Microbial chain elongation represents an attractive alternative to chemical synthesis for the production of medium-chain fatty acids (MCFAs), enabling the upgrading of short-chain organic acids and alcohols through reverse ß-oxidation. Clostridium kluyveri typically utilizes ethanol as an electron donor and acetate as a carbon acceptor; however, the potential of alternative substrates derived from industrial and biorefinery waste streams, such as succinic acid, remains poorly investigated. This study investigated the feasibility of using succinic acid as an alternative electron acceptor in C. kluyveri fermentation for MCFA production. Anaerobic batch fermentations were conducted in sealed serum bottles (40 mL working volume) at 37°C and initial pH 6.8, evaluating the co-utilization of succinate and ethanol. The effects of succinate on metabolic activity, product distribution, and product yields were assessed. The results demonstrated that succinate can sustain C. kluyveri metabolism under anaerobic conditions, resulting in hexanoic acid concentrations of approximately 4.3–4.7 g/L when ethanol was supplied as the reducing equivalent source. The co-utilization of succinate and ethanol promoted butyrate and hexanoate formation, confirming the activation of chain elongation pathways and highlighting the strong dependence of succinate conversion on ethanol availability. Overall, these findings demonstrate the metabolic flexibility of C. kluyveri and support the integration of succinate-rich waste streams into MCFA bioproduction processes, provided sufficient reducing equivalents are available.
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