EFFECT OF HEAT PRE-TREATMENT ON BIOHYDROGEN PRODUCTION BY STARCH-DEGRADING BACTERIA SITI AMIRAH BINTI MOHD ARIS A dissertation submitted in partial fulfilment of the requirements for the award of the degree of Master of Science Specialization Biotechnology Faculty of Biosciences and Medical Engineering Universiti Teknologi Malaysia APRIL 2018
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EFFECT OF HEAT PRE-TREATMENT ON BIOHYDROGEN PRODUCTION BY
STARCH-DEGRADING BACTERIA
SITI AMIRAH BINTI MOHD ARIS
A dissertation submitted in partial fulfilment of the
requirements for the award of the degree of
Master of Science Specialization Biotechnology
Faculty of Biosciences and Medical Engineering
Universiti Teknologi Malaysia
APRIL 2018
iii
This dissertation is dedicated to my beloved mother Hajah Fatimah binti Haji Bachik.
iv
ACKNOWLEDGEMENT
Thanks to Allah s.w.t for His blessing for allowing me the opportunity to
complete this Dissertation entitled “Effect of Heat Pre-Treatment on Biohydrogen
Production by Starch-Degrading Bacteria”. Firstly, I would like to express my highest
gratitude to my research supervisor, Dr. Mohd Firdaus bin Abdul Wahab for his
exemplary guidance, monitoring and constant encouragement throughout my research
study.
Much thanks to my colleagues for all the invaluable help, information and
advice offered during the entire of my research. I also wish to thank all the staffs in
the Faculty of Biosciences and Medical Engineering (FBME) for all assistance in my
research project. Lastly, I would like to thank my family and friends for their
continuous support and invaluable assistance throughout this research.
v
ABSTRACT
The combustion of fossil fuels emits majority parts of greenhouse gases thus
contributing to global warming. In addition, global economic development leads to the
increasing demand for fossil fuels as a major source of energy which causes this non-
renewable resource to become depleted. Therefore, hydrogen was discovered as an
alternative source of energy with carbon-neutral and renewable energy because it only
produces water vapour and heat upon combustion and can be generated from waste
materials. The low hydrogen yield is major challenges in biohydrogen production by
the mixed culture via dark fermentation approach. Hence, this study was conducted to
study the effect of heat pre-treatments on biohydrogen production of pre-treated
cassava sludge at 80oC, 90oC and 100oC for 60 minutes. The sludge was pre-treated to
select spore-forming hydrogen-producing bacteria and at the same time inhibit non-
spore hydrogen-consuming bacteria. Batch tests were conducted to investigate and
compare biohydrogen formation of untreated and pre-treated sludge at 80oC, 90oC and
100oC over 96 hours utilising cassava starch wastewater as substrate with an initial pH
5.5 at 30oC. Based on the experimental results, only hydrogen and carbon dioxide was
detected throughout the experiment without the presence of methane. No biohydrogen
was recorded for untreated sludge, 80oC and 100oC pre-treated sludge but biohydrogen
was detected only by 90oC pre-treated sludge with 0.0883 mL of cumulative hydrogen
formation. The biohydrogen production had the maximum biohydrogen production,
maximum biohydrogen production rate, starch utilisation, the yield of biohydrogen per
substrate and yield of biohydrogen per cell were 0.0012 mol H2, 0.0002 mol H2/h,
13.9%, 0.0050 mol H2/g and 0.0002 mol H2/g respectively. In conclusion, heat pre-
treatment by boiling at 90oC was suitable for enriching biohydrogen-producing
bacteria from cassava-processing sludge.
vi
ABSTRAK
Pembakaran bahan api fosil menghasilkan sebahagian besar gas rumah hijau
yang menyumbang kepada pemanasan global. Disamping itu, pembangunan ekonomi
global menjadikan permintaan bahan api fosil sebagai sumber tenaga utama semakin
meningkat lalu menyebabkan sumber tenaga yang tidak boleh diperbaharui ini menjadi
semakin berkurang. Oleh itu, gas hidrogen telah dikenalpasti sebagai sumber tenaga
alternatif dengan tenaga karbon neutral dan boleh diperbaharui kerana pembakarannya
hanya menghasilkan wap air dan haba serta boleh dihasilkan daripada bahan buangan.
