United States
Environmental Protection
Agency
ineering Issue
In Situ and Ex Situ Biodegradation Technologies
for Remediation of Contaminated Sites
Index
1.0 PURPOSE
2.0 INTRODUCTION
3.0 TECHNOLOGY DESCRIPTION
3.1 In Situ Bioremediation
3.1.1. Intrinsic In Situ Bioremediation
3.1.2. Enhanced In Situ Bioremediation
3.2 Ex Situ Bioremediation
3.2.1. Solids
3.2.2. Solid-Liquid Mixtures
3.2.3. Liquids
4.0 TECHNOLOGY SELECTION FACTORS
5.0 SUMMARY
6.0 ACKNOWLEDGEMENTS
7.0 REFERENCES
1.0 PURPOSE
The U.S. Environmental Protection Agency (EPA) Engineering Is-
sues are a new series of technology transfer documents that summa-
rize the latest available information on selected treatment and site re-
mediation technologies and related issues. They are designed to help
remedial project managers (RPMs), on-scene coordinators (OSCs),
contractors, and other site managers understand the type of data and
site characteristics needed to evaluate a technology for potential ap-
plicability to their specific sites. Each Engineering Issue document is
developed in conjunction with a small group of scientists inside the
EPA and with outside consultants and relies on peer-reviewed litera-
ture, EPA reports, Internet sources, current research, and other perti-
nent information. For this Engineering Issue paper, the reader is as-
sumed to have a basic technical background and some familiarity with
bioremediation. Those readers interested in a more basic discussion of
bioremediation should consult the A Citizen's Guide to Bioremediation
(EPA, 200la).
The purpose of this Engineering Issue paper for bio degradation tech-
nologies is to summarize current information on bioremediation and
to convey that information clearly and concisely to site managers. The
Table of Contents indicates the types of information covered in this
Engineering Issue paper, and this information relies, wherever feasible,
on independently reviewed process performance information. In an
effort to keep this Engineering Issue paper short, important informa-
tion is summarized, while references and Internet links are provided
for readers interested in additional information; these Internet links,
verified as accurate at the time of publication, are subject to change.
2.0 INTRODUCTION
Bioremediation is a grouping of technologies that use microbiota
(typically, heterotrophic bacteria and fungi) to degrade or transform
hazardous contaminants to materials such as carbon dioxide, water,
inorganic salts, microbial biomass, and other byproducts that may
be less hazardous than the parent materials. Biological treatment has
been a major component for many years in the treatment of mu-
nicipal and industrial wastewaters. In recent years, biological mecha-
nisms have been exploited to remediate contaminated ground water
and soils (EPA, 1998a; EPA, 2000). This Engineering Issue paper fo-
cuses on bioremediation technologies for treating contaminated soils,
sediments, sludges, ground water, and surface water since these are
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the matrices typically found at contaminated sites. Treat-
ments for municipal wastewater, industrial wastewater,
and storm water will not be discussed.
During the late 1970s, 1980s, and early 1990s, wastewa-
ter treatment1 and composting technologies were adapted
to new applications. At that time, bioremediation of any
hazardous constituents or waste was considered innova-
tive. However, numerous applications of bioremediation
are now widely accepted as a remedial alternative and are
in wide use at sites contaminated with petroleum prod-
ucts and/or hazardous wastes. Some bioremediation tech-
nologies, such as cometabolic bioventing, are still in de-
velopment and should be considered innovative. Other
bioremediation technologies, such as anaerobic biovent-
ing, are current topics of research. Since bioremediation
technologies that fall into the innovative or research cat-
egory have limited field implementation and effectiveness
data, additional site assessment and treatability studies
may be needed to confirm that a selected technology will
be effective at a specific site.
According to the EPA Office of Solid Waste and Emer-
gency Response annual treatment technology status re-
port (10th Edition) covering the period of Fiscal Year
(FY) 1982 through FY 1999, bioremediation was planned
or implemented for source control and ground water re-
mediation for 105 Superfund Remedial Action and 51
Superfund Removal Action projects. In some cases, bio-
remediation was applied at multiple operable units on a
site, each of which is included as a project (EPA, 2001b).
The following contaminants have been bioremediated
successfully at many sites:
Halogenated and non-halogenated volatile organic
compounds (VOCs)
Halogenated and non-halogenated semi-volatile or-
ganic compounds (SVOCs).
Contaminants with a more limited bioremediation per-
formance include:
Polycyclic aromatic hydrocarbons (PAHs)
Organic pesticides and herbicides
Polychlorinated biphenyls (PCBs).
Although the applications of bioremediation under the
EPA Superfund Program, as discussed above, involve
some of the most difficult sites and well-documented site
surveys, these examples represent only a fraction of the
bioremediation applications nationwide and worldwide.
Larger numbers of sites are handled under the Resource
Conservation and Recovery Act (RCRA), state-led re-
mediation programs, leaking underground storage tank
(LUST) programs, and state voluntary cleanup programs.
For example, a survey in 2001 showed that biodegrada-
tion technologies, such as land farming and biopiles, are
applied at 33% of the soil LUST sites. The same survey
showed that in situ bioremediation, biosparging, and tech-
nologies that may use bioremediation, such as monitored
natural attenuation, are used on 79% of the ground water
plumes at LUST sites (Kostecki and Nascarella, 2003).
However, applications of various bioremediation technol-
ogies at other site types are not summarized, so their use
is difficult to characterize.
Bioremediation remains an active field of technology re-
search and development at both the laboratory and field
scale. For example, applications to chlorinated aliphatic
hydrocarbons (CAHs), perchlorate, and methyl tert-bu-
tyl ether (MTBE) were developed rapidly in recent years.
Contaminants with a more limited bioremediation per-
formance record include:
Applications to additional contaminants and ma-
trices, such as (but not limited to) trinitrotoluene
(TNT), hexahydro-l,3,5-trinitro-l,3,5-triazine
(RDX), pesticides and herbicides, and dense non-
aqueous phase liquids (DNAPLs)
Delivery of treatment in difficult media (i.e., frac-
tured bedrock or tight clays)
Refinement of strategies for cost-effective system de-
sign and operation.
The field of bioremediation can be divided into several
broad categories. For example, bioremediation technolo-
gies may be applied to in situ or ex situ media. In situ
processes treat soils and ground water in place, without
removal. This approach may be advantageous since the
costs of materials handling and some environmental im-
pacts may be reduced. However, in situ processes may be
limited by the ability to control or manipulate the physi-
cal and chemical environment in place. An example of
an in situ bioremediation technology is aerobic biovent-
ing, which has been used at many sites to treat subsurface
soils contaminated by fuels. In aerobic bioventing, air is
typically injected into the subsurface to facilitate aerobic
metabolism of hydrocarbons. Ex situ processes involve the
removal of the contaminated media to a treatment area.
Historical information on the development of bioremediation can be found in Martin and Gershuny, 1992; Section 2.3 of EPA, 1993a; and Bradley, 2003.
Engineering Issue
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Examples of ex situ processes include land treatment and
composting. In these processes, soils are excavated, mixed
with amendments, and operated in a manner that facili-
tates degradation of the contaminants of concern.
Another way to divide the bioremediation field is based
on additives to environmental media. Intrinsic bioremedia-
tion depends on indigenous microflora to degrade con-
taminants without any amendments (EPA, 2000). This
approach is used in situ and takes advantage of pre-exist-
ing processes to degrade hazardous wastes. Intrinsic bio-
remediation requires careful site assessment and monitor-
ing to make sure that the ongoing processes are protective
of environmental receptors. Temperature, pH, and other
factors may also be adjusted and monitored to enhance
bioremediation. Alternatively, enhanced bioremediation
facilitates biodegradation by manipulating the microbial
environment, typically by supplying chemical amend-
ments such as air, organic substrates or electron donors,
nutrients, and other compounds that affect metabolic re-
actions (EPA, 2000). Enhanced bioremediation may also
be called biostimulation when only chemical amendments
are added. Examples of biostimulation include biovent-
ing, land farming or land treatment, biopiles, composting,
and sometimes anaerobic reductive dechlorination. Bio-
stimulation technologies may be applied to in situ or ex situ
situations and may be used to treat soil and other solids,
ground water, or surface water. In some cases, bioaugmen-
tation, which involves the addition of microbial cultures,
is used to enhance biotreatment. Bioaugmentation may
be needed for specific contaminants that are not degraded
by the indigenous organisms. Bioaugmentation is almost
always performed in conjunction with biostimulation. For
example, bioaugmentation has been used at some chlori-
nated solvent sites as a modification of anaerobic reductive
dechlorination when indigenous microbes were unable to
completely dechlorinate the contaminants of concern.
In bioremediation, fundamental biological activities are
exploited to degrade or transform contaminants of con-
cern. The biological activity to be exploited depends on
the specific contaminants of concern and the media where
the contamination is located. For example, in aerobic en-
vironments, many microbes are able to degrade organic
compounds, such as hydrocarbons. These microbes gain
energy and carbon for building cell materials from these
biochemical reactions. At many sites with fuel contami-
nation, the amount of oxygen present limits the extent of
biotreatment. Thus, by adding oxygen in the form of air,
contaminant degradation proceeds directly.
