ABSTRACT
Isolation and molecular characterization of hydrocarbon degrading bacteria from Okulu river in Eleme Local Government area, Rivers state were studied. Wastewater samples were randomly collected at four different points in the river using a 2 litre plastic hydrobios water sampler and aseptically transferred into sterile 2-litre plastic containers. Standard methods were adopted for the determination of microbiological and physico-chemical characteristics. The identification of bacteria isolates were carried out using both morphological and biochemical characteristics. Also isolates were molecularly characterized. The abilities of the bacterial isolates to degrade total petroleum hydrocarbon were determined using Gas chromatographic method. The total heterotrophic bacteria count in Okulu River ranged from 1.8×106 - 2.2×107 cfu/ ml. Hydrocarbon utilizing bacteria count recorded ranged from 1.0×103 – 4.2×104 cfu/ ml. The predominant bacterial genera identified were Bacillus, Micrococcus, Escherichia, Staphylococcus, Pseudomonas, Enterobacter, Klebsiella, Streptococcus, Pectobacterium, Bevibacillus, Serratia and Providencia species. The pH values in Okulu water samples ranged from 6.11 - 6.65, with mean value of 6.35±01.8. The temperature values ranged from 28.3 - 32.7 oC, with mean value of 31.04±1.72 oC. The turbidity ranged from 17.7 - 112 NTU, with mean value of 54.69±31.55 NTU. Values for total dissolved solids ranged from 22.1 - 58.9 mg/l, with mean value of 44.08 mg/l±12.63. Total suspended solids ranged from 6.01 – 8.77 mg/l, with mean value of 6.98mg/l±1.18. Biological oxygen demand ranged from 20.25 - 46.50 mg/l, with mean value of 32.85±10.62 mg/l. The chemical oxygen demand showed ranges of 29.37 - 66.52 mg/l, with mean value of 50.84±14.89 mg/l. Dissolved oxygen ranged from 2.28 - 4.37 mg/l, with mean value of 3.19 ±0.70 mg/l. Salinity ranged from 15.0 - 22.5 mg/l, with mean value of 18.23 ±2.65 mg/l. Conductivity values had range of 3.53 - 3.88 µS/cm, with mean value of 3.71±0.09 µS/cm. Nitrate values ranged from 2.18 - 4.21 mg/l, with mean value of 3.02±0.66 mg/l. Values of phosphate ranged from 2.58 - 4.11 mg/l, with mean value of 3.47 ±0.47mg/l. Total organic carbon had range of 1.12 - 2.70 %, with mean value of 1.92±0.55 %. Total hydrocarbon content values ranged from 13.27 - 17.70 mg/l, with mean value of 15.52 ±1.87 mg/l. For the heavy metals, lead values in the samples ranged from 2.0107 - 2.0857 mg/l, with mean value of 2.03553±0.02 mg/l. Values of cadmium ranged from 0.1639 - 0.3157 mg/l, with mean value of 0.233508±1.72 mg/l. Arsenic values in Okulu water samples ranged from 0.0154 - 0.0381 mg/l, with mean value of 0.0345±1.72 mg/l. The obtained 16S rRNA sequence of the bacteria produced close relatedness to their relatives from the NCBI data base. Based on their 16S rRNA sequence, the bacteria were identified as Lysinibacillus fusiformis, Alcaligenes faecalis, Enterobacter cloacae, Lysinibacillus sphaericus and Lysinibacillus macroides. The bioremediation potential of the bacteria isolates gave positive results singularly and in combination in terms of degradation of hydrocarbon content of the water sample. These bacteria isolated from Okulu River can be used for the remediation of polluted environment. In view of the results obtained, the test bacterial species can be applied in the remediation of polluted environment especially hydrocarbon-polluted environment
