19

DNA-based Evaluation of Biological Mercury Methylation
Potential in Waste Landfill
Daisuke INOUE1*, Jing YANG2, Masaki TAKAOKA2, Kazunari SEI3 and Tomonori ISHIGAKI4
Division of Sustainable Energy and Environmental Engineering, Graduate School of Engineering, Osaka University
2-1, Yamadaoka, Suita-shi, Osaka 565-0871, Japan2
Department of Environmental Engineering, Graduate School of Engineering, Kyoto University
C-cluster, Katsura, Nishikyo-ku, Kyoto 615-8540, Japan3
Department of Health Science, School of Allied Health Sciences, Kitasato University
1-15-1, Kitazato, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan
4
Center for Material Cycles and Waste Management Research, National Institute for Environmental Studies (NIES)
16-2, Onogawa, Tsukuba, Ibaraki 305-8506, Japan*E-mail: d.inoue@see.eng.osaka-u.ac.jp*1

Abstract
Waste landfills are a potential mercury (Hg) sink due to disposal of wastes containing mercury. In
addition, anaerobic environments created under the surface in landfills facilitate Hg methylation, which
produces methyl Hg (MeHg), the most potent mercury neurotoxin. Although Hg methylation in theenvironment is primarily mediated by microorganisms, little is known about the occurrence of Hg-methylating
microorganisms in landfills. Therefore, this study aimed to assess the potential of biological Hg methylationin waste landfills by applying a DNA-based monitoring tool. First, useful polymerase chain reaction (PCR)
primers targeting hgcAB genes that are involved in Hg methylation were selected from previously-developed
ones, and PCR detection conditions were optimized. Then, the occurrence of hgcAB genes was investigated incore samples collected from landfills in Japan. Among a total of 20 samples from five landfill sites, hgcAB
genes were detected in five samples. In addition, the abundance of hgcAB genes was estimated in three of thefive positive samples by most probable number-PCR. The results indicated not ubiquitous but only sporadic
occurrence of biological Hg methylation potential in landfill sites even under anaerobic conditions favorable
for Hg-methylating microorganisms.
Key words :	biological mercury methylation potential, hgcAB gene, polymerase chain reaction, waste landfill

1. Introduction
Mercury (Hg) is a ubiquitous, naturally occurring
element in the environment. Despite being an industriallyvaluable resource, Hg is a global pollutant that seriously
affects human and ecosystem health (Driscoll et al.,2013). Among Hg compounds, methyl Hg (MeHg) is themost toxic form, and can affect the central nervous
system by crossing the blood-brain barrier. MeHg canalso bioaccumulate and biomagnify in the food chain. To
reduce the risks of Hg to humans and the environment,
the Minamata Convention on Hg was adopted in 2013,
entering into force in August 2017. Adequate
management of Hg (especially MeHg) emissions into thenatural environment is an urgent issue.
MeHg, the most potent mercury neurotoxin, is
generated in the environment predominantly by anaerobic
microorganisms (Parks et al., 2013; Poulain & Barkay,
Global Environmental Research
24/2020: 019-025
printed in Japan井上.indd

192013), although abiotic (chemical) Hg methylation is also
possible if suitable methyl donors are present (Celo et al.,2006). Microorganisms capable of Hg methylation
include sulfate-reducing bacteria, iron-reducing bacteria,
syntrophic bacteria and methanogenic archaea, which are
distributed in diverse phylogenetic groups, such as
δ-Proteobacteria, Firmicutes, Chloroflexi andMethanomicrobia (Compeau & Bartha, 1985; Kerin etal., 2006; Hamelin et al., 2011; Gilmour et al., 2013; Baeet al., 2014).
Mercury has been used in a wide range of industrial
products such as batteries, paints, electrical and electronic
devices, thermometers, fluorescent and energy-savinglamps. Consequently, wastes containing Hg used forindustrial and domestic purposes are disposed in waste
landfills. A previous study estimated that 11–24 tons of
Hg are annually disposed of in landfill sites in Japan(Takaoka, 2015). Indeed, the occurrence of Hg in landfills©2020 AIRES

