UNIVERSITI PUTRA MALAYSIA

HYDROLYSIS OF SAGO STARCH BY ASPERGILLUS AWAMORI FOR THE PRODUCTION OF A GENERIC FERMENTATION MEDIUM

SOBRI BIN MOHD AKHIR

IB 2004 5

HYDROLYSIS OF SAGO STARCH BY ASPERGILLUS AWAMORI FOR THE PRODUCTION OF A GENERIC FERMENTATION MEDIUM

By

SOBRI BIN MOHD AKHIR

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia in Fulfilment of the Requirements for the Degree of Master of Science

December 2004

Specially dedicated

Alhamdulillah

To my most beloved parents

Mohd Akhir b. Awang Isa and Fatimah bt. Hanafiah

Your care, support and endless love “My success is only for you”

To my wife

Che Norma bt. Mat Taib

“Thanks for your caring and loving support”

To my son

Muhammad Danish Irfan b. Sobri

“Anak baba”

To my father and mother in-law and all my family

“Thanks for your support”

ii

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirements for the degree of Master of Science

HYDROLYSIS OF SAGO STARCH BY ASPERGILLUS AWAMORI FOR

THE PRODUCTION OF A GENERIC FERMENTATION MEDIUM

By

SOBRI BIN MOHD AKHIR

December 2004 Chairman: Professor Arbakariya Ariff, Ph.D. Institute: Bioscience The efficiency of several amylolytic-enzyme-producing fungi such as Aspergillus

niger, Aspergillus awamori and Aspergillus flavus to hydrolyse cooked sago starch

has been carried out in shake flask cultures. Subsequently, the performance of

microbial hydrolysis of sago starch by A. awamori, which was an industrial strain

used for glucoamylase production, into fermentable sugar was studied in shake flask

culture and 2 L stirred tank fermenter. The influence of medium composition

(different types and concentrations of starch and nitrogen sources) on the rate of

hydrolysis and fermentable sugar production was first carried out using shake flask

cultures. The performance of microbial hydrolysis was also carried out in different

modes of bioreactor operation (batch, repeated batch and continuous operation) using

freely suspended cell system. Beside rate and degree of starch conversion, the

enzymes produced during growth were also determined. For comparison, two-steps

enzymatic hydrolysis which involve liquefaction and saccharification steps, was also

carried out in batch and continuous processes.

The microbial hydrolysis of gelatinised sago starch to fermentable sugar by three

strains of amylolytic-enzyme-producing fungus (A. awamori, A. flavus and A. niger)

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was successfully carried out at low temperature (30oC) in a single step process.

However, the performance of the hydrolysis by A. awamori, in term of yield (0.59 g

glucose/g starch) and overall productivity (0.42 g glucose/L.h), was higher than other

strain. In addition, the yield of microbial hydrolysis of the different starches (sago,

potato, corn and tapioca starch) by A. awamori was more or less the same, ranged

from 0.56 to 0.59 g glucose/g starch. From this study it was also found that the

individual concentration of nitrogen and starch influenced glucoamylase production,

and thus the efficiency of starch hydrolysis to fermentable sugar, to a greater extent

than the carbon to nitrogen (C/N) ratio. The optimized medium formulation for high

performance of starch hydrolysis was by using sago starch ranged from 50 to 100

g/L, yeast extract as the sole nitrogen source and C/N ratio of 17.13.

