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REMOVAL OF OIL AND GREASE FROM AGRO-FOOD INDUSTRIAL
EFFLUENT USING Serratia marcescens SA30 AND ITS KINETIC STUDY
SHAKILA BT ABDULLAH
UNIVERSITI TEKNOLOGI MALAYSIA
REMOVAL OF OIL AND GREASE FROM AGRO-FOOD INDUSTRIAL
EFFLUENT USING Serratia marcescens SA30 AND ITS KINETIC STUDY
SHAKILA BINTI ABDULLAH
A thesis submitted in fulfilment of the
requirements for the award of the degree of
Degree of Philosophy (Civil Engineering)
Faculty of Civil Engineering
Universiti Teknologi Malaysia
JUNE 2016
iii
This thesis is dedicated to:
Special dedication to my beloved father and mother, Abdullah bin Ibrahim
and Hasnah bt. Mohd Yatim, who have been with me throughout the hard time of my
study. Thanks for the du’a and prayer, guidance, continuous support and tremendous
of love throughout my course of study.
For my beloved auntie, Hajah Samsiah bt. Ibrahim, Norashikin bt.Mohd
Yatim and Sarina bt. Mohd Yatim, thanks for the du’a, advices and constant support
for every step of my PhD.
and lastly
Special thanks to my best friends Wan Haslinda bt. Wan Ahmad and Azila bt. Md
Sudin who always keep my spirit in many moments of crisis, her encouragement and
helping me to succeed and instilling in me the confidence to be a strong and capable
person.
iv
ACKNOWLEDGEMENTS
In the name of Allah S.W.T, the Most Gracious, the Most Merciful,
Alhamdulillah and thanks to Allah, He who has given me the strength and patience to
complete this research.
I would like to express my heartiest gratitude goes to my supervisors: Assoc.
Prof. Dr. Mohamad Ali Fulazzaky and Prof. Dr. Mohd Razman Salim for his
willingness to help, listen and assist in every way, in the midst of his heavy
responsibilities and duties and thank you for the advice, guidance, ideas, criticism
and encouragement throughout the research study.
A special note of thanks and appreciation to all staff in Centre for
Environmental Sustainability & Water Security (IPASA), Research Institute of
Sustainable Environment (RISE), Resources Sustainability Research Alliance,
Environmental Engineering, Faculty of Civil Engineering, Chemistry Department,
Faculty of Science, UTM, Chemistry Department, Faculty of Science, UM and
Institute Medical Research for allowing me to use their laboratory facilities.
Last but not least, I would like to acknowledge and thank to my research
colleagues: Hairul, Mimi, Irena, Budi, Ain, Syahrul, Shikin, Thana, Maria, Anis,
Hudai and Biotechnology lab members for their constructive comments and constant
support.
v
ABSTRACT
Agro-food industrial effluent (AFIE) may contain high concentration of oil
and grease (O&G), which poses a major threat to aquatic environments, killing or
adversely affecting fish and other aquatic organisms. Even though biosorption
techniques are commonly used to remove inorganic and organic matters from
wastewater, the kinetics and mechanisms of O&G removal from AFIE by Serratia
marcescens SA30 immobilised in a packed-bed column reactor (PBCR) need to be
verified. The aims of this study were to perform characterisation of beneficial strain
of biosurfactant-producing bacteria in order to investigate their ability to remove
O&G from water, to develop kinetic models for predicting the efficiency of O&G
removal from AFIE and to apply modified mass transfer factor models for assessing
the mechanisms and mass transfer resistance for the biosorption of O&G from AFIE
by Serratia marcescens SA30. The performance of PBCR achieved 91% of
efficiency using Serratia marcescens SA30 as oil-degrading bacteria. The best
performance of nearly 100% efficiency can be achieved by experiments run at a
fixed volumetric flow rate of 0.18 L h-1
, even during treatment using two different
concentrations of O&G at 26.9 and 33.5 g L-1
to feed the reactor. The results show
the applicability of linear and logarithmic equations with high validity. The
resistance to mass transfer could be dependent on intracellular accumulation at the
beginning and then on film mass transfer at the final stage of O&G biosorption by
Serratia marcescens SA30. The well verified experimental data of kinetic models
and mass transfer mechanisms give significant contributions to the development of
biosorption theory and an insight of using new approaches to improve environmental
quality. This study would provide a green and sustainable pathway for removing
O&G from water.
vi
ABSTRAK
Efluen perindustrian agro-makanan (AFIE) mungkin mengandungi kepekatan
minyak dan gris (O&G) yang tinggi dan boleh memberikan ancaman besar kepada
persekitaran akuatik, membunuh atau memberi kesan buruk kepada ikan dan
organisma akuatik yang lain. Walaupun teknik biopenjerapan sering digunakan untuk
menyingkirkan bahan tak organik dan organik daripada air sisa, kinetik dan
mekanisma penyingkiran O&G daripada AFIE oleh Serratia marcescens SA30 yang
dipegunkan dalam reaktor turus terpadat tunggal (PBCR) perlu disahkan. Tujuan
kajian ini adalah untuk melakukan pencirian ke atas bakteria pengeluar biosurfaktan
bagi menyiasat keupayaan bakteria tersebut untuk menyingkirkan O&G dari air,
untuk membina model kinetik bagi menganggarkan kecekapan penyingkiran O&G
daripada AFIE dan menggunakan model faktor pemindahan jisim yang telah
diubahsuai untuk menilai mekanisma dan rintangan pemindahan jisim bagi
biopenjerapan O&G daripada AFIE oleh Serratia marcescens SA30. Prestasi PBCR
boleh mencapai 91% kecekapan menggunakan Serratia marcescens SA30 sebagai
bakteria pendegradasi minyak. Prestasi terbaik dengan kecekapan hampir 100%
boleh dicapai apabila eksperimen dijalankan pada kadar alir isipadu tetap 0.18 L h-1
,
walaupun pada rawatan menggunakan dua kepekatan O&G berbeza pada 26.9 dan
33.5 g L-1
untuk ditambah ke dalam reaktor. Keputusan menunjukkan kebolehgunaan
persamaan linear dan logaritma dengan kesahihan yang tinggi. Rintangan
pemindahan jisim boleh bergantung kepada pengumpulan intrasel pada permulaan
dan kemudian pada pemindahan jisim filem di peringkat akhir biopenjerapan O&G
oleh Serratia marcescens SA30. Data eksperimen model kinetik dan mekanisma
pemindahan jisim yang telah disahkan boleh memberikan sumbangan yang besar
kepada pembanggunan teori biopenjerapan dan memberi pandangan menggunakan
pendekatan baru untuk meningkatkan kualiti alam sekitar. Kajian ini akan
memberikan laluan hijau dan lestari untuk menyingkirkan O&G dari air.
