studies on microbial electrochemical cells using...
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Studies on Microbial Electrochemical Cells
Using Different Anode Respiring Bacteria
By
Qaiser Farid Khan
Department of Microbiology
Faculty of Biological Sciences
Quaid-I-Azam University
Islamabad, Pakistan
2016
Studies on Microbial Electrochemical Cells
Using Different Anode Respiring Bacteria
A thesis submitted in partial fulfillment of the requirements for
the degree of
DOCTOR of PHILOSOPHY
In
Microbiology
By
Qaiser Farid Khan
Department of Microbiology
Faculty of Biological Sciences
Quaid-I-Azam University
Islamabad, Pakistan
2016
IN THE NAME OF ALLAH, THE MOST BENEFICIENT, THE MOST MERCIFUL
To my Mother, who has loved and taken
care of me for all my life. I attribute all my
success in life to the moral and intellectual
education I received from her.
Declaration
The research work presented in this thesis is my original work conducted at Quaid-I-
Azam University, Islamabad, Pakistan and Swette Center for Environmental
Biotechnology, The biodesign institute at Arizona State University, USA. It is part
of US funded project by Office of Naval Research (grant N000141210344) and NSF
CAREER (Award 1053939). Additional support was provided by Higher Education
commission of Pakistan (HEC) under IRSIP program. I have not previously
presented any part of this work elsewhere for any other degree.
Qaiser Farid Khan (E-mail: [email protected])
LIST OF CONTENTS Page
LIST OF FIGURES…………………………………………………... …………....i
LIST OF TABLES..…………………………………………………... …………....iii
LIST OF APENDIX………………………………………………………………...iv
LIST OF ABBREVIATIONS ………………………………………………………v
ACKNOWLEDGMET……………………………………………………………..vi
ABSTRACT………………………………………………………………………...ix
CHAPTER 1
INTRODUCTION………………………………………………………………... 1
1.1 Background…………………………………………………………….. 2
1.1.1. Pakistan’s Energy Crisis……………………………………...2
1.1.2 Renewable Energy…………………………………………… 3
1.2 Microbial Electrochemical Cells………………………………………. 3
1.2.1 Anode Respiring Bacteria……………………………………. 6
1.2.2 Electron Transfer Mechanisms……………………………..... 6
1.2.3 Substrate Range……………………………………………… 6
1.3. Applications…………………………………………………………… 7
1.3.1 Remote Sensors………………………………………………. 7
1.3.2 Bioremediation……………………………………………….. 7
1.3.3 Valuable End Products……………………………………….. 8
1.3.4 Hydrogen Gas Production……………………………………. 8
1.3.5 Medical Devices Powered by MXCs………………………… 9
1.4 Current Affair and on Ongoing Research………………………………..9
1.5 Research Objectives…………………………………………………… 10
CHAPTER 2
REVIEW OF LITERATURE…………………………………………………… 11
2.1 Thermophilic Anode Respiring bacteria (ARB)……………………….. 13
2.1.1 Substrate Selection…………………………………………….14
2.1.2 Temperature Range……………………………………………14
2.1.3 Thermophilic MXC operation with Single Strain ARB……… 14
2.1.4 Thermophilic MXC operation using bacterial consortia………16
2.2 Enrichment of mesophilic ARB from diverse natural environments……17
2.2.1 Substrate selection for mesophilic ARB……………………… 17
2.2.2 Inoculum sources to enrich electroactive anodic biofilms…… 17
2.2.2.1 Aquatic Sediments…………………………………. 18
2.2.2.2 Water Sources……………………………………… 19
2.2.2.3. Garden Compost…………………………………… 20
2.2.3 Enriched Communities of ARB………………………………..20
2.2.4 Characterization tools to study enriched ARB on anode…….. 21
CHAPTER 3
CHARACTERIZATION OF THERMOPHILIC BACTERIUM
THERMOANAEROBACTER PSEUDETHANOLICUS FOR
ELECTRICAL CURRENT GENERATION USING MICROBIAL
ELECTROCHEMICAL CELLS………………………………………………... 24
3.1 Introduction…………………………………………………………….. 25
3.2 Materials and Methods…………………………………………………. 27
3.3 Results………………………………………………………………….. 34
3.4 Discussion……………………………………………………………… 52
3.4 Conclusions…………………………………………………………….. 55
CHAPTER 4
MICROBIAL DIVERSITY AND METAGENOME PREDICTION
APPROACHES TO CHARACTERIZE ELECTROCHEMICALLY
ACTIVE BIOFILMS DERIVED FROM DIFFERENT INOCULA
IN SINGLE-CHAMBER MICROBIAL ELECTROLYSIS
CELLS…………………………………………………………………………….. 57
4.1 Background…………………………………………………………….. 58
4.2 Materials and Methods…………………………………………………. 60
4.3 Results………………………………………………………………….. 66
4.4 Discussion……………………………………………………………….83
4.5 Conclusions……………………………………………………………...87
RECOMMENDATIONS FOR FUTURE WORK………………………………… 88
REFERENCES…………………………………………………………………….. 90
APPENDIX……………………………………………………………………….. 113
LIST OF PUBLICATIONS………………………………………………………... 119
i
List of Figures
Figure Page
1.1 Schematic illustration of dual-chamber microbial electrochemical
Cell (MXC) 5
3.1 Illustration of experimental setup for microbial electrochemical cell 29
3.2 Schematic illustration of spike experiments performed to evaluate
Current generation capabilities of T. pseudethanolicus using acetate 31
3.3 Thermophilic MXC reactor showing mature T. pseudethanolicus
Biofilm on anode surface 33
3.4 Initial growth phase in the xylose fed MEC operated in batch mode
Containing Thermophilic Thermoanaerobacter Pseudethanolicus 35
3.5a Xylose reactor CV at 1mV-sec
with observed data (blue) and
Nernst-monod fit at n = 1.0 35
3.5b Derivative of the LSCV at 1 mV s-1
for the xylose-fed electrochemical
Cell 36
3.6 Effect of pH on current density normalized to the maximum value
Of 2.7 A m-2
37
3.7a Current production by batch microbial electrochemical cell (MXC)
Using xylose inoculated with thermophile T. pseudethanolicus
Operated for ~77 days at 60ºC 39
3.7b Results for the xylose-fed electrochemical cell operated in batch
For ~66 days containing T. pseudethanolicus biofilm 40
3.8a Current production by batch microbial electrochemical cell (MXC)
Using glucose inoculated with thermophile T. pseudethanolicus
Operated for ~48 days at 60ºC 42
3.8b Results for the glucose-fed electrochemical cell operated in batch
For ~55 days containing T. pseudethanolicus biofilm 43
3.9a Current production by batch microbial electrochemicalcell (MXC)
Using cellobiose inoculated with thermophile T. pseudethanolicus
Operated for ~82 days at 60ºC 45
3.9b Results for the cellobiose-fed electrochemical cell operated in batch
for ~11 days T. pseudethanolicus biofilm 46
ii
3.10 Current increase shown after 10 mM acetate was spiked into a
xylose-fed reactor containing T. pseudethanolicus that had reached
a current density of 0.1 A m-2 48
3.11 SEM images of an anodic biofilm of Thermoanaerobacter
Pseudethanolicus grown on 40-mM xylose 50
3.12 Representative CLSM images for a LIVE/DEAD assay for the
Anode biofilm Thermoanaerobacter pseudethanolicus grown
On 40mM xylose 51
4.1 Illustration of single chamber microbial electrolysis cell (MEC)
Used to enrich electroactive anode biofilms (AEM) 61
4.2 Schematic illustration of experimental set-up used for enrichment
Of ARB from diverse environments 63
4.3 Current density profiles of sediment sample collected from BSDL 67
4.4 Current density profiles of sediment sample collected from BOHW 69
4.5 Current density profiles of sediment sample collected from BOHL 71
4.6 Cyclic voltammetry analysis of biofilms in acetate-fed MEC
Containing different sediment samples 74
4.7 Phylotype distribution of sediments and enriched biofilms from
Diverse environments at the class level 78
4.8 PCoA analysis of environmental and biofilm enriched bacterial
Communities 80
4.9 PICRUSt predictions of genes that code for enzymes involved in
Cellular and energy processes 82
iii
List of Tables
Table Page
Table 4.1 Bacterial diversity indices on anodic biofilms with different
sediments samples 76
iv
List of Appendix
Appendix Page
S1 Carbon and energy metabolism of Thermoanaerobacter strains 113
S2 Initial growth of current density of the xylose-fed electrochemical cell 114
S3 A reactor fed with 10 mM acetate operated in batch for 13 days
Containing T. pseudethanolicus biofilm showed no significant
Current density 115
S4 Fraction of electrons captured as current, acetate, lactate and initial
Substrate are shown as a percentage of the total electrons present in
The initial substrate (xylose) 116
S5 Fraction of electrons captured as current, acetate, lactate and initial
Substrate are shown as a percentage of the total electrons present in the
Initial substrate (Glucose) 117
S6 Fraction of electrons captured as current, acetate, lactate and initial
Substrate are shown as a percentage of the total electrons present in the
Initial substrate 118
v
List of Abbreviations
AFM Atomic Force Microscope
ARB Anode Respiring Bacteria
BES Bioelectrochemical Systems
BOHW Biofilm Ohio Wetland
BOHL Biofilm Ohio Lake
BSDL Biofilm Scottsdale Lake
CA Chronoamperometry
CE Coulombic Efficiency
CLSM Confocal Laser Scanning Microscopy
CV Cyclic Voltammetry
DET Direct Electron Transfer
DGGE Denaturing gradient Gel Electrophoresis
DNA Deoxyribonucleic acid
EAB Electro active anode biofilms
EET Extracellular Electron Transfer
E-In Enriched Inocula
EKA Midpoint Potential
ETC Electron Transport Chain
FISH Fluorescence in situ hybridization
J Current Density
LSCV Low Scan Cyclic Voltammetry
MEC Microbial Electrolysis Cell
MFC Microbial Fuel Cell
MXC Microbial Electrochemical Cell
OTUs Operational Taxonomic Units
PCR Polymerase Chain Reaction
PCoA Principal coordinate analysis
PICRUSt Phylogenetic Investigation of Communities by Reconstruction of
Unobserved States
QIIME Quantitative Insights into Microbial Ecology
qPCR Quantitative Polymerase Chain Reaction
RT-qPCR Quantitative reverse transcription PCR
rRNA Ribosomal Ribonucleic Acid
SIP Stable Isotope Probing
SEM Scanning Electron Microscopy
SOHW Soil Ohio Wetland
SOHL Soil Ohio Lake
SSDL Soil Scottsdale Lake
TEM Transmission Electron Microscopy
T-RFLP Terminal Restriction Fragment Length Polymorphism
vi
Acknowledgment
All praises to almighty ALLAH, whose blessings and love enabled me to stand on my
feet and yield higher ideas in life and blessed me an opportunity to stay on the way of
knowledge to seek my recognition in universe. He helped me to complete this
research work and thesis also the great blessings to His Prophet Muhammad (PBUH)
possessing not only such an immaculate personality who builds the height of standard
of character for all over the world but guided His Ummah to seek knowledge from
cradle to grave.
I would like to thank Chairperson Dr. Fariha Hasan, Department of
Microbiology, Quaid-i-Azam University, Islamabad, Pakistan for her support, ever
inspiring attitude and help towards postgraduate affairs.
My special appreciation goes to my supervisor, Professor Dr. Abdul Hameed,
Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan for
supervision and constant support. I am thankful to Dr. Naeem Ali, for his whole-
hearted help, trust, provoking guidance, invaluable suggestions and constructive
criticism. His invaluable help of constructive comments and suggestions throughout
the experimental and thesis work have contributed to the success of this research.
An overwhelming thank you goes to my foreign research advisors, Dr. Bruce
E. Rittmann and Dr. Cesar I. Torres, Swette Center for Environmental Biotechnology,
The Biodesign Institute, Arizona State University, USA, for their support and
guidance during research work. I feel very fortunate to have them as my foreign
advisors, to have had the opportunity to work with them. I am thankful to Dr. Prathap
Parameswaran, my mentor, who not only was a fantastic scientist but the most
gentlemanly scientist, I have ever met. Additionally, Dr. Bradley G. Lusk and Dr.
Esra Ilhan and Dr. Joe Miceli not only have you been my lab mate, but also wonderful
friends. I cannot thank you enough for all of your advice, kind words, and strength.
I have shared so many wonderful memories, laughs, tears, coffee breaks, with
Sudeep Popat, Megha, Daniel Masters, Burcu Yavuz, Sara Bermudez, Ornella, Diana,
vii
Devyn, Esra AL, Laura Rango, Nancy Meyer, Mia, Mohsin Bashir, Tariq Sheikh and
Waqas Ellahi that I will always laugh when I think about some of our conversations. I
am thankful would also like to thank Diane Hanger, my lab manager at SCEB, ASU
for her motherly behavior and support during my research work.
I also am fortunate to have a core group of friends from different parts of my
life. These friends have structured discrete stages of my life and as such kept me out
of trouble as well as gotten me into trouble. The impact of these friendships continues
today, nurturing and encouraging me every day. Zeeshan Ahmed, Pervez Ali, Ghulam
Mujtaba, Umair Seemab, Sulaiman Nawaz and Arsalan Khalid: I am inspired and
defined by you.
It is rare that I find people that I can share so much of myself with. At
Washington State University, USA I found two such people, Vi Tran and Afshin
Khan and Koey Kay Wong, whom I can share the spectrum of myself with from
scientific details to nuisances of life. Thank you for every moment and all of your
support. I look forward to a lifetime of sharing adventures, science, and laughs.
Certainly, this journey was not easy, through struggles and successes my
fellow group members. Thus, I would like to extend my gratitude to my group
members: Sadaf Dar, Lubna Tahir, Iffat Naz, Muttiullah, Farhan Chishti, Mohsin
Barq. Also special thanks to Zeeshan Khan, Junaid Yasin, Awais Rasheed, Hamid
Majeed and Jabir Shah, for their moral support. I also offer words of thanks to lab
attendants, technical and clerical staff for their friendly attitude, support and presence
all the time.
I am grateful to Higher Education commission of Pakistan for financial
support under Indigenous PhD Fellowship and IRSIP program to conduct research
abroad. I am also thankful to SCEB at Biodesign Institute, Arizona State University,
USA for additional funds.
Last but not least, my deepest gratitude goes to my beloved parents without
their love, kindness, prayers and support I wouldn’t be able to complete this degree. I
am also grateful for their unconditional love and support wherever I was; I am
viii
thankful to my brothers, for advising me, showing me the path towards success, and
for their patience. My special love and prayers are extended to my sweet nieces
specially Hania whom I miss a lot during my studies.
Qaiser Farid Khan
ix
Abstract
Microbial electrochemical cell (MXC) technology is a source of sustainable
energy which comes from microorganisms. Recent advances in the fields of
electromicrobiology and electrochemistry with focus on microbial electrolysis cells
(MECs) has earned this technology its name as alternate “green energy”. Despite
advances, this technology is still facing challenges to address low power and current
density output.
Thermoanaerobacter pseudethanolicus 39E (ATCC 33223), a thermophilic,
Fe(III)-reducing, and fermentative bacterium, was evaluated for its ability to produce
current from four electron donors xylose, glucose, cellobiose, and acetate with a fixed
anode potential (+ 0.042 V vs SHE) in a microbial electrochemical cell (MXC).
Under thermophilic conditions (60 °C), T. pseudethanolicus produced high current
densities from xylose (5.8 ± 2.4 Am−2
), glucose (4.3 ± 1.9 A m−2
), and cellobiose (5.2
± 1.6 A m−2
). It produced insignificant current when grown with acetate, but
consumed the acetate produced from sugar fermentation to produce electrical current.
Low-scan cyclic voltammetry (LSCV) revealed a sigmoidal response with a midpoint
potential of −0.17 V vs SHE. Coulombic efficiency (CE) varied by electron donor,
with xylose at 34.8% ± 0.7%, glucose at 65.3% ± 1.0%, and cellobiose at 27.7% ±
1.5%. Anode respiration was sustained over a pH range of 5.4−8.3, with higher
current densities observed at alkaline pH values. Scanning electron microscopy
showed a well-developed biofilm of T. pseudethanolicus on the anode, and confocal
laser scanning microscopy demonstrated a maximum biofilm thickness (Lf) greater
than ~150 μm for the glucose-fed biofilm.
