An Undergraduate Technical Research Submitted to the College of

Engineering, Architecture and Technology

Palawan State University
Puerto Princesa City

As Partial Fulfilment of the Requirements

For Degree of
Bachelor of Science in Petroleum Engineering

December 16, 2019

Potential of Carabao Grass (Paspalum Conjugatum) as

Bioethanol Feedstock

By

JAYHAZEL M. ROSIE
KENNETH REY S. CUNADO
JOHN GABRIEL A. ONDIANO
ANDRE JOSE B. SERINAS

College of Engineering, Architecture and Technology

Palawan State University
Puerto Princesa City
 ABSTRACT

Global demand for energy continues to grow due to rapidly expanding human

population and increase of the industrial prosperity in developing countries. However

increased the level of greenhouse gasses in the earth’s atmosphere along with the

inevitable depletion of the world’s energy supply, and unstable oil market have renewed

the interest of society in searching for alternative fuels. Ethanol has long been considered

as a suitable alternative to fossil fuels either as a sole fuel in cars with dedicated engines

or as an additive in fuel blends. According to the Alternative Fuels Data Center (2018),

regardless of from which ethanol is produced, its chemical formula never alters. Due to

this case, the researchers of this study will use carabao grass (Paspalum Conjugatum) as

ethanol feedstock. No previous studies yet have documented the potential of carabao

grass as a source of bioethanol.

To investigate its potential, the researchers fermented carabao grass extract,

obtained through dilute acid hydrolysis, for 12 days, 8 days, and 4 days using active dry

yeast (Saccharomyces cerevisiae). Then filtered and sent the fermented samples for Gas

Chromatography to determine the ethanol content produced.

The result shows that carabao grass (Paspalum Conjugatum) was able to produce

ethanol. All samples are subjected to Gas Chromatography which resulted to an ethanol

content of 0.48%, 0.97%, and 0.50% of 1L of fermented sample for 12, 8, 4 days

respectively. 100g of carabao grass produced about 0.0048L, 0.0097L, and 0.0050L of

ethanol thus needing 2083.33 kg, 1030.92 kg, and 2000 kg of carabao grass fermented for

12, 8, and 4 days respectively to produce 100L of ethanol. The result shows that carabao

grass can produce and is a potential alternative feedstock for bioethanol production.
SIGNATURE PAGE
ACKNOWLEDGEMENT
 TABLE OF CONTENTS

Contents
TITLE PAGE………………………………………………………………………………i
ABSTRACT .................................................................................................................... ii
SIGNATURE PAGE ...................................................................................................... iii
ACKNOWLEDGEMENT .............................................................................................. iv
TABLE OF CONTENTS ................................................................................................ v
LIST OF TABLES ......................................................................................................... vi
LIST OF FIGURES ....................................................................................................... vii
Chapter 1: INTRODUCTION.......................................................................................... 1
1.1 Background of the Study ................................................................................... 1
1.2 Statement of the Problem .................................................................................. 2
1.3 General and Specific objectives of the Study ..................................................... 3
1.4 Scope and Delimitation of the Study ...................................................................... 3
1 .5 Significance of the Study ...................................................................................... 3
1.6 Assumptions of the Study ...................................................................................... 4
Chapter 2: REVIEW OF RELATED LITERATURE ....................................................... 5
2.1 R.A. 9367 – Biofuels Act of 2006...................................................................... 5
2.2 Ethanol .............................................................................................................. 5
2.3 Ethanol Production in the Philippines ................................................................ 6
2.4 Sources of ethanol ............................................................................................. 6
2.5 Making of ethanol ............................................................................................. 8
2.6 Carabao Grass as an Alternative Bioethanol Feedstock .......................................... 8
2.7 Raw Material ......................................................................................................... 9
2.7.1 Characteristic of Lignocellulosic Biomass ....................................................... 9
2.7.2 Carabao Grass ............................................................................................... 10
2.7.3 Contents of Carabao Grass ............................................................................ 11
2.7.4 Contents of other Bioethanol Grass Feedstock ............................................... 12
2.8 Pretreatment......................................................................................................... 15
2.9 Acid Hydrolysis ................................................................................................... 15
2.10 Fehling Test ....................................................................................................... 15
2.11 Yeast ................................................................................................................. 16
2.12 Fermentation ...................................................................................................... 16
 2.14 Gas Chromatography Test .................................................................................. 17
2.15 Related Studies .................................................................................................. 18
Chapter 3: CONCEPTUAL FRAMEWORK ................................................................. 19
3.1 Conceptual Framework ........................................................................................ 19
3.2 Definition of Terms ............................................................................................. 21
Chapter 4: MATERIALS AND METHODS .................................................................. 23
4.1 Material, Toold and Equipment Used ................................................................... 23
4.1.1 Laboratory Apparatus.................................................................................... 23
4.1.2 Reagents ....................................................................................................... 23
4.1.3 Materials ....................................................................................................... 24
4.2 Experimental Procedure ....................................................................................... 25
4.2.1 Preparation of Sample ................................................................................... 25
4.2.2 Dilute Acid Hydrolysis.................................................................................. 25
4.2.3 Fehling’s Test ............................................................................................... 26
4.2.4 Activation of yeast ........................................................................................ 27
4.2.5 Fermentation ................................................................................................. 28
4.2.6 Filtration ....................................................................................................... 28
4.2.7 Gas Chromatography .................................................................................... 29
Chapter 5: RESULTS AND ANALYSIS ....................................................................... 30
5.1 Preparation of Sample .......................................................................................... 30
5.2 Acid Hydrolysis ................................................................................................... 30
5.3 Fehling’s Test ...................................................................................................... 30
5.4 Yeast Activation .................................................................................................. 31
5.5 Fermentation........................................................................................................ 31
5.6 Filtration .............................................................................................................. 32
5.7 Gas Chromatography ........................................................................................... 32
5.8 Ethanol Yield from Carabao Grass ....................................................................... 33
Chapter 6: SUMMARY, CONCLUSION AND RECOMMENDATION ....................... 35
6.1. Summary ............................................................................................................ 35
6.2 Conclusion........................................................................................................... 36
6.3 Recommendations................................................................................................ 36
BIBLIOGRAPHY ......................................................................................................... 37
GLOSSARY OF TECHNICAL TERMS ....................................................................... 40
RESEARCH WORK PLAN .......................................................................................... 44
APPENDICES .............................................................................................................. 47
 LIST OF TABLES

Table 2.4.1: List of major potential biomass materials for ethanol production in the

Philippines …………………………………………………………………………...…....7

Table 2.4.2: Required kg from various feedstocks to produce 100 liters of ethanol…...….7

Table 2.7.3.1: Table of chemical composition and nutritional value of Carabao grass....11

Table 2.7.4.1.1: Contents of Elephant Grass………………………….…………...……..12

Table 2.7.4.2.1: Contents of Cogon Grass……………………………………………….13

Table 2.7.4.3.1: Contents of Purple Guinea Grass……………………………………….14

Table 2.7.5.1: Comparison of Contents……………………………....…………………..14

Table 4.1.1.1 Laboratory Apparatus…………………….…………………..……………23

Table 4.1.2.1 Reagents…………………………………………………………………...23

Table 4.1.3.1 Materials………………………………………………………..….………24

Table 5.3.1: Results of Fehling’s Test of Samples …………………...........…………….31

Table 5.5.1: Duration of Fermentation…………………………………...………………32

Table 5.7.1: Result of Gas Chromatography for Samples 12D………....………………..32

Table 5.7.2: Result of Gas Chromatography for Samples 8D…………...……………….32

Table 5.7.3: Result of Gas Chromatography for Samples 4D……………..……………..33

 LIST OF FIGURES

Figure 3.1.1 Independent and Dependent Variables..........................................................19

Figure 3.1.2 Schematic Diagram of Bioconversion of Carabao Grass to Ethanol............20

Figure 4.2.1.1 Preparation of sample................................................................................25

Figure 4.2.2.1 Dilute Acid Hydrolysis..............................................................................25

