start-up methods for the development of carbon …
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The Pennsylvania State University
The Graduate School
College of Engineering
START-UP METHODS FOR THE DEVELOPMENT OF CARBON DIOXIDE FIXING
AND BIOFUEL PRODUCING BIOCATHODES
IN BIOELECTROCHEMICAL SYSTEMS
A Thesis in
Environmental Engineering
by
Zehra Zaybak
2012 Zehra Zaybak
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Master of Science
August 2012
The thesis of Zehra Zaybak was reviewed and approved* by the following:
Bruce E. Logan
Kappe Professor of Environmental Engineering
Thesis Advisor
John M. Regan
Associate Professor of Environmental Engineering
William D. Burgos
Professor of Environmental Engineering
Peggy Johnson
Professor of Civil Engineering
Head of the Department of Civil and Environmental Engineering
*Signatures are on file in the Graduate School
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ABSTRACT
New alternative sources of energy and fuels are needed to meet increasing demands. In
microbial electrosynthesis carbon dioxide is converted into organic compounds for potential
biofuel production. Here, two start-up methods were developed and further improved for the
enrichment of CO2 fixing and biofuel producing biocathodes. In the pre-enrichment method,
anaerobic cultures inoculated with bog sediment were initially heterotrophically grown on
glucose then inoculated into bioelectrochemical system (BES) cathode chambers. Next, CO2 was
provided as sole carbon source and electron acceptor to select for those bacteria able to switch to
autotrophic growth. Following the 14 day heterotrophic start-up period, recorded current density
uptake gradually increased and was sustained over a period of about two months with
concomitant decreases in cathode chamber headspace CO2. Maximum current density consumed
was −34 ± 4 mA/m2 (maximum current consumption of −50 ± 6 µA) at a set potential of −0.4 V
[versus the standard hydrogen electrode (SHE)]. Production of hydrogen gas along with organic
acids (acetate: 1.90 ± 0.73 g/L, propionate: 2.09 ± 0.56 g/L, and butyrate: 2.25 ± 0.20 g/L) and
biofuels (butanol: 26.82 ± 0.00 mg/L and ethanol: 16.04 ± 0.01 mg/L) were detected by using
either HPLC or GC measurements.
Bacterial community analysis based on 16S rRNA gene clone libraries after 19 days of
operation, revealed Trichococcus palustris strain DSM 9172 with 99% sequence identity as the
prevailing species in both replicate biocathode communities. Oscillibacter sp. (99% sequence
identity) and Clostridium butyricum (99% sequence similarity) were also dominant groups
detected in both replicate biocathode communities. A total of 26 isolated bacterial strains were
obtained by cultivating cathode scrapings on agar lacking organic nutrients under anoxic,
autotrophic growth conditions with CO2 as sole carbon source and H2 as electron donor. Isolate
ZZ16 showed 99% sequence identity to Clostridium propionicum. Isolate ZZ25 had highest
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sequence identity (98%) with Tissierella sp.S2. Isolates ZZ21, ZZ22, and ZZ23 were assigned
with 97–99% sequence identity to the species Clostridium celerecrescens, a bacterium known for
biocorrosion of steel surfaces.
The second biocathode start-up method tested concerned a recently developed inversion
technique in which a sediment microbial fuel cell (sMFC) anode is electrically converted into a
microbial electrolysis cell (MEC) biocathode. This inversion method was tested here using a
freshwater bog inoculum incubated at 30°C or 48°C without additional organic substrate during
sMFC operation in either open circuit or closed circuit mode. After 3 months of sMFC operation,
sMFC anodes were inverted to act as electron accepting biocathodes in anaerobic two-chamber
BESs. Linear sweep voltammetry (LSV) results showed that electrodes operated initially as
closed circuit sMFC outperformed electrodes operated at open circuit. During long term
operation, biocathodes started as sMFC anodes at an elevated temperature of 48°C outperformed
those at 30oC. Maximum current density uptake of −13 mA/m
2 (maximum current uptake of −19
µA) and maximum power density consumption of −16 mW/m2
were obtained for BES containing
an inverted anode obtained from a closed circuit sMFC (48°C). Cathode chamber headspace gas
analyses showed CO2 significantly decreased only for these BES reactors operated at 48°C,
suggesting explorations in temperature beyond the mesophilic range typically studied may enrich
novel electrotrophs. Acetate accumulation was only detected in these 48°C reactors, further
substantiating microbial electrosynthesis via reduction of CO2. These results show the glucose
pre-enrichment method as well as the elevated temperature inversion method can be successfully
employed for enrichment of CO2 fixing and biofuel/biochemical producing biocathodes.
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TABLE OF CONTENTS
LIST OF FIGURES .................................................................................................................... vii
LIST OF TABLES ....................................................................................................................... xi
ACKNOWLEDGEMENTS ........................................................................................................ xii
Chapter 1 Introduction ................................................................................................................. 1
1.1 World Energy Demand and Carbon Dioxide Emissions ..................................................... 1 1.2 Renewable Energy ............................................................................................................ 3 1.3 Bioelectrochemical Systems .............................................................................................. 4 1.4 Objectives ......................................................................................................................... 6
Chapter 2 Literature Review ......................................................................................................... 8
2.1 Biocathodes and Microbial Electrosynthesis ...................................................................... 8 2.2 Anaerobic Biocathodes, Bioelectrosynthetic Products, and Current Uptake ..................... 10 2.3 Methods for Enrichment of Biocathodes .......................................................................... 13
Chapter 3 Pre-Enrichment Method for Development of Anaerobic Biocathodes .......................... 15
Abstract ...................................................................................................................................... 15 3.1 Introduction ..................................................................................................................... 16 3.2 Materials and Methods .................................................................................................... 17 3.2.1 Pre-Enrichment with Glucose ...................................................................................... 17 3.2.2 BES Construction and Operation ................................................................................. 18 3.2.3 Chemical Analyses ...................................................................................................... 19 3.2.4 16S rRNA Gene Clone Library Analysis ..................................................................... 20 3.2.5 Construction of Phylogenetic Tree .............................................................................. 21 3.2.6 Isolation of Autotrophic Bacterial Strains .................................................................... 21 3.3 Results ............................................................................................................................ 22 3.3.1 Consumption of Current .............................................................................................. 22 3.3.2 Chemical Analysis ...................................................................................................... 23 3.3.3 Microbial Community Analysis .................................................................................. 25 3.3.4 Identification of Isolates .............................................................................................. 28
Chapter 4 Inversion of Freshwater Sediment MFC Anodes into MEC Biocathodes .................... 30
Abstract ...................................................................................................................................... 30 4.1 Introduction ..................................................................................................................... 31 4.2 Materials and Methods .................................................................................................... 32 4.2.1 Set-up and Operation of sMFCs .................................................................................. 32 4.2.2 Inversion of sMFC Anodes into MEC Biocathodes ..................................................... 33 4.2.3 Voltammetry ............................................................................................................... 34 4.2.4 Chemical Analyses ...................................................................................................... 34 4.3 Results ............................................................................................................................ 35 4.3.1 Linear Sweep Voltammetry ......................................................................................... 35 4.3.2 Current Density Uptake ............................................................................................... 36 4.3.3 Chemical Analysis ...................................................................................................... 38
vi
Chapter 5 Discussion ................................................................................................................. 40
Chapter 6 Conclusions and Future Directions ............................................................................. 47
References .................................................................................................................................. 49
Appendix A Preliminary Biocathode Experiments at Different Set Potentials............................. 56
Appendix B Preliminary Pre-Enrichment Experiments with Different Inocula ........................... 58
Appendix C Supplemental Materials - Chapter 3 ........................................................................ 59
Appendix D Supplemental Materials – Chapter 4 ....................................................................... 65
Appendix E Pure Culture Test Using Trichococcus palustris DSM9172 .................................... 69
Appendix F Pure Culture Test Using Desulfobacterium autotrophicum ATCC43914 ................. 70
vii
LIST OF FIGURES
Figure 1.1. Share of renewable energy in global energy consumption in 2009 according to
“Renewables 2011— Global Status” report, REN2011 [4]. ........................................... 3
Figure 1.2. Two traditional modes of BES are shown as two-chamber systems with
proton-exchange membrane (PEM). Dominant reactions on electrodes are
illustrated. (a) MFC with aerated chamber for oxygen reduction at the cathode using
platinum as catalyst. (b) MEC using platinum for catalysis of hydrogen evolution
reaction [16]. .................................................................................................................. 5
Figure 2.1. Schematic of a BES that is powered by renewable energy sources (i.e., solar,
wind) for storage of intermittent power production. The supplied power runs a
potentiostat that applies a set voltage at the cathode so that microbial
electrosynthesis can take place by microbial reduction of carbon dioxide into organic
chemicals, i.e. acetate. Abiotic water-splitting reaction on the anode provides
electrons to the system. .................................................................................................. 9
Figure 2.2. Schematic of standard electrode potentials (E’0) of selected redox couples
versus SHE at pH 7; adapted from [45]. PHB: poly-β-hydroxybutyrate. ........................ 12
Figure 3.1. Current density uptake (a) and electrons consumed (b) by BES inoculated
with pre-enriched cultures over two months. Error bars are obtained from duplicate
reactors. Current uptake and consumption of electrons started after approximately
two weeks after inoculation at applied potential of −0.4 V. During start-up period
(14 days) glucose was added into cathode compartment. ............................................... 23
Figure 3.2. Change in amount of headspace CO2 and H2 in mmol over a two-month time
period. Error bars are obtained from duplicate measurements and duplicate reactors
(biotic). During BES start-up period (first two weeks), amount of CO2 gas increased.
Steady decrease of CO2 gas was observed after start-up period. Hydrogen production
was detected during and after start-up period. ................................................................ 25
Figure 3.3. Clone libraries based on 16S rRNA gene obtained from duplicate reactors.
Samples were taken from cathode surface after start-up period on day 19 when
negative current production was established. Similar bacterial groups were obtained
for both replicates where all major groups belonged to the phylum of Firmicutes
(81-91%, n = 95 and 94). ................................................................................................ 26
Figure 3.4. Phylogenetic tree based on 16S rRNA gene sequence showing the position
and closest relatives of three clone sequences as representatives of the three major
groups obtained from bacterial community analysis. Marked species (*) are known
electrotrophic organisms. The tree was reconstructed from distance matrices using
the neighbor-joining method. Bar = 2 inferred nucleotide changes per 100
nucleotides. .................................................................................................................... 27
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Figure 4.1. (a) Set-up of sMFC and of (b) BES in MEC mode after inversion of sediment
anode into biocathode. Compartments of two-chamber system are separated by a
proton exchange membrane (PEM). Supposed reactions are shown in two-chamber
system. Reduction of CO2 into longer chain organic molecules occurs at the cathode.
At the anode water is split into oxygen molecules and protons. ..................................... 33
Figure 4.2. Ability of sMFC-enriched electrodes to accept electrons at negative applied
working electrode potentials determined by linear sweep voltammetry (LSV). ............. 36
Figure 4.3. Current uptake (a) and consumption of electrons (b) by BESs operated at
applied potential of −0.4 V after inversion of sMFC anodes into MEC cathodes.
Sediment MFCs and BESs were operated at 48°C. ........................................................ 37
Figure 4.4. Decrease of headspace CO2 over 14 days in BES with biocathodes obtained
from inversion of sediment anodes operated at 48°C. .................................................... 39
Figure A.1 Operation of duplicate reactors at applied potential of −0.089 V. Anodic
reactions were taking place at the working electrodes. ................................................... 56
Figure A.2 Operation of duplicate reactors at applied potential of −0.3 V. Cathodic
reactions were taking place at working electrodes. ......................................................... 56
Figure A.3 Comparison of duplicate reactors at applied potentials of (a) −0.3 V or (b)
−0.4 V at working electrodes. Cathodic reduction reactions were taking place at both
potentials. Consumption of current was higher at set potential of −0.4 V. ..................... 57
Figure B.1 Reactors operated after inoculation of cathode chambers with pre-enriched
culture. (a) Current density uptake and (b) consumption of electrons at applied
potential of −0.4 V (B1-PE and B2-PE duplicate reactors, pre-enriched bog
sediment; W1-PE pre-enriched wastewater). (c) Cyclic voltammetry after four
weeks of operation. ........................................................................................................ 58
Figure C.1 HPLC chromatograms obtained from analysis of cathode chamber solutions
from (a, b) biotic replicates and (c) control reactors to examine excreted metabolites.