Microbial electrochemical cells (MXCs) are devices powered by
microorganisms to generate electricity via oxidation of organic substrates. It is critical
to understand the significance of sediment inocula in forming anodic biofilms to
improve MEC performance. Five environmental samples were evaluated for electrical
current production using acetate-fed microbial electrolysis cells (MECs). Three of
these samples were able to produce significant current densities ranging between 3 to
6.3 Am-2
. 16S rDNA targeted deep sequencing comparisons of anodic biofilms and
sediment bacterial community structures revealed significant differences in bacterial
community structures. Bacterial community producing the highest current density
x
after enrichment was dominated by the class Bacteroidia, δ-proteobacteria and
Erysipelotrichi. Comparison of phylogenetic information of bacterial communities
with 7 previously reported enriched samples by reconstruction of unobserved states
(PICRUSt) analysis clearly distinguished the biofilm communities from the sediment
inocula in terms of higher abundance of genes related to anode respiration. Principal
Coordinate Analysis (PCoA) also indicated that the clustering of biofilm communities
was in accordance with the predominant genera in each sample, such as Geobacter
dominating one cluster of biofilms. All the sediments formed a single cluster, which
included the Carolina mangrove biofilm community which showed only minor
changes from its originating sediment community after enrichment. Predominantly,
high current densities are associated with the enrichment of a few microorganisms,
often within a single family; however, this organism can be different depending on the
inoculum source. Because the selective enrichment selects for just a few bacteria, the
biofilm community is significantly different from that of the sediment. While δ-
proteobacteria (or the family Geobacteraceae) is dominant in many samples
producing high current densities, other samples show communities with yet
unidentified ARB as the major fraction.
1
CHAPTER 1
General Introduction
2
1.1 Background
Currently, the population of the world is about 7.3 billion and it is expected to
rise 10 billion, with global energy demands expected to grow by 160 % from 60% by
the year 2050. Concerns regarding provision of adequate energy and clean water
supply have been envisaged in the energy and water sectors worldwide. This leads to
search for sustainable supply of renewable energy.
In recent years urbanization and increased fossil fuel burning have resulted in
high CO2 emission thereby causing global warming. The amount at which radiation
escapes earth’s atmosphere depends on the concentration of the green house gases
emissions. The earth’s biosphere has an average increase of 0.6°C ±0.2°C in
temperature over the last 100 years, and these rates of temperature increase may have
disastrous consequences in the future. Typically, the long term effects of CO2
emission may cause, ozone depletion, rise in sea level, abrupt changes in weather
patterns thereby effecting agriculture, natural aquatic and terrestrial ecosystems.
According to annual energy outlook (AEO)2014, an average increase of 0.9%/year in
energy related CO2 emissions is seen from 1980 to 2005, followed by decline of
0.2%/year from 2005 to 2040, possibly due to use of environment friendly alternate
energy resources.
1.1.1. Pakistan’s energy crisis
According to experts, energy crisis in Pakistan is the largest single factor
affecting economy, shaving off almost 2% of the annual domestic growth (EAW,
2013). The rise in economic growth in the past couple of years has raised demand for
energy, but no important steps have been taken to explore alternate energy sources.
Due to which demand exceeds supply and hence power outage can been seen
throughout the Pakistan (Noor ul Haq and Hussain, 2008). The gap between demand
and supply of energy is not the only concern that we are facing now. Problems like;
resource depletion, environmental pollution, climate change and lack of research for
alternate energy sources are seriously needed to be addressed to ensure eco-friendly
environment by using green energy (Bilgen et al. 2004).
3
1.1.2. Renewable Energy
Renewable energy is generated from natural resources, e.g., water, sunlight,
wind and rain. Global energy consumptions are summarized as; renewable energy
(16%), biomass (10%), and hydroelectricity (4%). However, recent renewable form of
energy from plant residue accounts 2.8% and is growing very rapidly (REN21, 2011).
Geographically Pakistan is blessed with variety of renewable energy sources viz.
hydel, solar, wind and bio-fuel. If these natural resources explored, exploited and
developed properly, they can significantly contribute to country’s economy.
Pakistan’s urban areas generate over 55000 ton of solid waste on daily basis (Pakistan
economic survey, 2010-11), however which at some point contributes to air, water
and soil pollution. If this waste can be handled properly, it could be converted to
biogas (16 million m3/day) and biofertilizer (21 million ton) (Absar Saleh, 2012).
Opportunity to convert organic waste into energy is in focus all over the world. Using
novel technologies such as microbial electrochemical cells (MXCs), where organic
waste can be treated and converted into electrical energy benefiting both environment
and community.
1.2. Microbial electrochemical cells (MXCs)
The concept of microbial electrochemical cells (MXCs) dates back to 1910,
when Potter (1910; 1911) reported electrical current generation by bacteria. Decade
ago scientists around the world started exploring electrochemical behavior of
microbes and now it’s a rapidly growing environmental technology in connection to
clean water and renewable energy (Logan and Rabaey, 2012; Torres et al., 2010).
MXC research is combination of new interdisciplinary fields which includes applied
microbiology, electrochemistry, materials science, electrical and chemical engineering
along-with other related areas.
Microbial electrochemical cell (MXC) is a promising device for electrical
current generation through chemical reactions. The basic concept in it is to use
bacteria for oxidation of biodegradable organic/inorganic compounds in the anode
compartment releasing electrons to the anode. Protons produced in anode
compartment are transfered to cathode comportment via conductive membrane. Two
types of membranes are normally used (1) for anions permeability: anion exchange
4
membrane (AEM), and for cations: cation exchange membrane (CEM). In microbial
electrolysis cell (MEC) external voltage source is used to drive current through an
electrode to effect a (bio)chemical conversion. However, microbial fuel cell (MFC)
uses the potential difference between an electrooxidation reaction (of a fuel) and an
electroreduction reaction (of an oxidant) to drive current through an external circuit.
Almost all MXCs share the same principal of microbial oxidation in the anode
chamber; however, the process of how to use electrons in cathode chamber is another
applied aspect of this technology, where X represent different applications e.g.
microbial electrolysis cells (MEC) (Torres et al., 2010).
To design and operate an MXC it requires considerable understanding at
scientific and engineering levels. For making an efficient microbial electrochemical
cell the most important areas to be considered are including: electrode material,
proton exchange system, pH buffer/electrolyte, as well as operating conditions in
anodic and cathodic compartments. MXCs can be of various designs (two chamber,
single chamber) but overall the concept is anodic and cathodic compartments with
electrodes separated by cation exchange membrane (CEM) (Fig. 1). Generally, when
electrons and protons make an exit from ETC they are passed to electron acceptor like
oxygen, and under anaerobic conditions electrons are transferred to anode in MXC. In
MXC, energy is determined by the redox potential difference between anode and
cathode.
5
Fig. 1.1. Schematic illustration of dual-chamber microbial electrolysis cell (MEC).
Anode respiring bacteria (ARB) oxidize organic substrate and transfer electrons to
anode. Reference electrode is used to poise anode potential at a fixed value with the
help of potentiostat. Ion exchange membrane is used to separate two chambers and for
migration of ions (H+ and OH
_) between anode and cathode.
A
n
o
d
e
C
a
t
h
o
d
e
e-
e-
H+
e-
OH-
2H2O
Anode Respiring
Bacteria (ARB)
e- e-
Ion Exchange
Membrane
Cathode Anode
Reference
Electrode
Applied Potential
H2+ 2OH-
e- donor
CO2
6
1.2.1 Anode respiring bacteria
Many researchers have been searching for specific electrogenic bacteria in
order to get the highest columbic yield in MXCs (Allen and Bennetto, 1993). The
most commonly used microorganisms in the anodic compartment of fuel cells are
those that oxidize glucose. There are numerous species of microorganisms that are
capable of metabolizing glucose these include: Escherichia coli (Park and Zeikus
2003), Proteus vulgarius (Allen and Bennetto, 1993), Bacillus subtilis (Choi et al.
2001), Lactobacillus plantarum, Streptococcous lactise, Erwinia dissolvens (Vega et
al. 1987), and Clostridium butyricum (Park et al. 2001).
1.2.2. Electron transfer mechanisms
Different mechanisms of electron transfer have been identified so far (1) direct
electron transfer (DET) between bacterial cell membrane to anode surface using
nanowires, conductive pili or excreted macromolecules, (2) Mediated electron transfer
(MET) where electrons are transferred by bacteria to anode using redox shuttles
produced by bacteria itself or externally added compounds like methyl blue and
neutral red. For direct electron transfer, bacteria should be physically attached to
anode for current generation (Wrighton and Coates, 2009). However, mediator-less
microbial electrochemical cell (MXC) does not indicate DET as the possible electron
transfer pathway. It rather indicates that no artificial mediators are used to facilitate
electron transfer (Schröder, 2007).
1.2.3. Substrate Range
Theoretically, MXCs can convert any biodegradable substrate into electrical
energy Pant et al., 2010. Electron donors can range from simple sugars to complex
substrates such as domestic and industrial wastewater, biomass waste from food
industry, inorganic wastes (ammonia) and acid mine drainage (Kuntke et al., 2012).
The question is how these complex substrates are degraded in MXCs? Usually, it
requires cooperation of two types of bacteria, one convert complex substrates like
cellulose into secondary metabolites such as hemicelluloses, amino acids, acetate,
alcohols, lactate etc. and other which oxidizes these simple organic products into
electrons and transfers it to electron acceptor (anode) (Parameswaran et al, 2009). In
terms of wastewater treatment MXCs represents a new generation of technology
7
which transforms conventional energy consuming treatment processes into
coordinated systems that will generate energy, clean water and other valueable
products (Liu and Logan, 2004; Logan et al., 2008; 2007; Virdis et al. 2011; Wang
and Ren, 2013).
Research on fermentation-based conversion of various kinds of biomass to
energy is expected to open a new way for future commercial applications. Challenges
in renewable energy generation requires two important components: 1) exploitation of
earth’s rich biodiversity of novel potential bacteria capable of producing electricity
from organic substrates and 2) novel methods to evaluate, characterize, and monitor
these processes.
1.3. Applications
1.3.1. Remote Sensors
MXCs as sensors in natural environment are nearing practical use. MXCs
powered by sediments in marine environments offer energy without recharging. The
operation includes graphite plate in the anoxic sediment (anode) and connected to air
cathode in aerobic water. These devices are used to monitor water quality in remote
places e.g. rivers, lakes and ocean (Rimbound et al., 2014; Liu et al., 2008).
1.3.2. Bioremediation
The use of different substrates mostly of organic nature in MXCs has
suggested the remediation of different contaminants from domestic and industrial
sources. Anodic compartment typically as anaerobic chamber has been used for
degradation of contaminants for generation of current. Likewise, cathodic chamber
viewed handy to clean contaminant via reductive processes (Gregory and Lovley,
2009). The term “green energy” fits well in such scenario where MXCs could
minimize the production cost and energy recovery for sustainable environment (Strik
et al., 2008). Currently, there is a market for microbial fuel cells which vary from size
to operating conditions. Private companies around the world are trying to consume
organic waste into hydrogen or electricity as well as valuable chemical compounds
(Shama and kundu, 2010). Researchers have used microbial fuel cell to treat azo dyes
(Li et al., 2010), slaughter house waste (Ghanapriya et al., 2012), cellulosic waste,
8
brewery waste (Feng et al., 2008) and sewage wastewater (Marshall et al., 2012; Ren
et al., 2008). This device can also be used to remove nitrate from wastewater without
producing ammonia. It has also been reported that MXCs has potential to remove
waste like uranium from contaminated soil and water (Thrash and Coats, 2008). In
addition, potential between the electrodes is also helpful to perform desalination using
microbial desalination cells (MDCs) (Cao et al., 2009; Jacobson et al., 2011; Luo et
al., 2011). For future application, this technology can be used to degrade/reduce toxic
metals, pesticides and herbicides.
1.3.3. Valuable end products
The miracles of MXC technology don’t end at power generation, waste
treatment and hydrogen production. Anode respiring bacteria plays a vital role when it
comes to degradation of organic compounds into valuable added products or biofuels.
It can produce valuable inorganic and organic compounds such as hydrogen peroxide
, methane gas, caustic soda (Rozendal et al., 2009; Pikaar et al., 2011; Marshall et al.,
2012; Cheng et al., 2009), organic compounds ethanol (Steinbusch et al., 2010),
butanol and propanol via fermentation. Efficient production of these compounds can
be achieved by using genetic engineering.
1.3.4. Hydrogen gas production
In the past few areas researchers have focused their interest on hydrogen
production through MXCs (Logan et al., 2008; Mehanna et al., 2008). Replacement of
fossil fuels with alternative fuel sources (biofuels) is not only economically affordable
but also environment friendly. In MEC, electric current generation is reversed to
produce hydrogen gas by providing an additional external power source (~250mV).
So, with high electrical potential, electrolysis of water occurs at cathode that results in
hydrogen gas production (Liu et al., 2005b; Liu et al., 2010). Rapid developments
have led scientists to achieve 100% hydrogen yields. Using microbial electrolysis cell
(MEC) energy production based on electrical energy input is many times higher than
conventional methods like water electrolysis (e.g. Solid oxide electrolysis cells
(SOECs) (Logan et al., 2008). The concept of hydrogen production using MECs is
similar to MFCs design except some changes in architechture and analytic methods
used to enhance performance. However, if we combine improved reactor design with
organic rich waste make this technology an attractive plan of action in future.
9
1.3.5. Medical devices powered by MXCs
Microbial electrochemical cells can also be used to power medical implant
devices by consuming blood glucose. An implanted MXC with ability to power
medical implant for indefinite time and will nullify the need of medical process (e.g.
surgery) to replace batteries. Likewise, scientists developed an abiotic fuel cell with
capability of generating energy from glucose present in the blood (Kerzenmacher et
al., 2008; Kim et al., 2003). Mingui et al. (2006) used electrons harvested from
human white blood cells (WBC) for anode. Similarly, Justin et al. (2005) generated
electrical current (1–3 μAcm−2
) using WBC and a ferric-cyanide cathode.
To improve the overall efficiency of the microbial electrochemical cells
research have been focused on 1) finding efficient anodic reactions which can
produce maximum number of electrons (Lee et al. 2002; Logan, 2004); 2) finding the
most efficient microorganisms capable of extracting maximum electrons from
substrate (Choi et al. 2003; Kim et al. 2002); 3) selecting effective redox mediators
(McKinlay and Zeikus, 2004); and 4) selecting effective electrode materials (Gregory
et al. 2004).
Till now, cost effectiveness and low energy output are the limiting factors in
MXC research. To overcome these issues low cost electrode materials can be tested to
obtain maximum energy output. Mutagenesis and rDNA technology has been
envisaged to obtain some “super bugs” for MXCs for high energy outputs. To produce
effective levels of electrical current from MXC, it requires substantial research and
developments. An understanding of bacterial respiration processes may lead to
unforeseen applications in nano-electronics (Reddy et al., 2010).
1.4. A current affair: Ongoing research and challenges
Limitations to the MXC research hardware has been linked with hardware and
operational issues. Additionally, the role of specific bacteria (discretely or in
consortium) with efficient metabolic activities and compatibility with cells has been
crucial. Despite further improvement by 10-100 fold in power out is essentially
required to make this technology marketable. Till this date MXC research has been
mostly conducted at lab scale and it is important to evaluate design, construction,
10
operation and microbial limitations for pilot scale applications. In the past few years,
few companies such as. Anheuser-Busch Inc. (USA) and Foster’s breweries
(Australia) tried to evaluate the pilot-scale MXC technology with focus on wastewater
treatment and electricity generation (Wrighton and Coates, 2009). Still large scale
economically viable solutions for this technology needs to be evaluated.
1.5. Research Objectives
The primary objective of the research presented in thesis was to identify novel
thermophilic anode respiring bacteria capable of electrical current generation in dual
chamber microbial electrochemical cell (MXC) using biological and electrochemical
techniques e.g. Chronoamperometry, cyclic voltammetry (CV), Scanning electron
microscopy (SEM), and confocal laser scanning microscopy (CLSM), presented in
chapter 3.
