Table of Contents

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

Bulk and cell-specific CO2 fixation and PO4 uptake from Atlantic Explorer cruise AE1524 (BATS validation cruise BV50), September 2015 in the North Atlantic subtropical gyre (September 2015), along a transect from Bermuda to Puerto Rico extending from 33°N to 22°N. Phosphate uptake rates were measured in Prochlorococcus, Synechococcus, pigmented eukaryotes, and unpigmented eukaryotes. Also reported are CO2 fixation rate by Prochlorococcus, Synechococcus, and pigmented eukaryotes. These data were published by Duhamel et al (2019), Table 3.

Seawater was collected into acid washed, ultra-pure water and sample rinsed, clear polycarbonate incubation bottles. PO4 assimilation rates were measured in triplicate 70-mL samples with ~259 kBq of added 33P-PO4 (Perkin-Elmer #NEZ08000; carrier free), incubated for 30 min to 1h. CO2 fixation rates were measured in duplicate 70-mL samples with ~17 MBq of added 14C-sodium bicarbonate (Perkin Elmer #NEC086H000, 1.6 GBq/mmol), incubated from dawn to dusk. Samples were incubated under simulated light and temperature conditions measured at the sampling site. A killed control sample was also prepared by adding paraformaldehyde (PFA, 2 % final concentration prepared with electron microscopy grade 16 % aqueous solution, Electron Microscopy Sciences) at least 15 minutes before introducing the radioisotope, in order to account for unincorporated radioactivity. At the end of incubation, samples were fixed with PFA (2% final, for 15-min in the dark), and triplicate 20-microliters aliquots were sampled to measure the total radioactivity added (with beta-phenylethylamine for 14C samples). The total microbial activity was determined by filtering a 3-mL aliquot through a 0.2-micron, pore-size polycarbonate membrane filter (Nuclepore). To reduce unincorporated 33P-PO4, the membrane filter was placed onto a filter type HA soaked in 100 mM KH2PO4, then rinsed three times with ~1 mL of 0.2-micron filtered seawater. To remove unincorporated 14C-sodium bicarbonate, the filter was acidified with 0.5 mL of 1N HCl for 24 h. To determine plankton groups specific uptake rates, a 20-mL aliquot was passed through a 0.2-micron polycarbonate membrane filter under gentle vacuum filtration, and the remaining volume from the 70-mL incubation bottle was passed through a 0.8-micron polycarbonate membrane filter. The 0.2-micron and 0.8-micron filters were stored in separate cryovials with 2 mL and 4 mL of the corresponding radiolabeled sample, respectively, vortexed to detach the cells from the filter, then flash frozen for later flow cytometric sorting (see below). The added radioactivity and total microbial activity were assayed by liquid scintillation counting in 7-mL plastic scintillation vials (Simport) with 4 mL of scintillation cocktail (Ultima Gold LLT, Perkin Elmer) added.

Turnover times (TPO4, h) were calculated by dividing the total radioactivity added (Bq L–1) by the rate of radiolabel uptake into the particulate fraction (Bq L–1 h–1). PO4 assimilation rates (nmol P L–1 h–1) were calculated by dividing PO4 concentration by TPO4. We used PO4 concentration estimated from a concentration series bioassays following the method of Wright and Hobbie (1966). Briefly, seawater samples were amended with non-radioactive PO4 to target additions of 0, 5, 10, 25, 50, 75, and 150 nmol PO4 L–1, spiked with 33P-PO4, incubated and sampled as described above. The resulting TPO4 values were plotted against a corresponding concentration of PO4, and extrapolated using linear regression (TPO4 = a x PO4 + b) to estimate the ambient concentration (Sn = b/a), which represents an upper estimate of ambient concentrations as detailed in Zubkov and Tarran (2005). Results from these bioassays were also used to calculate the Michaelis-Menten kinetic parameters for PO4 assimilation rates (Vmax, the maximum rate at saturating substrate concentration and Km, the half-saturation constant).

For cell sorting of Prochlorococcus, Synechococcus, pigmented and non-pigmented protists, the Influx flow cytometer was set at the highest sorting purity (1.0 drop single mode) and potential attached cells were discarded using a pulse width vs. forward scatter plot. The drop delay was calibrated using Accudrop Beads (BD Biosciences, USA) and verified manually by sorting a specified number of reference beads onto a glass slide and counting the beads under an epifluorescence microscope. Performance was validated as described in Duhamel et al. (2018). Three to four proportional numbers of cells from the same incubation sample were sorted for each target population. Sorted cells were assessed by liquid scintillation analysis following previously published protocols (Talarmin et al. 2011; Duhamel et al. 2012; Rii et al. 2016). The 14C-labeled samples were acidified with 1 mL of 2 mol L-1 HCl for 24 h to remove any unincorporated 14C-sodium bicarbonate.

