PRODUCTION OF BIOETHANOL FROM POMELO (Citrus maxima) PEEL

A Research

Presented to

The Faculty of the Chemical Engineering Department

College of Engineering, Eastern Visayas State University

Tacloban City

In Partial Fulfillment

of the Requirements for the course

CHE 362 (Methods of Research II)

Bachelor of Science in Chemical Engineering

by:

FIONA N. MANDIA

BSCHE-4A

2019
 APPROVAL SHEET

In partial fulfillment of the requirements for the degree of Bachelor of Science in

Chemical Engineering, this research entitled: “PRODUCTION OF BIOETHANOL
FROM POMELO (Citrus maxima) PEEL” has been prepared and submitted by FIONA
N. MANDIA. who is recommended for Oral Examination.

ENGR. ELVIRA DOLORES M. URGEL

Adviser

Approved by the Committee on Oral Examination with a rating of __________.

ENGR. EVELYN A. CARDOSO

Chairperson

ZENAIDA L. ANDRADE, Ph.D. MARIA LINA A. DOLLETE, Ph.D.

Member Member

ELVIRA DOLORES M. URGEL, Ph.D PROF. RICHARD S. BRUN

Member Member

Accepted in partial fulfillment of the requirements for the course, CHE 362
(Methods of Research II), Bachelor of Science in Chemical Engineering.

ENGR. EVELYN A. CARDOSO

Instructor, CHE 362 (Methods of Research II)
Eastern Visayas State University, Tacloban City

Date of Oral Examination

February 22, 2019

2
 ACCEPTANCE SHEET

This research hereto attached entitled: “PRODUCTION OF BIOETHANOL

FROM POMELO (Citrus maxima) PEEL” prepared and submitted by FIONA N.
MANDIA. in partial fulfillment of the requirements for the course, CHE 362 (Methods of
Research II), Bachelor of Science in Chemical Engineering, is hereby accepted.

ZENAIDA L. ANDRADE, Ph.D. MARIA LINA A. DOLLETE, Ph.D.

Member, Oral Examination Committee Member, Oral Examination Committee

PROF. RICHARD S. BRUN

Member, Oral Examination Committee

ENGR. EVELYN A. CARDOSO

Chairperson, Oral Examination Committee

Accepted in partial fulfillment for the requirements for the course, CHE 362
(Methods of Research II), Bachelor of Science in Chemical Engineering.

ENGR. EVELYN A. CARDOSO

Instructor, CHE 362 (Methods of Research II)

MARIA LINA DOLLETE, Ph.D.

Head, Chemical Engineering Department
Eastern Visayas State University, Tacloban City

ANNABELLE B. PILAPIL, Ph.D.

Dean, College of Engineering
Eastern Visayas State University, Tacloban City

3
 ACKNOWLEDGEMENT

First, the researcher would like to express her gratitude to the Almighty God for

His guidance and love all the time, especially when hard situations occurred on this

journey. The undying support of the researcher’s family is worthy to offer the success of

this research and friends who uplift and cheer whenever problems come.

The researcher also wants to give thanks to the mentors who played very important

roles in school life; To Engr. Evelyn Cardoso, Engr. Nida B. Lacaba and Engr. Zenaida L.

Andrade, and Engr. Elvira Dolores M. Urgel who helped and advised the researcher by

sharing their knowledge and things they can offer. To Engr. Ma. Lina A. Dollete, who

allowed me to conduct the experiments in the Chemistry Laboratory.

To Mr. Pascualito M. Ilagan, MRD, who allowed me to perform the experiments

and use the laboratory equipment in the Chemistry Laboratory.

To Mrs. Fe T. Piedad, R.Ch., who shared her knowledge and expertise about the
study.

To Mr. Darwin C. Gomez, R.Ch., I would like to express my heartfelt gratitude for
all the time, effort, sacrifice and for helping me all the way to make this study successful.
The study would not be positive without your help for I feel so lost at times and do not
know what to do anymore. Words are not enough to express how grateful I am for the
encouragement throughout.

To Blessel, Ada, Neil, Romeo, Jolie, Maura, Isip, Franz, Paul, and other 4 th year
BSChemistry students, who helped me I would like to thank all of you, even for just a short
time, I’m happy I made some friends there.

To everyone who was part on creating the study, I recognized all of you. Thank
You!

– FIONA

4
 Table of Contents

TITLE PAGE . . . . . . . . 1

APPROVAL SHEET . . . . . . . 2

ACCEPTANCE SHEET . . . . . . . 3

ACKNOWLEDGEMENT . . . . . . . 4

TABLE OF CONTENTS . . . . . . . 5

LIST OF FIGURES, TABLES AND APPENDICES . . . 7

ABSTRACT . . . . . . . . . 8

CHAPTER 1: INTRODUCTION

Background of the Study . . . . . . 10

Objectives of the Study . . . . . . 12

Null Hypothesis . . . . . . . 12

Conceptual Framework . . . . . . 13

Significance of the Study . . . . . . 15

Scope and Delimitation of the Study . . . . . 16

Definition of Terms . . . . . . . 17

CHAPTER II: RELATED LITERATURES AND STUDIES . . 20

CHAPTER III: METHODOLOGY

Research Design . . . . . . . 27

Research Subject and Sampling Procedure . . . . 28

Research Instrument . . . . . . . 29

Research Procedure . . . . . . . 30

Methods of Analysis . . . . . . . 32

5
 Statistical Treatment Tool . . . . . . 33

CHAPTER IV: RESULTS AND DISCUSSION . . . . 34

CHAPTER V: CONCLUSION AND RECOMMENDATIONS . . 38

BIBLIOGRAPHY . . . . . . . . 40

APPENDICES . . . . . . . . 47

6
 LIST OF TABLES, FIGURES AND APPENDICES

Figures

1.1 Conceptual Framework of the Study . . . . 14

3.1 Process flow of the experimental procedure of the study . 28

Tables

3.1 Chemical Composition of Raw Pomelo Peel . . . 23

3.2 Chemical Composition of Pomelo Albedo . . . 24

4.1 Summary of Experimental Conditions Using Hydrothermal Treatment 34

4.2 Summary of Experimental Conditions Using Hydrothermal Treatment 35

4.3 Results . . . . . . . . 36

Appendices

APPENDIX A: Raw Data . . . . . . 48

APPENDIX A2: Ethanol-Water Mixtures . . . . 53
APPENDIX B: Documentation on the Making of Rodenticide from
Color . . . . . . . . . 54
Odor . . . . . . . . . 55

