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EPA's Office of Research and Development
The Office of Research and Development (ORD)
conducts an integrated program of scientific research and
development on the sources, transport and fate processes,
monitoring, control, and assessment of risks and effects
of environmental pollutants. These activities are imple-
mented through its headquarters offices, technical sup-
port offices, and twelve research laboratories distributed
across the country. Research focuses on key scientific
and technical issues to generate knowledge supporting
sound decisions today and anticipating the complex
challenges of tomorrow. With a strong, forward-looking
research program, less expensive, more effective solu-
tions can be pursued and irreversible damage to the
environment prevented.
Front Cover:
Bioremediation has been an
effective treatment technique for tfie
reclamation of ocean beaches
contaminated as a result of crude oU
spills -
Alan Pitcairn/Grant Heilman Photography Photo
-------
The United States is the world leader in the
field implementation of bioremediation, an
attractive alternative to conventional methods
of cleaning up persistent hazardous wastes in
the environment.
Complex synthetic chemicals
and petroleum derivatives have
accumulated in the env ironment as
waste materials for decades. Con-
ventional treatments, such as exca-
vation followed by incineration,
have been used for some time to
clean hazardous waste sites, but can
be costly and inherently disruptive
to the environment. The United
States Environmental Protection
Agency's (EPA's) Office of Re-
search and Development plays a
major role in the basic science and
engineering involved in developing
and supporting innovative technolo-
gies that are cost-effective alterna-
tives to existing methods for
cleaning up hazardous waste sites
and oil spills. One of the most
promising of these new methods for
solving toxic waste cleanup prob-
lems is bioremediation.
Bioremediation technology can
be a non-disruptive, cost-effective,
and highly efficient method of de-
stroying many environmentally
persistent toxic chemicals. Al-
though the development of biore-
mediation has progressed rapidly
over the past several years, a great
deal must still be accomplished
before the technology can be fully
utilized. This is true in terms of
scientific research, the engineering
design of treatment systems, and
field evaluations. In response to
these needs, ORD has developed an
integrated Bioremediation Program
to advance the understanding,
development, and application of
bioremediation technologies to help
solve hazardous waste problems
threatening human health and the
environment. As these technologies
advance, ORD transfers information
on their use to groups who apply
them to treat specific sites.
What Bioremediation
Involves
Bioremediation technologies
typically use naturally occurring
microorganisms (bacteria or fungi) to
degrade hazardous wastes. Like all
living creatures, microbes need
nutrients, carbon, and energy to
survive and multiply. Such
organisms are capable of breaking
down toxic chemicals to obtain food
and energy, typically degrading them
into harmless substances consisting
Electron micrograph
ofPseudomonas
aeroginosa, oil-
degrading bacteria
(magnification at
X5,500).
Manfred Kage/Peter Arnold, Inc. Photo
Printed on Recycled Paper
-------
After the target
chemical has
been biode-
graded, most of
the microbial
population will die
off naturally since
there will no
longer be a suffi-
cient food or
energy source for
the microorgan-
isms to survive.
Two Alaskan beach
field test plots show
that a site where
fertilizer was
applied is much
cleaner than a site
where no fertilizer
was added.
mainly of carbon dioxide, water,
salts, other innocuous products, and
sometimes methane.
Microorganisms are present ev-
erywhere in nature, even in the deep
ocean, and are an integral part of the
earth's natural detoxification process.
Bioremediation technologies harness
this process by promoting the
growth of competent populations of
microorganisms that can biodegrade
contaminants.
Biostimulation and
Bioaugmentation
Some microbes capable of de-
grading target contaminants are often
already present at a hazardous waste
site, although not necessarily in the
numbers required to remediate the
site. In these cases, methods are de-
vised to stimulate the growth and
biodegradative activities of the exist-
ing microbial communities. Such
biostimulation usually involves add-
ing nutrients or oxygen to the con-
taminated material to help the
indigenous microorganisms flourish.
The greater the population of degrad-
ing microorganisms within the con-
trolled remediation area, the faster
and more efficient the biodegrada-
tion process. At present, most sites
being treated with bioremediation
use indigenous microorganisms.
During the summer following
the 1989 Exxon Valdez oil spill in
Alaska's Prince William Sound,
ORD initiated a bioremediation field
demonstration to determine the fea-
sibility of using nutrients to stimu-
late the indigenous microbial
degradation of oil on the Alaskan
shoreline.
The project involved applying
fertilizers containing nitrogen and
phosphorus (nutrients bacteria need
to utilize crude oil hydrocarbons as a
food source) to selected test plots on
oil-covered cobblestone and sand
and gravel beaches. Within two
weeks after applying the fertilizer to
the test plots, scientists began to
observe reductions in the amount of
oil on treated beach surfaces. Non-
treated plots remained as oiled as
they had been at the beginning of the
field study.
During the demonstration, sev-
eral sampling and field testing meth-
ods were used to observe changes in
the composition of the oil, monitor
the movement of added nutrients on
the test beaches, detect changes in
the number of bacteria present, and
assess the degradation of the oil.
This ORD study, which was the
largest project of its kind ever con-
ducted, clearly demonstrated the
capabilities of biostimulation tech-
niques to remediate oil spills in the
field. It has also provided a wealth
of data that will have far-reaching
implications for successfully miti-
gating the effects of future oil spills
worldwide.
