SCREENING, ISOLATION AND CHARACTERIZATION OF ANTIBIOTIC PRODUCING MICROORGANISMS FROM SOIL SAMPLES IN MOUAU

ABSTRACT

Soil is the major repository of microorganism that produce antibiotics. Clinically useful antibiotics have been isolated from several groups of soil microorganisms including bacteria (Streptomyces sp, Bacillus sp.).  Bacillus and actinomycetes are the most abundant microorganisms present in soil and inhibit the growth of other microorganisms present in soil and inhibit the growth of other microorganism.  In this research, Ten samples of soil collected from a cultivated Farmland within the premises of College of Crop and Soil Sciences were screened for antibiotic, producing microorganisms.  Ten fold serial dilutions of the soil samples were prepared and inoculated on Nutrient Agar and Saboraud Dextrose Agar.  A total number of Twenty three isolates were recovered.  Ten of the total isolates were subcultured on Nutrient agar and incubated at 37 for 24hrs.  Out of these ten isolates four are Micrococcus, four Bacillus species and two Actinomycetes were obtained.  Five different isolates was tested against four test organisms namely; Pseudomonas aeruginosa (ATCC), Staphylococcus aureus (ATCC) Escherichia coli (ATCC) and Mycobacterium smegmatis (ATCC28735) to observe their antimicrobial activity against the test organisms.  One of the isolates (CCSSH₁) that is actinomycete was able to inhibit growth of Staphylococcus aureus with a clear inhibition range with a clear inhibititon range of 0 – 14 mm, which indicates an antibacterial activity against S.aureus while others showed no antimicrobial activity against the test organisms.  This shows that the screening and isolation of CCSSH₁ from this study area may contribute to the discovery of new antibiotics with further extraction and purification of the isolate.  

TABLE OF CONTENTS

Title page                                                                                                         i

Certification                                                                                                    ii

Dedication                                                                                                      iii

Acknowledgements                                                                                        iv

Table of contents                                                                                             v

List of tables                                                                                                  viii

Abstract                                                                                                          ix

CHAPTER ONE

1.0 INTRODUCTION                                                                                    1

1.1 Aims and Objectives                                                                                4

CHAPTER TWO

2.0 LITERATURE REVIEW                                                                         5

2.1 Microbial Sources of Antibiotics-General Overview                               5

2.2 Antibiotic Formation by Actinomycetes                                                   7

2.2.1 Antibiotic Formation by Rare Actinomycetes                                        8

2.3 Industralization, Biostimulation and Bioaugmentation                             9

2.4 The Soil Rhizosphere                                                                                10

2.4.1 Soil Antimicrobial Agent Producing Microbes                                      11

CHAPTER THREE

3.0 MATERIALS AND METHODS                                                              14

3.1 Collection and Preparation of Soil samples                                              14

3.2 Sterilization of Materials                                                                           14

3.3 Media Used                                                                                               14

3.4 Serial Dilution                                                                                           15

3.4.1 Culturing                                                                                                15

3.4.2 Colonial Examination                                                                            16

3.4.3 Sub Culturing                                                                                         16

3.5 Gram Staining                                                                                           16

3.6 Biochemical Tests                                                                                     17

3.6.1    Hydrogen Sulfide Production Test                                                      17

3.6.2  Nitrate Reductase Test                                                                          18

3.6.3  Starch Hydrolysis                                                                                 18

3.7 Confirmation of Antimicrobial Activity                                                   19

3.8 Test Bacteria                                                                                             19

CHAPTER FOUR

4.0 RESULTS                                                                                                 20

CHAPTER FIVE

5.0 DISCUSSION, CONCLUSION AND RECOMMENDATION FOR

FURTHER STUDIES                                                                         24

5.1 Discussion                                                                                                 24

5.2 Conclusion                                                                                                25

5.3 Recommendation for further studies                                                        25

REFERENCES                                                                                               26

LIST OF TABLES

Table                                                Title                                                Page

1                          Microorganisms producing antibiotics                            8

2 Description of soil samples collected from locations of cultivate farmland and numbers of isolates.                                                                                   21

3 Colonial Characteristics and Biochemical Characterization of the Bacterial Isolates                                                                                        22

4: Zone of inhibition in millimeter (mm) of bacteria isolates against test organisms.                                                                                                           23

CHAPTER ONE

1.0 INTRODUCTION

The term soil refers to the outer loose material of the earth crust it maybe regarded as a three phases system composed of solids, liquids and gases, dispersed to form a heterogeneous matrix.  On the whole soil is composed of five major components, these includes; mineral water, mater, organic matter, air and living organisms.  The various component of the soil environment components constantly changed and the quantity of these constituents are not the same in all soil body includes small animals and microorganism but is generally considered that it’s in microorganisms that plays the most important role in the release of nutrient and carbondioxide for plant growth.  (Hanlon et al, 2007).  The bacteria are the most abundant group usually more numerous than the four combined.  Soil bacteria can be rod, (bacilli) cocci (spherical) spirilla of these bacillus, are more numerous than the others.  They are One of the major groups of soil bacteria population and are very widely distributed (Bhagabati et al, 2004).  The number and type of bacteria present in a particular soil would be greatly influenced by geographical location such as soil temperature, soil type, soil pH, organic matters contents, cultivation, aeration and moisture content (Davies, et al, 1999).

