ABSTRACT
The potentials of Saw dust, Rice husk and Groundnut shells were investigated for
bioethanol production. The substrates were pretreated using 2%, 4%, 6% and 8%
solution of dilute H2SO4 and HCl. The pretreated substrates were thermally treated at
1050C in order to detoxify them. The pretreated substrates obtained using different
concentrations of acids were fermented using Saccharomyces cerevisiae (Yeast). The
results show that Rice husk treated with 4% H2SO4 (2.689%dryweight) has the highest
result followed by the same sample treated with 2% H2SO4 (2.423%drywieght) then
followed by the Saw dust treated with 8% H2SO4 (1.802%dryweight) in which the least
result is observed with groundnut shells treated with 8% H2SO4 (0.862%dryweight). All
these were observed to be significantly different as calculated and checked between
individuals differences at 0.5% confidence limit using ANOVA. Also from the results,
it is clear that the samples treated with sulphuric acid treatments were better at reserving
the actual sugar (glucose) content. While samples treated with HCl has their sugar
content reduced; as this shows the effect of thermal treatment and detoxification
processes involved. Conclusively, individual substrates treated are said to be in different
acid measures. Thus, saw dust is optimum at 8% H2SO4 and 6% HCl while rice husk
shows its optimum progress at 6% in both H2SO4 and HCl. and ground nut shells are
good at 4% H2SO4 and 6% HCl acid treatments.
TABLE OF CONTENTS
TITLE PAGE ……………………………………………………………………………………………………… i
DEDICATION …………………………………………………………………………………………………… ii
CERTIFICATION …………………………………………………………………………………………….. iii
AKNOWLEDGEMENTS ………………………………………………………………………………….. iv
TABLE OF CONTENTS……………………………………………………………………………………. vi
LIST OF FIGURES …………………………………………………………………………………………… ix
LIST OF TABLES ……………………………………………………………………………………………… x
ABSTRACT……………………………………………………………………………………………………… xi
CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW
1.1 Introduction ………………………………………………………………………………………….. 1
1.2 Research Problems ………………………………………………………………………………… 4
1.3 Scope and Limitation …………………………………………………………………………….. 5
1.4 Aim and Objectives ………………………………………………………………………………. 5
1.5 Literature Review …………………………………………………………………………………. 6
1.5.1 Ethanol ………………………………………………………………………………………………… 6
1.5.2 Bioethanol ……………………………………………………………………………………………. 8
1.5.3 Natural Occurrence of Ethanol ……………………………………………………………… 10
1.5.4 Uses of Ethanol …………………………………………………………………………………… 10
1.5.5 Economic Importance of Bioethanol Production……………………………………… 10
1.5.6 Problems Associated with Ethanol Production ………………………………………… 11
1.5.7 Bioethanol Feedstocks …………………………………………………………………………. 12
1.5.8 Technology of Bioethanol Production ……………………………………………………. 13
1.5.9 Bioethanol Production from Cellulose Materials in Particular …………………… 16
1.5.10 Common Alcohol Processing Steps ……………………………………………………….. 20
1.5.10.1 Pretreatment Process ……………………………………………………………………………. 20
1.5.10.2 Cellulose Hydrolysis ……………………………………………………………………………. 21
1.5.10.3 Detoxification …………………………………………………………………………………….. 22
1.5.10.4 Fermentation ………………………………………………………………………………………. 23
1.5.10.5 Separation/Distillation …………………………………………………………………………. 24
1.5.11 Proximate Analysis ……………………………………………………………………………… 24
1.5.11.1 Determination of Moisture Content ……………………………………………………….. 25
vii
1.5.11.2 Determination of Ash Content ………………………………………………………………. 26
1.5.12 Applicable Methods of Analysis……………………………………………………………. 26
CHAPTER TWO: MATERIALS AND METHODS
2.1 Materials ……………………………………………………………………………………………. 27
2.1.1 List of Apparatus/Equipments ………………………………………………………………. 27
2.1.2 List of Reagents Used ………………………………………………………………………….. 28
2.1.3 Reagents Preparations ………………………………………………………………………….. 29
2.1.4 Sample Collection and Sample Treatment………………………………………………. 30
2.2 Methods …………………………………………………………………………………………….. 31
2.2.1 Pre-treatment Process ………………………………………………………………………….. 31
2.2.1.1 Acid Pre-treatment ………………………………………………………………………………. 31
2.2.2 Thermal Treatment ……………………………………………………………………………… 31
2.2.3 Detoxification …………………………………………………………………………………….. 32
2.2.4 Fermentation ………………………………………………………………………………………. 32
2.2.5 Distillation …………………………………………………………………………………………. 32
2.3 Proximate Analysis Test ………………………………………………………………………. 33
2.3.1 Determination of Moisture Content ……………………………………………………….. 33
2.3.2 Determination of Ash Content ………………………………………………………………. 33
2.3.3 Organic Matter Content and Carbon Content Composition ………………………. 33
2.3.4 Determination of Sugar Content ……………………………………………………………. 34
2.3.5 Determination of Ethanol Content …………………………………………………………. 34
2.4 Statistical Analysis ………………………………………………………………………………. 35
CHAPTER THREE: RESULTS AND DISCUSSION
3.1 Results ………………………………………………………………………………………………. 36
3.1.1 Physical Characteristics of the Samples …………………………………………………. 36
3.1.2. Proximate Composition of the Samples………………………………………………….. 37
3.1.3 Reducing Sugar Content Result ……………………………………………………………. 38
3.1.4 Ethanol Concentration Result ……………………………………………………………….. 39
3.2 Discussion ………………………………………………………………………………………….. 40
3.2.1 The Proximate Composition Analysis ……………………………………………………. 40
3.2.2 The Reducing Sugar Content ………………………………………………………………… 40
viii
3.2.3 The Bioethanol Content ……………………………………………………………………….. 42
CHAPTER FOUR: CONCLUSION AND RECONMENDATIONS
4.1 Conclusion …………………………………………………………………………………………. 45
4.2 Recommendations ……………………………………………………………………………….. 46
REFERENCES …………………………………………………………………………………… 47
APPENDICES ……………………………………………………………………………………. 51
ix
CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
1.1 Introduction
Biofuels (Bioethanol, Biodiesel, and Biogas) are fuels produced from biomass (a
biodegradable material) for heating, electricity generation and transport purposes etc
(Garba, 1999). Bioethanol can be produced from any biological feedstock’s that
contains appreciable amount of sugar/carbohydrate or materials that can be converted
into sugar such as starch or cellulose. Ethanol from renewable resources has been of
interest in recent decades as an alternative fuel to the current fossil fuels.
