Dataset: Attheya_sp_experiments
Deployment: lab_Fu

All methods below are described in King et al. 2011.

Organism source and culture conditions
Unialgal axenic Attheya sp. (CCMP207) stock cultures, isolated from the Bering Sea, were obtained from the Provasoli-Guillard Culture Collection of Marine Phytoplankton (Boothbay Harbor, ME) and were grown in microwave-sterilized media prepared from 0.2 mm-filtered natural seawater (collected using trace metal clean techniques). Media had Aquil concentrations of nitrate, phosphate and silicic acid with additions of 5 uM EDTA, 451 nM Fe, 80 nM Zn, 50 nM Co and no added Cd (Price et al., 1988/89). Stock cultures were incubated at 3 degrees C with a 12 h:12 h light–dark cycle and an incident photon flux density of 80 µmol photons per square meter per second.

Experimental treatments/conditions
Experimental treatments included trace metal clean vitamin B12 additions of 500 and 10 ng per liter (370 and 7 pM) for the B12-replete and B12-limited cultures, respectively. Vitamin and nutrient stocks were cleaned of trace metals before use by passing them through a column packed with Chelex-100 (Bio-Rad, Hercules, CA, USA; Price et al., 1988/89). For both B12 conditions, triplicate acid-washed polycarbonate bottles were equilibrated with commercially prepared air:CO2 mixtures at three different CO2 concentrations: 200, 370 and 670 ppm pCO2. In-line HEPA filters were used to avoid trace metal and bacterial contamination from the gas tanks or lines.

Trace metal clean semi-continuous culturing methods were used during acclimation periods and steady-state growth. Final sampling was carried out following 4 to 8 weeks of semi-continuous incubation after statistically invariant growth rates were recorded for at least three consecutive transfers.

Seawater medium pCO2 in the experimental bottles was calculated throughout the experiment using measurements of pH and total dissolved inorganic carbon according to Dickson and Goyet (1994). To ensure that CO2 levels remained constant during growth, the pH in each bottle was monitored daily using a microprocessor pH meter, calibrated with pH 4, 7 and 10 buffer solutions. Total dissolved inorganic carbon was measured via coulometry (model CM 140, UIC, Joliet, IL, USA). The calculated pCO2 values (using CO2SYS; http://www.cdiac.ornl.gov/ftp/co2sys/CO2SYS_calc_XLS) from the final day of the experiment were 201±13, 367±21 and 671±31 ppm (see table below); these treatments are referred to in the dataset and the literature using rounded-off values of 200, 370 and 670 ppm pCO2.

Seawater carbonate buffer system conditions (n=3):

Primary production and trace metal measurements
Primary production was measured in triplicate using 24 h incubations with 3.7 kBq per mL H14CO3 under the appropriate experimental growth conditions of light and temperature for each treatment, followed by filtration and scintillation counting. Aliquots for analysis of particulate organic C and N were filtered onto pre-combusted 25mm GF/F filters (Whatman, Maidstone, UK) and analyzed using a 4010 CHNS Elemental Combustion System (Costech, Valencia, CA, USA). Samples for particulate organic phosphorous were determined spectrophotometrically ( Fu et al. (2005, 2007).

Particulate samples for trace metal analysis were filtered onto acid-washed 3-um-pore-size polycarbonate filters (Millipore, Billerica, MA, USA), rinsed with oxalate reagent to remove extracellular trace metals (Tovar-Sanchez et al., 2003), and Fe, Zn, Co and Cd were determined with a magnetic sectorfield high-resolution inductively coupled plasma mass spectrometer (ICPMS) (Element 2, Thermo, Waltham, MA, USA). Procedural filter blanks were also subjected to the same storage, digestion, dilution and analysis processes, and these blank values were subtracted from sample measurements (Sañudo-Wilhelmy et al., 2001). The digestion protocol consisted of sequential additions of ultrapure HCl, HNO3 and HF (Omnitrace Ultra; VWR, Westchester, PA, USA) and heating to 100 degrees C (Eggimann and Betzer, 1976).

The measured concentrations of particulate elements (C, N, Fe, Co, Zn and Cd) were normalized to P. Cellular elemental 'quota' or 'content' are used interchangeably with 'requirement'. Trace metal:C ratios were used in conjunction with specific growth rates to calculate net use efficiencies (NUEs; specific growth rate divided by trace metal:C ratio), representing how efficiently trace metals are used by cells for growth and C fixation (Raven, 1991).