Measurements of nitrite production and nitrite-producing organisms in and around the primary nitrite maximum in the central California Current

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Full ) but were either below detection limits or 10 times lower than NH 3 oxidation rates around the PNM.One-dimensional modeling of water column NO − 2 profiles supported direct rate measurements of a net biological sink for NO − 2 just below the PNM.Residence time estimates of NO − 2 within the PNM were similar at the mesotrophic and oligotrophic stations and ranged from 150-205 d.Our results suggest the PNM is a dynamic, rather than relict, feature with a source term dominated by ammonia oxidation.

Nitrite (NO −
2 ) sits at the center of the nitrogen cycle.It fuels microbial metabolism as an intermediate in both nitrification and denitrification, and is a substrate for anaerobic ammonium oxidation (anammox).NO Introduction

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Full questions unanswered about what limits primary productivity, nitrogen remineralization, or both in the lower euphotic zone.Direct measurements of nitrate assimilation into phytoplankton (Dugdale and Goering, 1967), nitrate reductase enzyme activity (Eppley et al., 1969), and more recent nucleic acid-based methods for detecting nitrate reductase genes (Jenkins et al., 2006) (Paerl et al., 2008;Ward, 2008) and gene transcripts (Paerl et al., 2012) have all shown the importance and prevalence of nitrate-reducing activity in marine phytoplankton and the potential for nitrite production.None of these methods, however, address how much nitrite is released into the water column where it is detected in the dissolved phase, versus how much remains in the cell.High sensitivity, high precision techniques now allow access to stable isotope ratios of both nitrogen and oxygen in NO − 2 (δ 15 N NO2 and δ 18 O NO2 ) (McIlvin and Altabet, 2005;Casciotti and McIlvin, 2007).Understanding the sources and sinks of NO − 2 in the ocean are important to enabling natural abundance stable isotopes as a tracers of the balance between productive and consumptive pathways (Buchwald andCasciotti, 2010, 2013;Casciotti et al., 2010;Buchwald et al., 2012).
It has been recognized for some time that AOB are present at the depth of the PNM (Olson, 1981a;Ward et al., 1982).More recently, marine AOA abundance and AOA : AOB ratios have been correlated with [NO − 2 ] (Murray et al., 1999;Beman et al., 2010).Archaeal genes for ammonia oxidation (ammonia monooxygenase subunit A, amoA) have also been found in and around the PNM (Beman et al., 2010;Santoro et al., 2010;Newell et al., 2011).The photosynthetic community present near the PNM, at approximately the 1 % light depth, is composed predominantly of low light Prochlorococcus ecotypes (Rocap et al., 2003) and picoeukaryotes.Both of these groups have been recently implicated in active NO − 3 utilization and thus are a potential source of NO − 2 in the PNM.Metagenomic data (Martiny et al., 2009), flow cytometry coupled with stable isotope tracers (Casey et al., 2007) and natural abundance stable isotope measurements (Fawcett et al., 2011) suggest that some Prochlorococcus ecotypes are capable of using both nitrate and nitrite.Picoeukaryotes also appear to actively Introduction

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Full assimilate nitrate (Fawcett et al., 2011) and can grow at rapid rates in low-nitrogen open-ocean environments (Cuvelier et al., 2010).The purpose of this study was to determine the pathways of NO in and around the primary NO − 2 maximum (Olson, 1981a), in concert with quantification of relevant organisms, and high resolution NH + 4 and NO − 2 concentration measurements across a gradient in primary productivity.Water samples were collected at discrete depths using a 12-bottle rosette sampler.The sampler was equipped with a SBE 9 (Sea-Bird Electronics) to measure conductivity, temperature, and pressure as well as an In-Situ Ultraviolet Spectrophotometer (ISUS, Johnson and Coletti, 2002) for real-time NO samples were collected at 12 depths between 0 and 1000 m in 27 mL HDPE scintillation vials and stored frozen until analysis using standard colorimetric methods (Sakamoto et al., 1990).Samples for onboard nutrient analyses (described below) were collected in 60 or 125 mL polyethylene bottles that had been initially cleaned in Micro-90 cleaning solution (Andwin Scientific) and subsequently acid washed in 1.2 N HCl.Phytoplankton samples for shore-based flow cytometry were preserved in 0.25 % (final concentration) TEM grade glutaraldehyde (Tousimis) and fixed in the dark at room temperature for 20 min before flash freezing in liquid nitrogen.Samples for nucleic acid extraction were collected from the rosette in 2-4 L polycarbonate bottles.Cells were harvested by pressure filtration onto 25 mm diameter, 0.2 µm pore-size polyethersulfone membrane filters (Pall Supor 200) housed in Swinnex filter holders (Millipore) using a peristaltic pump and silicone tubing.For DNA extraction and analysis, 1-2 L sample volumes were collected at each station and depth and the filters were flash frozen in 2 mL gasketed bead beating tubes (Fisher Scientific) using liquid nitrogen.

