Clarkson University
Diversity and Abundance of Bacterial Species in Wetland Soil Samples with
Varying Concentrations of Mercury Contamination
A Thesis Proposal by
Lindsay R. Hoffman
Department of Civil and Environmental Engineering
March 2006
Advisor
Date
Abstract
Mercury is a pollutant that causes considerable harm to humans, other organisms, and the
environment. Industrial emissions, combustion of coal, eruption of volcanoes, and forest
fires have all contributed to atmospheric concentration and transport of mercury. When it
precipitates out of the atmosphere, mercury contaminates terrestrial and aquatic systems,
especially in the Northeastern United States. In methylmercuric form, mercury
bioaccumulates in various fish species and then biomagnifies through the food chain,
causing significant deleterious heath effects in humans. Many lakes in the Adirondacks
have been so polluted by mercury that people are directed not to eat fish caught in the
area because the levels of methylmercury the fish contain are high enough to be
dangerous.
Via the methylation process, bacteria convert the mercuric ion to methylmercury, which
affects biota in these ecosystems. However, it is unknown how the interactions of
mercury with element cycling and variations in redox (reduction-oxidation) conditions
might influence these microbial processes. Based on the literature search I conducted,
prior to this investigation there has not been a comprehensive review of the factors that
influence these microbial processes. I hope to determine how variations in redox
conditions influence the bioavailability of mercury in Huntington Forest in the
Adirondacks.
Chapter 1: Introduction
It is widely accepted that mercury is a pollutant of major importance; mercury has been
linked to a variety of detrimental effects on both the environment and all groups of
organisms, causing human illness and environmental damage (1). Mercury has also been
found to alter microbial communities in soil samples, as large concentrations of mercury
(>500 μg/g dw soil) reduce both microbial diversity and population size (2).
Mercury is a heavy metal that enters the environment from various sources, typically
from human activities like industrial applications and the disposal of waste products (2)
or from natural sources like mineral deposits, rock weathering, volcanoes, ocean
emissions, and forest fires (3). Mercury has caused significant pollution problems in the
Adirondacks. Power generation byproducts contribute approximately 37% of man-made
mercury to the environment; in fact, atmospheric deposition of mercury accounts for
more than 50% of its input to many waterbodies in the eastern United States, such as the
Chesapeake Bay and Lake Michigan (4). Mercury emitted from factories across the
country can be transported through the atmosphere over hundreds or thousands of miles
in gaseous, elemental, and particulate forms (4).
The biogeochemistry of mercury is significant because of the deleterious effects it can
have on the environment and for the significant role that microbes play in mercurial
cycling. After mercury has entered the environment, its fate and behavior depend on its
chemical state. Mercury is characteristically found in two chemical forms: elemental
mercury and methylmercury. Elemental mercury is found in the atmosphere and is
oxidized to the mercuric ion form, which absorbs easily to particulate matter and can be
metabolized by microorganisms (2, 5). Microbial activity yields methylmercury, a
particularly toxic form of mercury. This methylation has been shown to occur only under
sulfate-reducing conditions; however, it is unknown what organisms are actually
responsible for these transformations.
Methylmercury is the most dangerous type of mercury for humans and other living
organisms; it is 100 times more toxic than both elemental mercury and the mercuric ion
(5). Methylmercury accumulates in the liver, kidney, brain, and muscle of most benthic
and predatory pelagic species (3). See Appendix A for figures on mercury cycling and
bioaccumulation. In the Adirondacks, women of childbearing age and children younger
than 15 are advised to limit or cease consumption of certain species of fish from regional
waters because of methylmercury bioaccumulation problems (6). When humans eat
contaminated fish, they can absorb methylmercury concentrations that: damage the DNA
of blood cells; cause liver, kidney, or neurological damage; or are potentially toxic to a
fetus in a pregnant woman (3, 5). Another form of mercury, elemental mercury, can have
negative toxicological effects on the nervous system such as: tremors; memory loss;
emotional instability problems like irritability, confidence loss, nervousness, or shyness;
or headaches (7).
The goal of my research is to determine the effect of mercury and redox conditions on the
microbial diversity in wetland samples. The results should reveal the effects of mercury
and redox conditions on microbial diversity. My research should assist in the
identification of the microorganisms that are responsible for the transformations mercury
undergoes.
Chapter 2: Background
The National Science Foundation (NSF) has funded a project entitled “Atmospheric
Deposition, Transport, Transformations, and Bioavailability of Mercury across a
Northern Forest Landscape.” This project encompasses 4 years of study by researchers at
Clarkson University, Syracuse University, Rutgers University, and the University of
Massachusetts. I am part of this larger research team, working under Dr. Stefan
Grimberg of Clarkson University and Dr. Tamar Barkay of Rutgers University.
