CHAPTER 1 BIO AUGMENTATION FOR GROUNDWATER REMEDIATION: AN
OVERVIEW ........................................................ 1
1.1 Introduction ............................................... 1
1.1.1 Background: The Pollution Problem ................... 1
1.1.2 Definitions: General Bioremediation Terminology ..... 2
1.1.3 Chapter Overview .................................... 3
1.2 Development of Bioaugmentation for Groundwater
Bioremediation ............................................. 3
1.2.1 Historical Development of Bioaugmentation ........... 3
1.2.2 Recent Developments: Bioaugmentation with
Dehalococcoides for Reductive Dehalogenation of
Chlorinated Ethenes ................................. 4
1.3 Types of Bioaugmentation ................................... 6
1.3.1 Currently Practiced Methods ......................... 6
1.3.2 Potential Bioaugmentation Strategies ................ 9
1.4 Making the Decision to Bioaugment ......................... 12
1.4.1 Technical Analysis/Site Evaluation ................. 14
1.4.2 Select and Test Bioaugmentation Strategy ........... 15
1.4.3 Implement the Treatment ............................ 16
1.4.4 Monitoring Effectiveness ........................... 17
1.4.5 Other Considerations: Economics and Degradation
Kinetics ........................................... 19
1.5 Bioaugmentation Issues .................................... 19
1.5.1 Development of Effective Bioaugmentation Cultures .. 20
1.5.2 Successful Inoculum Delivery and Dispersion ........ 20
1.5.3 Inoculum Survival .................................. 20
1.5.4 Pollutant Bioavailability .......................... 21
1.5.5 Potential Undesirable Side-Effects ................. 21
1.6 Bioaugmentation to Remediate Chlorinated Compounds ........ 22
1.6.1 Chlorinated Aliphatic Hydrocarbons (CAHs):
Dehalococcoides and the Chloroethenes .............. 23
1.6.2 Applications for Other Chlorinated Compounds ....... 24
1.7 Bioaugmentation to Remediate Other Contaminants ........... 24
1.7.1 Petroleum and BTEX ................................. 25
1.7.2 Polycyclic Aromatic Hydrocarbons (PAHs) ............ 25
1.7.3 Methyl Tert-Butyl Ether (MTBE) ..................... 26
1.7.4 Pesticides ......................................... 26
1.7.5 Metals ............................................. 27
1.7.6 Mixed Pollutants ................................... 27
1.8 Summary ................................................... 28
References ................................................ 28
CHAPTER 2 DEHALOCOCCOIDES AND REDUCTIVE DECHLORINATION OF
CHLORINATED SOLVENTS ........................................... 39
2.1 Introduction .............................................. 39
2.1.1 The Chlorinated Ethene Problem ..................... 39
2.1.2 Anaerobic Microbial Degradation of Chlorinated
Ethenes ............................................ 43
2.1.3 Discovery of Dehalococcoides ....................... 46
2.2 Dehalococcoides Isolation and Cultivation Strategies ...... 49
2.2.1 General Considerations ............................. 49
2.2.2 Electron Acceptor .................................. 50
2.2.3 Electron Donor ..................................... 50
2.2.4 Carbon Source ...................................... 51
2.2.5 Reducing Agent (Reductant) ......................... 51
2.2.6 Incubation Conditions .............................. 52
2.2.7 Isolation .......................................... 52
2.3 Dhc Pure Cultures ......................................... 52
2.3.1 Isolation of Dhc mccartyi Strain 195 ............... 52
2.3.2 Isolation of Dhc sp. Strain CBDB1 .................. 53
2.3.3 Isolation of Dhc sp. Strain FL2 .................... 53
2.3.4 Isolation of Dhc Strains That Respire VC: Strains
BAV1, GT and VS .................................... 53
2.3.5 Isolation of Dhc Strain MB ......................... 54
2.4 Maintenance of Dehalococcoides Pure Cultures .............. 55
2.4.1 General Considerations ............................. 55
2.4.2 Growth Factors ..................................... 55
2.5 Dehalococcoides Morphology and Physiology ................. 56
2.6 Phylogeny of Dehalococcoides and Related Bacteria ......... 58
2.7 Dehalococcoides Genetics .................................. 61
2.7.1 Insights from Dehalococcoides Genomes .............. 61
2.7.2 Dehalococcoides Reductive Dehalogenases Gene
Operons ............................................ 63
2.8 Dehalococcoides Reductive Dehalogenases (RDASES) .......... 64
2.9 Biochemistry of Reductive Dechlorination by
Dehalococcoides ........................................... 65
2.10 Dehalococcoides Biomarkers ................................ 67
2.11 Dehalococcoides Evolution and Dissemination of Reductive
Dehalogenase Genes ........................................ 72
2.12 Dehalococcoides Biogeography .............................. 73
2.13 Dehalococcoides Ecology ................................... 74
2.14 Outlook ................................................... 75
2.15 Implications for Bioremediation Practice: Take Home
Messages .................................................. 76
