Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and beta-glucosidase activities 3- and 8 -fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3 -fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulase activity/g glucan was surprisingly effective when compared to the 5-15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme mixtures from the sequential fermentation could be one major reason for the more efficient enzyme hydrolysis that results when using the combined secretomes from A. niger and T. reesei. (C) 2016 Elsevier Inc. All rights reserved.