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D. INOUE et al.

has been reported in different countries (Ilgen et al.,2008; Lee et al., 2016; Tao et al., 2017; He et al., 2018;
Yang et al., 2018). Hg can be discharged via landfill gas
and leachate, which may pose potential risks to humans
and wildlife. In addition, methylation of Hg can occur inlandfills owing to the anaerobic environment beneath thesurface, which is favorable for Hg-methylating
microorganisms. Indeed, the occurrence of MeHg in
landfills has been observed in previous studies (Lee et al.,2016; Tao et al., 2017; Yang et al., 2018). Nevertheless,there is still limited knowledge on the occurrence of Hg
methylation in landfills. In particular, the distribution,
diversity and dynamics of Hg-methylating
microorganisms in landfills have hardly been investigatedat all, despite such information being required not only toevaluate the potential of Hg methylation in landfills but
also to control the potential risks associated with Hg.
Therefore, this study was conducted to assess thepotential for biological Hg methylation in waste landfills
in Japan by applying a DNA-based monitoring tool. The
hgcAB genes were applied as the target genes formonitoring Hg-methylating microorganisms. These genes
have recently been identified as involved in Hg
methylation (Parks et al., 2013). The hgcA gene encodes
the corrinoid protein that works as methyltransferase,while the hgcB gene encodes a 2[4Fe-4S] ferredoxin
which plays as an electron donor required for corrinoid
cofactor reduction. In this study, polymerase chain
reaction (PCR) primers to detect hgcAB genes inphylogenetically-diverse Hg-methylating microorganisms
were selected from previously-developed ones, andconditions for PCR amplification were optimized. Then,the occurrence of Hg-methylating microorganisms in coresamples collected from several landfills in Japan wasinvestigated by applying the optimized method.

2. Materials and Methods
2.1	Selection of PCR Primers and Optimization of
PCR Conditions for hgcAB Gene Detection
To select PCR primers for selective detection of
hgcAB genes, the effectiveness of two universal PCR
primers that had been previously designed were compared(Table 1). After selecting effective PCR primers, the PCR
thermal profile, which consists of denaturation, annealing
and extension steps, was optimized. The following four
strains were obtained from culture collection in Japan,
and applied as positive strains for PCR detection of
hgcAB genes: Methanocella palundicola NBRC 101707T,
Methanomethylovorans hollandica NBRC 107637T, andMethanospirillum hungatei NBRC 100397T, which aremethanogenic archaea and were provided by NITE
Biological Resource Center, Japan (Chiba, Japan); andDesulfosporosinus acidiphilus JCM 16185T, which is asulfate-reducing bacterium and was provided by RIKEN
BRC through the National Bio-Resource Project of the
Ministry of Education, Culture, Sports, Science andTechnology (MEXT), Japan (Ibaraki, Japan). DNA wasextracted from each strain using Cica Geneus DNA
extraction solution (Kanto Chemical, Tokyo, Japan). PCR
amplification was performed using the T100 thermal
cycler (Bio-Rad Laboratories, Hercules, CA, USA). The
PCR products were analyzed by electrophoresis on a
1.5% (w/v) agarose gel stained with SYBR Green I(Takara Bio, Shiga, Japan).
2.2	Detection and Quantification of hgcAB Genes in
Landfill Samples
The following two investigations were conducted toanalyze the occurrence of hgcAB genes at waste landfill
sites. In the first investigation, 14 core samples collectedfrom three landfill sites, H3S-2, Y2 and Y4, were used
(Table 2), while six core samples collected from two
landfill sites, A and B, were used in the second
investigation (Table 3). Among them, landfill sites H3S2, A and B were municipal landfills, while landfill sites
Y2 and Y4 were industrial landfills receiving incineration
residues from municipal and industrial wastes. In
addition, landfill sites Y2, Y4 and A had been closed
before our investigations, while landfill sites H3S-2 andB had been in active service.
Total microbial DNA was extracted from landfill
core samples of the first and second groups using ISOIL
Large for Beads kit ver. 2 (Nippon Gene, Tokyo, Japan)
and ISOIL for Beads Beating kit (Nippon Gene),
respectively. All the extracted DNA was purified using