References ................................................ 76
CHAPTER 3 PRODUCTION AND HANDLING OF DEHALOCOCCOIDES
BIOAUGMENTATION CULTURES ....................................... 89
3.1 Introduction .............................................. 89
3.1.1 Microbial Cultures Used for Bioaugmentation ........ 89
3.1.2 Why High Density Microbial Cultures Are Important .. 91
3.2 Growing Inocula ........................................... 91
3.2.1 Microbial Growth Options: Batch Versus Continuous .. 91
3.2.2 Culture Growth Protocol ............................ 93
3.3 Full-Scale Production Results ............................. 95
3.3.1 Factors Affecting Culture Growth ................... 99
3.4 Quality Assurance/Quality Control Considerations ......... 104
3.4.1 Pathogen Analysis ................................. 104
3.4.2 Dhc Concentrations ................................ 104
3.4.3 Specific Activity ................................. 105
3.4.4 Other QA/QC Considerations ........................ 106
3.5 Concentrating and Storing Inocula ........................ 106
3.5.1 Concentrating Cultures ............................ 107
3.5.2 Culture Stability and Storage ..................... 109
3.6 Shipping Cultures ........................................ 110
3.7 Onsite Handling .......................................... 110
3.7.1 Direct Injection .................................. 111
3.7.2 Dilution .......................................... 112
3.7.3 Mixing with Other Reagents Before Injection ....... 112
3.8 Summary .................................................. 113
References ............................................... 113
CHAPTER 4 BIOAUGMENTATION WITH DEHALOCOCCOIDES: DECISION
GUIDE ......................................................... 117
4.1 Introduction ............................................. 117
4.2 Need for Decision Guidance ............................... 118
4.3 Decision Guidance Overview ............................... 119
4.4 Is Complete Dechlorination Occurring? .................... 119
4.5 Are the Site Conditions Inhibitory? ...................... 121
4.6 Is the Site Highly Aerobic? .............................. 122
4.7 Will Biostimulation Work? ................................ 123
4.7.1 Laboratory Diagnostic Tests ....................... 124
4.7.2 Field Testing .................................... 128
4.8 How Valuable is Time? .................................... 131
4.9 Is the Risk of Exposure to Toxic Intermediates
Unacceptable? ............................................ 132
4.10 Economic Assessments of Bioaugmentation .................. 132
4.11 Summary and Recommendations .............................. 134
References ............................................... 135
CHAPTER 5 BIOAUGMENTATION CONSIDERATIONS ..................... 141
5.1 Introduction ............................................. 141
5.2 Effect of Site Conditions on Effectiveness of
Bioaugmentation .......................................... 141
5.2.1 Exposure to Oxygen ................................ 141
5.2.2 Temperature and pH ................................ 142
5.2.3 Competition for Electron Donor/Geochemical
Conditions ........................................ 142
5.2.4 Volatile Organic Compound (VOC) Concentration ..... 143
5.2.5 Inhibitory Constituents ........................... 143
5.2.6 Hydrogeology ...................................... 144
5.3 Field Methods ............................................ 144
5.3.1 Injection Infrastructure Considerations ........... 144
5.3.2 Preconditioning Requirements ...................... 145
5.3.3 Culture Requirements .............................. 148
5.3.4 Injection Techniques .............................. 148
5.3.5 Distribution Techniques ........................... 152
5.4 Bioremediation Configurations Employing Bioaugmentation .. 153
5.4.1 Active Recirculation Approach ..................... 153
5.4.2 Semi-Passive Approach ............................. 159
5.4.3 Passive Approach .................................. 160
5.5 Conclusions .............................................. 161
References ............................................... 162
Appendix 5A Background on Innoculum Density and
Dechlorination Rates .......................................... 162
CHAPTER 6 MICROBIAL MONITORING DURING BIOAUGMENTATION
WITH DEHALOCOCCOIDES .......................................... 171
6.1 Introduction ............................................. 171
6.2 MBTs for Chlorinated Ethene Biodegradation ............... 173
6.3 Developing a Monitoring Strategy ......................... 174
6.3.1 Defining Monitoring Objectives .................... 174
6.3.2 Temporal Considerations ........................... 174
6.3.3 Selection of Sampling Wells ....................... 174
6.4 МВТ Sampling Methods ..................................... 175
6.4.1 General Sampling Considerations ................... 175
6.4.2 Groundwater Sampling Protocol ..................... 177
6.5 Quantitative-PCR ......................................... 180
