UNIVERSITI PUTRA MALAYSIA

PRODUCTION OF SUGARS FROM SAGO HAMPAS BY TRICHODERMA SP. DURING SOLID SUBSTRATE FERMENTATION

ZAIZUHANA BINTI SHAHRIM.

FBSB 2006 18

PRODUCTION OF SUGARS FROM SAGO HAMPAS BY TRICHODERMA SP. DURING SOLID SUBSTRATE FERMENTATION

ZAIZUHANA BINTI SHAHRIM

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

March 2006

GOD You are m y Supreme Love

My beloved parents who brought me to this world, thanks for your love and care

My husband, Mohd. Nasir m y love, m y inspiration

My lecturers guidance and knowledge

My friends thanks for your support.

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

PRODUCTION OF SUGARS FROM SAGO HAMPAS BY TRICHODERMA SP. DURING SOLID SUBSTRATE FERMENTATION

BY

ZAIZUHANA BINTI SHAHRIM

March 2006

Chairman

Faculty

Professor Mohd. Ali Hassan, PhD

Biotechnology and Biomolecular Sciences

Advances in industrial biotechnology offer potential opportunities for economic utilisation

of agro-industrial residues such as sago hampas. Sago hampas, which is a complex

material, is one of the major by-product of the sago starch industry. It contains about

69.82% of starch and 13.88% lignocellulosic materials on dry weight basis. Due to its

abundant availability, it can serve as an ideal substrate for microbial processes for the

production of sugars. Application of solid substrate fermentation technology is an

attractive possibility for such bioconversions.

In this study, solid substrate fermentation (SSF) of sago hampas by indigenous isolated

Trichoderma sp. was carried out. In laboratory scale, SSF was conducted in a 250 mL

Erlenmeyer flask contains 5g of hampas was used as solid substrate and 10% (v/w) of

mycelia suspension was used as inoculum. Parameters optimised includes the initial

moisture content (60, 65, 70, 75 and 80%), mineral salts solution (10, 20 and 30% vlw),

urea concentration (0.5, 1.0 and 2.0% wlv), inoculum density (10, 20 and 30% vlw),

incubation temperature (25, 30, 35, 40, and 45 "C), incubation time (0, 12, 24, 36, 48, 60,

72, 84, 96, 108 and 120 h) and homogenisation speed (8,000, 9,500 and 13,500 rpm) and

time (1, 3 and 5 min) on reducing sugars recovery. Maximum reducing sugars obtained

after optimisation was 460 mglg substrate on 96 h incubation with 80% of initial moisture

content, 10% (vlw) of inoculum density, 1.0% of urea concentration in 20% (wlv) of

mineral salts solution and incubated at 30 + 2 OC. The solid culture was homogenised at

9, 500 rpm for 3 minutes for reducing sugars recovery. Meanwhile, the maximum enzyme

activities obtained were 3.19 UImL, 2.22 UImL, 1.66 UImL, 1.1 1 UImL and 1.48 UImL for

a-amylase, glucoamylase, carboxymethyl cellulase, filter paperase and Ij-glucosidase

respectively.

Bioconversion of sago hampas using a rotary drum was conducted by using a modified

cement mixer. Operating parameters such as temperature, moisture, agitation and aeration

via SSF were studied to achieve higher production of reducing sugar. After 96 h of

fermentation, maximum reducing sugar obtained was 380 mglg substrate. Maximum

enzyme activities achieved were 2.74 UImL, 2.19 UImL, 1.33 UImL, 1.12 UImL and 1.07

UImL for a-amylase, glucoamylase, carboxymethyl cellulase, filter Paperase and

I3-glucosidase, respectively.

