Enabling strong acetogenic growth on CO ₂ and H ₂ : H ₂ solubility limits Clostridium ljungdahlii growth on CO ₂ and H ₂

Due to their ability to convert CO ₂ , a greenhouse gas, into useful products, certain acetogenic bacterial species, such as Clostridium ljungdahlii , have been proposed as promising platform strains for renewable, carbon-negative chemical production. C. ljungdahlii , and similar acetogens, grows slowly and produce primarily acetate when grown on CO ₂ with H ₂ as the electron donor, but it grows quickly and can produce ethanol when grown on higher energy substrates, notably CO or fructose. Here, by utilizing different mixing strategies (and notably the first time use of roller bottles) to modulate the volumetric gas interfacial mass transfer coefficient (k _L a), we show that, under both mixotrophic (sugar and gas utilization) and autotrophic conditions, C. ljungdahlii growth and CO ₂ fixation are primarily electron-limited due to the low solubility of H ₂ relative to CO and CO ₂ . We demonstrate that, with sufficiently high H ₂ mass transfer, C. ljungdahlii can grow at similar high rates using CO ₂ as its sole carbon source compared to CO or fructose, a finding with significant implications for the use of acetogens in CO ₂ -negative biomanufacturing, especially because at least 50% of CO used is oxidized and released as CO ₂ . We also show that accumulation of fructose inhibits CO ₂ utilization by C. ljungdahlii under mixotrophic growth conditions, suggesting that a non-classical “catabolite repression” by fructose inhibits CO ₂ utilization.