FEMS Microbiology Letters, 367, 2020, fnaa196

doi: 10.1093/femsle/fnaa196
Advance Access Publication Date: 26 November 2020
Research Letter

R E S E A R C H L E T T E R – Environmental Microbiology

Production of methylmercury by methanogens in

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mercury contaminated estuarine sediments
Yuwei Wang1, *,† , Spencer Roth1,2,† , Jeffra K. Schaefer1 , John R. Reinfelder1
and Nathan Yee1
1
Department of Environmental Sciences, Rutgers University, New Brunswick, 14 College Farm Road, NJ 08901,
USA and 2 Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, 76 Lipman
Drive, NJ 08901, USA
∗
Corresponding author: Department of Environmental Sciences, Rutgers University, New Brunswick, 14 College Farm Road, NJ 08901, USA. Tel:
+7327894208; E-mail: [email protected]
One sentence summary: Methanogenic archaea play an important role in the production of MeHg in estuarine sediments contaminated with Hg(0).
†
These authors contributed equally to this work.
Editor: Robert Gunsalus

ABSTRACT
Anaerobic bacteria are known to produce neurotoxic methylmercury [MeHg] when elemental mercury [Hg(0)] is provided as
the sole mercury source. In this study, we examined the formation of MeHg in anaerobic incubations of sediment collected
from the San Jacinto River estuary (Texas, USA) amended with aqueous Hg(0) to investigate the microbial communities
involved in the conversion of Hg(0) to MeHg. The results show that the addition of the methanogen inhibitor
2-bromoethanesulfonate (BES) significantly decreased MeHg production. The mercury methylation gene, hgcA, was detected
in these sediments using archaeal specific primers, and 16S rRNA sequencing showed that a member of the
Methanosarcinaceae family of methanogens was active. These results suggest that methanogenic archaea play an
underappreciated role in the production of MeHg in estuarine sediments contaminated with Hg(0).

Keywords: sediment; anaerobic microorganisms; mercury transformation

INTRODUCTION Desulfovibrio desulfuricans ND132 (Wang et al. 2016), and the iron-
reducing bacterium Geobacter sulfurreducens PCA (Hu et al. 2013),
Liquid elemental mercury [Hg(0)] is a major environmental con-
the uptake of Hg(0) and its oxidation to Hg(II) precedes methy-
taminant (Stoffers et al. 1999) and its discharge from industrial
lation. Although pure cultures of bacteria have been shown to
plants (Biester and Scholz 1997; Lechler et al. 1997; Pestana et al.
mediate anaerobic Hg(0) oxidation and methylation, the impor-
2000; Boszke et al. 2008; Brooks and Southworth 2011; Miller
tance of these processes in the environment and the diversity of
et al. 2013) and gold mining areas (Pestana and Formoso 2003;
microorganisms that are able to carry them out are poorly under-
Alpers et al. 2005) can be an important source of mercury to
stood.
aquatic ecosystems. When released into the environment, Hg(0)
The unique physical and chemical properties of Hg(0) affects
may undergo biological transformations that lead to the forma-
its environmental interactions with sediments and microorgan-
tion of neurotoxic methylmercury (MeHg), and laboratory exper-
isms. While Hg(II) adsorbs and strongly partitions onto sedi-
iments have demonstrated that anaerobic bacteria can mediate
ment surfaces (Turner, Millward and Le Roux 2001; Hintelmann
the conversion of Hg(0) to MeHg (Colombo et al. 2013; Hu et al.
and Harris 2004), dissolved gaseous elemental mercury can
2013). In both the sulfate-reducing, Hg methylating bacterium
be transported in groundwater and migrate away from buried

Received: 24 August 2020; Accepted: 24 November 2020


C The Author(s) 2020. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail:

[email protected].