Low-level nutrient analyses
Measurements of low concentrations (< 1 µM) of NH + 4 , NO − 2 , and NO − 3 were made onboard the ship as soon as possible after collection.High-resolution (12 depths in the upper 200 m) nutrient measurements were made on the rosette cast immediately preceding the cast for incubation water collection.Nutrient measurements were also made at the incubation depths for rate determinations (see below).The NH + 4 analyzer was based on the method described in (Plant et al., 2009) but used a sequential injection platform.[NO − 2 ] and [NO − 3 ] were measured using standard colorimetric methods coupled with liquid waveguide capillary cells (LWCC) for detection (Zhang, 2000;Patey et al., 2008).The LWCC is composed of quartz capillary tubing (550 µm ID) covered by an outer surface cladding of AF-2400 Teflon resulting in a refractive index of 1.29 (World Precision Instruments).This refractive index is smaller than seawater (1.34) so that light is internally reflected through the water-filled waveguide to achieve path lengths of 1 (for [NO Introduction

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Full and sample processing manifold used for the low level NO − 3 and NO − 2 analyses were based on the chemistry (Cd reduction, followed by azo dye formation) and manifold of a rapid flow analyzer described in Sakamoto et al. (1990).Based on 3 × the standard deviation of low nutrient blanks, the low-level NH + 4 , NO − 2 , NO − 3 and measurements had detection limits of 10, 2, and 4 nM, respectively.Correlations among low-level nutrient concentrations and between nutrients and organism abundance were evaluated using Spearman non-parametric rank correlations implemented in MATLAB R2011b (Mathworks).

Flow cytometry
Fixed samples were analyzed on a flow cytometer equipped with a 200 mW 488 nM laser (InFlux, Becton Dickson).Autoclaved, 0.1 µm filtered 1 × phosphate buffered saline was used as sheath fluid.Fixed samples were thawed in a water bath in the dark, and fluorescent polystyrene beads (0.75 µm, Polysciences, Inc.) added, immediately prior to each run.Each sample was run for 2 min prior to data collection, and data were subsequently collected for 10 min at a flow rate of approximately 25 µL min −1 , measured by an inline flow meter (Sensirion SLG-1430 run with software designed by Jarred Swalwell, University of Washington).Sheath and sample pressure were adjusted as needed to maintain constant flow rate, to approximately 18.5 and 19.2 psi, respectively.Forward angle light scatter (FALS), pulse width, side scatter ( 90• angle; SSC), red (692 ± 40 nm band-pass filter) and orange (527 ± 27 nm band-pass filter) autofluorescence were recorded.Listmode files were analyzed in Winlist 6.0 (Verity Software House) to enumerate Prochlorococcus, Synechococcus, and small eukaryote populations that were defined based on natural autofluorescence and FALS characteristics.
While fixed samples were used for most analyses herein, unfixed samples were used to analyze oligotrophic surface Prochlorococcus populations (while at sea) that are better resolved when run live, and to verify that eukaryotic populations remained intact (numerically correct) with fixation and cryogenic storage.Live samples were run Introduction

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Full similarly to fixed samples, with slight modifications to accommodate the effect of the ship's motion.Specifically, because the ship's motion caused considerable movement of sheath fluid, the flow rate ranged from 12-30 µL min −1 in a time period of seconds.
To minimize this effect, the instrument was run with at least 5 L of sheath fluid at all times and total event rates only recorded when the flow rate was 25 µL min −1 (the average rate for all runs).Flow rate and cell count comparisons of samples run live and fixed showed that sheath pressure variability did not lower accuracy (data not shown).

Ammonia oxidation and nitrate reduction rate incubations
Rate measurements were conducted using stable isotope tracer additions ( 15 N) for five depths at each station targeting: the middle of the euphotic zone, just above the PNM, at the PNM, just below the PNM, and a depth well below the euphotic zone (500 m).For each depth, six 500 mL bottle incubations were conducted: two ammonia oxidation rate bottles (with 15 NH 15 N NO2 and δ 15 N NOx (0.2 µm syringe-filtered, frozen), as described below.

Stable isotope analyses
Analyses of δ 15 N of NO addition on δ 15 N NO2 determination of reference materials, as demonstrated previously (Casciotti and McIlvin, 2007).The isotope ratio of the original sample was then calculated by subtracting the concentration-weighted isotope ratio of the standard addition.