TABLE OF CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vii
List of Tables xi
List of Figures xii
Abstract v
CHAPTER 1: INTRODUCTION 1
1.1 Background of the Study 1
1.2 Justification of the Study 3
1.3 Statement of Problems 4
1.4 Aim of the Study 5
1.5 Scope of the Study 5
1.6 Objectives of the Study 5
CHAPTER 2:
LITERATURE REVIEW 6
2.1 Water
and its Importance 6
2.2 Pollution
of Water Bodies and Sources 8
2.2.1 Effects
of water pollution 12
2.3 Oil
Spillage 14
2.4 Hydrocarbons
16
2.4.1 Aliphatic hydrocarbons 17
2.4.2 Alicyclic
hydrocarbons 18
2.4.3 Aromatic hydrocarbons 18
2.4.4 Total petroleum hydrocarbon 18
2.4.5 Polycyclic aromatic hydrocarbons (PAHs) 19
2.5 Biodegradation
20
2.5.1 Bioremediation 21
2.5.2 Strategies to enhance bioremediation
efficiency 21
2.5.3 Principles of bioremediation 23
2.5.4 Hydrocarbon degrading microorganisms 24
2.5.5 Degradation
of petroleum hydrocarbons by aerobic and anaerobic
processes
26
2.6 Factors
Affecting Biodegradation 27
2.6.1 Temperature 27
2.6.2 pH 27
2.6.3 Oxygen availability 27
2.6.4 Nutrients 28
2.6.5 Type of pollutant / hydrocarbons 28
2.6.6 Site condition 28
2.6.7 Microbial communities 28
2.7 Molecular Characterization 29
CHAPTER 3:MATERIALS AND METHODS 30
3.1 Description of Study Area 30
3.2 Collection of Water Samples 30
3.3
Preparation of Sample Inocula (Water
samples) 30
3.4
Microbiological Analysis 31
3.4.1
Enumeration and isolation of bacteria 31
3.4.2
Enumeration and isolation of
hydrocarbon utilizing bacteria 31
3.5
Identification of Isolates 31
3.5.1 Bacterial isolates 31
3.6 Determination
of Physicochemical Properties of Samples 32
3.6.1
Determination of pH 32
3.6.2
Determination of temperature (oC) 32
3.6.3
Determination of turbidity (NTU) 32
3.6.4
Determination of total dissolved solids
(mg/l) 33
3.6.5
Determination of total suspended solid
(mg/l) 33
3.6.6
Biological oxygen demand (mg/l) 34
3.6.7
Determination of chemical oxygen demand
(mg/l) 35
3.6.8
Determination
of total hydrocarbon content (mg/l) 36
3.6.9
Determination of salinity (mg/l) 36
3.6.10
Determination of conductivity (µS/cm) 37
3.6.11 Determination of
nitrate (mg/l) 37
3.6.12 Determination of
phosphate (mg/l) 37
3.6.13 Determination of
total organic carbon (%) 38
3.6.14 Determination of
heavy metals (mg/l) 38
3.6.15 Determination of
total petroleum hydrocarbon (mg/l) 38
3.7 Molecular Identification 39
3.7.1 DNA extraction (boiling method) of bacteria 39
3.7.2 DNA
quantification of bacteria 39
3.7.3 16S rRNA amplification of
bacteria 40
3.7.4 Sequencing 40
3.7.5 Phylogenetic
analysis 41
3.8 Biodegradation Experiment 41
3.8.1 Preparation of bacterial inoculums 41
3.8.2 Composition of biodegradation set up 42
3.8.3 Biodegradation procedure 42
3.9
Statistical Analysis 43
CHAPTER 4:
RESULTS AND DISCUSSION 44
4.1 Results 44
4.2 Microbial Counts of Okulu Water Sample 44
4.3 Bacterial Species Isolated from Okulu
Water Samples 44
4.4
Percentage Occurrence of Bacterial
Isolates 44
4.5 Physicochemical Characteristics of Okulu
Water Samples 47
4.6: Heavy
Metal Concentrations of Water Samples of Okulu 47
4.7 Molecular
Identification of Isolates 50
4.8 Biodegradation
Results 55
4.8.1 Biodegradation of
total petroleum hydrocarbon (TPH) by single and
mixed isolates 55
4.9 Discussion 86
4.9.1 Microbial
