detection of hazardous heterocyclic hydrocarbon from water … of hazardous... · 2015-09-10 · os...

DETECTION OF HAZARDOUS HETEROCYCLIC HYDROCARBON FROM WATER SAMPLES

Yeap Kim Heng

QD

Y38 2 13 Bachelor of Science with Honours

(Resource BiotechnoJpgy) 2013

400

oS Pusat Khidmat Maklumat Akademik UNlVERSm MAlAYSIA SARAWAK

Detection of Hazardous Heterocyclic Hydrocarbon from Water Samples

Yeap Kim Beng (28678) PKHIDMAT MAKLUMAT AKADEMIK

111111111 IlimlIIII 11111 1000246625

A final project report submitted in partial fulfilment of the Final Year Project II (STF 3015) Course

Supervisor Dr Azham Bin Zulkharnain

Resource Biotechnology

Molecular Biology

Faculty ofResource Science and Technology

University Malaysia Sarawak

2013

ACKNOWLEDGEMENT

First of all I would like to thank the Department of Molecular Biology University

Malaysia Sarawak for giving me the opportunity to fulfil my Final Year Project I would

also like to express my deepest gratitude to my supervisor Dr Azham bin Zulkhamain to

have faith in me for letting me to learn valuable knowledge and experience from him

Thank you very much for all the useful advices and ideas that aided me in completing this

project and report writing

I would also like to thank all the master students of Molecular Genetic laboratory Kak

Jane Sebestian Taka Abang Farith Alvin and Simon for their generosity in helping and

give assistance throughout the project I would like to say thanks to all my friends for

sharing their ideas and advices as we shared most of the moment working together in the

laboratory Last but not least I would like to thank my family for their support and

advices for me when I am feeling discouraged

I

DECLARATION

I declared that this project entitled Detection of Hazardous Heterocyclic Hydrocarbon

from Water Samples is the result ofmy own research except as cited in the references

This project has not been accepted for any degree and is not concurrently submitted in

candidature ofany other degree

k Signature -robullbullbullbullbull bullbullbullbullbullbullbullbullbullbullbullbullbullbullbullbullbullbullbullbullbullbullbullbullbullbullbullbullbullbull bullbull

