Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Related Publications
• Parameters
• Instruments
• Project Information
• Funding

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.

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

v = ((R_n – R_f) × W) / (R × T)

Where v is the net uptake rate [mol L⁻¹ h⁻¹], R_n is the sample count [dpm] at time T, R_f 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).

NSF Award Abstract
Coccolithophores are single-cell algae that are covered with limestone (calcite) plates called coccoliths. They may make up most of the phytoplankton biomass in the oceans. Coccolithophores are generally considered to be autotrophs, meaning that they use photosynthesis to fix carbon into both soft plant tissue and hard minerogenic calcite, using sunlight as an energy source ("autotrophic"). However, there is an increasing body of evidence that coccolithophores are "mixotrophic", meaning that they can fix carbon from photosynthesis as well as grow in darkness by engulfing small organic particles plus taking up other simple carbon molecules from seawater. The extent to which Coccolithophores engage in mixotrophy can influence the transfer of carbon into the deep sea. This work is fundamentally directed at quantifying coccolithophore mixotrophy -- the ability to use dissolved and reduce carbon compounds for energy -- using lab and field experiments plus clarifying its relevance to ocean biology and chemistry. This work will generate broader impacts in three areas: 1) Undergraduate training: Two REU undergraduates will be trained during the project. The student in the second year will participate in the research cruise. 2) Café Scientifique program: This work will be presented in Bigelow Laboratory’s Café Scientifique program. These are free public gatherings where the public is invited to join in a conversation about the latest ideas and issues in ocean science and technology. 3) Digital E-Book: We propose to make a digital E-book to specifically highlight and explain mixotrophy within coccolithophores. Images of mixotrophic coccolithophores would be the primary visual elements of the book. The E-book will be publicly available and distributed to our educational affiliate, Colby College. The goal of the book is to further communicate the intricacies of the microbial world, food web dynamics, plus their relationship to the global carbon cycle, to inspire interest, education, and curiosity about these amazing life forms.

Coccolithophores can significantly affect the draw-down of atmospheric CO2 and they can transfer CO2 from the surface ocean and sequester it in the deep sea via two carbon pump mechanisms: (1) The "alkalinity pump" (also known as the calcium carbonate pump), where coccolithophores in the surface ocean take up dissolved inorganic carbon (DIC; primarily a form called bicarbonate, a major constituent of ocean alkalinity). They convert half to CO2, which is either fixed as plant biomass or released as the gas, and half is synthesized into their mineral coccoliths. Thus, coccolithophore calcification can actually increase surface CO2 on short time scales (i.e. weeks). However, over months to years, coccoliths sink below thousands of meters, where they dissolve and release bicarbonate back into deep water. Thus, sinking coccoliths essentially "pump" bicarbonate alkalinity from surface to deep waters, where that carbon remains isolated in the abyssal depths for thousands of years. (2) The "biological pump", where the ballasting effect of the dense limestone coccoliths speeds the sinking of organic, soft-tissue debris (particulate organic carbon or POC), essentially "pumping" this soft carbon tissue to depth. The biological pump ultimately decreases surface CO₂. The soft-tissue and alkalinity pumps reinforce each other in maintaining a vertical gradient in DIC (more down deep than at the surface) 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 coccolith particulate inorganic carbon (PIC) originates exclusively from dissolved inorganic carbon (DIC, as bicarbonate), not dissolved organic carbon (DOC). The goal of this proposal is to describe a) the potential uptake and assimilation of an array of DOC compounds by coccolithophores, b) the rates of uptake, and potential incorporation of DOC by coccolithophores into PIC coccoliths, which, if true, would represent a major shift in the alkalinity pump paradigm. This work is fundamentally directed at quantifying coccolithophore mixotrophy using lab and field experiments plus clarifying its relevance to ocean biology and chemistry. There have been a number of technological advances to address this issue, all of which will be applied in this work. The investigators will: (a) screen coccolithophore cultures for the uptake and assimilation of a large array of DOC molecules, (b) perform tracer experiments with specific DOC molecules in order to examine uptake at environmentally-realistic concentrations, (c) measure fixation of DOC into organic tissue, separately from that fixed into PIC coccoliths, (d) separate coccolithophores from other phytoplankton and bacteria using flow cytometry and e) distinguish the modes of nutrition in these sorted coccolithophore cells. This work will fundamentally advance the state of knowledge of coccolithophore mixotrophy in the sea and address the balance of carbon that coccolithophores derived from autotrophic versus heterotrophic sources.