mercury stable isotope fractionation during microbial reduction of hg(ii) to hg(0) kritee 1, m....

$: Mercury stable isotope fractionation during microbial reduction of Hg(II) to Hg(0) Kritee 1, M. Johnson 2, B. Bergquist 2, J. D. Blum 2, and T. Barkay$
Hg(II)

Hg0

Outer cell membrane Inner cell

membrane

Isotope Abundance196Hg 0.15 198Hg 9.97 199Hg 16.87 200Hg 23.10 201Hg 13.18 202Hg 29.86204Hg 6.87

} }Hg transport proteins

Hg(II) reductase

Fig. 3 Simplified schematic of bacterial proteins coded by mer (Hg resistance) operon in a bacterial cell

Do these proteins preferentially reduce lighter isotopes of Hg(II) compared to heavier ones?

Any isotope abundance changes?

Hg(II)

Hg0


membrane



Hg(II) reductase

}Hg transport proteins

Hg(II) reductase




Fig. 3 Hg resistance (mer) operon

Hg(II)

Hg0


membrane



Hg(II) reductase




Hg(II)

Hg0


membrane



Hg(II) reductase

}Hg transport proteins

Hg(II) reductase




Fig. 3 Hg resistance (mer) operon

Mercury stable isotope fractionation during 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

Email: [email protected]

1. Smith C. et al. (2005), Geology 33(10), 825-828

2. Jackson T. A. (2004), Env. Sci. & Tech. 38(10) 2813-2821

3. Hintelman and Lu (2003), Analyst 128, 635-638

4. Johnson C. M. et al. (Ed.) (2004), Geochemistry of non-traditional isotopes. Reviews in Mineralogy & Geochemsitry 55

5. Barkay T. et al. (2003), FEMS Microbiol. Rev. 27, 355-384

6. Lauretta et al. (2001), Geochim. Cosmochim. Acta 65, 2807-18

7. Barkay T. (1987), Appl. & Env. Microbiol. 53(12), 2725-32

Biogeochemical concern

• There are multiple sources (natural vs. anthropogenic, local vs. global) and transformations (microbial vs. abiotic) that can lead to buildup of methylmercury (Fig. 1).

• In order to design/implement effective remediation strategies, we need tools to track the actual causes of Hg accumulation in a given ecosystem.

Fig. 1 The Mercury Biogeochemical Cycle

Research Objectives

Broader research question

Can Hg isotope ratios serve as a tool to differentiate between different types of sources and transformation pathways?

To answer this question the scheme depicted in Fig. 2 is followed.

Objectives of this study

1. Does reduction of Hg(II) to Hg(0) by pure cultures of Hg resistant bacteria (Fig. 3) cause fractionation? (Stage 1 in Fig. 2)

Stable isotopes fractionation

Ratio of abundance of a heavier to a lighter stable isotope as compared to a standard - reported as delta () per mil (see methods).

• Measurement4 of isotope ratios of many elements (1H to 96Mo) has helped us determine:

Sources of pollutants or nutrients

Dominant pathways transforming the element

Paleo-environmental conditions (T, pH etc.)

• Living organisms preferentially uptake lighter isotopes leaving heavier isotopes in the environment.

Why does Life like lighter isotopes? It takes less energy to uptake/process lighter molecules.

• Hg has seven stable isotopes (Fig. 2). Significant Hg isotope ratio variations in natural samples from ores, sediment cores, and fish tissues have been reported1-3, but the processes leading to the fractionation have not yet been explored.

Funding was provided by the NSF and NJWRRI. We thank Drs. Bjoërn Klaue, John Reinfelder, Paul Falkowski & Ariel Anbar for their helpful inputs at different stages of this project and Matt Meredith for help with performing experiments at Rutgers.

Methods

To use Hg stable isotopes ratios as a successful bio-geochemical tool we need to:

• Address additional transformations (methylation, de-methylation and long range transport) in the Hg cycle including abiotic processes (Stages 1-2; Fig. 2).

• Improve instrumental sensitivity to get precise isotopic composition of natural samples with sub-ppb Hg concentrations.

• Mercury resistant bacteria prefer lighter isotopes when reducing Hg(II) to Hg(0) (Fig. 6 and 7).

• The similar values of observed for the experiments done with two bacterial genera (B. cereus; preliminary results not shown) and a natural community (Table 1) suggest that the isotopic signature produced during biological Hg(II) reduction is unique.

• Hg isotopes have the potential for distinguishing between different pathways leading to Hg(0) production based on the extent of fractionation and could help Hg remediation efforts.

• Hg is the heaviest element4 for which mass dependent biological fractionation has been documented to date (Table 2).

• The extent of fractionation observed per atomic mass unit (amu) is very high and is comparable to fractionation by much lighter elements (Table 2).

