ABSTRACT
Petroleum hydrocarbon-degrading bacteria are ubiquitous in nature and can utilize hydrocarbon as sources of carbon and energy. Bacteria with such capabilities are often exploited for the bioremediation of petroleum oil contaminated environments. Petroleum degradation potential of bacteria isolated from automobile mechanic workshop was carried out. A total of five samples of oil contaminated soil were collected weekly for five weeks. The samples were taken immediately to Microbiology laboratory of Madonna University Elele for analysis. The media used were Bushnell Haas agar, Mineral salt medium and Nutrient agar. The method used for isolation were serial dilution, pour plating and vapour phase transfer techniques. Colonial morphology, Gram staining and biochemical tests were carried out using the standard microbiological methods. Also genomic DNA were quantified, PCR was carried and the DNA were sequenced. Physicochemical parameters of the soil sample were also analyzed. The mean count log10 cfu/ mL of petroleum utilizing Bacteria ranges from 4.05±0.22 – 4.11± 0.22 for petrol, 4.06± 0.1 -4.11± 0.1 for diesel and 4.05± 0.2 – 4.11±0.2 for brake fluid. The total aerobic plate count mean count log10 cfu/ mL ranged from 5.05± 0.1 -5.16 ± 0.2. For physicochemical parameters analyzed, temperature ranged from 28.30- 29.60o C, pH ranged from 5.60 -6.96, Conductivity, 13.7 -860µs/cm, Total organic carbon 0.32-2.23%, Total nitrogen 0.13-0.19%, Phosphate 0.98-1.80 mg/kg,Iron 15.1-320 mg/kg , Potassium 6.00 -13.60mg/kg ,Calcium 108-690 mg/kg, Magnesium 152-388 mg/kg, Zinc 0.12-18.20 mg/kg, Manganese 9.20 -60.40 mg/kg and Copper <0.10- 1.70 mg/kg also Chromium ranged from 0.40 -10.50mg/kg, Lead <0.10-1.40 mg/kg, Barium < 0.10-0.18 mg/kg, Nickel < 0.10 -0.25 mg/kg, Cadmium<0.10 mg/kg, Mercury <0.10 mg/kg, Oil and grease 1.5-128 mg/kg, Total petroleum hydrocarbon 4.5-196 mg/kg and moisture 0.25 -0.39%. The hydrocarbon utilizing Bacteria isolated and their percentage occurrence were Pseudochrobactrum asaccharolyticum 24.5%, Empedobacter brevis28.6%, Lysinibacillus macroides24.5% and Neisseria weaveri 22.5%. The percentage occurrence of total aerobic plate count were Pseudochrobactrum asaccharolyticum 15.0%, %, Empedobacter brevis20.4%, Lysinibacillus macroides17.2% and Neisseria weaveri18.3%, Azotobacter sp 14.0% and Clostridium sp 15.1%. Statistical analysis of the results were done using ANOVA and least significant difference(LSD). Some bacteria such as those isolated in this research have the potential to utilize hydrocarbons as sole source of carbon and energy to survive in such environment thereby breaking down or degrading the hydrocarbon.
TABLE OF CONTENTS
Title
i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table
of Contents vii
List
of Tables x
List
of Figures xi
Abstract xii
CHAPTER
1: INTRODUCTION 1
1.1
Scope of the Study 4
1.2
Statement of Problem 4
1.3
Justification of the
Study 5
1.4
Objectives of the Study 5
CHAPTER
2: LITERATURE REVIEW
6
2.1 Petroleum 6
2.2 Uses
of Petroleum 6
2.3 The
Origin of Petroleum 7
2.4 The
Main Petroleum Producing Countries and their Exploration 8
2.5 Petroleum
Structure and Composition 9
2.6 Oil Structure and Concentration 9
2.7 Petroleum
Processing and Products 11
2.8 Octane
Number 13
2.9 The
Petrochemical Industry 13
2.10 Biodegradation 14
2.11 Etimology
of Biodegradable 15
2.12 Microbial
Biodegradation 16
2.13 Biodegradation
of Pollutants Aerobically 17
2.14 Bioavailability,
Chemotaxis and Transport of Pollutants 18
2.15 Oil
Biodegradation 18
2.16 Biocatalytic Application and Metabolic
Engineering Biodegradation 19
2.17
Postulates of Microbial Disintegration
of Petroleum Hydrocarbons
in the Environment. 20
2.18 Degradation
of Petroleum Hydrocarbons by Microbial Activity 21
2.19 Petroleum
Hydrocarbon Degradation Mechanism 27
2.20 The
role of Enzyme in Petroleum Hydrocarbons Degradation 28
2.21 Molecular
Identification of Microorganisms Involved in
Hydrocarbon
Degradation 29
2.22 Physicochemical
Properties of oil Contaminated Soil 30
CHAPTER 3: MATERIALS AND
METHODS 31
3.1 Study
Site 31
3.2 Collection
of Samples 31
3.3 Media
used and Preparation 31
3.4 Enumeration
of Bacteria 32
3.5 Isolation
of Petroleum Degrading Bacteria 32
3.6 Characterization
and Identification of Bacterial Isolates 33
3.7 Determination
of Petroleum Degrading Potential of Bacteria 33
3.8 Biochemical
Tests 33
3.9 Molecular
Identification of the Isolates 36
3.10 Sequence
Data Analysis for Identification of Bacterial Isolates 38
3.11 Physicochemical
assessment 38
3.12 Statistical
Analysis 41
CHAPTER
4: RESULTS
AND DISCUSSION 42
4.1 Results 42
4.2 Discussion 63
CHAPTER 5:
CONCLUSION AND RECOMMENDATION 67
5.1 Conclusion 67
5.2 Recommendation 67
References
Appendices
LIST OF TABLES
4.1: Mean count of bacteria isolated from soil
sample from mechanic
workshop 46
4.