Red arrows show to peak of acetic acid; blue arrows show to peak of propionic acid;
black arrows show to unknown peaks. ........................................................................... 59
Figure C.2 GC chromatograms obtained from analysis of cathode chamber solutions from
(a, b) duplicate reactors to examine excreted volatile fatty acids (VFAs). Green
arrows show to peak of ethanol; pink arrows show to peak of butanol; purple arrows
show peak of butyrate. ................................................................................................... 60
Figure D.1 Current produced by sMFC inoculated with bog sediment and operated at
48°C. .............................................................................................................................. 65
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Figure D.2 Current produced by duplicate sMFCs (a) sMFC1 and (b) sMFC2. A steady
increase of current produced over time was recorded. .................................................... 66
Figure D.3 Current density uptake (a) and consumption of electrons (b) by BES operated
at applied potential of −0.4 V after inversion of sMFC anodes into MEC cathodes.
After 30 days applied voltage was decreased to −0.5 V. Sediment reactors and
MECs were incubated at 30°C. ...................................................................................... 67
Figure D.4 HPLC chromatograms obtained from analysis of cathode chamber solutions
from two-chamber BES operated at 48°C with (a) biocathode obtained from former
sMFC (b) biocathode obtained from open circuit sMFC and (c) sterile control
reactors to examine excreted metabolites. Red arrows show to peak of acetic acid........ 68
Figure E.1 Current uptake by Trichococcus palustris DSM 9172, a strain that was shown
to be dominant in biocathode communities (see Chapter 3). A set potential of −0.5 V
was applied at 30°C. TP1 (green) and TP2 (blue) are duplicate reactors. Red curve is
a sterile control. .............................................................................................................. 69
Figure F.1 Desulfobacterium autotrophicum is a fully sequenced and well characterized
obligately anaerobic sulfate-reducing acetogen able to fix CO2 coupled with
oxidation of hydrogen gas. In this experiment hydrogen was replaced by a graphite
electrode as source of energy and reducing equivalents. Current uptake by BES
inoculated with Db. autotrophicum, is shown during fourth week of operation at an
applied potential of −0.5 V and incubated at 30°C. Reactors R1 and R2 have
electrodes that were acclimated to BES by addition of hydrogen and then transferred
to new reactors. Reactors R3 and R4 contain former acclimated culture of Db.
autotrophicum but contain new electrodes (recolonization test); reactor was
degassed with CO2:N2 (20:80) mix to remove residual hydrogen and to provide CO2
for autotrophic growth. Blue line is the control containing sterile medium and sterile
graphite electrodes. ........................................................................................................ 70
Figure F.2 Decrease of mmoles of CO2 headspace gas over 21 days. It can be seen that
greatest decrease in CO2 content occurred in reactor R4, which also showed greatest
current uptake. This might indicate CO2 fixation coupled with consumption of
electrons from cathode. .................................................................................................. 71
Figure F.3 HPLC chromatograms of control reactor (a) on day 0 and (b) on day 21. ............ 72
Figure F.4 HPLC chromatograms of reactor R1 (a) on day 0 and (b) on day 21. Increase
of acetate is detected. ..................................................................................................... 73
Figure F.5 HPLC chromatograms of reactor R2 (a) on day 0 and (b) on day 21. Increase
of acetate is detected. ..................................................................................................... 74
Figure F.6 HPLC chromatograms of reactor R3 (a) on day 0 and (b) on day 21. Increase
of acetate is detected. ..................................................................................................... 75
x
Figure F.7 HPLC chromatograms of reactor R4 (a) on day 0 and (b) on day 21. Increase
of acetate is detected. ..................................................................................................... 76
Figure F.8 Results of GC measurements on day 21. Acetate peaks are detected in all
cathode solutions inoculated with Db.autotrophicum except that of the sterile control
reactor. (Control) Control reactor. (R1) Reactor R1. (R2) Reactor R2. (R3) Reactor
R3. (R4) Reactor R4. ...................................................................................................... 77
xi
LIST OF TABLES
Table 3.1. Identities of 11 isolates showing their closest known relative resulting from
BLAST search. ............................................................................................................... 29
Table C.1 Number of clones belonging to major bacterial groups in duplicate reactors (n
=95 and n =94). .............................................................................................................. 61
xii
ACKNOWLEDGEMENTS
I would like to thank my adviser Dr. Bruce E. Logan for providing me the opportunity to pursue a
graduate degree at The Pennsylvania State University, and for his support and valuable
constructive feedback on this thesis. I would also like to thank Dr. Jay Regan and Dr. William
Burgos for their support and for serving as my committee.
I would like to thank all my lab mates in the Logan and Regan Lab. I would especially
like to thank Dr. Justin Tokash for his invaluable help in electrochemistry and in teaching me
various electrochemical techniques. In addition, I would like to acknowledge Dr. Rachel Wagner
and Dr. Doug Call for sharing their valuable experience in reactor construction and design, and
Dr. Xiuping Zhu for her help with Shimadzu HPLC/GC measurements. I would also like to thank
Dr. John Pisciotta for discussions on bioelectrochemical systems and microbiology and for
proofreading this thesis. I would like to acknowledge the King Abdullah University of Science
and Technology for funding my education and this project.
Finally, I would like to thank my family for their continuous support and love.
Chapter 1
Introduction
Mankind faces rapidly increasing energy demands associated with accelerating global
population growth and rising industrialization of developing countries. Emerging economies like
China, India, and Brazil bear together around 40% of the world’s population. As these countries
become increasingly developed, they will strive to eventually reach the same living standards and
energy demands as populations in industrialized countries like the United States. In the face of
dwindling resources, fossil fuel supplies will continue to diminish while their prices rise. This
situation represents a great threat for the Earth because of absent or not fully implemented
environmental regulations in many developing and emerging countries. This will inevitably lead
to greater air, soil, and water pollution. Alternative sources of energy and fuels for transportation
are needed to diminish these results of a swiftly growing world population and so secure the
living standards of all nations.
1.1 World Energy Demand and Carbon Dioxide Emissions
According to the International Energy Outlook 2011 (IEO2011), world energy
consumption will grow by 53% between 2008 to 2035 as total world energy use increases from
505 quadrillion British thermal units (Btu) in 2008 to 770 quadrillion Btu in 2035 [1]. The United
States accounts for 21% of the total energy consumption despite having only about 5% of the
world’s population [1]. However, the majority of the growth in energy consumption is predicted
to occur in countries outside the Organization for Economic Cooperation and Development
(OECD) resulting from a strong long-term economic growth in such non-OECD nations.
IEO2011 predicts also that energy use in non-OECD nations will increase by 85% compared to
2
an increase of 18% for the OECD nations [1]. Given the fact that most non-OECD nations lack
environmental policies, the consequences from such developments could be devastating for the
global climate and life on Earth.
Worldwide use of petroleum and other liquid, petroleum-based fuels is growing in
accordance with increased global energy demand. Indeed, IEO2011 predicts an increase from
85.7 million barrels per day in 2008 to 112 million barrels per day in 2035 [1]. Particularly in the
transportation sector, the use of liquid fuels will grow mainly from the increasing demand of
emerging economies. To meet the forecasted demands, liquid fuel production must be increased
via conventional as well as new technologies and resources. As economically recoverable
reserves of conventional fossil fuels dwindle and public concern rises over ecological disasters
such as the Deepwater Horizon, biofuels production will also become ever more feasible and
economically attractive.
One consequence of increasing energy and fuel consumption based on fossil fuels is the
concomitant rise in carbon dioxide emissions. Carbon dioxide is well known as a green house gas
(GHG) and regarded as a major contributor to global warming. IEO2011 predicts a tremendous
increase of 43% of world energy-related carbon dioxide emissions over the period from 2008 to
2035. Over this span CO2 emissions may increase from 30.2 billion metric tons in 2008 to 43.2
billion metric tons in 2035 [1]. The combustion of petroleum based fossil fuels for transportation
will account for 57% of this increase whereas electricity generation accounts for 49% of total
GHG emissions [2, 3]. In developing countries with strong economic growth, accelerating
reliance on hydrocarbon fuels will contribute to the global warming and on-going climate change.
Therefore, technologies are being developed for long term capture and storage of atmospheric
carbon dioxide. Other “carbon neutral” strategies aim to temporarily store CO2 as hydrocarbon
fuels. In 2007 the Intergovernmental Panel on Climate Change (IPCC) recommended a decrease
3
in global CO2 emissions greater than 50% [2]. To meet these aims economically attractive carbon
neutral fuels must be developed.
1.2 Renewable Energy
Currently more than 85% of the energy consumed in the United States is derived from
fossil fuels [3]. Only about 8% of the energy consumed in the United States came in 2009 from
renewable energy sources [1]. Renewable energy sources include wind power, solar
photovoltaics, solar thermal power, solar hot water/heating, biomass power and heat, biofuels,
geothermal power and heat, hydropower, and hydrokinetic energy [4]. In 2009, 81% of consumed
global energy came from fossil fuels. Renewable energy had a share of 16%; 2.8% came from
nuclear energy (Fig. 1.1) [4].
Figure 1.1. Share of renewable energy in global energy consumption in 2009 according to
“Renewables 2011— Global Status” report, REN2011 [4].
Biofuels are produced from renewable natural sources and considered as substitutes for
petroleum-based fuels. The most common two liquid biofuels are ethanol and biodiesel. Gaseous
biofuels include biogas (i.e., methane), biosyngas (gas mixture of carbon monoxide and
4
hydrogen/carbon dioxide), and biohydrogen. An example of solid biofuel is bio-char. Ethanol is
mainly produced from sugar cane, sugar beets, or corn and biodiesel from soy, rapeseed, palm, or
waste vegetable oil [4, 5]. The United States and Brazil accounted for 88% of world’s ethanol
production in 2010, whereas, the European Union was the major world biodiesel producer
accounting for nearly 53% of total output in 2010 [4]. Advanced technologies are being
developed for ethanol production from cellulosic biomass, such as wood and grasses [6]. An
advantage of production of ethanol from lignocellulose is the abundance and diversity of raw
materials compared to sources such as corn and sugar cane. These raw materials, however,
require greater amount of processing to make sugars available for microbial ethanol fermentation
[6-8]. Ethanol is also of considerably lower energy density than conventional gasoline and cannot
be used as a “drop in” gasoline replacement in most engines. Economically viable, carbon neutral
drop in petroleum replacement fuels are needed.
1.3 Bioelectrochemical Systems
Bioelectrochemical systems (BESs) are a promising technology for production of renewable
energy or biofuels [9]. In BESs, microorganisms catalyze heterotrophic oxidation reactions on the
anode (bioanode) and/or reduction reactions on the cathode (biocathode) [10-12]. Microbial fuel
cells (MFCs) and microbial electrolysis cells (MECs) represent two different modes of BES. In
MFCs, spontaneous reactions are associated with microbial metabolism at the anode that drive the
electrochemical cell. As a product electricity is generated by bioanodes containing
exoelectrogenic microorganisms that consume chemical energy by degradation of organic matter
(e.g., in wastewater) and use the anode as an external electron acceptor (Fig. 1.2a) [12, 13].
Microorganisms able to directly accept electrons from cathodes as electron source are referred to
as electrotrophs [14]. In MECs, on the other hand, additional power is supplied to the system
5
using either a potentiostat or power supply to drive reactions for biofuel production (e.g.,
hydrogen evolution) that would otherwise not be thermodynamically favorable. Organic wastes or
acetate are degraded by bioanodes but the main product is usually a desirable chemical product,
like hydrogen, which is generated abiotically or biotically at the cathode (Fig. 1.2b) [12, 15].