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

PENGHASILAN GULA DARIPADA HAMPAS SAGU OLEH TRICHODERMA SP. SEMASA PENAPAIAN SUBSTRAT PEPEJAL

Oleh

ZAIZUHANA BINTI SHAHRIM

Mac 2006

Pengerusi

Fakulti

Profesor Mohd. Ali Hassan, PhD

Bioteknologi dan Sains Biomolekul

Kejayaan dalam industri bioteknologi telah membuka peluang ekonomi melalui

penggunaan sisa agroindustri seperti hampas sagu. Hampas sagu ialah sejenis bahan yang

kompleks, antara sisa buangan terbesar dalam industri penghasilan kanji daripada sagu. Ia

mengandungi 69.82% kanji dan 13.88% bahan lignoselulos dalam berat kering. Oleh

kerana penghasilan hampas sagu yang berlebihan, ianya amat sesuai dijadikan substrat

dalam proses mikrobial untuk penghasilan gula. Justeru itu, penggunaan teknologi

penapaian substrat pepejal amat berpotensi bagi menjayakan proses biopenukaran ini.

Dalam kajian ini, penapaian substrat pepejal (SSF) oleh kulat tempatan yang telah

dipencilkan iaitu Trichoderma sp. telah dijalankan. Kajian diperingkat makrnal, SSF

dilakukan dengan menggunakan kelalang Erlenmeyer, 250 mL. 5g hampas sagu

digunakan sebagai substrat dan ampaian spora berusia tujuh hari pengkulturan digunakan

sebagai inokulum. Parameter yang dioptimumkan termasuk lembapan awal(60, 65, 70,75

and 80%), larutan garam mineral (10, 20 dan 30% bli), kepekatan urea (0.5, 1.0 dan 2.0%

bli), ketumpatan inokulum (10, 20 dan 30% bli), suhu inkubasi (25, 30, 35, 40, dan 45"C),

masa inkubasi (0, 12, 24, 36, 48, 60, 72, 84, 96, 108 and 120 h) kelajuan homogenasi

(8,000, 9,500 dan 13,500 rpm) dan masa homogenasi (1, 3 and 5 min). Gula penurun

maksima yang diperolehi pada 96 jam inkubasi ialah 460 mglg substrat dengan lembapan

awal 80%, 10% (vlw) ketumpatan inokulum, 1.0% (bli) kepekatan urea dalam 20% (bli)

larutan garam dan diinkubasi pada 30 2 2 "C. Pepejal kultur dihomogenasi pada 9,500 rpm

selama 3 minit. Aktiviti enzim maksima yang diperolehi ialah 3.19 UImL, 2.22 U ImL,

1.66 UImL, 1.1 1 UImL dan 1.48 Ulml untuk a-amilase, glukoamilase, karboksimetil

selulase, filter paperase dan P-glukosidase masing-masing.

Biopenukaran hampas sagu menggunakan drum berputar dijalakan dengan menggunakan

pengadun simen yang telah diubahsuai. Parameter operasi seperti suhu, lembapan,

pengadukan, pengudaraan dalam SSF dikaji untuk memperolehi hasil gula penurun yang

tinggi. Setelah 96 jam fermentasi, gula penurun maksima yang diperolehi ialah 380 mglg

substrat. Aktiviti enzim maksima yang diperolehi ialah 2.74 UImL, 2.19 UImL, 1.33

UImL, 1.12 UImL and 1.07 UImL untuk a-amilase, glukoamilase, karbosimetil selulase

(CMCase), Filter Paperase (FPase) and P-glukosidase (BGDase) masing-masing.

ACKNOWLEDGMENTS

All praise to supreme Almighthy Allah s.w.t the only creator, whose blessing and kindness

have enabled me to accomplish this project successfully.

I would like to convey my deepest appreciation and thanks to Prof. Dr. Mohd. Ali Hassan

from the Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia,

chairman of my supervisory committee for his endless advise, guidance, patience and

encouragement that lead to the completion of this thesis.

