Results from 14C-labeled uptake experiments determining uptake of specific dissolved organic compounds which showed high potential for osmotrophy

Website: https://www.bco-dmo.org/dataset/858771
Data Type: experimental
Version: 2
Version Date: 2021-10-05

Project
» Coccolithophore Mixotrophy (Cocco-Mix)
ContributorsAffiliationRole
Balch, William M.Bigelow Laboratory for Ocean SciencesPrincipal Investigator
Godrijan, JelenaContact
Drapeau, David T.Bigelow Laboratory for Ocean SciencesAnalyst
Rauch, ShannonWoods Hole Oceanographic Institution (WHOI BCO-DMO)BCO-DMO Data Manager

Abstract
This dataset includes results for 14C-labeled uptake experiments determining uptake of specific dissolved organic compounds which showed high potential for osmotrophy. Experiments used the BioLog Eco-plates (BioLog, Haywood, CA, U.S.A.) and were conducted at Bigelow Laboratory for Ocean Sciences, East Boothbay, ME.


Coverage

Spatial Extent: Lat:43.8597 Lon:-69.5802
Temporal Extent: 2017-09-06 - 2018-01-27

Acquisition Description

We investigated the uptake of specific dissolved organic compounds, which showed high potential for osmotrophy. We selected five ¹⁴C-labeled-DOC compounds based on results of the BioLog Eco-plates survey as well as commercial availability of radiotracer-labeled compounds. The selected compounds included sugar alcohols (glycerol and mannitol), carbohydrate (xylose), and amino-acid (arginine). Additionally, we selected acetate due to its biochemical importance and availability in marine ecosystems (Ho et al. 2002; Wu et al. 1997). Specific activities of the radiotracers were: glycerol - 160 µCi µmol⁻¹, mannitol - 57 µCi µmol⁻¹, xylose - 200 µCi µmol⁻¹, arginine - 338 µCi µmol⁻¹, and acetate - 52 µCi µmol⁻¹ (acetic acid sodium salt) (PerkinElmer, Inc. Waltham, MA). As a reference uptake compound we used ¹⁴C-bicarbonate (56 µCi µmol) (MP Biomedicals, LLC, Santa Ana, CA, USA) incubations in photosaturated light conditions. We performed radiolabel uptake experiments on axenic coccolithophore strains, CCMP289 Cruciplacolithus neohelis and CCMP3337 Chrysotila carterae (NCMA lists the strain as Pleurochrysis carterae). We maintained the cultures in media and light conditions as described above, and at 22°C (CCMP289) and at 16°C (CCMP3337).

For the survey of arginine and xylose net uptake in darkness, we prepared two 70 mL master samples (concentration of 1×10⁵ cells L⁻¹) of CCMP289 and CCMP3337 cultures in log phase growth. We measured cell concentrations using a haemocytometer on an American Optical Microscope (Spencer Lens Company, Buffalo, N.Y.) with polarization optics. We added unlabeled arginine or xylose to each strain’s master sample up to a 20 µM final concentration. From each master sample, 10 mL were then removed into separate borosilicate vials that were kept in the dark for subsequent cell counts over the duration of the experiment. To the remaining 60 mL culture samples containing unlabeled arginine or xylose, we added ¹⁴C-arginine or ¹⁴C-xylose, to a final concentration (labeled and unlabeled) of 20.25 µM and 20.83 µM, respectively. We withdrew 45 mL of the 60 mL sample and divided that into three 15 mL replicate vials. We transferred the remaining 15 mL into a fourth vial with buffered formalin as a formalin-killed control. Due to logistical issues in sample manipulation, the actual time of addition of the first ¹⁴C-labeled compound was 10±5 min after addition of formalin to the labeled control. We then subsampled and filtered all 16 vials (12 samples (triplicates of the two ¹⁴C-labeled compounds x two strains) and 4 formalin samples (two compounds x two strains)). After the first time point, we placed samples in the dark incubators at 22°C for CCMP289 and 16°C for CCMP3337. Subsampling for each time course experiment was performed at 3 h, 6 h, 24 h, and 48 h. For subsampling, we performed filtration of each 2 mL of culture subsamples onto each 0.4 µm pore-size, 25 mm diameter polycarbonate filter. Following filtration, filters were carefully rinsed three times with ASW (including a careful rim rinse) to remove any ¹⁴C-labeled, dissolved compound left on the filter. Each filter was then placed in the bottom of a clean scintillation vial, and scintillation cocktail was added (Balch et al., 2000).

