Iba (Averrhoa bilimbi) Fruit as Bioethanol Disinfectant

A Research Proposal

Presented to the Faculty of

San Remigio National High School

San Remigio, Cebu

In Partial Fulfillment of the Requirements

In Inquiries, Investigation, and Immersion

Cybelle Ann Familara

Charlyn Araneta

Daniela Rose Soliva

Dynna Khey Lepon

Evanne Boyose

Kristina Elaine Tubigon

April 2022
 CHAPTER I

Background of the Study

For the past few years, we have been fighting bacteria everywhere around us, particularly

those on frequently touched surfaces. Several disinfectants have evolved, especially now that

there is a global pandemic. As a result of panic buying, prices have risen and supplies have been

depleted. There has also been an increase in the number of people who are using fruits with

antibacterial qualities to create natural and efficient disinfectants.

Alcohol destroys microbes through denaturation, a simple chemical process.

Denaturation occurs when alcohol molecules break down the proteins in the structure of germs.

If the proteins break down and lose their structure, the cells will not function properly. They lose

the ability to preserve their membranes, become dehydrated, and die quickly. Denatured alcohol

is ethanol that has been tainted with chemicals that render it unfit for human consumption. It is

often known as "methylated spirits," containing chemicals that render it inedible. This type of

ethanol has a poor taste, and a nasty odor, and is dangerous if consumed. Alcohol molecules are

amphiphile chemical substances, which means they like both water and fat. Because bacterial

cell membranes contain a fat-based and a water-based side, alcohol molecules can interact with

and dissolve the protective barrier. When this happens, the bacteria's fundamental components

are exposed and dissolve, causing the bacteria to lose shape and cease to operate. The bacteria

dies swiftly as its organs essentially melt away. At the requisite quantities — between 60 and 90

percent — alcohol may destroy a wide spectrum of microorganisms, including bacteria, viruses,

and fungus. Alcohol, for example, has the ability to destroy microorganisms such as E. coli. .

Bacteria such as E. coli, Salmonella, and Staphylococcus aureus can all-cause sickness. Other

bacteria are becoming more resistant to the effects of alcohol-based disinfectants, such as

1 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

Enterococcus faecalis. Herpes, hepatitis B, HIV, influenza, rhinoviruses, and coronaviruses have

all been found to be killed by alcohol.

Bioethanol disinfectants derived from citrus fruits are now available. Citrus fruits are

commonly utilized due to their acidity, and acid pH is one of the elements that can prevent

bacterial development, causing the internal pH of bacterial cells to decrease and inhibiting

bacterial cell growth. Knowledge of fruits with antibacterial characteristics is valuable since such

knowledge may be useful in revealing a new disinfectant in the form of alcohol. Oranges and

lemons are the most common citrus fruits they use. Given that they are not native to our country,

these two citrus fruits are quite costly. That is why it is critical to choose a citrus fruit that is

prevalent in our area; Iba (Averrhoa bilimbi) is abundant in our area and often goes to waste due

to its abundance. This plant's fruits have antibacterial activity against E. coli, Salmonella typhi,

Staphylococcus aureus, and Bacillus cereus are all examples of bacteria.

With the high demand of alcohol during this time of pandemic and the discoveries for new

alternatives made by many researchers to address the issue paves a way for the researchers to conduct

their own study. Iba (Averrhoa bilimbi) fruit, a medicinal plant that is proven to have antimicrobial

properties and is commonly found in the area, made the researchers point their focus on the said

attributes. Which, as the outcome of this experience, leads to the idea that making bioethanol disinfectant

out of Iba fruit will address this issue.

2 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

Conceptual Framework

Independent Variable Dependent Variable

-Alcohol content of the bioethanol

Iba (Averrhoa bilimbi) as Bioethanol -Activity in inhibiting the growth of

microorganisms that are found in most
commonly touched places.

The bioethanol disinfectant made from Iba (Averrhoa bilimbi) fruit will be tested to

determine its effectiveness. To determine whether the disinfectant contains ethanol, it will be put

through a flammability test and a hydrometer analysis. Afterward, we will test its effectiveness

on the microorganisms that are found in commonly touched places; we will test its antimicrobial

susceptibility using the disk diffusion method by measuring its zone of inhibition and the time it

will take to prevent the growth of the microorganisms.

Statement of the Problem

The main goal of the study is to produce and test the effectiveness of the bioethanol

disinfectant using Iba (Averrhoa bilimbi) fruit. Specifically, this study intends to:

1) check for the presence of ethanol through:

a) flammability test; and

b) hydrometer analysis

2) check for its antimicrobial susceptibility using the disk-diffusion method:

a) measuring the zone of inhibition

3 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

 3) compare the produced bioethanol using Iba (Averrhoa bilimbi) fruit to the

commercially manufactured alcohol.

Research Hypothesis

Null Hypothesis: There is no significant difference between the effectiveness of the produced

bioethanol using Iba (Averrhoa bilimbi) fruit to the commercially manufactured alcohol.

Significance of the Study

The purpose of the research is to create a bioethanol disinfectant from the extract of the Iba

(Averrhoa bilimbi) fruit. People can use this disinfectant to sanitize commonly touched surfaces

in their homes to keep them safe, especially now that we are in the midst of a pandemic. With

this in mind, the findings of the study will be beneficial to the following specific community:

● Environment. The findings of this study will benefit the environment since people will

be motivated to plant not only Iba but also other plants that have antibacterial qualities

and have a high probability of becoming disinfectants due to the benefits they provide.

● Future researchers. They will benefit from the study's findings because they will know

that Iba fruit extract can be converted to alcohol, and then use the information to aid in

their research, whether they use the alcohol or are inspired to make alcohols from other

plants or fruits with antibacterial activity.

● Government. This will also benefit the government, particularly local governments

because they will be able to save money on commercially manufactured alcohol, which

they will give to public locations in the midst of a pandemic.

