InSitu Bioremediation Technonlogy

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In Situ Bioremediation: When Does it Work? (1993)
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In Situ Bioremediation: When Does it Work?
In Situ Bioremediation
When does it work?
Committee on In Situ Bioremediation

Water Science and Technology Board
Commission on Engineering and Technical Systems
National Research Council
NATIONAL ACADEMY PRESS

Washington, D.C. 1993
Copyright National Academy of Sciences. All rights reserved.
ii
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NOTICE: The project that is the subject of this report was approved by the Governing Board of the
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of Sciences, the National Academy of Engineering, and the Institute of Medicine. The members of
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approved by a Report Review Committee consisting of members of the National Academy of Sci-
ences, the National Academy of Engineering, and the Institute of Medicine.
Support for this project was provided by the U.S. Environmental Protection Agency under
Agreement No. CR 820730-01-0, the National Science Foundation under Agreement No.
BCS-9213271, the Electric Power Research Institute under Agreement No. RP2879-26, the Gas
Research Institute, the American Petroleum Institute, Chevron USA, Inc., and the Mobil Oil Corpo-
ration.
Library of Congress Cataloging-in-Publication Data
In situ bioremediation / Water Science and Technology Board, Commission on Engineering
and Technical Systems, National Research Council.
p. cm.
Includes bibliographical references and index.
ISBN 0-309-04896-6
1. In situ bioremediation—Evaluation. I. National Research Council (U.S.). Water Science and
Technology Board.
TD192.5.153 1993 93-5531
628.5'2—dc20 CIP
Copyright 1993 by the National Academy of Sciences. All rights reserved.
B-184
Cover art by Y. David Chung. Title design by Rumen Buzatov. Chung and Buzatov are graduates of
the Corcoran School of Art in Washington, D.C. Chung has exhibited widely throughout the coun-
try, including at the Whitney Museum in New York, the Washington Project for the Arts in Wash-
ington, D.C., and the Williams College Museum of Art in Williamstown, Massachusetts.
In brilliant colors, the cover art shows the amazing variety of unusual shapes found in bacterial
life forms.
Printed in the United States of America
First Printing, October 1993
Second Printing, December 1994

iii
COMMITTEE ON IN SITU BIOREMEDIATION
BRUCE E. RITTMANN, Chair, Northwestern University, Evanston, Illinois

LISA ALVAREZ-COHEN, University of California, Berkeley
PHILIP B. BEDIENT, Rice University, Houston, Texas
RICHARD A. BROWN, Groundwater Technology, Inc., Trenton, New Jersey
FRANCIS H. CHAPELLE, U.S. Geological Survey, Columbia, South Carolina
PETER K. KITANIDIS, Stanford University, Stanford, California
EUGENE L. MADSEN, Cornell University, Ithaca, New York
WILLIAM R. MAHAFFEY, ECOVA Corporation, Redmond, Washington
ROBERT D. NORRIS, Eckenfelder, Inc., Nashville, Tennessee
JOSEPH P. SALANITRO, Shell Development Company, Houston, Texas
JOHN M. SHAUVER, Michigan Department of Natural Resources, Lansing,
Michigan
JAMES M. TIEDJE, Michigan State University, East Lansing, Michigan
JOHN T. WILSON, Robert S. Kerr Environmental Research Laboratory, Ada,
Oklahoma
RALPH S. WOLFE, University of Illinois, Urbana
Staff
JACQUELINE A. MACDONALD, Study Director
GREGORY K. NYCE, Senior Project Assistant
GREICY AMJADIVALA, Project Assistant
WYETHA TURNEY, Word Processor
KENNETH M. REESE, Editorial Consultant
BARBARA A. BODLING, Editorial Consultant

iv
WATER SCIENCE AND TECHNOLOGY BOARD
DANIEL A. OKUN, Chair, University of North Carolina, Chapel Hill

A. DAN TARLOCK, Vice Chair, IIT Chicago-Kent College of Law, Chicago,
Illinois
J. DAN ALLEN, Chevron USA, Inc., New Orleans, Louisiana
KENNETH D. FREDERICK, Resources for the Future, Washington, D.C.
DAVID L. FREYBERG, Stanford University, Stanford, California
WILFORD R. GARDNER, University of California, Berkeley
DUANE L. GEORGESON, Metropolitan Water District of Southern California,
Los Angeles
LYNN R. GOLDMAN, California Department of Health Services, Emeryville
WILLIAM L. GRAF, Arizona State University, Tempe
THOMAS M. HELLMAN, Bristol-Myers Squibb Company, New York, New
York
ROBERT J. HUGGETT, College of William and Mary, Gloucester Point,
Virginia
CHARLES C. JOHNSON, Consultant, Bethesda, Maryland
JUDY L. MEYER, University of Georgia, Athens
STAVROS S. PAPADOPULOS, S. S. Papadopulos & Associates, Inc.,
Bethesda, Maryland
KENNETH W. POTTER, University of Wisconsin-Madison
BRUCE E. RITTMANN, Northwestern University, Evanston, Illinois
PHILIP C. SINGER, University of North Carolina, Chapel Hill
JOY B. ZEDLER, San Diego State University, San Diego, California
Staff
STEPHEN D. PARKER, Director
SARAH CONNICK, Senior Staff Officer
SHEILA D. DAVID, Senior Staff Officer
CHRIS ELFRING, Senior Staff Officer
GARY D. KRAUSS, Staff Officer
JACQUELINE A. MACDONALD, Staff Officer
JEANNE AQUILINO, Administrative Associate
ANITA A. HALL, Administrative Assistant
PATRICIA L. CICERO, Senior Project Assistant
GREGORY K. NYCE, Senior Project Assistant

COMMISSION ON ENGINEERING AND TECHNICAL

SYSTEMS
ALBERT R. C. WESTWOOD, Chair, Martin Marietta Corporation, Bethesda,

Maryland
NANCY CONNERY, Woolwich, Maine
RICHARD A. CONWAY, Union Carbide Corporation, South Charleston, West
Virginia
GERARD W. ELVERUM, JR., TRW Space & Technology Group, Banning,
California
E. R. (VALD) HEIBERG III, J. A. Jones Construction Services Company,
Charlotte, North Carolina
WILLIAM G. HOWARD, JR., Scottsdale, Arizona
JOHN McCARTHY, Stanford University, Stanford, California
ALTON D. SLAY, Slay Enterprises, Inc., Warrenton, Virginia
JAMES J. SOLBERG, Purdue University, West Lafayette, Indiana
CHARLES F. TIFFANY, Boeing Military Airplane Company, Yuma, Arizona
(Retired)
JOHN A. TILLINGHAST, TILTEC, Portsmouth, New Hampshire
PAUL TORGERSEN, Virginia Polytechnic Institute and State University,
Blacksburg
GEORGE L. TURIN, Teknekron Corporation, Menlo Park, California
JOHN B. WACHTMAN, JR., Rutgers University, Piscataway, New Jersey
BRIAN J. WATT, Joy Technologies, Inc., Houston, Texas
WILLIAM C. WEBSTER, University of California, Berkeley
ROBERT V. WHITMAN, Massachusetts Institute of Technology, Cambridge
Staff
ARCHIE L. WOOD, Executive Director
MARLENE BEAUDIN, Associate Executive Director
MARY FRANCES LEE, Director of Operations
ROBERT KATT, Associate Director for Quality Management
LYNN KASPER, Assistant Editor
TEREE DITTMAR, Administrative Assistant
SYLVIA GILBERT, Administrative Assistant

vi
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PREFACE vii
Preface
Bioremediation is a technology that is gaining momentum in technical,

policy, and popular circles. It also is a technology associated with mystery,
controversy, and "snake oil salesmen." When a representative of the U.S.
Environmental Protection Agency suggested in the fall of 1991 that the Water
Science and Technology Board conduct a study on bioremediation, it converged
with the board's internal initiative to "do something" in the area. Several high-
quality workshops and conferences had occurred in the previous year that
generated publications describing what is needed for bioremediation to fulfill its
potential. The board needed to design a study that would do more than repeat
what was already available, that would be completed in a time frame
commensurate with the urgent needs of those involved in bioremediation, and
that would meet the high standards expected of the National Academy of
Sciences. These criteria inevitably led to the subject of this report and to a unique
format for conducting the study.
The study's subject—"In Situ Bioremediation: When Does It Work?"—
narrows the focus to two critical facets of bioremediation. First, it addresses the
use of microorganisms to remove contamination from ground water and soils that
remain in place (i.e., in situ) during the cleanup. This focus distinguishes in situ
bioremediation of the subsurface from significantly different applications of
bioremediation, such as to treat oil tanker spills, wastewaters, or sludges. Second,
the

PREFACE viii
primary object of the study is to provide guidance on how to evaluate when an in

situ bioremediation process is working or has worked. This focus is most
important because the in situ environment is highly complex and very difficult to
observe. Therefore, tools from several scientific and engineering disciplines must
be used in a sophisticated manner if the success of a bioremediation effort is to be
evaluated. Guidance is acutely needed today because most people faced with
making decisions about bioremediation projects do not have the interdisciplinary
knowledge to integrate all of the necessary tools.
The format for this study was unique and designed to meet two criteria:
meaningful interdisciplinary interchange and timeliness. To gain interchange, a
committee of 14 was carefully chosen to include recognized leaders in academic
research, field practice, regulation, and industry. A balance was achieved between
those involved in research fundamentals and those involved in the practical
aspects of application, as well as between scientists and engineers. Once the
committee of interdisciplinary experts was assembled, meaningful interchange
was fostered by an intensive week-long workshop at the National Research
Council. The goals were to maximize opportunities for formal and informal
interchange among the committee members and to build a common purpose. Both
goals were achieved, directly leading to a consensus about the issues and what
were to be the committee's recommendations.
Timeliness was a prime consideration in designing the study's format. In
order to accelerate interdisciplinary communications, nine committee members
prepared seven background papers in advance of the week-long workshop. At the
workshop, the committee initially generated its own discussion topics and then
systematically discussed them. Key to timeliness and keeping the committee "on
target" was preparation of a draft report during the workshop. Near the end of the
workshop, the committee reviewed the draft report, which refocused the entire
group on exactly what it wanted to say.
Appearing first in this volume is the committee's report, which describes the
principles and practices of in situ bioremediation and provides practical
guidelines for evaluating success. The report's guidelines should be immediately
useful to regulators, practitioners, and buyers who are involved in decision-
making processes involving bioremediation. We envision that the report will
provide a commonly accepted basis for which all parties can agree to specific
evaluation protocols. Also included here are the seven background papers. These
papers will give the reader added insight into the different perspectives that were
brought to the committee. The entire report has been reviewed by a group other
than the authors, but only the committee

PREFACE ix
report was subjected to the report review criteria established by the National
Research Council's Report Review Committee. The background papers have been
reviewed for factual correctness.
Special acknowledgment must go to several individuals who contributed to
the committee's overall effort in special ways. First, Dick Brown and Jim Tiedje
joined me on the executive committee, which had the all-important tasks of
identifying and recruiting committee members and which also oversaw the
committee's management. Second, Eugene Madsen, the committee's rapporteur,
wrote the first draft of the report during the workshop and prepared an excellent
second draft after the workshop. Eugene did these crucial and grueling tasks with
skill and good humor. Finally, Jackie MacDonald, staff officer for the committee,
made this unique effort possible. She efficiently arranged all the logistics for the
workshop and for publishing the book. Even more importantly, she used her
exceptional technical and editorial skills to ensure that the report and the
background papers are logical, correct, understandable, and interesting to read.
The committee members owe Jackie a debt of gratitude for making us sound
more intelligent and better organized than we might actually be.
Finally, I want to mention two possible spin-off benefits of the study and
report. First, most of the principles and guidelines described here also apply to
evaluating bioremediation that does not occur in situ. Although the inherent
difficulties of working in an in situ environment make evaluation especially
challenging, other bioremediation applications also are subject to uncertainties
and controversy that can be resolved only with the kind of rational evaluation
strategies described here. Second, the format for the workshop might provide a
prototype for effective interdisciplinary communications, one of the most critical
needs for implementing bioremediation, as well as other technologies.
Bruce E. Rittmann, Chair

Committee on In Situ Bioremediation

PREFACE x

CONTENTS xi
Contents
EXECUTIVE SUMMARY 1
1 INTRODUCTION 12
2 PRINCIPLES OF BIOREMEDIATION 16
The Role of Microbes in Bioremediation 17
How Microbes Destroy Contaminants 17
How Microbes Demobilize Contaminants 22
Indicators of Microbial Activity 23
Complicating Factors 25
Contaminants Susceptible to Bioremediation 29
Petroleum Hydrocarbons and Derivatives 32
Halogenated Compounds 33
Nitroaromatics 34
Metals 34
Environments Amenable to Bioremediation 35
Two Types of Bioremediation: Intrinsic and Engineered 35
Site Conditions for Engineered Bioremediation 39
Site Conditions for Intrinsic Bioremediation 41
Impact of Site Heterogeneity on Bioremediation 42
Further Reading 43

CONTENTS xii
Boxes
Key Terms for Understanding Bioremediation 19
Intrinsic Bioremediation of a Crude Oil Spill—Bemidji, 37
Minnesota
Site Characteristics that Favor In Situ Bioremediation 40
3 THE CURRENT PRACTICE OF BIOREMEDIATION 47

Bioremediation Versus Other Technologies 48
Basics of Bioremediation Process Design 49
Engineered Bioremediation 50
Intrinsic Bioremediation 59
Integration of Bioremediation with Other Technologies 60
Good Practices 61
Box
Standards of Practice for Bioremediation Contractors 62
4 EVALUATING IN SITU BIOREMEDIATION 63

A Three-Part Strategy for ''Proving" In Situ Bioremediation 63
Techniques for Demonstrating Biodegradation in the Field 65
Measurements of Field Samples 65
Experiments Run in the Field 78
Modeling Experiments 80
Limitations Inherent in Evaluating In Situ Bioremediation 88
Boxes
Proving Engineered Bioremediation of Chlorinated Sol- 66
vents in a Field Test—Moffett Naval Air Station, Cali-
fornia
Proving Engineered Bioremediation of an Oil and Fuel Spill 71
—Denver, Colorado
Testing Bioremediation of PCBs in Hudson River Sedi- 77
ments—New York
Proving Intrinsic Bioremediation of a Spill at a Natural Gas 86
Manufacturing Plant—Northern Michigan
5 FUTURE PROSPECTS FOR BIOREMEDIATION 91

New Frontiers in Bioremediation 92
The Increasing Importance of Evaluating Bioremediation 93
Recommended Steps in Research 94
Recommended Steps in Education 95

CONTENTS xiii
BACKGROUND PAPERS 97
A Regulator's Perspective on In Situ Bioremediation 99
John M. Shauver
An Industry's Perspective on Intrinsic Bioremediation 104
Joseph P. Salanitro
Bioremediation from an Ecological Perspective 110
James M. Tiedje
In Situ Bioremediation: The State of the Practice 121
Richard A. Brown, William Mahaffey, and Robert D. Norris
Engineering Challenges of Implementing In Situ Bioremedia- 136
tion
Lisa Alvarez-Cohen
Modeling In Situ Bioremediation 153
Philip B. Bedient and Hanadi S. Rifai
Testing Bioremediation in the Field 160
John T. Wilson
APPENDIXES 185
A Glossary 187
B Biographical Sketches of Committee Members and Staff 195
INDEX 199

CONTENTS xiv

xv
In Situ Bioremediation When does it work?

xvi

EXECUTIVE SUMMARY 1
Executive Summary
The United States is investing billions of dollars in cleaning up polluted

ground water and soils, yet this large investment may not be producing the
benefits that citizens expect. Recent studies have revealed that because of
limitations of ground water cleanup technologies, there are almost no sites where
polluted ground water has been restored to a condition fit for drinking. While soil
cleanup efforts have come closer to meeting regulatory goals, the technologies
typically used to decontaminate soils often increase the exposure to contaminants
for cleanup crews and nearby residents.
The limitations of conventional ground water cleanup technologies and the
hazards of conventional soil treatment methods—along with the high costs of
both—have spurred investigations into alternative cleanup technologies,
including in situ bioremediation. In situ bioremediation uses microorganisms to
destroy or immobilize contaminants in place. The technology already has
achieved a measure of success in field tests and commercial scale cleanups for
some types of contaminants.
Proponents of in situ bioremediation say the technology may be less costly,
faster, and safer than conventional cleanup methods. Yet despite mounting
evidence in support of the technology, bioremediation is neither universally
understood nor trusted by those who must approve its use. Bioremediation is
clouded by controversy over what it does and how well it works, partly because it
relies on microorganisms,

EXECUTIVE SUMMARY 2
which cannot be seen, and partly because it has become attractive for "snake oil
salesmen" who claim to be able to solve all types of contamination problems. As
long as the controversy remains, the full potential of this technology cannot be
realized.
In this report the Committee on In Situ Bioremediation communicates the
scientific and technological bases for in situ bioremediation, with the goal of
eliminating the mystery that shrouds this highly multidisciplinary technology.
The report presents guidelines for evaluating in situ bioremediation projects to
determine whether they will or are meeting cleanup goals. The Committee on In
Situ Bioremediation was established in June 1992 with the specific task of
developing such guidelines, and it represents the span of groups involved in
bioremediation: buyers of bioremediation services, bioremediation contractors,
environmental regulators, and academic researchers. Included with the report are
seven background papers, authored by committee members, representing the
range of perspectives from which bioremediation may be viewed.
PRINCIPLES OF BIOREMEDIATION
The most important principle of bioremediation is that microorganisms
(mainly bacteria) can be used to destroy hazardous contaminants or transform
them to less harmful forms. The microorganisms act against the contaminants
only when they have access to a variety of materials—compounds to help them
generate energy and nutrients to build more cells. In a few cases the natural
conditions at the contaminated site provide all the essential materials in large
enough quantities that bioremediation can occur without human intervention—a
process called intrinsic bioremediation . More often, bioremediation requires the
construction of engineered systems to supply microbe-stimulating materials—a
process called engineered bioremediation. Engineered bioremediation relies on
accelerating the desired biodegradation reactions by encouraging the growth of
more organisms, as well as by optimizing the environment in which the
organisms must carry out the detoxification reactions.
A critical factor in deciding whether bioremediation is the appropriate
cleanup remedy for a site is whether the contaminants are susceptible to
biodegradation by the organisms at the site (or by organisms that could be
successfully added to the site). Although existing microorganisms can detoxify a
vast array of contaminants, some compounds are more easily degraded than
others. In general, the compounds most easily degraded in the subsurface are
petroleum hydrocarbons, but technologies for stimulating the growth of
organisms

EXECUTIVE SUMMARY 3
to degrade a wide range of other contaminants are emerging and have been
successfully field tested.
The suitability of a site for bioremediation depends not only on the
contaminant's biodegradability but also on the site's geological and chemical
characteristics. The types of site conditions that favor bioremediation differ for
intrinsic and engineered bioremediation. For intrinsic bioremediation, the key site
characteristics are consistent ground water flow throughout the seasons; the
presence of minerals that can prevent pH changes; and high concentrations of
either oxygen, nitrate, sulfate, or ferric iron. For engineered bioremediation, the
key site characteristics are permeability of the subsurface to fluids, uniformity of
the subsurface, and relatively low (less than 10,000 mg/kg solids) residual
concentrations of nonaqueous-phase contaminants.
When deciding whether a site is suitable for bioremediation, it is important
to realize that no single set of site characteristics will favor bioremediation of all
contaminants. For example, certain compounds can only be degraded when
oxygen is absent, but destruction of others requires that oxygen be present. In
addition, one must consider how the bioremediation system may perform under
variable and not perfectly known conditions. A scheme that works optimally
under specific conditions but poorly otherwise may be inappropriate for in situ
bioremediation.
THE CURRENT PRACTICE OF BIOREMEDIATION

Few people realize that in situ bioremediation is not really a "new"
technology. The first in situ bioremediation system was installed 20 years ago to
cleanup an oil pipeline spill in Pennsylvania, and since then bioremediation has
become well developed as a means of cleaning up easily degraded petroleum
products. What is new is the use of in situ bioremediation to treat compounds
other than easily degraded petroleum products on a commercial scale. The
principles of practice outlined here were developed to treat petroleum-based
fuels, but they will likely apply to a much broader range of uses for
bioremediation in the future.
Engineered Bioremediation
Engineered bioremediation may be chosen over intrinsic bioremediation
because of time and liability. Where an impending property transfer or potential
impact of contamination on the local community dictates the need for rapid
pollutant removal, engineered bioremediation

EXECUTIVE SUMMARY 4
may be a more appropriate remedy than intrinsic bioremediation. Because

engineered bioremediation accelerates biodegradation reaction rates, it requires
less time than intrinsic bioremediation. The shorter time requirements reduce the
liability for costs required to maintain and monitor the site.
Since many petroleum hydrocarbons require oxygen for their degradation,
the technological emphasis of engineered bioremediation systems in use today
has been placed on oxygen supply. Bioremediation systems for soil above the
water table usually consist of a set of vacuum pumps to supply air (containing
oxygen) and infiltration galleries, trenches, or dry wells to supply moisture (and
sometimes specific nutrients). Bioremediation systems for ground water and soil
below the water table usually consist of either a set of injection and recovery
wells used to circulate oxygen and nutrients dissolved in water or a set of
compressors for injecting air. Emerging applications of engineered
bioremediation, such as for degradation of chlorinated solvents, will not
necessarily be controlled by oxygen. Hence, the supply of other stimulatory
materials may require new technological approaches even though the ultimate
goal, high biodegradation rates, remains the same.
Intrinsic Bioremediation
Intrinsic bioremediation is an option when the naturally occurring rate of
contaminant biodegradation is faster than the rate of contaminant migration.
These relative rates depend on the type and concentration of contaminant, the
microbial community, and the subsurface hydrogeochemical conditions. The
ability of native microbes to metabolize the contaminant must be demonstrated
either in field tests or in laboratory tests performed on site-specific samples. In
addition, the effectiveness of intrinsic bioremediation must be continually
monitored by analyzing the fate of the contaminants and other reactants and
products indicative of biodegradation.
In intrinsic bioremediation the rate-controlling step is frequently the influx
of oxygen. When natural oxygen supplies become depleted, the microbes may
not be able to act quickly enough to contain the contamination. Lack of a
sufficiently large microbial population can also limit the cleanup rate. The
microbial population may be small because of a lack of nutrients, limited
availability of contaminants resulting from sorption to solid materials or other
physical phenomena, or an inhibitory condition such as low pH or the presence of a
toxic material.

EXECUTIVE SUMMARY 5
Integration of Bioremediation with Other Technologies

Bioremediation frequently is combined with nonbiological treatment
technologies, both sequentially and simultaneously. For example, when soil is
heavily contaminated, bioremediation may be implemented after excavating soils
near the contaminant source—a process that reduces demand on the
bioremediation system and the immediate potential for ground water
contamination. Similarly, when pools of contaminants are floating on the water
table, these pools may be pumped to the surface before bioremediation of
residual materials. Bioremediation may follow treatment of the ground water with
a conventional pump-and-treat system designed to shrink the contaminant plume
to a more manageable size. Bioremediation may also be combined with a vapor
recovery system to extract volatile contaminants from soils. Finally, it is possible
to follow engineered bioremediation, which cleans up most of the contamination,
with intrinsic bioremediation, which may be used for final polishing and
contaminant containment.
EVALUATING IN SITU BIOREMEDIATION

The inherent complexity of performing bioremediation in situ means that
special attention must be given to evaluating the success of a project. The most
elemental criterion for success of an in situ bioremediation effort is that the
microorganisms are mainly responsible for the cleanup. Without evidence of
microbial involvement, there is no way to verify that the bioremediation project
was actually a bioremediation—that is, that the contaminant did not simply
volatilize, migrate off site, sorb to the soil, or change form via abiotic chemical
reactions. Simply showing that microbes grown in the lab have the potential to
degrade the contaminant is not enough. While bioremediation often is possible in
principle, the more relevant question is, "Are the biodegradation reactions
actually occurring under site conditions?"
No one piece of evidence can unambiguously prove that microorganisms
have cleaned up a site. Therefore, the Committee on In Situ Bioremediation
recommends an evaluation strategy that builds a consistent, logical case for
bioremediation based on converging lines of independent evidence. The strategy
should include three types of information:
1. documented loss of contaminants from the site,

2. laboratory assays showing that microorganisms from site samples

EXECUTIVE SUMMARY 6
have the potential to transform the contaminants under the expected

site conditions, and
3. one or more pieces of information showing that the biodegradation
potential is actually realized in the field.
Every well-designed bioremediation project, whether a field test or full-scale

system, should show evidence of meeting the strategy's three requirements.
Regulators and buyers of bioremediation services can use the strategy to evaluate
whether a proposed or ongoing bioremediation project is sound; researchers can
apply the strategy to evaluate the results of field tests.
The first type of evidence—documented loss of contaminants from the site
—is gathered as part of the routine monitoring that occurs (or should occur) at
every cleanup site. The second type of evidence requires taking microbes from
the field and showing that they can degrade the contaminant when grown in a
well-controlled laboratory vessel. The most difficult type of evidence to gather is
the third type—showing that microbes in the field are actively degrading the
contaminant. There are two types of sample-based techniques for demonstrating
field biodegradation: measurements of field samples and experiments run in the
field. In most bioremediation scenarios a third technique, modeling experiments,
provides an improved understanding of the fate of contaminants in field sites.
Because none of these three techniques alone can show with complete certainty
that biodegradation is the primary cause of declining contaminant concentrations,
the most effective strategy for demonstrating bioremediation usually combines
several techniques.
Measurements of Field Samples

The following techniques for documenting in situ bioremediation involve
analyzing the chemical and microbiological properties of soil and ground water
samples from the contaminated site:
• Number of bacteria. Because microbes often reproduce when they

degrade contaminants, an increase in the number of contaminant-
degrading bacteria over usual conditions may indicate successful
bioremediation.
• Number of protozoans. Because protozoans prey on bacteria, an
increase in the number of protozoans signals bacterial population
growth, indicating that bioremediation may be occurring.
• Rates of bacterial activity. Tests indicating that bacteria from the
contaminated site degrade the contaminant rapidly enough to

EXECUTIVE SUMMARY 7
effect remediation when incubated in microcosms that resemble the field

site provide further evidence of successful bioremediation.
• Adaptation. Tests showing that bacteria from the bioremediation zone
can metabolize the contaminant, while bacteria from outside the zone
cannot (or do so more slowly), show that the bacteria have adapted to the
contaminant and indicate that bioremediation may have commenced.
• Carbon isotopes. Isotopic ratios of the inorganic carbon (carbon
dioxide, carbonate ion, and related compounds) from a soil or water
sample showing that the contaminant has been transformed to inorganic
carbon are a strong indicator of successful bioremediation.
• Metabolic byproducts. Tests showing an increase in the concentrations
of known byproducts of microbial activity, such as carbon dioxide,
provide a sign of bioremediation.
• Intermediary metabolites. The presence of metabolic intermediates—
simpler but incompletely degraded forms of the contaminant—in
samples of soil or water signals the occurrence of biodegradation.
• Growth-stimulating materials. A depletion in the concentration of
growth-stimulating materials, such as oxygen, is a sign that microbes are
active and may indicate bioremediation.
• Ratio of nondegradable to degradable compounds. An increase in the
ratio of compounds that are difficult to degrade to those that are easily
degraded indicates that bioremediation may be occurring.
Experiments Run in the Field

The following methods for evaluating whether microorganisms are actively
degrading the contaminant involve conducting experiments in the field:
• Stimulating bacteria within subsites. When growth-stimulating

materials such as oxygen and nutrients are added to one subsite within
the contaminated area but not another, the relative rate of contaminant
loss should increase in the stimulant-amended subsite. The contrast in
contaminant loss between enhanced and unenhanced subsites can be
attributed to bioremediation.
• Measuring the stimulant uptake rate. Growth-stimulating materials,
such as oxygen, can be added to the site in pulses to determine the rate
at which they are consumed. Relatively rapid loss of oxygen or other
stimulants in the contaminated area compared to an uncontaminated area
suggests successful bioremediation.

EXECUTIVE SUMMARY 8
• Monitoring conservative tracers. Tracer compounds that are not

biologically reactive can be added to the site to determine how much
contaminant (or growth-stimulating material) is disappearing through
nonbiological pathways and how much is being consumed by
microorganisms.
• Labeling contaminants. Contaminants can be labeled with chemical
elements that appear in metabolic end products when the contaminants
are degraded, providing another mechanism for determining whether
biodegradation is responsible for a contaminant's disappearance.
Modeling Experiments
A final set of techniques for evaluating whether bioremediation is occurring
in the field uses models—sets of mathematical equations that quantify the
contaminant's fate. Modeling techniques provide a framework for formally
deciding what is known about contaminant behavior at field sites. When
modelers have a high degree of confidence that the model accurately represents
conditions at the site, modeling experiments can be used to demonstrate field
biodegradation.
There are two general strategies for using models to evaluate
bioremediation. The first strategy, useful when biodegradation is the main
phenomenon controlling the contaminant's fate, is to model the abiotic processes
to determine how much contaminant loss they account for. Bioremediation is
indicated when the concentrations of contaminant actually found in field sites are
significantly lower than would be expected from predictions based on abiotic
processes (such as dilution, transport, and volatilization). The second strategy
involves directly modeling the microbial processes to estimate the biodegradation
rates. Direct modeling, while the intellectually superior approach, requires
quantitative information about the detailed interactions between microbial
populations and site characteristics. Because this information may be difficult to
obtain, direct modeling is primarily a topic of academic research and is seldom a
routinely applied procedure.
Four different types of models have been developed:
• Saturated flow models. These models describe where and how fast the
water and dissolved contaminants flow through the saturated zone.
• Multiphase flow models. These models characterize the situation in
which two or more fluids, such as water and a nonaqueous-phase
contaminant or water and air, exist together in the subsurface.

EXECUTIVE SUMMARY 9
• Geochemical models. These models analyze how a contaminant's

chemical speciation is controlled by the thermodynamics of the many
chemical and physical reactions that may occur in the subsurface.
• Biological reaction rate models. These models represent how quickly
the microorganisms transform contaminants.
Because so many complex processes interact in the subsurface, ultimately

two or more types of models may be required for a complete evaluation.
Limitations Inherent in Evaluating In Situ Bioremediation

Although microorganisms grown in the laboratory can destroy most organic
contaminants, the physical realities of the subsurface—the low fluid flow rates,
physical heterogeneities, unknown amounts and locations of contaminants, and
the contaminants' unavailability to the microorganisms—make in situ
bioremediation a technological challenge that carries inherent uncertainties. Three
strategies can help minimize these uncertainties: (1) increasing the number of
samples used to document bioremediation, (2) using models so that important
variables are properly weighted and variables with little influence are eliminated,
and (3) compensating for uncertainties by building safety factors and flexibility
into the design of engineering systems. These strategies should play important
roles in evaluating bioremediation projects.
While uncertainties should be minimized, it is important to recognize that no
strategy can entirely eliminate the uncertainties, even for the best-designed
systems. Given today's knowledge base, it is not possible to fully understand
every detail of whether and how bioremediation is occurring. The goal in
evaluating in situ bioremediation is to assess whether the weight of evidence from
tests such as those described above makes a convincing case for successful
bioremediation.
CONCLUSIONS: FUTURE PROSPECTS FOR

BIOREMEDIATION
Bioremediation integrates the tools of many disciplines. As each of the
disciplines advances and as new cleanup needs arise, opportunities for new
bioremediation techniques will emerge. As these new techniques are brought into
commercial practice, the importance of sound methods for evaluating
bioremediation will increase.
The fundamental knowledge base underlying bioremediation is sufficient to
begin implementing the three-part evaluation strategy

EXECUTIVE SUMMARY 10
the committee has recommended. However, further research and better education
of those involved in bioremediation will improve the ability to apply the strategy
and understanding of the fundamentals behind bioremediation.
Recommended Steps In Research

The committee recommends research in the following areas to improve
evaluations of bioremediation:
• Evaluation protocols. Protocols for putting the three-part evaluation

strategy into practice need to be developed and field tested through co-
ordinated efforts involving government, industry, and academia.
• Innovative site characterization techniques. Rapid, reliable, and
inexpensive site characterization techniques would simplify many of the
evaluation techniques this report describes. Examples of relevant site
measurements include distribution of hydraulic conductivities,
contaminant concentrations associated with solid or other nonaqueous
phases, native biodegradation potential, and abundance of different
microbial populations.
• Improved models. Improvements in mathematical models would
increase the ability to link chemical, physical, and biological phenomena
occurring in the subsurface and to quantify how much contaminant loss
occurs because of biodegradation.
Recommended Steps in Education

Steps need to be taken to improve the understanding of what bioremediation
is and what it can and cannot do. The committee recommends three types of
educational steps:
• Training courses that selectively extend the knowledge bases of the

technical personnel currently dealing with the uses or potential uses
of in situ bioremediation. This step explicitly recognizes that
practitioners and regulators who already are dealing with complicated
applications of bioremediation need immediate education about
technical areas outside their normal expertise.
• Formal education programs that integrate the principles and
practices for the next generation of technical personnel. This step
explicitly recognizes the need to educate a new generation of technical
personnel who have far more interdisciplinary training than is currently
available in most programs.

EXECUTIVE SUMMARY 11
• Means for effective transfer of information among the different

stakeholders involved in a project. Effective transfer requires that all
types of stakeholders participate, that all are invested in achieving a
common product (such as a design, a report, or an evaluation
procedure), and that sufficient time is allocated for sharing perceptions
and achieving the product. This step may involve more time and more
intensive interactions than have been the norm in the past.
In summary, in situ bioremediation is a technology whose full potential has

not been realized. As the limitations of conventional ground water and soil
cleanup technologies become more apparent, research into alternative cleanup
technologies will intensify. Bioremediation is an especially attractive alternative
because it is potentially less costly than conventional cleanup methods, it shows
promise for reaching cleanup goals more quickly than pump-and-treat methods,
and it results in less transfer of contaminants to other media. However,
bioremediation presents a unique technological challenge. The combination of the
intricacies of microbial processes and the physical challenge of monitoring both
microorganisms and contaminants in the subsurface makes bioremediation
difficult to understand, and it makes some regulators and clients hesitant to trust
bioremediation as an appropriate cleanup strategy. The inherent complexity
involved in performing bioremediation in situ means that special attention must
be given to evaluating the success of a project. Whether a bioremediation project
is intrinsic or engineered, the importance of a sound strategy for evaluating
bioremediation will increase in the future as the search for improved cleanup
technologies accelerates.

INTRODUCTION 12
1
Introduction
In the past decade the United States has spent billions of dollars trying to
cleanup contaminated ground water and soils, the legacy of an era in which
industry grew faster than knowledge about safe chemical disposal. Despite the
large financial investment, ground water cleanup efforts are falling short of public
expectations. Recent studies have revealed that, while conventional cleanup
technologies have prevented the contamination problem from spreading, in most
cases they are incapable of restoring the water to meet health-based standards in a
reasonable time frame. Soil cleanups have been more successful in meeting
regulatory standards. However, conventional soil cleanup methods may transfer
contaminants to the air, posing risks that are not always acceptable to residents
near the contaminated site. The limitations of conventional ground water cleanup
technologies and the hazards of conventional soil cleanup methods have spurred
investigations into in situ bioremediation, which uses microorganisms to destroy
or immobilize contaminants in place. Bioremediation is a promising alternative to
conventional cleanup technologies for both ground water and soil because it may
be faster, safer, and less costly.
Conventional methods for ground water cleanup rely on pumping water to
the surface and treating it there. Such pump-and-treat methods are slow; they
require the withdrawal of large volumes of water to flush contaminants from
aquifer solids, and they may leave

INTRODUCTION 13
behind reservoirs of contaminants that are lighter or denser than water and/or
have low solubilities. By treating the problem close to its source, in situ,
bioremediation speeds contaminant desorption and dissolution. Consequently, a
cleanup that might require decades using pump-and-treat methods could possibly
be completed in a few years with bioremediation. In addition, pump-and-treat
methods do not destroy contaminants but simply bring them to the surface for
treatment or disposal elsewhere. In situ bioremediation, on the other hand, can
transform contaminants to harmless byproducts such as carbon dioxide and
water.
Conventional methods for soil cleanup require digging up the contaminated
soil and either incinerating it or burying it at a specially designed disposal site.
Soil excavation and incineration may increase the exposure to contaminants for
both the workers at the site and nearby residents. Furthermore, excavation and
final disposal are extremely costly. By treating the soil in place, bioremediation
reduces both the exposure risk and the cleanup cost.
Because bioremediation shows promise as an alternative to conventional
environmental cleanup technologies, the number of vendors selling
bioremediation has increased dramatically in recent years. Bioremediation is one
of the fastest-growing sectors of the U.S. hazardous waste market. It is expected
to become a $500 million per year industry by the year 2000. Yet despite the
rapid growth in the use of this technology, bioremediation is not universally
understood or trusted by those who must approve its use, and its success is a hotly
debated issue.
A primary reason for the lack of understanding and mistrust of
bioremediation is that the technology requires knowledge not only of such fields
as environmental engineering and hydrology, which are important in
conventional cleanup methods, but also of the complex workings of
microorganisms. The potential for large profits, when combined with the
mysteriousness of applying microorganisms, makes bioremediation attractive for
''snake oil salesmen" who claim to be able to solve all types of contamination
problems. Many buyers of cleanup services and regulators who approve cleanup
plans lack the necessary background to evaluate whether a bioremediation
project has a feasible design. Furthermore, they may be unsure how to evaluate
whether an ongoing bioremediation project is progressing toward successful
completion. Consequently, some regulators and clients approach bioremediation
with skepticism, opting for more conventional technologies even when
bioremediation is the most appropriate technology for a particular site.

INTRODUCTION 14
The multidisciplinary nature of bioremediation presents problems not only

for clients and regulators but also for the vendors of environmental cleanup
services. These vendors face a challenge in integrating the wide range of
disciplines needed to carry out bioremediation in the field. Communications
among engineers, microbiologists, hydrologists, and chemists are complicated by
each discipline's highly specialized concepts, tools, and jargon.
Even when all parties are knowledgeable and competent, evaluating the
success of bioremediation (i.e., whether or not it is working) can cause
confusion. Part of the confusion comes from the inherent complexity of the sites.
Knowing with certainty the location and fate of all contaminants is impossible.
However, evaluating the success of in situ bioremediation is further complicated
by the multiple definitions of success set forth by those involved with the
cleanup:
• Regulators want cleanup standards to be met.

• Clients want the cleanup to be cost-effective.
• Bioremediation vendors and researchers want evidence that the cleanup
was caused by microbial action—that the contaminant did not, for
example, simply evaporate or migrate off site.
This report is designed for bioremediation clients, regulators, and vendors,

who need to agree on how to define success appropriately. The report emphasizes
ways to show that microorganisms aided cleanup efforts because the use of
microorganisms is what distinguishes bioremediation from other technologies.
Chapter 2 explains the principles of bioremediation, describing the
microbiological processes that can be employed in bioremediation and providing
practical guidance on what types of contaminants and site conditions are most
amenable to bioremediation. Chapter 3 reviews the current state of the art in in
situ bioremediation systems. These chapters provide the basis for understanding
when in situ bioremediation is likely to work. Chapter 4—the most critical part of
the report—presents methods and strategies for evaluating a bioremediation
project in the testing or implementation phase. Finally, Chapter 5 suggests
innovations that may improve the technology's capabilities in the future.
This report represents the opinions of the National Research Council's
Committee on In Situ Bioremediation. The National Research Council established
this committee in June 1992 and assigned it the specific task of preparing
guidelines for evaluating whether an in situ bioremediation project, either
proposed or in the implementation stage, is likely to reach cleanup goals. The
committee includes representatives of all groups with an interest in
bioremediation: buyers of bioremediation services, bioremediation contractors,
environmental

INTRODUCTION 15
regulators, and academic researchers. The committee developed the framework

for this report and the guidelines it presents at a one-week workshop in October
1992. Also included in this volume are seven background papers authored by
committee members to represent the range of perspectives from which
bioremediation can be viewed.

PRINCIPLES OF BIOREMEDIATION 16
2
Principles of Bioremediation
The key players in bioremediation are bacteria—microscopic organisms that

live virtually everywhere. Microorganisms are ideally suited to the task of
contaminant destruction because they possess enzymes that allow them to use
environmental contaminants as food and because they are so small that they are
able to contact contaminants easily. In situ bioremediation can be regarded as an
extension of the purpose that microorganisms have served in nature for billions
of years: the breakdown of complex human, animal, and plant wastes so that life
can continue from one generation to the next. Without the activity of
microorganisms, the earth would literally be buried in wastes, and the nutrients
necessary for the continuation of life would be locked up in detritus.
Whether microorganisms will be successful in destroying man-made
contaminants in the subsurface depends on three factors: the type of organisms,
the type of contaminant, and the geological and chemical conditions at the
contaminated site. This chapter explains how these three factors influence the
outcome of a subsurface bioremediation project. It reviews how microorganisms
destroy contaminants and what types of organisms play a role in in situ
bioremediation. Then, it evaluates which contaminants are most susceptible to
bioremediation in the subsurface and describes the types of sites where
bioremediation is most likely to succeed.

THE ROLE OF MICROBES IN BIOREMEDIATION

The goal in bioremediation is to stimulate microorganisms with nutrients and
other chemicals that will enable them to destroy the contaminants. The
bioremediation systems in operation today rely on microorganisms native to the
contaminated sites, encouraging them to work by supplying them with the
optimum levels of nutrients and other chemicals essential for their metabolism.
Thus, today's bioremediation systems are limited by the capabilities of the native
microbes. However, researchers are currently investigating ways to augment
contaminated sites with nonnative microbes—including genetically engineered
microorganisms—specially suited to degrading the contaminants of concern at
particular sites. It is possible that this process, known as bioaugmentation, could
expand the range of possibilities for future bioremediation systems.
Regardless of whether the microbes are native or newly introduced to the
site, an understanding of how they destroy contaminants is critical to
understanding bioremediation. The types of microbial processes that will be
employed in the cleanup dictate what nutritional supplements the bioremediation
system must supply. Furthermore, the byproducts of microbial processes can
provide indicators that the bioremediation is successful.
How Microbes Destroy Contaminants

Although bioremediation currently is used commercially to cleanup a limited
range of contaminants—mostly hydrocarbons found in gasoline—
microorganisms have the capability to biodegrade almost all organic
contaminants and many inorganic contaminants. A tremendous variety of
microbial processes potentially can be exploited, extending bioremediation's
utility far beyond its use today. Whether the application is conventional or novel
by today's standards, the same principles must be applied to stimulate the right
type and amount of microbial activity.
Basics of Microbial Metabolism

Microbial transformation of organic contaminants normally occurs because
the organisms can use the contaminants for their own growth and reproduction.
Organic contaminants serve two purposes for the organisms: they provide a
source of carbon, which is one of the basic building blocks of new cell
constituents, and they provide electrons, which the organisms can extract to
obtain energy.

