ETHANOL PRODUCTION FROM FOOD WASTE

Declaration
We declare that this project is based on our original work. We write this project by the
knowledge gets from the literatures. We have dually acknowledged and refereed all materials
used in this work. We understand that non-adherence to the principles of academic honesty and
integrity, misrepresentation/ fabrication of any idea/data/fact/source will constitute sufficient
ground for disciplinary action by the university and can also evoke penal action from the sources
which have not been properly cited or acknowledged thesis.

Advisor; signature

Mr. Melkamu birile ……………………………………………….

Name of student

Fanus melese………….………………………………………………

Abadi Brhanu………………………………………………………..

Ashenafi H/Michael………………………………………………..

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Acknowledgement
First of all, we would like to thank God for giving us the strength to finish our project. He gave
us the courage and wisdom to pursue this original and challenging idea and contribute a basic
knowledge foundation for the production of ethanol. Next, we would like to thank the School of
Chemical engineering for preparing this semester project program Special thanks to Mr.
melkamu our advisor, who relentlessly supported and advised us from the beginning of the
project and our organic lab assistance.

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Executive summery

Recently, research on the alcoholic fermentation of kitchen waste has been accelerating for both
ecological and economical reasons, primarily for ethanol use as renewable biofuel. Present work
deals with the fermentative production of ethanol from different starch based kitchen waste.
Kitchen waste from local students’ restaurant was separated by basic component as: potato,
wheat bread and pasta. Thermo-acidic pretreatment of these raw materials was conducted by the
addition of HCl up to pH of 0.5, 1 and 1.5 by autoclaving at 1200C and 1100c for 30 min. the best
hydrolyzing step was found at PH of 0.5 and temperature of 1200c. Commercial pure-culture yeast
Saccharomyces cerevisiae was used as a producing microorganism for ethanol fermentation. After
inoculation the flasks was fixed on a rotary shaker at shaking frequency 120 rpm and shaking
diameter 30 mm, and placed in a thermostat at 30oC. At the end of each batch fermented liquid
will be distilled

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Contents
Declaration ..................................................................................................................................................... i
Acknowledgement ........................................................................................................................................ ii
Executive summery ...................................................................................................................................... iii
List of Table .................................................................................................................................................. v
List of Figure................................................................................................................................................. v
CHAPTER ONE ........................................................................................................................................... 1
1.1 Introduction ......................................................................................................................................... 1
1.2 Statement of the problem .................................................................................................................... 3
1.3 Objective ............................................................................................................................................. 4
1.3.1 General objective ......................................................................................................................... 4
1.3.2 Specific objective ......................................................................................................................... 4
CHAPTER TWO .......................................................................................................................................... 5
LITRATURE REVIEW ............................................................................................................................ 5
CHAPTER THREE ...................................................................................................................................... 8
3.1 MATERIALS AND METHODS ........................................................................................................ 8
3.1.1 Materials ...................................................................................................................................... 8
3.2 Method ................................................................................................................................................ 9
CHAPTER FOUR....................................................................................................................................... 12
RESULTS AND DISCUSSION ............................................................................................................. 12
Conclusion and recommendation ................................................................................................................ 16
Conclusion .............................................................................................................................................. 16
Recommendation .................................................................................................................................... 16

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List of Table
Table 3.1 materials and equipment……..…………………………………………………………8

Table 3.2 chemicals ........................................................................................................................ 8

Table 4.1 standard quantity of food waste .................................................................................... 12
Table 4.2 result of iodine test and brix measurement ................................................................... 12
Table 4.3 fermentation time for different food waste ................................................................... 14
Table 4.4 ethanol characterization…………………………………………………………….....15

List of Figure
Figure 1.sieve analysis .................................................................................................................... 9
Figure 2-hydrolysis of food waste ................................................................................................ 10
Figure 3- addition of yeast for fermentation ................................................................................. 11
Figure 4-pH Vs brix Chart at 110oc .............................................................................................. 13
Figure 5-pH Vs brix Chart at 120oc .............................................................................................. 14
Figure 6-Time Vs Ethanol Yield Chart ......................................................................................... 15