An ecological niche is composed of many microhabitats; each microhabitat is composed of a microscopic diversity which includes bacteria, protozoa, fungi, and a macroscopic diversity that includes plants and insects. Soil is a complex medium in which one can encounter many kinds of microbial communities. Application of nucleic acid-based techniques to analyze soil microbial communities has revealed high prokaryote diversity (Pandey et al.. 2008). The microbial diversity or communities present in soil principally depend on the composition of the soil and many physical chemical properties that the medium posses.  Also the flora and decomposing organic matter on the surface of the soil will influence microbial diversity present.  For example, the fallen trees, barks and flowers provide nutrients both to the microbes and plants present, through microbial degradation of carbohydrates, lipids and proteins to sugars, fatty acids, glycerol and amino acids and respectively to mineralization. Besides providing these nutrients, plant secondary metabolites that are generally toxic to microorganisms will need to be degraded or detoxified by certain microbes. These degraders (microbes) are selectively pressured and ultimately evolve to produce novel secondary metabolites possibly to counteract the toxic plant secondary metabolites (Pandey et al., 2008). There are approximately 10⁶-10⁹ colony forming unit per gram of soil. Microbes present in medium posses advantages that will permit or facilitate their survival in that medium. For example research has demonstrated that in desert crust or in soil that has low water availability, gram positive and spore forming microbes are most abundant.  The gram-positive bacteria posses a thicker layer of murein in their cell wall which makes the cell less vulnerable to the limiting conditions present in these habitats.  Also, spore farming bacteria can resist long periods of desiccation  and limiting nutrient conditions since they compact and protect their genuine material in the bacteria spore, until conditions are favourable for sporulation to occur (Sadha  et al, 2011).  

The term antibiotic was first introduced by Selman Waksman in 1947 to describe, “A chemical substance derived from microorganisms, which has the capacity of inhibiting growth, and even destroying other microorganisms in dilute solutions” (Waksman, 1947). According to Gottlieb and Shaw (1967) antibiotics are “organic substances that are produced by microorganisms and are harmful at low concentrations to growth and metabolic activities of other organisms”. This definition was limited by Lancini and Parenti in 1982 to special inhibitory products of low molecular weight and excluded enzymes, lactic acid, ethanol and other similar substances that prevent growth of some microorganisms. Debate over the definition of antibiotics has continued. Forsdyke in 2000 included synthetic molecules like AZT in his definition on the basis that they are deriviates of microbial products.   According to him an antibiotic is “a chemical of natural or synthetic origin, which at low concentration inhibits microorganism of  some type within a host organism, while not unacceptably interfering with the life of that organism”. Carlberg (2000) opposed the use of „synthetic origin” in the definition and suggested that AZT and most of the other agents used to treat HIV infections should be referred to as antiviral agents, antiviral drugs or perhaps anti microbes, because they are synthetic molecules, and the term „antibiotic" should be reserved for such familiar compounds as penicillin and Streptomycin.

Most microbiologists are more comfortable with definitions that exclude any synthetic product (Lalitha et al,2011)

Antimicrobial agents are natural or synthetic chemical substances which are the capacity of inhibiting or terminating total metabolic cell activity. These chemical molecules are classified depending on their targets. They can also be referred to as broad or narrow spectrum depending on its strength of action towards their targets, principally including

a. Cell wall synthesis;

b. Protein synthesis and

c. DNA replication.

The major classes of antimicrobial agents are as

a. Lactams including penicillins, cephalosporins, monobactams, carbapenems;

b. Aminoglycosides;

c. Tetracyclines;

d. Sulfonamides;

e. Macrolides such as erythromycin;

f. Quinines; and

g. Glycopeptides such as vancomycin.

These secondary metabolites can affect many metabolic reactions in a cell in order to render effect. Penicillins and cephalosporins mode of action is the biosynthesis of the peptidoglycan present in the bacterial cell wall (Drews et al, 2000). They affect specifically the transpeptidase that forms the peptide cross-linking. Beta-lactams also affect peptidoglycan synthesis by forming covalent bonds with a specific group of proteins known as the penicillin binding proteins (Sainsbury et al., 2011). Both gram positive and gram negative microorganisms posses these proteins and other antimicrobials such as the quinolones and noviobiocin inhibit DNA replication by affecting enzymes, namely, DNA gyrase and the eukaryotic topoisomerase II (Han and Mar,2010).  Antimicrobials, known as 6-Aninicuracii, inhibits DNA polymerase III.Classes of antimicrobial like tetracyclines, chloramphenicol and Macrocides inhibits protein synthesis (Enright, 2003).  

1.1 Aims and Objectives

1. To isolate, identify and characterize antibiotics producing microorganism from soil samples of cultivated farmland within College of Crop and Soil Science of Michael Okpara University of Agriculture, Umudike.  

2. To determine if the antibiotic made by the antibiotic producer kills bacteria.  

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