Figure 1.2. Two traditional modes of BES are shown as two-chamber systems with proton-
exchange membrane (PEM). Dominant reactions on electrodes are illustrated. (a) MFC with
aerated chamber for oxygen reduction at the cathode using platinum as catalyst. (b) MEC using
platinum for catalysis of hydrogen evolution reaction [16].
To make BESs efficient, a catalyst is needed at the cathode surface due to slow reduction reaction
rates on carbon or graphite electrodes. Platinum is the most widely used chemical catalyst for
6
oxygen reduction or hydrogen evolution [17, 18]. Disadvantages are that it is expensive, non-
renewable, and also subject to poisoning by carbon monoxide or sulfide a common component of
wastewater that reduces its catalytic activity [19]. Therefore, on-going research is aimed at
finding a chemical or biological substitute for platinum [20-23]. Biological catalysts for cathodic
reduction processes are known as biocathodes and may be advantageous over chemical cathodes
because they are self-renewable and less costly (no need for precious metals or artificial electron
mediators) [14, 24].
1.4 Objectives
Electrotrophic bacteria can be used to produce a variety of useful products via a process
called microbial electrosynthesis; e.g. the bioelectrochemically-mediated reduction of carbon
dioxide into organic compounds by microbes on the cathode. Unfortunately, it is difficult to
produce anaerobic biocathodes using existing methods. Therefore, improved start-up methods for
the establishment of biocathodes are needed to enrich for electrotrophs able to fix CO2 into
valuable biofuels/biochemicals.
The objective of this study was to develop improved methods for the enrichment of
carbon dioxide fixing and biofuel producing biocathodes for use in BES. Two approaches are
presented. In Chapter 3, a novel heterotrophic pre-enrichment method is described, where an
anaerobic culture was first enriched on glucose then used as the inoculum for BES cathode
chambers. After a start-up period, a selective pressure was applied by providing CO2 as the sole
carbon source to enrich for bacteria able to grow autotrophically and produce biofuels or
biochemicals as metabolic end products. In Chapter 4, a recently developed sediment MFC
(sMFC) inversion method was used to test if electrotrophs can be enriched from freshwater
sediment samples without the addition of acetate during sMFC anode establishment. Chapter 4
7
further explored whether enrichment of exoelectrogens and electrotrophs capable of microbial
electrosynthesis might be enhanced by elevated temperature (48°C). In Chapter 5 results from
these two approaches are discussed and, finally, in Chapter 6 conclusions and future directions
are elucidated.
8
Chapter 2
Literature Review
2.1 Biocathodes and Microbial Electrosynthesis
In contrast to traditional chemically-catalyzed cathodes, biocathodes are self-renewable
and should be less expensive [14, 24]. However, biocathode biofilms are difficult to establish,
particularly under anoxic conditions, [25, 26]. Microorganisms that can directly accept electrons
from cathodes for reduction of terminal electron acceptors, such as oxygen, nitrate, sulfate, iron,
manganese, arsenate, fumarate, or carbon dioxide, are referred to as electrotrophs [14, 27-34].
Electrotrophs as cathode catalysts could conceivably be used to produce a variety of useful
products via microbial electrosynthesis. The term electrosynthesis is defined as the conversion of
inorganic chemicals into organic chemical compounds in an electrochemical cell; for example,
the conversion of CO2 into hydrocarbons [35]. Based on reaction thermodynamics, carbon
dioxide should be easily electrochemically convertible into various organic molecules, even under
atmospheric pressure [36, 37]. However, the kinetics are not favorable for product accumulation
without intensive energy input and high CO2 purity [38, 39]. Abiotic electrosynthesis of
chemicals from CO2 has also not been proven practical due to a lack of efficient and selective
catalysts [38, 40]. This problem may be circumvented through microbial electrosynthesis.
In microbial electrosynthesis, also called bioelectrosynthesis, microorganisms are used as
catalysts to convert simple substrates, like carbon dioxide, into various organic compounds that
may be released extracellularly in the BES [41-45] (Fig. 2.1). A critical advantage of using live
cells over purified enzymes or abiotic catalysts is the capacity of microorganisms for repair and
replication. In addition, microorganisms do not need high purity substances to function and are
selective in metabolite production. Another interesting aspect of using microorganisms as
catalysts lies in our modern ability of genetic engineering. This makes finding suitable
9
candidates, particularly fully sequenced and biochemically well characterized species a
paramount research objective. One of the promising applications for bioelectrosynthetically
created molecules is to serve as an organic energy storage means for intermittently produced
renewable power, such as from solar panels or wind turbines [14, 45] .
Figure 2.1. Schematic of a BES that is powered by renewable energy sources (i.e., solar, wind) for
storage of intermittent power production. The supplied power runs a potentiostat that applies a set
voltage at the cathode so that microbial electrosynthesis can take place by microbial reduction of
carbon dioxide into organic chemicals, i.e. acetate. Abiotic water-splitting reaction on the anode
provides electrons to the system.
10
2.2 Anaerobic Biocathodes, Bioelectrosynthetic Products, and Current Uptake
Microbial electrosynthesis of chemical products by biocathodes has been reported for
mixed as well as various pure cultures by using diverse electron acceptors (i.e., nitrate, formate,
protons, and carbon dioxide) and different applied cathode potentials. In early microbial
electrosynthesis studies, electrochemically induced shift of fermentation products of Clostridium
acetobutylicum was examined by addition of the mediator methyl viologen. With addition of
methyl viologen, Kim et al. [46] observed up to 26% more butanol production at a set cathode
potential of −2.5 V1, whereas Peguin et al. [47] obtained significant increase of butanol
production at an applied cathode potential of −0.56 V with maximum current uptake of around
−10 mA.
Nitrate was reduced by biocathodes to nitrite using mixed and pure cultures [27]. In this
study, a mixed consortium enriched from a sediment inoculum showed maximum current uptake
of greater than −0.8 mA at a set cathode potential of −0.389 V. A pure culture of Geobacter
metallireducens reduced nitrate to nitrite with a graphite electrode as sole electron donor at the
same applied cathode potential [27]. Furthermore, G. sulfurreducens reduced fumarate to
succinate with maximum current uptake of greater than −1.0 mA at a set cathode potential of
−0.389 V [27].
Hydrogen was produced by a mixed culture biocathode that was obtained by inversion of
a hydrogen fed anode [48]. Maximum current density was about −1.2 A/m2 at an applied potential
of −0.7 V [48]. In another study, hydrogen was produced by a pure culture of G. sulfurreducens
at set potentials ranging from −0.589 V to −0.789 V, where maximum current densities ranged
from −0.7 to −2.4 A/m2 with higher rates of H2 biosynthesis at more negative potentials [49].
Desulfitobacterium- and Dehalococcoides-enriched mixed culture biocathodes were shown to
1 All potentials are given versus the standard hydrogen electrode (SHE).
11
produce hydrogen in the presence of the mediator methyl viologen at an applied cathode potential
of −0.450 V [50]. Furthermore, this Desulfitobacterium- enriched culture catalyzed H2 production
at lower applied cathode potentials than −0.700 V with omission of mediators [50]. The
dominance of the species Desulfovibrio vulgaris was observed in a hydrogen producing
biocathode community that was originally enriched as a bioanode with a current density of 1.1
A/m2 at a set cathode potential of −0.700 V [51]. Subsequent pure culture tests with biocathodes
consisting of Desulfovibrio strain G11 likewise exhibited hydrogen gas formation with a
maximum current density of 0.76 A/m2 [51]. In yet another pure culture study with a
Desulfovibrio sp., namely D. caledoniensis, that was originally isolated from rust layers of steel,
biocatalysis of hydrogen evolution was demonstrated [52]. Here, at an applied cathode potential
of −0.610 V hydrogen production was associated with current uptake of greater than −0.8 mA
[52]. In a recent study, the species Desulfovibrio paquesii was also shown to produce hydrogen
with a current uptake of greater than −0.3 mA/cm2 at a poised cathode at −0.900 V [53].
Jeremiasse et al. [54] developed a hydrogen producing biocathode with addition of acetate for
start-up and obtained a maximum current uptake of 2.7 A/m2 at a set potential of −0.700 V. These
recent studies highlight the importance of sulfate reducing bacteria (SRB) in hydrogenogenic
biocathodes. However, the hypothesis that autotrophic SRB can be employed for microbial
electrosynthesis had never been tested.
Methane was produced by reduction of CO2 by a mixed culture dominated by the
methanogenic archeaon Methanobacterium palustre with a current of ca. 2.5 mA by applying a
cathode potential of −0.799 V [41]. In a recent study, Pisciotta et al. [55] developed a biogas
producing biocathode by inversion of a sediment MFC anode. Maximum current uptake was
around −0.45 mA using a poised cathode potential of −0.439 V [55]. Most prominent members of
this biocathode community were Eubacterium limosum, Desulfovibrio sp. strain A2,
Rhodococcus opacus, and Gemmata obscuriglobus [55].
12
Figure 2.2. Schematic of standard electrode potentials (Eʹ0) of selected redox couples versus SHE
at pH 7; adapted from [45]. PHB: poly-β-hydroxybutyrate.
The reduction of perchlorate by a pure culture of Dechlorospirillum sp. strain VDY in a
mediatorless cathode chamber has been reported for a set cathode potential of −0.301 V [56]. The
biocathodic catalysis of the reduction of trichloroethene (TCE) to cis-dichloroethene (cis-DCE),
vinyl chloride (VC), and ethene has been demonstrated for an anaerobic biocathode at a set
potential of −0.550 V [57]. Furthermore, Strycharz et al. [58] reported the reductive
dechlorination of 2-chlorophenol to phenol by the species Anaeromyxobacter dehalogenans with
a poised electrode at −0.300 V as the sole electron donor.
13
Bioelectrosynthesis of acetate and 2-oxobutyrate by biocathodic reduction of CO2 at an
applied cathode potential of −0.400 V has been reported for the acetogen Sporomusa ovata strain
DSM 2662 [42]. Maximum electrons consumed were about 700 Coulombs (C) after ca. 6 days
[42]. The same researchers showed biosynthetic activity of other known acetogens, namely
Sporomusa silvacetica (DSM 10669), Sporomusa sphaeroides (DSM 2875), Clostridium
ljungdahlii (DSM 13528), Clostridium aceticum (DSM 1496), and Moorella thermoacetica
(DSM 21394) [43]. Two Sporomusa species, C. ljungdahlii, C. aceticum, and M. thermoacetica
produced acetate along with 2-oxobutyrate and formate. For C. aceticum, S. sphaeroides, C.
ljungdahlii, and M. thermoacetica, the compound 2-oxobutyrate was found as the main product
[43]. After 6 days, C. ljungdahlii consumed >100 C, C. aceticum consumed < 100 C, S.
sphaeroides consumed ca. 60 C, M. thermoacetica consumed < 30 C, and S. silvacetica
consumed ca. 18 C [43].
2.3 Methods for Enrichment of Biocathodes
Various approaches have been developed for establishment of biocathodes. Methods for
biocathode development included the use of set potentials at cathodes and the addition of
hydrogen and/or organic compounds, e.g., acetate [27, 54] or trichloroethene (TCE) and cis-
dichloroethene (cis-DCE) [50]. The concept of electrical inversion of exoelectrogenic bioanodes
fed with hydrogen in order to obtain hydrogen-evolving biocathodes based on reversibility of
hydrogenases was first demonstrated by Rozendal et al. [48]. This approach has been shown to be
effective for the establishment of hydrogen-producing biocathodes, but required the use of
electrolytes that contain non-renewable and potentially toxic cyanide, namely ferricyanide as
catholyte and ferrocyanide as anolyte. A different inversion method was used by Cheng et al. [59]
to obtain biocathodes for oxygen reduction by inverting the polarity of a BES repeatedly by using
14
a potentiostat and by alternating exposure of electrode biofilm to oxygen and the addition of
acetate. The same researchers used a similar method to establish a methanogenic biocathode by
switching periodically the polarity of anode and cathode using a stack of rotatable conductive
disks that were one-half submerged in wastewater and one-half exposed to headspace gas [60].
