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UNIVERSITI PUTRA MALAYSIA

BIOREMEDIATION OF VEGETABLE OILY BALLAST WASTEWATER UNDER TEMPERATE CONDITION USING ANTARCTIC BACTERIA

MARYAM ABUBAKAR

FBSB 2018 9

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BIOREMEDIATION OF VEGETABLE OILY BALLAST WASTEWATER

UNDER TEMPERATE CONDITION USING ANTARCTIC BACTERIA

By

MARYAM ABUBAKAR

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfillment of the Requirements for the Degree of Master of Science

December 2017

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COPYRIGHT

All material contained within the thesis, including without limitation text, logos,

icons, photographs, and all other artwork, is copyright material of Universiti Putra

Malaysia unless otherwise stated. Use may be made of any material contained within

the thesis for non-commercial purposes from the copyright holder. Commercial use

of material may only be made with the express, prior, written permission of

Universiti Putra Malaysia.

Copyright © Universiti Putra Malaysia

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DEDICATION

This thesis is dedicated to my parents Engr Abubakar Magaji Gusau and Hajiya

Fatima Abubakar Magaji for their love, prayers and assistance towards the success of

thesis.

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment

of the requirement for the degree of Master of Science

BIOREMEDIATION OF VEGETABLE OILY BALLAST WASTEWATER

UNDER TEMPRATE CONDITION USING ANTARCTIC BACTERIA

By

MARYAM ABUBAKAR

December 2017

Chairman : Associate Professor Mohd Yunus Abd Shukor, PhD

Faculty : Biotechnology and Biomolecular Sciences

Spills of vegetable oily waste especially palm oil as a result of ballast water

discharge from vegetable oil tankers in temperate waters are of environmental

concern because they cause serious effects on marine life and coastal environments.

The ongoing reclassification of oil palm ballast waste as a hazardous substance by

the European Union will seriously affect the Malaysian economy. Biodegradation by

indigenous cold-tolerant microorganisms is an important and potentially remediating

process solving this current problem. This study aims to investigate the

biodegradability of vegetable oil (palm oil) under the influence of a cold-tolerant

bacteria (Rhodococcus erythropolis ADL36) previously isolated from Antarctica.

The strain was cultured at different oil concentrations, temperature, pH, salinity, and

inoculum size under simulated conditions of oily ballast waste water. Furthermore,

the influences of the independent variables were optimised using response surface

methodology (RSM). A Plackett-Burman screening was carried out prior to RSM.

Three factors namely temperature, oil concentration and inoculum size appears to be

the most significant factors among the five while two factors; pH and salinity show

non-significant effect on the degradation of palm oil. The results of the research have

shown that maximum growth and biodegradation occurred at 1% (v/v) of the oil, at

25oC, pH 6.8, 2% of NaCl and an inoculum size of 5% (v/v) after OFAT. The

difference in the peaks of the oil component was seen in the GCMS result.

Moreover, the results of RSM showed that oil concentration, temperature and

inoculum size showed significant effects on the biodegradation of the oil. Out of the

eight primary models utilize, the modified Gompertz model was the best in modeling

the bacterial growth. Based on the growth rate constants obtained from the primary

modelling, a secondary modelling was carried out using various models such as

Luong, Yano, Tessier-Edward, Aiba, Haldane, Monod, and Han and Levenspiel. The

best model was Haldane giving the calculated value for the Haldane’s constants such

as maximal growth rate ( max), half saturation constant for maximal growth (Ks) and

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growth inhibition constant (Ki) tolerated were 0.74±0.12 day-1, 1.23±0.14 palm oil

(% v/v), and 3.12±0.16 palm oil (% v/v), respectively. In conclusion, the results

indicated the efficiency of such a system as a potential treatment for oily ballast

wastewater (vegetable oil).

