Feeding microbial communities with both organic and inorganic substrates can improve sustainability and feasibility of chain elongation processes. Sustainably produced H 2 , CO 2 , and CO can be co‐fed to microorganisms as a source for acetyl‐CoA, while a small amount of an ATP‐generating organic substrate helps overcome the kinetic hindrances associated with autotrophic carboxylate production. Here, we operated two semi‐continuous bioreactor systems with continuous recirculation of H 2 , CO 2 , and CO while co‐feeding an organic model feedstock (lactate and acetate) to understand how a mixotrophic community is shaped during carboxylate production. Contrary to the assumption that H 2 , CO 2 , and CO support chain elongation via ethanol production in open cultures, significant correlations ( p < 0.01) indicated that relatives of Clostridium luticellarii and Eubacterium aggregans produced carboxylates (acetate to n ‐caproate) while consuming H 2 , CO 2 , CO, and lactate themselves. After 100 days, the enriched community was dominated by these two bacteria coexisting in cyclic dynamics shaped by the CO partial pressure. Homoacetogenesis was strongest when the acetate concentration was low (3.2 g L −1 ), while heterotrophs had the following roles: Pseudoramibacter , Oscillibacter , and Colidextribacter contributed to n ‐caproate production and Clostridium tyrobutyricum and Acidipropionibacterium spp. grew opportunistically producing n ‐butyrate and propionate, respectively. The mixotrophic chain elongation community was more efficient in carboxylate production compared with the heterotrophic one and maintained average carbon fixation rates between 0.088 and 1.4 g CO 2 equivalents L −1 days −1 . The extra H 2 and CO consumed routed 82% more electrons to carboxylates and 50% more electrons to carboxylates longer than acetate. This study shows for the first time long‐term, stable production of short‐ and medium‐chain carboxylates with a mixotrophic community.

Standard agarose gel electrophoresis is a widely used method to analyse diversity of nucleic acids. Certain conditions, however, may give rise to artefactual bands. We report on artefactual bands frequently occurring, especially when partially homologous nucleic acids, such as splicing variants of DNA transcripts, are analysed simultaneously. Interestingly, to some extent agarose concentration may influence the occurrence of artefactual bands.