BACKGROUND: The production of pullulan from synthetic medium by Aureobasidium pullulans P56 immobilized in Ca-alginate beads was investigated using batch and repeated batch fermentation systems. RESULTS: The highest pullulan concentration (19.52 +/- 0.37 g dm(-3)) was obtained with 2.0-2.4 mm. beads prepared from 2% sodium alginate solution. Pullulan production was mainly accomplished by immobilized fungal cells since leaked cells in the fermentation medium comprised 17.4% of the total fungal population at the end of fermentation. The pullulan proportion was 84.5% of the total polysaccharide in the fermentation medium. Response surface methodology was used to investigate the effects of three fermentation parameters (initial pH, agitation speed and incubation time) on the concentration of pullulan. Results of the statistical analysis showed that the fit of the model was good in all cases. The maximum pullulan concentration of 21.07 +/-0.48 g dm(-3) was obtained at the optimum concentrations of process variables (pH 7.31, agitation speed 191.5 rpm, incubation time 101.2 h). The gel beads produced pullulan under the optimized conditions for six consecutive batch fermentations without marked activity loss and deformation. CONCLUSION: The results of this study suggest that the immobilization of A. pullulans cells in Ca-alginate gel beads is suitable for batch and repeated batch production of pullulan. (C) 2007 Society of Chemical Industry.