Table Of ContentHandbook of
BIOREMEDIATION
NORRIS • HINCHEE • BROWN • MCCARTY
SEMPRM • WILSON • KAMPBELL
REINHARD • BOUWER • BORDEN • VOGEL
THOMAS • WARD
Project Officer
John E. Matthews
Chief, Applications and Assistance Branch
Robert S. Kerr Environmental Research Laboratory
Ada, Oklahoma
First published 1994 by Lewis Publishers
Taylor & Francis Group
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Reissued 2018 by CRC Press
© 1994 by Taylor & Francis
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FOREWORD
EPA is charged by Congress to protect the Nation's land, air and water systems. Under a mandate
of national environmental laws focused on air and water quality, solid waste management and the control
of toxic substances, pesticides, noise and radiation, the Agency strives to formulate and implement actions
which lead to a compatible balance between human activities and the ability of natural systems to support
and nurture life.
The RobertS. Kerr Environmental Research Laboratory is the Agency's center of expertise for
investigation of the soil and subsurface environment. Personnel at the laboratory are responsible for
management of research programs to: (a) determine the fate, transport and transformation rates of pollutants
in the soil, the unsaturated and the saturated zones of the subsurface environment; (b) define the processes
to be used in characterizing the soil and subsurface environment as a receptor of pollutants; (c) develop
techniques for predicting the effect of pollutants on ground water, soil, and indigenous organisms; and
(d) define and demonstrate the applicability and limitations of using natural processes, indigenous to the
soil and subsurface environment, for the protection of this resource.
In-situ bioremediation of subsurface environments involves the use of microorganisms to convert
contaminants to less harmful products and sometimes offers significant potential advantages over other
remediation technologies. This report provides the most recent scientific understanding of the processes
involved with soil and ground-water bioremediation and discusses the applications and limitations of the
various in-situ bioremediation technologies.
Clinton W. Hall
Director
Robert S. Kerr Environmental
Research Laboratory
iii
Dedicated to Richard L. Raymond, Sr.
Through his vision, commitment, and humanity,
he created the
subsurface bioremediation industry.
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CONTENTS
Foreword ..........................................................................................................................................iii
Figures .............................................................................................................................................xi
Tables .............................................................................................................................................xiii
Executive Summary...........................................................................................................................1
Section 1. Introduction ................................................................................................................13
Section 2. In-Situ Bioremediation of Soils and Ground Water Contaminated with
Petroleum Hydrocarbons
2.1. Introduction .....................................................................................................17
2.2. Fundamental Principles.....................................................................................17
2.3 Historical Perspective........................................................................................19
2.4 Repositories of Expertise..................................................................................21
2.5. General Designs................................................................................................21
2.6. Laboratory Testing............................................................................................22
2.7. Contamination Limits........................................................................................22
2.8. Site Characterization.........................................................................................24
2.9. Favorable Site Conditions.................................................................................25
2.9.1. Solubility ..........................................................................................25
2.9.2. Volatility ............................................................................................25
2.9.3. Viscosity ............................................................................................25
2.9.4. Toxicity .............................................................................................26
2.9.5. Permeability of Soils and Subsurface Materials..................................26
2.9.6. Soil Type............................................................................................26
2.9.7. Depth to Water....................................................................................26
2.9.8. Mineral Content..................................................................................26
2.9.9. Oxidation/Reduction Potential............................................................26
2.9.10. pH .....................................................................................................27
2.10. Infrastructure and Institutional Issues.................................................................27
2.11. Performance ......................................................................................................28
2.12. Problems ..........................................................................................................28
2.13. Site Properties vs Cost......................................................................................30
2.13.1. Mass of Contaminant..........................................................................30
2.13.2. Volume of Contaminated Aquifer.......................................................30
2.13.3. Aquifer Permeability/Soil Characteristics............................................30
2.13.4. Final Remediation Levels...................................................................30