Lignocelluloses biomass like wood and agricultural crops residue, e.g., straw and sugar
beet pulp are potential raw materials for producing several high-value products like fuel
ethanol and biodiesel (Yoswathana et al., 2010). Because of the recent increase in the
gas price and interest in environmental issues, the demand of ethanol as substitute of
gasoline is rapidly increasing. Basically, there are five (5) steps in ethanol production,
and these include; grinding, cooking, fermentation, distillation, and hydration. In each
step, there are several ideas to improve its productivity and benefits (Onuki, 2010).
For a large production of bioethanol; it is convenient to use cheaper and abundant
substrates always. So by using waste products from forestry, agriculture and industries,
the cost of feedstocks may be reduced; if we consider producing ethanol from
feedstocks such as maize, sugarcane, sweet potatoes, rice pulps etc; which constitutes a
larger percentage of the production cost (Energy Commision of Nigeria, 2010).
For the reduction of food competition it is necessary to use lignocelluloses (a complex
polymer made up of three components of carbohydrates; which are cellulose
2
((C6H10O5)n) hemicelluloses (Cx(H2nOn)y) and lignin (Paracoumaryl alcohol (C9H10O2,),
Coniferyl alcohol (C10H12O3,) and Sinapyl alcohol (C11H14O4))) which is considered as
an alternative and attractive feedstock for the production of ethanol due to its
availability in large quantities and low in cost (Cardona and Sanchez, 2007 cited in
Cardona et al., 2010).
O
O
O
O
HO
HO
HO OH
OH
HO OH
O
OH
OH
n
Figure 1 Cellulose Unit Structure (Onuki, 2010).
O O O
O
HO OH
O O
O
HO
OH
HO
HO
OH
OH
OH
HO HO
O
n
Figure 2: 1Hemicellulose Unit Structure (Shakhashiri, 2009).
3
OH
OH
1. Paracoumaryl Alcohol
OH
OH
H3CO
2. Coniferyl Alcohol
OH
OH
H3CO
OCH3
3. Sinapyl Alcohol
Figure 3: Lignin Structure (The three common monolignols: (1)Paracoumaryl Alcohol,
(2) Coniferyl Alcohol and (3)Sinapyl Alcohol) (Onuki et al., 2008).
Numerous studies for developing large scale production of ethanol from lignocelluloses
have been carried out in the world. However the main limiting factor is the higher
degree of complexity inherent to the processing of these feedstocks, which is related to
the nature and composition of the substrate biomass. Also, the content cellulose and
hemicelluloses by all means has to be broken down into fermentable sugars in order to
be converted to ethanol (Ali and Khan, 2014).
But these degradation process of hydrolysis and fermentation are complicated, energy
consuming and non completely developed (Sanchez and Cardona 2008 cited in Cardona
et.al., 2010). With the advent of modern genetics and other technology; the cost of
producing sugar from these wastes and converting them into products like ethanol can
4
be significantly reduced in the future in which high cost of production from these
feedstocks has been part of the major reason why ethanol has not made its breakthrough
yet (Cardona et al., 2010).
Many Lignocellulosic materials has been treated for bioethanol production and
reviewed as such; Nigeria produces large quantity of wastes in the form of agricultural
wastes during harvestings and food processing. Most of these wastes end up in the
environment thereby constituting environmental pollution and problems. However, the
wastes mostly are rich in carbohydrate content which can be processed into sugars and
subsequently fermented to ethanol (Ado et al., 2009).
1.2 Research Problems
Certain procedures exist in the quantitative production of ethanol which were adopted
and modified in such a way as to maximize yield and overcomes the challenges
involved in the ethanol production. So this work uses the existing procedures and tries
in finding out some of its challenges, which includes;
1. Among the Lignocellulosic plant materials used as substrates; which of the
plants parts (rice husk, saw dust and g.nut shells) will be best in optimizing the yield of
ethanol produced.
2. Find out among the most widely used acids (HCl and H2SO4) for pretreatment;
which of them is the best within the chosen substrates in the case of ethanol production.
3. Also try to find out in what concentration does the two most widely used acids
give an optimum yield in bioethanol production.
5
4. Also to find out whether factors like pH and temperature regulations affect and
reduce the effect of the facultative inhibitors like acetone, acetic acids etc in the
production of ethanol.
