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AFM images of EPS-K (a) and EPS-B (b) and 3D-EEM images of of EPS-K (c) and EPS-B (d) • EPS-K and EPS-B were extracted from pure strains in activated sludge. • Both EPS showed superb performances for Hg(II) immobilization. • FTIR, 3D-EEM, XPS, EDS, PT were used to characterize the adsorption. • EPS-K and EPS-B showed different characteristics and sorption mechanisms. • S and P play important roles in the Hg(II) complexation. Extracellular polymeric substances (EPS), with abundant functional groups, play important roles in heavy metal elimination in sludge systems. In this paper, EPS-K and EPS-B were extracted from Klebsiella sp. NT8 and Bacillus sp. NT10 in activated sludge, and evaluated for their potential in Hg(II) sorption. AFM observation showed EPS possessed huge surface area. The adsorption tests indicated EPS-K and EPS-B were both superb adsorbents with the maximum sorption capacity of 2597.62 and 2617.23 mg/g, respectively and the adsorption followed the pseudo-second-order kinetics and Langmuir isotherm model. FT-IR and 3D-EEM analysis showed aromatic and tryptophan-like proteins in EPS-B, while tryptophan-like protein and humic substance in EPS-K contributed to Hg(II) binding, with higher site binding number in EPS-B. EDS and XPS further highlighted the S- and P-based functional groups in the complexation with Hg besides N. This work provides new insights into microbial products from sludge with biosorbent potential for metal treatment. [ABSTRACT FROM AUTHOR]