The yield of glucose produced based on starch used in continuous microbial

hydrolysis using 2 L stirred tank fermenter was lower than that obtained in batch

hydrolysis (0.58 and 0.92 g/g, respectively) but the overall productivity was higher

than that obtained in batch hydrolysis (1.44 and 0.58 g/L.h, respectively). The yield

and overall productivity for microbial hydrolysis were lower than for enzymatic

hydrolysis (0.75 g/g and 4.4 g/L.h, respectively). However, substantially high

amount of biomass was also produced during microbial hydrolysis which can be used

as nitrogen sources for media formulation. The optimum dilution rate and starch

concentration for enzymatic hydrolysis using stirred tank was obtained at 0.125 h-1

and 300 g/L, respectively. On the other hand, the optimum dilution rate and starch

concentration for microbial hydrolysis was obtained at 0.022 h-1 and 30 g/L,

respectively. In addition, microbial hydrolysis was carried out at lower temperature

(30oC) than that required in enzymatic hydrolysis (60-95oC). Thus, large reduction of

iv

operating cost is possible. Once, sterilised the glucose produced in outflow of the

continuous microbial hydrolysis can be used as a generic medium for various

fermentation processes.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

HIDROLISIS KANJI SAGU OLEH ASPERGILLUS AWAMORI UNTUK

PENGHASILAN MEDIUM FERMENTASI

Oleh

SOBRI BIN MOHD AKHIR

Disember 2004 Pengerusi: Profesor Arbakariya Ariff, Ph.D. Institut: Biosains Keberkesanan beberapa kulat penghasil-enzim-amilolitik seperti Aspergillus niger,

Aspergillus awamori dan Aspergillus flavus untuk menghidrolisis kanji sagu yang

dimasak telah dijalankan di dalam kultur kelalang goncang. Seterusnya, hidrolisis

kanji sagu secara proses mikrobial oleh A. awamori yang merupakan sejenis strain

industri yang digunakan untuk penghasilan glukoamilase, kepada gula dijalankan di

dalam kultur kelalang goncang dan fermenter berpengaduk 2 L. Sebagai permulaan,

kesan komposisi media (jenis dan kepekatan kanji dan sumber nitrogen yang

berbeza) ke atas kadar hidrolisis dan penghasilan gula dijalankan di dalam kelalang

goncang. Hidrolisis secara mikrobial juga dijalankan pada mod operasi bioreaktor

yang berbeza (sesekumpul, sesekumpul berulang dan selanjar) menggunakan sistem

sel bebas terampai. Di samping kadar dan darjah penukaran kanji, enzim yang

terhasil semasa pertumbuhan kulat juga telah ditentukan. Sebagai perbandingannya,

hidrolisis kanji secara enzimatik dua-peringkat yang melibatkan proses likuifikasi

dan sakarifikasi juga dijalankan secara sesekumpul dan selanjar.

Hidrolisis kanji sagu kepada gula secara proses mikrobial oleh ketiga-tiga strain kulat

penghasil enzim amilolitik (A. awamori, A. flavus dan A. niger) dijalankan pada suhu

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yang rendah (30oC) menggunakan proses satu peringkat. Walau bagaimanapun,

hidrolisis kanji oleh A. awamori berdasarkan kepada hasil (0.59 g glukosa/g kanji)

dan produktiviti keseluruhan (0.42 g glukosa/L.j), adalah lebih tinggi berbanding

strain yang lain. Malahan, hasil daripada hidrolisis secara mikrobial ke atas kanji

yang berlainan (sagu, kentang, jagung dan ubikayu) oleh A. awamori adalah lebih

kurang sama, dalam julat 0.56 hingga 0.59 g glukosa/g kanji). Daripada kajian ini, di

dapati bahawa kepekatan nitrogen dan kanji mempengaruhi penghasilan

glukoamilase dan keberkesanan hidrolisis kanji kepada gula pada tahap yang lebih

besar daripada nisbah karbon kepada nitrogen (C/N). Formulasi media yang

dioptimakan untuk hidrolisis kanji yang tinggi perlaksanaannya adalah dengan

menggunakan kanji sagu yang berkepekatan antara 50 hingga 100 g/L, ekstrak yis

sebagai sumber nitrogen dan nisbah C/N pada 17.13.

