Polytechnic University of the Philippines

Maragondon Branch
Maragondon, Cavite

STUDY ABOUT BIOETHANOL PRODUCTION FROM COGON GRASS (IMPERATA

CYLINDRICA BEAV.) BY SEPARATE HYDROLYSIS AND FERMENTATION

A Thesis Presented to the Faculty of College of Engineering

Polytechnic University of the Philippines
Maragondon Branch
Maragondon, Cavite

In Partial Fulfillment of the Requirements in

MEEN 3282 – Mechanical Engineering Project Study 1

by:

BSME – V

Engr. John Jervic S. Barrera

Adviser

October 11, 2019

 Chapter 1

THE PROBLEM AND ITS SETTING

1.1 Introduction

According to Lin & Tanaka (2006) due to the rapid depletion of the world’s energy

supply, there is an increasing global interest in alternative energy sources. Ethanol from

biomass can provide a sustainable, albeit limited alternative to oil to mitigate the global

energy problem associated with fossil fuels exhaustion and greenhouse gas emissions

(Farrell et al., 2006). Currently, biomass-derived ethanol is produced at industrial scale

from sucrose and starch; however, this poses concerns about the potential competition

with food and feed supplies (Hahn-Hägerdal, Galbe, Gorwa-Grauslund, Lidén, & Zacchi,

2006; Field, Campbell, & Lobell, 2008).

The use of biomass is of significant interest to countries like the Philippines which

produces million tons of agricultural by-products annually. These lignocellulosic wastes

which include municipal solid wastes and many agricultural wastes like corn cobs, rice

stalks and weeds can be sources for low-cost biofuel such as ethanol (Y. Sun & J.Y.

Cheng, 2012).

Cogon grass (imperata cylindrica) also has been an agricultural problem in the

Philippines. It is a perennial, rhizomatous grass that grows from 2 to 4 feet in height and

is considered as ecological threat due to its inhibitory effect, making other plants nearly

impossible to coexist. It is a fast-growing weed which requires minimal water and grows

even in an unfertile soil (R.M. Brook, 2009).

Cogon grass’ viability as a substrate for ethanol production has not been

determined yet. The composition of the cogon grass can be approximated as that of the
 2

many other grasses which contain cellulose and hemicellulose, and is therefore a viable

ethanol source (R.M. Brook, 2009). Since it is considered as pest to many upland crops

because it consumes large amount of pesticide thereby increasing the inputs needed for

the land (S.K. McDonald et. al, 2016), utilizing it as raw material could lead to the decrease

in the amount of pesticide consumption, at the same time, minimize the use of food and

feed grade crops as substrates for bioethanol production.

The conversion of these lignocellulosic materials to ethanol has two main

processes, one is the pretreatment and hydrolysis wherein starch and cellulose are

hydrolyzed to fermentable sugars and other is the fermentation of sugars to ethanol (N.

Mosier et. al, 2015). This study investigates the potential of ethanol production from cogon

grass after applying separate hydrolysis and fermentation method.

1.2 Theoretical Framework

Figure 1. Hypothetical Structure

 3

1.3 Conceptual Framework

Figure 2. Flow of Producing Bioethanol from Cogon Grass

This represents the materials, concept and primary principle on how bioethanol is

produced through the selected method.

 4

1.4 Statement of the Problem

This study investigates the potential of ethanol production from cogon grass (imperata

cylindrica). The existing issues in the process of production of ethanol are listed below:

1. What are the main processes in converting lignocellulosic biomass to bioethanol?

2. Is it more efficient to extract bioethanol from cogon grass if the grass is shredded?

3. What is the method to be applied in converting cogon grass to bioethanol?

4. What type of hydrolysis is to be applied for converting cogon grass to bioethanol?

5. Is it feasible to produce bioethanol from cogon grass as a form of lignocellulosic

biomass?

6. What is the ethanol yield of the sample?

1.5 Hypothesis

Alternative Hypothesis

• If the cogon grass is shredded into tiny pieces, then it is easier to extract

bioethanol.

• If we add more sodium hydroxide, then it will convert more cellulose to glucose.

• If the sample was fermented in a long time, then it will produce more bioethanol.

Null Hypothesis

• If the cogon grass is shredded into tiny pieces, then it will be harder to extract

bioethanol.

• If we add more sodium hydroxide, then it will convert less cellulose to glucose.
 5

• If the sample was fermented in a long time, then it will produce less bioethanol.

1.6 Scope and Limitation of the Study

This study was made to investigate the potential of ethanol production from cogon

grass. The focus was extracting the bioethanol by pretreatment, fermentation and

distillation.

The researchers limited their study in proving the potential of cogon grass as a

source of bioethanol. Also the study is not intended for large scale production.

1.7 Significance of the Study

Cogon grass (imperata cylindrica), an invasive grass from Asia, is considered “the

seventh worst weed in the world”.

Cogon grass (imperata cylindrica) also has been an agricultural problem in the

Philippines. It is a perennial, rhizomatous grass that grows from 2 to 4 feet in height and

is considered as ecological threat due to its inhibitory effect, making other plants nearly

impossible to coexist (R.M. Brook, 2009).

The researchers aim to utilize cogon grass as a raw material for the production of

bio-ethanol which could lead to decrease the amount of pesticide consumption, at the

same time, minimize the use of food and feed grade crops as substrates for bioethanol

production.

1.8 Definition of Terms

For better understanding of the study, the researchers gave several terms that

were defined conceptually and operationally as used in the study.

Biomass - Plant matter of recent (nongeologic) origin.

 6

Cellulase - One or more enzymes that catalyze the reaction of water with cellulose to

release shorter glucose oligomers and ultimately monomeric glucose sugar.

Cellulose - A glucose polymer composed of up to about 15,000 glucose molecules

covalently joined by b 1–4 linkages in long, straight chains that can hydrogen bond with

parallel cellulose chains to form crystalline regions. About 35–50%ofthestructuralportion

of plants is cellulose.

Cellulosic biomass - Also known as lignocellulosic biomass, the structural part of plants

that is not edible by humans and contains cellulose, hemicellulose, pectin, and lignin.

Examples include grass, wood, and agricultural and forestry residues.

Cellulosic ethanol - Ethanol made from lignocellulosic biomass by biological, chemical, or

chemo biological processes.

Enzymes Proteins - produced by living cells or organisms that are able to catalyze

chemical reactions in organic substances.