1.3 Scope and Limitation
This study is intended to cover the successful determination of an optimum percentage
yield of ethanol produced from the saw dust, rice husk and groundnut shells using
acidic medium.
And this is limited to, the identification and determination of the preliminary factors of
investigations such as Moisture content, Ash content, carbon content and organic matter
content. Also the concentration of the sugar content determination and the concentration
of the Ethanol content after production.
1.4 Aim and Objectives
The main aim is to determine a simple way of attaining an optimum yield in the
production of ethanol through the conversion of Saw dust, Rice Husk and Ground-nut
shells into ethanol in a simultaneous saccharification and fermentation process using
Saccharomyces cerevisiae.
To achieve this production process of optimum yield the following objectives are
defined
The Proximate compositions which includes:
o The moisture content and ash content of the sample substrates.
o The organic matter and carbon content of the samples substrates under
study
6
Determination of the sugar content of the hydrolysates after pretreatment.
The ethanol concentration of the fermented hydrolysates.
1.5 Literature Review
1.5.1 Ethanol
Ethanol (ethyl alcohol, grain alcohol) is a clear, colorless liquid with a characteristic,
agreeable odor. In dilute aqueous solution, it has a somewhat sweet flavor, but in more
concentrated solutions it has a burning taste. Ethanol, (CH3CH2OH) is an alcohol, a
group of chemical compounds whose molecules contain a hydroxyl group, –OH,
bonded to a carbon atom (Onuki et al., 2008). Ethanol has been made since ancient
times by the fermentation of sugars. All beverage ethanol and more than half of
industrial ethanol is still made by this process. Simple sugars are the raw material.
Zymase, an enzyme from yeast, changes the simple sugars into ethanol and carbon
dioxide. The fermentation reaction, represented by the simple equation is actually very
complex, and impure cultures of yeast produce varying amounts of other substances,
including glycerine and various organic acids (Shakashiri, 2009).
C6H12O6 Yeast 2CH3CH2OH + 2CO2 ———————————–1.1
Starches from potatoes, corn, wheat, and other plants can also be used in the production
of ethanol by fermentation. However, the starches must first be broken down into
simple sugars. An enzyme released by germinating barley, diastase, converts starches
into sugars. Thus, the germination of barley, called malting, is the first step in brewing
beer from starchy plants, such as corn and wheat (Koseric et al., 1983).
Much ethanol not intended for drinking is now made synthetically either from
acetaldehyde made from acetylene or from ethylene made from petroleum (Onuki et al.,
7
2008). Ethanol can be oxidized to form first acetaldehyde and then acetic acid. It can be
dehydrated to form ether. Butadiene, used in making synthetic rubber, may be made
from ethanol, as can chloroform and many other organic chemicals. Ethanol is used as
an automotive fuel by itself and can be mixed with gasoline to form gasohol. Ethanol is
miscible (mixable) in all proportions with water and with most organic solvents. It is
useful as a solvent for many substances and in making perfumes, paints, lacquer, and
explosives. Alcoholic solutions of nonvolatile substances are called tinctures; if the
solute is volatile, the solution is called a spirit (Onuki et al., 2008).
Ethanol may also be produced industrially from ethene (ethylene), by hydrolysis of the
double bond in the presence of catalysts and high temperature (Allen et al., 1974).
C2H4 + H2O Catalyst C2H5OH ———————————-1.2
(Ethane) (Water) Δ (Ethanol)
The by far largest fraction of the global ethanol production, however, is produced by
fermentation. During ethanol fermentation, glucose and other sugars in the corn (or
sugarcane or other crops) are converted into ethanol and carbon dioxide (Allen et al.,
1974).
C6H12O6 Enzymes 2C2H5OH+ 2CO2 + heat ——————1.3
Like any fermentation reaction, the fermentation is not 100% selective and other side
products such acetic acid, glycols and many other products are formed to a considerable
extent and need to be removed during the purification of the ethanol. The fermentation
takes place in aqueous solution and the resulting solution after fermentation has an
ethanol content of around 15%. The ethanol is subsequently isolated and purified by a
combination of adsorption and distillation techniques. The purification is very energy
8
intensive. During combustion ethanol reacts with oxygen to produce carbon dioxide,
water, and heat (Onuki et al., 2008).
C2H5OH + 3O2 Energy Δ 2CO2 + 3H2O + heat —————–1.4
Starch and cellulose are molecules that are strings of glucose molecules. It is also
possible to generate ethanol out of cellulosic materials. However, a pretreatment is
necessary that splits the cellulose into glycose molecules and other sugars which
subsequently can be fermented. The resulting product is called cellulosic ethanol,
indicating its source (Onuki et al., 2008).
1.5.2 Bioethanol
Over the centuries; various forms of energy has been used by man in order to meet his
basic life essentials such as food, water and shelter. Starting with energy and sunlight,
he progressed to fuel-wood, draft animal power, water and wind power, then developed
engine power fuelled by wood, coal, petroleum and nuclear energy (Energy
Commission of Nigeria, 2010). As time passes; man developed industrially and in
status, more by producing and maintaining cars, generating plants and other form of
mechanical devices which operates on one form of fuel or the other; as a result of this,
there has been an increase in the uses and demands for fuel in terms of transportation
and power generation (Garba, 1999).
