ABSTRACT
Biodegradation of used engine oil contaminated soil using bacteria from such soils was undertaken. Three principal bacteria species, Staphylococcus, Bacillus and Pseudomonas were selected from among the soil isolates and tested for their potency as biodegraders of engine oil in the contaminated soil over a period of 28 days. Results of the preliminary soil analysis showed sand fractions in the range of 64.53% to 68.83% silt 9.50% to 18.33% and clay 16.67% to 22.93%. the soil was also rich in organic matter 3.57% to 4.04% and average moisture content in the range of 21.70% to 23.96% during bioremediation with Bacillus innocula, the bacteria load varied between 1.3x107 cfu/g to 2.5x107 cfu/g in the control soil of site A while the remediated soil for the same site were in the ranges of 1.3x107 cfu/g to 1.09x108 cfu/g at the end of the remediation with a reduction of soil content from 4.67% to 2.39% representing 48.8% remediation. Similarly, remediation of 38.21% (2.80% to 1.73%) and 48.47% (4.27% to 2.20%) were recorded in site B and C respectively. The levels of remediation with Pseudomonas species were 53.30% (4.67% to 2.19%), 40% (2.80% to 1.68%) and 50.40% (4.27% to 2.12%) in the soils of site A, B and C respectively. The bacteria load varied from 1.7x107 cfu/g to 3.4x107 cfu/g in the control soil of site A while the remediated soil for the same site was in the range of 1.7x107 cfu/g to1.55x108 cfu/g. the bacteria load of the test soil of site B varied from 1.3x107 cfu/g to 3.0x107 cfu/g while the remediated soil ranges from 1.3x107 cfu/g to 1.64x108 cfu/g of soil in the same site, while that of site C varied from 1.9x107 cfu/g to 3.1x107 cfu/g for control soil and 1.9x107 cfu/g to 1.71x108 cfu/g for the remediated soil. During bioremediation with Staphylococcus innocula, the bacteria load varied between 1.3x107 cfu/g to 2.4x107 cfu/g in the control soil of site A while the remediated soil for the same site was in the range of 1.3x107 cfu/g to 1.04x108 cfu/g while bacteria load varied between 1.2x107 cfu/g to 1.02x108 cfu/g in the control soil of site B while the remediated soil for the same site was in the range of 1.2x107 cfu/g to 1.32x108 cfu/g and the bacteria load of site C varied from 1.5x107 cfu/g to 3.5x107 cfu/g for the control soil while that of the remediated soil varied from 1.5x107 cfu/g to 1.23x108 cfu/g at the end of the remediation with a reduction of soil content from 4.67% to 3.46% representing 25% remediation for test soil from site A, similarly remediation of 31% (2.8% to 1.91%) and remediation of 33% (4.27% to 2.86%) were recorded for site B and C respectively. Slight but significant variations were recorded in the extent of bacteria biodegradation of the used engine oil contaminated soil with Pseudomonas species being more potent than the Bacillus species and Staphylococcus species as bioremediaters of the soil.
TABLE OF CONTENTS
Title
page i
Certification ii
Dedication
iii
Acknowledgements
iv
Table
of tables v
List of tables viii
List
of figures ix
Abstract
x
CHAPTER ONE
1.0
Introduction 1
1.1 Aim
and Objectives 3
CHAPTER TWO
2.0
Literature Review 4
2.1 Engine
Oil 4
2.2 Used
Engine Oil 4
2.3 Effects
of Used Engine Oil on Soil Physical Properties 5
2.4 Effects of Used Engine Oil on Soil Chemical Properties 6
2.5 Effects
of Used Engine Oil on Soil Health 8
2.6 Biodegradation
10
2.6 Hydrocarbon
Degrading Bacteria 12
2.6.1 Bacterial
Isolates 12
CHAPTER THREE
3.0
Materials and Methods 15
3.1 Source
of Materials 15
3.2 Sample
Preparation 15
3.2.1 Sterility
of glasswares 15
3.2.2 Preparation
of media 16
3.3 Methods
of Analysis 16
3.3.1 Isolation
of bacteria 16
3.3.2 Characterization
of bacteria isolates 16
3.3.2.1 Colony
features 16
3.3.2.2 Microscopic
features 17
3.4 Biochemical
features 17
3.4.1 Catalase
production test 17
3.4.2 Coagulase
production test 18
3.4.3 Urease
test 18
3.4.4
Citrate utilization test 18
3.4.5 Oxidase
test 18
3.4.6 Carbohydrate
Utilization test 19
3.4.7 Indole
Test 19
3.5 Identification
of Isolates 19
3.6 Determination
of Soil Physiochemical Parameters 19
3.6.1 Determination
of total organic carbon 20
3.6.2 Determination
of particle size distribution 20
3.6.3 Determination
of total exchangeable bases 20
3.6.4 Determination
of Ca2+ and Mg2+ by EDTA 21
3.6.5 Determination
of pH 21
3.7 Isolation
of Oil Utilizing Bacteria 21
3.8 Degradation
of Used Engine Oil 22
3.8.1 Determination
of residual oil in soil 22
3.8.2 Determination
of Bacteria Growth 23
CHAPTER
FOUR
4.0 Results 24
CHAPTER
FIVE
5.0 Discussion, Conclusion and
Recommendations 38
5.1 Discussion 38
5.2 Conclusion 39
5.3 Recommendations 40
References 41
Appendix 43
LIST
OF TABLES
Table
1: Physicochemical properties of used engine oil contaminated soil 24
Table
2: Macroscopic and microscopic characteristics of used engine oil degraders 25
Table
3: Biochemical characteristics of isolates 26
Table
4: Occurrence of bacteria isolated from used engine oil contaminated soil 27
Table
5: Changes in Hydrocarbon utilizing Bacteria count in Pseudomonas
remediated soil 34
Table
6: Changes in Hydrocarbon utilizing Bacteria count in the Bacillus
remediated
soils 35
Table 7: Changes
in Hydrocarbon utilizing Bacteria count in the Staphylococcus remediated soils
36
LIST
OF FIGURES
Figure 1: Change
in oil content (%) of used engine oil contaminated soil during remediation with Pseudomonas species 29
Figure 2: Change
in oil content (%) of used engine oil contaminated soil during remediation with Bacillus species 30
Figure 3: Change
in oil content (%) of used engine oil contaminated soil during remediation with Staphylococcus species 31
Figure 4: Total
heterotrophic count of bacteria remediated used of engine oil contaminated soil with Pseudomonas species 32
Figure 5: Total
heterotrophic count of bacteria remediated used of engine oil contaminated soil with Bacillus species 33
Figure 6: Total
heterotrophic count of bacteria remediated used of engine oil contaminated soil with Staphylococcus species 34
CHAPTER ONE
1.0 INTRODUCTION
Accidental
spills, illegal dumping and careless handling of spent lube oil in mechanic
workshop have been a significant source of environmental pollution because of
the predominantly unstructured practice of automobile vehicle repair services.
