UNIVERSITI PUTRA MALAYSIA

NG SIM HONG

IPTPH 2012 3

PRETREATMENT OF KENAF (Hibiscus cannabinus L.) CORE FIBRE FOR FERMENTABLE SUGAR

AND LACTIC ACID PRODUCTION

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PRETREATMENT OF KENAF (Hibiscus cannabinus L.)

CORE FIBRE FOR FERMENTABLE SUGAR

AND LACTIC ACID PRODUCTION

By

NG SIM HONG

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfillment of the Requirement for the Degree of Master of Science

December 2012

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment

of the requirement for the degree of Master of Science

PRETREATMENT OF KENAF (Hibiscus cannabinus L.) CORE FIBRE FOR

FERMENTABLE SUGAR AND LACTIC ACID PRODUCTION

By

NG SIM HONG

December 2012

Chair: Professor Paridah Md Tahir, PhD

Institute: Tropical Forestry and Forest Products

Kenaf (Hibiscus cannabinus L.) core which is mainly used for low range products

contains high cellulose content (46.9%) possesses great potential for fermentable

sugar production. Kenaf core can be converted into lactic acid (LA) through

enzymatic hydrolysis and fermentation process. However, pre-treatment is essential

prior to enzymatic hydrolysis for higher fermentable sugar yield. In this study, three

types of pre-treatments were applied: i) Physical pre-treatment (hammer milling,

HM); ii) Physical and thermal pre-treatment (HMTH) iii) Physical and chemical pre-

treatment (HMCH). The cellulase enzyme from Trichoderma reesei was used to

hydrolyze the pre-treated kenaf core. Lactic acid bacteria, Lactobacillus delbrueckii,

Lactoccocus lactis NZ9000 and Lactoccocus lactis MG 1363 were used for

fermentation in batch cultivation for 24 hours using kenaf core hydrolysate. The

chemical composition of kenaf core was analyzed according to TAPPI Standard

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methods and the concentration of fermentable sugar and LA yield was analyzed

using HPLC.

The study showed that NaOH pre-treated kenaf core recorded the highest alpha

cellulose content (91.5%) at >80 mesh among the pre-treatments. After hydrolysed

by cellulose, NaOH pre-treated kenaf core generated the highest amount of glucose

(11.64 g/L) compared to the untreated (40-60 mesh) kenaf core (0.04 g/L). In

fermentation by L. delbrueckii using kenaf core hydrolysate prepared from 2.0 mL

enzyme loading as a substrate, the highest lactic acid production with 11.80 g/L was

obtained at 12th hour of cultivation with 2.86 g/L of cell growth. However, the

maximum concentration of 12.78 g/L of lactic acid was obtained with 3.05 g/L of

maximum cell growth at 24 hours of cultivation. L. delbrueckii recorded the highest

cell efficiency to produce lactic acid at 4.24 g/g, and therefore, recorded the highest

productivity at 0.533 g/L/h. The results show that NaOH pre-treated kenaf core is

the most suitable pre-treatment to obtain the highest glucose yield by increasing the

alpha cellulose content. Subsequently, L. delbrueckii is the most suitable lactic acid

bacteria to produce lactic acid from kenaf core hydrolysates and can be used as a

potential lactic acid bacteria for other lignocellulose bioconversion to lactic acid.

Keywords: kenaf core, enzymatic hydrolysis, glucose, fermentation, Lactobacillus

delbrueckii, lactic acid

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains

PRAPENGOLAHAN KENAF (Hibiscus cannabinus L.) SERAT TERAS

UNTUK PENGHASILAN GULA TERFERMENTASI DAN ASID LAKTIK

Oleh

NG SIM HONG

Disember 2012

Pengerusi: Profesor Paridah Md Tahir, PhD

Institut: Perhutanan Tropika dan Produk Hutan

Bahagian teras Kenaf (Hibiscus cannabinus L.) yang biasanya digunakan untuk

produk julat rendah sebernarnya mengandungi kandungan celulosa yang tinggi

(47.7%) yang tinggi dan mempunyai potensi yang tinggi untuk pengeluaran gula

terfermentasi. Bahagian teras kenaf boleh ditukar menjadi asid laktik melalui proses

hidrolisis enzimatik dan fermentasi. Untuk penghasilan gula terfermentasi (glukosa)

yang lebih tinggi, pra-rawatan perlu dilakukan sebelum proses hidrolisasi enzimatik.

