TABLE OF CONTENTS
Cover Page i
Title Page ii
Certification Page iii
Dedication iv
Acknowledgement v
Table of Contents vi-viii
List of Tables ix
Abstract x
CHAPTER ONE
1.0 INTRODUCTION 1
1.1 Factors Affecting Soil Microbial Composition 1
1.2 Definition of Antibiotics 1
1.3 Bacillus spp. and antibiotics 3
1.4 Aims and Objectives 4
CHAPTER TWO
2.0 LITERATURE REVIEW 5
2.1` Soil and Antibiotics 5
2.2 Review on Different Soil Environment and Their Antibiotics-Producing Microbes Composition 6
2.3 Fungi and Antibiotics 7
2.4 Sources of Nutrients for Soil Microbes 9
2.5 Formation of Antibiotics in the Soil 9
2.6 Factors Influencing Natural Stimulation of Secondary Metabolite Production 9
2.7 Class: Actinomycetales 10
2.7.1 Actinomycetes 10
2.8 Estimation of Soil Microbial Flora 11
2.9 Distribution of the Actinomycetes in the Soil 13
2.10 Classification of Antibiotics 13
2.11 Streptomyces: Wide-range Antibiotics Producers 14
2.12 Ecological and Geographical Distribution of Microorganisms in Soils 14
2.13 Problems Encountered In Determining Soil Microbial Distribution 14
2.14 Factors Encouraging Antibiotics Production by the Streptomycetes 14
2.15 Effects of Isolation Media in Antibiotics Production 16
2.16 Techniques in Identifying Antibiotic Producers in Media Plates. 16
2.17 Failure of In-use Antibiotics and the need for Novel Antibiotics 16
2.18 Concentration, Inactivation, pH and Adsorption as Factors Affecting Effectiveness of Antibiotics in the Soil 17
2.19. The Need for Specific Isolation Media in Screening for Antibiotics Production 18
CHAPTER THREE
3.0 MATERIALS AND METHODS 20
3.1 Study Area and Period 20
3.2 Collection of Samples 20
3.3 Sterilization of Equipment and Preparation of Media 20
3.4 Preparation of Sample 21
3.5. Sample Inoculation, Incubation and Isolation of Microbes 21
3.6 Colonial Examination 22
3.7 Subculturing and Storage of Isolates 22
3.8 Testing for Antibiotic Production 22
3.9 Gram Staining 22
3.10 Biochemical Tests 23
3.10.1 Starch Hydrolysis Test 23
3.10.2 Hydrogen Sulphide Test 23
3.10.3 Sugar Utilization Test 24
CHAPTER FOUR
4.0 RESULTS 25
CHAPTER FIVE
5.1 Discussion 32
5.2 Recommendations 34
5.3 Conclusion 35
References 36
LIST OF TABLES
Table Title Page
1 The nature of the soil samples with the different number of distinct isolates from each site 26
2 List of positive isolates, their site number and isolation media and dilution coefficient 27
3 The sensitivities of the test organisms to the screened bacterial isolates 28
4 The sensitivities of the test organisms to the fungal isolates 29
5 Morphological characteristics, Gram status and Biochemical tests on the bacterial isolates 30
6 Morphological Characteristics of Positive Fungal Isolates 31
ABSTRACT
The soil environment is rich in microorganisms capable of antibiotics production but the rate of occurrence and distribution of these microorganisms at ecologically significant levels has not been much clear. In the past few decades, microorganisms have been demonstrated as synthsizers of varieties of antibiotics even under field conditions in the rhizosphere. In the present work, soil smples were collected randomly from the forest environment in Michael Okpara University of Agriculture, Umudike, in Abia-state, Nigeria and were screened for antibiotics-producing microorganisms by the agar sensitivity assay using paper discs impregnated with the isolates from the forest soil. Forty-eight (48) isolates were gotten on the total, out of which thirteen 13 (27.1%) isolates active against different test organisms (Salmonella typhi , Staphylococcus aureus ATCC 25923TM, Escherichia coli ATCC 25922TM, Pseudomonas aeruginosa ATCC 27893TM). Nine (9) (18.8%) bacterial isolates were active against the test pathogenic bacteria and they were identified by their cellular characteristics, colonial morphology and biochemical tests. These active isolates sort into the Genus Bacillus and Micrococcus and the ones that were not fully characterized yet are probably members of the Streptomyces genera. Four (4) (8.3%) fungal isolates were also active against the test bacteria and were identified based on their fungal morphology as probably Penicillium spp. and Aspergillus spp. Of all the isolates, isolate 36 which is probably a Streptomyces spp., has the highest range of antibacterial activity with 12mm zone of inhibition on Pseudomonas aeruginosa, 12mm on Staphylococcus aureus 5mm on Salmonella typhi and 4mm on Escherichia coli. Isolates 35 and 40 had the highest zones of inhibition, both on Salmonella typhi. The present work has once more contributed to the existing truth in cases of isolation of antibiotics-producing microorganisms from the forest soil and promises to contribute hugely in all enquiries into the antibiotics producing micro-flora in the forest environment.
CHAPTER ONE
1.0 INTRODUCTION
The soil harbours different kinds of life which include both plants, animals and micro-organisms, but it is considered that micro-organisms generally play the most important role in the release of nutrient and CO2 for the plant growth and other processes. The bacteria are the most abundant group of microorganism in the soil and they could be rod, cocci or spirilla but the bacilli are more numerous than others (Abdulkadir and Waliyu, 2012). The Baccilli are the major groups of soil bacteria and they are widely distributed (Bhagabati et al., 2009).