Ethanol - An alcohol with the formula C2H5OH that is a high octane (greater than 100

Motor Octane Number) transportation fuel and also used commercially in alcoholic

beverages, solvents, cosmetics, and other products. Ethanol can be made by microbial

fermentation of sugars derived from sugarcane, starch, or cellulosic biomass or made

catalytically from synthesis gas. Ethanol is hygroscopic (attracts water molecules) and

biodegradable.

Hemicellulose - An amorphous, non-crystalline polymer typically made up of some

combination of arabinose, galactose, glucose, mannose, and/or xylose sugars but also

containing less amounts of other compounds such as methoxyl, acetyl, and free carboxyl

groups. Hemicellulose joins with lignin to glue long cellulose fibers into a very strong
 7

composite material. Although the proportions can vary widely, hemicellulose often makes

up about 15–30% of the overall dry weight of cellulosic biomass.

Hydrolysis - The reaction of water with a sugar polymer or other compound to form other

compounds.

Lignin - A complex phenylpropanoic acid polymer chemically linked with hemicellulose to

bind cellulose chains together. Lignin oftenrepresentsabout7–35%of the dry weight of

cellulosic biomass. Lignin is more difficult to break down into its component molecules and

is not fermentable at an appreciable rate.

Sugars - Ring-shaped compounds consisting of five to six carbon atoms along with

hydrogen and oxygen at a ratio of two hydrogen atoms and one oxygen atom to one

carbon atom. Sugar is obtained from the juice of such plants as sugarcane and sugar

beets and can be obtained by hydrolysis of starch in corn and other starch plants and by

hydrolysis of cellulose and hemicellulose in cellulosic biomass. Sugars typically contained

in cellulosic biomass include arabinose, galactose, glucose, mannose, fructose,

rhamnose, and xylose. Sugars are typically sweet.

 Chapter 2

REVIEW OF LITERATURE AND STUDIES

This chapter presents the related literature and studies after the thorough and in-

depth search done by the researchers. Also this part of the thesis contains full analysis of

bioethanol production from lignocellulosic biomass

2. Ethanol from Cellulosic Materials

2.1 Bioethanol and its application as a fuel

Ethanol, also known as ethyl alcohol with the chemical formula C2H5OH, is a

flammable, clear, colorless and slightly toxic chemical compound with acceptable odor. It

can be produced either from petrochemical feedstocks by the acid-catalyzed hydration of

ethane, or from biomass feedstocks through fermentation. On a global scale, synthetic

ethanol accounts for about 3-4% of total production while the rest is produced from

fermentation of biomass – mainly sugar crops, e.g. cane and beet, and of grains (mainly

corn) (Licht, 2006).

Ethanol as a neat fuel or even in the blended form with gasoline has a long history

as automotive fuel. In 1860, German inventor Nicholas Otto used ethanol as a fuel in an

early prototype of an internal combustion engine because it was widely available

throughout Europe for use in spirit lamps. A few years later, Henry Ford built his first

automobile with an engine that could run on ethanol. In 1908, Ford unveiled his Model T

engine equipped with carburetors that could be adjusted to use alcohol, gasoline or a

mixture of both fuels (Solomon et al., 2007). Ethyl alcohol as "the fuel of the future" was

presented by him for the first time. In 1925, he told the New York Times: “The fuel of the

future is going to come from fruit like that sumac out by the road, or from apples, weeds,
 9

sawdust – almost anything… There is fuel in every bit of vegetable matter that can be

fermented. There's enough alcohol in one year's yield of an acre of potatoes to drive the

machinery necessary to cultivate the fields for a hundred years.” However, fossil fuels

were predominantly used for automobile transportation throughout the last century,

obviously due to their lower production cost. As an automotive fuel, hydrous ethanol can

be used as a substitute for gasoline in dedicated engines. Anhydrous ethanol, on the other

hand, is an effective octane booster when mixed in blends of 5 to 30% with no engine

modification requirement (Licht, 2006).

2.2. Environmental impacts

As long as global demand for energy continues to grow, oil prices are not likely to

decrease. Consequently, the demand for a renewable and environmentally benign fuel will

increase. Over the last 150 years, human activities have caused a dramatic increase in

the emission of a number of greenhouse gases such as CO2, which has led to changes in

the equilibrium of the earth’s atmosphere (Galbe and Zacchi, 2002). Fuel ethanol is

suggested as a sustainable fuel which can be produced from renewable resources and

led to maintain or even reduce the level of greenhouse gases. The net emissions of CO 2

are reported to be close to zero, since the CO2 released during ethanol production and

combustion is recaptured as a nutrient by the crops and plants which are the raw materials

for ethanol production. Ethanol in blend with gasoline increases octane and provides

oxygen to promote more complete combustion. Addition of ethanol or derivative such as

methyl tertiary butyl ether (MTBE) to gasoline as oxygenate reduces tailpipe emissions of

CO and unburned hydrocarbons, which can contribute to improving the urban air quality.

Unlike MTBE, which is not readily biodegradable and is known as a groundwater pollutant,

ethanol is a water-soluble and biodegradable compound and therefore is relatively

harmless to the environment, ground water and soil (Isenberg, 1999; Wyman, 1999).
 10

However, because in addition to solar energy, other energy inputs (often in the form of

fossil fuel) are required in the manufacturing and marketing of biofuel such as ethanol, the

entire process is not likely to be completely carbon-neutral (Granda Cesar et al., 2007).

A large number of assessments performed to estimate the environmental merit of

biofuels show contradictory results (Niven, 2005; von Blottnitz and Curran, 2007). The

reason may be that such assessments are generally related to the net energy value

(defined as difference in the energy content of ethanol and co-products with the fossil fuel

inputs) and to ethanol production routes including the type of raw materials and

technologies applied. The “greenness” of a biofuel like ethanol is therefore highly

dependent upon the efficiency of all stages in the process from raw material to the end

use of product and its avoided use of fossil fuels. While corn ethanol is claimed to have

either negative energy content or slightly positive value, environmental benefits of

cellulosic ethanol cannot be refuted as the corresponding estimated net energy is rather

high (Farrell et al., 2006; Granda Cesar et al., 2007; Kim and Dale, 2005).

