1

Efficiency of Enzymatic Extraction on Banana Peel (Musa acuminata) Substrate

Based on Ethanol Quantity and Quality Yield

Jeremie P. Capuli, Khristriana Ramos, Louis Jeinard V. Ramos, and Christian Jade G. Tancio

Senior High School Department, Holy Rosary Parochial Institute of Orani, Inc.

Research/Capstone Project

Rejun O. Buensuceso

May 7, 2021
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Introduction

Background of the Study

Dating back to the exact period when humans first produced bioethanol from various

solid-phase feedstock such as sugar cane, corn stover, and many more can be quite challenging

(Halder et al., 2019). However, it was dated back approximately 9000 years ago when bioethanol

production was first found in China. The most efficient and most commonly used method of

extracting from different fermented biomass was direct steam heating and stripping of biomass;

this ancient ethanol extraction method originated from the traditional Chinese liquor distillation

(Li & Li, 2020). Nonetheless, Galgano et al. (2012) stated that ethanol utilization was also a

significant part of the Brazilian car industry's history.

Bioethanol refers to "ethanol produced by microbial fermentation processes that can be

utilized as liquid fuel in internal combustion engines'' (Muleta, 2017, p. 1). They also added that

bioethanol is a renewable biofuel since it is derived from biomass like plants. It may also be

referred to as "an alternative fuel from biomass that contains a large amount of sugar that is

environmentally-friendly" (Zabochnicka-Świątek & Sławik, 2010, p. 237).

In the Philippines, biofuels were initiated due to the oil crisis in the 1970s (Gatdula et al.,

2021). However, they also averred that bioethanol implementation did not immediately pursue as

the domestic cost for biofuels was higher than importing oil. The Department of Energy in 2010

claimed that the Philippines compelled around 219 ml of bioethanol to comply with the

mandatory 5% by volume gasoline blending, which is anchored to Republic Act 9367 or the

Biofuel Act of 2006 (Borines et al., 2011). Therefore, it implies the need to investigate further a

much cost-efficient alternative method for bioethanol production using various agro-waste
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materials that will help satisfy the compelled amount in gasoline blending for efficient biofuel

utilization.

Moreover, the depletion of fossil fuels and the continuous global price increase of

petroleum-based fuels motivated researchers to tackle this topic. Specifically, this study attempts

to look for a cheaper alternative in producing bioethanol as some bioethanol processes require a

considerable sum of money in conducting one. Several studies asserted that biofuels could be

made from various agro-waste materials due to modern technological advancements, particularly

developing renewable and sustainable fuel sources. Additionally, a report by Corpuz (2017)

stated that experts have agreed to explore alternative feedstocks since climate change affecting

El Niño and La Niña may decrease the country's sugarcane production. Therefore, this study has

come up in generating biofuels made up of banana peels as its substrate. The researchers also

chose to utilize bananas because of an easy gathering of materials due to bananas having a large

production in the Philippines. The Philippine banana production was estimated at 2.40 MMT or

2.4 billion kg from October to December 2020 (Philippine Statistics Authority | Republic of the

Philippines, n.d.). Furthermore, the Philippines also ranked as the second major exporter of

bananas, with 2.83 MMT of bananas exported in 2017 (Department of Agriculture, 2018).

Consequently, several recommendations of previous studies to investigate further about

different extraction methods of various biomass substrates led the researchers to test the

efficiency of employing an alternative extraction method among agro-waste materials by

determining the quality and quantity variation of the ethanol yield. In response to the study of

Muleta (2017), the banana peels will be utilized as the substrate to determine the quality and

quantity of its ethanol yield, the efficiency of bioethanol made of banana peels, and the amount

of time it will take to derive ethanol.

Statement of the Problem

The researchers will investigate the quantity and quality of the ethanol yield from the

banana peel substrate to test the enzymatic extraction process’s efficiency. Hence, it aims to

answer the general problem, “How efficient is the enzymatic extraction in deriving ethanol from

the banana peel substrate based on its quantity and quality yield?”

