not only improve estimates of these N 2 O sinks, but also will improve estimates of N 2 O sources, two crucial variables to constrain in a changing ocean.

Sampling, incubations, and rate measurements
The sampling sites are within the ETNP OMZ, one of the three major OMZs in the world. We sampled at three stations (OMZ margin station PS1, open ocean OMZ station PS2, and coastal OMZ station PS3; Fig. 1a) in March and April 2018 on board R/V Sally Ride (Cruise ID: SR 1805). The three sampling stations represent a transect from offshore to onshore, along a gradient from low to high productivity, and from the oceanic edge of the OMZ to the intense ODZ of the coastal station. Station PS1 is on the margin of the ETNP OMZ, so oxygen intrusion events likely occur below the upper oxycline at this station. Station PS1 does not have a secondary nitrite maximum (SNM), while stations PS2 and PS3 both have SNM in their ODZs (Fig. S3). PS1 and PS2 are open ocean stations, and PS3 is a coastal station. Station PS3 has a very shallow oxycline close to the surface of the ocean. This shallow oxyline is probably due to the intense heterotrophic respiration fueled by the primary production (implied by chlorophyll concentrations of 3 µg/L at 10 m) at the surface. Twelve 30 L Niskin bottles on a rosette with a CTD profiler were used to collect seawater from different depths while recording temperature, pressure, salinity, chlorophyll, and in situ O 2 concentration with both Seabird (Sea-Bird SBE 9, Sea-Bird Electronics, Bellevue, NA) and STOX sensors [7]. Samples for measurements of in situ N 2 O concentration were collected from Niskin bottles into 160 mL bottles after overflowing three times and preserved with saturated HgCl 2 . N 2 O concentrations were measured on an isotope ratio mass spectrometer based on the major ion (m/z = 44) peak area [33]. Particulate material for microbial DNA and RNA analysis was collected by filtering up to 4 L seawater from Niskin bottles through Sterivex filters (0.22 µm). Filters were flash frozen in liquid N 2 on board, and then preserved at −80°C until DNA and RNA extraction in the lab. Seawater samples for determining N 2 O consumption rate were collected into 320 mL ground glassstoppered glass bottles after overflowing three times to minimize O 2 contamination. Seawater (8 mL) was then aliquoted into 12 mL exetainers inside a N 2 flushed glove bag to leave a 4 mL headspace for purging. After sealing in the glove bag, exetainers were purged with helium for 5 min to reach anoxia for depth profiles shown in Fig. 1 Fig. 4. A 50 nM N 2 O addition was made to ensure the produced N 2 was detectable. A set of 15 exetainers incubated in a time series (triplicates for each time point, 5 time points in total including three time zero bottles as abiotic controls) was used to determine a single rate. Incubations were sampled approximately every 12 h for 2 days and were terminated by adding 0.05 mL of 50% (w/v) ZnCl 2 following previous procedures [6]. The amount and isotopic composition of N 2 in each exetainer was measured on a mass spectrometer (Europa Scientific 20-20, Crewe, UK), and the rate of N 2 O consumption was calculated from the linear regression of the excess of 30 N 2 over the incubation time following the previous method [34].  were achieved prior to initiation of the experiment by varying flow rates of O 2 and helium gases using a custom-assembled gas flow manifold with two gas flow meters on board. The O 2 concentration for each set of exetainers was monitored by direct measurement using optical oxygen sensors with a detection limit of 0.06 µM (PyroScience GmbH, Aachen, Germany), and is shown on the x-axis in Fig. 3.

