ABSTRACT
Indiscriminate discharge of hydrocarbon waste, such as spent engine oil (SEO) in the environment has become a major source of concern due to their impacts on humans and other organisms. This study determined the effects of spent engine oil on soil physico-chemical properties and the biodegradation potentials of bacteria isolated from Ohiya automobile mechanic village in south eastern Nigeria. Soil samples were collected from five (5) different locations within the study area, these sampling point were constantly receiving spent engine oil and at each of these sampling point, hand held auger was used to bore holes of depths 0 – 15cm (top soil) and 15 – 30cm (subsoil). All the top soil (0-15cm) collected from the five workshops were homogenized in a clean bucket and a composite sample was drawn. Same process was repeated for the sub soil (15-30cm) and a control sample was collected few meters away from the study area free from contaminant. Physico-chemical properties of the soil samples were analyzed using standard techniques and heavy metals were analysed using Atomic Absorption Spectrophotometer (AAS). Hydrocarbon degrading bacteria were isolated and enumerated using pour plate method. Biodegradation ability of the bacterial isolates on used engine oil was studied in a shake- flask culture containing mineral salt medium broth (MSM) supplemented with 2% (V/v) engine oil and 5% (V/v) bacteria isolate and the residual hydrocarbon content was measured gravimetrically .The effect of temperature and pH was studied for a period of 14 days. The result revealed that spent engine oil had adverse effect on the soil physico-chemical properties and on bacteria population of the study area. Heterotrophic bacteria counts from the contaminated soil samples ranged from 1.70 x 105cfu/g (sub soil) to 1.80 x 105cfu/g (top soil) compared to the control sample 3.7 x 105cfu/g. Hydrocarbon utilizing bacteria counts ranged from 2.07 x 103(sub soil) to 2.25 x 103cfu/g (top soil) compared to the control sample 1.25 x 103cfu/g. Fifteen (15) isolates belonging to both Gram positive and Gram negative were obtained from the contaminated soil samples based on morphological and biochemical characteristics. The bacterial isolates belonged to the genera; Bacillus, Pseudomonas, Micrococcus, Citrobacter and Acinetobacter. The result of the biodegradation ability of the isolated bacteria strains revealed that the consortium of bacteria had higher percentage hydrocarbon reduction (89.27%) than the pure cultures: Bacillus (78.20%); Pseudomonas (74.82%); Micrococcus (56.28%); Acinetobacter (61.28%); and Citrobacter (50.66%). The ability of the consortium of bacteria to remove high percentage of engine oil components makes it potentially useful for bioremediation of site contaminated with petroleum hydrocarbon. The result of the effect of temperature and pH on biodegradation of spent engine oil revealed that the bacteria isolates gave the best degradation when incubated at 30°c and 40°c(optimal temperature) in MSM and at a pH range of 6.5- 7.5 (optimal pH). The study concluded that improper disposal of SEO has a negative effect on the soil physico-chemical properties, and that hydrocarbon utilizing bacteria can be isolated from the study area. The growth of these organisms in mineral salt medium suggest their hydrocarbon utilizing potential, hence, their possible use for the bioremediation of soils impacted with engine oil.
