Dataset: Colimitation Physiology

Culturing methods: Triplicate cultures of Crocosphaera watsonii strain WH0005 were grown under low iron (Fe, 5 nM) and low phosphorus (P, 2 uM) conditions for 3 months at 27C to acclimate to simultaneously low concentrations of both nutrients. Fe and P were then “added back” at different concentrations to create nutrient treatments to assess the physiological effects of Fe and P co-limitation. The treatments were:   Fe/P Replete (Replete, 250 nM Fe and 10 uM P), Fe-limited (Felim, 3 nM and 10 uM P), P-limited (Plim, 250 nM Fe and 0.15 uM P), Fe/P Co-limited (Colim, 3 nM Fe and 0.15 uM P). Phosphate was passed through an activated Chelex 100 resin column (BioRad Laboratories, Hercules, CA, USA) to remove contaminating iron. Aquil concentrations of vitamins and a modified trace metals mix were also added (1.21 x 10-7 M Mn, 7.97 x 10-8 M Zn, 1.00 x 10-7 M Mo, and 5.03 x 10-8 M Co).

Cultures were maintained under semi-continuous culturing conditions on a 12:12 light:dark cycle in temperature-controlled incubators at 150 μmol photons m^-2 s^-1 and diluted every three days to maintain steady-state exponential growth. Cultures were grown in microwave-sterilized media made with 0.2 micron-filtered Aquil-base artificial seawater (Sunda et al., 2005) for ~1 month and then grown for ~2 weeks (3-8 generations) in media made with Aquil artificial seawater passed through an activated Chelex 100 resin column to remove contaminating iron. All media was buffered with 25 uM EDTA. The limiting nutrient was directly added to the culture bottles during periodic dilutions.

Dilutions were conducted based on in-vivo fluorescence measured in real-time on a 10AU Fluorometer (Turner Designs, San Jose, CA), and growth rates were later validated with 1%, 0.2-μm filtered glutaraldehyde preserved cell samples.

Growth Rates & Cell size: The specific growth rate (μ) was then calculated using the equation μ = (ln N1 – ln N0) / t, where N refers to cell densities and t is time in days. The cell size was determined by measuring the cell diameters of at least 20 cells per sample using the CaptaVision Imaging Software (Commack, NY, USA).

Chlorophyll A: Chlorophyll concentrations were measured by filtering 30 mL of culture GFF filters (Whatman, Grade GF/F), extracted overnight at -20°C in 90% acetone (Welschmeyer 1994), and measured on a Trilogy fluorometer (Turner Designs).

Nitrogen fixation rates: Nitrogen-fixation rates were measured using the acetylene reduction assay during the night-time following previously described methods (Garcia et al., 2013). Briefly, 40 mL culture sub-samples were collected from the triplicate experimental cultures and 6 mL of acetylene was injected into 35 mL of headspace at the start of the dark period. All-night (~12 hours) accumulation of acetylene was measured at the end of the incubation period on a gas chromatograph GC-8a (Shimadzu Scientific Instruments, Columbia, Maryland), and the measured ethylene was converted to fixed nitrogen using a ratio of 3:1 and a Bunsen coefficient of 0.086. Converted nitrogen-fixation rates were then normalized to particulate organic nitrogen (N-specific rates) and cell counts (cell-specific rates).

Carbon fixation rates: To approximate net primary productivity, 10 ml sub-cultures from each experimental replicate were incubated for 3-5 hours with H14CO3 under the same experimental growth conditions (e.g. light, temperature, etc.). Samples were then filtered onto glass microfiber filters (GF/F) and stored in the dark overnight before analysis using a Wallac System 1400 liquid scintillation counter (Jiang et al., 2018). Carbon fixation rates were then normalized to particulate organic C (C-specific rates) and cell counts (cell-specific rates).

Elemental stoichiometry analysis: Particulate organic carbon and nitrogen (POC and PON) samples were filtered on pre-combusted glass microfiber filters (Whatman, Grade GF/F), dried in an oven at ~60C, and then pelleted and analyzed on a 4010 Costech Elemental Analyzer calibrated with methionine and acetanilide (Jiang et al., 2018). Particulate organic phosphorus (POP) samples were filtered on pre-combusted GF/F, dried, and analyzed following Fu et al., (2005). Briefly, the dried POP samples were combusted for 2 hours at 500C to convert organic phosphorus to inorganic orthophosphate, which was then measured spectrophotometrically.

Phosphorus Use Efficiency: Nitrogen-specific Phosphorus Use Efficiencies (NPUEs) were calculated by normalizing measured nitrogen-fixation rates to POP content (mol N fixed hr^-1 mol cellular P^-1). Similarly, Carbon-specific Phosphorus Use Efficiencies (CPUEs) were calculated by normalizing measured carbon fixation (C-fixation) rates to intracellular phosphorus (Jiang et al., 2018).