On the effect of prevalent carbazole homocoupling defects ...
ISOLATION OF GENE INVOLVED IN DEGRADATION OF … of Gene Involved in Degradation of Carbazole...
Transcript of ISOLATION OF GENE INVOLVED IN DEGRADATION OF … of Gene Involved in Degradation of Carbazole...
ISOLATION OF GENE INVOLVED IN DEGRADATION OF CARBAZOLE AND DIBENZOTHIOPHENE
BY MARINE BACTERIA
Nurul Nabila Humaira Binti Kamaruzaman
QR 106 N974
Bachelor of Science with Honours2013 (Resource Biotechnology)
2013
Acknowledgement
I would like to express my special gratitude and thanks to my supervisor, Dr. Azham Bin
Zulkharnain for giving me such attention and time in helped me to finish my final year project. I
am highly indebted with his guidance and constant supervision as well as providing necessary
information regarding to this final year project. My thanks and appreciations also go to my co
supervisor, Assoc. Prof. Dr. Awang Ahmad Sallehin Bin Awang Husaini for giving abundance
of information and suggestion to improve my final year project.
My sincere thanks also go to all postgraduate students from Molecular Genetic
Laboratory especially to Ms Azizah Ahmad for guidance with patient and giving information to
me. I really thanked them because they still trying to help me with all their effort even though
they are busy with their bench work as a postgraduate student.
I have taken all the effort to finish my final year project. However, without moral and
physical support from all course mates especially my final year project-mate, Ong Leng Hui,
there's might be hard for us to finish final year project without helping each other.
I thank almighty forgiving me all strength and blessing before, during and after finish my
final year project.
Lastly, I also take this opportunity to express a deep sense of gratitude to my family for
giving me supports in terms of money, times and advice.
Declaration
I hereby declare that the Final Year Project is based on my original work except for quotation
and citation which have been properly acknowledged. I also declared that it has not been
previously or concurrently submitted for any other degree in UNIMAS or other institutions.
(NURUL NABILA HUMAIRA BT KAMARUZAMAN)
Resource Biotechnology
Department of Molecular Biology
Faculty of Resource Science and Technology
University Malaysia Sarawak
Date:
ii
Khidmat Maklumat AkademikP tusa llAIH AYSIA SARAWAKUNlVERSm lY~
Table of Contents
Acknowledgement. ...................................................................................... i
Declaration................................... . ......... ...... ........ . ....... . ............................ ii
Table of Content. ................................................. ;................... ... .. .. ............. iii
List of Abbreviations .....................................................................................v
List ofTables and Figures ................ . ................................ .. ...........................vii
Abstract. ............................................................. .... ................................... 1
1.0 Introduction ...........................................................................................2
1.1 Objectives .................................................................................... 5
2.0 Literature Review ....................................................................................6
2.1 Carbazole Compound .......... ,. '" ............................... .. '" ....................6
2.2 Carbazole de.gradation pathway ...........................................................7
2.3 Genes and Enzymes involved in carbazole degradation ...............................9
2.4 Dibenzothiophene (DBT) compound ......................... . .......................... 12
2.5 Dibenzothiophene Desulfurization & Genes Involved ................................ 13
2.6 Bioremediation........... .. ............ . .................................... . ..............14
2.7 Heterocyclic-degrading bacteria ......................................................... 15
2.8 Primer Design ............................................................................... 18
3.0 Materials and Method ............................................................................... .20
3.1 Sample Collection .............. ... ....... . .................. . ... . ........... . ..............20
3.2 Enrichment Medium ......................................... . .............................20
3.3 primer Design ..............................................................................22
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.,.-
3.4 DNA Extraction ........................................................... .. .. ... ..........23
3.5 PCR..........................................................................................24
3.6 Agarose Gel Electrophoresis (AGE) ....................................................26
4.0 Results .... . .... . ................................................................. .. ................ .. .27
4.1 Primer Design ............ '" .... , .......... '" .............................................27
4.2 Total DNA Extraction ....................................................................37
4.3 PCR.........................................................................................38
5.0 Discussion.......................................................................................... 40
6.0 Conclusion .............................................. , ................ ... ........................44
References.............................................................................. . .................. 45
Appendices..............................................................................................49
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%
III
Acetyl-CoA
AGE
bp
CARDO
dH20
DNA
dNTP
et al
g
GC
kb
ml
NaCI
NADH
List of Abbreviations
Percentage
Degree Celcius
Microliter
Acetyl coenzyme A
Agarose Gel Electrophoresis
basepair
Carbazole 1,9-dioxygenase
distilled water
Deoxyribonucleic acid
deox ynucleotide triphosphate
et alii or et aUae
Gram
Guanine-Cytosine
Kilo base
milliliter
Sodium Chloride
Reduced Nicotinamide Adenine Dinucleotide
v
NCB!