Glukosa yang terhasil berasaskan kanji yang digunakan bagi hidrolisis kanji secara

mikrobial selanjar di dalam fermenter berpengaduk 2 L adalah lebih rendah daripada

yang diperolehi dari hidrolisis secara sesekumpul (masing-masing 0.58 dan 0.92 g/g)

tetapi produktiviti keseluruhannya adalah lebih tinggi daripada hidrolisis secara

sesekumpul (masing-masing 1.44 dan 0.58 g/L.j). Hasil dan produktiviti keseluruhan

proses hidrolisis secara mikrobial adalah lebih rendah daripada hidrolisis secara

enzimatik (masing-masing 0.75 g/g dan 4.4 g/L.j).

Walau bagaimanapun, amaun biomas yang agak tinggi yang dihasilkan semasa

proses hidrolisis secara mikrobial boleh digunakan sebagai sumber nitrogen untuk

formulasi media fermentasi. Kadar pencairan yang optimum dan kepekatan kanji

untuk hidrolisis secara enzimatik di dalam tangki berpengaduk adalah diperolehi

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pada kadar 0.125 j-1 dan 300 g/L. Manakala, kadar pencairan yang optimum dan

kepekatan kanji untuk hidrolisis secara mikrobial adalah diperolehi pada kadar 0.022

j-1 dan 30 g/L. Malahan, hidrolisis kanji secara mikrobial boleh dijalankan pada suhu

yang lebih rendah (30oC) berbanding suhu yang diperlukan untuk hidrolisis secara

enzimatik (60-90oC). Oleh itu, kos operasi untuk menjalankan proses hidrolisis dapat

dikurangkan dengan banyaknya. Apabila disterilkan, glukosa yang dihasilkan dari

hidrolisis selanjar kanji secara mikrobial boleh digunakan sebagai medium untuk

pelbagai proses fermentasi.

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ACKNOWLEDGEMENTS

I wish to express my deepest appreciation and sincere gratitude to my family

members, without their support and instinctive love, I could not become what I am

today. My deepest appreciations go to my father, mother and my wife, Che Norma

for their enormous support and sacrifices that given to me willingly. I would like to

express my hearty thanks to my dear Chairman Committee Supervisors: Prof. Dr.

Arbakariya Ariff, Prof. Dr. Mohammed Ismail Abdul Karim and Assoc. Prof. Dr.

Suraini Abd. Aziz for their financial support, numerous patient, constructive

suggestions and understanding throughout the years of my master study.

My special thanks go to members and staffs in Department of Bioprocess

Technology, Faculty of Biotechnology and Biomolecular Sciences – Encik Rosli,

Kak Aluyah and Renuga. Last but not least, special thanks goes to all my friends,

wherever you are I will always remember you, Insya-Allah.

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I certify that an Examination Committee met on 16th December 2004 to conduct the final examination of Sobri b. Mohd Akhir on his Master of Science thesis entitled "Hydrolysis of Sago Starch by Aspergillus awamori for the Production of a Generic Fermentation Medium" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows: RAHA ABDUL RAHIM, Ph.D. Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) ROSFARIZAN MOHAMAD, Ph.D. Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) LING TAU CHUAN, Ph.D. Faculty of Engineering Universiti Putra Malaysia (Member) SAHAID MOHD KHALIL, Ph.D. Associate Professor Faculty of Engineering Universiti Kebangsaan Malaysia (Independent Examiner)

_________________________________ GULAM RUSUL RAHMAT ALI, Ph.D.Professor / Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date :

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This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirements for the degree of Master of Science. The members of the Supervisory Committee are as follows: ARBAKARIYA ARIFF, Ph.D. Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) MOHAMMED ISMAIL ABDUL KARIM, Ph.D. Professor Kuliyyah of Engineering International Islamic University Malaysia (Member) SURAINI ABD AZIZ, Ph.D. Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member)

_________________ AINI IDERIS, Ph.D. Professor / Dean School of Graduate Studies Universiti Putra Malaysia Date :

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DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations, which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

