ABSTRACT
The problem of bioaccumulation and biomagnifications of Paraeforce in food chain and the attendant health hazards to human and farm animals as well as the cumbersome nature of the existing physicochemical methods of its degradation culminated in this study. The study is aimed at exploring the biodegradation potential of fungi on Paraeforce-impacted soil and the analysis of the associated enzymes and metabolites. Soil samples were collected from an agrarian soil previously exposed to Paraeforce herbicide. Physicochemical analysis was done on the homogenized soil sample. Fungal isolation was done using traditional plate culture and molecular techniques. The biodegradation potential of the isolates was determined using titrimetric method. The enzymes activity and metabolite concentration were measured spectrophotometrically. Statistical analysis was done on generated data using one way analysis of variance. The physicochemical analysis of the treated soil samples recorded a range of pH (5.1 to 5.9), temperature (31.9 to 33.3 0C), moisture content (52.28 to 62.5 %), nitrate (13.8 to 23.2 mg/kg), total organic carbon (5.3 to 25.1 mg/kg) and conductivity (0.09 to 0.18 µScm-1). Fungi isolated from the soil sample were Pichia kudriavzevii, Hanseniaspora opuntia, Aspergillus, Pichia cecembensis, Rhizopus, Candida, Fusarium and Penicillium. Of the above isolated microorganisms, Pichia kudriazevii, Hanseniaspora opuntia, Aspergillus spinosus and Pichia cecembensis were the only isolates capable of growth in Paraeforce medium. The degradation rate measured showed that Aspergillus spinosus was a better degrader, breaking down 36.8mg/kg of the Paraeforce in 90 days. There was a synergy in the degradative capacity of the mixed culture degrading 46.3 mg/kg in 90 days. At the end of 90 days, the rate of Paraeforce degradation was also high in treatments with Pichia kudriavzevii (27.5mg/kg), Hanseniaspora opuntia (22.4mg/kg) and Pichia cecembensis (23.1mg/kg). Natural attenuation showed a degradation rate of 41.37mg/kg in 90 days One way analysis of variance done on degradation showed there was a significant difference in the mean concentration of Paraeforce (F[6,42] = 4.338, p = 0.002) over a period of 90 days at 5% level of significance. When the sample was optimized using poultry wastes, the degradation rate improved. The mixed culture achieved 50mg/kg degradation of Paraeforce in 56 days. Natural attenuation achieved 50mg/kg degradation in 70 days while Aspergillus spinosus had 50mg/kg degradation in 84 days. Pichia kudriavzevii, Hanseniaspora opuntia and Pichia cecembensis had 36.7mg/kg, 29.5mg/kg and 26.08mg/kg respectively at the end of 90 days. The growth of fungi was influenced by pH, temperature, nitrate, total organic carbon and the Biochemical oxygen demand of the growth medium. Catalase, laccase, lignin peroxidase and manganese peroxidase were identified in the degradation process with associated formate and oxalate production. These metabolites were used to monitor the degradation process. Aspergillus spinosus and mixed culture of the isolates proved a more efficient tool in this study. The study showed that living cells of the four test fungi have great potential for the degradation of Paraeforce in an impacted soil and should be explored.
