biogas production and determination of methane producing bacteria

UNIVERSITI PUTRA MALAYSIA

BIOGAS PRODUCTION AND DETERMINATION OF METHANOGENS FROM DIGESTER - TREATED PALM OIL MILL EFFLUENT

MOHD RAFEIN BIN ZAKARIA @ MAMAT

FBSB 2008 9



MASTER OF SCIENCE

UNIVERSITI PUTRA MALAYSIA

2007


By


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

December 2007

DEDICATION

This piece of work is dedicated to my lovely parent and my wife, who has always been by my side and given me the encouragement and support that carries me

through my study period. Thanks for their undying love to me.

ii

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


By


December 2007

Chairman: Professor Mohd Ali Bin Hassan, PhD Faculty: Faculty of Biotechnology and Biomolecular Sciences Due to increasing awareness of the risk of the environmental pollution and emission

of green house gases (GHG) that caused global warming, a study of palm oil mill

effluent (POME) treatment using biological processes in close digesters has been

conducted. Potentially beneficial methane gas production from this treatment has

driven the objectives of this study to explore the methanogens from POME sludge

and their characteristic in order to improve the POME treatment efficiency. A pilot

plant digester with 500 m3 volume was operated for one year and biogas and

methane production profiles were monitored daily. Parameters used for monitoring

were pH, temperature, volatile fatty acids, chemical oxygen demand, biogas and

methane concentration during the treatment. Structure of the flocs formation and

methanogenic bacteria isolated from this digester was also carried out in order to

understand the microbiological characteristics of the bioprocess involved.

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Specific methanogenic activity test (SMA) and microscopic observation were carried

out to support the existence of the methanogens that were able to produce methane

gas. During POME treatment process, the highest methane concentration obtained

was 55 % (v/v) and chemical oxygen demand (COD) removal efficiency was up to

95%. Anticipated active biomasses retained in the digester were determined as total

solids (TS) and volatile suspended solids (VSS) at 2.5% and 1.5%, respectively.

SMA test for acetolastic methanogens was determined in the range of 0.05 - 1.3 g

COD/ g VSS/d. Microscopic observation of the sludge and isolated colonies have

shown that the digester systems were dominated by microorganism resemble to

Methanosaeta sp. and fluorescence microscope has proved that this microorganism

exhibited autofluorescence green color.

iv

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

PENGHASILAN BIOGAS DAN PENENTUAN METHANOGENS DARI TANGKI RAWATAN AIR PEMPROSESAN BUAH SAWIT

Oleh


Disember 2007

Pengerusi : Profesor Mohd Ali Bin Hassan, PhD Fakulti : Fakulti Bioteknologi dan Sains Biomolekul.

Kesedaran yang tinggi tentang risiko pencemaran alam dari pelepasan gas kesan

rumah hijau (GHG) yang menyebabkan pemanasan global telah mendorong kepada

penyelidikan tentang rawatan air buangan dari pemprosesan kelapa sawit (POME)

menggunakan kaedah proses biologi di dalam tangki tertutup. Secara potensinya,

kelebihan penghasilan gas metana dari sistem rawatan ini telah mencetuskan

matlamat kajian untuk mendalami ciri-ciri bakteria methanogens yang diperolehi dari

mendakan POME bagi meningkatkan kecekapan rawatan. Loji pandu biogas yang

berkapasiti 500 tan telah beroperasi selama satu tahun dan dipantau penghasilan

biogas dan gas metana. Suhu, pH, kandungan asid lemak meruap, keperluan oksigen

kimia (COD) juga adalah parameter yang dipantau semasa rawatan.

