Humus-gradient adaptive laboratory evolution remodels electroactive microbial consortia to achieve cold-resistant...

Low-temperature conditions trigger continuous humus accumulation in substrates, inhibiting extracellular electron transfer and reducing electrogenesis efficiency, which limits the practical performance and engineering application of constructed wetland-microbial fuel cells (CW-MFCs). Conventional physicochemical regulation strategies cannot fundamentally improve the intrinsic stress resistance of electroactive microbial consortia. Herein, a two-stage experimental workflow combining adaptive laboratory evolution (ALE) domestication and subsequent CW-MFC inoculation was established to obtain mixed microbial consortia with excellent cold-adapted electrogenic capacity. Electrochemical assays combined with 16S rRNA high-throughput sequencing demonstrated that ALE domestication remodelled biofilm community structure, enriching Geobacter-dominated core electroactive microorganisms and putative stress-tolerant Halomonas. SHapley Additive exPlanations (SHAP) interpretative modelling verified that microbial community composition acts as a major correlative factor governing electrogenic performance and revealed potential statistical associations between Geobacter and humus. Different from passive physicochemical external regulation, ALE converts humus-associated stress under low-temperature environments into controllable selective pressure to drive endogenous metabolic shifts within microbial consortia. This study clarifies the community-level response rules of electroactive consortia to combined low-temperature-humus stress and provides theoretical references for microbial bioaugmentation of low-temperature bioelectrochemical reactors. All experiments were carried out in laboratory-scale synthetic-wastewater systems and further validation under practical field conditions is required.