vii
TABLE OF CONTENTS
TITLE
CHAPTER
DECLARATION
ii
DEDICATION
iii
ACKNOWLEDGEMENTS
iv
ABSTRACT
v
ABSTRAK
vi
TABLE OF CONTENTS
vii
LIST OF TABLES
xiii
LIST OF FIGURES
xv
LIST OF ABBREVIATIONS
LIST OF APPENDICES
1
PAGE
xviii
xx
INTRODUCTION
1.1
1.2
1.3
Heavy metal and its impact on the environment
1
1.1.1 Toxicity of heavy metals
2
1.1.2 Chemistry of heavy metal
3
Chromium
5
1.2.1 Chemistry and physical properties
5
1.2.2 Chromium toxicity
7
Electroplating Industry
8
1.3.1 Electroplating process
8
1.3.2 Processes of Chromium Electroplating
8
1.3.3 Sources and regulations of Electroplating
Wastewater
1.4
10
Treatment Technologies for Cr (VI)
11
1.4.1
11
Conventional Treatments
viii
1.4.2
1.4.3
1.5.
Adsorption techniques
13
1.4.2.1 Activated carbon
13
1.4.2.2 Low-cost adsorbent
14
1.4.2.3 Microorganism as biosorbent
15
Physical Treatments
16
Bacterial biofilm
16
1.5.1
Mechanism of biofilm formation
17
1.5.2
Extracellular Polymeric Substances (EPS)
18
1.5.3
Functions of EPS
19
1.5.4
Observation of EPS
19
1.5.5
Factors enhancing biofilm formation
20
1.5.5.1 Surface properties of support
material
1.5.6
20
1.5.5.2 Bacterial cell surface
21
1.5.5.3 Hydrodynamics (fluid flow)
22
1.5.5.4 Temperature and pH
22
1.5.5.5 Types and amount of nutrients
23
Biofilm reactors in Cr(VI) wastewater
23
treatment
1.6
2
1.5.6.1 Support material
26
1.5.6.2 Bioreactor media
27
Microscopy techniques applied for monitoring the
development of biofilm
27
1.6.1
29
Scanning Electron Microscopy (SEM)
1.7
Problem statement
30
1.8
Objective of Thesis
30
1.9
Scope of Thesis
30
CHROMATE
REDUCTION
BY
Acinetobacter
haemolyticus UTILIZING MOLASSES AS CARBON
SOURCES
2.1
Introduction
2.1.1 Molasses
2.1.2
Composition of molasses
31
31
32
ix
2.1.3
2.2
Sugarcane processing
32
Materials and Methods
34
2.2.1
Bacteria
34
2.2.2
Preparation of Cr(VI) stock solution
34
2.2.3
Cr(VI)-containing electroplating
2.2.4
2.2.5
wastewater
34
Characteristics of molasses
34
2.2.4.1 Total Suspended Solid (TSS)
35
2.2.4.2 Total Carbohydrate
35
2.2.4.3 Total Nitrogen
36
2.2.4.4 Protein
36
2.2.4.5 Chemical Oxygen Demand (COD)
37
Growth of A. haemolyticus using molasses
as carbon source
37
2.2.5.1 A. haemolyticus grown in different
concentrations of molasses
37
2.2.5.2 Growth profile of A. haemolyticus
in molasses
2.2.6
Cr(VI) reduction study in batch system
38
38
2.2.6.1 Cr(VI) reduction by A. haemolyticus
grown in different concentrations of
molasses
2.2.7
38
Removal of Cr(VI)-containing wastewater
in laboratory-scale bioreactor
39
2.2.7.1 Laboratory-scale bioreactor setup
39
2.2.7.2 Immobilization of A. haemolyticus
onto bioreactor
2.3
39
2.2.7.3 Cr(VI) reduction system
40
2.2.7.4 Polishing unit
41
Results and Discussions
42
2.3.1
Characteristics of molasses
