A mixed culture system was considered in the present research where sugars such as glucose were converted to lactate by Lactobacillus delbrueckii and the lactate was converted to poly beta-hydroxybutyrate (PHB) by Ralstonia eutropha in one fermenter. Based on the experimental studies on the effect of lactate concentration on the cell growth of both microorganisms, the lactate concentration was controlled at less than 5 g/l using an on-line enzymatic lactate and glucose sensors with the FIA (flow injection analysis) system, and by manipulating the glucose feeding rate. Since L. delbrueckii prefers anaerobic conditions while R.eutropha prefers aerobic conditions, we studied the effect of DO concentration on fermentation characteristics of each microorganism. For the mixed culture, we considered the control scheme of a two inputs and three outputs multivariable system. It was experimentally shown that the periodic fermentation resulted in superior PHB yield with relatively high productivity as compared with the cases where DO concentration was controlled to be constant either at less than 1 ppm or 3 ppm.