continuous biological hydrolysis of sago starch by aspergillus
TRANSCRIPT
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.
v
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.
viii
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.
ix
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 :
x
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 :
xi
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
xiv
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
xvi
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