ESM enrichment contains 28.7 μM (final concentration in the medium) K2HPO4, but not in the Marine Art SF. In all acidification experiments, cells were grown in the artificial seawater containing EMS medium (MA/ESM medium) under constant illumination at 100 μmol photons m−2 s−1 and 20 °C (standard condition). To avoid large changes in the pH of the medium during culture, both HEPES and Tris-buffer (final concentration, 10 mM each) were added to the medium by considering those buffers’ buffering ability and pKa values. Bubbling cultures with air and air containing elevated concentration of CO2 Tanks containing

air with elevated concentrations of CO2, namely 406, 816 and 1192 ppm, were purchased from the company, Suzuki Shokan Ltd., Tsukuba, Japan. First, those gasses were bubbled through MA/EMS medium containing HEPES- and GSK872 price Tris-buffers (10 mM each) for 15 h as pre-bubbling for attaining equilibrium of CO2 between the bubbled gasses and the medium. The concentrations of respective DIC species in the medium shown in Fig. 1 and 6 were calculated according to Leuker et al. (2000) and CO2SYS, respectively. On the other hand, algal cells were grown

separately with air in the MA/ESM medium under constant illumination at 100 μmol m−2 s−1 and 20 °C for 3 days. And then, an selleck chemical aliquot of the algal suspension was transferred to the previously prepared medium of which pH

and pCO2 were already set by adding HCl or bubbling of air containing elevated CO2, as described above. Fig. 1 Effect of the acidification by HCl (a–e) and the ocean acidification conditions by elevating pCO2 (f–j) on the cell growth of the coccolithophore E. huxleyi. Before experiments, all cells had been grown at pH 8.2 under the bubbling of air containing 400 ppm CO2. Temperature was 20 °C. a, f, Change in turbidity; b, MEK inhibitor g change in cell number; c, h H in the medium. Initial pHs were set at 8.2 in a (closed circles), 7.7 in closed squares and 7.2 in closed triangles by HCl (a–c) and at 7.9 in closed circles, 7.6 in closed squares and 7.5 closed triangles by elevating pCO2 (f–h). d, i Specific growth rates (μ) calculated on the basis of cell number; e, j inorganic carbon concentrations in the medium at each pH and the elevated pCO2 concentration at 1 day. CO2 concentration was set at 15 μmol L−1 in all the conditions (right column). Solid (left) and stripe (middle) columns indicate total DIC and HCO3 − concentrations, respectively. DIC, bicarbonate and CO2 concentrations were calculated by a kind help of Dr. Midorikawa according to Leuker et al. (2000) Determination of the specific growth rate and microscopic observation Cell turbidity of the culture was determined by measuring OD750 using a spectrophotometer (UV-1700, Shimadzu, Kyoto, Japan).