New Biotechnology  Volume 00, Number 00  June 2015 RESEARCH PAPER

Mercury (II) removal by resistant

bacterial isolates and mercuric (II)

Research Paper
reductase activity in a new strain of
Pseudomonas sp. B50A
Q1
Patricia Giovanella1, Lucélia Cabral2, Fátima Bento1,
Clesio Gianello3 and Flávio Anastácio Oliveira Camargo3
1
Department of Microbiology – Institute of Health Science, Federal University of Rio Grande do Sul, 500 Sarmento Leite St, 90050-170 Porto Alegre – RS, Brazil
2
Centro Pluridisciplinar de Pesquisas Quı́micas, Biológicas e Agrı́colas (CPQBA/Unicamp), Rua Alexandre Cazelatto, 999, Caixa Postal: 6171, 13081-970 Vila Betel,
Paulı́nia – SP, Brazil
3
Department of Soil Science, Federal University of Rio Grande do Sul, 7712 Bento Gonçalves Ave, 91540-000 Porto Alegre – RS, Brazil

This study aimed to isolate mercury resistant bacteria, determine the minimum inhibitory
concentration for Hg, estimate mercury removal by selected isolates, explore the mer genes, and detect
and characterize the activity of the enzyme mercuric (II) reductase produced by a new strain of
Pseudomonas sp. B50A. The Hg removal capacity of the isolates was determined by incubating the
isolates in Luria Bertani broth and the remaining mercury quantified by atomic absorption
spectrophotometry. A PCR reaction was carried out to detect the merA gene and the mercury (II)
reductase activity was determined in a spectrophotometer at 340 nm. Eight Gram-negative bacterial
isolates were resistant to high mercury concentrations and capable of removing mercury, and of these,
five were positive for the gene merA. The isolate Pseudomonas sp. B50A removed 86% of the mercury
present in the culture medium and was chosen for further analysis of its enzyme activity. Mercuric (II)
reductase activity was detected in the crude extract of this strain. This enzyme showed optimal activity at
pH 8 and at temperatures between 378C and 458C. The ions NH4+, Ba2+, Sn2+, Ni2+ and Cd2+ neither
inhibited nor stimulated the enzyme activity but it decreased in the presence of the ions Ca2+, Cu+ and
K+. The isolate and the enzyme detected were effective in reducing Hg(II) to Hg(0), showing the potential
to develop bioremediation technologies and processes to clean-up the environment and waste
contaminated with mercury.

Introduction
Mercury (Hg) occurs naturally in the environment in different
Q4 chemical species with different solubility, reactivity and toxicity,
causing different impacts on ecosystems and human health [1].
Mercury is released into the environment by numerous natural
and anthropogenic sources through complex combinations in-
volving chemical, physical and biological reactions. The most
common anthropogenic sources of mercury are related to petro-
chemical, electronic and equipment (measurement, inks and
Q2
Corresponding author: Cabral, L. (luc.g.cabral@gmail.com, lucelia.cabral@hotmail.com)
chlorine – soda) industrial activities, besides its use in the extrac-
tion of gold and in amalgam [2].
Although mercury is toxic to both eukaryotic and prokaryotic
cells, some microorganisms have developed resistance mecha-
nisms to the metal. Of these, one can emphasize efflux pumps,
polymer chelation, precipitation, methylation and enzymatic re-
duction related to the mer operon [3]. Bacteria that possess the mer
operon are able to enzymatically reduce mercury (II) to the volatile
and less toxic form of mercury Hg(0) [4–6]. The gene merA is part of
an operon which comprises regulatory genes encoding transport
proteins [7,8]. In general, many mercury resistant isolates possess

