**4. Materials and Methods**

### *4.1. Experimental Strategy and Sampling*

The coastal centric diatom *Skeletonema marinoi* Sarno and Zingone (strain CCMP 2092) was used as the model species in this work for its high growth rate [16,64] and its use in aquaculture and biotechnology [18,19,65–68]. Additionally, the biology and photophysiology of this species are well known [16,63]. The strain (CCMP 2092) used in this work was collected from surface waters of the Northern Adriatic Sea, where this diatom provides the major contribution to the late winter blooms [69]. *S. marinoi* cultures were carried out in 4.5 L glass flasks under water movement, provided by an aquarium wave maker pump (Sunsun, JVP-110) at 20 ◦C, containing seawater previously prefiltered through a 0.7 μm GF/F glass-fiber filter (WhatmanTM) autoclaved and enriched with F/2 medium nutrients [70] with some modifications. In particular, to ensure enough nutrient supply, the content of essential nutrients (phosphate, dissolved silica, trace metals and vitamins) in the culture medium was doubled [63]. Light was provided by a custom-built LED illumination system, able to modulate light intensity and spectral composition (Patent number: EP 13196793.7) [14]. Light intensity was measured inside each flask by using a laboratory PAR4 π sensor (QSL 2101, Biospherical Instruments Inc., San Diego, CA, USA). The light spectral composition was kept constant during the preacclimation and all the experimental conditions were set up with a ratio blue:green:red = 50:40:10, usually found in the first layers of the photic zone at sea [14]. Preacclimation of cells was performed for a minimum of two weeks at a moderate light intensity, 12 h:12 h light:dark photoperiod, with a sinusoidal intensity distribution peaking at 150 μmol photons s<sup>−</sup><sup>1</sup> m<sup>−</sup><sup>2</sup> after 6 h from dawn (Sin150). Each experiment included three independent cultures and lasted 1–2 days, after the shift from the preacclimation condition to the experimental light condition. Both sinusoidal and square-wave light distributions were applied at different intensities in order to investigate the effect of different velocities of light increase on ovothiol biosynthesis [60]. Sinusoidal distribution is similar to the natural condition, with a succession of dawn, gradual light increase up to the midday intensity peak and gradual light decrease up to sunset. By contrast, the square-wave distribution—generally used in indoor algal cultivation systems—constitutes an unnatural condition, providing a fast increase of light intensity, kept constant for all the day phase (12 h) and then switched off for the night phase (12 h).

The seven experimental conditions were:


The Square300 condition provided the same daily light dose experienced by cells grown under Sin600, allowing us to compare square-wave and sinusoidal distributions of light, without any influence from different daily light doses [19]. All experimental conditions provided a 12 h:12 h light:dark photoperiod except for the conditions Square10 (very low intensity light was kept constant for all the duration of the experiment) and darkness (no "day phase" for all the duration of the experiment).

For each experimental condition, samplings were performed in the exponential growth phase, except for the control light condition (Sin150) in which samplings were done both in exponential and stationary growth phases.

In particular, for all the conditions samples were taken at predawn (time 0 h), at 6 h (midday peak) and 24 h (at the end of the night phase) from the light switch. Since high light is known to trigger rapid photoprotective processes in phytoplankton species [71] an additional sampling at 2 h was performed for Sin600, Square300 and Square600. In case of the square-wave light conditions (Square300 and Square600), in which cells experienced a very unnatural and fast increase of light intensity, samples were taken also at 0.2 h (12 min) to evaluate the short-term response. In case of very low sinusoidal and square-wave lights and darkness conditions, in which cells experienced different light:dark photoperiod cycles, samplings were done at 0 and 6 h for two consecutive days.