2: Hydrocarbon utilizing bacteria
isolated and their percentage
occurrence 47
4.3:
Total heterotrophic bacteria isolated
and their percentage
occurrence 48
4.
4: The
mean values of the physicochemical parameters of soil sample
from
mechanic workshop 49
4.5: Taxonomic report for isolate1(top 10 hits) 54
4. 6: Taxonomic report for isolate2(top 10 hits) 56
4.7: Taxonomic report for isolate 3(top 10 hits) 58
4.8: Taxonomic
report for isolate 4(top 10 hits) 60
LIST OF FIGURES
2.1 Factors affecting bio disintegration of
petroleum hydrocarbons. 22
2.2:
Hydrocarbon degradation rates in
soil, fresh water, and marine
environments 24
2.3: The
role enzyme in hydrocarbon degradation 29
4.1: Mean
value of petroleum degrading bacteria using diesel as the sole
source
of carbon and energy 50
4.2: Mean
value of petroleum degrading bacteria using petrol as the sole
source
of carbon and energy 51
4.3: Mean
value of petroleum degrading bacteria using brake fluid as the sole
source
of carbon and energy
52
4.4: Phylogenetic tree for isolate 1 top 10 blast
hits 53
4. 5: Phylogenetic tree for isolate 2 top 10 blast
hits 55
4.6: Phylogenetic tree for isolate 3top 10 blast
hits 57
4.7: Phylogenetic tree for isolate 4 top 10 blast
hits 59
4.8: Genomic DNA of bacteria isolated 61
4. 9: PCR amplicon
62
CHAPTER 1
INTRODUCTION
Petroleum is yellowish-to-blackish in colour, it a thick,
flammable and it’s a mixture of liquid, gaseous, and solid hydrocarbons which naturally
occurs below the surface of the earth. It can be separated into sections
involving asphalt, natural gas, naphtha, gasoline, kerosene, lubricating oils,
fuels, and paraffin wax which are utilized as raw material for a large variety
of derivative products(American Heritage Dictionary, 2016) .
The word petroleum originated from the Latin word Petra,
which means “rock,” and Oleum, which means “oil.” The oil company
groups "crude" by the position of its source and by virtue of its
weight or agglutination (light, intermediate or heavy). The relative content of
sulphur in oil deposited in nature results in referring to oil as
"sweet," meaning that it comprises of relatively little sulfur, or as
"sour," meaning that it comprises of large amounts of sulphur(American
Association of Petroleum Geologists, 2016).
Petroleum, along with oil and coal, is classified as a fossil
fuel. Fossil fuels are formed when sea plants and animals die, and the remains
become buried under several thousand feet of silt, sand or mud (American
Association of Petroleum Geologists, 2016).
Fossil fuels use millions of years
to establish, as such, petroleum is said to be a non-renewable source of energy.
. Petroleum collected in its natural state is commonly termed
crude oil, it may be clear,
green or black, also may either be thin-like gasoline or thick-like tar (American
Association of Petroleum Geologists, 2016).
The first known oil well was sank in Pennsylvania by Edwin
Drake in 1859. Since this time oil and petroleum production figure grew
exponentially (American Association of Petroleum Geologists, 2016).
Initially, petroleum was mainly used as a lighting fuel,
after distillation and conversion into kerosene. When the world's first
electricity generating plant headed by Edison commenced operation in 1882,
kerosene demand gradually dropped. (American Association of
Petroleum Geologists, 2016).
However, The demand for gasoline
increases when Henry Ford reveal to the world that the automobile would be the
most reliable form of transport for decades.
The First World War greatly
accelerated the production of petroleum, as petroleum was in continuous
production throughout the war than that which has been produced earlier.