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CHAPTER ONE

1.1 Introduction
During the last century, there has been a growing deterioration of the environment due to rapid
industrialization of human production. In addition, the dependence of countries on imported
fossil fuels has meant that satisfaction of their energy needs is not assured. These considerations
made the discovery of renewable fuels—which will be based on national resources—one of the
most important priorities for most countries worldwide [1–3].
Ethanol is an excellent candidate to replace gasoline, and it has traditionally been produced
mainly from sugars or starch [4,5]. The use of sugars and starch as raw materials for ethanol
production had a negative effect on their availability as human food, which raised their prices
globally and increased concern about the ethics of biofuel production [5–7]. This resulted in a
change of focus to the use of renewable raw materials such as lignocellulose biomass and waste.
The use of wastes as raw materials for the production of biofuels not only prevents the ―food
versus fuel‖ dilemma; it also counteracts accumulation of these wastes in the environment. Food
waste is an interesting potential substrate, as the amount of it in the EU is growing.
It has been estimated to increase from 89.3 Mt in 2006 (which resulted in the emission of 170
million tons of CO2) to 126.2 Mt by 2020, according to a report released by the European
Commission in 2010 [8].
Food waste can be derived from homes, manufacturing, food services, and the retail sector;
domestic food waste production is the largest part of them and accounts for 42% of the total. The
problem of food waste production is higher in Asia, where an increase in the total amount of
annual urban food waste is expected—from 278 Mt in 2005 to 416 Mt in 2025 [9].
The disposal of large amounts of such waste can be a challenge, and cause severe environmental
issues when used in landfill sites—such as uncontrolled gas emissions that contribute to the
greenhouse effect, and contamination of water underground [10,11]. It has been estimated that
approximately 125 m3 of gas is produced from each ton of food waste that is used for landfill,
with an average composition of 60%–65% methane and 35%–40% CO2, which is responsible for
8% of the total anthropogenic methane emissions [12]. In many Asian countries, landfill and
open dumping of food waste represent a large proportion of waste treatment methods [13].

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Other practices of food waste utilization, such as feeding animals, have raised serious hygiene-
related issues and the use of them as fertilizers can cause severe pollution of water [14,15].
Moreover, in recent years regulations about feeding animals with food waste have become
stricter. For example, in the EU, the EC law no. 1774/2002 prohibits the use of catering waste for
animal feed [9].
Finally, incineration is also a waste treatment method used for food waste. Despite the fact that
energy can be produced during incineration, the capital and operating costs are considered to be
too high and air pollution can also result [13]. It is obvious that these ―traditional‖ ways of food
waste manipulation have proven to be problematic from an environmental point of view and do
not help in reducing their total amount.
On the other hand, the rich composition of food waste—in carbohydrates, proteins, and
minerals—makes it an excellent raw material for the production of biofuels and bio-based
chemicals through microbial conversion [16,17]. The advantage of this ―alternative‖ use of food
waste is not only to solve the ―food versus fuel‖ dilemma and exploitation of low-cost raw
materials for the production of biofuels and chemicals; it would also lead to a decrease in the use
of landfill areas, minimizing greenhouse gas emissions and improving land use.
Biogas production is a well-established biological process regarding production of biofuels from
food wastes, and high yields of methane have already been reported [18–20]. These good yields
have resulted in the construction and operation of biogas plants, which make use of food waste
as raw material, either alone or in combination with other wastes such as municipal wastewater.
During the last years, there has also been increased interest in exploitation of food waste for the
production of ethanol [10, 15, 21,22].
Another interesting use of food waste is as a raw material for the production of different kinds of
enzymes. The rich and diverse composition of food wastes—which includes proteins, starch,
insoluble carbohydrates, and lipids—can facilitate the production of a diverse range of enzymes.
Some characteristic categories of enzymes that have already been produced from food wastes are
amylases [23,24], proteases [25,26], lignin cellulolytic enzymes [27,28], lipases [29,30], and
pectinolytic enzymes [31,32].
On the other hand, use of food wastes presents some challenges, such as the difficulty in
separating them from the whole waste mass and their easy degradability. The rich composition of
food wastes makes them easily contaminated by various microorganisms, which makes their

