CHAPTER ONE

INTRODUCTION

1.1 Background of Study

Energy consumption has increased during the last century due to the world
population development and growth (Sun et al., 2002). Nowadays, the
increasing problem of the CO2 emissions due to energy consumption, besides to
the future petroleum scarcity, has strengthened the interest in alternative,
nonpetroleum-based sources of energy.

One of the potential options to solve the environmental and energetic problems
is the use of bio-ethanol (Hamelinck et al., 2005). This is a renewable fuel,
which avoids the negative environmental impacts generated by petroleum-based
fuels.
Bio-ethanol can be produced by using different technologies. One of the most
important technology, fermentation, produces bio-ethanol by means of
biological transformation of natural starch and sugars resources such as energy-
rich crops, (first-generation biofuels) and lignocellulosic biomass (second-
generation biofuels).

The use of renewable biomass resources to produce liquid biofuels such as

bioethanol offers attractive solutions to reducing greenhouse gas (GHG)
emissions, decreasing reliance on foreign oils, addressing energy security
concerns, strengthening rural and agricultural economies, and increasing
sustainability of the world transportations system (Demirbas, 2007). Apart from
biofuels, many other valuable products for chemical and pharmaceutical
industry can be produced from organic byproducts through microbial
fermentation (Thomsen, 2005). Most current bioethanol production processes
(1st generation) utilize more easily degradable biomass feedstocks such as
cereals (corn or grain) and sugarcane juice. However, the utilization of these
agricultural crops exclusively for energy production is heavily conflicting with
food and feed production (Pimental et al., 2009; Wheals et al., 1999). Great
effort is enforced on advancing a cellulosic bioethanol concept (2 nd generation)
that utilizers lignocellulosic biomass. This is owing to the fact that they are
cheap, readily available and do not compete with food availability.

Cassava peels a waste product of cassava, yam peels a waste product of yam,
sugarcane bagasse a waste product of sugar cane, corn cob a waste product of
corn and rice husk a waste product of rice contains amount of sugars that can be
further utilized to produce various compounds [Cao et al., 1996; Adesanya,
D.A. and A.A. Raheem, 2009]. The bioconversion of lignocellulosic to biofuel
from cheap non-edible materials such as cassava peels, rice husk, corn cob, yam
peel and sugarcane bagasse for renewal energy is imperative.

1.2 Statement of Problem

In this study bioethanol will be produced from lignocelluloses material such as
rice husk, cassava peel, corn cob, sugarcane bagasse and yam peels. The
bioethanol produced will be characterized and the effect of the process variables
will be studied.

1.3 Aims and Objectives

1.3.1. Aims

The aim of this study was the production of ethanol from corn cob, rice husk,
cassava peels, sugarcane bagasse and yam peels.

1.3.2 Objectives
 Acid hydrolysis of corn cob and investigate the effects of process variables
such as hydrolysis time, concentration of H2SO4 and temperature in the
hydrolysis process.
 To ferment acid hydrolysis products
 Product characterization
 Study of the effect of process variables

1.4 Significance of study

This thesis has great significance in terms of assuring the production of

alternative forms of energy from cassava peels, rice husk corn cobs, yam peels,
sugarcane bagasse and in the determination of the potentials of the country in
supplying balancing feedstock for bioethanol production which is locally
available, abundant and no economical value .This study also highly contributes
in the substitute fossil fuel by biofuel. Fossil fuels are quickly being depleted
due to extensive and continuing over-utilization. If consumption goes in this
rate the fossil fuel reserve will be depleted completely within short period. In
addition to this, continuous burning of fossil fuel increase emission of green
house gasses to the atmosphere and causes global warming. Therefore, a
renewable and non-food competitive feedstock is desirable for the production of
alternative fuel such as bioethanol
Bioethanol production from agricultural corn cobs, cassava peels, yam peels,
rice husk and sugarcane bagasse are considered second generation biofuel
process since it has no direct conflict with human food, as it happens with the
first generation biofuels produced from agricultural crops, such as corn and
sugarcane for bioethanol production.

1.1 Scope of Study

In order to accomplish the objective, the scope of this research work revolves
around the production of Bioethanol from lignocelluloses material and its
characterization.

CHAPTER TWO

LITERATURE REVIEW

2.1 Bioethanol

Bioethanol a distilled colorless liquid fuel obtained from numerous potential

feedstock varieties such as sugar beet, wheat, corn, cassava, fruits, bagasse,
barley, molasses, skim milk (whey),potatoes, sorghum, switch grass and
cellulose biomass such as wood, paper, straw and other cellulose wastes such as
grasses, others includes municipal solid wastes. These various waste streams for
Ethanol production have their peculiar properties and generally differ.
Feedstocks prices and price of natural gas are predominant influential factors
that determine the cost of Ethanol Production.

Ethanol as an alternative fuel, offers a sustainable economy by reducing the use

of imported petroleum, emitting neutral CO 2 (g), boost economy providing
value added market opportunities for the Agricultural sector (Shell Global,
2001).

National energy security, energy sustainability and climate changes are the
primary reasons to find alternative, renewable and reliable resources to fulfill
energy demand. Currently, the world energy demand increases at an annual
growth rate of 1.6% up to 45% by 2030 (IEA, 2008). Figure 2.1 depicts the
world consumption of marketed energy from different fuel sources (EIA, U.S.
(2010)) and most of our energy demand depends on conventional fossil fuels.
However, fossil fuels are nonrenewable energy sources, and will be exhausted
in near future. In addition, the consumption of fossil fuels produces different
pollutants and causes environmental issues. These issues and depletion of fossil
fuel resources have led a rapid expansion of renewable resources. At present,
there are different renewable resources, namely wind, tide, hydropower and
biomass. These renewable resources can satisfy energy demand in power sector,
but transportation sector mainly depends on liquid fuels, which cannot be
produced from other renewable sources except biomass. The world energy
outlook projected to meet 5% of world demand for transportation fuel by
biofuels in 2030 (IEA, 2008).

Figure 2.1: World marketed energy use by fuel type, 1990-2035 (quadrillion Btu) (EIA, U.S.
(2010))
Biofuels, liquid fuels produced from biomass (FAO, 2008), are drawing
increasing attention globally as alternative sources to petroleum-derived
transportation fuels to help address high energy costs and global warming
concerns associated with fossil fuels. Currently, the use of biofuels is being
promoted as a sustainable means of increasing energy independence, reducing
greenhouse gas (GHG) emissions, contributing to farmer income, opening new
business opportunities, reducing poverty and promotion of rural development
(FAO, 2008; Howarth et al., 2008).

2.2 Category of Bioethanol

Biofuels can be categorized into three major groups, namely first-generation,
second-generation and third-generation types. The main distinction among them
is the type of feedstock used in the production process, their current and future
availability (OECD/IEA, 2008; UNEP, 2009).

First generation biofuels are currently produced in large commercial quantities

in many countries from agricultural crops such as sugarcane, maize, 2 soybean
and jatropha through well-established technologies such as hydrolysis,
fermentation and trans-esterification (UNEP, 2009). Bioethanol and biodiesel
are the two most well-known examples of first-generation biofuels used in the
transport sector and account for over 90% of global biofuel usage.

Second-generation biofuels may be produced from lignocellulosic biomass such

as agricultural crop residues, forestry and wood processing wastes, organic
components of municipal solid waste (MSW) and energy crops such as
Mischantus giganteus, using either thermochemical or biochemical processes
(UNDESA, 2007; OECD/IEA, 2008; UNEP, 2009).

Cellulosic ethanol or ethyl alcohol (CH3-CH2OH) and cellulosic butanol (CH3-

CH2-CH2-CH2OH) are two well-known examples produced from lignocellulosic
biomass through biochemical processes. They can be used as alternative sources
to petroleum-gasoline for transportation (OECD/IEA, 2008). While the
properties of cellulosic ethanol are similar to those of first-generation
bioethanol, the relative abundance and wide range of lignocellulosic biomass
used as feedstock, which is non-food, give them an advantage (Vantomme,
2006; UNDESA, 2007; UNEP, 2009).