We also examined the net uptake of ¹⁴C-arginine and ¹⁴C-xylose uptake in illuminated cultures over 24 hours. We added these ¹⁴C- labeled compounds to axenic cultures (CCMP289 or CCMP3337) to a final concentration of 0.37 µM for ¹⁴C-arginine and 1 µM for ¹⁴C-xylose. We sampled at T15 min and T24 h, stopping the incubation by filtration, and measured the ¹⁴C uptake as described above.

Furthermore, we examined the net uptake of ¹⁴C-acetate, ¹⁴C-glycerol, and ¹⁴C-mannitol in darkness over 24 h and compared it with ¹⁴C-bicarbonate net uptake (in light). Prior to addition of radiolabeled compounds, axenic cultures (CCMP289 or CCMP3337) were divided into separate vials and 5 mL of log-phase culture were removed for the enumeration of cell concentration. To correct for any effects due to EtOH solvent in the ¹⁴C-acetate, in one 5 mL sample we added only 0.0125 mL of EtOH as a control. We added ¹⁴C- labeled compounds to each separate vial to a final concentration of 4.81 µM of ¹⁴C-acetate, 1.49 µM of ¹⁴C-glycerol, 4.18 µM of ¹⁴C-mannitol, and for comparison we used ¹⁴C-bicarbonate to a final concentration of 2.6 mM of labeled and unlabeled form. Triplicate samples for uptake measurements were filtered after 15 min and 24 h of darkness.


Processing Description

Data Processing:
We calculated the ¹⁴C-labeled-compound net uptake rates following the equations of Parsons et al. (1984)

v = ((RnRf) × W) / (R × T)

Where v is the net uptake rate [mol L⁻¹ h⁻¹], Rn is the sample count [dpm] at time T, Rf is the formalin-killed control count [dpm], and W [mol L⁻¹] is the concentration of available compound in the sample. R is the total activity [dpm] of the added compound to a sample and T [h] is the number of hours of incubation.

BCO-DMO Processing:
- added column for species name; 
- converted dates to YYYY-MM-DD format;
- created date-time field in ISO8601 format (UTC).


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Related Publications

Balch, W. M., Drapeau, D. T., & Fritz, J. J. (2000). Monsoonal forcing of calcification in the Arabian Sea. Deep Sea Research Part II: Topical Studies in Oceanography, 47(7-8), 1301–1337. doi:10.1016/s0967-0645(99)00145-9 https://doi.org/10.1016/S0967-0645(99)00145-9
Methods
Godrijan, J., Drapeau, D., & Balch, W. M. (2020). Mixotrophic uptake of organic compounds by coccolithophores. Limnology and Oceanography, 65(6), 1410–1421. doi:10.1002/lno.11396
Results
Ho, T.-Y., Scranton, M. I., Taylor, G. T., Varela, R., Thunell, R. C., & Muller-Karger, F. (2002). Acetate cycling in the water column of the Cariaco Basin: Seasonal and vertical variability and implication for carbon cycling. Limnology and Oceanography, 47(4), 1119–1128. doi:10.4319/lo.2002.47.4.1119
Methods
Parsons, T. R., Maita, Y., & Lalli, C.M. (1984). A manual of chemical and biological methods for seawater analysis. Pergamon Press. doi:10.1016/c2009-0-07774-5 https://doi.org/10.1016/C2009-0-07774-5
Methods
Wu, H., Green, M., & Scranton, M. (1997). Acetate Cycling in the Water Column and Surface Sediment of Long Island Sound Following a Bloom. Limnology and Oceanography, 42(4), 705-713. Retrieved August 20, 2021, from http://www.jstor.org/stable/2839116
Methods

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Parameters

ParameterDescriptionUnits
CCMP_codeStrain code (CCMP) from the National Center for Marine Algae and Microbiota (NCMA) unitless
SpeciesSpecies name unitless
SubstrateSubstrate unitless
Light_conditionsLight conditions of the experiment unitless
LatitudeLatitude; positive values = North decimal degrees North
LongitudeLongitude; positive values = East decimal degrees East
DateSampling date; format: YYYY-MM-DD unitless
TimeSampling Time; format: hh:mm:ss unitless
Time_zoneTime zone unitless
Time_PointActual elapsed time hours
Cell_countCell count cells per milliliter (cell/ml)
UptakeUptake of 14C-labeled compounds moles per liter per hour (mol/L*h)
Avg_uptakeCalculated average of uptake moles per liter per hour (mol/L*h)
Stdev_uptakeCalculated standard deviation of uptake moles per liter per hour (mol/L*h)
Avg_Net_uptakeCalculated average of net uptake picomoles per cell per hour (pmol/cell*h)
Stdev_Net_uptakeCalculated standard deviation of net uptake picomoles per cell per hour (pmol/cell*h)
ISO_DateTime_UTCDate and time converted to ISO8601 format (UTC): YYYY-MM-DDThh:mm:ssZ unitless