Moreover, the researchers seek answers for the following specific problems:

1. How much ethanol can be extracted using the enzymatic extraction of the banana

peels based on the percentage by volume of the CO2 produced?

2. What would be the quality of the ethanol yield from the banana peel substrate

upon determining using the;

2.1. triiodomethane test; and

2.2. combustion test?

3. In what way does the enzymatic extraction method become efficient based on its

ethanol yield in terms of;

3.1. production cost;

3.2. fermentation periods; and

3.3. substrate materials?

Significance of the Study

This study aims to know the efficiency of using enzymatic extraction to yield ethanol

from banana peels. Moreover, this study caters to both technical and non-technical audiences.

Thus, this study will be beneficial to the following:

Energy Sector

It will benefit the sector through the potential of bioethanol from banana peels to be

blended to petroleum gasoline for much cost-efficient and sustainable production.

Science, Technology, Engineering, and Mathematics Students

This study can act as an awareness material to give STEM students in-depth information

about the feasibility of bioethanol to substitute conventional fossil fuels.

Educators

This study also contributes to the faculty, especially science educators, to give them more

information about biofuels, particularly bioethanol, whenever they discuss lessons related to this

topic.

Future Researchers

This study can be a basis for future researchers who want to tackle this topic. Future

researchers can expand this topic by using other alternative enzymatic extraction methods to

different banana varieties and agro-wastes materials with a larger sample to test its efficiency

further.

Scope and Limitations

This study will investigate the bioethanol yield from banana peels substrate through an

alternative enzymatic extraction method to test its efficiency. Hence, the scope of this study will

focus on determining the quality and quantity of ethanol yield. It will also determine the

efficiency of the enzymatic extraction by the following parameters such as production cost,

fermentation periods, and substrate materials. Specifically, this study will only be using Lakatan

(Musa acuminata) bananas as the substrate for conducting the enzymatic extraction method
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because this variety is familiar and typically consumed by most people in the area. Moreover, the

study will be conducted at Holy Rosary Parochial Institute of Orani, Inc., specifically in the

Science laboratory. This place will be the artificial setting for performing laboratory procedures

using the enzymatic extraction method. Additionally, the researchers hypothesized that the

enzymatic extraction used on banana peels would be efficient, and its ethanol yield, as evaluated

by several tests. Contrarily, the researchers also hypothesized the possibility of using enzymatic

extraction being not efficient and time-consuming. These hypotheses will serve as tentative

answers to the general problem while the researchers carry out the study.

However, the possible limitations of this study have been identified. Since the study is an

experimental type of research, the researchers noticed that the bioethanol's enzymatic extraction

is not time-bound for the researchers to accomplish the procedure and get the findings

immediately. Moreover, the study will not be using other varieties of bananas as substrate.

Additionally, the researchers have noticed the difficulty in employing enzymatic extraction for

bioethanol production due to the limited laboratory equipment needed to execute the method.

These limitations affect the reporting of the study's findings approach, particularly in

determining the quality and quantity yield of bioethanol from banana peel substrate.

Nevertheless, the researchers hope that future studies acknowledge these limitations mentioned

above that respond to the research problem.

Review of Related Literature

This chapter presents the assessment of related literature and studies about bioethanol

production, extraction methods, and applications from agro-waste materials and its feasibility

to be a viable substitute to gasoline from fossil fuels.

