THE MANUFACTURE OF BIOETHANOL THROUGH THE

MOLASE FERMENTATION PROCESS

Name : Fannisa Hapsari

Student ID : B1B015027
Entourage : III
Group :2
Assistant : Wahyu Dwi Saputra

BIOLOGY OF MICROSCOPIC FUNGI PRACTICAL WORK REPORT

MINISTRY OF RESEARCH, TECHNOLOGY, AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGY
PURWOKERTO

2018
 I. INTRODUCTION

A. Background

The history of yeast association with human society is synonymous with the
evolution of bread, beer and wine as global food and beverage commodities, originating
some 5,000 years ago. The microbial science of these products commenced in the mid-
1600s with the first observations of yeast cells being reported by Antonie van
Leeuwenhoek (The Netherlands). The significance of these findings laid dormant until
the classic studies of Pasteur (France) and Hansen (Denmark) during 1850–1900, which
heralded the beginnings of the disciplines of microbiology and biochemistry. Subsequent
studies by Guilliermond (France) and Kluyver (The Netherlands) in the early 1900s
established yeasts as a unique group of microorganisms that had a major role in food and
beverage production. Since the 1950s, several classic texts have specifically highlighted
the commercial and social significance of yeasts in foods and beverages. Today the
impact of yeasts on food and beverage production extends beyond the original and
popular notions of bread, beer and wine fermentations by Saccharomyces cerevisiae. In
a positive context, they contribute to the fermentation of a broad range of other
commodities, where various yeast species may work in concert with bacteria and
filamentous fungi. Many valuable food ingredients and processing aids are now derived
from yeasts. Some yeasts exhibit strong antifungal activity, enabling them to be
exploited as novel agents in the biocontrol of food spoilage. The probiotic activity of
some yeasts is another novel property that is attracting increasing interest (Fleet, 2006).
The energy crisis is a very basic problem in Indonesia. In modern life, energy
cannot be separated from daily life. The high dependence on fossil fuels, especially
petroleum (about 47%), coal (27%) and natural gas (20%) resulted in the depletion of
fossil fuels. The fossil resource is a non-renewable natural resource and will eventually
be exhausted if it is explored continuously. So it needs the development of renewable
energy such as ethanol productivity (Poernomo, 2014).
Ethanol is one type of alternative energy resembling the premium that is included
in Indonesia's new renewable energy development plan to become one of the priorities
and efforts of the government to answer the issue of energy crisis in Indonesia. And
ethanol is also one of the most widely used products in the industry, whether as a
solvent, cosmetic industry or pharmaceutical industry, besides ethanol can also be used
as a mixture with gasoline better known as gasohol, or the use of ethanol entirel (Rizki
et. al, 2016).

B. Objective

The objective of this practical activity is to know the manufactures of bioethanol

