Methane and carbon dioxide emissions from wastewater treatment units linked to DOM stabilization and phosphonate-scavenging microbiomes.

Municipal wastewater treatment plants (WWTPs) are major engineered facilities for urban carbon removal, yet methane (CH4) formation and source mechanisms in downstream stages after aeration and biological nutrient removal remain poorly resolved. Process resolved monitoring at a full-scale WWTP showed that CH4 emissions were concentrated upstream, while measurable fluxes persisted in downstream sedimentation and denitrification units. Dissolved CH4 profiles showed strong attenuation of influent derived CH4 during upstream treatment, followed by a local increase after secondary clarification. Carbon dioxide (CO2) emissions peaked in the biochemical tank, consistent with rapid oxidation of labile organic carbon. Fluorescence and molecular analyses revealed a shift in dissolved organic matter (DOM) from protein like to more humic and processed molecules, while community assembly remained predominantly deterministic despite greater stochasticity in later stages. The C-P lyase catalytic core was enriched in these units, accompanied by higher phnJ transcript abundance, candidate organophosphonate features, and genomic potential. Together with BES insensitive CH4 formation in independent microcosms, these observations supported C-P lyase mediated organophosphonate utilization as a contributing pathway to local CH4 formation. Integrated evidence indicated that DOM stabilization, deterministic community filtering, potential succinate mediated cross feeding, and phosphonate scavenging jointly shaped this process. These findings show that advanced treatment units are not CH4 hotspots, but neither are they CH4 inactive zones; process resolved GHG assessments should therefore consider persistent local CH4 generation and its association with substrate restructuring and alternative phosphorus acquisition.