Biorremediacion Estim, Ulada

United States Solid Waste and EPA 542-R-00-008
Environmental Protection Emergency Response July 2000 (revised)

Agency (5102G) www.epa.gov
clu-in.org
EPA Engineered Approaches to

In Situ Bioremediation of
Chlorinated Solvents:
Fundamentals and Field Applications
EPA 542-R-00-008
July 2000 (revised)
ENGINEERED APPROACHES TO IN SITU BIOREMEDIATION

OF CHLORINATED SOLVENTS:
FUNDAMENTALS AND FIELD APPLICATIONS
U.S. Environmental Protection Agency

Office of Solid Waste and Emergency Response
Technology Innovation Office
Washington, DC 20460
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents
NOTICE AND DISCLAIMER
This document was prepared by the U.S. Environmental Protection Agency’s Technology Innovation Office under
EPA Contract Number 68-W-99-003. Information in this report is derived from a variety of references (including
personal communications with experts in the field), some of which have been peer-reviewed. This report has
undergone EPA and external review by experts in the field. Key terms used in this report are defined in a glossary,
and shown in bold print. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use. For more information about this project, please contact: Linda Fiedler, U.S.
Environmental Protection Agency, Technology Innovation Office, Ariel Rios Building, 1200 Pennsylvania Avenue,
N.W. (MS 5102G), Washington, D.C., 20460; (703) 603-7194; e-mail: fiedler.linda@epa.gov. For technical
questions concerning in situ bioremediation, please contact: Ann Azadpour-Keeley, U.S. Environmental Protection
Agency, Office of Research and Development, National Risk Management Research Laboratory, Subsurface
Protection and Remediation Division, P.O. Box # 1198, Ada, Oklahoma, 74820; (580) 436-8890; e-mail:
keeley.ann@epa.gov.
This document may be obtained from EPA’s web site at www.epa.gov/tio/, or at clu-in.org. A limited number of
hard copies of this document are available free of charge by mail from EPA’s National Service Center for
Environmental Publications, at the following address (please allow 4 to 6 weeks for delivery):
U.S. EPA/National Service Center for Environmental Publications

P.O. Box 42419
Cincinnati, OH 45242
Phone: (513) 489-8190 or (800) 490-9198
Fax: (513) 489-8695
ACKNOWLEDGMENTS
Special acknowledgment is given to the following individuals for their thoughtful suggestions and support in
preparing this report: Lewis Semprini, Oregon State University; David Steckel, Edwards Air Force Base; Perry
McCarty, Stanford University; Edward Bouwer, The Johns Hopkins University; Kurt Gerdes, the U.S. Department of
Energy; David W. Anderson and Susan Litherland, Roy F. Weston, Inc.; Daniel L. Jacobs, ARCADIS Geraghty &
Miller; Willard Murray, Harding Lawson Associates; Mick Mikula, Dover Air Force Base; and David Ellis, E.I. du
Pont de Nemours and Company.
i
FOREWORD
Halogenated volatile organic compounds, including chlorinated solvents, are the most frequently-
occurring type of soil and groundwater contaminant at Superfund and other hazardous waste sites in the
United States. The U.S. Environmental Protection Agency (EPA) estimates that, over the next several
decades, site owners will spend billions of dollars to clean up these sites. New technologies that are less
costly and more effective are needed to accomplish hazardous waste site remediation. As these new and
innovative technologies are being developed and used, site managers require information on how they
work, their performance to date, and how to evaluate their application at a particular site.
This report provides an overview of the fundamentals and field applications of in situ bioremediation to
remediate chlorinated solvents in contaminated soil and groundwater. In situ treatment is increasingly
being selected to remediate sites because it is usually less expensive, and does not require waste
extraction or excavation. In addition, in situ bioremediation is more publicly acceptable than above-
ground technologies because it relies on natural processes to treat contaminants.
This document presents information at a level of detail intended to familiarize federal and state project
managers, permit writers, technology users, and contractors with in situ bioremediation. The report
describes how chlorinated solvents are degraded, how to enhance the process by the addition of various
materials and chemicals, design configurations, and the typical steps taken to evaluate technology
feasibility at a specific site. It also includes a list of technology vendors and nine case studies of field
applications.
It is important to note that this report cannot be used as the sole basis for determining this technology’s
applicability to a specific site. That decision is based on many factors and must be made on a case-by-
case basis. Technology expertise and sometimes treatability studies also are required to make a final
remedy decision.
ii
TABLE OF CONTENTS
Section Page
1.0 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1
2.0 BIOREMEDIATION MECHANISMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1
2.1 TYPICAL CAHs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1

2.2 PHYSICAL AND CHEMICAL PROPERTIES OF CAHs . . . . . . . . . . . . . . . . . . . . . . 2-1
2.3 TRANSPORT PROCESSES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5
2.4 DEGRADATION MECHANISMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7
2.4.1 Aerobic Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-13

2.4.2 Anaerobic Reductive Dechlorination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-15
2.4.3 Abiotic Degradation Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-17
3.0 IN SITU BIOREMEDIATION TECHNOLOGIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1
3.1 TECHNOLOGIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1

3.2 DESIGN APPROACHES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3
3.3 VENDORS OF IN SITU BIOREMEDIATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7
4.0 SELECTION AND IMPLEMENTATION OF IN SITU BIOREMEDIATION

TECHNOLOGIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1
4.1 TECHNOLOGY SELECTION AND IMPLEMENTATION . . . . . . . . . . . . . . . . . . . . 4-1
4.1.1 Site Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1

4.1.2 Site Conditions and Engineering Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5
4.1.3 Primary Reactants and Additives Typically Employed . . . . . . . . . . . . . . . . . . 4-6
4.1.4 Treatability (Bench-Scale) Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-7
4.1.5 System Design, Field Testing, and Implementation . . . . . . . . . . . . . . . . . . . . . 4-8
4.2 ADDITIONAL SELECTION FACTORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-11
5.0 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-1
6.0 ADDITIONAL INFORMATION SOURCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1
iii
TABLE OF CONTENTS (continued)
APPENDIX A CASE STUDIES AND SUMMARIES OF SITES USING IN SITU

BIOREMEDIATION TECHNOLOGIES FOR CAHs . . . . . . . . . . . . . . . . . . A-1
Case Study # 1
Aerobic Degradation Field Demonstration at Moffett Naval Air Station,
Mountain View, California . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-15
Case Study # 2
Aerobic Degradation Field Demonstration at Site 19, Edwards Air Force Base,
California . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-22
Case Study #3
Methane Enhanced Bioremediation Field Demonstration Using Horizontal Wells
at Savannah River Site, Aiken, South Carolina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-29
Case Study #4
Full-Scale Enhanced Bioremediation at the Texas Gulf Coast Site,
Houston, Texas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-35
Case Study #5
Pilot and Full-Scale Molasses Injection at the Avco Lycoming Superfund Site,
Williamsport, Pennsylvania . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-42
Case Study #6
Pilot and Full-Scale Anaerobic In Situ Reactive Zone at an Abandoned
Manufacturing Facility, Emeryville, California . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-49
Case Study # 7
Sequential Anaerobic/Aerobic Biodegradation of PCE Field Demonstration
at Watertown, Massachusetts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-56
Case Study # 8
Bioaugmentation (Accelerated Anaerobic Bioremediation) Field Demonstration
at Area 6 of the Dover Air Force Base, Dover, Delaware . . . . . . . . . . . . . . . . . . . . . A-62
Case Study # 9
Cometabolic Bioventing Field Demonstration at Building 719,
Dover Air Force Base, Dover, Delaware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-71
iv
LIST OF EXHIBITS
2-1 CAHs Commonly Identified as Environmental Contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2

2-2 Molecular Structures of Common CAHs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3
2-3 Chemical and Physical Properties of CAHs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4
2-4 Phase Equilibrium Mechanisms and Defining Properties of CAHs . . . . . . . . . . . . . . . . . . . . . . 2-5
2-5 Example CAH Subsurface Transport Processes (DNAPL Source) . . . . . . . . . . . . . . . . . . . . . . 2-6
2-6 Microbial Biodegradation and Ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-8
2-7 Redox Zones of a Typical Petroleum Plume in an Aerobic Aquifer (Areal View) . . . . . . . . . . 2-9
2-8 Selected Information on CAH Degradation Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-10
2-9 Biodegradation Mechanisms Typically Occurring During Enhanced In Situ Bioremediation
of CAHs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-12
2-10 Constituents Involved in Biodegradation Mechanisms for Enhanced In Situ Bioremediation
of CAHs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-12
2-11 Aerobic Oxidation (Direct) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-13
2-12 Aerobic Oxidation (Cometabolic) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-14
2-13 Anaerobic Reductive Dechlorination of PCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-16
2-14 Example of Hydrolysis Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-18
2-15 Example of Elimination Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-18
2-16 Example of Abiotic Reductive Dechlorination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-18
3-1 Components of In Situ Bioremediation Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2

3-2 Bioremediation System Configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5
3-3 In Situ Bioremediation System Configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6
3-4 Vendors Listed in EPA REACH IT That Provide In Situ Bioremediation Services . . . . . . . . . 3-8
4-1 Typical Selection and Implementation Steps for In Situ Bioremediation . . . . . . . . . . . . . . . . . 4-2
4-2 Common Site Characterization Parameters Relevant to In Situ Bioremediation . . . . . . . . . . . . 4-3
4-3 Range of Hydrogen Concentrations by Terminal Electron Accepting Process . . . . . . . . . . . . . 4-5
4-4 Generally Favorable and Unfavorable Site Conditions for In Situ Bioremediation of CAHs
and Typical Engineered Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6
4-5 Primary Reactants and Additives Typically Employed for Engineered
In Situ Bioremediation of CAHs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-7
A-1 Summary of Case Studies of In Situ Bioremediation Technology Applications . . . . . . . . . . . A-2

A-2 Summary of Full-Scale Applications and Pilot Studies Identified in Proceedings of
The Fifth International In Situ and On-Site Bioremediation Symposium . . . . . . . . . . . . . . . . A-4
A-3 Summary of Full-Scale Applications and Pilot Studies Identified in the Bioremediation
in the Field Search System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-10
v
GLOSSARY OF KEY TERMS
Abiotic Nonbiological process; also used to refer to nonbiological degradation

process.
Adsorption Removal of a substance from air or water by collecting the substance on the
surface of a solid material; process used in pollution control systems such as
activated carbon adsorption systems.
Advection The process of transfer of fluids (vapors or liquid) through a geologic
formation in response to a pressure gradient that may be caused by changes
in barometric pressure, water table levels, wind fluctuations, or infiltration.
Aerobic Condition in which oxygen is present; also used to refer to a type of
microbe that requires oxygen to live and reproduce.
Aerobic oxidation Microbial breakdown of a contaminant during which a contaminant is
(cometabolic) oxidized by an enzyme or cofactor produced during microbial metabolism
of another compound with oxygen. In such a case, the oxidation of the
contaminant does not yield any energy or growth benefit for the microbe
mediating the reaction.
Aerobic oxidation Microbial breakdown of a compound during which the compound serves as
(direct) an electron donor and as a primary growth substrate by the microbe
mediating the reaction. Electrons that are generated by the oxidation of the
compound are transferred to oxygen.
Air- or bio-sparging The process of injecting pressurized air beneath the water table to promote
mass transfer of volatile organic compounds out of the groundwater and
mass transfer of oxygen into the groundwater.
Anaerobic Condition in which no oxygen is present; also used to refer to a type of
microbe that is able to live and reproduce in the absence of oxygen.
Anaerobic reductive A biodegradation reaction in which a chlorinated hydrocarbon is reduced by
dechlorination an enzyme or cofactor produced during microbial metabolism of another
(cometabolic) compound in an environment devoid of oxygen. In such a case,
biodegradation of the chlorinated compound does not yield any energy or
growth benefit for the microbe mediating the reaction.
Anaerobic reductive A biodegradation reaction in which bacteria gain energy and grow as one or
dechlorination more chlorine atoms on a chlorinated hydrocarbon are replaced with
(direct) hydrogen in an environment devoid of oxygen. In the reaction, the
chlorinated compound serves as the electron acceptor and hydrogen serves
as the direct electron donor. Hydrogen used in this reaction typically is
supplied indirectly by the fermentation of organic substrates. The reaction
is also referred to as halorespiration or dehalorespiration.
vi
Bioaugmentation The addition of microbes to the subsurface where organisms able to degrade
specific contaminants are deficient. Microbes may be “seeded” from
populations already present at a site and grown in aboveground reactors or
from specially cultivated strains of bacteria having known capabilities to
degrade specific contaminants.
Bioenergetics The energy and mass transfer kinetics that are defined by microbial cell
metabolism.
Biomass All the living material in a given area.
Bioremediation A process by which microorganisms, fungi, and plants degrade pollutant
chemicals through use or transformation of the substances.
Capillary forces Forces that govern fluid flow through small diameter pathways, such as in
subsurface soil particles.
Chlorinated aliphatic Manmade, chlorine-containing organic compounds widely used as solvents
hydrocarbons and degreasers in various industries. Typical CAHs include
(CAHs) tetrachloroethene (PCE), trichloroethene (TCE), dichloroethene (DCE), and
vinyl chloride (VC).
Confining layer Impermeable layer (such as clay) that impedes the vertical migration of
groundwater or NAPL.
Degradation Chemical or biological breakdown of a complex compound into simpler
compounds. The breakdown may occur as a result of a single reaction or
multiple reactions.
Dense non-aqueous Chlorinated solvents that are minimally soluble in water, more dense than
phase liquids water, and present in concentrations large enough to form pools of free
(DNAPL) liquid. DNAPLs tend to sink and accumulate on a non-permeable layer
(aquitard) at the bottom of a confined aquifer.
Diffusion The movement of suspended or dissolved particles (or molecules) from an
area of higher concentration to one in which concentrations are lower. This
process tends to distribute the particles or molecules more uniformly.
Dispersion The process by which a substance or chemical spreads and dilutes in
flowing groundwater or soil gas.
Electron acceptor A compound capable of accepting electrons during oxidation-reduction
reactions. Microorganisms obtain energy by transferring electrons from
electron donors, such as organic compounds (or sometimes reduced
inorganic compounds, such as sulfide), to an electron acceptor. Electron
acceptors are compounds that are reduced during the process and include
oxygen; nitrate; iron (III); manganese (IV); sulfate; carbon dioxide; or, in
some cases, chlorinated aliphatic hydrocarbons, such as carbon
tetrachloride, PCE, TCE, DCE, and VC.
vii
Electron donor A compound capable of supplying (giving up) electrons during oxidation-
reduction reactions. Microorganisms obtain energy by transferring
electrons from electron donors, such as organic compounds (or sometimes
reduced inorganic compounds, such as sulfide), to an electron acceptor.
Electron donors are compounds that are oxidized during the process and
include fuel hydrocarbons and native organic carbon.
Enhanced Bioremediation of organic contaminants by microbes supplemented by
bioremediation increasing the concentration of electron acceptors, electron donors, or
nutrients in groundwater, surface water, and soil.
Exogenous bacteria Bacteria that have been obtained from a source other than the native site.
(also called non-
indigenous)
Groundwater A closed-loop, hydraulically-contained system based on a design of down-
recirculation gradient extraction and upgradient injections wells; sometimes referred to as
treatment system a recirculating treatment cell.
Hydrophobicity Tendency to repel water.
Metabolic Having to do with the energy producing processes conducted in cells.
Methanogenic Referring to the formation of methane by certain anaerobic bacteria during
the process of anaerobic fermentation.
Monitored natural Use of natural subsurface processes, such as dilution, volatilization,
attenuation (also biodegradation, adsorption, and chemical reactions with subsurface
known as Passive materials to reduce contaminant concentrations.
bioremediation)
Nutrients Elements required for microbial growth. In bioremediation, the term
generally refers to elements other than carbon, hydrogen, and oxygen that
are required to promote the growth of bacteria. Typical nutrients include
nitrogen and phosphorus.
Sorption The action of soaking up or attracting substances; a general term used to
encompass the processes of absorption, adsorption, ion exchange, and
chemisorption.
Substrate A source of energy or molecular building block used by a microorganism to
carry out biological processes and reproduce.
Volatilization The process of transfer of a chemical from the aqueous or liquid phase to
the gas phase.
viii
LIST OF ACRONYMS
ADP Adenosine diphosphate

ATP Adenosine triphosphate
BFSS Bioremediation in the Field Search System
CA Chloroethane
CAHs Chlorinated aliphatic hydrocarbons
CERCLA Comprehensive Environmental Response, Compensation, and Liability Act

CF Chloroform
CFR Code of Federal Regulations
CM Chloromethane
CO2 Carbon dioxide
CT Carbon tetrachloride
DCA Dichloroethane
DCE Dichloroethene
DNAPL Dense non-aqueous phase liquid
EPA U.S. Environmental Protection Agency
EPA REACH IT EPA REmediation And CHaracterization Innovative Technologies
Fe0 Zero-valent iron

Fe2+ Iron (II) ion
GW Groundwater
H2O2 Hydrogen peroxide
HCl Hydrogen chloride

HRC Hydrogen release compound
HS- Hydrogen sulfide ion
LDR Land Disposal Restrictions
LNAPL Light non-aqueous phase liquid
MC Methylene chloride
MNA Monitored natural attenuation
NAD Nicotinamide adenine dinucleotide
NAPL Non-aqueous phase liquid
ORC Oxygen release compound
ix
PCE Tetrachloroethene
PRB Permeable reactive barrier
RCRA Resource Conservation and Recovery Act
TCA Trichloroethane
TCE Trichloroethene
UIC Underground Injection Control

UST Underground storage tank
VC Vinyl chloride
x
1.0 INTRODUCTION
Halogenated volatile organic compounds (VOCs), including chlorinated aliphatic hydrocarbons
(CAHs)1, are the most frequently occurring type of contaminant in soil and groundwater at Superfund
and other hazardous waste sites in the United States. The U.S. Environmental Protection Agency (EPA)
estimates that cleanup of these sites will cost more than $45 billion (1996 dollars) over the next several
decades (EPA, 1997). Innovative technologies, including in situ bioremediation, are being developed
and implemented in an effort to reduce the cost and time required to clean up those sites. In situ
bioremediation is increasingly being selected to remediate hazardous waste sites because, when
compared to above-ground technologies, it is usually less expensive, does not require waste extraction or
excavation, and is more publicly acceptable as it relies on natural processes to treat contaminants.
This report provides an overview of the fundamentals and field applications of in situ bioremediation of
CAHs in contaminated soil and groundwater, including a summary of currently-available information on
the mechanisms and technologies used to implement in situ bioremediation. The report is intended to
familiarize those involved with hazardous waste site cleanups, including site project managers,
contractors, and other technology users, with in situ bioremediation. As such, the level of detail included
in the report about bioremediation mechanisms, technologies, and implementation is meant to provide
basic information about the technology, rather than providing an in depth primer about in situ
bioremediation. Therefore, the report should be used for informational purposes, but should not be used
as the sole basis for determining the use of this technology at a specific site. Such decisions must be
made on a case-by-case basis, considering site-specific factors. Information included in this report is not
intended to revise EPA policy or guidance concerning site clean up.
Section 2 of this report provides a description of bioremediation mechanisms, including the fate and
transport processes and degradation mechanisms. Section 3 describes the types of technologies used for
in situ bioremediation, including approaches used to design remedial systems. Information about vendors
of in situ bioremediation at sites contaminated with CAHs is also provided. Section 4 discusses the steps
involved with the selection and implementation of in situ bioremediation as a site remedy, including
factors typically considered. The references used in preparing this report are presented in Section 5.
Section 6 includes a list of additional information sources providing further detail about in situ
bioremediation.
Appendix A provides nine case studies of applications of in situ bioremediation of CAHs at Superfund
and other sites. Of the eight groundwater projects, three involved field demonstrations of aerobic
oxidation (cometabolic), three were full-scale projects that used anaerobic reductive dechlorination,
one was a field demonstration that used a combination of aerobic oxidation (cometabolic) and anaerobic
reductive dechlorination, and one was a field demonstration of bioaugmentation. The one soil project
was a field demonstration of aerobic oxidation (cometabolic).
1
Key terms used in this report are defined in a glossary, and shown in bold print.
1-1
1.0 INTRODUCTION
Halogenated volatile organic compounds (VOCs), including chlorinated aliphatic hydrocarbons
(CAHs)1, are the most frequently occurring type of contaminant in soil and groundwater at Superfund
and other hazardous waste sites in the United States. The U.S. Environmental Protection Agency (EPA)
estimates that cleanup of these sites will cost more than $45 billion (1996 dollars) over the next several
decades (EPA, 1997). Innovative technologies, including in situ bioremediation, are being developed
and implemented in an effort to reduce the cost and time required to clean up those sites. In situ
bioremediation is increasingly being selected to remediate hazardous waste sites because, when
compared to above-ground technologies, it is usually less expensive, does not require waste extraction or
excavation, and is more publicly acceptable as it relies on natural processes to treat contaminants.
This report provides an overview of the fundamentals and field applications of in situ bioremediation of
CAHs in contaminated soil and groundwater, including a summary of currently-available information on
the mechanisms and technologies used to implement in situ bioremediation. The report is intended to
familiarize those involved with hazardous waste site cleanups, including site project managers,
contractors, and other technology users, with in situ bioremediation. As such, the level of detail included
in the report about bioremediation mechanisms, technologies, and implementation is meant to provide
basic information about the technology, rather than providing an in depth primer about in situ
bioremediation. Therefore, the report should be used for informational purposes, but should not be used
as the sole basis for determining the use of this technology at a specific site. Such decisions must be
made on a case-by-case basis, considering site-specific factors. Information included in this report is not
intended to revise EPA policy or guidance concerning site clean up.
Section 2 of this report provides a description of bioremediation mechanisms, including the fate and
transport processes and degradation mechanisms. Section 3 describes the types of technologies used for
in situ bioremediation, including approaches used to design remedial systems. Information about vendors
of in situ bioremediation at sites contaminated with CAHs is also provided. Section 4 discusses the steps
involved with the selection and implementation of in situ bioremediation as a site remedy, including
factors typically considered. The references used in preparing this report are presented in Section 5.
Section 6 includes a list of additional information sources providing further detail about in situ
bioremediation.
Appendix A provides nine case studies of applications of in situ bioremediation of CAHs at Superfund
and other sites. Of the eight groundwater projects, three involved field demonstrations of aerobic
oxidation (cometabolic), three were full-scale projects that used anaerobic reductive dechlorination,
one was a field demonstration that used a combination of aerobic oxidation (cometabolic) and anaerobic
reductive dechlorination, and one was a field demonstration of bioaugmentation. The one soil project
was a field demonstration of aerobic oxidation (cometabolic).
1
Key terms used in this report are defined in a glossary, and shown in bold print.
1-1
2.0 BIOREMEDIATION MECHANISMS

This section provides background information about in situ bioremediation at sites contaminated with
CAHs, including:
• Typical CAHs (Section 2.1)

• Physical and chemical properties of CAHs (Section 2.2)
• Processes that transport CAHs through the subsurface (Section 2.3)
• Biological and chemical mechanisms that can degrade CAHs (Section 2.4)
More detailed information about the physical and chemical characteristics of CAHs and the subsurface
transport processes of CAHs can be found in the references listed in Section 6.0 of this report.
2.1 TYPICAL CAHs
CAHs are manmade organic compounds. They typically are manufactured from naturally occurring
hydrocarbon constituents (methane, ethane, and ethene) and chlorine through various processes that
substitute one or more hydrogen atoms with a chlorine atom, or selectively dechlorinate chlorinated
compounds to a less chlorinated state. CAHs are used in a wide variety of applications, including use as
solvents and degreasers and in the manufacturing of raw materials. CAHs include such solvents as
tetrachloroethene (PCE), trichloroethene (TCE), carbon tetrachloride (CT), chloroform (CF), and
methylene chloride (MC).
Historical management of wastes containing CAHs has resulted in contamination of soil and
groundwater, with CAHs present at many contaminated groundwater sites in the United States. TCE is
the most prevalent of those contaminants (U.S. Air Force 1998). In addition, CAHs and their
degradation products, including dichloroethane (DCA), dichloroethene (DCE), and vinyl chloride (VC),
tend to persist in the subsurface. Exhibit 2-1 lists the CAHs most commonly identified as environmental
contaminants, their abbreviations, their common names, and the types of waste from which they
commonly originate. Exhibit 2-2 presents the molecular structures of those CAHs.
2.2 PHYSICAL AND CHEMICAL PROPERTIES OF CAHs
The physical and chemical properties of CAHs govern their fate and transport in the subsurface
environment. The number of substituted chlorine atoms on the CAHs directly affects their physical and
chemical behavior. As the number of substituted chlorine atoms increases, molecular weight and density
generally increase, and vapor pressure and aqueous solubility generally decrease. Exhibit 2-3 lists
pertinent physical and chemical data for the CAHs commonly identified as subsurface contaminants.
2-1
Exhibit 2-1: CAHs Commonly Identified as Environmental Contaminants
Name Common Name(s) Abbreviation1 Common Waste Sources

CHLORINATED ETHENES
Tetrachloroethene(-ethylene) Perchloroethene PCE Solvent waste
Trichloroethene(-ethylene) None TCE Solvent waste, degradation product of
PCE
cis-1,2-Dichloroethene(-ethylene) Acetylene dichloride cis-DCE Solvent waste, degradation product of
PCE and TCE
trans-1,2-Dichloroethene Acetylene dichloride trans-DCE Solvent waste, degradation product of
(-ethylene) PCE and TCE
1,1-Dichloroethene(-ethylene) Vinylidene chloride 1,1-DCE Solvent waste, degradation product of
1,1,1-TCA
Chloroethene(-ethylene) Vinyl chloride VC Polyvinyl chloride production waste,
degradation product of PCE and 1,1,1-
TCA
CHLORINATED ETHANES
1,1,1-Trichloroethane Methyl chloroform 1,1,1-TCA Solvent waste
1,1,2-Trichloroethane Vinyl trichloride 1,1,2-TCA Solvent waste
1,2-Dichloroethane Ethylene chloride 1,2-DCA Solvent waste, degradation product of
1,1,2-TCA
1,1-Dichloroethane Ethylidene chloride 1,1-DCA Degradation product of 1,1,1-TCA
Chloroethane None CA Refrigerant waste, tetraethyl lead
manufacturing waste, degradation product
of 1,1,1-TCA and 1,1,2-TCA
CHLORINATED METHANES
Tetrachloromethane Carbon CT Solvent waste, fire extinguisher waste
tetrachloride
Trichloromethane Chloroform, CF Solvent waste, anesthetic waste, waste
methane trichloride degradation product of CT
Dichloromethane Methylene chloride, MC Solvent waste, degradation product of CT
methylene dichloride
Chloromethane Methyl chloride, CM Refrigerant waste, degradation product of
monochloromethane CT
Notes:
1
Abbreviations are based on the names in bold italic type.
Sources: Sawyer and others 1994; Merck 1989
2-2
Exhibit 2-2: Molecular Structures of Common CAHs
Source: Modified from Sawyer and others 1994
2-3
Exhibit 2-3: Chemical and Physical Properties of CAHs
Number of
Substituted Molecular Aqueous Log KOW Henry’s Law
Chlorine Weight Liquid Density (g/ml Solubility (mg/L Vapor Pressure (Octanol/Water Constant
Compound Atoms (g/mole)1 @ 20 °F/4 °C)1 @ approx. 25°C)2 (mm Hg @ 25 °C)2 Partition Coefficient)2 (atm-m3/mol)3
Chlorinated Ethenes
PCE 4 165.8 1.62 150 17.8 2.60 0.0153
TCE 3 131.4 1.46 1,100 57.9 2.38 0.0091
cis-DCE 2 96.9 1.28 3,500 208 0.70 0.0037
trans-DCE 2 96.9 1.28 6,300 324 0.48 0.0072
1,1-DCE 2 96.9 1.21 2,250 600 1.84 0.018
VC 1 62.5 gas 2,670 2,660 1.38 0.315 (5)
Chlorinated Ethanes
1,1,1-TCA 3 133.4 1.34 1,500 123 2.50 0.008
1,1,2-TCA 3 133.4 1.44 4,500 30 2.47 0.0012
1,2-DCA 2 99.0 1.26 8,520 64 1.48 0.00098
1,1-DCA 2 99.0 1.18 5,500 182 1.79 0.0059
CA 1 64.5 gas 5,700 1,064 1.52 to 2.16 (4) 0.0085
Chlorinated Methanes
CT 4 153.8 1.59 757 90 2.64 0.0304
CF 3 119.4 1.48 8,200 151 1.97 0.00435
MC 2 84.9 1.33 20,000 362 1.30 0.00268
CM 1 50.5 gas 6,500 4,310 0.95 0.0452 (5)

Notes:
1. Data from Merck Index 1989
2. Data from EPA 1986
3. Data from Gossett 1987
4. Data from EPA 1998
5. Data from EPA 1998a
“Gas” is indicated for liquid density of VC, CA, and CM because they are pure compounds that are gasses under typical environmental conditions.
2-4
2.3 TRANSPORT PROCESSES
A CAH released to the subsurface as a pure organic liquid (commonly referred to as non-aqueous phase
liquid [NAPL] in the subsurface) will seek phase equilibrium (a condition in which all acting influences
are canceled by others, resulting in a stable, balanced, or unchanging system). The CAH will remain as a
NAPL, adsorb to soil, dissolve in groundwater, or volatilize into soil gas to the extent defined by the
physical and chemical properties of the individual CAH and the subsurface environment. Partition
coefficients, which are related to the hydrophobicity and aqueous solubility of a CAH, define the extent
to which a CAH will partition between NAPL, adsorb to soil, and dissolve in groundwater. The vapor
pressure of a CAH defines the extent to which it will partition between NAPL or NAPL adsorbed to soil
and the soil gas. CAHs dissolved in groundwater will also partition themselves between the dissolved
phase and the vapor phase, as defined by their Henry’s Constant. Exhibit 2-4 shows those mechanisms
by which CAHs transfer phases in an attempt to reach equilibrium conditions and their related properties.
Exhibit 2-4: Phase Equilibrium Mechanisms

and Defining Properties of CAHs
Source: Modified from Huling, S.G. and J.W. Weaver 1991
Most of the CAH NAPLs discussed in this report are denser than water (referred to as dense non-
aqueous phase liquids [DNAPLs]). The exceptions are vinyl chloride, chloroethane, and
chloromethane, which are gaseous in their pure phase under standard conditions. DNAPLs tend to sink
through both unsaturated and saturated permeable soils until they reach the lowest point on the top of a
confining layer. NAPLs that are less dense than water (referred to as light non-aqueous phase liquids
[LNAPL]) will sink through unsaturated permeable soils and float on the water table, migrating to the
lowest water table elevation. In addition, capillary forces can trap NAPLs in porous media above or
below the water table.
In addition to transferring phases in an attempt to reach equilibrium conditions, CAHs can migrate in the
subsurface in their non-aqueous, aqueous, and vapor phases by both active and passive processes. In
active processes, such as advection and dispersion, CAHs migrate along with the flow of the
groundwater or soil gas to which they are partitioned. Passive processes, such as diffusion, are the result
of concentration gradients, which cause the CAH to seek phase and concentration equilibrium with its
surrounding environment. The extent of subsurface migration is a function of the volume of CAH
2-5
released, the area over which the release occurs, the duration of the release, and the chemical and
physical properties of both the CAH and the subsurface environment.
Typically, releases of CAHs to the groundwater result in the formation of a plume; releases to soil result
in subsurface soil contaminated with CAH constituents. In soil, CAHs typically are transported by the
flow of DNAPL or diffusion in soil-gas vapor. In groundwater, advective transport (the movement of
contaminants by flowing groundwater) is one of the most important processes that affect the transport of
dissolved CAHs. In general, the more soluble the compound, the further it will be transported in the
subsurface. For example, based on solubility data provided in Exhibit 2-3, MC and CF would be
transported more readily in groundwater that PCE and CT. Exhibit 2-5 presents an example of
subsurface transport processes.
Exhibit 2-5: Example CAH Subsurface Transport Processes (DNAPL Source)
Source: Modified from Sims and others 1992
2-6
2.4 DEGRADATION MECHANISMS
Bioremediation of CAHs can occur through natural mechanisms (intrinsic bioremediation) or by

enhancing the natural mechanisms (enhanced bioremediation).1 For a few CAHs (for example, 1,1,1-
TCA and CT), degradation also can also occur by abiotic (nonbiological) mechanisms. In most systems,
biological degradation tends to dominate, depending on the type of contaminant and the groundwater
chemistry (EPA 1998). Although a number of biological degradation mechanisms have been identified
theoretically and observed on a laboratory scale, the bioremediation mechanisms carried out by bacteria
that typically are used for enhanced bioremediation of CAHs generally can be classified into one of the
following mechanism categories:
& Aerobic oxidation (direct and cometabolic)