2.13.5. Depth to Water....................................................................................31
2.13.6. Monitoring Requirements...................................................................31
2.13.7. Contaminant Properties.......................................................................31
2.13.8. Location of Site..................................................................................31
2.14. Previous Experience with Costs.........................................................................32
2.15. Regulatory Acceptance......................................................................................33
2.16. Knowledge Gaps...............................................................................................34
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Section 3. Bioventing of Petroleum Hydrocarbons
3.1. Fundamental Principles........................................................................................39
3.1.1. Review of the Technology....................................................................39
3.1.2. Maturity of the Technology...................................................................41
3.1.3. Repositories of Expertise.......................................................................41
3.2. Contamination that is Subject to Treatment.........................................................43
3.3. Special Requirements for Site Characterization...................................................44
3.3.1. Soil Gas Survey.....................................................................................44
3.3.2. Soil Gas Permeability and Radius of Influence.....................................44
3.3.3. In-Situ Respiration.................................................................................45
3.4. Impact of Site Characteristics on Applicability...................................................47
3.5. Process Performance............................................................................................48
3.5.1. Case Study: Hill AFB Site....................................................................50
3.5.2. Case Study: Tyndall AFB Site...............................................................52
3.5.3. Performance of Other Sites...................................................................54
3.6. Problems Encountered with the Technology........................................................55
3.7. Costs ....................................................................................................................55
3.8. Regulatory Acceptance.........................................................................................55
3.9. Knowledge Gaps and Research Opportunities.....................................................55
Section 4. Treatment of Petroleum Hydrocarbons in Ground Water by Air Sparging
4.1. Introduction .....................................................................................................61
4.2. Development of Air Sparging...........................................................................62
4.3. Principles of the Technology............................................................................63
4.4. Benefits of Air Sparging...................................................................................65
4.5. Dangers of Air Sparging...................................................................................68
4.6. Barriers to Flow...............................................................................................68
4.7. Control of Flow...............................................................................................70
4.8. Summary of Limitations...................................................................................71
4.9. System Application and Design........................................................................73
4.9.1. Nature and Extent of Site Contaminants.............................................73
4.9.2. Hydrogeologie Conditions...................................................................74
4.9.3. Potential Ground-Water and Vapor Receptors.....................................74
4.10. Field Pilot Testing.............................................................................................74
4.11. Design Data Requirements...............................................................................75
4.12. System Elements..............................................................................................77
4.13. System Examples..............................................................................................79
4.14. Cost Factors......................................................................................................82
4.15. Conclusion ......................................................................................................83
Section 5. Ground-Water Treatment for Chlorinated Solvents
5.1. Introduction .....................................................................................................................87
5.2. Biotransformation of CaHs............................................................................................89
5.2.1. Primary Substrates and Cometabolism.......................................................89
5.2.2. CAH Usage as Primary Substrates..............................................................92
5.2.3. Anaerobic Cometabolic Transformations of CAHs...................................92
5.2.4. Aerobic Microbial Transformation of Chlorinated
Aliphatic Hydrocarbons................................................................................93
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5.3. Processes Affecting Chemical Movement and Fate............................................95
5.3.1. Effect of Sorption................................................................................95
5.4. Field Pilot Studies of CAH Transformation.......................................................97
5.4.1. Results with Methanotrophs.................................................................98
5.4.2. Results with Phenol Utilizers........................................................... 100
5.4.3. Comparison Between the Methane and Phenol Studies..................... 102
