Effects of manganese (11) on Bacillus licheniformis ATCC 9945A physiology and 7-poly(glutamic acid) formationt Anne-Marie Cromwick and Richard A. Gross* University of Massachusetts Lowell, Department of Chemistry, 1 University A venue, Lowell, MA 01854, USA Received 12 October 1994; revised 19 December 1994 Bacillus licheniformis ATCC 9945A was cultivated in shake flasks using citrate (1 2 g 1-1), glutamate (20g1-1) and glycerol (80g1-1) as carbon sources for cell growth and 7-poly(glutamic acid) (~-PGA) production. The effect of the MnSO4 concentration in the medium over a range from 0.0 to 615 ~M was studied. The number of viable cells increased for all concentrations of MnSO4 from approximately 105 to 109 colony-forming units (cfu) m1-1 by the early stationary phase (24h). However, after 50 h, the cell viability decreased rapidly for relatively lower MnSO4 concentrations (0.615 and 0/~M). The utilization of carbon sources by B. licheniformis was greater for cultures containing 33.8 and 615 #M MnS04 relative to cultures with no added MnSO4. For example, cultures with 615 #M MnSO4 utilized 37, 54 and 93% and cultures with no added MnS04 utilized 19, 10 and 17% of glutamate, glycerol and citrate, respectively. The 7-PGA volumetric yield increased from approximately 5 to 17 g 1-1 for corresponding increases in MnS04 concentration from 0 to 33.8 #M and then decreased at higher MnS04 concentrations. The stereochemical content of 7-PGA was found to vary inversely with MnS04 concentration, and ranged from 59 to 10% L-glutamate units for MnS04 concentrations of 0 and 615 #M, respectively. For all of the MnS04 concentrations investigated, the 7-PGA molecular weights decreased rapidly as the 7-PGA volumetric yield simultaneously increased for cultivation times from 24 to ~ 50 h. M~ and M, values after ~50h cultivation times, determined by gel permeation chromatography (GPC), were 1.3 to 1.6 and 0.5 to 0.8 million g tool -1, respectively. A complex 7-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/tM MnSO4 at extended cultivation times. A high-molecular-weight fraction and the unfractionated 7-PGA sample from the 33.8/~M MnS04 culture contained 13_+ 4 and 30 4-1% L-repeat units, respectively. A relationship between the product molecular weight and its stereochemical composition was thus established. Keywords: 7-poly(glutamic acid); manganese; Bacillus licheniformis ATCC 9945A 7-Poly(glutamic acid) (7-PGA) is an unusual bacterially synthesized water-soluble polyamide. This polymer may be classified as a pseudo-poly(amino acid) that contains only glutamate repeat units. 7-PGA differs from the well-known protein-like primary structure in that the glutamate repeat units are linked between the a-amino and 7-carboxylic acid functional groups1: O * To whom correspondenceshould be addressed This paper is part of the thesis dissertation of Anne-Marie Cromwick. University of Massachusetts Lowell, 1994 7-PGA produced from B. lichen(formis is believed to consist of homopolymers of t)- and L-glutamate repeat units 2. Furthermore, it has been reported by Leonard et al. that the stereochemistry of 7-PGA produced by B. subtilis 9945A (later reclassified as B. lichen~rmis) can be modulated by varying the manganese concentration in the medium 3. Specifically, the percentage of L- glutamate polymer repeat units was found to vary inversely with the concentration of manganese. Troy later reported that modulation of 7-PGA stereochemistry by variation of the manganese concentration in the medium could not be confirmed for B. lichen(formis 9945A 1"4. The nutritional requirements fof ~,-PGA production may vary according to the strain used. Medium E, adopted for the studies presented here, was partially optimized by Leonard et al. and contains three carbon 0141-8130/95/$09.50'('i 1995ElsevierScienceB.V.All rights reserved Int. J. Biol. Macromol. Volume 1 7 Number 5 1 995 259