vii
TABLE OF CONTENTS
CHAPTER TITLE PAGE
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENT iv
ABSTRACT v
ABSTRAK vi
TABLES OF CONTENTS vii
LIST OF TABLES xiii
LIST OF FIGURES xv
LIST OF ABBREVIATIONS xx
LIST OF SYMBOLS xxi
LIST OF APPENDICES xxiv
1 INTRODUCTION
1
1.1 Background 1
1.2 Problems Statement 2
1.3 Objectives 4
1.4 Scope of the study 4
1.5 Significance of the study 6
1.6 Thesis outline 7
viii
2 LITERATURE REVIEW 8
2.1 Industrial wastewater 8
2.1.1 Food processing industrial wastewater 9
2.1.2 Discharged regulations for oil and grease in
several countries
11
2.2 Oil and grease 13
2.2.1 Type and classification of oil-water mixture 15
2.2.2 Oily wastewater 15
2.2.3 Impacts of oily wastewater on the
environment
17
2.3 Oil-degrading bacteria 18
2.3.1 Biosurfactant 18
2.4 Supporting materials 27
2.5 Oily wastewater treatment processes 28
2.5.1 Physical treatment processes 28
2.5.1.1 Filtration 28
2.5.1.2 Flotation 31
2.5.1.3 Adsorption 32
2.5.2 Physico-chemical treatment processes 34
2.5.2.1 Coagulation 34
2.5.2.2 Ozonation 35
2.5.3 Biological treatment processes 36
2.5.3.1 Anaerobic Digestion 36
2.5.3.2 Aerobic digestion 37
2.5.3.3 Biosorption 44
2.5.3.3 Advanced biological process 47
2.6 Kinetic and mass transfer models 56
2.6.1 Adsorption kinetic models 56
2.6.2 Growth kinetic models 58
2.6.3 Mass transfer factor models 59
3 MATERIALS AND METHODS 63
3.1 Materials 63
ix
3.1.1 Glassware and apparatus 63
3.1.2 Growth media 63
3.1.2.1 Nutrient broth medium 63
3.1.2.2 Nutrient agar medium 64
3.1.2.3 Tween peptone agar medium 64
3.1.2.4 Blood agar medium 64
3.1.2.5 Basal salts medium 64
3.1.2.6 Oil agar medium 65
3.1.2.7 Luria bertani (LB)-glycerol stock
solution
65
3.1.2.8 Buffer solution 65
3.1.2.9 Active culture in Erlenmeyer
flask
66
3.1.2.10 Bacterial growth on nutrient agar
plate
66
3.2 Agro-food industrial effluent 67
3.2.1 Sampling locations and procedure 67
3.2.2 Characterisation of agro-food industrial
effluent
68
3.2.2.1 Determination of oil and grease 68
3.3 Isolation of oil-degrading bacteria 69
3.3.1 Isolation of single colony 69
3.3.2 Physico-chemical test for characterising
oil-degrading bacteria
69
3.3.2.1 Zeta potential test 71
3.4 Identification of bacterial gene using 16s rRNA
gene sequence analysis
71
3.5 Batch experiments for optimisation of
environmental conditions
71
3.5.1 Bacterial acclimatisation with synthetic
oily-wastewater
71
3.5.2 Test of oil and grease removed from
synthetic-oily wastewater
72
x
3.5.3 Bacterial adaptation with agro-food
industrial effluent
73
3.5.4 Test of oil and grease removed from agro-
food industrial effluent
73
3.6 Microscopic identification for Serratia marcescens
SA30
74
3.6.1 Scanning electron microscopy 74
3.6.2 Transmission electron microscopy 74
3.7 Packed-bed column reactor 75
3.7.1 Supporting materials 75
3.7.2 Characterisation of supporting materials 75
3.7.3 Packed-bed column reactor and
experimental procedure
75
3.7.4 Experimental runs by packed-bed column
reactor
77
4 DEGRADATION OF OIL AND GREASE FROM
AGRO-FOOD INDUSTRIAL EFFLUENT
78
4.1 Overview 78
4.2 Characteristics of the agro-food industrial effluent 79
4.2.1 Composition of fatty acids in the agro-food
industrial effluent
81
4.3 Morphological features of biosurfactant-producing
bacteria
84
4.4 Results of screening the beneficial oil-degrading
bacteria
86
4.4.1 Growth and pH profiles of the isolated
bacteria
86
4.4.2 Bacterial growth in minimal medium 88
4.4.3 Opaque halo formation 89
4.4.4 Microbial adherence to hydrocarbon 90
4.5 Characteristics of biosurfactant-producing bacteria 93
4.5.1 Biosurfactant production and formation of
xi
oil droplets 93
4.5.2 Biosurfactant activity 95
4.5.3 Haemolytic activity 96
4.5.4 Emulsification index (E24) 97
4.5.5 Surface tension 97
4.5.6 Zeta potential 98
4.6 Bacterial gene 99
4.7 Environmental condition of batch reactor 101
4.7.1 Synthetic wastewater for conditioning the
experimental runs
100
4.7.2 Agro-food industrial wastewater for
conditioning the experimental runs
111
4.8 Electron microscopy analysis for Serratia
marcescens SA30
119
4.8.1 Analysis of image from scanning electron
microscopy
119
4.8.2 Analysis of image from transmission
electron microscopy
121
4.9 Summary 123
5 DEVELOPMENT OF LINEAR AND LOGARITHMIC
EQUATIONS FOR PREDICTING THE OIL AND
GREASE REMOVAL EFFICIENCY
124
5.1 Overview 124
5.2 Basis of developing the linear and logarithmic
models
124
5.3 Objectives of developing the linear and logarithmic
models
125
5.4 Models Development 127
5.5 Validation of the models 130
5.5.1 Kinetic models of loading rate and
substrate utilisation
130
5.5.2 Logarithmic model for predicting the
xii
PBCR performance 135
5.6 Summary 138
6 APPLICATION OF THE MASS TRANSFER
MODELS FOR ASSESSING THE KINETIC
MECHANISMS FOR THE BIOSORPTION OF OIL
AND GREASE FROM AGRO-FOOD INDUSTRIAL
EFFLUENT BY Serratia marcescens SA30
140
6.1 Overview 140
6.2 Basis of the application of the mass transfer factor
models
140
6.3 Data processing 142
6.4 Application of the mass transfer factor models 145
6.4.1 Linear regression analysis 145
6.4.2 Global mass transfer and kinetics 149
6.4.3 External mass transfer and kinetics 151
6.4.4 Internal mass transfer and kinetics 153
6.5 Summary 156
7 CONCLUSIONS 157
7.1 Conclusions 157
7.2 Recommendations 159
REFERENCES 160
Appendix 196-197
xiii
LIST OF TABLES
TABLE NO. TITLE PAGE
2.1 Environmental Quality (Industrial Effluent) Regulation 2009 12
2.2 Acids of the fats and oils 13
2.3 Acid contents of fats and oils in percent 14
2.4 Types of oil 14
2.5 Concentrations of O&G presented in the different sources of
wastewater 16
2.6 Types of biosurfactant-producing by the microorganisms 20
2.7 Biosurfactant sources and their applications 24
2.8 Carbon sources using vegetable oil 26
2.9 Characteristics of support materials for cell immobilization of
bacteria 28
2.10 Typical characteristics of membrane processes 29
2.11 Oily wastewater treatment by flotation 32
2.12 Comparison of O&G degradation ability of oil-degrading
microorganisms 39
2.13 Comparison of hydrocarbon degradation ability of oil-
degrading microorganisms 42
2.14 Features of biosorption/bioaccumulation 46
2.15 Solid carriers used to immobilise microbial cells for use in
biodegradation 51
3.1 Proportion of monobasic and dibasic solution for specified pH 66
3.2 Water quality parameters for characterisation of the AFIE 68
3.3 Medium conditions for bacterial acclimatisation in synthetic
oily-wastewater 72
3.4 Medium conditions for bacterial adaptation with AFIE 73
xiv
4.1 Main characteristics of the AFIE 80
4.2 Percentage of fatty acid composition in AFIE 82
4.3 Characteristics features of the bacteria colonies isolated from
AFIE 84
4.4 Gram staining 85
4.5 Biosurfactant activity of AF01-O strain and Tween 80 95
4.6 Emulsification index of AF01-O strain and Tween 80 97
4.7 Surface tension for non-adapted and adapted AF01-O strains 98
4.8 Surface charge values of adapted and non-adapted bacteria
AF01-O and biosurfactant production at pH 7 99
4.9 Identification of Serratia marcescens SA30 by 16S rRNA gene
sequence analysis 100
5.1 Variations of O&G concentration in raw and treated AFIE
pursuant to time 126
5.2 Results obtained from the linear regression analysis of plotting
ln(q) versus t 131
5.3 Results obtained from the linear regression analysis of plotting
the accumulation of O&G onto OPF against the accumulation
of O&G loading the PBCR 134
5.4 Results obtained from the logarithmic regression analysis of
plotting E versus t 138
6.1 Values of β and B for different values of C0, obtained from
slope and interception, respectively, of plotting ln(q) versus
ln(t) 145
6.2 Values of α and γ for different values of C0, obtained from
logarithmic slope and biomass yield rate constant, respectively,
of plotting [kLa]g versus ln(Cs/Co)
149
xv
LIST OF FIGURES
FIGURES NO. TITLE PAGE
2.1 Agro-food processing production 10
2.2 Classification and size range of oil droplets 15
2.3 General structure of biosurfactant to form micelle and
reverse micelle 19
