This work aimed to identify the key operational factors that significantly affect succinate production by the high succinate producing Escherichia coli strain SBS550MG (pHL413), which bears mutations inactivating genes adhE 1dhA iclR ackpta:: Cm-R and overexpresses the pyruvate carboxylase from Lactococcus lactis. The considered factors included glucose concentration, cell density, CO2 concentration in the gas stream, pH, and temperature. The results showed that high glucose concentrations inhibited succinate production and that there is a compromise between the total succinate productivity and succinate specific productivity, where the total productivity increased with the increase in cell density and the specific productivity decreased with cell density, probably due to mass transfer limitation. On the other hand, a CO2 concentration of 100% in the gas stream showed the highest specific succinate productivity, probably by favoring pyruvate carboxylation, increasing the OAA pool that later is converted into succinate. A full factorial design of experiments was applied to analyze the pH and temperature effects on succinate production in batch bioreactors, where succinate yield was not significantly affected by either temperature (37 to 43 degrees C) or pH (6.5 to 7.5). Additionally, the temperature effect on succinate productivity and titer was not significant, in the range tested. On the other hand, a pH of 6.5 showed very low productivity, whereas pH values of 7.0 and 7.5 resulted in significantly higher specific productivities and higher titers. The increase on pH value from 7.0 to 7.5 did not show significant improvement. Then, pH 7.0 should be chosen because it involves a lower cost in base addition. (C) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 1225-1231, 2011