# Quantifying N2 fixation and its contribution to export production near the Tonga-Kermadec Arc using nitrogen isotope budgets

Heather Forrer, Sophie Bonnet, Rachel Thomas, Olivier Grosso, Cecile Guieu, Angela Knapp

► To cite this version:

Heather Forrer, Sophie Bonnet, Rachel Thomas, Olivier Grosso, Cecile Guieu, et al.. Quantifying N2 fixation and its contribution to export production near the Tonga-Kermadec Arc using nitrogen isotope budgets. Frontiers in Marine Science, 2023, 10, 10.3389/fmars.2023.1249115. [https://doi.org/10.3389/fmars.2023.1249216]

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OPEN ACCESS

EDITED BY

Margarita Fernández Tejedor,
Institute of Agrifood Research and
Technology (IRTA), Spain

REVIEWED BY

Dengzhou Gao,
East China Normal University, China
Annie Bourbonnais,
University of South Carolina, United States

*CORRESPONDENCE

Heather J. Forrer

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hforrer@fsu.edu

RECEIVED 28 June 2023

ACCEPTED 12 October 2023

PUBLISHED 27 October 2023

CITATION

Forrer HJ, Bonnet S, Thomas RK, Grosso O, Guieu C and Knapp AN (2023) Quantifying N₂ fixation and its contribution to export production near the Tonga-Kermadec Arc using nitrogen isotope budgets.
*Front. Mar. Sci.* 10:1249115.
doi: 10.3389/fmars.2023.1249115

COPYRIGHT

© 2023 Forrer, Bonnet, Thomas, Grosso, Guieu and Knapp. This is an open-access article distributed under the terms of the [Creative Commons Attribution License (CC BY)](https://creativecommons.org/licenses/by/4.0/). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Quantifying N₂ fixation and its contribution to export production near the Tonga-Kermadec Arc using nitrogen isotope budgets

Heather J. Forrer¹*, Sophie Bonnet², Rachel K. Thomas¹, Olivier Grosso², Cecile Guieu³ and Angela N. Knapp¹

¹Earth, Ocean, and Atmospheric Science Department, Florida State University, Tallahassee, FL, United States, ²Aix Marseille University, Université de Toulon, CNRS, IRD, MIO Marseille, France, ³Laboratoire d'Oceanographie de Villefranche, CNRS, Sorbonne University, Villefranche-sur-Mer, France

The spatial distribution of marine di-nitrogen (N₂) fixation informs our understanding of the sensitivities of this process as well as the potential for this new nitrogen (N) source to drive export production, influencing the global carbon (C) cycle and climate. Using geochemically-derived δ¹⁵N budgets, we quantified rates of N₂ fixation and its importance for supporting export production at stations sampled near the southwest Pacific Tonga-Kermadec Arc. Recent observations indicate that shallow (<300 m) hydrothermal vents located along the arc provide significant dissolved iron to the euphotic zone, stimulating N₂ fixation. Here we compare measurements of water column δ¹⁵N*NO₃⁺*NO₂ with sinking particulate δ¹⁵N collected by short-term sediment traps deployed at 170 m and 270 m at stations in close proximity to subsurface hydrothermal activity, and the δ¹⁵N of N₂ fixation. Results from the δ¹⁵N budgets yield high geochemically-based N₂ fixation rates (282 to 638 µmol N m⁻² d⁻¹) at stations impacted by hydrothermal activity, supporting 64 to 92% of export production in late spring. These results are consistent with contemporaneous ¹⁵N₂ uptake rate estimates and molecular work describing high *Trichodesmium* spp. and other diazotroph abundances associated with elevated N₂ fixation rates. Further, the δ¹⁵N of sinking particulate N collected at 1000 m over an annual cycle revealed sinking fluxes peaked in the summer and coincided with the lowest δ¹⁵N, while lower winter sinking fluxes had the highest δ¹⁵N, indicating isotopically distinct N sources supporting export seasonally, and aligning with observations from most other δ¹⁵N budgets in oligotrophic regions. Consequently, the significant regional N₂ fixation input to the late spring/ summer Western Tropical South Pacific results in the accumulation of low-δ¹⁵N*NO₃⁺*NO₂ in the upper thermocline that works to lower the elevated δ¹⁵N*NO₃⁺*NO₂ generated in the oxygen deficient zones in the Eastern Tropical South Pacific.

KEYWORDS

N₂ fixation, nitrate d15N, Tonga Arc, South Pacific, hydrothermal vents, d15N budget
# 1 Introduction

The biological fixation of dinitrogen (N₂) gas, mediated primarily by marine prokaryotes (“diazotrophs”), is the largest source of newly fixed nitrogen (N) to the global ocean (Gruber, 2004; Landolfi et al., 2018), fertilizing primary productivity and supporting carbon (C) export (Dugdale and Goering, 1967; Karl et al., 1997; Capone, 2001; Capone et al., 2005). The global rate and distribution of marine N₂ fixation remains uncertain, although geochemical and biological observations indicate significant N₂ fixation rates occur in both the Tropical Atlantic (Gruber, 2004; Capone et al., 2005; Mahaffey, 2005; Marconi et al., 2017) as well as the Western Tropical South Pacific (WTSP) (Berthelot et al., 2017; Bonnet et al., 2017; Caffin et al., 2018; Knapp et al., 2018). These high rates of N₂ fixation in the Tropical Atlantic are consistent with locations of elevated rates of atmospheric dust deposition (Jickells et al., 2005; Mahowald et al., 2005; Conway and John, 2014; Xu and Weber, 2021), while emerging evidence in the WTSP describes the significance of hydrothermally sourced iron (Fe) (Guieu et al., 2018; Bonnet et al., 2023b) meet the high Fe requirements of diazotrophs (Berman-Frank et al., 2001; Kustka et al., 2003) in the region. Indeed, Fe and phosphorus availability are thought to primarily influence the spatial distribution of marine N₂ fixation (Moore et al., 2009; Monteiro et al., 2011; Dutkiewicz et al., 2012; Weber and Deutsch, 2014).

A long-standing goal of marine N₂ fixation research is to better characterize the marine diazotroph community and their sensitivity to environmental fluctuations, while relating these to their regional distributions and consequential N₂ fixation fluxes (Mahaffey, 2005; Moisander et al., 2010; Sohm et al., 2011). Diazotrophs are identified by the nitrogenase (*nifH*) gene that encodes for the Fe binding protein of the *nifH* operon (Zehr et al., 1998; Zehr and Turner, 2001; Turk-Kubo et al., 2012). Common marine proteobacterial (e.g., alpha-, beta- gamma-, delta-) and cyanobacterial diazotroph types include: 1) non-heterocystous filamentous (e.g., *Trichodesmium* spp.), 2) heterocystous filamentous (e.g., *Richelia*), and 3) unicellular (e.g., *Crocosphaera* spp.) (Capone et al., 1997; Luo et al., 2012; Moisander et al., 2014). Historically, high rates of N₂ fixation by these and other diazotrophs have been associated with warm (>25 °C), nitrate (NO₃⁻)- and ammonium (NH₄⁺)-depleted, Fe-rich surface waters (Kustka et al., 2003; Staal et al., 2003; Bonnet et al., 2009). The impact Fe has on the intra-basin distribution of N₂ fixation rates is particularly evident in the South Pacific, with low Fe supply associated with low rates of N₂ fixation in the Eastern Tropical South Pacific (ETSP) (Dekaezemacker et al., 2013; Knapp et al., 2016a). While historically atmospheric deposition has been considered the primary source of Fe fueling marine diazotrophy, the WTSP surface waters appear relatively unique, with the primary Fe supply thought to originate from shallow (≤300 m) hydrothermal vents, particularly in the Lau Basin (Guieu et al., 2018; Tilliette et al., 2022; Bonnet et al., 2023b).

Biological tools have been used to calculate short-term N₂ fixation rates (e.g., Montoya et al., 2004; Capone et al., 2005;

Mulholland et al., 2019) and identify marine diazotrophic potential (Turk-Kubo et al., 2012; Stenegren et al., 2018; Meiler et al., 2022). However, the contribution of N₂ fixation to export production is primarily estimated using a geochemically-derived “δ¹⁵N” budget over short timescales (Casciotti et al., 2008; Bourbonnais et al., 2009; White et al., 2013) as well as annual cycles (Böttjer et al., 2017). The “δ¹⁵N” budget uses a two end-member mixing model to compare the isotopic composition (δ¹⁵N) of exported particulate organic matter captured in a sediment trap (PNsink) to the δ¹⁵N of N₂ fixation inputs (-1‰) (Hoering and Ford, 1960; Minagawa and Wada, 1986; Carpenter et al., 1997) and subsurface NO₃⁻ (measured at each location, where δ¹⁵N (‰ vs. air) = [(($^{15}$N/$^{14}$N)sample/($^{15}$N/$^{14}$N)AIR)-1]x1000) (Altabet, 1988; Karl et al., 1997; Dore et al., 2002; Casciotti et al., 2008; Bourbonnais et al., 2009; White et al., 2013). Most prior δ¹⁵N budgets indicate that N₂ fixation supports <20% of export production in oligotrophic regions. In the North Pacific and ETSP, ≤25% of export production is estimated to be supported by N₂ fixation (Casciotti et al., 2008; Knapp et al., 2016a; Böttjer et al., 2017), although that 25% is likely not equally distributed over an annual cycle. Specifically, summertime stratification is believed to promote N₂ fixation-supported export, while the deepening of the wintertime mixed layer promotes NO₃⁻-supported export production (Casciotti et al., 2008; Böttjer et al., 2017). In contrast to other δ¹⁵N budgets from oligotrophic regions, a recent study from the WTSP describes particularly large contributions of N₂ fixation to export production (>50%) during the late summer and early autumn (Knapp et al., 2018). However, it remains unclear whether N₂ fixation supports a meaningful fraction of N export in the WTSP annually. Here we apply a δ¹⁵N budget to samples collected in shallow (170 m and 270 m), short-term drifting sediment traps to evaluate the importance of N₂ fixation-supported export production during the late spring. Additionally, we use sinking material collected over the course of a year in a deep (1000 m), moored sediment trap to evaluate seasonal trends in the δ¹⁵N of exported particulate organic matter relative to the δ¹⁵N for sources of new N to surface waters. We compare the geochemically-derived N₂ fixation rates from the short-term δ¹⁵N budgets with $^{15}$N₂ incubation-based rates and estimates of diazotroph abundance, and evaluate these results in the context of previous regional and global N₂ fixation rate estimates, as well as seasonal trends extracted from the deep, moored trap.

# 2 Methods

## 2.1 Sample collection

Sample collection was conducted as part of the GEOTRACES TONGA (shallow hydroThermal sSources of trace elemeNts: potential impacts on biological productivity and the bioloGicAl carbon pump) research cruises (doi.org/10.17600/18000884) aboard the R/V *L'Atalante* in November 2019 and R/V *Alis* in
October 2020, with both cruises leaving from and returning to Nouméa, New Caledonia. The 2019 primary cruise collected samples at 13 stations along a roughly zonal transect at ~20° S, sampling Melanesian waters (MW), the Lau Basin (LB), and crossing the Tonga-Kermadec Arc into the deeper South Pacific Gyre (SPG). In 2020, samples were collected at four stations in MW and the LB (Figure 1). On both cruises, water column samples (n=200) for nitrate + nitrite ([NO₃⁻ + NO₂⁻]) concentration and δ¹⁵N analysis were collected from Niskin or GoFlo bottles deployed on conductivity, temperature, and depth (CTD), TOW (small, classical CTD with 12 Niskin bottles), or trace metal clean (TMC) rosettes equipped with sensors. At discrete depths from each of the casts, 60 mL of 0.2 µm filtered seawater were collected in duplicate and stored in acid and deionized water-washed, sample-rinsed (three times) high-density polyethylene bottles. These samples were then immediately frozen at -20° C and subsequently sent to Florida State University for post cruise analysis.

