Conidia of Aspergillus niger 200sm producing extracellular inulinase were immobilized on pumice stones or polyurethane sponge and used in repeated-batch processes. Some factors affecting inulinase biosynthesis by the mycelium A. niger immobilized on pumice stones were investigated. Maximal inulinase production occurred in 50 ml of medium containing 0.5 g of carrier at 30 degrees C, pH 6.0 and at an agitation speed of 200 rpm. This procedure enabled repeated-batch enzyme production and as many as six subsequent 24 h batches could be fermented by using the same carrier. This is the first report on inulinase biosynthesis by mycelium of A. niger immobilized on polyurethane sponge using unconventional oxygenation of culture which ensures that the dissolved oxygen concentration remains constant. (c) 2005 Elsevier Inc. All rights reserved.