In order to get an overall picture of the environmental impact of biofuel, emissions

other than CO2 including nitrogen oxides (NOx), carbon monoxide (CO), sulfur oxides

(SOx), total particulate matter (TPM), and volatile organic compounds (VOCs), which

could be hydrocarbon emissions, as well as other VOCs such as aldehydes, alcohols,

ethers, esters, and other organics, need to be considered as well. Although ethanol (used

as E85) generally generates less emissions in tailpipe (Wu et al., 2004), life-cycle analysis

covering entire routes from crop to wheel may give opposite results. Even though the

greenhouse gas emissions are lowered when ethanol from different routes is used and

compared to gasoline, it may result in high increases of TPM, NOx and SOx emissions.

Cellulose-based ethanol substantially decreases greenhouse gas emissions and most

other pollutants, but VOCs and NOx emissions may increase by substitution of gasoline
 11

with cellulosic ethanol. Nevertheless, cellulosic ethanol seems to be a promising choice

from the perspectives of both net energy gain and overall emissions of contaminants

(Granda Cesar et al., 2007; von Blottnitz and Curran, 2007).

2.3. Raw materials for ethanol production

2.3.1. Lignocelluloses

Lignocellulosic materials such as agricultural and forest residues, crops and

herbaceous materials in large quantities are available in many countries with various

climatic conditions, making them suitable and potentially cheap feedstocks for sustainable

production of fuel ethanol. The global production of plant biomass, with over 90%

lignocellulose content, is estimated to be about 200×109 tons/year, where about 8-

20×109 tons of primary biomass remain potentially accessible annually (Lin and Tanaka,

2006). Over the last few decades, extensive attention has been devoted to research on

the conversion of lignocellulosic materials to ethanol (Chandrakant and Bisaria, 1998;

Prasad et al., 2007). Lignocelluloses are complex mixtures of carbohydrate polymers,

namely cellulose, hemicellulose, lignin, and a small amount of compounds known as

extractives.

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. 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 (Sjoström, 1981).

 12

Figure #. 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 phenyl propane 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.

Figure #. The building units of lignin (Fengel and Wegener, 1989).

 13

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).

The compositional structure of common agricultural residues and wastes is shown in Table

#.

Table #

Cellulose, hemicellulose and lignin content in common agricultural wastes

The technology of ethanol production from biomass feedstocks consists of several

steps, and varies depending on the type of raw materials used. It becomes more

sophisticated as the raw materials turn from sugars to starches and cellulosic materials.

Unlike starch, the specific structure of cellulose favors the ordering of the polymer chains

into tightly packed, highly crystalline structures that are water-insoluble and resistant to

depolymerization. For production of ethanol from cellulosic feedstocks, four major unit

operations are required: pretreatment, hydrolysis, fermentation, and separation/

purification (Mosier et al., 2005; Solomon et al., 2007; Taherzadeh and Karimi, 2007).
 14

2.4. Pretreatment

One of the main problems in application of lignocellulosic materials is their

resistance against enzymatic depolymerization. The carbohydrate polymers in

lignocellulose are tightly bound to lignin mainly by hydrogen bonds as well as by some

covalent bonds which make it a recalcitrant substrate for hydrolysis and ethanol

production. Thus, delignification is a crucial step prior to depolymerization and

fermentation steps which can highly increase the rate of subsequent hydrolysis reaction.

Delignification liberates cellulose and hemicellulose from their complex with lignin (Duff

and Murray, 1996; Lin and Tanaka, 2006; Szczodrak and Fiedurek, 1996). Citrus wastes

are more susceptible to enzymatic hydrolysis, probably due to absence of lignin in their

structure. However, presence of cellulose, hemicellulose and pectin polymers bound to

each other in three-dimensional structures makes them relatively resistant materials for

hydrolysis (Grohmann et al., 1995).

The objective of pretreatment is therefore to increase the surface area and porosity

of the substrate, reduce the crystallinity of cellulose and disrupt the heterogeneous

structure of cellulosic materials. This process makes the carbohydrate polymers

accessible for depolymerization. Pretreatment and hydrolysis of lignocellulosic materials

result in a number of fermentable pentose and hexose sugars, leaving lignin as a by-

product which can be used as fuel to produce heat or electricity.

2.5. Hydrolysis

Hydrolysis of cellulosic materials includes the processing steps that convert the

carbohydrate polymers e.g. cellulose and hemicellulose into monomeric sugars. Cleavage

of these polymers can be catalyzed enzymatically by cellulases or chemically by acids

such as sulfuric acid (Mosier et al., 2005). The factors that have been identified to affect
 15

the hydrolysis of cellulosic biomass include porosity or accessible surface area, cellulose

fiber crystallinity, and the content of lignin and hemicellulose (Prasad et al., 2007).

2.5.1 Enzymatic hydrolysis

Hydrolysis of cellulosic materials can be catalyzed by a class of enzymes known

as cellulases. These enzymes are mainly produced by fungi, bacteria, and protozoans

that catalyze the cellulolysis or hydrolysis of cellulose. At least three major groups of

enzymes including exo-glucanase, endo-glucanase and β-glucosidase are involved in

depolymerization of cellulose to glucose. β-glucosidase catalyzes cleavage of cellobiose,

which plays a significant role in the hydrolysis process, since cellobiose is an end-product

inhibitor of many cellulases including both exo- and endo-glucanases (Galbe and Zacchi,

2002; Lee, 1997; Rabinovich et al., 2002; Sun and Cheng, 2002) β-glucosidase, in turn,

is inhibited by glucose and, therefore, enzymatic hydrolysis is sensitive to the substrate

concentration (Philippidis et al., 1993). In addition to substrate concentration, pretreatment

of cellulosic materials and hydrolyzing conditions such as temperature and pH are among

factors influencing the effectivity of enzymatic hydrolysis. Most cellulase enzymes show

an optimum activity at temperatures and pH in the range of 45-55°C and 4-5, respectively

(Duff and Murray, 1996; Galbe and Zacchi, 2002).

The enzymatic hydrolysis process can be accomplished using different strategies,

of which the most important ones include separate hydrolysis and fermentation (SHF) and

simultaneous saccharification and fermentation (SSF). In SHF, hydrolysis and

fermentation are carried out in separate vessels under their own optimal conditions;

however, end-product inhibition of enzymes’ activity and contamination problems are

associated with this process. In order to eliminate drawbacks of the SHF process, SSF

that combines hydrolysis and fermentation in one vessel has been developed. Sugars
 16

produced during hydrolysis are immediately fermented into ethanol and thus, problems

associated with sugar accumulation and enzyme inhibition as well as contamination can

be avoided (Galbe and Zacchi, 2002; Ohgren et al., 2007; Philippidis et al., 1993; Wyman

et al., 1992). The main drawback of SSF is the different optimum temperatures of the

hydrolysis and fermentation processes. Most fermenting yeasts have an optimal

temperature around 30-35 ºC while hydrolyzing enzymes show optimal activities around

50 ºC (Kadar et al., 2004).