It is this urgeness of mans alternative energy option to live that has stimulated
researchers to bend into various ways of finding more economically and
environmentally acceptable alternative source of energy such as biogas, bioethanol,
biodiesel etc (Subashini et al., 2011). There were numerous research efforts directed
9
towards the development of alternative energy sources, such as ethanol production from
agricultural products (Nagashima et al., 1984 in Subashini et al., 2011).
Generally, biofuels are defined as solid, liquid or gas fuels produced from biomass for
general purposes applications (Koseric et al., 1983). They are produced from
agricultural and forest products which are the biodegradable organic portions of
industrial and municipal wastes. Generally, biofuels are renewable energy resources
derived from recent biological materials, which distinguishes them from fossil fuels that
are derived from long dead biological materials. There are various forms of biofuels
which includes the bioethanols, biodiesel, biogas and other forms of fuels made from
biomass (Energy Commission of Nigeria, 2010).
The emphasis of this generation on bioethanol promotion is driven not only by the
energy security it provides but also the environmental benefits. Bioethanol just like
other renewable energy sources produces considerably lower emissions on combustion
and it only releases the same amount of carbon dioxide as plants take up for its life
process. This helps to reduce greenhouse gas emissions; the world energy policy,
therefore, is targeted at promoting the use of bioethanol and other biofuels (Energy
Commission of Nigeria, 2010).
Bioethanol is an important renewable energy source that is now being used as fuel or
fuel additive; though, using bioethanol is not a new idea it has being in process since
1872 when gasoline is not available; in particular ethanol make an excellent motor fuel
(Keating et al. 2006). The reason alcohol fuel has not been fully exploited is that, up till
recently, gasoline has been cheap, available and easy to produce. However crude oil is
getting rare and historic price differential between alcohol and gasoline is getting
narrower (Energy Commission of Nigeria, 2010).
10
1.5.3 Natural Occurrence of Ethanol
Ethanol is a byproduct of the metabolic process of yeast. As such, ethanol will be
present in any overripe fruit and yeast habitat (Dudley, 2004). Ethanol produced by
symbiotic yeast can be found in bertam palm blossoms. Although some species such as
the pentailed treeshrew exhibit ethanol-seeking behaviors, most show no interest or
avoidance of food sources containing ethanol (Cynthia, 2008). Ethanol is also produced
during the germination of many plants as a result of natural anerobiosis (Sylva et al.,
1974).
1.5.4 Uses of Ethanol
Uses of ethanol are many; among these applications includes.
I. It is used as a fuel and gasoline additive in automobiles
II. Used also in the productions of alcoholic drinks such as vodka among others
III. Widely used as solvent in productions of paints, vanish, drugs among others
IV. Used in thermometers and as an anti freezing agent
V. Also as a disinfectant in laboratories and hospitals also in preserving biological
specimens.
1.5.5 Economic Importance of Bioethanol Production
Certain factors can be put into considerations; which may likely include an important
factor which is reducing the cost of bioethanol production in the use of larger industrial
facilities, instead of small scale laboratory productions; where by the cost per-invested
unit price falls off. Also improving such productions; economics of ethanol may be the
application of energy integration of the already existing plant of production. Also,
beside cost of production is the cost of feedstocks which represents of about 40% of the
11
total production cost and the enzymes available on the ongoing process (Energy
Commission of Nigeria, 2010).
Ethanol represents closed carbon dioxide cycle because after burning of ethanol, the
released CO2 is recycled back into plant material because plants use CO2 to synthesize
cellulose during photosynthesis cycle (Wyman, 1999). Ethanol incineration process
only utilizes energy from renewable energy sources; no net CO2 is added to the
atmosphere, making ethanol an environmentally beneficial energy source. In addition,
the toxicity of the emissions from ethanol is lower than that of petroleum sources (Ali,
and Khan, 2014). Ethanol derived from biomass is the only liquid transportation fuel
that does not contribute to the green house gas effect (Foody, 1988 cited in Ali, and
Khan, 2014).
1.5.6 Problems Associated with Ethanol Production
Ethanol supply is constrained by arable (cultivatable land) land availability.
Competition with food production for land use could drive possible increases in both
ethanol and food prices. Ethanol markets still have a regional structure (Energy
Information Administration, 2007). Transport of biomass remains a logistics barrier that
limits the size of ethanol production plants and economies of scale. Conversely,
producing more biofuels from conventional feedstocks could conflict with conservation
of biodiversity and call for increased amounts of water, pesticides and fertilizers, thus
raising sustainability issues. In scenarios having 25% of transport fuels derived from
biomass, the use of fertilizers increases by about 40%. On a fuel-cycle basis, ethanol,
with its high vapour pressure, reduces NOx and volatile organic compound (VOC)
emissions but this is partly offset from increased N2O emission from increased use of
nitrogenous fertilizers. Developing cost-effective ethanol production from lignocellulose
via enzymatic hydrolysis would therefore increase the variety and availability
12
of feedstocks and hence expand the production of biofuels. Other ethanol drawbacks
include miscibility with water, aldehyde emissions, compatibility issues with some
plastics or metals (Al-alloys, brass, zinc, lead) and high latent vaporization heat (cold
start issues). Ethanol use in compression ignition engines needs additives due to the low
octane number and is impractical (Energy Information Administration, 2007).
1.5.7 Bioethanol Feedstocks
Ethanol is commonly derived from biological feedstocks utilizing fermentation
processes. During these processes, monosaccharides are fermented to ethanol by yeast
or bacteria. There are a variety of carbohydrate-containing feedstocks that yield
monosaccharides for fermentation, such as corn grain, sugarcane, wheat, sugar beet and
other biomass (Stefan et al., 2009).
