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PRODUCTION OF ETHANOL FROM TAPIOCA SUGARS (24pgs)
PRODUCTION OF ETHANOL FROM TAPIOCA SUGARS (24pgs)
PRODUCTION OF ETHANOL FROM TAPIOCA SUGARS (24pgs)
A thesis submitted in partial fulfilment of the requirement for the degree of Bachelor of
Science with Honour (Resource Biotechnology)
I hereby declare that no portion of the work referred in this project has been submitted in
support of an application for another degree qualification of this or any other university or
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ACKNOWLEDGEMENTS
First of all, I would like to deliver my appreciation to my supervisor Professor Dr. Kopli bin
Bujang for his dedicated supervision, patience and advice throughout this project. Also,
special thanks to my co-supervisor Assoc. Prof. Dr. Cirilo for his guidance.
Resource Science and Technology especially to Miss Rubena Malfia Kamal, Miss Sarina,
Miss Nur Jannah and also Miss Komathi for their assistance in this project.
Next, my sincere gratitude to my family for their encouragement, motivation and rendered
support during the development of this project. Last but not least, my greatest appreciation to
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TABLE OF CONTENTS
Declaration i
Acknowledgements ii
Table of contents iii
List of Figure v
List of Tables vi
List of Abbreviations vii
Abstract 1
1.0 INTRODUCTION
1.1 Background of study 2
1.2 Objectives 3
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4.0 RESULTS AND DISCUSSION
4.1 Characterization of fresh tapioca (FT) and tapioca flour (TF) 18
4.2 Enzymatic hydrolysis of fresh tapioca (FT) and tapioca flour (TF)
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to sugars
4.3 Batch ethanol fermentation from tapioca sugars 23
4.3.1 Utilization of tapioca flour sugar (TFS) at different
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concentrations
4.3.1.1 50g/L of TFS 23
4.3.1.2 100g/L of TFS 25
4.3.1.3 150g/L of TFS 26
4.3.2 Utilization of fresh tapioca sugar (FTS) at different
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concentrations
4.3.2.1 50g/L of FTS 29
4.3.2.2 100g/L of FTS 30
4.3.2.3 150g/L of FTS 32
iv
LIST OF FIGURES
v
LIST OF TABLES
vi
LIST OF ABBREVIATIONS
α alpha
β beta
°C Degree celcius
CO2 Carbon Dioxide
C2H5OH Ethanol
C6H12O6 Glucose
C6H10O5 Starch
H2O Water
DE Dextrose equivalent
FE Fermentation Efficiency
OD Optical density
DCW Dry cell weight
HPLC High Performance Liquid Chromatography
KI Potassium Iodide
RM Ringgit Malaysia
TF Tapioca Flour
TFS Tapioca Flour Sugar
FT Fresh Tapioca
FTS Fresh Tapioca Sugar
cm centimeter
g Gram
kg Kilogram
g/L Gram per liter
L Liter
mg microgram
mL Milliliter
mm Millimeter
μl microlitre
rpm Revolution per min
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Production of Ethanol from Tapioca Sugars
ABSTRACT
Tapioca (Manihot esculenta) is an excellent source of starch which can be hydrolyzed to produce reducing sugars
utilizing bacterial enzymes and subsequently fermented to produce ethanol. The objectives of this study are to
maximize the production of ethanol from hydrolyzed tapioca sugars (mainly glucose) and to study the effects of sugar
concentrations on the productivity of ethanol. Enzymatic hydrolysis was carried out in 2 stages namely liquefaction
and saccharification with the help of enzymes Termamyl SC and Dextroxyme under optimum conditions. Then, the
tapioca sugar syrup was used in batch fermentation using Saccharomyces cerevisiae, a type of baker yeast obtained in
the market. The glucose recovery resulted from tapioca flour was higher compared to fresh tapioca in a range of 63-
68% and 39-45%, respectively. Three different concentrations (50, 100 and 150 g/L) of tapioca sugar from tapioca
flour and fresh tapioca were tested in shake-flask fermentation for 24 hours to observe the optimum sugar
concentration to generate highest yield of ethanol. The ethanol obtained at 12 hours was 25.70 g/L from 50 g/L of TFS
which represented 50.2% of ethanol yield compared to 47.59 g/L from 100 g/L glucose and 58.53 g/L from 150 g/L
glucose with 48.0% and 38.3% of ethanol yield, respectively. Meanwhile, ethanol obtained at 12 hours from FTS was
25.89 g/L, 40.18 g/L and 50.19 g/L from 50 g/L, 100 g/L and 150 g/L of glucose which represented 51.1%, 40.8% and
33.3% of ethanol yield, respectively. Moreover, 150 g/L resulted a residual glucose at the end of 24 hours process for
both types of sugar. Therefore, 50 g/L is selected as the best glucose concentration of ethanol fermentation for
economical reason and to minimize the residual glucose.