Bioethanol or ethyl alcohol is a biofuel produced by microbial fermentation processes,

unlike synthetic ethanol made from petrochemical sources (Muleta, 2017). Regarding this

matter, Danmaliki et al. (2016) averred that bioethanol is known to be a clean fuel for

combustion engines, which is readily available to substitute as an alternative for fossil fuels

since it can be produced from plant-based and some agricultural residues. Similarly, Sarkar et

al. (2012) concluded that ethanol could be a valuable replacement for gasoline in the transport

fuel market. Moreover, bioethanol has been known for its various practical applications, such

as in the industry, transportation, and energy sectors (Casabar et al., 2019). However,

bioethanol production is more expensive than fossil fuel production (Sarkar et al., 2012). To

exemplify, Rabiu et al. (2019) listed challenges in using bioethanol, such as increased cost of

crops, needing advanced technological development and techniques for global scale bioethanol

production, and unsupportive policies in investing in bioethanol production. Contrarily, Muleta

(2017) asserted that bioethanol is cost-efficient when blended into gasoline since crude oil

prices are continuously increasing. Nonetheless, Vasić et al. (2021) contended that the

biodegradability of bioethanol reduces its toxicity.

Using different biomass as a primary substrate is considerably the main advantage of

biofuels over fossil fuels. Because of this, Demirbas (2011, as cited in Choi et al., 2015)

suggested that cheap and abundant materials must be used as an alternative to biomass

feedstocks, and efficient processes must be developed to be able to convert this biomass into
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biofuels. Hence, bioethanol can be an alternative to gasoline fuels because of its multifaceted

benefits and sustainable production from various biomass.

To illustrate, Gupta and Verma (2015) stated that people are approaching renewable

energy sources (e.g., biofuels) due to overconsumption and exploitation of non-renewable

energy resources. Subsequently, excessive consumption of non-renewable energy sources raises

the prices of oil and aggravates the environment. Thus, reducing the people's reliance on using

these non-renewable sources is a promising shift in utilizing alternative, sustainable, and

renewable energy sources, particularly bioethanol, to support the need for the world's fuel

shortly. Additionally, Koh and Ghazoul (2008, as cited in Dragone et al., 2010) asserted that an

increasing number of nations consider biofuels as vital energy sources. It reduces their reliance

on foreign oil, lowers the emissions of greenhouse gases (GHG), specifically carbon dioxide

(CO2) and methane (CH4), and for meeting rural development goals. In this sense, the utilization

of biomass and agricultural residues for biofuel production widens the reach of sustainability in

alternative energy production across the world (Gabhane et al., 2013).

However, since bioethanol is the most current commercially available among other

biofuels, many countries have already obligated their transportation sectors to blend

bioethanol into their petroleum products. Therefore, current bioethanol production

improvement must be raised to meet the global demand (Tatel & Medrazo, 2020). Since

bioethanol comes from agro-waste materials, there is quite a challenging production to

consider catering to the global market demand.

On the other hand, microalgae shape a life-form expected to be sufficient to produce

third-generation bioethanol (Martin & Grossmann, 2013, as cited in Simas-Rodrigues et al.,

2015). Moreover, microalgae are unicellular life-forms identified as separate species. They are
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photosynthetic species that also react to the conversion of carbon dioxide and the secretion of

oxygen gas in the ecosystem; however, heterotrophic microalgae are also present (Lee, 2008,

as cited in Simas-Rodrigues et al., 2015). Due to the high concentration of carbohydrates

provided by some plants, potential bioethanol production forms have been established (John et

al., 2011, as cited in Simas-Rodrigues et al., 2015). Additionally, Dragone et al. (2010)

enumerated advantages of utilizing microalgae for biofuels: it grows at high rates and

accumulate large quantities of neutral lipids, it is capable of production all year round, it only

needs less water than most crops, it does not need herbicides or insecticides for cultivation, it

reduces emissions of significant greenhouse gas, it cleans wastewater by bioremediation, it

can be cultivated in salt or brackish water on non-arable land or land not suitable for farming,

and it may also be extracted with an extensive range of fine chemicals and products in various

industrial sectors. Therefore, Oncel (2013) suggested that microalgae production's system and

facilities must be planned carefully for a suitable production strategy.

