Enzymatic hydrolysis, and the SSF process for optimal conditions
The enzymatic hydrolysis and the SSF experiments were conducted for optimal
pretreatment condition. The operating conditions of the enzymatic hydrolysis were
50 ◦C and pH 4.8 (0.05 M sodium citrate buffer) on a shaking incubator at 150 rpm
for 48 h. Enzyme loading amount was 30 FPU/g cellulose (Cellic® CTec II), and 15%
Cellic® HTec II (this value was based on the amount of loaded Cellic® CTec II). 10 g
of pretreated biomass was loaded for enzymatic hydrolysis, and total liquid and
solid was set to 100 mL. To investigate the effects of pretreatment on SSF, the same
amount of enzyme as used for enzymatic hydrolysis and 7% (v/v) yeast (Saccharomyces
cerevisiae CHY 1011) were added to each sample with a working volume
of 100 mL. The operating conditions for SSF were at 32 ◦C and pH 4.8 on a shaking
incubator at 150 rpm for 72 h. Samples were periodically analyzed for glucose
and ethanol concentration using high performance liquid chromatography (HPLC;
Waters, Milford, MA, USA) using a Bio-Rad Aminex HPX-87P column (Hercules, CA,
USA), and a refractive index detector. The mobile phase was deionized water at a
flow rate of 0.6 mL/min at 85 ◦C.