In cometabolism, microbes do not gain energy or carbon
from degrading a contaminant. Instead, the contaminant
is degraded via a side reaction. Technologies based on co-
metabolism are more difficult to use since the microbes do
not benefit from the desired reactions. Cometabolic bio-
venting is an example of cometabolism. In this technol-
ogy, microbes may be fed propane, and they degrade tri-
chloroethylene (TCE) or less chlorinated ethenes as well.
Depending on the contaminant of concern and the me-
dia, a technology may exploit aerobic or anaerobic metab-
olism. Aerobic metabolism is more commonly exploited
and can be effective for hydrocarbons and other organic
compounds. Many organisms are capable of degrading
hydrocarbons using oxygen as the electron acceptor and
the hydrocarbons as carbon and energy sources. In some
cases, contaminants are aerobically degraded to carbon
dioxide, water, and microbial biomass, but in other cases,
the microbes do not completely degrade contaminants.
Aerobic technologies may also change the ionic form of
metals. If a site contains mixed metal and organic wastes,
it is necessary to consider whether the oxidized forms of
the metal species (such as arsenic) will be environmen-
tally acceptable.
Anaerobic metabolism involves microbial reactions occur-
ring in the absence of oxygen and encompasses many pro-
cesses including fermentation, methanogenesis, reductive
dechlorination, sulfate-reducing activities, and denitrifica-
tion. Depending on the contaminant of concern, a subset
of these activities may be cultivated. In anaerobic metabo-
lism, nitrate, sulfate, carbon dioxide, oxidized metals, or
organic compounds may replace oxygen as the electron ac-
ceptor. For example, in anaerobic reductive dechlorination,
chlorinated solvents may serve as the electron acceptor.
Phytoremediation or phytotechnology, which involves
the use of plants to remediate contaminated media, is
not discussed in this Engineering Issue paper because this
technique can involve a number of physical and chemical
processes in addition to, or in place of, bioremediation.
More information on phytoremediation can be found
at the EPA Web site (http://www.clu-in.org/techfocus/
default.focus/sec/Phytoremediation/cat/Overview/) or at
the Interstate Technology & Regulatory Council (ITRC)
Web site (http://www.itrcweb.org/gd Phyto.asp).
When selecting a bioremediation technology, it is impor-
tant to consider the contaminants of concern, contami-
nated matrix, potential biological pathways to degrade
a contaminant, and current conditions at a site. For ex-
In Situ and Ex Situ Biodegredation Technologies
Engineering Issue
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ample, TCE can be degraded via aerobic and anaerobic
mechanisms. If ground water is contaminated with TCE,
current ground water conditions may be helpful in decid-
ing which biological mechanism to exploit. If ground wa-
ter is already anaerobic, then anaerobic reductive dechlo-
rination may be the best approach. However, if the TCE
plume is diffuse and the ground water is aerobic, it may
be possible to use cometabolic technologies.
A key concept in evaluating all bioremediation technolo-
gies is microbial bioavailability. Simply stated, if the con-
taminant of concern is so tightly bound up in the solid
matrix (either chemically or physically) that microorgan-
isms cannot access it, then it cannot be bioremediated.
However, low microbial bioavailability does not imply an
absence of risk; compounds may be available to environ-
mental receptors depending on the receptor and routes
of exposure. For example, if a child ingests contaminated
soil, digestive processes may release contaminants that
were not available to microbes.
Many of the guidance documents discussed in this Engi-
neering Issue paper address bioavailability from a practi-
cal engineering perspective. A more theoretical summary
of the implications of bioavailability is presented in Luthy
et al. (1997) and Alexander (2000). Implementation of
enhanced bioremediation technologies can, in some cases,
transfer mass from the solid phase to the mobile (i.e., wa-
ter) phase through a number of mechanisms, including
the generation of biosurfactants.
Thus, when selecting a bioremediation technology for a
specific site, it is prudent to consider the contaminants of
concern, potential degradation intermediates and residu-
als of the contaminants, co-contaminants, environmental
receptors, routes of exposure, and buffer zones between
contamination and receptors. Bioremediation technolo-
gies have proven to be protective and cost-effective solu-
tions at many sites. However, conditions at a specific site
may not be appropriate. In addition, worker safety issues
are a consideration in selection, design, and operation of
bioremediation technologies. These technologies may in-
volve the use of strong oxidants and/or highly reactive,
potentially explosive chemicals.
3.0 TECHNOLOGY DESCRIPTION
This section discusses in situ and ex situ bioremediation.
Technologies within each of these broad categories are
presented, including representative process schematics or
illustrations. Note that there are multiple vendors of some
technologies, each applying proprietary components or
processes to their particular technology. The continually
changing nature of bioremediation and the space limita-
tions of this Engineering Issue paper preclude compre-
hensive presentation of such information. Additional in-
formation on remediation technologies and links to other
sources are available on the World Wide Web, including:
Federal Remediation Technologies Roundtable
(FRTR) at http://www.frtr.gov/
Air Force Center for Environmental Excellence (AF-
CEE) Technology Transfer Program at
http://www.afcee.brooks.af.mil/products/techtrans/
treatmenttechnologies.asp
ITRC guidance documents and case studies at
http://www.itrcweb.org/gd.asp and
http://www.itrcweb.org/successstories.asp.
For information on specific compounds, a Biocatalysis/
Biodegradation Database developed by the University of
Minnesota may be helpful and can be found at
http://umbbd.ahc.umn.edu/.
3.1 In Situ Bioremediation
There are two major types of in situ bioremediation: in-
trinsic and enhanced. Both rely on natural processes to
degrade contaminants with (enhanced) or without (in-
trinsic) amendments.
In recent years, in situ bioremediation concepts have been
applied in treating contaminated soil and ground water.
Removal rates and extent vary based on the contaminant of
concern and site-specific characteristics. Removal rates also
are affected by variables such as contaminant distribution
and concentration; co-contaminant concentrations; indig-
enous microbial populations and reaction kinetics; and
parameters such as pH, moisture content, nutrient sup-
ply, and temperature. Many of these factors are a function
of the site and the indigenous microbial community and,
thus, are difficult to manipulate. Specific technologies may
have the capacity to manipulate some variables and may be
affected by other variables as well; these specific issues are
discussed with each technology in the following sections
(AFCEE, 1996; EPA, 1998a; EPA, 2000; FRTR, 2003).
When in situ bioremediation is selected as a treatment, site-
monitoring activities should demonstrate that biologically
mediated removal is the primary route of contaminant
removal. Sampling strategies should consider appropriate
analytes and tests, as well as site heterogeneity. In some
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In Situ and Ex Situ Biodegradation Technologies
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cases, extensive sampling may be required to distinguish
bioremediation from other removal mechanisms or statisti-
cal variation. Small-scale treatability studies using samples
from the contaminated site may also be useful in demon-
strating the role that biological activity plays in contami-
nant removal (EPA, 1995b; EPA, 1998a; EPA, 2000).
3.1.1 Intrinsic In Situ Bioremediation
Intrinsic bioremediation relies on natural processes to de-
grade contaminants without altering current conditions
or adding amendments. Intrinsic bioremediation may
play a role in monitored natural attenuation (MNA) sites,
which is a broader term defined by the National Research
Council (NRC) and EPA as "biodegradation, dispersion,
dilution, sorption, volatilization, radioactive decay, and
chemical or biological stabilization, transformation or de-
struction of contaminants" (NRC, 2000; EPA, 1999).
Natural attenuation (NA) relies on natural physical,
chemical, and biological processes to reduce or attenuate
contaminant concentrations. Under favorable conditions,
NA will reduce the concentration, mass, toxicity, mobil-
ity, and/or volume of contaminants in soil and ground
water. Natural processes involved in NA include dilution,
dispersion, sorption, volatilization, chemical reactions
such as oxidation and reduction, biological reactions, and
stabilization. EPA prefers those processes that degrade
contaminants and expects that NA will be most appropri-
ate for subsurface plumes that are stable. Some processes
have undesirable results, such as creation of toxic degrada-
tion products or transfer of contaminants to other media
as noted in the Seminar Series on Monitored Natural At-
tenuation for GroundWater (EPA, 1998a).
Implementing MNA requires a thorough site assessment
and development of a conceptual model of the site. After
determining the presence of a stable or shrinking plume,
site-specific, risk-based decisions using multiple lines of
evidence may facilitate implementation of MNA at a
site. While MNA is somewhat passive in that nothing is
being added to the contamination zone, MNA requires
active monitoring, which should be included as part of
the design plan for a site. In some cases, such long-term
monitoring may be more expensive than active remedia-
tion. MNA is only applicable to carefully controlled and
monitored sites and must reduce contaminant concentra-
tions to levels that are protective of human health and
the environment in reasonable timeframes (EPA, 1998a).
