Abstract
The production of medium‐chain carboxylates (MCC) by mixed culture fermentation of complex organic feedstocks is often constrained by the availability of reducing equivalents. Therefore, mixotrophic fermentation processes, which integrate diverse carbon and reducing equivalents sources, are gaining more attention. However, the microbial communities evolving in such complex environments, where organic and inorganic substrates interact, remain insufficiently understood. To identify the key microorganisms involved in mixotrophic MCC production, two bioreactors were initially fed with brewer's spent grain as organic feedstock and supplemented daily with H 2 and CO 2 as inorganic substrates. Time‐course analyses of net metabolite production rates and microbial community composition identified Megasphaera spp. (likely M. elsdenii and M. hexanoica ) as the main chain elongators in the system. The results suggest that these Megasphaera species metabolize short‐chain carboxylates, H 2 and CO 2 , with potential cross‐feeding interactions with acetogens (likely Clostridium ljungdahlii and Clostridium luticellarii ). The homoacetogenic acetate produced is subsequently elongated, likely by Megasphaera species, using H 2 and CO 2 as electron and carbon sources for production of elongated carboxylates. This study paves the way for the development of MCC production strategies that integrate organic waste valorization with direct CO 2 utilization, using H 2 as an electron donor, a process that aligns with low‐carbon‐footprint technologies.