Cultures of Emiliania huxleyi were obtained from the National Center for Marine Algae and Microbiota at Bigelow Laboratories (all CCMP strains), or from Dr. D. Iglesias-Rodriguez at UC Santa Barbara (strain NEZH) and maintained in the Strom laboratory at Shannon Point Marine Center.

Batch cultures were grown in 50-100 ml volumes of seawater (salinity = 30) amended with f/50 nutrients, at a temperature of 15 deg C and an irradiance of 140-300 umol photons m-2 s-1 on a 12L:12D light cycle. Replicate cultures (n=3) were subsampled for chemical measurements at cell densities of 1.4 to 2.7 x 10^5 cells ml-1 (DMSP) and 3.6 to 12.8 x 10^5 cells ml-1 (H2O2). Different chemical and size measurements reported for a given strain were made over the course of several separate experiments.

Dimethylsulfoniopropionate (DMSP) contained within E. huxleyi cells was measured using a Shimadzu GC-14A gas chromatograph and flame photometric detection, following the methods of Wolfe et al. (2002 J. Phycol. 38: 948-960). Cells were captured on 25 mm glass fiber filters (effective pore size 0.7 um) and placed into 3 ml 5N NaOH for hydrolysis. Method was standardized using ultrapure DMSP-Cl (standard range 0.625 to 50 nM; r2 ≥0.998).

Hydrogen peroxide (H2O2) released into the dissolved phase by E. huxleyi was measured using the Amplex Red – horseradish peroxidase method, using a kit from Molecular Probes (now part of Thermo Fisher Scientific) according to kit directions and to Suggett et al. (2008 J Phycol 44: 948-956). Fluorescent reaction product was quantified in a BioTek Synergy M plate reader (565 nm excitation, 585 nm emission). True reagent blanks were obtained by catalase treatment of E. huxleyi culture filtrate (50 U ml-1, 45 min, room temperature) following Shaked et al. (2010 Environ Sci Technol 44: 3238-3244). Method was standardized using ultrapure H2O2 (standard range 0.025 to 0.5 uM; r2 = 0.98)

E. huxleyi cell size was obtained by imaging live cells (n = 23-29) at 1000x magnification on a Leica DM5500 B microscope, and sizing them with associated image analysis software. Calcification (i.e. whether a strain harbored coccoliths) was also confirmed during microscopy. Note that the sample of strain CCMP3266 used for size measurement comprised a mixture of calcifying and non-calcifying cells.

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