United States

Environmental Protection

Agency

Solid Waste and 

Emergency Response 

(5102G)

EPA 542-R-00-008

July 2000 (revised)

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

i

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

ii

FOREWORD

Halogenated volatile organic compounds, including chlorinated solvents, are the most frequentlyoccurring 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 aboveground 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-bycase basis. Technology expertise and sometimes treatability studies also are required to make a final

remedy decision.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

iii

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
TABLE OF CONTENTS (continued)

Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

iv

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
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

v

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
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

vi

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 

(cometabolic)

Microbial breakdown of a contaminant during which a contaminant is

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 

(direct)

Microbial breakdown of a compound during which the compound serves as

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 

dechlorination

(cometabolic)

A biodegradation reaction in which a chlorinated hydrocarbon is reduced by

an enzyme or cofactor produced during microbial metabolism of another

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 

dechlorination

(direct)

A biodegradation reaction in which bacteria gain energy and grow as one or

more chlorine atoms on a chlorinated hydrocarbon are replaced with

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

vii

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 

hydrocarbons

(CAHs)

Manmade, chlorine-containing organic compounds widely used as solvents

and degreasers in various industries. Typical CAHs include

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 

phase liquids

(DNAPL)

Chlorinated solvents that are minimally soluble in water, more dense than

water, and present in concentrations large enough to form pools of free

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

viii

Electron donor A compound capable of supplying (giving up) electrons during oxidationreduction 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

Bioremediation of organic contaminants by microbes supplemented by

increasing the concentration of electron acceptors, electron donors, or

nutrients in groundwater, surface water, and soil.

Exogenous bacteria

(also called nonindigenous) 

Bacteria that have been obtained from a source other than the native site.

Groundwater 

recirculation

treatment system

A closed-loop, hydraulically-contained system based on a design of downgradient extraction and upgradient injections wells; sometimes referred to as

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

attenuation (also

known as Passive

bioremediation)

Use of natural subsurface processes, such as dilution, volatilization,

biodegradation, adsorption, and chemical reactions with subsurface

materials to reduce contaminant concentrations.

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

ix

LIST OF ACRONYMS

ADP

ATP

BFSS

CA

CAHs

Adenosine diphosphate

Adenosine triphosphate

Bioremediation in the Field Search System

Chloroethane

Chlorinated aliphatic hydrocarbons

CERCLA

CF

CFR

CM

CO2

CT

Comprehensive Environmental Response, Compensation, and Liability Act

Chloroform

Code of Federal Regulations

Chloromethane

Carbon dioxide

Carbon tetrachloride

DCA

DCE

DNAPL

EPA

EPA REACH IT

Dichloroethane

Dichloroethene

Dense non-aqueous phase liquid

U.S. Environmental Protection Agency

EPA REmediation And CHaracterization Innovative Technologies

Fe0

Fe2+

GW

H2O2

Zero-valent iron

Iron (II) ion

Groundwater

Hydrogen peroxide

HCl

HRC

HSLDR

LNAPL

Hydrogen chloride

Hydrogen release compound

Hydrogen sulfide ion

Land Disposal Restrictions

Light non-aqueous phase liquid

MC

MNA

NAD

NAPL

ORC

Methylene chloride

Monitored natural attenuation

Nicotinamide adenine dinucleotide

Non-aqueous phase liquid

Oxygen release compound
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

x

PCE

PRB

RCRA

TCA

TCE

Tetrachloroethene

Permeable reactive barrier

Resource Conservation and Recovery Act

Trichloroethane

Trichloroethene

UIC

UST

VC

Underground Injection Control

Underground storage tank

Vinyl chloride
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

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).

Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

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).
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

2-1

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

2-2

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 

(-ethylene)

Acetylene dichloride trans-DCE Solvent waste, degradation product of

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

tetrachloride

CT Solvent waste, fire extinguisher waste

Trichloromethane Chloroform, 

methane trichloride

CF Solvent waste, anesthetic waste, waste

degradation product of CT

Dichloromethane Methylene chloride,

methylene dichloride

MC Solvent waste, degradation product of CT

Chloromethane Methyl chloride,

monochloromethane

CM Refrigerant waste, degradation product of

CT

Notes: 1

Abbreviations are based on the names in bold italic type.

Sources: Sawyer and others 1994; Merck 1989 
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

2-3

Exhibit 2-2: Molecular Structures of Common CAHs

Source: Modified from Sawyer and others 1994
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

2-4

Exhibit 2-3: Chemical and Physical Properties of CAHs

Compound

Number of

Substituted

Chlorine

Atoms

Molecular

Weight

(g/mole)1

Liquid Density (g/ml

@ 20 °F/4 °C)1

Aqueous

Solubility (mg/L

@ approx. 25°C)2

Vapor Pressure

(mm Hg @ 25 °C)2

Log KOW

(Octanol/Water

Partition Coefficient)2

Chlorinated EthenesPCE 4 165.8 1.62 150 17.8 2.60 TCE 3 131.4 1.46 1,100 57.9 2.38 cis-DCE 2 96.9 1.28 3,500 208 0.70 trans-DCE 2 96.9 1.28 6,300 324 0.48 1,1-DCE 2 96.9 1.21 2,250 600 1.84 VC 1 62.5 gas 2,670 2,660 1.38 Chlorinated Ethanes1,1,1-TCA 3 133.4 1.34 1,500 123 2.50 1,1,2-TCA 3 133.4 1.44 4,500 30 2.47 1,2-DCA 2 99.0 1.26 8,520 64 1.48 1,1-DCA 2 99.0 1.18 5,500 182 1.79 CA 1 64.5 gas 5,700 1,064 1.52 to 2.16 (4) Chlorinated MethanesCT 4 153.8 1.59 757 90 2.64 CF 3 119.4 1.48 8,200 151 1.97 MC 2 84.9 1.33 20,000 362 1.30 CM 1 50.5 gas 6,500 4,310 0.95 

Notes:1. Data from Merck Index 19892. Data from EPA 19863. Data from Gossett 19874. Data from EPA 19985. 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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

2-5

Exhibit 2-4: Phase Equilibrium Mechanisms

 and Defining Properties of CAHs

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.

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 nonaqueous 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
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

2-6

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

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.

Source: Modified from Sims and others 1992
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

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

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

2-8

For the most part, the microorganisms that carry out the

bioremediation of the CAHs are single-celled procaryotic

bacteria. As living organisms, the bacteria require a

source of food to survive and propagate. This

requirement, or the bioenergetics of a bacterial system, is

defined by the thermodynamics of the processes used by

the microbes to derive energy and raw materials from

substrates and to use them to carry on biological processes

and reproduce. The figure below depicts the basic

bioenergetics of a typical microbial system.

ADP = adenosine diphosphate

ATP = adenosine triphosphate

In general, the mediating bacteria collect energy in the

form of electrons by a chemical reduction-oxidation

(redox) reaction (or photosynthesis). The energy is

generated in a redox reaction from the transfer of

electrons from an electron donor (the organic contaminant

in aerobic oxidation (direct)) to an electron acceptor

(oxygen in an aerobic reaction). The energy gained is

stored as high energy compounds, such as ATP and lowenergy compounds, such as nicotinamide adenine

dinucleotide (NAD). A portion of the stored energy is

used to conduct the biological processes necessary for cell

maintenance and reproduction. In addition, cell buildingblock materials are required in the form of carbon and

other nutrients (such as nitrogen and phosphorus). The

thermodynamics of a given system defines the energy that

is available from a substrate, the energy transfer efficiency

losses that will occur, and the portion of the available

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. 

Typically, bacteria that are involved in the

biodegradation of CAHs in the subsurface are

chemotrophs (bacteria that derive their energy from

chemical redox reactions) and use organic compounds as

electron donors and sources of organic carbon

(organoheterotrophs). However, lithotrophs (bacteria

that use inorganic electron donors) and autotrophs

(bacteria that use carbon dioxide as a carbon source) also

may be involved in degradation of CAHs.

CAH-degrading bacteria are classified further by the

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

determined

by relative

energy

generation

Bacteria

Electron

Acceptor

Class

Predominant

CAH

Biodegradation

Mechanism

Approximate

Redox

Potential

(volts)1

Most

dominant

|

|

|

|

|

|

|

|

|

\/

Least

dominant

Oxygenreducing

(aerobes)

Aerobic

oxidation +0.82

Nitratereducing

Reductive

dechlorination

+0.74

Mangane

se (IV)-

reducing

+0.52

Iron (III)-

reducing -0.05

Sulfatereducing -0.22

Carbon

dioxidereducing

(methanatrophs)

-0.24

1

 Standard redox potentials at pH of 7

Exhibit 2-6: Microbial Biodegradation and Ecology

Sources: Anderson, R.T., and D.R. Lovley 1997; McCarty, P.L. 1971; EPA 1998
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

2-9

Exhibit 2-7: Redox Zones of a Typical Petroleum Plume in an Aerobic

Aquifer (Areal View)

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.

