production of ethanol from tapioca sugars farah … of ethanol from tapioca sugars... · tapioca...

PRODUCTION OF ETHANOL FROM TAPIOCA SUGARS

FARAH ASILAH BINTI AZRI

(23463)


FARAH ASILAH BINTI AZRI

Bachelor of Science with Honours

(Resource Biotechnology)

2012

Faculty of Resource Science and Technology


FARAH ASILAH BINTI AZRI (23463)

A thesis submitted in partial fulfilment of the requirement for the degree of Bachelor of

Science with Honour (Resource Biotechnology)

Supervisor: Prof. Dr. Kopli Bin Bujang

Co-Supervisor: Assoc. Prof. Dr. Cirilo N. Hipolito

Resource Biotechnology Programme

Department of Molecular Biology


Universiti Malaysia Sarawak

i

DECLARATION

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

institution of higher learning.

(Farah Asilah Binti Azri)

Resource Biotechnology

Department of Molecular Biology



ii

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.

Then, millions of thanks to postgraduate students of the Biochemistry Laboratory, Faculty of

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

my course mates and friends.

iii

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

2.0 LITERATURE REVIEW

2.1 Biology and physiology of tapioca 4

2.2 Utilization of tapioca starch 4

2.3 Enzymatic hydrolysis of starch 5

2.4 Ethanol for biofuel 7

2.5 Glycolysis 8

2.6 Ethanol fermentation 9

3.0 MATERIALS AND METHODS

3.1 Materials

3.1.1 Fresh tapioca (FT) 10

3.1.2 Tapioca flour (TF) 10

3.1.3 Microorganism 11

3.2 Methods

3.2.1 Preparation and extraction of starch 11

3.2.2 Enzymatic hydrolysis of tapioca starch 12

3.2.3 Fermentation medium 14

3.2.4 Batch fermentation 14

3.2.5 Sampling 15

3.2.6 Analytical Techniques 15

3.2.6.1 Total starch determination 15

3.2.6.2 Moisture content 16

3.2.6.3 Ethanol determination 17

3.2.6.4 Fermentation efficiency 17

iv

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)

to sugars 20

4.3 Batch ethanol fermentation from tapioca sugars 23

4.3.1 Utilization of tapioca flour sugar (TFS) at different

concentrations 23

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

concentrations 29

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

5.0 CONCLUSION AND RECOMMENDATION 41

6.0 REFERENCES 42

Appendix A 45

Appendix B 46

v

LIST OF FIGURES

Figure Title Page

1 Fermentation of ethanol via EMP pathway 8

2 Fresh Tapioca (Manihot esculenta), unskinned 10

3 Commercial tapioca flour 10

4 Baker’s yeast, Saccharomyces cerevisiae 11

5 Deskinning of tapioca tubers 12

6 The white tuber of tapioca after deskinned 12

7 Small cubes and slices of tapioca tubers. 12

8 Starch slurries 12

9 Liquefaction 13

10 Saccharification 13

11 Filtration using vacuum pump 13

12 Before and after filtration 13

13 Ethanol fermentation from sugar syrup 14

14 The dried fresh tapioca in crucibles 17

15 Residual biomass of fresh tapioca which was not liquefied after hydrolysis 22

16 Different colour of hydrolyzed sugar (TFS and FTS) 22

17 Bubbles indicate air produced during fermentation (CO2). 23

18 Ethanol fermentation of 50g/L TFS 24



21 Ethanol yield (%) for 50, 100 and 150g/L of TFS 28

22 Ethanol fermentation of 50g/L FTS 30



25 Ethanol yield (%) for 50, 100 and 150g/L of FTS 34

26 Glucose consumption over time at different concentrations for FTS and TFS 37

27 Ethanol production over time at different concentrations for FTS and TFS 39

28 Standard curve of starch 45

vi

LIST OF TABLES

Table Title Page

1 Comparison between characteristics of fresh tapioca (FT) and tapioca flour

(TF) 19

2 Comparison result from this project with previous works 19

3 Analysis sample of 50g/L TFS 24



6 Analysis sample of 50g/L FTS 29



9 Tapioca sugars (glucose) consumption at different concentrations 36

10 Ethanol production from sugars at different concentrations 38

11 Comparison of current result with previous study 40

12 OD readings on starch 45

vii

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

1

Production of Ethanol from Tapioca Sugars

Farah Asilah Binti Azri

Program Resource Biotechnology



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

1.1 Background of study

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

xylem consists chiefly of living cells containing starch (Holttum, 1969).

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

tapioca starch yields 720 L or 95% ethanol theoretically.

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

environmental conditions or parameters namely temperature, agitation speed, pH value,

dissolved oxygen and nutrient.

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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

Malaysia economy and productivity.

1.2 Objectives

The main objective is to maximize the production of ethanol from fermentable sugars of fresh

tapioca. The general objectives of this study are to:

a. Study the process of enzymatic hydrolysis of tapioca to produce fermentable sugars.

b. Study the effects of sugar concentrations on the productivity of ethanol in shake flask

fermentation trials.

4

2.0 LITERATURE REVIEW

2.1 Biology and physiology of tapioca

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

Shepherd, 1987 and Hammer et al., 1987).