4 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

 ● Medical personnel. This will benefit medical personnel because it will provide them

with more ethanol disinfectant supplies, which they will need because it is commonly

utilized in their fields. This will also save money on the ethanol disinfectant, which is

especially important since the pandemic is still ongoing.

● Schools. The findings of this study will assist the school by allowing us to make our

bioethanol disinfectant and save money. It will also assist students and teachers because

they will be able to add an extra activity that will potentially boost the learner's learning.

Scope and Limitation of the Study

The scope of the study is limited to the production of a disinfectant in the form of alcohol

using Iba fruit (Averrhoa bilimbi). This study is conducted to know if the produced alcohol will

be able to disinfect the commonly touched surfaces from microorganisms. It considers the anti-

bacterial activity of Iba (Averrhoa bilimbi) fruit on one of the commonly found bacteria which is

Staphylococcus aureus. 

The materials that we are going to use are Iba (Averrhoa bilimbi) fruit, distilled water,

yeast, white sugar, wine bottles, cork, glass, pitcher, ladle, strainer, juicer, and funnel. However,

because the time is limited due to the restrictions of the pandemic the fermentation process for

each solution, wherein solution 1 is fermented for a month and solution 2 for two weeks, will be

conducted in the researchers’ home, while the rest of the process will be conducted in San

Remigio High School’s DOST Chemistry Laboratory. The study started in February 2022 and

will be accomplished in July 2022. 

5 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

Definition of Terms

● Alcohol refers to a commonly used disinfectant and the positive control of the study

● Antimicrobial Susceptibility Test refers to the measurement of the susceptibility of bacteria

to antibiotics

● Bioethanol Disinfectant refers to the resulting alcohol after the fermentation process of Iba

(Averrhoa bilimbi) fruit extract.

● Disk-diffusion Method refers to an antimicrobial susceptibility test in which

microorganisms are cultivated in an agar plate and bioethanol disinfectant is applied to see

how it reacts with the microorganisms

● Flammability Test refers to a test to determine whether the Iba fruit bioethanol disinfectant

contains ethanol by lighting it with matches and it will produce a smokeless blue flame

● Hydrometer Analysis refers to a test to determine how high the concentration of ethanol in

the bioethanol disinfectant

● Iba fruit refers to a citrus fruit that is the main component of the disinfectant

● Solution 1 refers to an Iba fruit bioethanol disinfectant fermented for 2 weeks

● Solution 2 refers to an Iba fruit bioethanol disinfectant fermented for a month

● Zone of Inhibition refers to an area where bacteria are unable to grow in this section of the

agar plate, due to the presence of the bioethanol disinfectant

6 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

 CHAPTER II

Review of Related Literature

Theoretical Background

Disinfectant 

The review of this study focuses on the benefits of disinfectants. Disinfectants are an

agent used to destroy microbes on inanimate items, such as creosote or alcohol Disinfectants and

antiseptics are both germicidal; however, antiseptics are used primarily on live tissue. The ideal

disinfectant would kill bacteria, fungi, viruses, and protozoans quickly, would not corrode

surgical instruments, and would not destroy or discolor the materials on which it is used. These

commonly found bacteria on surfaces are Staphylococcus aureus, Salmonella, and E. coli

(Escherichia coli) which can cause several diseases and infections. (Petruzzello, M., et al.,

2021). 

Denaturation 

Denaturation, a basic chemical process, is how alcohol kills microorganisms. When

alcohol molecules tear down the proteins in the structure of germs, this is known as denaturation.

The cells can't operate properly if the proteins break down and lose their structure. They lose

membrane protection, become dehydrated, and perish soon. Either ethanol (ethyl alcohol) or

isopropanol are the most often utilized alcohol-based sanitizers (isopropyl alcohol). Ethanol is

chemically identical to alcoholic beverages. Isopropanol is sometimes referred to as rubbing

alcohol. Both are reasonably good at removing bacteria and viruses from your skin and various

surfaces. Although it depends on the type of microorganism you wish to kill, ethanol is a more

potent trustee Source than isopropanol in general (Vandergriendt, C., 2021). 

7 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant
             

Alcohol

Bacteria are deterred by the use of alcohol. Ethyl alcohol (70%) is a potent broad-

spectrum germicide that is usually thought to be superior to isopropyl alcohol. Alcohol is

frequently used to disinfect tiny surfaces (such as the rubber stoppers of multiple-dose

prescription bottles and thermometers) and, on rare occasions, the exterior surfaces of equipment

(e.g. stethoscopes and ventilators) (World Health Organization, 2014). 

Ethanol, commonly known as alcohol, ethyl alcohol, or grain alcohol, is a colorless,

transparent liquid that is used to make alcoholic beverages such as beer, wine, and brandy.

Ethanol is used in a variety of items, from personal care and cosmetic products to paints and

varnishes to gasoline, because it dissolves easily in water and other organic molecules. Ethanol is

a naturally occurring byproduct of plant fermentation that can be made by hydrating ethylene.

Ethanol is a frequent ingredient in hand sanitizers because it is good at killing microorganisms

such as bacteria, fungus, and viruses. In cases where soap and water are not accessible, the US

Centers for Disease Control and Prevention (CDC) suggests using hand sanitizers. Hand

cleanliness is also a critical component of preventing the spread of COVID-19. SARS-CoV-2,

the coronavirus strain that causes COVID-19, can be inactivated with hand sanitizers or alcohol-

based hand rubs (ABHR), (Department of Health and Human Services, 2021).

Staphylococcus Aureus

Staphylococcus aureus is a gram-positive bacterium that causes a variety of illnesses,

including skin infections, bacteremia, endocarditis, pneumonia, and food poisoning. The

bacterium was once a major nosocomial pathogen, but epidemiologically separate clones began

8 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

to arise in community settings. S. aureus produces a number of virulence factors that aid in the

establishment of infection by aiding tissue adhesion, tissue invasion, and immune evasion. S.

aureus is unique in that it can develop resistance to various drug classes. aureus, a difficult

infection to eradicate. S. aureus's emergence and spread Methicillin-resistant Some strains of S.

aureus are those that are resistant to the antibiotic methicillin. MRSA (methicillin-resistant

Staphylococcus aureus) was responsible for significant illness, death, and treatment costs

(Gnanamani et. al. 2017).

