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Isolation and Ecology of Bacterial Populations Involved in Reductive Dechlorination of Chlorinated Solvents

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Isolation and Ecology of Bacterial Populations Involved in Reductive Dechlorination of Chlorinated Solvents ISOLATION AND ECOLOGY OF BACTERIAL POPULATIONS INVOLVED IN REDUCTIVE DECHLORINATION OF CHLORINATED SOLVENTS A Dissertation Presented to The Academic Faculty By Youlboong Sung In Partial Fulfillment of the Requirements of the Degree Doctor of Philosophy in Environmental Engineering School of Civil and Environmental Engineering Georgia Institute of Technology August 2005 ISOLATION AND ECOLOGY OF BACTERIAL POPULATIONS INVOLVED IN REDUCTIVE DECHLORINATION OF CHLORINATED SOLVENTS Approved by: Dr. Frank E. Löffler, Advisor Dr. Joseph B. Hughes School of Civil and Environmental Engineering School of Civil and Environmental Engineering Georgia Institute of Technology Georgia Institute of Technology Dr. Ching-Hua Huang Dr. Robert A. Sanford School of Civil and Environmental Engineering School of Geology Georgia Institute of Technology University of Illinois Dr. Patricia Sobecky School of Biology Georgia Institute of Technology Date Approved: July 14, 2005 ACKNOWLEDGEMENTS I would first like to thank my advisor, Dr. Frank E. Löffler, for his constant faith in me during the Ph.D. program. He is not only an educational mentor but also a friend to me. His talented guidance, advice, encouragement and humor enabled me to finish Ph.D. work at Georgia Tech successfully. I would like to thank all my Ph.D. committee members, Drs. Joseph B. Hughes, Ching- Hua Huang, Robert A. Sanford, and Patricia Sobecky, for their valuable comments and suggestions that helped to improve this dissertation. Especially, I would like to express my special appreciation to Dr. Robert A. Sanford for spending a lot of time discussing for sample analysis and data interpretation. I would like to thank all past and present students and postdoctoral fellows in the Löffler lab, Jianzhong, Ben, Ryoung, Sara, Rosy, Johnathan, Rosa, Ivy, Dr. Ritalahti and Dr. Wu for their friendship and helpful discussion. My sincere appreciation is also given to faculty, staff, and friends I met in Georgia Tech’s group Environmental Engineering, especially Dr. Zhu, Ms. Andrea Be, and Ms. Therese Rehkopf for their generosity and support. I want to express gratitude to my parents and brother for their love, understanding and support. Finally, I express my deepest appreciation to my wife and beloved son, Hyunkue Bryan Sung. Without them, none of my accomplishments would have been possible. I dedicate this thesis to my family and wife, Jihyun Lee. iii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS iii LIST OF TABLES ix LIST OF FIGURES x SUMMARY xii CHAPTER I Introduction 1 CHAPTER II Literature Review 4 2.1. Natural sources of halogenated compounds 4 2.2. Anthropogenic sources of halogenated compounds 4 2.3. Microbial oxidation of chlorinated ethenes under aerobic conditions 5 2.4. Microbial oxidation of chlorinated ethenes under anaerobic conditions 7 2.5. Microbial reductive dechlorination of chlorinated ethenes under anaerobic conditions 8 2.6. Reductive dehalogenases 11 2.7. Reductive dechlorination in the presence of other terminal electron acceptors 12 2.8. Metal and radionuclide contamination in environments 13 2.9. Microbial reduction of metal and radionuclide 13 2.10. References 16 CHAPTER III Environmental Distribution of Chloroethene-Dechlorinating Populations and Quantification of Dehalococcoides-Derived Reductive Dehalogenase Genes 25 3.1. Introduction 26 3.2. Materials and Methods 27 3.2.1. Chemicals 27 3.2.2. Sample collection 27 iv 3.2.3. Microcosms and enrichment cultures 28 3.2.4. Ampicillin treatment 31 3.2.5. Qualitative PCR 31 3.2.6. Quantitative Real-Time PCR (RTm PCR) 32 3.2.7. Analytical methods 33 3.3. Results 35 3.3.1. Microcosms and enrichment cultures 35 3.3.2. Reductive dechlorination in the presence of ampicillin 37 3.3.3. Reductive dechlorination of PCE in the absence of H2 37 3.3.4 Identification of chlorinated ethene degrading populations 37 3.3.5. Detection of tceA, bvcA, and vcrA 39 3.3.6. Quantification of tceA, bvcA, and vcrA 39 3.3.7. Development of Bio-Dechlor INOCULUM TM for bioaugmentation 42 3.4. Discussion 45 3.5. References 49 CHAPTER IV Characterization, Description and Specific Detection of Geobacter lovleyi Strain SZ sp. nov., a Novel Uranium-Reducing and Tetrachloroethene (PCE)-Dechlorinating Bacterium 53 4.1. Introduction 55 4.2. Materials and Methods 56 4.2.1. Isolation procedure and growth conditions 56 4.2.2. 