2. Materials and methods
2.1. Substrate preparation
BSGwas obtained from microbrewery of the Engineering College of Lorena.
As soon as obtained the material was washed with water until neutral pH, and
dried at 50±5 ◦C to attain 10% moisture content (untreated material).
The untreated material was submitted to a reaction with dilute sulfuric acid
under conditions previously optimized for an efficient hemicellulose removal
from BSG [16], which consisted in the use of 1.25% (w/v) H2SO4 solution in
a 1:8 g:g solid:liquid ratio, 120 ◦C for 17 min. After reaction the resulting solid
material (cellulignin) was separated by centrifugation, washed with water until
neutral pH, and dried at 50±5 ◦C to attain 50% moisture content.
The cellulignin was treated with 2% (w/v) sodium hydroxide solution in
a solid:liquid ratio of 1:20 g:g, 120 ◦C for 90 min, conditions previously optimized
for an efficient lignin removal from BSG [17]. At the end of the reaction,
the residual solid material (cellulose pulp) was separated by filtration in 100%
polyester cloth, washed with water to remove the residual alkali, and dried at
50±5 ◦C to attain 10% moisture content. The chemical composition of the three
BSG samples used in the enzymatic assays is given in Table 1.