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 pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Gluconobacter frateuri. The indigenous fungi were Mucor racemosus, Saccharomyces 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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