From clinical samples taken from the study of “Biochemical and Molecular Analysis of

Staphylococcus aureus Clinical Isolates from Hospitalized Patients”, a total of 100

Staphylococcus aureus isolates were collected. Different biochemical assays were used to

identify the probable Staphylococcus aureus clinical isolates phenotypically. PCR was used to

identify the bacteria using species-specific 16S rRNA primer pairs, and 100 isolates were

determined to be positive for Staphylococcus aureus. Several microbiological diagnostic assays,

including gelatin hydrolysis, protease, and lipase tests, were used to further examine the screened

isolates. Gelatin hydrolysis, protease, and lipase activities were detected in 78 percent, 81

percent, and 51 percent of the isolates, respectively (Karmakar et. al. 2016).

Bioethanol

Bioethanol is an alcohol derived from cereals or sugar beets, as well as agricultural waste.

There are enormous tanks in a bioethanol factory where yeasts convert the sugar-containing

biomass to ethanol (Graf, P., Et al., 2020). 

A research study acquired from the University of Malaya Research Grant (UMRG) was

conducted by Hossain, S., Yusoff, W.M., and Veettil, V.. This study is about the development of

biomaterials produced from fruit biomass (bio-antiseptics, bio solvents, biofilms, and biofuels)

9 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

that stimulates the widespread usage of biomass. As a result, fruit wastes can be recycled to

produce bioethanol, which can be used as an antiseptic and a bio-solvent. The goal of the study

was to look into optimizing bioethanol production and evaluating bioethanol as an anti-fermenter

and antiseptic. Through a fermentation bioprocess involving yeast, rotten banana, grape, and date

biomass were employed. The samples were washed completely in distilled water, sliced using a

sterile knife, and blended in a sterilized automatic juice blender. At 3 g L–1 yeast concentration

and 30°C, bioethanol yield was higher in dates biomass than grape and banana biomass. The

bioethanol produced from dates of biomass had the lowest pH. In the bioethanol made from

banana biomass, the TSS and glucose levels were lower. At 3 mg L–1 yeast concentration in

dates of biomass, the lowest viscosity, and the acid value was discovered. Chemical elements

such as Ca, P, Fe, Pb, Cu, and Si also met the standard specification's requirements. Grape juice

mixed with bioethanol was anti-fermented for two days, but the control juice began to rot faster

on the first day. In the dates bio, the lowest bacterial colony development was detected. The

findings revealed that the bioethanol produced was of good quality and may be used as an

antimicrobial and bio-solvent made from fruit biomass.

Iba Fruit (Averrhoa bilimbi)

Iba (Averrhoa bilimbi) is a medicinal plant in the Oxalidaceae family. The genus

Averrhoa was named after Ibn Rushd, an Arab philosopher, physician, and Islamic jurist who

was also known as Averroes A. Averrhoa carambola is linked to bilimbi (carambola, starfruit).

Its origins may be traced back to Southeast Asia. Iba is a tiny tree that may reach a height of 15

meters and has sparsely distributed branches. It bears compound leaves that are 5–10 cm long

and have twenty to forty leaflets apiece. The hairy leaves have pinnate forms and form clusters at

10 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

the ends of the branches. Cauliflorous trees have 18–68 blooms in panicles that grow on the

trunk and other branches. The blooms are heterostylous, with petal lengths ranging from 10–30

m and colors ranging from yellowish green to reddish-purple. The fruits form on the bare stem

and trunk. The fruits are greenish in hue, with solid and luscious flesh that softens as it ripens.

The fruit juice is exceedingly sour and acidic. It has a high value in alternative medicine, as

proven by a large quantity of study on it. As a result, we set out to create an up-to-date and

comprehensive assessment of A. bilimbi's traditional and folk medicinal applications,

phytochemistry, and pharmacology are all covered. (Alhassan, A. M., Ahmed, Q. U., 2016).

A research study acquired from Isabela School of Arts and Trades-Technical Education

and Skills Development Authority conducted by Madarang, K.. The role of housekeeping in the

hospitality and health industry is to eliminate hazards and reduce the risk in the workplace.

However, commercial toilet bowl cleaners, bleaches, and muriatic acid, which are toilet cleaning

agents used by housekeepers, have a strong chemical content that can be harmful to the health of

the workers. Hence, the researcher came up with a solution that will create a new disinfecting

agent at the same time stain removal that is natural and less harmful to workers and guests in

hotels and hospitals. To make the natural cleaning agent, the fruit is manually extracted with the

use of a mechanical presser. The extracted juice is sprayed onto the stained toilet bowls and tiles.

The effectiveness was observed by soaking the stains with the extracts for different durations of

time. The stains on the tiles, that were used as a reference are observing the potency of the

product, were removed in not less than 10 minutes. The efficacy of the natural cleaner depends

on the bilimbi fruit that was used. The younger the extracted fruit, the stronger and faster it can

remove stubborn dirt and stains on the tiles. The product came out effective as the commercial

toilet cleaner for tiles and toilet bowls. Moreover, many studies have shown that “Averrhoa

11 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

bilimbi”, the scientific name for Cucumber Tree (Kamias), has an antimicrobial effect and stain

removal qualities. The product is eco-friendly. It doesn't contain toxic chemicals that would harm

the environment.

Ethanol Fermentation 

Alcoholic fermentation is a biotechnological process that converts glucose into ethyl

alcohol and carbon dioxide using yeast, bacteria, or a few other microbes. Alcoholic

fermentation begins with yeasts breaking down carbohydrates to generate pyruvate molecules, a

process that is known as glycolysis (Buratti, S., Benedetti, S., 2016).