through molasses fermentation process.
 II. LITERATURE REVIEW

Ethanol is a colorless liquid and soluble in water. This type of alcohol often
called sebaalcohol grains. Actually the fermentation of the material which contains
carbohydrates like grapes, molasses of rice, potatoes will produce ethanol. Ethanol can
also be produced from the hydration of ethylene which derives derivatives from
petroleum and coal. This non-fermentation process takes place by adding water at high
temperatures (Winarno, 1990). According to Jamaliah (2011), 68% of ethanol in the
world is used as fuel. Ethanol production is widely developed with agricultural
commodities through fermentation. Ethanol production by fermentation can be produced
from 3 kinds of carbohydrates that are materials containing sugar such as cane sugar,
beet sugar, molasses (drops), juice and others.
Bioethanol is an alcohol compound with a hydroxyl group (OH), two carbon
atoms (C), with a chemical formula C2H5OH that made by fermentation sugar using
yeast. The compound can also be obtained by means of synthetic ethylene (CH2 = CH2),
which is more commonly called ethanol alone. Meanwhile, ethanol with sugar raw
materials is called bioethanol because the sugar comes from biological sources.
Bioethanol is produced using biological raw materials, therefore bioethanol in the event
of pollution does not provide significant environmental impact (Megawati, 2015).
Bioethanol can be obtained from all types of plants or biological materials which
contains sugar or starch. Bioethanol originally made from sugar and starch obtained
from sugar cane, corn, cassava, and others. Sugar from these various plants can be
directly fermented by yeast into ethanol. Ethanol made from sugar is in addition called
fermentation ethanol also called first generation bioethanol. Which means, ethanol from
the biological source found first person. As energy needs increase, the second, third, and
fourth generation bioethanol is present. For second, third, and fourth generation
bioethanol this is more difficult and longer processing to become ethanol. The second
bioethanol is bioethanol whose raw material uses a plant that is lignosellulose,
containing cellulose, hemicellulose, and lignin. Bioethanol from cellulose which is often
also called cellulosic ethanol is in the series of the manufacturing process there is also
the fermentation stage, but must go through the process of 7 pretreatment in advance
because it can not be directly done fermentation process. Until now no microorganisms
have been found that can directly ferment the cellulosic sugar polymer into a more
economical and efficient ethanol (Megawati, 2015).
The productivity of ethanol by using glucose can be done by using free cell
fermentation process by using Saccharomyces cerevisiae yeast, glucose
(monosaccharide) converted to ethanol and carbon dioxide (Prihandana, 2007). This
fermentation process is done by mixing the yeast Saccharomyces cerevisiae with sugar
substrate into glass erlemeyer shaken on top of shaker with certain condition. However,
this method has several disadvantages such as the difficulty in separating the products
produced with yeast cells in use (Sebayang, 2006).
Fermentation can be interpreted as a gradual change by several enzymes bacteria,
yeasts and fungi. Examples of chemical changes from fermentation include milk
acidification, starch decomposition and sugar to alcohols and carbon dioxide, as well as
the oxidation of organic nitrogen compounds. In the process of fermentation for more
than 3 days there is a change of sugar into alcohol, will be able to cause alcoholic
beverage juice. The fermentation process involves several enzymes produced by the
mold, so that the highest live cell count is present in the 3-day fermentation period and
the fermentation of fungi activity decreases (Hidayat, 2006).
Sugar is a common ingredient in fermentation. Some examples of fermented
products are ethanol, lactic acid, and hydrogen. However, some other components may
also be produced from fermentation such as butyric acid and acetone. Fermentation to
produce bioethanol by yeast is a simple change of sugar hexose into bioethanol and Co2
anaerobically, air is not needed during the fermentation process. According to Hadi
(2013), in the fermentation process occurs breakdown of parent compounds, where 1
molecule of glucose will produce 2 molecules of bioethanol, 2 molecules of Co2 and
energy liberation. Theoretically, 1 gram of sugar will be converted to 0,51 gram of
bioethanol (51% bioethanol) and 0,49 gr CO2 (49% CO2) (Chairul & Silvia, 2013).

Alcohol fermentation is the process of making alcohol by utilizing yeast activity.

The fermentation process is anaerobic, which converts glucose into alcohol, but in the
manufacture of starter requires an aerobic atmosphere where oxygen is required for cell
culture. The reactions are as follows:
a) breakdown of glucose in an aerobic atmosphere
C6H12O6 + 6O2 → 6CO2 + H2O ........................ (2.2)
b) Anaerobic glucose breakdown
C6H12O6 → 2C2H5OH + 2Co2 ........................ (2.3)
The process of breaking up glucose with the help of yeast is one of the enzymatic
processes because it produces an enzyme and can be simply formulated as follows:
C6 H12O6 → 2C2H5OH + 2Co2 + 2ATP + 57kCal ............. (2.4)
If the culture is too young or the incubation time is too short, it is possible that
the culture is still in the adaptation phase, so the growth is not optimal, but if the
incubation time is too long the possibility of the culture has reached the stationary phase,
therefore the best culture is in the log phase namely the most optimal growth phase
(Wahyudi, 1997).
Fermentation may occur due to microbial activity that causes fermentation in the
appropriate organic substrate. Factors affecting fermentation include:
a) Acidity (pH)
Foods that contain acid can be long-lasting, but if enough oxygen and mold can
grow and fermentation continues, then the durability of the acid will be lost. Acidity is
very influential in bacterial development. The good acidity condition for bacteria is 4.5-
5.5.
b) Microbes
Fermentation is usually done with pure culture produced in laboratories. This
culture may be kept dry or frozen.
c) Temperature
The fermentation temperature greatly determines the dominant microbe types
during fermentation. Each of the microorganisms has a maximum growth temperature,
minimal growth temperature, and the optimum temperature is the temperature that gives
the best and the fastest self-propagation.
d) Oxygen
 Air or oxygen during fermentation should be managed as best as possible to
multiply or inhibit the growth of certain microbes. Each microbe requires different
amounts of oxygen for growth or forming new cell cells and for fermentation. For
example, bread yeast (Saccharomycess cereviseae) will grow better in aerobic state, but
will continue to ferment the sugar much faster with anaerobic state.
e) Time
The rate of bacterial propagation varies according to species and growing
conditions. Under optimal conditions, bacteria will divide once every 20 minutes. For
some bacteria to choose the time generation that is the time interval between divisions,
can be achieved for 20 minutes. If the generation time is 20 minutes under suitable
conditions a cell can produce several million cells for 7 hours (Megawati, 2015).
Molasses are sided product derived from sugar cane making (Saccharum
officinarum L). Sugar cane is a viscous liquid and is obtained from the sugar crystal
separation stage. Molasses can no longer be formed into sucrose but still contain high-
grade sugars 50-60%, amino acids, and minerals. The high content of sugar in molasses
is very potential to be used as a bioethanol feedstock. Molasses still contain enough
sugar to produce ethanol by fermentation, usually a molasses pH ranging from 5.5 to
6.5. Molasses that still contain sugarcane around 10-18% have yielded satisfactory
results in the manufacture of ethanol (Tangkuman et. al, 2010).
 III. MATERIAL AND METHODS

