Mercury stable isotope fractionation during W-76
microbial reduction of Hg(II) to Hg(0)
Kritee1, M. Johnson2, B. Bergquist2, J. D. Blum2, and T. Barkay1
1Rutgers University, 76 Lipman Drive, New Jersey 08901, 2University of Michigan, 1100 N. University Avenue, Michigan 48109
Fig 2. Development of a stable isotope ratio based tool. Fig. 7 Fractionation of Hg isotopes by Hg(II)
Introduction resistant microbes from a natural source
Total Hg resistant Reactor Trap
Biogeochemical concern • Quantify fractionation during transformations by 1.E+04 1
202/198 Hg (‰)
pure cultures of microbes and abiotic processes.
• Determine isotope ratios for representative sources.
• There are multiple sources (natural vs.
anthropogenic, local vs. global) and Stage 1
transformations (microbial vs. abiotic) that can 1.E+01 -2
lead to buildup of methylmercury (Fig. 1). 1.E+00 -3
0 1 2 3 4 1 0.9 0.8 0.7 0.6
• In order to design/implement effective • Effect of change of environmental conditions Time (Days) Fraction of added Hg(II) remaining (f)
(T, pH, redox) on individual transformations
remediation strategies, we need tools to track the • Fractionation by natural microbial community
actual causes of Hg accumulation in a given • Kinetic vs. Equilibrium change A. Enrichment of Hg(II) resistant B. Isotopic composition of the
Stage 2 microbes in a natural water Hg(II) remaining in the
ecosystem. sample (Increase in % of Hg(II) reactor and Hg(0) produced
resistant colony forming units per by enriched microbes.
ml (CFU/ml) with time7).
Fig. 1 The Mercury Biogeochemical Cycle
Measure isotope ratios of an element in a natural ecosystem
• Identify dominant sources & pathways
Summary of 202/198 values obtained from linear regression of isotope data
• Date evolution of element’s microbial transformation
• Determine environmental conditions at the time of deposition
Conditions Based on reactor (Eq. 1) Based on trap (Eq. 2)
Pure Culture of E. coli JM109/pPB117
Temp. Reactor size Slope SE n* R2 Slope SE Intercept SE n* R2
370C 1L 1.0017 0.0002 5 0.969 1.0020 0.0000 1.0020 0.0000 5 0.999
1L 1.0014 0.0001 5 0.997 1.0018 0.0001 1.0021 0.0001 5 0.993
100 ml NA# 2 1.000 1.0020 0.0002 1.0020 0.0001 5 0.969
300C 1L 1.0018 0.0001 10 0.984 1.0022 0.0001 1.0020 0.0001 9 0.980
220C 100 ml 1.0020 0.0000 2 1.000 1.0031 0.0004 1.0026 0.0002 10 0.899
Natural microbial consortium
Fig. 4 Schematic of experimental set up
Hg resistant 1L 1.0016 0.0002 4 0.945
Control 1L 1.0005 0.0000 4 0.984
* Since fractionation was found to be suppressed at f < 0.3, the number of data points used for regression (n) is lower
Hg stable isotope for some experiments # Not applicable; One of the two data points available corresponds to f = 0.08
Air pump ratios analysis
Stable isotopes fractionation
• Mercury resistant bacteria prefer lighter isotopes
Ratio of abundance of a heavier to a lighter when reducing Hg(II) to Hg(0) (Fig. 6 and 7).
stable isotope as compared to a standard - Hg(II)
reported as delta () per mil (see methods). NIST 3133 Trap1: 0-40 min • The similar values of observed for the
0.05M KMnO4+ 5% H2SO4
experiments done with two bacterial genera (B.
Hg resistant E. coli or B. cereus (in M9 based media) cereus; preliminary results not shown) and a
OR Enriched Natural community (in site water)
• Measurement4 of isotope ratios of many natural community (Table 1) suggest that the
elements (1H to 96Mo) has helped us determine: isotopic signature produced during biological Hg(II)
Fig. 5 Measuring fractionation during Hg reduction is unique.
Sources of pollutants or nutrients
reduction by naturally occurring microbes • Hg isotopes have the potential for distinguishing
Dominant pathways transforming the element between different pathways leading to Hg(0)
Collect natural water sample
Paleo-environmental conditions (T, pH etc.) from an uncontaminated source production based on the extent of fractionation and
could help Hg remediation efforts.