Ethanol fermentation is one of the biotechnology industry's oldest and most essential

fermentation methods. Annually, over 4.5 billion gallons of ethanol are generated from corn in

the United States alone. Many microorganisms, including bacteria and yeasts, may ferment

carbohydrates to create ethanol as the primary product. Currently, yeast is used mostly in

industrial ethanol production. (Yang, S.T., 2013)

The cellular lipid content of yeast was altered by lowering the temperature, which

increased the degree of unsaturation at the start of fermentation and decreased the chain length as

fermentation progressed. Medium-chain fatty acids, primarily dodecanoic acid, were also

discovered in the cell phospholipids. Wines made from grapes must be more fragrant and contain

less volatile acidity than those made with a synthetic medium (Rozes, N. 2012).

Yeast

Yeast is a single-celled creature known as Saccharomyces cerevisiae that requires food,

temperature, and moisture to survive. Through fermentation, it turns its food—sugar and starch

12 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

—into carbon dioxide and alcohol. Carbon dioxide is what causes baked items to rise

(Pellegrinelli, C., 2021)

The role of yeast in food fermentation includes the production of alcohol, the

improvement of texture through leavening, preservation through acidification and the production

of the killer toxins, the improvement of nutritive values and the removal of anti-nutritional

factors, the production of value-added bioactive peptides, and the production of vitamins (Rai, A.

K., 2017). 

Distillation

Distillation is a technique for selective evaporation and condensation to separate the

component ingredient from a miscible fluid mixture. Many microfluidic applications, such as

food preparation, gas/liquid separation, and biochemistry, rely on distillation. However, at the

microscale, surface tension forces outnumber gravitational and hydrodynamic forces, limiting the

efficacy of standard gravity-based distillation processes. As a result, in recent years, a variety of

new ways for separating miscible fluid mixtures at the microscale have been developed, based on

techniques such as vacuum forces, capillary forces, centrifugal forces/gravity effect, and others.

(Yang, R.J., et al., 2017).

A distillation apparatus, often known as a still,' consists of a vessel for plant material and

water, a condenser to cool and condense the vapor produced, and a collection method, or

receiver.' In the distillation vessel, material from the appropriate area of the plant for extraction is

immersed in water. This is then heated to the boiling point, at which time the volatile oils are

carried away by steam (water vapor). Because the temperature will not surpass 100 degrees

Celsius, the water protects some components by preventing overheating (the boiling point of

13 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

water at normal pressure). However, distillation is a time-consuming procedure, and the water

may harm other molecules (Clarke, S., 2018).

14 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

Flammability Test

The capacity of a chemical to burn or ignite, resulting in fire or combustion, is referred to

as flammability. Fire testing determines the degree of difficulty necessary to induce a chemical

to burn. Materials are often classified as extremely flammable, flammable, or non-flammable

(Pro, L., 2016)

          The ease with which a material can be ignited, the intensity with which it burns and

releases heat once ignited, its propensity to spread fire, and the rate at which it produces smoke

and harmful combustion products during gasification and burning are all considered

flammability factors. Data from many laboratory tests, sometimes paired with some type of

analysis or modeling to appropriately interpret the results, may be required for a full evaluation

of a material's overall flammability (Lautenberger, C., et al., 2017). Flammability testing

examines the ease with which a material or completed product may ignite or burn when exposed

to or utilized near fire or heat (Biswas, S., 2021). 

Ethanol fires also called "invisible fires," are extremely dangerous. Ethanol has a blue

flame, emits no smoke, and is nearly imperceptible to the human eye. If you can't see the fire,

you could easily become engulfed in it before you realize it (Firecritic, 2013).

Hydrometer Analysis 

Hydrometers are instruments that use the principles of buoyancy and liquid displacement to

measure liquid displacement. Hydrometers are hollow-body items that have been calibrated across a

specific range and have a consistent volume. The weight of a solution displaced by the hydrometer is

directly proportional to the density of the displaced liquid (beer, wort, or alcohol solutions); readings are

subject to errors due to the presence of suspended solids and gases; readings are subject to errors due to

the presence of suspended solids and gases. When employing such instruments, liquid surface tension

15 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

effects with hydrometer cylinders and the hydrometers themselves must also be considered. Temperature

compensation is also essential when using hydrometers due to the thermal expansion of liquid volumes

and glass, and temperature correction tables are included with each calibrated hydrometer (MEBAK,

2013).

Hydrometer readings become progressively imperfect measurements of sugar level as

fermentation occurs. This is due to the effect of alcohol on specific gravity values. However, in dry table

wines, specific gravity can still be used to accurately predict the end of fermentation. Correction tables

are required in the case of sweet, fortified wines (Jackson, R., 2020). 

Antimicrobial Susceptibility Test

Antimicrobial susceptibility testing (AST) is a laboratory procedure used by medical

technologists (clinical laboratory scientists) to determine which antimicrobial regimen is best for

each patient. It facilitates the evaluation of treatment services offered by hospitals, clinics, and

national infectious disease control and prevention programs on a broader scale. Due to alterations

in bacterial DNA, researchers have now had to develop continuous surveillance efforts for

resistance patterns. Depending on the laboratory test menu that they offer, clinical laboratories

use a variety of methodologies. The disk diffusion and minimum inhibitory concentration (MIC)

methods are two of these methods. Commercial technologies that use both phenotypic and

genotypic assessment of bacterial resistance are already available in health centers and hospitals.

(Bayot, M., Bragg, B. 2021).

Nutrient Agar

Nutrient agar is a multipurpose medium that can be used to grow a variety of non-fibrous

organisms. Nutrient agar is widely used because it promotes the development of a wide range of

16 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

bacteria and fungi and contains many of the nutrients required for bacterial growth. Before

biochemical or serological testing, nutrient agar can be employed for quality control and purity

verification. It's a basic recipe that offers the nutrients required for the replication of a large

number of low-demand bacteria (Aryal, S., 2018).

