2008). The method makes use of the relatively slow chemical conversion between the CO2 and HCO3 − in the absence of CA (Johnson 1982), allowing for a differential labeling of these Ci species with 14C. This method is typically performed at pH of 8.5 (“”assay pH”"), deviating strongly from most natural in situ values and even more from the pH

values applied in OA-experiments (“”acclimation pH”"). In this study, we aimed to disentangle Foretinib in vivo the short-term effect of assay pH from the long-term effect of acclimation history on the photosynthetic Ci source of E. huxleyi. To this end, we grew haploid and diploid life-cycle stages at present-day (380 μatm) and elevated pCO2 (950 μatm), and measured the responses in growth, elemental composition, and production rates. These low and high pCO2-acclimated cells were then tested for their preferred Ci source by applying the 14C disequilibrium method, with assay conditions set to a range of ecologically relevant pH values (pH 7.9–8.7). The reliability of this new approach was tested by performing sensitivity studies. Methods pCO2 acclimations Haploid and diploid cells of E. huxleyi (strains RCC 1217 and RCC 1216, obtained from the Roscoff culture collection) were grown at 15 °C as dilute batch incubations. North Sea seawater medium (salinity 32.4) was sterile-filtered (0.2 μm) and enriched with vitamins

and trace metals according to F/2 (Guillard and Ryther 1962), as well as phosphate and nitrate (100 and 6.25 μmol L−1). Cells were exposed to a light:dark cycle (16:8 h) and saturating selleck light (300 μmol photons m−2 s−1) PD0325901 molecular weight provided by daylight lamps (FQ 54W/965HO, OSRAM, Munich, Germany). Light intensity was monitored with the LI-6252 datalogger (LI-COR,

Lincoln, NE, USA) using a 4π-sensor (US-SQS/L, Walz, Effeltrich, Germany). Culturing was carried out in sterilized 2.4 L borosilicate bottles (Duran Group, Mainz, Germany) on roller tables to avoid sedimentation. Prior to experiments, cells were acclimated to the respective pCO2 and light conditions for at least 7 days (i.e., more than 10 generations). Prior to initiating cultures, medium was pre-aerated for at least 36 h with humidified, 0.2 μm-filtered air comprising pCO2 values of 380 or 950 μatm (equivalent to 38.5 and 96.3 Pa, or ~15 and ~35 μmol kg−1, respectively). Aprepitant Gas mixtures were created by a gas flow controller (CGM 2000 MCZ Umwelttechnik, Bad Nauheim, Germany) using pure CO2 (Air Liquide Deutschland, Düsseldorf, Germany) and CO2-free air (CO2RP280, Dominick Hunter, Willich, Germany). Sampling and measurements were done 4–8 h after the beginning of the light period (i.e., at midday) in exponential growth at densities of 40,000–60,000 cells mL−1. Cultures showing a pH drift of > 0.05 were excluded from further analyses. The carbonate system (Table 1) during the acclimations was assessed based on measurements of pH and total alkalinity (TA).