Figure 4.2.4.1 Fehling’s Test............................................................................................26

Figure 4.2.4.1 Activation of Yeast....................................................................................27

Figure 4.2.5.1 Fermentation..............................................................................................28

Figure 4.2.6.1 Filtration of Samples.................................................................................28

Figure 5.9.1 Comparison of kg from various types of feed stock to produce 100 L of

ethanol..............................................................................................................................34
 1

Chapter 1

INTRODUCTION

1.1 Background of the Study

Global demand for energy continues to grow due to rapidly expanding human

population and increase of the industrial prosperity in developing countries. The major

energy demand is still supplied from conventional fossil fuels such as oil, coal and natural

gas. Utilization of fossil fuels over the last century and following years has drastically

increased the level of greenhouse gasses in the earth’s atmosphere. These facts along with

inevitable depletion of the world’s energy supply, and unstable oil market have renewed

the interest of society in searching for alternative fuels (Ballesteros et al., 2006). Ethanol

has long been considered as a suitable alternative to fossil fuels either as a sole fuel in cars

with dedicated engines or as an additive in fuel blends with no engine modification

requirement when mixed up to 30%. Today, bioethanol is the most dominant biofuel and

its global production showed an upward trend over the last 25 years with a sharp increase

from 2000 (Licht, 2006).

Sugar and starch based materials such as sugarcane and grains are two groups of raw

materials currently used as the main resources for ethanol production. The third group is

lignocellulosic materials representing the most viable option for production of ethanol.

Growing demand for human food, as it is for energy, and considering the priority for

starving human society could make the first two groups of raw materials potentially less

competitive and perhaps expensive feedstocks in the near future compared to

lignocellulosic materials. Cellulosic ethanol offers promise because cellulose fibers, a

major and universal component in plant cells walls, can be used to produce ethanol
 2

(Taherzadeh and Karimi, 2007). According to the International Energy Agency (IEA),

cellulosic ethanol could allow ethanol fuels to play a much bigger role in the future.

According to Sugar Regulatory Administration SRA of the Philippines (2017), it is

said that the country’s total bioethanol output on the said year is only 280 million liters,

lower than the mandated 570 million liters bioethanol required by the Department of

Energy (DOE). DOE requires high amount of ethanol also because of the Biofuels Act of

2006, stating that all liquid fuels for motors and engines sold in the Philippines should

contain locally sourced biofuel components. Oil companies were required to sell gasoline

with at least 10 percent ethanol blend. Oil companies, however, find it difficult to comply

with the provisions of the law due to the lack of locally produced ethanol.

Lignocellulosic materials obtained from grasses, energy crops, wood and agricultural

residues, represent the most abundant global source of renewable biomass (Lin and Tanaka,

2006). Carabao grass, with the scientific name Paspalum Conjugatum is a spreading

perennial grass which it grows in many types of soil. It is usually found abundantly in

Southeast Asia (Sauerborn et.al, 1988).

According to the Alternative Fuels Data Center (2018), regardless of from which ethanol

is produced, its chemical formula never alters. Due to this case, the researchers of this study

will use carabao grass as ethanol feedstock.

1.2 Statement of the Problem

To aid the possible future increase in ethanol blending in petrol, the Philippines should

utilize another alternative feedstock to continue the production of the ethanol needed to be

added to gasoline.
 3

1. Will Carabao grass fermented with Saccharomyces cerevisiae be capable of

producing ethanol?

2. What will be the effect of fermentation time to the ethanol yield?

3. Will Carabao grass be viable alternative source of bioethanol compared to other

feedstocks?

1.3 General and Specific objectives of the Study

The research has a general objectives of determining the potential of carabao grass to

produce bioethanol. The specific objective of this study is to determine the amount of

volume of ethanol produced per unit mass of carabao grass.

1.4 Scope and Delimitation of the Study

The study will focus on the ethanol production from carabao grass, determination of the

volume of ethanol produced, the quality of the ethanol produced is not intented to be

determined. The Gas Chromatography Test will be conducted by the University of the

Philippines Los Banos Biotech (UPLB).

The research methodology for this ethanol production was delimited to the availability

of equipments and facilities of Palawan State University laboratories. Moreover, the

students expertise, financial aspects and time allocation.

1 .5 Significance of the Study

The study aspires to contribute to research directed at exploring potential sources of

bioethanol. This study will be beneficial for ethanol production in the country as an

alternative source of ethanol. It might increase the value of carabao grass to have more use

to the society and provides economic growth and additional income in the society especially
 4

on rural areas. Furthermore, the results of the study would be beneficial to other researchers

who will conduct studies on extracting ethanol from other materials.

1.6 Assumptions of the Study

Since there are two types of pretreatment hydrolysis for breaking down sugars used for

fermentation, namely acid hydrolysis and enzymatic hydrolysis (Woiciechowski, Nitsche,

Pandey, & Soccol, 2002), the researchers will be using acid hydrolysis as it is the cheaper

alternative, assuming that the results will be the same if this study used the more expensive

alternative, enzymatic hydrolysis.

 5

Chapter 2

REVIEW OF RELATED LITERATURE

2.1 R.A. 9367 – Biofuels Act of 2006

As indicated by R.A. 9367, Section 1, this demonstration ought to be known as the

"Biofuels Act of 2006". It is stated that the dependence on imported fuels should be reduced

for the safety of public health, the environment and natural ecosystems, in line with the

sustainable economic growth of the country, which would increase livelihood

opportunities. The yearly complete volume of fuel sold and circulated by oil organizations

in the nation will contain no less than five percent (5%) ethanol in which all privately

conveyed diesel and gas is commanded to a mix of 2% in biodiesel and 10% in ethanol

separately.

According to the Biofuels Annual Report, compliance with the current mandated 10

percent ethanol-gasoline blend is challenging due to the inadequacy of capacity and

competitiveness of existing sugarcane distillers. In addition, imported ethanol is expected

to meet the gap between local production and mandated blend requirements due to trade

liberalization commitments under the ASEAN Economic Community (AEC). Based on

DOE reports, the 10 percent blend will be increased to 20 percent by 2020. According to

the rough estimate of DOE, 15 additional bioethanol plants will be needed to meet this

ambitious target for domestic supplies. (Corpuz, 2017)

2.2 Ethanol

Ethanol, also commonly referred to as ethyl alcohol, pure alcohol, grain alcohol,

and drinking alcohol, is most known as the alcohol present in alcoholic beverages. Ethanol,
 6

which can also be abbreviated as EtOH, is a colorless liquid with a slight odor, and it is

soluble in water. It is flammable and volatile, so it evaporates easily when left in an open

container. Ethanol's chemical formula is C2H6O. This chemical formula can also be written

as CH3CH2OH or C2H5OH. It is made of nine atoms that include two carbon (C) atoms, six

hydrogen (H) atoms, and one oxygen (O) atom (Garcia N, 2018).

2.3 Ethanol Production in the Philippines

Ethanol fermentation is one of the oldest and most important fermentation processes

used in the biotechnology industry. Many microorganisms, including bacteria and yeasts,

can produce ethanol as the major fermentation product from carbohydrates (Frigon&Guiot,

2010).

In the Philippines, according to the Department of Energy DOE there are ten accredited

bioethanol producers in the country which amounts to a total of 282.12 million liters per

year as of 2017. The country’s ethanol production on 2018 could increase by 8.3 percent to

325 million liters from the estimated 300 ML on 2017, according to a Global Agricultural

Information Network (Gain) report. The Gain report projected the Philippine ethanol

production on year 2018 would increase by 2.19 percent to 280 ML, from the expected

output of 274 ML on 2017.

2.4 Sources of ethanol

Ethanol can be produced from the fermentation of carbohydrates in plant, there

are three main types of biomass raw materials for ethanol production: a) sugar bearing

materials or saccharine plants such as sugarcane, molasses, sorghum, etc. which contain

carbohydrates in sugar form; b) starches such as cassava, corn, sweet potatoes, etc.; and c)

cellulose such as wood, agricultural residues, etc. for which the carbohydrates molecular

form is more complex (DENR, 1990-1993).