For each group, at least three samples were sorted and regression analysis between the number of cells sorted and the radioactivity taken up by the sorted cells was used to calculate the per cell activity (dpm cell−1). Radioactivity in sorted cells from the PFA-killed control samples (dpm cell−1) was deduced from radioactivity in the sorted cells from the respective samples (dpm cell−1). The cell-specific assimilation rate (nmol cell-1 h-1) was calculated by dividing the radioactivity per cell (dpm cell−1) by the total microbial activity (dpm L−1) measured in the same sample, and then multiplied by the total microbial assimilation rate at ambient substrate concentration (nmol L−1 h−1).

Michaelis– Menten kinetic parameters were determined using the Michaelis–Menten model in Prism 6.

BCO-DMO Processing Notes:
- added conventional header with dataset name, PI name, version date
- modified parameter names to conform with BCO-DMO naming conventions
- added cruise column

This project is an NSF Collaborative Research Project.

Description from NSF award abstract:
Protists are mostly single-celled, eukaryotic microorganisms, including algae and protozoans. They are ubiquitous, diverse, and major contributors in oceanic food webs. Determining their taxonomic identity and the extent to which they contribute to carbon and nutrient cycles (whereby carbon and minerals are continuously changed chemically in the environment and reincorporated in living organisms) are among the major goals of this study. Moreover, the investigators will study how they respond to environmental change, one of the most important and challenging current problems in oceanography. Answering these questions is fundamental to understanding how living organisms in the ocean environment interact with one another and contribute to the health and productivity of the ocean. The main goal of the project is to investigate biotic interactions of small-sized protists with very tiny cyanobacteria also known as picocyanobacteria, which represent the most abundant photosynthetic organisms in the ocean. These studies will be done both in ocean environments and in laboratory experimental settings. Considering the limited knowledge on this topic, the work planned in this project promises important and exciting discoveries. Two early career female scientists will lead this project. In addition, one postdoctoral scholar, one graduate student, and at least three undergraduate summer interns will participate in the proposed research activities. The principal investigators will create a strong public outreach program that will engage middle school students in hands-on activities related to ocean sciences, and will produce a video in collaboration with the Education Department at the American Museum of Natural History. The video will summarize the major findings of the proposed research. It can be used in schools and in informal learning settings, including access by the public on the Internet through the Museum's Science Bulletins web page.

Single-celled eukaryotic microorganisms or protists, though largely outnumbered by picocyanobacteria (Prochlorococcus and Synechococcus), contribute significantly to ocean carbon biomass and primary productivity, partially by virtue of their larger cell size. In addition, small planktonic protists can regulate picocyanobacteria abundance through grazing. The main goal of this project is to investigate biotic interactions of planktonic pico- and nano-sized eukaryotes with picocyanobacteria, both in the field and in laboratory settings. A set of field- and culture-based experiments will be conducted, using state-of-the-art methodologies, including fluorescence-activated cell sorting, isotope and fluorescent stain labeling, and next-generation molecular sequencing to address the research objectives.

Operationally, this project is structured around two objectives:

Objective 1 is to assess the contribution of small protists to carbon and nutrient cycling through measurement of primary production, bacterivory, mixotrophy and phosphorus uptake in major microbial groups, and evaluate the role of nutrient availability in controlling mixotrophy.

Objective 2 will focus on assessing the distribution and diversity of small-sized protists that feed on picocyanobacteria and further evaluate the role of nutrient availability among the protists that are mixotrophic.

To reach these objectives field-based experiments will be conducted in contrasted environments: the North Pacific subtropical gyre (phosphorus replete, dominated by Prochlorococcus at Sta. ALOHA) and the North West Mediterranean sea (phosphorus deplete, dominated by Synechococcus at Sta. DYFAMED). Complementary experiments using model protists and picocyanobacteria will be conducted using controlled cultures in the lab. The work will provide critical new information on the phylogenetic diversity and function of marine microbial eukaryotes, with emphasis on their ecological role as predators (phagotrophy, mixotrophy) on, and competitors with, the picoyanobacteria Prochlorococcus and Synechococcus.