population 86
4.9.2 Physicochemical
parameters 87
4.9.3 Molecular identification 94
4.9.4 Biodegradation activity 94
CHAPTER
5: CONCLUSION AND RECOMMENDATIONS 98
5.1 Conclusion 98
5.2 Recommendations
99
5.3 Contributions to Knowledge 99
References
Appendices
LIST OF TABLES
3.1: Biodegradation set up 42
4.1: Percentage
occurrence of bacteria from Okulu water samples 46
4.2: Monthly values of physicochemical parameters
of water samples obtained from Okulu 48
4.3.: Monthly
values of heavy metals of Okulu water samples 49
4.4:
Isolates and accession numbers 54
4.5:
Counts of bacteria (cfu/ml)
from biodegradation experimental
set-up 57
4.6 Biodegradation of TPH by single and mixed
bacterial isolates 58
LIST OF FIGURES
4.1. Monthly
total heterotrophic bacterial counts of Okulu water samples 45
4.2: Showing
the nucleotides
base sequence from the isolates 52
4.3: Phylogenetic
tree showing the evolutionary distance between the
bacterial
isolates 53
4.4: GC profile of total petroleum hydrocarbon
(TPH) in Okulu
water sample on day 1 60
4.5: GC profile of total petroleum hydrocarbon
(TPH) in Okulu water
sample in control on day 42 62
4.6: GC profile of total petroleum hydrocarbon
(TPH) in Okulu water
sample by Alcaligenes
faecalis on day 14 63
4.7: GC profile of total petroleum hydrocarbon
(TPH) in Okulu water
sample by Alcaligenes
faecalis on day 28 64
4.8: GC profile of total petroleum hydrocarbon
(TPH) in Okulu water
sample by Alcaligenes
faecalis on day 42 65
4.9: GC profile of total petroleum hydrocarbon
(TPH) in Okulu water
sample by Enterobacter
cloacae on day 14 65
4.10: GC profile of
total petroleum hydrocarbon (TPH) in Okulu water
sample by Enterobacter
cloacae on day 28 68
4.11: GC profile of
total petroleum hydrocarbon (TPH) in Okulu water
sample by Enterobacter
cloacae on day 42 69
4.12: GC profile of
total petroleum hydrocarbon (TPH) in Okulu water
sample by Lysinibacillus
sphaericus on day 14 71
4.13: GC profile of
total petroleum hydrocarbon (TPH) in Okulu water
sample by Lysinibacillus
sphaericus on day 28 73
4.14: GC profile of
total petroleum hydrocarbon (TPH) in Okulu water
sample by Lysinibacillus
sphaericus on day 42 74
4.15: GC profile of
total petroleum hydrocarbon (TPH) in Okulu water
sample by Lysinibacillus
macroides on day 14 75
4.16: GC profile of
total petroleum hydrocarbon (TPH) in Okulu water
sample by Lysinibacillus
macroides on day 28 77
4.17: GC profile of
total petroleum hydrocarbon (TPH) in Okulu water
Sample by
Lysinibacillus macroides on day 42 78
4.18: GC profile of
total petroleum hydrocarbon (TPH) in Okulu water
sample by Alcaligenes
faecalis + Lysinibacillus
sphaericus on
day 14 80
4.19: GC profile of
total petroleum hydrocarbon (TPH) in Okulu water
sample by Alcaligenes
faecalis + Lysinibacillus
sphaericus on
day 28 81
4.20: GC profile of
total petroleum hydrocarbon (TPH) in Okulu water
sample by Alcaligenes
faecalis + Lysinibacillus
sphaericus on
day 42 82
4.21: GC profile of
total petroleum hydrocarbon (TPH) in Okulu water
sample by Lysinibacillus
macroides + Enterobacter
cloacae on
day 14 83
4.22: GC profile of
total petroleum hydrocarbon (TPH) in Okulu water
sample by Lysinibacillus
macroides + Enterobacter
cloacae on
day 28 84
4.23: GC profile of
total petroleum hydrocarbon (TPH) in Okulu water
sample by Lysinibacillus
macroides + Enterobacter
cloacae on
day 42 85
A1.
