*3.4. Pigment Analysis*

Routinely, the 0.5–1.0 mL aliquots of cells suspension were extracted with dimethyl sulfoxide (DMSO). The cells were pelleted by centrifugation (5 min, 12,000 rpm), the supernatant was removed and the cells were incubated with 1.5 mL of DMSO at 75 °C for 15 min, and the cells were removed by centrifugation; then the extraction was repeated. Chlorophyll (Chl) *<sup>a</sup>*, Chl *b* and total carotenoids (Car) in the extract were assayed spectrophotometrically [35] with an Agilent Cary 300 (Agilent, Santa Clara, CA, USA) spectrophotometer in standard 1-cm cuvettes. 

For the fatty acid (FA) and pigment assay, the extraction by Folch was used [36]. In this case Chl *a*, *b,* and total Car contents were determined in lower (chloroform) fraction [37]. The pigments were initially separated by TLC on silica gel plates (*Sulifol*, Kavalier, Prague, Czech Republic). The hexane:benzene:chloroform (5:5:2, by volume) and hexane:chloroform:benzene (10:20:1, by vol.) were used for separation of "green" and "red" cell extracts, respectively. The spots obtained after separation of the "red" cell extracts were gently scrapped from the plate by scalpel and eluted with chloroform. 

Both the total pigment extracts and eluted fractions were subjected to HPLC analysis using an Alliance 2995 separation module (Waters, Milford, MD, USA) equipped with a 150 × 4.5-mm Prontosil RP C-18 column (Knauer, Berlin, Germany) maintained at 25°C and a Waters e2695 DAD detector (Waters, Milford, MD, USA). The gradient elution of pigments was achieved at a flow rate of 1 mL·minƺ1 using (A) acetonitrile, (B) water, and (C) ethyl acetate mixtures (vol. %): for the "red" cell extracts, 98:2:0 (2 min), 40:0:60 (10 min), 0:0:100 C (2 min) followed by 6-min reequilibration of the column; for the "green" cell extracts: 98:2:0% B (5 min); 48.5:  1.2:50, 0:0: 100 (3 min) followed by 6-min re-equilibration. Eluted component spectra were monitored in the range 400–700 nm. Free pigments were identified and quantified using authentic standards (Sigma, St. Louis, MS, USA). Astaxanthin esters were identified by their chromatographic mobility and quantified as free Ast. 
