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AND WASTEWATER TREATMENT BY MICROBIAL FUEL CELL

BY

AYOUB AHMED ALI

A dissertation submitted in fulfilment of the requirement for the degree of Master of Science (Biotechnology

Engineering)

Kulliyyah of Engineering

International Islamic University Malaysia

May 2019

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ABSTRACT

Microbial fuel cells (MFCs) are devices that use prokaryotic microorganism to produce electrical bioenergy from a wide range of organic substrates. MFCs provide a promising sustainable energy production as well as, simultaneous degradation of organic waste in the substrate. The active microorganisms capable of producing electricity by transferring an electron to the electrode are called Electrochemically Active Bacteria (EAB). In this study, the substrate used was water and mud collected from Malaysia's shrimp pond at Perak. A special U-shaped MFC reactor with carbon-based electrodes was used for screening and isolation the electrochemically active bacteria. In addition, most of the EAB isolated from MFC electrode are known as dissimilatory metal- reducing bacteria (DMRB), thus agar nutrient containing metal has been used for isolation. DMRB based isolation methods limited the potential isolation of diverse electrochemical bacteria. Therefore, in this study agar nutrient has been used for the isolation of the EAB from the anode electrode of the U-tube MFC. Where the pure culture obtained was Bacillus cereus strain cc-1 identified by using 16s rRNA genes.

Next, the Bacillus cereus strain cc-1 was mutated using gamma irradiation in order to increase the activity of bacteria. The optimum dose to increase the bacteria potential has been determined using one factor at time (OFAT) method. Results show that 63Gy gamma dose irradiation increased the cell voltage to 280 mV with 33% of chemical oxygen demand removal (COD) while the maximum voltage of the wild strain was154 mV with 55.7% of COD removal. The successful effect of gamma radiation dose on the increase of the MFC's bioelectricity and organic matter removal indicates that gamma rays are a way to boost the ability of the electrically active bacteria. Further investigation on the whole genome sequencing analysis should provide the full picture of the gamma radiation-induced effect.

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iii

ثحبلا ةصلاخ

ABSTRACT IN ARABIC

( ةيبوركيلما دوقولا يالاخ MFCs

تانئاكلا اهمدختست تيلا يالالخا يه ) ةقاطلا جاتنلإ ةاونلا ةيئادب ةقيقدلا

نم ةيربك ةعوممج مادختسبا ةيئبارهكلا ةيويلحا ةيبوركيلما دوقولا يالاخ لمعت ثيح ةيوضعلا داولما

( MFCs هدالما مادختسبا كلذو هددجتلما ةمادتسلما ةقاطلا جاتنإ ىلع )

ىقبتمو ةيساسلاا ةيوضعلا

اتهياافن

؛ٍدحاو ٍنآ فى لقن قيرط نع ءبارهكلا جاتنإ ىلع ةرداقلا ةطشنلا ةقيقدلا تانئاكلا تيسم كلذلو

لا بطقلا لىإ نوتركللإا ( ًايئبارهك ةطشنلا يايرتكبلبا يئبارهك

لك تمدختسُا ةساردلا هذه فى .) EAB

قاولا يبرملجا ةكرب نم ًاعم ينطلاو ءالما نم .يازيلام كايرب ةظفامح في ةع

لعافم مادختسا تم (

MFC )

ىلع صاخ لكش

U ايئبارهك ةطشنلا يايرتكبلا لزعو صحفل ىلع ةمئاقلا نوبركلا باطقأ عم ةفاضلإبا .

لىإ ا مظعم نإف ،كلذ ( ًايئبارهك ةطشنلا يايرتكبل

EAB ( ةيبوركيلما دوقولا يالاخ نم هلوزعلماو ) MFC

)

ام ًةداعو ا مسبا فرعُت

( نداعملل ةلزتخلما يايرتكبل DMRB

يوتتح تيلا تياذغلما مادختسا تم كلذلو ،)

لا عون ساسأ ىلع تممص كلت لزعلا قرط نا ثيح ؛يئبارهكلا لزعلا دنع ندعلما ىلع هلزتخلما يايرتكب

