KINETIC AND ATP MAINTENANCE STUDIES OF A METABOLICALLY ENGINEERED Zymomonas mobilis FERMENTING GLUCOSE AND XYLOSE MIXTURES

The growth (glucoseand xylose-associated processes) and non-growth-mediated (maintenance energy requirements) characteristics of a metabolically engineered strain of Zymomonas mobilis capable of fermenting both glucose and xylose to ethanol have been investigated in mixed sugar fermentations. Ideally such a microorganism will also tolerate acetic acid and other inhibitory components typically present in biomass hydrolyzates. We have measured substrates and products concentrations as well as intracellular adenosine-5’triphosphate (ATP) concentrations across a 2-factor, 3-level factorial design using the methods of analysis, techniques, and calculations described previously (1). Batch fermentations were conducted using a 10% (w/v) total sugar concentration (5% glucose/5% xylose mixture) at a temperature of 30 °C at pH 5.0, 5.3, or 6.0 in the presence of varying initial amounts of acetic acid (0, 4, and 8 g/L). Results show large differences in Z. mobilis fermentation kinetics but only modest changes in intracellular ATP levels across the experimental design space. Ethanol process yields varied between 56.6% and 92.3% of theoretical depending upon the test conditions, and illustrate how pH strongly influences the inhibitory effect of acetic acid on fermentation kinetics. For example, maximum specific growth rates varied between a low of 0.06 h observed at pH 5 in the presence of 8 g/L acetic acid to a high of 0.20 h obtained at pH 6 without any acetic acid present. Reflecting this trend, calculated rates of ATP consumption for growth-mediatedprocesses ranged from a low of 6.2 g ATP/g DCM-h at pH 5 and 8 g/L acetic acid to a high of 20.9 g ATP/g DCM-h at pH 6 without acetic acid. ATP requirements for maintenance showed an opposite pattern, with the highest maintenance requirement, 12.9 g ATP/g DCM-h, obtained at pH 5 and 8 g/L acetic acid, and the lowest, 0.2 g ATP/g DCM-h, at pH 6 without acetic acid present. Despite the more than sixty-fold difference in maintenance requirements observed across the design space, maximum levels of free intracellular ATP only varied within a narrow range of 1.5 to 3.8 mg ATP/g DCM. These findings provide quantitative information about how pH and acetic acid concentration affect fermentation kinetics. A mathematical model is being developed to describe how the rate of ATP consumption for maintenance varies as a function of the concentrations of undissociated acetic acid and dissociated acetate ion, which are modulated by pH (hydronium ion concentration), as well as the concentration of ethanol. Preliminary results suggest that in this system the concentration of undissociated acetic acid is the strongest determinant of maintenance requirements.