TBT 3013 BIOTECKNOLOGY

MINI PROJECT

FOOD WASTE AS ALCOHOL PRODUCER

NAME MATRIX NUMBER

Yii Ee Weng D20081032237
Wan Mohd Syahiran bin Wan Sabarudin D20081032317
Muhamamd Shakir bin Che Soh D20081032384
Lee Tack Hooi D20081032349

LECTURER’S NAME: DR HANIZA HANIM MOHD ZAIN

 FOOD WASTE AS ALCOHOL PRODUCER

ABSTRACT

Restaurants will produce a lot of food waste from their daily food
preparation for the customer. Some of them are the fresh vegetables parts
that are not served as food, the excessive rice which cannot be finish for
the day and the useless blended fruit. These food waste are usually seen as
rubbish and do not has other good use. This study will provide us a new
view about the food waste as it has shown that food wastes can produce
alcohol by the means of yeast fermentation in this study.

1.0 INTRODUCTION

Nowadays, the communities living in the big city or the metropolitan usually do not eat at home.
The editor of Ayushveda magazine had stated that the people living in the city or metros prefer
to eat outside in the Oktober 20, 2008 edition (http://www.ayushveda.com/magazine/side-
effects-of-eating-outside/). One of the reason might be that they are too busy working and do not
have much time to spend in cooking or prepare the food for their family.

This had caused fast and stable development of the restaurant industry in our country as
we can see that there are a lot of new restaurants being opened in the city. As there are more
restaurants out there, the food waste produce will increase as well. Food waste is defined as
uneaten portion of meals and trimming from food preparation activities in kitchens, restaurants
and cafeterias (Chaz Miller, 2004). Other than that, Chaz Miller (2004) also had stated that food
waste is the third-largest component of generated waste by weight. However, food waste has a
low composting rate and this had made them the largest component of discarded waste by weight.

Thus, we would like to propose a method in managing the food waste from their kitchen
that can conventionally carried out and produce a beneficial products. In this study, we will
concentrate on leftover or excessive rice which is staple food stuff in the everyday diet of
Malaysians and is a symbol of traditional Malay culture (http://www.nationsencyclopedia.com).
Hence, rice is also the main food waste produced from most of the kitchens in Malaysia. Most of
us will choose to finish the dish rather than the rice when we started to feel full during our meals.
This will produce much leftover rice in the end of the day. Other than that, we will also focus on
blended-fruit waste produced by most of the restaurants.

In facing these two food wastes from kitchen, we proposed a food waste managing
method of using yeast fermentation to degrade these food wastes. This method is conventional
and able to be carried out in the ethanol producing industry. Yeast or the ragi tapai which is
available in the market and the price of the yeast is quite cheap and affordable. As we know,
yeast is the eukaryotic micro-organisms classified in the kingdom of Fungi
(www.en.wikipedia.org/wiki/yeast) and it has the ability to use up the glucose in the anaerobic
condition to produce water and ethanol. This anaerobic degradation of glucose molecule is so
called yeast fermentation.

Actually, yeast fermentation is one of the oldest chemical processes known to man and is
used to make variety of products (http://www.andrew.cmu.edu). However, the agriculture
products from fermentation process command a higher price as foods and others are
uneconomical. This is due to the high cost of transportation and the food itself. Thus, we should
not just litter the food waste from our kitchen. Instead, we can collect or gather them and use it
as the raw materials in yeast fermentation to produce ethanol.

Fermentation process will occur with the presence of yeast and any materials that
containing sugar. The product of this process is ethanol. There are many and variable of raw
materials used in manufacturing ethanol via fermentation and they are classified into three types
that are sugar, starches and cellulose. The sugar is usually from the sugar cane, sugar beets,
molasses and fruits while starches are from grains, potatoes and other root crops. However,
starches used for fermentation needed to be hydrolyzed into fermentable sugar before the
fermentation process can be carried out. Celluloses are from wood, agricultural residues, water
sulfite liquor from pulp and paper mills. The celluloses used need to be hydrolyzed by mineral
acid before it can be ferment into alcohol.