Short-term Particle Interceptor Traps [PIT, collecting area of 0.0085 m², aspect ratio of 6.7, and filled with 0.2 µm filtered seawater with added formaldehyde brine (5% formaldehyde, final concentration) buffered with sodium tetraborate (pH 8)] were deployed on a drifting mooring in close proximity to the hydrothermal vents at 170 m and 270 m at station 5a-2019 for five days and at 270 m at station 10a-2019 for four days during the 2019 cruise, collecting sinking particulate N ("PNsink"). Additionally, a long-term Technicap PPS5 trap (1 m² collecting area, aspect ratio of 5.3) was deployed (containing the same buffered formaldehyde brine solution described above) at station 12-2019/4-2020 at 1000 m, collecting samples every 14 days (bimonthly) for 12 months from November 2019 to October 2020. Although sediment traps are a standard tool used to capture sinking particles, uncertainties remain in their collection efficiency within the water column and between trap designs (Buesseler et al., 2007; Baker et al., 2020; Tilliette et al., 2023).

## 2.2 NO₃⁻ + NO₂⁻ concentration and δ¹⁵N analysis

For the 2019 samples, [NO₃⁻ + NO₂⁻] was determined using colorimetric analysis (Aminot and Kerouel, 2007) with a detection limit of 0.05 µM and a standard deviation (S.D.) of ± 0.1 µM. Furthermore, the [NO₃⁻ + NO₂⁻] for the 2020 samples was measured by chemiluminescence (Braman and Hendrix, 1989) using a Thermo 42i NOₓ analyzer at Florida State University. Briefly, samples were injected into a heated, acidic vanadium (III) solution that reduces NO₃⁻ + NO₂⁻ quantitatively to nitric oxide gas (NOₓ). The NOₓ then reacts with ozone inside the analyzer to produce light, the intensity of which is quantitatively related to the amount of NOₓ in the sample and thus the original [NO₃⁻ + NO₂⁻]. The range of detection of the instrument was adjusted according to the concentrations of the samples. Sample [NO₃⁻ + NO₂⁻] was calibrated using a standard curve that bracketed the range of samples with a lower reporting limit of 0.1 µM and an average S.D. of ± 0.1 µM.

The nitrogen (N) isotopic composition of NO₃⁻ + NO₂⁻ (δ¹⁵NNO₃+NO₂) was determined using the “denitrifier” method (Sigman et al., 2001; Casciotti et al., 2002; McIlvin and Casciotti, 2011; Weigand et al., 2016). This analysis was performed when sample [NO₃⁻ + NO₂⁻] ≥ 0.3 µM. The δ¹⁵NNO₃+NO₂ values were reported when the standard deviation of replicate analyses was <0.5‰. Samples were calibrated with IAEA N3 and USGS 34 as described in McIlvin and Casciotti (2011).

## 2.3 Sinking particulate N flux and δ¹⁵N measurements

The bulk PNsink mass flux, and its associated isotopic composition, "δ¹⁵NPNsink", collected by the sediment traps, was

<figure><img src="image_4.png" /><figcaption>FIGURE 1</figcaption></figure>

Bathymetry of the southwest Pacific region with stations from the TONGA research cruises shown in larger filled circles with black outlines (2019), and smaller filled circles with white outlines (2020); gray represents areas above sea-level. Stations 2-2019, 3-2019 and 1-2020 were sampled in Melanesian waters (MW), stations 4-2019, 5-2019, 10-2019, 12-2019, 2-2020, 3-2020, and 4-2020 were sampled in the Lau Basin (LB), and stations 6-2019, 7-2019, and 8-2019 were sampled in the South Pacific Gyre (SPG). Stations 5 (a, b, c, d)-2019 and 10 (a, b)-2019 were proximal to the shallow hydrothermal vents 'Panamax' and 'Simone', respectively. The South Equatorial Current (SEC) and branches thereof are indicated by the white arrows.
measured using an Elementar Analyser - Isotope Ratio Mass Spectrometer (EA-IRMS) at the Mediterranean Institute of Oceanography (SERCON INTEGRA 2). The Quantification Limit was 7 µg N and the precision was between ± 0.3‰ for highest masses and ± 3.5‰ for masses close to Quantification Limit (k = 2).

## 2.4 δ¹⁵N budget calculations

A two end-member mixing model was used to evaluate the δ¹⁵N budgets. This model assumes two quantitatively important “new” N sources to surface waters, subsurface NO₃⁻+NO₂⁻ and biological N₂ fixation, as well as one loss term, PNₙₘₐₗ. The isotopic composition of N₂ fixation inputs, “δ¹⁵Nₙₐₗ” was assumed to be -1‰ (Hoering and Ford, 1960; Minagawa and Wada, 1986; Carpenter et al., 1997), while the δ¹⁵N of subsurface NO₃⁻+NO₂⁻ and PNₙₘₐₗ were measured. The relative contribution of N₂ fixation to export production, “fₙₘₐₗ”, is calculated by the following (Knapp et al., 2018):

$$ \delta^{15}\text{N}_{\text{PNsink}} = f_{\text{nfix}}(-1\%) + (1 - f_{\text{nfix}})(\delta^{15}\text{N}_{\text{NO3+NO2}}) \quad (1) $$

which can be rearranged to solve for fₙₘₐₗ:

$$ f_{\text{nfix}} = \frac{\left[ \left( \delta^{15}\text{N}_{\text{NO3+NO2}} \right) - \left( \delta^{15}\text{N}_{\text{PNsink}} \right) \right]}{\left[ 1 + \left( \delta^{15}\text{N}_{\text{NO3+NO2}} \right) \right]} \quad (2) $$

The depth from which subsurface NO₃⁻+NO₂⁻ is sourced likely varies and is difficult to constrain; therefore, the short-term δ¹⁵N budgets are evaluated over a range of subsurface δ¹⁵Nₙₐₗ+NO₂⁻ source values. These values include the shallowest δ¹⁵Nₙₐₗ+NO₂⁻ minima and the δ¹⁵Nₙₐₗ+NO₂⁻ of the sample collected immediately below the minima (Knapp et al., 2021). The long-term δ¹⁵N budgets are evaluated using the shallowest average δ¹⁵Nₙₐₗ+NO₂⁻ minima at station 12-2019/station 4-2020 as well as the average δ¹⁵Nₙₐₗ+NO₂⁻ of South Pacific Sub-tropical Under Water (SPSTUW, 150m-250m depth) at station 4-2020. Note, sampling resolution at station 12-2019 did not include SPSTUW. These δ¹⁵Nₙₐₗ+NO₂⁻ end-members for the moored trap δ¹⁵N bugdet were chosen to encompass the range of NO₃⁻+NO₂⁻ likely entrained to surface waters over an annual cycle (Moutin et al., 2018). This seasonality is also observed at the station ALOHA, Hawaii (Casciotti et al., 2008; Böttjer et al., 2017). Once the fraction of export supported by N₂ fixation is calculated, an N₂ fixation rate, “Rₙₘₐₗ” can be calculated by multiplying fₙₘₐₗ by the PNₙₘₐₗ mass flux, yielding a geochemically-derived N₂ fixation rate.

## 2.5 Bathymetric data

Bathymetric data from the sampling region were considered to better understand the bathymetric-induced current steering as well as the proximity of the stations to the shallow hydrothermal vents. These bathymetric data were downloaded from NOAA's National

Centers for Environmental Information page (https://www.ngdc.noaa.gov/mgg/global/). The ETOPO1 Global Relief Model was used with the grid version ETOPO1 Bedrock.

## 3 Hydrography

Water masses along the TONGA transect were identified using temperature, salinity, and potential density (σ₀) (Figure 2) and align with those reported in Tilliette et al. (2022). Plots of the water column profiles in temperature-salinity space indicate that all stations were largely influenced by the same water masses (Figure 2), reflecting the dominance of the westward flowing SEC, which impacts waters from 400 to 1000 m across this region (Figure 1) (Talley et al., 2011; Guieu et al., 2018; Tilliette et al., 2022). The SEC divides into branches, particularly in the LB, due to the bathymetric blocking by islands and deep-sea ridges (Webb, 2000; Tilliette et al., 2022). Many of these branches in the LB are observed to have an overall southwestern trajectory before returning to a western trajectory over the center of the LB and maintaining this westward trajectory in MW (Figure 1) (Tilliette et al., 2022). Additionally, the Tonga-Kermadec Arc acts as a physical barrier to deep water masses entering the LB from the SPG, influencing circulation downstream (Tilliette et al., 2022). Surface waters across this transect were turbulent down to ~150 m with a σ₀ of 23.7 ± 0.2, temperature of 24.4 ± 0.6°C, and salinity of 35.4 ± 0.5, aligning with previous studies (Table 1) (Tilliette et al., 2022). Below that, in the thermocline and extending to ~700 m, two major water masses were present: SPSTUW and Western South Pacific Central Water (WSPCW) (Talley et al., 2011; Lehmann et al., 2018; Tilliette et al., 2022) (Figure 2) (Table 1). Across the transect, SPSTUW had an average (± 1 S.D.) σ₀ of 25.0 ± 0.2, temperature of 22.5 ± 0.8°C and was further recognized by its characteristic salinity maximum of 35.7 ± 0.1 (Talley et al., 2011; Lehmann et al., 2018; Tilliette et al., 2022) between 150 and 250 m (Table 1). Between 250 and 500 m, WSPCW was identified by a linear temperature-salinity relationship, with an average (± 1 S.D.) σ₀ of 26.4 ± 0.2, temperature of 12.6 ± 0.9°C and salinity of 34.9 ± 0.2, comparable to values reported by Lehmann et al. (2018) and Tilliette et al. (2022). Notably, between 380 and 400 m South Pacific Subtropical Mode Water (SPSTMW), a subsidiary of WSPCW, was identified by its characteristic σ₀ of 26.0 (Talley et al., 2011), with an average (± 1 S.D.) temperature of 14.9 ± 0.5°C and salinity of 35.2 ± 0.1 (Table 1). Finally, deep water masses were observed in the SPG east of the Tonga-Kermadec Arc. In particular, Antarctic Intermediate Water (AAIW) was observed at station 8-2019 and station 2-2020 between 630 and 700 m where a σ₀ of 26.9 ± 0.1 temperature of 6.5 ± 0.1°C and salinity of 34.4 ± 0.0, aligning with previous studies (Table 1) (Talley et al., 2011; Tilliette et al., 2022).