2.4 Mechanism of O&G degradation by microorganisms in the
presence of a biosurfactant 22
2.5 Membrane filtration processes 30
2.6 Interaction mechanism between gas bubbles and oil droplets
during flotation 31
2.7 Biosorption mechanisms 44
2.8 Technique of immobilisation 49
2.9 Schematic representation of the steps involved in biofilm
formation 54
2.10 The transport limitations in a diffusion controlled biofilm 55
2.11 Schematic of the flowing model 60
3.1 Sampling points for collection of the AFIE 67
3.2 Schematic diagram of laboratory PBCR 77
4.1 Growth and pH profile of isolated bacteria 86
4.2 OD600 growth in minimal medium 89
4.3 Opaque halo formation of bacterial colony AF01-O; (a)
identified by a streak plate technique (b) identified by a
spread plate technique 90
4.4 Degree of hydrophobicity of a) non-adapted bacteria AF01-
O b) adapted bacteria AF01-O towards organic phase 92
4.5 Production of biosurfactant (a) without added AF01-O strain 94
xvi
at zero-day, (b) with added AF01-O strain at zero-day, (c)
without added AF01-O strain after 7 days, and (d) with
added AF01-O strain after 7 days of incubation
4.6 Clear zone producing by the AF01-O strain in BA medium 96
4.7 Acclimatisation in SOCWW 102
4.8 Effect of different concentration of SOCWW on O&G
removal and CFU mL-1
by Serratia marcescens SA30 (a) 1%
(v/v) (b) 3% (v/v) and (c) 5% (v/v) 105
4.9 Effect of concentration of SOCWW on OD600 measurement
for Serratia marcescens SA30 106
4.10 Effect of time on the removal of O&G for Serratia
marcescens SA30 in SOCWW 106
4.11 Effect of different pH of SOCWW on O&G removal and
CFU mL-1
by Serratia marcescens SA30 (a) pH 5 (b) pH 6
(c) pH 7 and (d) pH 8 109
4.12 Effect of pH on OD600 for Serratia marcescens SA30 110
4.13 Acclimatisation in AFIE 111
4.14 Effect of concentration in AFIE on OD600 by Serratia
marcescens SA30 114
4.15 Effect of different AFIE concentration on O&G removal and
CFU mL-1
by Serratia marcescens SA30 (a) 16424 mg L-1
(b) 25000 mg L-1
(c) 26072 mg L-1
and (d) 33548 mg L-1
. 115
4.16 Effect of time on the removal of O&G by Serratia
marcescens SA30 116
4.17 Effect of different pH of AFIE on O&G removal and CFU
mL-1
by Serratia marcescens SA30 (a) pH 5 (b) pH 6 (c) pH
7 and (d) pH 8
118
4.18 Effect of pH on OD600 by Serratia marcescens SA30 119
4.19 SEM micrographs of (a) Serratia marcescens SA30 without
O&G (control) and (b) Serratia marcescens SA30 in the
presence of O&G; red arrow indicates pore formation and
lysis; yellow arrow indicates formation of sticky 120
4.20 TEM micrographs of Serratia marcescens SA30 growing (a) 122
xvii
without oil droplets (control) and (b) in the presence of oil;
arrow indicates the presence of translucent globules of oil
droplets
5.1 Linear lines of plotting ln(q) versus t to represent the
experimental data 132
5.2 Linear lines of plotting the accumulation of O&G onto
OPF against the accumulation of O&G loading the PBCR to
represent the experimental data 133
5.3 Logarithmic lines of plotting E versus t to represent the
experimental data 136
6.1 Curves of plotting ln(q) versus ln(t), with (1) C0 = 16424 mg
L-1
, (2) C0 = 26072 mg L-1
and (3) C0 = 33548 mg L-1
146
6.2 FTIR spectra of OPF as support materials 148
6.3 Curves of plotting [kLa]g versus Cs/Co ratio, with (a) C0 =
16424 mg L-1
, (b) C0 = 26072 mg L-1
and (c) C0 = 33548 mg
L-1
150
6.4 Curves of plotting (1) [kLa]g, (2) [kLa]f and (3) [kLa]d against
Cs/Co ratio, with (a) C0 = 16424 mg L-1
, (b) C0 = 26072 mg
L-1
and (c) C0 = 33548 mg L-1
152
6.5 TEM images of Serratia marcescens SA30 to show that an
intracellular accumulation of O&G exists within cell
membrane 156
xviii
LIST OF ABBREVIATIONS
ADMI - American Dye Manufacturer Institute
AFIE - Agro-food industrial effluent
ALR’s - Airlift reactors
APHA - Standard Method for Examination of Water and Wastewater
ASTM - American Society for Testing and Materials
BA - Blood agar
BET - Brunauer, Emmett and Teller
BOD - Biochemical oxygen demand
BS - Basal salt
COD - Chemical oxygen demand
CSH - Cell surface hydrophobicity
CSTR - Continuous stirred-tank reactors
DAF - Dissolved air flotation
DispAF - Dispersed-air flotation
DI - Deionised water
DO - Dissolved oxygen
DOE - Department of Environment
DW - Distilled water
E24 - Emulsification index
EGSB - Expanded granular sludge bed
EMT - External mass transfer
EPS - Extracellular polymeric substances
FBR - Fluidised bed reactors
FTIR-ATR - Fourier Transform Infrared Attenuated Total Reflection
GMT - Global mass transfer
IMT - Internal mass transfer
LB - Luria Bertani
xix
MATH - Microbial adherence to hydrocarbon
MEL’s - Manosylerythritol lipids
MF - Microfiltration
MM - Minimal medium
MTF - Modified mass tranfer
NA - Nutrient agar
NB - Nutrient broth
NF - Nanofiltration
OA - Oil agar
OD600 - Optical density at 600 nm
O&G - Oil and grease
OPF - Oil palm frond
O/W - Oil in water
PAH’s - Polycyclic aromatic hydrocarbon
PBCR - Packed-bed column reactor
PCB’s - Polychlorinated biphenyls
PM - Particulate surfactant
PO - Palm oil
RO - Reverse osmosis
SEM - Scanning electron microscopy
SOCWW - Synthetic oily-contaminated wastewater
SS - Suspended solid
TAG - Triacylglycerol
TDS - Total dissolved solids
TEM - Transmission electron microscopy
TOC - Total organic carbon
TPA - Tween peptone agar
Tween 80 - Oleic acid monoester of polyoxyethylene sorbitan
UASB - Upflow anaerobic sludge blanket
UF - Ultrafiltration
USEPA - United States Environmental Protection Agency
VFR - Volumetric flow rate
W/O - Water in oil
xxi
LIST OF SYMBOLS
[kL]f - Film mass transfer coefficient or external mass transfer
coefficient (m h-1
)
[kLa]d - Porous diffusion factor or internal mass transfer factor
(h-1
)
[kLa]f - Film mass transfer factor (h-1
)
[kLa]g - Global mass transfer factor (h-1
)
a - Surface of interfacial liquid-solid (m-1
)
a - Slope to determine the ratio of the O&G consumption
by bacterial activity to O&G loaded from the AFIE
under steady state conditions (dimensionless)
b - Interception of curve Y versus X to define the initial
O&G needs of bacterial growth and metabolism until it
reaches a steady-state value (g)
B - Potential mass transfer index relating to driving force
of mass transfer (mg g-1
)
c - Initial accumulation rate constant relying the amount of
O&G has been accumulated onto the OPF required by
the weight of microorganisms to reach a steady state
condition (dimensionless)
C - Concentration of the adsorbate in bulk liquid (mg L-1
)
Ce - O&G concentration present in the treated AFIE
monitored at outlet of the PBCR treatment system (mg
L-1
)
Ci - Concentration at initial
Co - Concentration of the adsorbate to entry to the column
(mg L-1
)
C0 - O&G concentration present in the raw AFIE monitored
xxii
at inlet of the PBCR treatment system (mg L-1
)
Cs - Concentration of the adsorbate to depart from the
column (mg L-1
)
Ct - effluent dye concentration (mg L-1
)
C* - Concentration of the adsorbate in film zone (mg L-1
)
(C - C*) - Driving force (mg L-1
)
E - O&G removal efficiency (%)
k - Biochemical accumulation rate coefficient (h-1
)
ki - inhibition constant (mg L-1
)
ks - half-velocity concentration (mg L-1
)
kAB - kinetic constant (mL mg-1
.min-1
)
kTh - Thomas rate constant, (ml min-1
.mg-1
)
kYN - rate constant (min-1
)
L - linear velocity (cm min-1
)
NA - Quantity of the solute A transferred per unit of time
or molar flux of the solute A (mg h-1
)
No - Saturation concentration (mg mL-1
)
q - Accumulative quantity of the solute adsorbed onto
absorbent (mg g-1
)
q - Amount of O&G accumulated onto the OPF migrating
from AFIE to support the needs of bacterial growth and
metabolism in the column (g)
q - Cumulative quantity of the O&G to accumulate into the
biomass attached the OPF (mg g-1
)
qo - Amount of O&G accumulated onto the OPF migrating
from AFIE to support the needs of bacterial growth and
metabolism in the packed-bed column at time zero (g)
qo - Maximum solid-phase concentration of solute, (mg g-1
)
Q - Flow rate (L h-1
)
r - Biochemical accumulation rate (g h-1
)
S - Surface area (m2)