Table 1 Comparison of previously-designed PCR primers targeting hgcAB genes.
ReferencePrimer sequence (5'-3')*

Amplicon size (bp) Sequences used to design primers

hgcA_261F: CGGCATCAA YGTCTGGTGYGC~650
Schaefer et al., 2014
hgcA_912R: GGTGTAGGGGGTGCAGCCSGTRWARKTδ-Proteobacteria: 12
Firmicutes: 6
Methanomicrobia: 5

hgcA_F: GGNRTYAAYRTNTGGTGYGC
hgcB_R: CADGCNCCRCAYTCVATRCA

δ-Proteobacteria: 23
Chloroflexi: 1
Firmicutes: 6
Methanomicrobia: 10

Bae et al., 2014ca. 900*Degenerate nucleotide: D = A, G, T ; K = G, T ; N = A, C, G, T ; R = A, G ; S = C, G ; V = A, C, G ; W = A, T ; Y = C, T.

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Occurrence of Hg methylation genes in landfills

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Table 2 Characteristics of landfill core samples analyzed in the first investigation.
Landfill site

Sample No.

Depth
(m)

Hg concentration(mg/kg)Main components

H3S-2

Y2

Y4

hgcAB gene

71

‒4.5

51.16

Metals

‒

74

‒6.0

ND*

Ash

‒

81

‒6.7

7

Iron and steel slag

+

88

‒5.5

ND

Ash

‒

90

‒5.7

2

Ash

‒

91

‒5.2

1

Ash

‒

99

‒4.0

5

not determined

‒

104

‒4.1

1

Solidified ash

‒

107

‒0.8

4

Foundry sand

+

108

‒0.9

12

Foundry sand

‒

110

‒0.6

ND

not determined

‒

111

‒1.2

8

Slag

‒

112

‒1.4

12

Foundry sand

‒

113

‒1.6

2

Mixed wastes‒* ND: not detected.

Table 3 Characteristics of landfill core samples analyzed in the second investigation.
Landfill site

Sample No.

Depth (m)Main component

Hg concentration(mg/kg)B

10‒9 ~ ‒10

Incineration residue, fiber

0.102±0.020

11

0 ~ ‒1

Incineration residue, sand

4.45±0.799

22‒1 ~ ‒2

Incineration residue

0.503±0.130

43‒7 ~ ‒8

Iron wire with much rust

18.6±0.935

53‒7 ~ ‒8

Tiny stones

0.143±0.037

63‒7 ~ ‒8

Stone, less brick0.440±0.024

A

MagExtractor-PCR&Gel Clean-up kit (Toyobo, Tokyo,
Japan). The hgcAB genes in the extracted DNA weredetected by PCR amplification with the following
optimized thermal profile: initial denaturation at 94°C for
5 min; six cycles of denaturation at 94°C for 30 s,
annealing at 60°C (touching down to 55°C with ‒1°C/
cycle) for 30 s and extension at 72°C for 1 min; 30 cycles
of denaturation at 94°C for 30 s, annealing at 55°C for
30 s and extension at 72°C for 1 min; and a final
extension at 72°C for 5 min.
In addition to normal PCR detection, hgcAB geneabundance was determined by the most probable numberPCR (MPN-PCR; Picard et al., 1992). MPN-PCR has
been applied to quantify functional genes possessed bymultiple microbial groups, such as nitrifying anddenitrifying genes (Sang et al., 2008; Inoue et al., 2012),
the methyl coenzyme M reductase gene associated withmethanogenesis (Sang et al., 2009) and catabolic genes
associated with degradation of aliphatic alkanes (Sei etal., 2003) and aromatic compounds (Sei et al., 2004). For
quantification of hgcAB gene abundance by MPN-PCR,
the extracted DNA was serially diluted 10-fold, andtriplicate samples of each 10-fold dilution series were井上.indd

21

subjected to PCR amplification. The abundance of hgcAB
genes was determined with Cochran’s table (Cochran,1950) based on the combination of positive numbers
within triplicate samples of three consecutive dilution
series.

