Transcript Document

M-207
Reduction of Hg(II) to Hg(0) by
dissimilatory metal reducing bacteria
Heather Wiatrowski and Tamar Barkay
Department of Biochemistry and Microbiology, Rutgers University, Cook College
[email protected]
Abstract
The reduction of mercuric mercury (Hg[II]) to elemental (Hg[0]) affects the
bioavailability and mobility of mercury in the environment.
In surface
waters, mercury may be reduced by sunlight or by mercury resistant
bacteria that possess a mer system.
Figure 2: Reduction of Hg(II) to Hg(0) by
dissimilatory metal reducing bacteria
Shewanella oneidensis MR-1 and Geobacter
sulfurreducens PCA
N2
reduction of Hg(II) occurs at nM to µM concentrations of Hg(II), these
processes are irrelevant in ground water aquifers that are contaminated
with lower concentrations of mercury. Yet, presence Hg(0) in groundwater
has been documented and the mechanisms leading to its formation are
presently unknown. We therefore examined if dissimilatory metal reducing
bacteria (DMRB), which are often found in subsurface sediments and are
KMnO4,
H2SO4
cells,
Hg(II)
known for their ability to reduce toxic metals and radionuclides, also reduce
Hg(II). We show that Shewanella oneidensis MR-1 reduced 67.73.69% of
S. oneidensis MR-1
anaerobically with fumarate as a terminal electron acceptor. Under the
20
Hg (nmol)
which lacked either resulted in activity not significantly different from that
for reduction of Hg(II) of 3.14  0.25
protein-
1
16
15
12
10
8
5
4
0
0
Time (h)
and 3.07  0.35 nmol min-1 mg
DMRB, Geobacter sulfurreducens PCA and Geobacter metallireducens GS15, reduced Hg(II) at comparable rates to MR-1. However, Hg(II) reduction
is not universal among DMRB or anaerobic bacteria, as the iron reducing
bacterium Anaeromyxobacter dehalogenans 2CP-C and the denitrifier
Pseudomonas stutzeri OX1 did not reduce Hg(II).
Our observations of
constitutive reduction of Hg(II) by DMRB suggest a mechanism that may
affect mercury speciation, and thus bioavailability and environmental
mobility, in environments contaminated with a low level of mercury that do
not receive day light.
Unless otherwise noted, Hg(II) was added as HgCl2 at a
concentration of 300 nM.
Mercury was analyzed using Cold Vapor Atomic Absorbance
Spectroscopy on a Leeman Labs Hydra AA Mercury analyzer or
by Liquid Scintillation Counting using 203Hg (provided by Delon
Barfus) as a tracer.
1
ii
ii
ii
ii
ii
0
8
6
i
G. metallireducens
GS-15
4
live cells
2
inoculated
0
3
live
0
3
heat-killed
0
5
live
0
5
heat-killed
autoclaved cells
ii
ii
ii
ii
ii
FeOOH
+
+
+
+
+
FeOOH
- - +
+ +
none
- - +
+ +
FeOOH
-
Conclusions
• S. oneidensis MR-1, G. sulfurreducens PCA, and
A comparison of endogenous reduction of
Hg(II) by MR-1 to reduction of Hg(II) by the
mer system
G. metallireducens GS-15 are able to reduce
Hg(II) (Figs. 1-5).
To facilitate comparison, a mer system was
introduced into MR-1
mercury (Table 1) and does not require induction.
By contrast, the mer system is inducible and is
effective at high concentrations of Hg(II).
• A spontaneous rifampicin resistant mutant was selected for.
This strain was called MR-1H
• This strain was mated to Pseudomonas aeruginosa PAO1
containing a mer system on the plasmid R388::Tn501,
Materials and Methods
live cells
autoclaved cells
ii
TEA
preincubation
autoclaved
respectively. Reduction by MR-1 was enhanced five fold in iron
reducing conditions relative to fumarate reducing conditions. Two other
2
G. sulfurreducens PCA
of autoclaved cells. Unlike mer-mediated Hg(II) reduction, this activity is
not inducible, as exposed cells and unexposed cells had a specific activity
3
0
same conditions, uninoculated media removed 6.433.57% of the added
electron donor and an electron acceptor, as incubation of cells in media
G. sulfurreducens
PCA
i
ii
an initial concentration of 150 nM HgCl2 in 24 hours, when grown
HgCl2. Reduction of Hg(II) showed a dependence on the presence of an
4
Cells and mercury were added to
growth media and bubbled with
nitrogen for the indicated time,
allowing Hg to be collected in a trap of
acidified permanganate. Mercury was
analyzed by CVAAS both in the growth
media and in the trapping solution
before and after bubbling. For strain
MR-1, fumarate was used as an
electron acceptor and ferric
oxyhydroxide was used as an electron
acceptor for strain PCA.
Hg(0)
Because mer-mediated microbial
Figure 5: Reduction of Hg(II) by Geobacter
spp. in the presence of ferric oxyhydroxide
generating a transconjugate called MR-1H/R388::Tn501
MR-1 is not resistant to mercury
• MR-1 is inhibited by 0.5 mM Hg(II)
• MR-1H/R388::Tn501 can grow in the presence of 25 mM
Hg(II)
• This reduction occurs at low concentrations of
• Endogenous reduction of Hg(II) by MR-1 requires
presence of an electron donor and acceptor, occurs
in aerobic conditions, fumarate reducing
conditions, and is enhanced in iron reducing
conditions (Figs. 1, 3, 4).
