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    Experimental results: Chlorophyll-a, POC, and cell volume of E. huxleyi at 3 pCO2 levels, 2011-2012 (E Hux Response to pCO2 project)
    
  
  
    
    

Experimental results: Chlorophyll-a, POC, and cell volume of E. huxleyi at 3 pCO2 levels, 2011-2012 (E Hux Response to pCO2 project)

Website: https://www.bco-dmo.org/dataset/521347
Data Type: experimental
Version: 2014-07-03

Project
» Planktonic interactions in a changing ocean: Biological responses of Emiliania huxleyi to elevated pCO2 and their effects on microzooplankton (E Hux Response to pCO2)
ContributorsAffiliationRole
Olson, Brady M.Western Washington University - Shannon Point Marine Center (SPMC)Principal Investigator, Contact
Love, BrookeWestern Washington University (WWU)Co-Principal Investigator
Strom, SuzanneWestern Washington University - Shannon Point Marine Center (SPMC)Co-Principal Investigator
Wuori, TristenWestern Washington University (WWU)Student
Copley, NancyWoods Hole Oceanographic Institution (WHOI BCO-DMO)BCO-DMO Data Manager


Dataset Description

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 CN content
Emiliania huxleyi dilution calculations
Emiliania huxleyi DMSP
Emiliania huxleyi growth rates


Acquisition Description

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.


Processing Description

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

Relevant References:

Wuori, T. 2012. The effects of elevated pCO2 on the physiology of Emiliania huxleyi. M.S. Thesis, Western Washington University. http://cedar.wwu.edu/wwuet/235/


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Parameters

ParameterDescriptionUnits
strainEmiliania huxleyi stain id unitless
datedate of analysis yyyy-mm-dd
samplesample id unitless
Fofluorometric reading of the non acidified sample unitless
Fafluorometric reading of the acidified sample unitless
vol_filtvolume filtered ml
chl_achorophyll-a concentration ug/ml
Fo_to_Faratio of chlorophyll-a to phaeopigment based on fluorometric readings of a non acidified (Fo) and acidified (Fa) samples unitless
daysdays since start of experiment days
cell_concentrationEmiliania huxleyi cell concentration cells/ml
chl_a_cellchorophyll-a concentration per E.hux cell picograms chla/cell
chla_meanmean chorophyll-a concentration per E.hux cell for the pCO2 level picograms chla/cell
chla_stdevstandard deviation of chorophyll-a concentration per E.hux cell for the pCO2 level picograms chla/cell
pcent_changepercent change of chl-a from the control pCO2 level percent
change_avgmean percent change of chl-a from the control to treatment pCO2 level percent
change_stdevstandard deviation of the percent change of chl-a from the control to treatment pCO2 level percent
vol_cellaverage cell volume of replicates um3
fg_chla_um3chlorophyll-a per unit volume fg chla/um3
mean_fg_chla_um3average chlorophyll per unit volume fg chla/um3
stdev_fg_chla_um3standard deviation of the average chlorophyll per unit volume fg chla/um3
pcent_change_fg_chla_um3percent change of the chlorophyll per unit volume between the control and treatment pCO2 percent
mean_pcent_change_fg_chla_um3average of the percent change of the chlorophyll per unit volume between the control and treatment pCO2 percent
stdev_pcent_change_fg_chla_um3standard deviation of the percent change of the chlorophyll per unit volume between the control and treatment pCO2 percent
POC_cellparticulate organic carbon per cell pg C/cell
pg_chla_pg_Cratio of chlorophyll-a to carbon unitless
mean_pg_chla_pg_Caverage ratio of chlorophyll-a to carbon unitless
stdev_pg_chla_pg_Cstandard deviation of ratio of chlorophyll-a to carbon unitless
pcent_change_pg_chla_pg_Cpercent change of chlorophyll-a per carbon between control and treatment pCO2 levels percent
mean_pcent_change_pg_chla_pg_Caverage percent change of chlorophyll-a per carbon between control and treatment pCO2 levels percent
stdev_pcent_change_pg_chla_pg_Cstandard deviation of percent change of chlorophyll-a per carbon between control and treatment pCO2 levels percent
fmol_chla_cellchlorophyll-a per cell fmol chl/cell
fmol_POC_cellparticulate organic carbon per cell fmol POC/cell
fmol_chl_POCratio of chlorophyll-a to particulate organic carbon unitless
mean_fmol_chl_POCaverage ratio of chlorophyll-a to particulate organic carbon unitless
stdev_fmol_chl_POCstandard deviation of ratio of chlorophyll-a to particulate organic carbon unitless
commentscomments unitless


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Instruments

Dataset-specific Instrument Name
Fluorometer
Generic Instrument Name
Fluorometer
Dataset-specific Description
Turner Designs 10-AU fluorometer
Generic Instrument Description
A fluorometer or fluorimeter is a device used to measure parameters of fluorescence: its intensity and wavelength distribution of emission spectrum after excitation by a certain spectrum of light. The instrument is designed to measure the amount of stimulated electromagnetic radiation produced by pulses of electromagnetic radiation emitted into a water sample or in situ.