TABLE OF CONTENTS
Title page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi
List of Tables ix
List of Figure x
Abstract xi
CHAPTER
1
INTRODUCTION
1.1 Background
of the Study 1
1.2 Statement
of Research Problem 4
1.3 Justifications
of the Study 5
1.4 Research
Aim and Objectives 6
1.5 Research
Questions 7
1.6 Significance
of the Study 7
CHAPTER 2
LITERATURE REVIEW
2.1 Petroleum
Formation 8
2.1.1 Classification of petroleum
hydrocarbons 9
2.1.1.1 Aliphatic
hydrocarbons 9
2.1.1.2 Heterocyclic hydrocarbons 10
2.1.1.3 Aromatic hydrocarbons 10
2.1.1.4 Monoaromatics – BTEX 11
2.1.1.5 Polycyclic aromatic hydrocarbons (PAHs) 11
2.2 Engine Oil Composition 13
2.3 Oil Pollution in Nigeria Environment 13
2.4 Effect of Spent Engine Oil on Physical and
Chemical Properties of Soil 15
2.5 Effect
of Spent Engine Oil on the Development of Crop 17
2.6 Petroleum
Hydrocarbon Contamination in Developing Countries 18
2.7 Environmental
and Health Impact of Used Engine Oil 20
2.8 Isolation
of Petroleum Hydrocarbon-Degrading
Bacteria 21
2.8.1 Restriction
of physical contact among bacteria and petroleum
hydrocarbons 23
2.9 Principles
of Biodegradation 26
2.10 Strategies
of Biodegradation 30
2.10.1 In-situ
bioremediation of soil 30
2.10.2 Natural
attenuation 30
2.10.3 Engineered
bioremediation 30
2.10.4 Biostimulation
31
2.10.5 Bioventing
and biosparging 32
2.10.6 Injection
of hydrogen peroxide 33
2.10.7 Bioaugmentation 33
2.10.8 Ex-situ
bioremediation 32
2.10.8.1 Ex-situ remediation techniques 34
2.10.8.2 Advantage of ex-situ bioremediation
processes 36
2.10.9 Land farming
36
2.10.10 Composting 37
2.10.11 Biopiles 37
2.10.12 Bioreactors 37
2.11 Mechanism
of Biodegradation of Hydrocarbon 38
2.11.1 Microbial mechanisms of catabolism of
hydrocarbons 38
2.11.2 Enzymes
involved in hydrocarbon degradation 40
2.12 Environmental
Factors Influencing Biodegradation of Petroleum
Hydrocarbon 41
2.12.1 Physical
state of the oil Pollutant 41
2.12.2 Chemical
composition of the oil pollutant 42
2.12.3 Concentration
of the oil pollutant 43
2.12.4 Temperature
43
2.12.6 Nutrients
45
2.12.7 pH 45
2.12.8 Water
activity/moisture 46
2.12.5 Oxygen 44
CHAPTER 3
MATERIALS AND
METHODSS
3.1 Study Area 48
3.2 Sample Collection and Preparation 49
3.3 Determination of Soil Physico-Chemical
Parameters 50
3.3.1 Hydrogen
ion concentration (pH) 50
3.3.2 Total
nitrogen 50
3.3.3 Phosphorus
51
3.3.4 Exchangeable
bases (Mg, Ca, K and Na) 51
3.3.5 Total
organic carbon and organic matter 51
3.3.6 Heavy
metal analysis 52
3.4 Microbial
Analysis 52
3.4.1 Total
heterotrophic count 52
3.4.2 Hydrocarbon
utilizing bacteria count 52
3.4.3 Characterization
and identification of bacteria isolate
53
3.4.4 Biochemical
characteristics 53
3.4.4.1 Gram staining 53
3.4.4.2 Oxidase test 53
3.4.4.3 Catalase test 54
3.4.4.4 Citrate utilization test 54
3.4.4.5 Urease test 54
3.4.4.6 Methyl
red 55
3.4.4.7
Voges-proskauer 55
3.4.4.8 Indole
test 55
3.4.4.9 Triple
sugar iron agar test 55
3.4.5 The ability of bacteria isolates to utilize
used engine oil 56
3.4.6 Extraction
of residue oil 56
3.5 The
Effect of pH on Biodegradation of Used Engine Oil 57
3.6 The
Effect Of Temperature On Biodegradation Of Used Engine Oil 57
3.7 Statistical Analysis 57
CHAPTER 4
RESULTS AND
DISCUSSION
4.1 Effect
of Spent Engine Oil on Soil Physicochemical Properties 58
4.2 Isolation of Total Heterotrophic Bacterial
and Hydrocarbon Utilizing
Bacterial Counts from Engine
Oil Contaminated Soil 61
4.3 Biodegradation
Potential of Isolated Bacteria Strain and A Mixed
Microbial Consortium 65
4.4 Effect
of Temperature on Biodegradation Potential of Isolated Bacteria
Strain 68
4.5 Effect of pH on
Biodegradation potential of isolated
bacteria strain 70
CHAPTER 5
CONCLUSION AND RECOMMENDATIONS
5.1 Conclusion
72
5.2 Recommendation
and Proposed Future Research 73
References 74
Appendix I 90
Appendix II
LIST OF TABLES
2.1: Some
Contaminants Potentially Suitable for Bioremediation 28
4.1: Mean and
standard deviation of the physicochemical parameter of soil
samples 58
4.2: Mean and
standard deviation of heavy metals of soil samples 58
4.3: Colony
Characteristics and Microscopic observation of bacterial
Isolates 61
4.4: Biochemical tests and identification of
hydrocarbon degrading bacteria 62
4.5: Total heterotrophic bacterial and hydrocarbon
utilizing bacteria counts 62
LIST OF FIGURES
2.1: Average composition of crude oil along with some representatives of
each class of compounds 9
2.10: Environmental factors influencing the rate of
biodegradation in the
environment 47
2.2: BTEX
compounds 11
2.3: Structure of representative PAHs 12
2.6: Biodegradation of contaminants in the environment: Their
intrinsic
interactions. 29
2.8: Proposed putative pathway for microbial catabolism of PAHs 40
2.9: Hydrocarbon degradation rates in soil, fresh
water, and marine
Environments 44
3.1: GIS map of Umuahia South
showing the study site, Ohiya mechanic
village 49
4.1: Biodegradation potentials of isolated bacteria strain and a mixed
microbial
consortium 65
4.2: Effect of temperature on
biodegradation potential of isolated bacteria
Strain 68
4.3: Effect of pH on biodegradation potential of isolated bacteria strain 70
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
The
oil and gas industry with an anticipated ascent in world oil utilization from
85 million barrels in 2006 to 106.6 million barrels by 2030 has been one of the
most developing enterprises over the past 50 years (Igunnu and Chen, 2012).
