Automated anaerobic cultivation and phenotyping of diverse chain-elongating bacteria

Anaerobic bacteria perform diverse metabolic functions with considerable biotechnological potential, yet their physiology remains poorly characterized because conventional anaerobic cultivation is low throughput. Here, we developed a Platform for Automated Cultivation of Microbial Anaerobes (PACMAN) and applied it to systematically characterize the physiology of diverse chain-elongating bacteria (CEB) that produce medium-chain carboxylic acids (MCCAs). High-throughput screening across nine CEB and 20 potential electron donors revealed extensive metabolic diversity, including previously unreported conversion of glycerol to octanoate by Pseudoramibacter alactolyticus and xylose to hexanoate by Caproiciproducens sp. 7D4C2. Comparison with genome-derived metabolic pathways further revealed widespread discrepancies between predicted and observed substrate utilization. Across diverse CEB and electron donors, decreasing the acetate-to-electron-donor ratio consistently shifted product distributions toward longer-chain carboxylates, revealing a general principle governing chain-length specificity, although maximum attainable chain length remained strain dependent. Finally, screening CEB across increasing carboxylate concentrations revealed substantial variation in MCCA tolerance, with Gram-positive MCCA producers exhibiting greater resilience than Gram-negative and butyrate-producing bacteria. Together, these findings reveal organizing principles underlying the metabolic versatility and product specificity of CEB and establish automated anaerobic phenotyping as a scalable approach for discovering microbial functions relevant to biomanufacturing.