Coupling gas purging with inorganic carbon supply to enhance biohydrogen production with Clostridium thermocellum

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.