MICROBIAL DETOXIFICATION OF MERCURY-CONTAMINATED
Lisa Jean Snelling
B.S., University of Colorado at Denver, 2004
A thesis submitted to the
University of Colorado at Denver and Health Sciences Center
in partial fulfillment
of the requirements for the degree of
Master of Science
This thesis for the Master of Science
Lisa Jean Snelling
has been approved
Snelling, Lisa Jean (M.S. Biology)
Microbial Detoxification of Mercury-Contaminated Museum Materials
Thesis directed by Associate Professor Timberley M. Roane
Historically, pesticides containing metal compounds, such as mercuric chloride and
arsenic trioxide, were applied to artifacts to prevent pest degradation. These artifacts,
many of which are housed in museum collections across the United States, are
deemed hazardous to health and environment. While use of these pesticides ceased in
the 1970s, since metals are non-degradable and are long-lived these metal-based
pesticides continue to pose toxicity concerns. Under the Native American Graves
Protection and Repatriation Act (NAGPRA), enacted in 1990, cultural museum
objects may be earmarked for return to Native American tribes. Yet, treated museum
artifacts pose health risks to museum personnel and native peoples. Current
remediation strategies, such as complexation using UV light or washing/scrapping of
the artifact are considered too harsh and culturally incompatible, especially given the
sacredness of some tribal beliefs. However, metal-resistant, mercury-reducing
microorganisms have been characterized and identified in a variety of metal-
contaminated and pristine environments and are being investigated in the
bioremediation of contaminated soil and water systems. In the research presented
here, the microorganisms associated with mercury-contaminated museum artifacts
have not been identified or characterized in terms of their mercury-resistance. This
study investigated the bacterial populations found on the surface of mercury-treated
artifacts from the ethnographic collections in the Arizona State Museum (ASM).
Twenty artifact-associated bacterial isolates were identified using 16S rDNA
sequencing. Twelve isolates were able to grow in mercury concentrations ranging
from pg/L to mg/L mercury concentrations. One isolate in particular, Arthrobacter
sp. 2604, was able to grow in up to 50 mg/L mercury. Further examination revealed
that Arthrobacter sp. 2604 was able to reduce the mercury concentrations associated
with 10 mg/L amended tryptic soy broth, tryptic soy agar and paper substrates by
20%, 30%, and 20%, respectively, following a 7 day incubation period at 28C.
When the substrate utilization pattern for Arthrobacter sp. 2604 was examined, the
isolate showed a preference for organic acids as a source of carbon and energy,
indicating a possible relationship to artifact degradation. Another mercury-reducing
isolate, Cupriavidus metallidurans CH34 from a metal-contaminated soil, was able to
remove up to 40%, 50%, and 60% of the mercury from 10 mg/L amended nutrient
broth, nutrient agar and paper, respectively. Results demonstrate the potential use of
mercury-resistant bacteria in the removal of mercury from contaminated museum
This abstract accurately represents the content of the candidates thesis. I recommend
Timbefley M. Roane
I dedicate this thesis work to everyone in my life (you know who you are) that helped
me through my years of growing up, education experiences, and believing in me and
to Aubrey (my daughter) for being such a good sport. I thank you all so very much
for all of your encouragement, support, and love. You all have touched my heart in
some way, shape, or form. All my learning and success has come from all of you and
I will continue to pursue a life long journey in education, research, and teaching.
Thank you all so very much!
Live each day to its fullest, for tomorrow may be your last.
Dearest thanks to all of the friends in the Johansen lab that I hung out with while at
UCDHSC, especially Cory that I worked closely with...teaching microbiology,
drawing on beer coasters (molecular and microbiology mechanisms) at Old
Chicagos, grading tests, lab notebooks, papers, and long night discussions in the
research laboratory about various future research projects. Someday we might be
working together...You are my best-friend! I also would like to thank Dr. Audesirk,
Dr. J., James, and Jeff Boon for all of your support while working on this project. In
addition, I would like to thank Dr. Roane for being such a great advisor, mentor, and
friend through my years here at UCDHSC, especially through my trials and
tribulations. Thank you for showing me the ropes of the University system and
encouraging me to do the best at everything that I set forth it pays off. I would like to
thank the National Park Service for funding this research. Huge thanks to the faculty
for an outstanding job of teaching and encouragement. Last, I would like to thank my
daughter for being supportive, loving, caring, and keeping me on my toes at all times!
You will be a great scientist someday!
CHAPTER 1 LITERATURE REVIEW________________________________________________1
Toxicology of Mercury....................................................6
Mercury found in Museums.................................................9
Pest Management and Museum Remediation..................................15
Microbial Mercury-Resistant Mechanisms..................................18
Rationale and Objectives................................................25
CHAPTER 2 MANUSCRIPT......................................................30
Materials and Methods...................................................34
Mercury-Contaminated Museum Materials................................35
Isolation of Material-Associated Surface Bacteria....................35
Bacterial Maximum Mercury-Resistance Level (MRL).....................37
Growth of Arthrobacter sp. 2604 in the Presence of Mercury...........38
Ability of Mercury-Resistant Bacteria to Remove Mercury from Different
Analytical Mercury Concentration Determination.......................40
Results and Discussion..................................................41
Mercury-Contaminated Museum Collections..............................41
Isolation and Identification of Material-Associated Bacteria.........44
Maximum Mercury-Resistance Level.....................................49
Growth of Arthrobacter sp. 2604 in the Presence of Mercury.......49
Analysis of mercury-Resistant Bacteria to Remove Mercury from Different
CHAPTER 3 FUTURE DIRECTION............................................59
APPENDIX A INEFFECTIVE PROTOCOLS......................................61
Growth Optimization Experiments.....................................62
LIST OF FIGURES
1.1 Bacterial Metal-Resistance Mechanisms.........................................18
1.2 Bacterial Mercury-Reduction Mechanism.........................................20
2.1 Arthrobacter sp. 2604 Growth Curve............................................54
2.2 Mercury Removal by Arthrobacter sp. 2604.....................................55
2.3 Mercury Removal by C. metallidurans CH34.....................................56
LIST OF TABLES
1.1 Types of Chemicals Applied to Museum Artifacts.....................................10
2.1 Isolate Identification and Mercury Maximum Resistance Levels......................44
2.2 Museum Isolate Identification using 16S rDNA Sequencing...........................48
2.3 Substrate Utilization Pattern for Arthrobacter sp. 2604...........................58
Mercury has been used in various industries, including in the production of
batteries, cosmetics, synthetic materials, and pesticides. For example, mercuric
chloride (HgCL) used to be applied as a seed treatment to control fungal growth on
grain crops. Elemental mercury (Hg) is used in some folk medicines, such as
Santeria, an herbal remedy that is sprinkled in homes or cars, burned in candles, or
used with bathing to cure various ailments (www.ATSDR.cdc.gov, 1999). Ethyl-
mercury (CH3CH2Hg+) is used as a fungicide and an antibacterial preservative in
some vaccines (thimersol). Inorganic mercury, consisting of mercuric salts such as
HgCb, is used in skin lightening creams, ointments, and as a reagent in etching
Regardless of its use, mercury is most commonly found in the Earths crust as
poorly soluble cinnabar (HgS) with a solubility of 10'6 g/lOOmL of water, occurring
in background concentrations ranging from 21-56 ppb. Cinnabar can also occur in
much higher levels when associated with concentrated metal ores. While cinnabar is
considered relatively non-toxic, mercury can naturally be found as or transformed
into a variety of chemical states with varying toxicities, such as the elemental
mercury (Hg) released from volcanic activity; the HgS in mineral deposits; and the
organomercury forms associated with anaerobic sediments. Once transformed, there
are a number of factors that affect the toxicity and spread of mercury in the
environment, including temperature, pH, water content, soil type, mineral
composition, and biological activity. Consequently, mercury is a complex element
occurring as metallic or elemental mercury, inorganic mercury (Hg2+, HgCh, HgS),
and organomercury (CH3Hg+, CH3CH2Hg+) species.
An example of the environmental complexity of mercury is seen in the
Amazon Basin. The result of gold mining and deforestation, mercury levels in soils
in the Amazon Basin are increasing. In a study by Dolbec et al. (2001), hair samples
of locals analyzed for mercury levels correlated with season. Lower amounts of
mercury were associated with the diet during the rainy season versus the dry season,
because villagers consume more fish than plant material during the wet season and
vice-versa during the dry season. Scientists determined that local rains solubilized
the mercury in the nutrient-poor soils of the Basin, thereby increasing plant uptake of
the mercury. During the dry season, the mercury was less soluble and therefore less
susceptible to plant absorption. It was also found that the atmospheric deposition of
mercury to the soil was greater during the rainy season and also increased plant
uptake of mercury (Guimaraes et al., 2000; Morel et al., 1998). In a similar study,
Zillioux et al. (1993) found that the introduction of oxygen to wetland sediments,
through physical disturbance such as dredging, increased organic matter degradation
releasing bound mercury, thus mobilizing mercury in the environment and making it
more available to organisms.
Mercury is a silvery liquid with a molecular weight of 200.59 and a density of
13.53 g/cm3, capable of forming three oxidation states Hg2+, Hg1+, and Hg. The
toxicity of mercury has proven useful to medicine and agriculture as a biocide.
Mercurys high surface tension and electrical conductivity has proven useful to
industry as a malleable additive in, for example, gold extraction and metal amalgams.
Today, mercury can be found in thermometers, fluorescent light bulbs, and electrical
devices. It is also associated with fossil fuel plants, chlor-alkali plants, and waste
incineration facilities. Over the decades, the burning of fossil fuels to generate
electricity has increased anthropogenic mercury in the environment. Records from
lake sediment and peat samples show 2-5 orders of magnitude increase in
anthropogenic mercury since the beginning of the industrial period (Boening, 1999).
