vii
CHAPTER
1
2
TABLE OF CONTENTS
TITLE
DECLARATION
DEDICATION
ACKNOWLEDGEMENT
ABSTRACT
ABSTRAK
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
LIST OF ABBREVIATIONS
LIST OF APPENDIX
INTRODUCTION
1.1 Background of Study
1.2 Statement of Problem
1.3 Objectives of Study
1.4 Scope of Study
1.5 Significance of Study
LITERATURE REVIEWS
2.1 Heavy Metal Pollution
2.2 Chromium
2.2.1 Chromium Chemistry and Its Speciation
2.2.1.1 Hexavalent Chromium
2.2.1.2 Trivalent Chromium
2.2.2 Chromium in Industries
PAGE ii vii xiii xv xix xxi iii iv v vi
2
3
1
3
4
6
6
5
7
9
11

3 viii
2.2.3 Chromium Toxicity
2.2.4 Chromium Transport and Accumulation
3.2.1 Growth of A. haemolyticus in NB and LB
medium
3.2.2 Cr(VI) Resistance by A. haemolyticus in
LB Medium
3.2.3 Cr(VI) Reduction by A. haemolyticus
12
13
2.2.6 Regulations of Chromium
2.3 Treatment Technologies for Cr(VI) Remediation
2.2.1 Conventional Methods
2.2.2 Biological Methods
2.2.2.1 Microbial Cr(VI) Reduction
2.2.2.2 Enzymatic Cr(VI) Reduction
2.2.2.3 Immobilization of Micobial Enzyme for
Cr(VI) Bioremediation
17
24
29
14
15
15
17
2.2.2.4 Support Materials for Enzyme
EXPERIMENTAL
Immobilization
31
3.1 Materials
3.1.1 Glassware
3.1.2 Acinetobacter haemolyticus
3.1.3 Culture Media
35
35
35
3.1.3.1 Nutrient Broth
3.1.3.2 Nutrient Agar Plates
3.1.3.3 Luria-Bertani Broth
3.1.3.4 Luria-Bertani Agar Plates
36
36
36
36
3.1.3.5 Luria-Bertani Cr(VI) Agar Plates
3.1.4 Maintenance of Bacterial Culture
37
37
37
37 3.1.5 Preparation of Active Culture
3.1.6 Spread Plate Technique
3.1.7 Chromium(VI) Stock Solution
3.1.8 Buffer Solutions
3.2 Cr(VI) Resistance and Reduction by A. haemolyticus
38
38
38
39
39
39
40

3.2.3.1 Cr(VI) Reduction by Growing Cells of
A. haemolyticus
3.2.3.2 Cr(VI) Reduction by Resting Cells of
A. haemolyticus
3.2.3.3 Cr(VI) Reduction by Permeabilized
Cells of A. haemolyticus
3.2.3.4 Determination of Cr(VI) Concentration
3.2.3.5 Analysis of Total Chromium
3.2.4 Mechanism of Cr(VI) Reduction by A. haemolyticus using Instrumental Analysis
3.2.4.1 Field Emission Scanning Electron
Microscope coupled with Energy
Dispersive X – Ray (FESEM-EDX)
3.2.4.2 Fourier Transform – Infra Red (FTIR)
3.2.4.3 Transmission Electron Microscopy
(TEM)
3.2.4.4 Electron Spin Resonance (ESR)
3.3 Isolation and Characterization of Crude Enzyme from
A. haemolyticus
3.3.1 Sub-cellular Fractionation of A. haemolyticus
3.3.2 Enzymatic Assay
3.3.2.1 Protein Assay using Bradford Method
3.3.2.2 Chromate Reductase Assay
3.3.3 Localization of Chromate Reductase Activity of A. haemolyticus
3.3.4 Characterization of Chromate Reductase
Activity in Crude Enzyme (Crude-Cell Free
Extracts – CFE) of A. haemolyticus
3.3.4.1 Effect of pH on Crude Enzyme
Activity and Stability
3.3.4.2 Effect of Temperature on Crude
Enzyme Activity ix
40
44
44
45
43
43
45
42
42
43
41
41
47
46
46
46
46
47
47

