Mercury stable isotope fractionation during bacterial reduction of HgII to Hg0

1
Mercury stable isotope fractionation during
bacterial reduction of Hg(II) to Hg0
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 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. 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). Plausible
explanation 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. 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).
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. Plausible explanation 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 non-specific mechanisms
like reduction by light or weak organic acids
results in mixed/weaker signal.
Introduction 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.

• Research Questions
• 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?
• If yes, is it mass dependent? Is it kinetic
  fractionation? What is the value of alpha? What
  is the effect of changing temperature?
• Is this fractionation phenomenon limited to pure
  cultures grown under laboratory condition or does
  it occur when naturally occurring bacterial
  consortium reduce Hg(II)?
• Methods
• 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.
• 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.
• MC-ICPMS analysis3
• Sample introduction Cold vapor generation was
  employed using Sn(II) reduction. The cold vapor
  sample introduction has a gt99 efficiency and
  generates a signal of 600 mV/ppb at a sample
  consumption rate of 0.75 mL/min.
• 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
  d202Hg/198Hg (hereafter d202Hg). Typical in-run
  precision of better than 0.05 (2s) and external
  reproducibility of d202 between NIST 3133 and a
  secondary standard was 0.08 (2s).
• The kinetic fractionation factor (?) was
  determined from the results of our experiments
  using the Rayleigh Distillation Equation
• RVi/RLo (1/?) f (1/? -1)

• Conclusions
• Systematic Hg stable isotope fractionation does
  happen, both in pure cultures of bacteria and
  naturally occurring bacterial consortia!
• 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.

Figure 5. 5a. Extent of isotopic fractionation
(in per mil) measured in the trapping solution
vs. exposure 5b. ? factor/amu calculated
assuming kinetic isotope fractionation vs.
exposure 5c. Percentage of colony forming units
(CFU) after 4 days of exposure which are Hg
resistant vs. Hg exposure 5d. Hg0 produced (in
ppt) per Hg(II) resistant colony forming unit
(HgR CFU) vs. Hg exposure.
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 3. Lauretta et al.
(2001) Geochim.Cosmochim. Acta 65, 2807-2818 4.
Barkay T. (1987) Appl. Env. Microbiology
53(12), 2725-2732 5. Anbar(2004) Earth Planet.
Sci. Lett. 217, 223-236 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.