Pleurochrysis carterae growth cycle culture dynamics analyzed at Bigelow Laboratory from 2013 (OA Copes Coccoliths project)

Data Type: experimental
Version: 1
Version Date: 2016-09-29

» Effects of ocean acidification on Emiliania huxleyi and Calanus finmarchicus; insights into the oceanic alkalinity and biological carbon pumps (OA_Copes_Coccoliths)

» Science, Engineering and Education for Sustainability NSF-Wide Investment (SEES): Ocean Acidification (formerly CRI-OA) (SEES-OA)
Balch, William M.Bigelow Laboratory for Ocean SciencesPrincipal Investigator, Contact
Fields, DavidBigelow Laboratory for Ocean SciencesCo-Principal Investigator
White, MeredithBigelow Laboratory for Ocean SciencesContact
Ake, HannahWoods Hole Oceanographic Institution (WHOI BCO-DMO)BCO-DMO Data Manager

Pleurochrysis carterae growth cycle culture dynamics analyzed at Bigelow Laboratory from 2013 (OA Copes Coccoliths project)


Temporal Extent: 2014-04-22 - 2014-05-06

Dataset Description

Growth cycle (14 day) culture dynamics of Pleurochrysis carterae (NCMA strain 645). It was isolated from 41.525 degrees North, 70.6736 degrees West (Woods Hole, Massachusetts USA), but has been maintained in culture since 1958.

Acquisition Description

Cultures: Pleurochrysis carterae cultures were maintained in exponential growth phase under axenic conditions in semi-continuous batch culture using L1-Si media prepared on 0.2 um-filtered, UV-sterilized, autoclaved seawater.  Cultures were acclimated to one of three pCO2 treatments for > 9 generations before experiments were performed.  Cultures were maintained in an incubator at 16.5 +/- 0.5 degrees C and 470 umol photons/m-2/s PAR on a 14-10 light-dark cycle where the lights turned on at 6 am and turned off at 8 pm.

pCO2 Treatments: Carbonate chemistry was manipulated by bubbling cultures and prepared media with 500 mL/min with 0.2 um-filtered 280, 380, or 750 ppm pCO2 air.  The pCO2 levels of the treatment air were established using two mass flow controllers (Aalborg, Orangeburg, NY, USA) for each treatment to precisely mix in-house compressed air and pure CO2 (Maine Oxy, Auburn, ME, USA).  The in-house compressed air was stripped of CO2 to less than 10 ppm CO2 using a Puregas VCD CO2 Adsorber (Puregas, LLC, Broomfield, CO, USA).  The pCO2 of the gas mixtures was stable to +/- 8 ppm.  pCO2 values of the cultures may be different than the target levels due to biological activity.

Growth Cycle (14 d) Culture Dynamics Monitoring:  To understand the chemical and biological culture dynamics over an entire growth cycle (14 days, where days 1-9 represented exponential growth), the investigators took measurements of one culture from each pCO2 treatment every day at approximately the same time of day for one full growth cycle.  Our pH electrode malfunctioned on days 7 and 8, and the total alkalinity sample from the 750 ppm treatment on day 8 was accidentally dropped and broken, so calculations of full carbonate chemistry parameters for days 7 and 8 were not possible.

pH Measurements:  The pH of the cultures was measured using an OrionTM ROSSTM electrode connected to an Orion StarTM A211 Benchtop pH meter (ThermoFisher Scientific, Waltham, MA, USA), calibrated with NBS buffers (EK Industries, Inc., Joliet, IL, USA) and corrected to the total scale using weekly spectrophotometric pH measurements of culture samples.  Spectrophotometric pH measurements of 0.2 um-filtered culture samples were made with 20 mM m-Cresol purple sodium salt indicator dye (Alfa Aesar, Ward Hill, MA, USA) using a Hitachi U-3010 spectrophotometer (Hitachi High-Technologies, Clarksburg, MD, USA) equipped with a water circulated cell holder connected to a VWR 1160 water bath (VWR, Radnor, PA, USA) set at 16.5 degrees C, holding a 1 cm quartz cell.  The method followed the procedure described by Clayton and Byrne (1993) and Dickson et al. (2007), using the refit equation of Liu et al. (2011), resulting in a resolution of +/- 0.004 pH units.  

Temperature: Temperature measurements were made with an OrionTM ROSSTM electrode connected to an Orion StarTM A211 Benchtop pH meter (ThermoFisher Scientific, Waltham, MA, USA).