In 2018, Shokrkar et al., as cited in Vasić et al. (2021), developed a kinetic model of

enzymatic hydrolysis using microalgal cellulose since marine algae are quite attractive and

exciting to use as an alternative substrate for bioethanol production due to their rapid growth

rate. Moreover, several studies, including the study of Shokrkar et al. (2018), have already

used enzymatic hydrolysis to produce third-generation biofuels (Vasić et al., 2021). However,

they concluded that the current enzymatic hydrolysis process is still a narrow way to efficient

bioethanol production due to expensive enzymes and the inhibitory properties of compounds

that reduce glucose production effectiveness.

According to Danmaliki et al. (2016), banana peels are lignocellulosic agricultural

waste that can boost biofuel as a power generation. They investigated the feasibility of
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bioethanol production from banana peels as a source of lignocellulosic biomass. Pre-treatment

and hydrolysis of lignocellulosic materials are critical steps in the development of bioethanol.

Furthermore, biomass lignocellulose is distinguished by its carbohydrate content, which is a

requirement for the development of ethanol. Similarly, the studies of both Hossain et al.

(2011) and Itelima et al. (2013) showed high ethanol production from fermented bananas.

Therefore, they concluded that banana waste could be used for effective bioethanol production

and used in engines for transportation with less emission produced as the product has a good

quality. Additionally, to avoid the environmental problem due to excessive waste materials,

wastes from banana peels can be a significant renewable energy source as bioethanol.

Moreover, Muleta (2017) concluded that the bioethanol yield from blended banana

peels and microalgae is proven as the substrate to produce bioethanol. It is also supposed that

the substrate concentration and inoculums concentration are directly proportional to biofuel

production.

Overall, bioethanol derived from biomass substrates using different ethanol extraction

methods can be a valuable and sustainable substitute for commercial fuels. Nonetheless, many

studies like Vasic et al. (2021) recommended that producing bioethanol be further studied to

develop a much cost-efficient production using agro-waste materials. The studies of both

Dragone et al. (2010) and Simas-Rodrigues et al. (2015) also suggested using cheap and

abundant materials as the substrate while using reasonable bioethanol processes. Moreover,

Muleta (2017) recommended that banana wastes and microalgae be used as bioethanol

production materials due to their high ethanol yield. They also recommended alternative

extraction methods such as enzymatic extraction to determine the quality and quantity of

ethanol yield from agro-wastes materials as the substrate for further investigation.
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In response to these recommendations, particularly to Muleta (2017), the researchers

attempt to investigate the efficiency of an alternative extraction method in yielding ethanol

from a cheap biomass substrate. Thus, the study will focus on conducting enzymatic

extraction among agro-waste materials using banana peels in determining the quality and

quantity of its ethanol yield to test the efficiency of the alternative extraction method.
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Methodology

This chapter describes the experimental plan of determining the quantity and quality of

bioethanol produced from banana peel substrate's enzymatic extraction method to assess its

efficiency. Moreover, it presents the detailed procedures of the enzymatic extraction method,

including the research design, research instrument, materials, laboratory equipment, chemical

solutions, and several tests to be used.

The study will be experimental research. It utilizes a quantitative approach to generally

describe the quantity and quality yield of bioethanol from the data obtained throughout the

enzymatic extraction process of the banana peel substrate that will indicate its efficiency.

Furthermore, the researchers aim to test the efficiency of the enzymatic extraction process by

determining the quantity and quality yield of bioethanol from bananas, as Muleta (2017)

recommended by using enzyme cultivation and biological pretreatment of the substrate.

Accordingly, different processes are involved in banana peel substrate's cellulose

polymer conversion to ethanol, as Danmaliki et al. (2016) indicated. The first process is the

pretreatment of the substrate. Several pretreatment methods are usually employed to separate the

mixture of polymers of lignin, hemicellulose, and cellulose from equipping the amount of sugars

needed for the hydrolysis and the fermentation processes. According to Danmaliki et al. (2016),

the banana waste composition is 20.31% lignin, 57.76% holocellulose, and 13.63% ash.

