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EFFECTS OF SURFACTANT TRITON X-IOO ON DEGRADATION OF HETEROCYCLIC HYDROCARBON BY

MARINE BACTERIA

Susan Ling Lin Fei

Bachelor of Science with Honours (Resource Biotechnology)

2014

Effects of Surfactant Triton X-I00 on Degradation of Heterocyclic Hydrocarbon by Marine Bacteria

Susan Ling Lin Fei (33156)

A thesis submitted in partial fulfilment of the requirement of the degree of Bachelor of Science with Honours

(Recourse Biotechnology)

Supervisor Dr Azham Zulkharnain

Resource Biotechnology Programme Department of Molecular Biology

Faculty of Resource Science and Technology University Malaysia Sarawak

2014

ACKNOWNLEDGEMENT

I would like to express my gratitude thanks to my honorable supervisor Dr Azham

Zulkhamain Dr is a very hardworking supervisor and a kind person I sincerely thank for

his guidance and encouragement throughout the course of this study I am pleased to do my

Final Year Project under his supervision

A special thanks to our final year project coordinator Dr Mickey whose give full

effort on guiding us by sending the guideline for writing the reports to our email

I am extremely thankful to our labs master students whose also give a lot of

suggestion and excellent guidance when we are doing our lab work

I would like to thank my lab mate Li Hie Ming for her cooperation and help I am

appreciative to all my batch mates for giving suggestion and moral support during my Lab

work

Last but not least I would like to thanks the Almighty God and my family I could

never complete this Final Year Project without the belief I have in you the Almighty

UNIVERSITI MALAYSIA SARAWAK

Grade____ _

Please tick ltjgt Final Year Project Report o Masters D PhD D

DECLARATION OF ORIGINAL WORK

This declaration is made on the l~ day of June 2014

Students Declaration

I SUSAN LING LIN FEI 33156 Faculty of Resource Science and Technology hereby declare that the work entitled Effects of Surfactant Triton X-I00 on Degradation of Heterocyclic Hydrocarbon by Marine Bacteria is my original work I have not copied from any other students work or from any other sources except where due reference or acknowledgement is made explicitly in the text nor has any part been written for me by another person

~~tIl~ SUSAN -11( UN F~( ~ 3rb

Date submitted Name of the student (Matric No)

Supervisors Declaration

A ~c 2v-[ Ictolt-- ho I I______1-_____ ~_________________________________________ ---- (SU~ERVISOR S NAME~Mer~ptrertlfies that

the work entitled ftt-1l~~~~tl~~~-Y-~~~~L~-H~~~~~~~-~~~-~--j--(TiTLE) as prepared

by the above named student and was submitted to the FACULTY as a partialfull fulfillment for the conferment of __~~h~JII_~_J~___~~~C___~__ Jl~_~~_~~__t~~~~_~~~~31J) (pLEASE INDICATE THE DEGREE) and the aforementioned work to the best of my knowledge is the said students work

Received for examination by (Nam f the supervisor)

4thAM z1I ~ VlMt)l1A

Or Azham Zulkhamain Pensyarah Kanan

Fakulti Sains dan Teknologi Sumber UNlVERSm MAlAYSIA SARAWAr

11

I declare that Projecttrhesis is classified as (Please tick (~))

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research was done) D OPEN ACCESS

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I therefore duly affirm with free consent and willingly declare that this said ProjectThesis shall be placed officially in the Centre for Academic Information Services with the abiding interest and rights as follows

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bull This ProjectThesis or any material data and information related to it shall not be distributed published or disclosed to any party by the student except with UNIMAS permission

Student signature __~K_____-- ~1 (Date) 11~ 20lLf

___ Supervisor signature

~2~laquof Dr Azham Zulkharnain

Pensyarah KananCurrent Address Fakulti Sains dan Teknologi SUmberIt ~ll2 bVV 1i-Y) ~vvn tD~ I 000 ~~ ~Wklvpound uNWiFt5m MALAYSIA SARAWAK

Notes If the ProjectThesis is CONFIDENTIAL or RESTRICTED please attach together as annexure a letter from the organisation with the period and reasons of confidentiality and restriction

[The instrument is duly prepared by The Centre for Academic Information Services]

iii

- - --- -- -- --~

T ABLE OF CONTENTS

ACKNOWNLEDGEMENT

IIDECLARATION

IVTABLE OF CONTENTS

viLIST OF ABBREVIATIONS

VIILIST OF TABLES

viiiLIST OF FIGURES

ABSTRACT

210 INTRODUCTION

420 LITERATURE REVIEW

21 Heterocyclic hydrocarbons 4

22 Bacteria degradation of heterocyclic hydrocarbons 6

623 Marine bacteria

724 Surfactants

241 Characteristics of surfactants 7

242 Ionic surfactant 9

243 Zwitterionic or amphoteric surfactants 9

244 Non -ionic surfactants 9

245 Application of surfactants in remediation 10

25 Effect of surfactants on biodegradation of heterocyclic hydrocarbon 11

251 Toxicity of surfactants 11

252 Biodegradation of surfactants 12

26 Spectrophotometric analysis 13

30 METHOD AND MATERIALS 14

31 Chemical and Media 14

32 Marine bacteria source 14

IV

33 Media preparation 14

34 Bacteria culture 15

35 Surfactants toxicity test 16

36 Effect of surfactants toward degradation 17

361 Growth preparation 17

362 Harvesting bacteria 17

363 Spectrophotometer analysis 18

40 RESULT 19

41 Utilization of aromatic hydrocarbon 19

42 Effect of Triton X-I00 on bacterial growth 20

421 CFU counting 20

422 Turbidity test 21

43 Biodegradation of DBT and DBF 23

50 DISCUSSION 26

60 CONCLUSION 31

REFERENCES 31

v

LIST OF ABBREVIATIONS

CMC

CAR

DBF

DBP

FLN

PAHs

NAPLs

o

S

N

H

(vv)

