Pretreatment and enzymatic hydrolysis
Alkali and acid pretreatments and biomass enzymatic hydrolysis
were performed as previously described by Xu et al. (2012) with
minor modifications on chemical concentrations as described
below.
2.6.1. One-step pretreatment by H2SO4 and NaOH
The well-mixed biomass powders were pretreated by H2SO4
and NaOH. For one-step pretreatment with acid, the biomass sample
was added with H2SO4 at various concentrations (%, w/w) in a
plastic centrifuge tube, and treated at 121 C for 20 min in an autoclave.
The tube was then cooled down, and shaken at 150 rpm and
50 C for 2 h. The sample was finally centrifuged at 3000g for
5 min, and the supernatant was collected for determination of free
hexoses and pentoses while the residues were collected for plant
cell wall composition analysis. For one-step pretreatment with
alkali, the biomass sample was added with NaOH at various concentrations
(%, w/v) in a plastic centrifuge tube, and shaken under
150 rpm at 50 C for 2 h. The treated sample was centrifuged at
3000g for 5 min, and the supernatant was collected for assay of
hexoses and pentoses, and the residues were collected for plant
cell wall composition analysis. The biomass sample added with
distilled water was shaken for 2 h at 50 C as the control, and all
samples were carried out in biological triplicate.