Microbial iron limitation in the ocean’s twilight zone

Microbial iron limitation in the ocean’s twilight zone | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Physical Sciences - Article Microbial iron limitation in the ocean’s twilight zone Daniel Repeta, Jingxuan Li, Lydia Babcock-Adams, Rene Boiteau, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3749755/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Sep, 2024 Read the published version in Nature → Version 1 posted You are reading this latest preprint version Abstract One of the major advances in ocean biogeochemistry achieved over the past three decades is an understanding of how nutrients, primarily nitrate, phosphate and iron (Fe), combine in complex patterns to limit and shape primary production in the surface ocean 1-3 . Below the surface ocean, remineralization of sinking organic matter rapidly regenerates nutrients, and microbial metabolism in the upper mesopelagic “twilight zone” (200-500 m) instead appears to be limited by the delivery of labile organic carbon 4,5 . In contrast to the large number of studies describing nutrient limitation in ocean surface waters, nutrient limitation of microbial production in the mesopelagic has been unexplored. Here we report the distribution and uptake of siderophores, biomarkers for microbial Fe limitation 6 , across a meridional section of the eastern Pacific Ocean. Siderophore concentrations were high in chronically Fe limited surface waters, but they were also surprisingly high in the twilight zone underlying the North and South Pacific subtropical gyres, two key ecosystems for the global carbon cycle. Bacterial Fe deficiency due to low Fe availability is likely characteristic of the twilight zone in several large ocean basins, greatly expanding the region of the marine water column where nutrients limit microbial metabolism with potentially significant impacts on ocean carbon storage. Earth and environmental sciences/Biogeochemistry/Element cycles Earth and environmental sciences/Ocean sciences/Marine chemistry Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Primary production in the sunlit portion of the ocean is regulated by the supply of key nutrients needed by phytoplankton to fix carbon dioxide into biomass 1–3 . A fraction of this production sinks into the ocean’s interior, delivering organic substrates that fuel the metabolism of deep-sea food webs 7–9 . The vast majority of sinking organic matter escaping the surface is respired by heterotrophic bacteria 10,11 inhabiting the upper mesopelagic “twilight zone”, the region of the water column between 200 and 500 m 5,12,13 . Respiration attenuates the flux of organic carbon through the twilight zone and regenerates nutrients such that concentrations of dissolved nitrate, phosphate, and essential trace metals (iron, cobalt, zinc, etc .) increase rapidly from low or limiting concentrations in surface waters to replete values in the upper mesopelagic. However, nutrients are released from sinking particles at different rates. Due to slow remineralization or intense scavenging 14,15 , iron (Fe), a micronutrient essential for a number of cell functions, increases in concentration much more slowly with depth than nitrate or phosphate 16–18 . While many studies have described the impact of Fe, nitrate and phosphate on carbon cycling in the sunlit surface ocean, few studies have examined the role of nutrients in shaping microbial production in the mesopelagic. Fe is critical to a suite of enzymes that catalyze the metabolism of organic matter, and culture studies show that the cellular Fe requirements of heterotrophic bacteria are relatively high 19,20 . The slow increase in dissolved Fe (DFe) concentrations with depth together with the high Fe requirements of heterotrophic bacteria raise the potential for Fe limitation of bacterial metabolism in mesopelagic waters, which in turn could have consequences for the transfer of carbon from the surface into the deep ocean. Nearly all DFe in seawater is complexed to ligands that act to both elevate DFe concentrations and affect Fe bioavailability 21,22 . Most ligands are by products of organic matter degradation and have low binding affinities for Fe 23,24 . However, as Fe concentrations approach limiting values or as Fe becomes less bioavailable, some bacteria secrete siderophores, low molecular weight metabolites synthesized specifically for their high Fe affinity 6 . Siderophores form strong complexes with DFe and facilitate Fe uptake through specialized transporters that recognize the Fe-siderophore complex. Siderophore production and uptake however comes with a steep fitness cost to bacteria inhabiting low energy environments such as the mesopelagic ocean. Carbon and energy that would otherwise be allocated to biomass production and growth are instead allocated to iron acquisition. Metabolic models and cultivation experiments show siderophore biosynthesis and uptake is only favorable when Fe limits growth 25 . Siderophores are therefore good biomarkers for microbial Fe limitation in-situ . As part of the U.S. GEOTRACES GP15 expedition, we measured the distribution of siderophores and DFe across a section of the Eastern Pacific Ocean extending from 56 °N to 20 °S along 152 °W (Fig. 1a). The GP15 section traverses several biogeochemical provinces, including Fe rich productive coastal waters along the South Alaskan Shelf, the chronically Fe limited subpolar gyre and eastern equatorial upwelling regions, and two large oligotrophic subtropical (North and South Pacific) gyres. Overall, the section offered the opportunity both to compare the distribution of siderophores with DFe and to identify regions of microbial Fe limitation across the different nutrient regimes that characterize the Pacific Ocean. Iron limitation in the twilight zone The most striking feature of the GP15 section was the high concentrations of siderophores in the chronically Fe limited surface waters of the subpolar gyre (Stations 4-10; Fig. 1b) and equatorial upwelling areas (Stations 25-34), and throughout the upper mesopelagic twilight zone (200-500 m) underlying both the North and South Pacific subtropical gyres (Stations 12-23 and 35-39 respectively). Concentrations of siderophores in mesopelagic waters reached values of 68 pM, equal to their maximum concentrations in Fe limited surface waters (66 pM). In laboratory culture experiments siderophores are produced in response to low DFe 26,27 , but siderophore concentration across GP15 did not correlate with DFe; in fact, siderophores were undetectable (< 0.5 pM) in many samples with very low concentrations of DFe ( 800 pM) also had high concentrations of siderophores (> 10 pM; Fig. 2a). The distribution of DFe and siderophores across the east Pacific Ocean indicates that DFe concentration alone is not a good indicator for Fe limitation of bacteria (Fig. 2a) 28 . In chronically Fe limited surface waters, nutrients are supplied from deeper waters that are relatively enriched in nitrate and depleted in DFe (DFe:nitrate ratio of < 30 µmol/mol) 18 . The supply of Fe to these regions is not sufficient to support the complete drawdown of all available nitrate through biological production, leading to Fe limitation 29 . Heterotrophic bacteria collected from Fe limited surface waters of the subpolar Pacific Ocean had a cellular Fe:N ratio of 50 µmol/mol similar to bacteria grown in low DFe laboratory cultures, which had minimum cellular Fe:N ratios of 2-50 µmol/mol 19,20 . When siderophore concentrations across GP15 were plotted against DFe:nitrate, 205 of 253 samples in which siderophores were detected and 32 of 33 samples with high (> 20 pM) siderophore concentrations were associated with DFe:nitrate values < 50 µmol/mol (Fig. 2b). As predicted from the DFe:nitrate ratio, siderophore concentrations were high in the sunlit euphotic zone of the Fe limited subpolar gyre and equatorial upwelling regions (Fig. 1b). In contrast, surface waters of the North and South Pacific subtropical gyres have an average DFe:nitrate ratio of ~1800 µmol/mol, and siderophores were only occasionally detected in low concentrations in these samples. However, siderophore concentrations were high in the mesopelagic zone (200-500 m) underlying the North and South Pacific subtropical gyres, which were also characterized by DFe:nitrate < 50 µmol/mol. Deeper in the ocean, below 500 m, as the supply of labile organic carbon from sinking particles decreases, and there is a concomitant reduction in microbial Fe demand. Siderophores were occasionally detected at depths > 500 m, but only in low concentrations (~2 pM). Nearly all siderophores measured across the GP15 section were identified as Fe-amphibactins and Fe-marinobactins, two families of amphiphilic siderophores with a 4-5 amino acid peptide that complexes Fe, linked to a suite of lipids that differ in carbon number and degree of unsaturation (Extended Data Figs. 1, 2) 30,31 . The amphiphilic character of amphibactins and marinobactins allows them to associate with cell membranes. This association facilitates uptake after Fe binding and mitigates the diffusive loss of the siderophores into the environment 32,33 . However, we did not detect amphibactins or marinobactins in samples of suspended particulate matter collected concurrently with our water samples, even in mesopelagic samples with high concentrations of dissolved amphibactins and marinobactins (Extended Data Table 1). Although we cannot rule out that siderophore-membrane associations disintegrated during particulate matter collection, our measurements point to siderophores as part of the dissolved (< 0.2 µm) fraction of marine iron. Rapid Fe uptake from siderophores In seawater, siderophores complex Fe from weaker ligands (Fig. 3). The Fe-siderophore complex then binds to siderophore-specific membrane transporters, which deliver the complex into the cell 34,35 where Fe is recovered 36 . The Fe free siderophore is then returned to the environment to repeat the cycle (Fig. 3) 37 . To confirm that bacteria inhabiting the mesopelagic use this mechanism to acquire Fe, we measured the uptake of siderophores labeled with the rare 57 Fe isotope in a profile of samples collected between 75 and 450 m near Station ALOHA, a long-term study site with biogeochemical characteristics representative of the North Pacific Subtropical Gyre 39 . The study site (Fig. 1a) is located ~ 370 km west of GP15 Station 18, a station characterized by high siderophore concentrations and DFe:nitrate ratios between 15-75 µmol/mol in the upper mesopelagic. Unfiltered seawater samples were amended with picomolar concentrations of 57 Fe labeled amphibactins and marinobactins and incubated in the dark for 5 days, after which dissolved siderophore concentrations were compared to sterile filtered controls. If bacteria inhabiting the mesopelagic were Fe limited, we expected rapid uptake of the 57 Fe-siderophore amendment. For samples collected within the mesopelagic zone between 200-400 m, 88-100% of the added 57 Fe-siderophore was lost during incubation, indicating rapid uptake by the bacterial community (Fig. 4, Extended Data Table 2). To measure the fraction of uptake allocated to Fe acquisition, inorganic 57 Fe was added to the sample extracts and the extracts reanalyzed. Any metal-free siderophores secreted by bacteria following Fe uptake will complex with the newly added 57 Fe and be measured as an increase in the concentration of 57 Fe-siderophores. Addition of 57 Fe to the mesopelagic samples increased siderophore concentrations to ~ 63% of the original amendment (Extended Data Table 3). No metal-free siderophores were detected in the 75 m sample, indicating that microbes inhabiting the euphotic zone near Station ALOHA use other pathways to acquire iron. At 150 m, intermediate between the euphotic and mesopelagic twilight zones, 25% of the original amendment was taken up to acquire Fe while at 450 m 19% of the siderophore amendment was used to acquire Fe. The slower Fe uptake at this depth compared to 400 m was most likely due to a sharp decrease in Fe demand in response to the rapid attenuation of sinking organic carbon with depth. Between 11-37% of the siderophore amendment was not recovered even following