IDENTIFICATION OF DIESEL-DEGRADING BACTERIA FROM OIL-CONTAMINATED SOIL SAMPLES THAT WERE TREATED...

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

Mahidol University International College Senior Project / vii

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


5.4 Bac. 4 Gram Positive 29




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.


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.


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


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.


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)


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.


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


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


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).


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


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.


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


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


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.


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.


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,


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


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


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.


Figure 4.5 Liquid-liquid extraction procedure


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.


Figure 4.6 Multi-function Evaporator


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).


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


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.


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


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).


Figure 5.1 Bac. 1 Gram Negative

Figure 5.2 Bac. 2 Gram Positive



Figure 5.4 Bac. 4 Gram Positive


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


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


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


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.


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).


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.


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.


References

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microorganisms (11th ed.,International ed.). Upper Saddle River, N.J.:

Pearson/Prentice Hall.

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broth-lb-and-luria-agar-la-media-and-their-uses-protocol

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(2009). Isolation and characterization of a novel native Bacillus strain capable

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Technology, 6(3), pp.435--442.

7. Chagas-Spinelli, A., Kato, M., de Lima, E. and Gavazza, S. (2012).

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environmental management, 113, pp.510--516.

8. Palittapongarnpim, M., Pokethitiyook, P., Upatham, E. and Tangbanluekal, L.

(1998). Biodegradation of crude oil by soil microorganisms in the tropic.

Biodegradation, 9(2), pp.83--90.

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255.


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: [email protected]

IDENTIFICATION OF DIESEL-DEGRADING BACTERIA FROM OIL-CONTAMINATED SOIL SAMPLES THAT WERE TREATED...

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