Penghasilan hidrogen yang rendah adalah cabaran utama dalam penghasilan
biohidrogen oleh kultur campuran melalui pendekatan penapaian gelap. Justeru, kajian
ini dijalankan adalah untuk mengkaji kesan pra-rawatan terhadap pengeluaran
biohidrogen oleh kultur campuran dari sisa mendapan ubi kayu pada suhu 80oC, 90oC
dan 100oC selama 60 minit. Tujuan pra-rawatan haba terhadap sisa mendapan ubi kayu
adalah untuk memilih spora bakteria yang menghasilkan hidrogen dan pada masa yang
sama membantutkan aktiviti bukan spora bakteria yang menggunakan hidrogen
sebagai sumber tenaga. Pendekatan fermentasi kelompok telah dijalankan untuk
menyelidik dan membandingkan pembentukan biohidrogen daripada sisa mendapan
tanpa pra-rawatan dan pra-rawatan pada 80oC, 90oC dan 100oC selama 96 jam dengan
menggunakan air sisa buangan kanji ubi kayu sebagai substrat bermula dengan pH 5.5
pada suhu 30oC. Berdasarkan keputusan eksperimen, hanya gas hidrogen dan karbon
dioksida yang telah dikesan sepanjang eksperimen tanpa kehadiran gas metana. Tiada
biohidrogen telah direkodkan dari sisa mendapan tanpa pra-rawatan dan pra-rawatan
pada suhu 80oC dan 100oC tetapi hanya dikesan oleh pra-rawatan sisa mendapan pada
90oC dengan kumulatif pembentukan hidrogen sebanyak 0.0883 mL. Pengeluaran
biohidrogen ini mempunyai pengeluaran biohidrogen maksima, kadar pengeluaran
biohidrogen maksima, penggunaan kanji, hasil biohidrogen pada substrat dan hasil
biohidrogen pada sel adalah 0.0012 mol H2, 0.0002 mol H2/j, 13.9%, 0.0050 mol H2/g
and 0.0002 mol H2/g mengikut turutan. Kesimpulannya, kaedah pra-rawatan haba pada
suhu 90oC adalah sesuai untuk memperkayakan bakteria yang menghasilkan
biohidrogen dari sisa mendapan ubi kayu.
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TABLE OF CONTENTS
CHAPTER TITLE PAGE
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENT iv
ABSTRACT v
ABSTRAK vi
TABLE OF CONTENTS vii
LIST OF TABLES x
LIST OF FIGURES xi
LIST OF ABBREVIATIONS xii
LIST OF SYMBOLS xiv
LIST OF APPENDIX xv
1 INTRODUCTION 1
1.1 Research Background 1
1.2 Problem Statement 3
1.3 Objectives of the Research 5
1.4 Scope of the Study 5
1.5 Significance of Research 6
2 LITERATURE REVIEW 7
2.1 Hydrogen as an Alternative Source 7
2.2 Starch 9
2.2.1 Cassava Starch 10
2.3 Process of Hydrogen Production 11
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2.3.1 Thermochemical Process 11
2.3.2 Electrolytic Process 12
2.3.3 Photolytic Process 12
2.3.4 Biological Process 13
2.4 Fermentation 14
2.4.1 Dark Fermentation 15
2.4.2 Bacterial Communities of Mixed Culture
in Dark Fermentation
18
2.4.3 Factors Affecting of Fermentation
Process on Biohydrogen Production
20
2.5 Pre-treatment Methods of Sludge 21
2.5.1 Heat Pre-treatment 22
3 MATERIALS AND METHODS 25
3.1 Research Design 25
3.2 Sample Collection 27
3.3 Preparation of Medium Cassava Starch Broth
Medium
28
3.4 Heat Pre-treatment of Starch Processing Sludge at
Different Temperature
28
3.5 Biohydrogen Production by the Pre-treated
Sludge
29
3.6 Analysis Procedures 29
3.6.1 Biohydrogen Measurements 29
3.6.2 Determination of Starch Concentration 30
3.6.3 Determination of Glucose 31
3.6.4 Determination of α-amylase Activity 31
3.6.5 Determination of Biomass Concentration 32
3.7 Kinetic Analysis of Biohydrogen Production and
Starch Utilisation of Pre-treated Sludge
33
3.7.1 Yield of Coefficient 33
ix
4 RESULTS AND DISCUSSION 34
4.1 Application of Different Temperature of Heat Pre-
Treatments on Starch Processing Sludge
34
4.2 Effect of Different Heat Pre-Treatments on
Biohydrogen Production
36
4.3 Effect of Different Heat Pre-Treatments on Starch
Concentration, Glucose Concentration, Amylase
Activity and Biomass Concentration
39
4.3.1 Effect of Untreated Cassava Sludge 40
4.3.2 Effect at 80oC Heat Pre-Treatments of
Cassava Sludge
42
4.3.3 Effect at 90oC Heat Pre-Treatments of
Cassava Sludge
44
4.3.4 Effect at 100oC Heat Pre-Treatments of
Cassava Sludge
48
4.4 Kinetic Analysis of Biohydrogen Production and
Starch Utilisation Using Pre-Treated Sludge
51
5 CONCLUSION AND FUTURE WORK 54
5.1 Conclusion 54
5.2 Future Work 55
REFERENCES 56
APPENDICES 66
x
LIST OF TABLES
TABLE NO. TITLE PAGE
2.1 Biohydrogen yield of different temperature and duration
of heat pre-treatments.
23
4.1 Summary for kinetic parameters of biohydrogen
production and substrate utilization by untreated and pre-
treated sludge.
52
xi
LIST OF FIGURES
FIGURE NO. TITLE PAGE
2.1 Structure of amylose and amylopectin 9
2.2 General dark fermentation pathways 16
3.1 The overall flow of this study 26
3.2 A drain behind of Layang Food Sdn. Bhd where the
sludge sample was collected.
27
4.1 Cumulative biohydrogen production of untreated and
different temperatures of heat pre-treatment methods