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ANALYTICAL ASSESSMENT OF HETEROCYCLIC COMPOUND DEGRADING ABILITY OF BIPHENYL

DEGRADING MARINE BACTERIA.

Nurul Farahidayu Binti Ab Hadi

QD 480.S S9S Bachelor of Science with Honours N974 (Resource Biotechnology) 2012 2012

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ACKNOWLEDGEMENT

Apart from the efforts of me, the success of any project depends largely on the encouragement

and guidelines of many others. I take this opportunity to express my gratitude to the people

who have been instrumental in the successful completion of this project.

First and foremost, I would like to thank to my supervisor Dr. Azham Zulkharnain for the

valuable guidance and advice. This research project would not have been possible without the

support from him who was abundantly helpful. He also inspired me greatly in this project and

his willingness to motivate me contributed tremendously to my project. Besides, I also would

like to thank him for showing me some example that related to the topic of my project.

Deepest gratitude are also due to the co-supervisor Dr. Tay Meng Guan, without his

knowledge and assistance this study would not have been successful. I also would like to show

my greatest appreciation to postgraduate Miss Jane Sebastian Taka. I can't say thank you

enough for her tremendous support and help. Without her encouragement and guidance this

project would not have materialized. I am grateful for her endless patience teaches me from

the beginning till this project was completed.

My thanks and appreciations also go to my laboratory mates in developing the project and

, people who have willingly helped me out with their abilities. Finally, yet importantly, I would

like to express my heartfelt thanks to my beloved parents for their blessings, my friends/

course mates for their help and wishes for the successful completion of this project.

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DECLARATION

I hereby declare that no portion of the work referred in this project has been submitted in

support of an application for another degree qualification of this or any other university or

institution of higher learning.

(Nurul Farahidayu Binti Ab Hadi) Resource Biotechnology Department of Molecular Biology Faculty of Resource Science and Technology University Malaysia Sarawak

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Pusst Khidmat Maklumat Akademik UNlVEltSm MALAYSIA SARAWAK

Table of Contents

Pages

Acknowledgement ..... .. .......... ..... .... ......................... ......... ............... .......... ... .. .

Declaration....................................... .............................................. .................. 11

Abstract ............................... . ........................................ . ...... .

Table of Contents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. III

List of Abbreviations.................................................................. v

List of Tables ... ............... . ............... . ................................ .. ..... VI

List of Figures ... ........................................................... .................................... vii

1.0 Introduction..... . .................... . ................................... . .... . ... 2

2.0 Literature Review.............. . ................................. . ............... 4

2.1 Biphenyl and Polychorinated Biphenyl (PCBs) .................. .. 4

2.2 Biphenyl Degrading Bacteria ........................... ... ..... . ..... . 6

2.3 Biphenyl Degradation Pathway . ....................................... . 7

2.4 Gas Chromatography .................................................................. . 9

3.0 Materials And Methods........... ..... . ..................... . .... . ............. 10

3.1 Growth Confirmation ..................................................... .. 10

3.2.1 Grow On Liquid Medium CFMM With Biphenyl......... 10

3.2.2 Grow On Solid Medium (Agar) CFMM With Biphenyl. 10

3.2 Analytical Assessment of Biphenyl Degradation ............ . .... .. 11

3.2.1 Extraction of Residual Biphenyl from Growth Media ..... 11

3.2.2 Microbial Count during Biphenyl Degradation ............... 12

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3.2.3 Assessment Using Different Concentration of Biphenyl . 12

3.2.4 Growth Test Using Different Heterocylic Compounds .. , 12

4.0 Results. ................................................................................. 13

4.1 Growth Confirmation ................................................................. .. 13

4.1.1 Growth Confirmation of Isolate on Liquid Medium 13

CFMM with Biphenyl ....................................... ............. ..

4.1.2 Growth Confirmation of Isolate on Solid Medium 13

(Agar) with Biphenyl ........................................... .. ......... .