the addition of 57 Fe, indicating that at all depths a fraction of the siderophore amendment was metabolized as labile carbon substrate. Accompanying the loss of the 57 Fe-siderophores in the mesopelagic samples, we noted increases in the concentrations of 27 Al-siderophores (Extended Data Table 2). For example, at 250 m, the total concentration of 57 Fe-marinobactin-C and 57 Fe-amphibactin T (Peak C, Fig. 4) decreased by 81 pM (89%), while concurrently the concentration of 27 Al-peak C increased by 3.4 pM (Extended Data Table 2). Siderophores are known to form weak complexes with Al 40 , and suites of Al-marinobactins and Al-amphibactins were found alongside their Fe homologues across the GP15 section. The increase in Al-siderophore concentration with incubation provides further evidence that bacteria acquired Fe from amphibactins and marinobactins and secreted the siderophores back into the environment (Fig. 3). The zone of rapid Fe-siderophore uptake in the 57 Fe experiment aligns with the region of the North and South Pacific mesopelagic with high siderophore concentrations and low DFe:nitrate ratios, confirming that the microbial communities inhabiting these regions use siderophores to acquire Fe and are Fe limited. The absence of siderophore mediated Fe uptake in the euphotic zone aligns with the low siderophore and high DFe:nitrate region of the GP15 upper water column. Microbes inhabiting this region use other uptake pathways to acquire Fe and are likely Fe replete. Between the Fe replete euphotic zone and the Fe limited twilight zone, there is a transition zone characterized by slow uptake of Fe-siderophores, indicating with some Fe deficiency. Iron limitation in the twilight zone of the global ocean Iron bioavailability likely shapes and limits microbial metabolism in the mesopelagic across much of the global ocean. Using the DFe:nitrate ratio < 50 µmol/mol as a proxy for bacterial siderophore iron acquisition, the 200-500m twilight zones of the South Pacific and Southern Oceans (DFe:nitrate 14±1 µmol/mol), the Tropical Pacific (DFe:nitrate 20±18 µmol/mol), Arctic (DFe:nitrate 35±14 µmol/mol), Indian Ocean (DFe:nitrate 31±22 µmol/mol), and the South Atlantic (DFe:nitrate 28±11 µmol/mol) oceans could all be Fe limited even when microbial communities in the euphotic zone do not necessarily experience Fe deficiency (Extended Data Fig. 4). In contrast, the mesopelagic of the North Atlantic Ocean has a dFe:nitrate ratio of 59±64 µmol/mol, and Fe limitation of heterotrophic bacteria may be restricted to only certain geographic regions of this basin (Extended Data Fig. 4). Results from the emerging field of marine metaproteomics support this view. A recent survey of the bacterial metaproteome across a zonal section of the eastern tropical Pacific Ocean reported that spectral counts of outer membrane proteins characteristic of Fe-siderophore transporters were not detectable in surface waters but were very high in the 200-400 m twilight zone 41 , exactly where we also found high concentrations of siderophores. Most of the organic matter sinking below the euphotic zone is respired by heterotrophic bacteria 10,11 , which act as a key lever on the ocean’s biological carbon pump, the process by which carbon is transported either as dissolved or particulate matter from surface waters into the deep ocean. Mesopelagic bacterial Fe limitation can affect the export flux of carbon to the deep ocean in two ways. First, field and laboratory culture experiments show that Fe deficiency significantly decreases bacterial growth rates 19,42–44 . Slower uptake of organic carbon substrates could lead to higher steady-state concentrations and greater persistence of mesopelagic dissolved organic carbon, a significant reservoir of marine carbon. Second, although bacteria can restructure their metabolism in multiple ways to mitigate Fe deficiency 20,45 , field and laboratory experiments again suggest that Fe limitation significantly decreases bacteria growth efficiency 19,46,47 , the ratio of carbon allocated to biomass production relative to the total carbon uptake. If these studies are representative of the environment, a decrease in bacterial growth efficiency stimulated by Fe limitation would increase the amount of organic matter respired to carbon dioxide relative to biomass production and decrease the strength of the biological carbon pump below 500 m 48 . At the global scale, mesopelagic Fe limitation vastly expands our view of where oceanic microbial production is influenced by this essential trace metal 2,3 . While the complex and interdependent role of macro- (nitrogen, phosphorus and silica) and micro-nutrients (bioactive trace metals and vitamins) in regulating primary production and carbon exchange in surface waters has been a major theme of ocean biogeochemical research 3,49–51 , stimulating numerous mesoscale nutrient enrichment experiments 29,52 , the development of global biogeochemical models 49,53–55 , and most recently renewed interest in Fe fertilization as a means of carbon capture 4,56,57 , the role of nutrients in shaping microbial metabolism within the twilight zone is poorly understood. Mesopelagic bacterial carbon cycling plays a critical role in determining the strength of the ocean’s biological carbon pump and Fe availability may be an unrecognized factor in carbon cycling within this region. Future investigations of iron fertilization as a means to enhance ocean carbon storage therefore need to consider the consequences of mesopelagic nutrient limitation on carbon export into the deep ocean. Declarations Data availability Siderophore concentration data for the GP15 transect are publicly available through the BCODMO data archive at ( https://www.bco-dmo.org/dataset/875210 ). Dissolved Fe data for the GP15 transect have been deposited in BCODMO ( https://www.bco-dmo.org/dataset/883862 and https://www.bco-dmo.org/dataset/884673 ). Nitrate data have been deposited in BCODMO ( https://www.bco-dmo.org/dataset/777951 and https://www.bco-dmo.org/dataset/824867). The nitrate data is from Hawaii Ocean Time-series (HOT) data archive at the University of Hawai'i at Manoa (https://hahana.soest.hawaii.edu/hot/methods/llnuts.html). Siderophore concentration data for the uptake experiment are provided in the Extended Data. Acknowledgements We thank Phoebe Lam, Karen Casciotti, Greg Cutter, Brent Summers, Laramie Jensen for their organization, assistance and support of the GP15 expedition. Dr. Peter Morton provided assistance for ICP-MS analyses at Texas A&M University. We also thank Tim Burrell, Ryan Tabata, Brandon Brenes, Dave Karl, Angel White, Phil Kong and Miranda Seixas for their support and assistance of the PARAGON 2022 cruise on the R/V Kilo Moana . Funding for this work was provided by National Science Foundation Chemical Oceanography Program (NSF awards OCE-1736280 and OCE-2045223 to D.J.R.; OCE-1737167 to J.N.F.; OCE-1737136 to T.M.C.; and OCE-1736896 to S.G.J.). Support was also provided for the Simons Collaboration on Ocean Processes and Ecology program (SCOPE award 721227 to D.J.R. and 721221 to M.J.C), a Simons Foundation Early Career Investigator Award in Marine Microbial Ecology and Evolution (Life Sciences award 618401 to R.M.Bundy), and a Simons Foundation Postdoctoral Fellowship in Marine Ecology (award 729162 to L.E.M.). Author Contribution J.L., L.B.A. and D.J.R. designed the project. J.L and L.B.A. collected and processed the GP15 samples. J.L., L.B.A., and M.R.M measured and characterized siderophores in GP15 and PARAGON 2022 samples. R.M. Boiteau and R.M. Bundy developed the LC-MS methods and data interrogation algorithms, and purified marinobactins for the PARAGON 2022 experiments. J.L., L.E.M., I.M.S., B.N.G., and M.J.C. conducted the siderophore uptake experiments during the PARAGON 2022 cruise. M.S., N.T.L., X.B., T.M.C., J.N.F. and S.G.J. measured dissolved Fe in the GP15 samples. 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D. et al. The biological carbon pump in CMIP6 models: 21st century trends and uncertainties. Proc. Nat. Acad. Sci. USA doi:10.1073/pnas (2022). Methods Sample collection and preparation Seawater samples were collected on the US GEOTRACES cruise GP15, Pacific Meridional Transect (R/V Revelle , RR1814 and RR1815) from September 18 to November 24, 2018. MODIS-Aqua level 3 seasonal (September 21 to December 20, 2018) chlorophyll concentration products were downloaded at about 4 km spatial resolution as standard mapped images from the NASA Ocean Color website (https://oceancolor.gsfc.nasa.gov). Each water sample was filtered directly from the trace metal clean GTC rosette/Go-Flo bottle sampler through a 0.2 µm Pall Acropak-200 Supor cartridge into a trace metal grade acid-cleaned 4 L polycarbonate bottle for siderophore analysis or an acid cleaned 2 or 4 L Nalgene LDPE bottle for dissolved Fe analysis. Samples for siderophores were pumped at 20 mL/min through Bond-Elut ENV solid phase extraction (SPE) columns (1 g, 6 mL, P/N 12255012, Agilent Technologies) that had been previously activated by passing 6 mL each of distilled methanol (MeOH, Optima LCMS grade, Fisher Scientific) and ultrapure water (qH 2 O, 18.2 MΩ) through the column. SPE columns were frozen (-20 °C) immediately after sample collection and returned to the laboratory for processing. Filtered water samples for dissolved Fe concentration analysis were acidified to pH ~2 with the addition of the equivalent of 1 mL of 12 N teflon-distilled HCl back on shore and left for at least six months before processing 58 . Dissolved Fe processing and analysis Dissolved Fe concentrations were measured in GP15 samples via a Neptune multi-collector inductively coupled plasma mass spectrometry (ICPMS) in the Tampa Bay Plasma Facility at the University of South Florida using the isotope dilution technique, following the methods of Conway et al. (2013) and Sieber et al., (2021) 58,59 . Briefly, acidified seawater samples were amended with a 57 Fe- 58 Fe double spike solution and 1 mL of 10 mM H 2 O 2 , buffered to pH ~6 with ammonium acetate. Dissolved Fe was extracted and purified via Nobias PA-1 and AGMP-1 chelating resins before being analyzed by ICPMS. The procedural blank is equivalent to 2 pmol kg -1 in a 4 L sample 59 . The precision and accuracy of this method for dissolved concentrations has been demonstrated previously 58,60,61 , and we express uncertainty (1SD) on Fe concentrations as 2%, based on full replicate analysis of separate GP15 seawater samples collected at the same depth from different bottles (n = 26). Siderophore Processing SPE columns were thawed and washed with 6 mL qH 2 O (to reduce salts) and the qH 2 O wash was discarded. Ligands were then eluted with 6 mL distilled MeOH into acid-cleaned 10 mL polypropylene tubes. Process blanks were prepared in parallel by eluting activated SPE columns with 6 mL qH 2 O followed by 6 mL MeOH. The methanol fraction was collected as the process blank. For consistency among samples, the qH 2 O wash and MeOH extraction were performed by a trace metal clean liquid handler (model GX271, Gilson). A 10 µL stock solution of 2.2 µM Ga-Desferrioxamine-E (Ga-DFOE) was added to each sample as an internal standard. The sample was concentrated to ~500 µL by vacuum centrifugation (SpeedVac, Thermo Scientific; 35 °C, 5 hrs). A 100 µL aliquot of the sample was taken, mixed with 100 µL of qH 2 O, and immediately analyzed by LC-MS. To prepare the Ga-DOFE internal standard, 0.5 mg desferrioxamine-E (DFOE; Biophore Research) was dissolved with sonication in 1 mL distilled MeOH. Then, 10 µL of 200 mM gallium nitrate in qH 2 O adjusted to pH 1 with nitric acid (Optima grade, Fisher Scientific) was added to complex DFOE. The solution was diluted with 4 mL qH 2 O to make 5 mL of standard. To remove excess Ga, 500 µL of the solution was applied to a SPE column (C18; 100 mg, 1 mL, Agilent Technologies), which had been previously activated with 2 mL each of distilled MeOH and qH 2 O. The column was washed with 2 mL qH 2 O to remove excess Ga, and the Ga-DFOE eluted with 2 mL MeOH. The MeOH eluant was collected and then diluted with qH 2 O to a final volume of 20 mL. Quantitative analyses of siderophores Chromatographic analyses were performed on a bioinert