Contaminations of soil and ground water
have been imminent from the continuous disposal of used engine oil which could
lead to a great health problem. Used engine oil contains metals and heavy
polycyclic aromatic hydrocarbons (PAHs) and these could contribute to chronic
hazards including mutagenicity and carcinogenicity (Ogunbayo, et al., 2014)
As
engine oil is used in automobile, it picks up number of additional compounds
from engine wear; these include iron, steel, copper, zinc, lead barium,
cadmium, sulphur, dirt and ash because of the additives and contaminants. Used
motor oil disposal can be more environmentally damaging than crude oil
pollution. These additives and contaminants may cause both short and long term
effect if they are allowed to enter the environment through water ways or soil.
Once engine oil is drained off an engine it is no longer clean because it has
picked up materials, dirt particles and other chemicals during engine operation
thus such lubricating oil is now classified as spent engine oil (Uchendu and
Ogwo, 2014).
Waste
engine oil which is also known as used motor oil is produced when fresh engine oil
(or motor oil) is subjected to high temperature and high mechanical strain
during running of the vehicle for a stipulated time. It is a brown-to-black
liquid mixture consisting of low to high molecular weight (C16 to C36)
aliphatic and aromatic hydrocarbons, poly chlorinated biphenyls,
chlorodibenzofurans, lubricative additives along with heavy metal contaminants
such as zinc, lead and chromium, coming from engine parts thousand million
gallons of waste engine oil are generated annually from mechanical workshops which is not
recycled but spilled and dumped by automobile and generator mechanics into
runoff, gutter, water drains and open vacant plots and farm lands (Munna and
Dipa, 2014).
In
most countries of the world, oil spills
at auto-mechanic workshops have been left uncared for over the years and its
continuous accumulation is of serious environmental concern because of the
hazard associated with it. The physiochemical treatment technologies currently
in use are expensive and not environmentally friendly. In addition, some of
these technologies only transfer the contamination from one place to another.
In recent times, a lot of effort have been made towards reducing environmental
pollution by using natural processes to treat environmental pollution, these techniques
include: bioremediation (use of
micro-organism to degrade pollutants) and phytoremediation (use of
plants to clean pollutants by bioaccumulation into the plant tissues (Eniola, et al., 2014) .
Petroleum
products such as engine oil, petrol, diesel and kerosene are used daily in
various forms in mechanical workshops. These products tends to harden and
change the colour of the soil, which may have untold health hazards on the
technicians and artisans. Their soles tends to harden which may alter their
movement (Udeani, et al., 2009).
Both
fungi and bacteria are known to degrade aromatic hydrocarbons. Fungi perform
oxidation reaction as a prelude to the detoxification and excretion of
hydrocarbons rather than using these compounds as a carbon sources for growth
(Francese, et al, 2001).
The
bacteria metabolize the oil in such a way that human converts food into energy.
The soil is habitat to many living organism, any change in their number of form
may upset or cause a total collapse in the ecosystem. The effect of oil spill
on soil leads to enrichment of the soil degrading microbial population. No
single micro-organism has been found to be able to completely degrade a
petroleum hydrocarbon molecule; however
different species of strains of the same species maybe capable of degrading
different groups of hydrocarbon found in oil. Different naturally occurring
species of Pseudomonas is known to
contain plasmid with relevant genes for degradation of different hydrocarbon
(Umar, et al., 2013). A large number
of Pseudomonas strains capable of
degrading polycyclic aromatic hydrocarbon have been isolated from soil. Other
petroleum hydrocarbon degraders include
Alcaligenes species, Cyanobacteria
species and Bacillus species. Other micro-organism such as fungi
are also capable of degrading the hydrocarbons in engine oil to certain extent.
However, they take longer period of time to grow compared to their bacterial
counter parts (Udeani, et al., 2009).
1.1 Aim
and Objectives
The
aim of this study is to biodegrade used engine oil contaminated soil using
bacteria.
The
objectives are:
1. This
study is intended to isolate bacteria capable of effectively degrading and
cleaning up used engine oil contaminated soil.
2. To
identify bacteria which can utilize used engine oil as carbon and energy source
from used engine oil contaminated soil.
3. To
use the bacteria isolated and identified to biodegrade used engine oil
contaminated soil.
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