Salinity:  Salinity was measured using an Acorn SALT 6 handheld salinity meter (Oakton Instruments, Vernon Hills, IL, USA) with a resolution of +/- 0.1 ppt.

in vivo Fluorescence:  Fluoresence was measured using a Turner 10-AU fluorometer (Turner Designs, Sunnyvale, CA, USA).

Cell density and cell diameter: Culture density and mean cell diameter were measured using a Moxi Z mini automated cell counter (ORFLO Technologies, Ketchum, ID, USA), which has a coefficient of variation of 4%.

Particulate Inorganic Carbon:  Bulk culture PIC analyses followed the technique of Fernandez et al. (1993): 10 mL culture samples were filtered onto 0.4 um polycarbonate filters and rinsed with potassium borate buffer with the pH adjusted to 8.0 to remove seawater calcium chloride.  Filters were carefully moved to trace-metal free centrifuge tubes and digested with 5 mL of 5% nitric acid.  The calcium concentration was measured using a Jobin Yvon Ultima C inductively coupled plasma-atomic emission spectrometer (ICP-AES, HORIBA, Ltd., Kyoto, Japan).  Bulk culture PIC measurements were corrected to PIC/cell using the corresponding cell density measurements.

Particulate Organic Carbon:  To determine the bulk culture POC concentration, 10 mL of culture were filtered onto a pre-combusted Whatman GF/F filter, which was then fumed in 10 % HCl to remove inorganic carbonates.  The dried filters were then analyzed on an ECS 4010 CHNSO Analyzer (Costech Analytical Technologies, Valencia, CA, USA) by Bigelow Analytical Services, East Boothbay, ME, USA.  Bulk culture POC measurements were corrected to POC/cell using the corresponding cell density measurements.

Nutrients:  Culture samples were filtered to 0.2 um to remove all algal cells and coccoliths, and samples were frozen prior to analysis.  Total N (nitrate + nitrite), nitrite, phosphate, and silicate were measured by Continuous Flow Analysis by Bigelow Analytical Services using a SEAL AutoAnalyzer 3 HR (SEAL Analytical Inc., Mequon, WI, USA).  

Total Alkalinity:  Culture samples were filtered to 0.2 um to remove all algal cells and coccoliths.  Total alkalinity was measured via titration with 0.01 N HCl using a Metrohm Titrando 888 controlled by Tiamo software (Metrohm, Riverview, FL, USA) to perform automated Gran titrations of 4 mL samples.  Titrations were corrected to Certified Reference Materials (supplied by the laboratory of Andrew Dickson, Scripps Institution of Oceanography, La Jolla, CA, USA).

Processing Description

Nitrate:  To determine the nitrate concentration of our cultures, Total N (nitrate+nitrite) and nitrite concentrations were measured and the nitrate concentration was calculated as:

Nitrate = Total N - Nitrite

Carbonate Chemistry Calculations:  Using the measured values of pH (total scale), total alkalinity, temperature, salinity, phosphate, and silicate, the investigators used CO2SYS software (Pierrot et al. 2006) to calculate dissolved inorganic carbon (DIC), pCO2, [HCO3-], [CO32-], [CO2], and calcite using the first and second dissociation constants (K1 and K2) of carbonic acid in seawater from Mehrbach et al. (1973), refit by Dickson and Millero (1987); KHSO4 from Dickson (1990); and [B]T from Uppstrom et al. (1974).  Full carbonate chemistry could not be calculated for days 7 and 8, since the pH meter malfunctioned.

DMO notes:
- added underscores and removed spaces and units from column names
- changed column names to comply with BCO-DMO standards.
- replaced all "na" with "nd"

