A co-culture of Clostridium beijerinckii and Geobacter metallireducens with AH2QDS produced hydrogen from lignocellulosic hydrolysates (biomass of Miscanthus prepared by hydrothermal treatment with dilute acids). This co-culture system enhanced hydrogen production from lignocellulosic hydrolysates by improving substrate utilization and diminishing acetate accumulation, despite the presence of fermentation inhibitors in the hydrolysates. The improvements were greater for xylose-rich hydrolysates. The increase in maximum cumulative hydrogen production for hydrolysates with glucose:xylose mass ratios of 1:0.2, 1:1 and 1:10 g/g was 0%, 22% and 11%, respectively. Alternative extracellular electron shuttles (EES), including indigo dye, juglone, lawsone, fulvic acids and humic acids, were able to substitute for AH2QDS, improving hydrogen production in the co-culture system using xylose as model substrate. Increased utilization of xylose-rich hydrolysates and substitution of alternative EES make the co-culture with EES system a more attractive strategy for industrial biohydrogen production.