CHAPTER I

THE PROBLEM AND ITS BACKGROUND

Introduction

Gasoline is one of the most commonly used necessities in the world. It is

toxic and highly flammable by product of petroleum industries that is used in

cars. Regardless of its extensive uses, it remains a contributor to air pollution due

to the emission of different substances when it is burned such as Carbon

Monoxide, Nitrogen Oxide, particulate matter and unburned hydrocarbons. For

this, almost all gasoline is blended with ethanol which reduces its harmful

emissions.

In Philippines, it is required for companies to add 10% ethanol in all

gasoline to reduce hydrocarbon emission (using sugarcane, sweet sorghum,

corn, and cassava as feedstock for ethanol). And using these feedstocks causes

the decrease of agricultural lands used for food production. Hence, this can

cause competition between agrofuel industry and food industry.

On the other hand, Bioethanol is a clear colorless liquid and a renewable

fuel from some plant materials known as biomass which is domestically

produced. Most plant-based material can be a raw material for ethanol

production. It is either from plants containing sugar and starch or from non-food

based including crop residues and other wastes.

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 Furthermore, Calamansi is normally used for food production, as a

condiment and can also be used as a bleaching agent, stain remover, shampoo

or even insect repellant. Along with these uses, its peels are often treated as a

residual waste and no longer used after its juice is extracted. These peels

comprises of different properties such as pectin, cellulose, hemicellulose and D-

limonene as its major compound which is known for its anti-carcinogenic

properties. These different properties make Calamansi peel suitable for ethanol

production plus its richness in carbohydrates and low lignin content. In line with

this, the researchers came up with the proposal to evaluate the Calamansi Peel

as a possible alternative biofuel additive to gasoline.

Objectives of the Study

The main objective of this study is to evaluate Calamansi as a possible

alternative bio fuel additive for gasoline. Particularly, this study aims:

1. Preparation of the calamansi peels including:

1.1. Drying

1.2. Grinding

2. Optimization of process parameters (by exposing to acid catalyzed steam

pretreatment) such as:

2.1. Solid loading

2.2. Time of exposure

2.3. Sulfuric acid concentration

2
3. Extraction by enzymatic hydrolysis using Aspergillus Niger Enzymes

4. Fermentation of hydrolysate using baker's yeast

5. Perform distillation process

6. Perform series of experiments to obtain:

6.1. Production Rate

6.2. Production Yield

7. Evaluation of properties of the ethanol produced and compare to the standard

in terms of:

7.1. Ethanol concentration

7.2. Auto-ignition Temperature

7.3. Lower heating Value

7.4. Density at specific temperature

7.5. Appearance and odor

8. Perform blending process considering

8.1. 5% ethanol, 95% gasoline

8.2. 10% ethanol, 90% gasoline

9. Develop an experiment manual for production of biofuel additive to gasoline

from Calamansi peel

Significance of the Study

This study aims to produce alternative biofuel additive for gasoline from

the peel of Calamansi. And if this study will be proven potent and provident, this

will beneficial to the following:

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 First, this study will be of great help at different sectors like the fuel sector

as it set an additional alternatives for fuel . Aside from helping to lessen the

waste produced by companies after extracting its juice, it will be useful to the

agricultural sector also as it will be used as a solution to the growing demand of

biofuel causing shortage of food and feed supplies. And to the environment

sector, this study will help environmental problems be reduced as blending of the

produced bioethanol in gasoline will lower harmful emissions which is damaging

to our health.

To Petroleum Engineering Department, the study shall assist the students

and faculty in their specialization by providing them data and understanding

about the properties of Calamansi peel as an additive. Also, the concepts and

principles applied in the study will enable them to be knowledgeable about the

properties of the said raw material.

Lastly, to the future researchers conducting such like study may use this

as a reference.

Scope and Delimitations of the Study

This study will focus on the evaluation of Calamansi peel as a possible

alternative biofuel additive for gasoline. Moreover, this study will cover the

determination of properties of Calamansi peel that may affect the production of

bioethanol. Also, knowledge of fermentation using yeast is needed. The

Calamansi peels that will be used in the whole experiment will be collected from

juice extracting companies together with other establishment using Calamansi's

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extracted juice. Also, percent yield will be evaluated in this study. Properties of

produced ethanol will be analyzed as well as the percent blending of produced

bioethanol with gasoline.

On the other hand, this study will be limited only on the evaluation of

calamansi peel as a possible alternative biofuel additive to gasoline and will not

cover the determination of the properties and the consumption rate of gasoline

that the researchers will be using.

