Molecular insights into microbial transformation of dissolved organic matter from a wastewater treatment plant

Dissolved organic matter (DOM) in treated wastewater influences effluent reactivity and ecological risks. How biological treatment configurations reshape DOM at the molecular level remains unclear. This study integrated molecular characterization, metagenomic microbial community analysis and metatranscriptomic functional profiling, machine-learning interpretation, and dynamic Partial Mantel Distance network analysis to compare DOM transformation in full-scale anaerobic-anoxic-oxic process (A2O) and oxidation ditch process (OD). The A2O exhibited a stronger redox-stage-dependent DOM remodeling than the OD. In the A2O, the final aerobic effluent showed a reduced mean molecular weight of 411.45, higher O/C, and lower H/C, with lipid-like and protein-like compounds decreasing from 35.13% to 15.39% and from 35.70% to 21.44%, respectively, whereas the lignin/CRAM-like fractions increased to 47.64%, indicating preferential removal of labile aliphatic and proteinaceous molecules and enrichment of refractory DOM. The OD retained a higher final molecular weight of 451.43 and a lipid-like fraction of 33.94%, suggesting gradual stabilization under continuous circulation and mixed redox microenvironments. The microbial communities differed significantly between the processes, whereas the enzyme profiles showed weaker separation, indicating functional redundancy. The Random Forest models effectively predicted the DOM molecular fate in both processes, with macro AUC values of 0.8913 and 0.8923 for the A2O and OD, respectively. The dPMD networks revealed intermediate-mediated cascade transformations. The treatment configuration governed DOM persistence, microbial ecological control, and the refractory character of the residual effluent DOM. This study provides a molecular-level theoretical basis for optimizing biological wastewater treatment processes, regulating refractory DOM, and reducing the ecological risks associated with treated wastewater discharge.