It is technically feasible to make ethanol from a wide variety of available feedstocks.
Fuel ethanol could be made from crops which contain starch such as feed grains, food
grains, and tubers, such as potatoes and sweet potatoes. Crops containing sugar, such as
sugar beets, sugarcane and sweet sorghum also could be used for the production of
ethanol. In addition, food processing byproducts, such as molasses, cheese whey, and
cellulosic materials including grass and wood, as well as agricultural and forestry waste
could be processed to ethanol (Shapouri et al., 2006). For easier references the
feedstocks can be grouped under the following;
(1). First generation feedstock i. Saccharine materials (i.e. sugar containing)
ii. Starch materials
13
(2). Second generation feedstocks are the cellulose materials which includes
• The agricultural plant wastes such as rice straws,
• The plant wastes from industrial processes
• Forest wastes such as non edible plants, dead trees and leaves etc.
• Energy crops grown specifically for fuel production such as, jatropha etc
• Municipal solid wastes (Garba 1999).
(3). Third generation feedstocks which includes basically the fungi and the bacteria or
other microbes.
The majority of the first generation of feedstock for bioethanol production includes
those feedstocks which are widely grown and used for food and animal feed, hence
induces the current international debate, “food or fuel” (Energy Commission of Nigeria,
2010).
1.5.8 Technology of Bioethanol Production
Certain materials require less processing than others for example, small scale
production from saccharine materials is the easiest and most economical in terms of
labour and energy consumption. On the other hand starch materials usually produce the
most alcohol on a weight per weight basis while cellulose materials are the cheapest
because most of them are currently regarded as waste (Ado et al., 2009).
Ethanol production is usually accomplished by chemical synthesis of petrochemical
substrates and microbial conversion of carbohydrates present in agricultural products.
Owing to depleting reserves and competing industrial needs of petrochemical
feedstocks, there is global emphasis in ethanol production by microbial fermentation
14
process (Ali and Khan, 2014). Increased yield of ethanol production by microbial
fermentation depends on the use of ideal microbial strain, appropriate fermentation
substrate and suitable process technology (Brooks, 2008).
The ethanol produced by fermentation ranges in terms of concentration from a few
percent up to about 14 percent. Above the 14 percent, ethanol destroys the enzyme
(zymase) and fermentation stops. Ethanol is normally concentrated by distillation of
aqueous solutions, but the composition of the vapor from aqueous ethanol is 96 percent
ethanol and 4 percent water (Shakhashiri, 2009).
Ethanol is produced when yeast ferments 6-carbon sugars (mainly glucose) via the
glycolytic pathway, where the glucan is converted to glucose by enzyme hydrolysis and
fermentation of the glucose to ethanol by yeast. The mash is fermented using natural
yeast and bacteria in a process that takes up to 40 hours. The fermented mash is
separated into ethanol and residues (for feed production) via distillation. (Ali and Khan,
2014) The substrate, in this case, is starch that has been gelatinized (i.e., pretreated) by
cooking it in water (Kriz and Larkins, 2009).
The Technology of Ethanol Production in Large Scale entails a process of either wet or
dry milling processes to be used for ethanol production from a cereals starch. A drygrind
process entails grinding the cereals into a fine powder, which is then cooked,
hydrolyzed, and fermented. In a wet-milling plant, the number of co-products is higher
and more flexible, with processing consisting of steeping and separation of the cereals
kernel into germ, starch, and other components (Kriz and Larkins, 2009).
Wet Milling: – The wet milling process fully fractionates the cereals grain into
carbohydrates, lipids, and protein. These can be efficiently recovered and purified for
the production of value-added products. When the starch is converted to fuel ethanol,
15
the processing steps of saccharification, fermentation, and recovery are similar to those
in a dry grind (Kriz and Larkins, 2009).
Operation of the wet milling machine:- The first step in the wet milling process is
steeping, where the cereals kernel is placed in an aqueous solution of 0.1–0.2% SO2 and
allowed to cook at 48–520C for 30–50 hours. This facilitates downstream fractionation
by hydrolyzing disulfide bonds in proteins so that they are more soluble. The cereals are
then ground in its wet state and oil, fiber, and gluten are separated from the starch for
further processing into value-added co-products. During saccharification, enzymes
break down the starch into glucose. In the fermentation step, yeast grown in seed tanks
is added to the cereals mash to ferment the simple sugars (glucose) to ethanol. Finally,
ethanol is separated from the water by means of distillation and dehydration. Cereals
fiber contains cellulose and hemicellulose which cannot be used for producing ethanol
in wet mill facilities, since these facilities do not currently incorporate cellulose
conversion technology. Fiber is a potential feedstock for additional ethanol production
if lignocellulosic conversion technologies are applied (Graboski, 2002).
Dry Milling: – Dry milling technology produces high ethanol yields at lower capital
investment than wet milling. However, the only major co-products, other than CO2, are
the fermentation residuals which are sold as animal feed. These products are commonly
known as distillers’ grains (DG) and dried distillers’ grains with soluble (DDGS)
(Stefan et al., 2009).