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Related Publications

Clayton, T. D., & Byrne, R. H. (1993). Spectrophotometric seawater pH measurements: total hydrogen ion concentration scale calibration of m-cresol purple and at-sea results. Deep Sea Research Part I: Oceanographic Research Papers, 40(10), 2115–2129. doi:10.1016/0967-0637(93)90048-8
Dickson, A. G. (1990). Standard potential of the reaction: AgCl(s) + 1/2 H2(g) = Ag(s) + HCl(aq) and the standard acidity constant of the ion HSO4− in synthetic sea water from 273.15 to 318.15 K. The Journal of Chemical Thermodynamics, 22(2), 113–127. doi:10.1016/0021-9614(90)90074-z
Dickson, A. G., & Millero, F. J. (1987). A comparison of the equilibrium constants for the dissociation of carbonic acid in seawater media. Deep Sea Research Part A. Oceanographic Research Papers, 34(10), 1733–1743. doi:10.1016/0198-0149(87)90021-5
Dickson, A.G., Sabine, C.L. and Christian, J.R. (Eds.) 2007. Guide to best practices for ocean CO2 measurements. PICES Special Publication 3, 191 pp. ISBN: 1-897176-07-4. URL:
Fernández, E., Boyd, P., Holligan, P., & Harbour, D. (1993). Production of organic and inorganic carbon within a large-scale coccolithophore bloom in the northeast Atlantic Ocean. Marine Ecology Progress Series, 97, 271–285. doi:10.3354/meps097271
Liu, X., Patsavas, M. C., & Byrne, R. H. (2011). Purification and Characterization of meta-Cresol Purple for Spectrophotometric Seawater pH Measurements. Environmental Science & Technology, 45(11), 4862–4868. doi:10.1021/es200665d
Mehrbach, C., Culberson, C. H., Hawley, J. E., & Pytkowicx, R. M. (1973). MEASUREMENT OF THE APPARENT DISSOCIATION CONSTANTS OF CARBONIC ACID IN SEAWATER AT ATMOSPHERIC PRESSURE1. Limnology and Oceanography, 18(6), 897–907. doi:10.4319/lo.1973.18.6.0897
Pierrot, D. E. Lewis,and D. W. R. Wallace. 2006. MS Excel Program Developed for CO2 System Calculations. ORNL/CDIAC-105a. Carbon Dioxide Information Analysis Center, Oak Ridge National Laboratory, U.S. Department of Energy, Oak Ridge, Tennessee. doi: 10.3334/CDIAC/otg.CO2SYS_XLS_CDIAC105a.
Uppström, L. R. (1974). The boron/chlorinity ratio of deep-sea water from the Pacific Ocean. Deep Sea Research and Oceanographic Abstracts, 21(2), 161–162. doi:10.1016/0011-7471(74)90074-6

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pCO2_treatmentThe independent variable; one of three pCO2 levels (280 ppm, 380 ppm, or 750 ppm). These treatment levels are nominal values as they represent the target pCO2 for each treatment. Within each pCO2 treatment there are 14 days worth of culture measurements for the algal cultures. Measurements of the media (without algae) were made on days 0 and 14. Only chemical measurements were made on the media not biological measurements. parts per million (ppm)
dateThe date the measurement was taken; YYYY/mm/dd unitless
dayThe time elapsed since the beginning of the growth cycle. unitless
timeThe actual time that the measurements for that timepoint began; HH:MM unitless
pHpH measured on the total scale. pH
temperatureTemperature celsius
salinitySalinity practical salnity unit (PSU)
inVivo_fluorescencefluorescence to indicate relative chlorophyll-a concentration. relative fluorescence units
cell_densitycell density of the culture, as measured by the Moxi Z Automated Cell Counter. cells per milliliter (cells/mL)
mean_cellDiametermean cell diameter as measured by the Moxi Z Automated Cell Counter. microns (um)
fluorescencePerCellDensityFluorescence divided by cell density to give an estimate of fluorescence per cell. cells per milliliter (cells/mL)-1
PIC_ugCPerLParticulate inorganic carbon concentration ugC/L
PIC_pgCPerCellParticulate inorganic carbon concentration with unit conversion to pg/mL and divided by the cell density to give pg C per cell. pgC/cell
POC_ugCPermLParticulate organic carbon concentration ugC/mL
POC_pgCPerCellParticulate organic carbon concentration with unit conversion to pg/mL and divided by the cell density to give pg C per cell. pgC/cell
NO2Nitrite concentration. umol/kg
NO3_NO2Nitrate + Nitrite concentration. umol/kg
NO3Nitrate concentration calculated from the total N and nitrite measurements. umol/kg
PO4Phosphate concentration. umol/kg
SiO4Silicate concentration. umol/kg
mean_SiO4Silicate concentration estimated as the average of the values from all the measured values. Biologically there should be no change in silicate concentration because coccolithophores do not take up silicate. However silicate values are needed to calculate the carbonate chemistry parameters in CO2sys so the average value was used for days when silicate concentrations were not measured. umol/kg
TATotal alkalinity ueq/kg
DICDissolved inorganic carbon concentration calculated using CO2sys. umol/kg
pCO2Partial pressure of carbon dioxide in the water calculated using CO2sys. This calculated value more accurately represents the pCO2 of the culture than the nominal treatment value which represents the pCO2 of the air bubbled into the culture. uatm
HCO3Bicarbonate concentration calculated using CO2sys. umol/kg
CO3Carbonate concentration calculated using CO2sys. umol/kg
CO2Carbon dioxide concentration calculated using CO2sys. umol/kg
omega_calciteSaturation state of calcium carbonate with respect to calcite calculated using CO2sys. unitless
ISO_DateTime_UTCDate/Time (UTC) ISO formatted; YYYY/mm/dd;HH:MM:SS unitless

