Post on 02-Feb-2023
Senior Project
entitled
IDENTIFICATION OF DIESEL-DEGRADING BACTERIA FROM
OIL-CONTAMINATED SOIL SAMPLES THAT WERE TREATED
WITH EITHER BIOAUGMENTATION OR BIOSTIMULATION
was submitted to the Mahidol University International College, Mahidol University
for the degree of Bachelor of Science (Biological Sciences), Biomedical Science
Concentration
on
July 21st, 2014
……………….………….…..………
Narattapat Watcharapriyapat
Candidate
………………….…..………….……
Assoc. Prof. Prayad Pokethitiyook, Ph.D.
Advisor
……………………….….…..………
Assoc. Prof. Saovanee Chancharoensin,
Ph.D. Chair of Science Division
Mahidol University International College
Mahidol University
……………………….….…..………
Asst. Prof. Mr. Laird B. Allan. Program Director
Bachelor of Science in Biological
Sciences
Mahidol University International College
Mahidol University
iii
ACKNOWLEDEGEMENTS
First of all, I would like to express my most sincere gratitude to my advisor of
this project, Assoc. Prof. Prayad Pokethitiyook for his constant supervision, guidance
and encouragement throughout the research. His genuine support has led to the
completion and success of this project.
I also would like to extend my most sincere gratitude to all members of the
committee in the Science Division of Mahidol University International College as well
as the Faculty of Science of Mahidol University for their genuine collaboration in
making the ideas in this project becomes reality.
Furthermore, I would to give special thanks to my laboratory supervisors for
their technical and social support in demonstrating the uses of equipment and inspiring
the importance of biological sciences. Their guidance and company greatly contributed
to the success of this project.
Last but not least, I would like express my deepest gratitude to my family and
friends who were always there to support and motivate me in all aspects of life as a
student and also as a human being.
Narattapat Watcharapriyapat
Mahidol University International College Senior Project / iv
IDENTIFICATION OF DIESEL-DEGRADING BACTERIA FROM OIL-
CONTAMINATED SOIL SAMPLES THAT WERE TREATED WITH EITHER
BIOAUGMENTATION OR BIOSTIMULATION
NARATTAPAT WATCHARAPARIYAPAT 5580081
B.Sc. (BIOLOGICAL SCIENCES) BIOMEDICAL SCIENCE CONCENTRATION
SENIOR PROJECT ADVISOR: ASST. PROF. PRAYAD POKETHITIYOOK, Ph.D.
ABSTRACT
The petroleum chemicals that are used for energy consumption, once entered
into the environment such as lands and oceans, become dangerous to the survival of
animals and harmful to human health. This concern has led directly to the engagement
of this research in which the discovery of bacterial ability, known as biodegradation, to
degrade petroleum chemicals was pursued and the efficiency of the bacteria to degrade
such chemicals was to be determined. From this research, two piles of oil-contaminated
soils, that were treated using the application of biotechnology known as bioremediation,
were sampled to find out the bacteria. Seven different strains of bacteria were found to
possess such ability. Out of seven, three strains were present in both piles of treated soils.
Only one of the three showed the best growth ability after the screening and isolation
processes, and thus was chosen to be investigated for its diesel removal efficiency. It
was found that after 15 days of incubation, the chosen bacteria was able to degrade 49.78%
of the tested diesel oil. The bacteria was then identified using gene sequencing
technology. The strain of the bacteria was found to be Ochrobactrum sp.
KEY WORDS: BIODEGRADATION/ BACTERIA/ BIOREMEDIATION/ SOILS/
DIESEL OIL/ SCREENING PROCESS/ ISOLATION PROCESS/ IDENTIFICATION
45 pages
Mahidol University International College Senior Project / v
CONTENTS
Page
ACKNOWLEDGEMENTS ....................................................................................... iii
ABSTRACT ................................................................................................................. iv
LIST OF TABLES ....................................................................................................... vi
LIST OF FIGURES .................................................................................................... vii
CHAPTER 1 INTRODUCTION AND RATIONALE ............................................ 1
CHAPTER 2 OBJECTIVES ..................................................................................... 2
CHAPTER 3 LITERATURE REVIEW .................................................................. 3
3.1 Bioremediation ................................................................................................... 3
3.2 Screening Process ............................................................................................... 4
3.3 Diesel Oil ............................................................................................................ 5
3.4 Removal Efficiency Testing ............................................................................... 6
CHAPTER 4 MATERIALS, EQUIPMENT AND METHODS............................. 7
4.1 Materials ............................................................................................................. 7
4.2 Equipment........................................................................................................... 8
4.3 Methods .............................................................................................................. 9
4.3.1 Culture Medium Preparation and Incubation Procedure ...................... 12
4.3.2 Transfer of Culture Medium ................................................................. 12
4.3.3 Pour-Plate Technique ........................................................................... 13
4.3.4 Serial Dilutions ..................................................................................... 14
4.3.5 Agar-Plate Spreading Technique .......................................................... 15
4.3.6 Plate Streaking Technique .................................................................... 16
4.3.7 Gram Staining Procedure ..................................................................... 16
4.3.8 Removal Efficiency Testing Procedure ................................................ 17
4.3.9 Identification of bacteria by gene sequencing ...................................... 23
CHAPTER 5 RESULTS .......................................................................................... 24
CHAPTER 6 DISCUSSION .................................................................................... 36
CHAPTER 7 CONCLUSION ................................................................................. 38
REFERENCES ........................................................................................................... 39
BIOGRAPHY ............................................................................................................. 40
Mahidol University International College Senior Project / vi
LIST OF TABLES
Table Page
5.1 Types of bacterial colonies and their morphological characteristics 24
5.2 Types of bacterial colonies present in each pile of treated soil 25
5.2 Growth patterns of all identifiable bacterial colonies 26
5.4 Measurements of diesel biodegradation by Bac. 4 32
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LIST OF FIGURES
Figure Page
4.1 Preparation of LB solution 10
4.2 Incubator with automatic rotator 12
4.3 Transfers of bacterial cultures during the enrichment period 13
4.4 Addition of diesel oil 18
4.5 Liquid-liquid extraction procedure 20
4.6 Multi-function Evaporator 22
5.1 Bac. 1 Gram Negative 28
5.2 Bac. 2 Gram Positive 28
5.3 Bac. 3 Gram Negative 29
5.4 Bac. 4 Gram Positive 29
5.5 Bac. 5 Gram Negative 30
5.6 Bac. 6 Gram Negative 30
5.7 Bac. 7 Gram Negative 31
5.8 Weights comparison between the control and the average non-degraded diesel 33
5.9 Calculations of Percentages of Diesel Oil Removal Efficiencies 34
Mahidol University International College Senior Project / 1
Chapter 1 Introduction and Rationale
The rapidly expanding level of consumption of natural resources has given rise to
the need for society to exploit the amount of usage of petrochemical products or
petroluems (Scragg, 2004). Almost all nations are invloved with the manufactoring
processes and transportations of almost all types of commodities, and these activites
all require the use of protroleum as the main source of energy and raw materials.
