The use of immobilization matrix in bio-processing is a promising approach to immobilize a catalytic strain for production of biomolecules. In this study, a novel immobilization system of agarose-alginate cryogel was developed in the format of beads and characterized to facilitate the effective cell immobilization followed by enhanced solvent production compared to other immobilization matrix. Cryogel beads showed macroporous internal architecture and nano-range grooves on outer periphery. Study suggests that these grooves facilitate the convective medium transport throughout the cryogel beads in order to eliminate the substrate and product inhibition and also prevent cell leakage. The immobilization study was carried out on a typical anaerobic system of Clostridium acetobutylicum ATCC 824 for butanol production. The experiment was carried out in three different sets (A, B and C) with varying medium and substrate concentration. The adsorption of cells on agarose-alginate cryogel beads produced 11.79 g/l of butanol and 21.64 g/l total ABE (acetone, butanol and ethanol), while entrapment of cells on agarosealginate cryogel beads showed high glucose consumption, high butanol and total ABE production that was 92.16%, 14.47 g/l and 27.80 g/l, respectively, which was much higher than the control and other matrices. (C) 2010 Elsevier Inc. All rights reserved.