6.5.1 Description and General Methodology ............... 180
6.5.2 Standards ......................................... 182
6.5.3 Limitations ....................................... 182
6.5.4 Dhc Analysis ...................................... 184
6.5.5 Conclusion ........................................ 185
6.6 Fluorescent In Situ Hybridization ........................ 185
6.6.1 Introduction ...................................... 185
6.6.2 Description and General Methodology ............... 186
6.6.3 Limitations ....................................... 187
6.6.4 Conjunctive Technologies .......................... 188
6.6.5 Conclusion ........................................ 188
6.7 Community Profiling ...................................... 189
6.7.1 Gel Electrophoresis ............................... 189
6.7.2 Cloning and Sequencing ............................ 189
6.7.3 Terminal-Restriction Fragment Length
Polymorphism ...................................... 190
6.7.4 Denaturing Gel Gradient Electrophoresis ........... 190
6.7.5 Temperature Gel Gradient Electrophoresis .......... 190
6.7.6 Microarrays and High-Throughput Sequencing ........ 191
6.7.7 Conclusion ........................................ 192
6.8 Data Evaluation and Interpretation of MBTs ............... 193
6.9 Future Research Needs .................................... 194
References ............................................... 194
CHAPTER 7 BIOAUGMENTATION FOR AEROBIC DEGRADATION OF
CIS-1,2-DICHLOROETHENE ........................................ 199
7.1 Introduction ............................................. 199
7.2 Polaromonas sp. Strain JS666 ............................. 200
7.2.1 Isolation ......................................... 201
7.2.2 Kinetics, Thresholds and Tolerances to cis-DCE
and Oxygen ........................................ 201
7.2.3 Insight About Metabolic Pathways from Genomics
and Proteomics .................................... 202
7.2.4 Cometabolism of Other Chlorinated Solvents ........ 204
7.2.5 Development of a Molecular Probe for Process
Monitoring ........................................ 205
7.2.6 Development of Strategy for Growth of Inocula ..... 206
7.3 Microcosm Assessment of Site-Suitability ................. 207
7.3.1 Microcosm Preparation ............................. 207
7.3.2 Previous Experiences with Microcosm Assessment .... 208
7.4 Field Demonstration ...................................... 208
7.4.1 Test Site Selection ............................... 208
7.4.2 Preliminary Microcosm Study ....................... 209
7.4.3 Titration Studies with SJCA Groundwater ........... 210
7.4.4 Field Test ........................................ 210
7.5 Summary and Future Prospects ............................. 212
References ............................................... 213
CHAPTER 8 BIOAUGMENTATION FOR THE IN SITU AEROBIC
COMETABOLISM OF CHLORINATED SOLVENTS .......................... 219
8.1 Introduction ............................................. 219
8.2 Aerobic Cometabolic Processes ............................ 219
8.3 Aerobic Cometabolism by Indigenous Microorganisms ........ 222
8.3.1 Microcosm Studies with Indigenous Microorganisms .. 222
8.3.2 Field Studies with Indigenous Microorganisms ...... 222
8.4 Bioaugmentation Approaches ............................... 228
8.4.1 Bioaugmentation Approach I ........................ 228
8.4.2 Bioaugmentation Approach II ....................... 237
8.4.3 Bioaugmentation Approach III ...................... 242
8.4.4 Bioaugmentation Approach IV ....................... 248
8.5 Summary .................................................. 249
References ............................................... 251
CHAPTER 9 BIOAUGMENTATION WITH PSEUDOMONAS STUTZERI КС FOR
CARBON TETRACHLORIDE REMEDIATION .............................. 257
9.1 Introduction and Rationale ............................... 257
9.2 Physiological Function of PDTC Production ................ 258
9.3 CT Transformation by P. Stutzeri КС as a Novel
Dechlorination Reaction .................................. 259
9.3.1 Pathway of PDTC-Promoted CT Dechlorination ........ 260
9.3.2 Transition Metal Chelation of PDTC ................ 261
9.4 Genetic Requirements for PDTC Production ................. 263
9.5 PDTC-Mediated CT Transformation .......................... 265
9.5.1 Trace Metals ...................................... 266
9.5.2 Cell and CT Concentration ......................... 266
9.5.3 Cell Membrane Components .......................... 268
9.5.4 An Overall Model for CT Transformation by
Pseudomonas stutzeri КС ........................... 269
9.6 Bioaugmentation with P. Stutzeri КС: Transport, Growth
and Competition .......................................... 269
9.6.1 Inoculation and Transport ......................... 270
9.6.2 Growth and Competition ............................ 271
9.7 pH Adjustment ............................................ 273
9.8 Field Experience: Pilot- and Demonstration-Scale
Testing .................................................. 275