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Dataset-specific Instrument Name
SEAL AutoAnalyzer 3 HR
Generic Instrument Name
Nutrient Autoanalyzer
Dataset-specific Description
Continuous flow analysis performed by Bigelow Analytical Services (SEAL Analytical Inc., Mequon, WI, USA).
Generic Instrument Description
Nutrient Autoanalyzer is a generic term used when specific type, make and model were not specified. In general, a Nutrient Autoanalyzer is an automated flow-thru system for doing nutrient analysis (nitrate, ammonium, orthophosphate, and silicate) on seawater samples.

Dataset-specific Instrument Name
Orion ROSS electrode
Generic Instrument Name
pH Sensor
Dataset-specific Description
Orion ROSS electrode was connected to an Orion Star A211 Benchtop pH meter (ThermoFisher Scientific, Waltham, MA, USA)
Generic Instrument Description
An instrument that measures the hydrogen ion activity in solutions. The overall concentration of hydrogen ions is inversely related to its pH.  The pH scale ranges from 0 to 14 and indicates whether acidic (more H+) or basic (less H+). 

Dataset-specific Instrument Name
Metrohm Titrando 888
Generic Instrument Name
Automatic titrator
Dataset-specific Description
Automatic titrations controlled by Tiamo software (Metrohm, Riverview, FL, USA).
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
Hitachi U-3010 spectrophotometer
Generic Instrument Name
Dataset-specific Description
Spectrophotometric pH measurements were taken of culture samples
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
Acorn SALT 6 handheld salinity meter
Generic Instrument Name
Salinity Sensor
Dataset-specific Description
Salinity measured using this instrument with a resolution of +/- 0.1 ppt (Oakton Instruments, Vernon Hills, IL, USA)
Generic Instrument Description
Category of instrument that simultaneously measures electrical conductivity and temperature in the water column to provide temperature and salinity data.

Dataset-specific Instrument Name
Aalborg Mass Flow Controller
Generic Instrument Name
Mass Flow Controller
Dataset-specific Description
Indicate and control set flow rates of gases. Manufactured in Orangeburg, NY USA.
Generic Instrument Description
Mass Flow Controller (MFC) - A device used to measure and control the flow of fluids and gases

Dataset-specific Instrument Name
Jobin Yvon Ultima C
Generic Instrument Name
Inductively Coupled Plasma Optical Emission Spectrometer
Dataset-specific Description
Calcium concentration measured (ICP-AES, HORIBA, Ltd., Kyoto, Japan).
Generic Instrument Description
Also referred to as an Inductively coupled plasma atomic emission spectroscope (ICP-AES). These instruments pass nebulised samples into an inductively-coupled gas plasma (8-10000 K) where they are atomised and excited. The de-excitation optical emissions at characteristic wavelengths are spectroscopically analysed. It is often used in the detection of trace metals.

Dataset-specific Instrument Name
Puregas VCD CO2 Adsorber
Generic Instrument Name
CO2 Adsorber
Dataset-specific Description
Instrument stripped compressed air of CO2
Generic Instrument Description
CO2 Adsorber - an instrument designed to remove CO2 and moisture from compressed air.

Dataset-specific Instrument Name
Moxi Z Automated Cell Counter
Generic Instrument Name
Automated Cell Counter
Dataset-specific Description
Measures culture density
Generic Instrument Description
Automated Cell Counter (ACC) - a tool used for counting live and/or dead cells in a culture.  It can also be used to size particles.

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lab Bigelow
Start Date
Laboratory located at Bigelow Laboratory for Ocean Sciences

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Project Information

Effects of ocean acidification on Emiliania huxleyi and Calanus finmarchicus; insights into the oceanic alkalinity and biological carbon pumps (OA_Copes_Coccoliths)

Coverage: Laboratory experiments; East Boothbay, Maine

(Extracted from the NSF award abstract)

