Table of Contents

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

In this study, one diatom and three bacteria species were grown and measured.  Minutocellus polymorphus were incubated with or without the addition of bacteria in flasks and sampled throughout their growth (representative of bloom conditions), to determine the production of transparent exopolymeric particles (TEP) and the formation of micro-aggregates, and in roller tanks to investigate the formation of sinking aggregates (representative of end of bloom conditions).  Additional details are in Cruz & Neuer, 2022.  

Two growth experiments were carried out independently:
1. Growth experiment 1 with the addition of Marinobacter adhaerens
2. Growth experiment 2 with the addition of Pseudoalteromonas carrageenovora and Vibrio thalassae 

Stock cultures of marine Minutocellus polymorphus (CCMP497, National Center for Marine Algae and Microbiota, NCMA) were maintained in L1 medium prepared in artificial seawater and incubated in an environmental growth chamber (Conviron) at 23 ± 1 °C.   Stock cultures of Vibrio thalassae (DSM102810, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH), Pseudoalteromonas carrageenovora (DSM6820, DSMZ), and Marinobacter adhaerens HP15 were maintained on Marine Agar (BD Difco 2216, Becton Dickinson, NJ; ZoBell, 1941) plates at 23 ± 1 °C. 

Triplicate cultures of Minutocellus polymorphus (CCMP497) with and without the addition of known particle-associated marine bacteria (M. adhaerens, P. carrageenovora, and V. thalassae) were sampled every other day for 19-23 days for the quantification of:

• single cells [Cell Abundance dataset]
• suspended microaggregates (aggregates ca. 5-60 μm) [this dataset]
• TEP (Transparent Exopolymeric Particles) [TEP Concentration dataset]

This dataset contains data on suspended microaggregates, while the other measurements can be found in the Related Datasets below. 

Volume concentrations of micro-aggregates “suspended” in cultures (i.e., non-sinking particles with an equivalent spherical diameter [ESD] of 5–60 µm) were determined at every sampling period using a Multisizer 3 Particle Counter (Beckman Coulter, CA). Prior to fixation with glutaraldehyde, duplicate samples were diluted to a 1–10% final particle concentration with Isoton II diluent (Beckman Coulter, CA) and aggregates were measured and quantified with a 100 µm aperture tube. The volume concentration of aggregates was calculated in µm3 per mL.

Aggregation in roller tanks
To investigate the formation of visible aggregates, cultures of the nanodiatom Minutocellus polymorphus (CCMP497) with and without the addition of known particle-associated marine bacteria (Marinobacter adhaerens HP15, Pseudoalteromonas carrageenovora, and Vibrio thalassae) were incubated in  cylindrical1.25 L Plexiglass roller tanks with artificial seawater (ASW) for 7 days in the dark at 3.5 revolutions per minute (RPM) after the methodology of Shanks and Edmondson (1989). 

Two sets of experiments were performed:

1. cultures were inoculated to cell concentrations simulating natural conditions in the Sargasso Sea (10^{^3} cells per mL), and the experiment was performed with the addition of M. adhaerens alongside bacteria-only and diatom-only controls, as well as with the addition of microbeads
2. cultures were inoculated at higher cell concentrations (10^{^5} cells per mL).

All treatments were performed in triplicate (n = 3 tanks) in the 10^{^5} cells mL^-1 experiments and in duplicate (n = 2 tanks) in the 10^{^3} cells mL^-1 experiments. Aggregates formed were quantified, sized, and their sinking velocities were determined.  Suspended microaggregates (i.e., non-sinking particles with an equivalent spherical diameter of 5-60 microns) were quantified using a Multisizer 3 Particle Counter.  TEP concentrations were determined as in Bittar et al. (2018). The stocks of Alcian-Blue dye used for TEP quantification had calibration factors (or f-factors) of 81.70 for experiments with M. adhaerens and 83.83 for experiments with P. carrageenovora and V. thalassae.

Only the axenic Minutocellus 10^5 experiment had hardy-enough aggregates for analysis.  Please refer to the Supplemental document.  