In cases where insufficient in-
digenous microorganisms are
present at a site to degrade the target
hazardous wastes even with
-------
biostimulation,
non-indigenous
microorganisms
known to me-
tabolize the pol-
lutants can be
added to the af-
fected material.
Adding species
that are known to
work in concert
with resident
microorganisms
(bioaugmentation)
can result in
faster or more
complete waste
degradation.
For example,
one ORD study
conducted by EPA's Environmental
Research Laboratory in Gulf
Breeze, Florida, demonstrated the
ability of selected non-indigenous
microorganisms to facilitate biodeg-
radation of ground water contami-
nated with creosote and penta-
chlorophenol (PCP). The ground
water was taken from the American
Creosote Works Superfund site in
Pensacola, Florida. Results obtained
from the addition of non-indigenous
microorganisms were compared to
those obtained using only indig-
enous organisms. During the study,
more than 99% of creosote constitu-
ents in the samples and 87% of PCP
were removed by the non-indig-
enous bacteria. When indigenous
organisms were used alone, biodeg-
radation was much less successful.
Bioremediation Potential
The potential use of
bioremediation technologies is sig-
nificant, as federal and state govern-
ments, private industry, and others
responsible for environmental
cleanup efforts add it to their arse-
nals of methods for environmental
reclamation.
To date, bioremediation projects
are in planning stages, undergoing
treatability studies, or in full-scale
operation under federal or state regu-
latory authority at more than 150
sites across the United States. These
include sites identified for cleanup
under the Comprehensive Environ-
mental Response, Compensation, and
Liability Act (CERCLA, otherwise
known as Superfund), the Resource
Conservation and Recovery Act
(RCRA), the Toxic Substances Con-
trol Act (TSCA), and Underground
Storage Tank (UST) regulations. Of
the approximately 1,240 National
Priority List (NPL) sites already
identified for cleanup, many are pos-
sible candidates for bioremediation.
An estimated fifteen percent of
the nation's four to five million un-
derground storage tanks containing
petroleum, heating oil, and other haz-
ardous materials are leaking, con-
taminating the soil around them and
threatening or already contaminating
ground water supplies. As many as
Bioremediation
projects are being
studied, planned, or
are already
implemented in
thirty-six states.
-------
10CH
11
Petroleum Wood Solvents Pesticides Other
Preserving
Wastes
Various types of
contamination are
being treated using
bioremediation at
more than 150 sites
under federal or
state regulatory
authority.
Typical
operational
expenses
(without
operator
labor).
15,000 oil spills occur each year re-
quiring cleanup of contaminated
soils and waters. Additionally, thou-
sands of RCRA facilities are con-
taminated with solvents, wood
preservatives, halogenated aromatic
hydrocarbons (HAHs), and pesticide
wastes. And more than 10,000 pesti-
cide dealerships throughout the
country evidence contamination of
soil and/or ground water. Bioreme-
diation can play a significant role in
the remediation of many of these
sites.
Advantages
Bioremediation has some signifi-
cant advantages when compared to
other remediation technologies. The
most important is the ability of mi-
croorganisms to detoxify hazardous
substances instead of merely trans-
Chemicals 4%
Power for Equip. 36%
Equipment 60%
Source: Nytr, E. K. Ground*** Treatment Technology,
Van Noatrand R«lnhokl Company, Inc. New York.
ferring contaminants from one envi-
ronmental medium to another (such
as from water to the air during air
stripping).
Another major advantage, par-
ticularly for in situ (in place) treat-
ment of soils, sludges, and ground
water, is that bioremediation is usu-
ally less disruptive to the environ-
ment than other technologies used to
remediate hazardous wastes, such as
excavation followed by incineration
and landfilling. And since treatment
is normally accomplished on site,
there is typically no need to trans-
port hazardous materials to another
location.
Finally, the cost of treating a
hazardous waste site using bioreme-
diation technologies can be consid-
erably lower than with other
treatment methods. For example, the
cost of soil bioventing (discussed
later) by a field-scale system has
been estimated at less than $50 per
ton, while incineration costs are
typically more than ten times that
amount.
Limitations
The use of bioremediation is
limited by the need for a greater
understanding of biodegradation
processes, their appropriate applica-
tions, their control and enhancement
in the environment, and engineering
techniques required for broader ap-
plication of the technology. The
EPA recognizes that comprehensive
mechanistic process control, engi-
neering design, and cost data are
also necessary for the full accep-
tance and use of bioremediation by
the technical and regulatory commu-
nities.
Bioremediation Involves More
than Microbes
Although using microorganisms
to degrade hazardous wastes may
-------
seem fairly straightfor-
ward, the technology of
bioremediation is actually
multifaceted and com-
plex. It is based on exten-
sive research to
determine the biochemi-
cal capabilities of specific
microorganisms to inter-
act with specific waste
compounds to success-
fully degrade them.