Rate calculations
To calculate rates of nitrification, NH 3 oxidation, and NO − 3 reduction, we modeled the 15 N and 14 N contents of the receiving pool (Table 1) as a box, with inputs from the labeled pool and outputs through NO x or NO − 2 uptake using Eq. ( 1): This approach has been described in detail previously (Santoro et al., 2010).The initial atom fraction 15 N in the labeled pool (at in ) was calculated by mass balance from the ambient nutrient concentrations and the 15 N tracer addition.Initial atom fraction 15 N in the NH x production, with units µmol L −1 h −1 ) and k (the rate constant for NO − 2 or NO − x assimilation, with units h −1 ) were calculated using a non-linear least squares curve fitting routine, implemented in MATLAB R2011b with the Optimization Toolbox; results were then converted to units of nmol L −1 d −1 .Fractionation factors for NO − 2 or NO x assimilation (α) were assumed to be 1.001 and 1.005, respectively.Standard error in the fit coefficients was calculated by approximating the covariance matrix, and using the square root of the diagonal to calculate the standard error.
The detection limit for each rate measurement is dependent on the initial atom percent 15 N enrichment in the substrate pool and the concentration of the product poolhigh initial atom fraction enrichment in the substrate pool and low concentrations in the Introduction

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Full product pool result in greater sensitivity.The theoretical detection limit, a rate which we can be reasonably certain is significantly different than zero, was calculated individually for each rate and depth as the NO − 2 production rate necessary to cause a 2 ‰ increase in δ 15 N NO2 from the initial value (including the carrier), i.e. twice the precision of the measurement.Detection limits for ammonia oxidation at the mesotrophic station (67.70) ranged from 0.3 to 1.7 nmol L −1 d −1 at 33 m.At the oligotrophic station (67.155), ammonia oxidation rate detection limits ranged from 0.02 to 0.86 nmol L −1 d −1 .Rate determinations of NO − 2 production from NO − 3 were generally less sensitive than ammonia oxidation rates, due to lower 15 N enrichments in the NO − 3 pool, with detection limits at 67.70 ranging from 0.1 at 55 m to 10.4 nmol L −1 d −1 within the PNM.At 67.155, detection limits ranged from 0.01 at 50 m to 9.8 nmol L −1 d −1 at 500 m.

Nucleic acid extraction
Nucleic acids (DNA and RNA) were extracted as described previously (Santoro et al., 2010), with modifications.Briefly, cells on the filters were lysed directly in the bead beating tubes with sucrose-EDTA lysis buffer and 1 % SDS.Prior to mechanical lysis, filter samples were subject to three freeze-thaw cycles of 5 min in liquid nitrogen and 5 min in a 65 • C water bath.Tubes were then agitated in a FastPrep bead beating machine (MP Biomedicals) for 1.5 min at speed 5.5, and proteinase K (Invitrogen) was added to a final concentration of 0.5 mg mL −1 .Filters were incubated at 55 • C for approximately 4 h and the resulting lysates were purified with the DNeasy kit (Qiagen) using a slightly modified protocol (Santoro et al., 2010).The purified nucleic acids were eluted in 200 µL of DNase, RNase-free water (Gibco) and quantified using a fluorometer (Qubit and Quanti-T BR reagent, Invitrogen Molecular Probes).Plus real-time PCR machine (PE Applied Biosystems).Unless noted otherwise below, each reaction contained 12.5 µL Failsafe Green Real-Time PCR PreMix E (Epicentre Biotechnologies), 400 nM each primer, 1.25 U Failsafe Real-Time Enzyme Blend (Epicentre Biotechnologies), and ROX passive reference dye at the concentration recommended by the manufacturer.AOA group-specific assays for "shallow" water column ecotype A (WCA) and "deep" water column ecotype B (WCB) (Mosier and Francis, 2011) used TaqMan Environmental Mastermix (Life Technologies) chemistry as described below.Detection limits for all SYBR green assays were 10 copies mL −1 or better; detection limit for TaqMan assays was 1 copies mL −1 or better.
All reactions were run in triplicate with a standard curve spanning approximately 10 1 -10 5 templates, run in duplicate.Plasmids containing cloned inserts of the target gene (TOPO pCR4 vector, Invitrogen) were used as standards as indicated below.Standards were linearized with the restriction enzyme NotI (New England Biolabs), purified (DNeasy, Qiagen), quantified by fluorometry (Quanti-T HS reagent, Invitrogen), and stored at −80 • C. Fresh standard dilutions were made from frozen stocks for each day of analysis.A minimum of three negative control qPCR reactions to which no DNA template was added were run with every assay.A melting curve analysis was performed after each qPCR run with plate reads at a temperature increment of 0.3 for the standard curves (cycle threshold, Ct, vs. log 10 copy number) were 0.98 or better for all runs.Efficiency was calculated relative to a theoretical standard curve slope of 3.32.
The betaproteobacterial amoA qPCR assay used the amoA1F/2R primer set (Rotthauwe et al., 1997) and the following thermal profile: of 94 • C for 3 min followed by 35 cycles of 95 • C for 45 s, 56 • C for 30 s, 72 • C for 50 s, and a plate reading step at 82 • C for 10 s.The standard used for this assay was a marine Nitrosospira-like amoA gene generated using amoA genes PCR-amplified from Monterey Bay; average qPCR efficiency was 96 %.Total archaeal amoA genes were quantified using the primers Arch-amoAF/Arch-amoAR (Francis et al., 2005) with an additional 2 mM MgCl 2 added to the reaction chemistry described above, and the following thermal profile: 94  (Santoro and Casciotti, 2011); average qPCR efficiency was 94 %.Group-specific amoA assays used the WCA-amoA-F/R and WCB-amoA-F/R primer sets (Mosier and Francis, 2011) and the following thermal profile: 95 • C for 10 min, followed by 40 cycles of 95 • C for 30 s and 55 • C for 30 s.