5.4.4. Anaerobic Transformation of Carbon Tetrachloride.......................... 102
5.5. Procedures for Introducing Chemicals into Ground Water.............................. 103
5.6. The Effect of Site Conditions on Remediation Potential................................. 107
5.6.1. Microorganism Presence.................................................................. 109
5.7. Summary ....................................................................................................... 110
Section 6. Bioventing of Chlorinated Solvents for Ground-Water Cleanup
through Bioremediation
6.1. Fundamental Principles.................................................................................. 117
6.2. Maturity of the Technology........................................................................... 118
6.3. Primary Repositories of Expertise................................................................... 118
6.4. Contamination Subject to Treatment............................................................... 119
6.5. Special Requirements for Site Characterization............................................... 120
6.6. Site Characteristics that are Particularly Favorable......................................... 121
6.7. Site Characteristics that are Particularly Unfavorable..................................... 121
6.8. Performance under Optimal Conditions.......................................................... 122
6.8.1. Importance of the Rate Law............................................................ 122
6.8.2. Importance of Partitioning............................................................... 122
6.9. Problems Encountered with the Technology................................................... 125
6.10. Relevant Experience with System Design....................................................... 125
Section 7. In-Situ Bioremediation Technologies for Petroleum Derived Hydrocarbons
Based on Alternate Electron Acceptors (other than molecular oxygen)
7.1. Fundamental Principles.................................................................................. 131
7.1.1. Comparison of Oxygen and Alternate Electron Acceptor Based
In-Situ Bioremediation Technologies................................................ 131
7.1.2. Hydrocarbon Transformation Based on Alternate
Electron Acceptors........................................................................... 133
7.1.2.1. Laboratory Studies........................................................... 133
7.1.2.2. Large Scale Bioremediation Studies Using Nitrate........... 136
7.1.3. Maturity of the Technology.............................................................. 136
7.1.4. Primary Respository of Expertise...................................................... 137
7.2. Contamination that is Subject to Treatment.................................................... 138
7.2.1. Chemical Nature.............................................................................. 138
7.2.2. Range of Concentration................................................................... 138
7.3. Requirements for Site Characterization and Implementation of
the Technology ............................................................................................. 138
7.4. Favorable Site Characteristics......................................................................... 139
7.5. Unfavorable Site Characteristics..................................................................... 139
7.5.1. Chemical and Physical Nature of the Contamination....................... 139
7.5.2. Site Hydrogeology and Source Characteristics................................. 139
7.5.3. Infrastructure and Institutional Issues............................................... 139
7.6. Optimal Site Conditions................................................................................. 139
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7.7. Problems Encountered with the Technology................................................... 140
7.8. Properties of Site and Contaminants Determining the Cost of Remediation.... 140
7.9. Previous Experience with Cost of Implementing the Technology................... 141
7.10. Factors Determining Regulatory Acceptance of the Technology..................... 141
7.11. Primary Knowledge Gaps and Research Opportunities.................................... 141
7.11.1. Degradation Under Ideal Conditions................................................ 142
7.11.2. Degradation Under Ground-Water and Soil Conditions..................... 142
7.11.3. Degradation at Sites........................................................................ 142
7.11.4. Degradation at the Hydrocarbon/Water Interface and Within the
Nonaqueous Phase........................................................................... 143
7.11.5. Methods for Monitoring Performance of Bioremediation Process .... 143
7.11.6. Design of Optimal Nutrient and Electron Acceptor Systems............ 143
Section 8. Bioremediation of Chlorinated Solvents Using Alternate Electron Acceptors
8.1. Introduction .................................................................................................. 149
8.2. Metabolism and Alternate Electron Acceptors................................................ 150
8.3. Biotransformation of Chlorinated Solvents in the Presence of Alternate
Electron Acceptors........................................................................................ 151
8.3.1. Carbon Tetrachloride Biotransformation.......................................... 155
8.3.2. Tetrachloroethene and Trichloroethene Biotransformation................. 155
8.4. Approaches for Treatment.............................................................................. 156
8.5. Field Experience............................................................................................. 158
8.6. Sequential Anaerobic/Aerobic Transformations of Chlorinated Solvents........ 158
8.7. Performance .................................................................................................. 159
8.7.1. Physical/Chemical Properties........................................................... 159
8.7.2 Concentration Range........................................................................ 160
