Methodology:

Sampling and analytical procedures:

Metal quotas

Cellular metal quotas were measured by inductively coupled plasma mass spectrometry (ICP-MS). Biomass from replicate 25 mL double addition cultures of Chaetoceros RS19 were pooled upon entering stationary phase and were centrifuged at 11,000 RPM (14,610 x g) for 40 minutes at 4°C. The cell pellet was resuspended in ~1 mL media and transferred to an acid-cleaned microcentrifuge tube. Cultures were centrifuged again for 30 min at 14,100 RPM (13,336 x g) at 4°C before the supernatant was discarded. Half of the remaining cell pellet was acidified in 800 mL of 5% nitric acid (Optima) containing 1 ppb indium for at least seven days while the other half was retained for proteomic analysis. Solids were removed by centrifugation. No attempt was made to remove extracellular metals by washing cells with additional metal chelators in order to minimize processing blanks. Quota determinations therefore include contributions from both intracellular and extracellular pools. Process blank digestions containing acid but no cells were performed in parallel. Digests were diluted by a factor of 9 with 5% nitric acid 1 ppb indium solution before being analyzed in duplicate on a Thermo ICAP-Q plasma mass spectrometer calibrated to a multi-element standard curve (Spex Certiprep) over a range of 1 – 20 ppb. Samples were analyzed in KED mode after an 85s sample uptake window and element mass windows were scanned 3 times during measurements. The 1 ppb indium internal standard was used to correct for variation in sample delivery and plasma suppression between samples. Process blanks were subtracted from measured concentrations. Phosphorus concentrations were also measured by ICP-MS simultaneously and were calibrated to a standard curve ranging from 100 – 3,200 ppb using a 1 ppm certified P stock (Alfa Aesar Specpure). The seawater media base used for all growth experiments was similarly analyzed via ICP-MS using a 1:10 dilution of media base into 5% nitric acid 1 ppb indium and analyzed as above to determine background media concentrations of total Zn and Co (0.7 nmol L-1 and 0.1 nmol L-1, respectively).