Chapter 1

The Problem and the Setting

Introduction

The concept of producing bioethanol from Mangifera indica peelings came from

the notion that Mangifera indica peelings have considerable amounts of glucose. This

proves that Mangifera indica peelings can be a potential source of bioethanol since the

sugar found in its cellulosic material is the chief raw material of bioethanol production.

Moreover, a recent study entitled “Ethanol Production fromAlfalfa Fiber Fractions”

shows that simultaneous saccharification and fermentation produced the greatest yield

of ethanol from Alfalfa fiber fractions. Results further show that said simultaneous

saccharification and fermentation is the best method to be used for extracting

bioethanol. This study looks into the feasibility of producing greater yield of ethanol from

Mangifera indica peelings using the simultaneous saccharification and fermentation

process. While preliminary studies have been conducted researching the extraction of

bioethanol from various root crops, cellulosic material and algae, none have specifically

targeted Mangifera indica peelings as a source of bioethanol.

The world is currently undergoing an oil crisis. Due to our continuous annual oil

consumption, our reserves are quickly being depleted, with scientists predicting that at

our current rate of consumption, in just 40 years, our entire fossil fuel supply will run

completely out. However, oil consumption rates are increasing rather than decreasing,

thereby using up even more of this finite resource.

 Furthermore, while oil has a number of uses in the global community, burning it

increases the trace gas concentration in the atmosphere and causes significant

environmental problems such as global warming.

Additional, the Philippines is experiencing a problem in waste management.

Millions of tons of garbage are being thrown away by Filipinos each year, and the

accumulated waste takes up valuable real estate and is a source of land pollution.

The Mangifera indica is one of the leading products in the Philippines, as it

thrives in tropical climates. However, the Mangifera indica peelings have no

commercial value, and are therefore merely thrown away, contributing to the

Philippines’s waste problem.

This study aims to utilize Mangifera indica peelings as a raw material to obtain

bioethanol, thereby significantly contributing to the reduction of both the Philippines’s

total waste count and the growing global oil crisis.

Statement of the Problem

The study aims to extract bioethanol from Mangifera indica peelings through the

simultaneous saccharification and fermentation process using the yeast

Saccharomyces cerevisiae (Baker’s yeast).

Specifically, the study attempts:

• Will Mangifera indica peelings be a feasible source of bioethanol?

 • If variations in the simultaneous saccharification and fermentation process in

terms of the buffer levels and length of the fermentation time are made, then

there will be a significant effect on the net bioethanol yield

Hypotheses

The following hypotheses were formulated in this study:

a) Alternative Hypotheses

If the Mangifera indica peelings are subjected to the simultaneous

saccharification and fermentation process using the yeast Saccharomyces

cerevisiae, then a significant amount of bioethanol can be extracted.

• There is a significant difference in the average amount of ethanol

produced when fermentations times are subjected to two varieties.

• There is a significant difference in the average amount of ethanol

produced when different amounts of buffer are used.

• There is a significant difference in the percentage yield of bioethanol that

uses simultaneous saccharification and fermentation process and the

yield of the controlled group.

b) Null Hypotheses
 If the mangifera indica peelings are subjected to the simultaneous

saccharification and fermentation process using the yeast Saccharomyces

cerevisiae, then there’s no significant amount of bioethanol can be extracted.

• There is no significant difference in the average amount of ethanol

produced when fermentations times are varied.

• There is no significant difference in the average amount of ethanol

produced when different amounts of buffer are used.

• There is no significant difference in the percentage yield of bioethanol that

uses simultaneous saccharification and fermentation process and the

yield of that of the controlled group.

Significance of the Study

The world is currently undergoing an oil crisis. However, oil consumption rates

are increasing rather than decreasing, thereby using up even more of this finite

resource. Utilizing Mangifera indica peelings as a raw material to obtain bioethanol is

therefore significantly contributing to the reduction of both the Philippines’s total waste

count and the growing global oil crisis.

Assumption

The Mangifera indica peelings are rich in cellulosic ethanol. This research

assumes that Mangifera indica peelings will be an effective source of bioethanol.

Scope and Delimitation

This study is delimited in the amount of production of bioethanol from Mangifera

indica peelings as a source of bioethanol. Other products that Mangifera indica peelings

will produce are no longer covered in this research.

