Methodology:

Sampling and analytical procedures:

Media and culturing techniques

Chaetoceros RS19 cultures were maintained in a 4°C incubator under constant fluorescent lighting (30 µmol photon m-2 s-1). All cultures were randomly repositioned each day to avoid any effect of subtle variation in light intensity on growth. Chaetoceros RS19 was originally isolated by D. Moran from the Ross Sea, Antarctica, and cultures were maintained in the Saito laboratory culture collection at the Woods Hole Oceanographic Institution. All cultures were axenic and maintained by sterile technique until needed. Polycarbonate and plastic bottles were cleaned to remove trace metal contaminants before use. This procedure involved, at minimum, a 72h soak in <1% Citranox detergent, five rinses in Mili-Q water, a 7 day soak in 10% HCl, and five rinses with dilute acid (HCl, pH 2). Cultures were grown in microwave-sterilized 28 mL polycarbonate centrifuge tubes and all solutions were pipetted after a tip rinse procedure consisting of three rinses with 10% HCl followed by three rinses with sterile dilute HCl (pH 2). All culture work was conducted in a Class 100 clean room and transferring of cultures was conducted in a laminar flow hood within the clean room.

Culture media was prepared after that used by Sunda and Huntsman for trace metal experimentation (Sunda and Huntsman 1995). Microwave sterilized, 0.2 µm-filtered Equatorial Pacific surface seawater collected at station 14 of the 2016 ProteOMZ expedition (10.5600°S, 146.3133° W; cruise code FK160115) was used as the media base. Macronutrients were added to this sterile base to a final concentration of 88.2 µM NaNO3, 41.5 µM NaH2PO4, and 106 µM Na2SiO3 and were chelexed before use. Added vitamins included 2 nM biotin, 0.37 nM B12 as cyanocobalamin, and 300 nM thiamine and were also chelexed before use. Trace metals were added to final media concentrations of 10-7 M FeCl3, 4.8 x 10-8 M MnCl2, 4.0 x 10-8 M CuSO4, 10-7 M NiCl2, and 10-8 M Na2O3Se within a 10-4 M ethylenediamine tetraacetic acid disodium salt (EDTA, Acros Organics, C10H14N2Na2O8) metal ion buffer system. All media amendments were sterile filtered through acid-rinsed 0.2 µm filters before addition to final media, and final media equilibrated for at least 12h before inoculation.

Established cultures were first acclimated in low-metal media containing 1 nM total added Zn or less for at least three transfers. These acclimated cultures were used to inoculate initial cultures at 1% volume. All growth media was chilled at 4°C prior to inoculation. Zn or Co limitation experiments were first performed using a range of added Zn concentrations with Co omitted and vice versa. We refer to growth rate experiments using media amended with Zn or Co (while omitting the other) as “simple limitation” experiments. Growth rate experiments in which one metal was held at a constant total added value while varying the added concentration of the other metal were also conducted—we refer to these as “double addition” experiments. Growth of all experiment cultures was monitored by relative chlorophyll fluorescence using a Turner TD-700 fluorometer, calibrated prior to measurement with a solid standard. All cultures were grown in 28 mL acid cleaned, microwave sterilized polycarbonate tubes (Nalgene) that are compatible with the fluorometer, which enabled measurements to be taken without exposing cultures to contaminating metals. Growth rates were calculated as the slope of the natural logarithm of the increase in chlorophyll fluorescence over a four-measurement (usually 4 day) period during exponential growth. Computed ratios of [Zn2+] and [Co2+] to total concentrations, whose values are 10-3.99 and 10-3.63 respectively, were used to convert total added metal concentrations to free ion concentrations and are the same as those used by Sunda and Huntsman (Sunda and Huntsman 1995).