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研究生:范妮妮
研究生(外文):Vanita Roshan Nimje
論文名稱(外文):MICROBIAL FUEL CELL: SUBSTRATE EFFECTS AND MICROBIAL DYNAMICS FOR SUSTAINABLE BIOELECTRICITY GENERATION.
指導教授:陳建易陳浩仁
指導教授(外文):Chen, Chien-YenChen, Hau-Ren
口試委員:陳建易陳浩仁李文乾曾銘仁潘冠宇簡錦樹陳建成
口試委員(外文):Chen, Chien-YenChen, Hau-RenLee, Wen-ChienTseng, Min-JenPan, Kuan-YeuJean, Jiin-ShuhCheng, Chen-Chien
口試日期:2011-06-20
學位類別:博士
校院名稱:國立中正大學
系所名稱:分子生物研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:英文
論文頁數:308
外文關鍵詞:Microbial fuel cellWastewaterCyclic voltammetryBacillus subtilisNitrate reductionGlycerolShewanella oneidensis
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The Microbial Fuel Cell (MFC) is a new technology, in which microorganisms produce electricity from a renewable energy source in the form of biomass. MFCs are receiving increasing scientific and more recently commercial attention as their potential for alternative energy production, wastewater treatment and bioremediation of contaminated environments is steadily realized. In MFCs, bacteria convert chemical energy to electrical energy via the catalytic breakdown of organic substrates. The electrons are then transferred to a terminal electron acceptor (TEA- oxygen, nitrate, and sulfate) which is reduced by the electrons. As an emerging technology, MFCs shows great potential for the simultaneous generation of electricity and the treatment of wastewater.
However, this thesis aims to study the development of microbial fuel cell with emphasis on different bacterial cultures and utilization of different wastewater as a carbon/food source in MFC. To achieve this aim, different experiments has been conducted. Initial study deals with the establishment of double chamber MFC with Bacillus subtilis as a model organism. The Gram-positive aerobic bacterium B. subtilis has for the first time been employed in a double chambered MFC. A glucose-fed MFC with M9 minimal medium in the anode chamber was operated for 3 months. Despite its aerobic growth pattern, sustainable electricity generation was achieved with a maximum current of 0.4 mA. The simultaneous production of electricity while utilizing complex substrate, i.e. glycerol was examined for a continuous period of around 26 days. Results suggests that glycerol, a byproduct of transesterification process, can be used as a suitable carbon source for electricity generation in MFC. Maximum current generation of 0.5 mA was primarily attributed to the consumption of glycerol by the bacteria attached to the anode. While, B. subtilis achieved highest glycerol degradation efficiency at neutral pH of 7. Due to the aerobic growth physiology of B. subtilis, we developed a MFC in which microorganisms at the cathode performed a nitrate reduction by using electrons supplied by microorganisms oxidizing glucose at the anode. Electricity generation with simultaneous nitrate reduction in a single-chamber MFC without air cathode was studied, using glucose (1 mM) as the carbon source and nitrate (1 mM) as the final electron acceptor employed by Bacillus subtilis under anaerobic conditions. A power density of 0.0019 mW/cm2 was achieved at an Rext of 220 . Nitrate as the terminal electron acceptor was found to be successfully achieved in the single chamber MFC without utilization of oxygen. This results demonstrated that high-efficiency electricity generation is possible from wastewater containing nitrate, and this represents an alternative technology for the cost-effective and environmentally benign treatment of wastewater.
Enterobacter cloacae, hydrogen gas producing bacterium was also evaluated to study the performance of double chamber MFC in to variations in anodic pH microenvironment based on current generation, maximum power density, electrochemical losses, internal resistance, electrochemical activity, COD removal, and coulombic efficiency. Considering pH as an important parameter for microbial growth and physiology to produce electrons in MFC, community wastewater as a substrate was utilized by adjusting the pH between 6.5 and 9.5. Polarization and power curves obtained with respect to individual pH, deduced the maximum current, power density, internal resistance and electrochemical losses. Maximum power density obtained from the polarization curve was observed of 0.0042 mW/cm2 for pH 7.4. The excellent performance of MFC at pH 7.4 and 6.5 indicates the efficacy of higher dehydrogenase activity of E. cloacae to served effectively at near neutral pHs. However, these results demonstrate the influence of acidic to alkaline wastewater on the current generation and wastewater treatment by E. cloacae.
Shewanella oneidensis as a most electroactive bacterium to harvest electron from different substrates was selected to utilize in the MFC. We demonstrated electricity production by agriculture (AWW), wastewater, domestic wastewater (DWW), paper wastewater (PWW) and food/dairy (FDWW) wastewater. Current generation were evaluated and compared in combination with three inoculums: wastewater endogenous microbes (MFC1), S. oneidensis (MR-1) (MFC2), and wastewater endogenous microbes with MR-1 (MFC3) in a single chamber microbial fuel cell (MFC). All the inoculums studied, varied differently to produce current output with and without the presence of S. oneidensis.
Overall results depicts the feasibility of utilizing different substrates i.e., organic substrate like glucose and wastewaters and myriads of bacteria to generate electricity in MFC.