The second objective was to enrich electrochemically active microbial
communities in single chamber MXC using environmental samples from diverse
geographical locations, presented in chapter 4. Different biological, electrochemical
and computational techniques (e.g. Chronoamperometry, cyclic voltammetry,
Pyrosequencing, PCoA, and PICRUSt) were used to characterize enriched mixed
culture bacterial communities on anode surface.
11
CHAPTER 2
Review of Literature
12
MFC is an innovative bio-electrochemical approach toward generating
electricity, gases and valuable chemical compounds using biomass (Butler et al.,
2010, Lovely and Nevin, 2011; Rabaey and Rozendal, 2010). It is one of those
contemporary green approaches which are under way of development (Cheng et al.,
2009). The primary role in MXC is played by bacteria, although there are many other
factors that affect performance and operation of MXC e.g. substrate types and
concentrations, temperature, pH, presence of alternate electron acceptors, type and
nature of electrodes.
Bacteria used in MXC belongs to a unique group of microorganisms
commonly referred as electrochemically active microbes (EAB), anode respiring
bacteria (ARB) exo-electrogens or electricigens (Torres et al., 2009; Lovely, 2006;
Logan 2009). Microbes like ARB can fully degrade organic compounds to CO2 by
transferring electrons onto the electrode (anode) of electrochemical cell (Lovley,
2006). Early designs of microbial electrochemical cells make use of fermentable
substrates as carbon source and were operated using fermentative microorganisms
(Katz et al. 2003; Shukla et al. 2004; Lovley, 2006).
As discussed earlier, anode respiring bacteria (ARB) have capability of
transferring electrons directly through cell membrane known as direct electron
transfer (DET), or out of cell membranes to the electrodes, with the help of
membrane-bound protein arrangements known as “pilli”, c-type cytochromes and or
mediators for indirect electron transfer (IET) (Torres et al., 2009; Logan, 2009).
Pohlschroder and Esquivel (2015) discussed archeal type IV pili and their
involvement in biofilm formation, in addition to their role to transfer electrons
extracellularly (Reguera et al., 2005). Elaborating the subject, recent studies revealed
that Geobacter sulfurreducens has ability of cell-to-cell electron conduction via
specialized nanowires and c-type cytochrome OmcZ for electron transfer onto
electrode (Lovely et al., 2011). Similar study was conducted by Ishii et al. (2005)
where flagellum was seen between Pelotomaculum thermopropionicum and
Methanothermobacter thermautotrophicus. Likewise, nanowires have been reported
by researchers in various bacterial species e.g. Synechocystis PCC6803, P.
thermopropionicum (Gorby et al., 2006) and S. oneidensis MR-1, expressing filament
like structure closely related to pilus, and thin filaments produced by Geobacter
13
sulfurreducens (Reguera et al.,2005). In contrast, if we take example of Shewanella
species, it uses both direct (via conductive filaments) and indirect electron transfer
mechanisms (Marsili et al., 2008; Canstein et al., 2008). Bacteria also use externally
supplied artificial mediators (neutral red, methyl blue, thionine, methyl viologen) for
EET (Rabaey et al., 2005; Park and Zeikus, 2000). However, Pseudomonas sp.
produce electron shuttles which facilitates transfer of electrons between cells to the
electrode (Scott and Murano, 2007).
2.1. Thermophilic anode respiring bacteria (ARB)
Thermoanaerobacter sp. has been studied to efficiently produce ethanol at
commercial scale using different substrates e.g. glucose and xylose (Hemme et al.,
2011). The possible reasons to use Thermoanaerobacter for biofuel production is (I)
the simplification of nutrient requirement because of de novo cofactor (vitamin B12),
(II) elevated growth temperatures which facilitates handling of ethanol, (III) low risk
of contamination, (IV) ability of fermenting both hexose and pentose sugars to
ethanol, (V) Complete substrate application, (VI) easy to grow in microbial consortia,
(VII) a unique pathway to produce biofuel where lineage-specific alcohol
dehydrogenase enzymes are involved for fermentation (Peng et al., 2008; Burdette et
al., 1996). It’s not unusual that Thermoanaerobacter sp. have great biotechnological
potential to degrade cellulosic waste. This sp. is well studied at genetic, physiological
and biochemistry levels. This information leads me to evaluate this microbe as a
potential ARB.
Thermophiles have capability of high metabolic rates which will eventually
result in higher electric current generation, and are more stable than their mesophilic
counterparts in serve conditions which are common to industry. Bioenergy can be
cheaply and effectively produced at high temperatures (Lynd et al., 2005), for
example, hydrogen fuel cells and wastewater treatment processes are carried out at
elevated temperatures and often at low pH (Jong et al., 2006), which makes
thermophilic bacteria suitable candidates to carry out such processes.
14
2.1.1. Substrate selection
Researchers reported electricity generation using acetate as carbon source in
thermophilic MXCs (Jong et al., 2006; Marshal and May, 2009; Carver et al., 2011;
Hussain et al., 2012). Where Zavarzina et al. (2007) and Parameswaran et al. (2013)
described the possibility of using acetate as substrate and insoluble iron as an electron
acceptor to develop Thermincola ferriacetica biofilms on anode. Mathis et al. (2008)
generated electricity with cellulose and acetate in thermophilic MXC. Choi et al.
(2014) tested glucose and lactose to generate electricity, and concluded that it is
possible to construct thermophilic MFC when right choice is made towards substrate
selection and operating conditions.
2.1.2. Temperature range
Bacteria which thrive in extreme conditions may serve as better catalysts due
to high metabolic rate, greater stability, longer life and wide range of substrate
consumption. Microbial fuel cell performance was observed to increase when
thermophilic bacteria were grown at 50-60ºC. Similar trend was observed by Jong et
al. (2006), Fu et al. (2015a), Wrighton et al. (2011) and Mathis et al. (2008).
However, they also reported rapid deterioration in MFC performance when
temperature was increased to 70ºC. But, Fu et al. (2015b) successfully generated
electricity using hyperthermophilic microbial fuel cell operated at 80ºC with
improved reactor performance when temperature was raised to 95ºC, which concluded
that reactor performance can increase or decrease at elevated temperatures depending
on type of thermophile being used to produce electrical current.
2.1.3. Thermophilic MXC operation with single strain ARB
Only few thermophilic ARB capable of exo-electrogenic activity have been
reported to generate electricity e.g. Thermincola potens strain JR (Wrighton et al.,
2008), Thermincola ferriacetica (Marshall and May, 2009; Parameswaran et al.
2013), Caloramator australicus (Fu et al., 2013a) and Calditerrivibrio nitroreducens
(Fu et al., 2013b). Whereas, still use of G+ bacteria in MXC and yet their role is
unknown (Aelterman et al., 2006; Choi et al., 2004; Milliken and May, 2007; Park et
al., 2001; Rabaey et al., 2007).
15
Wrighton et al. (2008) studied role of Firmicutes in thermophilic microbial
fuel cells. Experiments were conducted at 55ºC, with maximum power output of
37mW m-2
and coulombic efficiency of 89%. They were able to isolate first
thermophilic organism Thermincola spp. capable of current production (0.42mA) as
well as direct anode reduction. Wrighton et al. (2011) reaffirmed that gram positive
thermophilic bacterium Therminocla potens strain JR can perform direct electron
transfer. Wrighton et al. (2011) explained that biofilm thickness dosen’t contribute to
current production, and presented evidence based on physiological and genomic
results that c-type cytochromes play a significant role in transferring electrons across
the bacterial cell wall, which was also confirmed by Carlson et al. (2012), who
studied dissimilarity metal reduction in T. potens and described role of surface
multiheme c-type cytochromes in respiratory metal reduction. Byrne-Bailey et al.
(2010) sequenced genome of anode respiring Thermincola potens strain JR.
Parameswaran et al. (2013) characterized different aspects of thermophile T.
ferriacetica e.g. Kinetic, electrochemical and microscopic and reported current
density of 7-8Am-2
. First effort to run thermophilic MXC using a redox mediator was
reported by choi et al. (2004), where G+ Bacillus licheniformis and Bacillus
thermoglucosidasius were used to generate electrical current.
Fu et al. (2013a) reported Caloramator australicus as a new electrochemically
active thermophilic exoelectrogen. For confirmation, cyclic voltammetry was
performed to evaluate electron transfer mechanism, however, no evidence of mediated
electron transfer mechanism was found, which suggested DET to the anode by
attached thermophilic microbial biofilm. Fu et al. (2013b) identified another
thermophilic gram negative bacterium, Calditerrivibrio nitroreducens, capable of
electrical current generation upto 823mW-2
. This study was first example of
bioelectrochemical characterization of thermophilic gram-negative exo-electrogen.
Furthermore, Fu et al. (2014) also developed a thermophilic biocathode system
capable of accelerating electromethanogensis process. High methane production
(1103 mmol m-2
day-1
) was observed with current production efficiency >90%, which
makes thermophiles a promising candidates to serve as biocatalysts. Fu et al. (2014)
concluded that Methanothermobacter-related methanogens got enriched during
acclimation. This study provides core information on use of thermophiles in
electromethanogenic bioelectrochemical systems that may be extended to other BESs.
16
2.1.4. Thermophilic MXC operation using bacterial consortia
There are few studies which discusses role of thermophilic inocula derived from
different environments for electricity generation in MXCs. Jong et al. (2006) might be
first to conduct a study using enriched thermophilic microbial consortium derived
from thermophilic anaerobic digester effluent for electricity production. Similarly,
Carver et al. (2011) used thermophilic compost as consortia to inoculate anode
chamber, he also proposed a new thermophilic MFC design that prevents evaporation,
which is indeed a major concern when it comes to operational conditions. Hussain et
al. (2012) operated a MFC using inoculum derived from mesophilic anaerobic
granular sludge treating agricultural wastes. Mathis et al. (2008) generated electrical
current using single chamber fuel cell (1,000-Ω resistor) enriched with thermophilic
anode respiring bacteria from marine sediment of a temperate lake, and claimed that
marine sediments are comparatively better source of electrochemically active
thermophilic bacteria. Fu et al. (2015b) used two-chamber MFC reactor (external
ressitance from 10,000 to 30ῼ) to enrich ARB from inocula native petroleum
reservoir.
Thermophilic consortia used in MXCs showed dominance of ARB e.g.
Coprothermobacter sp. and Thermodesulfovibrio sp. (Jong et al., 2006); microbial
consortium TC60 (Carver et al., 2011); Methanothermobacter wolfeii,
Methanobrevibacter arboriphilicus, Acetobacterium wieringae (Hussain et al., 2012);
Thermincola carboxydophila (Mathis et al. 2008); Caldanaerobacter subterraneus
and Thermodesulfobacterium commune (Fu et al., 2015b) capable of generating
electricity in the range of ~22 to~250mA/m2.
Jong et al. (2006) achieved maximum power density of 340mW/m2
during
treatment of wastewater using thermophilic MFC design. Ultimate goal of the study
was to convert agricultural wastewater into electricity, which was proven
successfully. Hussain et al. (2012) operated microbial fuel cell at 50ºC with maximum
volumetric output of 30–35 mWL R −1
. Likewise, Carver et al. (2011) achieved
maximum current density of 35mWm-2
. Fu et al. (2015b) observed fourfold increase
in current density (upto 165mWm-2
) when temperature was increased from 75ºC to 95
ºC. Similarly, Mathis et al. (2008) reported current generation upto 450 mA/m2, with
17
tenfold increase in electric current when cells were grown at 60ºC (254 mA/m2) vs
cells grown at 22ºC (22 mA/m2). Cho
2.2. Enrichment of mesophilic ARB from diverse natural
environments
2.2.1. Substrate selection for mesophilic ARB
Substrate selection is an important factor when it comes to grow mesophilic or
thermophilic ARB in anode chamber. Mostly, acetate has been used as a carbon
source for anaerobic bacteria by several researchers (Bond and Lovley, 2005; Logan
and Regan, 2006; Cercado et al., 2013), specifically in MFCs (Min and Logan, 2004;
Liu et al., 2005; Lee et al., 2003; Rozendal et al. 2006) producing high current
densities (Liu et al. 2005; Miceli et al., 2012). Sacco et al. (2012) reported that acetate
as an easily consumable substrate, which promotes bacterial growth in sediments as
well as in MFC. Acetate has also been found to be more efficient electron donor than
other substrates e.g. glucose, starch and butyrate. Researchers achieved high
coulombic efficiencies using acetate, ranging upto 65 to 92% (Min and Logan, 2004;
Rozendal et al., 2006). Lee et al. (2003) used acetate to enrich electrochemically
active bacteria from activated sludge in MFCs. 16S rDNA sequence analysis revealed
that diverse microbial communities were enriched when MFC was fed with acetate as
carbon source. Logan and Regan (2006) reported that acetate impacts on the
composition of microbial communities on anode surface of MFCs. Similarly Yang et
al. (2012) also claimed that microscopic analysis of enriched biofilms showed
different type of cells when grown separately with glucose and acetate which
indicates that ARB evolve differently when different substrates are used. Phung et al.
(2004) also used glucose along with glutamate to generate electricity using enriched
biofilms in MXC.
2.2.2. Inoculum sources to enrich electroactive anodic biofilms (EAB)
A wide range of geographically and naturally diverse environments have been
found hosting ARB in MXCs (Chabert et al., 2015; Ruiz et al., 2014), which are
discussed as under:
18
2.2.2.1 Aquatic sediments
In past few years studies have been focused on exploitation of organic rich
aquatic sediments to generate electricity using anode respiring bacteria in MXCs
(Phung et al., 2004; Sun et al., 2009; Pisciotta et al., 2012; Reimers et al., 2001).
Chaudhari and Lovely (2003) reported electrochemically active bacteria found in
sediments capable of direct oxidation of organic acids. Similarly, Martins et al. (2010)
and Sun et al. (2012) discussed the importance of use of lake sediments as inoculum
source to enrich ARB on anode surface. Cordova-Kreylos et al. (2006) explained that
ARB in sediments collected from different locations may show variance because of
different local environmental conditions. Holmes et al. (2004) studied importance of
microbial incoula and its influence on how microbial community evolves in MFC.
Schaetzle et al. (2008) also confirmed that the selection of inoculum is of great
importance in MFCs. Whereas, Nielsen et al. (2012) suggested that electrochemically
active bacteria as a part of microbial community in sediments play significant role in
the biogeochemical cycles.
Using sediments from diverse environments as a source of inoculum
containing novel ARB gives us better understanding of microbial ecology which leads
us to development of benthic microbial fuel cell (BMFC), which is a technology
where anode is fixed in anoxic sediments with air cathode (Reimers et al., 2004).
Benthic microbial fuel cells were first described by Tender et al. 2002 and Bond et al.
(2002), where marine sediments were used because of better ion conductivity between
MFC electrodes and saline environments, and were also used by Li and Nealson
(2015) to enriched electrochemically active microbial communities capable of
electricity generation. Mohan et al. (2008) reported use of lake sediments to operate
BMFCs. The predominance of the bacteria depends on the source such as
Desulfuromonas sp. is more common in marine sediments, while Geobacter sp. is
commonly found in freshwater sediments (Holmes et al., 2004). A previous study by
our group Miceli et al. (2012) addressed lack of bacteria capable of forming biofilms
and electron transfer to anode. Miceli et al. (2012) enriched anode respiring bacteria
enriched from diverse environments like marshes, lake sediments, saline microbial
mats and anaerobic soils using microbial electrochemical cell (MXC). Another study
conducted by Miceli et al. (2014) demonstrated electrical current generation using
sediments from marine hydrothermal vents (Guerrero-Barajas and García-Peña,2010)
19
to enrich ARB on anode surface when combined with sludge form a treatment plant (
Parameswaran et al.,2009; Torres et al. 2009) using butyrate. Mangrove sediments
have also been used to enrich potential ARB in MXC by Rivalland et al. (2015) and
Salvin et al. (2012).