Name YEAP KIM HENG

Date 2 JUL Y 2013

II

Pusat Khidmat Maklumat Akademik UNlVERSm MALAYSIA SARAWAK

TABLE OF CONTENTS

ACKNOWLEDGEMENT I

DECLARATION 11

TABLE OF CONTENTS III

LIST OF ABBREVlA TION VI

LISTS OF TABLES VIII

LIST OF FIGURES X

ABSTRACT 1

10 INTRODUCTION 2

20 LITERATURE REVIEW 5

21 Bioremediation 5

22 Marine Hydrocarbon Degrading Bacteria 6

23 Heterocyclic Hydrocarbon 7

231 Biphenyl and Carbazole 11

24 Heterocyclic Hydrocarbon Detection Method 13

241 Spectrophotometer 13

25 Factors Affecting the Bioremediation ofHeterocyclic Hydrocarbon 14

30 MATERIALS AND METHODS 15

31 Source of Bacteria 15

32 Media Preparation 16

III

321 PreparationofONR7aMedia 16

322 Preparation of Substrate Concentration 17

323 Preparation of ONR7a (Liquid Media) with Substrate 17

324 Preparation ofONR7a (Solid Media) with Substrate 17

33 Cultivation ofBacteria 18

34 Evaluation ofHeterocyclic Hydrocarbon Detection Kit 18

341 Alteration of the pH of the Media 18

342 Determination of Detection Limit 18

35 Production of Heterocyclic Hydrocarbon Detection KiL 19

36 Bacterial Degradation using Different Heterocyclic Hydrocarbon 19

40 RESULTS 20

41 Enrichment Culture in Marine Agar 20

42 Growth on ONR7a Liquid Media 21

43 Isolation and Subculture of Bacteria in Selective Media Agar 22

44 Identify the Degree of Sensitivity and Effectiveness of Bacteria in Different

Substrate Concentration 23

441 Fonnation ofHOPDA using SEMOl 23

442 Formation ofHOPDA using PSOl 24

443 Formation ofHOPDA using MTOl 24

444 Formation ofHOPDA using MOl 25

IV

445 Formation of HOADA using M02 25

45 Identify the Optimum pH for the Enzyme to Undergo Biodegradation of

Heterocyclic Hydrocarbon 26

451 Formation ofHOPDA using SEM01 27

452 Formation of HOPDA using PSOI 28

453 Formation ofHOPDA using MT01 29

454 Formation of HOPDA using MOl 30

455 Formation ofHOADA using M02 31

46 Development ofa Heterocyclic Compound Detection System 32

50 DISCUSSION 36

60 CONCLUSION 43

61 Conclusion 43

62 Recommendations 44

REFERENCES 45

APPENDIX 49

v

LIST OF ABBREVIATION

HOADA

HOPDA

PAH

TSAN

run

HPLC

GC

FID

HCI

NaOH

g

ml

DMSO

ONR7a

rpm

~L

2-Hydroxy-6-oxo-(2-aminophenyl)-hexa-2E4Z-dienoate

2-Hydroxy-6-oxo-6-phenylhexa-24-dienoate

Polycyclic Aromatic Hydrocarbon

Total Surface Area I Volume

nanometer

High Performance Liquid Chromatography

Gas Chromatography

Flame Ionization Detector

Hydrochloric acid

Sodium Hydroxide

gram

millilitre

Dimethyl Sulphoxide

artificial sea water

rotation per minute

microliter

VI

I

MgCh Magnesium Chloride

K Potassium

Mg Magnesium

Degree Celcius

vn

LISTS OF TABLES

Table 1 Heterocyclic Hydrocarbon Degrading Bacteria 15

Table 2 Recipe ofONR7a Media 16

Table 3 OD reading of HOPDA (biphenyl intennediate) produced using biodegradation

of SEMOI 49


ofPSOl 49

Table 5 OD reading of HOPDA (biphenyl intermediate) produced using biodegradation

ofMTOl 50


ofMOl 50

Table 7 OD reading of HOADA (carbazole intennediate) produced using biodegradation

ofM02 51


ofSEMOl 51


ofPSOl 52


ofMTOl 52

VIII

Table II OD reading of HOPDA (biphenyl intermediate) produced using biodegradation

of MOl 53

Table 12 OD reading of HOADA (carbazole intermediate) produced using

biodegradation ofM02 53


of MOl at 36 hours 54


ofMTOI at 36 hours 54


ofPSOI at 36 hours 54


ofSEMOI at 36 hours 54


biodegradation ofM02 at 36 hours 54

IX

I

LIST OF FIGURES

Figure I Molecular Structure of Biphenyl Dibenzofuran Carbazole and

Dibenzothiophene 8

Figure 2 Bacteria enrichment culture in Marine Agar 20

Figure 3 Bacteria culture in ONR7a media with its respective heterocyclic

hydrocarbon 21

Figure 4 Bacteria culture in ONR7a agar with its respective heterocyclic hydrocarbon 22

Figure 5 The formation of HOPDA using SEMOI at different substrate

concentration 23

Figure 6 The formation of HOPDA using PSOI at different substrate concentration 24

Figure 7 The formation ofHOPDA using MTOI at different substrate concentration 24

Figure 8 The formation of HOPDA using MOl at different substrate concentration 25

Figure 9 The formation ofHOADA using M02 at different substrate concentration 25

Figure 10 The formation of HOPDA at different pH using SEMOI 27

Figure II The formation of HOPDA at different pH using SEMOI at 36 hours 27

Figure 12 The formation of HOPDA at different pH using PSOI 28

Figure 13 The formation of HOPDA at different pH using PSQ] at 36

hours 28

Figure 14 The formation ofHOPDA at different pH using MTOI 29

X

Figure 15 The formation of HOPDA at different pH using MT01 at 36 hours 29

Figure 16 The formation of HOPDA at different pH using MOl 30

Figure 17 The formation of HOPDA at different pH using MOl at 36 hours 30

Figure 18 The formation of HOADA at different pH using M02 31

Figure 19 The formation of HOADA at different pH using M02 at 36 hours 31

Figure 20 Heterocyclic hydrocarbon detection system (using different strains of bacteria

to detect different types ofheterocyclic hydrocarbon) 32


to detect heterocyclic hydrocarbon at different concentration) 33

Figure 22 Heterocyclic hydrocarbon detection system (using PS01 to detect biphenyl at

different concentration and pH) 33

Figure 23 Heterocyclic hydrocarbon detection system (using MOl to detect biphenyl at


Figure 24 Heterocyclic hydrocarbon detection system (using SEM01 to detect biphenyl

at different concentration and pH) 34

Figure 25 Heterocyclic hydrocarbon detection system (using MT01 to detect biphenyl at


