ABSTRACT
This purpose of this experiment was to characterize wild strains of yeasts from
different sources from north eastern Nigeria that have unique traits that will be
applicable in the bioethanol production industry. The yeast characterization of these
yeast strains were based on the yeast strain ability to grow at different temperature
conditions, their ability to grow in various pH condition and their ability ferment
different sugars. 10 yeast strains were tested. These yeast strains were isolated from
coconut, kunu (millet drink), sugar cane juice among others.
vii
Table of Contents
CHAPTER ONE…………………………………………………………………………………………………. 1
INTRODUCTION ………………………………………………………………………………………………. 1
1.0 Introduction …………………………………………………………………………………………………. 1
1.1 Bioethanol …………………………………………………………………………………………………… 2
1.12 Bioethanol production ………………………………………………………………………………… 3
1.13 Bioethanol and fossil fuel ……………………………………………………………………………. 9
1.14 Fossil fuel and Bioethanol position in the economy ………………………………………. 11
1.15 Disadvantages of fossil fuel on health and environment………………………………… 13
1.16 Need for bioethanol ………………………………………………………………………………….. 14
1.17 Yeast ………………………………………………………………………………………………………. 15
1.18 Research hypothesis …………………………………………………………………………………. 16
1.19 Objectives ……………………………………………………………………………………………….. 16
CHAPTER 2………………………………………………………………………………………………… 17
MATERIALS AND METHODS ……………………………………………………………………. 17
2.1 Collection of samples and isolation ………………………………………………………………. 17
2.2 Characterization based on growth at different temperature conditions …………… 18
2.3 Characterization based in the ability of yeast strain to ferment different sugars . 20
2.4 Characterization based on growth at different pH conditions …………………………. 21
CHAPTER 3………………………………………………………………………………………………… 23
RESULTS ……………………………………………………………………………………………………. 23
3.1 Characterization based on growth at different temperature conditions …………… 23
Table 3.12 showing the yeast strains grown in 45oC (Trial One) ……………………………. 23
Table 3.13 showing the SECOND TRIAL …………………………………………………………… 24
3.2 Characterization based in the ability of yeast strain to ferment different sugars . 25
3.21 TEST WITH 5% ARABINOSE SUGAR SOLUTION …………………………………. 25
3.22 TEST WITH 5% MALLITOL ………………………………………………………………….. 25
3.23 TEST WITH 5% FRUCTOSE…………………………………………………………………… 27
3.24 TEST WITH 5% SUCROSE …………………………………………………………………….. 28
3.25 TEST IN 5% GLUCOSE ………………………………………………………………………….. 29
3.3 Characterization based on growth at different pH conditions …………………………. 30
3.31 Test in pH 2 …………………………………………………………………………………………….. 30
3.32 Test in pH4 ……………………………………………………………………………………………… 31
3.33 Test in pH6 ………………………………………………………………………………………………. 32
3.34 Test in pH 8 …………………………………………………………………………………………….. 33
3.35 Test in pH10…………………………………………………………………………………………….. 34
3.35 Test in pH 12……………………………………………………………………………………………. 35
viii
CHAPTER 4………………………………………………………………………………………………… 36
DISCUSSION ………………………………………………………………………………………………. 36
4.1 Summary of results ……………………………………………………………………………………. 36
4.13 Characterization based on growth at different temperature conditions …………. 36
4.2 Characterization based in the ability of yeast strain to ferment different sugars . 37
4.3 Characterization based on growth at different pH conditions …………………………. 37
4.4 Inconsistencies and errors …………………………………………………………………………… 38
4.5 Challenges …………………………………………………………………………………………………. 38
4.6 Solutions …………………………………………………………………………………………………… 39
4.7 Practical application …………………………………………………………………………………… 39
4.8 Improvement of results ……………………………………………………………………………….. 39
CHAPTER 5 ………………………………………………………………………………………………………. 41
5.1 Conclusion …………………………………………………………………………………………………. 41
References………………………………………………………………………………………………………….. 42
TABLE OF FIGURES
FIGURE 1 BIOFUEL, WASTE AND BIOMATERIAL CYCLE …………………………… 2
FIGURE 2 GENERAL STRUCTURE OF CELLULOSE, HEMICELLULOSE
AND LIGNIN ………………………………………………………………………………………….. 6