Song et al. (2012) collected sediment samples from different sites in fresh
water lake, and reported that sediments with high Fe(III) contents produced maximum
voltage (580mV) in MFCs. However, sediment samples with lowest Fe(III) content
produced less voltage (30mV). This study helps researchers to make scientific
decisions regarding the selection of exo-elctrogenic bacteria using fresh water
sediments, and also how to collect, transport and prepare sediment sample for
inoculation. Sacco et al. (2012) studied performance of cylindrical graphite electrodes
in sedimentary microbial fuel cell (Samples were collected from the shore of Rio de
La Plata River, South America).
2.2.2.2. Water sources
Researchers have reported use of different water sources to enrich potential
ARB in MXCs. Like, Phung et al. (2004) used river water to feed and enrich
oligotrophs in oligotrophic MFC. Purpose of the study was to identify enriched
electrochemically active oligotrophs, which normally are hard to cultivate in
laboratory conditions. Oligotrophs are microorganisms tend to grow in low nutrient
environments such as oceans, clear lakes and groundwater aquifers. Analysis of
enriched microbial diversity on anode revealed dominance of Deltaproteobacteria,
Acidobacteria, Chloroflexi and Verrucomicrobia.
It is important to test other water sources in MXC such as treated wastewater
effluents, which could possibly be containing bacteria capable of electrical current
generation and at the same time wastewater treatment (Doyle and Marsili, 2015).
Ketep et al. (2013) explained importance of sampling site when it comes to forming a
bacterial biofilm on anode surface, by inoculating anode compartment with samples
collected from inlet and outlet of aerated lagoon of Kraft pulp mill effluent. Primary
biofilms from the anode surface were scrapped and used to develop second generation
biofilms in new reactors. It was concluded that with each re-inoculation current
density was increased. Microbial community analysis of anodic biofilms showed
20
presence of Geobacter and Desulfuromonas sp. Authors claimed it to be the first
study to observe microbial community difference in different sampling locations of
the same effluent. Brewery wastewater, domestic wastewater and wastewater from
treatment plants have been used to enrich ARB for electricity generation (Larrosa-
Guerrero et al., 2010; Sun et al., 2015).
2.2.2.3. Garden Compost
Parot et al. (2008a) used garden compost to form electrochemically active
microbial biofilms on anode surface capable of electricity generation. It took 1 to
approx 10 days to produce current using enriched microbial biofilms. In our case
biofilms were developed within 2-5 days of initial inoculation. The current increased
after lag phase and remained stable for several days. Bacteria used organic matter in
the garden compost as carbon source which resulted in maximum current densities
achieved were upto 385mA/m2. Parot et al. (2008a) confirmed that soils are rich
source of anode respiring bacteria (ARB) which are “available everywhere, free of
charge”, which justifies report of Niessen et al. (2006). Parot et al. (2008b) conducted
another study to accelerate electrochemical performance of bacterial biofilms derived
from garden compost. Current densities were increased upto 545 mA/m2 after addition
of acetate. It was concluded that bacterial cells showed slow oxidation of acetate and
fast electron transfer between bacterial cell and the electrode surface. Similarly,
Cercado et al. (2013) also demonstrated that Inoculum originated from garden
compost lead to bacterial anodes with potential-independent characteristics. ARB
from different locations will help us to understand how bacteria react in ARB
consortia, and to comprehend the ubiquity process of anode respiration.
2.2.3. Enriched communities of ARB
Bacterial communities that develop in MXCs range from delta-Proteobacteria
which normally belongs to sediment MXCs to alpha, beta, or gamma-Proteobacteria
(Holmes et al., 2004; Tender et al., 2002; Bond et al., 2002; Logan and Regan, 2006).
It was believed that anode respiring bacteria (ARB) in MXCs belongs to iron reducers
Geobacter and Shewanella (Bond et al., 2002), but analysis of bacterial communities
revealed that there is much greater variety of microbes than these model iron reducers
(Rabaey et al., 2004). Sacco et al 2012 characterized anode samples and reported
dominance of sequences mostly related to Shewanella spp., Pantoea spp.,
21
Pseudoalteromonas spp., and the arctic bacteria R-11381. However, absence of
Geobacter spp. related sequences were possibly because of oxygen diffusion from the
cathode. Miceli et al. (2012) claimed presence of genus Geoabcter in lake sediments
achieving maximum 10.77 A/m2. Cercado et al. (2013) confirmed presence of
microbial groups related to Geobacter, Anaerophaga and Pelobacter on anode surface
derived from garden compost. Dennis et al. (2013) observed dominance of Geobacter
spp. along with Bacteroidales on anode surface. Ruiz et al. (2014) discussed that
efficiency of MXC improved when anode surface was dominant with microbial
groups belonging to Proteobacteria, Firmicutes and Bacteroidetes and Arcobacter.
Another study by Ortega-Martinez et al. (2013) identified electrochemically active
bacteria Geovibrio ferrireducens dominant on anode surface in enriched inocula (E-
In) .
2.2.4. Characterization tools to study Enriched ARBs on Anode surface
To study molecular based systematics of bacterial biofilms on electrode
surface, pre and post genomic techniques are important. Pre-genomic techniques
include 16S rRNA based phylogeny and metagenomics; while, post-genomics include
metatranscriptomics to help researchers characterize anodic biofilms and their
potential genomic expressions. Phylogenetic analysis could further elaborate
taxonomic evidence to bacterial metabolisms. Electrochemical, phylogenetic,
metagenomic and post-metagenomic techniques offer better indulgent of the EET
processes, which will contribute towards optimization of power output in MFCs (Zhi
et al., 2014; Rittmann et al., 2008; Ruiz et al., 2014, Gorby et al., 2006; Rabaey et al.,
2004; Schloss and Handelsman, 2005; Yates et al., 2012; Wang et al., 2013; Ishii et
al., 2013).
Zhi et al. (2014) discussed methods to understand bacterial community
structure and functions in microbial electrochemical cells. Techniques used by various
researchers includes: TEM and AFM (to study cell structures), 16S rRNA gene
sequencing (for identification) and polarization techniques: cyclic voltammetry (CV),
low scan cyclic voltammetry (LSCV), electrochemical impedance spectroscopy (EIS)
(for characterization of electrochemically active biofilms). Techniques to study
bacterial community include; quantitative polymerase chain reaction (PCR, qPCR),
22
fingerprinting methods (DGGE and T-RFLP), fluorescent in situ hybridization
(FISH), Pyrosequencing, and DNA microarray (Phylochip). However, to study
community function techniques like stable isotope probing (SIP),
metatranscriptomics, reverse transcription quantitative polymerase chain reaction
(RT-qPCR) have been considered.
While studying electrochemically active bacterial biofilms it is important to
recognize the genetic functionality and adaptability to change in environment like
understanding of EET mechanisms. As discussed earlier, EET is process through
which microbes breathe by transferring electrons out of the cell and solid phase
electron acceptors (Lovely D.R. 2012). Ishii et al. (2013) successfully unified
bioelectrochemical, metagenomic and metatranscriptomics data sets to identify EET-
related genes in diverse community. The basic objective was to discuss EET-related
bacteria and genes within a diverse community and monitor the response to changing
extracellular electron transfer conditions. Likewise, Ishii et al. (2015) studied
microbial metabolics in electroactive bacterial biofilms derived from stimulus-
induced metatranscriptomics approach. It’s well known that bacteria mostly exist as
mixed bacterial groups in natural environments. How bacterial communities respond
to environmental change is not yet been achieved. Meta-omics approach gives us an
understanding of complex microbial roles within a community and how community
members respond to the environmental limitations.
Langille et al. (2013) used computational method to predict marker gene data
and a database of reference genomes. PICRUSt software uses ancestral-state
reconstruction algorithm to predict gene families and to estimate the composite
genome. This tool has been successfully used in the human genome project and it to
predict the gene families, with quantifiable uncertainty. Statistic approaches have
been used to interpret MXC data in more comprehensive ways. In this context, El-
Chakhtoura et al. (2014) studied bacterial community structure in air-cathode MFCs
by using Pyrosequencing results to produce a principal coordinate analysis (PCoA)
plot. PCoA helps in understanding how communities in the MXCs evolve and
converge at the end of the experiments irrespective of the inoculum source.
23
Microbial electrochemical cells (MXCs) will contribute to the future energy sector.
Improved MXC reactor design has applications to wastewater treatment, hydrogen
production and valuable chemical compounds.
24
CHAPTER 3
Characterization of thermophilic bacterium
Thermoanaerobacter pseudethanolicus for
electrical current-generation using microbial
electrochemical cell (MXC)
25
3.1. Introduction
Anode-respiring bacteria (ARB) catalytically convert chemical energy stored
in organic compounds into electrical current via extracellular respiration at an
insoluble anode. In nature, these bacteria are known to reduce Fe(III) and Mn(IV)
oxides (Badalamenti et al., 2013), and potentially perform direct interspecies electron
transfer (DIET) (Rotaru et al., 2015), by using simple organic compounds (acetate,
lactate) as electron donors; however, in a microbial electrochemical cell (MXC), these
oxides are replaced with an anode having a set potential. Anode respiration has been
achieved in over 30 metal-reducing bacterial isolates from various genera, including
Shewanella, Geobacter, Pseudomonas, Thermincola, and Rhodoferax, but only a
select few ARB, including Geobacter sulfurreducens (Bond and Lovley, 2003),
Thermincola ferriacetica (Parameswaran et al., 2013), Geoalkalibacter
ferrihydriticus, and Geoalkalibacter subterraneus (Badalamenti et al., 2013) among
others, are able to produce high current densities (j) through the formation of a
biofilm that enables efficient extracellular electron transport (EET) (Lovley, 2008).
G. sulfurreducens and S. oneidensis are the two ARB most commonly studied
over the past decade (Bond and Lovely, 2003; Gorby et al., 2006). Both
microorganisms are mesophilic, Gram-negative, and from the Proteobacteria phylum.
The identification of Thermincola ferriacetica and Thermincola potens, two ARB
from the Firmicutes phylum, was an important physiological discovery, as these
microorganisms are thermophilic and Gram-positive (Marshall and May, 2009;
Wrighton et al., 2011). The discovery of new ARB is essential for the technological
applications envisioned for microbial electrochemistry. For example, thermophilic
ARB in the Thermincola family are known to consume only acetate and H2 as
electron donors (Parameswaran et al., 2013; Marshall and May, 2009; Carlson et al.,
2012), but conversion of organic waste streams into useful products using MXCs
demand microbial communities (Mathis et al., 2008) or ARB capable of producing j
from complex organic materials, such as sugars (Bond and Lovley, 2005; Luo et al.,
2013; Chaudhuri and Lovley, 2003, Weber et al., 2006). Mesophilic Gram-negative
ARB capable of converting sugars to current production in MXCs have been reported
using Geothrix fermentans (Bond and Lovley, 2005), Tolumonas osonensis (Luo et
al., 2013), and Rhodoferax ferrireducens (Chaudhuri and Lovley, 2003). However,
26
none of these studies produced more than 0.5 Am−2
from the sugars tested. T.
pseudethanolicus is the first fermenter capable of producing high current densities
from sugars and the most versatile in terms of the complexity of sugars utilized.
Thermophilic bacteria including Thermoanaerobacter pseudethanolicus,
Thermoanaerobacter ethanolicus, Clostridium thermocellum, and Clostridium
thermohydrosulfuricum contain thermozymes (enzymes that are stable at elevated
temperatures) capable of fermenting lignocellulosic materials or their hydrolysates
into ethanol, lactate, or acetate (Mathis et al., 2008; Cook et al., 1993; Demain et al.,
2005; Roh et al., 2002; Thomas et al., 2014). Some of these fermentative bacteria also
perform dissimilatory metal reduction (Roh et al., 2002); thus, they may be able to
convert xylose, glucose, and cellobiose into simple acids, including acetate, for
consumption and current production in MXC technology (Appendix fig. S1). T.
pseudethanolicus (ATCC 33223), a thermophilic, Gram positive, rod-shaped
bacterium, can grow with acetate in the presence of Fe(III) oxides and produces
acetate from fermentation of xylose, glucose, and cellobiose, (Roh et al., 2002; He et
al., 2009; Hemme et al., 2011; Hniman et al., 2011; Onyenwoke et al., 2007) making
it an ideal candidate for use as an ARB on an anode.
In this study, the ability of T. pseudethanolicus to respire to an anode was
evaluated using different monomeric organic compounds like xylose, glucose,
cellobiose, or acetate as an electron donor. For the first time, detailed chemical,
electrochemical, and microscopic evaluations were carried out on the T.
pseudethanolicus biofilm anode to understand its simultaneous fermentation and
anode-respiration capabilities. Thermoanaerobacter represents only the second
thermophilic ARB family to be studied in monoculture in MXCs, and it is one of the
first ARB shown to be capable of sugar fermentation (Bond and Lovley, 2005; Luo et
al., 2013; Chadhuri and Lovley, 2003). This discovery opens the possibility of using
fermentative bacteria that are also dissimilatory metal reducers as the primary ARB in
microbial electrochemistry, especially to capture the energy in the carbohydrate
fraction of biomass.
27
3.2. Materials and methods
3.2.1. Growth media and culture conditions
ATCC Medium 1118: Methanobacteria medium (ATCC medium 1045) was
used to grow T. pseudethanolicus 39E (ATCC 33223): K2HPO4 (0.45 g L−1
),
(NH4)2SO4 (0.3 g L−1), NaCl (0.6 g L−1
), MgSO4·7H2O (0.12 g L−1
), CaCl2·2H2O
(0.08 g L−1
), yeast extract (0.2 g L−1), and Wolfe’s Mineral Solution (10 mL L−1
).
Media was prepared in a condenser apparatus under N2:CO2 (80:20) gas conditions.
Medium was brought to boil and allowed to boil for 15 min L−1. Medium was then
stored in 100 mL serum bottle and autoclaved for 15 min at 121 °C. Wolfe’s Vitamin
Solution (10 mL L−1
) and Na2CO3 (4.2 g L−1
) were added after autoclaving. Substrate
was added after autoclaving, either glucose (1.8 g L−1
), xylose (3.0 and 6.0 g L−1
),
cellobiose (0.34 and 3.4 g L−1
), or acetate (0.82 g L−1
) (Cook et al., 1993; Onyenwoke
et al., 2007). Reducing agent, including sodium sulfide and cysteine, was excluded
from the media to minimize its background effect on electrochemical observations. T.
pseudethanolicus stock cultures were grown under fermentative conditions with
glucose as an electron donor in batch mode in 100 mL serum bottles on an Excella
E24 Incubator Shaker (New Brunswick Scientific) at 60 °C and 150 rpm.
3.2.2. H-Type MEC construction
Eight H-type reactors were constructed and operated in batch mode. Each
reactor consisted of two 350 mL compartments separated by an anion exchange
membrane (AMI 7001, Membranes International, Glen Rock, NJ). For all reactors, the
operating temperature was 60 °C. Each reactor was fed with a different substrate at
the concentrations mentioned above. All reactors contained two cylindrical graphite
anodes (anode surface area for glucose and xylose MXCs = 5.2 cm2; cellobiose and
acetate MXC = 5.0 cm2) and an Ag/AgCl reference electrode (BASi MF-2052).
Reference potential conversion to a standard hydrogen electrode (SHE) was
conducted by constructing a two chambered cell with one chamber containing
modified ATCC Medium 1118 and the other containing 1 M KCl (Parameswaran et
al., 2013; Greely et al., 1960). The anode potential was poised at 0.042 V vs SHE, and
the j was monitored continuously every 2 min using a potentiostat (Princeton Applied
Research, model VMP3, Oak Ridge, TN). The anode chambers were kept mixed via
28
agitation from a magnetic stir bar at 200 rpm. The cathode consisted of a single
cylindrical graphite rod (0.3 cm diameter and a total area of 6.67 cm2). Cathode pH
was adjusted to 12 via addition of NaOH. Gas collection bags were placed on the
anode compartments to collect volatile fermentation products and on the cathode to
collect hydrogen.
