Effect of different environmental and nutritional factors on succinic acid production by batch fermentation of Enterococcus flavescens and enzymes involved in the acid production through reverse tricarboxylic acid cycle (TCA) cycle was investigated. An overall seven-fold increase in succinic acid production (from 0.92 gl(-1) in 72 h initially to 6.7 gl(-1) in 48 h) was achieved in 300-ml of optimized medium having 3% sucrose, 1:0.5 ratio of tryptone and ammonium hydrogen phosphate, 15 mM MgCO3 at pH 6.5 when inoculated with 4% (v/v) of seed inoculum and incubated at 39 degrees C for 48 h, as against initial un-optirnized medium. Subsequent scale-up in a 10-1 bioreactor using these optimized fermentation conditions under controlled pH and continuous CO2 supply resulted in 14.25 gl(-1) of succinic acid in 30 h. Optimization of the environmental and nutritional parameters resulted in a maximum production of succinic acid by affecting the levels of the enzymes involved in its production via the reverse TCA cycle. A linear relationship was observed between succinic acid production and in the enzyme activities. The enzyme activity was found to be increase in order phosphoenol pyruvate carboxykinase < malate dehydrogenase < fumarase furnarate reductase < phosphoenol pyruvate carboxylase. (c) 2006 Elsevier Inc. All rights reserved.