isolation and characterization of antibiotic and enzyme producing bacteria from soil and temite gut

100 Pages·2013·1.58 MB·English

INFLUENCE OF CULTURE MEDIA AND INCUBATION
CONDITIONS ON THE POPULATION AND DIVERSITY OF
MICRORGANISMS CULTIVATED FROM SOILS AND TERMITE GUTS

REGINA MONGINA NTABO

MASTER OF SCIENCE
(Botany)

JOMO KENYATTA UNIVERSITY OF
AGRICULTURE AND TECHNOLOGY

2009
i
Influence of Culture Media and Incubation Conditions on the Population and
Diversity of Microorganisms Cultivated from Soils and Termite Guts

Regina Mongina Ntabo

A thesis submitted in partial fulfillment for the Degree of Master of Science in
Botany in the Jomo Kenyatta University of Agriculture and Technology.

2009

ii
Declaration
This thesis is my original work and has not been presented for a degree in any other university.
Signature…………………………………..Date………………………..
Regina Mongina Ntabo

This thesis has been submitted for examination with our approval as university supervisors
Signature…………………………………..Date………………………..
Prof. Hamadi Iddi Boga
JKUAT, Kenya

Signature…………………………………..Date……………………….
Prof. Anne W. T. Muigai
JKUAT, Kenya

iii
Dedication
This work is dedicated to my Father Mr. Thomas Ntabo who laid the foundation, supported and
encouraged me to attain even greater heights in the academic ladder.
iv
Acknowledgements
I would like to thank Jomo Kenyatta University of Agriculture and Technology (Research
production and Extension Division) through Professor Hamadi Iddi Boga for the research grant
without which it would not have been possible to do this work.

I sincerely thank Kenya Wildlife Service at Kakamega Forest for giving me permission to collect
samples from the forest. I would like to thank the department of Botany (JKUAT) for the
assistance provided throughout my study period. I am deeply indebted to all my supervisors Prof.
Hamadi Iddi Boga and Prof. Anne Muigai for the support, guidance, encouragement and
correction that they provided throughout the study period. I also wish to thank Mr. Romano
Mwirichia for the technical assistance in molecular work.

