Distinct Acetate Utilization Strategies Differentiate Butyrate and Medium-Chain Carboxylic Acid Producing Chain-Elongating Bacteria

Chain elongating bacteria (CEB) are a unique guild of anaerobes that upcycle organic waste into valuable short- and medium-chain carboxylic acids (MCCAs), enabling a circular bioeconomy. However, the metabolic rules that determine product chain length have remained elusive. Here, we combine 13C isotope tracing, proteomics, enzyme assays, and metabolic modelling to show that distinct acetate utilization strategies underlie the divergence between butyrate- and MCCA-producing CEB. MCCA-producing strains recycle acetate to maximize lactate use under acetate limitation, but at the cost of slower growth. In contrast, butyrate-producing strains grow faster by favoring acetate assimilation, at the cost of restricted lactate utilization when acetate is scarce. These physiological trade-offs are encoded in the substrate specificity of coenzyme A transferase, the terminal enzyme in reverse {beta}-oxidation. Our findings uncover a fundamental constraint shaping chain-length selectivity in CEB and offer new strategies to optimize MCCA production from organic waste streams.