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University of Oklahoma Graduate College UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE METABOLITE-PROFILING TO ASSESS IN SITU ANAEROBIC MICROBIAL HYDROCARBON DEGRADATION IN DIVERSE ENVIRONMENTS A DISSERTATION SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY By VICTORIA ALEXANDRA PARISI Norman, Oklahoma 2010 METABOLITE-PROFILING TO ASSESS IN SITU ANAEROBIC MICROBIAL HYDROCARBON DEGRADATION IN DIVERSE ENVIRONMENTS A DISSERTATION APPROVED FOR THE DEPARTMENT OF BOTANY AND MICROBIOLOGY BY __________________________ Dr. Joseph M. Suflita, Chair __________________________ Dr. Lee R. Krumholz __________________________ Dr. Paul A. Lawson __________________________ Dr. Michael J. McInerney __________________________ Dr. Mark A. Nanny Copyright by VICTORIA ALEXANDRA PARISI 2010 All Rights Reserved. Dedication I dedicate this work to my parents, Kim and Don Broomhall. I would not be half the person I am today, without your unconditional love and support. Acknowledgements I would like to thank Dr. Joseph M. Sulfita, my advisor. You have been instrumental in my growth as a young scientist, and will still be an important part of the researcher I have yet to become. I have been afforded opportunities that have encompassed more than I could have ever hoped for. Beyond the lab, you and Taylor also provided me with a home away from home when needed with food, volleyball and lots of advice. I would also thank my committee members for their advisement and guidance in my career at OU: Dr. Lee R. Krumholz, Dr. Paul A. Lawson, Dr. Michael J. McInerney and Dr. Mark A. Nanny, I am grateful for all you have taught me. I appreciated not only your insight with respect to my coursework, but my research as well. To Dr. Lisa Gieg, Dr. Irene Davidova and Dr. Kathleen Duncan: Thank you for everything you taught me at the bench, at the GC-MS, when hashing out manuscripts, or sampling on the North Slope of Alaska. I am forever in debt to you, and your contributions to my growth as a scientist. Lisa: I never imagined I would spend so much time clicking through TIC peaks. Thank you for teaching me everything you knew, my dissertation is what it is today because of the mass spectral knowledge you provided to me. Also, you were there every day to listen, lend guidance, and even jog or swim with me. To Dr. Mark Morrison, Karen Morrison and the girls. Thank you Mark for giving me an opportunity to work in anaerobic microbiology. I feel that I am where I am today because you encouraged me to go to graduate school. Also a big thank-you iv to Karen and the girls, always wonderful and gracious and supportive, and provided a place to stay when traveling around the world. To Dr. Kristen Savage-Ashlock, Dr. Elaine Hamm, Dr. Kelli Palmer, Keisha Beasley and Cat Isom: Each one of you helped me in different ways. I wouldn’t be half the woman scientist, microbiologist, Pilates-loving, rock-climbing, jogger, cycling fool I am today if it wasn’t for all of you. You have picked me up during hard times, and have been the family I needed when mine was so far away. There will never be enough pages I could fill to thank you for all you have done for me. Finally, and most importantly, I thank my all of family. Where do I begin? Firstly, I thank them for being so understanding for all the missed holidays and birthdays. Secondly, I am lucky enough to have my grandparents in my life. I thank them for all their advice, love and support during my time here at OU. Above all, I thank my Mom, and my step-dad, Don. I know that I am no shrinking violet, and since I was a little girl you have loved me and stuck by me through all my decisions (crazy or not). You supported me as I followed my dream to have a career, not just a job. Hopefully, this is just the beginning. v Table of Contents Acknowledgments………………………………………………………. iv List of Tables……………………………………………………………. ix List of Figures…………………………………………………………… xi Preface…………………………………………………………………… xiv Abstract……………………………………………………………..…… xxi Chapter 1. Field metabolomics and laboratory assessments of anaerobic intrinsic bioremediation of hydrocarbons at a petroleum contaminated site Abstract………………………………………………………………….. 1 Introduction……………………………………………………………… 2 Results…………………………………………………………………… 5 Discussion……………………………………………………………….. 9 Experimental Procedures………………………………………………… 15 References……………………………………………………………….. 20 Chapter 2. Biocorrosive thermophilic microbial communities in Alaskan North Slope oil facilities Abstract…………………………………………………………………… 31 Introduction……………………………………………………………….. 31 Materials and Methods …………………………………………………… 33 Results and Discussion …………………………………………………… 37 vi References………………………………………………………………… 48 Chapter 3. Detection of polar compounds as putative metabolic intermediates as in situ evidence for petroleum biodegradation in Alaskan North Slope oil reservoirs Abstract…………………………………………………………………… 57 Introduction……………………………………………………………….. 