Redox-active biomolecular networks drive extracellular selenate reduction by Shewanella putrefaciens CN-32.

Selenate [Se(VI)] reduction plays a key role in selenium (Se) biogeochemical cycling, with microbially mediated processes primarily driving this cycling. However, the relative contributions of enzymatic and extracellular processes remain poorly understood. Here, we demonstrated that Se(VI) can be reduced to Se nanoparticles (SeNPs) in the extracellular matrix of Shewanella putrefaciens CN-32 (CN-32), both in whole-cell systems and in cell-free extracellular polymeric substances (EPS). CN-32 cells achieved 69.3% reduction after 84 h, with over 90% of SeNPs localized extracellularly. EPS removal reduced reduction efficiency by 8.8%, whereas 23% reduction rates were observed with EPS supplementation. Se(VI) reduction was primarily driven by cytochrome-mediated electron transfer and intracellular metabolism, while electron shuttles restored activity in cytochrome-deficient mutants. Spectroscopic and kinetic analysis identified aldehyde as key electron donors and demonstrated that low molecular weight EPS fractions (<3 kDa) exhibited superior electron transfer efficiency due to enhanced accessibility of redox-active moieties. These results establish a dual-pathway framework for microbial Se(VI) reduction, highlighting EPS as an active extracellular redox matrix. Given the widespread occurrence of Se contamination in the environment, these findings provide basis for harnessing EPS-mediated reduction pathways in the design of more effective bioremediation strategies for selenium-impacted environments. SYNOPSIS: This study demonstrated that low-molecular weight molecules in extracellular polymeric substances play the key role for the microbial reduction of selenate to insoluble elemental selenium nanoparticles.