One-dimensional NO − 2 modeling
A one-dimensional, bulk mixed layer model (Price, Weller, Pinkel (Price et al., 1986); PWP) was used to approximate physical processes in the mixed layer.The present version of the model was implemented in MATLAB using code developed by (Glover et al., 2011) and modified by (Martz et al., 2009).The PWP model was initially developed to model diurnal heating and wind-driven mixing over a few days.Subsequent work has extended the model to run times from months to years (Archer et al., 1993;Mathieu and Deyoung, 1995;Plueddemann et al., 1995;Babu et al., 2004;Vage et al., 2008).

Location and magnitude of the PNM along the cruise transect
The cruise transect traversed productive, high chlorophyll waters in Monterey Bay (Chl a 5.2 µg L −1 ) to oligotrophic, open-ocean conditions (surface Chl a < 0.1 µg L −1 , Fig. 1d).As observed in previous occupations of this line, equatorward flow of the California Current causes upward tilting of isopycnal surfaces toward the coast (east) bringing relatively cold, saline, and high NO − 3 waters to the surface near the coast (Fig. 1b, c).The low-salinity core of the California Current was west of 126 • W (Fig. 1b), relatively far offshore.NO − 3 in the surface waters decreased along the transect from a high of 7 µmol L −1 within Monterey Bay down to below detection (< 4 nmol L −1 ) at 67.155.A subsurface deep chlorophyll maximum (DCM) layer became apparent at 126.3 • W.
We observed a primary nitrite maximum (PNM) at depths below the chlorophyll maximum at all stations.The PNM deepened offshore, concurrent with a deepening of the mixed layer and the appearance of the DCM (Fig. 1f).The magnitude of the PNM also decreased offshore, ranging from 0.53 µmol L −1 within Monterey Bay (31-40 m depth) to 0.17 µmol L −1 offshore (station 67.155 at 128 m depth).A distinct ammonium maximum was also observed near the base of the euphotic zone, below the DCM but above the PNM (Fig. 1e).The magnitude of the NH

Organism distribution in relation to the PNM
The shift from cool, nutrient-rich waters along the coast to oligotrophic offshore conditions was reflected in the phytoplankton community (Fig. 2).At the coast, Introduction

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Full Synechococcus and photosynthetic picoeukaryotes were abundant in surface waters within Monterey Bay, reaching abundances of 8.72 × 10 4 cells mL −1 and 2.66 × 10 4 cells mL −1 , respectively, where Procholorcoccus cells were undetectable.Both Synechococcus and picoeukaryotes were most abundant in surface waters and decreased in abundance with depth.At any one of these stations picoeukaryotes were present deeper in the water column than Synechococcus.Synechococcus abundance was correlated with water temperature (r = 0.27, p = 0.01), but peak abundance was observed in 16 • C waters.
Offshore, between 125.6 • and 126.3 • W, there was a shift in the photosynthetic community coincident with a transition to ∼ 17 • C waters (Fig. 2a, b).Procholorococcus cells became increasingly abundant west of this transition, reaching high abundances within distinct subsurface peaks, with local maxima of 1.53 × 10 5 to 2.09 × 10 5 cells mL −1 .
Procholorococcus abundance was inversely related to [NO − 3 ] (r = −0.83,p < 0.001) and positively related to water temperature (r = 0.68, p < 0.001).Organisms from all three groups of phytoplankton were generally present at the depth of the PNM (Fig. 3d, h), though with local minima in cell abundance.
amoA genes were quantified at two stations along the transect and used to infer the abundance of ammonia-oxidizing organisms.Archaeal amoA genes were detectable at both stations at nearly all depths.At 67.70 abundances ranged from 220 amoA copies mL −1 at the surface to a maximum of 1.4 × 10 4 copies mL −1 at 55 m depth (Fig. 3b), just below the PNM.At 500 m depth, there were 3.1 × 10 3 copies per mL −1 -less abundant than at the base of the euphotic zone, but more abundant than in the surface.At station 67.155, archaeal amoA abundance ranged from undetectable in the shallowest sample (50 m), to a maximum of 9.5 × 10 3 copies mL −1 at 128 m depth, coincident with the PNM (Fig. 3f).Water column group "A" amoA genotypes, thought to represent a shallow water ecotype (Francis et al., 2005;Hallam et al., 2006;Beman et al., 2008), accounted for 98 % or greater of the archaeal amoA genes at both stations at all depths except 500 m at station 67.155,where they were they were less than 5 % of the amoA ecotypes (Table 2).Introduction

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Full Bacterial amoA gene abundance at 67.70 ranged from below detection limits in the two shallowest samples to 610 copies mL −1 at 55 m, coinciding with the peak in AOA abundance.Bacterial ammonia oxidizers were less abundant at the oligotrophic station, ranging from undetectable at 50, 150, and 500 m, to near the detection limit above the PNM. with the greatest decreases in the euphotic zone bottles.

