Ultimate 3000 basic automated system

The concentration of glucose, formate, and methanol was measured in culture supernatants in a high-performance liquid chromatography (HPLC) instrument (UltiMate 3000 Basic Automated System ThermoFisher Scientific Co) equipped with an Aminex HPX-87P column (Bio-Rad) and a refractive index detector Shodex RI-101 (Showa Denko America Inc., NY, USA). Specific glucose (q_G), formate (q_F), and methanol (q_M) consumption rates were normalized to the cell dry weight (CDW) with the following equation
where q_S corresponds to the biomass-specific substrate consumption rate (mmol g_CDW⁻¹ h⁻¹), X is the average biomass concentration between two sampling time points (g_CDW L⁻¹), ∆S is the difference in substrate concentration between two sampling time points (mM), and ∆t refers to the time between two sampling points (h). The q_G,q_F, and q_M values correspond to an average of the values individually determined in three biological replicates.

The carotenoid profile was determined by reverse phase HPLC using an Ultimate 3000 Basic Automated System (Thermo Fisher Scientific Inc., Waltham, MA, USA) fitted with a diode arrangement detector. The chromatographic separation was performed on a Beckman Ultrasphere C18 250 × 4.6 mm silica column (5 μm dia. spheres). Elution was conducted using a gradient of methanol/acetonitrile (B: 35% 5 min, B: 35–10% 10 min, B: 10–35% 5 min, B: 35% 10 min) at a flow rate of 1 ml min⁻¹, and peak detection was performed at 480 nm. The peaks derived from the samples were identified by comparison to the spectra and retention time from astaxanthin and β-carotene standards (Sigma-Aldrich). All other reagents were purchased from J.T. Baker (Center Valley, PA, USA) unless otherwise specified.