& Anaerobic reductive dechlorination (direct and cometabolic)
While aerobic oxidation and anaerobic reductive dechlorination can occur naturally under proper
conditions, enhancements such as the addition of electron donors, electron acceptors, or nutrients help to
provide the proper conditions for aerobic oxidation or anaerobic reductive dechlorination to occur. In
general, highly chlorinated CAHs degrade primarily through reductive reactions, while less chlorinated
compounds degrade primarily through oxidation (Vogel and others 1987b). Highly chlorinated CAHs
are reduced relatively easily because their carbon atoms are highly oxidized. During direct reactions, the
microorganism causing the reaction gains energy or grows as the CAH is degraded or oxidized. During
cometabolic reactions, the CAH degradation or oxidation is caused by an enzyme or cofactor produced
during microbial metabolism of another compound. CAH degradation or oxidation does not yield any
energy or growth benefit for the microorganism mediating the cometabolic reaction.
Exhibit 2-6 presents a summary of the nomenclature of microbial biodegradation and ecology. As shown
in Exhibit 2-6, biodegradation involves the production of energy in a redox reaction within a bacterial
system. This includes respiration and other biological functions needed for cell maintenance and
reproduction. Ecology involves the different types of bacteria electron acceptor classes, such as oxygen-,
nitrate-, manganese-, iron (III)-, sulfate-, or carbon dioxide-reducing, and their corresponding redox
potentials. Redox potentials provide an indication of the relative dominance of the electron acceptor
classes.
1
Although the subject is not covered in this report, CAHs can also be degraded or otherwise removed from soil and
groundwater by larger organisms (such as trees), in a process referred to as phytoremediation. References and
additional information sources for phytoremediation are listed in Sections 5.0 and 6.0 of this report, respectively.
2-7
Exhibit 2-6: Microbial Biodegradation and Ecology
For the most part, the microorganisms that carry out the Typically, bacteria that are involved in the
bioremediation of the CAHs are single-celled procaryotic biodegradation of CAHs in the subsurface are
bacteria. As living organisms, the bacteria require a chemotrophs (bacteria that derive their energy from
source of food to survive and propagate. This chemical redox reactions) and use organic compounds as
requirement, or the bioenergetics of a bacterial system, is electron donors and sources of organic carbon
defined by the thermodynamics of the processes used by (organoheterotrophs). However, lithotrophs (bacteria
the microbes to derive energy and raw materials from that use inorganic electron donors) and autotrophs
substrates and to use them to carry on biological processes (bacteria that use carbon dioxide as a carbon source) also
and reproduce. The figure below depicts the basic may be involved in degradation of CAHs.
bioenergetics of a typical microbial system.
CAH-degrading bacteria are classified further by the
ADP = adenosine diphosphate electron acceptor that they use, and therefore the type of
zone that will dominate in the subsurface (for example,
an aerobic zone will dominate when aerobes are present).
The typical electron-acceptor classes of bacteria are listed
below in the order of those causing the largest energy
generation during the redox reaction to those causing the
smallest energy generation during the redox reaction. A
bacteria electron acceptor class causing a redox reaction
generating relatively more energy will dominate over a
bacteria electron acceptor class causing a redox reaction
generating relatively less energy.
Dominance
(as Predominant Approx-
determined Bacteria CAH imate
by relative Electron Biodegrada- Redox
energy Acceptor tion Potential
generation Class Mechanism (volts)1
Oxygen-
ATP = adenosine triphosphate Aerobic
reducing +0.82
Most oxidation
(aerobes)
In general, the mediating bacteria collect energy in the dominant
form of electrons by a chemical reduction-oxidation Nitrate-
+0.74
(redox) reaction (or photosynthesis). The energy is | reducing
generated in a redox reaction from the transfer of | Mangane
electrons from an electron donor (the organic contaminant | se (IV)- +0.52
in aerobic oxidation (direct)) to an electron acceptor | reducing
|
(oxygen in an aerobic reaction). The energy gained is Iron (III)-
| -0.05
stored as high energy compounds, such as ATP and low- | reducing Reductive
energy compounds, such as nicotinamide adenine | dechlorination
Sulfate-
dinucleotide (NAD). A portion of the stored energy is | -0.22
reducing
used to conduct the biological processes necessary for cell \/
maintenance and reproduction. In addition, cell building- Carbon
block materials are required in the form of carbon and Least dioxide-
other nutrients (such as nitrogen and phosphorus). The dominant reducing -0.24
(methana-
thermodynamics of a given system defines the energy that
trophs)
is available from a substrate, the energy transfer efficiency 1
losses that will occur, and the portion of the available Standard redox potentials at pH of 7
energy that will be used for reproduction versus the
portion that will be used for cell maintenance.
Bacteria generally are categorized by 1) the means by

which they derive energy, 2) the type of electron donors
they require, or 3) the source of carbon that they require.
Sources: Anderson, R.T., and D.R. Lovley 1997; McCarty, P.L. 1971; EPA 1998
2-8
Exhibit 2-7 shows the redox zones of a typical petroleum plume in an aerobic aquifer, showing the
progression from the source area to the edge of the plume. A plume moving with groundwater flow
typically will develop distinct redox zones (bacteria will use the electron acceptor that causes the most
energy to be generated during the redox reaction when compared with the energy generated from redox
reactions using other available electron acceptors). As Exhibit 2-7 shows, once an electron acceptor is
depleted, a new redox reaction with the electron acceptor that will result in the next largest generation of
energy during the redox reaction will dominate. The dominant redox reaction will determine the type of
bacteria that typically will exist in a particular zone and determine the CAH biodegradation mechanisms
that may occur.
Exhibit 2-7: Redox Zones of a Typical Petroleum Plume in an Aerobic

Aquifer (Areal View)
Source: Modified from Anderson, R.T. and D.R. Lovley 1997
Exhibit 2-8 provides a summary of information available for each degradation mechanism. The
degradation mechanisms that typically occur in the biodegradation of each CAH are summarized in
Exhibit 2-9, while Exhibit 2-10 presents a summary of the constituents involved in the redox reactions
that support each mechanism. Information presented in Exhibit 2-10 was derived from a number of
literature sources and from the case studies included in Appendix A of this report.
The remainder of this section presents a more detailed discussion about each of the mechanisms
including the specific CAHs that they can degrade, the types of conditions (aerobic or anaerobic) under
which they occur, and information about the biology or chemistry of each mechanism.
2-9
Exhibit 2-8: Selected Information on CAH Degradation Mechanisms
Degradation
Mechanism CAH Conditions Reported Bacteria Reported Rate Data Product Source
AEROBIC OXIDATION
Aerobic oxidation DCE, VC Aerobic Not reported Observed 2nd-order Michaelis-Menton CO2 Bradley and
(direct) kinetics (Vmax = 5.1 and 12.4 Chapelle 1998
umol/L/D for DCE and VC,
respectively)
DCE, VC, DCA, Aerobic Not reported Not reported CO2 RTDF 1997
CA, MC, CM
Aerobic oxidation TCE Aerobic, electron donor (phenol, Burkholderia cepacia G4, Not reported CO2 Munakata-Marr
(cometabolic) toluene, benzene) PR1301 1997; McCarty
and others 1998
TCE Aerobic, electron donor (toluene) Not reported 1 st order Not Petrovskis and
0.26-0.4 mol/d reported others 1995
TCE, DCE, VC, Aerobic, electron donor Not reported Not reported CO2 RTDF 1997
TCA, CF, MC (methane, aromatics, ammonia)
ANAEROBIC REDUCTIVE DECHLORINATION
Anaerobic reductive PCE, TCE, DCE, Anaerobic, electron donor PER-K23, Dehalospirillium Zero-order dechlorination kinetics for Ethene, Hollinger 1993;
dechlorination VC, DCA (hydrogen or fermentive multivorans, Dehalobacter PCE, TCE, cis-DCE and first-order ethane Smatlak 1996;
(dehalorespiration) hydrogen source), relatively low restrictus, Dehalococcus for trans-DCE and VC; total Tandol 1994;
Hydrogen partial pressure ethenogenes degradation of ~4.6 umol PCE/mg Yager 1997;
VSS/day ITRC 2000
TCE Anaerobic, electron donor Alcaligenes; (for example, Michaelis-Menton kinetics Ethene Harkness and
(lactate, methanol butyrate, hydrogenopheya present in others 1999
glutamate 1,2-propanediol, culture derived from soil)
toluene)
PCE, TCE, c- Anaerobic, electron donor Not reported Michaelis-Menton kinetics Not Ballapragada and
DCE, VC (Hydrogen, propionate or lactate) reported others 1997
PCE Anaerobic, electron donor Not reported 1.24 mg PCE mg /volatile suspended Not DeStefano 1992
(methanol) solids (VSS)/d reported
TCE Not reported Not reported 1st order 0.2-4.8 yr-1 Not Wilson and
reported others, 1996
DCE Not reported Not reported 1st order 0.5-9.4 yr-1 Not Wilson and others
reported 1996
2-10
Exhibit 2-8: Selected Information on CAH Degradation Mechanisms (continued)
Degradation
Mechanism CAH Conditions Reported Bacteria Reported Rate Data Product Source
Anaerobic reductive VC Anaerobic, iron-reducing Not reported Michaelis-Menton Vmax = 0.76 Not Bradley and
dechlorination (direct) conditions in aquifer umol/L-d reported Chapelle 1996
(dehalorespiration)
(continued)
VC Anaerobic, methanogenic Not reported Michaelis Menton Not Bradley and

conditions in aquifer Vmax = 0.19 umol/L-d reported Chapelle 1997
Anaerobic reductive PCE, TCE, DCE, Anaerobic, electron acceptor Methanosarcina barkeri, 0.84 and 2.34 nmol PCE/mg Ethene, Fathepure 1987
dechlorination VC, DCA (nitrate, sulfate), electron donor Desulfomonile tiedjei protein/day converted to TCE ethane
(cometabolic) (hydrogen)
PCE, TCE, CT Anaerobic, electron acceptor Methanogens, denitrifiers, Not reported Ethene, Workman 1997;
(nitrate, sulfate), electron donor sulfate reducers methane Yager 1997
(hydrogen)
CT Anaerobic, electron acceptor Shewanella putrefaciens Not reported CF, MC Petrovskis and
(Fe[III]) MR-1 others 1995
ABIOTIC MECHANISMS
Reductive dechlorination CT, CF Anaerobic, metal cofactor, Abiotic Ten-fold rate increase over Methane Workman 1997
(abiotic) corrinoid, or porphyrin catalyst cometabolic reductive dechlorination
(reduced vitamin B12 )r
Hydrolysis TCA, CA, CM Not reported Abiotic Not reported Acetic RTDF 1997; EPA
acid, 1998
ethanol
Hydrolysis TCA Highly Abiotic Half life of approximately 2 years Possibly McNab and Nara-
oxidized DCE, simhen 1994
groundwater acetic acid
Elimination TCA, CA, CM Not reported Abiotic Not reported DCE RTDF, 1997;
EPA, 1998
Elimination, hydrolysis TCA Abiotic 1st order (at each temperature) DCE, Haag and Mill,
10 oC 0.058-0.06 yr-1 acetic acid 1988; Cline and
15 oC 0.137-0.145 yr-1 Delfino 1989;
20 oC 0.31-0.34 yr-1 Jeffers and others
1989
Elimination, hydrolysis TCA Abiotic Avg. Half Life (at each temperature) DCE, Haag and Mill,
10 oC 12 yr acetic acid 1988; Cline and
15 oC 4.9 yr Delfino, 1989;
20 oC 0.95 yr Jeffers et al., 1989
2-11
Exhibit 2-9: Biodegradation Mechanisms Typically Occurring

During Enhanced In Situ Bioremediation of CAHs
Aerobic Oxidation Anaerobic Reductive Dechlorination

CAH Direct Cometabolic Direct Cometabolic
CHLORINATED ETHENES
PCE X X
TCE X
cis-DCE X
trans-DCE X
1,1-DCE X
VC
1
CHLORINATED ETHANES
1,1,1-TCA X X
1,2-DCA X X
1,1-DCA X X
CA X X X X
CHLORINATED METHANES
CT X X X
CF X X
MC
CM X X
1
Insufficient information was available for 1,1,2-TCA.
KEY:
Typically occurring
X Not typically occurring
Sources: RTDF 1997; ITRC 1998; EPA 1998
Exhibit 2-10: Constituents Involved in Biodegradation Mechanisms for

Enhanced In Situ Bioremediation of CAHs
Electron
Biodegradation Electron Donor Acceptor
Mechanism Carbon Source (Reductant) (Oxidant) Comments
Aerobic oxidation CAH CAH Oxygen Only less chlorinated CAHs
(direct) can be degraded
Aerobic oxidation Organic carbon Cometabolite (e.g., Oxygen CAH oxidized by
(cometabolic) toluene, methane cometabolic mechanism
propane)
Anaerobic reductive Organic carbon or Hydrogen CAH Greater chlorinated CAHs
dechlorination (direct) CO2 are more readily available
Anaerobic reductive Other organic carbon Hydrogen Cometabolic CAH reductively
dechlorination or CO2 electron acceptor dechlorinated by
(cometabolic) cometabolic mechanism
Sources: RTDF 1997; ITRC 1998; EPA 1998
2-12
2.4.1 Aerobic Oxidation
In aerobic zones of the subsurface (zones of the subsurface where oxygen is present), certain CAHs can
be oxidized to carbon dioxide, water, and chloride by direct and cometabolic mechanisms (Hartman and
DeBont 1992; McCarty and Semprini 1994; Malachowsky and others, 1994; Gerritse and others, 1995;
Bielefeld and others 1995; Hopkins and McCarty 1995). Direct mechanisms are more likely to occur
with the less chlorinated CAHs (mono- and di-chlorinates). In general, the more chlorinated CAHs can
be oxidized by cometabolic mechanisms, but no energy is provided to the organism. Incidental oxidation
is caused by enzymes intended to carry out other metabolic functions. Generally, direct oxidation
mechanisms degrade CAHs more rapidly than cometabolic mechanisms (McCarty and Semprini 1994)
(refer to the following case studies in Appendix A: Aerobic Degradation in Field Demonstration at
Moffett Naval Air Station, Mountain View California [Moffett Field]; Aerobic Degradation Field
Demonstration at Site 19, Edwards Air Force Base, California[Edwards AFB]; Methane Enhanced
Bioremediation Using Horizontal Wells at Savannah River Site, Aiken, South Carolina[SRS]; and
Cometabolic Bioventing at Building 719, Dover Air Force Base, Dover, Delaware[Dover Building 719]).
Aerobic Oxidation (Direct)
Aerobic oxidation (direct) is the microbial breakdown of a compound in which the compound serves as
an electron donor and as a primary growth substrate for the microbe mediating the reaction. Electrons
that are generated by the oxidation of the compound are transferred to an electron acceptor such as
oxygen.
In addition a microorganism can obtain energy for cell maintenance and growth from the oxidized
compound (the compound acts as the reductant). In general, only the less chlorinated CAHs (CAHs with
one or two chlorines) can be used directly by microorganisms as electron donors. CAHs that can be
oxidized directly under aerobic conditions include DCE, DCA, VC, CA, MC, and CM (Bradley 1998;
RTDF 1997; Harkness and others 1999). The CAHs are oxidized into carbon dioxide, water, chlorine,
and electrons, in conjunction with the reduction of oxygen to water. Exhibit 2-11 shows an example of
aerobic oxidation (direct) of a CAH.
Exhibit 2-11: Aerobic Oxidation (Direct)
Source: Modified from Hartmans and DeBont 1992
2-13
Aerobic Oxidation (Cometabolic)
Aerobic oxidation (cometabolic) is the microbial breakdown of a contaminant in which the contaminant
is oxidized incidentally by an enzyme or cofactor produced during microbial metabolism of another
compound. In such a case, the oxidation of the contaminant does not yield any energy or growth benefit
for the microorganism involved in the reaction.
The CAHs that have been observed to be oxidized cometabolically under aerobic conditions include
TCE, DCE, VC, TCA, DCA, CF, and MC (Munakata-Marr 1997; McCarty and others 1998; RTDF 1997;
Edwards and Cox 1997; McCarty 1997a; Bradley and Chapelle 1998; Travis and Rosenberg 1997). The
electron donors observed in aerobic oxidation (cometabolic) include methane, ethane, ethene, propane,
butane, aromatic hydrocarbons (such as toluene and phenol), and ammonia. Under aerobic conditions, a
monooxygenase (methane monooxygenase in the case of methanotrophic bacteria) enzyme mediates the
electron donation reaction. That reaction has the tendency to convert CAHs into unstable epoxides
(Anderson and Lovley 1997). Unstable epoxides degrade rapidly in water to alcohols and fatty acids,
which are readily degradable. Exhibit 2-12 shows an example of aerobic oxidation (cometabolic) of a
CAH.
Exhibit 2-12: Aerobic Oxidation (Cometabolic)
Source: Modified from McCarty and others 1998
Wilson and Wilson (1985) were the first to observe that the simultaneous addition of methane and
oxygen can stimulate biodegradation by aerobic oxidation (cometabolic) of TCE in aquifer material.
Subsequently, that approach was tested in the field at Naval Air Station (NAS) Moffett Field, California.
Intermittent pulses of oxygen and methane were provided to the subsurface, bringing about the in situ
stimulation of biodegradation of TCE, c-DCE, and VC in a contaminated aquifer (Semprini and others
1990). The strategy has been applied successfully to biodegradation of CAHs at a variety of other sites
(McCarty and others 1991; Travis and Rosenberg 1997).
Although the studies have demonstrated that addition of methane is an effective means of stimulating
cometabolic biodegradation of CAHs, additional field studies at the Moffett test site have shown that
toluene and phenol can be more effective electron donors than methane in the stimulation of cometabolic
biodegradation of TCE, c-DCE, and VC in groundwater (Hopkins and others 1993; Hopkins and
McCarty 1995).
2-14
2.4.2 Anaerobic Reductive Dechlorination
Under anaerobic conditions, reductive dechlorination mechanisms can effectively biodegrade CAHs.
Reductive dechlorination generally involves the sequential replacement of a chlorine atom on a CAH
with a hydrogen atom (that is, converting PCE to TCE to DCE, and so on) and has been observed to
occur both directly and cometabolically. In anaerobic reductive dechlorination (direct), the mediating
bacteria use the CAH directly as an electron acceptor in energy-producing redox reactions. Anaerobic
reductive dechlorination (cometabolic) occurs when bacteria incidentally dechlorinate a CAH in the
process of using another electron acceptor to generate energy. Reductive dechlorination theoretically is
expected to occur under most anaerobic conditions, but has been observed to be most effective under
sulfate-reducing and methanogenic conditions (EPA 1998). As in the case of aerobic oxidation, the
direct mechanisms may biodegrade CAHs faster than cometabolic mechanisms (McCarty and Semprini,
1994) (refer to the following case studies in Appendix A: Enhanced Bioremediation at the Texas Gulf
Coast Site, Houston, Texas[Texas Gulf Coast]; Molasses Injection at the Avco Lycoming Superfund Site,
Willimasport, Pennsylvania[Avco Lycoming]; Anaerobic In Situ Reactive Zone at an Abandoned
Manufacturing Facility, Emeryville, California[Emeryville]; Sequential Anaerobic/Aerobic
Biodegradation of PCE at Watertown, Massachusetts[Watertown]; and Bioaugmentation (Accelerated
Anaerobic Bioremediation) at Area 6 of the Dover Air Force Base, Dover, Delaware [Dover Area 6]).
Anaerobic Reductive Dechlorination (Direct)
Anaerobic reductive dechlorination (direct) is a biodegradation reaction in which bacteria gain energy
and grow as one or more chlorine atoms on a chlorinated hydrocarbon are replaced with hydrogen
(McCarty 1997b; Fennel and others 1997; Mayo-Gatell and others 1997; Gerritse and others 1999). In
that reaction, the chlorinated compound serves as the electron acceptor, and hydrogen serves as the direct
electron donor (Fennel and others 1997). Hydrogen used in the reaction typically is supplied indirectly
through the fermentation of organic substrates. The reaction is also referred to as halorespiration or
dehalorespiration (Gossett and Zinder 1997).
Anaerobic reductive dechlorination (direct) has been observed in anaerobic systems in which PCE, TCE,
DCE, VC, and DCA are used directly by a microorganism as an electron acceptor in their energy-
producing redox reactions (Neumann and others 1994; Scholz-Muramatsu and others 1995; Freedman
and Gossett 1989; Yagi and others 1994; Hollinger and Schumacher 1994; Major and others 1991;
McCarty 1997b; Gossett and Zinder 1996; Gerritse and others 1996; DeBruin and others 1992; Maymo-
Gatell and others 1997; Sharma and McCarty 1996; Hollinger 1993; Smatlak 1996; Tandol 1994; Yager
and others 1997). The mechanism generally results in the sequential reduction of a chlorinated ethene or
chlorinated ethane to ethene or ethane. Exhibit 2-13 shows the step-by-step dechlorination of PCE.
The anaerobic reductive dechlorination of the more chlorinated CAHs (PCE and TCE) occurs more
readily than the dechlorination of CAHs that already are somewhat reduced (DCE and VC); for that
reason, DCE and VC may accumulate in anaerobic environments. It also has been observed that, while
VC can be effectively dechlorinated, the presence of PCE in groundwater may inhibit the anaerobic
reductive dechlorination of VC (Tandol and others 1994).
VC is more commonly remediated using aerobic mechanisms than anaerobic mechanisms. In anaerobic
environments in which VC accumulates, enhanced aerobic bioremediation can be implemented to
degrade the VC. Recent studies have demonstrated significant anaerobic oxidation of VC to carbon
dioxide under Fe(III)-reducing conditions (Bradley and Chapelle 1998b) and of DCE to VC and VC to
carbon dioxide under humic acid-reducing conditions (Bradley and Chapelle 1998a). These studies
suggest the possibility of alternative biotransformation mechanisms under anaerobic conditions.
2-15
Exhibit 2-13: Anaerobic Reductive Dechlorination of PCE
Source: Modified from DeStephano and others 1992
Hydrogen has been observed to be an important electron donor in anaerobic reductive dechlorination
(Fennell and others 1997). The presence of hydrogen establishes a competition between the bacteria that
mediate the anaerobic reductive dechlorination (such as Dehalococcus ethenogenes and Dehalospirillium
multivorans) and methanogenic bacteria that also use hydrogen as an electron donor (ITRC 2000).
However, it has been observed that the dechlorinating bacteria can survive at a partial pressure of
hydrogen ten times lower than that at which the methanogenic bacteria can survive (Smatlak and others
1996), thus providing an opportunity to support the dechlorinating bacteria by providing hydrogen at a
slow rate. (Hydrogen addition at a slow rate has been demonstrated with the fermentation of butyric or
propanoic acid) (Fennell and others 1997). In addition, in some subsurface environments, competition
from nitrate or sulfate-reducing bacteria may limit both methanogenic activity and the extent of anaerobic
reductive dechlorination (RTDF 1997).
Studies have shown that anaerobic reduction of CAHs can occur by reductive dechlorination in a variety
of environmental conditions (Beeman and others 1994; Semprini and others 1995). A review of the
transformation of halogenated compounds has shown that the theoretical maximum redox potential for
transformation of PCE to TCE is +580 millivolts and for TCE to DCE is +490 millivolts (Vogel and
others 1987). Therefore, the anaerobic reductive dechlorination of the compounds is thermodynamically
possible under manganese- or iron-reducing conditions. No peer-reviewed reports of the transformation
of PCE to TCE under aerobic conditions were identified. However, the efficiency of the anaerobic
dechlorination processes at high redox potential values is limited; efficiency improves as the redox
potential decreases.
Pilot studies have been conducted at a variety of sites to examine the feasibility of stimulating in situ
anaerobic reductive dechlorination by providing to the subsurface simple organic substrates, such as
lactate, butyrate, methanol, ethanol, and benzoate (Harkness and others 1999; Freedman and Gossett
1989; Gibson and Sewell 1992; Buchanan and others 1997; Becvar and others 1997; Sewell and others
1998; Litherland and Anderson 1997; Spuij and others 1997).
Anaerobic Reductive Dechlorination (Cometabolic)
Anaerobic reductive dechlorination (cometabolic) is a biodegradation reaction in which a chlorinated

hydrocarbon is fortuitously degraded by an enzyme or cofactor produced during microbial metabolism of
another compound. In such a case, biodegradation of the chlorinated compound does not appear to yield
any energy or growth benefit for the microorganism mediating the reaction (Gossett and Zinder 1997).
2-16
Several CAHs have been observed to be reductively dechlorinated by cometabolic mechanisms. In those
instances, the enzymes that are intended to mediate the electron-accepting reaction “accidentally” reduce
and dehalogenate the CAH. Anaerobic reductive dechlorination (cometabolic) has been observed for
PCE, TCE, DCE, VC, DCA, and CT under anaerobic conditions (Fathepure 1987; Workman 1997; Yager
and others 1997).
In pilot- and full-scale applications, it is generally difficult to distinguish between direct and cometabolic
anaerobic reductive dechlorination reactions. Both biodegradation mechanisms are referred to more
generally as anaerobic reductive dechlorination. In laboratory-scale applications, direct and cometabolic
anaerobic reductive dechlorination reactions can be distinguished. The role played by anaerobic
reductive dechlorination (cometabolic) in relation to anaerobic reductive dechlorination (direct) remains
under study.
Anaerobic Reductive Dechlorination Combined with Aerobic Oxidation
Several investigators have suggested that the most efficient bioremediation of CAHs will occur in
aquifers that are characterized by an upgradient anaerobic zone and a downgradient aerobic zone
(Bouwer 1994; Carter and Jewell 1993; Gerritse and others 1995; Fathepure and others 1987). In the
upgradient aerobic zone, anaerobic reductive dechlorination of PCE might degrade to TCE, and
eventually to VC. VC could then be degraded by aerobic oxidation (direct) downgradient in the aerobic
zone of the CAH plume (the leading-edge fringe of the plume). Stratified redox conditions in the field
may provide the best opportunities, other than engineered remedies, for intrinsic biodegradation of
CAHs.
Generally, the substrate requirement for direct metabolism is relatively less than that for cometabolism.
In cometabolism, often the amount of primary substrate required is a factor of 100 to 1,000 times the
amount of the CAH. In direct metabolism (respiration with only the chlorinated solvent as the electron
acceptor), the stoichiometry is much more favorable, and a much smaller amount of supplemental
chemical is required (Bouwer 1994).
2.4.3 Abiotic Degradation Mechanisms
Abiotic degradation mechanisms involve chemical reactions to treat CAHs without biological processes.
These mechanisms include hydrolysis, elimination, and abiotic reductive dechlorination. In general, the
rates of abiotic degradation may be slow relative to biological mechanisms. However, the abiotic
mechanisms may play a significant role in the overall remediation of a site at which CAH contamination
is present, depending on the specific site conditions (for example, a site at which the contaminant plume
is moving slowly) (EPA 1998). Hydrolysis and elimination reactions are generally independent of redox
conditions, while abiotic reductive dechlorination is highly dependent on redox conditions.
Hydrolysis is a substitution reaction in which a CAH may react with water to substitute a chlorine atom
with a hydroxyl group, producing organic alcohols, acids, or diols, such as the formation of acetic acid
from 1,1,1-TCA (Exhibit 2-14). Generally, less chlorinated CAHs are more susceptible to degradation
by hydrolysis. Hydrolysis rates have been reported that have half-lives ranging from days for
monochlorinated alkanes to thousands of years for tetrachloromethane.
2-17
Exhibit 2-14: Example of Hydrolysis Reaction
Cl3C-CH3 + 2H2O H3C-COOH + 3HCl

1,1,1-TCA Acetic Acid
Hydrolysis is a common transformation mechanism for 1,1,1-TCA, chloroethane, and chloromethane,

producing acetate, ethanol, and methanol, respectively (Vogel and McCarty 1987).
Elimination reactions involve the removal of a hydrogen and a chlorine atom (sometimes referred to as
dehydrohalogenation) from a chlorinated alkane, with the formation of the corresponding alkane (Exhibit
2-15). In contrast to hydrolysis reactions, elimination reactions become more effective as the CAHs
become more chlorinated. Assuming that elimination rates for monochlorinated CAHs are negligible, the
abiotic conversion of TCA to DCE at 20C has been reported to exhibit relatively rapid first-order
kinetics, with a rate constant of approximately 0.04 ± 0.003 year-1 (Vogel and McCarty 1987).
Exhibit 2-15: Example of Elimination Reaction
Cl3C-CH3 Cl2C=CH2 + HCl

1,1,1-TCA 1,1-DCE
Abiotic reductive dechlorination of several CAHs also has been observed (Reinhard and others 1990;
Gillham and O’Hannesin 1994; Workman and others 1997). Abiotic reductive dechlorination occurs in
the presence of an extremely strong reductant (for example, zero-valent iron or reduced vitamin B12).
When the reductant present is sufficiently strong, the more chlorinated (and, therefore, more oxidized) of
the CAHs (PCE, TCE, CT, and CF) can be reduced to less chlorinated species without the mediation of
bacteria. As in the case of biologically mediated reductive dechlorination, abiotic reductive
dechlorination becomes less effective or ineffective for the less chlorinated CAHs (which already are
reduced somewhat). Exhibit 2-16 shows the general mechanism of abiotic reductive dechlorination
(using zero-valent iron as the reducing agent).
Exhibit 2-16: Example of Abiotic Reductive Dechlorination
CT + 2Fe0 MC + 2Fe+2
carbon tetrachloride zero-valent iron methylene chloride ferrous iron
Iron Oxidation State 0 +2
Carbon Oxidation State +4 0
2-18
3.0 IN SITU BIOREMEDIATION TECHNOLOGIES

In situ bioremediation technologies are used to enhance the mechanisms that degrade CAHs in
contaminated soil and groundwater (discussed in Section 2). Technologies include bioaugmentation
and the addition of nutrients, electron donors (substrates such as toluene, propane, and methane), and
electron acceptors (such as oxygen). Design configurations of in situ bioremediation systems include
direct injection, groundwater recirculation, installation of permeable reactive barriers (PRBs), and
bioventing.
3.1 TECHNOLOGIES
Generally, in situ bioremediation technologies employ engineered systems to heighten the effects of
naturally occurring degradation mechanisms. The engineered systems are designed to include one or
more of the following general classes of technologies: the addition of bacteria (bioaugmentation), the
addition of nutrients, the addition of electron donors, or the addition of electron acceptors. Each of the
technologies is discussed below in more detail. Exhibit 3-1 presents a summary of information about
each technology, including an example of how each may be applied, a discussion of the biodegradation
mechanisms generally supported by each, a discussion of the typical CAHs targeted through the use of
each technology, and a summary of how the enhancement technologies have been applied at the case
study sites included in Appendix A of this report.
Bioaugmentation – involves the addition of supplemental microbes to the subsurface where organisms
able to degrade specific contaminants are deficient. Microbes may be “seeded” from populations already
present at a site and grown in aboveground reactors or from specially cultivated strains of bacteria known
to degrade specific contaminants. The application of bioaugmentation technology is highly site-specific
and highly dependent on the microbial ecology and physiology of the subsurface (EPA 1998).
Nutrient addition – involves the addition of key biological building blocks, such as nitrogen and
phosphorus and other trace nutrients necessary for cell growth. Addition of nutrients generally is applied
as a supplement to bioaugmentation or addition of electron donors or electron acceptors, so that
concentrations of nutrients in the subsurface do not become a limiting factor for an in situ bioremediation
application.
Electron donor addition – involves the addition of a substrate that acts as a reductant in the redox
reaction used by the CAH-degrading microbe to produce energy. A substrate such as toluene, propane,
or methane may be added to act as a cometabolic oxidant, when the CAH also is oxidized. A substrate
such as hydrogen, a source of hydrogen, or a hydrogen release compound may be added to act as a direct
reductant, when the CAH is reduced.
Electron acceptor addition – involves the addition of oxygen (for aerobic mechanisms) or an anaerobic
oxidant such as nitrate (for anaerobic mechanisms), which is used by the CAH-degrading microbes
present in the subsurface.
As Exhibit 3-1 shows, one or more of the technologies were used at several of the case study sites. For
example, bioaugmentation was used at Dover Area 6, while addition of nutrients was used at SRS, Texas
Gulf Coast, Watertown, and Dover Area 6. Addition of electron donors, such as toluene, propane, or
methane, and an electron acceptor (oxygen) for aerobic cometabolic oxidation were used at the following
five sites: Moffett Field, Edwards AFB, SRS, Watertown, and Dover Building 719. Addition of an
electron donor in the form of a hydrogen source, such as methanol, molasses, or lactate, for anaerobic
3-1
Exhibit 3-1: Components of In Situ Bioremediation Technology1
Biodegradation Mechanisms
Component Example Supported Targeted CAHs Case Study Sites1
Bioaugmentation Seed the subsurface with non-native, Aerobic oxidation (cometabolic and TCE, DCE, TCA, None
CAH-degrading bacteria direct) DCA, CA, CT, CF
Anaerobic reductive dechlorination TCA, DCA, CA, CT, Dover Area 6

