Dataset: Minutocellus polymorphus TEP and Microaggregate Formation: Suspended Microaggregates

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