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UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE AN ASSESSMENT OF MICROBIAL COMMUNITIES AND THEIR POTENTIAL ACTIVITIES ASSOCIATED WITH OIL PRODUCING ENVIRONMENTS A DISSERTATION SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY By HEATHER SUE NUNN Norman, Oklahoma 2015 AN ASSESSMENT OF MICROBIAL COMMUNITIES AND THEIR POTENTIAL ACTIVITIES ASSOCIATED WITH OIL PRODUCING ENVIRONMENTS A DISSERTATION APPROVED FOR THE DEPARTMENT OF MICROBIOLOGY AND PLANT BIOLOGY BY ______________________________ Dr. Bradley S. Stevenson, Chair ______________________________ Dr. Paul A. Lawson, Co-Chair ______________________________ Dr. Lee R. Krumholz ______________________________ Dr. Joseph M. Suflita ______________________________ Dr. Andrew S. Madden © Copyright by HEATHER SUE NUNN 2015 All Rights Reserved. Dedication To my parents, Joe and Linda Drilling, for their unconditional love and never ending support. Acknowledgements I am grateful for the indispensible guidance and advice of my mentor, Bradley S. Stevenson, because it was essential to my development as a scientist. I would like to thank the rest of my graduate committee, Drs. Paul A. Lawson, Lee R. Krumholz, Joseph S. Suflita, and Andrew S. Madden, for their direction and insight in the classroom and the laboratory. To the members of the Stevenson lab past and present, Lauren Cameron, Dr. Michael Ukpong, Blake Stamps, Brian Bill, James Floyd, and Oderay Andrade, thank for your assistance, discussions, suggestions, humor and friendship. My time as a graduate student was made better because of all of you. I am thankful to my parents, Joe and Linda, my sister, Holly, and the rest of my family for all of their love and support they have always given me. Finally, I am thankful to my husband, Anthony, for being my constant source of love, friendship, encouragement and strength. iv Table of Contents Acknowledgements ......................................................................................................... iv List of Tables .................................................................................................................. vii List of Figures ................................................................................................................ viii Abstract ............................................................................................................................ xi Chapter 1 Summary .......................................................................................................... 1 References .................................................................................................................. 9 Chapter 2 Microbial Communities in Bulk Fluids and Biofilms of an Oil Facility Have Similar Composition but Different Structure ..................................................... 11 Abstract ..................................................................................................................... 11 Introduction .............................................................................................................. 12 Experimental procedures .......................................................................................... 15 Results. ..................................................................................................................... 24 Discussion ................................................................................................................. 29 Acknowledgements .................................................................................................. 35 References ................................................................................................................ 36 Chapter 3 New Insights into the Ecology of Oil Production Facilities Using Microbial Communities, Metagenomes and Hydrocarbon Metabolites ............................. 49 Abstract ..................................................................................................................... 49 Introduction .............................................................................................................. 50 Experimental Procedures .......................................................................................... 53 Results. ..................................................................................................................... 59 Discussion ................................................................................................................. 64 v Conclusion ................................................................................................................ 69 References ................................................................................................................ 71 Appendix A. Supplementary Information ...................................................................... 83 Chapter 2 .................................................................................................................. 83 Chapter 3 .................................................................................................................. 86 vi List of Tables Chapter 2 Table 1. Number of observed OTUs……………………………...……………………44 Table 2. Significantly different relative abundances for taxa between the microbial communities of the separator (SEP) and PIG in a pairwise comparison………………………………………47 Appendix A Chapter 2 Table S1. PCR and DGGE primers used in this study……………………………...….83 Table S2. List of 8 nucleotide barcodes used for parallel pyrosequencing of multiple libraries…………………………………………...84 Chapter 3 Table S1. Sample and site characteristics………………………………………...….....86 Table S2. PCR primers used in this study………………………………………...……87 Table S3. List of 8 nucleotide barcodes used for parallel pyrosequencing of multiple libraries……………………………………….…..88 Table S4. Proteins involved in anaerobic hydrocarbon degradation …...……...………89 Table S5. Metagenome overview…………………………………..……………...….108 Table S6. CheckM results for binned genomes showing genome completeness, contamination, and strain heterogeneity……………..109 vii List of Figures Chapter 1 Figure 1. Diagram of the approach taken to investigate field samples to identify the structure of microbial assemblages and their activities in the environment…………………………………………………….3 Chapter 2 Figure 1. Schematic diagram of the sampled oil production facility…………………..41 Figure 2. DGGE of bacterial 16S rDNA PCR fragments from the PIGb…………..….42 Figure 3. Relative abundance (%) of genera in (A) bacterial and (B) archaeal 16S rRNA clone libraries from the pig sample..……………...………………..43 Figure 4. Rarefaction analysis of OTUs………………………………………………..45 Figure 5. Pyrosequencing analysis of 16S rRNA gene libraries……………………….46 Figure 6. Copy number of 16S rRNA genes per mL of sample………………………..48 Chapter 3 Figure 1. Comparison of 16S rRNA gene libraries…………………………………….77 Figure 2. A dendogram (A.) showing the relatedness of samples based on presence/absence of metabolites, and a heatmap (B.) of putative metabolites detected (black boxes) or not,detected are (white boxes) are shown……………………………………………………78 Figure 3. Neighbor-joining tree showing the phylogenetic relationship of assA and bssA genes from reference strains and sequences viii with homology to known assA genes…………………………………………..79 Figure 4. Partial amino acid sequence alignments of BssA, AssA, and other predicted catalytic subunits of glycyl radical enzymes………...……80 Appendix A Chapter 3 Figure S1. 454 vs Metagenome 16S rRNA Classifications………………………….110 Figure S2. Neighbor-joining tree showing the phylogenetic relationship with representative sequences of α-subunits of the benzoate family of reiske non-heme iron oxygenases from reference strains and those from binned genomes (in bold)……………………………………………….111 Figure S3. Neighbor-joining tree showing the phylogenetic relationship with representative sequences of biphenyl/naphthalene family of reiske non-heme iron oxygenases from reference strains and those from binned genomes (in bold)…………………………………………112 Figure S4. Neighbor-joining tree showing the phylogenetic relationship with representative sequences of extradiol dioxygenases of the vicinal oxygen chelate superfamily, where characterized enzymes typically have a preference for bicyclic substrates, from reference strains and those from binned genomes (in bold)……………………………..113 ix Figure S5. Neighbor-joining tree showing the phylogenetic relationship with representative sequences of extradiol dioxygenases of the viccinal oxygen chelate superfamily, where characterized enzymes typically have a preference for monocyclic substrates, from reference strains and those from binned genomes (in bold)……………………………..114 Figure S6. Neighbor-joining tree showing the phylogenetic relationship with representative sequences of the homoprotocatechuate family of LigB superfamily of extradiol dioxygenases from reference strains and those from binned genomes (in bold)…………………………………….115 x Abstract Microbial populations have been found in oil-associated environments as early as the 1920s. The proliferation and metabolic activities of these microorganisms can have profound deleterious effects on the infrastructure associated with oil reservoirs, production, transport and storage. Biodegradation of hydrocarbons by reservoir microorganisms can lead to the formation of ‘heavy oil’ that is of lower economic value and is more difficult to recover. Some members of reservoir microbial communities also participate in microbial

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