BIODEGRADATION OF CORN WASTE FOR BIOETHANOL PRODUCTION BY USING SIMULTANEOUS SACCHAROMYCES FERMENTATION

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ABSTRACT

The production of bioethanol from corn waste was studied. The composition of the corn cob was determined using Association of Official and Analytical Chemists methods. Physicochemical analyses such as pH, temperature, total dissolved solids, total suspended solids, titratable acidity, specific gravity and reducing sugar were determined. One hundred grams (100g) of corn cob was pretreated and hydrolyzed using 1000ml of water and 20ml of 5% concentrated sulphuric acid. Ten grams (10g) of the ground corn waste was added as source of indigenous organisms. The fermentation was carried out for 7days using indigenous organisms and combination of indigenous organisms with starter culture. Ethanol yield was measured using a volumetric flask. The concentration of ethanol was done using spectrophotometric method. The indigenous bacteria and fungi were isolated using standard microbiological procedures as well as molecular identification techniques. The composition of corn cob was 6.02%, 2.21%, 0.54%, 2.28%, 0.30%, 44.02%, 32.72% and 11.30% for moisture, ash, starch, protein, fat, cellulose, hemicellulose and lignin, respectively. The physicochemical parameters tested varied with time. The pH decreased from 6.5-3.0 in both fermentation broths. Temperature was 300C in both fermentation broths. Total dissolved solids decreased from 889.00-0.80 in the fermentation broth that contained only indigenous organisms and 889.00-0.40 in fermentation broths that contained combination of indigenous organisms with starter culture. Total suspended solids increased from 104.50-314.50 and 104.50-365.50 in the fermentation broths that contained indigenous organisms and indigenous organisms with starter culture, respectively. Titratable acidity increased from 0.02-0.07 and 0.02-0.08 in fermentation broths that contained indigenous organisms and indigenous organisms with starter culture respectively. The specific gravity decreased from 1.0110-0.8281 and 1.0121-0.6120 for fermentation broths that contained indigenous organisms, and indigenous organisms with starter culture, respectively. Total reducing sugar was 38.30g and 40.98g for fermentation broths that contained indigenous organisms and indigenous organisms with starter culture, respectively. The ethanol yield using indigenous organisms were 0.0g/l, 3.0g/l, 7.0g/l, 10.0g/l, 9.0g/l, 8.5g/l and 7.5g/l while the yield using indigenous organisms with starter culture were 2.0g/l, 5.0g/l, 8.0g/l, 13.0g/l, 12.5g/l, 12.0g/l and 10.0g/l. The concentration of bioethanol produced using indigenous organisms were 0.7%, 3.2%, 11.3%, 10.3%, 9.0% and 8.4% while the concentration of bioethanol produced using indigenous organisms with starter culture were 0.3%, 1.7%, 6.5%, 13.2%, 12.0%, 10.3% and 9.0%. The indigenous bacteria were Lactobacillus casei, Klebsiella pneumoniaeEscherichia coliPseudomonas aeruginosa and Gluconobacter frateuri. The indigenous fungi were Mucor racemosusSaccharomyces cerevisiae and Mucor circinelloides. There is variation in the genera and total number of bacteria and fungi with increase in time during ethanol production. Lactobacillus casei was able to survive in the medium even when the pH was low. This study shows that indigenous organisms in corn waste can be used in the production of bioethanol and the process is recommended as a means of generating wealth from waste.






TABLE OF CONTENTS

Title Page                                                                                                                    i

Declaration                                                                                                                  ii

Certification                                                                                                                iii

Dedication                                                                                                                  iv

Acknowledgements                                                                                                    v

Table of Contents                                                                                                       vi

List of Tables                                                                                                              x

List of Figures                                                                                                             xi

Abstract                                                                                                                      xii

 

CHAPTER 1: INTRODUCTION                                                                          1

1.1       Background of the Study                                                                               1

1.2        Statement of the Problem                                                                               4

1.3       Justification of the Study                                                                               4

1.4       Aim of the Study                                                                                            5

1.5       Specific Objectives                                                                                         5

 

CHAPTER 2: LITERATURE REVIEW                                                              7

2.1       Corn Waste                                                                                                     6

