ISOLATION OF LIPASE PRODUCING BACTERIA FROM PALM PLANTATION SOIL; A CASE OF OPTIMIZATION

ABSTRACT

Isolation and optimization of Lipase producing bacteria from palm plantation soils of Michael Okpara University of Agriculture, Umudike was carried out using culture techniques. Microorganisms of four genera were isolated from the three samples collected from the areas under study. Bacillus, Staphylococcus and Pseudomonas occurred (100%) in all the samples which suggests them to be most prevalent while Enterobacteria sp occurred in only two samples (67%). Optimization was carried out using Pseudomonas  which showed highest clearance zone in the isolation medium. Maximal lipase production was achieved at the optimum incubation time of 48hours (25.90µ/mol), temperature of 40oC (24.41 µ/mol) and pH of 7.0 (24.59 µ/mol) using olive oil as the standard substrate. The effect of various substrate for optimum production of lipase was also investigated and palm oil was observed to have the peak enzyme activity. (22.07 µ/mol) That is to say that the search for novel gene for enzyme production should be channeled to soil sample exploitation.

TABLE OF CONTENTS

Preliminary Pages

Title Page                                                                                                                    i

Certification                                                                                                               ii

Dedication                                                                                                                  iii

Acknowledgement                                                                                                      iv

Table of Content                                                                                                         v

List of Tables                                                                                                              vi

List of Figures                                                                                                             viii

Abstract                                                                                                                      ix

Chapter One

1.1       Introduction                                                                                                    1

1.2       Aim of Study                                                                                                  4

1.3       Objective                                                                                                        4

Chapter Two

2.0       Literature Review                                                                                           5

2.1       Lipases                                                                                                            5

2.2       Sources of Lipases                                                                                          6

Chapter Three

3.0       Materials And Methods                                                                                  10

3.1       Area of Study                                                                                                  10

3.2       Sample and Sampling                                                                                     10

3.3       Isolation of Lypolytic Bacteria                                                                      10

3.4.0    Characterization of Lypolytic Bacteria Isolates                                             11

3.4.1    Colony Morphology and Characteristics                                                       11

3.4.2    Microscopic Morphological Characteristics                                                  11

3.4.3    Biochemical Reaction Test                                                                             12

3.4.4    Identification of Lypolytic Bacteria Isolate                                                   12

3.5.0    Optimization of Parameters for Lypolytic Bacteria Activity                         12

3.5.1    Optimization of Temperature for Lipase Activity                                         12

3.5.2    Optimization of pH for Lipase Activity                                                         13

3.5.3    Optimization of Incubation Time                                                                   13

3.5.4    Optimization of Substrates for Lipase Activity                                             13

3.6       Extraction of Crude Enzyme                                                                          14

3.7       Determination of Lipase Activity                                                                   14

Chapter Four

4.0       Results                                                                                                            15

Chapter Five

5.0       Discussion                                                                                                       26

5.1       Conclusion                                                                                                      29

5.2       Recommendation                                                                                           30

            References   

LIST OF TABLES

Table 2.1         Bacteria and Fermentation Techniques For Lipase Production

Presented in Resent Literature                                                           9

Table 4.1         Occurrence of Lypolytic Bacteria Isolates                                         16

Table 4.2         Characteristics of Lypolytic Bacteria Isolates                                    17

Table 4.3         Effect of Incubation Time on Lipace Activity Using Pseudomonas (µ/mol)                           18

Table 4.4         Effects of PH for Lipase activity using Pseudomonas (µ/mol)                        19

Table 4.5         Effect of temperature On Lipase Activity using Pseudomonas (µ/mol)                                                                    20

Table 4.6         Effects of Substrate on Lipase activity Using Pseudomonas

(µ/mol)                                                                       21

LIST OF FIGURES

FIG. 4.1           Effect Of Incubation Time On Lipase Activity                                  22

FIG. 4.2           Effect Of Various pH Ranges In Lipase Activity                              23

FIG. 4.3           Effect Of Temperature In Lipase Activity                                         24

FIG. 4.4           Effect Of Various Substrates On Lipase Activity                              25

CHAPTER ONE

1.1    INTRODUCTION

Lipases are by definition water soluble enzymes which have the ability to hydrolyze triacylglycerol to release free fatty acids and glycerol. They constitute a major group of biocatalyst that has immense biotechnological applications. Lipases have been isolated and purified from fungi, yeast, bacteria, plant and animal sources (Joseph, Rantekeand and Thomas, 2008). Of all these, bacterial lipases are more economical and stable (Snellman et al., 2002).