 Chapter 2

Review of Related Literature

This chapter contains the different related literature and related studies that the

research deals to.

Philippine Mangoes

Philippine mango (the national fruit) is known worldwide as the best tasting

variety of Carabao mangoes in the world. Philippine mangoes have a distinct rich taste,

no turpentine taste, not fibrous and high nutritional value compared to other cultivars.

Philippine carabao mangoes are available year-round but the best tasting fruits are

those picked during the summer months.

Pico originated from India, Burma and Malaya. Smaller than the carabao variety.

Kidney shaped with round apex and base, which is more, flattened. Light yellow orange

when ripe, thick and tough. Has fibrous orange to yellow orange flesh. Sweeter than the

carabao variety but not melting.

Bioethanol

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.

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

crops. These crops are grown specifically for energy use and include corn, maize and

wheat crops, waste straw, willow and popular trees, sawdust, reed canary grass, cord

grasses, jerusalem artichoke, myscanthus and sorghum plants. There is also ongoing

research and development into the use of municipal solid wastes to produce ethanol

fuel.

Ethanol or ethyl alcohol (C2H5OH) is a clear colourless liquid; it is

biodegradable, low in toxicity and causes little environmental pollution if spilt. Ethanol

burns to produce carbon dioxide and water. Ethanol is a high octane fuel and has

replaced lead as an octane enhancer in petrol. By blending ethanol with gasoline we

can also oxygenate the fuel mixture so it burns more completely and reduces polluting

emissions. The most common blend is 10% ethanol and 90% petrol (E10). Vehicle

engines require no modifications to run on E10 and vehicle warranties are unaffected

also. Only flexible fuel vehicles can run on up to 85% ethanol and 15% petrol blends

(E85).

Bioethanol has a number of advantages over conventional fuels. It comes from a

renewable resource i.e. crops and not from a finite resource and the crops it derives

from can grow well in the UK (like cereals, sugar beet and maize). 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 UK’s 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. 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. Bioethanol is produced

using familiar methods, such as fermentation, and it can be distributed using the same

petrol forecourts and transportation systems as before.

Bioethanol Production

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

processes. Biomass wastes contain a complex mixture of carbohydrate polymers from

the plant cell walls known as cellulose, hemi cellulose and lignin. In order to produce

sugars from the biomass, the biomass is pre-treated with acids or enzymes in order to

reduce the size of the feedstock and to open up the plant structure. The cellulose and

the hemi cellulose portions are broken down (hydrolysed) by enzymes or dilute acids

into sucrose sugar that is then fermented into ethanol. The lignin which is also present

in the biomass is normally used as a fuel for the ethanol production plants boilers. There
are three principle methods of extracting sugars from biomass. These are concentrated

acid hydrolysis, dilute acid hydrolysis and enzymatic hydrolysis.

Sugar Fermentation Process

The hydrolysis process breaks down the cellulostic part of the biomass or corn

into sugar solutions that can then be fermented into ethanol. Yeast is added to the

solution, which is then heated. The yeast contains an enzyme called invertase, which

acts as a catalyst and helps to convert the sucrose sugars into glucose and fructose

(both C6H12O6).

The chemical reaction is shown below:

The fructose and glucose sugars then react with another enzyme called zymase,

which is also contained in the yeast to produce ethanol and carbon dioxide. The

fermentation process takes around three days to complete and is carried out at a

temperature of between 250C and 300C.

Baker’s Yeast (Saccharomyces cerevisiae)

Saccharomyces when translated means “sugar fungus”. That is what this yeast

uses for food. They are found in the wild growing on the skins of grapes and other fruits.

Saccharomyces cerevisiae is in the fungi kingdom. The reasons for this classification

are because it has a cell wall made of chitin, it has no peptiodglycan in its cell walls, and
its lipids are ester linked. It also uses DNA template for protein synthesis and it has

larger ribosomes. It is then consider a yeast because it is a unicellular organism so it

can not form a fruiting body; like other fungi.

Saccharomyces cerevisiae has adapted in several important ways. One is the

fact that they are able break down their food through both aerobic respiration and

anaerobic fermentation. They can survive in an oxygen deficient environment for a

period. Another adaptation they have is their ability to have both sexual and asexual

reproduction. Very few other Ascomycota can do both processes. And very few

organisms can do all four of these processes. This allows this species to live in many

different environments. (Madigan, 457)

Saccharomyces cerevisiae gets its energy from glucose.

