TABLE OF CONTENTS
Content Pages
Title page i
Certification ii
Dedication iii
Acknowledgement iv
Table of Contents v
List of Tables ix
List of Plates xi
List of Figures xii
Abstract xiii
INTRODUCTION 1
LITERATURE REVIEW 4
MATERIALS AND METHODS 61
Soil sample collection 61
Isolation of soil actinomycetes 61
Selection of antagonistic strain 61
Taxonomic studies 62
Growth characterization 62
v
Micromorphological characterization of isolates 62
Physiological and biochemical characterization of isolates 63
Melanin production by isolates 63
Carbon utilization by isolates 63
Resistance towards sodium chloride 63
Casein hydrolysis
Gelatin liquifaction 64
Starch hydrolysis 64
Nitrate reduction 65
Primary selection of antibiotic production medium 65
Antibiotic assay 65
Effect of different concentrations of glucose
on antibiotic production by Streptomyces SP-76 66
Effect of different concentrations of soy bean
on antibiotic production by Streptomyces SP-76 66
Effect of surfactants on antibiotic production
by Streptomyces SP-76 67
Effect of growth factors on antibiotic production
by Streptomyces SP-76 67
vi
Effect of pH on antibiotic production by
Streptomyces SP-76 68
Time course of pH and antibiotic production by
Streptomyces SP-76 68
Paper chromatography of the culture filtrate 68
RESULTS 70
Selection of antagonistic strain 70
Taxonomic studies 70
Growth characterization 70
Micromorphological characterization of isolates 87
Physiological and biochemical characterization of isolates 87
Carbon utilization by isolates 87
Primary selection of antibiotic production medium 92
Effect of different concentrations of glucose
on antibiotic production by Streptomyces SP-76 92
Effect of different concentrations of soy bean
on antibiotic production by Streptomyces SP-76 92
Effect of surfactants on antibiotic production
by Streptomyces SP-76 98
Effect of growth factors on antibiotic production
vii
by Streptomyces SP-76 98
Effect of pH on antibiotic production by
Streptomyces SP-76 98
Time course of pH and antibiotic production by
Streptomyces SP-76 104
Paper chromatography of the culture filtrate 104
DISCUSSION AND CONCLUSION 108
DISCUSSION 108
CONCLUSION 113
REFERENCES
114
viii
LIST OF TABLES
Table Title Pages
1a. Antimicrobial Activity of Isolates against Gram
negative organisms 83
1b. Antimicrobial activity of isolates against Gram
positive organisms 84
2. Cultural characteristics of isolates 85
3. Physiological and biochemical property of isolates 90
4. Carbon utilization by isolates 91
5. Primary selection of antibiotic production medium 93
6. Effect of different concentrations of glucose on
antibiotic production by Streptomyces SP-76. 96
7. Effect of different concentrations of soybean on
antibiotic production by Streptomyces SP-76 87
8a. Effect of different concentrations of Tween 80
on antibiotic production by Streptomyces SP-76 99
8b. Effect of different concentrations of oleic acid
on antibiotic production by Streptomyces SP-76 100
8c. Effect of different concentrations of stearic acid
on antibiotic production by Streptomyces SP-76 101
8d. Effect of different concentrations of palmitic acid
on antibiotic production by Streptomyces SP-76 102
9. Effect of growth factors on antibiotic production
by Streptomyces SP-76 103
10. Effect of pH on antibiotic production by Streptomyces
ix
SP-76 103
11. Rf values of the culture filtrate of Streptomyces
SP-76 and different antibiotics. 107
x
LIST OF PLATES
Plate Title Page
1a. Spore chains of isolate MP-75 on starch casein
nitrate agar Incubated at 300C for 14 days. 88
1b. Spore chains of isolate SP-76 on starch casein
nitrate agar incubated at 300C for 14 days 88
1c. Spore chains of isolate QP-100 on starch casein
nitrate agar incubated at 300C for 14 days 89
2. Inhibition of E. coli and Bacillus sp. by the culture
filtrate of isolate MP-75 and SP-76. 95
xi
LIST OF FIGURES
Figure Title Page
1. Time course of pH and antibiotic production by
Streptomyces SP-76 using medium C 105
2. Paper chromatogram of culture filtrate of
Streptomyces SP-76 and some purified antibiotics. 106
xii
ABSTRACT
A total of 106 actinomycetes isolated from the rhizosphere of plants in abbatoir and
refuse dumps in Awka and Onitsha were investigated for the production of
antimicrobial substances. Five of them were found to show antimicrobial activity
against Gram positive and Gram negative bacteria as well as fungi on solid media.