S - Biomass concentration after time t (mg L-1
)
S - Concentration of the limiting substrate for growth
xxiii
So - Initial biomass concentration present in the column
under steady state conditions (mg L-1
)
t - Accumulative time of feeding water into the column
(h)
V - Volume of the treated water (L)
Veff - Volume of effluent (mL)
x - Amount of adsorbent in the column (g)
X - Accumulation of O&G loading the column (g)
Y - Accumulation of O&G attached onto the OPF (g)
Z - bed depth of the column (cm)
β - Adsorbate–adsorbent affinity parameter (g h mg-1
)
μ - Specific growth rate of the microorganisms (h-1
)
μmax - Maximum specific growth rate of the microorganisms
(h-1
)
τ - time required for 50 % adsorbate breakthrough (min)
xxiv
LIST OF APPENDICES
APPENDIX TITLE PAGE
A List of publication 196
1
CHAPTER 1
INTRODUCTION
1.1 Background
Oil and grease (O&G) is one of the major contaminants present in industrial
wastewaters (Karhu et al., 2013). Statistics indicate that global production of O&G
topped the chart at 170.01 million metric tons in 2014 (Statistics Portal, 2014). The
major O&G pollution would be coming from the agro-food processing industries
(Dumore and Mukhopadhyay, 2012). The agro-food industrial effluent (AFIE), as
typically referred to food processing effluent, containing high levels of O&G could
be dependent on a series of the industrial processes that include the raw material
storing, cleaning, shelling, slicing, washing, frying, salting, picking, coating and
packing (Kobya et al., 2006a). O&G is exposed to atmospheric oxygen and moisture
at a high temperature of about 180°C for extended periods of time during the frying
process, precipitating hundreds of chemical reactions in the frying oil, which
produces a number of harmful compounds, drastically changing the characteristics
and the quality of the resulting oil (Kumar et al., 2012; Mendick, 2015).
The effluent from an agro-food processing industry discharged into rivers and
lakes without treatment can cause the problems of water pollution, human health and
disequilibrium of the ecological systems (Qasim and Mane, 2013). Based on a report
by the Malaysian Department of Environment (DOE), the quantity of scheduled
wastes generated by O&G amounts to 154113.37 MT/year (9.02%) (Department of
Environment, 2012). To control the amount of discharge being released into the
environment, all industries must abide by the Environmental Quality (Industrial
2
Effluent) Regulation 2009, where the discharge limit for O&G is 1 mg/L for standard
A, and 10 mg/L for standard B (Environmental Quality (Industrial Effluent)
Regulation 2009).
Currently, there are many physical, chemical and biological treatment
processes have been proposed to remove O&G from industrial wastewaters, such as
electrocoagulation, advanced oxidation process and membrane technologies (Kobya
et al., 2006b; Karageorgos et al., 2006; Matos et al., 2008). However, the use of
these treatment processes requires high operational costs and might generate by-
products with an excessive amount of such as gas and sludge production (Kumar et
al., 2011). Because of the increased environmental awareness and the toughening of
governmental policies, it has become necessary to develop new environmentally
friendly ways to clean up contaminants using low-cost methods (Dors et al., 2013).
Biological treatment is a simple technique that complements existing technologies,
due to the fact that it is cost effective, eco-friendly, relatively effective (Zhang et al.,
2014) and can be used to remove O&G and other contaminants from industrial
wastewater.
1.2 Problems Statement
The removal of O&G from wastewaters represents one of the most critical
environmental challenges. The treatment and disposal of oily wastewaters, such as
agro-food processing effluent, is presently one of the most serious contributors to
environmental problems. AFIEs have been taken care of the environmental pollution
over the past two decades (Gao and Zhang, 2010), but their effects on the
environment are more noticeable now. The acceleration of agro-food processing
industries in Malaysia, particularly in the rural areas, is compelled to delicately
balance environmental sustainability and economic growth. This is due to the lack of
alternative technologies that can be used to treat the AFIE. It is also known that the
current treatment processes involving in the removal of O&G from wastewaters are
not complying with standards A and B of the Malaysian Environmental Quality
(Industrial Effluent) Regulation Year 2009.
3
Even though many physical, chemical and biological treatment processes are
available for the removal of O&G from industrial effluents, they suffer from inherent
disadvantages of being economically unfavourable and difficult to control, due to
high energy consumption and their by-products, such as chemical sludge, which lead
to secondary pollution (Qasim and Mane, 2013). Therefore, the aim of this work is to
scrutinise the ability of Serratia marcescens SA30 for removing O&G from effluent
of agro-food processing industry in both the batch reactor and the packed-bed
column reactor (PBCR). It is due to the newly proposed methods could be effective
and environmentally friendly biological treatment processes. However, the cellular
response to the pollutant exposure in these processes is not very well understood, due
to it being metabolically mediated by microorganisms.
For more detail on the removal of O&G by biosorption of using Serratia
marcescens SA30 must be scrutinised the kinetics as well as the global, external and
internal mass transfer. Understanding the bacterial growth rate and mass transfer
kinetics would be crucial towards up scaling the treatment processes from a
laboratory testing to industrial applications. It helps provide an explicit framework
for further improvement of biosorption capacity and specific growth rate on biomass
yield (Fulazzaky et al., 2013c). It is well recognised that biosorption is divided
according to the location into: (1) extracellular precipitation/accumulation, (2) cell
surface sorption and (3) intracellular accumulation (Saravanan et al., 2013). This
study suggests that the development of linear and logarithmic equations can predict
the efficiency of O&G removal and the application of the modified mass transfer
factor models (Fulazzaky et al., 2013c; Fulazzaky et al., 2014) may help determine
the resistance of mass transfer for biosorption of O&G by Serratia marcescens SA30
applied to a hydrodynamic column.
4
1.3 Objectives
The objectives of this study are as follows:
1. To determine and characterise the suitable strains of biosurfactant-
producing bacteria and to investigate the feasibility of the potential
bacteria isolated from AFIE for the removal of O&G.
2. To develop the linear and logarithmic equations for predicting the
efficiency of O&G removal by a biological treatment process with the
variables of concentration and flow rate.
3. To assess the application of the modified mass transfer factor models
for the removal of O&G by Serratia marcescens SA30 applied to a
PBCR treatment system and to determine the resistance of mass
transfer for the biosorption of O&G from AFIE.
1.4 Scope of the study
1) Characterisation of the AFIE
Samples of the AFIE were collected from agro-food industry located at
Rengit, Batu Pahat, 88.8 km from Johor Bahru the state capital of Johor, Malaysia.
The parameters of temperature, pH, dissolved oxygen (DO), total dissolve solids
(TDS) and ammonium (NH4+) were analysed in-situ at the location of agro-food
industry. The parameters of colour, suspended solids (SS), biochemical oxygen
demand (BOD), chemical oxygen demand (COD), total organic carbon (TOC) and
O&G were analysed at the Laboratory of Centre for Environmental Sustainability
and Water Security, Universiti Teknologi Malaysia, Skudai, Johor Bahru, Malaysia.
The analytical methods for the measurements of SS and O&G adhered to the
Standard Methods for Examination of Water and Wastewater (APHA, 2005).