3.2.3. MXC Batch Experimental Setup
All MXC reactors were operated as microbial electrolysis cells (MECs),
inoculated with 3 or 6 mL stock culture from serum bottles and grown under
fermentative conditions in batch mode. Coulombic efficiency (CE) was calculated
based on initial and final TCOD and the current measurement on the potentiostat
according to previous literature (Parameswaran et al., 2009). Yeast extract TCOD was
included in CE analysis since yeast extract utilization has been reported in previous
literature (Roh et al., 2002). Low scan cyclic voltammetry (LSCV) was conducted at
scan rates of 1 mVs−1
and 10 mVs−1
, when the j of the xylose reactors reached ~7.5 A
m−2
(pH 7.58), to measure the midpoint potential (EKA). Nernst-Monod equation (1)
was used as a model for anode potential losses in an ARB biofilm anode (Marcus et
al., 2007, Torres et al., 2008b). For Nernst-Monod fitting I used average EKA and the
jmax of each curve.
(1)
Where,
j = Current density (Am-2
)
jmax = Maxium current density (Am-2
)
R = Ideal gas contant
F = Faraday Constant (96,485 Coulomb per mole-e)
T = Temperature
EKA = Potential at which j = ½ jmax
29
Fig 3.1. Illustration of experimental setup for microbial electrolysis cell (a) muli-
channel potentiostat (b) EC-Lab software used for data collection and to run various
electrochemical techniques (c) H-type batch MEC placed in incubator at 60ºC
connected to potentiostat.
Potentiostat
H-Type Microbial Electrolysis Cell
EC-Lab Software
30
3.2.4. High pressure liquid chromatography
During operation of the reactors, the pH was measured every 2−3 days, and 1
mL of medium was collected and stored in an amber high pressure liquid
chromatography (HPLC) vial for tracking fermentation products including: ethanol,
acetate, lactate, propionate, isopropionate, butyrate, iso-butyrate, valerate, iso-
valerate, xylose, glucose, cellobiose, and sucrose. All HPLC samples were filtered
using a 0.2 μm membrane filter (Life Sciences Acrodisc 4450T) and stored at −20 °C
until analysis was conducted using a model LC-20AT HPLC (Shimadzu) equipped
with an AMINEX HPX/87H column (Bio-Rad Laboratories, Hercules, CA,1997) as
described earlier (Parameswaran et al,. 2009).
3.2.5. pH-Effect Experiments
To determine the effect of pH on j, two separate glucose-fed MECs were
constructed and operated under the same conditions as the previous reactors. After
achieving steady state conditions, pH was altered by either the addition of HCl or
NaOH. Results are shown as a ratio of the highest j achieved vs the j of a given pH.
Results were used to obtain a pH range for operation in MECs and also to understand
the role of pH in j.
3.2.6. Acetate Spike Experiment
A mature biofilm was grown on a xylose-fed MEC over ~70 days. The MEC
was then stopped and media was replaced with media deplete of an electron donor.
The MEC was then resumed and a stationary phase (~0.1 A m−2
) was achieved. Once
the reactor reached a stationary phase, a 1 mL injection containing 0.82 g L−1
acetate
was added to the reactor.
31
Fig 3.2: Schematic illustration of spike experiments performed to evaluate current
production capability of T. pseudethanolicus using acetate
32
3.2.7. Microscopic Analysis of Thermoanaerobacter pseudethanolicus Biofilm
Scanning electron microscopy (SEM) and confocal laser scanning microscopy
(CSLM) were accomplished by establishing a separate H-type batch reactor fed 20
mM xylose with the same operating conditions. After 60 days of operation, the two
anodes had developed mature biofilms that were sacrificed for imaging purposes.
Preparation for SEM followed the protocol from (Parameswaran et al., 2013). An FEI
XL-30 environmental SEM (Philips) was used with an accelerating voltage of 5−20
kV and a working distance of 8−10 mm. CSLM was used to measure biofilm
thickness (Lf) per the protocol from Parameswaran et al. (2013). Confocal images
were acquired using an upright Leica SP5 CSLM after applying LIVE/DEAD (Bac-
Light Cell vitality kit, Invitrogen) staining of the biofilm anode.
33
Fig 3.3. (a) Thermophilic MXC reactor showing T. Pseudethanolicus mature biofilm
on anode surface in H-type MEC (b) harvested biofilm used for CSLM and SEM
analysis.
34
3.3. Results
H-type microbial electrochemical cells were used under thermophilic
conditions to assess the potential of T. Pseudethanolicus for electrical current
generation. As mentioned above, four substrates were tested with reproducible results
discussed as follow:
3.3.1. Initial growth
A representative H-type electrochemical cell containing T. pseudethanolicus,
with its anode potential set at +0.042 V vs SHE, was allowed to ferment xylose into
acetate and then establish a biofilm over 5 days, when it reached a stationary current
density (j) of 6.5 A m−2
, as shown in Figure 3.4. (See Figure S2 in appendix for
results with the duplicate reactor.)
After the j reached stationary phase (day 4), CVs at a scan rate of 1.0 mVs−1
and 10 mVs−1
(data not shown) were performed. The midpoint potential (EKA) was
−0.17 V vs SHE, and the maximum current density (jmax) was ~7 A m−2
at pH 7.6, as
shown in Figure 3.5a. (For the derivative plot, see Figure 3.5b) The data fit the
Nernst-Monod equation with n = 1 with only slight deviation at the highest anode
potential. Figure 3.6 represents how j depended on the medium pH in the range of
5.4−8.27. At pH values lower than 5.40 or higher than 8.27, j dropped to ~0 A m−2
.
35
Figure 3.4. Initial growth phase in the xylose fed MEC operated in batch mode
containing Thermophilic Thermoanaerobacter Pseudethanolicus.
Figure 3.5a: Xylose reactor CV at 1mV-1
with observed data (blue) and Nernst-
monod fit at n = 1.0 (black dotted line).
j
-2
0
2
4
6
8
-0.5 -0.3 -0.1 0.1 0.3 0.5
Cu
rren
t D
ensi
ty (
Am
-2)
Anode Potential (V vs SHE)
N-M fit
n = 1
EKA = -0.168 V
36
Figure 3.5b. Derivative of the LSCV at 1 mV s-1
for the xylose-fed electrochemical
cell.
37
Figure 3.6. Effect of pH on current density normalized to the maximum value of 2.7
A m-2
(at pH 8.27)
38
3.3.2. Xylose Fermentation
At the end of the low-scan cyclic voltammetry (LSCV) experiments, the
reactor medium was replaced with new medium containing 20 mM xylose and left in
batch mode for over 75 days. The results can be seen in Figure 3.7a. (For the results
of the duplicate reactor, see Figure 3.7b) The sharp decrease in j from day 0 shows the
loss of current production, and acetate built up in parallel out to about day 8. Once the
acetate concentration reached ~20 mM, the j sharply increased, after which it
decreased again. Around day 29, the j recovered up to ~7.5 A m−2
and remained
relatively stable until it began a slow decrease as acetate concentration declined to
<10 mM. pH values remained relatively stable over the course of the experiment.
Coulombic efficiency (CE) was calculated to be 34.8% ± 0.7%.
39
Figure 3.7a. Current production by batch microbial electrochemicalcell (MXC) using
xylose inoculated with thermophile T. pseudethanolicus operated for ~77 days at
60ºC. Graph shows track of pH throughout the experiment, current density (Am-2
)
achieved using xylose as substrate, and concentration (mM) of by-products produced
during fermentation. An LSCV was conducted at time 0, just prior to replacing the
medium.
40
Figure 3.7b. Results for the xylose-fed electrochemical cell operated in batch for ~66
days containing T. pseudethanolicus. Orange circles indicate pH, black line indicates
current density, the purple square indicates xylose, red squares indicate lactate, and
blue diamonds indicate acetate.
41
3.3.3. Glucose fermentation
A grown T. pseudethanolicus biofilm containing no electron donor and
producing only a decay j (~0.5 A m−2
) was fed with new medium containing ~10 mM
glucose and operated in batch mode for over 45 days. The results can be seen in
Figure 3.8. (For results of the duplicate reactor, see Figure 3.8b) Complete glucose
fermentation occurred within 5 days, and lactate we fermented by day 12. Once the
acetate concentrations reached ~25 mM, the j sharply increased (up to ~4.8 A m−2
),
after which the j gradually decreased in parallel with the acetate concentration for the
remainder of the experiment. The CE for glucose was 65.3% ± 1.0%.
42
Figure 3.8a. Current production by batch microbial electrochemicalcell (MXC) using
glucose inoculated with thermophile T. pseudethanolicus operated for ~48 days at
60ºC. Graph shows track of pH throughout the experiment, current density (Am-2
)
achieved using glucose as substrate, and concentration (mM) of by-products produced
during fermentation.
43
Figure 3.8b. Results for the glucose-fed electrochemical cell operated in batch for
~55 days T. pseudethanolicus. Orange circles indicate pH, black line indicates
current density, yellow dashes indicate glucose, red squares indicate lactate, and blue
diamonds indicate acetate.
44
3.3.4. Cellobiose fermentation
A grown T. pseudethanolicus biofilm at stationary phase (~0.15 A m−2
)
containing no electron donor, was fed with new medium containing ~7.5 mM
cellobiose and left in batch mode for over 80 days. The results for the cellobiose-fed
electrochemical cell can be seen in Figure 3.9a. (For duplicate reactor, see Figure
3.9b) Complete cellobiose fermentation occurred within 10 days of reactor operation
producing j upto 3.5 Am-2
. Once the acetate concentrations reached ~25 mM, the j
sharply increased, after which it gradually decreased with acetate concentration for
the remainder of the experiment. Acetate and minimal lactate production was
observed in both cellobiose-fed electrochemical cells. The CE was calculated to be
27.7% ± 1.5%.
45
Figure 3.9a. Current production by batch microbial electrochemicalcell (MXC) using
cellobiose inoculated with thermophile T. pseudethanolicus operated for ~82 days at
60ºC. Graph shows track of pH throughout the experiment, current density (Am-2
)
achieved using cellobiose as substrate, and concentration (mM) of by-products
produced during fermentation.
46
Figure 3.9b. Results for the cellobiose-fed electrochemical cell operated in batch for
~11 days T. pseudethanolicus. Orange circles indicate pH, black line indicates
current density, green boxes with Xs indicate cellobiose, red squares indicate lactate,
and blue diamonds indicate acetate.
47
3.3.5. Acetate Consumption in T. pseudethanolicus Biofilm Anode
To determine whether or not a mature biofilm was able to produce current
from acetate, 10 mM acetate was spiked into a xylose fed electrochemical cell that
had developed a mature biofilms and was deplete of electron donors. The acetate
spike generated j of around 1.2 A m−2
(Figure 3.10).
48
Figure 3.10. Current increase shown after 10 mM acetate was spiked into a xylose-
fed reactor that had reached a current density of 0.1 A m-2
.
49
3.3.6. Morphological Characterization of Biofilm
Scanning Electron Microscopy (SEM) images (Figure 3.11a−d) reveal biofilm
morphology consistent with previous literature reports on T. pseudethanolicus: a
drumstick-shaped structure emanating from a terminal, round mother cell (see white
squares in Figure 3.11b−d) (Lee et al., 1993; Zeikus et al., 1980). The bacteria appear
to be 2−3 μm in length, but some are branched or form chains of bacteria that are
stacked in a network. CLSM images (Figure 3.12) reveal a biofilm at least 150 μm
thick and with many peaks and valleys. CLSM images also show that live and dead
bacteria are relatively evenly distributed throughout the biofilm, but with a higher
number of dead cells closest to the anode.
50
Figure 3.11. SEM images of an anodic biofilm of Thermoanaerobacter
pseudethanolicus grown on 40-mM xylose. [White squares indicate drumstick shaped
structures. (a) A broad overview of the biofilm at 1000X. (b) Complex biofilm
morphology at 5000X magnification. (c) Multiple layers of cells extending from the
anode of the MEC taken at 5000X magnification. (D) Drumstick structure extending
from the cells at 15000X]
51
Figure 3.12. Representative CLSM images for a LIVE/DEAD assay for the anode
biofilm Thermoanaerobacter pseudethanolicus grown on 40mM xylose. Shown is a
cross section of the z-axis with the cylindrical anode on the bottom black part of the
image while the top part is the media. (a) Red shows the thickness (Lf) of DEAD
cells within the biofilm. (b) Green shows the Lf of LIVE cells within the biofilm. (c)
An overlay of red and green to show a holistic representation of LIVE/DEAD
distribution within the biofilm.
52
3.4. Discussion
T. pseudethanolicus was initially grown using acetate as a carbon source in H-
type microbial electrochemical cell (Fig. 3.4). The increase in current follows an
exponential increase, similar to those observed by G. sulfurreducens and T.
ferriacetica (Parameswaran et al., 2013; Marsili et al., 2010). The rate of current
increase was faster than that of G. sulfurreducens (Marsili et al., 2010), possibly due
to the faster kinetic growth rates under thermophilic conditions and the fact that T.
pseudethanolicus can also grow under fermentative conditions. The data fit the
Nernst-Monod equation with n = 1 with only slight deviation at the highest anode
potential (Fig. 3.5a). The good fit suggests that, similar to G. sulfurreducens, the rate-
limiting step for T. pseudethanolicus kinetics is an enzymatic step that is involved in
an electron transfer (Torres et al., 2010). The EKA for T. pseudethanolicus, however,
is slightly lower than that usually observed for G. sulfurreducens, ~ −0.15 V vs SHE
(Marsili et al., 2010; Srikanth et al., 2008), and T. ferriacetica, another Gram-positive
thermophilic ARB, at −0.128 V vs SHE (Parameswaran et al., 2013).
Current density is dependent on the medium pH (Fig. 3.6). As with other
ARB, including G. sulfurreducens (Srikanth et al., 2008), T. pseudethanolicus
generated larger j at higher pH values, although the trend became less pronounced
above pH 7. The strong effect of pH at lower pH likely was due to a proton-transport
limitation. Although the pH is that of the bulk, it is reasonable to assume that the pH
of the outer layer of the biofilm is similar to that of the bulk and that the pH gradually
decreased throughout the biofilm due to diffusion limitations (Marcus et al., 2011).
Proton diffusion out of the biofilm should have been enhanced by a higher diffusion
coefficient of the transporting buffer with thermophilic conditions (Parameswaran et
al., 2013). T. pseudethanolicus generated current over a wider pH range (pH 5.40−
8.27) than that observed for G. sulfurreducens (pH 5.8− 8.0) (Torres et al., 2008a;
Franks et al., 2009).
To further characterize T. pseudethanolicus as an anode respiring bacteria
(ARB), different carbon sources (xylose, cellobiose, glucose, and acetate) were
evaluated for electrical current generation in MXC. Maximum current densities
ranged from ~3.9 to ~6.5 Am-2
(See duplicate results 3.7b, 3.8b, 3.9b). The mature
53
(~77 days) T. pseudethanolicus biofilm did not produce current directly from xylose,
but instead fermented xylose into acetate (almost exclusively), eventually producing j
up to ~7.5 A m−2
from the fermentation products (Fig. 3.7a). The pH was stable
throughout the experiment, although the pH was lowered initially as a result of acetate
accumulation (Hosono et al., 1995) and increased toward the end of the experiment as
a result of acetate depletion. The anion exchange membrane (AEM) (AMI 7001,
Membranes International, Glen Rock, NJ) is thermostable up to 90 °C, and is rated for
pH < 10. The pH increase observed after j reached 0 Am−2
(Fig. 3.7a) may be the
result of OH− leakage through the membrane from the cathode to the anode.
A mature T. pseudethanolicus biofilm did not produce current directly from
glucose, but instead fermented glucose into lactate and acetate, producing j up to ~4.8
A m−2
from these fermentation products (Fig. 3.8a). Lactate production was much
more significant during fermentation than it was with xylose fermentation, as was
reported in a previous study (He et al., 2009). The pH remained relatively stable over
the course of the experiment, although it was initially lowered as a result of acetate
and lactate production and increased at the end. The increase in pH at the end of the
experiment is partly a result of acetate depletion; however the increase prior to
complete acetate depletion may be the result of OH− leakage from the cathode to the
anode. CE for glucose was significantly higher than for xylose and similar to previous
observations with T. ethanolicus, a closely related bacterium (Lacis and Lawford,
1991).
As with the other substrates, T. pseudethanolicus did not produce current
directly from cellobiose, but fermented it first into acetate and other volatile acids and
subsequently produced j up to ~3.5 A m−2
from these fermentation products (Fig.