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

2-10

Exhibit 2-8: Selected Information on CAH Degradation Mechanisms

Degradation

Mechanism CAH Conditions Reported Bacteria Reported Rate Data P

AEROBIC OXIDATION

Aerobic oxidation(direct)

DCE, VC Aerobic Not reported Observed 2nd-order Michaelis-Menton

kinetics (Vmax = 5.1 and 12.4

umol/L/D for DCE and VC,respectively)

CO

DCE, VC, DCA,CA, MC, CM

Aerobic Not reported Not reported CO

Aerobic oxidation(cometabolic)

TCE Aerobic, electron donor (phenol,

toluene, benzene)

Burkholderia cepacia G4,PR1301

Not reported CO

TCE Aerobic, electron donor (toluene) Not reported 1st order

0.26-0.4 mol/d

Norep

TCE, DCE, VC,TCA, CF, MC

Aerobic, electron donor(methane, aromatics, ammonia)

Not reported Not reported CO

ANAEROBIC REDUCTIVE DECHLORINATION

Anaerobic reductivedechlorination(dehalorespiration)

PCE, TCE, DCE,VC, DCA

Anaerobic, electron donor(hydrogen or fermentivehydrogen source), relatively low

Hydrogen partial pressure

PER-K23, Dehalospirilliummultivorans, Dehalobacterrestrictus, Dehalococcusethenogenes

Zero-order dechlorination kinetics forPCE, TCE, cis-DCE and first-orderfor trans-DCE and VC; totaldegradation of ~4.6 umol PCE/mgVSS/day

Etheth

TCE Anaerobic, electron donor

(lactate, methanol butyrate,glutamate 1,2-propanediol,toluene)

Alcaligenes; (for example,hydrogenopheya present inculture derived from soil)

Michaelis-Menton kinetics Eth

PCE, TCE, cDCE, VC Anaerobic, electron donor (Hydrogen, propionate or lactate) Not reported Michaelis-Menton kinetics No

rep

PCE Anaerobic, electron donor

(methanol)

Not reported 1.24 mg PCE mg /volatile suspended

solids (VSS)/d

Norep

TCE Not reported Not reported 1st order 0.2-4.8 yr-1 No

rep

DCE Not reported Not reported 1st order 0.5-9.4 yr-1 No

rep
Exhibit 2-8: Selected Information on CAH Degradation Mechanisms (continued)

Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

Degradation

Mechanism CAH Conditions Reported Bacteria Reported Rate Data P

2-11

Anaerobic reductivedechlorination (direct)(dehalorespiration)(continued)

VC Anaerobic, iron-reducing

conditions in aquifer

Not reported Michaelis-Menton Vmax = 0.76

umol/L-d

Norep

VC Anaerobic, methanogenic

conditions in aquifer

Not reported Michaelis Menton

Vmax = 0.19 umol/L-d

Norep

Anaerobic reductivedechlorination(cometabolic)

PCE, TCE, DCE,VC, DCA

Anaerobic, electron acceptor(nitrate, sulfate), electron donor(hydrogen)

Methanosarcina barkeri,Desulfomonile tiedjei

0.84 and 2.34 nmol PCE/mgprotein/day converted to TCE

Etheth

PCE, TCE, CT Anaerobic, electron acceptor

(nitrate, sulfate), electron donor(hydrogen)

Methanogens, denitrifiers,sulfate reducers

Not reported Eth

me

CT Anaerobic, electron acceptor

(Fe[III])

Shewanella putrefaciens

MR-1

Not reported CF

ABIOTIC MECHANISMS

Reductive dechlorination(abiotic)

CT, CF Anaerobic, metal cofactor,

corrinoid, or porphyrin catalyst (reduced vitamin B12r

)

Abiotic Ten-fold rate increase over

cometabolic reductive dechlorination

Me

Hydrolysis TCA, CA, CM Not reported Abiotic Not reported Ac

acieth

Hydrolysis TCA Highly 

oxidizedgroundwater

Abiotic Half life of approximately 2 years Po

DCace

Elimination TCA, CA, CM Not reported Abiotic Not reported DCElimination, hydrolysis TCA

10 o

C

15 o

C

20 o

C

Abiotic 1st order (at each temperature)

0.058-0.06 yr-1

0.137-0.145 yr-10.31-0.34 yr-1

DCace

Elimination, hydrolysis TCA

10 o

C

15 o

C

20 o

C

Abiotic Avg. Half Life (at each temperature)

12 yr4.9 yr0.95 yr

DCace
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

2-12

Exhibit 2-9: Biodegradation Mechanisms Typically Occurring

During Enhanced In Situ Bioremediation of CAHs

CAH

Aerobic Oxidation Anaerobic Reductive Dechlorination

Direct Cometabolic Direct Cometabolic

CHLORINATED ETHENES

PCE X X  

TCE X   

cis-DCE X   

trans-DCE X   

1,1-DCE X   

VC    

CHLORINATED ETHANES 1

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

Biodegradation

Mechanism Carbon Source

Electron Donor

(Reductant)

Electron

Acceptor

(Oxidant) Comments

Aerobic oxidation

(direct)

CAH CAH Oxygen Only less chlorinated CAHs

can be degraded 

Aerobic oxidation

(cometabolic)

Organic carbon Cometabolite (e.g.,

toluene, methane

propane)

Oxygen CAH oxidized by

cometabolic mechanism

Anaerobic reductive

dechlorination (direct)

Organic carbon or

CO2

Hydrogen CAH Greater chlorinated CAHs

are more readily available

Anaerobic reductive

dechlorination

(cometabolic)

Other organic carbon

or CO2

Hydrogen Cometabolic

electron acceptor

CAH reductively

dechlorinated by

cometabolic mechanism

Sources: RTDF 1997; ITRC 1998; EPA 1998
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

2-13

Exhibit 2-11: Aerobic Oxidation (Direct)

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.

Source: Modified from Hartmans and DeBont 1992
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

2-14

Exhibit 2-12: Aerobic Oxidation (Cometabolic)

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. 

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). 
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

2-15

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 energyproducing 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; MaymoGatell 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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

2-16

 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).
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

2-17

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

2-18

Cl3C-CH3 + 2H2O  H3C-COOH + 3HCl

1,1,1-TCA Acetic Acid

Exhibit 2-14: Example of Hydrolysis Reaction

Cl3C-CH3  Cl2C=CH2 + HCl

1,1,1-TCA 1,1-DCE

Exhibit 2-15: Example of Elimination Reaction

CT + 2Fe0  MC + 2Fe+2

carbon tetrachloride zero-valent iron methylene chloride ferrous iron

 Iron Oxidation State 0  +2

 Carbon Oxidation State +4  0

Exhibit 2-16: Example of Abiotic Reductive Dechlorination

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).

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).
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

3-1

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 
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

1 Case studies provided in Appendix A of this report are cited as examples of each of the technologies.

3-2

Exhibit 3-1: Components of In Situ Bioremediation Technology1

Component Example

Biodegradation Mechanisms

Supported Targeted CAHs Ca

Bioaugmentation Seed the subsurface with non-native,

CAH-degrading bacteria

Aerobic oxidation (cometabolic anddirect)

TCE, DCE, TCA,DCA, CA, CT, CF

None

Anaerobic reductive dechlorination(cometabolic and direct)

TCA, DCA, CA, CT,CF, CM

Dover Area 6

Addition ofNutrients

Add nitrogen, phosphorus, or othergrowth factors that may be deficient in thesubsurface

Aerobic oxidation (cometabolic anddirect)

TCE, DCE, TCA,DCA, CA, CT, CF

SRS; Watertow

Anaerobic reductive dechlorination(cometabolic and direct)

TCA, DCA, CA, CT,CF, CM

Texas Gulf; Do

Addition ofElectron Donors

Add a substrate, such as toluene, propane,or methane

Aerobic oxidation (cometabolic) TCE, DCE, TCA,

CF, MC

Moffett Field; Watertown; Do

Add hydrogen, a hydrogen source, or ahydrogen release compound 

Anaerobic reductive dechlorination(cometabolic and direct)

PCE, TCE, DCE,VC, TCA, DCA, CA,CT, CF, MC

Texas Gulf; AvWatertown; Do

Addition ofElectronAcceptors

Add oxygen by bioventing, biosparging,or adding an oxygen source such ashydrogen peroxide

Aerobic oxidation (direct) TCE, DCE, VC,

TCA, DCA, CA, CE,MC, CM

Moffett Field; Watertown; Do

Add an anaerobic reductant such as nitrate Anaerobic reductive dechlorination

(cometabolic)

PCE, TCE, DCE,VC, DCA, CT

None

Source: ITRC, 1998; Leeson, 1999; Sewell, 1998; U.S. Air Force, 1998
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

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

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
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

3-4

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
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

3-5

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 

Configuration Purpose

Case Study

Examples

Applicability/

Advantages Potential Limitations

Direct injection To enhance the

biodegradation of

contaminants in place

in soil and

groundwater

SRS, Avco Lycoming,

Emeryville, Dover

Building 719

& Less aboveground

equipment needed

than for ex situ

systems

& Difficult to control

dispersion of

amendments in

aquifer

& Regulatory

concerns about

discharge of

chemicals to

groundwater

Groundwater

recirculation 

To contain the

contaminated

groundwater plume

and enhance the

biodegradation of

contaminants in the

recirculation area

Moffett Field,

Edwards AFB, Texas

Gulf Coast,

Watertown, Dover

Area 6

& Can provide

containment of the

plume

& Allows controlled

amendment of

groundwater

& Reinjection of

contaminated

groundwater may be

complicated

because of

regulatory concerns

& Silt build up in

recirculation wells

reduces

effectiveness of

systems

PRB To contain the

contaminated

groundwater plume

and degrade

contaminants in

groundwater that pass

though the barrier

None & Less aboveground

equipment needed

than for ex situ

systems

& Treats only

groundwater that

passes through the

barrier

Bioventing To enhance the

biodegradation of

contaminants in the

vadose zone

Dover Building 719 & Less aboveground

equipment needed

than for ex situ

systems

& Treats

contaminated soil

& Bioventing must be

coupled with a

groundwater

treatment

technology (such as

biosparging) to

remediate any

contaminated

groundwater

Source: ITRC, 1998; Leeson, 1999; Sewell, 1998; U.S. Air Force, 1998
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

3-6

Direct Injection

Groundwater

Recirculation

Permeable

Reactive Barrier

Exhibit 3-3: In Situ Bioremediation System Configurations

Source: Modified from USAF 1998
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

3-7

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
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

3-8

Exhibit 3-4: Vendors Listed in EPA REACH IT That Provide In Situ Bioremediation Service

Vendor Contact

Trade Name or

Technology

Enhanced

Bioremediation

Amendments2

# of Full-Scale

Units

Technology

Performance/ 

Vendor Claims

Spec

degr

Mec

Em

Design

Construction

Completion 

ABB EnvironmentalServices, Inc.www.abb.com

Jaret Johnson, P.E.Team LeaderPetroleum/Chemical TeamPhone: (781) 245-6606Fax: (781) 246-5060[no e-mail addressidentified]

Two-Zone Plume -InterceptionTreatmentTechnology

Biological,Nutrient,Oxidant (O),Oxidant (R),Reductant(O)