(wv)

min

ml

lmiddottI

rpm

J1ID

mm

M

CFUml

Critical micelle concentration

Carbazole

Dibenzofuran

Dibenzothiophene

Fluorene

Polycyclic aromatic hydrocarbons

Non-aqueous phase liquids

Oxygen

Sulfur

Nitrogen

Hour

Percentage of volume per volume of substrate

Percentage of weight per volume of substrate

Percentage

Minute

Celsius

Milliliter ~

Microliter

Revolution per minute

Micrometer

Millimeter

Meter

Colony-fonning units per milliliter

VI

LIST OF TABLES

Table 1 Example of specific bacteria strain degrade heterocyclic hydrocarbon 7

Table 2 Example of surfactants (Source I vankovic amp Hrenovic 20 I 0) 10

Table 3 ONR7a composition 15

Table 4 Glass tube with label and contents 18

Vll

LIST OF FIGURES

Figure 1 Chemical structure of dibenzofuran dibenzothiophene fluorene and 5 carbazole (Source Bressler amp Fedorak 2000)

Figure 2 Fonnation of micelle and micellar solubilization (Source Li amp Chen 8 2009)

Figure 3 Color changes occur in culture media containing (a)DBT and (b)DBF 19

Figure 4 Effect of absence and presence ofTriton X-I 00 at different 20 concentration on the growth of bacterial strain FNSOI (CFU counting)

Figure 5 Effect of absence and presence of Triton X-I 00 at different 22 concentration on the growth of bacterial strain FNSOI (turbidity test)

Figure 6 Effect of absence and presence of Triton X-I 00 at different 25 concentration on the biodegradation of dibenzothiophene (DBT)

Figure 7 Effect of absence and presence ofTriton X-I 00 at different 25 concentration on the biodegradation of dibenzofuran (DBF)

viii



Resource Biotechnology Department of Molecular Biology


ABSTRACT

Heterocyclic hydrocarbon contaminated environment has caused negative impact toward human health and ecosystem Bioremediation can be used to decontaminate heterocyclic hydrocarbon from the environment However biodegradation rate was limited due to the low bioavailability of heterocyclic hydrocarbon toward the microorgani m Thus surfactant is needed to enhance the biodegradation process This study was to determine the effects of different concentration of Triton X-IOO on degradation of aromatic hydrocarbon Bacterial strain FNSOI from previous study was cultured in ONR7a medium supplemented with heterocyclic hydrocarbon incubated at room temperature on rotary shaker until the growth is confirmed Toxicity effects of Triton X-IOO on bacteria growth was determined by CFU counting and turbidity test The average growth rate for control ample was the highest with 0591 Abslday compared to sample with addition of surfactant Triton X-IOO at concentration 01 (vv) or beyond it the bacteria density decreases Few concentration of Triton X-IOO was chosen to determine the effect on the degradation rate by spectrophotometric analysis From this study strain FNSOI degraded OBT and OBF Addition of higher concentration of Triton X-IOO does not enhance the growth of the strain FNSOI and degradation rate of OBT and OBF In conclusion Triton X-IOO is not appropriate to be used together with bacterial strains FNSO I in biodegradation of OBT and OBF

Key words Heterocyclic hydrocarbon Bioremediation Bioavailability Surfactants Biodegradation

ABSTRAK

Persekitarall lercemar dellgall hidrokarbollJHerosiklik telah mellyebabkall kesan negatif terhadap mamtSia dan ekosistem Bioremediasi boleh digunalWi untuk menghilangkan hidrokarbon dari alam sekitar tetapi kadar biodegradasi menjadi terhad disebabkan keterbiosediaan hidrokarbon heterosiklik yang rendah Oleh itll suifaktan diperlukan untuk meningkatkan proses biodegradasi Kajian ini adalah untuk menentukan kesan Triton X-JOO kepada degradasi hidrokarbon Strain FNSOJ dikultur di dalam ONR7a yang dibekalkan dengan hidrokarbon heterosiklik dieram pada suhu bilik di penggongcang putar sehingga pertllmbllhan disahkan Kelan ketoksikan Triton X-JOO pada pertumbuhan bakteria telah ditentukan dengan CFU dan ujian kekeruhan Kadar purata periumbuhan bagi sampel kawalan adalah tertinggi dengan 059J Abslhari Triton X-JOO pada kepekatan 01 (vlv) atau lebih ketumbuhan bakteria berkllranagatl Beberapa kepekatan Triton X-JOO telah dipilih untuk penentukan kesan ke atas kadar degradasi dengan mellgglillakan analisis spektrofotometri Dalam kajian ini strain FNSOJ dapat mendegradasi DBT dan DBF Penambahan kepekatan Triton X-JOO yang lebih tinggi telah menyebabkan penurunan bilangan strain FNS01 dan kadar biodegradasi Kesimpulannya Triton X-JOO tidak sesui digunakan bersama dengan jenis bacteria FNSOJ dalam biodegradasi DBT dan DBF

K y words Hidrokarbon heterosiklik Bioremediasi Keterbiosediaan Suifaktan Biodegradasi

10 INTRODUCTION

Heterocyclic hydrocarbons are compound in which the ring structure consists of more than

one type of atom According to AI-Majed et al (2012) petroleum oil containing

hydrocarbon is primary source of energy in modem era Thus the risk of spill or leaks

often occur in the environment due to the exploration transportation production and

storage of these natural resources by human being (AI-Majed et al 2012) Marine and

terrestrial environment contaminated with these heterocyclic hydrocarbon pollutants have

become a worldwide problem which considerable global concern It has brought a great

impact toward human health marine and terrestrial ecosystems Although several

traditional treatments have been carry out for oil spill cleanup but these methods cannot

degrade the crude oil completely (AI-Majed et al 2012) For instance collection by using

floating booms skimmers and adsorption by natural or synthetic materials usually can

decontaminate less than 15 of spilled oil (Thavasi et al 2011 AI-Majed et al 2012)