Moreover, the pretreatment phase aims to destroy the lignin shell that protects the cellulose and

hemicellulose inside the organic materials. However, the researchers will only employ a

biological pretreatment among the three pretreatment methods (chemical pretreatment, physical

pretreatment, and biological pretreatment) included in Danmaliki et al. (2016) since it is cost-

effective but extensive. They further emphasized that biological pretreatment uses
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microorganisms that include white-rot fungi, brown-rot fungi, Bacillus, Trichoderma,

Aspergillus, and others essential in bioethanol production. Various components condition the

efficacy of a biological pretreatment. It involves substrate-related parameters such as biomass

composition and other factors such as inoculum concentration, aeration intensity, water content,

incubation period, temperature, pH, and the form of microorganism involved (Sindhu et al.,

2016, as cited in Ummalyma et al., 2019). Moreover, the pretreatment time will be based on

Valencia and Meitiniarti’s (2017) study, as cited in Taufikurahman and Delimanto (2018),

showing that the biodelignification of Aspergillus niger continues to increase until the 7th day.

Indeed, they averred that the biological pretreatment byproduct does not harm the environment

when carried out to pretreat the substrate.

The following process is the cellulose conversion into glucose units through hydrolysis or

also called as saccharification process. Furthermore, the researchers will utilize the enzymatic

extraction method in the hydrolysis step, in which polysaccharides will break down into

monomers for much efficient fermentation and extraction. Moreover, the researchers will use

Benedict’s test to check for reducing sugars, mainly glucose, vital for the fermentation step. The

sugars produced from the hydrolysis process are then converted into ethanol through

Saccharomyces cerevisiae, Brewer's yeast in layman's term, fermentation, which is the

succeeding process. However, the researchers will set up the substrate in three samples with

varying fermentation periods. The period of fermentation influences the development of

microorganisms (Azhar et al., 2017). They concluded that shorter fermentation time results in

inefficient fermentation due to insufficient microorganism development. However, longer

fermentation time has a toxic impact on microbial growth, especially in batch mode, due to the

high ethanol concentration in the fermented broth. Moreover, the researchers will test each
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sample with varying fermentation periods to determine which period best contributes to much

efficient ethanol yield in the enzymatic extraction method.

The last part of the procedures will be the distillation of the fermented banana peel

substrate. The samples will undergo subsequent evaporation and condensation to separate and

purify the ethanol from the substrate. Since the fermented banana substrate is ethanol-water

composition, the ethanol component will be vaporized first before the water because water

(100 °C) has a higher boiling point than ethanol (78 °C).

After the actual enzymatic extraction process and its underlying procedures, the

researchers will now determine the quantity and quality of ethanol yield to test the efficiency of

the process. There are various procedures and tests to be employed to assess the effectiveness of

the enzymatic extraction method. The quantitative analysis of the ethanol yield will be

determined by multiplying the volume of gas distillate by the density of ethanol to obtain the

percentage by volume of the ethanol yield (Nwabanne & Aghadi, 2018). On the other hand, the

researchers will employ two different tests that indicate various qualities of ethanol yield: a

triiodomethane test and a combustion test. The triiodomethane test will be used to check the

presence of carbonyl groups in the substance. Subsequently, the combustion test will determine

the ethanol's capability to burn for a chemical reaction to evaluate the functioning of ethanol.

The basic steps in the enzymatic extraction method are presented in the following figure.
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Figure 1

Flowchart Process of Enzymatic Extraction

For the sample substrate collection, the researchers will purposely select nearly rotten

Lakatan (Musa acuminata) banana peels weighing 100 g; however, Saccharomyces cerevisiae

(Brewer's yeast) and other equipment and solutions needed will be purchased online. These

banana peels will undergo several steps in deriving ethanol.

