Enzyme-Assisted Microbial Electrosynthesis of Poly(3-hydroxybutyrate) via CO₂ Bioreduction by Engineered Ralstonia eutropha

Abstract

Microbial electrosynthesis (MES) is a promising technology to reduce carbon dioxide using inward electron transfer mechanisms to synthesize value-added chemicals with microorganisms as electrocatalysts and electrons from cathodes as reducing equivalents. To enhance CO 2 assimilation in Ralstonia eutropha, a formate dehydrogenase (FDH) assisted MES system was constructed, in which FDH catalyzed the reduction of CO 2 to formate in the cathodic chamber. Formate served as the electron carrier to transfer electrons derived from cathodes into R. eutropha. To enable efficient formation of formate from CO 2 , neutral red (NR) was used to facilitate the extracellular regeneration of NADH, the cofactor of FDH. Meanwhile, NR also played an essential role as electron shuttle to directly deliver electrons from cathodes into R. eutropha to increase the level of intracellular reducing equivalents, thus facilitating the efficiency of MES. On the other hand, the Calvin−Benson−Bassham (CBB) cycle was further engineered by the heterologous expression of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in R. eutropha, which strengthened the CBB pathway for CO 2 fixation. Upon application of the cathode potential at −0.6 V (vs Ag/AgCl) in the MES system with the genetically engineered R. eutropha, 485 ± 13 mg/L poly(3-hydroxybutyrate) (PHB) was obtained, which was ∼3 times that synthesized by the control (165 ± 8 mg/L), i.e., the wild-type R. eutropha in the absence of FDH and NR.

What would it take for renewably powered electrosynthesis to displace petrochemical processes?

“…There has been some initial promising work in this area, interfacing biological systems with inorganic systems for solar fuels and fertilizer production (86,87). The current state of the art couples water-splitting electrocatalysts with engineered bacteria to convert CO 2 into polymers and alcohols (88,89) or nitrogen into ammonia (90). These efforts have focused mainly on the electrochemical production of H 2 or acetate as input for bacteria (87,91).…”

Modular engineering to increase intracellular NAD(H/+) promotes rate of extracellular electron transfer of Shewanella oneidensis

“…Extracellular electron transfer (EET) of electroactive microorganisms involves bi-directional electron flow and exchange between intracellular and extracellular redox-active electron donors and acceptors 1 – 3 . EET underlies a number of bio-electrochemical systems (BES) for many environments and energy applications 4 , 5 , including microbial fuel cells (MFCs) for simultaneous biodegradation of organic wastes and bioelectricity harvest 6 , 7 , microbial electrolysis cells (MEC) for hydrogen production 8 , microbial desalination cells (MDC) for seawater desalination 9 , unbalanced electro-fermentation for production of biofuels 10 – 12 , and microbial electrosynthesis (MES) for production of valuable chemicals and biofuels from electro-reduction of CO 2 13 – 17 . However, the low rate of EET remains a crucial bottleneck preventing the use of BES in industrial applications.…”

Stabilization of Formate Dehydrogenase in a Metal–Organic Framework for Bioelectrocatalytic Reduction of CO2

“…Formate dehydrogenase (FDH) from the yeast species Candida boidinii has been widely studied not only for its ability to sequester CO 2 but also to transform this carbon source into biomass for the production of biofuels . Despite its high efficiency and selectivity, the FDH reaction rate depends heavily on the environment, and FDH activity decreases significantly when the pH is lower than 6 .…”