ABSTRACT
Bacterial biodegradation of xenobiotics has been seen as one of the biological means of bioremediation of polluted site. This study was designed to isolate bacteria from soil and to evaluate their ability to biodegrade glyphosate which is a water soluble, non-selective herbicide used to kill weeds, especially annual broadleaf weeds and grasses known to compete with commercial crops grown around the globe. The bacteria that were able to grow in the presence of glyphosate were isolated using culture techniques and were identified as Bacillus sp, and Pseudomonas sp. All the isolates recorded the highest growth rate in the presence of glyphosate at the concentration of 7.2 mg/ml and least growth rate at concentration of 200 mg/ml. The growth rate decreased with increase in glyphosate concentration. The Monod constants, half saturation constant (ks) and maximum growth rate (µmax) for Bacillus sp were determined as 7.15 mg/ml and 0.59 h¯1, that of Pseudomonas sp, 6.15 mg/ml and 0.62 hˉ1 respectively. The Monod constants for the consortium, half saturation constant (ks) and maximum specific growth rate (µmax) of Bacillus and Pseudomonas spp were 3.65 mg/ml and 0.65 hˉ1 respectively. This study demonstrates that the organisms were more effective in degrading glyphosate when used as consortium than when they are used separately.
TABLE OF CONTENTS
Title Page i
Approval Page ii
Dedication Page iii
Acknowledgement iv
Abstract v
Table of Content vi
List of Figure xi
List of Abbreviation xii
CHAPTER ONE: INTRODUCTION
1.1 Pesticide toxicology 2
1.2 Biomarkers in exotoxicology 3
1.3 Roundup 4
1.3.1 Glyphosate 4
1.3.2 Glyphosate trade names 6
1.3.3 Uses of glyphosate 6
1.4. Physicochemical properties of glyphosate 7
1.4.1 Method of application of glyphosate 7
1.4.2 Mode of action 8
1.4.3 Glyphosate metabolism 11
1.4.4 Adsorption of glyphosate 13
1.4.5 Chemical decomposition of glyphosate 13
1.5. Behaviours of glyphosate in the environment 13
1.5.1 Soil 13
1.5.2 Water 14
1.5.3 Vegetation 14
1.6.0 Persistence and movement of glyphosate in the soil 15
1.6.1 Glyphosate and water contamination 16
1.7.0 Glyphosate toxicity 16
1.7.1 Acute toxicity of glyphosate 17
1.7.1.1 Acute toxicity of glyphosate to laboratory animals 17
1.7.1.2 Acute toxicity of glyphosate to humans 17
1.7.2 Subchronic toxicity of glyphosate 18
1.7.3 Chronic toxicity of glyphosate 18
1.7.3.1 Glyphosate and carcinogenicity 18
1.7.3.2 Genotoxicity and mutagenicity of glyphosate 19
1.7.3.3 Chronic toxicity of glyphosate on mammalian enzymes 22
1.7.3.4 Chronic toxicity of glyphosate on endocrine system 23
1.7.3.5 Effect of glyphosate on the reproductive system 26
1.8. Effects of glyphosate on nontarget animals 26
1.8.1 Effects of glyphosate on beneficial insects 26
1.8.1.2 Effect of glyphosate on other insects 27
1.8.1.3 Effects of glyphosate on arthropods 27
1.8.1.4 Effects of glyphosate on earthworms 27
1.8.1.5 Effect of glyphosate on birds 28
1.8.1.6 Effect of glyphosate on small mammals 28
1.8.1.7 Effect of glyphosate on fish 29
1.8.2 Effects of glyphosate on nontarget plants 30
1.8.3 Effect of glyphosate on seed quality 30
1.8.4 Effect of glyphosate on nitrogen fixation 30
1.8.5 Effect of glyphosate on soil microorganism 31
1.8.5.1 Glyphosate and mycorrhizal fungi 32
1.8.6 Glyphosate and plant diseases 32
1.8.7 Glyphosate and weed resistance 33
1.9 Aim and objectives 33
CHAPTER TWO: MATERIALS AND METHODS
2.1 Material 34
2.1.1 Soil sample 34
2.1.2 Herbicide 34
2.1.3 Location 34
2.1.4 Equipment 34
2.2 METHODS 35
2.2.1 Preparation of soil samples 35
2.2.2 Preparation of isolation medium 35
2.2.3 Isolation of glyphosate utilizing bacteria 35
2.2.4 Identification of the isolated bacteria 35
2.2.5 Morphological characteristics of the isolates 36
2.2.5.1 Colony morphology 36
2.2.5.2 Cell morphology 36
2.2.6 Gram staining 36
2.2.7 Spore staining 36
2.2.8 Biochemical characteristics of the isolates 37
2.2.8.1 Catalase test 37
2.2.8.2 Oxidase test 37
2.2.8.3 Indole test 37
2.2.8.4 Coagulase test 37
2.2.8.5 Sugar fermentation test 37
2.2.8.6 Citrate utilization test 38
2.2.8.7 Starch hydrolysis test 38