3. Results and Discussion
3.1	Optimization of PCR Conditions for hgcAB Gene
Detection
Among the two previously-designed PCR primers
for detection of hgc genes (Table 1), the primer set[hgcA_261F, hgcA_912R] (Schaefer et al., 2014) targeted
only the hgcA gene, and was designed based on thealignment of 23 hgcA gene nucleotide sequences
including 12 from δ-Proteobacteria, six from Firmicutesand five from Methanomicrobia. The forward and reverse
primers include low numbers (2 and 5, respectively) of
degenerate nucleotides. However, the sequence of reverse
primers has many mismatches to the aligned sequences,
especially those from Firmicutes and Methanomicrobia,
due to great diversity in the hgcA sequences. On the other
hand, to avoid the problem for the reverse primer, the2020/07/30

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D. INOUE et al.

forward and reverse primers were respectively designed
in hgcA and hgcB genes in the other primer set [hgcA_F,
hgcB_R] (Bae et al., 2014). This PCR primer set was
designed based on the alignment of 50 sequences
including 23 from δ-Proteobacteria, one from
Chloroflexi, six from Firmicutes and 10 from
Methanomicrobia. In addition, the forward and reverse
primers include seven and six degenerate nucleotides,
respectively, due to the sequence diversity andconsequently they have no or only one mismatch to thealigned sequences. Because little information wasavailable regarding microorganisms capable of Hg
methylation in the landfill environment, the PCR primer
set [hgcA_F, hgcB_R], which should detect more diverse
hgc genes, would be appropriate to the objective of thisstudy.
Therefore, PCR amplification of hgc genes with theselected primer set was performed using genomic DNA
from four known hgc gene-carrying strains, M.
Tpalundicola NBRC 101707 , M. hollandicaT
NBRC107637 , M. hungatei NBRC100397T and D.
acidiphilus JCM 16185T. However, the anticipatedamplicons of approximately 900 bp were not amplified
with the PCR thermal profile described in Bae et al.
(2014). Therefore, optimization of the PCR thermal
profile, especially the annealing step, was conducted.
Consequently, the anticipated amplicons ofapproximately 900 bp were detected from the four test
strains with the optimized PCR thermal profile, andunspecific PCR amplicons were also detected (Fig. 1).
Generation of unspecific PCR amplicons also indicated

M

N

A

B

C

D

Fig. 1 Detection of hgcAB genes from 4 hcgAB gene-possessing
strains by optimized PCR conditions. Lane M, 100 bp DNA
ladder; Lane N, negative control; Lane A, M. palundicola
NBRC 101707T; Lane B, M. hollandica NBRC 107637T;
Lane C, M. hungatei NBRC 100397T; Lane D, D.
acidiphilus JCM 16185T. Arrows in lanes A to D indicate
the PCR amplicons with the anticipated size (ca. 900 bp).

