The aim of this study was to assess the synergistic effects of co-fermentation of glucose, starch, and cellulose using anaerobic digester sludge (ADS) on the biohydrogen (H-2) production and the associated microbial communities. Yields of H-2 were 1.22, 1.00, and 0.15 mol H-2/mol hexose-added in fermentation reactions containing glucose, starch, and cellulose as mono-substrates. The H-2 yields were greater by an average of 27 +/- 4% than expected in all the different co-substrate conditions, which affirmed that co-fermentation of different substrates improved the H-2 potential. Glucose addition to starch and/or cellulose favored acetate synthesis, while cellulose degradation was associated with the propionate synthesis pathway. Illumina sequencing technology and bioinformatics analyses revealed operational taxonomic units (OTUs) in the Phyla Bacteroides, Chloroflexi, Firmicutes, Proteobacteria, Spirochaetes, Synergistes, and Thermotogae were common in mono- and co-substrate batches. However, OTUs in the Phyla Acidobacteria, Actinobacteria, and Bacteroidetesee were unique to only the co-substrate conditions. Copyright (c) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.