 7

Table 2.4.1. List of major potential biomass materials for ethanol production in the
Philippines and their expected yields (Zayco and Rosario, 1980)

Yield Biomass and Ethanol for Some Agricultural Crops

Crop Cycle Yield of Fresh Crops Ethanol Yield
Crop
(days) t/ha/season t/ha/yr li/ton li/ha/yr
Saccharine Sources
Sugarcane 360 50 – 100 50 – 100 67 3350 – 6700
Nipa sap 252 83 21000
Coconut sap 60 83 5000
Sorghum 120 30 60 80 4800
Starchy Sources
Cassava 300 15 – 40 18 – 48 180 3240 – 8640
Sweet Potato 100 15 – 40 54 – 144 125 6750 –
18000
Corn (maize) 110 1.0 – 5 3.3 – 16 400 1320 – 6400
Rice 120 1.8 – 6 5.4 – 18 420 2270 – 7560
Pineapple 104 11568

Production of alcohol from sugarcane, corn, nipa sap, rice straw, cassava, sweet

potato, sugarcane bagasse, molasses, sapal, sweet sorghum, and even from banana peelings

and guava, etc. was studied here in the Philippines and found that only sugarcane, cassava,

and sweet potato were found to be attractive raw materials for ethanol fuel production

among all root crops (Villanueva, 1980).

Another table that is also showing the list of sources of ethanol as an additive in

gasoline, and the amount of feed it takes to produce 100 liters of ethanol is listed below

(Piyachomkwan, 2012).

Table 2.4.2 Required kg from various feedstocks to produce 100 liters of ethanol

To produce 100 liters of ethanol it takes… Kg

of cheese whey 4,000 kg
of sweet sorghum
1,400 kg
of sugarcane 1,270 kg
of Jerusalem artichoke 1,250 kg
of sugar beet 1,030 kg
of potatoes 850 kg
 8

of cassava 545 kg
of wood 385 kg
of molasses 360 kg
of maize (wet milling) 368 kg
of maize (dry milling) 258 kg
of wheat 260 kg
of millet 230 kg
of paddy of rice 225 kg

2.5 Making of ethanol

Ethanol, or ethyl alcohol, is a chemical that is volatile, colorless, and flammable. It can be

produced from petroleum via chemical transformation of ethylene, but it can also be

produced by fermentation of glucose, using yeast or other microorganisms; current fuel

ethanol plants make ethanol via fermentation (Carey, 2019).

The basic formula for making ethanol from sugar glucose is as follows:

C6H12O6 =Fermentation=> 2(CH3CH2OH) + 2(CO2) + Energy (which is stored in ATP)

Sugar (Glucose) => Alcohol (Ethyl alcohol) + Carbon dioxide gas + Energy

Fermentation produces only 12-15% alcohol in the solution because any higher

concentration is toxic to yeast cells. In order to raise the ethanol content to 95%, the solution

must be distilled. However, this method is expensive in making ethanol.

2.6 Carabao Grass as an Alternative Bioethanol Feedstock

Due to the rapid population growth and industrialization in the country, there has been

an gradually increase in the fossil fuel consumption and expected to rise continuously for

the next years (Corpuz, 2017). According to the Global Agricultural Information Network

(GAIN) report, the country’s ethanol imports on year 2018 has increased by 8.3 %

compared to the year 2017 but despite of increasing locally produced ethanol, it would still

not meet the demand for bioethanol in the country as per the Department of Energy (DOE)
 9

who mandated 570 million liters on 2018 but the country only produced approximately 300

million liters on the said year.

2.7 Raw Material

2.7.1 Characteristic of Lignocellulosic Biomass

Lignocellulosic materials including agricultural wastes, forestry residues, grasses

and woody materials have great potential for bio-fuel production. Typically, most of the

agricultural lignocellulosic biomass is comprised of about 10% - 25% lignin, 20% - 30%

hemicellulose, and 40% - 50% cellulose (Iqbal et.al, 2011). Cellulose is a major structural

component of plant cell walls, which is responsible for mechanical strength and chemical

stability to plants. While, hemi-cellulose macromolecules are often repeated polymers of

pentoses, and hexoses. Due to the genetic variability among different sources hemicellulose

macromolecules are also vary in structural composition. Lignin contains three aromatic

alcohols (coniferyl alcohol, sinapyl alcohol and p-coumaryl alcohol) produced through a

biosynthetic process and forms a protective seal around the other two components i.e.,

cellulose and hemicelluloses (Jiang et.al, 2010).

Plant biomass contains 40% to 50% of cellulose molecules which are fibrous in nature,

insoluble, crystalline polysaccharide. Being the most abundant and easily available

carbohydrate polymer all around the earth which is a major polysaccharide constituent of

plant cell wall (Himmel et.al, 2007). The second most abundant polymer after cellulose is

hemicellulose which is heterogeneously branched in nature. The backbone of the

hemicellulose polymer is built up by sugar monomers like xylans, mannans and glucans,

with xylans and mannans being the most common (Wyman et.al, 2005). Lignin is generally

the most complex and smallest fraction, representing about 10% to 25% of the biomass. It

has a long-chain, aromatic polymer composed largely of phenyl propane units. Lignin acts
 10

like a glue by filling the gap between and around the cellulose and hemicellulose

complexion with the polymers. It is present in almost all kind of cellulosic plant biomass

and acts as a protective sheet against cellulosic and hemicellulosic components of the

biomass materials. Lignin is consists of multifarious and large polymer of phenyl-propane,

methoxy groups and non-carbohydrate poly phenolic substance, which bind cell walls

constituent together (Hamelinck et.al, 2005).

2.7.2 Carabao Grass

According to the study entitled “An investigation of the germination of six tropical

arable weeds” by Sauerborn et.al, 1988 it is defined and descibe that carabao grass, with

the scientific name “Paspalum Conjugatum” is a spreading perennial grass which it grows

in many types of soil. It is usually found abundantly in Southeast Asia. By botanical

observation, these grass has a deep green blades, up to 20 cm long and 1 cm wide. Leaves

are narrow lanceolate, flat and thin, glabrous, 8 to 20 centimeters long, 5 to 15 millimeters

wide. Spikes are two, terminal, slender and 6 to 12 centimeters long. It grows in open waste

places, along trails and streams. It is considered as weed but sometimes It is usually used

for pasturing herbivore animals and suitable for lawn grass. According to the Livestock

Resource Center Palawan (2018), carabao grass is the 2nd most common grass in Palawan

next to paragrass.
 11

2.7.3 Contents of Carabao Grass

2.7.3.1 Table of chemical composition and nutritional value of Carabao grass (Heuzéet et

al., 2016.)

Crude fibre is mainly refers to one type of dietary fibre, it is primarily consist of cellulose

and lignins. The crude fiber residue contains no soluble fiber and retains only about 15%

of the total hemicelluloses, 50–100% cellulose, and 10–50% lignin. (Mongeau&Brooks,

2003)

Acid Detergent Fiber (ADF) is the residue of the raw plant material is contacted with dilute

acid and washed with solvents to remove starch, pectin, hemicellulose, fats, oils, protein,

free sugars, and soluble minerals. It contain cellulose, lignins, and insoluble minerals

(Holtzapple, 2003).

Neutral detergent Fiber (NDF) is fraction of the cell walls, considered to be roughly

equivalent to hemicellulose, true cellulose and lignin. The difference between NDF and

ADF is a measure of hemicellulose content (Holtzapple, 2003).

 12

2.7.4 Contents of other Bioethanol Grass Feedstock

2.7.4.1 Elephant Grass

Napier grass (Pennisetum purpureum) has been recognized to meet the

requirement of lignocellulosic bioethanol production, because it has low lignin-content

and a relatively high herbage mass per year and per area. Therefore, pretreatment,

saccharification, and fermentation processes for ethanol production from Napier grass

have been extensively studied. The ethanol yield reached 74.1%. Thus, Napier grass

was thought to be a promising biomass for ethanol production (Yasuda et al., 2015).

