vii
TABLE OF COTET
CHAPTER
I
TITLE
PAGE
DECLARATIO
ii
DEDICATIO
iii
ACKOWLEDGEMETS
iv
ABSTRACT
v
ABSTRAK
vi
TABLE OF COTETS
vii
LIST OF TABLES
xiv
LIST OF FIGURES
xvi
LIST OF ABBREVIATIOS
xxi
LIST OF APPEDICES
xxiv
ITRODUCTIO
1.1
The scenario of the electrical and electronic
1
sector in Malaysia
1.1.1 The Printed Circuit Board Industry
1.1.1.1 Printed circuit board manufacturing
3
3
process
1.1.1.2 Printed circuit board waste
6
description
1.1.1.3 Current treatment employed by the
8
PCB industry
1.2
Metal and its role
10
viii
1.3
1.2.1 Copper essentiality and toxicity
11
1.2.2 Nickel essentiality and toxicity
14
Alternative technology for heavy metal removal
16
1.3.1 Biological waste material as adsorbent
16
1.3.2 Microorganism as biosorbent
18
1.3.2.1 Mechanism of metal uptake by
20
microorganism
1.3.2.2 Kinetic of biosorption
20
1.3.2.3 Advantages and disadvantages
23
of biosorption process
1.3.3 Preparation of biosorbent
25
1.3.4 Medium formulation and supplementation
25
1.3.5 Application of immobilized cells in
30
biosorption process
II
1.4
Objective of Thesis
36
1.5
Scope of Thesis
36
CULTIVATIO OF BACTERIA I CHEAP
GROWTH MEDIUM AD ITS TOLERACE
LEVEL TOWARDS Cu(II) and i(II) IOS
2.1
Introduction
37
2.1.1 Growth medium for cultivation of
37
bacteria
2.2
2.1.2 Liquid pineapple waste
39
Materials and methods
41
2.2.1 Bacteria
41
2.2.2 Liquid pineapple waste (LPW)
41
2.2.3 Brown sugar
42
2.2.3.1 Sugar determination – Dubois
43
Method
2.2.3.2 Total Nitrogen Test
44
2.2.3.3 Determination of metal content
44
using ICP-MS and pH
ix
2.2.4 Growth of bacteria in LPW
2.2.4.1 Bacterial growth in LPW and LPW
45
45
added with Cu(II) and Ni(II)
2.2.4.2 Cu(II) and Ni(II) tolerance of
45
bacteria in LPW
2.2.4.3 Survival of mixed bacterial
46
consortium in LPW treated with
ethanol
2.2.5 Growth of bacteria in brown sugar
2.2.5.1 Effect of brown sugar
47
47
sterilisation on growth of
Acinetobacter haemolyticus
2.2.5.2 Growth of Acinetobacter
48
haemolyticus in different
concentrations of brown sugar
2.2.5.3 Growth of A. haemolyticus in
48
brown sugar supplemented with
different nitrogen sources
2.2.5.4 Growth of A. haemolyticus in
49
brown sugar supplemented with
amino acid as organic nitrogen
source
2.3
Results and discussions
50
2.3.1 Characterization of brown sugar
50
2.3.2 Bacterial growth in LPW
51
2.3.2.1 Growth profile of bacteria in
51
LPW
2.3.2.2 Bacterial growth in LPW
53
supplemented with Cu(II) and
Ni(II)
2.3.2.3 Bacterial tolerance towards Cu(II)
55
and Ni(II)
2.3.2.4 Survival of bacteria in ethanol-
58
x
treated LPW
2.3.3 Bacterial growth in brown sugar (BS)
2.3.3.1 Effect of brown sugar sterilisation
59
59
on growth of A. haemolyticus
2.3.3.2 Growth of A. haemolyticus in
61
different concentrations of brown
sugar
2.3.3.3 Growth of A. haemolyticus in
63
brown sugar supplemented with
different nitrogen sources
2.4
III
Conclusion
67
REMOVAL OF Cu(II) AD i(II) FROM
WASTEWATER USIG RUBBER WOOD
SHAVIGS
3.1
Introduction
69
3.2
Materials and methods
70
3.2.1 Adsorbent
70
3.2.2 Reagents
71
3.2.3 Buffers
71
3.2.4 Wastewater from the electronic industry
71
(EW)
3.2.5 The Cu(II) and Ni(II) uptake by RWS
3.2.5.1 Effect of heat and chemical
73
73
treatments
3.2.5.2 Effect of contact time, initial metal
73
concentration and adsorbent dosage
3.2.6 Cu(II) and Ni(II) uptake by RWS from
74
EW
3.2.7 Metal uptake determination
3.3 Results and discussion
75
75
3.3.1 Effect of RWS pretreatment
75
3.3.2 Effect of contact time
78
xi
3.3.3 Effect of initial metal concentration
80
3.3.4 Effect of adsorbent dosage
88
3.3.5 Cu(II) and Ni(II) uptake by RWS from
91
EW
3.4 Conclusion
IV
92
REMOVAL OF Cu(II) AD i(II) FROM
WASTEWATER USIG STROTIUM
ALGIATE IMMOBILIZED Acinetobacter