Dalam kajian ini, tiga jenis pra-rawatan diterapkan: i) Pra-rawatan fizikal (HM); ii)

pra- rawatan fizikal dan terma (HMTH) iii) pra-rawatan fizikal dan kimia (HMCH).

Enzim selulosa dari Trichoderma reesei telah digunakan untuk menghidrolisis bahan

kenaf teras selepas pra-rawatan. Bakteria asid laktik, Lactobacillus delbrueckii,

Lactoccocus lactis NZ9000 dan Lactoccocus lactis MG 1363 telah digunakan untuk

fermentasi yang berkelompok selama 24 jam. Kesan pra-rawatan komposisi kimia

pada teras kenaf dianalisis berdasarkan kaedah Piawai TAPPI dan kepekatan

glukosa (g/L) serta asid laktik (g/L) yang terhasil telah dianalisis menggunakan

HPLC.

Kajian in menunjukkan bahawa, teras kenaf yang dipra-rawat dengan NaOH

mencatatkan kandungan alfa selulosa tertinggi (91.5%) dengan >80 mesh saiz.

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Selepas hidrolisi berenzim, teras kenaf yang dipra-rawat dengan NaOH

menghasilkan glukosa tertinggi iaitu (11.63 g/L) manakala teras kenaf yang tidak

dirawat (40-60 mesh) memberi hasil (0.04 g/L). Dalam fermentasi oleh

Lactobacillus delbrueckii dengan glukosa yang didapati daripada 2.0 mL enzim,

pengeluaran asid lactic yang tertinggi tercatat pada 11.80 g/L dan diperolehi pada

jam ke-12 dengan 2.86 g/L pertumbuhan sel. Kepekatan maximum asid laktik

sebanyak 12.78 g/L telah diperolehi pada jam ke-24 dengan 3.05 g/L pertumbuhan

sel maximum. Lactobacillus delbrueckii telah mencatatkan kecekapan sel untuk

menghasilkan asid laktik yang tertinggi pada 4.24 g/g dan, dengan ini, telah

mencatatkan produktiviti tertinggi pada 0.533 g/L/h. Keputusan dalam kajian ini

menunjukkan bahawa teras kenaf yang dipra-rawat dengan NaOH adalah pra-

rawatan yang paling sesuai untuk mendapatkan hasil glukosa yang tinggi dengan

meningkatkan kandungan alfa selulosa. Lactobacillus delbrueckii adalah bakteria

asid laktik yang paling sesuai bagi menghasilkan asid laktik daripada glukosa yang

didapati daripada teras kenaf dan juga berpotensi digunakan untuk sumber

lignoselulosa yang lain.

Kata kunci: bahan teras kenaf, hidrolisis enzimatik, glukosa, Lactobacillus

delbrueckii, asid laktik

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ACKNOWLEDGEMENTS

During this period, I received guidance, assistance and encouragement from my

families, friends, supervisory committee and Institute of Tropical Forestry and

Forest Products (INTROP) staff. With their help and support, I finally result a thesis

which is richer and wider in scope. The list of all contributors is much too long to be

included here. However, I wish to acknowledge several people. Special thank are

due to Prof. Dr. Paridah Md Tahir, Prof. Dr. Luqman Chuah Abdullah and Assoc.

Prof. Dr. Rosfarizan Mohamad for their tireless supervision and encouragement. I

also would like to express my sincere appreciation to my husband Wong Thye Seang

for his never-ending support, encouragement and patience throughout my study

period. My friends Ms. Juhaidah, Ms. Hafiz, Ms. Janet Sayu Ling and Ms. Farina,

Ms. Jovy and Mr. Palie from BioTech 3 have also been a source of encouragement

for me, thank you very much! Sincere thanks to EPU for financial funding to support

my research studied. Lastly I would like to dedicate my deepest gratitude to my

family members for their support and encouragement and thank you Su God for all

the blessing and guidance.

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This thesis was submitted to the Senate of Universiti Pura Malaysia and has been

accepted as fulfilment of the requirement for the degree of Master of Science. The

members of the Supervisory Committee were as follows:

Paridah Md. Tahir, PhD

Professor

Institute of Tropical Forestry and Forest Products

Universiti Putra Malaysia

(Chairman)

Luqman Chuah Abdullah, PhD

Professor

Faculty of Engineering

Universiti Putra Malaysia

(Member)

Rosfarizan Mohamad, PhD

Associate Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Member)

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and citations

which have been duly acknowledged. I also declare that it has not been previously,

and is not concurrently, submitted for any other degree at Universiti Putra Malaysia

or at any other institution.

