Transcript Slide 1

Mercury stable isotope fractionation during bacterial reduction of Hg(II) to
0
Hg
K. Kritee1 , B. Klaue2 , J. D. Blum2, T. Barkay1
1 Rutgers University, 76 Lipman Drive, New Jersey 08901, 2 University of Michigan, 1100 N. University Avenue, Michigan 48109
Results
Introduction
Figure 2.
Figure 1. Bacterial Hg resistance proteins
The extreme toxicity of mercury (Hg) compounds warrants the search
for new methods that can be used to track the sources of Hg and the
dominant pathways leading to its bioaccumulation. Hg has seven stable
isotopes (Fig. 1; 0.15 – 30% abundance; mass spread of 4%), is redox
sensitive, and its compounds have a high degree of covalent character.
Moreover, in recent years, a number of groups have reported significant
and measurable Hg isotope ratio variation in natural samples1,2 from
hydrothermal ores, sediment cores and fish tissues--but the causes of
fractionation are not clear. Thus Hg seems to be undergoing stable
isotopic fractionation and the isotopic signatures of Hg may attest to its
origin and/or redox history. This study investigates the naturally
occurring processes that cause Hg to undergo reproducible and
systematic mass dependent stable isotopic fractionation.
At 370C, Hg(II) undergoes mass dependent (Fig. 3b) Rayleigh
fractionation (Fig. 3a) with fractionation factor () = 1.0006 +/- 0.00005
per amu during its reduction to Hg0 by E. coli.
Figure 2. Simplified
schematic
the experimentalsetup
setup
Simplified
schematic
of theofexperimental
Do these proteins and enzymes coded preferentially reduce
Air pump
lighter isotopes of Hg(II) to Hg0 compared to heavier ones?
Isotope Abundance
196Hg
0.15
9.97
16.87
23.10
13.18
29.86
6.87
198Hg
199Hg
200Hg
201Hg
202Hg
204Hg
}
}
Hg(II)
Hg transport proteins
8
Hg(II) reductase
(NIST 3133)
Hg0
6
At 220C, modeled isotope ratios (& corresponding ) based on  (~
1.0015) estimated by using Equation 1 (see methods & Fig. 3d) does not
match with measured isotope ratios (Figure 3c).
10
7
T5
T4: 130
T3:70-100
Plausible explanation:
T2:40-70
Hg(II)
NIST 3133
Outer cell
membrane
Inner cell
membrane
Hg0
Trap1: 0-40 min
0.05M KMnO4+ 5% H2SO4
A constant offset (~0.0009) between measured ratios and ratios modeled
assuming rayleigh fractionation could mean that net Hg fractionation at
lower temperature is a result of combination of kinetic fractionation by
Hg(II) reductase and equilibrium fractionation by Hg transport proteins
(Fig. 1). Hg(II) transport across bacterial cell could be the rate limiting
step in the reduction of Hg(II) at 220C and not at 370C due to increased
rigidity of cell membrane at lower temperature.
E. coli in M9 based defined media OR
Any isotope
abundance
changes?
Natural bacterial community following
enrichment in site water
Figure 3a.
Evidence of kinetic fractionation following Rayleigh distillation model
1.2
9
Figure 3B. Evidence of mass dependent fractionation during
Hg(II) reduction by E.coli at 37oC: δ200Hg/198Hg vs.
δ202Hg/198Hg
4
3
II. If yes, is it mass dependent? Is it kinetic fractionation? What is the
value of alpha? What is the effect of changing temperature?
0.6
2
198
delta/amu modelled based on Rayleigh fractionation with alpha = 1.00063
Hg/
0.3
1
200
0.0
For Manipulated naturally occurring bacteria: When Hg0 was produced
after being pre-exposure to Hg(II) conc. of 250 & 175 ppb: 100% of
surviving bacterial cells were Hg resistant (Fig. 5c) &  ~ 1.0006 (similar
to pure culture) was observed (Fig 5b).
2
1
δ
delta/amu per mil
I. Is there any fractionation associated with the reduction of Hg(II) to
Hg0 by the mercuric reductase, an enzyme found in a broad range
of Hg-resistant bacteria from diverse environments?