I will analyze various wetland soil samples from Sunday Lake in the Adirondacks, which
have already been collected. After separating DNA from these samples using a
commercial DNA extraction kit, I will use PCR and DGGE methods to perform an
analysis of the abundance and diversity of microbial species (8). More information about
these methods can be found in Chapter 3. After analyzing DGGE results, I expect to find
a decrease in the abundance of populations of microbial species as the level of mercury
contamination increases. The populations of remaining organisms should be resistant to
the presence of mercury. I will attempt to determine whether the species present contain
mercury detoxification (mer) genes or respond to redox (reduction-oxidation) conditions.
Scientists are unsure how methylmercury production is influenced by redox-sensitive
microbial transformations (1), and a goal of my research will be to ascertain how
methylation is affected by redox conditions.
The effects of bacteria on mercury cycling are considerable. Microbes are responsible for
transforming the mercuric ion into methylmercury, which can have harmful
consequences for humans and other species via bioaccumulation (9). As discussed in
Chapter 1, mercury is released into the air from a variety of sources: industrial
applications like coal combustion or natural processes like volcano eruptions (3).
Mercury enters the atmosphere primarily in its elemental gaseous state and is transported
around the world within the atmosphere (4). Elemental mercury is oxidized to the
mercuric ion photochemically; most of the mercury that ultimately enters aquatic
environments is the mercuric ion because it binds to particulate matter, which is removed
from the air by wet and dry precipitation or is metabolized by bacteria (5). Bacteria
methylate mercury to form methylmercury. Methylation occurs via the donation of
methyl groups from methyl-B12, forming methylmercury, which begins to enter the food
chain and bioaccumulate (10, 5). Bacteria continue the methylation process under certain
levels of mercury contamination or pH levels, forming the volatile compound
dimethylmercury (10).
Mercury detoxification (mer) genes are commonly present in the DNA of bacteria located
in mercury-contaminated soils. It has been noted that mer genes are the consequence of
mercury stress and are associated with increased rates of mercury speciation (12). Mer
genes are found in bacteria that reduce the mercuric ion (11). One mer gene of
significance is merA, which produces the enzyme mercuric reductase. Bacteria with the
merA gene transfer two electrons to the mercuric ion, reducing it to elemental mercury,
which is volatile but is generally nontoxic to organisms (5). Mer genes are significant to
mercury cycling because the bacteria that possess them prevent methylation of the
mercuric ion to methylmercury, instead producing less-harmful elemental mercury.
Previous studies of the effects of mercury on soil bacterial communities have determined
that the number of species present is significantly less in soil that is heavily contaminated
with mercury compared to relatively untainted soil. A study by A. K. Müller from the
University of Copenhagen found that the number of DGGE bands was highest in soil
only slightly contaminated with mercury and lowest in greatly contaminated soil.
Culturable bacteria in mercury-contaminated soil possess a high tolerance for mercury
(2), which is what I expect to find in my research. A survey of bacteria in the Idrija
River, a system contaminated with mercury from a mining operation, found mer genes in
locations in the river stressed by mercury contamination (12). Since the samples I will
examine are from an area in the Adirondacks that is fairly heavily contaminated, I hope to
find the presence of merA genes.
Based on my literature search, it is unknown how mercury interacts with other element
cycles and under various redox conditions, making it difficult to predict the fate and
transport of mercury during cycling. My research should provide more information
regarding the transport and bioavailability of mercury in aquatic systems in the
Adirondacks.
Chapter 3: Methodology
Wetland soil samples were collected at the Integrated Forest Study (IFS) plot in
Huntington Forest, located in Newcomb, NY. Three pits were excavated at the IFS plot,
and five replicate soil samples were collected at each pit. Samples were then stored at
4°C with minimal light exposure.
My work will be composed of 4 basic tasks: separation of DNA from the soil samples,
Polymerase Chain Reaction (PCR) to amplify DNA sequences, denaturing gradient gel
electrophoresis (DGGE), and analysis of the results of these methods.
Task 1: Separation of DNA
In order to identify the species of bacteria present in the soil, I will need to separate DNA
molecules from the soils, metals, humic substances, and other components of the sample.
After using a commercially available DNA extraction kit, I will have a sample that
contains a mixture of genomic DNA from all microorganisms that are present in the soil.
In order to identify the microbial species, PCR and DGGE will need to be performed
next.
Task 2: PCR
Polymerase chain reaction (PCR) is a technique I will use to amplify the DNA molecules
found in the soil, even if only very small quantities of DNA are present. PCR does not
require an organism to be grown in the lab, making it possible to reveal the diversity of
species present in the sample (8). During PCR, any DNA present in the sample is copied,
so each cycle effectively doubles the amount of DNA present in the sample (5). PCR
utilizes specifically designed primers to isolate target DNA and the enzyme DNA
polymerase to copy the DNA molecules, doubling the content of each original target
strand of DNA (5). Following this procedure, DGGE will then be used to separate the
PCR products and identify the species present.