Operation process of the dry mill: In a dry mill, cleaned cereals are first ground in
hammer mills, which breaks the tough outer coating of the seed and grinds the cereals
into a fine powder. During the liquefaction process, water and enzymes are added to the
ground cereals in order to create slurry. The gelatinized starch feedstock is easier to
16
hydrolyze into monomeric sugars than uncooked cereals, although processes that avoid
the cooking step are being considered for ethanol plants. Saccharification and
fermentation are similar to the processes performed in a wet mill. Ethanol is obtained
from the water slurry via a number of complex steps including distillation and
dehydration. A co-product of the dry milling process, heavy stillage, leaves the bottom
of the first distillation column. The heavy stillage is centrifuged to remove the majority
of the solids. The thin stillage is partly recycled to the liquefaction step. The centrifuged
solids are referred to as wet cake or wet distiller’s grains (35–40% solids). These are
further dried to give DDGS (Stefan et al., 2009).
1.5.9 Bioethanol Production from Cellulose Materials in Particular
Cellulose materials are similar to starchy materials in that they must be converted
through a set of pretreatment processes prior to fermentation. Cellulose is converted by
either enzymes or acid hydrolysis. Special enzymes such as “cellulast” and “cellubiase“
are used for fermenting cellulose to glucose (Brooks 2008). The acid process involves
either strong acid and low temperatures or weak acid and high temperatures, relatively.
The strong acid process has the problem that the glucose is destroyed almost as fast as it
is formed unless the contact time with the acid is very brief. The weak acid process
requires pressure proof cooking equipment. Again, for the obvious reasons, these
methods are not recommended on a small scale (Ali and Khan, 2014).
The only alternate to dissolving the lignin is to reduce the cellulose material to as fine
state as possible so that at least the cellulose can be recovered. This is done by
powdering, grinding and processing. The yield of cellulose is directly proportional to
how finely the starting material is reduced. After powdering the material is mixed with
17
as little water as possible to make a thick, soupy mass. The pH is adjusted to between
4.5 –6.0 and the enzyme is added (Energy Commission of Nigeria, 2010).
Generally dry cellulose materials such as wood, shells among others will have the
lowest yields. This is because the cellulose is enclosed in lignin, and the amount that is
ultimately accessible to the enzymes is proportional to how finely the material is
divided. Materials containing cellulose will have the next highest yields. This is partly
because the lignin content is lower and partly due to the presence of some fermentable
sugars in it. The highest yields will come from materials that are almost pure in
cellulose (Karimi et.al., 2006).
And as such the common processing steps for breaking cellulose materials includes
The first step is dilution: – dilution simply refers to as the addition of water to adjust the
amount of sugar in the mash or the amount of alcohol in the beer. It is necessary
because of the following reasons
. The yeast, used later in the fermentation process, can die when the concentration
of alcohol is high
. To make the mash easier to stir and handle (i.e to reduce the thickness of the
mash to easy handling)
The objective of dilution is to end up with a beer as close to 10% alcohol when
fermentation is complete. The optimum dilution, then, is a compromise between the
highest alcohol concentration and the point where the particular yeast strain being used
will die (Energy Commission of Nigeria, 2010).
18
A rule of thumb for an unknown material is the final alcohol concentration will be about
half the sugar content prior to fermentation (Ado et al., 2009). So it is good to
determine the amount of fermentable sugar in a mash which is best done in a laboratory
or estimated through the use of hydrometer. Sugar content of a solution can also be
determined with the use of an optical instrument called sugar refractometer (Energy
Commission of Nigeria, 2010).
The second step involved pH control: – controlling pH during mashing and fermentation
process is important for two reasons
. The growth of harmful bacteria is retarded by the acid solution and
. Yeast will grow only in an (slightly) acidic solution.
Most grain mashes naturally have a mild acid pH of 5.4 – 5.6 after malting or
conversion has been accomplished. Other materials, notably saccharine substrates like
molasses and fruits pressings have naturally alkaline pH and must be acidified prior to
fermentation (Koseric et al., 1983). Mineral acids such as sulphuric acids can be used to
reduce the pH. The acids should be added cautiously, the mash stirred, and the pH
checked, the pH can also be checked through the addition of natural acids left from
previous distillation; it (the pH) can also be raised with sodium hydroxide solution
(caustic soda) or with ordinary lime (Ado et al., 2009).
The third step involved fermentation: – all that is necessary for fermentation to begin is
to add the activated yeast with the cooled, pH adjusted mash in the fermentation broth.
When the fermentation begins, carbon dioxides gas will be given off. At this time of
fermentation; the mash will literally boil from the carbon dioxide produced. The
reaction produces some heat. The optimum temperature for the fermentation process is
19
between 200 – 300C and it is advisable not to let the temperature go much above 32 –
350C (Energy Commission of Nigeria, 2010). Because there will be an escape of the
volatile molecules of the carbon sources involved and thus the enzyme involved will be
degraded and might even perish in such condition of high temperature.
It is also advisable to use a fermentation lock to prevent alcohol vapors from escaping
out the of the fermentation broth. Otherwise, this can reduce the volume of the alcohol
produced. It is allowed to open the fermentation broth to check the progress of the
process and take samples for pH analysis, as long as care is taken not to introduce
bacteria that could contaminate the mash (Ado et al., 2009).
Bioethanol is produced through the conversion of sugars (Fermentation) to ethanol. The
biomass that exists as a complex sugars (Polysaccharides) are however first broken
down into fermentable sugars through a chemical process of chemical reaction called
hydrolysis. The simplified fermentation reaction equation for the simple 6 carbon
sugars, glucose is.