I would also like to express my thanks and gratitude to Prof. Dr Mohamed Ismail Abdul

Karim (UIA), Prof. Dr. Vikineswary Sabaratnam from the Institute of Biological Sciences,

Faculty of Science, Universiti Malaya and Assoc. Prof. Dr. Suraini Abdul Aziz from the

Faculty of Biotechnology and Biomolecular Sciences (UPM) for their constructive

suggestions and guidance, which has helped to sharpen my thinking.

In addition, I would like to express my appreciation to all my lab-mates and fermentation

laboratory staffs for your moral support, cooperation and willingness to help me

throughout this study. My heartiest thanks go to my husband Mohd. Nasir Abd. Aziz and

both of my parents for their endless love, support and encouragement.

vii

1 certify that an Examination Committee has met on 7'h March 2006 to conduct the final examination of Zaizuhana Shahrim on her Master of Science thesis entitled "Production of Sugars from Sago Hampas by Trichoderma sp. During Solid Substrate Fermentation" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 198 1. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:

Mohd. Arif Syed, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman)

Mariana Nor Shamsudin,PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner)

Suhaimi Mustafa, PhD Lecturer Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner)

Ainon Hamzah, PhD Professor School of Biosciences and Biotechnology Universiti Kebangsaan Malaysia (External Examiner)

~ r o f e s s o r / ~ e ~ u t ~ ~ e a n School of Graduate Studies Universiti Putra Malaysia

Date: 18 MAY 2006

... Vll l

This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee are as follows:

Mohd. Ali Hassan, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman)

Mohamed Ismail Abdul Karim, PhD Professor Kuliyyah of Engineering International Islamic University Malaysia (Member)

Vikineswary Sabaratnam, PhD Professor Faculty of Science Universiti Malaya (Member)

Suraini Abdul Aziz, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member)

AINI IDERIS, PhD ProfessorIDean School of Graduate Studies Universiti Putra Malaysia

Date: 0 8 JUN 2006

DECLARATION

I hereby declare that the thesis is based on 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.

ZAIZ H A BINTI SHAHRIM s Date: 7th March 2006

TABLE OF CONTENTS

Page

DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS

CHAPTER

I

I1

INTRODUCTION

LITERATURE REVIEW Solid Substrate Fermentation

Definition and Characteristics Substrate for Solid Substrate Fermentation

Agricultural Origin of Substrates Macromolecular Structure Substrate Preparation Substrate Heterogeneity

Cellulose Degrading Enzymes Starch Degrading Enzymes Environmental Parameters

Moisture Content Temperature and Heat Exchange

Bioreactor Operation for SSF Factors Affecting Reactor Operation

Agitation Aeration Heat Removal Measurement and Control of Parameters Drum Bioreactors

Sago Hampas Generated Composition of Sago Hampas

Trichoderma General Characteristics Trichoderma Physiology Pathogens Controlled Uses of Trichoderma

. . 11 . . .

111

v vii . . .

Vl l l

xvi xvii xxi

GENERAL MATERIALS AND METHODS General Plan of the Experimental Work Chemical Reagents Substrate Collection and Preparation Microorganism and Preparation Maintenance of Fungal Strain Inoculum Preparation Solid Substrate Fermentation (SSF)

Extraction Method Bioreactor Operation Using Rotary Drum Analytical Methods

Determination of Soluble Protein Determination of Moisture Content Determination Reducing Sugars Determination of Sugars Total Kjeldhel Nitrogen Measurement of Alpha-amylase Activity Measurement of Glucoamylase Activity Measurement of Filter Paparase Activity (FPase) Measurement of Carboxymethyl Cellulase Activity (CMCase) Measurement ofJ-D-Glucosidase Activity (BGDase) Determination of Cellulose, Hemicellulose and Lignin Content Determination of Neutral Detergent Fiber (NDF) Determination of Acid Detergent Fiber (ADF) Determination of Lignin Content Determination of pH Starch Content Determination of Total Ash Determination of Crude Fat Sample Preparation for Scanning Electron Microscopy (SEM)