Conceptual Framework

To describe the overall process and concepts regarding this study, the

researchers provide a research paradigm (Table 1.0) to show the process of

evaluating calamansi peel as a possible alternative biofuel additive to gasoline.

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6
Definition of Terms

The following terms were defined based on the actual denotative meaning

and based on the way they were used in this study in order to understand some

of the complicated terms.

Auto Ignition Temperature. A property of bioethanol which is the lowest

temperature it will ignite without a source of ignition such as flame and spark.

Bioethanol. It is alcohol which has low toxicity used as a fuel blended to

gasoline causing less harmful emission and normally from crop rich in starch.

Yeast. It is use in fermentation, it transforms sugar to alcohol and carbon dioxide.

D-limonene. It is a natural compound found in orange and lemon peels. It is

considered to have fairly low toxicity.

Enzymatic Hydrolysis. It is a process used to convert biomass into glucose by

adding chemicals. It also plays an important part in the digestion of foods.

Enzymes. It is any of various protein, as pepsin, originating from living cells and

capable of producing certain chemical changes in organic substance by catalytic

action, as in digestion. In the study, different enzymes were used to aid in

enzymatic hydrolysis.

Fermentation. It is a metabolic process in which an organism converts a

carbohydrate into a alcohol or an acid by chemical breakdown of a substance

(Helmenstine, 2016). In the study, it refers to the action of clostridium

Acetobutylicum to the reducing sugar to produce alcohol.

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Peel. It is the outer layer of a fruit or vegetable that is peeled off. Calamansi Peel

is called hesperidiu, it protects the inner part of the fruit or vegetable.

Production Yield. It is the difference between the actual output and the standard

output of the manufacturing or production process. It is caused at standard cost

and generally unfavorable.

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 CHAPTER II

REVIEW OF LITERATURE

This chapter presents the conceptual literature, research literature, and

synthesis that were considered to be vital in the pursuance of the research study.

Conceptual Literature

This contains the literal section from the different sources and unfinished

works which gave necessary information related to the study.

Fruit Peeling

Fruit peeling or fruit skin is the outer covering in fruits that protects the

underlying edible portion of fruit from microorganism and macro organisms as

well as from harsh environment factors. According to the study of Sanchez-

Orozco et al., fruit peeling are a remarkable source of sugar among the

agroindustrial residues that makes them an interesting candidate for the

production of value added products, such as butanol. The peel comprises 7 to

24% of the fresh fruits.

TABLE 1
Average Waste Produced of Fruits in the Philippines

Rank Fruit Average Waste (Metric Tons)

1 Banana 353, 541

2 Pineapple 121, 726

3 Mango 48, 645

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 TABLE 1
Average Waste Produced of Fruits in the Philippines

4 Calamansi 10, 066

5 Papaya 9, 815

6 Watermelon 7, 302

7 Durian 5, 148

8 Mandarin 4, 517

9 Jackfruit 2, 756

10 Orange 1, 818

Source: Philippine Statistic Authority, 2015

The Philippine Statistics Authority Releases a publication last 2015 on the

supply utilization accounts of selected agricultural commodities which was

referenced on the years 2012 to 2014.

Calamansi

Calamansi or calamondin (Citrus Macrocarpa Bunge) belongs to the

Citrus family. The tree is a low set, spreading and well-branched. the leaves are

oval, broad and pale green and dark green on the lower and upper surfaces,

respectively. The fruit is usually round and small, may be thick or thin. Calamansi

is a rich source of Vitamin C used as an ingredient in juices, syrups, concentrates

and purees, while the peels are processed into jams, candies, and marmalades.

The pulp can be utilized into beverages, syrups, and concentrates. (Food and

Agribusiness Yearbook, 2000).

Calamansi is native to China and thought to have been taken in early

times to Indonesia and in the Philippines. It is the most important Citrus juice

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source in the Philippine Islands and is widely grown in India and throughout

Southern Asia and Malaysia (Calamunding, 2006).

It is a citrus fruit indigenous to the Philippines and is mainly appreciated

for its characteristics of a good source of Vitamin C, its attractive color and

distinctive flavor. It has a Vitamin C content of 43 mg per 100 gms. It has a

diameter of 4 cm or less with color ranging from yellowish to green and the peak

season for fruit production is from June to October. In terms of production, it has

a higher production rate than the other citrus fruits since it is grown in commercial

scale in some areas (Calamansi Fact Sheet, 2003). Table 2 shows the

composition of Calamansi as to peel, pulp, seeds and juice.

