Radiation crosslinking of a bacterial medium-chain-length poly(hydroxyalkanoate) elastomer from tallow.

Pseudomonas resinovorans produces a medium-chain-length poly(hydroxyalkanoate) (MCL-PHA) copolymer when grown on tallow (PHA-tal). This polymer had a repeat unit composition ranging from C4 to C14 with some mono-unsaturation in the C12 and C14 alkyl side chains. Thermal analysis indicated that the polymer was semi-crystalline with a melting temperature (T(m)) of 43.5 +/- 0.2 degrees C and a glass transition temperature (Tg) of -43.4 +/- 2.0 degrees C. The presence of unsaturated side chains allowed crosslinking by gamma-irradiation. Irradiated polymer films had decreased solubility in organic solvents that indicated an increase in the crosslinking density within the film matrix. The addition of linseed oil to the gamma-irradiated film matrix enhanced polymer recovery while minimizing chain scission. Linseed oil also caused a decrease in the enthalpy of fusion (delta Hm) of the films (by an average of 60%) as well as enhanced mineralization. The effects of crosslinking on the mechanical properties and biodegradability of the polymer were determined. Radiation had no effect on the storage modulus (E') of the polymer. However, radiation doses of 25 and 50 kGy did increase the Young modulus of the polymer by 129 and 114%, and the tensile strength of the polymer by 76 and 35%, respectively. Finally, the formation of a higher crosslink density within the polymer matrix decreased the biodegradability of the PHA films.

Effects of pH and aeration on gamma-poly(glutamic acid) formation by Bacillus licheniformis in controlled batch fermentor cultures.

Bacillus licheniformis ATCC 9945A was grown on Medium E in batch fermentations in which the pH was maintained at 5.5., 6.5, 7.4, and 8.25. The effects of pH on cell growth, carbon source utilization, and gamma-polyglutamic acid (gamma-PGA) production, molecular weight, and polymer stereochemistry were determined. The gamma-PGA yield was highest (15 g/L, 96 h growth time) at pH 6.5. The increase in gamma-PGA formation at pH 6.5 corresponded with a relatively high specific production rate at high gamma-PGA concentration (0.09 h(-1), approximately 15 g/L gamma-PGA). In contrast, the specific gamma-PGA production rates at fermentor pH values of 5.5 and 7.4 decreased significantly for gamma-PGA fermentor yields > approximately 5 g/L. Interestingly, alteration of the medium pH had little to no significant effects on the product quality as measured by stereochemical composition and molecular weight. While glutamate and glycerol utilization were similar as a function of pH, citrate consumption increased at pH 6.5, indicating that the formation of gamma-PGA from citrate at pH 6.5 was of increased importance. The effect of aeration was evaluated by increasing the agitation speed (250 to 800 rpm) and aeration rate (0.5 to 2.0 L/min) at pH 6.5, the pH of maximal gamma-PGA production. Increased aeration resulted in doubling of the cell dry weights (2 to 4 g/L), increasing gamma-PGA yields (6.3 to 23 g/L by 48 h) and increasing in the maximum gamma-PGA-specific production rate (0.09 to 0.11 h(-1)). Other effects of increased agitation included a rapid depletion of glutamate and citrate (by 50 h) and a decrease in product molecular weight. Despite the increase in agitation and aeration, oxygen limitation of the culture was not avoided, because the partial pressure decreased to <1.0% by 29 h.

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 gl-1), glutamate (20 gl-1) and glycerol (80 gl-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 microns 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 microns). The utilization of carbon sources by B. licheniformis was greater for cultures containing 33.8 and 615 microns MnSO4 relative to cultures with no added MnSO4. For example, cultures with 615 microns 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 gl-1 for corresponding increases in MnSO4 concentration from 0 to 33.8 microns 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 microns, 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 approximately 50 h. Mw and Mn values after approximately 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 microns MnSO4 at extended cultivation times. A high-molecular-weight fraction and the unfractionated gamma-PGA sample from the 33.8 microns 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.

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.