ياتركبلا طاشنل ةجيتن ةثودح عقوتم عونت ىا ثودح عنلم لماكلا مكحتلا تتح نوكت ثيبح نداعملل ك ةطشنلا لزعو ومنل ةفورعم ةيئاذغ هدامك نىغلا راجلاا صلختسم مادختسا تم ،كلذل .ًايئايميكوره

( ًايئبارهك ةطشنلا يايرتكبلا EAB

خ نم دونلأا يئبارهكلا بطقلا نم ) يىبوركيلما ءاذغلا يالا

MFC

لكش وذ بوبنأ مادختسبا U

يه اهيلع لوصلحا تم تيلا ةيقنلا ياديلما تناك ثيبح Bacillus

cereus strain cc-1 مادختسبا اهديدتح تم تيلاو

ًانيج 16 rRNA هللاس نم ياتركب ريوتح تمو .

Bacillus cereus strain cc-1 لجا ديدتح تمو اماغ ةعشأ مادختسبا

تنااكمإ ةديازل ةيلاثلما ةعر

تكبلا ( دحاو تقو في دحاو لماع مادختسبا ياير OFAT

نأ لىإ جئاتنلا تحضوا ايرخاو .) 63

اماغ

لىإ ةيللخا دهج نم تداز عيعشتلا ةعرج نم 280

mV ةيبرلا ةللاسلل ىصقلأا دهلجا ناك امنيب 4

15

mv ةيسيطانغمورهك ةدياز ىلع اماج ةعشأ يرثتأ حانج نأ . MFC

نأ ىلع رشؤي ةيوضعلا داولما ةلازإو

ق ديزت نأ نكيم تيلا قرطلا نم اماج ةعشأ ةسلس ىلع ةساردلا نم ديزلما .ايئبارهك ةطشنلا يايرتكبلا ةرد

.يزيفحتلا اماج ةعشأ رثلأ لشمأ روصت يرفوت ىلع دعاسي نأ نكيم ةيلكلا مونيلجا

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APPROVAL PAGE

I certify that I have supervised and read this study and that in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Master of Science (Biotechnology Engineering)

………..

Azura Bt Amid Supervisor

………..

Azlin Suhaida Azmi Co-Supervisor

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Master of Science (Biotechnology Engineering)

………..

Nassereldeen Ahmed Kabbashi Internal Examiner

………..

Noor Illi Bt Mohamad Puad Internal Examiner

This dissertation was submitted to the Department of Biotechnology Engineering and is accepted as a fulfilment of the requirement for the degree of Master of Science (Biotechnology Engineering)

………..

Nor Fadhillah Mohamed Azmin Head, Department of

Biotechnology Engineering This dissertation was submitted to the Kulliyyah of Engineering and is accepted as a fulfilment of the requirement for the degree of Master of Science (Biotechnology Engineering)

………..

Ahmad Faris bin Ismail

Dean, Kulliyyah of Engineering

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DECLARATION

I hereby declare that this dissertation is the result of my own investigations, except where otherwise stated. I also declare that it has not been previously or concurrently submitted as a whole for any other degrees at IIUM or other institutions.

AYOUB AHMED ALI

Signature ... Date ...

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INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA DECLARATION OF COPYRIGHT AND AFFIRMATION OF

FAIR USE OF UNPUBLISHED RESEARCH

ENHANCEMENT OF ELECTRICITY GENERATION AND WASTEWATER TREATMENT BY MICROBIAL FUEL CELL

I declare that the copyright holders of this dissertation are jointly owned by the student and IIUM.

Copyright © 2019 AYOUB AHMED ALI and International Islamic University Malaysia. All rights reserved.

No part of this unpublished research may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without prior written permission of the copyright holder except as provided below

1. Any material contained in or derived from this unpublished research may be used by others in their writing with due acknowledgement.

2. IIUM or its library will have the right to make and transmit copies (print or electronic) for institutional and academic purposes.

3. The IIUM library will have the right to make, store in a retrieved system and supply copies of this unpublished research if requested by other universities and research libraries.

By signing this form, I acknowledged that I have read and understand the IIUM Intellectual Property Right and Commercialization policy.

Affirmed by AYOUB AHMED ALI

……..……….. ………..