PCR
pmol/L
rDNA
rpm
TCA
Tm
UV
vol
National Centre for Biotechnology Information
Polymerase Chain Reactions
Picomoles per Liter
Ribosomal Deoxyribunucleic Acid
Rotations per minute
Tricarboxyclic Acid Cycle
Annealing Temperature
Ultra-violet
Volume
vi
List of Tables and Figures
Table of Contents ....... .. .................... .. .................... ........................... ii
Table I ... ... .................................... ................................................ 18
Table 2 ....... . ............. . . . .................................. . .. . . . . . ...... . ..... . ........... 19
Table 3 ........................... .............. . ...... . ............................ . ...... . ..... 23
Table 4 ......... ........................................... ................................. . .... 25
Table 5 .......................................................................................... 25
Table 6 ........... . ...... ................................................... ..................... 36
Figure I .................................................. . ....................................... 7
Figure 2 ........................................ ............................. ..................... 8
Figure 3 ....................... . ..................... . ............................................ 11
Figure 4 ........................................................ . ...... . .......................... 12
Figure 5............... ... ................ .......................... .. ............................ 17
Figure 6............... ...........................................................................21
Figure 7 ..........................................................................................28
Figure 8................ . ... . .....................................................................29
Figure 9.. ........................................................... .................. ........... 30
Figure 10............................................ . .............. ..............................31
Figure 11 ............................ . ...... . ....... .. ......................... .. .................32
Figure 12................................ . .......... ...................................... .. ......33
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"...
Figure 13 .........................................................................................34
Figure 14.........................................................................................35
Figure 15 ......................... . ............... . ...............................................37
Figure 16.........................................................................................38
Figure 17..................................................................................., .....39
Figure 18.........................................................................................57
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Abstract
Bioremediation is a technique that has been proven to be an effective way to clean polluted area including heterocyclic compound polluted area such as seawater, marine, lake or estuaries. Thalassospira profundimaris and Pseudomonas stutzeri are known bacteria that can degrade carbazole and dibenzothiophene compound respectively are used in this study. These bacteria utilized compound as a sole of carbon and sulfur compound respectively for their growth, with converting compound into other types of compound by-product which is safer in environment. Total DNA extraction was done to identify the genes responsible in degradation activity, followed by direct Polymerase Chain Reaction (PCR) and DNA sequencing. Specific degenerative primer was designed based on previously reported genes in GenBank to detect carA, carB and carC gene that involved in respective carbazole and DBT-A gene in both heterocyclic compound degrading-bacteria. The most conserve region was chosen as forward and reverse primer during alignment using ClustalW2. Five different strains of carbazole degrading bacteria are chosen to design primer for carbazole gene. Four closely related species are chosen to design primer for DBT gene. Expected PCR product size for CarA, CarB, CarC and DBT-A are 1050 bp-, 700 bp-, 610 bp- and 1200 bp- respectively. Results from PCR showed there are product band for DBTA gene for both primer DBTA-Fl and DBTA-F2. Whereas, no carA, carB and carC gene was detected using all six set of designed primer.
