EVALUATION OF THE EFFECT OF VARIED CULTURE CONDITIONS AND NUTRITIONAL REQUIREMENTS IN THE PRODUCTION OF ANTIMICROBIAL METABOLITES BY STREPTOMYCES SPECIES

ABSTRACT

Streptomyces species was isolated from soil samples using spread plate method on Streptomyces agar while Streptomyces broth was used for fermentation and optimization. The varied culture conditions evaluated were pH, Mineral salt concentration, speed of agitation and nutritional components tested were Carbon and Nitrogen sources. The production of antimicrobial metabolite was authenticated by antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa. Amongst the carbon sources, maximum production of antimicrobial metabolite was observed with the medium containing Galactose anhydride evident by high inhibition zone of 32mm and 23mm (Staphylococcus aureus and Pseudomonas aeruginosa) respectively. This was followed closely by the medium incorporated with glycerol and glucose. Maximum productivity of antimicrobial metabolite was also achieved with the medium containing malt extracts evident with an inhibition zone of 30mm and 27mm for Staphylococcus aureus and Pseudomonas aeruginosa. This was also followed by the medium containing yeast extract. For pH, maximum metabolite production was attained in the medium adjusted to 10, with an inhibition zone of 30mm and 33mm for Staphylococcus aureus and Pseudomonas aeruginosa respectively, which was closely followed by pH 9.  For the medium incorporated with additional mineral salts, best metabolite production was obtained at a concentration of 0.30mol/ml evident by an inhibition diameter of 30mm and 27mm for the respective test organisms. Best antimicrobial metabolite production was also observed with the medium whose agitation speed was 250rpm. This gave an inhibition diameter of 26mm and 23mm respectively against the test organisms. Finally, for better productivity of antimicrobial metabolite by Streptomyces species, carbon source such as Galactose anhydride, Nitrogen source such as malt extract, mineral concentration of 0.30mol/ml, pH of 10 and agitation speed of 250rpm, should be used.

TABLE OF CONTENTS

Certification                                                                                                                           ii

Dedication                                                                                                                              iii

Acknowledgements                                                                                                                iv

Table of Contents                                                                                                                   v

List of Tables                                                                                                                          viii

List of Figures                                                                                                                                    ix

Abstract                                                                                                                                  x

Chapter One

1.0.          Introduction                                                                                                               1

1.1       Aim                                                                                                                            3

1.2       Objective                                                                                                                    4

Chapter Two

2.0       Literature review                                                                                                        5

2.1       Antimicrobial metabolites produced by Streptomyces species                                    5

2.1.1    History                                                                                                                        5

2.1.2    Synthesis                                                                                                                     6

2.1.3    Composition                                                                                                               6

2.1.4    Spectrum of activity                                                                                                   7

2.1.5    Mechanism of action                                                                                                  7

2.1.6    Clinical uses                                                                                                                8

2.2.      Streptomycetes                                                                                                           9

2.2.1    Habitat and reproduction                                                                                            12

2.2.2    Genome                                                                                                                                                    14

2.2.3    Ecology                                                                                                                       14

2.2.4.   Streptomyces as human pathogens                                                                             16

Chapter Three

3.0       Materials and method                                                                                                 17

3.1       Collection of samples                                                                                                 17

3.2       Sterilization of apparatuses and equipment                                                                17

3.3.      Preparation of culture and fermentative media                                                          17

3.4.      Isolation of Streptomyces                                                                                           18

3.5       Test organisms used in screening                                                                               18

3.6       Confirmation of the test isolates                                                                                 18

3.7.      Antimicrobial metabolites extraction                                                                         18

3.8.      Antimicrobial screening of the metabolites                                                               19

3.9.      Determination of the effect of varied nutritional requirements on metabolite Production                                                                                                                  19

3.9.1.   Carbon sources                                                                                                           19

3.9.2.   Nitrogen sources                                                                                                         19

3.10.    Determination of the effect of varied culture condition on metabolite production.            20

3.10.1.   pH                                                                                                                             20

3.10.2. Sodium chloride concentration                                                                                 20

3.10.3. Speed of agitation                                                                                                       20

Chapter Four

4.0.      Results                                                                                                                        29

Chapter Five

5.0.      Discussion and Conclusion                                                                                        30

5.1.      Discussion                                                                                                                   32

5.2.      Conclusion                                                                                                                  33

REFERENCE                                                                                                                        37

APPENDIX

LIST OF TABLES

TABLE         TITLE                                                                   PAGE

Table 1: Colours of substrate mycelium and soluble pigment occurring in Streptomycetes.                       11   

Table 2:   Isolation and identification of streptomyces species from the different soil samples that were collected.      23

Table 3:   Confirmation of the test organisms Staphylococcus aureus and Pseudomonas aeruginosa.           24

                                                     LIST OF FIGURES

FIGURE  TITLE                                                                PAGE

Figure 1: Showing the effects of varied carbon sources on the production of Antimicrobial metabolite by Streptomyces species.                25

Figure 2: Showing the effects of varied nitrogen sources in the production of antimicrobial metabolites by Streptomyces species.                  26

Figure 3: Showing the effects of varied pH condition in the production of Antimicrobial metabolites by Streptomyces species.                  27

Figure 4: Showing the effects of varied mineral salt concentration in the production of antimicrobial metabolites by Streptomyces species.                              28