Key words: Tapioca, enzymatic hydrolysis, tapioca flour sugar (TFS), fresh tapioca sugar (FTS), ethanol
ABSTRAK
Ubi kayu (Manihot esculenta) merupakan salah satu sumber kanji yang terbaik di mana boleh dihidrolisiskan kepada
gula penurun dengan menggunakan bantuan enzim dari bakteria dan seterusnya boleh difermentasikan untuk
menghasilkan etanol. Objektif pengajian ini adalah untuk memaksimakan penghasilan etanol daripada gula ubi yang
telah dihidrolisiskan (terutama glukosa) dan juga untuk mengenalpasti kesan kepekatan gula kepada penghasilan
ethanol melalui fermentasi. Hidrolisis berenzim dijalankan dengan 2 tahap iaitu pencairan dan pensakarifikasikan
melalui enzim-enzim Termamyl SC dan Dextrozyme dalam keadaan optima. Selepas itu, air gula ubi tersebut
digunakan dalam fermentasi kaedah berkelompok menggunakan sejenis yis segera iaitu, Saccharomyces cerevisiae.
Pemulihan glukosa dari tepung ubi kayu adalah lebih tinggi berbanding ubi kayu segar iaitu dalam julat 63 – 68%
dan 39 – 45%, masing-masing. Tiga kepekatan gula yang berbeza (50, 100 dan 150 g/L) dari ubi kayu segar dan
tepung diuji menggunakan kelalang goncangan selama 24 jam untuk melihat kepekatan optimum demi menghasilkan
etanol yang maksima. Terdapat 25.70 g/L dari 50 g/L GTU pada 12 jam iaitu 50.2% penghasilan etanol berbanding
47.59 g/L dari 100 g/L glucosa and 58.53 g/L dari 150 g/L glucosa dengan 48.0% dan 38.3% penghasilan etanol,
masing-masing. Manakala, etanol yang didapati pada 12 jam dari GUS adalah 25.89 g/L, 40.18 g/L dan 50.19 g/L
untuk 50 g/L, 100 g/L dan 150 g/L glukosa, mempersembahkan 51.1%, 40.8% dan 33.3% penghasilan etanol, masing-
masing. Tambahan lagi, 150 g/L menghasilkan lebihan glukosa walaupun selepas 24 jam proses fermentasi bagi
kedua-dua jenis gula. Oleh itu, 50 g/L dipilih sebagai kepekatan gula terbaik untuk fermentasi etanol di atas alasan
ekonomi dan meminimakan lebihan glukosa.
Kata kunci: Ubi kayu, hidrolisis berenzim, gula tepung ubi (GTU), gula ubi segar (GUS), etanol
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1.0 INTRODUCTION
Tapioca (Manihot esculenta), also known as cassava, manioc or yucca is a plant that
synthesized starch and can be consumed as food with benefits in various industrial processes
(Tonukari, 2004). Tapioca is one of the main food crops that demands less nutrients and able
to attune to drought condition (Burell, 2003). The roots serve as food storing tubers since the
A typical composition of the tapioca root is moisture (70%), starch (24%), fiber (2%),
protein (1%) and 3% of other components, being a potential raw material for in the production
of ethanol for fuel (Tonukari, 2004). Ethanol is generally produced by fermentation of sugars,
cellulose or converted starch. According to International Starch Trading (2003), one ton of
Hydrolyzed tapioca starch generates ethanol through fermentation with the aid of
Brewer’s or Baker’s yeast, Saccharomyces cerevisiae. The yeast is widely used in ethanol
fermentation due its high ethanol yield and productivity, no oxygen requirement and high
ethanol tolerance. In addition, in terms of economic value, yeast is inexpensive and widely
available in market. The success of fermentation depends upon the existence of defined
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Currently, tapioca is a crop that have been overlooked and considered to be without
any commercial value in ethanol production since there are more crops to be undertaken such
as palm oil, sugar cane, maize and wheat. Research done in UNIMAS by Bujang et al., since
1998 until recent on the production of sugars, ethanol and lactic acid from sago starch has
been successful. Therefore, tapioca is believed to have the same potential value as sago, in
producing starch, reducing sugars (glucose), lactic acid and ethanol. This study aims to
produce an alternative bio-fuel from tapioca natural plant sources subsequently will lead to a
safer environment, in an effort to reduce air pollution as well as smoothness of the engines.
