Chavali, Sri SannihitaSri SannihitaChavali2026-09-232026-09-232026-09-23https://hdl.handle.net/11420/64347Cupriavidus necator is a highly adaptable chemolithoautotroph and a promising chassis for sustainable bioproduction from CO₂. Coupling its metabolism to renewable electricity through microbial electrosynthesis (MES) requires the organism to form stable, electroactive biofilms on cathodes. This step is hindered under oxic conditions (oxic MES, oMES) by electrochemically generated reactive oxygen species (ROS), mass-transfer limitations and poor biofilm stability. Systematic strategies to improve C. necator biofilm formation on cathodes have been lacking. Here we used adaptive laboratory evolution (ALE) to address this gap. The progenitor strain colonised graphite cathodes only after an initial heterotrophic phase and formed spatially uneven biofilms whose distribution mirrored the local flow field, as resolved by micro-particle tracking velocimetry. Populations were then selected over five passages under controlled, oxygen-limited cathodic conditions. Resequencing revealed that the recovered population was genetically heterogeneous, and its dominant isolate, strain 890, was characterised in a microfluidic oMES platform. Strain 890 reached the target electrode coverage in about one day instead of six and maintained a robust biofilm at the progenitor's steady-state biovolume, whereas the multi-strain pool peaked higher but proved unstable. Whole-genome sequencing linked this accelerated, biofilm-forward phenotype to only two loci, both potentially central regulators of the planktonic-to-biofilm transition. The steady-state biovolume itself remained bounded most probably due to a mass-transfer ceiling set by the counter-directional supply of H₂ and O₂. This work shows that ALE renders the initial-attachment barrier of cathodic biofilm formation genetically tractable, providing strain 890 as a robust chassis and a well-defined starting point for oMES-based CO₂ valorisation.https://creativecommons.org/publicdomain/zero/1.0/Cupriavidus necatormicrobial electrosynthesisbiofilm formationadaptive laboratory evolutionCO₂ valorisationgenetic variantsTechnology::660: Chemistry; Chemical Engineering::660.6: BiotechnologyDataset10.15480/882.17914Chavali, Sri SannihitaSri SannihitaChavaliGescher, JohannesJohannesGescherChavali, Sri SannihitaSri SannihitaChavaliZhou, YitianYitianZhouNissen, Jan HendrickJan HendrickNissenLapp, Christian JonasChristian JonasLappChavali, Sri SannihitaSri SannihitaChavaliGescher, JohannesJohannesGescherLapp, Christian JonasChristian JonasLappNissen, Jan HendrickJan HendrickNissenHochschule für Angewandte Wissenschaften Hamburg