TABLE OF CONTENTS
Title
Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table
of Contents vi
List
of Tables x
List
of Figures xi
List
of Plate
xii
Abstract xiii
CHAPTER 1:
INTRODUCTION
1.1
Background to the Study 1
1.2
Statement of the Problem 3
1.3 Aim of the Study 5
1.4 Specific Objectives of the Study 5
1.6
Significance of the Study 5
CHAPTER 2:
LITERATURE REVIEW
2.1 Literature
Review 6
CHAPTER 3: MATERIALS AND METHODS
3.1
Study Area 21
3.2
Soil Sample Collection 21
3.3
Point of Analysis 21
3.4
Processing of Materials 22
3.4.1
Culture media 22
3.4.2
Paraeforce 22
3.5
Determination of the Physicochemical
Properties of the Soil Sample 22
3.5.1
Measurement of temperature 22
3.5.2
pH measurement 22
3.5.3
Determination of nitrate concentration 23
3.5.4
Determination of phosphate
concentration 23
3.5.5 Determination of biochemical oxygen demand 24
3.5.6
Determination of dissolved oxygen 24
3.5.7 Determination of electrical conductivity 25
3.5.8 Determination of total organic carbon 25
3.6
Isolation and Characterization of
Fungal Species 26
3.6.1 Isolation of fungal species 26
3.6.2
Purification of the fungal culture 27
3.6.3 Wet
preparation 27
3. 6.4 Gram stain reaction 27
3.6.5 Colonial
morphology 28
3.6.6 Sugar fermentation tests 28
3.6.7 Citrate utilization 28
3.6.8
Indole production 28
3.6.9 Total heterotrophic fungal counts 29
3.7 Screening
of Fungal Isolates for Herbicides Degrading Potential and
Molecular Characterization of Screened Fungal Isolates 29
3.7.1 Screening of fungal isolates for herbicides
degrading potential 29
3.7.2 Molecular method for characterization of
screened fungal isolates 29
3.7.2.1 Genomic DNA preparation 29
3.7.2.2 DNA extraction 29
3.7.2.3 TAE (Tris-acetate-EDTA) agarose gel electrophoresis 31
3.7.2.4 Polymerase
chain reaction: amplification of DNA 31
3.7.2.5
Sequencing 32
3.7.2.6
Phylogenetic analysis 32
3.7.3
Growth and tolerance of fungal isolates on paraeforce medium 32
3.8 Determination of the Effect of Different
paraeforce Concentration
on Fungal Growth. 33
3.9
Determination of the Rate of Biodegrading Activity of the Isolates 33
3.9.1 Experimental design 33
3.9.2
Rate of paraeforce degradation by fungal
isolates 34
3.9.2.1 Soil
extraction procedures 34
3.9.2.2
Paraeforce degradation test 34
3.9.2.3
Optimization of the fungal degradation process using poultry wastes 35
3.10 Determination of the Effect of Different
paraeforce Concentrations on
Soil Nitrate
and Total Organic Carbon 35
3.10.1
Assessment of the effect of different paraeforce concentration on
soil nitrate 35
3.10.2 Assessment of the effect of different
paraeforce concentration on soil
organic
carbon 36
3.11
Assay of the Fungal Enzymes and Metabolites 36
3.11.1 Screening of fungal
species for enzymes’ production 36
3.11.1.1 Catalase production 36
3.11.1.2 Laccase production 37
3.11.1.3 Lignin peroxidase assay 38
3.11.1.4 Manganese peroxidase assay 39
3.11.2 Analysis of some metabolites 40
3.11.2.1 Determination of oxalate concentration 40
3.11.2.2 Determination of formate 41
3.12 Statistical Analyses of Data 42
CHAPTER 4: RESULTS AND DISCUSION
4.1 Results 43
4.2 Discussions 92
CHAPTER 5:
CONCLUSION AND RECOMMENDATIONS 102
5.1
Conclusion 102
5.2 Recommendations 103
5.3
Contribution to knowledge 102
References
Appendices
LIST OF TABLES
4.1 Physicochemical
characteristics of the soil samples 44
4.2 Colonial
and cell morphology of the fungal isolates 45
4.3 Biochemical
characteristics of yeast isolates 46
4.4 Identity
of the fungal isolates 55
LIST OF FIGURES
1.1 Structure
of paraeforce 1
2.1 Pathway
for paraeforce degradation in soil 17
4.2 Phylogenetic tree showing evolutionary
relationship between fungal
isolates
and close relatives 51
4.3 Turbidimetric
measurement of fungal growth on paraeforce medium. 56
4.4 Measurement
of growth of Pichia kudriavzevii on
paraeforce medium 58
4.5 Measurement of growth of Hanseniaspora opuntiae on paraeforce
medium
60
4.6 Measurement of growth of Aspergillus spinosus on paraeforce
medium 62
4.7 Measurement of growth of Pichia cecembensis on paraeforce medium 64
4.8 Effect
of paraeforce concentration on fungal growth 67
4.9 Rate of
paraeforce degradation by fungal isolates 70
4.10 Optimization
of paraeforce degradation using poultry wastes 73
4.11 Effect