In addition there are a lot of idle lands owned by villagers in Sarawak, namely in Kota
Samarahan and Kuching areas. As a future thought, this study can also provide opportunity
for the villagers to use their land by planting tapioca and supply them to researchers or
manufacturers especially in pilot scale production of ethanol. Consequently, the villagers will
be able to earn extra money by working by themselves and simultaneously contribute to the
1.2 Objectives
The main objective is to maximize the production of ethanol from fermentable sugars of fresh
b. Study the effects of sugar concentrations on the productivity of ethanol in shake flask
fermentation trials.
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2.0 LITERATURE REVIEW
Tapioca or also known as ‘Ubi Kayu’ among Malaysian originally comes from tropical
America (Holttum, 1969). Generally, tapioca is one of the tuber plants that able to tolerant
drought conditions and low fertility soils. This is due to rapid stomata closure under water
stress (EL-Sharkawy and Cock, 1990) thus, let the plant productive both in humid and dry
environment. Moreover, tapioca plant can grow at low fertility soil that has fewer nutrients
such as phosphorus. Therefore, it helps to reduce the depletion of natural resources in land
(Hershey and Jennings, 1992). In addition, tapioca can be plant simply by half bury a stem
into the ground (Holttum, 1969). The starch content in the storage roots of tapioca plant is
higher during the period of lower vegetative growth rates (Keating et al., 1982; Hobman and
Starch is a complex carbohydrate which is not completely dissolved in water. Starch is widely
pharmaceuticals and building materials (FAO, 2004). In 2004, FAO reported that the global
demand for tapioca starch can rise up at annual rate of 3.1% and expected to be for Asia
(4.2%), Latin America (3.4%) and Africa (2.3%). In addition, sole cropping is very influential
2002). Malaysia harvests about 400,000 t.year-1 of tapioca from an area of about 39,000 ha
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(Hillocks et al., 2002). However, attempt to penetrate the street market will only succeed if
they have sufficient capital to back the venture and can supply reliable source of starch that
meet the consumer’s specification at a competitive price (FAO, 2004). Modification of starch
by altering their physic-chemical characteristics will diversify their scope of utilization and
intensify their value in comparison with native starch (Tan and Khatijah, 1984).
Starch extracted from plants either sago, potato, corn or tapioca is mainly used in food
production. Tapioca starch acts as a thickener and stabilizer in fruit pies, soups, pudding,
breads, sauces, soy and meat products (International Starch Trading, 2003). In addition,
tapioca is also involved in the production of snack foods such as oil-fried crisps and crackers
(Tan and Khatijah, 1984) and processing into chips and pellets for animal feeding (Hillocks et
al., 2002). Moreover, modified tapioca starch is used as colloid stabilizer and sweetener
Enzymatic hydrolysis process is more preferable in fermentation because less unwanted by-
products will be formed and therefore yield more products (Aiyer, 2005). This process
involves two steps namely liquefaction and saccharification (Bujang et al., 2000). Starch is
made by granules in most plant cells and is referred to native when in this particular granular
state. The starch granule is opened and allows access of the enzymes in the gelatinization step
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The purpose of liquefaction is to provide the partially hydrolyzed starch low viscosity
to be easier saccharified. According to Bujang et al., (2000) the enzyme that require in this
catalyzes the hydrolysis of the α-1, 4 glycosidic bond of starch (Aiyer, 2005). Moreover, it
also helps to reduce viscosity and induce partial hydrolysis of starch. Eventually, high glucose
syrup can be obtained from this step as well (Tucker and Woods, 1991).