Depending on site-specific conditions, MNA may be a
reasonable alternative for petroleum hydrocarbons as well
as chlorinated and non-chlorinated VOCs and SVOCs
(EPA, 1999a; EPA, 1999b).
Good overviews of natural attenuation are provided by:
Natural Attenuation for Groundwater Remediation
(NRC, 2000)
The MNA page of the AFCEE Technology Transfer
Program Web site at
http://www.afcee.brooks.af.mil/products/techtrans/
monitorednaturalattenuation/default.asp.
ITRC in situ bioremediation publications at
http://www.itrcweb.org/gd ISB.asp.
Detailed protocols for evaluation of natural attenuation at
sites with petroleum hydrocarbon and CAH contamina-
tion can be found at
http://www.afcee.brooks.af.mil/products/techtrans/
monitorednaturalattenuation/Protocols.asp.
3.1.2 Enhanced In Situ Bioremediation
Enhanced bioremediation can be applied to ground wa-
ter, vadose zone soils, or, more rarely, aquatic sediments.
Additives such as oxygen (or other electron acceptors),
nutrients, biodegradable carbonaceous substrates, bulk-
ing agents, and/or moisture are added to enhance the ac-
tivity of naturally occurring or indigenous microbial pop-
ulations (FRTR, 2003).
3.1.2.1 Vadose Zone Soil Remediation
While the fundamental biological activities exploited by
in situ bioremediation may occur naturally, many sites
will require intervention to facilitate cleanup. For exam-
ple, the addition of organic substrates, nutrients, or air
may provide the appropriate environment for specific mi-
crobial activities or enhanced removal rates. In general,
hydrocarbons and lightly chlorinated contaminants may
be removed through aerobic treatment. Highly chlorinat-
ed species are degraded primarily through anaerobic treat-
ment. Both anaerobic and aerobic treatment may occur
through direct or cometabolic pathways (EPA, 2000).
The primary in situ biological technology applicable to
the unsaturated zone is bioventing, which is categorized
as either aerobic, cometabolic, or anaerobic depending on
the amendments used.
In Situ and Ex Situ Biodegredation Technologies
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3.1.2.1.1 Aerobic Bioventing
Bioventing has a robust track record in treating aerobi-
cally degradable contaminants, such as fuels. In aerobic
bioventing, contaminated unsaturated soils with low oxy-
gen concentrations are treated by supplying oxygen to fa-
cilitate aerobic microbial biodegradation. Oxygen is typi-
cally introduced by air injection wells that push air into
the subsurface (see Figure 3-1); vacuum extraction wells,
which draw air through the subsurface, may also be used.
When building foundations or similar structures are close
to the site, the extraction mode may be used to avoid the
buildup of contaminated, and possibly explosive, vapors
in the building basements. Extracted gases may require
treatment since volatile compounds may be removed from
the ground. Compared with soil vapor extraction (SVE),
bioventing employs lower air flow rates that provide only
the amount of oxygen required to enhance removal. Op-
erated properly, the injection of air does not result in the
release of the contaminants to the atmosphere through
volatilization because of these low flow rates (AFCEE,
1996; EPA, 2000; FRTR, 2003).
Bioventing is designed primarily to treat aerobically bio-
degradable contaminants, such as non-chlorinated VOCs
and SVOCs (e.g., petroleum hydrocarbons), that are lo-
cated in the vadose zone or capillary fringe (EPA, 2000;
FRTR, 2003). The U.S. Air Force Bioventing Initiative
and the EPA Bioremediation Field Initiative demonstrat-
ed that bioventing was effective under a wide variety of
site conditions at about 125 sites. The experience from
bioventing demonstrations at these sites was condensed
into a manual, Bioventing Principles and Practice (EPA
1995a), which provides information about the applicabil-
ity of bioventing and outlines its use and design (AFCEE,
1996). Data collected during the bioventing demonstra-
tions also provide information about the rates of con-
taminant removal observed. In addition to the variables
Figure 3-1. Aerobic bioventing in injection mode. (Adapted from
EPA, 2004c)
discussed initially, bioventing rates and system design are
affected by soil gas permeability, soil water content, depth
to contamination, and oxygen supply and radius of in-
fluence (EPA, 2000; FRTR, 2003). The costs for aerobic
bioventing are about $50/cubic yard (AFCEE, 1996).
Based on experience gained to date in applying bioventing
to fuels, site heterogeneity is a principal impediment to
establishing that biological activity is the principal mode
of removal at these sites. Measurements of the rate and
amount of contaminant removed, oxygen supply, and car-
bon dioxide generation, as well as mass balances relating
to these three amounts, may be useful in establishing bio-
remediation as the primary mechanism of removal. For
sites where other contaminants are to be treated by bio-
venting, other factors may be considered in establishing
biological activity as the primary mechanism of removal
(AFCEE, 1996; EPA, 2000; FRTR, 2003).
Regulatory acceptance of this technology has been ob-
tained in 30 states and in all 10 EPA regions (FRTR,
2003). The use of this technology in the private sector is
increasing following the U.S. Air Force Bioventing Initia-
tive and the EPA Bioremediation Field Initiative.
In addition to fuels treatment, aerobic bioventing has
treated a variety of other contaminants including non-
halogenated solvents such as benzene, acetone, toluene,
and phenol; lightly halogenated solvents such as 1,2-di-
chloroethane, dichloromethane, and chlorobenzene; and
SVOCs such as low-molecular-weight PAHs. Since the
experience with these other types of contaminants is more
limited, laboratory- and pilot-scale studies may be needed
to evaluate effectiveness, design the bioventing system,
and estimate treatment times.
Bioventing has proven to be a useful technology at many
sites under a variety of conditions, but like all technolo-
gies, bioventing has some limitations. One set of biovent-
ing limitations involves the ability to deliver oxygen to
the contaminated soil. For example, soils with extremely
high moisture content may be difficult to biovent because
of reduced soil gas permeability. Similarly, low-perme-
ability soils also may pose some difficulties for biovent-
ing because of a limited ability to distribute air through
the subsurface. In both cases, the design of the bioventing
system may be able to compensate for low permeability.
Additionally, sites with shallow contamination can pose a
challenge to bioventing because of the difficulty in devel-
oping a system design that can minimize environmental
release and achieve sufficient aeration. In this situation,
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operating the system in the extraction mode may circum-
vent the difficulty (AFCEE, 1996; FRTR, 2003).
Another limitation is that bioventing will not stimulate
contaminant bioremediation if the contaminated zone is
aerobic. If a soil gas survey measures soil oxygen levels
consistently above 2-5%, then the soil is sufficiently aer-
ated for biodegradation to occur and oxygen is not limit-
ing degradation. Bioventing will not enhance bioreme-
diation in this situation. This situation is unusual and, if
encountered, may indicate that some other contaminants,
such as metals, are inhibiting degradation (AFCEE, 1996;
EPA, 1998a).
While bioventing is relatively inexpensive, bioventing can
take a few years to clean up a site depending on contami-
nant concentrations and site-specific removal rates. If a
quicker cleanup is needed, more intensive ex situ tech-
nologies may be more appropriate (AFCEE, 1996; EPA,
2000; FRTR, 2003).
3.1.2.1.2 Cometabolic Bioventing
Cometabolic bioventing has been used at a few sites to
treat chlorinated solvents such as TCE, trichloroethane
(TCA), and dichloroethene (DCE). The equipment used
in cometabolic bioventing is similar to aerobic bioventing,
but cometabolic bioventing exploits a different biological
mechanism. Similar to bioventing, cometabolic bioventing
involves the injection of gases into the subsurface; howev-
er, cometabolic bioventing injects both air and a volatile
organic substrate, such as propane. The concentrations in
this gas mixture should be well below the lower explosive
limit (LEL), and should be monitored in soil gas (AFCEE,
1996; EPA, 1998a; EPA, 2000; FRTR, 2003).
Cometabolic bioventing exploits competitive reactions
mediated by monooxygenase enzymes (EPA, 2000). Mo-
nooxygenases catalyze the oxidation of hydrocarbons, of-
ten through epoxide intermediates, but these enzymes can
also catalyze the dechlorination of chlorinated hydrocar-
bons. Thus, by supplying an appropriate organic substrate
and air, cometabolic bioventing can elicit the production
of monooxygenases, which consume the organic substrate
and facilitate contaminant degradation (AFCEE, 1996;
EPA, 1998a).
Cometabolic bioventing has been used to treat lightly
chlorinated compounds in the vadose zone or capillary
fringe. In addition to the variables discussed in the previ-
ous aerobic bioventing section, the degradation rate and
design of cometabolic bioventing systems are dependent
on many factors including soil gas permeability, organ-
ic substrate concentration, type of organic substrate se-
lected, and oxygen supply and radius of influence. Unlike
many variables that are determined by site conditions, the
selection and concentration of the organic substrate are
controllable and can be important to the removal rate.