2.2.8.8 Methyl red test 38
2.2.8.9 Vorges-proskauer test 39
2.2.9 Storage of pure bacteria isolates 39
2.3 Inoculum’s preparatios 39
2.4 Determination of glyphosate utilization patterns of the isolates 39
2.5 Determination of the role of glyphosate as carbon or phosphorus source of the isolates 39
CHAPTER THREE: RESULTS
3.1 Isolation of Glyphosate utilizing bacteria 41
3.2 Identification of the isolates 43
3.3 Effect of glyphosate as carbon or phosphorus source on the isolates 44
3.3.1 Effect of glyphosate as a carbon or phosphorus source on Bacillus sp44
3.3.2 Effect of glyphosate as a carbon or phosphorus source on Pseudomonas46
3.4 Effect of different concentrations of glyphosate on the isolates 48
3.4.1 Effect of different concentrations of glyphosate on pseudomonas sp48
3.4.2 Effect of different concentrations of glyphosate on the Bacillus sp 50
3.4.3 Effect of different concentrations of glyphosate on the consortium 52
3.5 Determination of kinetic parameters of Bacillus sp 54
3.5.1 Determination of kinetic parameters of Pseudomonas sp 56
3.5.2 Determination of kinetic parameters of the Consortium 58
CHAPTER FOUR: DISCUSSION
4.1 Discussion 60
4.2 Conclusion 63
References 64
LIST OF FIGURES
Fig. 1: Different structures of glyphosate 5
Fig. 2: Inhibition of shikimic acid pathway by glyphosate 10
Fig. 3: Degradation routes of glyphosate in soil 12
Fig. 4: Isolation of glyphosate utilizing bacteria using 7.2mg/ml of glyphosate 42
Fig. 5: Effect of glyphosate as a carbon or phosphorus source on Bacillus sp 45
Fig. 6: Effect of glyphosate as a carbon or phosphorus source on Pseudomonas sp 47
Fig 7: Growth curve of Pseudomonas sp in different concentrations of glyphosate 49
Fig 8: Growth curve of Bacillus sp in different concentrations of glyphosate 51
Fig 9: Growth curve of the consortium in different concentrations of glyphosate 53
Fig 10: Lineweaver-Bulk plot for cell growth and glyphosate utilization of Bacillus sp55
Fig 11: Lineweaver-Bulk plot for cell growth and glyphosate utilization of
Pseudomonas sp 57
Fig. 12: Lineweaver-Bulk plot for cell
growth and glyphosate utilization of the consortium 59
LIST OF ABBREVIATIONS
ACHe: Acetylcholinestrase
Ae: Acid equivalent
ai: Active ingredients
ALT: Alkaline phosphatase
AMPA: Aminomethylphosphonic acid
AST: Aspartate aminotranferase
ATP: Adenosine Triphosphate
DAHP: Skimate -3-deoxy-d-arabinoheptulose-7- phosphate
DNA: De-oxy Ribonucleic acid
EPA: Environmental Protection Agency
ESPS: 5-enolpyruvylshikimate-3-phosphate synthase
G6PD: Glucose-6-phosphate dehydrogenase
HCE: Hairy cell leukemia
LDH: Lactate Dehydrogenase
MSM: Mineral salt Media
NADPH: Nicotinamide adenine dinucleotide phosphate
NMR: Nuclear M010agnetic Resonance
NTP: National Toxicology Program
PEP: Phosphoenol pyruvate
Pi: Inorganic phosphate
POEA: Polyoxyethyleneamine
S3P: Shikimate -3-phosphate
StAR: Steriodogenic acute regulatory protein
WHO: World Health Organisation
CHAPTER ONE
INTRODUCTION
The need to feed the world’s increasing population has prompted the use of agrochemicals to increase food production and ensure the continuation of the human race. Such agrochemicals include pesticides like 2, 4-diphenoxyacetic acid, (2, 4-D), several formulations of inorganic fertilizer and the subject of this study Roundup. The increased use of pesticides in agricultural soils causes the contamination of the soil with toxic chemicals. When pesticides are applied, the possibilities exist that these pesticides may exert certain effects on non-target organisms, including soil microorganisms (Simon-Sylvestre and Fournier, 1979; Wardle and Parkinson, 1990). The microbial biomass plays an important role in the soil ecosystem where they play a crucial role in nutrient cycling and decomposition (De-Lorenzo et al., 2001). During the past four decades, a large number of herbicides have been introduced as pre and post-emergent weed killers in many countries of the world. In Nigeria, herbicides have since effectively been used to control weeds in agricultural systems (Adenikinju and Folarin, 1976). As farmers co
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