42
2.3.2
Effect of varying concentrations of molasses
on growth of A. haemolyticus
43
x
2.3.3
2.3.4
Growth profile of A. haemolyticus in
different concentrations of molasses
45
Cr (VI) reduction
47
2.3.4.1 Cr(VI) reduction in batch system
47
2.3.4.2 Removal of Cr(VI)-containing
wastewater in laboratory-scale
bioreactor
2.3.5
2.4
3
Performance of the polishing treatment unit
Conclusion
48
50
53
APPLICATION
OF
RESPONSE
SURFACE
METHODOLOGY (RSM) FOR OPTIMIZING
REMOVAL OF Cr(VI) SOLUTIONS USING Cr(VI)
REDUCING BIOFILM SYSTEMS
3.1
Introduction
54
3.2
Pineapple waste
55
3.3
Response Surface Methodology (RSM)
56
3.3.1
Introduction
56
3.3.2
Application of RSM
57
3.4
Materials and methods
59
3.4.1
Simulated Cr (VI) solution preparation
59
3.4.2
Liquid Pineapple waste
59
3.4.3
Laboratory-scale bioreactor
59
3.4.4
Evaluation of dislodging methods
60
3.4.5
Immobilization of A. haemolyticus in
bioreactor
3.4.6
61
3.4.5.1 Effect of contact time
61
3.4.5.2 Effect of nutrient supplementation
62
3.4.5.3 Number of bacteria
63
Experimental design for optimization of
63
parameters
3.4.7 Cr(VI) reduction process
63
64
3.4.8 Optimization and verification of Cr(VI)reducing biofilm system
66
xi
3.5
Results and discussion
67
3.5.1 Characteristic of Liquid Pineapple Waste
(LPW)
3.5.2
67
Effect of dislodging methods on bacterial
counts
3.5.3
3.5.4
3.5.5
68
Immobilization of A. haemolyticus onto
bioreactor
69
3.5.3.1 Effect of contact time
69
3.5.3.2 Effect of nutrient supplementation
70
ANOVA analysis and fitting of quadratic
model
73
Effects of parameters
77
3.5.5.1 Effect of initial Cr(VI)
concentration and flowrate
77
3.5.5.2 Effect of flowrate and nutrient
supplementation
78
3.5.5.3 Effect of nutrient supplementation
and Cr(VI) concentration
3.6.
4
79
3.5.6 Optimization and verification of models
81
Conclusion
82
PERFORMANCE AND DEVELOPMENT OF Cr(VI)REDUCING BIOFILM SYSTEM FOR TREATING
Cr(VI) FROM ELECTROPLATING WASTEWATER
4.1
Introduction
4.2
Materials and methods
83
84
4.2.1 Cr(VI)-containing Electroplating
Wastewater
4.2.2
Laboratory-scale bioreactor
4.2.3
Immobilization of A. haemolyticus on the
wood husk
4.2.4 Cr (VI) removal system
4.2.5
Polishing unit
4.2.6 Analysis on the development of biofilm on
84
84
84
85
86
xii
Wood Husk
4.3
87
Results and Discussions
88
4.3.1 Characteristics of electroplating wastewater
88
4.3.2
Performance monitoring of Cr (VI)
reduction system
89
4.3.3 Coagulation/flocculation and filtration of
the bioreactor effluent
4.3.4
92
Assessment of biofilm morphology and
architecture in Cr(VI)-reducing bioreactor
by FESEM
4.4
5
Conclusion
93
98
CONCLUSION
99
5.1
Conclusion
99
5.2
Suggestions for future works
101
REFERENCES
102
APPENDICES
113
xiii
LIST OF TABLES
TABLE NO.