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Struktur gumpalan yang terbentuk dan bakteria yang menghasilkan gas metana juga

dikaji semasa proses rawatan POME dengan tujuan mengetahui dengan lebih

mendalam tentang sifat-sifat mikrobiologi terhadap bioproses yang terlibat. Kajian

SMA dan pengamatan menggunakan mikroskop juga dilakukan bagi mengesahkan

kewujudan bakteria tersebut di dalam loji pandu biogas. Bacaan tertinggi gas metana

yang diperolehi semasa kajian ini adalah sebanyak 55% dan kadar penurunan

keperluan oksigen kimia (COD) adalah lebih dari 95%. Dianggarkan sebanyak 2.5%

jumlah pepejal (TS) dan 1.5% pepejal terampai meruap (VSS) dapat dikekalkan di

dalam sistem loji pandu. Kajian SMA bagi methanogens yang menggunakan asetik

asid bagi penghasilan gas metana adalah dianggarkan sebanyak 0.05 – 1.3

gCOD/VSS/d. Pengamatan menggunakan mikroskop untuk mendakan POME

menunjukkan loji pandu biogas didominasi oleh bakteria yang menyerupai spesis

Methanosaeta dan ianya dibuktikan dengan pengamatan mikroskop fluoresen dimana

bakteria tersebut menunjukkan warna hijau fluoresen.

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ACKNOWLEDGEMENTS

Alhamdulillah, great thanks to Allah for His mercy and guidance, I have completed

my thesis as required. I would like to express my gratitude to my main supervisor,

Prof. Dr. Mohd Ali Bin Hassan, co-supervisors, Prof. Dr. Mohamed Ismail Bin

Abdul Karim from International Islamic University and Prof. Dr. Yoshihito Shirai

from Kyushu Institute of Technology, Japan, for their dedicated effort, guidance

and encouragement through out the research. Their experiences are valuable and as a

pathway to my successful research.

To my parent, I would like to say that without your sacrifice, encouragement and

understanding I may not able to complete this study. To my wife, Ros , your support

and undying love taught me about life, discipline and working hard towards our goal.

Not forgetting to all my friends, I would like specially say thank you to Zulkifli,

Munir, Fadly, and Sharfan sharing a joyful moment with me. Words could not

express my gratitude to all of you.

Last but not least, special thanks to my seniors, juniors and lab assistants Cheong,

Kim Ying, Pooi Tie, Wong, Syahrakbah and Syahman, Azhari, Hidayah and Ily for

their support, co-operation, sharing ideas and guidance to me. Million thanks to lab

officer Mr. Rosli for his technical assistance and wonderful life during my many

hours in the microbiology laboratory.

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I certify that an Examination Committee met on 22 December 2007 to conduct the final examination of Mohd Rafein Bin Zakaria @ Mamat on his Master of Science thesis entitled “Methane Production and Determination of Methanogens from Digester Treating Palm Oil Mill Effluent” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The committee recommends that the candidate be awarded the degree of Master of Science. Members of the Examination Committees were as follows: Tong Chow Chin, PhD Associate Professor Faculty of Biotechnology and Biomolecular Science Universiti Putra Malaysia (Chairman) Rosfarizan Mohamad, Ph.D. Faculty of Biotechnology and Biomolecular Science Universiti Putra Malaysia (Internal Examiner) Nor `Aini Abdul Rahman, PhD Faculty of Biotechnology and Biomolecular Science Universiti Putra Malaysia (Internal Examiner) Vikineswary Sabaratnam, PhD Professor Faculty of Science Universiti Malaya (External Examiner)

__________________________________ HASANAH MOHD GHAZALI, Ph.D.

Professor and Deputy Dean School of Graduate Studies

Universiti Putra malaysia

Date :

viii

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: Mohd Ali bin Hassan, PhD Professor Faculty of Biotchnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Mohamed Ismail bin Abdul Karim, PhD Professor Faculty of Biotechnology Engineering International Islamic University Malaysia (Member)

Yoshihito Shirai, PhD Professor Graduate School of Life Science and Systems Engineering Kyushu Institute of Technology, Japan (Member)

______________________ AINI IDERIS, PhD

Professor and Dean School of Graduate Studies

Universiti Putra Malaysia

Date : 21st February 2008

ix

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 institution.