A great deal of infor-
mation is also required
about the characteristics
of the individual waste
site, including types and distribu-
tion of contaminants, properties of
the contaminated media (soil, sedi-
ments, water, sludge), numbers and
species of indigenous microorgan-
isms, and topographical and subsur-
face geological properties. Once all
of this is known, and it has been
determined that the site is biologi-
cally treatable, a remediation plan is
devised and engineering systems
designed and implemented to ac-
commodate the remediation process
and measure the results.
The study of bioremediation
requires a hybrid of several scien-
tific and-technical disciplines, in-
cluding microbiology, ecology,
biochemistry, analytical chemistry,
chemical engineering, environmen-
tal engineering, geology, mathemat-
ics, statistics, civil engineering, and
risk management. The EPA applies
expertise in all of these fields to
enhance the capabilities of the tech-
nology and match its promise as a
major factor in decontaminating
hazardous waste sites worldwide.
Ability of Microorganisms to
Degrade Wastes
Individual strains of microbes
have the capacity to degrade only
An important part of bioremediation is
identifying the various microbial
species present at a site. A
preliminary step in this process can be
to determine the total number of
strains present by placing diluted
sample material from the site onto an
enriched culture medium. Different
colors appearing on the culture (white,
yellow, and orange in photo) indicate
Hie presence of colonies of different
strains. Colonies can then be
collected from the culture and
subjected to biochemical screening
tests to identify the individual strains.
certain types of compounds. So,
while a given species may very effec-
tively degrade one compound, it may
have no ability to degrade another.
For this reason, understanding the
specific metabolic capabilities of
individual species is important to
effectively match the right microor-
ganisms or group of microorganisms
with the target compound to be reme-
diated.
Bacteria are, on average, one to
two micrometers (millionths of a
meter) in length. At this size, they
interact with hazardous waste com-
pounds on the molecular scale.
Metabolism occurs when the micro-
organisms make contact with
compound molecules and separate
and absorb those useful for their
nutritional and energy needs. Thus,
microorganisms can degrade a toxic
compound by systematically disman-
tling and consuming individual com-
ponents of its molecular structure
until there is nothing left but carbon
dioxide, water, and other innocuous
products.
The breakdown and digestion of
waste compounds during the meta-
bolic process are the result of bio-
chemical reactions catalyzed by
enzymes produced by the microor-
-------
Bioremediation
rate-limiting factors
are different for a
porous beach
environment than
for a wetland or
marsh.
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ganisms. Enzymes, which are com-
plex proteins, are highly specific in
their catalytic behavior, so a given
enzyme is effective for only a par-
ticular type of chemical reaction.
Because of this, the ability of a
microorganism to degrade a particu-
lar substance depends upon its ability
to produce enzymes capable of cata-
lyzing the necessary biochemical
reactions. ORD conducts ongoing
research to identify additional en-
zyme systems and to characterize the
full range of activities of enzymes
already identified for use in bioreme-
diation applications.
EPA research on microbial meta-
bolic processes also provides impor-
tant information about the types of
additional nutrients and energy
sources that individual strains re-
quire for growth, cell division (re-
production), and metabolism to
biodegrade hazardous wastes.
Factors Limiting
Biodegradation
While microorganisms are often
described as microscopic biochemi-
cal reactors, their activities are inti-
mately connected to and shaped by
their external environment. Because
of this, any number of environmental
conditions can slow or stop a biodeg-
radation process even when the
microorganisms have the ability to
otherwise degrade the target com-
pound. For example, the contami-
nated area may be too acidic or
alkaline or the moisture conditions
unfavorable for sufficient microbial
metabolic activity to occur. Some
microorganisms require the presence
of oxygen to live (aerobic), while
others live only in the absence of
free oxygen (anaerobic). In other
cases, the concentration of the target
waste compound may be so high in
the treatment area that it is toxic to
the microorganisms.
ORD research on the external
physical and chemical factors influ-
encing microbial metabolism and
growth is critical for developing effi-
cient and cost-effective bioreme-
diation technologies. A thorough
understanding of such factors is im-
portant for creating optimum envi-
ronmental conditions to stimulate the
metabolic activities of microbial
communities to degrade toxic
wastes.
Before they can design proper
treatment techniques, scientists and
engineers must first determine which
Grant Heilman/Grant Heilman Photography Photo
-------
The parent compound of a PCP congener is comprised of five chlorine molecules (Cl) and one hydroxide
molecule (OH) in a carbon ring structure. In biodegradation, certain aerobic microbes break down the ring
by metabolizing selected individual chlorine molecules, resulting in intermediate products and ultimately
carbon dioxide, water, and chloride.
OH OH OH OH
CI(^S||ClH2O»CI(^Ss||CI ^fc01!^?! 2H > X^jl
Cl OH OH OH
Parent
Intermediate
Intermediate
Intermediate
End Products
factors at a site would likely limit
the rate and extent of biodegrada-
tion. The biodegradation of oil, for
example, requires significant
concentrations of nutrients and
oxygen to proceed at a rate useful
for bioremediation. The effects of
these factors on oil biodegradation
are quite different in a porous beach
environment than a wetland or
marsh. On a porous beach, which is
constantly exposed to high
concentrations of dissolved oxygen
from tidal flows, oil biodegradation
is likely to be limited by an
insufficient supply of nutrients. A
marsh, which is usually rich in
organic carbon and nutrients, is
likely to be limited by insufficient
oxygen. Designing laboratory
studies and microcosm systems to
study methods for optimizing the
rate and extent of biodegradation
are important and cost-effective
components of this ORD research.