Kinetics models and in situ rate estimation
Half-saturation constant (K m ) and the maximum rate (V m ) were determined by fitting N 2 O consumption rate and N 2 O concentration data to the Michaelis-Menten equation (Eq. (1)). K m is the N 2 O concentration at which the rate (V) equals half of V m . Fitting was performed by the curve fitting tool in Matlab. The 95% confidence interval was used to determine whether a parameter is significantly different from zero. The half-inhibition constant (K i ), analogous to K m , is the O 2 concentration that causes half of the potential maximum inhibition (I m ). K i was calculated from fitting an inhibition curve (Eq. (2)). The unit of V or V m is nM d −1 , the unit of [N 2 O] or K m is nM, the unit of [O 2 ] or K i is µM and I m is unitless.
The in situ N 2 O consumption rate was estimated from in situ O 2 concentration, in situ N 2 O concentration, and calculated K m and V m . First, K m in Fig. 4, measured rates (V) and measured [N 2 O] were used to calculate V m for each depth based on Eq. (1). K m and V m determined in samples from the oxic layer were used to calculate in situ rates in oxic seawaters, and those determined in samples from the ODZ were used in ODZ rate estimation. Then, in situ rates were set to zero if in situ O 2 concentrations were above a threshold level of 4.5 µM. This threshold was chosen because it was the highest oxygen concentration at which N 2 O consumption was dependent upon N 2 O concentration i.e., N 2 O consumption showed similar kinetics in response to N 2 O concentration under undetectable O 2 concentration and at 4.5 µM O 2 (Fig. 4e), but not at higher O 2 concentrations. This is a conservative threshold because we did not determine the absolute highest O 2 level that allowed N 2 O consumption.
In situ N 2 O production rate was estimated by subtracting the advection and diffusion of N 2 O from in situ consumption rates assuming a steady state (Eq. (3)). v (1 × 10 −7 m s −1 ) is advection coefficient and D (2 × 10 −5 m 2 s -1 ) is the diffusivity coefficient. Coefficients and the steady-state 1-D model follow (Babbin et al.) [6].
A mechanistic 1-D biogeochemistry model [6] was updated based on the experimental results in this study to assess the effect of different O 2 thresholds on N 2 O predictions (Fig. S6). Briefly, the model was built upon the balance of physical and biological processes at steady state. Physical processes in the model include advection, diffusion, and gravitational sinking. Biological processes include aerobic respiration (i.e., remineralization fueled by O 2 ), nitrification, and denitrification. The production of N 2 O from both nitrification and denitrification, and the consumption of N 2 O from denitrification are considered. The DNA and RNA extraction, quantitative PCR (qPCR) assays, and nosZ microarray These experiments were performed as previously described [15]. Briefly, DNA and RNA were extracted from Sterivex filters including four filters collected from a previous cruise in the Arabian Sea OMZ [34]. Each DNA or RNA copy number value corresponds to one Sterivex. RNA was reverse transcribed into cDNA. qPCR was used to estimate the abundance of total and transcribed nosZ assemblages using the nosZ1F (5′ -WCSYTGTTCMTCGACAGCCAG-3′) and nosZ1R (5′-ATGTCGATCARCTGVKCRTTYTC-3′) primer set [35]. The qPCR products were purified from agarose gels and then used as targets for microarray experiments following a previous protocol [36]. The detection limit of qPCR is 18.1 copies mL −1 . The microarray contains 114 nosZ archetype probes and the sequences of all probes are published in the supplementary dataset in a previous study [15]. The fluorescence ratio of each archetype on the microarray is defined as the ratio of Cy3 to Cy5 fluorescence. Normalized fluorescence ratio (FRn) was calculated by dividing the fluorescence ratio of each archetype by the maximum fluorescence ratio on the same microarray. FRn is used as a proxy for the relative abundance of each nosZ archetype. FRn was used to determine the top five most abundant archetypes in each sample. Detrended correspondence analysis was performed on FRn to analyze the community composition of N 2 O-consuming assemblages using the vegan package in R (version 3.6.0). Identification of nosZ sequences is limited by the probe selection on the array and the larger database of nosZ sequences now available might help identify the oxic nosZ more precisely. Nonetheless, the limited database represented on the array sufficed to detect significant differences among samples and to identify phylogenetic affinities of OMZ nosZ genes.
Author contributions XS and BBW conceptualized the research. XS, BBW, and JCT sampled and performed incubation experiments on board. CLK and KLC sampled and measured N 2 O concentrations. AJ collected microbial samples, extracted DNA, and performed qPCR and microarray experiments. XS and EW assisted with DNA extraction and qPCR experiments. XS, AJ, and BBW analyzed microbial data. XS measured N 2 O consumption rates, analyzed the data, and modeled the rates. XS and BBW wrote the paper with inputs from all authors.

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