For additional Methods details, see Cruz & Neuer, 2022.  
For size and velocity of sinking aggregates from roller tank experiments, see Supplemental Files section

BCO-DMO Processing Notes: 
- Excel sheet "M.polymorphus_Microaggregates_and_TEP- Suspended Microaggregates.xlsx " was processed and saved as a CSV file
- added a conventional header with dataset name, PI name, version date
- modified parameter names to conform with BCO-DMO naming conventions
- blank values in this dataset are displayed as "nd" for "no data."  nd is the default missing data identifier in the BCO-DMO system.

NSF abstract:

Marine phytoplankton are microscopic algae that live in the sunlit zone of the ocean. They play an important role in the uptake of carbon dioxide from the atmosphere through photosynthesis, similar to what plants do on land, and are the basis of the marine food web. However, instead of storing this organic carbon in leaf tissue and roots, marine phytoplankton are grazed by planktonic animals, or die and subsequently sink out of the sunlit zone in the form of aggregates, also called "Marine Snow". These particles not only export the organic carbon contained in their cells to the deep ocean, but also serve as food for animals and bacteria that live in the deep. A considerable portion of these phytoplankton are extremely small, among the tiniest of all organisms known. These extremely small cells have not been thought to play an important role in the formation and sinking of marine snow; however, recent findings challenge this view. This project will investigate how the smallest of these phytoplankton contribute to the rain of sinking particles from the sunlit surface to the deep ocean. This research is important because, in some of the largest expanses of the open oceans, these minute cells dominate the phytoplankton community, and larger plankton organisms are very sparse. The project, through a combination of work in the laboratory and at a field station, will shed light on how these tiny phytoplankton cells make aggregates, which ultimately enable them to sink as "Marine Snow". The project also provides unique opportunities for undergraduate students at Arizona State University, a land-locked public university, to gain experience in working with marine research. The project will serve to educate one PhD student, one MS student in an accelerated BS-MS program, and 8-10 undergraduate students/semester in a unique, inquiry based learning effort termed Microbial EducatioN Training and OutReach (MENTOR). The undergraduate students will also participate in Arizona State University (ASU)'s School of Life Sciences, Undergraduate Research Program (SOLUR), which seeks to increase the participation of minorities in science. They will also contribute towards developing web and classroom materials, based on this project, which will then be distributed through a partnership with the award-winning ASU-sponsored Ask A Biologist K-12 web site.

The oceanic "biological carbon pump", the photosynthetically mediated transformation of dissolved inorganic carbon into particulate and dissolved organic carbon and its subsequent export to deep water, functions as a significant driver of atmospheric carbon uptake by the oceans. The traditional view of the biological carbon pump in the ocean is that of sinking of large aggregates (marine snow) or fecal pellets, which are made up of large, mineral ballasted cells of phytoplankton. However, recent evidence, stemming from in situ investigations of particulate matter, trap studies and modelling studies, have shown that micron-sized phytoplankton such as picocyanobacteria as well as picoeukaryotes can contribute significantly to the sinking of particulate matter. The specific mechanisms behind the sinking of these micrometer sized cells remain elusive as the cells are too small to sink on their own, and mesozooplankton is likely unable to ingest single cells. Intriguingly, recent research by the investigators has shown that the ubiquitous picocyanobacteria Synechococcus are able to form aggregates and sink at velocities comparable to those of marine snow. They found that the matrix of the Synechococcus aggregates was made of Transparent Exopolymeric Particles (TEP), and that TEP production was enhanced under nutrient limited culture conditions. Interaction with clays and presence of heterotrophic bacteria also enhanced aggregation and sinking velocity. This study aims to further investigate aggregation of other common picoplankton in the laboratory and aggregation occurring in natural settings at an oligotrophic open ocean site, the Bermuda Atlantic Time-series Site (BATS). Ultimately, this project will increase and refine our understanding of the role of the smallest phytoplankton in aggregation and sinking - information vital to understanding carbon cycling processes in the oceans.