H0751

The melanin-producing ability of strain GR-TSA-9^T was confirmed by growth on TSA medium supplemented with 0 and 10mg/ml of l-tyrosine followed by incubation at 25°C. Production was performed in TSB consisting of 0, 0.2, 0.4, 0.6, 0.8, and 1.0mg/ml l-tyrosine, and after inoculation with 1ml of the cell suspension (OD₆₀₀=1.0) in 100ml TSB, shaking at 25°C and 150rpm for 1–7days, and melanin production in the broth was measured at OD₄₀₀ using the standard synthetic melanin (Sigma-Aldrich, M8631) calibration curve method (Singh et al., 2018 (link)). Melanin was purified as previously described (El-Naggar and El-Ewasy, 2017 (link)). Briefly, the supernatant was centrifuged at 8,000rpm to remove the cells, followed by adjustment of the pH of the supernatant to 2.0 using 6M HCl; the samples were then allowed to stand for 4h and centrifuged at 8,000rpm to collect the precipitate. The melanin pellets were washed with distilled water three times and then centrifuged at 8,000rpm for 10min to obtain melanin. The purified melanin was freeze-dried for further use. In vitro-synthesized pyomelanin was produced by auto-oxidation of 10mM HGA (Sigma-Aldrich, H0751) solution at pH 10 with constant stirring for 3days as described by Schmaler-Ripcke et al. (2009) (link).

Homogentisic acid (HGA) concentration was estimated in the supernatant after biomass separation by UPLC/MS, using an Agilent 1290 Affinity chromatograph linked to an Agilent 6550 Q-ToF mass spectrometer. Separation was achieved using an Agilent Zorbax Eclipse Plus C18 column (2.1×50mm, 1.8μm) and an acetonitrile gradient of 0% for 2 minutes then an increase to 98% over 0.5 minutes at a flow rate of 0.3mL/min. Mass spectral data was acquired in negative ion mode from m/z 90 to 1000 at the rate of 3 spectra per second throughout the separation. 0.2μL was injected from both sample wells and standard solutions prepared from commercially available HGA (H0751, Sigma-Aldrich).

Homogentisic acid (HGA) concentration was estimated in the supernatant after biomass separation by UPLC/MS, using an Agilent 1290 Affinity chromatograph linked to an Agilent 6550 Q-ToF mass spectrometer. Separation was achieved using an Agilent Zorbax Eclipse Plus C18 column (2.1 × 50 mm, 1.8 µm) and an acetonitrile gradient of 0% for 2 minutes then an increase to 98% over 0.5 minutes at a flow rate of 0.3 mL/min. Mass spectral data was acquired in negative ion mode from m/z 90 to 1000 at the rate of 3 spectra per second throughout the separation. 0.2 µL was injected from both sample wells and standard solutions prepared from commercially available HGA (H0751, Sigma-Aldrich).