Table 2.7.4.1.1 Contents of Elephant Grass

2.7.4.2 Cogon Grass

Imperata Cylindrical or cogon grass is one of the abundant low-value by-products of

agriculture waste in the world. The research had provided a discussion of how this non-

valuable agriculture waste can be managed to become an alternative bioresource for

bioethanol production. The highest ethanol yield was obtained with the Imperata cylindrical

pretreated with sulphuric acid was 45.42 % (Wong & Lim, 2017).
 13

Table 2.7.4.2.1 Contents of Cogon Grass

2.7.4.3 Purple Guinea Grass

Purple guinea grass (Megathyrsus maximus) is one of the popular forage plants grown

in Thailand that gives a very high yield (9.4 to 25.0 tons /hectare) for 10 years or longer, is

easy to harvest, and self-regenerates after harvesting. In addition, purple guinea grass has

a low agrochemical consumption and requires less intensive agricultural management. The

high ethanol production yield (96% of theoretical), including a high cellulose content

(41.7% w/w), along with the high productivity, ease of harvesting and self-regeneration

after harvesting, a low agrochemical consumption, and low agricultural management

requirement, means that purple guinea grass, currently farmed as a forage crop, is also a

promising herbaceous energy crop (Ratsamee et al., 2012).

Table 2.7.4.3.1 Contents of Purple Guinea Grass

 14

2.7.5 Comparison of other Bioethanol Grass Feedstock to Carabao Grass

Feedstock Crude NDF ADF Lignin

fiber (% DM) (%DM) (%DM)

(%DM)

Carabao 30.2 68.1 35.6 4.4

grass

Elephant 35.6 71.1 41.9 5.8

grass

Cogon Grass 39.4 73.3 45.7 6.6

Purple 37.3 72.3 43.4 6.1

guinea grass

Table 2.7.5.1 Comparison of Contents

Crude fibre is mainly refers to one type of dietary fibre, it is primarily consist of cellulose

and lignins. The crude fiber residue contains no soluble fiber and retains only about 15%

of the total hemicelluloses, 50–100% cellulose, and 10–50% lignin. (Mongeau&Brooks,

2003)

Acid Detergent Fiber (ADF) is the residue of the raw plant material is contacted with dilute

acid and washed with solvents to remove starch, pectin, hemicellulose, fats, oils, protein,

free sugars, and soluble minerals. It contain cellulose, lignins, and insoluble minerals

(Holtzapple, 2003).

Neutral detergent Fiber (NDF) is fraction of the cell walls, considered to be roughly

equivalent to hemicellulose, true cellulose and lignin. The difference between NDF and

ADF is a measure of hemicellulose content (Holtzapple, 2003).

 15

2.8 Pretreatment

Particle size reduction increases specific surface area and reduces crystallinity to

improve accessibility of biomass to chemical and biological catalyst. It can take the form

of knife milling, hammer milling or ball or wet disk milling. Particle size reduction to pieces

0.5 - 1 cm in length would be ideal for achieving relatively high yield of ethanol (bohdan

et, al).

2.9 Acid Hydrolysis

Acid hydrolysis is a process used to convert lignocellulosic materials into a simple sugar.

The process is carried out by addition of water and little amount of oxidixing acid like HCl

or dilute H2SO4. The acid acts as a catalyst by providing H+ ions to facilitate the intake of

H2O molecules. It can be hydrolysed to the respective acid and alcohol by boiling with

water containing a little HCl or sulphuric acid (Kumaraswami, 2015).

2.10 Fehling Test

Fehling's solution is a chemical reagent used to test for reducing sugars and non-reducing

sugars (Prem Prakash et al., 2016). Fehling's solution is prepared by combining two

separate solutions, known as Fehling's A and Fehling's B. Fehling's A is aqueous Copper

sulphate solution, which is deep blue. Fehling's B is a colorless solution of

aqueous potassium sodium tartrate (also known as Rochelle salt) made in a strong alkali,

commonly with sodium hydroxide. The Fehling’s reagent is prepared fresh by mixing equal

volumes of solution A and B. A blue colored resulting solution means that the test was

negative and red-brown colored confirms that there was a glucose content

(PremPrakash&Neelu., 2017).
 16

2.11 Yeast

Bioethanol has been identified as the mostly used biofuel worldwide since it

significantly contributes to the reduction of crude oil consumption and environmental

pollution. It can be produced from various types of feedstocks such as sucrose, starch,

lignocellulosic and algal biomass through fermentation process by microorganisms.

Compared to other types of microoganisms, yeasts especially Saccharomyces cerevisiae is

the common microbes employed in ethanol production due to its high ethanol productivity,

high ethanol tolerance and ability of fermenting wide range of sugars. Yeasts can directly

ferment simple sugars into ethanol while other type of feedstocks must be converted to

fermentable sugars before it can be fermented to ethanol. The common processes involves

in ethanol production are pretreatment, hydrolysis and fermentation. Production of

bioethanol during fermentation depends on several factors such as temperature, sugar

concentration, pH, fermentation time, agitation rate, and inoculum size (Mohd Azhar et, al.

2017).

2.12 Fermentation

Ethanol fermentation is the anaerobic pathway carried out by yeasts in which simple

sugars are converted to ethanol and carbon dioxide. The basic equation for alcohol

fermentation shows that yeast starts with glucose, a type of sugar, and finishes with carbon

dioxide and ethanol. The process of alcohol fermentation can be divided into two parts. In

the first part, the yeast breaks down glucose to form 2 pyruvate molecules. This part is

known as glycolysis. In the second part, the 2 pyruvate molecules are converted into 2

carbon dioxide molecules and 2 molecules of ethanol, otherwise known as alcohol. The

main purpose of alcohol fermentation is to produce Adenosine Triphosphate (ATP), the

energy currency for cells, under anaerobic conditions. So from the yeast's perspective, the
 17

carbon dioxide and ethanol are waste products.(Campbell et al., 2017) . In the study Bio-

Conversion of Sweet Potato Peel Waste to BioEthanol Using Saccharomyces Cerevisiae

by Elizabeth et al. (2018), The yield of ethanol from sweet potato peel waste was increasing

with time for but after the maximum production of ethanol the yield of ethanol began to

decline. In another study Effect of Time on the Fermentation and Storage of Candanasava

by Muzaffer et al. (2011), it showed that the prolonged incubation did not increase ethanol

yield but resulted to a loss of yield after the peak of ethanol production.

This is one parameter for determining the acid level of ethanol in process of

alcohol fermentation. According to Jean Sloat Morton, PhD on her article “Glycolysis and

alcoholic fermentation” the best way to extract ethanol from carbohydrates is when it is

acidic. The pH values of ethanol produced by the process of fermentation ranges from 4 to

6. Yeast survives in a slightly acidic environment with pH ranges from 4 to 6.

2.14 Gas Chromatography Test

Gas chromatography (GC) is a common type of chromatography used in analytical

chemistry for separating and analyzing compounds that can be vaporized without

decomposition. Typical uses of GC include testing the purity of a particular substance, or

separating the different components of a mixture. In some situations, GC may help in

identifying a compound with its respective quantity in the solution. In preparative

chromatography, GC can be used to prepare pure compounds from a mixture (Pavia, 2005).
 18

2.15 Related Studies

A study entitled “Bioethanol Production from Elephant Grass (Pennisetum

Purpureum)” by Stanley et, al. uses physical preatreatment which reduced the size of the

grass into 0.5-1cm, then subjected to acid hydrolysis using hydrochloric acid to convert

lignocellulose into simple reducing sugars, then the hydrolized solution was subjected to

Fehlings test in order to confirm the presence of fermentable sugars. Once the presence of

fermentable sugar is confirmed it was fermented via Saccharomyces cerevisiae yeast.