Biodegradation Pathway
Microorganisms do not con-
sume all of the digestible molecular
constituents of a toxic compound at
once. Instead, they selectively re-
move and metabolize individual
components until a nondegradable
product is formed or the compound
has been completely degraded. Each
time a molecular component is re-
moved, the nature of the compound
The complexity of the biodegradation process necessitates
innovative tools to accurately and cost-effectively assess
any health and environmental impacts during and after
bioremediation treatment. Scientists from ORD's Health
Effects Research Laboratory in Research Triangle Park,
North Carolina, and the Environmental Monitoring Systems
Laboratory in Cincinnati, Ohio, are developing rapid, novel
bioassays to estimate toxicity to humans and animals
without the need for extensive chemical analysis. The
Environmental Research Laboratory at Gulf Breeze,
Florida, is performing similar studies to assess potential
toxic effects on ecological systems.
Bioassays can be used to examine the effects of any
intermediate and end products produced as a result of
biodegradation when specified microorganisms are grown
on the waste material of interest in a culture medium.
Scientists can use these tests to accurately detect such
toxicological properties as carcinogenicity and
mutagenicity. The estimation of toxicity through the use of
bioassays provides a powerful, cost-effective tool for the
development of bioremediation applications that protect
human health and the environment
Some bioassays test the possible
mutagenicity of target chemicals
using selected indicator bacteria. If a
chemical is mutagenic to these
organisms, there is a probability that
it could also be mutagenic or
carcinogenic to humans and animals.
In one bioassay developed by ORD,
a chemical testing positive (mu-
tagenic) is indicated by the formation
of o-nitrophenol which produces a
yellow color. The more intense yellow
indicates a higher degree of DNA
damage. No color is produced from
chemicals testing negative.
-------
River and lake
bottom sediments
contaminated with
chlorinated aro-
matic compounds
can be treated us-
ing both anaerobic
and aerobic
biotreatments.
is altered, resulting in the formation
of a new substance. Consequently,
metabolism involves a succession of
new substances being formed (inter-
mediates), beginning with the parent
(original) and ending with the final
product (end product). This is known
as the biodegradation pathway.
An important concern of EPA's
bioremediation research is under-
standing the chemical and biochemi-
cal reactions or pathways occurring
during microbial degradation of
hazardous waste compounds. This
allows scientists to ensure that the
intermediate and end products of the
metabolic process are not more toxic
than the original pollutant. Research
has shown that intermediates and end
products of biodegradation are most
often less toxic than the parent com-
pound.
Biodegradation by Microbial
Communities
Some toxic compounds resistant
to complete biodegradation by one
strain of microorganism may be
completely metabolized by a number
of species working in concert. For
example, one species may have the
enzymatic machinery to only par-
tially metabolize the parent com-
pound, resulting in an intermediate
product. Another species may be
able to metabolize the intermediate
product of the first species but lack
the enzymes needed to metabolize
the parent compound.
By themselves, neither species
could totally degrade the toxic sub-
stance. But the combined metabolic
activity of the two results in success-
ful degradation of the compound.
Microbial consortia consisting of
two or more strains of microorgan-
isms are typically required for the
degradation of hazardous wastes.
ORD research in this area is di-
rected toward identifying participat-
ing microbial species, the interactive
and sequential roles played by the
microorganisms, and any solubiliz-
ing agents they may produce to fa-
cilitate biodegradation. A more
thorough understanding of the bio-
chemistry, physiology, and ecology
of these systems will lead to addi-
tional capabilities using consortia
and sequential treatments to detoxify
hazardous compounds.
The coupling of anaerobic
dechlorination with aerobic metabo-
lism has been suggested as a pos-
sible method for reducing the levels
of highly chlorinated polychlorinated
biphenyls (PCBs) in the environ-
ment, and is a good example of us-
ing the combined activity of
different microbial communities to
detoxify wastes. Results from bench-
scale studies at the Environmental
Research Laboratory in Athens,
Georgia, using sediments collected
from the Saginaw River, Ashtabula
River, and the Sheboygan Harbor
and bay area, suggest that PCBs can
be biodegraded under both aerobic
and anaerobic conditions. Aerobic
bacteria can usually degrade only
congeners (members of a family of
Thomas Hovland/Gram Heilman Photography Photo
-------
compounds) with one to five chlo-
rine atoms, while anaerobic bacteria
can degrade only the more highly
chlorinated congeners. This study is
examining the effective biodegrada-
tion of highly chlorinated PCBs in
sediments by sequential anaerobic
and aerobic treatments.
Bioremediation and Genetic
Engineering
Understanding the genetic and
biochemical basis for microbial
biodegradation reactions can lead to
the innovative construction of mi-
crobial gene combinations useful
for degrading persistent toxic
chemicals.
A gene is essentially the
equivalent of a computer program
containing information that controls
specific biological functions of an
organism in relation to its environ-
ment. Manipulating this genetic
machinery can result in an organism
that is better able to degrade a
chemical under specific environ-
mental conditions.
Genetic engineering techniques
can accomplish this manipulation.
