Microbial Vanillin Tolerance Assay

LB medium was used for Escherichia coli, CM2B medium (10 g/L polypeptone, 10 g/L yeast extract, 5 g/L NaCl, 10 μg/L biotin, pH 7.0 adjusted with KOH) for C. glutamicum, and YPD medium (10 g/L yeast extract, 20 g/L Bacto peptone, 20 g/L glucose) for Saccharomyces cerevisiae. Culture temperature was set as 30 °C (S. cerevisiae), 31.5 °C (C. glutamicum), or 37 °C (E. coli). A 20 μL aliquot of each glycerol stock of the strains was applied to each agar medium and cultured for 20 h as pre-culture. The obtained cells were washed and suspended in sterile physiological saline. The optical density (OD) at 620 nm (OD_620nm) of the cell suspension was measured, and the cell suspension was inoculated into an L-shaped test tube containing 4 mL medium (initial OD_620nm: 0.02 for “Comparison of vanillin tolerance” and “Identification of the aromatic aldehyde reductase (AAR) that converts vanillin to vanillyl alcohol”, and 0.2 for “Evaluation of vanillin tolerance of FKFC14 strain”). Culturing was performed using a culture apparatus equipped with an automatic OD measurement function (TVS062CA ADVANTEC). OD_660nm was measured every 15 min. For “Comparison of vanillin tolerance”, 0, 1, or 2 g/L vanillin was added to the medium before inoculation. For “Identification of the aromatic aldehyde reductase (AAR) that converts vanillin to vanillyl alcohol”, 1 g/L vanillin was added to the medium before inoculation. For “Evaluation of vanillin tolerance of FKFC14 strain”, 0, 3, or 6 g/L of vanillin was added to the medium in the middle of the log phase. Specific growth rates (μ) were calculated according to the following equation: ln X_t = ln X₀ + μ, where X_t and X₀ are optical density measurements at time t and time 0, respectively.