A novel immobilization method of microorganisms was developed based on hybrid entrapment-encapsulation technique. Entrapment allows high mechanical strength, but contains some disadvantages such as cell leakage and cell loading limitation. However, even though encapsulation, high cell loading can be achieved, but the capsules are still very weak. Therefore, in this study, the hybrid entrapment-encapsulation system was explored to overcome the problems faced in each entrapment and encapsulation method. The hybrid immobilization was achieved using polyvinyl alcohol for entrapment and xanthan gum and Tween 20 for encapsulation. The clean spherical beads were formed at the optimal concentrations of these additives. Ochrobactrum anthropi SY509 was immobilized by this hybrid method and its biological denitrification was investigated in batch or packed-bed reactor. This hybrid immobilization increased the cell loading capacity and decreased the cell leakage from the beads, resulted in higher activity of immobilized cells compared with the single immobilization method. The denitrification efficiency of immobilized cells by the hybrid method was found to be higher 2.7 times than that of the traditional entrapped cells in batch reactor and the hybrid immobilized beads showed the high denitrification efficiency in the packed-bed reactor. (c) 2005 Elsevier Inc. All rights reserved.