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
CHAPTER ONE
2.0 INTRODUCTION
1.1 Introduction
This genus Aspergillus includes over 185 species and about 20 species have so far been reported as causing opportunistic infections in man (Prescott et al 2005; Tortora et al., 2004). Among the species, A. fumigatus is the most commonly isolated species followed by A. flavus (Liang et al., 2005). A niger, A. clavatus, A. glancus, A. oryzae, A. terreus, A. ustus and A. versicolor are among the other species that are not commonly isolated and are considered as no non opportunistic pathogens (Brock and Madigan, 1991; Brock et al., 1994). Several reports have been published by many researchers showing amylase production with Aspergilus species (Ellaiah et al., 2002; Ahmed et al., 2010). A niger in fermentation of oriental food products or industrial application of hydrolytic enzymes. Among mold species producing high levels of amylase, Aspergillus niger is used for commercial production of alpha amylase.
Alpha amylase (α-1, 4-D-glucan glucanohydrolase) is a starch digesting enzyme that randomly cleaves α-1, 4-D-glucosidic linkages in starch molecules. It releases short chain oligosaccharides and alpha limit dextrin, in addition to maltotriose and maltose. Neither terminal glucose residues nor α-1,6 linkage can be cleaved by alpha amylase. Alpha 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 in a wide variety of industries such as food, textile, paper, detergent as representing approximately 30% of the world enzyme production (Calik and Ozdamar, 2001).
It can be used in field related with biotechnology such as; removing environmental pollutant, conversion of starch to desired substrate by many micro-organisms, infiltration of waste contains starch and production by chemical materials with helping starch substrate. With the advent of new frontiers in biotechnology, the spectrum of amylase application has expanded into amylase application has expanded into many other fields, such as clinical, medicinal and analytical chemistry. Interestingly, the first enzyme produced industrially was an amylase from a fungal source in 1894, which was used as a pharmaceutical aid for the treatment of digestive disorders (Pandy et al., 2006).
Alpha amylase can be obtained from animals, plants and micro-organisms. However, the amount of enzymes synthesized by plants and animals is so low that it cannot meet the demands of the market. In contrast, adequate amount of alpha amylase can be obtained from native microbial sources. This has therefore necessitated the screening of micro-organisms like bacteria and fungi for α-amylase production (Shibuya et al., 1992; Ivanova et al., 2001). Bacterial sources of α-amylase are mainly derived from the genus Bacillus. Bacillus stearothermophilus, Bacillus amyloliquefaciens are known to be good producers of Thermostable α-amylase and these have been widely used for commercial production of the enzyme for various application (Prakash and Jaiswal, 2009). Most reports about alpha amylase have been limited to few species of mesophilic fungi and attempts have been made to specify the cultural conditions and to select superior strains of the fungus to produce on a commercial scale (Gupta et al., 2003).
Fungal sources are confined to terrestrial isolates, mostly to Aspergillus and Penicillium (Kathiresan and Manivannan, 2006). Among the filamentous genera of fungi that serve as a source of alpha amylase, Aspergillus and Penicillium are most significant and several other fungi like Trichoderma specie, Thermomyces lanuginosus, Fusarium moiliformis, Actinomyces and Alternaria specie have the ability to produce large quantity of extracellular enzymes making them suitable for enzyme production at industrial scale (Poornima et al., 2008). The Aspergillus species produce a large variety of extracellular enzymes and amylases are the ones with most significant industrial importance (Hermander et al., 2006).
Filamentous fungi, such as Aspergillus oryzae and Aspergillus niger, produce considerable quantities of enzymes that are used extensively in the industry. A. oryzae has received increased attention as a favourable host for the production of haterologus proteins because of its ability to secrete a vast amount of high value proteins and industrial enzyme example alpha amylase (Jin et al., 1998).Aspergillus niger has important hydrolytic capacities in the alpha amylase production and due to it`s tolerance of acidity (pH<3), it allows the avoidance of bacterial contamination (Djekrif-Dakhmouche et al., 2006). Filamentous fungi are suitable micro-organisms for solid state fermentation (SSF), especially because their morphology characteristics that allows them to colonize and penetrate the solid substrate (Rajagopalan and Krishnan, 2008). The fungal alpha amylases are preferred over other microbial sources due to their more accepted GRAS (Generally Recognized As Safe) status (Gupta et al., 2003).
Alpha amylase can be produced both by sing solid and submerged fermentation techniques (Rahardle et al., 2005). Solid State Fermentation (SSF) systems appear promising due to the natural potential and advantages they offer. SSF resembles the natural habitat of micro-organism and is therefore, the preferred choice for micro-organisms to grow and produce useful value added products. Submerged fermentation (SmF) can be considered as a violation of their natural habitat, especially of fungi (Singhania et al., 2009). Fungi and yeast were termed as suitable micro-organisms for SSF according to the theoretical concept of water activity, whereas bacteria have been considered unsuitable. However, experience has shown that bacterial cultures can be well managed and manufactured for SSF processes (Pandey, 2003). There are other advantages of SSF over SmF, including superior productivity, simpler technique, lower capital investment, lower energy requirement and less water output, better product recovery and lack of foam build up, besides it is reported to be the most appropriate process for developing countries. Recently, research evaluated whether SSF is the best system for producing enzymes and higher yields can be obtained when compared to SmF (Couto and Sanroman, 2006; Tanyildizi et al., 2007).
Agro industrial wastes such as wheat bran, rice bran have been reportedly used as good substrates for the effective cost cutting in the production of alpha amylases (Pandey et al., 1995). In a study using different agro industrial waste such as wheat bran, rice bran, corn, rice straw, sugar cane bagasses and green gram husk showed that highest enzyme production was obtained with wheat bran as the substrate followed by rice bran. (Murali et al., 2012; Satyanagalakshmi et al., 2014). Rice bran was used in this study because of its availability , low cost and also it is a good carbon and starch source which has the ability to produce alpha amylase. The availability and the cost of the raw materials are two important parameters that have to be considered while selecting a raw material in solid state fermentation (Pandey et al., 2000).
Optimization of process parameters is of utmost importance for better production of alpha amylase. The purpose of optimization is to find out conditions under which the fungus can flourish luxuriantly and produce alpha amylase in maximum amount which paves the way to its effective and fruitful commercialization. The determination of optimum time and temperature is important for the better yield of the enzyme. Power of H+ (pH) is an important factor that determines the growth of the enzyme producing micro-organisms as well as the extent to which they can produce alpha amylase production, the determination of the initial pH of the medium becomes obligatory in the process of optimization.
1.2 AIMS AND OBJECTIVES
(1) To investigate the production of alpha amylase by Aspergillus niger grown in growth medium containing rice as carbon and growth source for industrial purposes.
(2) To determine the optimal fermentation time, pH and temperature for alpha amylase production by Aspergillus niger using Solid State Fermentation of agro-industrial waste.
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