All the raw materials stated above will consume a very high cost and it might also waste
the food product especially those fermentation using sugars and starches. Thus, we carried out
this study to investigate the successiveness of using the food waste which containing sugars and
starches as the raw materials in producing the ethanol. At the same time, we will also discuss this
method as a conventional way to manage the abundant food wastes from the kitchen.
2.0 METHODOLOGY

2.1 Yeast Suspension for Fermentation

Yeast suspension in this experiment was prepared using two types of sources. One of the sources
is the instant yeast while the other source is the ragi tapai. The instant yeast is available in the
market and it is manufactured by many companies. The instant yeast that had been used in this
study was manufactured by the AB MAURI MALAYSIA SDN BHD. Each of the instant yeast was
packet in a small vacuumed sachet. Each of the sachets has the weight of 11g. The instant yeast
in each sachet was in yellowish brown colour and they are in tiny sphere shape with rough
surface.

Ragi tapai is also another source of yeast used in this study. The ragi tapai used in this
study was obtained from one of the outlets in Tanjong Malim that sell chinese herbs. Ragi tapai
actually is a dried yeast suspension with other materials. There are various ways and methods in
preparing the ragi. One of the common way in preparing the ragi is as follow (Susono et al., 1974;
Susono et al., 1986): rice flour is mixed with grounded spices such as garlic (Allium sativum),
roots of the plant Alpinia galangal, white pepper (Piper frutescens), cinnamon (Cinamon
burmani), the fruit “addas” (Foeniculum vulgare), cane sugar (Saccharum officinarum), lemon
(Citrus aurantiacum var. fusca),and coconut water (Cocos nucifera).

Water is added to the mixture to make thick dough which is then molded into small
circular flat cakes, the size of 3 cm in diameter and 1 cm thick. Some coconut water is sprinkled
(not always) over the cakes, or sometimes mixed in the dough. The cakes are then placed on
bamboo trays which are lined with banana leaves and then on top covered again with banana
leaves. The trays are kept in a certain wind free place or room for 2-3 days. This is the natural
“fermentation” incubator. Then the rather dry cakes are sundried and turned over several times
until they are really dry. The dry ragi is put in jars or directly into polyvenil bags of the size the
numbers of the cakes to be stored in it (Gandjar et. al., 1983). This is the ragi that available and
is used to carry out fermentation process.

2.2 Yeast Fermentation Preparation

A few series of yeast fermentation namely Series A, Series B and Series C had been carried out
in this study. Each series of the fermentation is investigating different variable. The following is
the method in preparing the food waste samples and the yeast suspension for the fermentation
process. There is another series of fermentation using the bacteria replacing the yeast in series D
too.

Series A

In series A, we had prepared three types of food waste that are the leftover rice, leftover
glutinous rice and the dried sugar cane waste. Both types of rice were purchased in small
quantities from one of the restaurant in Tanjong Malim. The rice was left in open space for 24
hours. The dried sugar cane waste was obtained from the hawker selling sugar cane juice. He had
produced large quantity of dried sugar cane waste daily as he sells the juice by the road side. The
dried sugar cane waste was cut into smaller pieces to provide a larger surface area for the
fermentation process to occur.

After the food waste samples were ready, approximately 10 grams of each food waste
samples were weighed using the electronic balanced into three different air-tight containers. The
air-tight containers were labelled as A1-Y, A2-Y and A3-Y where A1-Y contains leftover rice,
A2-Y containing leftover glutinous rice and A3-Y containing the dried sugar cane waste. A same
weight of each food waste sample was weighed into another three different air-tight containers
labelling A1-R, A2-R and A3-R. Hence, all together there are 6 air-tight containers where both
containing the same weight of the same sample type food waste samples.

Then, 5 grams of the instant yeast stated above in 2.1 Yeast Sample for Fermentation was
weighed using the electronic balance into the air-tight container of A1-Y, A2-Y and A3-Y which
already containing the food waste sample in it. The instant yeast which is powdery was spread
evenly on the food waste sample in those three air-tight containers. By using a glass rod, the food
waste sample and the yeast was mixed evenly and thoroughly. This is to ensure that all the food
samples will be reacted and not just the surface of the food sample which contact with the instant
yeast. When it is done, the container was closed with its lid tightly.

The other three air-tight containers A1-R, A2-R and A3-R in this series were mixed with
approximately 5 grams of ragi tapai(2.1 Yeast Sample for Fermentation) each. The flat round
cake of ragi tapai was firstly smashed using the spatula into powdery form. Then, it was weighed
using the electronic balance and being spread onto the food waste sample evenly. Then, the food
waste and the ragi tapai were mixed evenly and thoroughly using the glass rod before the air-
tight container was closed with its lid. After that, all these six air-tight containers were wrapped
in the towel and stored in the dark place of room temperature for one week or seven days period.
The solution obtained for these samples later were sent for gas chromatography analysis.