___________________________________

MOHD RAFEIN ZAKARIA @ MAMAT

Date:

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TABLE OF CONTENTS

Page DEDICATION ii ABSTRACT iii ABSTRAK v ACKNOWLEDGEMENTS vii APPROVAL viii DECLARATION x LIST OF TABLES xiv LIST OF FIGURES xv LIST OF ABBREVIATIONS xvi CHAPTER

1.0 INTRODUCTION 1 1.1 Objectives 5

2.0 LITERATURE REVIEW 6 2.1 Palm Oil Industry 6 2.2 POME Treatment 9 2.2.1 Pond/ Lagoon Systems 9 2.2.2 Open Digester Tank (ODT) 10 2.2.3 Closed Digester Tank (CDT) 11 2.3 Anaerobic Digestion of POME 12 2.3.1 Hydrolysis 13 2.3.2 Acidogenesis 14 2.3.3 Acetogenesis 14 2.3.4 Methanogenesis 15 2.4 Methane Producing Bacteria 16 2.5 Characteristics of Methanogens 19 2.5.1 Cell Wall 19 2.5.2 Antibiotic Resistance 20

2.5.3 Compound F420 21 2.5.4 Lipid Composition 22 2.6 Nutrients and Growth Requirements 22 2.6.1 Substrates and Supplements 22 2.6.2 pH 24 2.6.3 Oxygen Requirement 24 2.6.4 Agitation 25 2.6.5 Temperature 26 2.7 Taxonomy and Morphology 27 2.8 Ecology of Methanogens 31

xi

2.8.1 Soil and Aquatic Environments 31 2.8.2 Digester 33 2.8.3 Within Living Organisms 33

3.0 METHANE PRODUCTION DURING PALM OIL MILL 35

EFFLUENT (POME) TREATMENT BASED ON ORGANIC LOADING RATE (OLR) PATTERN

3.1 Introduction 35 3.2 Materials and Methods 39 3.2.1 Description of Closed Digester Tank (CDT) 39 3.2.2 Analytical Methods 42 3.2.3 VFA tests 43 3.3 Results and Discussion 44 3.3.1 Performance of CDT systems 44 3.3.2 COD Removal Efficiency 45 3.3.3 VFA Concentration 46 3.3.4 Methane Concentration 48 3.3.5 pH 50 3.3.6 Hydraulic Retention Time (HRT) 51 3.3.7 Microorganisms 53 3.3.8 Feeding Pattern 55

3.3.9 Mixing 56 3.3.10 Temperature 57 3.4 Conclusion 58

4.0 SPECIFIC METHANOGENIC ACTIVITY (SMA) TEST 59 DURING POME TREATMENT 4.1 Introduction 59 4.2 Materials and Methods 62 4.2.1 Sludge Sampling 62 4.2.2 Media 62 4.2.2.1 Anaerobic Technique 63 4.2.3 Preparation of Media 64 4.2.4 Experimental Procedure 65 4.2.5 Analytical Methods 66 4.3 Results and Discussion 66 4.3.1 Substrates 71 4.3.2 Sludge / Microorganism 73 4.4 Conclusion 75

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5.0 DETERMINATION OF ACETATE CONSUMING 76 METHANOGENS FROM CDT TREATING POME 5.1 Introduction 76 5.2 Materials and Methods 79 5.2.1 Media Components 79 5.2.2 Anaerobic Technique 80 5.2.3 Preparation of Media 80 5.2.4 Agar Tubes 81 5.3 Screening of Acetoclastic Methanogens 82 5.3.1 Sample Collections 82 5.3.2 Screening Procedures 82 5.3.3 Enrichment 84 5.3.4 Isolation and Purification 85 5.4 Microscopy 86 5.4.1 Light Microscope 87 5.4.2 Fluorescence Microscope 87 5.4.3 Scanning Electron Microscope (SEM) 88 5.4.4 Direct UV Observation 88 5.5 Results and Discussion 91 5.5.1 Acetate Enrichment 91 5.5.2 Methanol Enrichment 94