## 4 Results

### 4.1 NO₃⁻+NO₂⁻ concentration and δ¹⁵N

The [NO₃⁻+NO₂⁻] in the upper 100 m of the WTSP was ≤0.1 µM except at stations influenced by the hydrothermal vents, i.e., stations 5-2019 and 10-2019, where the [NO₃⁻+NO₂⁻] was 0.2 ± 0.0 µM to 0.7 ±
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>FIGURE 2
Temperature-salinity plot of samples from the 2019 and 2020 TONGA cruises as well as the prevalent water masses across the transect. The dotted grey lines indicate potential density ($\sigma_0$) isopycnals. The water masses include surface waters, South Pacific Subtropical Under Water (SPSTUW), Western South Pacific Central Water (WSPCW), South Pacific Subtropical Mode Water (SPSTMW), a subsidiary of WSPCW and Antarctic Intermediate Water (AAIW).</figcaption></figure>

0.0 µM (average ±1 S.D.) between 60 m and 100 m (Figures 3A, C). At 400 m, the [NO₃⁻+NO₂⁻] increased to between 10 and 15 µM, corresponding to δ¹⁵Nₙₐ₃+ₙₐ₂ ranging from 6 to 8‰ (Figures 3B, D). This is consistent with the presence of WSPCW (Tilliette et al., 2022) and more particularly, SPSTMW (Lehmann et al., 2018) and aligns with other studies in the region (Knapp et al., 2018). At station 8-2019, the [NO₃⁻+NO₂⁻] increased to 26.7 µM at 630 m with a corresponding δ¹⁵Nₙₐ₃+ₙₐ₂ of 6.9 ± 0.1‰ (Figures 3A, C), consistent with AAIW (Talley et al., 2011; Lehmann et al., 2018; Tilliette et al., 2022). The majority of stations within MW and the LB (i.e., stations 1-2019 to 5-2019, 10-2019, and 12-2019) had δ¹⁵Nₙₐ₃+ₙₐ₂ ranging from 4 to 6‰ at 200 m, decreasing shallower in the water column to 2 to 4‰ at 150 m associated with the transition between SPSTUW and surface waters (Figures 3B, D). The SPG stations (stations 6-2019, 7-2019 and 8-2019) had an average δ¹⁵Nₙₐ₃+ₙₐ₂ of 6.8 ± 0.0‰ at ~200 m characteristic of SPSTUW. This δ¹⁵Nₙₐ₃+ₙₐ₂ was significantly higher than the δ¹⁵Nₙₐ₃+ₙₐ₂ at 200 m in MW and LB samples, where the average δ¹⁵Nₙₐ₃+ₙₐ₂ at 200 m was 5.0 ± 0.6‰ and

5.1 ± 0.6‰, respectively (p<0.5 and p<0.01, respectively, evaluated with the Kruskal-Wallis test; Kruskal and Wallis, 1952). Furthermore, the elevated δ¹⁵Nₙₐ₃+ₙₐ₂ in SPG SPSTUW corresponded to significantly lower [NO₃⁻+NO₂⁻] at 200 m (2.4 ± 0.8 µM) compared to the LB, where the average [NO₃⁻+NO₂⁻] at 200 m was 3.3 ± 0.6 µM (p<0.01, Kruskal and Wallis, 1952) (Figures 3A, C). Within the surface waters (upper 150 m), the δ¹⁵Nₙₐ₃+ₙₐ₂ at hydrothermal station 5-2019 was significantly lower between the four casts (5a – d), 1.8 ± 0.9‰, compared to the average δ¹⁵Nₙₐ₃+ₙₐ₂ in the upper 150 m at hydrothermal station 10 (10 a, b), 4.4 ± 0.6‰ (p<0.01, Kruskal and Wallis, 1952) (Figures 3B, D). The lowest δ¹⁵Nₙₐ₃+ₙₐ₂, 0.7 ± 0.1‰, was observed at station 5c-2019 at 100 m, above which δ¹⁵Nₙₐ₃+ₙₐ₂ increased to 1.0 ± 0.0‰ at 95 m (Figures 3A, C). The δ¹⁵Nₙₐ₃+ₙₐ₂ reported here for the TONGA study are publicly available (Knapp and Forrer, 2023).

## 4.2 PN<sub>sink</sub> flux and δ¹⁵N

The PN<sub>sink</sub> flux collected in the shallow short-term drifting traps was calculated to be 350 µmol N m⁻² d⁻¹ (170 m, station 5a-2019), 436 µmol N m⁻² d⁻¹ (270 m, station 5a-2019), and 693 µmol N m⁻² d⁻¹ (270 m, station 10a-2019) (Table 2). Further, the average (± 1 S.D.) δ¹⁵N<sub>PNsink</sub> was -0.5 ± 3.5‰ and -0.2 ± 1.9‰ at 170 m and 270 m at station 5a-2019, respectively, and -0.6 ± 2.3‰ at 270 m at station 10a-2019 (Table 2) (Figure 4). In comparison, the annual average PN<sub>sink</sub> flux collected in the long-term 1000 m PPS5 moored trap at station 12-2019/4-2020 was an order of magnitude lower, 16.5 ± 14.3 µmol N m⁻² d⁻¹, and had a higher annual mass-weighted average (± 1 S.D.) δ¹⁵N<sub>PNsink</sub>, 3.4 ± 1.9‰, compared to the shallower, short-term traps (Table 2) (Figure 4).

The seasonal average (± 1 S.D.) PN<sub>sink</sub> fluxes collected in the deep, moored trap were higher in the austral summer and autumn, at 30.9 ± 11.0 µmol N m⁻² d⁻¹ and 17.0 ± 15.4 µmol N m⁻² d⁻¹, respectively, compared to the average austral winter and spring PN<sub>sink</sub> fluxes of 8.0 ± 9.0 µmol N m⁻² d⁻¹ and 8.5 ± 8.5 µmol N m⁻² d⁻¹, respectively (Table 2) (Figure 4). The average, mass-weighted (± 1 S.D.) summer δ¹⁵N<sub>PNsink</sub> from the 1000 m PPS5 moored trap, 1.5 ± 0.7‰, was lower than the wintertime average of 5.9 ± 1.1‰, while the average (± 1 S.D.) spring and autumn mass-weighted δ¹⁵N<sub>PNsink</sub>, 2.9 ± 0.5‰ and 3.3 ± 1.7‰, respectively, were intermediate between summer and winter values.

## 4.3 Results of the δ¹⁵N budget

The δ¹⁵N budget described above compares the δ¹⁵N of the primary form of N exported from the euphotic zone, δ¹⁵N<sub>PNsink</sub>, with the δ¹⁵N of the two input terms, subsurface NO₃⁻+NO₂⁻ and N₂ fixation. This provides a geochemically-derived estimate of the fractional contribution of N₂ fixation to export production (f<sub>nfix</sub>) as well as rate of N₂ fixation (R<sub>nfix</sub>). Given that the subsurface δ¹⁵Nₙₐ₃+ₙₐ₂ source is difficult to constrain, we evaluate the shallow δ¹⁵N budgets using the shallowest subsurface δ¹⁵Nₙₐ₃+ₙₐ₂ minima as well as the sample immediately below the minima (Knapp et al., 2021), while the δ¹⁵N budget using the deep trap PN<sub>sink</sub> flux uses the average subsurface
<table><caption>TABLE 1 Water masses identified from the 2019 and 2020 TONGA cruises and their average (± 1 S.D.) hydrographic and $NO_3^-$ + $NO_2^-$ properties and number (n) of samples from each water mass.</caption><thead><tr><th>Water Mass</th><th>Depth (m)</th><th>Temperature (°C)</th><th>Salinity</th><th>σ<sub>θ</sub></th><th>[NO<sub>3</sub><sup>-</sup> + NO<sub>2</sub><sup>-</sup>] µM</th><th>δ<sup>15</sup>N<sub>NO3+NO2</sub> (‰ vs. N<sub>2</sub> in air)</th></tr></thead><tbody><tr><td>Surface waters (97)</td><td>0 – 150</td><td>24.4 ± 0.6<sup>1</sup></td><td>35.4 ± 0.5<sup>1</sup></td><td>23.7 ± 0.2</td><td>0.2 ± 0.2<sup>1</sup></td><td>2.4 ± 1.5<sup>4</sup><br>4.3 ± 1.7<sup>5</sup></td></tr><tr><td>SPSTUW (38)</td><td>150 – 250</td><td>22.5 ± 0.8<sup>1,3</sup></td><td>35.7 ± 0.1<sup>1,3</sup></td><td>25.0 ± 0.2</td><td>2.4 ± 0.8</td><td>4.9 ± 1.4</td></tr><tr><td>WSPCW (30)</td><td>250 – 550</td><td>12.6 ± 0.9<sup>1,2</sup></td><td>34.9 ± 0.2<sup>1,2</sup></td><td>26.4 ± 0.2<sup>2</sup></td><td>7.8 ± 2.5<sup>1</sup></td><td>7.0 ± 0.7<sup>2</sup></td></tr><tr><td>SPSTMW (4)</td><td>380 – 400</td><td>14.9 ± 0.5<sup>3</sup></td><td>35.2 ± 0.1<sup>3</sup></td><td>26.0 ± 0.0<sup>3</sup></td><td>9.0 ± 0.5</td><td>7.4 ± 0.4</td></tr><tr><td>AAIW (2)</td><td>> 600</td><td>6.5 ± 0.1</td><td>34.4 ± 0.0<sup>1,3</sup></td><td>26.9 ± 0.1<sup>2,3</sup></td><td>26.7 ± 1.2<sup>1*</sup></td><td>6.9 ± 0.1<sup>2*</sup></td></tr></tbody></table>

$δ^{15}N_{NO3+NO2}$ minima at station 12-2019/4-2020 and the average SPSTUW $δ^{15}N_{NO3+NO2}$ at station 4-2020. At the shallow trap stations in close proximity to the hydrothermal vents, the subsurface $δ^{15}N_{NO3+NO2}$ ranged from 1.2 to 2.2‰ and 3.6 to 4.7‰ at stations 5-2019 and 10-2019, respectively, while at the deep mooring (station 12-

2019/4-2020) the average subsurface $δ^{15}N_{NO3+NO2}$ minima and SPSTUW $δ^{15}N_{NO3+NO2}$ were 2.4‰ and 4.6‰, respectively (Table 2). Additional uncertainty in the $δ^{15}N$ budgets includes the standard deviation of the $δ^{15}N_{PNsink}$ analysis (Table 2). The $δ^{15}N$ value for N₂ fixation, ($δ^{15}N_{Nfix}$) of -1‰ is based on literature reports of diazotrophic

<figure><img src="image_7.png" /><figcaption>FIGURE 3
Water column profiles of [NO₃⁻ + NO₂⁻] and $δ^{15}N_{NO3+NO2}$ vs. depth of samples from the 2019 and 2020 TONGA cruises (A, B) and vs. potential density (C, D). Error bars represent ±1 S.D. and are often smaller than the symbol size.</figcaption></figure>

<footnote n="1">¹Tilliette et al., 2022</footnote>

<footnote n="2">²Lehmann et al., 2018</footnote>

<footnote n="3">³Talley et al., 2011</footnote>

<footnote n="4">⁴ Average for hydrothermal vent stations only (station 5-2019 and station 10-2019).</footnote>

<footnote n="5">⁵ Average for all non-hydrothermal vent stations (i.e., all stations except station 5-2019 and station 10-2019).</footnote>

<footnote>*n = 1 for this measurement.</footnote>

<footnote>These data align with the studies indicated in superscript.</footnote>
biomass δ¹⁵N (Hoering and Ford, 1960; Minagawa and Wada, 1986; Carpenter et al., 1997) (Table 2). Comparing these values with the δ¹⁵NPNsink we calculate that at station 5-2019, the f_nfix ranged from 77 to 84 ± 159% for the 170 m trap, while the f_nfix ranged from 64 to 76 ± 86% at the 270 m trap (Table 2). At station 10-2019, the f_nfix at the 270 m trap ranged from 90 to 92 ± 50% (Table 2). We further calculate a geochemically derived R_nfix of 282 ± 550 µmol N m⁻<fnref n="2" /> d⁻<fnref n="1" /> and 331 ± 375 µmol N m⁻<fnref n="2" /> d⁻<fnref n="1" /> for the 170 m and 270 m traps deployed at station 5-

2019, respectively, and 638 ± 347 µmol N m⁻<fnref n="2" /> d⁻<fnref n="1" /> for the 270 m trap deployed at station 10-2019 (Table 2). The δ¹⁵N budget calculated for the moored trap at station 12-2019/4-2020 (note that the trap is deeper, i.e. 1000 m instead of 170 – 270 m for the drifting ones) yielded a mass-weighted f_nfix of 12 to 64 ± 29% (average = 42 ± 13%) for the summer, while the spring and autumn had similar but lower mass-weighted f_nfix of 0 to 37 ± 21% (average = 0 ± 29%) and 0 to 43 ± 69% (average = 12 ± 36%), respectively, and winter had a mass-weighted f_nfix of 0 ± 18%.

