Application of statistical experimental methods to optimize production of poly(γ-glutamic acid) by Bacillus licheniformis CCRC 12826

Abstract

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(γ-glutamic acid) (γ-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 γ-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 γ-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 γ-PGA production was 19.62±1.07 g/l (average of three repeats). The γ-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 γ-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 2×10 6 Da by GPC.