2.1.1    Corn production in Nigeria                                                                             7

2.2       Bioethanol                                                                                                       7

2.2.1    Characteristics of bioethanol                                                                          8

2.2.2    Generations of Bioethanol                                                                              8

2.2.3    Production of bioethanol using lignocellulosic substrate feedstock               9

2.2.4    Processes in bioethanol production                                                                 10

2.3       Factors that Affect Bioethanol Production                                                    14

2.4       Benefits of Using Corn Waste for Bioethanol Production                             17

2.4.1    Advantages of using corn waste for bioethanol production                           17

2.4.2    Disadvantages of using corn waste for bioethanol production                      17

2.5       Impact of Bioethanol to the Environment                                                      18

2.6       Indigenous Organisms                                                                                    19

2.6.1    Bacteria                                                                                                           19

2.6.2    Ethanol producing bacteria                                                                             22

2.6.3    Fungi                                                                                                               22

2.6.4    Ethanol producing fungi                                                                                 23

2.6.5    Starter culture                                                                                                 24

2.6.6    Characteristics of ethanol producing bacteria and fungi                                25

2.6.7    Effect of ethanol on bacteria and fungi                                                          25

2.6.8    Bioethanol production habitat                                                                        26

 

CHAPTER 3: MATERIALS AND METHODS                                                   27

3.1       Sample Collection and Preparation                                                                 27

3.2       Source of Starter Culture                                                                                27

3.2.1    Yeast Inoculum Preparation                                                                           27

3.3       Compositional Analysis                                                                                  28

3.4       Physicochemical Analysis                                                                               33

3.5       Biodegradation of Corn Waste for Ethanol Production                                 36

3.5.1    Ethanol production process                                                                            36

3.5.2    Pretreatment with acid hydrolysis                                                                  37

3.5.3    Fermentation using only indigenous organisms                                              38

3.5.4    Fermentation using both indigenous organisms with starter culture              38

3.5.5    Fractional distillation                                                                                      38

3.6       Determination of Quantity and Concentration of Bioethanol Produced

by Indigenous Organisms and Indigenous Organisms With Starter Culture 39                   

3.6.1    Determination of quantity of bioethanol produced                                        39

3.6.2    Determination of concentration of bioethanol produced                               39

3.7       Isolation and Identification of Indigenous Bacteria and Fungi present in

Corn Waste                                                                                                     40

3.7.1    Isolation and characterization of indigenous bacteria                                    40

3.7.2    Isolation and characterization of indigenous fungi                                        42

3.9       Molecular Identification and Characterization of the Isolates                       43

3.10     Statistical Analysis                                                                                          43

                       

CHAPTER 4: RESULTS AND DISCUSSION                                                    44

4.1       Composition of Corn Cob                                                                              44

4.2       Physicochemical Parameters Tested During Biodegradation of Corn

waste for Ethanol production                                                                                     46

 

4.3       Quantity and Concentration of Bioethanol Produced                                    54

4.4       Isolation and Identification of Indigenous Bacteria and Fungi Present in

 Corn Waste                                                                                                    57

4.5       Enumeration of Bacteria During Biodegradation of Corn Waste for            60

Ethanol Production

 4.6      Enumeration of Fungi during Biodegradation of Corn waste for                  61

 Ethanol Production

4.7       Statistical Analysis                                                                                          67

4.8       Discussion                                                                                                       67


CHAPTER 5: CONCLUSION AND RECOMMENDATIONS                         75

5.1       Conclusion                                                                                                      75

5.2       Recommendations                                                                                          75

References                                                                                                      77

Appendices                                                                                                     86






LIST OF TABLES

                                                                                                                                PAGES

 

2.1       Bacterial cell structure with their roles                                                           21

4.1       Total dissolved solids and total suspended solids of the      

biodegrading corn waste using indigenous organisms, and both

indigenous organisms and starter culture                                                        51

4.2       Specific gravity of the biodegrading corn waste using indigenous

organisms, and both indigenous organisms and starter culture                      52

4.3       Total reducing sugar (Brix level) of the final products                                   53

4.4       Characterization and identification of indigenous bacterial isolates              58                                                                                           

4.5       Characterization and identification of indigenous fungal isolates                 59       

4.6       Enumeration of bacteria on nutrient agar plate during biodegradation of corn

waste for Ethanol Production                                                                         63

