CO2 availability as process tool to enhance isobutyric acid production in methanol fermentation by Clostridium luticellarii.

The bioconversion of CO2‑derived methanol into higher‑value chemicals offers an attractive route for hybrid catalytic-biotechnological carbon capture and utilization (CCU). Clostridium luticellarii is one of the few acetogens able to produce isobutyric acid. However, operational and metabolic factors driving its production are poorly understood. This work investigates how CO2 availability shapes the product spectrum of C. luticellarii during methylotrophic growth and assesses whether CO2 supply can be used as a process lever to promote isobutyric acid formation. Batch experiments with varying initial bicarbonate concentrations revealed that conditions leading to CO2 limitation (i.e., DIC depletion at ≤ 30 mM NaHCO3) redirected carbon and electron fluxes away from acetic acid toward butyric and isobutyric acids, with the latter accounting for up to 41% of total products. This metabolic switch was not observed when CO2 was in excess (>45 mM). High acetic acid supplementation (100 mM) triggered isobutyric acid production even while CO2 was still available, indicating a combined regulation of dissolved inorganic carbon (DIC) and acetic acid availability. Net acetic acid consumption took place in all isobutyric acid-producing experiments. These observations were reproduced in 3-L bioreactors and further exploited through a fed‑batch strategy in which an initial acetic‑acid‑accumulating phase was followed by CO2‑limited feeding. This approach achieved complete conversion of methanol and CO2 and yielded an isobutyric acid titer of 2.70 ± 0.04 g·L-1. Controlling CO2 availability is a viable operational tool to steer C. luticellarii metabolism toward isobutyric acid production, in interaction with electron acceptor availability.