Crude oil currently Nigeria's and absolutely the world's most essential energy
source (Moffat and Linden, 2005). Oil industry is one of the significant sectors
in Nigeria and it's the pillar of the nation’s economy (Ojumu et al., 2004). Crude oil in its standard
state is alluded to as unrefined petroleum (Ukoli, 2003). The oil handling
plant industry changes raw petroleum into increasingly refined items, such as,
liquefied gas, gas, lamp oil, aeronautics fuel, diesel fuel, fuel oils,
greasing oils and feed stocks for petrochemical industry (Abdulkarim et al., 2005).
Contamination
of soil by utilized engine oil is quickly expanding because of universal
increment in the use of oil based goods. Ecological pollution with crude oil
and petrochemical items has attracted much attention in recent times. The
existence of various kinds of automobile and machines has brought about an
expansion in the utilization of greasing oil. Release of utilized engine oils;
diesel or jet fuel contaminates our natural habitat with hydrocarbon. Hydrocarbon
pollution of the air, soil and freshwater particularly by polycyclic aromatic
hydrocarbons (PAHs) attracts public attention in light of the fact that
numerous PAHs are harmful, mutagenic, and cancer-causing (Mandri and Lin,
2007). Utilized/Spent motor oil is characterized as utilized greasing oils
expelled from the crankcase of internal combustion engines (Jain et al., 2009). Before they are utilized,
they comprised hydrocarbons, (80 to 90% by volume) and execution upgrading
added substances (10 to 20% by volume). Motor oils are changed during use by
vehicles, engine bicycles, generators and other machines due to the breakdown
of added substances, defilement with the products of combustion and the
introduction of metals from the wear and tear of the engine. It is perceived
that the significant segments of spent motor oil comprises aliphatic and
aromatic hydrocarbons, such as, phenol, naphthalene, benz (a)antracene, benzo
(a)pyrene, fluoranthene, lead, cadmium and other conceivably lethal metals
(Jain et al., 2009). Utilized engine
oil can result to extraordinary harm to sensitive environs and soil
microorganisms. Significant volumes of soil have been debased by utilized oil
in numerous nations of the world, particularly industrialized countries. High
concentration of aliphatic, polycyclic aromatic hydrocarbon and heavy metals
contribute to the inherent toxicity of utilized oil (Vasquez–Duhalt and Bartha,
1989). A lot of utilized motor oil are freed into the earth when the oil from
engine vehicles, engine bicycles, generators, and so on is changed and
discharged into canals, water channels, open empty plots and farmlands, a
typical practice by engine and generator mechanics (Odjegba and Sadiq, 2002).
Furthermore, the oil is additionally discharged into the earth from the fumes
framework during motor use and from engine leaks (Anoliefo and Edegbai, 2000;
Osubor and Anoliefo, 2003).
Spent
motor oil, when present in the soil creates an unsatisfactory condition for life
in the soil, which is due to the poor aeration it causes in the soil,
immobilization of soil supplements and bringing down of soil pH (Atuanya,
1987). Different contaminants, for example, utilized motor oil and heavy metals
have been found to modify soil organic chemistry, which incorporates
modification in soil microbial properties: pH, O2 and nutrient
accessibility (Atuanya, 1987; Brookes, 1995 and Odjegba and Sadiq, 2002).