____________________________ SOBRI B. MOHD AKHIR Date :

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TABLE OF CONTENTS

DEDICATION ii ABSTRACT iii ABSTRAK vi ACKNOWLEDGEMENTS ix APPROVAL x DECLARATION xii LIST OF TABLES xvi LIST OF FIGURES xviiiLIST OF ABBREVIATIONS xix CHAPTER

1. INTRODUCTION 1 2. LITERATURE REVIEW

5

2.1 Properties of Starch 5 2.2 Industrial Applications of Starch 8 2.3 Starch Hydrolysis Products 10 2.3.1 Glucose Production 10 2.3.2 Applications of Glucose Syrup in Industrial and Fermentation

Processes 11

2.4 Hydrolysis of Starch 13 2.4.1 Acid Hydrolysis 13 2.4.2 Enzymatic Hydrolysis 14 2.4.3 Microbial Hydrolysis 23 2.5 Filamentous Fungi: Aspergillus awamori 27 2.5.1 Properties 27 2.5.2 Recombination of Fungi 29 2.6 Fungal Growth 32 2.6.1 Basic Aspects of Fungal Growth 34 2.7 Protein Production from Fungi 35 2.8 Glucoamylase 38 2.8.1 Properties 38 2.8.2 Isolation of Amylolytic Producing Microorganism 40 2.8.3 Production of Enzyme 43 2.8.4 Applications of Enzyme 45 2.9 Summary 47 3. THE PERFORMANCE OF STARCH HYDROLYSIS BY SEVERAL

AMYLOLYTIC FUNGUS

50

3.1 Introduction 50 3.2 Materials and Methods 53 3.2.1 Source of Starches 53

3.2.2 Microorganism, Culture Maintenance and Inoculum Preparation 54

xiii

3.2.3 Medium Composition and Preparation 55 3.2.4 Microbial Hydrolysis Process 56 3.3 Analytical Procedures 57 3.3.1 Cell Concentration 58 3.3.2 Glucose Concentration 58 3.3.3 Glucoamylase Activity 59 3.3.4 Calculation of C/N Ratios 59 3.4 Results and Discussion 60

3.4.1 Performance of Microbial Hydrolysis of Sago Starch by 60 Different Types of Fungus 3.4.2 Effect of Different Types of Starch 62 3.4.3 Effect of Different Nitrogen Sources and C/N Ratios 65 3.5 Conclusions 69 4. THE PERFORMANCE OF SAGO STARCH HYDROLYSIS USING

ENZYME AND MICROBIAL CELLS

70

4.1 Introduction 70 4.2 Materials and Methods 72 4.2.1 Microbial Hydrolysis of Sago Starch 72 4.2.2 Enzymatic Hydrolysis of Sago Starch 75 4.3 Analytical Procedures 78 4.4 Results and Discussion 79 4.4.1 Microbial Hydrolysis of Different Concentrations of Sago 79 Starch 4.4.2 Enzymatic Hydrolysis of Sago Starch 87 4.4.3 Comparison of the Performance 90 4.5 Conclusions 94 5. IMPROVEMENT OF MICROBIAL HYDROLYSIS OF SAGO

STARCH TO GLUCOSE BY Aspergillus awamori USING DIFFERENT MODES OF BIOREACTOR OPERATION

95

5.1 Introduction 95 5.2 Materials and Methods 96 5.2.1 Microorganism and Medium Composition 96 5.2.2 Microbial Hydrolysis of Sago Starch in Different Modes of 97 Bioreactor Operation 5.3 Analytical Procedures 100 5.4 Theory of Continuous Culture 100 5.5 Results and Discussion 104 5.5.1 Repeated Batch 104 5.5.2 Continuous Microbial Hydrolysis of Sago Starch 109 5.5.3 Continuous Enzymatic Hydrolysis of Sago Starch 112 5.5.4 Comparison of the Performance 114 5.6 Conclusions 117 6. CONCLUSIONS AND SUGGESTIONS FOR FURTHER WORK