I wish to express my sincere gratitude to my husband Enoch Karakacha for the much support
accorded during my study. Lastly, I thank all my colleagues and staff Botany laboratory and
institute for biotechnology Research for all their assistance and for encouraging a friendly
working environment in the laboratory during this work.
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Table of contents
Declaration .................................................................................................................. iii
Dedication ..................................................................................................................... iv
Acknowledgements ........................................................................................................ v
Table of contents .......................................................................................................... vi
List of tables.................................................................................................................. xi
List of figures ............................................................................................................... xii
List of plates................................................................................................................xiii
List of appendices ....................................................................................................... xiv
Abbreviations .............................................................................................................. xv
Abstract ...................................................................................................................... xvi
CHAPTER ONE ............................................................................................................ 1
1.0 INTRODUCTION ............................................................................................. 1
1.1 Soil microorganisms..................................................................................... 1
1.2 Termites ....................................................................................................... 2
1.3 Cultivation independent molecular approaches .................................................. 3
1.4 Cultivation dependent approaches ................................................................ 3
1.5 Statement of the problem ............................................................................. 4
1.6 Justification .................................................................................................. 5
1.7 Main objective ............................................................................................. 6
1.8 Specific objectives ....................................................................................... 6
1.9 Hypotheses................................................................................................... 6
1.10 Expected outputs ........................................................................................... 7
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CHAPTER TWO........................................................................................................... 8
2.0 LITERATURE REVIEW .................................................................................. 8
2.1 Soil feeding termites .................................................................................... 8
2.1.1 Microbial diversity in soil feeding termites gut ............................................. 9
2.2 Microbial diversity in soil .......................................................................... 12
2.3 Detection of Microbial diversity in Soils and termite guts using Molecular tools 13
2.4 Strategies for Increasing Cultivation of Microorganisms .................................... 15
2.5 Microorganisms as sources of natural products ........................................... 16
2.5.1 Enzyme production .................................................................................... 16
2.5.2 Antibiotic production ................................................................................. 17
CHAPTER THREE .................................................................................................... 18
3.0 MATERIALS AND METHODS ..................................................................... 18
3.1 Kakamega Forest ....................................................................................... 18
3.1.1 Study site ................................................................................................... 18
3.1.2 Climate ...................................................................................................... 18
3.1.3 Soils ........................................................................................................... 19
3.1.4 Vegetation .................................................................................................. 19
3.1.5 Kakamega Soil and Termite sampling ........................................................ 19
3.2 Juja ............................................................................................................ 21
3.2.1 Study site ................................................................................................... 21
3.2.2 Climate ...................................................................................................... 22
3.2.3 Soils ........................................................................................................... 22
3.2.3.1 Shallow clay soils (Murram). ............................................................................. 22
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3.2.3.2 Deep clay soils (Vertisols). ................................................................................ 23
3.2.3.3 Soil associations and complexes ........................................................................ 23
3.2.4 Vegetation .................................................................................................. 23
3.2.5 Juja soil sampling ....................................................................................... 24
3.3 Dry weight determination .................................................................................. 26
3.4 Cultivation and Isolation of Microorganisms ...................................................... 26
3.4.1 Termite gut ........................................................................................................ 26
3.4.2 Mounds and soil................................................................................................. 27
3.5 Statistical analysis .............................................................................................. 27
3.6 Isolation of pure cultures .................................................................................... 28
3.7 Stocking of the pure culture........................................................................ 28
3.8 Screening for antibiotic producing isolates ......................................................... 29
3.9 Screening for enzyme activity in isolates ........................................................... 30
3.9.1 Starch hydrolysis ........................................................................................ 30
3.9.2 Gelatin liquefaction .................................................................................... 30
3.9.3 Casein hydrolysis ....................................................................................... 30
3.9.4 Cellulose hydrolysis ................................................................................... 31
3.10 Characterization of isolates ........................................................................ 31
3.10.1 Morphological characterization .................................................................. 31
3.10.2 Biochemical characterization .......................................................................... 32
3.10.2.1 Indole production and Hydrogen sulfide production ........................................ 32
3.10.2.2 Nitrate reduction test ....................................................................................... 32
3.10.2.3 Urease test ...................................................................................................... 33
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3.10.2.4 Catalase test .................................................................................................... 33
3.10.2.5 Ability of isolates to grow in 7% sodium chloride ........................................... 33
3.10.2.6 Substrate utilization of isolates ........................................................................ 33
3.10.3 Molecular characterization ................................................................................. 34
3.10.3.1 DNA isolation protocol ................................................................................... 34
3.10.3.2 DNA purification Quick Clean 5M Gel Extraction Kit .................................... 36
3.10.3.3 PCR amplification of bacteria 16S rRNA gene ................................... 36
3.10.3.4 Purification of PCR products .............................................................. 37
3.10.3.5 Phylogenetic data analysis .................................................................. 38
CHAPTER FOUR ....................................................................................................... 39
4.0 RESULTS ......................................................................................................... 39
4.1 Enumeration of microorganisms from sample units under different conditions ... 39
4.1.1 Impact of incubation temperature on number of cultivable microorganisms in soil, gut and
mound samples .............................................................................................................. 39
4.1.2 Impact of heat shock method on number of cultivable microorganisms ................ 41
4.1.3 Impact of length of incubation period on number of cultivable microorganisms . 42
4.2 Isolation ............................................................................................................. 43
4.3 Screening for antibiotic producing bacteria ........................................................ 44
4.4 Screening for enzymatic activities of bacteria isolates ........................................ 47
4.5 Biochemical tests ............................................................................................... 49
4.6 Morphological characterization of isolates ......................................................... 50
4.7 PCR amplification of 16S rRNA gene from isolates ........................................... 52
4.8 Phylogenetic cluster analysis of sequences ......................................................... 53
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CHAPTER FIVE ......................................................................................................... 58
5.0 DISCUSSION, CONCLUSION & RECOMMENDATION .......................... 58
5.1 DISCUSSION ................................................................................................... 58
5.1.1 Comparison of number of cultivable microorganisms in soil and gut samples .... 58
5.1.2 Impact of cultivation temperature on number of cultivable microorganisms ....... 59
5.1.3 Impact of incubation period on number of cultivable microorganisms ................ 61
5.1.4 Impact of heat shock method on number of cultivable Microorganisms.............. 61
5.1.5 Characterization and identification of isolates .................................................... 63
5.1.6 Antibiotic production from Bacterial isolates ..................................................... 66
5.1.7 Enzyme production ............................................................................................ 66
5.2 CONCLUSIONS .............................................................................................. 68
5.3 RECOMMENDATIONS ................................................................................. 69
REFERENCES ............................................................................................................ 71
APPENDICES ............................................................................................................. 82

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