Series B

In Series B, two types of fruits (apple and oranges) and a potato was used as the food
waste sample. All these three types of samples were blended as if preparing its juice. Then, the
blended samples were dry filter by pressing them on a filter to squeeze out most of the water.
The leftover, partially dry blended fruit was used as the food waste sample to conduct the study
in Series B fermentation.

For each of the dry smashed sample, approximately 10 grams of each sample were
weighed using the electronic balance into three different air-tight containers. Each of the samples
was mix with 5 grams of ragi tapai. Once the dried blended sample was mixed thoroughly with
the ragi tapai, the air-tight container was closed with its lid and wrapped with the towel. All of
the three containers were kept the same ways like the fermentation in Series A for one week or
seven days. The solution or liquid produce later was sent for gas chromatography analysis.

Series C

As for Series C, we had prepared the same type of leftover rice as stated above in Series
A. When the leftover rice was ready, approximately 50 grams of leftover rice was weighed using
the electronic balance into each of four different Erlenmeyer flasks (volume of each Erlenmeyer
flask is 250ml). The four flasks were labelled as C1, C2, C3 and C4. Each of the Erlenmeyer
flask with the leftover rice was mixed with different weights of yeast as follow:

Erlenmeyer flask Weight of yeast mixed with the leftover rice (g)
C1 2
C2 4
C3 6
C4 8
Table 1: The amount of yeast used in each set up for yeast fermentation
 After the leftover rice in each of the Erlenmeyer flask was mixed thoroughly with the ragi,
the opening of each of the Erlenmeyer flask was closed tightly with the wax film strip. Then,
each of the Erlenmeyer flask was wrapped with the towel and stored in the dark place of room
temperature for one week. The product of the fermentation after one week was distilled in order
to get the purer ethanol for gas chromatography analysis.

Series D

Another series of fermentation had been carried out. In this series, the raw material used
for fermentation was common rice. We had measure 50 grams of common leftover rice into four
different Erlenmeyer flasks labelled D1, D2, D3 and D4. Each of the Erlenmeyer flask was
added with 100mL of distilled water.

Each of the Erlenmeyer flask will be inoculate with different types of bacteria available
in the laboratory. The D1 flask was inoculated with the bacteria Aspergillus niger, D2 with B.
spenzinni, D3 with the bacteria E. coli while D4 without any bacteria inoculated and as a control.

The set up was left in the water bath of 37°C for seven days. The liquid sample from each
flask were filtered and injected into the gas chromatography for analysis to detect any production
of ethanol.

2.3 Distillation of Fermentation Product

Distillation is a step carried out to separate the mixture of substances based on the differences in
their volatilities in a boiling point mixture. The set-up for simple distillation is shown in figure 1.

The liquid produced from the fermentation was placed into the round-bottomed
distillation flask and heated until the boiling point around 80-100 ۫C. As the liquid boils, vapours
rise into the distillation head and condensed liquid will be seen dripping from the thermometer
bulb. Eventually the vapours enter the side arm of the distillation head and continue into the
condenser. Once in the condenser the vapours are cooled due to the water circulating in the outer
jacket; the vapours condense back to a liquid that runs down the condenser and is collected in the
receiving flask. Then, the distillate collected was sent for gas chromatography analysis to
determine whether the fermentation product is ethanol or non-ethanol substance.
 Figure 1: Simple distillation set-up

2.4 Gas Chromatography Analysis

Gas chromatography (GC) is a technique used to analyze mixtures. The instrument allows
mixtures to be separated and the amount of each component to be determined. One advantage of
this technique is that very small (a few micro liters) samples can be analyzed.

Samples to be analyzed in a gas chromatograph must be volatile, that is, they must
vaporize easily. Once vaporized, the sample is carried through a long tube (called a column)
containing a porous material. A nonreactive gas, such as helium is used to carry the components
of the mixture through the column. Not all components of a mixture travel through the column at
the same rate. Thus, some components will arrive at the detector at the end of the column before
others. As the components pass over the detector, the detector sends a signal to a recorder and a
graph (chromatogram) is produced.

By using the chromatogram, the percent composition (amount) of each component in the
mixture can be determined. The percent composition is directly related to the area of each peak
in the chromatogram.
 In this investigation we will use gas chromatography to separate the contents of the
fermentation product and then determine the components in the product.