FIGURE 2-1 Microbes degrade contaminants because in the process they gain
energy that allows them to grow and reproduce. Microbes get energy from the
contaminants by breaking chemical bonds and transferring electrons from the
contaminants to an electron acceptor, such as oxygen. They "invest" the energy,
along with some electrons and carbon from the contaminant, to produce more
cells.
Microorganisms gain energy by catalyzing energy-producing chemical

reactions that involve breaking chemical bonds and transferring electrons away
from the contaminant. The type of chemical reaction is called an oxidation-
reduction reaction: the organic contaminant is oxidized, the technical term for
losing electrons; correspondingly, the chemical that gains the electrons is reduced.
The contaminant is called the electron donor, while the electron recipient is called
the electron acceptor. The energy gained from these electron transfers is then
"invested," along with some electrons and carbon from the contaminant, to
produce more cells (see Figure 2-1). These two materials—the electron donor and
acceptor—are essential for cell growth and are commonly called the primary
substrates. (See Box 2-1 and the glossary for definitions of these and other key
terms.)
Many microorganisms, like humans, use molecular oxygen (O2) as the
electron acceptor. The process of destroying organic compounds with the aid of O2
is called aerobic respiration. In aerobic respiration, microbes use O2 to oxidize
part of the carbon in the contaminant to carbon dioxide (CO2), with the rest of the
carbon used to produce new cell mass. In the process the O2 gets reduced,
producing

BOX 2-1 KEY TERMS FOR UNDERSTANDING

BIOREMEDIATION
Microorganism: An organism of microscopic size that is capable of
growth and reproduction through biodegradation of "food sources," which
can include hazardous contaminants.
Microbe: The shortened term for microorganism.
Oxidize: The transfer of electrons away from a compound, such as an
organic contaminant. The coupling of oxidation to reduction (see below)
usually supplies energy that microorganisms use for growth and
reproduction. Often (but not always), oxidation results in the addition of an
oxygen atom and/or the loss of a hydrogen atom.
Reduce: The transfer of electrons to a compound, such as oxygen,
that occurs when another compound is oxidized.
Electron acceptor: The compound that receives electrons (and
therefore is reduced) in the energy-producing oxidation-reduction reactions
that are essential for the growth of microorganisms and bioremediation.
Common electron acceptors in bioremediation are oxygen, nitrate, sulfate,
and iron.
Electron donor: The compound that donates electrons (and therefore
is oxidized). In bioremediation the organic contaminant often serves as an
electron donor.
Primary substrates: The electron donor and electron acceptor that are
essential to ensure the growth of microorganisms. These compounds can
be viewed as analogous to the food and oxygen that are required for human
growth and reproduction.
Aerobic respiration: The process whereby microorganisms use
oxygen as an electron acceptor.
Anaerobic respiration: The process whereby microorganisms use a
chemical other than oxygen as an electron acceptor. Common "substitutes"
for oxygen are nitrate, sulfate, and iron.
Fermentation: The process whereby microorganisms use an organic
compound as both electron donor and electron acceptor, converting the
compound to fermentation products such as organic acids, alcohols,
hydrogen, and carbon dioxide.

Cometabolism: A variation on biodegradation in which microbes

transform a contaminant even though the contaminant cannot serve as the
primary energy source for the organisms. To degrade the contaminant, the
microbes require the presence of other compounds (primary substrates)
that can support their growth.
Reductive dehalogenation: A variation on biodegradation in which
microbially catalyzed reactions cause the replacement of a halogen atom on
an organic compound with a hydrogen atom. The reactions result in the net
addition of two electrons to the organic compound.
Intrinsic bioremediation: A type of bioremediation that manages the
innate capabilities of naturally occurring microbes to degrade contaminants
without taking any engineering steps to enhance the process.
Engineered bioremediation: A type of remediation that increases the
growth and degradative activity of microorganisms by using engineered
systems that supply nutrients, electron acceptors, and/or other growth-
stimulating materials.
water. Thus, the major byproducts of aerobic respiration are carbon dioxide,
water, and an increased population of microorganisms.
Variations on Basic Metabolism

In addition to microbes that transform contaminants through aerobic
respiration, organisms that use variations on this basic process have evolved over
time. These variations allow the organisms to thrive in unusual environments,
such as the underground, and to degrade compounds that are toxic or not
beneficial to other organisms.
Anaerobic Respiration. Many microorganisms can exist without oxygen,
using a process called anaerobic respiration. In anaerobic respiration, nitrate
(NO3-), sulfate (SO42-), metals such as iron (Fe3+) and manganese (Mn4+), or even
CO2 can play the role of oxygen, accepting electrons from the degraded
contaminant. Thus, anaerobic respiration uses inorganic chemicals as electron
acceptors. In addition to new cell matter, the byproducts of anaerobic respiration
may include nitrogen gas (N2), hydrogen sulfide (H2S), reduced forms of metals,
and methane (CH4), depending on the electron acceptor.

Some of the metals that anaerobic organisms use as electron acceptors are
considered contaminants. For example, recent research has demonstrated that
some microorganisms can use soluble uranium (U6+) as an electron acceptor,
reducing it to insoluble uranium (U4+). Under this circumstance the organisms
cause the uranium to precipitate, decreasing its concentration and mobility in the
ground water.
Inorganic Compounds as Electron Donors. In addition to organisms that use
inorganic chemicals as electron acceptors for anaerobic respiration, other
organisms can use inorganic molecules as electron donors. Examples of inorganic
electron donors are ammonium (NH4+), nitrite (NO2-), reduced iron (Fe2+),
reduced manganese (Mn2+), and H2S. When these inorganic molecules are
oxidized (for example, to NO2-, NO3-, Fe3+, Mn4+, and SO42-, respectively), the
electrons are transferred to an electron acceptor (usually O2) to generate energy
for cell synthesis. In most cases, microorganisms whose primary electron donor is
an inorganic molecule must obtain their carbon from atmospheric CO2 (a process
called CO2 fixation).
Fermentation. A type of metabolism that can play an important role in
oxygen-free environments is fermentation. Fermentation requires no external
electron acceptors because the organic contaminant serves as both electron donor
and electron acceptor. Through a series of internal electron transfers catalyzed by
the microorganisms, the organic contaminant is converted to innocuous
compounds known as fermentation products. Examples of fermentation products
are acetate, propionate, ethanol, hydrogen, and carbon dioxide. Fermentation
products can be biodegraded by other species of bacteria, ultimately converting
them to carbon dioxide, methane, and water.
Secondary Utilization and Co-metabolism. In some cases, microorganisms
can transform contaminants, even though the transformation reaction yields little
or no benefit to the cell. The general term for such nonbeneficial
biotransformations is secondary utilization, and an important special case is
called co-metabolism. In co-metabolism the transformation of the contaminant is
an incidental reaction catalyzed by enzymes involved in normal cell metabolism
or special detoxification reactions. For example, in the process of oxidizing
methane, some bacteria can fortuitously degrade chlorinated solvents that they
would otherwise be unable to attack. When the microbes oxidize methane, they
produce certain enzymes that incidentally destroy the chlorinated solvent, even
though the solvent itself cannot support

microbial growth. The methane is the primary electron donor because it is the
organisms' primary food source, while the chlorinated solvent is a secondary
substrate because it does not support the bacteria's growth. In addition to
methane, toluene and phenol have been used as primary substrates to stimulate
co-metabolism of chlorinated solvents.
Reductive Dehalogenation. Another variation on microbial metabolism is
reductive dehalogenation. Reductive dehalogenation is potentially important in
the detoxification of halogenated organic contaminants, such as chlorinated
solvents. In reductive dehalogenation, microbes catalyze a reaction in which a
halogen atom (such as chlorine) on the contaminant molecule gets replaced with a
hydrogen atom. The reaction adds two electrons to the contaminant molecule,
thus reducing the contaminant.
For reductive dehalogenation to proceed, a substance other than the
halogenated contaminant must be present to serve as the electron donor. Possible
electron donors are hydrogen and low-molecular-weight organic compounds
(lactate, acetate, methanol, or glucose). In most cases, reductive dehalogenation
generates no energy but is an incidental reaction that may benefit the cell by
eliminating a toxic material. However, researchers are beginning to find
examples in which cells can obtain energy from this metabolic process.
Microbial Nutritional Requirements for Contaminant Destruction

Regardless of the mechanism microbes use to degrade contaminants, the
microbes' cellular components have relatively fixed elemental compositions. A
typical bacterial cell is 50 percent carbon; 14 percent nitrogen; 3 percent
phosphorus; 2 percent potassium; 1 percent sulfur; 0.2 percent iron; and 0.5
percent each of calcium, magnesium, and chloride. If any of these or other
elements essential to cell building is in short supply relative to the carbon present
as organic contaminants, competition for nutrients within the microbial
communities may limit overall microbial growth and slow contaminant removal.
Thus, the bioremediation system must be designed to supply the proper
concentrations and ratios of these nutrients if the natural habitat does not provide
them.
How Microbes Demobilize Contaminants

In addition to converting contaminants to less harmful products, microbes
can cause mobile contaminants to be demobilized, a strategy

useful for containing hazardous materials. There are three basic ways microbes
can be used to demobilize contaminants:
• Microbial biomes can sorb hydrophobic organic molecules. Sufficient

biomass grown in the path of contaminant migration could stop or slow
contaminant movement. This concept is sometimes called a biocurtain.
• Microorganisms can produce reduced or oxidized species that cause
metals to precipitate. Examples are oxidation of Fe2+ to Fe3+, which
precipitates as ferric hydroxide (FeOH3(s)); reduction of SO42- to sulfide
(S2-), which precipitates with Fe2+ as pyrite (FeS(s)) or with mercury
(Hg2+) as mercuric sulfide (HgS(s)); reduction of hexavalent chromium
(Cr6+) to trivalent chromium (Cr3+), which can precipitate as chromium
oxides, sulfides, or phosphates; and, as mentioned previously, reduction
of soluble uranium to insoluble U4+, which precipitates as uraninite
(UO2).
• Microorganisms can biodegrade organic compounds that bind with
metals and keep the metals in solution. Unbound metals often precipitate
and are immobilized.
Indicators of Microbial Activity

In the process of degrading or demobilizing contaminants, microbes cause
changes in the surrounding environment that are important to understand when
evaluating bioremediation.
Chemical Changes
Bioremediation alters the ground water chemistry. These chemical changes
follow directly from the physiological principles of microorganisms outlined
above. Microbial metabolism catalyzes reactions that consume well-defined
reactants—contaminants and O2 or other electron acceptors—converting them to
well-defined products.
The specific chemical reactants and products can be determined from the
chemical equations for the reactions the microbes catalyze. These equations are
familiar to anyone with a basic understanding of microbiology. For example, the
chemical equation for the degradation of toluene (C7H8) is:
Thus, when bioremediation is occurring, the concentration of inorganic

carbon (represented by CO2) should increase as the concentrations of toluene and
oxygen decrease. Another example is the dechlorination

of trichloroethane (C2H3Cl3, or TCA) to dichloroethane (C 2H4Cl2, or DCA) by

hydrogen-oxidizing anaerobic bacteria:
Here, TCA and hydrogen (H2) decrease as DCA, hydrogen ion (H+), and
chloride ion (Cl-) increase. The formation of hydrogen ion may cause the pH to
decrease, depending on the ground water chemistry.
In general, under aerobic conditions, one should expect to observe a drop in
the O2 concentration when microbes are active. Similarly, under anaerobic
conditions, concentrations of other electron acceptors— NO3-, SO42-, Fe3+, Mn4+
—will decrease, with a corresponding increase in the reduced species of these
compounds (N2, H2S, Fe2+, and Mn2+, respectively). Under both types of
conditions the inorganic carbon concentration should increase, because organic
carbon is oxidized. The inorganic carbon may take the form of gaseous CO2,
dissolved CO2, or bicarbonate ion (HCO3-).
Adaptation by Native Organisms

In addition to producing chemical changes in the ground water,
bioremediation can alter the metabolic capabilities of native microorganisms.
Often, microorganisms do not degrade contaminants upon initial exposure, but
they may develop the capability to degrade the contaminant after prolonged
exposure. Several mechanisms have been proposed to explain metabolic
adaptation, including enzyme induction, growth of biodegrading populations, and
genetic change. However, these proposals remain largely speculative because
methodological limitations usually preclude rigorous understanding of how
microbial communities develop, both in laboratory tests and at field sites.
Regardless of the mechanisms, adaptation is important because it is a critical
principle in ensuring the existence of microorganisms that can destroy the myriad
new chemicals that humans have created and introduced into the environment.
Adaptation occurs not only within single microbial communities but also
among distinct microbial communities that may evolve a co-operative
relationship in the destruction of compounds. One community may partially
degrade the contaminant, and a second community farther along the ground water
flow path may complete the reaction. This type of coupling occurs naturally in
anaerobic food chains that convert plant-derived organic compounds to methane.
Such coupling has obvious applications for bioremediation of sites bearing
contaminant compounds whose complete metabolism may require alternation
between anaerobic and aerobic processes.

Growth of Predators
Although bacteria are the agents for biodegradation during bioremediation,
other organisms that prey on bacteria also may grow as a result of
bioremediation. Protozoa are the most common bacterial predators. Just as
mammalian predators, such as wolves, can only be supported by certain densities
of their prey, microbial protozoan predators flourish only when their bacterial
prey are in large, rapidly replenished supplies. Thus, the presence of protozoa
normally signifies that enough bacteria have grown to degrade a significant
quantity of contaminants.
Complicating Factors
The basic principles of how microbes degrade contaminants are relatively
straightforward. Yet many details of microbial metabolism are not yet
understood, and the successful use of microbes in bioremediation is not a simple
matter. A range of factors may complicate bioremediation. Some of the key
complicating factors are the unavailability of contaminants to the organisms,
toxicity of contaminants to the organisms, microbial preference for some
contaminants or naturally occurring chemicals over other contaminants, partial
degradation of contaminants to produce hazardous byproducts, inability to
remove contaminants to very low concentrations, and aquifer clogging from
excessive biomass growth.
Unavailability of Contaminants to the Organisms

Readily biodegradable contaminants may remain undegraded or be
biodegraded very slowly if their concentrations in the ground water are too low.
The problem of too low concentrations usually is caused by unavailability, in
which the contaminant is sequestered from the microorganisms. Sequestering of
organic contaminants can occur when the contaminant is dissolved in a
nonaqueous-phase liquid—a solution that does not mix easily with water and
therefore travels through the ground separately from the ground water.
Sequestering of organic contaminants can also occur if the contaminant is
strongly adsorbed to soil surfaces or is trapped in pores too small for circulating
ground water to penetrate easily. In these cases, almost all of the contaminant is
associated with the solid, the nonaqueous-phase liquid, or the pores, and the very
small concentrations that dissolve in the water support very small or zero
biodegradation rates.

Sequestering of metals and other inorganic contaminants occurs most

frequently when they precipitate.
One possible strategy for overcoming the unavailability problem is to add
chemical agents that mobilize the contaminants, causing them to move with the
ground water. Such chemical agents are already used at some sites to increase the
efficiency of conventional pump-and-treat ground water cleanup systems.
However, their use to facilitate bioremediation is more complex than their use for
pump-and-treat systems because the mobilizing agents not only affect the
physical properties of the contaminants but may also affect the activity of the
microorganisms.
Organic contaminants can be mobilized by adding surfactants. When only
small surfactant concentrations are applied, the surfactant molecules accumulate
at solid surfaces, reduce the surface tension, and, in principle, increase the
spreading of organic contaminants. This spreading might improve contaminant
transfer to the water and thereby accelerate bioremediation, but evidence is not
clear for actual subsurface conditions. When large concentrations of surfactant
are added, the surfactant molecules join together in colloids, called micelles.
Organic contaminants dissolve into the micelles and are transported with the
water inside them. However, biodegradation usually is not enhanced by
contaminant transfer into the micelles because the true aqueous-phase
concentration is not increased.
Metals can be mobilized by adding chemicals called complexing agents, or
ligands, to which the metals bond. The formation of metalligand bonds dissolves
precipitated metals, increasing their mobility. However, the effectiveness of
strong ligands, such as ethylene-diaminetetra-acetic acid (EDTA), in enhancing
biodegradation is not yet proven. One potential limitation of using ligands to
mobilize metals is that microbes may degrade the ligands, releasing the metals
and causing them to precipitate again.
In some cases, bacteria produce their own surfactants and ligands that are
useful in mobilizing trapped contaminants. In these cases the main purpose of the
microorganisms is to produce mobilizing agents, not to biodegrade the
contaminants. Bacterially mediated mobilization makes trapped contaminants
more accessible for cleanup with pump-and-treat technology; it is potentially less
costly than injecting commercial surfactants.
Toxicity of Contaminants to the Organisms

Just as contaminant concentrations that are too low can complicate
bioremediation, high aqueous-phase concentrations of some contaminants

can create problems. At high concentration, some chemicals are toxic to

microbes, even if the same chemicals are readily biodegraded at lower
concentrations. Toxicity prevents or slows metabolic reactions and often prevents
the growth of the new biomass needed to stimulate rapid contaminant removal.
The degree and mechanisms of toxicity vary with specific toxicants, their
concentration, and the exposed microorganisms. Microbial cells cease to function
when at least one of the essential steps in their myriad physiological processes is
blocked. The blockage may result from gross physical disruption of cell structure
or competitive binding of a single enzyme essential for metabolizing the toxicant.
Presence of Multiple Contaminants and Natural Organic Chemicals

Frequently, contaminated sites contain a combination of several man-made
organic contaminants and naturally occurring organic chemicals from decayed
plant and animal matter. When such mixtures of organics are present, microbes
may selectively degrade the compound that is easiest to digest or that provides the
most energy. Microbiologists have long been aware that complex mechanisms
regulating microbial metabolism may cause some carbon compounds to be
ignored while others are selectively used. This phenomenon, known as diauxy,
could have serious implications for bioremediation efforts if the targeted
contaminant is accompanied by substantial quantities of preferred growth
substrates.
Mixtures do not always cause problems and sometimes can promote
bioremediation. For example, biomass that grows primarily to degrade one type
of organic compound may also degrade a second compound present at a
concentration too low to support bacterial growth by itself.
Incomplete Degradation of Contaminants

In some cases, contaminants may not be fully degraded by the organisms.
Partial degradation may diminish the concentration of the original pollutant but
create metabolic intermediates that in some cases are more toxic than the parent
compound. There are two main reasons why intermediates build up. In one case a
so-called dead-end product is produced. Dead-end products may form during
cometabolism, because the incidental metabolism of the contaminant may create a
product that the bacterial enzymes cannot transform any further. For example, in
the cometabolism of chlorinated phenols, dead-end products such as
chlorocatechols, which are toxic, some

times build up. In the second case the intermediate builds up even though the
compound can be fully degraded, because some of the key bacterially mediated
reactions are slow. For example, vinyl chloride, a cancer-causing agent, may
build up during trichloroethylene (TCE) biodegradation. The bacteria can convert
TCE to vinyl chloride relatively quickly, but the subsequent degradation of vinyl
chloride usually occurs slowly.
Inability to Remove Contaminants to Low Concentrations

Microorganisms may sometimes be physiologically incapable, even when
environmental conditions are optimal, of reducing pollutant concentrations to
very low, health-based levels, because the uptake and metabolism of organic
compounds sometimes stops at low concentrations. This may be caused by the
cells' internal mechanisms for regulating what reactions they perform or by an
inability of the capable microbial populations to survive given inadequate
sustenance. Regardless of the mechanism, if the final contaminant concentration
fails to meet the cleanup goal, other cleanup strategies (microbiological or other)
may have to be implemented to effectively reduce the concentration to acceptable
levels. Research on augmenting sites with nonnative microbes and controlling
cells' genetic capabilities and internal regulation may lead to means for
overcoming this limitation.
Aquifer Clogging
Stimulating the growth of enough microorganisms to ensure contaminant
degradation is essential to in situ bioremediation. However, if all the organisms
accumulate in one place, such as near the wells that supply growth-stimulating
nutrients and electron acceptors, microbial growth can clog the aquifer. Clogging
can interfere with effective circulation of the nutrient solution, limiting
bioremediation in places that the solution does not reach. Protozoan predators
may help mitigate clogging. In addition, two engineering strategies can help
prevent clogging: (1) feeding nutrients and substrates in alternating pulses and (2)
adding hydrogen peroxide as the oxygen source. Pulse feeding prevents excessive
biomass growth by ensuring that high concentrations of all the growth-stimulating
materials do not accumulate near the injection point. Hydrogen peroxide prevents
excessive growth because it is a strong disinfectant, until it decomposes to oxygen
and water.

CONTAMINANTS SUSCEPTIBLE TO BIOREMEDIATION

A critical factor in deciding whether bioremediation is the appropriate
cleanup remedy for a site is whether the contaminants are susceptible to
biodegradation by the organisms at the site (or by organisms that could be
successfully grown at the site). Some compounds are more easily degraded by a
wide range of organisms than others, and systems for encouraging biodegradation
are better established for some compounds than for others. Table 2-1 provides an
overview of classes of compounds and their inherent suitability for
bioremediation. The table is intended to deliver a broad perspective on how
chemical and microbiological properties jointly affect prospects for
bioremediation, and the judgments it presents are generalities that, of course,
have exceptions. The table shows that bioremediation treatment technology is
well established for certain classes of petroleum hydrocarbons but that the
technologies for treating all other classes are still emerging. Commercial
development of bioremediation technologies for these other compounds is
possible, but it will require further research and the scaling up of lab discoveries
for application in the field.
The table's first column shows the contaminant's frequency of occurrence at
hazardous waste sites. It indicates the magnitude of the problem the contaminant
poses. The second column indicates the state of development of bioremediation
technologies for cleaning up the contaminant. In this column, ''established" means
that bioremediation of the contaminant has been tried successfully many times at
the commercial scale. "Emerging" means that the concepts underlying
bioremediation of the contaminant have been tested in the laboratory and, in some
cases, tested successfully at a limited number of field sites under controlled
conditions. "Possible" means that evidence from lab tests indicates future
potential for bioremediation to successfully cleanup the compound. The third
column presents the evidence leading the committee to believe that the
contaminant can be cleaned up successfully with bioremediation in the future,
even though established bioremediation technology does not yet exist. It indicates
what types of organisms can degrade the contaminant and how easily they can
act. The fourth column describes contaminant properties that may limit
bioremediation. The key limiting properties are the contaminant's tendency to
sorb to subsurface solids and to partition into a nonaqueous-phase that travels
separately from the ground water. As discussed previously in this chapter, both of
these properties—sorption and nonaqueous-phase formation—decrease the
amount of contaminant available to the microorganisms, slowing

TABLE 2-1 Contaminant Susceptibility to Bioremediation
Chemical Class Frequency of Occurrence Status of Bioremediation Evidence of Future Success Limitations
Hydrocarbons and
Derivatives
Gasoline, fuel oil Very frequent Established Forms nonaqueous-phase
liquid
Polycyclic aromatic Common Emerging Aerobically biodegradable Sorbs strongly to
hydrocarbons under a narrow range of subsurface solids
conditions
Creosote Infrequent Emerging Readily biodegradable under Sorbs strongly to
aerobic conditions subsurface solids; forms

nonaqueous-phase liquid
Alcohols, ketones, esters Common Established
Ethers Common Emerging Biodegradable under a narrow
range of conditions using
aerobic or nitrate-reducing
microbes
Halogenated Aliphatics
Highly chlorinated Very frequent Emerging Cometabolized by anaerobic Forms nonaqueous-phase
microbes; cometabolized by liquid
aerobes in special cases
Less chlorinated Very frequent Emerging Aerobically biodegradable Forms nonaqueous-phase
under a narrow range of liquid
conditions; cometabolized by
anaerobic microbes

30
Halogenated Aromatics
Highly chlorinated Common Emerging Aerobically biodegradable under a Sorbs strongly to subsurface solids;
narrow range of conditions; forms nonaqueous phase—solid or
cometabolized by anaerobic microbes liquid
Less chlorinated Common Emerging Readily biodegradable under aerobic Forms nonaqueous phase—solid or
conditions liquid
Polychlorinated Biphenyls
Highly chlorinated Infrequent Emerging Cometabolized by anaerobic microbes Sorbs strongly to subsurface solids
Less chlorinated Infrequent Emerging Aerobically biodegradable under a Sorbs strongly to subsurface solids
narrow range of conditions
Nitroaromatics Common Emerging Aerobically biodegradable; converted to

innocuous volatile organic acids under

anaerobic conditions
Metals (Cr, Cu, Ni, Pb, Hg, Cd, Zn, etc.) Common Possible Solubility and reactivity can be changed Availability highly variable—
by a variety of microbial processes controlled by solution and solid
chemistry

31
bioremediation. In general, the least biodegradable contaminants are those

with the strongest tendency to sorb.
The table groups contaminants into five classes: petroleum hydrocarbons and
derivatives, halogenated aliphatics, halogenated aromatics, nitroaromatics, and
metals. Each class is discussed in more detail below.
Petroleum Hydrocarbons and Derivatives

Petroleum hydrocarbons and their derivatives are naturally occurring
chemicals that humans have exploited for a wide range of purposes, from fueling
engines to manufacturing chemicals. The representative types of petroleum
hydrocarbons and derivatives listed in Table 2-1 are gasoline, fuel oil, polycyclic
aromatic hydrocarbons (PAHs), creosote, ethers, alcohols, ketones, and esters.
Each of these chemicals has a broad range of industrial applications. For
example, PAHs are released when crude oil is refined and from the manufacture
of petroleum products such as plastics. Creosote is used in wood preservatives.
Ethers, esters, and ketones are components of chemicals ranging from perfumes,
to anesthetics, to paints and lacquers, to insecticides.
Gasoline, fuel oil, alcohols, ketones, and esters have been successfully
bioremediated at contaminated sites via established bioremediation procedures.
Gasoline, in particular, has been the focus of substantial biodegradation and
bioremediation research. The gasoline components benzene, toluene,
ethylbenzene, and xylene (together known as BTEX) are relatively easy to
bioremediate for several reasons:
• They are relatively soluble compared to other common contaminants and

other gasoline components.
• They can serve as the primary electron donor for many bacteria widely
distributed in nature.
• They are rapidly degraded relative to other contaminants shown in
Table 2-1.
• The bacteria that degrade BTEX grow readily if oxygen is available.
Under many circumstances, ether bonds show considerable chemical

stability and therefore resist microbial attack. High-molecular-weight compounds
such as creosotes and some PAHs are also only slowly metabolized—partly as a
result of their structural complexity, low solubility, and strong sorptive
characteristics. Thus, bioremediation techniques for these latter classes of
petroleum derivatives are still emerging.

Halogenated Compounds
Halogenated compounds are compounds with halogen atoms (usually
chlorine, bromine, or fluorine) added to them in place of hydrogen atoms.
Although halogenated organic compounds have been found in nature, these are
not significant compared to the synthetic chemicals listed in the middle portion of
Table 2-1. When halogen atoms are introduced into organic molecules, many
properties, such as solubility, volatility, density, and toxicity, change markedly.
These changes confer improvements that are valuable for commercial products,
such as solvents used for degreasing, but they also have serious implications for
microbial metabolism. The susceptibility of the chemicals to enzymatic attack is
sometimes drastically decreased by halogenation, and persistent compounds often
result. Consequently, bioremediation technologies for these chemicals are still
emerging.
There are two broad classes of halogenated chemicals: halogenated
aliphatics and halogenated aromatics.
Halogenated Aliphatics
Halogenated aliphatic compounds are compounds built from straight chains
of carbon and hydrogen with varying numbers of hydrogen atoms replaced by
halogen atoms. Halogenated aliphatics are effective solvents and degreasers and
have been widely used in manufacturing and service industries, ranging from
automobile manufacturing to dry cleaning. Some highly chlorinated
representatives of this class, such as tetrachloroethene, are completely resistant to
attack by aerobic microbes but are susceptible to degradation by special classes
of anaerobic organisms. In fact, recent evidence shows that certain anaerobes can
completely dechlorinate tetrachloroethene to the relatively nontoxic compound
ethene, which is readily decomposed by aerobic microbes.
As the degree of halogenation in aliphatics diminishes, susceptibility to
aerobic metabolism increases. The less halogenated ethenes may be destroyed by
cometabolism when certain aerobic microbes are supplied with methane, toluene,
or phenol, as described earlier in this chapter. Thus, a common treatment
rationale for the highly chlorinated aliphatics is to remove the chlorine atoms
anaerobically, with methanogens, and then complete the biodegradation process
using aerobic cometabolism. However, routine procedures for implementing
anaerobic/aerobic sequencing to bioremediate sites contaminated with chlorinated
aliphatic materials are not yet established at the commercial scale.

Halogenated Aromatics
Halogenated aromatics are compounds built from one or more halogen-
bearing benzene rings. Examples include chlorinated benzenes, used as solvents
and pesticides; pentachlorophenol, used in fungicides and herbicides; and
polychlorinated biphenyls (PCBs), once widely used in electrical transformers
and capacitors. The aromatic benzene nucleus is susceptible to aerobic and
anaerobic metabolism, although the latter occurs relatively slowly. Overall,
however, the presence of halogen atoms on the aromatic ring governs
biodegradability. A high degree of halogenation may prevent aromatic
compounds from being aerobically metabolized, as is the case for highly
chlorinated PCBs. However, as discussed above for the aliphatic compounds,
anaerobic microbes can remove chlorine atoms from the highly halogenated
aromatics. As the halogen atoms are replaced by hydrogen atoms, the molecules
become susceptible to aerobic attack. Thus, a possible bioremediation scenario
for treating soils, sediments, or water contaminated with halogenated aromatic
chemicals is anaerobic dehalogenation followed by aerobic destruction of the
residual compounds. It should be noted, however, that when certain substituent
groups accompany the halogens on the aromatic ring, aerobic metabolism may
proceed rapidly, as is the case for pentachlorophenol.
Nitroaromatics
Nitroaromatics are organic chemicals in which the nitro group (–NO2) is
bonded to one or more carbons in a benzene ring. A familiar example is
trinitrotoluene (TNT), which is used in explosives. Laboratory research has
shown that both aerobic and anaerobic microbes can convert many of these
compounds to carbon dioxide, water, and mineral components. Recent field tests
have confirmed that anaerobic microbes can transform nitroaromatics to
innocuous volatile organic acids, like acetate, which then may be mineralized.
Metals
The metals listed in Table 2-1 are common pollutants inadvertently released
during the manufacture of various industrial products, from steel to
pharmaceuticals. Microorganisms cannot destroy metals, but they can alter their
reactivity and mobility. Schemes for using microorganisms to mobilize metals
from one location and scavenge the metal from another location have been
applied to mining operations. Microbes produce acids that can leach metals, like
copper,

from low-grade ores. This same approach should be feasible for bioremediation
purposes, but it has not been proven. Microorganisms can also demobilize metals
by transforming them to a form that precipitates (see
"How Microbes Demobilize Contaminants," earlier in this chapter).
ENVIRONMENTS AMENABLE TO BIOREMEDIATION

The suitability of a site for bioremediation depends not only on the
contaminant's biodegradability but also on the site's geological and chemical
characteristics. The ideal site for in situ bioremediation is one that is as
controllable and easy to interpret as the small, laboratory-incubated flask
experiments used to test pollutant biodegradation. The site most amenable to
bioremediation, like the lab flask, has favorable chemical characteristics and
relatively uniform geology. Site characteristics are rarely ideal, however. Each
site is a unique section of landscape that presents an unpredictable variety of
environmental conditions. Properties such as soil type, geological strata, and
water chemistry vary not only from site to site but also within an individual site.
Furthermore, site complexity and lack of site data commonly obscure the true
type and severity of the contamination. It is normal in implementing
bioremediation—or any other cleanup technology—to revise cleanup plans
continually as more information becomes available during the remediation.
It is important to realize that no single set of site characteristics will favor
bioremediation of all chemical contaminants. For example, certain compounds
can only be metabolized under anaerobic conditions, while metabolism of others
requires oxygen. When the degradation mechanisms for two co-occurring
contaminants are mutually exclusive, difficult choices need to be made or
sequential treatment schemes need to be devised.
Two Types of Bioremediation: Intrinsic and Engineered

A principal concern in determining whether the site environment is
appropriate for in situ bioremediation is the type of bioremediation to be
implemented. Bioremediation can be grouped into two broad types: intrinsic and
engineered. Figure 2-2 illustrates the differences between the two.
Intrinsic bioremediation manages the innate capabilities of naturally
occurring microbial communities to degrade environmental pollutants without
taking any engineering steps to enhance the process. It differs from no-action
alternatives in that it requires thorough documentation

of the role of native microorganisms in eliminating contaminants via tests

performed at field sites or on site-derived samples of soil, sediment, or water.
Furthermore, the effectiveness of intrinsic bioremediation must be proven with a
site-monitoring regime that routinely analyzes contaminant concentrations. The
terms "natural," "passive," and ''spontaneous" bioremediation and
"bioattenuation"
FIGURE 2-2 The differences between intrinsic and engineered bioremediation.

In intrinsic bioremediation, left, native subsurface microbes degrade the
contaminants without direct human intervention. In the close-up view, the native
microbes use iron (Fe3+) as an electron acceptor to degrade toluene (C7H8), a
representative contaminant, and convert it to carbon dioxide (CO2). In
engineered bioremediation, right, oxygen (O2), nitrogen (N), and phosphorus (P)
are circulated through the subsurface via an injection and extraction well to
promote microbial growth. In this case the microbes use oxygen as the electron
acceptor, converting it to water (H2O) as they degrade the toluene. Note that, as
pictured in the close-up view, considerably more microbes are present in the
engineered bioremediation system than in the intrinsic system. Consequently,
contaminant degradation occurs more quickly in the engineered system. Intrinsic
bioremediation requires extensive monitoring to ensure that the contaminant
does not advance more quickly than the native microbes can degrade it.

BOX 2-2 INTRINSIC BIOREMEDIATION OF A CRUDE OIL

SPILL— BEMIDJI, MINNESOTA
In August 1979 an oil pipeline burst near Bemidji, Minnesota, spilling
approximately 100,000 gallons of crude oil into the surrounding ground
water and soil. In 1983 researchers from the U.S. Geological Survey
(USGS) began monitoring the site carefully to determine the crude oil's
fate. They discovered that, although components of the crude oil initially
migrated a short distance, native microorganisms capable of degrading the
oil have prevented widespread contamination of the ground water. The
microbes went to work with no human intervention, showing that intrinsic
bioremediation can be effective for containing spills of petroleum products.
In the years following the spill, portions of the crude oil dissolved in the
flowing ground water and moved 200 m from the original spill site. The
undissolved crude oil itself migrated 30 m in the direction of ground water
flow, and crude oil vapors moved 100 m in the overlying soil. However, the
USGS researchers' detailed monitoring shows that the contaminant plume
has not advanced since 1987, and the researchers have attributed this halt
to intrinsic bioremediation.
Three types of evidence convinced the researchers that intrinsic
bioremediation was largely responsible for containing the crude oil. First,
modeling studies showed that if the oil were not biodegradable, the plume
would have spread 500 to 1200 m since the spill (see Figure 2-3). Second,
the concentrations of Fe2+ and CH4 increased dramatically in the portion of
the contaminant plume where oxygen was not present—evidence of an
increase in activity by anaerobic organisms capable of degrading certain
crude oil components, such as toluene. Third, concentrations of the crude
oil components benzene and ethylbenzene, which are susceptible to
aerobic degradation but less susceptible to anaerobic degradation,
remained relatively stable in the anaerobic portion of the plume but
decreased dramatically at the outer edges of the plume, where mixing with
oxygenated water allowed aerobic degradation to occur.
The evidence from this site shows that, in hydrologic settings where
intrinsic bioremediation rates are fast relative to hydrologic transport rates,
native microbes can effectively confine contaminants to near the spill source
without further human intervention. However, it is essential for such sites to
have detailed, long-term monitoring plans to ensure that the contamination
is, indeed, contained. At some sites, the rates of hydrologic transport
outpace the rates of intrinsic bioremediation, and additional engineering
steps to contain or remove the contamination will be necessary.

FIGURE 2-3 Concentrations of the crude oil components toluene,

ethylbenzene, and benzene at various distances from the center of the Bemidji,
Minnesota, oil spill. These concentrations have remained relatively stable at the
levels shown here since 1987. Note that the contaminant concentrations are
very high near the center of the plume but that they drop dramatically within
100 m of the spill. If the contaminants were not biodegradable, this
concentration drop would not occur, and the contamination would have spread
much farther, as shown by the hypothetical concentration of a nondegradable
solute (called a "conservative solute") pictured here. Thus, at this site, intrinsic
bioremediation has effectively confined the contamination to a small region.
SOURCE: Baedecker et al. (in press), reprinted with permission.
References
Baedecker, M. J., D. I. Siegal, P. E. Bennett, and I. M. Cozzarelli. 1989. The Fate and Effects of
Crude Oil in a Shallow Aquifer: I. The Distribution of Chemical Species and Geochemical
Facies. U.S. Geological Survey Water-Resources Investigations Report 88-4220. Reston,
Va.: U.S. Geological Survey.
Baedecker, M. J., I. M. Cozzarelli, D. I. Siegal, P. E. Bennett, and R. P. Eganhouse. In press. Crude
oil in a shallow sand and gravel aquifer: 3. Biogeochemical reactions and mass balance
modeling in anoxic ground-water. Applied Geochemistry.
Cozzarelli, I. M., R. P. Eganhouse, and M. J. Baedecker. 1991. Transformation of monoaromatic
hydrocarbons to organic acids in anoxic ground-water environment. Environmental Geology
and Water Sciences 16(2):135-141.
Hult, M. F. 1984. Ground-Water Contamination by Crude Oil at the Bemidji, Minnesota Research
Site. U.S. Geological Survey Water-Resources Investigations Report 84-4188. Reston, Va.:
U.S. Geological Survey.

have also been used to describe intrinsic bioremediation. Box 2-2 describes a
Minnesota site where researchers documented that intrinsic bioremediation
prevented the further spreading of crude oil contamination.
Engineered bioremediation is the acceleration of microbial activities using
engineered site-modification procedures, such as installation of wells to circulate
fluids and nutrients to stimulate microbial growth. The principal strategy of
engineered bioremediation is to isolate and control contaminated field sites so
that they become in situ bioreactors. Other terms used to describe engineered
bioremediation include "biorestoration" and "enhanced bioremediation."
As summarized in Box 2-3 and described below, the site conditions that
influence a bioremediation project's success differ for intrinsic and engineered
bioremediation.
Site Conditions for Engineered Bioremediation

Because engineered bioremediation uses technology to manipulate
environmental conditions, the natural conditions are less important for engineered
than for intrinsic bioremediation. For engineered bioremediation, the critical
property influencing success is how well the subsurface materials at the site
transmit fluids. For systems that circulate ground water, the hydraulic
conductivity (the amount of ground water that moves through a unit section of the
subsurface in a given time) in the area containing the contaminant should be on
the order of 10-4 cm/s or greater (the precise value is site specific). For systems
that circulate air, the intrinsic permeability (a measure of how easily fluids flow
through the subsurface) should be greater than 10-9 cm2. For both types of
systems, the contaminated area will be much more difficult to treat if it has
crevices, fractures, or other irregularities that allow channeling of fluids around
contaminated material. Land near river deltas, floodplains of large rivers, and
areas where sand and gravel aquifers were formed from the melting of glaciers
can be uniform over large areas. On the other hand, braided stream channels can
contain a substantial number of irregularities that complicate bioremediation
system design.
At high concentrations, contaminants (including petroleum products and
chlorinated solvents) that form a nonaqueous-phase liquid can exclude water or
air from pores in the subsurface. Nonaqueous-phase liquids restrict access of the
remedial fluids and gases and complicate engineered bioremediation. In most
cases such contaminants at residual concentrations of less than 8000 to 10,000
mg/kg of soil do not significantly affect water or air flow, because at this level

BOX 2-3 SITE CHARACTERISTICS THAT FAVOR IN SITU

BIOREMEDIATION
Engineered bioremediation requires installing wells and other
engineering systems to circulate electron acceptors and nutrients that
stimulate microbial growth. Key site characteristics for engineered
bioremediation are:
• Transmissivity of the subsurface to fluids:
• hydraulic conductivity greater than 10-4 cm/s (if the system circulates
water)
• intrinsic permeability greater than 10-9 cm2 (if the system circulates air)
• Relatively uniform subsurface medium (common in river delta deposits,
floodplains of large rivers, and glacial outwash aquifers)
• Residual concentration of nonaqueous-phase contaminants of less than
10,000 mg/kg of subsurface solids.
Intrinsic bioremediation destroys contaminants without human
intervention, as the population of native microbes capable of degrading the
contaminant increases naturally. The process requires thorough site
monitoring to demonstrate that contaminant removal is occurring. Key
characteristics of sites amenable to intrinsic bioremediation are:
• Consistent ground water flow (speed and direction) throughout the
seasons:
• seasonal variation in-depth to water table less than 1 m
• seasonal variation in regional flow trajectory less than 25 degrees
• Presence of carbonate minerals (limestone, dolomite, shell material) to
buffer pH
• High concentrations of electron acceptors such as oxygen, nitrate,
sulfate, or ferric iron
• Presence of elemental nutrients (especially nitrogen and phosphorus)

the contaminants are essentially nonmobile and occupy much less pore space
than the water. The specific concentration value at which nonaqueous-phase
contaminants begin interfering with fluid circulation varies depending on the
contaminant (the value is higher for denser contaminants) and the soil.
Site Conditions for Intrinsic Bioremediation

If intrinsic bioremediation is the only option, ambient site conditions must
be accepted as constraints for meeting cleanup goals, because intrinsic
bioremediation by definition occurs without adding anything to the site. Only a
fraction of the contaminated sites offer naturally occurring hydrogeochemical
conditions in which microorganisms can degrade contaminants quickly enough to
prevent them from spreading without human intervention.
The critical site characteristic for intrinsic bioremediation is predictability of
ground water flow in time and space. Predictable water flow is essential for
determining whether the native microbes will be able to act in all the places
where the contaminant might travel in all seasons and for determining whether
the microbes can act quickly enough to prevent the contamination from spreading
with the flowing ground water. The hydraulic gradient and trajectory of ground
water flow should be consistent through the seasons and from year to year. To
ensure predictability of flow, the fluctuation in the water table should not vary
more than about 1 m, although the precise number is site specific. In addition, the
trajectory of regional flow should not change by more than about 25 degrees from
the primary flow direction. These circumstances are more likely in upland
landscapes with humid, temperate climates. In contrast, contaminant plumes in
estuaries or the flood plains of large rivers often behave unpredictably.
Another valuable characteristic is the presence in the aquifer of minerals
such as carbonates to buffer pH changes that would otherwise result from
biological production of carbon dioxide or other acids or bases. Carbonates in the
aquifer matrix can be expected when limestone or dolomite are the parent
material or when limestone dust or sand occurs in glacial outwash. Carbonates
can also occur as shell material in beach deposits.
Intrinsic bioremediation is more extensive when the ambient ground water
surrounding the spill has high concentrations of oxygen or other electron
acceptors. The importance of ambient concentrations of nitrate, sulfate, and ferric
iron as potential electron acceptors that can stimulate microbial growth in the
absence of oxygen is too often

ignored. Most ground waters have more nitrate and sulfate than oxygen. This is
particularly true in agricultural areas that have been overfertilized and in arid
regions where gypsum is dissolved in the ground water.
The concentration of electron acceptors required to ensure bioremediation
varies with the contaminant's chemical characteristics and the amount of
contamination. More soluble contaminants and large contaminant sources require
larger electron acceptor concentrations. Natural ground water circulation
conditions at the site also influence the required amount of electron acceptor. The
circulation patterns should provide enough mixing between contaminated water
and surrounding water that the organisms never consume all of the electron
acceptors within the bioremediation region. If the electron acceptor supply
becomes depleted, bioremediation will slow or cease.
Also necessary for intrinsic bioremediation is the presence of the elemental
nutrients that microbes require for cell building, especially nitrogen and
phosphorus. Although nutrients must be present naturally for intrinsic
bioremediation to proceed, the quantity of nutrients required is much less than the
quantity of electron acceptors. Therefore, a nutrient shortage is less likely to limit
intrinsic bioremediation than an inadequate electron acceptor supply.
Impact of Site Heterogeneity on Bioremediation

Observation of the geological cross section at a typical excavation site
reveals a complex patchwork of layers, lenses, and fingers of different materials.
Indeed, two overriding characteristics of the subsurface are that it is intricately
heterogeneous and difficult to observe. The patterns of variability of the
properties that govern the flow of water and the transport of chemicals are so
complex that it is not possible to predict these properties quantitatively or even to
interpolate them with accuracy from sparse observations. In practice, estimation
of subsurface hydrogeochemical properties depends on site-specific
measurements from water or soil samples and well tests. However, the inherent
unobservability of the system means that there is usually insufficient information
to characterize the site with certainty.
A consequence of this complexity and heterogeneity, in combination with
the poor observability of the subsurface, is that completely reliable prediction of
chemical transport and fate is out of reach in most real-world cases. In evaluating a
proposed intrinsic or engineered bioremediation scheme, one must consider how
it may perform

under variable and not perfectly known conditions. A scheme that works
optimally under specific conditions but poorly otherwise may be inappropriate
for in situ bioremediation.
FURTHER READING
While this chapter has briefly reviewed the principles underlying successful
bioremediation, the references listed in Table 2-2 provide more thorough
coverage of the key disciplines related to bioremediation. The list is not
exhaustive. The references it provides were selected to represent the diversity of
attitudes, perspectives, and paradigms that are pertinent to understanding
bioremediation.