<table><caption>TABLE 2 The mass and isotopic composition of the sinking particulate N (PNsink) flux captured in the short-term PIT and long-term PPS5 traps, and results of the δ¹⁵N budgets.</caption><thead><tr><th></th><th>Stn.</th><th>Lon.</th><th>Trap depth (m)</th><th>Time (days)*</th><th>Season<fnref n="1" /></th><th>Av. PNsink flux (µmol N m<sup>-2</sup> d<sup>-1</sup>)<sup>2</sup></th><th>Av. δ<sup>15</sup>NPNsink<sup>2</sup> (‰)</th><th>δ<sup>15</sup>NNO3 +NO2 end-member range<sup>3</sup> (‰)</th><th>f<sub>nfix</sub> <sup>4</sup> (%)</th><th>R<sub>nfix</sub> <sup>4</sup> (µmol N m<sup>-2</sup> d<sup>-1</sup>)</th><th>Bottle-based av. N<sub>2</sub> fix rate<sup>2</sup> (µmol N m<sup>-2</sup> d<sup>-1</sup>)</th></tr></thead><tbody><tr><td rowspan="8">TONGA (this study)</td><td>5-2019 (LB)</td><td>175.9° W</td><td>170</td><td>5</td><td>Late Spring</td><td>350</td><td>-0.5 ± 3.5</td><td>1.2 – 2.2</td><td>77 to 84 ± 159</td><td>282 ± 550</td><td>1942 ± 1212<sup>6</sup></td></tr><tr><td>5-2019 (LB)</td><td>175.9° W</td><td>270</td><td>5</td><td>Late Spring</td><td>436</td><td>-0.2 ± 1.9</td><td>1.2 – 2.2</td><td>64 to 76 ± 86</td><td>331 ± 375</td><td>1942 ± 1212<sup>6</sup></td></tr><tr><td>10-2019 (LB)</td><td>175.2° W</td><td>270</td><td>4</td><td>Late Spring</td><td>693</td><td>-0.6 ± 2.3</td><td>3.6 – 4.7</td><td>90 to 92 ± 50</td><td>638 ± 347</td><td>2047 ± 566<sup>6</sup></td></tr><tr><td rowspan="4">12-2019/ 4-2020 <sup>5</sup> (LB)</td><td rowspan="4">177.9° W</td><td rowspan="4">1000</td><td rowspan="4">360</td><td>Spring</td><td>8.5 ± 8.5</td><td>2.9 ± 0.5<sup>9</sup></td><td rowspan="4">2.4<sup>7</sup> – 4.6<sup>8</sup></td><td>0 to 37 ± 21<sup>9</sup></td><td><sup>-10</sup></td><td><sup>-10</sup></td></tr><tr><td>Summer</td><td>30.9 ± 11.0</td><td>1.5 ± 0.7<sup>9</sup></td><td>12 to 64 ± 29<sup>9</sup></td><td><sup>-10</sup></td><td><sup>-10</sup></td></tr><tr><td>Autumn</td><td>17.0 ± 15.4</td><td>3.3 ± 1.7<sup>9</sup></td><td>0 to 43 ± 69<sup>9</sup></td><td><sup>-10</sup></td><td><sup>-10</sup></td></tr><tr><td>Winter</td><td>8.0 ± 9.0</td><td>5.9 ± 1.1<sup>9</sup></td><td>0 ± 18<sup>9</sup></td><td><sup>-10</sup></td><td><sup>-10</sup></td></tr><tr><td rowspan="3">OUTPACE (Knapp et al., 2018)</td><td>A (MW)</td><td>163.6 °E</td><td>150</td><td>5</td><td>Late summer</td><td>303</td><td>0.6 ± 1</td><td>7.0 – 8.4</td><td>80 to 83 ± 13</td><td>254 ± 50</td><td>593 ± 5<sup>11</sup></td></tr><tr><td>B (SPG)</td><td>170.7° W</td><td>150</td><td>5</td><td>Autumn</td><td>30</td><td>3.1 ± 1</td><td>7.2 – 8.3</td><td>50 to 56 ± 12</td><td>16 ± 6</td><td>706 ± 302<sup>11</sup></td></tr><tr><td>C (SPG)</td><td>165.8° W</td><td>150</td><td>5</td><td>Autumn</td><td>47</td><td>7.7 ± 1</td><td>7.0 – 8.4</td><td>0 to 8 ± 11</td><td>5 ± 5</td><td>59 ± 16<sup>11</sup></td></tr></tbody></table>

<footnote n="1">¹ Austral seasons divided into: Spring (September, October, November), Summer (December, January, February), Autumn (March, April, May), Winter (June, July, August).</footnote>

<footnote n="2">² average ± 1 S.D.</footnote>

<footnote n="3">³ Subsurface δ¹⁵NNO3+NO2.</footnote>

<footnote n="4">⁴ See Methods section 2.6.</footnote>

<footnote n="5">⁵ Samples collected bimonthly, then seasonally averaged.</footnote>

<footnote n="6">⁶ Lory et al., 2023.</footnote>

<footnote n="7">⁷ Shallow subsurface δ¹⁵NNO3+NO2 minima end-member for station 12-2019 and 4-2020.</footnote>

<footnote n="8">⁸ Average SPSTUW δ¹⁵NNO3+NO2 end-member at station 4-2020. Note, sampling resolution at station 12-2019 did not include SPSTUW.</footnote>

<footnote n="9">⁹ Mass-weighted average values for each season.</footnote>

<footnote n="10">¹⁰ No data.</footnote>

<footnote n="11">¹¹ Caffin et al., 2018</footnote>

<footnote>The table includes the range in the δ¹⁵NNO3+NO2 end-member, fraction of export production supported by N₂ fixation ("f_nfix") and N₂ fixation rate determined by multiplying the PNsink flux by f_nfix for the TONGA (this study) and OUTPACE (Knapp et al., 2018) projects. Uncertainty in f_nfix and R_nfix reflects both the range in δ¹⁵NNO3+NO2 end-member as well as the standard deviation of the PNsink δ¹⁵N measurements.</footnote>

<footnote>*Trap deployment time.</footnote>
<figure><img src="image_8.png" /><figcaption>FIGURE 4</figcaption></figure>

Bimonthly measurements of PN<sub>sink</sub> flux and δ<sup>15</sup>N<sub>PNsink</sub> from the 1000 m trap deployed at station 12-2019/station 4-2020 between November 2019 to October 2020. Circle color corresponds to the season when the majority of the PN<sub>sink</sub> was collected over bimonthly sampling intervals and circle size corresponds to PN<sub>sink</sub> flux magnitude (µmol N m<sup>-2</sup> d<sup>-1</sup>). Each measurement is plotted at the end date of the two-week sampling interval. The δ<sup>15</sup>N<sub>Nfix</sub> end-member (-1‰) is represented by the dotted black line and the low δ<sup>15</sup>N<sub>NO3+NO2</sub> end-member at station 12-2019 and 4-2020 (2.4‰) and average SPSTUW δ<sup>15</sup>N<sub>NO3+NO2</sub> end-member at station 4-2020 (4.6‰) are represented by the dashed and solid lines, respectively. Data available in Supplementary Table S1.

These f<sub>nfix</sub> values indicate that the highest contribution of N<sub>2</sub> fixation to export production collected at 1000 m was during the summer, followed by autumn and spring. Due to significant PN<sub>sink</sub> flux attenuation with depth (Martin et al., 1987), we do not calculate a R<sub>nfix</sub> for the moored trap data.

# 5 Discussion

## 5.1 TONGA δ¹⁵N budgets reflect high rates of N₂ fixation in the WTSP

The results of the short-term δ¹⁵N budgets suggest that N₂ fixation rates were high (282 to 638 µmol N m⁻² d⁻¹) near the hydrothermal vents at the time of the 2019 cruise. While there are notable differences in the magnitude of these geochemically-derived N₂ fixation rates and the bottle-based ¹⁵N₂ uptake rates reported by Lory et al., 2023, we consider these results to be broadly consistent with one another, as well as consistent with estimates of diazotroph abundance measured contemporaneously (Bonnet et al., 2023b). Specifically, bottle-based ¹⁵N₂ uptake rates were 3 to 7 times higher than the R<sub>nfix</sub> estimated from the δ¹⁵N budget (Table 2); however, both rate estimates are at the upper-end typically reported by each technique (Gruber, 2004; Capone et al., 2005; Casciotti et al., 2008; Luo et al., 2012; Knapp et al., 2016a) and align with previous work from the region (Montoya et al., 2004; Berthelot et al., 2017; Bonnet et al., 2017; Knapp et al., 2018; Shao et al., 2023). Prior work investigating N₂ fixation's contribution to export production has attributed discrepancies between these two metrics to potential sediment trap under collection of exported material (Knapp et al., 2016a; Böttjer et al., 2017, Knapp et al., 2018), alternate sources of fixed N to the euphotic zone including horizontal advection (Böttjer et al., 2017), phytoplankton bloom stage as well as temporal delay

between organic matter formation and capture by the sediment trap (de Verneil et al., 2017; Caffin et al., 2018; Knapp et al., 2018), and bottle-based ¹⁵N₂ incubations with their associated methodological considerations (i.e., bottle effects) (White et al., 2020). Additionally, our δ¹⁵N budgets should be considered a lower bound for estimated N₂ fixation rates because of the mixing-model's inherent assumption that the only fate of newly fixed N is to be balanced by the sinking flux, and that no newly fixed N is released to the dissolved pool, which is likley unrealistic (Capone et al., 1994; Glibert & Bronk, 1994; Mulholland and Capone, 2004; Bonnet et al., 2016; Knapp et al., 2016b). Further, the trap depth used here, 170 m, is below the base of the mixed layer, and thus underestimates new/export production. While euphotic zone nitrification is a source of low-δ¹⁵N<sub>NO3</sub> that could lead to an overestimation of N₂ fixation supported export production, rates of euphotic zone nitrification from this and other similar oligotrophic regions are low (<10 nmol L⁻¹ d⁻¹) (Smith et al., 2014; Raes et al., 2020) compared to the very high rates of N₂ fixation using multiple metrics in this study. This therefore suggests that N₂ fixation is the dominant mechanism generating the low-δ¹⁵N<sub>PNsink</sub> signal observed.

