Impact of the Age OF particulates on the Bioremediation of Hydrocarbon Poluted Soil

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.

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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.

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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

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DEDICATION This project work is dedicated to God Almighty.

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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.

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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.

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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

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LIST OF TABLES Table 2.1

Elementary Composition of Crude Oil

7

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

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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.

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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

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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.

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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).

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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.

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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

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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

B

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

B

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).

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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 (β-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

5

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 SPDC’s 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). D’el 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 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 n–hexane 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

B

28

C

42

D

126

0

1

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

7

8

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

B

C

D

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) Χ 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

B

28

C

42

D

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 2.2Χ10 6 cfu/g and A having 3.0Χ106 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.

REFERENCES Abdul, A.S., Gibson T.L., and Rai D.N. (1990): Selection of surfactants for the removal of petroleum products from shallow sandy aquifers Ground Water, 28: 920-926. 62

Adekunle, A.A. and Adebambo, O.A. (2007): Petroleum hydrocarbon utilization by fungi isolated from Detarium senegalense (J. F Gmelin) seeds. Journal of American Science, 3(1): 69-76. Agbogidi, O.M. (2011): Effects of Crude Oil Contaminated Soil on Biomass Accumulation in Jatropha Curcas L. Seedlings. Journal of Ornamental and Horticultural Plants 1 (1) 43-49. Adams, P.J. (1996): Bioremediation of oil spills: Theory and Practice. Proceedings of the 8th. Biennial International NNPC Seminar. In: The Petroleum Industry and the Nigeria Environment, Port Harcourt, Nigeria, 183-203. Akonye, L.A. and I.O. Onwudiwe, 2007. Effects of certain soil amendment agents on Lead (Pb) uptake by plants grown on oil polluted soil. Scientia Africana. 6 (1) 85-93. Akpoveta O., Egharevba F., Medjor O., Osaro K. and Enyemike E. (2011): Microbial Degradation and its Kinetics on Crude Oil Polluted Soil. Research Journal of Chemical Sciences, Vol. 1(6), 8-14. American Petroleum Institute Information Bulletin, (1958) No 11, Philadelphia Pa. Atlas, R. M. (1991): Microbial hydrocarbon degradation: bioremediation of oil spills. J. Chem. Technol. Biotechnol. 52: 149-156.

63

Ayotamuno M.J., Olatunji O.M., Ikari S.T.(2013): Petroleum Contaminated Soil Remediation Using Poultry Feaces and Plantain Stem. International Journal of Scientific & Engineering Research Volume 4, Issue3. Baird, J. (2010): Oil’s shame in Africa. Newsweek No. 27. Balba M.T., Al-Awadhi N., Al-Daher R. (1998): Bioremediation Of OilContaminated Soil: Microbiological Methods for Feasibility Assessment And Field Evaluation Journal Of Microbiological Methods 32: 155–164. Barathi, S. and Vasudevan N. (2003): Bioremediation of crude oil contaminated soil by bioaugmentation of Pseudomonas fluorescens NS1. J Environ Sci Health A Tox Hazard Subst Environ Eng. 38 (9) 1857-66. Bartha, R., Pramer, D. (1965): Features of a flask and method for measuring the persistence of and biological effects of pesticides in soil. Soil Sci. 100, 86–170. Baruah, D., Das N.J. (1994): Industrial pollution control, 10 (2) 139-44. Benyahia F., Abdulkarim M., Zekri A., Chaalal O. and Hassan H. (2005): Bioremediation of Crude Oil Contaminated Soils; A Black Art Or An Engineering Challenge? Trans IChemE Part B, Process Safety and Environmental Protection, 83 (B4) 364-70. Bickelhaupt, D. (2013): Soil pH: what it means. Environmental Information Series. Black, C.A. (1957): Soil Plant Relationship. John Wiley and Sons. Inc., New York. 64

Boopathy, R. (2000): Factors Limiting Bioremediation Technologies. Bioresource Technology 74, pp63-67 Boopathy, R. And Manning, J. (1999): Surfactant-Enhanced Bioremediation Of Soil Contaminated With 2,4,6-trinitrotoluene In Soil Slurry Reactors. Water Environ. Res. 71: 119-24. Brian, K. (1977): Soil Processes. 1st Edition. Allen George Unwin. London. Braun, Robert, L., Burnham, lan, K. (June 1993): Chemical Reaction Model for Oil and Gas Generation from Type I and Type II Kerogen. Lawrence Livermore National Laboratory.

