1

INTRODUCTION

Background of the Study

The principle fuel used as a petrol substitute for road transport vehicles is

bioethanol. Bioethanol fuel is mainly produced by the sugar fermentation process,

although it can also be manufactured by the chemical process of reacting ethylene with

steam. Bioethanol is biodegradable and far less toxic that fossil fuels. The excessive

consumption of fossil fuels which contributed largely high levels of pollution have been a

problem. The use or addition of biofuels to gasoline which reduces emission of carbon

monoxide and unburned hydrocarbons that form smog has widely been enforced in the

recent years (Wyman, 1994). Converting a renewable non-fossil carbon, such as organic

wastes, and biomass consisting of all growing organic matter (plants, grasses, fruit wastes

and algae) to fuel would assure a continual energy supply (Wyman, 1996).

Ethanol can be produced from biomass by the hydrolysis and sugar fermentation

processes. The main sources of sugar required to produce ethanol come from fuel or

energy crops. Biomass wastes consist of a complex mixture of carbohydrate polymers

such as cellulose, hemi cellulose and lignin. Biomass is pre-treated with acids or allowed

to react with enzymes to reduce the size of the feedstock and produce sugars. There is a

great significance of reducing waste in a location by using the wastes for an

environmental and industrial use.

Moreover, the economics of ethanol production by fermentation are significantly

influenced by the cost of the raw materials, which accounts for more than half of the
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production costs (Classen, et. al., 1999). To achieve a lower production cost, the supply

of cheap raw material is thus a necessity. Production of value added products from agro-

industrial and food processing wastes is now a focusing area, as it reduces pollution in the

environment in addition to energy generation.

This experiment aimed to use the agricultural wastes, guava fruit wastes as

specifically used in this study, to produce bioethanol gas. The agricultural wastes used in

the experiment make use of these wastes in a very beneficial purpose to many.

Objectives of the Study

The objective of this study is to produce bioethanol gas from agricultural waste

(reject guava fruits) by hydrolysis, fermentation and distillation.

Significance of the Study

Bioethanol has many uses to so many industries and a number of advantages over

conventional fuels. The experiment done will open opportunities to reducing the city’s

solid waste production while making use of these wastes into profitable cause. It will also

be beneficial to oil industries.

Scope and Limitation of the Study

The experiment was limited to bioethanol production using only guava fruit

wastes gathered from the public market. Percentages of the Baker’s yeast used were 4, 6,

8, 10% by weight of the hydrolysate. To determine the volume of recovery, distillation

process was used to extract ethanol. The study was conducted in the chemistry laboratory in

the University of Southeastern Philippines – Tagum Campus. The study lasted for 2

months; from hydrolysis, fermentation to distillation with repetitions.

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Definition of Terms

Bioethanol – It is a high octane fuel and has replaced lead as an octane enhancer in
petrol.

Hydrolysate – It is a substance produced by hydrolysis.

Hydrolysis – It is a reaction involving the breaking of a bond in a molecule using water.

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REVIEW OF RELATED LITERATURES

Bioethanol

Bioethanol has a number of advantages over conventional fuels.

First, it comes from a renewable resource. Another benefit over fossil fuels is the

greenhouse gas emissions. The road transport network accounts for 22% of all

greenhouse gas emissions and through the use of bioethanol, some of these emissions will

be reduced as the fuel crops absorb the CO2 they emit through growing. Also, blending

bioethanol with petrol will help extend the life of the diminishing oil supplies and ensure

greater fuel security, avoiding heavy reliance on oil producing nations. By encouraging

bioethanol’s use, the rural economy would also receive a boost from growing the

necessary crops. Blending ethanol with petrol oxygenates the fuel mixture so that it burns

completely and reduces harmful emissions.

Bioethanol is also biodegradable and far less toxic that fossil fuels. In addition, by

using bioethanol in older engines can help reduce the amount of carbon monoxide

produced by the vehicle thus improving air quality. Another advantage of bioethanol is

the ease with which it can be easily integrated into the existing road transport fuel system.

In quantities up to 5%, bioethanol can be blended with conventional fuel without the need

of engine modifications.

The domestic production of ethanol improves the energy supply during potential

crises and reduces the dependency on oil-related fuels. According to studies made by the

University of Eastern-Finland, the carbon balance of the contemplated bioethanol plant is

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60 % less than the one of fossil gasoline, considering the modern agriculture and the

usage of wood chips in the steam energy production. Mixing bioethanol with gasoline

does not affect the car motors and does not increase noticeably the fuel consumption.

Currently even a mixing ratio of 10 % is possible. The new mix ratio can be used with the

current cars without problems.