TABLE 2
Components of Calamansi Fruit

Components Percentage(%)

Juice 31

Pulp 20

Seed 19

Peel 30

Source: Science and Technology Journal (1995)

Bioethanol

Bioethanol is a petrol additive and derived from alcoholic fermentation of

sucrose or simple sugar, which are produced from biomass by hydrolysis

process. The biomass undergoes fermentation, during which certain species of

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yeast digest the sugar contained in the biomass. The process releases

bioethanol and carbon dioxide. It is commonly blended with gasoline, used as an

additive.

Bioethanol is by no means a new invention. Henry Ford thought it was “the

fuel of the future” and designed his famous Model T to be able to run on pure

bioethanol, which also made it easy for the first car-owners to refuel at home

straight from their own “moonshine” distillery.

Process of Producing Bioethanol from Calamansi Peel as Biofuel Additive

1.1 Physical Pretreatment

Coarse size reduction, chipping, shredding, grinding, and milling is

amongst the different mechanical size reduction methods that have been used to

enhance the digestibility of lignocellulosic biomass (Palmowski and Muller, 1999).

These treatments increase the available specific surface area, and reduce both

the degree of polymerization (DP) and cellulose crystallinity (Sun and Cheng,

2002). Grinding and milling can reduce the particle size to 0.2-2 mm. Grinding

and milling are more effective at reducing the particle size and cellulose

crystallinity that chipping probably as result of the shear forces generated during

milling. (Agbor et al., 2011)

1.2 Chemical Pretreatment

Pretreatment is done to break the complex lignocellulosic structure of the

biomass. The lignin structure is broken down to make the cellulose and

hemicelluloses accessible for hydrolysis.

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1.3 Hot Water Pretreatment

Hot water pretreatment is also known as “auto hydrolysis.” It is

advantageous in the fact that it is less expensive, less corrosive to equipment,

less xylose degradation and thus has fewer byproducts including inhibitory

compound in the extracts. Hot water under pressure can break the cell structure

of biomass, hydrate cellulose, and remove hemicellulose, thus effectively

improving the enzymatic digestibility of biomass cellulose.

1.4 Acid Pretreatment

The use of acid hydrolysis for the conversion of cellulose to glucose is a

process that has been studied for the last 100 years. Generally, there are two

types of acid hydrolysis: dilute and concentrated, each having unique properties

and effects on biomass, and each having advantages and disadvantages in

terms of economics.

2. Enzymatic Hydrolysis

A multi-step occurring in a heterogeneous reaction in which insoluble

cellulose is initially broken down at the solid-liquid interface via synergistic action

is accompanied by further liquid-phase hydrolysis of soluble intermediated, that

is, short celluloligosaccharides and cellobiose, which are catalytically cleaved to

produce glucose by the action of b-glucosidase (Biofuels, 2011).

3. Fermentation

Fermentation is a metabolic process that converts sugar to acids, gases,

or alcohol. It occurs I'm yeast and bacteria, and also in oxygen-starved muscle

cells. Fermentation is also used more broadly to refer to the bulk growth of

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microorganisms on a growth medium, often with goal of producing a specific

chemical product.

4. Distillation

Distillatin is a widely used method for separating mixtures based on

differences in the condition required to change the phase of components of the

mixture. to separate a mixture of liquids, the liquids can be heated to force

components, which have different boiling points, into gas phase. The gas is then

condensed back into liquid form and collected. Repeating the process on the

collected liquid to improve the purity of the product is called double distillation.

Although the term is most commonly applied to liquids, the reverse process can

be used to separate gases by liquefying components using changes in

temperature and/or pressure.

Research Literature

Presented here are studies parallel to the proponents’ target of exploring

more about producing solvents from biomass.

Local Studies

A study by Arcan et al., (2012) entitled “Enzymatic Saccharification of

Lignocellulosic Component of Indian Mango Peel for Bioethanol Production”. The

sun drying of mango peels was done for a maximum of 10 hours per day. The

Indian mango peel were powdered and mixed with water to form slurry. 1-L of tap

water was added to 200g of macerated peelings in wide mouth open glass

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container. Then, the prepared slurry was weighted and placed in a flask.

Hydrogen peroxide was added, having 2% concentration at pH 11.5. The pH can

be adjusted by adding NaOH pellets. The solutions allowed to soak for 48 hours.

