The steam-exploded material was drained in a Buchner funnel and the pretreatment solids were used for enzymatic hydrolysis and fermentation. The liquid fraction of this filtration procedure was named primary water solubles, whereas the solid retentate was designated as unwashed (UW) steam-exploded substrate. A portion of the solid retentate was washed with water to remove any possible substances that would still inhibit both hydrolysis and fermentation. For the washing procedure, 5% (w/v) of the pretreated biomass was dispersed in distilled water and stirred at
600 rpm for 1 h at ambient temperature. Then, the suspension was filtered through a polyester fabrics and the excess of liquid was removed via vacuum filtration. In this case, the liquid fraction and solid retentate of this filtration procedure were named sec- ondary water solubles and water-washed (W) steam-exploded substrate, respectively. A portion of the solid retentate (approximately 2 g) was removed for determining its moisture content while the rest was accommodated in plastic bags and stored at
4 C for further use.