Sáez Miranda, Juan C.

Profile Picture

Filters

20061
Reset filters

Settings

Citations

Publication Search Results

Now showing 1 - 1 of 1
  • Thumbnail Image
    Publication
    Kinetic, intracellular ATP growth and maintenance studies, and modeling of metabolically engineered Zymomonas mobilis fermenting glucose and xylose mixtures
    (2006) Sáez Miranda, Juan C.; Saliceti Piazza, Lorenzo; College of Engineering; Díaz, Emilio; Colucci Ríos, José A.; McMillan, James D.; Department of Chemical Engineering; Aponte, María
    Intracellular adenosine-5’-triphosphate (ATP) levels were measured in a metabolically engineered Zymomonas mobilis strain 8b over the course of batch fermentations of glucose and xylose mixtures. Recombinant Z. mobilis, a gram-negative bacterium that distinctively uses the Entner-Doudoroff pathway (net yield of only one mole ATP per mole of glucose or, putatively, xylose consumed), is capable of efficiently converting both glucose and xylose to ethanol as well as to moderately tolerate acetic acid and other inhibitory components typically present in biomass hydrolyzates. Fermentations were conducted over a range of pH (5–6) in the presence of varying initial amounts of acetic acid (0–8 g/L) using a 10% (w/v) total sugar concentration (glucose only, xylose only, or 5% glucose/5% xylose mixtures). Specifically, when xylose was one of the sugar substrates or the sole substrate, acetic acid and/or low pH strongly affected maximum specific intracellular ATP production rates (7.3–26.0 g ATP/g DCM-h), accumulation rates (0.1–1.0 mg ATP/g DCM-h), accumulation levels (1.5–3.8 mg ATP/g DCM), maximum specific cellular growth rates (0.10 ± 0.01–0.33 ± 0.03 h-1), and ethanol process yields (56.6–95.4 ± 1.6%). Specific ATP accumulation rates (mg ATP/g DCM-h) are several orders of magnitude lower than the rates of ATP production (g ATP/g DCM-h), indicating that ATP accumulation and dilution terms can be neglected to enable a simplified intracellular ATP balance wherein the specific rate of ATP production is balanced by the total rate of ATP consumption (i.e., for cell growth and maintenance). Findings suggest that reductions in ATP production and consumption rates under increased stress conditions are associated with the uncoupling phenomenon. A novel improved flocculating recombinant strain 8bF of Z. mobilis was developed and isolated. The superiority of Z. mobilis strain 8bF in enabling more rapid and efficient fermentation of glucose and xylose to ethanol was demonstrated in both batch and continuous cultures. Specifically, in uncontrolled pH shake-flask batch fermentations, the flocculent Z. mobilis strain 8bF exhibited better pH stability (kept pH ≥ 4), grew to a higher final cell mass concentration (≥ 2 g/L of DCM), and achieved higher glucose and xylose mixture conversions (3–4-fold higher sugar utilization percentages) and higher ethanol yields (2–3-fold higher metabolic yields) than the non-flocculating parental strain 8b. During the chemostat runs, 8bF was able to maintain high cell mass concentrations (~8 g/L of DCM at µ = 0.47 h-1), high glucose and xylose mixture conversions, high ethanol productivities (~3-fold higher than 8b), and high ethanol yields at dilution rates well above 0.15 h-1. Advantages of Z. mobilis 8bF in comparison to other ethanologenic bacteria include: (i) the ability to flocculate, (ii) better pH stability, (iii) the ability to ferment sugars at 4 ≤ pH ≤ 5, (iv) biomass concentrations above 7 g/L of DCM at 0.19 h-1 ≤ µ ≤ 0.47 h-1, (v) high sugar mixture conversions achieved, (vi) high specific and volumetric ethanol productivities, (vii) high ethanol process and metabolic yields, and (viii) potential of facilitated and lower costs during cell separation and ethanol recovery steps. An intracellular ATP balance-based method is described to quantify glucose and xyloseassociated growth and non-growth (maintenance) energy requirements. Zymomonas mobilis strain 8b continuous fermentations were conducted at different pH values (5.0, 5.3, 5.5, or 6.0) and initial acetic acid concentrations (0, 2, and 4 g/L) while maintaining a constant temperature (30°C), agitation rate (300 rpm), and the sugar mixture feed concentration (5% glucose/5% xylose) supplemented with or without acetic acid. ATP requirements for cell growth and maintenance were calculated, and a significant dependence upon pH was observed, with the concentration of acetic acid exerting the strongest effect on maintenance ATP requirements. Specifically, calculated ATP maintenance rates varied significantly across the experimental design space tested (3.29–12.94 g ATP/g DCM-h). The highest maintenance requirement, 12.94g ATP/g DCM-h, was obtained at pH 5.3 and 4 g/L of initial acetic acid (higher stress condition), and the lowest, 3.29 g ATP/g DCM-h, at pH 6 with 2 g/L of initial acetic acid (low stress condition). A response surface linear model accurately predicted these specific ATP maintenance requirements across the experimental design space studied. Cell mass yields on ATP (6.03 ± 1.51g DCM/mol ATP) did not vary significantly with stress levels. A mathematical model based on calculations of a maintenance-related rate (rATP,m-H+) was developed to predict the pH-dependent uncoupling effect of acetic acid in steady state Z. mobilis 8b continuous cultures. Calculations and model predictions demonstrated that the uncoupling of the plasma membrane potential in Z. mobilis 8b chemostat cultures increased the maintenancerelated ATP consumption and became more severe in the presence of high initial acetic acid concentrations. Specifically, the highest uncoupling effect was obtained when the initial acetic acid concentration was increased to 4 g/L and using pHext values of 5.3 or 6 (rATP,m-H+ = ~0.6–0.8mmol ATP/g DCM-h). The model predicts accurately that the larger the ∆pH established across the plasma membrane in Z. mobilis 8b continuous fermentations, the higher the values of rATP,mH+ (and hence, higher observed inhibitory effects) are required at different culture conditions. Model validation resulted more challenging, predictions of validation runs were modest at pHext values below or above 5.5. Overall, a substantially better understanding of recombinant Z. mobilis strains 8b and 8bF batch and continuous mixed sugar (glucose and xylose) ethanol fermentations was accomplished. Specifically, the ability to accurately characterize fermentation kinetics, determine intracellular ATP levels (accumulation dynamics), quantify ATP production and consumption rates, and mathematically model a maintenance-related rate (rATP,m-H+) to predict the pH-dependent uncoupling effect of acetic acid in Z. mobilis strain 8b cultivations was achieved. More research is needed to identify the actual magnitude of the specific ATP consumption rates for H+ export (rATP,m-H+, maintenance-related rate) under increased stress conditions and to further validate the mathematical model.