ABSTRACT THE MICROBIAL COMMUNITY COMPOSITION OF CINCINNATI WASTEWATER TREATMENT PLANTS AND EUTROPHIC FRESHWATER LAKES by Keely Marie Icardi Communities of microorganisms play an important role in nutrient cycling within aquatic environments as they transform nutrients from organic and dissolved forms to inorganic forms. In wastewater treatment plants, this role is utilized to remove excess nutrients and clean the wastewater. In eutrophic freshwater lakes, microorganisms support the higher food web through the microbial loop. In this study, the microbial communities in the aeration tank of seven wastewater treatment plants as well as in the water column and sediment of two eutrophic lakes are determined and compared using next generation sequencing techniques. The microbial communities in the wastewater treatment plants are very similar, indicating the same microorganisms are carrying out the same processes. The microbial communities in the sediments and water column of both lakes are different from each other while overlap could be detected between the communities in the water columns and sediments of the different lakes. While the wastewater treatment plants and eutrophic lakes are high nutrient aquatic environments, the environmental factors in each are different and result in different microbial communities adapted to the conditions in each environment. THE MICROBIAL COMMUNITY COMPOSITION OF CINCINNATI WASTEWATER TREATMENT PLANTS AND EUTROPHIC FRESHWATER LAKES A Thesis Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Master of Science by Keely Marie Icardi Miami University Oxford, Ohio 2018 Advisor: Annette Bollmann Reader: Rachael Morgan-Kiss Reader: Xin Wang ©2018 Keely Marie Icardi This Thesis titled THE MICROBIAL COMMUNITY COMPOSITION OF CINCINNATI WASTEWATER TREATMENT PLANTS AND EUTROPHIC FRESHWATER LAKES by Keely Marie Icardi has been approved for publication by The College of Arts and Science and Department of Microbiology ____________________________________________________ Annette Bollmann ______________________________________________________ Rachael Morgan-Kiss _______________________________________________________ Xin Wang Table of Contents Page List of Tables iv List of Figures v Dedication vi Acknowledgements vii Chapter 1 Introduction 1 Chapter 2 Microbial communities in Wastewater Treatment Plants in Cincinnati, Ohio 8 Chapter 3 Microbial communities in two eutrophic Freshwater Lakes in Ohio 35 Chapter 4 Conclusions 71 References 75 Appendix A Filamentous Organisms 85 Appendix B Potential Pathogens 86 Appendix C Phyla less than 10% in Wastewater Treatment Plant communities 87 Appendix D Phyla less than 15% in Eutrophic Lake Sediment communities 88 Appendix E Phyla less than 15% in Eutrophic Lake Water Column communities 91 Appendix F Summary of phyla in Environments 94 iii List of Tables Page Table 1: Cincinnati wastewater treatment plant information 19 Table 2: Lake Acton and Burr Oak history 42 Table 3: Location of Lake sample collection 43 iv List of Figures Page Figure 1: Anthropogenic water use cycle 7 Figure 2: Map of Cincinnati wastewater treatment plants 12 Figure 3: Relative abundance of top 5 wastewater phyla 20 Figure 4: Top 5 core genera 21 Figure 5: Shannon Index of the Wastewater Treatment Plant communities 22 Figure 6: Chao1 Index of the Wastewater Treatment Plant communities 23 Figure 7: Weighted UniFrac of communities 24 Figure 8: Relative abundance of filamentous bacteria 25 Figure 9: Relative abundance of the Ammonia and Nitrite Oxidizing bacteria 26 Figure 10: Relative abundance of Phosphorus Accumulating Organisms 27 Figure 11: Jaccard of potential pathogens 28 Figure 12: Map of Ohio eutrophic lakes 41 Figure 13: Relative abundance of top 5 lake sediment phyla 48 Figure 14: Relative abundance of AOO in lake sediment 49 Figure 15: Relative abundance of top 5 lake water phyla 50 Figure 16: Shannon Index of Lake Acton sediments 51 Figure 17: Shannon Index of Burr Oak lake sediments 52 Figure 18: Chao1 of Lake Acton sediment 53 Figure 19: Chao1 of Burr Oak lake sediment 54 Figure 20: Shannon Index of Lake Acton water column 55 Figure 21: Shannon Index of Burr Oak lake water column 56 Figure 22: Chao1 of Lake Acton water column 57 Figure 23: Chao1 of Burr Oak lake water column 58 Figure 24: Weighted UniFrac of Incubated vs. Nonincubated sediment 59 Figure 25: Weighted UniFrac of the sediment and water column communities 60 Figure 26: Weighted UniFrac of the sediment communities 61 Figure 27: Weighted UniFrac of the water column communities 62 Figure 28: Jaccard of Actinobacteria in the water column 63 Figure 29: Jaccard of Cyanobacteria in the water column 64 Figure 30: Jaccard of Ammonia Oxidizing Organisms in the sediment 65 Figure 31: Jaccard of