ISOLATION AND EVALUATION OF MICROORGANISMS FROM DUMP SITES IN UMUAHIA METROPOLIS FOR ANTIBIOTIC PRODUCTION

ABSTRACT

This study was carried out to isolate and screen soil microorganisms that have potential to produce antibiotics. Twenty (20) soil samples were collected from four (4) different dump sites in Umuahia metropolis and analyzed for antimicrobial producing microorganisms. The organisms were isolated after serially diluting the soil samples and plating using the spread plate method of inoculation on Tryptone Soy agar and Sabouraud Dextrose agar. A total of thirty four (34) were recovered. Antibiotic activity screening of the isolates using the paper disc diffusion technique reveals that thirteen (13) of the isolates showed antibacterial activity against different strain of test bacteria with zone of inhibition ranging between 9mm and  18mm. isolate D31 produced the largest zone of clearing (18mm) against Staphylococcus aureus. Other isolates were active against at least two of the test organisms with isolate C27 recording zones of clearance in the range of 14mm, 10mm and 11mm against Staphylococcus aureus, Salmonella typhi and Enterococcus faecalis respectively. Most of these isolates were identified to be in the genus Bacillus and Micrococcus. Therefore, it is confirmed that soil sampes collected from different dump sites in Umuahia metropolis were found to be a potential source of novel antibiotics.

TABLE OF CONTENTS

Title page                                                                                                                                 i

Certification                                                                                                                                 ii

Dedication                                                                                                                               iii

Acknowledgments                                                                                                                   iv

Table of contents                                                                                                                     v

List of tables                                                                                                                           vii

Abstract                                                                                                                                   viii

CHAPTER ONE

1.0       Introduction                                                                                                                1

1.1       Meaning of Microorganism                                                                                        2

1.1.1    Meaning of soil Microorganisms                                                                                3

1.1.2    Factors that affects soil microorganisms                                                                     4

1.1.3    Secondary metabolites                                                                                                 6

1.1.4    Meaning of antibiotics                                                                                                6

1. 2      Aims and Objectives                                                                                                   8

CHAPTER TWO

2.0       Literature review                                                                                                        9

2.1       Dump sites                                                                                                                  10

2.2       Occurrences of antibiotic producing microorganisms from dump sites                 11

2.3       Factors considered in selection of antibiotics                                                             12

2.4       antibiotics and its classification                                                                                  13

2.5       Physiology of antibiotics production                                                                          14

2.6       Factors affecting antibiotics production                                                                     14

2.7       Fermentation conditions                                                                                             17

2.8       Biology and pathogenicity of some test organisms                                                    18

CHAPTER THREE

3.0       Collection of sample                                                                                                    21

3.1       Media used                                                                                                                  21

3.2       Media preparation                                                                                                       21

3.3       Isolation of microorganisms                                                                                       22

3.4       purification of isolates                                                                                                22

3.5       Preparation of inoculums of test organisms                                                               23

3.6       Gram-staining                                                                                                             23

3.7       Biochemical identification of bacteria isolates                                                          24

3.8       Biochemical test                                                                                                         24

3.9       Confirmation of antibacterial activity                                                                        26

CHAPTER FOUR

4.0       Results                                                                                                                        27

CHAPTER FIVE

5.0       Discussion                                                                                                                   31

5.1       Conclusion                                                                                                                  32

References     

LIST OF TABLES

CHAPTER ONE

1.0      INTRODUCTION

Microorganisms are of universal occurrence in nature and are widely distributed in natural and man-made environments. They are found in large numbers in soils, fresh waters, lake, river bottoms, manures, composts and dust as well as on plant residues and food products. However, the diversity and distribution of micro-organism that produce secondary metabolites can be determined by different physical, chemical and geographical factors  (Gurung et al., 2009; Ogunmwonyi et al., 2010).

