Isolation and Characterization of Extreme
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ISOLATION AND CHARACTERIZATION OF EXTREME HALOALKALIPHILIC BACTERIA AND ARCHAEA FROM LAKE MAGADI, KENYA. EVANS MANYARA NYAKERI MASTER OF SCIENCE (Biotechnology) JOMO KENYATTA UNIVERSITY OF AGRICULTURE AND TECHNOLOGY 2013 Isolation and Characterization of extreme haloalkaliphilic Bacteria and Archaea from Lake Magadi, Kenya. Evans Manyara Nyakeri A Thesis submitted in partial fulfilment for the Degree of Master of Science in Biotechnology 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:................................................ Evans Manyara Nyakeri This thesis has been submitted for examination with our approval as University supervisors: Signature:........................................... Date:................................................ Prof. Hamadi Iddi Boga JKUAT, Kenya Signature:........................................... Date:................................................ Dr. Romano Mwirichia JKUAT, Kenya ii DEDICATION This work is dedicated to my beloved wife Susan, who despite the loneliness occasioned by my absence, not only trusted and believed in me and gave me the impetus to go on. To my Lovely children Bless Evan Misiga, Praise Moraa and Gift Favour, for whom I do endeavour to live and strive. To my loving parents Emmanuel and Bilha, who encouraged and supported me all through to this level of education. To uncle Edward for hosting me and being patient with me even when no progress was forthcoming. Above all to God, the creator of all beings, who provided the strength, health and favor to enable me see this output. iii ACKNOWLEDGEMENTS I am deeply indebted to all my supervisors, Prof. Hamadi Boga and Dr. Romano Mwirichia, for the guidance, encouragement and correction that they provided throughout the study period. I thank all my colleagues at Institute for Biotechnology Research, especially Dan Kwalimwa and Kevin Omolo Mbogo, for helping me out on occassions that called for giving up and creating a friendly working environment in the laboratory during the carrying out of this work. I thank Dr. Romano Mwirichia, and Mr Richard Rotich, for their invaluable guidance on molecular work. To you all, I say God speed. iv TABLE OF CONTENTS DECLARATION..................................................................................................................II DEDICATION.....................................................................................................................III ACKNOWLEDGEMENTS................................................................................................IV TABLE OF CONTENTS.....................................................................................................V LIST OF TABLES................................................................................................................X LIST OF FIGURES............................................................................................................XI LIST OF PLATES.............................................................................................................XII LIST OF APPENDICES..................................................................................................XIII LIST OF ABBREVIATIONS..........................................................................................XVI ABSTRACT........................................................................................................................XV CHAPTER ONE....................................................................................................................1 1.0 INTRODUCTION...........................................................................................................1 1.1 Extremophilic microorganisms..............................................................................1 1.2 Halophilic habitats.................................................................................................2 1.3 Halophilic microorganisms....................................................................................3 1.4 Haloalkaliphiles.....................................................................................................5 1.5 Adaptations of extreme halophiles and haloalkaliphiles.......................................6 1.6 Biotechnological applications of halophiles and haloalkaliphiles.........................8 1.7 Statement of the problem.....................................................................................11 1.8 Justification of the study......................................................................................11 v 1.9 Hypothesis...........................................................................................................13 1.10 Objectives..........................................................................................................13 1.10.1 General objectives....................................................................................13 1.10.2 Specific objectives....................................................................................13 CHAPTER TWO................................................................................................................14 2.0 LITERATURE REVIEW.............................................................................................14 2.1 Soda lake enviroments........................................................................................14 2.2 Microbial biodiversity and productivity of soda lakes........................................15 2.3 The role of haloalkaliphiles in soda lakes............................................................19 CHAPTER THREE............................................................................................................23 3.0 MATERIALS AND METHODS..................................................................................23 3.1 Study site: Lake Magadi......................................................................................23 3.2 Sampling..............................................................................................................24 3.3 Enrichment and isolation.....................................................................................25 3.4 Characterization and identification of isolates....................................................26 3.4.1 Morphological characterization............................................................26 3.4.2 Physiological characterization..............................................................27 3.4.2.1 Salt tolerance test............................................................................27 3.4.2.2 Temperature tolerance test..............................................................27 3.4.2.3 Growth at different pH values........................................................28 3.5 Screening for enzyme production........................................................................28 vi 3.5.1 Cellulase production.............................................................................28 3.5.2 Xylanase production.............................................................................29 3.5.3 Amylase production..............................................................................29 3.5.4 Lipase production.................................................................................30 3.5.5 Protease production..............................................................................30 3.5.6 Esterase production...............................................................................31 3.6 Biochemical characterization...............................................................................31 3.6.1 Catalase test..........................................................................................31 3.6.2 Oxidase test...........................................................................................32 3.6.3 Nitrate reduction...................................................................................33 3.6.4 Gelatin liquefaction/hydrolysis.............................................................33 3.6.5 Amino acid utilization..........................................................................34 3.6.5.1 L-arginine.......................................................................................34 3.6.5.2 L-lysine...........................................................................................34 3.6.5.3 L-ornithine......................................................................................35 3.6.6 Methyl Red Voges-Proskauer (MR-VP) test........................................35 3.6.7 Indole production and hydrogen sulfide production tests.....................36 3.6.8 Citrate utilization..................................................................................37 3.6.9 Urease test.............................................................................................37 3.7 Molecular characterization..................................................................................38 3.7.1 DNA extraction.....................................................................................38 vii 3.7.2 Polymerase chain reaction....................................................................39 3.7.3 Purification PCR products....................................................................39 3.7.4 Phylogenetic data