IMPACT OF THE AGE OF PARTICULATES ON THE

BIOREMEDIATION OF CRUDE OIL POLLUTED SOIL

BY
AGUELE PRECIOUS OSATOHAMHEN
ENG0801529

DEPARTMENT OF CHEMICAL ENGINEERING

FACULTY OF ENGINEERING
UNIVERSITY OF BENIN
BENIN CITY, EDOSTATE.

FEBRUARY, 2014.

IMPACT OF THE AGE OF PARTICULATES ON THE

BIOREMEDIATION OF CRUDE OIL POLLUTED SOIL.

BY
AGUELE PRECIOUS OSATOHAMHEN
ENG0801529

THIS PROJECT IS SUBMITTED TO THE DEPARTMENT OF

CHEMICAL ENGINEERING, FACULTY OF ENGINEERING,
UNIVERSITY OF BENIN, BENIN-CITY, EDO STATE, IN
PARTIAL FULFILMENT OF THE REQUIREMENTS FOR
THE AWARD OF THE DEGREE OF BACHELOR OF
ENGINEERING (B.ENG) IN CHEMICAL ENGINEERING.

FEBRUARY, 2014.

CERTIFICATION
This is to certify that this project work was done by AGUELE PRECIOUS
OSATOHAMHEN in the Department of Chemical Engineering, Faculty of
Engineering, University of Benin, Benin-City, Edo State.

_______________________
PROF. OBAHIAGBON K.O.
(SUPERVISOR)

_____________________
DATE

________________________
PROF. ALUYOR E.O.
(HEAD OF DEPARTMENT)

______________________
DATE

_______________________
ENGR. AKHABUE C.E.
(PROJECT COORDINATOR)

_____________________
DATE

_______________________
EXTERNAL EXAMINER

_____________________
DATE

DEDICATION
This project work is dedicated to God Almighty.

ACKNOWLEDGEMENT
Profound gratitude goes to my supervisor, Prof Obahiagbon K.O. who
despite his tight schedule always attended to me, also for his motivation and
encouragement during the course of this project work. I also want to say a big
thank you to my lecturers, Dr.Owabor C.N., Mr.Amenaghawon N.A,
Mr.Osazuwa U.O.Mr Osadolor A.O., as well as my H.O.D., Prof. Aluyor
E.O.who contributed in one way or the other to this study.
I am highly indebted to my parents, Prof and Mrs. L.I Aguele for their
love and support morally, financially, emotionally and academically. I will ever
remain grateful to them.

Special thanks to my siblings; Osas, Nosa and

Princewill. I will not fail to acknowledge the families of Pastor and Dcns Paul
Ephraim and Prof. and Dr. (Mrs.) E. Ukpeborfor their continuous support and
care.
Thanks to my wonderful friends Nneka, Ebele, Epsi, Lizzy, Kara, all my
course mates amongst whom are, Mavis, Felix, Henry, Obiwan, IK,
William,Chuks.

ABSTRACT
This study was conducted to investigate the effect of the age of poultry manure
particulate on the bioremediation process of crude oil contaminated soil. pH,
Total Hydrocarbon Content(THC) and Total Microbial Count(TMC) were
measured to monitor the performance of four soil samples weighing 4kg each.
These samples were polluted with 0.2kg of crude oil per kilogram soil; and
amended with particulates and fertilizer of 0.2kg and 0.08kg per kilogram soil
respectively. The age of particulate used in each sample varied in no particular
order from 3days to 126 days. The study lasted 8weeks. Results obtained
showed 99.17 %(84.10-0.70mg/kg), 98.07%(82.90-1.60mg/kg), 98.69%(84.101.10mg/kg) and 97.72% (83.40-1.90mg/kg) drop in Total Hydrocarbon Content
for samples A,B,C and D respectively.THC for all samples fell below the FEPA
limit of 10mg/l on closure. Sample A had the highest TMC 3.0x10 6cfu/g; while
D had the least, 2.2x106cfu/g. The pH of all samples at the end of the study
(6.5-6.9) fell within the range specified by FEPA (6-9). The overall data
suggested that lower aged poultry manure particulates did best and be employed
in the amendment of crude oil polluted soil.

TABLE OF CONTENTS
Title Page
Certification
Dedication
Acknowledgement
Abstract
Table of Contents
List of Tables
viii
List of Figures

i
ii
iii
iv
v
vi
ix

CHAPTER ONE
1.0 Introduction
1.1 Background
1.2 Objective of Study
1.3 Scope of Study
1.4 Relevance of Study

1
1
3
3
4

CHAPTER TWO
2.0 Literature Review
2.1 Crude Oil
2.1.1 Formation
2.1.2 Composition of Crude Oil
2.1.3 Physical properties of Crude Oil
2.1.4 Products obtained from Crude Oil and the Uses
2.2 Oil Spillage
2.2.1 Definition
2.2.2 Effect of Crude Oil to the Soil
2.3 Soil Remediation
2.4 Bioremediation
2.4.1 Advantages of Bioremediation over other methods
2.4.2 Limitations of Bioremediation
2.4.3 Factors Affecting Bioremediation
2.5 Bioremediation of Crude Oil Contaminated Soil
2.5.1 Types of Bioremediation of Crude Oil Contaminated Soil
2.5.1.1
Phytoremediation
2.5.1.2
Microbial Remediation
2.5.2 Methods for Bioremediation Assessment

5
5
6
7
8
9
10
10
11
14
16
18
18
19
21
22
22
23
30

CHAPTER THREE
7

3.0
3.1
3.2
3.3
3.4
3.5
3.6
3.6.1
3.6.2
3.6.3

Materials and Method

Sample Collection
Chemicals and Reagents
Apparatus/ Equipment
Sample Preparation
Soil Treatment
Bioremediation Analysis
Soil pH
Total Hydrocarbon Content
Total Microbial Count

33
33
33
33
35
37
38
38
38
39

CHAPTER FOUR
4.0 Results and Discussions
4.1 Soil pH
4.2 Total Hydrocarbon Content
4.3 Total Microbial Count

41
41
44
48

CHAPTER FIVE
5.0 Conclusion and Recommendations
5.1 Conclusion
5.2 Recommendations

52
52
52

REFERENCES

53

LIST OF TABLES
Table 2.1

Elementary Composition of Crude Oil

Table 2.2

Some distillation components of Nigerian Brass crude oil

10

Table 2.3

Causes of Oil Spill and the Percentage

11

Table 2.4

Physico-Chemical Properties of Crude Oil Spilled Areas

12

Table 2.5

Major Factors Affecting Bioremediation

19

Table 2.6

Elements Contained Poultry Manure

25

Table 3.1

Reagents and their Uses

33

Table 3.2

Apparatus, Equipment and the Uses

34

Table 4.1

pH Values over Time for the Bioremediation of Crude Oil

Contaminated Soil with Particulates of Different Ages.

41

Total Hydrocarbon Content of Crude Oil Contaminated Soil

Bioremediated with Poultry Manure of Different Ages

44

Table 4.2
Table 4.3

Table 4.4

Percentage Reduction in Total Hydrocarbon Content (THC)

of Soil Bioremediated with Poultry Manure Particulates of
Different Ages.

45

Total Microbial Count of Crude Oil Contaminated Soil

Bioremediated with Poultry Manure of Different Ages.

48

LIST OF FIGURES
Figure 3.1

Compartments where Bioremediation Study was carried out

36

Figure 3.2

Compartments where Bioremediation Study was carried out

37

Figure 3.2

Standard THC Graph

39

Figure 4.1

Variation of soil pH with Bioremediation Time and Particulate

age.

41

Figure 4.2

Variation of Total Hydrocarbon Content with Time And Age of

Particles during the Bioremediation Process.
44

Figure 4.3

Variation of Total Microbial Count with Time during the

Bioremediation process.