4 x
3.3.4.3 Effect of Electron Donors on Crude
Enzyme Activity
47
3.3.4.4 Effect of Metal Ions, Metabolic
Inhibitor and Protein Denaturant
48
3.3.4.5 Time Course and Kinetic Studies 48
3.4 Preliminary Studies on Immobilization of Crude CFE of 49
A. haemolyticus for Cr(VI) Reduction
3.4.1 Screening of Support Matrices for
Immobilization of Crude CFE of A. haemolyticus
3.4.2 Optimization of Cr(VI) Reduction using Crude
49
50
Enzyme Immobilized in Ca-alginate Beads
3.4.2.1 Effect of Initial pH of Cr(VI)
3.4.2.2 Reusability of CFE-immobilized in
Ca-alginate beads
RESULTS AND DISSCUSSION
4.1 Cr(VI) Resistance and Reduction by
4.1.1 Growth of
medium
A. haemolyticus
A. haemolyticus
in NB and LB
4.1.2 Cr(VI) Resistance by A. haemolyticus in LB
51
51
50
50
52
Medium
4.1.3 Cr(VI) Reduction by A. haemolyticus
4.1.3.1 Cr(VI) Reduction by Growing Cells of
A. haemolyticus
4.1.3.2 Cr(VI) Reduction by Resting and
Permeabilized Cells of A. haemolyticus
4.1.4 Mechanism of Cr(VI) Reduction by
A. haemolyticus using Instrumental Analysis
4.1.4.1 FESEM-EDX Analysis
4.1.4.2 FTIR Analysis
4.1.4.3 TEM Analysis
4.1.4.4 ESR Analysis
57
59
55
55
59
61
64
66

xi
4.2 Isolation and Characterization of Crude Enzyme from
A. haemolyticus
4.3.1 Screening of Support Matrices for
Immobilization of Crude CFE of A. haemolyticus
4.3.2 Cr(VI) reduction using Crude CFE Immobilized
in Ca-alginate Beads
4.3.1.1 Effect of Initial pH
4.3.1.2 Effect of Agitation Speed, Reaction
Time, Bead’s Dosage and Initial Cr(VI)
Concentration
4.3.1.6 Reusability of CFE-immobilized in Ca-
alginate beads
4.3.3 Comparison between Free and Immobilized
Crude CFE for Cr(VI) Reduction
68
4.2.1 Localization of Chromate Reductase Activity of A. haemolyticus
68
4.2.2 Characterization of Chromate Reductase 70
Activity in Crude Cell-Free Extracts of A.
haemolyticus
4.2.2.1 Effect of Temperature on Cr(VI)
Reduction
70
4.2.2.2 Effect of pH on Activity and Stability of 71
Chromate Reductase
4.2.2.3 Effect of Electron Donors on Cr(VI) 72
Reduction
4.2.2.4 Effect of Metal Ions
4.2.2.5 Effect of Metabolic Inhibitor and
Protein Denaturant
4.2.2.6 Kinetic and Time Course Studies of 79
Chromate Reductase Activity
4.3 Preliminary Studies on Immobilization of Crude CFE of 84
A. haemolyticus for Cr(VI) Reduction
74
77
84
92
86
86
88
93

