Deciphering 17β-estradiol-3-sulfate biotransformation: From microbial interactions to strain-level biotransformation

While biodegradation shows promise for removing 17β-estradiol-3-sulfate (E2–3S) from wastewater, its underlying mechanisms remain poorly understood. We systematically elucidated E2–3S biodegradation from both microbial community and individual strain perspectives by constructing microbial co-occurrence networks across diverse reaction environments and isolating degrading bacteria from activated sludge. The results showed that activated sludge achieved E2–3S removal greater than 90 %, though estrogenic activity reduction varied with reaction environments. Network analysis, connectivity and centrality analyses identified Pseudomonas species as the keystone taxon driving estrogenic activity reduction, supported by synergistic interactions with Azonexus , Dechloromonas and Obscuribacter species to maintain redox homeostasis and nutrient availability. We further isolated non-pathogenic Pseudomonas CE2S, which demonstrated exceptional E2–3S degradation (> 90 %) and estrogenic activity reduction (> 70 %) across concentration gradients. Two major degradation pathways were proposed for strain CE2S, including deconjugation, hydroxylation, and oxidative ring cleavage reactions, ultimately yielding low-estrogenic-activity products. To our knowledge, this work provides the first comprehensive mechanistic understanding of E2–3S biodegradation spanning community to strain levels, and reports the first isolation of an activated sludge bacterium capable of simultaneous E2–3S degradation and estrogenic activity reduction. These findings provide critical insights for understanding E2–3S biodegradation and developing bioaugmentation strategies for optimal E2–3S removal.