isolation and characterization of extreme haloalkaliphilic bacteria and archaea from lake magadi ...

129 Pages·2014·1.52 MB·English

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

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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.

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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.

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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
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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
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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
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3.7.2 Polymerase chain reaction....................................................................39
3.7.3 Purification PCR products....................................................................39
3.7.4 Phylogenetic data analysis....................................................................40
CHAPTER FOUR...............................................................................................................41
4.0 RESULTS.......................................................................................................................41
4.1 Physical characteristics at sampling site..............................................................41
4.2 Isolation...............................................................................................................41
4.3 Morphological characterization...........................................................................42
4.4 Physiological characterization.............................................................................44
4.4.1 Salinity tolerance..................................................................................44
4.4.2 Temperature tolerance..........................................................................46
4.4.3 pH tolerance..........................................................................................47
4.5 Biochemical tests of the isolates..........................................................................48
4.6 Enzyme screening................................................................................................50
4.7 Molecular characterization..................................................................................52
4.7.1 PCR amplification of 16s rDNA genes from isolates...........................52
4.7.2 Phylogenetic analysis of sequences......................................................53
CHAPTER FIVE.................................................................................................................57
5.0 DISCUSSION.................................................................................................................57
5.1 Culturing and isolation........................................................................................57
5.2 Morphological characterization of the isolates....................................................59
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5.3 Physiological characterization of the isolates......................................................60
5.3.1 Growth at different salinity levels........................................................60
5.3.2 Growth at different temperatures..........................................................62
5.3.3 Growth at different pH levels...............................................................63
5.4 Biochemical characterization of the isolates.......................................................64
5.5 Screening for enzyme production........................................................................66
5.6 Phylogeny and identification of isolates..............................................................69
CHAPTER SIX....................................................................................................................77
6.0 CONCLUSION..............................................................................................................77
RECOMMENDATIONS....................................................................................................78
REFERENCES....................................................................................................................79
APPENDICES....................................................................................................................107

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