More recently Pisciotta et al. described a sediment MFC electrode inversion technique to
facilitate enrichment of anaerobic exoelectrogenic bacteria from sediments that subsequently
selects for electrotrophic bacteria by inversion of the sMFC anode into a biocathode [55].
Elements of many of these approaches were incorporated and tested in the current study.
Reported absolute values of current density for biocathodes are considerably lower than
those obtained for bioanodes. For example, a CO2 fixing and biogas producing biocathode
consumed a current of −2 mA/m2 using a poised cathode at −0.439 V [55], whereas a bioanode
consisting of the pure culture G. sulfurreducens strain KN400 produced a current density of 7.6
A/m2
[61]. Thus, improved start-up methods for CO2 fixing and biofuel producing biocathodes
are needed to establish more effective biocathodes with higher performances.
15
Chapter 3
Pre-Enrichment Method for Development of Anaerobic Biocathodes
Abstract
A two-step method was developed for enrichment and isolation of electrotrophic bacteria
capable of CO2 fixation and biofuel production in a two chamber BES. Anaerobic enrichment
cultures were first heterotrophically grown on glucose then used to inoculate BES cathode
chambers. After the two weeks heterotrophic start-up, carbon dioxide was provided as sole
electron acceptor and carbon source to select for bacteria able to switch to electroautotrophic
growth. Biocathodic uptake of electric current increased and was sustained over a period of about
two months. Maximum current density consumed was −34 ± 4 mA/m2 (maximum current
consumption: −50 ± 6 µA) at a set potential of −0.4 V. Production of organic acids (acetate: 1.90
± 0.73 g/L, propionate: 2.09 ± 0.56 g/L, and butyrate: 2.25 ± 0.20 g/L) and volatile fatty acids
(butanol: 26.82 ± 0.00 mg/L and ethanol: 16.04 ± 0.01 mg/L) were detected by either HPLC or
GC measurements. Production of biohydrogen was detected during and after start-up. These
metabolic end products may have resulted from microbial fermentation (electrochemically
assisted) during start-up and through microbial electrosynthesis. Bacterial community analysis
based on 16S rRNA gene clone library after 19 days of operation, revealed Trichococcus
palustris strain DSM 9172 with 99% sequence similarity as the prevailing species in both
replicate biocathode communities. Oscillibacter sp. (99% sequence similarity) and Clostridium
butyricum (99% sequence similarity) were also dominant groups detected. These results suggest
facultative autotrophic microorganisms (ex. acetogens) may have been attributable for
consumption of electrons and CO2 electrosynthesis.
16
3.1 Introduction
Capturing nature’s abundant energy from sun and wind through efficient photovoltaic
devices or wind turbines as renewable energy sources is an elegant carbon neutral way to meet
increasing energy demands. However, the intermittent feature of these energy sources still has to
be addressed by finding adequate energy storage systems [62]. Microbial electrosynthesis, a
process through which microorganisms reduce inorganic compounds into stable, energy dense
organic molecules in an electrochemical cell, is one strategy to solve this storage problem.
Conversion of the greenhouse gas CO2 into higher organic molecules as precursors of value-
added chemicals or transportation fuels that can be easily stored and distributed within the
existing infrastructure is an attractive option [42, 63].
Acetogenic bacteria are able to reduce CO2 and H2 into acetate and other organic
compounds by utilizing the reductive acetyl-CoA (Wood-Ljungdahl) pathway [64-66].
Microorganisms from this group have recently been shown to be potential electrotrophic
candidates in conversion reactions of CO2 into organic compounds in BES by replacing hydrogen
with an electrode as the energy and electron source [42, 43]. Since already known and isolated
acetogenic bacteria (i.e., Sporomusa sp., Clostridium sp.) were tested in previous studies [42, 43,
67], it would be of interest to enrich related microorganisms from their natural environment and
to select for the best candidate in a BES. Therefore, it was attempted using a two-step method
first to enrich bacteria heterotrophically grown on glucose based on the fact that acetogens are
facultative autotrophs [65, 66], then acclimated the culture to the BES. Finally, carbon dioxide
was provided as sole electron acceptor and carbon source to select for those bacteria that are able
to switch to autotrophic growth. This novel method was hypothesized to potentially provide an
easy way to isolate biofuel producing electrotrophs from various inoculum sources.
17
3.2 Materials and Methods
3.2.1 Pre-Enrichment with Glucose
The inoculum was sediment from a bog (Black Moshannon Park, Philipsburg, PA) collected in
May 2011 from the subsurface and transferred into plastic containers completely filled to avoid
any gas headspace. Samples were stored at 4°C in the dark and processed the next day.
Approximately 54 g (~20 mL) sediment slurry samples were transferred into three sterile 150-mL
serum bottles using a sterile spatula under anoxic conditions in an anaerobic glove box (Coy,
Laboratory Products, Grass Lake, MI) with N2:H2 (95:5, v/v) atmosphere. Anaerobic basal
medium (80 mL) was added into each culture bottle containing 1.5 g/L KH2PO4, 2.9 g/L K2HPO4,
0.5 g/L NH4Cl, 0.18 g/L MgCl2 ∙ 6 H2O, 0.09 g/L CaCl2 ∙ 2 H2O, 0.3 g/L Na2S∙ 9 H2O, 8 g/L
NaHCO3 supplemented with 10 mL/L minerals solution (SL10) [68]. In addition, 2 mL of
Wolfe’s vitamins solution [69], 2 mL glucose solution (from 1 M stock), and 2 mL sodium-2-
bromoethane-sulfonate (from 500 mM stock solution) to inhibit growth of methanogens [70-72]
were injected into each serum bottle sealed with a butyl rubber stopper (Chemglass, Vineland,
NJ) and an aluminum crimp top. Subsequently, the headspace was degassed for 10 minutes with a
sterile N2:CO2-gas-mix (80:20, v/v). Final pH was adjusted to 7.3. Enrichment cultures were
incubated in the dark at 30°C and agitated on a shaker (at 125 rpm). After a 7-day incubation, 20
mL of the enrichment culture were used to inoculate new serum bottles containing 80 mL fresh
anaerobic basal medium. To each serum bottle 2 mL vitamins, 2 mL glucose, and 1 mL sodium-
2-bromoethane-sulfonate were supplemented from stock solutions mentioned above. Cultures
were incubated under same conditions for another week.
18
3.2.2 BES Construction and Operation
Two-chamber H-type BESs were constructed from two 250-mL media bottles each
jointed together with a glass tube (2.5 cm diameter, 4.5 cm length; 325 mL total volume of each
chamber, Penn State glass workshop, University Park, PA). The two chambers were separated by
a pretreated cation exchange membrane (Nafion 117, Dupont Co., Newmark, DE) as previously
described [73, 74]. Electrodes were made of carbon fiber rods (7.5 cm length, 0.6 cm diameter;
0.00147 m2 surface area; McMaster-Carr), polished using sandpaper (grit type 400) and treated in
1 M HCl over night as described before [75]. Titanium wire (0.06 cm diameter; McMaster-Carr)
was cut to 12-cm length and polished with sandpaper (aluminum oxide; 3M, St. Paul, MN) to
remove any oxide layer. It was then attached to the carbon rod as described before [75]. The wire
of the electrodes was threaded through rubber stoppers and autoclaved along with reactors filled
with DI water and capped loosely with lids. Reference electrodes (Ag/AgCl, RE-5B,
Bioanalytical Systems, Inc.; E(Ag/AgCl) = E(SHE) − 0.211 V) [76] were checked for accuracy,
sterilized with 70% (v/v) ethanol in the laminar airflow cabinet and threaded through sterile
stoppers attached with electrodes determined for being the working electrode (cathode).
Autoclaved and emptied two-chamber reactors were then closed with stoppers attached with
respective electrodes for cathode and anode chamber and transferred into the anaerobic glove
box. Then 200 mL of anaerobically prepared sterile basal medium (see above), supplemented
with 2 mL/L vitamin solution and 2 mL/L sodium-2-bromoethane-sulfonate solution (1 mM final
concentration), was added into each BES chamber. Both chambers were inoculated by addition of
50 mL pre-enriched culture (working volume = 250 mL; headspace = 75 mL). The sterile control
was injected with 50 mL sterile-autoclaved pre-enriched culture into each chamber. The
headspace of all reactor chambers was purged with sterile N2:CO2-gas-mix (80:20, v/v) for 15
minutes to provide CO2. For chronoamperometric and chronocoulometric measurements the
19
BESs were connected to a potentiostat (model MPG2; Bio-Logic - Science Instruments, Claix,
France) by applying a potential of −0.400 V (versus SHE) to the cathode. During BES start-up
period, 1.5 mL glucose (6 mM final concentration) were added every second day into the cathode
compartment of each reactor until day 14 of BES operation. Statistical calculations are based on α
= 0.05. All potential values are reported against the standard hydrogen electrode (SHE).
3.2.3 Chemical Analyses
Hydrogen, nitrogen, and methane were examined using a gas chromatograph (GC, model
2601B; SRI Instruments, CA) equipped with a 3-m Molsieve 5A 80/100 column (Altech
Associates, Inc., Bannockburn, IL) with argon as carrier gas at 80°C. Carbon dioxide was
analyzed using GC (model 310; SRI Instruments, CA) using a 1-m silica gel column (Restek
Corporation, Bellefonte, PA) with helium as the carrier gas at 60°C. Both GCs were equipped
with thermal conductivity detectors (TCDs) with a detection limit of 0.01%. To examine
metabolites excreted into solution, 1 mL of cathode solution was filtered through a 0.22-µm-pore-
size filter and analyzed by a Shimadzu ultra-high-performance liquid chromatograph (HPLC;
Restek Corporation, Bellefonte, PA) (250 mm by 4.6 mm; Allure organic acids; 5-µm column)
using a phosphate buffer eluent (6.8 g/L KH2PO4, pH 2.3) and a photodiode array detector.
Adjustment of pH of the HPLC mobile-phase was carried out with 50 mM KH2PO4, using H3PO4.
To analyze for volatile fatty acids (VFAs), 1 mL of cathode solution was filtered through a 0.22-
µm-pore-size filter and 50 µL of formic acid (50%, v/v) was added. Samples were then examined
by a GC (Shimadzu GC-2010 Plus) equipped with a 5-µL SGE syringe injector (5F-S-0.63,
Restek, Bellefonte, PA; inlet temperature: 225°C) and a Stabilwax-DA column (30 m x 0.32 mm
x 0.5 µm, Restek, Bellefonte, PA). Helium was used as the carrier gas at 12 psi and 60°C.
Measurements were carried out in split mode (split ratio 10:1, 25 mL/min). The Shimadzu GC
20
was equipped with a flame ionization detector (FID; detector gases: H2/air; makeup gas: He;
detector temperature: 250°C).