PREFACE ………………………………………..…………………………...……..i
ABSTRACT….…………………………………………………………...……...…iii
ACKNOWLEDGEMENTS……………………………………………….……....vii
LIST OF FIGURES …….…………………………………………………….....xviii
LIST OF SCHEMES …….………………………...…………………………....xxiii
LIST OF TABLES ……………………………………………………………....xxiv
ABBREVIATIONS………………………………………………………….…. xxiv


CHAPTER 1: INTRODUCTION AND AIM

1.1 Energy consumption is linked to income………...……….…………..1
1.2 Energy: sources, consumption & efficiency …………………………3
1.3 The damage fossil fuels can do.……………...………………………7
1.4 Renewable energy sources..…..…………..………………………..…8
1.4.1 Biomass: an important renewable energy source …………..10
1.4.2 Biomass to energy conversion: Alternatives……….…..…...13
1.5 Aim of the thesis…..………………….…………...……………..…16
1.5.1 Description of chapters…..………………...……………..…17

CHAPTER 2: FUEL CELLS: BACKGROUND

2.1 History ……………………………………………...………………21
2.2 Types…………...…..……………………………………………….22
2.3 Microbial Fuel Cell: Bacteria-based renewable
electricity production……………………………………………….25
2.3.1 Overview…………………………………...……………….25
2.3.2 Biochemical basis …….…………………………………….28
2.3.3 Microbial Fuel cell design ………………………….………30
2.3.3.1 Double chambered microbial fuel cells............…30
2.3.3.2 Single chambered microbial fuel cells…..........…31
2.3.3.3 Anode compartment.………………….……....…32
2.3.3.4 Microbial cultures……………………..……...…33
2.3.3.5 Redox mediators and Electron transfer…...…..…34
2.3.3.6 Cathode compartment….….………….……....…36
2.3.3.7 Exchange membrane. ……..………….……....…37
2.4 Evaluation and performance of MFC ……….……………………...38
2.4.1 Polarization and power density curves………………....…...39
2.4.2 Chemical oxygen demand and coulombic efficiency ….…...43
2.4.3 Cyclic voltammetry.……………………………………..….44