Monthly hydrocarbon utilizing
bacterial counts of Okulu water samples 145
A2
Mean heterotrophic bacterial counts
of Okulu water samples during
dry
and rainy season 146
A3
Mean hydrocarbon utilizing bacterial
counts of Okulu water samples
during
dry and rainy season 146
A4: Monthly pH values of Okulu water samples 147
A5: Monthly temperature values of Okulu water
samples 147
A6: Monthly turbidity values of Okulu water
samples 148
A7: Monthly values of total dissolved solid of
Okulu water samples 148
A8: Monthly values of total suspended solid of
Okulu water samples 149
A9: Monthly values of biological oxygen demand
of Okulu water samples 149
A10: Monthly values of chemical oxygen demand of
Okulu water samples 150
A11: Monthly dissolved oxygen values of Okulu water
samples 150
A12: Monthly values of salinity of Okulu water
samples 151
A13: Monthly values of total hydrocarbon content of
Okulu water samples 151
A14: Monthly conductivity values of Okulu water
samples 152
A15: Monthly nitrate values of Okulu water samples 152
A16: Monthly phosphate values of Okulu water
samples 153
A17: Monthly Calcium values of Okulu water samples 153
A18: Monthly Magnesium values of Okulu water
samples 154
A19: Monthly Na (sodium) values of Okulu water
samples 154
A20: Monthly Potassium values of Okulu water
samples 155
A21: Monthly sulphate values of okulu water samples 155
A22: Monthly values of total organic carbon of
Okulu water samples 156
A23: Monthly TOM values of Okulu water samples 156
A24: Monthly concentrations of Lead (Pb) in Okulu
Water Samples 157
A25: Monthly
Concentrations of Cadmium (Cd) in Okulu Water Sample 157
A26: Monthly
Concentrations of Arsenic (As) in Okulu Water Samples 158
CHAPTER 1
INTRODUCTION
1.7 BACKGROUND OF THE STUDY
Water
is very important for agriculture, manufacturing, transportation and many other
human activities. Despite its importance, water is the most poorly managed
resource in the world (Chutter, 1998). The availability and quality of water
always have played an important role in determining the quality of life. Water
quality is closely linked to water use and to the state of economic development
(Chennakrishnan et al., 2008).
Various factors may pollute ground and surface waters. Careless disposal of
industrial effluents and other pollutants in metropolitan areas may add
significantly to detoriation of water quality (Mathuthu et al., 1997).
Most
of the water bodies in the areas of the developing world are the end points of
effluents discharged from industries. Despite the fact that petroleum has played important roles in the
economy of the country, over the past three decades, However, some Nigerian
ecosystems have been subjected to untold levels of pollution due to crude oil
spillage and other forms of effluents discharge resulting from various economic
operational activities (Adeniyi and Afolabi, 2002). The effluents' constituents
have severe toxicological consequences for both the aquatic lives and humans.
When oil-containing factory effluents are released into a water body, it could
lead to the lowering of dissolved oxygen. This could be worsened by the
products of microbial decomposition activities. The insufficiency of oxygen,
the products of decomposition and the presence of non-decomposed waste as a result
of low level of oxygen availability could lead to eutrophication. Such
eutrophication could lead to further loss of aquatic lives (Beeby, 1993). Effluents
from petroleum industries are disposed into the streams almost exclusively
without adequate treatment, which is likely to affect the water quality of the
receiving streams. The changes in the nutrient concentrations of water may lead
to harmful effects to humans and aquatic life. The majority of heavy metals
found in pools of water are frequently linked to industrial emissions (Mdamo,
2001), and nearly all heavy metals found in industrial effluents have
compounded toxicities that harm aquatic life. The kind and variety of aquatic
biota, as well as the water quality and pollution, are reflected in the
physical-chemical characteristics of a water body (Birley and Lock, 1999). The
pollution of much of the Niger Delta's creeks, marshes, and rivers with
hydrocarbons, and dispersant chemicals has become something of great concern.
Rivers
State forms a significant part of the Niger Delta estuary and one of the
largest oil producing State in Nigeria. Consequently, the State is grossly
subjected to environmental pollution or environmental deterioration which is
associated with health hazards. Over the years, the recurrence of oil spills
and the presence of untreated waste that are discharged into Delta area water bodies
have led to various health and economic problems of the indigenes (Nduka and
Orisakwe, 2009).The fate of petroleum in water bodies could be affected by
water currents, aquatic temperature, the suns irradiation, the residential
microbodies, the abilities to utilize the petroleum pollutants as a sole carbon
source and the prevailing pH range (Nikolopoulou and Kalogeraki, 2010).