of different paraeforce concentration on soil nitrate 75
4.12 Effect
of different paraeforce concentration on soil organic carbon 78
4.13 Catalase
production by the fungal isolates 81
4.14 Laccase
production by the fungal isolates 83
4.15 Lignin
peroxidase production by fungal isolates 85
4.16 Manganese
peroxidase production by the fungal isolates 87
4.17 Determination
of oxalate concentration in paraeforce degradation 89
4.18 Determination
of formate concentration in paraeforce degradation 91
LIST OF PLATE
4.1 Agarose
gel electrophoresis and sequencing of the extracted fungal DNA 49
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND TO THE STUDY
Microbial biodegradation
of xenobiotics is attracting attention worldwide including the break down of Paraeforce
which is a toxic nitrogen-based compound from paraquat group of herbicides. Paraeforce
is a household herbicide used for weed control in crop farming. It has a
chemical name (IUPAC) 1, 1-dimethyl-4, 4-bipyridinium dichloride. It is a green
coloured liquid with a boiling point of 175–180 OC and is very
soluble in water. Paraeforce is relatively stable at normal temperature and
pressure, and at about neutral pH. These properties enable it to persist longer
in the soil. Figure 1.1 shows the chemical structure of a Paraeforce
Figure 1.1: Structure of Paraeforce
Paraeforce which is
mainly used in agronomy to control weeds in farms has contributed so much to world
food crop production. The use of Paraeforce in crop farming is an augumentation
process aimed at improving crop yield. The use of Paraeforce and other
pesticides helps to confront challenges faced by farmers in their farming
business arising from invasion of many common weeds whose growth is supported
by favorable environmental conditions in the tropical countries. These
environmental conditions include adequate rainfall, favourable temperature, sufficient
sunlight, fertile soil and so on. As such, different types of herbicides are
found in the market. A greater population of the rural farmers are either
uninformed or illiterates and may not be able to read and understand
instructions on herbicides’ use, yet they use same for weed control. The wrong
application of these herbicides results in the contamination of some essential
natural resources such as soil, ground water, rivers and surface runoffs. These
contaminations expose both target and non-target organisms, the environment and
human beings to danger and health hazards. The effectiveness of these
herbicides including Paraeforce in controlling weeds in farms has resulted in
their use by most farmers.
The fate of herbicides applied into the soil is determined by the
processes of transfer and degradation. The transfer processes are usually in
form of percolation into the soil, surface runoffs, flora and fauna uptake. In
all these, the applied chemicals may persist in the soil environment for a
relatively long time. These processes determine the persistence of herbicides,
their effectiveness and the potential for soil and ground water contamination.
Currently
some physicochemical methods are being used in the detoxification of herbicides
aimed at restoration of contaminated soil to its original state. These methods
include among others evaporation, precipitation, electroplating, and
photocatalysis; and are seen to be tasking and expensive and may leave behind
toxic metabolic products that further contaminate the environmnent. Besides,
pesticides applied into the soil can cause environmental problems, affecting the
biochemical and microbial aspects of soil properties. Consequently, there is
the need for less cumbersome and less problematic methods. The new approach
currently being canvassed and is attracting attention in detoxification of Paraeforce
and similar persistent compounds is biodegradation involving living organisms.
Biodegradation brings about complete breakdown of these complex organic
compounds to simpler forms; carbon dioxide and water. Most microorganisms can detoxify
these recalcitrant compounds, mineralize them and/or use them for their growth.
This process is achieved by action of microbial enzymes. The benefits of
microorganisms in this new technology are based on their ability to withstand
unfavourable environmental conditions.
