SCREENING AND ISOLATION OF AMYLOLYTIC BACTERIA INVOLVED IN BIOGAS PRODUCTION

ABSTRACT

The Isolation and characterization of amylolytic bacterial strains with hydrolytic profile during anaerobic digestion of the mixture of cow rumen, dried cassava peels and poultry droppings was the subject of this research. Hydrolysis is important to biogas production as it involves breakdown of polysaccharides present in substrates. The experiment lasted for 30 days using a 10 litre scale bio-digester. Hydrolytic amylase producing bacteria isolated were Micrococcus luteus, Pseudomonas aeruginosa, Klebsiella oxytoca, Aspergillus spp, and Bacillus subtilis. The total anaerobic plate count ranged from 2.57cfu/g to 3.53cfu/g and the observed pH ranged from 5.12 to 6.19. The study reveals that the mixture of cassava peels, poultry droppings and cow rumen has the potential of producing biogas. In conclusion, further investigation of hydrolytic groups of bacteria beyond 30 days of anaerobic retention period should be investigated.

TABLE OF CONTENTS

Title Page                                                                                                                                i

Certification                                                                                                                           ii

Dedication                                                                                                                              iii

Acknowledgement                                                                                                                  iv

Table of Contents                                                                                                                   v

List of Tables                                                                                                                          ix

Abstract                                                                                                                                  x

Chapter One                                                                                                   

1.0              Introduction                                                                                                                1

1.1       Aim and Objectives                                                                                                    2

Chapter Two                                                                                     

2.0       Literature Review                                                                                                       3

2.1       What is Biogas?                                                                                                          4

2.2       Biogas Plants                                                                                                              5

2.3       Typical Composition of Biogas                                                                                  6

2.4       Use and Application of Biogas                                                                                   6

2.5       Microorganisms Involved in Biogas Production                                                        6

2.6       Amylolytic Bacteria                                                                                                   7

2.7       Biomass and Biogas Formation                                                                                  8

2.8       Factors Affecting the Hydrolytic Activity In Biogas Plants: A Brief Overview           10

2.8.1    Temperature                                                                                                               10

2.8.2    pH                                                                                                                               10

2.8.3    Substrate Related Factors                                                                                           11

2.8.3.1 Particle Size                                                                                                               11

2.8.3.2  Substrate Accessibility and Substrate Structure                                                        12

2.9       Decomposition of Organic Compounds in the Biogas Process                                     12

2.9.1    Stage 1. Hydrolysis                                                                                                     13

2.9.1.1    Hydrolysis of Polysaccharides                                                                                14

2.9.1.2     Hydrolysis of Proteins                                                                                15

2.9.1.3     Hydrolysis of Fats                                                                                       16

2.9.2    Stage 2. Fermentation                                                                                     16

2.9.3    Stage 3. Anaerobic Oxidation                                                                                    19

2.9.4    Stage 4. Methane Formation                                                                                      21

2.10     Alternative Methane Production Pathway from Acetate                                                23

2.11     Diversity of Microorganisms                                                                                      24

2.12     Interactions between the Various Microbial Groups                                                  25

Chapter Three                                                                                   

3.0       Materials and Methods                                                                                               26

3.1       Sample Collection                                                                                                      26

3.2       Isolation and Assessment of Bacterial Populations                                                    26

3.3       Characterization and Identification of the Isolates                                                     26

3.3.1    Identification of Bacteria                                                                                          26

3.3.2    Biochemical Tests                                                                                                      27

3.3.2.1 Catalase Test                                                                                                              27

 3.3.2.2 Coagulase Test                                                                                                          27

3.3.2.3 Oxidase Test                                                                                                              27

3.3.2.4 Citrate Utilization Test                                                                                              28

3.4       Bio-Digester Design And Loading                                                                             28

Chapter Four                                                                                    

4.0.   Results                                                                                                                           30

Chapter Five                                                                                     

5.1.    Discussion                                                                                                                     33

5.2.   Conclusion                                                                                                                     34

5.3.   Recommendation                                                                                                          34

References                                                                                                                              35

LIST OF TABLES

S/N                                   TABLE                                  PAGE NO

4.1 Total Anaerobic count (TAC) of Fresh cassava peels, cow rumen and poultry droppings   31

4.2 physicochemical parameter of fresh cassava peels, cow rumen and poultry droppings               32

4.3 Characteristics feature of isolated bacteria                                                                             33

4.4 Biochemical Characteristics of the isolated biogas production                                              34

CHAPTER ONE

1.0       INTRODUCTION

Biogas production technology through anaerobic digestion is an alternative form of fuel and a good fertilizer source. Biogas production is achieved via anaerobic digestion. The digestion process encompasses four stages namely: hydrolysis, acidogenesis, acetogenesis and methanogenesis Zieminski et al. ( 2012).

Cellulolytic” and “Amylolytic” are two common terms referring to anything capable of breaking down cellulose and amylose respectively. Enzymes responsible for the breakdown of cellulose and amylose are called “cellulase” and “amylase” respectively. Common cellulolytic and amylolytic bacteria in the hydrolytic stage of biogas production include the genera Bacteriodes, Clostridium and Acetivibrio (Cirne et al., 2007; Doi, 2008).

Amylolytic enzymes are widely distributed among microbes. They have been extracted from several fungi, yeasts and bacteria, however, enzymes from Fungal and bacterial sources have dominated applications in industrial Sectors. Bacillus and Aspergillus spp. are known as the most active amylase Producers. With the advent of new Boundaries in biotechnology, the amylase Family of enzymes and particularly those From microbial sources, have found Potential application in a number of Various fields, such as analysis (glycogen Determination), industries (including Brewing, baking, starch processing, Detergent, textiles, paper, food) and Clinical or pharmaceutical applications (diagnostic aids) (Pandey et al., 2000; Gupta et al., 2003). The major advantages of using Microorganisms for the production of Amylases are their economical bulk Production capacity and stability with respect to pH and temperature. Furthermore, microbes are easy to Manipulate in regard to obtaining enzymes with the desired characteristics. Amylolytic enzymes play an important Role in the degradation of starch, and are Produced in bulk from microorganisms, Representing about 25-33% of the world Enzyme market (Ramesh and Lonsane, 1990; Konsula and Liakopoulou- Kyriakides, 2004; Sivaramakrishnanet al., 2006). In the case of energy costs, Worldwide interest has been growing in Recent years regarding the acquisition of Desirable amylases that are capable of Digesting raw starches during the process Of starch liquefaction, but without the need For heating, inevitably leading to the Effective utilization of biomass and Reduction in the total cost of starch Processing (Wang et al., 1999; Robertson Et al., 2006). The microbial amylases that are used for Industrial purposes are derived mainly From Bacillus licheniformis, Bacillus Amyloliquefaciens and Aspergillus oryzae. Accordingly, native amylolytic microbial Strains especially, have abundant Applications to relevant industries in Biotechnology (Nigam and Singh. 1995; Crabb and Mitchinson. 1997).

1.1       AIM AND OBJECTIVES

To screen and isolate amylolytic bacteria involved in biogas production while the specific objectives are:

·       To isolate bacteria from wastes (cow rumen, cassava peels and poultry droppings)

·       To screen the isolate for amylase activity

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