3.2.4 16S rRNA Gene Clone Library Analysis
Biofilm samples from the cathode were taken by scraping material from the surface using
a sterile razor blade and transferring scrapings directly into appropriate tubes of the total DNA
extraction kit used (Power Soil, MO BIO Laboratories Inc., Carlsbad, CA). Extracted total DNA
was quantified using a spectrophotometer (model 2000C Nanodrop spectrophotometer; Thermo
Scientific, Waltham, MA). Then, 16S rRNA genes were amplified using the bacteria specific
primer set 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-
GGTTACCTTGTTACGACTT-3’) [77] and the archaea specific primer set 4Fa (5’-
TCCGGTTGATCCTGCCRG-3’) and 1492R [78, 79]. Purification of PCR products was
performed using the Qiagen PCR Purification Kit (Qiagen, Valencia, CA). Clone libraries were
constructed using a TOPO TA cloning kit (pCR®2.1-TOPO
®, Invitrogen Corp., Grand Island,
NY). PCR products were ligated with TOPO® vector and transformed into chemically competent
E. coli cells by heat shock transformation according to manufacturer’s instructions. Cells were
streaked onto LB agar plates containing 50 µg/mL ampicillin plus X-Gal (5-bromo-4-chloro-3-
indolyl-β-D-galactopyranoside) and incubated at 37°C overnight. White colonies were selected
from blue-white-screened transformants and subcultured overnight in LB broth with ampicillin in
96 deep well plates. Plasmids were isolated using an EZ Fastfilter Plasmid Kit (Omega Bio-Tek,
Inc., Norcross, GA). Sequencing of 16S rRNA gene inserts was carried out using M13r primers at
the Huck Institute of the Life Sciences Genomics Core facility at the Pennsylvania State
University. Obtained sequences were analyzed by alignments with sequences deposited in
21
GenBank (nucleotide collection) using the BLASTn algorithm of the National Center for
Biotechnology Information (NCBI).
3.2.5 Construction of Phylogenetic Tree
Representative sequences of three clones from major groups of the 16S rRNA gene clone
library analysis were aligned with sequences of closest relatives that were downloaded from
GenBank (http://www.ncbi.nlm.nih.gov/genbank) using the MEGA 4.0 software
(http://www.megasoftware.net/). Distance matrices were constructed from the aligned sequences
and corrected for multiple base changes at single positions by the method of Jukes and Cantor
[80]. A phylogenetic tree was finally reconstructed by the neighbor-joining method [81]. The
resultant tree topology was evaluated by bootstrap analyses based on 1000 resamplings.
3.2.6 Isolation of Autotrophic Bacterial Strains
Bacterial isolates were obtained by streaking cathode scrapings onto agar plates containing
anaerobic basal medium (see above) amended with 10 mL/L Wolfe’s vitamin solution and 9 g/L
agar (BactoTM
Agar, Difco) under anoxic conditions in an anaerobic glove box. Incubation of agar
plates was carried out in the dark at 30°C in an anaerobic jar (vented, BD GasPakTM
100 System,
Franklin Lakes, NJ) purged with N2:CO2 (80:20, v/v) and containing an anaerobe sachet plus an
anaerobic indicator strip to confirm anaerobic conditions (BD GasPakTM
EZ Container Systems,
Franklin Lakes, NJ). Through a one-way valve, pressurized H2 was supplied providing reducing
equivalents for bacterial growth. Single colonies were restreaked every 2 weeks onto fresh agar
medium until pure cultures were obtained.
22
3.3 Results
3.3.1 Consumption of Current
In preliminary studies, where reactors were directly inoculated with bog sediment no
increased current uptake was detected after about 42 days (see Appendix A, Fig. A.3b). Pre-
enrichment with glucose shortened highly start-up time after which steady current uptake
commenced (14 days vs > 42 days). Preliminary pre-enrichment experiments with different
inocula (wastewater vs. bog sediment) revealed also higher current and electron uptake for
reactors inoculated with bog sediment (Appendix B, Fig. B.1).
In the present study, biocathodic activity (i.e. negative current) in both duplicate reactors
commenced after approximately two weeks of BES operation at set a potential of −0.4 V when
current uptake and consumption of electrons started to increase steadily (Fig. 3.1a, b). Maximum
current density uptake was −34 ± 4 mA/m2 (maximum current consumption: −50 ± 6 µA) and
maximum electrons consumed were 186 ± 19 Coulombs over 68 days of operation. The sustained
negative current density that was recorded after approximately 25 days was on average −29 ± 6
mA/m2 and not significantly different for the replicate reactors (t-test; p = 0.005).
The highest power consumed was −58 ± 8 mW/ m2. For the sterile control reactor, there
was no appreciable change in current density uptake and electron consumption throughout the
course of BES operation (Fig. 3.1a, b).
During the bioelectrochemical reactor start-up period, glucose was added to the cathode
chambers to acclimate bacteria to BES and to accelerate biofilm formation on the cathode
surface. After day 14, addition of substrate was ceased to induce onset of autotrophic metabolism
and eventually to select for those bacteria able to fix CO2. After cessation of substrate addition
into cathode chamber, consumption of current and electrons from cathodes of biotic reactors
23
continued for another 54 days indicating biocathodic activity due to autotrophic metabolism after
consumption of glucose.
Figure 3.1. Current density uptake (a) and electrons consumed (b) by BES inoculated with pre-
enriched cultures over two months. Error bars are obtained from duplicate reactors. Current uptake
and consumption of electrons started after approximately two weeks after inoculation at applied
potential of −0.4 V. During start-up period (14 days) glucose was added into cathode
compartment.
3.3.2 Chemical Analysis
Analysis of headspace gas composition showed that during the start-up period CO2 gas
accumulated due to microbial decomposition of glucose. On day 12, an amount of 2.30 ± 0.35
mmol carbon dioxide was detected in the biotic replicate reactors compared to 0.66 ± 0.01 mmol
in the control (Fig. 3.2). After start-up period, a steady decrease of CO2 in all biotic replicate
-50
-40
-30
-20
-10
0
Cu
rre
nt
De
nsity (
mA
/m2)
Biotic Reactor
Control Reactor
0
50
100
150
200
250
0 10 20 30 40 50 60 70
Time (Days)
Ele
ctr
ons C
onsum
ed (
C)
a
b
-50
-40
-30
-20
-10
0
Cu
rre
nt
De
nsity (
mA
/m2)
Biotic Reactor
Control Reactor
0
50
100
150
200
250
0 10 20 30 40 50 60 70
Time (Days)
Ele
ctr
ons C
onsum
ed (
C)
a
b
24
reactors was recorded over 54 days of continuous operation. On day 68, the headspace contained
0.48 ± 0.03 mmol CO2 in biotic replicate reactors. Compared to the sterile control, this was 20 ±
4% less than the 0.60 ± 0.01 mmol CO2 recorded for the control reactor on day 68 (Fig. 3.2). This
decrease in headspace carbon dioxide in both biotic reactors indicates that over the course of BES
operation an autotrophic community was established and that this microbial community was
attributable for CO2 consumption observed.
Hydrogen production in biotic reactors was observed on day 3 during start-up period
that had an amount of 0.16 ± 0.14 mmol, whereas no hydrogen production was observed in the
control. On day 19 (5 days after start-up period ended), hydrogen production was detected in
biotic reactors in the amount of 0.97 mmol in one replicate and 0.15 mmol in the other. This
shows that biofuel hydrogen was produced in biotic BES. However, hydrogen was probably
consumed by other microorganisms that thrived in BES so that no further accumulation was
observed.
To examine organic metabolites excreted into catholyte solution, HPLC and GC analyses
of organic acids and volatile fatty acids (VFAs) were performed. Both replicate reactors showed
similar peak profiles resulting from HPLC and GC measurements. At the end of BES operation,
1.90 ± 0.73 g/L acetate, 2.09 ± 0.56 g/L propionate and several unknown peaks were detected
based on HPLC analysis (Appendix C, Fig. C.1). In addition, 2.25 ± 0.20 g/L butyrate, 26.82 ±
0.00 mg/L butanol, and 16.04 ± 0.01 mg/L ethanol were detected based on GC measurements of
VFAs (Appendix C, Fig. C.2). These organic metabolites could be attributable either to glucose
fermentation during start-up phase or to microbial electrosynthesis by reduction of CO2 into
organic compounds by an established biocathodic community.
25
Figure 3.2. Change in amount of headspace CO2 and H2 in mmol over a two-month time period.
Error bars are obtained from duplicate measurements and duplicate reactors (biotic). During BES
start-up period (first two weeks), amount of CO2 gas increased. Steady decrease of CO2 gas was
observed after start-up period. Hydrogen production was detected during and after start-up period.
3.3.3 Microbial Community Analysis
Genomic analysis by 16S rRNA gene clone libraries of biocathode scrapings after 19
days of BES operation showed similar microbial community profiles for both replicate reactors
(Fig. 3.3a, b). Both bacterial communities were dominated (47 to 60%) by species most closely
related to Trichococcus palustris strain DSM 9172 (99% sequence similarity) followed by species
most closely related to Oscillibacter sp. NML 061048 (GenBank accession number EU149939;
99% sequence similarity) with dominance of 10 to 23%.
26
Figure 3.3. Clone libraries based on 16S rRNA gene obtained from duplicate reactors. Samples
were taken from cathode surface after start-up period on day 19 when negative current production
was established. Similar bacterial groups were obtained for both replicates where all major groups
belonged to the phylum of Firmicutes (81-91%, n = 95 and 94).
The third group present on biocathodes of both replicate reactors showed closest relation
to Clostridium butyricum (GenBank accession number AY442812; 99% sequence similarity) with
a range of 4 to 21%. In contrast to the first replicate reactor, the clone library analysis of
biocathode community of the second replicate showed also dominance of a group (7%) that was
closest related to Anaerofilum agile strain F (GenBank accession number NR029315; 97%
sequence similarity) (Fig. 3.3b). All members of dominating groups belonged to the phylum of
Trichococcus
palustris (47%)
Trichococcus
palustris (60%)
Oscillibacter sp.
(23%)
Clostridium sp.
(21%)
Clostridium sp.
(4%)
Anaerofilum sp.
(7%)
Other
(19%)
Other
(9%)a
b
Oscillibacter sp.
(10%)
Trichococcus
palustris (47%)
Trichococcus
palustris (60%)
Oscillibacter sp.
(23%)
Clostridium sp.
(21%)
Clostridium sp.
(4%)
Anaerofilum sp.
(7%)
Other
(19%)
Other
(9%)a
b
Oscillibacter sp.
(10%)
27
Firmicutes. The genera Clostridium, Oscillibacter, and Anaerofilum belong also to the same order
of Clostridiales. Only Trichococcus sp. that was predominant in both biocathode communities
belongs to the order of Lactobacillales. Members of the Firmicutes phylum are very diverse in
their metabolic abilities. Known electrotrophs that have been shown to reduce CO2 to organic
compounds by electrosynthesis, like Sporomusa ovata or Clostridium ljungdahlii are members of
the phylum of Firmicutes [42, 43].
Figure 3.4. Phylogenetic tree based on 16S rRNA gene sequence showing the position and closest
relatives of three clone sequences as representatives of the three major groups obtained from
bacterial community analysis. Marked species (*) are known electrotrophic organisms. The tree
was reconstructed from distance matrices using the neighbor-joining method. Bar = 2 inferred
nucleotide changes per 100 nucleotides.
Representative 16S rRNA clone sequences were aligned with those of their closest
relatives to construct a phylogenetic tree. All three clones were relatively closely related to each
other (Fig. 3.4). Clone 1 that was most closely related to Trichococcus palustris DSM 9172 build
28
a cluster with the electrotrophic bacterium Sporomusa ovata DSM 2662 which indicates a close
phylogenetic relation. Clone 3 most closely related to Clostridium butyricum CB TO-A build a
cluster with the electrotroph C. ljungdahlii DSM 13528. This shows that organisms closely
related to known electrotrophic microorganisms were enriched on both replicate biocathodes.
Analysis of biocathode scrapings with primers specific to archaea produced no detectable
PCR products with varied DNA concentrations. This indicates that the use of sodium-2-
bromoethane-sulfonate during the pre-enrichment and also during BES operation inhibited the
growth of methanogens and other archaea.