Keywords: Bioremediation, heterocyclic compound, marine bacteria, design primer, PCR
Abstrak
Biopemulihan adalah teknik yang telah terbukti berkesan untuk membersihkan kawasan tercemar termasuk kawasan dicemari heterocyclic kompaun seperti air laut, laut, tasik atau muara. Thalassospira profundimaris dan Pseudomonas stutzeri dikenali bakteria yang boleh merendahkan kompaun karbazol dan dibenzothiophene digunakan dalam kajian ini. Bakteria ini digunakan bahan tunggal karbon dan kompaun sulfur untuk pertumbuhan mereka, dengan menukar kompaun ke dalam lain-lainjenis kompaun yang lebih selamat dalam persekitaran. Jumlah pengekstrakan DNA dilakukan untuk mengenal pasti gen yang bertanggungjawab dalam aktiviti kemusnahan, diikuti oleh Polymerase Chain Reaction (PCR) dan penjujukan DNA. Primer degen era tifkhusus yang telah direka berdasarkan laporan sebelum ini dalam GenBank untuk mengesan gen CarA, CarB dan CarC yang terlibat dalam karbazol dan DBT-A gen dalam kedua-dua sebatian heterocyclic merendah-bakteria. Rantau yang paling menjimatkan telah dipilih sebagai primer ke hadapan dan belakang dalamjajaran menggunakan ClustalW2. Lima jenis bakteria yang berbeza dipilih untuk mereka bentuk primer untuk gen carbazole. Empat spesies berkait rapat dipilih untuk mereka bentuk primer untuk gen DBT Dijangka saiz produk peR untuk CarA, CarB, CarC dan DBT-A adalah masing-masing1050 bp -, 700 bp -, 610 bpdan 1200 bp -. Basil daripada PCR menunjukkan terdapat band produk untuk gen DBTA untuk kedua-dua primer DBTA-F1 dan DBTA-F2. Manakala, tiada gen CarA, CarB dan CarC dikesan menggunakan 6 set primer yang telah direka.
Kala Kunci: Bioremediasi,bahan heterosiklik, bakteria marin, mereka primer, PCR
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1.0 INTRODUCTION
Heterocyclic compounds are any substance that has changes for oxygen, sulphur,
phosphorus and nitrogen in aromatic rings (Rajini, 2012). Heterocyclic compounds are related
closely to P AHs, which can be found in environment spontaneously and well distributed in soil,
marine, seawater (Yoon et al., 2002) and urban air (Rajini, 2012). PARs being used in many
areas due to extensive exploration of hydrocarbon including medicines, pesticides, fuels, dyes
and product of particular chemical process such as combustible gas production (Tsao et al.,
2010). There are two classes of heterocyclic compound including aliphatic compound and
aromatic compound. Amines, ethers and amides are the examples of cyclic analog for aliphatic
heterocyclic with the influence of strain in the ring. In contrast, aromatic heterocyclic compound
has several chemically similarities with benzene.
Dibenzothiophene (DBT) is the organosulphur compound with high molecular weight
where central thiophene ring are attached with two benzene rings. This compound commonly
found in petroleum-contaminated samples. This compound has many alkyls homologous and this
is important because within an aromatic series, acute toxicity of P AHs tends to increase with
increasing of alkyl substitution on the aromatic nucleus. DBT shows colorless solid is chemically
similar to anthracene, a solid polycyclic aromatic hydrocarbon (Irwin, 1997). DBT is one of the
compounds that may contribute to serious health problem and environment generally due to
toxicity and mutagenicity of compound. This compound also involved in acid rain since the
compound releasing sulfur dioxide once it ignited (Cara et al., 2006). Biodesulfurization (BDS)
is currently known as an effective alternative to hydrodesulfurizing technique since the BDS
technique do not affect the carbon interaction in removing the sulfur in desulfurization (Izumi &
Oshiro, 2001). Thus, the value of fuel does not affect.