5
2) Characterisation of biosurfactant-producing bacteria
The biosurfactant-producing bacterial strains isolated from AFIE were
examined according to the guideline of the Bergey’s Manual of Determinative
Bacteriology, which is based on the morphological features and gram staining of
using a microscopic technique to determine gram type of the bacteria. The bacterial
strains were screened to identify their oil-degrading characteristics that have a good
potential for the removal of O&G from AFIE. The characteristics of the bacteria
includes: cell surface hydrophobicity (CSH), biosurfactant production and activity,
haemolytic activity, emulsification index (E24), surface tension and zeta potential.
The potential strain of oil-degrading bacteria was identified based on the separation
of polymerase chain reaction-amplified fragments of genes coding for16s rRNA
analysis.
3) Acclimatisation of Serratia marcescens SA30
The aim of the acclimatisation of Serratia marcescens SA30 strains is to
adapt them to synthetic oily-contaminated wastewater (SOCWW) and AFIE. Firstly,
the nutrient broth (NB) as a rich medium was used for growing such strains of
biosurfactant-producing bacteria. Then secondly the medium was changed gradually
from NB to a new condition of using either SOCWW or AFIE with increasing of the
concentration of O&G in each selected new medium. Therefore, the number of
Serratia marcescens SA30 were counted (in CFU mL-1
) from every medium using a
colony counting method in a nutrient agar (NA) plate in order to having an insight of
the potential growth of such bacterial strains for removing O&G from AFIE.
4) The experiments in batch reactor
The removal of O&G using the most efficient oil-degrading bacteria was
carried out in a batch reactor. Factors driving the oil-microbe interactions that might
affect the performance of O&G removal in batch experiment of such as pH, contact
time and O&G concentration were evaluated. The images of scanning electron
microscope (SEM) and transmission electron microscope (TEM) were analysed to
investigate the oil-microbe interactions after degrading O&G from SOCWW and
AFIE by Serratia marcescens SA30.
6
5) Characterisation of oil palm frond and immobilisation of Serratia marcescens
SA30
The chemical characteristics of oil palm frond (OPF) were determined based
on the measurements of surface chemical functional groups. The OPF acted as a
supporting material in a fragmented form, each measuring 1-2 cm, which was then
washed with deionised water until it was free from any impurities, and then dried
overnight at 65ºC prior to being used in PBCR. The OPF in PBCR was rinsed with
deionised water using a suitable flow rate to having a hydrophilic-charged OPF for
allowing the immobilisation of Serratia marcescens SA30. The supplement of NB
into PBCR for 2 days was carried out to ensure the initial growth of the bacterial
strains.
6) The experiments in PBCR
The PBCR treatment system was set-up to remove O&G from AFIE. Factors
that might affect the PBCR performance such as pH, retention time, flow rate and
O&G concentration were verified. The water samples were collected at inlet and
outlet of the PBCR and analysed for the concentrations of O&G. The biosorption of
O&G from an AFIE can lead to the attachment of biomass onto OPF, which would
be controlled by either external or internal mass transfer; therefore, the use of the
modified mass transfer factor models could be useful to describe the mechanisms of
O&G removal by Serratia marcescens SA30 in a PBCR.
1.5 Significance of the study
The significance of this research in the field of environmental science and
engineering are:
1. Identification and characterisation of the potential isolated bacterial
strains can provide an insight on the ability of using the typical
biosurfactant-producing bacteria for degrading O&G from AFIE,
while the verification of physical and chemical properties for OPF can
7
justify the use of an appropriate supporting material for immobilising
the bacterial strains in a PBCR treatment system.
2. The development of linear and logarithmic equations based on a
laboratory-scale study can be used for predicting the design
parameters and the performance of PBCR for future wastewater
treatment applications in industrial scale.
3. Application of the modified mass transfer factor models can be useful
to assess the behaviours of external and internal mass transfer and to
determine the resistance of mass transfer for the biosorption of O&G
from AFIE by Serratia marcescens SA30 applied to a hydrodynamic
column.
1.6 Thesis Outline
This thesis is divided into seven chapters. Chapter 1 briefly mentions the
background, problem statement, objectives, scope of study and the significance of
the study. Chapter 2 presents the review of the literatures regarding the sources of
O&G released into the environment, biosurfactant-producing bacteria and their
holding materials, modelling and application of the kinetic models, and certain
technical approaches used to remove O&G. Chapter 3 describes the materials and
methods used for both the experiments in batch reactor and in PBCR. Chapter 4
presents the experimental data discussing on characteristics of the AFIE, supporting
material and potential bacterial strain, screening and identification of oil-degrading
bacteria, batch study for the removal of O&G, and analysis of surface morphology
and intracellular structure for biosurfactant-producing bacteria. Chapter 5 develops
the linear and logarithmic equations for predicting the efficiency of O&G removal by
Serratia marcescens SA30 applied to a hydrodynamic column. Chapter 6 applies the
modified mass transfer factor models to scrutinise the behaviours of mass transfer
and to determine the resistance of mass transfer for the biosorption of O&G
assimilation by Serratia marcescens SA30 in a PBCR. Chapter 7 provides the take-
home messages of this study and the recommendations for the future researches.
8
8
CHAPTER 2
LITERATURE REVIEW
2.1 Industrial wastewater
The increase in global population has resulted in increased industrial
activities, including the agricultural and manufacturing sectors. Such industrial
activities might generate enormous amounts of wastewater, which require an
appropriate treatment method prior to being discharged into the environment.
Discharging of untreated or partially treated wastewaters into an aquatic ecosystem
can create a major ecological problem of the receiving water due to the increased
volume of domestic and industrial wastewaters being discharged into such as lakes
and rivers can cause ecosystem disequilibrium and chaotic behaviour (Naidoo and
Olaniran, 2014).
Industrial wastewater is one of the important sources to cause the
environmental pollution. Highly released industrial wastewaters are coming from
food processing, pulp and paper, textile, chemical, pharmaceutical, petroleum,
tannery and manufacturing industries. The pollution indicators of industrial
wastewater can be classified into physical, physicochemical and biological
parameters (Karthik et al., 2014). The physical parameters are commonly measured
for temperature, pH, colour and odour, which are commonly observed in-situ, while
the physicochemical parameters such as NH4, NO2, NH3, SS, phosphorus, COD,
TOC, O&G and heavy metals are commonly analysed in a laboratory (Lin et al.,
2012). Typically, BOD represents biological parameter because the measurement of
such a parameter requires oxygen to oxidise organic biodegradable compounds by
9
9
aerobic bacteria (Ranade et al., 2014). The measurement of these parameters are
needed for both industrial wastewater and industrial wastewater treatment plant
effluent as the proper control for assessing the suitability quality of either wastewater
and treated wastewater to be discharged into the environment.
Industrial wastewaters may have a high inorganic and organic strength and
probably contain synthetic and natural non-biodegradable substances that may be
toxic to microorganisms so it can inhibit the biological treatment processes. Heavily
polluted industrial wastewaters would impart negative long-term effect to the
groundwater and surface water, hence potentially threatening the well-being of urban
and rural residents along with the destruction of the equilibrium ecosystem,
particularly aquatic life forms and hydrosphere. In order to minimise the
environmental damages and health impacts on the human, the presence of the
excessive amount pollutants in industrial wastewater needs to be brought down to
permissible limits via an appropriate treatment process for the safe disposal of treated
wastewater (Girish, 2014). Hence, it is imperative for all industrial activities to
practice the proper industrial wastewater management practices (Naidoo and
Olaniran, 2014).
2.1.1 Food processing industrial wastewater
Agro-food industry is one of the major contributors to environmental
pollution. Approximately 65-70% of the organic pollutants released into the water
bodies are coming from agro-product industrial activities (Rajagopal et al., 2013).
