2.7. Analytical methods
Cellulose, hemicelluloses and lignin of the solid fraction of the
slurry were determined according to NREL protocols (Sluiter
et al., 2011). The slurry was first centrifuged at 4000g for 5 min
to separate the solid and the liquid fractions. The solid fraction
was washed with 40 mL deionized water thrice to neutral pH
and then freeze-dried (Labconco, USA) at 52 C until its moisture
content was reduced to less than 10%. It was then hydrolyzed in
two steps using 72% H2SO4 in a shaker water bath at 30 C for
60 min, followed by hydrolysis by 4% H2SO4 in an autoclave at
121 C for 60 min. Acid soluble lignin (ASL) was determined using
a UV spectrophotometer (Libra S60, Biochrom, England) at 283 nm.
Acid insoluble lignin (AIL) was gravimetrically determined as
residual solid after hydrolysis corrected with ash content. The
ash content was determined in the muffle furnace at 575 C overnight.
Monomeric sugars contained in the hydrolysis liquid were
determined by HPLC.