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Carbazole is one of the heterocyclic aromatic compounds that produce during incomplete
combustion of nitrogen-containing organic matter. Carbazole are widely used in synthesis of
dyes, pharmaceuticals and plastics. This compound has been tested for carcinogenicity and
claimed as one of the carcinogenic substance (Miyata, et at,. 1985). In term of environmental
fate when carbazole released into atmosphere, vapor-phase carbazole is rapidly degraded by
photochemically produced hydroxyl radicals and the adsorbing substrate influence the
degradation rate of carbazole. Limitation and photolysis problem by the substrate adsorb
carbazole may happen if this compound release into soil. Specific degrading bacteria may require
for biodegradation process to occur. Carbazole is being produce as a product and is carcinogenic
and toxic to ecosystem. New Jersey Department of Health and Senior Services (2001) reported
that carbazole can cause cancer that effect the animal's liver and stomach.
The rapid expansion in chemical industry over century has meant that there are increasing
in amount and complexity of toxic waste effluent which is related to environmental problems.
Technique used to reduce or removed all the pollutants and contaminants in the environment by
using specific microorganism or fungi depends on the behavior of that particular species on
certain substrate are known as bioremediation. Commonly, bacteria work by breaking down the
materials into organic matter, nutrients or use the compound as the sole of energy source for
growth. There are three types of bioremediation including biostimulation, bioaugmentation and
intrinsic bioremediation. All are used to remove toxic substances and contaminants from
environment whether they are river or crude oils.
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Many bacterial species have been reported to degrade specific heterocyclic compounds
based on their specific metabolic pathway. Heterocyclic compound degrading-bacteria is an
effective way to control the environment pollutant since that it is cost-effective, non-hazardous
instead of using another physical nor chemical substances to degrade the compounds (Adeline et
at., 2009;Seo et aI., 2009). Psedomonas sp. and Sphingomonas sp. are well known as the best
carbazole-degrading bacteria based on previous research by Sato et al., (1997), Yoon et al.,
(2002) Hong et at., (2005) and Adeline et at. (2009). This bacteria contain gene that encoded
enzyme that can degrade carbazole by dioxygenation pathway by converted this compound into
catechol (Sato et at, 1997; Yoon et at, 2002). While Gordona (Rhee et al., 1998), Lysinibacillus
(Bahugana et at, 2011) and Rhodococcus (Denome et al., 1993) are example of bacteria from
different sources for DBT desulfurization have been studied.
Degradation of DBT by bacteria occurs in desulfurization process. Catabolism of DBT is
catalyzed by distinct enzymes in two pathways. Flavin-containing monooxygenases, noted as
DszA and DszB or in other bacteria known as SoxA and SoxB, are widely distributed in bacteria
and catalyzed a consecutive addition of single oxygen atom. Consecutive desulfurization is
achieved by desulfinase. Catabolism of carbazole is well dominated among nitrogen
heterocyclic. A well-known genes involves in carbazole degradation is CARDO gene that
initiates the degradation of carbazole compound by bacteria. P AH degrading bacteria includes in
the genus Myeobacterium, Acinetobacter, Arthrobacter and Burkholderia (Seo, Keum & Li,
2009).
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Pusat Kbidmat Maklumat Akademik IINlVF.Rsm MALAYSIA SARAWAK
Using of natural microorganisms in PAHs-contaminated environment however is not
guaranteed to remove all types of PAHs (Hong et al., 2005). Thus, there are need to identify and
isolate other strain of bacteria that can play an important role in the degradation process.
Commonly, bacteria will use this heterocyclic compound as energy sources in order for them to
grow. Effectiveness of certain bacteria in bioremediation can be seen through the utilization of
bacteria on hydrocarbon sources (Mittal & Sigh, 2009). The decreasing of hydrocarbon or
increasing of bacteria growth can be seen throughout the experiment.