Regardless of the mechanism(s) driving these discrepancies, both the δ¹⁵N budget and ¹⁵N₂ uptake rate estimates are also consistent with the elevated diazotroph abundances (Bonnet et al., 2023a; Lory et al., 2023) observed on the 2019 TONGA cruise (Figure 5). Here we compare averages of biological and geochemical metrics associated with N₂ fixation for the three hydrographic regions, MW, the LB, and the SPG (defined in Section 2.1). While results from the qPCR analysis targeting *nifH* genes indicate that *Trichodesmium* spp. and UCYN-A dominated the diazotroph assemblage in the upper 50 m across the TONGA transect, *Trichodesmium* spp. were most abundant in the LB near the hydrothermal vents (average 1.0x10⁷ gene copies L⁻¹), followed by UCYN-A (average 3.0x10⁶ gene copies L⁻¹) (Figures 5A, B). These
exceptionally high abundances of *Trichodesmium* spp. and UCYN-A were on the order of one to four times higher than previous studies (e.g., Zehr & Turner, 2001; Moisander et al., 2010; Turk-Kubo et al., 2012; Moisander et al., 2014; Benavides et al., 2018; Benavides et al., 2020; Confesor et al., 2022). Notably, the highest regionally-averaged, trapezoidally-integrated upper 50 m ¹⁵N₂ uptake rates were found in the LB (1038 ± 600 µmol N m⁻² d⁻¹), where *Trichodesmium* spp. were most abundant and where the lowest regionally-averaged δ¹⁵Nₐ₄₃⁺ₐ₄₂⁻ subsurface minima of 2.8 ± 1.5‰ (Figure 5F) was observed between 100 and 200 m. This underscores the potential significance of *Trichodesmium* spp. supporting N₂ fixation near the hydrothermal vents. UCYN-A was the dominant diazotroph in the upper 50 m in MW and the SPG, with regional averages of 2.5x10⁸ gene copies L⁻¹ and 3.1x10⁸ gene copies L⁻¹, respectively, followed by *Trichodesmium* spp., with regional averages of 3.0x10⁵ and 3.7x10⁵ gene copies L⁻¹, respectively. Average abundances of UCYN-B (2.6x10⁴ to 3.2x10⁵ gene copies L⁻¹), UCYN-C (4.3x10¹ to 1.7x10² gene copies L⁻¹) and Gamma proteobacteria (5.4x10³ to 9.9x10³ gene copies L⁻¹) remained comparatively low across the transect (Figures 5C-E), but were similar in magnitude to previous studies in the region (Moisander et al., 2014; Benavides et al., 2018; Benavides et al., 2020). Although UCYN-A have extremely high gene abundances and dominate the upper 50 m in MW and the SPG, the trapezoidally-integrated upper 50 m ¹⁵N₂ uptake rates were lower than in the LB, 713 ± 691 and 537 ± 629 µmol N m⁻² d⁻¹,

respectively, and were associated with a higher regional average δ¹⁵Nₐ₄₃⁺ₐ₄₂⁻ subsurface minima of 4.4 ± 1.2‰ and 5.5 ± 1.9‰, respectively, observed between 100 to 200 m (Figure 5F). While care should be taken when relating *nifH* gene copies to diazotroph biomass, these gene copy abundances broadly correspond to elevated diazotroph abundances (Meiler et al., 2022) and confirm the significance of diazotrophy in the region.

## 5.2 N₂ fixation is an important source of N supporting export production in the WTSP

The agreement between the geochemically-derived N₂ fixation rates, ¹⁵N₂ uptake rates and diazotroph abundances together indicate that export production in the WTSP at the time of this study was driven by N₂ fixation (Bonnet et al., 2023b). This is in contrast to prior work in other oligotrophic regions where the majority of export was supported by subsurface NO₃⁻, even when N₂ fixation inputs were high (Casciotti et al., 2008; Bourbonnais et al., 2009; White et al., 2013; Böttjer et al., 2017). The shallow sediment traps deployed for the TONGA project indicate that N₂ fixation supports a majority of export production (fₙₐₗ = 64 to 92%) near the hydrothermal vents, at least in the late spring (Table 2; Figures 6A, B). These fₙₐₗ values are similar to those calculated from the previous OUTPACE campaign during the late summer/early autumn in MW (station A, 80 to 83 ± 13%), and near the Tonga-

<figure><img src="image_9.png" /><figcaption>FIGURE 5
Box plots of the upper 50 m *nifH* gene abundances (gene copies L⁻¹) from Bonnet et al., 2023a and Lory et al., 2023 for the hydrographic regions of MW, the LB and the SPG for (A) *Trichodesmium* spp., (B) UCYN-A, (C) UCYN-B, (D) UCYN-C, and (E) Gamma proteobacteria, as well as (F) box plots of the regional average, trapezoidally-integrated upper 50 m ¹⁵N₂ fixation rates from Lory et al., 2023, with the corresponding regional average 90 to 195 m δ¹⁵Nₐ₄₃⁺ₐ₄₂⁻ (blue triangles). The open circles associated with the box plots indicate outliers.</figcaption></figure>
Kermadec Arc (station B, 50 to 56 ± 12%) (Table 2; Figure 6C) (Knapp et al., 2018). The high $f_{nfix}$ values reported here from the short-term traps deployed at TONGA Stations 5 and 10 replicate a geochemical signature that has not been observed outside of the WTSP, underscoring the significance of $N_2$ fixation regionally (Bonnet et al., 2023b). We also emphasize the potential for the $f_{nfix}$ from our $\delta^{15}$N budgets to underestimate the importance of $N_2$ fixation to export due to the mixing-model's inherent assumption that the only fate of newly fixed N is the $PN_{sink}$ flux captured by the sediment traps, as opposed to being released and persisting as dissolved organic nitrogen (Capone et al., 1994; Glibert and Bronk, 1994).

While the results of the short-term $\delta^{15}$N budgets from both the TONGA and OUTPACE campaigns found that $N_2$ fixation supports the majority of export production in the late spring, late summer, and early autumn in the LB and MW, we also consider the $\delta^{15}$N$_{PN_{sink}}$ collected in the deep, moored trap at station 12-2019/4-2020 in the LB to evaluate this trend over an annual cycle (Figure 4). Unsurprisingly, the mass flux collected in the shallower, short-term PIT traps was higher and the $\delta^{15}$N$_{PN_{sink}}$ was lower than that collected in the deeper, moored trap. However, we note that the moored trap was deployed ~200 km west of the shallow traps and hydrothermal vents (Table 2) (Figures 1, 4), and the conical shape of the PPS5 has been observed to undersample (Baker et al., 2020; Tilliette et al., 2023). The $\delta^{15}$N$_{PN_{sink}}$ in the shallow PIT traps collected in the late spring at stations 5-2019 and 10-2019 ranged from -0.2 to -0.6‰ compared to a seasonal average $\delta^{15}$N$_{PN_{sink}}$ of 2.9 ± 0.5‰ and 1.5 ± 0.7‰ observed in the moored PPS5 trap during the spring and summer, respectively. We expect that the higher $\delta^{15}$N$_{PN_{sink}}$ found in the deeper moored trap likely resulted from the collection of $PN_{sink}$ from a larger surface area than the shallow short-term traps, where export production may have been supported by a mixture of N sources with higher $\delta^{15}$N (Siegel and Deuser, 1997; Siegel et al., 2008). Additionally, horizontal advection of particles generated at locations not impacted by the shallow hydrothermal vents potentially decouples the euphotic zone diazotrophic abundance and/or importance from the PN collected in the moored trap (Waniek et al., 2000). Indeed, the exported material in the deep trap was observed to be compositionally different from that captured in the shallow, short-term traps, where the hydrothermal signature of the particles was less evident due to organic matter remineralization while being transported to depth (Tilliette et al., 2023). The associated distance and time components of sinking PN to the deep trap potentially underestimates the importance of $N_2$ fixation to export production. Similar flux and isotopic composition offsets have been observed between shallow and deep sediment traps in the ETSP (Berelson et al., 2015; Knapp et al., 2016a; Tilliette et al., 2023).

Since aerosol inputs to this region are minimal (Guieu et al., 2018), we expect that the seasonal variability of $\delta^{15}$N$_{PN_{sink}}$ in the deep, moored trap reflects shifts in the importance of $N_2$ fixation and subsurface $NO_3^-$ for supporting export production over seasonal timescales. In the deep trap, annual $PN_{sink}$ fluxes peaked

in the summer (30.9 ± 11.0 µmol N m⁻² d⁻¹), coinciding with the lowest average mass-weighted $\delta^{15}$N$_{PN_{sink}}$, 1.5 ± 0.7‰, while the lowest $PN_{sink}$ fluxes were observed in the winter (8.0 ± 9.0 µmol N m⁻² d⁻¹) and coincided with the highest average mass-weighted $\delta^{15}$N$_{PN_{sink}}$, 5.9 ± 1.1‰, indicating that isotopically distinct N sources support export seasonally. Since there are steep gradients in both [NO₃⁻ + NO₂⁻] and $\delta^{15}$N$_{NO₃+NO₂}$ with depth (Figures 3, 6), a higher winter $\delta^{15}$N$_{PN_{sink}}$ may reflect entrainment of a deeper NO₃⁻ + NO₂⁻ source (likely SPSTUW) with a higher $\delta^{15}$N$_{NO₃+NO₂}$ due to winter mixing (Moutin et al., 2018). The net effect of a higher subsurface $\delta^{15}$N$_{NO₃+NO₂}$ end-member would be to raise the estimated $f_{nfix}$ (Table 2) (Böttjer et al., 2017). As a result, the mass-weighted seasonal $f_{nfix}$ values for the deep trap range from 12 to 64 ± 29% in the summer and 0 ± 18% in the winter, describing a largely $N_2$ fixation supported export system in the summer. Further, since the majority of annual export is focused in the summer, and was supported by low-$\delta^{15}$N N sources, we attribute an important fraction of annual export production and deep (>1000 m) long-term C sequestration to $N_2$ fixation at station 12-2019/4-2020 (Figure 4; Table 2).

Considering the elevated chlorophyll *a* concentrations observed over a large area in this region ranging up to 360,000 km² (Bonnet et al., 2023b), and given the high $R_{nfix}$ and $f_{nfix}$ values estimated at station 5a-2019 and 10a-2019, along with the large fraction of $N_2$ fixation supported export production at station 12-2019/4-2020 over an annual timescale, the otherwise oligotrophic WTSP appears to be biogeochemically unique where $N_2$ fixation supports a large fraction of annual export production as a result of the influence of shallow hydrothermal vents. The significance of these regional $N_2$ fixation inputs in the WTSP are further pronounced in the gradients of water column $\delta^{15}$N$_{NO₃+NO₂}$ both zonally as well as with depth (Figures 3, 6). In particular, $\delta^{15}$N$_{NO₃+NO₂}$ between 150 and 400 m decreases from east to west (SPG to MW) across the zonal transect, and also decreases from ~400 m to shallower depths. These isotopic gradients reflect the accumulation of low-$\delta^{15}$N N inputs in the upper thermocline to the west along this transect that are presumably associated with the remineralization of diazotrophic inputs (Casciotti et al., 2008; Knapp et al., 2008). This accumulation of low-$\delta^{15}$N$_{NO₃+NO₂}$ in the upper 400 m of the WTSP erodes the elevated $\delta^{15}$N$_{NO₃+NO₂}$ originating from dissimilatory NO₃⁻ reduction occurring in the oxygen deficient zones of the ETSP (Bourbonnais et al., 2015; Peters et al., 2018; Casciotti et al., 2013), a geochemical signature that reflects basin-scale compensation of N losses in the east with N inputs in the west that is consistent with peloceanographic records (Brandes and Devol, 2002; Deutsch et al., 2004; Weber and Deutsch, 2014; Knapp et al., 2018).

