UNIVERSITI PUTRA MALAYSIA

BIOHYDROGEN PRODUCTION DURING GROWTH OF AN INDIGENOUS BACTERIUM, OCHROBACTRUM PUTRANENSIS EB2,

IN PALM OIL MILL EFFLUENT

CHEONG WENG CHUNG

FBSB 2006 36

BIOHYDROGEN PRODUCTION DURING GROWTH OF AN INDIGENOUS BACTERIUM, OCHROBACTRUM PUTRANENSIS EB2,

IN PALM OIL MILL EFFLUENT

CHEONG WENG CHUNG

DOCTOR OF PHILOSOPHY UNIVERSITI PUTRA MALAYSIA

2006

BIOHYDROGEN PRODUCTION DURING GROWTH OF AN INDIGENOUS BACTERIUM, OCHROBACTRUM PUTRANENSIS EB2,

IN PALM OIL MILL EFFLUENT

By

CHEONG WENG CHUNG

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

October 2006

Specially dedicated to,

My beloved parents who brought me to this world,

my brothers who gave me the encouragement and laughter,

and friends for their invaluable advice and morale support.

Sharon, thank you for being understanding throughout my study

and loving me…………………….

ii

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the Degree of Doctor of Philosophy

BIOHYDROGEN PRODUCTION DURING GROWTH OF AN INDIGENOUS BACTERIUM, OCHROBACTRUM PUTRANENSIS EB2,

IN PALM OIL MILL EFFLUENT

By

CHEONG WENG CHUNG

October 2006

Chairman: Prof. Mohd. Ali Hassan, PhD Faculty: Biotechnology and Biomolecular Sciences

The study was on the production of hydrogen from palm oil mill effluent using

indigenous bacteria. Three potential bacteria (WL1, EB2, and DT1) were isolated

from wetland soil, termite’s gut and anaerobic digester tank, respectively. All three

isolates were Gram-positive, spore-forming, facultative anaerobe rods and exhibited

motility by means of peritrichous flagella. All of the isolates were short straight rods,

appeared in-pairs, clumped-together and formed biofilm on the agar surface. Using

the 16s rRNA identification method, EB2 belonged to the Brucellaceae family and is

closely related to Ochrobactrum sp., thus, designated as Ochrobactrum putranensis

EB2; while WL1 and DT1 isolates were taxonomically positioned as Bacillus cereus.

These bacteria differed by 30-35% in the 16s rRNA identification method from the

existing bacteria recorded in the gene library. Since EB2 is different from other

bacteria in the database, it is regarded as a new bacteria. In this study, the EB2

bacteria was used throughout the study to enhance the production of hydrogen.

The study on the optimization of hydrogen production was done on the

Ochrobactrum putranensis EB2 and the parameters chosen were pH 4, 5 and 6; at

iii

temperatures 30°C, 37°C and 45°C. Comparisons were done with VFA and hydrogen

production as well as COD reduction. The Ochrobactrum putranensis EB2 was able

to grow steadily in POME and produced hydrogen at the pH of 5.5 and temperature

37°C. The highest µmax obtained was 0.384 h-1. An average of 1300 mL of

accumulated hydrogen volume was obtained within 72 hours of batch fermentation

from 1 L of POME in the optimization study.

Production of hydrogen from palm oil mill effluent (POME) by Ochrobactrum

putranensis EB2 was investigated in 2.0 L bioreactor with working volume of 1.0 L

at 37°C and pH of 5.5 ± 0.3. The optimized conditions were applied in the hydrogen

production study. The average hydrogen volume accumulated in the system was

1100 mL with 58.0% COD removal. Repeated experiments done on the batch

fermentation confirmed the reproducibility of the results. Continuous fermentation

were started after 72 hours of batch fermentation when there was no hydrogen

production.