10

48

CHAPTER ONE
INTRODUCTION
1.1 BACKGROUND
Over the years, a supposed natural resource, crude oil, has turned out to be a
threat, to the environment, when spillage into soil or water occurs. Spilled
crude-oil is denser than water, thus reduces and restricts permeability; also it
contains hydrocarbons which fill the soil pores and expel water and air, thereby
depriving the plant roots the much needed water and air (Brian, 1977).
Depending on the degree of pollution, the oil polluted soil may remain
unsuitable for use for months or even years (Nwandigwe and Onyeidu, 2012).
Oil spillage has caused untold hardship on those residing in areas where this
natural resource is in abundance, as it has deprived them of portable water;
being that the surface and ground water gets contaminated when oil spills. It has
also deprived most of them of their economic source of livelihood: the farmers
and fishermen especially.
Furthermore crude oil spillage tends to render the environment unhealthy, since
it contains several hydrocarbons that have negative effects when inhaled. Apart
from the aforementioned crude oil, other hydrocarbon sources, which most
times are refined products of it; hydrocarbon sources such as diesel, gasoline,
lubricating oil, etc tend to have similar effects as their parent material on the

11

soil and water bodies. This increases the number of geographical locations in
which spillage can occur.
Due to high demand on land and water, it is imperative that the soil and water
bodies affected be rehabilated in time for use; therefore natural methods like
bush fallowing (for soil), cannot always be relied on. This has given rise to
several techniques of remediation. This work focuses on the remediation of
polluted soil.
Amidst the various techniques that can be utilised in replenishing soil nutrients,
as has been developed over the years, bioremediation seems to be most thriving.
This is because, unlike the physiochemical, and other methods of remediation,
there is no destruction of site material, or its natural existing flora and fauna
before achieving complete degradation of the organic pollutants from the crude
oil (Timmis and Pieper, 1999).
Bioremediation involves degrading contaminants through the use of naturally
occurring microorganisms and microbes. Crude oil in a sense is a natural
product formed from high temperature anaerobic conversion of biomass, and all
natural organic compounds degrade, thus it could be subject to bioremediation
(Yehuda

C.,

2002).

The

two

approaches

to

bioremediation

are

Bioaugumentation and Biostimulation.

Bioaugumentation is the addition of the bacterial culture to the contaminated
soil. Biostimulation is adding microbial nutrients needed to increase microbial

12

activities of indigenous microbial flora and fauna to a contaminated soil (Odu et

al., 2005).
Poultry droppings act as a biostimulant, like the N.P.K. 15.15.15., ferrous
sulphate as well as other organic and inorganic fertilizers, under ambient
conditions, tend to reduce the hydrocarbon content of contaminated soil, thus
leading to increase in soil fertility. They achieve this through several natural
processes as will be discussed.

1.2 OBJECTIVE OF STUDY.

To determine how crude oil contaminated soil can be reclaimed.
To find environmentally safe means of utilizing poultry waste.
To use parameters such as Total Hydrocarbon Content (THC), soil pH,
Total Microbial Count (TMC) to evaluate the performance of
contaminated soil being remediated with poultry manure of different
ages.

1.3 SCOPE OF STUDY

Collection of samples (soil, crude oil, poultry droppings, N.P.K

fertilizer).
Contamination of soil with crude oil.
Addition of amendments for remediation to occur.
Collection of samples for laboratory analysis weekly, for 8 weeks.
Evaluating the performance of the process based on the results obtained
from the laboratory analysis.
13

1.4 RELEVANCE OF STUDY

The petroleum industry is an economic backbone to most countries today, hence
its production, which is continuous will be for a very long time. In view of this,
there is the need to investigate into cheap and environmentally conducive
methods of remediating soils affected by the accident (spillage) that comes
along with the production, transportation, etc of this petroleum; which in the
long run is inevitable. Bioremediation of contaminated soil with poultry manure
aids in utilizing such waste and providing an environmentally acceptable way
of disposal. It will also help boost economic activities as those utilizing the soil
for agricultural purposes will not be deprived of it. Community hostilities that
could hamper economic activities of the petroleum industry are minimized.
Most importantly, it safeguards the environment; being that it solves the
problem of land pollution in a natural and environmentally friendly way.

14

CHAPTER 2
LITERATURE REVIEW
2.1 CRUDE OIL
Crude oil, petroleum, fossil fuel, black gold and other various names by which
it is known is a naturally occurring, dark, viscous liquid found under the
ground. It is a non-renewable source of energy. Crude oil is a dark, sticky
naturally-occurring liquid, classified scientifically as a hydrocarbon found in
certain rock formations in the earth, highly flammable and can be burned to
create energy, may or may not contain non-metallic elements such as oxygen
and sulphur (OPEC).
Crude oil is a naturally occurring flammable liquid consisting of a complex
mixture of hydrocarbons of various molecular weights and other liquid organic
compounds, that are found in geologic formations beneath the earth's surface.
It is a mixture of hydrocarbons that exists in liquid phase in natural
underground reservoirs and remains liquid at atmospheric pressure after passing
through surface separating facilities.
Crude oil is a complex biodegradable substance containing a large variety of
hydrocarbons such as straight, branched and cyclic aliphatics, aromatic and
heterocyclic compounds (Obahiagbon et al., 2009).

15

2.1.1 Formation
Crude oil is formed from the remains of remains of organisms; such as the algae
and zooplankton, allowed to decay over a long period of time, hence it is a
fossil fuel. These remains, in large quantity mix with various sediments on
settling to the bottom of water bodies, and are buried in the absence of air. As
more layers of these fossils and sediments settle on previous ones, the
temperature and pressure increases in the lower regions, causing a change in the
organic matter. Through a process known as catagenesis, under very high
temperature, hydrocarbons are formed from an initial highly viscous material,
kerogen, first formed. Under simile condition of high temperature and pressure,
hydrocarbon pyrolysis occurs and crude oil (petroleum) is formed (Braun et al.,
1993; Keith and Kvenvolden, 2006). The oil also occurs in bands of spongy
rock, and is sometimes visible.
This process occurs over 6000meters below the earth surface, hence the need to
drill to obtain it. It is the pumped through pipelines to refineries where it is
refined into very useful products.

16

2.1.2 Composition of crude oil.

Crude oil is composed elementarily of carbon, hydrogen, sulphur and nitrogen.
It is a complex mixture of several polycyclic aromatic compounds and other
hydrocarbons (Domask, 1984).
Table 2.1: Elementary Composition of Crude Oil.
Element
Carbon
Hydrogen
Sulphur
Nitrogen

Percent by weight
84-87
11-14
0-3
0-0.6
Source: Gary and Handwerk, (2001).

Metals, such as vanadium, nickel, chromium are sometimes present in crude oil.
From table 2.1, it can be seen that crude oil is mainly composed of
hydrocarbons. These hydrocarbons are in three classes; they are:
Paraffins: They are characterised with a single carbon bond and have the
general formula CnH2n+2. The lower members of this class, C 1-C4 are gases;
higher than C4 go from liquid to solids.
Naphthenes: This group is the cyclic of the former (paraffins), thus they are
also referred to as the cycloparaffin. Examples of naphthenes present in crude
oil are cyclopentane, methylcyclopentane, decalin, cyclohexane, etc.
Aromatics: These are the unsaturated hydrocarbons, they usually have the
benzene, C6H6, ring. Benzene, xylene, ethylbenzene, etc are some of the
aromatics in crude oil (Gary and Handwerk, 2001).
2.1.3 Physical properties of crude oil.
17

There are certain properties that are most times used to classify crude oil. They
sometimes serve as basis for tests (Gruse and Stevens , 1960). These properties
include:
API Gravity: This is used in place of density for crude oil. It is obtained from
its specific gravity at 60oF, thus:
(0 )=

141.5
131.5
specific gravity (at 600 F )
API Gravity

It is used to classify crude oil into light to heavy crude.