In
Nigeria, contamination of the surface and underground water by oil and solid
wastes is widespread,thereby rendering water unsuitable for man's utilization
(Bakare et al., 2000). The hydrocarbon atoms that make up unrefined petroleum
and oil based commodities are profoundly lethal to many living things,
including people (Adekunle et al.,
2007). The contamination in the wastewater incorporates: aromatics, phenols,
polycyclic aromatic hydrocarbons (PAHs) and heavy metals (Bako et al.,
2008). The wastewater produced from the oil business finds its way into soil
and water bodies (Domde et al., 2007). The wastewater might be
treated by physicochemical or biological techniques. Biological treatment is
favored over physicochemical as the former is cost effective, efficient and
environmentally friendly (Hamza et al., 2008).
Indiscriminate
discharge of motor oil into canals, water channels, open empty plots and
homesteads is a typical practice in Nigeria particularly by engine mechanics
(Okonokhua et al., 2007). Heavy metals, such as, Vanadium, Lead, Aluminum, Nickel
and Iron usually below detectable limits
in unused engine oil have been reported by Whisman et al. (1971) to give
high qualities (ppm) in utilized oil. These metals might be held in soils as
oxides, hydroxides, carbonates, interchangeable cation, as well as bound to
organic matter in soil (Yong et al.,
1992). There is a growing global concern because of the numerous health risks
to animals and humans following exposure.
Pollution
is an undesirable change in the physical, chemical and biological
characteristics of all the component of an environment (Abioye, 2011). Oil pollution
is increasingly becoming a common theme in the world today and this has
resulted in the degradation of the environment particularly in the oil
producing area of the world (Obahiagbon et
al., 2009). Environmental contamination resulting from petroleum product
pollution typically occurs through accidental release of crude oil and from the
large quantities of oil sludge produced in refineries during the separation of
oil from water as well as the oil materials present at the bottom of crude oil
storage tanks (Kishore and Mukherjee, 2006).
In
auto-mechanics workshops, there is a ceaseless change in the soil micro-organism
because of deliberate discharged of utilized motor oil. These modify the
biomass and nature of the soil to such an extent that both microbial networks
and grasses can no longer grow on the soil spots. The colour and texture of the
soil are affected; this leads to different microbial flora creation in an
attempt to remedy the petroleum product discharged (Megharaj et al., 2000). Albeit a few
investigations have demonstrated that, oil-contaminated soils are oppressed by
Gram negative microscopic organisms (McNaughton et al., 1999; Kaplan and Kitts, 2004), the prevailing culturable
hydrocarbon consuming bacteria from the soil samples were made up of gram
positive Bacillus and Micrococcus and furthermore gram
negative Pseudomonas and Serratia.
Biodegradation
by microorganisms connote one of the essential components by which oil and
other hydrocarbon contaminants can be removed from the earth (Okoh, 2003). In
bioremediation, debasement of lethal toxins was done either through
intracellular amassing or by means of enzymatic change to less or nontoxic
compounds (Brar et al., 2006). Be that as it may, single
cultures of microbes have been observed to be superior to blended cultures
(Okerentugba and Ezeronye, 2003) and of late, bacteria have been observed to be
preferred degraders of oil over conventional bioremediation methods including
fungi (Batelle, 2000). Despite the expanding number of auto-mechanic workshops
in Umuahia, with their attendants indiscriminately dumping waste motor oil in
the environment, we are not aware of any
study that has attempted to isolate and identify bacteria present in used
engine oil contaminated soil environment. The present study was therefore
undertaken with a view to isolate bacteria in soil samples from the study area
and assesses their biodegradation potential.
1.2 STATEMENT OF RESEARCH PROBLEM
There are generally a lot of hydrocarbons in spent
motor oil including Polycyclic Aromatic Hydrocarbons (Wang et al., 2000). In Nigeria, it is common for automobilemechanics to
discharge utilized motor oil into canals, water drain and soil (Okonokhua et al., 2007). Ekundayo et al. (1989) have demonstrated that a
marked change in the properties happen in soils contaminated with hydrocarbons
influencing the physical, chemical and microbial properties of the soil. Oil
contamination prompts a development of fundamental organic nutrients, for
example, carbon, phosphorus, calcium and magnesium and non-essential Lead,
Zinc, Iron, Copper and Cobalt. These elements if in abundance will prompt
translocation of plant tissues.