ISOLATION AND SCREENING OF SAGO HAMPAS DEGRADING FUNGUS Introduction Materials and Methods

Materials Isolation of Sago Hampas Degrading Fungi Primary Screening for Sago Hampas Degrading Fungus Secondary Screening for Sago Hampas Degrading Fungus Radial Growth Estimation Identification of Isolate Growth Profile of Selected Fungus via SSF on Reducing Sugars Production Extraction Method Growth Profile Estimation Analytical Techniques

xii

Determination of Reducing Sugars Soluble Protein and pH Measurement Glucoamylase and Carboxymethyl Cellulase Activities

Results and Discussions Isolation and Identification of Isolates Macroscopic and Microscopic Features

Trichoderma EBOO 1 Aspergillus EB004

Primary Screening Analysis of Sago Hampas Degrading Fungi Secondary Screening for Sago Hampas Degrading Fungi Growth Profile of Selected Fungus via SSF on Reducing Sugars Production

Soluble Protein, Weight Loss and pH Profile Enzymes Activities and Reducing Sugars Production

Conclusions

OPTIMISATION OF REDUCING SUGAR PRODUCTION VIA SSF OF SAGO HAMPAS WITH Trichoderma sp. 8 9 Introduction 8 9 Materials and Methods 90

Substrate Collection 90 Substrate Preparation and Pretreatment 90 Microorganism and Maintainance 90 Inoculum Preparation 9 1 Solid Substrate Fermentation 9 1 Extraction Method 9 1 Reducing Sugars and Glucose Determination 9 1 Alpha-amylase and Glucoamylase Activities 9 1 Cellulases Activities 9 1 Soluble Protein and pH Measurement 92 Optimization of Process Parameters 92

Effect of Initial Moisture Content on Reducing Sugars Production Effect of Mineral Salts Solution on Reducing Sugars Production Effect of Urea Concentration on Reducing Sugars Production Effect of Inoculum Density on Reducing Sugars Production Effect of Incubation Temperature on Reducing Sugars Production Effect of Incubation Time on Reducing Sugars Production 94 Effect of Homogenization Time and Speed on Reducing Sugars Recovery 95

xiii

Statistical Analysis Results and Discussions

Sago Hampas Characterisation Pretreatment of Sago Hampas Effect of Initial Moisture Content on Reducing Sugars Production Effect of Mineral Salts Solution on Reducing Sugars Production Effect of Urea Concentration on Reducing Sugars Production Effect of Inoculum Density on Reducing Sugars Production Effect of Incubation Temperature on Reducing Sugars Production Effect of Incubation Time on Reducing Sugars Production Effect of Speed of Homogenization on the Recovery of Reducing Sugars Effect of Time of Homogenization on the Recovery of Reducing Sugars Reducing Sugars Production and Enzymes Activities During SSF Amylolytic Activity During SSF Cellulases Activities During SSF Soluble Protein and pH Profile during SSF

Conclusions

BIOCONVERSION OF SAGO HAMPAS TO REDUCING SUGARS USING ROTARY DRUM Introduction Materials and Methods

Microorganism and Maintenance Substrate and Inoculum Preparation Solid Substrate Fermentation Bioreactor Configuration

Analytical Methods Temperature and pH Measurement Reducing Sugars and Glucose Determination Soluble Protein and pH Measurement Starch Content Amylases Activities Cellulases Activities Extraction Method

Statistical Analysis Results and Discussion

Performance of SSF Using Rotary Drum Growth Observation During SSF

xiv

Moisture Content of Medium During SSF Temperature During SSF Aeration During SSF Mixing During SSF Sterility and Conduct Soluble Protein and pH During SSF Reducing Sugars and Enzymes Activities

Amylolytic Activities Cellulolytic Activity

Degradation of Starch Content Conclusions

VII GENERAL DISCUSSIONS, CONCLUSIONS AND FUTURE SUGGESTIONS

PERWSTAKAAN SULTAN ABDUL S w UNIVERSITI WTRA MALAYSIA

REFERENCES APPENDICES BIODATA OF THE AUTHOR

LIST OF TABLES

Table

Proximate composition of sago hampas % (per gram of hampas) dry weight.