2.5.2 Acid hydrolysis

Interest in wood hydrolysis dates back to 1819 when Braconnot discovered that

cellulose could be dissolved in concentrated acid solutions and converted to sugar. Acid

hydrolysis can be performed with various types of acids including sulfuric, sulfurous,

hydrochloric, phosphoric, nitric acid, etc. Acid hydrolysis is subdivided into concentrated-

and dilute acid hydrolysis. Through the concentrated-acid hydrolysis, the biomass is

treated with high concentration of acids at near ambient temperatures, which results in

high yield of sugars. However, this process has drawbacks including high acid and energy

consumption, equipment corrosion and longer reaction time (Galbe and Zacchi, 2002;

Harris et al., 1945; Taherzadeh and Karimi, 2007).

Dilute-acid hydrolysis, on the other hand, uses low-concentration acids e.g. 0.5-

1% H2SO4 and high temperatures. High temperature is required to attain acceptable rates

of cellulose conversion to glucose. Despite low acid consumption and short reaction time

in dilute-acid hydrolysis, application of high temperatures in this method accelerates the

rate of sugar decomposition and increases equipment corrosion (Galbe and Zacchi, 2002;

Taherzadeh and Karimi, 2007). Decomposition of sugars not only lowers the ultimate yield

of sugars in dilute-acid process, but also produces a number of by-products that show
 17

severe inhibiting effects on the subsequent fermentation step (Klinke et al., 2004; Luo et

al., 2002; Taherzadeh et al., 1997a). Although many of these inhibitors have been

identified and their effects on fermentation either individually or in combination have been

vastly investigated using synthetic medium (Delgenes et al., 1996; Martin and Jönsson,

2003; Taherzadeh et al., 1999a; Taherzadeh et al., 2000; Taherzadeh et al., 1997b), the

composition of dilute-acid hydrolysis remains far more complex than these synthetically

made media. Thus, a synthetic medium containing even all known inhibitors similar to a

sample of dilute-acid hydrolysate may show different results in the fermentation step.

Nevertheless, parallel investigation on both synthetic and dilute-acid hydrolysates for

many years has provided a great extent of valuable information regarding potential effects

of these toxic compounds on baker’s yeast. Based on this information, the main inhibiting

compounds are classified in three groups: furans, phenolic compounds, and carboxylic

acids (Clark and Mackie, 1984).

In order to decrease the amount of sugar degradation and consequently formation

of inhibitors, a two-stage process has been developed where hemicellulose sugars are

released in the first stage under milder conditions. The solid residue, mainly consisting of

cellulose, is then separated from the liquid hydrolysate and subjected to the second-stage

hydrolysis which is performed under harsher conditions. 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).

2.6 Fermentation

2.6.1 Microorganisms

Microorganisms play a significant role in production of ethanol from renewable

resources and thus, selection of suitable strain is essential for the individual process.
 18

Ethanol production is much more challenging and difficult when lignocellulosic and/or

cellulosic materials are to be used as raw materials. Unlike the starch-based materials,

pretreatment and hydrolysis of lignocellulosic materials produce a mixture of pentoses and

hexoses along with other inhibiting compounds, causing many problems in the

fermentation step. Therefore, capability of consuming both pentose and hexose sugars,

high tolerance against substrate, ethanol as well as inhibiting compounds, high ethanol

yield and minimum nutrient requirements are the essential features of an ideal

microorganism (van Zyl et al., 2007). Although no microorganism has been found yet to

meet all these requirements, development of a desirable strain is the focus of many

studies. Thus far, wide varieties of microorganisms including yeasts, bacteria and fungi

have been exploited offering different advantages and disadvantages (Olsson and Hahn-

Hägerdal, 1993). However, the most frequently used microbe has been yeast and among

the yeasts, S. cerevisiae which can tolerate ethanol concentration as high as ca. 20% of

fermentation medium is the preferred strain (Lin and Tanaka, 2006). Some species of

bacteria such as Zymomonas mobilis and the genetically engineered Escherichia coli can

produce ethanol at higher yields, but they are less resistant to the end product (ethanol)

and other compounds present in the hydrolysates when compared to the yeast (Lawford

and Rousseau, 1998; Olsson and Hahn-Hägerdal, 1993; Sprenger, 1996).

During the evolution of yeast S. cerevisiae, the ability to override the glucose

repression circuit that suppresses respiration of glucose and other hexose sugars above

20-40 mM threshold concentration in the presence of oxygen was developed. This

phenomenon, known as the ‘Crabtree effect’, provided the ancestor of S. cerevisiae with

an advantage over its competitors because high ethanol levels (>4% v/v) are toxic to most

other microorganisms (van Zyl et al., 2007; Verstrepen et al., 2004). In contrast with many

advantages offered by using yeast in ethanol production, it lacks the mechanism to take
 19

up pentose sugars as substrate. Attempts to add this ability by genetic manipulation are

still at the laboratory stage (Jeffries, 2006). Putting advantages and disadvantages

together, S. cerevisiae still remains the prime microorganism for ethanol production (Bai

et al., 2008).

2.6.2. Fermentation

A sugar such as glucose is directly metabolized by the yeast cells through the

glycolysis pathway to gain energy for biosynthesis. Under anaerobic conditions, the overall

reactions produce two moles of ethanol and CO2 per mole of glucose consumed.

Fermentation can be carried out with different types of industrial operation as batch, fed

batch, or continuous process. The most suitable choice depends on the kinetic properties

of the microorganism as well as process economics. Batch cultivations need low

investment cost and lower requirements for process control. The fed-batch operation,

sometimes regarded as a combination of the batch and continuous operations, involves

addition of feed at constant intervals while effluent is removed discontinuously. When the

substrate has inhibitory effects, this method is advantageous because the microorganism

is exposed to low concentration of substrate. Continuous operation offers ease of control

and high ethanol productivity, but contamination is a serious issue to be considered

(Olsson and Hahn-Hägerdal, 1996; Prasad et al., 2007).

 20

Chapter 3

METHODOLOGY

This chapter presents the conversion process, and comparison of methods of

producing ethanol from lignocellulosic material. Also the chapter shows materials and

methods that will be used to perform the experiment.

