The production of succinic acid from biodiesel based glycerol was carried out using Escherichia coli K-12 MG1655 strain. The contribution of every parameter was evaluated and optimized using statistical modeling. A 2 6-1 fractional factorial design (FFD) was applied to screen the effect of substrate concentration, tryptone concentration, sodium carbonate concentration, inoculums density, pH and incubation period for Escherichia coli reaction on succinic acid production. The result of first order factorial design showed that glycerol concentration, tryptone concentration, incubation period and pH had significant positive effect (p>0.001) to the reaction. Meanwhile, inoculums density and Na 2 CO 3 concentration exhibited insignificant effect in the reaction. In addition, interaction between inoculums density of substrate concentration and tryptone concentration, glycerol concentration and Na 2 CO 3 concentration, tryptone concentration and pH, Na 2 CO 3 concentration and incubation period and inoculums density with pH gave significant effects (p<0.001) to succinic acid production.