Table of Contents

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

BioLog Eco-plates contained 96-wells, prefilled with: (1) a colorless tetrazolium dye that reduces to a violet formazin if the substrate is oxidized, (2) triplicates of 31 different organic compounds in equimolar quantities, and (3) triplicate water blanks as a control for airborne bacterial contamination. We used a single coccolithophore strain in each plate. We started each experiment by inoculating a 96-well plate with 100 μL of log-phase cell suspension from each log phase culture. We carried out all inoculations under subdued light and all incubations in complete darkness. At time-zero (T0), 24 h, 48 h, and 72 h, we measured the reduced violet tetrazolium dye absorption on a FilterMax F5 multimode plate reader (Molecular Devices, LLC, San Jose, CA, U.S.A.) for absorption at 595 nm. We interpreted the increasing optical density at 595 nm as heterotrophic metabolism of the organic compound by the coccolithophore cultures. (Note: For two strains, CCMP298 and CCMP 3337, T72 was not performed, but the measurement was instead taken at T120.)

Several limitations to the BioLog Eco-plates should be noted (Stefanowicz 2006). One limitation is that the technique assumes that the uptake of each substrate is independent from any other (i.e., two substrates are not used in a synergistic fashion). The technique also assumes the concentrations of the substrates in each well of the microtiter plate are optimal for the organism in question, rather than too high (i.e., inhibitory) or low (limiting) based on the organism's specific uptake kinetic parameters. Therefore, we could not make any inferences on the uptake kinetics of DOC utilization using the microtiter plates. Furthermore, ions in seawater (specifically Ca++) can cause false positives in BioLog Eco-plates (Pierce et al. 2014). It is critical to lower the Ca++ concentration from 10 mmol L⁻¹ (normal concentration in seawater) to ~ 2.5 mmol L⁻¹ in order to eliminate false positives (Pierce et al. 2014). The BioLog Company suggests doing this with chelators. An alternative method was suggested by Tuchman et al. (2006) in which the cultures were centrifuged and pelleted to separate them from the nutrient-rich media, which might contain other growth-inducing substrates. Then the pellet should be resuspended in nutrient-free saline solution. We followed these latter recommendations and resuspended the coccolithophores in ASW with 2.5 mmol L⁻¹ calcium.

Data Processing:
The potential utilization of each organic compound was expressed as the average compound color development (ACCD). We averaged the absorption observed at five points across the center of each well at each time point and subtracted the average water control well. We then calculated the mean absorbance of replicate wells for each compound. The ACCD was computed as the percentage of the absorbance of each compound over the sum of absorbance of all compounds on a plate.

BCO-DMO Processing:
- concatenated data from separate Excel sheets into one dataset;
- joined the strain information from original file "CCMP_cocco_strains.xlsx" to the rest of the dataset, matching on the Strain_code field;
- renamed fields to conform with BCO-DMO naming conventions;
- replaced 'n.d.' with 'nd' (no data);
- converted dates to YYYY-MM-DD format;
- removed directionals from latitude and longitude column to allow for column to be typed as numeric;
- removed comma in D,L-α-Glycerol-Phosphate in Substrate column to allow for download as a .csv;
- replaced spaces in Species column and Collection_Site_Sea column with underscores to allow for sorting;
 - rounded data as requested by data provider.

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.