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains

BIOREMEDIASI MINYAK SAYURAN AIR SISA BUANGAN DIBAWAH

KEADAAN SUHU SEJUK DENGAN MENGGUNAKAN BAKTERIA

ANTARTIKA

Oleh

MARYAM ABUBAKAR

Disember 2017

Pengerusi : : Profesor Madya Mohd Yunus Abd Shukor, PhD

Fakulti : Bioteknologi dan Sains Biomolekul

Pencemaran dari tumpahan sayur-sayuran berminyak terutamanya kelapa sawit

akibat pelepasan air balast dari kapal pembawa minyak sayuran di perairan yang

sejuk menjadi kebimbangan kepada alam sekitar kerana ia menyebabkan kesan yang

serius terhadap kehidupan laut dan persekitaran pantai. Pengkelasan semula bahan

buangan kelapa sawit yang berterusan sebagai bahan berbahaya oleh Kesatuan

Eropah akan memberi kesan serius kepada ekonomi Malaysia. Biodegradasi oleh

mikroorganisma yang toleran kepada suhu sejuk adalah proses pemulihan yang

penting dan berpotensi untuk menyelesaikan masalah semasa ini. Kajian ini

bertujuan untuk mengkaji kebolehpenguraian minyak sayuran (minyak kelapa sawit)

di bawah pengaruh bakteria yang toleran pada suhu sejuk (Rhodococcus sp. strain

ADL36) yang sebelum ini dipencilkan dari Antartika. Strain ini ditumbuhkan pada

kepekatan minyak, suhu, pH, saliniti dan saiz inokulum yang berbeza di bawah

keadaan simulasi air sisa balast berminyak. Selain itu, pengaruh pembolehubah

bebas dioptimumkan menggunakan kaedah permukaan respon (RSM). Penyaringan

Plackett-Burman telah dijalankan sebelum RSM. Tiga faktor iaitu suhu, kepekatan

minyak dan saiz inokulum menjadi faktor yang paling penting di antara lima

manakala dua faktor; pH dan kemasinan menunjukkan kesan yang tidak signifikan

terhadap penguraian minyak kelapa sawit. Keputusan menunjukkan bahawa

pertumbuhan maksimum dan biodegradasi berlaku pada 1% (v /v) minyak, pada

suhu 25oC, pH 6.8, 2% NaCl dan saiz inokulum 5% (v /v) selepas RSM. Perbezaan

di puncak-puncak komponen minyak dilihat dalam pemerhatian berasaskan GCMS.

Selain itu, keputusan RSM menunjukkan kepekatan minyak, suhu dan saiz inokulum

menunjukkan kesan yang ketara terhadap biodegradasi minyak. Daripada lapan

model utama yang digunakan, model Gompertz yang diubahsuai adalah yang terbaik

dalam memodelkan pertumbuhan bakteria. Berdasarkan pemalar kadar pertumbuhan

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yang diperoleh dari pemodelan primer, pemodelan sekunder dilakukan dengan

menggunakan pelbagai model seperti Luong, Yano, Tessier-Edward, Aiba, Haldane,

Monod, dan Han dan Levenspiel. Model terbaik adalah Haldane yang memberikan

nilai-nilai pemalar Haldane seperti kadar pertumbuhan maksima (max), pemalar

ketepuan tepu untuk pertumbuhan maksimum (Ks) dan kepekatan yang merencat

pertumbuhan (Ki) pada 0.74 ± 0.12 hari-1, 1.23 ± 0.14 minyak sawit (% v /v), dan

3.12 ± 0.16 minyak sawit (% v /v), masing-masing. Kesimpulannya, hasil kajian

telah menunjukkan kecekapan bakteria ini yang berpotensi dalam merawat air

kumbahan balast berminyak (minyak sayuran).

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ACKNOWLEDGEMENTS

In the name of Allah, the Most Gracious and Most Merciful. All thanks and praises

are due to Allah (SWT) for giving me the opportunity to successfully conduct this

research. May all blessings be upon His Prophet and Messenger, Muhammad

(SAW).

