Microbial electrosynthesis (MES) has been highlighted as a means to valorize inorganic gaseous carbon, such as CO2, into value-added chemicals using electricity as the reducing power. Electrode-based electron transfer delivers respiratory electrons to a live-cell biocatalyst through a biofilm matrix or via electron shuttle molecules. The addition of artificial mediators, such as neutral red (NR) and 2-hydroxy-1,4-naphthoquinone (HNQ), increased acetate synthesis significantly, suggesting that these mediators improve the electron transport capability between the suspended cells and electrode. Regular media replacement also improves MES by adapting mediator-utilizing species in the reactor. MES without a mediator initially produced acetate at a reasonable rate (5.1 ± 0.2 mmol/l/day), but the rate became negligible in the later stage. In contrast, MES with NR or HNQ showed a higher acetate production rate (4.6 ± 0.4 vs. 7.4 ± 0.4 mmol/l/day, respectively) as the media replacement progressed. Confocal laser scanning microscopy and 3D imaging showed that the biofilm matrix consisted of live and dead cells, while the composition was different in the MES with and without a mediator. Microbial community analysis by next-generation sequencing (NGS) showed that acetogenic Acetobacterium (in suspension) and Sporomusa (in both suspension and biofilm) were dominant during the 96 days of operation. The biofilm and planktonic community interacted dynamically under MES conditions. This result provides a realistic model of biofilms and planktonic cells in the MES. The interaction of the suspended cells with the biofilm-forming electrode via electron shuttles could improve volumetric acetate production and stabilize the MES performance.

Clostridium thermocellum is a desirable biocatalyst for biohydrogen production, with a native ability to simultaneously saccharify cellulose and to metabolize released cellodextrins for hydrogen production. During fermentation with C. thermocellum, partial pressures of two gases - CO2 and H2 - are critical drivers of overall reaction kinetics. Biohydrogen production is enhanced by maintaining a low hydrogen partial pressure, while high concentrations of dissolved CO2 promote microbial biomass synthesis. Our study evaluates the inherent trade-offs between hydrogen stripping and inorganic carbon supply for optimized biohydrogen synthesis. We find that nitrogen sparging at low flow rates increases hydrogen production when compared with an equivalent nitrogen overlay, but that high rates of nitrogen sparging inhibit cell growth and hydrogen production. Decreasing dissolved hydrogen partial pressure via nitrogen sparging also lowers the production of reduced metabolites, including lactate and ethanol. To address potential stripping of inorganic carbon from the production medium, we supplemented CO2 to the sparging gas and co-optimized for gas flow rate and for the CO2 fraction of the sparging gas. Total hydrogen production increased from 50 mmol∙L−1 in the base condition, when the bioreactor was sparged with 0.1 LPM of pure nitrogen, to 181.3 mmol∙L−1 when sparged with 1.3 LPM of 33 % CO2, demonstrating that biohydrogen production is highly sensitive to both parameters. Fine sensitivity of biohydrogen production to sparging conditions highlights the critical importance of bioreactor design and operation to achieve maximum H2 removal without compromising inorganic carbon supply to bacterial central metabolism.