UNIVERSITI PUTRA MALAYSIA

GLYCEROL AS ALTERNATIVE SUBSTRATE FOR BIOETHANOL PRODUCTION USING FREE AND IMMOBILIZED

Escherichia coli SS1

NUR AMELIA AZREEN BINTI ADNAN

FBSB 2014 11

© COPYRIG

HT UPM

iii

GLYCEROL AS ALTERNATIVE SUBSTRATE FOR BIOETHANOL PRODUCTION USING FREE AND IMMOBILIZED

Escherichia coli SS1

By

NUR AMELIA AZREEN BINTI ADNAN

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

Science

June 2014

© COPYRIG

HT UPM

iv

COPYRIGHT

All material contained within the thesis, including without limitation text, logos, icons, photographs and all other artwork, is copyright material of Universiti Putra Malaysia unless otherwise stated. Use may be made of any material contained within the thesis for non-commercial purposes from the copyright holder. Commercial use of material may only be made with the express, prior, written permission of Universiti Putra Malaysia

Copyright © Universiti Putra Malaysia

© COPYRIG

HT UPM

i

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

GLYCEROL AS ALTERNATIVE SUBSTRATE FOR BIOETHANOL PRODUCTION USING FREE AND IMMOBILIZED

Escherichia coli SS1

By

NUR AMELIA AZREEN BINTI ADNAN

June 2014

Chairman : Phang Lai Yee, PhD Faculty : Biotechnology and Biomolecular Sciences

Increase in the crude oil price and the concern about climatic change has resulted in the rapid increase of biodiesel production. In the production of this biofuel, glycerol will essentially generate as by-product. As a result, the production of glycerol has also increased. Biofuel from glycerol can be categorized as second generation of biofuels where it can be used to replace the first generation of biofuel including glucose and vegetable oils as feedstock to reduce the competition between biofuel and food production. Thus the aim of this study is to carry out bioethanol production using glycerol in batch and continuous fermentation by free and immobilized Escherichia coli SS1. Bioethanol production was carried out using both free and immobilized cells in 2 liter bioreactor with 800 mL working volume. Glycerol concentration of 20 g/L, 35 g/L and 45 g/L were used as initial substrate concentration in batch free cells fermentations. Dilution rate of 0.1/h and 0.2/h were used and was selected based on the maximum specific growth rate from batch fermentation. Immobilization of E.coli SS1 was done by using sodium alginate and calcium chloride as crossed link agent. Optimization study was done to determine the stability and rigidity of alginate beads. Parameters involved in this optimization study were sodium alginate concentration, calcium chloride concentration, beads diameter and initial pH of medium. The optimum conditions for cell immobilization were 0.2 M calcium chloride, at pH 7 with 3% concentration of sodium alginate and beads diameter of 3 mm. Results showed that high glycerol concentration did not affect the yield of ethanol with the yield was closed to theoretical yield; 1 mol ethanol per 1 mol glycerol. Dilution rate of 0.1/h was the optimum dilution rate to be used in this fermentation where glycerol consumption and ethanol production was similar to batch fermentation by yielding 1 mol ethanol per mol glycerol. Continuous fermentation of immobilized E. coli SS1 was done by using the optimized beads with the dilution rate of 0.2/h and 20 g/L. The results showed that immobilized cells can last up to 3rd cycle of continuous fermentation. Ethanol production obtained was 6.17 g/L by utilizing approximately 19 g/L glycerol. The yield achieved was 0.65 mol ethanol per

© COPYRIG

HT UPM

ii

mol glycerol. Compare with other studies, the result in this experiment was slightly lower where other managed to obtain about 0.6 to 0.8 mol ethanol per mol glycerol, respectively.