118

6.1 Conclusions 118

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6.2 Suggestions for Further Work

121

REFERENCES 124 APPENDICES 150 BIODATA OF AUTHOR 158

xv

LIST OF TABLES

Table

Page

1 Comparative characteristics of amylose and amylopectin

6

2 Distribution of amylose and amylopectin in various types of starch

7

3 Product of enzymatic hydrolysis from various types of starch

15

4 Industrially important starch-degrading enzymes

16

5 Comparative advantages of intracellular and extracellular enzyme processes

32

6 Composition of microorganisms of interest in biomass production

36

7 Properties of sago starch

53

8 Other types of starch which used as the carbon source

54

9 Nutrient composition for fermentation experiments using different types of starches

55

10 Effect of different nitrogen sources and C/N ratios on glucose production by A. awamori in shaking flask fermentation

57

11 Comparison of hydrolysis of different concentrations of sago starch by different types of fungus (A. awamori, A. flavus and A. niger) in shake flask

61

12 Comparison of hydrolysis of different types of starch by A. awamori in the shaking flask culture

65

13 Comparison of hydrolysis of sago starch by A. awamori using different C/N ratios in the shaking flask culture

68

14 Geometrical dimensions of the bioreactor

75

15 Comparison of hydrolysis of different concentrations of sago starch by A. awamori in shake flask and 2 L fermenter fermentation

86

16 Comparison of production of reducing sugar during the liquefaction process using different concentrations of sago starch in 2 L fermenter

89

17 Comparison of glucose production from the saccharification process using different concentrations of sago starch in 2 L fermenter

90

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18 Comparison of glucose and glucoamylase production from the hydrolysis of sago starch by A. awamori in repeated batch fermentation

106

19 The performance of continuous microbial hydrolysis of sago starch (initial feed concentration was 30 g/L) by A. awamori at different dilution rates

112

20 The performance of continuous enzymatic hydrolysis of sago starch (initial concentration was 300 g/L) at different dilution rates

114

21 Comparison of the performance of enzymatic and microbial hydrolysis of sago starch using different modes of bioreactor operation

116

xvii

LIST OF FIGURES

Figure

Page

1 Starch processing using enzymes

20

2 A schematic diagram of the bioreactor set-up for continuous culture

26

3 Time courses of hydrolysis of different types of starch by A. awamori in batch process

63

4 Time courses of hydrolysis of sago starch by A. awamori using different nitrogen sources and C/N ratios in batch process

67

5 Photograph of 2L stirred tank fermenter

74

6 Schematic diagram of liquefaction and saccharification process of sago starch

77

7 Time courses of microbial hydrolysis of different concentrations of sago starch by A. awamori in shake flask

81

8 Time courses of microbial hydrolysis of different concentrations of sago starch by A. awamori in 2 L fermenter

82

9 Time courses of enzymatic hydrolysis of different concentrations of sago starch in 2 L fermenter during liquefaction and saccharification process

88

10 Comparison of the performance of the microbial hydrolysis of sago starch by A. awamori with two steps enzymatic reaction

91

11 Diagrammatic representation of bioreactor set-up for continuous liquefaction process

101

12 Diagrammatic representation of bioreactor set-up for continuous saccharification process

102

13 Diagrammatic representation of a fermentation process for a single stage bioreactor

103

14 Time courses of hydrolysis of different concentrations of sago starch by A. awamori in repeated batch fermentation

105

15 Continuous microbial hydrolysis of sago starch by A. awamori at different dilution rates

111

16 Time courses of continuous enzymatic hydrolysis of sago starch at different dilution rates.

113

xviii

xix

LIST OF ABBREVIATIONS

AG Novo amyloglucosidase unit BSPs Biomass support particles C Carbon C/N Carbon/Nitrogen CaCl2 Calcium chloride concn Concentration D Dilution DE Dextrose equivalent DNS 3,5 - dinitrosalicylic acid DOT Dissolved oxygen tension DP Degree of polymerization DW Dry weight Eq. Equation F Flow rate FeSO4.7H2O Ferum sulfate heptahydrate GAmax Maximum glucoamylase