3
Measured delta/amu in Hg(0) relative to NIST
Hg
Research Questions
0.9
0
-0.3
-1
-1
-0.6
1
III. Is this fractionation phenomenon limited to pure cultures grown
under laboratory condition or does it occur when naturally occurring
bacterial consortium reduce Hg(II)?
0.8
0.6
0.4
Average fraction of Hg(II) remaining (f)
0.2
0
-2
-3.0
-1.5
0.0
1.5
Figure 3c. Relative isotope ratios (RVi/RLo) vs. Hg(II)
E.coli at 22 C
Modelled relative ratios
based on Rayleigh
fractionation with alpha =
1.0015
Linear (Measured relative
isotope ratios in Hg(O)
relative to NIST 3133
standard)
0.9996
0.9993
0.999
0.9987
0.9984
0.7
0.8
0.9
1
1.1
Fraction of Hg(II) remaining
Mass bias correction: Addition of thallium (NIST 997) to the Hg
vapor using a desolvating nebulizer.
Precision: Fractionation was measured relative to the NIST 3133 Hg
standard run before and after each sample and data are presented
as δ202Hg/198Hg (hereafter δ202Hg). Typical in-run precision of better
than ±0.05‰ (2σ) and external reproducibility of δ202 between
NIST 3133 and a secondary standard was ±0.08‰ (2σ).
1/ = 1 + [Slope of ln (RVi/RLo) vs. ln(f)]
(Equation 1)
Conclusions
-0.00015
y = -0.0015x - 0.0004
R2 = 0.9249
0
 Systematic Hg stable isotope fractionation does happen, both in pure
cultures of bacteria and naturally occurring bacterial consortia!
0.00015
-0.4
-0.35
-0.3
-0.25
-0.2
-0.15
-0.1
-0.05
0
Trap Hgo produced in 20 hours &
determine Hg isotopic composition
using MC-ICPMS
Add NIST 3133 at level equal
to pre-exposure conc.
Figure 5a
-0.40
Table 1.
of the extent of fractionation observed for Hg
with other redox-sensitive elements undergoing fractionation5,6.
Avg. Mol.
Weight
% mass
spread
Maximum
Range of 
(‰/amu)
Maximum
reported
/amu
56
7
2**
1.0015**
1.003#
Se
80
10
3#
Mo
96
8
1.7##
1.002##
Hg
200
4
2
1.0015
** Maximum range of isotopic variation (relative to standard) reported for low temperature
processes occurring either in nature & under laboratory conditions. Eg. 56/54Fe in natural
samples varies from ~-3 to +1 making the max. range ~2‰/amu5. Max.  for 56/54Fe is
1.003 for non biological redox eqm. of Fe(III) and Fe(II)5.
# Max.  ~13‰ for 80Se/76Se during various Se transformations (or 3.25‰/amu).
 = 1000*( -1)(Johnson & Bullen6).
## 97/95Mo varies between -0.9 to +2.5 for natural samples (Anbar6).
4
Extent
of
isotopic
fractionation (in per mil)
measured in the trapping
solution vs. exposure
Figure 5b
1.00065
5b.
1.0004
1.00015
Blank (no
Cells
cells)
enriched
at 100
ppb
Cells
enriched
at 175
ppb
Figure 5c
Comparison*
Fe
3
0.00
2
-0.20
0.9999
Harvest cells. Resuspend
in filter sterilized source water
5a.
-0.60
1
Pre-expose sample in 4 different
bottles to 0,100,175 & 250 ppb Hg(II)
delta/amu (per mil)
Collect natural water sample
from an uncontaminated source
Figure 5.
Alpha/amu
Figure 4. Scheme followed to determine extent of fractionation
during Hg(II) reduction by naturally occurring bacteria
* This is a crude comparison & does not include fractionation due to amplifying processes
such as iterative distillation or chromatgraphy.
