UNIVERSITI PUTRA MALAYSIA BIOHYDROGEN PRODUCTION DURING GROWTH OF AN INDIGENOUS BACTERIUM, OCHROBACTRUM PUTRANENSIS EB2, IN PALM OIL MILL EFFLUENT

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1 UNIVERSITI PUTRA MALAYSIA BIOHYDROGEN PRODUCTION DURING GROWTH OF AN INDIGENOUS BACTERIUM, OCHROBACTRUM PUTRANENSIS EB2, IN PALM OIL MILL EFFLUENT CHEONG WENG CHUNG FBSB

2 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

3 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

4 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

5 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: Faculty: Prof. Mohd. Ali Hassan, PhD 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

6 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 µ max obtained was 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 H 2 /L per day for HRT 0.5 day, 13.0 ml H 2 /L per day for HRT 1.0 day and 12.6 ml H 2 /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 ml H 2 /g COD and was the highest recorded in batch fermentation reducing to 8 ml H 2 /g COD at the end of the batch fermentation. The highest productivity obtained in the continuous fermentation was 1.07 mol H 2 /mol glucose, was good in comparison with those reported earlier, mol H 2 /mol glucose. The high productivity was excellent as most of the COD iv

7 was converted to gas, based on the theoretical yield of 4 mol-h 2 /mol-glucose. As such, HRT 0.5 day gave the best result compared to other HRT done in the study. v

8 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: Fakulti: Prof. Mohd. Ali Hassan, PhD 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. Ketigatiga 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

9 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 µ max yang paling tinggi diperolehi dalam kajian ini adalah 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

10 Semasa fermentasi selanjar, penghasilan gas hidrogen ialah 34.2 ml H 2 /L sehari untuk HRT 0.5 hari, 13.0 ml H 2 /L sehari untuk HRT 1.0 hari dan 12.6 ml H 2 /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 H 2 /g COD tersingkir, dan menurun kepada 8 ml H 2 /g COD di akhir fermentasi dalam fermentasi selanjar. Produktiviti tertinggi ialah 1.07 mol H 2 /mol glukos sehari adalah baik berbanding nilai yang dilaporkan oleh penyelidik yang lain pada mol H 2 /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. viii

11 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

12 I certify that an Examination Committee met on 30 th 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 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

13 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 xi

14 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

15 TABLE OF CONTENTS Page DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS ii iii vi ix x xii xvi xviii xxii CHAPTER 1 INTRODUCTION Hydrogen Energy Environmental Impact of Palm Oil Industry Objectives 3 2 LITERATURE REVIEW 2.1 Palm oil Palm oil mill effluent (POME) Anaerobic decomposition Mechanism Interspecies hydrogen transfer Microbiology Hydrogen gas The properties of hydrogen Hydrogen pathway Bioreactor operation Rate-limiting processes ph, acidity and alkalinity Temperature Nutrients Cations Continuous operation Plasmid profile Polymerase chain reaction (PCR) Ochrobactrum Biohydrogen: future directions Hydrogen economy Differences between Bio-Methane and Bio-Hydrogen 42 xii

16 3 GENERAL MATERIALS AND METHODS Chemical reagents Palm oil mill effluent (POME) Microorganisms and preparation Source of samples Isolation of the hydrogen producing bacteria Inoculum preparation and maintenance Fermentation process using selected bacteria General study of hydrogen producing bacteria Hydrogen production using glucose as substrate Optimization study on POME as main substrate for hydrogen production Continuous fermentation study Analytical methods Organic acids determination BOD COD determination Total solids Suspended solids (SS) Total Kjeldahl nitrogen analysis Ammoniacal nitrogen (AN) Gas analysis 59 4 ISOLATION AND SCREENING OF HYDROGEN PRODUCING BACTERIA Introduction Materials and methods POME sludge Insect s gut Soil Bacteria from collection Heat treatment on the samples Screening of local strains bacteria Operation of batch reactor Analysis of fermentation products Sequencing of 16S rdna Growth profile of best hydrogen producing bacteria The selection of bacteria strain for further study Results and discussion Isolation of bacteria from various sources Durham tube test Hydrogen production using glucose by selected bacteria containing medium Hydrogen production using POME by selected bacteria Hydrogen productivity for different HRT 91 xiii

17 S rrna identification of bacteria selected from termite s gut Conclusion 96 5 OPTIMIZATION OF HYDROGEN PRODUCTION USING Ochrobactrum putranensis EB Introduction Kinetics of cell growth in batch culture Materials and methods POME Analysis of POME Microorganism Organic acids and sugar determination Optimization study on POME as main substrate for hydrogen production Characteristics of POME Results and discussion The growth of the Ochrobactrum putranensis EB Effect of ph and temperature on the hydrogen production Other parameters in batch fermentation COD removal and BOD yield Conclusion HYDROGEN GAS PRODUCTION AND PRODUCTION OF BY-PRODUCT BY Ochrobactrum putranensis EB Introduction Materials and methods POME Characteristics of POME Microorganism Organic acids determination Operation of batch fermentation Operation of continuous reactor Hydrogen analysis Results Batch fermentation Conversion of COD and BOD to organic acids Continuous fermentation COD Removal and BOD Conversion Discussions Conclusions 146 xiv

18 7 SUMMARY, CONCLUSION AND SUGGESTION FOR FUTURE WORK Summary Conclusion Suggestion for future work 151 REFERENCES/BIBLIOGRAPHY 152 APPENDICES 170 BIODATA OF THE AUTHOR 194 LIST OF PUBLICATIONS 195 xv

