AMYLASE PRODUCTION BY SOLID STATE FERMENTATION OF AGRO-INDUSTRIAL WASTE USING BACILLUS SPECIES

ABSTRACT

This study evaluated amylase production by Bacillus species employing the solid state fermentation method, agro-industrial waste were used as the substrate. Five species were tested for amylase production abilities. Bacillus subtilis showed the highest amylase production ability after incubation. Corn chaff gave maximum enzyme production (3.25U/ml) at 300C, while the least enzyme was recorded on groundnut husk (2.35U/ml) at 250C Potato peel had maximum enzyme production by Bacillus subtitlis (3.05U/ml) at ph 7.0,while the least enzyme production was from groundnut husk (2.84U/ml) at pH 4.0.Thus there was an increase in enzyme production with corresponding increase in substrate concentration. The results obtained in this study support the suitability of using agro-industrial wastes as solid for high production of amylase, and is a means of solving pollution problems, thus making solid state fermentation (SSF) an attractive method.

TABLE OF CONTENTS

Title Page                                                                                                                                i

Certification                                                                                                                           iii

Dedication                                                                                                                              iv

Acknowledgement                                                                                                                  v

Table of Contents                                                                                                                   vi

List of Tables                                                                                                                          vii

Abstract                                                                                                                                  viii

CHAPTER ONE

1.0       Introduction                                                                                                                1

1.1       Aim and Objectives                                                                                                    4

CHAPTER TWO

2.0       Literature Review                                                                                                       5

2.1       Solid-State Fermentation                                                                                            5

2.2       Microorganisms Associated with Solid State Fermentation                                      7

2.3       Substrates for Solid State Fermentation                                                                     8

2.3.1    Lignocellulose                                                                                                            9

2.3.2    Pectins                                                                                                                         10

2.3.3    Lignin                                                                                                                         10

2.3.4    Starch                                                                                                                          10

2.4       Environmental Factors Affecting Solid State Fermentation                                      11

2.4.1    Moisture Content and Water Activity (Aw)                                                               11

2.4.2    Temperature and Heat Transfer                                                                                  12

2.4.3    Control of pH and Risks of Contamination                                                               13

2.4.4    Oxygen Uptake                                                                                                           13

2.5       Optimization of Agro-residues for Αlpha-Amylase Production by

            Bacillus subtilis and its Application in Detergent Industry                                        14

2.5.1    Optimization of Agro-Residues for Αlpha-Amylase Production                                    14

2.5.2    Optimization of Process Parameters                                                                           15

2.5.3    Amylase Assay                                                                                                           16

2.5.4    Partial Purification of Αlpha-Amylase                                                                       16

2.6       Amylase Production by Solid-State Fermentation of Agro-Industrial

            Wastes Using Bacillus spp                                                                                         17

2.7       Bio-processing of Food Industrial Waste for Amylase Production by

            Solid State Fermentation                                                                                            18

2.8       Solid Substrate Fermentation Using Agro Industrial Waste for Amylase

            Production by Bacillus Licheniformis                                                                        20

2.8.1    Agro-Waste as Substrates for Amylase Production                                                   20

2.8.2    Fermentation Medium                                                                                                21

CHAPTER THREE

3.1       Materials and Methods                                                                                               22

3.2       Collection of Substrate                                                                                               22

3.3       Test Bacterial Used                                                                                                     22

3.4       Screening of Test Bacterial                                                                                        22

3.5       Solid State Fermentation Technique                                                                          22

3.5.1    Enzyme extraction                                                                                                      23

3.6       Amylase Enzyme Assay                                                                                             23

3.7       Optimization of Fermentation Parameters for Amylase Activity                                    23

3.7.1    Effect of Incubation Period on Amylase Activity                                                      24

3.7.2    Effect of Incubation Temperature on Amylase Activity                                            24

3.7.3    Effect of pH of the medium on amylase Activity                                                      24

3.7.4    Effect of Substrate Concentration on Amylase Activity                                            24

3.8       Statistical Analysis                                                                                                     24

CHAPTER FOUR

4.0       Results                                                                                                                        25

CHAPTER FIVE

5.0       Discussion and Conclusion                                                                                        31

