Mercury
in contaminated subsurface soils may be leached to the saturated
zone where its reduction to the elemental form, Hg(0), may enhance
its mobilization. Therefore, microbial transformations that reduce
the mercuric ion, Hg(II), to Hg(0) are of key importance to the remediation
of mercury in contaminated subsurface sediments. Microbes may
reduce Hg(II) by the activity of the inducible mercuric reductase (MR),
the gene product of merA, and this process is well understood
in a broad range of aerobic bacteria. Recently described processes
suggest that Hg(II) may be reduced by electrons that are produced during
respiratory activities of sensitive microbes and that these activities
may be of significant in environments with low levels of mercury contamination. To
date little is known about the reduction of Hg(II) by any mechanism
under anoxic conditions. Thus, we propose that:
In saturated sediments with high levels of mercury contamination
Hg(II) resistant bacteria can potentially mobilize mercury by the
activity of MR, whereas in less contaminated aquifers, sensitive
microbes with the potential to reduce Hg(II) co-metablically dominates. To
test this hypothesis we will compare mer operon abundance
and diversity in contaminated and clean subsurface sediments and
in anaerobic enrichments with various electron donors and acceptors.
Novel mer genes and operons are
found among anaerobic microbes. To test this hypothesis
we will examine the molecular characteristics of merA genes
in environmental DNA extracts, of mer operons and their
genomic location in cosmid libraries obtained from sediments and
enrichment culture DNA, and of mer operons and their activities
in Hg(II) resistant anaerobic isolates.
Metal and Hg(II) reducing Hg(II) sensitive anaerobes are more
abundant in background as compared to contaminated sediments, and
more significantly contribute to Hg(II) reduction in enrichments
at low as compared to high Hg(II) concentrations. To
test this hypothesis we will enumerate Hg(II) sensitive/reducing
anaerobes in sediments and enrichment cultures and follow production
of Hg(0) in enrichments with varied Hg(II) levels.
The results of these studies will be the first comprehensive evaluation
of microbial Hg(II) reduction in anaerobic environments, and of the presence,
molecular organization, and activities specified by the mer operon
among anaerobic bacteria. In addition, a study on the ecology of
microbes that reduce Hg(II) by an alternative mechanism will be initiated. The
results will contribute to the body of knowledge that support remedial
action aimed at the immobilization of mercury in subsurface sediments.
The proposed research addresses the need to understand how natural gene transfer
could be used to help naturally-occurring microbial communities adopt resistance
to specific environmental stresses such as heavy metals that inhibit their ability
to reduce and immobilize metals and radionuclides. Nickel will be used as a model
system to demonstrate how a metal resistance marker can be introduced into both
single species and naturally-occurring microbial communities in contaminated
sediments collected from the Oak Ridge Reservation Field Research Center (ORR
FRC). The overall objective of this work is to demonstrate the feasibility of
applying natural gene transfer to improve the performance of natural microbial
communities under conditions imposed by metal stress, using Ni toxicity and resistance
as a model system. We will determine if natural gene transfer of a broad host
nickel resistance marker will help naturally-occurring microbial communities
adapt to nickel toxicity imposed stress. The specific objectives of this work
are to (1) identify individual species that accept the nickel resistance operon
(nre), (2) evaluate how the introduced nickel resistance marker affects
community composition and structure, (3) demonstrate applicability of natural
gene transfer to improve community function under increased levels of toxic metal
stress, and (4) demonstrate ability to enhance uranium immobilization in ORR
FRC sediments by indigenous microorganisms that have adopted the nickel resistance
marker through natural gene transfer. We anticipate that this work will lead
to improved microbial community performance in the presence of nickel as measured
by the ability of the community to reduce and immobilize uranium, and in general,
this research will improve our understanding of how natural gene transfer can
be incorporated into bioaugmentation strategies to immobilize metals and radionuclides.