3.9a). Consistent with the xylose and glucose-fed reactors, pH remained relatively
stable over the course of the experiment, with a pH drop during the initial phase
resulting from the accumulation of acetate (Hosono et al., 1995). The Lag phase (~10
days) observed in Figures 3.7a, 3.8a and 3.9a are the result of a preference for T.
pseudethanolicus to ferment all possible substrates prior to anode respiration.
Fermentation of xylose, glucose, and cellobiose to acetate is more energetically
favorable in terms of Gibbs free energy, than is oxidation of acetate for anode
respiration, even at 0.42 V vs SHE. Thus, it is reasonable that T. pseudethanolicus
54
would perform anode respiration when there are no fermentable sugars remaining in
the MXC. This phenomenon is observed under all conditions investigated in this
study. It is observed that, in all MXCs, j appears to rise and fall during operation. This
may be caused by large pH gradients that develop in thick biofilms once anode
respiration occurs, driving j production to lower values. More investigation is needed
to confirm the limitations in T. pseudethanolicus biofilms.
Results indicated that T. pseudethanolicus performed fermentation (mostly to
acetate), not direct anode respiration from the fermentable substrates. This may be due
to the thermodynamic advantage of ATP production from the fermentation of xylose,
glucose, and cellobiose to acetate compared to the ATP-production capacity provided
by the oxidation of acetate by anode respiration with a fixed potential of 0.042 V vs
SHE. All reactors had accumulation of fermentation byproducts, primarily acetate,
prior to consumption for anode respiration. This accumulation likely was due to the
limited size of anode surface area (either 5.0 or 5.2 cm2 per 350 mL). Future research
can investigate the effects of anode surface area, and a larger specific surface area
should minimize acetate accumulation by being a faster acetate sink. While the T.
pseudethanolicus biofilms consumed acetate derived from fermentation, anode
respiration was not established in an acetate-fed batch MXC. Replicate trials yielded
almost no current generation, indicating that T. pseudethanolicus cannot develop
mature biofilms in electrochemical cells without an initial fermentation stage.
(Appendix S3 shows a representative run.). This phenomenon is likely due to the
limited inoculum size coupled with a minimal anode surface area. Without a
fermentable substrate, T. pseudethanolicus may not have achieved the biomass
necessary to attach to the anode. Theoretically, T. pseudethanolicus has more energy
to gain from fermenting cellobiose, glucose, and xylose to acetate than it does from
merely oxidizing acetate for anode respiration, even at 0.42V vs SHE. This being the
case, it is understandable that this microorganism would first ferment as much sugar
as possible before resorting to anode respiration
The ability of anode respiring bacteria to convert electrons into current is
known as coulombic efficiency (CE) (Razaei, 2008). It is an important consideration
when it comes to assess MXC performance. CE values varied among substrates:
glucose at 65.3% ± 1.0% > xylose at 34.8% ± 0.7% > cellobiose at 27.7% ± 1.5%. No
55
hydrogen or methane gas was observed in the gas phase of the anode chamber in any
of the reactors. For comparison, previous reports for T. ethanolicus, a closely related
thermophile, had 22−26% of electrons from xylose and 12−18% of electrons from
glucose being utilized for cell yield (Lacis and Lawford, 1991). It is likely that a
significant fraction of electrons not counted in the CE were contained in biomass
and/or EPS. Previous literature using fermentative MXCs (Lee et al., 2008) reported
the fraction of electrons used for growth by ARB in the biofilms anode were as much
as 1.5- to 2-fold higher than those used for fermentative growth. Substrate-specific
differences in CE also may have been caused by differences in end-product formation
as a result of the various metabolic pathways associated with transporting and
metabolizing each substrate (Hemme et al., 2011; Lacis and Lawford, 1991;
Stouthamer, 1979).
Characterization of the biofilm anode with light and electron microscopy, in concert
with electrochemical observations, showed that high j was achieved from xylose,
glucose, and cellobiose in a single step by a bacterial monoculture capable of forming
thick biofilms and transferring electrons to an anode (Fig. 3.11 and 3.12). These
findings facilitate the exploration of new EET mechanism(s) used by an increasingly
diverse set of thermophilic Gram-positive ARB.
3.5. Conclusions: Outlook of the Physiological and Practical
Implications of Current Production by Thermoanaerobacter
pseudethanolicus
The lag phase (~10 days) observed in Figures 3.7a, 3.8a, and 3.9a is the result
of a preference for T. pseudethanolicus to ferment all possible substrates prior to
anode respiration. Fermentation of xylose, glucose, and cellobiose to acetate is more
energetically favorable in terms of Gibbs free energy, than is oxidation of acetate for
anode respiration. Thus, it is reasonable that T. pseudethanolicus would perform
anode respiration when there are no fermentable sugars remaining in the MXC. This
phenomenon is observed under all conditions investigated in this study. (For fraction
of electrons accumulated in each substrate, see Figure S4, S5 and S6 in the
Appendix.) It is observed that, in all MXCs, j appears to rise and fall during operation.
This may be caused by lower pH that develops in the biofilm toward the end of the
56
fermentation process, which negatively affects the energetics of anode respiration.
More investigation is needed to confirm the limitations in T. pseudethanolicus
biofilms. Our results show that T. pseudethanolicus 39E was able to produce j
comparable to other ARB through the sequential fermentation of sugars and anode
respiration of the fermentation products. T. pseudethanolicus joins a cohort of fewer
than 10 ARB isolates, including Geobacter sulfurreducens (Bond and Lovley, 2003),
Thermincola ferriacetica (Parameswaran et al., 2013), Geoalkalibacter ferrihydriticus
(Badalamenti et al., 2013), Geoalkalibacter subterraneus (Badalamenti et al., 2013),
and Tolumonas osonensis (Luo et al., 2013) capable of high j (>2 A m−2
) (Torres et
al., 2014). It is the third thermophilic ARB isolated and the only one capable of
growing by fermentation (Parameswaran et al., 2013; Wrighton et al., 2011). The
capability to simultaneously ferment sugars and convert fermentation products to
current opens up new MXC applications related to using bacterial monocultures for
the thermophilic conversion of cellulosic and lignocellulosic byproducts into energy-
rich products or electrical power. For this conversion, T. pseudethanolicus may be
used alone or combined with other efficient cellulose degraders. T. pseudethanolicus
biofilms are thick and are limited by proton diffusion; however, very little is known
about the EET mechanisms used by Gram-positive bacteria (Carlson et al., 2012),
making future studies to elucidate these mechanisms of particular interest.
57
CHAPTER 4
Microbial diversity and metagenome prediction
approaches to characterize electrochemically
active biofilms derived from different inocula in
single-chamber microbial electrolysis cells
58
4.1. Background
Anode respiring bacteria (ARB) grow as biofilms on electrodes and are
capable of converting a wide range of biodegradable substrates into energy and
valuable by-products (Logan et al., 2006; Lovley, 2006; Torres et al., 2010). Recent
efforts by our group and other researchers have reported the identification of novel
ARB from diverse environments e.g. sediments, saline microbial mats, marshes, and
compost (Miceli et al., 2012; Parot et al., 2008a; Parot et al., 2008b; Ribot-Llobet et
al., 2014; Gregory et al., 2004; Bond et al., 2002). Zhang and Angelidaki (2012)
showed generation of electricity using a bio-electrode system with soil sediment
inoculum from eutrophic lakes. Song et al. (2012) used sediment inoculum collected
from a fresh water lake to produce current in single-chamber MECs. ARB that
perform direct oxidation of organic acids or sugars have also been identified from
sediments (Chaudhuri and Lovely, 2003).
Previously, studies that employed deep-sequencing techniques such as
Pyrosequencing (Parameswaran et al., 2010; Yang et al., 2012; Ruiz et al., 2014)
reported that less abundant microorganisms in the inoculum are selectively enriched
to higher abundance in the community during current generation. Current densities
have been almost always positively correlated with the presence of well-characterized
ARB belonging to the genus Geobacter. Nonetheless, Miceli et al. (2012) reported
that Geobacter dominated only in two high current producing biofilm communities
out of seven and the rest of the samples contained other electrochemically active
bacteria. Other studies also document that benthic MXC selectively enriched for
predominant electroactive bacteria belonging to δ- and ɣ-proteobacteria (Martins et
al., 2010), while Angelidaki et al. (2012) demonstrated electroactivity from
Flavobacterium and uncultured Rhizobium sp. isolated from an autotrophic lake.
Even though sediment enrichments of anode biofilm communities have been
characterized before, a comprehensive analysis of anodic microbiota structure and its
functional potential relevant to current generation have not been fully explored. The
use of advanced metagenome prediction tools such as phylogenetic information of
communities by reconstruction of unobserved states (PICRUSt) (Langille et al., 2013)
and microbial community distribution analysis over a landscape of key parameters
59
using Principal Component Analysis (PCoA) have strengthened the current
understanding of environmental niches such as the human and animal gut
microbiomes, anaerobic digesters, activated sludge processes etc. (Angenent et al,
2011; Zhou et al, 2015). Ishii et al. (2013) analyzed the metagenomes and
metatranscriptomes of anode microbial communities to evaluate indicators of anode
respiration and other relevant metabolic potential. They identified the occurrence of
core genes responsible for extracellular electron transfer (EET) within a complex
microbial community and its correlation with MXC performance.
The goals of study were: 1) enrich ARB from novel inocula sources, 2)
determine if enriched biofilms anodes are similar to other known communities in
ways other than presence of Geobacter, and 3) analyze metagenome prediction to
identify common gene pathways across high current producing communities. With a
wide variety of unidentified bacteria, it is important to look for microorganisms which
are more stable and efficient for domestic or industrial applications. I targeted lake
sediments because of their high nutrient availability which provides a suitable
environment for bacterial growth. Most of the efficient strains of ARB have been
isolated from sediments and aquatic environments. Mixed culture communities have
the ability to consume a wide range of substrates and fermentation products, which
indicates that mixed microbial communities can serve to be a good research tool for
MEC scale-up. I used sequencing data previously published by our group (Miceli et
al., 2012) (http://pubs.acs.org/doi/abs/10.1021/es301902h) and combined it with data
generated in this study to generate a heat map of gene presence during anode
respiration and electron transfer in electrochemically active microbial communities.
60
4.2. Materials and Methods
4.2.1. Sampling
Samples from three lake sediments were collected aseptically in sterile 50 ml
falcon tubes from (1) The Wilma H. Schiermeier Olentangy River Wetlands Research
Park, Columbus, Ohio (BOHW) (40°01'09.54"N: 83°01'06.90"W) (2) Broadmeadows
Park Lake, Columbus, Ohio (BOHL) (40°04'25.76"N: 83°02'00.93"W) and (3) a
representative sample of three lakes e.g. McKellips Lake, Vista Del Camino Lake and
Eldorado Lake, connected with each other via water channels situated in Scottsdale,
Arizona (BSDL) (33°27'33.01"N:111°54'46.19"W) (4) Tempe Town lake, Arizona (5)
Camp Verde, Arizona. Samples were collected from 15-20 cm depth below the
sediment surface, sealed immediately upon sampling to ensure anaerobic conditions,
and transported to lab and stored at 4 ºC.
4.2.2. MEC Design and Operation
Single chamber Microbial electrolysis cells (MEC) made from glass bottles
(volume 320ml), consisting of two cylindrical rod-shape graphite electrodes (anode
and cathode) with surface area of 6.1 cm2 along with sterilized Ag/AgCl reference
electrode (Bioanalytical Systems Inc.) were used to investigate the relation of
bacterial community structure with their ability to generate electric current (Fig. 4.1).
Before assemblage of reactor, anaerobic media was prepared by sparging it with
80:20 N2:CO2 for 30 min, autoclaved and then transferred to glass bottles along with
enrichment culture in an anaerobic glove box (Coy Laboratory, Michigan, USA). The
atmosphere of anaerobic glove box was maintained at ~ 97% nitrogen: 3% hydrogen.
All experiments were conducted in a constant room temperature (30°C).
61
Fig. 4.1. Illustration of single chamber microbial electrolysis cell (MEC) used to
enrich electroactive anode biofilms (AEM)
A
n
o
d
e
C
a
t
h
o
d
e
POTENTIOSTAT
Gas Bag
Cathode Anode
Reference Electrode
Anode Respiring
Bacteria (ARB)
Liquid Inlet/Outlet
Growth Media
Single Chamber Microbial Electrolysis Cell (MEC)
62
4.2.3. Electrochemical Set-up
All experiments were conducted using a potentiostat (BioLogic VMP3),
equipped with 12 independent channels. The anodes were poised at -0.2 V (vs)
Ag/AgCl reference electrode and later on values were converted to (vs) SHE
assuming a conversion factor of -0.205 V between Ag/AgCl and SHE. Cyclic
voltammetry (CV) was performed during turnover conditions at a scan of 1 mV s-1
.
4.2.4. Bacterial inoculum and growth medium
The growth media was prepared as reported by Kim et al., 2005. It contained
NH4Cl (0.33 g L−1
), KCl (0.33 g L−1
), trace mineral (10 mL/L) and vitamin (10 mL/L)
solutions, Na2HCO3 (50mM) and FeCl2 (1mL/L) (Balch et al., 1979). For the
establishment of biofilms 5g sediment, as recommended by Delgado et al. (2014), and
for successful anaerobic enrichments was inoculated into the pre-autoclaved reactors
containing 320ml of culture media (~pH 7.2) and 25mM acetate as the electron donor
for environmental samples operated at 30°C. Selective enrichment of the biofilm
community was done by more than three successive transfer’s cultivations in which
90% medium in the anode chamber was replaced by fresh medium containing carbon
source. The spent medium was refilled regularly and acetate consumption was
monitored through High Performance Liquid Chromatography using a previously
described method (Parameswaran et al., 2009). Anode biofilms after the 4th
fed batch
cycle were used for cyclic voltammetric experiments.
63
Fig. 4.2. Schematic illustration of experimental set-up used for enrichment of ARB
from diverse environments. (a) Anaerobic growth media prepared and stored in
serum bottles for later use in single chamber electrolysis cells (MECs) (b) Sediment
samples were inoculated to grow possible ARB on anode surface (c) mature biofilms
were scraped and shifted to sterile electrochemical cells to form biofilm and to get rid
of unwanted bacteria (d) after achieving maximum current densities biofilms were
removed for DNA extraction to identify bacterial communities on anode.
Growth Media in
Serum Bottles
Replaced (3-4)
times
Biofilm
Scraped
Anode
Biofilm
DNA Extraction
Single Chamber Microbial
Electrochemical Cell
Sediment
Inocula
(a)
(b)(c)
(d)
64
4.2.5. Gas Chromatography
We analyzed gas samples using gas chromatograph (GC 2010, Shimadzu)
equipped with a thermal conductivity detector and packed column (ShinCarbon ST
100/120 mesh, Restek Corporation, Bellefonte, PA) for separation of sample gases.
Samples were collected from headspace of the MEC using gas-tight syringe (SGE 500
μL, Switzerland). Carrier gas (N2) was fed at constant flow rate of 10ml/min.
Temperature conditions for the injection, column and detector were 110, 140, and 160
°C, respectively (Parameswaran et al., 2011).
4.2.6. DNA extraction
After two months of enrichment using MEC, graphite rods were cut and the
biofilm samples were scrapped from the anode using sterile scissors and syringe
needle. The biofilm samples was rinsed with sterile deionized water and centrifuged
at 10,000 rpm to remove the supernatant. Approximately, 0.30g pellet was used for
DNA extraction. PowerSediment DNA isolation kit (MoBio laboratories, Inc.,
Carlsbad, CA) was used to extract total DNA. The quality of extracted DNA was
measured by using a nanodrop spectrophotometer at 260 and 280nm (Ruiz et al.,
2014).