A study conducted by Gaddafi et, al. titled “Bioethanol Production from Banana Peels”

also employs the same method which reduces the size of the peels before conducting acid

hydrolysis using hydrochloric acid to convert polysaccharides sugar molecules into

monosaccharides (simple) sugar molecules which is ready for fermentation then subjected

to Benidicts test to confirm the presence of sugar. Fermentation was also done via

Saccharomyces cerevisiae yeast to convert the monosaccharides and some disaccharides

produced from hydrolysis into ethanol.

The researchers opted for the variation of methods used in the study of Stanley et.al, and

Gaddafi et.al, since it was a proven method that was able to produce positive and reliable

results in terms of measuring the potential of organic sources as ethanol feedstocks.

 19

Chapter 3
CONCEPTUAL FRAMEWORK

3.1 Conceptual Framework

A literature review from the previous chapter was presented to familiarize the

researchers as well as the readers with the content and the concept related to this

study. To better understand the objectives and the methods used in this study, a

conceptual framework is shown below.

Independent
Dependent
Variables
Variables
 Amount of feed 
Ethanol Produced
Ethanol Produced

 ph 
Sample A (unripe)
Fermentation days
Sample B (ripe)
 Sugar Content

Figure 3.1.1 Independent and Dependent Variables

 20

Preparation of
sample

Acid
Hydrolysis QUANTITATIVE
TEST
FOR THE
PRESENCE

Fehlings Test OF ETHANOL AT

BIOTECH, U.P LOS
BAÑOS GAS
CHROMATOGRAPH
Y
Fermentation

Filtration

Ethanol

Figure 3.1.2 Schematic Diagram of Bioconversion of Carabao Grass to Ethanol

 Preparation of Sample - The Carabao grass are gathered at Livestock

Resource Center at Barangay Irawan. The gathered grass are then washed
 21

and dried, then it was cut to approximately 0.5-1 cm and weighed 100g per

sample.

 Acid Hydrolysis - Acid hydrolysis is a process used to convert

lignocellulosic materials into a simple and fermentable sugars.

 Fehling’s Test - Test for the presence of fermentable sugars

 Fermentation – Fermentation is a process of converting the fermentable

sugars into ethanol at anaerobic condition.

 Filtration – A process used to remove solid particles from the solution.

 Gas Chromatography Test – A test for determining the presence of ethanol

and its quantity.

3.2 Definition of Terms

Acid Hydrolysis - the chemical process in which a 3 M Hydrochloric acid and 3 M

NaOH base is used to convert lignocellulose to simple sugar.

Active Dry Yeast - (Saccharomyces Cerevisiae) yeast used for fermentation process.

Ethanol - the end product of the study derived from the fermentation of glucose

from Carabao Grass.

Fehling’s Test - test done to determine the presence of reducing sugar that contains

numerous units of glucose. A positive result will show a formation of red precipitate

upon the addition of Fehling’s solution.

Filtration - physical method of separating liquid fraction of the slurry.

Glucose - refers to the fermentable sugar that will produce ethanol.

 22

Fermentation - refers to a biological process in which glucose (sugar) is converted

into ethanol.

pH – a figure expressing the acidity or alkalinity of the solution.

 23

Chapter 4

METHODOLOGY

The methods employed in this study were taken from the variations of the methodology

of the studies entitled “Bioethanol Production from Elephant Grass (Pennisetum

Purpureum)” by Stanley et, al. and “Bioethanol Production from Banana Peels” by Gaddafi

et, al.

4.1 Material, Tools and Equipment Used

4.1.1 Laboratory Apparatus
Table 4.1.1.1 Laboratory Apparatus
APPARATUS QUANTITY, pcs UNIT SIZE

Weighing Scale 1 Kilogram 30

Beaker 1 Milliliters 500

Test Tube 3 Milliliters

Thermometer 1 Degree Celcius 110

Pipet 1 Milliliters 5

Aspirator 1

Test Tube Stand 1

Filter Paper 1

pH Meter 1 pH 14

4.1.2 Reagents
Table 4.1.2.1 Reagents

Process Regeants Unit Amount

Acid Hydrolysis HCl (29.1%) Liters 1

Naoh (99.99%) Kilograms 1

 24

Fehling’s Test Fehling’s A Milliliters 4.5

Fehling’s B Milliliters 4.5

Fermentation Active Dry Yeast Milligrams 270

4.1.3 Materials
Table 4.1.3.1 Materials

MATERIALS QUANTITY, pcs UNIT SIZE

Garbage Bag 9

(transparent)

Knife 1

Scissors 3

Chopping Board 1

1.5 Liters Plastic 9 Liters 1.5

Bottles

4 Liters Plastic 9 Liters 4

Bottles

Distilled Water Liters 40

Sugar Tbsp. 9

Reagent Bottles 2 Milliliters 500

Funnel 1

Cloth 1

Rubber Band 9

Steel Container 2
 25

4.2 Experimental Procedure

4.2.1 Preparation of Sample

 Weighing of Sample

 Cutting

 Washing

Figure 4.2.1.1 Preparation of sample

The preparation of sample is done in order to know the exact volume and resize the

sample that will have used in the process.

1 Collected Carabao Grass

2 Gathered carabao grass were washed.

3 The grasses were cut into smaller size (approximately 0.5cm - 1cm)

4 Weighed the grass (100g/sample).

4.2.2 Dilute Acid Hydrolysis

Figure 4.2.2.1 Dilute Acid Hydrolysis

 26

1. 3M of HCl and 3M of NaOH. The 3M of HCl was prepared l by mixing 320 ml of

muriatic acid (29.1% concentration) into 680 ml of distilled water. And the 3M of

NaOH was prepared by mixing 120g of caustic soda into 1L of distilled water

(exothermic reaction) and left to cool to room temperature.

2. Mixed 750 ml of the 3M HCl and NaOH into a steel container (exothermic

reaction).

3. Poured and mixed the shredded carabao grass into the steel container and left it for

30 minutes mixing it every 5 minutes to remove the build-up of bubbles.

3. The mixture is then hydrolysed to 60-100°C for 10 minutes and mixed if there is a

build-up of bubbles.

4. The mixture is then cooled to room temperature and adjusted the pH level to 4-6.

4.2.3 Fehling’s Test

This test is done in order to confirm the presence of glucose in the solution. The reduction

of the deep blue solution to a red precipitate indicated the presence of glucose.

Figure 4.2.4.1 Fehling’s Test

 27

1. Mix the 0.5 mL of Fehling’s solution A with 0.5 ml of Fehling’s solution B in the

beaker.

2. Keep the solution in a sterilized container.

3. This mixture was added to an empty test tube.

4. 3 drops of the hydrolysed sample was added to the test tube.

5. The solution was placed in a water-bath at 60℃.

6. The changes in color to red-brown precipitate means that the sugar reducing

components are present and are ready to be fermented.

4.2.4 Activation of yeast

Figure 4.2.4.1 Activation of Yeast

1. Warmed distilled water (not exceeding 40°C) in a different steel container

2. Weighted 30g of yeast and then poured the yeast into 300ml of warm distilled water.

3. A pinch (1/8tsp) of white sugar was added into the solution to feed the bacteria for

easier activation and then mixed the solution.

 28

4. The steel container is the covered and left for 20 minutes for the yeast to activate.

4.2.5 Fermentation

Figure 4.2.5.1 Fermentation

1. The yeast is then mixed into the sample.

2. The sample is then transferred into a sterilized 4L container.

3. The sample is left open for 24 hours due to the pressure build up at early stage

3. Seal the container’s opening with plastic, tie it with a rubber band to create an

anaerobic condition.

4. Will then be fermented for 3 different fermentation time: 4, 8 and 12 days

respectively.

4.2.6 Filtration

Figure 4.2.6.1 Filtration of Sample

 29

The fermented sample will be filtered to filter the accumulated solids, yeast

and foams and placed into 1.5L containers to be sent for gas chromatography.

4.2.7 Gas Chromatography

The sample was sent to UPLB Biotech for the gas chromatography test to

determine the amount of ethanol produced.

 30

Chapter 5
RESULTS AND ANALYSIS

This chapter includes the results of all the laboratory procedures and experiments.
Discussions are arranged base on the order of the methodology used in this study.