Primary screening analysis of starch degrading ability on width of clear zones formed by each isolates. Results are in triplicates determination.

Page

Primary and secondary screening of celluose degrader fungi. Values are mean + S.D. of triplicates determinations. 8 1

Proximate analysis of sago hampas. Values are mean + S.D. of triplicates determinations.

xvi

LIST OF FIGURES

Figures Page

Three days old Trichoderma EBOOl on PDA plate incubated at 30 + 2 "C. White web mycelia observed with green ring of sporulation.

Five days old Trichoderma EBOOl on PDA plate incubated at 30 + 2 " C shows dense sporulation.

Light Microscopic observation of five days old Trichoderma EBOOl stained with methylene blue (1 00 X magnification). 74

Scanning Electron Micrograph of five days old Trichoderma EB001. 74

Clear zones formed by five days old Trichoderma EBOOl on RSS(a) and RH(b).

Secondary screening measurement of reducing sugars produced by isolated fungi; RSS (H ) and RH ( W ) and control ( ) in Czapek-Dox medium. Values are mean + S.D. of triplicates determinations.

Three days old Trichoderma EBOOl on CMC incorporated in basal medium agar incubated at 30 t 2 "C.

SSF conducted in 250 ml Erlenmeyer flask at 0 hour of cultivation at 30 2 2 "C. Experiments were done in triplicates.

(a) Spreading of white mycelial web over the entire surface of the hampas culture after 12 hours; (b) Mycelia changed from white to dark greenish colour after 120 hours of incubation at 30 2 2 "C via SSF.of incubation at 30 2 2 " C via SSF.

Profiles of pH, weight loss of substrate and soluble protein during SSF. Values are mean + S.D. of triplicates determinations (errors not shown were within the heights of the symbols).

Enzymes activities profiles, glucoamylase, CMCase and reducing sugars production via SSF. Values are mean + S.D. of triplicates determinations (errors not shown were within the heights of the symbols).

Fresh sago hampas collected after the extraction of starch with 90% of moisture content.

Dried and treated hampas using 0.5% (wlv) of NaOH with 14% of moisture content.

Scanning Electron Microscopic micrograph of raw sago hampas.

Comparison of treated and untreated hampas on reducing sugars production by Trichoderma EB001 via SSF cultivated at 30 + 2OC for 120 h. Values are mean + S.D. of triplicates determinations.

Effect of different initial moisture content of solid substrate medium on reducing sugars production by Trichoderma EB001 via SSF. Values are mean 2 S.D. of triplicates determinations (errors not shown were within the heights of the symbols).

Effect of different concentration of mineral salts solution on reducing sugars production by Trichoderma EBOOl via SSF. Values are mean + S.D. of triplicates determinations (errors not shown were within the heights of the symbols).

Effect of different concentration of urea on reducing sugars production by Trichoderma EBOOl via SSF. Values are mean + S.D. of triplicates determinations (errors not shown were within the heights of the symbols).

Effect of different inoculums densities on reducing sugar production by Trichoderma EBOOl via SSF. Values are mean 2 S.D. of triplicates determinations (errors not shown were within the heights of the symbols).

Effect of incubation temperatures ("C) on reducing sugar production by Trichoderma EBOOl via SSF. Values are mean + S.D. of triplicates determinations (errors not shown were within the heights of the symbols).

Effect of different incubation time on reducing sugars production by Trichoderma EBOO1 via SSF. Values are mean + S.D. of triplicates determinations (errors not shown were within the heights of the symbols).