Unfortuantely for the society, many of the petrochemical products are not being
efficiently used by the various industries. These petrochemical products are
occasioanlly being evidently seen to be spilled into the natural enviroment, causing
the contaiminations of both soil and water. As a result, damages are being done not
only to the natural ecosystems but also to the lands used for producing food needed
by the people in the society where these industries are responsible for. However, to
solve this problem, we cannot rely solely on the assumption that the industries held
responsible will be able to rectify their own mistakes. Viable solutions to any
problem, such as this one, that affects our society in the large scale need to be
provided.
On the other hand, because the human knowledge is constantly becoming more
vast and critical and the information regarding sceince and technology is being more
and more demanded for discussion and analysis by both intellectuals and the general
public. This makes us become more aware of different types of environmental
problems and issues. As such, we can use the available information to study what
types of applications science and technology has for us to solve the environmental
problems. Bioremediation is one of the applications that resulted from the study of
environmental science. This particular application involves the use of
microorganisms as a source of treatment to treat the soil and water that are
containminated with organic and inorganic pollutants. Therefore, in our case,
whereby the petrochemical products are the concerned pollutants, this project will
be invastigating the efficency of using diesel-degrading bacteria as the treatments
for various oil-contaimminated soils.
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Chapter 2 Objectives
2.1 To screen and isolate for bacteria capable of performing diesel biodegradation
from contaminated soil.
2.2 To test for the efficiencies of diesel biodegradation of the newly isolated
bacteria.
2.3 To identify the strain(s) of bacteria with the best growing ability in both
biostimulated and bioaugmented soils.
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Chapter 3 Literature Review
3.1 Bioremediation
Definition
The biological method of contaminant degradation and removal from
contaminated sites, such as soil and water, is known as a bioremediation (Scragg,
2005). The contaminants that can be degraded and removed by bioremediation can
either be in an organic or an inorganic form. Moreover, it is more cost-effective to
use bioremediation to degrade and remove the contaminants of lower concentrations
than to use the conventional chemical or physical removals; although the period of
the treatment may be longer. The general principle of bioremediation relies upon the
presence of the indigenous microbial population that thrive in soils and water; and
the stimulation to encourage the growth of this presence. Soils alone contain a large
variety of microorganisms capable of breaking down contaminants and use them as
their own carbon sources for growth and development. This method of employing
biological treatment on soil is known as bioremediation on land. If the biological
treatment is performed on-site it is often referred to as an in situ treatment. If the
treatment occurs off-site, then, it is often referred to as an ex situ treatment. (Scragg,
2005) Land farming, bioventing, biosparging, bioaugmentation and biostimulation
are all well-known methods of the in situ process of bioremediation. Some examples
of the ex situ process are composting, biopile process, and bioreactors (Scragg,
2005). Since this project will be investigating only the bioaugmentation and
biostimulation methods of an in situ process, the rest of the methods will not be
mentioned.
Bioaugmentation
This method involves the addition of external microorganisms to the contaminated
soil in order to further supplement the soil’s indigenous microbial population and
hence to speed up the biodegradation process (Scragg, 2005).
Biostimulation
The method refers to the addition of nutrients to the contaminated soil in order to
stimulate the growth of the soil’s indigenous microbial population. However, this
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method can be combined together with bioaugmentation in which the combined
method of adding both microorganisms and nutrients into the same soil is known as
bioaugmentation/stimulation (Scragg, 2005).
3.2 Screening Process
The screening method is one common method used in any molecular biology
laboratory to find out the variety of microorganisms present in the product of
interest, which in this case is the oil-contaminated soil obtained from the site of
contamination. This method involves several main procedures all of which are vital
to the successfulness and effectiveness of the final outcomes.
3.2.1 Culture Medium
Culture medium (plu. media) is nutrient-rich chemical solution used to grow
microorganisms in the laboratory (Madigan and Martinko, 2006). It is crucial that
attention must be paid when it comes to selecting the types of media used, because
different media contain different necessary nutrients for different types of
microorganisms to utilize. For this project, we will be using the Luria-Bertani (LB)
Broth, a type of liquid medium, which is well-known among biologists because it
can support the growths and can give excellent growth yields of many species of
bacteria (MacWilliams and Liao, 2006). Another type of medium we will be using
is called the Luria-Bertani (LB) Agar, which is simply the solidified version of the
LB broth whereby the agar is added to the medium solution to allow the medium to
be made into an agar plate.
3.2.2 Enrichment Period
The enrichment period describes the vital step in encouraging the growth of
bacteria suspended in liquid medium under specific laboratory settings, namely a
constant incubation temperature of 30°C with continuous unidirectional circular
motions of mixing. It is within this period that we will be magnifying the number of
bacteria great enough to be used in the later procedures.
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3.2.3 Gram Staining Method
After a certain number of bacteria, usually in the form of colonies, can be obtained
from the laboratory, it is time to identify the types of bacteria based on the
differences in their cell wall structures and compositions. This very common
technique of identification of bacteria is known as the gram stain. (Madigan and
Martinko, 2006) Based on the differential bacterial cell wall morphologies, the types
of bacteria can be divided into 1) Gram positive and 2) Gram negative. The Gram
negative bacteria has the cell wall that consists of one innermost layer of plasma
membrane, one inner layer of periplasmic space, one middle layer of peptidoglycan,
one outer layer of periplasmic space, and one outermost layer called outer membrane
composing of lipopolysaccharides and proteins. Similarly, the Gram positive
bacteria has almost all of the above components in its cell wall; only two layers that
are not present in the Gram Positive bacterial cell wall which are the outer layer of
periplasmic space and the outer membrane. After the gram stain, the Gram positive
bacteria will appear purple under the investigation using the compound light
microscope. Whereas, the Gram negative bacteria will appear pink or red instead of
purple. (Madigan and Martinko, 2006)
3.3 Diesel Oil
Physical & Chemical Properties
Diesel oil is a type of organic compound capable of providing energy source
once it is broken down through the process of combustion. It is a complex mixture
consisting of paraffinic, olefinic, and aromatic hydrocarbons, plus small additional
quantities other substances that contain sulfur, nitrogen, metals and oxygen
(Seelakhan, 2012). Common diesel oil can be produced from fractional distillation
of crude oil at the temperatures between 200°C (392°F) and 350°C (662°F), at the
atmospheric pressure. It has the molecular weight of approximately 200; densities
of 6.7 to 7.4 lb/gallon at 60°F; boiling temperatures of 370°F to 650°F; freezing
temperatures of -40°F to 30°F; a Reid vapor pressure of 0.2 psi; viscosities of 2.6 to
4.1 at 60°F; an auto-ignition temperature of approximately 600°F. (Seelakhan, 2012)
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Environmental Toxicology of Diesel Oil
Diesel oil and other petrochemical products derived from crude oil that are
released on land comes from the intentional disposal of waste motor oil, the
accidental leaking of storage tanks and spillages and other accidents during
transportation (Scragg, 2005). The leaks of this kind, accidental or not, is of a
particular interest because of the high content of BTEX (benzene, toluene, ethyl
benzene, and xylene) compounds present in diesel oil. Although BTEX is not known
to be miscible in water, but when spilled on land it can contaminate the ground
water. Furthermore, some components that are volatile can become airborne if the
spillage occurs close to the surface of the ground. (Scragg, 2005) The contamination
of ground water by BTEX compounds or compounds derived from crude oil poses
many hazards upon human health. Acute benzene exposure at 7500 ppm for 30
minutes is known to be fatal to humans. Also, chronic exposure of benzene can lead
to bone marrow injuries which can be irreversible. (Katzung, 2009) Another
example would be an exposure of toluene in which it can act as a depressant of the
central nervous system capable of causing severe fatigue, ataxia, and if
concentration is high enough it can lead to loss of consciousness (Katzung, 2009).