5.5.3 Screening and Isolation of Acetoclastic Methanogens 95

5.5.3.1 Light and SEM Microscopy 99 5.6 Conclusion 105

6.0 CONCLUSION AND SUGGESTIONS FUTURE RESEARCH 106

REFERENCES 110 APPENDICES 121 BIODATA OF STUDENTS 125

xiii

LIST OF TABLES

Tables Page

2.1 Types of palm biomass being utilized for value by-products 7

2.2 Chemical properties of POME 8

2.3 General differences and similarities among bacteria, archaea 18

and eukaryotes

2.4 Characteristics of methanogenic Archaea 28

3.1 General guidelines for biomethanation of POME 41

4.1 Media for SMA tests 63

4.2 Details of controlled parameters for AMA test 70

5.1 Enrichment media composition 79

5.2 Viable counts media composition 79

xiv

LIST OF FIGURES

Figures Page

2.1 Diagram of anaerobic degradation 15

2.2 Typical structures of murein and pseudomurein 20

2.3 Structure of F420 compounds 21

2.4 Phylogeny of methanogens, domain Archaea 30

3.1 Closed anaerobic digester tanks (CDT) 40

3.2 Organic loading rate and COD removal efficiency 47

3.3 VFA accumulation and Methane production 48

3.4 Organic loading rate and Hydraulic retention time 52

3.5 Total solids of fresh and treated POME 54

4.1 Effect of organic loading rate changes on acetoclatic 64

5.1 Research strategies for screening and isolation of 83

5.2 Enrichment of POME sludge in serum vials 89

5.3 Viable counts in Hungate tubes for isolation study 89

5.4 Effect of calcium carbonate on sedimentation 90

5.5 Biogas production from acetate enrichments 92

5.6 Methane production from acetate enrichment 92

5.7 Biogas and methane production from methanol 94

5.8 Colonies detection under ultra violet lamp 97

5.9 Fluorescence color exhibited from sludge and pure 99

5.10 Gram stain of sludge and enrichment cultures 100

5.11 Scanning electron micrograph of POME flocs 102

xv

LIST OF ABBREVIATIONS

AMA Acetoclastic Methanogenic Activity

ATP Adenine Triphosphate

BOD Biological Oxygen Demand

CDM Clean Development Mechanism

CDT Closed Digester Tank

CER Certified Emission Reduction

CFC Chlorofluorocarbon

CH4 Methane

CO Carbon Monoxide

CO2 Carbon Dioxide

CSTR Continuous Stirred Tank Reactor

d Day

DNA Deoxyribonucleic Acid

COD Chemical Oxygen Demand

CPO Crude Palm Oil

DOE Department of Environment

EFB Empty Fruit Bunch

FFB Fresh Fruit Bunch

g Gram

GHG Green House Gas

HOM Hydrogen Oxidizing Methanogens

HRT Hydraulic Retention Time

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H2 Hydrogen gas

L Liter

MABR Modified Anaerobic Baffled Reactor

NHOM Non-Hydrogen Oxidizing Methanogens

ODT Open Digester Tanks

OLR Organic Loading Rate

PCR Polymerase Chain Reaction

POME Palm Oil Mill Effluent

RABR Reversible Anaerobic Baffle Reactor

RNA Ribonucleic Acid

SMA Specific Methanogenic Activity

TMA Total Methanogenic Activity

TS Total Solid

UAF Upflow Anaerobic Filter

UASB Upflow Anaerobic Sludge Blanket reactor

UASFF Upflow Anaerobic Sludge Fixed-Film

VFA Volatile Fatty Acid

VS Volatile Solids

VSS Volatile Suspended Solid

CHAPTER 1

INTRODUCTION

1.0 Introduction

Since the global industrial revolution, environment has become polluted and

unhealthy for living organisms. These are attributed to deforestation, discharge of