5.1 Preparation of Sample

The Carabao grasses were collected from the compound of Livestock Resource
Center in Irawan, Puerto Princesa, Palawan. 300-400g of carabao grass (stem and leaves)
was collected before starting the experiment for each fermentation days so that the samples
are fresh. The grasses were cut into smaller sizes manually using scissors to approximately
0.5 to 1cm in length to increase the surface area of the grasses. After cutting 300g of carabao
grass was weighted used for the experiment for each sample.

5.2 Acid Hydrolysis

Acid hydrolysis was done to convert glucose within the sample into reducing sugar
to speed up the fermentation process and help increase the expected yield of ethanol.

The carabao 300g were divided into 3 samples weighing 100g each. After the
process the pH level was measured using a pH meter. At the end of the acid hydrolysis,
sample 12D-A, 12D-B, and 12D-C had a pH level of 2.53, 2.26, 2.04, sample 8D-A, 8D-
B, 8D-C had a pH level of 2.83, 2.05, and 2.34, and sample 4D-A, 4D-B, and 4D-C had a
pH level of 2.53, 2.97, and 2.45 respectively. The 3M of NaOH solution was slowly added,
constantly measuring the pH level until the acidity reached 4-6pH.

5.3 Fehling’s Test

Fehling’s test was done to confirm the effectiveness of acid hydrolysis. It was
conducted to ensure the presence of reducing sugar.
 31

Table 5.3.1 Results of Fehling’s Test of Samples

Samples Fehlings test Result

12D-A Red Precipitate

12D-B Red Precipitate
12D-C Red Precipitate
8D-A Red Precipitate
8D-B Red Precipitate
8D-C Red Precipitate
4D-A Red Precipitate
4D-B Red Precipitate
4D-C Red Precipitate
From the results presented in Table 5.3.1, all samples have presence of reducing
sugars so the samples can proceed to fermentation.

5.4 Yeast Activation

The yeast (30mg) was added to 300mL of warm water (40°C) then added a pinch
of sugar (about 1/8 tbsp) and left for 20 minutes to activate. Foam accured after 20 minutes
which signify that the yeast was activated.

5.5 Fermentation

To start the fermentation process, 300 mL of activated yeast was added to each of
the solution. The solution was kept on sterilized vessels and kept uncovered for 24 hours at
normal room temperature. After the first 24 hours, tall foam occurred signifying that the
yeast is alive. Sample 12D was fermented 4 days before sample 8D and sample 8D was
fermented 4 days before sample 4D .
 32

Table 5.5.1 Duration of Fermentation

Sample Days of Fermentation (Days)

12D 12
8D 8
4D 4

5.6 Filtration

The sample is filtered to filter out the grass, yeast, and foam. After filtration we
measured 1L of each sample and placed it into a 1.5L sterilized plastic container to be sent
for gas chromatography.

5.7 Gas Chromatography

A total of 9 L of sample, 3L for Sample 12D, 3L of Sample 8D and another 3L of

Sample 4D, were sent to UPLB Biotech.

Table 5.7.1 Result of Gas Chromatography for Samples 12D

Sample (12D) Total Volume % Ethanol (v/v)

12D-A 1L 0.54
12D-B 1L 0.55
12D-C 1L 0.35
Average 0.48

Table 5.7.2 Result of Gas Chromatography for Samples 8D

Sample (8D) Total Volume % Ethanol (v/v)

8D-A 1L 1.07
8D-B 1L 1.26
8D-C 1L 0.57
Average 0.97
 33

Table 5.7.3 Result of Gas Chromatography for Samples 4D

Sample (4D) Total Volume % Ethanol (v/v)
4D-A 1L 0.69
4D-B 1L 0.37
4D-C 1L 0.43
Average 0.50

The amount of sample that has undergone Gas Chromatography is 1 L for each
sample. It is shown in the Table 5.7 the ethanol content for Sample 12D is 0.48% (v/v)
which resulted in an ethanol yield of 0.0048L of ethanol from 1L of Sample 12D, the
ethanol content for Sample 8D is 0.97% (v/v) which resulted in an ethanol yield of 0.0097L
of ethanol from 1L of Sample 8D, and the ethanol content for Sample 4D is 0.50% (v/v)
which resulted in an ethanol yield of 0.0050L of ethanol from 1L of Sample 4D.

The maximum alcohol production (0.97%) was reached at 8 days of fermentation;

this is due to low sugar content which results in shorter fermentation time for the yeast to
consume the sugar present in the sample. With the progress of time beyond 8 days there
was a loss in ethanol yield as shown in the sample for 12 days in fermentation

5.8 Ethanol Yield from Carabao Grass

Based on the result of this study, 100g of carabao grass produced 0.0048L of ethanol
for sample 12D, 0.0097L for sample 8D and 0.0050L for sample 4D thus needing
2083.33kg, 1030.92kg, and 2000kg of carabao grass produce 100L of ethanol for Sample
12D, 8D, and 4D respectively.
 34

Cheese Whey 4000

Carabao Grass (12 days) 2083.33
Carabao Grass (4 days) 2000
Sweet Sorghum 1400
Sugar Cane 1270
Jerusalem Artichoke 1250
Carabao Grass (8 days) 1030.92
Sugar Beet 1030
Crop

Potatoes 850
Cassava 545
Wood 385
Molasses 360
Maiz(wet) 368
Maiz(Dry) 258
Wheat 260
Millet 230
Paddy Rice 225

Mass (kg)

Figure 5.8.1 Comparison of kg from various types of feed stock to produce 100 L of ethanol

The data shown in Figure 5.8.1 the mass of each sample is used for the comparison
to different various feed stock to produce 100 L. The Sample with the most amount of yield
requires less mass to produce the same amount of alcohol than sugar cane which is the most
commonly used ethanol feedstock which shows the viability of carabao grass as a
feedstock.
 35

Chapter 6

SUMMARY, CONCLUSSION AND RECOMMENDATIONS

6.1. Summary

The researchers tested the ethanol potential of carabao grass (Paspalum Conjugatum)
and its ethanol yield base on our produced sample. Carabao grass (Paspalum conjugatum)
has identified present of reducing sugar base on the Fehling test we had performed, it
indicates a red precipitate to a test sample solution, which has a potential to produce an
ethanol. The fermented solution shown bubbles which an indication of reaction of yeast to
the solution and fermentation process of the solution. The three variation of fermented
samples 4-days, 8-days, 12-days had undergone of Gas Chromatography test at
BIOTECH, University of the Philippines Los Baños, which contains an amount of 1L for
each sample and has an average result of 0.50% v/v, 0.97% v/v, 0.48% v/v respectively
and yields 0.005 L, 0.0097 L, 0.0048 L of ethyl alcohol respectively.. The maximum
alcohol production (0.97%) was reached at 8 days of fermentation; this is due to low
sugar content which results in shorter fermentation time for the yeast to consume the
sugar present in the sample. With the progress of time beyond 8 days there was a loss in
ethanol yield as shown in the sample for 12 days in fermentation. As in the study Effect
of Time on the Fermentation and Storage of Candanasava by Muzaffer et al. (2011). The
prolonged incubation did not increase ethanol yield but resulted to a loss of yield after the
peak of ethanol production. Based on the ethanol yield from carabao grass 100g of
carabao grass produced 0.0048L of ethanol for sample 12days, 0.0097L for sample 8days
and 0.0050L for sample 4days thus, it requires 2083.33kg, 1030kg, and 2000kg of
carabao grass to produce 100L of ethanol for Sample 12days, 8days, and 4days
respectively.
 36

6.2 Conclusion

Through the methods employed, the researchers successfully extracted ethanol

from carabao grass. Thus, Carabao grass that is fermented with Saccharomyces cerevisiae
is proven capable to produce ethanol.
The peak of the fermentation time in this study is at 8D, then after that time period
the amount of ethanol yield will start to decline.
Based on the results, carabao grass will be able to produce 100L of ethanol with
only 1030kg of grass. Comparing to other feedstocks, we can say that carabao grass is
viable to be an alternative source of bioethanol.

