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(g lutamate) (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 mole cular weight directly using culture filtrates. For GPC volumetric yiel d measurements, a calibration curve was generated using purified gamma -PGA to relate the gamma-PGA GPC peak area and polymer weight. Purifie d gamma-PGA was characterized by elemental analysis, H-1- and C-13-NMR spectroscopy. Cultures of B. licheniformis using all three carbon sou rces showed the following characteristics: cell growth mainly during t he first 24h; largest gamma-PGA volumetric productivity (similar to 0. 12 g l(-1) h(-1)) between 48 and 96 h; 11 g l(-1) gamma-PGA volumetric cultivation time from 80 to 45 g l(-1) yield by 96 h: reduction (util ization) of glycerol, glutamate and citrate during a 96 h 18 to 10 g l (-1) and 12 to similar to 1 g l(-1) respectively; a decrease in pH fro m 7.4 to similar to 5.5 by 42 h cultivation; acetic acid secretion int o the medium at a maximum level of similar to 4.5 g l(-1) and detectio n of the metabolite 2,3-butanediol (as acetoin) as a fermentation by-p roduct at similar to 42 h and through a 96 h cultivation period. The p resence of 2,3-butanediol indicated that the level of oxygen in the me dium no longer supported a fully aerobic mode of metabolism. When the medium formulation was altered by removal of either citrate, L-glutama te 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. Vari ation of the medium ionic strength by the addition of up to 4% (wlv) N aCl led to the formation of gamma-PGA of relatively higher molecular w eight but lower volumetric yield. Studies carried out on 5-day-old B. licheniformis cultures suggested that gamma-PGA depolymerase is intrac ellularly located or cell-bound. Culture filtrates showed no significa nt gamma-PGA depolymerase activity.