At
an early-stage light fractions of oil are naturally removed – mostly by
evaporation, thence by photo-oxidation and by geo-chemical reactions. Oil
degrading bacteria are capable of using organic substances, naturally or
synthetic as sources of nutrients and energy hence exhibiting remarkable range
of degradative capabilities (Dua et al.,
2002). Oil degrading microorganisms are ubiquitous in the environment,
particularly in the oil polluted sites. Bioremediation is said to be the best
approach for environmental cleanup because it is a cost effective and an
eco-friendly strategy. The increasing knowledge of gene sequences
and the attendant development of new culture independent molecular techniques
are providing new and effective tools for the characterization of the diversity
of microbial communities (Boucher et al.,
2003). This is different from conventional studies on classical culture
isolation techniques and subsequent identification based on morphology,
physiological and biochemical characteristics of microbial populations in natural ecosystems,
(Elijah, 2013). Molecular techniques employ the use of DNA from natural
population. These DNA molecules can be extracted and fragmented. The fragments could
be cloned into vectors to form a library. Each clone in the library represents
a single piece of DNA. Various molecular methods are employed in bacterial
characterization depending on its discriminatory power, reproducibility, ease
of use and ease of interpretation. These include 16S rRNA gene technology for
bacterial species, 18S rRNA gene technology for fungi and algae, DNA-DNA
hybridization and the next generation systems (Tindall et al., 2010).
1.8 JUSTIFICATION OF THE STUDY
Over
the years, the issue of environmental contamination and pollution by oil spills
in the Niger Delta region of the country has been of public concern and the
need for environmental cleanup has arisen. The primary sources of natural water
contamination are effluents discharged from residential and industrial
operations. Untreated wastes from processing plants in the study region are
released into water bodies, leading to the emission of repulsive odors, is
water discolorations thus leading to sticky oil, nature of water bodies. Therefore,
it has become for a detailed study to be carried out on how to solve the very
serious problem. The first part of call of such a study will be the isolation
of species of microorganisms associated with such hydrocarbon polluted
environment and the determination of their abilities to use the hydrocarbon as
sole carbon sources. It is anticipated that such carbonoclastic microorganisms
could thus play major roles of clean up exercises in the Niger Delta Area of
Nigeria.
1.9 STATEMENT OF PROBLEMS
Domestic
and industrial wastes as well as sewages are discharged into Okulu River. This practice
has led to the pollution of the river which might have resulted into serious
health and economic problem for the inhabitants of the zone. There is scanty
information on species of bacteria associated with the polluted Okulu River in
Eleme Local Government Area, Rivers State and this has been of major concern to
the indigenous environmental microbiologists and public health practitioners. There
is dire new for a comprehensive database on oil-degrading bacteria for
professional biodegradetionists for a functional clean-up exercise in the Niger
Delta region. High population density of both chemical and petrochemical
industry along the coast of this river contributes to increased levels of the
river pollution. However, apart from oil spills from vandalization of oil
distribution pipes, there is also the presence of effluents from Indorama
petrochemical plant in the form of wastewater and waste chemicals. There are
also pollutants from chemical fertilizer and other chemical processing plants
in the area. there are also discharges from motor mechanic workshops, electric
generators which release used engine oils, motor oils, used diesels,
transmission oils into the aquatic habitats in the said Region. All these affect the quality, population,
distribution and diversity of microorganisms in the polluted aquatic environment.
1.10
AIM
OF THE STUDY
The
aim of this study was to; Isolate and identify hydrocarbon utilizing bacteria
molecularly, from polluted river in Eleme LGA, Rivers State, Nigeria with potential
to biodegrade total petroleum hydrocarbon pollutants.
1.11
SCOPE
OF THE STUDY
The
study was limited to the creek or shore site in Okulu River, in Eleme LGA,
Rivers State. Samples were collected monthly for a period of twelve months and
transported to Environmental Microbiology Laboratory, University of Port
Harcourt for analysis. The study was limited to bacterial isolation, screening,
identification and characterization using molecular methods.
1.12
OBJECTIVES
OF THE STUDY
- To
enumerate hydrocarbon degrading bacteria associated with the Okulu River
in Eleme LGA, Rivers State.
- To
determine the physicochemical characteristics of the Okulu River
samples
- To
identify bacterial isolates using
molecular methods.
- To
determine the ability of the molecularly identified bacterial isolates to
biodegrade petroleum
hydrocarbon.
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