58 Experimental Procedures.…………………………………………………. 61 Results…………………………………………………………………….. 63 Discussion ………………………………………………………………… 67 References…………………………………………………………………. 73 Chapter 4. An alternate hypothesis for the anaerobic oxidation of methane Abstract……………………………………………………………………. 84 Introduction………………………………………………………………... 84 Materials and Methods...…………………………………………………... 85 Results and Discussion.……………………………………………………. 86 References…………………………………………………………………. 89 Appendix A: Biodegradation of low molecular weight alkanes under mesophilic, sulfate-reducing conditions: metabolic intermediates, and community patterns. Abstract…………………………………………………………………… 93 Introduction……………………………………………………………….. 94 vii Materials and Methods……………………………………………………. 97 Results and Discussion……………………………………………………. 101 References………………………………………………………………… 109 Appendix B: Supplementary Tables and Figures Chapter 1………………………………………………………………….. 120 Chapter 2………………………………………………………………….. 121 Supplementary References………………………………………………... 141 Chapter 3…………………………………………………………………... 142 viii List of Tables Chapter 1 Table 1. Signature anaerobic metabolites of microbial hydrocarbon decay detected in groundwater monitoring wells…………………………. 26 Table 2. Microbial biomass levels and community physiological status from trans- to cis- fatty acid ratios as well as and cyclopropyl fatty acids to their monoenoic precursors in selected incubations as determined by PLFA analysis…………………………………………….. 29 Table 3. Relationship between field profiling for metabolites, endogenous hydrocarbon decay and COC amended incubations for MW-439………… 30 Chapter 2 Table 1. Metabolites associated with the anaerobic biodegradation of C1-C4 hydrocarbons in Alaskan North Slope (ANS) oil field samples………….. 53 Chapter 3 Table 1. Description and characteristics of samples collected from oil Field A. Samples were collected from well heads and processing facilities……………………………………………………………………. 78 Table 2. Description and characteristics of samples collected from oil Field B. Samples were collected from well heads and processing facilities……………………………………………………………………. 79 Table 3. List of polar organic compounds differentially associated with the anaerobic biodegradation of hydrocarbons……………………………. 80 Chapter 4 Table 1. Methylsuccinate detected as a trimethylsilane derivative in various field and laboratory samples……………………………………… 90 ix Table 2. Comparison of predicted 13C-NMR shifts for putative AOM metabolites based on the pathway predicted in Figure 1 with those actually observed……………………………………………… 91 Appendix A Table 1. Differences in the carbon isotope ratios (δ C13) of propane and carbon dioxide in active propane-degrading, sulfate amended enrichments compared with the propane (from tank), propane-fed sterile control and a propane-fed enrichment without sulfate…………………………… 115 Table 2. Community composition of sub-cultured propane (101) and pentane (10-3) degrading enrichments……………………………………. 117 Appendix B Chapter 2 Table S1. Summary information regarding samples collected and types of analyses performed……………………………………………... 121 Table S2A. Sequence similarity and taxonomic relationships of bacterial representative small subunit partial rRNA gene sequences (OTUs at 97% similarity)………………………………………………… 122 Table S2B. Sequence similarity and taxonomic relationships of archaeal representative small subunit partial rRNA gene sequences (OTUs at 97% similarity)………………………………………………… 130 Table S3. Measures of genetic diversity and species richness…………………… 134 Table S4. Core and dominant bacterial OTUs (97% similarity) and physiologies………………………………………………………………. 135 Table S5. Dominant archaeal OTUs (97% similarity) and physiologies………… 136 x List of Figures Chapter 1 Figure. 1. Monitoring well locations (boxes) used for groundwater sampling at the former refinery site near the North Platte River in Casper, WY……………………………………………………………. 25 Figure 2. Rates of sulfate reduction in aquifer incubations. The average rate of sulfate reduction for the first sulfate amendment occurred at time zero until approximately 134 d…………………………………………….. 27 Figure 3. Select hydrocarbon loss in substrate-unamended incubations by GC-MS analysis (after a 250 d incubation)……………………………. 28 Chapter 2 Figure 1. Relative abundances of sequences from five bacterial 16S rRNAgene libraries……………………………………………………….. 54 Figure 2. Distribution of bacterial sequences from 2L, PS, and CO illustrating the number of sequences from taxa found in all three libraries……………………………………………………………………. 55 Figure 3. Relative abundances of sequences from three archaeal 16S rRNA gene libraries……………………………………………………….. 56 Chapter 3 Figure
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