4.2.7. Pyrosequencing Analysis
Extracted DNA from anodic biofilms and lake sediments were sent to MR
DNA, Molecular Research Laboratory located in Shallowater, TX for Pyrosequencing
with Genome Sequencer FLX-Titanium system to fingerprint the bacterial community
(Miceli et al., 2012). I used “blue” primers (104F, 530R) targeting V2-V3 regions of
the bacterial 16S rRNA gene (Miceli et al., 2012). The sequences were submitted to
NCBI Sequence Read Archive (SRA) and can be accessed under following accession
numbers; NCBI:SAMN03196398, NCBI:SAMN03196399, NCBI:SAMN03196400,
NCBI:SAMN03196401,NCBI:SAMN03196402,NCBI:SAMN03196403,NCBI:SAM
N03278675,NCBI:SAMN03278678,NCBI:SAMN03278687,NCBI:SAMN03278685,
NCBI:SAMN03278689,NCBI:SAMN03278683NCBI:SAMN03278672,NCBI:SAM
N03278682,NCBI:SAMN03278688,NCBI:SAMN03278680,NCBI:SAMN03278690,
NCBI:SAMN03278686, NCBI:SAMN03278676 and NCBI:SAMN03278674. For the
identification of community structure and diversity, sequences were analyzed using
Quantitative Insights into Microbial Ecology (QIIME) suite version 1.7.0 (Caporaso
65
et al., 2010). The numbers of sequences obtained per sample (after filtering for quality
control--minimum sequence length 250bp) were as follows: BSDL (3279), BOHL
(5926), BOHW (8730), SSDL (3446), SOHL (4459) and SHOW (5210), respectively.
In order to reduce bias, I rarefied each sample to 3251 sequences before calculating
diversity indices. I discarded the sequences with at least one of the following
properties: shorter than 250 bp, with primer mismatches, with homopolymers of more
than 6 base pairs or a quality score of <25. After screening I performed clustering and
defined operational taxonomic units (OTUs) at 97 % sequence similarity with
UCLUST (Edgar, 2010) and assigned taxonomy to the OTUs by using the RDP
classifier (Wang et al., 2007). For α- and β- diversity calculations, I generated the
phylogenetic tree using Fastree (Price et al., 2009) and calculated Phylogenetic
Distance Whole Tree index (Faith, 1992), Chao1 index (Chao, 1987), and the Unifrac
distance (Lozupone et al., 2006). The values represent average of 10 iterations and ±
sign represents the standard deviation. In order to identify which microorganisms
possibly contribute to current generation, I calculated Pearson’s correlation coefficient
and validated the strength of the relationship if P value was smaller than 0.05.
4.2.8. PICRUSt Analysis
Sequencing data from sixteen samples (thirteen samples from Miceli et al.
(2012) and three samples from the present study) was used to predict metagenomes
from 16S rDNA using Phylogenetic Investigation of Communities by Reconstruction
of Unobserved States (PICRUSt) (Langille et al., 2013) to determine in silico gene
abundances relevant to electron transport.
4.2.9. Principal Coordinate Analysis (PCoA)
EMPeror: a tool for visualizing high-throughput microbial community data
was used in present study to visualize similarities or dissimilarities of data in the form
of principal coordinates based on taxa from enriched as well as environmental
samples. Each sphere represents a single community (Vazquez-Baeza et al., 2013).
66
4.3. Results
4.3.1 Current Generation using enriched biofilms
A total of five environmental sediment samples as inocula were tested to
develop electroactive biofilms on anode using single chamber microbial electrolysis
cells. Biofilms developed within ~3-6 days after inoculation in three out of five
environmental samples during batch operation of MEC. After reaching maximum a
stable current density, low scan cyclic voltammetry (LSCV) was performed on the
three successful samples. Maximum current densities achieved by the respective soil
samples after multiple media replacements were: BSDL ~6.3 Am-2
, BOHW ~7Am-2
,
and BOHL ~3 Am-2
(Fig. 4.3, 4.4 and 4.5). At the same time, current density from
other samples such as Tempe Town Lake, AZ and Camp Verde, AZ did not reach
current densities higher than 0.1 A/m2 (data not shown) and hence were discarded
from the list of candidate samples. Coulombic efficiency (%) i.e., number of circuited
electron equivalents over the total electron equivalents of acetate consumed in the
anode, was: BOHW 45, BSDL 52, BOHL 8.
67
0
1
2
3
4
5
6
7
0 2 4 6 8 10 12 14
Cu
rren
t D
en
sity
Am
-2
Duration of batch operation (Days)
Inoculation
Transfer # 1
Transfer # 2
Transfer # 3
Fig. 4.3. Current density profiles of sediment sample collected from BSDL (After
replacing growth media with fresh medium approx. 2-3 times, bacterial biofilms were
scrapped from anode surface and transferred to new reactors). Inoculation- biofilm
formed using sediment inoculum (light green line), Transfer 1- scrapped biofilm
(orange line), transfer 2- media replacement (dark red line), and transfer 3- media
replacement (blue line).
68
Bacterial communities present in sediment sample collected from lake in
Scottsdale, Arizona (BDSL) started forming biofilm on third (3rd
) day of inoculation
(Fig. 4.3). Reactor was allowed to operate (10days) until maximum stable current
density (~1.2 Am-2
) was achieved. Bacterial biofilm was scrapped and transferred to a
new sterile reactor to get rid of unwanted bacteria. Increase in current density was
observed each time media was replaced. (see Fig. 4.3: transfer-1, transfer-2 and
transfer-3). Transfer-2 produced ~2.6 Am-2
of current density, before going into
decline phase. However, Transfer-3 showed log phase for almost three days followed
by stationary phase producing a current density of ~3.8 Am-2
.
69
Fig. 4.4. Current density profiles of sediment sample collected from BOHW (after
replacing growth media with fresh medium approx. 2-3 times, bacterial biofilms
were scrapped from anode surface and transferred to new reactors for enrichment
purpose). Inoculation- biofilm formed using sediment inoculum (Dark red line),
Transfer 1- media replacement (light blue line), transfer 2- media replacement (light
green line), and transfer 3- scrapped biofilm (blue line).
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 14 16
Cu
rren
t D
esn
ity
Am
-2
Duration of batch operation (Days)
Inoculation
Transfer # 1
Transfer # 2
Transfer # 3
70
Bacterial communities in sediment sample collected from wetland located in
Columbus, Ohio started forming biofilm on anode surface after 3 days of inoculation
and produced a maximum current density of ~7Am-2
. Log phase was observed
between ~8 to 11 days, followed by gradual decrease in current density. Growth
media was replaced with fresh medium supplied with 25mM acetate (transfer-2).
During batch operation, methane gas production was observed in the BOHW MEC
(transfer-2), indicating the presence of methanogens in the reactor. To avoid further
growth of methanogens, the biofilm was scraped and inoculated into a new sterile
reactor in order to enrich only ARB (transfer-3). Bacterial communities took (transfer-
3) one day to form biofilm on anode surface and generated a maximum current
density of 5.9 Am-2
. Transfer-3 MEC showed similar trends in current density
including a log phase followed by decline phase.
71
Fig. 4.5. Current density profiles of sample collected from BOHL (after replacing
growth media with fresh medium approx. 2-3 times, bacterial biofilms were scrapped
from anode surface and transferred to new reactors). Inoculation- biofilm formed
using sediment inoculum (purple line) (Purple line), transfer 1- media replacement
(blue line), transfer 2- scrapped biofilm (light green line), and transfer 3- media
replacement (dark red line).
0
0.5
1
1.5
2
2.5
3
3.5
0 2 4 6 8 10 12 14 16 18
Cu
rren
t D
esn
ity
Am
-2
Duration of batch operation (Days)
Inoculation
Transfer # 1
Transfer # 2
Transfer # 3
72
Microbial electrolysis cell (MEC) inoculated with sediment sample collected
from lake in Columbus, Ohio, produced current density of ~0.2 Am-2
(Fig. 4.5).
Initially reactor didn’t perform well in terms of current generation, so, media was
replaced after ~16 days of inoculation. Transfer-1 MEC showed a lag phase of 6 days,
followed by log phase which remained for ~ 5 days producing a current density of
~1.8Am-2
. Biofilm was scarped and transferred to new sterile reactor (transfer-2),
which produced maximum current density of ~3.0Am-2
. Media was replaced when
current density dropped to below 1.0Am~. Transfer-3 MEC showed immediate
response to fresh media by generating current density upto ~2.3Am-2
.
4.3.2. Electrochemical Characterization of anodic biofilms
After multiple enrichments, cyclic voltammetry (CV) was performed when
biofilms (BOHW, BSDL and BOHL) reached maximum steady current.. Mid-point
potentials (EKA) of electroactive anode biofilms (EABs) was determined which is
basically the potential at which half the maximum current is produced when sweeping
the potential and depends on the electron transfer mechanism of the ARB (Miceli et
al., 2012). Mid-point potential (EKA) for the anode biofilm was determined by
derivatives of the LSCVs were; BSDL -0.211V vs SHE, BOHW -0.201V vs SHE and
BOHL -0.226V vs SHE at pH 7.2 (Fig. 4.6).
73
(a)
b)
-0.5
0
0.5
1
1.5
2
2.5
-0.6 -0.4 -0.2 0 0.2 0.4
Cu
rren
t D
en
sity
(A
m-2
)
Anode Potential (vs) SHE
N-M fit
n = 1
EKA = -0.211 V
-0.3
0.2
0.7
1.2
1.7
2.2
2.7
-0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2
Cu
rren
t D
en
tist
y (
Am
-2)
Anode potential (vs) SHE
N-M fit
n = 1
EKA = -0.226 V
74
C)
Fig. 4.6. Cyclic voltammetry analysis of biofilms in acetate-fed MEC containing
different sediment samples: (A) BOHL, (B) BSDL (C) BOHW
-2
0
2
4
6
8
10
-0.6 -0.4 -0.2 0 0.2 0.4
Cu
rren
t D
en
sity
(A
m-2
)
Anode potential vs SHE (V)
N-M fit
n = 1
EKA = -0.201 V
75
4.3.3. Comparative bacterial community structure analysis
The diversity estimating parameters were calculated for richness: Chao1, for
diversity: PD whole tree, and for evenness: equitability (Table 4.1). The richness and
diversity indices indicated that sediments were more diverse than biofilm samples in
an agreement with (Miceli et al., 2012), and as expected the enrichment technique
reduced both microbial richness (Chao1 richness from 726-801 to 100-224) and
diversity (PD whole tree diversity from 38-47 to 6.7-9.9). Both richness and diversity
indices of Chao1 and PD Whole Tree negatively correlated with current density
(Pearson correlation coefficient = 1.00, P < 0.01).
76
Table 4.1: Bacterial diversity indices on anodic biofilms with different sediments
samples
Diversity
index
Olentangy River Wetlands
Research Park Broadmeadows Park Lake Scottsdale Sample
Sediment
(SOHW)
Biofilm
(BOHW)
Sediment
(SOHL)
Biofilm
(BOHL)
Sediment
(SSDL)
Biofilm
(BSDL)
Chao1 Richness 801.2±9.9 224.7±12.4
785.8±8.0 100.9±8.7
726.5±5.1 122.9±0.6
Phylogenetic
Diversity
Whole Tree
Diversity 45.3±0.3 9.9±0.3
38.7±0.3 6.7±0.2
47.5±0.1 7.3±0.0
Equitability Evenness
0.9±0.0
0.6±0.0
0.9±0.0
0.4±0.0
0.9±0.0
0.6±0.0
77
Pearson’s correlation was performed to find out if there is any linear
relationship between microbial groups and power generation. (Fig. 4.7) δ -
proteobacteria showed significant correlation (r=0.78) towards higher current
generation in BSDL and BOHW, as expected given that they are low-potential
anaerobic respirators. On the contrary, ɣ-proteobacteria (r=-0.99) and β-
proteobacteria (r=-1) which are high-potential anaerobic respirators showed a
negative correlation, thereby these bacterial groups may have negatively impacted
current generation (Grüning et al. 2015). This can be seen in BOHL sample where
Clostridia was abundant and produced only 3.0 Am-2
(Fig.3.), and when they are
lower in abundance, other organisms were capable of better current production are
present (BOHW 7.0 Am-2
).
78
Fig.4.7. Phylotype distribution of sediments (top) and enriched biofilms (bottom)
from diverse environments at the class level. The scale bars show the relative
abundance of each class within a sample. Minor phyla accounting for <0.5% of total
sequences are summed in the group ‘other’. Right corner shows the Pearson’s
correlation coefficient between current density and community members.
79
4.3.4. Bacterial population dynamics using principal coordinate analysis (PCoA)
The environmental and biofilm enriched bacterial communities from this study
were combined with the communities reported by Miceli et al. (2012), creating a
library of 10 high-current producing biofilm enrichments. The weighted Unifrac
distances among these communities are visualized using PCoA(Fig. 4.8). All of the
environmental samples clearly cluster together and away from the majority of the
biofilm samples. The biofilm samples on the other hand did not show a single
clustering pattern even though they were distant from the sediment samples. The four
sediments including BSDL, Superior biofilm, Cuzdrioara biofilm, and BOHW
indicated significant concentrations of Geobacter related sequences forming a cluster
on the bottom left of the plot after enrichment. The two Geoalkalibacter dominated
biofilms, Kochin Shoreline and Playa Sucia Mangrove, clustered nearly on top of
each other. Mayaguez and BOHL appeared up to the top, forming a cluster of the two
heavy communities of Clostridia. The Salt Flat biofilm was seen close to the cluster
of environmental samples, while, the Carolina Mangrove biofilm sits in the middle of
them due to the high diversity found in both samples and the relatively minor changes
in community structure which Carolina Mangrove underwent during enrichment.
80
Fig 4.8. PCoA analysis of environmental and biofilm enriched bacterial communities,
based on weighted UNIFRAC distance metric, including communities developed in
this study as well as those developed in Miceli et al. 2012.
81
4.3.5. Predicted genes encoding proteins of anode related metabolism using
PICRUSt
Based on metagenome predictions from 16S rDNA gene abundances using
PICRUSt, metabolic pathways were predicted and analyzed for key gene families
related to anaerobic respiration; biosynthesis of c-type cytochromes, cell division,
carbohydrate metabolism and transporters. The 16S rDNA gene sequencing data from
present study was compared with the data of Miceli et al. (2012) (Fig. 4.9). The
relative abundances of the predicted genes encoding for proteins involved in
anaerobic respiration were greater in the biofilm samples compared to the bacterial
communities in sediment samples. Two out of the three highest current generating
Geobacter-related biofilms (Cuzdrioara and Kochin Shoreline biofilms) had higher
abundances of predicted genes coding pilin, NADH dehydrogenase, and F- type H+
transporting ATPases related to “respiratory metabolism” compared to other biofilms
samples. However, lower gene expression for c-type cytochromes related to EET
pathways were observed in Geobacter-related high current generating biofilms.
Mangle Carolina, the non-Geobacter biofilm generated highest current density
(10.77 A/m-2
), even though showed less abundance of certain proteins which were
abundant in Geobacter-related biofilms. However, low expressions of c-type
cytochrome genes were observed in Mangle Carolina possibly belonging to other
bacterial groups. Likewise, low current producing non-Geobacter biofilm
“Mayaguez” showed abundance of NADH quinone oxidoreductase E, electron
transport flavoproteins (etfA1, etfB1) and low abundance of type IV pilus assembly
proteins (figure 4.9).
Predicted abundant genes belonging to Geobacter sulfurreducens found in highest
current producing biofilms were encoding for atpB and atpX, atpF, F-type H+-
transporting ATPase subunit b. Other Geobacter-related predicted genes included
nuoE-1 and nuoG-1, NADH dehydrogenase I subunit E (K00334) and NADH
dehydrogenase I subunit G (K00336). Type IV pilus assembly protein PilB (K02652)
belonging to Geobacter sp. was also found abundant in highest current producing
biofilms (Pohlschroder and Esquivel, 2015). Butler et al. (2010) explained evolution
of electron transfer out of the cell by studying genomics of six Geobacter genomes.
82
Fig. 5. PICRUSt predictions of genes that code for enzymes involved in cellular and energy processes.
Fig. 4.9. PICRUSt predictions of genes that code for enzymes involved in cellular and energy processes
83
4.4. Discussion
Single chamber microbial electrolysis cells (MEC) inoculated with three different
lake sediments were monitored for electricity generation using acetate as carbon source.