The light crude has an API value of about 35 o API, while the heavy crude oil is
at 25oAPI (ICCT, 2011).
Sulphur content: It is important to know the sulphur content, because high
sulphur content negatively affects refining and cracking processes. This is used
to classify crude oil into sweet and sour crude. If the sulphur content is greater
than or equal to 0.5%, it will require special processing and it is referred to as
sour crude; when less than 0.5%, it is called sweet crude and does not require
any special processing technique.
Reid vapour pressure: The vapour pressure of crude oil is measured with the
reid vapour pressure. Here, the vapour pressure is measured at 100 oF with
boiling point above 32oF.
Pour point (oF): Under standard test conditions, this is the lowest temperature
at which oil will flow. It is a measure of the degree of aromacity or paraffinicity

18

of the crude oil. The lower the pour point, the lower the paraffin content (Gary
and Handwerk, 2001).
Metal content: Due to the adverse effects of metals, such as vanadium and
nickel on the catalysts used in cracking petroleum, it is imperative that their
content in the oil is determined (Gruse and Stevens, 1960). The metallic content
may be reduced by solvent extraction with propane or similar solvents as the
organometallic compounds are precipitated with the asphaltenes and resins.
Carbon residue: This is usually expressed by the conradson and ramsbottom
ASTM test procedures. The lower the carbon residue, the more valuable the
crude oil.
2.1.4 Products Obtained From Crude Oil And Their Uses.
There are over 2000 products obtained from crude oil, according to a survey
carried out by the American Petroleum Institute, (API) (API bulletin, 1958).
Over 4000 petrochemical products can be obtained from crude oil (OPEC).
These products are obtained during the fractional distillation of the petroleum,
and at different temperatures, i.e. the desired boiling points, they are withdrawn.
The uses of these products range from providing heat energy for transport, to
cooking, etc, as well as lubricating, as chemical solvents, pigments, etc. Some
of the petroleum products produced in Nigeria include gasoline, diesel, lube oil,
asphalt, kerosene and other jet fuels, methane, ethane, butane, etc.
Table 2.2: Some distillation components of Nigerian Brass crude oil
Distillation fraction

Composition (%)

Chain length

Gasoline

11.2

C4 -C10

19

Naphtha

18.1

C4-C10

Kerosene

16.9

C10 C20

Gas oil

15.7

C15-C40

Heavy gas
25.8
Source: Adams P.J., (1996).

C40 and above

2.2 OIL SPILLAGE

2.2.1 Definition
Crude oil could be introduced into the soil accidentally, operationally or
intentionally, during production, while transporting, processing, released as
waste (Obahiagbon and Audu, 2002). In most cases, it is introduced
accidentally into soil and water bodies during drilling operations, production
processes, when pipelines are corroded or vandalised, leakage from storage
tanks, or certain other reasons caused by general laxity on the part of petroleum
companies.
The worst man made environmental disaster that has ever occured is as a
result of crude oil spillage. This was during the 1991 Gulf War, where destroyed
wells led to the release of millions of gallons of crude oil being into
surrounding land, forming more than 330 oil lakes, covering an area of 49
square kilometres (Salam, 1996).
Table 2.3: Causes of Oil Spill and the Percentage
Cause
Corrosion
Sabotage

Percent (%)
50
28
20

Production operations
Non-functional production equipment
Source: Nwilo and Badejo . (2001).

21
1

2.2.2 Effect of Crude Oil to the Soil

It is a known fact that petroleum hydrocarbon adversely affects soil ecology and
fertility. Crude oil changes the physico-chemical properties of soil. It increases
the level of toxins, such as zinc and iron in the soil (Udo and Fayemi, 1975) and
reduces the amount of nutrients available. There is high accumulation of
aluminium and manganese ions, which are toxic to plant growth, due to the
anaerobic condition in the soil,the water logging and acid metabolites created
by the crude oil (Onwurah et al., 2007). Furthermore, there is reduction in the
amount of oxygen diffused to the root system if the oil is stranded on the plant
shoot, thus affecting the soil indirectly. A study carried out in Assam, North
Eastern India yielded the result shown below.
Table 2.4: Soil Physico-Chemical Properties of Crude Oil Spilled Areas
Physical properties
Study

Colour

Soil texture

Soil moisture

Soil porosity

WHC of Soil

Dark black

Cl=06.67 03.47

35.67 5.12

35.83 4.76

26.67 4.83

43.67 7.54

50.88 1.89

48.86 1.15

35.83 6.50

35.27 4.51 2

26.84 4.44

area
A

Si=21.87 09.78
Sa=71.54 00.86
UA

Black

Cl=20.90 04.00
Si=17.52 05.84
Sa=61.57 05.22

Dark black

Cl=05.94 03.28
Si=24.52 04.61
Sa=69.57 01.91

21

UB

Black

Cl=25.70 02.06

44.33 7.63

50.90 1.71

48.78 1.32

Organic

Extractible

Exchangeable

carbon(%)

phosphorus(ppm)

potassium(ppm

Si=13.46 04.07
Sa=60.84 02.89
Chemical properties
Study

Ph

Total nitrogen(%)

areas
A

5.80 0.80

0.12 1.005

8.05 0.99

4.47 1.20

)
39.63 2.45

UA

6.40 0.20

0.074 .007

1.08 0.15

16.00 3.27

29.10 2.25

5.72 0.30

0.13 0.007

8.03 0.95

4.45 1.09

41.21 3.06

UB

6.36 0.20

0.73 0.008

1.07 0.13

16.40 3.17

28.80 3.01

Source: Debojit et al., (2011).

Key
A- area A contaminated with crude oil

UA- unpolluted area A

B- area B contaminated with crude oil

UB- unpolluted area B

Cl-clay

Si-silt

Sa-sand

WHC- Water holding capacity

From table 4 above, it can be seen that physical properties such as soil moisture,
water holding capacity, porosity, pH and exchangeable phosphorus reduce,
while soil nitrogen, organic carbon content and exchangeable potassium
increase. The pH of the polluted soil being more acidic is as a result of the
formation of toxic acids in the spilled oil (Udo and Fayemi, 1975). The low soil
water contents of the crude oil contaminated soil could be due to reduce soil
moisture recharge caused by hydrophobic nature of crude oil contaminated soil
(Baruah and Das, 1994).

22

Crude oil may also slow down the rate of seed germination, reduce height, stem
density, photosynthetic rate and biomass, most times resulting in complete
mortality (Tanee and Akonye, 2009). A possible reasons for inhibition of
germination in crude oil contaminated soil is insufficient aeration due to
decrease in air filled space and increased demand of oxygen by oil
decomposing microorganisms (Clarkson et al., 1980).
It causes morphological aberrations, reduction in biomass, to cellular and
stomatal abnormalities (Wallace 1951). A study carried out in Asaba, Delta
State, showed that crude oil contamination on the soil significantly reduces the
biomass accumulation in Jatropha curcas seedlings (Agbogidi, 2011). Another
research in Calabar by Ekpo et al., 2012 showed that crude oil pollution reduces
the germination and growth of the soybean plant, especially at higher pollution
rates. Black (1957) reported the inhibition of root growth due to acidity, which
was caused by crude oil contamination. He also demonstrated that growth and
development of plants are adversely affected by crude oil pollution, ranging
from wilting, chlorosis, tissue and cell maceration, blotching and the collapses
of marginal necrotic spots, which have eventually resulted in the death of
plants.
Fresh crude oil has a coagulatory effect on the soil, it binds the soil particles
into a water impregnable soil block thus, seriously impairing water drainage
and oxygen diffusion and seeds fail to germinate . Oil spillage that occurred in
Nigeria on dry land between 1978 and 1979, grossly affected farmlands where
23

crops like rice, yam, plantain, cassava were cultivated (Onwurah, 1999). The
depletion of the vegetative cover and mangrove ecosystem of the Niger Delta
area of Nigeria is as a result of the oil spillages that have occurred in the area
over the years (Odu, 1987). Hence, crude oil in soil makes the soil condition
unsatisfactory for plant growth
In view of this methods have to be developed to make the soil suitable for
farming again, as well as water bodies conducive for aquatic life.