Dionex Ultimate 3000 liquid chromatograph (LC) system fitted with a loading pump, a nano pump, and a 10-port switching valve 62 . During the loading phase, 200 µL of sample were withdrawn into the sample loop, then applied to a C18 trap column (3.5 μm, 0.5 mm x 35 mm, P/N 5064-8260, Agilent Technologies) by the loading pump at 25 μL/min for 10 min. The loading solvent is a mixture of 95% solvent A (5 mM aqueous ammonium formate, Optima, Fisher Scientific) and 5% solvent B (5 mM methanolic ammonium formate). During the elution phase, the solvent was delivered by a nano pump at 10 µL/min, and the trap column outflow directed onto two C18 columns (3.5 μm, 0.5 mm x 150 mm, P/N 5064-8262, Agilent Technologies) connected in series. Samples were separated with an 80 min linear gradient from 95% solvent A and 5% solvent B to 95% solvent B, followed by isocratic elution at 95% solvent B for 10 minutes. Meanwhile, the loading pump solvent was switched to 100% qH 2 O, the flow rate increased to 35 µL/min and directed as a post column make-up flow, which was infused with the column eluant into the ICPMS 62 . The high aqueous content of the combined flow serves to minimize the effect of changes in solvent composition (in this case increasing methanol content during the analysis) on the detector response to Fe, Ga, and Al 63 . For Station 39, the HPLC eluant at 10 µL/min was directed into the ICPMS without post column infusion of qH 2 O. The combined flow from the LC (45 μL/min) was analyzed using a Thermo Scientific iCAP Q ICPMS fitted with a perfluoroalkoxy micronebulizer (PFA-ST, Elemental Scientific), and a cyclonic spray chamber cooled to 4 °C 64 . Measurements were made in kinetic energy discrimination (KED) mode, with a helium collision gas flow of 4-4.5 mL/min to minimize isobaric 40 Ar 16 O + interferences on 56 Fe. Oxygen was introduced into the sample carrier gas at 25 mL/min to prevent the formation of reduced organic deposits onto the ICPMS skimmer and sampling cones. Isotopes monitored were 56 Fe (integration time 0.05 s), 54 Fe (0.02 s), 57 Fe (0.02 s), 69 Ga (0.05 s), 71 Ga (0.02 s) and 27 Al (0.02 s). The Fe detector response was calibrated using the siderophore ferrichrome, which elutes at ~40 min in our chromatographic analysis. Stock solutions of 250 µM ferrichrome were diluted to prepare standards with 2 nM, 5 nM, 10 nM, 20 nM, and 40 nM of the siderophore. Then, 5 µL of 2.2 µM Ga-DFOE was added to 995 µL of each standard, a 100 µL aliquot was taken, mixed with 100 µL of qH 2 O, and analyzed by LC-ICPMS. A plot of the ratio 56 Fe(ferrichrome): 69 Ga (Ga-DFOE) peak areas against ferrichrome/Ga-DFOE concentration yields a linear relationship (r 2 ~0.999) for the response of the ICPMS detector to Fe between 0.2-4 pmoles of ferrichrome. Calibrations and process blanks were made for every 10-20 samples analyzed, with only small changes (RSD ~30%) were observed in the slope of the calibration relationship over the course of the two years of sample analysis. Concentrations of Fe ligands in each sample were measured by plotting the FeL/Ga-DFOE peak area on the appropriate calibration curve. Identification of siderophores To assign Fe-Ls to known siderophores, select samples were analyzed by LC-electrospray ionization mass spectrometry (ESIMS). The eluant from the LC, without qH 2 O infusion, was coupled to a Thermo Scientific Orbitrap Fusion mass spectrometer equipped with a heated electrospray ionization source. ESI source parameters were set to a capillary voltage of 3500 V, sheath, auxiliary and sweep gas flow rates of 5, 2, and 0 (arbitrary units), and ion transfer tube and vaporizer temperatures of 275 °C and 20 °C. MS 1 scans for a m/z range of 150-1900 were collected in high resolution (450K) positive ion mode. The LC-ESIMS data was converted from raw file format to mzXML (Msconvert) 65 , imported to Matlab, and aligned with ICPMS data using the retention time of Ga-DFOE, which was obtained by monitoring m/z of 667.26 by ESIMS and 69 Ga by ICPMS. The m/z and intensity from each scan were extracted and ordered by scan number into a scan number/mass (m/z)/intensity matrix, which was interrogated by mass search algorithms 62,64 . The algorithms find pairs of co-eluting peaks with a mass difference of 1.995 D (ΔD = 56 Fe - 54 Fe) and an intensity ratio of 15.7, the crustal abundance ratio of 56 Fe and 54 Fe. Assignments of iron ligands as known compounds were made by comparing our measured masses to those in a library of 367 known siderophores in the Chelomex siderophore database 66 , and in some cases by comparison with amphibatins and marinobatins isolated from laboratory culture. Preparation of 57 Fe labeled amphibactins and marinobactins Amphibactins were produced by Vibrio 1F53 culture under Fe limitation induced by the addition of desferrioxamine B 62 . One liter of culture was pumped at 20 mL/min through a 0.2 µm PES capsule filter (Millipore), and Bond-Elut ENV solid phase extraction (SPE) column (1 g, 6 mL, Agilent Technologies) that had been previously activated by passing 6 mL each of distilled methanol (MeOH, Optima LCMS grade, Fisher Scientific) and ultrapure water (qH 2 O, 18.2 MΩ) through the column. After extraction, the column was washed with 6 mL qH 2 O and the qH 2 O wash was discarded. Amphibactins were then eluted with 6 mL distilled MeOH into acid-cleaned 10 mL polypropylene tubes. Marinobactins were produced by Alteromonas 2E5 and Pseudoalteromonas 2E11 culture 67 . For each culture, 25 mL of media was pumped through 0.2 μm PES Sterivex (MilliporeSigma), and C18 SPE columns (0.5 g, Biotage). The SPE column was rinsed with qH 2 O and eluted with 5 mL MeOH. The MeOH extracts were concentrated to ~300 µL under a stream of nitrogen. Amphibactins produced by Vibrio are dominated by non-metallated (apo) siderophores, due to Fe limitation induced by the presence of 10 nM desferrioxamine B in the culture media 62 . To prepare isotopically labeled amphibactins, 57 Fe oxide (Isotope enrichment > 95%, Cambridge Isotope Laboratories) was dissolved in concentrated HCl (Optima, Fisher Scientific) as a stock solution of 33.9 mM 57 Fe (180 µL HCl per 1 mg 57 Fe 2 O 3 ). Five microliters (5 µL) of this 57 Fe stock solution was added to a 500 μL aliquot of the amphibactin containing MeOH extract. After one hour, the mixture is diluted to 0.1% MeOH with the addition of 500 mL of qH 2 O. The solution was passed through a Bond-Elut ENV column (1 g, 6 mL, Agilent Technologies) at 20 mL/min, to remove excess 57 Fe. The SPE columns were then washed with 6 mL of MQ and eluted with 6 mL of MeOH. The MeOH extract was concentrated to approximately 500 μL by vacuum centrifugation (SpeedVac, Thermo Scientific), and used as amphibactin stock solution. Marinobactins produced by Alteromonas and Pseudoalteromonas were recovered as 56 Fe-siderophores, due to the higher concentration of Fe (125 nM), and the absence of desferrioxamine B in the growth media 67 . To label marinobactins with 57 Fe, 100 μL of 57 Fe stock solution was added to 10 mL of qH 2 O to create an 57 Fe stock solution of pH ~1, which facilitates the isotope exchange from 56 Fe to 57 Fe. Then, 500 μL of MeOH extract of Alteromonas and Pseudoalteromonas were combined and added to the 57 Fe stock solution. After two days, the mixture was diluted with 1000 mL of qH 2 O, neutralized with 10 mL 100 mM NaHCO 3 , and extracted by a Bond-Elut ENV column. Excess 56 Fe and 57 Fe passes through the column with the water wash. Then, the column was washed with qH 2 O and eluted with MeOH. The MeOH extract was concentrated to approximately 500 μL by vacuum centrifugation and used as marinobactin stock solution. Validation of 57 Fe-siderophore stock solution The integrity of the 57 Fe-siderophere amendment was established by measuring the chromatographic and isotopic properties of the amphibactin or marinobactin mixture by LC-ICPMS (Extended Data Fig. 2). Fe-chromatograms show that 56 Fe-siderophore accounted for 5-10% of the 57 Fe-siderophore concentration. In addition, both siderophore stock solutions show no Fe eluting at the solvent front on LC-ICPMS, confirming that the stock solution does not contain inorganic 57 Fe or 56 Fe. The baseline is low for both 57 Fe and 56 Fe, suggesting that the concentration of other Fe ligand complexes in the stock solutions is also low. Therefore, the iron added to the incubation by the amendment was dominated by 57 Fe-siderophores, with little contamination from other Fe ligands, inorganic Fe, or 56 Fe-siderophores. When amphibactins and marinobactins were mixed, some siderophores coeluted under the LC conditions used for this study, and multiple siderophores appear as a single peak on ICPMS. For example, peak A on the chromatogram of amphibactin stock solution (Amphibactin-C 10:1 ) and the peak A on the chromatogram of marinobactin (Marinobactin-A) stock solution coeluted (Extended Data Fig. 3). Using our chromatographic conditions, ten different peaks were resolved in the 57 Fe-chromatogram of the amphibactin/marinobactin amendment, representing > 20 different siderophores. Each peak includes an Fe-amphibactin and an Fe-marinobactin. All ten peaks were quantified before and after the incubation, but the discussion in the text focuses only on the four major peaks as representative of all siderophores in the mixture. The bold letters A-D were used to identify the four major peaks, representing 75% of the total siderophores in the amendment. Peak A is a combination of 57 Fe-Amphibactin-C 10:0 . and 57 Fe-Marinobactin-A. Amphibactin-C 10:0 is a novel amphibactin that has not been previously reported in the literature. Amphibactin-C 10:0 differs from Amphibactin-T by -C 2 H 4 - on the fatty acid chain. Peak B is a combination 57 Fe-Amphibactin-C 12:1 and 57 Fe-Marinobactin-B. Amphibactin-C 12:1 is also a novel amphibactin that differs from Amphibactin-T by a double bond. Peak C is a combination 57 Fe-Amphibactin-T and 57 Fe-Marinobactin-C. Peak D is a combination of 57 Fe-Amphibactin-S and 57 Fe-Marinobactin-D. These siderophores were identified by comparing their exact masses to those in the Chelomex siderophore database. To confirm our identifications, high-energy collision-induced dissociation (HCD) MS 2 spectra for 57 Fe-marinobactins and collision-induced dissociation (CID) MS 2 spectra for 57 Fe-amphibactins were collected on the Orbitrap mass analyzer. Ions were trapped using a quadrupole isolation window of 1.6 m/z and were then fragmented using an HCD collision energy of 30% or CID collision energy of 35%. The MS 2 of 57 Fe-Amphibactin-T (m/z = 858.384; Extended Data Fig. 3) has major fragments of m/z 486.12, 581.26 and 668.29. The fragment at m/z 486.12 represents the cleavage of a peptidic bond on the head group, while retaining 57 Fe. The fragments at m/z 581.26 and 668.29 represent the cleavage of another two peptidic bonds on the head group that do not retain 57 Fe. These fragmentation patterns are characteristic of amphibactins 68,69 . The MS 2 fragmentation spectrum of 57 Fe-Marinobactin-C ( m/z = 1014.437) shows major fragments at m/z 486.12, 573.15 and 743.22. For 57 Fe-Marinobactin-C, the fragment at m/z 486.12 represents diagnostic cleavage of a peptide bond 70 , and a further loss of H 2 O ( m/z 18). The fragment at m/z 486.12 was also found in the MS 2 of 57 Fe-Amphibactin T, due to the same structure of Fe-Marinobactin-C and Fe-Amphibactin-T (N 5 -acyl N 5 -hydroxy ornithine, serine, N 5 -acyl N 5 -hydroxy ornithine) after the neutral loss. Similarly, the fragment at m/z 573.15 represents a diagnostic cleavage of an ornithine -serine peptidic bond, and a further loss of H 2 O. Incubation experiments with 57 Fe-labeled siderophores Samples for Fe uptake experiments were collected onboard the R/V Kilo Moana at 23.29 °N, 155.32 °W near Station ALOHA (22.45 °N, 158.0 °W) during the SCOPE PARAGON II expedition in August 2022. Seawater was collected using a Niskin bottle rosette equipped with a conductivity, temperature, depth package (SBE 911Plus; Sea-Bird Scientifc) along with fluorescence, oxygen and transmissometer sensors. Two liters (2 L) of unfiltered seawater were sampled from nine depths between 75-450 m into acid cleaned 2 L polycarbonate bottles. Additional samples of filtered and unfiltered seawater were taken at 200 m and 400 m for experimental controls and measurements of the initial conditions. For the filtered control, samples were filtered directly from the Niskin bottle through an in-line 0.2 µm Acropak-1500 Supor cartridge (Pall). For each incubation sample, 20 µL of amphibactin stock solution and 20 µl of marinobactin stock solution were added. The bottles were wrapped in 4 mil black plastic, and placed in a temperature-controlled water bath incubator at 25 °C. After 5 days, the samples were filtered and extracted onto SPE columns, frozen (-20 °C) immediately, and returned to the laboratory for processing. References 58. Conway, T. M., Rosenberg, A. D., Adkins, J. F. & John, S. G. A new method for precise determination of iron, zinc and cadmium stable isotope ratios in seawater by double-spike mass spectrometry. Anal Chim Acta 793 , 44–52 (2013). 