ABSTRACT
The
yield of bioethanol from cassava peels hydrolysed with Aspergillus niger and crude enzymes (amylase, cellulase and
pectinase) was examined in this study. Cassava peels were pretreated by soaking
and a combination of soaking and boiling which removes 67% of cyanide. After
hydrolysis, Zymomonas mobilis and Saccharomyces cerevisiae were used to
ferment the hydrolysates separately at room temperature for five (5) days. The
result showed that pretreatment by soaking and boiling for up to 120 minutes
removed the highest amount of cyanide and increased carbohydrate but reduced
the fibre content (37.04±0.01mg/g, 71.42±0.02%, and 10.38±0.42%). Hydrolysis
using Aspergillus niger yielded up to
95.44±0.11mg/g reducing sugar while hydrolysis using enzymes yielded up to
72.38±0.06mg/g reducing sugar. Highest
ethanol yield when the hydrolysates were fermented with Zymomonas mobilis and Sacchromyces
cerevisiae were 3% and 2% respectively. Proximate composition of the
fermented waste showed 6.37±0.08, 2.71±0.03, 25.49±0.01, 8.97±0.02 and
34.41±0.16% ash, fat, protein, crude fibre and carbohydrate respectively while
nitrogen, phosphorus and potassium of the residue was 4.08±0.50, 0.40±0.28
and1.84±0.15% respectively. The work reveals that cassava peels is a suitable
agricultural waste for bioethanol production and fermentation with Zymomonas mobilis yielded high ethanol
compare to Saccharomyces cerevisiae.
The work also proved that the waste product of fermentation may be used as
animal feedstock or fertilizer to enrich the soil for plant growth.
TABLE OF CONTENT
Title
page i
Declaration ii
Dedication iii
Acknowledgement iv
Abstract
v
Table
of content vi
CHAPTER ONE 1
INTRODUCTION 1
1.1 QUEST FOR ALTERNATIVE ENERGY
SOURCE 2
1.2 JUSTIFICATION 2
1.3 STATEMENT OF
RESEARCH PROBLEM 2
1.4 RESEARCH AIM
AND OBJECTIVES 2
CHAPTER TWO 4
LITERATURE REVIEW 4
2.1 CASSAVA PRODUCTION IN AFRICA 4
2.2 CASSAVA PEELS 4
2.3 CYANIDE IN CASSAVA 5
2.4 LIGNOCELLULOSIC BIOMASS 6
2.4.1 SECOND GENERATION FEEDSTOCKS 7
2.4.2 STRUCTURE OF LIGNOCELLULOSIC BIOMASS 8
2.5 STRUCTURE AND PROPERTIES OF
STARCH 12
2.6 PRETREATMENT OF LIGNOCELLULOSIC
BIOMASS 14
2.6.1 MECHANICAL PRETREATMENT 16
2.6.2 PHYSICAL PRETREATMENT 16
2.6.3 PHYSICOCHEMICAL PRETREATMENT 17
2.6.4 CHEMICAL PRETREATMENT 17
2.6.5 BIOLOGICAL PRETREATMENT 18
2.7 HYDROLYSIS
AND FERMENTATION OF LIGNOCELLULOSIC
BIOMASS 19
2.8
BIOETHANOL 20
2.8.1
GLOBAL BIOETHANOL PRODUCTION 21
2.8.2 PRODUCTION AND UTILIZATION OF ETHANOL
INNIGERIA 22
2.9 MICROORGANISMS
EXPLORED FOR ETHANOL PRODUCTION 22
2.9.1
MICROORGANISMS OF INTEREST IN THIS RESEARCH WORK 23
2.9.2 Aspergillus niger 23
2.9.3
Zymomonas mobilis 24
2.9.4
THE METABOLIC PATHWAY OF Zymomonas
mobilis 25
2.9.5 Saccharomyces cerevisiae 27
2.10 MECHANISM OF YEAST
FERMENTATION 28