2.3 SOIL REMEDIATION

Several attempts have been made to correct or improve the condition of the soil.
Various methods have thus far been developed, they includes excavation,
vapour extraction, stabilization and solidification, containment in secured
landfills, solvent extraction, thermal desorption, incineration and vitrification
(US EPA, 1988). Although these processes have been proven capable of treating
the soil, several regulatory requirements also have to be satisfied before it could
be used on the field.
It was classed as physical/chemical treatment, heating treatment and biotreatment, others (land fill and natural purification). Remediation could be
classified as mechanical, physical, chemical and biological (Tanee and Albert,
2011).
Chemical remediation: In this form of remediation, photolysis, oxidation
(Onwurah, et al., 2007), solvent extraction, soil aeration (PEC, Kuwait, 1999).
24

Surfactants could be used for bioremediation. Examples of these surfactants are

sodium dedocyle sulphate (SDS), sodium carbonate, sodium metasilicate, water
and alcohol ethoxylate. They work based on mass transfer between solid, liquid
and micellare phases, also the solubility of the hydrocarbons depends on the
flow rate of the surfactant solution (Abdul et al., 1990). The organic molecules
are absorbed on soil and caught in the pores, as well as their encapsulation
within the micelle, formed at concentrations greater than the critical micelle
concentration (Khalladi et al.,2009).
Biological: This includes the use of processes or practices such as bio-reactors
(solid and slurry), landfarming, composting and bio-venting (PEC, Kuwait,
1999), as well as bioremediation. These practices help provide a breeding
ground for microorganisms. These organisms in turn degrade the hydrocarbon
contaminants, although complex hydrocarbon structures are very difficult to
degrade, and the rate of degradation also reduces (Atlas, 1995).
o Bioventing: Here, oxygen is drawn into the soil to increase the microbial
activities.
o Composting: The contaminated soil is mixed with a bulking agent, such as
aerated and static piles.
o Biofilters: This involves the use of stripping columns.
Mechanical: This usually involves the use of booms, skimmers and barriers.

2.4 BIOREMEDIATION
The first thing that comes to mind at this word bio and remediation, is
correcting a situation with living things. Thus, one could say bioremediation is
25

the use of microorganism metabolism to remove pollutants. It is a technology

that exploits the abilities of microorganisms and other natural habitat of the
biosphere to improve environmental quality for all species, including man
(Onwurah et al., 2007). It could also be defined as a process that utilizes
microorganisms or their enzymes to restore a contaminated environment to its
original state. It uses the natural ability of microorganisms to reduce the
concentration and toxicity of various chemical substances.
Bioremediation could also be said to be a process that involves the use of
microorganisms to degrade environmental contaminants. For bioremediation to
occur there has to be the contaminant, an electron acceptor and a
microorganism that has the ability to biodegrade the contaminant. Therefore it
is affected by the optimal conditions and limit of tolerance of the
microorganism involved, factors like pH, moisture content, temperature,
oxygen and nutrient availability (Atlas, 1991). Its effectiveness is determined
by the identification of the rate limiting factors and optimizing them. It is
applied in the cleanup of soil, ground water, lagoons, process waste streams and
sludges (Boopathy and Manning, 1999). Bioremediation can be done via natural
attenuation (intrinsic bioremediation), bioaugumentation or via biostimulation.
The path of the hydrocarbon biodegradation starts with the oxidation (by
molecular oxygen) of the substrate by oxygenases. Alkanes are converted to
carboxylic acids, which are biodegraded via the central metabolic pathway for
the utilization of fatty acids from lipids, which yields acetate which enter the
26

tricarboxylic acid cycle (-oxidation). Diols and catechols are formed from the
hydroxylation and cleavage of the aromatic rings present, these are
subsequently degraded to intermediates of the tricarboxylic acid cycle. Fungi
and bacteria form intermediates with differing stereochemistries. Fungi form
trans-diols, whereas bacteria almost always form cis-diols. Unlike fungi,
bacteria does not produce any carcinogen, and its more available naturally, thus
it is more often used. The complete biodegradation of hydrocarbons produces
the non-toxic end products carbon dioxide and water, as well as cell biomass
which can be safely assimilated into the food web (Atlas, 1991).

2.4.1 Advantages of bioremediation over other methods.

Bioremediation utilizes natural process of degradation for clean up and
completely destroys the hazardous compounds. It does not require much fund,
labour and energy as other methods, hence, it is cost effective for large areas.
It is common and hydrocarbons are broken down in a relatively short time
(Erdogan and Karaca, 2011). There is minimum formation of secondary waste
or by-products, leading to virtually no impact on the environment (Maletic S. et
al., 2013), being that the process changes the harmful chemicals into small
amounts of water and gas, carbon (IV) oxide.
Also, bioremediation has the advantage of being carried out along with other
physical and chemical methods of remediations. It can be used to treat several
27

organic compounds ranging from VOCs, simple PAH, BTEX compounds,

phenolic to nitroaromatic compounds (EPA, 2005)
It is safer, aids computation of material balance and process controllability.
2.4.2 Limitations of Bioremediation.
The use of bioremediation is not suitable for treating soils with high level of
heavy metals, chlorinated hydrocarbons and other chlorinated compounds,
complex PAHs, and radioactive elements, being that they are not susceptible to
biodegradation.
Also the fact that it is site specific, i.e. the procedure, stimulant or nutrient has
to fit the conditions of the site in which it is intended to be done (Boopathy,
2000). Bioremediation takes a long period of time for very effective results to
be obtained. Furthermore, it is highly susceptible to climatic and hydrological
conditions (Maletic S. et al., 2013).
There is also the problem of releasing toxic substances into the environment;
especially

when

fungi

which

produce

carcinogens

on

biodegrading

hydrocarbons is involved (Atlas, 1995).

2.4.3 Factors Affecting Bioremediation.
There are several factors; scientific, human and economic that affects the
process of bioremediation. Some of these are shown in the table 6. Other factors
affecting bioremediation are complex environmental laws and the slow pace in
enacting these rules by the Government, lack of trained personnel since it is

28

quite new, also the fact that it does not produce any physical or immediate
product discourages people from investing, especially as a new venture.

Table 2.5: Major Factors Affecting Bioremediation.

Microbial
Growth until critical biomass is reached
Mutation and horizontal gene transfer
Enzyme induction
Enrichment of the capable microbial populations
Production of toxic metabolites
Environmental
Depletion of preferential substrates
Lack of nutrients
Inhibitory environmental conditions
Physico-chemical bioavailability of pollutants
Equilibrium sorption
Irreversible sorption
Incorporation into humic matters
Substrate
Too low concentration of contaminants
Chemical structure of contaminants
Toxicity of contaminants
Solubility of contaminants
Growth substrate versus co-metabolism
Type of contaminants
Concentration
Alternate carbon source present
Microbial interaction (competition, succession, and predation)
Biological aerobic versus anaerobic process
29

Oxidation/reduction potential
Availability of electron acceptors
Microbial population present in the site
Mass transfer limitations
Oxygen diffusion and solubility
Diffusion of nutrients
Solubility/miscibility in/with water
Source: Boopathy, (2000).

2.5 BIOREMEDIATION OF CRUDE OIL CONTAMINATED SOIL.

As stated in the previous section, bioremediation can be used for the cleanup of
crude oil contaminated soil. Crude oil is natural product, thus it is susceptible to
degradation by naturally occurring microflora (Okeimen and Okeimen, 2005),
and like it is generally known, the performance of plants in soils not suitable for
their growth is enhanced when the soil is augmented with manure.
This clean up of the contaminated soil can be done in two methods, namely insitu and ex-situ bioremediation.
The in-situ bioremediation process has the advantage of being carried out
without disturbing normal site operations, since there is no need for excavation.
The treatment of the contaminated soil is done at the site of contamination.
Ex-situ bioremediation involves the physical removal of the contaminated
material into large contained spaces where they are treated (Boopathy, 2000). In
engineered biopile systems especially, it has the advantage of easier
controllabity, safe operations and facilitated material balance, being that the
30

cabon(iv)oxide generation rates and humidification rates are easily measured

(Benyahia F. et al., 2005).
2.5.1 Types of bioremediation of crude oil contaminated soil
There are two types of the bioremediation of crude oil contaminated soil; the
microbial remediation and phytoremediation (Tang et al., 2010).
2.5.1.1 Phytoremediation
This utilizes plant to degrade, stabilize and remove soil contaminants. These
plants decontaminate polluted soil through three mechanisms
direct uptake of petroleum hydrocarbons into their tissues
Release

of

enzymes

and

exudates

that

stimulate

the

activity

of

hydrocarbonoclastic microbes.
direct biochemical transformation (enzymes) of petroleum hydrocarbons
(Schnoor et al., 1995).
Green plants have effective filtering system endowed with measurable loading
degradative and fouling capacities. Examples of plants capable of
pytoremediating are salt marsh plants, black poplar, willows, miscanthus grass
(elephant grass). In the marsh environment Spartina patens, Sagittaria
lancifolia, Spartina alterniflora and Juncus roemeriannus are considered
ecologically and economically important in phytoremediation (Gerhadardta et
al., 2009). Phytoremediation can accelerate the reduction of oil concentration
in both surface and deep soil, and thus restore crop sustaining potential and

31

reducing marsh erosion after a spill. This method of phytoremediation is most

commonly used when the contaminants contain heavy metals such as cadmium,
or lead; being that they are not easily degraded by microorganisms. The upper
parts of the plants used bioaccumulate the contaminants, which are then
removed after harvesting (Meagher, 2000).
It is a common practice to combine both approaches aforementioned in treating
contaminated soil. This technique is referred to rhizoremediation; it involves
both plants and their associated rhizosphere microbe, or it can be actuated using
specific microbes (Gerhadardta et al., 2009).

