Synergistic acetate-butyrate co-utilization enhances caproate production by Clostridium kluyveri CICC 24509 under high substrate loads

Microbial chain elongation presents a compelling circular economy strategy for converting low-value waste streams into valuable chemicals like caproate. However, realizing its full potential is hampered by challenges, including limited microbial tolerance to industrially relevant high substrate concentrations and suboptimal electron donor-acceptor utilization. This study addresses these limitations by evaluating the performance of a previously uncharacterized chain-elongating bacterium, Clostridium kluyveri CICC 24509, under high-substrate conditions using ethanol as the electron donor. We systematically investigated the impact of ethanol concentration, total carbon loading (up to 1370 mM C), and electron acceptor composition. While acetate alone supported robust production (13.7 g L^-1 caproate at 20 g L^-1 ethanol), a remarkable synergistic effect was observed with the co-utilization of acetate and butyrate as electron acceptors. This dual-acceptor strategy yielded the highest reported caproate concentration for a pure C. kluyveri culture at 16.0 g L^-1, achieving a rapid production rate of 4.0 ± 0.1 g L^-1 d^-1. This approach significantly enhanced short-chain fatty acid conversion efficiency compared to single-acceptor systems. These findings underscored the robust biocatalytic capabilities of Clostridium kluyveri CICC 24509 and demonstrated a promising strategy for efficiently converting high-organic-loading industrial fermentation residues into caproate, paving the way for scalable resource recovery.