Dataset: Picocyanobacteria and picoeukaryote growth under different HOOH concentrations

Cultures:
The strains used in this study included axenic cultures of Prochlorococcus MIT9215, Synechococcus WH7803, Synechococcus CC9605, Micromonas commoda RCC299, Micromonas pusilla CCMP1545, Ostreococcus lucimarinus CCMP2972, and Alteromonas macleodii EZ55. All phytoplankton strains were routinely assayed for heterotrophic contaminants by staining cells with purity test broth as previously described (Morris, et al. 2008).

Cultivation methods for mono- and co-cultures:
Experiments were conducted at the University of Tennessee, Knoxville (Tennessee, USA). All cyanobacterial stock cultures were maintained in an artificial seawater medium, AMP-A (Morris and Zinser, 2013). Picoeukaryote stock cultures were maintained and all experiments were performed using an AMP-A derivative, AMP-PE (for& Pico-Eukaryotes), which allowed for efficient and consistent growth of all photosynthetic microbes in mono- and coculture. This medium has an identical recipe and preparation as AMP-A except for the following alterations: 10x addition of trace metal working stock, 1.06e⁻⁴ molar (M) silica, 2.96e⁻⁷ M thiamine, 2.05e⁻⁹ M biotin, and 3.69e⁻¹⁰ M cyanocobalamin. Stocks of these nutrients were filter sterilized and added after sterilization of the base saltwater medium. Axenic heterotrophic bacteria Alteromonas macleodii strain EZ55 was grown in 5 milliliters (mL) YTSS (Sobecky et al., 1996) and incubated shaking at 140 RPM at 24° Celsius (C) overnight. Before inoculation into experimental cultures, the heterotroph was washed three times in 1.5 mL microcentrifuge tubes by centrifugation at 8,000 RPM for two minutes in a tabletop microcentrifuge and resuspension in 1 mL AMP-A. All experiments were carried out in unshaken test tubes containing 20 mL cultures incubated at 24°C in Percival I36VLX incubators (Percival, Boone, IA) that allowed for gradual increase and decrease of cool white light to simulate sunrise and sunset with peak midday light intensity of 150 millimoles quanta per square meter per second (mmol quanta m⁻²s⁻¹) on a 14 hour:10 hour light:dark cycle (Zinser et al. 2009).

Sampling:
Cultures and uninoculated controls were monitored periodically during the incubations by removing small volumes (<1 mL) of culture to determine total cell concentrations with flow cytometry and hydrogen peroxide (HOOH) concentrations with the acridinium luminescence method.

HOOH concentration assay:
The concentration of HOOH in the medium and cultures was measured on an Orion L Microplate Luminometer (Titertek Instruments Inc, Berthold Detection Systems, Pforzheim, Germany) using an acridinium ester (Cayman Chemical Company, Ann Arbor, MI) chemiluminescence method (Morris et al. 2011). Concentrations in cultures were adjusted via both instantaneous and incremental (over 14-hour period) addition to achieve specific exposure conditions.

Cell concentration assay:
Abundances of cyanobacteria were quantified by flow cytometry using a CytoFLEX S flow cytometer (Beckman Coulter, Brea, CA) with populations of Prochlorococcus and Synechococcus differentiated in co-cultures by their red (675 nanometers (nm)) and red / yellow (675 nm / 578 nm) fluorescence, respectively (Cavender-Bares et al. 1998; Morris et al. 2008). Picoeukaryotes were quantified by red (675 nm) and far red (770 nm) fluorescence. Detection of red and yellow fluorescence was achieved after excitation with a blue (488 nm) laser, while detection of far-red fluorescence required excitation by a yellow (565 nm) laser. Quantification of Prochlorococcus in coculture was achieved by observing events determined by red fluorescence after events that corresponded to the fluorescence properties of either Synechococcus (red / yellow) or picoeukaryotes (red / far red) were removed from abundance calculation.