5 CONCLUSION AND RECOMMENDATION
5.1 Conclusion
5.2 Recommendation for Future Work
REFERENCES
APPENDIX xii
95
95
96
97
123

xiii

4.1
4.2
4.3
4.4 xiv
81 Kinetic Parameters (K m
and V max
) for Cr(VI) reduction by crude CFE of A. haemolyticus at different reaction time
Kinetic parameters (K m
and V max
) and optimum conditions for chromate reductase activity by crude cellfree extracts of various microorganisms
Beads Integrity after Chromate Reduction for Various
Matrices
Structural integrity for Ca-alginate beads (with and without immobilized crude CFE) at different pH values
83
85
87

xv
2.2
2.3
2.4
2.5
2.6
2.11
4.1
LIST OF FIGURES
TITLE FIGURE NO.
2.1
2.9
2.10
4.2
4.3
Compliance Status to the Environmental Quality
(Industrial Effluent) Regulations (Department of
Environment, 2010)
E h
–pH Profile for Chromium
Structure of (a) CrO
4
2-
ion and (b) Cr
2
O
7
2-
ion
Schematic Diagram of Toxicity and Mutagenicity of
Cr(VI), (modified from Vincent, 1994).
Possible mechanisms of enzymatic Cr(VI) reduction under aerobic and anaerobic conditions (Wang and Shen, 1995).
Methods of Enzymes Immobilizaion (Sato and Tosa,
2002)
Chemical structure of Na
+
alginate. G, guluronic acid; M, mannuronic acid (Smidsrod and Braek, 1990).
Chemicals structure of (a) chitosan and (b) the binding of metal anions on chitosan (Naja and Volaesky, 2011).
The overall reaction of PVA–alginate bead formation
Growth profile of A. haemolyticus in NB and LB medium
OD
600
(  ), CFU ( ) and LB medium; OD
600
(

), CFU
( )
Growth of A. haemolyticus in LB broth supplemented with 30 -200 mg/L of Cr(VI); (a) OD
600
and (b) CFU/mL
Survival of A. haemolyticus supplemented with 30 -200 mg/L of Cr(VI) in (a) LB broth and (b) LB agar at 24 hour, 48 hour and 96 hour
PAGE
6
8
9
13
26
29
31
32
33
51
53
54

4.11
4.12
4.13
4.14
4.7
4.8
4.4
4.5
4.6
4.9
4.10 xvi
Cr(VI) Reduction in LB medium for (a) abiotic and (b) biotic by A. haemolyticus
Cr(VI) reduction by Resting and Permeabilized Cells of A. haemolyticus
FESEM micrographs of A. haemolyticus cells grown in
LB broth (a, b) without Cr(VI) (control) and (c, d) with
100 mg/L Cr(VI); magnification (a, b) 10k and (c, d) 25k.
EDX spectra with elemental compositions of A. haemolyticus cells grown in LB broth (a) without Cr(VI)
(control) and (b) with 100 mg/L Cr(VI).
FTIR spectra of Acineobacter haemolyticus cells grown in
LB broth (a) without Cr(VI) (control) and (b) with 100 mg/L Cr(VI).
The cell wall structure and composition of gram-negative bacteria (a), peptidoglycan stucture (b) (Kotrba et al .,
2011) and a schematic diagram of Cr(VI)-bacterial cells;
Cr
6+
initially binds with functional groups of bacterial cell wall and then reduced to Cr
3+
(c) (Das and Guha, 2007).
TEM micrographs of A. haemolyticus cells grown in LB broth (a, b) without Cr(VI) (control) and (c, d) with 100 mg/L Cr(VI); bar represents 1000 nm - a and c, 100 nm – b and d; arrow represents chromium accumulation in the cell (membrane fraction and cytoplasm) and outside the cells.
ESR spectrum from a solid-state of A. haemolyticus incubated with 100 of mg/L Cr(VI) in LB broth after 48 hour; (a) K
2
Cr
2
O
7
, (b) CrCl
3
·6H
2
O and (c) sample
Proposed mechanism for Cr(VI) reduction by A. haemolyticus (Modified by Li et al ., 2008)
Chromate reductase activity in different cell fractions of
A. haemolyticus.
Effect of temperature on chromate reductase activity of
A.haemolyticus
56
58
59
61
62
64
65
67
68
69
70