On the other hand, saccharification is a step where it is further hydrolyzed into simpler
sugars. According to Bujang et al., (2000) the enzyme called Dextrozyme (a mixture of
utilized here to remove β-glucose units of starch by catalyzing the hydrolysis of both α-1, 4
and α-1, 6 glycosidic bond. The hydrolysis of tapioca flour has been proposed in the
Ulibarri and Hall, 1997). Tapioca flour production was considered as simpler and more
Starch from maize, barley, wheat, oats, rye, rice, potatoes, tapioca, grain sorghum
(Rose & Harison, 1993) and sago (Adeni & Bujang, 1998) can be used to produce ethanol
ethanol can be produced from a ton of glucose. Initial studies done by Bujang et al., (2010),
tapioca is the third in a ranking of starch with the highest glucose recovery by 76% DE as
sago starch in the first place with 99% DE and followed by corn starch on the second place,
84% DE. Therefore, this study is in a path to explore the ability of tapioca starch in the
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2.4 Ethanol for biofuel
Ethanol (ethyl alcohol, C2H5OH) is an alcohol made by fermenting and distilling simple
fructose. Ethanol can be fermented from various sources of biomass via hydrolysed sugar
from crops such as wheat, sugar cane, sago and tapioca. Recently, the depleting petroleum
source is due to the increased in world populations and number of vehicles. Therefore, ethanol
production from renewable sources has received a great attention as the alternative fuel.
same goes to corn ethanol in United States (David and Patzek, 2005), ethanol from cassava
(tapioca) in Thailand and bio-ethanol from potato waste in Finland (Liimatainen et al., 2004)
and India (Ramesh et al., 2010). In addition, compared with gasoline, sugar cane fossil fuel
input some 10% - 12% of the final energy and up to 90% CO2 reduction while corn resulted
higher energy input and lesser CO2 reduction at about 15-25% (IEA, 2007). This is because,
ethanol has decreased the need for other octane booster such as benzene, which is toxic and
often carcinogenic. The oxygen reduces emissions of burned hydrocarbons and carbon
Moreover, oxygen permits low temperature combustion with reduction of CO2 and
oxygen in combustion and hence obtains better combustion efficiency. Since the
14.5% (Rasskazchikova, 2004). Nguyen et al., (2008) has stated that the ethanol has lower
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environmental impact than petrol in terms of the combustion by-products and greenhouse
gases.
2.5 Glycolysis
pyruvate as the final product. However, under aerobic condition, the dominant product in
most tissue is pyruvate or pyruvic acid and the pathway is known as aerobic glycolysis.
The fate of pyruvic acid differ in distinct organism namely in yeast, where the pyruvic
acid is decarboxylated and reduced by NADH to form a molecule of carbon dioxide and
ethanol. This process is called alcoholic fermentation and is energetically wasteful because a
lot of free energy of glucose remains in the alcohol to produce good fuel (John, 2011).
Figure 1 below shows the mechanism or pathway of glycolysis, starting from glucose until
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2.6 Ethanol fermentation
Standard batch fermentation was used in ethanol production process by utilizing Baker’s
yeast, Saccharomyces cerevisiae under optimum condition such as pH, temperature and
substrate concentration where hydrolyzed tapioca starch is a source of carbon that acts as
substrate. The overall reaction of the starch converted to ethanol and carbon dioxide via
growth and for secondary metabolite production. Saccharomyces cerevisiae converts sugars
into ethanol under anaerobic condition. Yeast extract contains all the necessary cofactors such
magnesium and calcium that trigger biomass to activate several enzymes (Cysewki and
Wilke, 1978).
substrate utilization, growth and fermentation rate, optimum pH and temperature, stability to
chemicals and physical stress, ethanol yield, freedom from other end-products, osmotolerance
and tolerance to glucose and ethanol (Rose and Harrison, 1993). Saccharomyces cerevisiae is
the only yeast that can rapidly grow either under aerobic or anaerobic conditions. Besides
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3.0 MATERIALS AND METHODS
3.1 Materials
Fresh tapioca (Figure 2) was obtained directly from vendors at the local market (Pasar Satok,
Commercial tapioca flour (Figure 3) obtained from a local supermarket (UNACO) at a price
of RM 3.00/kg.