Researches have shown that the degree of pollution of soil and
ground water with these herbicides depends on their persistence, the quantity
and frequency of use; and their toxicity. It will be imperative to know that
most of these herbicides are formulated and designed to persist long enough to
be able to achieve the desired effect on the weeds. Paraeforce posses the property
of binding tightly to clay particles making it very difficult to be removed
once attached to the soil particles. This makes the herbicides biologically
unavailable for detoxification. The ability to bind tightly to soil particles
protects residual herbicides from photocatalysis and from biodegradation,
accounting for its long environmental half-life. In soils containing low amount
of clay, Paraeforce residues can be mobilized due to their water solubility. Herbicides contamination could be long-term and
has a significant impact on decomposition processes and thus nutrient cycling
and their degradation could be expensive and hard to achieve. The need to understand the basic factors affecting the processes
of biodegradation of Paraeforce is very important in order to adopt effective
methods to reduce its persistent period within the soil.
1.2 STATEMENT OF THE PROBLEM
The world-wide use of Paraeforce in
agronomy is an essential part of improving crop yields. This is because it
reduces competition with the invading weeds for limited nutrients and increases
crop yield. The indiscriminate use of it, however, leads to environmental
pollution and its associated health hazards. As farmers continue to see the
need for herbicides usage, large quantities of these chemicals are being
applied to the soil. As such, the fate of the herbicide deposited in the soils
has become a concern and of increasing importance since it could be leached to
contaminate the groundwater (Greer et
al., 2016). This Paraeforce could accumulate to toxic levels in soil and
ground water and become harmful to microorganisms, plants, wild life and man
(Amakiri, 2010; Asogwa and Dongo, 2009; Oluwole and Cheke, 2009).
Paraeforce
is a known carcinogen (EPA, 2003) causing so many health challenges such as
breast cancer, birth defects, heart failure, liver failure and disruption of
endocrine system
(Kumar et al., 2016; Baran et al., 2007).
In rats and rabbits, it causes genetic mutations, benign brain cancer and birth
defects (EPA, 2003).
The conventional flocculation and
coagulation treatment technology cannot adequately remove Pareforce in water
and soil; it requires a more expensive chemical treatment (EPA, 2003;
Cantavenera et al., 2007; Florencio et al., 2004). This is grossly
cumbersome and beyond the reach of peasant rural farmers.
There
is alteration of soil biological activities by regulating its protein synthesis
and enzyme activation (da Silva, 2010). Also, Paraeforce
application influences the dynamics of soil microbial population and diversity
due to death of some sensitive soil microbes (Devashree et al., 2014). Soil pollution with
uncontrolled use of Paraeforce has also affected the soil bioenergetic cycles
by inhibiting or completely eliminating essential energy fixing microbial
population in the soil (Karas et al.,
2011).
1.3
AIM OF THE STUDY
The
aim of this study is to investigate the biodegradation
potential of fungi on Paraeforce (herbicides) impacted soil and the analysis of
some associated enzymes.
1.4
SPECIFIC
OBJECTIVES OF THE STUDY
The specific objectives are to:
1. Determine the physicochemical
properties of the soil samples
2. Isolate and characterize fungal
species from contaminated soil
3. Determine the effect of different
concentrations of Paraeforce on fungal growth.
4. Determine the rate of biodegradation
activity of the isolated fungal species.
5. Optimize the biodegradation process
using poultry wastes
6. Determine the effect of different
concentrations of herbicides on soil nitrate and total organic carbon.
7. Evaluate some fungal enzymes on Paraeforce
degradation.
1.6 SIGNIFICANCE
OF STUDY
This
study is expected to contribute immensely towards enriching the available
technologies employed in the remediation of Paraeforce-impacted soils by use of
fungal living cells or the enzyme extracts. It
will be beneficial to the entire human race in that the health hazard
associated with the use of this herbicide would be reduced to infinitesimal
level or completely eliminated.
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