2.2 Utilization of tapioca starch

Starch is a complex carbohydrate which is not completely dissolved in water. Starch is widely

used in the production of food, paper, textile, adhesive, beverage, confectionary,

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

in commercialized production especially in Thailand, Malaysia and Sumatra (Hillocks et al.,

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

(source of fermentable sugar) in brewery application (International Starch Trading, 2003).

2.3 Enzymatic hydrolysis of starch

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

just before the hydrolysis occur (Aiyer, 2005).

6

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

step is Termamyl SC (thermostable α-amylase from Bacillus licheniformis). This enzyme

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

glucoamylase from Aspergillus niger and pullulanase from Bacillus acidopullulyticus) is

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

production of glucose in an enzymatic hollow-fiber reactor with 97.3% conversion (Lo’pez

Ulibarri and Hall, 1997). Tapioca flour production was considered as simpler and more

economic compared to tapioca starch production (Oscar and Carlos, 2007).

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

through fermentation process. According to Bujang et al., (2000), approximately 500 kg of

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

production of biofuel ethanol by which it is seen to have a huge opportunity.

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2.4 Ethanol for biofuel

Ethanol (ethyl alcohol, C2H5OH) is an alcohol made by fermenting and distilling simple

sugars. Ethanol is produced by the fermentation of monosaccharide such as glucose and

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.

In Brazil, production of ethanol from sugar-cane is dominant their biofuel industry,

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

monoxide, particularly for older vehicles that tend to burn rich.

Moreover, oxygen permits low temperature combustion with reduction of CO2 and

NOx emissions (IEA, 2007). As an oxygenated compound, ethanol provides additional

oxygen in combustion and hence obtains better combustion efficiency. Since the

completeness of combustion is increased by the present of oxygenated fuels, the emission of

carbon monoxide is reduced by 32.5% while the emission of hydrocarbon is decreased by

14.5% (Rasskazchikova, 2004). Nguyen et al., (2008) has stated that the ethanol has lower

8

environmental impact than petrol in terms of the combustion by-products and greenhouse

gases.

2.5 Glycolysis

Glycolysis is also known as Embden-Meyerhof-Parnas or EMP Pathway which involves the

oxidation of glucose. Throughout this mechanism, glucose is oxidized to either lactate or

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

the final product, ethanol.

Figure 1: Fermentation of ethanol via EMP pathway.

9

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

glucose can be expressed as:

The medium composition is critical to provide essential nutrients for microbial

growth and for secondary metabolite production. Saccharomyces cerevisiae converts sugars

into ethanol under anaerobic condition. Yeast extract contains all the necessary cofactors such

as amino acids, purines, pyrimidines and other minerals. It consists of phosphorus,

magnesium and calcium that trigger biomass to activate several enzymes (Cysewki and

Wilke, 1978).

Selection of Baker’s yeast, in ethanol production is made by considering its

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

that, baker’s yeast is inexpensive and widely available.

(C6H10O5)n + (n-1) H2O nC6H12O6 2nCH3CH2OH + 2nCO2

Starch Water Glucose Ethanol Carbon Dioxide

10

3.0 MATERIALS AND METHODS

3.1 Materials

3.1.1 Fresh tapioca (FT)

Fresh tapioca (Figure 2) was obtained directly from vendors at the local market (Pasar Satok,

Kuching) at an average price of RM 2.00/kg.

3.1.2 Tapioca flour (TF)

Commercial tapioca flour (Figure 3) obtained from a local supermarket (UNACO) at a price

of RM 3.00/kg.

Figure 2: Fresh Tapioca (Manihot esculenta), unskinned.

Figure 3: Commercial tapioca flour.

11

3.1.3 Microorganism

The baker’s yeast (Figure 4), Saccharomyces cerevisiae (Bunga Raya brand) was purchased

from a supermarket at RM 3.50 per box of 55 g.

Figure 4: Baker’s yeast, Saccharomyces cerevisiae.

3.2 Methods

3.2.1 Preparation and extraction of starch

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

growth of fungi and minimize changes of the moisture content.

12

3.2.2 Enzymatic hydrolysis of tapioca starch

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

Figure 5: Deskinning of tapioca tubers. Figure 6: The white tuber of tapioca

after deskinned.

Figure 7: Small cubes and slices of

tapioca tubers.

Figure 8: Starch slurries.

13

of starch was added to the slurries once reached 60˚C. The saccharification (Figure 10) was

done in 4 hours to convert dextrin to glucose by using glucoamylase. This is an established

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

the sample becomes clearer after filtration (Figure 12).

Figure 9: Liquefaction. Figure 10: Saccharification.

Figure 11: Filtration using vacuum pump. Figure 12: Before and after filtration.

14

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.

3.2.4 Batch fermentation

Approximately, 10 g of baker’s yeast, Saccharomyces cerevisiae was added into the

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

tapioca flour sugar (TFS).

15

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

performed with strict aseptic technique to minimize contamination.

3.2.6 Analytical techniques

3.2.6.1 Total starch determination

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.

Figure 13: Ethanol fermentation from sugar syrup.

production of ethanol from tapioca sugars farah … of ethanol from tapioca sugars... · tapioca...

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