32

2.5.1.2 Microbial remediation

It involves correcting the soil condition via the activities of microorganisms.
Biostimulation or bioaugumentation, or intrinsic approaches could be used in
this regard.
Bioaugmentation: It involves the addition of adapted microbial hydrocarbon
degraders. These new microbes help improve the ability of the naturally
occurring microbes to degrade hydrocarbons. It has the advantage of greater
respiration rate, thus shorter bioremediation period and most times better result
(Benyahia F. et al., 2005). Even after these microbes have been added to the
medium, there is the need to continue to cater for their nutritional need, thus the
correct quantity of fertilizer and water has to be applied.
Intrinsic bioremediation (natural attenuation): This is an unassisted method.
Bacteria are allowed to grow in the contaminated soil without interference of
any type. Only regular monitoring is done (Boopathy, 2000).
Biostimulation: This is the most widely used method; it involves the addition
of several nutrients, capable of making up for the lost inorganic nutrients
caused by the contamination with hydrocarbons. Organic, inorganic fertilizers,
such as N-P-K or oleophilic fertilizers could be used for this purpose of
stimulating indigenous microbial population in contaminated soils. These
nutrients help increase the bioavailability in the medium. It can also be
achieved by addition and aeration. It is the most commonly used method for insitu bioremediation (Baba M.T. et al., 1998).
33

o N-P-K 15.15.15.: This is an inorganic fertilizer that contains in equal

proportion, nitrogen, phosphorus and potassium.
o Poultry manure: The nutrient composition of poultry manure varies depending
on the type of bird, feed, litter and proportion of litter to droppings. This
manure is usually a mixture of the poultry droppings, beddings or litters (they
are usually wood chips, wheat straw, sawdust, rice and peanut hulls, paper, etc),
feathers and wasted feed.
Poultry manure is known to have very low moisture content but contains a high
proportion of micro and macro nutrients; especially Nitrogen, Phosphorus and
Potassium. The low moisture content is due to the fact that moisture from the
droppings is absorbed by the beddings. It is the most commonly used manure,
due to its availability. Apart from providing nutrients for crop production,
poultry litter applications build soil organic reserves. The organic matter
benefits crop production by increasing soil water-holding capacity, water
infiltration rates, cation exchange capacity, structural stability, and soil tilt.

34

Table 2.6: Elements Contained Poultry Manure

Element

Mean value (g/kg material)

Water

657

Total C

289

Total N

46

NH4-N

14

NO3-N

0.4

P
K

21
21

Cl

24.5

Ca

39

Mg

Na

4.2

Cu

0.304

Fe

0.320

Mn

0.053

Zn

0.354

As

0.029

Source: Edwards and Daniel, (1992).

Certain microorganisms can decompose the hydrocarbons present in crude oil
and its products, with each organism decomposing only a type of chemical
(Jelena et al., 2009).
Some of the micro organisms known to use petroleum and its products for
metabolic activities are bacteria (Escherichia coli, Pseudomonas sp.,
Clostridium, yeasts Candida) and molds (Aspergillus niger, Penicillium)
(Adekunle and Adebambo, 2007; Obahiagbon and Owabor, 2008).

35

The efficiency of the bioremediation process of crude oil contaminated soil is

dependent on the number of microorganisms capable of degrading the
hydrocarbons in the crude oil, present in the soil. The growth of these
organisms in turn depends on pH, temperature, moisture content, oxygen,
nitrogen and phosphorus availability. Also, the soil type in which the process
occurs influences the biodegradation rate .Temperature also affects the rate of
biodegradation as well as the chemical and physical composition of the
hydrocarbons.
The rate of degradation of hydrocarbons are however site dependent and limited
by the metabolic capabilities of the hydrocarbon degrading population, as well
as other environmental factors (Baba M.T., 1998). Apart from biodegradation,
there are several other physical and chemical processes, such as dilution,
sorption, volatilization and abiotic transformations are also involved.
Top soil bacteria, identified as Pseudomonas sp. and Bacillus sp., were used to
investigate the efficacy of bioaugumentation of crude oil contaminated soil.
Poultry manure was also used during the same research to study the efficiency
of biostimulation as well. Soybean was planted, for the influences on vegetative
and reproductive parameters to be monitored. The experiment was carried out
under natural conditions of rainfall and sunshine. At the end of the three
months, it was found that both bacteria and the poultry manure were capable of
remediating the soil, although bioaugumentation proved to be a better and faster
method (Nwandinigwe and Onyeidu, 2012).
36

Okeimen and Okeimen (2005) reported that laboratory bioremediation

experiments carried out at 290C on crude oil polluted soil by applying varying
amounts of natural rubber sludge and poultry manure. The process was
monitored by using the spectrophotochemical method to measure at weekly
intervals, the Total Hydrocarbon Content content of the soil samples. It was
seen that there was about a 100% decrease in TPH content of the treated soil
when compared to the untreated one.
Biodegradation rate of crude oil contaminated soil using fertilizer or cow dung
was analysed by Obahiagbon and Audu, (2002). The experiment lasted 8 weeks,
after which, results showed that the sample amended with moderate amount of
fertilizer did best. Bioremediation of a crude oil contaminated soil via
augmentation with cow dung was carried out with the growth and performance
of soybean used to monitor the study (Kelechi L. et al., 2008). Constant amount
of loamy soil was used, with varying amounts of crude oil, obtained from
SPDCs Health Safety and Environment laboratory, Port Harcourt. The
experiment lasted for a period of 15 weeks, with samples collected every
3weeks for analysis. With time, it was seen that the performance of the crops
improved, the chlorophyll content, the pod production and leaf area, suggesting
that toxicity of crude oil to the crop reduced.
A research carried out by Jelena, et al., (2009) on the bioremediation of soil
heavily contaminated with crude oil and its products showed that after
biostimulating and bioventilating for about 5 months, gram- positive bacteria,
37

from actinomycete- Nocardia group, which was most dominant in the soil was
able to remediate the soil; being that the total petroleum hydrocarbons reduced
by 89% (from 29.8 to 3.29g/kg).
Ebere et al., (2011) carried out a study using N.P.K., saw dust and poultry
manure over a 42 day period to remediate crude oil polluted soil. Parameters
such as potassium concentration, organic carbon content, pH, and Total
Hydrocarbon Content was used to monitor the degradation of the total
petroleum hydrocarbon. At the end, samples treated with saw dust did best.
The efficiency of pseudomonas fluorescens bioaugmented to stimulate in-situ
bioremediation of crude oil contaminated soil with different amendments in
treatment units. The study was for 35 days, at the end of which, it was found
that the addition of wheat bran as bulking agent compared to other treatment
units enhanced the bioremediation of the contaminated soil (Barathi and
Vasudevan, 2003).
Del Arco and Franca (1999) carried out a research where soil sediment
contaminated with Arabian crude oil was bioremediated. It was found that the
bacteria which got adapted to pH, carbon and other nutrient sources, during the
land farming process produced a better bioremediation performance than the
indigenous soil bacteria.
The possibility of treating diesel polluted soil in an alpine glacier area by
bioremediation was studied by Margesin and Schinner (2001). The intrinsic
bioremediation as well as the biostimulation were examined by comparing an
38