The liquid from the fermentation of the food waste was filtered using a 5ml syringe
attached with 0.5µm filter. The liquid filtered was collected into the sample vials. A Varian 1400
GC with a flame ionization detector was used. A 6-ft. X 1/~-inch copper column packed with 3 %
Carbowax 600 on 40/60-mesh Chromosorb T was used. (A 21uidized drying technique was used
to prepare the packing.) Column temperature was 80°C, with injector and detector temperatures
of 120 and 125°C, respectively. Helium carrier flow was 110 cc/minute. Electrometer attenuation
was 1 on the 10 -~; range. Sample size injected was 0.5 t~l. Peak areas and retention times were
calculated and printed out in digital form on paper automatically by a Varian 480 electronic
integrator.

Prior analyzing the liquid of the fermentation product, the pure ethanol was injected and
analysis using the gas chromatography. This will enable us to determine whether the
fermentation product is ethanol or non-ethanol substance by comparing the retention time of the
sample with the pure ethanol.

Figure 2: Gas chromatography analysis

3.0 RESULT AND DISCUSSION

Fermentation is defined by the biochemists as the process of breaking down the organic
compound to generate energy in the anaerobic condition. This reaction is usually carried out by
the microbes. One of them is yeast or scientifically known as Saccharomyces cevevisiae. Yeast is
able to have both the aerobic and anaerobic respiration depending on the availability of oxygen
of the environment. The yeast will use the substrate which can be sugars, starches or cellulose. In
this study, we had common rice and glutinous rice and blended potato as the starch type substrate.
We also had the fruit as the source of sugar substrate. As for cellulose, we had used the dries
sugar cane. All of this substrate used is potential source of food waste from the kitchen.

The product of the yeast fermentation on these listed substrates will be the same that is
the alcohol, water and energy. This chemical reaction of the yeast fermentation is as follow:

CARBOHYDRATES + ZYMASE → ALCOHOL + WATER + ENERGY (ATP)

Energy produced by the yeast fermentation process will be used by the yeast for its metabolic
process. The alcohol or more specific ethanol will be collected for other usages. The ethanol
produced will be collected using the method of distillation. Distillation process will be able to
separate the mixture of the fermentation into its component based on the boiling point of each
substance. However, in this study, we do not carried out distillation for Series A and B. We
collected the liquid in the syringe and pump it through the 0.5µm filter. The liquid filtered will
then used as the liquid samples.

In this study, we will investigate whether the food waste that produced from the kitchen
can be used as the substrate or raw material for yeast fermentation in producing ethanol. Thus,
filtered liquid samples from each series of fermentation set up will be injected it into the gas
chromatography apparatus to be analysed and to qualify the presence of the ethanol in the liquid.
The liquid produce is actually the ferment from the yeast fermentation and theoretically it is a
mixture of the ethanol and water. The liquid samples will be proved containing ethanol by the
gas chromatogram analysis when the result of the analysis (in the chromatogram form) show a
peak at the retention time around 7.342 minutes.

The liquid of 95% ethanol had being analysed by the gas chromatography apparatus
before our sample liquids of the yeast fermentation were analysed. The chromatogram of the 95%
ethanol shows two peaks; one peak (Peak 1) has the retention of 6.750 minutes while the other
 peak (Peak 2) has the retention time at 7.342 minutes. Peak 2 with the retention time of 7.342 is
determined as the peak of ethanol because Peak 2 has the highest voltage compare to the Peak 1.
Other than that, Peak 2 also has the largest peak area consisting of 95.619% compare to the area
of Peak 1 which is just comprises of 4.381%. The gas chromatography analysis also provides
reading for the height of each peak. The chromatogram shows that the height of Peak 2 is higher
than Peak 1. Thus, we conclude that if the liquid samples of the yeast fermentation containing
ethanol, the chromatogram will show a peak at the retention time of around 7.342 minutes. The
area and the height of the peak at this retention time will be used to show the relative
concentration of ethanol in the liquid samples.