4.7       Enumeration of bacteria on MRS agar plate during biodegradation of corn

waste for Ethanol Production                                                                         64

4.8       Enumeration of bacteria on MacConkey agar plate during biodegradation of

Corn Waste for Ethanol Production                                                               65

4.9       Enumeration of fungi during biodegradation of corn waste for ethanol Production                                                                                                 66

 

 

 

 

 

 

 

LIST OF FIGURES

                                                                                                                                PAGES

 

2.1       Pretreatment effect on lignocellulosic material                                               10

2.2       Hydrolysis pathways                                                                                      13

4.1       Composition of corn cob                                                                                45

4.2       pH of the biodegrading corn waste during ethanol production using

indigenous organisms, and both indigenous organisms and starter

culture                                                                                                             49

4.3       Titratable acidity of the biodegrading corn waste during ethanol

production using indigenous organisms, and both indigenous organisms

and starter culture                                                                                           50

4.4       Quantity of bioethanol produced using indigenous organisms, and both

indigenous organisms and starter culture                                                        55

4.5       Concentration of bioethanol produced from corn waste using indigenous

organisms, and both indigenous organisms and starter culture                      56

  

 

 

 

 

 

CHAPTER 1

INTRODUCTION

 

1.1           BACKGROUND OF THE STUDY

The growing demand for sources of alternative energy has brought about improvement on new technologies for biofuel production. Microbial biotechnology is among the technology that is mostly established, permitting the production and improvement of several various biofuels from substrates like wastes and effluents. Through this, the cost for processing is greatly reduced, thereby improving their economic competitiveness as well as reducing the environmental load for waste disposal (Zoppellari and Bardi, 2013).

Activities of man generate large amounts of waste such as crop residues, solid waste from agriculture and municipal waste. The wastes are sources of pollution and can harbor pathogenic microorganisms that cause health problems therefore, it is necessary to handle these wastes judiciously (Ledward et al., 2003). Corn wastes are great sources of energy. A good amount of the wastes are generated by man annually and are underutilized in Nigeria. The management method is to burn them or allow the waste to decay in the environment. However, researches have shown that these wastes could be processed into liquid fuel such as bioethanol and biogas, or combusted to produce electricity and heat (Soltes, 2000).

Corn is a staple food in Nigeria with an annual production of 10.8 million metric tonnes (Mojeed and Muktar, 2021). The cobs serve as organic fertilizer during farming. Latif and Rajoka (2001) reported that, modern technology uses lignocellulosic substrates like the cobs to produce chemicals and fuels, using microorganisms. Microorganisms have the ability to produce enzymes such as cellulases and hemicellulases that can hydrolyze pretreated lignocelluloses like corn cob (Aro et al., 2005). The corn wastes which include corn cob, stalk and leaves can be changed to fermentable sugar with cellulose processing technology that consists of pretreatment, hydrolysis and fermentation using yeast or other microorganisms (Stefan et al., 2009).

Bioethanol is a liquid that is produced through fermentation of plants that have sugar and starch (Crop Energies AG, 2016). Ethanol production processes derive energy from renewable sources such as corn cob. Also, ethanol that is produced from biomass is the only liquid fuel that does not cause greenhouse gas effect. The major advantage of using biomass to produce ethanol is the decrease in the emission of greenhouse gases (Anuj et al., 2007). Ethanol production using biomass is also important for global demand in reducing greenhouse gas emission from fossil fuels. The combustion of ethanol emits low volatile organic compounds, nitrogen oxide and carbon monoxide (Akin-Osannaiye et al., 2008).

Ethanol is commonly produced from biological substrates through fermentation processes. In the course of the process, glucose is converted to ethanol by bacteria and yeast. There are different carbohydrate containing substances that produce glucose used for fermentation such as sugarcane and grains (Stefan et al., 2009). The fermentation is an anaerobic process that is catalyzed by enzymes (Braide et al., 2016). Several advantages are been offered by renewable energy like, they are indigenous, increase in security of supply and as well reduction in dependency on oil importation (Jegannathan et al., 2011) and enhanced cleaner environment (Chaudhary and Qazi, 2006).