concentration Gmax Maximum glucose concentration Glu Glucose HCl Hydrochloric acid HFCS High fructose corn syrup KH2PO4 Kalium dihydrogen phosphate Km The concentration of substrate

that gives “half maximal activity” M Molar MgSO4.7H2O Magnesium sulfate heptahydrate MW Molecular weight N Nitrogen NaOH Sodium hydroxide (NH4)2SO4 Ammonium sulfate NU Novo α-amylase unit P Productivity PU Polyurethane SCP Single cell protein SSF Solid-state fermentation t Time μ Specific growth rate V Volume x Cell concentration Xmax Maximum cell concentration YE Yeast extract YG/S Ratio of glucose to substrate YX/S Ratio of cell to substrate

CHAPTER 1

INTRODUCTION

One of the problems in the development of fermentation process for the production

of various valuable products is the availability of cheap local carbon sources. Starch

is a polymer of glucose (Kennedy et al., 1988) and can be used as an alternative for

less expensive carbon sources. Several types of starch such as sago, tapioca, corn,

barley, wheat, potato and rice starches are available in the market. Sago palm can

produce 25 tonnes of starch/hectare in swampy areas that unsuitable for any other

crop (Doelle, 1998). The production of sago starch in Peninsular Malaysia is about

11, 000 tonnes which is from an area of only 4, 400 hectares. While Sarawak’s

export is 43, 000 tonnes of quality flour from an area of 19, 720 hectares (Chew and

Shim, 1993). The total area planted with sago in Malaysia is estimated at 35, 000

hectares (mainly in Sarawak and Sabah) and the yield is about 20 tonnes of dry

starch per hectare of sago between the age of 10-15 years (Westphal and Jansen,

1986). At present, amongst other similar agricultural feedstock (eg. cellulose), starch

is most readily available and most readily convertible to fermentable sugars (Reczey

et al., 1986).

Hydrolysis of starch can be carried out by enzymatic and/or chemical routes. With

the availability of commercial amounts of starch-degrading enzymes, traditional

methods using hydrochloric and sulfuric acid have decreased considerably. The

availability of enzymes has been followed by a rapid growth in the starch industry.

Starch-degrading enzymes and their role in the starch industry have already been

1

well reviewed (Fogarty, 1983; Kennedy et al., 1987; Maldonado and Lopez, 1995).

Existing processes were improved with greater efficiency and better quality of

products and the production of a wide variety of starch hydrolysates with well-

defined physical properties and carbohydrate profiles was also made possible

(Slominska, 1993). Biological-based technique is one of the oldest and most

powerful processing tools ever known. So, it is common practice to use

carbohydrates as the carbon sources in microbial fermentation processes and convert

it to a variety of chemicals.

Products of starch hydrolysis, such as maltodextrins, corn, glucose and high-fructose

syrups have wide application in the food, textile, brewing and pharmaceutical

industries. These products are derived mainly from corn, barley, wheat or potato. It

should be possible to obtain similar products from sago starch in addition to other

functional ingredients. One possible area that interesting is in the use of raw sago

starch for the production of glucose. This product can be further converted to

fermentation medium (fructose, gluconic acids, fatty acid esters or polyols) or high

value added products (penicillin).