APPENDIX C:
Documentation on Producing Ethanol from Pomelo Peels in a Laboratory Scale
. . . . . . . . . . 56

7
 ABSTRACT

PRODUCTION OF BIOETHANOL FROM POMELO (Citrus Maxima) PEEL

FIONA N. MANDIA
Eastern Visayas State University
Tacloban City

ENGR. ELVIRA DOLORES M. URGEL

Adviser

Energy is vital for life. Many billion people left cold and hungry without it. Due to

global energy consumption and dependence on non-renewable energy, the cost of transport

and environmental problems has increased. There was an urgent need for a clean,

renewable and economical energy source. This study presents an alternative raw material

for bioethanol production to address energy source problems. The pomelo peels were

processed to reduce size, treat hydrothermal and then fermented with alkaline peroxide.

Commercial ethanol and sucrose setup were designed to serve as the experiment's control

variable. The fermentation process lasted two days and was then distilled, the distillate

was tested using gravimetric analysis from different pretreatments. The distillate density

from commercial ethanol, hydrothermal and alkaline-peroxide treatment was 0.9926,

8
0.9915 and 0.9963. The hydrothermal treatment yielded 11.7 percent and 4.92 percent in

the treatment of alkaline-peroxide. The distillate pH from commercial treatment with

ethanol, hydrothermal and alkaline peroxide was 3.39, 3.41 and 3.40. The results show that

the density and pH of the distillates with commercial ethanol differs slightly. There is a

qualitative difference between distillate and commercial ethanol. Ethanol was therefore

obtained from pomelo peels for this study.

9
 CHAPTER I
INTRODUCTION

Background of the Study

Energy is vital for life. Many billion people left cold and hungry without it.

The main supply of energy comes from fossil fuels, which is why most industrialized and

developing countries are currently using fossil fuels such as oil, coal and gas. Burning fossil

fuels is considered the most cost-effective way to generate electricity, but it can lead to

serious environmental problems such as air pollution and is a limited and non-renewable

energy source. Due to global energy consumption and dependence on non-renewable

energy, the cost of transport and environmental problems has increased. There was an

urgent need for a clean, renewable and economical energy source. Ethanol was a promising

renewable source, according to Farrell et al. (2006). Bioethanol has gained more attention

as a replacement for fossil fuels as a clean and renewable fuel (P. Wei et al., 2014). It can

be produced from different types of raw materials classified into three categories of

agricultural raw materials: feedstocks containing sucrose, starch and lignocellulosic

materialsLignocellulosic materials are used for the production of second-generation

biofuel as a cheap and abundant feedstock compared to sugar and starch-based materials.

(Balat, 2011). Lignocellulose mainly consists of cellulose, hemicellulose and lignin, which

can be converted into fermentable sugars. These sugars are used by fermenting

microorganisms to produce ethanol as a metabolism by-product (Garrote, 2002).

A large amount of citrus waste is generated with grown citrus production and

consumption, which accounts for approximately 50 percent of the fruit weight. The waste

includes fruit peel, membrane residues and other by-products traditionally disposed of

10
through a burning process that leads to severe environmental pollution and large waste. It

is highly desirable to develop green and cost-effective ways of dealing with citrus waste

and using it to produce staple or added value products. Citrus waste consists mainly of

soluble mono-and disaccharides (e.g. glucose, fructose or sucrose), insoluble

polysaccharides (e.g. cellulose, pectin), and lignin. This chemical composition makes

waste a potential candidate for biofuels and other chemicals based on sugar (Huang et al.,

2014).

The aim of this study is to determine the viability of pomelo peel as a potential raw

material source for the production of bioethanol. The researcher decided to carry out a

study on one of the citrus fruits that was thought to produce ethanol. It contains abundant

waste of pectin-rich biomass and can be used as a fuel source. Citrus maxima, also known

as Pomelo, is the said fruit peel chosen by the researcher as the object of the study. Raw

pomelo peel consists of cellulose, hemicellulose, pectin, lignin and soluble sugars, making

it an interesting choice for appropriate organisms to produce metabolites such as ethanol.

However, an individual or combination of mechanical, chemical and biological treatments

is required to break down cellulose, hemicellulose and pectin polymers in the cell walls of

pomelo peels and convert them into monomers of sugars then fermented into ethanol.

11
Objectives of the Study

This study aims to utilize pomelo peels as an alternative raw material for the

production of bioethanol; specifically the study seeks to answer the following:

1. Determine the percent yield of ethanol obtained.

2. Determine the quality of ethanol from pomelo peels.

a. Color

b. Odor

c. Density

d. pH

3. Determine the significant difference between the quality of ethanol from pomelo peels

and commercial ethanol.

a. Color

b. Odor

c. Density

d. pH

Null Hypothesis

Pomelo peel is not a sustainable bioethanol raw material. In particular, the quality

of ethanol from pomelo peels and the quality of commercially produced ethanol do not

differ statistically significantly.

12
Conceptual Framework

The Republic Act No. 9367, known as the “Biofuels Act of 2006” is an act to direct

the use of biofuels to reduce dependence on imported fuels as an alternative to fossil fuels

(Republic Act No. 9367, 2007). The Republic Act No. 9513 known as the “Renewable

Energy Act of 2008” passed and strengthened R.A. 9367. It declared the State’s policy to

achieve energy security by reducing reliance on fossil fuels and minimizing exposure to

price fluctuations in oil markets (Sta. Lucia, 2015).