CHAPTER 3: MATERIALS AND METHODS

3.1 Experimental details ..…………………………………..……………50
3.1.1 MFC setup ….………………………………………………50
3.1.2 Microorganism and culture conditions …..…………..……..51
3.1.3 Data acquisition, electrochemical technique
and calculations ……………….…………….……………..52
3.1.4 Cyclic voltammetry ………………………….……..………54
3.1.5 Scanning electron microscopy (SEM)….……..…………….55
3.2 Experimental details ………...….………………………..…..………56
3.2.1 MFC setup and operation…………….….…………….……56
3.2.2 Microorganism and culture conditions……….…………..…57
3.2.3 Data acquisition, electrochemical
technique and calculations…………………………………..58
3.2.4 Cyclic voltammetry ………………………….……..………59
3.3 Experimental details ………...….…………………………..…….…60
3.3.1 MFC setup ……………..…………….….…………….……60
3.3.2 Microorganism and culture conditions and substrate….....…61
3.3.3 Analytics……………………………………………….……62
3.3.4 Experimental performance…………………………………..62
3.3.5 Data acquisition, electrochemical
technique and calculations………….……………………….63
3.3.6 Cyclic voltammetry ………………………….……..………65
3.4 Experimental details ………...….…………………………...…….…65
3.4.1 MFC setup ……………..…………….….…………….……65
3.4.2 Cultivation of bacteria…………………………………....…66
3.4.3 Nitrate measurement..………………………………….……66
3.4.4 Experimental performance…………………………………..62
3.4.5 Data acquisition, electrochemical
technique and calculations………….……………………….67
3.4.6 Cyclic voltammetry ………………………….……..………68
3.5 Experimental details..………...….…………………………..…….…69
3.5.1 MFC setup.……………..…………….….…………….……69
3.5.2 Microorganism and culture conditions and substrate…....…69
3.5.3 Substrates, their sources and analysis
Of various parameters………………………………….……72
3.5.4 Data acquisition, electrochemical
technique and calculations………….……………………….72
3.5.5 Cyclic voltammetry.………………………….……..………73


CHAPTER 4: STABLE AND HIGH ENERGY GENERATION BY PURE STRAIN OF BACILLUS SUBTILIS IN MICROBIAL FUEL CELL

4.1 Introduction…………………………………………………………76
4.2 Results and discussion ……..……….………………………………80
4.2.1 Stable and long-term power generation…………………….80
4.2.2 Polarization curve……..…….………………………………83
4.2.3 Cyclic voltammograms…………………….…………..……85
4.2.3.1 Electrochemical activity of the bacterial
growth phase………….....……...…………...………85
4.2.3.2 Verifcation of soluble redox compounds
secreted by bacteria…………………....…….………89
4.2.3.3 Electrochemical behavior of the biofilm anode….….91
4.3 Ribosomal Intergenic Spacer Analysis of Bacillus subtilis…………92
4.4 Conclusion……………………………………….………….………93

CHAPTER 5: GLYCEROL DEGRADATION IN SINGLE-CHAMBER MICROBIAL FUEL CELLS

5.1 Introduction……………………………………………….…………96
5.2 Results and discussion ……………………………………...….….100
5.2.1 MFC acclimation and maximum current generation…..….100
5.2.2 Performance evaluation of the MFC from polarization
curves obtained in different cycles…………………….…..103
5.2.3 pH and its effect on current output………………....….….107
5.2.4 Electrochemical activity in relation to pH change…….......109
5.2.5 Electrochemical activity and electron
transfer mechanisms……………………………………….112
5.2.6 Performance of MFC based on substrate
conversion and coulombic efficiency…..……………...…..115
5.3 Conclusion………………………………………………….....…..116






CHAPTER 6: EFFECT OF ANODIC PH MICROENVIRONMENT ON CURRENT, POWER DENSITY, INTERNAL RESISTANCE AND ELECTROCHEMICAL LOSSES IN MICROBIAL FUEL CELL OF ENTEROBACTER CLOACAE

6.1 Introduction………………...………………………..……….…….120
6.2 Results and discussion…………………………………….……….124
6.2.1 Effect of different anodic pH on current generation……....124
6.2.2 Effects of pH on Internal Resistance………………….…...128
6.2.3 Evaluation of MFC performance…………………….…….131
6.2.4 Electrochemical evaluation of MFC……………………....135
6.2.5 E cloacae wastewater organic matter removal
and coulombic efficiency……………………………..……137
6.3 Conclusion……………………………………………………...….…..138

