AbstractThe objective of this study was to optimize the fermentation parameters by using response surface methodology for increasing pullulan production of Aureobasidium pullulans AZ-6 in synthetic fermentation medium. The optimum conditions were determined as follows: the initial sucrose concentration (X1): 100 g/L; the initial peptone concentration (X2): 11.31 g/L; the initial pH (X3): 6.48; and the temperature (X4): 24.2 °C. In the optimum conditions, the maximum exopolysaccharide (EPS) concentration (Y1), the maximum pullulan concentration (Y2), the specific growth rate (Y3), the maximum specific pullulan formation rate (Y4), and the pullulan yield (Y5) were expected to be obtained as 37.078 and 35.372 g/L, 0.062 h−1, 0.021 [g pullulan/(g mo.h)], and 53.681%, respectively. The observed values of Y1, Y2, Y3, Y4, and Y5 were 36.950 g/L, 35.470 g/L, 0.064 h−1, 0.036 [g pullulan/(g mo.sa)], and 54.480%, respectively, as a result of validation experiments. EPS samples were characterized by Fourier-transform infrared spectroscopy and scanning electron microscope analyses. The optimization process caused more than a 50% increase in EPS and pullulan concentrations. This study showed that the color-variant A. pullulans AZ-6 strain could become a significant industrial strain if it is explored further in the future to produce pullulan on a larger scale.