Figure 5: Showing the effects of varied speeds of rotation in the production of  antimicrobial metabolites by Streptomyces species.        29

CHAPTER ONE 

INTRODUCTION

The search for isolating novel antibiotics, effective against pathogenic and resistant pathogenic microorganisms from unexplored habitats around the world, continues to be an important sector of research. The discovery and development of antibiotics has played a major role in clinical medicine, agricultural research works and generally all works of life (Moses, 2015). Antibiotics are chemical substances with a molecular weight generally below 2 kilo Dalton (Moses, 2015) that either inhibits or kills viruses, bacteria or fungi. If the antibiotic kill, it is referred to as cidal (Bactericidal, viricidal and fungicidal) and if it inhibits, it is referred to as static (Bacteristatic, viristatic and fungistatic).

Antibiotics are grouped into three groups, based on their source. This includes:

a)     Synthetic antibiotics

This includes all antibiotics produced purely from chemical processes which involves no microorganism in its production procedures. This group of antibiotics are not produced as antimicrobial metabolites or products of microorganisms but instead are produced by chemical processes. Examples include amoxicillin and all its derivatives, oxytetracycline, chloramphenicol, chlortetracycline, minocycline etc.

b)    Semisynthetic antibiotics

This includes antibiotics that are produced by microorganisms but their side chains have been modified so as to improve their efficacy. This entails chemical modification of their side chains to improve the spectrum activity of the antibiotics. Examples includes Penicillin derivatives; Penicillin G, Penicillin V, Methicillin, Ampicillin, Ticarcillin etc.

c)     Non- Synthetic antibiotics

This group of antibiotics are purely direct products of microorganisms. They are direct microbial antimicrobial metabolites without any modifications attached to them. Examples includes Streptomycin produced by Streptomyces species and penicillin Produced by Penicillum notatum.

            Most antibiotics are antimicrobial metabolites of microorganisms. Antimicrobial metabolites are produced by microorganisms in a closed system cultivation, when they have exhausted all the nutrients available for their growth. Since in a close system cultivation of microorganisms, there is no replacement of spent nutrients, the microorganisms are then forced to produce these metabolites which in some cases, may even be toxic to the producer microorganism.

Streptomycetes have been the most and generally evaluated source of antibiotics since the discovery of actinomycin D, grisein, streptothricin, and streptomycin in the 1940s by Waksman and co-workers (Waksman, 1948). Streptomycetes synthesize a large variety of chemically different but stable compounds, many of them acting as antibiotics, cytostatics, fungicides, or as modulators of immune responses (Wolfbang et al., 2015). Consequently, the study of Streptomyces, are very numerous in number and each of this genus member produces one or more antibiotics.

They produce these antibiotics as antimicrobial metabolites. These antimicrobial metabolites production is controlled by genes. Many of these gene sets are present in one genome, but not in others. At times, the clusters may be in same position in different species but can be occupied by different secondary metabolism. This is the sole reason why these group of microorganisms produce a vast range of antimicrobial metabolites (Waksman, 1948). For example, the pks1 cluster of Streptomyces avermitilis MA-4680T is replaced in Streptomyces ambofaciens ATCC 23877T by a different secondary metabolism cluster of 28 genes and in Streptomyces coelicolor A3 (2) by a 31-gene insertion (Wolfbang et al., 2015). The subtelomeric chromosome arms often contain gene clusters for secondary metabolism, especially those that are species-specific. The more abundant genes for secondary metabolism, such as those for the production of pentalenolactone, different siderophores, and the odour compound geosmin, typically fall in syntenous locations within the central core region. It is interesting that the genes for the biosynthesis of antibiotics are frequently located near one or more genes mediating resistance to the corresponding antibiotic. Asides the production of streptomycin, and the other pre-listed antibiotics, the following antibiotics are also produced by Streptomyces species. They include Actinorhodin, Daunorubicin, Frenolicin, Granaticin, Griseusin B, Jadomycin B, Mithramycin, Tetracycline, Macrolides, Peptide antibiotics, Cyclopentenoid antibiotics, Aminoglycosides

These antibiotics produced by the genus Streptomyces cannot be categorized into one group of antibiotics, due to the vast the nature of the antibiotics produced.  Their spectrum of activity is broad ranged and their mode of action ranges from cell wall synthesis inhibition to protein synthesis inhibition (50s and 30s ribosomal distortion). The various physical and chemical appearance and representation of these antibiotics depends on the group of antibiotics they fall into (Nzubechi, 2015).

1.1       AIM

The aim of the study was:

·       To evaluate the effect of varied culture condition and nutritional requirement in the production of secondary metabolite by Streptomyces species.

1.2       OBJECTIVES

The objectives of the study was:

·       To isolate Streptomyces species from soil samples.

·       To determine the effect of varied carbon sources in the production of antimicrobial metabolites by Streptomyces species. 

·       To ascertain the effect of varied nitrogen sources on antimicrobial metabolites production by Streptomyces species.

·       To determine the effect of varied pH values in the production of antimicrobial metabolites by Streptomyces species.

·       To evaluate the effect of varied mineral salts concentration in the production of antimicrobial metabolites by Streptomyces species.

·       To ascertain the effect of varied speed of agitation on antimicrobial metabolites production by Streptomyces species.

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