During continuous fermentation, hydrogen gas production was at 34.2 mL H2/L per

day for HRT 0.5 day, 13.0 mL H2/L per day for HRT 1.0 day and 12.6 mL H2/L per

day for HRT 1.5 days and these show the HRT-dependent characteristics of

hydrogen production. The efficiency of Ochrobactrum putranensis EB2 to convert

the POME into hydrogen gas was about 31.67 mL H2/g COD and was the highest

recorded in batch fermentation reducing to 8 mL H2/g COD at the end of the batch

fermentation. The highest productivity obtained in the continuous fermentation was

1.07 mol H2/mol glucose, was good in comparison with those reported earlier, 1.00-

2.36 mol H2/mol glucose. The high productivity was excellent as most of the COD

iv

v

was converted to gas, based on the theoretical yield of 4 mol-H2/mol-glucose. As

such, HRT 0.5 day gave the best result compared to other HRT done in the study.

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Doktor Falsafah

PENGHASILAN BIOHIDROGEN SEMASA PERTUMBUHAN BAKTERIA, OCHROBACTRUM PUTRANENSIS EB2, DALAM EFLUEN KILANG SAWIT

Oleh

CHEONG WENG CHUNG

Oktober 2006 Pengerusi: Prof. Mohd. Ali Hassan, PhD Fakulti: Bioteknologi dan Sains Biomolekul

Kajian ini melibatkan penggunaan bakteria tempatan dalam penghasilan gas hidrogen

menggunakan efluen kilang sawit (POME). Tiga bakteria yang berpotensi menghasilkan

gas hidrogen, WL1, EB2 dan DT1, telah disaringkan daripada tanah di kawasan paya,

perut anai-anai dan tangki anaerobik sistem rawatan efluen kilang sawit. Ketiga-tiganya

merupakan bakteria Gram-positif, mempunyai keupayaan membentuk spora, berbentuk

rod, fakultatif anaerobik, keupayaan bergerak dengan flagella dan membentuk biofilem di

atas permukaan agar. Dengan menggunakan kaedah pengenalpastian 16s rRNA, EB2

digolongkan dalam keluarga Brucellaceae dan hampir serupa dengan spesis

Ochrobactrum. Dengan itu, bakteria itu dinamakan Ochrobactrum putranensis EB2.

Manakala, WL dan DT1 digolongkan secara taksonomi sebagai Bacillus cereus. Ketiga-

tiga bakteria adalah berbeza daripada bakteria-bakteria yang dilaporkan dalam

perpustakaan gen sebanyak 30–35% dalam proses pengenalpastian bakteria dengan

kaedah 16s rRNA. Disebabkan kelainan bakteria EB2 daripada bakteria yang sedia ada,

vi

ia dianggap sebagai bakteria yang baru untuk dikaji. Dengan itu, Ochrobactrum

putranensis EB2 digunakan dalam kajian seterusnya.

Penyelidikan untuk mengoptimumkan penghasilan hidrogen dilakukan ke atas

Ochrobactrum putranensis EB2 dengan menggunakan nilai-nilai parameter (pH 4, 5 and

6; suhu 30°C, 37°C and 45°C). Perbandingan dilakukan dengan penghasilan VFA dan

hidrogen bersama penurunan COD dalam media. pH 5.5 dan suhu 37°C merupakan

parameter yang optimum untuk penghasilan hidrogen dan Ochrobactrum putranensis

EB2 dapat membiak di dalam POME. Nilai µmax yang paling tinggi diperolehi dalam

kajian ini adalah 0.384 h-1. Secara purata sebanyak 1300 mL gas hidrogen telah dapat

dikumpulkan dalam jangkamasa 72 jam selepas fermentasi dengan 1 L POME.

