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Geobacter species are common in iron-rich environments and can contribute to formation of methylmercury (MeHg), a neurotoxic compound with high bioaccumulation potential formed as a result of bacterial and archaeal physiological activity. Geobacter sulfurreducens can utilize various electron acceptors for growth including iron hydroxides or fumarate. However, it remains poorly understood how the growth on these compounds affects physiological properties of bacterial cells in biofilms, including the capacity to produce MeHg. The purpose of this study was to determine changes in the biochemical composition of G. sulfurreducens during biofilm cultivation in media containing iron hydroxide or fumarate, and to quantify mercury (Hg) methylation capacity of the formed biofilms. Biofilms were characterized by Fourier-transform infrared spectroscopy in the attenuated total reflection mode (ATR-FTIR), Resonance Raman spectroscopy and confocal laser scanning microscopy. MeHg formation was quantified by mass spectrometry after incubation of biofilms with 100 nM Hg. The results of ATR-FTIR experiments showed that in presence of fumarate, G. sulfurreducens biofilm formation was accompanied by variation in content of the energy-reserve polymer glycogen over time, which could be cancelled by the addition of supplementary nutrients (yeast extract). In contrast, biofilms cultivated on Fe(III) hydroxide did not accumulate glycogen. The ATR-FTIR results further suggested that Fe(III) hydroxide surfaces bind cells via phosphate and carboxylate groups of bacteria that form complexes with iron. Furthermore, biofilms grown on Fe(III) hydroxide had higher fraction of oxidized cytochromes and produced two to three times less biomass compared to conditions with fumarate. Normalized to biofilm volume, the content of MeHg was similar in assays with biofilms grown on Fe(III) hydroxide and on fumarate (with yeast extract and without). These results suggest that G. sulfurreducens biofilms produce MeHg irrespectively from glycogen content and cytochrome redox state in the cells, and warrant further investigation of the mechanisms controlling this process.

Introduction: Mercury (Hg) is a major environmental pollutant that accumulates in biota predominantly in the form of methylmercury (MeHg). Surface-associated microbial communities (biofilms) represent an important source of MeHg in natural aquatic systems. In this work, we report MeHg formation in biofilms of the iron-reducing bacterium Geobacter sulfurreducens.

Methods: Biofilms were prepared in media with varied nutrient load for 3, 5, or 7 days, and their structural properties were characterized using confocal laser scanning microscopy, cryo-scanning electron microscopy and Fourier-transform infrared spectroscopy.

Results: Biofilms cultivated for 3 days with vitamins in the medium had the highest surface coverage, and they also contained abundant extracellular matrix. Using 3 and 7-days-old biofilms, we demonstrate that G. sulfurreducens biofilms prepared in media with various nutrient load produce MeHg, of which a significant portion is released to the surrounding medium. The Hg methylation rate constant determined in 6-h assays in a low-nutrient assay medium with 3-days-old biofilms was 3.9 ± 2.0 ∙ 10−14  L ∙ cell−1 ∙ h−1, which is three to five times lower than the rates found in assays with planktonic cultures of G. sulfurreducens in this and previous studies. The fraction of MeHg of total Hg within the biofilms was, however, remarkably high (close to 50%), and medium/biofilm partitioning of inorganic Hg (Hg(II)) indicated low accumulation of Hg(II) in biofilms.

Discussion: These findings suggest a high Hg(II) methylation capacity of G. sulfurreducens biofilms and that Hg(II) transfer to the biofilm is the rate-limiting step for MeHg formation in this systems.

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.