3.3.4 Identification of Isolates
Under autotrophic growth conditions using hydrogen as the source of reducing
equivalents to replace the cathode as electron donor, 26 isolates were obtained of which 6 isolates
could be assigned to their closest known relatives based on 16S rRNA sequencing results and
alignments with deposited sequences in GenBank (Tab. 3.1). Sequence results from isolates
ZZ12, ZZ13, and ZZ26 showed Clostridium butyricum as closest relative. However, maximum
sequence similarities were only between 78 to 82%, which might indicate that these isolates
belong to a group of an unknown Clostridium sp. Isolate ZZ16 showed 99% sequence similarity
to C. propionicum (accession no. AB649276). Isolate ZZ17 was assigned to the genus
Anaerofilum with Anaerofilum agile strain F as closest known species. However, a low sequence
similarity of 88% may also indicate here that ZZ17 belongs to a group of an unknown
Anaerofilum sp. Isolate ZZ25 had highest sequence identity (98%) with Tissierella sp.S2 (C.
hastiforme). Identities of isolates ZZ18 and ZZ20 could not be found. Isolates ZZ21, ZZ22, and
ZZ23 were assigned to the species Clostridium celerecrescens with 97 to 99% sequence
29
similarity. Interestingly, the same isolates showed high sequence similarities (>97%) to
Desulfotomaculum sp., a genus, which contains sulfate-reducing acetogens.
Table 3.1. Identities of 11 isolates showing their closest known relative resulting from BLAST
search.
Isolates Closest known relative
Max.
Sequence
Identity (%)
GenBank
Accession No.
ZZ12,
ZZ13,
ZZ26
Clostridium butyricum
82, 80, 78
FR734081
FR734082
ZZ16 Clostridium propionicum 99 AB649276
ZZ17 Anaerofilum agile strain F 88 NR029315
ZZ18 Swine fecal bacterium RF2B-Cel14 83 FJ753845
ZZ20 Uncultured bacterium clone SW93 86 FJ809710
ZZ21,
ZZ22,
ZZ23
Clostridium celerecrescens 97, 99, 98 AJ295659.2
ZZ25 Tissierella sp.S2 98 EF202592
30
Chapter 4
Inversion of Freshwater Sediment MFC Anodes into MEC Biocathodes
Abstract
A recently developed “sMFC inversion technique” for producing biocathodes was tested
at different temperatures to determine if electrotrophs can be enriched from freshwater sediment
without additional organic substrate. Biocathodes were created by first enriching exoelectrogenic
microorganisms from freshwater bog sediments on buried sMFC anodes, then converting these
electrodes to biocathodes by applying a set potential of −0.4 V with a potentiostat. Separately
operated closed or open circuit reactors were prepared for each incubation temperature (30 and
48°C). After 3 months of sMFC operation without addition of any organic substrate, sMFC
anodes were inverted to act as electron accepting biocathodes in anaerobic two-chamber BESs.
LSV results showed that electrodes operated in closed circuit during sMFC start-up outperformed
electrodes operated at open circuit directly following inversion. Biocathodes operated for 14 days
at the elevated temperature (48°C) displayed better long term current uptake than mesophilic
samples (30°C), even at more negative applied potentials. Maximum consumed current density
was −13 mA/m2 (max. current uptake: −19 µA) for the biocathode produced from the closed
circuit sMFC at 48°C. Its maximum power density was −16 mW/m2. Headspace gas analyses
showed that CO2 decreased in both BES reactors operated at 48°C but not 30°C. BES reactors
containing inverted electrodes from sMFCs at 48°C (open and closed circuit) showed production
of acetate (1.9 mg/L), indicating enhanced microbial electrosynthesis of acetate from CO2 by an
established electrotrophic community at elevated temperature. These results show that the sMFC
inversion method holds great potential for enrichment of electrotrophic microorganism at
elevated temperatures
31
4.1 Introduction
The concept of electrical inversion of exoelectrogenic bioanodes into electrotrophic
biocathodes was first demonstrated by Rozendal et al. [48], but this approach required the use of
electrolytes that contain non-renewable and potentially toxic cyanide. A different inversion
method was used by Cheng et al. [59] to obtain biocathodes for oxygen reduction by inverting the
polarity of a BES repeatedly by using a potentiostat and by alternating exposure of electrode
biofilm to oxygen and the addition of acetate. The same researchers used a similar method to
establish a methanogenic biocathode by switching periodically the polarity of anode and cathode
using a stack of rotatable conductive disks [60].
Most recently Pisciotta et al. [55] designed a novel electrode inversion technique by
which a simple method is provided to ease the enrichment of obligate anaerobic exoelectrogenic
bacteria by concomitantly maintaining strictly anoxic conditions using a sediment MFC (sMFC)
and subsequently to select for electrotrophic bacteria by inversion of the sediment anode into a
biocathode installed in a two-chamber BES and by applying a set potential [55]. It was
successfully shown that this method is useful to enrich for bioelectrosynthetically active
electrotrophs from marine sediment able to convert CO2 into biogas. Here, it was examined if
electrotrophs can be enriched from freshwater sediment samples without the addition of acetate
during establishment of exoelectrogenic sMFC anodes and to test if the enrichment of
exoelectrogens and electrotrophs capable of microbial electrosynthesis of organic compounds by
CO2 conversion is enhanced at elevated temperatures, i.e. 48°C.
32
4.2 Materials and Methods
4.2.1 Set-up and Operation of sMFCs
Sediment MFCs were constructed from a 500-mL glass medium bottle that was filled
with 500 mL sediment from a bog with 110 mL water phase at the sampling site in November
2011 (Black Moshannon Park, Philipsburg, PA). The anode was constructed of a graphite rod (7.8
cm length, 0.6 cm diameter; 0.00153 m2 surface area; McMaster-Carr), polished using sandpaper
(grit type 400) and treated in 1 M HCl over night as described before [75]. Titanium wire (0.08
cm diameter; McMaster-Carr) was cut to 28-cm length and polished with sandpaper (aluminum
oxide; 3M, St. Paul, MN) and then attached to the graphite rod as described before [75]. It was
insulated at the upper part with Teflon to avoid short circuiting. The air-cathode was carbon cloth
(0.0025-m2 projected area; 30% polytetrafluoroethylene (PTFE) wet proofing, Fuel Cell Earth
LLC, Wakefield, MA) coated on both sides with platinum catalyst layer (0.5 mg of 10% platinum
on Vulcan, XC-72, Fuel Cell Store Inc., Boulder, CO) as previously described [82, 83]. Polished
titanium wire (ca. 8 cm length) was threaded through the prepared air-cathodes and resistance
was checked. The air-cathode was then positioned at the air-water interface and connected
through a 1,000-Ω resistor to the anode (Fig. 4.1a) [55]. For each temperature, one anode in a
separate sediment reactor operated at open circuit was not connected to an air-cathode. Reactors
were incubated in the dark at 30°C in a constant-temperature room or at 48°C in a water-bath. A
digital multimeter (model 2700; Keithley Instruments Inc., Cleveland, OH) was connected to
sMFCs to acquire voltage data at 20 min intervals, with the current calculated using Ohm’s law (I
= E/R). Deionized (DI) water was periodically added to replace water lost to evaporation. All
reactors were operated for 3 months.
33
4.2.2 Inversion of sMFC Anodes into MEC Biocathodes
After three months of operation of sediment reactors, electroactive bioanodes were
removed from the sediment and gently rinsed with sterile anaerobic basal medium in an anaerobic
chamber (Coy, Laboratory Products, Grass Lake, MI) with N2:H2 (95:5, v/v) atmosphere. Each
graphite sediment anode was then transferred to a sterile two-chamber H-type BES (Glasstron
Inc., Vineland, NJ) with four sampling ports at each side. The former anode was then operated as
the working electrode (cathode) by applying a potential of −0.4 V using a potentiostat (model
MPG2; Bio-Logic - Science Instruments, Claix, France) (Fig. 4.1b).
Figure 4.1. (a) Set-up of sMFC and of (b) BES in MEC mode after inversion of sediment anode
into biocathode. Compartments of two-chamber system are separated by a proton exchange
membrane (PEM). Supposed reactions are shown in two-chamber system. Reduction of CO2 into
longer chain organic molecules occurs at the cathode. At the anode water is split into oxygen
molecules and protons.
Each of the two chambers contained sterilized anaerobic basal medium (120-mL working
volume; 70 mL headspace) consisting of 1.5 g/L KH2PO4, 2.9 g/L K2HPO4, 0.5 g/L NH4Cl, 0.18
g/L MgCl2 ∙ 6 H2O, 0.09 g/L CaCl2 ∙ 2 H2O, 0.3 g/L Na2S∙ 9 H2O, 8 g/L NaHCO3 supplemented
with 10 mL/L minerals solution (SL10) [68], 10-7
M (final concentration) of Na2SeO3 solution,
34
and 2 mL/L of Wolfe’s vitamins solution [69]. The two chambers were separated by a pretreated
cation exchange membrane (Nafion 117, Dupont Co., Newmark, DE) as previously described
[73, 74]. The counter electrodes (anodes) were graphite rods (7.8 cm length, 0.6 cm diameter;
0.00153 m2 surface area; McMaster-Carr) prepared as described before [75]. Reference electrodes
(Ag/AgCl, RE-5B, Bioanalytical Systems, Inc.; E(Ag/AgCl) = E(SHE) − 0.211 V) [76] were
checked for accuracy, sterilized with 70% (v/v) ethanol in the laminar airflow cabinet and pushed
through punched sterile butyl stoppers attached at the cathode chamber. The solution in each
chamber was bubbled with sterile N2:CO2-gas-mix (80:20, v/v) for 10 minutes to remove oxygen
and provide CO2. Reactors were incubated in the dark at 30°C in a constant-temperature room or
at 48°C in a water-bath.
4.2.3 Voltammetry
Linear sweep voltammetry (LSV) was performed after transferring each sediment anode
to a separate two-chamber BES reactor in order to examine the ability to accept electrons before
applying a negative set potential of −0.4 V. Current density uptake was measured at a scan rate of
1 mV/s and a range from −0.3 V to −0.7 V.
4.2.4 Chemical Analyses
Hydrogen, nitrogen, and methane were examined using a gas chromatograph (GC, model
2601B; SRI Instruments, CA) equipped with a 3-m Molsieve 5A 80/100 column (Altech
Associates, Inc., Bannockburn, IL) with argon as carrier gas at 80°C. Carbon dioxide was
analyzed using GC (model 310; SRI Instruments, CA) using a 1-m silica gel column (Restek
Corporation, Bellefonte, PA) with helium as the carrier gas at 60°C. Both GCs were equipped
with thermal conductivity detectors (TCDs) with a detection limit of 0.01%. To examine
35
metabolites excreted into solution, 1 mL of cathode solution was filtered through a 0.22-µm-pore-
size filter and analyzed by a Shimadzu ultra-high-performance liquid chromatograph (HPLC;
Restek Corporation, Bellefonte, PA) (250 mm by 4.6 mm; Allure organic acids; 5-µm column)
using a phosphate buffer eluent (6.8 g/L KH2PO4, pH 2.3) and a photodiode array detector.
Adjustment of pH of HPLC mobile-phase was carried out with 50 mM KH2PO4, using H3PO4. To
analyze volatile fatty acids (VFAs), 1 mL of cathode solution was filtered through a 0.22-µm-
pore-size filter and 50 µL of formic acid (50%, v/v) was added. Samples were then examined by a
gas chromatograph (Shimadzu GC-2010 Plus) equipped with a 5-µL SGE syringe injector (5F-S-
0.63, Restek, Bellefonte, PA; inlet temperature: 225°C) and a Stabilwax-DA column (30 m x 0.32
mm x 0.5 µm, Restek, Bellefonte, PA). Helium was used as the carrier gas at 12 psi and 60°C.
Measurements were carried out in split mode (split ratio 10:1, 25 mL/min). The Shimadzu GC
was equipped with a flame ionization detector (FID; detector gases: H2/air; makeup gas: He;
detector temperature: 250°C).
4.3 Results
4.3.1 Linear Sweep Voltammetry
Sediment MFCs were operated over 3 months at which closed circuit sMFCs produced
electrical current that increased over time indicating successful enrichment of exoelectrogenic
microorganisms (Appendix D, Fig. D.1, D.2a, b). After transferring sediment anodes to two-
chamber BES reactors and switching them to cathodes, former anodes were analyzed in their
ability to accept electrons by LSV analysis. Electrodes previously operated as anodes in closed
circuit system showed a steep increase in current uptake starting at around −0.4 V at which the
curve of the sMFC anode incubated at 48 °C exhibited a peak at ca. − 0.575 V (Fig. 4.2).
