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.