(cometabolic and direct) CF, CM
Addition of Add nitrogen, phosphorus, or other Aerobic oxidation (cometabolic and TCE, DCE, TCA, SRS; Watertown
Nutrients growth factors that may be deficient in the direct) DCA, CA, CT, CF
subsurface
Anaerobic reductive dechlorination TCA, DCA, CA, CT, Texas Gulf; Dover Area 6
(cometabolic and direct) CF, CM
Addition of Add a substrate, such as toluene, propane, Aerobic oxidation (cometabolic) TCE, DCE, TCA, Moffett Field; Edwards AFB; SRS;
Electron Donors or methane CF, MC Watertown; Dover Building 719
Add hydrogen, a hydrogen source, or a Anaerobic reductive dechlorination PCE, TCE, DCE, Texas Gulf; Avco Lycoming; Emeryville;
hydrogen release compound (cometabolic and direct) VC, TCA, DCA, CA, Watertown; Dover Area 6
CT, CF, MC
Addition of Add oxygen by bioventing, biosparging, Aerobic oxidation (direct) TCE, DCE, VC, Moffett Field; Edwards AFB; SRS;
Electron or adding an oxygen source such as TCA, DCA, CA, CE, Watertown; Dover Building 719
Acceptors hydrogen peroxide MC, CM
Add an anaerobic reductant such as nitrate Anaerobic reductive dechlorination PCE, TCE, DCE, None
(cometabolic) VC, DCA, CT
Source: ITRC, 1998; Leeson, 1999; Sewell, 1998; U.S. Air Force, 1998
1
Case studies provided in Appendix A of this report are cited as examples of each of the technologies.
3-2
reductive dechlorination was used at the following five sites: Texas Gulf Coast, Avco Lycoming,
Emeryville, Watertown, and Dover Area 6 (refer to these case studies in Appendix A).
3.2 DESIGN APPROACHES
The components of in situ bioremediation technologies components described above can be implemented
in several different general configurations: direct injection, groundwater recirculation, permeable
reactive barriers (PRBs), and bioventing. In addition, in situ bioremediation may occur naturally, without
the application of enhancement technologies. The latter approach is one component of the approach EPA
refers to as monitored natural attenuation (MNA)2. Because MNA does not use enhancement
technologies, it is not discussed in detail in this report.
Exhibit 3-2 includes a summary of the purpose, advantages, and potential limitations of direct injection,
groundwater recirculation, PRB, and bioventing systems, described below, and lists the case study sites
included in Appendix A at which the configuration was used. Exhibit 3-3 shows the general layouts of
the configurations, often referred to as amendment delivery systems. The configurations include use of
vertical wells, horizontal wells, and trenches for both injection and extraction of groundwater, or for
injection of amendments. Biological, nutrient, electron donor, or electron acceptor amendments are
injected in a liquid or a gaseous phase.
Direct injection system - degradation is enhanced through the addition of microbes, nutrients, oxidants,
or reductants directly into the aquifer at injection points or directly into the soil. The natural flow of the
groundwater generally is not impeded, but is monitored to determine that the degradation of the
contaminants and their daughter products is completed within an acceptable distance from the source.
The case study sites at which direct injection into groundwater was used are SRS, Avco Lycoming, and
Emeryville. At SRS, methane (gas) and air were injected below the water table using a “lower”
horizontal well located at a depth of 175 ft below ground surface (bgs). An “upper” horizontal well,
located at a depth of 80 ft bgs, was used to extract air and contaminated vapors from the vadose zone. At
Avco Lycoming, a molasses solution was injected through 20 four-inch diameter wells completed in the
overburden. Molasses was added twice each day at various concentrations and rates, as the results of
monitoring the system indicated were appropriate.
Groundwater recirculation - extracts contaminated groundwater from the site, adding to or amending the
extracted water ex situ, and reinjecting the “activated” water to the subsurface, generally upgradient of
2
On April 21, 1999, EPA issued a final policy on the use of MNA at Superfund, RCRA corrective action, and
underground storage tank (UST) sites (Directive Number 9200.4-17P) available at
http://www.epa.gov/swerust1/directiv/d9200417.htm. The purpose of the directive was to clarify EPA’s policy
regarding the use of MNA for the remediation of contaminated soil and groundwater at sites regulated under the
Superfund, RCRA, and UST programs.
As defined in the directive, MNA is the reliance on natural attenuation processes (within the context of a carefully
controlled and monitored site cleanup approach) to achieve site-specific remedial objectives within a time frame
that is reasonable, compared with that offered by other, more active methods. The processes that are at work in
such a remediation approach include a variety of physical, chemical, or biological processes that, under favorable
conditions, act without human intervention to reduce the mass, toxicity, mobility, volume, or concentration of
contaminants in soil or groundwater. Such in situ processes include biodegradation; dispersion; dilution;
sorption; volatilization; and chemical or biological stabilization, transformation, or destruction of contaminants.
Other terms associated with MNA in the literature include “intrinsic remediation,” “intrinsic bioremediation,”
“passive bioremediation,” “natural recovery,” and “natural assimilation.”
3-3
the contaminated zone. As an alternative, extraction and injection are performed at different elevations
in a single well, creating vertical circulation. A groundwater recirculation configuration may be used to
provide containment of a plume or to allow the addition of amendments in a more controlled
environment.
The case study sites at which groundwater recirculation was used are Moffett Field, Edwards AFB, Texas
Gulf Coast, Watertown, and Dover Area 6. The project at Moffett Field was one of the earliest field
demonstrations of in situ bioremediation. The Edwards AFB project was conducted by the group of
researchers who had conducted the Moffett Field demonstration, who built upon the results obtained from
the earlier project. At Moffett Field, groundwater was extracted from one well, amended chemically, and
reinjected at another well located 6 meters (m) from the extraction well. The wells, screened in a sand
and gravel layer approximately 4 to 6 m bgs, created induced-gradient conditions in the aquifer. At
Edwards AFB, a two-well recirculation system was constructed; the system created separate bioactive
zones in upper and lower aquifers. One well was used to extract groundwater from the lower aquifer,
amend it chemically, and reinject the groundwater into the upper aquifer. The other well was used to
extract groundwater from the upper aquifer, amend it chemically, and reinject the groundwater into the
lower aquifer. The wells were spaced 10 m apart, and screened between approximately 6 and 24 m bgs.
At Texas Gulf Coast, the project used a recirculation system that consisted of an alternating series of four
extraction and four injection trenches spaced 100 ft apart. Extraction trenches were completed to a depth
of approximately 20 to 22 ft bgs, and injection trenches to a depth of approximately 10 ft bgs. At
Watertown, a recirculation cell that covered a surface area of approximately 10 ft by 20 ft was
constructed; the cell had three extraction wells and three injection wells, each screened from 13 to 20 ft
bgs. The extraction wells were located at the downgradient end of the cell, and the injection wells at the
upgradient end. At Dover Area 6, a hydraulically-controlled cell that covered a surface area of
approximately 40 ft by 60 ft was constructed; the cell had three extraction wells and three injection wells,
each screened from 13 to 20 ft bgs. The field demonstration conducted at Dover Area 6 included the
addition of an aqueous culture of non-indigenous microbes to the groundwater (bioaugmentation).
PRBs - an active bioremediation zone is created by such methods as backfilling a trench with nutrient-,
oxidant-, or reductant-rich materials, or by creating a curtain of active bioremediation zone through direct
injection or groundwater recirculation at the toe of a plume. PRBs contain a contaminant plume by
treating only groundwater that passes through it. PRBs are an emerging design approach for use of in
situ bioremediation. To date, application of PRBs to in situ bioremediation of CAHs has been limited to
demonstration tests (ITRC 1997).
None of the case studies included in this report involved the use of a PRB for in situ bioremediation of
CAHs; however, research has been conducted on the use of such configurations. At the Waterloo Center
for Groundwater Research at the University of Waterloo, in Ontario, Canada, a treatment system
consisting of a trench (backfilled with sand) was used in a demonstration test at a site with groundwater
contaminated with CAHs. In that system, water was extracted from the pore spaces of the wall, amended
with nutrients and substrate, and reinjected into the wall over a short period of time (a few hours). After
reinjection had been completed, the pumps were shut off, and the nutrients were transported out of the
wall under natural groundwater flow conditions (as a “slug”). The slug of amended groundwater mixed
with surrounding groundwater, and a zone developed in which microorganisms received a continuous
supply of the nutrients required to support biodegradation (Devlin and Barker 1994).
Bioventing - the process of aerating soils to stimulate in situ biological activity and promote
bioremediation. In this process, oxygen is delivered to unsaturated soils by forced air movement (either
extraction or injection of air) to increase oxygen concentrations and stimulate biodegradation.
Bioventing uses low air flow rates to provide only enough oxygen to sustain microbial activity, with
3-4
oxygen most commonly supplied through direct air injection. Bioventing is commonly used for treatment
of fuel-contamination in the vadose zone (EPA 1995).
At Dover Building 719, an air-sparge blower was used to inject a mixture of air and propane through
three injection wells screened to a depth of 10 ft bgs. The Dover Building 719 site is a field
demonstration of bioventing, at which treatment is limited to the soil above the water table.
Exhibit 3-2: Bioremediation System Configurations
Case Study Applicability/

Configuration Purpose Examples Advantages Potential Limitations
Direct injection To enhance the SRS, Avco Lycoming, & Less aboveground & Difficult to control
biodegradation of Emeryville, Dover equipment needed dispersion of
contaminants in place Building 719 than for ex situ amendments in
in soil and systems aquifer
groundwater & Regulatory
concerns about
discharge of
chemicals to
groundwater
Groundwater To contain the Moffett Field, & Can provide & Reinjection of
recirculation contaminated Edwards AFB, Texas containment of the contaminated
groundwater plume Gulf Coast, plume groundwater may be
and enhance the Watertown, Dover complicated
biodegradation of Area 6 & Allows controlled because of
contaminants in the amendment of regulatory concerns
recirculation area groundwater & Silt build up in
recirculation wells
reduces
effectiveness of
systems
PRB To contain the None & Less aboveground & Treats only
contaminated equipment needed groundwater that
groundwater plume than for ex situ passes through the
and degrade systems barrier
contaminants in
groundwater that pass
though the barrier
Bioventing To enhance the Dover Building 719 & Less aboveground & Bioventing must be
biodegradation of equipment needed coupled with a
contaminants in the than for ex situ groundwater
vadose zone systems treatment
technology (such as
& Treats biosparging) to
contaminated soil remediate any
contaminated
groundwater
Source: ITRC, 1998; Leeson, 1999; Sewell, 1998; U.S. Air Force, 1998
3-5
Exhibit 3-3: In Situ Bioremediation System Configurations
Direct Injection
Permeable
Reactive Barrier
Groundwater
Recirculation
Source: Modified from USAF 1998
3-6
3.3 VENDORS OF IN SITU BIOREMEDIATION
This section presents summary information about vendors of in situ bioremediation technologies,
including contact information and a brief overview of the technology, including information about
biodegradation mechanisms and full-scale units. Vendors of in situ bioremediation technologies were
identified from EPA REACH IT (<www.epareachit.org>), a comprehensive database maintained by EPA
about innovative site characterization and remediation technologies. Vendors voluntarily submit
information for inclusion in the database. As such, this list of vendors reflects those firms that are
currently participating in EPA REACH IT, and may not include all vendors of in situ bioremediation
technologies. It is also important to note that the information in Exhibit 3-4 is based on information
provided by the vendors, and was not independently verified for this report.
As Exhibit 3-4 shows, EPA REACH IT lists 18 in situ bioremediation vendors. These vendors offer
methods using several types of in situ bioremediation mechanisms and technologies at sites contaminated
with CAHs:
& Aerobic oxidation (cometabolic) or anaerobic reductive dechlorination

biodegradation mechanisms
& Direct injection or bioventing bioremediation technologies
In addition, more than 130 full-scale units were reported in design, 30 units were being constructed, and
120 units were completed. These units were used to treat media contaminated with CAHs and other
contaminants, including petroleum hydrocarbons. Each of the 18 vendors shown in Exhibit 3-4 indicated
that a patent is pending for its technology. In addition, 8 vendors have registered technologies, 3 have
exclusive licenses, and 3 have patented technologies. To use a patented process, it may be necessary that
the user obtain design or construction services directly from the patent holder or purchase a license to
provide the technology to others. Interested parties should contact the individual vendors to discuss
licensing terms and patent provisions.
The information included in Exhibit 3-4 is current as of June 2000. To search for more current
information about in situ bioremediation vendors in EPA REACH IT, the following query can be used:
• Technology Type - Bioremediation (in situ) - biosparging

Bioremediation (in situ) - groundwater
Bioremediation (in situ) - lagoon
Bioremediation (in situ) - other OR
Bioventing
AND
• Contaminant Group - All chlorinated ethanes, ethenes, and methanes that were
listed individually or in groups under this search
criterion were selected for this search
3-7
Exhibit 3-4: Vendors Listed in EPA REACH IT That Provide In Situ Bioremediation Services1
# of Full-Scale
Units
Construction
Enhanced
Completion
Bioreme- Specific Bio-
diation Technology degradation
Design
Trade Name or Amend- Performance/ Mechanisms Patent
Vendor Contact Technology ments2 Vendor Claims Employed Information
ABB Environmental Jaret Johnson, P.E. Two-Zone Plume - Biological, 2 1 3 & Treatment designs include Anaerobic reductive Patent
Services, Inc. Team Leader Interception Nutrient, recirculation of dechlorination pending
Petroleum/Chemical Team Treatment Oxidant (O), groundwater or creation of (direct)
www.abb.com Phone: (781) 245-6606 Technology Oxidant (R), biological barriers for
Fax: (781) 246-5060 Reductant interception of a plume Aerobic oxidation
[no e-mail address (O) & Treats BTEX, PAHs, or (cometabolic)
identified] any contaminant that
aerobic bacteria can
degraded readily
& Removal effectiveness can
approach 100 percent
B&S Research, Inc. H.W. Lashmett Bioremediation (in Biological, 0 6 0 & Degrades hydrocarbons, Not specified Registered
CEO situ) - groundwater Nutrient chlorinated solvents, PCBs, trademark;
[No Web page Phone: (218) 984-3757 [No trade name fertilizers, pesticides, and vendor has
identified] Fax: (218) 984-3212 given] other hazardous organic exclusive
[No e-mail address compounds in groundwater license;
identified] and converts contaminants technology
to harmless products patented; and
& Treatment from surface to patent
90 feet bgs pending
3-8
# of Full-Scale
Units
Construction
Enhanced
Completion
Design
Billings & Rick Billings Groundwater/ Reductant 100 20 40 & Installed rapidly Aerobic only Registered
Associates, Inc. Vice President Subsurface (O) & Uses readily available trademark and
Phone: (505) 345-1116 Volatilization and pumps and pipe valves patent
Fax: (505) 345-1756 Ventilation System & Installed with standard pending
[No Web page [No e-mail address Remediation drilling equipment
identified] identified] Technology & Advantages include use of
air to avoid water
treatment, ability to
manipulate air flows and
pressures, and ability to
direct air to selected areas
& Air emissions can be held
within permitted levels by
manipulation of flows and
use of a biological filter
& Has not been applied in
treating inorganic wastes
Bio-Genesis Victor Coukoulis Bioremediation (in Biological INP INP INP & Treats groundwater Not specified Patent
Technologies President situ) - groundwater contaminated with pending
Phone: (602) 990-0709 [No trade name petroleum hydrocarbons,
Fax: (602) 990-7745 given] PAHs, aromatics, alcohols,
www.biogti.com info@biogti.com ketones, phenols, PCBs,
solvents, carbohydrates,
and pesticides
& Advantages include:
products are composed of
natural biological
ingredients that are non-
hazardous, no safety
equipment is needed, and
by-products of the process
are non-hazardous
3-9
# of Full-Scale
Units
Construction
Enhanced
Completion
Design
Clayton John F. Vargas Bioventing Nutrient, 2 0 1 & Ease of design and Aerobic only Patent
Environmental Senior Project Manager [No trade name Reductant construction pending
Consultants Phone: (714) 431-4106 given] (O) & Can be incorporated into a
Fax: (714) 825-0685 remedial design of soil
[No Web page [No e-mail address vapor extraction system
identified] identified] & Effective at removing
heavier, semivolatile
petroleum hydrocarbons
such as diesel fuel, fuel oil,
kerosene, and JP-4 jet fuel
Ecology Pete Condy FYREZYME Biological, 1 1 2 & Effective in accelerating Aerobic only Registered
Technologies President additive Nutrient bioremediation of organic trademark and
International, Inc. Phone: (916) 939-2397 contaminants in an patent
Fax: (916) 939-2449 environmentally friendly, pending
[No Web page PKcondy@aol.com scientifically sound, and
identified] cost-effective manner
& Advantages include: ease
of use, permanent solution
to contamination, and
affordability
3-10
# of Full-Scale
Units
Construction
Enhanced
Completion
Design
ENSR Consulting Dan Groher Anaerobic Nutrient, 2 0 2 & Combines bioremediation, Anaerobic reduction Registered
and Engineering Principal Bioremediation Biotransformation Oxidant (R) steam injection, SVE, trademark and
Specialist with Steam Injection groundwater extraction, patent
www.ensr.com Phone: (508) 635-9500 and air-stripping pending
Fax: (508) 635-9180 & Effective in removing
[No e-mail address chlorinated solvents, such
identified] as TCA and TCE present
as DNAPL; effective for
LNAPL
& Requires removal of fewer
volumes of groundwater,
shortens remediation, and
therefore reduces costs
& Does not treat metals
& Injection of substrate and
nutrients into groundwater
may require a permit
Bioventing Nutrient, 6 3 5 & Nutrients may be provided Aerobic only Patent

[No trade name Reductant in liquid phase through pending
given] (O) surface infiltration or in
vapor phase through
injection by vent wells
& Advantages include low
cost and demonstrated
effectiveness
& Low pressure, long-term,
low-energy treatment
ENVIROGEN, Inc. Michael Shannon Ph.D. Bioventing Nutrient, 2 0 10 & Results in speedier and Aerobic only Patent
Technical Manager [No trade name Reductant more cost-effective pending
www.envirogen.com Phone: (609) 936-9300 given] (O) remediation
Fax: (609) 936-9221 & Not applicable for metals
[No e-mail address or nonvolatile or inorganic
identified] contaminants
3-11
# of Full-Scale
Units
Construction
Enhanced
Completion
Design
EOD Technology, Matt Kaye Bioremediation (in Not specified INP INP INP & Combined with on-site Not specified Patent
Inc. Director of Marketing and situ) - groundwater/ groundwater treatment to pending
Finance [no trade name enhance the rate of
[No Web page Phone: (423) 690-6061 given] contaminant degradation
identified] Fax: (423) 690-6065
eodtmk@aol.com
In-Situ Fixation, Inc. Richard P. Murray Dual Auger System Biological, 0 0 1 & Increases the quality and Aerobic only Registered
President Nutrient, acceleration of trademark;
www.insitufixation Phone: (602) 821-0409 Reductant biodegradation in deep vendor has
.com Fax: (602) 786-3184 (O) contaminated soils exclusive
info@insitufixation.com & No excavation is required, license;
and no residues or wastes technology
are generated in the patented; and
process, since treatment is patent
performed beneath the pending
ground surface
IT Corporation Dr. Duane Graves Restore brand Nutrient INP INP INP & Information not provided Aerobic only Patent
Process Development amendments pending
www.itcorporation Supervisor
.com Phone: (423) 690-3211
x7418
Fax: (423) 690-9573
[No e-mail address
identified]
3-12
# of Full-Scale
Units
Construction
Enhanced
Completion
Design
Micro-Bac Terry Nelson Bac-Terra Remedial Biological, 8 0 20 & Cost effective, accelerates Aerobic and Registered
International, Inc. Phone: (512) 310-9000 Technology Nutrient, remedial program, and is anaerobic trademark;
Fax: (512) 310-8800 Oxidant (O), adaptable to a broad mechanisms vendor has
www.micro.com mail@micro-bac.com (Three vendors Oxidant (R), spectrum of field exclusive
market the same conditions license;
Harry Christensen product) technology
Microbial Phone: (714) 666-0110 patented; and
International Fax: (714) 538-5134 patent
micro@webworldinc.com pending
Midwest Microbial, Del Christensen

L.C. Operating Manager
Phone: (402) 493-8880
Fax: (402) 496-9269
[No e-mail address
identified]
REGENESIS Shruti Gohil Bioremediation (in Reductant 0 0 23 & Information not provided Aerobic-ORC, Registered
Bioremediation Assistant Marketing situ) - groundwater/ (O), ORC, Anaerobic-HRC trademark;
Products Manager oxygen release HRC patent
Phone: (949) 443-3136 compound (ORC), pending
www.regenesis.com x27 hydrogen release
Fax: (949) 443-3145 compound (HRC)
orc@regenesis.com
3-13
# of Full-Scale
Units
Construction
Enhanced
Completion
Design
Richards Dr. Sheril D. Burton Rimlab Biological, 2 1 6 & Treats water saturated with Aerobic oxidation Patent
Laboratories Senior Microbiologist Nutrient, or containing free products (direct) pending
Phone: (800) 453-1210 Reductant of gasoline, diesel fuel,
[No Web page Fax: (801) 785-2521 (O) benzene, toluene,
identified] [No e-mail address monochlorobenzene,
identified] dichlorobenzene, crude oil
from production wells,
hydrocarbons from oil
recycling facilities, oil
refinery discharges, airport
deicing discharges,
alcohols from natural gas
pipelines, or similar
industrial pollutants; a
modified consortium
degrades TCE
& Not applicable for
treatment of water
contaminated with heavy
asphaltines
& Germicidal agents must be
neutralized
SBP Technologies, Director of Business UVB, KGB, BLK Biological 10 5 16 & Specially selected Direct mechanisms Patent
Inc. Development microorganisms patented only pending
Phone: (914) 694-2280 for treatment of water that
[No Web page Fax: (914) 694-2286 contains organic wood
identified] [No e-mail address preservatives, namely
identified] creosote and PCP, and
chlorinated solvents,
especially TCE
Terra Vac, Inc. Joseph Pezzullo BIOVAC® Nutrient, 0 0 11 & Significantly more cost- Aerobic oxidation Registered
Phone: (609) 371-0070 Reductant effective than excavation (direct) trademark and
www.terravac.com Fax: (609) 371-9446 (O) and disposal patent
[No e-mail address pending
identified]
3-14
# of Full-Scale
Units
Construction
Enhanced
Completion
Design
Waste Stream Jim Hyzy Bioremediation (in Biological, 0 2 8 & Costs are reduced by its Not Specified Patent
Technology, Inc. Director of Research and situ) - groundwater Nutrient rapidity, predictability, and pending
Development [No trade name technical merit
www.wastestream Phone: (716) 876-5290 given]
.com Fax: (716) 876-2412
wasstream@aol.com
Yellowstone Mary M. Hunter BIOCAT-II (TM) Biological, INP INP INP & Treats a variety of wastes, Not specified Registered
Environmental President Nutrient, including aromatic trademark and
Science (YES), Inc. Phone: (406) 586-2002 Oxidant (O), hydrocarbons and patent
Fax: (406) 586-8818 Oxidant (R), halogenated hydrocarbons pending
www.yestech.com Reductant & Advantages include:
yes@yestech.com (O) natural pH control and
immobilization of metals to
protect enriched population
of methanogens,
conversion of breakdown
products of toxic organics
to methane that can be
used to thermally enhance
biotransformations, and
elimination of releases of
VOC
1
Information included in this table, including aspects of configuration, number of units, points of contact, list of performance claims, and patent information, was extracted from EPA
REACH IT (<http://www.epareachit.org>) in June 2000. Information is shown as provided by technology vendors in EPA REACH IT and was not modified for this report.
2
Biological - Bioaugmentation Oxidant (O) - Oxidant addition for oxidation application
HRC - Hydrogen release compound Oxidant (R) - Oxidant addition for reductive dechlorination application
Nutrient - Nutrient addition ORC - Oxygen release compound
INP - Information Not Provided Reductant (O) - Reductant addition for oxidation application (oxygen unless otherwise specified)
Reductant (R) - Reductant addition for reductive dechlorination (cometabolic)
3
At an EPA Region 9 meeting on September 23, 1999, Regenesis Bioremediation Products claimed to have used its ORC product at more than 5,000 sites and its HRC product at more
than 100 sites.
3-15
4.0 SELECTION AND IMPLEMENTATION OF IN SITU

BIOREMEDIATION TECHNOLOGIES
This section discusses the general steps involved in selecting and implementing in situ bioremediation
technologies, and additional factors relevant to using this technology. As discussed in Section 1, this
report is intended to familiarize those involved with hazardous waste site cleanups, including site project
managers, contractors, and other technology users, with in situ bioremediation. As such, the level of
detail included in the report about bioremediation mechanisms, technologies, and implementation is
meant to provide basic information about in situ bioremediation. Therefore, the report should be used for
informational purposes, but should not be used as the sole basis for determining the use of this
technology at a specific site. Decisions about the use of in-site bioremediation must be made on a case-
by-case basis, considering site-specific factors. Information included in this report is not intended to
revise EPA policy or guidance concerning site clean up.
4.1 TECHNOLOGY SELECTION AND IMPLEMENTATION
The steps typically followed in the selection and implementation of an in situ bioremediation system at a
site contaminated with CAHs are generally the same as the steps taken to implement other types of
remedial systems. However, special attention is typically given to identifying the degradation
mechanisms that may be used to remediate the site and the enhancement technologies that could be
beneficial for use at the site. Exhibit 4-1 shows the typical steps in selection and implementation of in
situ bioremediation, which are:
& Evaluate site characteristics

& Identify general site conditions and engineering solutions
& Identify primary reactants and possible additives
& Perform treatability (bench-scale) testing
& Perform system design, field testing, and implementation
4.1.1 Site Characteristics
Site characteristics relevant to in situ bioremediation of CAHs include physical, chemical, and biological
parameters. Exhibit 4-2 summarizes the parameters that are commonly evaluated for a site where in situ
bioremediation of CAHs is being considered. These parameters are also relevant to the design and
implementation of the technology, as discussed later in this section.
Physical Parameters - Physical parameters determine how and at what rate liquids and gases move
through soils, aquifers, and other geologic units. Common physical parameters that are relevant to in situ
bioremediation include porosity, hydraulic conductivity, and hydraulic gradient of the geologic unit, and
the organic and moisture content of the soil. Because these parameters affect the flow rate of fluids, they
also are considered in determining the delivery method for any amendments that are used.
Hydrogeologic studies help determine information about several physical parameters such as
groundwater flow, and contaminant fate and transport, and might include aquifer parameter testing, tracer
tests, and hydrogeologic flow and transport modeling. Aquifer parameter tests include either slug tests or
down hole velocity measurements. Tracer tests have been conducted using constituents such as sodium
bromide, added at 100 times its detection limits. According to ITRC, the most commonly used flow
model is the U.S. Geological Survey model MODFLOW, which is often coupled with transport models
such as RT3D or MT3D, and a particle tracking module such as MODPATH. (ITRC, 1998)
4-1
Exhibit 4-1: Typical Selection and

Implementation Steps for In Situ Bioremediation
Chemical Parameters - Chemical parameters, along with biological parameters, affect the type of
degradation mechanisms that are likely to occur and the rate of degradation. Common chemical
parameters that are relevant to in situ bioremediation include concentrations of CAHs and daughter
products, oxygen content, pH, redox potential, concentrations of electron donors and acceptors, and
nutrient concentrations. Such parameters provide information about the baseline contamination at the
site, whether the natural conditions at the site are aerobic or anaerobic, whether sufficient electron donors
and acceptors are present to support biodegradation, and whether and how much intrinsic biodegradation
(without enhancements) may be occurring at the site. Several of these parameters are discussed in more
detail below.
CAH concentrations affect the specific degradation mechanisms that may be occurring at the site and the
substrate levels for direct degradation. In addition, the presence of co-contaminants may affect
biodegradation. For example, organic compounds such as toluene, methane, or phenol may augment the
performance by providing a substrate for oxygen depletion or for cometabolic degradation.
Alternatively, biodegradation may be limited by high concentrations of metals or other toxic compounds
that may inhibit microbial activity.
4-2
Exhibit 4-2: Common Site Characterization Parameters Relevant to In Situ Bioremediation
Parameter Relevance Typical Measurement Method Typical Units

PHYSICAL PARAMETERS
Soil porosity To determine the extent to which soil gas diffusion Soil sampling Percent
(natural or forced) can take place
Hydraulic conductivity To determine the potential rate of groundwater flow Aquifer testing; permeability testing Length per time
through soil or in an aquifer
Hydraulic gradient To determine the speed at which groundwater travels Water level measurements Elevation difference per length
(when combined with hydraulic conductivity)
Organic content of soil To determine the extent to which contaminants may Volatile solids analysis Percent by weight
adsorb to soil, rather than migrate with groundwater;
provide a source of carbon for biodegradation reactions
Moisture content of soil To determine whether sufficient moisture is present in Dry weight measurement Percent by weight of water
soil to support degradation processes
CHEMICAL PARAMETERS
CAH concentrations To determine the baseline level of contamination, Gas chromatography with flame Amount of analyte per volume of
specific degradation mechanisms that may be ionization detector or mass water (e.g., µg/L) or per mass of soil
applicable, and substrate levels for direct degradation spectrophotometric methods (e.g., mg/kg)
Concentrations of CAH To provide a measure of degradation mechanisms Gas chromatography with flame Amount of analyte per volume of
degradation products (that is, taking place naturally ionization detector or mass water (e.g., µg/L) or per mass of soil
less chlorinated CAHs or non- spectrophotometric methods (e.g., mg/kg)
chlorinated products)
Concentrations of organic To determine the potential for cometabolic degradation Gas chromatography with flame Amount of analyte per volume of
electron donors (for example, mechanisms without enhancements ionization detector or mass water (e.g., µg/L) or per mass of soil
toluene, methane, phenol, or spectrophotometric methods; oxygen (e.g., mg/kg)
organic acids) uptake analysis
Concentrations of inorganic To determine the potential for degradation mechanisms Direct electrode; wet chemistry methods; Amount of analyte per volume of
electron donors (for example, without enhancements atomic adsorption methods water (e.g., µg/L) or per mass of soil
hydrogen, iron, or ammonia) (e.g., mg/kg)
Oxygen content To determine whether aerobic or anaerobic conditions Direct electrode Percent oxygen or concentration by
are present volume
4-3
Exhibit 4-2: Common Site Characterization Parameters Relevant to In Situ Bioremediation
Parameter Relevance Typical Measurement Method Typical Units

CHEMICAL PARAMETERS (continued)
Redox potential To determine the approximate size and location of a Direct electrode Volts relative to standard electrode
plume; determine whether aerobic or anaerobic (more negative is a more reducing
conditions are present; identify microbes that are likely environment and more positive is a
to be present more oxidizing environment)
Concentrations of other To determine whether electron acceptors are sufficient Direct electrode; wet chemistry methods; Amount of analyte per volume of
electron acceptors (for for aerobic degradation of CAHs, or may cause atomic adsorption methods water (e.g., µg/L) or per mass of soil
example, nitrate, sulfate, carbon unwanted competition in anaerobic dehalogenation (e.g., mg/kg)
dioxide) processes
Concentrations of nutrients (for To determine whether nutrient levels are sufficient for Wet chemistry methods; atomic Amount of analyte per volume of
example, boron, calcium, microbial activity or whether nutrient addition is adsorption methods water (e.g., µg/L) or per mass of soil
magnesium, manganese, needed (e.g., mg/kg)
nitrogen, potassium, or
phosphorus)
pH To determine whether conditions are most beneficial Direct electrode Standard pH units
for microbial growth
BIOLOGICAL PARAMETERS
Presence and concentration of To estimate microbial abundance Direct microscopic analysis; electronic Concentration of total solid organic
non-specific microbes particle counting; volatile suspended material in terms of total organic
solids analysis; total kjeldahl nitrogen carbon
analysis; protein analysis
Presence and concentration of To determine the presence and concentration of Heterotrophic plate count; MPN tube Count of specific indigenous
specific microbes targeted type of organism counts bacteria per volume of culture
media
Microbial activity To quantify the rate of activity of targeted organisms Oxygen uptake rate analysis; Amount of oxygen consumed
dehydrogenate activity analysis during a specified time period
Sources: Gossett, Microbiology for Environmental Engineers 1997; EPA 1998
4-4
The presence, concentration, and distribution of daughter products often is used as an indicator that
biodegradation may be taking place in situ. For the CAH TCE, an increase in concentration of DCE in
groundwater (a daughter product of TCE), along with a decrease in TCE concentrations can be used as an
indicator that biodegradation is occurring. The presence of VC, a degradation product of DCE, and
ethene, a degradation product of VC, can be used as indicators of the biodegradation process.
The concentration of dissolved hydrogen in the subsurface can be used as an indicator of the type of
terminal electron-accepting process is occurring. Exhibit 4-3 provides data on the hydrogen
concentration for five processes - denitrification, iron (III) reduction, sulfate reduction, reductive
dechlorination, and methanogenesis. The type of terminal electron-accepting process helps to determine
the dominant types of microbes present at a site.
Exhibit 4-3: Hydrogen Concentrations by

Terminal Electron Accepting Process
Terminal Electron Accepting Process Typical Hydrogen Concentration

(nanomoles/L)
Denitrification < 0.1
Iron (III) reduction 0.2 to 0.8
Sulfate reduction 1 to 4
Reductive dechlorination >1
Methanogenesis 5 to 20
Source: EPA 1998
Redox potential is commonly used to determine whether the subsurface environment is more reducing or
more oxidizing. In addition, redox potential can be used to determine the approximate size and shape of
a reducing zone at a site. For example, at Avco Lycoming, redox potential was used to identify the size
and shape of the reducing zones within the plume. Before operation of the system, redox potential was
used to show that there were only two relatively small reducing zones, located near the edges of the
plume. After 18 months of operation of the system, data on redox potential were used to show that a
relatively large reducing zone covered the majority of the plume.
Biological Parameters - In addition to chemical parameters, there are several biological parameters that
affect the type of degradation mechanisms that are likely to occur and the rate of degradation. For in situ
bioremediation of CAHs, common biological parameters include the presence of specific and non-
specific microbes and microbial activity. The presence and concentration of non-specific microbes,
generally measured as total organic carbon, is used to estimate the abundance of microbes at a site. The
presence and concentration of specific microbes is used to determine the concentration of the targeted
type of organism at the site. Microbial activity, measured in terms of oxygen uptake rate or
dehydrogenate activity, is used to quantify the rate of activity of the targeted microbes.
4.1.2 Site Conditions and Engineered Solutions
In evaluating sites for in situ bioremediation, data on hydrogeologic and general aquifer chemistry can be
used to identify sites as having generally favorable conditions for in situ bioremediation of CAHs.
Exhibit 4-4 provides a summary of the generally favorable and unfavorable conditions for in situ
bioremediation, and typical engineered solutions for unfavorable conditions. As shown in Exhibit 4-4,
4-5
examples of generally favorable conditions include highly permeable, homogenous hydrogeology with
sufficient nutrients and primary reactants present in the aquifer. Conversely, examples of generally
unfavorable conditions include low permeability, highly stratified hydrogeology with insufficient
nutrients and primary reactants present in the aquifer.
For sites with unfavorable conditions for in situ bioremediation of CAHs, engineered solutions may be
available to improve suitability of the hydrogeologic conditions and/or aquifer chemistry of the site for in
situ bioremediation. Examples of engineered solutions, shown in Exhibit 4-4, include hydrofracturing to
increase permeability, and the addition of nutrients and reactants to promote biodegradation. It is
important to note that an engineered solution may not be available to modify every unfavorable site
condition (identified as “none typically employed” in Exhibit 4-4). In addition, the conditions in Exhibit
4-4 are intended to provide an overview of the types of site conditions that are generally favorable or
unfavorable to the use of in situ bioremediation. As with any technology, the site-specific conditions
should be fully evaluated, along with potential engineered solutions, in determining the applicability of in
situ bioremediation.
Exhibit 4-4: Generally Favorable and Unfavorable Site Conditions for In Situ Bioremediation of
CAHs and Typical Engineered Solutions
Conditions Typical Engineered Solution for

Favorable Unfavorable Unfavorable Conditions
Hydrogeologic
Granular porous media Fractured rock None typically employed
High permeability Low permeability Hydrofracturing and pneumatic
(K > 10-4 cm/s) (K < 10-4 cm/s) fracturing
Saturated media Unsaturated media Water application
Minimal heterogeneity Highly stratified deposits None typically employed
Aquifer Chemistry
Minimal NAPL in target area Significant NAPL in target area Source containment, treatment, or
removal
pH between 6 and 8 pH extremes Chemical additives (NaHCO3 as a
buffer)
Nontoxic contaminant Toxic contaminant concentrations Dilution by injection of water or
concentrations bioremediation additives
Simple contaminant mixtures Complex contaminant mixtures None typically employed
Moderate to high microbial Little microbial activity or Bioaugmentation
activity of appropriate microbes inappropriate microbes
Sufficient nutrients present Insufficient nutrients present Addition of nutrients
Sufficient primary reactants Insufficient primary reactants Add reactants needed to employ
specific mechanism (refer to
Exhibit 4-5)
Sources: RTDF 1997; Munakata-Marr and others 1997; ITRC 1998; USAF 1998
4.1.3 Primary Reactants and Additives Typically Employed
Exhibit 4-5 summarizes the primary reactants and additives that are typically employed for engineered in
situ bioremediation systems. As shown in the exhibit, the types of reactants and additives vary by
specific engineered bioremediation mechanism (direct aerobic oxidation, cometabolic aerobic oxidation,
and anaerobic reductive dechlorination), and by the targeted CAHs.
4-6
For direct aerobic oxidation, primary reactants include oxygen and CAH and possible additives include
air, oxygen, hydrogen peroxide (H202) and magnesium peroxide. For cometabolic aerobic oxidation,
additional primary reactants include organic or anthropogenic carbon and additional additives include
methane, propane, butane, and ammonia. For anaerobic reductive dechlorination, primary reactants
include hydrogen, organic carbon, carbon from a contaminant source (anthropogenic), and CAH while
possible additives include lactate, methanol, hydrogen, and molasses. The additives listed in Exhibit 4-5
could be used in any of the configurations described in Section 3, as determined by the specific
requirements of the site.
Exhibit 4-5: Primary Reactants and Additives Typically Employed for Engineered
In Situ Bioremediation of CAHs
Engineered Typical Primary Reactants and Additives in Engineered Systems