1.1 Factors Affecting Soil Microbial Composition
The number and type of bacteria present in a particular soil is greatly influenced by the geographical location and factors such as soil temperature, soil type, soil pH, organic matter content, cultivation, aeration and moisture content. Usually, the density of micro-organisms is less in cultivated soil than non- cultivated/virgin land and the population decrease with soil acidity. (Mishra et al., 2010).
1.2 Definition of Antibiotics
Broadly defined, antibiotics include a chemically heterogeneous group of small organic molecules of microbial origin that, at low concentrations, are deleterious to the growth or metabolic activities of other microorganisms (Thomashow and Weller, 1995).
Antibiotics are group of chemically heterogenous small organic molecules of microbial origin that at low concentrations are deleterious to the growth or metabolic activities of other micro-organisms (Ahmed et al; 2013). The soil is rich in microorganisms capable of producing antibiotics, but the frequency with which synthesis occurs at ecologically significant levels in nature has been much less clear (Brun and Skimkets, 2000). Antibiotics possess a lot of industrial importance and are best known to be produced by Actinomycetes (Wike et al., 2007; Hansen et al., 2006; Goldstein et al.; 2005). The Actinomycetes produce an enormous variety of bioactive molecules like actimicrobial compounds, e.g one of the first antibiotics used, which is streptomycin was produced by Streptomyces griseus. In the past 55 years, more than 12,000 antibiotics have been discovered and the actinomycetes yields about 70% of these and the remaining 30% are products of filamentous fungi and non-actinomycetes bacteria, (Goldstein et al., 2005; Bodosa et al., 2005; Collatz et al., 2007; Rasche et al., 2006). Also the experiment by the British scientist, Alexander Fleming, (who is credited to be the first to notice bacterial inhibition by another organism) which was carried out in 1928 provided another evidence of inhibition of Staphylcoccus aureus by a mold (Fungus) contaminant in his plate. The mold was later identified as Penicillium notatum and the antibiotic was isolated after a short time which is known as penicillin (Abdulkadir and Waliyu, 2012). All these evidence provide useful information that most antibiotics are produced by Actinomycetes and fungi.
The soil is considered a major source of these antibiotic producing organisms because the diversity of soil micro-organisms were of great significance as a factor promoting the daily discovery of antibiotics (Arifuzzaman et al, 2011). Some antibiotics like Erythromycin, penicillin and methicillin that used to be one-time effective in the treatment of infectious diseases are now less effective because bacteria have become more resistant to such antibiotics, to combat this drift, replacement of such existing antibiotics is necessary, and the development of novel drugs against drug resistant pathogens is significant for today. (Raja et al., 2010; Ilie et al; 2010). Thus finding and producing new antibiotics as well as using combined antibiotic therapy have been shown to delay the emergence of microbial resistance and can also produce desirable synergistic effects in the treatment of microbial infections (Abebe et al; 2013).
1.3 Bacillus spp. and antibiotics
According to stachelhaus et al, 1995, Bacillus spp. has diverse chemical structure and are able to produce secondary metabolites. Some of the important examples of these antibiotics produced by Bacillus spp used in medical treatments are bacitracin, gramicidin, polymyxin and tyrotricidin (Drabloset al; 1999). The genus Bacillus is characterized by being Gram positive, spore forming rods and it has been demonstrated that they produce agents in some stages of their growth curve, for example, Bacillus subtilis can produce non-ribosomal oligopeptides with antifungal and antimicrobial properties such as surfactins, inturinics and bacilysin and histatin also sysnthesizes Ribosomal antibiotics like subalanun and subticosin (Thajuddin and Subramanian, 2005).
Another genera related to the genus Bacillus are the lactobacillus, which are bacteria containing non-sporing rods and sometimes collobacilli, they lack catalase and cytochromes, some are usually facultative anaerobic or microaerophilic and produce lactic acid as their main product of fermentation, with complex nutrient requirement (Abdulkadir and Waliyu, 2012 ). Lactobacillus species produce many kinds of antibiotics which share a full range of antimicrobial activity, example of which is risin which is produced by lactobacillus lactis and active against Gram positive organism such as Corynebacter spp., Clostridia spp. and anaerobic cocci (Waites et al; 2008).
Research in finding newer antibiotics and increasing productivity of such agents has been a very important activity (Sundaramoorthi et al., 2011; Retinowati, 2010). This is because important drugs are expensive and/or have side effect to the host, also some microbes have no potent antibiotics against them while others are developing multidrug resistance (Abebe et al., 2013). These situations require more attention to find solutions by searching for and producing new and effective antibiotics from microbes like Bacillus spp. Actinomycete and Fungi.
As this search for most potent antibiotic-producing micro organisms proceed, the present work may provide some information on antibiotic production and the control of microbial strains since there has not been any such scientific report on antibiotic producing microbes in soil samples collected from the forest environment of Michael Okpara University of Agriculture, Umudike (MOUAU).
1.4 AIMS AND OBJECTIVES
The objectives of this study are:
1. To isolate soil microorganisms from soil samples collected from forest environment in Michael Okpara University of Agriculture, Umudike.
2. To screen the isolated microorganisms for antibiotic production.
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