Gamma-poly(glutamic acid) formation by Bacillus licheniformis 9945a: physiological and biochemical studies

Cryogenically frozen vegetative cells of Bacillus licheniformis 9945a derived from young mucoid colonies were used to inoculate gamma-poly(glutamate) (gamma-PGA) production media containing L-glutamate, citrate and glycerol as carbon sources. A gel permeation chromatography (GPC) method was developed to determine gamma-PGA volumetric yield and molecular weight directly using culture filtrates. For GPC volumetric yield measurements, a calibration curve was generated using purified gamma-PGA to relate the gamma-PGA GPC peak area and polymer weight. Purified gamma-PGA was characterized by elemental analysis, 1H- and 13C-NMR spectroscopy. Cultures of B. licheniformis using all three carbon sources showed the following characteristics: cell growth mainly during the first 24 h; largest gamma-PGA volumetric productivity (approximately 0.12 gl-1 h-1) between 48 and 96 h; 11 g l-1 gamma-PGA volumetric yield by 96 h; reduction (utilization) of glycerol, glutamate and citrate during a 96 h cultivation time from 80 to 45 g l-1, 18 to 10 g l-1 and 12 to approximately 1 g l-1, respectively; a decrease in pH from 7.4 to approximately 5.5 by 42 h cultivation; acetic acid secretion into the medium at a maximum level of approximately 4.5 g l-1 and detection of the metabolite 2,3-butanediol (as acetoin) as a fermentation by-product at approximately 42 h and through a 96 h cultivation period. The presence of 2,3-butanediol indicated that the level of oxygen in the medium no longer supported a fully aerobic mode of metabolism. When the medium formulation was altered by removal of either citrate, L-glutamate or glycerol in shake flask experiments where pH was not controlled, 2.3, 9.0 and 4.0 g l-1, respectively, of gamma-PGA were formed. Variation of the medium ionic strength by the addition of up to 4% (w/v) NaCl led to the formation of gamma-PGA of relatively higher molecular weight but lower volumetric yield. Studies carried out on 5-day-old B. licheniformis cultures suggested that gamma-PGA depolymerase is intracellularly located or cell-bound. Culture filtrates showed no significant gamma-PGA depolymerase activity.