A. Material

The tools used in this practical activity are beaker glass, bucket with capacity 25
liters, water dipper, stirring rod (spatula), distillator, and alcohol meter.
The materials used in this practical activity are molasses, yeast (Saccharomycess
cerevisiae), and clean water.

B. Method

A. Molasses dilution
Molasses with the volume 150ml are diluted with clean water until the volume reach
300ml.
B. Making the fermentation starter
The yeast are prepared as much as 4,5 pieces, then the yeast are diluted into the
molasses that have volume 1 liter and incubate it for 24 hours.
C. Fermentation process
The 24 hours incubation result are dilute with water until the volume reach 1 liter,
stir the molasses everyday and incubate it for 5 days.
D. Alcohol measurement
The incubation result are distilled with distillator and the substance from distillation
process are taken and measure the alcohol content with alcohol meter.
 IV. RESULT AND DISCUSSION

A. Result

Table 4.1. Observation The Manufacture of Bioethanol Through The Molase

Fermentation Process in Entourage I
Entoura Sample Observation of Molase Fermentation Alcohol
ge/ 1st day 2nd day 3rd day 4th day 5th day level
Group
I/1 Yeast 3 The smell The smell The smell like The smell The smell like 68%
pcs. like a soy like a soy a soy sauce, like a soy a soy sauce,
sauce, sauce, have have brown sauce, have have brown
have brown color, color, a lot of brown color, color, a lot of
brown a little bit foam, a lot of foam,
color like a foamy presence the foam, presence the
soy souce. activity of presence the activity of
microbe activity of microbe
microbe
I/2 Yeast 4 The smell The smell The smell like The smell The smell 65%
pcs. like a soy like a soy a soy sauce, similar like similar like
sauce, sauce, have have brown tapai, have tapai, have
have brown color color, a little brown color, brown color,
brown bit foamy, a little bit more foamy,
color, a presence the foamy presence the
little bit activity of presence the activity of
foamy microbe activity of microbe
microbe
I/3 Fermipan The smell The smell The smell like The smell The smell like 58%
25 gr like a soy like a soy a soy sauce, like a soy a soy sauce,
sauce, sauce, have have brown sauce, have have brown
have brown color, color, little bit brown color, color, not
brown foamy foamy little bit foamy
color, foamy
foamy
I/4 Fermipan The smell The smell The smell The smell The smell like 65%
20 gr like a soy lsimilar like lsimilar like lsimilar like a soy sauce,
sauce, tapai, have tapai, have tapai, have have brown
 have brown color, brown color, brown color, color, little bit
brown foamy foamy foamy foamy
color,
foamy

Table 4.2. Observation The Manufacture of Bioethanol Through The Molase

Fermentation Process in Entourage II
Entoura Sample Observation of Molase Fermentation Alcohol
ge/ 1st day 2nd day 3rd day 4th day 5th day level
Group
II/1 Yeast 5 The smell The smell The smell The smell The smell 60%
pcs like a soy like a soy similar like similar like similar like
sauce, sauce, have tapai, have tapai, have tapai, have
have brown color, brown color, brown color, brown color,
brown not foamy foamy foamy foamy
color, not
foamy
II/2 Yeast 6 The smell The smell The smell The smell 55%
pcs like a soy similar like similar like similar like
sauce, tapai, have tapai, have tapai, have
have brown color, brown color, brown color,
brown foamy foamy foamy
color, not
foamy
II/3 Fermipan The smell The smell The smell like The smell The smell 70%
15 gr like a soy like a soy a soy sauce, similar like similar like
sauce, sauce, have have brown tapai, have tapai, have
have brown color, color, foamy brown color, brown color,
brown foamy foamy foamy
color,
foamy
II/4 Fermipan The smell The smell The smell The smell The smell 65%
10 gr like a soy like a soy similar like similar like similar like
sauce, sauce, have tapai, have tapai, have tapai, have
have brown color, brown color, brown color, brown color,
 brown foamy foamy foamy foamy
color,
foamy