8.7.3. Favorable Redox Conditions............................................................ 161
8.8. Biotransformation Stoichiometry.................................................................... 163
8.9. Biotransformation Rates................................................................................ 166
8.10. Limitations ................................................................................................... 167
8.11. Research Needs.............................................................................................. 169
8.12. Concluding Remarks...................................................................................... 169
Section 9. Natural Bioremediation of Hydrocarbon-Contaminated Ground Water
9.1. General Concept of Natural Bioremediation.................................................. 177
9.2. Hydrocarbon Distribution, Transport and Biodégradation in the Subsurface ... 178
9.2.1. Petroleum Hydrocarbon Biodégradation.......................................... 179
9.2.2. Subsurface Microorganisms............................................................. 179
9.2.3. Use of Different Electron Acceptors for Biodégradation.................. 180
9.2.3.1. Aerobic Biodégradation.................................................. 180
9.2.3.2. Biodégradation via Nitrate Reduction.............................. 181
9.2.3.3. Biodégradation using Ferric Iron..................................... 181
9.2.3.4. Biodégradation via Sulfate Reduction and
Methanogenesis ............................................................. 182
9.2.4. Effect of Environmental Conditions on Biodégradation................... 182
9.3. Natural Bioremediation of a Hydrocarbon Plume............................................ 183
9.4. Case Studies of Natural Bioremediation......................................................... 185
9.5. Site Characterization for Natural Bioremediation............................................ 186
9.5.1. Is the Contaminant Biodegradable?.................................................. 187
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9.5.2. Is Biodégradation Occurring in the Aquifer?..................................... 187
9.5.3. Are Environmental Conditions Appropriate for Biodégradation?...... 188
9.5.4. If the Waste Doesn’t Completely Biodegrade, Where Will It Go? ... 188
9.6. Monitoring Natural Bioremediation Systems..................................................... 189
9.7. Performance of Natural Bioremediation Systems.............................................. 190
9.8. Predicting the Extent of Natural Bioremediation............................................... 190
9.9. Issues That May Affect the Costs of This Technology..................................... 192
9.10. Knowledge Gaps and Research Opportunities.................................................. 192
Section 10. Natural Bioremediation of Chlorinated Solvents
10.1. Summary .......................................................................................................201
10.2. Fundamental Principles..................................................................................201
10.3. Chemical Reactions........................................................................................203
10.4. Microbiological Reactions..............................................................................204
10.4.1. Aerobic ..........................................................................................204
10.4.2. Anaerobic ........................................................................................206
10.5. Predictions of Product Distribution................................................................208
10.6. Rationale for Technology...............................................................................211
10.7. Practical Implications.....................................................................................215
10.8. Special Requirements for Site Characterization...............................................216
10.9. Favorable Site Characteristics.........................................................................216
10.10. Unfavorable Site Characteristics.....................................................................218
10.11. Cost Evaluations.............................................................................................220
10.12. Knowledge Gaps............................................................................................220
10.13. Conclusion ....................................................................................................220
Section 11. Introduced Organisms for Subsurface Bioremediation
11.1. Fundamental Principles of the Technology..............................................................227
11.1.1. Review of the Development of the Technology.....................................227
11.1.2. Matrix Properties that Affect Transport...................................................228
11.1.3. Properties of Organisms that Affect Transport......................................230
11.1.4. Operational Factors that Affect Transport..............................................231
11.1.5. Environmental Factors that Affect Survivability of
Added Organisms.........................................................................................233
11.1.6. Field Demonstrations of Microbial Transport.........................................233
11.1.7. Inoculation to Enhance Biodégradation of Hydrocarbons.....................235
11.1.8. Inoculation to Enhance Biodégradation of Chlorinated
Compounds ..................................................................................................236
11.2. Maturity of the Technology.......................................................................................237
11.3. Primary Repositories of Expertise..............................................................................238
11.4. Other Factors Concerning Application.....................................................................238
11.5. State of the Art of Transport of Microorganisms with Specialized
Metabolic Capabilities and Research Opportunities..............................................239
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