Integrated multi-omics and metabolic modeling links structure to function in high-performing electrosynthetic biofilm...

Microbial electrosynthesis (MES) is a promising technology for the valorization of CO₂ into industrially relevant building blocks. The high-performing MES systems in terms of production rates of acetate (12.5-19.7 mmol L^-1_catholyte day^-1), butyrate (1.9-12.2 mmol L^-1_catholyte day^-1), and caproate (0.6-0.9 mmol L^-1_catholyte day^-1) discussed in this study consist of mixed microbial communities. However, the microbial community members, metabolic pathways, and interactions driving product formation in MES communities remain poorly understood. To overcome these challenges, we conducted a comprehensive characterization of three high-performing MES communities, combining multi-omics with metagenome-scale metabolic modeling. Using a high-resolution metagenomic pipeline, we reconstructed high-quality genomes of 25 metagenome-assembled genomes present in our reactors, including six fully circular genomes. We report the presence of Clostridium aromativorans for the first time in a gas-fermenting system. In particular, our findings identified three acetogenic species, Eubacterium limosum, Sporomusa sphaeroides, and C. aromativorans, as key contributors to the production of acetate, butyrate, and caproate via the Wood-Ljungdahl and the reverse β-oxidation pathways. In addition, we found genes related to lactate and ethanol production from acetyl-CoA, along with proteomic evidence of lactate production. This paves the way for investigating the role of cross-fed metabolites such as lactate and ethanol as electron donors in chain elongation. Finally, meta-genome-scale metabolic modeling suggests that the communities might be sustained by the cross-feeding of specific cofactors such as pyridoxine, pantothenate, biotin, and thiamin. This study provides key insights into the structure and function of electrosynthetic communities, bringing us closer to the rational engineering of MES systems.IMPORTANCEMicrobial electrosynthesis (MES) offers a promising route to transform CO₂ and renewable electricity into valuable platform chemicals. However, the microbial ecology governing the assembly and function of these systems remains poorly understood, limiting our ability to engineer them. By integrating high-resolution multi-omics with metabolic modeling, this study provides a systems-level framework to dissect the structure and function of electrosynthetic microbial communities. Understanding which organisms drive carbon fixation and chain elongation, how electrons and intermediates are transferred within the biofilm, and how metabolic dependencies structure these communities is essential for improving productivity and product specificity in MES. More broadly, this work highlights how integrated multi-omics approaches can resolve the functional organization of complex microbial ecosystems and provides methodological advances that are broadly applicable to the study and engineering of microbial communities in autotrophic, electricity-driven biotechnologies.