Figure 26 Heterocyclic hydrocarbon detection system (using M02 to detect carbazole at


XI

I

r I

Detection ofHazardous Heterocyclic Hydrocarbon from Water Samples

Yeap Kim Heng (28678)

Resource Biotechnology Molecular Biology

Faculty ofResource Science and Technology University Malaysia Sarawak

ABSTRACT

Environmental pollution is getting more severe with the contaminations caused by chemicals such as heterocyclic hydrocarbons from oil spills industrial activities and the use of chemicals which reside in the environment for extensive period of time In this study the feasibility of detection method for hazardous heterocyclic bydrocarbon using degrading bacteria in water sample is tested Sample can be detected and analyse for the concentration of the heterocyclic hydrocarbon in the water source by using the visible light spectrophotometer Several parameters such as pH and substrate concentration for the detection of heterocyclic hydrocarbon have been successfully indentified Results show that all the marine bacteria used in this study are less efficient at detecting heterocyclic hydrocarbon at pH that are lower than 7 except for ThaJassospira profundimaris which can degrade carbazole at the highest rate pH 65 and not able to degrade heterocyclic hydrocarbon at pH 95 It is also found that the system is able to detect the presence of heterocyclic hydrocarbon at a substrate concentration as low as 01 ppm

Keywords Bioremediation Carbazole heterocyclic hydrocarbon degrading bacteria heterocyclic

hydrocarbon

ABSTRAK

Pencemaran alom sekilar semakin leruk diakiballcan daripada pencemaran yang disebablcan oleh bahan kimia seperli hidrolcarbon heterosiklik disebablcan oleh tumpahan minyak aklivili perindustrian dan pengguflQQ1l bahan kimia yang ditinggal pada masa yang panjang Dalam lcajian ini lcaedah pengesanan hidrokarbon heerosiik yang berhazad menggunalcan bacteria dalam sam pel air lelah dilcaji Sampel dianalisis lenlang 1cepelcalan hidrolcarbon heterosiklik dalam sumber air lelah dijalanlcan dengan menggunalcan spelctrofotometer Beberapa parameter seperti pH dan konsentrasi substraJ unluk mengesan hidrokorbon helerosiIcJik telah berjaya dikenalpasli Kepulusan menunjukJcan bahawa semua bacleria laul yang digunalcan daam lcajian ini adalah kurang berkesan untuk mengesan hidrolcarbon helerosiklik pada pH yang rendah daripada 7 kecuali Thalassospira profundimaris yang boleh mendegradasilcan lcarbazol dengan Icadar terlinggi pada pH 65 dan tidak mampu untuk mendegradasilcan hidrolcarbon heterosikik pada pH 95 la juga mendapali bahawa system ini dapat mengesan kehadiran hidrolcarbon helerosiklik pada kepelcalan substrat serendah 01 ppm

Kala cunei Biopemulihan lcarbazol bakteria degradasi hidrolcarbon heterosiklik hidrolcarbon

helerosiIcJik

1

10 INTRODUCTION

Heterocyclic hydrocarbons are compound that consists of at least two different

kinds of elements in its ring This compound containing nitrogen sulphur or oxygen

have been detected in air soil sewage sludge marine environments and freshwater

sediments (Eisentraeger et al 2008) The presence of large numbers of compounds in

the environment will either benefits or harm to the environment According to

Eisentraeger et al (2008) the environmental effects of these compound are extensively

researched to understand its toxicity mechanism but still the data on its toxicity is scarce

It was found that heterocyclic hydrocarbon has the potential to cause ecotoxic (tested

with algae and dapbnids) cancer (tested with Salmonella and micosome test) and

mutation to an organism if is exposed in high concentration or a long period of time

In 1989 there were more than 200000 barrels of crude oil spill from the oil tanker

of Exxon Valdez in Prince William Sound Alaska (Atlas 1995) In addition the recent

pollution case in Mexico the blowout of the Deepwater Horizon (DH) that has happened

due to oil spillage has led to the major breakthrough in the heterocyclic hydrocarbon

bioremediation and a total of 24 bacterial strains were isolated from the oiled beach and

confinned as a oil-degrading microorganisms (Kostka et al 2011) Due to these

incidents the negative environmental effects of the heterocyclic hydrocarbon were

discovered For example it was found out that polycyclic aromatic hydrocarbon (PAH)

bas the ability to act as endocrine disruptor and cause the hormonal level of an organism

unregulated (Swedenborg et al 2009) Due to the knowledge that has been obtain by

understanding the effect that heterocyclic compounds have on the environment it is

