In Situ Bioremediation

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


    National Academy Press • 2101 Constitution Avenue, N.W. • Washington, D.C. 20418

    NOTICE: The project that is the subject of this report was approved by the Governing Board of the National Research Council, whose members are drawn from the councils of the National Academy of Sciences, the National Academy of Engineering, and the Institute of Medicine. The members of the committee responsible for the report were chosen for their special competences and with regard for appropriate balance.

    This report has been reviewed by a group other than the authors according to procedures approved by a Report Review Committee consisting of members of the National Academy of Sciences, 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-93213271, 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 Corporation.

    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.I53   1993
    628.5'2—dc20
    93-5531
    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 country, including at the Whitney Museum in New York, the Washington Project for the Arts in Washington, 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


    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





    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

    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




    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 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 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





Contents




    EXECUTIVE SUMMARY

    1  INTRODUCTION

    2  PRINCIPLES OF BIOREMEDIATION

      The Role of Microbes in Bioremediation
       How Microbes Destroy Contaminants
       How Microbes Demobilize Contaminants
       Indicators of Microbial Activity
       Complicating Factors
      Contaminants Susceptible to Bioremediation
       Petroleum Hydrocarbons and Derivatives
       Halogenated Compounds
       Nitroaromatics
       Metals
      Environments Amenable to Bioremediation
       Two Types of Bioremediation: Intrinsic and Engineered
       Site Conditions for Engineered Bioremediation
       Site Conditions for Intrinsic Bioremediation
       Impact of Site Heterogeneity on Bioremediation
      Further Reading
      Boxes
       Key Terms for Understanding Bioremediation
       Intrinsic Bioremediation of a Crude Oil Spill—Bemidji, Minnesota
       Site Characteristics that Favor In Situ Bioremediation

    3  THE CURRENT PRACTICE OF BIOREMEDIATION
      Bioremediation Versus Other Technologies
      Basics of Bioremediation Process Design
       Engineered Bioremediation
       Intrinsic Bioremediation
      Integration of Bioremediation with Other Technologies
      Good Practices
      Box
       Standards of Practice for Bioremediation Contractors

    4  EVALUATING IN SITU BIOREMEDIATION
      A Three-Part Strategy for "Proving" In Situ Bioremediation
      Techniques for Demonstrating Biodegradation in the Field
       Measurements of Field Samples
       Experiments Run in the Field
       Modeling Experiments
      Limitations Inherent in Evaluating In Situ Bioremediation
      Boxes
       Proving Engineered Bioremediation of Chlorinated Solvents in a Field Test—Moffett Naval Air Station, California
       Proving Engineered Bioremediation of an Oil and Fuel Spill—Denver, Colorado
       Testing Bioremediation of PCBs in Hudson River Sediments—New York
       Proving Intrinsic Bioremediation of a Spill at a Natural Gas Manufacturing Plant—Northern Michigan

    5  FUTURE PROSPECTS FOR BIOREMEDIATION
      New Frontiers in Bioremediation
      The Increasing Importance of Evaluating Bioremediation
       Recommended Steps in Research
       Recommended Steps in Education

    BACKGROUND PAPERS

      A Regulator's Perspective on In Situ Bioremediation
       John M. Shauver

      An Industry's Perspective on Intrinsic Bioremediation
       Joseph P. Salanitro

      Bioremediation from an Ecological Perspective
      James M. Tiedje

      In Situ Bioremediation: The State of the Practice
      Richard A. Brown, William Mahaffey, and Robert D. Norris

      Engineering Challenges of Implementing In Situ Bioremediation
      Lisa Alvarez-Cohen

      Modeling In Situ Bioremediation
      Philip B. Bedient and Hanadi S. Rifai

      Testing Bioremediation in the Field
      John T. Wilson

    APPENDIXES

    A  Glossary

    B  Biographical Sketches of Committee Members and Staff

    INDEX


    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, 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 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 clean up 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 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.



    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 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 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.

      • 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 inter actions 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.

      • 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 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 coordinated 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.

      • 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.


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