59. Sieber, M. et al. Isotopic fingerprinting of biogeochemical processes and iron sources in the iron-limited surface Southern Ocean. Earth Planet Sci Lett 567 , (2021). 60. Middag, R. et al. Intercomparison of dissolved trace elements at the Bermuda Atlantic time series stationMid. Mar Chem 177 , 476–489 (2015). 61. Ellwood, M. J. et al. Distinct iron cycling in a Southern Ocean eddy. Nat Commun 11 , (2020). 62. Li, J. et al. Element-Selective Targeting of Nutrient Metabolites in Environmental Samples by Inductively Coupled Plasma Mass Spectrometry and Electrospray Ionization Mass Spectrometry. Front Mar Sci 8 , (2021). 63. Boiteau, R. M., Fitzsimmons, J. N., Repeta, D. J. & Boyle, E. A. Detection of Iron Ligands in Seawater and Marine Cyanobacteria Cultures by High-Performance Liquid Chromatography–Inductively Coupled Plasma-Mass Spectrometry. Anal Chem 85 , 4357–4362 (2013). 64. Boiteau, R. M. & Repeta, D. J. An extended siderophore suite from Synechococcus sp. PCC 7002 revealed by LC-ICPMS-ESIMS. Metallomics 7 , 877–884 (2015). 65. Chambers, M. C. et al. A cross-platform toolkit for mass spectrometry and proteomics. Nature Biotechnology 30, 918–920 (2012). 66. Baars, O., Morel, F. M. & Perlman, D. H. ChelomEx: Isotope-assisted discovery of metal chelates in complex media using high-resolution LC-MS. Analytical chemistry. Anal Chem 86 , 11298–11305 (2014). 67. Boiteau, R. M. Molecular determination of marine iron ligands by mass spectrometry . (MIT/WHOI, 2016). 68. Vraspir, J. M., Holt, P. D. & Butler, A. Identification of new members within suites of amphiphilic marine siderophores. BioMetals 24 , 85–92 (2011). 69. Boiteau, R. M. et al. Structural characterization of natural nickel and copper binding ligands along the US GEOTRACES eastern Pacific zonal transect. Front Mar Sci 3 , (2016). 70. Kem, M. P. & Butler, A. Acyl peptidic siderophores: Structures, biosyntheses and post-assembly modifications. BioMetals 28 , 445–459 (2015). 71. Schlitzer, R. et al. The GEOTRACES Intermediate Data Product 2017. Chem Geol 493 , 210–223 (2018). 72. GEOTRACES Intermediate Data Product Group 2021. The GEOTRACES Intermediate Data Product 2021 (IDP2021). NERC EDS British Oceanographic Data Centre NOC. doi:10.5285/cf2d9ba9-d51d-3b7c-e053-8486abc0f5fd (2021). 73. Xiang, Y. & Lam, P. J. Size-Fractionated Compositions of Marine Suspended Particles in the Western Arctic Ocean: Lateral and Vertical Sources. J Geophys Res Oceans 125 , (2020). Additional Declarations There is NO Competing Interest. Supplementary Files ExtendedData12132023Final.docx extended Data 1 Cite Share Download PDF Status: Published Journal Publication published 25 Sep, 2024 Read the published version in Nature → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3749755","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Physical Sciences - Article","associatedPublications":[],"authors":[{"id":267370829,"identity":"1d263a08-bd5a-450f-8218-f64d3af00b61","order_by":0,"name":"Daniel 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18:00:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3749755/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3749755/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41586-024-07905-z","type":"published","date":"2024-09-25T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49777522,"identity":"c661ad76-2253-4551-8ffb-be120c8f9bb3","added_by":"auto","created_at":"2024-01-17 21:31:22","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":288591,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh siderophore concentrations in the mesopelagic of the eastern Pacific Ocean.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e) Cruise track of the\u003cstrong\u003e \u003c/strong\u003eGP15 section showing the locations of sampling stations (1-39) overlaid on Aqua-MODIS-derived average sea surface chlorophyll during the time of the cruise (October-December 2018). Station S3, the location of \u003csup\u003e57\u003c/sup\u003eFe-siderophore uptake experiments, is shown in red. \u003cstrong\u003eb\u003c/strong\u003e) The distribution of total Fe-siderophores in the upper 1000 m of GP15.\u0026nbsp;Sample locations appear as black circles on the plot. Station numbers appear above the plot.\u0026nbsp; Total concentrations of Fe-siderophores were calculated by summing all Fe-siderophores with a concentration \u0026gt; 0.5 pM. The white contour line indicates where DFe:nitrate transitions from values \u0026gt; 50 µmol/mol at depths shallower than the contour, to values \u0026lt; 50 µmol/mol below it. For Stations 1-7, the region with DFe:nitrate \u0026gt; 50 µmol/mol lies within the contour.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3749755/v1/c4cea616fb246b6e34e19234.jpg"},{"id":49777518,"identity":"7995c740-badd-4f2b-8cec-2bd785b0a587","added_by":"auto","created_at":"2024-01-17 21:31:22","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":173455,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNutrient regulation of siderophore distribution. a\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eLog-linear plot of total\u003cstrong\u003e \u003c/strong\u003eFe-siderophore concentration against dissolved iron (DFe) shows DFe is not a good indicator of where siderophores will occur in the GP15 samples. \u003cstrong\u003eb\u003c/strong\u003e) Log-linear plot of total Fe-siderophore concentration against the DFe:nitrate ratio in GP15 samples shows that most samples in which siderophores were detected had a DFe:nitrate ratio \u0026lt; 50 µmol/mol (vertical dashed line), the Fe quota of heterotrophic bacteria in chronically Fe limited surface waters\u003csup\u003e19\u003c/sup\u003e. The color of the symbols in \u003cstrong\u003eb\u003c/strong\u003e represents concentrations of DFe for each sample.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3749755/v1/78f6f0d85c959a8c83b777c9.jpg"},{"id":49777519,"identity":"e02c9b09-2f12-4505-9acf-95a328e0cf55","added_by":"auto","created_at":"2024-01-17 21:31:22","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":254587,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFe-siderophore cycling in the mesopelagic ocean.\u003c/strong\u003e\u0026nbsp;Marine bacteria acquire iron and cycle siderophores between the cell and the environment through several different species dependent pathways\u003csup\u003e34\u003c/sup\u003e. In the generalized scheme shown here, metal-free siderophores bind Fe from weaker organic ligands dissolved in seawater and the Fe-siderophore complex is then transported through the outer membrane of gram-negative bacteria\u0026nbsp;\u003cem\u003evia\u003c/em\u003e\u0026nbsp;Ton-B dependent transporters (TBDT).\u0026nbsp;Siderophores can also bind aluminum thereby decreasing the efficiency of the siderophore Fe acquisition pathway. \u0026nbsp;After passing through the outer membrane the Fe-siderophore complex binds to a periplasmic binding protein for transport into the cytoplasm where iron is recovered. \u0026nbsp;Siderophores in the cytoplasm are exported into the environment through major facilitator subtype (MFS) and ToLC protein complexes\u003csup\u003e35\u003c/sup\u003e. Siderophore mediated Fe acquisition is active in the mesopelagic at depths where the DFe:nitrate ratio (inset)\u003csup\u003e38\u003c/sup\u003e\u0026nbsp;falls below the minimum Fe:N quota of heterotrophic bacteria\u003csup\u003e19,20\u003c/sup\u003e\u0026nbsp;and where there is sufficient labile carbon substrate to fuel Fe demand. \u0026nbsp;\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3749755/v1/007c347ace3994d7c17fe12b.jpg"},{"id":49777521,"identity":"cc24bbf2-fbe9-4b08-91d5-cfcad0d266a1","added_by":"auto","created_at":"2024-01-17 21:31:22","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":216971,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRapid cycling of siderophores for iron in the twilight zone of the North Pacific Subtropical Gyre. a\u003c/strong\u003e)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAllocation of siderophores in\u0026nbsp;\u003csup\u003e57\u003c/sup\u003eFe-siderophore amendment Peak C (shown in panel\u0026nbsp;\u003cstrong\u003eb\u003c/strong\u003e) with depth after 5 days of incubation. The black vertical dashed line indicates the average concentration of siderophore Peak C in the filtered control. The light blue shaded region traces the\u0026nbsp;\u003csup\u003e57\u003c/sup\u003eFe-siderophores that remained in the sample at the end of the experiment while the dark blue shaded region is the Fe-siderophores used for Fe acquisition. The gray shaded region shows the siderophore metabolized as labile carbon substrate and not recovered after the incubation.\u0026nbsp;\u003cstrong\u003eb\u003c/strong\u003e)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eOverlay of\u0026nbsp;\u003csup\u003e57\u003c/sup\u003eFe-chromatograms for the filtered control at 200 m, the unfiltered sample at 200 m after 5 days of incubation, and the same sample after addition of\u0026nbsp;\u003csup\u003e57\u003c/sup\u003eFe to the sample extract. The increase in peak concentrations after\u0026nbsp;\u003csup\u003e57\u003c/sup\u003eFe addition is due to the complexation of\u0026nbsp;\u003csup\u003e57\u003c/sup\u003eFe by Fe-free siderophores in the sample extract. \u0026nbsp;Each peak is a combination of a\u0026nbsp;\u003csup\u003e57\u003c/sup\u003eFe-amphibactin and a\u003csup\u003e\u0026nbsp;57\u003c/sup\u003eFe-marinobactin (Extended Data Fig. 3).\u0026nbsp;\u003cstrong\u003ec\u003c/strong\u003e)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eOverlay of\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003csup\u003e27\u003c/sup\u003eAl-chromatograms for the filtered control and unfiltered sample from 200 m after 5 days of incubation showing the increase in Al-siderophores during incubation due to the complexation of dissolved aluminum by metal-free siderophores excreted after Fe-uptake.\u0026nbsp;\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3749755/v1/e1987c802800b46824634ede.jpg"},{"id":65331249,"identity":"4fb7c04c-8c41-4064-a841-f440d859cef6","added_by":"auto","created_at":"2024-09-26 07:09:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1750620,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3749755/v1/adcb30d3-2b74-4185-83b1-95408398ab0d.pdf"},{"id":49777520,"identity":"7f91e9bb-5ab1-478c-b4d9-4278d43637e4","added_by":"auto","created_at":"2024-01-17 21:31:22","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2575903,"visible":true,"origin":"","legend":"extended Data 1","description":"","filename":"ExtendedData12132023Final.docx","url":"https://assets-eu.researchsquare.com/files/rs-3749755/v1/1ab36ce0ae290e9d7c8b58c2.