EFFECTS OF MANGANESE(II) ON BACILLUS-LICHENIFORMIS ATCC-9945A PHYSIOLOGY AND GAMMA-POLY(GLUTAMIC ACID) FORMATION

Bacillus licheniformis ATCC 9945A was cultivated in shake flasks using citrate (12 g l(-1)), glutamate (20 g l(-1)) and glycerol (80 g l(-1)) as carbon sources for cell growth and gamma-poly(glutamic acid) (gamma-PGA) production. The effect of the MnSO4 concentration in the medium over a range from 0.0 to 615 mu M was studied. The number of viable cells increased for all concentrations of MnSO4 from approximately 10(5) to 10(9) colony-forming units (cfu) ml(-1) by the early stationary phase (24 h). However, after 50 h, the cell viability decreased rapidly for relatively lower MnSO4 concentrations (0.615 and 0 mu M). The utilization of carbon sources by B. licheniformis was greater for cultures containing 33.8 and 615 mu M MnSO4 relative to cultures with no added MnSO4. For example, cultures with 615 mu M MnSO4 utilized 37, 54 and 93% and cultures with no added MnSO4 utilized 19, 10 and 17% of glutamate, glycerol and citrate, respectively. The gamma-PGA volumetric yield increased from approximately 5 to 17 g l(-1) for corresponding increases in MnSO4 concentration from 0 to 33.8 mu M and then decreased at higher MnSO4 concentrations. The stereochemical content of gamma-PGA was found to vary inversely with MnSO4 concentration, and ranged from 59 to 10% L-glutamate units for MnSO4 concentrations of 0 and 615 mu M, respectively. For all of the MnSO4 concentrations investigated, the gamma-PGA molecular weights decreased rapidly as the gamma-PGA volumetric yield simultaneously increased for cultivation times from 24 to similar to 50 h. M(w) and M(n) values after similar to 50 h cultivation times, determined by gel permeation chromatography (GPC), were 1.3 to 1.6 and 0.5 to 0.8 million g mol(-1), respectively. A complex gamma-PGA molecular weight distribution that appeared bimodal by GPC analysis due to the presence of a low-molecular-weight product fraction was observed in cultures containing 33.8 and 61.5 mu M MnSO4 at extended cultivation times. A high-molecular-weight fraction and the unfractionated gamma-PGA sample from the 33.8 mu M MnSO4 culture contained 13 +/- 4 and 30 +/- 1% L-repeat units, respectively. A relationship between the product molecular weight and its stereochemical composition was thus established.