Signature Date

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To Shamis Hersi and Ahmed Ali, my beloved mother and father

To my country and our ministry of high education and research who gave me the opportunity to pursue my master’s degree at IIUM and all my lectures specially my

supervisor

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ACKNOWLEDGEMENTS

In the name of ALLAH, the most Merciful, the Most Compassionate.

Before all, I express my deepest appreciation to my Lord; the Almighty ALLAH (SWT) that has granted me the health and the strength to fulfil this work. May the blessing and peace of ALLAH be upon the messenger of ALLAH, Muhammed (SAW).

First, I would like to express my sincere gratitude to my supervisor Prof. Dr.

Azura Bt Amid who has given me the guidance, knowledge, advice, which made the successful completion of this thesis. I also express my sincere gratitude to my Co- supervisor Assistant Prof. Dr. Azlin Suhaida bt Azmi for her kind advice and academic support throughout my academic journey.

My heartiest gratitude to my Djiboutian brothers and sisters who was my only family during my study in Malaysia, specially Habib Said and Ibrahim Gamiye for their moral support, closest presence and countless help.

My special thanks to my colleagues in Bioprocess and Molecular Engineering Research Unit, the Department of Biotechnology engineering, laboratory technician and SBH Marine Industries for their sample. Again, I am thankful to our great university IIUM, for the support and the provided facilities that allowed me to carry out this study.

Finally, I gratefully acknowledge the continuous support and encouragement from my Mother, Father and family members and my love ina Moudaneh. I wish to express my appreciation and thanks to those who provided their time, effort and support for this project. To the members of my dissertation committee, thank you for sticking with me.

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TABLE OF CONTENTS

Abstract ... ii

Abstract in Arabic ... iii

Approval Page ... iv

Declaration ... v

Acknowledgements ... viii

Table of Contents ... ix

List of Tables ... xii

List of Figures ... xiii

List of Abbreviations ... xv

List of Symbols ... xvii

CHAPTER ONE: INTRODUCTION ... 1

1.1 Background ... 1

1.2 Problem Statement ... 4

1.3 Research Objectives... 5

1.4 Research Scope ... 5

1.5 Research Methodology ... 6

1.6 Dissertation Organization ... 7

CHAPTER TWO: LITERATURE REVIEW ... 8

2.1 Introduction... 8

2.2 Microbial Fuel Cell (MFC) ... 8

2.2.1 MFC Principle ... 8

2.3 Factors Affecting MFC Performance ... 10

2.3.1 Design ... 10

2.3.2 Electrode ... 11

2.3.3 Separator ... 14

2.3.4 Chemical Factor ... 15

2.3.5 Electrochemically Active Bacteria ... 18

2.4 Mechanism of Bio-Electrogenesis ... 21

2.4.1 Direct Electron Transfer ... 22

2.4.2 Indirect Electron Transfer (IET) ... 25

2.5 Factor Causing the Decrease of Voltage ... 26

2.5.1 Mass Transfer Limitation ... 26

2.5.2 Bacterial Limitation and Research Gap ... 26

2.5.3 Electron Transfer ... 27

2.5.4 Ohmic Resistance ... 27

2.6 Bacterial Dna Isolation ... 28

2.6.1 Dna Extraction ... 28

2.7 Bacteria Identification ... 29

2.7.1 Morphological Test ... 29

2.7.2 Biochemical Test... 30

2.7.3 Resistance Test and Immunological Methods ... 31

2.7.4 Molecular Test ... 31

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2.8 Bacteria Performance... 34

