Fermentation
A yeast strain was initially pre-cultured (30 °C, 150 rpm) using YPD
medium until the optical density (OD600nm) reached
around 50. Cells were cultivated (30 °C, 90 rpm) in a 100-mL Erlenmeyer
flask containing 12 mL of YPD medium supplemented with 50
g∙L-1 sterilized calcium carbonate (Sigma-Aldrich).
For fermentation without a neutralizing agent, however, the medium was
not supplemented with calcium carbonate.
Furthermore, recombinant yeast was also cultivated using
sterile-filtered sugarcane bagasse hydrolysate obtained from a
pre-treatment process that mixed liquefied C6 and C5 fractions obtained
from sugarcane bagasse following treatment with hot water. This medium
(pH 4.5) contained 50 g·L-1 glucose and 25
g·L-1 xylose with inhibitory chemical compounds (ICC)
of furfural (4.5 mM), 5-HMF (3.4 mM), acetic acid (66 mM), formic acid
(7.2 mM), and lactic acid (7 mM). After pre-culturing (30 °C, 150 rpm),
cells were cultivated (30 °C, 90 rpm) in a 100-mL Erlenmeyer flask
containing 12 mL of sterile hydrolysate without the addition of calcium
carbonate.