3.1. Research Design

 21

3.1. Process Flowchart

Figure 5. Flowchart of the Research

 22

The figure shows the project development or flow of the research design. It is the

end-to-end process of conceptualizing and delivering a research given a set of resources

and constraints. This typically involves the following project stages with the yes/no flow

chart:

First step is the initiation where the process of developing a project concept and

an initial set of goals takes up. It may include the topic selection that maps out the benefits

of the project with assumptions, constraints and estimates of required resources. At this

stage much is unknown and the project may be nothing more than a high level goal.

Next is research where the project typically begins with many unknowns. This often

involves a research stage whereby data is collected from various related studies and

literatures. Then estimates where the mapping of design requirements takes up and then

estimating the effort and cost required.

After that the knowledge and materials will be gathered that will make the question

of satisfying the design requirements. If yes, then the collected requirements will be

reviewed. If no, then it will need to search and choose for other requirements that will be

reviewed later to proceed to the next stage.

Project planning is one of the vital stages in the research. This is the process of

prioritizing work, identifying dependencies and creating a work breakdown structure and

schedule. Planning maybe a backlog to designing. If there was no plan created, you will

search for plans then choose that will satisfy the requirements. If there was a plan it will

be reviewed and may proceed.

 23

Then procuring the requirements and plans to advance to the designing of the

project where that can be used to implement a solution to a problem. After that the design

will be implemented to make the project happen.

Testing will be the process of controlling quality and confirming that what you have

implemented was working. This will serve as topic for another question regarding if the

project passed the test procedures. If yes then it will proceed to the deployment of the

project. If no, then the requirements needs to be reviewed.

Finally the deployment where the project will be operationalize. Then the finalizing

or conclusion of the process and communicating the close of a project. This often includes

the lessons learned that captures any useful knowledge related to the project

3.1. Biochemical Conversion Process

Lignocellulosic material is characterized by its strength and complexity due to a

network formed between hemicellulose and cellulose in close association with lignin. A

number of processing steps is required to overcome this complex structure to make it

suitable for fermentation.

3.1.1. Feedstock Size Reduction

Before pretreatment, the first stage in the production of ethanol from biomass is

cleaning followed by mechanical comminution combines chipping, grinding, and milling to

break the lignocellulosic materials down to 0.2 to 2 mm and reduce the crystallinity of the

materials (Mosier et al., 2005). Size reduction is necessary to provide pumpable slurry

and to increase the biomass surface area so that mass transfer effects are minimized

during the downstream processes. Techniques for size reduction include hammer, disk

and knife milling and are well established (Taherzadeh and Karimi, 2008).
 24

3.1.2. Pre-Treatment

. In 2005, Mosier et al., in their study reported that the first step in producing

cellulosic ethanol is biomass handling where the size of the lignocellulose is reduced to

make handling easier and ethanol production more efficient. During pretreatment cellulose

structure is disrupted, the lignin seal is broken, and the hemicellulose is partially removed.

This increases the specific surface area that is accessible to enzymes.

Figure #. Effect of Pre-Treatment to the Lignocellulose Structure

Depending on the nature of the pretreatment technology selected, this step can

also include solubilization of the lignin or the hemicellulose component. Various

pretreatment options are available now to fractionate, solubilize, hydrolyze and separate

cellulose, hemicellulose, and lignin components. These include physical,

physicochemical, chemical and biological pretreatment (Avira et al., 2010).

Table #

Common pretreatment methods for lignocellulosic materials

 25

Adapted from: Duff and Murray (1996), McKendry (2002), Prasad et al. (2007), Sun and Cheng (2002),
Szczodrak and Fiedurek (1996).

3.1.3. Hydrolysis

In the hydrolysis reaction, the complex chains of sugars that make up the hemi-

cellulose are broken, releasing simple sugars. The complex hemi-cellulose sugars are

converted to a mix of soluble five-carbon sugars, xylose and arabinose, and soluble six-

carbon sugars, mannose and galactose. The rest of hemicelluloses are degraded to weak

acids, furan derivates, and phenolics. These compounds, however, are potential

fermentation inhibitors. By the action of dilute acids, concentrated acids, and/or enzymes

(Cellulase), the glucose yields of cellulose hydrolysis often exceed 90%, but hydrolysis

without preceding pretreatment yields typically less than 20% only (Sun and Cheng,

2002). The cellulose hydrolysis reactions can be simply represented as:

(C5H8O4) n + n H2O→ n (C5H10O5) (C6H10O5) n + n H2O → n (C6H12O6)

3.1.3.1. Acid Hydrolysis

According to Harris E.E. et al. (1945), in the book Industrial and Engineering

Chemistry, pp. 12-23 stated that acid hydrolysis is perhaps currently seen as the most

technologically matured method of sugar release from biomass. Traditional methods

developed in the 19th century and at the beginning of the 20th century, produced glucose
 26

from cellulose by usage of acid. No pretreatment is required if the end product (glucose)

is to be fermented to alcohol. Depending on the concentration of the acid and the other

parameters can be determined i.e. dilute acid (H2SO4) maybe used at high temperature

and pressure while concentrated acids maybe used at very low temperature and pressure.

In the case when sulfuric acid (H2SO4) can be concentrated (25-80%) or dilute (3-8%),

measured as the weight of acid in the weight of acidified aqueous solution that is present

with the biomass.

3.1.4. Fermentation

This is the chemical transformation of organic substance into simpler compounds

by the action of enzymes. Originally the term fermentation was used to mean the

enzymatic breakdown of carbohydrates in the absence of air. In industrial practice,

fermentation refers to any process by which raw materials are transformed by the

controlled action of carefully selected strains of organisms into definite products. Louis

Pasteur used the term in a narrower sense to describe changes brought about by micro-

organisms growing in the absence of air (Bertilsson M., Feb 2007). However, for the cause

of this thesis it is a biological method of producing ethanol. The fermentation reaction is

caused by yeast or bacteria which feed on simple sugars. The glucose produced from the

hydrolysis described above is fermented with yeast to produce ethanol. Carbon-dioxide is

also produced as glucose is consumed. The simplified reaction equation is:

C6H12O6----------------------------------------------→ 2C2H5OH + 2CO2

3.1.4.1. Difference of Type of Yeast

In the study of Mussatto and Teixeira, in 2010, they stated that fermentation of,

hydrolyzed product, glucose into ethanol can be carried out using a biocatalyst, called
 27

