ANALYTICAL ASSESSMENT OF HETEROCYCLIC … Assessment of Heterocyclic... · analytical assessment of...
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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 4.2 Serial dilution day 2 15
Table 4.3 Serial dilution day 4 15
Table 4.4 Serial dilution day 6 15
Table 4.5 Serial dilution day 8 15
Table 4.6 Serial dilution day 10 16
Table 4.7 Serial dilution day 12 16
Table 4.8 Serial dilution day 14 16
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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-.\'~ ~'l>"" -.\,,-,~ t>.' .;...vc)"'~~'-o . ~~~ 'i:,.....oto~.....'- " . '< ~c~ ~~ -o<':-'.:!J'~ . _,:;..C >..<.0'1- 01>" .....~..... ,~o~:i;:.-oc -o<.o~_'..,:.'t> ~~~?>-<.c.'-':i • ..«:>~:(.~'" . ~i;:§:.""v .A<:; .~""-:~' ,,0 -,v. &' '-> , <:...... •........ ~~ :s' ~ ~, -0" -oc~ ,",<..~-......'b' .A-<:-'~~ '< ...... ,-v '-~ " t>.' "'..... t>.~-' 'V,'..... o-.....~v~~..... .:..... "'c.... -""" '\.~' c~..:::;: '\.- ,~c'" ~c' .-§,"
'"" I Upper Pathway
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"""-. "11 BphB """-. I 011 BpbC BphD 6~ + ~OIITI' I --~~~~.~ r ~ ... # II ,LrJOII2' I ~ 6' ~ T" N.ADH3' ...-9 5' I ...-9 NAD+ 1...-9 o;! VISP(rcd) ISP(o:x) r VI-I I·r .... H~() ./ 1/4' BphA I/BphA2 H HI
~~ 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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+ HI TCA cycle
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,
« Tar~..-( » Lin\...tl:2 R. ~rim(': 7 .()90(Sc.~~u}#:4191 ~f~\.~!'Pcnk.....:260 Rawl\1odt>:Av\."fi\gcd 7.0tol~-7 .I.>95{.... 18-110) Ba.......~~.a.k: 15..... 1(~ 131240, fiG 1\.1od."":Calc. from Peak Group 1 - Event I J(Xhr-------------------rr..---~----------------------------------------------_.
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Cllml'Namc: Biphe"yl $.~ J, j'. Bil'lk' "yl $$ Bib,,,",,,,,., $.'i; Diplk'.nyl :',$ Ph'~lylt>;,nh'l'" :;'$ 1. I '· Dip"""),1 S$ u"mll,,,,nc $$ Pl,cn.,,1or·X $'
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,
15
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
16
Table 4.3 Serial dilution day 4
Days 4 (11 April 2012)
Dilution I Dilution 2 Dilution 3 Dilution 4 Dilution 5
Table 4.4 Serial dilution day 6
Days 6 (13 April 2012)
Dilution I Dilution 2 Dilution 3 Dilution 4 Dilution 5
Table 4.5 Serial dilution day 8
Days 8 (15 April 2012)
Dilution I Dilution 2 Dilution 3 Dilution 4 Dilution 5
17