The filtered liquid samples from the fermentation of Series A had being analysed and the
result obtained was as follow:

TYPE OF FOOD
RICE GLUTINOUS RICE DRIED SUGAR CANE
WASTE
SAMPLE A1-Y A1-R A2-Y A2-R A3-Y A3-R
RETENTION TIME 7.333 7.333 7.350 7.342 7.350 7.350
AREA 4336280 5203367 2203940 2501421 956503 1523178
HEIGHT 610810 707910 249767 364695 130971 210038
VOLTAGE (µV) 0.615 0.710 0.268 0.372 0.149 0.218
Table 2: Result of analysis of the filtered liquid of Series A yeast fermentation

From the result, all of the liquid samples are assumed to contain ethanol even though the
peak of the liquid samples do not show the exact retention time of 7.342 minutes except liquid
sample of A2-R. The retention time of the peak other liquid samples were either at 7.333 minutes
and 7.350 minutes. The peaks at these retention time are assumed to be ethanol. The deviation of
the retention time of these peaks might due to the presence of contaminate or solid particle. Thus,
the peak at retention time of 7.333 minutes (varied 0.012 minutes from the retention time of 95%
ethanol) and 7.350 minutes (varied 0.008 minutes from the retention time of 95% ethanol) are
still assumed as the peak of ethanol. This will prove that the food waste actually can be used as
the substrate for yeast fermentation to produce ethanol.
 The ethanol produced from this food waste is quite high. All of the liquid samples have a
smaller peak area compare to the peak area of 95% ethanol. At the same time, the height of the
peak of each sample liquids also has the same trend too. This can be seen in the graphs below:

AREA
8000000
7000000
6000000
5000000
4000000
3000000
2000000 AREA
1000000
0

Graph 1: The peak area of each liquid samples and the peak area of 95% ethanol

HEIGHT
1200000
1000000
800000
600000
400000
HEIGHT
200000
0

Graph 2: The height of the peak of each liquid samples and the height of the peak of 95% ethanol

From the graphs, the ethanol produced from the leftover rice actually has a larger peak
area and higher peak height compare to the ethanol produce by the glutinous rice and the dried
sugar cane. The yeast fermentation of the sugar cane produces the least ethanol. This might be
because the dried sugar cane composed mostly of cellulose. Thus, the cellulose needs to be
breakdown to simple sugar before the yeast can carry out the fermentation process. This process
of breaking down will take some times and thus will cause the low production of ethanol.

When comparing the ethanol production of the rice and glutinous rice, yeast fermentation
of the rice will produce more ethanol compare to the glutinous rice. The different between the
glutinous rice and the common rice is that the glutinous rice does not have amylose whereas the
common rice is a mixture of amylase (10% to 20%) and amylopectin (80% to 90%). Since the
glutinous rice do not has amylose, it appears to be very sticky or having the gluiest properties.
The amylopectine actually is a more complex for of starch compare to amylose. Amylopectin is a
branced polymer with 1-6 linkages at the branch point. Amylose is a simpler starch without
branching and the glucose molecule link together by 1-4 linkage.

Since common rice has the amylose and amylopectin, the process of yeast fermentation
can take place easier and faster as there are availability of the unbranch starch polymer (amylose).
Glutinous rice consists of only the amylopectine. Hence, it will take some times for the yeast
fermentation to occur. That might had cause the result of higher ethanol being produced by the
common rice.

Another finding that can be observed from the result is that the yeast in the form of ragi
tapai can ferment more efficiently compare to the instant yeast. This can be seen from the graph
below as all of the three liquid samples of yeast fermentation using ragi tapai produce higher
amount of ethanol.

ETHANOL PRODUCTION BY THE FERMENTATION USING

RAGI TAPAI AND INSTANT YEAST
6000000
5000000
4000000
Axis Title

3000000
2000000
1000000
0
A1 A2 A3
INSTANT YEAST 4336280 2203940 956503
RAGI TAPAI 5203367 2501421 1523178

Graph 3: Ethanol production by the fermentation using ragi tapai and instant yeast
 Ragi tapai is more efficient in yeast fermentation might because it is not just contain the
yeast only. As explained in part 2.1 Yeast Suspension, the ragi tapai actually is the dried mixture
of yest with other material. One of the materials is the cane sugar. The presence of the sugar in
the yeast suspension will boost up the yeast budding before the real fermentation occurs. Thus,
the ragi tapi will provide more yeast for the fermentation to happen later. Compare to the instant
yeast, the population of the yeast increases as the process of fermentation proceed. Thus, it will
produce less ethanol.

However, the main aim of this series of yeast fermentation being carried out is to prove
that the food waste is able to be used as the substrate or the raw material for yeast alcohol
fermentation. Among all the leftover food studied in this series, the rice will produce the most
amount of ethanol. Thus, the leftover rice from the kitchen shall not be litter away jusy like that.
It can be collected and used as the substrate for the yeast alcohol fermentation rather than using
the food material for the process of yeast fermentation to obtain alcohol.

