Optimization for the production of Pest an was followed by the Plackett-Burman Design, using modified Czapek-dox medium as the starting point. At the flask level, $K_2HPO_4$, $MgSO_4{cdot}7H_2O$, and aeration variables positively affected the Pestan production, DCW (dry cell weight), apparent viscosity, and flocculating activity response. KCI and $FeSO_4{cdot}7H_2O$ negatively affected the Pestan production, DCW, apparent viscosity, and flocculating activity response. Aeration variable was shown to have a positive effect on only the flocculating activity response among Pestan production, DCW, and apparent viscosity responses. In comparison of the positive and negative variables media conditions, Pestan production and flocculating activity differed by about 9 and 125 times, respectively. In particular, at the jar fermentor level, the aeration variable was the most important factor of the all responses (pestan production, DCW, apparent viscosity, flocculating activity, and anionic charge density). The flocculating activity and apparent viscosity of Pestan were closely related to the molecular chain length and charge density.