BIODEGRADATION KINETICS OF THE HERBICIDE, ROUNDUP BY SOME SOIL MICROORGANISMS

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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

  • 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

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

BIODEGRADATION KINETICS OF THE HERBICIDE, ROUNDUP BY SOME SOIL MICROORGANISMS