Alcohol was produce in bulk in the industry by using the yeast fermentation. The raw
material used as the substrate is the sugar cane, field corn or cheap cereal grains. These raw
materials were not able to be fermented by yeast. They have to be added with dilute sulphuric
acids or fungal alpha amylase enzyme to break them into simple sugar before fermentation
process can occur (http://en.wikipedia/wiki/Yeast). Since the leftover rice can be used as the raw
material for yeast fermentation, why not it is being collected from the restaurants and home
kitchen for the mass production of ethanol? This might save up a lot of food source.

After the liquid samples from the fermentation of series B being analysed, we had found
that all of the liquid samples contain ethanol. Well, in yeast fermentation series B, we had used
two types of fruit that are the orange and apple. Fruit fermentation by yeast has being known for
centuries in production of beverage. For example is the red wine which is produced by
fermenting the grapes. We also have the apple wine in the market but so far there is no wine
being made from the orange. Both of these fruits are the major fruit juice that available in most
of the restaurants. At the same time, they are also the major food waste of fruit category. The
orange and apple juice was produce by blending the fruit using a blender and the juice was
extracted and the dried blended fruit was eliminated as food waste.

Thus, in this series of yeast fermentation, we would like to know whether this dried
blended fruit will be able to be used as the raw material for yeast fermentation to produce ethanol.
 At the same time, this series also investigate the ability of the potatoes in producing ethanol in
yeast fermentation as potato is the main stalk of dietary in the western country. The analysis of
gas chromatography of these three samples was as follow:

TYPE OF FOOD DRIED BLENDED DRIED BLENDED DRIED BLENDED

WASTE APPLE ORANGE POTATO
SAMPLE B1 B2 B3
RETENTION TIME 7.333 minutes 7.333 minutes 7.333 minutes
AREA 3875297 2847624 1029236
HEIGHT 439052 319424 113799
VOLTAGE (µV) 0.445 0.333 0.122
Table 3: Result of analysis of the filtered liquid of Series B yeast fermentation

From the result, all of the liquid samples analysed show a peak on the same retention
time of 7.333 minutes. This had proved that all of the samples of food waste are able to produce
ethanol. Although the retention time the ethanol peak of these three samples deviate 0.009
minutes from the retention time of the peak of 95% ethanol, it is still assumed as ethanol. The
very obvious different between the gas chromatography analysis these three liquid samples with
the liquid samples in series A is that these three samples produce more peaks or the
chromatogram is noisier. This means that there are mixtures of many other substances in the
liquid samples.

AREA
4500000
4000000
3500000 3875297
3000000
2500000 2847624
AREA
2000000
1500000 Linear (AREA)
1000000
1029236
500000
0
B1 B2 B3

Graph 4: Peak area of the three samples in Series B

 HEIGHT
500000
450000
400000 439052
350000
300000
319424
250000 HEIGHT
200000
Linear (HEIGHT)
150000
100000
113799
50000
0
B1 B2 B3

Graph 5: The height of each sample analysed

From graphs above, the ethanol peak of apple has the largest area follow by oranges and
potato. This also means that apple has the highest production of ethanol compare to oranges and
potatoes. From the result, it has shown that dried blended fruits (apple and orange to be specific)
are able to be used as the raw product or the substrate for the yeast fermentation. Thus, the
blended fruit from the restaurant can be collected and used for yeast fermentation rather than
lettering them away into the rubbish dump.

From these two series of fermentation conducted, we had concluded that the food waste
actually can be made of good used if collected as the raw material for yeast fermentation. These
food wastes also have the ability to produce ethanol like the other foods that being processed and
used as the substrate for yeast fermentation in industry. This food waste is more conventional
and it can help to save up food sources as we do not need to use the cereals, sugar cane or any
sources of starches that can be our food source to be fermented by the yeast. Other than that, by
using food waste as substrate, we can actually reduce the rubbish at the dumping site because
food waste has the low composting rate. It is also the major contributor of rubbish weight at the
dumping site. By using them as the substrate for yeast fermentation, it will be break down into
simpler molecule and used up by the yeast. Thus, it will help to convert this low composting
material into other useful product in our daily life.
 In the series C of yeast fermentation, we had manipulated the amount of yeast in each set
up of fermentation. We would like to prove the existing theory that the increasing amount of
yeast will actually increase the ethanol production of yeast also applicable on when using the
food waste as raw materials. The result obtained from the analysis of this manipulation is shown
below:

WEIGHT OF
2 grams 4 grams 6 grams 8 grams
INSTANT YEAST
SAMPLE C1 C2 C3 C4
RETENTION TIME 7.350 minutes 7.358 minutes 7.350 minutes 7.350 minutes
AREA 190825 181111 1309647 1721510
HEIGHT 26132 24634 152842 186608
VOLTAGE (µV) 0.029 0.032 0.160 0.194

Table 4: Result of analysis of the liquid sample in series C fermentation

The ethanol production show increment when the amount of yeast increases from sample
C1 to sample C4. This is can be seen from the graph below:

2000000 AREA
1800000
1600000
1400000
1200000
1000000
800000 AREA
600000
400000
200000
0
C1-2g C2-4g C3-6g C4-8g

Graph 6: Peak area of the three samples in series C

 HEIGHT
200000
180000
160000
140000
120000
100000 HEIGHT
80000
Linear (HEIGHT)
60000
40000
20000
0
C1-2g C2-4g C3-6g C4-8g

Graph 7: Height of peak of the three samples in Series C

The low content of the ethanol in the distillate collected (liquid sample) is because the
simple distillation are not able to condensed the vaporised ethanol from the ferment. Thus, the
ethanol vapours escape to the environment. We collected the first drop of distillate at the
temperate of 95°C. Thus, we do not really have a very pure ethanol as the boiling point of
ethanol is 78.3°C (http://www.ucc.ie/academic/chem/dolchem/html/comp/ethanol.html).
Although the pure ethanol was not successfully collected at its boiling point, the liquid was still
injected into the gas chromatography for analysis. The results show a very low ethanol content in
each liquid sample.

Hence, we deduct that the simple distillation method was not suitable in collecting the
ethanol from the ferment. It is not efficient enough to collect the ethanol as we do not really
know the boiling point of other material or substances in the mixture of the ferment although
theoretically the ferment supposedly have water and ethanol only. We suggest using the
fractional distillation to collect the ethanol from the ferment is a better solution for this problem.

Fractional distillation is the more or less like the simple distillation set up. The different
is that the present of the fractionating column. This fractionating column will enable the
condensate vapour to be heated and vaporised again. Thus a purer substance will be able to
collect at the correct boiling point. This might be the better way of collecting the ethanol from
the yeast fermentation.
 When we put all the gas chromatography result together, we see that the rice will have
the highest production of ethanol when fermenting it using the ragi tapai. Thi can be seen in the
graph below:

AREA
8000000
7000000 95%ETHANOL
6000000 A1-Y
5000000
A1-R
4000000
3000000 A2-Y
2000000 A2-R
1000000
A3-Y
0
A3-R
B1
B2

Graph 8: The collection of the result analysed from the liquid sample (parameter – peak area)

Actually, we can roughly estimate the percentage of concentration of the ethanol in each
liquid sample. By using the peak area of 95% ethanol as the standard, we can actually determine
the concentration of each liquid samples relatively. The formula used is shown below:

PERCENTAGE OF ETHANOL IN PEAK AREA OF LIQUID SAMPLE

LIQUID SAMPLE = PEAK AREA OF 95% ETHANOL
X 95% X 100

From the formula, the concentration of ethanol in each liquid sample is estimated and the
result is in the table below:

INSTANT YEAST ESTIMATED

SAMPLE SUBSTRATE PEAK AREA
/ RAGI TAPAI CONCENTRATION (%)
95% ethanol - - 7180521 95.00
A1-Y Rice Instant yeast 4336280 57.37
A1-R Rice Ragi tapai 5203367 68.84
A2-Y Glutinous rice Instant yeast 2203940 29.16
A2-R Glutinous rice Ragi tapai 2501421 33.09
 Dried sugar
A3-Y Instant yeast 956503 12.65
cane
Dried sugar
A3-R Ragi tapai 1523178 20.15
cane
Dried blended
B1 Ragi tapai 3875297 51.27
apple
Dried blended
B2 Ragi tapai 2847624 37.67
orange
Dried blended
B3 Ragi tapai 1029236 13.62
potato
C1 Rice Instant yeast 190825 2.52
C2 Rice Instant yeast 181111 2.40
C3 Rice Instant yeast 1309647 17.33
C4 Rice Instant yeast 1721510 22.77
Table 5: The estimated percentage of concentration of ethanol in each liquid sample

The result obtain was transferred into the bar chart as follow:

ESTIMATED CONCENTRATION OF ETHANOL(%)