2.8.1 Mutation ... 34

2.9 Application of Mfc ... 37

2.9.1 Wastewater Treatment ... 37

2.9.2 Bioremediation ... 38

2.9.3 Nitrogen Removal ... 39

2.9.4 H₂ Production ... 40

2.9.5 Other Applications ... 41

2.10 Summary ... 41

CHAPTER THREE: MATERIALS AND METHOD ... 42

3.1 Introduction... 42

3.2 Materials ... 44

3.2.1 Raw Material ... 44

3.2.2 Chemicals and Reagent ... 44

3.2.3 Catholyte ... 44

3.2.4 Nutrient Agar & Luria-Bertani (LB) ... 44

3.3 MFC Device... 45

3.4 Reagent Preparation ... 45

3.4.1 50x TAE Preparation ... 46

3.4.2 Ethidium Bromide ... 46

3.4.3 Iodine Solution ... 46

3.4.4 Safranin Solution... 46

3.4.5 Crystal Violet ... 47

3.5 Experimental Procedure... 47

3.5.1 Isolation of Bacteria from the Anode ... 47

3.5.2 Media and Equipment Sterilization... 47

3.5.3 Screening of Electrochemical Bacteria ... 48

3.5.4 Morphology Characteristic: Gram Staining ... 48

3.5.5 Isolation and Identification ... 49

3.5.6 Bacterial Radiation ... 55

3.6 Analytical Method ... 56

3.6.1 Bacterial Growth ... 56

3.6.2 Power Measurement ... 56

3.6.3 DNA Quantification ... 57

3.6.4 One Factor At A Time Optimization of Gamma Radiation ... 57

3.6.5 Total Chemical Oxygen Demand (COD)... 58

3.7 Summary ... 59

CHAPTER FOUR: RESULTS AND DISCUSSION ... 60

4.1 Introduction... 60

4.2 Bioelectrogenesis ... 60

4.3 Screening of Electrochemical Bacteria ... 62

4.3.1 Polarization Study ... 63

4.3.2 Single vs Mixed Culture ... 64

4.3.3 Relation between Bacterial Growth and Electricity Generation ... 66

4.4 Bacterial Identification ... 67

4.4.1 Morphology Characters... 67

4.4.2 Molecular Identification ... 68

4.5 Optimization of Gamma Rays Dose: One Factor At A Time (OfAT) ... 71

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4.5.1 The Effect of Mutation ... 74

4.5.2 Electricity Performance ... 76

4.5.3 The Effect of COD Removal and Strain Relations ... 78

4.6 Summary ... 79

CHAPTER FIVE: CONCLUSIONS AND RECOMMENDATIONS ... 80

5.1 Conclusions ... 80

5.2 Main Objective of the Study ... 81

5.3 Recommendations... 81

REFERENCES ... 83

APPENDIX A ... 93

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LIST OF TABLES

Table 1.1 The effect of dissolved oxygen on the shrimp grows 2 Table 2.1 Comparison between different anodes in MFCs 11 Table 2.2 Different substrate used to generate MFC 16

Table 2.3 Comparison of mixed and pure culture 19

Table 3.1 Optimal guideline for amplification 51

Table 3.2 Protocol for a 25 µl reaction 52

Table 3.3 Agarose concentration in gel for separation of different range of linear DNA molecules (Ylmaz et al. 2012).

52 Table 4.1 The characteristics of the colony observed on nutrient agar

plate

68 Table 4.3 Experimental Data of OFAT with the result 72

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LIST OF FIGURES

Figure 2.1 Sketch of MFC System 9

Figure 2.2 Comparison of Oxygen and Potassium Ferricyanide as Electrolyte using Diluted Restaurant Wastewater

17

Figure 2.3 Pure and Mixed Culture in MFC over a Period of 30 Days 20 Figure 2.4 Mechanism of Electron Transfer; Extracellular Electron

Transfer (EET), Direct Electron Transfer (DET), Mediator Electron Transfer (MET), Indirect Electron Transfer (IET)

22

Figure 2.5 Model of Electron Flow along Microbial Nanowires; (A) Metal-Like Conductivity and (B) Electron Hopping or Tunneling

24

Figure 2.6 Polymerase Chain Reaction Principal 32

Figure 2.7 Variation trend of the number of papers on index journals 39 Figure 3.1 Flow Diagram of Experimental Design for Electricity

Generation

43 Figure 4.1 Power Density and Power of Mixed Cultures in Function of

Time

61

Figure 4.2 Electricity Production of Pure Culture 63

Figure 4.3 Polarization Curve of the best Pure culture 64

Figure 4.4 Comparison of Electricity Generation Between Mixed and Pure Culture

65 Figure 4.5 Electricity Generation over Bacterial Growth 66

Figure 4.6 Gram Staining Results 67

Figure 4.7 Colony Characteristics on Nutrient Agar Plate 68 Figure 4.8 PCR Product of AA6 Strain, Where M is The DNA Ladder,