1.2 Objectives
The objective of this study:
1) Design primers for detection and isolation of genes related to degradation of carbazole
compound
2) Isolate genes using direct peR method
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I
2.0 LITERATURE REVIEW
2.1 Carbazole Compound
Carbazole is one of the heterocyclic compounds with molecular formula C 12H9N that is
abundant in nature including in river sediments, groundwater, and atmospheric environment
(Yoon et aI., 2002). Carbazole has N-heterocyclic aromatic compound (Maeda et aI., 2010) as
shown in Figure 1 and has dioxin shape that has planar structure (Schwartz, 2009) originated
from shale oil, coal tar production with high temperature (Maeda et aI., 2010; Tsao, Ouyang &
Chen, 2010) and from coal gas production by combustion process (Tsao, Ouyang & Chen, 2010).
According to Ramos and Filloux (2007), since that carbazole has the dioxin structure, the
bacteria also might have dioxin-degrading enzyme in order to degrade the carbazole compound.
Varieties of product production are derived from carbazole such as dyes, medicines, pesticides
and reagents.
Excessive exploration and use of carbazole meanwhile contribute to environment
pollution that is hazardous to biotic and abiotic condition. Consequences due to chemical
pollutants gave worst effect without consciousness. Besides, carbazole has been reported in
dictionary entitled The Dictionary of Substances and their Effects (DOSE) by Gangoli (1999)
and New Jersey Department of Health and Senior Services (2001) as carcinogenic and mutagenic
compound that has the ability to infect the liver and stomach and may cause cancer.
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N H
Figure I: Carbazole structure
2.2 Carbazole degradation Pathway
Carbazole degradation basicaHy consists of three stages or main pathway as shown in
Figure 2. The three are dioxygenation, meta-cleavage and spontaneous separation (Sato et ai,
2012; Maeda, Ito, Iwata & Omori, 2010). Crabazole is deoxygenated at angular (9a) and adjacent
(l) carbon atoms to produce an unstable hemiaminal (l-hydro-l ,9a-dihydroxycarbazole). Its five
member ring is spontaneously cleaved to form 2' -aminobiphenyl-2, 3-diol, which is converted to
anthranilic acid via meta-cleavage and subsequent hydrolysis. Antranilic acid has been identified
as a main metabolite from the culture extract of several carbazole degrader, suggesting that
bacteria genera Pseudomonas, Resinovorans, and Sphingomonas. Degrade carbazole through a
similar pathway.
Anthranilic acid is known as a biotic compound, and is fonned by the degradation of
trptophan in various organisms and is fonned by the degradation of tryptophan in various
organisms living. In addition, anthranilic acid is known as an important intennediate in the
metabolism of many N-heterocyclic compounds, including Pseudomonas quinolone sinal, which
is involved in quorum sensing in Pseudomonas aeruginosa cells. In the degradation pathway of
carbazole, anthranilic acid is converted to catechol by dioxygenation at the CI and C2 positions,
and spontaneous deamination and decarboxylation. Fonned catechol is converted to a
tricarboxylic acid (TCA)-cycle intennediate via ortho- and/or meta-cleavage pathways.
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The initial dioxygenation for carbazole is a distinct reaction because hydroxylation
occurs at the ring-fused position. This novel type of dioxygenation was termed 'angular
dioxygenation'. Once angu1ar dioxygenation and subsequent spontaneous ring cleavage occurs
for carbazole, the formed 2' -amino-biphenyl-2, 3-diol is degraded by analogous biphenyl
degradation pathways.
CHaCOC02H XI
02H 02H?Ha ......1--_ 9Ha ~ I 9 .. I 9 COSCoA c.t' CHO cue HO~O CuD ~OH
XIII XII
II
VIII
x IX
IVIII
~H~ CMS
VII
Figure 2: Degradation pathway of CAR in Pseudomonas sp. strain CAlO. CNojiri et at., 2001).
