EFFECT OF VARIED CULTURE CONDITIONS ON BACITRACIN PRODUCTION FROM BACILLUS SUBTILIS

ABSTRACT

Bacillus subtilis capable of producing bacitracin was isolated from soil and screened for the production of bacitracin on nutrient media against test pathogenic organisms viz: Micrococcus luteus, and Staphylococcus aureus. Bacitracin was produced by submerged fermentation from the isolated bacteria and checked for their antimicrobial activity by agar well diffusion method. By optimizing the media composition and culture conditions, the effect of different pH (5, 7, 8 and 9), time (24, 48,72 and 96 hours), temperature (25, 27, 37 and 40 °C) and salinity (0% w/v (no salt), 1% w/v, 2% w/v and 5% w/v ) on the production of bacitracin were investigated by agar diffusion assay as detected by the size of zones of inhibition. Peak bacitracin production was observed judging by their zones of inhibition after incubating at 40 °C of pH 7.0 for 96 hours at 0% w/v salinity.

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 Objectives 3

CHAPTER TWO

2.0 Literature review 4

2.1 Bacitracin 4

2.1.1 History 4

2.1.2 Synthesis 4

2.1.3 Composition 4

2.1.4 Spectrum of activity and susceptibility data 5

2.1.5 Mechanism of action 5

2.1.6 Clinical use 5

2.2 Bacillus subtilis 6

2.2.1 Habitat 7

2.2.2 Reproduction 7

2.2.3 Chromosomal replication 8

2.2.4 Genome 9

2.2.5 Transformation 9

2.2.6 Uses 10

2.2.7 Safety 12

2.2.7.1 In humans 12

2.2.7.1 In animals 14

CHAPTER THREE

3.0 Materials and methods 15

3.1 Source of material and collection 15

3.2 Preparation of culture media.   15

3.3 Sterilization of materials 15

3.3 Isolation of bacillus subtilis 15

3.3.1 Morphological Appearance 16

3.3.2 Gram reaction 16

3.3.3 Endospore stain 16

3.3.4 Biochemical characteristics 17

3.3.5 Growth at 55^Oc 19

3.4 Screening of bacitracin production 19

3.5 Variation of culture conditions 19

3.5.1 Variation of incubation time 20

3.5.2 Variation of temperature 20

3.5.3 Variation of ph 20

3.5.4 Variation of salinity 21

3.6 Effect of varying culture conditions on bacitracin activity 22

CHAPTER FOUR

4.0 Results 23

CHAPTER FIVE

5.0 Discussion and Conclusion 30

5.1 Discussion 30

5.2 Conclusion 31

REFERENCES 32

APPENDIX 35

LIST OF TABLES

LIST OF FIGURES

CHAPTER ONE

1.0 INTRODUCTION

The discovery and use of antibiotics, which has been produced by several microorganisms through secondary metabolic pathways has been one of the major scientific achievements in the earliest of 20th century (Singh et al., 2004) and these compounds can fight against various diseases. Generally antibiotic is a chemical substance, possessing a molecular weight lesser than 2 kilo Dalton and used to kill or prevent growth of any other type of microorganisms at a lower Dosage (Chopra et al., 1996). Antibiotics are molecules that kill, or stop the growth of, microorganisms, including both bacteria and fungi. Antibiotics have many classes which include;

1. β-Lactam antibiotics examples: penicillins (e.g. amoxicillin), cephalosporins, carbapenems, monobactams, etc.

2. Tetracyclines example: tetracycline

3. Macrolide antibiotics example: erythromycin

4. Aminoglycosides examples: Gentamicin, Tobramycin, Amikacin

5. Quinolones example: Ciprofloxacin (a fluoroquinolone)

6. Cyclic peptides examples: Vancomycin, Streptogramins, Polymyxins

7. Lincosamides example: clindamycin

8. Oxazolidinoes example: Linezolid (Zyvox)

9. Sulfa antibiotics example: sulfisoxazole

Most of the peptide antibiotics are produced by bacilli that are active against gram-positive bacteria; however, compounds such as polymyxin, colistin and circulin exhibit activity almost exclusively upon gram-negative bacteria, whereas bacillomycin, mycobacillin and fungistatin are effective against molds and yeasts (Hammes and Frank, 1979).

Bacitracin is derived from cultures of Bacillus subtilis. It is a white to pale buff, hygroscopic powder, odorless or having a slight odor. It is precipitated from its solutions and inactivated by many of the heavy metals. Bacitracin is a mixture of several polypeptides differing in their amino acid composition (Ohki, 2003) and functions as an inhibitor of cell wall biosynthesis. Bacitracin of other micro-organism is an antibiotic as well as non-ribosomally produced by Bacillus licheniformis (Jyothi et al., 2010). Bacitracin affects protein synthesis, cell wall synthesis and membrane functions. Bacitracin interferes with the dephosphorylation of C₅₅-isoprenyl pyrophosphate, a molecule that carries the building blocks of the peptidoglycan bacterial cell wall outside of the inner membrane. This places it in the class of β-Lactam antibiotics. Studies on antibiotics and antibiotics with enzyme combination have been made by Neeraj et al, 2010 and Meshcer, 1974. It is a potent antibiotic directed primarily against gram positive organisms like Bacillus spp, Staphylococcus sp. Streptococcus sp. Clostridium sp. as well as some Archaebacteria (Arky, 1997). It is one of the important antibiotic used in human medicine and also used in animal husbandry for the prevention and control of diseases existing in farm animals. It is an in gradient in several commercially available topical triple antibiotic ointments such as Polysporin and Neosporin that are used to prevent infections in minor cuts and burns. Bacitracin is an important feed supplement for a number of animal species. It improves the weight gain and feed efficiency when added in a concentration of 5-100 ppm.

1.1 AIM

The aim of the study was:

· To determine the optimum condition for bacitracin production by Bacillus subtilis.

1.2 OBJECTIVES

The objectives of the study was:

· To isolate and characterize Bacillus subtilis.

· To observe the effect of varied culture conditions on bacitracin production by Bacillus subtilis.

· To determine the optimum culture conditions for the production of bacitracin by Bacillus subtilis.

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