The U.S. Grains Council delegation visited ethanol plants and talked with ethanol

producers in the Philippines about the country’s plans to increase the national ethanol-

blending mandate to E20 by 2020. The movement “E20 by 2020” is an upward transition

that will result in increased domestic ethanol production as well as the potential for

additional imports (Council Shares U.S. Ethanol Experience In The Philippines, South

Korea, 2018).

13
P Ethanol from Pomelo Peels Commercial Ethanol
A
R
Color Color
A
M
Odor Odor
E
T
Density Density
E
R
pH pH
S

Figure 1.1 Conceptual Framework

14
Significance of the Study

Together with the pollution and waste generation abundant in the environment due

to the disposal of cellulosic wastes (e.g. pomelo peels), recycling these cellulosic wastes

for the production of ethanol would lead to a more efficient and eco-friendly substitute type

of energy source.

To the Researcher who conducted the study. It is to improve the proficiency and

skills in the field of the researcher, which can be useful in the line of work. The researcher

aims to help everyone to minimize the effects of pollution.

To the Chemical Engineering Field. This research may provide additional

information and data that can be used for the future generation that is relevant to the study

and production of energy from sustainable sources.

To the Fuel Industries. This study may be of help to them since fossil fuel is

rapidly depleting, to produce ethanol to produce ethanol from a cheap source that can also

minimize the polluting effects of the usual energy sources.

To the Community. This research may give insights and lead everyone not only

to attract them to use the product but also to challenge them in joining our cause to help

the environment. They can also make their own bioethanol at home for their vehicles to

reduce expenses. There are many people producing bioethanol at home for their own use

and many of them opt for a homemade “still” for the production process (makebiofuel,

2018)

To the Environment. The aim of this research is to minimize the emission of

greenhouse gases since ethanol burns clearly compared to pure gasoline. Under current

15
conditions, use of ethanol-blended fuels as E85 (85% ethanol and 15% gasoline) can reduce

the net emissions of greenhouse gases by as much as 37.1%. Ethanol-blended fuel such as

E10 (10% ethanol and 90% gasoline) reduces greenhouse gases by up to 3.9%. The

environmental implications of feedstock production associated with the production of

ethanol for fuel are biological renewability, sustainable agriculture, and energy balance

(Environmental Benefits of Ethanol, 2000).

To the Future Researchers. The data and information from this study can be

utilized for further researchers and future applications.

Scope and Delimitation of the Study

This study is limited on the viability of pomelo peel as a possible source for the

production of bioethanol. The study is also limited in terms of determining the percent

yield of ethanol obtained, the quality of ethanol from pomelo peels, and determining if

there is a significant difference between the quality of ethanol from pomelo peels and

commercial ethanol. The parameters checked for the produced ethanol are color, odor,

density, and pH and these characteristics are compared with the commercially produced

ethanol. It is limited on alkaline-peroxide and hydrothermal treatment as the process used

in the study. The study is restricted in gravimetric analysis as the technique used to

determine the density of ethanol in the distillate. The study is limited in the survey of five

respondents for their opinion on color and odor of the distillate and commercial ethanol.

Pretreatment conditions used in the study were limited to mechanical, hydrothermal, and

alkaline-peroxide techniques.

16
 This study is limited on determining the viability of pomelo peel as a source of

bioethanol by using Saccharomyces cerevisiae as a catalyst in the fermentation process.

The study will utilize the available facilities located within the Eastern Visayas State

University-Main Campus, Tacloban City. The pomelo peels will be taken from the

Tacloban City.

Definition of Terms

The following terms are defined for the better understanding of the reader with the

study at hand:

Alkaline-peroxide treatment. This form of pretreatment utilizes alkaline solutions

such as NaOH, KOH, NH4OH, or Ca(OH)2 (Taherzadeh and Karimi, 2008). Sodium

hydroxide is the most commonly studied pretreatment alkali and is seen as an alternative

to sulfuric acid (Silverstein et al., 2007; Kumar et al., 2009). This is one of the methods

applied to produce ethanol in the study.

Bioethanol. It is ethanol that is produced from agricultural products (such as sugar

cane or corn) and that is used as a fuel supplement (Merriam-Webster, 2018).

Color. It is the aspect of the appearance of objects and light sources that may be

described in terms of hue, lightness, and saturation (Merriam-Webster, 2018).

Distillation. The process of purifying a liquid by successive evaporation and

condensation (Merriam-Webster, 2018). It is the process used in the study to separate

ethanol from water.

17
 Ethanol. A colorless volatile flammable liquid C2H5OH that is the intoxicating

agent in liquors and used as a solvent and in fuel (Merriam-Webster, 2018).

Fermentation. An enzymatically controlled anaerobic breakdown of an energy-

rich compound (such as a carbohydrate to carbon dioxide and alcohol or to an organic acid)

(Merriam-Webster, 2018). In this study, the process takes place in which the glucose is

broken down to ethanol by the action of enzymes in the yeast used.

Gravimetric analysis. It is the process of producing and weighing a compound or

element in as pure form as possible after some form of chemical treatment has been carried

out on the substance to be examined (Gravimetric Analysis, 2019). The method used to

determine the specific gravity of the distillate.

Hydrothermal treatment. It refers to a thermochemical process for decomposing

carbonaceous materials such as coal and biomass with water in a high temperature and high

pressure condition (Yuliansyah & Hirajima, 2012). This is one of the methods applied in

the study to produce ethanol.

Lignocellulosic Biomass. It is a renewable and abundant resource suitable for the

production of bio-based materials such as biofuels and chemicals and is mainly composed

of cellulose, hemicellulose and lignin (Dutra, et al., 2017).

Odor. A characteristic or predominant quality (Merriam-Webster, 2018).

Percent Yield. The percent ratio of actual yield to the theoretical yield (Geankoplis,

1993). In this study, it refers to the percent ratio of the mass of ethanol to the mass of the

pomelo peels.

18
 Saccharification. It is the process of breaking a complex carbohydrate such as

starch or cellulose into its monosaccharide components (Saccharification, 2019). In this

study, this is one of the process that the experiment underwent.