36
Sediment MFCs operated in open circuit, on the other hand, showed significantly less current
uptake where a slight peak at around − 0.575 V was visible also for the open circuit anode
incubated at 48 °C. Abiotic graphite that was used as a negative control showed least current
density uptake (Fig. 4.2).
Figure 4.2. Ability of sMFC-enriched electrodes to accept electrons at negative applied working
electrode potentials determined by linear sweep voltammetry (LSV).
4.3.2 Current Density Uptake
After inverting sMFC anodes into BES cathodes and applying a potential of −0.4 V,
highest and relatively constant current uptake was recorded initially for the BES reactor with
biocathodes obtained from open circuit sMFC at 48°C (Fig. 4.3a). This observation was
somewhat surprising since LSV results indicated a reduced ability of current uptake for open
circuit sMFC electrodes compared to closed circuit sMFC electrodes (Fig. 4.2). However, over
the course of operation, BES with closed circuit sMFC electrode exhibited a steep decrease in
37
current uptake after ca. 8 days, and consumed towards the end of operation more current than the
BES containing electrode from open circuit sMFC (Fig. 4.3a).
Figure 4.3. Current uptake (a) and consumption of electrons (b) by BESs operated at applied
potential of −0.4 V after inversion of sMFC anodes into MEC cathodes. Sediment MFCs and
BESs were operated at 48°C.
A maximum consumed current density of −13 mA/m2 (max. current uptake: −19 µA) was
obtained for biocathode produced from the closed circuit sMFC at 48°C, compared to −9 mA/m2
(max. current uptake: −13 mA) for the BES containing biocathode obtained from the open circuit
sMFC at 48°C. Maximum power density uptake was −16 mW/m2 for the BES containing
38
biocathode produced from the closed circuit sMFC at 48°C, and −10 mW/m2 for the BES
containing biocathode obtained from the open circuit sMFC at 48°C. The largest total charge
consumed was recorded for the BES with biocathode obtained from the open circuit sMFC with a
value of 12 C, compared to 7 C for the BES containing electrode produced from the closed circuit
sMFC (Fig. 4.3b). The negative control BES containing a sterile graphite rod as cathode did not
show appreciable current density uptake and consumption of electrons (only ca. 1 C).
BES reactors containing biocathodes obtained from closed or open circuit sMFCs
operated at 30°C did not exhibit a substantial decrease of current density over an operation period
of 60 days even after applying a more negative voltage of −0.5 V at the cathode (see Appendix D,
Fig. D.3a, b).
4.3.3 Chemical Analysis
Headspace gas analyses showed that moles of CO2 decreased over time in both BES
reactors operated at 48°C that contained biocathodes originally operated in an open or closed
circuit sMFC. In the BES with electrode from the open circuit sMFC, CO2 decreased slightly
from 0.60 ± 0.01 to 0.58 ± 0.00 mmol, whereas the BES with electrode from the closed circuit
sMFC had a higher decrease of CO2 (drop from 0.62 ± 0.00 to 0.55 ± 0.00 mmol) (Fig. 4.4). The
decrease of carbon dioxide in both BES might indicate microbial autotrophic growth.
39
Figure 4.4. Decrease of headspace CO2 over 14 days in BES with biocathodes obtained from
inversion of sediment anodes operated at 48°C.
To examine organic metabolites excreted into catholyte solution, HPLC and GC analyses
of organic acids and volatile fatty acids (VFAs) were performed. BES reactors containing
inverted electrodes operated at open or closed circuit mode in sMFCs and incubated at 48°C
showed production of acetate in trace amounts (1.9 mg/L) (Appendix D, Fig. D.4a, b, c). This
might indicate microbial electrosynthesis by reduction of CO2 into acetate by an electrotrophic
community.
40
Chapter 5
Discussion
Electrosynthetic biocathodes are self-renewable, inexpensive and require no artificial
chemical mediators or precious metals, in contrast to conventional chemical catalysts. In
microbial electrosynthesis CO2 is converted into various chemical compounds by electrotrophic
bacteria which serve as biocatalysts [14]. Besides removing CO2 and the synthesis of organic
products, another application concerns renewable energy storage in the form of stable chemical
bonds (i.e., for intermittent renewable solar or wind energy) [41, 42]. Unfortunately, the
development of biocathodes for anaerobic reduction reactions has proven challenging and usually
requires long start-up periods [25, 44]. Here, two novel start-up methods were introduced that
hold potential for accelerated establishment and screening of CO2-fixing and biofuel producing
biocathodes in BES.
The glucose pre-enrichment method, presented in Chapter 3, consisted of two main steps.
First an anaerobic enrichment culture was grown heterotrophically on glucose, based on the fact
that acetogens are facultative autotrophs [65, 66]. Next this was used to inoculate cathode
chambers of BES reactors operated at a set potential of −0.4 V. After the heterotrophic
acclimation period of two weeks, carbon dioxide that accumulated in headspace served as the sole
electron acceptor and carbon source to select for electrotrophic bacteria able to switch from
heterotrophic to autotrophic growth. The increased current uptake after heterotrophic acclimation
suggests enrichment of heterotrophic electrotrophs. Current uptake was sustained for 54 days
with maximum current density consumption of −34 ± 4 mA/m2 (maximum current uptake: −50 ±
6 µA, maximum electrons consumed: 186 ± 19 C) (Fig. 3.1). A concomitant decrease in
accumulated CO2 gas was detected in the cathode chamber that reached 0.48 ± 0.03 mmol on day
68, an amount 20 ± 4 % less than that detected in the control reactor (Fig. 3.2). This sustained
41
current uptake and associated decrease in CO2 indicates metabolically active, facultative
autotrophic microorganisms (i.e. acetogens) were enriched on biocathodes. Biocathodic CO2
reduction in a BES was previously reported for a phototrophic mixed culture [84] and for a mixed
electromethanogenic community [41]. Electrotrophic reduction of carbon dioxide into acetate and
other organic compounds was also shown for pure cultures of diverse acetogenic bacteria, i.e.,
Sporomusa ovata, Clostridium ljungdahlii, and Moorella thermoacetica [42, 43]. In more recent
studies, indirect consumption of electrons from cathodes via electrochemically generated formate
was associated with isobutanol and 3-methyl-1-butanol production by genetically engineered
Ralstonia eutropha H16 [63], or with hydrogen, methane and biomass production from a mixed
culture obtained from inversion of an exoelectrogenic marine sediment bioanode [55]. In the
current study, hydrogen gas, butanol, ethanol, acetate, propionate, and butyrate were detected,
among other unknown organic compounds, in all biotic reactors. Based on stoichiometric
calculations of the 186 ± 19 C consumed, 88.7 ± 0.0 % of detected butanol, 54.1 ± 0.1 % of
detected ethanol, 3.05 ± 1.17 % of detected acetate, or 1.95 ± 0.52 % of detected propionate could
have been produced by microbial electrosynthesis. Since glucose was added during start-up and
afterwards the accumulated CO2 was consumed over time, detected products were either
produced by electrochemically assisted fermentation [46] or by microbial electrosynthesis [42,
43, 45].
Bacterial community analysis based on 16S rRNA gene clone library after 19 days of
operation, revealed Trichococcus palustris strain DSM 9172 with 99% sequence similarity as the
prevailing species in biocathode communities (Fig. 3.3). T. palustris (former Ruminococcus
palustris) is a facultative anaerobic, fermentative bacterium that is Gram-positive and non-spore-
forming, and was first isolated from a swamp [85, 86]. T. palustris ferments glucose to lactate,
acetate, ethanol, formate, and isobutyrate [85]. T. pasteurii is next closest relative of T. palustris
and has been reported to be able to form acetate from methanol [87]. Interestingly, T. pasteurii
42
was found as a dominant member of a biocathode community in a BES that was able to reduce
chromium(VI) to chromium(III) with NaHCO3 as sole carbon source [88]. This suggested that T.
palustris might be capable of electroautotrophic growth.
Oscillibacter sp. (99% sequence similarity) and Clostridium butyricum (99% sequence
similarity) were also dominant groups detected in both replicate biocathode communities.
Members of the genus Oscillibacter are described as fermentative, strictly anaerobic, Gram-
negative-staining, non-sporulating, and motile; the type species is Oscillibacter valericigenes that
produces n-valeric acid as main fermentation end product from glucose [89]. Clostridium
butyricum is the type species of the genus of Clostridium [90]. C. butyricum can be isolated from
various sources including soil, freshwater and marine sediment. It is a Gram-positive, anaerobic,
spore-forming, bacterium that is able to fix atmospheric N2 and to produce H2 [90]. C. butyricum
ferments glucose to acetate, butyrate, CO2, and H2 [91]. Park et al. isolated an exolectrogenic
bacterium most closely related to C. butyricum that was able to produce electricity in a
mediatorless MFC [92]. In the current study, all members of the predominant groups belonged to
the phylum of Firmicutes, which has previously been shown to include several electrotrophs
capable of reduction of CO2 into organic compounds by electrosynthesis, like Sporomusa ovata
or C. ljungdahlii [42, 43].
Phylogenetic analysis based on obtained 16S rRNA sequence data revealed that three
representative clones from the dominant groups of biocathode communities were closely related
(Fig. 3.4). Interestingly, clone 1 that was most closely related to Trichococcus palustris DSM
9172 clustered with the electrotrophic bacterium Sporomusa ovata DSM 2662, indicating a close
phylogenetic relation [42]. Clone 3 was most closely related to Clostridium butyricum CB TO-A
and clustered with the electrotroph C. ljungdahlii DSM 13528 [43]. Thus, microorganisms
closely related to known electrotrophic microorganisms were successfully enriched on both
biocathodes using the described method. The fact that samples for community analysis were
43
taken at a relatively early time point following the heterotrophic start-up period of BES operation
might explain why biocathode communities still contained heterotrophs. In future applications the
acclimation period could be shortened to more rapidly reach conditions that favor autotrophic
growth. Another option would be to transfer electrodes after the start-up period to new reactors
containing fresh autotrophic medium.
Twenty six bacterial isolates were obtained via autotrophic cultivation of cathode
scrapings in anaerobic jars with carbon dioxide as sole carbon source and hydrogen as the source
of reducing equivalents. Identities of several of these isolates were determined by 16S gene
sequencing. Isolate ZZ16 showed 99% sequence similarity to Clostridium propionicum, a
bacterium that is obligately anaerobic, fermentative, spore-forming, motile, and that produces
hydrogen gas [90, 93]. C. propionicum converts lactate to propionate using acrylate as an
intermediate, but to my knowledge autotrophic metabolism has not been reported [94-96]. C.
propionicum was recently examined in a BES using chemical mediators for its ability to produce
electricity and concurrently accumulate the industrial valuable compound acrylic acid, used in
many paints and plastics, by delivering electrons to an anode rather than dumping electrons onto
its fermentation end product propionate [97]. Isolate ZZ25 had highest sequence identity (98%)
with Tissierella sp.S2. Members belonging to the genus of Tissierella are obligately anaerobic,
Gram-positive, non-sporeforming, and fermentative but also here to my knowledge autotrophy
has not been reported [98-100]. To prove if isolates ZZ16 and ZZ25 are (electro-) autotrophs,
follow up growth tests in liquid autotrophic medium with CO2 as sole carbon source and H2 or
cathodes as sole electron donor should be performed.