1
 2 FEMS Microbiology Letters, 2020, Vol. 367, No. 23

wastes (Walvoord et al. 2008). Hg(II) also strongly binds to organic in pure culture (Ungerfeld et al. 2004) as well as environmen-
matter (Ravichandran 2004; Skyllberg 2012) and sulfides (Skyll- tal samples (Oremland and Polcin 1982; Compeau and Bartha
berg 2012), which can hinder the cellular uptake of mercury 1985). Sterile controls were prepared by autoclaving (121◦ C for
for methylation (Jonsson et al. 2012; Chiasson-Gould, Blais and 45 min), sitting at room temperature for 48 h, then autoclav-
Poulain 2014). In contrast, dissolved Hg(0) is highly mobile and ing (121◦ C for 45 min) a second time to sterilize any spores that
can diffuse into pore spaces and microenvironments that are may have grown. Additional control incubations were conducted
not accessible to Hg(II). Furthermore, because of its neutral with no addition of Hg(0) to determine the background levels
charge, Hg(0) readily crosses cell membranes, potentially ren- of MeHg production. Each treatment was performed in tripli-
dering it more bioavailable to mercury methylating organisms cate. The sediment slurries were purged with ultrapure nitro-
in the environment (Colombo et al. 2013; Wang et al. 2016). gen gas (N2 ) to remove oxygen. An acid-cleaned silicone per-
In this study, we investigated the production of MeHg in meation tube containing a small (∼30 mg) droplet of Hg(0) with
estuarine sediments when Hg(0) was provided as the sole mer- knots on both ends was added to each slurry. Incubations were