Analyze Hg(0) in traps
Pre-expose sample to 250 ppb Hg(II)
• Hg is the heaviest element4 for which mass
• Living organisms preferentially uptake lighter and Hg(II) remaining in dependent biological fractionation has been
the reactor by MC-ICPMS
isotopes leaving heavier isotopes in the documented to date (Table 2).
environment. In 4 days: Enrichment
of Hg resistant bacteria • The extent of fractionation observed per atomic
Why does Life like lighter isotopes? It Add 250 ppb NIST 3133 mass unit (amu) is very high and is comparable to
takes less energy to uptake/process lighter fractionation by much lighter elements (Table 2).
molecules. Harvest cells. Re-suspend
in filter sterilized source water
• Hg has seven stable isotopes (Fig. 2). Multiple collector inductively coupled plasma mass
Comparison of the extent of fractionation observed
Significant Hg isotope ratio variations in natural spectrometry (MC-ICPMS)1,6 for Hg with other redox-sensitive elements*4.
samples from ores, sediment cores, and fish Avg. Mol. % mass Maximum Maximum
tissues have been reported1-3, but the processes • δ202Hg = [(202Hg/198Hg)Sample - 1] * 1000 ‰ (per mil)
Weight spread Range of
leading to the fractionation have not yet been
Fe 56 7 2 1.0015
explored. (202Hg/198Hg)NIST 3133 Standard
Se 80 10 3 1.003
Mo 96 8 1.7 1.002
Research Objectives • Sample introduction: Cold vapor generation using Hg 200 4 2 1.0015
Sn(II) reduction. * This is a crude comparison & does not include fractionation due to amplifying processes
such as iterative distillation, chromatography or high temperature processes.
Broader research question • Mass Bias correction: Thallium (NIST 997) added to
Hg(0) using desolvating nebulizer.
Can Hg isotope ratios serve as a tool to Future research
differentiate between different types of • Precision: Typical internal precision < ±0.01‰ (2 SE)
sources and transformation pathways? • Fractionation factor:
To use Hg stable isotopes ratios as a successful bio-
To answer this question the scheme 202/198 = [(202Hg/198Hg)reactor/(202Hg/198Hg)trap] geochemical tool we need to:
depicted in Fig. 2 is followed.
• Rayleigh Equation: • Address additional transformations (methylation,
Objectives of this study R(Reactor at t = i)/R(Reactor at t = 0) = f (1/ -1) (1) de-methylation and long range transport) in the Hg
cycle including abiotic processes (Stages 1-2; Fig. 2).
RTrap/R(Reactor at t = 0) = (1/) f (1/ -1) (2)
1. Does reduction of Hg(II) to Hg(0) by pure • Improve instrumental sensitivity to get precise
cultures of Hg resistant bacteria (Fig. 3) isotopic composition of natural samples with sub-ppb
cause fractionation? (Stage proteins coded
Fig. 3 Simplified schematic of bacterial 1 in Fig. 2) by Hg concentrations.
mer (Hg resistance) operon in a bacterial cell • Mass dependence: Multiple Hg isotope ratio
Fig. 3 Hg resistance (mer) operon
Do these proteins preferentially reduce lighter isotopes of Hg(II) compared to heavier ones? (200Hg/198Hg, 204Hg/202Hg) measurement
0.15 1. Smith C. et al. (2005), Geology 33(10), 825-828
Hg(II) Results 2. Jackson T. A. (2004), Env. Sci. & Tech. 38(10) 2813-2821
200Hg 23.10 } Hg transport proteins 3. Hintelman and Lu (2003), Analyst 128, 635-638
Fig. 6 The isotope data plotted as δ202/198Hg vs. f 4. Johnson C. M. et al. (Ed.) (2004), Geochemistry of non-traditional
isotopes. Reviews in Mineralogy & Geochemsitry 55
204Hg 6.87 E. coli JM109/pPB117 at 370C [A] and 300C [B]
5. Barkay T. et al. (2003), FEMS Microbiol. Rev. 27, 355-384
Outer cell 4 3 6. Lauretta et al. (2001), Geochim. Cosmochim. Acta 65, 2807-18
membrane Inner cell
membrane Any isotope
A 2 B 7. Barkay T. (1987), Appl. & Env. Microbiol. 53(12), 2725-32
202/198 Hg (‰)
changes? 1 1
0 0 Acknowledgements
2. What is the effect of changing incubation Reactor -2 Funding was provided by the NSF and NJWRRI. We thank Drs.
Trap Trap Bjoërn Klaue, John Reinfelder, Paul Falkowski & Ariel Anbar for their
temperature on fractionation? (Stage 2) -4 -3 helpful inputs at different stages of this project and Matt Meredith for
1 0.8 0.6 0.4 0.2
1 0.8 0.6 0.4 0.2 0 help with performing experiments at Rutgers.
3. Do naturally occurring microbes fractionate Fraction of added Hg(II) remaining (f) Fraction of added Hg(II) remaining (f)
Hg when reducing Hg(II)? (Stage 2)