Mercury stable isotope fractionation during microbial reduction of

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					                                              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
                                                                                                                                              Email: kritee@eden.rutgers.edu
                                                                                                                   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
                                                                                                                                                                                                                                                      1.E+05                                                                         2
                                                                                                                                                                                                                                                                               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.




                                                                                                                                                                                                                                             CFU/ml
                                                                                                                                                                                                                                                      1.E+03                                                                         0
                                                                                                                                        • Determine isotope ratios for representative sources.
• There are multiple sources (natural vs.
anthropogenic,      local  vs.     global)   and                                                                                                                                                                Stage 1
                                                                                                                                                                                                                                                      1.E+02                                                                         -1

transformations (microbial vs. abiotic) that can                                                                                                                                                                                                      1.E+01                                                                         -2
                                                                                                                                                                                                                                                                                                                                                                                        B
                                                                                                                                                                                                                                                                                                             A
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
                                                                                                                                                                                                                                                                                                                Table 1
                                                                                                                                    • 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)


                                                                                                                                                                                                                      Stage 3
                                                                                                                                                                                                                                                           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


                                                                                                                                                             Methods
                                                                                                                                                                                                                                                                             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
                                                                                                                                                                                                using MC-ICPMS




                                                                                                                                                                                                                                                                                           Conclusions
                                                                                                                                                             Hg0

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


                                                                                                                                                                                                                                                                                                                   Table 2
• 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 
                                                                                                                                                                                                                                                                                                                                               (‰/amu)
                                                                                                                                                                                                                                                                                                                                                                      reported
                                                                                                                                                                                                                                                                                                                                                                       /amu
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
                                                                                                                                                                                                                                                                                                                References
   Isotope    Abundance
   196Hg



                          }
               0.15                                                                                                                                                                                                                                              1. Smith C. et al. (2005), Geology 33(10), 825-828
   198Hg
   199Hg
               9.97
              16.87
                               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
   201Hg
   202Hg
               13.18
                                                                   Hg(II) reductase
                                                                                                                            Fig. 6 The isotope data plotted as δ202/198Hg vs. f                                                                                  4. Johnson C. M. et al. (Ed.) (2004), Geochemistry of non-traditional
               29.86
                                                                                                                                                                                                                                                                    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
                                                                                                                   3
                                                                                                                             A                                                        2       B                                                                  7. Barkay T. (1987), Appl. & Env. Microbiol. 53(12), 2725-32
                                                                     Hg0                                           2
                                                                            abundance
                                                                                                 202/198 Hg (‰)




                                                                                                                                                                            Hg (‰)




                                                                            changes?                               1                                                                  1
                                                                                                                   0                                                                  0                                                                                                            Acknowledgements
                                                                                                                                                                           202/198




                                                                                                                   -1                                                                -1
                                                                                                                   -2                                                                                                           Reactor
  2. What is the effect of changing incubation                                                                                                            Reactor                    -2                                                                             Funding was provided by the NSF and NJWRRI. We thank Drs.
                                                                                                                                                                           




                                                                                                                   -3
                                                                                                                                                          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)