FIGURE 3 A HAEMACYTOMETER ………………………………………………………………. 19
FIGURE 4 CC43 AND SC9 SHOWING NO YEAST CELL GROWTH IN 45
DEGREES …………………………………………………………………………………………….. 45
FIGURE 5 SC3 PLATE SHOWING NO YEAST CELL GROWTH AT 45
DEGREES …………………………………………………………………………………………….. 46
FIGURE 6 PLATES WITH YEAST CELL GROWTH AFTER 2 DAYS OF
INCUBATION ………………………………………………………………………………………. 46
LIST OF TABLES
TABLE 1 POLYMER COMPOSITION OF LIGNOCELLULOSIC BIOMASS …….. 5
TABLE 2 SUMMARY OF PRETREATMENT METHODS FOR
LIGNOCELLULOSIC BIOMASS …………………………………………………………….. 8
TABLE 3 SOURCES OF YEAST STRAINS ……………………………………………………… 17
1
CHAPTER ONE
INTRODUCTION
1.0 Introduction
During the industrial age, the beginning of the world we have today, fossil fuels are
the main source of energy and power for different kinds of machines. The refining of
this fuel brings about petroleum and other by products (Van Maris et al, 2006).
Although this fossil fuel has been the source of energy for machines for a long period
of time, bioethanol is a breath of fresh air. Bioethanol is a new form of fuel that has
taken the world by storm. Ethanol is an alkyl alcohol that can be used in engine that
are spark ignition oriented, which is the type of car people use today. It has adequate
octane levels and it can either be mixed with petrol or used a lone fuel with cars that
will be built solely for its purpose (Xuan, 2010). The knowledge of global warming
is becoming more rampant and people are looking for ways to live a more
sustainable life by recycling, producing less waste, using hybrid cars and the use of
bioethanol. This global warming is one of the side effects of the over usage of fossil
fuels. The reservoirs for fossil fuel are diminishing because the fossil fuel is not
being restored as much as it is being used. This is known as diminishing returns in
economics and with the rate at which it is fossil fuel is being used, it is evident that it
will not be able to satisfy the ever-increasing needs and therefore, needs a substitute
(Dahida and Akangbe, 2013).
2
1.1 Bioethanol
Bioethanol is produced from mostly waste products containing high levels of sucrose
or starch and materials which have larger sugars and need to be fermented to be
broken down (Cardona & Sanchez, 2007). Bioethanol has started coming into the
limelight as an alternate fuel option. Example of an alternate fuel is biofuel. Biofuel
is made from material gotten from living organisms such as plants animals and their
by-products; this is called biomass. The use of biomass as fuel is a project that is
being considered because of the availability of materials and its positive impact on
the environment. Bioethanol is an example of a biofuel. Hence, it is safe to say that
Bioethanol is renewable because unlike fossil fuel, it can be renewed in our time.
Figure 1 BIOFUEL, WASTE AND BIOMATERIAL CYCLE
(Ragaukas et al, 2006; Bhatia, Johri and Rumana, 2012).
3
1.12 Bioethanol production
Materials needed for the production of bioethanol are divided in two. The first
generation materials and the second generation materials (European Renewable
Energy Council, 2006).
The first generation materials used for biofuels include corn (maize), sugarcane and
sugarcane waste known as bagasse, waste products from starch-based materials like
rice and wheat among others. Sugarcane is sometimes preferable because it does not
have to go through long processes before it used for fermentation to produce ethanol
and it also has a very high content of sucrose. Corn is the main product used in the
production of ethanol at this point in time because it is very high in starch although it
has to go through a process called enzymatic hydrolysis for it to be able to produce
sugars that can ferment (Wackett, 2008; Wilkie et al., 2000). Some countries in the
world already use this as a standard fuel together with hydrogen fuel.
Hydrogen fuel is a type of biofuel that is produced from biomass in a sustainable way
(Urbaneik, Freidl, Huisisngh and Claassen, 2010).