Saccharomyces cerevisiae has both asexual and sexual reproduction.

In asexual reproduction the haploid of the yeast under goes mitosis and forms more

haploid yeasts. There is an a and ά strain of these haploids. Then these haploid yeasts,

one from each strain, can fuse together and become on cell. Then the nuclei of both cell

fuses together and this cell is now the zygote. These diploid cells can go through

mitosis, which they call budding, and four more zygotes or they can undergo meiosis

and from an ascus which will split into four ascospores. These haploids can then

undergo germination and become haploid yeast again. (Madigan, 457)

Saccharomyces cerevisiae is one of the most important fungi in the history of the

world. This yeast is responsible for the production of ethanol in alcoholic drinks and is

the reasons your mother’s bread dough rises in the pan. That is where the names
brewer’s and baker’s yeast come from. The process in which it produces ethanol is one

way this yeast converts glucose into energy. There are two ways Saccharomyces

cerevisiae breaks down glucose. One way is through aerobic respiration. This process

requires the presence of oxygen. When oxygen is not present the yeast will then go

through anaerobic fermentation. The net result of this is two ATP, and it also produces

two by products; carbon dioxide and ethanol. So if this yeast is allowed to grow in a

container lacking oxygen it will produce ethanol (alcohol). Humans have been isolating

this process since the beginning of history. The yeast helps in the rising of bread with

it’s other by-product carbon dioxide. The gas that is produce inside the dough causes it

to rise and expand. Both of these processes use the haploid of this yeast for this

process. In industry they isolate one strain, either a or ά, of the haploid to keep them

from undergoing mating. (Madigan, 457) In the baker’s yeast they have a strain were

the production of carbon dioxide is more prevalent then ethanol and vice versa for

brewing. (Tomvolkfungi.net) Another importance is that “live yeast supplementation to

early lactating dairy goats significantly increased milk production”.

Ethanol fermentation

Ethanol fermentation, also referred to as alcoholic fermentation, is a biological

process in which sugars such as glucose, fructose, and sucrose are converted into

cellular energy and thereby produces ethanol and carbon dioxide as metabolic waste

products. Because yeasts perform this process in the absence of oxygen, ethanol

fermentation is classified as anaerobic. Ethanol fermentation occurs in the production

of alcoholic beverages and ethanol fuel, and in the rising of bread dough.
Saccharification

Saccharification is the process of breaking a complex carbohydrate (as starch)

into simple sugars.

Ethanol Production from Alfalfa Fiber Fractions by Saccharification and

Fermentation

This work describes ethanol production from alfalfa fiber using separate

hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation

(SSF) with and without liquid hot water (LHW) pretreatment. Candida shehatae FPL-702

produced 5 and 6.4 g/l ethanol with a yield of 0.25 and 0.16 g ethanol/g sugar

respectively by SHF and SSF from alfalfa fiber without pretreatment. With LHW

pretreatment using SSF, C. shehatae FPL-702 produced 18.0 g/l ethanol, a yield of 0.45

g ethanol/g sugar from cellulosic solids or ‘raffinate’. Using SHF, it produced 9.6 g/l

ethanol, a yield of 0.47 g ethanol/g sugar from raffinate. However, the soluble extract

fraction containing hemicelluloses was poorly fermented in both SHF and SSF due to

the presence of inhibitors. Addition of dilute acid during LHW pretreatment of alfalfa

fiber resulted in fractions that were poorly saccharified and fermented. These results

show that unpretreated alfalfa fiber produced a lower ethanol yield. Although LHW

pretreatment can increase ethanol production from raffinate fiber fractions, it does not

increase production from the hemicellulosic and pectin fractions.

 Chapter 3

Research Methodology

This chapter contains the materials and procedures used for extraction of bioethanol from
the two varieties of Mangifera indica peelings through simultaneous saccharification and
fermentation using the Saccharomyces cerevisiae

Materials and Equipment

One hundred seventy grams of each of the variety of Mangifera indica peelings (carabo
and piko) was used for the total sugar determination and for simultaneous saccharafication and
fermentation. One hundred mL of Glucose-Yeast-Peptone, 20 grams of glucose and 10 grams of
peptone in 1L of distilled water were used in dilution. Five mL suspension of Saccharomyces
cerevisiae was used for the saccharafication. Twenty five grams of powdered cellulose and
xylanase were diluted in 200 mL of sodium hydroxide (NaOH).
Blender was used to make the Mangifera indica peelings susceptible to enzyme attack.
Eight flasks were used to be a container of the samples.