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Please cite this article in press as: Giovanella, P. et al., Mercury (II) removal by resistant bacterial isolates and mercuric (II) reductase activity in a new strain of Pseudomonas sp. B50A, New
Biotechnol. (2015), http://dx.doi.org/10.1016/j.nbt.2015.05.006
 NBT 794 1–8
RESEARCH PAPER
the merR, merP and merA genes encoding proteins for regulatory
function, transport and extracellular binding, and mercuric (II)
reductase, respectively [9].
Mercuric (II) reductase (MerA) is a flavin oxidoreductase
(120 kDa), which acts as a dimer and is composed of three
domains. The three-dimensional structure of the enzyme indicates
that the active site is formed by the interaction of the central
domain of a subunit with another C-terminal domain [6]. The
central domain, described as a pyridine nucleotide oxidoreductase
disulfide group, is where catalysis and the transfer of two electrons
from NADPH to Hg(II) via FAD, occurs. In this case, there is a pair
Research Paper
of active redox cysteines that carry out the charge-transfer reac-
tion. The core of the MerA enzyme is a C-terminal domain found
only in the mercuric reductase enzyme, together with the central
domain [10]. An N-terminal domain has the function of directing
the Hg(II) to the active site of MerA. This domain is essential in
cells with low concentrations of sulfide agents, where the supply of
thiolated Hg(II) may be limited. Thus the mer system has evolved
to perform metal regulation, transport and enzymatic catalysis,
converting the highly reactive form of the mercury cation to the
relatively inert monoatomic form of volatile Hg(0) [6].
Remediation of environments contaminated with toxic metal
ions has conventionally employed physicochemical techniques
including precipitation, filtration and electrochemical recovery, as
well as membrane separation, excavation, waste disposal in land-
fills or just burying the contaminated site. These techniques are
ineffective, time and money costly and often produce additional
waste with greater contamination potential [11]. Thus the study of
alternative decontamination methods for this metal, as well as the
investigation of the kinetic properties of the enzymes involved in
the reduction of Hg(II), may contribute to understanding the
processes involved in the enzymatic reduction of Hg and assist
in improving the performance of microbial strains used in mercury
clean-up strategies.
To evaluate this contribution, this study aimed to isolate mer-
cury resistant bacteria; determine the minimum inhibitory con-
centration for Hg; estimate mercury removal by selected isolates;
explore the merA genes systems; and detect and characterize the
activity of the enzyme mercuric (II) reductase produced by a new
strain of Pseudomonas sp. B50A.
Material and methods
Soil sampling
Soil samples were collected from landfarming and landspreading
areas used to treat petrochemical waste contaminated with mer-
cury. Soil sub-samples (n = 4–5/sample) were taken to a depth of
10 cm at 5 m intervals using a grid measuring 10 m  10 m and a
Dutch auger (5 cm diameter; Eijkelkamp, The Netherlands). The
sub-samples were combined and the resulting composite sample
weighed approximately 2–3 kg. Chemical characterization of the
soils and sludge sewage was carried out as described in Tedesco
et al. (1995) [12].
Isolation of mercury resistant bacteria
Mercury resistant bacteria were isolated using the primary enrich-
ment culture method. Soil (0.1 g) samples were added to 20 mL of
Luria Bertani broth medium (LB) (g/L: peptone, 10; yeast extract, 5;
NaCl, 10) containing different doses of mercury (250 and 500 mM)
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New Biotechnology  Volume 00, Number 00  June 2015
as HgCl2 and incubated for 24 h at 308C [13]. An aliquot of 1 mL of
the enriched culture medium was inoculated into LB broth con-
taining the same concentrations of mercury described above.
These tubes were incubated for 24 h at 308C. The inoculation
and incubation procedures were repeated three times. An aliquot
of 30 mL of the enrichment culture was then spread on LB agar
supplemented with 125, 250 and 500 mM of mercury, followed by
incubation at 308C for 24 h. Colonies showing different morpho-
logical characteristics were purified using three consecutive streaks
and then submitted to 16S rDNA sequencing.
Molecular identification of mercury resistant bacteria
The bacterial genomic DNA was extracted using a Wizard Genomic
DNA Purification Kit (Promega, WI). The 16S rDNA was amplified
in a PCR reaction using the following universal primers for bacte-
ria: 27F (50 -AGATTTGATCMTGGCTCAG-30 ) and 1492R (50 -TACG-
GYTACCTTGTTACGACTT-30 ). The amplification reaction was
based on Sambrook and Russel [14]: buffer (50 mM Tris–HCl,
pH 9.0, 50 mM KCl, 2.5% Triton X 100), dNTPs (200 mM of each),
0.2 mM MgCl2, 0.25 pmol of each primer, 0.8 ng/mL of the DNA
sample and 0.02 U of Taq DNA polymerase. The amplification was
carried out in a thermal cycler (MJ Research Inc., Watertown, MA,
USA) and the program consisted of 35 cycles (initial denaturation
at 958C for 5 min, subsequent denaturation at 958C for 30 s,
annealing at 508C for 1 min, extension at 728C for 90 s and final
extension for 5 min at 728C). The PCR products were purified by a
standard precipitation method with PEG 8000 (polyethylene gly-
col). For the sequencing reactions of the PCR fragments, labeled
terminator kits (GE Healthcare) with the primer 519r (50 -GWAT-
TACCGCGGCKGCTG-30 ) were used. All the sequences generated
were submitted to the Genbank/NCBI database. The sequences of
the 16S rDNA genes from each isolate were used as a query to
determine the genus and species of its most closely related pro-
karyote using BLASTN [http://www.ncbi.nlm.nih.gov/BLAST].
PCR detection of the merA genes
In order to detect the merA gene, the DNAs of all the bacterial
isolates were obtained as described above for the amplification of
the 16S rDNA genes. The PCR reaction for merA was carried out
using the primers: 3F (CGT SAA CGT SGG STG CGT GCC STC CA)
and 3R (CGA GCY TKA RSS CYT CGG MCA KSG TC) [15]. Each
reaction mixture contained 5 mL of 10 PCR buffer, 1.5 mL of
25 mM MgCl2, 1 mL of 10 mM dNTPs, 0.02 U of Taq DNA poly-
merase (Denville, NJ), 0.5 mL of 20 pmol mL1 of each primer
(forward and reverse) and 16.75 mL of ultrapure water,. The fol-
lowing PCR conditions were used for merA: hot start at 958C for
5 min, followed by 35 cycles of 958C for 1 min, 648C for 60 s, 908C
for 72 s and a final extension for 10 min at 728C. Each PCR reaction
included a negative control that was prepared under identical
conditions as described above, except for the omission of the
DNA template. The reactions were run in a GeneAmp PCR System
9700 (Applied Biosystems, CA). The products of the PCR reactions
were separated on 1% agarose gel (Fisher Scientific, MA) and
observed using a Geldoc 2000 system (Bio-Rad, CA).
Minimal inhibitory concentrations (MIC)
All bacterial isolates were evaluated for their ability to grow in
LB medium containing different concentrations of mercury as
