UNIVERSITI PUTRA MALAYSIA

SIREN ANAK LINGGANG

FBSB 2012 46

PRODUCTION OF ACETONE-BUTANOL-ETHANOL FROM SAGO PITH RESIDUES

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PRODUCTION OF ACETONE-BUTANOL-ETHANOL FROM SAGO PITH

RESIDUES

By

SIREN ANAK LINGGANG

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

Fulfilment of the Requirements for the Degree of Master of Science

September 2012

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DEDICATED TO MY PARENT, SISTER AND BROTHER

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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 ACETONE-BUTANOL-ETHANOL FROM SAGO PITH

RESIDUES

By

SIREN ANAK LINGGANG

September 2012

Chairman : Suraini Abd. Aziz, PhD

Faculty : Biotechnology and Biomolecular Sciences

Sago pith residues are one of the abundant lignocellulosic residues that left behind

after starch extraction process and contains significant amount of starch (58%),

cellulose (23%), hemicellulose (9.2%) and lignin (3.9%). This residue has a great

potential as cheap and affordable substrate for the production of enzymes,

fermentable sugars and biofuel due to their high content of cellulose and

hemicellulose. This study was carried out to produce fermentable sugars from sago

pith residues using crude cellulases produced from local fungal isolates namely

Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2 either individual

or in combination. The fermentable sugars produced were then converted to acetone-

butanol-ethanol (ABE) by Clostridium acetobutylicum ATCC 824. In this study,

crude cellulases produced from Aspergillus fumigatus UPM2 exhibited high specific

cellulases activity (0.77 U/mg FPase, 47.76 U/mg CMCase and 1.56 U/mg β-

glucosidase) as compare to crude cellulases from Trichoderma asperellum UPM1

and mixed cultures. Therefore, crude cellulases produced from Aspergillus fumigatus

UPM2 was used for further study. Enzymatic hydrolysis using crude cellulases

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produced by Aspergillus fumigatus UPM2 based on 23.4 I.U of β-glucosidase as rate-

limiting released approximately 21 g/L fermentable sugars from 5% (w/v) sago pith

residues at pH 5.0 and temperature of 50ºC. The hydrolysis yield obtained was 73%

which were comparable with the hydrolysis yield of maize straw (66%) and rice

straw (70%) as reported in earlier studies by other researchers. High Performance

Liquid Chromatography (HPLC) analysis on sago pith residues hydrolysate showed

that it consisted mainly of glucose (10 g/L) and cellobiose (4.26 g/L). The total

acetone-butanol-ethanol (ABE) production by Clostridium acetobutylicum ATCC

824 using concentrated sago pith residues hydrolysate with addition 0.5 g/L yeast

extract was 8.84±0.20 g/L (5.41±0.10 g/L of butanol) after 72 hours of fermentation.

Yield and productivity of the total acetone-butanol-ethanol were 0.30 g ABE/g

glucose and 0.12 g/L/h, respectively. This result was almost comparable to the result

obtained using synthetic glucose (40 g/L) for ABE production by Clostridium

acetobutylicum ATCC 824 which the yield and productivity were 0.32 g ABE/g

glucose and 0.15 g/L/h, respectively.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Sarjana Sains

PENGHASILAN ASETON-BUTANOL-ETANOL DARIPADA SISA

EMPULUR SAGU

Oleh

SIREN ANAK LINGGANG

September 2012

Pengerusi : Suraini Abd. Aziz, PhD

Fakulti : Bioteknologi dan Sains Biomolekul

Sisa empulur sagu atau hampas sagu adalah salah satu daripada sisa lignoselulosa

yang banyak terhasil daripada proses pengekstrakan kanji dan mengandungi 58%

kanji, 23% selulosa, 9.2% hemiselulosa dan 3.9% lignin. Sisa ini mempunyai potensi

yang besar sebagai substrat yang murah dan berpatutan untuk penghasilan enzim,

gula terfermentasi dan biotenaga kerana mempunyai kandungan selulosa dan

hemiselulosa yang tinggi. Kajian ini telah dijalankan untuk menghasilkan gula

terfermentasi daripada sisa empulur sagu dengan menggunakan enzim selulase yang

dihasilkan oleh kulat pencilan tempatan iaitu Trichoderma asperellum UPM1 dan

Aspergillus fumigatus UPM2 sama ada secara individu atau gabungan kedua-dua

kultur. Gula terfermentasi yang dihasilkan ditukarkan kepada aseton-butanol-etanol