Research Design
The Fermentation Time of Mangifera indica

For carabao mango

SIMULTANEOUS
SACCHARIFICATION LEVEL OF BUFFER (mL)
AND
FERMENTATION
50.0 mL 100.0 mL
TIME (hours) 150 mL
A% of B% of A% of B% of A% of B% of
Time A the the the the the the
50mL 50mL 100mL 100mL 100mL 100mL
A% of B% of A% of B% of A% of B% of
Time B the the the the the the
50mL 50mL 100mL 100mL 100mL 100mL

For pica mango

 SIMULTANEOUS
SACCHARIFICATION LEVEL OF BUFFER (mL)
AND
FERMENTATION
50.0 mL 100.0 mL
TIME (hours) 150 mL
A% of B% of A% of B% of A% of B% of
Time A the the the the the the
50mL 50mL 100mL 100mL 100mL 100mL
A% of B% of A% of B% of A% of B% of
Time B the the the the the the
50mL 50mL 100mL 100mL 100mL 100mL

Dependent Variables:
Level of buffer (mL) in 50 mL
Level of buffer (mL) in 100 mL
Independent Variable:
Fermentation time A
Fermentation time B
Extraneous Variables:
2 Varieties of mango, specifically carabao and piko

Procedure

Waste peelings were collected from Mangifera indicas. These were then ground into pulp
using a blender, placed in a sterile container, and stored for the subsequent sugar concentration
analysis.
The container was then labelled for identification in preparation for the total sugar
determination. Fifty grams of the pulp was subjected to automatic chemical analysis of its total
sugar content. The test determined that the glucose content of the Mangifera indica peelings was
suitable for saccharification and fermentation.
Having determined the glucose content of the peelings to be used for simultaneous
saccharification and fermentation process, 120g of peelings were cut into smaller particles and
ground into pulp for fifteen minutes using a blender to make the samples more susceptible to
enzyme attack.
Two- 100mL flasks of Glucose-Yeast-Peptone (GYP) medium were prepared by diluting
20 grams of glucose and 10 grams of peptone in 1L of distilled water and sterilized for 20
minutes at 121˚C.
Five mL suspension of Saccharomyces cerevisiae strain was obtained and inoculated into
the prepared broth. The broth was then incubated at room temperature on a rotary shaker at 200
rpm for 24 hours before inoculation into the fermentation medium.
Twenty-five grams of powdered cellulase and xylanase enzymes were weighed and
transferred into two separate flasks. Each was diluted with 200mL of prepared sodium hydroxide
(NaOH) buffer. Each flask was vigorously shaken through mechanical means for thorough
incorporation of the enzyme with the liquid buffer.
Eight flasks were used for the experiment and labeled depending on the type of
simultaneous saccharification and fermentation manipulation they would undergo. The flasks
were then divided into two groups, A and B. Each subgroup is composed of four flasks each,
containing 15g of pulp per flask. Fifty mL of prepared 2.0% sodium hydroxide (NaOH) buffer
were distributed to each flask of group A, and 100mL of the same buffer were distributed to each
flask of group B. Groups A and B were inoculated with 5mL of cellulase and xylanase solution
and 5mL suspension of yeast cells.
The first two flasks of the first group, labelled A1, were allowed to ferment for 24 hours,
while the second pair of flasks from the same group, labelled A2, was allowed to ferment for 72
hours.
Likewise, the first two flasks of the second group, labelled B1, were allowed to ferment
for 24 hours, while the second pair of flasks of the same group, labelled B 2, was allowed to
ferment for 72 hours.
Bioethanol evaporation was prevented and aerobic conditions were maintained by placing
cotton plugs on all flasks. After their respective fermentation times, the pulp broth was filtered
through ordinary filter paper, and the filtrate of each flask was immediately submitted for
chemical analysis.
All the filtrates of the flasks of the two groups were sent for chemical analysis to
determine the bioethanol content of the liquid filtrate determined through gas chromatography.
Samples of each liquid filtrate in each of the flasks were loaded to the gas chromatogram for
chemical analysis, and the results of the test were recorded and analyzed.