NO
NO − 2 production from ammonia oxidation at station 67.70 ranged from below detection limits at 500 m to 39 nmol L −1 d −1 at 55 m, just below the PNM (Fig. 3a).Rates of nitrification ranged from below the detection limit at the surface and 500 m to 34 nmol L −1 d −1 at 55 m.At a given depth, rates of ammonia oxidation to NO − 2 and nitrification were similar, except at 55 m where the average nitrification rate between the two incubation bottles (33 nmol L −1 ) was greater than the average ammonia oxidation rate (23 nmol L −1 d −1 ) .
The NH + 4 and NO − 2 profiles are the result of both physical and biological processes.If the nutrient profiles are assumed to be in a steady state on the scale of days, and the physical changes can be accounted for, then the residual differences can be attributed to biological activity.Here, a one-dimensional mixed layer model (PWP) was used simulate the observed distributions of NH + 4 and NO − 2 .At steady state, the rates of NO − 2 production needed to sustain the peak concentration against the rates of mixing, and the rates of NO − 2 consumption needed to prevent mixing from broadening the peak can be computed (Fig. 5).Modeled rates were compared against net NO  1e, f).Co-localization of these features has been observed in isolated profiles (Olson, 1981a;Lipschultz et al., 1996;Beman et al., 2012), and across the South Pacific Gyre (Raimbault et al., 2008).The localization of sequential maxima in Chl a, NH  (Dore and Karl, 1996a).This rate distribution has previously led to the proposal that reduction of NO − 3 by phytoplankton was the source of NO − 2 in the upper PNM.However, direct measurements of NO − 2 production via this pathway had not been made.Our results indicate that NO − 3 reduction contributed minimally to NO − 2 dynamics at these sites.
In contrast to data from Station ALOHA (Dore and Karl, 1996b), we did not observe a "double peaked" PNM, though we have observed this feature on previous occupations of line 67 (Santoro et al., 2010).Dore and Karl observed an upper PNM (UPNM) and lower PNM (LPNM) that they attributed to NO − 3 reduction and nitrification, respectively.They also detected NO − 2 below the depth of the LPNM; 2-5 nmol L −1 NO − 2 was present as deep as 800 m.We did not find evidence of deep NO − 2 , though measurements at 500 m and 1000 m were not made at every station.As suggested by Dore and Karl (1996b), this "tail" may be a result of episodic organic matter export events from the mixed layer not occurring during our cruise.
The patterns observed here in photosynthetic picoplankton distribution are consistent with previous observations from both the Atlantic (Zubkov et al., 2000;Cavender-Bares et al., 2001;Johnson et al., 2006) and Pacific.Zubkov et al. (2000) suggested that a high abundance of Synechococcus marked the transition between temperate and oligotrophic waters, consistent with the data presented here showing a high abundance of Synechococcus in 16 • C waters.Deeper distribution of picoeukaryotes relative to picocyanobacteria has been observed at ALOHA (Campbell and Vaulot, 1993;Campbell et al., 1997), consistent with observations presented here.Due to the strong inverse correlation between temperature and [NO − 3 ], the inverse correlation between

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Full 3 uptake by Prochlorococcus, but instead likely reflects the strong influence of temperature on the distribution of these organisms (Zubkov et al., 2000;Johnson et al., 2006;Zinser et al., 2007).Previous work in the Sargasso Sea (Zinser et al., 2007) did not find a correlation between Synechococcus abundance and temperature as we report here.
The abundance of AOA relative to AOB was greater at station 67.155 (AOA : AOB > 2000) in the oligotrophic waters of the North Pacific gyre than at the upwelling station 67.70 (AOA : AOB ∼ 10-400) supporting previous speculation that AOA in the ocean are oligotrophic specialists (Martens-Habbena et al., 2009).The high relative abundance of the water column A amoA ecotype suggests this ecotype is responsible for the majority of the ammonia-oxidizing activity detected in our incubations.At present the physiological characteristics differentiating the two ecotypes is unknown (Beman et al., 2008;Santoro et al., 2010).Sharp gradients were observed in nitrification rates, with apparent maxima just below the PNM at both stations.At 67.70 the maximum nitrification rate was coincident with the maximum AOA abundance, whereas at 67.155, the abundance of AOA peaked just above the PNM.Beman et al. (2012) also observed peaks in nitrification rates just below the PNM in the Eastern Tropical North Pacific using high resolution measurements, particularly within the Gulf of California.Accumulation of NO − 2 in the water column is not necessarily directly correlated to NO − 2 production, but rather reflects the balance of production and consumption of NO not as good at station 67.155 (Fig. 5b) where low [NO − 2 ] and relatively lower sampling resolution through the PNM contribute to uncertainty in the model.There is also a high degree of uncertainty in the net NO − 2 production calculation, as error from all 3 rate determinations is propagated through the calculation.
The mechanism that allows remineralization products (NH + 4 and NO − 2 ) to accumulate is still uncertain.Differential light inhibition of the two nitrifier groups is often proposed (Olson, 1981b), whereby NO − 2 oxidizers are inhibited at lower light levels (are more light sensitive) than ammonia oxidizers.Our data are consistent with this hypothesis, however, it seems unlikely that photochemical stress would be significant at that depth, where irradiance is between 0.1-1 % of surface irradiance.Laboratory data suggest that AOA are also extremely light sensitive (Merbt et al., 2012), which argues against the differential light inhibition hypothesis.However, the extent to which these laboratory results reflect responses in nature is unclear, as extreme light sensitivity does not appear consistent with widespread presence of AOA amoA genes and transcripts in stratified Pacific surface waters (Church et al., 2010;Santoro et al., 2010).We were unable to sample light profiles consistently at every station during our cruise because some stations were occupied at night.
Gradients in several other growth factors with "nutrient-like" depth distributions are seldom discussed, but equally plausible mechanisms for the accumulation of NO − 2 at the PNM.Iron (Fe), in particular, could be limiting at the depth of the PNM.Non steadystate Fe limitation is associated with NO − 2 excretion in diatoms (Behrenfeld et al., 2006) and has been invoked to explain a potential phytoplankton source of NO − 2 within the PNM (Lomas and Lipschultz, 2006).Fe could also play an underexplored role in a nitrification-sourced PNM.Our results indicate that AOA are both the most abundant and active nitrifiers at the PNM, as suggested previously (Mincer et al., 2007;Santoro Introduction

