(60c) Enhancing ‘Dark’ CO2 Fixation in Succinate Fermentations Via Hollow Fiber Membrane Carbonation and Strain Engineering

Although photoautotrophic microbes can naturally fix CO₂ to biomass and useful chemicals via the Calvin-Benson-Basham Cycle, overall rates are limited by slow carboxylation kinetics by RuBisCO, low cell densities and growth rates, and poor light penetration. In contrast, ‘dark’ fermentations offer the potential to fix CO2 in both a RuBisCO and light-independent manner, by employing alternative electron donors and carboxylases. Heterotrophic fermentations represent a promising option here, due to the availability of many optimized strains that are capable of growing quickly and achieving high product yields. Due to low energy costs and desirable kinetic parameters, the highest potential for heterotrophic fixation of inorganic carbon (C_i) occurs during anaerobic fermentations via the reductive TCA (rTCA) cycle. In the rTCA cycle, C_i is combined with phosphoenolpyruvate (PEP) by PEP carboxylase (ppc) or PEP carboxykinase (pck) to form oxaloacetate (OAA); the key precursor to succinate, along with malate, fumarate, aspartate and other bioproducts of interest. That said, to provide the needed C_i, industrial succinate fermentations currently require the media be supplemented with significant levels (up to 20 – 50 g/L) of bicarbonate (HCO₃^-) or carbonate (CO₃^2-) salts; levels far above what is required by the cells, with the excess going unused or even released as CO₂. Moreover, production of HCO₃^- and CO₃^2- salts from CO₂ or natural ores is thermodynamically unfavorable and thus energetically costly, which reduces the overall sustainability of the fermentation process. To address this, we have recently been investigating how Hollow Fiber Membranes (HFM) carbonation can instead be used to achieve efficient, bubble-free, and on-demand delivery of CO₂ in support of succinate fermentation by Escherichia coli and other microbes. Other benefits of the HFM delivery system include increased energy efficiency, high specific surface area to volume ratios, little off gassing, reduced shear stress (previously from bubbles passing the cells), prevention of foam formation, and reduced stripping of water and other volatile media components.

Here, using composite HFMs comprised of a nonporous polyurethane core and a microporous polyethylene outer layer we first demonstrate how net gas delivery rates can be controlled by modulating internal gas pressure within the membrane lumen and/or membrane surface area. Then, based on this, novel HFM fermenters were designed and constructed using optimized membrane surface areas to ensure that biofixation rates are adequately met. Further optimization of the HFM fermenter design and the development of a dynamic pressurization schedule was investigated to further enhance titer, rate, and yield (TRY) metrics. In fermentations with engineered E. coli, succinate titers and yields remained on par with published results using HCO₃^- or CO₃^2- salts, whereas experimental CO₂ fixation rates reached up to 125 mg/L/h: a 1.5 - 12.5 fold increase relative to typical, photosynthetic processes. Importantly, meanwhile, HFMs showed little sign of biofouling, suggesting that they should be well-suited for continued reuse in long-term applications. Lastly, multiple strategies were also investigated to enhance strain performance through both transporter and enzyme engineering. Overall, HFM-based CO₂ delivery represents a more sustainable alternative to the use of HCO₃^- or CO₃^2- salts in succinate fermentations, and likely also other ‘dark’ fermentations.