Saccharomyces cerevisiae yeast or Zymomonas mobilis bacteria. Saccharomyces

cerevisiae and related species have the ability to utilize a wide range of hexoses such as

glucose, fructose, sucrose, galactose, maltose and maltotriose to produce a high yield of

ethanol. The fermenting of the biomass is conducted under standard fermenting conditions

and will utilize all the major biomass. Yeasts are minute, often unicellular, fungi. The yeasts

used are typically brewers' yeasts. Examples of yeast capable of fermenting the decaying

biomass include, but are not limited to, Saccharomyces cerevisiae and Saccharomyces

uvarum. Non-Saccharomyces yeasts, also known as non-conventional yeasts, are also

used to make a number of commercial products. Some examples of non-conventional

yeasts include Kuyberomyces lactis, Yarrowia lipolytica, Hansenula polymorpha and

Pichia pastoris (Kuhad et al., 2010). Microorganisms other than yeast can also be useful

in making fermentation products. For example, cellulosic ethanol production also utilizes

fungi and bacteria. Examples of these cellulolytic fungi include Aspergillus niger,

Trichoderma reesei, Trichoderma longibrachiatum and Trichoderma viride. One example

of a bacteria used in cellulosic ethanol production is Clostridium thermocellum, Clostridium

cellulovorans and Clostridium Ijungdahlii. Mid- to long-term technology under

development are expected to improve the fermentation efficiency of the organism,

producing higher yields in less time, and an organism requiring less detoxification of the

hydrolysate. This process has the advantage of being able to maintain a much higher cell

density in the fermenter, thereby increasing ethanol productivity (Begum and Alimon,

2011)

3.1.5. Distillation

Separation of ethanol from the fermentation solution refers to the stage in which

once ethanol begins to form during fermentation, it is isolated from the fermentation

solution. This is a separation of mixtures based on the volatilities (boiling points) of the
 28

individual components that make up the mixture. Distillation is often used only if one

product is required. The product which is of low volatility is called distillate while the

substances of high boiling point that remain in the flask is called residue or bottoms. In

acid hydrolysis, part of the acid and water is recovered in distillation (Wyman et al., 2005).

3. 2. Comparison of Separate Hydrolysis and Fermentation (SHF) and Simultaneous

Saccharification and Fermentation (SSF)

In the study of Kingsley O. in March 2012, the sawdust was used to carry out the

experiment from the production of ethanol and two methods were considered: SHF

(Separate Hydrolysis and Fermentation) and SSF (Simultaneous Saccharication and

Fermentation).

3.2.1 Experiment 1: Separate Hydrolysis and Fermentation (SHF)

Culturing saccharomyces cerevisiae: Saccharomyces cerevisiae is used to

ferment the sugar in the ethanol and has to be cultured 48 hours before commencing the

experiment. 10 g of potato dextrose Agar (PDA) is dissolve completely in 250ml water in

a conical flask. The mixture is covered with cotton wool and foil paper and then sterilized

in an autoclave at 1210 C for 5minutes. On removal it is allowed to cool and then poured

into petri dishes and is set aside and allowed to solidify. The Saccharomyces cerevisiae

are then introduced into the petri dishes with the aid of a sterilized inoculating loop. The

petri dishes are then sealed and kept in incubator for 48 hours at a temperature of 250 C.

Figure #. Simplified flow chart on the SHF production of ethanol from sawdust
 29

Sawdust is sieved to create uniformity of particles. The sawdust is then dried for

12hours to remove moisture. A 250 ml beaker is filled with 100 g of dry sawdust and 100

ml of 18M H2SO4 (sawdust to acid (w/v) ratio is 1:1) is added to it at standard conditions.

The reaction is spontaneous producing lignin (lignin is the substance that bonds sugar

molecules to make cellulose out of them) which is seen as black residues, the conical flask

also immediately become very hot and bubbles due to air pocket in sawdust. However,

the pH is very low and Saccharomyces cerevisiae cannot function at this condition, it would

function optimally at a pH of 4.5-6.0. Thus, there is need to increase the pH. In a 1000ml

beaker add 200 ml of water (water at pH of 9.7) and pour acidic solution into it and stir

thoroughly. The pH was read to be 1.37. After which 100ml water was added again to the

solution and stirred thoroughly, the pH was then read to be 1.91. 100ml water was added

again to the solution and the pH read 2.35. However allowable dilution factor with water is
 30

1:4. From the equation below it can be seen that 9M NaOH is required to form salt thus

pH 7 and less will be required for an optimal reaction.

H2SO4 + 2NaOH ---------------------------→ Na2SO4 + 2H2O

8.5M NaOH solution is prepared and added drop by drop until pH of 4.87 was

attained. The solution is filtered bringing out the cellulose substrate as filtrate and lignin

as residue using a Buchner funnel. Using a DMA 35 the sugar produced is measured to

32.4g. The cultures of saccharomyces cerevisiae in the agar slant tubes were dissolved

with 10ml of distilled water containing a drop of tween 80. 10 ml of the solution is then

added to the cellulose substrate to ferment it. On a four-hourly basis the sample is tested

for sugar content to determine rate of conversion of sugar to ethanol thereby determining

the time required for fermentation and rate of fermentation. Ethanol fermentation was

performed in a shaker incubator at 150rpm for 48-72 hours at 300 C to allow it to ferment

completely. Bubbles of CO2 are seen to appear.

This 100ml of the filtrate is then distilled using a distillation bath. 5.9ml of ethanol

is distilled at 780 C and water can be distilled at 1000 C.

3.2.2. Experiment 2: Simultaneous Saccharification and Fermentation (SSF)

Figure #. Simplified flow chart on the SSF production of ethanol from sawdust
 31

100g of fine sawdust was added to 100ml conical flask and 100ml of 0.4M H2SO4

was added to it. The pH of the mixture was 3.1, thus, 0.01M Ca (OH) 2 was prepared and

added in drops until pH of 4.61 was attained. The mixture was put in an autoclave and

was subjected to a temperature of 120C for 10minutes. The mixture was then removed but

because the temperature was too high for enzymes to be added, so it was cooled in a

refrigerator until a temperature of about 300C was attained. The sugar content was tested

for to be 24.7g. After which the 2.5g of cellulose was added and Saccharomyces

cerevisiae was added. The mixture was kept in a shaker incubator at 150rpm for 48 – 72

hours at 300 C to allow it to ferment completely. On a four hourly basis, the mixture was
 32

tested for sugar content to determine rate of fermentation with time and the time required.