Treatability or bench-scale testing can be useful in select-
ing the organic substrate and concentration for a site. In
addition, small-scale testing can demonstrate that full de-
chlorination is observed at a site (AFCEE, 1996; EPA,
1998a; EPA, 2000; FRTR, 2003).
Establishing cometabolic bioventing as the primary mech-
anism of removal in the field is challenging. Unlike aerobic
bioventing, the oxygen use and chlorinated solvent remov-
al are not related stoichiometrically because the metabo-
lism of added organic substrates also consumes oxygen.
As a result, measurements of oxygen use, carbon dioxide
generation, and contaminant removal cannot be linked
stoichiometrically. Indirect measures, such as measuring
chloride ion accumulation in the soil and correlating that
accumulation to contaminant removal, have been useful
at some sites. In addition, collecting data to demonstrate
degradation of the organic substrate (by a shutdown test)
in the field may be helpful, especially in conjunction with
laboratory testing using contaminated soil from the site
(AFCEE, 1996; EPA, 1998a; FRTR, 2003).
Cometabolic bioventing has been successfully demon-
strated at a few sites. The Remediation Technology De-
velopment Forum (RTDF) Bioremediation Consortium
has conducted cometabolic bioventing demonstrations at
Dover and Hill Air Force Bases. At Dover, a field dem-
onstration of cometabolic bioventing was conducted at
Building 719a site contaminated with fuel and solvents
during engine inspection and maintenance operations.
The targeted contaminants of the demonstration and
their concentrations were TCE, as high as 250 mg/kg;
1,1,1-TCA, 10-1,000 mg/kg; and cis-l,2-DCE, 1-20
mg/kg. Laboratory tests were used to select propane as
the cometabolic substrate and to predict that a substrate
acclimation period would be needed. The test plot was ac-
climated to propane addition through pulsed propane/air
injections for three months, then the test plot was oper-
ated for 14 months with continuous propane injection.
Concentrations of TCE, TCA, and DCE were reduced
to less than 0.25, 0.5, and 0.25 mg/kg, respectively. Soil
chloride accumulation analysis confirmed biodegradation
as the mechanism of removal (EPA, 1998a).
In Situ and Ex Situ Biodegredation Technologies
Engineering Issue
-------
As with aerobic bioventing, difficulty in distributing gases
in the subsurface may make the application of cometa-
bolic bioventing more complicated. In some cases, such as
high moisture content or low-permeability soils, the de-
sign of the cometabolic system may compensate for poor
permeability. In the case of shallow contamination, de-
signing a cometabolic bioventing system that minimizes
environmental release and achieves sufficient aeration and
organic substrate distribution may be difficult (AFCEE,
1996; EPA, 1998a; EPA, 2000; FRTR, 2003).
Another limitation to cometabolic bioventing is the lack
of experience with the technology. Since cometabolic bio-
venting has been demonstrated at a limited number of
sites, the technology is not as well understood as aerobic
bioventing. Researchers are still studying which contam-
inants are amenable to this type of biodegradation and
what removal rates can be expected. Establishing that bio-
logical processes are the primary mechanism for contami-
nant removal is also more difficult. Finally, regulatory and
public acceptance is not as strong for cometabolic bio-
venting as for aerobic bioventing. However, treatability
testing of samples from the contaminated site and pilot-
scale testing may alleviate many of these limitations and
concerns (EPA, 1998a). As more sites are remediated us-
ing cometabolic bioventing, these limitations may ease.
3.1.2.1.3 Anaerobic Bioventing
While aerobic and cometabolic bioventing are useful for
degrading many hydrocarbons and lightly chlorinated
compounds, some chlorinated species are not effectively
treated aerobically. Microbes may degrade these contami-
nants directly via anaerobic reductive dechlorination or
through anaerobic cometabolic pathways. Anaerobic re-
ductive dechlorination is a biological mechanism typically
marked by sequential removal of chlorine from a molecule.
Microbes possessing this pathway do not gain energy from
this process. Anaerobic cometabolism is similar to aero-
bic cometabolism in that microbes fortuitously degrade
contaminants while reducing other compounds (come-
tabolites). Anaerobic bioventing may use both biological
mechanisms to destroy the contaminants of concern.
Anaerobic bioventing uses the same type of gas delivery
system as the other bioventing technologies, but injects
nitrogen and an electron donor, instead of air, to establish
reductive anaerobic conditions. The nitrogen displaces
the soil oxygen, and small amounts of an electron do-
nor gas (such as hydrogen and carbon dioxide) produce
reducing conditions in the subsurface, thereby facilitat-
ing microbial dechlorination. Volatile and semi-volatile
compounds may be produced during anaerobic biovent-
ing. Some of these compounds may be slow to degrade
under anaerobic conditions. These compounds may be
treated in two ways. Volatile compounds may diffuse into
the soils surrounding the treatment zone, where aerobic
degradation may occur. SVOCs and VOCs remaining in
the treatment zone may be treated by following anaerobic
bioventing with aerobic bioventing. Since aerobic and an-
aerobic bioventing share similar gas delivery systems, the
switch can be made by simply changing the injected gas.
Anaerobic bioventing is an emerging technology that has
been demonstrated in several laboratory and field stud-
ies. This process may be useful in treating highly chlori-
nated compounds such as tetrachloroethene (PCE), TCE,
RDX, pentachlorophenol, and pesticides such as lindane
and dichlorodiphenyltrichloroethane (DDT). Due to the
limited experience with this technique, laboratory, pilot,
and field demonstrations are recommended to confident-
ly apply this technology to remediate a site.
As with the other bioventing technologies, the ability to
deliver gases to the subsurface is important. Soils with
high moisture content or low gas permeability may re-
quire careful system design to deliver appropriate levels of
nitrogen and the electron donor. Sites with shallow con-
tamination or nearby buildings are also a challenge since
this technology is operated by injecting gases. In addition,
anaerobic bioventing can take a few years to clean up a
site depending on the contaminant concentrations and
site-specific removal rates. If a quicker cleanup is needed,
other technologies may be more appropriate.
3.1.2.2 Surficial Soil Remediation
If contamination is shallow, soil may be treated in place
using techniques similar to land treatment or compost-
ing. Variations of these technologies involve tilling shal-
low soils and adding amendments to improve aeration
and bioremediation. This process is similar to the land
farming and composting discussed later in the Ex Situ
Bioremediation section of this Engineering Issue paper,
except that the soils are not excavated.
Since these treatments do not include an impermeable
sublayer, contaminant migration may be a concern de-
pending on the contaminants of concern and treatment
amendments. A more prudent approach would be to ex-
cavate soils and treat them in lined beds.
Engineering Issue
In Situ and Ex Situ Biodegradation Technologies
-------
This technology will generally require special permission
from the applicable regulatory agency. Frequently, some
type of monitoring for contaminant migration is required.
3.1.2.3 Ground Water and Saturated Soil Remediation
In situ bioremediation techniques applicable to ground
water and saturated soil include dechlorination using an-
aerobic reducing conditions, enhanced aerobic treatment,
biological reactive barriers that create active remediation
zones, and bioslurping/biosparging techniques that pro-
mote aerobic degradation.
3.1.2.3.1 Anaerobic Reductive Dechlorination
Anaerobic reductive dechlorination has been used at
many sites where the ground water has been contami-
nated with chlorinated solvents, such as TCE or PCE. In
this treatment, organic substrates are delivered to the sub-
surface where they are fermented. The fermentation cre-
ates an anaerobic environment in the area to be remedi-
ated and generates hydrogen as a fermentation byproduct.
The hydrogen is used by a second microbial population
to sequentially remove chlorine atoms from chlorinated
solvents (AFCEE, 2004). If PCE were degraded via re-
ductive dechlorination, the following sequential dechlo-
rination would be observed: PCE would be converted to
TCE, then to DCE, vinyl chloride (VC), and/or dichlo-
roethane (EPA, 1998a).
Anaerobic dechlorination may also occur via cometabo-
lism where the dechlorination is incidental to the meta-
bolic activities of the organisms. In this case, contaminants
are degraded by microbial enzymes that are metabolizing
other organic substrates. Cometabolic dechlorination
does not appear to produce energy for the organism. At
pilot- or full-scale treatment, cometabolic and direct de-
chlorination may be indistinguishable, and both process-
es may contribute to contaminant removal. The microbial
processes may be distinguished in the more controlled en-
vironment of a bench-scale system (EPA, 1998a).
Anaerobic reductive dechlorination is primarily used to
treat halogenated organic contaminants, such as chlorinat-
ed solvents. As well as the variables discussed initially, the
treatment rate and system design are dependent on sev-
eral factors including site hydrology and geology, type and
concentration of organic substrates, and site history. As
with cometabolic bioventing, the selection of organic sub-
strate and the concentration used are controllable and can
be important to the removal rate. Treatability or bench-
scale testing can be useful in selecting the best organic sub-
strate and concentration for a site. In addition, small-scale
testing can demonstrate that full dechlorination is possible
at a site. In some cases, dechlorination may stall at DCE
despite the presence of sufficient electron donors. If a site
does not demonstrate full dechlorination (either as part
of site assessment or in microcosm testing), a combined
treatment strategy, such as anaerobic treatment followed
by aerobic treatment, may be successful. Alternatively,
bioaugmentation may improve the dechlorination rate
(AFCEE, 1996; EPA, 1998a; EPA, 2000; FRTR, 2003).