The successful completion of this research was made possible with the support and

assistance of many important people. First and foremost, I would like to express

profound gratitude to my supervisor Assoc Prof. Dr. Mohd Yunus Abd Shukor and

my co-supervisors; Dr Adeela Yasid and Dr. Siti Aqlima Ahmad Whom accepted

me as their student and offered me mentorship, moral support and care. This work

would not have been possible without their guidance and involvement. Equally, I

wish to express my appreciation to all my colleagues in the Bioremediation Lab for

the contributions they rendered during the conduct of this research.

Words cannot express my gratitude to my husband Dr. Abbas Sani Dahiru for the

support and encouragement towards the success of this research. He was always

beside me during the happy and hard moments to motivate me. I thought that it is

impossible to continue, you helped me to keep things in perspectives. I greatly value

his contributions and deeply appreciate his belief in me. Equally, my profound

gratitude goes to my brothers, sisters, friends and family members for their concern

and prayers which serves as a source of inspiration to me. My heart felt regard goes

to my father inlaw, mother in law for their love, prayers and support.

Last and not the least is my lovely daughter Ramlat I really appreciate you for

bearing with me and making the house lively despite my busy academic schedules.

Maryam Abubakar, 2018.

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This thesis was submitted to the Senate of the Universiti Putra Malaysia and has

been accepted as fulfilment of the requirement for the degree of Master of Science.

The members of the Supervisory Committee were as follows:

Mohd Yunus Shukor, PhD

Associate Professor

Faculty Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairperson)

Nur Adeela Yasid, PhD

Senior Lecturer

Faculty of Biotechnology

Universiti Putra Malaysia

(Member)

Siti Aqlima Ahmad, PhD

Senior Lecturer

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Member)

ROBIAH BINTI YUNUS, PhD

Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date :

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Declaration by graduate student

I hereby confirm that:

this thesis is my original work;

quotations, illustrations and citations have been duly referenced;

this thesis has not been submitted previously or concurrently for any other degree

at any institutions;

intellectual property from the thesis and copyright of thesis are fully-owned by

Universiti Putra Malaysia, as according to the Universiti Putra Malaysia

(Research) Rules 2012;

written permission must be obtained from supervisor and the office of Deputy

Vice-Chancellor (Research and innovation) before thesis is published (in the

form of written, printed or in electronic form) including books, journals,

modules, proceedings, popular writings, seminar papers, manuscripts, posters,

reports, lecture notes, learning modules or any other materials as stated in the

Universiti Putra Malaysia (Research) Rules 2012;

there is no plagiarism or data falsification/fabrication in the thesis, and scholarly

integrity is upheld as according to the Universiti Putra Malaysia (Graduate

Studies) Rules 2003 (Revision 2012-2013) and the Universiti Putra Malaysia

(Research) Rules 2012. The thesis has undergone plagiarism detection software

Signature: Date:

Name and Matric No : Maryam Abubakar, GS44411

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Declaration by Members of Supervisory Committee

This is to confirm that:

the research conducted and the writing of this thesis was under our supervision;

supervision responsibilities as stated in the Universiti Putra Malaysia (Graduate

Studies) Rules 2003 (Revision 2012-2013) were adhered to.

Signature:

Name of Chairman

of Supervisory

Committee:

Associate Professor

Dr. Mohd Yunus Shukor

Signature:

Name of Member

of Supervisory

Committee:

Dr. Nur Adeela Yasid

Signature:

Name of Member

of Supervisory

Committee:

Dr. Siti Aqlima Ahmad

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TABLE OF CONTENTS

Page

ABSTRACT i

ABSTRAK iii

ACKNOWLEDGEMENTS v

APPROVAL vi

DECLARATION viii

LIST OF TABLES xii

LIST OF FIGURES xiii

LIST OF ABBREVIATIONS xv

CHAPTER

1 INTRODUCTION 1

1.1 Research Background 1

1.2 Problem statement 3

1.3 Objectives 3

2 LITERATURE REVIEW 4

2.1 Biodegradation 4

2.2 Degradation of Fats and Oils by Lipases 4

2.3 Oil-Degrading Bacteria 5

2.4 Aerobic Biodegradation 5

2.5 Vegetable Oil 6

2.5.1 Properties of Vegetable Oil 7

2.5.2 Industrial Uses of Vegetable Oils 7

2.5.3 Biodegradation Characteristics of Vegetable Oils 8

2.6 Marine Pollution 9

2.6.1 Vegetable oil pollution 9

2.7 Remedies for Oil Spill in Marine Water 11

2.7.1 Physical Remediation Methods 11

2.7.2 Chemical Remediation Methods 13

2.7.3 Thermal Remediation Method 14

2.7.4 Bioremediation Method 14

2.8 Remedies for Oil Spill in Arctic Waters 15

2.8.1 Mechanical Recovery 15

2.8.2 Use of Dispersants 16

2.8.3 In-situ Burning 16

2.9 Methods of Optimisation of Vegetable Oil-Degrading Capacity

in Microorganisms 17

2.9.1 One Factor at a Time 17

2.9.2 Experimental Designs and Statistical Optimisations 17

2.10 Bioremediation of Ballast Water through Biodispersion 19

2.11 Growth Kinetics of Bacteria 20

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3 MATERIALS AND METHODS 28

3.1 Experimental Approach 28

3.2 Source and Maintenance of bacteria 28

3.3 Media Preparation 29

3.4 Growth Determination 29

3.5 Determination of Oil Degradation 30

3.6 Gas Chromatographic Analysis of Palm Oil Degradation 30

3.7 Optimization of Factors Affecting Oil Degradation Using OFAT 31

3.7.1 Optimization of Substrate Concentration 31

3.7.2 Optimization of Inoculum Size 31

3.7.3 Optimization of the Medium pH 31

3.7.4 Optimization of Temperature 31

3.7.5 Optimization of the Salinity 32

3.8 Optimization Using Statistical Approach 32

3.8.1 Plackett – Burman Factorial Design (PBFD) 32

3.8.2 Central Composite Design (CCD) and Response

Surface Methodology (RSM) 32

3.9 Modelling the Kinetics of Growth on palm oil 33

3.9.1 Fitting of the data 33

3.9.2 Statistical analysis 33

4 RESULTS AND DISCUSSIONS 35

4.1 One Factor at a Time (OFAT) 35

4.1.1 Effect of initial Substrate Concentration 35

4.1.2 Effect of Temperature 36

4.1.3 Effect of pH 37

4.1.4 Effect of Salt Concentration 38

4.1.5 Effect of Inoculum Size 39

4.2 GCMS Analysis 40

4.3 Statistical Optimizations 44

4.3.1 Selection of Significant Variables by Plackett–Burman

Design 44

4.3.2 Response Surface Methodology (RSM) 46

4.4 Kinetic Modelling of Growth 53

4.4.1 Growth on palm oil modelled using primary models 53

4.4.2 Growth on palm oil modelled using secondary models 60

5 SUMMARY, CONCLUSION AND RECOMMENDATIONS 65

66

84

87

REFERENCES

APPENDICES

BIODATA OF STUDENT

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LIST OF TABLES

Table Page

2.1 Bacterial growth models used in this study 22

2.2 Various mathematical models developed for degradation kinetics

involving substrate inhibition

24

4.1 GCMS analysis of 1% palm oil 42

4.2 GCMS analysis of control palm oil 43

4.3 Coded and actual values of significant factors used in plackett-

Burman factorial design

44

4.4 Experimental sdesign matrix of Plackett-Burman and the response 45

4.5 Analysis of ANOVA for the Model of Factors in Placket-Burman 45

4.6 Coded and actual values of significant factors used in central

composite design (CCD)

46

4.7 CDD experimental matrix generated by Design expert and

corresponded responses (actual and predicted)

46

4.8 Analysis of variance (ANOVA) for Response Surface Quadratic

Model

47

4.9 Comparison of the optimized condition between OFAT and RSM of

4% palm oil

53

4.10 Statistical analysis of the various fitted models 58

4.11 Bacterial growth coefficients at various palm oil as modelled using the

modified Gompertz model

59

4.12 Statistical analysis of kinetic models 64

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LIST OF FIGURES

Figure Page

2.1 Hydrolysis of TAG by lipase (Edmund, 2001) 4

2.2 Global vegetable oil production. Source: USDA, AMI 6

3.1 A schematic overview of the experimental stages 28

4.1 Effect of Palm oil Concentration (sole carbon source) on the growth

of Rhodococcus erythropolis ADL36. Error bars represent mean ±

standard deviation of triplicates.