Collect natural water sample from an uncontaminated source

Analyze Hg(0) in traps and Hg(I I ) remaining in the reactor by MC-ICPMS

In 4 days: Enrichment of Hg resistant bacteria

Harvest cells. Re-suspend in filter sterilized source water

Add 250 ppb NIST 3133

Pre-expose sample to 250 ppb Hg(I I )

Collect natural water sample from an uncontaminated source

Analyze Hg(0) in traps and Hg(I I ) remaining in the reactor by MC-ICPMS

In 4 days: Enrichment of Hg resistant bacteria

Harvest cells. Re-suspend in filter sterilized source water

Add 250 ppb NIST 3133

Pre-expose sample to 250 ppb Hg(I I )

Fig. 5 Measuring fractionation during Hg reduction by naturally occurring microbes

Fig. 4 Schematic of experimental set up

Air pump

Hg(II)NIST 3133

Hg0

Trap1: 0-40 min 0.05M KMnO4+ 5% H2SO4

Hg resistant E. coli or B. cereus (in M9 based media)

OR Enriched Natural community (in site water)

Hg stable isotope ratios analysis

using MC-ICPMS Air pump

Hg(II)NIST 3133

Hg0






Hg(II)NIST 3133

Hg0






Hg(II)NIST 3133

Hg0






Hg(II)NIST 3133

Hg0






Hg(II)NIST 3133

Hg0





using MC-ICPMS

Results

Multiple collector inductively coupled plasma mass spectrometry (MC-ICPMS)1,6

Introduction

Conclusions

Future research

References

Acknowledgements

• δ202Hg = [(202Hg/198Hg)Sample - 1] * 1000 ‰ (per mil)

(202Hg/198Hg)NIST 3133 Standard

• Sample introduction: Cold vapor generation using Sn(II) reduction.

• Mass Bias correction: Thallium (NIST 997) added to Hg(0) using desolvating nebulizer.

• Precision: Typical internal precision < ±0.01‰ (2 SE)

• Fractionation factor:

202/198 = [(202Hg/198Hg)reactor/(202Hg/198Hg)trap]

• Rayleigh Equation:

R(Reactor at t = i)/R(Reactor at t = 0) = f (1/ -1) (1)

RTrap/R(Reactor at t = 0) = (1/) f (1/ -1) (2)

• Mass dependence: Multiple Hg isotope ratio (200Hg/198Hg, 204Hg/202Hg) measurement

2. What is the effect of changing incubation temperature on fractionation? (Stage 2)

3. Do naturally occurring microbes fractionate Hg when reducing Hg(II)? (Stage 2)

A1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

0 1 2 3 4

Time (Days)

CF

U/m

l

Total Hg resistant

B

-3

-2

-1

0

1

2

3

0.20.40.60.81

Fraction of added Hg(II) remaining (f)

20

2/19

8 Hg

(‰

)

Reactor Trap

A

-4

-3

-2

-1

0

1

2

3

4

00.20.40.60.81Fraction of added Hg(II) remaining (f)

202

/198

Hg

(‰

)

ReactorTrap

B. Isotopic composition of the Hg(II) remaining in the reactor and Hg(0) produced by enriched microbes.

Fig. 7 Fractionation of Hg isotopes by Hg(II) resistant microbes from a natural source

A. Enrichment of Hg(II) resistant microbes in a natural water sample (Increase in % of Hg(II) resistant colony forming units per ml (CFU/ml) with time7).

Fig. 6 The isotope data plotted as δ202/198Hg vs. f

E. coli JM109/pPB117 at 370C [A] and 300C [B]

Stage 2

Fig 2. Development of a stable isotope ratio based tool.

Stage 3

Stage 1

Measure isotope ratios of an element in a natural ecosystem

• Identify dominant sources & pathways • Date evolution of element’s microbial transformation• Determine environmental conditions at the time of deposition

• Effect of change of environmental conditions (T, pH, redox) on individual transformations• Fractionation by natural microbial community• Kinetic vs. Equilibrium change

• Quantify fractionation during transformations by pure cultures of microbes and abiotic processes. • Determine isotope ratios for representative sources.

B -3

-2

-1

0

1

2

0.60.70.80.91

Fraction of added Hg(II) remaining (f)

202

/198 H

g (

‰)

Reactor Trap

Summary of 202/198 values obtained from linear regression of isotope data

Conditions Based on reactor (Eq. 1) Based on trap (Eq. 2)

Pure Culture of E. coli JM109/pPB117

Temp. Reactor size Slope SE n* R2Slope SE Intercept SE n* R2

370C 1 L 1.0017 0.0002 5 0.969 1.0020 0.0000 1.0020 0.0000 5 0.9991 L 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 1 L 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

Hg resistant 1 L 1.0016 0.0002 4 0.945Control 1 L 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 for some experiments # Not applicable; One of the two data points available corresponds to f = 0.08

Table 1

Table 2

Comparison of the extent of fractionation observed for Hg with other redox-sensitive elements* 4.

4

8

10

7

% mass spread

1.0021.796Mo

Maximum reported /amu

Maximum Range of (‰ /amu)

Avg. Mol. Weight

1.00152200Hg

1.003380Se

1.0015256Fe

4

8

10

7

% mass spread

1.0021.796Mo

Maximum reported /amu

Maximum Range of (‰ /amu)

Avg. Mol. Weight

1.00152200Hg

1.003380Se

1.0015256Fe

* This is a crude comparison & does not include fractionation due to amplifying processes such as iterative distillation, chromatography or high temperature processes.

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mercury stable isotope fractionation during microbial reduction of hg(ii) to hg(0) kritee 1, m....

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