CHAPTER 7: SIMULTANEOUS NITRATE REDUCTION AND ELECTRICITY GENERATION IN A SINGLE-CHAMBER MICROBIAL FUEL CELL WITHOUT AN AIR CATHODE

7.1 Introduction………………………….……………………….…….142
7.2 Results and discussion……………………………….………….…146
7.2.1 Nitrate respiration and current generation…………...…....146
7.2.2 Effect of nitrate addition in different batch cycles
and its relationship with current generation and
nitrate reduction………………………………………..…..148
7.2.3 Polarization curve…………………………………....…….151
7.2.4 Electrochemical activity as a result of change
in electron acceptor …………………………………….…153
7.2.5 Electrochemical activity during different fed batch
cycles and electron transfer mechanisms…………………..155
7.2.6 Performance of the MFC at the end of the batch
test ………………...…………………………………….…158
7.2.7 Coulombic efficiency……………..……………………..…159
7.3 Conclusion…………………………….……………………….…..160

CHAPTER 8: COMPARATIVE BIOELECTRICITY PRODUCTION FROM VARIOUS WASTEWATERS IN MICROBIAL FUEL CELLS USING MIXED CULTURES AND A PURE STRAIN OF SHEWANELLA ONEIDENSIS

8.1 Introduction……………………………………….………….…….164
8.2 Results and discussion……………………………………….…….169
8.2.1 Utilization of different wastewaters and
microbial inoculums for current generation ……….….......169
8.3 Conclusion………………………………………………….……...…..195

CHAPTER 9: CONCLUSIONS
9.1 Brief conclusion……………………………….…….…………….197

REFERENCES

APPENDICES

APPENDIX -A

Supplementary material for Ribosomal Intergenic Spacer
Analysis (RISA) analysis of Bacillus subtilis………………………….……….….221



APPENDIX -B

Supplementary material for volatile fatty acid determination…………………………………………...……..……….………….226

APPENDIX -C

Supplementary material for nitrate determination……………….………………...232

APPENDIX -D

CHAPTER 10: MICROBIAL INDUCED CALCIUM CARBONATE PRECIPITATION AND CRYSTAL MORPHOLGY IN MONOCULTURE AND BINARY CULTURE EXPERIMENTS

10.1 Literature review…………………………….……….…………….238
10.1.1 Microbial induced precipitation
of carbonates (MICP)………………………………….….239
10.1.2 MICP by urea hydrolysis………………………………….242
10.1.3 Biogrout process, its requirements and limitations……….243
10.1.4 Objective of this project…………………………………...245
10.2 Introduction ………………………………………………..……...246
10.3 Experimental details ……………………………………...……….249
10.3.1 Microorganisms culture conditions ………………..……...249
10.3.2 Optical density ………………………………….…...…….249
10.3.3 Column Parameters and Sampling……………………...…250
10.3.4 Monitoring Methods …………………………………...….251
10.3.4.1 Conductivity as a measure of urease activity….251
10.3.4.2 Conversion of conductivity (mS) to
urea hydrolyzed (mM)…………………...…….252
10.3.4.3 Ammonium determination……………..….…..254
10.3.4.4 Calcium concentration……………………...….256
10.3.4.5 Calcium carbonate content…………………….258
10.3.4.6 Flushed Volume…………………..………...….259
10.3.4.7 Analysis of crystal properties……………….....259
10.4 Results and Discussion…………………………………….………260
10.4.1 Optical density and urease activity……………….………..261
10.4.2 Solution chemistry……………………………….………...263
10.4.3 CaCO3 profile along the column………………….….…….269
10.4.4 Comparison of CaCO3 crystal morphology
induced by biological factors……………….…………..….270
10.4.5 Comparison of XRD patterns among monoculture
and biculture experiments……………………………...….274
10.5 Conclusion………………………………………….……….……..277

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