In this work, a carbohydrate-rich microalga, Chlorella vulgaris ESP6, was grown photoautotrophically to fix the CO2. The resulting microalgal biomass was hydrolyzed by acid or alkaline/enzymatic treatment and was then used for biohydrogen production with Clostridium butyricum CGS5. The C. vulgaris biomass could be effectively hydrolyzed by acid pretreatment while similar hydrolysis efficiency was achieved by combination of alkaline pretreatment and enzymatic hydrolysis. The biomass of C. vulgaris ESP6 containing a carbohydrate content of 57% (dry weight basis) was efficiently hydrolyzed by acid treatment with 1.5% HCl, giving a reducing sugars (RS) yield of nearly 100%. C. butyricum CGS5 could utilize RS from C. vulgaris ESP6 biomass to produce hydrogen without any additional organic carbon sources. The optimal conditions for hydrogen production were 37 degrees C and a microalgal hydrolysate loading of 9 g RS/L with pH-controlled at 5.5. Under the optimal conditions, the cumulative H-2 production, H-2 production rate, and H-2 yield were 1476 ml/L, 246 ml/L/h, and 1.15 mol/mol RS, respectively. The results demonstrate that the C. vulgaris biomass has the potential to serve as effective feedstock for dark fermentative H-2 production. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.