19 LIST OF TABLES Table Page 2.1 Types of POME Available And Their Chemical Composition Characteristics of Palm Oil Mill Effluent (POME) Non-methanogenic bacteria Characteristics of Palm Oil Mill Effluent (POME) Results of the screened bacteria on the Nutrient agar (Heat Treatment) Morphological and biochemical characteristics of isolated hydrogen producing bacteria H 2 yield and productivity using glucose as substrate with the 4 isolated bacteria Amount of hydrogen produced from POME at 37 C Hydrogen yield reported in the literatures (continuous system using glucose) 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 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 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 COD removal during anaerobic treatment of POME BOD yield during steady state of anaerobic treatment of POME Comparison COD removal during anaerobic treatment of POME 129 xvi

20 6.2 Comparison BOD conversion to VFA after anaerobic treatment of POME Hydrogen yield reported in the literature (continuous system using glucose) Comparison COD removal during anaerobic treatment of POME BOD Conversion to VFA in anaerobic treatment of POME 142 xvii

21 LIST OF FIGURES Figure Page 2.1 Schematic flow diagram of processing carried out in a palm oil mill Schematic diagram of anaerobic digestion of organic compounds Fate of electrons on reduced pyridine nucleotides in many fermentative Metabolic steps and microbial groups involved in the anaerobic digestion Representative pathways of fermentative hydrogen evolution Experimental overview of the bio-hydrogen research Experimental design of screening process for hydrogen producing bacteria Experimental design of glucose substrates for Hydrogen production. (Preliminary study) Experimental design of optimization study using POME as main substrate in Hydrogen production Experimental design of optimization of HRT for hydrogen production using POME as main substrate The set-up using POME as substrate for hydrogen production fermentation The program for hydrogen analysis in gas chromatography Wet Heat Treatment on POME Sludge Experimental design of glucose substrates for Hydrogen production. (Preliminary study) (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) The DT1 and DT2 bacteria on microscope view (1000X) shows that rod shape (similarity) with Gram staining 71 xviii

22 4.5 The DT2 bacterium forming film on the agar after 24 hours (First streaking) The DT2 bacterium forming yellow film on the agar after 36 hours incubation at 37 C (subsequent streaking) (A) WT1 Bacterium covering the nutrient agar with white film/slime (B) The flowery pattern of WT1 bacterium growth after incubation Light micrograph (1000X) of WL (A) FP1 bacterium on the first streaking formed yellow film (B) FP1 bacterium on second streaking after 2 hours of incubation Light micrograph of FP1 (1000X) IG1 bacterium forming white film on nutrient agar Light micrograph of the IG1 bacteria (1000X) SEM micrograph of the IG1 bacteria (5000X) SEM micrograph of the IG1 bacteria (10,000X) Durham tube test on the 4 bacteria locally isolated 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 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 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 Setup of 250mL bioreactor using glucose for hydrogen production experiment mL bioreactor with glucose in the broth Gas scrubber unit Gas outlet to inverted 20% NaOH solution 84 xix

23 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 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 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 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 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 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 Phylogenetic tree of the Ochrobactrum putranensis and its close relatives in family of Ochrobactrum, based on 676 nucleotides in the 16S rrna sequence Experimental design of optimization study using POME as main substrate in Hydrogen production Setup of bioreactor The Ln X t /X o against time graph of Ochrobactrum putraensis EB CDW, Ammoniacal Nitrogen and VFA content profiles of batch fermentation using glucose as substrate at ph 5.5 ± ph influence (ph 4.0, 5.0 and 6.0) on hydrogen production at 30 C in batch fermentation ph influence (ph 4.0, 5.0 and 6.0) on hydrogen production at 37 C in batch fermentation 110 xx

24 5.7 ph influence (ph 4.0, 5.0 and 6.0) on hydrogen production at 45 C in batch fermentation Total VFA, Total Nitrogen, COD concentration, TS, SS, ph, Ammoniacal Nitrogen and Microbial Count on batch fermentation using POME at 37ºC temperature BOD and COD Profiles On The Batch Fermentation with ph 6.0 and temperature 37ºC Diagram setup of anaerobic continuously stirred tank reactor 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 COD and BOD profiles in batch fermentation with ph 5.5 and temperature 37 C 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 Conversion efficiency in the POME fermentation process using Ochrobactrum putranensis EB2 with HRT 1.5 day, ph 5.5 and temperature 37 C 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 Conversion efficiency in the POME fermentation process using Ochrobactrum putranensis EB2 with HRT 1.0 day, ph 5.5 and temperature 37 C 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 Conversion efficiency in the POME fermentation process using Ochrobactrum putranensis EB2 with HRT 0.5 day, ph 5.5 and temperature 37 C 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 Gaseous evolved on the top of the working level with whitish slime in the POME 142 xxi

25 6.12 Whitish slime breakdown to tiny particles after fermentation in HRT 0.5 day 143 xxii

IDENTIFICATION AND CHARACTERIZATION OF BACTERIA FROM THE SKIN OF JACKFRUIT CHITRA A/P SELVARATNAM

IDENTIFICATION AND CHARACTERIZATION OF BACTERIA FROM THE SKIN OF JACKFRUIT CHITRA A/P SELVARATNAM A dissertation submitted in partial fulfilment of the requirements for the award of the degree of Master