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cury source. The objective was to determine which microor- sealed with a Teflon stopper, crimp-sealed and the headspace of
ganisms in the sediments were involved in mercury trans- each incubation was purged again with ultrapure N2 for 30 min
formation when exposed to Hg(0). Sediment samples from before initiating the experiment. A negligible amount of Hg(0)
the San Jacinto River estuary were collected and incubated was removed in the N2 purging step. Slurries were incubated
with an elemental mercury source. Experiments with metabolic in the dark for three to seven days at room temperature. The
inhibitors and molecular analyses were performed to character- initial rate of Hg(0) release was 1.2 ppb/h and dissolved Hg(0)
ize the microbial communities involved in methylmercury for- saturation was achieved within 48 h at approximately 40 ppb.
mation. The results indicate that in these estuarine sediments, After a 3 or 7-day incubation period, each bottle was sacrificed,
methanogenic archaea play an important role in the production opened and purged with ultrapure N2 to remove Hg(0) gas. One
of MeHg. gram of slurry from two randomly selected bottles of the three
replicates for each treatment was immediately sampled for DNA
and RNA extraction. The remaining sediment slurries were pre-
METHODS served by freeze-drying for non-purgeable mercury (NP-Hg) and
MeHg analyses.
Field sampling
Sediment samples were collected on November 10, 2017 Mercury analyses
from the San Jacinto River at a site (location: 29◦ 46’53.5‘N,
95◦ 06’04.8’W) in Channelview, Texas (USA), approximately 3 km Slurry remained in each reactor was transferred to falcon tubes,
east of the City of Houston. They were therefore collected a lit- freeze dried and 1 g of dried sediment from each reactor was
tle over 10 weeks after the record rainfall and major flooding digested in 10 mL mixed HCl and HNO3 (4:1) for 24 h and
in the Houston metro area associated with Hurricane Harvey then diluted with 30 mL 18 megaohm of water prior to anal-
in August, 2017. The salinity and pH of water at the sampling ysis. Total Hg (THg) in field sediment and non-purgeable Hg
site were measured 8 and 7.04, respectively. Triplicate sediment (NP-Hg) in sediment slurry samples were measured by MERX-
samples spaced 50 to 100 cm apart were collected using an acid- T (Brooks Rand) automated total Hg analysis system with cold
cleaned plastic scoop and transferred to 500 mL acid-cleaned vapor atomic fluorescence spectroscopy (CVAFS) detector. Using
glass jars. Sediment-filled jars were placed in plastic bags and the NIST Standard Reference Material 1944 (New York/New Jer-
immediately stored on ice. Subsamples of sediment were freeze sey Waterway Sediment), a recovery of 104% of the certified
dried and analyzed for total Hg (53.3 ± 10.1 ng/g) and MeHg (0.2 ± value (3.4 ± 0.5 μg/g) was obtained. To analyze MeHg, 1 g of
0.1 ng/g) prior to the Hg(0) transformation experiments. dried sediment was distilled using a Tekran 2750 Methylmer-
cury Distillation System and measured using a Tekran 2700
Automated Methylmercury Analysis System (EPA method 1630).
MeHg recovery was tested by spiking 2 ng MeHgCl (Brooks Rand)
Hg(0) transformation experiments
into the sediments, and the recovery was 109% ± 10%. ANOVA
Laboratory experiments were conducted using sediment slurries was applied to examine the significance of differences among
to examine the production of MeHg when Hg(0) was provided as treatments and student’s t-test was used to determine whether
a mercury source. Sediment slurries were prepared in aluminum two treatments were statistically different.
foil-wrapped serum vials (25 ml) by combining 10 g sediment
and 5 mL artificial brackish water (7.5 g/L NaCl, 2.75 g/L MgCl2 –
Microbial analyses
6H2 O, 0.375 g/L CaCl2 –2H2 O, 0.38 g/L KCl, 0.14 g/L Na2 SO4 and
2.1g/L MOPS with pH adjusted to 7). Slurries were untreated (con- Two samples from each treatment was selected using excel
trol) or treated with either sodium molybdate (2 mM) [molyb- random selection function for molecular analyses. Both RNA
date:sulfate = 2:1] or 2-bromoethanesulfonate (BES) (15 mM) and DNA were extracted from slurry incubations using Qia-
(Oremland and Polcin 1982) to inhibit the activity of sulfate- gen RNeasy Powersoil Total RNA kit and RNeasy Powersoil
reducing bacteria or methanogens, respectively. Molybdate is a DNA Elution kit, following manufacturer’s protocols. Total RNA
structural analog to sulfate and has been shown to inhibit sul- was DNase-treated using Turbo DNase Kit (Thermo Fisher) and
fate reduction in pure culture (Biswas et al. 2009) and in environ- converted to cDNA using Superscript III First-strand cDNA Kit
mental samples for greater than 50 days (Sela-Adler et al. 2017) (Thermo Fisher), according to manufacturer’s protocols. The V4
by inhibiting ATP sulfuylase (Peck 1959, 1962) and sulfate uptake region of the 16S rDNA and rRNA was sequenced using Illumina
(Newport and Nedwell 1988), and increasing concentrations of MiSeq (Mr. DNA Lab, Shallowater, TX). Sequences were imported
molybdate have abolished sulfate reduction (Fleming et al. 2006). and demultiplexed in QIIME2. Primer adapter sequences were
BES inhibits methanogenesis as an analog of coenzyme M (Gun- removed using the cutadapt plugin, and paired-end reads were
salus, Romesser and Wolfe 1978) and inhibits methanogenesis joined using the VSEARCH plugin (Rognes et al. 2016). Quality
 Wang et al. 3

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Figure 1. Formation of MeHg in control, inhibitor treated (MoO4 , BES) and autoclaved incubations of estuarine sediments after (A) 3 days and (B) 7 days in which
Hg(0) was provided as a Hg source. Results for incubations without added Hg(0) are also shown. Concentrations of MeHg were normalized to the mass of freeze-dried
sediment. The three bars represent triplicate bottles in the order of individual replicates A, B, C for each incubation condition, thus each bar represents an independent
incubation.