Enzyme designations: CarAaAcAd, carbazole 1,9a-dioxygenase; CarBaBb, 2'-aminobiphenyl
2,3-diol 1,2-dioxygenase; CarC, 2-hydroxy-6-oxo-6-(2' -aminophenyl)-hexa-2,4-dienoic acid
(meta-cleavage compound) hydrolase; CarD, 2-hydroxypenta-2,4-dienoate hydratase; CarE, 4
hydroxy-2-oxovalerate aldolase; CarF, acetaldehyde dehydrogenase (acylating); AntABC,
anthranilate 1,2-dioxygenase; CatA, catechol 1,2-dioxygenase; CatB, cis,cis-muconate
lactonizing enzyme; CatC, muconolactone o-isomerase.
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2,
Compounds: I, CAR; II, 2' aminobiphenyl-2,3-diol; III, 2-hydroxy-6-oxo-6-(2'-aminophenyl)
hexa-2,4-dienoic acid (meta cleavage compound); IV, anthranilic acid; V, catechol; VI, cis,cis
muconic acid; Vll, muconolactone; VIIl, p-ketoadipic acid enol-lactone; IX, 2-hydroxypenta
2,4-dienoic acid; X, 4 hydroxy-2-oxovaleric acid; XI, pyruvic acid; XII, acetaldehyde; XIII,
acetyl coenzyme A.
2.3 Genes and enzyme involved in carbazole degradation
The nucleotide sequences of the 27,939-bp-Iong upstream and 9,448-bp-Iong downstream
regions ofthecarAaAaBaBbCAc(ORF7)Ad genes of carbazole-degrading Pseudomonas sp. strain
CAlO were detennined (Nojiri et al., 2001) as shown in Figure 3. Thirty-two open reading
frames (ORFs) were identified, and the car gene cluster was consequently revealed to consist of
10 genes (carAaAaBaBbCAcAdDFE) encoding the enzymes for the three-step conversion of
carbazole to anthranilate and the degradation of2-hydroxypenta-2, 4-dienoate.
Gene cluster consists of CarAaAcAd, encoded for the enzyme carbazole 1,9a
dioxygenase (CARDO), which is act on 9a position in carbazole compound fonning highly
unstable hemiaminallater divided into 2-aminobiphenyl-2, 3-diol. 2'-aminobiphenyl 2,3-diol 1,2
dioxygenase enzyme encoded by CarBaBb gene converted into 2-hydroxy-6-oxo-6-(2'
aminophenyl)-hexa-2,4-dienoic acid. CarC encoded 2-hydroxy-6-oxo-6-(2'-aminophenyl)-hexa
4-dienoic acid (meta-cleavage compound) hydrolase enzyme convert compound into
anthranilic acid. AntABC gene encoded anthranilate 1, 2-dioxygenase enzyme to convert
anthranilic acid into catechol which is further convert into cis-cis muconic acid by catechol 1, 2
dioxygenase encoded by catA gene. Muconolactone will produce as further process by action of
cis,cis-muconate lactonizing enzyme encoded by catB gene. Gene catC encoded muconolactone
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....
o-isomerase enzyme further convert muconolactone into lactone which is later proceed into TeA
cycle.
Anthranilic acid also divided into 2-hydroxypenta-2, 4-dienoic acid which is converted
into 4 hydroxy-2-oxovaleric acid by 2-hydroxypenta-2, 4-dienoate hydratase encoded by card
gene. 4 hydroxy-2-oxovaleric divided into acetaldehyde by 4 hydroxy-2-oxovalerate aldolase
encoded by care gene and pyruvic acid. Acetaldehyde further converted into acetyl coenzyme A
by carF gene encoded acetaldehyde dehydrogenase (acylating).
