To remove the inhibitory ligneous material, samples were pretreated with 2% NaOH (20 mL/g of biomass) for 80 min at 121 C [16].
After this alkali-mediated pretreatment, extensive washing was performed with distilled water to remove all the traces of alkali material and its byproducts, until the effluent pH reached 7.0.
Once the effluent became neutral, the pretreated substrate was recovered through filtration and subsequently soaked in a buffer solution (pH 5.0) overnight.
The pretreated biomass was then recovered from the buffer solution and dried at 60 C overnight to reach a stable dryness.
All chemicals and reagents used in the study are of analytical grade and purchased from Sigma-Aldrich (Seoul, Korea).