3.4 Removal Efficiency Testing
Since the biodegradation of organic contaminants is related to the growth and
the overall metabolism of microorganisms, hence any of the factors that can affect
microbial growth will also affect the rate of biodegradation (Scragg, 2005). This is
why it is important to find out the differences in removal efficiencies of diesel oil
deployed by different species of the microorganisms. For this experiment, after the
screening process is completed, the strain(s) of bacteria that displays the best
growing ability in the treated soils will be identified, and its percentages of removal
efficiencies will be calculated to find out their potential uses in the treatments of
diesel oil-contaminated soils.
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Chapter 4 Materials, Equipment and Methods
4.1 Materials
Culture media
o Luria-Bentani Broth (LB)*
o Mineral Salt Media (MSM)**
Bacto™ Agar
Grade II deionized water (dH2O)
Grade I deionezed water (dH2O)
85% NaCl Solution
95% Ethyl Alcohol (EtOH)
Diesel oil
Crystal Violet solution
Iodine solution
Fuchsine counterstain
Acetone
Hexane
Sodium Sulfate
Bioaugmented & Biostimulated soils
*Luria-Bentani Broth (LB) formula:
- Bacto lyptone 10 g
- Yeast extract 5 g
- NaCl 10 g
- dH2O 1000 ml
** Mineral Salt Media (MSM) formula:
- K2HPO4 6 g/1000 ml
- KH2PO4 4 g/1000 ml
- NH4Cl 40 g/1000 ml
- MgSO4∙7H2O 2 g/1000 ml
- NaCl 1 g/1000 ml
- FeSO4∙7H2O 0.1 g/1000 ml
Mahidol University International College Senior Project / 8
4.2 Equipment
Autoclave machine
Laminar flow cabinet
Evaporator
Separatory funnels for Liquid-liquid extraction
Laboratory scale
Weighing paper
Filter papers
Incubator with automatic rotary shaker
Compound light microscope
Alcohol Burner
Micropipettes
Micro-tubes
Volumetric flasks (125 ml)
Glass bottles (500 ml)
Glass tubes
Petri dishes
Graduated cylinder (500 ml)
Graduated cylinder (100 ml)
Spatula
Glass droppers
Small and medium beakers
Cotton cloth balls & Aluminum foil
Parafilm® M
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4.3 Methods
4.3.1 Culture Medium Preparation and Incubation Procedure
The successful preparation of culture medium is the first step in making sure that
the experiment will proceed with as few errors as possible. The medium that was
used in this experiment was the Luria-Bentani Broth or LB, and the reason for this
choosing was because it contained necessary ingredients for the growth of many
species of bacteria. First, the LB medium that was used came in the form of a
powder containing a standardized mixture of all common ingredients used to make
the LB solution. Then, for the LB solution to be successfully prepared, the
laboratory scale that had a precision of four decimal places was used to measure up
the LB powder, so that exactly 12.50 g of LB powder could be obtained. After that,
the 12.50 g of LB powder was transferred into a clean 500 ml glass bottle, followed
with exactly 500 ml of Grade II dH2O. The steps of mixing the LB powder with
Grade II dH2O occurred two more times, to yield exactly three bottles each
containing 500 ml of LB solution (Figure 4.1).
Mahidol University International College Senior Project / 10
To make sure that the mixture between the LB powder and the Grade II dH2O
would not form any precipitate, the mixtures in all three bottles were shaken
thoroughly by hands until the mixtures became completely homogenous. Then, a
100 ml graduated cylinder was used to transfer 50 ml of the LB solution into a clean
125 ml volumetric flask. This step was repeated fifteen times until 16 flasks all
containing exactly 50 ml of LB solution were obtained. After the LB medium was
prepared and distributed into 16 flasks, it must be sterilized before it could be used
to grow the bacterial cultures. To sterilize the medium, all of the 16 flasks were
capped tightly with cotton cloth balls and aluminum foil, and then were arranged
into the autoclave machine that was capable of producing high vapor pressure. The
autoclave machine was set up to sterilize the flasks at 121°C for 20 minutes. The
whole sterilization process took about 2 hours to complete because the machine had
to warm up and warm down to complete the sterilization cycle. The autoclave
machine was stopped when its display showed 80°C, and then all of the 16 flasks
Figure 4.1 Preparation of LB solution
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were removed and cooled down in the laboratory at room temperature. After all of
the flasks had cooled down, they were then labeled accordingly before being used.
The 16 flasks were labeled according to the periods and the piles of the two different
soils that had undergone either biostimulation or bioaugmentation. From now on,
let Pile 1 (P1) denotes the biostimulated soil while Pile 2 (P2) denotes the
bioaugmented soil. Because there must be two trials for each period and each pile
of the treated soils, therefore two flasks were used for each of the circumstances. In
details, the flasks were labeled from Day 0 (D0)/Pile 1 (P1) to Day 60 (D60)/Pile 2
(P2); hence D0P1, D0P1*, D15P1, D15P1*, D30P1, D30P1*, D60P1, D60P1*,
D0P2, D0P2*, D15P2, D15P2*, D30P2, D30P2*, D60P2, D60P2*; whereby the
‘*’ signs indicate the flasks that accommodate the 2nd trials of the experiment. After
all of the flasks were labeled, exactly 5 g of treated soil from Pile 1 were added to
each of the flasks that were labeled from D0P1 to D60P1*. This step was repeated
with the treated soil from Pile 2 and the other eight remaining flasks that were
labeled from D0P2 to D60P2*. Then, all of the prepared flasks were put into the
incubator with automatic rotary shaker that was set at 150 rpm and 30°C (Figure
4.2). This marked the first enrichment period that took 3 consecutive days.
