Quasi-SSFs were carried out in 250-mL Erlenmeyer flasks using
a shaker incubator (Thermo Fisher Scientific, Model 4450, Waltham,
MA) at 35 C and 90 rpm with 10% solid substrate (water
insoluble). The enzyme loadings were the same as for enzymatic
hydrolysis described above. The solid substrate was first liquefied
at 50 C and 200 rpm for 120 min on the shaker incubator before
adding the yeast S. cerevisiae FPL-450. Initial cell concentration
for all SSF experiments was 0.4 mg dry cell g1 substrate. No nutrients
were added for all fermentation experiments. Samples of the
fermentation broth were taken every 24 h and centrifuged at
10,000 g for 5 min and stored at 4 C until analyzed for sugar
and ethanol. Reported results are the average of duplicates with
an average relative standard deviation of about 4%.