Disk-diffusion Method

The disk diffusion technique (DDM) is classed as an agar diffusion method (ADM),

because the plant extract to be tested diffuses from its reservoir across the agar medium seeded

with the test microorganism. A filter paper disk is typically used as the reservoir, which is placed

on top of an agar surface. After incubation, if the studied plant extracts or isolated chemicals are

microbiologically active, an inhibitory zone forms around the filter paper disk. The antibacterial

efficacy of plant extracts or specific components is best described by the diameter of the

inhibition zone. The diffusion of the water-insoluble EO and its components from a filter paper

disk into the agar medium is insufficient, hence DDM is not an acceptable approach for

lipophilic extracts (e.g., EOs) (Horvath, Gy. 2016).

Zone of Inhibition

The Zone of Inhibition is a circular area surrounding the antibiotic's active site where

bacteria colonies do not develop. The zone of inhibition can be used to determine a bacteria's

sensitivity to an antibiotic. Image processing can be used to automate the process of measuring

the diameter of this Zone of Inhibition. In this paper, a computer vision-based technique is

created to detect the bacteria's inhibitory zones. This study offers a useful method for estimating

the radius of the Zone of Inhibition by sketching contours and selecting the appropriate threshold

17 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

value. Using the computed Zone of Inhibition and the provided standard values, this study also

indicates whether a particular bacteria is susceptible or resistant to the applied antibiotic.

(Bhargav, H.S., et al., 2016).

18 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

 Chapter III

METHODOLOGY

This chapter describes the procedures to be utilized to carry out this research study. This

chapter will discuss the material collection and preparation, extract preparation, fermentation,

filtration, and distillation processes, alcohol content testing, and antimicrobial susceptibility

testing that the researchers propose to be used as data collection methods for hypothesis testing

and data analysis.

I. COLLECTING OF MATERIALS

The ripe 2960 grams of Iba fruit samples will be collected in the researchers' area and

transported to one of the researchers' homes for fermentation. The researchers will also provide

the necessary materials for this process, such as a fermentation vessel, a cork stopper (which will

act as an airlock), a temperature scanner, cheesecloth, and a blender or a juicer. The laboratory

equipment will be borrowed from San Remigio NHS Chemistry Laboratory; which includes, a

distilling flask, glass tubing, rubber tubing, Erlenmeyer flask, graduated cylinder, evaporating

dish, petri dish, and an alcohol lamp.

II. PREPARING OF MATERIALS

The ripe Iba fruit samples will be washed using distilled water to ensure that there are no

contaminants. The materials that will be used will also be decontaminated using hot distilled

19 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

water to prevent any foreign particles which can affect the results. And then the ripe Iba fruit will

be weighed.

III. PREPARING OF EXTRACT

A total of 1480 grams of ripe Iba fruit will be trimmed and then grounded using a blender

or a juicer. Using a cheesecloth, the ground Iba will be filtered to eliminate the seeds and pulp.

IV. FERMENTATION PROCESS

A. Preparing of Solvent for Fermentation

The 1000 ml of Iba extract will be brought to a boiling order to eliminate other foreign

bodies which may affect the results. 300 grams of white sugar will be added gradually to the

solvent until dissolved. The sugar will make the yeast ferment faster and more efficiently than

with no sugar. And then the mixture will be cooled down to room temperature.

B. Activating of Yeast

The 3.5 grams of yeast will be added to a 50 mL room temperature distilled water. It will

be stirred gently, and a little sugar will be added to test if the yeast is activated (if bubbles form).

C. Combining of Mixtures

The room temperature Iba mixture will be placed into a fermentation vessel. Then, the

activated yeast will be added gradually into the mixture and stirred until they are well combined.

20 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

D. Preparing for Fermentation

i. Solution One

The distilled cork stopper will be placed on the rim of the vessel acting as an

improvised airlock. The solution will then be stored in a warm dark place and will

be fermented for two weeks.

ii. Solution Two

The distilled cork stopper will be placed on the rim of the vessel acting as an

improvised airlock. The solution will then be stored in a warm dark place and will

be fermented for at least a month.

V. Filtration Process

The fermented solution will be filtered using filter paper or a strainer to get rid of the

excess yeast.

VI. Distillation Process

A. Preparing of Improvised Distillation Apparatus

A distilling flask will be attached to an iron stand using iron clamps and alcohol lamps

will be prepared. The glass tubing will be cut into the proper size. Using an alcohol lamp as a

source of heat, the glass tubing will be heated up and bent to the proper angle needed. One end

of the glass tubing will be inserted into a cork with a hole on top and a cork will be used to seal

the Erlenmeyer flask. The other end of the glass tubing will be connected to the distilling flask

using rubber tubing.

21 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant
B. Distillation

The fermented solution will be placed inside the distilling flask while the top will be

sealed with a cork. The solution will then be boiled. A damp cool cloth will be placed on the

distilling flask’s arm to condense the vapor created.

VII. Testing for Alcohol Content

A. Hydrometer Analysis

The fermented solution will be placed into a 100 mL graduated cylinder and will be

stirred slowly to get rid of any bubbles formed. The hydrometer will be slowly placed into the

solution until it floats. If most of the hydrometer is submerged, it means that there is a higher

alcohol content than sugar in the solvent when its specific gravity reads slightly below 1.000.

B. Flammability Test

The solution will be placed in an evaporating dish, then placed in a lit match to test if it

will combust. Ethanol is a colorless, volatile liquid with a faint odor. It should produce a

smokeless blue flame that is not always visible in natural light.

VIII. Antimicrobial Susceptibility Test

A. Agar Plate Preparation

The culture medium will be prepared by dissolving 15.4 grams of Muller-Hinton agar into 400mL

of distilled water. Normal saline solution (NSS) will also be prepared for the inoculum. All prepared

22 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

materials will be sterilized using an autoclave at 121°C for 15 minutes. Petri dishes will be sterilized

using the dry oven at 160°C for 2 hours.  