(ABE) oleh Clostridium acetobutylicum ATCC 824. Di dalam kajian ini, keputusan

menunjukkan bahawa enzim selulase yang dihasilkan oleh Aspergillus fumigatus

UPM2 mempamerkan aktiviti spesifik enzim selulase (0.77 U/mg FPase, 47.76 U/mg

CMCase and 1.56 U/mg β-glucosidase) yang lebih tinggi berbanding dengan enzim

selulase yang dihasilkan oleh Trichoderma asperellum UPM1 dan gabungan kedua-

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dua kultur. Oleh itu, enzim selulase yang dihasilkan oleh Aspergillus fumigatus

UPM2 telah dipilih untuk kajian selanjutnya. Hidrolisis berenzim menggunakan

enzim selulase yang dihasilkan oleh Aspergillus fumigatus UPM2 berdasarkan

aktiviti β-glucosidase sebanyak 23.4 I.U sebagai kadar pengehad, menghasilkan kira-

kira 21 g/L gula terfermentasi daripada 5% sisa empulur sagu pada pH 5.0 dan suhu

50ºC. Kadar hasil hidrolisis yang dicapai adalah 73% dimana ianya adalah setanding

dengan hasil hidrolisis jerami jagung (66%) dan jerami padi (70%) seperti yang

dilaporkan dalam kajian terdahulu oleh penyelidik lain. Analisa menggunakan

kromatografi cecair berprestasi tinggi (HPLC) terhadap hidrolisat sisa empulur sagu

menunjukkan bahawa ianya terdiri daripada glukosa (10 g/L) dan selobiosa (4.26

g/L). Jumlah penghasilan aseton-butanol-etanol (ABE) oleh Clostridium

acetobutylicum ATCC 824 menggunakan hidrolisat sisa empulur sagu yang

dipekatkan dengan penambahan 0.5 g/L yis ekstrak adalah 8.84±0.20 g/L (5.41±0.10

g/L butanol) selepas 72 jam fermentasi. Hasil dan produktiviti aseton-butanol-etanol

masing-masing adalah 0.30 g ABE/g glucose dan 0.12 g/L/h. Keputusan ini adalah

setanding dengan keputusan yang diperolehi dengan menggunakan glukosa sintetik

(40 g/L) untuk penghasilan ABE oleh Clostridium acetobutylicum ATCC 824 iaitu

hasil dan produktiviti masing-masing adalah 0.32 g ABE/g glucose dan 0.15 g/L/h.

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ACKNOWLEDGEMENTS

First of all, I would like to thanks The Almighty God, for blessing and grace, I am

able to complete this research project at Faculty of Biotechnology and Biomolecular

Sciences, Universiti Putra Malaysia, Serdang.

I would like to extend my sincere gratitude and appreciation to my main supervisor,

Prof. Dr. Suraini Abd. Aziz for giving the opportunity to do research work in UPM. I

really appreciate her guidance, concern, supports and comments throughout this

study and during paper writing. My sincere thanks and appreciation also goes to my

co-supervisor, Dr. Phang Lai Yee and Dr. Helmi Wasoh, for the guidance and

assistance throughout my research and during paper writing.

I also would like to express my sincere thanks to Mrs. Zuraida, Mrs. Nurul Kartini,

Miss Ezyana, Mr. Mohd Faizal, Mr. Mohd Nafis and all Environmental

Biotechnology group members for their motivation and generous guidance during the

difficult moment of the project. It had been a great experience working with all of

them.

Last but not least, my deepest thanks to my beloved parents, Mr. Linggang and Mrs.

Betty for their endless love, sacrifice and encouragement that will remain in my mind

forever.

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I certify that a Thesis Examination Committee has met on 27 September 2012 to

conduct the final examination of Siren anak Linggang on her thesis entitled

“Production of Acetone-Butanol-Ethanol from Sago Pith Residues” in accordance

with the Universities and University College Act 1971 and the Constitution of the

Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The committee

recommends that the student be awarded the Master of Science.

Members of the Thesis Examination Committee were as follows:

Umi Kalsom binti Md Shah, PhD Associate Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Shuhaimi bin Mustafa, PhD Associate Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Internal Examiner)

Wan Azlina binti Wan Abdul Karim Ghani, PhD Associate Professor

Faculty of Engineering

Universiti Putra Malaysia

(Internal Examiner)

Darah Ibrahim, PhD Professor

School of Biological Sciences

Universiti Sains Malaysia

(External Examiner)

_______________________

SEOW HENG FONG, PhD

Professor and Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

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This thesis was submitted to the Senate of Universiti Putra 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:

Suraini Abd. Aziz, PhD

Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Phang Lai Yee, PhD

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Member)

Helmi Wasoh, PhD

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: 10 January 2013

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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 institutions.