2

crucial to create an efficient and cheap way for the detection of heterocyclic hydrocarbon

to detect the heterocyclic hydrocarbon present in the water

In this research water samples that are taken from various sources in Kuching in

order to detect the hazardous heterocyclic hydrocarbon The heterocyclic hydrocarbons

such as carbazole dibenzofuran dibenzothiophine fluorene and biphenyl that are going

to be studied because of its toxicity characteristic that is harmful to the environment

Furthermore the detection of the heterocyclic hydrocarbon is important in order to

determine the threshold level in that source This is because if the concentration is high

then treatment should have taken pace to reduce the damage to the habitat of that area In

addition this study uses marine bacteria that are able to uptake the harmful heterocyclic

hydrocarbon as an energy source for its own use Most bioremediation studies on the

decomposition of hannful heterocyclic hydrocarbon use terrestrial bacteria and few of

them uses marine bacteria Therefore the water source that is polluted with this

compound can use the marine bacteria to treat or reduce the concentration

Due to arising issue on pollution and oil spillage around the global people are

finding ways to reduce and avoid this issue Hazardous heterocyclic hydrocarbon not

only brings harm to the balance of ecosystem but also to the health of the hwnan being

who exposed to the substances for a long period of time or in high concentration In

addition there is need to be aware of the negative effect that it may cause such as

increase the viscosity of the sea reduce the rate of oxygen diffusion in to the sea and the

health of sea organisms If the problem is not taken care seriously the food and water

sources soon will become a problem to us as well Furthermore the measurement of the

concentration of heterocyclic hydrocarbon in an area also need to be known and assure

3

that it is safe to do any activity in that area by public This is why it is very important to

develop analytical methods for monitoring the presence of heterocyclic hydrocarbon in

the environment Thus the development of a cost effective heterocyclic compounds

detection system need to be done and there is a need to detect pollution level in water

sample quickly in order to monitor the condition of the water source periodically

The objectives of this study are to

- develop a heterocyclic compound detection system using marine bacteria

- optimize the reaction for detecting heterocyclic compounds

- identify factors affecting the reaction for detecting heterocyclic compounds

4

Pusat Khidmat Maklumat Akademik UNIVERSm MALAYSIA SARAWAK

10 LITERATURE REVIEW

11 BioremediatioD

Bioremediation is a biological process that widely used to reduce or degrade the

environmental pollution that is caused by human activity accident and natural disaster

This technique of reducing the pollution is gaining its reputation for being efficient and

environmentally friendly by using degrading bacteria to speed up the rate of degrading

the harmful compound However this process required very explicit information on the

types of mechanism that can be performed by the bacteria because not all the bacteria

able to up the pollutants as their source and the rate of degradation is still consider slow

for a large scale clean up (Atlas 1995) Besides in order to be able to do full scale c1eanshy

up in an area the condition of the area need to be examined and compatible to the

capability of the bacteria to adapt the environment (Balha et al~ 1998) Thus many

people nowadays use this technique for cleaning marine oil spillage although there are

still many things need to be improved Although many terrestrial bacteria that has the

ability to degrade heterocyclic compound has been identified only a few of marine

bacteria was found or examined by the scientist for this aspect (Nagashima et aI 2009)

5

22 Marine Hydrocarbon Degrading Bacteria

Hydrocarbon degrading bacteria present in soil and marine environment According

to Atlas (1995) the population of hydrocarbon degrading bacteria constitutes less than

1 of the total microbial community but when the presence of hydrocarbon in an area

the population of these bacteria will shoot up to 10 as compared to the total microbial

community One of the advantages of the marine hydrocarbon degrading bacteria over

the terrestrial is the capability to survive in the seawater which the terrestrial cannot adapt

to the conditions such as the pH salinity temperature changes and water current

Besides according to Takacs et al (1964) marine bacteria are also able to maintain the

osmotic pressure at the high salinity by maintain the sodium ion and potassium ions

concentration at the membrane which most of the terrestrial microbes cannot Thus

many scientists are trying to fmd marine bacteria that have the ability to degrade harmful

heterocyclic compound The marine bacteria that are used are Pseudomonas

Pachastrellae strain M03 Thalassospira profundimaris strain MOl and M02

Chromohalobacter morismortui strain SEMO 1 and SEM02 and Thalassospira

Xwmenensis strain MTO1 and MT02 in this research

6

23 Heterocyclic Hydrocarbon

Heterocyclic hydrocarbons such as Carbazole Dibenzofuran Dibenzothiophene

and Biphenyl as shown in figure 1 are compound that consists of at least two different



