## 2 Materials and Methods
### HPLC Analysis  
HPLC analyses were performed on cultures during both their green and red stages of growth. Cultures were harvested on cultivation day 14 (prior to astaxanthin induction) and at day 21 (7 days following induction). In both instances, cells were centrifuged at 3000\(\times\)\(g\) for 15 min; pellets of sub-samples were then flash-frozen in liquid nitrogen, stored at \(-\)80 \({}^{\circ}\)C, and then freeze-dried (ALPHA 1-2 LD plus freeze dryer, Christ).  
The lyophilised biomass was weighed, and the pigments extracted by employing a modified protocol by [PERSON] (2006) [PERSON] (2006). Briefly, 3 mg of dry weight (DW) biomass was resuspended in 1 mL of 2N HCl and incubated at 70 \({}^{\circ}\)C for 2 min. The pigment extraction was facilitated by using glass beads while vortexing (diameter of 0.25-0.5 mm, Roth, Karlsruhe, Germany). Samples were then cooled at \(-\)80 \({}^{\circ}\)C for five minutes, to exploit the thermic stress to favour the cell breakage. After thawing, samples were then centrifuged at 5000 rpm for 3 min (Thermo Fisher Sorvall Legend X1R Refrigerated Benchtop Centrifuge), the supernatant was removed, and the HCl-treated samples were washed twice in 2 mL deionized water to neutralise the pH, pelletized, and finally resuspended in 1 mL of extraction solvent (95% methanol, buffered with 2% ammonium acetate with the addition of the internal standard) and stored at \(-\)20 \({}^{\circ}\)C for 15 min. The extraction procedure was repeated until the algal cell debris was almost colourless and several short (30 s) vortexing steps were alternated between each resuspension in the extraction solvent.  
Supernatants were collected after centrifugation (5000 rpm, 3 min, at 4 \({}^{\circ}\)C) and syringe filtered (porosity, 0.47 um). The extracts were injected into a high-performance liquid chromatography (HPLC) system (Waters Alliance 2695) equipped with a cooled auto-sampler and a photodiode array detector (Waters PDA 2998). Chromatographic separation was carried out using a 4.6 mm \(\times\) 100 mm column (Sunfire, Waters) equipped with a 3.9 mm \(\times\) 5 mm pre-column (Sunfire, VanGuard, Waters) for reverse-phase chromatography during a 30 min elution program. The elution gradient was run as follows with solvent A (80/20 \(v/v\) methanol and ammonium acetate 1 M) and solvent B (60/40 \(v/v\) methanol and acetone); the time gradient was 0 min 80% A, 20% B; 10 min 0% A, 100% B; 20 min 0% A, 100% B; 30 min 80% A, 20% B with a flow rate of 1.0 mL/min and a sample volume of 100 \(\upmu\)[PERSON] (2006). The synthetic carotenoid trans-\(\beta\)-Apo-8\({}^{\prime}\)-carotenal (Sigma) was used as an internal standard in each sample to improve the precision of the quantification and to detect eventual instrumental errors.  
A chromatographic and spectral library was created from standard pigments (DHI lab, Hersholm, Denmark). Pigments in the extracts were identified by comparison of chromatographic and spectral data recorded with standard pigments, using Empowered software (Waters). In the absence of the pigment standard, canthaxanthin and carotenoidlike pigments were quantified by the ISTD method (internal standard analysis), relating the ratio of the standard peak area and the pigment peak area to their respective amounts, as the standard concentration did not change. The absorbance spectra (400-800 nm) and retention times were also checked to validate the output given by the software, and the pigments' concentrations were obtained from the signals in the photodiode array detector at 440 nm [29; 30]. For some pigments, concentrations were summed: chlorophyll \(a\) and its allomeric and epimer forms. Values are expressed as mean values \(\pm\) standard error of the triplicates.