Chikere, C.B., Okpokwasili, G.C. and Chikere B.O. (2009):Bacterial diversity in a tropical crude oil-polluted soil undergoing bioremediation African Journal of Biotechnology Vol. 8 (11), pp. 2535-2540. Clarkson, D.T., Hanson, J.B., Ann Rev (1980): Plant Physiol. 31: 239-98. D’el Arco j.p. and franca f.p. (1999): Biodegradation of crude oil in sandy sediment. International biodeterioration and biodegradation 44:87-92. Department Of Petroleum Resources, DPR (1991): Environmental Guidelines and Standards for the Petroleum Industry in Nigeria. Department of Petroleum Resources, Ministry of Petroleum and Mineral Resources, Lagos, Nigeria. Domask, W. G. (1984). Introduction to petroleum hydrocarbons. Chemistry and composition in relation to petroleum derived fuels and solvents. Effects of petroleum hydrocarbons. Adv. Nod. Environ. Tox. 8: 1-26.

65

Ebere, J.U., Wokoma, E.C. and Wokocha, C.C. (2011): Enhanced Remediation of a Hydrocarbon Polluted Soil. Research Journal of Environmental and Earth Sciences 3 (2) 70-74. Erdogan E.E. and Karaca A. (2011): Bioremediation of crude oil polluted soils. Asian Journal of Biotechnology, 3: 206-213. Edwards, D.R., and Daniel, T.C. (1992): Environmental impacts of on-farm poultry waste disposal—A review. Bioresource Technology 41:9–33. Ellis, R., Adams, R.S. (1961): Contamination of Soil by Petroleum Hydrocarbons. Advanced Agronomy, Vol. 13, 197 – 216.

Eneje R.C., Nwagbara C., and Uwumarongie-Ilori E.G. (2012): Amelioration of chemical properties of crude oil contaminated soil using Compost from Calapoigonium mucunoides and poultry manure. International Research Journal of Agricultural Science and Soil Science Vol. 2(6) pp. 246-251. Essien, O.E., and John I.A. (2010): Impact of Crude-Oil Spillage Pollution and Chemical Remediation on Agricultural Soil Properties and Crop Growth. Journal of Applied Science and Environmental Management, Volume 14 (4), 147-154. Federal Environmental Protection Agency (FEPA) (1997): Guidelines and Standards for Environmental Impact Assessment in Nigeria, pp. 87-95. Gary, J.H. and Handwerk, G.E. (2001): Petroleum Refining and Technology and Economics, fourth edition, Marcel Dekker, Inc. pages 21-35.

66

Gerhardta, K. E., Huang, X. D., Glicka, B. R., and Greenberg, B. M. (2009): Phytoremediation and rhizoremediation of organic soil contaminants: potential and challenges, Plant Sci., 176 (5) 20–30. Gruse, W.A and Stevens, D.R. (1960): Chemical Technology of Petroleum, 3rd Edition, McGraw-Hill Book Company, New York, pp. 424–472. http://www.opec.org/pressroom/FAQ/crudeoil Jelena, S.M., Vladimir, P.B., Mila, V.I., Samira A.M.A., Gordana Đ.G.C., and Miroslav M.V. (2009): Bioremediation of Soil Heavily Contaminated With Crude Oil and Its Products: Composition of the Microbial Consortium. Journal of Serbian Chemical Society 74 (4) 455-60. Khalladi, R., Benhabiles, O., Bentahara,F. and Moulai- Mostefa, N. (2009): Surfactant remediation of diesel fuel polluted soil. J. Hazardous Mater., 164: 1179-1184. Kelechi L.N., Modupe O.A. and Bola O.O. (2008): Growth and Performance of Glycine max L. (Merrill) Grown in Crude Oil Contaminated Soil Augmented With Cow Dung Nature and Science, 6(1). Kuwait Petroleum Energy Research Centre, (1999): Survey of Technology for Remediation of Oil-Contaminated Soil in Kuwait. Survey 8. Kvenvolden, Keith, A. (2006): Organic geochemistry – A retrospective of its first 70 years. Organic Geochemistry 37 (1).

