γ-Poly(glutamic acid) formation by Bacillus licheniformis 9945a: physiological and biochemical studies.

Abstract Cryogenically frozen vegetative cells of Bacillus licheniformis 9945a derived from young mucoid colonies were used to inoculate γ-poly(glutamate) (γ-PGA) production media containing l -glutamate, citrate and glycerol as carbon sources. A gel permeation chromatography (GPC) method was developed to determine γ-PGA volumetric yield and molecular weight directly using culture filtrates. For GPC volumetric yield measurements, a calibration curve was generated using purified γ-PGA to relate the γ-PGA GPC peak area and polymer weight. Purified γ-PGA was characterized by elemental analysis, 1 H- and 13 C-NMR spectroscopy. Cultures of B. licheniformis using all three carbon sources showed the following characteristics: cell growth mainly during the first 24 h; largest γ-PGA volumetric productivity (∼0.12 gl −1 h −1 ) between 48 and 96 h; 11 gl −1 γ-PGA volumetric yield by 96 h; reduction (utilization) of glycerol, glutamate and citrate during a 96 h cultivation time from 80 to 45 gl −1 , 18 to 10 gl −1 and 12 to ∼1 gl −1 , respectively; a decrease in pH from 7.4 to ∼5.5 by 42 h cultivation; acetic acid secretion into the medium at a maximum level of ∼4.5 gl −1 and detection of the metabolite 2,3-butanediol (as acetoin) as a fermentation by-product at ∼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 gl −1 , respectively, of γ-PGA were formed. Variation of the medium ionic strength by the addition of up to 4% (w/v) NaCl led to the formation of γ-PGA of relatively higher molecular weight but lower volumetric yield. Studies carried out on 5-day-old B. licheniformis cultures suggested that γ-PGA depolymerase is intracellularly located or cell-bound. Culture filtrates showed no significant γ-PGA depolymerase activity.