Dataset-specific Instrument Name
camera
Generic Instrument Name
Camera
Dataset-specific Description
Photometrics CoolSNAP camera, networked to microscope
Generic Instrument Description
All types of photographic equipment including stills, video, film and digital systems.

Dataset-specific Instrument Name
CHN_EA
Generic Instrument Name
CHN Elemental Analyzer
Dataset-specific Description
CE Elantech Flash EA 1112 elemental analyzer
Generic Instrument Description
A CHN Elemental Analyzer is used for the determination of carbon, hydrogen, and nitrogen content in organic and other types of materials, including solids, liquids, volatile, and viscous samples.

Dataset-specific Instrument Name
Flow Cytometer
Generic Instrument Name
Flow Cytometer
Dataset-specific Description
BD FACSCalibur flow cytometer
Generic Instrument Description
Flow cytometers (FC or FCM) are automated instruments that quantitate properties of single cells, one cell at a time. They can measure cell size, cell granularity, the amounts of cell components such as total DNA, newly synthesized DNA, gene expression as the amount messenger RNA for a particular gene, amounts of specific surface receptors, amounts of intracellular proteins, or transient signalling events in living cells. (from: http://www.bio.umass.edu/micro/immunology/facs542/facswhat.htm)

Dataset-specific Instrument Name
Gas Chromatograph
Generic Instrument Name
Gas Chromatograph
Dataset-specific Description
Shimadzu GC-14A gas chromatograph
Generic Instrument Description
Instrument separating gases, volatile substances, or substances dissolved in a volatile solvent by transporting an inert gas through a column packed with a sorbent to a detector for assay. (from SeaDataNet, BODC)

Dataset-specific Instrument Name
Automatic titrator
Generic Instrument Name
Automatic titrator
Dataset-specific Description
Metrohm 888 Titrando with a Metrohm Ecotrode combined electrode, calibrated with TRIS and AMP buffers on the total H+ ion pH scale.
Generic Instrument Description
Instruments that incrementally add quantified aliquots of a reagent to a sample until the end-point of a chemical reaction is reached.

Dataset-specific Instrument Name
spectrophotometer
Generic Instrument Name
Spectrophotometer
Dataset-specific Description
Agilent 5480 UV-VIS spectrophotometer (+/- 0.02)
Generic Instrument Description
An instrument used to measure the relative absorption of electromagnetic radiation of different wavelengths in the near infra-red, visible and ultraviolet wavebands by samples.

Dataset-specific Instrument Name
compound microscope
Generic Instrument Name
Microscope-Optical
Dataset-specific Description
Olympus CH30 compound microscope networked to a Photometric CoolSNAP camera
Generic Instrument Description
Instruments that generate enlarged images of samples using the phenomena of reflection and absorption of visible light. Includes conventional and inverted instruments. Also called a "light microscope".

Dataset-specific Instrument Name
MFC
Generic Instrument Name
Mass Flow Controller
Dataset-specific Description
Sierra Instruments
Generic Instrument Description
Mass Flow Controller (MFC) - A device used to measure and control the flow of fluids and gases

Dataset-specific Instrument Name
CO2 Analyzer
Generic Instrument Name
CO2 Analyzer
Dataset-specific Description
Licor 820: a non-dispersive infrared CO2 sensor
Generic Instrument Description
Measures atmospheric carbon dioxide (CO2) concentration.


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Deployments

Lab_Olson_B

Website
Platform
WWU
Start Date
2011-03-31
End Date
2016-09-15
Description
laboratory experiments


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Project Information

Planktonic interactions in a changing ocean: Biological responses of Emiliania huxleyi to elevated pCO2 and their effects on microzooplankton (E Hux Response to pCO2)


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.


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Funding

Funding SourceAward
NSF Division of Ocean Sciences (NSF OCE)

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This document is created by info v 4.1f 5 Oct 2018 from the content of the BCO-DMO metadata database.    2020-02-21  22:19:50