213 & Treatment designs include

recirculation ofgroundwater or creation ofbiological barriers forinterception of a plume 

& Treats BTEX, PAHs, or

any contaminant thataerobic bacteria candegraded readily

& Removal effectiveness can

approach 100 percent

Anaerobdechlori(direct)Aerobic (cometab

B&S Research, Inc.[No Web pageidentified]

H.W. Lashmett CEOPhone: (218) 984-3757Fax: (218) 984-3212[No e-mail addressidentified]

Bioremediation (insitu) - groundwater [No trade namegiven]

Biological,Nutrient

060 & Degrades hydrocarbons,

chlorinated solvents, PCBs,fertilizers, pesticides, andother hazardous organiccompounds in groundwaterand converts contaminantsto harmless products

& Treatment from surface to

90 feet bgs

Not spec
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

Exhibit 3-4: Vendors Listed in EPA REACH IT That Provide In Situ Bioremediation Service

Vendor Contact

Trade Name or

Technology

Enhanced

Bioremediation

Amendments2

# of Full-Scale

Units

Technology

Performance/ 

Vendor Claims

Spec

degr

Mec

Em

Design

Construction

Completion 

3-9

Billings &Associates, Inc.[No Web pageidentified]

Rick Billings Vice PresidentPhone: (505) 345-1116Fax: (505) 345-1756[No e-mail addressidentified]

Groundwater/SubsurfaceVolatilization andVentilation SystemRemediationTechnology

Reductant(O)

100 20 40 & Installed rapidly

& Uses readily available

pumps and pipe valves 

& Installed with standard

drilling equipment

& Advantages include use of

air to avoid watertreatment, ability tomanipulate air flows andpressures, and ability todirect air to selected areas

& Air emissions can be held

within permitted levels bymanipulation of flows anduse of a biological filter

& Has not been applied in

treating inorganic wastes

Aerobic 

Bio-GenesisTechnologieswww.biogti.com

Victor CoukoulisPresidentPhone: (602) 990-0709Fax: (602) 990-7745info@biogti.com

Bioremediation (insitu) - groundwater [No trade namegiven]

Biological INP INP INP & Treats groundwater

contaminated withpetroleum hydrocarbons,PAHs, aromatics, alcohols,ketones, phenols, PCBs,solvents, carbohydrates,and pesticides

& Advantages include:

products are composed ofnatural biologicalingredients that are nonhazardous, no safetyequipment is needed, andby-products of the processare non-hazardous

Not spec
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

Exhibit 3-4: Vendors Listed in EPA REACH IT That Provide In Situ Bioremediation Service

Vendor Contact

Trade Name or

Technology

Enhanced

Bioremediation

Amendments2

# of Full-Scale

Units

Technology

Performance/ 

Vendor Claims

Spec

degr

Mec

Em

Design

Construction

Completion 

3-10

ClaytonEnvironmentalConsultants[No Web pageidentified]

John F. Vargas Senior Project ManagerPhone: (714) 431-4106Fax: (714) 825-0685[No e-mail addressidentified]

Bioventing[No trade namegiven]

Nutrient,Reductant(O)

201 & Ease of design and

construction

& Can be incorporated into a

remedial design of soilvapor extraction system

& Effective at removing

heavier, semivolatilepetroleum hydrocarbonssuch as diesel fuel, fuel oil,kerosene, and JP-4 jet fuel

Aerobic 

EcologyTechnologiesInternational, Inc.[No Web pageidentified]

Pete Condy PresidentPhone: (916) 939-2397Fax: (916) 939-2449PKcondy@aol.com

FYREZYMEadditive

Biological,Nutrient

112 & Effective in accelerating

bioremediation of organiccontaminants in anenvironmentally friendly,scientifically sound, andcost-effective manner

& Advantages include: ease

of use, permanent solutionto contamination, andaffordability

Aerobic 
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

Exhibit 3-4: Vendors Listed in EPA REACH IT That Provide In Situ Bioremediation Service

Vendor Contact

Trade Name or

Technology

Enhanced

Bioremediation

Amendments2

# of Full-Scale

Units

Technology

Performance/ 

Vendor Claims

Spec

degr

Mec

Em

Design

Construction

Completion 

3-11

ENSR Consultingand Engineeringwww.ensr.com

Dan Groher Principal BioremediationSpecialistPhone: (508) 635-9500Fax: (508) 635-9180[No e-mail addressidentified]

AnaerobicBiotransformationwith Steam Injection

Nutrient,Oxidant (R)

202 & Combines bioremediation,

steam injection, SVE,groundwater extraction,and air-stripping

& Effective in removing

chlorinated solvents, suchas TCA and TCE presentas DNAPL; effective forLNAPL

& Requires removal of fewer

volumes of groundwater,shortens remediation, andtherefore reduces costs

& Does not treat metals& Injection of substrate and

nutrients into groundwatermay require a permit

Anaerob

Bioventing[No trade namegiven]

Nutrient,Reductant(O)

635 & Nutrients may be provided

in liquid phase throughsurface infiltration or invapor phase throughinjection by vent wells 

& Advantages include low

cost and demonstratedeffectiveness

& Low pressure, long-term,

low-energy treatment

Aerobic 

ENVIROGEN, Inc.www.envirogen.com

Michael Shannon Ph.D.Technical ManagerPhone: (609) 936-9300Fax: (609) 936-9221[No e-mail addressidentified]

Bioventing[No trade namegiven]

Nutrient,Reductant(O)

2 0 10 & Results in speedier and

more cost-effectiveremediation

& Not applicable for metals

or nonvolatile or inorganiccontaminants

Aerobic 
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

Exhibit 3-4: Vendors Listed in EPA REACH IT That Provide In Situ Bioremediation Service

Vendor Contact

Trade Name or

Technology

Enhanced

Bioremediation

Amendments2

# of Full-Scale

Units

Technology

Performance/ 

Vendor Claims

Spec

degr

Mec

Em

Design

Construction

Completion 

3-12

EOD Technology,Inc.[No Web pageidentified]

Matt Kaye Director of Marketing andFinancePhone: (423) 690-6061Fax: (423) 690-6065eodtmk@aol.com

Bioremediation (insitu) - groundwater/ [no trade namegiven]

Not specified INP INP INP & Combined with on-site

groundwater treatment toenhance the rate ofcontaminant degradation

Not spec

In-Situ Fixation, Inc.www.insitufixation.com

Richard P. Murray PresidentPhone: (602) 821-0409Fax: (602) 786-3184info@insitufixation.com

Dual Auger System Biological,

Nutrient,Reductant(O)

001 & Increases the quality and

acceleration ofbiodegradation in deepcontaminated soils

& No excavation is required,

and no residues or wastesare generated in theprocess, since treatment isperformed beneath theground surface

Aerobic 

IT Corporationwww.itcorporation.com

Dr. Duane Graves Process DevelopmentSupervisorPhone: (423) 690-3211 

x7418

Fax: (423) 690-9573[No e-mail addressidentified]

Restore brandamendments

Nutrient INP INP INP & Information not provided Aerobic 
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

Exhibit 3-4: Vendors Listed in EPA REACH IT That Provide In Situ Bioremediation Service

Vendor Contact

Trade Name or

Technology

Enhanced

Bioremediation

Amendments2

# of Full-Scale

Units

Technology

Performance/ 

Vendor Claims

Spec

degr

Mec

Em

Design

Construction

Completion 

3-13

Micro-BacInternational, Inc.www.micro.comMicrobialInternationalMidwest Microbial,L.C.

Terry NelsonPhone: (512) 310-9000Fax: (512) 310-8800mail@micro-bac.comHarry ChristensenPhone: (714) 666-0110Fax: (714) 538-5134micro@webworldinc.comDel Christensen Operating ManagerPhone: (402) 493-8880Fax: (402) 496-9269[No e-mail addressidentified]

Bac-Terra RemedialTechnology(Three vendorsmarket the sameproduct)

Biological,Nutrient,Oxidant (O),Oxidant (R),

8 0 20 & Cost effective, accelerates

remedial program, and isadaptable to a broadspectrum of fieldconditions

Aerobic anaerobimechani

REGENESISBioremediationProductswww.regenesis.com

Shruti Gohil Assistant MarketingManagerPhone: (949) 443-3136 

x27

Fax: (949) 443-3145orc@regenesis.com

Bioremediation (insitu) - groundwater/oxygen releasecompound (ORC),hydrogen releasecompound (HRC)

Reductant(O), ORC,HRC

0 023 & Information not provided Aerobic-

Anaerob
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

Exhibit 3-4: Vendors Listed in EPA REACH IT That Provide In Situ Bioremediation Service

Vendor Contact

Trade Name or

Technology

Enhanced

Bioremediation

Amendments2

# of Full-Scale

Units

Technology

Performance/ 

Vendor Claims

Spec

degr

Mec

Em

Design

Construction

Completion 

3-14

RichardsLaboratories[No Web pageidentified]

Dr. Sheril D. Burton Senior MicrobiologistPhone: (800) 453-1210Fax: (801) 785-2521[No e-mail addressidentified]

Rimlab Biological,

Nutrient,Reductant(O)

216 & Treats water saturated with

or containing free productsof gasoline, diesel fuel,benzene, toluene,monochlorobenzene,dichlorobenzene, crude oilfrom production wells,hydrocarbons from oilrecycling facilities, oilrefinery discharges, airportdeicing discharges,alcohols from natural gaspipelines, or similarindustrial pollutants; amodified consortiumdegrades TCE

& Not applicable for

treatment of watercontaminated with heavyasphaltines

& Germicidal agents must be

neutralized

Aerobic (direct)

SBP Technologies,Inc.[No Web pageidentified]

Director of BusinessDevelopmentPhone: (914) 694-2280Fax: (914) 694-2286 [No e-mail addressidentified]

UVB, KGB, BLK Biological 10 5 16 & Specially selected

microorganisms patentedfor treatment of water thatcontains organic woodpreservatives, namelycreosote and PCP, andchlorinated solvents,especially TCE

