ISOLATION AND IDENTIFICATION OF MICROBES INVOLVED IN PRODUCTION OF BIOGAS USING CASSAVA PEEL AND SEWAGE

ABSTRACT

The research study was aimed at investigating microorganisms associated with biogas production. Using sewage and cassava peel, as substrates. Sewage was collected from Ibrahim Babanginda hostel in Michael Okpara University of Agriculture, Umudike, Abia state while cassava peels were collected in Amawom, Umuahia, Abia state. Standard microbiological methods were used to isolate the organisms and anaerobic bio-digester was constructed to house the waste substrates for biogas production. Anaerobic bacteria isolated were identified as, Pseudomonas sp., Escherichia coli, Bacillus sp., Micrococcus sp., Proteus vulgaris, Citrobacter sp., Clostridium sp., Streptococcus bovis, Enterobacterium cloacae and Klebsiella sp., while fungi isolated were identified as Fusarium sp., Mucor sp., Aspergillus sp., Rhizopus sp. Methanogenic bacteria isolated were identified as Methanococcoides methyilutens, Methanoculleus bourgense, M. hungatei, Methanobacterium formicicum, M. hungatei, M. voltaei from media using anaerobic chamber for isolation . Analysis from this research revealed that the temperature of cassava peels and sewage ranged between 26°C and 30°C while the pH varied between 4.00 and 6.80 during digestion. The volume of biogas produced during the course of this project was between 1.6 and 8.2 ml and values for dissolve oxygen decrease from 10.4 to 0.0 mg/l.  However, the study has shown that the role of Methanogens and other complementing bacteria and fungi in biogas production is indispensable.

TABLE OF CONTENTS

Title page                                                                                                                                    i

Certification                                                                                                                                ii

Dedication                                                                                                                                   iii

Acknowledgement                                                                                                                      iv

Table of Contents                                                                                                                        v

List of Tables                                                                                                                              viii

List of Figures                                                                                                                             ix

Abstract                                                                                                                                        x

CHAPTER ONE

1.0 Introduction                                                                                                                        1

1.1 Aim and Objective                                                                                                              3

CHAPTER TWO

LITERATURE REVIEW

2.0 Brief history of biogas                                                                                                         4

2.1 Process/Principles of biogas production                                                                              5

2.1.1 Hydrolysis                                                                                                                         8

2.1.2 Acidogenesis (acidification phase)                                                                                   8

2.1.3 Acetogenesis                                                                                                                     9

2.1.4 Methanogenesis                                                                                                                10

2.2 Microorganisms involved in biogas processes                                                                    11

2.2.1 Acidogens                                                                                                                         11

2.2.2 Syntrophic Acetogens                                                                                                       12

2.2.3 Methanogens (Archaea)                                                                                                       13

2.3 Cassava peel and Sewage  as substrates for biogas production                                              16

2.3.1 Sewage                                                                                                                                 16

2.3.1.1 Types of Sewage                                                                                                               18

2.3.1.2 Reuse of Sewage                                                                                                               18

2.3.2 Cassava peels                                                                                                                        19

2..3.3 Pretreatment of cassava peel and sewage for efficient activity of microbial and biogas production             21

2.3.3.1  Pre-separation                                                                                                                   21

2.3.3.2 Size reduction and disinfectant                                                                                         21

2.4 Factors affecting anaerobic degradation                                                                                  22

2.4.1 Temperature                                                                                                                          22

2.4.2 pH and Alkalinity                                                                                                                  23

2.4.3 Oxygen                                                                                                                                  24

2.4.4 Ammonia Concentration                                                                                                       25

2.4.5 Hydraulic Retention Time and Organic Loading Rate                                                         27

2.4.6  Inhibitor                                                                                                                                28

 2.4.7 Mixing                                                                                                                                  32

2.5 Importance of co-digestion                                                                                                      33

2.6 Types of digesters                                                                                                                    33

2.6.1 Plug flow digesters                                                                                                               33

2.6.2 Fixed dome digesters                                                                                                            34

2.6.3 Floating drum digesters                                                                                                         35

CHAPTER THREE

3.0 Materials and Methods                                                                                                             37

3.1 Biogas digester construction                                                                                                    37             

3.2 Sample collection and preparation                                                                                          38

3.3 Microbiological analysis                                                                                                         38

3.3.1 Isolation of microorganisms(Bacteria and fungi)                                                                 38

3.3.2 Identification of bacterial and fungal isolates                                                                      39

3.3.2.1 Morphological and biochemical identification of bacteria and fungi isolates                  39

3.5 Biochemical test                                                                                                                 40-43

3.6 Analysis of physico-chemical parameters                                                                               43

3.7 Quantification of gas analysis                                                                                                  43

CHAPTER FOUR

4.0 Results                                                                                                                                44-51

CHAPTER FIVE

5.0 Discussion                                                                                                                                52

5.1 Conclusion                                                                                                                               57

5.2 Recommendation                                                                                                                     57