Targeted
Bioremediation
CAHs Typical Additives (primary reactant
Mechanism Primary Reactants
supplemented)
Aerobic oxidation DCE, VC, Oxygen, CAH Air, oxygen, hydrogen peroxide, magnesium
(direct) DCA, CA, peroxide (oxygen)
MC, CM
Aerobic oxidation TCE, DCE, Oxygen Air, oxygen, hydrogen peroxide, magnesium
(cometabolic) VC, TCA, peroxide (oxygen)
CF, MC Organic carbon or Methane, propane, butane, ammonia (organic
carbon from a carbon)
contaminant source
(anthropogenic)
Anaerobic PCE, TCE, Hydrogen, organic Lactate, methanol, hydrogen, molasses
reductive DCE, VC, carbon, or carbon from (electron donor)
dechlorination TCA, DCA, a contaminant source
CT, CF, MC (anthropogenic)
Sources: Anderson and Lovley 1997; McCarty 1994; McCarty and others 1998; USAF 1998; EPA 1998; Yager and others 1997
4.1.4 Treatability (Bench-Scale) Testing
Treatability (bench- or laboratory-scale) testing is generally conducted after site characteristics,

degradation mechanisms, and potential enhancements have been identified. Treatability testing is used to
evaluate the effectiveness of degradation mechanisms and enhancements that are being considered for the
site. For example, results of treatability testing can be used to determine the conditions under which
degradation products are produced, the rates of degradation, and the paths of degradation in order to
identify the specific formulation that supports the most complete and rapid biodegradation of the targeted
CAHs. Examples of treatability tests for in situ bioremediation of CAHs include microcosm bottle
studies and soil column studies. Typically, samples of the media to be treated at the site (groundwater,
sediment, or soils) are used in the treatability tests. Because microbial populations usually are
heterogeneous in the subsurface and the type of plume may vary across the site, treatability tests are often
conducted using samples from several areas of a site.
In addition, data from treatability testing can help identify the parameters to be used for field-scale
testing and implementation. It should be noted that rates of biodegradation observed during bench-scale
microcosm studies typically will be higher than those observed in the field and that in situ residence
times will require adjustment accordingly (U.S. Air Force 1998).
4-7
The specifics of treatability testing for in situ bioremediation can be complex, and are influenced by site-
specific conditions. Additional details on designing and conducting treatability tests for in situ
bioremediation can be found in the references listed in Sections 5 and 6 of this report.
4.1.5 System Design, Field Testing, and Implementation
Information from the first four steps in the selection and implementation of in situ bioremediation (shown
in Exhibit 4-1) is used in designing the system, testing the system in the field, and implementing the
technology on a full-scale basis. As discussed above, treatability testing is generally used to determine
the specific formulation (combination of substrates or nutrients as amendments) that supports the most
complete and rapid biodegradation of the targeted CAHs. The technology is then scaled-up for field or
pilot-scale testing, and configured on a full-scale basis for the conditions specific to the site.
System Design - In the system design stage, a system configuration such as groundwater recirculation or
direct injection (see Section 3) that is appropriate for the site conditions and remedial goals is paired with
one or more enhancement technologies. Major considerations for system design include the type and size
of extraction and injection systems, the arrangement of plumbing and other infrastructure, the method
and schedule for addition of amendments, monitoring system equipment, and monitoring schedule.
Additional considerations include design of above-ground components, such as storage containers,
pumps, mixers, and flow meters. (ITRC, 1998)
In general, for a groundwater recirculation system, well spacing is designed to be at a distance sufficient
to observe measurable changes in contaminant concentrations between two or more wells. Residence
time or period is the amount of time required for a pollutant molecule to pass through the contaminated
area, and is measured with a variety of techniques. For example, bromide tracer studies are used to
determine actual travel time between the injection and the extraction wells.
Pilot/Field-Scale Testing - Pilot- or field-scale testing involves installation of an extraction and/or

injection system at a site, and operation of that system over time. While pilot/field-scale systems vary in
size, they are usually constructed on a smaller scale than a full-scale system.
For example, a small pilot-scale test of a groundwater recirculation system can be designed using the
ESTCP technical protocol. Their design includes a system with three 2-inch injection wells spaced along
12-inch centers, one 2-inch extraction well down-gradient from the injection wells, and one 4-inch
extraction well further down-gradient. This design indicates that the system be oriented parallel to the
natural flow direction, and that the spacing between injection and extraction wells be the distance that
groundwater would travel in 35 - 40 days under natural flow conditions. The design also includes three
rows of monitoring wells between the injection and extraction wells. (ESTCP, 1998)
An important aspect of pilot/field-scale testing is the evaluation of the results from testing to determine
system effectiveness. AFCEE has suggested three lines of evidence for performance evaluation:
1. Reduction in contaminant mass - this includes temporal and spatial reductions in

concentrations; integration of extrapolated concentration measurements for the system;
comparison of concentrations through multiple recirculation cycles and addition of
amendments; and comparison of mass leaving injection points and arriving at extraction points
2. Microbiological activity linked to degradation - this includes microcosm or column studies

which demonstrate metabolic activity; demonstrations showing that calculated field
4-8
degradation rates are consistent with microcosm or column studies; and correlation of biomass
in the field with zones of contaminant depletion
3. Contaminant disappearance linked to cometabolic system (only for aerobic cometabolic

systems) - correlation of contaminant depletion with substrate depletion; correlation of temporal
changes in contaminant concentration with addition of substrate; and evidence showing aerobic
conditions, such as high oxidation reduction potential
To evaluate pilot/field test results, amended groundwater can be sampled and the results compared with
up-gradient contaminated conditions as it passes each monitoring point, or once steady-state conditions
have been achieved and for several residence periods. Frequently, control plots with unamended
groundwater are sampled to provide a basis of comparison with the amended groundwater plots.
When multiple formulations are being pilot-tested, parallel plots can be used to allow simultaneous
testing of alternative formulations. Parallel plots also can be used to determine the lateral extent of
biodegradation, especially from monitoring locations perpendicular to groundwater flow near a test plot,
also referred to as diffusion degradation.
For some in situ bioremediation applications, site remedial goals might be achieved after the conclusion
of field testing. In other cases, the pilot system is scaled up to a full-scale system to remediate the site.
Because of the variability of subsurface conditions, field testing of an in situ bioremediation process may
be iterative, requiring several preliminary designs and field tests to determine the best configuration for a
full-scale system. It is also possible that an enhancement technology shown to be effective in the
laboratory is ineffective in the field, possibly requiring additional site characterization and re-evaluation
of appropriate in situ bioremediation enhancement technologies.
Example of Field Testing and Implementation - Field testing and implementation for in
situ bioremediation systems for cleanup of CAHs is illustrated in the following example, as
shown in the case studies discussed in Appendix A of this report.
At the Dover Air Force Base Building 719 site, a pilot test that used cometabolic bioventing was
conducted over 14 months. Before the pilot test began, laboratory tests were performed on soils
from the area of Building 719 to evaluate candidate substrates. Propane was identified as the
preferred substrate to be tested in the pilot study because of its ability to stimulate cometabolic
activity affecting both TCA and TCE, the two contaminants of interest at the site.
Full-Scale Implementation - Once pilot/field testing is complete, the next step in the process is the
design (scale-up) of the full-scale system. As discussed earlier, the full-scale system is configured
according to the conditions specific to the site. Other considerations in designing the full-scale system
include aerial extent and depth of contamination to be remediated, types and concentrations of
contaminants and the remedial goals, other regulatory considerations, timeframe for remediation to be
completed, and cost.
The first step in a full-scale application is to perform system start-up, where the activity of the microbial
populations are acclimatized to the added amendments. After start-up, routine operation and
maintenance is generally performed, including monitoring of system performance. Many of the same
types of data gathered and evaluated during a pilot/field-scale test are collected for full-scale
applications, including information about reductions in contaminant mass and microbiological activity
linked to degradation.
4-9
Clogging of injection and extraction wells is a particularly important consideration during full-scale
implementation of an in situ bioremdiation system. Clogging occurs when there is a buildup of biomass
on or near the well screens, making it difficult to inject or extract fluids from the subsurface. Techniques
that have been used to reduce the impacts from clogging include pulsed addition of substrates, use of
reduced concentrations of substrates, and routine well cleaning. Pulsed addition of substrates (for a
cometabolic system) has the added advantage of providing limited slugs of substrate that can be
periodically depleted, thus reducing the competitive inhibition of the primary substrate on the targeted
chlorinated compound. (ITRC, 1998)
Additional information about full-scale design and implementation of in situ bioremediation can be found
in sections 5 and 6 of this report. Example resources include the AFCEE’s guidance manual and
screening software for in situ bioremediation and ITRC’s document about technical and regulatory
requirements for enhanced in situ bioremediation, described briefly below.
• Aerobic Cometabolic In Situ Bioremediation Technology Guidance Manual and Screening

Software User’s Guide; AFCEE; June 1998 (available on the Internet at
http://en.afit.af.mil/env/insitubio.htm). This guide presents the principles of aerobic cometabolic
in situ bioremediation, the mathematical models used to describe the technology, and a
discussion of the applicability and limitations of the technology. In addition, a software program
that may be used to help determine whether the technology is appropriate for implementation at a
given site is available at the Internet site listed above. The steps needed to design and implement
the technology, a discussion of regulatory acceptance, and case studies of use of the technology
also are presented.
• Technical and Regulatory Requirements for Enhanced In Situ Bioremediation of Chlorinated

Solvents in Groundwater; ITRC; In Situ Bioremediation Subgroup; December 23, 1998
(available on the Internet at http://itrcweb.org/reports/isb.htm). This document provides
technical and regulatory information that state and federal regulators need to support decisions
about whether to proceed with field studies of enhanced in situ bioremediation, and to implement
such pilot tests successfully.
Example of Full-Scale Implementation - Full-scale implementation is illustrated in the

following example, as shown in the case studies in Appendix A of this report.
At the abandoned manufacturing facility site in Emeryville, California, a pilot study was
conducted for approximately six months to determine whether the rate of anaerobic
dechlorination could be enhanced by the addition of a molasses solution to the groundwater. The
pilot study demonstrated that the rate was enhanced through addition of the molasses, and the
technology subsequently was used on a full-scale basis. Groundwater monitoring data collected
after the system had operated for 18 months showed that the pilot-scale system had been a good
indicator of the performance that the system could achieve.
4-10
4.2 ADDITIONAL SELECTION FACTORS
Additional factors relevant to the selection of in situ bioremediation as a treatment for CAHs include
(ITRC 1998):
& Advantages and potential limitations of the technology

& Regulatory considerations
Advantages and Potential Limitations of the Technology
In situ bioremediation is used at sites at which soil or groundwater is contaminated with CAHs.
Advantages of using the technology include:
& Capability to degrade CAH contaminants to relatively less toxic end products
& Generation of relatively small amounts of remediation wastes, compared to ex situ
technologies
& Reduced potential for cross-media transfer of contaminants commonly associated with ex situ
treatment
& Reduced risk of human exposure to contaminated media, compared to ex situ technologies
& Relatively lower cost of treatment compared to excavation and disposal, ex situ treatment, or
conventional pump-and-treat systems
& Potential to remediate a site faster than with use of conventional technologies (EPA 1999)
Potential limitations of using in situ bioremediation include:
& A perceived lack of knowledge about biodegradation mechanisms

& Specific contaminants at a site may not be amenable to biodegradation
& Enhancement technologies, when needed, may be costly or their implementation may be
technologically challenging
& The toxicity of transformation (daughter) products may exceed that of parent compounds
Regulatory Considerations
As with any technology, there are a number of regulatory considerations that may impact the selection
and implementation of in situ bioremediation as a remedy for a site. The following presents general
information about some of the regulations that are relevant to in situ biormediation. It is important to
note that the determination of what regulations (federal, state, and local) apply to a specific technology
application must be made on a case-by-case basis.
Because in situ bioremediation typically involves the direct injection of chemicals into the subsurface or
the pumping and reinjecting of “activated” groundwater, the Underground Injection Control (UIC)
regulatory program under the Safe Drinking Water Act is often a consideration. UIC regulations prohibit
the injection of fluid that contains a contaminant into an underground source of drinking water if the
presence of the contaminant may cause a violation of any primary drinking water regulation under 40
Code of Federal Regulations (CFR) Part 142 or otherwise have an adverse effect on the health of
persons. Under 40 CFR 144.11, any underground injection, except into a well authorized by rule or
except as authorized by permit issued under the UIC program, is prohibited. Under 40 CFR 144.12(a),
no owner or operator shall construct, operate, maintain, convert, plug, abandon, or conduct any other
injection activity in a manner that allows the movement of fluid containing any contaminant into
underground sources of drinking water, if the presence of that contaminant may cause a violation of any
primary drinking water regulation under 40 CFR 142 or may otherwise adversely affect the health of
4-11
persons. UIC regulations define five “classes” of injection wells (see 40 CFR 144.6). Under the federal
UIC regulations, the injection of hazardous waste (as might be the case in a groundwater recirculation
system) into a formation that is within or above an underground source of drinking water (deemed a
Class IV well) generally is prohibited, unless the injection is part of an approved RCRA corrective action
or CERCLA response (see 40 CFR 144.13). In such cases, it may be necessary to use other means of
groundwater reinjection (such as recharge trenches). In cases in which the injected material is not a
hazardous waste or in which hazardous waste is being injected into an aquifer that is not an underground
source of drinking water, a Class V injection well permit may be required3. In addition, establishment of
a “containment area” is sometimes required to demonstrate that injected products are not migrating from
the site.
Contaminated soil or groundwater may be identified as a RCRA hazardous waste either because it is a
listed hazardous waste (such as F001 through F005, spent solvents) or because it exhibits a characteristic
of hazardous waste (such as the Toxicity Characteristic [TC]). Under RCRA, the injection of a
hazardous waste into an aquifer is defined as “land disposal”, and therefore is subject to the RCRA Land
Disposal Restriction (LDR) regulations under 40 CFR Part 268. These restrictions may prohibit the
injection of hazardous wastes into an aquifer until the groundwater is treated (1) to specified
concentrations, or (2) using a prescribed technology. However, section 3020(b) of RCRA provides a
statutory waiver for compliance with LDRs that may permit reinjection of contaminated groundwater that
is a hazardous waste. In order to qualify for this waiver, the reinjection process must meet all of the
following criteria:
& Must be performed under CERCLA response or RCRA corrective action authorities.
& Can occur only after the groundwater has been treated to “substantially reduce hazardous
constituents” prior to reinjection.
& Must be part of a cleanup process that will, upon completion, be sufficient to protect human
health and the environment.
RCRA section 3020(a) bans hazardous waste disposal by underground injection into or above an
underground source of drinking water (within one-quarter mile of the well). However, as discussed
above, section 3020 (b) exempts from the ban the reinjection of contaminated groundwater if certain
conditions are met. To facilitate the use of in situ bioremediation of contaminated groundwater, EPA has
clarified the applicability of RCRA section 3020(b) to in situ bioremediation technologies. In a
December 1999 letter to an official in the California hazardous waste program, titled Applicability of
RCRA 3020(B) to In Situ Bioremediation Technologies (available at http://clu-in.org), EPA stated that the
addition of nutrients or other products designed to promote in situ bioremediation is considered
“treatment” under section 3020(b), and that the substantial reduction of hazardous constituents required
may occur before or after reinjection. This clarification allows for in situ bioremediation of groundwater
without an above-ground pump-and-treat system in place, as long as the other conditions of section
3020(b) discussed above are met.
3
Permits are not required for on-site CERCLA response actions. However, the substantive requirements of
permits (that is, closure requirements governing closure of injection wells) must be met for CERCLA actions.
4-12
5.0 REFERENCES
Anderson, R.T. and Lovley, D.R. 1997. Ecology and Biogeochemistry of In Situ Groundwater
Remediation. Advances in Microbial Ecology, Volume 15. Plenum Press. New York. Pages 289-350.
Becvar, E. C. and others. 1997. “In situ dechlorination of solvents in saturated soils.” In Situ and On-
Site Bioremediation: Vol. 3. Battelle Press. Columbus, Ohio. Pages 39-44.
Beeman, R.E. and others. 1994. “A field evaluation of in situ microbial reductive dehalogenation by the
biotransformation of chlorinated ethenes.” Bioremediation of Chlorinated and Polycyclic Aromatic
Hydrocarbon Compounds. Lewis Publishers. Boca Raton. Pages 14-27.
Bielefeldt, A.R., H.D. Stensel, and S.E. Strand. 1995. “Cometabolic Degradation of Trichloroethene and
Dichloroethene Without Intermediate Toxicity.” Journal of Environmental Engineering. Volume 121.
Page 791. November.
Bouwer, E.J. 1994. “Bioremediation of chlorinated solvents using alternate electron acceptors.”
Handbook of Bioremediation. Lewis Publishers, Inc. Boca Raton, Florida.
Bradley, P.M. and F.H. Chapelle. 1998. “Effect of contaminant concentration on aerobic microbial
mineralization of DCE and VC in stream-bed sediments.” Environmental Science and Technology.
Volume 32. Number 5. Pages 553-557.
Buchanan, Jr., R.J. and others. 1997. “Aerobic reductive dehalogenation pilot design for Dover AFB.”
In Situ and On-Site Bioremediation: Vol. 3. Battelle Press. Columbus, Ohio. Pages 289-290.
Carter, S.R. and W.J. Jewell. 1993. “Biotransformation of tetrachloroethylene by anaerobic attached-
films at low temperatures.” Water Research. 27. April.
DeBruin, W.P. and others. 1992. “Complete biological reductive transformation of tetrachloroethene to
ethane.” Applied and Environmental Microbiology. Volume 58. Pages 1996- 2000.
DeStefano, T.D., J.M. Gossett, and S.H. Zinder. 1992. “Hydrogen as an electron donor for
dechlorination of tetrachloroethene by an anaerobic mixed culture.” Applied and Environmental
Microbiology. Volume 58. Number 11. Pages 3622-3629.
Devlin, J. F. and J. F. Barker. 1994. “A semi-passive nutrient injection scheme for enhanced in situ
bioremediation.” Ground Water. Volume 3. Pages 374-380.
Edwards, E.A. and E.E. Cox. 1997. “Field and laboratory evidence of sequential aerobic chlorinated
solvent biodegradation.” In situ and On Site Bioreclamation: Volume 3. Battelle Press. Columbus,
Ohio.
Engineered Approaches for In Situ Bioremediation of Chlorinated Solvent Contamination. 1999.

Leeson, A. and B.C. Alleman, eds. Proceedings, The Fifth International In Situ and On-Site
Bioremediation Symposium; San Diego, California, April 19-22, 1999. Battelle Press. Columbus, Ohio.
Fathepure, B.Z. and others. 1987. “Anaerobic bacteria that dechlorinate perchloroethene.” Applied and
Environmental Microbiology. Volume 53. Pages 2771-2674.
5-1
Fennell, D.E. and others. 1997. “Comparison of bytyric acid, ethanol, lactic acid, and propionic acid as
hydrogen doors for the reductive dechlorination of trichlorothene.” Environmental Science and
Technology. Volume 31. Pages 918-925.
Freedman, D.L. and J.M. Gossett. 1989. “Biological reductive dechlorination of tetrachloroethylene and
trichloroethylene to ethylene under methanogenic conditions.” Applied and Environmental
Microbiology. Volume 55. Pages 2144-2151.
Gerritse, J. and others. 1995. “Complete degradation of trichloroethene by combining anaerobic

dechlorinating and aerobic methanetrophic enrichment cultures.” Applied Microbiology and
Biotechnology. Volume 43. Page 920. October.
Gerritse, J. and others. 1996. “Desulfitobacterium sp. Strain PCE1, an anaerobic bacterium that can
grow by reductive dechlorination of tetrachloroethene or ortho-chlorinated phenols.” Archives of
Microbiology. Volume 165. Pages 132-140.
Gerritse, J. and others. 1999. “Influence of different electron donors and acceptors on dehalospiration of
tetrachloroethene by Desulfitobacterium Frappiere TCE1.” Applied and Environmental Microbiology.
Volume 65. Pages 5212-5221.
Gibson, S.A. and G.W. Sewell. 1992. “Stimulation of reduction dechlorination of tetrachloroethene in
anaerobic aquifer microcosms by addition of short chain organic acids or alcohols.” Applied and
Gillham, R.W. and S.F. O'Hannesin. 1994. “Enhanced degradation of halogenated aliphatics by zero-
valent iron.” Ground Water. Volume 32. Number 6. Pages 958-967.
Gossett, J.M. 1987. “Measurement of henry's law constants for C1 and C2 chlorinated hydrocarbons.”
Environmental Science and Technology. Volume 21. Page 202. February.
Gossett, J.M. 1997. Microbiology for Environmental Engineering. Cornell University. Ithaca, New
York.
Gossett, J.M., and S.H. Zinder. 1996. “Microbiological aspects relevant to natural attenuation of
chlorinated ethenes.” Proceedings, US EPA Symposium in Natural Attenuation of Chlorinated Organics
in Ground Water. Dallas, Texas. Sept 11-13. 1996. EPA/540/R-97/504/1997. Pages 12-15.
Harkness, M.R. and others. 1999. “Use of bioaugmentation to stimulate complete reductive
dechlorination of trichloroethene in Dover soil columns.” Environmental Science and Technology.
Hartman, S. and J. DeBont. 1992. “Aerobic vinyl chloride metabolism in Mycobacterium arurum Li.”
Applied and Environmental Microbiology. Volume 58. Page 1220.
Hollinger, C and W. Schumacher. 1994. Antonie van Leeuwenhoek. Volume 66. Pages 239-246.
Hollinger, C. and others. 1993. “A highly purified enrichment culture couples the reductive
dechlorination of tetrachloroethene to growth.” Applied and Environmental Microbiology. Volume 59.
Pages 2991-2997.
5-2
Hopkins, G.D., J. Munakata, and L. Semprini. 1993. “Trichloroethylene concentration effects on pilot
field-scale in-situ groundwater bioremediation by phenol-oxidizing microorganisms.” Environmental
Science and Technology. Volume 27. Page 2542.
Hopkins, G.D. and P.L. McCarty. 1995. “Field evaluation of in situ aerobic cometabolism of
trichloroethylene and three dichloroethylene isomers using phenol and toluene as the primary substrates.”
Environmental Science and Technology. Volume 29. Page 1628.
Huling, S.G. and J.W. Weaver. 1991. Groundwater Issue: Dense Nonaqueous Phase Liquids. EPA
Office of Solid Waste and Emergency Response, Office of Research and Development. Washington,
D.C. EPA/540/4-91-002. March.
Interstate Technology and Regulatory Cooperation Working Group (ITRC). 1997. Regulatory Guidance
for Permeable Barrier Walls Designed to Remediation Chlorinated Solvents, Final. Permeable Barrier
Walls Work Team Regulatory Guidance Project. December.
ITRC 1998. Technical and Regulatory Requirements for Enhanced In Situ Bioremediation of
Chlorinated Solvents in Groundwater. In Situ Bioremediation Group. December 23.
ITRC. 2000. Presentation on Natural Attenuation of Chlorinated Solvents in Groundwater. March.
Litherland, S.T., and D.W. Anderson. 1997. In Situ and On-Site Bioremediation: Volume 5. Battelle
Press. Columbus, Ohio. Pages 425-430.
Major, D.W., W.W. Hodgiuns, and B.J. Butler. 1991. “Field and laboratory evidence of in situ
biotransformation of tetrachloroethene to ethene and ethane at a chemical transfer facility in North
Toronto.” On Site Bioreclamation. Hinchee, R.E. and R.F. Olfenbuttel, eds. Publisher. San Diego,
California.
Malachowsky, K.J. and others. 1994. “Aerobic mineralization of trichloroethylene, vinyl chloride, and
aromatic compounds by Rhodococcus species.” Applied and Environmental Microbiology. Volume 60.
Page 542. February.
Maymo-Gatell, X. and others. 1997. “Isolation of a bacterium that reductively dechlorinates

tetrachloroethene to ethene.” Science. Volume 276. Pages 1568-1571.
McCarty, P.L. 1971. “Energetics and bacterial growth.” Organic Compounds in Aquatic Environments.
Faust, S. and J.V. Hunter, eds. Marcel Dekker, Inc. New York. Pages 495-512.
McCarty, P.L. 1994. “An overview of anaerobic transformation of chlorinated solvents.” Proceedings,
Symposium on Natural Attenuation of Ground Water. September.
McCarty, P.L. 1997a. “Biotic and abiotic transformations of chlorinated solvents in ground water.” EPA
Proceedings, The Symposium in Natural Attenuation of Chlorinated Organics in Ground Water. Dallas,
Texas. September 11-13, 1996. EPA/540/R-97/504/1997. Pages 7-11.
McCarty, P.L. 1997b. “Breathing with chlorinated solvents.” Science. Volume 276. Page 1521.
McCarty, P.L. and L. Semprini. 1994. “Ground-water treatment for chlorinated solvents.” Handbook of
Bioremediation. Lewis Publishers. Boca Raton, Florida. Pages 87-116.
5-3
McCarty, P.L. and others. 1991. “In situ methanotrophic bioremediation for contaminated groundwater
at St. Joseph, Michigan.” Proceedings, International Symposium on In Situ and On Site Bioreclamation.
San Diego, California.
McCarty, P.L., and others. 1998. “Full-scale evaluation of in situ cometabolic degradation of
trichloroethylene in groundwater through toluene injection.” Environmental Science and Technology.
Merck & Co., Inc. 1989. The Merck Index, An Encyclopedia of Chemicals, Drugs, and Biologicals,
Eleventh Edition. Merck & Co., Inc. Rahway, New Jersey.
Munakata-Marr, J. and others. 1997. “Long-term biodegradation of trichloroethylene influenced by

bioaugmentation and dissolved oxygen in aquifer microcosms.” Environmental Science and Technology.
Neumann, A., H. Scholz-Muramatsu, and G. Diekert. 1994. “Tetrachloroethene metabolism of

Dehalospirillum multivorans.” Archive of Microbiology. Volume 162. Pages 295-301.
Reinhard and others. 1990. Abiotic Reductive Dechlorination of Carbon Tetrachloride and
Hexachloroethane by Environmental Reductants: Project Summary. EPA/600/S2-90/040. September.
Remediation Technologies Development Forum. 1997. Natural Attenuation of Chlorinated Solvents in

Groundwater: Principles and Practices. Version 3.0. August.
Sawyer, C.N. and others. 1994. Chemistry for Environmental Engineering, Fourth Edition. McGraw-
Hill, Inc. New York.
Scholtz-Muramatsu and others. 1995. “Isolation and characterization of Dehalospirillum multivorans

gen sp. Nov., a tetrachloroethene-utilizing, strictly anaerobic bacterium.” Archive of Microbiology.
Semprini, L., P.V. Roberts, and G.D. Hopkins. 1990. “A field evaluation of in-situ biodegradation of
chlorinated ethenes: results of biostimulation and biotransformation experiments.” Ground Water.
Semprini, L. and others. 1995. “Anaerobic transformation of chlorinated aliphatic hydrocarbons in a

sand aquifer based on spatial chemical distributions.” Wat. Res. Res. Volume 31. Number 4. Pages
1051-1062.
Sewell, G.W. and others. 1998. “Designing and applying treatment technologies.” Remediation of
Chlorinated and Recalcitrant Compounds. Battelle Press. Columbus, Ohio. Pages 9-14.
Sharma, P.K. and P.L. McCarty. 1996. “Isolation and characterization of a facultatively aerobic
bacterium that reductively dehalogenates tetrachloroethene to cis-1,2-dichloroethene.” Applied
Sims, J.L. and others. 1992. Groundwater Issue: In-Situ Bioremediation of Contaminated Ground
Water. EPA Office of Solid Waste and Emergency Response, Office of Research and Development.
Washington, D.C. EPA/540/S-92/003. February.
5-4
Smatlak, C.R. and others. 1996. “Comparative kinetics of hydrogen utilization for reductive
dechlorination of tetrachloroethene and methanogenesis in an anaerobic enrichment culture.”
Environmental Science and Technology. Volume 30. Pages 2850-2858.
Spuij, F. A. and others. 1997. In Situ and On Site Bioremediation, Volume 5. Battelle Press. Columbus,
Ohio. Pages 431-437.
Tandol, V. and others. 1994. “Reductive dehalogenation of chlorinated ethenes and halogenated ethanes
by a high-rate anaerobic enrichment culture.” Environmental Science and Technology. Volume 28.
Pages 973-979.
Travis, B.J. and N.D. Rosenberg. 1997. “Modeling in situ bioremediation of TCE at Savannah River:
Effects of product toxicity and microbial interactions on TCE degradation.” Environmental Science and
Technology. Volume 31. Pages 3093-3102.
U.S. Air Force. 1998. Installation Restoration Program, Aerobic Cometabolic In Situ Bioremediation
Technology Guidance Manual and Screening Software User's Guide. Center for Environmental
Excellence, Environmental Restoration Division. Brooks Air Force Base, Texas. June.
<http://en.afit.af.mil/env/insitubio.htm>.
U.S. Environmental Protection Agency. Bioremediation in the Field Search System (BFSS) (Version
2.1).
U.S. Environmental Protection Agency. 1986. Superfund Public Health Evaluation Manual. Office of
Solid Waste and Emergency Response. Washington, D.C. October.
U.S. Environmental Protection Agency. 1995. Bioventing Principles and Practices (Volume 1).
September.
U.S. Environmental Protection Agency. 1997. Cleaning Up the Nation’s Hazardous Waste Sites:
Markets and Technology Trends, 1996 Edition. Office of Solid Waste and Emergency Response,
Technology Innovation Office. Washington, D.C. EPA-524-R-96-005, BP96-178041. April.
U.S. Environmental Protection Agency. 1998. Technical Protocol for Evaluating Natural Attenuation of
Chlorinated Solvents in Ground Water. Office of Research and Development. Washington, D.C.
September. <http://www.epa.gov/ada/reports.html>
U.S. Environmental Protection Agency. 1998a. Human Health Risk Assessment Protocol for Hazardous
Waste Combustion Facilities, Appendix A: Chemical-Specific Data (Peer Review Draft). EPA Region 6.
September 17.
U.S. Environmental Protection Agency. 1999. Directive Number 9200.4-17P, Use of Monitored Natural
Attenuation at Superfund, RCRA Corrective Action, and Underground Storage Tank Sites. Office of
Solid Waste and Emergency Response. Washington, D.C. April 21.
U.S. Environmental Protection Agency. 1999a. Yuma Marine Corps Air Station Fact Sheet. January.
Vogel, T. M. and P.L. McCarty. 1987. “Rate of abiotic formation of 1,1-dichloroethylene from 1,1,1-
trichloroethane in groundwater.” Journal of Contaminant Hydrology. Volume 1. Pages 299-308.
5-5
Vogel, T.M., C.S. Criddle, and P.L. McCarty. 1987b. “Transformations of halogenated aliphatic
compounds.” Environmental Science and Technology. Volume 22. Pages 722-736.
Wilson, J.T. and B.H. Wilson. 1985. “Biotransformation of trichloroethylene in soil.” Applied and
Workman, D.J. and others. 1997. “Microbial reduction of Vitamin B12 by Shewanella alga Strain BrY
with subsequent transformation of carbon tetrachloride.” Environmental Science and Technology.
Yager, R.M. and others. 1997. “Metabolic and in situ attenuation of chlorinated ethenes by naturally
occurring microorganisms in a fractured dolomite aquifer near Niagara Falls, New York.”
Environmental Science and Technology. Volume 31. Pages 3138-3147.
Yagi, and others. 1994. “Bioremediation of trichloroethylene-contaminated soils by a methane-utilizing

bacterium Methylocystis.” Bioremediation of Chlorinated and Polycyclic Aromatic Hydrocarbon
Compounds. Lewis Publishers. Boca Raton, Florida. Pages 28-36.
5-6
6.0 ADDITIONAL INFORMATION SOURCES

Alexander, M. 1994. Biodegradation and Bioremediation. Academic Press. San Diego, CA.
American Society of Microbiology. 1997. Manual of Environmental Microbiology. Hurst, C.J. and
others, eds. Washington, D.C.
Arcangeli, J-P. E. Arvin, and H. Moller-Jensen. 1995. “Cometabolic biodegradation of trichloroethylene

in a biofilm reactor.” Bioremediation of Chlorinated Solvents. R.E. Hinchee, A. Leeson, and L.
Semprini, eds. Battelle Press. Columbus, Ohio. Page 203.
Ballapragada, B.S. and others. 1997. “Effect of hydrogen on reductive dechlorination of chlorinated
ethenes.” Environmental Science and Technology. Volume 31. Pages 1728-1734.
Biology of Anaerobic Microorganisms. 1988. Zehnder, A.B.J., ed. Wiley Interscience. New York.
Bradley, P.M. and F.H. Chapelle. 1997. “Kinetics of DCE and VC mineralization under methanogenic
and Fe(III)-reducing conditions.” Environmental Science and Technology. Volume 31. Pages 2692-
2696.
Bradley, P.M. and F.H. Chapelle. 1996. “Anaerobic mineralization of vinyl chloride in Fe(III) -
reducing aquifer sediments.” Environmental Science and Technology. Volume 30. Pages 2084-2086.
Chapelle, F.H. 1993. Ground Water Microbiology and Geochemistry. John Wiley Sons. New York.
Chappell, J. 1997. Phytoremediation of TCE in Groundwater using Populus. U.S. Environmental

Protection Agency Office of Solid Waste and Emergency Response, Technology Innovation Office.
Washington, D.C. August. <http://clu- in.org>.
Cline, P.V. and J.J. Delfino. 1989. “Effect of subsurface sediment on hydrolysis of haloalkanes and
epoxides.” Biohazards of Drinking Water Treatment. Larson, R.A., ed. Lewis Publishers, Inc. Chelsea,
Michigan. Pages 47- 56.
Cohen, R. and Mercer, J. 1993. DNAPL Site Evaluation. EPA. Washington, D.C. EPA/600/3/002,
BP93-150217. February.
Environmental Security Technology Certification Program. 1998. A Treatability Test for Evaluating the
Potential Applicability of the Reductive Anaerobic Biological In Situ Treatment Technology (RABITT) to
Remediate Chloroethenes, Draft Technical Protocol. February.
Federal Remediation Technologies Roundtable. 1998. Remediation Case Studies: Innovative

Groundwater Treatment Technologies, Volume 11. EPA 542-R-98-015. September.
Fetter, C.W. 1999. Contaminant Hydrogeology, 2nd Edition. Prentice Hall. Oshkosh, Wisconsin.
Fogel, M.M. and others. 1986. “Biodegradation of chlorinated ethenes by a methane-utilizing mixed
culture.” Applied and Environmental Microbiology. Volume 51. Number 4. Pages 720-724.
6-1
Fountain, J.C. 1998. Technologies for Dense Nonaqueous Phase Liquid Source Zone Remediation.
Ground-Water Remediation Technologies Analysis Center. Pittsburgh, Pennsylvania. December.
<http://www.gwrtac.org>.
Haag, W.R. and T. Mill. 1988. “Transformation kinetics of 1,1,1-trichloroethane to the stable product
1,1-dichloroethene.” Environmental Science and Technology. Volume 22. Pages 658-663.
Handbook of Bioremediation. 1994. X. Norris and others, eds. Lewis Publishers. Boca Raton, Florida.
Howard, P. H., and others. Handbook of Environmental Degradation Rates. Lewis Publishers. Boca
Raton, Florida.
Jeffers, P. and others. 1989. “Homogeneous hydrolysis rate constants for selected chlorinated methanes,
ethanes, ethenes, and propanes.” Environmental Science and Technology. Volume 23. Number 8.
Pages 965-969.
Madsen, E.L. 1997. Manual of Environmental Microbiology. Hurst, C.J and others, eds. American
Society of Microbiology. Washington., D.C. Pages 709-720.
McCarty, P.L. 1972. “Energetics or Organic Matter Degradation. Water Pollution Microbiology.
Mitchell, R., ed. John Wiley & Sons. New York. Pages 91-118.
McNab, W.W. and T.N. Narasimhan. 1994. “Degradation of chlorinated hydrocarbon and groundwater
geochemistry: A field study.” Environmental Science and Technology. Volume 28. 769-775.
Mercer, J.W. and R.M. Cohen. 1990. “A review of immiscible fluids in the subsurface: properties,
models, characterization and remediation.” Journal of Contaminant Hydrology. Volume 6. Pages 107-
116.
Miller, R.R. 1996. Phytoremediation. Ground-Water Remediation Technologies Analysis Center.