According to Zhang et al (2005) biodegradation is proposed as the most effective

method to decontaminate spilled oil from the environment During biodegradation

microorganisms are used to breakdown hydrocarbon pollutant permanently into carbon

dioxide and water or into less toxic compounds (Noor amp Hardjito 2008) However the

heterocyclic hydrocarbon biodegradation is limited due to the low bioavailability of this

low solubility hydrophobic compound to bacteria (Zhang et al 2005) The use of

surfactants has been found to be useful in bioremediation of hydrocarbon polluted sites

since they enhance the crude oil or other hydrocarbon degradation by increasing the

bioavailability of these weekly soluble hydrophobic compounds (Bautistaet al 2009)

Many studies have been conducted to enhance the biodegradation of hydrocarbon by

ing surfactants to decrease the surface tension between heterocyclic hydrocarbon and

the water interphase When surfactant concentration is beyond critical micelle

2

concentration (CMC) surfactants molecule will cluster and form micelles Micelles

provide hydrophobic area in the core region of micelles which increase the solubility of

heterocyclic hydrocarbon

Non-ionic surfactants have positive effect on biodegradation of heterocyclic

hydrocarbon (Louvado et aI 2010) However some of the studies have reported that

surfactants have negative effects due to the competition of surfactants with the

hydrocarbon pollutants and some surfactants are toxic toward heterocyclic hydrocarbon

degrading bacteria (Bautista et aI 2009 Li amp Chen 2009) Thus the use of surfactants in

biodegradation of heterocyclic hydrocarbon process should be optimized for each of the

factors that influence biodegradation in order to reduce the toxic effect of surfactants

(Bautista et aI 2009)Types and concentration of surfactants type of the bacteria used in

the degradation process and also heterocyclic hydrocarbon specificity should be taken in

consideration before a surfactant is chosen for remediation (Bautista et aI 2009 Li ampChen

2009)

The objectives of this present study are

1 To determine the utilization of difference aromatic hydrocarbons by bacterial strain

FNSOI

11 To determine toxicity of surfactant Triton X-I 00 towards bacterial strain FNSO1

iii To determine the effect of the surfactant Triton X-IOO on the degradation of

aromatic hydrocarbon

3


21 Heterocyclic hydrocarbons

Heterocyclic hydrocarbons are the compound with a cyclic structure containing at least one

different atom such as oxygen (0) nitrogen (N) and sulfur (S) They have low solubility in

water These various types of heterocyclic aromatic hydrocarbon have been detected in

aquatic and terrestrial environment due to the increasing consumption of fossil fuel by

human being (Gai et al 2007 Seo et al 2009) The pollutants that face great attention

today are heterocyclic aromatic compound and polycyclic aromatic hydrocarbon (Makkar

amp Rockne 2003) According to Bressler and Fedorak (2000) crude oils creosote and shale

oil contain fluorene (FLN) dibenzofuran (OBF) dibenzothiophene (OBT) and carbazole

(CAR) These compounds are detected in the environment due to the spills of creosote

(Bressler amp Fedorak 2000) Some of the heterocyclic hydrocarbons are persistent in the

environments which can bioaccumulate via the food web and cause severe environmental

and health problem (Seo et al 2009)

Carbazole is a nitrogen heterocyclic aromatic compound (Gai et al 2007)It has a

tricyclic structure with the two benzene rings with six membrane fused on either side of the

five membrane ring containing nitr~ as shown in Figure 1 According to Seo et al

(2009) it is component of petroleum and creosotes which and always deposited with other

polycyclic aromatic hydrocarbons (P AHs) and other aromatic compounds into the

environment CAR and its derivatives are reported to be toxic (OBrien et al as cite in Li

et al 2(06) and mutagenic (Reddy amp Randerath as cited in Li et al 2006)

Oibenzofuran is one of the oxygen heterocyclic aromatic compounds It is an

matic compound with two six membrane benzene rings fused on either side of the five

membrane ring containing oxygen as shown in Figure I It is chemically stable and

4

j

damaged tankers

Dibenzofuran

9 1 8~

7~_~6 0 4a

5

Fluorene

6 5 4

8 1a 9

-

2

3

insoluble in water (Huff et aI 1991) Incomplete combustion of biomass fuel and coal

can cause the discharge of DBF into environment According to Breessler and Fedorak

(2000) it has been widely used as an insecticide

Dibenzothiophene is sulfur heterocyclic hydrocarbon (Gai et aI 2007) It is an

aromatic compound with two six membrane benzene rings fused on either side of the five

membrane ring containing sulfur as shown in Figure 1 According to Van Afferden et a1

(1990) DBT belong to recalcitrant group It can persist in the environment (Seo et aI

2009) The discharge of DBT into aquatic environment is due to the oil spills from

Fluorene is a polycyclic aromatic hydrocarbon (PAH) It consists of three rings as

shown in Figure I and is a major component of fossil fuels and coal derivatives Some

fluorene degradation bacteria from genera Arthrobacter sp (Grifoll as cited in Seo et aI

2009) Mycobacteria sp (Boldrin et a1 as cited in Seoet aI 2009) Pseudomonas sp

(Yanget a1 as cited in Seo et aI 2009) Staphylococcus sp (Monna et aLas cited in Seo et

aI 2009) are able to use FLN as a carbon and energy source have been discovered

Dibenzothiophene

9 1 8~2

7~_~3 6 S 4a 44

5

Carbazole

3 ~3 7~-~270=02

1 8 N 9a 1 I

H

re 1 Chemical structure of dibenzofuran dibenzothiophene fluorene and carbazole (Source Bressler amp Fedorak 2000)

5

I

22 Bacteria degradation of heterocyclic hydrocarbons

Since some of these heterocyclic hydrocarbon compound are toxic mutagenic and

carcinogen which cause severe impact toward human health so several research have been

conducted to examine the biodegradation of these compounds and the use of bacteria in

remediation in order to reduce their concentrations in the environment (Gai et al 2007)

According to Vignesh et al (2011) the use of microorganism to decontaminate

environment polluted by hazardous compounds is known as bioremediation Bacteria are

able to produce dioxygenase enzymes (Breessler amp Fedorak 2000) The connecting atom

o and N atom in DBF and CAR respectively has high electronegativity which serve as

substrate for anguJar dioxygenases enzyme whereas the Sand C connecting atom in the

DBT and FLN respectively have lower electronegativity which must be oxidized first

before enzyme dioxygenases attack them (Breesser amp Fedorak 2000)