Penghasilan hidrogen daripada efluen kilang sawit (POME) menggunakan Ochrobactrum

putraensis EB2 dalam 2.0 L bioreactor (1.0 L POME dengan suhu 37°C dan pH 5.5 ±

0.3) dikaji dengan cara fermentasi sesekelompok. Eksperimen fermentasi sesekelompok

dijalankan dengan menggunakan parameter yang optimum untuk penghasilan hydrogen

dan didapati tiada perubahan ketara. Sebanyak 1100 mL hidrogen dapat dikumpulkan dan

penurunan kepekatan COD 58.0% dicatatkan. Eksperimen yang dibuat berulang kali

menunjukkan keputusan yang serupa. Fermentasi selanjar dilakukan selepas 72 jam

fermentasi sesekelompok setelah tiada penghasilan hidrogen dikesan.

vii

viii

Semasa fermentasi selanjar, penghasilan gas hidrogen ialah 34.2 mL H2/L sehari untuk

HRT 0.5 hari, 13.0 mL H2/L sehari untuk HRT 1.0 hari dan 12.6 mL H2/L sehari untuk

HRT 1.5 hari. Ini menunjukkan penghasilan gas hidrogen menggunakan bakteria adalah

bergantung kepada faktor HRT. Tahap kecekapan tertinggi dalam fermentasi

sesekelompok Ochrobactrum putranensis EB2 untuk menukarkan POME kepada gas

hidrogen ialah 67 mL H2/g COD tersingkir, dan menurun kepada 8 mL H2/g COD di

akhir fermentasi dalam fermentasi selanjar. Produktiviti tertinggi ialah 1.07 mol H2/mol

glukos sehari adalah baik berbanding nilai yang dilaporkan oleh penyelidik yang lain

pada 1.00-2.36 mol H2/mol glukos. Produktiviti yang tinggi ini adalah nilai yang

menunjukkan kebanyakan COD telah ditukarkan kepada gas. HRT 0.5 hari memberikan

nilai yang terbaik berbanding keputusan HRT yang lain dalam kajian ini. HRT 0.5 hari

memberikan nilai yang terbaik berbanding keputusan HRT yang lain dalam kajian ini.

ACKNOWLEDGEMENTS

I would like to express my deepest gratitude and appreciation to my main supervisor,

Prof. Dr. Mohd. Ali Hassan and members of the supervisory committee; Prof. Dr.

Mohamed Ismail Abdul Karim, Prof. Vikineswary Sabaratnam, Assoc. Prof. Dr.

Suraini Abdul Aziz and Prof. Dr. Yoshihito Shirai for their invaluable advice and

support, encouragement and willingness to share their views throughout the project.

I sincerely thank Prof. Dr. Mohd Ali Hassan for giving me the chance to further my

study in a doctorate program and also for his immeasurable support, advice and ideas

throughout my study.

In addition, I would like to express my appreciation to my labmates and fermentation

laboratory staff, Mr. Rosli Aslim, Madam Renuga a/p Panjamurti and

Madam Aluyah Marzuki, thank you for your moral support, cooperation and

willingness to teach me.

I am grateful to my beloved parents and brothers for their patience, support and

encouragement. Acknowledgement is also due to all who were involved directly and

indirectly in the completion of this study. Last but not least, to Sharon, thank you for

sharing the sweet and sour throughout my studies and for loving me.

ix

I certify that an Examination Committee met on 30th October 2006 to conduct the final examination of Cheong Weng Chung on his Doctor of Philosophy thesis entitled “Biohydrogen Production During Growth Of An Indigenous Bacterium, Ochrobactrum Putranensis Eb2, in Palm Oil Mill Effluent” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulation 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the examination Committee are follows: Arbakariya Ariff, PhD Professor Faculty of Graduate Studies Universiti Putra Malaysia (Chairman) Mohd Arif Syed, PhD Professor Faculty of Biotechnology and Molecular Sciences Universiti Putra Malaysia (Internal Examiner) Fakhrul Razi-Ahmadun, PhD Assoc Prof, Faculty of Engineering Universiti Putra Malaysia (Internal Examiner) Abdul Jalil Abdul Kader, PhD Professor Faculty of Science and Technology Universiti Kebangsaan Malaysia Putra Malaysia (External Examiner) ________________________________________ HASANAH MOHD. GHAZALI, PhD Professor/Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date: 22 MARCH 2007