TABLE 2-2 Recommended Sources for Obtaining In-Depth Information About the Disciplines Pertinent to Bioremediation
Discipline Reference Synopsis
Environmental microbiology Chapelle, F. H. 1993. Ground-Water Microbiology and Reviews how the growth, metabolism, and ecology of
Geochemistry. New York: John Wiley & Sons. microorganisms affect ground water chemistry in both
pristine and chemically contaminated aquifer systems.
Gibson, D. T. 1984. Microbial Degradation of Organic Provides a detailed survey of how microorganisms
Compounds. New York: Marcel Dekker. metabolize organic compounds. Each chapter, written by a
different expert, focuses on a different class of compounds.
Madsen, E. L. 1991. Determining in situ biodegradation: Reviews principles and limitations of environmental
facts and challenges. Environmental Science and microbiology as they apply to determining in situ
Technology 25:1661-1673. biodegradation. Proposes useful approaches, especially as

applicable to academic research.

Madsen, E. L., and W. C. Ghiorse. 1993. Ground water Reviews major concepts and methodological developments
microbiology: subsurface ecosystems processes. Pp. that determine our understanding of microorganisms and
167-213 in Aquatic Microbiology: An Ecological the processes they carry out in subsurface ecosystems.
Approach, T. Ford, ed. Cambridge, Mass.: Blackwell
Scientific Publishers.
VanLoosdrecht, M. C. M., J. Lyklema, W. Norse, and A. J. Provides a critical and cross-disciplinary review of how
B. Zehnder. 1990. Influences of interfaces on microbial surfaces affect microbial activity and substrate availability.
activity. Microbiologic Reviews 54:75-87.

44
Hydrobiology Dominic, P. A., and F. W. Schwartz. 1990. Physical and Reviews principles and practice of ground water hydrology,
Chemical Hydrogeology. New York: John Wily & Sons. with emphasis on environmental applications.
Freeze, R. A., and J. A. Cherry. 1979. Groundwater. Provides a comprehensive presentation of the theory,
Englewood Cliffs, NJ.: Prentice-Hall. principles, and practice of hydrogeology.
Environmental engineering McCarty, P. L. 1988. Bioengineering issues related to in situ Discusses engineering issues relevant to in situ
remediation of contaminated soils and groundwater. Pp. bioremediation.
143-162 in Environmental Biotechnology: Reducing Risks
from Environmental Chemicals Through Biotechnology, G.
S. Omenn, ed. New York: Plenum Press.
Rittmann, B. E., A. J. Valocchi, E. Seagren, C. Ray, and B. Provides a comprehensive critical review of the
Wrenn. 1992. A Critical Review of In Situ Bioremediation. microbiological, engineering, and institutional possibilities
Chicago: Gas Research Institute. and restrictions for in situ bioremediation.
Statistics ASCE Task Committee. 1990. Review of geostatistics in Discusses geostatistical techniques and how they can assist
geohydrology, parts I and II. ASCE Journal of Hydraulic in the solution of estimation problems, including
Engineering 116(5):612-658. interpolation, averaging, and network design.

45
Discipline Reference Synopsis
Contaminant fate and transport Fetter, C. W. 1993. Contaminant Hydrogeology. New Gives a comprehensive treatment of ground water
York: Macmillan Publishing Co. contaminants and their transport, retardation, and
transformation in the subsurface. Particularly strong on
the chemistry of organic contaminants.
National Research Council. 1990. Ground Water Models. Provides a thorough review of the theory, use, limitations,
Washington, D.C.: National Academy Press. and applications of computer modeling applied to the
subsurface.
Sahwney, B. L., and K. Brown, eds. 1989. Reactions and Provides a thorough review of sorption-desorption
Movement of Organic Chemicals in Soils. Madison, behavior of contaminants in soil. Includes chapters on
Wisc.: Soil Science Society of America. contaminant movement and transformation.

Commercial application Hinchee, R. E., and R. F. Olfenbuttel, eds. 1992. In Situ Papers in this compendium discuss field and research
Bioreclamation: Applications and Investigations for studies of in situ and on-site bioremediation.
Hydrocarbon and Contaminated Site Remediation.
Boston: Butterworth-Heinemann.

46
THE CURRENT PRACTICE OF BIOREMEDIATION 47
3
The Current Practice of Bioremediation
Increasingly, in situ bioremediation is being heralded as a promising ''new"

alternative ground water cleanup technology. In fact, however, bioremediation is
not new. It has been used commercially for more than 20 years. The first
commercial in situ bioremediation system was installed in 1972 to cleanup a Sun
Oil pipeline spill in Ambler, Pennsylvania.
Since 1972, bioremediation has become well developed as a means of
cleaning up spills of gasoline, diesel, and other easily degraded petroleum
products. In general, in situ bioremediation has not developed to the point where
it can be used on a commercial scale to treat compounds other than easily
degraded petroleum products. However, although in situ bioremediation of
petroleum-based fuels is the only common use of the technology now, in the
future bioremediation will likely be used to treat a broad range of contaminants.
Recently, research has intensified on bioremediation of less easily degraded
compounds, such as chlorinated solvents, pesticides, and polychlorinated
biphenyls (PCBs). Bioremediation of many such recalcitrant compounds has been
successfully field tested (see the case examples in Boxes 4-1 and 4-3, in
Chapter 4).
This chapter describes the state of the practice of in situ bioremediation as
used today. Although the current uses of bioremediation apply primarily to
petroleum-based fuels, the principles of practice

outlined here extend to a much broader range of uses for the technology in the
future.
BIOREMEDIATION VERSUS OTHER TECHNOLOGIES

For the past decade, the method of choice for ground water cleanup has been
pump-and-treat systems. These systems consist of a series of wells used to pump
water to the surface and a surface treatment facility used to clean the extracted
water. This method can control contaminant migration, and, if recovery wells are
located in the heart of the contaminant plume, it can remove contaminant mass.
However, recent studies have shown that because many common contaminants
become trapped in the subsurface, completely flushing them out may require the
pumping of extremely large volumes of water over very long time periods. In situ
bioremediation, because it treats contaminants in place instead of requiring their
extraction, may speed the cleanup process. Consequently, bioremediation is likely
to take a few to several years compared to a few to several decades for pump-
and-treat technology. Thus, while capital and annual operating costs may be
higher for bioremediation, its shorter operating time usually results in a reduction
of total costs. Factors contributing to cost reductions in bioremediation compared
to pump-and-treat systems include reduced time required for site monitoring,
reporting, and management, as well as reduced need for maintenance, labor, and
supplies. Furthermore, the surface treatment methods that are part of pump-and-
treat systems typically use air stripping and/or carbon treatment to remove
contaminants from the water—processes that transfer the contaminant to another
medium (the air or the land) instead of destroying it. Bioremediation, on the other
hand, can completely destroy contaminants, converting them to carbon dioxide,
water, and new cell mass.
For cleaning up contaminated soils, in situ bioremediation is only one of
several possible technologies. Alternatives include (1) excavation followed by
safe disposal or incineration, (2) on-site bioremediation using land-farming or
fully enclosed soil cell techniques, (3) low-temperature desorption, (4) in situ
vapor recovery, and (5) containment using slurry walls and caps. In situ methods
(desorption, vapor recovery, containment, and bioremediation) have the
advantages of being minimally disruptive to the site and potentially less
expensive. Because ex situ methods require excavation, they disrupt the
landscape, expose contaminants, and require replacement of soils. For these
reasons, ex situ methods sometimes are impractical. Potential advantages of
bioremediation compared to other in situ methods

include destruction rather than transfer of the contaminant to another medium,

minimal exposure of on-site workers to the contaminant, long-term protection of
public health, and possible reduction in the duration of the remediation process.
BASICS OF BIOREMEDIATION PROCESS DESIGN

Biological and nonbiological measures to remedy environmental pollution
are used the same way. All remediation techniques seek first to prevent
contaminants from spreading. In the subsurface, contaminants spread primarily as
a result of partitioning into ground water. As the ground water advances, soluble
components from a concentrated contaminant pool dissolve, moving forward with
the ground water to form a contaminant plume. Because the plume is mobile, it
could be a financial, health, or legal liability if allowed to migrate off site. The
concentrated source of contamination, on the other hand, often has settled into a
fixed position and in this regard is stable. However, until the source can be
removed (by whatever cleanup technology), the plume will always threaten to
advance off site.
Depending on the nature of the site, the types of contaminants, and the needs
of the parties responsible for the contaminated site, the treatment technologies
administered may vary. The source area and the ground water plume may be
treated by engineered bioremediation, intrinsic bioremediation, a combination of
the two, or a mixture of bioremediation with nonbiological treatment strategies.
Contaminant concentrations in ground water plumes are typically much lower
than in the source area. Because of this concentration difference, management
procedures for the source area and the plume may be quite different. When the
source area is highly contaminated, aggressive containment and treatment are
often required to bring the site under control.
Selection and application of a bioremediation process for the source or the
plume require the consideration of several factors. The first factor is the goals for
managing the site, which may vary from simple containment to meeting specific
regulatory standards for contaminant concentrations in the ground water and soil.
The second factor is the extent of contamination. Understanding the types of
contaminants, their concentrations, and their locations is critical in designing in
situ bioremediation procedures. The third factor is the types of biological
processes that are effective for transforming the contaminant. By matching
established metabolic capabilities with the contaminants found, a strategy for
encouraging growth of the proper

organisms can be developed. The final consideration is the site's transport

dynamics, which control contaminant spreading and influence the selection of
appropriate methods for stimulating microbial growth.
Once site characteristics have been discerned, strategies for gaining
hydrologic control and for supplying the requisite nutrients and electron
acceptors for the microorganisms can be developed. If there is a sufficient natural
supply of these substances, intrinsic bioremediation may be effective. On the
other hand, if these biochemical or environmental requirements must be
artificially supplied to maintain a desired level of activity, engineered
bioremediation is the desired course. The ultimate consideration is if and when
the targeted cleanup goal can be achieved.
Engineered Bioremediation
Engineered bioremediation may be chosen over intrinsic bioremediation
because of time and liability. Because engineered bioremediation accelerates
biodegradation reaction rates, this technology is appropriate for situations where
time constraints for contaminant elimination are short or where transport
processes are causing the contaminant plume to advance rapidly. The need for
rapid pollutant removal may be driven by an impending property transfer or by
the impact of contamination on the local community. A shortened cleanup time
means a correspondingly lower cost of maintaining the site, as more rapid
remediation reduces the long-term sampling and analysis costs. Actions implicit
in engineered bioremediation also address the political and psychological needs
of a client or community that has been affected by the contamination. The
construction and operation of engineered bioremediation systems can
demonstrate to the local community that the party responsible for the
contamination has a responsive "good neighbor" attitude.
Engineered bioremediation can take a number of forms. The different
applications vary according to both the context of the contamination (site
geology, hydrology, and chemistry) and the biochemical processes to be
harnessed. Regardless of site conditions, however, certain principal parameters
guide the design, depending on whether the system is to treat soil or water.
Bioremediation Systems for Unsaturated Soils

When subsurface contamination exists substantially or entirely above the
water table (in what is known as the unsaturated, or vadose,

This test plot in Kalkaska County, Michigan, is being used to demonstrate a

simple form of engineered bioremediation for soil. The soil is contaminated with
crude oil from an oil well. Tilling provides oxygen, and periodic irrigation
maintains the soil moisture. The black tarp shown here helps warm the soil,
which speeds microbial activity and prevents rain from infiltrating the site and
contaminating deeper levels of the subsurface. Cleanup began in September
1991. Within a year, total petroleum hydrocarbons dropped from tens of
thousands of parts per million to less than 400 ppm. Researchers expect that the
concentrations will drop further, to nondetectable levels, within one more
growing season. Based on the success at this site, the state of Michigan plans to
approve bioremediation as a cleanup method for soils contaminated with crude
oil. CREDIT: John M. Shauver, Michigan Department of Natural Resources.
zone), the treatment system relies on transport of materials through the gas
phase. Thus, engineered bioremediation is effected primarily through the use of
an aeration system, oxygen being the electron acceptor of choice for the systems
used so far to treat petroleum contamination. If the contamination is shallow,
simple tilling of the soil may accelerate oxygen delivery sufficiently to promote
bioremediation. For deeper contamination, aeration is most commonly provided
by applying a vacuum, but it may also be supplied by injecting air. In either case
the three primary control parameters are, in order of importance, oxygen supply,
moisture maintenance, and the supply of nutrients and other reactants. Figure 3-1
shows an

engineered bioremediation system for unsaturated soils. It indicates the types of

systems used to supply oxygen and nutrients and to maintain moisture.
FIGURE 3-1 Engineered bioremediation system for treating soil above the water
table (indicated by triangles at the bottom of the drawing). The vacuum pumps
circulate air to supply oxygen. The infiltration gallery in the center of the diagram
supplies water to replace lost moisture and nutrients to stimulate microbial
growth.
The design and implementation of an effective vacuum or injection system

for oxygen delivery require knowledge of the vertical and horizontal location of
the contaminants and the geological characteristics of the contaminated zones.
Because air flow is proportional to the permeability characteristics of each
geological stratum, aeration points must be separately installed at depths that
correspond to every contaminated geological unit. For effective oxygen delivery,
the spacing of the aeration points within a geological unit is a function of the soil
permeability and the applied vacuum (or pressure). Determination of spacing
should be based on field data and/or computer models. In some clay-rich soils the
circulation of sufficient oxygen to promote bioremediation is extremely difficult
because such soils are relatively impermeable. In these soils hydraulic fracturing
or another engineered approach may be required to facilitate air flow.
The passage of air through the subsurface will remove moisture.

This can cause drying that, if severe enough, may impede biological
processes. Therefore, maintaining a proper moisture balance is critical to the
system's success. Moisture is added to the treatment area by spraying or flooding
the surface (if the surface is relatively permeable) or by injecting water through
infiltration galleries, trenches, or dry wells. Care must be taken that excess water
is not added, because it can leach contaminants into the ground water or decrease
the amount of air in the subsurface pores.
If inorganic nutrients or other stimulants are required to maintain the
effectiveness of the bioremediation system, they may be added in soluble form
through the system used for moisture maintenance, as shown in Figure 3-1. In
some cases, nutrients and stimulants could be added as gases. At some sites,
nitrogen has been added in the form of gaseous ammonia. Future applications of
bioremediation could add methane gas to stimulate the cometabolism of
chlorinated ethenes. Gaseous additives can be administered through wells or
trenches constructed parallel to the aeration system.
Bioremediation Systems for Ground Water

Bioremediation systems for treating ground water below the water table fit
two categories: water circulation systems and air injection systems. Most aquifer
bioremediation systems have used the former approach, but in the last few years
air injection systems have become increasingly common.
Water Circulation Systems. Water circulation systems work by circulating
water amended with nutrients and other substances required to stimulate
microbial growth between injection and recovery wells. This approach is
sometimes referred to as the Raymond Method, named after the scientist who
designed the system for the 1972 Sun Oil spill. The method has typically
incorporated an optional above-ground water treatment facility into the ground
water circulation system. Figure 3-2 shows a diagram of a water circulation
system, with oxygen supplied by hydrogen peroxide (H2O2) and the recovered
water treated with an air stripper to remove any remaining volatile contaminants.
Under normal operations, all of the ground water is recovered, and all or a
portion of the treated ground water is reinjected after being amended with
nutrients and a final electron acceptor. Recovery systems most frequently use
wells, although trenches can be used in some situations. Injection is commonly
achieved with wells, but several systems have used injection galleries. In some
systems all of the recovered water is discharged to an alternate reservoir, and
either

drinking water or uncontaminated ground water is used for injection. The injected
ground water moves through the saturated sediments toward the ground water
capture system. As the amended water moves through the contaminated portions
of the site, it increases microbial activity by providing the elements that limit
intrinsic biodegradation.
FIGURE 3-2 Water circulation bioremediation system for treating contaminated

ground water. Water containing H2O2 (as an oxygen source) and nutrients is
circulated through the site to stimulate microbial growth. An air stripper treats
the recovered water to remove remaining volatile contaminants.
At the Wexford County, Michigan, gas-processing plant pictured on the

right, a water circulation bioremediation system is being used to clean up ground
water contaminated with gasoline spilled from a tanker truck. The top photo
shows the system that supplies oxygen to the microbes. Pure oxygen from the
oxygen tank is bubbled into water moving through the pipes shown here to a
series of injection wells. Originally, the water was also amended with nutrients,
but tests showed no increase in the biodegradation rate with nutrient addition, so
nutrient addition was stopped. The cylinder in the center of the photo removes
iron from the water to prevent it from clogging the injection wells. The lower
photo shows some of the monitoring wells (the capped cylinders) used to test the
system's effectiveness. Treatment began in August 1991. Within 14 months,
pollutant concentrations dropped to levels that met state standards. CREDIT: John
M. Shauver, Michigan Department of Natural Resources.



Nutrients typically added are nitrogen and phosphorus, although other

minerals are occasionally used. Ammonium and nitrate salts are the most
common nitrogen sources. Orthophosphate and tripoly-phosphate salts are the
most common phosphorous sources. The electron acceptor is most commonly
oxygen in the form of air, pure oxygen, or hydrogen peroxide. Nitrate has also
been used as an electron acceptor in some commercial scale engineered
bioremediation systems.
Air Injection Systems. Historically, one of the major limitations of water
circulation systems has been the effective supply of an electron acceptor.
Delivery of oxygen, the most common electron acceptor, is difficult because
oxygen gas has limited water solubility and other oxygen vehicles (such as
hydrogen peroxide and liquid oxygen) are costly and have had limited
effectiveness. The difficulties associated with oxygen delivery have hampered the
performance of bioremediation technology.
In the past few years, U.S. contractors have adopted the European practice
of air sparging—the injection of air directly into ground water (see Figure 3-3).
Air sparging serves two purposes. First, it is an efficient method of delivering
oxygen to promote microbial growth. The injected air displaces water in the
subsurface, creating pores temporarily
At the Hanahan, South Carolina, petroleum tank farm pictured on the left, a
water circulation bioremediation system is being used to cleanup extensive
ground water contamination from leaks in storage tanks and disposal of tank
bottoms. The site contains a mixture of a wide variety of petroleum
hydrocarbons, including aliphatic hydrocarbons, polycyclic aromatic
hydrocarbons (PAHs), and gasoline components (benzene, ethylbenzene,
toluene, and xylene, or BTEX). The treatment system consists of infiltration
galleries used to circulate water amended with nitrate, which serves as an electron
acceptor.
The top photo shows the tank farm. The lower photo shows construction of
an infiltration gallery. The perforated pipe in the gravel-lined trench will be used
to deliver the nutrients and water. The trench will be packed with gravel and
capped with sand after the pipe is installed.
The treatment goals at this site are to destroy PAHs and aliphatic
hydrocarbons sorbed to the soil and to decrease the BTEX concentration in the
water. Researchers will use a subsite in which water has been infiltrated but no
nitrate has been added as a control. The control subsite will be used to determine
the effect of nitrate addition, and the resultant stimulation of microorganisms, on
the rate of contaminant destruction.
CREDIT: Don A. Vroblesky, U.S. Geological Survey.

filed with air that is easily available to the microbes. Second, air sparging can
help remove volatile contaminants. As the injected air sweeps upward through the
contaminated zone, it can carry volatile contaminants to the soil above the water
table for capture by a vapor recovery system.
FIGURE 3-3 Air-sparging system for treating contaminated ground water.

Vacuum pumps circulate air to promote the growth of aerobic microbes and to
extract volatile contaminants. An infiltration system supplies nutrients.
Essential to the proper use of air sparging is delineation of the extent of

contamination and the subsurface geological profile. These requirements must be
met to ensure that air can move readily and uniformly through the area to be
treated. If there are geological barriers that can trap or channel the air flow, the
use of air sparging may be precluded. The spacing of wells and the injection
pressure for the sparging system need to be determined by field testing and/or
modeling.
Addition of nutrients or other amendments, if necessary, can be
accomplished through the use of injection wells or infiltration galleries (as shown
in Figure 3-3). The movement of air through the subsurface provides a mixing
function that helps disperse nutrients through the water column. Gaseous
reactants, such as methane, that may be required for cometabolic bioremediation
strategies could be added through the sparge wells.

In most situations, sparging is conducted with a ground water capture system

to prevent migration of dissolved contaminants. When volatile compounds are
present, air recovery systems are used to prevent contamination of the air above
the system and contaminant transfer to adjacent areas.
Intrinsic Bioremediation
Because intrinsic bioremediation relies on the innate capabilities of naturally
occurring microbial communities, the capacity of the native microbes to carry out
intrinsic bioremediation must be documented in laboratory tests performed on
site-specific samples. These tests must be carried out before intrinsic
bioremediation can be proposed as a legitimate cleanup strategy. In addition, the
effectiveness of intrinsic bioremediation must be proven with a site-monitoring
regime that includes chemical analysis of contaminants, final electron acceptors,
and/or other reactants or products indicative of biodegradation processes (as
explained in Chapter 4).
Intrinsic bioremediation may be used alone or in conjunction with other
remediation techniques. For instance, soils may be excavated for disposal or
treatment, with intrinsic bioremediation used to eliminate residual contamination.
Similarly, this process may be implemented after a pump-and-treat or engineered
bioremediation system has reduced the potential for migration of contaminants
off site.
For intrinsic bioremediation to be effective, the rate of contaminant
biodegradation must be faster than the rate of contaminant migration. These
relative rates depend on the type of contaminant, the microbial community, and
the subsurface hydrogeochemical conditions. Frequently, the rate-controlling step
is the influx of oxygen. Lack of a sufficiently large microbial population can also
limit the cleanup rate; this can be caused by a lack of nutrients or an inhibitory
condition, such as low pH or the presence of a toxic material.
Prior to implementation of intrinsic bioremediation, the site must be
thoroughly investigated. Parameters of concern include the type, mass, and
distribution of contaminant; the contaminant's susceptibility to biodegradation by
microorganisms at the site; the flow of ground water under nonpumping
conditions (including seasonal fluctuations); historical data on plume migration;
and the closeness and sensitivity of potential receptors that may be adversely
affected if reached by the contaminant. If information on all of these parameters
is available, a mathematical model can be used to predict the rates of migration
and biodegradation. Thus, prospects for expansion or recession of the
contaminated area can be assessed.

For regulators to approve intrinsic bioremediation, the regulatory agency

must be provided with supportive data to ensure that the public health will be
adequately protected. The implementation plan must include a site-monitoring
program to confirm that intrinsic bioremediation is performing as expected. If
performance falls short of expectations and the contamination spreads, further
corrective action will likely be required.
INTEGRATION OF BIOREMEDIATION WITH OTHER

TECHNOLOGIES
As a contaminated site is cleaned up, the biological reactions promoted in
bioremediation affect site chemistry in ways that may make the site more
amenable to cleanup with nonbiological technologies. For example, biological
activity may speed contaminant desorption from solids, making it easier to
extract the contaminants with a pump-and-treat system. Similarly, nonbiological
cleanup technologies can affect microbial activity at a site, sometimes promoting
bioremediation. For example, techniques designed to vent volatile contaminants
may increase the oxygen supply, encouraging microbial growth. Such synergistic
effects can maximize rates of contaminant loss. Thus, bioremediation is
frequently integrated with other technologies, both sequentially and
simultaneously. Some examples follow:
• When contaminant concentrations are high and affected zones are

accessible, bioremediation frequently follows excavation of soils near
the contaminant source. Excavated soils may be disposed of off site or
treated by a surface (ex situ) bioremediation system or by a thermal
method. Removal of heavily contaminated soils reduces the demands on
an in situ technology and immediately reduces the potential for impact
on the ground water.
• Where residual pools of contaminants are present in the subsurface, these
pools may be removed prior to implementation of other remediation
technologies by a process known as free product recovery. For pools of
contaminants that are less dense than water, such as gasoline and diesel
fuel, free product recovery is accomplished by pumping the liquids from
wells or trenches. This removes the contaminant mass in the most
concentrated form and reduces the demand for nutrients and electron
acceptors during bioremediation procedures that may follow. No good
recovery methods exist for pools of contaminants that are more dense
than water, such as chlorinated solvents, because these tend to sink deep
into the subsurface, where they are difficult to locate.

• Bioremediation may follow the use of a pump-and-treat system. The

pump-and-treat system may be used to shrink the contaminant plume
and, along with a free product recovery system, help remove
contaminant mass. Once a sufficient quantity of the contaminant has
been removed, the pump-and-treat system may be augmented with or
converted to an in situ bioremediation system.
• Frequently, in situ bioremediation for cleaning up ground water is
conducted in conjunction with in situ vapor recovery for cleaning up
regions above the water table. The in situ vapor recovery system uses a
series of recovery wells or trenches to extract air and volatile
contaminants from above the water table. In addition to withdrawing
volatile contaminants, the wells and trenches provide oxygen for
biodegradation. Hence, the process has become commonly known as
bioventing. By a combination of volatilization and biodegradation,
contaminant levels above the water table are reduced, thus decreasing
the potential for the contaminants to leach into the ground water.
Further, as the water table drops during dry seasons, more subsurface
sediments are exposed to air movement; thus, contamination is reduced
within the zone of water table fluctuation. Alternatively, air sparging
may be used along with vapor extraction procedures to reduce
contamination below the water table.
• It is possible to follow engineered bioremediation with intrinsic
bioremediation. After removal of free product contaminants, engineered
bioremediation may be used to eliminate the majority of residual
contaminants. Then, after an asymptotic decline of contaminants in the
plume, final polishing and containment may be accomplished using
intrinsic bioremediation. In this case, microbial activity will have been
stimulated and the biodegradation process at the site will be well
understood. (See Box 4-2, in Chapter 4, for an example.)
GOOD PRACTICES
The general approach required to earn credibility in the bioremediation
industry is the same as for any technical business: work only within areas of
expertise, be aware of the general limitations of the technology, pay attention to
details, and work closely with clients. In general, buyers of bioremediation
services can determine whether a bioremediation contractor is competent to do
the job by reviewing the contractor's credentials. Competent contracting firms
have employees and consulting experts with credentials in the scientific and
engineering fields important to bioremediation. And, like any other successful
business, a bioremediation firm should have a

track record of reliable performance that can be determined by reviewing the

firm's references. Box 3-1 lists standards of practice that all contractors should
follow.
BOX 3-1 STANDARDS OF PRACTICE FOR BIOREMEDIATION

CONTRACTORS
• Contractors should employ experts in the many scientific and engineering
fields important in bioremediation, including environmental engineering,
hydrogeology, microbiology, and chemistry.
• Contractors should be frank and open with the client about all
uncertainties in the process.
• Before treatment begins, the contractor should negotiate with all involved
parties (clients, regulators, and the affected community) the standards
that will be used to evaluate process performance. Agreeing on
performance standards will prevent conflicts resulting from transient or
trace amounts of contaminants found at sites after the treatment is
completed and will give the clients a realistic picture of what to expect
after the project is finished.
• The contractor should develop a clearly defined engineering design for
the treatment program. The design should provide a clear course toward
achieving a specific endpoint.
• The contractor's design should be based on site-specific data.
• The design should include a clearly defined monitoring program.
• The design should leave room for flexibility based on operational data
that indicate a need for adjustments, especially if the process is
innovative. Operators should be informed of the need to adjust the
system.

EVALUATING IN SITU BIOREMEDIATION 63
4
Evaluating In Situ Bioremediation
Showing that a bioremediation project is working requires evidence not only

that contaminant concentrations have decreased but also that microbes caused the
decrease. Although other processes may contribute to site cleanup during a
bioremediation, the microbes should be critical in meeting cleanup goals.
Without evidence of microbial involvement, there is no way to verify that the
contaminant did not simply volatilize, migrate off site, sorb to subsurface solids,
or change form via abiotic chemical reactions. This chapter discusses a strategy
for evaluating the effectiveness of in situ bioremediation projects, based on
showing that microbes were responsible for declining contaminant
concentrations. Regulators and buyers of bioremediation services can use the
strategy to evaluate the soundness of a proposed or ongoing in situ
bioremediation system. Researchers can apply the strategy to evaluate the results
of field tests.
A THREE-PART STRATEGY FOR ''PROVING" IN SITU

BIOREMEDIATION
To answer the question "What proves in situ bioremediation?", one must
recognize that only under rare circumstances is proof of in situ bioremediation
unequivocal. In the majority of cases the complexities of contaminant mixtures,
their hydrogeochemical settings, and competing abiotic mechanisms of
contaminant loss make it a

challenge to identify biodegradation processes. Unlike controlled laboratory

experiments in which measurements can usually be interpreted easily, cause-
and-effect relationships are often difficult to establish at field sites. Furthermore,
certain data that may convince some authorities of in situ bioremediation may not
convince others.
Although proving microbial involvement in cleanup with complete certainty
is seldom possible, the weight of the evidence should point to microbes as key
actors in the cleanup. Because one measurement is seldom adequate, the
evaluation strategy must build a consistent, logical case that relies on convergent
lines of independent evidence taken from the field site itself. The general strategy
for demonstrating that in situ bioremediation is working should include three
types of evidence:
1. documented loss of contaminants from the site,

2. laboratory assays showing that microorganisms in site samples have
the potential to transform the contaminants under the expected site
conditions, and
3. one or more pieces of evidence showing that the biodegradation
potential is actually realized in the field.
The strategy applies not only to bioremediation projects in the

implementation phase but also to those in the testing phase. The strategy is not
just for research purposes. Every well-designed bioremediation project should
show evidence of meeting the strategy's three requirements. Thus, regulators and
buyers of bioremediation services can use the strategy to evaluate whether a
proposed or ongoing bioremediation project is sound.
The first type of evidence in the strategy—showing decreasing contaminant
concentrations—comes from standard sampling of the ground water and soil over
time as cleanup progresses. The second type of evidence—showing the potential
for microorganisms to degrade the contaminants—is also relatively simple to
provide. In most cases it requires taking microbes from the field and showing
that they can degrade the contaminant when grown in a well-controlled laboratory
vessel. For some cases, lab studies may not be needed when a body of peer-
reviewed published studies documents that the compounds are easily and
commonly biodegraded.
The most difficult evidence to gather is the third type—showing that the
biodegradation potential demonstrated in the laboratory is being realized in the
field. Evidence of field biodegradation is essential: data showing that organisms
are capable of degrading the contaminant in the laboratory are not sufficient
because the organisms may not perform the same tasks under the less hospitable
field conditions.

A variety of techniques, explained below, exist for demonstrating field

biodegradation.
TECHNIQUES FOR DEMONSTRATING BIODEGRADATION

IN THE FIELD
The goal of the techniques for demonstrating field biodegradation is to show
that characteristics of the site's chemistry or microbial population change in ways
that one would predict if bioremediation were occurring. The environmental
changes measured in these tests should correlate to documented contaminant loss
over time. No one technique alone can show with complete certainty that
biodegradation is the primary reason for declining contaminant concentrations in
the field. The wider the variety of techniques used, the stronger the case for
successful bioremediation. As an example, Box 4-1 describes a site where several
tests were combined.
There are two types of sample-based techniques for demonstrating field
biodegradation: measurements of field samples and experiments run in the field.
In most bioremediation scenarios a third technique, modeling experiments,
provides an improved understanding of the fate of contaminants. Examples of
field measurements, field experiments, and modeling experiments are described
below. These examples provide general guidance about which types of tests are
appropriate. Detailed experimental protocols for carrying out the tests need to be
developed and will vary depending on the types of contaminants present, the
geological characteristics of the site, and the level of rigor desired in the
evaluation.
Measurements of Field Samples

A number of techniques for documenting in situ bioremediation involve
removing samples of soil and water from the site and bringing them to the lab for
chemical or microbiological analysis. Many of these techniques require
comparing conditions at the site once bioremediation is under way with site
conditions under baseline circumstances, when bioremediation is not occurring.
Baseline conditions can be established in two ways. The first method is to
analyze samples from a location that is hydrogeologically similar to the area
being treated but is either uncontaminated or is outside the zone of influence of
the bioremediation system. The second method is to gather samples before
starting the bioremediation system and compare them with samples gathered at
several time points after the system is operating. This second method applies only
to engineered

BOX 4-1 PROVING ENGINEERED BIOREMEDIATION OF

CHLORINATED SOLVENTS IN A FIELD TEST— MOFFETT
NAVAL AIR STATION, CALIFORNIA
Researchers at Stanford University conducted a field demonstration to
evaluate the potential for using cometabolism for in situ bioremediation of
chlorinated solvents. The work done in this field demonstration shows how a
variety of tests can be combined to evaluate whether new bioremediation
processes researched in the lab can be applied successfully in the field.
The demonstration site was a highly instrumented and well-
characterized confined sand-and-gravel aquifer at Moffett Naval Air Station
in Mountain View, California. To this site the researchers purposely added
chlorinated solvents in a carefully controlled way that ensured that the
solvents would not migrate beyond the research plot. Chlorinated solvents
cannot support microbial growth on their own, but, if supplied with methane,
a special class of organisms can destroy the contaminants through
cometabolism (see Chapter 2). Thus, at this site, researchers stimulated
native organisms by adding oxygen and methane. When stimulated, the
organisms destroyed significant quantities of the chlorinated solvents. The
researchers evaluated the success of their work using tests that meet the
three criteria discussed in this chapter:
1. Documented loss of contaminants: The researchers documented that

95 percent of the vinyl chloride, 85 percent of the trans-1,2-
dichloroethylene, 40 percent of the cis-1,2-dichloroethylene, and 20
percent of the trichloroethylene added to the site were transformed.
2. Laboratory assays showing that microorganisms have the potential
to degrade the contaminants: When cores of the aquifer removed to
the lab were exposed to methane and oxygen, the methane and
oxygen were used up, showing that the cores contained bacteria that
thrive on methane (methanotrophs). Previous research had shown
that methanotrophs can cometabolize chlorinated solvents.
3. Evidence that biodegradation potential is realized in the field: The
researchers used a variety of methods to demonstrate biodegradation
in the field. First, they showed that before the methanotrophs were
stimulated with methane and oxygen, destruction of trichloroethylene
was minimal. Second, they performed conservative tracer tests with
bromine to show that the methane and oxygen added to the site were
not disappearing by physical transport but were being used by
microorganisms. Third, they identified microbial breakdown
products from the solvents in aquifer samples. Fourth, they used
models to show that theoretical estimates of biodegradation rates
could account for contaminant loss in the field.

References
Roberts, P. V., G. C. Hopkins, D. M. Mackay, and L. Semprini. 1990. A field evaluation of in situ
biodegradation of chlorinated ethenes: Part 1—Methodology and field site characterization.
Groundwater 28:591-604.
Semprini, L., P. V. Roberts, G. D. Hopkins, and P. L. McCarty. 1990. A field evaluation of in situ
biodegradation of chlorinated ethenes: Part 2—The results of biostimulation and
biotransformation experiments. Groundwater 28:715-727.
bioremediation systems, because the "starting time" of intrinsic

bioremediation occurs whenever the contaminant enters the subsurface and
therefore cannot be controlled.
Following are several types of analyses that may be performed on samples
removed from the field.
Number of Bacteria
When microbes metabolize contaminants, they usually reproduce. (In
general, the larger the number of active microbes, the more quickly the
contaminants will be degraded.) Thus, samples correlating contaminant loss with
an increase in the number of contaminant-degrading bacteria above the normal
conditions provide one indicator that active bioremediation may be occurring in
the field. When contaminant biodegradation rates are low, such as when
contaminant levels are low or biodegradable components are inaccessible,
increases in the number of bacteria may not be great enough to detect above
background levels, given the error in sampling and measurement techniques.
Thus, the absence of a large increase in bacterial numbers does not necessarily
mean that bioremediation is unsuccessful.
The first issue for determining the size of the bacterial population is what to
sample. In principle, the best samples include the solid matrix (the soil and rock
that hold the ground water) and the associated pore water. Because most
microorganisms are attached to solid surfaces or are trapped in the intersticies
between soil grains, sampling only the water normally underestimates the total
number of bacteria, sometimes by as much as several orders of magnitude. In
addition, water samples may misrepresent the distribution of microbial types,
because a water sample may contain only those bacteria easily dislodged from
surfaces or that can be transported in the moving ground water.
While obtaining soil samples from the earth's surface is not difficult,

subsurface sampling is expensive and time consuming. Subsurface samples are

obtained by removing cylindrical cores from below ground. A major effort is
required to prevent microbial contamination of the sample during the coring
operation and while handling the sample. Whenever possible, sampling
equipment should be sterilized before use. Contamination from extraneous
material, including the air, soil, and human contact, should be prevented.
Although ground water samples have important deficiencies, they have a
role as semiquantitative indicators of microbial numbers. Major increases in the
number of bacteria in the ground water usually correlate to large increases in the
total number of bacteria in the subsurface. The main advantages of ground water
samples are that they can be taken repeatedly from the same location and that
they are relatively inexpensive.
The second issue for determining bacterial numbers is how to assay for the
bacteria. Several standard and emerging techniques, each of which has
advantages and disadvantages, are available:
• Direct microscopic counting is a traditional technique that involves using

a microscope to view the sample and count the bacteria, which are
distinguished from solid debris based on their size and shape.
Microscopic counting is greatly aided by the use of the acridine orange
stain and epifluorescence microscopy, which make intact bacteria stand
out from other particles. Microscopic enumeration can be tedious and
requires technician experience, particularly when the sample contains
solids. The technique provides data on total bacterial counts but does not
give information on cell types or metabolic activity.
• The INT activity test can enhance direct microscopic counting by
identifying only those bacteria active in electron transport, the main
force behind all metabolism. If the sample (or bacteria harvested from
the sample) is incubated with a tetrazolium salt under controlled
conditions, actively respiring bacteria transfer electrons to the
tetrazolium salt, forming purple INT crystals that can be observed
microscopically inside the metabolically active bacteria.
• Plate counts, another standard technique, can be used to quantify the
number of bacteria able to grow on a prescribed set of nutrients and
substrates immobilized in a solid medium. The solid medium is created
from a liquid solution with the appropriate nutrients and substrates,
solidified with agar to form a gel. A sample containing the bacteria of
interest is spread thinly over the surface of the gel. After the plate is
incubated, visible bacterial colonies form, and the colonies can be
counted to indicate the number of metabolically active

bacteria in the original sample. Because plate counting requires

significant growth to form visible colonies, the method often
underestimates the number and diversity of bacteria. On the other hand,
considerable information on the metabolic capability of bacteria can be
obtained by using a range of growth substrates to prepare the media.
• The most-probable-number (MPN) technique also relies on significant
growth in prescribed media, but enumeration is carried out through a
statistical analysis. Instead of counting colonies for a few incubations on
solid media, the MPN technique uses a large number of incubations from
portions of the sample diluted to prescribed levels in a nutrient solution.
Based on simple statistics and the number of diluted liquid samples that
show evidence of bacterial growth, the number of bacteria in the
original undiluted sample can be calculated. Although details of the
MPN and plate-counting methods differ, both techniques have the same
general advantages and disadvantages.
Modern tools of biochemistry and molecular biology are becoming available

to provide more precise ways of identifying and enumerating bacteria in site
samples. The tools exploit the growing understanding of genetically determined
characteristics of particular cell components:
• Oligonucleotide probes are small pieces of deoxyribonucleic acid

(DNA) that can identify bacteria by the unique sequence of molecules
coded in their genes. The small DNA probe bonds with a complementary
region of the target cell's genetic material, and the amount of bound
probe can be quantified and correlated with the number of cells.
Advanced probing techniques to count the cells in intact samples are
under development. Probing is a very powerful technique for identifying
which types of bacteria are present, as long as the genetic sequences are
known for the target bacteria. This knowledge is available for some
common types of bacteria and often is known for genetically engineered
microorganisms or other specialized microorganisms that might be used
as part of a bioaugmentation strategy. Probing also can be used to
determine whether the gene for a particular biodegradation reaction is
present. The drawbacks of probing are that it requires considerable prior
knowledge of the cells' genetic sequences, it is only semiquantitative in
its current state, and it requires specialized equipment and knowledge.
• Fatty acid analysis is a second new bacterial identification technique; it
uses the characteristic "signature" of fatty acids present in the
membranes of all cells. The distribution of fatty acids is unique

and stable among different bacteria and therefore can be used as an

identifying signature. Like gene probing, fatty acid analysis requires
specialized knowledge and equipment. It is limited in its quantification
ability and may have sensitivity limitations for small populations.
With gene probing and fatty acid analysis, it is unnecessary to grow the
bacteria in the laboratory to detect what kinds and how many are present in a
sample. While holding great promise, these new methods are still in the
development and testing stages.
Number of Protozoans
Because protozoans prey on bacteria, an increase in the number of
protozoans suggests a major increase in the number of bacteria. Therefore,
samples correlating contaminant loss with growth in the protozoan population can
provide further evidence of active bioremediation. The protozoan population can
be counted using a statistical MPN technique similar to that used for bacteria. The
MPN technique for counting protozoans requires growing various dilutions of the
soil or water sample in cultures containing a large number of bacterial prey.
Whether protozoans grow to feed on these prey can be determined by viewing the
diluted samples through a microscope.
Rates of Bacterial Activity

While an increase in bacterial numbers usually is a key sign that
bioremediation is working, the stronger measure of success is that potential
biotransformation rates are great enough to remove the contaminant rapidly or to
prevent contaminant migration. Therefore, measurements demonstrating that the
bacteria are capable of performing the desired reactions at significant rates help to
provide evidence of successful bioremediation.
The most direct means for estimating biodegradation rates is to construct
laboratory microcosms with environmental conditions as close as possible to
those from which the sample was taken. (See Box 4-2 for an example of the use
of microcosms.) To these microcosms, field samples and the accompanying
microbes (or other microbes that could be released into the field) are added.
Microcosms are useful because substrate concentrations and environmental
conditions can be controlled and the loss of the contaminant or other markers of
biodegradation can be measured relatively easily. For many pollutants (including
BTEX and PCBs), versions labeled with carbon-14 are

BOX 4-2 PROVING ENGINEERED BIOREMEDIATION OF AN

OIL AND FUEL SPILL—DENVER, COLORADO
In Denver, Colorado, a temporary holding tank under a garage used to
service vehicles leaked crankcase oil, diesel fuel, and gasoline. The leak
contaminated the surrounding soil and created a plume of benzene,
toluene, ethylbenzene, and xylene (BTEX) in the ground water below. An
engineered bioremediation system was installed at the site in 1989. The
soil was cleaned by removing the remaining pools of leaked contaminants
and by venting to supply oxygen and promote biodegradation. The ground
water was treated by circulating oxygen (in the form of hydrogen peroxide),
phosphorus (in the form of phosphate), and nitrogen (in the form of
ammonium chloride) to promote bioremediation.
By March 1992, after three years of treatment, the dissolved plume of
contaminants had been nearly eliminated from the ground water. However,
tests revealed that the aquifer contained a small layer that had trapped
considerable quantities of BTEX. This layer is relatively impermeable and
therefore had been bypassed by the fluids circulated to promote
bioremediation. When the bioremediation system was shut down in 1992,
long-term monitoring began to ensure that the native microbial community
could act quickly enough to degrade any contamination that might leak from
this layer.
Although the engineered bioremediation system at this site was unable
to eliminate all of the contamination, it succeeded in reducing the amount
and risk of the contamination to acceptable levels. Furthermore, it is likely
that microbes at the periphery of the remaining contamination will provide
effective intrinsic bioremediation that will prevent the reemergence of a
contaminant plume.
The cleanup using the engineered bioremediation met the three criteria
set forth in this report:
1. Documented loss of contaminants: At the monitoring well closest to
the gallery used to deliver oxygen and nutrients to the site, the BTEX
concentration dropped from 2030 µg/l before bioremediation to 6
µg/l after bioremediation. At other monitoring wells, the
concentration dropped more than an order of magnitude, to less than
46 µg/l.
to degrade the contaminants: Studies showed that microorganisms in
the transmissive layers adjacent to the trapped contaminants could
consume as much as 7 mg/l of oxygen per day, resulting in the
potential destruction of as much as 2 mg/l of hydrocarbons perday.