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4.3.2 Transfer of Culture Medium
After the first enrichment period, the bacterial cultures must continue to be
incubated for another 3 consecutive days, but they must also be transferred into the
new medium to ensure optimal growth. As such, the new set of LB solution was
made according the steps described earlier. The new set of 16 volumetric flasks
were then prepared with the new LB solution. Using a micropipette, a 5 ml of
solution from the previous D0P1 flask was transferred into the new D0P1 flask that
contained the new LB solution. This step was repeated fifteen times so that the
cultures from each of the previous 16 flasks were transferred into a new set of 16
flasks containing the new LB solution (Figure 4.3). Then, after transferring, the new
set of 16 flasks underwent the enrichment period for 3 consecutive days in the same
incubator that was also set at 150 rpm and 30°C.
Figure 4.2 Incubator with automatic rotator
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4.3.3 Pour-Plate Technique
This pour-plate procedure allowed the bacterial cultures to be grown on petri
dishes or plates that contained the mixture of Bacto™ Agar and LB medium. The
LB medium provided necessary nutrients for the bacteria while the Bacto™ Agar
allowed the LB solution to turn into a semi-solid substrate inside the plate called
the agarose gel and the agar plate. To make the agarose gels and the agar plates for
this experiment, a set of two 500 ml glass bottles and about 65 petri dishes were
used. First, a mixture of 15 g of Bacto™ Agar and 25 g of LB powder was made
into a solution using 1000 ml Grade II dH2O. The solution was then distributed
equally into two 500 ml glass bottles. To make this Agar-LB solution become
homogenous, a microwave was used to heat up the mixtures inside the bottles so
that the Bacto™ Agar could dissolve completely in the Agar-LB solution. When
the mixtures in both bottles became homogenous, they were sterilized inside the
autoclave machine before being used. At the same time, the petri dishes were also
Figure 4.3 Transfers of bacterial cultures during the enrichment period
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being sterilized. Once sterilized, the bottles were allowed to cool down just so that
the bare hands would not burn upon coming into contacts. Then, pour-plate
procedure began inside the laminar flow cabinet. However, the cabinet was
sterilized using UV radiation bulbs for 20 minutes prior being used for this
procedure. Inside the cabinet, the petri dishes were arranged in the manner that they
could be easily accessed by the experimenter. The homogenous and sterilized Agar-
LB solution was then poured directly into each of the petri dishes at the thickness
of about 5 mm. Then, the solutions in the petri dishes were allowed inside the
cabinet to completely cool down until they formed semi-solid agarose gels, which
took about 30 minutes. Then, the finished agar-plates were now ready to be used.
4.3.4 Serial Dilutions
The serial dilution took place right after the end of the enrichment period. The
point of this procedure was to ensure an appropriate amount of bacteria from the
enrichment period were allowed to grow on the agar plates made in the above
procedure. Each of the 16 flasks that underwent the enrichment period were used as
the starting materials for the serial dilution. Using a micropipette, 1 ml of the
enrichment solution was withdrawn from the D0P1 flask and then transferred into a
sterilized microtube. This D0P1 microtube now contained only 1 ml of bacterial
solution. Then, 0.1 ml of the bacterial solution was taken out to be mixed with 0.9 ml
of 85% NaCl solution inside a second microtube. The second microtube now
contained 10-1 ml of bacteria per 1 ml of solution. Then, from the second microtube,
another 0.1 ml of the solution was taken out to be mixed with another 0.9 ml of 85%
NaCl solution inside a third microtube. The third microtube now contained 10-2 ml
of bacteria per 1 ml of solution. This procedure of diluting the bacterial
concentrations in a series of 0.1 ml bacteria /0.9 ml NaCl was carried out until the
bacterial concentration from the D0P1 microtube was reduced to 10-5 and 10-6 ml of
bacteria per 1 ml of solutions. Only these two concentrations would be used to
perform the agar-plate spreading technique. Once, the desired concentrations of
D0P1 was obtained, the procedure continued until the desired concentrations from
the remaining D0P1* to D60P2* flasks were also obtained.
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4.3.5 Agar-Plate Spreading Technique
The purpose of the agar-plate spreading technique was to separate the different
types of bacteria that were present in each of the treated soils, collectively known as
bacterial consortia (sing. consortium), into different bacterial colonies that possessed
different morphological characteristics. This must be done so that the number of
types of bacterial colonies could be identified. The bacterial solutions inside the
D0P1 to D60P2* microtubes made in the previous procedure were used as the starting
materials for this procedure along with the agar plates that were made in 4.3.3. First,
two sets of the agar plates were labeled from D0P1 10-5 to D60P2* 10-5; the first set
would the first trial and the second set would be the second trail of the same
procedure. Then, another two sets of the agar plates were labeled from D0P1 10-6 to
D60P2* 10-6, for the exact same reason as the previous set. Once the agar plates were
labeled, an alcohol burner was prepared along with a medium beaker filled with
enough ethanol for a glass rod spreader to be submersed in. Then, using a
micropipette, 0.5 ml of the bacterial solution was withdrawn from the D0P1 10-5
microtube. It was then transferred onto the substrate of the D0P1 10-5 agar plate
followed with the temporary covering of the agar plate by its own lid to prevent
contaminations. Then, the glass rod spreader was taken out of the beaker and
sterilized with the flame from the ignited alcohol burner. Once the alcohol on the
spreader was burnt out and allowed to cool for 3 seconds, the lid of D0P1 10-5 agar
plate was lifted and the spreader was immediately placed inside the plate to start the
spreading of the bacterial solution. The spreading occurred in a unidirectional
circular motion to cover all of the surface of the agar plate’s substrate. The spreading
was completed as soon as a slight friction could be felt while spreading. The spreader
was then put into the alcohol beaker and the D0P1 10-5 agar plate was lidded and
stored up-side-down to prevent the substrate coming into contact with moisture. This
entire procedure was repeated until the rest of the labeled agar plates underwent the
spreading technique. The already spread agar plates where then put into an incubating
cabinet for 24 hours at 30°C. After the incubation, the agar plates were examined for
a variety of different bacterial colonies. The number of different bacterial colonies
and their morphological characteristics were recorded and presented in the results
section of this report.
