The present study deals with the optimization of pretreatment conditions followed by thermophilic dark fermentative hydrogen production using Anabaena PCC 7120 as substrate by mixed microflora. Different airlift photobioreactors with ratio of area of down-comer and riser (A(d)/A(r)) in range of 0.4-3.2 were considered. Maximum biomass concentration of 1.63 g L-1 in 9 d under light intensity of 120 mu E m(-2) s(-1) was observed at A(d)/A(r) of 1.6. The mixing time of the reactors was inversely proportional to A(d)/A(r). Maximal H-2 production was found to be 1600 mL L-1 upon pretreatment with amylase followed by thermophilic fermentation for 24 h compared to other methods like sonication (200 mL L-1), autoclave (600 mL L-1) and HCl treatment (1230 mL L-1). The decrease of pH from 6.5 to 5.0 during fermentation was due to the accumulation of volatile fatty acids. Amylase pretreatment gave higher reducible sugar content of 7.6 g L-1 as compare to other pretreatments. Thermophilic fermentation of pretreated Anabaena biomass by mixed bacterial culture was found suitable for H-2 production. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.