Cellular Surface Hydrophobicity Assay

The cellular surface hydrophobicity (CSH) was determined by a two-phase system following previous reports [26 (link)]. In brief, yeasts were grown in SD broth at 28 °C for 24 h. Then, cells were washed with sterile saline buffer and 0.5% Tween 20 was added; they were resuspended in 0.05 M PBS (pH 7.2) at a final concentration of 2 × 10⁶ cells/mL. The cell suspension was transferred to a glass tube containing 500 μL octane (Sigma Aldrich, Saint Louis, MO, USA). The mixture was vortexed for 1 min and maintained at room temperature for phase separation. After the two phases had been separated, the aqueous phase was measured at OD600. The group without the octane overlay was used as the control. Relative CSH was calculated as follows: [(OD600 of the control-OD600 after octane overlay)/OD600 of the control] × 100. The value for each strain was the average of three independent biological replicates.