## 6 Conclusions

Here we report results of $\delta^{15}$N budgets that compare subsurface $\delta^{15}$N$_{NO₃+NO₂}$ with the $\delta^{15}$N$_{PN_{sink}}$ captured in short-term, shallow (170 and 270 m) PIT deployed near the hydrothermal vents of the Tonga-Kermadec Arc and long-term, deep (1000 m) moored PPS5 sediment
<figure><img src="image_10.png" /><figcaption>FIGURE 6</figcaption></figure>

The fraction of N₂ fixation-supported export production in the austral spring at (A) TONGA station 5-2019, and, (B) TONGA station 10-2019, compared with (C) results from the austral late summer/early autumn at OUTPACE stations (A-C) Each panel includes water column profiles of δ¹⁵N*NO₃⁻*+*NO₂* for samples from the 2019 and 2020 TONGA cruises in color and the OUTPACE cruise in grey and the associated δ¹⁵N budget terms, including the sediment trap δ¹⁵N*PNsink* (inverted triangles and open circle and square), the δ¹⁵N*Nfix* (vertical dashed line) and ranges in subsurface δ¹⁵N*NO₃⁻*+*NO₂* which are represented by the orange shaded region in (A), blue shaded region in (B), and steel-grey shaded region in (C) (Table 2). The fractional contribution of N₂ fixation to export production is indicated by the grey shading and the corresponding fraction is indicated along the bottom of each panel.

traps deployed ~200 km west of the Arc. These results are evaluated in the context of ¹⁵N₂ uptake rates (Lory et al., 2023) and *nifH* gene abundances (Bonnet et al., 2023b) collected contemporaneously, as well as with prior work from the region (Knapp et al., 2018). Results from the short-term, shallow δ¹⁵N budgets indicate that N₂ fixation supports the majority, 64 to 92%, of export in late spring in the Lau Basin, while the mass-weighted, seasonally-averaged δ¹⁵N budgets from deeper traps suggest that N₂ fixation supports 12 to 64% of export production and thus long-term C sequestration in the summer when the highest PNsink fluxes are observed. As the seasons progress into winter, export production becomes increasingly supported by subsurface NO₃⁻. The observations from this cruise as well as from the OUTPACE study (Knapp et al., 2018) are in contrast to other regions explored so far, where even significant N₂ fixation inputs do not support the majority of export (Casciotti et al., 2008; Bourbonnais et al., 2009; Böttjer et al., 2017), underscoring the significance of diazotrophy in the WTSP. While diazotroph abundance was high across the transect, *Trichodesmium* spp. *nifH* gene copies were highest in the vicinity of the hydrothermal vents, which appear shallow enough to meet the considerable Fe demands of primary productivity in general (Tilliette et al., 2022), and N₂ fixation in particular, in the region (Bonnet et al., 2023b), highlighting the sensitivity of N₂ fixation to Fe availability. These results suggest that the significant N₂ fixation inputs to the WTSP in the late spring, summer, and early autumn work to lower the elevated upper thermocline δ¹⁵N*NO₃⁻*+*NO₂* originating from dissimilatory NO₃⁻ reduction in the oxygen deficient zones of the Eastern Tropical South Pacific.

## Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

## Author contributions

SB, CG, and AK designed the study. SB and CG carried out the sampling. HF and RT carried out the δ¹⁵N*NO₃⁻*+*NO₂* sample analysis and data aquisition. SB and OG carried out the δ¹⁵N*PNsink* sample analysis and data aquisition. HF and AK wrote the first manuscript draft, which was then revised by all authors. All authors contributed to the article and approved the submitted version.

## Funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research is a contribution to the TONGA project (Shallow hydroThermal sSources of trace elemeNts: potential impacts on biological productivity and the biological carbon pump; TONGA cruise https:// doi.org/10.17600/18000884) funded by the Agence Nationale de la Recherche (grant TONGA ANR-18-CE01-0016), the LEFE-CyBER
program (CNRS-INSU), the A-Midex foundation, the TGIR Flotte océanographique française, the Institut de Recherche pour le Développement (IRD). HF acknowledges funding from the Winchester Fund at Florida State University's EOAS Department. AK acknowledges NSF OCE-1829797 for supporting the nitrate isotopic analyses.

## Acknowledgments

The authors warmly thank the captain and crew of the R/V L'Atalante and R/V Alis (both TGIR Flotte, operated by IFREMER) for outstanding shipboard operations. Vincent Taillandier and Nagib Bhairy are thanked for the CTD rosettes management, and data processing, Sandra Nunige for nutrient analyses of the 2019 samples, Mary Dennis for her assistance with the 'denitrifier method' at Florida State University, Caroline Lory for her assistance with the ¹⁵N₂ fixation experiments and data acquisition and Nathalie Leblond (Cellule Piège INSU) for the treatment/ splitting of the moored trap.

## References

Altabet, M. A. (1988). Variations in nitrogen isotopic composition between sinking and suspended particles: implications for nitrogen cycling and particle transformation in the open ocean. Deep Sea Research Part A. Oceanographic Res. Papers 35(4), 535-554. doi: 10.1016/0198-0149(88)90130-6

Aminot, A., and Kerouel, R. (2007). Dosage automatique des nutriments dans les eaux marines: méthodes en flux continu, Ifremer, Plouzané.

Baker, C. A., Estapa, M. L., Iversen, M., and Lampitt Buesseler, R. K. (2020). Are all sediment traps created equal? An intercomparison study of carbon export methodologies at the PAP-SO site. Prog. Oceanography 184, 1023-1021. doi: 10.1016/j.poccean.2020.102317

Benavides, M., Martias, C., Elifantz, H., Berman-Frank, I., Dupouy, C., and Bonnet, S. (2018). Dissolved organic matter influences N2 fixation in the New Caledonian lagoon (Western tropical south pacific). Front. Mar. Sci. 5, 89. doi: 10.3389/fmars.2018.00089

Benavides, M., Duhamel, S., Van Wambeke, F., Shoemaker, K. M., Moisander, P. H., Salamon, E., et al. (2020). Dissolved organic matter stimulates N2 fixation and nifH gene expression in *Trichodesmium*. FEMS Microbiol. Lett. 367 (4), fnaa034. doi: 10.1093/femsle/fnaa034

Berelson, W. M., Haskell, W. Z., Prokopenko, M., Knapp, A. N., Hammond, D. E., Rollins, N., et al. (2015). Biogenic particle flux and benthic remineralization in the Eastern Tropical South Pacific. Deep Sea Res. Part I: Oceanographic Res. Papers 99, 23-34. doi: 10.1016/j.dsr.2014.12.006

Berman-Frank, I., Cullen, J. T., Shaked, Y., Sherrell, R. M., and Falkowski, P. G. (2001). Iron availability, cellular iron quotas, and nitrogen fixation in *Trichodesmium*. Limnology Oceanography 46 (6), 1249-1260. doi: 10.4319/lo.2001.46.6.1249

Berthelot, H., Benavides, M., Moisander, P. H., Grosso, O., and Bonnet, S. (2017). High-nitrogen fixation rates in the particulate and dissolved pools in the Western Tropical Pacific (Solomon and Bismarck Seas). Geophysical Res. Lett. 44 (16), 8414-8423. doi: 10.1002/2017GL073856

Bonnet, S., Biegala, I. C., Dutrieux, P., Slemons, L. O., and Capone, D. G. (2009). Nitrogen fixation in the western equatorial Pacific: Rates, diazotrophic cyanobacterial size class distribution, and biogeochemical significance: N₂ FIXATION IN THE EQUATORIAL PACIFIC. Global Biogeochemical Cycles 23 (3). doi: 10.1029/2008GB003439

Bonnet, S., Berthelot, H., Turk-Kubo, K., Fawcett, S., Rahav, E., L'Helguen, S., et al. (2016). Dynamics of N₂ fixation and fate of diazotroph-derived nitrogen in a low-nutrient, low-chlorophyll ecosystem: results from the VAHINE mesocosm experiment (New Caledonia). Biogeosciences 13 (9), 2653-2673. doi: 10.5194/bg-13-2653-2016

Bonnet, S., Caffin, M., Berthelot, H., and Moutin, T. (2017). Hot spot of N₂ fixation in the western tropical South Pacific pleads for a spatial decoupling between N₂ fixation and denitrification. Proc. Natl. Acad. Sci. 114 (14). doi: 10.1073/pnas.1619514114

Bonnet, S., Benavides, M., Le Moigne, F. A. C., Camps, M., Torremocha, A., Grosso, O., et al. (2023a). Diazotrophs are overlooked contributors to carbon and nitrogen export to the deep ocean. ISME J. 17 (1), 47-58. doi: 10.1038/s41396-022-01319-3

## Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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## Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmars.2023.1249115/full#supplementary-material

Bonnet, S., Guieu, C., Taillandier, V., Boulart, C., Bouruet-Aubertot, P., Gazeau, F., et al. (2023b). Natural iron fertilization by shallow hydrothermal sources fuels diazotroph blooms in the ocean. Science 380 (6647), 812-817. doi: 10.1126/science.abq4654

Böttjer, D., Dore, J. E., Karl, D. M., Letelier, R. M., Mahaffey, C., Wilson, S. T., et al. (2017). Temporal variability of nitrogen fixation and particulate nitrogen export at Station ALOHA. Limnology Oceanography 62 (1), 200-216. doi: 10.1002/lno.10386

Bourbonnais, A., Lehmann, M. F., Waniek, J. J., and Schulz-Bull, D. E. (2009). Nitrate isotope anomalies reflect N₂ fixation in the Azores Front region (subtropical NE Atlantic). J. Geophysical Res. 114 (C3), C03003. doi: 10.1029/2007JC004617

Bourbonnais, A., Altabet, M. A., Charoenpong, C. N., Larkum, J., Hu, H., Bange, H. W., et al. (2015). N-loss isotope effects in the Peru oxygen minimum zone studied using a mesoscale eddy as a natural tracer experiment: N-LOSS ISOTOPE EFFECTS IN AN EDDY. Global Biogeochemical Cycles 29 (6), 793-811. doi: 10.1002/2014GB005001

Braman, R. S., and Hendrix, S. A. (1989). Nanogram nitrite and nitrate determination in environmental and biological materials by vanadium(III) reduction with chemiluminescence detection. Analytical Chem. 61 (24), 2715-2718. doi: 10.1021/ac00199a007

Brandes, J. A., and Devol, A. H. (2002). A global marine-fixed nitrogen isotopic budget: Implications for Holocene nitrogen cycling: NITROGEN ISOTOPIC BUDGET. Global Biogeochemical Cycles 16 (4), 67-1-67-14. doi: 10.1029/2001GB001856