2.10.1
INHIBITION OF ETHANOL PRODUCTIVITY IN YEAST CELLS 29
2.11 HYDROLYTIC ENZYMES 30
2.11.1
MEASUREMENT OF ENZYME ACTIVITY 31
2.11.2 AMYLASE 31
2.11.3 CELLULASE 32
2.11.4 PECTINASE 33
2.12 FERMENTED CASSAVA PEELS AS
ANIMAL FOOD SUPPLEMENTS 34
CHAPTER THREE 35
MATERIALS
AND METHODS 35
3.1
Collection of sample (Cassava peels) 35
3.2
Microorganisms 35
3.3
Identification of microorganisms 35
3.3.1
Aspergillus niger 35
3.3.2
Saccharomyces cerevisiae 35
3.4
Isolation of Zymomonas mobilis from
fresh sweet Palm Wine 36
3.5
Pretreatment of Cassava peels 36
3.6
Determination of cyanide in pretreated cassava peels 36
3.7
Determination of crude fibre in pretreated cassava peels 37
3.8 Determination of Carbohydrate 37
3.9
PRODUCTION OF ENZYMES (Amylase, Cellulase, and Pectinase) 37
3.10
Assay for Cellulase activity 38
3.11Assay
for pectinase activity 38
3.12
Assay for amylase activity 39
3.13
Enzyme Hydrolysis 39
3.14
Microbial Hydrolysis 39
3.15
Determination of Reducing Sugar 40
3.16
Fermentation of the Hydrolysate 40
3.17
Distillation 40
3.18 Determination of Percentage Ethanol Yield 40
3.19
PROXIMATE ANALYSIS OF THE HYDROLYSATE 41
3.19.1 Total ash 41
3.19.2
Crude fat 41
3.19.3 Crude fibre 41
3.19.4 Crude Protein 42
3.20
Determination of Phosphorus in the Waste after fermentation 42
3.21
Determination of Nitrogen in the Waste after fermentation 42
3.22
Determination of potassium 43
3.23
Analysis of data 43
CHAPTER
FOUR 44
4.1
Result 44
4.2
Discussion of result 52
CHAPTER
FIVE 56
CONCLUSION 56
RECOMMENDATION 56
REFERENCE 57
APPENDIX 77
LIST OF TABLES
Table
2.1: Cyanogenic glycosides (CNp) in different tissues of the cassava plant 6
Table 2.2: Composition of some
common sources of biomass 12
Table
4.1 Morphological and biochemical
characterization of isolates from palm wine 44
Table 4.2: Assay
for activities of enzymes produced from Aspergillus
niger 46
LIST OF FIGURES
Figure 2.1: Location of cyanogenic
glycoside and the enzyme linamarase in the cell 5
Figure 2.2: Hydrolysis of linamarin, in
β-glucosidase, and in α-hydroxynitrile lyase 6
Figure 2.3: structure of
plant cell wall components 8
Figure 2.4: Structure of lignin 9
Figure 2.5: Structure of Hemicellulose 10
Figure 2.6: Structure of cellulose 11
Figure2.7: The structure of Amylose 13
Figure2.8: Structure of Amylopectin 13
Figure2.9:
Schematic representation on biomass pretreatment 15
Figure
2.10: The Entner-Doudoroff (ED) pathway and ethanologenesis 26
Figure 2.11: Metabolic pathway for ethanolic
fermentation in S. cerevisiae cells 27
Figure 2.12: Possible target sites for ethanol
inhibition in yeast cells 29
Figure 4.1: The carbohydrate, crude fibre
and cyanide contents of the pretreated cassava peel 45
Figure 4.2: Reducing sugar yield from
hydrolysis of treated cassava peels using A.