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Microbial iron limitation in the ocean’s twilight zone","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePrimary production in the sunlit portion of the ocean is regulated by the supply of key nutrients needed by phytoplankton to fix carbon dioxide into biomass\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e. A fraction of this production sinks into the ocean\u0026rsquo;s interior, delivering organic substrates that fuel the metabolism of deep-sea food webs\u003csup\u003e7\u0026ndash;9\u003c/sup\u003e. The vast majority of sinking organic matter escaping the surface is respired by heterotrophic bacteria\u003csup\u003e10,11\u003c/sup\u003e inhabiting the upper mesopelagic \u0026ldquo;twilight zone\u0026rdquo;, the region of the water column between 200 and 500 m\u003csup\u003e5,12,13\u003c/sup\u003e. Respiration attenuates the flux of organic carbon through the twilight zone and regenerates nutrients such that concentrations of dissolved nitrate, phosphate, and essential trace metals (iron, cobalt, zinc, \u003cem\u003eetc\u003c/em\u003e.) increase rapidly from low or limiting concentrations in surface waters to replete values in the upper mesopelagic. However, nutrients are released from sinking particles at different rates. Due to slow remineralization or intense scavenging\u003csup\u003e14,15\u003c/sup\u003e, iron (Fe), a micronutrient essential for a number of cell functions, increases in concentration much more slowly with depth than nitrate or phosphate \u003csup\u003e16\u0026ndash;18\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWhile many studies have described the impact of Fe, nitrate and phosphate on carbon cycling in the sunlit surface ocean, few studies have examined the role of nutrients in shaping microbial production in the mesopelagic. Fe is critical to a suite of enzymes that catalyze the metabolism of organic matter, and culture studies show that the cellular Fe requirements of heterotrophic bacteria are relatively high\u003csup\u003e19,20\u003c/sup\u003e. The slow increase in dissolved Fe (DFe) concentrations with depth together with the high Fe requirements of heterotrophic bacteria raise the potential for Fe limitation of bacterial metabolism in mesopelagic waters, which in turn could have consequences for the transfer of carbon from the surface into the deep ocean.\u003c/p\u003e\n\u003cp\u003eNearly all DFe in seawater is complexed to ligands that act to both elevate DFe concentrations and affect Fe bioavailability\u003csup\u003e21,22\u003c/sup\u003e. Most ligands are by products of organic matter degradation and have low binding affinities for Fe\u003csup\u003e23,24\u003c/sup\u003e. However, as Fe concentrations approach limiting values or as Fe becomes less bioavailable, some bacteria secrete siderophores, low molecular weight metabolites synthesized specifically for their high Fe affinity\u003csup\u003e6\u003c/sup\u003e. Siderophores form strong complexes with DFe and facilitate Fe uptake through specialized transporters that recognize the Fe-siderophore complex. Siderophore production and uptake however comes with a steep fitness cost to bacteria inhabiting low energy environments such as the mesopelagic ocean. Carbon and energy that would otherwise be allocated to biomass production and growth are instead allocated to iron acquisition. Metabolic models and cultivation experiments show siderophore biosynthesis and uptake is only favorable when Fe limits growth\u003csup\u003e25\u003c/sup\u003e. Siderophores are therefore good biomarkers for microbial Fe limitation \u003cem\u003ein-situ\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eAs part of the U.S. GEOTRACES GP15 expedition, we measured the distribution of siderophores and DFe across a section of the Eastern Pacific Ocean extending from 56 \u0026deg;N to 20 \u0026deg;S along 152 \u0026deg;W (Fig. 1a). The GP15 section traverses several biogeochemical provinces, including Fe rich productive coastal waters along the South Alaskan Shelf, the chronically Fe limited subpolar gyre and eastern equatorial upwelling regions, and two large oligotrophic subtropical (North and South Pacific) gyres. Overall, the section offered the opportunity both to compare the distribution of siderophores with DFe and to identify regions of microbial Fe limitation across the different nutrient regimes that characterize the Pacific Ocean.\u003c/p\u003e"},{"header":"Iron limitation in the twilight zone ","content":"\u003cp\u003eThe most striking feature of the GP15 section was the high concentrations of siderophores in the chronically Fe limited surface waters of the subpolar gyre (Stations 4-10; Fig. 1b) and equatorial upwelling areas (Stations 25-34), and throughout the upper mesopelagic twilight zone (200-500 m) underlying both the North and South Pacific subtropical gyres (Stations 12-23 and 35-39 respectively). Concentrations of siderophores in mesopelagic waters reached values of 68 pM, equal to their maximum concentrations in Fe limited surface waters (66 pM). In laboratory culture experiments siderophores are produced in response to low DFe\u003csup\u003e26,27\u003c/sup\u003e, but siderophore concentration across GP15 did not correlate with DFe; in fact, siderophores were undetectable (\u0026lt; 0.5 pM) in many samples with very low concentrations of DFe (\u0026lt; 100 pM), while a few samples with high DFe (\u0026gt; 800 pM) also had high concentrations of siderophores (\u0026gt; 10 pM; Fig. 2a). The distribution of DFe and siderophores across the east Pacific Ocean indicates that DFe concentration alone is not a good indicator for Fe limitation of bacteria (Fig. 2a)\u003csup\u003e28\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn chronically Fe limited surface waters, nutrients are supplied from deeper waters that are relatively enriched in nitrate and depleted in DFe (DFe:nitrate ratio of \u0026lt; 30 \u0026micro;mol/mol)\u003csup\u003e18\u003c/sup\u003e. The supply of Fe to these regions is not sufficient to support the complete drawdown of all available nitrate through biological production, leading to Fe limitation\u003csup\u003e29\u003c/sup\u003e. Heterotrophic bacteria collected from Fe limited surface waters of the subpolar Pacific Ocean had a cellular Fe:N ratio of 50 \u0026micro;mol/mol similar to bacteria grown in low DFe laboratory cultures, which had minimum cellular Fe:N ratios of 2-50 \u0026micro;mol/mol\u003csup\u003e19,20\u003c/sup\u003e. When siderophore concentrations across GP15 were plotted against DFe:nitrate, 205 of 253 samples in which siderophores were detected and 32 of 33 samples with high (\u0026gt; 20 pM) siderophore concentrations were associated with DFe:nitrate values \u0026lt; 50 \u0026micro;mol/mol (Fig. 2b).\u003c/p\u003e\n\u003cp\u003eAs predicted from the DFe:nitrate ratio, siderophore concentrations were high in the sunlit euphotic zone of the Fe limited subpolar gyre and equatorial upwelling regions (Fig. 1b). In contrast, surface waters of the North and South Pacific subtropical gyres have an average DFe:nitrate ratio of ~1800 \u0026micro;mol/mol, and siderophores were only occasionally detected in low concentrations in these samples. However, siderophore concentrations were high in the mesopelagic zone (200-500 m) underlying the North and South Pacific subtropical gyres, which were also characterized by DFe:nitrate \u0026lt; 50 \u0026micro;mol/mol. Deeper in the ocean, below 500 m, as the supply of labile organic carbon from sinking particles decreases, and there is a concomitant reduction in microbial Fe demand. Siderophores were occasionally detected at depths \u0026gt; 500 m, but only in low concentrations (~2 pM).\u003c/p\u003e\n\u003cp\u003eNearly all siderophores measured across the GP15 section were identified as Fe-amphibactins and Fe-marinobactins, two families of amphiphilic siderophores with a 4-5 amino acid peptide that complexes Fe, linked to a suite of lipids that differ in carbon number and degree of unsaturation (Extended Data Figs. 1, 2)\u003csup\u003e30,31\u003c/sup\u003e. The amphiphilic character of amphibactins and marinobactins allows them to associate with cell membranes. This association facilitates uptake after Fe binding and mitigates the diffusive loss of the siderophores into the environment\u003csup\u003e32,33\u003c/sup\u003e. However, we did not detect amphibactins or marinobactins in samples of suspended particulate matter collected concurrently with our water samples, even in mesopelagic samples with high concentrations of dissolved amphibactins and marinobactins (Extended Data Table 1). Although we cannot rule out that siderophore-membrane associations disintegrated during particulate matter collection, our measurements point to siderophores as part of the dissolved (\u0026lt; 0.2 \u0026micro;m) fraction of marine iron.\u003c/p\u003e"},{"header":"Rapid Fe uptake from siderophores","content":"\u003cp\u003eIn seawater, siderophores complex Fe from weaker ligands (Fig. 3). The Fe-siderophore complex then binds to siderophore-specific membrane transporters, which deliver the complex into the cell\u003csup\u003e34,35\u003c/sup\u003e where Fe is recovered \u003csup\u003e36\u003c/sup\u003e. The Fe free siderophore is then returned to the environment to repeat the cycle (Fig. 3)\u003csup\u003e37\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo confirm that bacteria inhabiting the mesopelagic use this mechanism to acquire Fe, we measured the uptake of siderophores labeled with the rare \u003csup\u003e57\u003c/sup\u003eFe isotope in a profile of samples collected between 75 and 450 m near Station ALOHA, a long-term study site with biogeochemical characteristics representative of the North Pacific Subtropical Gyre\u003csup\u003e39\u003c/sup\u003e. The study site (Fig. 1a) is located ~ 370 km west of GP15 Station 18, a station characterized by high siderophore concentrations and DFe:nitrate ratios between 15-75 \u0026micro;mol/mol in the upper mesopelagic. Unfiltered seawater samples were amended with picomolar concentrations of \u003csup\u003e57\u003c/sup\u003eFe labeled amphibactins and marinobactins and incubated in the dark for 5 days, after which dissolved siderophore concentrations were compared to sterile filtered controls. If bacteria inhabiting the mesopelagic were Fe limited, we expected rapid uptake of the \u003csup\u003e57\u003c/sup\u003eFe-siderophore amendment.\u003c/p\u003e\n\u003cp\u003eFor samples collected within the mesopelagic zone between 200-400 m, 88-100% of the added \u003csup\u003e57\u003c/sup\u003eFe-siderophore was lost during incubation, indicating rapid uptake by the bacterial community (Fig. 4, Extended Data Table 2). To measure the fraction of uptake allocated to Fe acquisition, inorganic \u003csup\u003e57\u003c/sup\u003eFe was added to the sample extracts and the extracts reanalyzed. Any metal-free siderophores secreted by bacteria following Fe uptake will complex with the newly added \u003csup\u003e57\u003c/sup\u003eFe and be measured as an increase in the concentration of \u003csup\u003e57\u003c/sup\u003eFe-siderophores. Addition of \u003csup\u003e57\u003c/sup\u003eFe to the mesopelagic samples increased siderophore concentrations to ~ 63% of the original amendment (Extended Data Table 3). No metal-free siderophores were detected in the 75 m sample, indicating that microbes inhabiting the euphotic zone near Station ALOHA use other pathways to acquire iron. At 150 m, intermediate between the euphotic and mesopelagic twilight zones, 25% of the original amendment was taken up to acquire Fe while at 450 m 19% of the siderophore amendment was used to acquire Fe. The slower Fe uptake at this depth compared to 400 m was most likely due to a sharp decrease in Fe demand in response to the rapid attenuation of sinking organic carbon with depth. Between 11-37% of the siderophore amendment was not recovered even following the addition of \u003csup\u003e57\u003c/sup\u003eFe, indicating that at all depths a fraction of the siderophore amendment was metabolized as labile carbon substrate.\u003c/p\u003e\n\u003cp\u003eAccompanying the loss of the \u003csup\u003e57\u003c/sup\u003eFe-siderophores in the mesopelagic samples, we noted increases in the concentrations of \u003csup\u003e27\u003c/sup\u003eAl-siderophores (Extended Data Table 2). For example, at 250 m, the total concentration of \u003csup\u003e57\u003c/sup\u003eFe-marinobactin-C and \u003csup\u003e57\u003c/sup\u003eFe-amphibactin T (Peak C, Fig. 4) decreased by 81 pM (89%), while concurrently the concentration of \u003csup\u003e27\u003c/sup\u003eAl-peak C increased by 3.4 pM (Extended Data Table 2). Siderophores are known to form weak complexes with Al\u003csup\u003e40\u003c/sup\u003e, and suites of Al-marinobactins and Al-amphibactins were found alongside their Fe homologues across the GP15 section. The increase in Al-siderophore concentration with incubation provides further evidence that bacteria acquired Fe from amphibactins and marinobactins and secreted the siderophores back into the environment (Fig. 3).