Task 3: DGGE
Denaturing gradient gel electrophoresis (DGGE) is a method that separates DNA
fragments of similar lengths based on differences in sequence composition (5). PCR
generates bands that appear to be purely the same DNA fragments, but they are
sometimes merely highly related and not actually identical genes. DGGE separates the
PCR products by differentiating between genes that appear similar and are the same size
because these similar genes vary in denaturing because of their sequences are not
identical (5). Separation occurs as each PCR product denatures because electrophoretic
mobility changes; as DNA fragments move through the increasing gradient of
denaturants, they stop migrating through the gradient gel, theoretically leaving distinct
bands for each species (8). The bands can be detected from digital images of the gel and
the species quantified by using an image analysis software program like Bio-Rad or
Quantity One 4.0.1 (2). The bands can then be analyzed to determine what microbial
species are present in the sample.
Task 4: Analysis
After I complete the previous procedures, I will examine the microbial diversity and
abundance of each species. I will compare the results for various contamination levels
with that of the control group, which is a sample from the area with an extremely low
concentration of mercury. I expect to find changes in the diversity and abundance of the
bacterial species present; there should be a decreased number of species overall, with a
smaller size of the remaining populations (2). I also believe that the species present in
the highly contaminated soil will most likely be resistant to mercury and will probably
contain merA genes, which are found in bacteria stressed by high concentrations of
mercury.
Chapter 4: Future Work
I plan to visit Rutgers University over Spring Break (March 13th – 17th) to work with Dr.
Barkay and her graduate students. They have developed methods of PCR and DGGE
analysis that are similar to the methods I will use. I hope to become accustomed to the
procedures enough to be able to repeat the experiments myself while I am at Clarkson
University over the summer.
After I return from Rutgers, I will spend the remainder of the semester adjusting the
methods used at Rutgers to fit the experiments I need to conduct. I may need to use
different chemicals or make other modifications I cannot foresee at this time. Over the
summer, I will use Dr. Woodworth’s laboratory to conduct the experiments discussed in
Chapter 3.
Chapter 5: Timeline
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January - March 2006: finish proposal, outline procedure
March 13-17, 2006: visit Rutgers to learn extraction, PCR, and DGGE methods
March – May 2006: gather necessary supplies and equipment, identify the
procedure I will use, possibly begin separating DNA from soil samples
May - July 2006: perform analysis of soil samples, identify microbes present in
soil, begin preliminary analysis of diversity of communities compared to mercury
contamination as well as diversity vs. mer expression and diversity vs. redox
conditions
Fall 2006: continue analysis and begin writing thesis
Spring 2007: finish thesis and submit first draft, make any necessary revisions to
thesis, give thesis presentation, and if results and time permit, write manuscript
for publication
References
1. Project Description of NSF Grant Proposal: Atmospheric Deposition, Transport,
Transformations, and Bioavailability of Mercury across a Northern Forest
Landscape.
2. Müller, A.K. et all. The Effect of Long-term Mercury Pollution on the Soil
Microbial Community. FEMS Microbiology Ecology 36 (2001) 11-19.
3. Renzoni, A. et al. Mercury Levels along the Food Chain and Risk for Exposed
Populations. Env. Research Section A 77 (1998) 68-72.
4. Overview of the Human Health and Environmental Effects of Power Generation:
Focus on Sulfur Dioxide (SO2), Nitrogen Oxides (NOX) and Mercury.
http://www.epa.gov/air/clearskies/pdfs/overview.pdf. 2002.
5. Madigan, M.T. et al. Brock Biology of Microorganisms. 10th edition. Prentice
Hall: Upper Saddle River, NJ:, 2003.
6. 2005-2006 Health Advisories: Chemicals in Sportfish and Game.
http://www.health.state.ny.us/nysdoh/fish/fish.htm. 2005.
7. U.S. EPA, 1997. Mercury Study Report to Congress, Volume V: Health Effects of
Mercury and Mercury Compounds. EPA-452/ R-97-004. Washington, DC: U.S.
Environmental Protection Agency.
8. Hurst, C.J. et al. Manual of Environmental Microbiology. ASM Press:
Washington, D.C., 1997.
9. Mercury Interactions: The Effect of Redox Conditions on Mercury Partitioning in
Subsurface Systems. PowerPoint presentation by S. Grimberg on April 21, 2005 at
Rutgers University.
10. Schnoor, J.L. Environmental Modeling. Wiley: New York, 1996.
11. Schlesinger, W.H. Biogeochemistry: An Analysis of Global Change. Academic
Press: London, 1997.
12. Hines, M.E. et al. Mercury Biogeochemistry in the Idrija River, Slovenia, from
above the Mine into the Gulf of Trieste. Env. Research Section A 83 (2000) 129139.
Appendix A: Mercury Cycling and Bioaccumulation
Taken from http://www.epa.gov/mercury/exposure.htm.
Figure 1. Mercury cycling from emission to human dose-response.
Taken from http://www.ec.gc.ca/MERCURY/EN/bf.cfm.
Figure 2. Bioaccumulation of Methylmercury in Organisms.