Graduate Theses, Dissertations, and Problem Reports 2013 Spatial Relationship between the Trichloroethylene Degrading Bacteria Dehalococcoides Sp., Sulfate Reducers and Archaea during Reductive Dechlorination Pujya Wagle Gautam West Virginia University Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Gautam, Pujya Wagle, "Spatial Relationship between the Trichloroethylene Degrading Bacteria Dehalococcoides Sp., Sulfate Reducers and Archaea during Reductive Dechlorination" (2013). Graduate Theses, Dissertations, and Problem Reports. 580. https://researchrepository.wvu.edu/etd/580 This Thesis is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Thesis in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you must obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Thesis has been accepted for inclusion in WVU Graduate Theses, Dissertations, and Problem Reports collection by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. Spatial Relationship between the Trichloroethylene Degrading Bacteria Dehalococcoides Sp., Sulfate Reducers and Archaea during Reductive Dechlorination Pujya Wagle Gautam Thesis submitted to the College of Benjamin M Statler College of Engineering and Mineral resources at West Virginia University in partial fulfillment of the requirements for the Degree of Masters of Science in Civil and Environmental Engineering Jennifer L Weidhaas, Ph.D., P.E. Committee Chairperson Radhey S. Sharma, Ph.D. Lian-Shin Lin, Ph.D., P.E. Department of Civil and Environmental Engineering Morgantown, West Virginia 2013 Key words: bedrock fracture, bioremediation, DHC, sandy aquifer, silt aquifer, reductive cometabolism, qPCR, TCE. ABSTRACT Spatial Relationship between the Trichloroethylene Degrading Bacteria Dehalococcoides Sp., Sulfate Reducers and Archaea during Reductive Dechlorination Pujya Wagle Gautam Trichloroethylene (TCE) released to the environment is of great concern due to its toxicity and carcinogencity. The microorganisms involved in bioremediation of TCE such as methanogens (organisms within the domain Archaea), sulfate reducing bacteria (SRB) and Dehalococcoides sp. (DHC) are of particular interest in this study. Three different types of bench scale reactors were constructed to model different aquifer types such as sandy, silty and fractured bedrock aquifers (hereafter type 1, 2 or 3 reactors, respectively). This study evaluated the effect of TCE concentration in different types of reactors on the distribution of selected microorganisms with distance from the source of the TCE. It also examined the spatial relationship between Dehalococcoides sp., Archaea and SRB with respect to reducing equivalents (e.g., food) in different types of aquifer environment contaminated with TCE. The DNA analysis was performed by using quantitative polymerase chain reaction (qPCR) and fluorescent in situ hybridization (FISH). In this study concentrations of Archaea were higher in all reactors than other microorganisms under study. In the type 1 reactor, with increasing concentrations of TCE, DHC concentrations and SRB concentrations increased. In type 2 and 3 reactors, there were no observed correlations between initial concentrations of TCE and the concentration of the studied microbes. The highest DHC concentration was present near the food source in type 1 and 2 reactors. In type 3 reactors the concentration of microorganisms was higher outside the tube (e.g., fracture) than inside. The spatial relationship between Dehalococcoides sp. and various microorganisms that compete for bioremediation substrates supplied is helpful to understand when and where to bioaugment an aquifer undergoing bioremediation of TCE with Dehalococcodies sp. Acknowledgements My greatest gratitude goes to my supervisor Jennifer L Weidhaas, Ph.D, P.E. who offered her continuous advice, encouragement and undertook tremendous responsibility in supervising the completion of my research and thesis. I thank her for the systematic guidance, patience, motivation, enthusiasm, great effort and immense knowledge she put into training me in the scientific field. I could not have imagined having a better advisor and mentor for my study. It was with her encouragement and help that I could be confident in doing the whole job. I also would like to thank Dr. Lian-Shin Lin and Dr. Radhey Sharma for their valuable time and serving on my committee. I acknowledge David Turner, Senior Lab Instrumentation Specialist, Civil and Environmental Engineering, for contributing his time to make the reactors to support this study. Also I would like to thank Dr Jianbo Yao, Division of Animal & Nutritional Sciences and his students for allowing me to use nanodrop quantification spectrophotometer to complete analysis at his laboratory. My thanks go to the chemistry department for allowing me to use the Gas chromatography instrument and Dr Karen Martin, Imaging facility of Mary Babb Randolph Cancer Center at Health science department for training me in image analysis. I am thankful to Dr. Ryan Dupont (USU) and Dr. Paul Hatzinger (Shaw) for providing Dehalococcoides cultures studies. Finally, I take this opportunity to express the profound gratitude to my dear husband Lekhnath Gautam, my beloved parents, siblings and my son Prithul Gautam for their love, encouragement, understanding and continuous support during all this years. I really appreciate the help of my friends and fellow students: Thomas, Autumn, Isabel, Dong Yang (Sunny) and Chenjie. Last but not least, my thanks go to all the other faculty and staff in Civil and Environmental Engineering at WVU especially, Dr. Karen Buzby, and Research Assistant Will Ravenscroft for their help. ii Table of contents ABSTRACT............................................................................................................................... i Acknowledgements ................................................................................................................... ii Table of contents ..................................................................................................................... iii List of Tables .............................................................................................................................v List of figures ........................................................................................................................... vi List of Abbreviations ............................................................................................................... ix CHAPTER 1 INTRODUCTION ..............................................................................................1 1.1 Background .............................................................................................................................. 1 1.2 Exposure and environmental effects of TCE ............................................................................. 2 1.3 Current treatment methods and issues ....................................................................................... 2 1.4 Research objectives and hypotheses ......................................................................................... 5 1.5 Significance of the research ...................................................................................................... 5 CHAPTER 2 LITERATURE REVIEW ..................................................................................7 2.1 Trichloroethylene production, use, properties and treatment ..................................................... 7 2.1.1 Physical and chemical properties: ..................................................................................... 8 2.2 Physical and chemical TCE treatment technologies .................................................................. 9 2.3 Biological TCE treatment ....................................................................................................... 10 2.3.1 Aerobic oxidation of TCE ............................................................................................... 11 2.3.2 Anaerobic reductive dechlorination................................................................................. 12 2.3.3 Reductive co-metabolism ............................................................................................... 15 2.4 Electron acceptor/electron donor and redox potential .............................................................. 16 2.5 Dehalococcoides sp. (DHC) ................................................................................................... 23 2.6 Sulfate reducing bacteria (SRB) ............................................................................................. 26 2.7 Archaea/Methanogens ............................................................................................................ 26 2.8 Molecular screening techniques .............................................................................................. 27 2.8.1 PCR/qPCR ..................................................................................................................... 27 2.8.2 Fluorescence in situ hybridization (FISH)