Experimental setup:

The experiments were designed to test the combined effects of four temperatures, and eight light intensities on growth and photophysiology of the diatom T. pseudonana CCMP1335 in a multifactorial design. Four temperatures were tested: 15°C, 18°C, 22°C, and 26°C. Within each temperature, eight light levels were tested: 30, 40, 70,90,105,125,140 and 265 µmol photons · m-2 · s-1. All lights were set at a 12 h day: 12 h dark cycle. For logistical reasons, experiments were partially conducted in series.

Experiments were conducted in Multicultivator MC-1000 OD units (Photon Systems Instruments, Drasov, Czech Republic). Each unit consists of eight 85 ml test-tubes immersed in a thermostated water bath, each independently illuminated by an array of cool white LEDs set at specific intensity and timing. A 0.2µm filtered ambient air was bubbled through sterile artificial seawater, and the humidified air was supplied to each tube Each experiment was split into two phases: An acclimation phase spanning 3 days, was used to acclimate cultures to their new environment. Pre-acclimated, exponentially-growing cultures were then inoculated into fresh media and incubated through a 4-day experimental phase during which assessments of growth, photophysiology, and nutrient cycling were carried out daily. All sampling started 6 hours into the daily light cycle to minimize effects of diurnal cycles.

Experiments were conducted with artificial seawater (ASW) prepared using previously described methods (Kester et. al 1967), and enriched with 50mL per liter of UV sterilized natural seawater and nitrate (NO3), phosphate (PO4), silicic acid (Si[OH]4), at levels ensuring that the cultures would remain nutrient-replete over the course of the experiment. Trace metals and vitamins were added as in f/2 (Guillard 1975). he pH of the growth media was measured spectrophometrically using the m-cresol purple method (Dickson 1993), and adjusted using 0.1N HCl or 0.1M NaOH.

Flow cytometry:

Samples were fixed in Hexamethylenetetramine-buffered formaldehyde (final concentration 1% v/v) and stored at 4°C in the dark for a maximum of 4 days. Cell counts were confirmed to be unaffected over storage for up to a week. Samples were analyzed on a Guava easyCyte HT Benchtop Flow Cytometer (Millipore-Sigma, USA). All data acquisitions were done with logarithmic signal amplification. Cytometer sample flow rates were kept low (0.24 µL · s-1) to accommodate high cell concentrations. Diatoms were identified based on size and chlorophyll autofluorescence using the forward scatter channel (FSC) and Red-FL (695/50 nm) channel respectively. Growth rates were derived by fitting an exponential curve to cell concentrations vs. time for a 48-hour period during which cells exhibited exponential growth in the experimental phase. Growth rates in treatments where cells did not grow, or declined in abundance were listed as 0. Particle sizes (equivalent spherical diameter in µm, ESD) were derived from FSC using size-calibration beads of known diameters ranging from 2 µm to 10 µm (Particle Size standard kit, Spherotech Inc.).

Cell sizes varied between temperature treatments, which were conducted weeks apart for logistical reasons. Presumably cell size differences are thus due to differences in the overall lifecycle of the population and not a direct consequence of the temperature and irradiance regime.