Presently, petroleum is seen as a costly commodity, traded all over the world
as well as gold and diamond. Majority seem to believe that petroleum in the
form of gasoline or petrol is used mainly in powering internal combustion
engines. Despite the fact that our automobiles and other forms of transport
utilize the highest amount of petroleum, it has an enormous array of
applications.
The thickest form of petroleum,
which is nearly black is called bitumen, it can be used for roofing, making
pavement in roads, forming the blacktop, it also serves as first-class water
repellant.
Petroleum is also an
important chemical constituent of many plastics and synthetics. Possibly the
most outstanding use of petroleum for many, is in its outlook in foodstuffs
like beer and in drugs like aspirin(American Association of Petroleum
Geologists, 2016).
The supply of petroleum in the world
is less, present calculations has revealed that humanity will totally exhaust
this costly natural resources within few decades. Although measures have been
employed to ensure the availability of affordable, renewable fuel options put
in place for the eventuality, it is clear that humanity are still faced with
serious challenges when the supply of petroleum is exhausted (Vassiliou, 2009).
Bacteria
are incredible microorganisms that are in existence. They are ubiquitous, can
be found in the air, water, soil and living organisms. Bacteria in addition
have the capability to degrade both synthetic and natural materials (Habe et al., 2001). This process is known as
Biodegradation. Although fungi play a significant role in wood degradation,
some bacteria do also degrade cell walls containing lignin and also wood
fibres. Ideonella sakaiensis (a
bacterium) have ability to degrade plastic as sole source of carbon and energy(Yoshida et al.,2016). There are other bacteria
that can degrade plastic polymers namely, Pseudomonas
stutzeri, Alcaligenes faecalis, Streptomyces sp., and Brevibacillus borstelensis(Ghosh et al., 2013). Almost all petroleum hydrocarbon found in the environment
are metabolized or degraded by bacteria
following their carbon and energy needs for reproduction and growth as
well as the need to relieve physiological stress as result of the presence of
the petroleum hydrocarbons in the environment(Hazen,2010).
Biodegradation is
the natural way through which wastes are recycled, or it is the breakdown of organic
substance into nutrients that are beneficial to other organisms.
"Degradation" simply means decay, and the prefix "bio-"
means that the decomposition is performed by a large collection of fungi,
bacteria, insects and other organisms that consume non-living or dead material
thereby recycling them into new forms.
There is no waste
product in nature since everything undergoes recycling. One organism’s waste
product is a source of food for others, supplying energy and nutrients while
the organic waste matters are broken down. The breakdown of some organic
materials are faster while others are slower, but eventually, all will
decay.
By
tackling biodegradation, there might be reduction in waste and some kind of
environmental contaminants cleaned up. Through composting,
natural biodegradation is accelerated and organic wastes transformed into
precious resources. Microorganisms are
employed in cleaning up oil spills and some other forms of organic pollution.
Composting and bioremediation provide many possibilities for researchers (Das
and Chandran,2011). Hydrocarbon microbial degradation pathways aim to
illustrate wide substrate specificity, being controlled by various oxido reductase
enzymes involving mono oxygenases, dioxygenases, peroxidases and laccases, and
take place either in an aerobic or anaerobic way (Haritash and Kaushik,2009).
1.1 SCOPE OF THE STUDY
This
research work is limited to petroleum degradation potential of some bacteria
isolated from a mechanic (automobile) workshop along NTA road Mgbuoba, Port
Harcourt, Rivers State.
1.2 STATEMENTS OF PROBLEM
The
problem posed by regular oil spillage in the Niger-Delta region of Nigeria is a
thing of concern due to its adverse
effect on both human and animal lives. There is therefore necessary to proffer solution to this
problem.
1.3 JUSTIFICATION
Oil
spill is a very big challenge in Nigeria, especially in the Niger Delta region.
This is because of the activities of companies especially oil and gas which are
located there. The Companies are sited because the area is endowed with such
natural resources. The spillage of these petroleum products is detrimental to
agricultural products both in the waters and on the land. The soil in the
Mechanic workshop is a typical example of soil contaminated with petroleum
products (oil spillage). There has being efforts by Government in cleaning up
oil spills so as to return the soil and water to their natural state. Therefore,
the isolation of bacteria that have the potential to degrade petroleum is a
right step in the use of biological method to control oil spillage.
1.4 OBJECTIVES OF THE STUDY
·
To isolate and
characterize bacteria species from soil sample from Mechanic workshop.
·
To determine the
hydrocarbon (Petroleum) degrading potentials of the isolates.
·
To compare the rate of
degradation by the isolates and to identify the predominant oil-degrading bacteria that may
be used as model hydrocarbon degrader.
·
To determine the Total
Petroleum Hydrocarbon (TPH) of samples from the soil.
·
To ascertain and monitor
the physicochemical attributes of the sampled soil.
·
To statistically analyze
the result obtained.
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