NG SIM HONG

Date: 4 December 2012

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TABLE OF CONTENTS

Page

ABSTRACT ii

ABSTRAK iv

ACKNOWLEDGEMENTS vi

APPROVAL vii

DECLARATION ix

LIST OF TABLES xii

LIST OF FIGURES xiii

LIST OF ABBREVIATIONS xv

CHAPTER

1 INTRODUCTION 1

1.1 Introduction 1

1.2 Problem statement and justification 6

1.3 Objectives 7

2 LITERATURE REVIEW 9

2.1 Lignocellulosic materials for bioproducts 9

2.1.1 Chemical nature of lignocellulose 9

2.1.2 Biomass availability 13

2.1.3 Kenaf (Hibiscus cannabinus L.) 18

2.1.4 Chemical properties of kenaf 20

2.2 Production of biochemicals from biomass 20

2.2.1 Pre-treatment technologies 21

2.2.1.1 Physical pre-treatment 26

2.2.1.2 Thermal pre-treatment 27

2.2.1.3 Chemical pre-treatment: acid and alkaline 27

2.2.1.4 Combination of pre-treatments 28

2.2.2 Enzymatic hydrolysis 28

2.2.2.1 Cellulolytic enzyme 30

2.2.2.2 Cellulase from Trichoderma reesei 31

2.2.2.3 Limiting factors of enzymatic hydrolysis 31

2.2.3 Fermentation 32

2.2.3.1 Production of lactic acid 35

2.2.3.2 Lactic acid bacteria 38

3 MATERIALS AND METHODS 41

3.1 Experimental design 41

3.2 Chemicals and material used 42

3.2.1 Chemicals 42

3.2.2 Raw materials preparation 42

3.3 Chemical composition determination 43

3.3.1 Cold water solubility determination 44

3.3.2 Hot water solubility determination 44

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3.3.3 Alcohol-acetone solubility 45

3.3.4 Acid insoluble lignin determination 45

3.3.5 Holocellulose determination 46

3.3.6 Alpha cellulose determination 46

3.3.7 Ash determination 47

3.4 Pre-treatment 48

3.4.1 Physical pre-treatment (hammer milling, HM) 48

3.4.2 Physical and thermal pre-treatment (HMTH) 48

3.4.3 Physical and chemical pre-treatment (HMCH) 49

3.5 Microscopic observations of pre-treated kenaf core particles 49

3.6 Kenaf core hydrolysates for lactic acid production 50

3.6.1 Preparation of kenaf core hydrolysates 50

3.6.2 Glucose concentration 50

3.7 Preparation of lactic acid from kenaf core hydrolysate 51

3.7.1 Inoculum preparation 51

3.7.2 Growth medium for lactic acid production (Fermentation) 52

3.8 Determination of cell concentration and lactic acid concentration 53

3.8.1 Determination of cell concentration 53

3.8.2 Lactic acid concentration 54

3.9 Statistical analysis 54

4 RESULTS AND DISCUSSION 56

4.1 Chemical constituents of kenaf core 56

4.2 Effect of pretreatment on kenaf core fibre 57

4.2.1 Effect of pre-treatment on schemical

constituents of pre-treated of kenaf core 57

4.2.2 Effect of pre-treatments on the surfaces of kenaf 60

core particles

4.3 Effect of kenaf core pre-treatment on fermentable sugar 66

production

4.4 Production of lactic acid in batch cultivation of lactic acid 69

bacteria using kenaf core hydrolysate

4.4.1 Lactobacillus delbrueckii 70

4.4.2 Lactococcus lactis NZ 9000 73

4.4.3 Lactoccoccus lactis 1363 75

4.4.4 Comparison of the growth performance of lactic acid 77

bacteria in batch cultivation using kenaf core

hydrolysate as a substrate

5 CONCLUSIONS AND RECOMMENDATIONS 82

5.1 Conclusions 82

5.2 Recommendation 83

REFERENCES 85

APPENDICES 92

BIODATA OF STUDENT 110

LIST OF PUBLICATIONS 111