These include countries like the United States of America, Brazil and others. The
problem with using these crops is that they are staple foods in many parts of the
world and the amount of produce needed is far too much. The more this produce is
needed, the more its demand in the world and more demand means an increase in the
price of these goods because more demand will cause suppliers to hoard and increase
prices. This will affect the poor countries because they are the countries with
populations that eat these staple foods the most. This problem also lowers the
availability of land for the cultivation of crops like millet because the land that could
4
have been used to cultivate, millet for food for people to consume is now being used
to grow corn for bioethanol production instead of food and this will in turn make
millet scarce which will cause and increase in the millet price. (Frow et al., 2009).
The second-generation material has been suggested for the production of bioethanol
due to the side effects of using feedstock such as wheat and corn to produce
bioethanol. These materials are known as lignocellulosic biomass. Lignocellulosic
biomass are very promising materials looking at its availability, the low cost and its
richness in polysaccharide content (Fujii, Fang, Inoue and Murakami, 2009).
The polysaccharides in lignocellulosic materials are cellulose, hemicellulose, lignin
and pectin. Cellulose is the major ‘stakeholder’ in the make up of lignocellulose. It is
composed of thousands of D-glucose units. These are held together by B(1-4)
glycosidic bonds. Hemicellulose is comprised of xylose, arabinose, mannose,
glucose, galactose and uronic acids. It has a lower molecular weight than cellulose
and it is easily broken down to its various components (Perez et at al). Lignin
comprises of phenolic residues (Kumar, Perez, van Maris). Lignin is the molecule
(component) that gives support and helps with resistance to attack by microbes. This
is what also makes it hard for enzymes to pass through. This molecule is also
hydrophobic. Pectins are structural polysaccharides that serve as the cement for the
cellulose structure in the cell wall (Blanco, 1999).
5
Polymers Content in Lignocellulose
(%)
Major components
Cellulose 33-51 Glucose
Hemicellulose 19-34 Xylose, glucose, mannose,
galactose, arabinose,
rhamnose
Lignin 20-30 Aromatic alcohols
Pectins (when present) 2-20 Galacturonic acid and
rhamnose
Table 1 POLYMER COMPOSITION OF LIGNOCELLULOSIC BIOMASS
(van Maris, 2006; Kuloyo, 2012)
6
Figure 2 general structure of cellulose, hemicellulose and lignin
(Chang, 2007; Kuloyo, 2012)
Lignocellosic materials go through 3 main processes before it produces bioethanol.
These three processes are namely; pretreatment, enzymatic hydrolysis and
fermentation. The pretreatment is divided into four parts; physical treatment,
chemical treatment, physiochemical treatment and biological treatment.
7
Pretreatment
methods
Pretreatment
process
Advantages Limitations and
disadvantages
Physical treatment Mechanical
comminution
Pyrolysis
Ozonolysis
Dilute acid
Reduces cellulose
crystallinity and
increases biomass
surface
areaıProduces gas
and liquid products
Reduces lignin
content; toxic
substances are not
produced
Energy required
usually higher than
inherent biomass
energy
High temperature;
ash production
Expensive; ozone
required in large
amounts
High cost;
corrosion of
equipment; forms
inhibitors
Chemical
treatment
Alkali
Hydrogen peroxide
Organosolv
Hydrolyzes
hemicellulose to
xylose and other
sugars; alters lignin
structure Removes
hemicellulose and
lignin; increases
biomass surface
area
Solubilises lignin;
does not produce
inhibitors
Hydrolyses lignin
and hemicellulose
High cost;
corrosion of
equipment; forms
inhibitors
Long residence
times;
irrecoverable salts
formed and
incorporated into
biomass; not
effective on
softwoods
Hydrogen peroxide
decomposes at
high temperature,
causing a decrease
in lignin and
hemicellulose
solubilization
High cost; solvents
need to be
recovered and
recycled
8
Physio-chemical
treatment
Steam pretreatment
AFEX
Causes
hemicellulose
degradation and
lignin
transformation;
short residence
time; cost effective
Increases biomass
surface area;
removes
hemicellulose and
lignin to an extent;
inhibitory
compounds are not
formed
Destroys a portion
of the xylan
fraction;
incomplete
destruction of the
lignincarbohydrate
matrix; formation
of toxic
compounds
Not effective for
biomass with a
high lignin content;
ammonia is
expensive and
hazardous
Biological
treatment
Fungal
dilignification
Degrades lignin and
hemicellulose;
requires low energy
Slow reaction rate;
loss of cellulose
Table 2 summary of pretreatment methods for lignocellulosic biomass
(Kuloyo, 2012)
Enzymatic hydrolysis of cellulose involves using particular cellulases under mild
conditions with glucose and other sugars as products (Sun and Cheng, 2002).