x

xi

This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. 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 Faculty of Engineering International Islamic University Malaysia (IIUM) (Member) Suraini Abdul Aziz, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) Vikineswary Sabaratnam, PhD Professor Faculty of Science Universiti Malaya (Member) Yoshihito Shirai, PhD Professor Kyushu Institute of Technology (KIT) Kitakyushu, Japan (Member) ________________________ AINI DERIS, PhD Professor / Dean School of Graduate Studies Universiti Putra Malaysia

Date: 12 APRIL 2007

DECLARATION I hereby declare that the thesis is based on my original work except for quotations and citations, which have been duly acknowledge. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions. _______________________________ Name : CHEONG WENG CHUNG

Date : 27 FEBRUARY 2007

xii

TABLE OF CONTENTS

Page

DEDICATION ii ABSTRACT iii ABSTRAK vi ACKNOWLEDGEMENTS ix APPROVAL x DECLARATION xii LIST OF TABLES xvi LIST OF FIGURES xviii LIST OF ABBREVIATIONS xxii CHAPTER 1 INTRODUCTION 1 1.1 Hydrogen Energy 1

1.2 Environmental Impact of Palm Oil Industry 1 1.3 Objectives 3

2 LITERATURE REVIEW

2.1 Palm oil 4 2.2 Palm oil mill effluent (POME) 5 2.3 Anaerobic decomposition 9

2.3.1 Mechanism 9 2.3.2 Interspecies hydrogen transfer 13

2.3.3 Microbiology 15 2.4 Hydrogen gas 19

2.4.1 The properties of hydrogen 20 2.4.2 Hydrogen pathway 22

2.5 Bioreactor operation 25 2.5.1 Rate-limiting processes 25 2.5.2 pH, acidity and alkalinity 27 2.5.3 Temperature 28 2.5.4 Nutrients 30 2.5.5 Cations 31 2.5.6 Continuous operation 32

2.6 Plasmid profile 33 2.6.1 Polymerase chain reaction (PCR) 33

2.7 Ochrobactrum 36 2.8 Biohydrogen: future directions 38 2.9 Hydrogen economy 41 2.10 Differences between Bio-Methane and Bio-Hydrogen 42

xii

3 GENERAL MATERIALS AND METHODS 44 3.1 Chemical reagents 44 3.2 Palm oil mill effluent (POME) 45 3.3 Microorganisms and preparation 46

3.3.1 Source of samples 46 3.3.2 Isolation of the hydrogen producing bacteria 46 3.2.3 Inoculum preparation and maintenance 47

3.4 Fermentation process using selected bacteria 47 3.4.1 General study of hydrogen producing bacteria 47 3.4.2 Hydrogen production using glucose as substrate 50 3.3.3 Optimization study on POME as main substrate

for hydrogen production 51 3.3.4 Continuous fermentation study 52

3.4 Analytical methods 55 3.4.1 Organic acids determination 55 3.4.2 BOD 55 3.4.3 COD determination 56 3.4.4 Total solids 57 3.4.5 Suspended solids (SS) 57 3.4.6 Total Kjeldahl nitrogen analysis 58 3.4.7 Ammoniacal nitrogen (AN) 59 3.4.8 Gas analysis 59

4 ISOLATION AND SCREENING OF HYDROGEN

PRODUCING BACTERIA 62 4.1 Introduction 62 4.2 Materials and methods 63

4.2.1 POME sludge 63 4.2.2 Insect’s gut 63 4.2.3 Soil 63

4.2.4 Bacteria from collection 64 4.2.5 Heat treatment on the samples 64

4.2.6 Screening of local strains bacteria 65 4.2.7 Operation of batch reactor 65

4.2.8 Analysis of fermentation products 65 4.2.9 Sequencing of 16S rDNA 66 4.2.10 Growth profile of best hydrogen

producing bacteria 67 4.2.11 The selection of bacteria strain for further study 69