TITLE
PAGE
1.1
Toxicity and exposure limits of metals to human body
3
1.2
Toxic metals in industrial effluents
5
1.3
Characteristics of Cr(VI) and Cr(III)
6
1.4
Comparison between L and H carbon
14
1.5
Functions of EPS
19
1.6
Lists the treatment of Cr(VI)-containing wastewater using
biofilm reactors
1.7
24
The summary of microscopy techniques applied for
monitoring the development of biofilm
28
2.1
Composition of molasses.
32
2.2
Characteristics of molasses
42
2.3
CFU/mL of A.haemolyticus grown at different molasses
concentrations (0.25-2.5 %)
46
2.4
Metal analysis of effluents using ICP-MS
51
2.5
Metals concentration after filtration process
52
3.1
Composition of Liquid Pineapple Wastes
56
3.2
The use of RSM in different types of experiments
58
3.3
Independent variables: Coded and real value center
composite rotatable design
3.4
Central Composite Design (CCD) for three variables
together with the observed response
3.5
63
65
Optimum values of the variables for maximum percentage
Cr(VI) removal
66
3.6
Characteristics of Liquid Pineapple Waste
67
3.7
Effect of nutrient supplementation on the Cr(VI) reduction
72
xiv
3.8
ANOVA table (partial sum of squares) for response surface
quadratic model
3.9
73
Predicted and experimental value for the responses at
optimum condition.
81
4.1
Composition of Electroplating wastewater
88
4.2
Attached bacterial cells on wood husk in the bioreactor
during Cr(VI) reduction process
4.3
91
Profile for removal of pollutants at initial Cr(VI) wastewater
of 105 mg/L
92
xv
LIST OF FIGURES
FIGURE NO.
1.1
TITLE
PAGE
Department of Environment: Enviromental Quality (Sewage
and Industrial Effluent Regulations 1979, compliance status,
2008
2
1.2
Electroplating Industry Conventional Wastewater Treatment
12
1.3
Development model for biofilm formation
17
1.4
Bound and soluble EPS
18
1.5
E. coli PHL628 observed by SEM. Arrows indicate some
curli and EPS (a) dense EPS formation (b)
1.6
20
Prevalent concept regarding the effect of substratum
roughness on bacterial adhesion. Pits and appendages were
considered to provide bacteria place of reduced fluid shear
(Bryers, 2000).
21
2.1
Production of raw sugar (Malaysian Standard, 1973).
33
2.2
Schematic diagram of laboratory scale bioreactor for Cr
(VI) Reduction System
2.3
39
Schematic diagram of experimental setup for Cr (VI)
reduction system: 1-mixing tank, 2a- external carbon
source: molasses, 2b- Cr(VI) wastewater 3- peristaltic
pump, 4- bioreactor, 5- biological treated effluent, 6coagulation/flocculation tank 6a- Alum solution, 6bpolyacrylamide,
7-
sedimentation
(activated carbon/ sand)
tank
8-
filtration
40
xvi
2.4
A. haemolyticus grown in different concentrations of
molasses (0.25-3.25%).
2.5
44
Growth profile of A. haemolyticus in different
concentrations of molasses
2.6
45
Cr(VI) reduction percentage using A. haemolyticus grown in
varying molasses concentration.
2.7
47
Profile for Cr(VI) reduction by the bioreactor; peaks
indicate initial Cr(VI) concentration; bullets in – between
peaks indicate number of cycles needed to complete the
Cr(VI) reduction process for each batch of wastewaters.
2.8
49
Cr (VI) reduction profile by the biofilm system at Cr (VI)
solutions containing 50-200 mg/L using glucose as a carbon
source.
2.9
50
Effluent from different ports; 1: mixing tank, 5: treated
effluent from Cr(VI)-reducing bioreactor, 6: effluent from
precipitation process.
52
3.1
Pineapple canning process
55
3.2
Laboratory-scale bioreactor setup for Cr(VI) reduction
process
60
3.3
Evaluation of dislodging methods
68
3.4
Distribution
of
A.haemolyticus
cells
in
down-flow
bioreactor.