Cellulases are enzymes mainly from three groups; endoglucanases, exoglucanase and
beta glucosidase. Endoglucanase creates free chain ends by targeting and breaking
down regions that have low crystalinity. Exoglucanase removes cellobiose from the
free chain ends made by endo glucanase. Beta-glucosidase hydrolyzes cellobiose to
produce two glucose molecules (Duff and Murray, 1996).
Fermentation of the biomass hydroltaes is the last step in the processing of
lignocellulosic biomass to produce bioethanol. Fermentation is the proess used to
produce ethanol from sugars produces after enzymatic hydrolysis has taken place.
9
Fermentation occurs in two steps; Separate hydrolysis and fermentation and
simultaneous saccharification and fermentation. Separate hydrolysis and
fermentation is the process whereby the biomass undergoes enzymatic hydrolysis
before fermentation while simultaneous saccharification and fermentation is a
process in which hydrolysis and fermentation take place simultaneously to enable the
sugars produced in hydrolysis, to be completely used up by the organism carrying
out the fermentation process (Kuloyo, 2012). Organism used in the fermentation
process is yeast.
The difference between first generation and second-generation materials for
bioethanol production is that the second-generation materials require a more
complicated process than the first generation materials.
1.13 Bioethanol and fossil fuel
As explained before, bioethanol has been getting a lot of attention lately because of
global warming. This is because Biofuel does not produce ancient carbon. Bioethanol
is a carbon neutral fuel because it is produced from newly captured carbon. Ethanol
is a type of Fossil fuel is the fuel that is being used all over the world. Fossil fuel is
fuel that is gotten from decaying matter under the earth . Fossil fuel has been used for
millions of years in form of coal but the industrialization period heightened its usage
because it was used to run the industrial machines. It is used because it has a lot of
energy stored inside it. Although it is the most preferable sort of fuel, it is also the
most dangerous to the individuals, the society and the world at large. Nuclear energy
is also famous source of energy in some countries but is very expensive to produce.
The extraction of fossil fuel is a long and very expensive project and once a site is
depleted, it is very difficult to get another source. During this process, trees are
10
destroyed because the site has to be cleared for the excavation of the oil to begin.
This is the causing deforestation, which causes the release of the carbon dioxide
stored in the ground by the trees to be released. This thereby increases the amount of
carbon in the atmosphere. After getting the crude oil, it has to be taken to the refinery
to be processed into petroleum and other kinds of products. The refining of these
fuels releases toxic chemicals into the atmosphere and this causes global warming
because of the increase of green house gases in the atmosphere. The burning of
petroleum and the other products made from this crude oil release toxic materials
such as carbon soot and carbon monoxide. The release of these chemicals may cause
very serious health problems such as chronic breathing problems.
Biofuel on the other hand, is as safe as it gets. Biofuels are made from purely natural
material, which means the waste produced is not too harmful although the feedstock
used in the production of this biofuel could be grown on already contaminated soil,
which will be incorporated in the bioethanol if used. Bioethanol is an example of a
biofuel. The process used to produce bioethanol, which is fermentation does not need
any long procedures. Extracting the sugar and adding the yeast for the fermentation
process which yield ethanol. The production as well as the usage does not release
ancient carbon dioxide, which is more harmful. The production of this does not cost
as much as that of fossil fuel because this does not require the use very expensive
sources. The use of biofuels will cut down dependence on fossil fuel. The potential
of biofuel to become an economic good is very low. The production of biofuels is
still too small globally and the raw material needed for them is too high for it to start
having good market value.
