Mercury Methylation by Dissimilatory Iron-Reducing Bacteria

The Hg-methylating ability of dissimilatory iron-reducing bacteriain the genera Geobacter, Desulfuromonas, and Shewanella wasexamined. All of the Geobacter and Desulfuromonas strains testedmethylated mercury while reducing Fe(III), nitrate, or fumarate.In contrast, none of the Shewanella strains produced methylmercuryat higher levels than abiotic controls under similar cultureconditions. Geobacter and Desulfuromonas are closely relatedto known Hg-methylating sulfate-reducing bacteria within theDeltaproteobacteria.

INTRODUCTION

Top

Introduction

Methylmercury (MeHg) concentrations in most sediments are controlledby in situ net microbial methylation (1, 13). Environmentalmercury methylation is an anaerobic microbial process generallydriven by dissimilatory sulfate-reducing bacteria (DSRB) (1).However, recent research suggests that dissimilatory iron-reducingbacteria (DIRB) may play a role in environmental methylation(10, 27). Further, Fleming et al. (10) demonstrated Hg methylationby a Geobacter strain isolated from Clear Lake, CA. Consequently,we designed an experiment to screen a phylogenetically diversegroup of DIRB cultures for Hg-methylating capability in orderto develop insight into in situ biological methylation controlsand to further investigate the phylogenetic distribution ofmethylating bacteria.

Two studies have demonstrated net MeHg production in sedimentswhere iron was the dominant terminal electron acceptor (10,27), but another demonstrated inhibition of methylation by iron(23). Iron could potentially influence Hg methylation rateseither through changes in DIRB activity or via the impact ofiron on Hg complexation and bioavailability. In a study of estuarinewetland sediment slurries from San Francisco Bay, CA, Mehrotraand Sedlak (23) observed decreases in Hg methylation rates withthe addition of 30 mM Fe(III), and they suggested that thiseffect was caused by decreases in dissolved Hg and sulfide dueto complexation with iron. However, Warner et al. (27) foundmeasurable methylation in sediments where iron reduction wasthe dominant terminal electron acceptor, although rates of methylationwere lower than those observed in sulfate-reducing or methanogenicsediments. Similarly, in sediments from Clear Lake, CA (10),where microbial Fe(III) reduction was apparent, chemical inhibitionof sulfate reduction did not result in complete inhibition ofHg methylation. This decoupling of Hg methylation from sulfatereduction suggests that another process (i.e., iron reduction)may be responsible for some amount of in situ Hg methylation.Mercury methylation by a Geobacter strain isolated from ClearLake (10) further supports this hypothesis.

Phylogenetic relationships between members of the Geobacteraceaeand the Hg-methylating DSRB also suggest a possible role forDIRB in environmental Hg methylation. The Geobacteraceae arefound in the Deltaproteobacteria, branching phylogeneticallybetween the orders Desulfovibrionales and Desulfobacterales(15), both of which contain DSRB with methylating capability(2, 3, 9, 16). A wide variety of bacteria and archaea are capableof dissimilatory Fe(III) reduction (19, 20, 22), including Shewanella,which is in the ${gamma}$ subclass of Proteobacteria. This phylogenicdistribution of DIRB implicates Geobacteraceae as possible Hgmethylators and provides strains that are phylogenetically distantfrom the DSRB, which may give insight into the phylogeneticdistribution of Hg methylation.

To assess the role of DIRB in Hg methylation, pure culturesof Desulfuromonas palmitatis SDBY-1 (8), Geobacter hydrogenophilus(7), Geobacter metallireducens GS-15 (21), Geobacter sulfurreducens(6), Shewanella alga BrY (5), Shewanella oneidensis MR-1 (26),and Shewanella putrefaciens CN-32 (18) were tested for the abilityto methylate inorganic Hg while growing on a variety of electrondonors and acceptors, including Fe(III), nitrate, and organicsubstrates (Table 1). Cultures were grown in media modifiedfrom the work of Bond and Lovley (4) with electron donors andacceptors as described in Table 1, using previously describedtrace elements and vitamins (17).

View this table:
[in this window]
[in a new window]

TABLE 1. Electron donors and acceptors used in Hg methylation tests

MeHg production was assayed by measuring the amount of MeHgproduced from an inorganic Hg spike during batch culture growththrough stationary phase. All Hg methylation assays were conductedin 20-ml Hungate tubes with butyl-rubber stoppers under strictlyanaerobic conditions at 30°C and pH 7.0. Assays using G.metallireducens, G. sulfurreducens, S. putrefaciens, and S.oneidensis were conducted using an enriched stable Hg isotope,added as ²⁰¹HgCl₂, at a final concentration of 10 ng/ml. Assaysusing D. palmitatis, G. hydrogenophilus, and S. alga were conductedwith natural isotopic abundance HgCl₂ at the same concentration.For each strain and growth condition, triplicate assays andabiotic controls were prepared. Abiotic controls were composedof autoclaved medium spiked with inorganic HgCl₂.

