A considerable amount of volatile solids (VS) contained in the biomass of microalgae makes it promising for use as feedstock in fermentation processes. In this study, a biomass of microalga Chlorella sp. was used as a sole substrate for hydrogen production in an anaerobic solid-state fermentation (ASSF). Optimization of the process was investigated on the selected critical variables, i.e., total solid (TS) content, initial pH, and feed to inoculum (F/I) ratio (on a VS basis) using response surface methodology (RSM) with central composite design (CCD). TS content and F/I ratio were found to have statistically significant effects on hydrogen production. Maximal hydrogen production of 165 +/- 12 mL H-2, equivalent to 18.58 mL H-2/g VS and 0.28 L H-2/L reactor d, was achieved under the optimal conditions of 38.83% TS, pH 6.03, and an F/I ratio of 4.33. Acetic and butyric acids were found to be main soluble microbial products (SMPs) in the fermented biomass. Based on the compositions of the biomass, an equation for theoretical bioconversion of Chlorella sp. biomass to hydrogen was proposed. (C) 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.