haemolyticus
4.1 Introduction
94
4.2 Materials and Method
95
4.2.1 Preparation of A. haemolyticus biomass
95
4.2.2 Preparation of Cu(II) and Ni(II) stock
95
solution
4.2.3 Buffers
96
4.2.4 Immobilization of bacteria in alginate
96
4.2.5 Biosorption of Cu(II) and Ni(II) using
97
strontium alginate immobilized bacteria
4.2.5.1
Effect of different form of
97
bacterial biomass immobilized
in strontium alginate
4.2.5.2
Effect of different biomass
98
loading in alginate
4.2.5.3
Effect of bead’s concentration
99
4.2.5.4
Effect of contact time and initial
99
metal concentration
4.2.5.5
Biosorption of Cu(II) and Ni(II)
100
from EW
4.3 Results and Discussion
4.3.1
Effect of different form of bacterial
biomass immobilized in strontium
alginate
100
100
xii
4.3.2
Effect of different biomass loading in
103
alginate
4.3.3
Effect of bead’s concentration
105
4.3.4
Effect of contact time
107
4.3.5
Effect of initial metal concentration
109
4.3.6
Cu(II) and Ni(II) uptake by SAIB using
115
EW
4.4 Conclusion
V
116
ITEGRATIO OF TWO STAGE
BIOSORPTIO PROCESS AD SURFACE
STUDY O METAL–BACTERIA
ITERACTIO
5.1 Introduction
117
5.2 Materials and Method
118
5.2.1
Sampling and characterization of
118
electronic wastewater
5.2.2
Integration of two–stage biosorption
118
system using RWS and SAIB
5.2.2.1
Two stage Removal of Cu(II)
119
and Ni(II) using simulated
effluent
5.2.2.2
Two Stage Removal of Cu(II)
119
and Ni(II) using EW
5.2.2.3
Single Stage Removal of Cu(II)
119
and Ni(II) by RWS and SAIB
from EW
5.2.3
Characterization of bacteria-metal
120
interaction
5.2.3.1
FTIR Analysis
120
5.2.3.2
Field–Emission Scanning
120
Electron Microscope Analysis
5.3 Results and Discussion
121
xiii
5.3.1
EW Characteristics
121
5.3.2
Integration of two stage biosorption
122
system using RWS and SAIB
5.3.3
Characterization of metal-bacteria
125
interaction
5.3.3.1
FTIR Analysis
125
5.3.3.2
Scanning Electron Microscope
128
Analysis
5.4 Conclusion
VI
131
COCLUSIO
6.1 Conclusion
132
6.2 Suggestions for future work
133
REFERECES
135
APPEDICES
149
xiv
LIST OF TABLES
TABLE O.
1.1
TITLE
PAGE
Types of waste streams generated in the Printed Circuit
7
Board Manufacturing Industry
1.2
Discharge limit of sewage and industrial effluents
8
1.3
Example of copper-containing enzymes and their
13
function
1.4
Example of nickel-containing enzymes and their
15
function
1.5
The different types of adsorbent used for metal removal
17
1.6
Advantages and disadvantages of biosorption by non-
24
living biomass
1.7
Elemental composition of microbial cell
28
1.8
Recent application of cell entrapment in the metal
34
removal studies
2.1
The composition of 200 g L-1 brown sugar
50
2.2
The number of bacterial colonies after growing for 12
58
hours in LPW treated with 5% (v/v)
2.3
Effect of different sterilization techniques of brown
59
sugar on growth of A. haemolyticus
3.1
Some characteristics for the *EW from Johor Bahru,
72
Johor
3.2
Equilibrium constants for the single metal uptake of
85
Cu(II) and Ni(II) by NaOH–treated RWS
3.3
Equilibrium constants for the uptake of Cu(II) in
88
xv
mixed–metal solution by RWS
4.1
Calculated equilibrium constant for adsorption of
112
single–metalof Cu(II) and Ni(II) on SAIB
4.2
Calculated equilibrium constant for adsorption of
114
mixed-metal of Cu(II) and Ni(II) on SAIB
5.1
Characteristics of *EW
121
5.2
Changes of the FTIR bands of A.haemolyticus before
125
and after biosorption of Cu(II)
5.3
Changes of the FTIR bands of A.haemolyticus before
127
and after biosorption of Ni(II)
5.4
Gross composition of bacterial cells (Naumann, 2000)
127
5.5
EDAX analysis of the surface of A. haemolyticus cell
131
after exposure to 200 mg L-1 Cu(II) and Ni(II)
xvi
LIST OF FIGURES
FIGURE O.
