'Dark' CO₂ fixation in succinate fermentations enabled by direct CO₂ delivery via hollow fiber membrane carbonation

Anaerobic succinate fermentations can achieve high-titer, high-yield performance while fixing CO₂ through the reductive branch of the tricarboxylic acid cycle. To provide the needed CO₂, conventional media is supplemented with significant (up to 60 g/L) bicarbonate (HCO₃^-), and/or carbonate (CO₃^2-) salts. However, producing these salts from CO₂ and natural ores is thermodynamically unfavorable and, thus, energetically costly, which reduces the overall sustainability of the process. Here, a series of composite hollow fiber membranes (HFMs) were first fabricated, after which comprehensive CO₂ mass transfer measurements were performed under cell-free conditions using a novel, constant-pH method. Lumen pressure and total HFM surface area were found to be linearly correlated with the flux and volumetric rate of CO₂ delivery, respectively. Novel HFM bioreactors were then constructed and used to comprehensively investigate the effects of modulating the CO₂ delivery rate on succinate fermentations by engineered Escherichia coli. Through appropriate tuning of the design and operating conditions, it was ultimately possible to produce up to 64.5 g/L succinate at a glucose yield of 0.68 g/g; performance approaching that of control fermentations with directly added HCO₃^-/CO₃^2- salts and on par with prior studies. HFMs were further found to demonstrate a high potential for repeated reuse. Overall, HFM-based CO₂ delivery represents a viable alternative to the addition of HCO₃^-/CO₃^2- salts to succinate fermentations, and likely other 'dark' CO₂-fixing fermentations.