Pittsburgh, Pennsylvania. October. <http://www.gwrtac.org>.
Pankow, J.F. and J.A. Cherry. 1996. Dense Chlorinated Solvents and Other DNAPLs in Groundwater.
Petrovskis, E.A. and others. 1995. “Transformation of tetrachloromethane by Shewanella putrefaciens

MR-1.” Bioremediation of Chlorinated Solvents. Hinchee, R.E., A. Leeson, and L. Semprini, eds.
Battelle Press. Columbus, Ohio.
Sewell, G.W. and S.A. Gibson. 1997. “Microbial ecology of adaption and response in the subsurface.”
Proceedings, Symposium on Natural Attenuation of Chlorinated Organics in Ground Water. Washington,
D.C. EPA/540/R-97/504/May. Pages 16-18.
Stotzky, G. and J-M. Bollag. 1996. Soil Biochemistry. Marcel Dekker. New York.
U.S. Environmental Protection Agency. 1996. A Citizen's Guide to Phytoremediation. Technology

Innovation Office. Washington, D.C. September.
U.S. Environmental Protection Agency. 1997b. Proceedings of the Symposium on Natural Attenuation
of Chlorinated Organics in Ground Water. EPA/540/R-97/504.
6-2
Wang, T.C. and C.K. Tan. 1990. “Reduction of halogenated hydrocarbons with magnesium hydrolysis
process.” Bulletin of Environmental Contamination and Toxicology. Volume 45. Pages 149-156.
Wilson, J.T., D.H. Kampbell, and J.W. Weaver. 1996. US EPA Symposium in Natural Attenuation of
Chlorinated Organics in Ground Water. Dallas, Texas. Sept 11-13. 1996. EPA/540/R-96/509/1996.
Pages 124-127.
Zhang, X. and others. Mathematical Models for Biodegradation of Chlorinated Solvents: I. Model
Framework.
6-3
APPENDIX A
CASE STUDIES AND SUMMARIES OF SITES USING IN SITU

BIOREMEDIATION TECHNOLOGIES FOR CAHs
Appendix A includes nine case studies prepared by EPA of sites which have used, or are in the process of
using, in situ bioremediation for treatment of CAHs in contaminated soil and groundwater. In addition,
summary information about other full-scale and pilot-scale applications of in situ bioremediation based
on information from the proceedings of the Fifth International and On-Site Bioremediation Symposium
and from the Bioremediation the Field Search System (BFSS) is presented.
The nine case studies, summarized in Exhibit A-1, contain information about site history and source of
contamination, geology/hydrogeology/contaminant characterization, technology description, technology
performance and cost, summary observations and lessons learned, contact information, and references
used in the preparation of the case studies. The case studies include three full-scale applications of
anaerobic reductive dechlorination (Texas Gulf Coast, Avco Lycoming, and Emeryville) and two field
demonstrations of anaerobic reductive dechlorination (Watertown and Dover Area 6). Four case studies
address field demonstrations of aerobic oxidation (Moffett Field, Edwards AFB, and SRS for groundwater
and Dover Building 719 for soil).
To provide additional information about full-and pilot-scale in situ bioremediation applications, summary
information was obtained from the proceedings of the Fifth International In Situ and On-Site
Bioremediation Symposium and from the BFSS. While not comprehensive, the summary information
presented in Exhibits A-2 and A-3 provides an overview of the types of sites using in situ bioremediation.
& Exhibit A-2 presents a summary of information about 8 full-scale applications and 14
pilot studies that are described in the proceedings of the Fifth International In Situ and
On-Site Bioremediation Symposium. This exhibit presents the site name and location,
technology, media, contaminants, period of performance, and points of contact for each
of the in situ bioremediation full-scale applications and pilot studies identified in the
proceedings. Most of the projects involved treatment of chlorinated solvents by either
anaerobic reductive dechlorination, aerobic oxidation, or a combination of those two
technologies.
& Exhibit A-3 provides summary information about remediation efforts at 8 sites that are
described in the BFSS, including site name and location, technology, media,
contaminants, project status, and points of contact for the applications.
A-1
Exhibit A-1: Summary of Case Studies of In Situ Bioremediation Technology Applications
Site Name, Technology Matrix Contaminants Period of

# Location Mechanism(s) Technology/Configuration Scale Treated Targeted1 Operation
1 NAS Moffett Field, Aerobic oxidation Electron acceptor (EA) addition (oxygen Field demon- Ground- TCE, 9/86-11/88
Mountain View, (cometabolic and and hydrogen peroxide) stration water cis-DCE, trans-
California direct) Electron donor (ED) addition (methane, DCE, VC
toluene, and phenol)
Groundwater recirculation
2 Edwards Air Force Aerobic oxidation EA addition (oxygen and hydrogen Field demon- Ground- TCE 2/5/96-4/1/97
Base (AFB), (cometabolic and peroxide) stration water
California direct) ED addition (toluene)
Groundwater recirculation
3 U.S. Department of Aerobic oxidation Nutrient addition Field demon- Sediment TCE, PCE 2/26/92-
Energy Savannah (cometabolic and EA addition (oxygen) stration and 4/30/93
River Site, Aiken, direct) ED addition (methane) Ground-
South Carolina* Direct injection water
4 Texas Gulf Coast Anaerobic reductive Nutrient addition Full Ground- TCE, cis-1,2-DCE, Ongoing (data
Site (site name dechlorination ED addition (methanol) water VC available from
confidential) (cometabolic and Groundwater recirculation 6/95 - 12/98)
Houston, Texas** direct)
5 Avco Lycoming Anaerobic reductive ED addition (molasses) Pilot and full Ground- TCE, DCE, VC, Pilot study
Superfund Site dechlorination Direct injection water Cr+6, Cd 10/95-3/96;
Williamsport, (cometabolic and Full scale
Pennsylvania direct) Ongoing (data
available from
1/97 - 10/97)
6 Abandoned Anaerobic reductive ED addition (molasses) Pilot and full Ground- TCE, Cr+6 Pilot study
Manufacturing dechlorination Direct injection water 8/95-2/96;
Facility Emeryville, (cometabolic and Full scale
California direct) Ongoing (data
available from
4/97 - 10/98)
A-2
Exhibit A-1: Summary of Case Studies of In Situ Bioremediation Technology Applications (continued)
Site Name, Technology Matrix Contaminants Period of

# Location Mechanism(s) Technology/Configuration Scale Treated Targeted1 Operation
7 Watertown, Anaerobic reductive Nutrient addition Field demon- Ground- PCE, TCE Anaerobic,
Massachusetts** dechlorination ED addition (lactate) stration (SITE water 11/96-7/97;
(cometabolic and Groundwater recirculation test) Aerobic,
direct) ongoing (data
available from
Aerobic oxidation EA addition (oxygen)
8/97 - 10/97)
(cometabolic and ED addition (propane)
direct) Groundwater recirculation
8 Dover AFB Area 6 Anaerobic reductive Bioaugmentation Field demon- Ground- TCE 9/96 - 3/98
Dover, Delaware dechlorination Nutrient addition stration (proof water
(cometabolic and ED addition (lactate) of technology
direct) Groundwater recirculation test)
9 Dover AFB Aerobic oxidation EA addition (oxygen) Field demon- Soil TCE, TCA, DCE 5/98 - 7/99
Building 719 (cometabolic and ED addition (propane) stration (pilot
Dover, Delaware direct) Direct injection test)
* Remediation efforts at this site also are included in Exhibit A-2 (Summary of Full-Scale Applications and Pilot Studies Identified in Proceedings of the Fifth International In
Situ and On-Site Bioremediation Symposium) and Exhibit A-3 (Summary of Full-scale Applications and Pilot Studies Identified in the Bioremediation in the Field Search
System [BFSS]).
** Remediation efforts at these sites also are included in Exhibit A-2 (Summary of Full-Scale Applications and Pilot Studies Identified in Proceedings of the Fifth International
In Situ and On-Site Bioremediation Symposium).
1
Contaminant Key: Cd = Cadmium, Cr = Chromium, DCE = dichloroethene, PCE = tetrachloroethene, TCA = trichloroethane, TCE = trichloroethene, VC = vinyl chloride
A-3
Exhibit A-2: Summary of Full-Scale Applications and Pilot Studies Identified in Proceedings of The Fifth International In Situ and
On-Site Bioremediation Symposium
Period of Page No. in

Site Name, Location Technology Media Contaminants1 Performance Points of Contact Proceedings
FULL-SCALE APPLICATIONS
Site name not provided Aerobic oxidation; Groundwater PCE, TCE, 1,2- June 1998 - Peter I. Dacyk 1
(commercial laundry) injection of propane DCE pilot test William D. Hughes
southwestern Ohio September 1998 Parsons Engineering Science, Inc.
- begin full 13 Triangle Park Drive, Suite 1302
scale; end date Cincinnati, Ohio 45246
not provided
Site name not provided Aerobic oxidation Groundwater VC March 1998 - Peter L. Tacy, Jr. 15
(aluminum coating facility) (oxygen release injection STS Consultants, Ltd.
location not provided compound [ORC] 1502 Randolph Street, Suite 100
(midwestern state) slurry injection) Detroit, Michigan 48226-2216
Site name not provided Anaerobic reductive Groundwater PCE Not provided John K. Sheldon 61
(dry cleaning site) dechlorination Montgomery Watson
Wisconsin (injection of 11153 Aurora Avenue
glycerol polylactate Des Moines, Iowa 50322-7238
hydrogen release Kenneth J. Quinn, Montgomery Watson,
compound [HRC]) Madison, Wisconsin
Stephen S. Koenigsberg and Craig A.
Sandefur, Regenesis, San Juan Capistrano,
California
Site name not provided Aerobic oxidation Groundwater PCE, TCE March 1998 - Mark S. Nelson, Williams Gas Pipeline- 113
(natural gas pipeline (Methanotrophic ongoing (end Transco, Houston, Texas
compressor station) Treatment date not Robert Legrand and Andrew J. Morecraft
Virginia Technology [MTT], provided) Radian International
injection of 8501 N. Mopac Blvd
methane, air, P.O. Box 201088
nitrous oxide, and Austin, Texas 78720-1088
triethylphosphate) (and Raleigh-Durham, North Carolina)
John A. Harju, Gas Research Institute,
Chicago, Illinois
A-4
Exhibit A-2: Summary of Full-Scale Applications and Pilot Studies Identified in Proceedings from The Fifth International In Situ and
On-Site Bioremediation Symposium (continued)

FULL-SCALE APPLICATIONS (continued)
Site name not provided Anaerobic reductive Groundwater PCE, TCE, DCE, February 1998 - Maureen A. Dooley and Willard A. Murray, 121 **
Watertown, Massachusetts dechlorination VC January 1999 Harding Lawson Associates
(addition of HRC to 107 Audubon Road, Suite 300
a recirculation cell) Wakefield, Massachusetts 01880
Stephen Koenigsberg, Regensis, San Juan
Capistrano, California
Site name not provided Anaerobic reductive Groundwater TCE June 1995 - Susan Tighe Litherland, P.E. and David W. 157 **
(manufacturing facility) dechlorination December 1998 Anderson
Texas Gulf Coast, Texas (addition of Roy F. Weston, Inc.
methanol to Building 1, Suite 100
recirculation 5300 Bee Caves Road
system) Austin, Texas 78746-5225
Blake A. Dinwiddie, Roy F. Weston, Inc.,
Houston, Texas
Site name not provided Aerobic oxidation Groundwater TCE May 1997 - end Jian Xing and Richard M. Raetz 217
Lansing, Michigan (cometabolic) date not Global Remediation Technologies, Inc.
(phenol addition provided (data 1235 Woodmere
with pressurized available for 15 Traverse City, MI 49686
fluidized bed months of
reactor); operation)
Groundwater
recirculation;
Biosparging
Evenblij site, Hoogeveen, Aerobic oxidation Groundwater PCE, TCE Not provided M.J.C. Henssen 225
The Netherlands (acetate and lactate Bioclear Environmental Biotechnology
addition); Groningen, The Netherlands
Anaerobic reductive C. Hubach and R. Blokzijl
dechlorination; DHV Environmental & Infrastructure
Groundwater Europaweg 33/2
recirculation 9723 AS Groningen, The Netherlands
J. Mourik, Logisticon Water Treatment,
Groot-Ammers, The Netherlands
E. Meijerink, Province of Drenthe, Assen,
The Netherlands
A-5

PILOT STUDIES
Hill Air Force Base, Bioventing Soil 1,2- One year (dates James T. Gibbs, Battelle 7
Operable Unit 1, Chemical Dichlorobenzene not provided) 505 King Avenue
Disposal Pit 1 Columbus, Ohio 43201
Ogden Utah R. Kenneth Crowe, TRW Inc., Tyndall AFB,
Florida
Jon Ginn, USAF, Hill AFB, Utah
Site name not provided Aerobic oxidation; Groundwater cis-1,2-DCE, VC July 1998 - Liselotte Ludvigsen 81
Copenhagen, Denmark injection of ongoing; end HOH Water Technology, Greve, Denmark
methane and air date not Kim Broholm, VKI
provided Agern Alle 11
DK-2970 Horsholm, Denmark
Lars Deigaard, Scanrail Consult,
Copenhagen, Denmark
U.S. Department of Energy Aerobic oxidation; Groundwater TCE, cis-1,2- September 1996 Robin L. Brigmon 107 **
Savannah River Site, injection of DCE, VC Westinghouse Savannah River Company
Sanitary Landfill methane, air, Savannah River Technology Center
Aiken, South Carolina triethyl phosphate, P.O. Box 616
and nitrous oxide Aiken, South Carolina 29808
Terry C. Hazen, Lawrence Berkeley National
Laboratory, Berkeley, California
Al W. Bourquin, Camp Dresser and Mckee
Inc., Denver, Colorado
Site name not provided Anaerobic reductive Groundwater PCE, TCE, cis- April 1997 - James J. Reid, P.E. and Denis Balcer 135
(Superfund site) dechlorination 1,2-DCE, VC August 1998 ARCADIS Geraghty & Miller
Location not provided 4700 Lakehurst Court, Suite 100
Dublin, Ohio 43016
A-6

PILOT STUDIES (continued)
Site name not provided Anaerobic reductive Soil PCE, TCE, cis- 80 days (dates H. Slenders 141
(industrial cleaning dechlorination 1,2-DCE, VC not provided) TNO Institute of Environmental Sciences,
company) Energy Research and Process Innovation
Arnhem, The Netherlands Business Park E.T.V.
Laan van Westenenk 501
P.O. Box 342
7300 AH Apeldoorn, The Netherlands
S. Hofstra, IWACO, Environmental
Consultants, The Netherlands
H.de Sain, BdS, Management Consultancy,
The Netherlands
R. Hetterschijt, Netherlands Institute of
Applied Geoscience TNO
H. de Kreuk, BioSoil R&D
Idaho National Engineering Anaerobic reductive Groundwater TCE November 1998 Kent S. Sorenson, Jr. and Lance N. Peterson 147
and Environmental dechlorination - ongoing; end Lockheed Martin Idaho
Laboratory, Test Area date not Idaho Falls, Idaho
North provided Roger L. Ely, University of Idaho, Moscow,
Idaho Idaho
Naval Air Station Point Anaerobic reductive Groundwater Chlorinated Not provided Christian D. Johnson 165
Mugu, IRP Site 24, dechlorination ethenes; volatile Battelle PNWD
California (Phase 1 – lactate organic acids P.O. Box 999
addition; Phase 2 – Richland, Washington 99352
nutrient injection); Daniel P. Leigh and Lisa A. Bienkowski, IT
groundwater Group, Pleasanton, California
recirculation Steve Granade, U.S. Navy, Naval
Construction Battalion Center, Port
Hueneme, California
Bryan Harre, U.S. Navy, Port Hueneme,
California
Todd Margrave, U.S. Navy, San Diego,
California
A-7

Site name not provided Anaerobic reductive Groundwater PCE, TCE, 1,1,1- July 1997 – I. Richard Schaffner, Jr., GZA 171
(former wastewater dechlorination TCA, 1,1-DCE, November 1998 GeoEnvironmental, Inc.
treatment facility) (yeast extract cis/trans-1,2- 380 Harvey Road
Location not provided injection) DCE, 1,1-DCA, Manchester, New Hampshire 03103
1,2-DCA, VC
Site name not provided Anaerobic reductive Groundwater TCE, cis-1,2- March 1998 – Madeline Wu 177
Florida dechlorination DCE, VC June 1998 Water Restoration Inc.
(addition of HRC) Fort Lauderdale, Florida
Site name not provided Anaerobic reductive Groundwater PCE, TCE, cis- April 1998 – S. Kallur, Environmental Strategies & 181
New Jersey dechlorination 1,2-DCE June 1998 Applications, Inc.
(addition of HRC) Somerset, New Jersey
S. Koenigsberg, Regenesis
1011 Calle Sombra
San Clemente, CA 92672
Offutt Air Force Base, Fire Anaerobic reductive Groundwater cis-1,2-DCE, November 1998 R. Todd Fisher and Charles J. Newell , 185
Protection Training Area 3 dechlorination BTEX Groundwater Services, Inc., Houston, Texas
Nebraska (direct addition of Patrick E. Haas, Air Force Center for
hydrogen); Environmental Excellence, Brooks AFB,
groundwater Texas
recirculation Joseph B. Hughes, Rice University,
Houston, Texas
Site name not provided Chemical oxidation, Groundwater TCE Not provided Christopher H. Nelson, IT Corporation, 199
(industrial site) with addition of Englewood, Colorado
Adelaide, South Australia permanganate Craig S. Barker, IT Environmental
(Australia) Pty Ltd, Adelaide, South
Australia
A-8

ITT Industries Night Aerobic oxidation Groundwater TCE, TCA, Not provided Gregory L. Carter, Earth Tech 255
Vision (injection of acetone, 5320 Peters Creek Road, Suite D
Roanoke, Virginia methane, air, isopropanol Roanoke, Virginia 24019
nitrous oxide, and Jennifer C. Vincent and Barbara B. Lemos,
triethylphosphate) Earth Tech, Concord, Massachusetts
Rosann Kryczkowski, ITT Industries Night
Vision, Roanoke, Virginia
Rickenbacker Air National LasagnaTM Soil TCE January 1997 - Wendy J. Davis-Hoover et. al., EPA 263
Guard Base (combination of November 1998 CHL, EPA Facilities
Ohio aerobic 26 W. Martin Luther King Drive
cometabolic- Cincinnati, Ohio 45268
bioremediation Taras Bryndzia, Shell Oil, Houston, Texas
[with addition of Michael H. Roulier and Lawrence C.
methane], Murdoch, Clemson University, Clemson,
electroosmosis, and South Carolina
hydraulic Mark Kemper and Philip Cluxton, Cluxton
fracturing) Instruments, Inc., Martinsville, Ohio
Souhail Al-Abed and William Slack, FRX,
Inc., Cincinnati, Ohio
* These full-scale applications and pilot studies are described further in the following document: Andrea Leeson, A. and B. C. Alleman. Engineered Approaches for In
Situ Bioremediation of Chlorinated Solvent Contamination 5(2): The Fifth International In Situ and On-Site Bioremediation Symposium; San Diego, California, April
19-22, 1999; Battelle Press; 1999. Page numbers shown in Exhibit A-2 refer to the pages of the Battelle document in which the information can be found.
** These full-scale applications and pilot studies are also included in Exhibit A-1: Summary of Case Studies of In Situ Bioremediation Technology Applications.
*** This pilot study is also included in Exhibit A-3: Summary of Full-Scale Applications and Pilot Studies Identified in the Bioremediation in the Field Search System
(BFSS)
1
Contaminant Key: DCA = dichloroethane, DCE = dichloroethene, PCE = tetrachloroethene, TCA = trichloroethane, TCE = trichloroethene, VC = vinyl chloride
A-9
Exhibit A-3: Summary of Full-Scale Applications and Pilot Studies Identified in the Bioremediation in the Field Search System (BFSS)
Site Name,
Location Technology Media Contaminants1 Project Status Points of Contact
A. B. Dick Inland Environmental Bio- Soil TCE, cis-1,2-DCE, TCA Full-scale operations were Gregory C. Weeks
Niles, Illinois Treatment Process. An anaerobic, completed on 8/23/95. The Inland Environmental, Inc.
in-situ soil bioremediation remediation was completed 3921 Howard St.
technology. A proprietary liquid ahead of schedule (three months Skokie, IL 60076
blend (patent applied for) of versus one year) and within the Inland@Inland-Env.com
nutrients, stimulants, and mobilizing budget. All treatment goals (847) 677-7500
agents is injected into the soil and were met and the site is now
stimulates the natural anaerobic considered to be remediated.
bio-degradation of contaminants to
mineralization
Barber In situ, aerobic process. Oxygen is Soil (vadose), PCE, 1,1-DCA, 1,1,1-TCA, Full-scale operations were Eric Portz
Greene introduced into the groundwater groundwater TCE completed on 7/18/97. Illinois EPA
North through air sparging. Vacuum 1021 N. Grand Ave. East
Aurora, extraction provides air movement Springfield, IL 62702
Illinois through the soil to enhance epa4204@epa.state.il.us
groundwater bioremediation (217) 782-6761
(bioventing for vadose soil).
Chevron Aerobic in situ groundwater and soil Soil (vadose), soil The system is designed to The full-scale system began Robert Huntoon
Chemical bioremediation process. Treatment (saturated), treat 1,1,1-TCA, 1,2-DCE, operating on 1/1/95. Chevron Chemical
Company - process involves pumping groundwater and TCE. Other Mechanical problems caused by Company
Berkeley groundwater, adding nutrients contaminants include PCBs microorganism growth occurred. P.O. Box 6012
Heights, New (nitrogen and phosphorus) and and heavy metals. San Ramon, CA 94583
Jersey amendments (hydrogen peroxide RBHU@
and oxygen) above ground, and CHEVRON.COM
reinjecting the groundwater into the (925) 842-5576
soil. The reinjected water will help
to remediate the contaminated soils. Glenn Savary
Groundwater that meets cleanup New Jersey Department of
criteria will be sent to a POTW. Environmental Protection
and Energy
(609) 633-1408
A-10
(continued)
Site Name,
NAS Fallon Aerobic bioslurping process. A Soil (vadose), soil System is designed to treat The full-scale system is Ron Hoeppel
Fallon, vacuum is pulled in dewatering (saturated), DCE, PCE, operational. Naval Facilities
Nevada wells to promote the rapid aeration groundwater trichloroethylene, and Engineering Service Center
of subsurface vadose zone soils by cis-1,2-DCE. Other (805) 982-1655
movement of air into soil pores. contaminants include RHOEPPE@
Since the vacuum (slurper) tubes are perchloroethene and NFESC.NAVY.MIL
situated at the interface of the free trichloroethene (in vadose
fuel and the groundwater, free fuel zone) and arsenic and David Chesmore
is removed (vacuum assisted), while borates (natural State of Nevada Federal
simultaneously promoting aerobic contaminants). Facilities Coordinator
biodegradation of fuel in the vadose (702) 687-5872
zone through bioventing.
Doug Bonham
NAS Fallon Public Works
Department
4755 Pasture Rd.
Fallon, NV 89496-5000
(702) 426-2772
Naval Aerobic, in situ groundwater and Groundwater The system is designed to The full-scale system has been John Garner
Submarine soil bioremediation process. treat 1,1-DCA, 1,2-DCA, completed, and no problems Naval Submarine Base
Base Bacteria, nutrients, and water were cis-1,2-DCE, PCE, have been reported. One of the Kings Bay
Kings Bay, injected periodically into the trans-1,2-DCE, and TCE. sites (“generator” site) is Kings Bay, GA 31547
Georgia groundwater. Three sites were BTEX is also present in the operational. jgarner@
contaminated with petroleum groundwater. subase.kb.navy.mil
hydrocarbons (heating fuel, diesel (912) 673-2001
fuel, and lubricating oil).
J.A. Jones
(912) 673-2001
Garland Creech
Naval Submarine Base
Kings Bay
(912) 673-2001
A-11
(continued)
Site Name,
Pine Bend In situ groundwater bioremediation Groundwater The system is designed to The full-scale system is in the Joe Julik
Landfill process. A pilot-scale system that treat TCA, DCA, DCE, predesign phase. Treatment Minnesota Pollution
Inner Grove consists of three injection wells and perchloroethylene. currently is in the feasibility Control Agency
Heights, currently is being installed at the study stage. The pilot study 520 Lafayette Rd.
Minnesota site. The wells will deliver fructose evaluation is expected to St. Paul, MN 55155-4194
corn syrup to the groundwater to continue for 18 months. joe.julik@pca.state.mn.us
induce anaerobic conditions and (612) 296-8454
stimulate the anaerobic groundwater
microbial consortia to effectively Neil Wilson
dehalogenate highly chlorinated Minnesota Pollution
contaminants in groundwater. Control Agency
Groundwater that retains (612) 296-8596
contaminants may be treated
aerobically downgradient through
installation of a sparging curtain.
A-12
(continued)
Site Name,
Savannah Full-Scale System Groundwater The system treats PCE and Full-scale operations were Ed Wilde
River Site Biosparging, in situ soil flushing, TCE. Copper is also completed. No date provided. Westinghouse Savannah
Aiken, South vacuum extraction. Aerobic present in the groundwater. River Company
Carolina ** process. Indigenous microbes are Pilot-scale operations were Savanna River Technology
encouraged to degrade contaminants completed on 4/1/93. The full- Center
by injecting air in the aquifer. Air is scale system design was P.O. Box 616
injected via horizontal wells in the completed on 9/30/96. Aiken, SC 29808
aquifer. Air is vacuum-extracted ed.wilde@srs.gov
simultaneously through a parallel (803) 557-7049
horizontal well just above the water
table. Off-gas is treated by catalytic James A. Wright
oxidation. DOE
(803) 725-5608
Pilot-Scale System
Biosparging, in situ methane, in situ
soil flushing, vacuum extraction.
Aerobic process. Nutrients are
methane, nitrous oxide, gas (triethyl
phosphate). Methane in air (1 to 4
percent) is injected through
horizontal wells in the aquifer. Air
is extracted simultaneously through
a parallel horizontal well just above
the water table. Off-gas is treated
by catalytic oxidation. Methane is
added to stimulate indigenous
methanotrophs (bacteria) to
biodegrade chlorinated solvents
through cooxidation.
A-13
(continued)
Site Name,
Technical Inland Environmental Bio- Soil (vadose) The system is designed to Full-scale operations were Gregory C. Weeks
Products, Treatment Process. In situ, treat TCE, 1,2-DCE, and completed on 12/30/96. Inland Environmental, Inc.
Inc. anaerobic process. The site was 1,2-DCA. Numerous other 3921 Howard St.
Chicago, excavated to remove tanks and the contaminants are also Skokie, IL 60076
Illinois soil was replaced in deep lifts. present. The concentration Inland@Inland-Env.com
Inland Environmental's proprietary of all of the unidentified (847) 677-7500
bio-stimulating solution (nutrients, compounds decreased
biostimulants, and surfactants) was along with the
sprayed onto each lift. concentrations of the
regulated compounds.
* BFSS can be downloaded from the following Web site: <http://www.clu-in.org/PRODUCTS/MOREINFO/Bfss.htm>. BFSS is also available on diskette from EPA’s
Center for Environmental Research Information (CERI) at (513) 569-7562. The BFSS was queried in January 2000 to obtain pertinent data.
** Remediation efforts at this site also are included in Exhibit A-1 (Summary of Case Studies of In Situ Bioremediation Technology Applications) and Exhibit A-2
(Summary of Full-Scale Applications and Pilot Studies Identified in Proceedings of the Fifth International In Situ and On-Site Bioremediation Symposium).
1
Contaminant Key: BTEX = benzene, toluene, ethylbenzene, and xylenes; DCA = dichloroethane; DCE = dichloroethene; PCB = polychlorinated biphenyl;
PCE = tetrachloroethene; TCA = trichloroethane; TCE = trichloroethene
A-14
Case Study # 1
Aerobic Degradation Field Demonstration
at Moffett Naval Air Station, Mountain View, California
Summary Information [1,4,12]
Site Name, Location Moffett Naval Air Station, Mountain View, CA
EPA ID Number CA2170090078
Mechanism(s) Aerobic Oxidation (Cometabolic and Direct)
Technology Electron Acceptor Addition (Oxygen and

Hydrogen Peroxide)
Electron Donor Addition (Methane, Toluene, and
Phenol)
Configuration Groundwater Recirculation
Technology Scale Field Demonstration
Media/Matrix Treated Groundwater
Contaminants Targeted TCE, cis-DCE, trans-DCE, VC
Period of Operation September 1986 to November 1988 (methane

addition studies)
Site History/Source of Contamination [1,4,6,7]
Moffett Naval Air Station (Moffett), used for aircraft operations and maintenance, operated from 1933 to
1994. In 1994, the Navy ceased operations and the airfield was transferred to the National Aeronautics
and Space Administration (NASA). Moffett is located 35 miles south of San Francisco in Santa Clara
County. Soil and groundwater at the site are contaminated with petroleum products and chlorinated
aromatic hydrocarbons (CAHs) such as tetrachloroethene (PCE) and trichloroethene (TCE). Moffett is
adjacent to other Superfund sites in the Middlefield-Ellis-Whisman (MEW) study area, and a large
groundwater plume crosses Moffett from off-site sources. This site was added to the National Priorities
List (NPL) on July 22, 1987 and is being addressed through Federal actions. Several Records of
Decision (RODs) have been signed for this facility, including RODs for OU 1 (Sites 1 and 2 Landfills),
dated August 1997; OU 2 (East Side Soils), dated December 1994; and OU 5 (East Side Aquifers), dated
June 1996. In addition, for the West Side Aquifers, the Navy adopted an adjacent site’s ROD, dated
1989.
Moffett was selected by researchers from Stanford University for a field demonstration of in situ aerobic
degradation to treat groundwater contaminated with CAHs. A series of experiments was conducted
between September 1986 and November 1998 to evaluate native bacteria enhanced through addition of
methane, toluene, and phenol in degrading CAHs, including PCE and TCE.
A-15
Geology/Hydrogeology/Contaminant Characterization [3,5,11,12]
As shown in Figure 1, the demonstration site (test zone) was approximately 4 to 6 meters (m) below
ground surface (bgs), located in a shallow, confined aquifer (1.5 m thick)consisting of sands and gravels.
The groundwater velocity ranged from 1.5 to 3 m/day and the hydraulic conductivity of the aquifer was
0.11 cm/sec. In addition, indigenous methanotrophic bacteria were reported to be present in the aquifer.
Figure 1. Cross-section of Well Field Used at Moffett [4,5,12]
The CAHs present in the test zone prior to the demonstration included 1,1,1-trichloroethane (TCA) and
1,1-dichloroethane (DCA). However, TCE, cis-dichloroethene (cis-DCE), trans-dichloroethene (trans-
DCE), and vinyl chloride (VC)were not detected in the groundwater in the test zone. As described
below, regulatory approval was obtained to inject TCE, cis- and trans-DCE, and VC into the groundwater
for the demonstration.
Matrix Characteristic Value [11,12]

Soil Type sand and gravel
Depth to Groundwater 4 to 6 m bgs
Thickness of Aquifer(s) 1.5 m
Fraction of Organic Carbon 0.00112 ± 0.00020
Hydraulic Conductivity 0.11 cm/sec
pH 6.5
DNAPL Present None identified
Nitrogen 30 to 60 milligrams per liter (mg/L) (as nitrate)
Phosphorus <0.1 mg/L (as phosphate)
Groundwater Velocity 1.5 to 3 m/day
A-16
Technology Description [4,5,7,12]
The demonstration of aerobic degradation was performed under induced-gradient conditions created by
the extraction and injection of groundwater. As shown in Figure 1, groundwater was extracted at well P,
amended chemically, and injected at wells SI and NI, located 6 m from extraction well P (information
about the construction and operation of the wells was not provided). Regulatory approval was obtained
for injecting TCE, cis- and trans-DCE, and VC into the groundwater.
Table 1 presents a summary of the nine experiments that were conducted over three seasons of the
demonstration, including the period of operation, groundwater extraction and injection rates, chemical
amendments, and processes studied. The experiments included biostimulation (Biostim) to stimulate the
activity of native methane-using bacteria, and biotransformation (Biotran) to transform TCE into lower
chlorinated compounds. Tracer experiments, using bromide, were performed to evaluate organic
transport and “Decmeth” experiments were performed to evaluate methane addition.
Concentrations of CAHs, methane, DO, and bromide were monitored using the wells shown in Figure 1.
An automated data acquisition and control system was used to provide as many as six sets of analyses per
day at each of the sampling locations.
Additional experiments performed at the site included using phenol and toluene (alternative electron
donors) as substrates in place of methane, and using hydrogen peroxide as an alternative to oxygen.
Table 1. Summary of Experimental Conditions Used at Moffett [4]
Extraction
(E) and
Injection (I) Chemical Amendments
Experiment Rates Duration (average mg/L) Processes Studied
First Season
Biostim 1 E: 8 L/min 9/5/86 - Methane: 5.9 Biostimulation of native methane-using

I: 1 L/min 9/30/86 DO: 20.8 bacteria. Alternating pulse injection of
Bromide: 166 methane and DO.
Biotran 1 E: 8 L/min 9/30/86 - Methane: 5.7 Biotransformation of TCE with active

I: 1 L/min 10/21/86 DO: 22.2 biostimulation. Nonsteady-state
TCE: 0.097 conditions.
Biotran 4 E: 8 L/min 12/10/86 - Methane: 5.2 Biotransformation of TCE with active

I: 1 L/min 12/31/86 DO: 23 biostimulation. Steady-state conditions.
Bromide: 159
TCE: 0.051
Second Season
Tracer 8 E: 10 L/min 7/6/87 - DO: 14.3 Transport and breakthrough of bromide,