36

4.2 Effect of temperature on the growth of Rhodococcus erythropolis

ADL36. Error bars represent mean ± standard deviation of triplicates

37

4.3 Effect of pH on the growth of Rhodococcus erythropolis ADL36.

Error bars represent mean ± standard deviation of triplicates

38

4.4 Effect of Salinity on the growth of Rhodococcus erythropolis ADL36.

Error bars represent mean ± standard deviation of triplicates

39

4.5 Effect of Inoculum size on the growth of Rhodococcus erythropolis

ADL3. Error bars represent mean ± standard deviation of triplicates

40

4.6 Inoculated Gas chromatographic analysis of fatty acids from

utilization of 1% (v/v) palm oil by the culture of Rhodococcus

erythropolis ADL36 after 7 days of inoculation with (a) uninoculated

(control) and (b) treated

41

4.7 Model diagnostic plots (a) predicted versus actual (b) studentized

residue versus predicted (c) normal plots of residue (d) outlier T

versus run

49

4.8 3D and 2D surface response view showing the interaction between oil

concentration and temperature

50

4.9 3D and 2D surface response view showing the interaction between

temperature and inoculum size

51

4.10 3D and 2D surface response view showing the interaction between oil

concentration and inoculum size

52

4.11 The bacterial growth curves of Rhodococcus sp. strain ADL36 at

various concentrations of palm oil over time after RSM optimization.

The error bars represent mean ± standard deviation of three replicates

54

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4.12 Growth profile of Rhodococcus sp. strain ADL36 on palm oil fitted

according to the Huang model

54

4.13 Growth profile of Rhodococcus sp. strain ADL36 on palm oil fitted

according to the Baranyi-Roberts model

55

4.14 Growth profile of Rhodococcus sp. strain ADL36 on palm oil fitted

according to the modified Gompertz model

55

4.15 Growth profile of Rhodococcus sp. strain ADL36 on palm oil fitted

according to the Buchanan-3-phase model

56

4.16 Growth profile of Rhodococcus sp. strain ADL36 on palm oil fitted

according to the modified Richards model.

56

4.17 A. Growth profile of Rhodococcus sp. strain ADL36 on palm oil

fitted according to the modified Schnute model

57

4.18 Growth profile of Rhodococcus sp. strain ADL36 on palm oil fitted

according to the modified Logistics model

57

4.19 Growth profile of Rhodococcus sp. strain ADL36 on palm oil fitted

according to the von Bertalanffy model

58

4.20 Fitting experimental data with the Luong model 60

4.21 Fitting experimental data with the Yano model 61

4.22 Fitting experimental data with the Teissier model 61

4.23 Fitting experimental data with the Aiba model 62

4.24 Fitting experimental data with the Haldane model 62

4.25 Fitting experimental data with the Monod model 63

4.26 Fitting experimental data with the Han-Levenspiel model 63

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LIST OF ABBREVIATIONS

% Percent

CCD Central composite design

cm Centimetre

dH2O Distilled water

et al and friends

G Gram

h Hour

kpa Kilopascal

L Liter

M Molar

min Minute

mL Mililiter

MSM Minimal salt medium

NA Nutrient agar

sec Second

ºC Degree Celcius

OD Optical density

RPM Rotation per minute

RSM Response surface method

µL Microlitre

µm Micrometer

mm Millimeter

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CHAPTER 1

1 INTRODUCTION

1.1 Research Background

More than 80% of commercial goods shipments are transported through Sea globally

(Ibrahim & El-Naggar 2012). In addition to the intentional shipment, over 12 billion

tons of ballast water is moved through massive coastal and sea areas annually

(Wattayakorn, 2012). Ships are made purposely to the transportation of goods such

as oil through aquatic. However, if the ship is travelling without the goods or has

discharged goods in one port on its way to other port of call, In other to achieve the

required safe, functional conditions that include keeping the ship sufficiently in the

water to make sure effective propeller and rudder procedure also to avoid the bow

developing from the water, mostly in heavy oceans, Ballast may be carried on board.