井上.indd

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that purification of PCR amplicons with anticipated size(ca. 900 bp) may be needed for further analysis ofsequence diversities. It was also indicated that the primer
set applied here would not be suitable for real-time PCR
quantification, and thus MPN-PCR, which can estimate
the abundance of target genes independently from thegeneration of unspecific PCR amplicons, is suitable forquantification of hgc genes with the applied primer set.
3.2	Occurrence of hgcAB Genes in Landfill Samples(First Investigation)
To clarify the occurrence of Hg-methylating
microorganisms in landfills and their relationship withthe presence and level of Hg, the first investigation was
conducted with 14 core samples collected from threelandfill sites, H3S-2, Y2 and Y4 (Table 2). The hgcAB
genes were detected in two samples, No. 81 from landfill
Y2 and No. 107 from landfill Y4. The target genes were
not found in the other 12 samples. It was quite surprisingthat hgcAB genes were not detected even in samples
Nos. 108, 111 and 112. These three samples werecollected from the same core sample as sample No. 107
at similar depth (10 – 60 cm distance), and had higher
concentrations of Hg than sample No. 107. In addition,hgcAB genes were not detectable even in sample No. 71
from landfill H3S-2 which had the highest Hg
concentration (51.16 mg/kg) among the 14 samples.
Based on these results, it is suggested that the Hg
methylation potential is inherent in the landfill
environment, and that the occurrence of Hg methylation
potential might not be necessarily correlated with the Hg
pollution level.
3.3	Occurrence of hgcAB Gene in Landfill Samples(Second Investigation)
The second investigation of the occurrence of hgcAB
genes in landfills was conducted with six core samples
collected from two landfill sites, A and B (Table 3). The
anticipated PCR amplicons of approximately 900 bp weredetected in three samples, Nos. 43, 53 and 63, collectedfrom landfill site A, but not in the other three samples
collected from landfill site B. Although many unspecific
PCR amplicons were also detected, they disappeared
when the DNA extracts were serially diluted withultrapure water prior to use in PCR amplification (Fig. 2).
This confirmed that the PCR amplicons of approximately
900 bp were derived from hgcAB genes, and consequently
the occurrence of hgcAB genes in the three samples(Nos. 43, 53 and 63) from landfill site A. By contrast, thelack of detection of hgcAB genes in samples from landfillsite B would be due to strongly basic conditions in thislandfill site that are not favorable for survival of
microorganisms. Based on the results of the first andsecond investigations, it is indicated that the hgcAB
genes, namely the biological Hg methylation potential,
occur not ubiquitously but sporadically in landfill sites.

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Occurrence of Hg methylation genes in landfills

23

Dilution ratio
M

N

P

10

102

103

104

Fig. 2 PCR detection of hgcAB genes from landfill sample No. 53. The extracted DNA from the sample was diluted 10-104-fold, andsubjected to PCR. Lane M, 100 bp DNA ladder; Lane N, negative control; Lane P, positive control.

To estimate the abundance of hgcAB genes in thethree positive samples, MPN-PCR was conducted. The
results showed the abundance of hgcAB genes to be
2.8×102 MPN-copies/g-wet in sample Nos. 43 and 63,
and 2.9×104 MPN-copies/g-wet in sample No. 53 (Fig. 3).
No clear correlation was found between hgcAB geneabundance and Hg concentration (Fig. 3, Table 3). Thus,
it was suggested that the occurrence of hgcAB genes (Hgmethylation potential) in landfills would not be affectedby Hg pollution levels. A previous study applying another
PCR primer set targeting hgcA genes also found nocorrelation between hgcA gene abundance and total Hg
and MeHg contents in landfill core samples (Yang et al.,2018). Similar results were obtained in an investigationof core samples collected from natural sediment and soil
(Christensen et al., 2019). These previous findings
corroborated the results of this study. Such lack ofcorrelation would be reasonable, because known Hgmethylating microorganisms are distributed among
sulfate- and iron-reducing bacteria and methanogens, andtheir existence is primarily governed not by Hg pollution
at low levels but by their fundamental growth parameters(e.g., carbon sources, nutrients, temperature, pH, salinity,etc.). By contrast, our results indicate that Hg methylation
might occur at sites where both Hg and Hg methylation
potential occur at certain high levels. To suppress
biological MeHg production in waste landfills, it is ofprimary importance to dispose of Hg-containing wastes
in a manner that avoids contact with Hg-methylating
microorganisms (e.g., concrete solidification and separate
landfilling). In addition, controlling environmental
conditions in waste landfills to inhibit the growth andactivity of major Hg-methylating microorganisms might
be another way to mitigate biological MeHg emissions

井上.indd23

hgcAB gene abundance（MPN-copies/g-wet）

1.0 ×105
1.0 ×104
1.0 ×103
1.0 ×102
1.0 ×101
1.0 ×100

ND

ND

ND

11

12

22

43

53

63

Sample No.
Fig. 3 Abundance of hgcAB genes in six core samples fromlandfills A and B, estimated by MPN-PCR. ND: notdetected.

from previously disposed Hg-containing wastes.