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PselldomOllos-typ(>
CAIO IIIHWOlII~Hllnlllllll~ cnrAn An Bn Bb C Ac Ad
J3 <GIilJ(}--1 cnrR Btl
CAR-SF ctlrAn Btl Bb
Spllillgolllonas-type
K..i\.l ctlrRI AnI BtlI BbI CI Ad
C Ac GTINll
cnrR An Btl Bb
Oth(>(' types
CB3 carAa Ab Ac Ad B c D
ICI'7 <Q]]JIIUUIIIIII.r_cnrR! Aa C Btl Bb Ac Ad
OC7 11I11nlllll~ carAa C Btl Bb
OC9 carAc R _,(tl c Btl Bb
• TtrmiDaJ O~'gtDa~t larg~ subunit ~ Ttnninal oxygtnllst smaliliubunit ~ FtrrtdoDn B1 FfITtdoDn rtducta~ @ Dthydrognallst
• IIIIfII-CltaYllgt tnzymt largt subUDit B IINItII-Clell'l1lgt t~"1Ilt small subUDit H~'drolast ~ Transcriptional rtgUlator I
Figure 3: The genetic structures of Pseudomonas-type (Pseudomonas resinovorans CA I 0, Janthinobacterium sp.
strain 13, and Neptuniibacter sp. strain CAR-SF), Sphingomonas-type (Novosphingobium sp. strain KAI and
Sphingomonas sp. strain GTINII), and other types (Sphingomonas sp. strain CB3, Nocardioides aromaticivorans
le177, Lysobacter sp. strain OC7, and novel genus strain OC9) of car gene clusters.
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.....
2.4 Dibenzothiophene compound (DBT)
Dibenzothiophene (DBT) is the organosulphur compound, which is organic compound
that contain sulfur (Monticello, 2000). DBT consists of central thiophene ring fused by two
benzene rings as in Figure 4. DBT is a colorless solid and occur widely in petroleum fraction.
Petrochemical sector has highly several of chemical pollutants including polycyclic aromatic
hydrocarbon, organ sulfur compound and heavy metal while these pollutants may contribute to
serious health problem due to high toxicity and mutagenicity. DBT also shows high potential to
have dangerous effect on health and ecosystem in general (Morales et al., 2010). DBT also one
of the substance that contribute in acid rain once it ignited, due to releasing of sulfur dioxide
(S02) (eara et aI., 2006). Sulfur content such as in DBT need to be reduced to the lowest ever
and refiners has used hydrodesulfurizing technique which is much cost, energy intensive and less
effective in removing of hydrocarbons. This method do not successful to reduce the sulfur
content to low- sulfur fuels (Rhee et al., 1998).
Therefore, biodesulfurization (BDS) or the technique used microbial for desulfurization
process has been an attention that attract people which the technique is much lower cost and
effective alternative. The microbes desulfurize the compound by help in sulfur metabolism
without affect the molecule intact energy source (Izumi & Ohshiro, 2001). Thus the values of
fuel do not affect. DBT is preferred as fossil fuel biological desulfurization model and for
persistent compound studies.
Figure 4: DBT structure compound
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2.S Dibenzothiophene (DBT) desulfurization and genes involved.
Organic compounds consist of sulfur (organosulfur compound) such as DBT that
desulfurize by microbial systems has potential to be used to remove sulfur from fossil fuels.
Sulfur removing process occurs in catabolic pathway without affect the carbon interaction itself
that may convert organic carbon to carbon dioxide. DBT desulphurization by Rhodococcus
rhodochrons IGTS8 shows the process undergo 2 pathways that resulted in 2-hydroxybiphenyl or
2,2' -dihydroxybiphenyl. Growth or undergrowth condition used for desulphurization process
affect either two of end result. 2-hydroxybiphenyl was resulted under non-growth condition
which conversion occurs via 2-hydroxybiphenyl-2-sulfinate.