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New Biotechnology  Volume 00, Number 00  June 2015
HgCl2. All tests were carried out in 96-multiwell plates, each well
being filled with 200 mL of the growth medium, inoculated with
5 mL of overnight cultures, and incubated at 308C for 48 h. The
lowest concentration of mercury and other metals for which no
visible growth was observed was considered as the minimum
inhibitory concentration (MIC) [16,17]. Non-inoculated LB medi-
um served as the negative control and inoculated LB medium with
no mercury or other metals served as the positive control.
Capacity to remove mercury from the growth medium
Preparation of cells for mercury removal assays
Each isolate showing resistance to mercury was grown overnight in
LB containing HgCl2 (10 mM) at 30 8C for 24 h. The optical density
was adjusted by dilution with fresh LB medium to produce a density
of 2  107 CFU mL1. The flasks with non-inoculated LB medium
served as negative controls, and were incubated at 308C and 100 rpm
for 24 h. Mercury removal from inoculated LB medium was mea-
sured and related to cell growth (OD), the optical density readings
being taken at 600 nm (A600) using a Spectronic-20, GENESYSTM
(Spectronic Analytical Instruments, Rochester, NY, USA).
Mercury removal by the isolates
The assay was carried out according to Kannan & Krishnamoorthy
(2006). Briefly, approximately 2  107 CFU mL1 of the bacterial
isolates were inoculated into LB medium containing 10 mM of
Hg(II) and incubated at 308C for 24 h. The performance of the
most resistant isolate (B50A) with respect to mercury removal was
evaluated under the same conditions as described previously, but
taking readings after zero, 4, 8, 12 and 24 h in a medium contain-
ing 350 mM of mercury (II). The samples were then homogenized
and the growth determined by reading the OD600, and the mercury
removal capacity determined from the (%) mercury remaining.
Mercury analysis by cold vapor atomic absorbance spectroscopy
The concentrations of mercury remaining in the homogenized
medium inoculated with the isolates and in the non-inoculated
medium were determined following digestion according to the EPA
7471B method [18]. All glassware used during the Hg(II) analyses was
washed in 30% HNO3 and rinsed several times in ultrapure water
prior to use. An aliquot of supernatant (0.1 mL) was collected and
added to tubes containing 5 mL concentrated H2SO4 and 2 mL of
concentrated HNO3. An aliquot of 10 mL of 5% KMnO4 (potassium
permanganate) was then added to the tubes, which were autoclaved
for 15 min at 1218C. The remaining potassium permanganate was
reduced by adding 6 mL of 12% hydroxylamine hydrochloride. The
mercury was quantified in a cold vapor atomic absorption spectrom-
eter (Analyst 100, Perkin Elmer, Waltham, MA, USA).
Detection and characterization of mercuric reductase
The crude extracts (CE) of the more resistant isolates (B50A and V1)
were obtained according to the Ghosh methodology [19], with
modifications, inoculating 2  107 CFU mL1 of the different iso-
lates into LB medium containing 20 mM HgCl2, and incubated for
4 h at 30 8C and 100 rpm. A further 20 mM of HgCl2 were added after
4 h incubation, and maintained under the same conditions for 20 h.
The culture was then centrifuged for 10 min at 10,000 rpm and 4 8C,
the supernatant discarded, and the cells resuspended in (50 mM, pH
7.35) phosphate buffer. This procedure was repeated four times to
Please cite this article in press as: Giovanella, P. et al., Mercury (II) removal by resistant bacterial isolates and mercuric (II) reductase activity in a new strain of Pseudomonas sp. B50A, New
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RESEARCH PAPER
wash the cells and the final cell pellet resuspended in 20 mL phos-
phate buffer (50 mM, pH 7.35) and b mercaptoethanol (1 mM). The
cells were then subjected to six ultrasound (35 W) pulses in an ice
bath to promote cell disruption, and the cell extract centrifuged for
45 min at 10,000 rpm to remove cell debris and obtain the super-
natant. The supernatant was subsequently used as the enzyme
source for the enzymatic analyses under different conditions (crude
extract). The mercuric reductase activity was determined in a spec-
trophotometer at 340 nm by monitoring the decrease in absorbance
resulting from the oxidation of NADH. The total reaction volume
was 3 mL, containing: 89.75 mM HgCl2, 5 mM Na2EDTA, 1 mM b
Research Paper
mercaptoethanol, 0.15 mM NADH, sodium phosphate buffer
(50 mM, pH 7.35), 0.2 mM MgSO4 and 200 mL crude extract. The
reaction mixture was pre-incubated for 10 min at 378C and the
reaction initiated by the addition of crude extract and NADH. The
specific activity was expressed in units (U) mg1 protein, in which
1 U mercuric reductase is the amount of enzyme per mg1 protein
capable of oxidizing 1 mmol NADH per min1 in the presence of
HgCl2. The soluble protein content was determined by the Lowry
method [20] using Folin–Ciocalteau reagent, comparing with a
standard curve prepared using bovine serum albumin.
Statistical analyses
The experiments were carried out in triplicate and the means and
standard deviations (N  1) calculated. The results were submitted
to the Tukey’s means test using the statistical program (Systat11).
The kinetic parameters of the mercuric (II) reductase (Km and Vmax)
were obtained by adjusting the results to a hyperbolic Michaelis-
Menten equation (vo = Vmax [S]/[S]1/2 + [S]; where [S]1/2 = Km).
Results and discussion
Isolation and identification of the microorganisms
The analysis of the 16S rRNA gene sequence proved that all the
bacteria isolated were Gram-negative, with six isolates belonging
to the genus Pseudomonas and two to the genus Enterobacter
(Table 1). Several studies have shown that bacteria of the genus
Pseudomonas are highly resistant to the different forms of mercury
[21–25], and have been reported in great abundance in wastes
containing high Hg concentrations [25,26]. Mercury resistance is
common amongst Gram-negative microorganisms. Nakamura
et al. [27] isolated 55 mercury resistant bacteria and found that
only four isolates were Gram positive, and of the Gram-negative
bacteria, 13 were from the genus Enterobacter.
Minimum inhibitory concentration (MIC)
All isolates showed high resistance, but with different tolerance
capabilities (Table 1), indicating that the efficiency of the mercury
detoxification systems can be distinct for different microorganisms.
Isolates exhibiting the lowest mercury resistance were able to grow at
a concentration of 250 mM for the divalent inorganic form, and
400 mM for the organic form of mercury acetate. Most isolates had a
MIC between 450 and 920 mM, as observed by other authors
[17,24,28].
Mercury removal capacity of the isolates
All isolates were capable of removing mercury from the culture
medium after 24 h of incubation (Table 1). Seven isolates did not
differ in their ability to remove Hg, showing a percentage removal
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RESEARCH PAPER New Biotechnology  Volume 00, Number 00  June 2015