Analysis of total MeHg was performed via distillation/ethylation(11)/cold vapor atomic fluorescence (CVAF), using a Tekran 2500atomic fluorescence detector. For CVAF analysis, the methoddetection limit was determined by the method blank, which wasgenerally <20 pg/sample. For analysis of a 20-ml culturesample at 10 ng Hg/liter, this yields a blank equivalent toroughly 0.01% methylation. Analysis of Me²⁰¹Hg was performedby distillation/ethylation/ICP-MS (inductively coupled plasmamass spectometry), with isotope dilution (14), using a PerkinElmer ELAN 6100 DRCII. Me²⁰⁰Hg (96.4% purity) was used as theisotope dilution standard. The concentration of Me²⁰⁰Hg wasdetermined by reverse isotope dilution analysis against certifiedstandards. Me²⁰⁰Hg was synthesized from ²⁰⁰HgCl₂ using an aqueousmethylcobalamine method (14). All enriched isotopes were purchasedfrom Oak Ridge National Labs as HgO. Method detection limitsusing isotope dilution-ICP-MS were generally <1 pg Me²⁰¹Hg/sample,or <0.001% methylation.

Phylogenetics of DIRB Hg-methylating capability.

Top

Phylogenetics of dirb hg...

Methylation of inorganic mercury significantly above that inuninoculated controls (t tests, P < 0.05, two-tailed, unequalvariances) was observed on Fe-reducing medium in G. metallireducens,G. sulfurreducens, G. hydrogenophilus, and D. palmitatis butnot S. alga or S. putrefaciens (Fig. 1). While growing withelectron acceptors other than Fe(III), both G. metallireducensand G. sulfurreducens produced MeHg above abiotic control levels,while S. oneidensis and S. putrefaciens did not. The small percentagesof methylation observed in abiotic controls are attributed toabiotic formation of MeHg in the experiment or during analysis(12).

View larger version (18K):
[in this window]
[in a new window]

FIG. 1. Observed MeHg production by pure cultures of DIRB, expressed as the percentage of added inorganic Hg methylated. "Other electron acceptors" refers to cultures that were grown with either nitrate or fumarate as an electron acceptor; "iron medium" refers to cultures grown with Fe(III) citrate as an electron acceptor (see Table 1). Significant differences (P < 0.05) in MeHg production in cultures relative to matched abiotic controls are indicated by stars. Error bars represent the standard deviations for three separately prepared tubes for each sample. Culture and growth conditions that were not assayed for MeHg production are labeled "NT" (not tested).

These results, in combination with the observation by Fleminget al. (10) of methylation by a Geobacter isolate, suggest thatthe ability to methylate Hg may be common among the Geobacteraceae.However, the observed lack of methylating capability among theShewanella strains tested (all Gammaproteobacteria) shows thatthe ability to methylate Hg is not ubiquitous among Fe(III)-reducingbacteria. To date, essentially all strains for which Hg methylationhas been demonstrated fall in the Deltaproteobacteria (2, 9,16, 24). These include DSRB from the orders Desulfovibrionalesand Desulfobacterales. However, it is important to note thatthe ability to produce MeHg is not ubiquitous among DSRB inthese families. Further studies are needed to ascertain whetherthe Hg-methylating capability is randomly distributed amongProteobacteria or related to phylogeny. Improved understandingof the phylogenetic distribution of Hg methylation capabilitymay provide insight into the biochemical process of MeHg productionwithin cells.

It is important to note that the Geobacter strains tested producedMeHg during growth with Fe(III) and with other electron acceptors(nitrate and fumarate). This indicates that active Fe(III)-reducingelectron-transport chains are not necessary for Hg methylationin these strains. However, this experiment was not designedto quantify the effect of electron acceptors and donors on methylationrates. Further studies would be needed to quantify these effects.

Environmental significance of MeHg production by DIRB.

Top

Environmental significance of...

The observation of Hg methylation by DIRB has implications forthe prediction of in situ MeHg production. Due to the importanceof DSRB as methylators, current models for methylation are basedon relationships between methylation and sulfate reduction (1).However, the finding that DIRB can produce MeHg suggests thatHg methylation may be important in sediments and soils wherethese organisms are dominant, e.g., iron-rich sediments withlow concentrations of sulfate (25). Iron can affect methylationby altering the chemistry of Hg (and hence its bioavailability)or by changing the activity of DIRB versus other groups of organisms,particularly DSRB (10, 23, 25, 27). The influence of iron onboth Hg complexation and microbial activity will need to beconsidered in order to resolve how Hg methylation by DIRB willchange the paradigm for in situ MeHg production.

ACKNOWLEDGMENTS

This work was supported through National Science FoundationDivision of Environmental Biology grant 0451345, Division ofOcean Sciences grants 0351050 and 0550547, and the SmithsonianInstitution. E. J. Kerin was supported by a Chesapeake BiologicalLaboratory Graduate Student Fellowship. J. D. Coates was supportedthrough grant DE-FG02-98ER63592 from the Department of Energy,Natural and Accelerated Bioremediation Program.

We thank Tyler Bell, Georgia Riedel, and Sarah Werner for labassistance at SERC, Christopher Conaway for editorial assistance,and Mandy Ward and Chad Saltikov for providing Shewanella cultures.

FOOTNOTES

* Corresponding author. Mailing address: Smithsonian Environmental Research Center, P.O. Box 28, 647 Contees Wharf Road, Edgewater, MD 21037. Phone: (443) 482-2498. Fax: (443) 482-2380. E-mail: gilmourc{at}si.edu.

Published ahead of print on 20 October 2006.

REFERENCES

Top