\u003c/p\u003e\n\u003cp\u003eThe zone of rapid Fe-siderophore uptake in the \u003csup\u003e57\u003c/sup\u003eFe experiment aligns with the region of the North and South Pacific mesopelagic with high siderophore concentrations and low DFe:nitrate ratios, confirming that the microbial communities inhabiting these regions use siderophores to acquire Fe and are Fe limited. The absence of siderophore mediated Fe uptake in the euphotic zone aligns with the low siderophore and high DFe:nitrate region of the GP15 upper water column. Microbes inhabiting this region use other uptake pathways to acquire Fe and are likely Fe replete. Between the Fe replete euphotic zone and the Fe limited twilight zone, there is a transition zone characterized by slow uptake of Fe-siderophores, indicating with some Fe deficiency.\u003c/p\u003e"},{"header":"Iron limitation in the twilight zone of the global ocean","content":"\u003cp\u003eIron bioavailability likely shapes and limits microbial metabolism in the mesopelagic across much of the global ocean. Using the DFe:nitrate ratio \u0026lt; 50 \u0026micro;mol/mol as a proxy for bacterial siderophore iron acquisition, the 200-500m twilight zones of the South Pacific and Southern Oceans (DFe:nitrate 14\u0026plusmn;1 \u0026micro;mol/mol), the Tropical Pacific (DFe:nitrate 20\u0026plusmn;18 \u0026micro;mol/mol), Arctic (DFe:nitrate 35\u0026plusmn;14 \u0026micro;mol/mol), Indian Ocean (DFe:nitrate 31\u0026plusmn;22 \u0026micro;mol/mol), and the South Atlantic (DFe:nitrate 28\u0026plusmn;11 \u0026micro;mol/mol) oceans could all be Fe limited even when microbial communities in the euphotic zone do not necessarily experience Fe deficiency (Extended Data Fig. 4). In contrast, the mesopelagic of the North Atlantic Ocean has a dFe:nitrate ratio of 59\u0026plusmn;64 \u0026micro;mol/mol, and Fe limitation of heterotrophic bacteria may be restricted to only certain geographic regions of this basin (Extended Data Fig. 4). Results from the emerging field of marine metaproteomics support this view. A recent survey of the bacterial metaproteome across a zonal section of the eastern tropical Pacific Ocean reported that spectral counts of outer membrane proteins characteristic of Fe-siderophore transporters were not detectable in surface waters but were very high in the 200-400 m twilight zone\u003csup\u003e41\u003c/sup\u003e, exactly where we also found high concentrations of siderophores.\u003c/p\u003e\n\u003cp\u003eMost of the organic matter sinking below the euphotic zone is respired by heterotrophic bacteria \u003csup\u003e10,11\u003c/sup\u003e, which act as a key lever on the ocean\u0026rsquo;s biological carbon pump, the process by which carbon is transported either as dissolved or particulate matter from surface waters into the deep ocean. Mesopelagic bacterial Fe limitation can affect the export flux of carbon to the deep ocean in two ways. First, field and laboratory culture experiments show that Fe deficiency significantly decreases bacterial growth rates\u003csup\u003e19,42\u0026ndash;44\u003c/sup\u003e. Slower uptake of organic carbon substrates could lead to higher steady-state concentrations and greater persistence of mesopelagic dissolved organic carbon, a significant reservoir of marine carbon. Second, although bacteria can restructure their metabolism in multiple ways to mitigate Fe deficiency\u003csup\u003e20,45\u003c/sup\u003e, field and laboratory experiments again suggest that Fe limitation significantly decreases bacteria growth efficiency\u003csup\u003e19,46,47\u003c/sup\u003e, the ratio of carbon allocated to biomass production relative to the total carbon uptake. If these studies are representative of the environment, a decrease in bacterial growth efficiency stimulated by Fe limitation would increase the amount of organic matter respired to carbon dioxide relative to biomass production and decrease the strength of the biological carbon pump below 500 m\u003csup\u003e48\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAt the global scale, mesopelagic Fe limitation vastly expands our view of where oceanic microbial production is influenced by this essential trace metal\u003csup\u003e2,3\u003c/sup\u003e. While the complex and interdependent role of macro- (nitrogen, phosphorus and silica) and micro-nutrients (bioactive trace metals and vitamins) in regulating primary production and carbon exchange in surface waters has been a major theme of ocean biogeochemical research\u003csup\u003e3,49\u0026ndash;51\u003c/sup\u003e, stimulating numerous mesoscale nutrient enrichment experiments\u003csup\u003e29,52\u003c/sup\u003e, the development of global biogeochemical models\u003csup\u003e49,53\u0026ndash;55\u003c/sup\u003e, and most recently renewed interest in Fe fertilization as a means of carbon capture\u003csup\u003e4,56,57\u003c/sup\u003e, the role of nutrients in shaping microbial metabolism within the twilight zone is poorly understood. Mesopelagic bacterial carbon cycling plays a critical role in determining the strength of the ocean\u0026rsquo;s biological carbon pump and Fe availability may be an unrecognized factor in carbon cycling within this region. Future investigations of iron fertilization as a means to enhance ocean carbon storage therefore need to consider the consequences of mesopelagic nutrient limitation on carbon export into the deep ocean.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSiderophore concentration data for the GP15 transect are publicly available through the BCODMO data archive at ( https://www.bco-dmo.org/dataset/875210 ). Dissolved Fe data for the GP15 transect have been deposited in BCODMO ( https://www.bco-dmo.org/dataset/883862 and https://www.bco-dmo.org/dataset/884673 ). Nitrate data have been deposited in BCODMO ( https://www.bco-dmo.org/dataset/777951 and https://www.bco-dmo.org/dataset/824867). The nitrate data is from Hawaii Ocean Time-series (HOT) data archive at the University of Hawai\u0026apos;i at Manoa (https://hahana.soest.hawaii.edu/hot/methods/llnuts.html). Siderophore concentration data for the uptake experiment are provided in the Extended Data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Phoebe Lam, Karen Casciotti, Greg Cutter, Brent Summers, Laramie Jensen for their organization, assistance and support of the GP15 expedition. Dr. Peter Morton provided assistance for ICP-MS analyses at Texas A\u0026amp;M University. We also thank Tim Burrell, Ryan Tabata, Brandon Brenes, Dave Karl, Angel White, Phil Kong and Miranda Seixas for their support and assistance of the PARAGON 2022 cruise on the R/V \u003cem\u003eKilo Moana\u003c/em\u003e. Funding for this work was provided by National Science Foundation Chemical Oceanography Program (NSF awards OCE-1736280 and OCE-2045223 to D.J.R.; OCE-1737167 to J.N.F.; OCE-1737136 to T.M.C.; and OCE-1736896 to S.G.J.). Support was also provided for the Simons Collaboration on Ocean Processes and Ecology program (SCOPE award 721227 to D.J.R. and 721221 to M.J.C), a Simons Foundation Early Career Investigator Award in Marine Microbial Ecology and Evolution (Life Sciences award 618401 to R.M.Bundy), and a Simons Foundation Postdoctoral Fellowship in Marine Ecology (award 729162 to L.E.M.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.L., L.B.A. and D.J.R. designed the project. J.L and L.B.A. collected and processed the GP15 samples. J.L., L.B.A., and M.R.M measured and characterized siderophores in GP15 and PARAGON 2022 samples. R.M. Boiteau and R.M. Bundy developed the LC-MS methods and data interrogation algorithms, and purified marinobactins for the PARAGON 2022 experiments. J.L., L.E.M., I.M.S., B.N.G., and M.J.C. conducted the siderophore uptake experiments during the PARAGON 2022 cruise. M.S., N.T.L., X.B., T.M.C., J.N.F. and S.G.J. measured dissolved Fe in the GP15 samples. J.L. and D.J.R. performed the data analysis and wrote the first draft of the paper. All authors contributed to writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArrigo, K. R. Marine microorganisms and global nutrient cycles. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e437\u003c/strong\u003e, 349\u0026ndash;355 (2005).\u003c/li\u003e\n\u003cli\u003eMoore, C. M. \u003cem\u003eet al.\u003c/em\u003e Processes and patterns of oceanic nutrient limitation. \u003cem\u003eNat Geosci\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 701\u0026ndash;710 (2013).\u003c/li\u003e\n\u003cli\u003eBrowning, T. J. \u0026amp; Moore, C. M. 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Adv\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 341\u0026ndash;369 (2019).\u003c/li\u003e\n\u003cli\u003eBuesseler, K. O. \u003cem\u003eet al.\u003c/em\u003e Environment: Ocean iron fertilization - Moving forward in a sea of uncertainty. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e319\u003c/strong\u003e, 162 (2008).\u003c/li\u003e\n\u003cli\u003eWilson, J. D. \u003cem\u003eet al.\u003c/em\u003e The biological carbon pump in CMIP6 models: 21st century trends and uncertainties. \u003cem\u003eProc. Nat. Acad. Sci. USA \u003c/em\u003edoi:10.1073/pnas (2022).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eSample collection and preparation \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeawater samples were collected on the US GEOTRACES cruise GP15, Pacific Meridional Transect (R/V \u003cem\u003eRevelle\u003c/em\u003e, RR1814 and RR1815) from September 18 to November 24, 2018. MODIS-Aqua level 3 seasonal (September 21 to December 20, 2018) chlorophyll concentration products were downloaded at about 4\u0026thinsp;km spatial resolution as standard mapped images from the NASA Ocean Color website (https://oceancolor.gsfc.nasa.gov).\u003c/p\u003e\n\u003cp\u003eEach water sample was filtered directly from the trace metal clean GTC rosette/Go-Flo bottle sampler through a 0.2 \u0026micro;m Pall Acropak-200 Supor cartridge into a trace metal grade acid-cleaned 4 L polycarbonate bottle for siderophore analysis or an acid cleaned 2 or 4 L Nalgene LDPE bottle for dissolved Fe analysis. Samples for siderophores were pumped at 20 mL/min through Bond-Elut ENV solid phase extraction (SPE) columns (1 g, 6 mL, P/N 12255012, Agilent Technologies) that had been previously activated by passing 6 mL each of distilled methanol (MeOH, Optima LCMS grade, Fisher Scientific) and ultrapure water (qH\u003csub\u003e2\u003c/sub\u003eO, 18.2 M\u0026Omega;) through the column. SPE columns were frozen (-20 \u0026deg;C) immediately after sample collection and returned to the laboratory for processing. Filtered water samples for dissolved Fe concentration analysis were acidified to pH ~2 with the addition of the equivalent of 1 mL of 12 N teflon-distilled HCl back on shore and left for at least six months before processing\u003csup\u003e58\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDissolved Fe processing and analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDissolved Fe concentrations were measured in GP15 samples via a Neptune multi-collector inductively coupled plasma mass spectrometry (ICPMS) in the Tampa Bay Plasma Facility at the University of South Florida using the isotope dilution technique, following the methods of Conway et al. (2013) and Sieber et al., (2021)\u003csup\u003e58,59\u003c/sup\u003e. Briefly, acidified seawater samples were amended with a \u003csup\u003e57\u003c/sup\u003eFe-\u003csup\u003e58\u003c/sup\u003eFe double spike solution and 1 mL of 10 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, buffered to pH ~6 with ammonium acetate. Dissolved Fe was extracted and purified via Nobias PA-1 and AGMP-1 chelating resins before being analyzed by ICPMS. The procedural blank is equivalent to 2 pmol kg\u003csup\u003e-1\u003c/sup\u003e in a 4 L sample\u003csup\u003e59\u003c/sup\u003e. The precision and accuracy of this method for dissolved concentrations has been demonstrated previously\u003csup\u003e58,60,61\u003c/sup\u003e, and we express uncertainty (1SD) on Fe concentrations as 2%, based on full replicate analysis of separate GP15 seawater samples collected at the same depth from different bottles (n = 26).