TABLE 1
Sampling sites, isolates, genetic similarity index based on the partial 16S rRNA sequence, accession number, Hg2+ removed and
minimum inhibitory concentration (MIC) of mercury resistant bacteria.
Isolate Genetic similarity index (%) Accession number Hg2+ removed (%) MIC (mM)
MC MA
A25B Enterobacter sp. (100%) AB098582.1 75.76  1.66 400 550
A50A Pseudomonas entomophila (100%) CT573326.1 80.60  1.74 250 550
B50A Pseudomonas sp. (99%) KF312471.1 82.24  2.15 920 550
B50B Pseudomonas sp. (100%) AB076857.1 88.21  2.01 450 900
B50C Enterobacter sp. (99%) AB098582.1 64.50  3.32 250 490
Research Paper

B50D Pseudomonas sp. (100%) AM08847.1 85.36  1.91 822 890

B100A Pseudomonas entomophila (100%) CT573326.1 89.96  0.02 450 490
V1 Pseudomonas putida (100%) AE015451.1 83.67  1.25 920 400
Values in columns followed by different letters indicate significant differences between treatments according to Tukey’s test (p < 0.05).
MC: mercury chloride (HgCl2).
MA: mercury acetate (C4H6HgO4).

TABLE 2
Effect of Hg2+ concentration on the growth (OD 600 nm) and percentage of mercury removal by the isolates Pseudomonas sp. B50A and
Pseudomonas putida V1 after 24 h incubation at 308C  2.
Hg2+ concentration Isolates
Pseudomonas sp. B50A Pseudomonas putida V1
350 mM Percentage of Hg2+ removed 51.08  0.93 a 54.30  4.06 a
Absorbance (OD 600 nm) 1.41  0.03 a 1.41  0.09 a
10 mM Percentage of Hg2+ removed 82.24  2.15 b 83.67  1.26 b
Absorbance (OD 600 nm) 1.69  0.01 b 1.55  0.02 b
Values in columns followed by different letters indicate significant differences between treatments according to Tukey’s test (p < 0.05).

of approximately 80%. Sadhukham et al. [29] evaluated mercury
resistant microorganisms and obtained similar results with respect
to mercury removal after 24 h of incubation. In the same study, it
was found that the ability of the microorganisms to remove
pollutants was related to higher MIC values, confirming the results
of Ghosh et al. [30] and Cabral et al. [31]. However, this relation-
ship was not proven in the present study. The opposite was
observed with the isolate A50A, which, although presenting the
lowest tolerance to Hg, removed about 80% of the mercury from
the culture medium.
The most resistant isolates (Pseudomonas sp. B50A and Pseudo-
monas putida V1) were evaluated in a high mercury concentration.
The mercury removing ability of the isolates selected in from
landfarming (B50A) and landspreading (V1) sites was significantly
reduced by 30% because of the increase in Hg concentration in the
culture medium (Table 2). Ghosh et al. [30] found that the ability
of microorganisms to remove mercury from the culture medium
was affected by high concentrations of HgCl2, suggesting that it
may be due to sequestering of intracellular Hg by cell components
that bind to the metal.
At high concentrations of the metal, the Hg binding sites of the
MerP may be saturated, so the removal rate is reduced at high Hg
concentrations. MerP is the most abundant protein synthesized by
the mer system, because of the role it plays in the transport of Hg
into the intracellular medium. This protein binds to the mercury
present in the periplasm that leads to the MerT. Finally, the
MerT leads to the interior of the cell across the inner membrane
of Gram-negative microorganisms, and thus the mercury is sub-
jected to the action of the mercury reductase [5]. Therefore, even if
the MerP is being abundantly transcribed in the high Hg concen-
trations, the volatilization rate is reduced.
Another possibility concerns MerA, since one of the factors
affecting the rate of a reaction catalyzed by enzymes is the amount
of substrate. The maximum reaction rate of an enzyme is achieved
when almost all the molecules are in the enzyme/substrate form
and the concentration of free enzyme is insignificant. The enzyme
is saturated with substrate and the reaction rate does not increase,
even though the substrate is in the reaction medium [32]. There-
fore, we can assume that the Hg present in the culture medium is in
excess relative to the number of active sites on MerA available for
the reaction. Consequently, a larger amount of mercury could
have been removed if these microorganisms had been submitted
to longer incubation times.
The growth of the two isolates Pseudomonas sp. B50A and
Pseudomonas putida V1 in 350 mM mercury was approximately
15% lower when compared to their growth in a concentration
of 10 mM (Table 2). The statistical analysis showed significant
differences in the optical densities of V1 and B50A in the presence
of 350 and 10 mM Hg. Other authors [29,33] obtained similar
results when assessing the optical density of the isolates at low
and high mercury concentrations, and concluded that the dispar-
ity observed at high and low mercuric chloride concentrations
was not significant. Taking into consideration that the isolates
evaluated in this experiment were subjected to sub-lethal Hg

