1290 affinity chromatograph

All succinic acid production strains were picked into 6 mL of synthetic minimal (SD) medium and grown at 30 °C, 250 rpm overnight. The next day, optical density was measured in a spectrophotometer (WPA Biowave II) and cultures were diluted to OD₆₀₀ = 0.05 in 1 mL SD media, with and without 1 µM aTc (Alfa Aesar, J66688-MB). Cultures were grown in 48-deep-well-plates (Agilent, 201238-100) at 30 °C in an Infors HT Multitron, shaking at 700 rpm. After 2 days, plates were spun down at 4000 × g, 4 °C for 10 min. Then, 300 µL of the supernatant was sampled for each well. The same day, supernatant samples were measured directly by LC-MS alongside a succinic acid standard, as follows: succinic acid was detected and measured 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 min then an increase to 98% over 0.5 min 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. In total, 0.2 µL was injected from both sample wells and standard solutions. Succinic acid concentrations were calculated from a succinic acid standard curve in Microsoft Excel.

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).