Fossil fuel burning releases airborne metallic mercury that has a residence time of up
to a year before being oxidized by O3 radicals to Hg2+ (Morel et al., 1998). The Hg2+
can then precipitate out of the atmosphere through wet deposition and adsorption to
soot particles to be deposited in soils and aquatic systems. Once mercury is oxidized,
60% of atmospheric mercury will be re-deposited on land and 40% to water (Morel et
While the mercury re-deposited on soil can undergo a number of chemical
reactions, including adsorption and reduction, the Hg deposited into aquatic
environments is generally reduced by chemical and biological reactions back to
gaseous mercury that re-enters the atmosphere. The remaining Hg2+ that is not
readily reduced can be biologically transformed into methylmercury (CH3Hg+) in
anaerobic sediments by sulfur-reducing bacteria (SRB) (Morel et al., 1998).
Methylmercury is one of the most toxic forms of mercury since it bioaccumulates in
the food chain.
One of the major contributors to mercury pollution is coal burning.
According to the Environmental Protection Agency (EPA), data complied from 1994
indicates that 51 tons of elemental mercury were generated by coal-burning plants,
accounting for 13 to 26 percent of the total airborne emissions of mercury.
Approximately 1 percent (0.9 metric tons/year) of elemental mercury is transformed
to divalent mercury (Hg2+) by troposphere ozone that then becomes adsorbed to
particulates for surface deposition, contributing to the global cycle of mercury. At
approximately 36.5 metric tons/year, divalent mercury accounts for 70% of the total
mercury returned to the Earths surface through wet and dry deposition from the
atmosphere (EPA, 1997).
While coal burning is a major contributor to pollution, chlor-alkali plants are
major users of elemental mercury and thus also contribute significant amounts of
mercury pollution (Boening, 1999). In the chlor-alkali process, liquid elemental
mercury acts as a cathode for the separation of ions. As brine flows through an
electrochemical cell, sodium (Na+) and chlorine (Cf) ions dissociate. The Cl' ions are
oxidized by the anode to chlorine gas, and the Na+ ions are reduced to NaOH and
hydrogen gas at the mercury cathode. The end-products are chlorine gas, which is
used extensively in wastewater treatment plants during the disinfection process,
hydrogen gas, and sodium hydroxide solution. However, in the chlor-alkali process
the elemental mercury is readily volatile and small amounts of mercury gas are
released into the environment. The release of mercury as a contaminant in the chlor-
alkali process is significant enough that the EPA has established a specific guideline
for the process. No more than 5 lbs of elemental mercury per 24 hour period can be
released. This standard also applies to the release of gaseous mercury in the mining
Due to the widespread use of mercury and its prevalence in everyday products
and our environment, we are regularly exposed to mercury. As such, standards for
some types of mercury exposure have been established. Mercury in drinking water
should not exceed 0.002 mg/L or 144 ng/L in lakes, rivers, and streams. Mercury in
freshwater fish and shellfish is not to exceed 1 mg/Kg for interstate commerce;
however, regulatory levels are not set for fish caught in-state and sold in local
markets. Minimal risk levels are set at 0.007 mg/Kg/day (acute) and 0.002
mg/Kg/day (intermediate) for mercury chloride exposure and 0.0002 mg/m3 for
mercury inhalation within air (ATSDR, 2006). Despite our widespread exposure,
however, mercury toxicity is a function of a number of factors.
Toxicology of Mercury
Mercury exposure in humans can be diagnosed by blood, urine and hair
samples, which can be used to identify acute versus chronic exposures. For example,
in the 1970s, epidemiologists used hair samples to identify chronic mercury poisoning
in Iraqi families who consumed mercury-treated grain (Amin-Zaki et al., 1974).
Linked to kidney disease and central nervous system disorders, the toxicology of
mercury is determined by the type of mercury species, concentration (dose), and route
of exposure (ATSDR, 2006). For example, inhalation of elemental mercury can
cause nervous system damage, resulting in tremors, headaches, short-term memory
loss, weakness, and loss of appetite, as well as insomnia and excessive sweating.
However, gaseous elemental mercury is lipid soluble, and can be oxidized to Hg in
red blood cells by the enzyme catalase. Divalent mercury has a strong affinity for
sulfydryl groups, such as in glutathione produced in the kidneys, resulting in mercury
accumulation and kidney damage over time. Historically, there are several examples
of widespread elemental mercury exposure in people. In Hoboken, New Jersey (early
1990s), a group of artists purchased a building that was once owned by the General
Electric Company. The building housed a facility for the production of mercury
vapor lamps. The artists converted the building into 17 condominium units, in which
32 residents, including 6 children (ages 9-months to 8 years), occupied the units when
residents started to notice drops of elemental mercury. Upon examination by the
authorities, the building was found to contain high levels of elemental mercury
throughout the structure ranging from 5 pg/m to 888 pg/m Of the 32 residents, 29
had been tested and 20 (including 5 of the 6 children) had mercury levels of >20 pg/L
and 8 had mercury levels measured in blood, hair or urine of >56 pg/L (Fiedler et al.,
1999; CDC, 2007). On January 3, 1995, the Agency for Toxic Substances and
Disease Registry (ATSDR) condemned the building as an imminent health hazard
and all residents had to relocate.
In Catamarca, Peru, 2000, a truck carrying 300 pounds of elemental mercury
had leaked in villages where the truck driver had stopped. Villagers collected the
mercury to use in rituals. When homes were tested, several exceeded 1 mg/m Hg
exceeding the U.S. regulatory standard of 0.0002 mg/m3 by 5,000 times. Urine
analysis showed some individuals had up to 20 pg/L mercury in their urine.
According to U.S. standards, 5 pg/L of mercury in urine is the minimum risk level.
Exposure to organic mercury, such as methyl mercury (CH3Hg+), can occur
through the consumption of contaminated fish. Methyl mercury is one of the most
toxic forms of mercury due to its ability to bioaccumulate in adipose tissue and
readily cross cell membranes. Mercury biomagnification is the increased
concentration of mercury associated with biological systems higher up in the food
chain. According to the World Health Organization (1989), 50% of an organisms
total exposure to mercury will accumulate in adipose tissue. Symptoms of organic
mercury poisoning include nausea, abdominal pain, weight loss, kidney failure,
tremors and vision and hearing loss leading to ataxia. Pregnant women who consume
excess mercury in fish have a higher incidence of neurological disorders in their
children (World Health Organization, 1989).
The most disastrous example of organic mercury poisoning was in the 1960s
when thousands of people in Minamata, Japan, consumed mercury-contaminated fish
from Minamata Bay. When hundreds of people started becoming ill, it was found
that the fish from the Bay had elevated levels of mercury (> 10 mg/Kg Hg). The
mercury contamination originated from an acetaldehyde factory that had dumped
relatively nontoxic elemental mercury into the Bay. However, bacterial sulfate-
reducing activity in the sediment converted the elemental mercury into the highly
toxic and bioavailable methylmercury, which accumulated in the plankton that was
consumed by fish that were later consumed by people. Infants bom to mothers who
ate the contaminated fish had a higher incidence of profound mental retardation,
cerebral palsy, and blindness. The death toll from the Minamata Bay disaster is still
being determined 50 years after the mercury release (Gochfeld, 2003; and Futatsuka
et al., 2000) and is still the cause of considerable controversy regarding the ecological
and health effects of mercury.
Mercuric salts are associated with a variety of industries including mining,
coal burning, agriculture, and cosmetics. They are so prevalent and toxic that salts,
such as mercuric chloride (HgCL), are considered extremely hazardous substances
under Section 313 of Title III of the Superfund Amendments and Reauthorization Act
(SARA) of 1986 (EPA, 2006). Exposure to mercury salts generally occurs through
ingestion or absorption through the skin, possibly leading to nausea, abdominal pain,
decreased urination or kidney failure, and major neurological disorders.
For example, mercuric nitrate (HgNCb) was used to preserve beaver felt hats
in the 19th century. Pelts were saturated in a mercuric nitrate solution to soften the
coarse hair to felt. As a result their exposure to the mercury, workers in the beaver
factories often experienced neurotic behavior known as mad as a hatter disease.
Another example of using mercury salts to preserve materials was during Lewis and
Clarks expedition. Solutions were made with mercury salts to preserve the materials
they collected, many of which currently reside in museums. As a matter of fact,
many of the early collectors of ethnographic and zoological specimens treated their
materials with mercury (Hawks, 2001). These collections have recently been
identified as hazardous and are generating much concern over the handling and
mitigation of mercury now found in museums.
Mercury in Museums
Some collections housed in museums, including ethnographic, zoological, and
herbarium collections, were treated with fumigants and pesticides during the 19th and
20th centuries. Until the 1970s, collectors and museums used the application of these
chemicals to preserve artifacts from insect, microbial, and rodent damage. Table 1.1
shows a log of when specific chemicals were used.
Table 1.1 Summarizes types of chemicals applied to museum artifacts dating from
800 to 1950 (adapted from Go dberg, 1996).
Years Chemical Preservatives
1800s Inorganic arsenic, inorganic mercury
1850s Arsenic, mercury, tobacco, sulfur, camphor, heat
1900s Arsenic, mercury, strychnine, carbolic acid, naphthalene, wax/solvents, carbon disulfide
1950s Dichlorobenzene, hydrocyanide gas, aluminum silicate, DDT, ethylene dichloride, carbon tetrachloride, ethylene dibromide, dichlorvos, sulfuryl fluoride, freezing
However, in general, artifact preservation was poorly documented, and
information, such as which chemical was applied, in what amount, how often, and the
method of application, is often lacking. According to Goldberg (1996), collectors
would use poisons such as arsenic, corrosive sublimate (mercuric chloride),
camphor, and fumigating tobacco to preserve biological specimens during the
expedition. Records from the Smithsonian Institution, Washington, D.C., indicate
that dried specimens were painted, brushed, and immersed with arsenical or mercuric
compounds (Goldberg, 1996).