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Full suggesting a very high iron demand for this process (Kirstein and Bock, 1993;Spieck et al., 1998).Genomic information has only recently become available for relevant open ocean NO − 2 oxidizers, such as Nitrospina spp.(Mincer et al., 2007;Luecker et al., 2013) and is currently lacking for Nitrospira spp.
We measured low rates of NO − 2 production from NO − 3 at all stations and depths, arguing against a large role for NO − 2 excretion from phytoplankton in the formation of the PNM at these sites.Just below the PNM at 67.70, NO − 2 production from NO − 3 was 2 nmol L −1 d −1 compared with mean rates of ammonia oxidation and nitrification of 26 and 33 nmol L −1 d −1 , respectively.It should be stressed that low rates of NO − 2 production from NO − 3 measured using this technique should not be interpreted to mean there was little or no active assimilatory NO − 3 reduction, or that NO − 3 -based or "new" production is not important at these sites.Our data suggest only that any NO − 2 produced during assimilatory NO − 3 reduction does not escape the cell.Unfortunately, NO − 3 uptake rates were not directly measured in our study, and there are few published NO − 3 uptake data along Line 67.One possible explanation for the low rates of NO − 2 production from NO − 3 could be that phytoplankton NO − 2 excretion only occurs under Fe-limited conditions.Our experiments were not performed using trace metal clean conditions, but variable fluorescence data (F v /F m ) taken over the course of the experiment do not suggest a fertilization effect of the incubation conditions (data not shown).NO − 2 release by dinoflagellates has been observed in cells growing with both NH + 4 and NO − 3 (Flynn and Flynn, 1998).Thus, the low NO − 2 production rates observed in this study may indicate reliance on a single N source.
Previous studies that have attributed a large phytoplanktonic role in NO − 2 production (Vaccaro and Ryther, 1960;Kiefer et al., 1976;Mackey et al., 2011)  Our results offer some support for the suggestion that mixed-layer NO − 2 production shallower than the PNM (Al-Qutob et al., 2002) is the result of NO − 3 reduction from either photolysis or active phytoplankton reduction.The residence time of NO − 2 within the PNM can be approximated from the concentration and the total NO − 2 production rate, assuming steady state (Table 3).Residence times at the peak of the PNM are long -127-179 days at the mesotrophic station and over 200 days at the oligotrophic station.At the base of the PNM, residence times are much shorter -4 to 9 days at station 67.70 and less than a day at 67.155.Early investigations into the PNM (Kiefer and Kremer, 1981) suggested that the PNM is a relict feature of "old" NO − 2 excreted during rapid phytoplankton growth.Our results support previous findings (Olson, 1981a;Ward et al., 1982;Dore and Karl, 1996a) that portions of the PNM reflect active and dynamic N cycling processes, while the peak reflects relatively slow biological turnover (Buchwald and Casciotti, 2013).
In summary, the results presented here strongly suggest that most PNM NO − 2 in the central California Current and offshore waters originates from NH  Full  Full 4 ] was below the detection limit at all depths at station 67.155.
a substrate for both oxidative and reductive microbial metabolism.NO − 2 accumulates at the base of the euphotic zone in oxygenated, stratified open ocean water columns, forming a feature known as the primary nitrite maximum (PNM).Potential pathways of NO − 2 production include the oxidation of ammonia (NH 3 ) by ammoniaoxidizing bacteria or archaea and assimilatory nitrate (NO − 3 ) reduction by phytoplankton or heterotrophic bacteria.Measurements of NH 3 oxidation and NO − 3 reduction to NO − 2 were conducted at two stations in the central California Current in the eastern North Pacific to determine the relative contributions of these processes to NO − 2 production in the PNM.Sensitive (< 10 nmol L −1 ), high-resolution measurements of [NH the PNM at every station, with concentrations as high as 1.5 µmol L −1 .Within and just below the PNM, NH 3 oxidation was the dominant NO − 2 producing process with rates of NH 3 oxidation of up to 50 nmol L −1 d −1 , coinciding with high abundances of ammonia-oxidizing archaea.Though little NO − 2 production from NO − 3 was detected, potentially nitrate-reducing phytoplankton (photosynthetic picoeukaryotes, Synechococcus, and Prochlorococcus) were present at the depth of the PNM.Rates of NO Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |

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2 formation in the PNM of the eastern North Pacific Ocean by coupling high precision, high resolution measurements of NO − 2 and ammonium (NH + 4 ) concentrations with 15 N tracer experiments and enumeration of microbial groups.Ammonia oxidation (production of NO − 2 ), nitrification (production of NO − 2 + NO − 3 ), and NO − 3 reduction to NO − 2 were measured, as was abundance of potentially NO − 2 producing organisms in and around the PNM (photosynthetic eukaryotes, Synechococcus, and Prochlorococcus), AOB and AOA).These measurements allow a direct comparison of NO − 2 production from NO − 3 and from NH + 4 Fig.1a) were chosen for microbial characterization and geochemical rate measurements.Water samples were collected at discrete depths using a 12-bottle rosette sampler.The sampler was equipped with a SBE 9 (Sea-Bird Electronics) to measure conductivity, temperature, and pressure as well as an In-Situ Ultraviolet Spectrophotometer Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | for the experimental bottles was collected directly from the rosette into 500 mL acidcleaned polycarbonate bottles.Water for filtered controls was then 0.2 µm-pressure filtered into acid-clean bottles.Each bottle was spiked with 100 µL of 1mmol L −the "deep" depth at each station were spiked with 200 µL of [ 15 N]O − 3 to achieve a final label concentration of 400 nM.These 15 N additions corresponded to 0.5-13 × ambient [NH + 4 ] and 0.02-0.23 × ambient [NO − 3 ] (see below).Bottles were incubated in an on-deck circulating seawater incubator in neutral densityscreened bags calibrated to approximate the in situ light field at each depth using a photosynthetically active radiation sensor (Biospherical Instruments QSL-2200).Replicate 50 mL samples were removed from each bottle at time points of 0 h, 12 h, 24 h, and 36 h for onboard determination of [NH + ratio mass spectrometer outfitted with a custom purge and trap system.Samples were prepared from 5 nmol or 10 nmol of analyte using the azide(McIlvin and Altabet, 2005) and denitrifier methods(Sigman et al., 2001;McIlvin and Casciotti, 2011) for δ 15 N NO2 and δ 15 N NOx determination, respectively.δ 15 N NO2 values were calibrated against nitrite isotope reference materials N-23, N-7373, and N-10219(Casciotti and McIlvin, 2007)  analyzed in parallel.δ 15 N NOx values were calibrated against NO − 3 isotope reference materials USGS 32, USGS 34, and USGS 35, analyzed in parallel.Duplicate δ 15 N NO2 analyses were performed on each sample, while δ 15 N NOx measurements were performed once.Due to the necessary addition of carrier NO − 2 (see below), the δ 15 N NO2 analyses had mean standard deviations (for replicate samples) ranging from 0.3 to 1.1 ‰ .Precision of δ 15 N NO3 analysis using the denitrifier method was 0.5 ‰ or better (McIlvin and Casciotti, 2011).Handling of all 15 N-enriched samples was carried out in a laboratory dedicated for this purpose.[NO − 2 ] in all incubation samples was too low (< 1 µmol L −1 ) to allow direct δ 15 N NO2 measurements.Therefore, either 5 or 10 nmol of natural abundance NO − 2 of known isotopic composition was added (either reference material N-23, δ 15 N NO2 = −3.7 ‰, or a laboratory NO − 2 stock, δ 15 N NO2 = 1.1 ‰) to 5 mL of each incubation sample prior to preparation and analysis.To prevent potential 15 Discussion Paper | Discussion Paper | Discussion Paper |

+
4 additions ranged from 0.35 to 0.93.Initial atom fraction 15 N in the NO − 3 additions ranged from 0.02 to 0.19.Coefficients F in (the rate of NO − 2 or NO − Screen / Esc Printer-friendly Version Interactive Discussion Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |

2. 8
Quantitative PCR qPCR assays for total archaeal amoA (AOA) and betaproteobacterial amoA (AOB) were carried out in 25 µ L reactions using SYBR Green chemistry on a StepOne Introduction Discussion Paper | Discussion Paper | Discussion Paper | The model has 1 m vertical resolution down to 200 meters, a time step of 15 s, and a vertical eddy diffusivity of 1.5 × 10 −4 m 2 s −1 .PWP is forced at the surface using NCEP/NCAR Reanalysis 1 surface flux data(Kalnay et al., 1996).The 4 times daily data were provided by the National Oceanic and Atmospheric Administration (NOAA) Earth System Research Laboratory (http://www.esrl.noaa.gov/psd/).Upwelling velocities were calculated from 4 times daily wind stress curl, calculated using data from the NOAA Environmental Research Division (ERD) of the South West Fisheries Science Center (http://las.pfeg.noaa.gov/thredds/dodsC/Model/FNMOC/).Wind stress curl was calculated by ERD from analyzed fields of sea level pressure from the Fleet Numerical Meteorology and Oceanography Center (http://www.usno.navy.mil/FNMOC).Ekman depth and vertical velocity attenuation were calculated following the approach ofSignorini et al. (2001).Climate data were extracted for the 2 months prior to the profile date at each station position.The model was initialized with the 12-depth station [ to 0.014 µmol L −1 offshore, with a particularly high ammonium maximum during the second occupation of station 67.70 of 1correlated with depth-integrated Chl a (r = 0.86, p < 0.01; r = 0.63, p = 0.02, respectively).
Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | the same stations that we quantified ammonia-oxidizing microbial abundances (Figs.3, 4).[NO − 2 ] and [NO − 3 ] were constant in the incubation bottles during the 36 h of the incubation, varying only within the error of the measurements (data not shown).[NH + 4 ] decreased in nearly all incubation bottles, between 30 and 120 nmol L −1 from 0.03 at the surface to 50 nmol L −1 d −1 just below the PNM, albeit with large uncertainties at this depth (± 21nmol L a similar pattern, ranging from below detection limits at the surface to 8 nmol L −1 d −1 just below the PNM.Even accounting for large uncertainties, at 67.155 ammonia oxidation rates were significantly greater than NO − 2 + NO − 3 production rates at this activity maximum.just below the PNM and coincident with the highest rates of nitrification.At station 67.155, NO − 2 production from NO − 3 above detection limits was only observed at 50 m, the shallowest depth sampled, at a mean rate of 0.75 nmol L −1 d −1 sum of NO − 2 production from ammonia oxidation and nitrate reduction, minus NO − 2 losses from nitrification.At station 67.70, the model predicts net NO − 2 consumption below the PNM, which agrees with measured net NO − 2 consumption below the PNM.At station 67.155, modeled rates of net NO − 2 production and consumption were less than 10 mmol m −2 d −1 , lower than measured net NO − suggest that the PNM is a remineralization feature, whereby the sequential one above the other and are correlated in magnitude.A nitrification source of PNM NO − 2 in the California Current is further supported by the high rates of NO − 2 and not within the peak of the PNM, consistent with previous reports from the North Pacific Discussion Paper | Discussion Paper | Discussion Paper | Prochlorococcus abundance and [NO − 3 ] cannot be interpreted as a signal of NO −

− 2 .
Thus, a spatial offset between maximal rates of production NO − 2 concentration is not surprising.A one-dimensional model was used to estimate NO − 2 production rates from NO − 2 concentration profiles at station 67.70 and model the balance of biological production, consumption, and physical mixing (Fig. 5a, b).At station 67.70 (Fig. 5a), modeled net consumption just below the PNM agrees with net NO − 2 consumption measured in the 15 N rate measurements, where nitrification was slightly greater than NH 3 oxidation.The model output in the model is highly dependent on the choice of eddy diffusivity (1.5 × 10-4 m 2 s −1 used here).Agreement between the model and measurements was Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | have interpreted correlations between Chl a stocks and NO − 2 as evidence for phytoplankton production of NO − 2 without direct measurements of NO − 2 production from NO − Discussion Paper | Discussion Paper | Discussion Paper | correlations between Chl a and NO − 2 .
suggest that it occurs intermittently and therefore was not captured by our incubation experiments.Our results help constrain possible sources of NO − 2 within the PNM, but questions still remain about why NO − 2 accumulates within this feature and is not consumed by nitriteoxidizing bacteria.Accessing the natural abundance stable isotope ratios of NO − 2 is now possible and may provide further insight into the mechanisms controlling NO − 2 production and consumption in the PNM (Buchwald and Casciotti, 2013)Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |
followed by 35 cycles of 94 • C for 30 s, 58 • C for 45 s, and 72 • C for 50 s, and a plate read at 80 • C for 10 s.The standard used for this assay was an archaeal amoA gene amplified from the California Current • C for 3 min, Introduction Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |

Table 2 .
(Mosier and Francis, 2011)of ammonia-oxidizing archaea: water column A (WCA) and water column B (WCB) based on the abundance of archaeal amoA genes determined by quantitative PCR using ecotype-specific primers and probes(Mosier and Francis, 2011).

Table 3 .
Compilation of residence time estimates of NO− 2 in the primary nitrite maximum (PNM).When not reported in the original manuscript, residence time was determined as [NO − 2 ] /production rate.