67

Maletić S., Dalmacija B. and Rončević S. (2013): Petroleum Hydrocarbon Biodegradability in Soil – Implications for Bioremediation. Intech: Open science, open mind. Chapter 3, pp 43-62. Margesin, R. and Schinner, F. (2001): Bioremediation (Natural Attenuation and Biostimulation) of Diesel-Oil-Contaminated Soil in An Alpine Glacier Skiing Area. Applied and Environmental Microbiology, 67 (7) 3127-33. Mckee, K.G. and Mendelssohn, I.A., (1995): A review of the methods of ecological consequences of substrate aeration for the enhancement of oil bioremediation in wetland. Report to the Marine Spill Response Corporation. Meagher, RB (2000). "Phytoremediation of toxic elemental and organic pollutants". Current Opinion in Plant Biology 3 (2): 153–162. Mills A.I., Beuil C. And Cowell R.R.(1978): Enumeration Of Petroleum Degrading Marine And Estuarine Microorganisms By Most Probable Number Method, C and J. Microbiolx., 24, 552-557. Nester, M.T., Eugene, W., Denise, G.A., Roberts, C.E., Pearsall N.N., (2001): Microbiology: A Human Perspective. 3rd ed. New York: McGraw-Hill. Nwadinigwe, A.O., Onyeidu, E.G. (2012): Bioremediation of Crude OilPolluted Soil Using Bacteria and Poultry Manure Monitored through Soybean Productivity. Pol. J. Environ. Stud. 21 (1) 171-176. Nwilo, P.C., Badejo, O.T. (2001): Impacts of Oil Spills Along The Nigerian Coast. The Association for Environmental Health and Services.

68

Obahiagbon, K.O., Akhabue, C.E., Aluyor, E.O. (2009): Effect of varying concentration of sodium nitrate on biological oxidation of petroleum hydrocarbon polluted water. Journal of Engineering and Technology Research, 1(3): 50-55. Obahiagbon, K.O., and Audu T.O.K. (2002): Analysia of biodegradation rate of crude oil contaminated soil using fertilizer or cow dung.Nigerian Journal of Environmental Research and Development 1(1). Obahiagbon, K.O. and Owabor, C.N. (2008): Biotreatment of Crude Oil polluted water using mixed microbial populations of P. aureginosa, Penicillium notatum, E. coli and Aspergillus niger. Proceedings of the 2nd International Conference on Engineering Research and Development: Innovations, Benin City, Nigeria. Odu, E.A. (1987): Impact of Pollution in Biological Resources Within The Niger Delta. Technical Report on Environmental Pollution Monitoring of the Niger Delta Basin of Nigeria. Environmental Consultancy Group, University Of Ife 6: 69-121. Odu, C.T.I., Amadi, E.N., Okolo, J.C. (2005): Effect of soil treatments containing poultry manure on crude oil degradation in a sandy loam soil. Applied Ecological Environ. Res 3 (1) 47. Okeimen C.O and Okeimen F.E. (2005): Bioremediation of Crude Oil-Polluted Soil- Effect of Poultry Droppings and Natural Rubber Processing Sludge Application on Biodegradation of Petroleum Hydrocarbons. Environmental Sciences, MY Tokyo, 12 (1) 1-8.