23 Marine bacteria

In the past marine bacteria that are able to degrade heterocyclic hydrocarbon and P AHs

have been isolated from several marine environments at different location (Vignesh et al

2011) Marine bacteria have weaker de~y than soil bacteria (Maeda et al 2010) Besides

that specific media that contain similar component like seawater are needed for marine

bacteria isolation (Maeda et al 2010) Media such as ONR7a and natural seawater

medium are growth media thatc~m be used to isolate marine bacteria Some bacteria strain

have been isolated and is confirmed can degrade specific heterocyclic hydrocarbon are

shown in Table 1

6

Table 1 Example of specific bacteria strain degrade heterocyclic hydrocarbon

Bacteria Strain Type of heterocyclic hydrocarbon degradation

Study

Erythrobacter sp Lysobacter sp

IC114 OC7

CAR CAR

Inoue et ai (2005) Maeda et ai (2009)

Teabacter sp Sphingomonas sp

OBF63 HH69

OBF OBF

Fuse et ai (2003) Fuse et a1 (2003)

Sphingomonas sp XLON2-5 OBT Gai et ai (2007) Teabacter sp OBF63 FLN Habe et ai (2004)

24 Surfactants

241 Characteristics of surfactants

Surface active compounds also known as surfactants are amphiphilic molecule which

composed of at least two parts one part is hydrophobic part and another is hydrophilic part

(Makkar amp Rockne 2003 Singhet et aI 2007) The hydrophilic part is referred to polar

water-soluble head group whereas the hydrophobic part is nonpolar hydrocarbon tail group

(lvankovic amp Hrenovic 20 I 0) The hydrophobic part makes surfactants concentrate at

interface whereas the hydrophilic part makes them soluble in water (Makkar amp Rockne

2003) According to White and R sels study (as cited in Cloete amp Jacobs 2001)

surfactants are primary classified into four types include cationic anionic zwitterionic and

non-ionic surfactants depending on the nature ionic charge of the hydrophilic group either

ionic or non-ionic Surfactants dissolve in water and form monomer at low concentrations

as shown in Figure 2 (Ying as cited in Ivankovic amp Hrenovic 2010) The first

characteristic of surfactant is that it adsorbs at surfaces or interfaces thus decrease surface

ions (Makkar amp Rockne 2003) Besides that surfactant is able to form small

aggregates of surfactants molecule known as micelles (Volkering et aI 1997) In water

7

bull bull

micelles form at very low concentration The micelles consist of hydrophobic core and

hydrophilic shell The concentration at which micelles start to form is known as critical

micelle concentration (CMC) The CMC differ for every surfactant according to the

temperature type and structure of surfactant (Volkering et aI 1997) These surfactant

micelles allow the fusion of the hydrophobic hydrocarbon into the hydrophobic core of the

micelles thus increase the solubility of these compounds as shown in Figure 2 This

process is known as micellar solubilization According to Volkering et al (1997) micellar

solubilization of hydrophobic compound can be defined as separating of this compound

between hydrophobic cores of micelle and surrounding water Surfactants can be produced

either chemically or biologically which are used in oil recovery and bioremediation of

pollutants (Singh et aI 2007)

Increase (surfactant concentration to above C~IC1 1 111 i II 1 1 11

1-1__gt ~amp~ bull ~ bull bull~ ~ middotftshybull

surfactant monomer bull solute molecule ~ micelle

Figure 2 Formation of micelle and micellar solubihzation (Source Li amp Chen 2009)

8

242 Ionic surfactant

Ionic surfactant can be divided into anionic and cationic surfactant Some examples of

anionic and cationic surfactants are shown in Table 2 Anionic surfactant is a surfactant

that has negatively charge at its polar head Anionic surfactants are more widely used

because they are cheap to manufacture (Yu et aI 2008)

Cationic surfactant is a surfactant that has positively charge at its polar head Since

most of the surfaces are negatively charge including cell membrane so cationic surfactants

absorb strongly toward the bacteria cell membrane These surfactants will penetrate into

cell wall and react with cytoplasmic membrane (Cannona-Ribeiro amp de Melo Carrasco

2013) Thus they are more toxic compare to other surfactants

243 Zwitterionic or amphoteric surfactants

Zwitterionic surfactants have both positive and negative charge in the polar water-soluble

head group However the charge of the zwitterionic surfactant is pH dependence (Yu et aI

2008) The zwitterionic surfactant acts as anionic at high pH while it acts as cationic at low

pH which makes the properties of the zwitterionic surfactant change according to pH

change Example ofzwitterionic surf8H8nts is shown in Table 2

244 Non -ionic surfactaots

Nonionic surfactants are surfactant that lack of charge and widely used as wetting agents

emulsifiers and foam stabilization agent (Ivankovic amp Hrenovic 20 I 0) Non-ionic

lUlfactants have high hydrocarbon solubilizing power less toxic to the bacteria and weak

tion to the charged sites Examples of non-ionic surfactants are shown in Table 2

9

Table 2 Example of surf act ants (Source IvankoviC amp Hrenovic 2010)

Class Common name Abbreviations Anionic Sodium dodecyl sulphate

Alkyl sulphate Sodium lauryl sulphate Alkyl ethoxysulphate

SDS AS SLS AES

Cationic Quaternary ammonium compound Benzalkonium chloride Cetylpyridinium bromide Cetylpyridinium chloride Hexadecyl trimethylammonium bromide

QAC BAC CPB CPC

HDTMA Non- ionic Alkylphenol ethoxylate

Alcohol ethoxylate Fatty acid ethoxylate

APE AE

FAE

I

Amphoteric zwitterionic

Amine oxide AO

245 Application of surfactants in remediation

The degradation rate of heterocyclic hydrocarbons such as phenanthrene carbazole

dibenzofuran dibenzothiophene and fluorene in the environment is often limited due to the

low bioavailability of these compounds for microbial conversion (Rao 2009) Thus either

chemical synthetic surfactants or biosurfactants have been used to increase the

bioavailability of heterocyclic compounds in the environment for microbial conversion