100ml of sample was distilled in a distillation bath and 6.3ml of ethanol was distilled at

780C,

Safety consideration:

1. Hydrolysis reaction is an exothermic reaction; care was taken in handling the

reactor vessel.

2. Care was also taken in handling the H2SO4.

3. The acid was poured into water and not water into acid to avoid explosions of skin

burns.

4. Overalls, gloves and goggles were worn in the course of the experiment.

5. In removing the hot liquids from the autoclave care was taken.

3.2.3. Analysis of the SHF of Sawdust

The procedure for the production of ethanol from cellulose by SHF has been

discussed Where 100g of hardwood sawdust is used to produce ethanol by acid hydrolysis

using H2SO4 as the acid. 32.4g of sugar was obtained on completion of the hydrolysis;

however, theoretically 52.2g of sugar can be produce from 100g of sawdust. The yield

obtained by acid hydrolysis of hardwood sawdust can be calculated as follow:

𝑎𝑐𝑡𝑢𝑎𝑙 𝑣𝑎𝑙𝑢𝑒
1. 𝑌𝑖𝑒𝑙𝑑 = 𝑡ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑣𝑎𝑙𝑢𝑒 × 100

Thus the sugar yield is:

32.4
2. 𝑆𝑢𝑔𝑎𝑟 𝑦𝑖𝑒𝑙𝑑 = 52.2
× 100 = 62.07%

Also, the experiment entailed a test of the sugar concentration on a four hourly basis

to determine the rate of fermentation by checking the sugar disappearance and also to
 33

determine the exact time required for fermentation which is noticed when there is no

significant change in concentration. The raw data is shown below.

Brix is the sugar content in an aqueous solution

Figure #. Graph showing sugar content against time by SHF

From the graph above it can be deduced that fermentation is completed after

44hours. It can also be seen that 10.5g of sugar was converted to ethanol. Sugar

conversion can be calculated as follow:

𝑆𝑢𝑔𝑎𝑟 𝑝𝑟𝑜𝑑𝑢𝑐𝑒𝑑 − 𝑠𝑢𝑔𝑎𝑟 𝑢𝑛𝑐𝑜𝑛𝑣𝑒𝑟𝑡𝑒𝑑

× 100
𝑆𝑢𝑔𝑎𝑟 𝑝𝑟𝑜𝑑𝑢𝑐𝑒𝑑

32.4−10.5
Thus, Sugar conversion for SHF 32.4
× 100 = 67.59%

67.59% is converted by SHF

From experiment as described in section 3.2.1, 5.9ml of ethanol was produced

from 100ml of a 400ml solution the total ethanol contained is 5.9 × 4 = 23.6𝑚𝑙
 34

23.6
𝐸𝑡ℎ𝑎𝑛𝑜𝑙 𝑦𝑖𝑒𝑙𝑑 = 34.1
× 100 = 69.21%

The ethanol yield by SHF is 69.21%

Therefore, by Separate Hydrolysis and Fermentation using acid hydrolysis 23.6ml

of ethanol is produced from 100g of sawdust

3.2.4. Analysis of the SSF of Sawdust

The procedure for the production of ethanol by SSF is discussed in section 3.2.2,

SSF method is used to produce ethanol from 100g of hardwood sawdust. Cellulose

enzymes and yeast were used in the production. Theoretically 52.2g of sugar is present

in 100g of hardwood sawdust.

Thus the yield can be calculated as follow:

𝑎𝑐𝑡𝑢𝑎𝑙 𝑣𝑎𝑙𝑢𝑒
𝑌𝑖𝑒𝑙𝑑 = × 100
𝑡ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑣𝑎𝑙𝑢𝑒

Thus the sugar yield is:

36.6
1. 𝑆𝑢𝑔𝑎𝑟 𝑦𝑖𝑒𝑙𝑑 = 52.2
× 100 = 70.11%

Also, on a four hour interval the sugar concentration of solution was tested to

determine the rate of conversion of sugar to ethanol and also to determine the exact time

required in SSF for the production of ethanol. The raw data obtained is shown below:

Figure #. Graph showing sugar content against time by SSF

 35

From the graph above, it can be deduced that because of the two processes

occurring simultaneously there is an unsteady state which is responsible for the zigzag

nature of the graph between 0-20 hours. The maximum sugar was produced at 20hours

corresponding to 36.6 after which a steady state was obtained and only fermentation

occurred. The time required for the process was 52hours. After 52hours the sugar content

9.8g remained unconverted. Conversion of sugar can be calculated as follow:

𝑆𝑢𝑔𝑎𝑟 𝑝𝑟𝑜𝑑𝑢𝑐𝑒𝑑 − 𝑠𝑢𝑔𝑎𝑟 𝑢𝑛𝑐𝑜𝑛𝑣𝑒𝑟𝑡𝑒𝑑

× 100
𝑆𝑢𝑔𝑎𝑟 𝑝𝑟𝑜𝑑𝑢𝑐𝑒𝑑

36.6−9.8
Sugar conversion: 36.6
× 100 = 73.22%

73.22% is converted by SHF

6.3ml is distilled from 100ml of a 400ml solution. So the total ethanol content is 25.2ml.

25.2
𝐸𝑡ℎ𝑎𝑛𝑜𝑙 𝑦𝑖𝑒𝑙𝑑 = 34.1
× 100 = 73.9%

The ethanol yield by SHF is 73.29%

3.2.5. Synthesis of the Comparison of SSF and SHF

 36

This research work based on the extraction of sugar and subsequent fermentation

of the sugar from cellulose. Study was carried out on ethanol from starch and ethanol and

cellulose were compared. However for the experiment the cellulosic material or biomass

used is sawdust. Two methods were used to produce the cellulosic ethanol: SHF and SSF.

It can be inferred that the SHF is a dangerous method as highly concentrated acid

is being use for the hydrolysis. However it is less costly compared to SSF method due to

the use of cellulose enzymes. The SSF, however, produces more ethanol compared to

SHF but the difference in the ethanol production doesn’t account for the difference in cost

of production making the SHF more cost effective. This may not be applicable on a large

scale though.

The SHF proved more hazardous than SSF and also had waste products that are

hazardous to the environment. However, it is less costly. On the other hand SSF produced

more yield and the process took a longer duration compared to the SHF. Therefore it is

more preferable to perform Separate Hydrolysis and Fermentation (SHF) for the

researchers.

