ABSTRACT
Oil pollution is a worldwide threat to the environment and the remediation of oil contaminated soils, sediments and water is a major challenge for environmental research. Bioremediation is a useful method for soil remediation. The objective of this study was to isolate Actinomyces from the soil using membrane filter techniques and at the same time use the isolates for remediation. Results showed polluted soil contained average of 46.51% of total petroleum hydrocarbon (TPH) prior to remediation. Two Actinomyces isolates were obtained, characterized (phenotypically and genotypically) and identified as Streptomyces carpaticus and Streptomyces xiamenensis respectively and subsequently used for bioremediation trials. At an inoculation size of 1.0x108 cfu/ml and a trial period of 90 days. The total petroleum hydrocarbon concentration in the soil was reduced by 75.51% for Streptomyces carpaticus and by 75.96% for Streptomyces xiamenesis. Slight variations were recorded in the rate of degradation of TPH by the two isolates. The rate of remediation was in the range of 1.066% per day to 0.396% per day in treatment 1 (S. carpaticus) and 0.926% per day to 0.386% per day in treatment 2 (S. xiamenensis). Remediation rates decreased with time in both treatments. The efficiency of bioremediation process in treated soils increased with time from 31.74% (day 15) to 65.61% (day 90) in treatment 1 and from 27.04% (day 15) to 64.61% (day 90) in treatment 2. The Total Heterotrophic Bacteria Count (THBC) increased to maximum population of 6.13x108 cfu/g and 6.17x108 cfu/g in the soils respectively, there were however no significant reduction in the total petroleum hydrocarbon of the control soil and that was attributed to possible metabolism of the hydrocarbon by indigenous organism. The potentials of the two Actinomyces isolates for polluted soil was established by the finding of the work.
INTRODUCTION 1
1.1 Background of the Study 1
1.2 Justification of the Study 4
1.3 Significance of Study 2
1.4 Aims of the Study 3
1.5 Objectives of the Study 3
CHAPTER 2: LITERATURE REVIEW 4
2.1 Spilled Petroleum
Hydrocarbons 4
2.2 Ecological Impacts of Spilled Petroleum
Hydrocarbons 6
2.3 Crude oil Bioremediation Strategies 7
2.3.1 Land farming 7
2.3.2 Composting 8
2.3.3 Use of bioreactors 9
2.3.4 Bioventing/biosparging 9
2.3.5 Biostimulation 10
2.3.6 Bioaugmentation 11
2.4 Factors Affecting Bioremediation
Treatment 11
2.4.1 Nutrient availability 12
2.4.2 Temperature 12
2.4.3 Oxygen limitations 13
2.4.4 pH 13
2.4.5 Bioavailability of hydrocarbon 14
3: MATERIALS
AND METHODS 15
3.1 Study Area 16
3.2
Collection of
Soil Sample 16
3.3 Isolation of Actinomyces for Remediation 16
3.3.1 Membrane filter
culture technique 16
3.4 Isolation and
Characterization of Actinomyces
Isolates 17
3.4.1 Morphological
examination 17
3.4.2 Microscopic examination 17
3.4.3 Gram stain 17
3.5 Biochemical Tests 18
3.5.1 Catalase
production 18
3.5.2 Oxidase test 18
3.5.3 Test for urease activity 18
3.5.4 Indole production 19
3.5.5 Carbohydrate utilization test 19
3.6 Preparation of Contaminated Soil 19
3.6.1 Soil analysis-physical
properties 20
3.6.1.1 Determination
of soil texture 20
3.6.1.2 Determination of moisture
content 21
3.6.2 Determination of
chemical properties 22
3.6.2.1 Determination of pH 22
3.6.2.2 Determination
of soil nitrogen 22
3.7 Bioremediation Treatment (Experimental
Setup) 24
3.8 Bioremediation Monitoring 25
3.8.1 Determination of bacteria
load 25
3.8.2 Determination of
Total Petroleum Hydrocarbon 26
3.8.3 Determination of
Bioremediation Rate 26
3.9 Molecular
Identification 27
3.9.1 DNA extraction 27
3.9.2 DNA quantification 27
3.9.3 Phylogenetic analysis 28
3.10 Statistical Analysis 29
CHAPTER 4: RESULTS AND DISCUSSIONS 30
4.1 Results 30
4.2 Discussion 40
CHPATER 5: CONCLUSION AND
RECOMMENDATIONS 43
5.1 Conclusion 43
5.2 Recommendations 43
REFERENCES 44
APPENDICES 49
LIST OF TABLES
3.1 The Experimental Set up
Involved Three Treatments for each
Assessment 23
4.1: Initial
Analysis of the Contaminated Soil 31
4.2: Total
Petroleum Hydrocarbon (Tph) “G/Kg” Content of Polluted Soil
Remediated
With Actinomyces 33
4.3: Rate
of Remediation (G/Day) 35
4.4: Bioremediation Efficiency % 37
4.5: Total
Heterotrophic Bacteria Count (Thbc) of Actinomyces
Remediating
Crude Oil Polluted Soil (Cfu/G) 38
4.6: Showing Biochemical Test Result 39
LIST OF
FIGURES
3.1 Map of Eleme Local Government in Rivers
State showing Onne,
the Study Area 21
4.1 Total Petroleum Hydrocarbon (TPH) “G/KG”
Content of Polluted 40
Soil Remediated With Antinomies
(Bioremediations)
4.2 Rate of Remediation (G/Day) 41
4.3 Bioremediation Efficiency 42
INTRODUCTION
1.1 BACKGROUND
OF THE STUDY
Human activity has led
to the release of liquid petroleum hydrocarbon (also known as crude oil) into the
environment causing the pollution of farmlands, marine/coastal water,
shorelines etc. Liquid petroleum hydrocarbons are naturally-occurring fossil fuel,
formed from dead organic materials in the earth’s crust. (Kingston, 2002).