I: 1.5 L/min 8/15/87 Bromide: 78 TCE, cis- and trans-DCE without
TCE: 0.048 biostimulation.
cis-DCE: 0.110
trans-DCE: 0.112
A-17
Table 1. Summary of Experimental Conditions Used at Moffett [4] (continued)
Extraction
(E) and
Injection (I) Chemical Amendments
Experiment Rates Duration (average mg/L) Processes Studied
Biostim 2 E: 10 L/min 8/17/87 - Methane: 5.3 Simultaneous biostimulation and

I: 1.5 L/min 10/26/87 DO: 23.4 biotransformation of TCE, cis- and trans-
Bromide: 44 DCE.
TCE: 0.036
cis-DCE: 0.091
trans-DCE: 0.092
Decmeth 1 E: 10 L/min 10/27/87 - DO: 24.5 Test if active biotransformation occurs

I: 1.5 L/min 11/8/87 TCE: 0.045 without addition of methane.
cis-DCE: 0.136
trans-DCE: 0.095
Third Season
Tracer 11 E: 10 L/min 8/10/88 - Bromide: 72 Transport and breakthrough of bromide,

I: 1.5 L/min 10/10/88 TCE: 0.047 TCE, cis- and trans-DCE without
cis-DCE: 0.085 biostimulation.
trans-DCE: 0.050
Tracer 12 E: 10 L/min 10/10/88 - Bromide: 44 Transport and breakthrough of bromide

I: 1.5 L/min 10/20/88 TCE: 0.042 and VC while continuing injection of
cis-DCE: 0.100 TCE, cis- and trans-DCE.
trans-DCE: 0.054
VC: 0.044
Biostim 3 E: 10 L/min 10/20/88 - Methane: 6.6 Simultaneous biostimulation and

I: 1.5 L/min 11/23/88 DO: 21.3 biotransformation of TCE, cis- and trans-
Bromide: 45 DCE, and VC.
TCE: 0.046
cis-DCE: 0.100
trans-DCE: 0.052
Technology Performance [2,3,4,7,12]
The objective of the field demonstration was to collect data to be used in evaluating aerobic degradation
of CAHs under several different experimental scenarios. Specific remedial goals were not established
for this demonstration.
Several methods were used to evaluate the amount of CAHs that were biodegraded in these experiments,
including mass balances on the amounts of CAH injected and extracted, and comparison of breakthrough
concentrations using controlled experiments and bromide tracers. Results showed that active use of
methane in the treatment zone was required for biodegradation of CAHs, and that groundwater residence
times in the treatment zone of 1-2 days resulted in biodegradation of TCE at 20 - 30%, cis-DCE at 45 -
55%, trans-DCE at 80 - 90%, and VC at 90- 95%. The results indicated a similar degree of
biodegradation of TCE over the three seasons of field testing, suggesting that there was no apparent
increase in the ability of the bacteria to degrade TCE. In addition, results showed that an intermediate
biotransformation product, trans-DCE oxide, was produced in a manner consistent with the expected
transformation pathway for trans-DCE. Detailed analytical results for each of the nine experiments are
provided in reference 4 for this case study.
A-18
Table 2 summarizes the results from the third season of the methane addition experiments, and the
experiments with phenol and toluene as primary substrates. As shown in the table, the use of phenol and
toluene achieved higher percent removals of TCE (93 - 94%) compared with use of methane (19%).
Table 2. Summary of Results (% Removal) Using Different Substrates at Moffett [3]
% Removal by Constituent
Substrate
Primary Concentration Vinyl
Substrate (mg/L) TCE 1,1-DCE cis-DCE trans-DCE Chloride
Methane 6.6 19 NE 43 90 95
(third
season)
Phenol 12.5 94 54 92 73 >98
Toluene 9 93 NE >98 75 NE
Note:
NE - not evaluated
Hydrogen peroxide was found to achieve TCE removals similar to those achieved using oxygen. While
1,1-DCE was partially transformed in the study with phenol, the transformation products were found to
be toxic to the transforming bacteria.
Additional information and discussion about the experiments conducted using phenol and toluene are
provided in references 8 - 10 for this case study.
No exceptions to established quality assurance/quality control (QA/QC) protocols were noted in the
available information.
Technology Cost
No information was provided about the cost for the in situ bioremediation treatment system used at
Moffett.
Summary Observations and Lessons Learned [3,4,7-12]
The results of the field demonstration at Moffett showed that native bacteria enhanced with methane,
phenol, or toluene, plus oxygen or hydrogen peroxide was effective in degrading CAHs in groundwater.
Concentrations of CAHs were reduced by as much as 94% for TCE, 92% for cis-DCE, and 98% for VC.
Native bacteria enhanced with phenol and toluene achieved higher removal rates for TCE than bacteria
enhanced with methane. The results from the field experiments were consistent with the results from
batch soil column laboratory testing using aquifer solids from the test zones.
The presence of 1,1-DCE in the groundwater was found to be toxic to the bacteria, and should be
considered when evaluating this technology for use in other applications. However, the relatively low
concentration of phosphate in the groundwater did not limit the biodegradation of CAHs at this site.
According to the researchers, other phosphate minerals may have dissolved in the groundwater to
replenish this mineral as it was being removed by the bacteria.
A-19
During the field demonstration, the use of alternating pulsed addition of methane and oxygen minimized
biofouling in the area near the injection well.
Contact Information
EPA RPM:
Roberta Blank
U.S. EPA Region 9
75 Hawthorne Street, SFD-8-1
San Francisco, CA 94105
(415) 744-2384
e-mail: blank.roberta@epa.gov
Principal Investigator:
Dr. Lewis Semprini
Oregon State University
Department of Civil, Construction, and Environmental Engineering
202 Apperson Hall
Corvallis, OR 97331-2302
(541) 737-6895
fax: (541) 737-3099
e-mail: Lewis.Semprini@orst.edu
References
1. U.S. EPA. 1998. Fact Sheet. Moffett Naval Air Station, California, http://epainotes1.rtpnc.epa.gov:
7777/r9/nplpad.nsf/?opendatabase&start=59. December 15.
2. Lang, Margaret M., Paul V. Roberts, and Lewis Semprini. 1997. Groundwater Model Simulations in
Support of Field Scale Design and Operation of Bioremediation Based on Cometabolic Degradation.
July-August.
3. U.S. Air Force, Center for Environmental Excellence, Environmental Restoration Division. 1998.
Installation Restoration Program: Aerobic Cometabolic In Situ Bioremediation Technology
Guidance Manual and Screening Software User's Guide. Prepared for Air Force Research
Laboratory, Airbase and Environmental Technology Division, Tyndall AFB, Florida.
<http://en.afit.af.mil/env/insitubio.html>.
4. Semprini, Lewis, Paul V. Roberts, Gary D. Hopkins, and Perry L. McCarty. 1990. Groundwater,
Volume 28, Number 5. A Field Evaluation of In Situ Biodegradation of Chlorinated Ethenes: Part 2,
Results of Biostimulation and Biotransformation Experiments. September-October.
5. Roberts, Paul V., Gary D. Hopkins, Douglas M. Mackay, and Lewis Semprini. 1990. Groundwater,
Volume 28, Number 5. A Field Evaluation of In Situ Biodegradation of Chlorinated Ethenes: Part 1,
Methodology and Field Site Characterization. July-August.
6. Gill, Michael, U.S. EPA Region 9. 1999. Comments on Draft Case Study Report. E-mail Message
to Dawn Carroll, EPA\TIO. January 25.
A-20
7. Semprini, Lewis. Comments on Draft Case Study Report. E-mail Message to Dawn Carroll,
EPA\TIO. January 29.
8. Hopkins, D.G., L. Semprini, and P.L. McCarty. 1993a. Microcosm and In-situ Field Studies of
Enhanced Biotransformation of Trichloroethylene by Phenol-Utilizing Microorganisms. Applied
Environmental Microbiology. 59:2277-2285.
9. Hopkins, G.D. and P.L. McCarty. 1995. Field Observations of In situ Aerobic Cometabolism of
Trichloroethylene and Three Dichloroethylene Isomers Using Phenol and Toluene as Primary
Substrates. Environmental Science and Technology. 29:1628-1637.
10. Hopkins, G.D., J. Munakata, L. Semprini, and P.L. McCarty. 1993. Trichloroethylene
Concentration Effects on Pilot Field-Scale In-situ Groundwater Bioremediation by Phenol-Oxidizing
Microoganisms. Environmental Science and Technology. Vol. 27, no. 12. 2542-2547.
11. Roberts, P.V., L. Semprini, G.D. Hopkins, D. Grbic-Galic, P.L. McCarty, and M. Reinhard. 1989.
In-Situ Aquifer Restoration of Chlorinated Aliphatics by Methanatrophic Bacteria. EPA/600/2-
89/033. July.
12. P.L. McCarty. 1999. E-mailed comments on Case Study #1. September 30.
A-21
Case Study # 2
Aerobic Degradation Field Demonstration
at Site 19, Edwards Air Force Base, California
Site Name, Location Edwards Air Force Base, CA
EPA ID Number CA1570024504
Technology Electron Acceptor Addition (Oxygen and

Hydrogen Peroxide)
Electron Donor Addition (Toluene)
Contaminants Targeted TCE
Period of Operation February 5, 1996 to April 1, 1997
Site History/Source of Contamination [3,7]
Edwards Air Force Base (AFB), located on the western portion of the Mojave Desert, about 60 miles
north of Los Angeles, covers approximately 301,000 and is used for aircraft research and development.
From 1958 through 1967, engines for the X-15 rocket airplane were maintained in facilities at the site,
and trichloroethene (TCE) was used to clean the engines. The used TCE was disposed of at Site 19, an
area of about 53 acres on the west side of Rogers Dry Lake, resulting in groundwater contamination. The
contaminant plume extends approximately 3,200 ft down-gradient from the contamination source, and
nearly the same distance cross-gradient. This site was added to the National Priorities List (NPL) on
August 30, 1990, and is being addressed through Federal actions. A Record of Decision (ROD) had not
been signed for this facility at the time of this report.
A field demonstration of aerobic biodegradation was performed at Site 19. The area of the plume used
for this field demonstration was about 400 meters (m) east of the contamination source.
Geology/Hydrogeology/Contaminant Characterization [3,8,9,10]
The Site 19 demonstration area contains two relatively homogeneous aquifers. The upper, unconfined
aquifer is 8 m thick, and is separated by a 2 m aquitard from the lower confined aquifer. The lower,
confined aquifer is approximately 5 m thick and lies above weathered bedrock. At the demonstration
site, the concentration of TCE in the groundwater plume varied between 500 and 1,200 micrograms per
liter (µg/L), with average TCE concentrations in the upper and lower aquifer of 680 and 750 µg/L,
respectively. No 1,1-DCE was found at the site prior to the demonstration.
A-22
Matrix Characteristic Value

Soil Type fine to medium sized sand mixed with some silt
Depth to Groundwater 9 m below ground surface (bgs)
Thickness of Aquifer(s) 15 m (total)
Fraction of Organic Carbon 0.0001 to 0.0004
Hydraulic Conductivity 1.5 to 5.5 x 10-3 cm/sec to east/southeast
(average 3.4 x 10-3 cm/sec)
Groundwater Velocity 6.9 cm/day
Technology Description [3,4,6,8]
The in situ bioremediation treatment system used at this site, shown in Figure 1, was designed based on
the results from the demonstration at Moffett (case study No. 1), and consisted of two 8-in diameter,
PVC treatment wells installed approximately 24 m deep and spaced 10 m apart. Each treatment well was
screened in both the upper and lower aquifers (15 m and 10 m, respectively), and a submersible pump,
placed in each well, was used to draw contaminated water into the well through one of the screens. The
initial flow rate for the wells was 38 liters per minute (L/min) to limit drawdown in the upper aquifer and
pressure changes in the lower aquifer. The primary substrate (toluene) and oxygen were introduced into
the wells via feed lines and mixed with the water using static mixers inside the wells. The groundwater,
containing TCE, toluene, and oxygen, was discharged from the second screen into the aquifer, where a
treatment zone developed around the well. Treatment well 1 (T1) withdrew water from the upper aquifer
and discharged it into the lower aquifer, while treatment well 2 (T2) withdrew water from the lower
aquifer and discharged it into the upper aquifer. This process recirculated the water between the two
aquifers creating a bioreactive treatment cell.
Treatment system operation included groundwater pumping, pulsed addition of toluene, and addition of
dissolved oxygen (DO, as gaseous oxygen) and hydrogen peroxide (H2O2). The system was operated for
444 days. The demonstration included five phases, during which time the operating parameters were
varied as follows:
(1) pre-operational studies (days 0 - 33)

(2) establishment of a toluene-degrading consortium (days 34 - 55)
(3) pre-steady-state operation (days 56 - 136)
(4) steady-state operation (days 142 - 271)
(5) balanced flow operation (days 317 - 444)
Operating parameters for steady-state and balanced flow periods, which correspond to results provided
below, are shown in Tables 1 and 2.
A-23
Table 1. Operating Parameters for Steady-State Operation (Days 142 - 271) [3]
Groundwater Toluene Toluene H2O2

Treatment Pumping Rate Addition Addition - DO Addition Addition
Well (L/min) (mg/L) Pulses per Day (mg/L) (mg/L)
T1 25 0 - 11.6 0.67 - 1 44 17 - 117
T2 38 13.4 1 29 47 - 63
Table 2. Operating Parameters for Balanced Flow Operation (Days 317 - 444) [3]
Groundwater Toluene Toluene H2O2

Treatment Pumping Rate Addition Addition - DO Addition Addition
Well (L/min) (mg/L) Pulses per Day (mg/L) (mg/L)
T1 25 9.0 0.67 44 47
T2 25 9.0 0.67 44 47
Figure 1: Cross-section of two-well treatment system used at Edwards AFB [3]
A-24
Technology Performance [2,8,9,10]
The objectives of the pilot study at Edwards AFB were to evaluate the advantages and limitations of in
situ bioremediation for full-scale aquifer remediation. Specific remedial goals (contaminant
concentrations in groundwater) were not established for the demonstration.
An area of 480 m2 (0.12 acres) was monitored using 20 monitoring wells. Fourteen of the monitoring
wells surrounded treatment wells T1 and T2 in a diamond formation, and two wells were nested between
the treatment wells. Other wells were located at the “compass points” (North, South, East, West)
surrounding the site. The 14 diamond formation wells and three of the four compass point wells were
screened in both the upper and lower aquifers, allowing sampling from each aquifer independently.
10,500 samples were collected and analyzed automatically at the site throughout the course of the
demonstration.
Comparison of measured TCE concentrations at the treatment well discharge screens, and at monitoring
wells located 7.5 m away from the screens, allowed estimation of TCE removal in the bioactive treatment
zones surrounding the discharge screens. The results from these analyses, during steady-state and
balanced flow operation, are presented in Table 3.
Table 3. TCE Concentrations During Steady-state and Balanced Flow Operation [3]
Average TCE Average TCE TCE Removal %

Treatment Operating Concentration in Concentration in (average and
Well - Period Treatment Well Monitoring Well standard
Aquifer (Days) (g/L) 7.5 m Distant (g/L) deviation)
T1 - lower 145 - 204 80 17 79 ± 42
T1 - lower 212 - 271 63 26 59 ± 22
T1 - lower 365 - 444 107 18 83 ± 16
T2 - upper 145 - 204 304 46 85 ± 9
T2 - upper 212 - 271 254 29 89 ± 7
T2 - upper 365 - 444 171 24 86 ± 9
Table 3 shows that the average reduction of TCE during steady-state operation (days 145 - 271) was 87%
in the upper aquifer bioactive zone and 69% in the lower aquifer adjacent to treatment well T1 discharge
screen. During balanced flow operation (days 365 - 444), the average removal of TCE was 86% and 83%
in the upper and lower aquifer bioactive zones, respectively. Over the duration of the demonstration,
TCE concentrations were reduced by 97.7%, from 1,150 g/L (groundwater moving into the study area)
to 27 g/L (groundwater moving out of the study area) and toluene removal generally exceeded 99.98% .
According to the researchers the overall TCE concentration reduction of 97.7% is higher than the
removals reported in Table 3 as groundwater recirculated through the bioactive zone multiple times
during the overall demonstration. The dual-well system was found to be technically feasible for
remediation of TCE in a two aquifer system.
A-25
No information was provided about potential degradation products from this demonstration. The
researchers presumed that toluene degraded aerobically to carbon dioxide and water, and TCE was
cometabolized, ultimately producing carbon dioxide, water, and chloride ions. No exceptions to
established quality assurance/quality control (QA/QC) protocols were noted in the available information.
Technology Cost [5]
Table 4 provides the actual cost for the in situ bioremediation treatment system used for the
demonstration at Edwards AFB, including capital and operation and maintenance costs. Software is
available [5] for estimating costs of applying this technology at a site with specified characteristics.
These actual costs are provided as an example in the software user’s guide.
Table 4. Actual Costs for the Field Demonstration at Edwards AFB [5]
Cost Element Actual Cost (1995-1996 $)

Capital - Treatment wells (2) 30,000
Capital - Other treatment equipment - flow sensors and 32,707
controllers, static mixers, packing assembly, deionized
water system, pumps and ancillary equipment, tubing and
connectors, valves and fittings)
Capital - Monitoring wells 190,000
Capital - Monitoring equipment (pumps and ancillary 70,746
equipment, tubes and connectors, valves and fittings,
miscellaneous supplies)
Total Capital Costs 323,453

Annual O&M - Materials - Well Redevelopment 8,000
($4,000/well-year x 2 wells)
Annual O&M - Materials - Hydrogen Peroxide, 30% 4,633
Annual O&M - Materials - Toluene 47
Annual O&M - Materials - Oxygen Gas 1,674
Total Annual O&M Costs 14,354
Volume of Water in Test Area 1,160 m3

Volume of Water Pumped 12,132 m3 from upper to lower aquifer
16,063 m3 from lower to upper aquifer
A-26
Summary Observations and Lessons Learned [3]
The dual-well system met the objectives of the pilot study, and was found to be technically feasible for
remediation of TCE in a two aquifer system. In addition, this technology might be feasible for use in a
single aquifer system where low permeability layers separate lower and upper zones, and where vertical
hydraulic conductivity is significantly lower than horizontal conductivity. Alternatively, with a relatively
homogeneous single aquifer system, groundwater might be pumped to the surface from one location and
then reinjected at another location with chemical amendments added at the surface or down-well at the
injection location.
Prevention of well clogging was identified as an important operational consideration for application of
this technology. To control well clogging during this demonstration, site operators used well
redevelopment (three times in the upper and twice in the lower aquifer) and addition of hydrogen
peroxide, which increased the operational costs.
The extensive network of monitoring wells was a major capital cost component for this application. The
monitoring system was installed to allow a detailed evaluation of the treatment system’s performance.
Monitoring of this magnitude would likely not be required for a full-scale application.
Contact Information [2]
EPA RPM:
Richard Russell
U.S. EPA Region 9
75 Hawthorne Street, SFD-8-1
San Francisco, CA 94105
(415) 744-2406
e-mail: russell.richard@epa.gov
Air Force Project Manager:

David Steckel
AFFTC/EMR
5 East Popson Avenue, Building 2650A
Edwards Air Force Base, CA 93524-1130
(805) 277-1474
fax: (805) 277-6145
e-mail: david.steckel@edwards.af.mil
Principal Investigator:
Dr. Perry McCarty
Stanford University
Department of Civil and Environmental Engineering
Stanford, CA 94305-4020
(650) 723-4131
fax: (650) 725-9474
e-mail: mccarty@ce.stanford.edu
A-27
References
1. U.S. EPA. 1998. Fact Sheet. Edwards Air Force Base, California, http://epainotes1.rtpnc.epa.gov.
December 15.
2. Record of Telephone Conversation. 1998. Richard Weisman, Tetra Tech EM Inc. and David
Steckel, Edwards Air Force Base. December 1.
3. McCarty, Perry L., Mark N. Goltz, Gary D. Hopkins, Mark E. Dolan, Jason P. Allan, Brett T.
Kawakami, and T.J. Carrothers. 1998. Environmental Science Technology. Full-Scale Evaluation
of In Situ Cometabolic Degradation of Trichloroethylene in Groundwater Through Toluene Injection.
Volume 32, Number 1: 88-100.
4. Lang, Margaret M., Paul V. Roberts, and Lewis Semprini. 1997. Groundwater. Model Simulations
in Support of Field Scale Design and Operation of Bioremediation Based on Cometabolic
Degradation. Volume 35, Number 4: 565-573.
5. U.S. Air Force, Center for Environmental Excellence, Environmental Restoration Division. 1998.
Installation Restoration Program: Aerobic Cometabolic In Situ Bioremediation Technology
Guidance Manual and Screening Software User's Guide. Prepared for Air Force Research
Laboratory, Airbase and Environmental Technology Division, Tyndall AFB, Florida.
<http://en.afit.af.mil/env/insitubio.html>.
6. E-mail Transmittal. 1999. From David Steckel, Edwards Air Force Base, to Dawn Carroll, U.S.
EPA. Review of EPA Bio Document. January 25.
7. E-mail Transmittal. 1999. From David Steckel, Edwards Air Force Base, to Dawn Carroll, U.S.
EPA. Review of EPA Bio Document. January 29.
8. Fax Transmittal from Perry McCarty, Stanford University, to Dawn Carroll, U.S. EPA. 1999.
Suggested Changes to Case Study #2, Edwards Air Force Base. February 1.
9. Hopkins, Gary D. and Perry L. McCarty. 1995. Environmental Science Technology. Field
Evaluation of In Situ Aerobic Cometabolism of Trichloroethylene and Three Dichloroethylene
Isomers Using Phenol and Toluene as Primary Substrates. Volume 29: 1628-1637.
10. Dolan, Mark E. and Perry L. McCarty. 1995. Environmental Science Technology. Methanotrophic
Chloroethene Transformation Capacities and 1,1-Dichloroethylene Transformation Product Toxicity.
Volume 29: 2741-2747.
A-28
Case Study #3
Methane Enhanced Bioremediation Field Demonstration Using Horizontal Wells
at Savannah River Site, Aiken, South Carolina
Summary Information [1,4]
Site Name, Location Savannah River Site, Aiken, SC
EPA ID Number SC1890008989
Technology Nutrient Addition

Electron Acceptor Addition (Oxygen)
Electron Donor Addition (Methane)
Configuration Direct Injection
Media Treated Sediment and Groundwater
Contaminants Targeted TCE, PCE
Period of Operation February 26, 1992 to April 30, 1993
Site History/Source of Contamination [1,4,5]
The U.S. Department of Energy (DOE), Savannah River Site (SRS) is a 300 square mile facility located
in Aiken, South Carolina that has been used for a wide range of operations associated with the research
and production of nuclear materials. Area M at the facility was used for aluminum forming and metal
finishing operations. From the 1950's to the 1980's, wastewaters from Area M operations were
discharged to an unlined settling basin and a nearby stream, resulting in soil and groundwater in the area
becoming contaminated with high levels of chlorinated solvents, primarily trichloroethlyene (TCE) and
tetrachloroethene (PCE). Dense nonaqueous phase liquids (DNAPLs) have also been observed. In
September 1985, a full-scale pump and treat system began operating at the site. This site is was added to
the National Priorities List on November 21, 1989. A Record of Decision (ROD) had not been signed for
this facility at the time of this report.
DOE, as part of the volatile organic compound (VOCs) in Non-Arid Soils Integrated Demonstration
program, tested several innovative technologies to augment the pump and treat system in Area M. This
report focuses on the field demonstration of methane enhanced bioremediation using horizontal wells.
The demonstration site was located within the VOC groundwater plume, estimated to cover about 1200
acres and to be about 150-ft thick. Prior to the demonstration, concentrations of TCE and PCE in
groundwater ranged from 10 to 1,031 ug/L and 3 to 124 ug/l, respectively. Sediment TCE and PCE
concentrations ranged from 0.67 to 6.29 mg/kg and 0.44 to 1.05 mg/kg, respectively.
Geology/Hydrogeology/Contaminant Characterization [1,5]
The demonstration area was underlain by relatively permeable sands with thin lenses of clayey
sediments. The clay layers generally were relatively thin and discontinuous, with thicker clay layers
found at depths of 90 and 160 feet below ground surface (bgs). The water table occurred at depths
A-29
ranging from 120 and 135 feet bgs. The groundwater flow was radial, extending outward from a
groundwater plateau under the demonstration area. In addition, there was a moderate downward gradient
beneath the site, with vertical flow rate estimated to be 2 to 8 feet/year.

Soil Type sand, clay, and gravel
Depth to Groundwater ranges from 120 to 135 feet bgs
Thickness of Aquifer(s) 150 feet
Groundwater Velocity 15 to 100 feet/year (horizontal)
Technology Description [1,2,3]
Figure 1 presents a process schematic of the methane enhanced bioremediation (MEBR) system used for
the demonstration at the M area. The system included two horizontal wells. The “lower” horizontal well
was placed below the water table (saturated zone) at a depth of 175 feet bgs , with a screen length of 310
feet. The “upper” horizontal well was placed in the vadose zone at a depth of 80 feet bgs, with a screen
length of 205 feet. Air and gas were injected into the saturated zone through the lower horizontal well at
a rate of 200 scfm. Air and contaminants were then extracted from the vadose zone through the upper
horizontal well at a rate of 240 scfm. A thermal catalytic oxidizer, operated at 825° C, was used to treat
the extracted vapors, prior to discharge to the atmosphere.
Figure 1: Methane Enhanced Bioremediation (MEBR) [1]
The demonstration was performed in six different operational modes, as described in Table 1. These
included baseline tests of the vapor extraction and injection systems, a series of nutrient additions, a
tracer test, and an assessment of microbiological assays for monitoring performance.
A-30
Table 1. Modes for the Demonstration [1,3]
Operational
Mode Description of system operation
Baseline Test Initial vacuum extraction of vadose zone gases at a rate of 240 scfm
Baseline Test Addition of air sparging - simultaneous injection of air into the saturated zone
coupled with vacuum extraction of the vadose zone at a rate of 202 scfm (84% of
the first baseline test)
Nutrient Addition of 1% methane
Addition -1
Nutrient Addition of 4% methane
Addition - 2
Nutrient Pulsed 4% methane addition at a rate of 8 hr every two days
Addition - 3
Nutrient Continuous addition of a combination of nitrous oxide at 0.007% and triethyl
Addition - 4 phosphate at 0.07% in air in combination with pulses of 4% methane
Tracer tests Helium tracer tests to measure the amount of injected methane consumed by the
indigenous microbes
Microbiological Comparison of microbial assays for monitoring and control of in situ
Assays bioremediation
Technology Performance [1,2,3]
After 384 days of operation, concentrations of PCE and TCE in sediments were reduced to below
detectable limits, and concentrations of PCE and TCE in groundwater were reduced to below 5 ppb each
for PCE and TCE. In addition, soil gas concentrations decreased by more than 99%. The system
removed about 17,000 lbs of VOCs through a combination of vacuum extraction and biodegradation.
The concentration of TCE and PCE in the sediments before and after the demonstration were used to
calculate the mass of VOCs degraded. The vacuum component of the system removed 12,096 lbs of
VOCs and the biological component degraded 4,838 lbs of VOCs.
The addition of methane stimulated the growth of methanotrophs. During the 1% methane addition
phase, the population of methanotrophs increased by several orders of magnitude, to levels close to
100,000 MPN/ml. During the 4% methane addition phase, the population of methanotrophs increased
initially, then decreased as a result of nutrient depletion. The addition of nitrogen and phosphorous
nutrients with pulsed methane stimulated microbial activity, and was reported to have optimized
bioremediation and mineralization of TCE and PCE in groundwater and sediments. The results of the
helium tracer tests indicated that more than 50% of the injected methane was consumed by indigenous
microbes before it reached the extraction well. No results were provided from the microbiological
assays.
The zone of influence of the extraction well in the vadose zone was reported to be greater than 200 feet
based on pressure measurements. The sparge zone of influence in the saturated zone, measured using
electrical resistance tomography, was reported to be a “complex three-dimensional network of channels”
A-31
extending as far as 100 feet from the injection well. The system was operational 90% of the time and no
problems were reported during the demonstration.
Technology Cost [1,5,6]
Table 2 presents the projected costs for full-scale application of MEBR. The projected capital costs were
$452,407 (including equipment costs amortized over 10 years, and costs for well installation and
mobilization), and the projected operation and maintenance (O&M) costs were $236,465 (including
monitoring, consumables, and demobilization).
Table 2. Project Costs for Full-scale MEBR Application [1,5]
Element Cost ($)

Capital
Site cost 5,400
Equipment cost 9,200
Design and Engineering 10,000
Mobile equipment 18,000
Well Installation 183,000
Other fixed equipment 183,732
Mobilization 43,075
Total Capital Equipment and 452,407
Mobilization Cost
O&M Costs
Monitoring/maintenance 71,175
Consumables 122,215
Demobilization 43,075
Total O&M 236,465
Summary Observations and Lessons Learned [1,2]
The in situ bioremediation system demonstrated as SRS removed about 17,000 lbs of VOCs through
vacuum extraction (about 12,000 lbs) and through biodegradation (about 5,000 lbs). According to DOE,
the addition of nitrogen and phosphate nutrients in conjunction with 4% pulsed methane provided the
best results of the four nutrient addition campaigns tested.
No toxic intermediates were produced during the demonstration. However, the use of technical grade
methane was found to be growth inhibiting because it contained small amounts of acetylene which is
poisonous to the microbes.
Los Alamos National Laboratory completed a cost-benefit analysis that showed that in situ
bioremediation could reduce costs by more than 30% compared to a baseline technology of SVE/pump
A-32
and treat. According to DOE, in situ bioremediation could reduce the time required to remediate a site
by 5 to 7 years compared to SVE/pump and treat.
Contact Information
Principal Investigators:
Dr. Terry C. Hazen
Lawrence Berkeley National Laboratory
Center for Environmental Biotechnology
MS 70A-3317
One Cyclotron Road
Berkeley, CA
(510) 486-6223
(510) 486-7152 (fax)
tchazen@lbl.gov
Brian Looney
Westinghouse Savannah River Company
PO Box 616
Aiken, SC 29802
(803) 725-6413/(803) 725-3692
DOE Integrated Demonstration Manager:

Kurt Gerdes
U.S. DOE
Office of Environmental Management
Science & Technology Development
Office of Technology Systems
Cloverleaf Room 1135
Germantown, MD 20874
(301) 903-7289
(301) 903-7457 (fax)
References
1. U.S. Department of Energy, Office of Environmental Management, Office of Technology

Development. In Situ Bioremediation of Using Horizontal Wells. Demonstrated at M Area,
Savannah River Site, Aiken, SC. April 1996.
2. Methane Enhanced Bioremediation for the Destruction of Trichloroethylene Using Horizontal Wells.
Westinghouse Savannah River Company. Technology Catalog Site Remediation Profiles. July 1998.
3. Methane Enhanced Bioremediation for the Destruction of Trichloroethylene Using Horizontal Wells.
Technology and Systems Solutions. Em-50.em.doe.gov/BEST/techs/aa/tech0210.html. July 1998.
4. Superfund EPA. EPA National Priorities List. Savannah River Site (USDOE). July 1998.
A-33
5. Fax transmittal from Kurt Gerdes, U.S. DOE, to Richard Weisman, Tetra Tech EM Inc. 1999.
Review Comments on Preliminary Draft. February 4.
6. U.S. DOE/EM/OTD. 1995. In Situ Bioremediation Using Horizontal Wells; Innovative Technology
Summary Report. April.
A-34
Case Study #4
Full Scale Enhanced Bioremediation at the Texas Gulf Coast Site,
Houston, Texas
Site Name, Location Texas Gulf Coast Site, Houston, Texas (actual
site name confidential)
EPA ID Number Not available
Mechanism(s) Anaerobic Reductive Dechlorination

(Cometabolic and Direct)
Technology Nutrient Addition

Electron Donor Addition (Methanol)
Technology Scale Full
Contaminants Targeted TCE, cis-1,2-DCE, VC
Period of Operation Ongoing (data available from June 1995 to

December 1998)
The Texas Gulf Coast site (actual site name confidential) is an abandoned industrial manufacturing
facility located near Houston, Texas that operated between 1952 and 1985. Trichloroethene (TCE) was
used in facility operations until about 1978. In 1986, elevated levels of TCE were found in groundwater
at the site, and groundwater monitoring has been performed periodically since that time. Groundwater at
the site is being remediated through the State of Texas Voluntary Cleanup Program, administered by the
Texas Natural Resource Conservation Commission (TNRCC).
Monitoring data from 1986 to 1995 showed that TCE concentrations in the groundwater had decreased
from approximately 50 to 22 mg/L, and that TCE degradation products such as DCE were present in the
groundwater, indicating that natural attenuation was occurring at the site. In 1995, an enhanced
bioremediation system was installed to actively remediate the contaminated groundwater at the site to a
point where natural attenuation would prevent further migration of the plume. The TNRCC approved the
work plan for the use of in situ bioremediation at the site and for reinjection of water under the
Underground Injection Control (UIC) program.
Geology/Hydrogeology [1,3]
The area of groundwater contamination is approximately 600 ft by 700 ft, located in an unconsolidated
aquifer which occurs at a depth of approximately 12 - 20 ft below ground surface (bgs). The upper
aquifer is underlain by a 15-ft thick clay layer, below which is lies a deeper aquifer, which is not
contaminated. The natural flow direction for the upper aquifer is to the southeast. A flood control ditch
is located on the eastern edge of the area, and intercepts the upper two feet of the aquifer.
A-35