sediments (Eisentraeger et al 2008) The presence of a large number of compound in the

environment will somewhat affect the surrounding either it gives benefits or brings harm

to the surrounding The environmental effects of this compound are extensively

researched to understand its toxicity mechanism but still the data on its toxicity is still

scarce Based on the research of Eisentraeger et al (2008) that has been done it was

found that heterocyclic hydrocarbon has the potential to cause ecotoxic (tested with algae

and daphnids) cancer (tested with Salmonella and micosome test) and mutation to an

organism if is exposed in high concentration or a long period of time

7

Biphenyl dibenzofuran

Dibenzothiophene carbazole

Figure 1 Molecular Structure of Biphenyl Dibenzofuran

Carbazole and Dibenzothiophene

8

Carbazole dibenzofuran biphenyl and dibenzothiophene are heterocyclic

hydrocarbon that can contaminate the environment According to Bressler and Federak

(2000 p397) In the process of biodegrading these compounds bacteria oxidise those

compounds in three most susceptible ways

(i) the naphthalene-like attac~ in which one of the aromatic rings is oxidized to a

dihydrodiol

(ii) an angular dioxygenase attack in which the carbon bonded to the methylene group in

fluorene or to the heteroatoms in the analogs and the adjacent carbon in the aromatic ring

are both oxidized

(iii) the five-membered ring attac~ in which the methylene carbon atom in fluorene or

the sulfur atom in dibenzothiophene is oxidized

By understanding the method that the bacteria most probably will be using to

degrade the heterocyclic hydrocarbon the intennediate and final products can be

predicted much easily Thus we can identify the heterocyclic hydrocarbon by observing

the colour changes on the medium due to the intennediate or fmal product Furthennore

the concentration of the hydrocarbon present in the water sample can be determined by

running the mediwn in spectrophotometer

9

The study of Was sen berg et al (2009) shows that the combination of carbazole and

dibenzothiophene with the co-occurance ofPAH-type aryl hydrocarbon can enhance fish

embroyo toxicity although both the heterocyclic hydrocarbon is not embryo toxic itself

Thus this proves that the presence of these compounds in the water can bring damage to

the balance of ecosystem Although dibenzothiophene is not an embryotixic it is still the

most persistent and toxic P AH in marine environment and it was found in high

concentration from 22 different sampling sites in South China Sea (Yang et al 1998)

According to Yang et al (1998) the content of dibenzothiophene is found higher

nearshore than offshore sediments and is consistent to the distribution trend of organic

carbon in the sampling sites In addition to that it is showed that the concentration of

dibenzotiophene is correlated with the organic carbon distributed in the area using linear

regression (Yang et al 1998) Thus it is reckoned that the sources of dibenzothiophene

include terrestrial runoff oil pollution and the airborne particulates formed from

combustion processes (Yang et al 1998)

10

131 Biphenyl and Carbazole

Biphenyl is a two aromatic ring organic compound that is able to be degraded by all

the marine bacteria used in this experiment Although three enzymes are needed for the

degradation of biphenyl in order to obtain its intermediate (HOPDA) yet the degradation

rate of biphenyl is still much faster than degrading carbazole (Linden amp Sun 2011) lbis

is maybe due to its molecular and physical properties of the compound Although

biphenyl is insoluble in water the hydrophobic properties of biphenyl are not as strong as

carbazole This can be seen when same amount of carbazole and biphenyl is added into

the water but carbazole become a huge precipitate while biphenyl only insoluble in the

form of powder in water lbis allows larger TSAIV for the bacteria to come in contact

with biphenyl rather than carbazole

Although the degradation of carbazole only needs two enzyme to obtain the

intennediate product HOADA the molecular structure and the physical properties of

carbazole makes it hard for the enzyme to come in contact or approach the heterocyclic

hydrocarbon (f~ 2012) The insoluble in water properties of carbazole might also

cause the reduction of the chance of bacteria to come in contact with carbazole (Bastiaens

et aI 2(00) The bacteria might also need to figure a way to identify the conpound and

degrades it In addition to that carbazole has three aromatic rings that are arrange in such

a way that it is very stable and the bond are hard to be broken (Watts 1998) Moreover

the more aromatic ring presence in a compound the harder it is for it to be degraded