Generally, agro-food industry produces a lot of both solid and liquid wastes coming
from the different kinds of processes that include the raw material storing, cleaning,
shelling, slicing, washing, frying, salting, picking, coating and packing as shown in
Figure 2.1 (Ibrahim et al., 2013a). Amount of the wastewater and level of its
contamination released from industrial activities are very high, mostly including
carbohydrates, starches, proteins, vitamins, pectin and sugars, which are responsible
for high concentration of COD, BOD, SS and O&G (Shak and Wu, 2014). Its
10
10
accumulation in drain and water bodies is largely not recovered the polluted surface
water as a primary resource for drinking water production; however, it can cause the
critical environmental problems that treated naturally ecological systems and thus
becoming the most concern in many countries for handling industrial wastewaters
before rejected them into the environment (Qasim and Mane, 2013). Therefore,
wastewaters generated from the agro-food industry must be treated efficiently prior
to being discharged into the receiving water to comply with the stringent discharge
standards (Naidoo and Olaniran, 2014).
Figure 2.1: Agro-food processing production (Ibrahim et al., 2013a)
Storing and cleaning
Shelling
Cutting, slicing
Washing
Frying, boiling and
cooking
Salting
Picking
Coating and packing
Wastewater
Wastewater
Fats and oils
Wastewater
Wastewater & Fats and oils
11
11
2.1.2 Discharged regulations for oil and grease in several countries
Effluent discharge standards are the permissible concentration limits of
specific parameters in industrial wastewater that can be directly discharged into the
receiving water. All these concentration limits provide a simple mean of enforcing
control of discharges to the aquatic environment and are intended to promote a
consistent wastewater treatment approach towards the clean-up and prevention of
water pollution. This would ensure with the application of the best practicable
control technologies.
For the protection of the aquatic environment and public health, many
countries have been implemented their effluent standard for O&G discharger;
therefore, different concentration limits for the permissible discharge of O&G into
the environment may vary from one to another country. The United States
Environmental Protection Agency (USEPA) has set the maximum limit standards for
O&G to be 42 mg L-1
for the early daily allowable maximum concentration limit and
29 mg/L for the monthly concentration limit (Fakhru‟l-Razi et al., 2009). In
Malaysia, the DOE has limited the standard discharge of O&G designated by
Environmental Quality Regulation 2009 to 1 mg L-1
for standard A and to 10 mg L-1
for standard B, as shown in Table 2.1.
The monthly average concentration limit of O&G discharge allowed by the
People‟s Republic of China is 10 mg L-1
. In Indonesia, the effluent discharge
standards for O&G vary for different types of industry i.e., 5.0 mg L-1
for leather
tanning and textiles, 25 mg L-1
for oil refining and urea fertilizer and 30 mg L-1
for
palm oil industries. Singapore‟s effluent discharge standards for O&G range from 5-
30 mg L-1
, depending on the water use. The Thailand‟s effluent discharge standard of
O&G is 15.0 mg L-1
for the refinery and oil lubricant industry and is 5.0 mg L-1
for
all other industries, while Vietnam‟s standards for industrial wastewater range from 5
mg L-1
for mineral oils to 30 mg L-1
for animal-vegetable oils and fats (Tong et al.,
1999).
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12
Table 2.1: Environmental Quality (Industrial Effluent) Regulation 2009,
Amendment for Malaysia (Department of Environment, 2009)
Parameter Unit Standard
A* B**
Temperature
pH value
BOD at 20 oC
Suspended Solids
Mercury
Cadmium
Chromium, Hexavalent
Chromium, Trivalent
Arsenic
Cyanide
Lead
Copper
Manganese
Nickel
Tin
Zinc
Boron
Iron (Fe)
Silver
Aluminium
Selenium
Barium
Fluoride
Formaldehyde
Phenol
Free Chlorine
Sulphide
Oil and Grease
Ammoniacal Nitrogen
Colour ADMI***
oC
-
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
40
6.0 – 9.0
20
50
0.005
0.01
0.05
0.20
0.05
0.05
0.10
0.20
0.20
0.20
0.20
2.0
1.0
1.0
0.1
10
0.02
1.0
2.0
1.0
0.001
1.0
0.50
1.0
10
100
40
5.5 – 9.0
50
100
0.05
0.02
0.05
1.0
0.10
0.10
0.5
1.0
1.0
1.0
1.0
2.0
4.0
5.0
1.0
15
0.5
2.0
5.0
2.0
1.0
2.0
0.50
10
20
200
*Standard A means the effluent discharged at the upstream of a water supply intake,
**Standard B means the effluent discharged at the downstream of a water supply
intake and ***ADMI means American Dye Manufacturers Institute
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13
2.2 Oil and grease
O&G are essentially triglycerides consisting of straight-chain fatty acids,
attached by such as esters and glycerol (Wakelin and Forster, 1997), which are
commonly derived from animal and vegetable sources (Farhat et al., 2005). The
glycerides of fatty acids that are liquid at ordinary temperatures are called oils and
those that are solids are called fats (Sawyer et al., 2003). The fatty acids are
generally of 16 or 18-carbon atoms. The principal acids composing the glycerides of
fats and oils are shown in Table 2.2. The relative amounts of major fatty acids
contained in various fats and oils are shown in Table 2.3.
Table 2.2: Acids of the fats and oils (Sawyer et al., 2003)
Name Formula Mp, oC Source
Butyric C3H7COOH -5.7 Butter
Caproic C5H11COOH -3 Butter, coconut oil
Caprylic C7H15COOH 16.3 Palm oil, butter
Capric C9H19COOH 31.9 Coconut oil
Lauric C11H23COOH 43.2 Coconut oil, spermaceti
Myristic C13H27COOH 53.9 Nutmeg, coconut oil
Palmitic C15H31COOH 63.1 Palm oil, animals fats
Stearic C17H35COOH 69.6 Animal & vegetable fats,
oils
Arachidic C20H40O2 76.5 Peanut oil
Behenic C22H44O2 81.5 Ben oil
Oleic C18H34O2 13.4 Animal & vegetable fats,
oils
Erucic C22H42O2 34.7 Rape oil, mustard oil
Linoleic C18H32O2 -12 Cottonseed oil
Linolenic C18H30O2 -11 Linseed oil
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14
Table 2.3: Acid contents of fats and oils in percent (Sawyer et al., 2003)
Name Oleic Linolei
c
Linoleni
c
Stearic Myristi
c
Palmitic Arachidi
c
Butter 27.4 - - 11.4 22.6 22.6 -
Mutton
tallow
36.0 4.3 - 30.5 4.6 24.6 -
Castor oil 9.0 3.0 - 3.0 - 41.1- -
Olive oil 84.4 4.6 - 2.3 Trace 6.9 0.1
Palm oil 38.4 10.7 - 4.2 1.1 41.1 -
Coconut oil 5.0 1.0 - 3.0 18.5 7.5 -
Peanut oil 60.6 21.6 - 4.9 - 6.3 3.3
Corn oil 43.4 39.1 - 3.3 - 7.3 0.4
Cottonseed
oil
33.2 39.4 - 1.9 0.3 19.1 0.6
Linseed oil 5.0 48.5 34.1 - - - -
Soybean oil 32.0 49.3 2.2 4.2 - 6.5 0.7
Tung oil 14.9 - - 1.3 - 4.1 -
Mineral oil usually consists of mixtures of high molecular weight paraffins,
naphtene and aromatic hydrocarbons, with a certain mixture of tar and asphaltene
substances (Pushkarev et al., 1983). Characteristics of O&G derived from biological
sources are polar and biodegradable (Alade et al., 2011), whereas O&G originating
from petroleum (or minerals) has the characteristics of non-polar and bioresistant
(Patterson, 1989). Generally, O&G can be classified into five types as shown in
Table 2.4.
Table 2.4: Types of oil (Alther, 2008)
Types of oil Description
Mineral oil viscous liquid that is insoluble in water
but soluble in alcohol or ether and is
flammable
Petroleum made up of gaseous, liquid and solid
components and its viscosity varies
according to the mixture composition
Animal oil fatty acids or fixed oil. In solid form,
animal oils are known as fats
Vegetable oil primarily derived from kernel or many
other parts of plant materials
Essential oil complex, volatile liquid derived from
flowers, stems, etc
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15
2.2.1 Type and classification of oil-water mixture
Type of oil-water mixture can be classified as oil present as free oil, dispersed
oil, emulsified oil, or dissolved oil. Free oil is usually characterised by an oil-water
mixture with droplets greater than or equal to 150 µm in size, dispersed oil mixture
has a droplet size range between 20 and 150 µm, emulsified oil mixture has droplet
size ranged from 5 to 20 µm and soluble oil has a droplet size smaller than 5 µm, as
shown in Figure 2.2 (Rhee et al., 1987; Mohammed and Al-Gurany, 2010).