Third-generation biofuels are, however, produced from feedstocks such as

micro- and macro-algae, vegetable oils and biodiesel (OECD/IEA, 2008;
UNEP, 2009).
2.3Bioethanol application and its utilization

Ethanol or ethyl alcohol, has been identified as one of the most interesting
synthetic oxygen-containing organic chemical because of its unique
combination of properties as a solvent, a beverage, an antifreeze, and more
especially due to its versatility as a chemical intermediate for other chemicals.
Ethanol is an industrial chemical which has high significant utilization. It can be
used in the transportation sectors as well as in production of pharmaceutical
products, dyestuffs, perfumes and numerous products. Ethanol under ordinary
condition is a volatile, flammable, clear, colorless chemical compound. The
largest bioethanol producers in the world are the USA and Brazil, though they
utilize cornstarch and sugarcane juice as the main substrate for bioethanol
production, which is globally seen as unsustainable because of energy, food and
feed controversy (Pimental et al., 2009; Wheals et al., 1999). Bioethanol can be
blended with normal gasoline in various forms: low-level blends (E10), high-
level blends (E85 or E95) (Lynd, 1996). E10 (10% ethanol and 90% gasoline) is
the most common ethanol blend in USA, and this can be used in new vehicle
engines with non- engines. Most new cars sold in Brazil are flexible-fuel
vehicles (FFV) that can run on pure 100% anhydrous ethanol as well as blends
with up to 80% of gasoline. In Europe, a large volume of bioethanol is used in
blends with gasoline (5% ethanol and 95% gasoline) (Wheals et al., 1999).
However, the market potential for bioethanol is not just limited to transport fuel
or energy production but has a great potential to supply the existing chemical
industry. Ethanol is also used as an oxygenate additive for conventional
gasoline, as a replacement for methyl tertiary buthyl ether (MTBE), which is
normally mixed with gasoline as additive to improve the octane number. Due to
toxic properties associated with MTBE, which is also responsible for
groundwater contamination, it is therefore more frequently replaced by ethyl
tertiary butyl ether (ETBE) that is normally produced from bioethanol (Rutz and
Janssen, 2007; Twidell and Weir, 2006). Ethanol is therefore an excellent
additive for preventing engine knock and overheating of the engine valves
(Wyman, 1996). Ethanol has higher octane number (96- 113) than conventional
gasoline (86-87) and thus, when blended the octane number increases, thereby
reducing the need for toxic, octane enhancing additives (Mielenz, 2001). It
enables combustion engines to run at a higher compression ratio and therefore
provides a net performance gain of nearly 15% w/w (Wheals et al., 1999;
Knapp et al., 1998). As earlier mentioned, the main chemical industries that
patronize ethanol industry are: solvents and alcoholic for beverages.

According to Fukuda et al. (2009), about 73% of produced ethanol worldwide is

used as fuel ethanol, while the rest goes to the beverage and industrial sectors
(Figure 1).

Figure 2.2:- Worldwide ethanol application (millions of gallons) (Berg and

Licht 2004).

2.4 World Market of Ethanol

Today, bio-ethanol is the most dominant bio-fuel and its global production
showed an upward trend over the last 25 years with a sharp increase from 2000.
As of 2005, worldwide production capacity for bio-ethanol fuel was about 45
billion liters per year, with approximately 15% annual growth between 2000
and 2005. This value increased to 49 billion liters in 2006, when the Americans
produced 75% of the total world ethanol output, followed by Asia/Pacific and
Europe/Africa with respective values of 15 and 10% (Licht, 2006). The
industrial alcohol market showed a rather modest rate of growth similar to the
increase in Gross Domestic Product in many countries. The market for beverage
alcohol in most developed countries is stagnating, due to increased health
awareness.

In 2009, production of fuel ethanol reached an estimated 76 billion liters, an

increase of 10 percent over 2008. The United States and Brazil accounted for 88
percent of global ethanol production in 2009. Most of the increased production
occurred in the United States (Licht, 2006).

After a significant downturn in the U.S. fuel ethanol market in 2008, U.S.
production rose 16 percent to about 41 billion liters in 2009, accounting for
approximately 54 percent of global ethanol production. According to one
estimate, U.S. ethanol (which is mostly corn-based) displaced more than 360
million barrels of imported oil for gasoline production. The highest sugar prices
in years, combined with adverse weather conditions in a major producing
region, resulted in a drop in Brazil’s ethanol production from 27.1 billion liters
in 2008 to 26.3 billion liters in 2009. All ethanol produced in Brazil is from
sugar cane. All fueling stations in Brazil sell both pure ethanol and gasohol, a
25 percent ethanol/75 percent gasoline blend. Flex-fuel cars, which can use pure
ethanol, gasoline, or any blend of the two, provide the flexibility to choose fuel
based on price at the pump. They have been widely embraced by drivers and
represent more than 95 percent of all new cars sold in Brazil. In recent years,
significant global trade in fuel ethanol has emerged, with Brazil being the
leading exporter. However, Brazilian ethanol export declined by almost 31
percent in 2009. International demands declined in great part because of the
global economic crisis (Licht, 2006).

Table2.1:- World Fuel Ethanol Production by Country or Region (millions of Gallon)

Country 2007 2008 2009 2010 2011 2012 2013 2014

USA 6,521 9,309 10,938 13,298 13,948 13,300 14,300
13,300
Brazil 5,019 6,472 6,578 6,922 5,573 5,577 6,267 6,190
Europe 570 734 1,040 1,209 1,168 1,179 1,371 1,445
China 486 502 542 542 555 555 696 635

Canada 211 238 291 357 462 449 523 510

Rest of 315 389 914 985 698 752 1,272 1,490
World
WORLD 17,644 20,303 23,311 22,404 23,429 24,570
13,123 21,812

Data Source: F.O. Licht cited in Renewable Fuels Association, Ethanol Industry
Outlook 2008-2014 reports. Available at www.ethanolrfa.org/pages/annual-
industry-outlook

2.5 Feedstocks for Bio-ethanol Production

Application of agricultural wastes in bio-fuel industry is the focus of many

researches aimed at achieving an effective and efficient waste management
scheme.

Fossil fuel is a non renewable source of energy and the concern generated by its
sustainability, economic and environmental impact led to the use of renewable
sources for fuel (Schneider, S.H. (1989)). Energy from biomass is renewable
and can contribute to sustainable development. In addition, biomass resources
are often available locally and processing is feasible without high capital
investment. Furthermore, dependence on biomass energy can help reduce green
house gas emission (Lynd, L. R. (1996).) thereby impacting on public health.

The various raw materials used in the manufacture of ethanol via fermentation
are classified into three main types: sugars, starches and cellulose materials
(Lin, Y. and Tanaka, S. (2006))

2.5.1 Sugars

Fermentation involves microorganisms that use the fermentable sugars for food
and in the process produces ethanol and other byproducts. These
microorganisms can typically use the 6-carbon sugars, one of the most common
being glucose. Therefore, biomass materials containing high levels of glucose or
precursors to glucose are the easiest to convert to ethanol. However, since sugar
materials are in the human food chain, these materials are usually too expensive
to use for ethanol production. One example of a sugar feedstock is sugarcane.
Although fungi, bacteria, and yeast microorganisms can be used for
fermentation, specific yeast (Saccharomyces cerevisiae also known as Bakers’
yeast, since it is commonly used in the baking industry) is frequently used to
ferment glucose to ethanol. Theoretically, 100 grams of glucose will produce
51.4 g of ethanol and 48.8 g of carbon dioxide. However, in practice, the
microorganisms use some of the glucose for growth and the actual yield is less
than 100%. Other biomass feedstocks rich in sugars include sugar beet, sweet
sorghum, and various fruits. However, these materials are all in the human food
chain and, except for some processing residues are generally too expensive to
use for fuel ethanol production (J. Janick and P.C. Badger, 2002).

2.5.2 Starches
Another potential ethanol feedstock is starch. Starch molecules are made up of
long chains of glucose molecules. Thus, starchy materials can also be fermented
after breaking starch molecules into simple glucose molecules. Examples of
starchy materials commonly used around the world for ethanol production
include cereal grains, potato, sweet potato, and cassava. Cereal grains
commonly used in the US for ethanol production include maize and wheat.
Starchy materials require a reaction of starch with water (hydrolysis) to break
down the starch into fermentable sugars (saccharification). Typically, hydrolysis
is performed by mixing the starch with water to form slurry which is then stirred
and heated to rupture the cell walls. Specific enzymes that will break the
chemical bonds are added at various times during the heating cycle (J. Janick
and P.C. Badger, 2002).