Saccharomyces cerevisiae. It is a type of budding yeast that is able to ferment sugar

into carbon dioxide and alcohol and is commonly used in the baking and brewing industries

(Encyclopaedia Brittanica, 2018). It is the microorganism used in the fermentation process

of the study.

19
 CHAPTER II
REVIEW OF RELATED LITERATURE

This chapter presents the various literatures and studies reviewed by the

researchers, which provide more understanding and substance to this study.

Related Literature

The production of bioethanol from lignocellulosic biomass mainly involves two

processes: Hydrolysis of cellulose in lignocellulosic biomass to reduce sugar production

and fermentation sugars into ethanol (Sun & Cheng, 2002). However, the sugars necessary

for fermentation are trapped in the lignocellulose structure, so biomass pretreatment is

always necessary to remove the surrounding lignin and hemicellulose matrix before the

hydrolysis of polysaccharides (cellulose and hemicellulose) in the biomass. Pre-treatment

methods can generally be classified into three categories, including physical, chemical and

biological. After pretreatment, residues can be converted into ethanol by means of well-

established fermentation technologies when hydrolyzed into their component sugars

(Zheng, Pan, & Zhang, 2009). The sugar concentration is determined after the pre-

treatment and hydrolysis steps and the yield of ethanol after fermentation. Sugars are

quantified using spectrophotometric methods or chromatographic techniques, while

ethanol is determined by simple distillation followed by specific gravity analysis or liquid

chromatography.

20
Bioethanol

It is one of the most promising and environmentally friendly alternatives to fossil

fuels from renewable sources (Zabed, Sahu, Boyce, & Faruq, production of fuel ethanol

from lignocellulosic biomass: An overview of feedstocks and technological approaches,

2016). Bioethanol is produced from biomass containing free fermentable sugars or

complex carbohydrates, which can be converted to fermentable sugars. These feedstocks

can be divided into three groups of sugars, starchy crops and lignocellulosic biomass

(Zabed, Sahu, Boyce and Faruq, Production of fuel ethanol from lignocellulosic biomass:

An overview of feedstocks and technological approaches, 2016). The fermentation of raw

materials based on sugar is referred to as bioethanol of "first generation," while the use of

raw materials from lignocellulose is commonly referred to as bioethanol of "second

generation.". Algal bioethanol's "third generation" is at an early stage of investigation. The

main reasons for the increased development of bioethanol are its use as a favorable and

near-carbon-neutral renewable fuel, which reduces CO2 emissions and related climate

change; its use as an octane enhancer in unleaded gasoline; and its use as an oxygenated

fuel mix for cleaner gasoline combustion (Kang, Appels, Tan, & Dewil, 2014).

Lignocellulosic Materials

As a sustainable source of sugars and chemical platforms for conversion into

renewable fuels, fine chemicals and materials, lignocellulosic biomass is of particular

interest. It is also the only accessible non-fossil carbon source that can be processed into

liquids that are easily incorporated into the existing fuel infrastructure for transport (Xu &

Sun, 2016). It consists mainly of cellulose, hemicellulose and lignin, as well as small

fractions and ash extraction. It requires a stage of pre-treatment to convert it into energy

21
products. The main purpose of pretreatment is to disrupt the recalcitrance of the

lignocellulosic matrix and facilitate the separation of polysaccharides and lignin, which

leads to increased accessibility to enzymatic hydrolysis (Dutra, et al., 2017).

Hydrothermal Pretreatment

Pretreatments that mainly use steam or liquid water at high temperatures can

efficiently convert biomass into a form that enzymes can easily digest by facilitating

autohydrolysis reactions in biomass. Hydrothermal pretreatment is known as hot water or

steam processes as the primary chemical. Both forms of hydrothermal pre-treatment use

steam (explosion of steam) and water (pre-treatmentof liquid hot water). The pretreatment

of liquid hot water is the form of hydrothermal pretreatment used in the study. During the

pre-treatment of liquid hot water, water is present as a liquid instead of gas (Waldron,

2010).

Alkaline Hydrogen Peroxide Pretreatment

It is an oxidative pretreatment process that acts in the delignment of lignocellulosic

biomass, which allows for greater efficiency in the recovery of sugars in the liquid phase

of enzyme hydrolysis, since the presence of lignin makes it difficult for enzymes to attack

the substratum. This type of pretreatment is low-energy and does not produce inhibitors

such as hydroxymethylfurfural and furfural (Dutra, et al., 2017). Hydroxides of sodium,

potassium, calcium and ammonium are often used to treat alkaline reagents and can

improve the efficiency of lignin removal. Sodium hydroxide (NaOH) pretreatment is one

of the most common methods of bioconversion of lignocelluloses and has been extensively

22
studied. Treatment with NaOH is very effective in increasing the digestibility of low lignin

content hardwood and agricultural residues (Xu & Sun, 2016).

Pomelo Peels

Pomelo or Pummelo is a Philippine citrus fruit produced from Davao or popularly

known as the "Philippine Fruit Basket." The greenish-yellow rind of the pomelo peel is

thick and contains a thick layer of spongy pith. Instead of throwing the pomelo peel, use it

for bioethanol production.

Table 3.1 Chemical Composition of Raw Pomelo Peel

Composition Percentage

Cellulose 16.5%, based on dry matter

Hemicellulose 6.86%

Pectin 35.42%

Lignin 3.16%

Soluble sugars 12.62%

Ash 4.14%

Raw pomelo peel consists of cellulose (16.5 %), hemicellulose (6.86 %), pectin

(35.42 %), lignin (3.16 %), soluble sugar (12.62 %), and ash (4.14 %) (Renliang Huang,

2014). Pomelo peel is one of the under-used waste materials that can produce bioethanol

because it contains the above-mentioned bioethanol components and is a lignocellulosic

material. In terms of energy security, environmental concerns, employment opportunities,

agricultural development, foreign exchange savings, socio - economic issues, etc.,

23
lignocellulosic feedstocks do not interfere with food security and are necessary for each

rural and specific area (Zafar, Biofuels from Lignocellulosic Biomass, 2018).

