The Effects of Varying Redox Conditions on Bacterial
Communities in Mercury-Contaminated Soil
Lindsay Hoffman1 and Dr. Stefan Grimberg2
Mercury is a pollutant that causes considerable harm to humans, other organisms, and the
environment. Mercury is released into the atmosphere via anthropogenic sources like industrial
emissions, combustion of coal, and mining as well as from natural sources such as the eruption of
volcanoes and forest fires. Mercury is a global pollutant because it is transported worldwide through the
atmosphere. Mercury contaminates terrestrial and aquatic systems when it precipitates out of the
atmosphere; this pollution is particularly significant in the Adirondack Mountains, which have no local
sources of contamination and yet are highly polluted. Many lakes in the Adirondacks have been so
polluted by mercury that people are directed by the government not to eat fish caught in these lakes
because the levels of methylmercury in these fish contain could be toxic.
Mercury is emitted into the atmosphere primarily in its elemental gaseous form, which is then
transported worldwide and ultimately oxidized photochemically into its ionic form. Ionic mercury enters
terrestrial and aquatic systems as it is removed from the air by means of wet or dry deposition. Ionic
mercury is transformed into methylmercury by microbial processes; bacteria bind a methyl group donated
from methyl-B12 to ionic mercury, forming methylmercury. In methylmercuric form, mercury
bioaccumulates in the muscle and other tissue of animal species and then biomagnifies through the food
chain. Methylmercury is toxic to humans because it is able to be absorbed through the skin and causes
liver, kidney, and neurological damage.
Not much is known about the conditions bacteria are able to methylate successfully under or how
variations in redox (reduction-oxidation) conditions might influence methylation. It has previously been
determined that bacterial methylation occurs under sulfate-reducing conditions, but it is uncertain whether
other redox conditions may allow for methylation. Sulfate-reducing bacteria have been identified as
methylators, but it is uncertain whether other types of bacteria may methylate as well.
In order to assess the bacterial community structure for various redox conditions, four different
types of soil were analyzed: virgin, aerobic, sulfate-reducing, and iron-reducing. In order to establish
these redox conditions, soil columns were created in the laboratory. Soil samples were taken from two
1
Class of 2007, Department of Civil and Environmental Engineering, Clarkson University, Honors Program and
REU in Environmental Science and Engineering
2
Project Mentor, Department of Civil and Environmental Engineering, Clarkson University
sites in the Adirondacks: Huntington Forest and Sunday Lake. A combination of freeze-dried and fresh
soil was used to fill four columns (aerobic, sulfate-reducing, iron-reducing, and sterile sulfate-reducing).
These columns were then fed continuously with a solution of lake water and the desired redox couples.
The columns were run for approximately two weeks, which was assumed to be sufficient time for the
microbial population to alter due to the induced redox conditions.
A mixture of DNA from the microbes in the soil was removed from each column as well as from
the virgin soil using the commercially available PowerSoil DNA Isolation Kit from Mo Bio Laboratories.
This DNA was then amplified using the Polymerase Chain Reaction (PCR) process, which copies the
DNA and effectively doubles the amount present during each cycle. Figure 1 shows preliminary results
of DNA extraction and PCR amplification of virgin soil (lanes 1-3 and 7-9) from Huntington Forest and
Sunday Lake as well as the aerobic, sulfate-reducing, and sterile sulfate-reducing soil columns (lanes 4-6
and 10-12).
Figure 1. DNA extraction and PCR amplification products of both virgin soil from Huntington Forest and
Sunday Lake soil and soil columns. Lanes: 1. and 7. Huntington Forest upland soil extraction and
amplification, respectively; 2. and 8. Sunday Lake conifer extraction and amplification, respectively; 3.
and 9. Sunday Lake deciduous extraction and amplification, respectively; 4. and 10. Aerobic column
extraction and amplification, respectively; 5. and 11. Sulfate-reducing column extraction and
amplification, respectively; 6. and 12. Sterile sulfate-reducing column extraction and amplification,
respectively; 13. Positive control; 14. DNA molecular weight marker.
Figure 1 shows clear bands in lanes 1-5, which when compared to the DNA molecular weight marker,
indicate the presence of large strands of DNA after the initial extraction. Lane 6, the sterile sulfatereducing column, did not confirm the presence of any DNA, which is ideal because the column was
intended to be devoid of microbes, though the amplification shown in lane 12 indicates that there may
have been some DNA in the product. Lanes 7 and 9 did not show amplified DNA, but it is uncertain why
the extracted DNA did not amplify correctly. The rest of the PCR amplification products (lanes 8, 10,
and 11) showed strong bands weighing much less than the extraction products; this can be explained by
the PCR process, which utilizes primers that target a specific sequence of DNA, the V3 region of 16S
DNA. Figure 1 indicates a successful DNA extraction and, though there might have been an error during
the PCR process, the successful amplification of most of the extraction products.
The PCR products have been separated by species through Denaturing Gradient Gel
Electrophoresis (DGGE), which denatures the DNA and causes it to separate into bands for each species.
These bands will be analyzed by using a computer software package called ONE-Dscan from Scanalytics,
Inc. to evaluate digital images of the DGGE gel, yielding an assessment of the bacterial community size
and structure. DGGE bands will be removed from the gel and sent to a laboratory for sequencing so that
specific bacterial species can be identified.
It is expected that the community structure will vary for each soil type, which should ultimately
lead to fluctuations in mercury methylation rates. A confirmation that sulfate-reducing bacteria are the
primary methylators is expected as well, though it is thought that other types of bacteria may methylate as
well. Concurrent experiments regarding mercury methylation will provide a better understanding of
which bacterial species methylate. Overall, this research will increase the understanding of which
organisms are involved in mercury cycling and under which conditions methylation occurs; this further
understanding could lead to management practices that would ultimately cause a reduction in
methylmercury production.