THE ISOLATION OF BIOSURFACTANT PRODUCING BACTERIA IN FLORAL NECTAR, SCREENING OF THE ISOLATES USING THE DROP COLLAPSE METHOD AND CHARACTERIZATION OF THE ISOLATED STRAINS.

Abstract

Biosurfactants are amphiphilic molecules that have both hydrophilic and hydrophobic moieties which partition preferentially at the interfaces such as liquid/liquid, gas/liquid or solid/liquid interfaces. Such characteristics enable emulsifying, foaming, detergency and dispersing properties. Their low toxicity and environmental friendly nature and the wide range of potential industrial applications in bioremediation, health care, oil and food processing industries makes them a highly sought after group of chemical compounds. Interest in them has also been encouraged because of the potential advantages they offer over their synthetic counterparts in many fields spanning environmental, food, biomedical, petrochemical and other industrial applications. Their large scale production and application however are currently restricted by the high cost of production and by the limited understanding of their interactions with cells and with the abiotic environment.

TABLE OF CONTENTS

Certification i

Dedication ii

Acknowledgement iii

Table of contents iv

List of Tables vii

List of Figures viii

List of Plates    ix Indent                         x

Abstract xi

CHAPTER ONE

INTRODUCTION

1.1 Background of the study 1

1.2 Statement of the Problem 3

1.3 Objective of the Study 3

CHAPTER TWO

LITERATURE REVIEW

2.1 Microorganisms producing biosurfactant 4

2.2 Applications Of Biosurfactant 7

2.3 Biosurfactant Production Substrates 8

2.3.1 Agro-industrial waste 9

2.3.2 Vegetable Oils and Oil Wastes 10

2.3.3 Olive Oil Mill Waste Effluents (OMWE) 11

2.3.4 Starchy substrates 12

2.3.5 Industrial waste from animals and plant origin 13

2.4 Influence of culture medium composition on Biosurfactant production 15

2.4.1 Carbon source 15

2.4.2 Nitrogen source 16

2.5 Environmental factors affecting the production of Biosurfactant 17

2.6 Classes of Biosurfactant 20

2.7 Properties of Biosurfactant 23

CHAPTER THREE

MATERIALS AND METHODS

3.1 Sample Collection 26

3.2 Preparation and Sterilization Of Media 26 3.3 Isolation and Screening Of Bacterial Isolates 27

3.4 Morphological and Biochemical Characterization of Biosurfactant

Producing Isolates 27 

CHAPTER FOUR

RESULTS

 4.1 Isolation And Screening Of Isolates for  Biosurfactant Production 32

4.2 Morphological And Biochemical Characterization Of Biosurfactant

Producing Isolates 36

4.3 Degree of Occurrence of Biosurfactant Producing Microorganisms 38

CHAPTER FIVE

DISCUSSION, CONCLUSION AND RECOMMENDATION

5.1 Discussion 39

5.2 Conclusion 40

5.3 Recommendation 41

REFERENCES 42

LIST OF TABLES

Table     Title       Page

2.1  List of Biosurfactant producing organisms 4

3.1 Composition of Mineral Salt Medium(MSM) 26

4.1 Biosurfactant production by distinct isolates 32

4.2 Biochemical characterization of Biosurfactant producing isolates 38

4.3 Frequency of occurrence of Biosurfactant producing organisms 39

LIST OF FIGURES

Figure             Title             Page

2.1 Structure of glycolipids   20

2.2 Structure of mono and di rhamnolipid 18

2.3 Structure of Trehalose lipid 18

2.4 Structure of sl lactonized and acid form 19

2.5 Structure of aminolipopeptide and cyclic aminolipopeptide 20

LIST OF PLATES

Plate                                                        Title                                                      Page