Table 3.2 Chemical composition of Pomelo Albedo

Composition (% w/w, dry basis)

Carbohydrate 72.62 ± 0.42

Moisture 16.13 ± 0.16

Protein 6.27 ± 0.23

Ash 3.41 ± 0.05

Fat 1.56 ± 0.07

Cellulose 21.29 ± 1.90

Carbohydrate (72 %) consists mainly of dried pomelo albedo This value was

significantly higher than the content of carbohydrates found in lemon pulp (70 %)

(Brillouet JM, 1988) and lemon albedo (59 %) (Ros JM, 1996). Pomelo albedo also found

moisture and protein content at 16.13 percent and 6.27 percent. These values were

obviously higher than those recorded in albedo lemon, orange peel and orange pulp

(Sariçoban C, 2008) (Nassar AG, 2008).

Small amounts of ash (3.41 %) and fat (1.56 %) were observed in the meantime.

This is in accordance with Martin et al. (Marin FR, 2007), which described the amount of

ash in the citrus peel ranging from 2.56 to 8.09 percent, while the amount of fat is 1.51 to

4.00 percent. Meanwhile, the content of cellulose in pomelo albedo was 21.29 percent

significantly higher than 14.4 percent in orange peel (Bicu I, 2011). Pomelo albedo may be

a better source of cellulose than other types of citrus fruit peel (Nor Fazelin Mat Zain,

24
preparation and characterization of cellulose and nanocellulose from Pomelo (Citrus

Grandis) Albedo, 2014).

Related Studies

The following studies were conducted by the researchers – local and abroad, and

somehow have bearing to the present studies:

Abiertas (2018) conducted a study using carabao grass (Paspalum conjugatum). Its

result revealed that the condition used in the study failed to produce significant amount of

ethanol. It was determined that the conditions used were not suitable to produce ethanol by

using a flammability test (Abiertas, 2018).

In a study conducted by (Gatela Jr., 2018), peanut shell waste was used as a raw

material to produce ethanol. The result revealed that most of the characteristics of the

produced ethanol from peanut shells were quite similar to the commercial. Only the purity,

odor, and color of the ethanol produced had a significant difference from the commercial.

Huang (2014) utilized pomelo peel waste by integrated hydrothermal treatment,

multi-enzyme formulation, and fed-batch operation. The results show that hydrothermal

treatment (120 °C, 15 min) could significantly reduce the use of cellulase (from 7 to 3.8

FPU g−1) and pectinase (from 20 to 10 U g−1). A multienzyme complex, which consists of

cellulase, pectinase, β-glucosidase, and xylanase, was also proven to be effective to

improve the hydrolysis of pretreated pomelo peel, leading to higher concentrations of

fermentative sugars (36 vs 14 g L−1) and galacturonic acid (23 vs 9 g L−1) than those with

the use of a single enzyme. The results indicate

25
that the use of the fed-batch mode could alleviate the decrease in ethanol yield at high solid

loading, which is caused by significant mass transfer limitation and increased inhibition of

toxic compounds in the SSF process (Huang, et al., 2014).

26
 CHAPTER III

METHODOLOGY

This chapter presents the discussion on the research methodology of the study, the

research design, research subject and sampling procedure, research procedure, research

instrument, and statistical analysis used for accurate data analysis and interpretation.

Research Design

This study used an experimental design, which followed an adopted method from

various sources. Cellulosic ethanol was produced from pomelo peels. Two pretreatment

methods were used to ferment the sample and the distillate recovered from each method

was compared with the commercial ethanol.

Three major processes will be conducted and specifically centered on the conversion of the

cellulose substrate (pomelo peels) into fermentable sugar, converting the sugar to alcohol,

and distillation of alcohol solution to yield a high percentage of ethanol. In the first part of

the process, the pomelo is peeled and separated from its fruit. The pomelo peels will be

reduced to fine sizes and be introduced to the processes of hydrothermal treatment and

alkaline-peroxide treatment then autoclaved to obtain sugar (hexose compounds) from the

pomelo peels. A filter will be utilized to separate the aqueous solution with the solid

residue. The filtrate is the main ingredient in the microbial fermentation process in

producing ethanol. Yeast is the microorganism used for the fermentation. The fermented

solution will undergo distillation to acquire ethanol. The ethanol taken is introduced to tests

and analysis afterwards.

27
 Pomelo Peel

Size Reduction

10 g w/v solid

Hydrothermal
treatment

Filtrate Residue

Fermentati Alkaline-peroxide
on treatment
Distillation
Filtrate Residue
Ethanol
Fermentati
on
Distillation

Ethanol

Tests and Analysis

Figure 3.1 Process of the experimental procedure followed in the study

28
 The study will be conducted in the Chemistry Laboratory located on the third floor

of the Science Building (SB) of Eastern Visayas State University, Tacloban City. The

pomelo peels will be taken from Tacloban City, Leyte.

Research Subject and Sampling Procedure

Pomelo peels and yeast are the primary materials in making bioethanol. They will

undergo pretreatment, hydrolysis, filtration, fermentation, and distillation to obtain ethanol.

The bioethanol will then undergo tests and analysis to determine the quality of ethanol in

terms of its color, odor, density, and pH level. The quality of ethanol taken from pomelo

peels has been compared with the commercially produced ethanol to see the difference

between them.

Research Instruments

This refers to the materials or equipment used in the course of the study. The main

raw material in the study are pomelo peels. In the size reduction of pomelo peels, a knife

and scissors will be used. Furthermore, several materials and apparatuses such as plastic

bowls, beakers, reagent bottles, and Erlenmeyer flasks will be used for the pre-treatment

and fermentation.

In the ethanol production and tests, several raw ingredients such as hydrogen

peroxide, sodium hydroxide, yeast and distilled water will be used. Moreover, an autoclave

equipment for reducing the bacteria to minimal and an equipment composed of a container

for the liquid, a heat source, and a condenser will be needed for the distillation in the course

of the experiment.

29
 A thermometer, pH meter, burette and small Erlenmeyer flasks were used to test

the temperature, pH content, and specific gravity of the materials respectively.

