Genetic Diversity of Actinobacteria Isolated from Lake Magadi Regina Chemutai Ronoh A thesis submitted in partial fulfillment for the degree of Master of Science in Genetics in the Jomo Kenyatta University of Agriculture and Technology 2013. DECLARATION This thesis is my original work and has not been presented for a degree in any other University. Signature: --------------------------------------- Date: ------------------------ Regina Chemutai Ronoh. This thesis has been submitted for examination with our approval as university supervisors. Signature: ------------------------------------------ Date: ----------------------- Prof. Hamadi Boga JKUAT, Kenya Signature: ---------------------------------------- Date: -------------------------- Dr. Romano Mwirichia JKUAT, Kenya Signature-------------------------------------- Date: ------------------------- . Dr. Nancy Budambula JKUAT, Kenya ii DEDICATION I dedicate this thesis to my family for their moral support and words of encouragement. iii ACKNOWLEDGEMENTS First and foremost I thank God who makes all things possible. Without Him I cannot accomplish anything. My gratitude goes to my supervisors; Prof. Boga, Dr. Mwirichia and Dr. Budambula, for their academic and technical advice that brought this research work to maturity. I especially thank Prof. Boga for research grant that enabled me to do my project. I also thank the Institute of Biotechnology Research for allowing me to use their laboratory. I thank the Director and all the staff of the institute for assisting whenever possible. I also acknowledge my colleagues and all the staff of Botany Department for their support during my study. iv TABLE OF CONTENTS DECLARATION.................................................................................................. II DEDICATION.....................................................................................................III ACKNOWLEDGEMENTS ............................................................................... IV TABLE OF CONTENTS .....................................................................................V LIST OF TABLES .............................................................................................. IX LIST OF FIGURES ..............................................................................................X LIST OF PLATES .............................................................................................. XI LIST OF APPENDICES ...................................................................................XII ABBREVIATIONS AND ACRONYMS........................................................XIII ABSTRACT...................................................................................................... XIV CHAPTER ONE ....................................................................................................1 1.0 INTRODUCTION AND LITERATURE REVIEW..................................1 1.1 Introduction ...................................................................................................1 1.2 Literature review ...........................................................................................4 1.2.1 Life in Alkaline Environment.....................................................................4 1.2.2 Soda Lakes Ecology ...................................................................................7 1.2.3 Microbial Diversity of Soda Lakes ..........................................................10 1.2.4 Polyketide Synthase-I genes (PKS-I).......................................................11 v 1.2.5 Application of Actinobacteria in Enzymes Production............................14 1.3 Statement of the problem ............................................................................17 1.4 Justification .................................................................................................18 1.5 Hypothesis ...................................................................................................19 1.6 Objectives....................................................................................................19 1.6.1 General Objective.....................................................................................19 1.6.2 Specific objectives....................................................................................19 CHAPTER TWO .................................................................................................20 2.0 MATERIALS AND METHODS ...............................................................20 2.1 Sampling site ...............................................................................................20 2.2 Description of Sampling Points...................................................................20 2.4 Molecular Characterization of Isolates........................................................23 2.4.1 DNA Isolation ..........................................................................................23 2.4.2 Amplification of 16S rRNA Genes ..........................................................24 2.4.3 Sequencing and Phylogenetic Analysis....................................................25 2.5 Diversity of Polyketide Synthase Genes Type-1(PKS-I)............................26 2.5.1 Amplification of PKS-1 Genes.................................................................26 2.5.2 Sequence Analysis of Polyketide Synthase Genes...................................27 2.6 Physiological and Biochemical Characterization........................................27 2.6.1 Growth of Isolates at different pH............................................................27 2.6.2 Determination of optimum Growth Temperature ....................................28 vi 2.6.3 Determination of Sodium Chloride Requirement ....................................28 2.6.4 Heat Resistance ........................................................................................29 2.6.5 Lysozyme Resistance ...............................................................................29 2.6.6 Utilization of Carbohydrates ....................................................................29 2.6.7 Nitrate reduction.......................................................................................30 2.6.8 Urea Hydrolysis........................................................................................30 2.6.9 Catalase Test.............................................................................................31 2.6.10 Gelatine Hydrolysis................................................................................31 2.7 Test for Production of Extra-Cellular Enzymes ..........................................31 2.7.1 Test for Amylase. .....................................................................................32 2.7.2 Test for Protease.......................................................................................32 2.7.3 Test for Lipase..........................................................................................32 2.7.4 Test for Xylanase......................................................................................33 2.7.5 Test for Cellulase......................................................................................33 2.8 Antimicrobial Activity ................................................................................34 CHAPTER THREE.............................................................................................35 3.0 RESULTS ....................................................................................................35 3.1 Physical Parameters at the Sampling Stations.............................................35 3.2 Isolation of Cultures ....................................................................................35 3.3 Molecular Characterization of Isolates........................................................38 3.4 Phylogenetic Analysis of 16S rRNA Gene Sequences ...............................38 vii 3.5 Amplification of Polyketide Synthase Genes..............................................44 3.6 Polyketide Synthase Genes Sequence Analysis ..........................................45 3.7 Physiological Characterization....................................................................45 3.7.1 Growth of Isolates at Different pH Levels ...............................................45 3.7.2 Determination of Sodium Chloride Requirement ....................................46 3.7.3 Determination of Optimum Growth Temperature....................................48 3.8 Biochemical Tests .......................................................................................49 3.9 Bio-polymers Hydrolysis ............................................................................51 3.10 Antimicrobial Activity ..............................................................................53 CHAPTER FOUR................................................................................................55 4.0 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS .........55 4.1 Discussion ...................................................................................................55 4.2 Conclusions .................................................................................................62 4.3 Recommendations .......................................................................................63 REFERENCES.....................................................................................................65 APPENDICES......................................................................................................78