100
90 95
80
70
68.84
60
50 57.37
51.27
40 ESTIMATED CONCENTRATION OF
30 37.67 ETHANOL(%)
33.09
29.16
20
20.15 22.77
10 17.33
12.65 13.622.52 2.4
0

Graph 9: Estimated concentration of ethanol for each samples

 The last series (Series D) of alcohol fermentation was carried out using the bacteria as
listed in the methodology. The result shows that none of the liquid sample has the ethanol
content as there is no peak at the retention time of around 7.324 minutes. In other words, the
bacteria of Aspergillus niger, B. spenzinni and Escherichia coli was not functional in conducting
fermentation to produce ethanol. The result of the liquid sample analysis was as follow:

SAMPLE RETENTION TIME (MINUTES)

D1 6.242
D2 6.250 & 6.708
D3 6.725
D4 No peaks found
Table 6: Result of analysis of liquid sample of set up in series D

4.0 CONCLUSION

From the study carried out, we once again we would like to state that rice was the stalk in
our daily diet. Thus, it also the main food wastes in the food industry. There will be bundle of
collectable leftover rice from the restaurants. If we are able to collect this food waste and used it
as the raw material for fermentation, we can save up a lot of food resources that had been used in
the ethanol fermentation industry. Thus, we strongly recommend the usage of food waste
(leftover rice and dried blended fruit waste) as the substrate in yeast fermentation to collect
ethanol.

5.0 REFERENCES

Gandjar, I., D.S. Slamet & I. Rukmi. 1983. Brem Bali Fermentation.Proceedings of the
Symposium on Research in Biology and Biotechnology in Developing Countries,
26-28 NationalUniversity of Singapore. November 2-4, Singapore.

Susono, S., I. Gandjar, T. Basuki & H. Karsono. 1974. Mycoflora of ragi and some other
traditional fermented foods from Indonesia .Annales Bogorienses V: 187-204.

Saono, S., R.R. Hull & B. Dhamcharee. 1986 A Concise Handbook of Indigenous Fermented
Foods in the ASCA Countries . Indonesian Institute of Sciences, Jakarta,
Indonesia
The Nuffield Foundation and Royal Society of Chemistry 2010. (2009). Retrived October 28,
2010, from http://www.practicalchemistry.org/experiments/fermentation-of-
glucose-using-yeast,109,EX.html

G.D. Najafpour, J.K. Lim (2002), Evaluation and Isolation of Ethanol Producer Strain SMP-6,
Regional Symposium on Chemical Engineering 2002. Retrived October 28, 2010,
from
http://www.andrew.cmu.edu/user/jitkangl/Fermentation%20of%20Ethanol/Ferme
ntation%20of%20Ethanol.htm

Chromatography Online (2000-2009). Retrived October 28, 2010, from

http://www.chromatography-
online.org/quant/Chromatographic%20Data/Data%20Processing/Peak%20Area%
20Measurements.html

Danal O’Leary (2000) Ethanol. Retrived October 28, 2010, from

http://www.ucc.ie/academic/chem/dolchem/html/comp/ethanol.html

IPCS Inchem (1974) WHO Food Addictive Series No. 5. Retrived October 28,2010 , from
http://www.inchem.org/documents/jecfa/jecmono/v05je65.htm

Wikipedia, The Free Encyclopedia (2010) Yeast. Retrived October 28,2010 from
http://en.wikipedia.org/wiki/Yeast

Wikipedia, The Free Encyclopedia (2010) Apple Wine. Retrived October 28,2010 from
http://en.wikipedia.org/wiki/Apple_wine

Online Health and Lifestyle Magazine (2008) Side Effects of Eating Outside. Retrived October
28, 2010 from http://www.ayushveda.com/magazine/side-effects-of-eating-
outside
APPENDIX

Preparation of rice fermentation using ragi tapai (A1-R)

Preparation of the rice fermentation using instant yeast (A1-Y)

The already fermented glutinous rice using ragi tapai (A2-R)

The dried sugar cane used in the fermentation series A

 Preparation of dried blended apple for fermentation using ragi tapai

Preparation of dried blended oranges for fermentation using ragi tapai

Preparation of dried blended potatoes for fermentation using ragi tapai

The prepared set up for ragi tapai fermentation in Series B

 Ragi tapai used in the fermentation

Instant yeast used in the fermentation

Sample liquids collected from simple distillation in fermentation series C

The filtered liquid samples are collected into this small bottle before injected into the
chromatography analysis