Direct monly

Terra Vac, Inc.www.terravac.com

Joseph PezzulloPhone: (609) 371-0070Fax: (609) 371-9446[No e-mail addressidentified]

BIOVAC® Nutrient,

Reductant(O)

0 0 11 & Significantly more costeffective than excavation

and disposal

Aerobic (direct)
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

Exhibit 3-4: Vendors Listed in EPA REACH IT That Provide In Situ Bioremediation Service

Vendor Contact

Trade Name or

Technology

Enhanced

Bioremediation

Amendments2

# of Full-Scale

Units

Technology

Performance/ 

Vendor Claims

Spec

degr

Mec

Em

Design

Construction

Completion 

3-15

Waste StreamTechnology, Inc.www.wastestream.com

Jim Hyzy Director of Research andDevelopmentPhone: (716) 876-5290Fax: (716) 876-2412wasstream@aol.com

Bioremediation (insitu) - groundwater [No trade namegiven]

Biological,Nutrient

028 & Costs are reduced by its

rapidity, predictability, andtechnical merit

Not Spe

YellowstoneEnvironmentalScience (YES), Inc.www.yestech.com

Mary M. Hunter PresidentPhone: (406) 586-2002Fax: (406) 586-8818yes@yestech.com

BIOCAT-II (TM) Biological,

Nutrient,Oxidant (O),Oxidant (R),Reductant(O)

INP INP INP & Treats a variety of wastes,

including aromatichydrocarbons andhalogenated hydrocarbons

& Advantages include:

natural pH control andimmobilization of metals toprotect enriched populationof methanogens,conversion of breakdownproducts of toxic organicsto methane that can beused to thermally enhancebiotransformations, andelimination of releases ofVOC

Not spec

1 Information included in this table, including aspects of configuration, number of units, points of contact, list of performance claims, and patent informa

REACH IT (<http://www.epareachit.org>) in June 2000. Information is shown as provided by technology vendors in EPA REACH IT and was not m

2

Biological - Bioaugmentation Oxidant (O) - Oxidant addition for oxidation application HRC - Hydrogen release compound Oxidant (R) - Oxidant addition for reductive dechlorination applicationNutrient - Nutrient addition ORC - Oxygen release compoundINP - Information Not Provided Reductant (O) - Reductant addition for oxidation application (oxygen unless otherwise sp

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 sit

than 100 sites.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

4-1

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 caseby-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)
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

4-2

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.
Exhibit 4-2: Common Site Characterization Parameters Relevant to In Situ Bioremediation

Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

4-3

Parameter Relevance Typical Measurement Method 

PHYSICAL PARAMETERSSoil porosity To determine the extent to which soil gas diffusion

(natural or forced) can take place

Soil sampling Percent

Hydraulic conductivity To determine the potential rate of groundwater flow

through soil or in an aquifer

Aquifer testing; permeability testing Length p

Hydraulic gradient To determine the speed at which groundwater travels

(when combined with hydraulic conductivity)

Water level measurements Elevation 

Organic content of soil To determine the extent to which contaminants may

adsorb to soil, rather than migrate with groundwater;provide a source of carbon for biodegradation reactions 

Volatile solids analysis Percent b

Moisture content of soil To determine whether sufficient moisture is present in

soil to support degradation processes

Dry weight measurement Percent b

CHEMICAL PARAMETERSCAH concentrations To determine the baseline level of contamination,

specific degradation mechanisms that may beapplicable, and substrate levels for direct degradation

Gas chromatography with flameionization detector or massspectrophotometric methods

Amount water (e.(e.g., mg

Concentrations of CAHdegradation products (that is,less chlorinated CAHs or nonchlorinated products) 

To provide a measure of degradation mechanismstaking place naturally

Gas chromatography with flameionization detector or massspectrophotometric methods

Amount water (e.(e.g., mg

Concentrations of organicelectron donors (for example, toluene, methane, phenol, ororganic acids)

To determine the potential for cometabolic degradationmechanisms without enhancements

Gas chromatography with flameionization detector or massspectrophotometric methods; oxygenuptake analysis

Amount water (e.(e.g., mg

Concentrations of inorganicelectron donors (for example,hydrogen, iron, or ammonia)

To determine the potential for degradation mechanismswithout enhancements

Direct electrode; wet chemistry methods;atomic adsorption methods

Amount water (e.(e.g., mg

Oxygen content To determine whether aerobic or anaerobic conditions

are present

Direct electrode Percent o

volume
Exhibit 4-2: Common Site Characterization Parameters Relevant to In Situ Bioremediation

Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

Parameter Relevance Typical Measurement Method 

4-4

CHEMICAL PARAMETERS (continued)Redox potential To determine the approximate size and location of a

plume; determine whether aerobic or anaerobicconditions are present; identify microbes that are likelyto be present

Direct electrode Volts rel

(more neenvironmmore oxi

Concentrations of otherelectron acceptors (forexample, nitrate, sulfate, carbondioxide)

To determine whether electron acceptors are sufficientfor aerobic degradation of CAHs, or may causeunwanted competition in anaerobic dehalogenationprocesses

Direct electrode; wet chemistry methods;atomic adsorption methods

Amount water (e.(e.g., mg

Concentrations of nutrients (forexample, boron, calcium,magnesium, manganese,nitrogen, potassium, orphosphorus)

To determine whether nutrient levels are sufficient formicrobial activity or whether nutrient addition isneeded

Wet chemistry methods; atomicadsorption methods

Amount water (e.(e.g., mg

pH To determine whether conditions are most beneficial

for microbial growth

Direct electrode Standard 

BIOLOGICAL PARAMETERSPresence and concentration ofnon-specific microbes

To estimate microbial abundance Direct microscopic analysis; electronic

particle counting; volatile suspendedsolids analysis; total kjeldahl nitrogenanalysis; protein analysis

Concentrmaterial carbon

Presence and concentration ofspecific microbes

To determine the presence and concentration oftargeted type of organism

Heterotrophic plate count; MPN tubecounts

Count of bacteria pmedia

Microbial activity To quantify the rate of activity of targeted organisms Oxygen uptake rate analysis;

dehydrogenate activity analysis

Amount during a 

Sources: Gossett, Microbiology for Environmental Engineers 1997; EPA 1998
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

4-5

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 nonspecific 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,
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

4-6

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

(K > 10-4 cm/s)

Low permeability

(K < 10-4 cm/s)

Hydrofracturing and pneumatic

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

concentrations

Toxic contaminant concentrations Dilution by injection of water or

bioremediation additives

Simple contaminant mixtures Complex contaminant mixtures None typically employed

Moderate to high microbial

activity of appropriate microbes 

Little microbial activity or

inappropriate microbes

Bioaugmentation

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

4-7

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

Bioremediation

Mechanism

Targeted

CAHs

Typical Primary Reactants and Additives in Engineered Systems

Primary Reactants Typical Additives (primary reactant

supplemented)

Aerobic oxidation

(direct)

DCE, VC,

DCA, CA,

MC, CM

Oxygen, CAH Air, oxygen, hydrogen peroxide, magnesium

peroxide (oxygen)

Aerobic oxidation

(cometabolic)

TCE, DCE,

VC, TCA,

CF, MC

Oxygen Air, oxygen, hydrogen peroxide, magnesium

peroxide (oxygen) 

Organic carbon or

carbon from a

contaminant source

(anthropogenic)

Methane, propane, butane, ammonia (organic

carbon)

Anaerobic

reductive

dechlorination

PCE, TCE,

DCE, VC,

TCA, DCA,

CT, CF, MC

Hydrogen, organic

carbon, or carbon from

a contaminant source

(anthropogenic)

Lactate, methanol, hydrogen, molasses

(electron donor)

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).
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

4-8

The specifics of treatability testing for in situ bioremediation can be complex, and are influenced by sitespecific 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 
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

4-9

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. 
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

4-10

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

4-11

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
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

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

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

5-1

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 OnSite 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 attachedfilms 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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

5-2

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

Environmental Microbiology. Volume 58. Pages 1392-1393.

Gillham, R.W. and S.F. O'Hannesin. 1994. “Enhanced degradation of halogenated aliphatics by zerovalent 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. 

Volume 33. Pages 1100-1109.

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

5-3

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

5-4

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. 

Volume 32. Pages 88-100.

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. 

Volume 31. Pages 786-791.

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. McGrawHill, 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. 

Volume 163. Pages 48-56.

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. 

Volume 28. Pages 715-727.

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

Environmental Microbiology. Volume 62. Pages 761-765.

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

5-5

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

5-6

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

Environmental Microbiology. Volume 29. Pages 242-243.

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. 

Volume 31. Pages 2292-2297.