Microorganism provide many important bioactive substances that have high commercial value. Their ability to produce a variety of bioactive substances has been utilized in a comprehensive series of researches in numerous institutional and industrial laboratories. This has resulted in the isolation of certain agents, which have found application in combating a variety of human infections (Retinowati, 2010). That is why more than 70% of naturally occurring antibiotics have been isolated from different Micro-organism (Khanna et al., 2011). Out of these different genus, Streptomyces is the largest genus known for the production of many secondary metabolites, which have different biological activities, such as antibacterial, antifungal, antiparasitic, antitumor, anticancer and immunosuppressive actions (Maleki and Mashinchian, 2011).

Some antibiotics like penicillin, erythromycin, and methicillin which used to be one-time effective treatment against infectious diseases are now less effective because bacteria have become more resistant to such antibiotics (Jemimah et al., 2011). Antibiotic resistant pathogens such as methicillin and vancomycin resistant strains of Staphylococcus aureus (S. aureus) and others cause an enormous threat to the treatment of serious infections. To avoid this happening, immediate replacement of the existing antibiotic is necessary and the development of novel drugs against drug resistant pathogens is significant for today (Nonoh et al., 2010).

Thus, finding and producing new antibiotics as well as using combined antibiotic therapy have been shown to delay the emergency of microbial resistance and can also produce desirable synergistic effects in the treatment of microbial infection. Antibiotic synergisms between known antibiotics and bioactive extracts are a novel concept and have an important activity against pathogens and host cells (Raja et al., 2010).

1.1          Meaning of Microorganism

A Microorganism is a microscopic living organism, which may be single celled or multicellular. The study of Microorganisms is called microbiology, a subject that began with the discovery of Microorganisms in 1674 by Antonie van Leeuwenhoek, using a microscope of his own design.

Microorganisms are very diverse and include all bacterial, archaean and most of the protozoan species on the planet. This group also contains some species of fungi, algae, and certain animals, such as rotifers. Many macroscopic animals and plants have microscopic juvenile stages. Some microbiologists also classify viruses (and viroids) as microorganisms, but others consider these as nonliving.

Microorganisms live in every part of the biosphere, including soil, hot springs, seven miles deep in the ocean, 40 miles high in the atmosphere and inside rocks far down within the Earth's crust. Microorganisms, under certain test conditions, have been observed to thrive in the vacuum of outer space. The total amount of soil and subsurface bacterial carbon is estimated as 5 x 10¹⁷g, or the weight of the United Kingdom. The mass of prokaryote Microorganisms which includes bacteria and archaea, but not the nucleated Eukaryote Microorganisms may be as much as 0.8 trillion tons of carbon (of the total biosphere mass, estimated at between 1 and 4 trillion tons) (Vaisberg et al., 2015).

1.1.1     Meaning of Soil Microorganisms

Soil Microbiology is the study of organisms in soil, their functions, and how they affect soil properties. It is believed that between two and four billion years ago, the first ancient bacteria and microorganisms came about in Earth's oceans. These bacteria could fix nitrogen, in time multiplied and as a result released oxygen into the atmosphere. This led to more advanced microorganisms. Microorganisms in soil are important because they affect soil structure and fertility. Soil Microorganisms can be classified as bacteria, actinomycetes, fungi, algae and protozoa. Each of these groups has characteristics that define them and their functions in soil (Christner et al., 2008).

1.1.2   Factors That Affect Soil Microorganism

Temperature 

Temperature directly affects the activity of the soil biota by determining the rate of physiological activity such as enzyme activity and indirectly by affecting physcio-chemical properties such as diffusion and solubility of nutrients. Organisms have a specific range of temperature at which their biological activity operates. For common soil organisms the temperature range at which they can be active ranges from 0-60.

Soil PH

In a similar manner to temperature, Microorganisms have a range of PH at which they are active. Microorganisms can tolerate extremes but this is normally requires the cell to use energy to maintain internal cellular of 7.0 PH directly affects the solubility of elements. At acidic PH, aluminum becomes more soluble and hence more available to organisms with increased toxicity. Essential minerals can become unavailable at extremes of PH for example, phosphorous and manganese becomes increasingly unavailable at high PH.