Ocean acidification is one of the most pressing marine science issues of our time, with potential biological impacts spanning all marine phyla and potential societal impacts affecting man's relationship to the sea. Rising levels of atmospheric pCO2 are increasing the acidity of the world oceans. It is generally held that average surface ocean pH has already declined by 0.1 pH units relative to the pre-industrial level (Orr et al., 2005), and is projected to decrease 0.3 to 0.46 units by the end of this century, depending on CO2 emission scenarios (Caldeira and Wickett, 2005). The overall goal of this research is to parameterize how changes in pCO2 levels could alter the biological and alkalinity pumps of the world ocean. Specifically, the direct and indirect effects of ocean acidification will be examined within a simple, controlled predator/prey system containing a single prey phytoplankton species (the coccolithophore, Emiliania huxleyi) and a single predator (the oceanic metazoan grazer, Calanus finmarchicus). The experiments are designed to elucidate both direct effects (i.e. effects of ocean acidification on the individual organisms only) and interactive effects (i.e. effects on the combined predator/prey system). Interactive experiments with phytoplankton prey and zooplankton predator are a critical starting point for predicting the overall impact of ocean acidification in marine ecosystems. To meet these goals, a state-of-the-art facility will be constructed with growth chambers that are calibrated and have highly-controlled pH and alkalinity levels. The strength of this approach lies in meticulous calibration and redundant measurements that will be made to ensure that conditions within the chambers are well described and tightly monitored for DIC levels. Growth and calcification rates in coccolithophores and the developmental rates, morphological and behavioral effects on copepods will be measured. The PIC and POC in the algae and the excreted fecal pellets will be monitored for changes in the PIC/POC ratio, a key parameter for modeling feedback mechanisms for rising pCO2 levels. In addition, 14C experiments are planned to measure calcification rates in coccolithophores and dissolution rates as a result of grazing. These key experiments will verify closure in the mass balance of PIC, allowing the determination of actual dissolution rates of PIC within the guts of copepod grazers.

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Program Information

Science, Engineering and Education for Sustainability NSF-Wide Investment (SEES): Ocean Acidification (formerly CRI-OA) (SEES-OA)

Coverage: global

NSF Climate Research Investment (CRI) activities that were initiated in 2010 are now included under Science, Engineering and Education for Sustainability NSF-Wide Investment (SEES). SEES is a portfolio of activities that highlights NSF's unique role in helping society address the challenge(s) of achieving sustainability. Detailed information about the SEES program is available from NSF (

In recognition of the need for basic research concerning the nature, extent and impact of ocean acidification on oceanic environments in the past, present and future, the goal of the SEES: OA program is to understand (a) the chemistry and physical chemistry of ocean acidification; (b) how ocean acidification interacts with processes at the organismal level; and (c) how the earth system history informs our understanding of the effects of ocean acidification on the present day and future ocean.

Solicitations issued under this program:
NSF 10-530, FY 2010-FY2011
NSF 12-500, FY 2012
NSF 12-600, FY 2013
NSF 13-586, FY 2014
NSF 13-586 was the final solicitation that will be released for this program.

PI Meetings:
1st U.S. Ocean Acidification PI Meeting(March 22-24, 2011, Woods Hole, MA)
2nd U.S. Ocean Acidification PI Meeting(Sept. 18-20, 2013, Washington, DC)
3rd U.S. Ocean Acidification PI Meeting (June 9-11, 2015, Woods Hole, MA – Tentative)

NSF media releases for the Ocean Acidification Program:

Press Release 10-186 NSF Awards Grants to Study Effects of Ocean Acidification

Discovery Blue Mussels "Hang On" Along Rocky Shores: For How Long?

Discovery - National Science Foundation (NSF) Discoveries - Trouble in Paradise: Ocean Acidification This Way Comes - US National Science Foundation (NSF)

Press Release 12-179 - National Science Foundation (NSF) News - Ocean Acidification: Finding New Answers Through National Science Foundation Research Grants - US National Science Foundation (NSF)

Press Release 13-102 World Oceans Month Brings Mixed News for Oysters

Press Release 13-108 - National Science Foundation (NSF) News - Natural Underwater Springs Show How Coral Reefs Respond to Ocean Acidification - US National Science Foundation (NSF)

Press Release 13-148 Ocean acidification: Making new discoveries through National Science Foundation research grants

Press Release 13-148 - Video - News - Video - NSF Ocean Sciences Division Director David Conover answers questions about ocean acidification. - US National Science Foundation (NSF)

Press Release 14-010 - National Science Foundation (NSF) News - Palau's coral reefs surprisingly resistant to ocean acidification - US National Science Foundation (NSF)

Press Release 14-116 - National Science Foundation (NSF) News - Ocean Acidification: NSF awards $11.4 million in new grants to study effects on marine ecosystems - US National Science Foundation (NSF)

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Funding SourceAward
NSF Division of Ocean Sciences (NSF OCE)

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