ABSTRACT
The soil environment constitutes largely an untapped source of microorganisms with the potentiality of producing antibiotics. The soil microorganisms are responsible for a great percentage of discovered bioactive secondary metabolites notably antibiotics. In this study, 10 soil samples from different ecological sites were obtained and cultured in microbiological media to isolate antibiotic producing microorganisms. Ten fold serial dilution of each soil sample was carried out. 0.1ml of the serially diluted samples were plated on Nutrient agar and Sabouraud Dextrose agar (SDA) using the spread plate inoculation technique. A total of 25 isolates mostly Gram- Positive organisms (Bacillus sp, Micrococcus sp and Actinomycetes were recovered from the 10 samples analyzed. Of this number, 5 isolates was tested against four organisms (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27153 , Mycobacterium smegmatis ATCC 14468) for antibacterial activity. Out of the five isolates, only two (BL61 and BL42) exhibited inhibitory effect against one of the test organisms (Pseudomonas aeruginosa) with zones of inhibition of 9mm and 13mm respectively. The remaining isolates showed no antimicrobial activity against the test organisms. Considering the fact that soil harbours a wide range of microflora, there is the tendency of finding out new species and strains that have antimicrobial property. Hence this area of study needs an extensive and wide research on the soil micro environment to discover potential antibiotics that can be effective against microorganisms in the face of the current antibiotic resistance.
TABLE OF CONTENTS
Cover page i
Certification ii
Dedication iii
Acknowledgements iv
Table of Contents v
List of Tables vii
Abstract viii
CHAPTER ONE
1.0 Introduction 1
1.1 Aims and Objectives 3
CHAPTER TWO
2.0 Literature Review 4
2.1 Antibiotics 5
2.1.1 GENUS Streptomyces 6
2.1.2 Secondary metabolites and their functions 7
2.1.3 Genes for secondary metabolites 9
2.2 Microbial diversity in Soil 10
2.3 Microorganisms as Sources of Natural Products 12
2.3.1 Enzyme production 12
2.3.2 Antibiotic production 12
CHAPTER THREE
3.0 Material and methods 14
3.1 Collection and preparation of soil sample 14
3.2 Media used 14
3.2.1 Sterilization of materials 14
3.3 Sample preparation 14
3.3.1 Sub- Culturing 15
3.4 Gram Staining 15
3.4.1 Biochemical tests 16
3.4.1.1 Hydrogen sulfide test 16
3.4.1.2 Nitrate reduction test 16
3.4.1.3 Starch hydrolysis test 16
3.5 Primary Screening of Isolates 16
3.6 Test bacteria 17
CHAPTER FOUR
4.0 Results 18
CHAPTER FIVE
5.0 Discussion 22
5.1 Conclusion 23
5.2 Recommendation 23 References
LIST OF TABLES
TABLE 1: Gram reaction from all the soil samples and their percentage Occurrence 18
TABLE 2: Description of samples collected from different sites in Michael Okpara University of Agriculture, Umudike (MOUAU). 19
TABLE 3: Macroscopic and Microscopic examination of isolates and their percentage (%) occurrence 20
TABLE 4: Zone of inhibition in millimeter (mm) produced by isolates against test organisms. 21
CHAPTER ONE
1.0 INTRODUCTION
Soil is a highly heterogeneous environment (Alexander, 1977). It contains a high diversity of microorganisms (Liesack et al., 1997) which can be found as single cells or micro colonies developing around the soil particles (Torsvik, 2002). The numbers and kinds of microorganisms present in the soil are dependent on many environmental factors such as soil type, nutrient availability, degree of aeration, pH, temperature and soil moisture (Prescott et al., 1999). Soil is therefore regarded as heterogeneous with respect to conditions for microbial growth and for the distribution of microorganisms and matrix substances. As a result, there is a wide variety of microbial niches and a high diversity of soil microorganisms (Rolf, 2004). Soil microorganisms influence above ground ecosystems by contributing to plant nutrition, plant health, soil structure and soil fertility (O’Dounell et al., 2001). They are responsible for many of the key processes in the biogeochemical cycling on earth such as nitrogen, sulphur and carbon cycle (Lengeler et al., 1999). In addition they are a valuable sources of natural products providing important antibiotics for pharmaceuticals, important enzymes and bioactive compounds for industries (Strohl, 2000).
In recent years, the emergence of multidrug resistance pathogens has rekindled the need to discover new antimicrobials from the environment. The tremendous diversity of microorganisms could represent one of the richest sources of new antimicrobials (Keller and Zengler, 2004). However, numerous attempts made using traditional cultivation methods have failed to discover new antimicrobial products (Strohl, 2000). This is because isolate culture extracts often produced numerous previously described metabolites (Silva et al., 2001) and the rate of rediscovery of known antimicrobial has approached 99.9% (Zahner and Fiedler, 1995). It’s reported that only 1% of the greater than a million cells in each gram of soil seems able to form colonies on laboratory media implying that majority have eluded cultivation hence their biotechnology potential cannot be explored.
The continuing success of a biotechnologist in the search for microbial metabolites for use as antimicrobial compounds (antibiotics) is critical to combating human, animal and plant diseases. Microorganisms constitute an inexhaustible reservoir of compounds with pharmacological, physiological, medical or agricultural applications (Jian and Jian, 2005). Antibiotics continue to play a crucial role in the development of tissue culture techniques and basic screenings, primarily in biochemistry, molecular biology, microbiology and genetics (including genetic engineering) and to a lesser extent, pharmacology and organic chemistry. The actinomycetes are Gram positive organisms that tend to grow slowly as branching filaments. Many actinomycetes will grow on the common bacteriological media used in the laboratory, such as nutrient agar, trypticase soy agar, blood agar, and even brain-heart infusion agar. Actinomycetes encompass a wide range of bacteria. They have universal occurrence and play an active part in the cycle of nature (Antal et al., 2005). Antibiotics are the best known products of actinomycete. The morphology of an actinomycete growing on agar can provide useful and rapid clues to its identity, but viewing isolated colonies can give little worthwhile information. Morphological characters are still widely used for characterizing genera, for example, the presence or absence of spores on the substrate mycelium or the formation of zoospores in specialized spore vesicles or sporangia. The ability to produce motile spores is more widespread in the actinomycetes (Antal et al., 1996).
1.1 AIMS AND OBJECTIVES
To screen, isolate and characterize antibiotic producing bacteria from the soil of school hostels of Michael Okpara University of Agriculture, Umudike Abia State.
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