|dc.description.abstract||Succinate has a wide range of applications in the chemical, food, and pharmaceutical industries, and its production by the metabolically engineering Escherichia coli AFP111 was studied. The principal aims of this research were to understand the effects of pH, base counter-ion, CO2 concentration and flue gas components in two-phase fermentations using a defined medium. A pH of 6.4 yielded the highest specific succinate productivity, and the metabolic flux determined with 13C-labeled glucose showed that 61% of the PEP partitioned to oxaloacetate and 39% partitioned to pyruvate. Although a pH of 6.4 was optimal, the flux distribution at a pH of 6.8 was not significantly different. Ca(OH)2 was superior to NaOH or KOH as the base for controlling the pH.
A series of dual-phase fermentations showed that CO2 concentration influenced succinate production. The succinate specific productivity was increased from 1.89 mg/gh at 0% CO2 to 224.50 mg/gh at 50% CO2, and the yield was increased from 0.04 g/g to 0.75 g/g. Above 50% CO2, succinate production did not increase with increasing CO2 gas concentration. Using 13C-labeled glucose, the fraction of flux into the pentose phosphate pathway increased from 0.04 at 3% CO2 to 0.17 at 50% CO2. Also, the fractional flux through carboxylation at the PEP node increased slightly from 0.53 at 3% CO2 to 0.63 at 50% CO2. The increased flux into the pentose phosphate pathway is attributed to an increased demand for NADH with elevated CO2. A four-process explicit model to describe the CO2 transfer and utilization was proposed. The model predicted that at CO2 concentrations below about 40% the system becomes limited by gas phase CO2, while at higher CO2 concentrations the system is limited by PPC enzyme kinetics.
Finally, each of the other components of flue gas, oxygen (O2), nitrogen dioxide (NO2), sulfur dioxide (SO2) or carbon monoxide (CO), was studied for succinate production. Following aerobic cell growth, cells were exposed to 50% CO2 and 3-10% O2, and 50-300 ppm NO2, SO2 or CO during a succinate production phase. Although 3% O2 did not significantly affect succinate formation, 10% O2 reduced the final succinate concentration from 33 g/L to 17 g/L, specific succinate productivity from 1.90 to 1.13 mmol/g•h and the succinate yield from 1.15 to 0.81 mol/mol glucose. The effect of O2 correlated with the culture redox potential (ORP) with more reducing conditions favoring succinate production. The trace gases NO2 and SO2 also reduced the rate of succinate formation by as much as 50%, but led to a greater than two-fold increase in pyruvate formation. 100-500 ppm CO showed no effect on succinate production rate or yield. Using synthetic flue gas AFP111 could generate 12 g/L succinate with a succinate specific productivity of 0.73 mmol/g•h and a yield of 0.65 mol/mol.||