ABSTRACT
Petroleum – polluted sites are disturbed ecosystem with scanty plants. Study was carried out in such sites to assess the area of its rhizobacterial status, particularly comparing that of the rhizospheres and non-rhizospheres in the site. The samples were suspended and serially diluted in physiological saline and inoculated on nutrient media by spread plates method. After incubation and isolation, the isolates were characterized by morphological examination, gram staining, biochemical tests, biodegradation test, and motility test. The isolated bacteria were Acinetobacter species, Pseudomonas species, Micrococcus species, and Bacillus species. The total heterotrophic bacterial counts of rhizosphere samples were sample Ar 1.3 x 106 cfu/g, sample Br 2. 54 x 106 cfu/g, sample Cr. 2.96 x 106 cfu/g, and sample Dr. 2.37 x 106 cfu/g, while the total heterotrophic counts of non – rhyzosphere samples were sample An 1.01 x 106 cfu/g, Bn 1.12 x 106 cfu/g, sample Cn 1.11 x 106 cfu/g, sample Dn 1.02 x 106 cfu/g. All the isolates were able to grow in medium containing crude oil. But while Pseudomonas species showed heavy growth, Bacillus species and Acinetobacter species showed moderate growths, and Micrococcus species showed scanty growth. Petroleum – polluted soils contain rhizobacteria. But while rhizosphere of such soils contains more, non rhizosphere contains less. Therefore, rhizosphere will be very helpful in bioremediation of petroleum – polluted soils.
TABLE OF CONTENTS
Title Page i
Certification ii
Dedication iii
Acknowledgment iv
Table of Contents v
List of tables vi
Abstract viii
CHAPTER
ONE - INTRODUCTION
1.1 INTRODUCTION 1
1.2 Objectives:
2
CHAPTER TWO - LITERATURE REVIEW
2.1 Rhizobacteria 3
2.1.1 Nitrogen fixation 3
2.1.2 Symbiotic relationship 4
2.1.3 Promotion of plant growth 5
2.1.3.1 Mechanisms of action of plant growth promoting
rhizobacteria (PGPRs) 6
2.1.4 Biocontrol function of rhzibacteria 7
2.2 Petroleum polluted soil 8
2.2.1 Ecological impacts of hydrocarbon
contamination 9
2.2.1.1 Soil Quality 10
2.2.1.2 Plants 10
2.2.1.3 Microorganisms 11
2.3 Remediation of hydrocarbon-contaminated
soil 14
2.3.1 The aim of remediation 15
2.3.2 Bioremediation 15
2.3.3 Phytoremediation 17
CHAPTER 3 -
MATERIALS AND METHOD
3.1 Sample Collection 21
3.2 Materials Used 21
3.3 Preparation of culture media 22
3.3.1 Nutrient agar (NA) 22
3.3.2 Nutrient broth (NB) 22
3.3.3 Mineral salt broth (MSB) 22
3.4 Sterilization of Culture Media 22
3.5 Bacterial Isolation 23
3.6 Characterization of Bacterial Isolates 23
3.6.1 Morphological examination 23
3.6.2 Gram staining test 24
3.6.3 Biochemical test 24
3.6.4 Biodegradation test 26
3.6.5 Motility test 26
CHAPTER FOUR
4.0 Result 27
4.1 Morphological characteristics of the
isolated bacteria 27
4.2 Bilochemical
and characterization of the isolated bacteria 29
4.3 Screening
test for utilization of petroleum hydrocarbon by isolates 31
4.4 Isolated
rhizobacteria and the total heterotrophic bacterial counts
(THBC) of rhizosphere soil. 33
4.5 Isolated
rhizobacteria and the total heterotrophic bacterial counts
(THBC) of non rhizosphere soil. 35
CHAPTER FIVE
5.1 Discussion 37
5.2
Conclusion and Recommendation 38
References 39
LIST OF TABLE
Table
1: Morphological characteristics of
the isolated bacteria 28
Table
2: Biochemical and characterization
of the isolated bacteria 30
Table
3: Screening test for utilization of
petroleum hydrocarbon by isolates 32
Table
4: Isolated rhizobacteria and the
total heterotrophic bacterial
counts (THBC) of rhizosphere soil. 34
Table 5: Isolated
rhizobacteria and the total heterotrophic bacterial counts
(THBC) of non rhizosphere
soil. 36
CHAPTER ONE
1.1 INTRODUCTION
In an
environment, petroleum hydrocarbon accumulation can cause serious problems.
This can affect negatively the stability of many ecosystems. It can also cause
difficulties for animals and human health (Chekroun et al, 2014). While in an aquatic ecosystem, chemical contaminants
present may be immobilized and
accumulated in sediments or may be subject to transformation and activation
processes (Martinez – Jeronimo et al,
2008), remediation of soils containing organic pollutant can be enhanced by
plants by various processes (Kelechi and Yusuf, 2014). This is called
phytoremediation. It involves use of plants to degrade, stabilize, and /or
remove soil chemical contaminants. A type of phyteremediation is
rhizoremediation, which involves both plants and their associated rhizosphere
microbes. The microbes can occur naturally or by deliberate introduction by
humans. These microbes can be contaminant degraders and /or can promote plant
growth under stress conditions. Most plant have symbolic relationships with
soil microorganisms. The area around plant roots, otherwise called the
rhizosphere contains high populations, greater diversities and activities of
microorganisms (Brimecombe et al,
2007) than soil with no plants. Rhizosphere microorganisms are especially
critical for plant colonization of unfavourable soils, since they can alleviate
biotic and abiotic stress of plants. This has caused the emergence of a green
technology which employs the symbolic relationship between plants and rhizo-microorganisms
in the breakdown of contaminants to clean up the environment. This techniques
is referred to as rhizoremediation (Kuiper et
al, 2004). A plant can be considered to be a solar-driven biological pump
and treatment system, attracting water with its root system, accumulating water
soluble pollutant in the rhizosphere and concluding with the degradation or
translocation of pollutant (Liste and Alexander, 2000). In some case,
rhizosphere microbes are even the main contributors to the degradation process.
Plants release exudates into the soil ecosystem that increases the microbial
activity and aid the degradation of xenobiotic substances. The soluble root
exudates include enzymes, amino acids, sugars and low molecular weight
carbohydrates (Kelechi and Yusuf, 2014). The plant rhizosphere is recognized as
a niche rich in growth substrates in comparison with the surrounding bulk soil
(Dunfield and Germida, 2001). Rhizospheres are dynamic microorganisms in which
microbial communities have access to an elevated supply for carbon, oxygen and
energy rich materials from plant roots (Clegg and Murray, 2002). Rhizosphere
are also stable physically, avoiding the potentially adverse effects of
naturally occurring disturbances on microbial community composition or
activities (Piceno et al, 2000). This
stimulatory rhozosphere effect has been recognized for many years and was
described for the first time by hitlner in 1904 (Kuiper et al, 2004). In rhizoremediation, plant roots sustain the
degrading microflora by supplying them with nutrients other than pollutants,
and also help in spreading the degrading microorganisms to new sites in the
soil.
1.2 OBJECTIVES:
The main objective of this work is to assess and
characterize the rhizobacteria in petroleum polluted soils in Umuahia.
Specific objective are:
•
To isolate rhizobacteria in
petroleum polluted soil.
•
To enumerate the rhizobacteria in
petroleum polluted soils.
•
To characterize and identify the
rhizobacteria in petroleum polluted soils.
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