pollutants into rivers, lands and air. Some hazardous compounds such as

chlorofluorocarbon (CFC), nitrous oxide, carbon monoxide (CO), carbon dioxide

(CO2) and methane (CH4) are green house gases (GHG) which have strong infrared

absorption capacity and trap a large portion of the thermal radiation emitted from the

earth’s surface (Yang et al., 2003). Uncontrolled release of the pollutants into the air

has led to an unstable environment threat. Some negative effects of these pollutants

and massive concern causing worldwide are depletion of ozone layer, global

warming and significant raise of ocean level. Malaysia, like other developing

countries had achieved an outstanding performance of economic growth since

implementing mass industrial revolution. One of big and outstanding industries in

Malaysia is agriculture- based oil palm industry.

Oil palm industry was brought by Frenchman, Henri Fauconnier in the early 20th

century (Teoh, 2002). Since then mass planting of oil palm has been promoted, the

development and management of land has been expanded particularly in rural area,

setting up research and development in recognized institutions have fuelled the

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nation’s economy. The oil palm planted area has expanded from 55,000 hectares in

1960 to 3.5 million hectares by 2001, occupying 60% of the agricultural land in the

country with 380 palm oil mills of which about 70% are located in Peninsular

Malaysia (Khoo, 2001). Malaysia is the largest palm oil producer, contributing

approximately 11.8 million tonnes or 50.9% of total world production (Khoo, 2001).

Despite being known as world’s biggest palm oil producer, this industry also

generates large amounts of wastes and is among the most polluting in the country.

The wastes or residues generated from palm oil industry are in two forms, solid

wastes that consists of empty fruit bunch (EFB), shell, trunks, and fronds. Secondly

is the liquid waste mainly palm oil mill effluent (POME), which is highly polluting at

an average of 25,000 mg/L biochemical oxygen demand (BOD) and 50,000 mg/L

chemical oxygen demand (COD) (Ma, 1999). Conventionally, POME is being

treated using the cheapest technologies, open digester tanks and pond or lagoon

systems. According to the strict regulations of wastewater disposal amendment by

the Department of Environment (DOE) Malaysia, the POME should be treated first

or converted into by-products with added value before it`s disposed to appropriate

places. Generally, these systems need large land areas which release directly and

uncontrolled GHG particularly CH4 and CO2 gas to the atmosphere. In 2005, the

estimation methane of annual emission potential from POME was 5,000,000 tonnes

(Tong and Jaafar, 2005).

2

Studies by Yacob and co-workers (2005a and 2005b) on the methane emission from

lagoon and open digesting tanks treating POME have provided valuable information

in establishing GHG emission particularly in palm oil industry. These studies were

done in line with the initiatives laid out to reduce GHG emission under the Kyoto

Protocol. Briefly, Kyoto Protocol encourages the involvement of the developing

countries in reducing the climate problems on the way to development. Thus, there

have been keen overwhelming interests in the palm oil industry due to the huge

potential of untapped biogas and biomass as clean, green and renewable resources of

energy during the mitigation of GHG emission. By ratifying the Kyoto Protocol, the

implementation of the Clean Development Mechanisms (CDM) for palm oil industry

is now possible. The industry could now contribute to global emissions reduction via

CDM projects, in addition to earning economic benefits from Certified Emission

Reduction (CER) credits. The palm oil industry can derive new economic,

development and environment benefits through the implementation of CDM projects.

The increasing concerns for rapid depletion of the non-renewable fuel and the other

world’s environmental issues have resulted in searching for sustainable alternative

fuels. Anaerobic degradation of organic residues for biogas production is the best

option as a substitution for petrochemical fuel. Anaerobic degradation of organic

waste/ residues in closed digesters is not a new technology. European countries have

adopted and established this technology over than 50 years ago. However, this green

and clean technology is not well appreciated in treating and handling organic

residues in Malaysia. Quah and Gilles (1981) reported anaerobic degradation of

POME in closed digester tanks produced approximately 28.3 m3 gas per cubic meter

3

of POME digested where methane was found to be at the range of 54 -70% volume.