B. Culturing of Microorganisms

i. Preparation of Inoculum

24 hours of culture of Staphylococcus aureus will be obtained. The microorganism will

be inoculated to the sterile saline solution on tubes using an inoculating loop until it becomes

comparable to the 0.5 McFarland standard. The bacterial suspension will be performed using

aseptic techniques inside the biological safety cabinet level 2.

ii. Disk-diffusion Method

A sterile Muller-Hinton agar will be cooled to 50-55°C. The medium that will be

cooled was poured into the individual sterile Petri dishes. Inoculum of test bacteria which

is comparable to the 0.5 McFarland standard will be swabbed uniformly on solidified

sterile Muller-Hinton agar plates using sterile cotton swabs. Sterile disks of 6mm

diameter (Whatman no. 1) will be soaked with the given solvent. The disks will be placed

on the swabbed plates aseptically using sterile forceps. Forceps will be sterilized by

soaking them in 80% ethanol and will be passed onto the flame. The inoculated plates

will be incubated at 37°C for 24 hours for bacteria in an upright position and the zones of

inhibition that formed around the wall will be measured using a micro caliper. A brand X

alcohol will be introduced to the plates after the preceding procedure. Diameters of the

inhibition zone by solvent and the known alcohol will be compared. All procedures will

be done in three trials with three replicates at the Applied Microbiology Laboratory,

University of San Carlos, Talamban, Cebu City, Philippines. 

23 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

24 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant
 iii. Disposal of Culture Medium

All inoculated plates will then be disposed of by the decontamination process. It is

conducted by sterilizing the inoculated Muller-Hinton agar with bacteria in the autoclave

for 121°C for 15 minutes. 

25 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

TIMELINE
DATE ACTIVITIES

March 26, 2022 The 1480 grams of Iba fruit was extracted

to make a 1-liter juice ready for fermentation.

The solution 2 will be fermented for a month.

April 02, 2022 The 1480 grams of Iba fruit was extracted

to make a 1-liter juice ready for fermentation.

The solution 1 will be fermented for 2 weeks.

April 18-19, 2022 Solution 1 will undergo distillation so that

we will be left with the bio ethanol.

April 25-26 2022 Solution 2 will undergo distillation so that

we will be left with the bio ethanol.

April 28-29, 2022 Solution 1 and 2 will undergo different test

to determine whether we made a bio-ethanol

disinfectant. This test are the flammability test

and the hydrometer analysis.

First week of May Solution 1 and 2 will be brought to the

University of San Carlos for its Antimicrobial

26 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

 Susceptibility test.

27 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

REFERENCES

Alhassan, A.M. and Ahmed, Q.U. (2016). Averrhoa bilimbi Linn.: A review of its ethnomedicinal

uses, phytochemistry, and pharmacology. Accessed on March 07, 2022, from

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5314823/

Aryal, S. (2021, May 05). McFarland Standards- Principle, Preparation, Uses, Limitations. Retrieved

April 07, 2022, from https://microbenotes.com/mcfarland-standards/

Azhar, H., Abdulla, R., et al. (2017). Yeast in sustainable bioethanol production: A review. Accessed

on March 13, 2022, from

https://www.researchgate.net/publication/314263476_Yeasts_in_sustainable_bioethanol_producti

on_A_review

Bayot, M. & Bragg, B. (2021). Antimicrobial Susceptibility Testing. Accessed on April 01, 2022,

from https://www.ncbi.nlm.nih.gov/books/NBK539714/

Bhargav, H. S., et. al.,(2016). Measurement of the Zone of Inhibition of an Antibiotic. Accessed

on March 08, 2022, from https://ieeexplore.ieee.org/document/7544871?fbclid=IwAR3sMNIzK-

OUQaDB721a2QD-7pta_Mrhp6t_DRhb11SO5ew5pyOeyRiPajw

Bhargav, H.S., et al. (2016). Measurement of the Zone of Inhibition. Accessed on March 13, 2022,

from https://ieeexplore.ieee.org/document/7544871

Britannica, T. Editors of Encyclopaedia (2021). disinfectant. Encylopedia Britannica. Accessed on

March 07, 2022, from https://www.britannica.com/technology/disinfectant

28 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant
Buratti, S. and Benedetti, S., (2016). Alcoholic Fermentation Using Electronic Nose and Electronic

Tongue. Accessed on March 08, 2022, from https://www.sciencedirect.com/topics/agricultural-

and-biological-sciences/alcoholic-fermentation?

fbclid=IwAR1QwNPqHCwC5BZk6VXZQvyF1q2il6vD-

KXzJw0KReslDgO2bjJufpf929s#:~:text=Alcoholic%20fermentation%20is%20a

%20complex,properties%20of%20the%20fermented%20foodstuffs

Busic, A., et. al. (2018). Bioethanol Production from Renewable Raw Materials and Its Separation

and Purification. Accessed on March 07, 2022, from

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233010/?

fbclid=IwAR0MvF_4DskjXSyJ6BOZI36KoVv-_dlgB3Wpaqm2FJMW-9kJtpOb4qmfnH4

Cat, D. (2019). How To Make Homemade Lemon Vinegar Cleaning Spray. Accessed on March 07,

2022, from https://homesteadandchill.com/homemade-lemon-vinegar-cleaning-spray/

Corona: Bioethanol for disinfectants. Accessed on March 07, 2022, from

https://biooekonomie.de/en/news/corona-bioethanol-disinfectants

De Vasconcelos, J.N. (2015). Ethanol Fermentation. Accessed on March 07, 2022, from

https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/ethanol-fermentation

Distillation Apparatus. Accessed on April 03, 2022, from

http://jupiter.plymouth.edu/~wwf/distillation.htm

EcoLink. (n.d.). Denatured Alcohol vs Rubbing Alcohol. Retrieved April 06, 2022, from