2.5.3 Lignocellulosic Biomass

Agricultural residues are a great source of lignocellulosic biomass which is

renewable, chiefly unexploited, and inexpensive. Such resources include:
leaves, stems, and stalks from sources like corn cob, corn stover, sugarcane
bagasse, rice husk, woody crops, and forest residues. Also, other multiple
sources of lignocellulosic waste from industrial and agricultural processes
include citrus peel waste, sawdust, paper pulp, industrial waste, municipal solid
waste, and paper mill sludge. In addition, dedicated energy crops for biofuels
could include perennial grasses such as switchgrass and other forage feedstocks
such as Miscanthus giganteus, M. sinensis, Bermuda grass, elephant grass,
poplar etc. (Maki et al., 2009).

Cellulose materials represent the most abundant global source of biomass and
have been largely unutilized. The global production of plant biomass, of which
over 90% is lignocellulose, amounts to about 200×10 9 tons per year, where
about 8 to 20×109 tons of the primary biomass remains potentially accessible
(Lin et al., 2006).
Cellulose is not used for food and the biofuels industries that use lignocellulosic
materials do not compete for raw materials. Cellulosic biomass such as switch
grass and agricultural wastes are cheaper to produce and requires fewer inputs
in form of energy.

Cultivation of such plants improves soil fertility and is accompanied by less soil
erosion. Moreover, the process also represents a means of effective and efficient
waste management as a large proportion of agricultural and municipal wastes
are lignocellulosic. Another benefit of cellulosic ethanol is the reduction in
greenhouse gas emission. Compared to gasoline, ethanol burns cleaner with
greater efficiency thereby releasing up to 85% less carbon dioxide (Demain et
al., 2005). In contrast, ethanol from corn which frequently involve the use of
natural gas as energy source for processing may not reduce green house gas
emission at all (Maki et al., 2009).

Lignocelluloses are complex substrates composed of a mixture of carbohydrate

polymers i.e. cellulose and hemicelluloses tightly bound to lignin, a complex
aromatic polymer, mainly by hydrogen bonds but also by some covalent bonds.
Lignin interferes with cellulose hydrolysis because it acts as a physical barrier
that prevents the contact of cellulase to cellulose (Umamaheswari et al., 2010).
The first step in biofuels production from lignocelluloses therefore is
delignification to liberate cellulose and hemicelluloses from their complex with
lignin. It is a very crucial, rate limiting and difficult task (Demain et al., 2005).
Cellulose from wood, agricultural residues, etc must be converted to sugars,
either by acid or alkali hydrolysis, or by action of cellulase enzymes and the
sugar can be fermented to ethanol.
The chemical compositions (cellulose, hemicellulose and lignin content) of
various feedstocks for bioethanol production in Table2.1 show that, on average,
agricultural wastes with the highest cellulose content. Forest residues comprise
about 80% of the world’s biomass [Demirbas, A., 2005] and in the United
States alone 33.5-44.6 million metric tons of corn cob are available for harvest
each year [Zych, D., 2008].

Table 2.3:- The contents of cellulose, hemicellulose, and lignin in common

agricultural residues and wastes

Lignocellulosic material Cellulose Hemicellulose Lignin

(%) (%) (%)
Cassava peels 5.40 21.65 20.8-24.4
Rice husk 35 25 20
Sugarcane bagasse 41-55 20- 27.5 18- 26.3
Corn cobs 45 35 15
Yam peel 34.5 36.0 22.4
– 30
Paper 85 – 99 0 0 – 15
Wheat straw 30 50 15
Sorted refuse 60 20 20
Leaves 15 – 20 80 – 85 0
Cotton seed hair 80 – 95 5 – 20 0
News paper 40 – 55 25 – 40 18 – 30
waste paper from 60 – 70 10 – 20 5 – 10
chemical pulps
Primary wastewater 8 – 15 Na Na
solids
Solid cattle manure 1.6 – 4.7 1.4 – 3.3 2.7 – 5.7
Coastal bermudagrass 25 35.7 6.4
Switchgrass 45 31.5 12
Swine waste 6.0 28 Na

a
Source: Sun, Y. and Cheng, J. (2002).
2.6 Composition of Lignocellulosic Materials

Lignocellulosic materials do not contain monosaccharides that are readily

available for bioconversion but polysaccharides such as cellulose and
hemicellulose, lignin, extractives, and ashes. The polysaccharides need to be
hydrolysed by means of either enzymes or acids to fermentable sugars (Kalman,
2006).
Cellulose is an unbranched homopolysaccharide composed of β-D glucose units
linked by (1,4) glycosidic bonds. However, the basic building block of cellulose
is a dimer of two glucose units known as cellobiose (Fig. 2.2). Cellulose is the
most abundant material on Earth, and it is the main constituent of plants. It is
also present in bacteria, fungi, algae and even in animals (O'Sullivan, 1997). In
nature, cellulose chains have a degree of polymerization (DP) of approximately
10,000 and 15,000 glucopyranose units in wood and native cotton celluloses,
respectively (Sjostrom, 1981).

Fig. 2.3:- Chemical structure of cellulose.

Hemicellulose or polyose is a mixture of polymers comprising pentoses,
hexoses hexuronic acids and deoxy-hexoses. Hemicelluloses differ from
celluloses by a composition of various sugar units and by much shorter and
branched molecular chains.
In contrast to cellulose which is crystalline, strong, and resistant to hydrolysis,
hemicellulose has a random, amorphous structure with little strength. Therefore,
it is easily hydrolyzed by dilute acid or base, as well as hemicellulase enzymes
(Fengel and Wegener, 1989).
Lignin is a complex, hydrophobic, cross-linked, three-dimensional aromatic
polymer of phenylpropane building blocks. The mechanical strength properties
of plants are mainly due to incorporation of lignin into their cell walls, whereby
huge plants such as trees can remain upright. P-coumaryl alcohol (I), coniferyl
alcohol (II) and sinapyl alcohol (III) are the primary precursors and building
units of all lignins (Fig.2.3).

Fig.2.4:- The building units of lignin (Fengel and Wegener, 1989).

Lignin is one of the most complicated natural polymers with respect to its
structure and heterogeneity, which make it extremely resistant to chemical and
biological degradation (Lee, 1997).

Extractives: are woody compounds that are soluble in neutral organic solvents
or water. The extractives usually represent a minor fraction (between 1-5%) of
lignocellulosic materials. They contain a large number of both lipophilic and
hydrophilic constituents. The extractives can be classified in four groups: (a)
terpenoids and steroids, (b) fats and waxes, (c) phenolics constituents and, (d)
inorganic components (Taherzadeh, 1997).
2.7 Agricultural waste used for feedstock of Bioethanol

 Corn cobs

The maize plant comprise of the stalks, husks, shanks, silks, leaf blades, leaf
sheaths, tassels and cobs. The corn cob carries the grain and together with
associating husks, shanks and silks are harvested from the farm. The other parts
are left on the farm to rot [Kludze et al., 2010]. Corn cobs form about 30% of
maize agro-wastes [Rangkuti and Djajanegara, 1983]. The agricultural residues
from maize production are potential sources of sugar for ethanol production, in
addition to starch and by-products. When maize is harvested in the field, the
corn grain is separated from the cobs, stalks, and leaves. While the grain is
transported for storing and processing, the stover is currently not widely
collected. However, this biomass could be used for lignocellulosic ethanol
production. Corn stover includes stalks, leaves, and corn cobs. As a renewable
raw material, the corncobs from grain maize are a potential feedstock for the
production of biogas, biodiesel and bioethanol to fulfill the increasing demand
for biofuels (Ioannidou et al., 2009).

Before its use as a substrate for fermentation processes, the raw material has to
be pretreated. Pretreatment is one of the many steps in the cellulose-to-ethanol
process, but represents a currently critical step for hydrolysis. An effective
pretreatment is performed at conditions that avoid degradation of pentose from
hemicellulose, or glucose from cellulose, and limit formation of degradation
products that inhibit the growth of fermentative microorganisms (Mosier et al.
2005a). The lignocellulosic structure is destroyed by treatment with high
temperature and saturated steam in a reactor followed by a sudden pressure
decrease (Menon & Rao, 2012), (Eisenhuber et al., 2013). Corncobs are a
lignocellulosic material composed of cellulose, hemicellulose and lignin. These
polymeric fibres consist of monomeric molecules. Cellulose is built of C6
sugars; hemicellulose mainly of the C5 sugars xylose and arabinose. Lignin
consists of phenolic macromolecules. Corn cobs contain 32.3-45.6% cellulose,
39.8% hemicelluloses - mostly composed of pentosan and 6.7-13.9% lignin
[Zych, 2008; Sun and Cheng, 2002] and can be converted to fermentable sugar
for ethanol production.