4.15
4.16
4.17
4.18
4.19
4.20
4.21
4.22
4.23
4.24 xvii
Effect of pH on the activity and stability of Cr(VI) reductase from CFE; (a) specific chromate reductase activity and (b) percentage Cr(VI) reduction
Effect of electron donors on Cr(VI) reductase activity (a) total and specific chromate reductase activity, and (b)
Cr(VI) reduction
Effect of metal ions on chromate reductase activity, (a) specific chromate reductase activity and (b) percentage
Cr(VI) reduction .
Non-competitive inhibition of chromate reduction by Ag
+ salts. Crude CFE was assayed for the rate of reduction of
29 or 48 µM Cr(VI) in the absence or presence of 1-14 mM Ag
+
Effect of EDTA and azide, as protein denaturant and metabolic inhibitor, on Cr(VI) reductase activity in CFE of A. haemolyticus .
Specific chromate reductase activity of CFE for A. haemolyticus at different initial Cr(VI) concentrations and reaction time; (a) 3 min – 60 min and (b) 120 min – 360 min.
Effect of initial Cr(VI) concentration on chromate reduction (a) in the presence of 1 mM Co
2+
and (b)
Lineweaver–Burk plot for Cr(VI) reduction after 3 min.
Cr(VI) reduction by crude CFE of A. haemolyticus immobilized in various support matrices
Effect of Initial pH on Cr(VI) reduction of 48 µM of
Cr(VI) (5g wet weight beads, 24 hour reaction time) by crude CFE immobilized-alginate
Effect of Agitation Speed on Cr(VI) reduction of 48 µM of Cr(VI) (pH 3, 5g wet weight beads, 24 hour reaction time) by crude CFE immobilized-alginate
72
73
75
76
78
80
81
84
86
88

4.25
4.26
4.27
4.28
4.29 xviii
Effect of Reaction Time on the Cr(VI) Reduction of 48
µM of Cr(VI) (100 rpm, pH 3, 5g wet weight beads) by crude CFE immobilized-alginate
Effect of Bead Dosage on immobilized CFE-alginate of
48 µM of Cr(VI) (100 rpm, pH 3, 24 hour reaction time) by crude CFE immobilized-alginate
Effect of Cr(VI) concentrations on immobilized CFEalginate of 10 - 192 µM of Cr(VI) (100 rpm, pH 3, 24 hour reaction time) by crude CFE immobilized-alginate
Reusability studies of immobilized CFE-alginate beads of
48 µM of Cr(VI) (100 rpm, pH 3, 24 hour reaction time) by crude CFE immobilized-alginate
Total Activity and Cr(VI) Reduction of 10 - 192 µM of
Cr(VI) at 30 min reaction time by Free and Immobilized crude CFE on Ca-alginate
89
90
91
92
93

% kWh
L
M m
3 mg
NADH
NADPH ng
[Cr]
°C
µg
CFE
CFU
DPC
EDTA g h
K i
K m kPa
OD
600 ppm rpm v v/v
V max
LIST OF ABBREVIATIONS
- Percentage
- chromium Concentration
- Degree Celcius
- Microgram
- Cell free extract
- Colony forming unit
- 1,5 - diphenylcarbazide
- Ethylenediaminetetraacetic acid
- Gram
- Hour
- Dissociation constant for inhibitor binding
- Michaelis–Menten constant
- kilopascal
- kilo Watt hour
- Liter
- Molar
- Meter Cubic
- Miligram
- Nicotinamide adenine dinucleotide
- Nicotinamide adenine dinucleotide phosphate
- Nanogram
- Optical density at 600 nm
- Part Per Million
- Rotation per minute
- Volume
- Volume per volume
- maximum rate or maximum velocity xix

V o w w/v
λ
- initial velocity
- Weight
- Weight per volume
- wavelength xx

APPENDIX NO.
A xxi
LIST OF APPENDIX
TITLE PAGE
List of publications (journal/article/chapter in book/book), awards, paper presentation seminar and workshop during this project period (Jun 2010 to July 2012)
123