UNDERSTANDING THE RELATIONSHIP BETWEEN BACTERIAL COMMUNITY COMPOSITION AND THE MORPHOLOGY OF BLOOM-FORMING MICROCYSTIS A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Leighannah Akins December 2018 ©Copyright All rights reserved Except for previously published materials Dissertation written by Leighannah Akins B.S., Spring Hill College, 2003 M.S., University of Nebraska-Lincoln, 2009 Ph.D., Kent State University, 2018 Approved by Laura G. Leff, Ph.D. , Chair, Doctoral Dissertation Committee Xiaozhen Mou, Ph.D., Members, Doctoral Dissertation Committee Darren L. Bade, Ph.D. Joseph Ortiz, Ph.D. Alison Smith, Ph.D. Accepted by Laura G. Leff, Ph.D. , Chair, Department of Biological Sciences James L. Blank, Ph.D., Dean, College of Arts and Sciences TABLE OF CONTENTS TABLE OF CONTENTS..................................................................................................iii LIST OF FIGURES ..........................................................................................................v LIST OF TABLES ............................................................................................................vi PREFACE…......................................................................................................................vii ACKNOWLEDGEMENTS ............................................................................................ viii I. OVERVIEW CHAPTER..........................................................................1 II. COMPOSITION AND DIVERSITY OF CYANOBACTERIA- ASSOCIATED AND FREE-LIVING BACTERIAL COMMUNITIES DURING CYANOBACTERIAL BLOOMS............................................10 INTRODUCTION.....................................................................................10 METHODS................................................................................................12 RESULTS..................................................................................................18 DISCUSSION............................................................................................27 CONCLUSIONS.......................................................................................32 III. EXUDATES OF HETEROTROPHIC BACTERIA ENHANCE FREQUENCY AND SIZE OF MICROCYSTIS AERUGINOSA (CYANOPHYCEAE) COLONIES........................................................... 34 INTRODUCTION....................................................................................34 METHODS...............................................................................................37 RESULTS.................................................................................................43 DISCUSSION...........................................................................................55 CONCLUSIONS.......................................................................................60 IV. AI-2 QUORUM SENSING SIGNAL PROMOTES COLONY FORMATION IN MICROCYSTIS AERUGINOSA..................................61 INTRODUCTION....................................................................................61 METHODS...............................................................................................64 iii RESULTS.................................................................................................66 DISCUSSION...........................................................................................71 CONCLUSIONS......................................................................................73 V. INTERACTIVE EFFECTS OF BACTERIA AND NUTRIENTS ON MICROCYSTIS MORPHOLOGY............................................................75 INTRODUCTION....................................................................................75 METHODS...............................................................................................77 RESULTS.................................................................................................80 DISCUSSION...........................................................................................89 CONCLUSIONS.......................................................................................92 VI. SUMMARY AND GENERAL DISCUSSION........................................93 REFERENCES................................................................................................................102 APPENDIX A. Supplementary Table 1. Abundance of Bacterial Families.................................124 iv LIST OF FIGURES Figure 1. Study Sites .......................................................................................................13 Figure 2. Bacterial Community Diversity........................................................................19 Figure 3. Bacterial Community Compositional Similarity..............................................21 Figure 4. Variation in Abundance of Proteobacteria......................................................22 Figure 5. Variation in Abundance of Other Bacteria.......................................................23 Figure 6. Microcystis aeruginosa Colonies at 200x Magnification.................................44 Figure 7. Phylogeny of Colony-Promoting Isolates and Selected Other Bacteria...........46 Figure 8. Treatment Effects on Morphology....................................................................48 Figure 9. Correlations of Colony Frequency with Colony Size and EPS........................50 Figure 10. Components of Reflectance Across the Visible Spectrum .............................53 Figure 11. Treatment Differences in Reflectance Components.......................................54 Figure 12. Differences in Morphology at 24 Hours.........................................................69 Figure 13. Differences in Morphology and EPS at 48 Hours..........................................70 Figure 14. Morphological Effects of EI-23......................................................................85 Figure 15. Morphological Effects of EI-7........................................................................86 Figure 16. Effects of EI-23 on EPS..................................................................................87 Figure 17. Relationship of Colony Size and EPS Ratio...................................................88 v LIST OF TABLES Table 1. Physical and Chemical Conditions at Sampling Sites.......................................27 Table 2. Colony-Promoting Effects of Bacterial Isolates…............................................45 Table 3. Cyanobacterial Growth Rates Varying with Isolate Treatment….....................51 Table 4. Cyanobacterial Growth Rates Varying with AI-2 Treatment….........................67 Table 5. Main and Interactive Effects of Four Independent Variables ….......................83 vi Preface The second chapter of this dissertation was previously published under the title “Composition and Diversity of Cyanobacteria-Associated and Free-Living Bacterial Communities During Cyanobacterial Blooms” in Annals of Microbial Ecology Volume 68, Issue 8, 493-503. My co-author Paul Ayayee made substantive contributions to the selection of analysis methods for the metagenomic dataset and to the design of Figures 3, 4, and 5. My dissertation advisor Laura G. Leff, also a co-author of the manuscript, advised me extensively on experimental design, analysis, and writing for publication. The third chapter of this dissertation is a manuscript in preparation for submission for publication. My co-author Joseph Ortiz contributed greatly by advising me on the collection and analysis of reflectance data. My dissertation advisor Laura G. Leff, also a co-author, advised me on experimental set-up, presentation of figures and tables, and writing for publication. vii Acknowledgements I would like to thank my committee members, Laura G. Leff, Xiaozhen Mou, Darren Bade, and Joseph Ortiz for the all of time, effort, and expertise they have contributed to this project. I would also like to thank Paul Ayayee for his contributions to my first manuscript and to improving my understanding of genetic sequencing and statistical analysis of large datasets. Thanks to Mike Model for help with imaging many cyanobacterial cells and colonies, to Mahinda Gangoda for advice on analyzing microcystin, and to Chris Blackwood for statistical advice and the use of his lab’s instruments. Thanks to fellow graduate students Alescia Roberto, Jon Van Gray, Joe Taura, and Anjali Krishnan for help with lab work, coding, and moral support and to my undergraduate assistant Emily Senderak for her careful work with my cell cultures and her enthusiastic attitude during trying times. I thank my beloved mother Charlotte Ferrell for setting me on this journey and believing in me and encouraging me throughout it all. viii Chapter I: Overview The Growing Challenge of Cyanobacterial Blooms The geographic range and temporal duration of cyanobacterial harmful algal blooms (CyanoHABs) are expanding due to climate change and economic development, presenting challenges for ecological conservation and public health throughout the world (Paerl & Huisman 2009, Paerl & Paul 2012). CyanoHABs occur in warm, thermally stratified, eutrophic waters (Paerl & Huisman 2009, Paerl & Paul 2012, Reynolds 1981). During a bloom, buoyant cyanobacteria float at the surface, shading out other primary producers which have higher nutritional value for consumers in aquatic food webs (Paerl & Paul 2012). CyanoHABs have a negative impact on the