Mercuric and arsenic compounds were widely used as preservatives on
anthropological and ethnographic objects, preventing decay and pest damage.
According to records from the Smithsonian Institution, in 1887 Dr. Walter Hough, a
copyist and head curator of the Anthropology Department, designed a preservative
solution using mercury compounds. His recipe included dissolving mercuric salts in
alcohol (50%) with small amounts of naphtha (this prevented the mercury from re-
crystallizing). Other curators also used mercuric salts in alcohol and water for
materials such as feathers, hair, fur, and woolen cloth, as well as skins, wood, and
baskets (Goldberg, 1996). Collection records indicate that objects were either dipped
in or were painted with mercuric solutions. Scattering of mercuric salts were used in
comers of cabinets and on textiles. Due to their stability long after treatment,
mercury and arsenic compounds pose health concerns for museum personnel who
work around these objects. For instance, the National Museum and Galleries of
Wales, UK, has identified that the majority of their herbaceous collection has been
treated with mercury. Six samples from the herbarium examined using X-ray
fluorescence (XRF) had mercury concentrations up to 4905.8 pg/g (Purewal, 2001).
It should also be recognized that not all contaminated objects were directly treated
with mercury, however. Some artifacts associated with elevated levels of mercury
were simply housed with treated materials. As a matter of fact, because of this, many
of the original storage cabinets are also contaminated.
Recently, under the Native American Graves Protection and Repatriation Act
(NAGPRA), enacted in 1990, and amended in 1996, cultural collections in all U.S.
federally funded museums have to be returned to Native peoples upon request.
NAGPRA also requires that all federal agencies and museums inventory all Native
collections. Inventories are to be sent to tribes and posted with the National Park
Service for review. In 1996, NAGPRA was amended to include the legal informing
of tribes of any known treatments of their items that would render the material as a
potential health hazard to the person (Public law 101-601, NAGPRA, section 10.10
(4) e). Under NAGPRA, museum holdings under repatriation include: human
remains (e.g., tissue or bones), funerary objects (e.g., medicine bundles), unassociated
funerary objects (e.g., ceremonial masks), objects of cultural patrimony (e.g., war
gods and spirit representations), and other cultural items (e.g., feathers, pouches, and
headdresses). In addition to those above, other materials with associated chemical risk
include moccasins, war bonnets, baskets, masks, leggings, deerskin dresses, katsinas,
and wood carvings.
Based on a survey of museum conservators with a 45% response rate, analysis
of 77 ethnographic objects found six commonly used chemical agents including
arsenic, naphthalene, PDB (paradichlorobenzene), DDT
(dichlorodiphenyltrichloroethane), mercury compounds, and fumigants such as
methyl bromide and ethyl oxide (Lazar, 2000). According to Nason (2001), museum
collections are contaminated with chemicals of concern listed by the Environmental
Protection Agency (EPA) and the ATSDR. In order of appearance on the 1999
Comprehensive Environmental Response Compensation and Liability Act
(CERCLA) list, these are: (1) arsenic; (2) lead; (3) mercury; (12) DDT; (22)
chlordane; (24) aldrin; and (26) cyanide (Nason, 2001). Dr. Peter Palmer, a professor
of chemistry at San Francisco State University, analyzed 17 objects that had been
repatriated to the Hoopa tribe in California. Of the 17 objects, 7 were identified as
having greater than 1% of mercury associated with the material, and one set of eagle
feathers had a mercury level greater than 16% (Caldararo et al., 2001).
In 1998, the Arizona State Museum (ASM), Tucson, AZ, developed a
NAGPRA check list to address the repatriation of potentially contaminated materials:
1. Is there evidence of prior infestation? E.g., insect or rodent damage
2. Are residual pesticides indicated? E.g., the presence of white powder to
indicate possible mercury treatment
3. Is there evidence of museum repairs, restorations, and alterations?
4. Are there any written records that would suggest the use of pesticides?
5. Based on the past storage locations, what pesticides might typically have
been used on or near this object?
ASM conservators have compiled a list of contaminants commonly associated with
specific types of ethnographic materials, making this an efficient database for
repatriated materials. The database with 91 identified contaminants includes
information regarding chemical name, common name, Chemical Abstract Service
number, status and dates of use, methods of application, characteristics, target pests,
field half-life, and persistence (Odegaard and Sadongei, 2001).
The extent of the problem associated with chemically preserved museum
collections was not initially recognized upon the enactment of NAGPRA. It was not
until the physical repatriation of tribal artifacts that the toxicity associated with some
of these artifacts was recognized. In the early 1990s, the Hopi tribe of the
Southwestern U.S. was the first tribe to undergo repatriation. The Hopi community
initially reclaimed 60 sacred objects to be put back into cultural use. The Hopi
practice rituals that derive from prehistoric Puebloan traditions, such as religious
ceremonies held throughout the year to promote rain from the cloud spirits in hopes
of bountiful crops. All objects of the Hopi are considered sacred. For example, the
Hopi Katsina, which means friends, are considered living by the Hopi and bring
happiness and prosperity. Katsina priests care for the friends. Even before the 1996
amendment to NAGPRA, Hopi advisors, which included elders and Katsina priests,
identified several Hopi objects to be repatriated under NAGPRA. At the time, the
elders were not told of and so were not aware of the potential toxic chemicals
associated with their items. Upon repatriation some of the Hopi objects were placed
in homes and returned to ceremonial practice. In both situations, Hopi tribal
members were exposed to high levels of mercury that lead to physical illness. To this
day, some Hopi feel deceived by not being made aware of the potential health hazards
associated with their objects, and to know that Hopi children may have been exposed
to toxic chemicals is a great insult for the tribe (LomaOmvaya, 2001).
It is now realized that not only are tribal members at increased risk of
exposure to the toxic preservatives but so are the museum personnel who handle the
treated artifacts. Since metals are non-degradable and persist indefinitely in the
environment and are toxic to all living organisms, these museum materials pose long-
term health risks that need to be addressed.
Pest Management and Museum Remediation
Current pest control in museums takes the form of an Integrated Pest
Management (IPM) program that involves a non-destructive approach to preserving
the materials. The harsh pesticides of old, such as mercury and arsenic, are no longer
used. IPM programs involve methods such as freezing, heating, anoxic
environments, and the use of insect pheromones to lure insects away from collections
(Hawks, 2001; Odegaard and Sadongei, 2001; Osorio, 2001). While new chemicals
are no longer being used in museums, the presence of old chemicals still needs to
be addressed. As such, identifying and understanding the chemicals on materials
from past management practices are critical for museum personnel, as well as other
persons coming into contact with contaminated materials. Analytical techniques,
such as hand held X-ray fluorescence (XRF), are used for the detection of chemical
quantities associated with suspect artifacts. Chemical spot tests using kits containing
indicator strips for the detection of particular contaminants such as mercury and
arsenic are also used (Makos, 2001; Palmer, 2001; Sirois, 2001). Atomic Absorption
Spectroscopy (AAS) is a more destructive method for the detection of metals from
materials (since a small amount of material from an object must be removed);
however, this method is more accurate and precise than the spot tests due to lower
detection limits, less interference, and more reliable results (Palmer, 2001). A
common limitation to each of these techniques is that the chemical associated with
the entire object cannot be quantified. This leaves many unanswered questions as to
the extent of contamination and the necessary remediation required.
Current remediation approaches for contaminated museum materials that are
earmarked for repatriation include replacement (which has cultural limitations
depending on tribal beliefs), containment (which is not a solution for repatriated
items), washing/scraping (which can be too harsh for the material), heat and radiation
(which may not be accepted by tribes) (Odegaard, 2001). Object fragility is a large
concern for remediation attempts, as well as cultural sensitivity. For example, some
tribes consider their objects living spirits and as such these objects can not undergo
what are considered harsh remediation treatments, such as heat and UV radiation
exposure. In consequence, other methods are being examined. HEPA-filtered
vacuums were tested for the removal of DDT from a museum storage room at the
Australian National University and have been found effective (Odegaard, 2001).
However, the Danish National Museum in Denmark used compressed air to remove
contaminants from their collections and found that the pesticides remained in the
objects (Glastrup, 2001).
An approach to removing mercury that is considered less destructive and
culturally sensitive (having received approval from several tribes) is the use of
microorganisms. Some microorganisms have been found to detoxify contaminated
environments, such as soils and waters contaminated with various metals and organic
pesticides. These same microorganisms might also be found associated with
mercury-contaminated artifacts found in museums, and therefore could possibly be
used to bioremediate collections.
f. H Hg
Figure 1.1 Illustration of bacterial metal-resistance mechanisms (from Roane and
Microbial Mercury-Resistance Mechanisms
Some microorganisms have the ability to thrive in metal stressed
environments. These microorganisms have developed mechanisms to be metal-
resistant, thus protecting their cellular functions, while reducing the toxicity of the
metal to other organisms (Roane and Pepper, 2001). Microbial metal-resistance can
involve exopolymer sequestration, metal precipitation, metal volatilization, and
reduction of the metal. Different mechanisms may be used with different metals. For
example, lead is most often sequestered with the use of extracellular polymers, while
mercury readily undergoes reduction or volatilization (Roane and Pepper, 2000).
Mercury-resistance can involve reduction (conversion of Hg2+ to Hg) or
volatilization (conversion of Hg2+ and Hg to CH3Hg+). However, due to the energy
requirements required for mercury methylation, majority of the mercury-resistant
microorganisms identified use reduction as a means of mitigation.