• Reduction of Hg(II) by Geobacter spp. requires
the presence of an electron acceptor (Fig. 5).
Geobacter metallireducens GS-15 was grown in ATCC medium
1768 and Geobacter sulfurreducens PCA was grown in ATTC
medium 1957 with ferric citrate (20 mM) as an electron
acceptor.
In all experiments for which electron donating or electron
accepting conditions varied from the culture conditions, cells
were washed two times in media containing no electron donor
and/or electron acceptor. Care was taken to ensure that anoxic
conditions were maintained throughout the washes.
Cell concentrations were normalized to extractable protein, and
a typical inoculum was ~ 0.4 mg protein/ml, which corresponds
to approximately 105 cells/ml
Data represents means of three replicates, and all errors are
standard deviations. Means that are not significantly (p > .05)
are indicated with the same roman numeral.
catalase
elemental mercury
merA,
Fe(II) dependent reduction
by Acidithiobacilli
Hg(II)
sulfate
reducing
bacteria
ionic mercury
merB
specific activities of 3.14  0.25 and 3.07  0.35 nmol
Hg(II)/min/mg protein respectively.
Table 1: Endogenous reduction of Hg(II) is
effective at low concentrations of Hg(II), but
not at high concentrations
strain
MR-1H/R388::Tn501
MR-1H
MR-1
MR-1H, autoclaved
uninoculated media
a
Initial specific reduction rates (nmol/min/mg protein) in
mediuma containing Hg(II) at:
25 µmol L-1
0.3 µmol L-1
16.3 ± 1.3(i)
1.60 ± 0.32 (i)
1.2 ± 0.7 (ii)
0.44 ± 0.08 (ii)
2.0 ± 0.6 (ii)
2.56 ± 0.17 (iii)
0.7 ± 0.4 (ii)
0.28 ± 0.04 (ii)
0.4 ± 0.5 (ii)
0.10 ± 0.04 (ii)
Figure 3: Electron donors and electron
acceptors are required for Hg(II) reduction
by MR-1
ii
ii
i
1.0
ii
ii
+
+
+
-
hydrogen (ND)
fumarate (10 mM)
autoclaved
+
+
-
+
-
-
Figure 4: A higher reduction rate of Hg(II)
occurs with iron as compared to fumarate
as an electron acceptor for MR-1
fumarate
pregrown in
fumarate
3
2
100
i
1
Hg(II). Because reduction by MR-1 is
constitutive and functions at low Hg(II)
concentrations, it may be more applicable to
subsurface environments.
ii
50
TEA
preincubation
autoclaved
0
0 24
0 24
0 24
MR-1
sterile
media
MR-1
20
ii
i
-
-
-
+
15
ii
ii
5
ii
FeOOH FeOOH
+
-
-
203Hg
• Microbial community analysis on Hg(II) reducing
anoxic sediments
iii
Mercury was analyzed using
none FeOOH FeOOH
-
+
+
+
as a tracer.
• MR-1 reduces Hg(II) at ~5 X higher rate in iron
versus fumarate reducing conditions
• A 24h preincubation period in FeOOH was required
for Hg(II) reduction activity.
in intact sediment samples
conditions maximize mer-independent Hg(II)
reduction.
pregrown in
ferric citrate
0
fumarate FeOOH none fumarate
• Investigate mer-independent reduction of Hg(II)
• Determine what terminal-electron accepting
10
ii
0
sterile
media
available for methylation by anaerobic bacteria.
Future directions
150
0 24
• Reduction of Hg(II) to Hg(0) may make it less
New Jersey, and it is not likely due to pointsource contamination, and this process is a
potential cause.
+
+
+
+
Results
Fig 1: Shewanella oneidensis MR-1 causes
loss of Hg(II) from culture media
Time (h)
mobility, because Hg(II) sorbs to sediments and
Hg(0) is a volatile gas that is poorly soluble in
water.
• Hg(0) has been discovered in groundwater in
lactate (10 mM)
oxygen
• Reduction of Hg(II) to Hg(0) increases its
contaminated by metals and radionuclides is to
stimulate endogenous dissimilatory metal
reducing bacteria to reduce and immobilize
these contaminants.
1.5
0.0
ionic mercury
Environmental Relevance
• A strategy in development to remediate sites
2.0
methyl mercury
Can anaerobic microorganisms reduce Hg(II)?
Hg(II) through their electron transport chains.
• Many sites are contaminated with low levels of
Assays were performed in fumarate reducing conditio ns.
0.5
CH3Hg
• We hypothesize that these organisms reduce
• MR-1 cells pregrown in 300 nM Hg(II) and unexposed cells had
anaerobic microbes
Hg(0)
aerobic microbes
anaerobic microbes
aerobic microbes
Microbial transformations
of mercury
mer-independent reduction is not an
inducible process
Acknowledgements
We would like to thank D. Lovley for strains, Delon Barfus for
providing the 203Hg, and Costantino Vetriani and Jeffra Schaefer
for advice. This research was funded by the Natural and
Accelerated Bioremediation Research (NABIR) program,
Biological and Environmental Research (BER), U.S. Department
of Energy (Grant No. DE-FG02-99ER62864).