BACKGROUNDPullulan with high molecular weight (Mw) is attractive for industrial application. Previous studies indicated that high yield and high Mw of pullulan cannot be simultaneously obtained under constant agitation speeds or constant dissolved oxygen levels during batch pullulan fermentation. In this study, a two-stage agitation speed control strategy was developed to obtain pullulan with both high yield and high Mw. RESULTSBatch pullulan production at various constant agitation speeds ranging from 200 to 600rpm was carried out in a 5L stirred tank fermentor. Based on kinetic analysis of batch processes, activity assays of the enzymes associated with pullulan biosynthesis and biodegradation, and intracellular energy supply under different constant agitation speeds, a two-stage control strategy that shifted the agitation speed from 500 to 400rpm at 18h proved to effectively achieve simultaneous high yield of high Mw pullulan. CONCLUSIONA maximum pullulan concentration of 27.4gL(-1) and final Mw of 3.09x10(6)Da were obtained using the two-stage agitation speed control strategy, which were both greatly improved compared with those obtained at constant agitation speeds. (c) 2014 Society of Chemical Industry