This oxygen consumption rate was determined by placing a

dewatered core from the site in a sealed glass mason jar and
measuring the amount of oxygen the microbes in the core consumed
in 24 hours. No direct tests—other than measuring the oxygen
consumption rate—of the native microbes' ability to degrade BTEX
were performed. However, the ability of subsurface microorganisms
to degrade BTEX is well established (see Table 2-1), so direct lab
tests were not as important for this site as for sites with contaminants
for which bioremediation techniques are still emerging.
3. Evidence that biodegradation potential is realized in the field: At
this site, two types of tests provided evidence of biodegradation in
the field. First, the oxygen consumption rate in microcosms
constructed with cores from the site was highest when the cores came
from near the layer of trapped contaminants. Thus, microbes with
access to the largest supply of contaminants consumed oxygen most
rapidly, supporting the expectation that bacterial growth on the
hydrocarbons had been stimulated. Second, the ratio of BTEX to
total petroleum hydrocarbons (TPH) was lower in the bioremediated
area than in the contaminant source. Research has shown that
microorganisms prefer BTEX to other components of TPH, leaving a
TPH residual that is relatively low in BTEX after a successful
remediation.
References
Nelson, C., R. J. Hicks, and S. D. Andrews. In press. In situ bioremediation: an integrated system
approach. In Bioremediation: Field Experiences, P. E. Flathman, D. E. Jerger, and J. H.
Exner, eds. Chelsea, Mich.: Lewis Publishers.
available and can be used in microcosm tests to trace the pollutant's fate very
precisely. Comparing the microcosm-generated biodegradation rates under a
variety of conditions can provide valuable information concerning whether
environmental conditions in the field are conductive for high degradation rates.
The careful control and monitoring possible in microcosms make rate
determinations much less ambiguous than rates measured in the field.
Methods that rely on laboratory microcosms have uncertainties associated
with directly extrapolating the laboratory results to the field. The delicate balance
of chemical, physical, and biological relationships that influence bioremediation
can change rapidly with environmental disturbances, such as to oxygen
concentration, pH, and nutrient concentration. Research has shown that microbes
removed to the laboratory may behave differently from those in the field—

quantitatively and qualitatively. Thus, laboratory experiments may impose

artifacts that distort the interpretation of field conditions.
Bacterial Adaptation
Over time, bacteria at a contaminated site may develop the capability to
metabolize contaminants that they were unable to transform—or that they
transformed very slowly—when the contaminant was first spilled. Thus,
metabolic adaptation provides evidence of bioremediation in the field. Adaptation
can result from an increase in the number of bacteria able to metabolize the
contaminant or from genetic or physiological changes within the individual
bacteria.
Microcosm studies are well suited for assessing adaptation. An increase in
the rate at which microorganisms in the sample transform the contaminant in
microcosm tests provides evidence that adaptation has occurred and
bioremediation is working. The rate increase can be determined by comparing
samples from the bioremediation zone with samples from an adjacent location or
by comparing rates before and after bioremediation.
Developments based on tools used in molecular biology may provide new
methods for tracking whether bacteria have adapted to degrade certain
contaminants. Gene probes specifically targeting degradative genes can be
constructed and can, at least in principle, determine if that gene is present in a
mixed population. Using probes in this manner requires knowledge of the DNA
sequence in the degradative gene.
For the special case when a genetically engineered microorganism is applied
to a site for bioaugmentation, the engineered organism can be fitted with a
reporter gene that is expressed only when a degradative gene of interest also is
expressed. Thus, the protein product of the reporter gene signals (for example, by
emitting light) that the degradative gene is present and is being expressed in the
in situ population.
Inorganic Carbon Concentration

In addition to more microbes, bacteria produce inorganic carbon—usually
present as gaseous CO2, dissolved CO2 or HCO3-—when they degrade organic
contaminants. Therefore, samples showing enrichment of the water and gas
phases with inorganic carbon indicate active biodegradation. Gas chromatography
is the method of choice for determining gaseous CO2 concentrations; inorganic
carbon analysis is appropriate for water samples.

Monitoring changes in inorganic carbon is inaccurate where high

background bicarbonate concentrations or dissolution of calcareous minerals
masks respiratory production of inorganic carbon. In these cases, stable isotope
analysis of the carbon (described below) is a possible means to distinguish
bacterially produced inorganic carbon from mineral carbon, but these techniques
are still in the early research stages.
Carbon Isotope Ratios

One way of determining whether the CO2 and other inorganic carbon in a
sample is an end product of contaminant biodegradation or whether it originates
from some other source is to analyze the sample's carbon isotopes. Most of the
carbon will be present as the isotope 12C (having six protons and six neutrons in
its nucleus), but some will be present as 13C (having six protons and seven
neutrons in its nucleus, thus weighing slightly more than 12C). The 13C/12C ratio
of the inorganic carbon in a sample varies depending on where the carbon
originated—from contaminant degradation, degradation of other organic matter,
or mineral dissolution. Depending on the situation, 13C/12C ratios can be used in
one of two ways.
The first use of 13C/12C ratios is appropriate when the carbon in the organic
contaminant has a substantially different 13C/12C ratio than the inorganic carbon
derived from mineral dissolution. This situation is relatively common because
inorganic carbon from minerals contains substantially more 13C than carbon
derived from most organic contaminants. Although the 13C/12C ratio changes
somewhat when organic contaminants are biodegraded to CO2, inorganic carbon
produced from most organic contaminants remains substantially more enriched in
12C than inorganic carbon dissolved from mineral deposits. Thus, if the inorganic
carbon taken from site samples has a 13C/12C ratio much lower than the ratio for
carbon from mineral sources, it is likely that the carbon originates from
contaminant biodegradation.
The second type of application exploits isotope fractionation, in which
microbial metabolism usually creates inorganic carbon that is enriched in 12C,
while the remaining organic contaminant source becomes enriched in 13C. For
example, microorganisms degrade the lighter (12C) isotopic forms of petroleum
hydrocarbons more quickly than they degrade the heavier (13C) forms. As a
result, the petroleum hydrocarbons remaining in the subsurface become relatively
enriched in 13C as bioremediation proceeds. Thus, observation of a decreasing
13C/12C ratio in inorganic carbon, coupled with an increase in the

ratio for the organic source, usually provides evidence that the inorganic carbon
is being produced by contaminant biodegradation.
An exception to the typical trend of decreasing 13C/12C ratios in inorganic
carbon occurs during methanogenesis, in which the end product of contaminant
biodegradation is not CO2, but methane (CH4). Methanogenic organisms
consume CO2 by converting it to CH4. In the process the pool of CO2 becomes
depleted in 12C, while the methane generated by the organisms becomes enriched
in 12C. Thus, in methanogenic environments the 13C/12C ratio observed in
samples of inorganic carbon may increase, instead of decreasing. Meanwhile, in
the methane—the final sink for the carbon from the contaminant—the 13C/12C
ratio decreases.
The 13C/12C ratio can be determined by analyzing samples with a mass
spectrometer, a standard chemist's tool for separating isotopes and determining
the relative masses of chemical compounds. The procedures for determining
isotope ratios are elaborate, expensive, and only pertinent if the characteristic
13C/12C ratio of the contaminant source can be ascertained. Today, the 13C/12C
ratio is an experimental method that requires further development and evaluation

before it can be used as a definitive indicator of bioremediation. However, given
the proper circumstances, the approach is advantageous because there is no
requirement for sampling adjacent areas outside the bioremediation zone to
evaluate relative responses (although the contaminant's characteristic 13C/12C
signature must be determined from a sample representative of the source).
Another potential advantage is that sampling for inorganic carbon does not
require unusual precautions or equipment.
Electron Acceptor Concentration

In the process of transforming contaminants, bacteria consume electron
acceptors, usually O2, NO3-, or SO42-, as explained in Chapter 2. A depletion in
the electron acceptor concentration that occurs simultaneously with contaminant
loss is further evidence that bioremediation is occurring. The electron acceptor
concentration can be determined by standard analyses in wet chemistry. Sampling
for O2 must be carried out with extreme care to prevent increases in the sample's
dissolved O2 concentration due to contact with air.
Byproducts of Anaerobic Activity

Some of the key organisms useful in bioremediation are anaerobic—that is,
able to exist without oxygen. These anaerobes are valuable

because they are able to carry out many important biotransformation reactions
when the supply of oxygen is limited. In addition, certain anaerobes are best able
to carry out the initial dechlorination steps for highly chlorinated solvents and
PCBs (see Table 2-1). Increases in metabolic products produced by anaerobes can
signal an increase in anaerobic activity and indicate successful bioremediation
(see Box 2-2). Key byproducts of anaerobic respiration include methane,
sulfides, reduced forms of iron and manganese, and nitrogen gas. When
significant amounts of chlorinated compounds are biotransformed, increases in
chloride ion also may be observable. These measurements give the strongest
evidence when parallel measurements confirm an anaerobic (oxygen-depleted)
environment, loss of electron acceptors other than oxygen (for example, nitrate
and sulfate), and consumption of electron donors responsible for the loss of the
electron acceptors.
Intermediary Metabolite Formation

Microbiological processes may transform contaminants into unique
intermediary metabolites. For example, during cometabolic microbial
transformation of trichloroethylene, trans-dichloroethylene oxide may be
produced. Detection of such metabolites from field samples provides evidence
that in situ biodegradation is progressing (see Box 4-3 for an example).
Intermediary metabolites can be determined by using gas chromatography, high-
performance liquid chromatography, or one of these methods coupled to mass
spectrometry. To ensure validity of this approach, the intermediary metabolites
cannot have been present in the originally released contaminants, and they should
be absent in adjacent uncontaminated areas. Because some intermediary
metabolites degrade too quickly to be detected, an absence of intermediates does
not indicate that bioremediation is not occurring.
Ratio of Nondegradable to Degradable Substances

If a site contains mixtures of contaminants, a decrease in the ratio of
biodegradable to nonbiodegradable organic compounds over time can indicate
microbiological activity in the field (see boxes 4-2 and 4-3). For example,
phytane, a molecule that occurs in crude oil, is more resistant to microbial attack
than octadecane, another crude oil component. Phytane and octadecane have the
same molecular weight and similar volatility and transport characteristics and,
consequently, are likely to undergo nearly identical abiotic reactions. Therefore, a
decrease in the ratio of octadecane to phytane is evidence that microbes are
degrading the octadecane. A possible drawback of this

BOX 4-3 TESTING BIOREMEDIATION OF PCBS IN HUDSON

RIVER SEDIMENTS—NEW YORK
Laboratory studies have shown that polychlorinated biphenyls (PCBs)
—substances once thought highly resistant to microbial attack—can, in
fact, be biodegraded. In an effort to demonstrate the practical implications
of these studies, the General Electric Corporation sponsored a 10-week in
situ biodegradation field test. Researchers anchored six large adjacent
cylinders in shallow areas of the Hudson River where the sediments are
contaminated with lightly chlorinated PCBs. They tested the ability of native
microbes and PCB-degrading microbes brought to the site from the
laboratory to degrade the PCBs in place when stimulated with oxygen, a
complete mixture of nutrients, and biphenyl to stimulate PCB
cometabolism. While addition of the laboratory bacteria had no effect on in
situ PCB degradation, significant destruction of PCBs occurred, and the
researchers attributed the loss to biodegradation by the native microbes.
In demonstrating in situ bioremediation, the researchers provided the
three key types of evidence outlined in this report:
1. Documented loss of contaminants: Over the 10-week test, between
one-third and one-half of the PCBs were destroyed. The researchers
determined PCB losses by measuring PCB concentrations in 12 cores
in each cylinder at the beginning and end of the experiment.
to degrade the contaminants: For this type of evidence, the
researchers relied on several published laboratory studies showing
that lightly chlorinated PCBs are susceptible to aerobic
biodegradation.
3. Evidence that biodegradation potential is realized in the field: The
most important evidence of in situ biodegradation was tests showing
that chlorophenols—key intermediary metabolites in PCB
degradation—appeared in the test cylinders after the microbes were
supplied with the necessary nutrients, biphenyls, and oxygen. In
addition, the researchers showed that the ratio of degradable to
nondegradable PCBs decreased over time, indicating microbial
attack of the degradable portions.
References
Harkness, M. R., J. B. McDermott, D. A. Abramowicz, J. J. Salvo, W. P. Flanagan, M. L. Stephens,
F. J. Mondello, R. J. May, J. H. Lobos, K. M. Carroll, M. J. Brennan, A. A. Bracco, K. M.
Fish, G. L. Warner, P. R. Wilson, D. K. Dietrich, D. T. Lin, C. B. Morgan, and W. L.
Gately. 1993. In situ stimulation of aerobic PCB biodegradation in Hudson River sediments.
Science 259(Jan. 22):503-507.

approach is that naturally occurring microbes often eventually attack

phytane, causing the octadecane/phytane ratio to underestimate in situ
biodegradation rates. Another example of this strategy is differential removal of
volatile organic compounds that have roughly the same transport and
volatilization properties but that are degraded at different rates. For instance,
dichloroethane behaves nearly identically to trichloroethylene, but, unlike
trichloroethylene, dichloroethane is not readily degraded under anaerobic
conditions.
This approach is also useful for single contaminants having different forms,
one of which is biodegradable and the other of which resists biodegradation.
Some organic contaminants consist of mixtures of stereoisomers—molecules that
are formed of the same elements and the same bonds but that have different
spatial arrangements of the atoms. Hexachlorocyclohexane, for example, exists in
two different forms, only one of which is readily metabolized. Thus, chemical
analyses documenting selective disappearance of the degradable form of this
contaminant are evidence of bioremediation. This approach is contaminant
specific and requires substantial prior biochemical and physiological knowledge,
but it illustrates an important principle that in the future could be of practical
value in bioremediation projects.
Experiments Run in the Field

Several useful methods for evaluating whether microorganisms are actively
degrading the contaminant involve not just sampling the site but also conducting
active experiments in the field. These field experiments require adding various
chemicals to the subsurface in a strictly controlled manner to see if their fate is
consistent with what should occur during bioremediation.
Stimulating Bacteria Within Subsites

One type of field experiment involves adding materials that stimulate
biodegradation to subsites within the contaminated area. Addition of stimulants
such as electron acceptors, electron donors, and nutrients should speed
biodegradation but not abiotic contaminant removal processes. Thus, when
stimulants are added to one subsite but not another, the relative rate of
contaminant loss should increase in the stimulant-amended subsites. The contrast
in contaminant loss between enhanced and unenhanced subsites can be attributed
to microbial activity. Applying this approach requires a setting uniform enough to
have comparable subsites.

Measuring the Electron Acceptor Uptake Rate

A second field experiment involves alternately starting and stopping the
supply of oxygen or other electron acceptors to the site to determine the rate at
which the electron acceptors are consumed. This approach is particularly useful
with air sparging because the oxygen supply can be controlled rapidly and
independently of water flow. Immediately after stopping the flow of sparged
gases, an oxygen probe is lowered into ground water wells to measure the rate of
oxygen consumption. To distinguish oxygen used by contaminant-degrading
microbes from oxygen used by ordinary microbial activity, background oxygen
uptake rates should be measured in adjacent uncontaminated wells. Relatively
rapid oxygen loss in the contaminated area compared to the uncontaminated area,
coupled with a drop in the contaminant concentration, suggests successful
bioremediation.
Monitoring Conservative Tracers

A third type of field experiment requires adding a conservative tracer to the
site. Conservative tracers have chemical and transport properties similar to those
of microbiologically reactive chemicals but are not microbiologically reactive
themselves. Thus, conservative tracers can be used to distinguish abiotic
chemical changes—such as volatilization, sorption, and dilution—from chemical
changes caused by microorganisms.
One possible use of conservative tracers is to determine how much sparged
oxygen is being consumed by microbes and how much is disappearing through
abiotic routes, such as dilution. For this determination, helium gas (He) can be
used as a conservative tracer for O2. A known concentration of He is incorporated
into the sparging system used to supply O2 to the contaminated zone, and the
changing concentrations of both He and O2 are measured over time using a
portable gas chromatograph and oxygen meter or other appropriate instruments.
The rate of O2 depletion relative to He depletion indicates the rate at which
microbes are consuming O2. If O2 is being consumed at a rate related to the
contaminant consumption rate, it is likely that microorganisms are responsible for
contaminant disappearance. In some cases, O2 can be consumed abiotically, such
as by iron oxidation (converting Fe 2+ to Fe3+). When such a possibility exists, O2
depletion measured in comparison with a tracer should also be determined in
background uncontaminated zones to estimate the abiotic O2 consumption rate.
For sites where a dissolved chemical (such as NO3-, SO42-, or

dissolved O2) is the electron acceptor, bromide can be used as a conservative

tracer. In this approach the bromide is added to the water circulated through the
ground to supply the electron acceptor.
Although conservative tracers that mimic contaminant behavior often are
added to the site, they also may be fortuitously present in the contaminant. As
discussed under ''Measurements of Field Samples," some contaminants contain
mixtures of degradable and nonbiodegradable compounds that move through the
subsurface in similar ways. When the concentration of degradable compounds
drops faster than the concentration of conservative tracers, the difference can be
attributed to microbial activity in the field.
Labeling Contaminants
A fourth type of field experiment involves monitoring the fate of "labeled"
contaminants. Contaminants can be labeled by synthesizing versions in which the
contaminant molecules contain a known amount of a stable isotope, usually 13C
or deuterium (a hydrogen isotope). If the expected metabolic byproducts, such as
inorganic carbon and intermediary metabolites, carry the same relative amounts
of 13C and deuterium as the labeled contaminants, bioremediation is occurring.
This technique is useful primarily for field research and not commercial
bioremediation because it involves synthesizing a special version of the
contaminant, which is costly, and adding it to the site, which temporarily
increases the level of contamination. In addition, contaminant labeling is useful
only for situations in which the contaminant source can be located. Adding the
labeled compound to the wrong location may result in a false negative.
Modeling Experiments
A final type of technique for evaluating whether bioremediation is occurring
in the field uses models—sets of mathematical equations that quantify the
contaminant's fate. Models keep track of all the contaminant mass that enters the
subsurface, describing how much dissolves, how much sorbs to solids, how much
reacts with other chemicals, how much flushes out in the water, and how much
biodegrades. The goal of using models is to see whether predictions of
contaminant fate based on interpretation of the phenomena taking place during
the bioremediation, as described by the model, match what is happening in the
field, as determined by field sampling.
Contaminated field sites can be efficiently managed with the aid of models
because models provide a means for synthesizing all relevant

information. Furthermore, because models quantitatively link many types of

measurements, they assist in evaluating the significance of a limited number of
field observations. When models are sufficiently accurate, they may be powerful
tools for assessing bioremediation.
Depending on the type and amount of data, the stage of process
understanding, and the types of questions being asked, models can vary from very
simple to highly complex. For example, a conceptual model, which does not yet
have mathematical equations, may be appropriate when limited data are available
during initial site characterization. On the other hand, complex mathematical
models, solved on high-speed computers, become possible and more appropriate
as understanding of the site expands during design and operation of a
bioremediation project.
Types of Models
Because so many complex processes interact in the subsurface, four
different types of models have been developed: saturated flow, multiphase flow,
geochemical, and reaction rate models. Each model describes a different suite of
subsurface processes and is used in particular ways to evaluate bioremediation.
Ultimately, researchers often combine two or more types of models to do a
complete evaluation.
Saturated Flow Models. Saturated flow models start by describing where and
how fast the water flows through the saturated zone (the region below the water
table). These models are derived from basic principles of conservation of fluid
mass. Saturated flow is reasonably well understood, and the basic forms of these
models for water flow are relatively simple, accurate, and accepted.
Once the direction and velocity of water flow are known, saturated flow
models can be extended to describe the movement of dissolved contaminants.
These contaminant transport models are based on principles of conservation of
chemical mass. When the model contains no terms for reactions, it describes the
fate of a conservative tracer. The conservative material basically moves with the
water flow, although it is subject to processes that disperse, or mix, the
contaminants.
Sorption of contaminants to the solid matrix slows the movement of the
dissolved contaminants, compared to the water. Sorption effects often can be
modeled simply by incorporating "retardation factors" that reflect the slower rate
of transport of the contaminant relative

to the water. In other cases, sorption phenomena are more complex than can be
captured by a simple retardation factor and must be modeled using equations that
consider sorption and desorption rates.
In special cases, biodegradation reactions can be described by very simple
expressions (for example, first-order decay) that are easily incorporated into the
transport part of a saturated flow model. However, many biodegradation
phenomena are too complex to be incorporated so simply into a saturated flow
model. Special modeling tools are needed and are discussed in the section below
on biological reaction rate models.
Multiphase Flow Models. Whereas saturated flow models describe the flow
of only one fluid, the ground water, through a porous medium, multiphase flow
models describe the situation in which two or more fluids exist together in the
porous medium. The fluids can be liquids or gases. The most common multiphase
flow models predict the movement of water and contaminants above the water
table, where a gas phase is present. This situation is called unsaturated flow.
Addition of a light nonaqueous-phase liquid contaminant such as gasoline, which
resides at or near the top of the water table, is a further complication that may be
considered in models of unsaturated flow. Multiphase flow models also can
describe the flow of dense nonaqueous-phase liquids such as chlorinated
solvents, which move in a distinct mass separate from the ground water.
The phenomena controlling multiphase flow are not as well understood and
are much more difficult to represent mathematically than are those for water flow
in the saturated zone because they involve complex interactions among solids,
water, air, and nonaqueous phases. The accuracy of multiphase flow models for
water direction and velocity is limited by the large number of required transport
parameters. Furthermore, the modeling community has not yet reached a
consensus as to which modeling approach is most valid. Despite these
limitations, multiphase flow modeling provides a framework for conceptualizing
the movement of fluids in the subsurface and for making order-of-magnitude
estimates of fluid movement.
If the direction and velocity of fluid flows can be predicted, modeling
contaminant transport with multiphase flow models is similar to that for saturated
flow. However, contaminant transport is complicated by the multiple phases,
which introduce heterogeneities that affect dispersion and sorption.
Geochemical Models. At many contaminated sites, the contaminants are
subject to a significant number of different chemical reactions.

Geochemical models describe how a contaminant's chemical speciation is

controlled by the thermodynamics of the many types of chemical reactions that
may occur in the subsurface. Today, geochemical models are used primarily to
understand the fate of inorganic compounds. For example, these models can be
used to analyze the series of reactions that influence whether a particular metal
will precipitate. Geochemical models also can aid in determining the availability
(solubility) of nutrients and trace metals required for microbial metabolism.
Although they are valuable, geochemical models have had limited use for
assessing bioremediation. There are three reasons for the relatively low level of
use. First, existing commercial applications of bioremediation have focused on
aerobic biodegradation of petroleum hydrocarbons, a situation for which
inorganic geochemistry usually is not a crucial factor. As bioremediation is
applied to more complex sites, especially those containing contamination by
heavy metals, the need for geochemical modeling will increase. Second,
traditional geochemical models are founded on the principle of equilibrium
conditions—in other words, all possible reactions are assumed to occur to their
maximum possible extent. The equilibrium assumption typically is not valid for
bioremediation because the key reactions are almost always controlled by
kinetics—the rate at which a reaction moves toward equilibrium. Third,
traditional geochemical models are very complicated and expensive to use, even
when they are not connected to transport modeling. Therefore, their use has been
limited to evaluating possible changes in subsurface chemicals.
Biological Reaction Rate Models. Biological reaction rate models represent
how quickly the microorganisms transform contaminants. They are useful for
evaluating bioremediation systems because the rate at which the microbes work is
the key factor influencing how much time the cleanup will take.
The rate of biodegradation depends on the amount of active biomass
present; the concentrations of contaminants, electron acceptors, and other "food"
sources for the bacteria; and certain parameters that describe transport rates of
key chemicals to the bacteria and rates of enzyme-catalyzed reactions. All of this
information can be packaged into a rate expression of the form:
in which qmax describes the reaction rate per unit amount of biomass for
optimal conditions, X is the amount of active biomass, f(S1, S2 , .…) is a
mathematical function that describes how substrate transport

and concentration reduce the rate from the optimal rate, and S1, S2,. … represent
different substrates that participate in the reaction. The value of X is not
necessarily constant; it can change with time and location. Keeping track of X is
part of the model. The f(S1, S2,. …) function can range from very simple, such as
the concentration of just one substrate, to complex sets of equations involving
several substrates and rate parameters. Determination of appropriate rate
expressions and parameter values for those expressions is an active research area.
Combining Models. In many cases, evaluating bioremediation involves
combining two or more of the model types. For example, in situ bioremediation
of a chlorinated solvent may require a multiphase flow model coupled to a
sophisticated biodegradation rate model. The multiphase flow model tracks the
movements of the water and the solvent; once the flows are known, a transport
model uses a biodegradation rate model as a sink term.
Biodegradation rate models are most easily combined with flow models
when one rate-limiting material can be identified. The rate-limiting material often
is the primary electron donor or electron acceptor. For example, the
biodegradation rate of petroleum hydrocarbons often can be modeled with
dissolved oxygen as the rate-limiting substance. In several successful modeling
studies, overall biodegradation rates could be modeled by the rate at which
oxygen entered the bioremediation zone.
The major simplification achieved by assuming rate limitation solely by
oxygen should not be considered a general rule. It can be appropriate for
biodegradation of petroleum hydrocarbons (a process that is especially sensitive
to low oxygen concentrations) when the input rate for dissolved oxygen is low
compared to the amount of hydrocarbon present and the site is large. Because
these conditions are not true in many other situations, biodegradation rate
modeling may require different and more sophisticated approaches.
Except when the biodegradation or geochemical models are very simple,
coupling them with flow models requires more than an extension of the existing
contaminant transport models used for conservative tracers. Considerable
attention must be given to proper model formulation and to efficient and accurate
solution techniques. Otherwise, costs and computer time will be excessive.
How to Use Models

Models provide a framework for organizing information about

contaminated sites. They increase understanding of contaminant behavior by

requiring the model user to confront details such as the mass of contaminants,
their chemical properties, and their dynamic interactions with site
hydrogeochemical characteristics. When this required information is available
and integrated into the proper model, modeling predictions become useful tools
for managing field sites and evaluating bioremediation.
Models can be useful for evaluating in situ bioremediation in two ways. One
approach is to see if a model representing only abiotic mechanisms can or cannot
account for all of the contaminant loss. A second approach goes a step further and
evaluates if "reasonable" estimates of microbial processes, quantified through the
model, can explain contaminant losses (see Box 4-4). This second approach
requires detailed knowledge of rate coefficients describing how quickly the
microbes degrade the contaminant, in addition to parameters describing transport
and other abiotic phenomena.
Mass Losses. The first modeling approach requires analyzing whether
abiotic mechanisms (for example, dilution, transport, and volatilization) can
explain all of the losses of the contaminant mass. The approach recognizes that
biodegradation rate models often have greater uncertainty that do models of
abiotic processes. The uncertainty can be caused by poor understanding of the
biochemical reactions, difficulty estimating parameters, and inadequate site
characterization. Eliminating biological reactions from the model avoids this
uncertainty.
When the model of abiotic mass losses shows that some contaminant mass
remains after all the abiotic sinks are considered, there are two possible
explanations: (1) biodegradation processes are implicated as the sink for the
"missing" mass, or (2) the modeling parameters were improperly selected, have
led to inaccurate predictions, and are therefore misleading the modeler. Because
judgments about microbiological involvement in contaminant loss may be
contingent upon the selection of parameters used to describe abiotic losses, a
modeler must be vigilant—constantly scrutinizing the validity of decisions and
parameters that affect the modeling results. Adjustments in modeling parameters
can lead to vastly different predictions; therefore, it is prudent to give credence to
evidence for bioremediation only when the modelers have a high degree of
confidence in their results and when discrepancies between actual and modeled
contaminant behavior are unambiguous. Conclusions about effective
bioremediation should only be drawn when concentrations of contaminants found
in field sites are not simply lower but significantly lower than would be

BOX 4-4 PROVING INTRINSIC BIOREMEDIATION OF A SPILL

AT A NATURAL GAS MANUFACTURING PLANT—
NORTHERN MICHIGAN
At a plant in northern Michigan, waste products from natural gas
manufacturing leaked from a disposal pit into the surrounding ground
water. Having installed wells around the plant to prevent off-site migration
of contaminated water, the company in charge of the facility chose intrinsic
bioremediation to cleanup the contaminants (primarily benzene, toluene,
and xylene, or BTX). In demonstrating the effectiveness of bioremediation,
the company provided evidence that meets the three criteria discussed in
this report:
1. Documented loss of contaminants: The company began its extensive

site-monitoring program to follow the effectiveness of intrinsic
bioremediation in 1987. Since that time the benzene concentration
has dropped by approximately 90 percent and the contaminant plume
has shrunk considerably.
to degrade the contaminants: The company performed a series of lab
tests with soil cores retrieved from the field showing that the site's
native microbes could degrade BTX at a high rate—5 to 10 percent
per day—if supplied with adequate oxygen (1 to 2 ppm or more).
3. Evidence showing that biodegradation potential is realized in the
field: The company used a computer-based model, BIOPLUME II, to
demonstrate that the rate of contaminant loss that one would predict
if bioremediation were occurring closely matched the actual
contaminant loss rate in the field. In 1987 the company measured the
BTX and dissolved oxygen levels at various points in the plume.
These values were input into BIOPLUME II to predict how they
should change with time if bioremediation were occurring. The field
measurements of both the contaminant concentrations and the
dissolved oxygen levels taken since 1987 closely match the model's
predictions. In addition, the biodegradation rate predicted by the
model closely matches the rate measured in the field.
Monitoring at this site is still ongoing to demonstrate the long-term
effectiveness of intrinsic bioremediation.
References
Chiang, C. Y., J. P. Salanitro, E. Y. Chai, J. D. Colthart, and C. L. Klein. 1989. Aerobic
biodegradation of benzene, toluene, and xylene in a sandy aquifer: data analysis and
computer modeling. Groundwater 27(6):823-834.

expected from predictions based on abiotic processes. Thus, this approach

works well when biodegradation is the dominant sink and when the abiotic
processes are well characterized. Uncertainty in modeling the abiotic processes
makes this approach unreliable when biodegradation is not the dominant removal
mechanism.
Direct Modeling. When reasonable estimates of biological processes and
parameters are available, directly modeling the biodegradation process is the
superior modeling strategy. These estimates can be obtained from the scientific
literature, past experience with similar circumstances, laboratory experiments, or
field-scale pilot studies, depending on the site conditions and biodegradation
reactions.
One approach is to use the model to answer the question, "Does our best
representation of the biodegradation rates, when combined with the
simultaneously occurring abiotic rates, support the conclusion that biological
reactions are responsible for observed changes in contaminant levels or other
relevant observations?" If the answer is "yes," modeling provides a much greater
measure of confidence that observations supporting biodegradation are not
artifacts.
A second approach is to use direct modeling to predict the contaminant's
concentration at unsampled locations or to predict the future concentration. The
model then identifies sample locations and times that should yield particularly
definitive measurements. Subsequent sampling, if consistent with model
predictions, confirms the analyst's understanding of what is occurring in the
subsurface. Lack of agreement between model predictions and actual
developments forces a reevaluation of the model and improves understanding of
the site and the parameter values.
In some cases, direct modeling must involve highly sophisticated computer
codes that take into account the three-dimensional nature of the site,
heterogeneities, and complex reactions. These models are expensive to formulate
and run, but they are essential tools for investigators who require a detailed
description of what is happening at a site. Currently, these types of models are
viewed primarily as research tools appropriate for highly monitored research,
demonstration, or pilot sites.
In many practical applications, direct modeling can be greatly simplified by
eliminating all but the most essential phenomena. A good strategy is to compare
expected rates of all phenomena that might affect the bioremediation. For
example, the expected rate of contaminant loss due to biodegradation can be
compared with the expected contaminant loss rate due to volatilization.
Normally, a few of the possible phenomena will have expected rates much
greater

than those of the other phenomena, and the model can consider only the
phenomena having relatively high rates. If the biodegradation rate is high enough
that it should remain in the model, the model provides prima facie evidence that
bioremediation is working. Solution of the complete model can verify the
evidence.
Limitations of Models
Although a powerful tool, modeling has its shortcomings. One shortcoming
is that a model's validity must be established on a site-by-site basis, because no
"off-the-shelf" models are available for evaluating bioremediation on a routine
basis. Although a drawback in terms of time and cost, model validation may be a
net advantage because it results in a more complete understanding of the site.
Another limitation is that determining each of the many modeling parameters
(such as hydraulic conductivity, retardation factors, and biodegradation rate
parameters) may be as demanding and expensive as making the measurements
for other types of verification criteria. Thus, a trade-off may exist between better
modeling and more field measurements.
Despite its limitations, modeling should be a routinely used tool for
understanding the dynamic changes that occur in field sites during
bioremediation. Although the complexity and type of model can vary, modeling
is a valuable tool for linking conceptual understanding of the bioremediation
process with field observations and for giving weight to a limited set of data.
Even if site complexities preclude assembling a model that provides valid
quantitative predictions, models are valuable management tools because they
integrate many types of information relevant to the fate of contaminants.
LIMITATIONS INHERENT IN EVALUATING IN SITU

BIOREMEDIATION
Because the subsurface is complex and incompletely accessible, knowledge
of the fate of ground water contaminants always will be limited. This situation is
intensified for in situ remediation technologies of any type, because frequently
the amount, location, and type of contamination are unknown. Without knowing
the starting point for a remediation, defining the finishing point is difficult. Errors
in measurements, artifacts imposed by extrapolating lab results to the field, and
an inherent shortage of data further complicate the evaluation and create
uncertainty about the performance of a remediation process. For example, in
analyzing chemical concentrations in ground water, a large number of samples
from spatially different locations

may be gathered. Even assuming the laboratory results are completely error free,
uncertainty arises from extrapolating these point samples in an attempt to portray a
complete picture of how the water's chemical composition varies in space.
Because evaluation of bioremediation requires integrating concepts and
tools from very different disciplines, efforts to synthesize information from these
different disciplines can create problems. For example, microbiologists and
hydrogeologists use space and temporal scales that seldom match. The seconds
and micrometers characteristic of microbial processes are very much smaller than
the months and kilometers typical of hydrogeological descriptions of landscape
processes. Thus, the hydrologic data describing large-scale water flow do not
always meet a microbiologist's needs for understanding the small-scale
mechanisms that control microbial activity. For instance, models efficient for the
typical space scale of water movement (i.e., meters to kilometers) obliterate all of
the details of microbial reactions, which occur in distances of micrometers to
centimeters.
A prime example of the problem of trying to synthesize different scales is
illustrated by the problems encountered when trying to document major increases
in biomass during in situ bioremediation. Microorganisms often are highly
localized near their food sources. This localization makes it difficult to "find" the
organisms when only a few samples can be taken. Microbial numbers,
biodegradation rate estimates, or biodegradation potentials can vary
tremendously, depending on whether the sample was from a location of high
microbial activity or from a nearby location with low activity. Microbiological
variability occurs on a small scale compared to the scale represented by field
samples. Consequently, uncertainty in microbiological parameters always is a
risk.
Three strategies can help minimize uncertainty and should play important
roles in evaluating bioremediation: (1) increasing the number of samples, (2)
using models so that important variables are properly weighted and variables with
little influence are eliminated, and (3) compensating for uncertainties by building
safety factors into the design of engineering systems. Investigators can trade-off
these three strategies. For example, if gathering a large number of samples or
using sophisticated models is not possible, larger safety factors can cover the
resulting uncertainty. At small field research sites designed to investigate
bioremediation of contaminants not yet treated on a commercial scale, a large
number of samples and complex models may be possible—and necessary—to
draw detailed conclusions from the research results. On the other hand, at large
commercial sites, a similarly high density of samples may be cost prohibitive, and
it may

be more appropriate to rely on larger safety factors to account for the greater
uncertainties.
Uncertainties in evaluating bioremediation can be minimized but not
eliminated. Investigators cannot fully understand the details of whether and how
bioremediation is occurring at a site. The goal in evaluating in situ bioremediation
is to assess whether the weight of evidence from tests such as those described
above documents a convincing case for successful bioremediation.

FUTURE PROSPECTS FOR BIOREMEDIATION 91
5
Future Prospects for Bioremediation
In preparing this report the National Research Council's Committee on In

Situ Bioremediation sought to communicate the scientific and technological bases
for bioremediation. As the report has explained, the principle underlying
bioremediation is that microorganisms (mainly bacteria) can be used to destroy
hazardous contaminants or transform them into less harmful forms.
Microorganisms are capable of performing almost any detoxification reaction.
Nevertheless, the commercial practice of bioremediation today focuses primarily
on cleaning up petroleum hydrocarbons. The full potential of bioremediation to
treat a wide range of compounds cannot be realized as long as its use is clouded
by controversy over what it does and how well it works. By providing guidance
on how to evaluate bioremediation, the committee hopes this report will eliminate
the mystery that shrouds this highly multidisciplinary technology and pave the
way for further technological advances.
This chapter summarizes new research advances that the committee foresees
as expanding the future capabilities of bioremediation. It recommends steps that
will improve the ability to evaluate bioremediation technologies objectively,
whether the technologies are new or established.

NEW FRONTIERS IN BIOREMEDIATION

Bioremediation integrates the tools of many disciplines. As each of the
disciplines advances and as new cleanup needs arise, opportunities for new
bioremediation techniques will emerge.
Until now, three types of limitations have restricted the use of
bioremediation to cleanup contaminants other than petroleum hydrocarbons:
inadequate understanding of how microbes behave in the field, difficulty
supplying the microbes with stimulating materials, and problems with ensuring
adequate contact between the microbes and the contaminant. Consequently, only a
few of the myriad microbial processes that could be used in bioremediation are
applied in practice. Recent advances in science and engineering show promise for
overcoming these limitations, as illustrated by the following examples:
• Understanding microbial processes. As novel biotransformations

become better understood at ecological, biochemical, and genetic levels,
new strategies will become available for bioremediation. A recent
example is microbial dechlorination of polychlorinated biphenyls
(PCBs), which is being investigated by a group of researchers from
academia and industry. The researchers, studying PCBs in Hudson River
sediments, have documented that anaerobic microbes in the sediments
can transform highly chlorinated PCBs to lightly chlorinated PCBs,
which can be degraded completely by aerobic microbes (see Box 4-3).
This research may become the basis for commercial bioremediation of
PCBs—compounds once thought to be undegradable. Similar advances
are being made for the dechlorination of chlorinated solvents, also once
believed to resist biodegradation.
Advances in understanding microorganisms may also improve
bioremediation's effectiveness in meeting cleanup standards. As
explained in Chapter 2, uptake and metabolism of organic compounds
sometimes stop at concentrations above cleanup standards. Research on
bioaugmentation and direct control of the cell's genetic capability and/or
regulation is very active today and may lead to methods to overcome
such microbiological limitations.
• Supplying stimulating materials. Innovative engineering techniques
for supplying materials that stimulate microorganisms are pushing the
boundaries of bioremediation. For instance, the recent innovation of gas
sparging has substantially expanded capabilities for aerobically
degrading petroleum hydrocarbons. Research is ongoing into optimizing
ways to supply materials other than oxygen. Such research

will pave the way for emerging bioremediation applications, such as

degradation of PCBs and chlorinated solvents and demobilization of
metals, which are not necessarily controlled by oxygen.
• Promoting contact between contaminants and microbes. Research is
under way into engineering advances to increase the availability of
contaminants to microbes—advances that, if successfully applied, would
increase bioremediation's efficiency. New techniques for promoting
contaminant transport to the organisms include high-pressure fracturing
of the subsurface matrix, solubilization of the contaminants by injecting
heat (via steam, hot water, or hot air), and, perhaps, addition of
surfactants. Discovery of improved methods for dispersing the
microorganisms may also enhance microbial contact with the
contaminants and lead to more effective bioremediation.
THE INCREASING IMPORTANCE OF EVALUATING

BIOREMEDIATION
As new bioremediation techniques are brought from the lab into commercial
practice, the importance of sound methods for evaluating bioremediation will
increase. The Committee on In Situ Bioremediation has recommended a three-
part strategy for ''proving" that bioremediation has worked in the field. As
explained in Chapter 4, the three central parts of this strategy are (1) documented
loss of contaminants from the site, (2) laboratory assays showing that
microorganisms from site samples have the potential to transform the
contaminants, and (3) one or more pieces of information showing that the
biodegradation potential is actually realized in the field. The main goal of this
strategy is to show that biodegradation reactions that are theoretically possible are
actually occurring in the field, at fast enough rates and in appropriate locations to
ensure that cleanup goals are met.
While the three-part strategy provides a general framework for evaluating
bioremediation, the level of detail with which it should be applied depends on the
interests of those involved with the bioremediation. Each party involved must
realize that "success" may mean different things to the different parties.
Regulators are primarily concerned that legislated standards are achieved, clients
emphasize attaining cost-effective goals, and vendors have a vested interest in
demonstrating that their technology is effective and predictable. Clear
communications about everyone's goals and negotiations about specific criteria to
meet the different goals are critical to the project's perceived success and must
occur in advance of its implementation.
The current knowledge base is sufficient to allow implementation

of the three-part strategy. However, the specific experimental protocols for

carrying out the strategy need to be developed. In addition, further research and
better education of those involved in bioremediation will improve the ability to
implement the strategy as well as understanding of the fundamentals of
bioremediation.
Recommended Steps in Research

The committee recommends research in the following areas to improve
evaluations of bioremediation:
• Evaluation protocols. Protocols need to be developed for putting the

three-part evaluation strategy into practice. Consideration should be
given to evaluating a range of chemical contaminants (including
petroleum hydrocarbons, chlorinated solvents, PCBs, and metals) and
site characteristics (such as shallow and deep aquifers and sites with high
and low heterogeneity). These protocols should be field tested through
co-ordinated efforts involving government, industry, and academia and
should be subject to scientific and peer review.
• Innovative site characterization techniques. Rapid, reliable, and
inexpensive site characterization techniques would have a significant
impact on the ease of evaluating bioremediation. Examples of relevant
site measurements include distribution of hydraulic conductivities,
contaminant concentrations associated with solid or other nonaqueous
phases, native biodegradation potential, and abundance of different
microbial populations. Techniques to measure physicochemical
characteristics in situ are being developed and could revolutionize the
capability to do field assessments. Methods adapted from molecular
biology seem especially promising for augmenting current techniques
for assaying biodegradation potential and microbial populations.
Gathering more and better characterization data would diminish
uncertainties and reduce the needs for overdesign via safety factors.
• Improved models. Improvements in mathematical models are essential
because models link understanding of chemical, physical, and biological
phenomena. One particularly promising advancement is the use of
modeling as a key part or improved means for on-site management,
which requires an appreciation of the dynamic interactions among the
many phenomena. As field sampling becomes more rapid and accurate,
on-site decisions will be limited more by the ability to understand the
dynamic interactions than by turnover times between sampling and
analysis.

Recommended Steps in Education

Steps need to be taken to educate all components of society about what
bioremediation is and what it can and cannot do. Especially important is
improved education of the people who are in direct decision-making positions.
The committee recommends three types of education:
• Training courses that selectively extend the knowledge bases of the

technical personnel currently dealing with the uses or potential uses
of in situ bioremediation. This step explicitly recognizes that
practitioners and regulators who already are dealing with complicated
applications of bioremediation need immediate education about
technical areas outside their normal expertise.
• Formal education programs that integrate the principles and
practices for the next generation of technical personnel. This step
explicitly recognizes the need to educate a new generation of technical
personnel with far more interdisciplinary training than is currently
available in most programs.
• Means for effective transfer of information among the different
stakeholders involved in a project. Effective transfer requires that all
types of stakeholders participate, that all are invested in achieving a
common product (e.g., a design, a report, or an evaluation procedure),
and that sufficient time is allocated for sharing perceptions and achieving
the product. This step may involve more time and more intensive
interactions than have been the norm in the past.
In summary, in situ bioremediation is a technology whose full potential has

not yet been realized. As the limitations of conventional ground water and soil
cleanup technologies become more apparent, research into alternative cleanup
technologies will intensify. Bioremediation is an especially attractive alternative
because it is potentially less costly than conventional cleanup methods, it shows
promise for reaching cleanup goals more quickly, and it results in less transfer of
contaminants to other media. However, bioremediation presents a unique
technological challenge. The combination of the intricacies of microbial
processes and the physical challenge of monitoring both microorganisms and
contaminants in the subsurface makes bioremediation difficult to understand—
and makes some regulators and clients hesitant to trust it as an appropriate
cleanup strategy. The inherent complexity involved in performing bioremediation
in situ means that special attention must be given to evaluating the success of a
project. Whether a bioremediation project is intrinsic—

relying on the natural properties of the subsurface—or engineered—augmenting

subsurface properties to promote microbial activities—the importance of a sound
strategy for evaluating bioremediation will increase in the future as the search for
improved cleanup technologies accelerates.

