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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 y-polyglutamic acid (y-PGA) production, molecular weight, and polymer stereochemistry were determined. The y-PGA yield was highest (15 g/L, 96 h growth time) at pH 6.5. The increase in y-PGA formation at pH 6.5 corresponded with a relatively high specific production rate at high y-PGA concentration (0.09 h-', -15 g/L y-PGA). In contrast, the specific y-PGA production rates at fermentor pH values of 5.5 and 7.4 decreased significantly for y-PGA fermentor yields >-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 y-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 t o 2.0 L/min) at pH 6.5, the pH of maximal y-PGA production. Increased aeration resulted in doubling of the cell dry weights (2 t o 4 g/ L), increasing y-PGA yields (6.3 to 23 g/L by 48 h) and increasing in the maximum y-PGA-specific production rate (0.09 to 0.1 1 h-'). Other effects of increased agitation included a rapid depletion of glutamate and citrate (by 50 h) and a decrease in product molecularweight. 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. 0 1996 John Wiley & Sons, Inc.

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 y-polyglutamic acid (y-PGA) production, molecular weight, and polymer stereochemistry were determined. The y-PGA yield was highest (15 g/L, 96 h growth time) at pH 6.5. The increase in y-PGA formation at pH 6.5 corresponded with a relatively high specific production rate at high y-PGA concentration (0.09 h-', -15 g/L y-PGA). In contrast, the specific y-PGA production rates at fermentor pH values of 5.5 and 7.4 decreased significantly for y-PGA fermentor yields >-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 y-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 t o 2.0 L/min) at pH 6.5, the pH of maximal y-PGA production. Increased aeration resulted in doubling of the cell dry weights (2 t o 4 g/ L), increasing y-PGA yields (6.3 to 23 g/L by 48 h) and increasing in the maximum y-PGA-specific production rate (0.09 to 0.1 1 h-'). Other effects of increased agitation included a rapid depletion of glutamate and citrate (by 50 h) and a decrease in product molecularweight. 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.

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Conditions for transformation of the solventogenic anaerobe Clostridium beijerinckii NRRL B-592 with plasmid DNA via electroporation are described. Shuttle plasmid pHR106 and two derivatives constructed in this study were transferred and were expressed in this organism. One recombinant derivative of pHR106 was constructed by separately subcloning the clostridial tetracycline (tetP) resistance genes into pHR106. The second vector conferring erythromycin resistance was obtained via in-vivo recombination. The new constructs, termed pRZL and pRZE respectively, were then transferred to C. beijerinckii in order to evaluate their potential as shuttle vectors. The recombinant plasmids were shown to transfer to C. beijerinckii and were expressed as autonomously replicating vectors. The use of these plasmids as cloning and shuttle vectors for C. beijerinckii is discussed.