Unfortunately, this valuable finding was not expanded and widely implemented

because of several factors. The market demand for alternative fuels is insignificant

and industries are pleased with the available resources. Most important factor is lack

of enough motivation for improving and implementing of this technology as POME

is only resources available in Malaysia.

Now the application of anaerobic digestion of POME using closed system has

become attractive and expanded. There are a lot of researchers reported the potential

application of the anaerobic digester in laboratory and pilot scale studies. There are

varietion in the term of types of bioreactor design and system monitoring for

treatment of POME such as the modified anaerobic baffled bioreactor (Faisal and

Unno, 2001), anaerobic filter and anaerobic fluidized bed reactor (Borja and Banks,

1995), thermophilic upflow anaerobic filter (Mustapha, et al., 2003), continuous stir

tank reactor (Tong and Jaafar, 2005), and reversible anaerobic baffled reactor

(RABR) (Raof et al., 2005). However no study has been done on the microorganisms

and their contribution to the successful anaerobic POME degradation process.

Previous studies have shown a lot of obstacles have been faced during anaerobic

degradation of POME. High content of oils and fats that are not easy to hydrolyse,

uncertainties about quantity and quality of fresh POME, bioreactor design, washout

of active microorganisms especially methane producing bacteria are the factors that

have to be taken into consideration. Specific methanogenic activity tests, diversity of

characteristics and determination of methanogenic bacteria will help in

understanding the characteristic of biomass sludge in anaerobic digesters thus

4

maintain the stability of POME digestion performance. Recently, similar efforts have

been made by the largest palm oil company in the world, Federal Land and

Development Authority (FELDA), through the R&D collaboration with Universiti

Putra Malaysia (UPM) and Kyushu Institute of Technology (KIT), Japan to utilize

the biogas and biomass for new bioproducts. Three projects have been conducted

including; the generation of biogas for energy using 500 m3 CDT (Yacob et al.,

2006), production of organic acids and biodegradable plastics from POME (Nor

`Aini et al., 1999) and saccharafication of EFB to produce sugar (Hassan et al.,

2003). Those projects are examples for the mitigation methods to reduce GHG

emitted from palm oil industries.

1.1 Objectives

The objectives of this study were;

1. To study the methane production in the closed digester tanks (CDT) based on

organic loading rates (OLR) feeding pattern.

2. To study the microbial diversity of anaerobic fermentation in the CDT using

microscopy observations and Specific Methanogenic Activity (SMA) test.

5

CHAPTER 2

LITERATURE REVIEWS

2.1 OIL PALM INDUSTRY

Oil palm (Elaeis guineensis) is vastly cultivated as a source of oil in West and central

Africa where it is originated from, and in Malaysia, Indonesia and Thailand as well.

In Malaysia, oil palm is one of the most important commercial items and accounts

for 20% and 46% of the global oil and fat production and trade, respectively.

Malaysia is the world’s largest producer and exporter of palm oil with 11.80 million

tonnes or 50% of the world palm oil production. The oil palm planted area increased

to 3.5 million hectares by 2001, occupying 60% of the agricultural land in the

country and the industry still expanding corresponding to the growing world

population (Khoo, 2001).

Apart from edible oil, the palm oil industry also generates large amount of biomass.

The biomass can be divided into two forms of renewable biomass, solid biomass and

liquid biomass. Solid biomass comprises of empty fruit bunch (EFB), 53%, mesocarp

fibre, 32%, and palm kernel shell, 15%. There are many practices of EFB utilization

particularly, as it is rich in cellulose (50.4%), hemi-cellulose (21.9%), lignin (10%),

and ash (17.7%), (Umikalsom, et al., 1997). Among the potential application of EFB

are soil mulching (Weng, 1999), fiber board for the furniture industry (Tan and

Kang, 1993), and as boiler fuel (Chua, 1991).

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biogas production and determination of methane producing bacteria