Mercury reduction involves the conversion of mercury to elemental mercury
(Hg), which readily volatilizes to elemental mercury gas. This is carried out with the
use of mercury specific cellular proteins (Barkay et al., 2003). Bacteria that are able
to transform mercury are categorized as either broad-spectrum or narrow-spectrum.
Broad-spectrum refers to the ability of bacteria to transform inorganic and organic
mercury compounds such as Hg2+ and CHaHg+ to Hg. Narrow-spectrum refers to
bacteria that can only transform inorganic mercury to elemental mercury (Clark et al.,
1977). The ability to transform mercury depends on the mer genes that some
bacterial possess. Bacteria able to transform inorganic and organic mercury
compounds will have both merA and merB genes, while bacteria able to transform
inorganic mercury alone will only have the merA gene. The merA gene is the key to
transforming Hg2+ to elemental Hg. The merB gene codes for the MerB enzyme that
cleaves the C-Hg bond on organic mercury. Figure 1.2 summarizes the mercury
interactions inside a bacterial cell.
Figure 1.2-Reduction of mercury in microbial mercury resistance (Adapted from
Nies, 1999). Model of a Gram-negative bacterial cell that illustrates the mer genes.
MerP is a protein used to transport Hg2+ to MerT and MerC embedded within the
cytoplasmic membrane to transport Hg2+ inside to the cytoplasm where MerA
transforms Hg2+ to Hg. MerB cleaves C-Hg from organic mercury compounds
which are converted by MerA to Hg. The resulting Hg diffuses away from the cell.
The merA and merB genes are actually part of an operon that includes the
merC, merD, merP, merR, and merT genes. The mer genes can be found on
chromosomes, plasmids, and transposons (Silver and Walderhaug, 1992). The mer
genes are transcribed into a single mRNA (Prescott et al., 2005). Interestingly, it
costs the bacterial cell little to no energy to reduce Hg2+ to Hg, due to the abundance
of the reductant NADPH with the cytoplasm. Once mercury is in the cell, it takes the
cell seconds to start transcription of the mer genes (Ganbill and Summers, 1992).
The MerP protein, found in the periplasmic space, binds mercury and
transports it to the MerT protein, embedded in the cytoplasmic membrane, which
brings the mercury across the membrane into the cytoplasm. The MerC protein, also
embedded within the cytoplasmic membrane, also aids in the transport of Hg into
the cytoplasm. Once in the cytoplasm, the MerA protein, a mercuric reductase, binds
the mercury and reduces it to its elemental form. Once mercury is in its elemental
form, it readily volatilizes and can diffuse away from the cell.
Regulation of the mer operon uses a regulatory protein known as MerR. In
the presence of mercury, MerR activates expression of the mer genes, and in the
absence of mercury, it will repress the transcription of the mer genes. This protein
has its own promoter-operator and is transcribed opposite of the merTAPCD genes
(Nascimento and Chartone-Souza, 2003). This acts as a negative regulatory control
by turning off the genes used for reduction of mercury. Mercury itself acts as an
inducer causing a conformational change in the MerR protein, allowing RNA
polymerase to find the start site for transcription of the mer genes (Barkay et al.,
2003). The MerD protein regulates operon expression when small amounts of
mercury are present (Barkay et al., 2003; Iohara et al., 2001; Nies, 1999).
Mercury has a high affinity for thiol groups, and therefore readily binds to the
amino acid cysteine. MerR is a protein dimer containing cysteine residues on the C-
terminal ends. Mercury binds to Cysl 17 and Cysl26 on MerR before binding to the
promoter region (Zeng et al., 1998). In vitro studies show that only one mercury ion
will be bound at a time to the MerR dimer. Using western blotting and analytical
techniques, it was found that MerR not only binds mercury, but also zinc, cadmium,
lead, and arsenic. Interestingly, scientists are interested in using metal binding
domains, such as MerR, in genetically modified cells for remediation of sites
contaminated with more than one metal (Song et al., 2004).
The mer genes are of great interest in the genetic modification of other
organisms to carry out mercury reduction and to understand the gene regulation that
controls the proteins that could be coupled with other methods to enhance the
remediation of mercury-contaminated environments.
Bioremediation uses microorganisms to degrade organic pollutants or to
transform metal pollutants to less toxic forms. Microbial resistance mechanisms
include heavy metal resistance and degradation of organic chemicals, making the use
of microorganisms a cost effective and practical approach to remediating
contaminated environments. Microbial bioremediation has been applied in
wastewater treatment facilities and chlor-alkali processes. Chlor-alkali plants use
indigenous and genetically-engineered mercury-resistant bacteria to remove mercury
from their waste-streams. (Wagner-Dobler et al., 2000). This process uses a packed
bioreactor to enhance the formation of biofilms by supplying low levels of nutrients
for the organisms. Contaminated water then passes through the bioreactor where
indigenous microorganisms remove 10-50 pg/L mercury (with a 2000 L/hr flow
rate) by converting the mercury to Hg (Wagner-Dobler et al., 2000).
Microbial bioremediation strategies include three areas to consider when
remediating metal-contaminated environments: (1) immobilize and precipitate the
metal, (2) reduce the concentration of the metal and (3) convert the metal to a less
biologically available form through binding (Barkay and Schaefer, 2001). In order to
address and implement these strategies, bacterial responses to metal-contaminated
environments must be elucidated.
Bacterial mechanisms of metal resistance include using exopolymeric
substances, sequestration, efflux pumps, and reduction or volatilization to transform
metal species to a less toxic form (Figure 1.1). For example, mercury volatilization
transforms Hg2+ into CH3Hg+, which diffuses away from the cell as a gas, thus
protecting the cell. However, this conversion increases the bioavailability of
mercury, therefore increasing its overall toxicity. The use of exopolymeric
substances, on the other hand, bind the metal reducing its mobility, solubility and,
Several types of bacteria possess metal-resistant mechanisms, as well as some
fungi and yeast. Eukaryotic microorganisms rely on metallothioneins, which are a
group of intracellular proteins that sequester metals, thereby protecting cellular
functions. Bacteria, such as Cupriavidus metallidurans CH34, a common soil
organism, can contain mega plasmids that code for resistance to many different
metals. In the case of C. metallidurans CH34, the organism uses 2 megaplasmids,
pMOL28 (180 kb) and pMOL30 (240 kb), that code for resistance to nine metals,
including mercury, lead, cadmium and zinc (Nies, 2003). Desulfovibrio
desulfuricans, a sulfate-reducing bacterium (SRB), uses sulfide production to
precipitate metals out as sulfide salts. In addition, SRBs can have enzymes to
precipitate toxic metals such as uranium (U), chromium (Cr), and arsenic (As).
Thiobacillus ferrooxidans, an iron and sulfur oxidizing bacterium, is resistant to
aluminum (Al), copper (Cu), cobalt (Co), nickel (Ni), manganese (Mn), and zinc
(Zn), in addition to being resistant to pM mercury concentrations (Barkay and
Schaefer, 2001; Vails and Lorenzo, 2002). Pseudomonas putida strain can remove
90% of 40 mg/L Hg2+ solution within 24 hours (Okino, et al., 2000). Clostridium
cochlearium contains a plasmid coding for the cleavage of CfLHg to Hg which
reacts with hydrogen sulfide to form HgS (Osborn et al., 1997). Other studies show
genetically engineered microorganisms have the potential to be used for
bioremediation. For example, Deinococcus radiodurans, found in a variety of
environments, cloned with the mer operon could remediate sites that contained
radionuclides, Hg and organic contaminants (Daly, 2000). The success of
microbial mercury resistance has led to studies into the use of genetically engineered
plants, such as Arabidopsis thaliana containing the mer system of mercury resistance,
in the phytoremediation of mercury (Rugh et al., 1996).
Some bacteria have adapted to surviving in contaminated environments, such
as soils and wastewaters from industrial sites, as well as natural environments with
high metal concentrations. An environment that has yet to be looked at for metal-
resistant microorganisms is museums that contain artifacts that were once treated with
metals, such as mercury and arsenic, to prevent pest degradation. We would like to
use such museum originated metal-resistant microorganisms to detoxify mercury-
treated artifacts, especially those earmarked for repatriation.
Rationale and Objectives
Although little is known about the concentration of chemicals applied to
museum collections, current methods of detection ensure that metal-based pesticides
were applied to collections for preservation from insect and microbial degradation
(Goldberg, 1996). In the research proposed here, the potential to remediate these
contaminated artifacts relies on the use of non-pathogenic microorganisms that can
thrive on these artifacts that contain mercury. The presence of metals on museum
materials increases the risk of metal exposure for individuals handling these
materials, especially the ethnographic collections earmarked for repatriation. The
study of metal-resistant microorganisms cultured from mercury-contaminated
museum artifacts will be the key to detoxifying mercury from collections. The
application of mercury-volatilizing microorganisms to reduce mercury concentrations
is a novel strategy in the remediation of a poorly elucidated yet significantly toxic
Microbial diversity, biomass, and reduction of mercury will reflect the ability
to remediate the mercury present on mercury-contaminated museum materials. The
use of a mercury analyzer can quantify the mercury concentrations being reduced by
individual bacterial isolates. Coupled to this method is the use of molecular
techniques to identify the mercury-resistant microorganisms.
The proposed research here will investigate microbial activity in the presence
of mercury and the relationship to metal removal. Several biotic and abiotic factors
will be examined to assess this relationship.
a. ) Microbial activity
b. ) Metal concentrations
c. ) Metal-resistance through reduction of mercury
d. ) Remediation of mercury through reduction
Methods proposed for the above measurements include using microbiological
culturing and molecular techniques on bacterial isolates obtained from mercury-
contaminated museum artifacts from the Arizona State Museum ethnographic and
educational collections. These methods address three major objectives:
(1) Collect isolates from mercury-contaminated museum educational
and ethnographic collections to assess the ability of microorganisms to
(2) Identify of mercury-reducing microorganisms using the molecular
method of 16S rDNA sequencing.