Effect of different speed of homogenisation on the reducing sugars recovery by Trichoderma EBOOl via SSF. Values are mean + S.D. of triplicates determinations (errors not shown were within the heights of the symbols).

xviii

Effect of different time of homogenisation on the reducing sugars recovery by Trichoderma EBOOl via SSF. Values are mean + S.D. of triplicates determinations (errors not shown were within the heights of the symbols).

Alpha-amylase activity during SSF in optimised conditions by Trichoderma EB001. Values are mean + S.D. of triplicates determinations (errors not shown were within the heights of the symbols).

Glucoamylase activity during SSF in optimised conditions by Trichoderma EB001. Values are mean + S.D. of triplicates determinations (errors not shown were within the heights of the symbols).

Relationship between reducing sugar yield and amylases activities in optimised condition via SSF. Values are mean -t S.D. of triplicates determinations (errors not shown were within the heights of the symbols).

Cellulases activities in optimised condition via SSF; FPase ( + ); CMCase ( A ); BGDase ( + ); Control ( ). Values are mean + S.D. of triplicates determinations. 121

Relationship between reducing sugars yield and cellulases activities with optimised conditions via SSF. Values are mean 2 S.D. of triplicates determinations (errors not shown were within the heights of the symbols).

Soluble protein determination during SSF with optimal condition via SSF. Values are mean + S.D. of triplicates determinations (errors not shown were within the heights of the symbols).

pH determination during SSF with optimal condition via SSF. Values are mean + S.D. of triplicates determinations (errors not shown were within the heights of the symbols).

Spreading of white mycelial web over the entire surface of the hampas culture after 12 h of incubation at 30 5 2 O C during optimisation.

Mycelia change from white to dark green colour (dense sporulation) after 120h of incubation at 30 + 2 "C during optimisation. 125

Prepared sago hampas ( 5 kg) with 80% initial moisture content in the drum for solid substrate fermentation.

xix

Spreading of white mycelial web over the entire surface of the sago hampas after 24 h of SSF in the drum.

Mycelia change from white to dark green colour (dense sporulation) after 120 h of SSF.

The rotary drum with fixed baffles. : Internal drum was painted with antifungal paint.

Experimental Set-up; (I) Rotary drum; (2) Motor system; (3) Air Compressor; (4) Heat gun; (5) Bottle contains alcohol; (6) Sterile glass wool.

Schematic diagram from the front view of the rotary drum.

Schematic diagram from the side view of the rotary drum.

Soluble Protein Measurement during SSF. Values are mean 2 S.D. of triplicates determinations (errors not shown were within the heights of the symbols).

pH Measurement during SSF. Values are mean 2 S.D. of triplicates determinations (errors not shown were within the heights of the symbols).

Correlation between reducing sugars production and amylases activities via SSF during bioreactor operation. Values are mean 2 S.D. of triplicates determinations (errors not shown were within the heights of the symbols). 147

Correlation between reducing sugars production and cellulases activities via SSF using rotary drum. Values are mean + S.D. of triplicates determinations (errors not shown were within the heights of the symbols).

SSF

SmF

DNS

PDA

CMC

RSS

RSH

SEM

LIST OF ABBREVIATIONS

Solid Substrate Fermentation

Submerged Fermentation

Dinitrosalicylic acid

Potato Dextrose Agar

Carboxymethyl Cellulose

Raw Sago Starch

Raw Sago Hampas

Scanning Electron Microscopy

xxi

CHAPTER I

INTRODUCTION

Solid substrate fermentation (SSF) has been known for centuries and successfully used for

the production of oriental foods. SSF stimulates the growth of microorganisms in nature

on moist solids. More recently, it has gained much importance in the production of

microbial enzymes due to several economic advantages over conventional submerged

fermentation (SmF), because of its advantages such as less requirement of water, smaller

volumes of fermentation mash, less physical energy requirement, smaller capital

investment, fewer operating costs and lower space requirement furthermore reduced

reactor volumes, easier product recovery, less liquid water to dispose of and hence less

pollution problems. Several reports on SSF have been published in recent years on the

production of fine chemicals (Roukas, 1999; Vandenberghe, 2000) enzymes antibiotics

and immunosuppressant (Leangon et al., 1999).