3.3. Experiment for other Lignocellulosic Biomass (Banana Peels)

Figure #. The steps involved in the conversion lignocellulosic biomass to ethanol

3.3.1. Banana Peels Collection

 37

Lignocellulosic residues of banana peels were obtained from the student cafeteria

at Usmanu Danfodiyo University Sokoto (UDUS) and the residues were used as the

source of sugars for the bioethanol production. Sodium hydroxide, sulfuric acid, and yeast

were used for the alkaline pretreatment, acid hydrolysis, and fermentation respectively.

3.3.2. Pretreatment of Banana Peels

Pretreatment of lignocellulosic biomass prior to hydrolysis is a prerequisite for

bioethanol production because it determines the yield of bioethanol that would be obtained

after fermentation. The aim of pretreatment is to reduce the compactness, strength and

crystalline nature of cellulose aiding in hydrolyzing the lignocellulosic biomass to simple

sugar units. The alkaline pretreatment was carried out using electrically heated autoclave

by the use of 10 % (wt/wt) NaOH and liquor to fiber ratio of 6:1. They cooked the fiber at

120 °C for six hours prior to the discharge of pressure into the atmosphere. The peels

were washed with water and air-dried at 45 °C. They subjected the peels to water

pretreatment as a pretreatment process in a closed autoclave. The banana waste was

cooked at 120 ◦C using water to liquor ratio of 1:10 for six hours. The pressure was

released into the atmosphere and the pulped fiber was washed with water and air-dried at

45 °C. For acid pretreatment, 40 g of banana sample was mixed with 200 mL of five

percent H2SO4 and kept at 120 °C for six hours. The mixture was filtered to separate the

solid residues from the filtrate fraction. The solid residues were thoroughly washed with

tap water to neutral pH and dried at 45 °C. The setup we used for the pretreatment

techniques is depicted in the figure below.

Figure #. Setup for Pretreatment Techniques

 38

3.3.3. Hydrolysis of Pretreated Banana Peels

The aim of hydrolysis is to further degrade the polysaccharides present in the

pretreated lignocellulosic biomass of banana waste into monosaccharides subunits. The

monosaccharides that will be produced upon hydrolysis will enhance the fermentation

process by S. cerevisiae. This study used sulfuric acid due to its availability and ease of

handling. 10 % sulfuric acid was prepared and mixed with the lignocellulosic biomass of

banana waste produced from the various pretreatment processes. They used a sulfuric

acid to fiber ratio of 6:1. The set up was heated at a temperature of 120 °C for six hours

and allowed to cool. There was a color change observed after hydrolysis and the intensity

was pretreatment dependent. The image is given in the figure below.

Figure #. The color change observed after hydrolysis from different pretreatment

techniques

3.3.4. Fermentation of Banana Peels

 39

Fermentation is the final stage of bioethanol production. We used S. cerevisiae to

convert the monosaccharides and some disaccharides produced during hydrolysis into

ethanol with the help of invertase and zymase enzymes present in S. cerevisiae. The S.

cerevisiae cells were suspended in deionized water and the pretreated banana waste was

used as the only carbon source for the yeast cells. Out of the six bottles used, three served

as controls (deionized water plus banana peels but without yeast cells) while the remaining

three were supplemented with both banana peels and the yeast cells in the deionized

water. The figure below illustrates the yeast cells prior to activation and the fermentation

set up we applied. We allowed the fermentation process to continue for three days

because S. cerevisiae grows in three days, and finally, the samples were centrifuged and

bioethanol was analyzed from the filtrate using chromatography.

Figure #. Fermentation setup

According to the study the selection of a good pretreatment technique determines

the success of every bioethanol production, so it is imperative to select the best

pretreatment technique available. It helps open up and disintegrate the biomass

constituents into individual components i.e. cellulose, hemicellulose, and lignin. The

breakdown helps achieve an efficient hydrolysis and fermentation. They used three
 40

different pretreatment techniques to process the banana peels namely water, alkaline, and

acidic pretreatments using distilled water, sodium hydroxide and sulfuric acid respectively.

Hydrolysis is the second stage of processing bioethanol from lignocellulosic

biomass. Two different methods are commonly employed during hydrolysis: acid

hydrolysis and enzymatic hydrolysis. The sole aim of hydrolysis is to convert

lignocellulosic biomass constituents into accessible reducing sugars that will serve as

substrates during fermentation by S. cerevisiae. This study used 10 % sulfuric acid for the

hydrolysis; they autoclaved the mixtures for five hours at 120 °C and allowed the

hydrolysis to proceed for three days.

On our study we will follow the acid pretreatment used in this study because it

produced the highest reducing sugar concentration followed by alkaline pretreatment

technique and finally water pretreatment as confirmed by Benedict’s test.

3.3. Materials and Methods of Bioethanol Production from Cogon Grass

 41

3.3.1. Materials

Figure #. Sulfuric Acid 5%

Figure #. Sodium Hydroxide 10%

Figure #. Water
 42

Figure #. Beaker/Pan

Figure #. Burner

Figure #. Fractional Distillation Setup

 43

3.3.2. Sample collection

Lignocellulosic residues of cogon grass were obtained from……

Figure #. Cogon Grass

3.3.3. Pretreatment

Pretreatment of lignocellulosic biomass prior to hydrolysis is a prerequisite for

bioethanol production because it determines the yield of bioethanol that would be obtained

after fermentation. The aim of pretreatment is to reduce the compactness, strength and

crystalline nature of cellulose aiding in hydrolyzing the lignocellulosic biomass to simple

sugar units……

Figure #.

3.3.4. Hydrolysis

The aim of hydrolysis is to further degrade the polysaccharides present in the

pretreated lignocellulosic biomass of cogon grass into monosaccharides subunits.

This study used sulfuric acid due to its availability and ease of handling. 10 %

sulfuric acid was prepared and mixed with the lignocellulosic biomass of cogon grass

produced from the various pretreatment processes….

 44

The researchers used sulfuric acid to fiber ratio #:#.....

Figure #.

3.3.5. Fermentation

Fermentation is the final stage of bioethanol production. The researchers will use

“this kind of yeast”…

Figure below illustrates the yeast cells prior to activation and fermentation setup

the researchers applied….

The researchers will allowed the fermentation process to continue for three or more

days….

Figure #.

3.3.6. Ethanol Analysis

Ethanol analysis will be carried out using the gas chromatography technique…