Three of the very active isolates designated MP-75, SP -76 and QP-100 were further
investigated in submerged medium in a shake-flask experiment using glucose and soy
bean as carbon and nitrogen sources respectively. Isolates MP-75 and SP-76 were
found to produce antimicrobial substances. The antimicrobial substance produced by
isolates SP-76 showed the highest antimicrobial activity against Escherichia coli,
Pseudomonas aeruginosa, Klebsiella sp, Staphylococcus aureus and Bacillus sp. The
isolates were identified as Streptomyces species based on their characteristic features.
Activity of the antimicrobial substance produced by Streptomyces SP-76 was
maximum when 4% (w/v) glucose and 2% (w/v) soybean were used in the
fermentation process. Influence of surfactants on accumulation of antimicrobial
substance by Streptomyces SP-76 in the fermentation broth showed that Tween 80,
oleic acid, palmitic acid and stearic acid enhanced antimicrobial activity. The effect of
growth promoters on antibiotic production by Streptomyces SP-76 indicated that
peptone, casein and yeast extract stimulated antimicrobial activity against the test
organisms. The effect of varying pH on antibiotic production by Streptomyces SP-76
showed that there was maximum antimicrobial activity at pH 7. In a time course for
antibiotic production, maximum antimicrobial activity was obtained at 120h. Paper
chromatography of the culture filtrate of Streptomyces SP-76 indicated that it contains
more than one antimicrobial substance. From this, it can be seen that the growth and
subsequent production of bioactive metabolites by Streptomyces SP-76 isolated from
the soil
xiii
CHAPTER ONE
INTRODUCTION
For centuries, preparations derived from living matter were applied to wounds to
destroy infection. The fact that a microorganism is capable of destroying one another
was not established until the latter half of the 19th century, when Pasteur noted the
antagonistic effect of other bacteria on the anthrax organism and pointed out that this
action might be put to therapeutic use. Meanwhile, the German Chemist, Paul Ehrlich
developed the idea of selective toxicity; that certain chemicals that would be toxic to
some organisms like infectious bacteria, would be harmless to other organisms e.g
humans (Limbird, 2004).
In 1928, Sir Alexander Fleming, a Scottish biologist, observed that Penicillium
notatum, a common mold, had destroyed Staphylococcus bacteria in culture and in
1939, the American microbiologist Rene Dubois demonstrated that a soil bacterium
was capable of decomposing the starchlike capsule of the Pneumococcus bacterium,
without which the Pneumococcus is harmless and does not cause pneumonia.
Dubois then found in the soil a microbe, Bacillus brevis, from which he obtained a
product, tyrothricin, that was highly toxic to a wide range of bacteria (Limbird, 2004).
Tyrothricin, a mixture of the two peptides, gramicidin and tyrocidine, was also found
to be toxic to red blood and reproductive cells in humans but could be used to good
effect when applied as an ointment on body surfaces. Penicillin was finally isolated in
1939, and in 1994 Selman Waksman and Albert Schatz, American microbiologists,
isolated streptomycin and a number of other antibiotics from Streptomyces griseus
(Calderon and Sabundayo, 2007).
1
Discovery of new antibiotics produced by Streptomyces still continues. Today, due to
the increasing resistance of pathogenic bacteria to our current arsenal of antibiotics, a
great need exist for the isolation and discovery of new antibiotics and other drug
agents (Jarroff, 1994; Rice, 2003; Wenzel, 2004). Fifty years ago, it was easy to
discover new antibiotics by simply screening the fermentation broths of
actinomycetes and fungi. Today, it is much more challenging, but there are much
better tools to address this problem. Discovering new antibiotics, pharmacophores is a
long-term endeavor that requires deft orchestration and support of many innovative
sciences (Cuatrecasas, 2006). There are three approaches that can be used to improve
our chances of finding new antibiotic substances: new test methods, new organisms,
and variation of culture conditions. None of these three options guarantees success
alone and the chances are best if the three are combined. There is need for long-term
basic microbiological research, which should cover the following areas: methods of
isolating and cultivating microorganisms that have not yet been accessed or only with
great difficulty, studies on the transportation of antibiotics into the bacterial cell,
comparative biochemistry of prokaryotes and eukaryotes, mode of action of
antibiotics and pathogenicity factors. In addition to search for new antibiotics, longterm
strategies to prevent the development and spreading of resistant bacteria must be
developed (Fiedler and Zanher, 1995). Therefore, it is time to define natural product
discovery from actinomycetes and other microbes as a major priority for medical
sciences and to engage the most creative scientists in academia (Cuatrecasas, 2006) in
close collaboration with biotechnology and pharmaceutical companies, which would
elevate the science to a new level of achievement so as to be commensurate with past
successes and present demonstrated potential.
2
In this study, effort has been made:
– To isolate Streptomyces from the soil, capable of producing antimicrobial
substances
– To study the cultural conditions necessary for antibiotic production.
– To determine the time taken for the optimum production of antimicrobial
substance.
– To determine the type of antimicrobial substance(s) present in the culture
filtrate.
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