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4.3.6 Plate Streaking Technique
After the number of different bacterial colonies and their morphological
characteristics were determined, the plate streaking technique was used to isolate
each type of bacterial colony. The remaining non-spread agar plates were used as the
substrates for the colonial isolation procedure. First, the alcohol burner was ignited
and ready to be used. Then, a wire loop was sterilized with the flame from the burner
until the loop turned bright orange. When the loop cooled down for 3 seconds, it was
used to scoop up one colony of Bac. 1 which was the first type of bacterial colony
identified. Then, the Bac. 1 colony was immediately placed inside the new remaining
non-spread agar plate labeled Bac. 1. Using the wire loop, the Bac.1 colony was
streaked on the substrate in a zig-zag manner for several turns starting from one
corner of the agar plate down to another corner to cover 1/5 of the entire substrate
area. This became the first streaked area of the plate. Then, after sterilizing the wire
loop, a line was draw out from the first area into the new area whereby the second
streaking occurred, also in a zig-zag manner to cover about 1/6 of the entire substrate
area. This now became the second streaked area of the plate. Next, the third area of
streaking was performed the same way as the first and the second with an exception
then the area covered now became smaller. Then, from the third streaked area of the
plate, the sterilized wire loop was used to draw four separate straight lines out of the
third area half-way towards the first streaked area, making the four lines became
parallel with the second streaked area. This streaking technique was now completed
for Bac.1. After that, the same procedure was carried out until the remaining Bac. 2
to Bac. 7 colonies were isolated and streaked onto the new non-spread agar plates.
After all of the identified bacterial colonies were isolated, the streaked plates were
then put into the incubating cabinet for 24 hours at 30°C to allow the bacteria to grow.
4.3.7 Gram Staining Procedure
To complete the screening process of the bacteria that were able to perform
biodegradation with previously treated soils, the Gram staining was used to identify
the types of bacteria based on the structures of their cell walls. This procedure
allowed the bacteria to be divided into main types, which are ‘Gram Negative’
bacteria and ‘Gram Positive’ bacteria. To divide the bacteria into these two types,
Mahidol University International College Senior Project / 17
several steps were involved. First, a clean microscopic slide was prepared by putting
one drop of Grade I dH2O onto it. Then, using a disposable toothpick, a small colony
of bacteria from the streaked plate of Bac. 1 was selected and transferred onto the
drop on water on the slide. Then, a circular motion of mixing the colony with water
occurred using the toothpick until the water turned cloudy. Then, the cloudy water
on the slide was dried out by the indirect contact with the flame of the alcohol burner,
leaving only the dry sediments of bacteria behind. The slide was now considered
heat-fixed. After heat-fixing the slide, a drop of crystal violet was put onto the dry
sediments of bacteria and left for exactly 1 minute. Then, after 1 minute the slide was
rinsed with a gentle stream of water for 2-3 seconds. After that, a drop of iodine
solution was put onto the same area for exactly 1 minute, and then the slide was
rinsed again for 2-3 seconds. Next, a drop of ethanol was added to the area and left
for only 15 seconds to decolorize the previous dye. After 15 seconds the slide was
immediately rinsed with water. Then, a drop of the Fuchsine counterstain was added
to the area and then left for exactly 30 seconds. The slide was finally rinsed with
water one last time and was allowed to air dry. The same procedure was repeated
until the remaining Bac. 2 to Bac. 7 were stained. After Gram staining, Bac. 1 to Bac.
7 were investigated under the compound light microscope. The pictures of each
bacteria were taken and presented in the results section of this report.
4.3.8 Removal Efficiency Testing Procedure
4.3.8.1 Using diesel oil as the sources of nutrients for bacteria
The second last part of the experiment was to test for the diesel biodegradation
efficiencies of the chosen bacteria under different periods of time and to analyze the
efficiencies in terms of percentages to see the trend of the efficiencies. However,
before the efficiencies could be measured, the chosen bacteria must first be cultured
within the mineral salt medium or MSM, and with the presence of diesel oil as the
main source of nutrients. First, the MSM was prepared using 6 g of K2HPO4, 4 g of
KH2PO4, 40 g of NH4Cl, 2 g of MgSO4∙7H2O, 1 g of NaCl, and 0.1 g of FeSO4∙7H2O.
These ingredients were dissolved in the total of 1000 ml of Grade II dH2O in the
exact order as written in the previous sentence, to become the functional solution of
Mahidol University International College Senior Project / 18
medium. If the order of dissolving was ignored, there would be a certain degree of
precipitations and the solution would become useless. After the MSM was prepared,
a set of 12 sterilized volumetric flasks were labeled according to the periods in which
the incubation would take place. For this experiment, the periods of incubation were
at Day 0 (D0), Day 3 (D3), Day 7 (D7), and Day 15 (D15). Hence, the flasks were
labeled as such, in which three flasks were labeled as D0 control. D0, and D0*, for
the’*’ sign helped indicate the second trail of the same procedure. Then, the
remaining flasks were labeled in the exact same way as the flasks representing Day
0. After that, the labeled flasks were then used to accommodate the MSM, whereby
45 ml of MSM was transferred into each flask. Then, using a micropipette, 0.5 ml of
diesel oil was added to all of the flasks (Figure 4.4). Next, the flasks were then put
into the rotating incubator for the periods of time according to their labels. Thus, Day
0 flasks would require no incubation.
Figure 4.4 Addition of diesel oil
Mahidol University International College Senior Project / 19
4.3.8.2 Liquid-liquid Extraction Method
After the desired period of incubation, the diesel oils inside the flasks were now
ready to be extracted. The liquid-liquid extraction method was used to extract the
non-degraded diesel oils that the bacteria were not able to degrade and used up within
the allotted period of time. First, three sets of separatory funnels were set up on a
bench. Then, a mixture of solution containing 1 part Hexane per 1 part Acetone was
made inside a hooded cabinet. The mixture was transferred into a 500 ml glass bottle.
Then, the incubated flasks were taken out and put inside the hooded cabinet, ready
for the next step. Next, 50 ml of the 1:1 hexane-acetone solution was transferred to
each of the incubated flasks and was then mixed with the contents from the flasks for
2 minutes using a vortex machine. After that, each of the mixed solutions in each
flask was transferred into one of the three separatory funnels. This step was repeated
two more times so that all of the mixed solutions from the flasks were transferred
into all three sets of separatory funnels. After waiting for 2-3 minutes, the mixed
solution in the funnel was separated into two parts, in which the top part contained
most of the hydrophobic contents and the bottom part contained most of the
hydrophilic contents of the mixed solution. The contents of the bottom part were then
dispensed into the flask that was previously used in this procedure, living only the
contents of the top part inside the funnel (Figure 4.5). A new volumetric flask was
then used to collect the contents of the top part through a filter paper filled with water-
absorbing sodium sulfate. Then, the flask that contained the bottom part contents was
mixed with a solution containing only hexane. Once mixed, the solution was then
transferred into the same funnel so that more of the hydrophobic contents could be
further separated and extracted. After more hydrophobic contents appeared at the top
of the funnel, the extraction was carried out in the same way of when the mixture
previously contained the 1:1 hexane-acetone solution. This entire procedure was
repeated until the non-degraded diesel oils from all of the flasks of the particular
incubation period were extracted. Then, the extracted hydrophobic contents were
refrigerated until the last three flasks of Day 15 underwent the extraction method
described above.