Bioethanol is produced using familiar methods, such as fermentation, and it can

be distributed using the same petrol forecourts and transportation systems as before. The

carbohydrate polymers are broken down with the help of enzymes or dilute acids into

sucrose sugar and then fermented into bioethanol. However, the lignin present in the

biomass is used as a fuel for boilers in which bioethanol is produced. Enzymatic

hydrolysis, concentrated acid hydrolysis and dilute acid hydrolysis are the three basic

methods for extracting sugar from biomass.

Composition of Substrate (Guava)

Like most fruits, guava fruits contain mostly water (more than 70%), are rich in

sugars (more than 40% DM) and poor in protein (less than 8% DM). The whole fruits are

also rich in fibre, due to the peels and the presence of seeds and stone cells. The soluble

dietary fiber fraction (SDF) in guava consists of pectins, beta-glucans, gums, mucilages,

and some hemicelluloses and may be found at higher concentration in the skin than in the

seed of the fruit.

Guava waste is made of variable proportions of peels, seeds and stone cells. Seeds

are rich in fibre (42%) with moderate oil (14%) and protein (15%) concentrations. Stone
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cells are highly rich in lignin (37%) and cellulose (54%) (El Boushy et al., 2000). The

resulting wastes are, therefore, poor in protein (7-11% DM) and rich in fibre (ADF 48-

70% DM), and particularly in lignin (16-22% DM).

According to Dhawan, et al. (2003), starch was extracted from the sugarfree

residue (guava) by 52% perchloric acid and estimated using anthrone reagent with

glucose as standard. Starch content was calculated by multiplying glucose concentration

by a factor of 0.9 [14]. Cell wall polysaccharides, viz. hemicellulose, cellulose, and

lignin, were determined by the methods of Van-Soest. Total pectin was extracted by the

method of Ahmed and Labavitch and determined as uronic acid equivalents by following

the procedure of Blumekrantz and Asboe-Hansen.

In addition, a study of Camarena-Tello, et. al. (2015) showed results of the

chemical composition of the biomass of guava generated in the pruning practices. These

results are as follows: Runkel lignin (17.77-35.26 %), holocellulose (26.56 -69.49 %), α-

cellulose (15.53-35.36 %), hemicelluloses (11.02-34.12 %), tannins in aqueous extracts

(3.81-9.06 %), and tannins in ethanolic extracts (3.42-15.24 %).

Molasses Application

Molasses is undoubtedly the most valuable byproduct from the sugar mill. When

produced from sugar beet, molasses is a source of alcohol and other chemicals, and is an

important constituent of animal feeds (Cleasby, 1995). Molasses when produced from

sugarcane is a low glycemic, nutrient-rich sweetener that is a substitute to the cane sugar,

which is known for spiking blood sugar levels and leading to illnesses such as obesity

and diabetes among others (Ettinger, 2012).

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Molasses, and its various types, proved to have multiple uses and are continuously

gaining popularity in the agricultural industry. Taking for example, a study conducted by

the Nebraska On-Farm Research Network in Nebraska USA, applying a molasses-water

solution to crops resulted to an increase of beneficial insects such as ladybugs, lacewings

and more (Rees et. al, 2010).

Considerable quantities of molasses are still used in Mauritius and Australia. It is

evident from work in these, and other countries, that where soils are deficient in potash

and of poor structure, the application of molasses is extremely beneficial (Cleasby,1995).

In the Philippine context, the Department of Agrarian Reform Program

Beneficiaries Development Division (DAR-PBDD) pursued a concept that convert

agricultural crop waste and molasses into cattle feeds in Masbate to make up for the fast

dwindling grazing areas in the city (DAR-Ph, 2014).

Among the chemicals produced from molasses, bioethanol, an alternative fuel, is

currently sparking interest in different gas industries due to its friendly effects on the

environment. A number of distilleries in Pakistan are utilizing bioethanol produced from

molasses to overcome the country’s reoccurring energy crises (Parkash, 2015).

The United States of America is considered the world’s largest producer of

bioethanol in the year 2010 due to its multiple advanced biofuel companies. The USA-

based Gevo Advanced Biofuel Company partners with the India-based bioethanol

company Praj Industries to let Praj use Gevo’s proprietary isobutanol technology to

process and produce bioethanol from sugarcane juice and molasses (Advanced Biofuels

USA, 2017).
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In the Philippine context, the Biofuels Act of 2008 provides that all liquid fuels

for motors and engines sold in the Philippines shall contain locally sourced biofuels,

including bioethanol from molasses and other fuels produced from biomass (Valencia,

2015).

The Sugar Regulatory Administration of the Philippines (SRA) has tightened the

traceability requirements of imported molasses in the country to ensure locally produced

molasses would continue to be used in the manufacture of bioethanol in the Philippines

(Valencia, 2015).