Corn cob contains cellulose, hemicellulose, lignin and other constituents. The breakdown of cellulose and hemicellulose attracts the attention of biotechnologists and microbiologists for several years. The diversity of lignocellulosic and cellulosic substrates has helped to contribute to the difficulties found in enzymatic studies. Fungi are best known microorganisms that have the ability to breakdown these three polymers (Perez et al., 2002). Saccharomyces cerevisiae is extremely in use for the fermentation of sugar to ethanol which is used to produce industrial solvents, biofuels and beverages (Boboye and Dayo-owoyemi, 2009). Saccharomyces cerevisiae is seen as the world’s premier industrial microorganism in terms of both old and new biotechnologies (Noor et al., 2003). Saccharomyces cerevisiae is used to produce ethanol by industries because, it is capable of producing ethanol in high concentrations from hexoses and tolerance to high ethanol concentration and other compounds that can inhibit growth of microorganisms (Somda et al., 2011).

Succession of microorganisms, including fungi and bacteria, that exist together in a minute or very small environment is called micro-succession. Another word for micro-succession is serule. This type of succession exists in recently disturbed communities or newly available habitat. Microbial communities may change due to products secreted by the bacteria and fungi present. Changes in pH in a habitat could provide favorable conditions for a new species to inhabit the area. At times, the new species may not be able to withstand the present ones for nutrients leading to the primary demise (Franscisco et al., 2015).

Ethanol inhibits growth of yeast when the concentration is low by hindering cell division, reducing cell volume and some other growth rate, whereas, when the concentration of ethanol is high, it reduces cell life and cause cell death to increase (Birch and Walker, 2000). Accumulation of ethanol in a medium causes stress to the microorganisms as fermentation takes place (Geleote et al., 2001). The choice of strain for the production of bioethanol is based on some conditions such as their yield, ability to withstand ethanol, fermentation inhibitors as well as severe pH and temperature. pH has a significant effect on the growth of microorganisms. Most bacteria grow very well at pH 7 and they grow poorly or do not grow at all below pH 4. Some bacteria desire neutral pH (6.5-7.5). Fungi such as yeasts and mold grow in wider range but still desire pH 5 and 6. Yeast and mold also predominate in low pH substrates or solutions where bacteria cannot compete. Lactic acid bacteria are exceptions because they can grow in high acid solutions and produce acid as by-product (Cao et al., 2014). 

Therefore, development programmes are needed in order to get strains that can withstand ethanol for fermentation (Gunasekaran and Chandra, 2007). Furthermore, looking for effective fermenting bacteria and fungi that can make use of polymers in their metabolism to produce ethanol with minimal glycerol and foam formation is a vital factor in fermentation (Radhakumari et al., 2016).

 

1.2       STATEMENT OF THE PROBLEM

Depletion of crude oil reserves alongside rapid climate change caused by greenhouse gas emissions (GHG) has led researchers to pursue sources of renewable energy (Katsimpouras et al., 2016).

Corn waste causes environmental pollution and harbor microorganisms which are dangerous to human health.

The biodegradable, renewable fuels (bioethanol, biodiesel or biogas) should serve as a source of renewable energy with production of lesser pollutants like nitrous oxides, sulphur, carbon dioxide (Sanchez and Cardona, 2008).

 

1.3       JUSTIFICATION OF THE STUDY

The inoculation of fungi and bacteria in fermentation of sugars is an important aspect in the production of bioethanol. The use of corn waste for bioethanol production should be encouraged since the technology is environmentally friendly.

1.4       AIM OF THE STUDY

This work is designed to produce bioethanol from corn waste (corn cob) using indigenous organisms, and indigenous organisms with starter culture.

 

1.5       SPECIFIC OBJECTIVES

1.         To determine the composition of the corn waste (corn cob).

2.         To do the physicochemical analysis of the biodegrading corn waste (corn cob).

3.         To determine bioethanol production from corn waste (corn cob).

4.         To compare the quantity and concentration of bioethanol from corn cob using indigenous organisms, and indigenous organisms with starter culture.

5.         To isolate and identify indigenous bacteria and fungi present in corn waste (corn cob).

 

 

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