Glucose syrups are conventionally produced from gelatinised starches by acid and/or

enzyme hydrolysis (Hakulin et al., 1983). The main disadvantages of acid hydrolysis

are the relatively low yield and the excessive formation of by-products. On the other

hand, enzymatic hydrolysis requires starch to be gelatinised first, usually in a jet

cooker, producing a highly viscous slurry. Recent reports highlighted the use of twin

screw extrusion technology as a bioreactor (Chouvel et al., 1983; Hakulin et al.,

1983; Roussel et al., 1991). But these techniques required simultaneous high

2

temperature for enzymatic liquefaction of sago starch and then followed by

subsequent saccharification. So, this technique may require higher operational cost

and in some cases may not economically viable. To overcome such problems, the

microbial hydrolysis of sago starch by amylolytic enzyme-producing fungus such as

Aspergillus awamori and Aspergillus oryzae to fermentable sugar is one of the

possible approaches.

The mold such as A. awamori is able to grow in cooked starch by secreting

amylolytic enzymes (α-amylase and glucoamylase) at 30oC and as a result glucose is

accumulated in the culture broth. Limitation of nitrogen supply inhibits growth, so

that glucose produces is not consumes for further growth. The activity of this mold

can be exploited in continuous hydrolysis of cooked starch at room temperature for

the production of fermentable sugars. The outflow from the continuously operated

bioreactor, once sterilized, is an ideal fermentation medium. In addition, the biomass

produced during the process can also be autolysed chemically or by heat treatments

to extract nitrogen which can be used as nitrogen source in the formulation of the

fermentation medium.

In this study, the feasibility of using several amylolytic enzyme-producing fungus,

such as, A. awamori, A. niger and A. flavus in microbial hydrolysis of gelatinised

sago starch into fermentable sugars was investigated. Further study was also carried

out to find the optimize medium (starch concentration, type of nitrogen and C/N

ratio) for improvement of the microbial hydrolysis rate of gelatinised sago starch and

other starches (corn, potato and tapioca) by A. awamori in a single step batch

process. The use of continuous process for the improvement of microbial hydrolysis

3

4

in terms of yield and productivity was also explored. For comparison, two steps

enzymatic hydrolysis, liquefaction and saccharification processes, was also carried-

out.

The objectives of this study were as follows:

a) To study the feasibility of using amylolytic enzyme producing mold (A.

awamori, A. flavus and A. niger) in hydrolysing gelatinised sago starch for the

production of a generic fermentation medium.

b) To investigate the effect of sago starch concentration, type of nitrogen source and

C/N ratio on the performance of sago starch hydrolysis by A. awamori.

c) To compare the performance between microbial technique in hydrolyzing starch

to fermentable sugars with enzymatic technique.

d) To study the influence of the different modes of bioreactor operation (batch and

continuous) on the performance of microbial hydrolysis of sago starch by A.

awamori.

5

CHAPTER 2

LITERATURE REVIEW

2.1 Properties of Starch

In fermentation, the cost of the substrate is an important economic parameter. Instead

of using pure glucose, starch can be served as a cheap source of substrate for

fermentation processes. According to Andrzejczuk et al. (1985), various kinds of

starch such as potato, corn, wheat, rice and tapioca proved uniform as a carbon

source for microbial hydrolysis. Starch is formed as a reserve carbohydrate in plants

by the enzymatic condensation of glucose. It is the most abundant utilizable resource

in plant biomass and can be hydrolysed enzymatically into fermentable sugar

available to microorganisms. Pure starch is a white, amorphous, relatively tasteless

solid, which possesses no odour and naturally is water-insoluble granules. Starches

are polymers of anhydroglucose units with the general formula of (C6H10O5)n in

which these units are joined by α-glucosidic linkages.

Normally natural starches consist of two main polysaccharides, amylose and

amylopectin in a ratio of 20-30% to 70-80%, respectively (Shannon and Garwood,

1984). Both of which are based on 1,4-linked α-D-glucose chains. Amylose is an

essentially linear polymer having primarily α-1,4 linkages whereas amylopectin is

highly branched polymer which is also mainly α-1,4-linked units, but about 5% of

the bonds are α-1,6 branch points. Amylose is very unstable in water even in dilute

solutions of 1% or less. It is usually defined, on the basis of its interaction with