Table 2.4: - Chemical composition of corn cobs

Component Content (%)

Cellulose 32.3 – 45.6
Hemicellulose 39.8
Starch 0
Lignin 6.7 – 15
Volatile matter 78.7
Ash 0.9
Extractive 3

 Cassava peels

When cassava is harvested in the field, the peels are separated from the cassava,
while the cassava is transported for storing and processing. However, this
biomass could be used for lignocellulosic ethanol production. As a renewable
raw material, the cassava peel from cassava are potential feedstock for the
production of biogas, biodiesel and bioethanol to fulfill the increasing demand
for biofuels.

Before its use as a substrate for fermentation processes, the raw material has to
be pretreated. Pretreatment is one of the many steps in the cellulose-to-ethanol
process, but represents a currently critical step for hydrolysis. An effective
pretreatment is performed at conditions that avoid degradation of pentose from
hemicellulose, or glucose from cellulose, and limit formation of degradation
products that inhibit the growth of fermentative microorganisms (Mosier et al.
2005a). The lignocellulosic structure is destroyed by treatment with high
temperature and saturated steam in a reactor followed by a sudden pressure
decrease (Menon & Rao, 2012), (Eisenhuber et al., 2013). Cassava peel is a
lignocellulosic material composed of cellulose, hemicellulose and lignin.

 Sugarcane bagasse

Sugarcane is one of the most widely cultivated crops in the world, with the
major producing countries being in the tropics, including Brazil, India, China,
Thailand, Pakistan and Mexico.

The extraction of sugar from this crop generates several residues that are often
disposed improperly especially where sugar mills use basic process technology.
The huge quantities of solid waste are often destroyed or burned inefficiently
causing environmental pollution. Sugarcane solid residues include bagasse and
filter cake. Bagasse is the solid residue resulting after the juice extraction from
the sugarcane stalks and contains the fibrous lignocellulosic material of the
stalks. The precipitate in the form of sludge slurry after filtration of the
sugarcane juice is the filter cake. Every 1000 tons of processed sugarcane
generates about 270 tons of bagasse and 34 tons of cake .Approximately, 1.81
billion tons of sugarcane were produced worldwide in 2015, and this is expected
to reach more than 2.21 billion tons by 2024. Based on these values, the world’s
potential generation of sugarcane bagasse will reach 0.6 billion tons, which
could be valorized into bioenergy, biofuels, and other products.

The expected increase in bagasse availability is driven by the increasing

demand for sugar, and sugarcane is the most important source of sugar in the
world. However, sugar industries are one of the most polluting ones in view of
the generated solid wastes, wastewater, and gaseous emissions of carbon
monoxide, volatile organic compounds, and also greenhouse gases during crop
cultivation phase . Transforming all by-products obtained from sugar mills
(bagasse, filter mud, fly ash and molasses) into value-added products will
minimize the pollution to a large extent. Treating sugar industry effluent for
reuse in agriculture and other applications is another strategy to reduce the
environmental impacts. In summary, sugar industry wastes should be seen as
economic resources that can be converted into valuable products in progressing
toward resource recovery as a sustainable solution that could generate social
welfare and economic development from the sugarcane industry and its
residues.

Table 2.5: - Chemical composition of sugarcane bagasse

Component Content (%)

Cellulose 42
Hemicellulose 28
Polysaccharide 4.6
Lignin 20
Sacharose 3
Ash 24
Fiber 48

 Rice husk

The lignocellulosic residues generated in the processing of rice, such as husk

and chaff, which is the waste left in the collection site, are considered materials
of little value and in some cases are waste. It is important to highlight the
economic and social influence of rice cultivation in Nigeria, since more than
28000 farmers and their families directly depend on it. Rice cultivation is one of
the few short-cycle crops that have remained stable despite the variability of
Nigeria agriculture. The annual production of rice is more than two million tons,
generating as a byproduct around 400000 tons of husk. In Nigeria, the burning
of this waste is usually a quick strategy for its elimination, however, the
negative environmental impact that is generated must not be ignored, due to the
fact that, during the combustion process of the rice husk (RH), high levels of
greenhouse gases are emitted.

RH has a high content of polysaccharides such as cellulose and hemicellulose,

so this is considered a valuable resource in the production of sugars that can be
fermented. Because the national production of rice is considerable, large
quantities of husk are obtained that currently have little use, therefore, it is
important to find other applications, in addition to their use as fuel for the
ovens, among them, their use as raw material for the production of sugars and
its subsequent fermentation and distillation to obtain bioethanol.

The literature reports obtaining bioethanol from rice husk using Kluyveromyces
marcianus CK8. With respect to the use of the waste generated during the
production of rice, it is found the use of these residues to obtain sugars,
obtaining a carbonated alcohol beverage and obtaining lactic acid, among
others. Kinetic analysis of the thermal decomposition of four lignocellulosic
biomasses including rice husk and a literary review of the synthetic silica
production pathways, focusing on the possibility of using rice husk ash as a
possible raw material for reinforcement of polymeric materials are also
reported. In San José de Cúcuta (Norte de Santander) there are several
companies that process and market rice, among them Coagronorte Ltda,
Arrocera Agua Blanca S.A., and Arrocera Gelvez SAS, among others, which
generate as a byproduct a large amount of husk. These companies generally use
the RH as fuel for the ovens, generating problems for the environment due to
the emission of polluting smoke. With this, it is inferred that these companies
have no established plans to use or adequately dispose of the large quantities of
husk that are produced. For this reason, the proposal was made to produce
bioethanol from this agro industrial waste, taking advantage of its content of
cellulose and hemicellulose, which are broken down into sugars through
enzymatic hydrolysis with acid cellulase (CFB3S) and subsequent fermentation
of sugars obtained for the production of alcohol. Initially, bioethanol was
obtained on a laboratory scale in order to determine the appropriate conditions,
to then produce it on a pilot scale.

 Yam peel

Yam has the lowest moisture content (58.18 ±1.22 % w/w) and the highest
starch concentration (85.51 ± 1.21% w/w) in dry basis. Also, its starch
concentration surpasses that of corn and cassava in a dry basis (78 % and 35 %
w/w, respectively), making it a potential feedstock for ethanol production.

However, ethanol production from amylaceous crops requires a starch

hydrolysis process before fermentation, which increases direct costs. This has
been observed in feedstocks such as corn, with 35 % higher production costs per
liter of ethanol than sugarcane.

2.8 Overview of Ethanol Process

Ethanol can be produced in two different ways. Either chemically, by hydration

of ethylene, which is derived from crude oil or natural gas, or by fermentation
of sugar containing feeds, starchy feed materials or lignocellulosic materials.
About 5% - 10% of the ethanol produced in the world is a petroleum product.
Petroleum ethanol product is made by the catalytic hydration of ethylene with
sulfuric acid as the catalyst. It can also be obtained via ethylene or acetylene,
from calcium carbide, coal, oil gas, and other sources. The two primary ways of
producing fuel ethanol from cellulosic feedstock are: Biochemical conversion
process and Thermo chemical conversion process (Cellulosic ethanol, 2010) (as
cited by Wondale M, 2014).
 Ethanol

Fig2.6 Schematic diagram of cellulosic ethanol production

2.8.1 Biochemical Conversion

The biochemical conversion process is similar to the process currently used to

produce ethanol from agricultural waste. Enzymes or acids are used to break
down a plant’s cellulose into sugars, which are then fermented into liquid fuel.
Four key steps are involved. First, feedstock is pretreated by changing its
chemical makeup to separate the cellulose and hemicellulose from the lignin in
order to maximize the amount of available sugar. Second, hydrolysis uses
enzymes or acids to break down the complex chains of sugar molecules into
simple sugars for fermentation. Third, fermentation is used to convert the sugar
into liquid fuel. Fourth, the liquid fuel is distilled to achieve a 95% pure form
(fig. 2.6) (Zhu JY et al., 2009).

