The aim of this study was to develop a bioprocess for the production of beta-glucosidase and pectinase from the fungus Annulohypoxylon stygium DR47. Media optimization and bioreactor cultivation using citrus bagasse and soybean bran were explored and revealed a maximum production of 6.26 U/mL of pectinase at pH 4.0 and 10.13 U/mL of beta-glucosidase at pH 5.0. In addition, the enzymes extracts were able to replace partially Celluclast 1.5L in sugar cane bagasse hydrolysis. Proteomic analysis from A. stygium cultures revealed accessory enzymes, mainly belong to the families GH3 and GH54, that would support enhancement of commercial cocktail saccharification yields. This is the first report describing bioreactor optimization for enzyme production from A. stygium with a view for more efficient degradation of sugar cane bagasse. (C) 2014 Elsevier Ltd. All rights reserved.