control was performed using the Deblur plugin (Amir et al. 2017) Hg methylation, we treated slurries with sodium molybdate
and taxonomy was assigned using the feature-classifier plugin or BES to inhibit sulfate reduction or methanogenesis, respec-
trained on the V4 region of Silva database version 132 (Bokulich tively, and characterized the microbial communities. In BES-
et al. 2018). Taxonomic families that have member strains pre- treated slurries, MeHg formation was inhibited in all three repli-
viously shown to carry hgcAB were selected from the classi- cates after three days and continued to be significantly inhib-
fied data for Fig. 3. Sequences classified to methanogenic fami- ited after seven days when compared to the control slurries
lies were extracted from the dataset and a maximum likelihood (student’s t-test, P < 0.05). This suggests that methanogens
phylogenetic tree was constructed with reference sequences in were involved in Hg methylation in the estuarine slurries. The
MEGA v 7.0.26. Detection of the archaeal hgcA gene was con- involvement of methanogenic communities in MeHg produc-
ducted by PCR amplification as described by Christensen et al tion is consistent with previous studies that have identified
(Christensen et al. 2016) for 35 cycles. To validate the presence numerous archaeal species as carriers of the hgcAB genes for
of methanogenic archaea in the microcosms, mcrA, the gene Hg methylation (Gilmour et al. 2013; Yu et al. 2013; Podar et al.
encoding for methyl coenzyme M reductase, was detected by 2015; Gilmour et al. 2018). Notably, some methanogen species
PCR, using a previously described protocol for 30 cycles (Stein- are capable of producing MeHg at rates and yields that are
berg and Regan 2008). Genomic DNA from Methanospirillum hun- comparable to the well-known Hg-methylating sulfate-reducing
gatei was used as a positive control for the presence of mcrA. and iron-reducing bacteria (Yu et al. 2013; Gilmour et al. 2018).
Methanogenic communities have also been identified as impor-
RESULTS AND DISCUSSION tant Hg methylators in diverse habitats including freshwa-
ter environments (Avramescu et al. 2011; Gilmour et al. 2013;
The addition of Hg(0) to sediment slurries resulted in the produc- Bravo et al. 2018; Christensen et al. 2018), thawing Arctic soils
tion of non-purgeable mercury (NP-Hg) and MeHg after reacting (Podar et al. 2015), wetland soils (Schaefer et al. 2014), lake peri-
for three and seven days (Fig. 1, Fig. S1). Hg(0) was rapidly con- phyton (Hamelin et al. 2011) and rice paddies (Gilmour et al.
verted to 3074 ± 1520 ng/g and 7575 ± 1404 ng/g NP-Hg in Day 2013). Three sequence variants were identified as methanogenic
3 and Day 7, respectively (Fig. S1, Supporting Information). The archaea by 16S rRNA sequencing (RNA fraction) (Fig. S2, Sup-
concentration of NP-Hg in autoclaved slurries was not statisti- porting Information). One belonging to the order Methanosarci-
cally distinguishable from all other treatments based on ANOVA nales (Fig. 3; Fig. S2-‘Houston-2’, Supporting Information) was
analysis (P > 0.05). This indicates abiotic oxidation of Hg(0), and found in low relative abundance in ‘Control B’ and both BES
the rate is approximately 980 ng/g/day. However, biotic NP-Hg treated sediments (average relative abundance = 0.013% of total
formation may have also occurred in un-autoclaved slurries. 16S rRNA reads). Currently, 36 genomes have been sequenced
Both abiotic (Zheng, Liang and Gu 2012; Zheng et al. 2013) and in the Methanosarcinaceae family, 7 of which carry the hgcAB
biotic (Colombo et al. 2013; Hu et al. 2013) processes are known to gene. In addition, pure cultures of the strains Methanomethylovo-
oxidize Hg(0) to Hg(II) which may then be bioavailable for methy- rans hollandica and Methanolobus tindarius, which belong to the
lation. More than 12 ng/g methylmercury was formed in two Methanosarcinaceae family, have been demonstrated experimen-
of three control replicates after three days of incubation when tally to produce MeHg (Gilmour et al. 2013). The occurrence of
compared to the autoclaved and no Hg(0) slurries (Fig. 1A). After Methanosarcinaceae in the PCB-contaminated sediments of the
seven days of incubation, approximately 49 ± 17 ng/g of MeHg San Jacinto River (EPA Record of Decision) we sampled in estu-
was formed in the sediments when Hg(0) was provided as a Hg arine waters with a relatively high (for methanogens) salinity
source (Fig. 1B), suggesting biological Hg methylation. The toxic (8) is consistent with the metabolic diversity of these microor-
concentration of Hg for the microbes responsible for mercury ganisms. Methanosarcinaceae are known to occur in a variety
methylation is unknown. Nonetheless, the production of MeHg of anaerobic environments, such as sediments from freshwa-
indicates that the microorganisms were still active after 7 days. ter, high salinity wetlands, and organic waste treatment sys-
In order to determine the microorganisms responsible for tems (Kendall and Boone 2006). Their ability to obtain energy
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Figure 2. Microbial genes and transcripts in estuarine sediments incubated for 7 days. (A), Archaeal hgcA genes in DNA extracts of the same treatments and replicate
bottles. Archaeal hgcA was detected in the control and both inhibitor-treated incubations, but not in the autoclaved treatment. (B), mcrA in DNA and RNA extracts
from control, inhibitor treated (MoO4 , BES), and autoclaved (‘Autoclave’) incubations. mcrA genes and transcripts were present in control (bottles A and B) and sodium
molybdate (bottles A and B) incubations. However, mcrA genes, but not transcripts were detected in BES treated sediments (bottles A and C). mcrA genes and transcripts
were not detected in autoclaved incubations (bottles A and B). Positive and negative PCR controls for mcrA are displayed.