29 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

 https://ecolink.com/info/denatured-alcohol-vs-rubbing-alcohol/

Ekawati, E.R. and Darmanto W. (2019). Lemon (Citrus limon) Juice Has Antibacterial Potential

against Diarrhea-Causing Pathogen. Accessed on March 07, 2022, from

https://iopscience.iop.org/article/10.1088/1755-1315/217/1/012023/pdf

Fermentation. Accessed on March 07, 2022, from

https://www.nordzuckerireland.ie/ie/about-sugar/functional-properties-of-sugar/

fermentation#:~:text=A%20little%20sugar%20

Functional properties of sugar. Accessed on March 21, 2022, from

https://www.nordzuckerireland.ie/ie/about-sugar/functional-properties-of-sugar/

fermentation#:~:text=A%20little%20sugar%20

Gargalo, C., et al. (2014). Bioethanol Production. Accessed on March 07, 2022, from

https://www.sciencedirect.com/topics/chemistry/bioethanol-production#:~:text=The

%20bioethanol%20production%20relies%20on,stage%20where%20bioethanol%20is

%20produced

Gnanamani, A., Hariharan, P., & Paul- Satyaseela, M. (2017, March 08). Staphylococcus aureus:

Overview of Bacteriology, Clinical Diseases, Epidemiology, Antibiotic Resistance and

Therapeutic Approach. Retrieved April 07, 2022, from

https://www.intechopen.com/chapters/54154

Hall-Geisler, K. (2021). What’s the Difference Between Ethyl Alcohol and Isopropyl Alcohol?.

30 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

 Accessed on March 07, 2022, from https://science.howstuffworks.com/difference-between-ethyl-

and-isopropyl-alcohol.htm

Helmenstine, A. M., (2019). Hydrometer Definition in Science. Accessed on March 08, 2022,

from https://www.thoughtco.com/definition-of-hydrometer-605226?

fbclid=IwAR1ja8XGca2JizIvlZvTNyYXtjJPq3HC0zeOrP-znaaBBKyqyIyV_MUaBnk

Helmenstine, A. M., (2020). What Is Distillation? Chemistry Definition. Accessed on March

08, 2022, from https://www.thoughtco.com/what-is-distillation-601964#:~:text=Distillation

%20Definition,points%2C%20into%20the%20gas%20phase

Helmenstine, A.M., (2020). Understand How Distillation Works. Accessed on March 08,

2022, from https://www.thoughtco.com/what-is-distillation-601964#:~:text=Distillation

%20Definition,points%2C%20into%20the%20gas%20phase

Higuera, V. and Jewell, T (2020). Bacteria and viruses in the home. Accessed on March 07, 2022, from

https://www.healthline.com/health/germy-places

Horvath, G.Y., (2016). Disk Diffusion - an overview. Accessed on March 08, 2022, from

https://www.sciencedirect.com/topics/immunology-and-microbiology/disk-diffusion#:~:text=The

%20disk%20diffusion%20method%20

How to know the type of alcohol by colour of flame and duration of combustion?. Accessed on March

07, 2022, from http://rxmarine.com/ethanol-ipa-propanol-methanol-flame-test

https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/ethanol-fermentation

31 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

Karmakar, A., Dua, P., & Ghosh, C. (2016, April 24). Biochemical and Molecular Analysis of

Staphylococcus aureus Clinical Isolates from Hospitalized Patients. Retrieved April 07, 2022,

from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4904573/

Khandaker, M.M., et al. (2020). Bio-Ethanol Production From Fruit and Vegetable Waste. Accessed on

March 07, 2022, from https://www.intechopen.com/chapters/74050

Laboratory Methodologies For Bacterial Antimicrobial Susceptibility Testing. Accessed on March

24, 2022, from https://www.oie.int/fileadmin/Home/eng/Our_scientific_expertise/docs/pdf/

GUIDE_2.1_ANTIMICROBIAL.pdf

Levy, E. (2020). How to Use a Fermentation Airlock. Accessed on March 21, 2022,

from https://www.storeitcold.com/how-to-use-a-fermentation-airlock/

Loresca, K. (2020). Learn how to make your own alcohol. Accessed on March 07, 2022, from

https://www.youtube.com/watch?v=sPXdHP0DFyA

Lui, H., et al. (2017). Impact of Sucrose Addition on the Physiochemical Properties and Volatile

Compounds of “Shuangyou” Red Wines. Accessed on March 07, 2022, from

https://www.hindawi.com/journals/jfq/2017/2926041/

Mikell, M., et. al. (2021). Alcohol Fermentation: Definition, Equation & Process.

Accessed March 08, 2022, from https://study.com/academy/lesson/alcohol-fermentation-

definition-equation-process.html?fbclid=IwAR3lWUXkm-

32 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

 Jlr_vVL6qdABdLZKm4BYsOsclIknTAQpC0fq2Xi3y4SVXKLEo#:~:text=The%20main

%20purpose%20of%20alcohol,basic%20overview%20of%20alcohol%20fermentation

Morata A. and Loira I. (2017). Yeast: Industrial Applications. Accessed on March 07, 2022, from

https://books.google.com.ph/books?id=sGqQDwAAQBAJ&printsec=frontcover&dq=importance

%20of%20yeast%20in%20alcohol

%20making&hl=en&sa=X&ved=2ahUKEwjHsojWnrP2AhWTDd4KHZk5DWEQ6wF6BAgHE

AU#v=onepage&q=importance%20of%20yeast%20in%20alcohol%20making&f=false

Nasrin, B., et. al. (2012). In vitro antimicrobial and cytotoxicity screening of hexane, chloroform

and ethyl acetate extracts of Lablab purpureus (L.) leaves. Accessed March 07, 2022, from

https://scihub.org/ABJNA/PDF/2012/2/ABJNA-3-2-43-48.pdf

Papers for Bioethanol. Accessed on April 03, 2022, from

https://www.connectedpapers.com/search?q=bioethanol

Parker, N., et al. (2021). The Effects of pH and Temperature on Microbial Growth. Accessed on

March 23, 2022, from

https://bio.libretexts.org/Courses/Manchester_Community_College_(MCC)/Remix_of_Openstax

%3AMicrobiology_by_Parker_Schneegurt_et_al/08%3A_Microbial_Growth/

8.03%3A_The_Effects_of_pH_on_Microbial_Growth

Ramadan, N., Wessjohann, L. et al (2020). Nutrient and Sensory Metabolites Profiling of Averrhoa

Carambola L. (Starfruit) in the Context of Its Origin and Ripening Stage by GC/MS and

