Production of PHB From CO2-Derived Acetate With Minimal Processing Assessed for Space Biomanufacturing

Providing life-support materials to crewed space exploration missions is pivotal for mission success. However, as missions become more distant and extensive, obtaining these materials from in situ resource utilization is paramount. The combination of microorganisms with electrochemical technologies offers a platform for the production of critical chemicals and materials from CO2 and H2O, two compounds accessible on a target destination like Mars. One such potential commodity is poly(3-hydroxybutyrate) (PHB), a common biopolyester targeted for additive manufacturing of durable goods. Here, we present an integrated two-module process for the production of PHB from CO2. An autotrophic Sporomusa ovata (S. ovata) process converts CO2 to acetate which is then directly used as the primary carbon source for aerobic PHB production by Cupriavidus basilensis (C. basilensis). The S. ovata uses H2 as a reducing equivalent to be generated through electrocatalytic solar-driven H2O reduction. Conserving and recycling media components is critical, therefore we have designed and optimized our process to require no purification or filtering of the cell culture media between microbial production steps which could result in up to 98% weight savings. By inspecting cell population dynamics during culturing we determined that C. basilensis suitably proliferates in the presence of inactive S. ovata. During the bioprocess 10.4 mmol acetate L –1 day–1 were generated from CO2 by S. ovata in the optimized media. Subsequently, 12.54 mg PHB L–1 hour–1 were produced by C. basilensis in the unprocessed media with an overall carbon yield of 11.06% from acetate. In order to illustrate a pathway to increase overall productivity and enable scaling of our bench-top process, we developed a model indicating key process parameters to optimize.

The Sporomusa type Nfn is a novel type of electron-bifurcating transhydrogenase that links the redox pools in acetogenic bacteria

Flavin-based electron bifurcation is a long hidden mechanism of energetic coupling present mainly in anaerobic bacteria and archaea that suffer from energy limitations in their environment. Electron bifurcation saves precious cellular ATP and enables lithotrophic life of acetate-forming (acetogenic) bacteria that grow on H2 + CO2 by the only pathway that combines CO2 fixation with ATP synthesis, the Wood–Ljungdahl pathway. The energy barrier for the endergonic reduction of NADP+, an electron carrier in the Wood–Ljungdahl pathway, with NADH as reductant is overcome by an electron-bifurcating, ferredoxin-dependent transhydrogenase (Nfn) but many acetogens lack nfn genes. We have purified a ferredoxin-dependent NADH:NADP+ oxidoreductase from Sporomusa ovata, characterized the enzyme biochemically and identified the encoding genes. These studies led to the identification of a novel, Sporomusa type Nfn (Stn), built from existing modules of enzymes such as the soluble [Fe–Fe] hydrogenase, that is widespread in acetogens and other anaerobic bacteria.

Accelerated H2 Evolution during Microbial Electrosynthesis with Sporomusa ovata

Microbial electrosynthesis (MES) is a process where bacteria acquire electrons from a cathode to convert CO2 into multicarbon compounds or methane. In MES with Sporomusa ovata as the microbial catalyst, cathode potential has often been used as a benchmark to determine whether electron uptake is hydrogen-dependent. In this study, H2 was detected by a microsensor in proximity to the cathode. With a sterile fresh medium, H2 was produced at a potential of −700 mV versus Ag/AgCl, whereas H2 was detected at −500 mV versus Ag/AgCl with cell-free spent medium from a S. ovata culture. Furthermore, H2 evolution rates were increased with potentials lower than −500 mV in the presence of cell-free spent medium in the cathode chamber. Nickel and cobalt were detected at the cathode surface after exposure to the spent medium, suggesting a possible participation of these catalytic metals in the observed faster hydrogen evolution. The results presented here show that S. ovata-induced alterations of the cathodic electrolytes of a MES reactor reduced the electrical energy required for hydrogen evolution. These observations also indicated that, even at higher cathode potentials, at least a part of the electrons coming from the electrode are transferred to S. ovata via H2 during MES.

Porous nickel hollow fiber cathodes coated with CNTs for efficient microbial electrosynthesis of acetate from CO2 using Sporomusa ovata

Nickel porous hollow fibers coated with CNTs acted as both a gas transfer membrane for CO2 delivery and a cathode for providing electrons to microbial catalysts, achieving improved CO2 conversion to value-added products in microbial electrosynthesis.

Electrosynthesis of acetate from CO2by a highly structured biofilm assembled with reduced graphene oxide–tetraethylene pentamine

A highly structured biofilm assembled with reduced graphene oxide–tetraethylene pentamine and the bacteriumSporomusa ovataperformed high-rate microbial electrosynthesis.