The flask was placed in a magnetic stirrer at 100rpm to make sure that

substrates will come in contact with treatment solution (Srinokutara et.al., 2003).

After alkaline peroxide pretreatment, substrate was washed using tap water until

pH 7 is obtained. The neutralized substrate was hydrolyzed with (10mL, 15mL,

and 20mL) cellulase, 4.7mL glucoamylase, and 0.66mL pectinate. Constant

stirring was carried out for the proper mixing of the enzymes and to prevent the

settling of pwwling particles at the bottom of the flask. Saccharification was

performed in the water bath shaker at 50C for 72 hours. The supernatant was

separated from the mixture by centrifugation at 400 rpm for 30 minutes. The

supernatant was used for the determination of sugar content.

Marinay et al., (2013) conducted a study entitled “Bioethanol Production

from Pili (Canariumovatum) Fruit Coatings”. The researcher performed their

enzymatic saccharification using 10 grams in wet basis of pretreated pili. Sodium

citrate buffer was prepared prior to enzyme loading. The sodium citrate buffer

solution was prepared by diluting 7.69g of sodium citrate and 5.013 of citric acid

to 500mL of distilled water. The flask with 10g of pretreated pili fruit coating were

loaded with 150 mL of prepared sodium citrate buffer. The cellulase (25mL,

30mL, and 35mL) was loaded with a enzyme activity of 600U/mL, and

glucoamylase (4.7mL) with an enzyme activity 0f 1000U/mL was added

consecutively to the substrate. The prepared flash were placed in a water bath

15
shake at 50C for 84 hours. The samples were withdrawn after 60hrs, 72hrs, and

84 hrs and were centrifuged in a desktop centrifuge for the determination of total

sugar.

A. Ethanol from Orange Peels

A researcher Henry Daniell of the University of Central Florida, who naturally

turned to oranges, with a new method for producing ethanol for his biofuel

inspiration. The fact that Florida is swimming in oranges illustrates the unfolding

potential of the biofuel industry. In contrast to fossil fuels, which are often

shipped long distances to their point of use, biofuel production can be tailored to

take advantage of local resources, whether it’s biofuel from unmarketable

watermelons, algae, carbon monoxide harvested from factory emissions, or even

biofuel from microbes and sunlight. As for the connection between biofuel and

tobacco, that’s the “secret ingredient” behind Daniell’s success.

(https://cleantechnica.com/2010/02/25/a-sustainble-recipe-for-biofuel-ethanol-

from-orange-peels-and-tobacco/)

B. Production of ethanol and biomass from orange peel waste

Orange peels are the major solid by-product. Dried orange peels have a high

content of pectin, cellulose and hemicellulose, which make it suitable as

fermentation substrate when hydrolyzed. One problem when using orange peels

as raw material is its content of peel oils (mainly D-limonene), which has an

immense antimicrobial effect on many microorganism even at low

concentrations.(https://pdfs.semanticscholar.org/8912/2198dd281484fe2abfbac0

645c270880d7b6.pdf)

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C. Ethanol Production from citrus Peel Waste

Citrus peel waste is a valuable lignocellulosic feedstock for bioethanol

production due to its richness in fermentable sugars and low lignin content. Citrus

peel contains two major value-added products: d-limonene and pectin. d-

Limonene is widely used in food, cosmetics, and pharmaceutical industries.

However, it acts as a microbial growth inhibitor for yeast during the fermentation

process and hence it has to be removed prior to fermentation. Pectin is used as

thickening agent, gelling agent, and stabilizer in the food industry. Since pectin

increases the viscosity of the fermentation medium and makes fermentation

troublesome, it has to be either extracted or degraded into galacturonic acid

using pectinase enzyme. Thus, the removal and recovery of both D-limonene

and pectin from citrus peel are essential for better fermentation. For bioethanol

production, pretreatment plays a crucial role in the utilization of citrus peels since

the reduction of d-limonene concentration to less than 0.05% is necessary. This

review solely describes the potential of citrus waste for value added products

such as d-limonene and pectin and the production of bioethanol from citrus peel

waste is discussed in detail. (www.tandfonline.com)

Synthesis

Generally, the revised literature and studies greatly enriched the content

of this study. Despite the found similarities in the cited studies, this study is not a

duplication of their studies, thus, this study has a distinct personality of its own.

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 CHAPTER III

RESEARCH METHODS AND PROCEDURES

This chapter presents the methods and procedures used by the

researcher for the evaluation of Calamansi peel as a possible alternative biofuel

additive for gasoline.