Research methods used to establish that anaerobic dechlo-
rination is occurring at a site are similar to those discussed
in previous sections of this Engineering Issue paper.
Regulatory considerations for this technology involve the
Safe Drinking Water Act and RCRA hazardous waste reg-
ulations, as well as state and local regulations. At the time
that this Engineering Issue paper was written, judgments
about the applicability of this technology were made on
a case-by-case basis. The regulations can impact the de-
sign and operation of the treatment system as well as the
overall applicability. Engineered Approaches to In Situ Bio-
remediation of Chlorinated Solvents: Fundamentals and
Field Applications (EPA, 2000) provides more detailed
information about regulatory concerns and applicability
(EPA, 1998a).
Additional information on anaerobic reductive dechlori-
nation is available from the following source: Principles
and Practices of Enhanced Anaerobic Bioremediation of
Chlorinated Solvents (AFCEE, 2004).
3.1.2.3.2 Aerobic Treatment
Similar to bioventing, enhanced in situ aerobic ground
water bioremediation processes are used in situations
where aerobically degradable contaminants, such as fuels,
are present in anaerobic portions of an aquifer. In these
situations, air or other oxygen sources are injected into
the aquifer near the contamination (see Figure 3-2). As
the oxygenated water migrates through the zone of con-
tamination, the indigenous bacteria are able to degrade
the contaminants (EPA, 1998a; EPA, 2000).
Aerobic treatment may also be used to directly or cometa-
bolically degrade lightly chlorinated species, such as DCE
or VC. In the direct aerobic pathway, air is injected into
the aquifer. The microbes appear to generate energy by
oxidizing the hydrocarbon backbone of these contami-
nants, resulting in the release of chloride (EPA, 2000).
In Situ and Ex Situ Biodegredation Technologies
Engineering Issue
-------
This process has been used to complete contaminant re- elevation, amending it in the ground, and re-injecting it
moval following anaerobic treatment at several sites (EPA, into another elevation (EPA, 1998a; EPA, 2000).
1998a; EPA, 2000).
In addition to the variables discussed initially, the
treatment rates and system design are the result of
several factors including site hydrology and geol-
ogy, amendment to be added, solubility of air or
oxygen sources, and site history. The low solubil-
ity of air in water often limits reaction rates and
may make this process impractical if cleanup time
is short (AFCEE, 1996; EPA, 1998a; EPA, 2000;
FRTR, 2003).
Careful attention also should be given to co-con-
taminants, especially metals. When an aquifer en-
vironment is converted from an aerobic to an an-
aerobic environment, a variety of chemical species
may become soluble. Therefore, it is important to
check for changes in co-contaminants such as ar-
senic, which may be solubilized during the treat-
ment process (AFCEE, 1996; EPA, 1998a; EPA,
2000; FRTR, 2003).
a
&~
Air blower
i
Mutrient pH
djustment adjustment
Injection
well
i
JL.JIL
i.
0
Ground water
extraction wells
Contaminated
« o f ground water
I
,
To further
Mreatment discharge
or recharge
i
Vadose zone
1
-' f
Saturated zone
1
S 1
7
Submersible
pump
Figure 3-2. Aerobic treatment. (Adapted from FRTR, 2003)
Cometabolic aerobic treatment is founded on the same
biological principles as cometabolic bioventing and in-
volves the addition of oxygen and organic substrates,
such as methane, to the aquifer. As with other cometa-
bolic processes, these organic substrates are metabolized
by enzymes that incidentally degrade the contaminant. In
this treatment, sufficient oxygen must be present to fuel
the oxidation of both the substrate and contaminant (AF-
CEE, 1996; EPA, 1998a).
3.1.2.3.3 Amendment Delivery
In «Vw ground water treatment, either aerobic or anaero-
bic, may be configured as direct injection of air or aque-
ous streams or as ground water recirculation. In direct in-
jection, amendments, such as organic substrates, oxygen
sources, or nutrients, are directly injected into the aquifer.
For example, oxygen may be sparged into the aquifer as
a gas. Lactate or hydrogen peroxide may be injected as
a liquid stream; when using hydrogen peroxide, caution
should be used as it may act as a disinfectant. In some cas-
es, both liquids and gases are added. The ground water re-
circulation configuration involves extracting ground wa-
ter, amending it as needed, and then re-injecting it back
into the aquifer. Recirculation may also be conducted be-
low the ground surface by extracting ground water at one
3.1.2.3.4 Biological Reactive Barriers
Biological reactive barriers consist of an active bioreme-
diation zone created in the contamination zone. These
barriers may be constructed to exploit aerobic or anaero-
bic processes depending on the contaminant of concern
and site needs. A trench is excavated and filled with sand
pre-mixed with nutrient-, oxidant-, or reductant-rich
materials to form a bioremediation zone (see Figure 3-
3). Alternatively, a bioremediation curtain can be formed
by injection of amendments or recirculation of amended
ground water at the toe of the plumes (EPA, 2000). Con-
taminants biodegrade as they pass through the permeable
reactive barrier (PRB).
Trenches are dug with a backhoe or similar device and
are filled with permeable materials, such as sand or bark
mulch, that are mixed together prior to placement. Nu-
trients, degradable carbonaceous substrates (e.g., ma-
nure, compost, and wheat straw), and other additives
are introduced into the permeable layer. As ground wa-
ter flows through the treatment zone, indigenous mi-
crobes are stimulated to improve natural bio degradation
(NFESC, 2000). Biological PRBs have been studied by
the PRB RTDF, and further information, including case
studies, may be found at http://www.rtdf.org/public/
permbarr/pbar qa.htm and http://costperformance.org/
search.cfm.
10
Engineering Issue
In Situ and Ex Situ Biodegradation Technologies
-------
from biosparging require treatment. For this reason, bio-
sparging may be implemented along with SVE or biovent-
ing as a remedy for increased contaminant concentrations
in the unsaturated zone. The SVE wells are designed to
capture the introduced air and contaminant vapors (EPA,
2004b). Figure 3-4 depicts a typical biosparging system
with optional SVE system. Alternatively, a lower-flow
bioventing system may be added to facilitate bioremedia-
tion of volatilized contaminants in the vadose zone.
Figure 3-3. Permeable reactive barrier. (Adapted from EPA, 2000)
3.1.2.3.5 Biosparging and Bioslurping
Biosparging (similar to air sparging) involves the injection
of a gas (usually air or oxygen) and occasionally gas-phase
nutrients, under pressure, into the saturated zone to pro-
mote aerobic biodegradation (GWRTAC, 1996). In air
sparging, volatile contaminants also can be removed from
the saturated zone by desorption and volatilization into
the air stream. Emphasis on the biological degradation rate
over physical removal, as well as lower rates of air injection,
are what distinguishes this technology from air sparging.
Typically, biosparging is achieved by injecting air into a
contaminated subsurface formation through a specially
designed series of injection wells. The air creates an invert-
ed cone of partially aerated soils surrounding the injection
point. The air displaces pore water, volatilizes contami-
nants, and exits the saturated zone into the unsaturated
zone. While in contact with ground water, oxygen disso-
lution from the air into the ground water is facilitated and
supports aerobic biodegradation.
A number of contaminants have been successfully ad-
dressed with biosparging technology, including gasoline
components such as benzene, toluene, ethylbenzene, and
xylenes (BTEX) and SVOCs. Biosparging is most often
recommended at sites impacted with mid-weight petro-
leum hydrocarbon contaminants, such as diesel and jet
fuels. Lighter contaminants, such as gasoline, tend to be
easily mobilized into the unsaturated zone and physically
removed. Heavier contaminants, such as oils, require longer
remedial intervals because of reduced microbial bioavail-
ability with increasing carbon chain length (EPA, 2004b).
Care must be taken to determine whether contaminant
concentrations in soil gas and released vapors resulting
I I Vapor phase
I I Adsorbed phase
V//A Dissolved phase
₯ Water table
Atmospheric
discharge
Vapor
treatment
Figure 3-4. Biosparging system (used with soil vapor extraction).
(Adapted from NMED, 2004)
One specialized form of biosparging involves the injec-
tion of organic gases into the saturated zone to induce co-
metabolic biodegradation of chlorinated aliphatic hydro-
carbons, and this is analogous to cometabolic bioventing
discussed in this Engineering Issue paper. The injection
of gases below the water table distinguishes biosparging
from bioventing.