4.3 Results and discussion 70 4.3.1 Isolation of bacteria from various sources 70 4.3.2 Durham tube test 78 4.3.3 Hydrogen production using glucose by

selected bacteria containing medium 79 4.3.4 Hydrogen production using POME by

selected bacteria 86 4.3.5 Hydrogen productivity for different HRT 91

xiii

4.3.6 16S rRNA identification of bacteria selected from termite’s gut 93

4.4 Conclusion 96

5 OPTIMIZATION OF HYDROGEN PRODUCTION USING Ochrobactrum putranensis EB2 97

5.1 Introduction 97 5.1.1 Kinetics of cell growth in batch culture 99

5.2 Materials and methods 102 5.2.1 POME 102 5.2.2 Analysis of POME 102 5.2.3 Microorganism 103 5.2.4 Organic acids and sugar determination 103 5.2.5 Optimization study on POME as main substrate

for hydrogen production 103 5.2.6 Characteristics of POME 103

5.3 Results and discussion 106 5.3.1 The growth of the Ochrobactrum putranensis EB2 106 5.3.2 Effect of pH and temperature on the

hydrogen production 109 5.3.3 Other parameters in batch fermentation 114

5.3.4 COD removal and BOD yield 116 5.4 Conclusion 118

6 HYDROGEN GAS PRODUCTION AND PRODUCTION OF BY-PRODUCT BY Ochrobactrum putranensis EB2 120 6.1 Introduction 120 6.2 Materials and methods 122

6.2.1 POME 122 6.2.2 Characteristics of POME 122 6.2.3 Microorganism 122 6.2.4 Organic acids determination 122 6.2.5 Operation of batch fermentation 123 6.2.6 Operation of continuous reactor 124 6.2.7 Hydrogen analysis 125

6.3 Results 126 6.3.1 Batch fermentation 126 6.3.2 Conversion of COD and BOD to organic acids 127 6.3.3 Continuous fermentation 130 6.3.4 COD Removal and BOD Conversion 141

6.4 Discussions 144 6.5 Conclusions 146

xiv

xv

7 SUMMARY, CONCLUSION AND SUGGESTION FOR FUTURE WORK 148

7.1 Summary 148 7.2 Conclusion 150 7.3 Suggestion for future work 151

REFERENCES/BIBLIOGRAPHY 152 APPENDICES 170 BIODATA OF THE AUTHOR 194 LIST OF PUBLICATIONS 195

LIST OF TABLES

Table Page 2.1 Types of POME Available And Their Chemical Composition 7 2.2 Characteristics of Palm Oil Mill Effluent (POME) 7 2.3 Non-methanogenic bacteria 18 3.1 Characteristics of Palm Oil Mill Effluent (POME) 45 4.1 Results of the screened bacteria on the Nutrient agar (Heat Treatment) 70 4.2 Morphological and biochemical characteristics of isolated hydrogen producing bacteria 77 4.3 H2 yield and productivity using glucose as substrate with the 4 isolated bacteria 86 4.4 Amount of hydrogen produced from POME at 37°C 90 4.5 Hydrogen yield reported in the literatures

(continuous system using glucose) 92 5.1 Comparison of performance and the kinetic parameter values

of hydrogen production in batch culture by Ochrobactrum putraensis EB2 using constant pH 4 with different temperatures 107

5.2 Comparison of performance and the kinetic parameter values

of hydrogen production in batch culture by Ochrobactrum putraensis EB2 using constant pH 5 with different temperatures 107

5.3 Comparison of performance and the kinetic parameter values of hydrogen production in batch culture by Ochrobactrum putraensis EB2 using constant pH 6 with different temperatures 107

