For the production of poly(gamma-glutamic acid), a newly isolated Bacillus sp. RKY3 was phylogenetically identified as Bacillus subtilis based on its 16S rRNA gene sequence. The culture medium for the production of poly(gamma-glutamic acid) by B. subtilis RKY3 was optimized statistically. The parameters significantly affecting poly(gamma-glutamic acid) production were found to be glycerol, glutamic acid, yeast extract, and K(2)HPO(4). A further advanced statistical approach, central composite design, found the optimum levels of the screened variables as follows (g l(-1)): glycerol 17.6, glutamic acid 59.6; yeast extract 2.7; K(2)HPO(4) 2.3. The predicted response as poly(gamma-glutamic acid) production under the statistically optimized conditions was 48.5 g l(-1), which was only 0.4% different from the maximum poly(gamma-glutamic acid) concentration (48.7 g l(-1)) observed at the validation experiment using 7-1 lab-scale fermentor containing 31 of working volume. (C) 2010 Elsevier Ltd. All rights reserved.