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Please cite this article in press as: Giovanella, P. et al., Mercury (II) removal by resistant bacterial isolates and mercuric (II) reductase activity in a new strain of Pseudomonas sp. B50A, New
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New Biotechnology  Volume 00, Number 00  June 2015 RESEARCH PAPER

1.4 100 0.10

0.09
A a

Enzymatic activity (U/mg proteína)

1.2

Percentage of Hg2+ removed

80
0.08
1.0

from medium (%)

Biomass (OD600nm)

0.07 a
60
0.8 0.06

0.05
0.6
40
0.04
0.4
0.03
20
0.2 0.02

Research Paper
0.01 a
0.0 0 a a
0 4 8 12 18 0.00 a
a a
Time (hours) 0 15 30 45 60
Time (minutes)
0.10
FIGURE 1 B a
0.09
Dynamic removal of Hg2+ (squares) and growth of Pseudomonas sp. B50A in

Enzymatic activity (U/mg protein)

the presence of Hg (10 mM) (circles) and absence (triangles) of mercury. 0.08

Points represent the average of three replicates, and the error bars represent 0.07
the standard deviation. b
0.06

0.05 a

concentrations, with a reduction in the growth rate of around

15%, it can be assumed that, biologically, no difference was
observed.
The isolate Pseudomonas sp. B50A grew rapidly in four hours,
both in the presence and absence of Hg, maintaining this growth
until the final reading of the optical density. Mercury removal
from the culture medium was 86% in just 4 h, and after 8 h of
incubation, it was able to remove 93% Hg. The amount of mercury
remaining after 18 h was only 3% of the initial concentration
(Fig. 1). Other authors [34,35] also observed that the removal of
Hg(II) was preceded by high cell concentrations in the culture
medium.
Characterization of mercuric reductase in the crude extract (CE)
The PCR reaction was used to amplify and detect the merA gene
before characterizing the mercuric reductase. The B50A, B50B,
B50C, B50D and V1 isolates tested positive for the presence of
the merA gene. Subsequently, the crude extracts of the more
resistant isolates (B50A and V1) were evaluated for mercuric
reductase activity. Only the isolate B50A showed enzyme activity
in the crude extract (CE), which may be associated with the use of
NADH alone as the acceptor in the reaction. Mercuric reductase
requires NADPH or NADH, but with NADPH the enzyme exhibits
activity at much lower Hg concentrations. This behavior is char-
acteristic of the enzyme relative to the acceptor, and has no
connection with the preparation of the extract or the methodolo-
gy used to quantify the mercury [36].
The enzyme activity did not differ as a function of the incuba-
tion time of the isolate (Fig. 2a). This result corroborates the
performance of the isolate in removing approximately 90% of
the Hg present in the culture medium (10 mM) within 24 h. Since
the mer operon has no constitutive expression, but is induced by
the presence of mercury in the culture medium [37], the produc-
tion of the enzyme MerA may be suppressed by the deficiency or
absence of Hg in the culture medium after 24 h of incubation.
Volumes above 200 ml CE in the reaction negatively affected
enzyme activity, probably due to the absence of NADH, cofactors,
0.04 c
b a
0.03 a
b
0.02 c
c
b
0.01
c
0.00
0 15 30 45 60
Time (minutes)
FIGURE 2
Mercuric reductase activity in the crude extract (CE) of the Pseudomonas sp.
(B50A) at different reaction times and incubation periods: 24 h (squares) and
48 h (circles) (a); and effect of increasing the amount of crude extract on the
enzyme activity at different reaction times: 200 mL of crude extract (circles),
300 mL of crude extract (squares) and 400 mL of crude extract (triangles) (b).
Points represent the mean of three replicates and error bars represent the
standard deviation. Means with the same letter do not differ statistically
between reaction times according to Tukey’s test (p > 0.05).
and substrate in the presence of larger amounts of the enzyme. It is
also possible that larger amounts of CE result in a greater number
of inhibitors of the reaction. In both experiments, the most
suitable reaction time for estimating the maximum enzyme activ-
ity was 15 min (Figs. 2a and 3b).
Increasing the temperature to 458C had a positive effect on
enzyme activity (Fig. 3). Some authors have found that the mer-
curic reductase obtained from Gram-negative bacteria is stable at
high temperatures [39], and in this study, the optimum tempera-
ture was in the range from 37 to 458C. The increase in enzyme
activity occurring up to 458C may be due to the catalytic activity of
the highly temperature dependent enzymes [32,38]. When the
temperature increases, the kinetic energy of both the enzyme and
its substrate also increases, a greater number of collisions between
enzyme and substrate occurring, resulting in greater enzyme ac-
tivity [40]. There was a decrease in enzyme activity above 558C, but
at 758C, the enzyme still presented 50% of the activity shown at
the optimum temperature. This was because temperatures above
the ideal one affect the ionization state of the enzyme, leading to a