In contrast to cometabolic bioventing, the solubility of
organic gases in water limits delivery of the primary sub-
strate during cometabolic biosparging applications. This
solubility limitation affects the economics of cometabolic
biosparging applications since the interaction between
bacterial cometabolite consumption and cometabolite wa-
ter solubility directly determines the number of methane
biosparging injection wells required at a given site. Safe-
ty precautions similar to those required for cometabolic
bioventing apply to cometabolic biosparging (Sutherson,
2002;AFCEE, 1998).
In Situ and Ex Situ Biodegredation Technologies
Engineering Issue
11
-------
Bioslurping (also known as multi-phase extraction) is ef-
fective in removing free product that is floating on the wa-
ter table (Battelle, 1997). Bioslurping combines the two
remedial approaches of bioventing and vacuum-enhanced
free-product recovery. Bioventing stimulates aerobic bio-
remediation of contaminated soils in situ, while vacuum-
enhanced free-product recovery extracts light, nonaque-
ous-phase liquids (LNAPLs) from the capillary fringe and
the water table (AFCEE, 2005). Bioslurping is limited to
25 feet below ground surface as contaminants cannot be
lifted more than 25 feet by this method.
A bioslurping tube with adjustable height is lowered into
a ground water well and installed within a screened por-
tion at the water table (see Figure 3-5). A vacuum is ap-
plied to the bioslurping tube and free product is "slurped"
up the tube into a trap or oil water separator for further
treatment. Removal of the LNAPL results in a decline in
the LNAPL elevation, which in turn promotes LNAPL
flow from outlying areas toward the bioslurping well. As
the fluid level in the bioslurping well declines in response
to vacuum extraction of LNAPL, the bioslurping tube
also begins to extract vapors from the unsaturated zone.
This vapor extraction promotes soil gas movement, which
in turn increases aeration and enhances aerobic biodegra-
dation (Miller, 1996).
Recent improvements in bioslurping technology and ap-
plication assessments for this technology are contained in
reports by the U.S. Navy (NFESC, 2003) and the U.S.
Army Corps of Engineers (USAGE, 1999). It is widely
accepted in the industry that source removal, such as that
offered by proper application of the bioslurping technol-
ogy, should be part of most remedial strategies at sites in-
volving separate product phases.
3.2 Ex Situ Bioremediation
Ex situ bioremediation technologies can most easily be
classified by the physical state of the medium to which
they are typically applied. The following discussion is
organized accordingly, with descriptions of bioremedia-
tion processes for various solids, solid-liquid mixtures,
and liquids.
Also common to the ex situ remediation technologies are
the processes for removing contaminated materials for
treatment. Contaminated media are excavated or extract-
ed (e.g., ground water removal by pumping) and moved
to the process location, which may be within or adjacent
to the contamination zone. Special handling required for
excavation and preparation of sediments is described in
detail in Selecting Remediation Techniques for Contami-
nated Sediment (EPA, 1993b), as well as Physical Separa-
tion (Soil Washing) for Volume Reduction of Contaminated
Soils and Sediments: Processes and Equipment (Olin et al.,
1999).
Air discharge
Air treatment
Vacuum pump
Ground surface
Slurping tube
Casing
Aerobic
biodegradation
Filter pack
Hydrocarbon/
water separator Water
treatment
/
J^W
Hydrocarbon discharge
Water discharge
Surface seal
Backfill/grout
Bentonite seal
Liquid hydrocarbon layer
Movement of
hydrocarbon to well
Wall screen
Water table
3.2.1 Solids
The most common types
of solids bioremediation
are (1) land farming or
land treatment, (2) com-
posting, and (3) biopiles,
cells, or mounds. In prac-
tice, these types are not
rigidly divided, but the
subdivision is useful for
this discussion. Table 3-1
presents a comparison of
the characteristics of vari-
ous solid-phase bioreme-
diation technologies.
Figure 3-5. Bioslurping technology. (Adapted from USAEC, 2004)
Engineering Issue
In Situ and Ex Situ Biodegradation Technologies
-------
Table 3-1. Comparison of the Characteristics of £xS/ft/Bioremediation Technologies for Solids
Characteristic
Liner Utilized
Containerized
Bulking Agent
Mechanical
Aeration2
Nutrient3 Added
Temperature
Land Treatment
Yes
Bermed
Sometimes
Mobile Equipment
Mechanical Mixing
Sometimes
Ambient
Composting
Static Pile
Yes
Pad
Yes
No
Pressure
Yes
54°-65°C
In-Vessel
No
Yes
Yes
In-Vessel
Vacuum
Yes
54°
Windrow
Yes
Pad
Yes
Mobile Equipment
Mechanical
Yes
65°C
Biopiles
Yes
Pad
Sometimes
Mobile Equipment
Pressure/Vacuum
Yes
Ambient/Mesophilic
1 Mechanical mixing may be necessary to provide thorough distribution of nutrients and other additives, promote aeration, and enhance biodegradation. Mechanical
mixing may use mobile equipment (e.g., bulldozers) or may occur within a reactor vessel.
2 Aeration involves the introduction of oxygen into ex situ material to promote aerobic degradation. Aeration mechanisms may include mechanical mixing, pressure, and
vacuum, as well as natural draft. "Typical" aeration mechanisms are shown.
3 Nutrients may not be required or may be supplied by bulking agents alone, or additional nutrients may be supplied.
3.2.1.1 Land Treatment
Land treatment, also called land farming, is useful in treat-
ing aerobically degradable contaminants. This process is
suitable for non-volatile contaminants at sites where large
areas for treatment cells are available. Land treatment of
site-contaminated soil usually entails the tilling of an 8-
to 12-inch layer of the soil to promote aerobic biodeg-
radation of organic contaminants. The soils are periodi-
cally tilled to aerate the soil, and moisture is added when
needed. In some cases, amendments may be added to
improve the tilth of the soil, supply nutrients, moderate
pH, or facilitate bioremediation. Typically, full-scale land
treatment would be conducted in a prepared-bed land
treatment unit (see Figure 3-6)an open, shallow reac-
tor with an impermeable lining on the bottom and sides
to contain leachate, control runoff, and minimize erosion
and with a leachate collection system under the soil layer
(EPA, 1993). In some cases, hazardous wastes (such as
highly contaminated soils) or process wastes (such as dis-
tillate residues) may be treated in land treatment units. In
these cases, the waste may be applied to a base soil layer.
The performance of land treatment varies with the con-
taminants to be treated. For easily biodegradable contam-
inants, such as fuels, land treatment is inexpensive and
effective. Contaminants that are difficult to degrade, such
as PAHs, pesticides, or chlorinated organic compounds,
are topics of research and would require site-specific treat-
ability testing to verify that land treatment can meet de-
sired endpoints.
Existing
surface
T
Lea
collect
Microbes' Contaminated
nutrients soils
/ /
/
/
^bPfaX
X^l^iii3*e£fci^fc3i!X\ \
£ J&3 @
-------
face irrigation often is used to maintain moisture content.
Temperatures are controlled, to a degree, by mixing, ir-
rigation, and air flow, but are also dependent on the de-
gradability of the bulk material and ambient conditions
(FRTR, 2003).
There are three designs commonly applied for composting:
1. Aerated static pilesCompost is formed into piles
and aerated with blowers or vacuum pumps.
2. Mechanically agitated in-vessel compostingCompost
is placed in a reactor vessel, in which it is mixed and
aerated.
3. Windrow compostingCompost is placed in long,
low, narrow piles (i.e., windrows) and periodically
mixed with mobile equipment.
Windrow composting is the least expensive method,
but has the potential to emit larger quantities of VOCs
(FRTR, 2003). In-vessel composting is generally the most
expensive type, but provides for the best control of VOCs.
Aerated static piles, especially when a vacuum is applied,
offer some control of VOCs and are typically in an in-
termediate cost range, but will require offgas treatment
(FRTR, 2003).
Berms may also be needed to control runoff during com-
posting operations. Runoff may be managed by retention
ponds, provision of a roof, or evaporation.
Composting has been successfully applied to soils and
biosolids contaminated with petroleum hydrocarbons
(e.g., fuels, oil, grease), solvents, chlorophenols, pesti-
cides, herbicides, PAHs, and nitro-aromatic explosives
(EPA, 1998b; EPA, 1997; EPA, 2004b). For TNT, com-
plete mineralization has been difficult to demonstrate via
composting. TNT may bind to soil, resulting in low mi-
crobial bioavailability and apparent disappearance (Rodg-
ers and Bunce, 2001). Composting is not likely to be suc-
cessful for highly chlorinated substances, such as PCBs,
or for substances that are difficult to degrade biologically
(EPA, 1998b).
3.2.1.3 Biopiles
Biopiles involve the mixing of excavated soils with soil
amendments, with the mixture placed in a treatment area
that typically includes an impermeable liner, a leachate
collection system, and an aeration system. Biopiles are
typically 2-3 meters high, and contaminated soil is often
placed on top of treated soil (see Figure 3-7). Moisture,
nutrients, heat, pH, and oxygen are controlled to enhance
biodegradation. This technology is most often applied to
readily degradable species, such as petroleum contami-
nants. Surface drainage and moisture from the leachate
collection system are accumulated, and they may be treat-
ed and then recycled to the contaminated soil. Nutrients
(e.g., nitrogen and phosphorus) are often added to the
recycled water. Alkaline or acidic substances may also be
added to the recycled water to modify or stabilize pH to
optimize the growth of select microbes capable of degrad-
ing the contaminants of concern (FRTR, 2003).
