Various factors like amount of carbon source, concentration of nitrogen sources (like NaNO2 and yeast extract), initial pH, concentration of NaCl, concentration of MgSO4·7H2O and concentration of ZnSO4·5H2O were considered. After screening variables that significantly influenced the process, using Plackett–Burman fractional factorial design [results not shown], the levels of the screened variables were optimized to achieve maximum pullulan yield using Box–Behnken design of experiment.