This involves identifying and col-
lecting specific strands of DNA
from one or more existing microbial
species, splicing them together, and
inserting the recombinant DNA into
another strain. In this way, addi-
tional survival and metabolic capa-
bilities can be added to the recipient
strain, greatly enhancing its effi-
ciency to degrade target hazardous
compounds in a wide range of envi-
ronmental conditions. To be effec-
tive, the recombinant DNA must be
maintained in the bacterium and be
passed on to subsequent genera-
tions.
For bioremediation purposes,
genetically engineered microorgan-
isms (OEMs) are still in the re-
Will & Deni Mclntyre/Photo Researchers, Inc. Image
search and development stages. The
use of such organisms is regulated by
the Toxic Substances Control Act,
and all genetically altered microbes
undergo rigorous safety reviews to
evaluate any possible risk to human
health or the environment before they
are approved for use in the field. To
date, GEMs have not been used for
site cleanup in the United States.
Genetic engineering research is
an exciting and useful technology
that has significant future potential.
Such research by the EPA may
provide microorganisms and
biodegradation systems that can
destroy persistent, previously
undegradable hazardous toxic com-
pounds in the environment.
Hazardous Waste Site
Characterization
Site characterization identifies
any site-specific problems that must
be addressed in applying bioremedia-
tion cleanup technology. As previ-
ously stated, bioremediation is
affected by the types, levels, and dis-
tribution of contaminants and the
physical nature of the treatment site.
All such factors influence the selec-
tion of treatment constituents and the
engineering methodology for their
delivery and maintenance. Because
Deoxyribonucleic
acid (DNA) is the
heredity molecule.
DNA is a long,
threadlike macro-
molecule In which
purine and
pyrimidine bases
(red and yellow in
image) carry
genetic information
while sugar and
phosphate groups
(green) perform
structural roles.
-------
Scientists conduct
site characterization
activities at a
cleanup site prior to
selecting treatment.
Contour map of a
chlorinated aliphatic
compound plume
found at a National
Priority List
industrial site in St.
Joseph, Michigan.
Don Riepe/Peter Arnold, Inc. Photo
of this, site characterization is a criti-
cal phase of bioremediation technol-
ogy.
To perform site characterization,
scientists and engineers use special-
ized equipment and surveying, sam-
pling, and soil coring techniques to
ascertain all pertinent topographical,
structural, and geologic features of
the site. They also collect numerous
samples that are sent to the labora-
tory for analysis and classification.
Data resulting from these activities
are used to compile site
characterization reports that
present an accurate compos-
ite description of the site and
any contamination it con-
tains.
Defining site geological
conditions and contaminants
present are just part of a
comprehensive site charac-
terization. In subsurface soil
bioremediation, for example,
often a controlling factor is
the rate at which the treat-
ment constituents can be
successfully applied to the
contaminated zone. Soil per-
meability controls the flux of
air or remedial fluids into
the contaminated area. Soil
composition (for example,
clay and organic matter) has a strong
influence on both the rate and extent
of bioremediation during land treat-
ment. The capacity of geologic ma-
terials to hinder the passage of
nutrients often complicates the
implementation of bioremediation.
All such factors must be considered
in the selection of bioremediation
technologies. Sometimes the site
characterization shows physical or
chemical barriers that would prohibit
successful bioremediation.
10
-------
An important aspect of EPA's
research in this area involves devel-
oping innovative site characteriza-
tion methods and systems featuring
improved reliability, efficiency, and
cost-effectiveness. For example, the
only approach now available for
taking a core sample from the sub-
surface is to use a hollow stem au-
ger, extract the cores, and determine
the quantity of contamination by
analytical chemistry techniques.
Soil coring is very expensive and
carries the risk of spreading the
contamination at a site. In collabo-
ration with the U.S. Army Corps of
Engineers, ORD has made recent
progress toward replacing the hol-
low stem auger procedure for some
applications with a cone penetrom-
eter using fiber optic spectroscopy
and on-board computer interpreta-
tion for locating and analyzing sub-
surface waste materials.
ORD is also applying and
evaluating a mobile, hydraulically
driven soil gas and ground water
probe to measure concentrations of
hydrocarbons, oxygen, and carbon
dioxide. Coupled with established
analytical technology, such as field
gas chromatographs and infrared
cells, this probe will be useful for
monitoring and optimizing
bioremediation treatment for sites
selected for in situ bioremediation.
These tools will directly improve
site characterization by providing
efficient and affordable techniques
useful for three-dimensionally
mapping the distribution of
contaminants and measuring the
rate of remediation.
Treatment Design and
Implementation
Site treatment includes engi-
neering design and field implemen-
tation of bioremediation applications
to physically carry out site cleanup.
The techniques and equipment con-
figurations for the remediation of any
hazardous waste site are initially se-
lected in response to the various fac-
tors identified in the site
characterization. Treatment may be
required anywhere from the air above
the site to the deep subsurface, with
treatment in situ and/or in above
ground systems.