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSiderophore Processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSPE columns were thawed and washed with 6 mL qH\u003csub\u003e2\u003c/sub\u003eO (to reduce salts) and the qH\u003csub\u003e2\u003c/sub\u003eO wash was discarded. Ligands were then eluted with 6 mL distilled MeOH into acid-cleaned 10 mL polypropylene tubes. Process blanks were prepared in parallel by eluting activated SPE columns with 6 mL qH\u003csub\u003e2\u003c/sub\u003eO followed by 6 mL MeOH. The methanol fraction was collected as the process blank. For consistency among samples, the qH\u003csub\u003e2\u003c/sub\u003eO wash and MeOH extraction were performed by a trace metal clean liquid handler (model GX271, Gilson).\u003c/p\u003e\n\u003cp\u003eA 10 \u0026micro;L stock solution of 2.2 \u0026micro;M Ga-Desferrioxamine-E (Ga-DFOE) was added to each sample as an internal standard. The sample was concentrated to ~500 \u0026micro;L by vacuum centrifugation (SpeedVac, Thermo Scientific; 35 \u0026deg;C, 5 hrs). A 100 \u0026micro;L aliquot of the sample was taken, mixed with 100 \u0026micro;L of qH\u003csub\u003e2\u003c/sub\u003eO, and immediately analyzed by LC-MS.\u003c/p\u003e\n\u003cp\u003eTo prepare the Ga-DOFE internal standard, 0.5 mg desferrioxamine-E (DFOE; Biophore Research) was dissolved with sonication in 1 mL distilled MeOH. Then, 10 \u0026micro;L of 200 mM gallium nitrate in qH\u003csub\u003e2\u003c/sub\u003eO adjusted to pH 1 with nitric acid (Optima\u003csub\u003e \u003c/sub\u003egrade, Fisher Scientific) was added to complex DFOE. The solution was diluted with 4 mL qH\u003csub\u003e2\u003c/sub\u003eO to make 5 mL of standard. To remove excess Ga, 500 \u0026micro;L of the solution was applied to a SPE column (C18; 100 mg, 1 mL, Agilent Technologies), which had been previously activated with 2 mL each of distilled MeOH and qH\u003csub\u003e2\u003c/sub\u003eO. The column was washed with 2 mL qH\u003csub\u003e2\u003c/sub\u003eO to remove excess Ga, and the Ga-DFOE eluted with 2 mL MeOH. The MeOH eluant was collected and then diluted with qH\u003csub\u003e2\u003c/sub\u003eO to a final volume of 20 mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative analyses of siderophores \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChromatographic analyses were performed on a bioinert Dionex Ultimate 3000 liquid chromatograph (LC) system fitted with a loading pump, a nano pump, and a 10-port switching valve\u003csup\u003e62\u003c/sup\u003e. During the loading phase, 200 \u0026micro;L of sample were withdrawn into the sample loop, then applied to a C18 trap column (3.5 \u0026mu;m, 0.5 mm x 35 mm, P/N 5064-8260, Agilent Technologies) by the loading pump at 25 \u0026mu;L/min for 10 min. The loading solvent is a mixture of 95% solvent A (5 mM aqueous ammonium formate, Optima, Fisher Scientific) and 5% solvent B (5 mM methanolic ammonium formate). During the elution phase, the solvent was delivered by a nano pump at 10 \u0026micro;L/min, and the trap column outflow directed onto two C18 columns (3.5 \u0026mu;m, 0.5 mm x 150 mm, P/N 5064-8262, Agilent Technologies) connected in series. Samples were separated with an 80 min linear gradient from 95% solvent A and 5% solvent B to 95% solvent B, followed by isocratic elution at 95% solvent B for 10 minutes. Meanwhile, the loading pump solvent was switched to 100% qH\u003csub\u003e2\u003c/sub\u003eO, the flow rate increased to 35 \u0026micro;L/min and directed as a post column make-up flow, which was infused with the column eluant into the ICPMS\u003csup\u003e62\u003c/sup\u003e. The high aqueous content of the combined flow serves to minimize the effect of changes in solvent composition (in this case increasing methanol content during the analysis) on the detector response to Fe, Ga, and Al\u003csup\u003e63\u003c/sup\u003e. For Station 39, the HPLC eluant at 10 \u0026micro;L/min was directed into the ICPMS without post column infusion of qH\u003csub\u003e2\u003c/sub\u003eO.\u003c/p\u003e\n\u003cp\u003eThe combined flow from the LC (45 \u0026mu;L/min) was analyzed using a Thermo Scientific iCAP Q ICPMS fitted with a perfluoroalkoxy micronebulizer (PFA-ST, Elemental Scientific), and a cyclonic spray chamber cooled to 4 \u0026deg;C\u003csup\u003e64\u003c/sup\u003e. Measurements were made in kinetic energy discrimination (KED) mode, with a helium collision gas flow of 4-4.5 mL/min to minimize isobaric \u003csup\u003e40\u003c/sup\u003eAr\u003csup\u003e16\u003c/sup\u003eO\u003csup\u003e+\u003c/sup\u003e interferences on \u003csup\u003e56\u003c/sup\u003eFe. Oxygen was introduced into the sample carrier gas at 25 mL/min to prevent the formation of reduced organic deposits onto the ICPMS skimmer and sampling cones. Isotopes monitored were \u003csup\u003e56\u003c/sup\u003eFe (integration time 0.05 s), \u003csup\u003e54\u003c/sup\u003eFe (0.02 s), \u003csup\u003e57\u003c/sup\u003eFe (0.02 s), \u003csup\u003e69\u003c/sup\u003eGa (0.05 s), \u003csup\u003e71\u003c/sup\u003eGa (0.02 s) and \u003csup\u003e27\u003c/sup\u003eAl (0.02 s). \u003c/p\u003e\n\u003cp\u003eThe Fe detector response was calibrated using the siderophore ferrichrome, which elutes at ~40 min in our chromatographic analysis. Stock solutions of 250 \u0026micro;M ferrichrome were diluted to prepare standards with 2 nM, 5 nM, 10 nM, 20 nM, and 40 nM of the siderophore. Then, 5 \u0026micro;L of 2.2 \u0026micro;M Ga-DFOE was added to 995 \u0026micro;L of each standard, a 100 \u0026micro;L aliquot was taken, mixed with 100 \u0026micro;L of qH\u003csub\u003e2\u003c/sub\u003eO, and analyzed by LC-ICPMS. A plot of the ratio \u003csup\u003e56\u003c/sup\u003eFe(ferrichrome):\u003csup\u003e69\u003c/sup\u003eGa (Ga-DFOE) peak areas against ferrichrome/Ga-DFOE concentration yields a linear relationship (r\u003csup\u003e2\u003c/sup\u003e ~0.999) for the response of the ICPMS detector to Fe between 0.2-4 pmoles of ferrichrome. Calibrations and process blanks were made for every 10-20 samples analyzed, with only small changes (RSD ~30%) were observed in the slope of the calibration relationship over the course of the two years of sample analysis. Concentrations of Fe ligands in each sample were measured by plotting the FeL/Ga-DFOE peak area on the appropriate calibration curve.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of siderophores \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assign Fe-Ls to known siderophores, select samples were analyzed by LC-electrospray ionization mass spectrometry (ESIMS). The eluant from the LC, without qH\u003csub\u003e2\u003c/sub\u003eO infusion, was coupled to a Thermo Scientific Orbitrap Fusion mass spectrometer equipped with a heated electrospray ionization source. ESI source parameters were set to a capillary voltage of 3500 V, sheath, auxiliary and sweep gas flow rates of 5, 2, and 0 (arbitrary units), and ion transfer tube and vaporizer temperatures of 275 \u0026deg;C and 20 \u0026deg;C. MS\u003csup\u003e1\u003c/sup\u003e scans for a m/z range of 150-1900 were collected in high resolution (450K) positive ion mode. \u003c/p\u003e\n\u003cp\u003eThe LC-ESIMS data was converted from raw file format to mzXML (Msconvert)\u003csup\u003e65\u003c/sup\u003e, imported to Matlab, and aligned with ICPMS data using the retention time of Ga-DFOE, which was obtained by monitoring m/z of 667.26 by ESIMS and \u003csup\u003e69\u003c/sup\u003eGa by ICPMS. The m/z and intensity from each scan were extracted and ordered by scan number into a scan number/mass (m/z)/intensity matrix, which was interrogated by mass search algorithms\u003csup\u003e62,64\u003c/sup\u003e. The algorithms find pairs of co-eluting peaks with a mass difference of 1.995 D (\u0026Delta;D = \u003csup\u003e56\u003c/sup\u003eFe - \u003csup\u003e54\u003c/sup\u003eFe) and an intensity ratio of 15.7, the crustal abundance ratio of \u003csup\u003e56\u003c/sup\u003eFe and \u003csup\u003e54\u003c/sup\u003eFe. Assignments of iron ligands as known compounds were made by comparing our measured masses to those in a library of 367 known siderophores in the Chelomex siderophore database\u003csup\u003e66\u003c/sup\u003e, and in some cases by comparison with amphibatins and marinobatins isolated from laboratory culture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of \u003csup\u003e57\u003c/sup\u003eFe labeled amphibactins and marinobactins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmphibactins were produced by \u003cem\u003eVibrio\u003c/em\u003e 1F53 culture under Fe limitation induced by the addition of desferrioxamine B\u003csup\u003e62\u003c/sup\u003e. One liter of culture was pumped at 20 mL/min through a 0.2 \u0026micro;m PES capsule filter (Millipore), and Bond-Elut ENV solid phase extraction (SPE) column (1 g, 6 mL, Agilent Technologies) that had been previously activated by passing 6 mL each of distilled methanol (MeOH, Optima LCMS grade, Fisher Scientific) and ultrapure water (qH\u003csub\u003e2\u003c/sub\u003eO, 18.2 M\u0026Omega;) through the column. After extraction, the column was washed with 6 mL qH\u003csub\u003e2\u003c/sub\u003eO and the qH\u003csub\u003e2\u003c/sub\u003eO wash was discarded. Amphibactins were then eluted with 6 mL distilled MeOH into acid-cleaned 10 mL polypropylene tubes. Marinobactins were produced by \u003cem\u003eAlteromonas\u003c/em\u003e 2E5 and \u003cem\u003ePseudoalteromonas\u003c/em\u003e 2E11 culture\u003csup\u003e67\u003c/sup\u003e. For each culture, 25 mL of media was pumped through 0.2 \u0026mu;m PES Sterivex (MilliporeSigma), and C18 SPE columns (0.5 g, Biotage). The SPE column was rinsed with qH\u003csub\u003e2\u003c/sub\u003eO and eluted with 5 mL MeOH. The MeOH extracts were concentrated to ~300 \u0026micro;L under a stream of nitrogen. \u003c/p\u003e\n\u003cp\u003eAmphibactins produced by \u003cem\u003eVibrio\u003c/em\u003e are dominated by non-metallated (apo) siderophores, due to Fe limitation induced by the presence of 10 nM desferrioxamine B in the culture media\u003csup\u003e62\u003c/sup\u003e. To prepare isotopically labeled amphibactins, \u003csup\u003e57\u003c/sup\u003eFe oxide (Isotope enrichment \u0026gt; 95%, Cambridge Isotope Laboratories) was dissolved in concentrated HCl (Optima, Fisher Scientific) as a stock solution of 33.9 mM \u003csup\u003e57\u003c/sup\u003eFe (180 \u0026micro;L HCl per 1 mg \u003csup\u003e57\u003c/sup\u003eFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e). Five microliters (5 \u0026micro;L) of this \u003csup\u003e57\u003c/sup\u003eFe stock solution was added to a 500 \u0026mu;L aliquot of the amphibactin containing MeOH extract. After one hour, the mixture is diluted to 0.1% MeOH with the addition of 500 mL of qH\u003csub\u003e2\u003c/sub\u003eO. The solution was passed through a Bond-Elut ENV column (1 g, 6 mL, Agilent Technologies) at 20 mL/min, to remove excess \u003csup\u003e57\u003c/sup\u003eFe. The SPE columns were then washed with 6 mL of MQ and eluted with 6 mL of MeOH. The MeOH extract was concentrated to approximately 500 \u0026mu;L by vacuum centrifugation (SpeedVac, Thermo Scientific), and used as amphibactin stock solution. \u003c/p\u003e\n\u003cp\u003eMarinobactins produced by \u003cem\u003eAlteromonas\u003c/em\u003e and \u003cem\u003ePseudoalteromonas \u003c/em\u003ewere recovered as \u003csup\u003e56\u003c/sup\u003eFe-siderophores, due to the higher concentration of Fe (125 nM), and the absence of desferrioxamine B in the growth media\u003csup\u003e67\u003c/sup\u003e. To label marinobactins with \u003csup\u003e57\u003c/sup\u003eFe, 100 \u0026mu;L of \u003csup\u003e57\u003c/sup\u003eFe stock solution was added to 10 mL of qH\u003csub\u003e2\u003c/sub\u003eO to create an \u003csup\u003e57\u003c/sup\u003eFe stock solution of pH ~1, which facilitates the isotope exchange from \u003csup\u003e56\u003c/sup\u003eFe to \u003csup\u003e57\u003c/sup\u003eFe. Then, 500 \u0026mu;L of MeOH extract of \u003cem\u003eAlteromonas\u003c/em\u003e and \u003cem\u003ePseudoalteromonas\u003c/em\u003e were combined and added to the \u003csup\u003e57\u003c/sup\u003eFe stock solution. After two days, the mixture was diluted with 1000 mL of qH\u003csub\u003e2\u003c/sub\u003eO, neutralized with 10 mL 100 mM NaHCO\u003csub\u003e3\u003c/sub\u003e, and extracted by a Bond-Elut ENV column. Excess \u003csup\u003e56\u003c/sup\u003eFe and \u003csup\u003e57\u003c/sup\u003eFe passes through the column with the water wash. Then, the column was washed with qH\u003csub\u003e2\u003c/sub\u003eO and eluted with MeOH. The MeOH extract was concentrated to approximately 500 \u0026mu;L by vacuum centrifugation and used as marinobactin stock solution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation of \u003csup\u003e57\u003c/sup\u003eFe-siderophore stock solution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe integrity of the \u003csup\u003e57\u003c/sup\u003eFe-siderophere amendment was established by measuring the chromatographic and isotopic properties of the amphibactin or marinobactin mixture by LC-ICPMS (Extended Data Fig. 2). Fe-chromatograms show that \u003csup\u003e56\u003c/sup\u003eFe-siderophore accounted for 5-10% of the \u003csup\u003e57\u003c/sup\u003eFe-siderophore concentration. In addition, both siderophore stock solutions show no Fe eluting at the solvent front on LC-ICPMS, confirming that the stock solution does not contain inorganic \u003csup\u003e57\u003c/sup\u003eFe or \u003csup\u003e56\u003c/sup\u003eFe. The baseline is low for both \u003csup\u003e57\u003c/sup\u003eFe and \u003csup\u003e56\u003c/sup\u003eFe, suggesting that the concentration of other Fe ligand complexes in the stock solutions is also low. Therefore, the iron added to the incubation by the amendment was dominated by \u003csup\u003e57\u003c/sup\u003eFe-siderophores, with little contamination from other Fe ligands, inorganic Fe, or \u003csup\u003e56\u003c/sup\u003eFe-siderophores.