Subcellular Mechanisms of Se(IV) Reduction in Stenotrophomonas bentonitica: Linking Environmental Detoxification and Antimicrobial Activity of Se Nanostructures Within a Circular Economy Framework.

Selenium (Se) is an essential trace element whose toxicity depends on its oxidation state. Microorganisms detoxify Se(VI) and Se(IV) by reducing them to elemental selenium [Se(0)], forming selenium nanoparticles (SeNPs) with antimicrobial activity. Stenotrophomonas bentonitica BII-R7 exhibits remarkable tolerance and reduction capacity toward toxic Se oxyanions, making it a promising candidate for bioremediation and green nanotechnology. In this study, cells exposed to Se(IV) were fractionated into cytoplasmic and membrane components and analysed at 24, 168 and 720 h. Spherical SeNPs were observed in the cytoplasm, while irregular aggregates formed in the membrane fraction, suggesting compartment-specific reduction pathways. The delayed formation of SeNPs in membranes supports a time-dependent, multimodal mechanism. Homogeneous biogenic SeNPs (160-180 nm) produced by intact S. bentonitica cells exhibited antimicrobial activity against Escherichia coli CET101 and Staphylococcus aureus ATCC 25923. Flow cytometry revealed strong, time-dependent cytotoxicity. In E. coli, SeNPs induced 21.1% membrane depolarization, 62.8% ROS accumulation and DNA damage at 48 h, indicating a ROS-mediated mechanism. In contrast, S. aureus showed early membrane depolarization at 12 h, with only 4.37% active cells and minimal ROS levels, and a significant drop in viability at 24 h (31.3%), suggesting a ROS-independent mechanism driven by membrane disruption. These findings highlight the strain-specific toxicity of SeNPs and their potential as eco-friendly, broad-spectrum antimicrobials.