This paper studies the metabolic pathway of sucrose in pullulan fermentation by Aureobasidium pullulans. Because of its high pullulan production, sucrose proved to be the best carbon source for pullulan synthesis by A. pullulans CGMCC1234 (36.3 g/L). Compared to other carbon sources, A. pullulans cells reached the stationary phase more quickly in the presence of sucrose. The specific sugar types and concentrations occurring during pullulan fermentation were detected using High Performance Liquid Chromatography (HPLC). HPLC results revealed that sucrose did not simply break down into glucose and fructose in the medium employed. Kestose (22.69 g/L) also accumulated during early stages of fermentation (24 h), which reduced the osmotic pressure of the extracellular fluid and diminished the inhibition of cell growth and pullulan production. beta-Fructofuranosidase activity strongly depended on the carbon source. Sucrose was the best inducer of beta-fructofuranosidase production. However, beta-fructofuranosidase production did not directly and/or proportionally correlate with the growth of A. pullulans cells. (C) 2016 Elsevier Inc. All rights reserved.