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

6-1

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

6-2

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

6-3

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-1

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-2

Exhibit A-1: Summary of Case Studies of In Situ Bioremediation Technology Applications

#

Site Name,

Location Mechanism(s) Technology/Configuration

Technology

Scale

Matrix

Treated

Contam

Targe

1 NAS Moffett Field, 

Mountain View,California

Aerobic oxidation(cometabolic anddirect)

Electron acceptor (EA) addition (oxygenand hydrogen peroxide)Electron donor (ED) addition (methane,toluene, and phenol)Groundwater recirculation

Field demonstration Ground- water TCE, cis-DCE, tr

DCE, VC 

2 Edwards Air Force

Base (AFB),California

Aerobic oxidation(cometabolic anddirect)

EA addition (oxygen and hydrogenperoxide)ED addition (toluene)Groundwater recirculation

Field demonstration Ground- water TCE 

3 U.S. Department of

Energy SavannahRiver Site, Aiken,South Carolina*

Aerobic oxidation (cometabolic anddirect)

Nutrient additionEA addition (oxygen)ED addition (methane)Direct injection

Field demonstration Sediment and

Groundwater

TCE, PCE 

4 Texas Gulf Coast

Site (site nameconfidential)Houston, Texas**

Anaerobic reductivedechlorination (cometabolic anddirect)

Nutrient additionED addition (methanol)Groundwater recirculation

Full Groundwater TCE, cis-1

VC 

5 Avco Lycoming

Superfund SiteWilliamsport,Pennsylvania

Anaerobic reductivedechlorination (cometabolic anddirect)

ED addition (molasses)Direct injection

Pilot and full Groundwater TCE, DCE

Cr+6, Cd 

6 Abandoned

ManufacturingFacility Emeryville,California

Anaerobic reductivedechlorination (cometabolic anddirect)

ED addition (molasses)Direct injection

Pilot and full Groundwater TCE, Cr+6 
Exhibit A-1: Summary of Case Studies of In Situ Bioremediation Technology Applications (contin

Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

#

Site Name,

Location Mechanism(s) Technology/Configuration

Technology

Scale

Matrix

Treated

Contam

Targe

A-3

7 Watertown,

Massachusetts**

Anaerobic reductivedechlorination (cometabolic anddirect)

Nutrient additionED addition (lactate)Groundwater recirculation

Field demonstration (SITEtest)

Groundwater PCE, TCE 

Aerobic oxidation (cometabolic anddirect)

EA addition (oxygen)ED addition (propane)Groundwater recirculation

8 Dover AFB Area 6

Dover, Delaware

Anaerobic reductivedechlorination (cometabolic anddirect)

BioaugmentationNutrient additionED addition (lactate) Groundwater recirculation

Field demonstration (proofof technologytest)

Groundwater TCE 

9 Dover AFB

Building 719Dover, Delaware

Aerobic oxidation(cometabolic anddirect)

EA addition (oxygen)ED addition (propane)Direct injection

Field demonstration (pilottest)

Soil TCE, TCA

* Remediation efforts at this site also are included in Exhibit A-2 (Summary of Full-Scale Applications and Pilot Studies Identified in Proceedings 

Situ and On-Site Bioremediation Symposium) and Exhibit A-3 (Summary of Full-scale Applications and Pilot Studies Identified in the Bioremed

System [BFSS]).

** Remediation efforts at these sites also are included in Exhibit A-2 (Summary of Full-Scale Applications and Pilot Studies Identified in Proceedin

In Situ and On-Site Bioremediation Symposium).

1 Contaminant Key: Cd = Cadmium, Cr = Chromium, DCE = dichloroethene, PCE = tetrachloroethene, TCA = trichloroethane, TCE = trichloroeth
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-4

Exhibit A-2: Summary of Full-Scale Applications and Pilot Studies Identified in Proceedings of The Fifth Intern

On-Site Bioremediation Symposium

Site Name, Location Technology Media Contaminants1

Period of

Performance Points of Contact

FULL-SCALE APPLICATIONS

Site name not provided(commercial laundry)southwestern Ohio

Aerobic oxidation;injection of propane

Groundwater PCE, TCE, 1,2-

DCE

June 1998 -pilot testSeptember 1998- begin fullscale; end datenot provided

Peter I. DacykWilliam D. HughesParsons Engineering Science, Inc13 Triangle Park Drive, Suite 130Cincinnati, Ohio 45246

Site name not provided(aluminum coating facility)location not provided(midwestern state)

Aerobic oxidation(oxygen releasecompound [ORC]slurry injection) 

Groundwater VC March 1998 -

injection

Peter L. Tacy, Jr.STS Consultants, Ltd.1502 Randolph Street, Suite 100 Detroit, Michigan 48226-2216

Site name not provided(dry cleaning site)Wisconsin

Anaerobic reductivedechlorination(injection ofglycerol polylactatehydrogen releasecompound [HRC])

Groundwater PCE Not provided John K. Sheldon

Montgomery Watson11153 Aurora AvenueDes Moines, Iowa 50322-7238Kenneth J. Quinn, Montgomery WMadison, WisconsinStephen S. Koenigsberg and CraiSandefur, Regenesis, San Juan CaCalifornia

Site name not provided(natural gas pipelinecompressor station)Virginia

Aerobic oxidation(MethanotrophicTreatmentTechnology [MTT],injection ofmethane, air,nitrous oxide, andtriethylphosphate)

Groundwater PCE, TCE March 1998 -

ongoing (enddate notprovided)

Mark S. Nelson, Williams Gas PiTransco, Houston, TexasRobert Legrand and Andrew J. MRadian International8501 N. Mopac BlvdP.O. Box 201088Austin, Texas 78720-1088(and Raleigh-Durham, North CaroJohn A. Harju, Gas Research InstChicago, Illinois
Exhibit A-2: Summary of Full-Scale Applications and Pilot Studies Identified in Proceedings from The Fifth Inte

On-Site Bioremediation Symposium (continued)

Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

Site Name, Location Technology Media Contaminants1

Period of

Performance Points of Contact

A-5

FULL-SCALE APPLICATIONS (continued)

Site name not providedWatertown, Massachusetts

Anaerobic reductivedechlorination(addition of HRC toa recirculation cell)

Groundwater PCE, TCE, DCE,

VC

February 1998 -January 1999

Maureen A. Dooley and Willard AHarding Lawson Associates107 Audubon Road, Suite 300Wakefield, Massachusetts 01880Stephen Koenigsberg, Regensis, SCapistrano, California

Site name not provided(manufacturing facility)Texas Gulf Coast, Texas

Anaerobic reductivedechlorination(addition ofmethanol torecirculationsystem)

Groundwater TCE June 1995 -

December 1998

Susan Tighe Litherland, P.E. and AndersonRoy F. Weston, Inc. Building 1, Suite 1005300 Bee Caves RoadAustin, Texas 78746-5225Blake A. Dinwiddie, Roy F. WestHouston, Texas

Site name not providedLansing, Michigan

Aerobic oxidation(cometabolic)(phenol additionwith pressurizedfluidized bedreactor);Groundwaterrecirculation;Biosparging

Groundwater TCE May 1997 - end

date notprovided (dataavailable for 15months ofoperation)

Jian Xing and Richard M. RaetzGlobal Remediation Technologie1235 WoodmereTraverse City, MI 49686

Evenblij site, Hoogeveen,The Netherlands

Aerobic oxidation(acetate and lactateaddition);Anaerobic reductivedechlorination;Groundwaterrecirculation

Groundwater PCE, TCE Not provided M.J.C. Henssen

Bioclear Environmental BiotechnGroningen, The NetherlandsC. Hubach and R. BlokzijlDHV Environmental & InfrastrucEuropaweg 33/29723 AS Groningen, The NetherlJ. Mourik, Logisticon Water TreaGroot-Ammers, The NetherlandsE. Meijerink, Province of DrentheThe Netherlands
Exhibit A-2: Summary of Full-Scale Applications and Pilot Studies Identified in Proceedings from The Fifth Inte

On-Site Bioremediation Symposium (continued)

Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

Site Name, Location Technology Media Contaminants1

Period of

Performance Points of Contact

A-6

PILOT STUDIES

Hill Air Force Base,Operable Unit 1, ChemicalDisposal Pit 1Ogden Utah

Bioventing Soil 1,2-

Dichlorobenzene

One year (datesnot provided)

James T. Gibbs, Battelle505 King AvenueColumbus, Ohio 43201R. Kenneth Crowe, TRW Inc., TyFloridaJon Ginn, USAF, Hill AFB, Utah

Site name not providedCopenhagen, Denmark

Aerobic oxidation;injection ofmethane and air

Groundwater cis-1,2-DCE, VC July 1998 -

ongoing; enddate notprovided

Liselotte LudvigsenHOH Water Technology, Greve, DKim Broholm, VKIAgern Alle 11DK-2970 Horsholm, DenmarkLars Deigaard, Scanrail Consult,Copenhagen, Denmark

U.S. Department of EnergySavannah River Site,Sanitary LandfillAiken, South Carolina

Aerobic oxidation;injection ofmethane, air,triethyl phosphate,and nitrous oxide

Groundwater TCE, cis-1,2-

DCE, VC

September 1996 Robin L. Brigmon

Westinghouse Savannah River CoSavannah River Technology CentP.O. Box 616Aiken, South Carolina 29808Terry C. Hazen, Lawrence BerkelLaboratory, Berkeley, CaliforniaAl W. Bourquin, Camp Dresser aInc., Denver, Colorado

Site name not provided(Superfund site)Location not provided

Anaerobic reductivedechlorination

Groundwater PCE, TCE, cis1,2-DCE, VC April 1997 - August 1998 James J. Reid, P.E. and Denis Bal

ARCADIS Geraghty & Miller4700 Lakehurst Court, Suite 100Dublin, Ohio 43016
Exhibit A-2: Summary of Full-Scale Applications and Pilot Studies Identified in Proceedings from The Fifth Inte

On-Site Bioremediation Symposium (continued)

Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

Site Name, Location Technology Media Contaminants1

Period of

Performance Points of Contact

A-7

PILOT STUDIES (continued)

Site name not provided(industrial cleaningcompany)Arnhem, The Netherlands

Anaerobic reductivedechlorination

Soil PCE, TCE, cis1,2-DCE, VC 80 days (dates not provided) H. Slenders TNO Institute of Environmental S

Energy Research and Process InnBusiness Park E.T.V.Laan van Westenenk 501P.O. Box 3427300 AH Apeldoorn, The NetherlS. Hofstra, IWACO, EnvironmenConsultants, The NetherlandsH.de Sain, BdS, Management CoThe NetherlandsR. Hetterschijt, Netherlands InstitApplied Geoscience TNOH. de Kreuk, BioSoil R&D 

Idaho National Engineeringand EnvironmentalLaboratory, Test AreaNorthIdaho

Anaerobic reductivedechlorination

Groundwater TCE November 1998

- ongoing; enddate notprovided

Kent S. Sorenson, Jr. and Lance NLockheed Martin IdahoIdaho Falls, IdahoRoger L. Ely, University of IdahoIdaho

Naval Air Station PointMugu, IRP Site 24,California

Anaerobic reductivedechlorination(Phase 1 – lactateaddition; Phase 2 –nutrient injection);groundwaterrecirculation 