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storage and handling a considerable challenge. Moreover, the high water content of food wastes
results in high volumes—which must be stored and transferred. These properties lead to
difficulties in storage and a requirement for large cooling units, which in turn have a negative
impact on the cost of the raw material and its availability. Separation of food waste could be
improved by using a source separation system in houses. Moreover, drying of food waste in situ
could be one possible solution to improve their stability in storage and to reduce the total
volume.
Except for the readily available soluble carbohydrates, food waste also contains insoluble
carbohydrates such as cellulose, which makes the inclusion of an enzymatic saccharification step
important in order to liberate more glucose. Generally a pretreatment step before saccharification
could improve the saccharification yields, but the presence of soluble sugars makes it unsuitable
for application to food waste as the sugars can undergo degradation reactions, with formation of
inhibitors [21]. Also, incorporation of a saccharification step not only facilitates an increase in
soluble sugars but also leads to a decrease in the viscosity of the slurry, which in turn enables
better mixing of the microorganism [33].
Thermo stability of the cellulolytic enzyme(s) is an important consideration, as cellulose
hydrolysis takes place at high temperatures and the enzyme should be able to maintain high
levels of activity for the time required for the saccharification. For this reason, enzymes from
thermophilic microorganisms are considered to be more favorable for industrial applications, due
to their increased activity and stability at high temperatures [34]. Furthermore, when thermo
stable enzymes are used, less of them is needed—as they have higher specific activity at elevated
temperatures [35], thus reducing the cost of the process. Finally, higher temperatures result in
reduced viscosity of the slurry, which in turn enables the application of higher substrate
consistencies—with a positive impact on process economics [36].
Generally, Ethanol or ethyl alcohol is a volatile, flammable, colorless liquid with flash point 55
0
F, ignition temperature 7930F, specific gravity 0.79, vapor density 1.49, vapor pressure
44mmHg, boiling point 1730F, flammable rang 3.3% _19%, and high solubility.[37]

1.2 Statement of the problem

The disposal of large amounts of food wastes cause severe environmental issues when used in
landfill sites—such as uncontrolled gas emissions that contribute to the greenhouse effect, and
contamination of water underground. The convenient way of treatment such food wastes is

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converting to bioethanol which can substitute fossil fuels and expected to be environmental
friendly. using the food waste for ethanol production is not only to solve the ―food versus fuel‖
dilemma; it would also lead to a decrease in the use of landfill areas, minimizing greenhouse gas
emissions, improving land use and hygiene related issue.

1.3 Objective

1.3.1 General objective

Ethanol production from cafeteria food waste

1.3.2 Specific objective

a) To determine the effect of temperature and PH for pretreatment of cafeteria food waste
on ethanol production.
b) To evaluate the effect of starch concentration in each food waste samples on the ethanol
yield.
c) To know the effect of fermentation time on ethanol production.

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CHAPTER TWO

LITRATURE REVIEW
Radojka N.Razmovski et al (2014) recently, research on the alcoholic fermentation of kitchen
waste has been accelerating for both ecological and economical reasons, primarily for ethanol
use as renewable biofuel. Present work deals with the fermentative production of ethanol from
different starch based kitchen waste. Kitchen waste from local students restaurant was separated
by basic component as: peas, green beans, beans, rice, potato, wheat bread and corn. Thermo-
acidic pretreatment of these raw materials was conducted by the addition of HCl up to pH of 1,
and by autoclaving at 120 0C for 30 min. From the experimental result, maximum ethanol yield
was obtained from wheat bread (0.11 g/g). The highest ethanol yield per starch of 0.36 g/g,
which equals to 64% of the theoretical value, was obtained for peas. From the overall analysis,
the examined thermo-acid pretreatment was the most efficient for hydrolysis of wheat bread,
while it was least efficient for green beans.in order to enhance the efficiency of conversion of
starch from kitchen waste into ethanol, pH lower than 1 is highly recommended. The results
demonstrated the potential of different food waste as a promising biomass resource for the
production of ethanol.