Ensiling of Guava Fruit Wastes (as treatment)

Ensiling is a technique for preserving forage, in which the ensiled mass is

acidified under anaerobic conditions (Ramos, 2016).

The ensiling process is composed of four phases namely; Pre-seal, active

fermentation, stable phase and feed-out, converts perishable wet forage plants to a stable,

stored feed energy source. This involves controlled fermentation, the beneficial microbial

process and the riddance of the bad microbial process called respiration, which is

considered wasteful as it causes the loss of energy and dry matter in the crop. The goal is

to always produce good quality silage (Barnhart, 2008).

Guava (Psidium guajava Linn.) locally known as “Bayabas”, is a popular fruit in

the Philippines and found indigenous to the American tropics. It is one of the most

gregarious fruit tree species of the Myrtaceae family which has a great potential for

extensive commercial production because of its ease of culture, high nutritional value and
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popularity of processed products. It is usually grown in open areas, second-growth

forests, and backyard or as part of a mixed orchard (Production Guide for Guava, 2016).

However, mature guava is highly susceptible to damage even on slight pressure.

A large proportion of guava, particularly of the rainy season, has to be disposed of in a

limited peak season. Due to inadequate facilities for processing and transportation, large

quantities of guava perish at the site of the orchard. The diseases of the fruit also add to

the economic loss to the producer. With increased guava production, the proportion of

culled fruits has also increased. Therefore, their utilization in some form becomes

absolutely essential (Elsevier Science Ltd, 1997).

An alternative to the above-mentioned problem is to utilize excess guava fruits for

the production of ethanol. In response to the dwindling supplies of petroleum and with

the regular hike in the price of crude oil, considerable effort has been focused to develop

fermentation processes for the production of substitutes for petroleum-based fuels and

chemicals. Of all the substitutes, ethanol is of great interest since it can be easily

manufactured employing various fruits and agro-industrial by-products (Elsevier Science

Ltd, 1997).

In an experiment conducted by Elsevier Science Limited (1997) using guava fruit

waste, they followed the simple conventional method for the preparation of guava pulp in

which mixed local varieties of fruit were washed, cut into small pieces, blended and hand

pressed. The pulp was then sieved through muslin cloth and stored in plastic cans at -

18°C till further use.

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Three yeast strains: Isolate-l, Isolate-2 purified from the culled guava and

identified as Saccharomyces spp., and Saccharomyces cerevisiae MTCC 1972 procured

from the Institute of Microbial Technology, Chandigarh, India, were used. Yeast cultures

were maintained by fortnightly transfers on yeast extract peptone dextrose (YEPD) agar

slants and stored at 4_+ I°C.

The yeast inocula were added at the rate of 10% (v/ v) to sterilized guava pulp as

per the treatment. The ethanol fermentation efficiency of various yeast strains was

optimized for sugar concentration (10, 15, 20% w/v) and pH (3.5, 4.1, 5.0) of guava pulp

substrate. The sugar concentration above the natural 10% and pH of guava pulp were

adjusted using glucose and 1 N NaOH/1N HC1, respectively.

The experiment’s results were, the guava pulp used for ethanol production by

three yeast strains contained 10% (w/v) total sugars and was pH 4.1. Ethanol production

at the optimum sugar concentration of 10%, at pH4.1 and 30°C was 1.5%, 3.6% and

3.9% (w/v) by Saccharomyces cerevisiae MTCC 1972, Isolate-1 and Isolate-2,

respectively, at 60 h fermentation. Higher sugar concentrations at 15 and 20% were

inhibitory for ethanol production by all test cultures. The maximum production of ethanol

at optimum natural sugar concentration (10%) of guava pulp, was 5.8% (w/v) at pH 5.0

by Isolate-2 over 36 h fermentation, which was only slightly more than the quantity of

ethanol produced by Saccharomyces cerevisiae (5.0%) and Isolate-1 (5.3%) over 36 and

60h fermentation, respectively.

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METHODOLOGY

Materials Needed

The following materials needed for the bioethanol production:

 9 one-click bottles
 Pomelo peels
 Molasses
 Baker’s yeast
 Cheesecloth

Image 1. The 9 one-click bottles used in the ensiling.

Conversion of cellulose polymer to ethanol involves two different processes:

1. Converting cellulose to glucose units by hydrolysis and the sugars produced

from the hydrolysis process can then be converted to ethanol by

2. Fermentation Baker’s yeast.

Pre-treatments are normally applied to separate the mixed polymers of lignin,

hemicellulose and cellulose to provide the sugars needed for the hydrolysis and the
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fermentation processes. The basic steps involved in the conversion of lignocellulosic

biomass to ethanol are shown in Fig 1.