Background Papers


A REGULATOR'S PERSPECTIVE ON IN SITU BIOREMEDIATION 99
A Regulator's Perspective on In Situ

Bioremediation
John M. Shauver
Michigan Department of Natural Resources
Lansing, Michigan
SUMMARY
Bioremediation, like any technology applied to clean up a contaminated site,
must first be approved by government regulators who ultimately must agree that
the technology has a reasonable chance to reduce the contaminant(s) at the site to
acceptable levels. This paper describes the information that regulators need to
make their decision. Basically, this information comprises descriptions of the
site, the specific cleanup process, and the overall approach to site cleanup. The
paper also answers the questions of who, what, when, where, and how in the
context of bioremediation on the basis of my 24 years of experience as a
regulator.
INTRODUCTION
During the past 20 years, various companies and individuals have developed
or claim to have developed biological treatment processes that could cleanup
various wastes generated by human activities. These wastes include
polychlorinated biphenyls (PCBs), crude oil, refined crude oil products, crude oil
wastes, and DDT, to name a few. One problem that the proponents of such
treatment technology face is state and federal regulations. It is often hard for the
regulated community to understand what is required to ensure that the regulator
will approve a proposed treatment process.

This paper describes what a regulator looks for in a proposal to cleanup

(remediate) a site to legal standards. The guidance provided here is a
condensation of the requirements of the many statutes and regulations used by the
Michigan Department of Natural Resources. The paper reflects my view, after 24
years as a regulator, of what information needs to be routinely provided to
evaluate a cleanup technology before it is applied to a particular site. Complex
sites with unique or unusual features may have to be characterized in greater
detail before a cleanup technology can be chosen. Also, the regulated community
(potentially responsible parties) must realize that the cleanup process itself is but
one facet of the overall site cleanup. To gain approval for implementation of a
cleanup process, the responsible party should supply information that includes:
• a description in three dimensions of the site and of the type and extent of
contamination,
• a detailed description of the cleanup process(es) to be applied to the site,
and
• a detailed description of the approach to overall site cleanup.
SITE DESCRIPTION
The site description should specifically identify the types and amounts of
chemical(s) released to the soil and ground water and other phases of the site
environment. The description should also include estimates of the rate of
movement of the contaminants through the various phases of the environment and
of where they are likely to end up. The regulator's response to a given situation
depends strongly on the rate of transport and the likely fate of the contaminants.
The site is the three-dimensional area contaminated by the chemicals that
have been released. The site is not limited to legal property boundaries. In fact, it
usually involves more than one property owner, and the owners may not all be
responsible for the contamination. The site description should also include the
vertical, horizontal, and lateral extent of contamination, which includes:
• soil type(s), permeability, porosity of the soils and/or aquifer, and

concentrations of contaminants in soil;
• if appropriate, depth to ground water, rate and direction of flow,
concentrations of contaminants in ground water, and concentrations of
naturally occurring or other compounds (inorganic or organic) that may
interfere with the treatment process;
• if appropriate, concentrations of contaminants in the air, prevailing wind
direction, and nearest human receptors; and

• concentrations of contaminants in surface waters and sediments.
In any site description the regulator will place great emphasis on identifying
the location of the source(s) of contamination. Removal of these sources, or hot
spots (identified by an adequate site investigation), is the most effective way to
limit migration of chemicals off site. In addition, elimination of the source of the
contamination as early as possible is one of the most cost-effective ways to limit
future cleanup costs.
A site description should also describe the process that caused the release.
This is important because the regulator is required to determine the full extent of
the type of contamination at the site. If the material released is gasoline, for
example, it is very important to know whether it is leaded or unleaded and
whether it came from a hole in a tank; an overfilled tank; or faulty pipes, valves,
or other fittings. If the release is described as crude oil, it is important to know if
brine, condensate, or other materials are present as well. The description of the
cause of the release allows the regulator to identify its source and thus the most
highly contaminated areas of the site.
PROCESS DESCRIPTION
The responsible party should provide a detailed description of the treatment
process to be used. The engineer who is accustomed to describing an activated
carbon process should provide the same level of detail for a biological process.
The description should show how the process chosen will contain, destroy, or
remove the contaminants to meet legal standards. If biological treatment is
chosen, the regulator must be given data that show the ability of the organisms
present in or added to the contaminated area to safely and effectively treat the
chemical(s) on the site.
When living organisms are proposed to cleanup a site, the regulator expects
to see a detailed description of the organisms' requirements for oxygen, nutrients,
temperature, moisture, and pH. We must be sure the organism will thrive long
enough to treat the chemicals to legal cleanup standards. In addition, if an
anaerobic treatment scenario (such as one using iron or sulfur) is proposed, the
regulator needs to know that native microbes are capable of the proposed
metabolism and that ambient or added nutrients will be available in amounts
likely to allow effective treatment but not likely to cause rapid plugging of the
delivery wells and/or the soils.

We must be able to determine that the use of bacteria in the soils and ground
water (if unsuccessful) will not prevent other treatment technologies from being
applied. Use of organisms without adequate information or controls in the past
has resulted in severe plugging problems in ground water monitoring wells and/
or the aquifer itself. Such loss of permeability not only prevents delivery of the
nutrients and oxygen necessary to sustain biological activity to cleanup the soils
or aquifer but may seriously hamper use of other technologies.
OVERALL SITE CLEANUP DESCRIPTION

A very important part of the description of the overall approach to site
cleanup is the method(s) to be used to prevent movement of the contaminants
farther off site through the soil or to or through the ground water or other
medium. Containment to prevent further spread of the chemicals is as important
in the regulator's mind as any other part of the cleanup. The regulator needs a
complete description of the steps to be taken to prevent further movement of the
chemicals through the soil, air, ground water, or surface water.
For example, contaminated ground water may be moving down-gradient at
15 cm per day. Purge and capture wells would have to be installed to pump this
contaminated ground water back upgradient to the treatment system to prevent
further movement of the contaminants off site. If the water is discharged to the
ground surface via an infiltration bed, and if it contains volatile organic chemicals
(VOCs) that would be released, the responsible party needs to demonstrate
adequate control of VOC discharge to the air.
The description also should cover equipment necessary to achieve the
cleanup. With biological treatment systems, equipment may be needed for
adjustment of the pH of the ground water, removal of iron or other interfering
substances before treatment, oxygen/air delivery or oxygen reduction, and
identification and monitoring of tracers and nutrients added. For example, if the
proposal is to use aerobic bacterial decomposition of the contaminant(s) and the
contamination exists to a depth of 15 m below ground water surface in soils with a
permeability of 10-7 cm/s, the regulator will be interested in how the responsible
party intends to deliver oxygen or air and related nutrients to the organisms.
Also necessary is a description of the monitoring procedures to be used to
show that the cleanup system is operating properly. When using biological
systems, the responsible party must show that the organisms are, in fact, doing
the job. For example, if an aerobic process is used, the level of oxygen in and
around the plume of

contamination in the ground water will have to be monitored to ensure that the
organisms have sufficient oxygen to decompose the chemicals in the ground
water. This type of monitoring may be in addition to or in place of simply
monitoring for the contaminant itself. In addition, if nutrients are added, they may
also be contaminants and require monitoring. Nitrate, for example, is a chemical
of concern that may have to be added to a biological treatment system as a
nutrient or may be proposed as an electron acceptor in an anaerobic treatment
process. In Michigan the drinking water supplies may not contain more than 5
mg/1 of nitrate. Therefore, if nitrate is used, the regulatory agency will require
that it be monitored in addition to other monitoring requirements.
CONCLUSION
A regulator looks for the data necessary to determine that a proposed
treatment technology, if properly installed and operated, will reduce the
contaminant concentrations in the soil and water to legally mandated limits. In
this sense the use of biological treatment systems calls for the same level of
investigation, demonstration of effectiveness, and monitoring as any
conventional system.

AN INDUSTRY'S PERSPECTIVE ON INTRINSIC BIOREMEDIATION 104
An Industry's Perspective on Intrinsic

Bioremediation
Joseph P. Salanitro
Shell Development Company
Houston, Texas
SUMMARY
Laboratory and field evidence is now sufficient to demonstrate that soil
microorganisms in aquifers are responsible for a significant portion of the
attenuation of aromatic compounds—benzene, toluene, ethylbenzene, and
xylenes (BTEX)—from fuel spills to the subsurface environment. Most subsoils
contain indigenous microbes that can biodegrade low levels of BTEX (ppb to low
ppm), given enough dissolved oxygen in the ground water. With adequate site
characterization, analysis, and monitoring, this type of intrinsic bioremediation
can shrink plumes and control the migration of hydrocarbons. In situ
biodegradation processes, properly monitored, should be considered practical,
cost-effective alternatives for managing low-risk, hydrocarbon-contaminated
ground waters that are unlikely to affect drinking water wells.
PROBLEM IDENTIFICATION
Accidental releases of fuels from underground storage tanks over the past 10
to 20 years have been responsible for the presence of hydrocarbons, mainly
water-soluble aromatic compounds (benzene, toluene, ethylbenzene, and xylenes,
or BTEX), in aquifers. In most states, government agencies have required the
regulated industry to

restore ground water at such sites to drinking water (health) standards—for

example, 1 to 5 parts per billion (ppb) benzene (Marencik, 1991). Corrective
actions taken include removal of free product and contaminated soil, site
assessments (soil borings and monitoring wells), and determination of the extent
of contamination in subsoils and ground water. For a majority of the sites, the
ground water hydrocarbon (BTEX) levels are low, on the order of 100 to 1000
ppb. Higher levels are often associated with soil and ground water samples taken
near the spill area.
Technologies that have been used to control migration of hydrocarbon
plumes or to remediate subsurface soils include soil venting (vadose zone) and
sparging (saturated zone) and ground water extraction and treatment (pump and
treat) (Mackay and Cherry, 1989; Newman and Martinson, 1992). In addition to
these operations, extensive soil and ground water surveying must be done to
assess the extent of contamination and the effectiveness of the cleanup method.
Current estimates for site assessment and in situ or ex situ restoration of subsoils
and ground water to health standard criteria indicate that these operations may be
costly ($500,000 to $2 million per site) and not cost-effective and that they may
not achieve restoration within time periods of years or decades (Travis and Doty,
1990).
Many contaminated ground waters (e.g., at fuel service station sites) are in
shallow aquifer zones, are not used directly for human consumption, and do not
even affect downstream drinking water wells. Furthermore, good field evidence
indicates that plumes in these ground waters reach a stable condition in which
contaminants of concern (BTEX) are biodegraded at some rate by indigenous
hydrocarbon-utilizing soil bacteria. This type of unassisted in situ biodegradation
has been termed natural attenuation or intrinsic bioremediation.
Industry has been confronted with very large operating and cleanup costs for
subsoils and ground water in the restoration of underground fuel storage tank
sites to drinking water standards. Where thorough site characterization warrants
its use, intrinsic bioremediation offers a way to manage non-migrating or
shrinking BTEX plumes in low-risk aquifers that do not affect drinking water
wells. Evidence that this natural process is occurring has been obtained from
laboratory and field observations.
EVIDENCE FOR INTRINSIC BIOREMEDIATION OF

AROMATIC HYDROCARBONS IN
It is now widely recognized that the most significant factor in the time-
dependent decrease of BTEX compounds in aquifers is degradation

by soil microbes. Studies reported for laboratory slurry microcosms of subsoil

and ground water show that microbes in many soils inherently biodegrade
aromatic hydrocarbons at varying rates (5 to 50 percent per day) (Barker et al.,
1987; Chiang et al., 1989; Gilham et al., 1990; Hutchins et al., 1991;
Kemblowski et al., 1987; Major et al., 1988; and Thomas et al., 1990). These
biodecay rates are usually first order; they occur with low levels of hydrocarbon
(50 to 10,000 ppb); and they are rapid with adequate dissolved oxygen (e.g., 2 to 3
mg oxygen per milligram of hydrocarbon). Field estimates of hydrocarbon
biodegradation rates calculated from fate and transport models using data from
upstream and downstream monitoring wells have shown that plume BTEX
compounds usually decrease at rates of 0.5 to 1.5 percent per day (Barker et al.,
1987; Chiang et al., 1989; Kemblowski et al., 1987; Rifai et al., 1988; and Wilson
et al., 1991). Laboratory and field data suggest that in a well-studied sandy
aquifer a minimum, or threshold, level (`1 to 2 ppm) of dissolved oxygen may be
required to sustain hydrocarbon degradation (Chiang et al., 1989).
It should be emphasized that laboratory and field data have confirmed that
all BTEX compounds can be biodegraded under aerobic conditions (dissolved
oxygen in ground water) in aquifer subsoils in which oxygen is the terminal
electron acceptor. Soil microcosm experiments or enrichments of aquifer
material have shown that toluene and xylenes can be degraded by microbes under
iron-reducing, denitrifying, and sulfate-reducing (anaerobic or very low dissolved
oxygen) conditions when ferric ion (Fe3+), nitrate ion (NO3-), and sulfate ion
(SO42), respectively, serve as electron acceptors (Beller et al., 1992; Edwards et
al., 1992; Hutchins, 1991; Lovley et al., 1989; and Zeyer et al., 1986). Field
evidence is insufficient, however, to demonstrate that BTEX is biodegraded
under anaerobic conditions in an aquifer.
LEVELS OF INTRINSIC ATTENUATION IN GROUND WATER

Evidence from site characterization, ground water monitoring, and modeling
at field sites suggests that there may be two levels of intrinsic bioremediation.
Figure 1 shows these aspects of a plume in which one is stabilized (A) and the
other is reducing (B) in size and extent of contamination. In Figure 1A the
hydrogeological features indicate that ground water velocity (also BTEX and
dissolved oxygen) and recharge are slow because of low permeability of the
aquifer subsoil. Dissolved oxygen is low within the plume (e.g., <1 ppm). Oxygen
is detected in monitoring wells at the edges and is responsible

FIGURE 1 Levels of intrinsic bioremediation in aquifers.
for the biodegradation of low levels (ppb) of BTEX. Another indirect

indicator of soil microbial degradation in aquifers low in dissolved oxygen may
be the presence of dissolved ferrous ion (Fe2+) above background well levels. It is
known that various ferric oxides in soil can be used (as electron acceptors) by
anaerobic iron-reducing bacteria to completely metabolize some aromatic
compounds, such as toluene and phenol (Lovley et al., 1989). Therefore, when
dissolved oxygen is low, ferric iron may substitute for oxygen, and this
biodegradation process may result in elevated concentrations of ferrous ion in
ground water.
At the next level of intrinsic bioremediation, plumes noticeably shrink over
time, with significant decreases in shape and extent (Figure 1B). This type of
plume behavior is observed in aquifers that usually are more permeable (e.g.,
sandy subsoils), that exhibit higher ground water velocities, and that are higher in
dissolved oxygen (higher aquifer reaeration rate) in many monitoring wells.
Published examples of plumes undergoing significant intrinsic attenuation of
BTEX are those at the Borden (Barker et al., 1987), Traverse City (Rifai et al.,
1988; Wilson et al., 1991), and Michigan gas plant (Chiang et al., 1989) sites.
Monitoring wells at the periphery show significantly higher dissolved oxygen
(e.g., ` 1 ppm) and lower BTEX concentrations, which are consistent with a
predominantly biodegradation-driven mass reduction in the aquifer. Examination
of monitoring well BTEX levels within the flow path of upstream and downstream
segments may also match the biodecay rates (about 1 percent per day) calculated
from fate and transport models for BTEX and dissolved oxygen

(e.g., BIOPLUME II, Rifai et al., 1988). These plumes may initially shrink
(narrow) in the longitudinal direction because the high infiltration rate of oxygen
continues to enhance degradation of hydrocarbons to low concentrations at the
edges. Continued monitoring also indicates that because of the higher dissolved
oxygen, more BTEX is degraded and the plume may recede closer to the
hydrocarbon source. It should be emphasized that the degree to which these
reductions in plume BTEX occur depends on the removal of the free-phase and
sorbed hydrocarbons from the contaminated zones. For example, a fluctuating
water table could continue to flush more BTEX into the plume from the source
area. Removal and management of the contaminant source, therefore, are
important prerequisites for successfully implementing intrinsic bioremediation at
field sites.
FUTURE DIRECTIONS
Laboratory research and field research have contributed to our understanding
of intrinsic bioremediation of BTEX in aquifers as a viable option for managing
and controlling hydrocarbon plumes. Research in several areas, however, could
enhance the validity and overall regulatory acceptability of the plume
containment process. For example, important factors for understanding
contaminant behavior and predicting the time for remediation may include (1) a
better understanding of aquifer parameters (e.g., recharge and water table
fluctuations); (2) tools for quantifying subsoil sources of hydrocarbons and their
potential for transport into ground water; and (3) user-friendly ground water
models that use monitoring well, hydrogeological, and soil microbiology data to
predict the transport and fate of contaminants. Geochemical and biological
indicators of in situ biodegradation in addition to BTEX and dissolved oxygen,
such as the formation of carbon dioxide and other microbial metabolites as well
as ferrous ion, may also help verify intrinsic biodegradation processes in
aquifers. Information on the limits of degradation of soil contaminants (e.g.,
optimum BTEX and dissolved oxygen concentrations and supplemental nutrient
effects) and on the widespread occurrence of BTEX degraders in aquifers would
also improve our understanding of plume management. Finally, it is important
that demonstrated in situ biodegradation gain acceptance by the regulatory
authorities and that intrinsic bioremediation be considered a valid and cost-
effective means of controlling pollutant migration in low-risk aquifers.
Biodegradation in aquifers will continue to play a major role in the management
of low levels of soluble hydrocarbons from fuel spills to the subsurface.

REFERENCES
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sulfate-reducing conditions and the influence of iron on the process. Applied and
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Microbiology 52:944-947.

BIOREMEDIATION FROM AN ECOLOGICAL PERSPECTIVE 110
Bioremediation from an Ecological

Perspective
James M. Tiedje
Center for Microbial Ecology
Michigan State University
East Lansing, Michigan
SUMMARY
The ecological approach to bioremediation is distinctly different from the
traditional engineering approach: it focuses on such principles as microbial
natural selection rather than on mass balances of pollutants. Questions derived
from certain basic ecological principles, including specificity and diversity, can
serve as key guides in determining the feasibility of bioremediation at a particular
site. Similarly, certain kinds of evidence in the biological record, such as numbers
of organisms, are strongly indicative of successful bioremediation. A shift in
paradigm—emphasizing the ecological principles governing biodegradation
instead of contaminant mass balances—would greatly advance the understanding
of bioremediation.
INTRODUCTION
I suggest that there are at least two conceptual approaches to hazardous
waste bioremediation. In the dominant approach, derived from engineering, mass
balance and stirred tank reactor philosophy dominate. An alternative, or
ecological, approach focuses on such principles as microbial natural selection and
niche fitness characterization. Reliance on the engineering approach has brought
us to an impasse—namely, that nature is not a stirred tank reactor, and thus

the mass balance and predictive models of such systems are often inadequate or
too expensive. In ecology, however, one recognizes from the beginning that
nature is heterogeneous; to understand nature, one focuses on key principles
governing the behavior of populations and does not attempt to achieve mass
balances. Thus, I suggest that we consider a shift in paradigm—to consider the
important ecological principles governing biodegradation and reduce the
emphasis on achieving a mass balance for the pollutant.
This paper emphasizes the ecological approach and key questions related to
it. The differences in the philosophies underlying the ecological and engineering
approaches are substantial. As details of both approaches are developed, some of
the underlying factors may merge into the same issues. Nonetheless, the emphasis
in the ecological approach is not on quantification of pollutants but on whether
principles are met, since it is known that biological communities respond
according to these principles.
The first part of this paper reviews basic ecological principles important to
the evaluation and success of in situ bioremediation. The second part converts
these principles into key questions about the feasibility of bioremediation for a
particular site. Finally, the paper outlines ways to determine whether the
ecological principles, especially the principle of natural selection, are met.
BASIC PRINCIPLES
Specificity
An ecological approach recognizes a key principle in biology—specificity.
Specificity provides the fitness advantage in a niche. In terms of pollutant
degradation, this means that organisms are relatively specific for particular
substrates (chemicals) and for particular environmental conditions (the niche).
Oxidation by biological organisms is the extreme opposite of oxidation by
combustion. The former is specific for particular chemicals, while the latter is
entirely nonspecific. The specificity of biological organisms is conferred by such
features as membrane selectivity, permeases, regulatory proteins controlling
enzyme synthesis, and the structure of the enzyme-active site. There is too great a
tendency to generalize about bioremediation as a class of technology, like
combustion, which obscures the fact that biodegradability should always be
discussed together with the particular chemical.
Although specificity may seem to be a disadvantage for bioremediation, in
fact it provides one of the cost advantages of the

technique because resources are focused only on the target chemical. In

combustion, for example, external resources are needed to oxidize all organic
compounds, while in biodegradation resources go only to the compounds that can
reach the enzyme's catalytic site. In cometabolism, where external resources are
often needed, this feature is extremely important.
Microbial Diversity
Diversity, nature's counter to specificity, results from evolution, in which
organisms diversify from their progenitors to occupy new niches. Because of the
heterogeneity in nature, there are many niches and thus a naturally high degree of
biodiversity. For bacteria, diversity seems to be exceedingly high; there are likely
more than 10,000 species per gram of soil (Torsvik et al., 1990). Fungi also seem
to be very diverse, with an estimated 1.6 million species on earth (Hawksworth,
1991). Most of these organisms have never been isolated, let alone studied. For
example, Bergey's Manual, which describes all known bacteria, includes only
3000 to 4000 species, and most of these are not from soil or water (Holt, 1989).
This great diversity is important to bioremediation in two ways. First, it
means some diversity in the mechanisms that confer specificity. For example, a
small number of the organisms that degrade benzoate will also be able to degrade
chlorobenzoate or perhaps dichlorobenzoate, because the active site pocket is
slightly modified in these variants to allow access to the bulkier chlorine group.
This principle seems to be important in the metabolism of polychlorinated
biphenyls (PCBs), since the oxygenase of some toluene- and naphthalene-
degrading organisms can attack PCBs (Kuhm et al., 1991). Generally, the
principle applies to structurally similar chemicals or chemicals subject to the
same mechanism of attack. Thus, specificity is not absolute but usually limits the
range of substrates attacked to very few.
The second reason that diversity is important is that it is thought to lead to a
more robust and stable process because diverse species are likely to include
specialists for assimilating low and high pollutant concentrations; for tolerances
for different pHs, metals, and solvents; for different growth rates; and for
different resistances to phage infection or protozoan grazing. For example, among
benzene degraders in a gram of soil, there may be hundreds or even thousands of
indigenous strains that may vary in these other important ecological traits.
Original ecological dogma was that more diversity leads to stability, but current
evidence from macroecology suggests

that less complex systems are more stable (e.g., Begon et al., 1990). However, no
evidence exists on the relationship between stability and diversity for a microbial
process. In any case, higher diversity among pollutant degraders should lead to
emergence of the most fit organisms for the degradation and hence enhance
degradation performance.
If high diversity and large populations of pollutant degraders already exist in
the habitat, it becomes virtually impossible to successfully introduce an
inoculum. The native organisms both preemptively colonize the niche and are
likely more fit for the niche. Thus, super biodegraders, whether natural or
genetically engineered, stand little chance against a significant indigenous
population that can degrade the target chemical.
Biogeography of Biodegraders
Bacteria have been on earth for 3 billion years, an extremely long period of
time. Indeed, 85 percent of bacterial existence to date occurred before the
continental plates began to drift apart. Thus, the organisms have had a very long
time to evolve, adapt, and disperse. This long period likely also led to excellent
survival strategies, so that organisms can persist outside their optimum niches for
many years. A century ago, Beijerinck, a famous Dutch microbiologist, stated
that ''everything [bacterial types] is everywhere, the environment selects." This
remains the accepted dogma. Extended to biodegrading organisms, this dogma
suggests that biodegradative traits found in one soil or water would be found in
most other soils or waters around the world. The global distribution of such traits
has not yet been fully evaluated (and is the subject of research), but general
experience suggests that the dogma is true, at least at the level of the particular
activity, if not the identical strain. Hence, there may be some local variation, but
it likely occurs at the variety or strain level and is probably not apparent at the
process level. In other words, biodegradation proceeds on similar substrates and
at similar rates even though some of the strains are slightly different.
The importance of this biogeographical analysis to bioremediation is that it
suggests that biodegrading populations are similar at many sites. The portion of
biodegrading organisms in the total community at a given site may be somewhat
similar to that at other sites if selection has not already occurred. Thus, if the total
population is high, as in a fertile surface soil, the biodegrading population will be
high. In contrast, in the vadose zone and aquifer soil, which are impoverished in
organic matter, the total populations will be lower and hence

so will be the biodegrading populations. At sites where biodegrading populations

are likely to be large based on similarity to other sites, it would be difficult to
successfully inoculate a biodegrading organism.
Pollutants as Analogs of Natural Products

Biodegradation occurs when organisms have enzymes that can attack the
substrate. Natural selection throughout evolutionary history has maintained those
enzymes because they enhance fitness. Thus, pollutant degradation occurs
because this enzyme probably also metabolizes an analogous natural product in
order for selection to have preserved the gene sequence. It is often very difficult
to identify the natural substrate for the biodegrading enzyme without obvious
structural analogs. For example, halogenated chemicals are rare in nature, and the
natural substrates for enzymes involved in reductive dehalogenation are
completely unknown (Mohn and Tiedje, 1992).
The corollary of this situation is that bond types (or structures) not known in
nature are often not metabolized. Since these new substrates are a potential
energy resource, they exert selective pressure for organism variants to use them.
To acquire basically new enzymatic traits through natural evolution is thought to
take a very long time, probably hundreds or thousands of years. If one wants to
biodegrade these nonnatural chemicals in our lifetime to cleanup hazardous
waste, the task will likely involve protein and gene engineering, a process not
financially feasible in the foreseeable future.
Natural Selection
Ecological systems are driven by the resources available and the competition
for them among the community members. For pollutant degradation, the major
question is whether the pollutant is an energy resource—will an organism grow
on the chemical as a substrate? If so, there is strong selective pressure for the
degrading population to outgrow others, thereby amplifying the rate of
degradation. It is useful to group chemicals into two classes of biodegradability:
(1) those that support the growth of microbial populations and (2) those that are
cometabolized (in other words, they do not support growth but are partly
metabolized, usually through only a step or two of the complete metabolic
pathway). Organisms that carry out cometabolism are not naturally selected and,
therefore, are much more difficult to manage in nature. For this reason the
distinction of these two classes is important.

When pollutants are growth substrates, major advantages accrue: (1) the
catalyst grows logarithmically with no external input of resources; (2) the proper
growth, activity, and distribution of the microbial population (which is very
difficult to manage under other circumstances) is an inherent outcome of natural
selection for the primary energy substrate; and (3) growth substrates are almost
always completely oxidized to carbon dioxide, leaving no toxic intermediates.
Less than complete pollutant destruction by natural selection is usually due to
limitation by some other resource, most commonly the electron acceptor. Because
of these advantages, chemicals that are growth substrates have not and should not
become widespread pollution problems. This is because the limitations on natural
selection disappear as the chemical becomes more widely distributed. Examples
of chemicals that are growth substrates are benzene, toluene, xylenes,
naphthalene, chlorophenols, acetone, nitrilotriacetic acid, and 2,4-D. Whenever a
pollutant is a growth substrate, bioremediation should be seriously considered.
Even if the waste contains mixtures of chemicals, some of which are growth
substrates and others not, bioremediation may still be advantageous because it can
reduce other remediation costs, such as the amount of activated carbon needed.
Cometabolism usually results from relaxed specificity of an enzyme. No
sequential metabolic pathway or energy coupling to adenosine triphosphate
production typically occurs. Therefore, natural selection cannot be achieved
through this secondary (pollutant) substrate. If cometabolism is to be used, it
must be done by managing a primary substrate that selects for growth of active
organisms, induces the activity, and/or provides a necessary oxidant or reductant
to drive the reaction. Sometimes the primary and secondary substrates are
competitive inhibitors, which may require more sophisticated management, such
as pulse feeding or precise concentration control. Cometabolic processes typically
accumulate intermediates, some of which may be toxic.
Cometabolic reactions seem to be the only ones that show activity on many
of the recalcitrant chlorinated solvents, such as perchloroethylene (PCE),
trichloroethylene (TCE), carbon tetrachloride, and chloroform. Laboratory testing
and field testing are beginning to show that it may be possible to successfully
manage a cometabolic process in situ. Nonetheless, the experimentation, field
testing, and monitoring will all need to be more extensive than for pollutants that
are growth substrates.

THREE KEY QUESTIONS

I suggest that the following key questions, in the indicated order of priority,
are a basic guide to successful bioremediation:
1. Is the chemical degradable?

2. Is the environment habitable?
3. What is the rate-limiting factor and can it be modified?
Is the Chemical Biodegradable?

The first question is whether the chemical is biodegradable, because
bioremediation cannot be accomplished if no organism exists that can degrade the
chemical. Biodegradability must be established if it is not already well-
documented in the literature. Subquestions are whether the chemical is a growth
substrate, for the reasons discussed above, and whether the biodegrading organism
exists at the site.
A focus on the biodegradability of the pollutant is also important because it
suggests the time until application and the research needed for application, as
shown in Figure 1. In the figure, biodegradability is indicated by the frequency of
the biodegrading populations within the total soil community. The higher
frequency implies several benefits to bioremediation, including greater diversity
among the populations of degraders, less chance of encountering patches devoid
of organisms, and a rather global distribution of this biodegradative property at
most sites, which allows extrapolation of information among sites. If organisms
are widespread, they cannot be limiting to biodegradation. Hence, environmental
factors are then the focus for ensuring or enhancing bioremediation.
The time until field application of a bioremediation technology can also be
predicted by the biodegradability scale of Figure 1. When natural degrading
organisms are widespread, application is more immediate because conditions may
be met naturally or, if not, technology exists for removing some of the
environmental limitations. However, when organisms do not exist or are rare, the
time until application is more distant because successful addition or distribution
of organisms is difficult to achieve, especially in the subsurface (Harvey et al.,
1989). It is even more difficult to genetically engineer a new catalytic property;
this approach is far from any practical application to bioremediation.

FIGURE 1 Relationship of frequency of biodegraders in the community to

application of bioremediation.
Is the Environment Habitable?

The second question—is the environment habitable?—comprises two
issues. First, does the environment contain toxic chemicals that make it difficult
or impossible for microbes to live? Many polluted sites contain mixtures of
chemicals and metals, some at high concentrations, that may pickle the
environment so that bioremediation is not feasible. The second issue is the
availability of sufficient life-sustaining growth factors, such as nutrients,
particularly nitrogen and phosphorus; appropriate electron acceptors; and perhaps
other growth factors that might be contained in soil organic matter. Nutrient
supply can be evaluated by considering whether the proper carbon-nitrogen-
phosphorus (C:N:P) ratio is likely to be met by the soil environment. A C:N:P
ratio of 30:5:1 is needed for unrestricted growth of soil bacteria (Paul and Clark,
1989). Microbial growth in most subsoils is not limited by nitrogen and
phosphorus as long as the new carbon being provided is not in amounts greater
than tens of parts per million. This is often the case with pollutant chemicals.
Since nitrogen and phosphorus are inexpensive, however, they are often added as
insurance.
What Is the Rate-Limiting Factor and Can It Be Modified?

Too often in bioremediation there is a solution in need of a problem. Thus,
effort or money is spent to modify something that is not ratelimiting. To avoid
this waste, the rate-limiting parameter must first be defined. In doing so, it is
worthwhile to consider the ecosystem in its entirety and to recognize the three key
components: sub

strate (pollutant), biodegrading organism, and environment, as depicted in

Figure 2. Factors that reflect this interrelationship and that can limit
biodegradation are shown in parentheses in Figure 2.
FIGURE 2 Interrelationships of essential components that determine successful

bioremediation.
If biodegradability and habitability have been established, the most common

limiting parameter is oxygen, since it has relatively low solubility in water and is
in high demand as an oxidant for all biological respiration. Thus, schemes for
injection of oxygen or hydrogen peroxide into soil or aquifers are common. Such
treatments overcome a rate limitation if the site is anaerobic. Alternative electron
acceptors are possible, and nitrate is particularly attractive because of its high
electron-accepting capacity in water, its leachability in soils, its low toxicity, and
its low cost. Research on denitrification-driven bioremediation is in its infancy,
however. The frequency of this type of biodegrading population in soil is not
known, but it almost certainly is lower than for oxygen-respiring organisms.
Other treatments to meet physiological requirements include addition of
nutrients, adjustment of pH, and removal of toxicants by leaching, precipitation,
or some form of inactivation. As stated above, nutrient addition is common,
probably because it is easy and cheap and may occasionally provide some
benefit, not because it has been a well-documented requirement for many sites.
A second important limitation on biodegradation is the availability of the
chemical to the organisms, or bioavailability. Bioavailability is limited when the
pollutant is dissolved in organic matter or trapped in micropores in the soil
matrix. Substantial work is under way to attempt to understand and enhance the
bioavailability of water-in-soluble chemicals. The ecological approach to this
problem, however, would be to focus on ensuring that the local environment
contains

zones that would support natural selection if and when the chemical became
available, and not on the immediate (and impossible) recall of that chemical from
all microsites.
A related issue, but on a slightly larger scale, is the movement of the
chemical or organism so that the two come into contact. Mobility is not a
limitation for water-soluble chemicals, which move through soil easily, but it is a
severe problem for very insoluble chemicals. In this case, movement of
organisms is all that is feasible if physical mixing is not possible.
CONCLUSION
Returning to the ecological approach, the key point in determining whether
bioremediation is successful is to establish whether the conditions of natural
selection can be expected to be met within the site vicinity. The point is not to
determine pollutant mass balances; it is not to ensure that all heterogeneity can be
understood and accounted for; and it is not even to worry about local
concentrations above regulatory targets if conditions of the surrounding
environment ensure that natural selection will occur. This approach recognizes
that energy from organic matter is the key limitation for microbial growth and
that if the appropriate enzymes and required environmental conditions exist, there
is no way to prevent complete biodegradation. Thus, the first criterion for
successful bioremediation is documentation of the conditions for natural
selection, namely: (1) is the chemical a growth substrate for microbes? (2) is the
site habitable (nontoxic) for microbial life? (3) is there sufficient electron
acceptor? The ecological approach suggests that more emphasis should be placed
on documenting adequate electron acceptor supply and less on measuring the
actual pollutant.
A second line of evidence for a successful bioremediation is whether the
biological record suggests that natural selection has occurred. This evidence was
well illustrated by Madsen et al. (1990) for a plume from a coal tar site. Types of
evidence in the biological record include (1) increased rate of pollutant
mineralization; (2) increased populations of microorganisms (e.g., total microbial
populations, the biodegrading population, and grazers of those populations); and
(3) chemical gradients that show a discontinuity caused by respiratory
consumption of electron donors (pollutant) and electron acceptors. At
contaminated sites, this kind of evidence in the biological record would be
strongly indicative of successful intrinsic bioremediation and its persistence as
long as the conditions for natural selection can be ensured.

ACKNOWLEDGMENTS
The author's research on biodegradation has been funded by the U.S.
Environmental Protection Agency and the National Institute of Environmental
Health Sciences Superfund Program.
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Begon, M., J. L. Harper, and C. R. Townsend. 1990. Ecology: Individuals, Populations and
Communities. Cambridge, Mass.: Blackwell Scientific Publications.
Harvey, R. W., L. H. George, R. L. Smith, and D. R. LeBlanc. 1989. Transport of microspheres and
indigenous bacteria through a sandy aquifer: results of natural- and forced-gradient tracer
experiments. Environmental Science and Technology 23:51-56.
Hawksworth, D. L. 1991. The fungal dimension of biodiversity: magnitude, significance, and
conservation. Mycological Research 95:641-655.
Holt, J. G. 1989. Bergey's Manual of Systematic Bacteriology. Baltimore: Williams & Wilkins.
Kuhm, A. E., A. Stolz, and H. J. Knackmuss. 1991. Metabolism of naphthalene by the biphenyl-
degrading bacterium Pseudomonas paucimobilis Q1. Biodegradation 2:115-120.
Madsen, E. L., J. L. Sinclair, and W. C. Ghiorse. 1990. In situ biodegradation: microbiological
patterns in a contaminated aquifer. Science 252:830-833.
Mohn, W. W., and J. M. Tiedje. 1992. Microbial reductive dehalogenation. Microbiological Reviews
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Paul, E. A., and F. G. Clark. 1989. Soil microbiology and biochemistry. San Diego: Academic Press.
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IN SITU BIOREMEDIATION: THE STATE OF THE PRACTICE 121
In Situ Bioremediation: The State of the

Practice
Richard A. Brown
Groundwater Technology, Inc.
Trenton, New Jersey
William Mahaffey
ECOVA Corporation
Redmond, Washington
Robert D. Norris
Eckenfelder, Inc.
Nashville, Tennessee
SUMMARY
Since the pioneering work by Dick Raymond during the 1970s and early
1980s, in situ bioremediation has been widely used to cleanup aquifers
contaminated with petroleum hydrocarbons. A need for better performance led to
development of the use of hydrogen peroxide and direct injection of air into the
aquifer as sources of oxygen, which was a critical problem in bioremediation.
Bioremediation has developed in two branches. The first has been engineering
techniques and mathematical models for applying bioremediation to readily
degradable contaminants. The second branch has focused on ways to address
more recalcitrant contaminants such as chlorinated solvents, polychorinated
biphenyls, and pesticides. Work on these more challenging problems has met with
some success in the laboratory, but the techniques have yet to be
commercialized, largely because of failure to establish and maintain critical
control parameters in the subsurface. Continued improvements in the technology
will result from efforts in site delineation, engineering controls, use of
nonindigenous microorganisms, and field methods for evaluating the
microbiological processes.

INTRODUCTION
Bioremediation was first used commercially in 1972 to treat a Sun Oil
gasoline pipeline spill in Ambler, Pennsylvania (Raymond et al., 1977), and has
been used almost as long as simple pump-and-treat technology. In situ
bioremediation was one of the first technologies that was able to bring a site to
closure by significantly and permanently reducing soil and ground water
contamination, predating in situ processes such as soil vapor extraction and air
sparging.
The evolution of in situ bioremediation has had three important aspects:
microbiology, engineering, and applications. The microbiological aspects have
been concerned with basic metabolic processes and how to manipulate them.
Much of this work has been and continues to be laboratory scale and is currently
directed at recalcitrant substrates such as polychlorinated biphenyls (PCBs),
chlorinated solvents, and pesticides. The second aspect, the engineering of in situ
bioremediation, has been concerned with field-scale systems needed to provide
the substances required for the metabolic processes, such as oxygen, moisture,
and nutrients (Brown and Crosbie, 1989). The most difficult aspect of
development has been the translation of laboratory results to field applications.
Finally, specific types of bioremediation have been developed to treat specific
types of contaminants or matrixes. For example, a significant outgrowth of in situ
bioremediation has been the development of ex situ soil biotreatment (Brown and
Cartwright, 1990), which has become a cost-effective and widely applied on-site
technology. The engineering aspects of bioremediation have produced the
greatest successes in the commercial use of the method, leading to the
development of specific applications.
Bioremediation has been a successful technology when properly used. It is
also an oversold technology, having more promise than results. Understanding
the practice of in situ bioremediation—its legitimate uses and potential results—
requires an examination of historical developments in microbiology, the current
status of the practice of bioremediation, and new developments in
bioremediation. This examination illustrates the successes, limitations, and
continued needs of bioremediation technology.
HISTORICAL DEVELOPMENTS
The development of bioremediation has been predicated on an evolving use
of indigenous microorganisms to biodegrade a variety of organic compounds in
soils and wastewater. A large body of information about biooxidation
mechanisms and products and the

effects of reaction conditions was available before the technology was

commercialized. The microorganisms that could degrade various classes of
compounds under both aerobic and anaerobic conditions and the effects of and
requirements for pH, nutrients, oxygen, temperature, redox potential, and
moisture were all reasonably well established before in situ bioremediation was
practiced commercially.
Early studies in hydrocarbon metabolism were reported by Tausson (1927),
who isolated bacterial strains capable of oxidizing naphthalene, anthracene, and
phenanthrene. Subsequently, Sisler and Zobell (1947) demonstrated that marine
bacteria could rapidly oxidize benzo[a]anthracene to carbon dioxide. Senez and
co-workers (1956) were the first to suggest that normal alkanes were
enzymatically attacked at the first carbon atom (C1 position). Finally, Leadbetter
and Foster (1959) were the first to observe, define, and report on the co-oxidation
of hydrocarbons previously considered resistant to oxidation and assimilation.
Early in the development of bioremediation, oxygen availability was seen as
a critical factor (Floodgate, 1973; Zobell, 1973). The concept of introducing
water amended with nutrients and oxygen (using in well aeration) to promote
biodegradation was first tried by Dick Raymond in 1972 at the Ambler pipeline
spill mentioned earlier. This technology was patented by Raymond in 1974.
From 1975 to 1983, Raymond and co-workers (Jamison et al., 1975)
conducted several demonstration projects with the support of the American
Petroleum Institute (API). These studies demonstrated the feasibility of in situ
bioremediation; the observed reductions in soil and ground water contamination
were sufficiently encouraging to stimulate widespread interest in the technology.
This early work identified oxygen supply as crucial if the technology was to be
generally applicable. This finding led to the innovative use of hydrogen peroxide
as an oxygen carrier (Brown et al., 1984).
Laboratory tests at the Texas Research Institute (1982) demonstrated that
hydrogen peroxide could be a source of oxygen for bacteria and could be
tolerated at concentrations up to 1000 mg/l. API and FMC Corporation supported a
field test in Granger, Indiana, that demonstrated that hydrogen peroxide could be
used on a field scale (American Petroleum Institute, 1987). The use of hydrogen
peroxide as an oxygen source and as an agent for maintaining well performance
was subsequently patented (Brown et al., 1986).
During 1983-1986, several commercial in situ bioremediation projects using
hydrogen peroxide as the oxygen source were implemented and in some cases
reduced hydrocarbons (Frankenberger et al., 1989) and BTEX (benzene, toluene,
ethylbenzene, xylenes) (Norris and Dowd,

1993) to below detection limits. Because of the potential for more efficient
oxygen supply, the use of hydrogen peroxide expanded interest in
bioremediation. However, even though hydrogen peroxide did significantly
improve oxygen supply, it, too, had severe limitations: in the treatment of vadose
zone (unsaturated) soils and the instability of hydrogen peroxide in certain types
of soils (Britton, 1985), which can cause problems such as too rapid
decomposition and formation plugging.
The first change in the use of hydrogen peroxide came with the
development of soil vapor extraction (SVE), which is now recognized as a more
efficient supplier of oxygen for unsaturated soils and which has replaced the use
of hydrogen peroxide (Brown and Crosbie, 1989). While the focus of soil vapor
extraction has always been removal of volatiles, it was observed that the process
of vapor recovery could also result in substantially increased biodegradation rates
(Thornton and Wooten, 1982; Wilson and Ward, 1986). Several recent tests, such
as those conducted by the U.S. Air Force, have demonstrated a high degree of
biooxidation versus physical removal (Miller et al., 1990).
The development of SVE led to a broadening of remedial technology.
Because soil vapor extraction could physically remove volatile organics,
bioremediation became less of a stand-alone technology. Site remediation became
an integrated approach using SVE and bioremediation.
Concerns with hydrogen peroxide stability led to a search for other soluble
electron acceptors. Several tests were conducted to evaluate nitrate as an alternate
electron acceptor for degradation of monoaromatic (except benzene) and
polyaromatic compounds. Nitrate is inexpensive, is easily transported through the
formation, and appears to cause fewer problems than oxygen. However, nitrate
does not result in degradation of aliphatic compounds, and its use may be limited
by state and local regulations and concerns for nitrite formation and potential for
eutrophication.
The most recent innovation in bioremediation technology has been the use
of air sparging to oxygenate ground water (Brown and Jasiulewicz, 1992). Air
sparging involves injecting air below the water table to saturate the ground water
with air (and thus provide oxygen), as shown in Figure 1. The process can also
transfer volatile components to the unsaturated zone for capture by a vapor
recovery system. Currently, air sparging is receiving great attention because it is
relatively inexpensive and can distribute oxygen across the entire site at one time
rather than relying on an oxygen front moving across the site. In formations
where air sparging is applicable, it has supplanted hydrogen

peroxide. Air sparging provides the same benefits to saturated zone treatment
that soil vapor extraction has to vadose zone treatment.
FIGURE 1 Diagram of integrated remedial system.
CURRENT USES
The application of bioremediation is continually changing. Initially, the
technique was viewed as a primary treatment process—able, potentially, to treat a
wide range of organic compounds in soil and ground water. The advent of soil
vapor extraction and air sparging, however, has diminished the importance of
bioremediation as a stand-alone system for contaminants that are relatively
volatile and thus readily removed physically by sparging and venting. As a result,
bioremediation has evolved in two directions: as part of an integrated system for
treating highly mobile (volatile and/or soluble) and/or degradable substrates, such
as gasoline or diesel fuel, and as a primary system for treating nonmobile or
recalcitrant substrates such as heavier petroleum products and, potentially, PCBs
and pesticides.