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pH 0,10
4 5 6 7.35 8 9
a b
0.10 b

Enzymatic activity (U/mg protein)

b b
Enzymatic activity (U/mg protein)

c c 0,08
0.09 d
c a
c
0.08
0,06
0.07
b e
e
0.06 f
a 0,04
0.05 d
g
0.04
b
0.03 0,02
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0.02
a h 0,00
0.01
i 0 30 60 90 120 150
0.00
2+ -1
1 25 37 45 55 65 75 85 95 Hg (µM L )

Temperature (ºC)

FIGURE 3
Effect of temperature (circles) and pH (squares) on the activity of the mercuric
reductase in the crude extract (CE) of the Pseudomonas sp. B50A. Points
represent the average of three replicates and the error bars represent the
standard deviation. Means with the same letter are not significantly different
between treatments according to Tukey’s test (p > 0.05).
FIGURE 5
Effect of increasing the substrate concentration (Hg2+) as an inducer of
mercuric reductase activity in the crude extract (CE) of the Pseudomonas sp.
B50A. Points represent the mean of three replicates and error bars represent
the standard deviation. Means with the same letter do not differ between Hg
concentrations according to Tukey’s test (p > 0.05).

reduction in enzyme activity. In addition, high temperatures The enzyme showed higher activity between pH 6 and 8, and
increase the translational, vibrational and rotational energies of maximum activity at pH 8 (Fig. 4). These results are consistent with
the molecules involved in molecular bonding, which could break those of other authors [39,42] when evaluating Gram-negative and
some of the bonds that determine the tri-dimensional form of the Gram-positive microorganisms. Increasing the pH to the upper limit
proteins, resulting in enzyme denaturation [41]. The mercuric (8.0) resulted in increased enzyme activity, providing evidence that
reductase maintained approximately 50% of its activity at the more alkaline pH values are more appropriate for the performance of
lowest temperature tested (18C) indicating that low temperatures this enzyme. According to Ledwidge et al., 2010 [43] deprotonation
are not ideal for the full activity of this enzyme. Nevertheless, from of the thiol groups of the cysteines of mercuric reductase may
a practical point of view, this response suggests that it would still stabilize its negative charge and facilitate the reactions of the
be possible to use this enzyme to remove mercury under such enzyme mercuric reductase. However the enzyme activity was
temperature conditions. reduced by 50% at pH 9. The effects of pH on the enzyme are
due to changes in the ionization state of the system components
as the pH varies. Since enzymes are proteins and contain many
0.08 a
a ionizable groups, they exist in different ionization states, and their
Enzymatic activity (U/mg protein)

0.07 a
a a catalytic activity is restricted to a small range of pH [38].
a The activity of the mercuric reductase of Pseudomonas sp. B50A
0.06
was neither affected negatively nor positively by the presence of
b
0.05 b b the ions Ni2+, Cd2+, Sn2+, Ba2+ or NH4+, and hence it appears that
these ions are not necessary for the catalytic activity of this
0.04
enzyme. However the enzyme was partially inhibited by the ions
0.03 Ca2+, K+ and Cu2+, and so these ions may have bound to an
essential functional group of the mercuric reductase, resulting
0.02
in a conformational change, or simply occupied the active site.
0.01 Metal ions can also cause a reduction in catalytic activity, probably
due to partial denaturation of the enzyme [44]. Ray et al. [28]
0.00
ro
le
tCont. 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 4+ + 2+ 2+ KK
NiNi CdCd SnSn BaBa NHNH4 Ca Ca
++ + 2+
CuCu
investigated copper, nickel and cadmium in concentrations ten
n
Co times smaller than those used in the present study, and found
Íons
approximately 100% inhibition of the enzyme activity. Moreover,
Gachhuy et al. [45] detected no decrease in reductase activity after
FIGURE 4 the addition of nickel and cadmium, although calcium and copper
Effect of ions on the activity of the mercuric reductase in the crude extract partially inhibited the enzyme under study.
(CE) of the Pseudomonas sp. B50A. Points represent the average of three
replicates and the error bars represent the standard deviation. Means with
Concentrations above 30 mM Hg increased the activity of mer-
the same letter do not differ between treatments according to Tukey’s test curic reductase (Fig. 5). Significant differences were observed
(p > 0.05). when comparing the lowest concentration studied (30 mM) with

