The Combined Effect of Hg(II) Speciation, Thiol Metabolism, and Cell Physiology on Methylmercury Formation by Geobacter sulfurreducens

Abstract

The chemical and biological factors controlling microbial formation of methylmercury (MeHg) are widely studied separately, but the combined effects of these factors are largely unknown. We examined how the chemical speciation of divalent, inorganic mercury (Hg(II)), as controlled by low-molecular-mass thiols, and cell physiology govern MeHg formation by Geobacter sulfurreducens. We compared MeHg formation with and without addition of exogenous cysteine (Cys) to experimental assays with varying nutrient and bacterial metabolite concentrations. Cysteine additions initially (0-2 h) enhanced MeHg formation by two mechanisms: (i) altering the Hg(II) partitioning from the cellular to the dissolved phase and/or (ii) shifting the chemical speciation of dissolved Hg(II) in favor of the Hg(Cys)₂ complex. Nutrient additions increased MeHg formation by enhancing cell metabolism. These two effects were, however, not additive since cysteine was largely metabolized to penicillamine (PEN) over time at a rate that increased with nutrient addition. These processes shifted the speciation of dissolved Hg(II) from complexes with relatively high availability, Hg(Cys)₂, to complexes with lower availability, Hg(PEN)₂, for methylation. This thiol conversion by the cells thereby contributed to stalled MeHg formation after 2-6 h Hg(II) exposure. Overall, our results showed a complex influence of thiol metabolism on microbial MeHg formation and suggest that the conversion of cysteine to penicillamine may partly suppress MeHg formation in cysteine-rich environments like natural biofilms.

Keywords: anaerobe microorganisms; low molecular mass thiols; mercury methylation; mercury speciation.

Figure 1

Methylmercury formation by G. sulfurreducens cells in the presence of 30 nM Hg(II) and addition of exogenous cysteine in the (a) standard, (b) metabolite, and (c) nutrient assay over time (n = 3; ± standard deviation). 0 nM cysteine (black circles), 100 nM cysteine (squares open), and 600 nM cysteine (triangles open).

Figure 2

Composition of dissolved Hg(II)-species (bars) in the extracellular media of G. sulfurreducens in the (a, d, g) standard, (b, e, h) metabolite, and (c, f, i) nutrient assay with exogenous cysteine of 0, 100, and 600 nM after 0.5, 2, 6, and 24 h incubation time. All assay systems contained initially 30 nM of Hg(II). Dissolved Hg(II) species are Hg(Cys)₂ (dark gray), Hg(CysN)₂ (light gray), Hg(PEN)₂ (white), and other Hg(II) species (white dotted). Measured concentrations of MeHg (nM) formed by G. sulfurreducens in each assay system over time (same data as shown in Figure 1, red diamonds). Modeled MeHg concentrations (nM) with the species-specific Hg(II) rate model (red line) and total dissolved Hg(II) rate model (dotted red line) over time based on a first-order rate model as described in the Supporting Information.

Figure 3

Evolution over time of ATR-FTIR spectra collected from 0.5 to 24 h bacterial suspension of G. sulfurreducens cells with 30 nM Hg(II) in (a) standard, (b) metabolite, and (c) nutrient assays. Spectra were recorded after 0.5, 2, 6, and 24 h incubation times and indicated in black, red, blue, and green, respectively. Corresponding supernatants at each time point were used as the reference spectra after centrifugation of the sample. The spectra were baseline corrected and normalized to the amide II band at 1548 cm^–1. Principle assignments of the infrared bands are indicated in the spectra and based on Quilès et al.