unfertilized soil with a fertilized one. The study was carried out for three years,
the pH, total petroleum hydrocarbons, soil respiration, catalase and lipase
activities were monitored. The catalase activity was determined by noting the
amount of carbon dioxide, CO2 that evolved from a hydrogen peroxide,
phosphate soil suspension. TPH was measured with the German method, pH
was measured with a glass electrode. It was seen that there was a decrease in
the hydrocarbon content in the aforementioned by 50 and 70 percents
respectively. Also microbial activities like respiration, population also
increased. Over time, biostimulation no longer had effect on the naturally
existing soil microorganisms. The rate of biodegradation was very slow due to
the area (alpine glacier), but this went to show that bioremediation of
hydrocarbon polluted soil is quite versatile been that after at the long run, the
desired result was achieved.
Poultry and cow dung manures were used to bioremediate a contaminated soil
in which maize was used as test crop. It was found at the end of the 104 days
experiment that crude oil impaired the growth of the plant especially at
increased level of pollution, also that poultry manure was a better choice, since
it repaired the contaminated soil better.
A research was carried out by Tanee and Albert, (2011) at the University of Port
Harcourt, Rivers State. 5Kg of loamy top soil was polluted with 200ml of crude
oil and left for a week. Post pollution treatment was then carried out by the
addition of sawdust to each sample. The effect on plant growth was monitored
39

by planting two varieties of cassava on each. Plant growth parameters, shoot

height, fresh and dry weight yield (above and below ground levels) every four
weeks; soil parameters, pH (with pH and conductivity meters respectively),
TOC, THC (with a DR/300 spectrophotometer at 430nm) were measured every
8 weeks as well. At the end with the results obtained, it could be concluded that
crude oil polluted soil could be biostimulated with saw dust, being that the soil
nutrient as well as the performance of the cassava improved, also, the THC
reduced.
2.5.2 Methods for bioremediation assessment.
Several molecular, classical microbiological methods, as well as fatty acid
profiling can be used to monitor the individual microorganisms metabolic
activities. Some of these include:
Total Microbial Count (TMC): It is carried out before and after the
bioremediation process; this is to provide information on the biological
activities and how the microbial population has acclimated to the site condition.
Balba et al., (1998) reported a correlation between microbial count and
hydrocarbon degradation, it was found that TMC in the form of total colony
forming units (TCFU) increased by four orders of magnitude.
Soil respirometric test: This test could be used for testing the effect of nutrient
supplementation, microbial inoculation, confirming active hydrocarbon
degradation during full scale bioremediation. A simple respirometric flask could

40

be used for the respiration tests, by monitoring the oxygen and carbon dioxide
evolution rate (Baba et al., 1998).
Dehydrogenase activity: The soil microorganisms, which reflect its
biochemical activities, are most times determined by the several enzymic
systems which are involved in the dehydrogenase activity. The dehydrogenase
assay could be used to study the inhibitory effects of soil microbial activities
contaminants (Bartha and Pramer, 1965). These contaminants include toluene
and chloroform at very high concentrations (Page et al., 1982).
Microbial Survival Test And Tracking Of Genetically Engineered
Microorganisms (GEM): In order to increase the degradation of oil in the soil,
especially when high molecular weight polyaromatics are present; GEMs are
introduced. These microorganisms have certain abilities that the naturally
occurring ones do not possess, thus making them able to degrade several PAH
and their derivatives. For GEMs to be used successfully, techniques to ensure
their survival as well as tracking them, inorder to be able to study the progress
of the bioremediation process has to be put in place (Veal and Stokes, 1992).
Toxicity and ecological impact assessment: it is also important that apart from
measuring the effectiveness of the process, one also measures and ensure that
there is no undesired toxic release into the environment. Leachate migration out
of the zone of the experiment should be prevented by all means possible (Ellis
et al., 1990). Nitrate fertilizers should be avoided, especially that of sodium,
being that it not only contaminates ground water, nitrates also cause blue baby
syndrome in infants.
41

 Total Hydrocarbon Content: These are soil contaminants which may not be
generally regarded as hazardous. THC can be determined via gas
chromatography (GC), or with a spectrophotochemical method, also
gravimetric analysis and immunoassay could be used. The GC method of
measuring THC is highly preferred over the other methods because of its
sensitivity, selectivity, THC identification and it detects a wide range of
hydrocarbons. THC can be used in determining if there is a problem, assessing
the severity of contamination and following the progress of a remediation
effort. If THC data indicate that there may be significant contamination of
environmental media, other data can be collected so that harm to human health
can be quantitatively assessed (Wade, 1988 ). On bioremediating, the THC of
the polluted soil reduces.
Soil pH: this is the degree of acidity or alkalinity of the soil. It is also known as
soil reaction. The pH scale on which its measurement is based, ranges from 0 to
14. pH 7 is the neutral point while below 7 (<7) is acidic and above it (>7) is
alkalinic or basic. The optimum range of the soil for most plants is 5.5-7. It
affects the soil structure, availability of nutrients, presence of toxic element,
leaching, etc (Ronen, 2007).

CHAPTER THREE
MATERIALS AND METHODS
3.1 SAMPLE COLLECTION

42

The soil sample (loamy sandy) used for this study was obtained from several
spots in the Faculty of Engineering, University of Benin. The soil used was
devoid of any hydrocarbon pollution. Crude oil of API gravity 46.6 0, specific
gravity 0.7945 and water cut 1.9 was obtained from the Ologbo oil field in
oredo Local Government Area, Edo State.
The N.P.K.15:15:15 fertilizer used was purchased from the Agricultural
Development Project (ADP), Oko, Edo State. The poultry manure used for
biostimulation was obtained from a poultry farm in Ugbowo.

3.2 CHEMICALS AND REAGENTS

Table 3.1: Table Showing Reagents and their Uses
Reagent
Water
Phosphate

Uses
For making soil solution, when measuring its pH.
It is used to standardise the pH meter to 7.

solution
Forcados blend

It is the crude oil of known concentration used in preparing

crude oil
n-hexane
Methylene blue

the standard Total Hydrocarbon Content THC plot.

It is used as an extractant when determining THC.
It is used as a staining agent when carrying out

solution

enumeration of microbes.

3.3 APPARATUS/EQUIPMENT
Table 3.2: Apparatus, Equipment and the Uses
APPARATUS/EQUIPMENT
Stirrer
250ml measuring cylinder

USES
For stirring the soil solution
It is used to measure the volumes of
43

100ml beaker

liquids as required,
For holding sample solutions when

10ml pipette

measuring pH.
Used for measuring the required

100ml polyethylene bottle

volume of crude oil.

Used to hold soil solution in the
mechanical shaker when computing
absorbances for the standard Total

Dropper

Hydrocarbon Content (THC) plot.

For taking the required quantity of

100ml conical flask

100ml volumetric flask
weighing balance
Mechanical shaker

methylene blue.
For holding sample solutions
For measuring the volumes of liquid.
To measure the weight of soil samples
To agitate the soil and n-hexane

Cuvette

mixture during THC determination.

For holding samples in the

pH meter

spectrophotometer.
It is used for measuring the pH of soil

Spectrophotometer

solution.
It is used to determine the Total
Hydrocarbon Content of the soil.

3.4 SAMPLE PREPARATION

The soil sample was air dried for about two days, while the poultry droppings
were properly sun dried for 5days, ground and sieved, in other to increase
uniformity of particle size and surface area, hence facilitating contact. Four (4)
kilograms of the air dried soil was weighed into the four compartments earlier
44

prepared. 1liter of crude oil was added to achieve 20% pollution. 0.4liter of
water was also added to each compartment. This mixture was properly stirred.
For proper acclimatization of the soil indigenous microorganisms to the new
environment, the contaminated soil left to stand for a week.

45

Figure 3.1: Compartments where Bioremediation Study was carried out

Figure 3.2: Compartments where Bioremediation Study was carried out

3.5 SOIL TREATMENT

After the one week of acclimatization, 800g of poultry manure particulate and
320g fertilizer was added to the mixture. Water was added from time to time as
deemed necessary, while the polluted soil being remediated was stirred properly
at intervals of 2 days for the 8 weeks of the experiment. Samples were collected
every week and analysed to monitor the bioremediation process.