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Instruments

Dataset-specific Instrument Name
Tri-Carb 3110TR liquid scintillation analyzer
Generic Instrument Name
Liquid Scintillation Counter
Dataset-specific Description
Tri-Carb 3110TR liquid scintillation analyzer (PerkinElmer, Waltham, MA, USA)
Generic Instrument Description
Liquid scintillation counting is an analytical technique which is defined by the incorporation of the radiolabeled analyte into uniform distribution with a liquid chemical medium capable of converting the kinetic energy of nuclear emissions into light energy. Although the liquid scintillation counter is a sophisticated laboratory counting system used the quantify the activity of particulate emitting (ß and a) radioactive samples, it can also detect the auger electrons emitted from 51Cr and 125I samples.

Dataset-specific Instrument Name
haemocytometer
Generic Instrument Name
Hemocytometer
Generic Instrument Description
A hemocytometer is a small glass chamber, resembling a thick microscope slide, used for determining the number of cells per unit volume of a suspension. Originally used for performing blood cell counts, a hemocytometer can be used to count a variety of cell types in the laboratory. Also spelled as "haemocytometer". Description from: http://hlsweb.dmu.ac.uk/ahs/elearning/RITA/Haem1/Haem1.html.

Dataset-specific Instrument Name
American Optical Microscope
Generic Instrument Name
Microscope - Optical
Dataset-specific Description
American Optical Microscope (Spencer Lens Company, Buffalo, N.Y.) with polarization optics
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".


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Project Information

Coccolithophore Mixotrophy (Cocco-Mix)

Coverage: Partially lab-based, with field sites in Gulf of Maine and NW Atlantic between the Gulf of Maine and Bermuda


Coccolithophores are unicellular haptophyte algae generally thought of as photoautotrophs. They are covered with scales or "coccoliths" (made of calcium carbonate (particulate inorganic carbon, PIC)). Recent observations suggest that globally, haptophytes contribute more biomass than ubiquitous Prochlorococcus and Synechococcus. Coccolithophores can affect the draw-down of atmospheric CO2 and are involved in two fundamental "pump paradigms": (1) The alkalinity pump (also known as the carbonate, PIC, or CaCO3 pump) lowers total alkalinity (TA) and dissolved inorganic carbon (DIC) in the euphotic zone during calcification, and increases upper ocean and atmospheric CO2. Coccoliths eventually sink below the ocean’s lysocline (the depth where calcium carbonate dissolves), where they release the bicarbonate back into deep water. Thus, they essentially "pump" bicarbonate alkalinity from surface to benthic waters, where it remains isolated in the deep sea for thousands of years. (2) The biological pump in which the ballasting effect of the heavy coccoliths on sinking particulate organic carbon (POC) increases the magnitude of the soft tissue (POC) pump, which ultimately decreases surface CO2. The soft-tissue and alkalinity pumps reinforce each other in maintaining a vertical gradient in DIC but they oppose each other in terms of the air-sea exchange of CO2. Thus, the net effect of coccolithophores on atmospheric CO2 depends on the balance of their CO2-raising effect associated with the alkalinity pump and their CO2-lowering effect associated with the soft-tissue biological pump. It is virtually always assumed that the PIC found in coccoliths originates exclusively from DIC, not dissolved organic carbon (DOC). However, there is an increasing body of evidence that coccolithophores are mixotrophic (defined as a combination of growth fueled by autotrophy, uptake of DOC and phagotrophy of small particles (POC). This proposal is to describe the potential uptake and assimilation of an array of DOC compounds in the sea, the kinetics of their uptake and potential incorporation of organic carbon by coccolithophores into PIC coccoliths (which could significantly alter the alkalinity pump paradigm since calcite production in the surface ocean would not be at the expense of bicarbonate).

This work is fundamentally directed at quantifying coccolithophore mixotrophy in lab of technological advances to address this issue, all of which we will apply in this work. We will: (a) screen axenic coccolithophore cultures for the uptake and oxidation of a large array of potential DOC substrates, (b) perform radiolabel-uptake experiments with these molecules using high-specific activity substrates in order to provide the basic kinetic response at environmentally-realistic concentrations, (c) measure radio-labelled carbon fixed into organic tissue, separate from that fixed into PIC, (d) sort 14C-labelled coccolithophores free of the other free-living phytoplankton and bacteria using flow cytometry and e) distinguish the modes of nutrition in these sorted coccolithophore cells. This work will advance the state of knowledge of coccolithophore mixotrophy in the marine environment and address the balance of carbon that coccolithophores derived from autotrophic versus heterotrophic sources.



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Funding

Funding SourceAward
NSF Division of Ocean Sciences (NSF OCE)

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