According to Schipper et a1 (2000) study three approaches have been proposed to increase ~

the biodegradation of the heterocyclic compound by the bacteria in the presence of

surfactants Firstly the bacteria are able to direct uptake of heterocyclic hydrocarbon from

the hydrophobic core of micelles The second approach is that the bacteria are able to

uptake heterocyclic hydrocarbon from the aqueous phase after they released from micelle

cores Lastly surfactants facilitate the uptake of the heterocyclic hydrocarbon via direct

contact between the cell and non-aqueous phase liquids (NAPLs) Bautista et a1 (2009)

nducted a study to test the effect of different non-ionic surfactants on biodegradation

10

f polycyclic aromatic hydrocarbons (PAHs) by aerobic bacteria From this study

bicxlegradation rate increases by the action of Enterobacter sp in presence of the non-ionic

aurfactants such as Triton X-lOa or Tween-80 Besides that the control experiment

without adding any surfactants show the reduction in biodegradation rate due to decrease

ofbacteria cell growth rate From the study done by Bautista et al (2009) they conclude

that the solubility and bioavailability of weekly soluble P AHs reduced by the action of

bacteria in the absence of any surfactants

5 Effect of surfactants on biodegradation of heterocyclic hydrocarbon

wfactants have positive effect on biodegradation of hydrocarbon by increase the

solubility of hydrocarbon pollutants or enhance the mass transfer of pollutants toward

bacteria However some study show that surfactants reduce or inhibit biodegradation of

these hydrocarbon pollutants due to the toxicity of some surfactants toward the

heterocyclic degrading bacteria and surfactants biodegradability (Willumsen amp Karlson

1998 Bautista et aI 2009 Li amp Chen 2009)

51 Toxicity of surfactants

Some surfactants cause toxic effect toward bacteria They inhibit bacteria cell proliferate

and cause the rate of degradation reduced (Li amp Chen 2009) The surfactants presence in

high concentration is main factors that cause toxic effect toward bacteria Besides that

molecular structure of surfactant is also one of the important factors that cause toxic effect

toward bacteria According to Li and Chen (2009) and Louvado et al (20 10) non-ionic

surfactants are less toxic than ionic surfactants especially cationic surfactants This is

because positive charges present at the polar head of the cationic surfactants will react with

11

negatively charged bacterial cells surface (Li amp Chen 2009) The toxic effect of the

ufacfants on bacteria can be explained by two main factors Firstly surfactants form

bull cd micelles with membrane lipid when the concentration of surfactants above the CMC

aod cause the disruption of bacterial cell membrane (Volkering et aI 1997 Li amp Chen

2009 Louvado et aI 2010) Besides that some surfactant molecule react with the protein

1bat essential for the cell functioning Jin et al (2007) have done a study on the surfactant

toxicity toward bacteria cell and the phenanthrene degradation

2 2 Biodegradation of surfactants

urfactant biodegradability has become a vital factor before using the surfactant for

bioremediation (Li amp Chen 2009) If the surfactants are highly degraded by the bacteria

the biodegradation rate for hydrocarbon pollutants decreases thus reduces the

bioavailability enhancing effect of the surfactants This is because surfactants compete

with the primary substrates (hydrocarbon pollutants) for the bacteria in order to be

degraded Tiehm (as cited by Li amp Chen 2009) has reported that the highly biodegradable

of surfactant by bacteria can inhibit the biodegradation of the P AHs process Thus the

SUIfactants biodegradability and their influence on the biodegradation of pollutants process

should be balanced The involvement of surfactant in remediation has to be tested before it

is chosen for in situ remediation (Li amp Chen 2009) Factors that influence the

biodegradation process are type and concentration of surfactants heterocyclic hydrocarbon

specificity and the involvement of bacteria in biodegradation process (Li amp Chen 2009)

12

IIOJI18tic

pedrophotometric analysis

Spectrophotometer is an instrument that used to measure the amount of light absorbed

~ce) by a solution or mixture A spectrophotometer consist two part include a

apelCttlDDlleter and photometer Absorbance is another term for optical density (OD) From

Jeason et al (2003) study spectrophotometric turbidity test was performed to determine

bacterial growth at 600 nrn However the bacterial density was monitored by

moasuring the As60 according to Liu et al (1995) study According to Mohd Kamil et al

(2012) the absorbance values represent the samples turbidity where the degree of turbidity

reflects with the amount of bacteria in the sample Thus the highest absorbance value

ampbows the highest turbidity of the sample showing the highest growth of the bacteria Apart

Iiom that spectrophotometric analysis can be used to determine the degradation rate of

hydrocarbon by recording the absorbance value at certain wavelength and

perfono an absorbance against time graph Jenson et al (2003) reported that the increase in

absorbance value at the one of the specific wavelength indicate the accumulation of

metabolites from degradation process

13

Carbazole

2

bacterium

30 METHOD AND MATERIALS

CllemIeaI and Media

dibenzofuran dibenzothiophene and fluorene with 98 purity (Sigmashy

AldricbUSA) were employed in the degradation experiments Surfactants (Triton X-100)

ere be purchased from Sigma-Aldrich (USA) Marine broth powder agar powder were be

purcbasc from Sigma- Aldrich (USA)

riDe bacteria source

Bacterial strain FNSOI was obtained from previous study which has been isolated from

JIUID8IOve environment in Asajaya Sarawak Strain FNSO 1 was isolated as CAR degrading

33 Media preparation

The composition of ONR7a medium was shown in table 3 All the chemical powders

shows Table 3 were weighted using analytical weight balance and pour into a I L media

bottle with blue cap A volume of of distilled water was measured using measuring

cylinder The media bottle with media and magnetic rod inside were placed on magnetic

stirrer plate According to Sutiknowati (2010) study the pH should be maintained within

76 -78 The pH was adjusted using sodium hydroxide NaOH and measured using pH

meter Media of ONR7a agar was done by adding 15 of the volume of ONR7a media

The media bottle containing ONR7a agar in liquid form will be autoc1aved first before it

poured into petri dishes

14



A thesis submitted in partial fulfilment of the requirement of the degree of Bachelor of Science with Honours

