Pigment Extraction, Identification, and Quantification

The whole procedure of pigment extraction, identification and quantification is described in detail elsewhere [36 (link)]. Briefly, total pigments were extracted in acetone by disrupting roughly 20 mg lyophilized biomass between glass beads in a FastPrep-24 instrument (MP Biomedicals, Santa Ana, CA, USA). The acetonic extract was separated on a 1290 Infinity II LC System (Agilent Technologies, Santa Clara, CA, USA) equipped with an Acclaim C30, 3 µm, 2.1 mm × 100 mm column (Thermo Fisher Scientific, Waltham, MA, USA). The masses were detected by a 6545 LC/Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) operated in atmospheric pressure chemical ionization (APCI) positive mode. The pigments were separately identified by comparison of detected and theoretical masses in combination with characteristic spectral absorption of each substance in agreement to literature [51 (link)]. Carotenoid mono- and diesters were assigned to adonixanthin and astaxanthin according to the characteristic absorption spectra of the respective unesterified form [52 (link)]. Astaxanthin was quantified on a Vanquish Flex HPLC system with DAD at 450 nm (Thermo Fisher Scientific, Waltham, MA, USA) with standard calibration. All other carotenoids were reference to astaxanthin at 450 nm [36 (link)]. Chlorophyll a and b were analyzed photometrically [53 (link)].