food waste as alcohol producer

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

WASTE RICE GLUTINOUS RICE DRIED SUGAR CANE

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:

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

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

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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.

Graph 3: Ethanol production by the fermentation using ragi tapai and instant yeast

A1 A2 A3

INSTANT YEAST 4336280 2203940 956503

RAGI TAPAI 5203367 2501421 1523178

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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

WASTE

DRIED BLENDED

APPLE

DRIED BLENDED

ORANGE

DRIED BLENDED

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.

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

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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.

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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:

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:

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

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INSTANT YEAST 2 grams 4 grams 6 grams 8 grams

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

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.

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HEIGHT

HEIGHT

Linear (HEIGHT)

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:

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:

From the formula, the concentration of ethanol in each liquid sample is estimated and the

result is in the table below:

SAMPLE SUBSTRATE INSTANT YEAST

/ RAGI TAPAI PEAK AREA

ESTIMATED

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

010000002000000300000040000005000000600000070000008000000

AREA

95%ETHANOL

A1-Y

A1-R

A2-Y

A2-R

A3-Y

A3-R

B1

B2

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

PERCENTAGE OF ETHANOL IN LIQUID SAMPLE =

A3-Y Dried sugar

cane Instant yeast 956503 12.65

A3-R Dried sugar

cane Ragi tapai 1523178 20.15

B1 Dried blended

apple Ragi tapai 3875297 51.27

B2 Dried blended

orange Ragi tapai 2847624 37.67

B3 Dried blended

potato Ragi tapai 1029236 13.62

C1 Rice Instant yeast 190825 2.52




Table 5: The estimated percentage of concentration of ethanol in each liquid sample

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

Graph 9: Estimated concentration of ethanol for each samples

95

57.37

68.84

29.1633.09

12.6520.15

51.27

37.67

13.622.52 2.417.33

22.77

0

10

20

30

40

50

60

70

80

90

100

ESTIMATED CONCENTRATION OF ETHANOL(%)

ESTIMATED CONCENTRATION OF ETHANOL(%)

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

food waste as alcohol producer

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