Soil Type Silty sand
Depth to Groundwater 12 - 20 ft bgs
Thickness of Aquifer Approximately 8 ft
Fraction of Organic Carbon Not provided
Hydraulic Conductivity 1 x 10-4 to 4 x 10-4 cm/sec
pH Approximately 6.7 to 7.2
Nitrogen <0.1 mg/L
Sulfur (as Sulfate) Approximately 30 mg/L
Groundwater Velocity 4 - 18 ft/yr
Technology Description [1,3]
The in situ bioremediation treatment system being used at this site consists of an alternating series of four
extraction (1,800 linear ft total) and four injection (1,100 linear ft total) trenches set at a spacing of
approximately 100 ft, as shown in Figure 1. The furthest down-gradient trench is an extraction trench
that runs along the portion of the down-gradient property line intersected by the plume.
The extraction trenches were completed to a depth of at least one foot into the bottom clay layer (20 - 22
ft bgs), and were sloped to a sump. A perforated pipe was installed along the bottom of each trench and
the trenches were filled with gravel. The injection trenches were constructed in a manner similar to that
used for the extraction trenches; however, the perforated pipes were installed at a depth of approximately
10 ft bgs. Extracted groundwater is pumped to a holding tank in the control building. Water drains from
the holding tank to a wet well and then to the injection trenches. The above-ground equipment was
designed to minimize the introduction of air into the equipment.
A-36
Figure 1: System Layout, Extent of TCE Plume, and Groundwater Flow Directions for Texas Gulf
Coast Site [1,3]
System operation began in August 1995 with a circulation rate of 12 gallons per minute (gpm). Addition
of nitrogen and phosphorus nutrients began in September 1995. In June 1996, methanol was added,
along with nitrogen and phosphorus, to serve as a primary substrate and to further reduce the dissolved
oxygen and oxidation-reduction potential to levels that were thought to be more favorable to anaerobic
degradation of TCE. Nutrient addition was discontinued in May of 1997 because it appeared to be
preventing continued decrease in the redox potential within the treatment area; methanol addition
continued. As of January 1999, the recirculation rate averages 6 to 8 gpm, and a total of 12 million
gallons have been recirculated through the system (approximately 2.5 pore volumes). System operating
parameters are summarized in Table 1.
A-37
Table 1. Operating Parameters for the Enhanced Bioremediation System [1,3]
Parameter Value
Circulation Rate 12 gpm (August 1995 - January 1999)
6 to 8 gpm (starting in January 1999)
Methanol Concentration up to 500 mg/L (in January 1999)
Nitrate Concentration initially 9 mg/L (as potassium nitrate)
(discontinued in May of 1997)
Phosphate Concentration initially 9 mg/L (as potassium tripolyphosphate)
(discontinued in May of 1997)
Technology Performance [1,2,3]
The performance goals of this treatment system are to achieve stable or declining contaminant
concentrations and to remediate the groundwater to a point where natural attenuation can be used to
prevent future migration of the contaminant plume. Once these goals have been achieved, use of active
bioremediation will be discontinued and groundwater at the site will be monitored for a period of 2 to 5
years to determine that the plume has not migrated. No specific cleanup goals have been identified for
groundwater at this site.
Table 2 summarizes groundwater monitoring data from June 1995 (prior to system operation) to
December 1998 for five sampling events, including data for TCE, DCE, and VC, as well as other
parameters such as chloride, DO, and redox potential. The data presented are the average concentrations
measured in six wells located outside of the treatment zone (“Outside Wells”) and in eight wells located
within the treatment zone (“Inside Wells”), including one of the monitoring wells, well MW-40, that was
located within the apparent “source” area. The average concentrations for the TCE, DCE, and VC in the
“Inside Wells” were calculated two ways - one including the data from well MW-40, and one excluding
the data from well MW-40.
Results for the inside wells, including the source area well MW-40, show the average concentration of
TCE was reduced by about 88% (22.7 mg/L to 2.6 mg/L) and DCE by about 96% (1.91 mg/L to 0.682
mg/L). VC concentrations in the inside wells remained essentially unchanged (0.102 mg/L to 0.105
mg/L). When the results for well MW-40 are not included, the average concentration of TCE was
reduced by about 99% (11.8 mg/L to 0.12 mg/L); DCE by about 87% (1.28 mg/L to 0.165 mg/L); and
VC by about 30% (0.078 mg/L to 0.054 mg/L).
According to the site contractor, Well MW-40, located within the treatment area, has shown consistently
elevated concentrations of TCE. Recent excavation from within this area identified a potential source of
continuing release to the groundwater that was preventing the rate of decrease observed in the remaining
plume. Soil in the potential source area was excavated during October 1998 to allow volatilization of the
chlorinated organic compounds. Debris (concrete, piping, and trash) was excavated in this area;
however, no evidence of dense non-aqueous phase liquids (DNAPL) was identified. In addition, TCE
concentrations in portions of the plume have now decreased to below the detection limit (0.005 mg/L).
A-38
Table 2. Summary of Technology Performance Data

Average Concentrations (mg/L) [3]
Jun-95 May-96 Jun-97 Mar-98 Dec-98

Analytical Inside Outside Inside Outside Inside Outside Inside Outside Inside Outside
Parameter Methods* Wells Wells Wells Wells Wells Wells Wells Wells Wells Wells
Average including potential source well MW-40
TCE EPA 8260 22.7 0.070 16.7 0.07 4.02 0.748 2.80 0.046 2.60 0.065
cis-1,2-DCE EPA 8260 1.91 < 0.005 1.0 < 0.005 1.31 0.049 2.90 0.006 0.682 0.009
Vinyl Chloride EPA 8260 0.102 < 0.005 < 0.005 <0.005 0.084 < 0.002 0.222 < 0.002 0.105 < 0.002
Average excluding potential source well MW
TCE EPA 8260 11.8 - 9.68 - 3.24 - 2.42 - 0.119 -
cis 1,2-DCE EPA 8260 1.28 - 0.733 - 1.38 - 2.52 - 0.165 -
Vinyl Chloride EPA 8260 0.078 - 0.016 - 0.095 - 0.181 - 0.054 -
Chloride EPA 325.3 162 35 147 29 132 20 136 23 120 14
NO3-NO2 EPA 353.2 < 1.0 < 1.0 11 0.90 0.20 0.13 < 0.10 < 0.10 < 0.10 < 0.10
O-PO4 EPA 365.2 0.24 0.09 2.1 0.40 1.9 0.52 0.56 0.12 0.65 0.10
TKN EPA 351.2 < 1.0 < 1.0 3.6 1.6 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0
Ferrous Iron EPA 6010 - - - - 0.23 < 0.05 0.27 < 0.02 2.1 < 0.02
Total Iron EPA 6010 - - - - - - 3.4 0.25 4.4 0.07
Sulfate EPA 375.4 - - - - 170 28 146 24 42 38
Sulfide EPA 376.1 - - - - < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0
Methane EPA 3810 - - - - 0.69 0.007 2.8 0.009 3.6 0.85
Ethene EPA 3810 - - - - - - - - 0.09 0.02
DO 1.7 3.4 1.1 2.4 0.45 1.6 0.65 1.6 1.1 2.3
Redox (mV) +292 +319 +111 +126 +85 +178 -61 +332 -116 +245
pH (SU) 6.56 6.75 6.69 6.84 6.46 6.83 6.38 6.74 6.85 7.23
Notes: (-) Not Analyzed.

(*) Latest sampling event only.
A-39
The concentration of chloride was used to evaluate the potential impact of dilution on groundwater
quality. As shown on Table 2, there is a difference between the chloride concentrations in the inside
wells and those in the outside wells. According to the site contractor, assuming the chloride is due
exclusively to dechlorination of TCE (and its degradation products), the average TCE concentration at
project outset was estimated to be as high as 100 mg/L to 125 mg/L. During the active treatment period,
chloride in the outside wells has decreased from approximately 35 mg/L to 14 mg/L. The site contractor
applied the same assumptions about dilution to the treatment area and estimated that the December 1998
chloride concentration for the inside wells was approximately 65 mg/L. The differences between this
and the measured concentration (120 mg/L) was assumed by the site contractor to be due to additional
contributions from dechlorination of TCE (and the degradation products). Accounting for dilution, the
site contractor reported that TCE concentrations were reduced by approximately 2% per month during
the period of nutrient-only addition, and approximately 10% per month during the period of nutrient and
methanol (substrate) addition. Also, the ratio of cis-1,2-DCE to TCE increased from approximately
0.06:1 to 0.30:1 after addition of methanol, suggesting more active dechlorination associated with higher
concentrations of substrate.
According to the site contractor, current plans are to shut down the active bioremediation system by the
middle of 1999. Should this occur, this site will meet its design goal of completing the active
remediation period within 3-5 years (actual period of operation would be 4 years). It is anticipated that
the approval for system shutdown will be based on the following:
& The impacted aquifer has been designated as “low-yield” and, therefore, has less stringent
groundwater remediation standards.
& The concentration and size of the plume will have been reduced to a point that growth or
migration of the remaining plume will be controlled by natural attenuation.
Information is not provided about the analytical methods used in this application, however no exceptions
to established quality assurance/quality control (QA/QC) protocols were noted.
Technology Cost [3]
Capital costs for construction of the extraction/injection trenches and control building were approximately
$600,000. Annual costs for operation, maintenance and monitoring are approximately $100,000.
Methanol addition was found to increase the rate of biodegradation of TCE at this site, based on the
reduction of TCE concentration and increase in the ratio of cis-1,2-DCE to TCE. This site is planning to
stop using active bioremediation after four years of system operation (three years of methanol addition)
to allow use of natural attenuation. According to the site contractor, natural attenuation will be used to
prevent future migration of the plume, and to achieve stable or declining contaminant concentrations.
Excessive biomass formation, leading to a reduced flow rate, was found to be a concern for addition of
methanol. Excess biomass was not noted during the period when nutrients alone were added; however, a
significant increase in biomass formation was noted after addition of methanol. To remedy this, the site
contractor modified their methanol addition to a batch system.
The site contractor found that it was difficult to balance the system hydraulics between the extraction and
infiltration trenches, and that it required approximately one year of operating time to achieve a balance.
A-40
In addition, they found it difficult to interpret the treatment performance data because of the non-
homogeneous nature of the initial groundwater quality, and dilution due to recharge of rainwater and
clean water from beyond the planned treatment area.
Contact Information
Site Contractor:
Susan Tighe Litherland, P. E
David W. Anderson, P.E., P.G.
Roy F. Weston, Inc.
5300 Bee Caves Road, Suite 1-100
Austin, TX 78746
Telephone: (512) 329-8399
Fax: (512) 329-8348
E-mail: litherls@mail.rfweston.com
E-mail: andersod@mail.rfweston.com
References
1. Litherland, Susan Tighe, P. E., and David W. Anderson, P.E., P.G., Roy F. Weston, Inc. 1997. Full-
Scale Bioremediation at a Chlorinated Solvents Site. In Proceedings of 4th International In Situ and
On-Site Bioremediation Symposium. New Orleans, LA. Batelle Press.
2. Record of Telephone Conversation between David W. Anderson, P.E., P.G., Roy F. Weston, Inc.,
and Richard J. Weisman, Tetra Tech EM Inc. 1998. Bioremediation at Texas Gulf Coast Property.
December 21.
3. Fax transmittal from Susan Litherland, Roy F. Weston, Inc., to Dawn Carroll, U.S. EPA. 1999.
Draft Case Study - Revisions. January 28.
A-41
Case Study #5
Pilot and Full Scale Molasses Injection at the Avco Lycoming Superfund Site,
Williamsport, Pennsylvania
Summary Information [1-6]
Site Name, Location Avco Lycoming Superfund Site, Williamsport,

Pennsylvania
EPA ID Number PAD003053709

Technology Electron Donor Addition (Molasses)
Technology Scale Pilot and Full
Contaminants Targeted TCE, DCE, VC, hexavalent chromium, cadmium
Period of Operation Pilot study October 1995 to March 1996;

Full-scale system ongoing, data available from
January 1997 to July 1998
Site History/Source of Contamination [1,6,7,8]
The Avco Lycoming Superfund site (Lycoming) is a 28-acre facility located in Williamsport,
Pennsylvania. Since 1929, various manufacturing companies have operated at the site, including a
bicycle and sewing machine plant, a sandpaper plant, a tool and die shop, a silk plant, and an aircraft
engine plant, that is currently operating at the site. Past waste handling practices, including disposal of
wastes in a dry well and coolant well, spillage and dumping of wastes from metal plating areas, and
storing sludge in a holding lagoon, resulted in soil and groundwater contamination at the site.
In the mid-1980's, the state identified the Lycoming site as the source of volatile organic compound
(VOC) contamination in the local municipal water authority well field located 3,000 ft south of the site.
Contaminants include trichloroethene (TCE) and trans-1,2-dichloroethene (DCE), and metals, including
hexavalent chromium and cadmium. A pump and treat system was installed at the site in the late 1980's
to remediate on-site and off-site groundwater contamination. The Lycoming site was placed on the
National Priority List on February 21, 1990, and a Record of Decision (ROD) was issued for this site in
June 1991 requiring pump and treat for the shallow groundwater beneath the facility property, followed
by discharge to a nearby stream. Design of this system was suspended pending resolution of a permit for
the discharge.
In May 1995, the potentially responsible party (PRP) proposed an alternative for remediating the shallow
groundwater that involved using an in situ bioremediation system that included molasses injection and air
sparging/soil vapor extraction (SVE). Pilot studies were conducted from October 1995 to June 1996. A
new ROD was issued in December 1996 replacing the pump and treat with an in situ remedy. A full-
scale molasses injection system was installed and has been operating at the site since January 1997.
A-42
Although the air sparging/SVE system was pilot tested, and construction of a full-scale system begun in
October 1997, construction was suspended in the Spring of 1998, due to higher than anticipated water
levels at the site. The PRP is planning to submit a new proposal to EPA for remediation of the organic-
contaminated groundwater at the site. Therefore, this case study focuses on the molasses injection
technology.
Geology/Hydrogeology/Contaminant Characterization [1,3]
Site geology consists of a sandy silt overburden overlying a fractured bedrock and a fractured limestone.
The target area for the in situ treatment is the shallow overburden to approximately 25 ft below ground
surface (bgs), and covers approximately two acres. The maximum concentrations measured in this area
in late 1996 were TCE, 0.7 mg/L, hexavalent chromium, 3 mg/L, and cadmium, 0.8 mg/L.
Matrix Characteristics at Lycoming Superfund Site [1,3,8]
Parameter Value
Soil Type Sandy silt
Depth to Groundwater 10 to 15 ft bgs
Thickness of Aquifer 10 to 12 ft
Fraction of Organic Carbon Not available
DNAPL Present Not identified
Hydraulic Conductivity 0.2 to 24 ft/day
Groundwater Velocity 0.02 to 2.3 ft/day
Technology Description [1,7,8]
Pilot Study. The molasses pilot study was conducted from November 1995 to June 1996. In the pilot
study, an in situ reactive zone was created to reduce groundwater concentrations of hexavalent chromium
and cadmium. Monitoring data showed that chromium concentrations were reduced from 7 mg/L to less
than 0.05 mg/L in the test zone, and that the technology also created conditions needed to reductively
dechlorinate the CAHs. For example, during the pilot study, redox conditions were shown to be strongly
reducing, at less than -300mV. The air sparging/SVE pilot system took place from October 1995 to May
1996.
Full-scale System. The full-scale molasses injection system, a proprietary technology owned by
ARCADIS Geraghty & Miller, was constructed in late 1996 and began operating in January 1997. As
shown in Figure 1, the molasses injection system consists of 20 four-inch diameter injection wells,
ranging in depth from 19 to 30 ft, completed in the overburden. Each well is connected to a 10 ft square
treatment building by 3/4-inch diameter piping. Molasses is added two times each day at variable
concentrations and rates based on the results from system monitoring. Figure 2 shows the monitoring
wells network at the site. A programmable logic controller monitors and controls the feed rate and
frequency of substrate addition. Table 1 summarizes the operating parameters for the system.
A-43
Figure 1: Molasses Injection System Used at Lycoming Superfund Site [2]
Figure 2: Monitoring Well Network at the Site [7]
A-44
Table 1. Operating Parameters Used at Lycoming Superfund Site [1]
Operating Parameter Value

Substrate (molasses) addition Not provided
Redox potential < -300mV
Technology Performance [7,8]
The 1996 ROD specified the following cleanup goals for groundwater: TCE (5 ug/L), 1,2-DCE (70
ug/L), VC (2 ug/L), cadmium (3 ug/L), hexavalent chromium (32 ug/L), and manganese (50 ug/L).
Groundwater monitoring data are available for January 1997 to July 1998. Samples were collected from
16 wells, (GM-1 through GM-8; MW-3R, 4, 18,and 46; and PRW-7, 8, 9, and 10), as shown on Figure 1.
Figures 3 and 4 show the geochemical conditions of the groundwater in January 1997 and July 1998,
respectively, using the following indicator parameters - redox potential, sulfide, and total organic carbon
(TOC). The January 1997 results, collected prior to initiation of mollasses injections, were used as the
baseline conditions. The data showed that anaerobic and reducing conditions were present only near two
of the site monitoring wells (GM-3 and MW-18) located near the northeastern and southeastern corners
of the site.
Figure 3: Distribution of Groundwater Indicator Parameters, Baseline Conditions, January

1997 [2,8]
A-45
Figure 4: Distribution of Groundwater Indicator Parameters, Reactive Zone Established,

July 1998 [8]
Data collected in July 1998 (Figure 4) show that the redox levels have decreased to anaerobic conditions
in many of the wells that had previously indicated an aerobic environment, indicating that anaerobic and
reducing conditions have been expanded to include a majority of the eastern portion of the treatment
area. The presence of sulfide, the reduced product of sulfate, indicated that conditions were sufficient to
promote reductive dechlorination of chlorinated aliphatic hydrocarbons. Concentrations of TOC also
were increased in the treatment area. (DO data collected during the study were not collected using a
flow-through cell. The PRP contractor reported that these data were believed to be significantly biased
high, however, no additional information was provided).
As of July 1998, concentrations of TCE, DCE, and hexavalent chromium have been reduced to below
than their cleanup goals in many of the monitoring wells at the site. Concentrations of hexavalent
chromium been reduced by more than 99% from 1,950 ug/L to 10 ug/L. Figure 5 presents the results of
analyses for TCE, DCE, and VC between January 1997 and July 1998 for monitoring well GM-7, which
is located near the South Wall and within an area that was converted from aerobic to anaerobic during the
first 18 months of treatment. As shown on Figure 5, the concentration of TCE was reduced by 90% from
67 ug/L to 6.7 ug/L. The concentration of DCE initially increased from 7 ug/L to 100 ug/L after 10
months of treatment, indicating the successful dechlorination of TCE, then decreased to 19 ug/L by July
1998. Similarly, the initial concentration of VC increased from below the detection limit of <1 ug/L to 5
ug/L after 10 months of treatment, then decreased to below the detection limit by July 1998.
A-46
Figure 5: Analytical Results for Well GM-7 at Lycoming Superfund Site [8]
(January 1997 to July 1998)
While specific information about the analytical methods or data quality were not provided in the
available references, no exceptions to the quality assurance/quality control (QA/QC) protocols were
noted.
Technology Cost [3,8]
ARCADIS Geraghty & Miller, Inc. reported project costs for the full-scale molasses injection system at
the Lycoming site to be about $220,000 for construction and about $50,000 per year for operation and
maintenance. The cost for the pilot study at this site, including preparation of a work plan, was about
$145,000.
The use of molasses injection was shown to create an anaerobic reactive zone within an 18-month period,
with concentrations of TCE, DCE, and hexavalent chromium reduced to below the cleanup goals in many
of the wells.
This was one of the earliest full-scale applications of this technology at a Superfund site. According to
ARCADIS Geraghty & Miller, this technology was shown to save substantial resources when compared
to pump and treat.
The pilot study demonstrated the ability of the technology to create strongly reducing redox conditions
and to reduce concentrations of chlorinated aliphatic hydrocarbons and hexavalent chromium. The
results of the pilot study were used in the design and operation of the technology on a full-scale basis at
the site.
A-47
Contact Information
EPA RPM:
Eugene Dennis
U.S. EPA Region 3
1650 Arch Street (3HS21)
Philadelphia, PA 19103-2029
(215) 814- 3202
E-mail: dennis.eugene@epa.gov
PRP Contractor:
Daniel L. Jacobs
ARCADIS Geraghty & Miller, Inc.
3000 Cabot Boulevard, West, Suite 3004
Langhorne, PA 19047
Telephone: (215) 752-6840
Fax: (215) 752-6879
E-mail: djacobs@gmgw.com
References
1. Nyer, Evan, Frank Lenzo, and Jeffery Burdick, ARCADIS Geraghty & Miller, Inc. 1998. In Situ
Reactive Zones: Dehalogenation of Chlorinated Hydrocarbons. Groundwater Monitoring Review.
Spring.
2. ARCADIS Geraghty & Miller, Inc. Slides (2A and 2B) on Case Studies for Natural and Enhanced
Reductive Dechlorination for Groundwater Remediation. Not Dated.
3. ARCADIS Geraghty & Miller, Inc. Enhanced Reductive Dechlorination Through Reactive Zones
(Site 2 - Metal Manufacturing and Plating Site). Not Dated.
4. U.S. EPA. 1990. NPL Site Narrative at Listing. Avco Lycoming (Williamsport Division). February
21.
5. U.S. EPA. 1991. Record of Decision (ROD) Abstract. ROD Number: EPA/ROD/R03-91/112. In
Situ Metals Treatment; Williamsport, PA. June 28.
6. U.S. EPA. 1998. General Site Information. Avco Lycoming (Williamsport Division). February.
7. Eugene Dennis, U.S. EPA RPM. 1999. Comments on Draft Case Study Report. January 27.
8. Fax Transmittal from Dan Jacobs, ARCADIS Geraghty & Miller, to Dawn Carroll, U.S. EPA. 1999.
Comments on Draft Case Study Report. February 9.
A-48
Case Study #6
Pilot and Full Scale Anaerobic In-situ Reactive Zone at an Abandoned Manufacturing Facility,
Emeryville, California
Summary Information
Site Name, Location Abandoned Manufacturing Facility,

Emeryville, California
EPA ID Number Not Applicable
Mechanism(s) Anaerobic Reductive Dechlorination and
Metal Precipitation
Technology Electron Donor Addition (Molasses)
Technology Scale Pilot and Full
Contaminants Targeted TCE, hexavalent chromium
Period of Operation Pilot study – August 1995 to February 1996
Full scale system-ongoing, data available
from April 1997 to October 1998
Site History/Source of Contamination [1]
From 1952 until 1995, metal plating operations, including nickel plating, were performed at a
manufacturing facility located in Emeryville, California (actual site name confidential). Solvents were
used in degreasing operations until 1992, when they were replaced with a liquid-alkaline soak process.
Plating operations were discontinued in 1995, and the associated plating equipment has subsequently
been removed from the site. Operations at the site resulted in the groundwater becoming contaminated
with chlorinated solvents and metals.
Between 1977 and 1985, 24 groundwater monitoring wells were installed at the site and on adjacent
properties. Figure 1 shows the location of the 14 on-site monitoring wells. Elevated levels of chromium
and trichloroethene (TCE) was detected in groundwater in the late 1970s and early 1980s. The cleanup
of the site is being completed under a state voluntary cleanup program. In 1995, the site owner initiated a
pilot study to evaluate anaerobic reductive dechlorination and metals precipitation via an in-situ reactive
zone as a possible remedy for the site (as a potential alternative to a conventional pump and system).
The following case study primarily focuses on the reductive dechlorination of TCE; limited data on the
precipitation of hexavalent chromium was provided in the available references.
Geology/Hydrogeology/Contaminant Characteristics [1]
The geology of the site geology consists of interbedded sand and clay units. Groundwater is found at depths
of 3.5 to 8 feet below ground surface (bgs). Groundwater velocity is estimated to be 60 feet per year.
TCE and chromium are the primary contaminants in the groundwater at the site. TCE concentrations
from April 1995 (prior to initiation of the pilot study) were as high as 17,000 ug/L (Well MW-14).
Historical groundwater data from on-site wells indicated that, over the past 10 years, TCE concentrations
have been slowly decreasing. For example, TCE concentrations in Well MW-10 were 12,000 g/L
during a June 1985 sampling event and 10,000 g/L during an April 1995 sampling event.
A-49
Figure 1: Pre-Injection CAH Concentrations, Abandoned Manufacturing Facility, Emeryville,

California (April 1995) [1]
MW-4 MW-1
MW-16 PCE <50 PCE <0.5

TCE 4,000 TCE 13
DCE 410 DCE 1.2
VC <100 VC <1
LEGEND:
MW-5 MONITORING WELL
VOC CONCENTRATIONS: MW-20

(ALL CONCENTRATIONS SHOWN IN PCE <100
MICROGRAMS PER LITER (ug/L)) TCE 10,000
PCE TETRACHLOROETHENE DCE 900
TCE TRICHLOROETHENE VC <200 MW-10
DCE DICHLOROETHENE
VC VINYL CHLORIDE
PCE <500
NS NOT SAMPLED
TCE 17,000 FORMER TCE
DCE <500 DEGREASING
VC <1,000 AREA
PCE 6.2
TCE 65
PCE <50 DCE 17
TCE 280
MW-13 MW-14 PCE 15 VC <10
MW-17 DCE 62
TCE 95 FORMER
VC <100 CHROMIUM WASTE
DCE 8.8
STORAGE TANK PCE <0.5
VC <5
MW-5 TCE 1.1
PCE 10 MW-9 DCE 9.7
TCE 210 VC 4.6
FORMER MW-12
DCE 31 MW-11
BOILER
VC <10 FACILITY MW-3A MW-3C
MW-3B PCE <0.5
PCE <10 TCE 30
TCE 260 DCE 11
DCE 17 VC 2.2
VC <20
Matrix Characteristics at Abandoned Manufacturing Building [1]
Parameter Value
Soil Type Interbedded sand and clay units
Depth to Groundwater Approximately 3.5 to 8 feet
Thickness of Aquifer Not available
Fraction of Organic Carbon Not available
Hydraulic Conductivity Not available
Groundwater Velocity 60 feet per year
A-50
Pilot Study
The pilot study was conducted between August 1995 and February 1996. The pilot study was performed
to determine if the rate of TCE degradation and metals precipitation could be enhanced by an anaerobic
in-situ reactive zone. Groundwater monitoring data, collected prior to the start of the pilot study,
indicated that limited reductive dechlorination of TCE to cis-1,2-dichloroethene (DCE) was occurring,
but that the rate of dechlorination was limited due to the biogeochemical conditions at the site (the
organic carbon source was depleted or the environment was not sufficiently reducing). Vinyl chloride
(VC), the degradation product of cis-1,2-DCE, was either not detected or was sporadically detected in
many of the wells. According to the site contractor, DCE and VC may have been present in some wells
prior to start of the pilot study, but were not detected because of high method detection limits (e.g., 1,000
g/L for DCE and 2,000 g/L for VC).
To establish the anaerobic reactive zone, a mixture of molasses, biologically inoculated solution
(supernatent), and tap water was injected into the subsurface. Injection of the supernatent was needed
because of low plate counts observed in one well during a baseline sampling event. The supernatent used
for the pilot study was from the anaerobic treatment system of a local municipal authority.
The results of the pilot study indicated that the historical reductive dechlorination rate at the site could be
enhanced via the injection of the molasses solution. For example, TCE concentrations in Well MW-10
were reduced from 10,000 g/L in April 1995 to 4,200 g/L in February 1996.
Full-Scale System
In April 1997, ninety-one temporary injection points were installed at the site, as shown in Figure 2. The
injection points are located in two areas due to the location of existing buildings. Each injection points
was installed to a depth of 24 feet bgs.
The full-scale system has been operating since April 1997 and data are available through October 1998.
Two molasses injection events have been performed at the site, in April 1997 and in February 1998.
Each molasses injection event included a mixture of water, molasses, and a small amount of supernatent
(to provide additional bacteria capable of degrading TCE). During the first injection event, each
injection point received 25 gallons of molasses, 1 gallon of supernatent, and 125 gallons of water.
Information about the volume and composition of the solution used in the second injection event was not
available. The reagent was mixed on-site and manually injected into the subsurface using a centrifugal
pump.
A-51
Figure 2: Full-Scale Injection Area, Abandoned Manufacturing Facility, Emeryville, California [2]
MW-4 MW-1
MW-16
LEGEND:
APPROXIMATE BOUNDARY
INJECTION AREAS MW-20
MW-10
FORMER TCE
DEGREASING
AREA
MW-13 MW-14
MW-17 FORMER
CHROMIUM WASTE
STORAGE TANK
MW-5
MW-9
FORMER MW-12
BOILER MW-11
FACILITY
MW-3A MW-3C
MW-3B
Technology Performance [1,2]
Performance data are available through October 1998. Figure 3 presents the data on concentrations of
PCE, TCE, DCE, and VC in on-site wells as of October 1998. Figures 4 and 5 show the change in
concentrations of TCE, DCE, and VC from December 1996 through October 1998 for Wells MW-4 and
MW-14, respectively. Well MW-14 is located in the source area and MW-4 is in the mid-plume area.
Figure 6 shows the average TCE, DCE and VC concentrations in the on-site monitoring wells within the
remediation area (MW-4, MW-10, W-13, and MW-14).
As shown in Figure 3, the maximum contaminant concentrations measured in groundwater at the site as
of October 1998 were PCE (0.75 ug/L), TCE (17 ug/L), DCE (1,400 ug/L), and VC (180 ug/L). Figures
4 and 5 show that TCE, DCE, and VC concentrations in wells MW-14 and MW-4 were reduced to below
the detectable levels by October 1998. Initial DCE and VC concentrations increased following the first
reagent injection, but then declined by October 1998. According to the site contractor, the trends for
TCE degradation products (DCE and VC) indicate that TCE is being reductively dechlorinated to ethene.
As shown in Figure 6, concentrations of TCE in wells located within the remediation area have decreased
by 99% (3,040 g/L in April 1995 to 4 g/L in October 1998).
A-52
Figure 3: CAH Concentrations - October 1998, Abandoned Manufacturing

Facility, Emeryville, California [2]
MW-4 MW-1
MW-16 PCE <0.5 PCE <0.5

TCE 1.6 TCE 17
DCE 7.4 DCE 1.7
VC 19 VC <1
LEGEND:
VOC CONCENTRATIONS: MW-20

(ALL CONCENTRATIONS SHOWN IN PCE <1.2
MICROGRAMS PER LITER (ug/L)) TCE 11
PCE TETRACHLOROETHENE DCE 53
TCE TRICHLOROETHENE VC 14 MW-10
DCE DICHLOROETHENE
VC VINYL CHLORIDE
PCE <0.5
NS NOT SAMPLED
TCE <0.5 FORMER TCE
DCE 350 DEGREASING
VC <50 AREA
PCE 0.75
TCE 11
PCE <0.5 DCE 7.8
TCE 4.4
MW-13 MW-14 PCE <25 VC <1
MW-17 DCE 2.7
TCE <25 FORMER
VC 1.3 CHROMIUM WASTE
DCE 1,400
STORAGE TANK PCE NS
VC 180
MW-5 TCE NS
PCE <0.5 MW-9 DCE NS
TCE 3.5 VC NS
DCE 2.4 FORMER MW-12
BOILER MW-11
VC <0.5 FACILITY
MW-3A MW-3C
MW-3B PCE NS
PCE <2.5 TCE NS
TCE 13 DCE NS
DCE 8 VC NS
VC 16
Figure 4: Analytical Results for Well MW-4, Abandoned Manufacturing

A-53
Figure 5: Analytical Results for Well MW-14, Abandoned Manufacturing

Figure 6: Average Concentrations, On-Site Wells in Remediation Area,

Abandoned Manufacturing Facility, Emeryville, California [2]
A-54
In addition, the average concentrations of total chromium and hexavalent chromium in the injection area
have been reduced by approximately 98% and 99%, respectively, and some of the wells where historic
hexavalent chromium concentrations were in excess of 100,000 g/L are now less than the detection
limit (5 g/L).
Technology Costs [1]
The overall project cost is approximately $400,000. No further information was provided about the
components of this cost, such as a breakdown of capital or operations and maintenance (O&M) costs.
Summary Observation and Lessons Learned [1,2,3]
The injection of molasses reagent solution created conditions favorable for the reduction in TCE, DCE,
VC, and chromium concentrations in the subsurface. During an 18-month period of full-scale operation,
average concentrations of TCE were reduced by 99%, from more than 3,000 ug/L to 4 ug/L. Average
concentrations of hexavalent chromium were reduced by 99% to below detection levels.
The solution of molasses, supernatent, and water was injected through 91 temporary injection points
installed using a GeoprobeTM. According to the remediation contractor, the use of a GeoprobeTM allowed
the injection points to be installed relatively quickly and at low cost.
A pilot study was conducted prior to the full-scale operation. The pilot study showed that the rate of
reductive dechlorination could be enhanced with the use of an injected molasses solution.
Contact Information
Remediation Contractor:
Daniel L. Jacobs
ARCADIS Geraghty & Miller, Inc.
3000 Cabot Boulevard West, Suite 3004
Langhorne, PA 19047
Telephone: (215) 752-6840
Fax: (215) 752-6879
e-mail: Djacobs@gmgw.com
References
1. E-mail Transmittal from Dan Jacobs, ARCADIS Geraghty & Miller, to Richard Weisman, Tetra
Tech EM Inc. 1999. California Text - ECI EPA.doc. March 24.
2. E-mail Transmittal from Dan Jacobs, ARCADIS Geraghty & Miller, to Richard Weisman, Tetra
Tech EM Inc. 1999. California Text - ECI Figures.xls. March 24.
3. Fax Transmittal from Dan Jacobs, ARCADIS Geraghty & Miller, to Kathy Yager, EPA. 1998. Site 3
- Abandoned Manuafacturing Facility, Emeryville, California. May 18.
A-55
Case Study # 7
Sequential Anaerobic/Aerobic Biodegradation of PCE Field Demonstration
at Watertown, Massachusetts
Site Name, Location Watertown, Massachusetts
EPA ID Number Not provided
Mechanism(s) Anaerobic Reductive Aerobic Oxidation

Dechlorination (Cometabolic (Cometabolic and Direct)
and Direct)
Technology Nutrient Addition Electron Acceptor Addition
Electron Donor Addition (Oxygen)
(Lactate) Electron Donor Addition
(Propane)
Contaminants Targeted TCE, PCE
Period of Operation Anaerobic: November 1996 to July 1997;

Aerobic: August 1997 to ongoing (data available through
October 1997)
The Watertown site has been used since the late 1800's for a variety of operations, including a coal gas
manufacturing plant, which ceased operations in the 1930's, and a metal plating shop, which ceased
operations in 1990. The site is currently being used as a manufacturing facility for electric switch
assembly. Soil and groundwater at the site are contaminated with chlorinated solvents, including
trichloroethene (TCE) and tetrachloroethene (PCE), from past operations and waste disposal practices.
A field demonstration of the Two-Zone Plume-Interception Treatment Technology developed by Harding

Lawson Associates (HLA, formerly ABB Environmental Services, Inc.) was conducted at the Watertown
site under the Superfund Innovative Technology Evaluation (SITE) program. The field demonstration is
currently ongoing. This report addresses the results through October 1997.
Geology/Hydrogeology [1,2]
Soil at the Watertown site consists of about 13 feet (ft) of sand and gravel over approximately 7 ft of silty
sand. Glacial till about 5-ft thick acts as an aquitard and separates the sand layer from the underlying
bedrock (light grey, moderately weathered, amorphous Cambridge Argillite). Groundwater is
encountered approximately 8 ft below ground surface (bgs).
A-56
Matrix Characteristics for the Watertown Site [1]