Buesseler, K. O., Antia, A. N., Chen, M., Fowler, S. W., Gardner, W. D., Gustafsson, O., et al. (2007). An assessment of the use of sediment traps for estimating upper ocean particle fluxes. J. Mar. Res. 65 (3), 345-416. doi: 10.1357/002224007781567621

Caffin, M., Moutin, T., Foster, R.A., Bouruet-Aubertot, P., Doglioli, A.M., Berthelot, H., et al. (2018). N₂ fixation as a dominant new N source in the western tropical South Pacific Ocean (OUTPACE cruise). Biogeosciences 15 (8), 2565-2585. doi: 10.5194/bg-15-2565-2018

Capone, D. G. (2001). Marine nitrogen fixation: what's the fuss? Curr. Opin. Microbiol. 4 (3), 341-348. doi: 10.1016/S1369-5274(00)00215-0

Capone, D. G., Zehr, J. P., Paerl, H. W., Bergman, B., and Carpenter, E. J. (1997). *Trichodesmium*, a globally significant marine cyanobacterium. Science 276 (5316), 1221-1229. doi: 10.1126/science.276.5316.1221

Capone, D. G., Burns, J. A., Montoya, J. P., Subramaniam, A., Mahaffey, C., Gunderson, T., et al. (2005). Nitrogen fixation by *Trichodesmium* spp.: An important source of new nitrogen to the tropical and subtropical North Atlantic Ocean: NITROGEN FIXATION IN THE NORTH ATLANTIC. Global Biogeochemical Cycles 19 (2). doi: 10.1029/2004GB002331

Capone, D. G., Ferrier, M. D., and Carpenter, E. J. (1994). Amino acid cycling in colonies of the planktonic marine cyanobacterium *trichodesmium thiebautii*. Appl. Environ. Microbiol. 60 (11), 3989-3995. doi: 10.1128/aem.60.11.3989-3995.1994
Carpenter, E. J., Harvey, H. R., Fry, B., and Capone, D. G. (1997). Biogeochemical tracers of the marine cyanobacterium Trichodesmium. *Deep Sea Res. Part I: Oceanographic Res. Papers* 44 (1), 27–38. doi: 10.1016/S0967-0637(96)00091-X

Casciotti, K. L., Buchwald, C., and McIlvin, M. (2013). Implications of nitrate and nitrite isotopic measurements for the mechanisms of nitrogen cycling in the Peru oxygen deficient zone. *Deep Sea Res. Part I: Oceanographic Res. Papers* 80, 78–93. doi: 10.1016/j.dsr2.2013.05.017

Casciotti, K. L., Sigman, D. M., Böhlke, J.K., and Hilkert, A. (2002). Measurement of the oxygen isotopic composition of nitrate in seawater and freshwater using the denitrifier method. *Analytical Chem.* 74 (19), 4905–4912. doi: 10.1021/ac020113w

Casciotti, K. L., Trull, T. W., Glover, D. M., and Davies, D. (2008). Constraints on nitrogen cycling at the subtropical North Pacific Station ALOHA from isotopic measurements of nitrate and particulate nitrogen. *Deep Sea Res. Part II: Topical Stud. Oceanography* 55 (14–15), 1661–1672. doi: 10.1016/j.dsr2.2008.04.017

Confesor, K. A., Selden, C. R., Powell, K. E., Donahue, L. A., Mellett, T., Caprara, S., et al. (2022). Defining the realized niche of the two major clades of trichodesmium: A study on the west Florida shelf. *Front. Mar. Sci.* 9, 821655. doi: 10.3389/fmars.2022.821655

Conway, T. M., and John, S. G. (2014). Quantification of dissolved iron sources to the North Atlantic Ocean. *Nature* 511 (7508), 212–215. doi: 10.1038/nature13482

Dekaezemacker, J., Bonnet, S., Grosso, O., Moutin, T., Bressac, M., and Capone, D. G. (2013). Evidence of active nitrogen fixation in surface waters of the eastern tropical South Pacific during El Niño and La Niña events and evaluation of its potential nutrient controls: N₂ FIXATION IN THE ETSP. *Global Biogeochemical Cycles* 27 (3), 768–779. doi: 10.1002/gbc.20063

Deutsch, C., Sigman, D. M., Thunell, R. C., Meckler, A. N., and Haug, G. H. (2004). Isotopic constraints on glacial/interglacial changes in the oceanic nitrogen budget: GLACIAL/INTERGLACIAL NITROGEN BUDGET. *Global Biogeochemical Cycles* 18 (4). doi: 10.1029/2003GB002189

de Verneil, A., Rousselet, L., Doglioli, A. M., Petrenko, A. A., and Moutin, T. (2017). The fate of a southwest Pacific bloom: gauging the impact of submesoscale vs. mesoscale circulation on biological gradients in the subtropics. *Biogeosciences* 14 (14), 3471–3486. doi: 10.5194/bg-14-3471-2017

Dore, J. E., Brum, J. R., Tupas, L. M., and Karl, D. M. (2002). Seasonal and interannual variability in sources of nitrogen supporting export in the oligotrophic subtropical North Pacific Ocean. *Linnmology Oceanography* 47 (6), 1595–1607. doi: 10.4319/lo.2002.47.6.1595

Dugdale, R. C., and Goering, J. J. (1967). Uptake of new and regenerated forms of nitrogen in primary productivity: uptake of nitrogen in primary productivity. *Linnmology Oceanography* 12 (2), 196–206. doi: 10.4319/lo.1967.12.2.0196

Dutkiewicz, S., Ward, B. A., Monteiro, F., and Follows, M. J. (2012). Interconnection of nitrogen fixers and iron in the Pacific Ocean: Theory and numerical simulations: MARINE NITROGEN FIXERS AND IRON. *Global Biogeochemical Cycles* 26 (1). doi: 10.1029/2011GB004039

Glibert, P. M., and Bronk, D. A. (1994). Release of dissolved organic nitrogen by marine diazotrophic cyanobacteria, trichodesmium spp.t. *Appl. Environ. Microbiol.* 60, 3996–4000. doi: 10.1128/aem.60.11.3996-4000.1994

Gruber, N. (2004). "The dynamics of the marine nitrogen cycle and its influence on atmospheric CO₂ variations," in *The ocean carbon cycle and climate*. Eds. M. Follows and T. Oguz (Dordrecht: Springer Netherlands), 97–148. doi: 10.1007/978-1-4020-2087-2_4

Guieu, C., Bonnet, S., Petrenko, A., Menkes, C., Chavagnac, V., Desboeufs, K., et al. (2018). Iron from a submarine source impacts the productive layer of the eastern Tropical South Pacific (WTSP). *Sci. Rep.* 8 (1), 9075. doi: 10.1038/s41598-018-27407-z

Hoering, T. C., and Ford, H. T. (1960). The isotope effect in the fixation of nitrogen by azotobacter. *J. Am. Chem. Soc.* 82 (2), 376–378. doi: 10.1021/ja01487a031

Jickells, T. D., An, Z. S., Andersen, K. K., Baker, A. R., Bergametti, G., Brooks, N., et al. (2005). Global iron connections between desert dust, ocean biogeochemistry, and climate. *Science* 308 (5718), 67–71. doi: 10.1126/science.1105959

Karl, D., Letelier, R., Tupas, L., Dore, J., Christian, J., and Hebel, D. (1997). The role of nitrogen fixation in biogeochemical cycling in the subtropical North Pacific Ocean. *Nature* 388 (6642), 533–538. doi: 10.1038/41474

Knapp, A. N., DiFiore, P. J., Deutsch, C., Sigman, D. M., and Lipschultz, F. (2008). Nitrate isotopic composition between Bermuda and Puerto Rico: Implications for N₂ fixation in the Atlantic Ocean: SARGASSO SEA NITRATE ISOTOPES. *Global Biogeochemical Cycles* 22 (3). doi: 10.1029/2007GB003107

Knapp, A. N., Casciotti, K. L., Berelson, W. M., Prokopenko, M. G., and Capone, D. G. (2016a). Low rates of nitrogen fixation in eastern tropical South Pacific surface waters. *Proc. Natl. Acad. Sci.* 113 (16), 4398–4403. doi: 10.1073/pnas.1515641113

Knapp, A. N., Fawcett, S.E., Martínez-Garcia, A., Leblond, N., Moutin, T., and Bonnet, S. (2016b). Nitrogen isotopic evidence for a shift from nitrate- to diazotroph-fueled export production in the VAHINE mesocosm experiments. *Biogeosciences* 13 (16), 4645–4657. doi: 10.5194/bg-13-4645-2016

Knapp, A. N., McCabe, K.M., Grosso, O., Leblond, N., Moutin, T., and Bonnet, S. (2018). Distribution and rates of nitrogen fixation in the western tropical South Pacific Ocean constrained by nitrogen isotope budgets. *Biogeosciences* 15 (9), 2619–2628. doi: 10.5194/bg-15-2619-2018

Knapp, A. N., Thomas, R. K., Stukel, M.R., Kelly, T.B., Landry, M.R., Selph, K.E., et al. (2021). Constraining the sources of nitrogen fueling export production in the Gulf of

Mexico using nitrogen isotope budgets. *J. Plankton Res.* 44 (5), 692–710. doi: 10.1093/ plantk/fbab049

Knapp, A. N., and Forrer, H. J. (2023). Water column nitrate plus nitrite d15N measurements from seawater collected in November 2019 and November 2020 in the Western Tropical South Pacific. *Biol. Chem. Oceanography Data Manage. Office (BCO-DMO).* doi: 10.26008/1912/bco-dmo.869963.2

Kruskal, W. H., and Wallis, W. A. (1952). Use of ranks in one-criterion variance analysis. *J. Am. Stat. Assoc.* 47, 583–621. 40. doi: 10.1080/01621459.1952.10483441

Kustka, A., Saudo-Wilhelmy, S., Carpenter, E. J., Capone, D. G., and Raven, J. A. (2003). A revised estimate of the iron use efficiency of nitrogen fixation, with special reference to the marine cyanobacterium *trichodesmium* spp. (cyanophyta) ¹. *J. Phycology* 39 (1), 12–25. doi: 10.1046/j.1529-8817.2003.01156.x

Landolfi, A., Kähler, P., Koeve, W., and Oschlies, A. (2018). Global marine N2 fixation estimates: from observations to models. *Front. Microbiol.* 9, 2112. doi: 10.3389/ fmicb.2018.02112

Lehmann, N., Granger, J., Kienast, M., Brown, K. S., Rafter, P. A., Martínez-Méndez, G., et al. (2018). Isotopic evidence for the evolution of subsurface nitrate in the western equatorial pacific. *J. Geophysical Research: Oceans* 123 (3), 1684–1707. doi: 10.1002/ 2017J0C13527

Lory, C., Guieu, C., Rodier, M., Planquette, H., Gonzales Santana, D., Sarthou, G., et al. Environmental drivers of diazotrophs biography in the subtropical South Pacific Ocean. *Front Mar Sci* (2023). Available at: https://campagnes. flotteoceanographique.fr/campagnes/18000884/.