They are used to
synthesize plastics, fertilizers, pesticides and other petrochemical products.
In addition, they are refined to form fuels to run internal combustion engines of
cars and vehicles as well as heavy plants and machinery used by a wide range of
industries around the world. The domestic utilization of petroleum products in
households for heating, cooking lighting and electricity generation, has
increased for the liquid hydrocarbons. In 2008, the volume of petroleum
demanded globally was 85-62 million barrels per day (Ibrahim, 2008).
However,
liquid petroleum has become one of the most prevalent pollutants in
industrialized and developing countries (Josh and Pandy, 2011). Its
transportation and global usage has increased the tendency to pollute the
environment. The source of pollution is usually accidental spills, uncontrolled
landfills, leakage, underground storage tanks or improper storage of crude oil
(Plohl et al., 2002).
Oil spills pose serious
environmental challenge due to the possibility of air, water and soil
pollution. Large oil spills threaten both terrestrial and marine ecosystems:
hence, attention has been draw towards identifying eco-friendly and
cost-effective cleaning methods (Trindade et
al., 2005).
Actinomyces is a genus of the
actinobacteria class of bacteria. They are all Gram-positive Actinomyces species are facultatively
anaerobic (except Actinomyces meyeri
and Actinomyces israelli are obligate
anaerobes) and they grow best under anaerobic conditions. (Bowden and Baron,
1996). Actinomyces species may form endospores, and while individual bacteria
are rod-shaped, actinomyces colonies form fungus-like branched networks of
hyhae (Holt, 1994).
The aspect
of these colonies initially led to the incorrect assumption that the organism
was a fungus and to the name Actinomyces,
“ray fungus” (from Greek actis, ray or beam, and mykes, fungus). Actinomyces species are ubiquitous,
occurring in soil and in microbiota. They are known for the important role they
play in soil ecology, they produce a number of enzymes that help degrade
organic plant material, lignin and chitin. Thus, their presence is important in
the formation of compost. Certain species are commensal in the skin flora, oral
flora, gut flora and vaginal floral of humans and livestock (Petrova et al., 2015).
1.2 JUSTIFICATION
OF THE STUDY
It is imperative to note
that the increasing demand for liquid petroleum may likely not reduce the number
of oil spill occurrences. Therefore, oil spill accidents are prone to occur
considering the enormous pressure on oil companies/drilling firms to make
petroleum products readily available for global consumption. Whenever there is
an oil spill, shorelines, marine waters, ground water, soils (including farm
lands) lakes, rivers and creeks stand the risk of being severely polluted and
if not controlled within a short time frame, may lead to long term ecological
devastation. Several oil spill remediation techniques for the clean-up of
polluted terrestrial and marine environment have been established. However,
most of them have been proven to be cost-ineffective and
environmentally-unfriendly and hence unsustainable. As a result, a control
measure that will be swift efficient and sustainable is a necessity.
1.3 SIGNIFICANCE
OF THE STUDY
This study is intended
to identify the efficiency, cost-effectiveness and eco-friendliness of
Actinomyces species in bioremediation
of crude oil contaminated farm land and also to make recommendations, which are
likely to form the basis for new lines of research on how to overcome the
challenges of oil spillages or contaminations.
1.4 AIMS OF
THE STUDY
The aim of this study
were to isolate Actinomyces from the
soil using membrane filter technique and at the same time use the isolates for
remediation of crude oil contaminated soil.
1.5 OBJECTIVE
OF THE STUDY
The objectives of this
study were to:
1.
Determine
the total petroleum hydrocarbon (TPH)
2.
Evaluate
the bioremediation rates.
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