Synechococcus sp. WH8102 was grown in exponentially fed batch (EFB) continuous cultures under controlled laboratory conditions to maintain near steady-state physiology. Cultures were maintained at 26 °C on a 12:12 h light:dark cycle (250 µE m⁻² s⁻¹) in SN medium with three phosphorus treatments: High-P (36 µM), Mid-P (5 µM), and Low-P (1 µM). Continuous dilution rates were adjusted (0.35, 0.25, and 0.12 d⁻¹, respectively) to match growth rates and maintain stable biomass.
Samples were collected daily and processed immediately. Cell abundance was measured using a Coulter Counter, and growth rates were calculated from changes in in vivo fluorescence (measured by fluorometry), corrected for dilution. Subsamples were filtered onto 0.2 µm polycarbonate or GF/F filters for intracellular phosphorus analyses and stored at −80 °C or extracted immediately, depending on the assay.
Intracellular phosphorus pools were quantified across major macromolecular fractions, including ATP, DNA, RNA, phospholipids, and polyphosphate, using established biochemical and fluorometric methods. ATP was measured via bioluminescence, nucleic acids via fluorometric assays, polyphosphate via dye-based fluorometry, and phospholipid-associated P following extraction and colorimetric phosphate determination. Dissolved and particulate phosphorus fractions, including soluble reactive phosphorus (SRP), total particulate phosphorus (TPP), and intracellular inorganic phosphate (Pi), were measured using standard colorimetric methods. Intracellular phosphorus pools, including ATP, DNA, RNA, phospholipid-associated phosphorus (P_lipid), free inorganic phosphate (free_Pi), and polyphosphate (PolyP), as well as TPP, are reported as bulk concentrations in the culture (µM), phosphorus quotas per cell (fmol cell⁻¹), and phosphorus normalized to cell biovolume (fmol µm⁻³).
PolyP was extracted by enzymatic lysis and quantified fluorometrically using the JC-D7 dye following Yang et al. (Yang et al., 2024), calibrated against a 45-chain-length polyP standard
DNA and RNA were extracted by bead-beating and quantified fluorometrically with PicoGreen and RiboGreen assays following Mouginot et al. (Mouginot et al., 2015)
Intracellular ATP was extracted by boiling in Tris buffer and quantified by firefly bioluminescence following Holm-Hansen and Booth (Holm‐Hansen & Booth, 1966), with baseline drift corrected using a custom Python pipeline (available at https://github.com/DuhamelLab/ATP, DuhamelLab. (n.d.)).
Phospholipids were extracted using a biphasic isopropanol/ethyl acetate protocol adapted from Bielawski et al. (Bielawski et al., 2009), and phospholipid-bound P was quantified colorimetrically following Bartlett (Bartlett, 1959).
SRP and TPP were determined by standard phosphomolybdenum blue colorimetry (Grasshoff et al., 1983; Strickland & Parsons, 1972), and free intracellular phosphate was recovered by osmotic shock extraction following Raimbault and Mingazzini (Raimbault & Mingazzini, 1987).
Together, these approaches enabled quantification of cellular phosphorus allocation and nutrient dynamics under controlled phosphorus supply conditions.