Soil moisture

Soil moisture affects the soil biota in two ways .Biologically water is essential for life and for enzyme activity and metabolism and is a solvent for biological nutrients and other chemicals. Physically soil moisture affects soil temperature and soil aeration. The degree at which soil pores are filled affects the movement and predation of Microorganisms .In very dry soil, plants may not be able to extract sufficient water through the roots because of the energy it takes to remove water from the small pores. Fungi tend to be more resistant to water stress than bacteria although under extreme conditions.

Light

In soil, light directly affects those organisms on or just below the surface and indirectly by heating the soil surface. Prototrophs such as plants, algae and cyanobacteria use the energy from sunlight to synthesis carbohydrate. Parts of light spectrum are more damaging to organisms than others. Ultraviolet light can damage DNA which induce mutations in the organism resulting in death of the organism or biochemical changes through for example changes of enzyme and metabolic pathways.

1.1.3   Secondary Metabolites

Secondary metabolites are produced by some organisms such as bacteria, fungi, plants, actinomycetes and so forth. Among the various groups of organisms that have the capacity to produce such metabolites, the Microorganisms occupy a prominent place (Ramasamy et al., 2010). Secondary metabolites including antibiotics are produced in nature and serve survival functions for the organisms producing them. The antibiotics are   heterogeneous group, the functions of some being related to their antimicrobial activities. Secondary metabolites serve as a competitive weapon used against other bacteria, fungi, plants and insects. Although antibiotics are not obligatory for sporulation, some secondary metabolites stimulate spore formation and inhibit or stimulate germination.

1.1.4   Meaning of Antibiotics

The term “antibiotic” literally means “against life”. In our every day usage, however, we use the word to describe a set of chemicals that inhibit or kill bacteria. The British scientist Alexander Fleming is credited with being the first to notice that another organism could inhibit bacterial growth in 1928. He noticed that growth of the bacterium Staphylococcus aureus was inhibited by a mold (fungus) that had contaminated his plate. The mold was later identified as Penicillium notatum and the antibiotic, isolated a short time later, was named penicillin. The value of penicillin was immediately recognized, but it was not until 1940 before the first clinical trials of penicillin were tried on humans. The reason for the long delay was because of the difficulty in producing large enough quantities of pure penicillin. While research was progressing World War Two reached Britain and the entire research project was given the highest priority and moved to the United States for safety. This was before the time of Genetic Engineering, so one of the aspects of the research project was to find a mutant strain of  Penicillium that would produce massive amounts of penicillin.  Penicillium mold was collected wherever it was found and cultured to examine its antibiotic producing potential. Finally, a high-producing strain was found growing on a cantaloupe in a market in Peoria, Illinois. This strain finally enabled large-scale production of the antibiotic penicillin. Its impact was immediately appreciated. Largely because of penicillin, World War Two was the first war in history where more soldiers died from wounds than from disease. Penicillin was described as a wonder drug and it was widely believed that infectious diseases would never again be a dominant issue for mankind. Those who understood the mechanisms and potential of evolution should have known better. Since then, there have been numerous antibiotics discovered or developed and there have been also bacteria resistant to many of them. Several diseases, once thought to be controlled are re-emerging as potent public health hazards because of antibiotic resistance and a lax attitude toward the potential of infectious diseases. It has gotten to the point where many scientists and medical personnel fear we are teetering on the brink of disaster and infectious disease may once again inhibit our ability to enjoy life.

1.2      Aims and Objectives

1.    To screen soil samples collected from dump sites in Umuahia metropolis for isolation of antibiotic producing Microorganisms.

2.    To characterize the antibiotic producing Microorganisms isolated from the dump sites in Umuahia metropolis.

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