Soil Type Sand, gravel, and silt
Soil Permeability Not Provided
Depth to Groundwater 8 ft bgs
Fraction of Organic Carbon Not Provided
DNAPL Present None Identified
Hydraulic Conductivity Not Provided
pH Not Provided
Porosity Not Provided
Hydraulic Gradient Not Provided
Groundwater Velocity Not Provided
The technology demonstrated at the Watertown site is a “two-zone” enhanced bioremediation process
that uses sequential anaerobic and aerobic biodegradation processes to degrade PCE and TCE. The first
zone is designed to operate under highly reducing conditions to stimulate anaerobic bacteria to
dechlorinate solvents. While complete degradation can occur under these conditions, according to the
vendor, the process may be slow; whereas under aerobic conditions, completing the degradation of DCE
and VC is relatively fast. Therefore, a second zone, designed to operate under aerobic conditions, is used
to stimulate methanotrophic bacteria to complete degradation by oxidizing DCE and VC.
The field demonstration system, shown in Figure 1, consisted of three injection wells and three extraction
wells. The injection/extraction wells were used to develop a groundwater recirculating cell that covered
a surface area of approximately 10 ft by 20 ft. The wells were constructed of 4-inch diameter PVC pipe
and screened from 13 to 20 ft bgs. Five monitoring wells, with 5-ft screens at 15 to 20 ft bgs, were
located between the injection and extraction wells, and additional monitoring wells were located outside
of the treatment cell, as shown in Figure 2. Nutrients and a carbon source were injected into the
groundwater through the three “upgradient” wells and extracted through the three “downgradient” wells.
During the period of operation, a relatively constant recirculating flow rate of 0.25 gallons per minute
(gpm) was used along with an amendment injection rate of 10 mL/min or about four gallons per day
(approximately 1% of the recirculating flow). The system was operated under anaerobic conditions for
eight months (through late July 1997), then changed to aerobic conditions, as described below.
A-57
Figure 1: Process Diagram [1]
Figure 2: Well Locations [1]
A-58
Anaerobic conditions (November 1996 to July 1997):
Amendments were added to the recirculating flow (continuous addition) to enhance anaerobic biological
dechlorination. Initially, ammonia chloride (25 mg/L), potassium tripolyphosphate (25 mg/L), lactic acid
(100 mg/L), yeast extract (5 mg/L), and sodium hydroxide (at concentrations needed to neutralize the pH
of the amendment batch) were added. From April to June 1997, amendments were pulsed into the system
and the concentration of lactic acid was increased to 250 mg/L to lower redox conditions. In June 1997,
the concentration of lactic acid was increased to 350 mg/L and the system was operated under anaerobic
conditions until late July 1997.
Aerobic conditions (August 1997 to ongoing - data available through October 1997):
In July 1997, “socks” containing oxygen release compound (ORC) were suspended inside the three
injection wells to provide a continuous release of oxygen into the recirculating groundwater flow.
However, it took about a month before aerobic conditions were established. The reason for the lag
period was attributed to the presence of residual carbon in the system that had to be degraded before
aerobic conditions were established in the injection wells.
Because levels of methane in the groundwater had decreased from 150 to 200 ug/L at the beginning of
the demonstration to 50 to 100 ug/L at the start of the aerobic phase, methane was added to the system to
enhance aerobic bacteria activity. Methane was added on a weekly basis starting about two months after
the ORC socks were installed in the injection wells.
Technology Performance [1]
Anaerobic Conditions (November 1996 to July 1997):
During the first four months of operation under anaerobic conditions (November 1996 to February 1997),
limited degradation was observed. Data from February 11, 1997 showed that the concentrations and
relative ratio of PCE and TCE to DCE and VC were relatively uniform throughout the treatment cell.
After four to five months of operation (March to April 1997), significant increases in DCE were observed
along with decreases in TCE concentrations, indicating that reductive dechlorination was occurring.
However, no significant increases in VC concentrations were observed until July 1997, 8 months after
operations began.
Methane levels declined continuously during this period from 0.2 to 0.3 mg/L at the beginning of the
demonstration to 0.05 to 0.1 mg/L in July, 1997. These results indicate that methanotrophic conditions
were not achieved during the anaerobic phase; most of the reductive dechlorination was therefore
attributed to sulfate-reducing bacteria. Sulfide was detected in wells during the last two months of the
anaerobic phase.
By July 1997, TCE concentrations had been reduced from about 12 mg/L at the beginning of the
demonstration to less than 1 mg/L. In addition, there was an overall reduction of about 80% in the mass
of total VOCs, as measured in well IN-2 as of July 1997.
A-59
Aerobic Conditions (August 1997 to ongoing - data available through November 1997):
Data on the aerobic phase of the demonstration have been collected through November 1997. These data
show that VOC levels, primarily DCE and vinyl chloride, have started to decrease in the groundwater. In
addition, DCE epoxide, a transient biodegradation product of aerobic degradation of DCE, was detected
during two sampling events, indicating that aerobic VOC-degrading bacteria have been stimulated. HLA
is continuing to collect data on the aerobic phase of the demonstration. During the aerobic phase over a
period of about 3 months, the degradation half-life for vinyl chloride was approximately 45 days, and for
the latter month of this period was about 22 days.
Technology Cost [4]
The cost for the field-scale pilot study through November 5, 1997 was approximately $150,000. No
estimates were provided about the projected costs for a full-scale system using this technology.
Summary Observations and Lessons Learned [1,2,4]
Under anaerobic conditions, TCE in groundwater was reduced by reductive dechlorination (from 12
mg/L to less than 1 mg/L) and there was an overall reduction of about 80% of the total VOC mass in well
IN-2. Data indicate that methanogenic conditions were not achieved during the anaerobic phase and
most of the reductive dechlorination was attributed to sulfate-reducing bacteria.
During the anaerobic phase, there was a lag time of four to five months before significant reductive
dechlorination was observed; substantial increases in vinyl chloride were observed, eight months after
the demonstration began. Possible reasons for the lag time include: the redox conditions may have been
too high; there may have been insufficient electron donor present (lactic acid); or an acclimation period
may have been required before anaerobic degradation occurred.
A period of about one month was required to establish aerobic conditions after ORC socks were placed in
the wells. This lag time was attributed to the presence of residual carbon that had to be degraded before
aerobic conditions could be established. Initial results indicate that VOC levels, primarily DCE and vinyl
chloride, are decreasing. However, HLA is continuing collect data on the aerobic phase.
The EPA contact for this application stated that future applications should consider not starting in the
winter, start when the anaerobic process can go quickly, use a higher level of lactate, and drive the
oxidation potential down quickly. [4]
Contact Information
EPA Contact:
Dr. Ronald Lewis
U.S. Environmental Protection Agency
26 W. Martin Luther King Dr.
(573) 569-7856
lewis.ronald@epa.gov
A-60
Contractor:
Dr. Willard Murray
Harding Lawson Associates
107 Audubon Road Suite 25
Wakefield, MA 01880
(781) 245-6606
E-mail: wmurray@harding.com
References
1. Lewis, Ronald. Sequential Anaerobic/Aerobic Biodegradation of Chlorinated Solvents: Pilot-Scale

Field Demonstration. Presented at the University of Massachusetts Amherst Conference, October
1997.
2. Designing and Applying Treatment Technologies. Remediation of Chlorinated and Recalcitrant

Compounds. Presented at the First International Conference on Remediation of Chlorinated and
Recalcitrant Compounds. Monterey, California. May 1998.
3. SITE Technology Profile. ABB Environmental Services, Inc. Two-Zone, Plume Interception, In Situ
Treatment Strategy. August 1995.
4. E-Mail from Dr. Ron Lewis, U.S. EPA to Dawn Carroll, U.S. EPA. 1999. Comments on Bio
Document. January 21.
5. Fax Transmittal from Dr. Willard Murray, Harding Lawson Associates, to Richard Weisman, Tetra
Tech. 1999. Case Study #8, In Situ Bio of Chlorinated. February 21.
A-61
Case Study # 8
Bioaugmentation (Accelerated Anaerobic Bioremediation) Field Demonstration
at Area 6 of the Dover Air Force Base, Dover Delaware
Summary Information [1]
Site Name, Location Dover Air Force Base, Area 6, Dover, Delaware
EPA ID Number DE8570024010

Technology Bioaugmentation
Nutrient Addition
Electron Donor Addition (Lactate)
Technology Scale Field demonstration (pilot proof of technology

test)
Contaminants Targeted TCE
Period of Operation Proof of Technology Test: September 1996 to

March 1998 (subject of this case study report)
Testing for Technology Scale-up: April 1998 to
June 1999 (planned)
Full-scale System: Summer 1999 (planned)
Site History/Source of Contamination [1, 2, 5]
Dover Air Force Base (AFB), located in Dover, Delaware, is a 4,000 acre military installation that began
operating in 1941. An estimated 23,000 cubic feet of waste, including solvents, waste fuels and oils, and
a variety of other wastes, were disposed at the site from 1951 to 1970. Soil and groundwater at the base
were found to be contaminated with volatile organic compounds (VOCs), including TCE and PCE, and
with heavy metals, including arsenic and cadmium. In March 1989, the site was listed on the National
Priorities List. During a remedial investigation, “Area 6" was one of the areas at the base that was
determined to have been contaminated with chlorinated solvents; a plume of VOCs was identified in
groundwater in this area. Based on the results of that investigation as well as additional sampling, the
area was selected for pilot testing of a bioaugmentation process. Concentrations of PCE, TCE, cis-DCE,
and vinyl chloride in the pilot area before the test were 46 ug/L, 7,500 ug/L, 1,200 ug/L, and 34 ug/L,
respectively.
The remediation of Dover AFB is managed by EPA Region 3 and the Delaware Department of Natural
Resources and Environmental Control (DNREC). Records of Decision (RODs) have been signed for
nine of the 12 operable units at the site, including operable units within Area 6. Interim RODs were
signed in September 1995 that identify the following technologies for remediation at Dover: anaerobic
reductive dehalogenation, cometabolic bioventing, and monitored natural attenuation. The pilot test was
A-62
performed as part of the Bioremediation Consortium of the Remediation Technology Development

Forum.
Geology/Hydrogeology [1, 2, 5]
Dover AFB is underlain by glacial-fluvial deposits of sand, silt, and clay of the Pleistocene Columbia
Formation. Within the Area 6 pilot test area, the saturated portion of the formation consists of fine,
medium, and coarse sands and is about 38 feet thick. The aquifer acts as one unconfined unit that
includes three zones (approximately equal thickness) - an upper zone of fine sand (0 to 12 ft below
ground surface or bgs), an intermediate zone of medium sand (12 to 25 ft bgs), and a deep zone also of
medium sand (25 to 48 ft bgs). Groundwater is found in the intermediate and deep zones, starting at 10
to 12 ft bgs.
Matrix Characteristics for Area 6 of the Dover AFB Site [1, 5]

Soil Type Sand with varying amounts of clay, silt and gravel.
Fine-grained clay and silt to a depth of 5 ft bgs;
underlain by more permeable layer of silt and sand.
Soil Permeability Not provided
Fraction of Organic Carbon 0.2
Thickness of Aquifer 38 ft (saturated portion)
Hydraulic Conductivity 60 ft/day
pH 5.6 (average)
Porosity 30%
Hydraulic Gradient 0.001 ft/ft
Groundwater Velocity 60 ft/year (0.16 ft/day)
Technology Description [1, 2, 5]
Groundwater flow and three-dimensional transport models (MODFLOW and MT3D) were used in
designing the pilot system. The models were used to simulate groundwater flow under different test
scenarios and to simulate the three-dimensional transport of substrates.
The pilot system, shown in Figure 1, included three extraction or pumping wells and three injection
wells, each screened to a depth of 38 to 48 ft bgs. The pilot system was designed to operate as semi-
isolated or “closed-loop” recirculation cell and the wells formed a rectangular, hydraulically-controlled
cell that was 40 ft wide and 60 ft long.
A-63
Figure 1: Process Diagram of Accelerated Anaerobic Biodegradation at Dover AFB [3, 5]
The pumping wells were operated at a combined rate of 3.06 gpm (1.03 gpm each), providing a residence
time of about 60 days for groundwater from the deep zone of the aquifer.
Monitoring wells, shown in Figure 2, were located within the cell (wells 3D, 6D, 7D, 8D, 9D, and 18D)
and outside of the cell. Groundwater samples were collected from the monitoring wells and analyzed for
field parameters such as dissolved oxygen and temperature, water chemistry such as total organic carbon
and ammonia, injected amendments, and VOCs and degradation products (PCE, TCE, DCE, vinyl
chloride, ethene, ethane, and methane). The interior wells were sampled weekly; the exterior wells were
sampled monthly. Sampling frequency was determined based on the results of the groundwater
modeling. In addition, flow rate, total flow, and pressure were monitored. Prior to the start of the pilot
system in September of 1996, a tracer test was conducted to verify groundwater flow patterns, collect
data to calibrate and verify modeling results, and to verify system operation.
A-64
Figure 2: Well Locations [5]
40 ft
Key:
IW = injection well
EW = extraction well
AA = monitoring well
PZ = piezometer
The system was designed to allow operation with any combination of extraction pumps. The extracted
groundwater was combined into a single stream and passed through a filter to remove suspended solids
that could cause fouling problems in the injection wells. Using high precision, low volume metering
pumps, substrate and nutrients were injected into the combined groundwater stream downstream of the
filter. The substrate used in the pilot test was sodium lactate, and for most of the pilot test was delivered
as 200 mg/L as carbon to all of the injection wells. The nutrients added to the system were ammonia and
phosphate.
Terra Systems, Inc., of Wilmington, Delaware, operated the pilot system at Dover. For the pilot test, a
method of cycling substrate and nutrient injection was used to minimize biogrowth in the system tanks
and at the well screens. The substrate was fed into the system for 3.75 days, followed by 8 hours of
circulation with groundwater (unamended). The nutrient was then injected into the system for 2.75 days,
followed by 8 hours of circulation with unamended groundwater. This cycle was used throughout the
pilot test.
A-65
Technology Performance [1, 2, 5]
The goals of the pilot test, as specified in the design report, were to 1) demonstrate that TCE degradation
can be stimulated in the deep portion of an aquifer; 2) confirm that degradation will proceed to nontoxic
end products; 3) develop operation and cost data for a full-scale system; and 4) document the
methodology used in the pilot system.
The proof of technology portion of the pilot system was operated 18 months, from September 1996 to
March 1998. During the first five months of operation, the concentration of TCE in the groundwater
gradually decreased, while concentrations of cis-DCE increased slightly. There was no change in the
concentrations of vinyl chloride or ethene, indicating that limited dechlorination was occurring.
In January 1997, the substrate feed concentration was increased from 100 to 200 mg/L as carbon in an
attempt to stimulate dechlorination. In the monitoring well nearest the injection wells (well 6D), an
increase in DCE concentrations was observed almost immediately, along with a decrease in TCE
concentrations. Total organic carbon and lactate concentrations also reportedly increased in monitoring
wells near and downgradient of the injection wells. However, there was no evidence that dechlorination
beyond DCE to vinyl chloride or ethene was occurring. Analyses of the geochemistry of the aquifer
before and after test cell operation showed that the indigenous bacteria consumed the lactate; the test cell
area showed reductions in nitrate and sulfate concentrations to nondetectable levels and the generation of
methane.
In February 1997, a decision was made by the RTDF to evaluate the potential for bioaugmentation for
this pilot system. The bioaugmentation plan was approved by EPA Region 3 and DNREC. A number of
microcosm tests and column studies were performed with known dechlorinating strains from other sites
to evaluate candidate cultures. Based on the screening tests, a culture from the Department of Energy’s
Pinellas site in Largo, Florida was selected for use at Dover. The culture was grown in liquid media to
allow aqueous addition for the pilot test.
Between February and May 1997, TCE continued to degrade to DCE, but the DCE was not further
degraded to vinyl chloride. Consequently, the RTDF decided to implement bioaugmentation, in an
attempt to further degrade the DCE to vinyl chloride. On June 5, 1997, 180 liters of the aqueous culture
(augmenting solution) was injected into the cell through injection well no. 2. On June 20, 1997, another
171 liters of augmenting solution were injected through the same well. Substrate feed concentrations
were maintained at 200 mg/L as carbon after bioaugmentation.
For the first 90 days following bioaugmentation, TCE concentrations continued to decrease and DCE
concentrations continued to increase; however, there was no evidence of vinyl chloride or ethene in the
groundwater. At the beginning of September 1997, vinyl chloride and ethene began appearing in wells
closest to the injection wells (well 6D, 18D, and 7D), indicating that DCE degradation was occurring.
Vinyl chloride and ethene continued to be detected in downgradient wells within the recirculation cell
(wells 3D, 8D, and 9D), and by December 1997, these constituents had been detected in wells inside the
recirculation cell but outside the bioaugmented area of the recirculation cell (well 14D and 11D).
Between December 1997 and February 1998, the concentrations of ethene increased, while TCE and
DCE concentrations continued to decrease. By March 1998, all TCE and DCE in the groundwater were
converted to ethene and between 75 and 80% of the TCE and DCE had been recovered as ethene,
indicating that the bioaugmentation was successful in destroying TCE by reductive dechlorination.
Reasons for incomplete recovery included biodegradation of ethene or a decline in overall plume
contaminant concentrations during the pilot test. Data from a background well upgradient from the pilot
A-66
test showed an overall decline in VOC concentrations; however, there was no significant change in the
ratio of TCE to DCE in this location.
The proof of technology portion of the pilot test was completed in March 1998. From April 1998
through June 1999, the test is focusing on testing of parameters involved with technology scale up.
Well Plugging During Pilot Test [1]
During the operation of the pilot test, plugging of the injection wells was a problem. As early as 50 days
from the start of the system (mid-October 1996), the injection wells became plugged. Biogrowth in the
injection wells and on the well screens was the primary cause of the plugging problem. The injection
wells were redeveloped and operations resumed. In March 1997, the injection wells again became
clogged and another well redevelopment was performed. However, the redevelopment was not as
successful as the one performed in October. In an attempt to alleviate the plugging, the substrate was
changed to lactic acid in April 1997. However, no appreciable increase in the efficiency of the injection
wells was noted and the substrate feed was changed back to sodium lactate on June 8, 1997. Routine
brushing of screens and hydrogen peroxide treatments were implemented to keep the wells open.
Future Plans [4, 5]
Funding has been approved for a full-scale application of the technology at an area upgradient of the pilot
test. Treatment is scheduled to begin in the summer of 1999. The full-scale system is planned to be a
one pass flow through system, with wells used to control the flow of groundwater, directing it through
one to two “gates”.
The U.S. Army Corps of Engineers (USACE) and Dames and Moore will design the full-scale system.
Technology Cost [2]
Costs for in situ enhanced anaerobic biodegradation are presented in Table 1. The total capital costs were
$285,563. The total operating costs were $164,962 for the first three months of operation (through
November 30, 1996) and $522,620 for the first fifteen months of operation (through November 30,
1997).
According to the RTDF contact, a typical full-scale bioaugmentation system would cost substantially less
than the system used in the pilot test at Dover. The pilot system included oversized stainless steel wells,
wire-wrapped screens, duplicate control equipment, a monitoring well network that allowed for three-
dimensional modeling, a frequent sampling and analysis program, one full-time equivalent operator, and
a control building that was designed to be reused to store gymnasium equipment after the project was
completed.
A-67
Table 1. Costs for In Situ Enhanced Anaerobic Biodegradation at Dover AFB [2]
Element Cost ($)

Capital
Site preparation 5,742
Structures 39,818
Process equipment and appurtenances 24,067
Utility infrastructure hookups (electricity, gas, 50,716
sewer)
Installation labor 8,600
Monitoring wells 78,306
Dedicated pumps 18,224
Other equipment 5,690
Injection wells 30,400
Extraction wells 24,000
Total Capital Costs 285,563

Operating Costs
Period Startup through Startup through
11/30/96 11/30/97
Direct Labor 74,096 168,850
Materials:
Substrate 771 6,700
Nutrients 230 580
Supplies 2,272 10,140
Laboratory analysis 75,196 286,000
Sampling 2,018 11,000
Shipping 5,061 22,900
Overhead 975 7,000
Injection well maintenance 4,343 9,450
Total Operating Costs 164,962 522,620
A-68
The Dover AFB pilot project was the first successful bioaugmentation project using live bacteria from
another site to destroy TCE using reductive dechlorination.
Data from the pilot test indicated that an extended period of time was required for the bacteria to exhibit
functional dechlorination. At the start of bioaugmentation, lag periods of about 180 days between
bioaugmentation and complete reduction of TCE and DCE to ethene were observed, including a 90-day
lag period before vinyl chloride was first observed. The factors that likely contributed to the lag times
included slow growth under the conditions at Dover AFB and time required for acclimatization. In
addition, the initial mass of bacteria injected during the augmentation (about 35 grams) was relatively
small, and may have increased the time required to grow a bacteria population at Dover that was
sufficient to dechlorinate the target contaminants.
Laboratory studies are recommended to ensure that the bacterial culture selected will be effective given
the site conditions and contaminants. For the Dover pilot test, a number of laboratory studies were
performed using soil and groundwater from the Dover site to evaluate candidate cultures. Screening
parameters included growth in liquid culture, growth on lactate, and dechlorination to ethene.
Injection well plugging was a problem during the pilot test. Several methods were used to keep the wells
unplugged including cleaning the well screens with wire brushes and pumping out residue from the
screened interval, using hydrogen peroxide to clean the wells, and changing substrates from sodium
lactate to lactic acid. Hydrogen peroxide proved the most effective technique for keeping the wells from
clogging.
Contact Information [2,3]
Remedial Project Manager:

R. Drew Lausch
U.S. EPA Region 3
1650 Arch Street
Philadelphia, PA 191103
(215) 814-3359
email: lausch.robert@epa.gov
ITRC Contact:
Paul Hadley
ITRC In Situ Bioremediation Technical Task Team Leader
California Environmental Protection Agency
Department of Toxic Substances Control
PO Box 806
Sacramento, CA 95814
(916) 324-3823
A-69
RTDF Contact:
Dr. David Ellis
DuPont Engineering
Barley Mill Plaza 27-2234
P.O. Box 80027
Wilmington, DE 19880-0027
(302) 892-7445
email: david.e.ellis@usa.dupont.com
References
1. Ellis, David et al. Bioaugmentation for Accelerated In Situ Anaerobic Bioremediation.

Environmental Science and Technology. Volume 34. No. 11. Page 2254-2260. June 1, 2000.
2. Cost and Performance Reporting for In Situ Bioremediation Technologies. Prepared by the Interstate
Technology and Regulatory Cooperation Work Group. December 1997.
3. RTDF Update. May 1998.
4. Telephone Conversation. Richard Weisman, Tetra Tech EM Inc. and Mick Mikula, Dover AFB.
December 1, 1998.
5. Telephone Conversation. Richard Weisman, Tetra Tech EM Inc. and Dr. David Ellis, DuPont.
January 29 and February 24, 1999.
A-70
Case Study # 9
Cometabolic Bioventing Field Demonstration at Building 719,
Dover Air Force Base, Dover Delaware
Site Name, Location Dover Air Force Base, Building 719, Dover,
Delaware
EPA ID Number DE8570024010
Technology Electron Acceptor Addition (Oxygen)

Electron Donor Addition (Propane)
Technology Scale Field Demonstration (Pilot Test)
Media/Matrix Treated Soil
Contaminants Targeted TCE, 1,1,1-TCA, cis-1,2-DCE
Period of Operation Propane acclimation period: December 1997 to

April 1998
Bioventing operation: May 1998 to July 1999
Site History/Source of Contamination [1, 2, 3]
Dover Air Force Base (AFB), located in Dover, Delaware, is a 4,000 acre military installation that began
operating in 1941. Building 719 is a jet engine inspection and maintenance shop where a variety of
materials, including solvents, JP-4 fuel, and hydraulic fluids, have been used in shop operations. Until
the mid-1960s, wastes from the shop were discharged to a drainage ditch and sanitary sewer. In addition,
the northeast area of the building is the location of two former leaking underground storage tanks
(USTs), an oil/water separator, and a former neutralization tank. The results of investigations showed
that soil and groundwater in the area of the former USTs were contaminated with fuel (BTEX - benzene,
toluene, ethylbenzene, and xylene) and solvents. Results of samples of the vadose zone of Building 719
found concentrations of TCE as high as 250 mg/kg; TCA ranging from 10 to 1,000 mg/kg; and DCE
ranging from 1 to 20 mg/kg. TCE concentrations in groundwater were reported as high as 19,000 ug/L.
Dover AFB was listed on the National Priorities List in March 1989. The remediation of Dover AFB is
managed by EPA Region 3 and the Delaware Department of Natural Resources and Environmental
Control. Interim RODs were signed in September 1995 that identify the following technologies for
remediation at Dover: anaerobic reductive dehalogenation, cometabolic bioventing, and monitored
natural attenuation. The area in the vicinity of Building 719 was selected for a pilot test of cometablic
bioventing. The cometabolic bioventing pilot test was conducted for the In Situ Bioremediation of
Chlorinated Solvents Work Group of the Remediation Technology Development Forum (RTDF). The
RTDF also conducted a pilot test of bioaugmentation of groundwater at Dover AFB, which is the subject
of Case Study number 8.
A-71
Geology/Hydrogeology [1,2,7]
Dover AFB is underlain by glacial-fluvial deposits of sand, silt, and clay of the Columbian Formation.
The soil in the vicinity of Building 719 was sand with clay, silt, and gravel. Depth to groundwater was 6
to 10 feet (ft) below ground surface (bgs).
Matrix Characteristics for the Building 719, Dover AFB Site [1,6]

Soil Type Sand with varying amounts of clay, silt and
gravel. Fine-grained clay and silt to a depth of 5
ft bgs; underlain by more permeable layer of silt
and sand.
Soil Permeability 1.9x10-7 to 7.0x10-8 cm2
Hydraulic Conductivity 0.017 to 0.052 cm/sec
pH - Soil 6.0 to 11.0 (median 7.7)
Total Chloride 8 mg/kg (median)
Total Kjeldahl Nitrogen 42 mg/kg (median)
Total Phosphorus 30 mg/kg (median)
Total Organic Carbon 0.11% (w/w) (median)
Technology Description and Performance [1,2,4,5,7]
The primary objectives of the pilot test were to determine the efficiency and demonstrate the viability of
an in situ cometabolic bioventing process for CAHs under field conditions (benzene, toluene,
ethylbenzene, and xylene were not targeted for treatment). Prior to the pilot test, laboratory tests were
performed on soils from the area of Building 719 to evaluate candidate substrates. Propane was selected
because of its ability to stimulate cometabolic activity towards both TCA and TCE.
Based on the results of site investigations in the vicinity of Building 719, the location of the test plot was
identified as an area of high concentrations of chlorinated aliphatic hydrocarbons (CAHs). The test plot
was approximately 30 ft long, 20 ft wide, and 10 ft deep with a volume of 4,500 ft3 of soil . The mass of
soil in the test plot was estimated to be 450,000 lbs, based on an assumed density of 100 lbs/ft3. Prior to
the pilot test, a total of 80 soil samples were taken from the test plot to provide a contaminant profile and
to estimate the mass of contaminants in the test plot. This information was used to develop an order of
magnitude estimate of the mass of CAHs and BTEX in the soil for a total gross estimate of 26 lbs of
CAH and BTEX constituents in the subsurface soils of the test plot. 1,1,1-TCA made up approximately
70% of the total estimated mass of contaminants.
The pilot system includes three injection wells, screened to a depth of 10 ft bgs, which was the lowest
expected water table elevation. In addition, 13 soil gas monitoring points were installed to monitor soil
A-72
gas conditions throughout the demonstration. Each of the soil gas monitoring points was equipped with
two gas probes (one at a depth of 4-5 ft and one at a depth of 8-9 ft bgs). Another 11 “temporary” soil
gas monitoring points were installed for use during initial air permeability testing, and were used during
system operation to monitor soil gas. A blower and a mass flow controller were used to inject a mixture
of air and propane (300 ppm in air) through the three wells at a rate of 1 cfm.
Figure 1 shows histograms of initial and final concentrations of TCE, TCA, DCE and chloride from the
soil in the test plot, including reductions in the concentrations observed for TCE, TCA and DCE during
treatment. These reductions can be at least partly attributable to biodegradation by noting the large
increase in the soil chloride levels during treatment. Chloride is a product of the biodegradation of
chlorinated solvents.
Figure 1. Histograms of initial and final TCE, TCA, DCE and chloride concentrations from soil in
the cometabolic bioventing test plot [7]
80 80
T=0 T=0
70 70
T = Final T = Final
Frequency - No. of Samples

60 60
50 50
40 40
30 30
20 20
10 10
0
0
5
00
-5
-5
0
5
.5
0.
5
-1
10
10
-2
-1
2
-0
-1
<
5
1
0.
0.
5
>
>
25
0.
25
5
<
0.
Soil TCE Levels (mg/kg) Soil 1,1,1-TCA Levels (mg/kg)
80 70
T=0 T=0
70
T = Final T=Final
60
60
50
50
40
40
30
30
20 20
10 10
0 0
.5
-5
25
-1
20
-0
-2
0
20
20
00
0.
1
5
>
-4
-6
-8
12
0.
25
-1
-1
<
<
>
20
40
60
0.
80
0
10
Soil 1.2-cis DCE Levels (mg/kg) Soil Chloride Levels (mg/kg)
A-73
Technology Cost
Cost data were not available at the time of this report.
The researchers provided the following observations:
& Over a 14-month period of operation, cometabolic bioventing was successful in removing TCE,
TCA and DCE from soils in the test plot.
& Laboratory treatability testing identified propane as a useful cosubstrate to drive cometabolism of
TCE and TCA (DCE may have been biodegraded via cometabolism or by direct aerobic
bioprocesses). The lab studies also successfully predicted the need for a significant time period
(weeks) for the test plot to begin using propane after initial exposure. Thus, a cosubstrate
acclimation period may be a common element of cometabolic bioventing startup.
& In aerobic bioventing, the amount of fuel biodegraded during treatment can be estimated by
oxygen use. In cometabolism, oxygen use and chlorinated solvent biodegradation are not
stoichiometrically related. Thus, in cometabolic bioventing, indirect measures must be employed
to show that biodegradation is removing contaminant. These may include chloride accumulation
in soil, and previous lab studies using site-contaminated soil which have shown that
biodegradation of cosubstrate (which can be measured in the field using a shut down test) implies
biodegradation of the chlorinated solvent. There is a need for innovative approaches to proving
that biodegradation is occurring in the field.
Contact Information
Remedial Project Manager:

Darius Ostrauskas
U.S. EPA Region 3
1650 Arch Street (3HS50)
Philadelphia, PA 191103
(215) 814-3360
ostrauskas.darius@epa.gov
EPA Contact for Demonstration:

Dr. Gregory Sayles
U.S. EPA (mail stop 420)
26 W. Martin Luther King Drive
(513) 569-7607
Fax: (513) 569-7105
E-mail: sayles.gregory@epa.gov
A-74
References
1. Cometabolic Bioventing at Building 719. Dover Air Force Base, Dover, Delaware. Pilot Test Plan.
Remediation Technologies Development Forum. September 1997.
2. Sayles, G.D. at al, Abstract, Development of Cometabolic Bioventing for the In-Situ Bioremediation
of Chlorinated Solvents, April 1998.
3. U.S. EPA Region 3. Hazardous Site Cleanup Division. Dover AFB. August 1998.
4. RTDF Update. May 1998.
5. E-Mail from Diane Dopkin, EMS, to Dawn Carroll, U.S. EPA. Bioventing. February 18, 1999.
6. Fax Transmittal from Dr. Greg Sayles, U.S. EPA to Dawn Carroll, U.S. EPA. Comments on
Cometabolic Bioventing Case Study. February 22, 1999.
7. E-mail from Dr. Greg Sayles, U.S. EPA to Linda Fiedler, U.S. EPA. Comments on Case Study #9.
November 19, 1999.
A-75

Biorremediacion Estim, Ulada

Uploaded by

Copyright:

Available Formats

Biorremediacion Estim, Ulada

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Biorremediacion Estim, Ulada

Uploaded by

Copyright:

Available Formats

United States Solid Waste and EPA 542-R-00-008

Environmental Protection Emergency Response July 2000 (revised)

EPA Engineered Approaches to

ENGINEERED APPROACHES TO IN SITU BIOREMEDIATION

U.S. Environmental Protection Agency

NOTICE AND DISCLAIMER

U.S. EPA/National Service Center for Environmental Publications

1.0 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1

2.0 BIOREMEDIATION MECHANISMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1

2.1 TYPICAL CAHs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1

2.4.1 Aerobic Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-13

3.0 IN SITU BIOREMEDIATION TECHNOLOGIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1

3.1 TECHNOLOGIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1

4.0 SELECTION AND IMPLEMENTATION OF IN SITU BIOREMEDIATION

4.1 TECHNOLOGY SELECTION AND IMPLEMENTATION . . . . . . . . . . . . . . . . . . . . 4-1

4.1.1 Site Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1

4.2 ADDITIONAL SELECTION FACTORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-11

5.0 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-1

6.0 ADDITIONAL INFORMATION SOURCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1

TABLE OF CONTENTS (continued)

APPENDIX A CASE STUDIES AND SUMMARIES OF SITES USING IN SITU

2-1 CAHs Commonly Identified as Environmental Contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2

3-1 Components of In Situ Bioremediation Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2

A-1 Summary of Case Studies of In Situ Bioremediation Technology Applications . . . . . . . . . . . A-2

GLOSSARY OF KEY TERMS

Abiotic Nonbiological process; also used to refer to nonbiological degradation

ADP Adenosine diphosphate

CERCLA Comprehensive Environmental Response, Compensation, and Liability Act

Fe0 Zero-valent iron

HCl Hydrogen chloride

UIC Underground Injection Control

2.0 BIOREMEDIATION MECHANISMS

• Typical CAHs (Section 2.1)

2.1 TYPICAL CAHs

2.2 PHYSICAL AND CHEMICAL PROPERTIES OF CAHs

Exhibit 2-1: CAHs Commonly Identified as Environmental Contaminants

Name Common Name(s) Abbreviation1 Common Waste Sources

Exhibit 2-2: Molecular Structures of Common CAHs

Source: Modified from Sawyer and others 1994

Exhibit 2-3: Chemical and Physical Properties of CAHs

PCE 4 165.8 1.62 150 17.8 2.60 0.0153

TCE 3 131.4 1.46 1,100 57.9 2.38 0.0091

cis-DCE 2 96.9 1.28 3,500 208 0.70 0.0037

trans-DCE 2 96.9 1.28 6,300 324 0.48 0.0072

1,1-DCE 2 96.9 1.21 2,250 600 1.84 0.018

VC 1 62.5 gas 2,670 2,660 1.38 0.315 (5)

1,1,1-TCA 3 133.4 1.34 1,500 123 2.50 0.008

1,1,2-TCA 3 133.4 1.44 4,500 30 2.47 0.0012

1,2-DCA 2 99.0 1.26 8,520 64 1.48 0.00098

1,1-DCA 2 99.0 1.18 5,500 182 1.79 0.0059

CA 1 64.5 gas 5,700 1,064 1.52 to 2.16 (4) 0.0085

CT 4 153.8 1.59 757 90 2.64 0.0304

CF 3 119.4 1.48 8,200 151 1.97 0.00435

MC 2 84.9 1.33 20,000 362 1.30 0.00268

CM 1 50.5 gas 6,500 4,310 0.95 0.0452 (5)

2.3 TRANSPORT PROCESSES

Exhibit 2-4: Phase Equilibrium Mechanisms

Source: Modified from Huling, S.G. and J.W. Weaver 1991

Exhibit 2-5: Example CAH Subsurface Transport Processes (DNAPL Source)