\u003c/p\u003e\n\u003cp\u003eWhen amphibactins and marinobactins were mixed, some siderophores coeluted under the LC conditions used for this study, and multiple siderophores appear as a single peak on ICPMS. For example, peak A on the chromatogram of amphibactin stock solution (Amphibactin-C\u003csub\u003e10:1\u003c/sub\u003e) and the peak A on the chromatogram of marinobactin (Marinobactin-A) stock solution coeluted (Extended Data Fig. 3). Using our chromatographic conditions, ten different peaks were resolved in the \u003csup\u003e57\u003c/sup\u003eFe-chromatogram of the amphibactin/marinobactin amendment, representing \u0026gt; 20 different siderophores. Each peak includes an Fe-amphibactin and an Fe-marinobactin. All ten peaks were quantified before and after the incubation, but the discussion in the text focuses only on the four major peaks as representative of all siderophores in the mixture. The bold letters A-D were used to identify the four major peaks, representing 75% of the total siderophores in the amendment.\u003c/p\u003e\n\u003cp\u003ePeak A is a combination of \u003csup\u003e57\u003c/sup\u003eFe-Amphibactin-C\u003csub\u003e10:0\u003c/sub\u003e. and \u003csup\u003e57\u003c/sup\u003eFe-Marinobactin-A. Amphibactin-C\u003csub\u003e10:0\u003c/sub\u003e is a novel amphibactin that has not been previously reported in the literature. Amphibactin-C\u003csub\u003e10:0\u003c/sub\u003e differs from Amphibactin-T by -C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e- on the fatty acid chain. Peak B is a combination \u003csup\u003e57\u003c/sup\u003eFe-Amphibactin-C\u003csub\u003e12:1\u003c/sub\u003e and \u003csup\u003e57\u003c/sup\u003eFe-Marinobactin-B. Amphibactin-C\u003csub\u003e12:1\u003c/sub\u003e is also a novel amphibactin that differs from Amphibactin-T by a double bond. Peak C is a combination \u003csup\u003e57\u003c/sup\u003eFe-Amphibactin-T and \u003csup\u003e57\u003c/sup\u003eFe-Marinobactin-C. Peak D is a combination of \u003csup\u003e57\u003c/sup\u003eFe-Amphibactin-S and \u003csup\u003e57\u003c/sup\u003eFe-Marinobactin-D. These siderophores were identified by comparing their exact masses to those in the Chelomex siderophore database. \u003c/p\u003e\n\u003cp\u003eTo confirm our identifications, high-energy collision-induced dissociation (HCD) MS\u003csup\u003e2\u003c/sup\u003e spectra for \u003csup\u003e57\u003c/sup\u003eFe-marinobactins and collision-induced dissociation (CID) MS\u003csup\u003e2\u003c/sup\u003e spectra for \u003csup\u003e57\u003c/sup\u003eFe-amphibactins were collected on the Orbitrap mass analyzer. Ions were trapped using a quadrupole isolation window of 1.6 m/z and were then fragmented using an HCD collision energy of 30% or CID collision energy of 35%. The MS\u003csup\u003e2\u003c/sup\u003e of \u003csup\u003e57\u003c/sup\u003eFe-Amphibactin-T (m/z = 858.384; Extended Data Fig. 3) has major fragments of \u003cem\u003em/z\u003c/em\u003e 486.12, 581.26 and 668.29. The fragment at \u003cem\u003em/z\u003c/em\u003e 486.12 represents the cleavage of a peptidic bond on the head group, while retaining \u003csup\u003e57\u003c/sup\u003eFe. The fragments at \u003cem\u003em/z\u003c/em\u003e 581.26 and 668.29 represent the cleavage of another two peptidic bonds on the head group that do not retain \u003csup\u003e57\u003c/sup\u003eFe. These fragmentation patterns are characteristic of amphibactins\u003csup\u003e68,69\u003c/sup\u003e. The MS\u003csup\u003e2\u003c/sup\u003e fragmentation spectrum of \u003csup\u003e57\u003c/sup\u003eFe-Marinobactin-C (\u003cem\u003em/z\u003c/em\u003e = 1014.437) shows major fragments at \u003cem\u003em/z\u003c/em\u003e 486.12, 573.15 and 743.22. For \u003csup\u003e57\u003c/sup\u003eFe-Marinobactin-C, the fragment at \u003cem\u003em/z \u003c/em\u003e486.12 represents diagnostic cleavage of a peptide bond\u003csup\u003e70\u003c/sup\u003e, and a further loss of H\u003csub\u003e2\u003c/sub\u003eO (\u003cem\u003em/z\u003c/em\u003e 18). The fragment at \u003cem\u003em/z\u003c/em\u003e 486.12 was also found in the MS\u003csup\u003e2\u003c/sup\u003e of \u003csup\u003e57\u003c/sup\u003eFe-Amphibactin T, due to the same structure of Fe-Marinobactin-C and Fe-Amphibactin-T (N\u003csup\u003e5\u003c/sup\u003e-acyl N\u003csup\u003e5\u003c/sup\u003e-hydroxy ornithine, serine, N\u003csup\u003e5\u003c/sup\u003e-acyl N\u003csup\u003e5\u003c/sup\u003e-hydroxy ornithine) after the neutral loss. Similarly, the fragment at \u003cem\u003em/z\u003c/em\u003e 573.15 represents a diagnostic cleavage of an ornithine -serine peptidic bond, and a further loss of H\u003csub\u003e2\u003c/sub\u003eO. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIncubation experiments with \u003csup\u003e57\u003c/sup\u003eFe-labeled siderophores\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples for Fe uptake experiments were collected onboard the R/V \u003cem\u003eKilo Moana\u003c/em\u003e at 23.29 \u0026deg;N, 155.32 \u0026deg;W near Station ALOHA (22.45 \u0026deg;N, 158.0 \u0026deg;W) during the SCOPE PARAGON II expedition in August 2022. Seawater was collected using a Niskin bottle rosette equipped with a conductivity, temperature, depth package (SBE 911Plus; Sea-Bird Scientifc) along with fluorescence, oxygen and transmissometer sensors. Two liters (2 L) of unfiltered seawater were sampled from nine depths between 75-450 m into acid cleaned 2 L polycarbonate bottles. Additional samples of filtered and unfiltered seawater were taken at 200 m and 400 m for experimental controls and measurements of the initial conditions. For the filtered control, samples were filtered directly from the Niskin bottle through an in-line 0.2 \u0026micro;m Acropak-1500 Supor cartridge (Pall). \u003c/p\u003e\n\u003cp\u003eFor each incubation sample, 20 \u0026micro;L of amphibactin stock solution and 20 \u0026micro;l of marinobactin stock solution were added. The bottles were wrapped in 4 mil black plastic, and placed in a temperature-controlled water bath incubator at 25 \u0026deg;C. After 5 days, the samples were filtered and extracted onto SPE columns, frozen (-20 \u0026deg;C) immediately, and returned to the laboratory for processing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReferences\u003c/strong\u003e\u003c/p\u003e\n\u003cp class=\"MsoNormal\"\u003e\u003cspan lang=\"EN\"\u003e58. Conway, T. M., Rosenberg, A. D., Adkins, J. F. \u0026amp; John, S. G. 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Acyl peptidic siderophores: Structures, biosyntheses and post-assembly modifications. \u003cem\u003eBioMetals\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 445\u0026ndash;459 (2015).\u003c/span\u003e\u003c/p\u003e\n\u003cp class=\"MsoNormal\"\u003e\u003cspan lang=\"EN\"\u003e71. Schlitzer, R. \u003cem\u003eet al.\u003c/em\u003e The GEOTRACES Intermediate Data Product 2017. \u003cem\u003eChem Geol\u003c/em\u003e \u003cstrong\u003e493\u003c/strong\u003e, 210\u0026ndash;223 (2018).\u003c/span\u003e\u003c/p\u003e\n\u003cp class=\"MsoNormal\"\u003e\u003cspan lang=\"EN\"\u003e72. GEOTRACES Intermediate Data Product Group 2021. The GEOTRACES Intermediate Data Product 2021 (IDP2021). NERC EDS British Oceanographic Data Centre NOC. doi:10.5285/cf2d9ba9-d51d-3b7c-e053-8486abc0f5fd (2021).\u003c/span\u003e\u003c/p\u003e\n\u003cp class=\"MsoNormal\"\u003e\u003cspan lang=\"EN\"\u003e73. Xiang, Y. \u0026amp; Lam, P. J. Size-Fractionated Compositions of Marine Suspended Particles in the Western Arctic Ocean: Lateral and Vertical Sources. \u003cem\u003eJ Geophys Res Oceans\u003c/em\u003e \u003cstrong\u003e125\u003c/strong\u003e, (2020).\u003c/span\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3749755/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3749755/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOne of the major advances in ocean biogeochemistry achieved over the past three decades is an understanding of how nutrients, primarily nitrate, phosphate and iron (Fe), combine in complex patterns to limit and shape primary production in the surface ocean\u003csup\u003e1-3\u003c/sup\u003e. Below the surface ocean, remineralization of sinking organic matter rapidly regenerates nutrients, and microbial metabolism in the upper mesopelagic “twilight zone” (200-500 m) instead appears to be limited by the delivery of labile organic carbon\u003csup\u003e4,5\u003c/sup\u003e. In contrast to the large number of studies describing nutrient limitation in ocean surface waters, nutrient limitation of microbial production in the mesopelagic has been unexplored. Here we report the distribution and uptake of siderophores, biomarkers for microbial Fe limitation\u003csup\u003e6\u003c/sup\u003e, across a meridional section of the eastern Pacific Ocean. Siderophore concentrations were high in chronically Fe limited surface waters, but they were also surprisingly high in the twilight zone underlying the North and South Pacific subtropical gyres, two key ecosystems for the global carbon cycle. Bacterial Fe deficiency due to low Fe availability is likely characteristic of the twilight zone in several large ocean basins, greatly expanding the region of the marine water column where nutrients limit microbial metabolism with potentially significant impacts on ocean carbon storage.\u003c/p\u003e","manuscriptTitle":"Microbial iron limitation in the ocean’s twilight zone","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-17 21:31:17","doi":"10.21203/rs.3.rs-3749755/v1","editorialEvents":[],"status":"published","journal":{"display":false,"email":"[email protected]","identity":"nature","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nature","sideBox":"Learn more about [Nature](http://www.nature.com/nature/)","snPcode":"","submissionUrl":"","title":"Nature","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6f415ef2-006a-484d-8841-c6a40ea54fd1","owner":[],"postedDate":"January 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28166077,"name":"Earth and environmental sciences/Biogeochemistry/Element cycles"},{"id":28166078,"name":"Earth and environmental sciences/Ocean sciences/Marine chemistry"}],"tags":[],"updatedAt":"2024-09-26T07:09:43+00:00","versionOfRecord":{"articleIdentity":"rs-3749755","link":"https://doi.org/10.1038/s41586-024-07905-z","journal":{"identity":"nature","isVorOnly":false,"title":"Nature"},"publishedOn":"2024-09-25 04:00:00","publishedOnDateReadable":"September 25th, 2024"},"versionCreatedAt":"2024-01-17 21:31:17","video":"","vorDoi":"10.1038/s41586-024-07905-z","vorDoiUrl":"https://doi.org/10.1038/s41586-024-07905-z","workflowStages":[]},"version":"v1","identity":"rs-3749755","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3749755","identity":"rs-3749755","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}