Kara walker et al (2013) studied on the use of restaurant waste for production of ethanol. Food
wastes (corn, potatoes, and pasta) were converted to ethanol in a two-step process: a two-part
enzymatic digestion of starch using alpha-amylase and glucoamylase and then fermentation of
the resulting sugars to ethanol using yeast. Because of the low initial composition of starch in the
food waste, low ethanol concentrations were achieved: at best 8 mg/ml ethanol (0.8 % by mass).
Ethanol concentration increased with increasing enzyme dosage levels. Calculations were
conducted to evaluate whether waste heat from restaurant waste could be used to drive flash
vaporization to purify ethanol. If the solution produced by fermenting food waste is flashed at a
temperature of 99.7oC, 77% of the ethanol is recovered in a vapor stream with 1.14 mole%
ethanol (2.87 mass %). Waste heat could provide over a third of the energy for this vaporization
process. If 4 mole% ethanol could be produced in the fermentation step by increasing the initial
starch content in the waste solution and improving the fermentation process, then a single flash at
98.9oC will recover nearly 99% of the ethanol, giving a mass concentration of ethanol of 10.3%,
which is similar to that achieved in industrial grain fermentation.

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H.Le man et al(2009) studied on the production of ethanol from food waste. Research on the
production of ethanol from waste has been accelerating for both ecological and economical
reasons, primarily for its use as an alternative to petroleum based fuels. In this study, response
surface methodology based 23 -full factorial central composite design was employed to optimize
the parameters of ethanol production from Korean food waste leachate. The reducing sugar
concentration of the food waste leachate determined by the dinitrosalicylic acid method was 75
g/L. A second order polynomial model was developed to evaluate the quantitative effects of
temperature, pH and reducing sugar concentration in order to find an optimum condition for the
ethanol production from food waste leachate. From the experimental result, maximum ethanol
concentration of 24.17 g/L was obtained at the optimum condition of temperature (38 ºC), pH
(5.45) and reducing sugar concentration (75 g/L). The experimental value (24.17 g/L) agreed
very well with the predicted one (23.66 g/L), indicating the suitability of the model employed
and the success of response surface methodology in optimizing the conditions of ethanol
production from food waste leachate. Canonical analysis indicated that the stationary point was a
saddle point for the ethanol yield. Despite being a waste, an ethanol yield of 0.32 g ethanol/g
reducing sugar demonstrated the potential of food waste leachate as a promising biomass
resource for the production of ethanol.

Department of Biotechnology, DVR & Dr. HS MIC College of Technology, Kanchikacherla,

A.P. INDIA(2013) studied Production of Ethanol fuel from the garbage/kitchen waste was
carried out with the main purpose of converting the domestic waste into a useful material. The
conversion of food waste or garbage by acid hydrolysis was carried out to obtain fermentable
sugars, which was converted into ethanol by fermentation process using Pichia stipitis. The
present study indicated that at 36 h of incubation resulted in utilization of 29 g/L of glucose with
yield of 9.2 g/L ethanol. Compared to various sugars the glucose resulted in the production of
ethanol.

Leonidas Matsakas et al (2015), the environmental crisis and the need to find renewable fuel
alternatives have made production of biofuels an important priority. At the same time, the
increasing production of food waste is an important environmental issue. For this reason,
production of ethanol from food waste is an interesting approach. Volumes of food waste are
reduced and ethanol production does not compete with food production. In this work, we

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evaluated the possibility of using source-separated household food waste for the production of
ethanol. To minimize the cost of ethanol production, the hydrolytic enzymes that are necessary
for cellulose hydrolysis were produced in-house using the thermophillic fungus Myceliophthora
thermophila. At the initial stage of the study, production of these thermophilic enzymes was
studied and optimized, resulting in an activity of 0.28 FPU/mL in the extracellular broth. These
enzymes were used to saccharify household food waste at a high dry material consistency of
30% w/w, followed by fermentation at 60 0C for 8hrs. Ethanol production reached 19.27 g/L
with a volumetric productivity of 0.92 g/L·h, whereas only 5.98 g/L of ethanol was produced
with a volumetric productivity of 0.28 g/L·h when no enzymatic saccharification was used.