C6H12O6 Yeast 2CH3CH2OH + 2CO2 ————————– 1.5
(Glucose) (Ethanol) (Carbon Dioxide)
The last step involved is distillation: – the separation of alcohol and water by distillation
is made possible by the fact that alcohol boils at about 780C and water at 1000C. When
the mixture of water and alcohol boils, vapours with a greater concentration of alcohol
will be formed and liquid with a lesser concentration of alcohol will be left behind.
However, because water and alcohol do not form what is called ideal mixture, the
separation cannot be done in one clean step (Energy Commission of Nigeria, 2010).
20
1.5.10 Common Alcohol Processing Steps
Though new technology may eventually blur the distinction between them, ethanol is
produced by one of two processes: wet milling and dry milling (Rendleman and
Shapouri, 2007). So, in an overall process of ethanol processing steps there exist five
main steps which are
1. Biomass Pre-treatment,
2. Cellulose Hydrolysis,
3. Detoxification
4. Fermentation,
5. Separation/ Distillation and lastly,
6. Effluent treatment (Cardona et al., 2010).
1.5.10.1 Pretreatment Process
Pretreatment is intended to disorganize the crystalline structure of macro and micro
fibrils; in order to release the polymer chains of cellulose and hemicelluloses and
modify the process in the materials to allow the enzymes to penetrate into fibres to
render them useable to enzymatic hydrolysis (Keating et al., 2006). This pretreatment
enables more efficient enzymatic hydrolysis of the cellulose by removal of the
surrounding hemicelluloses, lignin and the cellulose along with the modification of the
structures (Koseric et al., 1983). Acid treatment, alkaline treatment, wet oxidation,
steam explosion, solvent extraction, thermal pretreatment using acids or bases,
biological treatment among others serves as some of the best pretreatment method we
have in this process. A several pretreatment methods have been investigated for
different lignocellulosic materials (Sanchez and Cardona, 2008). Lignocellulosic
materials do not contain monosaccharide available for bioconversion. Instead they
21
contain polysaccharides such as cellulose and hemicelluloses, which have to be
hydrolyzed by means of acids, bases or enzymes to fermentable sugars (Cardona et al.,
2010).
Remarkably, dilute sulphuric acid treatment has been successfully developed given that
high reaction rates can be achieved improving significantly the subsequent process of
cellulose hydrolysis; however the cost of this type of treatment is still higher (Cardona
et al., 2010).
Advantages of this process is the recovery of high amount of sugars that is derived from
the hemicelluloses but the reducing sugar concentration is relatively very low due to
high liquid to solid ratio also the formation of furan derivatives and other toxic products
(Brooks, 2008), and the need for additional concentration steps are included.
Likely alkaline treatment decreases the polymerization degree and crystallinity of
cellulose by the destruction of links between lignin and other polymers and break down
of lignin (Shakhashiri, 2009). Also its cost is too high that they become competitive for
large scale plants. Biological treatment process has a low energy requirements and mild
environmental conditions thus proves slower and limiting its application to industrial
level.
Cx(H2O)y + H2O Acid (C6H12O6)n + C6H12O6 ——1.6
(Cellulose Chain) (Water) Δ (Fructose) (Glucose)
1.5.10.2 Cellulose Hydrolysis
Cellulosic hydrolysis produces glucose and other six-carbon sugars (hexoses) from the
cellulose and five-carbon sugars (pentoses) from hemicellulose. The non-glucose sugars
must be fermented to produce ethanol, but are not readily fermentable by
Saccharomyces cerevisia, a naturally occurring yeast. However, they can be converted
22
to ethanol by genetically engineered (Saccharomyces Cereviciae) yeasts (Shapouri et
al., 2006). Cellulose (C6H10O5)n obtained from pretreatment are degraded into
saccharides (Glucose) using acids, base or enzymes. Prior to fermentation to help the
enzymes perform well and degrade the lignocelluloses efficiently, the fibres in the raw
materials need to be accessible to the enzymes. This method of cellulose hydrolysis is
needed to expose the fibres. As such if the treatment is too harsh, liberated sugars can
be degraded to enzyme and yeast inhibiting compounds lowering the overall yields.
likely if too weak pretreatment conditions are used will result in low enzyme
accessibility and the same drawbacks (Cardona et al., 2010).
O
O
O
O
HO
HO
HO OH
OH
HO OH
O
OH
OH
n
Figure 4: Structure of Cellulose (C6H10O5)n (Cardona et al., 2008).
Cellulose (Crystall) Endocellulose Cellulose Exocellulose
Glucose Cellobiase (β-Glucosidase) Cellobiase/Cellotettrose
Figure 5: Reation Pathway from Cellulose to Glucose (Cardona et al., 2010).
1.5.10.3 Detoxification
This is a process taken before fermentation of the substrate solution, during and after
pretreatment of the lignocellulosic materials. Several detoxification methods exists such
as neutralization, over liming with calcium hydroxide, activation with charcoal, ion
23
exchange resins (Carvalho et al., 2005), are known for removing various inhibitory
compounds from lignocellulosic hydrolysates (Cardona et al., 2010 ).
During pretreatment of lignocellulosic, in addition to sugars, aliphatic acids (acetic,
formic and levulinic acid), furan derivatives furfural and 5-hydroxymethylfurfural
(HMF) and phenolic acids are formed, the existence of this substances is more likely
when concentrated acid and/or high temperatures are used (Cardona et al., 2010). these
compounds are known to affect ethanol fermentation performance and are hereby
regulated.