5.1       Discussion                                                                                                                   31

5.2       Conclusion                                                                                                                  35

            References

LIST OF TABLES

CHAPTER ONE

1.0       INTRODUCTION

Amylase is one of the most widely used enzymes in the industry. It hydrolyses starch and is used commercially for the production of sugar syrups from starch which consist of glucose, maltose, and higher oligosaccharides (Hagihara et al., 2001). Amylases are of great significance in biotechnological applications ranging from food, fermentation, detergent, pharmaceutical, brewing and textile to paper industries (Kathiresan, and Manivannan, 2006). To meet the higher demands of these industries, low cost production of amylase is required.

The amylases can be derived from several sources, such as plants, animals and micro-organisms. Because of their short growth period, the enzymes from microbial sources generally meet industrial demands (Odee, et al., 2007). The first enzyme produced industrially was an amylase from a fungal source in 1994, which was used for the treatment of digestive disorders (Crueger, and Crueger, 2009).

Amylase is produced in bacteria, fungi, plants and animals, the major bacteria belong to Bacillus species and fungi such as Aspergillus niger, Penicillium sp., Cephalosporium and Rhizopus are the major α-amylase producing microorganisms (Suganthi et al., 2011). However, due to efficient production strategies, microorganisms have substantial potential to contribute to a number of industrial applications (Sodhi et al., 2005). Such industrially important microorganisms are found within the Bacillus species because of their rapid growth rates that lead to short fermentation cycles, their capacity to secrete proteins into extra cellular medium and general handling safety (Pandey et al., 2010).

Production of these α amylases has been investigated through submerged (SmF) and solid-state fermentation (SSF) (Perez-Guarre et al., 2003). However, the contents of a synthetic medium are very expensive and uneconomical, so they need to be replaced with more economically available agricultural and industrial byproducts, as they are considered to be good substrates for SSF to produce enzymes (Kunamnen et al., 2005). In recent years the technique of solid-state fermentation (SSF) process has been developed and used more extensively. It has advantages over SmF like simple technique, low capital investment, cheaper production of enzyme having better physiochemical properties, lower levels of catabolite repression and better product recovery (Baysal et al., 2003). The major factors that affect microbial synthesis of enzymes in a SSF system include selection of a suitable substrate and microorganism, particle size of the substrate, inoculums concentration and moisture level of the substrate. Thus it involves the screening of a number of agro-industrial materials for microbial growth and product formation (Sodhi, et al., 2005). Temperature and pH are known to be important parameters in the production of enzymes from bacteria; hence, the thermal and the Ph stability of the enzyme, which is a function of the exposure time, must also be taken into account.

Several terrestrial microorganisms have been used for enzyme production, for example, protease, xylanase, amylase, chitinase, cellulase, lipase, and inulinase, (De-Azeredo et al., 2001). However, limited studies are reported on amylase production from marine microbial strains (Chakraborty et al., 2009). Moreover, marine microorganisms are reported to produce enzymes with industrially-important properties, such as stability at elevated temperature and alkaline pH conditions (Ventosa, and Nieto, 2005). These bacterial characteristics are essentially important for amylase production using cost-effective materials as energy sources.

High fermentation medium cost is one of the major concerns in amylase production from microbial sources. Amylase demand is continuously increasing and researchers are attempting to establish economical fermentation processes. Several agro-industrial residues have been utilized for amylase production and many other value-added products. Researchers are looking for novel microbial strains to produce amylase enzymes with industrially-important properties, for example, alkaline pH-stable, thermo-stable, and surfactant-stable amylase. In addition, those microorganisms should utilize agricultural waste material as cost effective carbon and nitrogen sources. Therefore this present study focuses on the production of amylase enzyme by solid state fermentation of different agro-industrial wastes (corn cobs, potato peel and maize straw) using Bacillus spp.

1.1       AIM AND OBJECTIVES

To produce amylase by solid state fermentation of agro industrial wastes (corn cobs, potato peel and maize straw) using bacillus spp. while the specific objectives are;

1.     To screen the bacterial isolate for amylase production

2.     To determine the effect of different physiochemical parameters on enzyme activity

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