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NBT 794 1–8
New Biotechnology  Volume 00, Number 00  June 2015
concentrations above 90 mM. These results indicate that concen-
trations between 60 and 90 mM Hg would be sufficient for maxi-
mum activity of the enzyme, since the reaction rate was stabilized
at these concentrations. The kinetic parameters of the crude
extract of mercuric reductase were obtained directly by fitting
the results of the enzyme activity to the Hg concentration
using the hyperbolic Michaelis-Menten equation. It was found
that the data adequately fitted the equation (r2 = 0.98) with Vmax
and Km values of 0.0845 mmol min1 and 7.9085 mmol L1, re-
spectively. The low value of Km of the mercuric reductase of
Pseudomonas sp. B50A indicated the great affinity of the enzyme
for Hg2+.
Conclusions
The mercury-resistant bacterium Pseudomonas sp. B50A possesses
the merA genes and mercuric reductase activity, which enables it to
survive in the presence of mercury and to convert the toxic forms
of mercury to less toxic forms. This isolate removed 80% of
the mercury at low concentrations and 50% at sub-lethal
References
[1] UNEP. Global Mercury Assessment, Tech. rep., UNEP, Geneva, Switzerland,
www.chem.unep.ch/MERCURY/, (2002).
[2] Maxson P. Dynamics of mercury pollution on regional and global scales. In:
Pirrone N, Mahaffey KR, editors. Atmospheric processes and human exposures
around the world. New York: Springer; 2005. p. 25–50.
[3] Boening DW. Ecological effects, transport, and fate of mercury: a general review.
Chemosphere 2000;40:1335–51.
[4] Nies DH. Microbial heavy-metal resistance. Appl Microbiol Biotechnol
1999;51:730–50.
[5] Barkay T, Miller SM, Summers A. Bacterial mercury resistance from atoms to
ecosystems. FEMS Microbiol Rev 2003;27:355–84.
[6] Barkay T, Wagner-Döbler I. Microbial transformations of mercury: potentials,
challenges, and achievements in controlling mercury toxicity in the environ-
ment. Adv Appl Microbiol 2005;57:1–52.
[7] Summers AO. Untwist and shout. A heavy metal responsive transcriptional
regulator. J Bacteriol 1992;174:3097–101.
[8] Narita M, Chiba K, Nishizawa H, Jshij H, Huang CC, Kawabata Z, et al. Diversity
of mercury resistance determinants among Bacillus strains isolated from sedi-
ment of Minamata Bay. FEMS Microbiol Lett 2003;223:73–82.
[9] Silver S, Hobman JL. Mercury microbiology: resistance systems, environmental
aspects, methylation, and human health. Microbiol Monographs 2007;6:
357–70.
[10] Engst S, Miller SM. Alternative routes for entry of HgX2 into the active site of
mercuric ion reductase depend on the nature of the X ligands. Biochemistry
1999;38:3519–29.
[11] Camargo FAO, Bento FM, Jacques RJS. Uso de microrganismos para remediação
de metais. In: Ceretta CA, Silva LS, Reichert JM, editors. Tópicos em Ciência do
Solo. Minas Gerais: Sociedade Brasileira de Ciência do Solo; 2007. p. 468–96.
[12] Tedesco MJ, Gianello C, Bissani CA. Análises de solo, plantas e outros materiais.
In: Boletim Técnico2nd edn, Porto Alegre: Universidade Federal do Rio Grande
do Sul; 1995.
[13] Takeuchi F, Negishi A, Nakamura S, Kanao T, Kamimura K, Sugio T. Existence of
an iron-oxidizing bacterium Acidithiobacillus ferrooxidans resistant to organ-
omercurial compounds. J Biosci Bioeng 2005;99:586–91.
[14] Sambrook J, Russell DW. Molecular cloning – a laboratory manual, 3rd edn.,
New York: Cold Spring Harbor Laboratory Press; 2003.
[15] Wang Y, Boyd E, Crane S, Lu-Irving P, Krabbenhoft D, King S, et al. Environ-
mental conditions constrain the distribution and diversity of archaeal merA in
Yellowstone National Park, Wyoming, U.S.A.. Microbial Ecol 2011;62:739–52.
[16] Murtaza I, Dutt A, Ali A. Relationship between the persistance of mer operon
sequences in Escherichia coli and their resistance to mercury. Curr Microbiol
2002;44:178–83.
[17] Ball MM. Mercury resistance in bacterial strains isolated from tailing ponds in a
gold mining area near El Callao (Bolı́var State, Venezuela). Curr Microbiol
2007;54:149–54.
[18] EPA 7471B (2009) Mercury in solid or semisolid waste: manual cold-vapor tech-
nique http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/7471b.pdf.
[19] Ghosh S, Sadhukhan PC, Ghosh DK, Chaudhuri J, Mandal A. Purification and
properties of mercuric reductase from Azotobacter chroococcum. J Appl Microbiol
1999;86:7–12.
[20] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the
folin phenol reagent. J Biol Chem 1951;193:265–75.
Please cite this article in press as: Giovanella, P. et al., Mercury (II) removal by resistant bacterial isolates and mercuric (II) reductase activity in a new strain of Pseudomonas sp. B50A, New
Biotechnol. (2015), http://dx.doi.org/10.1016/j.nbt.2015.05.006
RESEARCH PAPER
concentrations, and is hence a promising candidate for applica-
tion in the bioremediation of environments contaminated with
high mercury concentrations. The enzyme mercuric reductase of
Pseudomonas sp. B50A maintained 50% of its activity at extreme
temperatures (1 and 758C) and at alkaline pH values (9), demon-
strating that adverse environmental conditions do not impede the
use of this enzyme in the bio-removal of Hg. Understanding these
capacities advances the knowledge of the physiological responses
and enhances our capacities to develop technologies for the
remediation of mercury-contaminated wastes.
Research Paper
Acknowledgements
The authors are grateful to Professor T. Barkay (Rutgers University)
for allowing us to carry out the PCR (gene merA). This project was
supported by CAPES (Coordination for the improvement of Higher
Education Staff), CNPq (National Council for Scientific and
Technological Development), FAPERGS (Foundation for the
Support of Research of the State of Rio Grande do Sul), Research
Project No. 2471 12-0, Brazil.
[21] Horn JM, Brunke M, Deckwer WD, Timmis KN. Pseudomonas putida strains
which constitutively overexpress mercury resistance for biodetoxification of
organomercurial pollutants. Appl Environ Microbiol 1994;60:357–62.
[22] Canstein H, Li Y, Timmis KN, Deckwer WD, Wagner-Döbler I. Removal of
mercury from chloralkali electrolysis wastewater by a mercury-resistant Pseudo-
monas putida strain. Appl Environ Microbiol 1999;65:5279–84.
[23] Fortunato R, Crespo JG, Reis MAM. Biodegradation of thiomersal containing
effluents by a mercury resistant Pseudomonas putida strain. Water Res
2005;39:3511–22.
[24] Mortazavi S, Rezaee A, Khavanin A, Vamazyar S, Jafarzadeh M. Removal of
mercuric chloride by a mercury resistant Pseudomonas putida strain. J Biol Sci
2005;5:269–73.
[25] Giovanella P, Bento F, Cabral L, Gianello C, Camargo FAO. Isolamento e seleção
de micro-organismos resistentes e capazes de volatilizar mercúrio. Quim Nova
2011;34:232–6.
[26] Nakamura K, Fujisaki T, Yoshisada S. Mercury-resistant bacteria in sediment of
Minamata Bay. Nippon Suisan Gakkaishi 1998;54:1359–63.
[27] Nakamura K, Iwahara M, Furukawa K. Screening of organomercurial-volatilizing
bacteria in the mercury-polluted sediments and seawater of Minamata Bay in
Japan. Clean Prod Processes 2011;3:104–7.
[28] Ray S, Pahan K, Gachhui R, Chaudhuri J, Mandal A. Studies on the mercury
volatilizing enzymes in nitrogen-fixing Beijerinckia mobilis. World J Microbiol
Biotechnol 1993;9:184–6.
[29] Sadhukhan PC, Ghosh S, Chaudhuri J, Ghosh DK, Mandal A. Mercury and
organomercurial resistance in bacteria isolated from freshwater fish of wetland
fisheries around Calcutta. Environ Pollut 1997;97:71–8.
[30] Ghosh S, Sadhukhan PC, Ghosh DK, Mandal AK, Chaudhuri J, Mandal A. Studies
on the effect of mercury and organomercurial on the growth and nitrogen
fixation by mercury-resistant Azotobacter strains. J Appl Bacteriol 1996;80:
319–26.
[31] Cabral L, Giovanella P, Gianello C, Bento FM, Andreazza R, Camargo FAO.
Isolation and characterization of bacteria from mercury contaminated sites in
Rio Grande do Sul, Brazil, and assessment of methylmercury removal capability
of a Pseudomonas putida V1 strain. Biodegradation 2013;24:319–31.
[32] Viloca MG, Gao J, Karplus M, Truhlar DG. How enzymes work: analysis by
modern rate theory and computer simulations. Science 2004;303:186–95.
[33] Kannan SK, Krishnamoorthy R. Isolation of mercury resistant bacteria and
influence of abiotic factors on bioavailability of mercury: a case study in Pulicat
lake north of Chennai, South East India. Sci Total Environ 2006;367:341–53.
[34] Vetriani C, Chew YS, Miller SM, Yagi J, Coombs J, Lutz RA, et al. Mercury
adaptation among bacteria from a deep-sea hydrothermal vent. Appl Environ
Microbiol 2005;71:220–6.
[35] Chatziefthimou A, Crespo-Medina M, Wang Y, Vetriani C, Barkay T. The
isolation and initial characterization of mercury resistant chemolithotropic
thermophilic bacteria from mercury rich geothermal springs. Extremophiles
2007;11:469–79.
[36] Ji G, Salzberg SP, Silver S. Cell-free mercury volatilization activity from three
marine Caulobacter strains. Appl Environ Microbiol 1989;55:523–5.
[37] Brown NL, Stoyanov JV, Kidd SP, Hobman JL. The MerR family of transcriptional
regulators. FEMS Microbiol Rev 2003;27:145–63.
[38] Whiteley CG, Lee D. Enzyme technology and bioremediation. Enzyme Microb
Technol 2006;38:291–316.
www.elsevier.com/locate/nbt 7
 NBT 794 1–8

