Chitosan was produced by Rhizopus oryzae 00.4367 in shake flask culture and a stirred tank fermenter. Synthetic medium, treated and untreated beet molasses were used as cultivation media in shake flask cultures. In the stirred tank fermenter, the cultivation media were synthetic medium and untreated beet molasses. Shake flask culture containing untreated molasses with a sugar concentration of 40 g dm(-3) produced the maximum chitosan yield (961 mg dm(-3)). Chitosan concentration reached its maximum value at the late exponential growth phase of R oryzae. In all experiments almost 8-10% of biomass and 32-38% of alkali-insoluble material was extracted as chitosan. A central composite design was employed to determine the optimum values of process variables (aeration rate, agitation speed and initial sugar concentration) leading to maximum chitosan concentration in the stirred tank fermenter. In all cases, the fit of the model was found to be good. Aeration rate, agitation speed and initial sugar concentration had a strong linear effect on chitosan concentration. Moreover, the concentration of chitosan was significantly influenced by the negative quadratic effects of the given variables and by their positive or negative interactions. A maximum chitosan concentration of 1109.32 mg dm(-3) was obtained in untreated molasses medium containing an initial sugar concentration of 45.37 g dm(-3) with an aeration rate and agitation speed of 2.10 vvm. and 338.93 rpm, respectively. (C) 2004 Society of Chemical Industry.