ANTIBACTERIAL PROPERTIES OF BACILLUS SPECIES ISOLATED FROM SOIL AND OPTIMIZATION OF GROWTH CONDITIONS

ABSTRACT

This research work was conducted to access the antibacterial activity of Bacillus species isolated from agricultural sites in two different communities (Umudike and Ndoru in Ikwuano local government area) in Abia State of Nigeria. The antibacterial activities of the isolated Bacillus spp was tested against three different bacterial strains (Staphylococcus aureus, micrococcus spp, Escherichia coli) by agar well diffusion method and incubated for 24hrs at 370C. The Research Results shows that most strains possess significant antibacterial potentials mostly against S. aureus and micrococcus sp but less activity against the Gram negative E. coli. Thereafter, Further investigations was carried out to determine the optimum growth conditions for these Bacillus species for maximum antibiotic production. Optimum activity was achieved using nutrient broth culture at different optimization conditions (temperatures; 300C., 370C. and 450C.   incubation time; 24hrs,48hrs and 72hrs and pH; 6, 7, 8), The medium was then centrifuged at 10,000rpm for 20minutes to get the cell free supernatant and a Bacitracin assay done on nutrient agar plates by measuring the diameter zones of inhibition in millimeters(mm) against the test organisms being used. And the highest zone of inhibition for incubation temperature was observed, (24mm) against Micrococcus spp at 370C, (27mm) at 72hrs incubation period and (24mm) at pH8, antibacterial activity was also produced maximally against S. aureus at optimized temperature, incubation time and pH as (18mm, 20mm and 14mm) respectively and zero effect was seen on E. coli. Therefore, antimicrobial compounds from these Bacillus species can be good candidates for future antibiotics production. Further screening for antimicrobial agents should be carried out in search of novel therapeutic compounds. 

TABLE OF CONTENTS

Title page ﾿ I

Certification ﾿ II

Dedication ﾿ III

Acknowledgement ﾿ IV

Table of content ﾿ V

List of Tables ﾿ VIII

Abstract ﾿ IX

 CHAPTER ONE

1.1 Introduction ﾿ 1

CHAPTER  TWO

2.0 Literature Review ﾿ 4

 2.1 Bacillus and its Taxanomy ﾿ 4

2.1.1 Bacillus Cereus Group ﾿ 4

2.1.2 Bacillus Subtilis Group ﾿ 7 ﾿

2.2 The Nature Of Bacillus Endospore ﾿ 8 ﾿

2.3  Bacillus Endospore Formation Process ﾿ 8

2.4 Structure, Biosynthesis And Mechanism Of Action Of Bacitracin ﾿ 10

2.4.1 Structure Of Bacitracin ﾿ 10

2.4.2  Biosynthesis Of Bacitracin ﾿ 11 

2.4.3 Mechanism Of Action Of Bacitracin ﾿ 11

2.5  Antimicrobial Spectrum Of Bacitracin ﾿ 11 ﾿

2.6  Uses Of Bacitracin ﾿ 12

CHAPTER THREE

3.0     Materials And Methods ﾿ 13

3.1     The Study Area ﾿ 13

3.2     Soil Sample Collection ﾿ 13

3.3 Sample Preparation ﾿ 14

3.4 Sterility ﾿ 14

3.5 Preparation Of Culture Media ﾿ 14

3.7 Collection And Confirmation Of Test Isolates ﾿ 15

3.7.1 Colonial Morphology ﾿ 15

3.7.2 Gram Staining ﾿ 15

3.7.3 Motility Test ﾿ 15

3.7.4 Biochemical Test ﾿ 16

3.8 Preliminary Screening For Bacitracin Producing Bacillus Spp ﾿ 18

3.9 Optimization Of Conditions For Bacitracin Production ﾿ 18

3.9.1 Optimization For Incubation Temperature ﾿ 19

3.9.2 Optimization For Time Of Incubation ﾿ 19

3.9.3 Optimization For pH Of Incubation ﾿ 19

3.10 Presentation And Analysis Of Data ﾿ 19

CHAPTER FOUR

4.0 Results ﾿ ﾿ 20

4.1 Results Presentation ﾿ 20

CHAPTER FIVE

5.0 Discussion, Conclusion And Recommendation ﾿ 28

5.1 Discussion  ﾿ 28

5.2 Conclusion ﾿ ﾿ 29

5.3 Recommendations ﾿ 30

REFERENCES

LIST OF TABLES

Table 1: Sample collection sites ﾿ 22 

Table 2: Morphological and biochemical characteristics of isolates ﾿ 23

Table 3: Preliminary screening for bacitracin ﾿ 24

Table 4:  Antimicrobial effect of crude extracts of Bacitracin against E. coli,

 S. aureus and Micrococcus spp at optimized incubation temperature ﾿ 25

Table 5:  Antimicrobial effect of crude extracts of Bacitracin against E. coli,

 S. aureus and Micrococcus spp at various incubation periods ﾿ 26

Table 6: Antimicrobial effect of crude extracts of Bacitracin against E. coli,

 S. aureus and Micrococcus spp at various optimized pH ﾿ 27