1.5.10.4 Fermentation
Fermentation is a biochemical reaction that breaks down complex organic molecules
(such as carbohydrates) in to smaller simpler materials (such as ethanol, carbondioxide
and water). Fermentation also is a process whereby starches and sugars are first broken
down to glucose which is then decomposed to ethanol and carbondioxide using
enzymes in a complex series of reactions (Nagashima et al., 1984). Fermentation of
Lignocellulosic hydrolysate is more difficult than the well established process of
ethanol production from, for example sugarcane juice or grains etcetera (Keating et al.,
2006).
All that is necessary to start fermentation is to mix the activated yeast and the cooled pH
adjusted mash in the fermentation container controlling the temperature in the process.
Once treatment of lignocelluloses is complete the solution harbours cellulose that is
assessable to acids or enzyme and as such should be fermented immediately for
conversion to ethanol within the time interval for complete fermentation process. The
most widely used enzyme is the Saccharomyces cerevisae (Yeast) which hydrolyses the
lignocellulosic hydrolysate to ethanol (Sanchez and Cardona, 2008).
24
Yeast is a facultative anaerobe. In an aerobic environment, it converts sugars into
carbon dioxide and water. In an anaerobic environment, it converts sugars into carbon
dioxide and ethanol (Onuki, 2010). When certain species of yeast (e.g., Saccharomyces
cerevisiae) metabolize sugar in reduced-oxygen conditions they produce ethanol and
carbon dioxide. The chemical equations below summarize the conversion:
C6H12O6 Yeast 2CH3CH2OH + 2CO2 ———————————- 1.7
(Cellulose) (Ethanol) (Carbondioxide)
C12H22O11 + H2O Yeast 4CH3CH2OH + 4CO2 ————————– 1.8
(Disaccharide) (Water) (Ethanol) (Carbondioxide)
Fermentation is the process of culturing yeast under favourable thermal conditions to
produce alcohol. This process is carried out at around 35–40 °C. Toxicity of ethanol to
yeast limits the ethanol concentration obtainable by brewing; higher concentrations,
therefore, are usually obtained by fortification or distillation.
1.5.10.5 Separation/Distillation
After fermentation, we have to make the purity of ethanol higher. Distillation is one of
the steps of the purifications. Distillation is the method to separate two liquid utilizing
their different boiling points (Onuki, 2006). 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.
1.5.11 Proximate Analysis
This type of analysis involves determination of moisture, volatile matter, ash and carbon
content of a sample. The advantage of this kind of analysis is that it is rapid and gives
ideas regarding the commercial classification and suitability of sample under
investigation, for different purposes. It is important to note that in the determination of
25
moisture, ash content, or volatile matter; the results depend upon several factors such as
size and shape of the crucibles used, the mode and period of heating etc. hence in order
to get reproducible results, experimental conditions have been specified and if the
stipulated conditions are followed, fairly reproducible results can be obtained (Verma,
2003).
1.5.11.1 Determination of Moisture Content
Moisture often refers to as the water content, present in sample vegetable. The
proportion of water in tissues varies with sample wastes; but is higher in natural
products and as such is useful to control the water content of all feedstocks in
investigation (Verma, 2003). Moisture content is often expressed as percentages of the
waste biomass. Thus is also the predominant constituent of every plant material; which
is reduced readily by drying. As medium moisture supports chemical reactions and is a
direct reactant in hydrolytic processes (Belitz et al., 2009)
Therefore, removal of moisture content from food retards many reactions and inhibits
the growth of microorganisms, thus improving the shelf lives of a number of foods
particles; through physical interaction with proteins, polysaccharides, lipids and salts
water contributes significantly to the texture of food (Belitz et al., 2009). Moisture is
determined by heating a known weight of sample at 105 – 1100C for 1hour and then
finding the weight loss of the sample as described below.
After sampling a measurable amount of the sample feedstock is weighed on an empty
dish and measured using a weighing balance. The weight is noted as W1. The weighed
samples are heated in a moisture analyzer heater thermoset at 1100C for 24hrs. The
dried samples were cooled for about 20-30mins under controlled ATP and weighed on a
balance and noted with label W2.
26
After both the readings and the procedures were taken/noted then calculation for the
average moisture content was done later using the formula below: (AOAC, 1991).
% moisture content =
𝑙𝑜𝑠𝑠 𝑖𝑛 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑊2−𝑤1)
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑤1)
𝑥 100 —————————-. 1.9
1.5.11.2 Determination of Ash Content
This is the amount of inorganic residues that remains after the removal of moisture and
organic matter using heat in the presence of oxidizing agent usually oxygen (O2) from
air. Ash content of a sample gives a measure of the total amount of minerals presents in
the sample. Low or high ash value in a sample indicates the quantity of essentials
minerals (Belitz et al., 2009).
A measurable amount of the sample after moisture analysis as completely oxidized
which is measured and recorded as X1 The weighed sample are then placed into an
electric lenton furnace at 5000c for 2hrs. The ashed sample was cooled in desiccators
and weighed and labeled X2. The result and the procedure were recorded and calculated
for average ash content (AOAC, 1991).
%Ash content=
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑎𝑠ℎ
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
𝑥 100 ——————————————– 1.10
1.5.12 Applicable Methods of Analysis
Both the Reducing sugars and the ethanol produced are essentially checked using
different methods. Technologically, the method of ligands symmetrical arrangements
using different indicators is preferable among the best techniques by the application of
the spectrophotometer or UV spectrophotometer (Electronic Spectra).
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