Molecular ecological network-guided consortium assembly reveals stage-dependent metabolic cooperation for enhanced...

Cephalexin (CLX), a typical β-lactam antibiotic, poses challenges to the biological treatment of antibiotic wastewater due to its environmental persistence and resistance risks. Bioaugmentation offers a green removal strategy, but single functional strains are often constrained by toxicity inhibition and intermediate accumulation. Here, a dual-strain synthetic consortium was rationally constructed from Glutamicibacter sp. S2 and Herbaspirillum sp. S8 based on the molecular ecological network (MEN) of native sludge, and its enhanced CLX removal performance and cooperative metabolic mechanism were systematically investigated. The consortium C2, assembled according to the key-node ratio (S2:S8 = 1:3), exhibited the best overall performance, removing 91.12% of CLX and 60.50% of TOC within 60 h. It also increased BOD₅/COD to above 0.3, with AOS and COS values reached 3.160 and 3.690, respectively. Structural and extracellular characterizations showed that C2 formed core-periphery aggregates and established an extracellular microenvironment enriched in a protein (5.097 mg/L)-polysaccharide (1.67 mg/L) matrices and abundant active functional groups. Metabolomic further revealed that S2 was associated with CLX transformation, whereas S8 provided a stable adsorption interface through membrane structural regulation and stress buffering. Their combination induced pantothenate-centered energy supply, sphingolipid-centered stress and signaling responses, and enrichment of β-lactam resistance- and cytochrome P450-associated pathways, jointly promoting sustained CLX attenuation and intermediate transformation. This study elucidates an adsorption enrichment-enzymatic transformation mechanism underlying efficient CLX removal by bioaugmentation consortium and provides a reference for designing functionally differentiated microbial inoculants for antibiotic wastewater treatment.