69

Onwurah, I. N. E. (1999): Restoring the crop sustaining potential of crude oil polluted soil by means of Azotobacter inoculation. Plant Prod. Res, Journal 4: 6-16. Onwurah, I.N.E., Ogugua, V.N., Onyike, N.B., Ochonogor, A.E. and Otitoju, O.F. (2007): Crude Oil Spills in the Environment, Effects and Some Innovative Clean-Up Biotechnologies, International Journal of Environmental Research 1 (4) 307-320. Page, A.L., Miller, R.H., Keeney, D.R. (1982): Methods of Soil Analysis, part II, 2nd ed., ASA-SSSA, Wisconsin, pp. 937–970. Perfumo A., Ibrahim M.B., Roger M., Luigi V., (2007): Thermally Enhanced Approaches

for

Bioremediation

of

Hydrocarbon-Contaminated

Soils.

Chemosphere 66:179-184. Rittmann, B.E., McCarty, P.L. (2001): Environmental Biotechnology: Principles and Applications, McGraw-Hill, New York, Page 695. Ronen, E. (2007):Micro-elements in Agriculture. Practical Hydroponics and Greenness. 39-45 Salam, A.A., 1996. Remediation and Rehabilitation of Oil-Lake Beds. In: AlAwadhi, N., Balba, M.T., Kamizawa, C. (Eds.), Environmental Disaster, Elsevier, Amsterdam, pp. 117–139. Schnoor, J. L., Licht, L. A., McCutcheon, S. C., Wolfe, N. L., Carreira, L. H., (1995): Phytoremediation of organic and nutrient contaminants. Environ. Sci. Technol., 29 (7), 318A-323A. 70

Stanton, R.E. (1996): Rapid methods of Trace Analysis for Geochemical Applications. Edward Arnold Ltd., London. Tanee, F.B.G., Albert, E. (2011): Biostimulation Potential Of Sawdust on Soil Parameters And Cassava (Manihot Esculenta; Crantz) yields in Crude Oil Polluted Tropical Soil. Advances in Environmental Biology. Tanee, F.B.G., Akonye, L.A. (2009):

Phytoremediation potential of Vigna

unguiculata in a crude oil polluted tropical soil of the Niger-Delta. Global Journal of Pure and Applied Sciences, 15 (1) 1-4. Tang, J., Wang, R., Niu, X., Wang, M., and Zhou Q. (2010): Characterization on the rhizoremediation of petroleum contaminated soil as affected by different influencing factors. Biogeosciences Discuss., 7: 4665–4688. The International Council on Clean Transportation, (2011): An Introduction to Petroleum Refining And Production Of Ultra Low Sulphur Gasoline And Diesel Fuel. Energy Economics Applied Optimization, pp 6-9. Timmis, K.N., Pieper, D.H. (1999). Bacteria designed for bioremediation. Trends Biotechnology 17: 201–4. Udo, E.J., Fayemi, J. (1975): the effect of pollution on germination and nutrient uptake of corn. J. Environ. Qual. 4: 537-40. United States Department of Agriculture, Natural Resources Conservation Services (2004): Use of Reaction (pH) In Soil Taxonomy. Soil Survey Laboratory Methods Manual, Version No. 4. Soil Survey Investigations Report No 42. 71

US environmental Protection Agency, 2006. Landfarming . Wade Weisman, (1998): Analysis of Petroleum Hydrocarbons in Environmental Media. Total Hydrocarbon Criteria Working Series, Vol1. Amherst Scientific Publishers. Wallace, T. (1951): Trace elements, in plant, physiology. International Union of Biological Sciences. Colloquia, No. 1, series B. 5-9. Xu J. (2012): Introduction to Enhanced Oil Recovery (EOR) Processes and Bioremediation of Oil-Contaminated Sites. Intech publishers. Pages 207-244. Yehuda, C. (2002): Bioremediation of oil by marine microbial mats. Int. Microbial 5: 189-93.

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