-Ve: Negative Control; and 1: The 16s rRNA of AA6 Strain Genome

69

Figure 4.9 Alignment of Blast Query 71

Figure 4.10 Response Curve, A: Voltage over Dose, B: Time over Dose 73

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Figure 4.11 SEM image of Carbon Cloth Anode after 3 Days of Electricity production in the U-tube MFC; A: Control X1500, B: Bacillus Cereus Strain Cc-1, C: Mutant 25 Gy, D: Mutant 63 Gy, E: Mutant 100 Gy

75

Figure 4.12 Comparison of Cell Potential Between the three Mutant Strain and The Wild Strain of Bacillus Cereus Cc-1

76 Figure 4.14 Comparison of the Total COD Removal Between the Mixed

Culture, the Three Mutant of Strain Cc-1 And the Wild Strain of Cc-1

79

Figure A1 Photograph (A) and schematic diagram (B) of U shaped MFC reactor

94

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LIST OF ABBREVIATIONS

AEM Anion Exchange Membrane

BOD Biological Oxygen Demand

BudR 5- bromodeoxyuridine

CE Columbic Efficiency

CEM Cation Exchange Membrane

COD Chemical Oxygen Demand

DET Direct electron transfer

DO Dissolved Oxygen

DOE Design of Experiment

DMRB Dissimilatory Metal Reducing Bacteria

DNA Deoxynucleic acid

DTT Dithiothreitol

EAB Electrochemically Active Bacteria FAO Food Agriculture Organization FDNB Fluor dinitro benzene

IET Indirect Electron Transfer MDC Microbial Desalination Cell MET Mediator Electron Transfer MEC Microbial Electrolysis Cell

MFC Microbial Fuel Cell

MID Minimum Inducing Dose

NASA US National Aeronautic Space Administration

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NCBI National Center for Biotechnology Information

OD Optical Density

OFAT One-factor-at-a-time

PAH Polycyclic Aromatic Hydrocarbons

PBS Phosphate buffer solution

PCR Polymerase Chain Reaction

PEM Proton Exchange Membrane

POME Palm Oil Mill Effluent

RAS Recirculating Aquaculture System rRNA ribosomal Ribonucleic Acid

SPEEK/cSMM Sulfonated Poly(ether ether Ketone)/charged Surface Modifying Micro- Molecules

SEM Scanning Electron Microscopy

SDS Sodium Dodecyl Sulfate

TSS Total Suspended Solid

UV Ultraviolet light

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LIST OF SYMBOLS

°C Degree Celsius

% Percentage

Ω Ohm

$ US dollar

A Adenine

A₂₆₀ Absorption 260 nm

Am Current Density

Bp Base pairs

C Cytosine

Cm Centimeter

Cm² Centimeter square

e¯ Electron

G Guanine

G Gram

g/l Gram per liter

Gy Gray

Kb Kilobase

kDa Kilodalton

Ma Milliampere

mA/cm² Milliampere per centimeter square

Min Minute

mM Millimolar

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mL Milliliter

mV Millivolt

mW/cm² Milliwatt per centimeter square

nm Nanometer

pd Power density

Ppt Parts per thousand

Rpm Revolution per minute

T Thymine

Ton Tonne

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CHAPTER ONE INTRODUCTION

1.1 BACKGROUND

Industrial revolutions, the process of shifting from an agrarian and handicraft economy to one ruled by industry and manufacturing have changed the history of human lifestyle.

The development of the technology helps to make our daily lives easier, in different area such as medicine, transportation, communication and others, but at same time, we are facing the worst challenge that the world has ever experienced. Climate change, overpopulation, the appearance of gigantic megacities around the world causing a threat of water, food and energy. Therefore, to provide solution for the food shortage, polluted water sources and demand for clean energy, enormous efforts have been made and aquaculture is one of the solutions.

Aquaculture is the farming of aquatic animals (FAO 1990). It is a growing food producing sector, where fish and shellfish are farmed in an artificial pond. The benefit of an aquaculture is not only for the production of food but also diminish the impact on the wild stocks, and it is more economical (Kautsky et al. 2000). Farming seafood is very popular in the indo-pacific country such as Vietnam, Indonesia, and Malaysia.

Malaysia cultivated 87,202 ton of shrimp in 2010 (Chowdhury 2014). Culturing shrimp, the most consumed crustacean after fish in an aquaculture need to be well handled.