16S rRNA gene sequencing and phylogenetic analysis 56 4.2.3. Restriction Fragment Length Polymorphism (RFLP) analysis of PCR amplified 16S rRNA genes 57 4.2.4. PCR-based detection of strain SZ 57 4.2.5. Substrate utilization and growth conditions 58 4.2.6. Growth linked uranium reduction by strain SZ 60 v 4.2.7. PCE dechlorination in phylogenetically related species 61 4.2.8. Determination of electron donor consumption threshold concentrations 61 4.2.9. Rate and yield measurements 62 4.2.10. Influence of different electron acceptors on PCE dechlorination 62 4.2.11. Induction studies 63 4.2.12. Microscopy 63 4.2.13. Analytical techniques 63 4.3. Results 65 4.3.1. Enrichment and isolation 65 4.3.2. Culture purity and phylogeny 65 4.3.3. Morphology of strain SZ 66 4.3.4. Substrate utilization 67 4.3.5. Growth linked uranium reduction by strain SZ 72 4.3.6. Electron donor consumption threshold concentrations 72 4.3.7. Induction studies 74 4.3.8. Dechlorination in presence of alternate electron acceptors 74 4.3.9. PCR-based detection of strain SZ 74 4.4. Discussion 77 4.5. Description of Geobacter lovleyi sp. nov. 82 4.6 References 84 CHAPTER V Quantitative PCR Confirms Purity of Strain GT, a Novel Trichloroethene-to-Ethene Respiring Dehalococcoides Isolate 89 5.1. Introduction 91 5.2. Materials and Methods 92 5.2.1. Chemicals 92 vi 5.2.2. Source of dechlorinating culture and isolation 93 5.2.3. Determination of substrate utilization 93 5.2.4. 16S rRNA gene analysis and presence of reductive dehalogenase genes 94 5.2.5. Quantitative Real-Time PCR (RTm PCR) 96 5.2.6. Analytical techniques 96 5.3. Results 98 5.3.1. Isolation of Dehalococcoides sp. strain GT 98 5.3.2. Physiological characteristics of strain GT 104 5.3.3. Detection of vcrA in strain GT 106 5.3.4. Growth-linked VC dechlorination 106 5.3.5. Environmental detection of vcrA containing Dehalococcoides species 106 5.4. Discussion 109 5.5 References 112 CHAPTER VI Effect of Sulfur Oxyanions and Sulfide on the Bacterial Reduction of Chlorinated Ethenes 115 6.1. Introduction 116 6.2. Materials and Methods 117 6.2.1. Chemicals 117 6.2.2. Organisms, medium and growth conditions 117 6.2.3. Detection of chloroethene dechlorinating populations 118 6.2.4. Growth conditions in defined co-culture experiments 119 6.2.5. Analytical methods 119 6.3. Results 120 6.3.1. Microbial reductive dechlorination in the presence of sulfate 120 6.3.2. Identification of the chloroethene-dechlorinating populations 124 vii 6.3.3. Effect of microbial reductive dechlorination in the presence of sulfide 124 6.3.4. Effect of sulfur oxyanions and sulfide on chloroethene degrading pure cultures 127 6.3.5. Influence of sulfide to defined coculture experiments 127 6.4. Discussion 131 6.5. References 136 CHAPTER VII Conclusions and Recommendations 139 APPENDIX A 16S rRNA gene sequence of Geobacter lovleyi strain SZ 143 APPENDIX B 16S rRNA gene sequence of Dehalococcoides sp. strain GT 144 APPENDIX C Ferrozine method for determination of Fe(II) and Total Fe 145 APPENDIX D Spectrophotometric determination of U(VI) 146 APPENDIX E DNA isolation from bacterial cell 147 APPENDIX F Dilution and extinction series (media and low melting agarose) 148 APPENDIX G Quantification of Geobacter 16S rRNA gene copies 149 APPENDIX H Preparation of MnO2 stock solution (0.125 M) 150 APPENDIX I Calculation of aqueous chloroethene concentration (Ce) 151 APPENDIX J Preparation of poorly crystalline Fe(III): FeOOH 152 APPENDIX K Chloroethene standards 153 APPENDIX L Spectrophotometric determination of hydrogen sulfide 154 APPENDIX M Spectrophotometric determination of Mn(IV) 155 viii LIST OF TABLES Page Table 3.1. Characteristics of contaminated aquifer samples and microcosm test results 29 Table 3.2. Characteristics of non-contaminated aquifer samples and microcosm test 30 results Table 3.3. Sequences of 16S rRNA gene and Dehalococcoides derived RDase gene targeted primers and PCR conditions 34 Table 3.4. Distribution of chlorinated ethene dechlorinating populations, Dehalococcoides RDase genes, dechlorination end products in the presence of ampicillin, and the dechlorination end products in the absence of H2 in ethene producing enrichment cultures 38 Table 3.5. Quantification of total bacterial and Dehalococcoides 16S rRNA gene copies, and chloroethene RDase genes in select PCE dechlorinating enrichment cultures 44 Table 4.1. Phenotypic differences between Geobacter thiogenes and Geobacter lovleyi 68 Table 4.2. Pairwise % similarity values for Geobacter lovleyi related species and environmental clone sequences 83 Table 5.1. Utilization profiles of chlorinated ethenes as an electron acceptor in defined Dehalococcoides cultures 110 Table 6.1. Detection of chloroethene dechlorinating populations using specific 16S rRNA gene targeted primers 125 Table 6.2. Reductive dechlorination end products in pure chloroethene dechlorinating cultures in the presence of sulfate or sulfide 129 ix LIST OF FIGURES Page Figure 3.1. Reductive dechlorination of PCE (A), TCE (B), cis-DCE (C), and VC (D) to ethene by the H7 culture amended with (5 mM) acetate and H2. 36 Figure 3.2. Proportion of Dehalococcoides in PCE dechlorinating enrichment cultures (A). Proportion of known and unidentified RDase genes as a fraction of the total Dehalococcoides population in PCE dechlorinating enrichment cultures (B). 41 Figure 3.3. Reductive dechlorination of TCE in bioaugmented microcosms. 44 Figure 4.1. Phylogenetic tree of strain SZ and related species and environmental clones based on 16S rRNA gene sequences.
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