11
Using biofuels can reduce the use of fossil fuels, which will reduce the greenhouse
gas emissions and also reduce pollution. There have been studies that prove that the
use of waste to produce biofuels will contribute to the reduction of global warming.
Biofuels can also be derived from waste products, solid waste to be exact. Solid
wastes such as crop residues and municipal waste. The disposal of municipal solid
waste has always been a problem in major cities around the world because they are
sources of pollution and sources as well as breeding sites for pathogenic organisms.
The production of biofuels from this waste will bring a way to move this waste from
the streets and the start the transition into a sustainable community instead of
discarding crop residues by burning, which will expel carbon dioxide. It is more
sustainable to use this waste for biofuels. There are some ways to convert this waste
to biofuels such as coal derived from sugarcane bagasse. Solid waste is generated in
large quantities almost everyday, so this is a great way to get rid of this waste. Solid
waste could be used to generate methane gas by burying.
1.14 Fossil fuel and Bioethanol position in the economy
The overall production of biofuels is increasing because sustainability is being more
embraced. The increasing oil prices have made biofuel production a more costeffective
business opportunity.
Nigeria is one of the largest oil producers in the world. It is the sixth largest producer
of oil to be exact. The economy of Nigeria depends heavily on the revenue derived
from the oil sector. This situation is however, depreciating because of the increase on
the cost of the dollar per barrel of oil that is sold (Badejo and Nwilo, 2006). Fossil
fuels are a key in the development of Nigeria. It is very crucial in the progress of the
12
country. The rising cost of fuel and its consequences on the environment are however
beginning to ignite interest of biofuel production. Bioethanol is still struggling
because it has not being well accepted in countries such as Nigeria. Nigeria does not
yet have machinery that run on bioethanol or any form of biofuel at all. Bioethanol
does not yet have a strong place in the economy in Nigeria because bioethanol is not
a widely known product. Unlike Nigeria, some European countries have accepted
bioethanol in a bid to reduce global warming. Such countries are Sweden; which is
the strongest in the bioethanol market with hundreds of bioethanol fueling stations
and the production of cars, which run solely on bioethanol, Germany, France, United
Kingdom, Ireland and Switzerland and also the United States of America. In Sweden,
there is growing preference for the use of bioethanol because it cheaper than
petroleum and it also helps in the march for a sustainable environment. Having a
sustainable environment is one of the Millennium Development Goals by the United
Nations Educational, Scientific and Cultural Organization (UNESCO). So, the use of
bioethanol is a step in the right direction (Okonko et al, 2009).
It has been said that the oil reserves in Nigeria would not last for another 50 years.
This reason is good enough for Nigeria to make the change that is needed.
Bioethanol production and use will cut the dependence of the revenues from fuel by
half and will help with income for farmers and will help provide jobs. Farmers will
be contracted to use fields to produce first generation materials for bioethanol
production. This will give income to the farmer and the industry where the
bioethanol will be produced, will provide jobs for people (Okonko, 2009).
13
1.15 Disadvantages of fossil fuel on health and environment
The constant use of fossil fuel has a very bad impact in the environment. The burning
of fossil fuels can cause the release of smoke into the atmosphere, which is very
harmful to the health of humans. This smoke can lead to and worsen cases COPD
(Chronic Obstructive Pulmonary Diseases). The people staying near areas of refining
are at risk too because they could die of suffocation from the smoke. The refining of
this fossil fuel and its transportation also pose a huge risk in the Nigerian
environment. During the refining process, there is the production of gas, which burns
and emits harmful substances. The burning of gas in this process is known as gas
flaring. Gas flaring has been a controversial subject for quite a while in Nigeria
especially in the Niger Delta area. This gas flaring occurs from the refinery or where
a pipe burst and the gas ignites. This gas flaring occurs mostly around farmland and
it aids in the degradation of the soil and anything that grows on it. Gas flaring has
lead to deforestation and great climate changes as it causes overheating, excessive
rainfall and due to deforestation, erosion. Gas that is being set on fire instead of
wasting could open another industry opportunity; a new market that would contribute
greatly to the gross domestic product of the country. Gas flaring releases large
amounts of carbon dioxide in the atmosphere, thereby increasing global warming
(Nwanya, 2011).