In doing the enzymatic extraction method, the following materials, chemical solutions,

and laboratory equipment are to be used by the researchers:

Materials

Substrate

● 100 g rotten banana peels (Musa acuminata)

● Coarse sand

● Brewer's yeast (Saccharomyces cerevisiae)

● Air-tight bag
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● Corks

● 200 g potatoes

● 16 g agar

● 20 g dextrose powder

● 1 ml sterile water

● 60 ml deionized water

● 800 ml distilled water

Chemical Solutions

● 1 ml sodium hydroxide solution in water

● 0.5 ml iodine solution (diluted in 0.2 ml potassium iodine solution in water)

● 2 ml Benedict’s reagent

Laboratory Equipment

● Conical flask

● Bunsen burner

● Distillation apparatus

● Laboratory grinder machine

● Laboratory thermometer

● Petri dish

● Pressure cooker

● Digital scale

● Eudiometer

● Test tube

● Pasteur pipette
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The bioethanol's quantity and quality yield of the banana peel substrate will be

determined as the researchers employ the following procedures in conducting the enzymatic

extraction method adapted from the studies of Danmaliki et al. (2016), Nichols (2016), American

Herbal Products Association (2017), and Nwabanne and Aghadi (2018). This is to ensure the

consistency of the process to limit external factors from influencing the results. The procedures

will be conducted thrice to maintain the results' consistency and overcome possible errors.

Moreover, each procedure involved in the enzymatic extraction process is detailed as follows.

I. Collection of Banana Peel and Preparation of the PDA Cultivation Medium and

Aspergillus niger Fungus

A. The researchers will obtain 100 g of nearly rotten banana peel waste. Some of the

samples will be kept isolated for the harvesting of the Aspergillus niger. In

contrast, the other samples will be washed and sun-dried for 2-3 days to obtain the

easily crushable material.

B. The dried samples will be milled using a laboratory grinder machine until it

becomes powder and will be sieved. It will be packed in a clean air-tight bag and

will be kept for the following procedure.

C. The researchers will prepare the Potato Dextrose Agar (PDA) cultivation medium

by peeling, mincing, and boiling 200 g of potatoes in 800 ml distilled water for 30

min. It will be strained to collect the decoction; then, it will be mixed with 20 g

dextrose powder and 16 g agar. The mixture will sterilize for 45 min using a

pressure cooker. It will be poured into a petri dish to solidify to obtain the PDA

cultivation medium as the isolation substrate for Aspergillus niger.

D. The researchers will isolate 0.1 g of rotten banana peels by diluting in 1 ml sterile

water and will be plated 0.1 ml of dilution on PDA substrate on a petri dish to

cultivate for 3 days to obtain the fungus.

II. Biological Pretreatment of the Banana Peel Substrate Using the Aspergillus niger for

Enzymatic Hydrolysis

A. Biological pretreatment

1. The researchers will use microorganisms such as the black rot Aspergillus

niger fungus obtained from samples to pretreat the substrate for 5-7 days

biologically. The biodelignification by the Aspergillus niger will continue

to increase until the 7th day.

2. The fungi will actively degrade lignin on a bunsen burner to extract the

cellulose needed to convert the substrate to glucose for 3 days.

B. Enzymatic hydrolysis and Benedict's test

1. The 5% inoculum will be prepared by transferring the pure and screened

Aspergillus niger from the PDA slant to a conical flask.

2. The 5% inoculums of Aspergillus niger will be combined with 11 g of

powdered banana peel sample in a conical flask and will be hydrolyzed at

120 ºC for 6 hrs using a bunsen burner and will allow it to cool.

3. The glucose solution will determine the concentration of reducing sugar

(glucose) through Benedict's test to assess the presence of reducing and

non-reducing sugars before the fermentation process.

4. The Benedict’s test will be done by heating 1 ml of the sample with 2 ml

Benedict's reagent inside a test tube in boiling water for 3-5 minutes.
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5. The colors yielded from Benedict’s test will indicate the level of glucose

concentration. Red color precipitate signifies reducing sugar, such as

glucose needed for the fermentation step.

III. Fermentation of the Processed Substrate by Using Yeast or Saccharomyces cerevisiae

A. The researchers will use the prepared activated Saccharomyces cerevisiae (yeast)

in fermenting the pretreated banana waste. The yeast cells will be suspended in

60 ml deionized water.