Shrimp growth requires good water quality at around pH 8, salinity 1.5% to 2.5% (15ppt to 25 ppt) and temperature (30-31ºC) and the an optimum dissolved oxygen of 4-7 mg/l (Kasnir 2014). Unfortunately, Widanarni et al. (2010) stated that the feed leftover and shrimp metabolic waste deteriorate the quality of the water in the pond. Moreover, dissolved oxygen in the pond is an extremely important factor that affect the growth

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and the health of shrimp (Table 1.1). Therefore, there is a need to use one kilowatt paddle-wheel in the artificial pond to increase the dissolved oxygen level.

Table 1.1The effect of dissolved oxygen on the shrimp grows (Re and Díaz 2011).

Dissolved oxygen (mg/l) Effects on shrimp

0.3 Shrimp die

1.0 Anoxia in Shrimp, it may die

2.0 Shrimp cannot grow up

3.0 Shrimp grows slowly

4.0 Shrimp cannot grow up

6 Shrimp grows healthy and rapidly

Furthermore, low water quality impacts the productivity of aquaculture. So, to maintain the quality of water, it is being discharged to the environment especially to the estuaries. Unfortunately, this practice depleted the environment where many investigation stated that the effluent from shrimp farming pond caused higher amount of chlorophyll a, dissolved oxygen (DO), biological oxygen demand (BOD), pH and salinity at the discharged site (Avnimelech 1999; Kautsky et al. 2000; Trott and Alongi 2000). Hence, more recent researchers expose closed recirculating aquaculture system (RAS), to eradicate the depletion of the environment and to maintain the quality of water in the pond (Krummenauer et al., 2014; Orellana et al., 2014). Unlike the old fashion, there is no water exchange in this system. In addition, the recirculation is carried out either by a complex and expensive water filtration, or by the integration of a secondary containment basin. The water in the secondary pond is purified by bio-flocs (Krummenauer et al. 2014), halophytic plant (Buhmann and Papenbrock 2013) or aquaponics (Martan 2008). Microbial fuel cell (MFC) can be used instead of bio-flocs, aquaponics, and others, because MFC is very cheap and address simultaneously energy and environmental problems (He et al. 2016).

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MFC is a popular technology which has a double shoot of benefit. It treats wastewater and simultaneously generates electricity from the waste. The phenomenon of bioelectricity was first observed by Italian physicist Luigi Galvani in 1790 (Piccolino 1997), but the principle that the production of electricity is associated with the microbial metabolism was first discovered by Michael C. Potter in 1911 (Potter 1911). Two decades later, Cohen reported a biological fuel cell device delivering 35 V and 2 mA confirming Potter’s observation (Schröder 2007). However, MFC gain its popularity when NASA (the US National Aeronautics and Space Administration) interested in harnessing the organic waste into electricity during long journey of space flights (Santoro et al. 2017). NASA’s interest in MFC was the ability of the electrochemical microbes capable of converting the chemical energy present in the wastewater (substrate) into electrical energy through the oxidation and reduction reaction. In an MFC, the biocatalyst is kept in the anode chamber filled by the substrate and separated from the electron acceptor (cathode) by a proton exchange membrane.

Further, many studies reported MFC using various substrates for electricity generation and wastewater treatment such as starch treatment (Chaturvedi and Verma 2014), swine waste (Min et al. 2005), domestic wastewater (Yu et al. 2012), sewage sludge (Jiang et al. 2009), and wastewater from brewery and leachate (Li et al. 2013;

Wen et al. 2010 respectively). Additionally, it has been reported that the organics of wastewater is considered as energy in an anaerobic condition (Feng et al. 2010). Where the bacterial growth yield in MFC is measured between 0.07–0.22 g of chemical oxygen demand (COD) biomass g⁻¹ COD substrate for a glucose-fed (Rabaey et al. 2004).

Unfortunately, MFC has low energy production (Kiely et al. 2010). Thus, to increase the energy output of this living battery, previous studies investigated on various factors

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such the electrode, membrane, substrate, configuration (Logan et al. 2006) but few have studies on the microorganism.