Spent engine oil causes great damage to soil and soil
microflora. It creates unsatisfactory condition for life in the soil due to
poor aeration, immobilization of soil nutrient and lowering of soil pH (Ugoh and Moneke, 2011). It has been
demonstrated that marked changes in properties occur in soil contaminated with hydrocarbon;
this affect the physical, chemical and microbiological properties of the soil
(Okonokhua et al., 2007). Low concentration of some of these metals is vital
micronutrient for plants; however at high concentration they can cause
metabolic disorder and inhibit growth.
1.3 JUSTIFICATIONS OF THE STUDY
Biodegradation, utilizing biological procedures to
‘ameliorate’ hydrocarbons from the environment, is as of now a viable technique
for tidying up soil and marine residue (Van Herwijnen et al., 2006; Das and Mukherjee, 2007). Biodegradation of
hydrocarbon-contaminated soils, which exploits the capacity of microorganisms
to degrade as well as detoxify organic contaminants, has been proposed as a
productive, economic and robust technique that suits local conditions and sites
(Mehrashi et al., 2003, Taki et al., 2007, Obayori et al., 2008). Bioremediation is one of
the types of biodegradation which entails the in situ utilization of plants and
related microorganisms for the remediation of polluted sites. It has been
assessed by a few research studies to remediate oil polluted soils (Merkl et al., 2005; Issoufi et al., 2006; Diab, 2008).
Extra research, including genetic engineering, is
being carried out to improve the natural abilities of microorganisms to perform
remediation activities and to explore different microorganisms with potential
bioremediation applications (Robertson et
al., 2007). As the technology matures and its utilization grows past
research labs and government-financed remediation, site owners and consultants
will need comparative information on bioremediation to ascertain its
appropriateness for a specific site (Arthur et
al., 2005). Growing awareness of the harm that toxins do to the soil just
as to the entire environmental chain has prompted more investigation into how
to tidy up polluted sites. Because of the extraordinary variety of pollutants,
nonetheless, there is no basic answer for settle a wide range of soil
pollution. Hence studies related to hydrocarbon pollution of soil and its
biological cleanup is of significant importance. The availability of a
practical bioremediation technique would provide a cost efficient plausible and
environmentally friendly choice for the remediation of hydrocarbon polluted
sites in Nigeria particularly in the south–south area of the nation.
1.4 AIM AND OBJECTIVES
OF THE STUDY
The aim of the study was to assess the hydrocarbon
biodegrading capability of bacteria isolated from used engine oil contaminated
soil at Ohiya mechanic village, Umuahia.
The Specific
Objectives are:
i. Determine
the physico-chemical parameters of soil samples in the study area.
ii. To
isolate, identify and characterize hydrocarbon degrading bacteria from the
study area.
iii. To
ascertain the capability of bacteria isolate to biodegrade used engine oil
contaminated
Soil.
iv. To
determine the impact of temperature and pH on the biodegradation capability of
the
Bacteria isolate.
1.5 RESEARCH QUESTIONS
This study was intended to address the accompanying
questions:
1. What
is the impact of spent engine oil on the soil physico-chemical properties and
bacteria population of the study
area?
2. Can
hydrocarbon degrading bacteria be isolated from the study area?
3. What
is the biodegradation potential of bacteria isolate on used engine oil
contaminated soil?
4. What
is the influence of temperature and pH on biodegradation capability of the
bacteria isolate?
1.6 SIGNIFICANCE OF THE STUDY
The discoveries in this study will be valuable in the
remediation of utilized engine oil contaminated soils and some other
hydrocarbon contaminated part of the nation. Bioremediation is as of late being
favored as a decent alternative for the remediation of polluted sites since it
utilizes economical tools, ecologically friendly and straightforward.
Bioremediation is cheap, environmentally and socially adequate and ecologically
acceptable. The technique under investigation will help limit the prohibitive cost
related with soil remediation, avert critical soil surface change, avoid the
exchange of toxins from one medium onto the next and guarantee a healthier
method for remediating heavy metals. This work will add to the database on
locally accessible bacterial diversity of oil polluted environment and their
ecology. This will likewise build information and knowledge of the bacteria
associated with biodegradation of oil in Umuahia, Abia State.
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