________________________

SIREN ANAK LINGGANG

Date: 27 September 2012

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TABLE OF CONTENTS

Page

ABSTRACT

ABSTRAK

ACKNOWLEDGEMENTS

APPROVAL

DECLARATION

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

ii

iv

vi

vii

ix

xii

xiv

xvi

CHAPTER

1 INTRODUCTION

1.1 Background

1.2 Research Objectives

1

3

2 LITERATURE REVIEW

2.1 Lignocellulosic Biomass

2.1.1Characteristics of Lignocellulosic Biomass

4

5

2.2 Sago Palm

2.2.1 Sago Industry in Malaysia

2.2.2 Sago Starch Processing

2.2.3 Sago Wastes

2.2.4 Potential Utilization of Sago Pith Residues

9

10

13

15

21

2.3 Cellulolytic Enzyme

2.3.1 Endo-β-1,4-glucanase

2.3.2 Exo-β-1,4-glucanase

2.3.3 β-glucosidase

2.3.4 Cellulolytic Microorganisms

2.3.5 Mode of Action of Cellulolytic Enzymes

2.3.6 Cellulases Fermentation

23

24

24

25

25

27

30

2.4 Acetone-Butanol-Ethanol (ABE) Fermentation

2.4.1 Acidogenic Pathways

2.4.2 Solventogenic Pathways

2.4.3 ABE Producing Microorganisms

2.4.4 Substrate for ABE Fermentation

2.4.5 Application of Acetone-Butanol-Ethanol

31

33

34

36

37

42

2.5 Concluding Remarks

45

3 MATERIALS AND METHODS

3.1 Substrate Preparation 47

3.2 Production of Crude Cellulases

3.2.1 Microorganisms

3.2.2 Inoculum Preparation

3.2.3 Submerged Fermentation of Sago Pith Residues

48

48

48

49

3.3 Saccharification of Sago Pith Residues

3.3.1 Effect of Enzyme Concentration

3.3.2 Effect of Substrate Concentration

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3.3.3 Effect of Temperature

3.3.4 Effect of pH Medium

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52

3.4 Production of Acetone-Butanol-Ethanol

3.4.1 Microorganism and Maintenance

3.4.2 Preparation of Inoculum Medium

3.4.3 Preparation of Production Medium

3.4.4 Inoculum Preparation

3.4.5 Acetone-Butanol-Ethanol (ABE) Fermentation

52

52

53

54

55

55

3.5 Analytical Methods

3.5.1 Proximate Analysis of Sago Pith Residues

3.5.2 Measurement of Enzyme Activities

3.5.3 Soluble Protein Assay

3.5.4 Total Reducing Sugars Assay

3.5.5 Simple Sugars Determination

3.5.6 Optical Density Analysis

3.5.7 Acetone-Butanol-Ethanol Analysis

3.5.8 Organic Acids Analysis

57

57

60

64

64

65

65

66

67

3.6 Summarized Methodology

68

4 RESULTS AND DISCUSSION

4.1 Chemical Characteristics of Sago Pith Residues

4.2 Submerged Fermentation of Sago Pith Residues

4.2.1 Crude Enzyme Production

69

70

70

4.3 Saccharification of Sago Pith Residues 74

4.3.1 Effect of Different Enzyme Concentration 77

4.3.2 Effect of Substrate Concentration 79

4.3.3 Effect of Temperature 82

4.3.4 Effect of pH Medium 84

4.3.5 Image of Sago Pith Residues Fiber under Scanning

Electron Microscopes (SEM)

86

4.4 Production of Acetone-Butanol-Ethanol by C.

acetobutylicum ATCC 824

89

4.4.1 Composition of Sago Pith Residues Hydrolysate 89

4.4.2 Production of ABE in Synthetic Medium and Sago

Pith Residues Hydrolysate

89

4.4.3 Effect of Sago Pith Residues Hydrolysate

Concentrations

96

4.4.4 Effect of Initial Culture pH 98

4.4.5 Effect of Different Concentration of Yeast Extract

100

5 CONCLUSIONS AND RECOMMENDATIONS FOR

FUTURE WORK

5.1 Conclusions 104

5.2 Recommendations for Future Work

105

REFERENCES

APPENDICES

BIODATA OF STUDENT

LIST OF PUBLICATIONS

107

123

138

139