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Metagenomic and Metatranscriptomic Analysis of Bacterial, Archaeal and Fungal Communities Within the Hot Springs of Lake Magadi in Kenya

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Metagenomic and Metatranscriptomic Analysis of Bacterial, Archaeal and Fungal Communities Within the Hot Springs of Lake Magadi in Kenya METAGENOMIC AND METATRANSCRIPTOMIC ANALYSIS OF BACTERIAL, ARCHAEAL AND FUNGAL COMMUNITIES WITHIN THE HOT SPRINGS OF LAKE MAGADI IN KENYA ANNE KELLY KAMBURA DOCTOR OF PHILOSOPHY (Biotechnology) JOMO KENYATTA UNIVERSITY OF AGRICULTURE AND TECHNOLOGY 2016 Metagenomic and Metatranscriptomic analysis of Bacterial, Archaeal and Fungal communities within the hot springs of Lake Magadi in Kenya Anne Kelly Kambura A Thesis Submitted in Fulfillment for the Degree of Doctor of Philosophy in Biotechnology in the Jomo Kenyatta University of Agriculture and Technology 2016 DECLARATION This thesis is my original work and has not been presented for a degree in any other University. Signature: _______________________ Date: ______________ Anne Kelly Kambura This thesis has been submitted for examination with our approval as university supervisors Signature: ______________________ Date: ______________ Prof. Hamadi Iddi Boga, (PhD) Taita Taveta University (TTU), Kenya Signature: _________________________ Date: ______________ Dr. Romano Mwirichia, (PhD) University of Embu (UoEm), Kenya Signature: __________________________ Date: ______________ Dr. Remmy Kasili, (PhD) JKUAT, Kenya ii DEDICATION This work is dedicated to my dear family; my husband Edward Nderitu, children; Shawn Karanja, Melissa Wangithi and Shayne Koome. I appreciate the support you have accorded me during the course of my studies. Without your encouragement and support this journey would be longer and tougher. iii ACKNOWLEDGEMENTS I thank God Almighty for bringing me this far from the commencement of this long and challenging journey. I wish to thank the following institutions for their assistance in this research. The National Commission for Science, Technology and Innovation (NACOSTI), for a research grant and permit; Higher Education Loans Board (HELB) for a partial scholarship to pursue this degree; Kenya Wildlife Service (KWS) for an access permit to the sampling area in Lake Magadi and Little Magadi; and Tata Chemicals Ltd for providing invaluable guides in this area. I wish to acknowledge the Director, Institute for Biotechnology Research (IBR), JKUAT; Prof. Bernard Nyende for allowing me to enroll for this degree in the Institute as a staff, and for providing an enabling environment for laboratory and research work for this study. This work would not have been accomplished without the wise counsel, professional acumen and research mentorship of Prof Hamadi Boga and Dr. Romano Mwirichia in microbial biotechnology. I truly appreciate my supervisors for their patience and for believing in me, even at the time when submission of papers for publication was very traumatic. Thank you for standing by me. My most sincere gratitude goes to Dr. Remmy Kasili for walking the research walk with me, the distances we covered to collect samples in Lake Magadi and Little Magadi. His presence both in the field and JKUAT made this research possible and the challenges encountered livable. iv In addition, no adjectives can describe the appreciation I have for Mr. Josephat Muthanga, for his intellectual ability and ensuring exact sampling techniques were achieved for this research. I am greatly indebted to him. The latter part of this work would not have been accomplished without the meticulous encouragement from the Dean, Taita Taveta University, School of Agriculture, Earth and Environmental Sciences, Dr. Marianne Maghenda. She burnt the midnight oil with me to make sure that this work was accomplished in time. I appreciate her wisdom. Particular recognition goes to my colleagues; Odilia Salano, Grace Mungai, Amanuel Menghs, Johnstone Neondo and Richard Wekesa in IBR, JKUAT; and friends (Rosaline Macharia - International Center for Insect Physiology and Ecology (ICIPE), Joyce Njuguna- International Livestock Research Institute (ILRI) and June Mwajuma – Technical University of Mombasa (TUM); Thank you for your assistance particularly in troubleshooting. Special appreciation is directed to Mr. Nashon Adero for his expertise in Geographical Information Systems (GIS) that has made the maps in this work exemplary; and to my special friends, Mrs. Leah Andisi and Ms. Valentine Malemba who invested their invaluable time and moral patronage in Ngerenyi School. I would like to register exceptional appreciation to my loving husband Edward Nderitu for his patience and personal sacrifices in providing field support and shouldering the cost of sample analysis in a foreign laboratory. I cannot thank him enough for being there for me. MAY GOD BLESS YOU ALL! v TABLE OF CONTENTS DECLARATION............................................................................................................. ii DEDICATION................................................................................................................ iii ACKNOWLEDGEMENTS............................................................................................iv TABLE OF CONTENTS................................................................................................vi LIST OF TABLES ..........................................................................................................xi LIST OF FIGURES ...................................................................................................... xii LIST OF APPENDICES ..............................................................................................xvi LIST OF ABBREVIATIONS AND ACRONYMS .................................................. xvii ABSTRACT................................................................................................................ xviii CHAPTER ONE ..............................................................................................................1 INTRODUCTION............................................................................................................1 1.2 General Introduction ...................................................................................................1 1.2 Distribution and features of soda lakes .......................................................................1 1.3 The formation of Soda lake environment....................................................................4 1.4 Microbial Diversity and productivity..........................................................................7 1.5 Statement of the problem ............................................................................................8 1.6 Justification .................................................................................................................8 1.7 Hypotheses ..................................................................................................................9 1.8 Objectives.....................................................................................................................9 vi 1.8.1 General Objective......................................................................................................9 1.8.2 Specific Objectives....................................................................................................9 CHAPTER TWO ...........................................................................................................10 GENERAL LITERATURE REVIEW.........................................................................10 2.1 Microbial diversity of soda lakes ..............................................................................10 2.2 Diversity of haloalkaliphiles in Lake Magadi...........................................................10 2.3. Molecular microbial ecology ....................................................................................14 2.4 Restrictions of 16SrRNA approach to diversity studies ...........................................14 2.5 Next Generation sequencing technology ..................................................................15 CHAPTER THREE .......................................................................................................18 GENERAL MATERIALS AND METHODS..............................................................18 3.1 Research authorization..............................................................................................18 3.2 Study site...................................................................................................................18 3.3 Measurements of physicochemical parameters.........................................................19 3.4 Sample Collection....................................................................................................21 3.5 Water analysis of samples from the hot springs of Lake Magadi and Little Magadi 22 3.6 Total DNA extraction................................................................................................23 3.7 Total RNA Extraction ...............................................................................................23 3.8 Synthesis of cDNA from 16S rRNA.........................................................................24 3.9 Amplicon Library Preparation and Sequencing........................................................25 3.10 Sequence analysis and taxonomic classification.....................................................25 3.11 Statistical analysis ...................................................................................................26 vii CHAPTER FOUR..........................................................................................................27 BACTERIA AND ARCHAEA DIVERSITY WITHIN THE HOT SPRINGS OF LAKE MAGADI AND
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