6.3 Recommendations
The following recommendations were formulated from the results of this study:

 Use distillation process and consider the use of repetitive distillation method or send
a sample to a distilling laboratory for more accurate ethanol yield result.

 Consider the use of quality test to classify and determine the samples’ ethanol grade;

 Combine acid hydrolysis and enzymatic hydrolysis that can be used when
performing the extraction of ethanol from Carabao grass. This would maximize the
extraction of reducing sugar from cellulose that may improve ethanol content of the
sample;

 Incorporate economic feasibility study or cost-benefit analysis in the entire study.

 37

BIBLIOGRAPHY

Amin, F. R., Khalid, H., Zhang, H., Rahman, S. U., Zhang, R., Liu, G., & Chen, C.
(2017). Pretreatment methods of lignocellulosic biomass for anaerobic digestion. AMB
Express, 7(1), 72. doi:10.1186/s13568-017-0375-4
AsmamawTesfaw and FassilAssefa, “Current Trends in Bioethanol Production
by Saccharomyces cerevisiae: Substrate, Inhibitor Reduction, Growth Variables,
Coculture, and Immobilization,” International Scholarly Research Notices, vol. 2014,
Article ID 532852, 11 pages, 2014. https://doi.org/10.1155/2014/532852.
Ballesteros, I., Negro, M.J., Oliva, J.M., Cabanas, A., Manzanares, P., Ballesteros,
M.,2006. Ethanol production from steam-explosion pretreated wheat straw. Applied
Biochemistry and Biotechnology 130, 496–508.
Bohdan Volynets, Farhad Ein-Mozaffari, Yaser Dahman (2017). Biomass processing into
ethanol: pretreatment, enzymatic hydrolysis, fermentation, rheology, and mixing. DOI:
https://doi.org/10.1515/gps-2016-0017
Danmaliki, Gaddafi & Usman, Bashir & M Muhammad, Auwal & Ahmad, Shamsuddeen.
(2016). Bioethanol Production from Banana Peels. IOSR Journal of Environmental
Science, Toxicology and Food Technology (IOSR-JESTFT). 10. 56-62. 10.9790/2402-
1006025662.
Dor´ee,The Methods of Cellulose Chemistry , Chapman &Hall,London, UK, 1947.
Dussan, K.,Virginio da Silva, D.,Moraes, E., Arruda, P.& Felipe, M. (2014).Dilute-acid
hydrolysis of cellulose to glucose from sugarcane bagasse.Chemical Engineering
Transactions. 38. 433. 10.3303/CET1438073.

Elizabeth, Ojewumi & Job, Akwayo & Taiwo, Olugbenga & Obanla, Oyinlola & Ayoola,
Ayodeji & Ojewumi, Emmanuel. (2018). Bio-Conversion of Sweet Potato Peel Waste to
BioEthanol Using Saccharomyces Cerevisiae. 8. 46-54.

Himmel, M.E., Ding, S.Y., Johnson, D.K., Adney, W.S., Nimlos, M.R., Brady, J.W. and
Foust, T.D. (2007) Biomass recalcitrance: engineering plants and enzymes for biofuel
production. Science, 315, 804-807.

Muzaffer, A. L. A., M., Rukmani, B., Shanmughadasan, K., & Purushothaman, K. (2011).
“Effect of time on the fermentation and storage of candanasava”
R. Kataria & S. Ghosh (2014) Physical Pretreatment and Enzymatic Hydrolysis
of Saccharum spontaneum for Reducing Sugars Production, Energy Sources, Part A:
Recovery, Utilization, and Environmental Effects, 36:9, 1028-
1035, DOI: 10.1080/15567036.2010.551268
Ratsamee, S., Akaracharanya, A., Leepipatpiboon, N., Srinorakutara, T., Kitpreechavanich,
V., Tolieng, V. (2012). Purple guinea grass: Pretreatment and ethanol fermentation.
BioResources. 7. 10.15376/biores.7.2.1891-1906.
 38

Siti HajarMohd Azhar, RahmathAbdulla, Siti AzmahJambo, Hartinie Marbawi, Jualang

Azlan Gansau, Ainol Azifa Mohd Faik, Kenneth FrancisRodrigues. (2017). “Yeasts in
sustainable bioethanol production”
Stanley, H.O. & Ezeife, C.O. & Onwukwe, Chimaobi. (2017). Bioethanol Production
from Elephant Grass (Pennisetum purpureum). Nigerian Journal of Biotechnology. 32. 1.
10.4314/njb.v32i1.1.
Wong, Y.C. & Lim, S.Y.. (2017). Production of Bioethanol from Cogon Grass (Imperata
cylindrical). Asian Journal of Chemistry. 29. 52-56. 10.14233/ajchem.2017.20128.
Yasuda, M., Ishii, Y., Ohta, K. (2015). Napier grass (Pennisetum purpureum Schumach) as
raw material for bioethanol production: Pretreatment, saccharification, and fermentation.
Biotechnology and Bioprocess Engineering. 19. 943-950. 10.1007/s12257-014-0465-y.

Arcalas, J. Y. (December 11, 2016). Ethanol output to hit 322M liters in 2017. Business
Mirror.
Arcalas, J. Y. (October 30, 2017). Ethanol imports seen growing by 8.3% in 2018.
Business Mirror.
Carey, F. A. (2019). Ethylene. Retrieved March 15, 2019, from
https://www.britannica.com/science/ethylene
Clifford B. C.,2018. Ethanol Production - General Information. Energy Institute, The
Pennsylvania State University.
Corpuz, P. (2017). Philippine Biofuels Situation and Outlook (Rep. No. RP1713).
Retrieved March 15, 2019, from Global Agriculture Information Center website:
https://gain.fas.usda.gov/Recent GAIN Publications/Biofuels
Annual_Manila_Philippines_10-18-2017.pdf
Corpuz, P. (2014). Philippine Biofuels Situation and Outlook (Rep.). Retrieved March 15,
2019, from Global Agriculture Information Center website:
https://gain.fas.usda.gov/Recent GAIN Publications/Biofuels
Annual_Manila_Philippines_10-18-2017.pdf
Frigon, Guiot (2010), Biofuels, Bioproducts&Biorefining, 4, 447-458.
Garcia N. 2018. Understanding Ethanol. Chapter 2. Related study materials.

Hamelinck, C.N., Hooijdonk, V. and Faaij, A.P.C. (2005) Ethanol from lignocellulosic
biomass: Techno-economic performance in short, middle and long-term. Biomass &
Bioenergy, 28, 384-410.

Heuzé V., Tran G., Baumont R., 2016. Buffalo grass (Paspalumconjugatum). Feedipedia,
a programme by INRA, CIRAD, AFZ and
FAO .https://www.feedipedia.org/node/407 Last updated on April 1, 2016, 15:40
Holtzapple,M.T.(2003), Encyclopedia of Food Sciences and Nutrition (Second Edition)
Horng LC, Leu LS, 1978. The effects of depth and duration of burial on the germination
of ten annual weed seeds. Weed Science, 26(1):4-10
 39

Iqbal, H.M.N., Ahmed, I., Zia, M.A. and Irfan, M. (2011) Purification and characterization
of the kinetic parameters of cellulase produced from wheat straw by Trichoderma viride
under SSF and its detergent compatibility. Advances in Bioscience and Biotechnology, 2,
149-156.

Jiang, G., Nowakowski, D.J. and Bridgwater, A.V. (2010) A systematic study of the
kinetics of lignin pyrolysis. Thermochimica Acta., 498, 61-66.