11

oS Pusat Khidmat Maklumat Akademik UNlVERSm MAlAYSIA SARAWAK

Detection of Hazardous Heterocyclic Hydrocarbon from Water Samples

Yeap Kim Beng (28678) PKHIDMAT MAKLUMAT AKADEMIK

111111111 IlimlIIII 11111 1000246625

A final project report submitted in partial fulfilment of the Final Year Project II (STF 3015) Course

Supervisor Dr Azham Bin Zulkharnain

Resource Biotechnology

Molecular Biology

Faculty ofResource Science and Technology

University Malaysia Sarawak

2013

ACKNOWLEDGEMENT













I

DECLARATION






Name YEAP KIM HENG

Date 2 JUL Y 2013

II


TABLE OF CONTENTS

ACKNOWLEDGEMENT I

DECLARATION 11




LIST OF FIGURES X

ABSTRACT 1

10 INTRODUCTION 2


21 Bioremediation 5










III











40 RESULTS 20










IV










50 DISCUSSION 36

60 CONCLUSION 43

61 Conclusion 43


REFERENCES 45

APPENDIX 49

v


HOADA

HOPDA

PAH

TSAN

run

HPLC

GC

FID

HCI

NaOH

g

ml

DMSO

ONR7a

rpm

~L





nanometer


Gas Chromatography


Hydrochloric acid

Sodium Hydroxide

gram

millilitre

Dimethyl Sulphoxide


rotation per minute

microliter

VI

I


K Potassium

Mg Magnesium

Degree Celcius

vn

LISTS OF TABLES




of SEMOI 49


ofPSOl 49


ofMTOl 50


ofMOl 50


ofM02 51


ofSEMOl 51


ofPSOl 52


ofMTOl 52

VIII


of MOl 53













IX

I

LIST OF FIGURES


Dibenzothiophene 8



hydrocarbon 21



concentration 23









hours 28


X




















XI

I

r I





ABSTRACT



hydrocarbon

ABSTRAK



helerosiIcJik

1

10 INTRODUCTION






















2
























3










4



11 BioremediatioD















5

















6














7





8






dihydrodiol



are both oxidized









9















10





















11

ACKNOWLEDGEMENT













I

DECLARATION






Name YEAP KIM HENG

Date 2 JUL Y 2013

II


TABLE OF CONTENTS

ACKNOWLEDGEMENT I

DECLARATION 11




LIST OF FIGURES X

ABSTRACT 1

10 INTRODUCTION 2


21 Bioremediation 5










III











40 RESULTS 20










IV










50 DISCUSSION 36

60 CONCLUSION 43

61 Conclusion 43


REFERENCES 45

APPENDIX 49

v


HOADA

HOPDA

PAH

TSAN

run

HPLC

GC

FID

HCI

NaOH

g

ml

DMSO

ONR7a

rpm

~L





nanometer


Gas Chromatography


Hydrochloric acid

Sodium Hydroxide

gram

millilitre

Dimethyl Sulphoxide


rotation per minute

microliter

VI

I


K Potassium

Mg Magnesium

Degree Celcius

vn

LISTS OF TABLES




of SEMOI 49


ofPSOl 49


ofMTOl 50


ofMOl 50


ofM02 51


ofSEMOl 51


ofPSOl 52


ofMTOl 52

VIII


of MOl 53













IX

I

LIST OF FIGURES


Dibenzothiophene 8



hydrocarbon 21



concentration 23









hours 28


X




















XI

I

r I





ABSTRACT



hydrocarbon

ABSTRAK



helerosiIcJik

1

10 INTRODUCTION






















2
























3










4



11 BioremediatioD















5

















6














7





8






dihydrodiol



are both oxidized









9















10





















11

DECLARATION






Name YEAP KIM HENG

Date 2 JUL Y 2013

II


TABLE OF CONTENTS

ACKNOWLEDGEMENT I

DECLARATION 11




LIST OF FIGURES X

ABSTRACT 1

10 INTRODUCTION 2


21 Bioremediation 5










III











40 RESULTS 20










IV










50 DISCUSSION 36

60 CONCLUSION 43

61 Conclusion 43


REFERENCES 45

APPENDIX 49

v


HOADA

HOPDA

PAH

TSAN

run

HPLC

GC

FID

HCI

NaOH

g

ml

DMSO

ONR7a

rpm

~L





nanometer


Gas Chromatography


Hydrochloric acid

Sodium Hydroxide

gram

millilitre

Dimethyl Sulphoxide


rotation per minute

microliter

VI

I


K Potassium

Mg Magnesium

Degree Celcius

vn

LISTS OF TABLES




of SEMOI 49


ofPSOl 49


ofMTOl 50


ofMOl 50


ofM02 51


ofSEMOl 51


ofPSOl 52


ofMTOl 52

VIII


of MOl 53













IX

I

LIST OF FIGURES


Dibenzothiophene 8



hydrocarbon 21



concentration 23









hours 28


X




















XI

I

r I





ABSTRACT



hydrocarbon

ABSTRAK



helerosiIcJik

1

10 INTRODUCTION






















2
























3










4



11 BioremediatioD















5

















6














7





8






dihydrodiol



are both oxidized









9















