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

iii

.,.-

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

iv

%

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

vii

"...

Figure 13 .........................................................................................34

Figure 14.........................................................................................35

Figure 15 ......................... . ............... . ...............................................37

Figure 16.........................................................................................38

Figure 17..................................................................................., .....39

Figure 18.........................................................................................57

viii

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

1

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.

2

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.

3

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).

4

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

5

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.

6

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.

7

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.

8

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

9

....

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).

10

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.

11

.....

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

12

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.

13

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).

14

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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