Bacterial Growth and Metabolite Analysis

Bacterial cell growth was followed by measuring the optical density at 600 nm using a UV/Vis spectrophotometer (Mecasys Co., Ltd., Korea). The glucose concentration in the culture medium was measured with glucose assay kit (Sigma-Aldrich, USA). Porphyrin intermediates and heme concentrations were measured with a high-performance liquid chromatography (HPLC) system (Waters Corporation, USA) with a UV detector (Waters 2487) at 400 nm [41 (link)]. The initial mobile phase composition was 20% solvent A (1:9 methanol:acetonitrile) and 80% solvent B (0.5% trifluoroacetic acid in water, pH 2.60). The solvent gradient consisted in 40 min linear change at 95% solvent A and 5% solvent B with 1.0 ml/min flow velocity at 40 °C. Biliverdin concentration was analyzed by HPLC system with a UV detector at 376 nm [54 ]. The initial mobile phase composition was 25% solvent C (methanol) and 75% (1 M ammonium acetate, pH 5.16). The solvent gradient consisted of 8 min linear change at 95% solvent C and 5% solvent D, and then 2 min at these conditions and 8 min at 25% solvent C and 75% solvent D with 1.5 ml/min flow velocity at 70 °C.