Mahidol University International College Senior Project / 20
Figure 4.5 Liquid-liquid extraction procedure
Mahidol University International College Senior Project / 21
4.3.8.3 Evaporation procedure to obtain non-degraded diesel oil
After the procedure of liquid-liquid extraction, there was a portion of hexane still
remained inside the extracted hydrophobic part which was responsible for dissolving
the non-degraded diesel oil. As such, to obtain only the non-degraded diesel oil, the
remaining portion of hexane must be taken out. An evaporator capable of producing
heat, vapor pressure and circular motion was used to take out the remaining portion
of hexane (Figure 4.6). First, 12 glass tubes were labeled according to the flasks in
4.3.8.1 and were weighed using a laboratory scale with the precision of four decimal
places. The weights of the empty glass tubes were recorded. After that, all of the
extracted hydrophobic parts from the previous procedure were transferred into the
labeled glass tubes. The tubes were then placed inside the evaporator that were set at
70°C±5, 360 mbar, and 6 rps. The process of evaporation for all of the tubes took
approximately 2 hours to complete. Once completed, the tubes were taken out and
were weighed again using the same laboratory scale. Because this time the non-
degraded diesel oil was the only content left inside each tube, therefore the weight of
the non-degraded diesel oil inside the tube would equal to the weight of the
evaporated tube subtracted by the weight the glass tube previously recorded. The
details of the weights of each tube, both before and after evaporation, were
summarized in the results section below.
Mahidol University International College Senior Project / 23
4.3.8.4 Calculation for Removal Efficiency
The removal efficiency of the bacterial ability to degrade diesel oil could be
expressed in terms of percentages by using the following equation for calculation;
% 𝑅𝑒𝑚𝑜𝑣𝑎𝑙 𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 =𝑊𝑒𝑖𝑔ℎ𝑡 (𝑐𝑜𝑛𝑡𝑟𝑜𝑙) − 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑊𝑒𝑖𝑔ℎ𝑡 (𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡)
𝑊𝑒𝑖𝑔ℎ𝑡 (𝑐𝑜𝑛𝑡𝑟𝑜𝑙) 𝑥 100
4.3.9 Identification of bacteria by gene sequencing
This procedure marked the final step of this experiment. The last of objective of
this experiment was to find out exactly the species of the bacteria that were chosen so
far to represent the best ability to degrade diesel oil in this experiment. The chosen strain
bacteria was sent to Mahidol University-Osaka University Collaborative Research
Center for Bioscience and Biotechnology, Faculty of Science, Mahidol University, to
be identified using the method of real-time PCR based bacterial identification. The
ribosomal RNA (rRNA), namely the 16S rRNA, of the chosen bacteria contained the
genetic sequences that once amplified and determined could be used to find out the
species of the chosen bacteria by comparing its genetic sequences with those provided
on the GeneBank database by the National Center for Biotechnology Information, U.S.
National Library of Medicine. This method of identifying various strains of bacteria can
also be seen in other projects whose experiments were also related to bioremediation
(Kebria et al., 2009).
Mahidol University International College Senior Project / 24
Chapter 5 Results
5.1 Screening Results of Bacterial Colonies
5.1.1 Screening and isolation of oil-degrading bacteria
The Identification of different types of bacterial colonies show exactly how many
different types of bacterial colonies exist after the procedure of the agar-plate spreading
technique. The types of bacterial colonies can be distinguished and differentiated by
their different appearances in morphological characteristics. The morphological
characteristics of different bacterial colonies are those that can describe the structures
of the colonies and the colors of the colonies. The structures of the colonies can be
divided into three main types, which are a clear defined margin, a defined margin, and
an undefined margin. A clear defined margin describes the appearance of a colony that
exhibits no coloration around the outermost part of its surrounding border while its
border is circularly shaped. A defined margin describes a border of a colony that is
circularly shaped but with a coloration and that coloration is of the same color as the
rest of the colony. An undefined margin describes a border of a colony that is not
circularly shaped or is shaped in a geometrically non-uniformed way. After the agar-
plate spreading technique, there were altogether seven different types of bacterial
colonies existed. Each type of bacterial colony was given an ID so that it can be easily
differentiated from one another. The bacterial colonies are summarized in Table 5.1.
Table 5.1 Types of bacterial colonies and their morphological characteristics
Bacterial ID Colonial Morphological Characteristics
Bac. 1 Defined margin and overall cloudy white
Bac. 2 Defined margin and overall cloudy yellow
Bac. 3 Clear defined margin with a pale-pink center
Bac. 4 Defined margin and overall cloudy pink
Bac. 5 Clear defined margin with a yellow center
Bac. 6 Clear defined margin with a yellow border surrounding an orange center
Bac. 7 Undefined margin and overall cloudy green
Mahidol University International College Senior Project / 25
Table 5.2 Types of bacterial colonies present in each pile of treated soil
Pile no. Bac. 1 Bac. 2 Bac. 3 Bac. 4 Bac. 5 Bac. 6 Bac. 7
1 G - G G - G G
2 - G G G G G -
G = growth is present
Table 5.2 represents the types of bacterial colonies that were found in each of the treated
soils. It could be seen that the number of types of bacterial colonies were the same for
both treated soils, but the types were different. Only three types, Bac. 3, Bac. 4 and Bac.
6, were present in both.
5.1.2 Growth patterns of different types of bacterial colonies
After the spread-plate technique was performed we would know exactly the
abilities of different types of bacteria to grow in each of the treated soils. As such, by
knowing their growth patterns we would be able to choose the bacteria with the best
growing ability to be tested for its efficiency in degrading diesel oil. However, before
we could perform an agar-plate spreading we must do the serial dilutions for every flask
from the previous enrichment period of each pile of treated soil, whereby the
concentrations of each enrichment flask were diluted to the maximum of 10-6 ml of
bacteria per 10 ml of NaCl solution before they were poured onto an agar plate.
However, after the spreading some plates that contained the concentrations of 10-5
showed successful growth patterns, whereas all of the plates that contained the
concentration of 10-6 did not show growth for both of the piles of treated soils. As such,
the growth patterns allowed us to see which bacterial colonies had the best chance of
growing and multiplying after they were isolated from each of the treated soils, and
hence allowed us to select the bacteria with the best growing ability for further testing
of its ability to degrade diesel oil. From Table 5.3, Bac. 4 showed us that not only it was
present in both piles (P1 and P2) of treated soils but also it was able to grow from Day
0 (D0) up until Day 60 (D60) of the enrichment periods. Therefore, Bac. 4 was
considered the bacteria with the best growing ability in both biostimulated and
bioaugmented soils.