from diverse substrates including methylated amines, acetate, In contrast to the untreated control, treatment with sodium
dimethyl sulfide, methanol, H2 /CO2 , methanethiol and carbon molybdate blocked MeHg formation after three days (Fig. 1A),
monoxide (Balch et al. 1979; Rosenberg et al. 2014), tolerate high implicating the activity of sulfate reducing bacteria in MeHg pro-
salinity and dehalogenate certain halogenated organic com- duction. Despite initial inhibition after three days, MeHg produc-
pounds (Rosenberg et al. 2014) illustrates how members of this tion in two of three molybdate-treated slurries fully recovered to
family have adapted to diverse anaerobic environments. concentrations seen in control slurries after seven days of incu-
The most abundant methanogenic family detected in the bation (Fig. 1B). At this time point, active sulfate-reducing bac-
slurries belonged to the order Methanobacterales at a highest teria were detected in the slurries through 16S rRNA sequencing
relative abundance of approximately 0.15% (Fig. S2-‘Houston- (RNA fraction). The 16S rRNA gene analysis showed that the fam-
1’ and ‘Houston-3’, Supporting Information). No representatives ilies Syntrophobacteraceae, Syntrophaceae, Desulfobulbaceae and
in the Methanobacterales have been shown to carry hgcAB genes, Desulfobacteraceae were the dominant hgcAB-containing microor-
however, an indirect role through syntrophy with Hg methylat- ganisms found in the slurries (Fig. 3). When the sediments were
ing secondary fermenters or more complex community dynam- treated with molybdate, the abundance of Desulfobacteraceae and
ics could occur (Schink 1997; Correia, Miranda and Guimarães Desulfobulbaceae decreased from 8.1% to 0.8% (P = 0.003) and 7.6%
2012; Yu et al. 2018). Syntrophy is normally observed in sul- to 2.6% (P = 0.0697), respectively, suggesting that MoO4 had an
fate limiting conditions, therefore it is not likely to have been inhibitory effect on the sulfate-reducing bacterial community
a major contributor to MeHg production, but its potential can- even after seven days (Fig. 3). While the decrease in SRB abun-
not be excluded. Low relative abundances of methanogen and dance was reflected by inhibition of MeHg formation after three
archaeal sequences from the slurries could have been the result days, it did not extend to seven days, when SRB abundance was
of sequencing primer bias or RNA and DNA recovery bias (Eloe- low but Hg methylation was observed in molybdate treated slur-
Fadrosh et al. 2016). The role of methanogens in MeHg forma- ries. This suggests that the SRB community was not the primary
tion observed in the incubations was therefore validated by methylating group in the sediments.
detection of archaeal hgcA genes and mcrA genes and tran- Several other metabolic guilds have been shown to be directly
scripts. Archaeal hgcA genes were detected in the control, BES- involved in Hg methylation including fermenters (Gilmour et al.
treated, and molybdate-treated samples (Fig. 2A), showing the 2013) and iron-reducing bacteria (Fleming et al. 2006). A small
genetic potential for Hg methylation by methanogens in the amount of MeHg was formed in BES treated sediments after
sediments. In the control and sodium molybdate treated sedi- three days and seven days when compared to the no-Hg(0)
ments, mcrA genes and transcripts were detected (Fig. 2B). The treatment (Fig. 1). This MeHg formation could be the result of
expression of mcrA transcripts after 7 days indicates that toxic metabolic guilds that we were unable to account for due to lack
levels of Hg were not reached in the experiments. Incubations of specific inhibitors. 16S rRNA sequencing shows an abundance
with BES contained detectable mcrA genes, but not transcripts, of Geobacteraceae in the incubations, as well as several fermenta-
suggesting that BES inhibited methanogenic communities tive guilds in lower abundances (Fig. 3). While the contribution
(Fig. 2B). to MeHg from these metabolic groups cannot be excluded, it was
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Figure 3. Relative abundances of families of microorganisms (based on the 16S rRNA gene) known to include potential Hg methylators (hgcAB-containing microorgan-
isms) in the active communities of control and inhibitor treated (MoO4 , BES) incubations of estuarine sediments after 7 days. A, B and C represent individual bottles
for each treatment.

