Fermentation
For early screening, the transformants obtained were cultivated overnight using 5 mL of YPD medium (30 °C, 150 rpm) in a test tube. The fermentation broths were sampled for further quantification, and the cells were observed using a microscope (BZ-X810, Keyence, Japan) equipped with an oil immersion lens (100x magnification). For cultivation with no inhibitors, the recombinant yeast colonies with the highest lactic acid accumulation were initially precultured using 12 mL of YPD medium (30 °C, 150 rpm) in a 100 mL flask for 1-2 days until reaching the target cell density. The initial optical cell density (OD600nm) was adjusted to 1 and 50 for low- and high-density cell cultures, respectively. The cells were washed with water and cultivated (30 °C, 90 rpm) using 12 mL of YPD medium in a 100 mL flask without the supplementation of a neutralizing agent. Due to the rapid cell coagulation in the flocculant strain, which causes difficulty in taking homogenous samples, the glucose consumption rate was measured, as opposed to the cell concentration, to monitor the progress of fermentation. Glucose concentration was monitored using a glucose CII-test kit (Fujifilm Wako Pure Chemical Corporation, Japan). For cultivation with 20% inhibitory chemical complexes (20% ICC), 12 mL of YPD medium containing 60 mM acetic acid, 30 mM formic acid, 60 mM furfural, 5 mM levulinic acid, and 10 mM 5-hydroxymethyl furfural (5-HMF) was used as the medium. The experiments were conducted with three biological replicates, and sampling points before complete glucose consumption were selected for further comparative analyses.