Three species of marine phytoplankton – Micromonas pusilla, Emiliania huxleyi, and Isochrysis galbana - were grown under four phosphorus (P) conditions. These include phosphate (Pi) replete and deplete conditions and the phosphonate conditions where cultures received either methylphosphonate (MPN) or 2-aminoethylphosphonate (2-AEPN) as the sole source of phosphorus at replete levels.

Axenic cultures of the pico-prasinophyte Micromonas pusilla (CCMP1545), the coccolithophore Emiliania huxleyi (CCMP2090), and the pico-prymnesiophyte Isochrysis galbana (CCMP1323) were obtained from the National Center for Marine Algae and Microbiota (Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine). The cultures remained axenic throughout the experiments as determined by SYTO-staining and flow cytometric counting on a BD FACSJazz cell sorter; all cultures were free of bacteria during these experiments. Phytoplankton were grown in artificial sea water amended with L1 media silica. The P source was added separately to achieve the desired growth conditions; Pi-replete media contained 36 µM PO43-, the Pi-deficient condition received 0.1 µM PO43-, and the phosphonate treatments received either 36 µM MPN or 2-AEPN. The Pi-deficient treatment (0.1 µM) represents a control for the low level of contaminating Pi measured in the phosphonate media; thus, an increase in growth in the MPN and 2-AEPN conditions above that measured in the Pi-deficient condition is due to phosphonate utilization. The potential for abiotic breakdown of phosphonate to Pi was previously investigated in media-only tubes exposed to the experimental temperature and light conditions for 10 days. Pi levels did not change throughout the experimental period (MPN average Pi = 0.11 µM ± 0.02; 2-AEPN average Pi = 0.10 µM ± 0.02), strongly supporting the notion of active enzymatic breakdown of phosphonates for growth. Cultures were acclimated to the four growth conditions described above as they had been maintained in each P treatment for a minimum of two transfers (20 days). Cultures were grown at 20°C in a 14-hour light/10-hour dark cycle at ~100 µE m-2 s-1 with a starting concentration of ~1x104 cells per mL in 25 mL culture volumes. Phytoplankton growth was monitored by fluorescence measurements using a Turner TD-700 fluorometer and cell counts analyzed by flow cytometry. Triplicate cultures were setup for each treatment and were harvested in the late exponential phase of growth for transcriptomic analysis.

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