1.1
TITLE
The process sequence of Printed Circuit Board
PAGE
4
Manufacturing
1.2
Principle of heavy metal precipitation (Khandpur,
9
2006)
1.3
Techniques of cell immobilization (Kourkoutas et al.,
31
2004)
1.4
The chemical structure of alginate. M, β-D-mannuronic
33
acid; G, α-L-guluronic acid (Rehm, 2006)
1.5
Alginate gel formation and cross-linking with Ca2+
35
ions. Gray oxygen atoms of guluronic acid (G) are
interacting with Ca2+ ions. M, β-D-mannuronic acid
(Rehm, 2006)
2.1
Waste processing in a pineapple canning plant located
40
in Skudai, Johor; A – waste from the production, B –
waste grinding process, C – extraction of juice from
grinded solid waste, D – liquid waste collection tank, E
– 2nd stage grinding process, F – small pieces of solid
waste after grinding process
2.2
Collection of LPW from the waste processing section
42
of the pineapple canning plant, Skudai; A – drain, B –
sedimentation tank
2.3
Growth profile of A.calcoaceticus, C.agropyri and
52
xvii
C.cellulans in LPW in the absence of metal
2.4
Colonies of C. cellulans on NA plates during the 24
53
hours growth phase
2.5
Growth profile of bacteria in LPW in the presence of
54
100 mg L-1 Cu(II), (◊) C. agropyri, (■) A.
calcoaceticus, (▲) C. cellulans, (×) A. haemolyticus
and (♦) Mixed culture
2.6
Growth profile of bacteria in LPW in the presence of
54
50 mg L-1 Ni(II), (◊) C. agropyri, (■) A. calcoaceticus,
(▲) C. cellulans, (×) A. haemolyticus and (♦) Mixed
culture
2.7
Growth of bacteria in LPW containing Cu(II); CC - C.
56
cellulans, AH - A. haemolyticus, AC - A. calcoaceticus,
CA - C. agropyri, Mixture - A. calcoaceticus, C.
agropyri and C. cellulans
2.8
Growth of bacteria in LPW containing Ni (II)
57
2.9
Growth of A. haemolyticus in different concentrations
61
of brown sugar; OD B4 – OD600 before incubation, OD
After - OD600 after 7 hours of incubation, CFU –
colony forming unit after 7 hours of incubation
2.10
Growth of A. haemolyticus in 25 g L-1 brown sugar
65
supplemented with different nitrogen sources; S1 – BS
supplemented with NH3, S2 – BS supplemented with
(NH4)2SO4, S3 - BS supplemented with NH4NO3, S4 BS supplemented with tryptone, S5 - BS only
2.11
Viable cell count of A. haemolyticus in 25 g L-1 brown
66
sugar supplemented with different nitrogen sources; S1
– BS supplemented with NH3, S2 – BS supplemented
with (NH4)2SO4, S3 – BS supplemented with NH4NO3,
S4 – BS supplemented with tryptone, S5 – BS only.
Data are mean value ± standard deviation of duplicate
experiment
2.12
Viable cell count of A. haemolyticus in 25 g L-1 brown
67
xviii
sugar supplemented with different amino acids as
organic nitrogen sources; S6 – BS supplemented with
methionine and S7 – BS supplemented with arginine.
Data are mean value ± standard deviation of duplicate
experiment
3.1
Thin rubber wood shavings (RWS) collected from
70
wood – finishing factory, Skudai.
3.2
Cu(II) and Ni(II), 50 mg L-1 uptake from single and
76
mixed metal solutions by the untreated, heat-treated,
CaCl2-treated and NaOH-treated RWS, 1.5 % (w/v).
