SYNTHESIS OF ANTIBACTERIAL SILVER NANOPARTICLES FROM ACTINOMYCETES ISOLATED FROM THIKA INDUSTRIAL WASTE DUMP SITES SOIL AND MOLECULAR IDENTIFICATION OF POTENTIAL ACTINOMYCETES ABEBE BIZUYE KASSAHUN DOCTOR OF PHILOSOPHY IN MOLECULAR BIOLOGY AND BIOTECHNOLOGY PAN AFRICAN UNIVERSITY INSTITUTE FOR BASIC SCIENCES, TECHNOLOGY AND INNOVATION 2018 SYNTHESIS OF ANTIBACTERIAL SILVER NANOPARTICLES FROM ACTINOMYCETES ISOLATED FROM THIKA INDUSTRIAL WASTE DUMP SITES SOIL AND MOLECULAR IDENTIFICATION OF POTENTIAL ACTINOMYCETES Abebe Bizuye Kassahun MB400-0005/15 A Thesis submitted to Pan African University Institute for Basic Sciences, Technology and Innovation in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular Biology and Biotechnology 2018 DECLARATION Statement by the student I, declare that this thesis submitted to the Pan African University Institute of Basic Sciences, Technology and Innovation in partial fulfillment of the requirements for the degree of doctor of philosophy in Molecular Biology and Biotechnology, is original research work done by me under the supervision and guidance of my supervisors, except where due acknowledgement is made in the text; to the best of my knowledge it has not been submitted to in this institution or other institutions seeking for similar degree or other purposes. Student name: Abebe Bizuye Kassahun Signature---------------Date--------------------- ID: MB400-0005/2015 This thesis has been submitted with our approval as university supervisors. 1. Professor Naomi Maina Signature------------------------Date------------------ JKUAT, Nairobi, Kenya 2. Professor Erastus Gatebe Signature-----------------------Date---------------- KIRDI, Nairobi, Kenya 3. Doctor Christine Bii Signature-----------------------Date------------------ KEMRI, Nairobi, Kenya iii DEDICATION I dedicated this work to my parents and family for their support and encouragement throughout my studies. May God bless you! iv ACKNOLEDGEMENTS It is a privilege to acknowledge my supervisors Professor Naomi Maina, Professor Erastus Gatebe and Doctor Christine Bii for their supervision, guidance and support by facilitating and connecting me with research and laboratory institutions. My acknowledgement goes to KIRDI, KEMRI, PAUSTI and JKUAT allowing me to use their laboratories. I thank African Union Commission (ADF/BD/WP/2013/68 to CNM) and Japanese International Co-operation Agency (JICA) (00025 to CNM) for funding this research work and University of Gondar, Department of Biology for giving me supportive materials and study leave. My acknowledgement goes to Mark Mongaka, Godfrey Mwangi, Evangelen Mathiu, Kamathi Muchuna and Taoheed Abddulkareem for their assistance during soil sampling, isolation of actinomycetes, bioassay, FTIR and PCR activities, respectively. My special thanks go to my family and colleagues who directly or indirectly supported and encouraged me towards the accomplishment of this work. Finally, thank you GOD for giving me wisdom, mental and physical health and strength to undertake and finish this work. v LIST OF ABBREVIATIONS AND ACRONYMS AgNPs Silver Nanoparticles ANOVA Analysis of Variance ATCC American type culture collection BLAST Basic Local Alignment Search Tool CFU/g Colony forming unit per gram ESBL Extended spectrum beta-lactamase FTIR Fourier Transform Infrared spectroscopy GPS Global Positioning System HTUs Hypothetical Taxonomic Units JKUAT Jomo Kenyata University of Agriculture and Technology KEMRI Kenya Medical Research Institute KRDI Kenya Research and Development Institute LCA Last Common Ancestor MEGA Molecular Evolutionary Genetics Analysis ML Maximum Likelihood MRSA Methicillin resistance Staphylococcus aureus MUSCLE Multiple Sequence Comparison by Log- Expectation NCBI National Center for Biotechnology Information NJ Neighbor Joining MSA Multiple Sequence Alignment OTUs Operational Taxonomic Units SPSS Statistical Package for Social Sciences PHYLIP PHYLogeny interference Package RDP Ribosomal Database Project vi rRNA gene ribosomal RiboNucleic Acid gene RNA Ribonucleic Acid UPGMA Unweighted Pair Group Method with Arithmetic Mean UV-vis spectroscopy Ultra Violet visible Spectroscopy vii TABLE OF CONTENTS DECLARATION .............................................................................................................................. iii DEDICATION .................................................................................................................................. iv ACKNOLEDGEMENTS ................................................................................................................... v LIST OF ABBREVIATIONS AND ACRONYMS .......................................................................... vi LIST OF TABLES ............................................................................................................................ xi LIST OF FIGURES .......................................................................................................................... xii LIST OF APPENDICES ................................................................................................................. xiii ABSTRACT ................................................................................................................................ xiv CHAPTER ONE: INTRODUCTION ................................................................................................ 1 1.1. Background ............................................................................................................................. 1 1.2. Statement of problem .............................................................................................................. 3 1.3. Justification ............................................................................................................................. 4 1.4. Research questions .................................................................................................................. 5 1.5. Objectives ................................................................................................................................ 5 1.5.1. General objective .............................................................................................................. 5 1.5.2. Specific objectives ............................................................................................................ 5 1.6. Significance of the study ......................................................................................................... 5 1.7. Scope, assumptions and limitation of the study ...................................................................... 6 CHAPTER TWO: LITERATURE REVIEW .................................................................................... 8 2.1. Antibacterial resistance bacteria and its impact ...................................................................... 8 2.1.1. The mechanism of resistance in bacteria .......................................................................... 9 2.1.2. The status and challenges of antibacterial resistant bacteria .......................................... 10 2.2. The status and approaches of antibacterial discovery ........................................................... 13 2.2.1. Isolation of actinomycetes from soil sample .................................................................. 14 2.2.2. Bioassay methods for screening of antibacterial producing actinomycetes ................... 15 2.3. Principles and mechanisms for antibacterial silver nanoparticle synthesis ........................... 17 2.3.1. Biological methods for silver nanoparticles synthesis ................................................... 18 2.3.2. Synthesis of antibacterial silver nanoparticles using actinomycete metabolites ............ 19 2.3.3. Characterization of actinomycetes metabolite mediated antibacterial silver nanoparticles .................................................................................................................................................. 20 2.4. Approaches for identification of actinomycetes .................................................................... 23 2.4.1. Phenotypic based identification of actinomycetes ......................................................... 24 2.4.2. Genotypic based identification of actinomycetes ........................................................... 25 2.4.3. Molecular phylogenetics analysis for actinomycetes ..................................................... 27 viii CHAPTER THREE: MATERIALS AND METHODS ................................................................... 32 3.1. Soil sample preparation and isolation of actinomycetes ....................................................... 32 3.1.1. Study period, area and sampling sites ............................................................................ 32 3.1.2. Study design, target groups and summery of research work flow ................................. 33 3.1.3. Soil sample collection and composite soil sample preparation ...................................... 35 3.1.4. The moisture content and pH analysis for composite soil samples ................................ 37 3.1.5. Composite soil sample pre-treatment for isolation ......................................................... 38 3.1.6. Isolation of actinomycetes from pretreated composite soil samples .............................. 39 3.2. Screening of actinomycetes isolates for antibacterial activity .............................................