Licht, F.O., 2006. World Ethanol Market: The Outlook to 2015, Tunbridge Wells, Agra
Europe Special Report, UK.
Mongeau,R. & Brooks, S.P.J.(2003), Encyclopedia of Food Sciences and Nutrition
(Second Edition).
Morton, J. S. 1980. Glycolysis and Alcoholic Fermentation. Acts & Facts.9 (12).
Pamplona PP, 1975. Studies on the biology, competition and control of sourgrass
(Paspalumconjugatum Berg.). Biotrop Newsletter, No. 12:13
PremPrakash, G., &Neelu, G. (2017).Chapter-43 Chromatography. In Essentials of
Practical Biochemistry(1st ed.).
Roach, J. (July 18, 2005). "9,000-Year-Old Beer Re-Created From Chinese
Recipe". National Geographic . Retrieved 2007-09-03.
Sanjukta, S., Rai, A., Ali, A. M., Kumaraswamy, J., Talukdar, N. C. (2015).Enhancement
of antioxidant properties of two soybean varieties of Sikkim Himalayan region by
proteolytic Bacillus subtilis fermentation.Journal of Functional Foods. 14. 650–658.
10.1016/j.jff.2015.02.033.
Sauerborn J, 1985. Studies on the segetal flora of taro (Colocasiaesculenta (L.) Schott)
and on the germination biology of selected weeds of Western Samoa. PLITS (Plant
Protection Information Tropics/Subtropics), 3(1):85 pp
Sauerborn J, Koch W, 1988. An investigation of the germination of six tropical arable
weeds. Weed Research, UK, 28(1):47-52
Sharmas, S. K., Kalar, K. L., and Grewal, H. S. 2002. Enzymatic saccharification of
pretreated sunflower stalks. Biomass Bioenergy 23:237–243.
Sun, S., Sun, S., Cao, X., & Sun, R. (2016). The role of pretreatment in improving the
enzymatic hydrolysis of lignocellulosic materials. Bioresource Technology , 48-49.

Taherzadeh, M.J., Karimi, K., 2007. Acid-based hydrolysis processes for ethanol from
lignocellulosic materials: a review. Bioresources 2, 472–499.
Wang J, Xi J and Wang Y, Recent Advances in the Catalytic Production of Glucose from
Lignocellulosic Biomass. Green Chemistry., 2015; 17(2): 737-751P
Wyman, C.E., Dale, B.E., Elander, R.T., Holtzapple, M., Ladisch, M.R. and Lee, Y.Y.
(2005) Coordinated development of leading biomass pretreatment technologies.
Bioresource Technology, 96, 1959-1966.

Y. P. Zhang and L. R. Lynd, Biotechnol. Bioeng., 2004, 88, 797–824.

 40

GLOSSARY OF TECHNICAL TERMS

Alcohol – a colorless volatile flammable liquid that is produced by the natural

fermentation of sugars and is the intoxicating constituent of wine, beer, spirits, and other

drinks, and is also used as an industrial solvent and as fuel

Anaerobic condition- is the condition that exist due to the absence of free oxygen

Biofuel - a fuel derived directly from living matter.

Biomass - is plant or animal material used for energy production, or in various industrial

processes as raw material for a range of products. It can be purposely grown energy crops,

wood or forest residues, waste from food crops, horticulture, food processing, animal

farming, or human waste from sewage plants.

Carbohydrate - any of a large group of organic compounds occurring in foods and living

tissues and including sugars, starch, and cellulose. They contain hydrogen and oxygen in

the same ratio as water (2:1)

Catalyst - a substance that increases the rate of a chemical reaction without itself

undergoing any permanent chemical change.

Cellulose - an insoluble substance that is the main constituent of plant cell walls and of

vegetable fibers such as cotton. It is a polysaccharide consisting of chains of glucose

monomers.

Dilute Acid Hydrolysis - is a process in which cellulose is hydrolyzed to glucose

monosaccharide by using an inorganic acid such as sulfuric acid or hydrochloric acid as

the catalyst.
 41

Distillation - the action of purifying a liquid by a process of heating and cooling

Enzyme - a substance produced by a living organism that acts as a catalyst to bring about

a specific biochemical reaction.

Enzymatic hydrolysis - is a process in which enzymes facilitate the cleavage of bonds in

molecules. It plays an important role in the digestion of food. It may be used to help

provide renewable energy, as with cellulosic ethanol.

Ethanol – is commonly called alcohol, spirits, ethyl alcohol, and drinking alcohol, is the

principal type of alcohol found in alcoholic beverages, produced by the fermentation of

sugars by yeasts

Ethene - is a hydrocarbon which has the formula C2H4 or H₂C=CH₂. It is a colorless

flammable gas with a faint "sweet and musky" odor when pure. It is the simplest alkene,

and the second simplest unsaturated hydrocarbon after acetylene.

Ethyl Alcohol - a colorless volatile flammable liquid that is the intoxicating constituent

of wine, beer, spirits, and other drinks, and is also used as an industrial solvent and as

fuel.

Feeds - refer to the amount of food commodity allotted for animals or livestock/poultry

during the reference period.

Feedstock - raw material to supply or fuel a machine or industrial process.

Fehling’s Test – is the test that uses a chemical reagent to differentiate between water

soluble carbohydrate and ketone functional groups, and as a test for monosaccharides.
 42

Fermentation - the chemical breakdown of a substance by bacteria, yeasts, or other

microorganisms, typically involving effervescence and the giving off of heat

Filtration - is a physical, biological or chemical operation that separates solid matter and

fluid from a mixture with a filter medium that has a complex structure through which only

the fluid can pass.

Fuel - material such as coal, gas, or oil that is burned to produce heat or power

Gasoline - refined petroleum used as fuel for internal combustion engines

Gas Chromatography - is a common type of chromatography used in analytical

chemistry for separating and analyzing compounds that can be vaporized without

decomposition. Typical uses of GC include testing the purity of a particular substance, or

separating the different components of a mixture.

Glucose - a simple sugar that is an important energy source in living organisms

Hydrolysis - the chemical breakdown of a compound into simpler substances

Lignocellulosic- any of several closely related substances constituting the essential part

of woody cell walls of plants and consisting of cellulose intimately associated with lignin

Monosaccharide - also called simple sugar, are the simplest form of sugar and the most

basic units of carbohydrates. They cannot be further hydrolyzed to simpler chemical

compounds.

pH value – is the measure of the acidity or alkalinity of a solution

 43

Polysaccharide - are long chains of carbohydrate molecules, specifically polymeric

carbohydrates composed of monosaccharide units bound together by glycosidic linkages.

Pretreatment - means any treatment, which is done before actual process.

Reagent - A reagent is a substance or compound added to a system to cause a chemical

reaction, or added to test if a reaction occurs.

Renewable Energy – energy from a source that is not depleted when used, such as wid or

solar power

Yeast - a microscopic fungus consisting of single oval cells that reproduce by budding,

and are capable of converting sugar into alcohol and carbon dioxide.

Yield - is the amount of product obtained in a chemical reaction.

 44

RESEARCH WORK PLAN

The table below shows the chronological work plan of the research for proposal stage and
experimental stage

Table W.1 Work Plan for Proposal Stage

November December January February March
Week
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
Selection of
Topic
Chapter 1

Chapter 2

Chapter 3

Chapter 4

Revision of Final
Paper
Proposal Defense
 45

Table W.2 Work Plan for Experimental Stage

August September October November December

Weeks

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

Preparation of

Materials,

Laboratory

Apparatus and

Reagents

Preparation of

Sample

Acid Hydrolysis

Fehling’s Test

Fermentation

Filtration

Gas

Chromatography

Final Defense
 46

Revision of

Final Paper
 47

APPENDICES

Appendix A. Biotech Laboratory Form and Results

Request for analysis/ Statement of Charges

 48

Appendix B. Gas Chromatography Analysis Result

 49

Appendix C. Documentation of Collection of Carabao grass at Livestock Resource

Center in Irawan, Puerto Princesa, Palawan

Appendix D. Preparation of the Carabao grass (washing and cutting)

 50

Appendix E. Documentation of Acid Hydrolysis

 51

Appendix F. Documentation of Fehling’s Test

Appendix G. Documentation of activation of yeast

 52

Appendix H. Documentation of Fermentation

Appendix I. Documentation of Filtration of Samples