PRODUCTION OF ALPHA AMYLASE BY Aspergillus niger USING SOLID STATE FERMENTATION OF AGRO-INDUSTRIAL WASTE (RICE BRAN)

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