Spot assay

Spot assay was performed using cells precultured for 24 h in YPD media. From these cultures, 4 series of 1:10 dilution were prepared, starting with an initial optical density of 1 (660 nm). We spotted 5 μl of each of the five suspensions gradually diluted on the different plates and let the cells grow for 3 days at 30 °C. We performed each spot assay in duplicate.

We prepared two different kind of solid media to perform spot assay: one with different concentrations of SBP hydrolysate and one with minimal medium, added with different concentrations of acetic acid and/or lactic acid. As a control, minima medium plates without inhibitors were prepared.

Solid media containing SBP were prepared by mixing agar 2X (40 g/L, autoclaved) with 10% SBP diluted as needed.

The minimal media we used is MeOL medium: 1 g/L of yeast extract, 1.31 g/L of (NH₄)₂SO₄, 0.95 g/L of Na₂HPO₄, 2.7 g/L of KH₂PO₄, 0.2 g/L of Mg₂SO₄·7H₂O and 20 g/L agar. After the pH was adjusted to 5.5 using NaOH 4 M and autoclaved. The medium was supplemented with a 100X trace mineral stock solution consisting of: 4 g/L CaCl₂·2H₂O; 0.55 g/L FeSO₄·7H₂O; 0.52 g/L citric acid; 0.10 g/L ZnSO₄·7H₂O; 0.076 g/L MnSO₄·H₂O; and 100μL 18 M H₂SO₄. Glucose and Xylose were added as additional carbon source with a final concentration of 10 g/L each. Acetic and lactic acid were added in MeOL media at the desired concentrations.