2.8.1.1 Pretreatment

Pretreatment is required to alter the biomass macroscopic and microscopic size

and structure as well as its submicroscopic chemical composition and structure
so that hydrolysis of carbohydrate fraction to monomeric sugars can be
achieved more rapidly and with greater yields (Sun and Cheng, 2002; Mosier et
al., 2005).

The effect of pretreatment of lignocellulosic materials has been recognized for a

long time. The purpose of the pretreatment is to remove lignin, reduce cellulose
crystalline and increase the porosity of the materials. Pretreatment must meet
the following requirements: Improve the formation of sugars or the ability to
subsequently form sugars by acidic or enzymatic hydrolysis; avoid the
degradation or loss of carbohydrate; avoid the formation of byproducts
inhibitory to the subsequent hydrolysis and fermentation processes. Physical,
chemical, physico-chemical, and biological processes have been used for
pretreatment of lignocellulosic materials (Sun, Y. and Cheng, J. (2002).

1 Physical Pretreatment

Mechanical Comminution: Waste materials can be comminuted by a

combination of chipping, grinding and milling to reduce cellulose crystallinity.
The size of the materials is usually 10–30 mm after chipping and 0.2–2 mm
after milling or grinding. Vibratory ball milling has been found to be more
effective in breaking down the cellulose crystallinity of spruce and aspen chips
and improving the digestibility of the biomass than ordinary ball milling. The
power requirement of mechanical comminution of agricultural materials
depends on the final particle size and the waste biomass characteristics (Sun, Y.
and Cheng, J. (2002).

Pyrolysis: Pyrolysis has also been used for pretreatment of lignocellulosic

materials. When the materials are treated at temperatures greater than 300°C,
cellulose rapidly decomposes to produce gaseous products and residual char.
The decomposition is much slower and less volatile products are formed at
lower temperatures. Mild acid hydrolysis (1 N H2SO4, 97 °C, 2.5 h) of 20 the
residues from pyrolysis pretreatment has resulted in 80–85% conversion of
cellulose to reducing sugars with more than 50% glucose .The process can be
enhanced with the presence of oxygen. When zinc chloride or sodium carbonate
is added as a catalyst, the decomposition of pure cellulose can occur at a lower
temperature (Sun, Y. and Cheng, J. (2002).

2 Physico-chemical pretreatment

Steam explosion (autohydrolysis):

Steam explosion (uncatalysed or catalysed) is one of the most applied

pretreatment processes owing to its low use of chemicals and limited energy
consumption. With this method high-pressure saturated steam is injected into a
batch or continuous reactor filled with biomass. During the steam injection, the
temperature rises to 160-260 ºC. Subsequently, pressure is suddenly reduced
and the biomass under-goes an explosive decompression with hemicellulose
degradation and lignin matrix disruption as result.
Results of steam-explosion pretreatment depend on residence time, temperature,
particle size and moisture content (Sun and Cheng, 2002). Studies have been
carried out to try to improve the results of steam explosion by addition of
chemicals such as acid or alkali (Cara et al., 2008). Limitations of steam
explosion include the formation of degradation products that may inhibit
downstream processes (Garcia-Aparicio et al., 2006).
Steam explosion is recognized as one of the most cost effective pretreatment
processes for hardwoods and agricultural residues, but it is less effective for
softwoods (Sun, Y. and Cheng, J. (2002).

Ammonia fiber explosion (AFEX)

In the AFEX process, biomass is treated with liquid ammonia at high

temperature and pressure (Teymouri et al., 2005). After a few seconds, pressure
is swiftly reduced. A typical AFEX process is carried out with 1-2 kg
ammonia/kg dry biomass at 90 °C during 30 min. It reduces the lignin content
and removes some hemicellulose while decrystallising cellulose. The cost of
ammonia and especially of ammonia recovery drives the cost of the
pretreatment (Holtzapple et al., 1991 & 1994), although ammonia is easily
recovered due to its volatility.

To reduce the cost and protect the environment, ammonia must be recycled after
the pretreatment. In an ammonia recovery process, a superheated ammonia
vapor with a temperature up to 200 oC was used to vaporize and strip the
residual ammonia in the pretreated biomass and the evaporated ammonia was
then withdrawn from the system by a pressure controller for recovery .The
ammonia pretreatment does not produce inhibitors for the downstream
biological processes, so water wash is not necessary .AFEX pretreatment does
not require small particle size for efficacy (Sun, Y. and Cheng, J. (2002).

CO2 explosion

Similar to steam and ammonia explosion pretreatment, CO 2 explosion is also

used for pretreatment of lignocellulosic materials. It was hypothesized that CO 2
would form carbonic acid and increase the hydrolysis rate. The yields were
relatively low compared to steam or ammonia explosion pretreatment, but high
compared to the enzymatic hydrolysis without pretreatment. Compared CO 2
explosion with steam and ammonia explosion, found that CO2 explosion was
more cost-effective than ammonia explosion and did not cause the formation of
inhibitory compounds that could occur in steam explosion (Sun, Y. and Cheng,
J. (2002).

3 Chemical pretreatment

Alkaline Pretreatment:

Some bases can also be used for pretreatment of lignocellulosic materials and
the effect of alkaline pretreatment depends on the lignin content of the
materials. The mechanism of alkaline hydrolysis is believed to be saponification
of intermolecular ester bonds cross linking xylan hemicelluloses and other
components, for example, lignin and other hemicellulose. The porosity of the
lignocellulosic materials increases with the removal of the crosslinks. Dilute
NaOH treatment of lignocellulosic materials caused swelling, leading to an
increase in internal surface area, a decrease in the degree of polymerization, a
decrease in crystallinity, separation of structural linkages between lignin and
carbohydrates, and disruption of the lignin structure. The digestibility of NaOH-
treated hardwood increased from 14% to 55% with the decrease of lignin
content from 24–55% to 20%. However, no effect of dilute NaOH pretreatment
was observed for softwoods with lignin content greater than 26% (Sun, Y. and
Cheng, J. (2002)

Dilute-sulfuric acid pre-hydrolysis (DAPH)

Acid pretreatment firstly developed in Germany in 1898. In this method

concentrated or dilute mineral acids like sulfuric acid are used in order to break
down hemicelluloses into monomeric sugars and simultaneously removing part
of the lignin. This method needs a small amount of water since a small amount
of energy is required to get an optimum temperature.

Some advantages of this method are: (1) High yield of hemicelluloses sugar, (2)
Remove of lignin and hemicelluloses in this method increases exposing of
cellulose to enzyme, (3) Remove of heavy metals in the raw materials.
Some disadvantages of this method are: (1) Neutralization of acids is necessary,
(2) Degradation of hemicelluloses sugar produce some inhibitors like acetic acid
and furfural, (3) High cost of reactor due to high pressure and temperature and
resistance to low PH (Sanchez, Pilcher et al. 2004).

Ozonolysis:

Ozone can be used to degrade lignin and hemicellulose in many lignocellulosic

materials such as wheat straw, bagasse, green hay, peanut, pine, cotton straw,
and poplar sawdust. The degradation was essentially limited to lignin and
hemicellulose was slightly attacked, but cellulose was hardly affected.
Enzymatic hydrolysis yield increased from 0% to 57% as the percentage of
lignin decreased from 29% to 8% after ozonolysis pretreatment of poplar
sawdust .Ozonolysis pretreatment has the following advantages: (1) it
effectively removes lignin; (2) it does not produce toxic residues for the
downstream processes; and (3) the reactions are carried out at room temperature
and pressure. However, a large amount of ozone is required, making the process
expensive (Sun, Y. and Cheng, J. (2002).

Oxidative delignification

Lignin biodegradation could be catalyzed by the peroxidase enzyme with the

presence of H2O2. The pretreatment of cane bagasse with hydrogen peroxide
greatly enhanced its susceptibility to enzymatic hydrolysis. About 50% lignin
and most hemicellulose were solubilized by 2% H 2O2 at 30 oC within 8 h, and
95% efficiency of glucose production from cellulose was achieved in the
subsequent saccharification by cellulase at 45 oC for 24 h used wet oxidation
and alkaline hydrolysis of wheat straw (20 g straw/l, 170 oC, 5–10 min), and
achieved 85% conversion yield of cellulose to glucose (Sun, Y. and Cheng, J.
(2002).