© COPYRIG

HT UPM

iii

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

GLISEROL SEBAGAI SUBSTRAT ALTERNATIF BAGI PENGHASILAN BIOETANOL MENGGUNAKAN SEL Escherichia coli SS1 BEBAS DAN

TERSEKAT-GERAK

Oleh

NUR AMELIA AZREEN BINTI ADNAN

Jun 2014

Pengerusi : Phang Lai Yee, PhD Fakulti : Bioteknologi dan Sains Biomolekul

Kenaikan harga minyak mentah dan kesedaran terhadap perubahan iklim telah mengakibatkan peningkatan dalam penghasilan biodiesel. Dalam penghasilan bahan api biologi ini, gliserol akan dijana sebagai bahan sampingan. Oleh sebab itu, kadar pengeluaran gliserol juga meningkat. Bahan api biologi dari gliserol boleh dikategorikan sebagai generasi kedua bahan api biologi di mana ia boleh digunakan untuk menggantikan generasi pertama bahan api biologi termasuk glukosa dan minyak sayur-sayuran sebagai bahan mentah untuk mengurangkan persaingan antara bahan api biologi dan pengeluaran makanan. Oleh itu, tujuan kajian ini adalah untuk menjalankan pengeluaran bioetanol daripada gliserol dalam fermentasi sesekelompok dan selanjar dengan menggunakan Escherichia coli SS1 bebas dan tersekat-gerak. Pengeluaran bioetanol telah dijalankan dengan menggunakan kedua-dua sel bebas dan tersekat-gerak dalam bioreaktor 2 liter dengan 800 mL isipadu kerja. Kepekatan gliserol 20 g/L, 35 g/L dan 45 g/L telah digunakan sebagai kepekatan substrat awal dalam fermentasi sesekelompok dengan menggunakan sel bebas. Kadar pencairan 0.1/h dan 0.2/h telah digunakan di mana ia dipilih berdasarkan kadar pertumbuhan spesifik maksimum daripada fermentasi sesekelompok. Penyekat-gerak sel E. coli SS1 telah dilakukan dengan menggunakan natrium alginat dan kalsium klorida sebagai ejen pautan bersilang. Kajian pengoptimuman telah dijalankan untuk menentukan kestabilan dan ketegaran manik alginat. Parameter yang terlibat dalam kajian pengoptimuman ini adalah kepekatan natrium alginat, kepekatan kalsium klorida, diameter manik dan pH awal medium. Keadaan optimum untuk penyekat-gerak sel adalah 0.2 M kalsium klorida, pada pH 7 dengan kepekatan 3% natrium alginat dan diameter manik 3 mm. Keputusan menunjukkan bahawa kepekatan gliserol tinggi tidak menjejaskan hasil bioetanol dimana nilainya berdekatan dengan nilai teori. Kadar pencairan sebanyak 0.1/h ialah kadar pencairan optimum untuk digunakan dalam fermentasi ini di mana penggunaan gliserol dan pengeluaran etanol adalah sama dengan fermentasi sesekelompok dengan menghasilkan 1 mol etanol kepada mol gliserol. Fermentasi selanjar sel pegun E. coli SS1 telah dilakukan dengan menggunakan manik yang telah

© COPYRIG

HT UPM

iv

dioptimumkan dengan kadar pencairan 0.2/h dan 20 g/L. Hasil kajian menunjukkan bahawa sel-sel pegun boleh bertahan sehingga kitaran ke-3 di dalam fermentasi selanjar. Pengeluaran bioetanol yang diperolehi ialah 6.17 g/L dengan menggunakan kira-kira 19 g/L gliserol. Hasil yang diperolehi adalah 0.65 mol etanol kepada mol gliserol. Jika dibandingkan dengan kajian-kajian lain, hasil eksperimen ini adalah lebih rendah sedikit di mana penyelidik lain berjaya menghasilkan antara 0.6-0.8 mol etanol kepada mol gliserol, masing-masing.

© COPYRIG

HT UPM

v

ACKNOWLEDGEMENTS

Alhamdulillah, all praise to Allah for granting me a life, a priceless opportunity for me to treasure the beautiful journey reside it, and this study has been part of the important chapters in my lifetime journey.

Foremost, I would like to express my sincere gratitude and appreciation to my main supervisor, Dr. Phang Lai Yee for giving me a valuable chance to be in her supervision. The experience I have gained during this study period has boosted my self-esteem and reliance over my previous, present and future accomplishment. Thanks for the continuous support, patient, advice, help and supervision that nurtured and matured me to be a better learner than before. I would also like to extend my deep gratitude to my co-supervisor, Professor Dr. Suraini Abd Aziz for always supporting my research works with her valuable comments and suggestions throughout my study.

In addition, a special thanks to lab staff Mrs Rosema, Mr. Rosli, Mrs Norazlina, Mrs Siti Noraisyah, Mrs Aluyah and Mrs Renuga and all other staffs in MTDC and Faculty of Biotechnology and Biomolecular Sciences, UPM for countless assistances and supports. I am indeed fortunate to be surrounded by colleagues and lab mates who are helpful and supportive. Warmest gratitude to Fikhri, Ain, Faiqah, Jauhar, Sheril, Azman, Yee Lian Ngit, Environmental Biotechnology group members and all my friends for sharing the joy moments during my mastery study in UPM. My special and warm gratitude definitely goes to my beloved parents, Adnan Abdullah and Sharifah Hashimah Syed Hassan for their prayers, always believing in me, for their continuous love and their supports in my decisions. Without whom I could not have made it here. Not to forget, all my family members for always relishing my ups and supporting my downs no matter when and where.

Last but not least, again, many thanks to everybody who have contributed directly and indirectly to the completion of my research work. May Allah bless all of you.

