Cultivating Emiliania huxleyi Strains

Emiliania huxleyi strain AWI1516, which produces coccoliths, and its closely related sister strain CCMP2090, which does not produce coccoliths, were grown in artificial seawater medium Aquil, at 18 °C and under 50 µE light in a 12/12-h light/dark cycle. The concentration of nitrate in the medium was 0.2 mM and of phosphate 10 µM. Under standard growth conditions, where the cells form calcite, the medium contained 10 mM CaCl₂ (std-Ca medium), while under low-Ca growth conditions, where the cells do not form calcite, it contained 0.1 mM CaCl₂ (low-Ca medium). In experiments with cells from low-Ca growth conditions, the cultures had been kept in a low-Ca medium for at least 2 weeks before use. For cells inoculated into low-Ca medium, the extracellular coccoliths were dissolved with EDTA, and cells were washed with Ca-free medium before inoculation, as previously described^{20 (link)}. Algal cell density was measured using a Beckman Coulter Z2 particle counter.