Hence, this study investigated the electrochemically active bacteria (EAB) isolated from a mix of water and mud collected from shrimp pond, followed by morphology and molecular identification of the EAB. The performance of this study has been compared to that reported by (Zuo et al. 2008) in which the same MFC reactor, carbon cloth and carbon fibers were used as an electrode. Then, to mutate the bacteria, low gamma irradiation dose was induced to the bacteria at Malaysia Nuclear Agency.

Thus, gamma irradiation induced mutation increased the ability of bacteria for electricity generation as well as organic matter removal.

1.2 PROBLEM STATEMENT

During last 3 decades, farming seafood in artificial ponds has exponentially increased due to the high demand of fish and shellfish on the market but also the rapid growth of aquatic animal which is more economical. Therefore, to maintain the quality of the water in the pond, adding antibiotic to kill the bad bacteria and fertilizer to grow microalgae was practiced. But, after a while, water in the pond losses its quality and need to be exchanged to maintain the growth of the seafood. Unfortunately, the growth of aquaculture seems to decrease recently because of the tough environmental sanction of water discharge to the estuaries. Henceforth, MFC is a promising solution because its electrochemically active bacteria that have the ability to remove the chemical oxygen demand (COD) as well as the Total Suspended Solid (TSS), Biological Oxygen Demand (BOD) and directly produce electricity.

Different substrates have been used to investigate the energy generation of MFC but so far none have tried the mud and water of shrimp culture pond. Therefore, this

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study explored the type of electrochemical in shrimp pond and its capacity to transfer electron as well as the wastewater treatment. Furthermore, to enhance the low electricity production of MFC previous studies reported the impact of various factor such as electrode, substrate, separator and configuration. But few have focused on the electrochemical microorganism which produces the electron from its metabolism pathways. Hence, the main focus of this research is to overcome the low electron transfer of the bacteria. Therefore, the active electrochemical bacteria were mutated by exposing gamma rays of nuclear radiation to increase the power density.

1.3 RESEARCH OBJECTIVES

Based on the current investigation of MFC, this research was inspired to develop or to increase the indigenous environmentally friendly energy and treat simultaneously wastewater from shrimp pond. The objectives of this research are:

1. To isolate electrochemical active bacteria from the anode of U-tube shape.

2. To identify the isolated bacteria through PCR of 16s rRNA and blast to the NCBI database sequencing

3. To mutate the identified bacteria to enhance the power output by gamma radiation.

1.4 RESEARCH SCOPE

The work of this research started with the construction of the U-tube MFC device and filling the anode and the cathode with the anolyte and catholyte respectively. After, the generation of the electricity was observed, the electrochemical bacteria present on the anode electrode were isolated and screened in order to the select the best strain producing the highest cell voltage. DNA of the selected bacteria were extracted in order

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to identify the strain’s identity. Therefore, PCR amplification have been used to amplify the 16s rRNA gene of the bacteria. Followed by sequencing the amplified sequence and then NCBI Database was used to find the analogy of the sequence. After completing the identification, one factor at a time (OFAT) was used where the radiation applied to the identified bacteria were optimized by varying gamma dose rays. Finally, the electricity generation and chemical oxygen demand of the radiated strains and the wild strain was compared.

1.5 RESEARCH METHODOLOGY

This study comprised of laboratory based experimental work in MFC device, and the procedures adopted were designed to increase the production of electricity from MFC using mud and water collected from shrimp pond. The research was debuted with the review of literature on the parameters affecting the microbial fuel cell such as the electrodes, substrate and configuration. It was followed by reviewing the various mode of electron transfer as well as the wide range of electrochemically active microorganism.

The experimental work started by collecting the substrate from Perak; Malaysia (SBH Marine Industries Sdn.Bhd) and constructing a U-tube MFC devices containing the electrode, membrane as well as the anolyte and catholyte. Henceforth, a mix culture of seven bacteria were isolated from the anode of the U-tube MFC, followed by screening and selecting the best bacteria that produce the highest electricity. The selected bacteria were identified using the 16s rRNA genes. Finally, the identified strain was radiated with gamma radiation. One factor at time (OFAT) of the Design of experiment (DOE) was used to optimize the radiation dose and to find the best dose that increase the electrochemical performance.

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DOKUMEN BERKAITAN

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