3.5
Effect of nutrient supplementation on the number of
immobilized cells in the bioreactor
3.6
69
70
Normal probability plot of residuals, residuals vs. predicted
response and predicted vs. actual and outlier T for % Cr(VI)
removal.
3.7
76
3D plot (a) and Contour plot (b) showing effect of initial
Cr(VI) concentration and flowrate at fixed nutrient
supplementation (15 %) on the Cr(VI) percentage removal
3.8
77
3D plot (a) and Contour plot (b) showing effect of flowrate
and nutrient suplementation at fixed Cr(VI) concentration
(70 mg/L) on the Cr(VI) percentage removal.
79
xvii
3.9
3D plot (a) and Contour plot (b) showing effect of nutrient
supplementation and Cr(VI) concentration at fixed flowrate
(4.5 mL/min) on the Cr(VI) percentage removal
80
4.1
Schematic diagram of Cr(VI) removal system.
85
4.2
Profile for Cr(VI) reduction by the bioreactor; peaks
indicate initial Cr(VI) concentration; bullets in – between
peaks indicate number of cycles needed to complete the
Cr(VI) reduction process for each batch of wastewaters.
4.3
Treated effluent after coagulation/flocculation techniques
using A: Lime/ polymer and B: Alum/polymer
4.4
93
FESEM micrographs of biofilm development a) at 0.45 h b)
24 h c) 48h d) 72 h and e) 98 h.
4.5
89
94
Adherence of A. haemolyticus grown in Nutrient broth to
the walls of plastic tubes. The test tubes were emptied and
stain with safranin. A) A. haemolyticus grown in NB,
duplicates sample and control: NB B) Slime-producing of
A.haemolyticus in NB.
4.6
95
FESEM micrographs of biofilm development in the
bioreactor during the Cr(VI) reduction at day A) 30 B) 60
and C) 90.
97
xviii
LIST OF ABBREVIATIONS
mg
-
miligram
g
-
gram
mL
- mililiter
L
- liter
mg/L
- miligram per liter
g/L
- gram per liter
ICP-MS
- Inductively coupled plasma-mass spectrometry
AAS
-
Atomic Absorption Spectrophotometer
FESEM
-
Field Emission Scanning Electron Microscopy
EDAX
-
Energy Dispersive X-ray
NB
-
Nutrient Broth
NA
-
Nutrient agar
TSS
-
Total Suspended Solid
SS
-
Suspended Solid
COD
-
Chemical Oxygen Demand
NaOH
- Sodium Hydroxide
Cr(VI)
-
Cr(III)
- Trivalent Chromium
A.haemolyicus
- Acinetobacter haemolyticus
LPW
- Liquid Pineapple Waste
EW
-
Electroplating Wastewater
ND
-
Not detected
REL
-
Recommended Exposure Limit
TLV
-
Threshold Limit Value
OSHA
-
Occupational Safety and Health Administration
REL
- Recommended Exposure Limit
Hexavalent Chromium
xix
ACGIH
- American Conference of Industrial Hygenists
TLV
-
Threshold Limit Value
OSHA
-
Occupational Safety and Health Administration
PEL
-
Permissible Exposure Level
TWA
-
Time weighted average
NIOSH
-
National Institute for Occupational Safety and Health
TSS
-
Total suspended solids
CFU
-
Colony forming unit
H2SO4
-
Sulphuric acid
HCl
- Hydrochloric acid
v/v
-
Volume per volume
OD
-
Optical density
OD600
-
Optical density at 600 nm
0
-
Degree celsius
-
Response Surface Methodology
-
1,5- diphenylcarbazide
-
milliliter per minute
EW
-
Electroplating Wastewater
ppm
-
part per million
C
RSM
DPC
mL min
-1
xx
LIST OF APPENDICES
APPENDIX
TITLE
A
List of publication (journal/ article), awards and
seminar/ paper presentation during Msc study period
between June 2008 to January 2011
PAGE
113