Methane Oxidizers in the sediment 66 Figure 32: Weighted UniFrac of lake sediment, water column and wastewater communities 74 v Dedication For my family and wonderful husband, their support has made this all possible. “The more that you read, the more things you will know. The more that you learn, the more places you’ll go.” – Dr. Seuss vi Acknowledgements First: thank you to the Microbiology department for the opportunity to complete my Masters degree. In particular, my advisor Dr. Annette Bollmann for challenging me in the laboratory and helping me gain many skills which have made me a successful scientist. The skills I was able to learn and utilize through my research have already proven to be an asset in my career. From my Undergraduate program I’d like to thank Dr. George Bullerjahn for taking me on as a researcher and for encouraging me to go further than I’d considered in my education. I’d also like to thank Dr. Kathy Durham for convincing me to check out Biology as a major and introducing me to the world of Nitrogen through molecular biology research, without her I’d still be struggling with theoretical physics instead. While my life has been touched by so many, it’s impossible to list them all here. Thank you to my fellow students, graduate and undergraduate, for sharing our lives together. Whether we just passed in the hallways, shared a class, laughed, cried, or had many heart to hearts, your influence in my life will not be forgotten. Lastly, thank you to my family. My parents who always supported my dreams and encouraged me to keep moving forward no matter what. My sisters for asking all sorts of questions that made me think. My grandparents for their continuous support. My wonderful husband for keeping me together through this long process and his continuous support. vii Chapter 1 – Introduction Microbial Communities Microbial communities are complex assemblages of unique organisms – viruses, bacteria, archaea, fungi, and protist – which live and interact with each other in a specified environment (Willey, 2008). Defining the environment of these microbial communities can be difficult due to the spatial scale of microbial interactions. Direct interactions between microbes occur on a very small scale (µm), however; the impact of microbes via nutrient use and by-product release can occur on a broader scale (km) (Furhman, 2009). The larger scale caused by nutrient use and by- product release is assisted by environmental factors present including the movement of larger organisms. The results of this collaboration of unique organisms is generally beneficial for macro-organisms within the environment as well as some other micro-organisms (Schink, 1981). Aquatic Environments The primary role of microbes in any environment is to act as a catalyst for biogeochemical cycles by mediating kinetically inhibited reactions that are thermodynamically favorable (Konopka, 2007). In freshwater lakes and wastewater treatment plants these microbes are primary producers, decompose organic matter, and are responsible for nitrogen and phosphorous transformations (McIlroy et al., 2015; Okuda et al., 2014). Freshwater lakes: In lakes, microbial communities are key in the flow of nutrients through the rest of the food chain via the microbial loop (Okuda et al., 2014; Sherr and Sherr, 1988). In many aquatic environments the microbial community is referred to as the microbial loop within the larger food web which encompasses larger consumers – fish, vertebrates, and zooplankton (Sherr and Sherr 1988). Organisms within the microbial loop not only include bacteria but also archaea, viruses and protozoa (Azam et al., 1983). These organisms remove dissolved organic matter from the water, decompose waste materials, and assimilate nutrients as biomass and export by-products which support other microorganisms within the environment (Pomeroy et al., 2007). The assimilated carbon then flows into the rest of the food chain by the consumption of zooplankton and larger consumers (Sherr and Sherr, 1988). Without the microbial loop, much of the carbon present in aquatic systems would be unavailable to higher 1 trophic organisms, as the carbon is transformed from dissolved organic carbon and photosynthesis into available forms (Sherr and Sherr, 1988). Wastewater treatment: Microorganisms are important in wastewater treatment plants as they are responsible for excess nutrient removal and chemical neutralization in the wastewater. Through the cycle of anthropogenic use of water, wastewater treatment plants and natural reservoirs – such as lakes – have the potential to affect the microbial communities of each other (Figure 1). Water is transported from a natural reservoir to be treated in a drinking water plant before