4.2 Analytical Assessment of Biphenyl Degradation ...................... .. 15

4.2.1 Extraction of Residual Biphenyl from Growth Media .... . 15

4.2.2 Microbial Count during Biphenyl Degradation .... ........... 16

4.2.3 Assessment Using Different Concentration of Biphenyl. 20

4.2.4 Growth Test Using Different Heterocylic Compounds ." 21

5.0 Discussions ................. . ................................................. . ..... 22

5.1 Growth Confirmation .................................................................. . 24

5.2 Analytical Assessment of Biphenyl Degradation ...................... .. 27

5.2.1 Extraction of Residual Biphenyl from Growth Media ..... 27

5.2.2 Microbial Count during Biphenyl Degradation ............... 29

5.2.3 Assessment Using Different Concentration of Biphenyl. 31

5.2.4 Growth Test Using Different Heterocylic Compounds ... 32

6.0 Conclusion ................ ... ....... .. ........ ...... .............................. ...... ...... ..... ....... 34

7.0 References................. .. ........ .. .... . ....................................... 37

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List of Abbreviations

CaCh'2H20 Calcium Chloride Dihydrate

Ca(NOJ)2 AH20 Calcium Nitrate

CFMM Carbon Free Minimal Medium

CFU Colony Forming Unit

DCM Dichloromethane

DNA Deoxyribonucleic Acid

FeS04 '6H20 Ferrous Sulphate Heptahydrate

GC Gas Chromatography

KH2 P04 Monopotassium Phosphate

MgS04 '7H2 0 Magnesium Sulfate Heptahydrate

NaCl Sodium Chloride

Na2HP04 Sodium Pyrophosphate

Na2HP04'7H20 Sodium Phosphate, Dibasic Heptahydrate

Na2 S04 Sodium Sulfate

(N~)2NOJ Ammonium nitrate

Na2S04 Sodium Sulphate

PAHs Polycyclic Aromatic Hydrocarbons

PCBs Polychlorinated Biphenyls

PHA Polyhydroxyaikanoates

Weight! Volume

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w/v

List of Tables

Page

Table 4.1 Serial dilution day 0 14








Table 4.9 Microbial count on days 10 according to concentration of biphenyl 18

(w/v)

Table 4.10 Degradation rate experiment using different heterocyclic compound 19

Table 5.1 Composition of CFMM in liquid media 23

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Analytical Assessment of Heterocyclic Compound Degrading Ability of Biphenyl Degrading Marine Bacteria.

Nurul Farahidayu Binti Ab. Hadi

Resource Biotechnology Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Biphenyl is a kind of a heterocyclic compound with some toxicity and may form during incomplete combustion of mineral oil and coal and may cause toxic thus pollute the environment. Due to this, bioremediation is the way to reduce the pollutant that caused by the biphenyl into the environment by introducing biphenyl-degrading marine bacteria (Asturias & Timmis, 1993). Analytical assessment of heterocyclic compound degrading ability of biphenyl degrading marine bacteria is to identify the degradation rate of biphenyl. In this study, a method that was used to identify the ability and examined the degradation rate of marine bacteria isolate for biphenyl degradation is to precultivated isolate and inoculated the culture for 14 days . Then the culture was analyzed by using analytical method such as GCIFID. Chromohalobacter marismortui which is biphenyl-degrading bacteria showed a concentration-dependent growth in all concentration of biphenyl they grew in and were visible changes color of the growth medium of the isolates during their incubation, suggesting the production of different substrates.

Key words: Bioremediation, biphenyl, biphenyl-degrading marine bacteria, GC/FID.

ABSTRAK

Bifenil adalah merupakan sejenis sebatian heterosiklik yang bertoksik dan terbentuk semasa proses pembakaran minyak mineral dan arang batu yang tidak lengkap dan seterusnya akan menyebabkan alam sekitar tercemar dan bertoksik. Disebabkan hal ini, bioremediasi adalah cara untuk mengurangkan pencemaran alam sekitar yang berpunca daripada bifenil dengan memperkenalkan bakteria marin yang dapat menguraikan bifenil (Asturias & Timmis, 1993). Penilaian analitikal keupayaan bakteria marin bifenil untuk menguraikan sebatian heterosiklik adalah untuk mengenal pasti kadar degradasi bifenil. Dalam kajian ini, kaedah yang telah digunakan untuk mengenal pasti keupayaan dan mengkaji kadar penguraian isolat bakteria marin untuk penguraian bifenil adalah dengan cara membiakkan semula isolat dan inokulat pembiakan selama 14 hari. Kemudian, hasil pembiakan telah dianalisis dengan menggunakan kaedah analisis seperti GCIF1D. Chromohalobacter marismortui merupakan bakteria penguraian bifenil telah menunjukkan species ini hanya dapa! membiak pada ketumpatan yang tertentu sahaja dan perubahan warna pada media pembiakan isolat jelas kelihatan semasa inkubasi sebatian heterosiklik yang berlainan.