Mahidol University International College Senior Project / 26
Table 5.3 Growth patterns of all identifiable bacterial colonies
Plate
no.
Pile
no.
Dilution
Conc.
(ml/ml)
Bac. 1 Bac. 2 Bac. 3 Bac. 4 Bac. 5 Bac. 6 Bac. 7
D0P1 1 10-5 G - G G - G -
D0P1* 1 10-5 G - G G - G -
D0P2 2 10-5 - G G G G G -
D0P2* 2 10-5 - G G G G G -
D15P1 1 10-5 - - G G - G -
D15P1* 1 10-5 - - - - - - -
D15P2 2 10-5 - G G G G G -
D15P2* 2 10-5 - G G G G G -
D30P1 1 10-5 - - G G - G -
D30P1* 1 10-5 - - G G - G -
D30P2 2 10-5 - G G G G G -
D30P2* 2 10-5 - G G G G - -
D60P1 1 10-5 - - - G - - -
D60P1* 1 10-5 - - - G - - -
D60P2 2 10-5 - - - G - - -
D60P2* 2 10-5 - - - G - - -
* Indicates the second trail of the same procedure
G = growth is present
Mahidol University International College Senior Project / 27
5.1.3 Gram staining of different bacterial colonies
The Gram staining is one of the common procedure involved in the screening
process of bacterial cultures. Although, this procedure cannot tell the specific species of
bacteria, it can directly tell the types of bacteria based on their differences in the
components of their cell walls. That being said, bacterial cell walls can be divided into
two types, which are called Gram Negative and Gram Positive. The Gram Negative
bacteria has the cell wall that consists of one innermost layer of plasma membrane, one
inner layer of periplasmic space, one middle layer of peptidoglycan, one outer layer of
periplasmic space, and one outermost layer called outer membrane composing of
lipopolysaccharides and proteins. Similarly, the Gram Positive bacteria has almost all
of the above components in its cell wall; only two layers that are not present in the Gram
Positive bacterial cell wall which are the outer layer of periplasmic space and the outer
membrane. These differences are important as they are indicators for differences in
properties and differences in growing conditions of the two types of bacteria. All of the
seven isolated bacterial colonies from the experiment underwent the Gram staining
procedure and their results are presented in the figures below. The Gram negative
bacteria will appear pink in color while the Gram Positive bacteria will appear purple or
blue (Figure 5.1 to Figure 5.7).
Mahidol University International College Senior Project / 28
Figure 5.1 Bac. 1 Gram Negative
Figure 5.2 Bac. 2 Gram Positive
Mahidol University International College Senior Project / 29
Figure 5.3 Bac. 3 Gram Negative
Figure 5.4 Bac. 4 Gram Positive
Mahidol University International College Senior Project / 30
Figure 5.5 Bac. 5 Gram Negative
Figure 5.6 Bac. 6 Gram Negative
Mahidol University International College Senior Project / 32
5.2 Removal efficiency of bacteria with the best growing ability
Table 5.4 Measurements of diesel biodegradation by Bac. 4
It can be seen from Table 5.4 that the weights of the non-degraded diesel oils
decreased gradually from Tube no. D0 and D0*, which presented Day 0 of incubation,
to Tube no. D15 and D15*, which represented Day 15 of incubation. From these data,
it could be interpreted that the longer periods of incubations resulted in more diesel oil
being degraded by the bacteria; hence living behind the decreasing amounts of non-
degraded diesel oils. Because the procedure was carried out twice so the two trials to
would minimize any potential error in the measurements, therefore we needed to find
out the average weight of each of the tubes that were incubated within the same period
of time, and then compare the average with the amounts of non-degraded diesels from
control trials to see if the results were consistent or not.
Tube no. Weight of Tube (g) Weight of Tube (g) +
non-degraded diesel (g)
Non-degraded
Diesel (g)
D0 control 31.0642 31.5365 0.4723
D0 31.6414 32.1470 0.5056
D0* 31.7942 32.1807 0.3859
D3 control 31.4519 31.7663 0.3144
D3 31.6430 31.9024 0.2594
D3* 31.7581 32.0261 0.2680
D7 control 31.3010 31.6873 0.3863
D7 31.3941 31.6241 0.2300
D7* 31.6833 31.9413 0.2580
D15 control 31.6798 32.0221 0.3423
D15 31.2670 31.4250 0.1580
D15* 31.7192 31.9050 0.1858
Mahidol University International College Senior Project / 33
From the graphical representation in Figure 5.8, almost all of the control trails,
except D0, contained noticeably more amounts of non-degraded diesel oils than the
treatment trials. The reason why at D0 the control and the treatment trials had similar
values was because at Day 0 incubation the bacteria had not yet begun to degrade the
diesel oil. Also, it appeared that the amounts of non-degraded diesel oils from all of the
four control trails showed some inconsistency, in which the output values of diesel oils
in the control trials should theoretically have been the same as the values of the input,
which was at 0.5 ml. The reason why this inconsistency occurred was because in the
actual events of experimentation not all of the amounts of diesel oils were able to be
collected from the separatory funnel. As the oil went through the filter papers that were
filled with sodium sulfate, some amounts of oils could easily be trapped inside the
clamped mass of the sodium sulfate and even inside the filter papers.
0.4723
0.3144
0.3863
0.3423
0.4458
0.26370.2440
0.1719
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
D0 D3 D7 D15
Wei
ght
(g)
Average Weight of Non-degraded Diesel
Control Average non-degraded diesel
Figure 5.8 Weights comparision between the control and the average non-degraded diesel
Mahidol University International College Senior Project / 34
Figure 5.9 Calculations of Percentages of Diesel Oil Removal Efficiencies
By using the equation given in 4.3.8.4, four different percentages of diesel oil
removal efficiencies were calculated. The calculated trend in Figure 5.9 appeared to be
consistent with the fact that as the period of incubation increased so did the percentage
of removal efficiency. However, it appeared that at D0 whereby no incubation took
place there was a 5.61% removal of diesel oil, which, in theory, this should not have
happened. This slight removal of diesel occurred because, as mentioned previously, a
small amount of diesel was removed by the clamped mass of sodium sulfate and by the
filter paper that were used to filter the diesel oil during the liquid-liquid extraction
procedure. As such, this kind of accidental removal of diesel oil could also have
occurred with other treatments from different periods of incubations.
5.61%
16.13%
36.84%
49.78%
0%
10%
20%
30%
40%
50%
60%
D0 D3 D7 D15
Rem
oval
Eff
icie
ncy
Per
centa
ge
Percentages of Diesel Oil Removal Efficiencies
Increasing Trend of Removal Efficiency
Mahidol University International College Senior Project / 35
5.3 Identification of bacteria with the best growing ability
The species of bacteria that was able to grow the best in both biostimulated and
bioaugmented soils from this experiment was identified to be ‘Ochrobactrum sp.’ This
result was based on the matching of the 16S rRNA gene sequence of the chosen bacteria
to the gene sequence of Ochrobactrum sp. recorded in the GenBank database.