(Recourse Biotechnology)

Supervisor Dr Azham Zulkharnain

Resource Biotechnology Programme Department of Molecular Biology


2014

ACKNOWNLEDGEMENT











work




Grade____ _
















11


















iii

- - --- -- -- --~

T ABLE OF CONTENTS

ACKNOWNLEDGEMENT

IIDECLARATION

IVTABLE OF CONTENTS


VIILIST OF TABLES

viiiLIST OF FIGURES

ABSTRACT

210 INTRODUCTION




623 Marine bacteria

724 Surfactants













IV








40 RESULT 19



421 CFU counting 20



50 DISCUSSION 26

60 CONCLUSION 31

REFERENCES 31

v


CMC

CAR

DBF

DBP

FLN

PAHs

NAPLs

o

S

N

H

(vv)

(wv)

min

ml

lmiddottI

rpm

J1ID

mm

M

CFUml


Carbazole

Dibenzofuran

Dibenzothiophene

Fluorene



Oxygen

Sulfur

Nitrogen

Hour



Percentage

Minute

Celsius

Milliliter ~

Microliter


Micrometer

Millimeter

Meter


VI

LIST OF TABLES





Vll

LIST OF FIGURES








viii





ABSTRACT



ABSTRAK



10 INTRODUCTION

























2














2009)



FNSOI




3

























4

j

damaged tankers

Dibenzofuran

9 1 8~

7~_~6 0 4a

5

Fluorene

6 5 4

8 1a 9

-

2

3















Dibenzothiophene

9 1 8~2

7~_~3 6 S 4a 44

5

Carbazole

3 ~3 7~-~270=02

1 8 N 9a 1 I

H


5

I













23 Marine bacteria








shown in Table 1

6



Study


IC114 OC7

CAR CAR



OBF63 HH69

OBF OBF



24 Surfactants
















7

bull bull
















8






















9




SDS AS SLS AES


QAC BAC CPB CPC



APE AE

FAE

I


Amine oxide AO















10























11





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14

ACKNOWNLEDGEMENT











work




Grade____ _
















11


















iii

- - --- -- -- --~

T ABLE OF CONTENTS

ACKNOWNLEDGEMENT

IIDECLARATION

IVTABLE OF CONTENTS


VIILIST OF TABLES

viiiLIST OF FIGURES

ABSTRACT

210 INTRODUCTION




623 Marine bacteria

724 Surfactants













IV








40 RESULT 19



421 CFU counting 20



50 DISCUSSION 26

60 CONCLUSION 31

REFERENCES 31

v


CMC

CAR

DBF

DBP

FLN

PAHs

NAPLs

o

S

N

H

(vv)

(wv)

min

ml

lmiddottI

rpm

J1ID

mm

M

CFUml


Carbazole

Dibenzofuran

Dibenzothiophene

Fluorene



Oxygen

Sulfur

Nitrogen

Hour



Percentage

Minute

Celsius

Milliliter ~

Microliter


Micrometer

Millimeter

Meter


VI

LIST OF TABLES





Vll

LIST OF FIGURES








viii





ABSTRACT



ABSTRAK



10 INTRODUCTION

























2














2009)



FNSOI




3

























4

j

damaged tankers

Dibenzofuran

9 1 8~

7~_~6 0 4a

5

Fluorene

6 5 4

8 1a 9

-

2

3















Dibenzothiophene

9 1 8~2

7~_~3 6 S 4a 44

5

Carbazole

3 ~3 7~-~270=02

1 8 N 9a 1 I

H


5

I













23 Marine bacteria








shown in Table 1

6



Study


IC114 OC7

CAR CAR



OBF63 HH69

OBF OBF



24 Surfactants
















7

bull bull
















8






















9




SDS AS SLS AES


QAC BAC CPB CPC



APE AE

FAE

I


Amine oxide AO















10























11





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14


Grade____ _
















11


















iii

- - --- -- -- --~

T ABLE OF CONTENTS

ACKNOWNLEDGEMENT

IIDECLARATION

IVTABLE OF CONTENTS


VIILIST OF TABLES

viiiLIST OF FIGURES

ABSTRACT

210 INTRODUCTION




623 Marine bacteria

724 Surfactants













IV








40 RESULT 19



421 CFU counting 20



50 DISCUSSION 26

60 CONCLUSION 31

REFERENCES 31

v


CMC

CAR

DBF

DBP

FLN

PAHs

NAPLs

o

S

N

H

(vv)

(wv)

min

ml

lmiddottI

rpm

J1ID

mm

M

CFUml


Carbazole

Dibenzofuran

Dibenzothiophene

Fluorene



Oxygen

Sulfur

Nitrogen

Hour



Percentage

Minute

Celsius

Milliliter ~

Microliter


Micrometer

Millimeter

Meter


VI

LIST OF TABLES





Vll

LIST OF FIGURES








viii





ABSTRACT



ABSTRAK



10 INTRODUCTION

























2














2009)



FNSOI




3

























4

j

damaged tankers

Dibenzofuran

9 1 8~

7~_~6 0 4a

5

Fluorene

6 5 4

8 1a 9

-

2

3















Dibenzothiophene

9 1 8~2

7~_~3 6 S 4a 44

5

Carbazole

3 ~3 7~-~270=02

1 8 N 9a 1 I

H


5

I













23 Marine bacteria








shown in Table 1

6



Study


IC114 OC7

CAR CAR



OBF63 HH69

OBF OBF



24 Surfactants
















7

bull bull
















8






















9




SDS AS SLS AES


QAC BAC CPB CPC



APE AE

FAE

I


Amine oxide AO















10























11





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14


















iii

- - --- -- -- --~

T ABLE OF CONTENTS

ACKNOWNLEDGEMENT

IIDECLARATION

IVTABLE OF CONTENTS


VIILIST OF TABLES

viiiLIST OF FIGURES

ABSTRACT

210 INTRODUCTION




623 Marine bacteria

724 Surfactants













IV








40 RESULT 19



421 CFU counting 20



50 DISCUSSION 26

60 CONCLUSION 31

REFERENCES 31

v


CMC

CAR

DBF

DBP

FLN

PAHs

NAPLs

o

S

N

H

(vv)