The initial aim of the study was to enrich electrochemically active anode respiring
bacteria from diverse environments on anode surface. Maximum current production using
sediment inoculum was observed in BOHW (~7Am-2
) following by BSDL (~6.3Am-2
)
and BOHL (~3Am-2
) (Fig. 4.3, 4.4, 4.5). BOHW showed maximum reactor performance
with initial biofilm derived from sediment inoculum (Fig. 4.4), but decrease in current
density (~5.9Am-2
) was observed in second generation biofilm derived from biofilm
sample developed in the first phase. Loss in reactor performance (~3.2 Am-2
) was also
observed when growth media was refreshed containing 25mM acetate. This justified
Rivalland et al. (2015) observation where he mentioned decrease in bacterial diversity
effects performance of MEC reactor. It indicated that species richness was an important
when it comes to bacterial communities functioning and its related current generation
ability (Bell et al., 2005). Results obtained from BOHW reactor confirmed report of
Rivalland et al. (2015), that current generation by first generation of electroactive anode
biofilm (EAB) was higher than further generations. However, EAB derived from BOHL
and BSDL opposed finding of Rivalland et al. (2015), when current production was
higher in further generations (Fig. 4.3, 4.5). Other possible reason for loss in reactor
performance in second or third generation EABs might be that different bacterial
communities in the MEC reactor used direct or indirect pathways to transfer electrons to
the electrode. In the first run (initial growth media), all endogenous mediators
accumulated by bacteria washed out when fresh media was supplied or replaced (Salvin
et al., 2012).
Liu et al. (2008) also discussed decrease in reactor performance using four
generation biofilms, where only the second generation EAB produced highest current
densities than the first EABs. As discussed earlier, it could be due to the presence of other
bacteria in the growth medium of MEC, playing role in electron transfer which got
removed during media replacement (Rimboud et al., 2014; Caporaso et al., 2011; Quast
et la., 2013). Other possible reasons for decrease in current density like observed in
84
BOHL MEC (3Am-2
) could be due to (1) biofilm loss during media replacement, (2) low
availability of bacteria in the lake sediments (Lin et al., 2013) (3) Aging of biofilm
(Bridier et al., 2015). Besides, the increase in thickness of biofilm sometime reduced
substrate diffusion or mass transfer rate to cells (Virdis et al., 2014; Renslow et al., 2013).
However, mixed culture biofilms and G. sulfurreducens follow different strategies so
cannot be compared directly. The resumption of similar current density after replacement
of growth media with fresh nutrient medium suggested that that planktonic cells or any
soluble electron shuttles were not playing any role for electricity generation, and current
was generated only by electroactive anode biofilm (EAB) (Badalamenti et al., 2013;
Zhang et al., 2012b) (Fig. 4.3, 4.4, 4.5).This was also justified by cyclic voltammetry
results (Fig. 4.6 a,b,c)
The gradual increase in current density was observed after reusing the previously
established biofilm of EAB on anode in the new reactor, Similar trend was observed in
MEC when run with BSDL (Fig. 4.3) and BOHL (Fig. 4.5) samples and was also
reported in a number of previous reports (Liu et al., 2008; Rabaey et al., 2013; Kim et al.,
2005, Blanchet et al., 2014; Baulder et al., 2014; Salvin et al., 2012). It took second
generation EAB an about 24 hrs to redevelop biofilm on anode surface and to regain cell
potential (Fig. 4.4, transfer 3) (Salvin et al., 2012). This might be due to enrichment and
acclimation of specific bacterial community in the acetate fed reactor (1st phase) that
regained its potential when allowed to reform in a new reactor (2nd
phase) rapidly. .
Similar observations were recorded in previous studies when they re-inoculated the
biofilm in subsequent reactors to develop second generation electroactive biofilms
(Miceli et al., 2012; Behera et al., 2013; Ghizelini et al., 2012).
Increase in current density (log phase ~10 days) after lag phase of ~4-6 days was
observed in BOHW and BOHL MEC, which closely related to results reported by Zhang
et al. (2012) (Fig. 4.4, 4.5). However, BSDL showed lag phase of 1~ day when second
generation biofilm was used (Fig. 4.3, Transfer 1) and current density remained stable for
~7 days (Fig. 4.3, Transfer 2). Current density restored rapidly within hours when anodes
were transferred to fresh growth medium (Fig. 4.3, transfer 2 and 3; Fig. 4.5, transfer 3).
85
Low coloumbic efficiency (CE) was observed in BOHL reactor fed with acetate (25mM)
might be due to utilization of the substrate for bacterial growth instead of electricity
generation (Sharma and Li, 2010; Liu et al., 2005).
The data fit the Nernst-Monod equation with n = 1 with only slight deviation at
the highest anode potential (Fig. 4.6 a, b and c). The majority of the CVs fit the Nernst-
Monod model equation (Marcus et al., 2007), with some samples showing higher
potential losses than expected through this model. Biofilms with Geobacter as dominant
bacterial community (e.g. BSDL) tend to be in line with our previous findings (Miceli et
al., 2012), where reported EKA value for a Geobacter dominated biofilm was (-0.22 V vs
SHE). Nonetheless, these EKA values are lower than reported for G. sulfurreducens in
pure cultures -0.15 V vs SHE (Srikanth et al., 2008). The biofilms with lower abundances
of Geobacter sp. showed more negative EKA values, e.g. BOHL (-0.226 vs SHE).
Sediment sample collected from Arizona was rich in Geobacter sp., consistent with
previous studies (Miceli et al., 2012), as compared to samples collected from Ohio, USA.
It is interesting to see linear correlations between current densities and EKA value; the
higher the current density, the less negative the EKA value. The trend for current densities
from highest to lowest BOHW>BSDL>BOHL is opposite to EKA trend from more
negative to less negative BOHL>BSDL>BOHW.
Bacterial community analysis suggested that lower bacterial community diversity
resulted in higher performance of MEC reactors (Fig. 4.7), which contradicts with study
conducted by Rivalland et al. (2015). However, results from present study were similar to
those of Miceli et al. (2012). Moreover, diversity might lead to a reduction in current
generation possibly due to the presence of bacteria which diverted electrons to microbial
processes other than current generation. This also suggested that the enrichment of a few
electroactive species is the key for good reactor performance.
Bacterial community structure of the sediment samples used as inoculum and
biofilms developed in MECs were analyzed at the class level. Major identified phyla for
the three MEC biofilm anode samples were Proteobacteria, Firmicutes, Bacteroides and
86
Spirochaetes, which were similar to those observed by Fung et al. (2006). As previously
reported by Miceli et al. (2012), Arizona sediment sample (BSDL) was rich in δ -
proteobacteria, with the specific genus being mostly Geobacter. Bacterial communities
that develop in MECs from other reports also revealed that MEC biofilm communities
were dominated by δ-proteobacteria, followed by members of α-, β-, ɣ-, proteobacteria
(Logan and Regan, 2006). I also observed other bacteria present in the overall bacterial
communities of the MEC anodes belonging to phyla Tenericutes, Actinobacteria and
other uncultured bacteria. As discussed earlier, low current densities were produced by
EAB in BOHL reactor (1.5 to ~3Am-2
) possibly because of presence of organisms from
the class Clostridia (Miceli et al., 2012), which is also confirmed by Zhu et al. (2011).
Abundance of Deltaproteobacteria (Geobacter sp.) have been observed when acetate is
used in MECs inoculated with different inocula, which justified our results showing
abundance of thesis bacteria in BSDL and BOHW (Sun et al., 2015)
Principal coordinate analysis showed clear clustering of all the sediment samples
away from the majority of the biofilm samples, likely due to the high diversity in these
bacterial communities and the strict enrichment strategy used in the MXCs (Ishii et al.,
2013b; Lu et al., 2015) (Fig. 4.8). This analysis backs up the fact that biofilm anode
communities do not stem from a single group of bacteria and points out the need for
further work searching for novel ARB.
PICRUSt analysis (Fig. 4.9.) confirmed phenotypical potential of the biofilm
communities that varied depending on the inocula. Our results were related to Ishii et al.
(2013), though he used metatranscriptomic approach to identify gene expression during
EET. Predicted genes coding for enzymes involved in anaerobic respiration in BOWL
and BSDL supported the fact that these cultures were enriched with δ-proteobacteria,
Bacteroidia, Erysipelotrichi, Bacteroides, Clostridia and Spirochaetes that could carry
out exoelectrogenic activities. Butler et al. (2010)
87
4.5. Conclusions
The present study provided considerable insight into bacterial community
structure in lake sediments before and after enrichment in MXCs. Our results reaffirmed
the ubiquity of metal reducing Geobacter phylotypes in biofilms performing anode
respiration. The enriched biofilms were often close to monocultures having dominant
ARB generating maximum current densities, but it was not always the case. PICRUSt
analysis has been used for the first time for genome predictions of ARB in MXCs
revealing presence of certain proteins indicative of efficient ARB. Still, presence of
certain bacteria may lead to lower current densities in the reactors. PCoA of the enriched
bacterial communities showed that communities responsible for high current production
are more varied than sediment communities from which they derive, indicating that
further work identifying and characterizing novel ARB remains to be done.
88
Recommendation for Future Research
Microbial electrochemical cell (MXC) technology has proved to be useful to
understand fundamentals of novel anode respiring bacteria (ARB). Future research can be
focused on discovery of novel gram-positive thermophilic bacteria capable of
dissimilatory metal reduction. Bacteria with such capabilities can be characterized using
different microbial and electrochemical techniques e.g. cyclic voltammetry (CV),
chronoamperometry (CA), and potentially electrochemical impedance spectroscopy
(EIS), SEM, TEM, and Pyrosequencing (Badalamenti et al., 2013). Genome of anode
respiring bacteria should be sequenced in order to characterize them at genetic level.
Furthermore, genetic data of ARB can be used to drive efficient energy recovery
processes from wastes. In addition, proteomic studies would help researchers to identify
which proteins are translated and expressed during anode respiration.
Thermoanaerobacter is capable of utilizing complex organic molecules (sugars),
convert these sugars into organic acids and produce high current densities from the
oxidation of these acids. One might argue that for this process Thermoanaerobacter
needs high temperatures (60°C), which restricts the applicability of the bacterium. But on
the other hand, these high temperatures lead to improved kinetic conditions (diffusion
rates, probably also the cathode reaction of a respective MFC/MEC) and offer the access
to high temperature waste streams (e.g., from food industry). Thermophilic ARB can also
be used to generate hydrogen gas or ethanol either as single or co-culture which can
degrade complex substrates (Jessen and Orlygsson, 2012; Koskinen et al., 2008). Future
directions should be emphasized on co-culture studies, where two bacterial isolated work
together to degrade complex chemical substrate. More work is needed towards discovery
of novel thermophilic ARB, which can be done by isolating bacteria from natural
environments or by identifying them from previously published literature by researchers.
Optimization of operational conditions to enhance energy recovery processes is also an
important aspect words commercialization of MXC technology.
Characterization of multifunctional electroactive anode biofilms (EAB) from
diverse environments is of great value. Bacteria with capability of anode respiration and
89
extracellular electron transfer (EET) can be isolated from regions yet unexplored by
researchers. Further these mixed culture bacterial communities can be used to treat or
convert variety of wastewaters. In terms of isolation of uncultured microbes, MXC
technology comes handy. Tools like Pyrosequencing, PICRUSt and PCoA can be helpful
to isolate and characterize anode respiring bacteria (ARB) using MXC technology.
Newly discovered ARB along with intelligently engineered MXC deign can be useful in
terms of renewable energy and waste treatment.
90
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Appendix
Figure S1. Central carbon and energy metabolism of Thermoanaerobacter strains
(Hemme et al., 2011)
H2/e-
Acetate
Cellobiose
Xylose
Glucose
Ethanol
114
Figure S2. Initial growth of current density of the xylose-fed electrochemical cell.
115
Figure S3. A reactor fed with 10 mM acetate containing T. pseudethanolicus operated in
batch for 13 days showed no significant current density.
116
Figure S4 (a). Fraction of electrons captured as current, acetate, lactate and initial
substrate are shown as a percentage of the total electrons present in the initial substrate.
A. shows the results for 20 mM xylose-fed electrochemical cell from Figure 3.7a in the
main text.
117
Figure S5. Fraction of electrons captured as current, acetate, lactate and initial substrate
are shown as a percentage of the total electrons present in the initial substrate. It shows
the results for 10 mM glucose-fed electrochemical cell from Figure 3.8a in the main text.
118
Figure S6. Fraction of electrons captured as current, acetate, lactate and initial substrate
are shown as a percentage of the total electrons present in the initial substrate. It shows
the results for 7.5 mM cellobiose-fed electrochemical cell from Figure 3.9a in the main
text.
119
List of Publications
Journal Research Articles
1. Qaiser Farid Khan and Bradley G. Lusk, Prathap Parameswaran, Abdul
Hameed, Naeem ali, Bruce E. Rittmann, Cesar I. Torres (2015). Characterization
of electric current generation capabilities of the thermophile Thermoanaerobacter
pseudethanolicus using xylose, glucose, cellobiose, or acetate with a fixed anode
potential. Environ Sci Technol. 2015 Nov 24.
2. Mia Mae Kimco, Erhan Atci, Qaiser Farid Khan, Abdelrhman Mohamed, Ryan
S. Renslow, Nehal Abu-Lail, Boel A. Fransson, Douglas R. Call, and Haluk
Beyenal (2015). Vancomycin and maltodextrin affect structure and activity of
Staphylococcus aureus biofilms. Journal of Biotechnology and Bioengineering,
DOI: 10.1002/bit.25681.
3. Hamid Majeed, Yuanyuan Bian, Barkat Ali, Anjum Jamil, Usman Majeed,
Qaiser Farid Khan, Khalid Javed Iqbal, Charles F Shoemaker and Fang Zhong
(2015). Essential oil encapsulations: Uses, Procedures, and Trends. RSC
Advances, DOI: 10.1039/C5RA06556A.
Abstracts/ Poster presentations
1. Qaiser F. Khan, Bradley G. Lusk, Prathap Parameswaran, Naeem Ali, Abdul
Hameed, Bruce E. Rittmann, and César I. Torres. Characterization of the
electrical current generation capabilities of the thermophile Thermoanaerobacter
presudethanolicus, using a Microbial Electrochemical Cell. 114th General
Meeting, American Society for Microbiology, May 17-20 2014, MA, USA.
(Poster Presentation)
2. Qaiser F Khan, Bradley G Lusk, Prathap Parameswaran, César I Torres.
Characterization of the simultaneous fermentation and anode respiration
capabilities of the thermophilic Thermoanaerobacter pseudethanolicus in a
Microbial Electrolysis Cell (MEC). NA ISMET, May 13-15 2014, Pennsylvania,
USA.
3. Jonathan P. Badalamenti, Oluyomi Ajulo, Qaiser F. Khan, Prathap
Parameswaran, Naeem Ali, Abdul Hameed, Rosa Krajmalnik-Brown, and César I.
Torres. Characterization of newly discovered anode respiring bacteria. 4th
International Microbial Fuel Cell Conference, 1-4 September 2013, Australia.
4. Mia Mae Kiamco, Erhan Atci, Qaiser Farid Khan, Ryan S Renslow, Nehal Abu-
Lail, Boel A Fransson, Douglas R Call, Haluk Beyenal. Combination of
hyperosmotic agent and antibiotic enhances oxygen penetration into
120
Staphylococcus aureus biofilms. The symposium on Advanced Wound Care
(SAWC Fall), September 26-28, 2015, Caesars Palace, Las Vegas, Nevada, USA.
5. H. Beyenal, M. Kiamco, Qaiser F. Khan, E. Atci, A. Mohamed, E. Davenport, B.
Fransson, D. Call, N. Abu-Lail. Oxygen profiling and its biokinetic effects in
wound biofilms with and without treatment. The symposium on Advanced
Wound Care (SAWC Fall), October 16-18, 2014, Caesars Palace, Las Vegas,
Nevada, USA. (Poster Presentation)
6. H. Beyenal, M. Kiamco, Qaiser F. Khan, B. Fransson, D. Call, N. Abu-Lail, R.
Renslow. Hyperosmatic agents can enhance antibiotic efficacy against MRSA
biofilms. Wound Healing Society. April 23-17 2014, OR, Florida, USA.