A novel and economical method was used to optimize bacterial growth and bioemulsan production. Acinetobacter calcoaceticus PTCC 1641 was applied in an innovated synthetic medium comprised whey and mineral salt medium (MSM) in ratio of 1:1 in a shaken flask bioreactor. The optimum inoculum size, shaking frequency rate and filling volume (V(L)) using ventilation flasks were investigated that resulted in 5 vol.%, 300 rpm and 15 ml), respectively. The surface tension decreased around 48% during bioemulsan production. Due to determine bioemulsan properties, the reliability of blood agar method was confirmed as well. (C) 2010 Elsevier Ltd. All rights reserved.