Abstract
The environmental risk posed by 17β-estradiol (E2) and tetracycline (TC) contamination within livestock wastewater has been widely concerned. Especially, the co-occurrence of these pollutants poses a tremendous challenge to their efficient bioremediation, highlighting the need for strains capable of simultaneous E2 and TC removal. In this study, a novel strain of Alkalibacterium sp. AEPI-S25 was successfully isolated from the sediments of Qinghai Lake, demonstrating the ability to remove 89.91% of 20 mg L-1 E2 and nearly 100% of 20 mg L-1 TC simultaneously within 5 days. AEPI-S25 exhibited remarkable environmental adaptability and maintained high simultaneous removal efficiency under various stress conditions and the presence of two typical livestock wastewater samples. Based on Ultra-Performance Liquid Chromatography coupled with Orbitrap High-Resolution Mass Spectrometry (UPLC-Orbitrap-HRMS) analysis, dehydrogenation and monooxygenation were identified as the key steps in the removal pathways of E2 and TC, respectively. Whole-genome sequencing further identified the potential E2/TC removal genes, and the detected potential E2-dehydrogenation (S25_gene0393) and TC-monooxygenation (S25_gene0878) genes were subsequently validated through transcription analysis and heterologous expression. Notably, S25_gene0878 exhibited significant differences from the well-characterized TC removal gene TetX, extending a new understanding of the bacterial TC removal mechanism. Overall, this study provides the first report of a single microbial strain capable of simultaneously removing E2 and TC, offering valuable insights for the application of microbial technologies in addressing typical E2-TC combined pollution in livestock wastewater. KEY POINTS: • AEPI-S25 can remove nearly 90% and 100% of 20 mg L-1 E2 and TC within 5 days • Key E2 (S25_gene0393) and TC (S25_gene0878) removal genes were cloned and expressed • A novel TC-monooxygenase gene distinct from TetX was discovered within the strain.