Electron‐transfer pathways occurring in biocathodes are still unknown. We demonstrate here that high rates of acetate production by microbial electrosynthesis are mainly driven by an electron flux from the electrode to carbon dioxide, occurring via biologically induced hydrogen, with (99±1) % electron recovery into acetate. Nevertheless, acetate production is shown to occur exclusively within the biofilm. The acetate producers, putatively Acetoanaerobium, showed the remarkable ability to consume a high H₂ flux before it could escape from the biofilm. At zero wastage of H₂ gas, it allows superior production rates and lesser technical bottlenecks over technologies that rely on mass transfer of H₂ to microorganisms suspended in aqueous solution. This study suggests that bacterial modification of the electrode surface (possibly via synthesis of Cu nanoparticles) is directly involved in the significant enhancement of the hydrogen production.

Extracellular polymeric substances (EPS) are core biofilm components, yet how they mediate interactions within and contribute to the structuring of biofilms is largely unknown, particularly for non-culturable microbial communities that predominate in environmental habitats. To address this knowledge gap, we explored the role of EPS in an anaerobic ammonium oxidation (anammox) biofilm. An extracellular glycoprotein, BROSI_A1236, from an anammox bacterium, formed envelopes around the anammox cells, supporting its identification as a surface (S-) layer protein. However, the S-layer protein also appeared at the edge of the biofilm, in close proximity to the polysaccharide-coated filamentous Chloroflexi bacteria but distal to the anammox bacterial cells. The Chloroflexi bacteria assembled into a cross-linked network at the edge of the granules and surrounding anammox cell clusters, with the S-layer protein occupying the space around the Chloroflexi. The anammox S-layer protein was also abundant at junctions between Chloroflexi cells. Thus, the S-layer protein is likely transported through the matrix as an EPS and also acts as an adhesive to facilitate the assembly of filamentous Chloroflexi into a three-dimensional biofilm lattice. The spatial distribution of the S-layer protein within the mixed species biofilm suggests that it is a “public-good” EPS, which facilitates the assembly of other bacteria into a framework for the benefit of the biofilm community, and enables key syntrophic relationships, including anammox.