The production of pullulan from hydrolysed potato starch waste by Aureobasidium pullulans P56 was investigated. The liquefaction of potato starch was done by Ca-alginate immobilized amyloglucosidase and pullulanase enzymes in a packed bed bioreactor. Various organic nitrogen sources were tested and none of the nitrogen sources gave pullulan concentrations as high as that obtained with yeast extract. Response surface methodology was used to investigate the effects of three factors (incubation time, initial substrate concentration and initial pH) on the concentration of pullulan in batch cultures of A. pullulans. No previous work has used statistical analysis on the optimization of process parameters in pullulan production from hydrolysed potato starch waste. Maximum pullulan concentration of 19.2 g/l was obtained at the optimum levels of process variables (incubation time 111.8 h, initial substrate concentration 79.4 g/l, initial pH 7.26). The optimization led to a 20% increase in pullulan concentration. (C) 2010 Elsevier Ltd. All rights reserved.