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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 (~))
~ONFIDENTIAL (Contains confidential information under the Official Secret Act 1972) p RESTRICTED (Contains restricted information as specified by the organisation where
research was done) D OPEN ACCESS
Validation of ProjectIThesis
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bull The Centre for Academic Information Services has the lawful right to make copies for the purpose of academic and research only and not for other purpose
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bull No dispute or any claim shall arise from the student itself neither third party on this ProjectThesis once it becomes the sole property of UNIMAS
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
Effects of Surfactant Triton X-I00 on Degradation of Heterocyclic Hydrocarbon by Marine Bacteria
Susan Ling Lin Fei (33156)
Resource Biotechnology Department of Molecular Biology
Faculty of Resource Science and Technology University Malaysia Sarawak
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
20 LITERATURE REVIEW
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
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 (~))
~ONFIDENTIAL (Contains confidential information under the Official Secret Act 1972) p RESTRICTED (Contains restricted information as specified by the organisation where
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 The Centre for Academic Information Services has the lawful right to make copies for the purpose of academic and research only and not for other purpose
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bull No dispute or any claim shall arise from the student itself neither third party on this ProjectThesis once it becomes the sole property of UNIMAS
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
Effects of Surfactant Triton X-I00 on Degradation of Heterocyclic Hydrocarbon by Marine Bacteria
Susan Ling Lin Fei (33156)
Resource Biotechnology Department of Molecular Biology
Faculty of Resource Science and Technology University Malaysia Sarawak
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
20 LITERATURE REVIEW
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
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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bull The Centre for Academic Information Services has the lawful right to make copies for the purpose of academic and research only and not for other purpose
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bull The Centre for Academic Information Services has the lawful right to make copies of the ProjectThesis for academic exchange between Higher Learning Institute
bull No dispute or any claim shall arise from the student itself neither third party on this ProjectThesis once it becomes the sole property of UNIMAS
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
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[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
Effects of Surfactant Triton X-I00 on Degradation of Heterocyclic Hydrocarbon by Marine Bacteria
Susan Ling Lin Fei (33156)
Resource Biotechnology Department of Molecular Biology
Faculty of Resource Science and Technology University Malaysia Sarawak
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
20 LITERATURE REVIEW
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
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 (~))
~ONFIDENTIAL (Contains confidential information under the Official Secret Act 1972) p RESTRICTED (Contains restricted information as specified by the organisation where
research was done) D OPEN ACCESS
Validation of ProjectIThesis
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
bull This ProjectThesis is the sole legal property of Universiti Malaysia Sarawak (UNIMAS)
bull The Centre for Academic Information Services has the lawful right to make copies for the purpose of academic and research only and not for other purpose
bull The Centre for Academic Information Services has the lawful right to digitalise the content for the Local Content Database
bull The Centre for Academic Information Services has the lawful right to make copies of the ProjectThesis for academic exchange between Higher Learning Institute
bull No dispute or any claim shall arise from the student itself neither third party on this ProjectThesis once it becomes the sole property of UNIMAS
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
Effects of Surfactant Triton X-I00 on Degradation of Heterocyclic Hydrocarbon by Marine Bacteria
Susan Ling Lin Fei (33156)
Resource Biotechnology Department of Molecular Biology
Faculty of Resource Science and Technology University Malaysia Sarawak
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
20 LITERATURE REVIEW
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
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research was done) D OPEN ACCESS
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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
Effects of Surfactant Triton X-I00 on Degradation of Heterocyclic Hydrocarbon by Marine Bacteria
Susan Ling Lin Fei (33156)
Resource Biotechnology Department of Molecular Biology
Faculty of Resource Science and Technology University Malaysia Sarawak
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
20 LITERATURE REVIEW
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
- - --- -- -- --~
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
Effects of Surfactant Triton X-I00 on Degradation of Heterocyclic Hydrocarbon by Marine Bacteria
Susan Ling Lin Fei (33156)
Resource Biotechnology Department of Molecular Biology
Faculty of Resource Science and Technology University Malaysia Sarawak
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
20 LITERATURE REVIEW
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
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
Effects of Surfactant Triton X-I00 on Degradation of Heterocyclic Hydrocarbon by Marine Bacteria
Susan Ling Lin Fei (33156)
Resource Biotechnology Department of Molecular Biology
Faculty of Resource Science and Technology University Malaysia Sarawak
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
20 LITERATURE REVIEW
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
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
Effects of Surfactant Triton X-I00 on Degradation of Heterocyclic Hydrocarbon by Marine Bacteria
Susan Ling Lin Fei (33156)
Resource Biotechnology Department of Molecular Biology
Faculty of Resource Science and Technology University Malaysia Sarawak
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
20 LITERATURE REVIEW
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
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
Effects of Surfactant Triton X-I00 on Degradation of Heterocyclic Hydrocarbon by Marine Bacteria
Susan Ling Lin Fei (33156)
Resource Biotechnology Department of Molecular Biology
Faculty of Resource Science and Technology University Malaysia Sarawak
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
20 LITERATURE REVIEW
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
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
Effects of Surfactant Triton X-I00 on Degradation of Heterocyclic Hydrocarbon by Marine Bacteria
Susan Ling Lin Fei (33156)
Resource Biotechnology Department of Molecular Biology
Faculty of Resource Science and Technology University Malaysia Sarawak
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
20 LITERATURE REVIEW
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
Effects of Surfactant Triton X-I00 on Degradation of Heterocyclic Hydrocarbon by Marine Bacteria
Susan Ling Lin Fei (33156)
Resource Biotechnology Department of Molecular Biology
Faculty of Resource Science and Technology University Malaysia Sarawak
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
20 LITERATURE REVIEW
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
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
20 LITERATURE REVIEW
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
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
20 LITERATURE REVIEW
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
20 LITERATURE REVIEW
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
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
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
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
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
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
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
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
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
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
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