1 Biosurfactant testing of isolates using drop collapse method 34

2 Culture plate showing physical characteristics of some isolates 35

APPENDIX

Appendix               Title  Page

1 Cross section of flower samples used for this work 54

2          Vaginalis flower specie 55

3 Hibiscus flower 56

CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND OF THE STUDY

 Bio-surfactant is a structurally diverse group of a surface-active molecule, synthesized during microbial growth. These bio-surfactants are extracellular products released by microorganisms growing on decaying hydrocarbons also, they are amphiphilic compounds consisting of hydrophilic and hydrophobic domains. The hydrophilic domain can be carbohydrate, amino acid, phosphate group or some other compounds whereas the hydrophobic domain usually is a long chain fatty acid (Lang, 2002). The majority of known biosurfactants are synthesized by microorganisms grown on water immisible hydrocarbons, but some have been produced on water soluble substrate such as glucose, glycerol and ethanol (Abu-Ruwaida et al.,1991). Microorganisms have been reported to produce several classes of biosurfactants such as glycolipids, lipopeptides, phospholipids, neutral lipids or fatty acids and polymeric biosurfactants (Franzetti et al., 2010; Banat et al., 2010).This bi-polar characteristic makes it energetically favourable for biological and synthetic surfactants to aggregate at fluid-fluid, fluid-air, and fluid-solid interfaces. Such aggregation determines the arrangement of liquid molecules at an interface. This subsequently influences interactions between hydrophilic and hydrophobic entities within the system. The overall result is a reduction of surface and interfacial tensions (Fiechter,1992; Jones,1997; Rouse et al., 1994; Shafi and Khanna, 1995).

Great emphasis has recently been given to the environmental impacts caused by chemical surfactants due to their toxicity and difficulty in being degraded in the environment( Van et al.,2006). Increasing environmental concerns, the advance in biotechnology and the emergence of more stringent laws have led to biosurfactants being a potential alternative to the chemical surfactants available on the market( Banat et al .,2000 and Henkel et al.,2012). Although biosurfactants have promising use in bioremediation processes, their  industrial scale production is currently difficult due to high raw material costs, high processing costs and low manufacturing output (Henkel et al.,2012). As a result, the current research challenges are to increase the yield and to reduce the cost of raw materials (Mukherjee,et al.,2006). The number of publications and patents involving biosurfactants has recently increased considerably (Müller et al.,2012). Although many biosurfactants and their manufacturing processes have been patented, only some of them have been commercialized. EC-601 (EcoChem Organics Company), a dispersive agent of water-insoluble hydrocarbons containing rhamnolipids, and PD5 (Pendragon Holdings Ltd), an additive for fuels based on a mixture of rhamnolipid biosurfactants and enzymes, are examples of biosurfactant-based products commercially available (Kronemberger et al.,2008)]. Several studies have aimed to optimize the biosurfactant production process by changing the variables that influence the type and amount of biosurfactant produced by a microorganism. Important variables are carbon and nitrogen sources (Santos et al.,2002), potential nutrient limitations and other physical and chemical parameters such as oxygen (Kronemberger et al.,2008), temperature and pH (Mukherjee et al.,2006) . Recent studies have also focused on in situ production from renewable substrates, resulting in the so called new generation of biosurfactant production (Henkel et al.,2012) as well as metabolic engineering strategies and strain improvements to enhance the metabolic fluxes towards the product (Mukherjee et al.,2006).Although the function of biosurfactants in microorganisms is not fully understood,it is known that these secondary metabolites can enhance nutrient transport across membrane,act in various host-microbe interactions and provide biocidal and fungicidal protection to the producing organisms(Lin,1996;Banat,1995a;Banat,1995b).

However,it is the ability of the biosurfactant producers to reduce interfacial surface tension,which has important tertiary oil recovery and bioremediation consequences. (Lin,1996;Rouse et al.,1994;Volkering et al.,1998).Many of the known biosurfactant producers are also hydrocarbon-degrading organisms (Rouse et al.,1994;Willumsen and Karlson,1997;Volkering et al.,1998). A drop collapsing test was advised for screening bacterial colonies that produce surfactants. (Bento et al.,2005) used the reduction of surface tension and the emulsify capacity to screen biosurfactant producing microorganisms. Hence, the study screened for the diversity and distribution of surface-active agent producing bacteria from contaminated soil and water that will be of potential use in the remediation of mixture of oils polluted areas.

1.2 STATEMENT OF THE PROBLEM

chemical surfactants cause environmental impacts due to their toxicity and difficulty in being degraded in the environment and this Increases environmental concerns ( Van et al.,2006) thereby the need for the production of non toxic surfactants.

1.3 OBJECTIVE OF THE STUDY

My study was based on the isolation of biosurfactant producing bacteria in floral nectar, screening of the isolates using the drop collapse method and characterization of the isolated strains.

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