Global warming which is partly caused by the burning of fossil fuel is threatening the
peace of Nigeria as well. Global warming is bringing about change in weather
patterns of the country (Nigeria). Changes in weather are having adverse effects on
the food production of developing countries such as Nigeria. Food insecurity will
cause uproar in the nation (Nigeria). Over 60% of the Nigerian population is said to
14
be living in poverty. Food insecurity among the individuals in this section of the
population will cause deaths and fights in such populations due to frustration and
aggression (Okonko, I.O, 2009).
Climate change is also a major result of global warming. Climate change results from
changes in solar activity, long-period changes, changes in eccentricity, obliquity of
the ecliptic, procession of equinoxes, increasing concentrations o carbon dioxide and
greenhouse gases. Climate is a major factor in making decisions for the world we
live in today. It determines the kind of food we will eat, the kinds of clothes to wear,
it determines when and when to travel among others. Climate is unpredictable. The
irregularity has caused many disasters such as floods, droughts or wildfires
(Ajetomobi and Abiodun, 2010; Dahida and Akange, 2013).
1.16 Need for bioethanol
The need for bioethanol production is on the rise. The world needs an alternative
energy source and needs to save the environment as well. The growth of human
civilization and population has sparked this interest and this is paramount in most
economies around the world. There is need to provide an alternative source for
energy supply because of the release of the carbon dioxide embedded in the fossil
fuel which is worsening the greenhouse effect. A large bulk of fossil fuel is used in
the transportation sector and there is an increasing the demand on this sector as man
has become more mobile and moves to more places (Okonko, 2009).
“The issue of development is one of the most divisive of our time and development
at what cost. Should it come at the expense of the environment, so that rapid
economic growth lays the seeds of future catastrophes?”
15
– G. Paschal Zachary
1.17 Yeast
The conversion of both first generation and second-generation materials for the
production of bioethanol required fermentation. The fermentation process is where
the ethanol is being produced and the organisms responsible for that are yeast. Yeast
cells are the most widely used organisms used in industrial fermentation. They still
have some limitations because of their lack of ability to withstand certain industrial
production stress.
Yeast has been with us since the old days. Wine came to be when the fermentation of
grapes in barrels produced slightly alcoholic wine; this phenomenon occurred only
because of the presence of yeast which fermented the sugar in the grape. This was
used by the Egyptians or noticed by the Egyptians rather. Yeast was also used to
make bread, which was and still is a favorite staple in all houses around the world.
These processes were thought to be magical at the beginning but now, they are
proving themselves day by day in many fields (Chambers and Pretorius, 2010).
There are two types of yeast used in the bioethanol production process which are
Saccharomyces cervisae and Kluveromyces marxianus (Kuloyo, 2012).
Yeasts are anaerobic unicellular fungi, which break down sugars to produce alcohol.
They do this by budding or division. The yeast used in this research is the budding
yeast also known as Saccharomyces cerevisae.
Saccharomyces cerevisae also known as baking yeast, is the most widely used yeast
in the bioethanol production process. This is because it produces a sufficient quantity
16
of bioethanol and has a good tolerance for ethanol. Saccharomyces has however, had
a problem with fermenting arabinose (Ruisjes et al, 2012).
There has been a search for microorganisms of unique abilities from regions with
rich diversity for biomolecules that will be substitutes for chemicals used in
industries. Breakthroughs in these researches, are dependent on the discovery of new
organism with unique abilities that can be used in the biotechnology industry. There
is scientific hypothesis though, that there are yeast strains in nature which have
unique abilities that can be applicable in the production of bioethanol.
1.18 Research hypothesis
This research hypothesis is that some yeast strains from North Eastern Nigeria have
unique abilities to increase the performance of yeast for bioethanol production
1.19 Objectives
Generally, this research is to isolate different yeast strains from different locations of
North Eastern Nigeria and physiologically characterize isolated yeast strains by
observing their ability to grow at elevated temperatures, to grow in various pH
conditions and their ability to ferment different sources of sugar.
Specifically, this research aims to find yeast strains from North Eastern Nigeria with
unique abilities that will help in the yeast role in bioethanol production process.
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