Soil vapor
monitoring probes
Air inlet/
exhaust
Leachate collection
and treatment (optional)
Contaminated soil
Air injection
(or extraction)
Nutrient and
moisture addition
Figure 3-7. Typical biopile system. (Adapted from EPA, 2004b)
An air distribution system is buried in the soil as the bio-
pile is constructed. Oxygen exchange can be achieved uti-
lizing vacuum, forced air, or even natural draft air flow.
Low air flow rates are desirable to minimize contaminant
volatilization. If volatile constituents are present in sig-
nificant concentrations, the biopile may require a cover
and treatment of the offgas.
Biopile treatment lasts from a few weeks to a few months,
depending on the contaminants present and the design
and operational parameters selected for the biopile (FRTR,
2003). Biopiles are typically mesophilic (10°-45°C).
Additional information on ex situ biological soil treatment
is available from the following sources:
U.S. Navy BiocellApplication Guidance (NFESC,
1998)
U.S. Navy Biopile Design and Construction Manual
(Battelle, 1996a)
U.S. Navy Biopile Operations andMaintenance Man-
ual (Battelle, 1996b)
U.S. Army Environmental Center Multiple Biotech-
nology Demonstration of Explosives-Contaminated Soils
(USAEC, 2005)
74
Engineering Issue
In Situ and Ex Situ Biodegradation Technologies
-------
Chapters 13 and 14 in Eiodegradation of Nitroaro-
matic Compounds and Explosives (Spain et al., 2000)
On-site Bioremediation of Oil and Grease Contami-
nated Soils (Vance, 1991).
3.2.2 Solid-Liquid Mixtures
Solid-liquid mixtures consist of materials such as slurries
and sludges. One technology for treating such mixtures is
discussed below.
3.2.2.7 Slurry Bioreactors
Slurry bioreactors are utilized for soil, sediments, sludge,
and other solid or semi-solid wastes. Slurry bioreactors are
costly and, thus, are likely to be used for more difficult
treatment efforts.
Typically, wastes are screened to remove debris and other
large objects, then mixed with water in a tank or other
vessel until solids are suspended in the liquid phase. If
necessary, further particle size reduction can be accom-
plished before the addition of water (by pulverizing and/
or screening the wastes) or after the addition of water
(through use of a sheering mixer). Suspension and mix-
ing of the solids may increase mass transfer rates and may
increase contact between contaminants and microbes ca-
pable of degrading those contaminants (EPA, 1990).
Mixing occurs in tanks or lined lagoons. Mechanical mix-
ing is generally conducted in tanks. Typical slurries are
10-30% solids by weight (FRTR, 2003). Aeration, with
submerged aerators or spargers, is frequently used in la-
goons and may be combined with mechanical mixing to
achieve the desired results. Nutrients and other additives,
such as neutralizing agents, surfactants, dispersants, and
co-metabolites (e.g., phenol, pyrene) may be supplied to
improve handling characteristics and microbial degrada-
tion rates. Indigenous microbes may be used or microor-
ganisms may be added initially to seed the bioreactor or
may be added continuously to maintain proper biomass
levels. Residence time in the bioreactor varies with the
matrix as well as the type and concentration of contami-
nant (EPA, 1990).
Once contaminant concentrations reach desired levels
on a dry-weight basis, the slurry is dewatered. Typical-
ly, a clarifier is utilized to dewater the slurry by gravity.
Other dewatering equipment may be used depending
on slurry characteristics and cost considerations (Olin
et al., 1999). Water, air emissions from all process steps,
and oversize materials may require additional treatment.
More information on this technology is available from the
Naval Facilities Engineering Service Center (NFESC) at
http://enviro.nfesc.navy.mil/erb/erb a/restoration/
technologies/tech transfer/ttweb.asp?id=3.
3.2.3 Liquids
Liquids, such as surface water, ground water, mine drain-
age, and effluent from other treatment operations, can
undergo ex situ bioremediation in constructed wetlands.
Note that surface water and ground water have important
differences, such as concentrations of contaminants and
degradable organic material, than may be found in waste
streams from other treatment operations.
3.2.3.7 Constructed Wetlands
Constructed wetlands provide for biological assimilation,
breakdown, and transformation of contaminants; chemi-
cal breakdown and transformation of contaminants; and
physical sedimentation and filtration (USDA and EPA
1994a), as shown in Figure 3-8. Biological processes asso-
ciated with wetlands include bioremediation (microbial-
ly-based remediation) and phytoremediation (plant-based
remediation). Microbes attached to the surfaces of plants,
plant litter, and the wetland substrate degrade and/or sorb
the organic substances present in the water undergoing
treatment (USDA and EPA, 1994a). Phyto remediation
uses plants to remove, transfer, stabilize, or destroy con-
taminants through biological, chemical, and physical pro-
cesses that are influenced by plants and their roots (i.e.,
rhizosphere) that include degradation, extraction through
accumulation in plant roots/shoots/leaves, metabolism
of contaminants, and immobilization of contaminants at
the interface of roots and soil (EPA, 2004a).
Gas
discharge
1
) ) )
Water \ ( \
inflow , \i/w ? ( vi/y
Algae uptake '' ".??^^.^_
and disposition ' ''":'; :i.-.-.;.
Man-made v
Sediment
recovery
Sediment
accumulation
j S*
wetlands
(organic soil, microbial fauna,
algae, plants, microorganisms)
Landfill
-t ''""''-
Plant
. uptake VVater
IM/, outflow
H .
r^;-\v^v;:.
Microbial
oxidation and
reduction
Figure 3-8. Constructed wetland. (Adapted from FRTR, 2003)
In Situ and Ex Situ Biodegredation Technologies
Engineering Issue
15
-------
Wetlands inherently have a higher rate of biological pro-
ductivity/activity than many other natural ecosystems and
are thus capable of efficiently and economically transform-
ing many common contaminants to harmless byproducts
(Kadlec and Knight, 1996). Constructed wetlands have
been applied successfully to remove contaminants such as
metals, petroleum hydrocarbons, and glycols; to decrease
metal concentrations via chemical or microbial precipi-
tation; and to neutralize acidity (ESTCP, 2004a; USDA
and EPA, 1994b). Recent research also has demonstrat-
ed applicability to explosive-contaminated water (Bader,
1999). However, wetlands are sensitive to high ammonia
levels, herbicides, and contaminants that are toxic to the
plants or microbes (USDA and EPA, 1994a).
Constructed wetlands are well suited for the treatment of
contaminated ground water emerging from surface and
mine seeps, pump-and-treat waste streams with low con-
centrations of easily biodegradable contaminants, and con-
taminated surface waters (EPA, 200 Ic). Constructed wet-
lands may also be used to pretreat contaminated water prior
to conventional treatment or to further treat a waste stream
prior to disposition or discharge (USDA and EPA, 1994b).
However, applicability to highly acidic waste streams may
not be cost-effective (USDA and EPA, 1994b).
Discharges must meet applicable effluent limitations and
related regulatory requirements. Discharges that do not
meet these requirements may be required to undergo fur-
ther treatment or may be found suitable for recycling into
the wetland as a supplemental water source (USDA and
EPA, 1994a).
There are various types of constructed wetlands, depend-
ing on the type of flow (surface or subsurface), contami-
nant of concern, or type of substrate, which can include
limestone, organic material such as compost, or gravel
(USDA and EPA, 1994a; USDA and EPA, 1994b; Bader,
1999). The chemical and microbial processes may pro-
ceed either in an anaerobic or aerobic environment.
Since constructed wetlands function both as macroscop-
ic and microscopic ecosystems to promote contaminant
treatment, the biological characteristics of the system
must be taken into account during the design phase. The
chemistry of the waste stream and how the passive chemi-
cal, physical, and biological processes affect this or are, in
turn, affected by the waste stream are important design
factors (USDA and EPA, 1994b). The chemical charac-
teristics of the waste stream can affect sizing of the sys-
tem for adequate retention time and whether the waste
stream may require pretreatment to (1) address concen-
tration, ammonia, nutrient, and organic loads that may
damage vegetation, or (2) remove solids or materials, such
as grease, that may clog the wetland (USDA and EPA,
1994a). In addition, pH adjustment may be necessary,
either prior to waste stream treatment or through use of
limestone substrate (USDA and EPA, 1994b).
Climatic and seasonal circumstances as well as waste
stream characteristics are important considerations when
selecting the types of plants to use in a constructed wet-
land. Salinity, either in the waste stream or as a result of
treatment, can harm or destroy the wetland vegetation if
the plants are not salt tolerant. In addition, cold weather
can reduce microbial activity, and hail or other weather
events can damage the plants (USDA and EPA, 1994a).
