Culturing:

Cultures of E. huxleyi Strain CCMP2668, were innoculated at low cell density into media prepared from autoclaved filtered seawater with nutrient amendments based on F/2 medium kit from the National Center for Marine Algae and Microbiota, with a 1:25 reduction in nutrient additions. These were allowed to acclimate for approximately five generations, until cell density neared levels likely to significantly change the pH/pCO2. Daily dilutions of cultures with pre-equilibrated media kept cell density low (<1x105 cells/ml), ensured cells remained in exponential growth phase and prevented excessive drawdown of nutrients and CO2. Cell density was determined by flow cytometry (Model) and each flask was diluted with media that was continuously sparged with air containing 400, 750 or 1000 ppm CO2. Air mixtures were created using CO2 free air (Powerex air compressor, and Twin Towers CO2 scrubber) and pure CO2 (Airgas) combined using a system of mass flow controllers (Sierra Instruments) and verified using a non-dispersive infrared CO2 sensor (Licor 820). Cultures were maintained in 1 liter polycarbonate flasks at 150 C under a 12/12 light dark cycle. Replicates (n=5) were placed in Plexiglas chambers which were supplied with a flow of the appropriate air mixture for each treatment. Preliminary experiments showed that gas exchange across the air/water surface significantly helped to maintain the target pCO2 in cultures without the mechanical disturbance of bubbling. Sedimentation was minimized by gentle mixing of the cultures by rotation of the bottles twice a day, during sampling and dilution. Cell densities ranged between about 30,000 cells/ml after dilutions to 80,000 cells/ml on the following day. The culture volume that was removed was used for analyses, and replaced with pre-equilibrated media. Cultures were maintained in this fashion for about 8 days. Since the first dilution occurred on day 4 after innoculation, this gives a total of 12 to 14 days in culture at experimental conditions. This experiment was carried out twice, in 2011 and 2012.

SEM:

On days 1 and 8, a few small volume of culture from each replicate was dropped onto SEM stubs and allowed to dry. The stubs were sputter coated with Palladium gold for approximately three minutes (2012) or one minute (2011). Five images of each replicate with three or more cells in each image were taken at 5000x magnification (Smith et al. 2012) using an FEI Quanta 450 Scanning Electron Microscope. To compose each image the field was zoomed out to about 200x magnification to reduce bias in finding clusters of three or more cells. When a cluster of three of more cells was found, the magnification was changed to 5000x magnification and the image was focused and captured.

Parameters including cell size, coccolith size, coccoliths per cell, and percentage of malformed coccoliths were measured from the SEM images. Cell size (control: n= 235, moderate: n= 234, high: n= 200) and coccolith size (control: n= 75, moderate: n= 72, high: n= 67) were calculated using the free hand tool in Image J. To determine the number of coccoliths per cell and the percentage of malformed coccoliths, images were loaded into Windows Photo Viewer (control: n= 1453, moderate: n= 1409, high: n= 1215). Coccoliths malfomation was assessed by the scheme of DeBodt et al, 2010.

Carbonate chemsitry: (THIS DATASET)

pH was measured photometrically using 1 cm cuvettes, m-cresol dye and an Agilent 5480 UV-VIS spectrophotometer (+/- 0.02). Alkalinity was measured by gran titration using a Titrando 888, and 0.1 N HCl titrant, in a temperature controlled titration vessel (+/1 5 ueq/kg). Other parameters were calculated with CO2sys. pCO2 conditions were the same in the two experiments with the exception that the moderate concentration had slightly more elevated pCO2 in 2011 (662 ppm compared to 602 ppm). (When day 1 is included for 2012, there is no significant difference; the difference in the Moderate treatment is present when only day 12 and 14 are included.)

Related dataset: Emiliania huxleyi SEM

carbonate.csv