(3) Apply these microorganisms to laboratory mercury-treated
materials (substrates) to assess the potential of mercury-reducing
microorganisms to remove mercury from contaminated materials.
(1) Collection of samples: The surfaces of materials were swabbed with
sterile cotton applicators and inoculated onto nutrient agar (a nutrient rich
growth medium) and R2A (a minimal growth medium); both media were
used to enhance the recovery of the isolates. Samples were incubated at
28C until growth appeared on the surface of the agar. Replicate samples
were collected from Arizona State Museum, Tucson, AZ.
(2) Culturing Techniques: Colonies from the samples were isolated into pure
colonies and grown in nutrient broth or tryptic soy broth prior to growth in
varying mercury concentrations.
(3) Mercury measurements: The reduction in mercury concentrations (with a
10 mg/L starting concentration) with time of incubation was determined
using a PS200II Leeman Labs Mercury analyzer and microwave acid
digestion using a modified EPA 3051 method. All analyses were run in
conjunction with a set of known mercury concentration standards.
(4) Bacterial Identification: Isolates were identified using the molecular
method of 16s rDNA sequencing. Results obtained from the University of
Colorado Health Sciences Center (UCHSC) DNA sequencing core were
entered into Gene Bank (NCBI databases) for identification.
(5) Evaluation of remediation of mercury: Mercury losses from treated (with
mercury and bacteria) materials were analyzed to determine the potential
for the use of the mercury-resistant bacteria in the remediation of museum
Historically, cultural collections were often treated with mercury as a
preservative against insect and microbial damage. Still contaminated, these materials
pose current health risks upon contact. An effective method of mercury mitigation
from treated cultural museum materials is currently not available. Methods that have
been proposed are too harsh for some culturally sensitive materials.
However, bioremediation technologies can be used to detoxify contaminants
associated with the cultural artifacts. Due to the nature of microbial resistance to
mercury and microbial activity in the environment, this study demonstrates a novel
approach to using mercury-resistant bacteria isolated from mercury-treated museum
materials. One isolate in particular was examined for this purpose, Arthrobacter sp.
2604. This isolate could remove 2 mg/L Hg from tryptic soy broth (TSB), 3 mg/L Hg
from tryptic soy agar (TSA), and 2 mg/kg Hg from paper after 7 days at 28C.
Arthrobacter sp. 2604 could grow in up to 50 mg/L Hg, and utilized various organic
acids, such as pyruvic acid methyl ester, acetic acid, and hydroxybutyric acid, as well
as sugars, such as lactose, galactose, trehalose, and melibiose, when substrate analysis
was performed. The overall goal of this project was to use mercury-resistant bacteria
to reduce the mercury from mercury-treated laboratory materials, such as paper. The
long-term goal is to apply mercury-resistant bacteria as a cell suspension onto
mercury-contaminated museum artifacts to reduce the mercury associated with these
Museum curators and collectors historically used a wide range of chemicals to
manage pest activity on ethnographic and zoological collections. Such chemicals
included dichlorodiphenyltrichloroethane (DDT), naphthalene, chlordane, and
dieldrin (Hawks, 2001). These chemicals also included the metal salts of arsenic and
mercury to prevent pest (microbial, insect, and rodent) infestations. Mercuric
chloride (HgCy along with arsenical soaps were used as preservatives on the skins of
birds, pelts and furs, feathers, clothing, ceremonial masks, pipes, leather, pottery,
wood, and hair (Goldberg, 1996). A study of mercury and arsenic contamination
conducted at five Natural History Museums in Canada found that 81% of natural
history collections from these museums tested positive for arsenic and 5% tested
positive for mercury (Sirois, 2001). Similar surveys in the United States are currently
underway; however, estimates of 20-80% of ethnographic and natural history
specimens are metal contaminated (Nancy Odegaard, personal communication).
Unfortunately, prior to the 1970s, clear records of which materials were
treated, how often they were treated, with what concentrations they were treated,
when they were treated and how they were treated were generally not kept. However,
pesticides applied to preserve museum materials continue to pose a major health
concern for museum personnel and Native peoples as items are repatriated.
Symptoms of mercury poisoning can include nausea, headaches, fatigue, speech and
coordination difficulties, brain damage, and lung damage (EPA, 2005).
While museum personnel are at risk of exposure, Native peoples run a greater
risk once the items are repatriated. Repatriated items may be used on a regular basis
for personal use and ceremonial practices. Many ceremonies involve smoking pipes,
wearing clothing, headdresses, and masks, and holding figurines, all of which, if
housed in a museum, could have been treated with metal-based pesticides. Heat,
sweat, deep inhaling, and repeated skin contact are thought to increase the risk of
metal exposure through inhalation, ingestion, or skin absorption (Odegaard and
Sadongei, 2001). Although current remediation practices in museums include
replacement, containment, washing or scraping, heat and radiation (Odegaard, 2001),
these methods may not be appropriate for fragile or culturally sensitive materials. For
example, some Native people consider some cultural objects living spirits and as such
these materials cannot undergo what are considered harsh remediation treatment.
Some microorganisms can survive and grow in the presence of metal toxicity.
These microorganisms have developed physiological and structural mechanisms for
the protection of cellular functions. In some cases, metal-resistant microbial activity
can reduce the toxicity and bioavailability of metals, making these organisms ideal
candidates for metal remediation (Roane and Pepper, 2001). These metal-resistance
mechanisms can include exopolymer sequestration, volatilization, efflux pumps, and
chemical reduction of the metal. For reasons that are not entirely understood, metal-
resistant bacteria are fairly ubiquitous occurring in pristine uncontaminated
environments. Yet, highly resistant organisms seem to be found only associated with
The same holds true for mercury-resistant bacteria; however, mercury is
prevalent in the environment which may explain their widespread distribution. The
most commonly observed bacterial response to mercury is reduction, producing
gaseous mercury. True volatilization involving the attachment of methyl, ethyl or
dimethyl groups can be energy intensive and so is less common. Many bacteria have
been isolated that are capable of volatilizing mercury, including Escherichia coli,
Desulfovibrio spp., Bacillus spp., Kebsiella spp., and Pseudomonas spp. (Klein and
Thayer, 1990). For example, studies conducted by Choi et al. (1994) identified
corrinoid-containing proteins, normally involved in the Acetyl-CoA pathway, as
responsible for the enzymatic methylation of mercury in Desulfovibrio desulfuricans.
Mercury reducing proteins within the cytoplasm are coded by the mer operon
in Escherichia coli, Cupriavidus necator, Salmonella spp., Streptomyces lividans, and
Thiobacillus ferrooxidans, to name a few (Iwahori et al., 2000; Ravel et al., 2000;
Simbahan et al., 2005; Smalla et al., 2000). The MerP protein, embedded in the
cytoplasmic membrane, binds mercury and transports it to the MerT protein, which
brings the mercury across the membrane into the cytoplasm. Once in the cytoplasm,
the MerA protein binds the mercury (Hg2+) and reduces it to its elemental form (Hg)
(Barkay et al., 2003; Nies, 1999; Taghavi et al., 1997). Although elemental mercury
has a low vapor pressure (0.00185 torr), it nevertheless vaporizes as a gas and
diffuses away from the cell.
Both mercury reduction and true volatilization are attractive for
bioremediation because both mechanisms result in conversion to a gas. Mercury gas
can either be collected, such as in a contained bioreactor (von Canstein et al., 2002),
or allowed to diffuse into the surrounding environment (Bohme et al., 2005). Noting
that mercury volatilization does not reduce the toxicity of the metal itself, mercury
diffusion away from the cell reduces the effective concentration of mercury
immediately surrounding the cell. With an improved understanding of mercury-
resistance and mercury-resistant bacteria, work into the microbial remediation of
mercury-contaminated sites is only now underway.
While contaminated air, soil, and water represent the vast majority of
contaminated environments, museums across the U.S. are concerned about mercury-
treated artifacts in their collections. With an estimated 20-80% of ethnographic and
zoological collections containing elevated levels of mercury and arsenic ranging from
parts per billion to parts per million range, and the enactment of the 1990 Native
American Graves Protection and Repatriation Act (NAGPRA) that requires Native
American collections be returned to tribes, museums and federal agencies are trying
to identify non-destructive, culturally-sensitive methods of artifact mercury
mitigation (Odegaard, 2001; Sirois, 2000).
Due to known microbial resistance to mercury, and microbial ubiquity in the
environment, we hypothesized that mercury-resistant bacteria could be found on the
surface of mercury-treated museum artifacts. The overall goal of this project is to use
museum isolated mercury-resistant bacteria to reduce the mercury concentrations
associated with mercury-treated museum materials. This approach will be applied in
the remediation of educational materials and ethnographic collections that are
earmarked for repatriation. The questions this research addresses include (1) whether
mercury-reducing microorganisms already exist on the surface of mercury-treated
museum materials, and (2) whether these microorganisms can be used to reduce the
mercury on mercury-treated laboratory materials. This environment has not been
microbially explored or characterized, making it a prime environment for the isolation
of mercury-reducing microorganisms.
Materials and Methods
Mercury-Contaminated Museum Materials
The Arizona State Museum in Tucson, AZ, provided access to several
mercury-treated artifacts in their anthropological and herbarium collections. The
surfaces of museum artifacts were examined for mercury by museum personnel using
a hand held X-ray fluorescence (XRF) detector (NITON Xli 700 Series Analyzer,
Thermo Scientific, Billerica, MA). Materials that tested positive for mercury were
selected for further microbial examination.