Bacterial lipases are used extensively in the food and dairy industry for the hydrolysis of milk, fat, cheese, ripening, flavor enhancement and lipolysis of butter fat and cream (Falch and Falch, 1991). Lipases are also used in detergent industry as additives in washing powder (Jaeger et al., 1994), in textile industry to increase fabric absorbency (Sharma and Christi 2001) for the synthesis of biodegradable polymers or compounds (Linko et al., 1998) and different transesterification reactions (Farha, Aamer, and Abdulhameed, 2006). In addition, the enzyme is used as a catalyst for the production of different products used in cosmetic industry (Eugene, 1974), in pulp and paper industry to improve quality (Bajpai, 1999) in the synthesis of biodiesel (Noureddini et al., 2005), in degreasing of leather (Nakamura and Nasu, 1990) and pHarmaceutical industry (Sirishaet al., 2010).

Currently, bacterial lipases are of great demand because of their potential industrial applications.

Lipases show stability and more activity in higher temperature and they are usually stable in the presence of chemicals (Castro-Ochra et al., 2005; Messias et al., 2009;Ultatree et al., 2010). High global demandfor lipases and billion-dollar business (more than 1000 ton each year which makes lipase the third largest group of enzymes based on total sales volume after protease and carbohydrate) has resulted in increased number of research to identify, isolate and introduce new lipase-producing microorganisms (Hasan et al., 2006; Shu et al., 2010; Treichel et al., 2010).

Majority of lipases currently used in the industry have microbial sources (Castro-Ochra et al., 2005; Hassan et al.,2006; Nthangein et al., 2001; Adiguzel et al., 2009). Some of theadvantages of these bacterial lipases include high and wide range conditions, stability, simplicity, ease of mass production and gene manipulation, activity in extreme conditions (high/low temperature and PH),no requirement for cofactors, high specificity and low waste production, the possibility of use in continuous operation, ease of recovery and re-use, cost benefit and low required downstream processing (Haba et al., 2000; Sheikhet al., 2003; Treichel et al., 2010).

One of the richest sources for identification and isolation of lipases producing organisms is the soil. Such studies introduce new lipases with stability in different temperature and PH, specificity of certain fatty acids and substrates and enantioselectivity. Some important lipase-producing genera include Pseudomonas,Bacillus, Burkholderia etc. Lipases as mentioned earlier are used in many industrial applications such as pharmaceuticals, food, detergent, paper and pulp, agro chemicals, surfactants and bioremediations (Pogori et al., 2007; Sharma et al., 2011). The main function of these enzymes is to hydrolyze triglycerides to glycerol and free fatty acids at an oil-water interface (Kulkarim and Garde et al.,2002; Palroja and Sethar, 2002). Lipases can be produced from various sources e.g. animals, plants and microorganisms.

However, for industrial applications, Lipases from microorganisms are more interesting because

1.     They can be produced in the high yields

2.     There are many varieties of catalytic activities which can be used in many applications

3.     The genetic manipulation is available (Hassan et al., 2006)

Several studies reposted the elevation of bacterial lipase production by addition of some metal ions such as Na⁺, Ca²⁺, Mg²⁺ in the culture medium (Josephet al., 2006; Kader et al., 2007; Sharma et al., 2009). It was explained that Ca ²⁺ may help the structure formation of active enzymes leading to higher enzyme activity. However, the mechanism of metal ions as a lipase enhancer are still not clearly understood.

Lipases in recent times have emerged as key enzymes in swiftly growing biotechnology, owing to their multi-facetted properties which find usage in wide varieties or array of industrial applications such as food technology, detergent, chemical industries and biomedical sciences (Jaeger et al., 1994,1999; Pandey et al., 1999). The natural substrates of lipases are long chain triacylglycerol which have very low solubility in water and the reaction is catalyzed at the lipid-water interface. Under micro-aqueous conditions, lipases possess the unique ability to carry out the reverse reaction leading to esterification, alcoholysis and acidolysis. Besides being lipolytic, lipases also possess esterolytic activity and thus have a very diverse substrate range, although they are highly specific as chemo-, region-, and enantioselective catalyst (Jaeger et al., 1994, 1999; Jaeger and Reetz 1998; Kazlauskas and Bornschew 1998; Pandey et al., 1999; Beisson et al., 2000; Gupta and Soin 2000; Jaeger and Eggert, 2002).

In this study, the lipase producing bacteria were isolated from palm plantation and soils within MOUAU. Isolate showing the highest lipase activity was identified and its culture conditions for maximal lipases activity was investigated.

1.2    AIM OF STUDY

The sole aim of this research work is to isolate lipase producing bacteria from palm plantation soils collected from areas within Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria and to determine the optimization conditions of the enzyme.

1.3    OBJECTIVES

The study has the following objectives;

·       To isolate and identify lipase producing bacteria from palm plantation soils

·       To determine the percentage occurrence of isolated bacteria

·       To determine the optimal conditions of the bacteria for maximum lipase production

·       To represent result graphically

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