Luo, Y.-W., Doney, S. C., Anderson, L. A., Benavides, M., Berman-Frank, I., Bode, A., et al. (2012). Database of diazotrophs in global ocean: abundance, biomass and nitrogen fixation rates. *Earth System Sci. Data* 4 (1), 47–73. doi: 10.5194/essd-4-47-2012

Mahaffey, C. (2005). The conundrum of marine N2 fixation. *Am. J. Sci.* 305 (6–8), 546–595. doi: 10.2475/ajs.305.6.8-546

Mahowald, N. M., Baker, A. R., Bergametti, G., Brooks, N., Duce, R. A., Jickells, T. D., et al. (2005). Atmospheric global dust cycle and iron inputs to the ocean: ATMOSPHERIC IRON DEPOSITION. *Global Biogeochemical Cycles* 19 (4). doi: 10.1029/2004GB002402

Marconi, D., Sigman, D. M., Casciotti, K. L., Campbell, E. C., Alexandra Weigand, M., et al. (2017). Tropical dominance of N₂ fixation in the north atlantic ocean: tropical lead of atlantic N₂ fixation. *Global Biogeochemical Cycles* 31 (10), 1608–1623. doi: 10.1002/2016GB005613

Martin, J. H., Knauer, G. A., Karl, D. M., and Broenkow, W. W. (1987). VERTEX: carbon cycling in the northeast Pacific. *Deep Sea Res. Part A: Oceanographic Res. Papers* 34 (2), 267–285. doi: 10.1016/0198-0149(87)90086-0

McIlvin, M. R., and Casciotti, K. L. (2011). Technical updates to the bacterial method for nitrate isotopic analyses. *Analytical Chem.* 83 (5), 1850–1856. doi: 10.1021/ac1028984

Meiler, S., Britten, G. L., Dutkiewicz, S., Gradoville, M. R., Moisander, P. H., Jahn, O., et al. (2022). Constraining uncertainties of diazotroph biography with nifH gene abundance. *Linnmology Oceanography* 67 (4), 816–829. doi: 10.1002/lno.12036

Minagawa, M., and Wada, E. (1986). Nitrogen isotope ratios of red tide organisms in the East China Sea: A characterization of biological nitrogen fixation. *Marine Chem.* 19 (3), 245–259. doi: 10.1016/0304-4203(86)90026-5

Moisander, P. H., Beinart, R.A., Hewson, I., White, A. E., Johnson, K. S., Carlson, C. A., et al. (2010). Unicellular cyanobacterial distributions broaden the oceanic N₂ fixation domain. *Science* 327 (5972), 1512–1514. doi: 10.1126/science.1185468

Moisander, P. H., Serros, T., Paerl, R. W., Beinart, R. A., and Zehr, J. P. (2014). Gammaproteobacterial diazotrophs and nifH gene expression in surface waters of the South Pacific Ocean. *ISME J.* 8 (10), 1962–1973. doi: 10.1038/ismej.2014.49

Monteiro, F. M., Dutkiewicz, S., and Follows, M. J. (2011). Biogeographical controls on the marine nitrogen fixers: CONTROLS ON MARINE NITROGEN FIXERS. *Global Biogeochemical Cycles* 25 (2). doi: 10.1029/2010GB003902

Montoya, J. P., Holl, C. M., Zehr, J. P., Hansen, A., Villareal, T. A., and Capone, D. G. (2004). High rates of N2 fixation by unicellular diazotrophs in the oligotrophic Pacific Ocean. *Nature* 430 (7003), 1027–1031. doi: 10.1038/nature02824

Moore Mills, M. M., Achterberg, E. P., Geider, R. J., LaRoche, J., Lucas, M. I., McDonagh, E. L., et al. (2009). Large-scale distribution of Atlantic nitrogen fixation controlled by iron availability. *Nat. Geosci.* 2 (12), 867–871. doi: 10.1038/ngeo667

Moutin, T., Wagener, T., Caffin, M., Fumenia, A., Gimenez, A., Baklouti, M., et al. (2018). Nutrient availability and the ultimate control of the biological carbon pump in the western tropical South Pacific Ocean. *Biogeosciences* 15, 2961–2989. doi: 10.5194/bg-2017-565

Mulholland, B., and Capone, D. (2004). Dinitrogen fixation and release of ammonium and dissolved organic nitrogen by Trichodesmium IMS101. *Aquat. Microbial Ecol.* 37, 85–94. doi: 10.3354/ame037085

Mulholland, M. R., Bernhardt, P. W., Widner, B. N., Selden, C. R., Chappell, P. D., Clayton, S., et al. (2019). High rates of N₂ fixation in temperate, western north atlantic coastal waters expand the realm of marine diazotrophy. *Global Biogeochemical Cycles* 33 (7), 826–840. doi: 10.1029/2018GB006130

Peters, B., Horak, R., Devol, A., Fuchsman, C., Forbes, M., Mordy, C. W., et al. (2018). Estimating fixed nitrogen loss and associated isotope effects using concentration and isotope measurements of NO₃⁻, NO₂⁻, and N₂ from the Eastern Tropical South Pacific oxygen deficient zone. *Deep Sea Res. Part II: Topical Stud. Oceanography* 156, 121–136. doi: 10.1016/j.dsr2.2018.02.011
Raes, E. J., van de Kamp, J., Bodrossy, L., Fong, A. A., Riekenberg, J., Holmes, B. H., et al. (2020). N2 fixation and new insights into nitrification from the ice-edge to the equator in the south pacific ocean. *Front. Mar. Sci.* 7, 389. doi: 10.3389/fmars.2020.00389

Shao, Z., Xu, Y., Wang, H., Luo, W., Wang, L., Huang, Y., et al. (2023). Global oceanic diazotroph database version 2 and elevated estimate of global oceanic N2 fixation. *Earth System Science Data* 15 (8), 3673–3709. doi: 10.5194/essd-15-3673-2023

Siegel, D. A., and Deuser, W. G. (1997). Trajectories of sinking particles in the Sargasso Sea: modeling of statistical funnels above deep-ocean sediment traps. *Deep Sea Res. Part I: Oceanographic Res. Papers* 44 (9–10), 1519–1541. doi: 10.1016/S0967-0637 (97)00028-9

Siegel, D. A., Fields, E., and Buesseler, K. O. (2008). A bottom-up view of the biological pump: Modeling source funnels above ocean sediment traps. *Deep Sea Res. Part I: Oceanographic Res. Papers* 55 (1), 108–127. doi: 10.1016/j.dsr.2007.10.006

Sigman, D. M., Casciotti, K. L., Andreani, M., Barford, C., Galanter, M., and Böhlke, J. K. (2001). A bacterial method for the nitrogen isotopic analysis of nitrate in seawater and freshwater. *Anal. Chem.* 73 (17), 4145–4153. doi: 10.1021/ac010088e

Smith, J. M., Chavez, F. P., and Francis, C. A. (2014). Ammonium uptake by phytoplankton regulates nitrification in the sunlit ocean. *PloS One* 9 (9), e108173. doi: 10.1371/journal.pone.0108173

Sohm, J. A., Webb, E. A., and Capone, D. G. (2011). Emerging patterns of marine nitrogen fixation. *Nat. Rev. Microbiol.* 9 (7), 499–508. doi: 10.1038/nrmicro2594

Staal, M., Meysman, F. J. R., and Stal, L. J. (2003). Temperature excludes N2-fixing heterocystous cyanobacteria in the tropical oceans. *Nature* 425 (6957), 504–507. doi: 10.1038/nature01999

Stenegren, M., Caputo, A., Berg, C., Bonnet, S., and Foster, R. A. (2018). Distribution and drivers of symbiotic and free-living diazotrophic cyanobacteria in the western tropical South Pacific. *Biogeosciences* 15 (5), 1559–1578. doi: 10.5194/bg-15-1559-2018

Talley, L. D., Pickard, G. L., Emery, W. J., and Swift, J. H. (2011). *Descriptive physical oceanography: an introduction. 6th ed* (Amsterdam; Boston: Academic Press).

Tilliette, C., Gazeau, F., Chavagnac, V., Leblond, N., Montanes, M., Leblanc, K., et al. (2023). Temporal and spatial variability in the hydrothermal signature of sinking particles and sediments in the Western Tropical South Pacific Ocean. *ESS Open Archive*. doi: 10.22541/essoar.167898492.26733124/v1

Tilliette, C., Taillandier, V., BouruetAubertot, P., Grima, N., Maes, C., Montanes, M., et al. (2022). Dissolved iron patterns impacted by shallow hydrothermal sources along a transect through the Tonga-kermadec arc. *Global Biogeochemical Cycles* 36, e2022GB007363. doi: 10.1029/2022GB007363

Turk-Kubo, K. A., Achilles, K. M., Serros, T. R. C., Ochiai, M., Montoya, J. P., Zehr, J. P., et al. (2012). Nitrogenase (nifH) gene expression in diazotrophic cyanobacteria in the Tropical North Atlantic in response to nutrient amendments. *Front. Microbiol.* 3. doi: 10.3389/fmicb.2012.00386

Waniek, J., Koeve, W., and Prien, R. D. (2000). Trajectories of sinking particles and the catchment areas above sediment traps in the northeast Atlantic. *J. Mar. Res.* 58 (6), 983–1006. doi: 10.1357/002224000763485773

Webb, D. J. (2000). Evidence for shallow zonal jets in the south equatorial current region of the southwest pacific. *J. Phys. Oceanography* 30, 15. doi: 10.1175/1520-0485 (2000)030<0706:EPSZJ1>2.0:CO₂

Weber, T., and Deutsch, C. (2014). Local versus basin-scale limitation of marine nitrogen fixation. *Proc. Natl. Acad. Sci.* 111 (24), 8741–8746. doi: 10.1073/pnas.1317193111

Weigand, M. A., Foriel, J., Barnett, B., Oleynik, S., and Sigman, D. M. (2016). Updates to instrumentation and protocols for isotopic analysis of nitrate by the denitrifier method. *Rapid Commun. Mass Spectrometry* 30 (12), 1365–1383. doi: 10.1002/ rcm.7570

White, A. E., Foster, R. A., Benitez-Nelson, C. R., Masqué, P., Verdeny, E., Popp, B. N., et al. (2013). Nitrogen fixation in the gulf of California and the eastern tropical north pacific. *Prog. Oceanography* 109, 1–17. doi: 10.1016/j.poccean.2012.09.002

White, A. E., Granger, J., Selden, C., Gradoville, M.R., Potts, L., Bourbonnais, A., et al. (2020). A critical review of the ¹⁵N₂ tracer method to measure diazotrophic production in pelagic ecosystems. *Linnology Oceanography: Methods* 18 (4), 129–147. doi: 10.1002/lom.3.10353

Xu, H., and Weber, T. (2021). Ocean dust deposition rates constrained in a data-assimilation model of the marine aluminum cycle. *Global Biogeochemical Cycles* 35 (9). doi: 10.1029/2021GB007049

Zehr, J. P., Mellon, M. T., and Zani, S. (1998). New nitrogen-fixing microorganisms detected in oligotrophic oceans by amplification of nitrogenase (nifH) genes. *Appl. Environ. Microbiol.* 64 (9), 3444–3450. doi: 10.1128/AEM.64.9.3444-3450.1998

Zehr, J. P., and Turner, (2001) *Nitrogen fixation: Nitrogenase genes and gene expression - ScienceDirect.* Available at: https://www.sciencedirect.com/science/article/ abs/pii/S0580951701300491?via%3Dihub (Accessed 14 February 2023).