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 55Oc ﾿ 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

Table ﾿ Title ﾿ Page

1a ﾿ Characterization and identification of bacillus subtilis from the samples collected. ﾿ 24

1b ﾿ Confirmation of the test organisms Staphylococcus aureus and 

Micrococcus lateus. ﾿ 25

LIST OF FIGURES

Figure ﾿ Title ﾿ Page

1 ﾿ The effect of temperature on bacitracin production ﾿ 26

2 ﾿ The effect of incubation time on bacitracin production ﾿ 27

3 ﾿ The effect of pH on bacitracin production ﾿ 28

4 ﾿ The effect of salinity on bacitracin production ﾿ 29