To enhance biohydrogen production, Clostridium beijerinckii was co-cultured with Geobacter metallireducens in the presence of the reduced extracellular electron shuttle anthrahydroquinone-2, 6-disulfonate (AH2QDS). In the co-culture system, increases of up to 52.3% for maximum cumulative hydrogen production, 38.4% for specific hydrogen production rate, 15.4% for substrate utilization rate, 39.0% for substrate utilization extent, and 34.8% for hydrogen molar yield in co-culture fermentation were observed compared to a pure culture of C. beijerinckii without AH2QDS. G. metallireducens grew in the co-culture system, resulting in a decrease in acetate concentration under co-culture conditions and a presumed regeneration of AH2QDS from AQDS. These co-culture results demonstrate metabolic crosstalk between the fermentative bacterium C. beijerinckii and the respiratory bacterium G. metallireducens and suggest a strategy for industrial biohydrogen production. Biotechnol. Bioeng. 2013; 110: 164172. (C) 2012 Wiley Periodicals, Inc.