Isolation of Material-Associated Surface Bacteria
In the museum, the surfaces of identified mercury-treated collections were
swabbed with sterile cotton applicators to collect bacteria. The bacteria were then
transferred onto R2 A plates (nutrient limiting growth medium for heterotrophic
bacteria) and nutrient agar plates (NA; a nutrient rich medium for heterotrophic
bacteria). Both types of media were used to enhance the recovery of the isolates with
different nutritional requirements. Different areas on each artifact were swabbed to
include various material types and topographies, such as leather, feathers,
indentations and cracks. The R2A and NA plates were incubated aerobically at 28C
until bacterial colony growth was apparent, approximately 21 days on average. Once
colonies appeared, each colony was sub-cultured onto R2A or NA to obtain pure
cultures for analysis. Pure cultures were stored at 4C on R2A or NA, respectively,
for short-term storage, and stored in 20% sterile glycerol at -80C for long-term
Isolates cultured from mercury-treated museum collections were identified
using 16S rDNA sequencing using universal primers F63 (5 cag gcc taa cac atg caa
gtc 3) and R1387 (5 ggg egg wgt gta caa ggc 3) (Lane, 1991; Marchesi et al.,
1998). The PCR master mix contained the following (50 fil final volume): 43 fil of
Platinum PCR Supermix (Invitrogen, Carlsbad, CA) containing: Taq polymerase,
deoxyribonucleotide triphosphates (dNTPs), buffer, and magnesium chloride; 0.25
/iM F63 final concentration, 0.25 fiM R1387 final concentration, and 3 fil of template
DNA (from cell lysis).
Cell lysis was achieved by heat lysing the cells at 98C for 10 minutes in a
Perkin-Elmer Eppendorf Mastercycler thermocycler (CA). PCR cycle conditions
included an initial denaturing step at 95C for 5 minutes, followed by 30 cycles of
95C (2 min.), 55C (45 sec), and 72C (2 min.), with a final extension at 72C for 10
min. PCR reactions were held at 4C until electrophoretic analysis. PCR products
were electrophoresed at lOOV/cm on a 1% agarose gel containing ethidium bromide
for gel visualization. A 1 Kb ladder was used to confirm a PCR product size of
Upon successful amplification, PCR products were purified using the
Millipore Genomics Montage kit for DNA purification (Millipore Corporation;
Bedford, MA) and sequenced at the University of Colorado at Denver and Health
Sciences Center Cancer Center DNA Sequencing Core (Denver, CO). The resulting
16S rDNA sequences were analyzed with the BLAST program from the National
Center for Biotechnology Information (NCBI) and compared to those from known
organisms for sequence identification.
Bacterial Maximum Mercury-Resistance Level (MRL1
To achieve high cell concentrations, individual isolates were grown in nutrient
broth (NB) or tryptic soy broth (TSB) at 28C at 150 rpm on a rotary incubator. Upon
growth to turbidity, 100 pi of culture was transferred to 25 mL of NB or TSB
respectively, amended with 10 mg/L Hgfforn a mercury chloride (HgCL) stock
solution of 1000 mg/L. The mercury stock was made using HgCl2 (MW 271.49) and
Type II water (milli-Q analytical water 18 Megohms). Mercury-amended cultures
were incubated at 25 C at 150 rpm under a chemical hood for 7 days and examined
for turbidity, indicative of growth in the presence of mercury. Isolates growing in the
presence of 10 mg/L Hg were then similarly examined for their ability to grow in 20-
100 mg/L Hg. Those isolates that did not grow in 10 mg/L Hg were re-examined at
10 /tg/L to <10 mg/L Hg.
Growth of Arthrobacter sp. 2604 in the Presence of Mercury
The growth of Arthrobacter sp. 2604 in the presence of 10 mg/L Hg was
examined using viable plate counts with time. An initial inoculum of 2.4 x 104
CFU/ml was used for 25 mL TSB amended with mercury. Cultures were incubated at
25C at 150 rpm. Growth was determined by dilution and plating onto TSA every 24
hours for 168 hours.
Ability of Mercury-Resistant Bacteria to Remove Mercury
from Different Substrates
Arthrobacter sp. 2604 was examined for its ability to remove mercury from
trypitic soy broth (TSB), tryptic soy agar (TSA) and paper amended with 10 mg/L
Hg. A known mercury-reducing bacterium containing the mer operon, Cupriavidus
metallidurans CH34 was used as a possible positive control for mercury removal
from nutrient broth (NB), nutrient agar (NA) and paper amended with 10 mg/L Hg.
Arthrobacter sp. 2604 was grown in 25 mL TSB (no mercury) and C. metallidurans
CH34 was grown in 25 mL of NB (no mercury) at 150 rpm for 18 hours prior to
inoculation. For the broth studies, a 100 pL of exponential culture was used to
inoculate 25 mL TSB and 25 mL NB amended with 10 mg/L Hg. Mercury-
containing cultures were incubated at 25C at 150 rpm for 7 days under a chemical
hood prior to analysis. Negative controls consisted of medium plus inoculum (no
mercury) and medium plus mercury (no inoculum).
For the agar studies, 10 mg/L of sterile TSA and NA were cooled to 50C
prior to amendment with 10 mg/L Hg and inoculation with 100 pL of 18 hour old
culture. The agar mixture was then poured into sterile Petri plates and incubated at
25C for 7 days under a chemical hood for mercury containment. Negative controls
contained: 1) agar plus inoculum (no mercury); and 2) agar plus mercury (no
To examine the ability of the bacteria to remove mercury from paper, Petri
plates containing 10 mL TSA medium and 10 mL NA medium were made. Sterilized
filter paper (Whatman 42 ashless circles 70mm) was weighed on an analytical
balance to the nearest 0.1 microgram prior to dipping in 10 mg/L Hg solution. In
each experiment, two Petri plates were set up as follows: 1)10 mL of sterile water
with 1 mL of culture, 2) 10 mL of 10 mg/L Hg solution with 1 mL of culture, and 3)
10 mL of 10 mg/L Hg solution with 1 mL of sterile water. Filter paper was dipped in
each treatment for 2 minutes to saturate. The filter paper was removed from the
solution, briefly drained, and then placed on 10 mL TSA plates and 10 mL NA plates.
All plates were incubated at 25C under chemical hood for 7 days. All treatments
(broth, agar, and paper) were performed in triplicate.
Analytical Mercury Concentration Determination
Mercury quantification was performed using microwave acid digestion on a
Floyd RMS-150 pressure control module microwave (Questron, Princeton, NJ)
followed by total mercury analysis using a PS200II Leeman Labs Cold Vapor-Atomic
Absorption Spectrometry (CV-AAS) instrument (Hudson, NH). Acid digestion both
releases bound mercury in the sample and oxidizes it to Hg2+ form. In CV-AAS, tin
chloride (SnCh) is used to reduce Hg2+ to Hg. Argon gas picks up the volatile
mercury passing through an absorption cell where the amount of energy (produced by
a mercury lamp) absorbed by the mercury is proportional to the amount present in the
sample. A modified EPA method 3051 and sample prep method from Duquesne
University (Walter et al., 2005) were used to program the microwave for total
mercury digestion. The following method was used: 0.5 gram filter paper was
digested in 10 mL of concentrated nitric acid in a Teflon vessel for 2.5 minutes at 600
Watts (100% power). Dilutions of the digestates were made using 2 mL concentrated
nitric acid and Type II water (milli-Q analytical water 18 Megohms) solution to a
final volume of 100 mL and read on the CV-AAS for Hg at 253.7 nm absorption
based on a standard curve (0, 5,10,15, 20, 25 parts per billion). All instrumentation
needed for mercury analysis was provided by the Shared Analytical Services
Laboratory at the University of Colorado Downtown Denver Campus.
Some of the isolates capable of growing in elevated mercury concentrations
were examined for substrate utilization. Isolates were grown on NA or TSA plates at
28C for 24 hours. After growth, the colonies were collected using a sterile cotton tip
applicator. The cells were then re-suspended in 15 mL of sterile water by agitating
the applicator in the water. Cells were pelleted at 4,000 rpm on an IEC Centra-4R
centrifuge (International Equipment Company; Needham Hts., MA) for 10 minutes.
The supernatant was removed; the pelleted cells were washed with 15 mL sterile
water and re-suspended, and then re-centrifuged. The cells were washed in this
manner three times.
A 150 /il aliquot of the washed, suspended bacterial cells was placed into each
well of a 96-well GN BIOLOG plate (BIOLOG; Hayward, CA) and allowed to
incubate at 28C for 24 hours. The BIOLOG plates were analyzed using a
Labsystems Multiskan Ascent plate reader (Helsinki, Finland) to determine substrate
usage. Coloration in a well was indicative of substrate usage.
Results and Discussion
Mercury-Contaminated Museum Collections
In the museum setting, X-ray fluorescence spectroscopy has only been
standardized for surface determinations of metals. The penetration of X-rays into
different material types, ranging from cloth to wood to ceramics, is unknown.
Consequently, XRF is currently being used to determine amounts of mercury per unit
area on the surfaces of items, as opposed to concentrations of mercury in the objects.
Table 2.1 lists the materials that were identified and the amounts of mercury
associated with them measured as fig/cm2. Mercury levels ranged between 93 pg/cm2
to 2000 fig/cm on materials that included headdresses, ceremonial leather pouches,
and even the felt lining of a Harvard cabinet that had housed mercury-treated
herbarium collections. Although an estimated 20-80% of museum collections is
thought to have some level of associated mercury or arsenic (and sometimes both),
research into appropriate methods for quantification of the mercury or arsenic in an
artifact is actively being conducted.
