Isolation and Characterization of bateria from Panteka stream, kaduna, Nigeria and their potential for bioremediation

ABSTRACT

Panteka stream is a flowing stream polluted with waste from the activities of

mechanics. Water samples collected at different sampling points of the stream were

analysed in order to determine the level of heavy metal contamination and bacteria

diversity with the view to elucidating the bioremediation potentials of the bacteria

isolates. Four bacteria species with high tolerance to lead, zinc, iron, nickel and

cadmium were identified by morphological and biochemical analysis as

Staphylococcus epidermidis, Serratia marcescens, Proteus mirabilis and Escherichia

coli. The 16S rRNA gene sequencing and Basic Local Alignment search tool

(BLAST) result confirmed E. coli and Staphylococcus spp as heavy metal tolerant

bacteria. Heavy metal tolerance analysis of the isolates exposed to nickel, zinc, lead,

cadmium and iron showed that the isolates had maximum tolerance to the four heavy

metals. Studies on bioremediation potential of the isolates to heavy metals in the

stream revealed that mixed bacteria culture completely removed lead, nickel, zinc and

cadmium. Analysis of pure isolates revealed S. epidermidis to be the most effective in

removing lead (100%), nickel (100%), cadmium (90.29%), zinc (84.95%) and iron

(54.82%). The results obtained from this study show that all four bacteria species

isolated from Panteka stream have potential for bioremediation of heavy metals in

contaminated water.

TABLE OF CONTENT

Dedication……………………………………………………………………...............i

Acknowledgement……………………………………………………..…………..….ii

Table of contents……………………………………………………………………..iii

List of figures……... ……………………………………………………………….vii

List of Tables……………………………………………..…………………............viii

List of plates……………………………………...…………………………………..ix

List of Appendixes………………………………………………………………….....x

Abstract…………………………………………………………………….................xi

Chapter One

1.0 Introduction…………………………………………………………………….1

1.1 Statement of the problem………………………………………........................3

1.2 Justification for the study………….……………………………………….......4

1.3 Aim……………………………………………………………………….…….5

1.4 Objectives………………………………………………………........................5

1.5 Research hypothesis………………….…………………..……………….…....6

Chapter Two

2.0 Literature review………………………………………………..……………....7

2.1 Water Quality………………………………………………………………..…7

2.2 Toxic heavy metals…………………….…………………………………….…7

2.2.1 Lead…………………………………………………………………….……..10

2.2.2 Cadmium………………………………………………………………….......11

iii

2.2.3 Nickel……………………………………………………………....................13

2.2.4 Iron…………………………………………………………………………....14

2.2.5 Zinc………………………………………………………………………........16

2.3 Bacterial Interactions with metals…..………………………………………...17

2.4 Bioremediation of heavy metals……………………………………………....19

2.4.1 Types of bioremediation……...……………………………………….............19

2.4.2 Biostimulation……………………………………….………………………..19

2.4.3 Bioaugmentation…………….………………………………………………..20

2.4.4 Role of biotechnology in bioremediation……...………...................................23

Chapter 3

3.0 Materials and Methods

3.1 Sample collection………………………………...……………………….......25

3.2 Isolation of heavy metals tolerant bacteria………….………...........................25

3.3 Identification of isolated bacteria…………………………………………..…25

3.4 Cultural and Biochemical Characterization of Bacteria Isolates……………..26

3.4.1 Cultural Morphology of Bacteria Isolates………………………………….....26

3.4.2 Gram staining…………………………………………………………….…...26

3.4.3 Oxidase test……………….……….………..………………………………....26

3.4.4 Catalase test……………………………………………………………….…...26

3.4.5 Motility indole ornithine agar………………………………………………...26

3.4.6 Urease test…………………………...………………...……...………………27

3.4.7 Methyl red test……………………………………………………...................27

3.4.8 Citrate test…………………………….…………………................................27

3.4.9 Nitrate Reduction……………………………………………………………..27

iv

3.4.10 Coagulase test…………………………………….…………………………..28

3.5 Molecular Characterization…………………………………………………..28

3.5.1 Isolation of genomic DNA……………………………..………………….…28

3.5.2 Amplification of 16S rDNA genes by polymerase chain reaction (PCR)……...….28

3.5.3 Nucleotide sequencing and alignment……………………...….......................29

3.6 Maximum Tolerance Concentration (MTC)………………………................29

3.7 Bioremediation Potentials of heavy metal tolerant isolates…………………..29

3.8 Statistical Analysis……………………………………………………..……..30

Chapter 4

Results

4.1 Identification of bacteria isolates…………….. ..………………………………..31

4.2 Heavy metal tolerance of isolated bacteria……………………............................37

4.3 Bioremediation of heavy metals by Bacteria isolates…………………………....43

Chapter 5

5.1 Discussion…………..……………………………………………………………49

5.2 Conclusion…………………………………………………….……....................51

5.3 Recommendation…….……………………………………….……………….....52

v

References…………………………………………………………………………...53

Appendices…………………………………………………………………………..68

vi

LIST OF FIGURES

Fig 1: Blast result of E. coli showing 99% similarity..…………...……..………….35

Fig 2: Blast result of Staphylococcus spp. showing 100% similarity……….............36

Fig 3: Effect of different Nickel concentrations on the growth rate of bacteria isolates

from Panteka stream……………………………………………………………..…..38

Fig 4: Effect of different Zinc concentrations on the growth rate of bacteria isolates

from Panteka stream……………………………………………………………..…..39

Fig 5: Effect of different Lead concentrations on the growth rate of bacteria isolates

from Panteka stream……………………….………………………………..…..…...40

Fig 6: Effect of different cadmium concentrations on the growth rate of bacteria

isolates from Panteka stream………………………………………….….………….41

Fig 7: Effect of different iron concentrations on the growth rate of bacteria isolates

from Panteka stream……………………………………..……………………..……42

Fig 8: Nickel depletion by bacteria isolates in Panteka Stream…….……………….44

Fig 9: Lead depletion by bacteria isolates in Panteka Stream………….…................45

Fig 10: Zinc reduction by bacteria isolates in Panteka Stream………...……............46

Fig 11: Cadmium reduction by bacteria isolates in Panteka Stream………..……….47

Fig 12: Iron reduction by bacteria isolates in Panteka Stream …………..……….…48

vii

LIST OF TABLES

Table 1: Morphological characterization of heavy metals tolerant

bacteria……………………………………………………………………….............32

Table 6: Biochemical characterization of heavy metals tolerant

bacteria……………………………………………………………………………….33

viii

LIST OF PLATES

Plate 1: PCR amplicons of rRNA genes in nine bacteria isolates from Panteka

stream………………………………………………………………………………...34

ix

LIST OF APPENDIX

Appendix 1:``Nucleotide sequence and chromatogram of E

``````.coli…………………………………..................................................................68

Appendix 2: Nucleotide sequence and Chromatogram of Staphylococcus

spp……………………………………………………………………………............69

Appendix 3: Maximum Tolerance Concentration (MTC) for nickel, zinc, cadmium,

Iron and Lead…………………………………………………...................................70

Appendix 4: Percentage removal of heavy metals from Panteka

stream…………………………………………………………………….…….…….72

Appendix 5: Student’s t – test comparison between mixed bacteria culture and

Staphylococcus epidermidis……………..………………………….……………..…72

Appendix 6: Picture of researcher watching, as an engine block is being washed in

Panteka stream……………………………………………………………………….74

Appendix 7: Experimental set-up for the determination of maximum tolerance

concentration (MTC) of the four bacteria isolates to iron…………………………...75