ABSTRACT

Solid State Fermentation (SSF) holds tremendous potentials for the production of the enzyme alpha amylase by Aspergillus niger. Agro-industrial wastes present a great potential and support the low production costs for amylase production under Solid State Fermentation by using Aspergillus strains. The substrate, rice bran was used in this research project because it is a good carbon and starch source which has the ability to produce amylase. And also because of its availability and low cost. The fungal alpha amylases are preferred over other microbial sources due to their more accepted GRAS (Generally Recognized As Safe). Among mould species producing high levels of amylase, Aspergillus niger is used for commercial production of alpha amylase. Alpha amylase is widely distributed in nature. It is the most important enzyme and is of great significance in the present day biotechnology. They have diverse applications such as food, textile, paper, detergents as representing approximately 30% of the world enzyme production. The effect of process parameters namely fermentation time, temperature and pH was examined. Cultures were carried out at different temperatures 25oC, 30oC, 35oC, 40oC, 45oC and 50oC. The study of pH was conducted with pH 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0. It was found that alpha amylase production was highest at 5th day (120 hours) with amylase activity of 58.37% of starch hydrolysis, temperature 35oC with activity of 37.63% of starch hydrolysis and pH 6.0 with activity of 58.02% of starch hydrolysis. This investigation suggests a means of production of amylase using rice ban for industrial purposes.

TABLE OF CONTENTS

Title page i

Certification page ii

Dedication iii

Acknowledgement iv

Table of Contents v

List of Tables viii

List of Figures ix

List of Plates x

List of Appendices xi

Abstract xii

CHAPTER ONE

1.0 INTRODUCTION 1

1.1 Introduction 1

1.2 Aims and Objectives 4

CHAPTER TWO

2.0 LITERATURE REVIEW 5

2.1 Production of Alpha Amylase 5

2.1.1 Sources 5

2.1.2 Production Methods 8

2.1.3 Process Parameters 9

2.1.3.1 Temperature 9

2.1.3.2 pH 10

1.1.3.1 Duration of Fermentation 10

2.1.3.4 Moisture 11

2.1.4 Substrate 12

2.1.4.1 Carbon Source 12

2.1.4.2 Nitrogen Source 12

2.1.5 Determination of Enzyme Activity 13

2.1.5.1 Dinitrosalicyclic Acid Method (DNS) 14

2.1.5.2 Nelson-Somogyi (NS) Method 14

2.1.5.3 Determination of Activity using Iodine 14

2.1.5.4 Dextrinizing Activity 15

2.1.5.5 Indian Pharmacopoeia Method 15

2.1.5.6 Reduction in Viscosity of Starch Suspension 15

2.1.6 Purification of Alpha Amylase 16

2.2 Review of other Literatures 17

CHAPTER THREE

3.0 MATERIALS AND METHODS 20

3.1 Materials 20

3.2 Methods 20

3.2.1 Preparation of Sabourad Dextrose Agar (SDA) 20

3.2.1.2 Preparation of Defined Mineral Medium 21

3.2.1.3 Preparation of Rice Bran Medium 21

3.2.2 Lactophenol Cotton Blues Staining of Aspergillus niger. 21

3.2.3 Preparation of Aspergillus niger inocula 22

3.2.4 Solid State Fermentation 22

3.2.5 Assessment of Enzyme Activity (amylase) 22

3.2.6 Optimization of Process Parameters 23

3.3.4.1 Fermentation Time 23

3.3.4.2 Optimization of Temperature 24

3.3.4.3 Optimization of pH 24

3.3.5 Statistical Analysis 24

CHAPTER FOUR

4.0 RESULTS 25

CHAPTER FIVE

5.0 DISCUSSION, CONCLUSION AND RECOMMENDATION 32

5.1 Discussion 32

5.2 Conclusion 32

5.3 Recommendations 35

REFERENCES 36

LIST OF TABLES

Table Title Page

1: Roles/Uses of Alpha Amylase in Biotechnology 19

2: Macroscopic and Microscopic Observation of Aspergillus niger 26

LIST OF FIGURES

Figure Title Page

 1: Optimization of fermentation time for amylase production 29

 2:  Optimization of temperature for amylase production. 30

 3: Optimization of pH for amylase production. 31

LIST OF PLATES

Plate Title Page

I: Standard Structure of Aspergillus niger 7

II: Aspergillus niger colonies 27

III: A microscopic photograph of radiate conidial head 

Aspergillus niger viewed under 40× objective. 28

LIST OF APPENDICES

Appendix Title Page

I: Statistical Analysis Table Results 43

II: One Way ANOVA for Temperature 44

III: One Way ANOVA for pH 46

IV: One Way ANOVA for Fermentation Time 49