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CHAPTER THREE

3.1 MATERIALS AND METHODS

3.1.1 Materials

Table 3.1 materials and equipment

Material and Function
equipment
Erlenmeyer flask To hold the
sample
Oven To calculate the
dry mass
Rotary shaker To rotate the
fermenting
sample
Autoclave To heat the
sample in
pretreatment
Grinder Size reduction
Sieve To determine
particle size
Bread, pasta and Samples to be
potato fermented
PH meter To measure the
PH value

Table 3.2 chemicals

Chemicals Function Chemical formula
Hydrochloric acid To make the medium acidic HCL
Sodium hydroxide To neutralize the acidic NaOH
medium

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Iodine To test the presence of starch I2

Water To wash the waste foods and H2O
equipment
Yeast(saccharomyces To ferment the food waste
cerevisiae)

3.2 Method
Starch based kitchen waste from local student’s restaurant was separated by basic component as:
pasta, potato and wheat bread. Each food waste sample was washed to remove the other
components found on it. The samples was dried by sun light .each sample was ground
using the grinder to obtain mash with diameter of particles smaller than 3 mm. food waste
samples (44.0 g dm) was poured into an Erlenmeyer flask (0.5l) and mixes with distilled water,
keeping the dry matter to water ratio (hydro module) at 1:5.

Figure 1.sieve analysis

Yeast strain
Commercial pure-culture yeast Saccharomyces cerevisiae was used as a producing
microorganism for ethanol fermentation. The yeast was activated by mixing 15g of agar with
500ml of water at 300c at incubator for three days.
Thermo-acidic pretreatment

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Thermo-acidic pretreatment of the FW mash samples was conducted by the addition of 1 M HCl
0
up to pH of 0.5, 1 and 2, by autoclaving at 110 C and 120 0C for 30 min. Afterwards, the
pretreated KW was cooled at room temperature and was neutralized with 1 M NaOH up to pH 5,
in order to obtain optimal pH of the fermentation medium.

Figure 2-hydrolysis of food waste

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Iodine Test
In order to test if starch hydrolysis is complete the iodine test which is a qualitative estimate of
starches present was conducted. Three drops of iodine was added to 1 ml of each neutralized
food waste sample to test for starches. The presence of starches is denoted by a blue color due to
the starch-iodine complex. Dextrin’s present produce a brown color. Brix meter was used to
determine the quantity of sugar produced.
Ethanol fermentation
After inoculation the flasks was fixed on a rotary shaker at shaking frequency 120 rpm and
shaking diameter 30 mm, and placed in a thermostat at 30 0C. At the end of each batch fermented
liquid was distilled at various time interval (18, 24 and 28) hours.

Figure 3- addition of yeast for fermentation

Fermentation parameters
The ethanol yield (Yp/fw, g/g) was calculated as grams of ethanol (P) produced per gram of
kitchen waste dry matter (DM g). The ethanol yield per starch (Yp/s, g/g) was calculated as
grams of produced ethanol (P) per gram of initial starch (Su).

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CHAPTER FOUR

RESULTS AND DISCUSSION

Standard quantity of food waste:

Table 4.1 standard quantity of food waste

Samples Dry matter (%) Starch (%dm) Protein (%dm)
Bread 68.2 69.9 13.1
Potato 16.4 74.9 6.2
Pasta 87.5 60.5 11.3

Analytical analysis and qualitative determination

Wheat bread= 44/0.682=64.516g
potato=44/0.164=268.3g
pasta=44/0.875=50.3g

Table 4.2 result of iodine test and brix measurement

PH at 0.5 and Temperature at 1200C
Samples Color Brix
Bread Brown 13.331
Potato Brown 14.321
Pasta Brown 19.561
PH at 1 and temperature at 1200c
Bread Blue black 7.621
Potato Blue black 9.741
Pasta Blue black 12.431
pH at 1.5 and Temperature at 1200c
Bread Blue 1.9
Potato Blue 3.334
Pasta Blue 6.12
pH at 0.5 and temperature at 1100c
Bread Blue black 9.321