5.4 COD removal during anaerobic treatment of POME 117

5.5 BOD yield during steady state of anaerobic treatment of POME 117

6.1 Comparison COD removal during anaerobic treatment of POME 129

xvi

xvii

6.2 Comparison BOD conversion to VFA after anaerobic treatment of POME 129

6.3 Hydrogen yield reported in the literature

(continuous system using glucose) 138 6.4 Comparison COD removal during anaerobic treatment

of POME 140 6.5 BOD Conversion to VFA in anaerobic treatment of POME 142

LIST OF FIGURES

Figure Page

2.1 Schematic flow diagram of processing carried out in a palm oil mill 6

2.2 Schematic diagram of anaerobic digestion of organic compounds 10

2.3 Fate of electrons on reduced pyridine nucleotides in

many fermentative 15

2.4 Metabolic steps and microbial groups involved in the anaerobic digestion 16

2.5 Representative pathways of fermentative hydrogen evolution 24

3.1 Experimental overview of the bio-hydrogen research 48 3.2 Experimental design of screening process for hydrogen

producing bacteria 49

3.3 Experimental design of glucose substrates for Hydrogen production. (Preliminary study) 50

3.4 Experimental design of optimization study using POME

as main substrate in Hydrogen production 52

3.5 Experimental design of optimization of HRT for hydrogen production using POME as main substrate 53

3.6 The set-up using POME as substrate for hydrogen

production fermentation 54

3.7 The program for hydrogen analysis in gas chromatography 60

4.1 Wet Heat Treatment on POME Sludge 64 4.2 Experimental design of glucose substrates for

Hydrogen production. (Preliminary study) 68

4.3 (A) The DT1 bacterium coating the surface of the plate after 24 hours incubation at 37˚C;

(B) The DT2 bacterium with tiny colonies beginning to form/joining to each other (less than 3 hours incubation

on subsequent streaking) 71

4.4 The DT1 and DT2 bacteria on microscope view (1000X) shows that rod shape (similarity) with Gram staining 71

xviii

4.5 The DT2 bacterium forming film on the agar after 24 hours (First streaking) 72

4.6 The DT2 bacterium forming yellow film on the agar after 36 hours incubation at 37˚C (subsequent streaking) 72

4.7 (A) WT1 Bacterium covering the nutrient agar with white

film/slime (B) The flowery pattern of WT1 bacterium growth after incubation 73

4.8 Light micrograph (1000X) of WL1 73 4.9 (A) FP1 bacterium on the first streaking formed yellow film

(B) FP1 bacterium on second streaking after 2 hours of incubation 74

4.10 Light micrograph of FP1 (1000X) 74 4.11 IG1 bacterium forming white film on nutrient agar 75 4.12 Light micrograph of the IG1 bacteria (1000X) 75 4.13 SEM micrograph of the IG1 bacteria (5000X) 76 4.14 SEM micrograph of the IG1 bacteria (10,000X) 76 4.15 Durham tube test on the 4 bacteria locally isolated 79 4.16 Hydrogen production, total VFA and CDW using isolated

DT1 bacteria. Glucose was used as substrate at initial pH 7.0 with 37°C in batch fermentation 80

4.17 Hydrogen production, total VFA and CDW using isolated

WT1 bacteria. Glucose was used as substrate at initial pH 7.0 with 37°C in batch fermentation 81

4.18 Hydrogen production, total VFA and CDW using isolated

FP1 bacteria. Glucose was used as substrate at initial pH 7.0 with 37°C in batch fermentation 82

4.19 Setup of 250mL bioreactor using glucose for hydrogen production experiment 82 4.20 250mL bioreactor with glucose in the broth 83 4.21 Gas scrubber unit 83 4.22 Gas outlet to inverted 20% NaOH solution 84

xix

4.23 Hydrogen production, total VFA and CDW using isolated IG1 bacteria. Glucose was used as substrate at initial pH 7.0 with

37°C in batch fermentation 85

4.24 COD concentration, hydrogen and methane production in batch fermentation of POME using bacteria isolated from termite’s gut (IG1) with temperature 37˚C and initial pH 7.0 87