According to Alther (2008), the physical existence of oil in water can be classified
into several types, as follows: (1) free oil is defined as the oil which rapidly rises to
the surface of water and has a droplet size of 150 µm or more, (2) mechanical
dispersion consists of fine droplets with a size range of 10-1000 µm. Such droplets
are electrostatically stable without the influence of emulsifying agents, (3)
chemically stabilised emulsified oil consists of fine droplets with their size range
between 5 and 20 µm and is stable with the presence of an emulsifying agent, (4)
dissolved oil consists of fine oil droplets of less than 5 µm and usually refers to the
light end of the spectrum of compounds, such as benzene, toluene or xylene and (5)
oil wet solids, this category includes oil that adheres to sediments and other
particulate matter which is common in wastewater.
Figure 2.2: Classification and size range of oil droplets (Rhee et al., 1987)
2.2.2 Oily wastewater
The presence of O&G in a wastewater can be traced back to industrial and
municipal sources. The major sources of O&G present in contaminated industrial
150 µm 40 µm 20 µm 5 µm
FREE OIL DISPERSED OIL EMULSIFIED
OIL SOLUBLE OIL
REMOVAL BY API SEPARATOR
REMOVAL BY FLOTATION
REMOVAL BY CARBON MEMBRANE FILTRATION OR CARBON
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16
wastewaters are coming from the petroleum, metal, agro-food processing and textile
industries (Ibrahim et al., 2009). For municipal sources, oily wastewater could be
originated from oil-used food preparation, cleaning of oil-contaminated kitchen and
garbage oil-contaminated disposal (Chen et al., 2000). Table 2.5 shows the ranges in
O&G concentration released from various sources of wastewater into the
environment as reported in the literatures.
Table 2.5: Concentrations of O&G presented in the different sources of wastewater
Sources of Wastewater Concentration (mg L-
1)
References
Bistro 140-410 (Chen et al., 2000)
Poultry slaughterhouse 1500-1800 (Kobya et al., 2006b)
Student canteen 415-1970 (Chen et al., 2000)
Restaurant and food processing
industry
3000 (Sugimori, 2009)
Palm oil mill 4000-6000 (Ahmad et al., 2005)
Chinese restaurant 120-172 (Chen et al., 2000)
Pet food industries 52000-114000 (Jeganathan et al., 2006)
University cafeteria 4000 (Matsuoka et al., 2009)
Vegetable oil processing 5000-10000 (Chen et al., 2000)
Locomotive washing and
maintenance
5066 (Rajakovic et al., 2007)
Metal industries 1080-3271 (Zhu et al., 1997)
Petroleum industry is one of the major industrial sources of oily wastewater,
which might be the result of production, refining, storing and transportation
(Fakhru‟l-Razi et al., 2009). In an agro-food industry, the major sources of O&G
contaminant are the results of processing raw materials, storing, cleaning, shelling,
slicing, washing, frying, salting, picking, coating and packing (Kobya et al., 2006a).
Most O&G contaminants released from metal industry would be generated from
metal working operations, where oil is commonly used as the coolant liquid to cool
process machinery and equipment, to lubricate the cutting/grinding process and to
dissipate heats during the rolling of metals strip (Zhu et al., 1997). In a vegetable oil
processing, the main sources of oily wastewater are coming from cleaning, screening
and crushing of the raw materials such as fruits, nuts and seeds, typically used for
extracting oils as an economic value. In many cases, the concentrations of O&G
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17
released into the environment are highly dependent upon size of the industry and the
technology used for manufacturing process.
2.2.3 Impacts of oily wastewater on the environment
Oily wastewater poses a problem not only to the environment surrounding
the location of the industrial activities, such as the petroleum, food, cosmetic and
pharmaceutical industry; it also can have the adverse effects of the aquatic ecosystem
located far from an industry due to a water flow can transport such a contaminant to
go away from its original source. Nowadays, the quality of surface water tends to
decline increasingly caused by oily wastewater contaminants; therefore, it can cause
irreversible effects on the aquatic living organisms. As a consequence, such a
phenomenon can affect the human health as it entering the food chain in an
ecosystem has been proven (Alade et al., 2011). The presence of O&G in surface
water can lead to the formation of oil layer, which causes significant pollution
problem, such as the reduction of light penetration and photosynthesis. It further
hinders oxygen transfer from the atmosphere to water, leading to decreased amounts
of DO in water, which can affect the survival of aquatic life (Agrawal and Sahu,
2009). Even though the composition of oily wastewater varies from one industry to
another, it is important to note that a significant part of the emulsified forms is
difficult to treat (Karhu et al., 2013). Two types of emulsion have been identified
depending on the kind of liquid formed continuous phase, i.e., (i) oil-in-water (O/W)
for oil droplets dispersed in water; (ii) water-in-oil (W/O) for water droplets
dispersed in oil. A stable oil emulsion occurs once oil presents in contact with water
in the presence of an emulsifying agent (Zhou et al., 2008). The term „stable‟ refers
to the capacity of oil droplets remained independent in a dispersion form (Zhu et al.,
1997). In case of the vegetable oil, the stable emulsion appears as a milky white
solution (Alter, 2001; Ibrahim et al., 2012).
The existence of emulsified oil in wastewater can lead to negative impacts on
the environment and can reduce the performance of a wastewater treatment process.
18
18
Alade et al. (2011) reported that the excessive amounts of O&G in a wastewater can
block the sewerage, pumping, screening, and filtering system of a wastewater
treatment plant, leading to increase maintenance cost. This would reduce the
performance of a biological treatment process due to the presence of O&G can
interfere the bacterial activities and the emergence of unpleasant odours (Baig et al.,
2003). Generally, choosing a suitable approach to treat oily wastewater depends on
four main factors, such that: (1) oil droplet size distribution, (2) droplet velocity, (3)
O&G concentration, and (4) emulsion formation (Kings, 1999). It is recognised that a
high concentration of O&G in wastewater may contain toxic compounds and thus
leads to an acute effect and carcinogenicity to human (Boesch and Rabalais, 1987;
Kings, 1999).
2.3 Oil-degrading bacteria
2.3.1 Biosurfactant
Biosurfactants are amphipathic compounds excreted as a metabolite by the
microorganisms that exhibit surface activity and have the ability to reduce the
interfacial surface tension of two different liquid phases (Ibrahim et al., 2013b). Such
amphiphilic compounds contain both hydrophobic and hydrophilic regions and
possess a polar (hydrophilic) head and nonpolar (lipophilic) tail. They are capable of
forming micelles and reverse micelles as shown in Figure 2.3.
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Biosurfactant molecules
Polar head
Non-polar tail
Micelle
Hydrophobic
oil or organic phase
Reverse micelle
Aqueous
phase
Figure 2.3: General structure of biosurfactant to form micelle and reverse micelle
(Daverey and Pakshirajan, 2011)
The hydrophobic moiety of a biosurfactant includes long-chain fatty acid,
hydroxy fatty acid, or α-alkyl β-hydroxy fatty acid, while the hydrophilic moiety can
be carbohydrate, amino acid, cyclic peptide, phosphate, carboxylic acid, or alcohol.
Microbial surfactants are complex molecules commonly consisting of peptides, fatty
acids, glycolipids, rhamnolipids, lipopeptides, and sophorolipids (Al-Araji et al.,
2007). High molecular weight biosurfactants are polyanionic heteropolysaccharides
containing both polysaccharides and proteins, while low molecular weight
biosurfactants are glycolipids. Several types of biosurfactant producing by the
microorganisms are presented in Table 2.6.
20
20
Table 2.6: Types of biosurfactant-producing by the microorganisms (Daverey and
Pakshirajan, 2011)
Biosurfactant type Producing Microbial Species
Glycolipids
Trehalose mycolates Rhodococcus erythropolis, Arthrobacter
paraffineu, Mycobacterium phlei,
Nocardia erythropolis
Trehalose esters Mycobacterium fortium, Micromonospora
sp., M.smegmatis, Rhodococcus
erythropolis
Trehalose mycolates of mono, di
trisaccharide
Corynebacterium diptheriae,
Mycobacterium smegmati, Arthrobacter
sp.
