The aim of this study was to investigate the combined effects of temperature and light on the photosynthetic parameters and lipid accumulation in the diatom Phaeodactylum tricornutum, a model organism widely used for studies on diatom physiology, ecology, and biotechnology. Our results highlight the importance of the interaction between temperature and light intensity in influencing growth rates, pigments and active photosystems content, photosynthetic efficiency, lipid production and fatty acid composition in P. tricornutum. Measurements of the maximum electron transport rate (rETRmax) and rETR at maximum PAR (830 µmol m−2 s−1) confirmed that P. tricornutum exhibits significantly higher light sensitivity as growth temperature increases under light/dark cycles at two light intensities (25–60 µmol m−2 s−1). However, this trend was reversed under continuous light (25 µmol m−2 s−1). Moreover, higher rETRmax values (up to double) were observed at higher irradiance, either in intensity or under continuous light regimes, at the two temperatures tested. On the other hand, increasing light intensity amplified the observed effect of temperature on photosystem I (PSI) activity under light/dark regimes, but not under continuous light conditions. This resulted in a greater deficiency in PSI activity, likely due to limitations in electron supply to this photosystem. Furthermore, increasing the culture temperature from 20 °C to 25 °C triggered an increase in the number and size of cytoplasmic lipid droplets under conditions of increased light intensity, with an even more pronounced effect under continuous illumination. Notably, the combination of 25 °C and continuous illumination resulted in a more than twofold increase in triacylglyceride (TAG) content, reaching approximately 17 mg L−1. This condition also caused a substantial rise (up to ≈90%) in the proportions of palmitoleic and palmitic acids in the TAG fatty acid profile.
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