B. The samples will be fermented using three 125 ml conical flasks. Each flask will

contain both banana peels and the yeast cells in the deionized water. Every bottle

will be labelled regarding its fermentation period.

C. For bottle A, the fermentation process will take 1-2 days; for bottle B, the

fermentation process will take 2-3 days; and for bottle C, the fermentation process

will take 3-4 days to allow the Saccharomyces cerevisiae (yeast) to grow and

ferment the pretreated banana waste.

D. Lastly, the fermented samples will be filtered to separate the solid particles from

the liquid in the substrate. The filtrate will be kept for the distillation step.

Moreover, the volume of gas (CO2) produced will be measured by a eudiometer

for quantity analysis.

IV. Distillation of the Pretreated and Fermented Banana Waste

A. The distillation apparatus will be assembled to the intended height and weight of

the fermented banana waste until the apparatus is filled.

B. After assembling the apparatus, add the fermented banana waste substrate to start

the circulation in the condenser.

C. Start heating the flask with coarse sand for calm boiling depending on its size and

the needed temperature. Ideally, the temperature requires the boiling point of

water, which is 100 °C to separate ethanol with 78 °C boiling point.

D. When the two substrate components (water and ethanol) have been separated and

purified, the distillation will be stopped by removing the heat source from the

distilling flask and keeping the liquid circulating in the condenser until the

substrate becomes warm.

V. Determination of the Quality and Quantity of Ethanol Yield

A. Quantity of ethanol yield

1. The measured volume of gas (CO2) produced during fermentation will be

converted into moles of CO2 by using the formula:

The volume of gas x 0.042-mole liter (the RTP or room temperature and 1

atm)

2. The ethanol produced is equivalent to the product calculated from the

formula expressed through moles of ethanol unit.

B. Quality of ethanol yield through a triiodomethane test and a combustion test

1. Triiodomethane test

a. The researchers will transfer the ten drops of the fermented and

distilled ethanol into a clean and dry test tube to test its quality

content.

b. Twenty-five drops of iodine solution and an adequate sodium

hydroxide solution will be added to the fermented ethanol to

remove iodine color. This will be mixed gently for a few minutes.
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c. The researchers will gently warm the mixture to form the pale

yellow precipitate of triiodomethane, indicating the positive quality

result when the carbon-containing group will be hydrogen.

2. Combustion test

a. The researchers will first transfer about 5 ml of the fermented

banana waste ethanol to a large test tube.

b. The coarse sand will be added to make the sample boil more

calmly.

c. Hold the test tube with its holder and heat it using a bunsen burner,

indicating until the mixture is boiling.

d. The researchers will ignite the ethanol vapors by holding the open

end of the test tube to the flame. When the ethanol burns, its

quality will be signified by a pale blue flame without smoke.

After several determination tests of the quantity and quality of the ethanol yield, the

multiple test data will be studied using the mean average and sample variance. These statistical

treatments will be applied, especially on the fermentation periods, to determine whether the

longer fermentation or shorter fermentation brings quality and quantifiable ethanol yield. It will

then show the efficiency of employing enzymatic extraction. Moreover, the mean average will

validate how precise the collected data are to each other per fermentation period.

𝛴𝑥
𝑥 =
𝑁

Where:

x̄ = mean

Σx = sum of x
 22

N = number of sample data

𝛴(𝑥 − 𝑥)2
𝑠 = '
𝑁

Where:

x̄ = mean

Σx = sum of x

N = number of sample data

On the other hand, the researchers will use the sample variance formula to indicate how

the collected data set is dispersed and varied to each other.

The researchers will examine if these several steps for bioethanol production will indicate

the natural touchstone and the quantity of the ethanol yield that will indicate the efficiency of

employing enzymatic extraction using banana peels as the substrate, as recommended by Muleta

(2017).
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