Organosolv process

In the organosolv process, an organic or aqueous organic solvent mixture with

inorganic acid catalysts (HCl or H2SO4) is used to break the internal lignin and
hemicellulose bonds. The organic solvents used in the process include
methanol, ethanol, acetone, ethylene glycol, and triethylene glycol and
tetrahydrofurfuryl alcohol. Organic acids such as oxalic, acetylsalicylic and
salicylic acid can also be used as catalysts in the organosolv process. At high
temperatures, the addition of catalyst was unnecessary for satisfactory
delignification. Usually, a high yield of xylose can be obtained with the addition
of acid. Solvents used in the process need to be drained from the reactor,
evaporated, condensed and recycled to reduce the cost. Removal of solvents
from the system is necessary because the solvents may be inhibitory to the
growth of organisms, hydrolysis, and fermentation (Sun, Y. and Cheng, J.
(2002).

4 Biological pretreatment

In biological pretreatment processes, microorganisms such as brown, white and

soft-rot fungi are used to degrade lignin and some hemicellulose in waste
materials. Brown rots mainly attack cellulose, while white and soft rots attack
both cellulose and lignin. White-rot fungi are the most effective basidiomycetes
for biological pretreatment of lignocellulosic materials.

The white-rot fungus P. chrysosporium produces lignin-degrading enzymes,

lignin peroxidases and manganese- dependent peroxidases, during secondary
metabolism in response to carbon or nitrogen. Both enzymes have been found
in the extracellular filtrates of many white-rot fungi for the degradation of wood
cell walls. Other enzymes including polyphenol oxidases, laccases, H 2O2
producing enzymes and quinone-reducing enzymes can also degrade lignin. The
advantages of biological pretreatment include low energy requirement and mild
environmental conditions. However, the rate of hydrolysis in most biological
pretreatment processes is very low (Sun, Y. and Cheng, J. (2002).

Figure 2.7 Schematic Representation on Biomass Pre-Treatment (Mosier et al.,

2005).

2.8.1.2 Hydrolysis
After the pretreatment process, there are two types of processes to hydrolyze the
feedstocks for fermentation into ethanol, most commonly used are acid (dilute
and concentrated) and enzymatic hydrolysis. In addition, there are some other
hydrolysis methods in which no chemicals or enzymes are applied. For instance,
lignocellulose may be hydrolyzed by gamma-ray or electron-beam irradiation,
or microwave irradiation. However, those processes are commercially
unimportant.

Both enzymatic and chemical hydrolyses require a pretreatment to increase the

susceptibility of cellulosic materials. (Demirbas, A. (2005)).

1 Acid hydrolysis

Dilute Acid Hydrolysis

The dilute acid process is conducted under high temperature and pressure, and
has a reaction time in the range of seconds or minutes, which facilitates
continuous processing. The combination of acid and high temperature and
pressure dictate special reactor materials, which can make the reactor expensive.
The first reaction converts the cellulosic materials to sugar and the second
reaction converts the sugars to other chemicals. Unfortunately, the conditions
that cause the first reaction to occur also are the right conditions for the second
to occur.

The biggest advantage of dilute acid processes is their fast rate of reaction,
which facilitates continuous processing. Since 5-carbon sugars degrade more
rapidly than 6-carbon sugars, one way to decrease sugar degradation is to have a
two-stage process. The first stage is conducted under mild conditions to recover
the 5-carbon sugars while the second stage is conducted under harsher
conditions to recover the 6-carbon sugars (Demirbas, A. (2005)). A range of
temperatures between 170-190°C in the first stage and 200-230°C in the second
stage is commonly used (Galbe and Zacchi, 2002).

Concentrated Acid Hydrolysis

Hydrolysis of cellulosic materials by concentrated sulfuric or hydrochloric acids

is a relatively old process. The concentrated acid process uses relatively mild
temperatures, and the only pressures involved are those created by pumping
materials from vessel to vessel. Reaction times are typically much longer than
for dilute acid (Demirbas, A. (2005)). This method generally uses concentrated
sulfuric acid followed by a dilution with water to dissolve and hydrolyze or
convert the substrate into sugar. This process provides a complete and rapid
conversion of cellulose to glucose and hemicelluloses to 5-carbon sugars with
little degradation. The critical factors needed to make this process economically
viable are to optimize sugar recovery and cost effectively recovers the acid for
recycling. The solid residue from the first stage is dewatered and soaked in a 30
to 40% concentration of sulfuric acid for one to four hours as a pre-cellulose
hydrolysis step. The solution is again dewatered and dried, increasing the acid
concentration to about 70%.

After reacting in another vessel for 1 to 4 h at low temperatures, the contents are
separated to recover the sugar and acid. The sugar/acid solution from the second
stage is recycled to the first stage to provide the acid for the first stage
hydrolysis.

The primary advantage of the concentrated acid process is the potential for high
sugar recovery efficiency. The acid and sugar are separated via ion exchange
and then acid is re-concentrated via multiple effect evaporators. The low
temperatures and pressures employed allow the use of relatively low cost
materials such as fiberglass tanks and piping. The low temperatures and
pressures also minimize the degradation of sugars (Demirbas, A. (2005)).
Unfortunately, it is a relatively slow process and cost effective acid recovery
systems have been difficult to develop. Without acid recovery, large quantities
of lime must be used to neutralize the acid in the sugar solution. This
neutralization forms large quantities of calcium sulfate, which requires disposal
and creates additional expense (Demirbas, A. (2005)).

2 Enzymatic hydrolysis

The hydrolysis of cellulose by cellulolytic enzymes has been investigated

intensively since the early 1970s, with the objective of developing a process for
the production of ethanol. Enzymatic hydrolysis methods have shown distinct
advantages over acid based hydrolysis. The very mild process conditions (pH
4.8 and temperature 45– 50 oC) give potentially higher yields, the utility cost is
low (no corrosion problems). Therefore, this is the method of choice for future
cellulosic-to-ethanol processes (Duff and Murray, 1996; Hsu, 1996). Enzymatic
hydrolysis involves soluble enzymes working on insoluble substrates, so a
better understanding of the action of cellulase enzyme systems and their
substrates is required as this complex reaction involves multiple cellulose-
hydrolyzing activities and substrate features.

Enzymatic hydrolysis of cellulose is carried out by cellulase enzymes which are

highly specific (Beguin and Aubert, 1994). The products of the hydrolysis are
usually reducing sugars including glucose. Both bacteria and fungi can produce
cellulases for the hydrolysis of lignocellulosic materials.

These microorganisms can be aerobic or anaerobic, mesophilic or thermophilic.

Because the anaerobes have a very low growth rate and require anaerobic
growth conditions, most research for commercial cellulase production has
focused on fungi (Duff and Murray, 1996). During the enzymatic hydrolysis,
cellulose is degraded by the cellulases to reducing sugars that can be fermented
by yeasts or bacteria to ethanol.
Due to the tough crystalline structure, the enzymes currently available require
several days to achieve good results. Since long process times tie up reactor
vessels for long periods, these vessels have to either be quite large or many of
them must be used. Either option is expensive. Currently the cost of enzymes is
also too high and research is continuing to bring down the cost of enzymes.

However, if less expensive enzymes can be developed enzymatic processes hold

several advantages: (1) their efficiency is quite high and their byproduct
production can be controlled; (2) their mild process conditions do not require
expensive materials of construction; and (3) their process energy requirements
are relatively low (J. Janick and P.C. Badger, 2002).

2.8.1.3 Fermentation

Lignocellulose is often hydrolyzed by acid treatment. The hydrolysate obtained

is then used for bioethanol fermentation by microorganisms such as yeast.
Because such lignocellulose hydrolysate contains not only glucose, but also
various monosaccharides, such as xylose, mannose, galactose, arabinose, and
oligosaccharides, microorganisms should be required to efficiently ferment
these sugars for the successful industrial production of bioethanol (Katahira et
al., 2006). In general, the conversion of lignocellulosic material to sugar and
then ethanol is governed below:
(C6H10O5) n + nH2O nC6H12O6 + yeast 2nC2H5OH + 2nCO2

According to the reactions, the theoretical maximum yield is 0.51 kg bioethanol

and 0.49 kg carbon dioxide per kg of xylose and glucose (Hamelinck et al.,
2003, 2005). The overall reaction of this fermentation of hexose sugar (glucose)
by yeast has been expressed by Gay-Lussac which forms the basis of calculating
fermentation efficiency as:
3C5H10O5 5C2H5OH + 5CO2,
C6H12O6 2C2H5OH + 2CO2

Fermentation involves microorganisms that use the fermentable sugars for food
and in the process produces ethyl alcohol and other byproducts. These
microorganisms can typically use the 6-carbon sugars, one of the most common
being glucose. Therefore, cellulosic biomass materials containing high levels of
glucose or precursors to glucose are the easiest to convert to bioethanol.

