Deciphering intrakingdom synergism in 17β-estradiol degradation through DNA-SIP coupled metagenomics: Metabolic cooperation and niche partitioning in bioaugmented soil microbiomes.

17β-Estradiol (17β-E2), a persistent endocrine-disrupting compound, threatens ecosystem health through bioaccumulation. While bioaugmentation offers promise for environmental remediation, mechanistic insights into interspecies interactions between exogenous and indigenous degraders remain underexplored. Here, a synthetic microbial consortium (EL) was constructed by combining Rhodococcus erythropolis D310-1 and Microbacterium oxydans ML-6, which reduced the 17β-E2 degradation half-life by 53.65 % compared with that of the noninoculated control while suppressing the accumulation of the toxic intermediate estrone (E1). Temporal 16S rRNA gene amplicon sequencing profiling with co-occurrence network analysis revealed that the consortium EL dynamically reinforced ecological synergies with indigenous functional microbiota, accelerating contaminant mineralization. DNA-stable isotope probing (DNA-SIP) coupled with metagenomics identified Rhodanobacter, Mycobacterium, Rhodococcus, Sphingomonas, and Microbacterium spp. as active 17β-E2 assimilators. Furthermore, high-performance liquid chromatography coupled with quadrupole timeflight mass spectrometry (HPLCQTOFMS) was used to predict three complementary degradation pathways in the assembled genomes, revealing related functional enzymes and addressing functional partitioning between exogenous inoculants and indigenous degraders. Two novel gene clusters responsible for 17β-E2 biodegradation were evaluated. This study pioneers DNA-SIP and metagenomics to track C-labelled 17β-E2 fate within bioaugmented soil microbiota, resolving intrakingdom bacteria collaborations that drive 17β-E2 biodegradation in soil. The identification of cross-consortium metabolic handoffs provides a blueprint for engineering syntrophic partnerships targeting steroidal estrogens (SEs) pollutants.