Mahidol University International College Senior Project / 36
Chapter 6 Discussion
It can be said that, by overall, the outcome of this project did successfully fulfill
the purposes or the specific objectives that were created since the beginning of the
experiment. The steps involved in each of the procedures that were needed to perform
the experiment were attempted and proceeded with extreme cautions, resulting in very
little errors. Some of the errors that occurred during the experiment were minor or not
significant enough to make the results of this experiment become obsolete. Some errors
that occurred includes the occasional mishandling of non-hazardous equipment such as
cracking some of the volumetric flasks while washing them after they were being used,
and the occasional over or under-approximation when using the graduated cylinder used
for the transfers chemical solutions from one container to another, resulting in having
to pour back-and-fourth the solutions a couple of times until the exact amounts were
finally obtained. Although, these types of human errors did not cause significant
numerical errors in the quantitative measurements of the experiment, they did cause the
increases in costs and time needed to complete the project. As such, more handling
practices and more precise measurements must be utilized in the future.
In details, the experiment done for this project did not involve some procedures
that were commonly performed by other experimenters who also attempted to
investigate the methods of bioremediation. Regardless of the other procedures that did
not take place in this project, the results from the attempted procedures were consistent
with the set objectives and some of the results showed similarity to the results from other
projects related to bioremediation done by other experimenters. Some of the procedures
that did not occur in this project were soil analysis via gas chromatography, soil pH
measurement and its effects on bacteria, and total petroleum hydrocarbons
determination via gas chromatography. All of the mentioned procedures were
occasionally carried out to gain the deeper and more elaborated understanding of
individual components of petroleum chemicals that were able to be degraded by
different strains of bacteria (Chagas-Spinelli et al., 2012). Another procedure that was
commonly performed to investigate, in details, the comparative growth of different
bacterial species under the presence of petroleum chemicals was the growth
measurement and the assessment of oil biodegradation (Palittapongarnpim et al., 1998).
Mahidol University International College Senior Project / 37
Because the objectives of experimentation vary from one project to another, which was
the reason that results presented within this project did not contain information
regarding those other common procedures.
Furthermore, if looking at the available results obtained from this project, it could
be said that the numbers of bacterial strains found after isolation, in each consortium of
each of the biostimulated soil and bioaugmented soils, appeared to be similar to the
results obtained from another project done in the past. From the results of this project,
both biostimulated soil and bioaugmented soil contained 5 different types of bacteria.
The number of types of bacteria found here was similar to those found after the isolation
procedure done by researchers at Mahidol University, in which 3 different strains of
bacteria were identified (Palittapongarnpim et al., 1998). Nevertheless, if taken the
results from other places around the world whereby the screening process was
performed in a similar manner, some differences could be seen. One bioremediation
related experiment done by researchers at the University of Rio Grande Sul and the
University of California showed that 12 strains of bacteria capable of producing
biosurfactants were found after an isolation procedure (Menezes Bento et al., 2005).
Therefore, further experimentations could be performed to investigate what factors
caused the differences in the populations of petroleum-degrading bacteria found in
different locations.
Apart from the screening process, the results from the removal efficiency testing
also showed that the ability of the chosen bacteria to degrade diesel oil that came from
the screening process was consistent with the fact that longer periods of incubation
would result in more contaminants being degraded by bacteria. From figure 15, it could
be seen that the removal efficiencies of diesel oil, expressed in percentages, started from
5.61% at Day 0 of incubation and then gradually increased to 49.78% at Day 15 of
incubation; a total increase of 44.17% within the period of 15 days; or at the rate of
about 2.94%/day. However, these percentages of removal efficiencies could only
accurately represent the ability of the chosen bacteria to degrade diesel at the laboratory
scale. For the chosen bacteria to be applied in a real situation, further experimentations
must be done to increase the parameters to a much larger scale.
Mahidol University International College Senior Project / 38
Chapter 7 Conclusion
Almost all of the commodities that are made for consumption by machinery
involve the use of petroleum chemicals as fuel sources for energy and transportation.
More of than not, these petroleum chemicals are exploited by the industries that acquire
and use them. When exploitation of these chemicals occur, the impacts it has upon the
environment that we live in become a disturbing issue. The petroleum chemicals that
are used for energy consumption, once entered into the environment such as lands and
oceans, can post tremendous threats to the survival of animals. They can also be
extremely hazardous to human health when they enter the living and farming quarters
of human communities. For these reasons, the research with the purpose to find a viable
solution to the problems must be pursued. The experiment done in this project
represented such the research. The experiment consisted of the discovery of bacterial
ability, known as biodegradation, to degrade a kind of petroleum chemical, namely
diesel oil, and its efficiency in degrading such chemicals. The main steps involved are
the screening and isolation processes, the diesel oil removal efficiency testing, and then
the specific identification of the bacterial strain. As a result, seven different strains of
bacteria were found to possess the ability to degrade diesel oil. Out of seven, three
strains were present in both piles of treated soils. However, only one of the three strains
showed the best growth ability after the screening and isolation processes, and thus was
chosen to be investigated for its diesel removal efficiency later on. For the removal
efficiency, it was found that after 15 days of incubation, the chosen bacteria was able to
degrade 49.78% of the tested diesel oil. Finally, this particular strain of bacteria was
then identified using a gene sequencing technology known real-time PCR. The strain of
the bacteria was found to be Ochrobactrum sp.
In addition, there were some unknown factors that arose from this experiment that
should be examined in the future by conducting research relating to bioremediation.
These unknown factors were most related to the differences in the numbers and types of
bacterial strains found in the contaminated soils that were treated with either
bioaugmentation or biostimulation. Another factor that needed an investigation was the
long-term effect Ochrobactrum sp. has on the soil quality after the oil was degraded.
Mahidol University International College Senior Project / 39
References
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Mahidol University International College Senior Project / 40
BIOGRAPHY
NAME Narattapat Watcharapriyapat
DATE OF BIRTH 28. NOVEMBER. 1986
PLACE OF BIRTH Bangkok, Thailand
INSTITUTIONS ATTENDED Debsirin School, Bangkok, Thailand
Ivanhoe Grammar School,
Melbourne, Australia
Huazhong University of Science &
Technology, Wuhan, Hubei, PRC
Mahidol University International College,
Nakhonpathom, Thailand
HOME ADDRESS 823/29 Soi Phran Nok 6, Phran Nok Rd.,
Banchanglor, Bangkoknoi District,
Bangkok, Thailand 10700
Tel. 0957614996, 0896159449
E-mail: narattapat@hotmail.com