To get an efficient fermentation severe inhibition should be avoided. There are

four different strategies to do this. These are modifying the hydrolysis process,
detoxification, in-situ detoxification and using less sensitive microorganisms to
inhibitors (Taherzadeh, M. (1999). Microorganisms, termed ethanologens,
presently convert an inadequate portion of the sugars from biomass to
bioethanol (Demirbas, 2005). There are a number of microorganisms that
produce significant quantities of bioethanol (Stewart, G. and Russell, I. (1987).

Xylose-fermenting microorganisms are found among bacteria, yeast and

filamentous fungi (Hahn-Hagerdal et al., 2006). Today, xylose fermenting
bacteria include both native and genetically engineered organisms, and many
have characteristics useful for simultaneous saccharification and fermentation
(Jeffries, T. and Jin, Y. (2000).

One of the most effective bioethanol producing yeasts, S. cerevisiae, has several
advantages owing to its high bioethanol production from hexoses and high
tolerance to bioethanol and other inhibitory compounds in the acid hydrolysates
of lignocellulosic biomass. However, because wild-type strains of this yeast
cannotutilize pentoses, such as xylose and arabinose, and celloligosaccharides,
bioethanol production from a lignocellulose hydrolysate is inadequate (Katahira
et al., 2006). For xylose-using S. cerevisiae, high bioethanol yields from xylose
also require metabolic engineering strategies to enhance the xylose flux (Hahn-
Hagerdal et al., 2006).
The microorganism is capable of growing at a pH as low as 5.0 and
temperatures as warm as 308 K. Natural xylose-fermenting yeasts, such as
Pichia stipitis, Candida shehatae, and C. parapsilosis, can metabolize xylose
via the action of xylose reductase (XR) to convert xylose to xylitol, and of
xylitol dehydrogenase (XDH) to convert xylitol to xylulose. Therefore,
bioethanol fermentation from xylose can be successfully performed by
recombinant S. cerevisiae carrying heterologous XR and XDH from P. stipitis,
and xylulokinase (XK) from S. cerevisiae (Katahira et al., 2006).

Microorganisms for bioethanol fermentation can best be described in terms of

their performance parameters and other requirements such as compatibility with
existing products, processes and equipment. The performance parameters of
fermentation are temperature range, pH range, alcohol tolerance, growth rate,
productivity, osmotic tolerance, specificity, yield, genetic stability, and inhibitor
tolerance (Demirbas, 2004). All the recombinant strains are mesophilic
organisms and function best between 303 and 311 K (Hettenhaus, 1998).
An organism must maintain fairly constant balance of pH to survive. Most
bacteria grow best in a narrow range of pH from 6.5 to 7.5 (Aminifarshidmehr,
1996).

The ability to lower pH below 4.0 offers a method for present operators using
yeast in less than aseptic equipment to minimize loss due to bacterial
contaminants. The majority of organisms cannot tolerate bioethanol
concentrations above 10–15 % (w/v) (Hettenhaus, 1998).

2.8.1.4 Distillation

Distillation is one of the steps of the purifications. Distillation is the method

used to separate two liquid based on their different boiling points. However, to
achieve high purification, several distillations are required. This is because all
materials have intermolecular interactions with each other, and two materials
will co-distill during distillation. This means that proportion between two
materials, in this case ethanol and water can be changed, and still, there are two
materials in layers, the liquid and the vapor layers (Onuki, S. (2005) .

Whatever method of preparation is used, the ethanol is initially obtained in a

mixture with water. The ethanol is then extracted from this solution by
fractional distillation. Although the boiling point of ethanol, 78.30C, is
significantly lower than the boiling point of water, 1000C, these materials
cannot be separated completely by distillation. Instead, an azeotrope mixture
(i.e. a mixture of 95% ethanol and 5% water) is obtained, and the boiling point
of the azeotrope is 78.150C. In a distillation, the most volatile material (i.e. the
material that has the lowest boiling point) is the first material to distill from the
distillation flask, and this material is the azeotrope of 95% ethanol which has
the lowest boiling point. If an efficient fractionating column is used, 95%
alcohol could be obtained first and then a small intermediate fraction of lower
concentration, and then water. But no matter how efficient the fractionating
column used, 95% alcohol cannot be further concentrated by distillation
because the vapor has exactly the same composition as the liquid; towards
distillation, then, 95% alcohol behaves exactly like a pure compound (Jackman,
1987).

2.8.1.5 Dehydration

After distillation, about 5% of water remains in ethanol. Especially, this water is

a big problem for fuel ethanol because the presence of this amount of water
enhances the molecular polarity of ethanol when it is mixed with gasoline.
Consequently, they separate into two phases, ethanol phase and gasoline phase.
It is easy to imagine that this inhomogeneous fuel is not acceptable. Thus,
dehydration can be another issue (Onuki, 2005).

For the ethanol to be usable as a fuel, water must be removed. Most of the water
is removed by distillation, but the purity is limited to 95-96% due to the
formation of a low boiling water-ethanol azeotrope. For blending with gasoline,
purity of 99.5 to 99.9% is required, depending on temperature, to avoid
separation. Currently, the most widely used purification method is a physical
absorption process using molecular sieves. Another method is azeotropic
distillation (Onuki, 2005).

CHAPTER THREE
 MATERIALS AND METHOD

3.1 Materials and chemicals

Cassava peels, rice husk, yam peels, corn cob and sugarcane bagasse were
bought from Otuocha market, Anambra State and grinded. The microorganisms,
which is a hybrid of yeasts (Saccharomyces cerevisiae), was gotten from a live
brewery in Onitsha, Anambra state. Distilled water which was used for the
entire process was obtained at Eke Awka market, Anambra state.

Chemicals: Sodium Hydroxide (NaOH, min. assay 17.5% BDH Chemicals Ltd
pool England cellulose), Sulphuric acid (H2SO4, (98%, England)), Dextrose
sugar, Yeast extract, Urea, MgSo4.7H2O, distilled water,Yeast (Saccharomyces
cerevisiae).

Equipments: Digital balances, Vacuum Filter (model =BN 3 STAATLICH,

Berlin), Sieves (mesh size of 2.0 mm, Sortmks-3332, PFEUFFR, Germany),
Shaking Incubator,Vertical Autoclave , pH- Meter , Ovens- Loading model 100
-800, Pycknometer, spectrophotometer (Perkin Elmer).

3.2 Method

3.2.1 Sample Preparation

The main aim of the research was ethanol production from corncob, cassava
peel, rice husk, yam peel and sugarcane bagasse. First the raw materials were
collected. The collected raw material was selected which is believed to
sufficient for this study. The feedstock’s were rinsed in water, drained and
sundried for two days. They were further treated by breaking to small pieces
with the aid of wooden mortar and pestle in such a way that it is suitable to be
dried and grind.
Then they were ground with grinding machine till they were 2 mm size. Next, it
was sieved. After it was sieved, flour which was found more that 2 mm while
sieving was again ground. Grinding of feedstock into powder form gives the
surface area of the sample increased which enhance the contact between
hemicellulose and cellulose with dilute acid to reduce cellulose crystallinity.

3.2.2 Pretreatment

The purpose of the pretreatment was to reduce cellulose crystallinity and

increase the porosity of the materials. Pretreatment must meet the following
requirements: improve the formation of sugar, avoid the degradation or loss of
carbohydrate, avoid the formation of by-product inhibitors and must be cost
effective.

