This study evaluated fermentative biohydrogen production from sucrose supplemented with dairy cattle manure at different sucrose:manure ratios. Hydrogen yields found in this study (2.9-5.3 M hydrogen/M sucrose) at ambient temperature are higher than literature results obtained at mesophilic temperatures. This study demonstrated that dairy cattle manure could serve as a buffering agent to maintain recommended pH levels; as a nutrient source to provide the required nutrients for hydrogen production; as a seed to produce hydrogen from sucrose; and as a co-substrate to improve the hydrogen yield. Based on an analysis of the net energy gain, it is concluded that positive net energy gains can be realized with non-thermal pretreatment and/or by combining dark fermentation with anaerobic digestion or microbial fuel cells to extract additional energy from the aqueous products of dark fermentation. (C) 2011 Elsevier Ltd. All rights reserved.