Corncobs pretreatment Corncobs (obtained from local market in Tokyo,
Japan) were sliced into small pieces and then sun-dried for 3 days before ball-milled
with P-6 (Fretsch Co. Ltd, Germany). Ball-milled corncobs with a final particle size of
about 5 mm were used throughout experiments. Corncobs were pretreated with H2SO4
solution at 0.5% and 5% (vol./vol.) and autoclaved at temperatures of 105 °C, 122 °C,
and 128 °C for 20 min and 2 h, respectively. After cooling at room temperature, the
pretreated corncobs were passed through the glass membrane filter to remove the
H2SO4 solution and were then washed with 50 mM citric buffer solution (pH 5.0) to
shift the substrate pH to around 5. The washed corncobs were soaked in 50 mM citric
buffer (pH 5.0) before the saccharification and fermentation.