The kinetic fractionation factor () was determined from the results of
our experiments using the Rayleigh Distillation Equation:
RVi/RLo = (1/) f (1/ -1)
-0.0003
ln (fraction Hg(II) remaining)
% Hg resistant cells
Sample introduction: Cold vapor generation was employed using
Sn(II) reduction. The cold vapor sample introduction has a >99%
efficiency and generates a signal of ~600 mV/ppb at a sample
consumption rate of 0.75 mL/min.
ln (Relative Isotope Ratio/amu)
Relative isotope ratios
0.9999
-0.00045
Cells
enriched
at 250
ppb
 factor/amu calculated
assuming kinetic isotope
fractionation vs. exposure
120
90
5c.
60
Percentage
of
colony
forming units (CFU) after
4 days of exposure which
are Hg resistant vs. Hg
exposure
30
0
1
2
3
4
Figure 5d
Hg(0)/HgR CFU
MC-ICPMS
Measured relative isotope
ratios in Hg(O) relative to
NIST 3133 standard
1.0002
At high exposures, reduction by bacteria which have a unique & efficient
(Fig. 5d) Hg reducing mechanism leads to fractionation similar to its
extent in pure cultures. At lower exposures, reduction by variable nonspecific mechanisms like reduction by light or weak organic acids results
in mixed/weaker signal.
0
In 4 days: Enrichment of Hg
resistant bacteria depending
on exposure.
analysis3
7.5
Figure 3d. ln (RVi/RL0) vs. ln(f) - Rayleigh fractionation by
1.0005
Hg reduction by naturally occurring bacteria : NIST 3133 was
added to water samples from an uncontaminated source after a 4
day long pre-exposure4 and Hgo produced was purged into a
trapping solution (See Fig. 2 & 4). 250 ppb NIST was added to the
control given no exposure.
6.0
Plausible explanation:
fraction remaining(f) during reduction by E.coli at 22 C
Hg(II) reduction by a pure culture NIST 3133 was used as a
source of 3 µM (600 ppb) Hg(II). Hg0 volatilized during the growth
of E.coli/pPB117 cells at 370C (or 220C) in M9-based minimal media
and was purged into a trapping solution by air stripping (Fig. 2). In
order to determine the change in isotopic composition as a function
of the extent of the reaction, traps were replaced every 30-40 min
for a period of 320 min (and every 90 minutes for a period of 900
minutes for the experiment at 220C) to collect products
corresponding to different stages of the reaction.
4.5
δ202Hg/198Hg
0
Methods
3.0
But at low or no pre-exposure: Much lower % of total cells (10%) were
Hg resistant & lower extent of fractionation (Fig. 5a and 5b) was
observed.
 Hg is the heaviest metal for which biological fractionation has been
detected to date. In spite of the reduced % mass spread of its isotopes
and increased molecular weight, the extent of fractionation found lies in
the same range as for much lighter elements (Table 1).
 Use of Hg isotope ratios for identifying sources and sinks, in situ
pathways leading to its toxicity, and/or the nature and evolution of redox
reactions in both modern and paleo environments is plausible.
 Future work will determine how the change in physico-chemical
parameters (T, pH, e- donor etc.) can change the extent of fractionation
during Hg(II) reduction and other Hg transformations.
References
1. Smith C. et al. (2004), Eos Trans. AGU, 85(47), Fall Meet. Suppl., V51A-0515
2. Xie Q. et al. (2004), Eos Trans. AGU, 85(47), Fall Meet. Suppl., V51A-0518
1.E+02
3. Lauretta et al. (2001) Geochim.Cosmochim. Acta 65, 2807-2818
1.E+00
4. Barkay T. (1987) Appl. & Env. Microbiology 53(12), 2725-2732
1.E-02
1.E-04
0 100 175 250
Hg(II) exposure (ppb)
5d.
5. Anbar(2004) Earth & Planet. Sci. Lett. 217, 223-236
Hg0 produced (in ppt) per
Hg(II) resistant colony
forming unit (HgR CFU) vs.
Hg exposure.
6. Johnson C. M. et al. (Ed.) (2004) Geochemistry of non-traditional isotopes. Reviews
in Mineralogy & Geochemsitry 55.
Acknowledgements
Authors wish to acknowledge funding by NSF and NJWRRI. We thank John Reinfelder,
Paul Falkowski, Robert Sherrell, Constantino Vetriani & Ariel Anbar for their helpful
inputs at different stages of this project.