However, the pursuit is complicated by the fact that many artifacts are
materially complex. For example, Headdress 2 (refer to Table 2.1) consists of
feathers, felt, cotton cloth, and leather, all of which potentially have to be treated for
mercury removal. Additionally, the mercury or arsenic associated with these
materials are not evenly distributed over or throughout the object. Surfaces readily
available for contact in storage or handling are predicted to have less associated
mercury. Recesses of an object where mercury accumulation can occur will have an
increased amount. Due to material complexities, mercury may also be bound to the
organics of the material. Finally, age may play a role in the availability of the metal;
however this is not clear at this point. The age of the material itself does not seem to
play a role in how much mercury is found, but material scientists hypothesize that the
degree of complexation between the mercury and material compounds may increase
Table 2.1 Isolate identification and mercury maximum resistance levels (MRL) from
mercury-treated museum artifacts.________________________________________________
Source Mercury on material (Mg/cm2) Isolate Identification (Mercury MRL)
Leather bag 93 Arthrobacter sp. 2604 (50 mg/L)
Turtle fetish - Bacillus megaterium (5 mg/L), Pseudomonas sp. (2 mg/L) Korea rosea (-) Bacillus sp. (-) Arthrobacter sp. (-) Pseudomonas tolassi (-)
Spear thrower 2147 Bacillus sp. (10 mg/L)
Headdress 1 280 Unknown (-)
Headdress 2 1076 Korea sp. (-)
Moccasin 23 Chelacoccus asaccharyorans (-), Arthrobacter sp. (100 /rg/L)
Harvard cabinet 300 Pseudomonas synxantha (1 mg/L) Kaistobacter koreensis (-) Arthrobacter sp. (100 fig/L) Unknown (-) Unknown (-) Unknown (-)
Leather pouch - Agrococcus jenensis (-)
Red textile 370 Unknown (100 /ig/L)
Isolation and Identification of Material-Associated Bacteria
The initial collection of bacteria from the surfaces of the museum materials
presented an interesting problem. Many of these materials are fragile and cannot be
rinsed or extracted with buffer, the most common way of collecting bacteria from
different media. For example, soil bacteria are collected with a series of charge-
neutralizing extractions to release the bacteria from the surface of the soil particles.
The resulting suspension containing the bacteria is then used for a variety of analyses.
In this case, however, due to potential damage to the materials, rinsing was not an
option. Our collection method could not damage or alter the artifacts in any way.
Consequently, a dry collection using a non-invasive cotton swab was used.
We were initially unsure how many bacterial isolates we were going to find
on these surfaces. Care was taken to collect bacteria from readily exposed surfaces,
as well as depressions within the surfaces. When complex materials were examined,
such as headdresses, all material types associated with the headdress were sampled,
including feathers, cloth, and leather. Twenty different bacterial isolates were
obtained from sampling, with 1-6 different isolates from a given artifact (Table 2.1).
Table 2.2 indicates the different isolates and their percent similarity to known
Microorganisms can be isolated from artwork, historic buildings, and chapels
with organic residues from environmental factors, such as soot. For example,
Agrococcus citreus spp. have been isolated from a mural painting in the chapel of the
castle of Herberstien in Styria, Austria (Wieser et al., 1999). Other microorganisms
found on historical stone artwork include Desulfovibrio desulfuricans, Desulfovibrio
vulgaris, Pseudomonas spp., Actinobacteria spp., and Bacillus spp. (Cappitelli et al.,
2006; McNamara and Mitchell, 2005). These microorganisms are currently being
characterized for their role in biodegradation of organics associated with artwork and
stone monuments in addition to understanding the process of biodeterioration of these
materials (Gonzalez and Saiz-Jimenez, 2005; Ranalli et al., 2004; Saiz-Jimenez,
1997; Zanardini et al., 1997).
Isolate identification was done to learn more about the unknown organisms on
museum materials and to ensure we did not continue to work with a potentially
pathogenic organism. Little is currently known about the microorganisms inhabiting
the surfaces of museum collections. Some information is available addressing
destructive microorganisms associated with paintings and statues (see above).
Ironically, it is against these very organisms that the mercury- and arsenic-based
pesticides were used. The microorganisms we identified in this study represent
commonly found bacteria, such as Arthrobacter, Bacillus, and Pseudomonas (Table
2.2). Members of many of these genera have been found to be mercury-resistant.
For example, Arthrobacter is a member of the Microcaceae family that is
commonly found in soil and water and associated with organic contaminants, such as
2,4-D (2,4-dichlorophenoxyacetic acid) and naphthalene. Arthrobacter is also
recognized as the third most abundant bacterium to be characterized for its
biotransformation reactions (www.cbs.umn.edu. 2006; Dore et al., 2003 and
Overhage et al., 2005). Moreover, Arthrobacter spp. isolated from historic buildings
were found to degrade hydrocarbons, such as steranes found in petroleum products
(Saiz-Jimenez, 1997). In addition to the ability to degrade organics, Arthrobacter
spp. have been found with mercury reductase genes located on their plasmids
(Bogdanova and Mindlin, 1988; Nies, 1999). While it is not known if Arthrobacter
sp. 2604 in this study contains mercury reductase, this will be determined in future
Several Gram-positive bacteria, such as Arthrobacter and Corynebacterium
spp., are resistant to mercury and are commonly found in mercury contaminated soil
and water systems, as well as hospital samples (Trajanovska et al., 1997). Also,
Escherichia coli, Staphylococcus aureus, and Pseudomonas spp. have been well
documented as mercury-resistant bacteria (Robinson and Tuovinen, 1984). Studies
show that Bacillus spp. isolated from Minamata Bay carry the mer operon to reduce
Hg2+ into Hg (Iohara et al., 2001; Osborn et al., 1997).
Table 2.2 Museum isolate identification using 16S rDNA sequencing showing
Museum Accession Number 16S rDNA Identification Similarity %
E2604-331 Arthrobacter sp. 98%
GP50054B-323 Unknown -
26033-330 Agrococcus jenensis 98%
16975-325 Bacillus sp. 98%
GP50057-316 Unknown -
74-22-2-336 Kocuria sp. 97%
20-350 Arthrobacter sp. 98%
20-350 Unknown -
20-350 Unknown -
20-350 Kaistobacter koreensis 98%
20-350 Unknown -
20-350 Pseudomonas synxantha 98%
E1487-332 Kocuria rosea 97%
E1487-332 Bacillus megaterium 96%
E1487-332 Pseudomonas sp. 98%
E1487-332 Arthrobacter sp. 85%
E1487-332 Bacillus sp. 99%
E1487-332 Pseudomonas tolassii 97%
E1493B-328 Arthrobacter sp. 99%
E1493B-328 Chelatococcus asaccharvorans 95%
Maximum Mercury-Resistance Level
Each of the twenty bacterial isolates identified from museum collections were
examined for their ability to grow in the presence of mercury to characterize the
maximum resistance level of each isolate. While 62% of the isolates appeared to
have little or no resistance to mercury (less than 10 ppb, the lowest concentration
examined), several had the ability to grow in up to 50 mg/L Hg. One isolate, in
particular, identified as Arthrobacter sp. 2604 was able to grow in up to 50 mg/L Hg.
This is an extraordinary level of mercury resistance. Most mercury resistant bacteria
can grow in pg/L to low mg/L levels. For example, Staphylococcus aureus can grow
in up to 10 pg/mL Hg, whereas some Escherichia coli strains can grow up to 10 mg/L
Hg (Robinson and Tuovinen, 1984). Yet, some strains isolated from the coast of
India can grow in up to 50 mg/L Hg (Ramaiah and De, 2003). Due to the
extraordinary mercury resistance of Arthrobacter sp. 2604, it was chosen for further
Growth of Arthrobacter sn. 2604 in the Presence of Mercury
In order to better understand the observed mercury-resistance of Arthrobacter
sp. 2604, a growth study was performed monitoring the growth of the isolate in 10
mg/L Hg. A mercury concentration of 10 mg/L was chosen to correlate with the
material studies conducted with broth, agar, paper amended with 10 mg/L Hg. In the
growth curve presented in Figure 2.1, the mercury was toxic due to the 5 order of
magnitude decrease in cell numbers within the first 24 hours. However, resistant
cells were evident within 48 hours, ultimately reaching almost the same cell
concentration as the zero mercury control within 120 hours.
The initial decrease in cell number upon mercury exposure was not
unexpected, even for a mercury-resistant population. Studies are finding that not all
members within a given metal-resistant population of cells are equally resistant
(Kassab and Roane, 2006). Kassab and Roane (2006) also found in response to
cadmium that there is often a time delay between metal exposure and activation of
bacterial metal-resistance mechanisms. This delay may also account for the observed
decrease in cell numbers. According to a study conducted by Ramaiah and De (2003)
indigenous mercury-resistant bacteria collected off the coast of India showed a
prolonged lag time when compared to no mercury controls indicating a physiological
adaptation to induce the mer system (Ramaiah and De, 2003). In our study, the
resistant cells recovered and showed rapid exponential growth with a similar growth
rate as the no mercury control culture, with 0.20 generations/hour as compared to
0.28 generations/hour, respectively
Analysis of mercurv-Resistant Bacteria to
Remove Mercury from Different Substrates
Two bacterial isolates were examined for their ability to remove mercury from
different substrates: Arthrobacter sp. 2604 and Cupriavidus metallidurans CH34.
C. metallidurans CH34 is well characterized in terms of its metal-resistance (Legatzki
et al., 2003; Nies and Silver, 1989). With two megaplasmids coding for a variety of
metal-resistance mechanisms, C. metallidurans CH34 is resistant to lead, cadmium,
zinc, copper, and mercury, using the mer operon for mercury resistance. While C.
metallidurans CH34 has not, as far as we know, been used to remove mercury from
solid materials before, the bacterium is being actively investigated for its ability to
remediate contaminated soils and waters (Mergeay et al., 2003).
