Saccharification of sugarcane bagasse pretreated at the pilot-scale with different processes (in combination with steam-explosion) was evaluated. Maximum glucan conversion with Celluclast 1.51 (15-25 FPU/g glucan) was in the following order: glycerol/HCl>HCl>H2SO4>NaOH, with the glycerol system achieving similar to 100% conversion. Surprisingly, the NaOH substrate achieved optimum saccharification with only 8 FPU/g glucan. Glucan conversions (3.6-6%) obtained with mixtures of endo-1,4-beta-glucanase (EG) and beta-glucosidase (beta G) for the NaOH substrate were 2-6 times that of acid substrates. However, glucan conversions (15-60%) obtained with mixtures of cellobiohydrolase (CBH I) and beta G on acidified glycerol substrate were 10-30% higher than those obtained for NaOH and acid substrates. The susceptibility of the substrates to enzymatic saccharification was explained by their physical and chemical attributes. Acidified glycerol pretreatment offers the opportunity to simplify the complexity of enzyme mixtures required for saccharification of lignocellulosics. (C) 2013 Elsevier Ltd. All rights reserved.