Groundwater Chlorinated

ethenes; volatileorganic acids

Not provided Christian D. Johnson

Battelle PNWDP.O. Box 999Richland, Washington 99352Daniel P. Leigh and Lisa A. BienGroup, Pleasanton, CaliforniaSteve Granade, U.S. Navy, NavalConstruction Battalion Center, PoHueneme, CaliforniaBryan Harre, U.S. Navy, Port HueCaliforniaTodd Margrave, U.S. Navy, San DCalifornia
Exhibit A-2: Summary of Full-Scale Applications and Pilot Studies Identified in Proceedings from The Fifth Inte

On-Site Bioremediation Symposium (continued)

Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

Site Name, Location Technology Media Contaminants1

Period of

Performance Points of Contact

A-8

PILOT STUDIES (continued)

Site name not provided(former wastewatertreatment facility)Location not provided

Anaerobic reductivedechlorination(yeast extractinjection)

Groundwater PCE, TCE, 1,1,1-

TCA, 1,1-DCE,cis/trans-1,2-DCE, 1,1-DCA,1,2-DCA, VC

July 1997 –November 1998

I. Richard Schaffner, Jr., GZAGeoEnvironmental, Inc.380 Harvey RoadManchester, New Hampshire 031

Site name not providedFlorida

Anaerobic reductivedechlorination(addition of HRC)

Groundwater TCE, cis-1,2-

DCE, VC

March 1998 –June 1998

Madeline WuWater Restoration Inc.Fort Lauderdale, Florida

Site name not providedNew Jersey

Anaerobic reductivedechlorination(addition of HRC)

Groundwater PCE, TCE, cis1,2-DCE April 1998 – June 1998 S. Kallur, Environmental Strategi

Applications, Inc.Somerset, New JerseyS. Koenigsberg, Regenesis1011 Calle SombraSan Clemente, CA 92672

Offutt Air Force Base, FireProtection Training Area 3Nebraska

Anaerobic reductivedechlorination(direct addition ofhydrogen);groundwaterrecirculation

Groundwater cis-1,2-DCE,

BTEX

November 1998 R. Todd Fisher and Charles J. Ne

Groundwater Services, Inc., HousPatrick E. Haas, Air Force Center Environmental Excellence, BrookTexasJoseph B. Hughes, Rice UniversitHouston, Texas

Site name not provided(industrial site)Adelaide, South Australia

Chemical oxidation,with addition ofpermanganate

Groundwater TCE Not provided Christopher H. Nelson, IT Corpor

Englewood, ColoradoCraig S. Barker, IT Environmenta(Australia) Pty Ltd, Adelaide, SouAustralia
Exhibit A-2: Summary of Full-Scale Applications and Pilot Studies Identified in Proceedings from The Fifth Inte

On-Site Bioremediation Symposium (continued)

Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

Site Name, Location Technology Media Contaminants1

Period of

Performance Points of Contact

A-9

PILOT STUDIES (continued)

ITT Industries NightVisionRoanoke, Virginia

Aerobic oxidation(injection ofmethane, air,nitrous oxide, andtriethylphosphate)

Groundwater TCE, TCA,

acetone,isopropanol

Not provided Gregory L. Carter, Earth Tech

5320 Peters Creek Road, Suite DRoanoke, Virginia 24019Jennifer C. Vincent and Barbara BEarth Tech, Concord, MassachuseRosann Kryczkowski, ITT IndustVision, Roanoke, Virginia

Rickenbacker Air NationalGuard BaseOhio

LasagnaTM(combination ofaerobiccometabolicbioremediation[with addition ofmethane],electroosmosis, andhydraulicfracturing)

Soil TCE January 1997 -

November 1998

Wendy J. Davis-Hoover et. al., EPCHL, EPA Facilities26 W. Martin Luther King DriveCincinnati, Ohio 45268Taras Bryndzia, Shell Oil, HoustoMichael H. Roulier and Lawrence Murdoch, Clemson University, CSouth CarolinaMark Kemper and Philip CluxtonInstruments, Inc., Martinsville, OSouhail Al-Abed and William SlaInc., Cincinnati, Ohio

* These full-scale applications and pilot studies are described further in the following document: Andrea Leeson, A. and B. C. Alleman. Engin

Situ Bioremediation of Chlorinated Solvent Contamination 5(2): The Fifth International In Situ and On-Site Bioremediation Symposium; Sa19-22, 1999; Battelle Press; 1999. Page numbers shown in Exhibit A-2 refer to the pages of the Battelle document in which the information 

** These full-scale applications and pilot studies are also included in Exhibit A-1: Summary of Case Studies of In Situ Bioremediation Technol

*** This pilot study is also included in Exhibit A-3: Summary of Full-Scale Applications and Pilot Studies Identified in the Bioremediation in th

(BFSS)

1

Contaminant Key: DCA = dichloroethane, DCE = dichloroethene, PCE = tetrachloroethene, TCA = trichloroethane, TCE = trichloroethene, 
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-10

Exhibit A-3: Summary of Full-Scale Applications and Pilot Studies Identified in the Bioremediation in the Field S

Site Name,

Location Technology Media Contaminants1 Project Status 

A. B. DickNiles, Illinois

Inland Environmental BioTreatment Process. An anaerobic,in-situ soil bioremediationtechnology. A proprietary liquidblend (patent applied for) ofnutrients, stimulants, and mobilizingagents is injected into the soil andstimulates the natural anaerobicbio-degradation of contaminants tomineralization

Soil TCE, cis-1,2-DCE, TCA Full-scale operations were

completed on 8/23/95. Theremediation was completedahead of schedule (three monthsversus one year) and within thebudget. All treatment goalswere met and the site is nowconsidered to be remediated. 

BarberGreeneNorthAurora,Illinois

In situ, aerobic process. Oxygen isintroduced into the groundwaterthrough air sparging. Vacuumextraction provides air movementthrough the soil to enhancegroundwater bioremediation(bioventing for vadose soil).

Soil (vadose),groundwater

PCE, 1,1-DCA, 1,1,1-TCA,TCE

Full-scale operations werecompleted on 7/18/97.

ChevronChemicalCompany -BerkeleyHeights, NewJersey

Aerobic in situ groundwater and soil

bioremediation process. Treatmentprocess involves pumpinggroundwater, adding nutrients(nitrogen and phosphorus) andamendments (hydrogen peroxideand oxygen) above ground, andreinjecting the groundwater into thesoil. The reinjected water will helpto remediate the contaminated soils.Groundwater that meets cleanupcriteria will be sent to a POTW.

Soil (vadose), soil(saturated),groundwater

The system is designed totreat 1,1,1-TCA, 1,2-DCE,and TCE. Othercontaminants include PCBsand heavy metals.

The full-scale system beganoperating on 1/1/95. Mechanical problems caused bymicroorganism growth occurred.
Exhibit A-3: Summary of Full-Scale Applications and Pilot Studies Identified in the Bioremediation in the Field S

(continued)

Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

Site Name,

Location Technology Media Contaminants1 Project Status 

A-11

NAS FallonFallon,Nevada

Aerobic bioslurping process. Avacuum is pulled in dewateringwells to promote the rapid aerationof subsurface vadose zone soils bymovement of air into soil pores. Since the vacuum (slurper) tubes aresituated at the interface of the freefuel and the groundwater, free fuelis removed (vacuum assisted), whilesimultaneously promoting aerobicbiodegradation of fuel in the vadosezone through bioventing.

Soil (vadose), soil(saturated),groundwater

System is designed to treatDCE, PCE,trichloroethylene, andcis-1,2-DCE. Othercontaminants includeperchloroethene andtrichloroethene (in vadosezone) and arsenic andborates (naturalcontaminants).

The full-scale system isoperational.

NavalSubmarineBaseKings Bay,Georgia

Aerobic, in situ groundwater andsoil bioremediation process. Bacteria, nutrients, and water wereinjected periodically into thegroundwater. Three sites werecontaminated with petroleumhydrocarbons (heating fuel, dieselfuel, and lubricating oil).

Groundwater The system is designed to

treat 1,1-DCA, 1,2-DCA,cis-1,2-DCE, PCE,trans-1,2-DCE, and TCE. BTEX is also present in thegroundwater.

The full-scale system has beencompleted, and no problemshave been reported. One of thesites (“generator” site) isoperational. 
Exhibit A-3: Summary of Full-Scale Applications and Pilot Studies Identified in the Bioremediation in the Field S

(continued)

Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

Site Name,

Location Technology Media Contaminants1 Project Status 

A-12

Pine BendLandfillInner GroveHeights,Minnesota

In situ groundwater bioremediation

process. A pilot-scale system thatconsists of three injection wellscurrently is being installed at thesite. The wells will deliver fructosecorn syrup to the groundwater toinduce anaerobic conditions andstimulate the anaerobic groundwatermicrobial consortia to effectivelydehalogenate highly chlorinatedcontaminants in groundwater. Groundwater that retainscontaminants may be treatedaerobically downgradient throughinstallation of a sparging curtain.

Groundwater The system is designed to

treat TCA, DCA, DCE, and perchloroethylene.

The full-scale system is in thepredesign phase. Treatmentcurrently is in the feasibilitystudy stage. The pilot studyevaluation is expected tocontinue for 18 months.
Exhibit A-3: Summary of Full-Scale Applications and Pilot Studies Identified in the Bioremediation in the Field S

(continued)

Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

Site Name,

Location Technology Media Contaminants1 Project Status 

A-13

SavannahRiver SiteAiken, SouthCarolina **

Full-Scale SystemBiosparging, in situ soil flushing,vacuum extraction. Aerobicprocess. Indigenous microbes are encouraged to degrade contaminantsby injecting air in the aquifer. Air isinjected via horizontal wells in theaquifer. Air is vacuum-extractedsimultaneously through a parallelhorizontal well just above the watertable. Off-gas is treated by catalyticoxidation.Pilot-Scale SystemBiosparging, in situ methane, in situ

soil flushing, vacuum extraction. Aerobic process. Nutrients aremethane, nitrous oxide, gas (triethylphosphate). Methane in air (1 to 4percent) is injected throughhorizontal wells in the aquifer. Airis extracted simultaneously througha parallel horizontal well just abovethe water table. Off-gas is treatedby catalytic oxidation. Methane isadded to stimulate indigenousmethanotrophs (bacteria) tobiodegrade chlorinated solventsthrough cooxidation.