Kata kunci: bioremediasi, bifenil, bakteria marin pengurai bifenil, GCIF1D.

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1.0 Introduction

A heterocyclic compound is a cyclic compound which has atoms of at least two

different elements as members of its rings. Biphenyl is a heterocyclic compound with two

different kinds of atoms in it rings one of them which are carbon and the other one is aromatic.

Biphenyl usually possesses a stable ring structure which does not readily hydrolyze or

depolymerize (Richter, 1947). However, this compound is volatile if in aqueous solution.

Biphenyl is also an aromatic hydrocarbon with some toxicity. Due to this, bioremediation is

the way to reduce the pollutant that caused by the biphenyl into the environment. These

methods can help by introducing specific microorganisms to a site and they can transfonn

hazardous contaminants to a less hannful fonn. A number of bacteria are able to initiate the

degradation of the compound by adding molecular oxygen to the ring. In the other word, the

pollution caused by these xenobiotic compounds can potentially be removed by biphenyl-

degrading bacteria used in bioremediation (Asturias & Timmis, 1993). After growth with

biphenyl, many bacteria can oxidize polychlorinated biphenyls (PCBs), a group of man-made

compounds composed of biphenyl molecules containing from 1 to 10 chlorines, and persistent

and toxic in biosphere. Besides, capital and operational costs for biological treatment is

cheaper compared to those mechanical and chemical remediation methods (Schultz, 2005). In

fact, marine biphenyl degrading bacteria is also able to degrade dioxins and PAHs (Atlas et

aI., 1981; Gundlach et aI., 1983). Since last three decades, many reports on the ability of

bacteria to utilize heterocyclic as source of carbon and energy to degrade crude oil have been

appearing (Horowitz et al., 1975). In addition, previous studies by Habe el aI., (200 I) and

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Widada et al., (2002) have reported biphenyl degrading bacteria have the ability in vivo

degradation for biphenyl and various other contaminants. Analytical assessment of

heterocyclic compound degrading ability of biphenyl degrading marine bacteria is to identify

the degradation rate of biphenyl. In this study, a Chromohalobacter marismortui is a moderate

halophiles belonging to the family Halamonadaceae (Antonio et al., 1998) was used as the

marine bacteria to utilize the biphenyl. These type of marine bacteria need nutrient, carbon and

energy that contain in biphenyl as a food and energy source then transforming biphenyl into

harmless substances consisting mainly of carbon dioxide, water and fatty acids. The technique

that were used in this study is to identify the ability and examine the degradation rate of

marine bacteria isolate for biphenyl degradation is by microbial count and using analytical

method such as GCIFID. Other than that, the concentration of the biphenyl and the rate

analysis for other substrates of heterocyclic compound were also determined.

The objectives of this project are:

I. To develop extraction method from growth culture for analytical assessment of biphenyl

degradation

2. To assess the number of bacterial cells during biphenyl degradation

3. To assess the biodegradation ability on different concentration of biphenyl and different

heterocyclic compound

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2.0 Literature Reviews

2.1 Biphenyl and Polychlorinated Biphenyl (PCBs)

Biphenyl (C I2HIO) has the following structural formula:

Figure 2.1 Structural formula of biphenyl. Picture taken from http://upload.wikimedia.org/wikipedialcommons/e/ec/Bifenyl.svg

The chemical is an aromatic hydrocarbon with a peculiar, strong odour similar to that of

geraniums (Safe, 1990). At room temperature, the substance is colorless. Because of its

significant vapour pressure (4 Pa at 20 QC) and low water solubility (4.45 mg/litre at 20 QC),

biphenyl shows considerable volatility from aqueous solutions (Safe, 1990). Biphenyl occurs

in varying concentrations in coal tar, crude oil (up to 0.4 mg/g oil), and natural gas (3- 42: g

/m3) (Safe, 1990). Because of its natural occurrence in coal tar and crude oil, biphenyl has also

been detected in products derived from these substances.