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Potato Blue black 11.433

Pasta Brown 16.876
pH at 1 and temperature at 1100c
Bread Blue 6.123
Potato Blue black 9.87
Pasta Blue black 10.932
pH at 1.5 and temperature at 1100c
Bread Blue 1.34
Potato Blue 3.783
Pasta Blue 3.991
Fermentation parameters obtained for different starch based food waste

pH Vs brix Chart at 110oc

18 16.87
16
Bread
14
11.433 potato
12 10.932
9.87 Pasta
9.321
brix

10
8
6.123
6
3.783 3.991
4
2 1.34

0
0.5 1 1.5
pH

Figure 4-pH Vs brix Chart at 110oc

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pH Vs brix Chart
25

19.561 Bread
20
potato
14.321 Pasta
15 13.331
12.431
brix

9.741
10
7.621
6.12
5 3.334
1.9

0
0.5 1 1.5
pH

Figure 5-pH Vs brix Chart at 120oc

Table 4.3 fermentation time for different food waste
For 3 days
Samples Ethanol yield(g) Ethanol yield per dry Ethanol yield per
matter(g/g) starch(g/g)
Bread 3.23 0.073 0.109
Potato 2.52 0.057 0.076
Pasta 20.43 0.464 0.769
For 5 days
Bread 4.440 0.101 0.144
Potato 3.32 0.075 0.1
Pasta 26.84 0.61 1.008
For 7 days
Bread 4.45 0.101 0.144
Potato 3.3 0.075 0.1
Pasta 26.93 0.612 1.01

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Time Vs Ethanol Yield Char at 120oc

30 Bread 26.84 26.93
potato
25
Pasta
Ethanol Yield(g/g)

20.43
20

15

10
4.44 4.45
5 3.23 2.52 3.32 3.3

0
3 5 7
Time(days)

Figure 6-Time Vs Ethanol Yield Chart

The results of iodine test and measurement of brix lead to the conclusion that the hydrolysis of
starch from all examined food waste samples to fermentable sugars was accomplished by the
thermo-acidic pretreatment at pH of 0.5 and temperature 1200c.the incomplete hydrolysis could
be due to the factors autoclaving, temperature and pH. In order to enhance the efficiency of
starch hydrolysis, pH<1 and temperature 1200c can be simple possibility. In conclusion, breaking
down the starch to fermentable sugars was a critical step. It is well known that the shape, size
and physicochemical characteristics of starch granules are particular to each plant species.
Hence, the efficiency of starch hydrolysis was different for each kind of examined food waste.
The optimum yield of ethanol from the fermentation could be obtained at 5 days. At this
fermentation time the amount of ethanol yield per dry mass is 4.44 g/g for bread, 26.84 g/g for
pasta and 3.32 g/g for potato. The further fermentation would not result on the yield of ethanol
Ethanol characterization
Ethanol was characterized by measuring its specific gravity, its viscosity and miscibility.

Table 4.4 ethanol characterization

Parameters Measurements Measured Standard value
value(result)
Specific gravity Hydro meter 0.82 0.79

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Viscosity Viscometer 0.005cp 0.001901cp

Miscibility - Miscible

Conclusion and recommendation

Conclusion
The maximum hydrolysis of starch from all examined food waste samples to fermentable sugars
was accomplished by the thermo-acidic pretreatment at pH of 0.5 and temperature 1200c. From
the experiment we could conclude that the factors which determine yield of ethanol are the
fermentation time and the hydrolysis of starch (conversion of starch into sugars). The amount of
starch present in each food waste has not proportional effect to the ethanol production.

Recommendation
The characteristic deviation of measured values of ethanol from the standard values is due to the
separation challenge which encountered us .If there were equipment which could separate the
two mixtures, the yield of ethanol and would be approached to its standard value. There is
shortage of equipment which can challenge the laboratory procedures. We would like
recommend the challenges to be alleviated soon.

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