The bars represent standard deviation of the mean, n=2
3.3
Speciation of Cu(II) as a function of pH (Elliot and
77
Huang, 1981)
3.4
Effect of contact time on the uptake of 50 mg L-1
79
Cu(II) and Ni(II) by 1.5 % (w/v) RWS, (a) single
metal solution (b) mixed metal solution, (♦) Cu(II), (■)
Ni(II). The bars represent standard deviation of the
mean, n=2
3.5
Uptake of (a) Cu(II) and (b) Ni(II) from single–metal
83
solutions by NaOH–treated RWS. Bar indicates
standard error of the mean, n = 2
3.6
Linearized (a) Langmuir adsorption isotherm (b)
84
Freundlich adsorption isotherm of single-metal of
Cu(II) and Ni(II) by rubber wood shavings
3.7
Uptake of (a) Cu(II) and (b) Ni(II) from mixed–metal
86
solutions by NaOH–treated RWS. Bar indicates
standard error of the mean, n = 2
3.8
Linearized (a) Langmuir adsorption isotherm (b)
87
Freundlich adsorption isotherm of mixed-metal of
Cu(II) and Ni(II) by rubber wood shavings
3.9
Linearized BET adsorption isotherm of mixed-metal of
88
Ni(II) by rubber wood shavings
3.10
The uptake of (a) Cu(II) and (b) Ni(II) in single–metal
89
xix
solutions by varying doses of RWS; (♦) % removal (■)
uptake. The bars represent standard deviation of the
mean, n=2
3.11
The uptake of (a) Cu(II) and (b) Ni(II) in mixed–metal
91
solutions by varying doses of RWS; (♦) % removal (■)
uptake. The bars represent standard deviation of the
mean, n=2
3.12
Cu(II) and Ni(II) uptake from EW using RWS. The
92
bars represent standard deviation of the mean, n=2
4.1
Effect of different form of bacterial biomass
101
immobilized in strontium alginate, SAIBD – dried Sralginate immobilized bacterial suspension, SAIBW –
wet Sr-alginate immobilized bacterial suspension, SAD
– dried Sr-alginate, SAW – wet Sr-alginate, SAIPD –
dried Sr-alginate immobilized bacterial pellet and
SAIPW – wet Sr-alginate immobilized bacterial pellet
4.2
The (a) Cu(II) and (b) Ni(II) uptake profiles in single
104
and mixed metal solution using Sr-alginate bead
immobilized with different biomass concentration,
Cu(II) and Ni(II) – 25 mg L-1, FC – free cells and
SAIB – Sr-alginate immobilized bacteria. The bars
represent standard deviation of the mean, n=2
4.3
The (a) Cu(II) and (b) Ni(II) uptake profiles in single
106
metal solution at varying SAIB concentrations, Cu(II)
and Ni(II) – 25 mg L-1, bars represent standard
deviation of the mean, n=2
4.4
Profiles for the (a) Cu(II) and (b) Ni(II) uptake from
107
mixed metal solution at varying SAIB concentrations,
Cu(II) and Ni(II) – 25 mg L-1, bars represent standard
deviation of the mean, n = 2
4.5
Effect of contact time on Cu(II) and Ni(II) uptake
from; (a) single metal solution (b) mixed metal
solution, using SAIB; (♦) Cu(II), (■) Ni(II). The bars
108
xx
represent standard deviation of the mean, n=2
4.6
Adsorption isotherm of (a) Cu(II) and (b) Ni(II) from
110
single metal solution using SAIB. Bar indicates
standard error of the mean, n=2
4.7
Linearized (a) Langmuir adsorption isotherm (b)
111
Freundlich adsorption isotherm of single-metal of
Cu(II) and Ni(II) by SAIB
4.8
Adsorption isotherm of (a) Cu(II) and (b) Ni(II) from
113
mixed metal solution using SAIB. Bar indicates
standard error of the mean, n=2
4.9
Linearized (a) Langmuir adsorption isotherm (b)
114
Freundlich adsorption isotherm of mixed-metal of
Cu(II) and Ni(II) by SAIB
4.10
Cu(II) and Ni(II) uptake from PCBW using SAIB. The
116
bars represent standard deviation of the mean, n=2
5.1
Removal of Cu(II) and Ni(II) from simulated effluent
123
using integration of RWS and SAIB
5.2
Removal of Cu(II) and Ni(II) from EW using
123
integration of RWS and SAIB
5.3
Removal of Cu(II) and Ni(II) from EW using RWS and
124
SAIB as a single stage system
5.4
SEM micrographs of inactive (autoclaved) A.