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PUsat Khidqat Maklumat Aka4lemik ~lVERSm MALAYSIA SAKAWA)(

PCBs (CI2HIO.xClx) has the following structural fonnula:

(COn

Figure 2.2 Structural formula of PCBs. Picture taken from http://upload. wi kimedia.orglwikipedialcommonsl 4/49/Polychlorinated _ biphenyl _structure .svg

PCBs belong to a broad family of man-made organic chemicals known as chlorinated

hydrocarbons. PCBs were domestically manufactured from 1929 until their manufacture was

banned in 1979 (Faroon et al., 2003). They have a range of toxicity and vary in consistency

from thin, light-colored liquids to yellow or black waxy solids. Due to their non-flammability,

chemical stabi l~ity, high boiling point, and electrical insulating properties, PCBs were used in

hundreds of industrial and commercial applications including electrical, heat transfer, and

hydraulic equipment.

Once in the environment, PCBs do not readily break down and therefore may remain for long

periods of time cycling between air, water, and soil (Schwarzenbach et aI., 1992). PCBs can

be carried long distances and have been found in snow and sea water in areas far away from

where they were released into the environment. As a consequence, PCBs are found all over the

world. In general, the lighter the fonn of PCB, the further it can be transported from the source

of contamination (Safe, 1994).

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2.2 Biphenyl Degrading Bacteria

Pseudomonas pulida is a Gram-negative biphenyl degrading soil bacterium that is commonly

found in most soil and water habitats where there is oxygen. Members of the genus

Pseudomonas often displays to be rod-shaped, have single or multiple polar flagella for

motility, and are aerobic and non-spore forming microorganisms (Krieg & Noel, 1984).

Pseudomonas pulida has a very diverse aerobic metabolism that is able to degrade organic

solvents such as toluene and also to convert styrene oil to biodegradable plastic

Polyhydroxyalkanoates (PHA) (Muller, 1992). This helps degrading the polystyrene foam

which was thought to be non-biodegradable. In bioremediation, most genes of Pseudomonas

pulida can break down aromatic and alipathic hydrocarbons which are hazardous chemicals

caused by burning fuel, coal, tobacco and other organic matter.

Besides, Pseudomonas sp., the Rhodococcus erylhropolis is also known as the biphenyl

degrading bacteria. Rhodococcus is a genus of non-motile, non-sporulating, aerobic Gram

positive filamentous rods of the phylum Actinobacteria. These organisms reside in soil and

water environments and are classified as one of the most industrial important organisms

(Asturias & Timmis, 1993). Its strain contains enzyme that can carry out biologically relevant

reactions such as degradation of polychlorinated biphenyl and utilization of wide variety of

other organic compounds as energy source (Wu et al., 2003). Rhodococcus sp. strain RHA I

has the ability to aerobically degrade polychlorinated biphenyls (PCBs) through

cometabolization by the bph pathway, which is responsible for the aerobic degradation of

biphenyl (McLeod et ai., 2006).

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2.3 Biphenyl Degradation Pathway

The major catabolic pathway is the most common pathway for biphenyl degradation (Catelani

et al., 1973; Hayase et al., 1990; Kimbara et al., 1989). The degradation of biphenyl has two

pathways which are Upper-Catabolic-Pathway and Lower-Catabolic-Pathway. In Upper

Catabolic-Pathway Metabolites, the biphenyl is an inducer of the enzymes. The biphenyl

degrading bacteria produce some defined metabolites such as chlorobenzoates when growing

on monochlorinated biphenyls.

In dehydrogenase activity, the product of the biphenyl molecule which is meta-cleavage can be

transfonned into a saturated side-chain compound. In Lower-Catabolic-Pathway, it influences

the upper-catabolic-pathway enzyme activity by further transfonned of the metabolites. For

examples, if the microorganism possesses the enzymes of both catabolic pathways, a molecule

can be transfonned to other organic or inorganic substances.

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Pseudomonas pseudoalcaligenes KF707 bph gene cluster

II RI II All A2 10':'/31 A3 I A4 liB II ell xo II Xl II X2 II' X3 II D ~

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'"" I Upper Pathway

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~~ BPhX~ ~>--< ...s::: s

FcrrcdoxinCox) Fcrrcdoxin(rcd) ~ H~O -TCA cycle I t-OOH ~ ~ BPbXY VII FCl'rc(.hlxin I-crrcdoxin n.:dUl.;t~lsc(rcd) l'I:ductasc(ox) ~

B .·hX') C··I':l ·I N 0 C)o . P . . ~ 'c' + o

CH3-C# CH.!-C~ t ('0011. ........11~ /

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Figure 2.3 Catabolic pathways for degradation of biphenyl and organization ofbph gene cluster in P. Psedoalcaligenes KF707 (Kensuke et al., 2004).