(wv)

min

ml

lmiddottI

rpm

J1ID

mm

M

CFUml


Carbazole

Dibenzofuran

Dibenzothiophene

Fluorene



Oxygen

Sulfur

Nitrogen

Hour



Percentage

Minute

Celsius

Milliliter ~

Microliter


Micrometer

Millimeter

Meter


VI

LIST OF TABLES





Vll

LIST OF FIGURES








viii





ABSTRACT



ABSTRAK



10 INTRODUCTION

























2














2009)



FNSOI




3

























4

j

damaged tankers

Dibenzofuran

9 1 8~

7~_~6 0 4a

5

Fluorene

6 5 4

8 1a 9

-

2

3















Dibenzothiophene

9 1 8~2

7~_~3 6 S 4a 44

5

Carbazole

3 ~3 7~-~270=02

1 8 N 9a 1 I

H


5

I













23 Marine bacteria








shown in Table 1

6



Study


IC114 OC7

CAR CAR



OBF63 HH69

OBF OBF



24 Surfactants
















7

bull bull
















8






















9




SDS AS SLS AES


QAC BAC CPB CPC



APE AE

FAE

I


Amine oxide AO















10























11





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14

- - --- -- -- --~

T ABLE OF CONTENTS

ACKNOWNLEDGEMENT

IIDECLARATION

IVTABLE OF CONTENTS


VIILIST OF TABLES

viiiLIST OF FIGURES

ABSTRACT

210 INTRODUCTION




623 Marine bacteria

724 Surfactants













IV








40 RESULT 19



421 CFU counting 20



50 DISCUSSION 26

60 CONCLUSION 31

REFERENCES 31

v


CMC

CAR

DBF

DBP

FLN

PAHs

NAPLs

o

S

N

H

(vv)

(wv)

min

ml

lmiddottI

rpm

J1ID

mm

M

CFUml


Carbazole

Dibenzofuran

Dibenzothiophene

Fluorene



Oxygen

Sulfur

Nitrogen

Hour



Percentage

Minute

Celsius

Milliliter ~

Microliter


Micrometer

Millimeter

Meter


VI

LIST OF TABLES





Vll

LIST OF FIGURES








viii





ABSTRACT



ABSTRAK



10 INTRODUCTION

























2














2009)



FNSOI




3

























4

j

damaged tankers

Dibenzofuran

9 1 8~

7~_~6 0 4a

5

Fluorene

6 5 4

8 1a 9

-

2

3















Dibenzothiophene

9 1 8~2

7~_~3 6 S 4a 44

5

Carbazole

3 ~3 7~-~270=02

1 8 N 9a 1 I

H


5

I













23 Marine bacteria








shown in Table 1

6



Study


IC114 OC7

CAR CAR



OBF63 HH69

OBF OBF



24 Surfactants
















7

bull bull
















8






















9




SDS AS SLS AES


QAC BAC CPB CPC



APE AE

FAE

I


Amine oxide AO















10























11





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14








40 RESULT 19



421 CFU counting 20



50 DISCUSSION 26

60 CONCLUSION 31

REFERENCES 31

v


CMC

CAR

DBF

DBP

FLN

PAHs

NAPLs

o

S

N

H

(vv)

(wv)

min

ml

lmiddottI

rpm

J1ID

mm

M

CFUml


Carbazole

Dibenzofuran

Dibenzothiophene

Fluorene



Oxygen

Sulfur

Nitrogen

Hour



Percentage

Minute

Celsius

Milliliter ~

Microliter


Micrometer

Millimeter

Meter


VI

LIST OF TABLES





Vll

LIST OF FIGURES








viii





ABSTRACT



ABSTRAK



10 INTRODUCTION

























2














2009)



FNSOI




3

























4

j

damaged tankers

Dibenzofuran

9 1 8~

7~_~6 0 4a

5

Fluorene

6 5 4

8 1a 9

-

2

3















Dibenzothiophene

9 1 8~2

7~_~3 6 S 4a 44

5

Carbazole

3 ~3 7~-~270=02

1 8 N 9a 1 I

H


5

I













23 Marine bacteria








shown in Table 1

6



Study


IC114 OC7

CAR CAR



OBF63 HH69

OBF OBF



24 Surfactants
















7

bull bull
















8






















9




SDS AS SLS AES


QAC BAC CPB CPC



APE AE

FAE

I


Amine oxide AO















10























11





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14


CMC

CAR

DBF

DBP

FLN

PAHs

NAPLs

o

S

N

H

(vv)

(wv)

min

ml

lmiddottI

rpm

J1ID

mm

M

CFUml


Carbazole

Dibenzofuran

Dibenzothiophene

Fluorene



Oxygen

Sulfur

Nitrogen

Hour



Percentage

Minute

Celsius

Milliliter ~

Microliter


Micrometer

Millimeter

Meter


VI

LIST OF TABLES





Vll

LIST OF FIGURES








viii





ABSTRACT



ABSTRAK



10 INTRODUCTION

























2














2009)



FNSOI




3

























4

j

damaged tankers

Dibenzofuran

9 1 8~

7~_~6 0 4a

5

Fluorene

6 5 4

8 1a 9

-

2

3















Dibenzothiophene

9 1 8~2

7~_~3 6 S 4a 44

5

Carbazole

3 ~3 7~-~270=02

1 8 N 9a 1 I

H


5

I













23 Marine bacteria








shown in Table 1

6



Study


IC114 OC7

CAR CAR



OBF63 HH69

OBF OBF



24 Surfactants
















7

bull bull
















8






















9




SDS AS SLS AES


QAC BAC CPB CPC



APE AE

FAE

I


Amine oxide AO















10























11





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14

LIST OF TABLES





Vll

LIST OF FIGURES








viii





ABSTRACT



ABSTRAK



10 INTRODUCTION

























2














2009)



