Nova pak silica column

The NMR data were recorded on Bruker Advance-III spectrometer (Bruker, Karlsruhe, Germany). Mass spectra were performed on the Waters 2767 LC-MS system using the Nova-pak silica column (Waters, Milford, CT, USA). HR-ESI-MS was recorded on Thermo Scientific Q Exactive HF Orbitrap-FTMS (Thermo Fisher, Waltham, MA, USA). Analytic HPLC was operated on Agilent 1200 series liquid chromatography with an Agilent XDB-C18 column (9.4 × 250 mm, 5 μm) and UV detector (Agilent, Santa Clara, CA, USA). Semi-preparative HPLC was operated on Welch Sail 1000 liquid chromatography with Prep RP-18 column (28 × 4 cm) and UV detector (Welch, Baltimore, MD, USA). Mid-press reverse phase chromatography was operated on Buchi Switzerland system with BUCHI RP-18 column (20 × 4 cm, 170 g) (Büchi, Uster, Switzerland). Column chromatography (CC) was performed with silica gel (300–400 mesh, Yantai Jiangyou silicon development Co., Ltd., Yantai, China) and Sephadex LH-20 (GE Healthcare, Chicago, IL, USA).

Pigments were quantified using a reverse-phase HPLC method (Shimadzu, Tokyo, Japan) with a photodiode array detector SPD-M20A (PDA, Shimadzu, Tokyo, Japan). 2 ml of culture were centrifuged (16,000×g; 3 min) and the pellet was resuspended in dichloromethane/methanol (1:3, v/v). Glass beads were added to the sample and cells were disrupted through horizontal agitation in a TissueLyser II (Qiagen, Hilden, Germany) for 2 × 5 min at 30 Hz, with 5 min break on ice between the two lysing steps. After centrifugation (16,000×g; 3 min) and 0.22 μm filtration, 40 μl of samples were injected on a Nova-Pak C18 column (Nova-Pak silica column, 3.9 × 150 mm, 4 μm particle size, Waters, Milford, MA, United States). Elution was performed during 25 min at 25°C with a flow of 1 ml.min^–1 using a gradient mode with three solvents: methanol 80% and ammonium acetate 100 mM (A), acetonitrile 90% (B), and ethyl acetate 100% (C). Gradient was: 0 min − 100% A; 0.5 min − 100% B; 1.1 min − 90% B + 10% C; 6.1 min − 65% B + 35% C; 11.5 min − 40% B + 60% C; 15.0 min − 100% C; 17.0 min − 100% A; 23.0 min − 100% A. Quantification was based on the peak area at 430 nm and compared to calibration curves obtained with pure pigments (DHI Lab Products, Denmark). Pigment content was then normalized on the dry biomass concentration.