3.6 BIOREMEDIATION ANALYSIS

46

The bioremediation process was monitored by measuring the pH, Total

Hydrocarbon Content and Total Microbial Count. These parameters were
obtained for each week for all four samples.
3.6.1 Soil pH.
20ml of distilled water was added to 20g of each polluted soil in a 100ml
beaker. The mixture was properly stirred and allowed to stand for 30 minutes.
The pH of the mixture was then obtained with a pH meter, which had already
been standardised to pH 7.0 by dipping in a phosphate solution of 2g of
phosphate powdered buffer dissolved in 200ml of distilled water.
3.6.2 Total Hydrocarbon Content (THC)
5g of contaminated soil was weighed into a 100ml polyethylene bottle. 25ml of
nhexane was added, shaken properly, covered and left to stand for 10 minutes.
The mixture was filtered and the absorbance of filtrate was read with a HACH
DR/2000 spectrophotometer at a wavelength of 460nm. The Total Hydrocarbon
Content (concentration) was extrapolated from a standard plot (Stanton, 1996).
The standard plot of absorbance (nm) against concentration (mg/l) utilized was
prepared by measuring the absorbances of crude oil of known concentration
(forcados blend), with n-hexane as extractant.
A pipette was used to measure 1.18 ml of crude oil, n-hexane was added to
make up to 1liter. From this mixture, 0, 10, 20, 40, 60, 80 and 100ppm working
standards by serial dilution was prepared. The absorbances of these solutions of

47

known concentration were obtained with the spectrophotometer at wavelength

of 460nm.

16

f(x) = 0.17x

14
12
10
Absorbance (nm)

8
6
4
2
0
0

20

40

60

80

100

120

Concentration(mg/l)

Fig 3.3: Standard THC graph

3.7.3 Total Microbial Count

The soil water extract was obtained by washing the sample with distilled water.
Dilutions of 10-1, 10-3, 10-6 was prepared with already made diluents. The
colony counting chamber was assembled by applying the cover glass. Few
drops of methylene blue solution were added to water and dilution samples. An
area of the counting chamber was ruled. A loop-full of water and the different
dilutions was then placed on the earlier ruled area. The chamber was allowed to
rest for 5minutes; after which the bacteria were enumerated randomly in 50-100
48

squares, so that the total number of bacteria obtained was 500 with a 4mm lens
microscope. A triplicate count was obtained for each sample. The number of
count was divided by the number of squares and the result was multiplied by
the dilution factor and a constant K. This yielded the number of organisms in a
millilitre of the given water sample (Mills et al., 1978).

49

CHAPTER FOUR
RESULTS AND DISCUSSION
4.1 pH
Table 4.1: pH Values over Time for the Bioremediation of Crude Oil
Contaminated Soil with Particulates of Different Ages.
SAMP
LE
A

AGE OF
SAMPLE(DA
YS)
3

28

42

126

TIME (WEEKS)
2 3 4 5 6

5.
9
6.
2
6.
3
6.
1

5.
7
6.
3
6.
1
6.
3

5.
8
6.
1
5.
9
6.
2

50

5.
9
5.
9
6.
1
6.
4

6.
2
6.
0
6.
2
6.
5

6.
3
6.
1
6.
4
6.
7

6.
5
6.
2
6.
4
6.
6

6.
5
6.
4
6.
5
6.
7

6.
6
6.
5
6.
7
6.
9

Figure 4.1: variation of soil pH with Bioremediation Time and Particulate

age.
It was observed from figure 4.1, a plot of pH as a function of bioremediation
time and age of particulates that pH progressively increased with time. The
same trend was observed with respect to the age of particles. However, at the
start of bioremediation, it could be observed that there was a slight fluctuation
in pH with particulate age. On pollution, i.e. week 0; all samples were slightly
acidic (5.9-6.3). Over time, an averagely increasing trend was noticed. This
suggests that the amendments tended to decrease the acidity of the soil samples.
This decrement could have resulted from the biodegradation of crude oil by
bacteria under anaerobic conditions in the soil pores (Essien and John, 2010); as
51

well as the ability of crude oil to act as a buffer for the polluted soil (Ellis et al.,
1961).This trend was also noticed by Ebere et al., (2011), where pH increased
averagely from 5.21 to10.1for crude oil polluted soil remediated with NPK, saw
dust and poultry manure. Essien and John ( 2010), reported similar results when
loamy soil polluted with crude oil was reclaimed with chemical degreasers and
detergents, the pH increased from 4.5 to 5.8.
Eneje et.al., (2012) also reported a decrease in pH acidity when the effect of
poultry manure and Calapoigonium mucunoides on crude oil contaminated soil
was studied in a completely random design. Samples were collected at different
depths, polluted by 30mg/l and water 50% of retention capacity was added.
Amendments were added, kept in a green house and allowed to stand for three
weeks. The pH of samples amended with poultry increased from 4.85 to 6.83
In this work, samples A and B had same pH in week 3, after which a
progressively increasing trend is observed for both, with A having a higher pH
at the end of the experiment. Sample C (containing poultry manure aged 42
days) had a higher pH value than samples A and B at the end of the process; its
pH initially decreased from 6.3 in week 0 to 6.1 in week 1 after amendments
were added. From thence, C showed a gradual increase in pH throughout the
experiment. Similarly, sample D, which contained the oldest poultry manure
particles of 126days had the highest pH value throughout the experiment,
except for the first week, despite fluctuations in the trend within weeks. The
decrease in pH of sample B as compared to the increasing trend of pH with age
52

of particles at the end of the remediation period could be attributed to climatic

conditions. Salt concentration is said to increase as the soil dries, thus under
such conditions, soluble cations are replaced by hydronium (H 30+) ions (USDA,
2004), hence sample B which received more heat from the sun as compared to
other samples returned the least increase in pH. The pH throughout the 8weeks
of bioremediation for all four samples was within the range optimum for plant
growth (5.5-7) (Ronen, 2007), as well as 6-9 as required by FEPA. This
indicates an effective bioremediation process.

4.2 TOTAL HYDROCARBON CONTENT

Table 4.2: Total Hydrocarbon Content of Crude Oil Contaminated Soil
Bioremediated with Poultry Manure of Different Ages
SAM
PLE
A

AGE OF
TIME (WEEKS)
1
2
3
4
5
6 7
SAMPLE( 0
DAYS)
3
84. 63. 48. 34. 25. 17. 10. 4.
10 90 90 80 30 40 20 3
0
28
82. 65. 49. 36. 26. 18. 11. 5.
90 80 20 80 70 90 10 4
0
42
84. 63. 48. 33. 25. 18. 11. 5.
10 50 20 90 70 30 40 2
0
126
83. 61. 47. 34. 26. 19. 12. 6.
53

8
0.
7
0
1.
6
0
1.
1
0
1.