Acid pretreatment

The method used by Utama et al. was adopted in this work with slight
modification. H2SO4 (98% analytical grade JHD) with 98.08 g/mol molecular
weight was used. 80 ml of the sulphuric acid was measured and transferred into
a 2000 ml volumetric flask and distilled water was used to make up the volume
to 2000 ml. 0.75 M solution of the sulphuric acid was thus obtained. The
prepared biomass (individual and combinations) were then soaked with the
prepared acid solution in batches and for each case, about 4 to 6 L of distilled
water was used alongside the 2 L of the 0.75 M solution of the H2SO4 based on
the absorbing capacity of the biomass. The mixture was then thoroughly mixed
in the vessel before being transferred to a pressure pot and allowed to boil for
about an hour. It was allowed to cool to ambient temperature before filtering
and then kept inside sampling plastics. The filtrate from each batch was also
stored separately.
3.2.3 Hydrolysis

The cellulose molecules which are composed of long chains are broken down to
simple sugar, before it was fermented for alcohol production.

Procedures for Acid Hydrolysis

Diluted sulfuric acid was added to the non soluble component from
pretreatment steps. The feedstock were then hydrolyzing in the reactor at three
levels of temperature (120,130, and 140), time of (30, 60 and 90min). Next
separate the solid particles from the liquid in the hydrolysate by vacuum
filtration (to remove the non fermentable lignin portion). After separating the
solid part, wash the solid part with distilled water two times. The washing was
performed in order to extract all soluble sugars from the solid feedstock.
Finally, mix the soluble component with the previously filter solution from the
pretreatment step for the next procedure.

pH Adjustment

Before addition of any micro-organism to the above prepared samples, pH of

these samples has to be adjusted. Otherwise the micro-organism will die in
hyper acidic or basic state. A pH of around 5.0 -5.5 was maintained.

Procedures in pH adjustment

Mix pretreated and hydrolyzed solution, shaken substrate primarily checked for
pH using a digital pH meter. The pH then adjusted to 5. The mixed samples
(pretreated and hydrolyzed) were acid hydrolyzed, so it needs highly basic
solution to bring the pH of 5. Sodium hydroxide solution was added drop wise
to the other flask with constant stirring until the pH reaches 5. Finally, suppose
the pH goes beyond 5 concentrated sulfuric acid was added drop wise to
maintain the pH.
3.2.4 Fermentation

Microorganism used for fermentation

Baker’s yeast, Saccharomyces cerevisae used for fermentation was cultured on

yeast extract agar. In order to prepare the media should have the favorable
condition for yeast growth or to supply the required amount of nutrients. Mix
the following nutrients in there proportion.

Fermentation Medium: 200ml of production medium was prepared according

to the requirements of Saccharomyces cerevisiae, containing 4 gm dextrose,
2gm dry yeast extract, 4gm peptone, 1 gm Urea, 1gm MgSO 4.7 H2O and 200 ml
make up distilled water. The pH was adjusted to 5 and autoclaved at 121°C to
maintain for 15 minutes.
After that, 1gm of yeast Saccharomyces cerevisiae (instant premium) was added
to the above 200 ml media in a 250 ml conical flask. Next the conical flasks
were properly covered with aluminum foil. Finally the conical flask was then
placed in a shaking incubator for 24 hours, a temperature of 30o C and 200rpm.

Sterilization

The reactor and all the equipments that were used for fermentation purposes
were sterilized (autoclaved). The sterilization was carried out at a temperature
of 121oC for 15 minutes.

The Procedure for Fermentation

Set shaking incubator at 30oC and 200 rpm and then mix the prepared sample
with the media prepared in the shaking incubator using sterilized funnel. The
parameters of fermentation i.e. fermentation time, yeast concentration (yeast
proportion) and fermentation temperature were set to be at 72 hour, 10% ( with
the proportion of 1:10 that is the prepared media and sample respectively) and
30oC respectively. And after 72 hours of fermentation, the samples were taken
out and distilled.

3.2.5 Distillation

Distillation was the last step in the production of ethanol from lignocelluloic
feedstock experiments. It is the purifications steps. Distillation is the method
used to separate two liquid based on their different boiling points. However, to
achieve high purification, several distillations are required. In this experiment
separation were used by simple distillation at a temperature of 85 oC.

3.3 Proximate Analysis

The proximate analysis was carried out on the samples to determine the
moisture content (MC), volatile matter content consisting of gases and vapours
driven off during pyrolysis , the fixed carbon content, the ash content (the
inorganic residue remaining after combustion of the sample).

Moisture Content

Samples were weighed accurately in clean preheated moisture crucible of

known weight by using sensitive balance. The sample and crucible were kept in
an oven 105oC for an hour. The crucible was covered and transferred to
desiccators, and weighed after reaching room temperature. The crucible was
heated in the oven for another two hours and was re-weighed. This was repeated
until constant weight was obtained.
The loss of weight was calculated as percent of weight and expressed as
moisture content.
Calculation:

Moisture content (%) = (W1- W2)* 100

Ws
Where:
W1= Weight of sample + crucible before oven drying

W2 = Weight of sample + crucible after oven drying

Ws = Weight of sample

Volatile Matter Content

A crucible was weighed empty, and then accurately samples were put in it. The
sample and the crucible were placed in a muffle furnace for 30min at 600 oC.
The crucible was removed from furnace and placed in a desiccators to cool, then
was reweighed. The process was repeated until constant weight was obtained.

Volatile Matter Content (%) = (W1- W2)* 100

Ws

Where:
W1= Weight of sample + crucible before oven drying

W2 = Weight of sample + crucible after oven drying

Ws = Weight of sample

Ash Content

A crucible was weighed empty, and then accurately samples were put in it. The
sample and the crucible were placed in a muffle furnace for 2 hours at 550 oC.
The crucible was removed from furnace and placed in a desiccators to cool, then
was reweighed. The process was repeated until constant weight was obtained.
Ash content (%) = (W2 – W1) *100

Ws

Where:
W1= weight of empty crucible
W2 = weight of crucible + sample after ashing
WS = weight of sample

Fixed Carbon Content

This is the residue left after the moisture, volatile and ash is given up. It is
deduced by subtracting from 100, the percentage of moisture, volatile matter
and ash content. The fixed carbon content (FC) is given as

FC = 100 – (%moisture + %volatile matter + %ash)

3.4 Chemical composition of feedstocks

Determination of extractives: oven dried sample was placed into Trimble

which is plugged with small amount of cotton and placed in a soxhlet extraction
tube. For the Soxhlet method, this is usually 6-24 hours. An exhaustive ethanol
extraction is usually complete in 24 hours using the Soxhlet method. An
exhaustive ethanol extraction is usually complete in 24 hours using the Soxhlet
method.

Extractive (%) = W1-W2/W1*100

W1 = oven dry sample
W2 = extracted residue

Determination of cellulose: The extractive free sample was treated with

alcoholic nitric acid solution under reflux during four cycles of 1hr. After each
cycle, the solution was removed for a fresh volume. The alcoholic nitric acid
solution consisted of mixing one volume of 68% (w/w) solution of nitric acid
with four volumes of 97% purity alcohol. At the end the cellulose was washed,
dried and weighed.

Cellulose (%) = W1-W2/W1*100

W1 = oven dry sample
W2 = extracted residue

Determination of lignin: Extractive free sample was placed in flask and 72%
sulfuric acid was added. The flask was kept in water bath at 30 oC during
dispersion of the material for 1hr. after that adding deionised water and placed
in autoclave for 121oC to 1hr. Next the insoluble material (lignin) was filter by
vacuum filtration. The lignin was washed until became acid free (with hot
water) then dry and weight.

Lignin (%) = W1-W2/W1*100

W1 = oven dry sample
W2 = extracted residue

Determination of hemicellulose content

The cellulose content, WH, was calculated by difference, assuming that

extractives, hemicellulose, lignin, ash, and cellulose are the only components of
the entire biomass (Blasi et al., 1999, Li et al., 2004, and Lin et al., 2010):

100 = WC + WH +WE + WL
WH = 100 - WC + WE + WL

3.5 Characterization of product

Concentration: This was done by using the specific gravity bottle method and
comparing with the standard chart of alcohol concentration in water. Several
other methods are available which are not easily accessible within NAU
environment.
𝐑𝐞𝐥𝐚𝐭𝐢𝐯𝐞 𝐃𝐞𝐧𝐬𝐢𝐭𝐲 =𝐌𝐚𝐬𝐬 𝐨𝐟 𝐄𝐭𝐡𝐚𝐧𝐨𝐥 𝐏𝐫𝐨𝐝𝐮𝐜𝐞𝐝\𝐌𝐚𝐬𝐬 𝐨𝐟 𝐰𝐚𝐭𝐞𝐫