Research Procedure

1. Selection and preparation of Pomelo Peels

Pomelo is bought in Tacloban City, Leyte and selected based on the greenish color

as a suitable raw material for its ripeness. The fruit is separated with its peels using a knife

and cut to pieces manually with a scissor. The small pieces of peels will now be the raw

material for the experiment.

2. Size reduction, Pretreatment, and Filtration

The chopped samples were oven-dried for 24 hours at 60˚C and pulverized using a

blender. Pulverized samples were sieved into a beaker and weighed using a top loading

balance. Four samples of 10 g pulverized pomelo peels were prepared. A buffer 2 solution

was prepared dissolving 1 f of urea and 1 g of potassium dihydrogen orthophosphate in a

1 liter distilled water. A 100 mL of buffer 2 solution was added to the 10 g fine powder of

pomelo peel in the beaker. The mixture is autoclaved at 121˚C, 15 psi for 1 hour and further

cooled to room temperature. The hydrolysate was filtered with a cheesecloth to separate

the juice and residue. The juice obtained from hydrothermal treatment was set aside in an

Erlenmeyer flask to be fermented. 2 g NaOH dissolved in a 400 mL buffer 2 solution and

mixed with a 13.3 mL H2O2 was poured to the residue collected. The mixture is subjected

to alkaline-peroxide pretreatment and autoclaved at 121˚C, 15 psi for 1 hour. It was cooled

30
to room temperature, filtered with a cheesecloth until the aqueous solutions are the only

components remaining in the Erlenmeyer flask. The pH was maintained at 4.55.

3. Fermentation Process and Distillation

Fermentation is the next step after filtering the hydrolyzed raw materials. Two

fermentation setups were made: the juice collected from hydrothermal treatment and

alkaline-peroxide treatment. The first setup was the juice obtained from hydrothermal

treatment, it was added with 150 mL buffer 2 and 2.5 g yeast to be fermented. The second

setup was the liquid obtained from the alkaline-peroxide treatment, it was added with 60

mL buffer 2 and 2.5 g yeast. Each setup consists of an Erlenmeyer flask where the aqueous

solutions are contained and sealed with a plastic wrap and pierced with a needle then

covered with a balloon. The balloon act as a pathway for the carbon dioxide product that

will be given off from the chemical reaction. The two setups were kept in an incubator at

a temperature of 33˚C. The optimum temperature range for yeast fermentation is between

32˚C to 35˚C (Temperature is key to fermentation success, 2013). The fermentation lasted

for two days.

After two days, ethanol in each fermentation product will be distilled using simple

distillation method. A 250 mL of fermentation product in each setup will be placed in a

distillation flask to begin the process. A 50 mL distillate will be collected in a volumetric

flask in each setup as a receiver in the distillation process.

31
 4. Tests and Checking

Gravimetric analysis was used to determine the density of the distillate. It is

concerned with the process of producing and weighing a compound or element in as pure

form as possible after some form of chemical treatment has been carried out on the

substance to be examined (Gravimetric Analysis, 2019).

Methods of Analysis

After distillation, the distillate from different pretreatments were put into different

tests and analysis. A gravimetric analysis was performed in both pretreatments to determine

the density of the distillate. A burette, small Erlenmeyer flasks, and analytical balance were

used to perform the method. No contamination should be present as much as possible

because it can affect the results. All the equipments used and distillates obtained were

stabilized at 20˚C. The burette was rinsed off with a small amount of the distillate then the

remaining were poured into the equipment. An analytical balance was used to get the initial

mass of the small Erlenmeyer flasks. The initial and final volume of the distillate was noted

on the burette. One Erlenmeyer flask is used as a receiver for the distillate from the burette

and the final mass was noted. The change in mass and volume were calculated for each

trial. The density was determined by the change in mass over the change in volume.

The testing of the odor and color of the ethanol produced from the study is surveyed.

Five respondents are approached to rate the odor and color of the ethanol solutions. The

categories for odor in the survey are 1 – favorable, 2 – slightly favorable, 3 – can’t decide

if favorable or unpleasant, 4 – unpleasant, and 5 – very unpleasant. The categories of the

32
color are 1 – colorless, 2 – slightly blurred white, 3 – slightly blurred yellow, 4 – light

yellow, and 5 – yellow.

Statistical Treatment Tool

Mean was used in quantitative measurements for determining the average results

from the data gathered in the experiments. For the comparative analysis of the odor and

color between the distillates of two pretreatments and commercial ethanol, Analysis of

Variance (ANOVA) of Two-Factor Without Replication was used.

33
 CHAPTER IV
RESULTS AND DISCUSSION

This chapter tackles about the different laboratory data, reports and results which

were obtained during the experiment. The gathered information throughout the course of

the experiment were tabulated and recorded. Such data were relevant and pertains to the

statement of the problem.

Cellulosic Ethanol from Pomelo Peels

Table 4.1 I and 4.2 presents the summary of experimental conditions used to

produce ethanol from pomelo peels. Findings revealed that the conditions used in the study

succeeded to produce significant amount of ethanol that can be detected by gravimetric

analysis. This implies that the conditions used were suitable to produce ethanol.

Table 4.1 Summary of Experimental Conditions Using Hydrothermal Treatment

Conditions Experiment

Pretreatment: size reduction Powder

Pretreatment: hydrothermal 121˚C autoclaved for 1 hr

Fermentation: ph; time 4.5; 2 days

Ethanol content: gravimetric analysis Detectable

34
 Table 4.2 Summary of Experimental Conditions Using Alkaline-Peroxide Treatment
Conditions Experiment

Pretreatment: size reduction Powder

Pretreatment: alkaline-peroxide 121˚C autoclaved for 1 hour

Fermentation: pH; time 4.5; 2 days

Ethanol content: gravimetric analysis Detectable

Pretreatment conditions used in the study were limited to mechanical,

hydrothermal, and alkaline-peroxide techniques. The pomelo peel samples were reduced

in size to increase the surface area of the raw materials. It has been reasoned that higher

surface area result in increased sugar yield due to more efficient sugar hydrolysis (Koullas

et al., 1992).

