Sulfide-dependent mercury speciation, bioavailability, and microbial methylation revealed by whole-cell biosensor, SEC-ICP-MS, and thermodynamic modelling

Methylmercury (MeHg) is predominantly produced via anaerobic microbial methylation, which efficiency is fundamentally governed by the bioavailability of inorganic Hg(II). Reduced sulfur species (S(-II)) is known to exert a non-linear regulatory effect on Hg methylation in sulfidic environments, yet the core mechanistic underpinnings of this relationship remain poorly constrained. Here, we integrated a Hg-specific whole-cell biosensor (Escherichia coli DH5α PRL), size-exclusion chromatography coupled with ICP-MS (SEC-ICP-MS), and PHREEQC thermodynamic modeling to systematically resolve how S(-II) modulates Hg bioavailability across a broad Hg/S ratio gradient (1:0 to 1:500). After 4 h, biosensor-derived intracellular active Hg (CellHg_act) increased significantly at Hg/S = 1:0.5 but decreased by 83.8-87.9% at Hg/S ≥ 1:10, relative to the sulfide-free control. SEC-ICP-MS speciation quantification verified that mild sulfide loading favors the formation of HgS nanoparticles (HgS_NP), which constituted up to 42.8% of total Hg and possessed high bioavailability for microbial uptake. By contrast, excessive S(-II) drives the dissolution of HgS_NP and concurrent formation of soluble charged Hg-S complexes (dominated by HgS₂H^-), which drastically diminish Hg bioavailability. Thermodynamic simulations incorporating a calibrated apparent solubility constant for HgS_NP (logK = -31.0) accurately reproduced the dynamic shift between HgS_NP formation and sulfide-triggered dissolution across the S(-II) gradient. Complementary methylation assays using Geobacter sulfurreducens PCA validated that MeHg production peaks at Hg/S = 1:0.5, consistent with the biosensor-derived bioavailability trend. Together, these experimental and modeling evidences reveal that S(-II) controls Hg bioavailability via the formation and dissolution of HgS_NP, providing a mechanistic framework bridging Hg speciation and microbial MeHg production in sulfidic environments.