0.22 μm ptfe membrane

The lutein and chlorophyll contents were determined following the report by Baroli [65 (link)]. The wet algal cell pellets obtained after centrifugation (3000× g, 5 min) were frozen at −70 °C for at least 1 h. The frozen cell pellets were then lyophilized for 36 h in a freeze dryer. Following lyophilization, the dry cell pellets were ground into a powder in a mortar. The pigments were extracted with acetone until the cell debris became colorless. Then the supernatant containing extracted pigments was filtered through a 0.22 μm PTFE membrane (Millipore, Burlington, MA, USA). Each sample (20 μL) was separated on a Waters Spherisorb 5 μm ODS 4.6 mm × 250 mm analytical column (Waters Corp., Milford, MA, USA). Samples were eluted at a flow rate of 1.0 mL/min with a linear gradient from 100% solvent A [acetonitrile: methanol: 0.1 M Tris-buffer, pH 8.0 (84:2:14, by vol.)] to 100% solvent B [methanol: ethyl acetate (68:32, by vol.)] for 15 min, followed by 10 min of solvent B. Individual pigments were identified by comparing their absorption spectra to those of standards (Sigma-Aldrich, St. Louis, MO, USA). The concentration of each pigment was calculated based on the corresponding standard curve. The contents of lutein and chlorophyll were described as the amount of pigments per dry weight (mg/g dry cell weight).