A good weight-to-volume ratio is taken in a detached or empty cargo tanks which

are used only for ballast water and when a container is parting a port any residue that

is pumped into the ballast tanks that may be disturbed up, is released again in the

next port when takings on cargo. Hazardous nature of oil spill can effectively be

tackled through enforcing proper regulations in the best good working condition, but

can never be removed completely.

Vegetable or petroleum oil spills are considered as serious contaminants in marine

environments, due to their toxicity to marine organisms (El-Masry et al. 2004). Only

few oil spills in Arctic waters have been reported (Singsaas & Lewis 2011). Though,

with an increase in the activities of oils such as production, exploration, and

transport there is an increased risk for future occurrence. Various Arctic areas are

remote with inadequate infrastructure to support spill response. Additionally, oil spill

response in an extremely low temperature and darkness is much difficult. The

physical treatment of fats containing wastewaters is considered inadequate and hence

is costly while biological treatment provides the most efficient means that eliminate

fats and oil using lipases enzyme (Manan et al. 2014). These enzymes otherwise

known as triacylglycerol acylhydrolases are produced by many microorganisms

where they catalyse the synthesis or hydrolysis of fats (Shabtai et al. 1992).

According to the U.S. Environmental Protection Agency (USEPA), the release of

edible fats, oils, and greases (FOGs) into the environment does not constitute danger

to the environment or human health, However, it is required by regulation that

FOGs be treated by the same safety and spill response practices applied to petroleum

oils due to its definition under the general definition of ‘‘oil’’ as specified by 40

CFR 112.2 and Clean Water Act Section 311(a)(1), (USEPA 1997). It is evident

from the literature that discharges of FOGs into open seas, coastlines, or river bodies

can be as harmful as spills of petroleum oil, resulting in environmental and economic

damage (Bucas and Saliot 2002).

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Therefore, it is important to identify effective bacteria that degrade lipids through

culture-dependent technique. The lipid- degrading bacteria usually produce

triacylglycerol acylhydrolases which influences the structural arrangements of fats

and oil such as oleic acid and Tween 20, olive and palm oils (El-Masry et al. 2004).

El-Bestawy et al. (2005) reported many pathways through which triacylglycerol

acylhydrolases are produced and secreted by microorganisms (Nizam & Zuhan,

2008). Margesin and Schinner (2001) stated that hydrocarbon polluted environments

are categorized based on extreme temperatures, pH, and salinity. Despite such

extreme environmental conditions, microorganisms thrive and adapt to the

environment thereby utilising the oils as potential energy means.

The vegetable oil spill is the most common form of environmental pollution in the

United States which attracts many scientific attentions in the field of its toxicology

and the probable degradation response under aerobic and anaerobic conditions (Al-

Darbi et al. 2005). Palm oil pollution in the European seas has reached the limelight

as it has been proven to be the cause of deaths of dogs and animals consuming the

cold precipitated washed palm oil residues from oily ballast wastewater discharge

into the sea (Cudmore, 2017). The rise in the number of pollution cases in the

European waters is an alarming issue as the European Union has recently tried to ban

the use of palm oil in biodiesel production and pollution cases involving palm oil

will only make the ban a reality in the near future (Tornero et al., 2016).