RESEARCH PAPER New Biotechnology  Volume 00, Number 00  June 2015

[39] Olson GJ, Porter FD, Rubinstein J, Silver S. Mercuric reductase enzyme from a
mercury-volatilizing strain of Thiobacillus ferrooxidans. J Bacteriol 1982;
15:1230–6.
[40] Bisswanger H. Enzyme kinetics – principles and methods. Germany: Wiley-
VCH; 2002, 268 pp.
[41] Somero GN. Proteins and temperature. Annu Rev Physiol 1995;57:43–68.
[42] Zeroual Y, Moutaouakkil A, Dzairi AZ, Talbi M, Chung PU, Lee K, et al. Purifica-
tion and characterization of cytosolic mercuric reductase from Klebsiella pneu-
monia. Annals Microbiol 2003;53:149–60.
Research Paper
[43] Ledwidge R, Hong B, Dötsh V, Miller SM. NmerA of Tn501 mercuric ion
reductase: structural modulation of the pKa values of the metal binding cysteine
thiols. Biochemistry 2010;49:8988–98.
[44] Tunga R, Banerjee R, Bhattacharyya BC. Optimization of n-variable biological
experiments by evolutionary operation-factorial design technique. J Biosci
Bioeng 1999;87:125–31.
[45] Gachhuy JR, Chaudhuri J, Ray S, Pahan K, Mandal A. Studies on mercury-
detoxicating enzymes from a broad-spectrum mercury-resistant strain of Flavo-
bacterium rigense. Folia Microbiol 1997;42:337–43.

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