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.