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       Â
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      Â
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
2.5Â Â Determination of the role of glyphosate as carbon or phosphorus source of the isolates 39
- Determination of the effect of different concentration of Roundup on the isolates                                                                40
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      Â
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 continue to realize the usefulness of herbicides, larger quantities are applied to the soil. However, the fate of these compounds in the soils is becoming increasingly important since they could be leached; in which case groundwater is contaminated or becomes immobile, and may persists on the top soil (Ayansinaet al., 2003). These herbicides could then accumulate to toxic levels in the soil and become harmful to microorganisms, plant, wild life and man (Amakiri, 1982).
Contamination of soil from pesticide mixing, loading, storage and rinsing at agricultural chemical dealership is a concern due to potential contamination of surface water and groundwater (Moormannet al., 1998). There is an increasing concern that herbicides not only affect the target organisms (weeds) but also the microbial communities present in soils, and these non-target effects may reduce the performance of important soil functions. These important soil functions include organic matter degradation, nitrogen cycle and methane oxidation (Hutsch, 2001). Roundup is the clear herbicide of choice for most illiterate farmers; it is used either alone or in combination with other herbicide preparations like 2, 4-Diphenoxyacetic acid apparently to achieve additive or synergistic action. It is mostly used in the rice farm to control post emergence weed. These farmers indulge in the use of Roundup and other herbicides to clear their farms prior to cultivation without cognizance to the obvious ecotoxicological impacts of such practices. The extensive use of Roundup and other herbicides by these farmers is attributable to the aggressive marketing strategies of the representative of the manufacturers in Nigeria who are able to demonstrate to these farmers the wonders their products could achieve, promising them less toiling on their farms with much better results. This is preached without commensurate caveat on the possible toxicity of these chemicals, and highlighting the possible danger of these chemicals to man and his environment
1.1Pesticide toxicology