Chemometric Analysis. Accessed on March 07, 2022, from

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7287910/

33 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

Ramirez, C., Singian, I., et al. Development of Spray-Dried Powdered Kamias (Averrhoa

bilimbi). Accessed on March 07, 2022, from https://research-manila.letran.edu.ph/read/52

Rozes, N. (2012). Effect of Fermentation Temperature and Culture Medium on Glycerol and Ethanol

during Wine Fermentation. Accessed on April 01, 2022, from

https://www.academia.edu/18916752/Effect_of_Fermentation_Temperature_and_Culture_Mediu

m_on_Glycerol_and_Ethanol_during_Wine_Fermentation

Rutala, W. Ph.D., et al. (2016). Chemical Disinfectants. Accessed on March 07, 2022,

from https://www.cdc.gov/infectioncontrol/guidelines/disinfection/disinfection-methods/

chemical.html#:~:text=The%20culture%20phase%20was%20more,the%20tissue%20phase

%20493%2C%20494

Science Buddies. (2014). Single-Celled Science: Yeasty Beasties. Accessed on March 13, 2022, from

https://www.scientificamerican.com/article/single-celled-science-yeasty-beasties/#:~:text=Yeasts

%20feed%20on%20sugars%20and,bread%20so%20soft%20and%20spongy

ScienceDirect, (2014). Disk Diffusion: An Overview. Accessed on March 08, 2022,

from https://www.sciencedirect.com/topics/immunology-and-microbiology/disk-diffusion?

fbclid=IwAR2EHMW26UNYsBxiU_UgtXURp1FKqfZydCKYsgk79Btj5lpT7AGgi1H31fw#:~:t

ext=The%20disk%20diffusion%20method%20

Sharif Hossain, A.B.M, et al. (2019). Bioethanol Production from Fruit Biomass as Bio-antiseptic and

Bio-antifermenter: Its Chemical and Biochemical Properties. Accessed on March 07, 2022, from

https://scialert.net/fulltext/amp.php?doi=jas.2019.311.318

34 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

Shu, H.G. (2019). Iba (Averrhoa bilimbi). Accessed on March 07, 2022, from

http://www.stuartxchange.org/Kamias

Siswanto, A.P. (2021). Utilization of Bioethanol Fermentation Waste Pineapple and Coconut Water as

Disinfectants With Bacteria Saccharomyces Cerevisiae. Accessed on March 13, 2022, from

https://www.eduvest.greenvest.co.id/index.php/edv/article/view/60

Spedding, G., (2016). Brewing Materials and Processes. Accessed on March 08, 2022,

from https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/hydrometers?

fbclid=IwAR2tdeKODCmAq4g8cKyKWjWSPYND5i8bOW_CsNMWBA-

34jZ3kWgX61VlYnU

Street, T. (2014). Antimicrobial Susceptibility. Accessed on April 01, 2022,

from https://emedicine.medscape.com/article/2103786-overview#:~:text=Antimicrobial

%20susceptibility%20tests%20are%20used,which%20are%20obtained%20much%20sooner

Tan, B., et. al. (2005). Anti-diabetic Activity of the Semi-Purified Fractions of Averrhoa

Bilimbi In High Fat Diet Fed-Streptozotocin-Induced Diabetic Rats. Accessed March 07, 2022,

from https://pubmed.ncbi.nlm.nih.gov/15808883/

Thermal Protective Clothing For Firefighters. (2017).Flammability - an overview. Accessed on April

04, 2022, from https://www.sciencedirect.com/topics/chemistry/flammability

Tse, T., et. al. (2018). Production of Bioethanol—A Review of Factors Affecting Ethanol Yield.

35 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

 Accessed on March 07, 2022, from https://www.mdpi.com/2311-5637/7/4/268?

fbclid=IwAR3CWnQ0CAlJki726xmJ5DBwGCGYWHiEUZGCtHixVnEMAZn180GRBB1lzfs

Types of Disinfectants. Accessed on March 07, 2022, from

https://www.nycoproducts.com/resources/blog/types-of-disinfectants-how-to-make-the-best-

choice-for-your-facility/

Vandergriendt, C., (2021).What to Know About Using Alcohol to Kill Germs. Accessed

March 08, 2022, from https://www.healthline.com/health/does-alcohol-kill-germs?

fbclid=IwAR123rRoALqOGkHRpOtzB5Fn8QjeOPKIEBY4-fvmz-936f6GjD-QfpPLkkc

Vandergrient, C. (2021, June 30). Does Alcohol Kill Germs? Using Isopropyl, Ethanol to Destroy

Germs. Retrieved April 06, 2022, from https://www.healthline.com/health/does-alcohol-kill-

germs

Why Is 70% Isopropyl Alcohol (IPA) a Better Disinfectant than 99% Isopropanol, and What Is IPA

Used For?. Accessed on March 13, 2022, from https://blog.gotopac.com/2017/05/15/why-is-70-

isopropyl-alcohol-ipa-a-better-disinfectant-than-99-isopropanol-and-what-is-ipa-used-for/

#:~:text=Ethanol%20is%20low%2Dlevel%20disinfectant,%2C%20display%20surfaces%2C

%20and%20enamels

World Health Organization (2014). Use of Disinfectants: Alcohol and Bleach. Accessed on March

07, 2022, from https://www.ncbi.nlm.nih.gov/books/NBK214356/

Yang, R.J., et al. (2016). A comprehensive review of micro-distillation methods. Accessed on April

36 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant

 01, 2022, from https://www.sciencedirect.com/science/article/abs/pii/S1385894716316011

Zabed, H., et. al. (2014). Bioethanol Production from Fermentable Sugar Juice. Accessed on

March 07, 2022, from https://www.hindawi.com/journals/tswj/2014/957102/?

fbclid=IwAR1izzvU9iQw9Gdy7MdriwmbLK_47Q1JRDUmOtLpcA3b8oyq20ETSXKwzWI

37 | Iba (Averrohoa bilimbi) Fruit as Bioethanol Disinfectant