4. Conclusions
The results of this study revealed the occurrence of
hgcAB genes in some of the investigated landfill coresamples. It was thus indicated that the potential forbiological Hg methylation exists but is not ubiquitously
distributed in landfill sites. However, given theheterogeneity of disposed wastes in waste landfills, themicrobial community should differ not only vertically but
also horizontally. Thus, further investigations of verticaland horizontal distributions would be needed to clarify
the overall biological Hg methylation potential in a waste
landfill. Evaluation of biological methylation potential by
the DNA-based monitoring applied in this study, in
combination with chemical analysis of Hg, could be

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D. INOUE et al.

helpful in predicting the possibility of MeHg production
in waste landfills.
However, the hgcAB gene monitoring tool applied
here cannot provide information on the composition of
Hg-methylating microorganisms directly. Hg methylation
ability differs among Hg-methylating microorganisms(Gilmour et al., 2013). Thus, to evaluate the Hgmethylation potential, including its strength, theabundance of each group needs to be differentiallymonitored. Thus, analysis of the composition in the PCR
amplicon obtained by the method presented above or the
combined use of group-specific hgcAB gene monitoring
tools would be required in the future for more correct
estimation of biological Hg methylation potential.
Acknowledgements
This study was supported by the Environment
Research and Technology Development Fund (3K143002
and 3K153004) of the Ministry of the Environment,
Japan.
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Occurrence of Hg methylation genes in landfills

Daisuke INOUE
Daisuke Inoue is an associate professor in the
Division of Sustainable Energy andEnvironmental Engineering, Graduate School ofEngineering, Osaka University, Japan. He earned
his Ph.D. in environmental engineering from
Osaka University, in 2005. He majored in biologicalenvironmental engineering, and his research
fields are relevant to the treatment of and resource recovery from wastes
and wastewater; the remediation of polluted environments using biological
functions including those of plants, microorganisms and their associations;and the monitoring of microbial community dynamics in the environment
using culture-dependent and -independent molecular techniques.

Jing YANG

25

Tomonori ISHIGAKI
Tomonori Ishigaki is a senior researcher at the
National Institute for Environmental Studies. He
serves as an editorial board member for theemission factor database and lead author of the
Guidelines for National Greenhouse Gas
Inventories for the Intergovernmental Panel on
Climate Change. He is also serving as a memberof the Technical Committee (Solid Recovered Fuels) of International
Organization for Standardization. His published works cover subjects suchas the mechanism of waste conversion in landfills, technology development
for emission reduction from landfills, assessment of the fate of hazardous
substances in the environment, and appropriate management of solid waste
in urban areas of Asia to prevent urban floods.
(Received 10 December 2019, Accepted 16 May 2020)

Jing Yang received her Ph.D. in environmental
engineering at the Graduate School ofEngineering, Kyoto University, Japan in 2018.
She has worked at the School of Geographic
Science, Nantong University, China as an
associate professor since 2019. Her research field
is mercury behavior in municipal waste andincineration residuals. Her current research interests are the separation andrecovery of mercury from spent fluorescent lamps and the removal of
mercury from flue gas using activated coke.

Masaki TAKAOKA
Masaki Takaoka is a professor in the Departmentof Environmental Engineering, Graduate Schoolof Engineering, Kyoto University, Japan. He is amember of the Minamata Convention Group ofTechnical Experts on Mercury Waste Thresholds
and a resource person under the UNEP Global
Mercury Partnership ― Waste Management
Partnership Area. His main research subject is management and treatmentof solid waste. He is particularly interested in controlling toxic substancessuch as heavy metals including mercury, persistent organic pollutants andradioactive nuclides in waste treatment processes and waste-to-energy
projects.

Kazunari SEI
Kazunari Sei is a professor of environmental
hygiene at the School of Allied Health Scienceand the Graduate School of Medical Sciences,
Kitasato University, Japan. He received his Ph. D.
from Osaka University in 2001. His research field
is environmental engineering, and he has beenworking on monitoring specific bacteria in naturalenvironments, wastewater treatment plants, waste landfill sites and theaquatic plant rhizosphere. Currently he is working on monitoring
antimicrobial resistant bacteria and their genes, and vectors of infectious
diseases in aquatic environments. He is director of general affairs of theJapan Society on Water Environment.

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