Statistical experimental methods (SEM) were employed to study the effects of glutamic acid, citric acid, glycerol and initial medium pH on the production of poly(gamma-glutamic acid) (gamma-PGA) by Bacillus licheniformis CCRC 12826 in shaken cultures. The results of first-order factorial design experiments showed that the liner terms of glutamic acid, citric acid and glycerol had significant positive effects, but the initial medium pH exhibited insignificant effect on gamma-PGA production. The effects decreased in the order of glycerol, glutamic acid and citric acid. In addition, the interaction term of glutamic acid-glycerol exhibited a significant positive effect. Based on the results of the first-order factorial design experiment, the optimum composition was then investigated by using a central composite design (CCD). The experimental results of CCD were fitted with a second-order polynomial equation by a multiple regression analysis. The coefficient of determination (R-2) was 0.9078, the Fisher F-test was significant at upper 5% level and the lack of fit was insignificant at 5% level. All of these indicated a good adequacy of the second-order polynomial model proposed to explain the data observed. The optimal gamma-PGA yield (19.80+/-1.59 g/l) was determined by the CCD experiments and was predicted to be at the regions where respective concentrations of citric acid, glutamic acid and glycerol were around 24.50, 57.30 and 157.11 g/l, respectively. When the strain was cultivated using the optimized medium predicted by the model, the yield of gamma-PGA production was 19.62+/-1.07 g/l (average of three repeats). The gamma-PGA production by B. licheniformis CCRC 12826 was increased significantly by 372%, from 5.27 to 19.62 g/l when the strain was cultivated in the optimal medium developed by SEM, as compared to conventional medium E used in the literature. The gamma-PGA thus produced was shown to be a homogeneous polymer of glutamic acid by thin-layer chromatography and amino acid analysis, and its molecular weight was over 2 x 10(6) Da by GPC.