Table of Contents

• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

These data show cellular characterizations of two strains of Emiliania huxleyi cultured semi-continuously over a period 13-14 days under three different pCO2 concentrations (400 ppmv, 750 ppmv, and 1000 ppmv).  Cellular characterization measurements were taken throughout the course of the experiments, resulting in a time course data set.  CO2 chemistry was also monitored over the course of the experiment.  Cellular characterizations included: intrinsic growth rate, cell volume, cellular particulate organic carbon and nitrogen, cellular particulate inorganic carbon, cellular chlorophyll a, and cellular particulate dimethylsulfoniopropionate.

Emiliania huxleyi strains:

Strain NCMA 2668, calcifying phenotype, isolated from Gulf of Maine 2002
Strain NCMA 374, non-calcifying phenotype, isolated from Gulf of Maine 1990

Related Datasets:
Emiliania huxleyi Chl-a, POC, cell volumes
Emiliania huxleyi CN content
Emiliania huxleyi dilution calculations
Emiliania huxleyi growth rates

Culturing conditions:

Cultures of E. huxleyi StrainNCMA 2668 and 374 were innoculated at low cell density into media prepared from autoclaved filtered seawater with f/50 nutrient ammendment.  Cell populations 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 described below) 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 15^°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 maintianed in this fashion for 14 days.  This experiment was carried out twice, in 2011 and 2012. 

CO₂ chemistry:

pCO₂ throughout the course of the experiment was calculated using CO2sys program, with pH and total alkalinity as variables and using Millero constants.  pH was measured using a Metrohm 888 Titrando with a Metrohm Ecotrode combined electrode calibrated with TRIS and AMP buffers on the total H⁺ ion pH scale.

Total alkalinity was measured with a Metrohm 888 Titrando with seawater buffers prepared by combining prepared sea salts and HCl with 2-amino-2-hydroxymethyl-1,3-propanediol and 2-aminopyridine.

Intrinsic growth rate:

Daily cell counts were made using a BD FACSCalibur flow cytometer.  Manual counts were done on select samples using a hemocytometer.  Manual counts were consistently within 5% of flow cytometry counts.  Intrinsic growth rate was calculated using exponential growth equation.

Additional results:
Stats testing differences in growth between strains: ANOVA

Cell size:

Live cells were imaged using an Olympus CH30 compound microscope networked to a Photometric CoolSNAP camera.  Cell diameter was measured using ImageJ software, and cell volume was calculated using standard geometric equations.

Cellular chlorophyll a:

Chlorophyll a samples were extracted for 24 h in acetone under -20 °C.  Chlorophyll a was measured using a Turner Designs 10-AU fluorometer.  The acidifying equations of Parsons were used to convert raw fluorescence into chlorophyll a concentration.

Cellular carbon and nitrogen:

Samples for cellular particulate carbon and nitrogen were analyzed using a CE Elantech Flash EA 1112 elemental analyzer.  In all analysis blanks were run, and internal standards were inserted between samples, and remained within 1% of standard curve.  For the calcifying strain (2668), samples were acid fumed for 24 h to drive off PIC.  Values of organic carbon were subtracted from total carbon to yield cellular particulate inorganic carbon.

Cellular particulate DMSP:

A Shimadzu GC-14A gas chromatograph was used to measure cellular particulate DMSP. Standards were prepared using DMSP Cl.

Data are compiled to show averages and standard deviations by day and treatment. 

Relevant References:

Wuori, Tristen, "The effects of elevated PCO2 on the physiology of Emiliania huxleyi" (2012). Western Washington University Masters Thesis Collection. Paper 235. http://cedar.wwu.edu/wwuet/235/

Description from NSF award abstract:
The calcifying Haptophyte Emiliania huxleyi appears to be acutely sensitive to the rising concentration of ocean pCO2. Documented responses by E. huxleyi to elevated pCO2 include modifications to their calcification rate and cell size, malformation of coccoliths, elevated growth rates, increased organic carbon production, lowering of PIC:POC ratios, and elevated production of the active climate gas DMS. Changes in these parameters are mechanisms known to elicit alterations in grazing behavior by microzooplankton, the oceans dominant grazer functional group. The investigators hypothesize that modifications to the physiology and biochemistry of calcifying and non-calcifying Haptophyte Emiliania huxleyi in response to elevated pCO2 will precipitate alterations in microzooplankton grazing dynamics. To test this hypothesis, they will conduct controlled laboratory experiments where several strains of E. huxleyi are grown at several CO2 concentrations. After careful characterization of the biochemical and physiological responses of the E. huxleyi strains to elevated pCO2, they will provide these strains as food to several ecologically-important microzooplankton and document grazing dynamics. E. huxleyi is an ideal organism for the study of phytoplankton and microzooplankton responses to rising anthropogenic CO2, the effects of which in the marine environment are called ocean acidification; E. huxleyi is biogeochemically important, is well studied, numerous strains are in culture that exhibit variation in the parameters described above, and they are readily fed upon by ecologically important microzooplankton.

The implications of changes in microzooplankton grazing for carbon cycling, specifically CaCO3 export, DMS production, nutrient regeneration in surface waters, and carbon transfer between trophic levels are profound, as this grazing, to a large degree, regulates all these processes. E. huxleyi is a model prey organism because it is one of the most biogeochemically influential global phytoplankton. It forms massive seasonal blooms, contributes significantly to marine inorganic and organic carbon cycles, is a large producer of the climatically active gas DMS, and is a source of organic matter for trophic levels both above and below itself. The planned controlled study will increase our knowledge of the mechanisms that drive patterns of change between trophic levels, thus providing a wider array of tools necessary to understand the complex nature of ocean acidification field studies, where competing variables can confound precise interpretation.