A metabolically engineered Escherichia coli strain SBS550MG (pHL413) was used in this study to investigate the impact of various culture operating conditions for improving the specific succinate production rate for better final titer while maintaining the theoretical succinate yield on glucose in multiphase fed-batch cultures. Previously, we reported that changes in the level of aeration during the cell growth phase significantly modified gene expression profiles and metabolic fluxes in this system (Martinez et al. 2010). Based on these observations, the examination of culture conditions was mainly focused on the aerobic growth phase. It was found that 2-5 h of low dissolved oxygen culture during the aerobic phase improves cell productivity, but pH control during the aerobic phase was not favorable for the system. Cell viability has been identified as a major limiting factor for succinate production. Supplementing LB medium and betaine, an anti-osmotic stress reagent, did not improve cell activity. A higher succinate titer (537.8 mM) using the current metabolic engineering E. coli strain was achieved, which can potentially be improved further by increasing cell viability.