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Assessment of Biodegradation and Toxicological Effects Of ASSESSMENT OF BIODEGRADATION AND TOXICOLOGICAL EFFECTS OF MICROCYSTINS A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Anjali Krishnan August, 2019 Copyright All rights reserved Except for previously published materials Dissertation written by Anjali Krishnan B.Sc., University of Mumbai, 2011 M.Sc., University of Mumbai, 2013 Ph.D., Kent State University, 2019 Approved by Xiaozhen Mou, Ph.D., Chair, Doctoral Dissertation Committee Gary Koski, Ph.D., Members, Doctoral Dissertation Committee Darren Bade, Ph.D. Joseph Ortiz, Ph.D. Michael Strickland, Ph.D. Accepted by Dr. Ernest Freeman, Ph.D., Chair, Department of Biological Sciences Dr. James L. Blank, Ph.D., Dean, College of Arts and Sciences TABLE OF CONTENTS . .. iii LIST OF FIGURES . .. v LIST OF TABLES . .. .viii ACKNOWLEDGEMENTS . .. .. ix I.GENERAL INTRODUCTION AND OVERVIEW . 1 REVIEW OF LITERATURE . 1 REFERENCES . .22 II. IDENTIFICATION AND CHARACTERIZATION OF MICROCYSTIN DEGRADING BACTERIA FROM LAKE ERIE. .38 PREFACE . 38 ABSTRACT . .38 INTRODUCTION . 39 METHODS . .41 RESULTS . .44 DISCUSSION . .. .. 44 ACKNOWLEDGEMENTS . 48 REFERENCES . 54 III TAXONOMIC AND FUNCTIONAL HETEROGENEITY OF BACTERIOPLANKTON IN LAKE ERIE. 59 PREFACE . 59 ABSTRACT . 59 INTRODUCTION . 62 METHODS . .. 65 RESULTS . .69 DISCUSSION . .72 iii REFERENCES . .87 IV. IDENTIFICATION OF NOVEL MICROCYSTIN DEGRADING PATHWAY ADOPTED BY BACTERIOPLANKTON FROM LAKE ERIE USING TRANSPOSON MUTAGENESIS AND LC-MS. .. 95 ABSTRACT . .95 INTRODUCTION . .96 METHODS . 99 RESULTS . .103 DISCUSSION . 105 CONCLUSION . 107 REFERENCES . 114 V.CHARACTERIZATION OF MICROCYSTIN-INDUCED APOPTOSIS IN HepG2 HEPATOMA CELLS. 117 ABSTRACT . 117 INTRODUCTION . .118 METHODS . 120 RESULTS . 123 DISCUSSION . 123 CONCLUSION . …. 127 ACKNOWLEDGEMENTS. 127 REFERNCES . .. .. .. 133 VI.SUMMARY CHAPTER . 139 GENERAL DISCUSSION . 139 GENERAL CONCLUSION . 144 REFERENCES . 145 APPENDIX . 147 iv LIST OF FIGURES Figure 1. Cyanobacterial blooms as seen in western basin of Lake Erie in the Summer of 2016.19 Figure 2. Schematic representation of mlr and xenobiotic degradation pathway . .. .. 20 Figure 3. Schematic representation of the extrinsic and intrinsic apoptotic pathway . …..21 Figure 4. Changes of MC-LR concentration (left axis, solid gray line) and optical density OD600 (right axis, dash black line) in BG11 growth media supplied with 1 µg/ml of MC-LR and bacterial isolates. .. .. .. .. 50 Figure 5. A neighbor-joining phylogenetic tree based on partial sequence of 16S rRNA gene showing the relationship sequences of isolated MC-degrading bacteria and their closely related relatives. Gray circles are to label previously identified MC-degrading bacteria. The GenBank accession numbers of the sequences are shown in parentheses. Bootstrap values that are higher than 50% are show at the branch nodes (1,000 resampling). The scale bar represents 0.02 nucleotide substitutions per position . .. .. 51 Figure 6. Effect of temperature on MC-LR degradation by MC- degrading bacteria. 52 Figure 7. Effects of pH on degradation of MC-LR by MC-degrading bacteria. .53 Figure 8. Microcystin degradation of the MC+ isolates as seen in the BIOLOG MT2 screening. Bacterial isolates which showed a significant increase in their optical density for MC-LR concentrations of 0.1, 1 and 10µg/ml were depicted in this bar graph . .. .79 Figure 9. Microscopic observation for gram staining performed for MC-degrading bacterial isolates with morphologies of bacilli (a) ,cocci (b) and coccobacilli (c) . 80 Figure 10. A neighbor-joining phylogenetic tree based on partial sequence of 16S rRNA gene showing the relationship sequences of isolated MC-degrading bacteria and their closely related v relatives. The GenBank accession numbers of the sequences are shown in parentheses. Bootstrap values that are higher than 50% are show at the branch nodes (1,000 resampling). The scale bar represents 0.02 nucleotide substitutions per position. .. .. .. .81 Figure 11: Gel image for the mlrA based amplification performed for MC+ isolates. mlrA amplification was observed for the positive control Sphingosinicella microcystinivorans (16998) and Paucibacter toxinivorans (19791) . 82 Figure 12: Effect of temperature on MC-LR degradation rate by MC-degrading bacteria as shown in a heat map. The gradient of colors from blue to red indicate the degradation rate for the MC+ isolates . ..
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