REFERENCES                                                                                                                       58-67

LIST OF TABLES

Table                                                      Title                                                                           Page

 2.1:        Various constituents of biogas generated from the anaerobic digestion process.        11

 2.2:         Synthrophic aceto-oxiding bacteria in association with hydrogentrophic methagens.           13 

 2.3:         Inhibitors in anaerobic decomposition processes and the concentrations at which     31    

                They become damaging.                                              

4.1:       Morphological characteristics of bacterial isolates.                                                         48

4.2:          Methane producing bacteria during the process of biogas production.                       49

4.3:          Biochemical characterization and identification of bacterial isolates                          50

4.4:          Characterization and identification of fungal isolates                                                  51

LIST OF FIGURES

   Figure                                            Title                                                                                Page

     2.1:        A schematic presentation of anaerobic digestion process.                                           6

     2.2:        A schematic presentation of anaerobic digestion process.                                           7

     2.3:        Schematic representation of a plug flow digester                                                      34

     2.4:        Schematic representation of a fixed dome digester                                                   35

     2.5:        Schematic representation of a floating drum digester                                                36

     3.1:       Locally made biodigester                                                                                             37

     4.1:     Shows pH variation of cassava peels and sewage during fermentation process.                    44

     4.2:       Shows the mesophilic temperature variation of the substrates                                    45

                  (cassava peels and sewage) before and during the digestion process.

     4.3:      Shows Dissolve oxygen decreasing from 10.4 - 0.0 during the digestion                    46

                 process indicating consumption of oxygen by facultative organisms.

     4.4:       Shows the quantity of biogas produced during anaerobic digestion.                          47

CHAPTER ONE

1.0 Introduction:

Millions of tons of wastes (liquid or solid) are generated each year from municipal, industrial and agricultural sources. Unmanaged organic waste fractions from farming, industry and municipalities etc. decompose in the environment, resulting to large-scale contamination of land, water and air, and also release green house gases. These wastes not only represent a threat to environmental quality, but also possess a potential energy value that is not fully utilized despite the fact that they are cheap and abundant in most parts of the world (Merlin Christy, et al., 2014). Biogas is one of the versatile renewable fuels gotten or prepared from the organic wastes which can be used for power and heat/cool production or it can be upgraded to biomethane to be used as vehicle fuel (Bhuvaneswari et al., 2014).

Anaerobic digestion is a suitable technology to treat organic wastes with aid of various diversity of microorganism for biogas production. Anaerobic digestion consists of several interdependent, complex sequential, and parallel biological reactions, during which the products from one group of microorganisms serve as the substrates for the next, resulting in transformation of organic matter mainly into a mixture of methane and carbon dioxide with minor quantities of nitrogen, hydrogen, ammonia and hydrogen sulfide etc (Ishmael et al., 2014; Merlin Christy et al., 2014). In nature, this process occurs in environments such as hot springs, swamps, paddy fields, lakes and oceans and the intestinal tract of animals like ruminants such as cattle etc. Biogas is produced by anaerobic digestion of biological wastes such as cattle dung, vegetable wastes, sheep and poultry droppings, municipal solid waste, industrial waste water and land fill etc to give mainly methane (50-70%), carbon dioxide (20-40%) and traces of other gases such as nitrogen, hydrogen, ammonia, hydrogen sulphide, water vapour etc. these gases are smokeless, hygienic and more convenient to use than other solid fuels (Gopinath et al., 2014).

     In biogas production, the conversion of complex organic matter to methane and carbon dioxide and other traces of gases are possible mainly by the actions of different group of microorganisms, with the microbial community of biogas comprised essentially of bacteria and fungi and other groups of protozoan.  The essential microbial complex is comprised of hydrolytic bacteria, fermenting bacteria, acetogenic bacteria and methanogenic bacteria and these groups of microorganisms have been reported to establish synthrophic relationships where the later members of the food chain depend on the previous for their substrate but may also have significant metabolic products. These microorganisms act at the different stages of the anaerobic process to bring about effective biogas production and are an integral component of nature’s waste management and are commonly found in soils and deep waters as well as land fill sites (Asikong et al., 2016).

The anaerobic digestion is characterized by a series of biochemical transformations of organic matter or wastes. The degradation steps are carried out by different consortia of microorganisms, which partly stand in syntrophic interrelation and place different requirements on the environment.

The whole process involves several distinct stages such as hydrolysis, acidogenesis, acetogenesis and the final stage methanogenesis. In stage 1, fats, complex carbohydrate and proteins are hydrolysed to their monomeric form by enzymes. In stage 2, the monomers are further degraded into short chain acids and these short chain acids are converted to hydrogen, carbon dioxide and acetate and in the final stage which is stage 3, the intermediate products are converted to methane and carbon dioxide by methanogens (Ishmael et al., 2014).

1.1 AIM AND OBJECTIVE:

1. To isolate and identify microorganisms from sewage and cassava peels responsible for   biogas production.

2. To produce biogas from sewage and cassava peels.

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