A process was developed for seed culture expansion (3.6 million-fold) using 5% of the hemicellulose hydrolysate from dilute acid pretreatment as the sole organic nutrient and source of sugar. Hydrolysate used for seed growth was neutralized with ammonia and combined with 1.0 mM sodium metabisulfite immediately before inoculation. This seed protocol was tested with phosphoric acid pretreated sugarcane and sweet sorghum bagasse using a simplified process with co-fermentation of fiber, pentoses, and hexoses in a single vessel (SScF). A 6 h liquefaction (L) step improved mixing prior to inoculation. Fermentations (L + SScF process) were completed in 72 h with high yields (>80 gal/US ton). Ethanol titers for this L + SScF process ranged from 24 g/L to 32 g/L, and were limited by the bagasse concentration (10% dry matter). (C) 2012 Elsevier Ltd. All rights reserved.