Butyrate production from the simultaneous utilization of carbon monoxide and acetate in Eubacterium-dominated enrichments

Abstract Syngas conversion to carboxylic acids can be achieved through the combined actions of acetogens and chain elongators. Mixed cultures enriched with these functional groups can serve as robust and resilient catalysts for bio-based production, but developing efficient and stable communities remains challenging. In this study, we aimed to enrich a mixed microbial culture for efficient syngas-driven chain elongation using carbon monoxide (CO) and acetate as substrates. Three strategies were employed: (i) conventional enrichment using anaerobic sludge only, (ii) sludge augmentation with known syngas fermenters (Clostridium autoethanogenum, Clostridium carboxidivorans, Eubacterium limosum, and Acetobacterium wieringae) to enhance carboxydotrophic activity, and (iii) sludge augmentation with chain elongator Clostridium kluyveri to stimulate chain elongation. Enrichments were performed in batch cultures with stepwise increases in CO partial pressure across successive transfers until a 100% CO (170 kPa) headspace was reached. Ultimately, all enrichments grew under a 100% CO headspace, simultaneously utilizing CO and acetate, with butyrate as main product. 16S rRNA gene and metagenomic analyses revealed that Eubacterium became predominant. Several genera (Aminobacterium, Anaerotignum, Oscillibacter, and Proteiniphilum) not known to ferment CO, were also enriched. Based on physiological and metagenomic analyses, we hypothesize that Eubacterium consumes CO and acetate, producing butyrate and extracellular polymeric substance (EPS)-like material. Metagenomic data further suggests that components derived from this EPS-like material may serve as substrate for the other enriched genera. This study demonstrates that augmentation can steer mixed microbial communities toward targeted product formation and highlights the critical role of initial culture conditions in shaping community composition and functionality. Key points • Mixed cultures converted CO and acetate mainly to butyrate under a 100% CO headspace. • Acetogen augmentation to a mixed community shortened lag-phase in the presence of CO. • Metagenomics suggests microbial use of sugars and amino acids from EPS-like material.