In situ Treatment Techniques
In situ bioremediation techniques
are designed to treat the contami-
nated media in place. Such treatment
for soil might include installing irri-
gation or sprinkler systems to deliver
liquid nutrient mixtures directly to
the contaminated region to stimulate
microorganism growth. If the con-
taminant is present in the top twelve
inches of soil, treatment may also
include tilling to aerate the soil. Con-
tamination beneath the surface,
where oxygen may be limited, can be
treated by installing a series of vent-
ing or air injection wells to force air
through the soil at low pressure to
add oxygen (bioventing). Because
bioventing equipment and wells are
A cost-effective
Cone Penetrometer
with fiber optics and
on-board computer
will replace hollow
stem auger soil
coring techniques
for some
applications.
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Surface
Low Monitoring
Rate for
.Air VOCs
Injection /
Biodegradation
n vapors
Soil Gas
Monitoring
Points
Bioventing
introduces oxygen
to contaminated soil
to stimulate aerobic
biodegradation.
Breakdown of the
various types of
media being treated
using bioremedia-
tion at more than
150 sites under
federal or state
regulatory authority.
relatively nonintrusive, this technol-
ogy is especially valuable for treat-
ing contaminated soils in areas
where buildings and underground
utilities cannot be disturbed. In other
cases, injection wells may be used to
introduce nutrients and additional
oxygen supply (such as hydrogen
peroxide) directly to contaminated
areas of underground water supplies.
ORD's Risk Reduction
Engineering Laboratory (RREL) in
Cincinnati, Ohio, is presently
conducting two in situ bioventing
research projects in collaboration
with the United States Air Force.
The first involves a field bioreme-
diation study at Hill Air Force Base
near Salt Lake City, Utah, treating a
150 n
Soil Ground Sediments Sludge Surface
Water Water
site with jet fuel-contaminated soil.
The second bioventing project
involves remediating a jet fuel spill
at Eielson Air Force Base near
Fairbanks, Alaska. These bioventing
studies are generating valuable pilot-
scale performance data and
operational experience for a
technology that can provide an
economic, non-intrusive means of in
situ cleanup of contaminated soils.
Above Ground Treatment
Techniques
EPA scientists and engineers
have also developed several above
ground bioremediation techniques to
treat hazardous wastes on site. These
techniques generally use confined
areas such as lined treatment beds or
enclosed vessels known as bioreac-
tors or biofilters. Bioreactors and
biofilters are engineered systems that
perform the various stages of the
treatment process, such as mixing,
nutrient addition, and separation of
water and solids. Microorganisms
are either freely dispersed or at-
tached as a film to a stationary or
moveable surface within the reactor
or filter. Treatment in reactors may
consist of single or multiple stages.
The particular design of a system
depends primarily on the characteris-
tics of the site, the type of material to
be treated, and the desired treatment
results.
Contaminated materials are
placed into or fed through these sys-
tems for treatment. Above ground
systems provide substantial control
of conditions that influence the level
of microbial growth and metabolism,
such as pH, temperature, oxygen
levels, and nutrient concentrations.
They also allow for maximum con-
tact between the toxic substance and
the microorganisms.
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ORD researchers
have recently devel-
oped an innovative
biofilter to control
volatile (capable of
changing from liquid
into gas) organic
compound (VOC)
emissions from
hazardous wastes and
contaminated liquid
and soil media. The
highly efficient
system produces
complete biode-
gradation of the VOCs
entering the biofilter
in as little as two to
six minutes under
aerobic conditions.
Anaerobic ex-
panded-bed reactors,
using granular
activated carbon
(GAC) as the
microorganism
support medium, have
been developed for
efficient removal of
chlorinated and non-chlorinated
hydrocarbons and other
representative hazardous organics
in contaminated aqueous wastes and
leachates.
Monitoring and Performance
Assessment
Built into the site remediation
plan is a system for continually
monitoring and assessing the
progress of the bioremediation
treatment and any residual impact
to human health or the environment.
Research is underway to develop
techniques that will streamline the
process and reduce costs by provid-
ing the most information with the
least effort. Such systems might
include strategically placed moni-
toring wells, various types of moni-
toring equipment, and numerous
sampling points. Scientists collect
and analyze data obtained from these
sources to continually assess the bio-
degradation of the target compounds.
ORD has
developed a
highly efficient Air
Biofilter system
that produces
complete
biodegradation of
VOCs under
aerobic condi-
tions.
Site personnel take
samples at
monitoring points to
measure bioreme-
diation progress.
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ORD has
developed an
integrated biore-
mediation research
program to advance
the technology.
Research is designed to improve
measures of biodegradation activity.
Based on data obtained from moni-
toring activities, site remediation
personnel can make any necessary
adjustments to the treatment process,
such as increasing the volume of
nutrient supplements, to maintain
optimal performance.
Bioremediation Used with
Other Technologies
Many hazardous waste sites con-
tain complex mixtures of persistent
organic and inorganic contaminants
that can be cleaned up only by a
combination of treatment techniques.
For example, highly chlorinated
wastes can be effectively treated
using chemical methods to dechlori-
nate the compounds followed by
bioremediation to complete detoxifi-
cation. ORD researchers are devel-
oping methods to combine various
physical, chemical, and biological
treatment technologies, and compar-
ing the effectiveness of the various
combinations.