Treatment of Degradable Mobile Contaminants

In the integrated treatment of hydrocarbon fuels or other mobile and
degradable substances, bioremediation, or biodegradation, has become an
effective incremental technology in conjunction with SVE and air sparging.
Biodegradation occurs readily during the aeration of petroleum hydrocarbons
(Miller et al., 1990). The degree to which biodegradation occurs relative to other
removal processes, such as volatilization, depends on the properties of the
contaminant and the rate of air flow and other environmental factors.
Biodegradation can be enhanced by adjusting air flow and moisture and by
adding nutrients. Physical removal is enhanced by increasing air flow.
The design of bioremediation strategies is highly site specific. It depends on
contaminant properties and distribution, lithology, infrastructure (buildings,
pavement, utilities, etc.), regulatory requirements, and client-specific issues such
as site usage and time requirements. For instance, soil permeability and layering
of highly permeable or very tight soils may preclude one or more technologies or
restrict design options. Generally, most in situ processes have had little success in
clay-based soils.
Many sites are now being remediated using multiple technologies. Where
free-phase hydrocarbons are present, it is almost always advisable to remove the
recoverable free-phase liquids. This typically leaves small pockets of free-phase
liquids as well as soils contaminated with several thousand parts per million of
adsorbed-phase organics. Pump-and-treat methods will satisfactorily remove only
those contaminants with water solubilities in excess of 10,000 mg/1. Thus,
remediation of most sites requires the incorporation of technologies that can
remove or destroy substantial quantities of contaminants.
For volatile biodegradable contaminants, a combination of in situ
bioremediation, air sparging, and/or vapor extraction may be the best strategy,
provided the soil properties and site infrastructure permit. Designs that emphasize
air sparging and vapor recovery are likely to lead to faster remediation than
systems that emphasize bioremediation. The latter, accomplished by using
intermittent or low air flow rates, offers the advantage of minimizing off-gas
treatment as a trade-off for speed of remediation.
Integration of technologies will typically provide the most cost-effective
remedial design. Thus, where unsaturated soils are contaminated by
biodegradable substances with vapor pressures exceeding approximately 1.0 mm
Hg, a combination of vapor recovery and bioremediation is likely to be used.
Where saturated zones are contaminated

largely by compounds with vapor pressures exceeding 1.0 mm Hg and with

Henry's Law constants exceeding 10-5 atm m3/mole, air sparging can be used to
provide oxygen and physically transfer contaminants to the unsaturated zone for
capture with a vapor extraction system.
For biodegradable contaminants with minimal volatility, bioremediation
may be a stand-alone technology. Polyaromatic hydrocarbons (PAHs), heavy
fuels, and plasticizers, for example, respond primarily to bioremediation alone.
The oxygen, however, may be provided by air sparging and/or vapor extraction
techniques. In fractured bedrock, highly stratified aquifers, or where the saturated
interval is no more than about 1 m, oxygen is more aptly provided through
recirculated ground water using hydrogen peroxide.
Resistant Organics
Recent years have seen continued progress with microbial degradation of
chlorinated solvents, pesticides, PCBs, and nitroaromatic compounds. In general,
however, the current state of technology does not permit these classes of
compounds to be treated on a commercial scale. Similarly, there is little evidence
that nonindigenous microorganisms have been used successfully on a
commercial scale for in situ bioremediation.
With highly degradable substances, intrinsic bioremediation can be used as
the final treatment when the contaminant load has been reduced to the point that
the ambient nutrient levels and oxygen diffusion are sufficient to support
biodegradation. With this unassisted bioremediation, treatment costs can be very
low.
FUTURE OF THE TECHNOLOGY

The engineering aspects of bioremediation have produced the greatest
successes in commercial use of the methods. This is due primarily to a
substantial body of information that existed on microbial use of petroleum
hydrocarbons as sources of carbon and energy for growth. Raymond's pioneering
efforts in the commercialization of bioremediation for petroleum hydrocarbons
were based on 45 years of research in biodegradation. In considering the future of
bioremediation it is wise to maintain perspective on the historical elements of
microbiology and biotechnology that support the engineering breakthroughs. One
must also acknowledge the current technical limitations of in situ bioremediation,
which fall into three major and highly interactive areas: physical/chemical,
microbiological, and site assessment.

Physical/Chemical Limitations
Major engineering advances have already been made in overcoming
physical/chemical constraints on in situ bioremediation systems, particularly in
the area of oxygenation. However, certain physical/chemical elements still
significantly affect the microbiological component of in situ bioremediation. Of
these, the molecular architecture of organic pollutant molecules has the greatest
implications.
Size and the extent and type of functional group substitution dictate the
bioavailability and biodegradability of a molecule. Bioavailability through
desorption is greatly reduced by solubility limitations as well as degree of
hydrophobicity, both of which depend on molecular size and functional group
substituents. Surfactants may improve bioavailability, but they are of no avail
where the microbial populations lack the catabolic capacity to biodegrade the
molecule(s) of concern.
Another important factor is that single-substance contamination is rare in
most polluted environments. Microbial biodegradation of multicomponent
mixtures is not as well understood as many would believe. Biodegradation of
complex mixtures is often assumed to occur if the contaminants are known to be
biodegradable and substrate interactions are known to be not important.
However, at least two studies involving gasoline (Barker et al., 1987; Wilson et
al., 1990) reported that some BTX (benzene, toluene, xylenes) constituents
persisted above regulatory action levels, even after stimulation of bioremediation
by addition of inorganic nutrients and various electron acceptors. A number of
investigators (Alvarez and Vogel, 1991; Arvin et al., 1989; Bouwer and Capone,
1988) have recognized and begun to investigate the importance of substrate
interactions.
Microbiological Limitations
The unpredictability of biodegradation adds to the importance of continued
research on metabolic processes such as adaptation, co-oxidation, diauxy,
catabolite repression, and competitive inhibition. Central requirements of in situ
bioremediation are that the contaminants are biodegradable, that the appropriate
microbial populations are present, and that the microbes are able to thrive. The
understanding of metabolic pathways in biodegradation and of the factors that
control microbial populations continues to grow, thus increasing the potential for
bioremediation.
Research into the biodegradation of chlorinated organics illustrates the
importance of continued microbiological research. Chlorinated solvents and many
other halogenated compounds (e.g., PCBs)

were previously thought to be recalcitrant both aerobically and anaerobically.

However, the early 1980s witnessed major advances in our fundamental
knowledge of the biodegradation of chlorinated organics. Bouwer et al. (1981)
demonstrated the anaerobic degradation of halogenated 1- and 2-carbon
compounds. Subsequent research on trichloroethylene (TCE) (Vogel and
McCarty, 1985) and perchloroethylene (Fathepure et al., 1987) demonstrated that
these compounds were cometabolized through a reductive dehalogenation
mechanism by a consortium of anaerobic organisms. Researchers at General
Electric Corporation (Bedard et al., 1987; Quensen et al., 1988) identified a
reductive dehalogenation mechanism for PCBs. Bedard and her co-workers
further demonstrated novel aerobic processes that degraded the more refractory
orthosubstituted PCB congeners and have isolated a number of bacterial strains
that are highly efficient in degrading the more highly chlorinated congeners. TCE
was shown to be co-oxidized by methanotrophic bacteria supplied with methane
(Wilson and Wilson, 1985) and by a strain of Pseudomonas cepacia (G4)
supplied with phenol or toluene (Nelson et al., 1987).
There has been a plethora of laboratory investigations to identify beneficial
microbial processes but relatively few field pilot studies demonstrating the
efficacy of in situ bioremediation for recalcitrant compounds and little
commercialization of novel microbial processes. Extensive field studies by
researchers at Stanford University used stimulation of methanotrophs to co-
oxidize TCE under nearly ideal field conditions. To date, the technology has not
been commercialized. Another in situ field study was performed by General
Electric Corporation in the summer of 1991. While limited in scope, this study
provided field-scale data for evaluating aerobic biodegradation of PCBs by
naturally occurring microorganisms. Despite these partially successful field
studies, there has been little progress toward commercialization of new
bioremediation processes for in situ application.
The disparity between research success and commercialization reflects the
difficulty of maintaining critical control parameters (e.g., the requirement of
methane for co-oxidation of TCE and the coincident competitive inhibition of
TCE degradation in the presence of excess methane). Further, many research
studies use highly adapted cultures that are not readily dispersed throughout the
formation or maintained in the presence of predators. To date, there has been only a
preliminary report suggesting that the injection of a specific degrader population,
P. cepacia strain G4, for co-oxidizing TCE may be effective under highly ideal
site conditions (Nelson et al., 1990). These results are currently being reevaluated
by further field pilot testing.

Site Assessment Limitations

An important element of any field pilot program is that the site be well
characterized and that statistically valid sampling plans be used during the site
investigation and remediation. Several critical elements of an environmental
sampling plan are:
• a definition of the time-space population(s) of interest;

• development of field-sampling designs and sample measurement
procedures that will yield representative data from the defined
populations; and
• assessment of the uncertainty of estimated quantities through means,
trends, and average values.
Evaluation of the applicability of bioremediation requires answers to some

basic questions, such as: What is the validity of assuming that the enumeration of
specific degrader populations can be used to assess the degradative potential at a
site or that these populations can be adequately stimulated to degrade the
pollutants? How many site samples must be analyzed by treatability methods to
demonstrate a biodegradative rate enhancement sufficient to achieve a regulatory
level? What are the acceptable standardized methods? How well do the data from
these test methods predict actual field results, and to these results justify the costs
of obtaining these data? The answers to these questions are likely to vary from
site to site and will be greatly influenced by experimental design. Field
performance can be predicted from laboratory experiments only through
development of appropriate mathematical models that are verified over time by
demonstrating good correlation of laboratory and field data.
Future Needs
The future of bioremediation lies in overcoming the limitations of the
technologies. Clearly, the enormous costs of site remediations and the goal of
eliminating future liability constrain the development of new technologies. The
most significant advances will be those that result in the development of
predictable, efficient, lower-cost methods of remediation. Some of the limitations
are physical/chemical and will be overcome by purely engineering methods;
other solutions will be uniquely biological. In addition to the identification of new
microbial capabilities for degrading chemical pollutants, other biotechnical
offshoots will evolve. These can be viewed as bioaugmentation, analytical
methods, and process innovations.

Bioaugmentation
Genetic engineering to improve catabolic capacity has enormous potential
for obviating cellular regulatory control over the expression of biodegradative
pathways. This technology offers the distinct advantage of constructing new
biodegradative pathways by eliminating misrouting of metabolites to end
products that inhibit further biodegradation of a pollutant (Reineke and
Knackmuss, 1990). The use of specially constructed strains to biodegrade a
heretofore recalcitrant pollutant would expand the range of compounds and
therefore the number of sites amenable to bioremedial technologies. However,
until the release of genetically engineered organisms is more acceptable from a
social and regulatory perspective, this technology will be of use only from an
academic perspective.
An alternative to classical genetic engineering is laboratory breeding of
organisms under appropriate selective pressures to enrich for strains with the
desired phenotypic characteristics. This process was effective in isolating a single
strain of bacteria capable of degrading chlorobenzenes from the coculture and in
the selective breeding of a bacterium that degrades toluene and one that degrades
chlorobenzoate. In addition to developing improved strains, a great deal must be
done in developing inoculation systems that assure that the desired strain(s)
compete effectively and establish residence long enough to achieve the remedial
objective.
Analytical Methods
Field analytical techniques for monitoring for the presence of specific
degrader populations or levels of contaminants that are as easy to use as home
pregnancy tests would revolutionize the environmental industry. Such methods as
nucleic acid probes and monoclonal antibody tests have been developed but are
not widely used because of their relatively high cost and low reliability. Are these
deficiencies inherent in the technology or is further development required?
It would seem that monitoring methods that could provide direct evidence of
the performance of in situ bioremediation processes would go a long way toward
validating treatment effects early in the remediation process and even provide the
mechanism for stimulus-response control of the process. Methods for on-line
analysis of general metabolic end results, such as carbon dioxide production and
oxygen consumption, are used fairly routinely. However, as the Stanford field
pilot program demonstrated, additional benefit can be gained by tracking the
levels of specific transient metabolic products of the biological

process. These observations beg the question of whether our fundamental

knowledge of biodegradative pathways can be used to suggest and/or develop
similar methods for classes of contaminants in addition to petroleum
hydrocarbons.
Process Innovations
A number of new technologies, biological and chemical, could be used to
enhance bioremediation. With increasing knowledge of anaerobic
biodegradation, it should not be long before we witness the use of this microbial
process to encourage in situ biorestoration of sites contaminated with chlorinated
solvents, PCBs, chlorinated pesticides, or other halogenated organics that
otherwise resist microbial degradation. On purely thermodynamic grounds, it is
not unreasonable to suppose that a treatment-train approach using both anaerobic
and aerobic biodegradation would be the most efficient way to handle such
compounds as PCE and PCBs.
A second possibility involves in situ soil flushing, a technology derived from
tertiary recovery of petroleum from oil fields. Surfactant/polymer floods are used
to essentially wash product or pollutants from the subsurface for above-ground
recovery. Typically, this process will leave behind residual contaminants and
polymer/surfactant. The potential of using in situ bioremediation to treat these
residuals (biopolishing) has received minimal investigation.
CONCLUSION
Bioremediation technology has evolved over 20 years of commercial life. It
started as one of the first primary treatment processes, able to address both soil
and ground water contamination. It has since become an incremental technology,
directed at accelerating the remediation of sites contaminated by petroleum
hydrocarbons and other degradable substrates.
The evolution of bioremediation has resulted primarily from engineering
work. Most advances in commercial application have been tied to improving
oxygen availability. The technology has evolved from simple in well aeration to
chemical carriers such as hydrogen peroxide or nitrate and, finally, to aeration
technology—soil vapor extraction and air sparging. In the course of this evolution
the importance of the biological pathway has declined as physical removal
processes have evolved.
The future of bioremediation lies in addressing those contaminants that are
not easily extracted physically, such as PAHs, PCBs,

and pesticides. This, approach, however, requires advances in the fundamental

knowledge of microbial ecology and biodegradation pathways. Application of
new microbial processes requires better monitoring and mathematical modeling
as well as improved subsurface engineering. Such advances will lead to better
understanding and use of natural or enhanced in situ bioremediation.
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ENGINEERING CHALLENGES OF IMPLEMENTING IN SITU BIOREMEDIATION 136
Engineering Challenges of Implementing In

Situ Bioremediation
Lisa Alvarez-Cohen
University of California
Berkeley, California
SUMMARY
The use of in situ bioremediation to destroy ground water contaminants
essentially requires the creation and management of a subsurface bioreactor.
Physical and chemical conditions within the subsurface environment can be
manipulated to optimize microbial growth by using hydrodynamic or gas-phase
controls. Requisite factors for successful application of in situ bioremediation
include adequate aquifer permeability; a suitable microbial population; sufficient
hydrodynamic control for plume containment and delivery of required electron
donors, electron acceptors, and/or nutrients; and a complete monitoring system.
Evaluating the progress of in situ bioremediation and proving that the
microbes are responsible for contaminant degradation can be challenging because
of the inaccessibility of the subsurface bioreactor, aquifer heterogeneities, and the
wide range of potential contaminant fates. However, overlapping lines of
evidence from a range of field-monitoring techniques may provide suitable
indication of successful in situ bioremediation.
INTRODUCTION
In situ bioremediation involves the stimulation of microorganisms within a
subsurface aquifer to degrade ground water contami

nants—that is, management of a subsurface bioreactor to carry out specific

biological degradations. Management of a subsurface bioreactor can be passive,
involving only the monitoring of naturally occurring microbial degradations, or it
can be active, involving engineered systems for the manipulation of physical and
chemical conditions within the subsurface environment. Subsurface conditions
can be effectively altered with hydrodynamic controls, using water as the delivery
and transport system, or with gas-phase control within the vadose zone
(unsaturated subsurface). Hydrodynamic controls involve manipulation of ground
water flow and may include injection wells or infiltration galleries for the
introduction of water to the subsurface along with production wells for ground
water withdrawal. Gas-phase controls may take the form of vacuum extraction or
venting systems, which may be accompanied by direct injection of gas to the
vadose zone or sparging of gas into the ground water. The subsurface bioreactor
or zone of biostimulation would then occur between the injection and withdrawal
systems. However, the inherent heterogeneity and inaccessibility of the
subsurface make in situ bioprocesses much more difficult to monitor and control
than above-ground engineered systems. The following discussion addresses some
of the unique engineering challenges associated with the use of in situ
bioremediation to treat contaminated aquifers.
SUBSURFACE BIOREACTOR REQUIREMENTS

Before applying in situ bioremediation to a contaminated aquifer, it is
necessary to evaluate the feasibility of engineering a subsurface bioreactor at the
specific site to carry out the biological degradations of interest. Engineering
feasibility depends on a number of factors; principal among them are aquifer
permeability, heterogeneity, and geochemical characteristics, as well as the nature
and distribution of the contaminants. As with above-ground bioreactors,
providing appropriate environmental conditions, residence times, and substrate
availability are fundamental requirements for promotion of efficient
biodegradation reactions.
Subsurface Investigation
The practicality of a subsurface bioreactor depends on aquifer characteristics
that can best be evaluated by a thorough site investigation, including tracer
studies. A combination of existing site data and both direct and indirect
measurements can be used to evaluate these characteristics and define the nature
and extent of subsurface

contamination. Boreholes and monitoring wells can permit direct sampling of

aquifer material and ground water, which is useful for aquifer characterization
and development of a three-dimensional assessment of contaminant distribution.
In determining well placement for site assessment, the cost associated with
each boring must be weighed against the information lost in the spaces between
borings, a balance that is highly dependent on the heterogeneity of the aquifer. It
is also necessary to place wells upgradient of the contamination source to provide
information on background water quality as well as downgradient for information
on the location and size of the pollutant plume. Preliminary information on
aquifer composition and plume location derived from remote sensing by
geophysical techniques can be useful for optimizing well placement and,
consequently, for decreasing the number of wells necessary for adequate
monitoring (Benson et al., 1988). Additionally, gas surveys (analyses of gas
samples from within the unsaturated zone) are useful for detecting volatiles that
diffuse up from the water table. Such surveys may be advantageous for
decreasing the number of monitoring wells since they offer additional information
on location and migration of volatile plumes (LaGrega et al., 1992).
Tracer tests are additional direct measurement tools that are useful for
estimating the direction and velocity of ground water flow and the hydraulic
conductivity, porosity, and dispersivity of the aquifer. Tracers also facilitate
estimation of contaminant residence times within the biostimulation zone, which
is useful for predicting degradation efficiency.
Permeability
Adequate permeability for the transport of solutions delivering nutrients or
other compounds required for stimulation of the desired microbial population
within the subsurface bioreactor is essential to in situ bioremediation.
Additionally, the aquifer must be sufficiently permeable that the increased
microbial mass and volume will not cause extensive plugging of the aquifer
pores, thus restricting further ground water movement. The proposed rule of
thumb (Thomas and Ward, 1989) is that aquifers with overall hydraulic
conductivities of 10-4 cm/s or greater would be most amenable to in situ
bioremediation (10-4 cm/s hydraulic conductivity corresponds roughly to an
intrinsic permeability of 10-9 cm2 for clean water at typical subsurface
temperatures). However, it has been shown that microbial growth in aquifer
material can cause permeabilities to decrease by a factor of 1000 (Taylor et al.,
1990). Additionally, modeling that considered

microbial growth, transport, and biofilm shearing has shown that high-porosity
media with widely distributed pore sizes in the small-diameter range are much
more susceptible to biofouling than high-porosity media with a narrow pore size
range. These results suggest that both permeability and pore size distribution
must be considered in determining the feasibility of in situ bioremediation
(Taylor and Jaffe, 1991).
Environmental Conditions
It is important to analyze the environmental parameters inside the intended
zone of biostimulation that could exert significant impact on microbial growth
and degradation potential. Microbial metabolism is substantially affected by
temperature: the metabolism of subsurface populations tends to accelerate with
increased subsurface temperatures within typical (nongeothermal) ranges.
Although temperatures within the top 10 m of the subsurface may fluctuate
seasonally, subsurface temperatures down to 100 m typically remain within 1° to
2°C of the mean annual surface temperature (Freeze and Cherry, 1979),
suggesting that bioremediation within the subsurface would occur more quickly
in temperate climates (Lee et al., 1988).
Additional factors that may limit microbial activity have been summarized
elsewhere (Ghiorse and Balkwill, 1985; Ghiorse and Wilson, 1988). They include
pH values outside the range of neutral (pH<6, pH>8), desiccating moisture
conditions, and extreme redox (reduction-oxidation) potentials. Each of these
factors may be mitigated or controlled within a desired range with varying levels
of success using hydrodynamic controls.
Monitoring
Monitoring of ground water and aquifer conditions over time is necessary
for assessing activity within the subsurface bioreactor and evaluating the progress
of the bioremediation. Monitoring wells typically are installed between the
injection and production wells so as to detect microbial growth and contaminant
degradation within the biostimulation zone. Additional wells installed upgradient
from the contamination provide background characterization data, while wells
installed beyond the downgradient contaminant boundaries are useful for
detection of plume expansion or migration. Factors that should be analyzed in the
monitoring samples include contaminant concentration, microbial numbers,
electron donor and acceptor concentrations, oxygen demand, degradation
products, pH, and major ion concentrations.

Sample collection and handling procedures have been summarized elsewhere

(Barcelona et al., 1990).
AQUIFER PREPARATION
Before applying in situ bioremediation, the source of contamination must be
detected and mitigated, major accumulations of free product must be removed,
and mechanisms for plume containment must be installed. Contaminants entering
the subsurface partition into different phases due to sorption, volatilization, and
dissolution processes. Contaminant partitioning impedes pump-and-treat removal
methods and may decrease the contaminant's availability to microbial
degradation.
Source and Free Product Removal

The first step in most aquifer remediation efforts is removal or mitigation of
the contaminant source: excavation of leaking underground storage tanks,
plugging or repairing leaking surface impoundments or landfill liners, restricting
intentional or unintentional land application, and similar measures. Removal
typically is achieved by excavating the most contaminated surface soils and
drilling wells to pump out the most concentrated source material. Liquid
contaminants, which often exist as nonaqueous-phase liquids, or free product, are
drawn into the subsurface by gravity and capillary action within the porous
media. Free product that is lighter than water, such as petroleum hydrocarbons,
tends to migrate downward through the unsaturated zone until hitting an
impermeable layer or the water table, where it spreads laterally. Free product that
is denser than water, such as many of the chlorinated solvents, would continue to
migrate downward through the water table and saturated zone until reaching an
impermeable barrier. Free product can be removed from an aquifer by direct
pumping using production wells alone or combinations of injection and
production wells to cause directional migration of the floating or sinking product.
However, most pumping strategies will be capable of removing only part of the
free product from the subsurface, leaving the remainder of the organic material
trapped in pores as residual free product, dissolved in the surrounding ground
water as a contaminant plume, sorbed onto the solid subsurface material, or
volatilized into the gas-filled pores of the unsaturated zone. Additionally, the
location and removal of sinking free product typically represent much more of an
engineering challenge than that of floating product, often resulting in low
recovery efficiency.

Plume Containment
In situ bioremediation typically requires times on the order of months to
years to reduce contaminants to acceptable levels. During that time the
contaminants must not be allowed to spread outside the bioremediation zone and
thereby escape treatment.
A contaminant plume can be contained by physical or hydrodynamic
controls or a combination of both. Physical controls include low-permeability
vertical walls installed to physically block the transport of the plume and/or to
inhibit the flow of clean ground water into the contaminated zone. The most
commonly used physical containment barrier is the slurry trench wall, which
typically is composed of a mixture of bentonite and soil or bentonite and cement.
A slurry wall keyed into a confining impermeable layer can significantly decrease
localized ground water flow and lengthen the ground water flow path. Grout
curtains, vibrating beam walls, and synthetic sheet curtains are also used on a
limited basis for physical containment. Physical barriers are most effective with
shallow aquifers underlayed by a solid confining layer of bedrock or clay
(LaGrega et al., 1992).
Hydrodynamic controls are used alone or in conjunction with physical
controls. They are especially suited for use with in situ bioremediation since
biostimulation amendments could be added with the control water.
Hydrodynamic controls typically consist of combinations of injection and
extraction wells and/or infiltration galleries that manipulate ground water flow in
order to prevent undesirable plume movement. Wells are situated so that their
radii of influence (area of water drawdown or mounding) overlap, allowing
control of water within the entire treatment zone as well as effective manipulation
of the level of the water table. Radii of influence are computed by iterative
application of steady pumping rates with drawdown equations appropriate to the
specific aquifer conditions. Plume direction, shape, and migration speed can each
be effectively manipulated by hydrodynamic controls, which regulate the
detention time and amendment delivery within the biostimulation zone
(Barcelona et al., 1990; Knox et al., 1986).
IN SITU BIOSTIMULATION
Stimulation of microbial populations within a subsurface bioreactor requires
an appropriate carbon source; electron donors/acceptors for energy production;
and inorganic nutrients such as nitrogen, phosphorus, and some trace metals. Also
required are proper conditions within the aquifer, such as appropriate pH,
temperature, moisture content, and redox potential.

Indigenous microbial populations from many aquifers have been shown to

be capable of degrading a wide range of organic contaminants, obviating the need
for introduction of exogenous cultures in most bioremediation applications (Lee
et al., 1988). However, in the absence of appropriate indigenous strains,
introduction of laboratory-enriched populations or even genetically engineered
microorganisms may be possible.
To detect the presence of microbial populations capable of degrading the
contaminant of interest, laboratory feasibility studies should be conducted. In
these studies an aseptically collected sample of subsurface material is exposed to
the contaminant of interest under simulated aquifer conditions and is analyzed for
contaminant degradation and the concurrent appearance of degradation products.
Aseptic aquifer samples are collected by withdrawing an uncontaminated section
of a drilling core using a sterile paring device (Lee et al., 1988). Radiotracers may
also be used as an analytical tool to confirm contaminant degradation and to trace
the degradation sequence. Additional information that may be collected from
feasibility studies includes the range of nontoxic contaminant concentrations,
nutrient and electron donor/acceptor requirements for optimizing cellular growth
and contaminant degradation, and estimates of microbial acclimation periods and
growth rates. Adequate detention time for the contaminants within the
biostimulation zone must be maintained to allow the degradation reaction to
reduce the contaminant concentrations to the desired levels, and acclimation
periods may be necessary before the indigenous population becomes capable of
carrying out the degradation reaction (Wilson et al., 1985).
Role of Nutrients, Electron Donors, Acceptors

Microorganisms produce energy by moving electrons between an electron
donor and an electron acceptor. These reactions can be carried out aerobically,
using oxygen as the electron acceptor, or anaerobically, using nitrate, sulfate,
carbon dioxide, or other oxidized species as the electron acceptor. Many organic
contaminants can be used as a primary substrate for microbial metabolism, in
which case the contaminant serves as an electron donor and sometimes also as the
major carbon source for the microbial cells. Therefore, some degradation
reactions produce energy and usable carbon, resulting in microbial growth.
Hence, bioremediation of a primary substrate has a built-in termination
mechanism: as the contaminant/substrate is consumed, resulting in depleted
concentrations, microbial growth slows and ceases. The hydrocarbons in gasoline
and other petroleum derivatives

can be aerobically degraded and used as a primary substrate for growth by a wide
range of naturally occurring microorganisms (Armstrong et al., 1991; Ridgway et
al., 1990). Bioremediation of gasoline-contaminated aquifers by indigenous
microflora has been successfully implemented a number of times (Jamison et al.,
1976; Lee et al., 1988; Thomas and Ward, 1989), although researchers have
sometimes reported that the indigenous microbial population required a period of
adaptation before degradation commenced (Armstrong et al., 1991).
Alternate Substrates
Because of the nature of contaminant partitioning within the subsurface,
contaminants may be transported through the aquifer in dilute ground water
plumes at concentrations that provide insufficient energy and/or carbon to
support microbial growth. Additionally, since aquifers may be used as drinking
water sources, the allowable levels of many ground water contaminants are set in
the range of micrograms per liter, requiring reduction of contaminants to
concentrations lower than those required for microbial reproduction. Under these
circumstances, it may be necessary to supply an alternate substrate for the
microorganisms in order to promote degradation by secondary metabolism.
Similarly, contaminants that do not benefit microorganisms by providing energy
or carbon can sometimes be degraded in the presence of an alternate microbial
substrate by cometabolism. Some examples of cometabolic degradations are those
catalyzed by the mono- or dioxygenase enzymes of methane-, propane-, or
toluene-oxidizing bacteria, which use the contaminant as electron donor and
oxygen as electron acceptor, or those carried out by anaerobic bacteria capable of
using the contaminant as the electron acceptor. A wide range of chlorinated
solvents, including trichloroethylene and vinyl chloride, have been shown to be
cometabolically degraded by methane- and toluene-oxidizing bacteria. For the
application of either secondary metabolism or cometabolism, use of an alternate
substrate presents an additional engineering challenge and potential limit on the
reaction rate. However, the use of an alternate substrate also enables the
degradation reaction to be maintained at contaminant concentrations below those
required to support microbial growth and much lower than those possible for
degradations in which the contaminant is used by the microorganisms as a
primary substrate. Therefore, alternate substrates can increase the potential for
attaining contaminant removal to regulatory levels.

Nutrient Delivery
Nutrients typically are delivered by controlling ground water flow using
injection wells or infiltration galleries coupled with downstream production
wells. In the most common configuration, ground water withdrawn from
production wells downgradient from the biostimulation zone is amended with the
nutrients required for biostimulation, treated if necessary to remove
contaminants, and reintroduced to the aquifer upgradient of the biostimulation
zone using the injection wells or infiltration galleries. Water from an external
source is required if the flow of withdrawn water is insufficient to control the
subsurface flow or if it is infeasible to reinject the withdrawn ground water. The
rate of nutrient delivery to the biostimulation zone, therefore, is often limited by
the solubility of the nutrients in water and the reinjection flow rate.
Alternately, gaseous nutrients or substrates such as oxygen or methane may
be delivered to the biostimulation zone by sparging, the direct injection of gas
into the saturated aquifer to effect in situ dissolution of the gas into solution.
However, mobilization of volatile contaminants into the gas phase may
necessitate additional gasphase controls.
When the limiting nutrients for microbial growth are added to the
subsurface, excessive microbial growth may occur around the injection zone,
causing significant plugging of the permeable media and limiting the reinjection
flow. Innovative methods for discouraging well plugging while promoting
dispersed microbial growth throughout the zone of contamination are required.
One such method, which has been shown in field studies to reduce localized
plugging associated with cometabolic bioremediation, is alternating pulses of
electron donor and electron acceptor in the reinjection water. Since both electron
donor and acceptor are required for microbial metabolism, advective and
dispersive processes within the aquifer must mix the nutrients before conditions
promote microbial growth, causing cells to grow dispersed throughout the aquifer
and producing a large biostimulation zone (Semprini et al., 1990).
Oxygen, Air, Hydrogen Peroxide

Because of the low solubility of oxygen in water, the major kinetic limitation
on aerobic bioremediation reactions is often the availability of oxygen (Lee et al.,
1988; Thomas and Ward, 1989). This is especially the case with high BOD
(biological oxygen demand) compounds such as petroleum hydrocarbons. Air
sparging of water can supply 8

mg/l dissolved oxygen, while sparging with pure oxygen can deliver 40 mg/l and
with hydrogen peroxide more than 100 mg/l oxygen. Therefore, while air
sparging is the simplest and most common oxygen delivery technique, the use of
oxygen or hydrogen peroxide may speed the bioremediation process and decrease
the pumping required. However, in some cases the increased cost and potential
explosion hazard associated with pure oxygen may more than offset its increased
delivery efficiency.
Application of hydrogen peroxide to in situ bioremediation is limited by its
toxicity to microbes and its potential for causing aquifer plugging. Two
molecules of peroxide are required to produce one molecule of oxygen:
Although this reaction can be catalyzed by microorganisms, it is also

catalyzed by naturally occurring compounds in aquifer material. The highly
reactive nature of hydrogen peroxide results in chemical oxidations of organic
and inorganic compounds, producing precipitants that may contribute to aquifer
plugging and may decrease the oxygen-carrying capacity of the water (Spain et
al., 1989). Additionally, both metal-catalyzed and microbially induced
decomposition of hydrogen peroxide may produce oxygen at concentrations
above water saturation, causing bubbles to form and further decreasing aquifer
permeability (Morgan and Watkinson, 1992; Pardieck et al., 1992). To mitigate
undesirable peroxide reactions, phosphate is sometimes added before the
peroxide to precipitate iron and thereby diminish the metal-catalyzed
decomposition. Additional chelating agents have been shown to decrease metal-
catalyzed decomposition; however, in a biologically active zone the majority of
peroxide decomposition would be expected to be biologically induced (Morgan
and Watkinson, 1992). Therefore, the dual actions of precipitation of oxidation
products and bubble formation typically limit the practical concentration for
addition of hydrogen peroxide in ground waters to 100 mg/l or less
(corresponding to 47 mg/l oxygen or less).
The reactivity of hydrogen peroxide in aquifers can be expected to vary
considerably from site to site and may not result in significant plugging problems
in very highly permeable soils and gravels. However, peroxide is also capable of
causing mobilization of undesirable metals such as lead and antimony, producing
additional ground water contamination. Therefore, it is important to do laboratory
feasibility studies before using hydrogen peroxide in an aquifer since the range of
potential adverse reactions is so great.

Finally, hydrogen peroxide can be significantly toxic to microbial

populations at relatively low concentrations. The toxicity of the compound
reportedly is species specific and depends on cell density (Lee et al., 1988;
Pardieck et al., 1992). Significant toxicity has been reported for peroxide
concentrations as low as 3 mg/l, whereas other studies have shown addition of
270 mg/l to exert no adverse effects. Additionally, acclimation of
microorganisms to slowly increasing concentrations of peroxide has been
reported with successful additions greater than 2000 mg/l (Pardieck et al., 1992).
Again, laboratory feasibility studies will be necessary to determine the tolerance
range of the indigenous microbial population.
The toxic effects of hydrogen peroxide on microbes have a side benefit that
can be exploited through careful control of the injection stream. A relatively high
concentration of hydrogen peroxide in the injection water may be useful for
controlling the growth of biofilms within the immediate vicinity of the injection
wells or infiltration galleries, providing the peroxide concentration decreases
sufficiently with migration through the aquifer to preclude toxic microbial effects
within the biostimulation zone, bubble formation, and precipitation of oxides.
Inorganic Nutrients
Studies of the effects of inorganic nutrient addition on bioremediation rates
have yielded varying results. Experiments using nitrogen and phosphorous
amendments have shown that they enhanced metabolic activities in some aquifer
samples while having no significant effect in other samples from the same
aquifer (Swindoll et al., 1988). Others reported that addition of nitrogen and
phosphorus enhanced in situ gasoline degradation (Jamison et al., 1976). A series
of microcosm and field studies suggest that enhancement of biodegradation by
addition of inorganic nutrients is extremely case specific (Baker and Herson,
1990). It has been further suggested that not only are inorganic nutrients not
always effective but in some cases they inhibit microbial degradation (Morgan
and Watkinson, 1992). Morgan and Watkinson have also shown that phosphate
addition in combination with hydrogen peroxide may cause precipitation of
insoluble salts during migration through the aquifer, decreasing the permeability
within the biostimulation zone.
Hence, the evidence indicates that chemical analysis is not adequate for
predicting necessary nutrient amendments. Laboratory or field studies with
aquifer material are needed to determine nutrient amendments required to
promote maximum cell growth and contaminant

degradation and to predict potential reactivity between the aquifer material and
the amendments.
Microbial Introduction
Although in situ bioremediation is a developing technology and is currently
the subject of many research studies, little is known about the movement of
microbial cells through the subsurface matrix or the feasibility of introducing a
stable mixed population of organisms into a contaminated site for the purpose of
remediation (Thomas and Ward, 1989). Microbial populations suitable for
introduction to a contaminated aquifer may have been selectively enriched in the
laboratory or genetically engineered to carry out specific degradation reactions, to
resist certain toxic effects, or to grow preferentially under specific environmental
conditions. However, while laboratory-enriched populations may be added to the
subsurface with little regulatory concern, the introduction of genetically
engineered cultures currently is not allowed in the United States.
Successful microbial introduction requires a range of factors: (1) the
population must be capable of surviving and growing in the new environment;
(2) the microorganisms must retain their degradative abilities under the new
conditions; (3) the organisms must come in contact with the contaminants; and
(4) the electron donors/acceptors and nutrients necessary for microbial growth
and contaminant degradation must be made available to the population (Thomas
and Ward, 1989). Once the microorganisms are injected into the aquifer, there
must be some mechanism for dispersing them throughout the biostimulation zone
before they attach to the solid matrix and carry out the degradation reaction of
interest. Cell transport within porous media is highly dependent on the
characteristics of both the solid media and the microbial cells. Experiments have
shown that the conditions that best promote microbial transport in porous media
include (in order of their importance) highly permeable media, ground water of
low ionic strength, and small-diameter cells (Fontes et al., 1991). To date, there
has been little convincing evidence for successful in situ remediation of aquifers
resulting from introduced microbial populations.
DETERMINING THE SUCCESS OF IN SITU

BIOREMEDIATION
Perhaps the biggest challenges associated with managing a subsurface
bioreactor for in situ bioremediation are evaluating its progress

in the field and determining when sufficient contaminant destruction has occurred
to warrant discontinuation of the biostimulation. For in situ bioremediation to be
deemed successful, it must be shown that the mass of contaminant in the aquifer
has been decreased to desired levels and that the microbial population caused the
decrease. Factors such as the heterogeneous nature and inaccessibility of
subsurface aquifers, together with competing concurrent processes that affect the
form and location of the contaminant (such as volatilization, sorption,
dissolution, migration, and dilution), all conspire to confound mass balance
analyses. Therefore, specific documentation of the successful application of in
situ bioremediation for the destruction of aquifer contaminants is extremely rare.
It has been asserted that true proof of in situ bioremediation requires convergent
lines of independent evidence of microbial degradation in the field (Madsen,
1991). These include diminished contaminant concentrations within both the
horizontal and the vertical dimensions of the plume; increased microbial growth
on the contaminant of interest in samples taken from the biostimulation zone; and
detection of metabolic products coupled with diminished substrate concentrations
(Madsen, 1991). These types of evidence may not be readily obtainable because
of the complexity of the concurrent physical, chemical, and biological processes
involved, aquifer heterogeneities, and site-monitoring limitations. However, a
range of innovative sampling techniques may be incorporated into the field-
monitoring methods in order to measure and quantify in situ bioremediation and
provide a preponderance of supporting evidence.
Field-Monitoring Methods
The following is a sampling of the diverse field methods that have been
applied with varying degrees of success to determine and quantify the success of
in situ bioremediation:
1. Two field studies in which in situ bioremediation was based on

cometabolism were performed within an initially uncontaminated
confined aquifer (Semprini et al., 1990, 1991). A series of tracer
studies were conducted using bromide to determine flow
characteristics and capture efficiency and chlorinated organics to
determine sorption and retardation factors and to evaluate the initial
degradation potential within the aquifer. Afterward the aquifer was
enriched for methane-oxidizing microorganisms that aerobically
degrade chlorinated organics in one field study and for denitrifying
organisms that anaerobically degrade chlorinated organics in the
other. Comparisons

of chlorinated organic concentrations recovered before and after

biostimulation, and experiments in which the required electron
donors and/or acceptors were eliminated, indicated that in situ
bioremediation was successful in both field studies. The transport
and degradation of the organics were quantified and the reaction
kinetics were calculated by fitting models to the experimental data.
The extensive instrumentation and monitoring facilities used in these
studies provided much more accurate mass balances than could be
expected for typical field applications.
2. Natural gradient tracer tests were used to compare methane oxidation
activities in pristine and sewage-contaminated aquifers (Smith et al.,
1991). Two inert ion tracers, chloride and bromide, and an inert
dissolved gas tracer, hexafluoroethane, were used to determine
advective and diffusive ground water characteristics. Methane
break-through curves were measured at both sites, and methane
oxidation was estimated from differences between tracer and
methane recoveries. Methane oxidation was confirmed by injecting
carbon-13-labeled methane and by recovering carbon-13-labeled
carbon dioxide. Quantification of the methane degradation was
possible, and a one-dimensional transport-and-decay model was used
with the field data to determine kinetic degradation parameters.
3. Push/pull tests were used in a field study to determine whether
oxygen addition was enhancing the subsurface degradation of
polynuclear aromatics (Borden et al., 1989). Ground water was
extracted from the aquifer, mixed with aromatics and chloride tracer,
oxygenated or deoxygenated, and then rapidly reinjected into the
bioactive zone. Samples from the reinjection well were analyzed
periodically to determine oxygen, aromatics, chloride, and
conductivity. The tracer was used to determine the recapture
efficiency and to help compensate for dilution factors. While a
comparison of results from the oxygenated and deoxygenated tests
suggested that oxygen enhanced aromatic degradation, thus proving
successful bioremediation, data from the push/pull test alone were
not sufficient to quantify the degradation.
4. Indirect evidence for remediation of coal tar constituents was
collected by conducting laboratory comparisons of degradation
activities and microbial distributions in contaminated and pristine
core samples (Madsen et al., 1991). Increased numbers of
contaminant-degrading microorganisms in the contaminated cores,
coupled with increased populations of protozoans within the
contaminant plume, provided evidence for in situ bioremediation but
did not permit quantification of the degradation.
5. In situ bioremediation of jet fuel was qualitatively demonstrated

in an actively vented region of the unsaturated zone by comparing

ratios of stable isotopes (carbon-13/carbon-12) associated with
carbon dioxide from atmospheric and vented gas samples (Hinchee
et al., 1991). Active jet fuel degradation was confirmed by assuming
that higher isotope ratios indicated atmospheric or plant respiratory
origin of the carbon dioxide, while lower ratios indicated petroleum
hydrocarbon degradation. However, while this approach may offer
qualitative evidence for in situ bioremediation, the results are
nonquantifiable.
CONCLUSIONS
In situ bioremediation is the management of a subsurface bioreactor to carry
out specific biological degradations of ground water contaminants. Successful
implementation should include a thorough aquifer characterization, removal of
contaminant source and free product, plume containment, laboratory feasibility
studies, installation and operation of biostimulation controls, and continuous
monitoring.
Although proving the success of in situ bioremediation is challenging, a
variety of field methods can be used to provide adequately convincing evidence
of success. Quantification of in situ bioremediation, however, is much more
difficult, requiring mass balances that may be achievable only under the most
controlled circumstances.
ACKNOWLEDGMENTS
This work was sponsored in part by the National Institute of Environmental
Health Sciences under grants P42-ES047905 and P42-ES04705 and by the U.S.
Department of Energy Junior Faculty Research Award Program administered by
Oak Ridge Associated Universities.
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MODELING IN SITU BIOREMEDIATION 153
Modeling In Situ Bioremediation
Philip B. Bedient and Handadi S. Rifai

Rice University
Houston, Texas
SUMMARY
The problem of quantifying biodegradation of subsurface pollutants can be
addressed by using models that combine physical, chemical, and biological
processes. Developing such models is difficult, however, for reasons that include
the lack of field data on biodegradation and the lack of numerical schemes that
accurately simulate the relevant processes. This paper reviews modeling efforts,
including BIOPLUME II.
INTRODUCTION
One of the aquifer remediation methods that has been gaining more
widespread attention recently is bioremediation, the treatment of subsurface
pollutants by stimulating the growth of native microbial populations. The purpose
is to biodegrade complex hydrocarbon pollutants into simple carbon dioxide and
water. The technology is not novel; biodegradation of organic contaminants has
been recognized and utilized in the wastewater treatment process for years.
Bioremediation is not without its problems, however. The most important
are the lack of well-documented field demonstrations, preferably quantitative, of
the effectiveness of the technology and its long-term effects, if any, on ground
water systems. Other problems include

the possibility that the biodegradation process will generate undesirable

intermediate compounds that are more persistent in the environment than the
parent compounds.
MODELING BIODEGRADATION PROCESSES

The problem of quantifying biodegradation in the subsurface can be
addressed by using models that combine physical, chemical, and biological
processes. Developing such models is not simple, however, because of the
complex nature of microbial kinetics, the limitations of computer resources, the
lack of field data on biodegradation, and the lack of robust numerical schemes
that can simulate the physical, chemical, and biological processes accurately.
Several researchers have developed ground water biodegradation models. The
main approaches used for modeling biodegradation kinetics are:
• first-order degradation models,

• biofilm models (including kinetic expressions),
• instantaneous reaction models, and
• dual-substrate Monod models.
These are described in more detail in the next section. A more thorough
discussion of models can be found in an earlier National Research Council report
(National Research Council, 1990).
Previous Modeling Efforts

McCarty et al. (1981) modeled the biodegradation process using biofilm
kinetics. They assumed that substrate concentration within the biofilm changes
only in the direction normal to the surface of the biofilm and that all the required
nutrients except the rate-limiting substrate are in excess. The model employs
three basic processes: mass transport from the bulk liquid, biodecomposition
within the biofilm, and biofilm growth and decay. The authors evaluated the
applicability of the biofilm model to aerobic subsurface biodegradation using a
laboratory column filled with glass beads. The experimental data and the model
predictions were relatively consistent.
Kissel et al. (1984) developed differential equations describing mass
balances on solutes and mass fractions in a mixed-culture biological film within a
completely mixed reactor. The model incorporates external mass transport
effects, Monod kinetics with internal determination of limiting electron donor or
acceptor, competitive and sequential reactions, and multiple active and inert
biological fractions that vary spatially. Results of hypothetical simulations
involving competition between heterotrophs that derive energy from an

organic solute and autotrophs that derive energy from ammonia and nitrite were
presented.
Molz et al. (1986) and Widdowson et al. (1987) presented one-and two-
dimensional models for aerobic biodegradation of organic contaminants in ground
water coupled with advective and dispersive transport. A microcolony approach
was used in the modeling effort: microcolonies of bacteria are represented as
disks of uniform radius and thickness attached to aquifer sediments. Associated
with each colony was a boundary layer of a given thickness across which
substrate and oxygen are transported by diffusion to the colonies. The authors'
results indicated that biodegradation would be expected to have a major effect on
contaminant transport when proper conditions for growth exist. Simulations of
two-dimensional transport suggested that under aerobic conditions microbial
degradation reduces the substrate concentration profile along longitudinal
sections of the plume and retards the lateral spread of the plume. Anaerobic
conditions developed in the center of the plume because of microbial
consumption and limited oxygen diffusion into the plume's interior.
Widdowson et al. (1988) extended their previous work to simulate oxygen-
and/or nitrate-based respiration. Basic assumptions incorporated into the model
include a simulated particle-bound microbial population comprised of
heterotrophic facultative bacteria in which metabolism is controlled by lack of an
organic carbon electron donor source (substrate), an electron acceptor (oxygen
and/or nitrate), a mineral nutrient (ammonium), or all three simultaneously.
Srinivasan and Mercer (1988) presented a one-dimensional, finite difference
model for simulating biodegradation and sorption processes in saturated porous
media. The model is formulated to accommodate a variety of boundary
conditions and process theories. Aerobic biodegradation was modeled using a
modified Monod function; anaerobic biodegradation was modeled using
Michaelis-Menten kinetics. In addition, first-order degradation was allowed for
both substances. Sorption was incorporated using linear, Freundlich, or Langmuir
equilibrium isotherms for either substance.
MacQuarrie and Sudicky (1990) used the model developed by MacQuarrie
et al. (1990) to examine plume behavior in uniform and random flow fields. In
uniform ground water flow, a plume originating from a high-concentration source
will experience more spreading and slower normalized mass loss than a plume
from a source of lower initial concentration because dissolved oxygen is more
quickly depleted. Large ground water velocities produced increases in the rate of
organic solute mass loss because of increased mechanical mixing of the organic
plume with oxygenated ground water.