One of the most promising and environmentally friendly biomass pretreatment

methods available is hydrothermal treatment since it makes the lignocellulosic biomass

vulnerable to enzymatic breakdown (Cybulska, Brudecki, & Lei, 2013). Hydrothermal

pretreatment greatly increases surface area of cellulose (by nonchemical swelling), which

significantly enhances possible enzyme access (Chang et al. 1981; Sun and Cheng 2002).

Characterization of Distillate

Gravimetric analysis was used to determine the density of the distillate. The

analysis revealed that the ethanol content can be determined gravimetrically. Downstream

analysis of distillate was performed in terms of its color, odor, density, and pH.

35
 Table 4.3 Results
Treatment Densitya Percent pH Color Odor
Yieldb
Hydrothermal 0.9915 11.7 3.41 Colorless Can’t decide
if favorable
or
unpleasant
Alkaline-Peroxide 0.9963 4.92 3.40 Slightly Can’t decide
blurred white if favorable
or
unpleasant
Commercial 0.9926 - 3.39 Slightly Favorable
Ethanol (5% v/v) blurred white
a determined at 20 °C
b calculated as mass ethanol (g)/gram dry weight of pomelo peels x 100

Based on the table, the density of the distillate in a hydrothermal and alkaline

peroxide treatment were 0.9915 g/mL and 0.9963 g/mL respectively. There is only a small

difference between them and compared with commercial ethanol (5% v/v) density of

0.9907 g/mL. The percent yield in a hydrothermal and alkaline-peroxide treatment is 11.7%

and 4.92%. It can be inferred in the data that the percent yield of a hydrothermal treatment

is higher than the alkaline-peroxide treatment because it hydrolyzed the fermentable sugars

in the sample. The hydrothermal treatment was the first method applied in the sample and

the residue collected was used for alkaline-peroxide treatment. The remaining soluble

sugars in the residue were hydrolyzed in the alkaline-peroxide treatment. The pH of the

distillate from a hydrothermal and alkaline-peroxide treatment were 3.41 and 3.40,

commercial ethanol has a pH of 3.39. There is only a 0.01 difference between the distillates

and commercial ethanol.

A survey was conducted with five respondents for the color and odor of the

distillate. It can be inferred from the mean data that hydrothermal treatment is colorless,

36
alkaline pretreatment is slightly blurred white and commercial ethanol is slightly blurred

white. In the odor of the distillate and commercial ethanol, the respondents cannot decide

if it is favorable or unpleasant. In the mean data of the respondents, the odor of commercial

ethanol is favorable.

37
 CHAPTER V
CONCLUSIONS AND RECOMMENDATIONS

This chapter presents the conclusions from the light of the study and a number of

recommendations to improve the study in different ways.

CONCLUSION

The main goal of the study was to analyze the potential of pomelo peels to produce

ethanol. The density and pH of the produced ethanol and ethanol distillate from

hydrothermal and alkaline treatment was almost similar. The color and odor of the ethanol

produced from pomelo peels have a significant difference from the commercial. The

following are the specific conclusions made from the study.

1. The percent yield of hydrothermal treatment is 11.7% and 4.92% in alkaline-

peroxide treatment.

2. The color of the product in a hydrothermal treatment is colorless and slightly

blurred white in the alkaline peroxide treatment and commercial ethanol.

3. The respondents cannot decide if the odor of the product in a hydrothermal and

alkaline-peroxide treatment are favorable or unpleasant. The odor of the

commercial ethanol is favorable.

4. The densities have a uniform value for the first two decimal places.

5. The pH of the distillates and commercial ethanol were measured using a pH meter.

There is a difference of 0.01 pH between the distillates and commercial ethanol.

6. There was a significant difference between the ethanol from hydrothermal

treatment and alkaline pretreatment with the commercial ethanol in terms of its

38
 color, odor, density, and pH. Therefore, it has been concluded that pomelo peel can

be a substitute raw material for commercial ethanol.

RECOMMENDATIONS

The specific recommendations with regards to the study are listed below:

1. For future researchers, it is recommended to try enzymes in the hydrolysis of the

biomass. Enzymatic hydrolysis process converts pre-treated substrate to

monomeric sugars, which could be further converted to ethanol (Enzyme

Hydrolysis, 2019).

2. It is recommended to try different solid loadings in an experiment. High solid

loadings offers many advantages over lower-solid loadings, including sugar and

ethanol concentration and decreased production and capital costs (AA & SE, 2012).

3. The researcher suggests to follow the procedure in the study but utilize the

remaining residue from alkaline peroxide with enzymatic hydrolysis to get the

soluble sugars the past two pretreatments failed to separate from cellulose,

hemicellulose, and lignin.

39
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46
APPENDICES

47
APPENDIX A
RAW DATA

Table 1

ETHANOL CONCENTRATION IN SUCROSE FERMENTATION

Replicate Mass of Volume of Specific Mean Ethanol

No. Solution (g) Solution Gravity* Specific Concentration
(mL)* Gravity*
1 14.7879 14.97 0.98784
2 14.7480 14.94 0.98715 0.98733 9.296 g/L
3 14.9037 15.10 0.98700
*determined at 20.0 ± 1.0 °C

Table 2

WATER

Replicate Mass of Volume of Solution Specific Gravity* Mean Specific

No. Solution (g) (mL)* Gravity*
1 14.4327 14.47 0.99742
2 14.9585 14.97 0.99923 0.99922
3 15.0952 15.08 1.00100
*determined at 20.0 ± 1.0 °C

Table 3

DISTILLED WATER

Replicate Mass of Volume of Solution Specific Gravity* Mean Specific

No. Solution (g) (mL)* Gravity*
1 15.05419 15.07 0.99895
2 14.6451 14.64 1.00034 0.99958
3 15.0116 15.02 0.99944
*determined at 20.0 ± 1.0 °C

48
 Table 4

ETHANOL CONCENTRATION IN ALCOHOL STANDARD (5%)