niger 47
Figure 4.3: Reducing sugar yield from
enzymatic hydrolysis of treated cassava peels 48
Figure 4.4: Ethanol yield by Z. mobilis and S. cerevisiae fermentation 49
Figure 4.5: Proximate analysis of dried
waste product of fermentation 50
Figure 4.6: Nitrogen, Potassium and
phosphorus in the waste product of fermentation 51
CHAPTER ONE
INTRODUCTION
Cassava
is one of the important food crops that survive conditions of low nutrient
availability and drought (John, 2004). History revealed that cassava originates
from Brazil and was introduced into West Africa by Portuguese and today, over
600 million people from Africa, Asia and Latin America depend on cassava for
food (Asegbeloyin and Onyimonyi, 2007). Cassava was introduced into Nigeria 300
years ago and became generally accepted and cultivated in late 1990s (Nigeria
Cassava Master Plan, 2006). Today, Nigeria is the highest producer of cassava
in the world, producing higher than Brazil, Thailand and Indonesia (Asegbeloyin
and Onyimonyi, 2007). Cassava has been a raw material globally for industrial
production of textiles, papers, adhesives, pharmaceuticals and various food
products because it is rich in carbohydrate with high energy density and has
generated great impact in world economics (Aigbe and Remison, 2010). Cassava
and it is by – products are renewable source of energy that pose no threat to
the environment and has been used by several researchers in the production of
biofuel (Musatto and Teixira, 2010). Interest in the production of biofuel from
agricultural wastes is driven by several reasons such as: global search for
alternative source of energy and transportation fuel to replace the depleting
fossil fuel. It was also because of several benefits derivable from the use of
ethanol. These benefits includes: the use of ethanol as solvents, germicides,
antifreeze and intermediates for other organic chemicals (Kingsley, 2012). Bioethanol
can be produced by fermenting sugars in plant materials as opposed to synthetic
ethanol production from petrochemical sources (Oyeleke a Jibrin, 2009). Ethanol
is pollution free, biodegradable, renewable, cause no climate change and the by
– product of fermentation can be used as animal feedstock (Adelekan, 2010;
Teerapatr, Lerdluk and La-aied, 2010). It has been estimated that 2.96 million
metric tons of cassava peels is generated annually in Nigeria from processing
cassava to various food products (Nwabueze and Otunwa, 2006). These enormous
wastes that constitute 20 – 35% of the weight of cassava tuber are discarded
with consequent implication of environmental pollution. In view of this, it has
become necessary to convert this waste to useful end products in other not to
pose threat to the environment (Nwabueze and Otunwa, 2006).
1.1
QUEST
FOR ALTERNATIVE ENERGY SOURCES
The
earth experienced an increase in the mean temperature in the 19th
century due to emission of greenhouse gases. Carbon dioxide has been the
largest greenhouse gas emitted through combustion of fossil fuel as coal, oil
and natural gases (Sun and Cheng, 2002). United States alone is responsible for
25% of global energy consumption and 25% of the world carbon dioxide emission. Researches
have shown that the deposit of crude oil in the earth crust is gradually
depleting with time. This reason couple with the continuous hike in the pump
price has driven global attention to the search for a renewable energy source
to serve as transportation fuel and power industrial machines (Jin, Michel,
Wyman and Dale, 2003). Bioethanol
produced from fermentation of sugars in plants has been discovered to be a
perfect replacement for gasoline in some advanced countries (Sharma, Kalra,
Oberoi and Bansal, 2007).
1.2 STATEMENT OF RESEARCH PROBLEM
Large tons of cassava wastes are discarded annually in
Nigeria when cassava is processed to various food products. This waste ends up
in open dumps or drainage systems, threatening both surface water and ground
water quality. It is therefore necessary to convert these wastes to useful end
products rather than allowing them to become nuisance to the environment.
1.3 JUSTIFICATION
The global search for alternative renewable
transportation fuel to replace the depleting fossil fuel is on the increase due
to hike in petroleum price and global warming. This research work is aimed at
investigating the possibility of transforming cassava peels to valuable
products like ethanol, animal feedstock or fertilizer, thereby contributing
toward alternative energy supply as well as creating employment opportunity.
1.4 RESEARCH AIM AND OBJECTIVES
AIM
The aim of this research work is to produce ethanol
from Cassava peels using Zymomonas
mobilis and Saccharomyces cerevisiae
and compare ethanol yield of the two organisms.
OBJECTIVES
The overall objectives of this work are:
1. To determine the
pretreatment method for cyanide removal in cassava peels.
2. To determine the
condition for enzyme hydrolysis of pretreated cassava peels.
3. To establish
fermentation condition for hydrolyzed cassava peel.
4. To determine the yield of ethanol produced from
fermentation by Zymomonas mobilis and
Saccharomyces cerevisiae
5. To assess the fermentation residue for possible
organic fertilizer or animal feedstock.
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