Experimental setup:

The experiments were designed to test the combined effects of two CO2 concentrations, four temperatures, and three light intensities on growth and photophysiology of the cyanobacterium S. elongatus CCMP1629 in a multifactorial design. Two CO2 concentrations were tested: 410 ppm, and 1000 ppm. For each CO2 concentration, four temperatures were tested: 20°C, 28°C, 36°C, and 44°C. Within each temperature, three light levels were tested: sub-optimum irradiance (SOI) intensity of 50 umol photons · m-2 · s-1, optimum irradiance (OI) intensity of 230 umol photons · m-2 · s-1 and extreme Irradiance (EI) intensity of 600 umol 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, with all light treatments at all four temperatures running simultaneously. This was repeated for each CO2 concentration.

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 0.2um filtered CO2-air mix (Praxair Distribution Inc.) was bubbled through sterile artificial seawater, and the humidified gas mix was supplied to each tube via gentle sparging through a 2um stainless steel diffuser. Flow rates were gradually increased over the course of the incubation to compensate for the DIC uptake of actively growing cells, and ranged from <0.04 Liters per minute (LPM) at the start of the incubations to 0.08 LPM in each tube after 2 days. For each CO2 and temperature level, replication was achieved by incubating three tubes at sub-optimum light intensities, two tubes at optimum light intensity, and three tubes at extreme light intensities. 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 3-day experimental phase during which assessments of growth, photophysiology, and nutrient cycling were carried out daily. All sampling started 5 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 nitrate (NO3), and phosphate (PO4), 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). The expected DIC concentration, and pH of the growth media was determined for the different pCO2 and temperatures using the CO2SYS calculator (Pierrot et al. 2006), with constants from Mehrbach et al. (1973, refit by Dickson & Millero 1987), and inputs of temperature, salinity, total alkalinity (2376.5 umol · kg−1), pCO2, phosphate, and silicic acid. DIC levels in ASW at the start of each phase of the experiments were manipulated by the addition of NaHCO3, and was then maintained by bubbling a CO2-Air mix through the cultures over the course of the experiments. The 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. The media was distributed into 75 ml aliquots and each aliquot was inoculated with the S. elongatus CCMP 1629 (SE1629) stock culture at the start of the experiments.

Photophysiology

Photophysiology was assessed daily using a handheld Pulse Amplitude Modulated (PAM) fluorometer (AquaPen-C AP-C 100, Photon System Instruments, Czech Republic). A sample was collected from each tube, 5 hours after the start of the daily light cycle, and placed in the dark for a minimum of 30 minutes prior to measurements. The dark-adapted sample was used to generate light curves that provide measurements of in-vivo chlorophyll autofluorescence (F0), the maximum quantum yield (QYmax or Fv/Fm), and relative photosynthesis rates based on PSII quantum yields at varying light intensities - using the instrument’s LC3 protocol. The LC3 protocol involves measurements of baseline and maximal fluorescence over seven 60-second phases, with each phase representing a light intensity from 10 to 1000 umol photons m-2 · s-1. Red light (620 nm) was used as actinic light in these experiments, and measurements were made at measuring illumination (f-pulse) intensity of 0.03 umol photons m-2 · s-1, and saturating (F-pulse) illumination of 2700 umol photons m-2 · s-1, and actinic illumination (A-pulse) controlled by the instrument’s protocol were set at 10, 20, 50, 100, 300, 500,and 1000 umol photons m-2 · s-1 (for each 60-second phase).