Thus, ways to remediate oily ballast wastewater containing palm oil is in urgent

demand. In addition to bio treatment, many techniques used to remedy marine oil

spills involve mechanical and chemical methods. Boben and Yanting (1996)

recommended using in-situ burning as the primary means of response to the major

oil spill occurrence. However, these methods are expensive and not practical to treat

oily ballast wastewater that contains mainly palm oil and small amounts of minerals

in seawater. The treatment of oily ballast wastewater tends to focus on tropical and

subtropical regions, and concerns on the reduction of invasive microorganisms

chiefly phytoplankton and hence treatment usually involve filtration and UV

treatment (Tsolaki & Diamadopoulos, 2009). With the increasing problem in oily

ballast wastewater containing toxic hydrocarbon and lipid wastes, the use of

microorganism is being developed (Ganti et al., 2003) but is still limited. Further

aggravating this issue is that biological treatment for oily ballast wastewater in cold

seas, especially in the European waters centring on palm oil, is absence due to the

difficulty in finding suitable microorganisms to treat the waste before being

discharged. The ongoing reclassification of oil palm ballast waste as a hazardous

substance by the European Union will seriously affect Malaysian economy (Höfer &

Mez 2008) being the second world most palm oil production and exportation after

Indonesia.

Considering the facts discussed above, this research was conducted to address the

issue of oily ballast wastewater treatment using microorganisms as an alternative to

physicochemical methods. More specifically, this study aims to investigate the

aerobic biodegradability of palm oil under a simulated oily ballast wastewater

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conditions. Furthermore, refined commercial palm oil was selected for this study

which is one of the main sources of vegetable oil (Przybylski et al., 2005).

1.2 Problem statement

This is the first attempt to use an Antarctic diesel-degrader as a candidate for the

subtropical conditions remediation of oily ballast wastewater-containing palm oil as

a major pollutant under marine environment. Major issues that can hamper the

successfulness of this study include the ability of the bacterium to degrade high

concentration of palm oil in the simulated oily ballast wastewater. Another issue is

whether the degradation of oil palm can be effective under high salts and cold

conditions that are present in the Atlantic seas. The degradation will be carried out

under controlled conditions that can be found in the oily ballast wastewater.

As of now, there is no attempt in studying the possibility of using cold region

microbes in remediating oily ballast wastewater from palm oil tankers. This is the

first such study, and the effect of environmental factors in palm oil biodegradation

needs to be studied and optimized under cold conditions.

1.3 Objectives

The general objective of the study is to remediate oily ballast wastewater under

temperate condition using Antarctic bacteria.

The specific objectives of the study are;

1. To investigate the biodegradability of palm oil under simulated oily ballast

wastewater conditions by Rhodococcus erythropolis ADL36 using one factor

at a time (OFAT).

2. To optimize the degradation condition using response surface methodology

(RSM).

3. To determine the growth parameters of the bacteria in utilizing the oil using

kinetic models.

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6 REFERENCES

AbdEl-Mongy, Shukor, M., A., M.,S., Hussein, S., Ling A., P., K., Shamaan, N., A.

& Shukor, M.,Y. (2015) Isolation and characterization of a molybdenum-

reducing, phenol- and catechol-degrading Pseudomonas putida strain amr-12

in soils from Egypt. Scientific Study & Research Chemistry & Chemical

Engineering, Biotechnology, Food Industry 16, 353–369.

Abou-Shanab R., A., I., Khalafallah, M., A., Emam, N., F., Aly, M., A., Abou-Sdera

S., A., Matter, I., A. (2012) Characterisation and identification of carbofuran-

utilising bacteria isolated from agricultural soil. Chemistry and Ecology 28,

193–203.

Adhvaryu, A., Erhan, S., & Perez, J. (2002). Wax appearance temperatures of

vegetable oils determined by differential scanning calorimetry: effect of

triacylglycerol structure and its modification. Thermochimica acta, 395, 191-

200.

Agarry, S., Audu, T., & Solomon, B. (2009). Substrate inhibition kinetics of phenol

degradation by Pseudomonas fluorescence from steady state and wash-out

data. International Journal of Environmental Science and

Technology:(IJEST), 6, 443.

Ahmad, S., A., Ahamad, K., N., E., K., Johari, W., L.,W., Halmi, M., I., E., Shukor,

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