Mostly 2,2' -hydroxybiphenyl should be resulted, but under growth condition, very little
2-hydroxybiphenyl-2-sulfonate only produces. In aqueous buffer, sulfinate to sulfonate oxidation
spontaneously occur. However, sulfinate also has possibility to be oxidized in enzyme-catalyzed
reaction. Different bacteria possess different name of gene involved rather than carbazole
degrading bacteria which is mostly has very common name such as carA, carB or carC. As an
example, for Rhodococcus sp strain IGTS8, the gene involved was written as dszA, dszB or
dszC. While other bacteria have soxA, soxB and others may contain dbdA and dbdB. However,
they still possess the same function in metabolization of process of bacteria.
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2.6 Bioremediation
Bioremediation is the natural solving problem involving of microorganisms to convert,
degrade, and break down toxicity compound to more useful and non-carcinogenic substances
(Alexander, 1999). The process also can be said to remove the contaminants from the site or
area. The process is biologically conversion of chemicals. Bioremediation is a superior
contribution to environment biotechnology since that it is cost-effective, easy to conduct after
some research is done and effective way than use other chemical to treat the contaminants.
Evolving in utilizing of hydrocarbon as sources to produce pharmaceuticals, dyes, industry and
others contribute to increasing of environment pollutants. Environment pollutants may include
air, soil and water pollutants. Heterocyclic compound presence in groundwater, soil sediment
and also in air samples (Y oon et, al., 2002)
Bioaugmentation is the process where selected bacteria are added on the contaminant site
such as soil or water to remove unwanted substances. The process also equivalent to increasing
the metabolic capabilities of the microbiota present in the soil. Increase of metabolic capabilities
actually the result of an enlargement of the genetic capacity present at the site (Lens, 2005).
Bioaugmentation is commonly used in municipal wastewater treatment to restart activated
sludge bioreactors. Bioaugmentation used to ensure that the in-situ microorgansims can
completely degrade the contaminants. As an example, at site where soil and groundwater are
contaminated with chlorinated ethenes such as tetrachloroethylene and tricholoroethylene,
bioaugmentation is done to degrade substance to non-toxic substance which is ethylene and
chlorine. Biostimulation is addition of electron donor usually done with bioaugmentation to
accomplished geochemical conditions in groundwater that favor growth of microorganisms in
the bioaugmentation culture ( Alvarez, 2005).
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There are plenty of eminent microorganisms in this world act as effective-bioremediation
microorganisms including mycobacterium tuberculosis ( Garcia et al. , 2011) that degrade the
cholesterol and other steroids contaminants and Pseudomonas, Burkholderia and
Sphingomonas (Koukkou & Vandera, 2011) involved in soil hydrocarbon contaminants.
Bacteria usually feed on contaminants as nutrition sources for them to grow and reproduce.
2.7 Heterocyclic-degrading bacteria
Heterocyclic degrading bacteria use compounds as a sole of carbon sources for them to
survive. They metabolized compounds to non-toxic substance. Bacteria involved in degrading
process posseses all specific enzyme responsible in complete degrading processcess. Usually
these bacteria widely distributed and easily can be found in oil-contaminated area such as
industry area,in freshwater, soil and marine environment. In the general bacterial degradation
system, the aromatic compounds are converted to the corresponding cis-dihydrodiol compounds
and then to the corresponding echol-type derivatives, after which they are, cleaved into the
meta- or ortho-ring fission compounds (Sato et al., 1997).
Naphtalene is widely used as a model to identify the degradation process of the P AHs
compound since naphthalene is the simplest and most soluble PAHs (Goyal & Zylstra, 1997).
Thus, the information about this compound has been used to understand and predict the
degradation pathway of other P AHs compound. Abundance of bacteria have been isolated and
studied regarding to utilization of naphthalene as sole of carbon sources belongs to genera
Alcaligenes, Burkholderia, Mycobacterium, Polaromonas, Pseudomonas, Ralastonia,
Rhodococcus, Sphingomonas, and Streptomyces ( Seo, Keum & Li, 2009). Bacteria use the
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