10





















11


TABLE OF CONTENTS

ACKNOWLEDGEMENT I

DECLARATION 11




LIST OF FIGURES X

ABSTRACT 1

10 INTRODUCTION 2


21 Bioremediation 5










III











40 RESULTS 20










IV










50 DISCUSSION 36

60 CONCLUSION 43

61 Conclusion 43


REFERENCES 45

APPENDIX 49

v


HOADA

HOPDA

PAH

TSAN

run

HPLC

GC

FID

HCI

NaOH

g

ml

DMSO

ONR7a

rpm

~L





nanometer


Gas Chromatography


Hydrochloric acid

Sodium Hydroxide

gram

millilitre

Dimethyl Sulphoxide


rotation per minute

microliter

VI

I


K Potassium

Mg Magnesium

Degree Celcius

vn

LISTS OF TABLES




of SEMOI 49


ofPSOl 49


ofMTOl 50


ofMOl 50


ofM02 51


ofSEMOl 51


ofPSOl 52


ofMTOl 52

VIII


of MOl 53













IX

I

LIST OF FIGURES


Dibenzothiophene 8



hydrocarbon 21



concentration 23









hours 28


X




















XI

I

r I





ABSTRACT



hydrocarbon

ABSTRAK



helerosiIcJik

1

10 INTRODUCTION






















2
























3










4



11 BioremediatioD















5

















6














7





8






dihydrodiol



are both oxidized









9















10





















11











40 RESULTS 20










IV










50 DISCUSSION 36

60 CONCLUSION 43

61 Conclusion 43


REFERENCES 45

APPENDIX 49

v


HOADA

HOPDA

PAH

TSAN

run

HPLC

GC

FID

HCI

NaOH

g

ml

DMSO

ONR7a

rpm

~L





nanometer


Gas Chromatography


Hydrochloric acid

Sodium Hydroxide

gram

millilitre

Dimethyl Sulphoxide


rotation per minute

microliter

VI

I


K Potassium

Mg Magnesium

Degree Celcius

vn

LISTS OF TABLES




of SEMOI 49


ofPSOl 49


ofMTOl 50


ofMOl 50


ofM02 51


ofSEMOl 51


ofPSOl 52


ofMTOl 52

VIII


of MOl 53













IX

I

LIST OF FIGURES


Dibenzothiophene 8



hydrocarbon 21



concentration 23









hours 28


X




















XI

I

r I





ABSTRACT



hydrocarbon

ABSTRAK



helerosiIcJik

1

10 INTRODUCTION






















2
























3










4



11 BioremediatioD















5

















6














7





8






dihydrodiol



are both oxidized









9















10





















11










50 DISCUSSION 36

60 CONCLUSION 43

61 Conclusion 43


REFERENCES 45

APPENDIX 49

v


HOADA

HOPDA

PAH

TSAN

run

HPLC

GC

FID

HCI

NaOH

g

ml

DMSO

ONR7a

rpm

~L





nanometer


Gas Chromatography


Hydrochloric acid

Sodium Hydroxide

gram

millilitre

Dimethyl Sulphoxide


rotation per minute

microliter

VI

I


K Potassium

Mg Magnesium

Degree Celcius

vn

LISTS OF TABLES




of SEMOI 49


ofPSOl 49


ofMTOl 50


ofMOl 50


ofM02 51


ofSEMOl 51


ofPSOl 52


ofMTOl 52

VIII


of MOl 53













IX

I

LIST OF FIGURES


Dibenzothiophene 8



hydrocarbon 21



concentration 23









hours 28


X




















XI

I

r I





ABSTRACT



hydrocarbon

ABSTRAK



helerosiIcJik

1

10 INTRODUCTION






















2
























3










4



11 BioremediatioD















5

















6














7





8






dihydrodiol



are both oxidized









9















10





















11


HOADA

HOPDA

PAH

TSAN

run

HPLC

GC

FID

HCI

NaOH

g

ml

DMSO

ONR7a

rpm

~L





nanometer


Gas Chromatography


Hydrochloric acid

Sodium Hydroxide

gram

millilitre

Dimethyl Sulphoxide


rotation per minute

microliter

VI

I


K Potassium

Mg Magnesium

Degree Celcius

vn

LISTS OF TABLES




of SEMOI 49


ofPSOl 49


ofMTOl 50


ofMOl 50


ofM02 51


ofSEMOl 51


ofPSOl 52


ofMTOl 52

VIII


of MOl 53













IX

I

LIST OF FIGURES


Dibenzothiophene 8



hydrocarbon 21



concentration 23









hours 28


X




















XI

I

r I





ABSTRACT



hydrocarbon

ABSTRAK



helerosiIcJik

1

10 INTRODUCTION






















2
























3










4



11 BioremediatioD















5

















6














7





8






dihydrodiol



are both oxidized









9















10





















11

I


K Potassium

Mg Magnesium

Degree Celcius

vn

LISTS OF TABLES




of SEMOI 49


ofPSOl 49


ofMTOl 50