Pomelo peels
Cellulose Sugar/Molasses
(Pretreatment Distillation Ethanol
(Hydrolysis) (Fermentation)
)

Figure 1. The steps involved in the conversion of lignocellulosic biomass to

ethanol

Pretreatment

Pretreatment of lignocellulosic biomass prior to hydrolysis is a prerequisite for

bioethanol production because it determines the yield of bioethanol that would be
obtained after fermentation. The aim of pretreatment is to reduce the compactness,
strength and crystalline nature of cellulose aiding in hydrolyzing the lignocellulosic
biomass to simple sugar units. The guava wastes were washed with water and air-dried.
Compaction was done inside the one-click bottles for ensiling. The wastes were treated
with sugar-cane molasses (10% of fresh weight) and were put into bottles.
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Hydrolysis

The aim of hydrolysis is to further degrade the polysaccharides present in the

pretreated lignocellulosic biomass of the guava waste into monosaccharides subunits. The
monosaccharides that will be produced upon hydrolysis will enhance the fermentation
process by the Baker’s yeast.

After a week, we extracted the hydrolysate. Baker’s yeast is added (4%, 6%, 8%
and 10% of 250 ml hydrolysate). It was then left for a week.

Image 2. shows the boiling of the hydrolysate before the addition of Baker’s
yeast.
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Image 3 Image 4

Image 5 Image 6

Images 3-6. shows the addition of Baker’s yeast to the hydrolysate in a

step-by-step process.
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Image 7 Image 8

Images 7 and 8 shows the sealing of the Erlenmeyer flasks with hydrolysate
and yeast concentration inside.
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Fermentation

Fermentation is the final stage of bioethanol production. We have used an Baker’s

yeast to convert the monosaccharides and some disaccharides produced during hydrolysis
into ethanol.

Distillation

Distillation is the process of separating the components or substances from a

liquid mixture by selective boiling and condensation. The mixture was heated in a flask.
Ethanol has a lower boiling point than water so it evaporates first. The ethanol vapor is
then cooled and condensed inside the condenser to form a pure liquid. The thermometer
shows the boiling point of the pure ethanol liquid. When all the ethanol has evaporated
from the solution, the temperature rises and the water evaporates. The temperature was
maintained into a degree not higher than 80degree Celsius.

Thermometer

Hydrolysate

Bioethanol

Figure 2 shows the distillation process of the hydrolysate with Baker’s yeast
concentration.
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Image 9 Image 10

Image 11 Image 12

Images 9-12 shows the testing of ethanol produced by ignition using a matchstick.
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RESULTS AND DISCUSSION

Volume of Volume of Ethanol

Percentage of Yeast
Bottle Hydrolysate per
applied Gathered (mL)
Bottle (mL)
#1 250 4 2.10
#2 250 6 2.15
#3 250 8 2.20
#4 250 10 2.15

Table 1. Volume of ethanol gathered (mL) per bottle with different levels of Baker’s
yeast applied.

Bioethanol
2.22
Volume of Ethanol Gathered, mL

2.2
2.18
2.16
2.14
2.12
2.1
2.08
2.06
2.04
1 2 3 4

Figure 3. Volume of ethanol gathered (mL) per bottle

It can be observed from Fig. 3 that the largest volume of ethanol produced was the

Bottle #3 which has 8% of yeast added to the hydrolysate with a volume of recovery of
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2.2mL. The least volume, on the other hand, was the Bottle #1 which has also the least of

Baker’s yeast added onto it, that was 4% of the volume of hydrolysate.
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CONCLUSION

Rotten fruits may serve as a good substrate as they contain sufficient amount of

carbohydrates naturally which can be used for production of bioethanol. From the data

above, we can conclude that the most efficient concentration of hydrolysate and yeast is

the 8% of yeast per 250mL bottle of hydrolysate. This considers guava waste as a source

of bioethanol but need further study for having higher recovery in processing it for more

bioethanol production.

In current time, the importance of alternative energy source has become even

more necessary not only due to the continuous deletion of limited fossil fuel stock but

also for the safe and better environment. Moreover, utilizing agricultural wastes to

produce bioethanol will be greatly beneficial in reducing the solid waste management

locally while boosting the oil industry.

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Wyman, CE. 1994. “Ethanol from lignocellulosic biomass: technology, economics, and
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Wyman, CE. 1996. “Handbook on Ethanol: Production and Utilization”. Taylor &
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Cleasby, T. (1959) Use of Molasses on Land: A Report of Four Experiments Being

Carried Out by the Tongaat Sugar Company, Ltd. South Africa

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www.organicauthority.com/health/how-and-why-to-use-molasses.html

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isobutanol-technology/

Valencia, C. (2015) SRA tightens rules on imported molasses.

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http://bpi.da.gov.ph/images/Production_guide/pdf/Guava%20Guide-reviewed