FNSOI




3

























4

j

damaged tankers

Dibenzofuran

9 1 8~

7~_~6 0 4a

5

Fluorene

6 5 4

8 1a 9

-

2

3















Dibenzothiophene

9 1 8~2

7~_~3 6 S 4a 44

5

Carbazole

3 ~3 7~-~270=02

1 8 N 9a 1 I

H


5

I













23 Marine bacteria








shown in Table 1

6



Study


IC114 OC7

CAR CAR



OBF63 HH69

OBF OBF



24 Surfactants
















7

bull bull
















8






















9




SDS AS SLS AES


QAC BAC CPB CPC



APE AE

FAE

I


Amine oxide AO















10























11





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14

LIST OF FIGURES








viii





ABSTRACT



ABSTRAK



10 INTRODUCTION

























2














2009)



FNSOI




3

























4

j

damaged tankers

Dibenzofuran

9 1 8~

7~_~6 0 4a

5

Fluorene

6 5 4

8 1a 9

-

2

3















Dibenzothiophene

9 1 8~2

7~_~3 6 S 4a 44

5

Carbazole

3 ~3 7~-~270=02

1 8 N 9a 1 I

H


5

I













23 Marine bacteria








shown in Table 1

6



Study


IC114 OC7

CAR CAR



OBF63 HH69

OBF OBF



24 Surfactants
















7

bull bull
















8






















9




SDS AS SLS AES


QAC BAC CPB CPC



APE AE

FAE

I


Amine oxide AO















10























11





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14





ABSTRACT



ABSTRAK



10 INTRODUCTION

























2














2009)



FNSOI




3

























4

j

damaged tankers

Dibenzofuran

9 1 8~

7~_~6 0 4a

5

Fluorene

6 5 4

8 1a 9

-

2

3















Dibenzothiophene

9 1 8~2

7~_~3 6 S 4a 44

5

Carbazole

3 ~3 7~-~270=02

1 8 N 9a 1 I

H


5

I













23 Marine bacteria








shown in Table 1

6



Study


IC114 OC7

CAR CAR



OBF63 HH69

OBF OBF



24 Surfactants
















7

bull bull
















8






















9




SDS AS SLS AES


QAC BAC CPB CPC



APE AE

FAE

I


Amine oxide AO















10























11





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14

10 INTRODUCTION

























2














2009)



FNSOI




3

























4

j

damaged tankers

Dibenzofuran

9 1 8~

7~_~6 0 4a

5

Fluorene

6 5 4

8 1a 9

-

2

3















Dibenzothiophene

9 1 8~2

7~_~3 6 S 4a 44

5

Carbazole

3 ~3 7~-~270=02

1 8 N 9a 1 I

H


5

I













23 Marine bacteria








shown in Table 1

6



Study


IC114 OC7

CAR CAR



OBF63 HH69

OBF OBF



24 Surfactants
















7

bull bull
















8






















9




SDS AS SLS AES


QAC BAC CPB CPC



APE AE

FAE

I


Amine oxide AO















10























11





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14














2009)



FNSOI




3

























4

j

damaged tankers

Dibenzofuran

9 1 8~

7~_~6 0 4a

5

Fluorene

6 5 4

8 1a 9

-

2

3















Dibenzothiophene

9 1 8~2

7~_~3 6 S 4a 44

5

Carbazole

3 ~3 7~-~270=02

1 8 N 9a 1 I

H


5

I













23 Marine bacteria








shown in Table 1

6



Study


IC114 OC7

CAR CAR



OBF63 HH69

OBF OBF



24 Surfactants
















7

bull bull
















8






















9




SDS AS SLS AES


QAC BAC CPB CPC



APE AE

FAE

I


Amine oxide AO















10























11





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14

























4

j

damaged tankers

Dibenzofuran

9 1 8~

7~_~6 0 4a

5

Fluorene

6 5 4

8 1a 9

-

2

3















Dibenzothiophene

9 1 8~2

7~_~3 6 S 4a 44

5

Carbazole

3 ~3 7~-~270=02

1 8 N 9a 1 I

H


5

I













23 Marine bacteria








shown in Table 1

6



Study


IC114 OC7

CAR CAR



OBF63 HH69

OBF OBF



24 Surfactants
















7

bull bull
















8






















9




SDS AS SLS AES


QAC BAC CPB CPC



APE AE

FAE

I


Amine oxide AO















10























11





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14

j

damaged tankers

Dibenzofuran

9 1 8~

7~_~6 0 4a

5

Fluorene

6 5 4

8 1a 9

-

2

3















Dibenzothiophene

9 1 8~2

7~_~3 6 S 4a 44

5

Carbazole

3 ~3 7~-~270=02

1 8 N 9a 1 I

H


5

I













23 Marine bacteria








shown in Table 1

6



Study


IC114 OC7

CAR CAR



OBF63 HH69

OBF OBF



24 Surfactants
















7

bull bull
















8






















9




SDS AS SLS AES


QAC BAC CPB CPC



APE AE

FAE

I


Amine oxide AO















10























11





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14

I













23 Marine bacteria








shown in Table 1

6



Study


IC114 OC7

CAR CAR



OBF63 HH69

OBF OBF



24 Surfactants
















7

bull bull
















8






















9




SDS AS SLS AES


QAC BAC CPB CPC



APE AE

FAE

I


Amine oxide AO















10























11





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14



Study


IC114 OC7

CAR CAR



OBF63 HH69

OBF OBF



24 Surfactants
















7

bull bull
















8






















9




SDS AS SLS AES


QAC BAC CPB CPC



APE AE

FAE

I


Amine oxide AO















10























11





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14

bull bull
















8






















9




SDS AS SLS AES


QAC BAC CPB CPC



APE AE

FAE

I


Amine oxide AO















10























11





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14






















9




SDS AS SLS AES


QAC BAC CPB CPC



APE AE

FAE

I


Amine oxide AO















10























11





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14




SDS AS SLS AES


QAC BAC CPB CPC



APE AE

FAE

I


Amine oxide AO















10























11





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14























11





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14





















12

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14

IIOJI18tic
















13

Carbazole

2

bacterium


CllemIeaI and Media


















14

Carbazole

2

bacterium


CllemIeaI and Media


















14

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