ofMOl 50


ofM02 51


ofSEMOl 51


ofPSOl 52


ofMTOl 52

VIII


of MOl 53













IX

I

LIST OF FIGURES


Dibenzothiophene 8



hydrocarbon 21



concentration 23









hours 28


X




















XI

I

r I





ABSTRACT



hydrocarbon

ABSTRAK



helerosiIcJik

1

10 INTRODUCTION






















2
























3










4



11 BioremediatioD















5

















6














7





8






dihydrodiol



are both oxidized









9















10





















11

LISTS OF TABLES




of SEMOI 49


ofPSOl 49


ofMTOl 50


ofMOl 50


ofM02 51


ofSEMOl 51


ofPSOl 52


ofMTOl 52

VIII


of MOl 53













IX

I

LIST OF FIGURES


Dibenzothiophene 8



hydrocarbon 21



concentration 23









hours 28


X




















XI

I

r I





ABSTRACT



hydrocarbon

ABSTRAK



helerosiIcJik

1

10 INTRODUCTION






















2
























3










4



11 BioremediatioD















5

















6














7





8






dihydrodiol



are both oxidized









9















10





















11


of MOl 53













IX

I

LIST OF FIGURES


Dibenzothiophene 8



hydrocarbon 21



concentration 23









hours 28


X




















XI

I

r I





ABSTRACT



hydrocarbon

ABSTRAK



helerosiIcJik

1

10 INTRODUCTION






















2
























3










4



11 BioremediatioD















5

















6














7





8






dihydrodiol



are both oxidized









9















10





















11

I

LIST OF FIGURES


Dibenzothiophene 8



hydrocarbon 21



concentration 23









hours 28


X




















XI

I

r I





ABSTRACT



hydrocarbon

ABSTRAK



helerosiIcJik

1

10 INTRODUCTION






















2
























3










4



11 BioremediatioD















5

















6














7





8






dihydrodiol



are both oxidized









9















10





















11




















XI

I

r I





ABSTRACT



hydrocarbon

ABSTRAK



helerosiIcJik

1

10 INTRODUCTION






















2
























3










4



11 BioremediatioD















5

















6














7





8






dihydrodiol



are both oxidized









9















10





















11

I

r I





ABSTRACT



hydrocarbon

ABSTRAK



helerosiIcJik

1

10 INTRODUCTION






















2
























3










4



11 BioremediatioD















5

















6














7





8






dihydrodiol



are both oxidized









9















10





















11

10 INTRODUCTION






















2
























3










4



11 BioremediatioD















5

















6














7





8






dihydrodiol



are both oxidized









9















10





















11
























3










4



11 BioremediatioD















5

















6














7





8






dihydrodiol



are both oxidized









9















10





















11










4



11 BioremediatioD















5

















6














7





8






dihydrodiol



are both oxidized









9















10





















11



11 BioremediatioD















5

















6














7





8






dihydrodiol



are both oxidized









9















10





















11

















6














7





8






dihydrodiol



are both oxidized









9















10





















11














7





8






dihydrodiol



are both oxidized









9















10





















11





8






dihydrodiol



are both oxidized









9















10





















11






dihydrodiol



are both oxidized









9















10





















11















10





















11





















11

detection of hazardous heterocyclic hydrocarbon from water … of hazardous... · 2015-09-10 · os...

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