Tunable and Efficient CO₂ Delivery Supports Robust Malic Acid Production by Escherichia coli in a Hollow Fiber...

Malic acid, a four-carbon dicarboxylic acid, represents a top value-added chemical from biomass and important building-block molecule for various industrial chemical processes. Currently, however, it is primarily produced from petrochemicals via an unsustainable process with high greenhouse gas intensity. Alternatively, malic acid can be produced from renewable biomass sugars via anaerobic fermentation by engineered Escherichia coli. While inorganic carbon (C_i) serves as an essential co-substrate in this carbon-negative process, its typical delivery methods suffer from several key limitations. To address these, we systematically developed a hollow fiber membrane (HFM) bioreactor for direct and efficient delivery of CO₂ gas in a bubbleless manner. A kinetic model for predicting CO₂ mass transfer rates to the HFM bioreactor was first developed and used to identify process conditions where rates of C_i delivery closely match those of biological demand, thereby minimizing wasteful oversupply. Ultimately, using a CO₂ delivery pressure of 2.5 psig and 8 fiber HFM (4.93 m^-1 specific surface area), a maximum malic acid titer of 38.2 ± 0.4 g/L was achieved at a yield of 0.94 ± 0.01 g-malic acid/g-glucose; both comparable to the performance of a conventional E. coli batch fermentation wherein (bi)carbonate salts were directly supplied, albeit with ~50% less CO₂ off-gassing from the headspace. By enabling direct and efficient CO₂ delivery while achieving greater overall carbon efficiency, the HFM bioreactor represents a promising and more sustainable design for malic acid bioproduction.