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Distribution and Dynamics of Pyrene-Degrading Mycobacteria in Freshwater Sediments Contaminated with Polycyclic Aromatic Hydrocarbons

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University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 8-2008 Distribution and dynamics of pyrene-degrading Mycobacteria in freshwater sediments contaminated with polycyclic aromatic hydrocarbons Jennifer M. DeBruyn University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Ecology and Evolutionary Biology Commons Recommended Citation DeBruyn, Jennifer M., "Distribution and dynamics of pyrene-degrading Mycobacteria in freshwater sediments contaminated with polycyclic aromatic hydrocarbons. " PhD diss., University of Tennessee, 2008. https://trace.tennessee.edu/utk_graddiss/424 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Jennifer M. DeBruyn entitled "Distribution and dynamics of pyrene-degrading Mycobacteria in freshwater sediments contaminated with polycyclic aromatic hydrocarbons." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Ecology and Evolutionary Biology. Gary S. Sayler, Major Professor We have read this dissertation and recommend its acceptance: Gary McCraken, Jennifer Schweitzer, Mark Radosevich Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a dissertation written by Jennifer M. DeBruyn entitled “Distribution and dynamics of pyrene-degrading Mycobacteria in freshwater sediments contaminated with polycyclic aromatic hydrocarbons.” I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Ecology & Evolutionary Biology. Gary S. Sayler Gary S. Sayler, Major Professor We have read this dissertation and recommend its acceptance: Gary McCraken Gary McCracken Jennifer Schweitzer Jennifer Schweitzer Mark Radosevich Mark Radosevich Acceptance for the Council: Carolyn R. Hodges Carolyn R. Hodges, Vice Provost & Dean of the Graduate School (Original signatures are on file with official student records.) DISTRIBUTION AND DYNAMICS OF PYRENE-DEGRADING MYCOBACTERIA IN FRESHWATER SEDIMENTS CONTAMINATED WITH POLYCYCLIC AROMATIC HYDROCARBONS A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville JENNIFER M. DEBRUYN AUGUST 2008 ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Gary Sayler, as well as my committee members Dr. Mark Radosevich, Dr. Gary McCracken, and Dr. Jennifer Schweitzer for their guidance throughout this project. I also want to thank Dr. L. McKay, Dr. V. Vulava, and Dr. F. Menn for their invaluable advice and assistance. This work would not have been possible without those that have directly assisted me in field sampling: D. Castle, C. Chewning, J. Rainey, J. Easter, M. Scholz, D. Williams, and C. Lalande all braved hot weather and voracious insects to retrieve malodorous mud samples from Chattanooga Creek. My gratitude goes to Dr. Johanna Rinta-Kanto who shared her sediment samples from Lake Erie. I am especially indebted to Chris Chewning and Tommy Mead for their patience and hard work with me in the laboratory. Thank you to my coworkers at the Center for Environmental Biotechnology and the University of Tennessee with whom I’ve had the honour to work. You provided advice, support, and friendship, and ensured that my time here was never boring. Last but not least, thank you to my family: To my father, Ed, for instilling an insatiable curiosity about the world around me. To my mother, Mary, and sisters Emily and Rebecca, for their love, friendship and support. And finally, to Steven, for his wisdom, inspiration, and unwavering belief in me. This work was supported by the Center for Environmental Biotechnology, Research Center of Excellence, University of Tennessee and the Waste Management Research and Education Institute at the University of Tennessee. ii ABSTRACT Microbial biodegradation of polycyclic aromatic hydrocarbons (PAHs) is the primary means of attenuation of these toxic and carcinogenic compounds from contaminated soils and sediments. The documented toxicity and carcinogenicity of many PAHs demands remedial action for PAH-contaminated soils and sediments. This is especially important for historically contaminated sites, where higher molecular weight PAHs (HMW) are recalcitrant. Recently, fast-growing Mycobacteria have been identified that can degrade HMW PAHs, such as pyrene and benzo[a]pyrene. These bacteria have been isolated from a variety of geographical locations, indicating a cosmopolitan distribution. This dissertation work was driven by the need for a better understanding of the ecology, distribution, and dynamics of indigenous microbial populations that can biodegrade PAHs, with an emphasis on Mycobacteria. Both culture-independent molecular approaches and cultivation were used to 1) determine the presence of pyrene-degrading Mycobacterium genotypes and compare dynamics to proteobacterial naphthalene-degradation genotypes; 2) determine the distribution of these genotypes across environments, including different geographical locations (Chattanooga Creek and Lake Erie) and different physical environments (sediments vs. suspended particles); and 3) provide a link between functional pyrene genotypes and phylogenetic identity of isolated pyrene-degrading organisms. The results of these studies indicate the pyrene-degrading Mycobacteria have broad, cosmopolitan distribution in contaminated sediments and suspended particles. Isolation of pyrene-degrading organisms from both Chattanooga Creek and Lake Erie has provided strong evidence for horizontal transfer of pyrene- degrading genes between diverse genera. This work demonstrates the prevalence of pyrene-degrading organisms in contaminated sediments and implicates an integral role in natural attenuation of HMW PAHs. iii TABLE OF CONTENTS PART I. ................................................................................................ 1 LITERATURE REVIEW ............................................................................................ 1 1. Biodegradation of Polycyclic Aromatic Hydrocarbons .......................... 2 1.1 Polycyclic aromatic hydrocarbons........................................................ 2 1.2 Microbial degradation of PAHs ............................................................ 5 1.3 Bioremediation and natural attenuation ................................................ 8 1.4 Ecology and biogeography of PAH-degrading organisms ................. 11 2. Diversity of PAH Dioxygenases: Applications in Ecology of Biodegrative Microorganisms ................................................................. 13 2.1 Ring-hydroxylating dioxygenase structure and specificity ................ 15 2.2 Genetic organization of PAH-catabolism genes ................................. 17 2.3 Diversity of PAH dioxygenases ......................................................... 18 2.4 Ecological insight from dioxygenase-based studies ........................... 22 3. PAH-Degrading Mycobacteria ............................................................... 25 3.1 Identification of pyrene-degrading organisms – Mycobacterium ...... 25 3.2 Distribution and ecology .................................................................... 27 3.3 Pyrene dioxygenase genes: nidA and nidA3 ..................................... 28 4. Methods in Molecular Microbial Ecology ............................................. 31 4.1 Extracting DNA from environmental samples ................................... 31 4.2 Real-time PCR .................................................................................... 32 4.3 16S rDNA Methods ............................................................................ 35 4.4 PCR bias ............................................................................................. 36 4.5 Community fingerprinting: T-RFLPs ................................................. 38 4.6 Statistical analysis of community fingerprinting data ........................ 43 5. General Objectives and Hypotheses ....................................................... 49 6. Study Areas............................................................................................... 52 6.1 Chattanooga Creek ............................................................................. 52 6.2 Lake Erie............................................................................................. 53 iv PART II. ............................................................................................ 56 COMPARATIVE QUANTITATIVE PREVALENCE OF MYCOBACTERIA AND FUNCTIONALLY ABUNDANT NIDA, NAHAC AND NAGAC DIOXYGENASE GENES IN COAL TAR CONTAMINATED SEDIMENTS ............................................................... 56 1. Abstract ....................................................................................................
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