Table 4.3. Observation The Manufacture of Bioethanol Through The Molase

Fermentation Process in Entourage III
Entoura Sample Observation of Molase Fermentation Alcohol
ge/ 1st day 2nd day 3rd day 4th day 5th day level
Group
III/1 Yeast The smell The smell The smell The smell The smell 50%
3,5 gr like a soy like a soy similar like similar like similar like
sauce, sauce, have tapai, have tapai, have tapai, have
have brown color, brown color, brown color, brown color,
brown foamy foamy have a lot have a lot
color, foam foam
foamy
III/2 Yeast The smell The smell The smell The smell The smell 60%
4,5 gr like a soy like a soy similar like similar like similar like
sauce, sauce, have tapai, have tapai, have tapai, have
have brown color, brown color, brown color, brown color,
brown foamy have a lot have a lot have a lot
color, foam foam foam
foamy
III/3 Fermipan The smell The smell The smell The smell The smell 60%
2,5 gr like a soy like a soy similar like similar like similar like
sauce, sauce, have tapai, have tapai, have tapai, have
have brown color, brown color, brown color, brown color,
brown foamy, have a lot have a lot little bit foam.
color, presence the foam, foam,
foamy activity of presence the presence the
microbe activity of activity of
microbe microbe
 Picture 1. Fermentation
starter

Picture 2. Picture 3.
Fermentation on Fermentation on third
second day day

Picture 4. Picture 5.
Fermentation on Fermentation on fifth
fourth day day
B. Discussion

The alcohol content that obtained from our molasses fermentation are 60% the
fermentation agent that we used is yeast as much as 4,5gr. On the first day of
fermentation the molasses is smells like a soy sauce, have brown color, foamy, and there
is no microbial activity yet. On the second day of fermentation the molasses is smells
like a soy sauce, have brown color, foamy, and there is microbial activity yet. On the
third day of fermentation the molasses is smells similar like tapai, have brown color,
and also have a lot foam. On the fourth day of fermentation the molasses is smells
similar like tapai, have brown color, and have a lot foam. On the fifth day of
fermentation the molasses is smell similar like tapai, have brown color, and have a lot
foam. So from the result shows that the microbial activity are begin from the third days
of fermentation that signed by changing of its smells and the presence of foam is
increase. According to Megawati (2015), If the culture is too young or the incubation
time is too short, it is possible that the culture is still in the adaptation phase, so the
growth is not optimal, but if the incubation time is too long the possibility of the culture
has reached the stationary phase, therefore the best culture is in the log phase namely the
most optimal growth phase. The higher the initial sugar concentration, the higher the
concentration of ethanol produced, this is due to the higher sugar content in the
substrate, the greater the chance of microorganisms to decompose sugar into products or
ethanol (Winarti, 1996). The ethanol concentration increases with time fermentation due
to the longer fermentation time the higher yeast activity in converting glucose to ethanol
(Rizki et. al, 2016).
Very high gravity (VHG) technology was employed on industrial scale to
produce ethanol from molasses (fermented) as well as by-products formation estimation.
The effect of different Brix◦ (32, 36 and 40) air-flow rates (0.00, 0.20, 0.40, and 0.60
vvm) was studied on ethanol production. The maximum ethanol production was
recorded to be 12.2% (v/v) at 40 Brix◦ with 0.2 vvm air-flow rate. At optimum level
aeration and 40 Brix◦ VHG, the residual sugar level was recorded in the range of 12.5–
18.5 g/L, whereas the viable cell count remained constant up to 50h of fermentation and
dry matter production increased with fermentation time. Both water and steam
consumption reduced significantly under optimum conditions of Brix◦ and aeration rate
with compromising the ethanol production. Results revealed VHG with continuous air
flow is viable technique to reduce the ethanol production cost form molasses at
commercial scale (Arshad et. al, 2017).
 V. CONCLUSION & SUGGESTION

A. Conclusion

Based on result above, can be concluded that the alcohol contained that obtain from
the molasses fermentation using 4,5gr yeast is 60%.

B. Suggestion

Suggestions for this practicum is in every observation, all practicans required to

present so that all practicans understand the steps and results that obtained.
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