PPIX standards purchased from Sigma-Aldrich (St. Louis, MO, USA). PPIX was dissolved in DMSO to obtain 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μg/L solutions, respectively. PPIX solutions of different concentrations (200 μL) were pipetted into a 96-well plate. A microplate reader was used to detect the fluorescence intensity of the solutions (excitation at 410 nm, emission at 633 nm). A standard curve was drawn (Additional file 5 : Figure S1). Fermentation broth was centrifuged to obtain the supernatant. After appropriate dilution by DMSO, the fluorescence intensity (excitation at 410 nm, emission at 633 nm) was detected in 96-well plates, and the PPIX concentration was determined from the standard curve [38 (link)].
Heme concentration was determined using a high-performance liquid chromatography (HPLC) system with a Discovery HS C18 column (250 × 4.6 mm, 5 μm; Supelco Inc., USA). Filtered samples were separated using a linear gradient of 20%–95% solvent A in B at 40 °C. Solvent A was a 10:90 (v/v) HPLC-grade methanol:acetonitrile mixture, and solvent B was 0.5% (v/v) trifluoroacetic acid in HPLC-grade water. The flow rate was 1 mL/min for 40 min, and the absorbance was measured at 400 nm [39 (link)]. When the heme concentration was < 2 mg/L, a Heme Colorimetric Assay Kit (BioVision, USA) was used for determination.
ALA and PBG concentrations were analyzed using modified Ehrlich’s reagent [36 (link)]. Porphyrin was determined by HPLC. The fermentation broth was extracted twice with 5% HCl and incubated at 37 °C for 30 min. After centrifugation, the supernatant was passed through a Bischoff Prontosil 120-5-C18 ace EPS chromatography column (125 mm × 4 mm; 5 μm) at a flow rate of 0.75 mL/min. Porphyrin was detected at 30 °C using a fluorescence detector (excitation 400 nm, emission 620 nm). Mobile phase A was 1 M ammonium acetate, pH 5.16, with 10 mL triethylamine and 100 mL acetonitrile in 1-L chromatographic-grade water; mobile phase B was methanol:acetonitrile (9:1, v:v). During the first 15 min, the proportion of mobile phase A was a linear decreased from 62 to 5%; the mobile phase A ratio was then maintained at 62% for 10 min.
Heme concentration was determined using a high-performance liquid chromatography (HPLC) system with a Discovery HS C18 column (250 × 4.6 mm, 5 μm; Supelco Inc., USA). Filtered samples were separated using a linear gradient of 20%–95% solvent A in B at 40 °C. Solvent A was a 10:90 (v/v) HPLC-grade methanol:acetonitrile mixture, and solvent B was 0.5% (v/v) trifluoroacetic acid in HPLC-grade water. The flow rate was 1 mL/min for 40 min, and the absorbance was measured at 400 nm [39 (link)]. When the heme concentration was < 2 mg/L, a Heme Colorimetric Assay Kit (BioVision, USA) was used for determination.
ALA and PBG concentrations were analyzed using modified Ehrlich’s reagent [36 (link)]. Porphyrin was determined by HPLC. The fermentation broth was extracted twice with 5% HCl and incubated at 37 °C for 30 min. After centrifugation, the supernatant was passed through a Bischoff Prontosil 120-5-C18 ace EPS chromatography column (125 mm × 4 mm; 5 μm) at a flow rate of 0.75 mL/min. Porphyrin was detected at 30 °C using a fluorescence detector (excitation 400 nm, emission 620 nm). Mobile phase A was 1 M ammonium acetate, pH 5.16, with 10 mL triethylamine and 100 mL acetonitrile in 1-L chromatographic-grade water; mobile phase B was methanol:acetonitrile (9:1, v:v). During the first 15 min, the proportion of mobile phase A was a linear decreased from 62 to 5%; the mobile phase A ratio was then maintained at 62% for 10 min.
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