Groundwater The system treats PCE and

TCE. Copper is alsopresent in the groundwater.

Full-scale operations werecompleted. No date provided.Pilot-scale operations werecompleted on 4/1/93. The fullscale system design wascompleted on 9/30/96.
Exhibit A-3: Summary of Full-Scale Applications and Pilot Studies Identified in the Bioremediation in the Field S

(continued)

Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

Site Name,

Location Technology Media Contaminants1 Project Status 

A-14

TechnicalProducts,Inc.Chicago,Illinois

Inland Environmental BioTreatment Process. In situ,

anaerobic process. The site wasexcavated to remove tanks and thesoil was replaced in deep lifts. Inland Environmental's proprietarybio-stimulating solution (nutrients,biostimulants, and surfactants) wassprayed onto each lift.

Soil (vadose) The system is designed to

treat TCE, 1,2-DCE, and1,2-DCA. Numerous othercontaminants are alsopresent. The concentrationof all of the unidentifiedcompounds decreasedalong with theconcentrations of theregulated compounds.

Full-scale operations werecompleted on 12/30/96.

* BFSS can be downloaded from the following Web site: <http://www.clu-in.org/PRODUCTS/MOREINFO/Bfss.htm>. BFSS is also availab

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 Applicatio

(Summary of Full-Scale Applications and Pilot Studies Identified in Proceedings of the Fifth International In Situ and On-Site Bioremediati

1 Contaminant Key: BTEX = benzene, toluene, ethylbenzene, and xylenes; DCA = dichloroethane; DCE = dichloroethene; PCB = polychlori

PCE = tetrachloroethene; TCA = trichloroethane; TCE = trichloroethene
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-15

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. 
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-16

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 (transDCE), 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
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-17

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]

Experiment

Extraction

(E) and

Injection (I)

Rates Duration

Chemical Amendments

(average mg/L) Processes Studied

First Season

Biostim 1 E: 8 L/min

I: 1 L/min 

9/5/86 -

9/30/86

Methane: 5.9

DO: 20.8

Bromide: 166

Biostimulation of native methane-using

bacteria. Alternating pulse injection of

methane and DO.

Biotran 1 E: 8 L/min

I: 1 L/min

9/30/86 -

10/21/86

Methane: 5.7

DO: 22.2

TCE: 0.097

Biotransformation of TCE with active

biostimulation. Nonsteady-state

conditions.

Biotran 4 E: 8 L/min

I: 1 L/min

12/10/86 -

12/31/86

Methane: 5.2

DO: 23

Bromide: 159 

TCE: 0.051

Biotransformation of TCE with active

biostimulation. Steady-state conditions.

Second Season

Tracer 8 E: 10 L/min

I: 1.5 L/min

7/6/87 -

8/15/87

DO: 14.3

Bromide: 78

TCE: 0.048 

cis-DCE: 0.110

trans-DCE: 0.112

Transport and breakthrough of bromide,

TCE, cis- and trans-DCE without

biostimulation.
Table 1. Summary of Experimental Conditions Used at Moffett [4] (continued)

Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

Experiment

Extraction

(E) and

Injection (I)

Rates Duration

Chemical Amendments

(average mg/L) Processes Studied

A-18

Biostim 2 E: 10 L/min

I: 1.5 L/min

8/17/87 -

10/26/87

Methane: 5.3

DO: 23.4

Bromide: 44

TCE: 0.036

cis-DCE: 0.091

trans-DCE: 0.092

Simultaneous biostimulation and

biotransformation of TCE, cis- and transDCE.

Decmeth 1 E: 10 L/min

I: 1.5 L/min

10/27/87 -

11/8/87

DO: 24.5

TCE: 0.045

cis-DCE: 0.136

trans-DCE: 0.095

Test if active biotransformation occurs

without addition of methane.

Third Season

Tracer 11 E: 10 L/min

I: 1.5 L/min

8/10/88 -

10/10/88

Bromide: 72

TCE: 0.047

cis-DCE: 0.085

trans-DCE: 0.050

Transport and breakthrough of bromide,

TCE, cis- and trans-DCE without

biostimulation.

Tracer 12 E: 10 L/min

I: 1.5 L/min

10/10/88 -

10/20/88

Bromide: 44

TCE: 0.042

cis-DCE: 0.100

trans-DCE: 0.054

VC: 0.044

Transport and breakthrough of bromide

and VC while continuing injection of

TCE, cis- and trans-DCE.

Biostim 3 E: 10 L/min

I: 1.5 L/min

10/20/88 -

11/23/88

Methane: 6.6

DO: 21.3

Bromide: 45

TCE: 0.046

cis-DCE: 0.100

trans-DCE: 0.052

Simultaneous biostimulation and

biotransformation of TCE, cis- and transDCE, and VC.

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. 
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-19

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]

Primary

Substrate

Substrate

Concentration

(mg/L)

% Removal by Constituent

TCE 1,1-DCE cis-DCE trans-DCE

Vinyl

Chloride

Methane

(third

season)

6.6 19 NE 43 90 95

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-20

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-21

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-22

Case Study # 2

Aerobic Degradation Field Demonstration

at Site 19, Edwards Air Force Base, California

Summary Information [1,2,3]

Site Name, Location Edwards Air Force Base, CA

EPA ID Number CA1570024504

Mechanism(s) Aerobic Oxidation (Cometabolic and Direct)

Technology Electron Acceptor Addition (Oxygen and

Hydrogen Peroxide)

Electron Donor Addition (Toluene)

Configuration Groundwater Recirculation

Technology Scale Field Demonstration

Media/Matrix Treated Groundwater

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-23

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

DNAPL Present None identified

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-24

Table 1. Operating Parameters for Steady-State Operation (Days 142 - 271) [3]

Treatment

Well

Groundwater

Pumping Rate

(L/min)

Toluene

Addition

(mg/L)

Toluene

Addition -

Pulses per Day

DO Addition

(mg/L)

H2O2

Addition

(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]

Treatment

Well

Groundwater

Pumping Rate

(L/min)

Toluene

Addition

(mg/L)

Toluene

Addition -

Pulses per Day

DO Addition

(mg/L)

H2O2

Addition

(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]
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-25

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]

Treatment

Well -

Aquifer

Operating

Period

(Days)

Average TCE

Concentration in

Treatment Well

(g/L)

Average TCE

Concentration in

Monitoring Well 

7.5 m Distant (g/L)

TCE Removal %

(average and

standard

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-26

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

controllers, static mixers, packing assembly, deionized

water system, pumps and ancillary equipment, tubing and

connectors, valves and fittings)

32,707

Capital - Monitoring wells 190,000

Capital - Monitoring equipment (pumps and ancillary

equipment, tubes and connectors, valves and fittings,

miscellaneous supplies)

70,746

Total Capital Costs 323,453

Annual O&M - Materials - Well Redevelopment

($4,000/well-year x 2 wells)

8,000

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
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-27

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
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-28

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-29

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

Mechanism(s) Aerobic Oxidation (Cometabolic and Direct)

Technology Nutrient Addition

Electron Acceptor Addition (Oxygen)

Electron Donor Addition (Methane)

Configuration Direct Injection

Technology Scale Field Demonstration

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
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-30

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. 

Matrix Characteristic Value

Soil Type sand, clay, and gravel

Depth to Groundwater ranges from 120 to 135 feet bgs

Thickness of Aquifer(s) 150 feet

DNAPL Present None identified

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-31

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 -1

Addition of 1% methane 

Nutrient

Addition - 2

Addition of 4% methane 

Nutrient

Addition - 3

Pulsed 4% methane addition at a rate of 8 hr every two days 

Nutrient

Addition - 4

Continuous addition of a combination of nitrous oxide at 0.007% and triethyl

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

Assays

Comparison of microbial assays for monitoring and control of in situ

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”
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-32

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

Mobilization Cost 

452,407

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
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-33

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-34

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-35

Case Study #4

Full Scale Enhanced Bioremediation at the Texas Gulf Coast Site,

Houston, Texas

Summary Information [1,2]

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)

Configuration Groundwater Recirculation

Technology Scale Full 

Media/Matrix Treated Groundwater

Contaminants Targeted TCE, cis-1,2-DCE, VC

Period of Operation Ongoing (data available from June 1995 to

December 1998) 

Site History/Source of Contamination [1,2,3]

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-36

Matrix Characteristic Value

Soil Type Silty sand

Depth to Groundwater 12 - 20 ft bgs

Thickness of Aquifer Approximately 8 ft

Fraction of Organic Carbon Not provided

DNAPL Present None identified

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-37

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-38

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). 
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-39

Table 2. Summary of Technology Performance Data

Average Concentrations (mg/L) [3]

Parameter

Analytical

Methods*

Jun-95 May-96 Jun-97 Mar-98 

Inside

Wells

Outside

Wells

Inside

Wells

Outside

Wells

Inside

Wells

Outside

Wells

Inside

Wells

Outsid

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 

cis-1,2-DCE EPA 8260 1.91 < 0.005 1.0 < 0.005 1.31 0.049 2.90 0.006 

Vinyl Chloride EPA 8260 0.102 < 0.005 < 0.005 <0.005 0.084 < 0.002 0.222 < 0.002 

Average excluding potential source well MW

TCE EPA 8260 11.8 - 9.68 - 3.24 - 2.42 - 

cis 1,2-DCE EPA 8260 1.28 - 0.733 - 1.38 - 2.52 - 

Vinyl Chloride EPA 8260 0.078 - 0.016 - 0.095 - 0.181 - 

Chloride EPA 325.3 162 35 147 29 132 20 136 23 

NO3-NO2 EPA 353.2 < 1.0 < 1.0 11 0.90 0.20 0.13 < 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 

TKN EPA 351.2 < 1.0 < 1.0 3.6 1.6 < 1.0 < 1.0 < 1.0 < 1.0 

Ferrous Iron EPA 6010 - - - - 0.23 < 0.05 0.27 < 0.02 

Total Iron EPA 6010 - - - - - - 3.4 0.25 

Sulfate EPA 375.4 - - - - 170 28 146 24 

Sulfide EPA 376.1 - - - - < 1.0 < 1.0 < 1.0 < 1.0 

Methane EPA 3810 - - - - 0.69 0.007 2.8 0.009 

Ethene EPA 3810 - - - - - - - - 

DO 1.7 3.4 1.1 2.4 0.45 1.6 0.65 1.6 

Redox (mV) +292 +319 +111 +126 +85 +178 -61 +332 

pH (SU) 6.56 6.75 6.69 6.84 6.46 6.83 6.38 6.74 

Notes: (-) Not Analyzed.