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2.4 Gas Chromatography

Gas chromatography (GC) is used to separate volatile and semi-volatile organic components

of a mixture and has been one of the most versatile applicable techniques leading the field of

analytical chemistry over the last forty years (McNair & Miller, 1998). In GC the analyte is

carried through the column by a mobile phase composed of an inert gas such as helium or

hydrogen. A carrier gas, such as helium, flows through the injector and pushes the gaseous

components of the sample onto the GC column. It is within the column that separation of the

components takes place. Molecules partition is between the carrier gas (the mobile phase) and

the high boiling liquid (the stationary phase) within the GC column. As the vaporized analyte

travels through the column it interacts with the liquid stationary phase (McNair & Miller,

1998).

Depending on the analytes solubility for the stationary phase, they will separate and elute,

from the column (McNair & Miller, 1998). Upon elution the analytes enter a detector, which

produces an electrical signal. This signal is sent to a data system that generates an image,

called a chromatogram, displaying the analyte peaks. Certain analytes, specifically nitrogen

containing compounds (i.e. aliphatic primary, secondary, and tertiary amines), can be difficult

to detect using GC because there is significant adsorption of the basic amines on the often

acidic column as well as decomposition of the analyte (Kataoka, 1996). It would be

advantageous to use GC in many applications that analyze amine-containing compounds if a

reproducible, reliable method could be developed. An application of particular interest

concerning amine analysis is the detection and quantification of biogenic amjnes.

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3.0 Material and Methods

3.1 Growth Confirmation

3.1.1 Growth on Liquid Medium CFMM with Biphenyl

The strain that was obtained from Genetic Molecular Laboratory collection was grew at room

temperature in carbon free minimal medium as described by Monna et al., (1993) [CFMM;

containing (per liter): 2.2 g Na2HP04, 0.8 g KH2P04, 0.2 g MgS04'7H20, 0.05 g

FeS04'7H20, 0.01 g CaCh '2H20 , 0.01 g yeast extract) and supplemented with biphenyl

Imglml. The culture was rotated on the rotary shaker for 140 rpm.

3.1.2 Growth on Solid Medium (Agar) CFMM With Biphenyl

The strain was grown at room temperature In carbon free minimal medium [CFMM;

containing (per liter): 2.2 g Na2HP04, 0.8 g KH2P04, 0.2 g MgS04 '7H20, 0.05 g

FeS04'7H20, 0.01 g CaCh'2H20, 0.01 g yeast extract and 5.25 g of Bacto agar/ 350ml of

CFMM were added. All the composition was mixed and after the CFMM media was

autoclaved the media was poured into the plates and was stored in refrigerator at 4°C. Besides,

13.09 g of marine broth was also used with 5.25 g of Bacto agar in 350 ml of distilled water.

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3.2 Analytical Assessment of Biphenyl Degradation

3.2.1 Extraction of Residual Biphenyl from Growth Media

Biphenyl was determined by gas chromatography flame ionization detector (GCIFIO) in the

following procedure modified from Larentis et al., (2011). The sample was extracted with

equal volume of dichloromethane (OCM) which is 2.5 ml to determine the concentration of

the remaining biphenyl and after that, was determined by a 2.5 ml aliquot will be sampled

from each flask every second day from day 0 till day 14 of the experiment. The sample was

extracted 3 times with 2.5 ml of ethyl acetate to recover the substrates and products, and then

was dried over anhydrous sodium sulfate (Na2S04)' After that, they were filtered through

Whatman Syringe Filters and were evaporated to dryness for 1 hour in laminar flow for

evaporation process. The samples were then redissolved in 2.5 ml dichloromethane (OCM)

and win be placed in 4 ml amber screw capped vials (Supelco, Mississauga, ON). Then, ten

microliters of the resulting solution was subjected to Gc. The operation conditions were as

follows: Oven initial temperature (50°C); Oven final temperature (270°C); Injection port

temperature (90°C); N2 carrier gas flow rate (30 mllmin); H2 gas pressure (1.5 kg/cm2); Air

pressure (2.0 kg/cm2); Chart speed (1 crn/min); and Volume of sample injected (0.5 ml).

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3.2.2 Microbial Count during Biphenyl Degradation

The microbial count was monitored by standard serial dilution technique using agar spread

plate method from 0.1 ml till 1 x 10-5 ml. The culture was collected every 2 days for 14 days

for analysis and was stored at room temperature.