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3.1.3 Microorganism
The baker’s yeast (Figure 4), Saccharomyces cerevisiae (Bunga Raya brand) was purchased
3.2 Methods
Fresh tapioca was deskinned by removing the brown outer skin (Figure 2) followed by the
pink layer to reveal the white tubers (Figures 5 and 6). The tubers were cut cubes and small
slices to ease the mashing process (Figure 7). Then, 500 g of the small cubes of tapioca was
pulverized in a high speed blender with 1 L of water. After that, the starch slurries (Figure 8)
were used in enzymatic hydrolysis. For storing purpose, 1 kg of sliced tapioca was packed in
a plastic bag and stored at 4˚C. The tapioca was stored in low temperature to prevent the
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Figure 5: Deskinning of tapioca tubers. Figure 6: The white tuber of tapioca
after deskinned.
Hot plate stirrer was used to carry out enzymatic hydrolysis process. Starch slurries in Figure
8 was adjusted to pH 6 – 6.5 and gelatinized at 80˚C for 10-20 minutes to allow the starch to
dissolve in water. Then, 0.5 µl Termamyl SC per gram of starch was added and the starch
slurries were maintained at temperature 80 – 90˚C for 2 hours. This liquefaction step (Figure
9) helps to convert starch granules into soluble dextrins by α-amylase. The second step which
is saccharification was carried out after cooling down the temperature of the slurries and
transferred into a bottle. Then, the pH was adjusted to 4 - 4.5 and 0.6 µl Dextroxyme per gram
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of starch was added to the slurries once reached 60˚C. The saccharification (Figure 10) was
procedure in our laboratory from the previous work of Bujang et al., (2000).
After the hydrolysis was done, the sugar syrup was centrifuged at 8000 rpm for 20 minutes
and the supernatant was removed. Then, sugar syrup was filtered by vacuum pump using 0.45
µm filter membrane (Figure 11) to separate the impurities in the glucose sample. The color of
Figure 11: Filtration using vacuum pump. Figure 12: Before and after filtration.
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3.2.3 Fermentation medium
The glucose content was determined using HPLC or glucose analyzer to calculate the volume
of hydrolysed sugar syrup to be used in the medium. M1V1=M2V2 formula was applied to
obtain 50 g, 100 g and 150 g concentration in 1 L fermentation medium which was later filled
up by distilled water up to a 1 L working volume. 5 g of yeast extract was added into the
medium as the growth promoter and the pH is set to a range of 6.0 – 6.5. The medium was
poured into a 2 L conical flask set up with a long silicon tube passed through the stopper. The
end of the tube and mouth of the flask was covered up by aluminum foil before autoclaving.
autoclaved fermentation medium. This step was done in a laminar chamber to minimize the
risk of contamination. Then, the flask was left on the hot plate stirrer at 34˚C and agitation
rate at 300 rpm. The fermentation was done for 24 hours. A syringe was connected at the end
of the tube for sampling purpose (Figure 13). The same procedure was repeated for other
concentration which at 50 g/L, 100 g/L and 150 g/L for both fresh tapioca sugar (FTS) and
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Figure 13: Ethanol fermentation from sugar syrup.
3.2.5 Sampling
Approximately, 10 ml sample was obtained every 6 hours, for 24 hours. The sample was
filtered using a vacuum pump and the cell collected on the filter paper was weighted and dried
in the oven at 60˚C. The filtrate was kept in refrigerator until further analyses. Sampling was
Iodine solution was used to determine the starch content in the tapioca. 2.0 g of potassium
iodide, KI was dissolved in 80 ml of distilled water before 0.2 g of iodine was added into the
concentrated solution of KI. The mixture was swirled until all the solid iodine dissolved.
Then, after all the iodine has dissolved, top-up the solution with distilled water up to 100 ml.
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