Replicate Mass of Volume of Specific Mean Ethanol

No. Solution (g) Solution Gravity* Specific Concentration
(mL)* Gravity*
1 14.969 15.09 0.99198
2 14.8911 15.02 0.99142 0.99229 4.124 g/L
3 15.0212 15.12 0.99347
*determined at 20.0 ± 1.0 °C

Table 5

ETHANOL CONCENTRATION IN POMELO PEEL FERMENTATION

USING ALKALINE-PEROXIDE PRETREATMENT

Replicate Mass of Volume of Specific Mean Ethanol

No. Solution (g) Solution Gravity* Specific Concentration
(mL)* Gravity*
1 13.9161 13.95 0.99757
2 14.0006 14.03 0.99790 0.99636 2.006 g/L
3 13.9402 14.03 0.99359
*determined at 20.0 ± 1.0 °C

Table 6

ETHANOL CONCENTRATION IN POMELO PEEL FERMENTATION

USING HYDROTHERMAL PRETREATMENT

Replicate Mass of Volume of Specific Mean Ethanol

No. Solution (g) Solution Gravity* Specific Concentration
(mL)* Gravity*
1 15.0455 15.14 0.99376
2 14.8719 14.98 0.99278 0.99158 4.788 g/L
3 14.8332 15.01 0.98822
*determined at 20.0 ± 1.0 °C

49
 ETHANOL ODOR SURVEY

Respondent Hydrothermal treatment Alkaline-peroxide treatment

Odor rating Odor rating
1 3 2
2 5 4
3 2 4
4 4 3
5 2 4
Sum 16 17
Mean average 3.2 3.4
Overall average 16.5

COMMERCIAL ETHANOL ODOR SURVEY

Commercial Ethanol
Respondent
Odor rating

1 1

2 1

3 1

4 1

5 1

Sum 5

Mean average 1

50
 ETHANOL COLOR SURVEY

Respondent Hydrothermal treatment Alkaline peroxide treatment

Color rating Color rating

1 1 1

2 1 1

3 1 2

4 1 3

5 1 1

Sum 5 8

Mean average 1 1.6

Overall average 1.3

51
 COMMERCIAL ETHANOL COLOR SURVEY

Respondent Commercial Ethanol

Color rating

1 2

2 2

3 2

4 2

5 2

Sum 10

Mean average 2

52
 APPENDIX A2

ETHANOL-WATER MIXTURES

Density % by weight % by volume Density % by weight % by volume

20˚C ethanol ethanol 20˚C ethanol ethanol
(g/cm3) (g/cm3)

1,00000* 0 0 0,91097 52 59,8

0,99813 1 1,3 0,90872 53 60,8

0,99629 2 2,5 0,90645 54 61,8

0,99451 3 3,8 0,90418 55 62,8

0,99279 4 5,0 0,90191 56 63,8

0,99113 5 6,2 0,89962 57 64,8

0,98955 6 7,5 0,89733 58 65,8

0,98802 7 8,7 0,89502 59 66,8

0,98653 8 10,0 0,89271 60 67,7

0,98505 9 11,2 0,89040 61 68,6

0,98361 10 12,4 0,88807 62 69,6

0,98221 11 13,6 0,88574 63 70,5

0,98084 12 14,8 0,88339 64 71,5

0,97948 13 16,1 0,88104 65 72,4

0,97816 14 17,3 0,87869 66 73,3

0,97687 15 18,5 0,87632 67 74,2

0,97560 16 19,7 0,87396 68 75,1

0,97431 17 20,9 0,87158 69 76,0

53
0,97301 18 22,1 0,86920 70 76,9

0,97169 19 23,3 0,86680 71 77,8

APPENDIX B

COLOR

Anova: Two-Factor Without Replication

SUMMARY Count Sum Average Variance

1 3 4 1.333333 0.333333
2 3 4 1.333333 0.333333
3 3 5 1.666667 0.333333
4 3 6 2 1
5 3 4 1.333333 0.333333

A 5 5 1 0
B 5 8 1.6 0.8
C 5 10 2 0

ANOVA
Source of
Variation SS df MS F P-value F crit
Rows 1.066667 4 0.266667 1 0.460905 3.837853
Columns 2.533333 2 1.266667 4.75 0.043672 4.45897
Error 2.133333 8 0.266667

Total 5.733333 14

54
 ODOR

Anova: Two-Factor Without Replication

SUMMARY Count Sum Average Variance

1 3 6 2 1
2 3 10 3.33333 4.33333
3 3 7 2.33333 2.33333
4 3 8 2.66667 2.33333
5 3 7 2.33333 2.33333

A 5 16 3.2 1.7
B 5 17 3.4 0.8
C 5 5 1 0

ANOVA
Source of Variation SS df MS F P-value F crit
Rows 3.06667 4 0.76667 0.88462 0.51455 3.83785
Columns 17.7333 2 8.86667 10.2308 0.00624 4.45897
Error 6.93333 8 0.86667

Total 27.7333 14

55
 APPENDIX C

Documentation on Producing Ethanol from Pomelo Peels in a Laboratory Scale

Drying of pomelo peels in the oven at 60

˚C for 24 hours The oven dried pomelo peels were
pulverized using a blender

A buffer 2 solution was prepared as a Samples that went through a

nutrient for the yeast hydrothermal treatment (autoclaved at
121˚C, 1 hr)

56
 The residue (left) and filtrate (right)
obtained from hydrothermal treatment

Samples that went through an

alkaline-peroxide treatment
(autoclaved at 121˚C, 1 hr)

The residue (left) and

filtrate (right) obtained
from alkaline-peroxide
treatment

57
58
 Distillation setup

The fermentation setup of two

pretreatments
Hydrothermal treatment (left)
Alkaline-peroxide treatment (right)

Samples Autoclaved at 121˚ for 1

hour

59
 Survey conducted for the odor
and color of each product

Fermentation setup in the incubator

Hydrothermal treatment (left)
Alkaline-peroxide treatment (right)

The distillates used for survey

A- hydrothermal treatment
B- alkaline-peroxide treatment
C- commercial ethanol

60