minor in comparison to the contribution by methanogens, as (Smith et al. 1998; Colombo et al. 2013; Wang et al. 2016). An
indicated by a 12-fold reduction in MeHg concentrations when important question for future investigation is the bioavailability
sediments were treated with BES (Fig. 1B). of Hg(0) to different microbial groups in anaerobic sediments.
The results of this study highlight the importance of It is currently unknown if Hg(0) is more readily accumulated
methanogenic archaea in the production of MeHg when high by methanogens than sulfate-reducing bacteria. For example,
sulfate estuarine sediments are contaminated with Hg(0). sulfidization of proximal organic matter and the cell surface
Although high sulfate environments favor the activity of SRB, sulfhydryl groups of sulfate reducing bacteria might oxidize and
our study shows the dominance of methanogens over SRB in the bind Hg(0), thereby limiting its entry into the cell. Detailed com-
production of MeHg when elemental Hg was provided as the sole parisons between archaeal methanogens and sulfate-reducing
mercury source. Sulfate-reducing bacteria have been shown to bacteria, as well as the associated effects on Hg(0) bioavailabil-
be major contributors to MeHg production in previous research ity, merit further study.
(Compeau and Bartha 1985; Compeau and Bartha 1987; King et
al. 2000) and our results do not exclude the role of SRBs in MeHg
formation. Rather, they highlight the potential importance of SUPPLEMENTARY DATA
methanogens, which have been largely excluded when consider-
Supplementary data are available at FEMSLE online.
ing Hg methylation in estuarine environments. While our results
in microcosms clearly show the involvement of methanogens in
Hg methylation, in situ MeHg formation may be influenced by
other microbial guilds and should not be overlooked. Previous FUNDING
research has shown that methanogens are able to grow in high
This work was supported by funds from the United States
sulfate sediments under sulfate reducing conditions (Oremland
National Science Foundation award number 1760534 and by
and Taylor 1978; Ferry and Lessner 2008; Sela-Adler et al. 2017).
the National Science Foundation Graduate Research Fellowship
Despite their relatively low abundance in the sediments exam-
under grant number DGE 1842213 (Author SR).
ined in this study, our results indicate that methanogens may
dominate an important biogeochemical transformation. Mer- Conflicts of interest. None declared.
cury transformation by methanogens when exposed to elemen-
tal mercury is not well understood. The conversion of Hg to
MeHg by methanogens may be catalyzed by direct uptake of dis-
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