Both Arthrobacter sp. 2604 and C. metallidurans CH34 were able to remove
mercury in the broth, agar, and paper experiments. Broth was initially used as a
screening method for removal. Since liquid media are easier to handle and use, the
broth experiments allowed us to optimize our digestion protocols to quantify the
mercury removal. Agar was used to represent a porous solid medium. We anticipate
that ultimate mercury removal from museum artifacts will require more than just
surface removal. Given that many of the materials found in the artifacts are porous to
differing degrees, the agar was used as an initial assessment of the ability of the
bacterial isolates to remove mercury throughout a material. Agar was also easy to
manipulate while method development was underway. With encouraging results with
both broth and agar, paper was used as a simulated museum material type.
When compared to the uninoculated controls, Arthrobacter sp. 2604 was able
to remove up to 2 mg/L Hg from broth (TSB), 3 mg/L Hg from agar (TSA), and 2
mg/Kg of Hg from paper within 7 days (Figure 2.2), from a starting concentration of
10 mg/L Hg. C. metallidurans CH34 also showed mercury removal with up to 4
mg/L Hg from broth (NB), 5 mg/L Hg from agar (NA), and 6 mg/Kg Hg from paper
removed within 7 days (Figure 2.3). Some abiotic loss of mercury was apparent and
may have been due to binding of mercury to the glassware or matrix interference
caused by the substrate. These results are very encouraging for the removal of
mercury from museum artifacts; however, optimization of mercury removal needs to
be performed. For the material experiments here, we found the highest amount of
mercury removed occurred with some humidity and when the cells were applied to
the materials in a dilute nutrient solution (Appendix A). Without the humidity (future
research will quantify this) and the nutrient solution, little mercury was removed.
Interestingly, in a variation of the currently used method for the paper experiment, the
mercury-amended inoculated paper was placed on top of an agar filled Petri plate.
When placed in a sealed chamber for incubation, the humidity was so high (water
droplets were present) that mercury removal was inhibited. Future research will work
to optimize humidity levels. Future optimization experiments will also address
nutrient availability, temperature effects, inoculum concentration, and application of
Figure 2.1 Effects of mercury on the growth of Arthrobacter sp. Cells (6.62 x
107cells/mL) were inoculated in TSB amended with 10 mg/L Hg.
Mercury Removal by Arthrobacter sp. 2604
Broth (mg/L) Agar (mg/L) Paper (mg/kg)
Figure 2.2 Mercury removal within 7 days by Arthrobacter sp. from broth (TSB),
agar (TSA), and paper amended with 10 mg/L of Hg. Uninoculated negative controls
containing 10 mg/L Hg were used to assess abiotic loss of mercury. Inoculum
concentration was approximately 107 CFU/mL. The bars represent the mean of 3
trials (with 2-replicates in each trial) with standard deviation amongst the means.
Mercury Removal by C. metallidurans CH34
_ 12 -
Broth (mg/L) Agar (mg/L) Paper (mg/kg)
Figure 2.3 Mercury removal within 7 days by C. metallidurans CH34 from broth
(NB), agar (NA), and paper amended with 10 mg/L of Hg. Uninoculated negative
controls containing 10 mg/L Hg were used to assess abiotic loss of mercury.
Inoculum concentration was approximately 107 CFU/mL. The bars represent the
mean of 3 trials (with 2-replicates in each trial) with standard deviation amongst the
To see if there was a correlation between the type of material the bacteria
were isolated from and their ability to use particular substrates to support their
metabolism, GN BIOLOG substrate utilization patterns were determined.
Arthrobacter sp. 2604 was tested for its ability to utilize different substrates within 24
hours with an inoculum of 107 cells/mL. Results found that Arthrobacter sp. 2604
could utilize four different types of sugars, three types of amino acids, and fourteen
different organic compounds (Table 2.3).
This indicates that Arthrobacter sp. 2604 isolated from a leather bag has the potential
to use different carbon sources to meet its metabolic requirements. Since many
museum artifacts are materially complex, this provides some insight into what
materials are possibly susceptible to degradation by this isolate and into possible
supplemental nutrients that might be co-applied with the organism to sustain
metabolic activity throughout the mercury removal process.
Table 2.3 Substrate utilization pattern for Arthrobacter sp. 2604 based on the GN
Sugars a-D lactose
D-galactose D-trehalose D-melibiose
Amino Acids L-alanine L-proline L-threonine
Organic Compounds Acetic acid Pyruvic acid methyl ester Quinic acid D-saccharic acid L-alanyl glycine Urocaric acid Inosine Propionic acid Bromosuccine acid Glucuronamide a-ketobutyric acid a-ketoglutaric acid a-ketovaleric acid /3-hydroxybutyric acid
With this research we now know more about the bacteria present on mercury-
contaminated museum artifacts and that these organisms are present even with the
treatment of mercury, initially used to prevent microbial growth on museum
collections. This particular environment has not before been microbially
characterized. These museum artifacts are also a source of mercury-resistant bacteria.
From the initial data Arthrobacter sp. 2604 isolated from a Native American leather
pouch could remove 2 mg/Kg Hg of 10 mg/Kg mercury within 7 days from
laboratory-treated paper. There is the potential to remove more mercury with further
optimization of the treatment conditions. An unexpected outcome was the mercury
removal performed by Cupriavidus metallidurans CH34, initially used as a known
mercury-reducing isolate. C. metallidurans CH34 removed up to 6 mg/Kg of 10
mg/Kg mercury within 7 days from paper.
Results from this study indicate a possible novel microbial strategy for the
removal of mercury from varied museum collections, providing an alternative
approach for repatriated artifacts under the Native American Graves Protection and
Repatriation Act (NAGPRA).
Additional characterization of the mercury-resistance genes for Arthrobacter
sp. 2604 is necessary to better understand this organisms transformation of mercury,
as well as its substrate usage in order to understand the role Arthrobacter sp. 2604
could play in the possible degradation of treated materials. Current data show that
this strain can remove up to 3 mg/L Hg within 7 days on laboratory materials meant
to simulate materials associated with the contaminated artifacts for remediation.
However, optimization of mercury removal including incubation time, inoculum
concentration, humidity, and nutrient supplementation is required for continued
success of this work. This optimization process needs to occur for various types of
materials likely to be encountered in the museum setting, for example, leather, cloth,
feathers and hair.
The lack of information on the microbial ecology of museum
microorganisms has led to current work in our group (not presented here)
characterizing the microbial community found on mercury-treated museum materials
using Denaturing Gradient Gel Electrophoresis (DGGE). This molecular method
allows for the identification of community DNA, even that from unknown or
unculturable bacteria. This approach may provide valuable information about
possible remediating bacteria but also about the natural microbial flora of artwork.
Finally, work to determine any possible degradation of microbially-treated
artifacts needs to be determined. Many tribal consultations indicate that some
degradation as the result of microbial treatment is acceptable for those artifacts
earmarked for repatriation under NAGPRA. Many tribes consider this type of
degradation part of the natural aging process for a given artifact, especially those
with spiritual meaning. However, for some items meant to stay in museums,
degradation is not a desired outcome (although it may be better than some alternatives
such as leaving the mercury on an item). The use of scanning electron microscopy
(SEM) will help determine if material degradation occurs during microbial treatment.
A specific method for the quantification of mercury associated with paper is
not available. Consequently, this research required the development of a method.
Modifications of existing protocols were attempted. A successful protocol was one
that recovered >90% of the mercury from uninoculated mercury controls.
The first protocol was a modified method from the EPA method 7471 A:
Mercury in Solid or Semisolid Waste. In this approach, following the 7 day
incubation period, 0.2g paper was placed in a biological oxygen demand (BOD)
bottle. Aliquots of 5 mL of reagent grade water and 5 mL of aqua regia (hydrochloric
acid and nitric acid) were added to the bottle, and then heated for 2 minutes in a 95C
water bath. Fifty mL of reagent grade water and 15 mL of potassium permanganate
(KMn04) were added to the solution, mixed thoroughly, and heated in the 95C water
bath for another 30 minutes. The solution was then cooled and 6 mL of sodium
chloride-hydroxylamine sulfate was added (this reduces the permanganate).
Quantitative dilutions were made using Type II water, and samples were quantified
using CV-AAS (cold vapor atomic absorption spectrometry) for mercury.
Undigested paper was visually apparent at the end of the protocol with <90% mercury
Another modification was made to the EPA method 7471A using 10 mL of
concentrated perchloric acid to cold digest 1 g paper at room temperature. Following
mixing and diluting, the mercury recovery was again suboptimal.
Growth Optimization Experiments
Experimental paper was treated with 10 mg/L Hg for 30 seconds and air dried
in a fume hood for 45 minutes. The treated paper was then inoculated with 10 mL of
exponential bacterial culture for 30 seconds by dipping the paper in the culture
suspension. Negative controls with no inoculum were similarly treated with 10 mL
sterile broth. The paper was then placed in empty sterile Petri plates and incubated in
a sealed jar with a beaker of sterile water to create and maintain a humid environment
for 7 days. At the end of the incubation period, bacterial growth was not apparent and
mercury removal did not occur under these conditions.
In another attempt, excess nitrogen was supplied as NH4NO3, which can
stimulate microbial growth on carbon rich substrates, such as the paper. Paper
treatments were dipped in 10 mg/L Hg using NH4NO3 and dried for 45 minutes.
Then 1 mL of inoculum was added to the paper in a sterile Petri plate. Plates were
incubated in an air tight container with a beaker of sterile water for 21 days. At the
end of the incubation period, microbial growth was apparent; however mercury
removal was not quantified.
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