Rhamnolipids Pseudomonas sp.
Sophorolipids Torulopsis bombicola/ apicola, Torulopsis
petrophilum, Candida sp.
Rubiwettins R1 and RG1 Serratia rubidaea
Diglycosyl digyycerides Lactobacillus fermenti
Schizonellins A and B Schizonella melanogramma
Ustilipids Ustilago maydis and Geotrichum
candidum
Amino acid lipids Bacillus sp.
Flocculosin Pseudomonas flocculosa
Phospholipid and Fatty acids
Phospholipids and Fatty acids Candida sp., Corynebacterium
sp.,Micrococcus sp., Acinetobacter sp.,
Thiobacillus thiooxidans, Asperigillus sp.,
Pseudomonas sp., Mycococcus sp.,
Penicillium sp.
Polymeric surfactants
Lipoheteropolysaccharide (Emulsan) Acinetobacter calcoaceticus RAG-1,
Arethrobacter calcoaceticus
Heteropolysaccharide (Biodispersan) Acinetobacter calcoaceticus A2
Polysaccharide protein Acinetobacter calcoaceticus strain
Manno-protein Saccharomyces cerevisiae
Carbohydrate-protein Candida petrophillium, Endomycopsis
lipolytica
Mannan-lipid complex Candida tropicalis
Mannose/ erythrose lipid Shizonella melanogramma, Ustiloga
maydis
Carbohydrate-protein-lipid-complex Pseudomonas fluorescences,
Debaryomyces polymorphus
Liposan Candida lypolytica
Alasan Acinetobacter calcoaceticus
Protein PA Pseudomonas aeruginosa
21
21
*Table 2.6 continued
Biosurfactant type Producing Microbial Species
Particulate biosurfactant
Membrane vesicles Acinetobacter sp. H01-N
Fimbriae, whole cells Acinetobacter calcoaceticus
Lipopeptides and lipoproteins
Gramicidins Bacillus brevis
Peptide lipids Bacillus licheniformis
Polymyxin E1 Bacillus polymyxa
Omithine-lipid Pseudomonas rubescens, Thiobacillus
thiooxidans
Viscosin Pseudomonas fluorescens
Serrawettin Serratia marcescens
Cerilipin Glucunobacter cerius
Lysine-lipid Agrobacterium tumefaciens
Surfactin, subtilysin, subsporin Bacillus subtilis
Lichenysin G Bacillus licheniformis IM 1307
Omithine lipid Pseudomonas sp., Thiobacillus sp.,
Agrobacterium sp., Gluconobacter sp.
Amphomycin Streptomyces canus
Chlamydocin Diheterospora chlamdosporia
Cyclosporin A Tolypocladium inflatum
Enduracidin A Streptomyces fungicidius
Globomycin Streptomyces globocacience
Bacillomycin L Bacillus subtilis
Iturin A Bacillus subtilis
Putisolvin I and II Pseudomonas putida
Artrofactin Arthobacter
Fengycin Bacillus thuringiensis CMB26
Mycobacillin Bacillus subtilis
Biosurfactants would be a heterogeneous group of the surface-active
chemical compounds produced by a wide variety of the microorganisms and have the
properties of low toxicity and high biodegradability. Due to having a mild production
condition and environmental compatibility, they can be used widely in industrial
applications (Kumar et al., 2008). Biosurfactants might accumulate at air–water, air-
oil and oil–water interfaces to forming allegiances with a compatible interaction of
the opposing regions.
Biosurfactants caused by their chemicals are advantageous due to having the
properties of biodegradability, low toxicity, ecological acceptability and
effectiveness at extreme temperatures and pH. Therefore, biosurfactants have a
22
22
Degrade
O&G Enzymes
Inside
Cell
Cell
membrane
BS micelle BS micelle
with O&G
Outside the
cell
Phospholipid
bilayer
Inside the cell
O&G
variety of the potential applications, such as in pharmaceutical, cosmetic, detergent
and food industries (Yin et al., 2009; Jain et al., 2012). Biosurfactants also play an
important role in the bioremediation of organic pollutants like polycyclic aromatic
hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), O&G and petroleum
hydrocarbons (Yin et al., 2009). Figure 2.4 shows the mechanism of O&G removed
by biosurfactant from waters. Firstly, formation of the micelle by which the
hydrophobic portion of biosurfactant attaches to O&G via hydrophobic interactions
to forming the micelle containing O&G. Secondly, such a micelle then performs the
contact with a highly hydrophilic cell membrane to cause an increase in membrane
porosity to possibly entering O&G into the cell. Once inside the cell, O&G is then
catalysed by an enzyme for its degradation (Daverey and Pakshirajan, 2011).
Figure 2.4: Mechanism of O&G degradation by microorganisms in the presence of a
biosurfactant (Daverey and Pakshirajan, 2011)
Biosurfactants may contribute to the formation of biofilm and play a
protective role for surviving the microorganisms when the presence of toxic
inorganics and organics is high. More biosurfactants produced by the bacteria in
form of micelle can allow the increased O&G uptake and thus potentially reduce
access to undesirable compounds, provided the microbial cell surface repels
23
23
biosurfactant molecules. For example, a cell secreting biosurfactants may display a
hydrophobic cell surface to avoid interacting with polar micelles or bioemulsifier
coated non-aqueous phases (Van Hamme and Urban, 2009). In a bacterial strain,
CSH may be modulated by changing lipid composition, expression of hydrophobic
surface proteins, incorporation of extracellular compounds such as alkanes, and
intercalation, as well as adsorption and action of biosurfactant molecules
(Sokolovska et al., 2003; Van Hamme and Urban, 2009). Lipopolysaccharide as a
key hydrophobic component in bacterial cell walls can be an important CSH
determinant because of the biosurfactant may strip lipopolysaccharide from the cell
walls when added exogenously with subsequent impacts on the overall CSH
conditions (Al-Tahhan et al., 2000; Van Hamme and Urban, 2009).
Extreme environments are bioresources of the potential microorganisms that
secrete new bioactive compounds and biosurfactants to possibly having a wide
adaptability and stability towards chronic toxic conditions. It makes the use of
biosurfactants suitable for the applications in bioremediation of vegetable oil,
hydrocarbon and toxic metals from contaminated soils and wastewaters (Jain et al.,
2012). Table 2.7 shows the types of biosurfactant, biosurfactant-producing
microorganisms and the applications of biosurfactant.
24
Table 2.7: Biosurfactant sources and their applications (Makkar et al., 2011)
Class Surfactant Microorganisms Application/activity
Trehalose lipids Arthrobacter paraffineus
Corynebacterium spp
Mycobacterium spp
Rhodococcus erythropolis
Nocardia sp
Cosmetics, antifungal, antiviral
Oil bioremediation and recovery
Rhamnolipids Pseudomonas aureginosa
Pseudomonas sp, Serratia rubidea
Oil recovery, metals removal,
hydrocarbon removal, PAH
removal
Glycolipids Sophorose lipids Candida apicola, Candida bombicola,
Candida lypolitica, Candida
bogoriensis,
Metals removal, Hydrocarbon
removal
Glycolipids Rhodococcus erythropolis,
Serratia marcescens, Tsukamurella sp
Oil bioremediation
Lipopolysaccharides Acinetobacter calcoaceticus (RAG1),
Pseudomonas sp., Candida lypolitica
Bioavalability of surfaces
Manosylerythritol
lipids (MEL‟s)
Pseudozyna sp Oil emulsification
Surfactin Bacillus Subtilis, Bacilus pumitus Metal Remediation
Arthrofactin Pseudomonas sp MIS38 Oil emulsification
Lichenysin A,
Lichenysin B
Bacillus licheniformis Metal Remediation
Lipopeptide Bamylocin Bacillus amyloliquefaciens Oil emulsification
Syringafactin A-F Pseudomonas syringae pv. Tomato
DC3000
Swarming motality
Ornithine lipids Myroides sp strain SM1 Oil emulsification
Ornithine, lysine
peptide
Thiobacillus thioxidans, Steptomyces
sioyaensis, Gluconobacter cerinus
Oil emulsification
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