Development and Application of BIOPLUME

Borden and Bedient (1986) developed the first version of the BIOPLUME
model. They developed a system of equations to simulate the simultaneous
growth, decay, and transport of microorganisms combined with the transport and
removal of hydrocarbons and oxygen. Simulation results indicated that any
available oxygen in the region near the hydrocarbon source will be rapidly
consumed. In the body of the hydrocarbon plume, oxygen transport will be rate-
limiting and the consumption of oxygen and hydrocarbon can be approximated as
an instantaneous reaction. The major sources of oxygen, this research concluded,
are transverse mixing, advective fluxes, and vertical exchange with the
unsaturated zone.
Rifai et al. (1987, 1988) expanded and extended the original BIOPLUME
and developed a numerical version of the biodegradation model (BIOPLUME II)
by modifying the U.S. Geological Survey (USGS) two-dimensional method of
characteristics model (Konikow and Bredehoeft, 1978). The basic concept used in
developing BIOPLUME II includes the use of a dual-particle mover procedure to
simulate the transport of oxygen and contaminants in the subsurface.
Biodegradation of the contaminants is approximated by the instantaneous
reaction model. The ratio of oxygen to dissolved contaminants consumed by the
reaction is determined from an appropriate stoichiometric model (assuming
complete mineralization). In general, the transport equation is solved twice at
every time step to calculate the oxygen and contaminant distribution:
where C and O are the concentration of contaminant and oxygen,

respectively; C' and O' are the concentration of contaminant and oxygen in a
source or sink fluid; n is the effective porosity; b is the saturated thickness; t is
time; xi and xj are Cartesian co-ordinates; W is the volume flux per unit area; Vi is
the seepage velocity in the direction of xi; Rc is the retardation factor for the
contaminant; and Dij is the coefficient of hydrodynamic dispersion.
The BIOPLUME II model simulates dissolved contaminant concentrations
vertically averaged over the thickness of the aquifer. The

two plumes are combined using the principle of superposition to simulate the
instantaneous reaction between oxygen and the contaminants, and the decrease in
contaminant and oxygen concentrations is calculated from:
where DCRC and DCRO are the calculated changes in the concentrations of
contaminant and oxygen, respectively, caused by biodegradation, and F is the
ratio of oxygen to contaminant consumed.
The only input parameters to BIOPLUME II that are required to simulate
biodegradation are the amount of dissolved oxygen in the aquifer prior to
contamination and the oxygen demand of the contaminant determined from a
stoichiometric relationship. Other parameters are the same as would be required
to run the standard USGS model in two dimensions (Konikow and Bredehoeft,
1978).
Borden et al. (1986) used the first version of the BIOPLUME model to
simulate biodegradation of polycyclic aromatic hydrocarbons at the Conroe
Superfund site in Texas. Oxygen exchange with the unsaturated zone was
simulated as a first-order decay in hydrocarbon concentration. The loss of
hydrocarbon because of horizontal mixing with oxygenated ground water and
resulting biodegradation was simulated by generating oxygen and hydrocarbon
distributions independently and then combining them by superposition. Simulated
oxygen and hydrocarbon concentrations closely matched the observed values at
the Conroe site.
Rifai et al. (1988) used BIOPLUME II to model biodegradation of aviation
fuel at the U.S. Coast Guard Station, Traverse City, Michigan (Figure 1).
Vertically averaged plume data came from 25 wells. The modeling results along
the centerline of the contaminant plume were good, and the BIOPLUME II
results matched the field observations except in an area between monitoring well
M30 and the pumping wells.
Chiang et al. (1989) used BIOPLUME II to characterize hydrocarbon
biodegradation in a shallow aquifer. They measured the soluble hydrocarbon
concentrations and dissolved oxygen levels in monitoring wells. Results from 10
sampling periods over 3 years showed a significant reduction in total benzene
mass with time. The natural attenuation rate was calculated to be 0.95 percent per
day. Spatial relationships between dissolved oxygen and total benzene, toluene,

and xylene (BTX) were shown to be strongly correlated by statistical analyses

and solute transport modeling using BIOPLUME II. The results were remarkably
consistent with field data on the presence of high or low levels of BTX and
dissolved oxygen in several monitoring well samples.
FIGURE 1 Aviation fuel plume at the Traverse City field site (quarter 2,1986).
SOURCE: Rifai et al. (1988). Journal of Environmental Engineering, Vol.
114:1021. Copyright © by the American Society of Civil Engineers (ASCE).
Reprinted with permission of ASCE.

REFERENCES
Borden, R. C., and P. B. Bedient. 1986. Transport of dissolved hydrocarbons influenced by reaeration
and oxygen limited biodegradation: 1. Theoretical development. Water Resources Research
22:1973-1982.
Borden, R. C., P. B. Bedient, M. D. Lee, C. H. Ward, and J. T. Wilson. 1986. Transport of dissolved
hydrocarbons influenced by oxygen limited biodegradation: 2. Field application. Water
Resources Research 22:1983-1990.
biodegradation of benzene, toluene, and xylene in a sandy aquifer—data analysis and
computer modeling. Ground Water 27(6):823-834.
Kissel, J. C., P. L. McCarty, and R. L. Street. 1984. Numerical simulation of mixed-culture biofilm.
American Society of Civil Engineers Journal of Environmental Engineering Division
110:393.
Konikow, L. F., and J. D. Bredehoeft. 1978. Computer Model of Two-Dimensional Solute Transport
and Dispersion in Ground Water: Automated Data Processing and Computations.
Techniques of Water Resources Investigations of the U.S. Geological Survey. Washington,
D.C.: U.S. Geological Survey.
MacQuarrie, K. T. B., and E. A. Sudicky. 1990. Simulation of biodegradable organic contaminants in
groundwater: 2. Plume behavior in uniform and random flow fields. Water Resources
Research 26(2):223-239.
MacQuarrie, K. T. B., E. A. Sudicky, and E. O. Frind. 1990. Simulation of biodegradable organic
contaminants in groundwater: 1. Numerical formulation in principal directions. Water
Resources Research 26(2):207-222.
McCarty, P. L., M. Reinhard, and B. E. Rittmann. 1981. Trace organics in groundwater.
Environmental Science and Technology 15(1):40-51.
Molz, F. J., M. A. Widdowson, and L. D. Benefield. 1986. Simulation of microbial growth dynamics
coupled to nutrient and oxygen transport in porous media. Water Resources Research
22:107.
National Research Council. 1990. Ground Water Models: Scientific and Regulatory Applications.
Washington, D.C.: National Academy Press.
Rifai, H. S., P. B. Bedient, R. C. Borden, and J. F. Haasbeek. 1987. BIOPLUME II Computer Model
of Two-Dimensional Contaminant Transport Under the Influence of Oxygen Limited
Biodegradation in Ground Water User's Manual Version 1.0. Houston: Rice University,
National Center for Ground Water Research.
Rifai, H. S., P. B. Bedient, J. T. Wilson, K. M. Miller, and J. M. Armstrong. 1988. Biodegradation
modeling at a jet fuel spill site. American Society of Civil Engineers Journal of
Environmental Engineering Division 114:1007-1019.
Srinivasan, P., and J. W. Mercer. 1988. Simulation of biodegradation and sorption processes in
ground water. Ground Water 26(4):475-487.
Widdowson, M. A., F. J. Molz, and L. D. Benefield. 1987. Development and application of a model
for simulating microbial growth dynamics coupled to nutrient and oxygen transport in
porous media. Pp. 28-51 in Proceedings of the Association of Ground Water Scientists and
Engineers/International Ground Water Model Center, Holcomb Research Center Institute
Conference on Solving Ground Water Problems with Models. Dublin, Ohio: National
Ground Water Association.
Widdowson, M. A., F. J. Molz, and L. D. Benefield. 1988. A numerical transport model for oxygen-
and nitrate-based respiration linked to substrate and nutrient availability in porous media.
Water Resources Research 24(9):1553-1565.

TESTING BIOREMEDIATION IN THE FIELD 160
Testing Bioremediation in the Field
John T. Wilson
U.S. Environmental Protection Agency
Robert S. Kerr Environmental Research Laboratory
Ada, Oklahoma
SUMMARY
An operational definition for success of in situ bioremediation at field scale
includes meeting regulatory goals for ground water quality in a timely fashion at a
predictable cost. Current practice for site characterization does not adequately
define the amount of contamination subject to bioremediation. As a result,
laboratory estimates of the requirements for electron acceptors and mineral
nutrients and of the time required for remediation have much uncertainty.
Another aspect of success is the capacity to continue to meet regulatory goals for
ground water quality after the active phase of remediation is complete. In
contrast to laboratory studies, the extent of remediation achieved at field scale is
influenced by dilution of compounds of regulatory concern in circulated water
and by partitioning of the regulated compounds between water and residual
nonaqueous-phase oily material. The extent of weathering of residual oily-phase
material and the hydrologic environment of the residual have a strong influence
on the potential for ground water contamination after active remediation ceases.

INTRODUCTION
Transfer of bioremediation laboratory research to the field is often a
frustrating and unsatisfying activity. Part of the problem has to do with the levels
of inquiry in the laboratory and in the field. Laboratory studies deal with
biochemical or physiological processes. Appropriate controls ensure that only one
mechanism is responsible for the phenomena under study. During field-scale
implementation of bioremediation technology, several processes operate
concurrently. They may involve several distinct mechanisms for biological
destruction of the contaminant, as well as partitioning to immobile phases,
dilution in ground water, and volatilization.
Experimental controls are usually unavailable during full-scale
implementation of in situ bioremediation because the technology is applied
uniformly to the contaminated area. As a result, performance monitoring that is
limited to the concentration of contaminants in ground water over time, and
perhaps the concentrations of nutrients and electron acceptors, cannot ensure that
the biological process developed in the laboratory was responsible for
contaminant removal at full scale.
The appropriate equivalent of experimental controls is a detailed
characterization of the site, the flow of remedial fluids, and the flux of
amendments. This characterization allows an assessment of the influence of
partitioning, dilution, or volatilization and provides a basis for evaluating the
relative contribution of bioremediation.
APPROPRIATE SITE CHARACTERIZATION

Most plumes of organic contamination in ground water originate from spills
of refined petroleum hydrocarbons, such as gasoline, or chlorinated solvents, such
as trichloroethylene. These substances enter the subsurface as nonaqueous-phase
oily liquids, traveling separately from the ground water. As long as the oily-phase
liquid is present in the subsurface, it can act as a continuing source of
contamination because contaminants contained in the nonaqueous phase will
dissolve in the ground water.
Traditionally, monitoring wells have been used to define the extent of
contamination in the subsurface environment. However, wells cannot determine
the extent of contamination by oily-phase materials. If monitoring well data are
the only data available, it is difficult to estimate the total contaminant mass
subject to remediation within an order of magnitude.
As an example, Kennedy and Hutchins (1992) estimated the mass

of alkylbenzenes released to a shallow water table aquifer from a pipeline spill of

refined petroleum products. The contaminated area was roughly circular with a
diameter of 150 m. Thirteen monitoring stations were located uniformly across
the spill. At each station a series of continuous cores was taken extending from
clean material above the spill, through the spill, to clean material below it. Then
monitoring wells were installed in the boreholes used to acquire the cores.
The cores were extracted and analyzed for the content of BTEX (benzene,
toluene, ethylbenzene, xylenes) and total petroleum hydrocarbons. Ground water
was collected and analyzed for the same parameters. Data from the 13 stations
were subjected to geostatistical analysis to estimate the total contaminant mass in
the aquifer and the total mass dissolved in the ground water (see Clark, 1979, for a
description of geostatistics). Kennedy and Hutchins (1992) used proprietary
computer software to estimate the total mass of contaminants. SURFER,
available from Golden Software, Denver, Colorado, supports linear kriging of
plan two-dimensional data following the trapezoidal rule, Simpson's rule, and
Simpson's 3/8 rule. Lacking information on the structure of the data, Kennedy and
Hutchins ran all three simulations and took the numerical average.
Of 320 kg of benzene in the aquifer, only 22 kg was dissolved in the ground
water; of 8800 kg of BTEX compounds, only 82 kg was dissolved; and of
390,000 kg of total petroleum hydrocarbons, only 115 kg was dissolved.
Monitoring well data grossly underestimated the extent of contamination. This
research shows the need for site characterization techniques that can accurately
estimate the total mass of contaminants subject to bioremediation.
Estimates of Total Contaminant Mass

Estimates of total contaminant mass in the subsurface are required to predict
the demand for nutrients and electron acceptor that must be met to complete the
remediation. If the total demand for nutrients and electron acceptor has been
estimated, the rate of supply of the limiting requirement can be used to estimate
the time required for remediation.
The most rigorous approach involves the collection of cores from the
contaminated regions of the subsurface environment, followed by extraction and
analysis of the cores for the contaminants of concern. A wireline piston sampler
(Zapico et al., 1987) makes it possible to collect representative continuous cores,
even in noncohesive material.

To preclude losses to volatilization and biodegradation, current good

practice recommends against shipping core samples to a laboratory for
extraction. When cores are subsampled and extracted in the field, the variability
between replicate subsamples is much smaller (Siegrist and Jenssen, 1990).
Standard operating procedure at the Robert S. Kerr Environmental Research
Laboratory uses a paste sampler to take replicate subcores of each core sample.
Each subcore weighs 10 to 15 g and represents 10 to 15 cm of vertical core. The
subcore is delivered to a 40-ml vial, containing 5 ml of methylene chloride, which
is sealed with a Teflon-faced septum. The sample is dispersed in the solvent to
begin extraction, preclude volatilization, and prevent biodegradation. The vials
are shipped to the laboratory for subsequent extraction and analysis.
Unless the cores are screened to identify those that deserve analysis, this
approach may be too expensive to be practical for general use. The data set
reported by Kennedy and Hutchins (1992) contained more than 400 analyses.
However, there are several techniques that can reduce the analytical burden.
Headspace analysis can be used to screen cores in the field to determine whether
the depth interval represented by a core is contaminated with oily-phase
hydrocarbons, so that field extraction and analysis of the core are justified.
Aquifer samples can be equilibrated with the headspace of a plastic bag
before analysis by a field gas chromatograph, organic vapor analyzer, or
explosimeter (Robbins et al., 1989). Alternately, a plug can be removed from a
core with a paste sampler and the inlet to an explosimeter or organic vapor
analyzer inserted directly into the cavity (Kampbell and Cook, 1992). These
techniques are inexpensive and generate data in real time, which allows the
screening information to be used to guide decisions about depth and location of
subsequent cores.
Often the meter's response in the field headspace analyses has a strong
correlation to the content of total petroleum hydrocarbons. In these cases, results
from a limited number of expensive core analyses can be extrapolated to a large
number of inexpensive field headspace analyses. Kampbell and Cook (1992)
compared the hydrocarbon vapor concentrations in the cavities left when plugs
were removed from cores for extraction to the content of total petroleum
hydrocarbons in the cores. The correlation coefficient between meter response
and hydrocarbon content was 0.957 on a set of 24 cores and 0.801 on a set of 64
cores.

Estimates of Contaminant Dilution in Ground Water

In the process of remediation, prodigious quantities of water may be
circulated through an oily-phase spill. The compounds of regulatory concern,
such as the BTEX compounds, are often more water-soluble than the other
components of refined petroleum hydrocarbons. As the circulated water sweeps
through the spill, the more water-soluble components partition into the water and
are diluted out. The concentration of regulated compounds will drop because of
simple dilution.
Downs et al. (1989, in press) quantitatively described this effect in a pilot-
scale demonstration of in situ bioremediation of a jet fuel spill using nitrate as the
electron acceptor. They used a tracer to estimate the volume of recirculated
water, and they cored the area perfused by ground water to estimate the quantity
of total hydrocarbons and the quantity of hydrocarbons of regulatory concern.
Simple partitioning theory was used to calculate the distribution of hydrocarbons
of concern between recirculating ground water and the residual jet fuel.
Estimate of Recirculated Volume of Water

An infiltration gallery sited above the spill effectively perfused a plan
surface area of 130 m2 with nitrate-amended ground water. The infiltrated water
was recovered in five purge wells. In Figure 1 a computer model predicts flow
paths from the infiltration gallery to the recovery wells. In Figure 2 a cross
section shows the relationship of the infiltration gallery, the contaminated
interval, and the recovery wells. A pulse of chloride was used to trace the flow of
water to the recovery wells. The volume of circulated water was considered to be
the pumping rate multiplied by the travel time of the chloride between infiltration
and the recovery wells. The arrival time at each well was weighted by its
pumping rate to calculate the overall residence time, and circulated volume,
between infiltration and recovery. Figure 3 shows breakthrough curves of the
chloride tracer.
In the demonstration the average residence time was 10 days and the
circulated volume was 10,900 m3.
Estimate of Partitioning Between Oil and Water

The area of the spill that was perfused by the infiltration gallery was cored to
determine the total quantity of jet fuel and the quantities of benzene, toluene,
ethylbenzene, and the xylenes. Smith et al.

(1981) reported empirical partition coefficients for the compounds between JP-4
jet fuel and water. To estimate the distribution of an individual BTEX compound
between fuel and water, the published partition coefficients were multiplied by
the ratio of the volume of JP-4 under the infiltration gallery to the volume of
water in circulation. The distribution between oil and fuel was used to calculate
the fraction of total material in oil or water. To predict the equilibrium solution
concentration of a BTEX compound in circulation, the quantity of the compound
originally in the fuel was multiplied by the fraction that should partition to water
and was divided by the circulated volume of ground water.
FIGURE 1 Hydraulic model of in situ bioremediation of a JP-4 spill at the

Traverse City demonstration site. Ground water amended with mineral nutrients
and nitrate as an electron acceptor was recharged through an infiltration gallery.
The model predicted flow lines from the infiltration gallery to pumping wells
(PP5 to PP9) that capture and recirculate ground water. To capture the infiltrated
water containing nitrate, more water was pumped than was delivered to the
gallery. As a result, some of the flow lines to the wells originate in
uncontaminated ground water upgradient of the spill.

FIGURE 2 Cross section of a JP-4 spill at the Traverse City demonstration site.
Ground water was amended with mineral nutrients and nitrate as an electron
acceptor. Water was recirculated to an infiltration gallery installed above the
JP-4 spill. It moved vertically across the spill, then laterally through the aquifer
to the recovery wells. Part of the recovered water was recirculated; part was
purged.
Table 1 compares the predicted dilution of benzene, toluene, and o-xylene to

the actual concentration in monitoring wells before recirculation of ground water.
Dilution alone produced at least a fivefold reduction in concentration.
To maintain hydraulic control over the spill, a fraction of the recovered
water was discharged to waste. This flow was replaced with clean water from the
aquifer. If the circulation system behaved as a completely mixed reactor, solutes
in the circulated water would be diluted at a first-order rate of 0.03 per day.
Dilution of a BTEX compound was estimated by multiplying the rate of dilution
of circulated water by the portion of the total mass that partitioned to the
circulated water.
Estimate of Bioremediation
The actual behavior of benzene is depicted in Figure 4. The dashed line is
the calculated equilibrium concentration of benzene in the recirculated water
based on partitioning and dilution. The solid line

shows concentrations in the recirculation well that captured the greatest portion
of infiltrated water. Concentrations rose slowly over time, overshot the prediction
at about two recirculation volumes, then showed good agreement with the
prediction for another recirculation volume. Then biological acclimation
occurred, and benzene was removed from the circulated water. Concentrations
dropped below the analytical detection limit over a 2-day period. Concentrations
of other BTEX compounds were not reduced (compare data for o-xylene in
Figure 5), which established that the removal was a biological process. If a
physical or chemical process had been responsible for the
FIGURE 3 Breakthrough of chloride from a tracer test conducted to evaluate the

hydraulic model of water flow. Chloride was added to water supplied to the
infiltration gallery. The concentration of chloride that breaks through is
proportional to the number of flow lines originating in the infiltration gallery,
compared to flow lines originating upgradient in the aquifer.

TABLE 1 Comparison of Concentrations (µg/1) of BTEX Compounds in Ground

Water After Bioremediation to the Concentrations Expected from the BTEX Content
of the Residual Petroleum Hydrocarbons
Compound Concentration Prior Concentration After Concentration
to Bioremediation Bioremediation Predicted from
Residual
Benzene 760 <1 2
Toluene 4500 <1 15
Ethylbenzene 840 6 6
m p-Xylene 2600 23 27
o-Xylene 1380 37 18
FIGURE 4 Bioremediation of a JP-4 jet fuel spill using nitrate. Comparison of

the depletion of benzene in circulated ground water to the depletion predicted
from dilution and partitioning.

FIGURE 5 Comparison of the depletion of o-xylene in circulated groundwater to

the depletion predicted from dilution and partitioning in a JP-4 jet fuel spill.
benzene removal, other BTEX compounds (such as o-xylene) would also

have been removed because these other compounds have physical and chemical
properties similar to those of benzene but are less readily biodegradable.
Note from Figure 4 that removal of benzene occurred before nitrate was
added to the system. This effect had not been predicted in the laboratory
treatability study conducted as part of the design of the pilot-scale demonstration
(Hutchins et al., 1991a) and has not been conclusively reproduced at laboratory
scale since then.
CRITERIA FOR SUCCESS AT FIELD SCALE

The criteria for success at each remediation site are unique, depending on the
particular requirements of state and federal regulators and the particular concerns
of the site owner. However, requirements at many sites can be generalized to the
following:

1. Concentrations of substances of regulatory concern in ground water

at the end of active bioremediation will be less than the cleanup
goals established by the regulatory authorities.
2. Concentrations of substances of regulatory concern in ground water
will not rise above the cleanup goals, within a prescribed period of
monitoring, after active bioremediation is concluded.
3. The site owner will enjoy beneficial use of the property during
remediation and will be allowed to sell or transfer it when
remediation has been complete.
The following considerations have a direct bearing on the first two

requirements.
Can Any Oily-Phase Residual Support a Plume?

Monitoring wells can provide a misleading picture of the course of
bioremediation. Pumping, or seasonal changes in regional water tables, can drop
ground water elevations below the depth interval occupied by oily-phase
contaminants. Water produced by monitoring wells may be clean, but
contamination will return when pumping stops or recharge raises the regional
water table elevation. Changes in the stage of nearby rivers or lakes, combined
with seasonal variations in recharge, may alter the slope of the water table
(hydraulic gradient), which will change the trajectory of the plume of
contamination. Plumes may actually move away from monitoring wells under
these conditions, then return to them later.
To supplement data from monitoring wells, many regulatory authorities
require a measure of residual oily-phase material left after bioremediation.
Cleanup goals are usually set with the conservative assumption that the relative
composition of oily-phase material does not change during remediation. As a
result, concentrations of oily-phase material that are determined to be protective
of ground water quality are low, on the order of 10 to 100 mg total petroleum
hydrocarbon per kilogram of aquifer material (Bell, 1990).
Bioremediation, particularly innovative bioremediation that uses an electron
acceptor other than oxygen, can remove the compounds of regulatory concern
from the subsurface while leaving significant amounts of oily-phase
hydrocarbons. The issue is whether any residual oily-phase hydrocarbon is
capable of producing a plume of contamination at concentrations that exceed the
cleanup goal.
The JP-4 bioremediation demonstration at Traverse City, Michigan, was
used to evaluate the importance of partitioning of contaminants between ground
water and residual oily material. The concentrations

of BTEX compounds in recirculated ground water were compared to

concentrations in the weathered oily-phase residual.
When infiltration with nitrate brought the concentrations of BTEX
compounds in the JP-4 spill below action levels, infiltration was stopped and
concentrations of BTEX compounds in the aquifer were measured under natural
conditions. The JP-4 contaminated interval was cored and analyzed for residual
total hydrocarbons and concentrations of BTEX compounds. The reduction in
concentration of total petroleum hydrocarbons was minimal, from 2000 mg/kg to
1400 mg/kg. The concentrations of BTEX compounds in the residual oil were
divided by the fuel-to-water partition coefficients of Smith et al. (1981) to predict
the capacity of the residual to contaminate ground water. The concentrations
measured in ground water under natural conditions were near or below the
predicted concentrations (Hutchins et al., 1991b; see Table 1).
Apparently ground water quality is controlled by the relative concentration
of organic contaminants in the weathered oily-phase residual and not by the
absolute amount of weathered total petroleum hydrocarbons. The relative
concentrations of organic contaminants can be used to predict the concentrations
in ground water in contact with the oily-phase residual.
Accounting for Spatial Heterogeneity

Bioremediation is difficult to assess in heterogeneous geological material.
Often, oily-phase material is associated with fine-textured material with low
hydraulic conductivity. Remedial fluids tend to pass around the fine-textured
material. Because the flux of nutrients and electron acceptor through the fine-
textured material is small, there is little opportunity for bioremediation, and
significant concentrations of contaminants can remain in subsurface material.
These relationships will be illustrated in a case history from an industrial site
in Denver, Colorado (Nelson et al., in press). At this site, a temporary holding
tank under a garage leaked used crankcase oil, diesel fuel, gasoline, and other
materials into a shallow water table aquifer. Figure 6 shows the relationship
between the garage, the work pit containing the leaking holding tank, and the
approximate area of the spill.
Remediation involved removal of separate oily phases, in situ
bioremediation with hydrogen peroxide and mineral nutrients, and bioventing.
Ground water flow under ambient conditions was to the north or northeast. The
flow of water during the remediation paralleled the natural gradient. Water was
produced from a recovery well

on the northeast side of the spill. The flow from the well was split; part of the flow
was amended with hydrogen peroxide and nutrients and recharged to the aquifer
in a nutrient recharge gallery on the south side of the spill (Figure 6). The
remainder of the flow was delivered to a ground water recharge gallery to the
south of the nutrient recharge gallery. From 3 to 6 gpm (11 to 22 l/min) was
delivered
FIGURE 6 Infrastructure at an in situ bioremediation project in Denver,

Colorado. A holding tank in a work pit under a garage leaked petroleum
hydrocarbons to the water table aquifer. Ground water was pumped from a
recovery well (RW-1) and filtered through activated carbon. The flow was split.
Part was amended with hydrogen peroxide and mineral nutrients and recharged
in a nutrient recharge gallery. The remainder was recharged in a ground water
recharge gallery. The system was designed to sweep hydrogen peroxide and
nutrients under the service building. MW-1, 2A, and 3 are monitoring wells, and
61A through 61J are boreholes for cores.

to the nutrient recharge gallery, and 4 to 8 gpm (15 to 30 l/min) was delivered to
the ground water recharge gallery, for a total flow of 9 to 11 gpm (34 to 42 l/
min). Figure 7 presents a mathematical model of the flow paths from the galleries
to the recovery well. The system was designed to sweep the ground water
containing hydrogen peroxide and mineral nutrients through the spill to the
recovery well.
FIGURE 7 Hydraulic model of flow from the nutrient recharge gallery to the
recovery well. Note that the spill is contained within the flow path from the
gallery to the well and that the well captures all the flow lines from the nutrient
recharge gallery.
The system was operated from October 1989 to March 1992. At a flow of 10
gpm (38 l/min), 10 to 15 pore volumes would have been exchanged in the area
between the nutrient recharge gallery and the recovery well.

Remediation of Ground Water at the Denver Site

Table 2 compares the reduction in concentrations of benzene and total BTEX
compounds in ground water that was achieved by in situ bioremediation at the
site in Denver. Monitoring wells MW-1 and MW-8 (shown in Figure 6) are in
areas with oily-phase hydrocarbons. Well MW-2A is just outside the region with
oily-phase hydrocarbon. Well MW-3 is a significant distance from the region
with oily-phase hydrocarbon; it sampled the plume of contaminated ground water
that moved away from the spill.
Before remediation, concentrations in wells MW-1 and MW-8 were
equivalent. Well MW-1 was closest to the nutrient recharge gallery, and the
aquifer surrounding MW-1 was completely remediated; BTEX compounds were
undetectable in ground water. In well MW-8, immediately adjacent to the point
of release, the concentration of benzene was reduced at least one order of
magnitude, and the concentrations of benzene and BTEX compounds in well
MW-3 were also reduced an order of magnitude.
It is of particular interest that significant concentrations of benzene or total
BTEX never developed in the pumped recovery well (RW-1). The BTEX
compounds were monitored twice a month from July 1989 to March 1992.
Benzene was detected only twice, at a concentration of 2 µg/1. The other BTEX
compounds were never detected. Water from contaminated flow paths sampled by
MW-3 was probably diluted by uncontaminated water from other flow paths to
RW-1 (compare Figure 7). This behavior illustrates the contrast in contaminant
concentrations between passive monitoring wells and pumped wells.
TABLE 2 Reduction in Concentration (µg/1) of Hydrocarbon Contaminants in Ground
Water Achieved by In Situ Bioremediation
Benzene Total BTEX
Well Before During After Before During After
MW-1 220 <1 <1 2030 164 <6
MW-8 180 130 16 1800 331 34
MW-2A ? 11 0.8 ? 1200 13
MW-3 11 5 2 1200 820 46
RW-1 <1 2 <1 <1 2 <1

Remediation of Subsurface Material at the Denver Site

Water in the monitoring wells and the recirculation well contained low
concentrations of contaminants by March 1992. Active remediation was
terminated, and the site entered a period of postremediation monitoring.
In June 1992 core samples were taken from the aquifer to determine the
extent of hydrocarbon contamination remaining and whether a plume of
contamination could return once active remediation ceased. The site was cored
along a transect downgradient of the release. The transect extended laterally from
clean material, through part of the spill, into clean material on the other side. In
each borehole, continuous cores extended vertically from clean material above
the spill, through the spill, to clean material below. The cores were extracted and
analyzed for total petroleum hydrocarbons and for the concentrations of
individual BTEX compounds.
The relationships between the land surface, the water table, the region
containing hydrocarbons, and the bedrock are presented in Figure 8. Significant
amounts of hydrocarbons remain within a narrow interval, approximately 0.6 m
thick, near the water table. The total saturated thickness of the aquifer was
approximately 6 m. At the time of sampling the elevation of the water table was
1610 m (5280.5 ft) above mean sea level, and all the hydrocarbons were below
the water table.
The highest concentrations of hydrocarbons at the Denver site were obtained
in samples from the borehole (D) closest to the work pit. Table 3 presents the
vertical distribution of BTEX compounds and total petroleum hydrocarbons
(TPH) in borehole D. The material in the interior of the spill had higher
proportions of BTEX compounds. Table 4 makes the same comparison at the
most contaminated depth interval along the transect. Material closer to the spill
had higher concentrations of TPH and greater relative proportions of BTEX
compounds.
Figure 9 plots the percentage of BTEX in the residual oil after
bioremediation against the total content of hydrocarbon. Obviously, the materials
with lower residual concentrations of hydrocarbons are more extensively
weathered.
Infiltration of hydrogen peroxide and mineral nutrients at an aviation
gasoline spill in Michigan preferentially removed BTEX compounds from the
oily-phase gasoline, leaving a total petroleum hydrocarbon residual low in
aromatic hydrocarbons (Wilson et al., in press). At the Denver site, apparently, a
cortex of material that has been physically

FIGURE 8 Cross section showing the vertical relationship of the land surface,
water table, residual hydrocarbon after bioremediation, and the lower confining
layer of the aquifer. The cross section runs through core boreholes depicted in
Figure 6.
TABLE 3 Vertical Extent of Total BTEX Compounds and Total Petroleum

Hydrocarbons (mg/kg) at Borehole D, the Most Contaminated Borehole in the
Transect (Figure 8)
Elevationa TPH BTEX Benzene Color and Texture
1609.711 to 1609.458 <44 <1 <0.2 Brown sand
1609.458 to 1609.354 227 5.1 <0.2 Brown sand
1609.354 to 1609.230 860 101 <0.2 Black sand
1609.230 to 1609.101 1176 206 4.3 Black sand
1609.101 to 1609.050 294 27 0.68 Black sand
1609.050 to 1608.949 273 7.4 0.26 Black sand
1608.949 to 1608.821 <34 <1 <0.2 Black sand
1608.821 to 1608.492 <24 <1 <0.2 Brown to yellow sand
a Meters above sea level.

TABLE 4 Lateral Distribution of Total BTEX Compounds and Total Petroleum

Hydrocarbons (mg/kg) Along the Transect (Figure 8) at the Most Contaminated Depth
Interval
Borehole TPH BTEX Benzene
B 167 0.8 <0.2
C 156 3.5 <0.2
D 1176 260 4.3
E 156 3.5 0.06
FIGURE 9 Relationship between extent of hydrocarbon contamination

(concentration of total petroleum hydrocarbons) and the extent of biological and
chemical weathering (reduction in percentage of BTEX compounds in total
petroleum hydrocarbons) in core material after bioremediation at the Denver
site.

and biologically weathered surrounds a central core of material that has not
been depleted of BTEX compounds.
The concentration of an individual petroleum hydrocarbon in solution in
ground water in contact with oily-phase hydrocarbon can be predicted by Raoult's
law. The solution concentration in water should be proportional to the mole
fraction of the hydrocarbon in the oily phase. Assume that the weathered material
is weathered because it is in effective contact with moving ground water that
supplied nutrients and electron acceptors, and the residual is not weathered
because it was not in effective contact and the supply of nutrients and electron
acceptors was inadequate. If partitioning between moving ground water and the
weathered oily residual controls the concentration of hydrocarbons in the water,
the 10-fold reduction in concentrations of benzene and BTEX compounds seen in
the weathered core material (Table 3) would produce the 10-fold reduction in
concentrations of benzene and BTEX compounds seen in the monitoring wells
(Table 2).
Do Mass Transfer Effects Limit Development of a Plume?

The usual expectation for in situ bioremediation is total removal of the
contaminant from the subsurface environment. The extent of remediation more
commonly achieved is removal of the contaminant from the circulated ground
water.
In situ bioremediation merely accelerates the natural physical and biological
weathering processes that occur in the subsurface. The oily material in most
intimate contact with the circulated ground water is weathered to the greatest
extent. After extensive remediation of the more transmissive regions, the release
of contaminants to the circulated ground water is controlled by diffusion and slow
advection from the subsurface material that still contains significant quantities of
contaminants. The relationships are presented in Figure 10.
In such circumstances the disposition of contamination in the ground water
can only be understood as a dynamic system. This release may best be described
through the chemical engineering concept of a mass transfer coefficient. As the
circulated water passes through the weathered spill, a certain quantity of
hydrocarbon is transferred to the water; the amount transferred is directly
proportional to the exposure time of the water in the contaminated area. If the
circulated water contains enough nutrients and electron acceptor to meet the
demand of the contaminants transferred from the fine-textured material, the
plume will be destroyed by biological activity as rapidly as it is produced.
When active remediation is stopped, the concentration of elec

FIGURE 10 Schematic representation of in situ bioremediation in heterogeneous

geological material. A fresh release (panel A) is rapidly weathered (panel B) due
to increased flow of water and increased concentration of electron acceptor. As
weathering progresses, aromatic hydrocarbons such as the BTEX compounds are
restricted to regions with low hydraulic conductivity (panel C). After
bioremediation the flux of aromatic hydrocarbons from the residual core to the
moving ground water is controlled by mass transport limitations. The extent of
the plume produced is controlled by the supply of electron acceptor (panel D).
Although greatly attenuated, the plume may be regenerated under ambient
conditions.

tron acceptor returns to ambient conditions in the aquifer, and the hydraulic
gradient returns to the normal condition. As a result, the residence time of water
in the spill area is longer, and the total amount of hydrocarbon transferred to the
water is greater, although the supply of electron acceptor for biological
destruction of the hydrocarbon is less (compare panels C and D in Figure 10).
TABLE 5 Contrast in Conditions During Active In Situ Bioremediation and
Conditions at the Termination of Remediation at a Spill from an Underground Storage
Tank in Denver Colorado
Parameter During Active Ambient Conditions
Remediation After Remediation
Introduced concentration 470 mg/l 5.5 mg/l
of oxygen
Hydraulic gradient 0.097 m/m 0.0012 m/m
Interstitial flow velocity 2.4 m/day 0.03 m/day
Travel time of water across 20 days to recovery well 1500 days to monitoring
the spill well
Maximum oxygen demand 20 mg/l per day 0.004 mg/l per day
supported
These relationships are well illustrated by the performance of bioremediation

at the Denver site (Table 5). The hydraulic conductivity in the depth interval
containing the hydrocarbons is approximately 8.5 m per day (David Szlag,
University of Colorado, Boulder, personal communication). The distance from
the nutrient recharge gallery to the recovery well is 45 m. Assume that the
distance from the upgradient edge of the spill, through the spill, to monitoring
wells downgradient at the property boundary is also 45 m. Assuming an effective
porosity of 0.35, Darcy's law can be used to estimate the interstitial flow velocity
of the ground water and its residence time along the flow path. When active
remediation was terminated, the residence time of water was 75 times longer, and
the supply of oxygen was 85 times less than conditions during active remediation
(compare Table 5).
In June 1992 core material from the spill was assayed for the potential rate
of oxygen consumption. Core material was dewatered by placing it in a Buchner
funnel and applying a partial vacuum. Then the core material was sealed in a
glass mason jar. After 24 hours, a sample of air in contact with the core material
was passed through an oxygen-indicating tube to estimate oxygen consumption
potential. Acclimated material exerted oxygen demands of 6 to more than 36
mg/kg core material per day (Table 6), equivalent to 40 to more than 240 mg/l
ground water per day. At these rates, the oxygen

supplied as hydrogen peroxide during active remediation would be consumed in 2

to 12 days.
TABLE 6 Relationship Between the Potential Oxygen Uptake Rate (mg oxygen per kg
per day) of Freshly Collected Core Material and the Location of the Core Material with
Respect to the Interval Contaminated with Hydrocarbons
Position of Borehole in the Transect
Location in Borehole A B C D E F
Just Above <4 7.4
Within 15.5 >30 >36 >34 23.5
Just Below 6.0 <3 5.7 7.3 21
In the assay the microbial consumption of oxygen was faster than the rate of
supply during active remediation. If the microbes in the aquifer expressed the
potential rate of oxygen consumption, oxygen would have been depleted before
the recharge water moved across the spill. In the absence of oxygen, BTEX
compounds would have partitioned to the ground water and should have been
detected in the monitoring wells. In fact, oxygen concentrations between 2 and 5
mg/l were always present in water produced by the recovery well, and BTEX
compounds were virtually absent. Oxygen consumption must have been limited
by mass transfer of hydrocarbon to the ground water circulated through the spill.
Relationship to Siting and Sampling Monitoring Wells

No established procedures exist for determining under ambient conditions
whether the mass transfer of hydrocarbons from oily residual material will exceed
the supply of oxygen or other natural electron acceptors. As a result, it is
impossible to predict if natural bioremediation will prevent the regeneration of a
plume, or if a plume of contaminated ground water will regenerate and at what
concentrations. Ground water moving under the natural gradient must be allowed
to travel all the way through the spill and then to the monitoring wells before it is
possible to determine whether mass transfer effects will reestablish a plume.
An assessment of natural hydrologic conditions at a site will be necessary to
intelligently locate compliance monitoring wells and determine an appropriate
schedule of monitoring. Required are an understanding

of the average natural hydraulic gradient and the hydraulic conductivity in the
depth interval containing residual hydrocarbon. This information can be used to
predict the velocity and trajectory of potential plumes of contaminated water. The
frequency of monitoring can be adjusted to reflect the expected time required for
ground water to travel through the area containing residual hydrocarbon to the
point of compliance.
CONCLUSIONS
• When oily-phase materials are to be remediated, core analyses are

required to estimate the total mass of contaminant subject to
remediation.
• Headspace analyses in the filed can be used to screen core samples to
identify those that deserve further analysis in the laboratory. If properly
benchmarked by a limited number of laboratory analyses, field
headspace techniques can provide a rapid and affordable estimate of
total contaminant concentrations.
• Simple ground water flow models can estimate the volume of water
circulated through a spill during in situ bioremediation. This information
can be coupled with simple partitioning theory to estimate the apparent
attenuation due to dilution.
• In situ bioremediation frequently leaves a residual of weathered oily-
phase material.
• Partitioning theory can be used to predict the concentrations of BTEX
compounds in ground water in contact with the weathered oily residual.
• After extensive in situ bioremediation, pockets of fine-textured material
may still contain high concentrations of contaminants.
• Mass transfer effects control the access of residual organic contaminants
to moving ground water.
• Under proper conditions, natural biodegradation supported by ambient
concentrations of electron acceptors and mineral nutrients may destroy
organic contaminants as fast as they escape from the oily-phase residual.
• At the present state of science, only long-term monitoring can determine
if natural biodegradation will prevent the regeneration of a plume of
contaminated ground water.
ACKNOWLEDGMENTS
This work was supported by the United States Air Force through Interagency
Agreement RW 57935114 between the Armstrong Laboratory

Environics Directorate (U.S. Air Force) and the R. S. Kerr Laboratory (U.S.
Environmental Protection Agency). This work was also supported by the U.S.
Environmental Protection Agency through the Bioremediation Field Initiative. It
has not been subjected to agency review and therefore does not necessarily
reflect the views of the agency, and no official endorsement should be inferred.
REFERENCES
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Remediation (Nov.-Dec.):10-16.
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Downs, W. C., S. R. Hutchins, J. T. Wilson, R. H. Douglas, and D. J. Hendrix. 1989. Pilot project on
biorestoration of fuel-contaminated aquifer using nitrate: part I—field design and ground
water modeling. Pp. 219-233 in Proceedings, Petroleum Hydrocarbons and Organic
Chemicals in Ground Water: Prevention, Detection, and Restoration, Nov. 15-17, 1989,
Houston. Dublin, Ohio: National Water Well Association.
Downs, W. C., S. R. Hutchins, J. T. Wilson, R. H. Douglas, and D. J. Hendrix. In press. Nitrate-
mediated biodegradation of BTEX in JP-4 contaminated soil and ground water. In
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Hutchins, S. R., G. W. Sewell, D. A. Kovacs, and G. A. Smith. 1991a. Biodegradation of aromatic
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Hutchins, S. R., W. C. Downs, J. T. Wilson, G. B. Smith, D. A. Kovacs, D. D. Fine, R. H. Douglass,
and D. J. Hendrix. 1991b. Effect of nitrate addition on biorestoration of fuel-contaminated
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Kampbell, D. H., and M. L. Cook. 1992. Core assay method for fuel contamination during drilling
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constraints of in situ BTEX bioremediation. Remediation 3(1):83-108.
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Exner, eds. Chelsea, Mich.: Lewis Publishers.
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Engineering and Services Laboratory, Air Force Engineering and Services Center.
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Jerger, and J. H. Exner, eds. Chelsea, Mich.: Lewis Publishers.
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(3):74-82.