4.25 COD concentration, hydrogen and methane production in

batch fermentation of POME using bacteria isolated from wetland’s soil (WL1) with temperature 37˚C and initial pH 7.0 87

4.26 COD concentration, hydrogen and methane production

in batch fermentation of POME using bacteria isolated from anaerobic digester tank (DT1) with temperature 37˚C and pH 7.0 88

4.27 pH profiles throughout the batch fermentation using:

(A) bacteria isolated from termite’s gut (IG1), (B) bacteria isolated from wetland’ soil (WL1), (C) bacteria isolated from anaerobic digester tank (DT1)

at initial pH 7.0 and temperature 37˚C 89

4.28 Hydrogen yield based on COD conversion to mol glucose using different HRT (0.5 day, 1.0 day and 1.5 day) with

temperature 37°C and initial pH 7.0 92 4.29 Phylogenetic tree of the Ochrobactrum putranensis and

its close relatives in family of Ochrobactrum , based on 676 nucleotides in the 16S rRNA sequence 95

5.1 Experimental design of optimization study using POME as main substrate in Hydrogen production 104 5.2 Setup of bioreactor 105 5.3 The Ln Xt/Xo against time graph of

Ochrobactrum putraensis EB2 106

5.4 CDW, Ammoniacal Nitrogen and VFA content profiles of batch fermentation using glucose as substrate at pH 5.5 ± 0.3 108

5.5 pH influence (pH 4.0, 5.0 and 6.0) on hydrogen production

at 30°C in batch fermentation 109

5.6 pH influence (pH 4.0, 5.0 and 6.0) on hydrogen production at 37°C in batch fermentation 110

xx

5.7 pH influence (pH 4.0, 5.0 and 6.0) on hydrogen production at 45°C in batch fermentation 111

5.8 Total VFA, Total Nitrogen, COD concentration, TS, SS, pH, Ammoniacal Nitrogen and Microbial Count on batch fermentation using POME at 37ºC temperature 114

5.9 BOD and COD Profiles On The Batch Fermentation

with pH 6.0 and temperature 37ºC 116 6.1 Diagram setup of anaerobic continuously stirred tank reactor 125 6.2 Hydrogen production, total VFA and COD concentration

using POME as substrate at pH 5.5 with 37°C in batch fermentation prior to continuous fermentation 126

6.3 COD and BOD profiles in batch fermentation with pH 5.5

and temperature 37°C 128 6.4 Hydrogen, COD concentration and VFA concentration in

POME fermentation with Ochrobactrum putranensis EB2 using HRT 1.5 day, pH 5.5 and temperature 37°C 131

6.5 Conversion efficiency in the POME fermentation process

using Ochrobactrum putranensis EB2 with HRT 1.5 day, pH 5.5 and temperature 37°C 132

6.6 Hydrogen, COD concentration and VFA concentration in POME fermentation with Ochrobactrum putranensis EB2 using HRT 1.0 day, pH 5.5 and temperature 37°C 134

6.7 Conversion efficiency in the POME fermentation process

using Ochrobactrum putranensis EB2 with HRT 1.0 day, pH 5.5 and temperature 37°C 135

6.8 Hydrogen, COD concentration and VFA concentration in POME fermentation with Ochrobactrum putranensis EB2 using HRT 0.5 day, pH 5.5 and temperature 37°C 137

6.9 Conversion efficiency in the POME fermentation process using Ochrobactrum putranensis EB2 with HRT 0.5 day, pH 5.5 and temperature 37°C 138

6.10 Hydrogen yield based on COD conversion to mol glucose

at different HRT (0.5, 1.0 and 1.5 day) at temperature 37˚C and pH 5.5 140

6.11 Gaseous evolved on the top of the working level with whitish

slime in the POME 142

xxi

xxii

6.12 Whitish slime breakdown to tiny particles after fermentation in

HRT 0.5 day 143