Sample collection and preparation:
Each water sample was filtered directly from the trace metal clean GTC rosette/Go-Flo bottle sampler through a 0.2-micrometer (µm) Pall Acropak-200 Supor cartridge into a trace metal grade acid-cleaned 4-liter (L) polycarbonate bottle for siderophore analysis or an acid-cleaned 2- or 4-L Nalgene LDPE bottle for dissolved iron (Fe) analysis. Samples for siderophores were pumped at 20 milliliters per minute (mL/min) through Bond-Elut ENV solid phase extraction (SPE) columns (1 gram (g), 6 milliliters (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 (qH₂O, 18.2 megaohm (MΩ)) through the column. SPE columns were frozen (-20 degrees Celsius (°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.

Siderophore Processing:
SPE columns were thawed and washed with 6 mL qH₂O (to reduce salts) and the qH₂O 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 qH₂O followed by 6 mL MeOH. The methanol fraction was collected as the process blank. For consistency among samples, the qH₂O wash and MeOH extraction were performed by a trace metal clean liquid handler (model GX271, Gilson).

A 10-microliter (µL) stock solution of 2.2 micromolar (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 hours). A 100 µL aliquot of the sample was taken, mixed with 100 µL of qH2O, and immediately analyzed by LC-MS.

To prepare the Ga-DOFE internal standard, 0.5 milligrams (mg) desferrioxamine-E (DFOE; Biophore Research) was dissolved with sonication in 1 mL distilled MeOH. Then, 10 µL of 200 mM gallium nitrate in qH2O adjusted to pH 1 with nitric acid (Optima grade, Fisher Scientific) was added to complex DFOE. The solution was diluted with 4 mL qH2O 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 qH2O. The column was washed with 2 mL qH2O to remove excess Ga, and the Ga-DFOE eluted with 2 mL MeOH. The MeOH eluant was collected and then diluted with qH2O 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. 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 millimeter (mm) x 35 mm, P/N 5064-8260, Agilent Technologies) by the loading pump at 25 microliters per minute (μL/min) for 10 minutes. The loading solvent is a mixture of 95% solvent A (5 millimolar (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 minute 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% qH₂O, 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 an inductively coupled mass spectrometer. 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. For Station 39, the HPLC eluant at 10 µL/min was directed into the ICPMS without post column infusion of qH₂O.

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. Measurements were made in kinetic energy discrimination (KED) mode, with a helium collision gas flow of 4-4.5 mL/min to minimize isobaric 40Ar16O+ interferences on 56Fe. 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 56Fe (integration time 0.05 seconds), 54Fe (0.02 seconds), 57Fe (0.02 seconds), 69Ga (0.05 seconds), 71Ga (0.02 seconds), and 27Al (0.02 seconds).

The Fe detector response was calibrated using the siderophore ferrichrome, which elutes at ~40 minutes in our chromatographic analysis. Stock solutions of 250 µM ferrichrome were diluted to prepare standards with 2 nanomolar (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 qH₂O, and analyzed by LC-ICPMS. A plot of the ratio 56Fe(ferrichrome):69Ga (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.