3.2.3 Assessment Using Different Concentration of Biphenyl

The sample from solid medium cultured was inoculated with different concentration of

biphenyl which are 0.1 % (w/v), 0.5% (w/v) , 1.0% (w/v) , 1.5% (w/v) and 2.0% (w/v)

respectively. 0.1 % (w/v) of biphenyl was used as control because it is the most suitable

biphenyl-degrading bacteria to grow. Then the microbial were counted by using standard serial

dilution technique same method as mention in the previous method in microbial count.

3.2.4 Growth Test Using Different Heterocylic Compounds

The sample from solid medium subculture was inoculated and was cultured into CFMM liquid

medium supplemented with different heterocyclic compounds which are dibenzothiophene,

carbazole, dibenzofuran, fluorine and biphenyl by using the same isolate . Biphenyl was used

as controlled. The culture was done in the suspension to view the changes and intensity of

color.

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4.0 Result

4.1 Growth Confirmation

The result of growth confinnation of isolate on liquid medium and solid medium with

biphenyl is summarized in Figure 4.1 to Figure 4.4.

4.1.1 Growth Confirmation of Isolate on Liquid Medium CFMM with Biphenyl:

Figure 4.1 Culture media without biphenyl-degrading Figure 4.2 Culture media with biphenyl-degrading bacteria bacteria

The culture media without biphenyl-degrading bacteria in Figure 4.1 was shown that the color

of the media was colorless with white precipitate while the culture media with biphenyl-

degrading bacteria in Figure 4.2 was golden yellowish in color. Besides, based on Figure 4.2

also it showed that the Chromohalobacter marismortui was degrade the biphenyl.

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4.1.2 Growth Confirmation ofIsolate on Solid Medium (Agar) With Biphenyl:

Figure 4.3 Solid medium (agar) CFMM with biphenyl-degrading bacteria

Figure 4.4 Solid medium Marine Agar with biphenyl-degrading bacteria

In Figure 4.3 above, the solid medium (agar) CFMM with biphenyl-degrading bacteria was

shown that its colony is too tiny to be seen and the color was light yellow while in Figure 4.4

the solid medium Marine Agar with biphenyl-degrading bacteria was golden yellowish in

color and the colony can clearly be seen with naked eye.

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4.2 Analytical Assessment of Biphenyl Degradation

4.2.1 Extraction of Residual Biphenyl from Growth Media

Before using ethyl acetate as an extraction solvent, acetonitrile was used to extract the residual

biphenyl from growth media but they do not dissolved the sample completely and form salt

crystal. Thus, ethyl acetate was used as extraction solvent and the result of mass spectrum with

retention time from the extraction of residual biphenyl from growth media by gas

chromatography flame ionization detector (GCIFID) is shown below,

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IOOl . . ,'f: ( ) ' t""P") I ~ ,. j

" .1'>1 ),1 'n I IN 10': I i!i l ~:-' .~ .......~- ....

iVY' i ,"1 r' ; , i '''r,+''''4 ,,",I i i; 1 i 14+1#P \ i ". ii' i 'j i 'i+t4f''rYi if Fty r+j Ii 1 i F fG f" r if' f ¥ f 19 Pi' ,&qT4, Ff4=F" iii ''114 20 50 SO 110 140 170 200 l~O 260 ~90 320 350 3110 410 44() 470

Figure 4.5 Mass spectrum of biphenyl-degrading bacteria

Mass spectrum of biphenyl-degrading bacteria above showed the peak at mlz 154 is the actual

product that must be obtained which is biphenyl. Thus, the method proved that it is the best

method to extract the residual of biphenyl from growth media,

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4.2.2 Microbial Count during Biphenyl Degradation

Degradation rate analysis by microbial count was monitored by standard serial dilution

technique using agar spread plate method from 0.1 ml till 1 x 10-5 ml was collected every 2

days for 14 days result is summarized below. The plate count method below shows that the

colony forming units were increasing until day 10 and decreasing on day 12 and day 14.

Table 4.1: Serial dilution day 0

Day 0 (7 April 2012)

Dilution 1 Dilution 2 Dilution 3 Dilution 4 Dilution 5

Table 4.2 Serial dilution day 2

Days 2 (9 April 2012)

Dilution 1 Dilution 2 Dilution 3 Dilution 4 Dilution 5

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Dilution I Dilution 2 Dilution 3 Dilution 4 Dilution 5







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