40

80

30

20

40

90

10

6
0

9
0

Figure 4.2: Variation of Total Hydrocarbon Content with Time And

Age of Particles during the Bioremediation Process.
The variation of the Total Hydrocarbon Content (THC) with age of manure
particulate and bioremediation time is presented in figure 4.2. It was observed
generally that THC decreased with time of bioremediation, but increased with
age of particulates. During the early period of remediation, precisely weeks 1-3;
samples C and D retuned better results, being that their THC values were
54

slightly lower than those of A and B for that period. However from the
following week, (4), there was a change as samples A and B returned THC of
lower values than C and D. No particular order for the decrease in Total
Hydrocarbon Content for each individual week with respect to the age of the
particles could be initially inferred as there was no noticeable trend till the last
two weeks of the experiment.
Table 4.3: Percentage Reduction in Total Hydrocarbon Content (THC) of
Soil Bioremediated with Poultry Manure Particulates of Different Ages.
Sample
A
B
C
D

THC reduction (%)

99.17
98.07
98.69
97.72

Table 4.3 shows a summary of the result presented in table 8. It shows the
percentage decrease in THC of the four samples. From the table it was deduced
that the younger the particulates the better as sample A (3 days) had the highest
percentage reduction and D (126 days) had the least percentage reduction value,
since the major aim of the bioremediation process is to reduce the hydrocarbon
content to levels recognised as safe by regulatory bodies(Obahiagbon et.al.,
2009). This conforms with the results obtained by Tanee and Albert, (2011);
showing a decrease in Total Hydrocarbon Content in a crude oil polluted soil
amended with saw dust,(similar to sample A) by 75%.
Ayotamuno et.al.,(2013) in a study where petroleum contaminated soil was
remediated with plantain stem and poultry feaces, observed a decrease in Total
55

Hydrocarbon Content as well. 400g of soil was polluted with 0.25litres of crude
oil, treatments of different quantities were added. The samples containing the
poultry dung performed best with the total petroleum hydrocarbon reducing
from 17, 284 mg/kg to 1,186 mg/kg at the end of the two months.
A similar decrease in total petroleum hydrocarbon on biostimulating crude oil
polluted soil in the university of port-harcout by Chikere et. al., (2009) was
observed. TPH decreased from 3666mg/kg on pollution to 135.01mg/kg(with
poultry manure) and 89.68mg/kg(with N.P.K fertilizer) after treatment for 157
days.
The general reduction in the Total Hydrocarbon Content could be attributed to
the ability of the particulates and fertilizer to foster the growth of micro
organisms (biostimulation), which in turn feed on the hydrocarbons. The lower
aged particles returned a better result in conformity to the growth curve of
micro organisms, which after the growth and stationary period, the death phase
sets in. Hence, while the lower aged particles will be harbouring organisms that
are still in their growth phase, the older ones will have organisms in their
stationary or death phase, leading to lower degradation rate as competition, age,
lack of nutrient would have led to the decline in microbial population. This also
explains why samples C and D initially did better. In thiswork, sample C
returned a slightly lower THC value than that of sample B, this was a deviation
from the trend. This deviation could be due to the fact that the sample B
compartment received more heat from the sun, this could have led to a decrease
56

in moisture content which invariably reduces the number of microorganisms in

that compartment. The heat also makes it difficult for the mesophillic bacteria,
(which is mainly responsible for the degradation of petroleum hydrocarbon) to
perform properly, limiting its habitat to only the cooler part of compartment C;
this is as suggested by Nester et al., (2001) and Perfumo et al., (2007).
It was also observed that the highest decrease in Total Hydrocarbon Content
occurred between week 0 and week 1; this was at the point at which the
amendments were initially added, after acclimatization had occurred, this was
probably because the micro organisms just entered their exponential growth
phase. The addition of NPK fertilizer also enhanced the hydrocarbon
degradation (Palmroth et al., 2006). This is supported from the above that
moderate fertilizer must be used for better THC degradation.
The decrease in THC from the time the amendments were added to the end of
the 8weeks bioremediation process was very high from a mean value of 83.6 to
1.3mg/kg (98% decrease). This was below the crictical level of 10 mg/l (FEPA)
or 50mg/kg (Department of Petroleum Resources, 2002).

4.3 Total Microbial Count (cfu/g) 106

Table 4.4: Total Microbial Count of Crude Oil Contaminated Soil
Bioremediated with Poultry Manure of Different Ages.
SAMPL
E
A

AGE OF
SAMPLE(DAYS)
3

0
1.
5

1
1.
8
57

TIME (WEEKS)
2
3
4
5
6
1. 2. 2. 2. 2.
9
2
5 4
6

7
2.
8

8
3.0

28

42

126

1.
6
1.
6
1.
8

1.
8
1.
9
1.
9

1.
8
1.
9
1.
8

2.
0
2.
1
1.
9

2.
1
2.
2
2.
0

2.
1
2.
3
2.
2

2.
2
2.
3
2.
2

2.
2
2.
4
2.
1

2.5
2.6
2.2

Figure 4.3: Variation of Total Microbial Count with Time and

particulate age during the bioremediation process.
Figure 4.3 is a plot of total micobial count as a function of time of
bioremediation and age of particulate. A general increase in microbial
population as well as a decrease with the age of particles was observed over the
period of bioremediation.

58

This increasing trend of colony forming units is in agreement with that

observed by Chikere et. al.(2009). In the study, the colony forming units of the
heterotrophic bacteria of the crude oil polluted soil increased from 1.20 X 10 6
cfug-1 to on amendment with 19.57cfug-1 poultry manure and 450cfug-1 on
amendment with N.P.K.; while that of the hydrocarbon utilizing bacteria
increased from 2.51 X 102cfug-1 to 0.257cfug-1 on treating with poultry manure
and 38cfug-1 with N.P.K fertilizer.
Nwandinigwe and Onyeidu, (2012), observed same trend when crude oil
contaminated soil was bioaugumented with pseudomonas and bacillus; and also
treated with poultry manure. Its effect monitored by the growth of soya bean. At
the end of the experiment on the soil there was a remediative effect.
The result of this present study revealed that the 3days old particle amended
series, from week 3 exhibited a continuous lead in the population of microbes,
samples B, C then D followed suit in the order (i.e. A having the highest and D
having the least each week), with D having 2.210 6 cfu/g and A having
3.0106 cfu/g in the last week; thus, implying that fresher particles provide
better breeding ground for microorganisms. As previously mentioned, this is in
line with the growth curve of microorganisms.
The increase in microbial population was via biostimulation, being that the
poultry waste and fertilizer provided nutrients for increased cell growth.
The growth in microbial population was also fostered by the crude oil as it
served as carbon and energy source as asserted by Akpoveta et al., (2011).
59

The range of pH observed in the study contributed immensely as well, to the

proper growth and reproduction of the microbes by providing the required
conditions for mineralization of hydrocarbons since most bacteria capable of
metabolizing hydrocarbons develop best at pH close to neutrality (Mckee and
Mendelssohn); and bacteria and fungi seems to be the microbes in this case.
Bacteria has high capacity to degrade petroleum hydrocarbon, Xu,
(2012),reported that it had a much higher hydrocarbon degradation rate when
compared to other microbes. Also nutrients such as nitrogen and phosphorus are
readily available in soil of pH range centered around 6.5 (Bickelhaupt, 2013).
The adequate supply of water to the soil aided the cell growth and function of
the microorganism, by controlling the diffusion of water and soluble nutrients
into and out of microorganism cells. However, excess moisture, such as in
saturated soil, reduces the amount of available oxygen for aerobic respiration,
hence making anaerobic respiration (which produces less energy for microbes
and slows the rate of biodegradation), the predominant process. Soil moisture
content between 45 and 85 percent of the water-holding capacity (field
capacity) of the soil or about 12 percent to 30 percent by weight is optimal for
petroleum hydrocarbon degradation (US EPA, 2006).
In all, the biodegradation rate was collectively enhanced by temperature,
moisture, oxygen level, nutrients pH, presence of microorganisms, etc. The
above listed parameters could negatively affect the rate of degradation as well if
not properly managed (Baba et al., 1998).
60

CHAPTER FIVE
CONCLUSION AND RECOMMENDATIONS
5.1 CONCLUSION

61

Inorganic fertilizers and poultry manure particles of different ages can

significantly reduce the level of hydrocarbons in the soils to tolerable limits as
observed in this study. The parameters with which the process was monitored
were pH, Total Hydrocarbon Content (THC) and Total Microbial Count (TMC).
At the end of the eight weeks bioremediation period, a biodegradation rate of
10.45 mg/kgwk,10.16 mg/kgwk,10.38 mg/kgwk and 10.19mg/kgwk

was

computed for samples A,B,C and D respectively. It is therefore safe to conclude

that lower aged poultry particles perform better than the older ones, in that they
yielded a higher biodegradation rate.

5.2 RECOMMENDATIONS
There is prospect in utilizing the biological waste obtained from poultry birds in
time, industrially, to ameliorate the menace left from oil spillage occurrences on
land by contravening bodies. This method is environmentally friendly as it
degrades the hydrocarbon present in the soil to less toxic substances.
Indigenous farmers can easily procure and utilize early aged particles in
rectifying the condition of the soil if contaminated with crude oil.

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