Quantifying Algal Carotenoid Production

To quantify the production of astaxanthin, zeaxanthin and canthaxanthin, samples comprising 1 mL of culture broth were centrifuged at 16,200 × g for 3 min, and the cell pellet was washed with sterile water. Subsequently, 750 μL of extraction solution (acetonitrile/methanol/dichloromethane, 21:21:8, v/v/v) was added to the pellet and ultrasonicated in an ice bath for 30 min, after which the resulting lysate was centrifuged at 16,200 × g for 3 min. The resulting supernatant was collected and another 750μL of extraction solution was added to repeat the extraction [27 (link)]. The supernatants of both extraction steps were combined, centrifuged at 16,200 × g for 3 min, and filtered through a 0.22 μm organic nylon filter before analysis by high-performance liquid chromatography (HPLC) using an Agilent Series 1200 system with a variable wavelength detector set at 476 nm and a Symmetry C18 column (250 mm × 4.6 mm, 5 μm, Waters, Ireland). The carotenoid detection method was the same as reported before [11 (link), 28 (link)]. The results represent the means of three independent experiments. The reference standards of the indicated compounds were purchased from Sigma (Sigma-Aldrich, USA). Dry cell weight (DCW) was calculated according to the empirical formula: 1 OD6₀₀ = 0.323 g DCW/L.