Lack of knowledge about the fundamental processes is the major limitation of successful

application of SSF for the large scale production, especially on the transport phenomena

occurring during SSF, and also effects of design and operation of bioreactors on SSF

performance itself (Conti et al., 2001). The intermittent mixed bed bioreactor has become

a popular choice for large-scale operation. It is a promising solution since many fungal

processes can tolerate infrequent mixing events without unduly deleterious effects on the

growth kinetics, if the mixing periods are relatively short. In fact, various authors have

worked successfully with intermittently mixed bioreactors at pilot scale, indicating the

potential of this operating strategy (Meien et al., 2004).

In Malaysia, the use of sago starch has been increasing, and it is presently being used for

the production of glucose (Suraini, 2002), on the other hand the sago starch processing

industry is now being confronted with solid and liquid waste disposal problems. One of

the major byproduct is sago hampas. Sago hampas (the fibrous pith residue) obtained

after starch extraction from the rasped sago pith consists of about 66% starch, 15% crude

fiber and 1% crude protein on a dry weight basis (Shim, 1992). The development of

appropriate technology for converting sago hampas into value-added product has been

undertaken by several researches (Tuen, 1994). Hampas, which contains large amounts of

trapped starch granules, has been studied by Rifat et al. (2003) and Vikineswary and

Nadaraj (1992) for its utilisation by amylolytic and cellulolytic fungi. Study by Shim

(1992) revealed high activity of cellulase and a-amylase during the growth of

Myceliopthora thermophila on sago hampas. None of these researches concentrate on the

production of sugars. Based on these findings, sago hampas is a preferable choice as solid

substrate for fermentable sugars production. This so called 'sugars syrup' as a product in

this study is an ideal substrate for all microorganisms, and thus can be used in making a

variety of other products such as biopolymers, antibiotic, acetone, butanol, citric acid,

lactic acid etc.

Fungi play a key role in solid substrate fermentation. Their hyphal development allows

them to effectively colonise and penetrate the solid matrix (Biesebeke et al., 2002). The

hyphal mode of growth gives a major advantage to filamentous fungi over unicellular

microorganisms in the colonization of solid substrate and for the utilisation of available

nutrients. Considering that fungi are suitable for wide range of industrial applications

(e.g. organic acids and enzymes) their exploitation to their full potential many times

associated with the use of heterogeneous fermentation media (Koutinas et al., 2003) such

as sago hampas. Members of the fungal genus Trichoderma have been extensively

studied, particularly due to their ability to secrete cellulose degrading enzymes (Zaldivar et

al., 2001). Most of the work has been carried out on strains of T. viride, T. reesei,

T. harzianum. These strains have been extensively studied in their ability to produce

extracellular cellulolytic enzymes, namely endoglucanases, exoglucanases and cellobiase,

which act synergistically in the conversion of cellulose to glucose (Zaldivar et al., 2001).

The use of Trichoderma sp. in SSF for the production of lytic enzymes such as cellulose

has tremendous impact for an industrial scale production (Nampoothiri et al., 2004).

Trichoderma sp. is also known of its amylases production as well and few studies on its

ability to produce amylases have been reported and among them by Azevedo et al. (2000).

The objectives of this study were:

a. to isolate and screen sago hampas degrading fungus and to study its growth profile via

SSF.

b. to optimise the production of reducing sugars from sago hampas via solid substrate

fermentation by Trichoderma EBOO 1.

c. to study the bioconversion of sago hampas to reducing sugars via SSF by Trichoderma

EBOO 1 in rotary drum bioreactor.