The low cost, passivity (i.e., lack of dependence on pow-
er or mechanical components), and efficacy for treating
many common contaminants are key advantages of con-
structed wetland treatment systems. Constructed wetlands
are often visually attractive, but can require more space
than other remedial systems. The wetlands should be sized
with an understanding that both plant-based and bacteri-
al-based remediation will decline during colder seasons. A
key design element is sizing to achieve adequate retention
time to enable the biological, chemical, and physical pro-
cesses to be effective (USDA and EPA, 1994a). Seasonal,
climatological, and waste stream factors that control the
water balance in the wetland also must be considered dur-
ing design to achieve project goals (FRTR, 2003). For ex-
ample, photosynthesis of TNT colors the treated water
red, which negatively impacts plant growth (Bader, 1999),
and pH affects the kinetics of the abiotic and biotic pro-
cesses, including solubility of metal oxides or hydroxides,
oxidation, and hydrolysis (USDA and EPA 1994b). In
addition, animals such as tadpoles or deer may defoliate
plant material, thereby affecting treatment (Bader, 1999).
Constructed wetlands require a continuous supply of wa-
ter. While tolerant of fluctuating flows, constructed wet-
lands cannot withstand complete drying. A slow water
flow must be maintained to prevent the development of
stagnant water that can lead to performance and vector
difficulties. Recycling wetland water can supplement in-
flow, but this can increase salinity over time, which can
affect design and cost (USDA and EPA, 1994a).
More information on constructed treatment wetlands is
available in the Interstate Technical and Regulatory Guid-
ance Documentor Constructed Wetlands (ITRC 2003).
16
Engineering Issue
In Situ and Ex Situ Biodegradation Technologies
-------
4.0 TECHNOLOGY SELECTION FACTORS
Table 4-1 summarizes the general applicability of each
technology type for the contaminant classifications dis-
cussed in this Engineering Issue. The table presents in-
formation for contaminant treatment in soil, sediment,
sludge, ground water, surface water, and leachate. The
analysis of technology applicability is based on published
literature and expert judgment. Note that the technolo-
gies may be applicable to some contaminants within a
contaminant group but not to others. Site- and contami-
nant-specific treatability studies may be required to de-
termine the actual efficacy of any one technology on the
site-specific soils, contaminants, and conditions.
Applicability of biologically based remedies is highly in-
fluenced by the type of microbiological community that
is best suited for the biodegradation of the particular con-
taminant or mix of contaminants. Of primary importance
in gaining an initial understanding of the applicability of
a biological remedy to contaminants of concern is the
baseline oxidation/reduction potential of the site to be re-
mediated. The presence or absence of oxygen is a signifi-
cant determining factor that defines the microbiological
community characteristics. Some contaminants are best
treated under anaerobic conditions, and others can only
be treated aerobically. Some contaminants can be treated
both anaerobically and aerobically, but there are techni-
cal implications to be considered and contaminant half-
lives may vary between the two processes. Therefore, the
baseline oxidation/reduction potential is of primary im-
portance to the practitioner who is evaluating a contami-
nated site for a biological remedy (Rottero et al., 2004).
Table 4-1. Demonstrated Effectiveness of Biological Treatment Technologies for Soil, Sediment, Bedrock, and Sludge
Contaminant
Type
Non-halogenated VOCs
HalogenatedVOCs
Non-halogenated SVOCs
HalogenatedSVOCs
Fuels
Inorganics
Radionuclides
Explosives
In Situ Treatment Technologies
Intrinsic
Soil/
Ground
Water
Monitored Natural
Attenuation
4
4
A
A
4
Enhanced
Vadose
Zone
Aerobic Bioventing
4
4
4
A
Cometabolic Bioventing
4
Anaerobic Bioventing
A
A
Surficial
Soil
Land Treatment
V
4
42
4
Composting
42
4
42
4
Ground Water/
Saturated Soil
Anaerobic Reductive
Dechlorination
4
A
Aerobic Treatment
4
4
4
A
Biological Reactive
Barriers
4
4
A
4
4
4
4
4
Biosparging/
Bioslurping
4
4
A
4
Ex Situ Treatment Technologies1
Solids
Land Treatment
42
4
42
4
Composting
42
4
42
4
in
_«
'n.
o
m
V
4
42
A
Cnlirl
Liquid
Mixtures
Slurry Bioreactors
V
4
42
4
Liquids
Constructed Wetlands
42
4
42
4
A
1 Not generally applicable to rocks and bedrock. Potential Adverse Effects: Adverse effects are documented at any scale,
2 Volatilization must be controlled. or expert opinion notes that the treatment technology may result in adverse
effects to the environment.
Demonstrated Effectiveness: Successfully treated at pilot or full scale and
verified by an independent agency.
Potential Effectiveness: Successfully treated at laboratory or bench scale, or
similar contaminanttypes have been successfully demonstrated at pilot or full
scale.
No Expected Effectiveness: No successful treatments documented at any
scale, and expert opinion notes thatthe contaminant in question is not likely to
be effectively treated by the technology.
Adapted from information in EPA (1998a, 2000,2004b, 2004c), FRTR (2003, 2004),
ESTCP (2001, 2004a, 2004b), ITRC (2004), and AFCEE (1996).
Site characterization and long-term monitoring are necessary to support
system design and sizing as well as to verify continued performance. There
are also regulatory requirements to be addressed regarding system design,
implementation, operation, and performance, including the disposition of liquid
effluents and other wastes resulting from the treatment process.
In Situ and Ex Situ Biodegredation Technologies
Engineering Issue
17
-------
5.0 SUMMARY
In situ and ex situ biodegradation technologies are in-
creasingly selected to remediate contaminated sites, ei-
ther alone or in combination with other source control
measures. Bioremediation technologies have proven effec-
tive in remediating fuels and VOCs and are often able to
address diverse organic contaminants including SVOCs,
PAHs, CAHs, pesticides and herbicides, and nitro-aro-
matic compounds (such as explosives), potentially at low-
er cost than other remediation options. Some bioreme-
diation techniques are also able to address heavy metal
contamination. Bioremediation continues to be an active
area of research, development, and demonstration for its
applications to diverse contaminated environments.
A unique feature of bioremediation is the diversity of its
application to solids, liquids, and liquid-solid mixtures,
involving both in situ and ex situ environments. Amend-
ments may be necessary to support or enhance the bio-
degradation processes to improve the timeframe involved
to achieve cleanup goals.
Site characterization and long-term monitoring are nec-
essary to support system design and sizing as well as to
verify continued performance. There are also regulatory
requirements to be addressed regarding system design,
implementation, operation, and performance, including
the disposition of liquid effluents and other wastes result-
ing from the treatment process.
6.0 ACKNOWLEDGMENTS
This Engineering Issue paper was prepared for the U.S.
Environmental Protection Agency, Office of Research
and Development, National Risk Management Research
Laboratory (NRMRL) by Science Applications Interna-
tional Corporation (SAIC) under Contract No. 68-C-02-
067. Doug Grosse served as the EPA Work Assignment
Manager. Paul McCauley (NRMRL) acted as the Tech-
nical Project Manager. Lisa Kulujian was SAIC's Work
Assignment Manager, and Jim Rawe and Virginia Hodge
served as SAIC's technical leads and primary authors
of this Engineering Issue paper, with additional techni-
cal input provided by Carolyn Acheson (NRMRL) and
Chris Lutes and David Liles from ARCADIS. Review
and comments were provided by the following members
of the EPA Engineering Forum: Jon Josephs (EPA Region
2), Ed Mead (USAGE), Bernie Schorle (EPA Region 5),
Charles Coyle (USAGE), Michael Gill (EPA Region 9),
and Jon Bornholm (EPA Region 4). David Reisman and
Ann Keely also reviewed drafts of this document.
For additional information, contact the ORD Engineer-
ing Technical Support Center (ETSC):
David Reisman, Director
U.S. EPA Engineering
Technical Support Center
26 W Martin Luther King Drive MLK-489
Cincinnati, OH 45268
(513) 487-2588
Reference herein to any specific commercial products,
process, or service by trade name, trademark, manufac-
turer, or otherwise, does not necessarily constitute or im-
ply its endorsement, recommendation, or favoring by the
United States Government. The views and opinions of
authors expressed herein do not necessarily state or reflect
those of the United States Government, and shall not be
used for advertising or product endorsement purposes.
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In Situ and Ex Situ Biodegredation Technologies
Engineering Issue
21
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SEPA
United States
Environmental Protection
Agency
Office of Research and Development
National Risk Management
Research Laboratory
Cincinnati, OH 45268
Official Business
Penalty for Private Use
$300
EPA/625/R-06/015
October 2006
www.epa.gov
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT No. G-35
Recycled/Recyclable
Printed with vegetable-based ink on
paper that contains a minimum of
50% post-consumer fiber content
processed chlorine free
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