(*) Latest sampling event only.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-40

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.

Summary Observations and Lessons Learned [1,3]

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. 
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-41

In addition, they found it difficult to interpret the treatment performance data because of the nonhomogeneous 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. FullScale 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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-42

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

Mechanism(s) Anaerobic Reductive Dechlorination

(Cometabolic and Direct)

Technology Electron Donor Addition (Molasses)

Configuration Direct Injection

Technology Scale Pilot and Full

Media/Matrix Treated Groundwater

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 fullscale molasses injection system was installed and has been operating at the site since January 1997. 
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-43

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 organiccontaminated 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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-44

Figure 2: Monitoring Well Network at the Site [7]

Figure 1: Molasses Injection System Used at Lycoming Superfund Site [2]
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-45

Figure 3: Distribution of Groundwater Indicator Parameters, Baseline Conditions, January

1997 [2,8]

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-46

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-47

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. 

Summary Observations and Lessons Learned [1,3]

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. 
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-48

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-49

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)

Configuration Direct Injection

Technology Scale Pilot and Full

Media/Matrix Treated Groundwater

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-50

VC <20

NS NOT SAMPLED

VINYL CHLORIDE

DICHLOROETHENE

TRICHLOROETHENE

TETRACHLOROETHENE

MICROGRAMS PER LITER (ug/L))

(ALL CONCENTRATIONS SHOWN IN 

PCE

TCE

DCE

VC

MW-5 MONITORING WELL

VOC CONCENTRATIONS:

LEGEND:

PCE <100

TCE 10,000

DCE 900

VC <200

PCE <10

TCE 260

DCE 17 VC 2.2

DCE 11

TCE 30

PCE <0.5

PCE <0.5

TCE 1.1

DCE 9.7

VC 4.6

VC <10

DCE 17

TCE 65

PCE 6.2

PCE 15

TCE 95

DCE 8.8

VC <5

VC <1,000

DCE <500

TCE 17,000

PCE <500

VC <1

DCE 1.2

TCE 13

PCE <0.5

PCE 10

TCE 210

DCE 31

VC <10

PCE <50

TCE 280

DCE 62

VC <100

VC <100

DCE 410

TCE 4,000

PCE <50

FORMER 

BOILER 

FACILITY

FORMER TCE 

DEGREASING 

AREA

FORMER 

CHROMIUM WASTE 

STORAGE TANK

MW-3C

MW-11

MW-3B

MW-12

MW-3A

MW-9

MW-20

MW-4 MW-1

MW-10

MW-13 MW-14

MW-5

MW-16

MW-17

Figure 1: Pre-Injection CAH Concentrations, Abandoned Manufacturing Facility, Emeryville,

California (April 1995) [1]

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

DNAPL Present None identified

Hydraulic Conductivity Not available

Groundwater Velocity 60 feet per year
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-51

Technology Description [1,3]

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-52

APPROXIMATE BOUNDARY

INJECTION AREAS

MW-5 MONITORING WELL

LEGEND:

FORMER 

BOILER 

FACILITY

FORMER TCE 

DEGREASING 

AREA

FORMER 

CHROMIUM WASTE 

STORAGE TANK

MW-3C

MW-11

MW-3B

MW-12

MW-3A

MW-9

MW-20

MW-4 MW-1

MW-10

MW-13 MW-14

MW-5

MW-16

MW-17

Figure 2: Full-Scale Injection Area, Abandoned Manufacturing Facility, Emeryville, California [2]

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).
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-53

VC 16

NS NOT SAMPLED

VOC CONCENTRATIONS:

(ALL CONCENTRATIONS SHOWN IN 

MICROGRAMS PER LITER (ug/L))

VC

DCE

TCE

PCE TETRACHLOROETHENE

TRICHLOROETHENE

DICHLOROETHENE

VINYL CHLORIDE

MW-5 MONITORING WELL

LEGEND:

PCE <0.5

TCE 1.6

DCE 7.4

VC 19

VC 1.3

DCE 2.7

TCE 4.4

PCE <0.5

VC <0.5

DCE 2.4

TCE 3.5

PCE <0.5

PCE <0.5

TCE 17

DCE 1.7

VC <1

PCE <0.5

TCE <0.5

DCE 350

VC <50

VC 180

DCE 1,400

TCE <25

PCE <25

PCE 0.75

TCE 11

DCE 7.8

VC <1

VC NS

DCE NS

TCE NS

PCE NS

PCE NS

TCE NS

DCE NS

DCE 8 VC NS

TCE 13

PCE <2.5

VC 14

DCE 53

TCE 11

PCE <1.2

FORMER 

BOILER 

FACILITY

FORMER TCE 

DEGREASING 

AREA

FORMER 

CHROMIUM WASTE 

STORAGE TANK

MW-3C

MW-11

MW-3B

MW-12

MW-3A

MW-9

MW-20

MW-4 MW-1

MW-10

MW-13 MW-14

MW-5

MW-16

MW-17

Figure 3: CAH Concentrations - October 1998, Abandoned Manufacturing

Facility, Emeryville, California [2]

Figure 4: Analytical Results for Well MW-4, Abandoned Manufacturing

Facility, Emeryville, California [2]
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-54

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

Facility, Emeryville, California [2]

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

Abandoned Manufacturing Facility, Emeryville, California [2]
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-55

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-56

Case Study # 7

Sequential Anaerobic/Aerobic Biodegradation of PCE Field Demonstration

at Watertown, Massachusetts

Summary Information [1,3]

Site Name, Location Watertown, Massachusetts

EPA ID Number Not provided

Mechanism(s) Anaerobic Reductive

Dechlorination (Cometabolic

and Direct)

Aerobic Oxidation

(Cometabolic and Direct)

Technology Nutrient Addition

Electron Donor Addition

(Lactate)

Electron Acceptor Addition

(Oxygen)

Electron Donor Addition

(Propane)

Configuration Groundwater Recirculation

Technology Scale Field Demonstration

Media/Matrix Treated Groundwater

Contaminants Targeted TCE, PCE

Period of Operation Anaerobic: November 1996 to July 1997;

Aerobic: August 1997 to ongoing (data available through

October 1997)

Site History/Source of Contamination [1,2,5]

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). 
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-57

 Matrix Characteristics for the Watertown Site [1]

Matrix Characteristic Value

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

Technology Description [1,2]

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-58

Figure 1: Process Diagram [1]

Figure 2: Well Locations [1]
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-59

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. 
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-60

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.

Cincinnati, OH 45268

(573) 569-7856

lewis.ronald@epa.gov
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-61

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-62

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

Mechanism(s) Anaerobic Reductive Dechlorination

(Cometabolic and Direct)

Technology Bioaugmentation

Nutrient Addition

Electron Donor Addition (Lactate)

Configuration Groundwater Recirculation

Technology Scale Field demonstration (pilot proof of technology

test)

Media/Matrix Treated Groundwater

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
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-63

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]

Matrix Characteristic Value

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

Depth to Groundwater 10 to 12 ft bgs

Fraction of Organic Carbon 0.2

Thickness of Aquifer 38 ft (saturated portion)

DNAPL Present None identified

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 semiisolated or “closed-loop” recirculation cell and the wells formed a rectangular, hydraulically-controlled

cell that was 40 ft wide and 60 ft long.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-64

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. 
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-65

N

40 ft

Key:

IW = injection well

EW = extraction well

AA = monitoring well

PZ = piezometer

Figure 2: Well Locations [5]

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-66

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
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-67

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 threedimensional 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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-68

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,

sewer)

50,716

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

11/30/96

Startup through

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
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-69

Summary Observations and Lessons Learned [1]

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
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-70

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.
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-71

Case Study # 9

Cometabolic Bioventing Field Demonstration at Building 719,

Dover Air Force Base, Dover Delaware

Summary Information [1,3,7]

Site Name, Location Dover Air Force Base, Building 719, Dover,

Delaware

EPA ID Number DE8570024010

Mechanism(s) Aerobic Oxidation (Cometabolic and Direct)

Technology Electron Acceptor Addition (Oxygen)

Electron Donor Addition (Propane)

Configuration Direct Injection

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. 
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-72

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]

Matrix Characteristic Value

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

Depth to Groundwater 6 to 10 ft bgs

DNAPL Present None identified

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
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-73

Soil TCE Levels (mg/kg)

Frequency - No. of Samples

0

10

20

30

40

50

60

70

80

T = 0

T = Final

< 0.25

0.25 - 0.5

0.5 - 1

1 - 5

5 - 10

> 10

Soil 1,1,1-TCA Levels (mg/kg)

Frequency - No. of Samples

0

10

20

30

40

50

60

70

80

T = 0

T = Final

< 0.5

0.5 - 5

5 - 25

25 - 100

> 100

Frequency - No. of Samples

0

10

20

30

40

50

60

70

T=0 

T=Final 

< 20

20 - 4040 - 6060 - 80

80 - 100

100 - 120

> 120

Soil Chloride Levels (mg/kg) Soil 1.2-cis DCE Levels (mg/kg)

Frequency - No. of Samples

0

10

20

30

40

50

60

70

80

T = 0

T = Final

< 0.25

0.25 - 0.5

0.5 - 1

1 - 5

5 - 20

> 20

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]
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-74

Technology Cost

Cost data were not available at the time of this report.

Summary Observations and Lessons Learned [7]

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

Cincinnati, OH 45268

(513) 569-7607

Fax: (513) 569-7105

E-mail: sayles.gregory@epa.gov
Engineered Approaches to In Situ Bioremediation of Chlorinated Solvents

A-75

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.