9.8.1 Design and Site Characterization .................. 275
9.8.2 pH of Adjustment, Inoculation and Biocurtain
Colonization ...................................... 275
9.8.3 Long-Term Maintenance of the Biocurtain ........... 279
9.9 Future use of Pseudomonas Stutzeri КС and PDTC ........... 283
References ............................................... 285
CHAPTER 10 BIOAUGMENTATION FOR MTBE REMEDIATION ............... 289
10.1 Introduction ............................................. 289
10.2 MTBE Use and Occurrence in Groundwater ................... 289
10.3 Scientific Basis for Bioaugmentation of MTBE and TBA ..... 290
10.3.1 MTBE Degrading Bacteria ........................... 290
10.3.2 MTBE and TBA Biodegradation in Microcosms ......... 293
10.3.3 Evaluating MTBE and TBA Biodegradation ............ 293
10.4 Bioaugmentation Pilot Testing ............................ 296
10.4.1 Bioaugmentation with Direct Degraders (MC-100
and SC-100) ....................................... 296
10.4.2 Bioaugmentation with Direct Degraders (PM1) ....... 302
10.4.3 Bioaugmentation with Propane Oxidizers (ENV 425) .. 302
10.5 Full Scale Bioaugmentation ............................... 303
10.5.1 MC-100 and SC-100 (Port Hueneme, California,
USA) .............................................. 303
10.5.2 MC-100 (Connecticut, USA) ......................... 305
10.5.3 MC-100 (California, USA) .......................... 305
10.5.4 Propane Oxidizing Bacteria (Camden, New Jersey,
USA) .............................................. 305
10.6 Lessons Learned .......................................... 305
10.7 Current Status ........................................... 307
10.8 Future Prospects for MTBE Bioaugmentation ................ 308
References ............................................... 308
CHAPTER 11 ECONOMICS AND VALUATION OF BIOAUGMENTATION ......... 313
11.1 Introduction ............................................. 313
11.2 Primary Cost Drivers ..................................... 313
11.2.1 Site Specific Testing to Evaluate
Bioaugmentation ................................... 314
11.2.2 Amount and Distribution of Active Organisms ....... 315
11.3 Costs, Value and Benefits of Bioaugmentation ............. 318
11.3.1 Costs for Bioaugmentation Culture and Injection ... 318
11.3.2 Value of Bioaugmentation Relative to a "Wait and
See" Approach to Degradation of DCE and VC ........ 319
11.4 Economics of Alternative Approaches ...................... 320
11.4.1 Costs for Purchase and Injection of Concentrate
Versus In Situ Growth and Distribution ............ 320
11.5 Estimated Costs for Template Scenarios ................... 321
11.5.1 Template Site Descriptions ........................ 321
11.5.2 Costs Categories and Components ................... 323
11.5.3 EISB Remediation Technology Description ........... 324
11.5.4 EISB Remediation Technology Costs ................. 326
11.6 Summary .................................................. 330
References ............................................... 331
CHAPTER 12 RESEARCH NEEDS FOR BIOAUGMENTATION ................. 333
12.1 Introduction ............................................. 333
12.2 Research Needs in Basic Science .......................... 333
12.2.1 Molecular Scale ................................... 334
12.2.2 Organismal Scale .................................. 335
12.2.3 Community Scale ................................... 339
12.2.4 Environmental/Ecosystem and Earth Scale ........... 339
12.3 Key Concepts for Bioaugmentation Research ................ 341
12.3.1 The Niche Concept and Its Importance for
Bioaugmentation ................................... 341
12.3.2 Hydrocarbons and Other Reduced Contaminants ....... 343
12.3.3 The Much-Maligned Microcosm and the Need for
Activity-Based Tests .............................. 345
12.3.4 The Enrichment Paradox ............................ 346
12.4 Applied Research Needs ................................... 349
12.4.1 Monitoring Tools .................................. 349
12.4.2 Production, Storage and Shipping .................. 350
12.4.3 Delivery and Mixing ............................... 350
12.4.4 Electron Donor Choice ............................. 350
12.4.5 Regulatory Considerations ......................... 351
12.4.6 Modeling of Sites, Dechlorination and Biological
Activity .......................................... 351
12.5 Future Perspectives ...................................... 351
12.5.1 Biosensors as MBTs ................................ 352
12.5.2 Designer Microbes and Synthetic Biology ........... 352
12.5.3 Bioaugmenting with Genes .......................... 353
12.5.4 Bioaugmenting with Viruses ........................ 354
12.6 Conclusions .............................................. 356
References ............................................... 356
APPENDIX A: List of Acronyms, Abbreviations and Symbols ....... 363
APPENDIX B: Unit Conversion Table ............................. 367
APPENDIX C: Glossary .......................................... 369
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