Research Design

This study utilizes the experimental method of research and also focused

on varying enzymatic hydrolysis and fermentation parameters to produced bio

ethanol from Calamansi peel which then will be used as an additive to gasoline.

The researchers provide a flow diagram (Figure 3.0) to show the procedure that

will be used.

COLLECTION AND
PREPARATION OF CALAMANSI
PEEL
ACID CATALYZED STEAM
PRE - TREATMENT

CELLULOSE CONTENT

D-LIMONENE CONTENT

ENZYMATIC HYDROLYSIS

FERMENTATION

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 DISTILLATION

EVALUATION OF PROPERTIES

BLENDING

(FIGURE 3.0) APPLICATION

It starts at the collection and preparation of Calamansi peel by drying and

grinding from various companies then it shall undergo a pre - treatment to allow

researchers separate biomass allowing the succeeding processes easy to put

through. Then, it will undergo processes including fermentation, distillation, and

evaluation of properties and blending of the produced biofuel with the gasoline.

Preparation and Collection of Raw Materials

Calamansi peels will be obtained from a company using calamansi for

extracting juices and treated its peel as a residue. These peels will be washed

using distilled water. These washed peels will be subjected to drying to reduce

excessive moisture content through open sun drying. Lastly, these peelings will

be grounded to a very small sized and storing to a covered container.

Pre - treatment of Calamansi Peel

Pre - treatment is a vital tool and is required in enzymatic hydrolysis to

make cellulosic substances accessible to enzymes that will be used. During

these, several factors will be needed to be optimized such as solid loading, time

19
of exposure, and sulfuric acid concentration using acid catalyzed pre - treatment

which needed lower operating temperature.

Determination of ethanol concentration

Produced ethanol will be sent and tested to private sectors for the

evaluation of different properties in terms of:

 Ethanol concentration

 Auto-ignition temperature

 Lower heating value

 Density at 15˚C and 20˚C

 Appearance and odor

Actual gathering of data

The possible biofuel additive for gasoline will be obtained through series of

experiment, set ups and data collection. Cautious observation will be used as

well as recording to document the result.

Evaluation of the performed experiment

The success of the experiment will be typically evaluated through percent

yield and production rate. Production yield is the difference between actual

output and standard output of a production or manufacturing process, based on

standard inputs of materials and labor.

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 𝑷𝑹𝑶𝑫𝑼𝑪𝑬𝑫 𝑬𝑻𝑯𝑨𝑵𝑶𝑳 𝑭𝑹𝑶𝑴 𝑨𝑪𝑻𝑼𝑨𝑳 𝑬𝑿𝑷𝑬𝑹𝑰𝑴𝑬𝑵𝑻
PY = 𝑻𝑯𝑬𝑶𝑹𝑬𝑻𝑰𝑪𝑨𝑳 𝑨𝑴𝑶𝑼𝑵𝑻 𝑶𝑭 𝑬𝑻𝑯𝑨𝑵𝑶𝑳

While production rate measures the average time needed for a manufacturing

process to produce its goods.

𝑷𝑹𝑶𝑫𝑼𝑪𝑬𝑫 𝑬𝑻𝑯𝑨𝑵𝑶𝑳 𝑭𝑹𝑶𝑴 𝑨𝑪𝑻𝑼𝑨𝑳 𝑬𝑿𝑷𝑬𝑹𝑰𝑴𝑬𝑵𝑻

PR = 𝑷𝑹𝑶𝑫𝑼𝑪𝑻𝑰𝑶𝑵 𝑻𝑰𝑴𝑬

Blending process

The table below represents blending ratio. (figure 3.1)

Ratio Ethanol Pure gasoline

• 5:95 • 25 ml • 475 ml
• 10:90 • 50ml • 450 ml
• 15:85 • 75ml • 425 ml
• 20:80 • 100 ml • 400 ml
• 25:75 • 125 ml • 375 ml

FIGURE 3.1

The figure shown above presents the blending ratio equivalent to its

volumetric unit (ml) and using 500 ml or half a liter as a constant volume.

21
Evaluation of the Performed Experiment

Evaluate the performance of blended gasoline in a grasscutter in terms of

exhaust emission compared to pure gasoline.

Statistical treatment

Analysis of variance is basically a statistical technique used to test

differences among two dissimilar means found in a statistics. This study will use

it to compare the total ethanol produced during fermentation and enzymatic

hydrolysis, varying fermentation time, and cellulose content.

22