© COPYRIG

HT UPM

© COPYRIG

HT UPM

vii

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: Phang Lai Yee, PhD Senior Lecturer Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Suraini Abd-Aziz, PhD 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:

© COPYRIG

HT UPM

viii

DECLARATION

Declaration by the student

I hereby confirm that: this thesis is my original work quotations, illustrations and citations have been duly referenced the thesis has not been submitted previously or comcurrently for any

other degree at any institutions intellectual property from the thesis and copyright of thesis are fully-

owned by Universiti Putra Malaysia, as according to the Universiti Putra Malaysia (Research) Rules 2012;

written permission must be owned from supervisor and deputy vice –chancellor (Research and innovation) before thesis is published (in the form of written, printed or in electronic form) including books, journals, modules, proceedings, popular writings, seminar papers, manuscripts, posters, reports, lecture notes, learning modules or any other materials as stated in the Universiti Putra Malaysia (Research) Rules 2012;

there is no plagiarism or data falsification/fabrication in the thesis, and scholarly integrity is upheld as according to the Universiti Putra Malaysia (Graduate Studies) Rules 2003 (Revision 2012-2013) and the Universiti Putra Malaysia (Research) Rules 2012. The thesis has undergone plagiarism detection software

Signature: Date:

Name and Matric No: Nur Amelia Azreen Binti Adnan (GS29109)

© COPYRIG

HT UPM

ix

DECLARATION

Declaration by Members of Supervisory Committee

This is to confirm that: the research conducted and the writing of this thesis was under our

supervision; supervision responsibilities as stated in the Universiti Putra Malaysia

(Graduate Studies) Rules 2003 (Revision 2012-2013) are adhered to.

Signature: Name of Chairman of Supervisory Committee: Phang Lai Yee, PhD

Signature: Name of Member of Supervisory Committee: Suraini Abd Aziz, PhD

© COPYRIG

HT UPM

x

TABLE OF CONTENT

Page ABSTRACT i ABSTRAK iii ACKNOWLEDGEMENT v APPROVAL vi DECLARATIONS viii LIST OF TABLES xiii LIST OF FIGURES xiv LIST OF ABBREVIATIONS xvi

CHAPTER

1.0 INTRODUCTION 1

2.0 LITERATURE REVIEW 4 2.1 Glycerol 4 2.2 Glycerol-fermenting microorganisms 6 2.3 Glycerol conversion in industrial microbiology 7 2.3.1 1,3-propanediol 7 2.3.2 Succinic acid 7 2.3.3 Dihyroxyacetone 8 2.3.4 Propionic acid 8 2.3.5 Bioethanol 8 2.4 Bioethanol production from glycerol 10 2.5 Microbial conversion of glycerol to ethanol 13

2.5.1 Escherichia coli 13 2.6 Basic modes of fermentation 14 2.6.1 Batch fermentation 15

2.6.2 Continuous fermentation 16 2.7 Factors affecting fermentation 20 2.7.1 Temperature 21 2.7.2 Initial pH 21 2.7.3 Initial cell concentration 21 2.7.4 Substrate concentration 22 2.8 Immobilized cells fermentation 22 2.8.1 Sodium alginate 25 2.8.2 Application of immobilization system 27

2.8.2.1 Ethanol industry 27 2.8.2.2 Food industry 28 2.8.2.3 Clinical industry 28 2.8.2.4 Bioremediation 28

2.8 Concluding remarks 29

© COPYRIG

HT UPM

xi

3.0 MATERIALS AND METHODS

3.1 Chemical reagents 30 3.2 Experimental set up 30 3.3 Bioethanol fermentation by free suspended cells 32

3.3.1 Microorganism and maintenance 32 3.3.2 Inoculum preparation 32 3.3.3 Batch fermentation and culture conditions 32 3.3.4 Continuous fermentation 34

3.4 Bioethanol fermentation by immobilized cells 35 3.4.1 Cells immobilization using sodium

alginate 35

3.4.2 Effects of immobilization parameters 37 3.4.3 Batch fermentation 37 3.4.4 Continuous fermentation 37

3.5 Analytical Methods 37 3.5.1 Cell determination for free suspended

Escherichia coli SS1 cells 37

3.5.2 Cell determination for immobilized Escherichia coli SS1 cells

38

3.5.3 Bioethanol Analysis 38 3.5.3.1 Sample preparation 38 3.5.3.2 Bioethanol determination 39

3.5.4 Glycerol Assay 39

4.0 RESULTS AND DISCUSSION

40

4.1 Glycerol fermentation using free Escherichia coli SS1 cells

40

4.1.1 Batch fermentation 40 4.1.2 Continuous fermentation 46

4.2 Glycerol fermentation using immobilized Escherichia coli SS1

53

4.2.1 Determination of the factors affecting the stability of sodium alginate beads

53

4.2.1.1 Effect of Sodium Alginate Concentration

53

4.2.1.2 Effect of Calcium Chloride Concentration

55

4.2.1.3 Effect of Initial pH 57 4.2.1.4 Effect of Beads Diameter 60

4.2.2 Morphology of alginate beads by SEM 62 4.2.3 Batch fermentation using immobilized

Escherichia coli SS1 66

4.2.4 Continuous fermentation using immobilized Escherichia coli SS1

70

4.3 Overall comparison between free suspended cells and immobilized cell

72

© COPYRIG

HT UPM

xii

5.0 SUMMARY, GENERAL CONCLUSIONS ANDSUGGESTIONS FOR FUTURE RESEARCH

74

REFERENCES 77 APPENDICES 94 BIODATA OF STUDENT 100 PUBLICATION 101