ORD Bioremediation
Program
The need to clean up ha/.ardous
waste sites as a national priority has
accelerated the development of
bioremediation technologies. ORD
has developed an integrated
Bioremediation Program to advance
the understanding, development, and
application of bioremediation. The
program has been designed to strike a
balance between basic research
activities leading to a fundamental
understanding of biodegradation
processes and engineering activities
leading to practical environmental
cleanup applications.
Research, development, and field
evaluations are implemented through
the efforts of a multi-disciplinary
staff of Agency scientists and engi-
neers and are coordinated and di-
rected by the Biosystems Technology
Development Steering Committee.
This committee, and the broader
group of scientists and engineers it
represents, constitutes a unique
resource in science, innovation.
14
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creativity, and responsiveness to
environmental cleanup needs. The
program is supplemented by
extramural research carried out in
concert with other federal agencies,
states, contractors, and academic
institutions under EPA-funded
cooperative agreements, contracts,
and interagency agreements.
Bioremediation Program
Objectives
The overall goals of ORD's
Bioremediation Research Program
include the following:
• Identify and characterize
biodegradation processes that
may be used in the treatment of
contaminated surface waters,
ground water, sediments, surface
and subsurface soils, and gases
• Define, evaluate, optimize, and
demonstrate engineering systems
necessary for application of the
technology to detoxify pollutants
in situ, on-site, or at centralized
treatment facilities
• Develop process-based
mathematical models to evaluate
potential treatment scenarios and
provide a basis for tailoring
bioremediation actions to variable
chemical contaminants and
environmental factors
• Provide protocols and technical
assistance to site cleanup
managers in selecting appropriate
bioremediation technologies for
different EPA, regional, state, and
local programs
• Formulate a waste site-directed
planning framework to
characterize sites as suitable for
bioremediation
• Develop feedback mechanisms
for integrating field information
The Bioremediation Research Program draws on
scientists and engineers from the following ORD
laboratories and organizations:
• Environmental Research Laboratory - Athens, GA
• Environmental Research Laboratory - Gulf Breeze, FL
• Health Effects Research Laboratory - Research
Triangle Park, NC
• Risk Reduction Engineering Laboratory -
Cincinnati, OH
• R.S. Kerr Environmental Research Laboratory -
Ada, OK
• Center for Environmental Research Information -
Cincinnati, OH
from ongoing hazardous waste
site cleanup efforts into the re-
search and development process
• Transfer research and technical
information to the user commu-
nity through investigators'
meetings, bioremediation work-
shops, comprehensive resource
documents, and a national data-
base on bioremediation field
applications
Additional Program
Components
Additional components of
EPA's Bioremediation Program are
the Bioremediation Action
Committee and Bioremediation Field
Initiative.
The Bioremediation Action
Committee (BAG)
ORD chairs and oversees the
Bioremediation Action Committee, a
working affiliation of experts from
government, industry, academia, and
the public dedicated to expanding the
use of bioremediation in the treat-
ORD provides
technical
assistance to EPA
regional offices
and individual
state regulatory
agencies
overseeing
bioremediation
projects or
considering the
use of bioreme-
diation. Technical
Support Centers
in Ada,
Oklahoma, and
Cincinnati, Ohio,
provide
assistance with
site character-
ization, treat-
ability study
design, and inter-
pretation of data.
15
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The Bioremediation Field Initiative Is
Evaluating Three Different Bioremediation
Technologies In Progress at a Superfund
Cleanup Site In Llbby, Montana
Soil and ground water at the Champion
International Superfund Site in Llbby, Montana, are
contaminated by creosote, PAHs, and PCP as a
result of past wood treatment operations. The total
estimated soil volume requiring treatment today is
45,000 cubic yards (uncompacted), while the
plume of contaminated ground water from one of
two aquifers at the site extends more than one
mile.
Three different bioremediation technologies have
been initiated to clean the site, including biological
treatment of soils in a prepared-bed land treatment
unit (LTU), oil/water separation of ground water
followed by biological treatment in a fixed-film
bioreactor, and in situ biotreatment of the aquifer.
Under the Bioremediation Field Initiative, ORD
scientists from EPA's R. S. Kerr Environmental
Research Laboratory in Ada, Oklahoma, provide
technical support for remedial activities at the Libby
Site. This includes technical direction and oversight
for planning, data interpretation, and reporting
results from performance evaluation studies being
conducted for each of the bioremediation
processes in operation.
ment, control, and prevention of en-
vironmental contamination. The
BAG serves as an important forum
for sharing information and collabo-
rative actions across diverse organi-
zations to advance the science and
practical field application of biore-
mediation.
BAC activities include promot-
ing the increased acceptance and use
of bioremediation, developing stan-
dard protocols for testing bioreme-
diation products and techniques, and
coordinating the incorporation of
bioremediation in oil and hazardous
substance contingency response
plans across the United States. The
committee also identifies priority
research needs, investigates pollu-
tion prevention applications of biore-
mediation, promotes education
curricula to adequately prepare sci-
entists and engineers for the field,
and facilitates information exchange
between EPA and other interested
parties on developments and issues
regarding federal regulations affect-
ing bioremediation.