haemolyticus cell: before biosorption (A1 and A2),
after Cu(II) biosorption (B1 and B2) and after Ni(II)
biosorption (C1 and C2) with magnification of 10 K
and 25 K
129
xxi
LIST OF ABBREVIATIOS
Å
-
Ångström (1 × 10-10 metre)
A. calcoaceticus
-
Acinetobacter calcoaceticus
A. haemolyticus
-
Acinetobacter haemolyticus
AAS
-
Atomic Absorption Spectroscopy
ATTC
-
American Type Culture Collection
b
-
constant related to the affinity of binding site
C. agropyri
-
Clavibacter agropyri
C. cellulans
-
Cellulosimicrobium cellulans
CaCl2
-
calcium chloride
Ceq
-
final (equilibrium) metal concentration (mg L-1)
CFU
-
colony forming unit
Ci
-
initial metal concentration
COD
-
chemical oxygen demand
Cr(VI)
-
chromium(VI)
Cu(II)
-
copper(II)
DNA
-
deoxyribonucleic acid
DSMZ
-
German collection of microorganisms and cell cultures
EDAX
-
Energy Dispersive X-ray
EDS
-
Electron Dispersive Spectroscopy
EW
-
electronic wastewater
FESEM
-
Field-Emission Scanning Electron Microscope
FTIR
-
Fourier-transform Infrared
g L-1
-
gram per liter
H2SO4
-
sulphuric acid
HCl
-
hydrochloric acid
xxii
HNO3
-
nitric acid
ICP-MS
-
Inductive Coupled Plasma-Mass Spectrometry
k
-
relative adsorption capacity
KBr
-
Potassium bromide
LPW
-
liquid pineapple waste
M
-
molar
mg g-1
-
milligram per gram
mg L-1
-
miligram per liter
MIC
-
minimum inhibitory concentration
mm
-
millimeter
mM
-
millimolar
n
-
affinity constant
NA
-
nutrient agar
NaOH
-
sodium hydroxide
NB
-
nutrient broth
Ni(II)
-
nickel(II)
ºC
-
degree Celsius
OD
-
optical density
OD410
-
optical density at 410 nm
OD485
-
optical density at 485 nm
OD600
-
optical density at 600 nm
PIPES
-
Piperazine - N,N’ - bis (2 - ethanesulfonic acid)
qe
-
uptake at equilibrium
-
maximum uptake at equilibrium
RNA
-
ribonucleic acid
rpm
-
rotation per minute
RWS
-
rubber wood shaving
SA
-
strontium alginate
SAD
-
dried strontium alginate
SAIB
-
strontium alginate immobilized bacterial suspension
SAIBW
-
wet strontium alginate immobilized bacterial
qe,
max
suspension
SAIP
-
strontium alginate immobilized bacterial pellet
xxiii
SAIPD
-
dried strontium alginate immobilized bacterial
suspension
SAIPD
-
dried strontium alginate immobilized bacterial pellet
SAIPW
-
wet strontium alginate immobilized bacterial pellet
SAW
-
wet strontium alginate
str.
-
stretching
USEPA
-
United States Environmental Protection Agency
v/v
-
volume per volume
w/v
-
weight per volume
w/w
-
weight per weight
XAFS
-
X-ray Absorption Fine Structure
XPS
-
X-ray Photoelectron Spectroscopy
µm
-
micrometer
xxiv
LIST OF APPEDICES
APPEDIX
A
TITLE
List of publications (journal/article), awards and
PAGE
148
seminar/paper presentation during MSc. study period
between July 2006 to Jun 2009
B
ICP-MS analysis of brown sugar (100× dilution)
151
C1
Elemental balances using NH3 as nitrogen source
152
C2
Elemental balances using ammonium salt as nitrogen
154
source
D
ICP-MS analysis of 1st sampling electronic
156
wastewater (EW)
E1
FTIR spectra of A.haemolyticus before and after
157
biosorption of Cu(II)
E2
FTIR spectra of A.haemolyticus before and after
158
biosorption of Ni(II)
F1
EDAX analysis of inactive (autoclaved) A.
159
haemolyticus cell: before biosorption
F2
EDAX analysis of inactive (autoclaved) A.
160
haemolyticus cell after Cu(II) biosorption
F3
EDAX analysis of inactive (autoclaved) A.
161
haemolyticus cell after Ni(II) biosorption
G
ICP-MS analysis of 2nd sampling electronic
wastewater (EW)
162