APPENDIXES 185
Appendixes

APPENDIXES 186

GLOSSARY 187
A
Glossary
Abiotic— Occurring without the involvement of microorganisms.

Aerobic Process whereby microorganisms use oxygen as an electron
respiration— acceptor to generate energy.
Air sparging— Injection of air into ground water to remove volatile chemicals
and deliver oxygen, which promotes microbial growth.
Air stripping— Above-ground process used to remove volatile contaminants
from water. It involves exposing the water surface to a large
volume of air, usually by flowing water through a tower in one
direction and air through the tower in the opposite direction.
Aliphatic A compound built from carbon and hydrogen joined in a linear
hydrocarbon— chain. Petroleum products are composed primarily of aliphatic
hydrocarbons.
Anaerobic Process whereby microorganisms use a chemical other than
respiration— oxygen as an electron acceptor. Common ''substitutes" for
oxygen are nitrate, sulfate, and iron.
Aquifer— An underground geological formation that stores ground water.
Aromatic A chemical formed from benzene rings, originally called
hydrocarbon— "aromatic" because of benzene's distinctive aroma. Solvents,
many types of pesticides, and polychlorinated biphenyls are
composed of aromatic hydrocarbons.
Bacterium— A single-celled organism of microscopic size. Bacteria

GLOSSARY 188
are ubiquitous in the environment, inhabiting water, soil,

organic matter, and the bodies of plants and animals.
Benzene— A chemical composed of six carbon atoms arranged in a
hexagonal ring, with one hydrogen atom attached to each
carbon.
Bioaugmentation The addition of nonnative microorganisms to a site.
—
Biocurtain— A large quantity of organisms grown underground specifically
to stop contaminant migration by creating localized clogging.
Biodegradation— Biologically mediated conversion of one compound to another.
Biomass— Total mass of microorganisms present in a given amount of
water or soil.
Bioremediation— Use of microorganisms to control and destroy contaminants.
Biotransformation Microbially catalyzed transformation of a chemical to some
— other product.
Bioventing— Circulation of air through the subsurface to remove volatile
contaminants and provide oxygen, which stimulates
microorganisms to degrade remaining contaminants.
BTEX— Acronym for benzene, toluene, ethylbenzene, and xylenes,
which are compounds present in gasoline and other petroleum
products, coal tar, and various organic chemical product
formulations.
Carbon treatment Above-ground process for removing contaminants from water
— or air. It involves contact between the water or air and activated
carbon, which adsorbs the contaminants, usually by flowing the
water or air through columns packed with carbon.
Carbonate— Any chemical containing the CO32- group; limestone and
dolomite are examples of rocks formed primarily from
carbonate minerals.
Chlorinated A hydrocarbon in which chlorine atoms substitute for one or
solvent— more hydrogen atoms in the compound's structure. Chlorinated
solvents commonly are used for grease removal in
manufacturing, dry cleaning, and other operations. Examples
include trichloroethylene, tetrachloroethylene, and
trichloroethane.
Cometabolism — A reaction in which microbes transform a contaminant even
though the contaminant cannot serve as an energy source for
the organisms. To degrade the contaminant, the microbes
require the presence of other compounds (primary substrates)
that can support their growth.
Complexing agent A chemical agent that chemically bonds with a
—

GLOSSARY 189
positively charged molecule, such as a metal. Complexing

agents can be used to dissolve precipitated metals.
Conservative A chemical that does not undergo microbiological reactions but
tracer— has transport properties similar to those of microbiologically
reactive chemicals (such as the contaminant and oxygen).
Dechlorinate— The removal of chlorine atoms from a compound.
Desorption— Opposite of sorption; the dissolution of chemicals from solid
surfaces.
Deuterium— Hydrogen isotope with twice the mass of ordinary hydrogen; it
contains one proton and one neutron in its nucleus.
Diauxy— Selective biodegradation of some organic compounds over
others, which sometimes occurs when the compounds are
present in mixtures.
DNA Substance within a cell that passes hereditary information from
(deoxyribonucleic one generation to the next.
acid)—
Electron— A negatively charged subatomic particle that may be transferred
between chemical species in chemical reactions. Every
chemical molecule contains electrons and protons (positively
charged particles).
Electron acceptor Compound that receives electrons (and therefore is reduced) in
— the energy-producing oxidation-reduction reactions that are
essential for the growth of microorganisms and bioremediation.
Common electron acceptors in bioremediation are oxygen,
nitrate, sulfate, and iron.
Electron donor— Compound that donates electrons (and therefore is oxidized) in
the energy-producing oxidation-reduction reactions that are
essential for the growth of microorganisms and bioremediation.
In bioremediation the organic contaminant often serves as an
electron donor.
Engineered Type of bioremediation that stimulates the growth and
bioremediation— biodegradative activity of microorganisms by adding nutrients,
electron acceptors, or other stimulants to the site using an
engineered system.
Enzyme— A protein created by living organisms to use in transforming a
specific compound. The protein serves as a catalyst in the
compound's biochemical transformation.
Enzyme induction Process whereby an organism synthesizes an enzyme in
— response to exposure to a specific chemical, the inducer.
Equilibrium— Condition in which a reaction has occurred to its maximum
extent.

GLOSSARY 190
Ex situ— Latin term referring to the removal of a substance from its

natural or original position.
Fermentation— Process whereby microorganisms use an organic compound as
both electron donor and electron acceptor, converting the
compound to fermentation products such as organic acids,
alcohols, hydrogen, and carbon dioxide.
Fixation— Process whereby microorganisms obtain carbon for building
new cells from inorganic carbon, usually carbon dioxide.
Free product Removal of residual pools of contaminants, such as gasoline
recovery— floating on the water table, from the subsurface.
Gas Instrument used to identify and quantify volatile chemicals in a
chromatograph— sample.
Gene probe— One class of oligonucleotide probes. Gene probes are used to
identify the presence of a particular gene (such as the gene
responsible for a particular biodegradative reaction) on the
cell's DNA.
Genetically An organism whose genes have been altered by humans. For
engineered example, researchers have used genetic engineering to give
organism— bacteria the capability to degrade hazardous chemicals that
normally resist biodegradation.
Glacial outwash— Materials (typically sand and gravel) deposited during the
melting of glaciers.
Halogenate— Replacement of one or more hydrogen atoms on a chemical
compound with atoms of a halogen, such as chlorine, fluorine,
or bromine.
High-performance Instrument used to identify and quantify contaminants in a
liquid sample.
chromatograph—
Hydraulic A measure of the rate at which water moves through a unit area
conductivity— of the subsurface under a unit hydraulic gradient.
Hydraulic Change in head (i.e., water pressure) per unit distance in a given
gradient— direction, typically in the principal flow direction.
Hydrocarbon— A chemical composed of carbon and hydrogen in any of a wide
variety of configurations. Petroleum products, as well as many
synthetic industrial chemicals, contain many different
hydrocarbons.
Hydrophobic A "water-fearing" compound, such as oil, that has low solubility
compound— in water and tends to form a separate phase.

GLOSSARY 191
In situ— Latin term meaning "in place"—in the natural or original

position.
Infiltration gallery Engineered system used to deliver materials that stimulate
— microorganisms in the subsurface. Infiltration galleries
typically consist of buried perforated pipes through which
water containing the appropriate stimulating materials is
pumped.
Inorganic A chemical that is not based on covalent carbon bonds.
compound— Important examples are metals, nutrients such as nitrogen and
phosphorus, minerals, and carbon dioxide.
Intrinsic A type of in situ bioremediation that uses the innate capabilities
bioremediation— of naturally occurring microbes to degrade contaminants
without taking any engineering steps to enhance the process.
Intrinsic A measure of the relative ease with which a liquid will pass
permeability— through a porous medium. Intrinsic permeability depends on
the shape and size of the openings through which the liquid
moves.
Isotope— Any of two or more species of an element in the periodic table
with the same number of protons. Isotopes have nearly
identical chemical properties but different atomic masses and
physical properties. For example, the isotope carbon 12 has six
protons and six neutrons, while the isotope carbon 13 has six
protons and seven neutrons. Both have atomic number 6 (the
number of protons), but carbon 13 is more massive than carbon
12 because it carries an extra neutron.
Isotope Selective degradation by microorganisms of one isotopic form
fractionation— of a carbon compound over another isotopic form. For
example, microorganisms degrade the 12C isotopes of
petroleum hydrocarbons more rapidly than the 13C isotopes.
Kinetics— Refers to the rate at which a reaction occurs.
Land farming— Above-ground process used to stimulate microorganisms to
degrade contaminants in soil. The process involves spreading
out the soil, adding nutrients, and tilling.
Ligand— See "complexing agent."
Mass spectrometerInstrument used to identify the chemical structure of a
— compound. Usually, the chemicals in the compound are
separated beforehand by chromatography.
Metabolic A chemical produced by one step in a multistep
intermediate— biotransformation.

GLOSSARY 192
Metabolism— The chemical reactions in living cells that convert food sources
to energy and new cell mass.
Methanogen— A microorganism that produces methane. Because they thrive
without oxygen, methanogens can be important players in
subsurface biotransformations, where oxygen is often absent.
Micelle— An aggregate of molecules, such as surfactant molecules, that
form a small region of nonaqueous-phase within an otherwise
aqueous matrix.
Microcosm— A laboratory vessel set up to resemble as closely as possible the
conditions of a natural environment.
Microorganism— An organism of microscopic or submicroscopic size.
Microorganisms can destroy contaminants by using them as
"food sources" for their own growth and reproduction.
Mineralization— The complete degradation of an organic chemical to carbon
dioxide, water, and possibly other inorganic compounds.
Most-probable- A statistical technique for estimating the number of organisms
number (MPN) present in a sample.
technique—
Nonaqueous- A liquid solution that does not mix easily with water. Many
phase liquid— common ground water contaminants, including chlorinated
solvents and many petroleum products, enter the subsurface in
nonaqueous-phase solutions.
Oligonucleotide A short piece of DNA that can be used to identify the genetic
probe— makeup of microorganisms in a sample and the reactions they
are capable of carrying out.
Organic A compound built from carbon atoms, typically linked in
compound— chains or rings.
Oxidization— Transfer of electrons away from a compound, such as an
organic contaminant. The oxidation can supply energy that
microorganisms use for growth and reproduction. Often (but
not always), oxidation results in the addition of an oxygen atom
and/or the loss of a hydrogen atom.
Petroleum A chemical derived from petroleum by various refining
hydrocarbon— processes. Examples include gasoline, fuel oil, and a wide
range of chemicals used in manufacturing and industry.
Plume— A zone of dissolved contaminants. A plume usually originates
from the contaminant zone and extends for some distance in the
direction of ground water flow.
Primary The electron donor and electron acceptor that are essential to
substrates— ensure the growth of microorganisms. These com

GLOSSARY 193
pounds can be viewed as analogous to the food and oxygen that

are required for human growth and reproduction.
Protozoan— A single-celled organism that is larger than a bacterium and
may feed on bacteria.
Pump-and-treat Most commonly used type of system for cleaning up
system— contaminated ground water. Pump-and-treat systems consist of
a series of wells used to pump contaminated water to the
surface and a surface treatment facility used to clean the
extracted ground water.
Rate-limiting Material whose concentration limits the rate at which a
material— particular process can occur.
Reduction— Transfer of electrons to a compound, such as oxygen. It occurs
when another compound is oxidized.
Reductive A variation on biodegradation in which microbially catalyzed
dehalogenation— reactions cause the replacement of a halogen atom on an
organic compound with a hydrogen atom. The reactions result
in the net addition of two electrons to the organic compound.
Reporter gene— A tool used with genetically engineered microorganisms. When
a reporter gene is incorporated into a microorganism's genetic
material, it provides a signal when the organism is present and
active. An example is a gene that produces a protein that causes
the microorganism to emit light.
Saturated zone— Part of the subsurface that is beneath the water table and in
which the pores are filled with water.
Secondary A chemical that can be transformed by microorganisms through
substrate— secondary utilization.
Secondary General term for the transformation of contaminants by
utilization— microorganisms when the transformation yields little or no
benefit to the organisms.
Slurry wall— A clay barrier constructed in the subsurface to prevent the
spread of contaminants by preventing water flow.
Soil vapor See "Vapor recovery."
extraction—
Sorption— Collection of a substance on the surface of a solid by physical
or chemical attraction.
Substrate— A compound that microorganisms can use in the chemical
reactions catalyzed by their enzymes.
Sulfate reducer— A bacterium that converts sulfate to hydrogen sulfide. Because
they can act without oxygen, sulfate-reducing bacteria can be
important players in the oxygen-limited subsurface.
Surfactant— Soap or a similar substance that has a hydrophobic and

GLOSSARY 194
a hydrophilic end. Surfactants can bond to oil and other

immiscible compounds to aid their transport in water.
Unavailability— Situation in which a contaminant is sequestered from the
microorganism, inhibiting the organism's ability to degrade the
contaminant.
Unsaturated zone Soil above the water table, where pores are partially or largely
— filled with air.
Vadose zone— See "Unsaturated zone."
Vapor recovery— A method for removing volatile contaminants from the soil
above the water table by circulating air through the soil.
Volatilization— Transfer of a chemical from the liquid to the gas phase (as in
evaporation).

BIOGRAPHICAL SKETCHES OF COMMITTEE MEMBERS AND STAFF 195
B
Biographical Sketches of Committee
Members and Staff
BRUCE E. RITTMANN, committee chair, is the John Evans Professor of

Environmental Engineering at Northwestern University and an active researcher
and teacher in the field of environmental biotechnology. His special interests
include biofilm kinetics, microbial ecology, in situ bioremediation, biological
drinking water treatment, and the fate of hazardous organic chemicals. Dr.
Rittmann was a member of the National Research Council committee that
authored Ground Water Models: Scientific and Regulatory Applications. He
recently served as president of the Association of Environmental Engineering
Professors.
LISA ALVAREZ-COHEN, assistant professor of environmental
engineering at the University of California, Berkeley, received a Ph.D. in
environmental engineering and science from Stanford University in 1991 and a
B.A. in engineering and applied science from Harvard University in 1984. Dr.
Alvarez-Cohen's research interests are modeling of microbial processes in porous
media, bioremediation of contaminated aquifers, innovative hazardous waste
treatment technologies, and application of cometabolic biotransformation
reactions.
PHILIP B. BEDIENT, Shell Distinguished Professor of Environmental
Science at Rice University, received a B.S. in physics in 1969, an M.S. in
environmental engineering in 1972, and a Ph.D. in environmental engineering
sciences in 1975 from the University of Florida.

His primary research interests include ground water pollutant transport modeling
and hazardous waste site evaluation.
RICHARD A. BROWN, vice president of remediation technology for
Groundwater Technology, Inc., in Trenton, New Jersey, received a B.A. in
chemistry from Harvard University and a Ph.D. in inorganic chemistry from
Cornell University. His responsibilities include the development and
implementation of remediation technologies such as bioremediation, soil vapor
extraction, and air sparging. Before joining Groundwater Technology, Dr. Brown
was director of business development for Cambridge Analytical Associates'
Bioremediation Systems Division and technology manager for FMC
Corporation's Aquifer Remediation Systems. Dr. Brown holds patents on
applications of bioreclamation technology, on the use of hydrogen peroxide in
bioreclamation, and on an improved nutrient formulation for the biological
treatment of hazardous wastes.
FRANCIS H. CHAPELLE, a researcher at the U.S. Geological Survey in
Columbia, South Carolina, received a Ph.D. in hydrology in 1984 from George
Washington University. He also holds a B.A. in music and a B.S. in geology from
the University of Maryland. Currently, he studies the impacts of subsurface
microbiology on ground water chemistry.
PETER K. KITANIDIS, professor of civil engineering at Stanford
University, received a B.S. in civil engineering in 1974 from the National
Technical University of Athens, Greece, an M.S. in civil engineering in 1976 from
the Massachusetts Institute of Technology, and a Ph.D. in water resources in
1978, also from MIT. His current research focuses on the use of geostatistical and
predictive ground water hydrology methods for designing water quality
monitoring networks. He is also conducting research on the design of nutrient
circulation systems for stimulating subsurface microorganisms to degrade ground
water contaminants.
EUGENE L. MADSEN, assistant professor in the Section of Microbiology,
Division of Biological Sciences, at Cornell University, received B.A., B.S., and
M.S. degrees from the University of California at Santa Cruz, Oregon State
University, and Cornell University, respectively. His Ph.D. from Cornell in 1985
is in soil science, microbiology, and ecology. Since 1989, as a researcher at
Cornell, he has pursued interests in ground water microbiology, microbial
metabolism of environmental pollutants, and developing criteria for proving in
situ biodegradation. Prior to returning to Cornell, he held research appointments
at Rutgers and Penn State universities and at an environmental restoration
company in Bozeman, Montana.
WILLIAM R. MAHAFFEY, vice president of the technology department

for ECOVA Corporation in Redmond, Washington, earned a B.S. in

microbiology in 1976 and an M.S. in microbial ecology in 1978 from the State
University of New York. He holds a Ph.D. in microbial biochemistry, earned in
1986, from the University of Texas. Dr. Mahaffey serves as technical principal on
all of ECOVA's bioremediation projects. He directs the activities of project
microbiologists, develops operating parameters for the use of enhanced
biodegradation in the field, and reviews all company projects involving
bioremediation.
ROBERT D. NORRIS, technical director of bioremediation at
Eckenfelder, Inc., in Nashville, Tennessee, received a B.S. in chemistry from
Beloit College and a Ph.D. in organic chemistry from the University of Notre
Dame. He has been involved since 1983 in the development and implementation
of a variety of bioremediation processes and holds 13 U.S. patents, including four
on various aspects of bioremediation. He has developed and conducted laboratory
and pilot tests as well as successful in situ and ex situ bioremediation under a
range of conditions.
JOSEPH P. SALANITRO, senior staff research microbiologist at Shell
Development Company in Houston, Texas, holds a Ph.D. in microbiology from
Indiana University. During his 21-year career with Shell, he has been involved in
both the chemical and oil sectors of environmental research, studying the aerobic
and anaerobic biodegradability of detergents, chemicals, pesticides and
petrochemical waste effluents, and the role of microbes and sour gas formation in
oil field waterfloods. His current research interests are in defining the potential
and limits of biodegradation of gasoline components in subsurface remediation.
JOHN M. SHAUVER, environmental enforcement manager for the
Michigan Department of Natural Resources, earned a B.S. in fisheries biology
from Michigan State University and did two years of postgraduate work in
geology. A 24-year veteran of the Department of Natural Resources (minus a
two-year absence for military duty from 1968 until 1970), he has worked as a
water quality investigator, hazardous waste cleanup specialist, aquatic biologist,
environmental law enforcement specialist, and environmental enforcement
manager.
JAMES M. TIEDJE, director of the Center for Microbial Ecology at
Michigan State University, received a B.S. in agronomy in 1964 from Iowa State
University. He received an M.S. in 1966 and a Ph.D. in 1968 in soil microbiology
from Cornell University. He is currently a professor in the Departments of Crop
and Soil Sciences and Microbiology and Public Health at Michigan State
University. His expertise is in microbial ecology, and he conducts research in
three focal

areas: denitrification, reductive dehalogenation, and the use of gene probes to

study community selection in nature.
JOHN T. WILSON, research microbiologist at the U.S. Environmental
Protection Agency's R. S. Kerr Laboratory in Ada, Oklahoma, earned a B.S. in
biology in 1969 from Baylor University, an M.A. in microbiology in 1971 from
the University of California at Berkeley, and a Ph.D. in microbiology in 1978
from Cornell University. His areas of expertise are bioremediation and subsurface
microbiology, with emphasis on quantitative description of the biological and
physical processes that control the behavior of hazardous materials in soils and
the subsurface.
RALPH S. WOLFE, a professor of microbiology at the University of
Illinois since 1955, received a Ph.D. at the University of Pennsylvania in 1953. A
member of the National Academy of Sciences, his major research interest has
been anaerobic microbial metabolism. He was attracted to the study of
methanogenic bacteria in 1961 because their biochemistry was unknown, and the
difficulty of isolating and cultivating these extremely oxygen-sensitive anaerobes
was legendary. Dr. Wolfe developed a system for mass culture of methanogens in
kilogram quantities, obtained the first formation of methane by a cell-free
extract, and evolved a simplified procedure for the routine culture of
methanogens in a pressurized atmosphere of hydrogen and carbon dioxide—a
technique that has played a pivotal international role in development of the field.
JACQUELINE A. MACDONALD, program officer at the National
Research Council's Water Science and Technology Board, served as study
director and managing editor for the Committee on In Situ Bioremediation. She
holds an M.S. in environmental science in civil engineering from the University
of Illinois and a B.A., magna cum laude, in mathematics from Bryn Mawr
College.
GREGORY K. NYCE, senior project assistant at the National Research
Council's Water Science and Technology Board, served as project assistant for
the Committee on In Situ Bioremediation. He received his B.S. in psychology
from Eastern Mennonite College.

INDEX 199
Index
A microscopic counting, 68
oligonucleotide probes, 69
Abiotic processes
sample selection, 67-68, 89-90
conservative tracers of, 79
in contaminant mass loss, 85-87
modeling, 8, 85-87
Adaptation
as evidence of bioremediation, 7, 73
by native organisms, 24
Aeration systems, 51-53
Aerobic respiration
modeling, 155
oxygen delivery for, 144-146
process, 18-20
Agricultural areas, 42
Air sparging, 57-59, 124-125, 126, 127
definition, 187
monitoring conservative tracers in, 79-80
monitoring electron acceptor uptake in,
79
oxygen delivery via, 144-145
Alcohols, 32
Alkylbenzenes, 161-162
Anaerobic respiration, 19, 20-21, 187
measuring byproducts of, 75-76
process innovations, 132
Aquifer
bioremediation systems for, 53-59
clogging, 28, 138-139
definition, 187
minerals in, 41
monitoring of, 137-140
permeability, 138-139
preparation for bioremediation, 140-141
Aromatic hydrocarbons, 187
B
Bacteria measurement
bacterial activity, rates of, 70-73
biogeography, 113-114
fatty acid analysis, 69-70
field evaluation, 67-70
metabolic adaptation, 73

INDEX 200
Baseline conditions, 65-67 estimating distribution in ground water,

Benzene, 32 164
See also BTEX estimating mass of, 161-163
Bioaugmentation, 17, 131, 188 halogenated, 22, 33-34, 128-129
Biocurtain, 23, 188 incomplete degradation of, 27-28
Biodiversity, 111-112 low concentrations of, 25-26
Biofilm kinetics, 154-155 metals, 20-21, 23, 26, 34-35
Biological reaction rate models, 83-84 microbial demobilization of, 22-23
BIOPLUME model, 156-158 microbial destruction of, 17-22, 48
Biopolishing, 132 mobilization of, 26
Bioremediation. See In situ bioremediation modeling subsurface behavior of, 81-88
BTEX (Benzene, toluene, ethylbenzene, multiple, 27, 128
xylenes), 32, 70, 128, 188 nitroaromatics, 34
conventional cleanup approaches, 105 plume containment, 141
engineered bioremediation of, case prevalence, 29
example, 71-72 sequestering of, 25-26
estimate of oil/water partitioning, source removal, 140
164-166 subsurface spreading of, 49
estimating distribution of, 164 susceptibility to bioremediation, 2-3,
extent of problem with, 104-105 29-35
intrinsic bioremediation of, 105-106 toxicity to microorganisms, 26-27
levels of intrinsic attenuation of, 106-108 See also Petroleum products
oily-phase residual, 170-171 Conventional cleanup technologies
practicality of bioremediation for, 104 for BTEX, 15
remediation in ground water, case exam- excavation-and-incineration, 13
ple, 174 integrated with bioremediation, 60-61,
remediation of subsurface material, case 126-127
example, 175-178 limitations of, 12, 48
research needs in bioremediation of, 108 in preparation for bioremediation, 139
pump-and-treat methods, 12-13, 48, 61,
140, 193
C Core samples, handling of, 163
Carbon-13/14 labeling, 70-72, 80, 149
Carbon isotopes, 7, 74-75
Carbon-nitrogen-phosphorous ratio, 117
Carbonates, in aquifer matrix, 41
Chlorocatechols, 27
Coal tar, remediation of, 149
Cometabolism, 20, 21-22, 188
dead-end products from, 27-28
in ecological perspective, 114, 115
principles of, 143
Commercial bioremediation
growth of, 13
standards of practice for, 61-62
status of, 129
Complexing agents, 26, 188-189
Conservative tracers, 79-80, 189
Contaminants
combined remediation strategies for,
126-127
designing bioremediation strategy for,
49-50

INDEX 201
Cost of remediation See also BTEX

of aromatic compounds, 104-105 Ethylene-diaminetetraacetic acid (EDTA),
time as factor in, 48 26
Creosote, 32 Evaluation of bioremediation
of carbon-nitrogen-phosphorous ratio,
117
D case examples, 66-67, 71-72, 77, 86,
Dead-end products, 27-28 148-150
Demobilization of contaminants, 22-23 difficulty in, 14, 148
Demonstration projects, evaluation of, 64 ecological perspective in feasibility stud-
Diauxy, 27, 189 ies, 116-119
Dichloroethylene, 78 evidence for, 5-6
feasibility studies, 142
field experiments for, 7-8
E field measurements for, 6-7
gas surveys in, 138
Ecological perspective, 110-111 individual site differences and, 88
biogeography, 113-114 in intrinsic bioremediation, 59-60
biological specificity in, 111-112 limitations, 9, 88-90, 148
feasibility evaluation in, 116-119 modeling techniques for, 8-9, 80-88,
microbial diversity, 112-113 153, 154
microbial natural selection in, 114-115 monitoring-well placement in, 138, 139,
successful bioremediation in, 119 181-182
Education/training, recommendations for, as multidisciplinary activity, 89
10-11, 95 principles of, 63-65, 139-140
Electron acceptor, 18, 19, 189 protocols for, 94
air injection for, 57-59 of rate-limiting factors, 117-119
in bioremediation mechanics, 142, 144 regulatory, 99-103
in ecological perspective, 119 research needs in, 10, 131-132
measuring concentration of, 75
measuring uptake of, 79
nitrate as, 124
in water circulation systems, 41-42, 57
Electron donor
in bioremediation mechanics, 142, 144
definition, 18, 19, 189
inorganic compound as, 21
in reductive dehalogenation, 22
Engineered bioremediation
air injection systems in, 57-59
definition, 2, 20, 189
determining baseline conditions, 65-67
followed by intrinsic bioremediation, 61
indications for, 3-4, 50
process innovations, 4, 53
proving, in case example, 71-72
site conditions for, 3, 39-41
systems for, 4
for unsaturated soils, 50-53
vs. intrinsic bioremediation, 35
water circulation systems in, 53-57
Esters, 32
Ethers, 32
Ethylbenzene, 32

INDEX 202
of residual oily-phase hydrocarbons, Gas chromatography, 73, 190

170-171 Gasoline, 32, 143
role of, 91, 93-94 Genetic engineering, 131, 190
See also Field evaluation; Geochemical models, 82-83
Measurement Ground water
air injection systems for, 57-59
bacteriological samples from, 67-68
F circulation systems, 53-57
Fatty acid analysis, 69-70 engineered bioremediation for, 4
Fermentation, 19, 21, 190 estimating contaminant concentration
Field evaluation in, 164-166
of bacterial adaptation, 73 estimating contaminant distribution in,
byproducts of anaerobic activity in, 75-76 164
carbon isotope ratios for, 74-75 estimating recirculated volume in, 164
of contaminant distribution, 164 evaluating processes in, 88-89
of co-oxidation of trichloroethylene, 129 in flow models, 81-84
degradable/nondegradable substance in intrinsic bioremediation, 41-42
ratio in, 76-78 tracer tests for, 138
difficulty of, 63-64, 161 Growth substrates, 114-116
of electron acceptor concentration, 75
electron acceptor uptake in, 79
H
establishing baseline conditions for,
65-67 Halogenated aliphatics, 33
evidence collection for, 6 Halogenated aromatics, 34
handling core samples for, 163 Halogenated compounds, 22, 33-34,
of hydrocarbon concentration in ground 128-129
water, 178 Headspace analysis, 163, 182
of inorganic carbon concentration, 73-74 Helium, as conservative tracer, 79
intermediary metabolites in, 76 Hexachlorocyclohexane, 78
labeling contaminants in, 80 Hudson River, 77, 92
laboratory microcosms for, 70-73 Hydraulic conductivity, 39, 190
monitoring conservative tracers in, 79-80
need for, 64-65
of number of bacteria, 67-70
of oil/water partitioning, 164-166
of polychlorinated biphenyls, 77
of postbioremediation processes, 178-181
of protozoa, 70
of rate of bacterial activity, 70-73
sample selection for, 67-68, 89-90
spatial heterogeneity in, 171-173
stimulating bacteria in subsites for, 78
techniques, 6-7, 148-150
of total contaminant mass, 161-163
See also Evaluation
Flow models, 8-9
estimating recirculated volume in, 164
multiphase, 82
saturated, 81-82
Free product recovery, 60, 190

INDEX 203
Hydrocarbon. See Petroleum products role of microbes in, 16-17

Hydrogen peroxide strategy selection, 49-50
in bioremediation mechanics, 145-146 technical developments in, 92-93
in controlling bioremediation, 28 vs. other technologies, 12-13, 48-49
development of, in bioremediation, INT activity test, 68
123-124 Intrinsic bioremediation
limitations of, 145-146 of aromatic hydrocarbons, 105-106
in water circulation system, 53 of crude oil spill, case example, 37-38
definition of, 2, 20, 191
following engineered bioremediation, 61
I indications for, 4
In situ bioremediation levels of attenuation in, 106-108
advantages of, 48-49 limitations of, 4, 59-60
bacterial measures as evidence of, 65-78 requirements for, 35-39, 59-60
biodiversity and, 112-113 site conditions for, 3, 39, 41-42
chemical changes in ground water in, vs. engineered bioremediation, 35
23-24 Intrinsic permeability, 39, 138-139, 191
as commercial industry, 13, 61-62, 129 Isotope fractionation, 74-75, 191
complicating factors in, 25-28
contaminants susceptible to, 29-35
J
current status of, 11, 29, 47-48,
121-122, 125-127 Jet fuel, evaluating bioremediation of,
defining success in, 14, 160, 169-170 149-150
determinants of success in, 49, 116-119,
136, 137, 150
ecological perspective of, 110-111 K
educational recommendations for, Ketones, 32
10-11, 95-96
effect on native organisms, 24-25
engineered, 2, 3-4, 20, 35, 39-41, 50-59,
61, 65-67, 71-72, 189
environments amenable to, 35-43
evaluation of, 5-9, 63-90
evolution of, 122-125
first application of, 3, 47, 122
good practices in, 61-62
integrated with nonbiological technolo-
gies, 5, 60-61, 126-127
intrinsic, 2, 3, 4, 20, 35-39, 41-42,
59-60, 105-108, 191
limitations of, 29-32, 127-130, 153-154
measuring microbial action as evidence
of, 78-80
multidisciplinary nature of, 9, 13-14,
44-46
natural selection and, 114-115, 119
preparation for, 140-141
principles of, 2-3, 49-50, 136-137
prospects for, 9-10, 95-96, 108, 127-133
proving, 63-65
regulatory assessment of proposal for,
99-103
research recommendations for, 10, 94-95
role of evaluation in, 91, 93-94
INDEX 204
L determinants of, in bioremediation, 16,

147
Labeling of contaminants, 8, 80 in evaluating bioremediation, 5-6, 63, 64
Laboratory microcosms, 70-73
evidence of, 67-70
Ligands. See Complexing agents
field evaluation of, 6-8, 65, 78-80
genetic engineering for, 131
M hydrogen peroxide as oxygen source
for, 123-124
Measurement incomplete degradation of contaminant
of anaerobic activity, 75-76 by, 27-28
of bacterial activity, 70-73 inorganic nutrients for, 146-147
of bacterial population, 67-70 intermediate metabolite formation in, 76
of contaminant mass loss, 85-87 intrinsic bioremediation requirements
of degradable/nondegradable substance for, 59-60
ratio, 76-78 intrinsic hydrocarbon biodegradation
establishing baseline conditions for, by, 105-106
65-67 laboratory breeding for, 131
interdisciplinary integration in, 89 limits to, 128-129
of labeled contaminants, 80 measuring rate of, 70-73
of metabolic byproducts, 7 on metals, 34-35
of microbiological field activity, 7-8, in multiple-contaminant environment,
23-24 27, 128
of microbiological field samples, 6-7, nutrient delivery for, 144
65-67 nutrients in water circulation for, 54-57
modeling techniques for, 8-9 nutritional requirements for, 22
of protozoa, 70 predator growth from, 25
research needs in, 10 prevented by toxicity of contaminant,
of subsurface hydrogeochemical proper- 26-27
ties, 42 principles of, in bioremediation, 142-143
See also Bacteria measurement reaction rate models of, 83-84
Metals, 34-35 stability in, and biodiversity, 112-113
in anaerobic respiration, 20-21 stimulants for, 92-93
mobilization of, 26
precipitation of, microorganisms for, 23
Methanotrophs, 129
Microbial action
adaptation and, 7, 24, 73
advances in understanding of, 92
aerobic stimulation of, 144-146
air sparging for, 57-59
alternate substrates for, 143
aquifer clogging from, 28, 138-139
availability of contaminants for, 25-28
basic metabolism in, 17-20
biological specificity in, 111-112
biostimulation of, 79, 92-93, 141-147
changes in ground water chemistry
from, 23-24
chemical indicators of, 23-25
in demobilizing contaminants, 22-23
description of, in bioremediation pro-
posal, 101-102
in destroying contaminants, 17-22

INDEX 205
See also Bacteria measurement Pesticides, 34, 127

Modeling techniques Petroleum products
biodegradation effects in, 82 degradable/nondegradable substances in
biofilm kinetics, 154-155 bioremediation of, 76-78
biological reaction rate models, 83-84 estimating ground water concentration
BIOPLUME model, 156-158 of, 178
for bioremediation evaluation, 8-9, 80-81 first bioremediation application to, 3
combining, 84 intrinsic bioremediation of crude oil
direct methods, 87-88 spill, 37-38
evolution of, 154-155 proving bioremediation of, 71-72
geochemical models, 82-83 spatial heterogeneity in bioremediation
in intrinsic bioremediation, 59 of, 171-173
limitations of, 88 susceptibility to bioremediation, 2, 32
measuring mass loss in, 85-87 types of, 32
multiphase flow models, 82 See also BTEX
research needs for, 10, 94-95 Phosphates
role of, 80-81, 84-85 in controlling hydrogen peroxide reac-
saturated flow models, 81-82 tions, 145
sorption effects in, 81-82 effects of, on bioremediation rate, 146
types of, 81-84, 154 Phytane, 76-78
Moffett Naval Air Station, 66-67 Plume containment, 141
Most-probable-number measures, 69, 70 evaluation of, 170
Multiphase flow models, 82 levels of intrinsic attenuation in, 106-108
modeling of, 155
postbioremediation, 178-182
N Polyaromatic hydrocarbons, 127
Natural gas, 86 Polychlorinated biphenyls, 34, 70, 127
Natural selection, 114-115, 119 anaerobic dechlorination of, 76, 92
Nitrate, 103 bioremediation of, case example,77
as electron acceptor, 118, 124 dehalogenation of, 129
Nitroaromatic compounds, 34, 127
Nonaqueous-phase formation, 192
contaminants susceptible to, 29-32
flow characteristics and, 39-41
in multiphase flow models, 82
as obstacle to bioremediation, 25, 29-30
removal, before bioremediation, 140
strategies for overcoming, 26
Nutrients
in air sparging, 58
delivery of, 144
in water circulation, 54-57
O
Octadecane, 76-78
Oligonucleotide probes, 69, 192
Oxidation-reduction reaction, 18
P
Pentachlorophenol, 34
Perchloroethylene, 129

INDEX 206
Polycyclic aromatic hydrocarbons, 32, 157 estimating total contaminant mass,

Polynuclear aromatics, 149 161-163
Primary substrates, 18, 19, 142-143, 192 ground water behavior, 41-42
in cometabolism, 115 heterogeneity, 42-43, 138
Protozoa indications for integrated cleanup
as evidence of bioremediation, 6 approach, 5, 60-61, 126-127
field evaluation of, 70 individual differences in, 3, 35, 88
growth in bioremediation, 25 for intrinsic bioremediation, 3, 39,
Pump-and-treat methods 41-42, 59-60
integrated with bioremediation, 61 multiple contaminants, 27, 128
limitations of, 12-13, 48 regulatory description of, 100-101
in preparation for bioremediation, 140 See also Soil conditions
process, 48, 193 Slurry wall, 141, 193
Push/pull tests, 149 Soil conditions
aeration systems and, 51-53
in air sparging, 58
R bioremediation contraindicated by, 126
Reductive dehalogenation, 20, 22, 193 hydraulic conductivity, 39
Regulatory assessment intrinsic attenuation of plume and, 107
information needed for, 99-100 permeability, 39, 138-139
proposed process description for, unsaturated, 50-53
101-102 See also Site conditions
site cleanup description for, 102-103 Solvents
site description for, 100-101 dechlorination of, 76, 127, 143
Research halogenated compounds as, 33, 34
in development of bioremediation, Stereoisomers, 78
122-124 Surfactants, 26, 128, 193-194
for evaluation protocol development, 94
for improving models, 94-95
on increasing microbe availability, 93
on microbial processes, 92, 128-129
needs, 108
in site characterization techniques, 94
on stimulating microbial action, 92-93
S
Saturated flow models, 81-82
Saturated zone, 81, 193
Secondary utilization/cometabolism, 21,
143, 193
Sequestering of contaminants, 25-26
Sewage contamination, 149
Site conditions
characterization of, 10, 94
in choosing bioremediation strategy,
49-50
contaminant concentrations, 25-27
determinants of bioremediation poten-
tial, 35, 126, 130, 137-138
electron receptor concentration, 41-42
for engineered bioremediation, 3-4,
39-41, 50

INDEX 207
T
Tetrachloroethene, 33
Time factors
in bioremediation vs. conventional
methods, 13, 48
in ecologically oriented bioremediation,
116
in engineered bioremediation, 3-4, 50
Toluene, 23, 32
See also BTEX
Tracer compounds, 8
Trichloroethane, 24
Trichloroethylene, 28, 129, 143
intermediary metabolites in transforma-
tion of, 76
Trinitrotoluene, 34
U
Unsaturated soils, 50-53, 194
oxygen delivery techniques for, 124-125
V
Vadose zone. See Unsaturated soils
Vapor recovery, 124, 194
integrated with bioremediation, 61,
126-127
Vinyl chloride, 28, 143
W
Water circulation systems, 53-57
X
Xylene, 32

OTHER RECENT REPORTS OF THE WATER SCIENCE AND TECHNOLOGY BOARD 208
Other Recent Reports of the Water Science

and Technology Board
Ground Water Vulnerability Assessment: Predicting Contamination Potential

Under Conditions of Uncertainty (1993)
Managing Wastewater in Coastal Urban Areas (1993)
Sustaining Our Water Resources: Proceedings, WSTB Symposium (1993)
Water Transfers in the West: Efficiency, Equity, and the Environment (1992)
Restoration of Aquatic Ecosystems: Science, Technology, and Public Policy
(1992)
Toward Sustainability: Soil and Water Research Priorities for Developing
Countries (1991)
Preparing for the Twenty-first Century: A Report to the USGS Water Resources
Division (1991)
Opportunities in the Hydrologic Sciences (1991)
A Review of the USGS National Water Quality Assessment Pilot Program
(1990)
Ground Water and Soil Contamination Remediation: Toward Compatible
Science, Policy, and Public Perception (1990)
Managing Coastal Erosion (1990)
Ground Water Models: Scientific and Regulatory Applications (1990)
Irrigation-Induced Water Quality Problems: What Can Be Learned from the San
Joaquin Valley Experience? (1989)
Copies of these reports may be ordered from
the National Academy Press
1-800-624-6242
202-334-3313

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Bioremediation is a technology that is gaining momentum in technical,

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primary object of the study is to provide guidance on how to evaluate when an in

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3 THE CURRENT PRACTICE OF BIOREMEDIATION 47

4 EVALUATING IN SITU BIOREMEDIATION 63

5 FUTURE PROSPECTS FOR BIOREMEDIATION 91

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In Situ Bioremediation When does it work?

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The United States is investing billions of dollars in cleaning up polluted

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THE CURRENT PRACTICE OF BIOREMEDIATION

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may be a more appropriate remedy than intrinsic bioremediation. Because

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Integration of Bioremediation with Other Technologies

EVALUATING IN SITU BIOREMEDIATION

1. documented loss of contaminants from the site,

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have the potential to transform the contaminants under the expected

Every well-designed bioremediation project, whether a field test or full-scale

Measurements of Field Samples

• Number of bacteria. Because microbes often reproduce when they

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effect remediation when incubated in microcosms that resemble the field

Experiments Run in the Field

• Stimulating bacteria within subsites. When growth-stimulating

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