Bioremediation Field Initiative
The Bioremediation Field Initia-
tive is a cooperative effort by ORD,
EPA's Office of Solid Waste and
Emergency Response (OSWER),
regional offices, other federal agen-
cies, state agencies, industry, and
several universities. The focus of
this program is to expand the
nation's field experience in bioreme-
diation techniques. Objectives are to
more fully assess and document the
performance of full-scale field appli-
cations and regularly provide infor-
mation on treatability studies,
design, operation, and costs of ongo-
ing bioremediation projects. Another
important objective is providing
technical assistance to site managers.
The initiative is currently tracking
16
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bioremediation activities at more
than 150 Superfund, RCRA, and
Underground Storage Tank sites.
Conclusion
The growing volume of persis-
tent, difficult-to-treat waste
materials accumulating in the
environment over the past several
decades has contributed to a
national pollution problem that
affects virtually every community.
The United States Environmental
Protection Agency is fully
committed to reducing, eliminating,
or preventing waste materials that
threaten human health and the envi-
ronment. In support of this commit-
ment, the Office of Research and
Development will continue its
efforts to conduct critical research
on cost-effective methods for
degrading such materials at their
source and in the environment.
Because bioremediation can be
an effective, non-disruptive, and
cost-efficient means of reducing or
eliminating many toxic materials, it
should have an ever-increasing role
in the successful remediation of
hazardous waste and chemical spill
sites around the world. The United
States is already the world leader in
the field implementation of biore-
mediation technologies. Through its
own concerted efforts and activities
in collaboration with other pro-
grams, ORD will continue to lead in
the development, application, and
assessment of the technology.
Collaborative Programs
Other programs contributing to the research and
development of bioremediation technologies include the
following:
Superfund Innovative Technology Evaluation
Program
EPA's Superfund Innovative Technology Evaluation
(SITE) Program supports the development of innovative
treatment technologies for hazardous waste remediation
and monitoring/measurement technologies for evaluating
the nature and extent of waste site contamination. These
include bioremediation technologies. Under the SITE
Program, EPA enters into cooperative agreements with
technology developers who refine their innovative
technologies at the bench-, pilot-, or field-scale and may
demonstrate them, with support from EPA, at hazardous
waste sites. The SITE Program collects and publishes
engineering, performance, and cost data to aid in future
decision-making for hazardous waste site remediation.
The program is administered by ORD and supported by
the Office of Solid Waste and Emergency Response.
Hazardous Substance Research Centers Program
The five cooperative, multi-university Hazardous
Substance Research Centers (HSRC) form an integrated
national program of basic and applied research,
technology transfer, and training. The centers focus on
managing hazardous substances and promoting long-
term exploratory research to find innovative ways to
remediate them. Numerous projects are currently
underway exploring bioremediation of contaminated soils
and ground water.
Federal Technology Transfer Act Program
The Federal Technology Transfer Act (FTTA) Program
supports cooperative research and development
agreements between federal laboratories, industry, and
academic institutions to develop and commercialize
innovative technologies. Such agreements have resulted
in the development and field application of several
bioremediation technologies and expanded the database
of chemicals known to be biodegradable.
17
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One of the pilot-scale technologies evaluated by EPA under the Superfund
Innovative Technology Evaluation Program utilizes anaerobic bacterial consort/a
in a bioreactor (bottom photo) to treat soils contaminated with the pesticide
dinoseb (encircled yellow material in soil-top photo). The technology can reduce
this pollutant to less than the detection limit in soils and has also been shown to
be effective for biodegrading trinitrotoluene (TNT).
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EPA Publications
The following reference materials provide more detailed information
about the subjects discussed in this document. Copies of these refer-
ences may be requested at no charge (while supplies are available)
from the EPA's Center for Environmental Research Information (CERI).
Once the CERI inventory is exhausted, clients will be directed to the
National Technical Information Service (NTIS) where documents may
be purchased.
Alaskan Oil Spill Bioremediation Project, EPA/600/8-89/073.
Bioremediation Field Initiative Fact Sheets, EPA/540/F-92/012A-J.
Bioremediation in the Field (quarterly newsletter-latest issue
August 1993, EPA/540/N-93/002).
Bioremediation of Hazardous Wastes (1990), EPA/600/9-90/041.
Bioremediation of Hazardous Wastes (1991), EPA/600/9-91/036.
Bioremediation of Hazardous Wastes (1992), EPA/600/R-92/126.
Guide for Conducting Treatability Studies Under CERCLA: Aerobic
Biodegradation Remedy Screening-Interim Guidance,
EPA/540/2-91/013A.
Microbial Decomposition of Chlorinated Aromatic Compounds,
EPA/600/2-86/090.
The Federal Technology Transfer Act-Opportunities for Cooperative
Biosystems Research and Development with the U.S. EPA,
CERI-90-114.
Understanding Bioremediation-A Guidebook for Citizens,
EPA/540/2-91/002.
Bioremediation Resource Guide, EPA/542/B-93/004*
* Distributed through the National Center for Environmental Publications and
Information (NCEPI): Telephone: (513) 891-6561; Fax: (513) 891-6685
Center for Environmental Research Information (CERI)
U.S. Environmental Protection Agency
26 W. Martin Luther King Drive
Cincinnati, OH 45268
Phone: (513) 569-7562 FAX: (513) 569-7566
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