Three isolates (ZZ21, ZZ22, and ZZ23) were assigned to the species C. celerecrescens
with 97 to 99% sequence similarity. C. celerecrescens is an anaerobic, Gram-positive, spore-
forming, motile, cellulolytic bacterium first isolated from a methanogenic, cellulytic culture
[101]. Interestingly, C. celerecrescens was recently isolated among known biocorrosion causing
44
sulfate-reducing bacteria, like Desulfovibrio vulgaris and Desulfovibrio desulfuricans, from a
corroded steel gas pipeline in a petroleum-producing facility [102-104]. C. celerecrescens was
examined for its biocorrosion ability on API XL 52 steel surface by polarization resistance and
electrochemical impedence spectroscopy techniques and found to have an effect on the corrosion
process [105]. Since certain biocorrosive bacteria have been shown to accept electrons when
grown on graphite, these findings might indicate that C. celerecrescens, as a biocorrosive
bacterium, has mechanisms for direct uptake of electrons from electrode surfaces and ZZ21,
ZZ22, and ZZ23 may be potential biocathodic isolates [51, 52]. Interestingly, these isolates
showed high sequence similarities (≥ 97%) to a Desulfotomaculum sp., a genus that contains
several acetogens able to fix CO2 by using the Wood-Ljungdahl pathway [106, 107]. Based on
these results, the heterotrophic pre-enrichment method for establishment of biocathodes holds
great potential for the enrichment of CO2 fixing and biofuel/biochemical producing
microorganisms in BES. This appears due to the detected substantial decrease in headspace CO2
over time and associated production of biohydrogen and biochemicals.
The inversion technique, presented in Chapter 4, is based on a novel method developed
by Pisciotta et al., in which a sMFC anode is converted to a MEC biocathode. This is a simple,
robust method for the enrichment of obligate anaerobic biocathodes [55]. Compared to previous
inversion methods it does not require the use of non-renewable and potentially toxic cyanide
compounds as soluble mediators nor the use of multi-step acclimation procedures using
complicated reactor designs [48, 59, 60]. Oxygen leakage through reactor seals and gaskets that is
common for certain reactor designs is especially a problem for the growth and enrichment of
strict anaerobes [108]. The sMFC reactor makes use of the fact that dissolved oxygen typically
does not penetrate more than ca. 1 cm below the surface [55, 109, 110]. Previously, it was
successfully shown that this method can enrich bioelectrosynthetically active electrotrophs from
marine sediment that convert CO2 into biogas [55]. Here, it was examined whether electrotrophs
45
could be enriched from freshwater sediment samples to explore if enrichment of microorganisms
capable of microbial electrosynthesis might be enhanced at elevated temperatures compared to a
more traditionally studied mesophilic temperature.
For the two different operational temperatures (48°C vs. 30°C), separate closed and open
circuit sMFCs were prepared. No acetate was added to run reactors only from organic matter in
the bog sediment. After 3 months of sMFC operation, the sMFC anodes were inverted to act as
electron accepting biocathodes in anaerobic two-chamber BESs. LSV results clearly showed that
electrodes established as a closed circuit sMFC bioanode outperformed electrodes established in
open circuit mode. Abiotic graphite, used as a sterile negative control, exhibited lowest electron
uptake capability (Fig. 4.2). Long-term operation of inverted bioanodes to biocathodes at a set
potential of −0.4 V did not confirm LSV predicted performances for sediment anodes operated in
closed sMFC system at 30°C. This suggests short term patterns of electron uptake (LSV results)
cannot definitively predict long term patterns of electron uptake in mixed culture biofilms.
Importantly, electrodes incubated throughout at an elevated temperature of 48°C
outperformed those incubated at 30°C, even if a more negative potential was applied. It is well
known that higher temperatures increase reaction rates and decrease activation energies for many
chemical and biological reactions. This suggests organic matter in the sMFC may have been
consumed and exhausted sooner in the elevated temperature samples, thus extending the period
during which conditions favorable for enrichment of autotrophs predominated, relative to the
mesophilic samples. It is widely established practice to add acetate for start-up of conventional
mesophilic BES bioanodes [48, 60, 111]. The lack of added acetate could help explain these
results. Headspace gas analyses showed that CO2 decreased over time in BES reactors operated at
48°C whether obtained from open or closed circuit sMFCs. BES reactors containing inverted
electrodes from sMFCs at 48°C (open and closed circuit) alone showed production of acetate.
These findings suggest that CO2 was reduced to acetate by an electrotrophic community at the
46
cathode. For example, stoichiometric calculations indicate that for the electrode from the closed
circuit sMFC incubated at 48°C, 42% of the electrons and 10% of the CO2 consumed in the BES
could have resulted in the formation of detected acetate. Existing knowledge about production of
acetate via microbial electrosynthesis by acetogenic bacteria is well established [42, 43]. As
demonstrated here, the sMFC inversion method holds significant potential for enrichment of
electrotrophic microorganism at elevated temperatures. The importance of acetate as a product
lies in the fact that it is formed from acetyl coenzyme A (acetyl-CoA). This molecule is an
essential intermediate used to produce a variety of cell metabolites and a precursor in the
production of energy dense drop in biofuels and diverse chemicals for chemical and
pharmaceutical industries [112-114]. A promising option is the biosynthesis of hydrocarbons or
butanol using genetically engineered acetogens while providing carbon dioxide and electrical
current instead of hydrogen [44, 115, 116]. Therefore, a paramount research objective is to
discover such suitable candidates, particularly fully sequenced and biochemically well
characterized microbial species, for future large-scale applications.
Engineering applications of biocathodes and microbial electrosynthesis are not only
conceivable in the production of biofuels, but also in energy storage systems for intermittent
renewable energy sources or in the production of biochemicals for pharmaceutical and chemical
industry [44, 45]. Furthermore, biocathodes and microbial electrosynthesis can be used in waste
water treatment [45] or in bioremediation of various inorganic and organic pollutants, e.g. heavy
metals, trichloroethene, or 2-chlorophenol [14, 28, 57, 58].
47
Chapter 6
Conclusions and Future Directions
Two novel start-up methods have been presented which demonstrated high potential for
accelerated establishment of CO2-fixing and biofuel producing biocathodes in BES. The
heterotrophic pre-enrichment method shortened the start-up time for biocathode establishment
(compared to direct cathode chamber inoculation with fresh bog sediment) and supported stable
and sustained current density uptake for nearly two months. An electrotrophic community was
established in replicate reactors that consumed CO2 and produced biohydrogen and biofuels, as
determined by HPLC and GC analysis. The heterotrophic start-up conditions could be, in the
future, optimized to improve or direct the selection of facultative autotrophic bacteria. This
method holds great potential to enrich for bacterial communities for microbial electrosynthesis. It
can be employed to enrich electrotrophic microorganisms with diverse metabolic capabilities and
is not strictly limited to the enrichment of acetogens.
The inversion method in which sediment anodes are converted into MEC biocathodes has
been previously shown to be successful in the enrichment of biofuel producing and CO2 fixing
electrotrophs. Here operational improvements related to temperature were tested and the
inversion method’s utility for enriching freshwater electrotrophs was explored. It was discovered
that a higher temperature of BES operation may facilitate organic product formation. The sMFC
inversion method is therefore confirmed as a promising technique, particularly for the enrichment
of anaerobic electrotrophs for microbial electrosynthesis from diverse aquatic settings.
Pure culture tests were initiated with Trichococcus palustris DSM 9172, a dominant
biocathode species (Appendix E). Another pure culture experiment was performed using the
sulfate-reducing acetogenic strain Desulfobacterium autotrophicum ATCC43914. Db.
autotrophicum was discovered to fix CO2 in association with acetate production and current
48
uptake from a graphite electrode (Appendix F). Db. autotrophicum is a promising candidate for
detailed biomolecular studies into the establishment and operation of electrosynthetic biocathodes
because it is fully sequenced and biochemically well characterized. This slow growing bacterium
could be suitable for genetic engineering in scaled-up systems. Future work will test these and
other promising pure cultures to gain insight into the biological pathways and electron transfer
mechanisms involved in establishment, growth and metabolism of electrotrophs.
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Appendix A
Preliminary Biocathode Experiments at Different Set Potentials
Figure A.1 Operation of duplicate reactors at applied potential of −0.089 V. Anodic reactions were
taking place at the working electrodes.
Figure A.2 Operation of duplicate reactors at applied potential of −0.3 V. Cathodic reactions were
taking place at working electrodes.
57
Figure A.3 Comparison of duplicate reactors at applied potentials of (a) −0.3 V or (b) −0.4 V at
working electrodes. Cathodic reduction reactions were taking place at both potentials.
Consumption of current was higher at set potential of −0.4 V.
58
Appendix B
Preliminary Pre-Enrichment Experiments with Different Inocula
Figure B.1 Reactors operated after inoculation of cathode chambers with pre-enriched culture. (a)
Current density uptake and (b) consumption of electrons at applied potential of −0.4 V (B1-PE and
B2-PE duplicate reactors, pre-enriched bog sediment; W1-PE pre-enriched wastewater). (c) Cyclic
voltammetry after four weeks of operation.
Appendix C
Supplemental Materials - Chapter 3
Figure C.1 HPLC chromatograms obtained from analysis of cathode chamber solutions from (a, b)
biotic replicates and (c) control reactors to examine excreted metabolites. Red arrows show to
peak of acetic acid; blue arrows show to peak of propionic acid; black arrows show to unknown
peaks.
60
Figure C.2 GC chromatograms obtained from analysis of cathode chamber solutions from (a, b)
duplicate reactors to examine excreted volatile fatty acids (VFAs). Green arrows show to peak of
ethanol; pink arrows show to peak of butanol; purple arrows show peak of butyrate.
61
Table C.1 Number of clones belonging to major bacterial groups in duplicate reactors (n =95 and n
=94).
Genus/ Species # of clones
(reactor 1)
# of clones
(reactor 2)
Trichococcus palustris 44 56
Oscillibacter sp. 22 9
Anaerofilum sp. 2 7
Clostridium sp. 20 4
Other 7 18
Total (n) 95 94
Sequencing Results of 11 Strains Isolated Under Autotrophic Growth Conditions
ZZ12
GTCGCTGCCTCGCTNNNNCGTNATCTCAAAAACTTTGGGTATTGNNAACTCTCGTGGTGTGAGGGGNGGTG
TGCACNGGGCCNGGGAACATTTTCACCGCCACTTTCTGATTCGNGATTACTAGCAACTCCCTCTTCGTGTG
NGAGAGTTTCAGCCTACTATCCGAAGTGATATNGGTTTTATTTTTGTGCTCTCTCTCGNGAGGTTGCCTCT
CTGTGTACCGAATATTGTANCACGTGTGTACCCCTACACATAGGGGGCATGAAGATTTGACGTCCTCCCCA
CCTTCCCCCGGGATAACCNGGGGAGTCTCNCTAGAGTGCTCAANTAAGNGGTCGCCACTAANAAAANGGGG
TGCGCTCGTTGNGAGATTTAACCCAATATCTCAACACACGATGTGAACACNACCGTGCCCCACCTGTCTTC
GTGCCACCAAAGGGGTTTCCTCCATTACAGAAAAATTCGAGANATGTCGAGTCTGGGTAAGGCTCTTCGCG
TTGCTTCAAATTAAACCATGNGCTCCTGTGCGTGTGGGGGCCCCCGTCTATTTCTTAGAGTTNAANTCGTG
CNACCGCACTCCCNANGCAGNATACTTAGTGCNTTAGCCGCGGCACAGATGTTNTGACCACCTCTACATCT
AGTACTTCTCGTNTAGNGTGNNNACTACNAGGGTATCTACNTGTGTGTGCTCCCCACTCTCTCGAGTCTCN
GTGTGATATAGNGTCCANANNNGCGCCTTCNACTGTGGTATTCTTTCTTATCTNNACATATTTCACCNNTA
CACTNNAAATTCTCTTTTCCTCTCGTGCACTCNAGATATCCAGTNNNNAGTGCAGCNCCCGTGATAANCCC
GANTATNTCANNTCNCANNTAAATATCCCNNNACTCTCNNTNNACCCNCNAGAAAANCNNNNACNACNNTN
GNCNNNTACNTANTNNNGNGTGNTNNNGNCACAGNNATANCNGGGGTTNTCCTNNGTGGGNCNACCGNTNT
TNATCNCCCCCAAAANACANNATTTTTAACACCCANNANGNCCGTCATCNNCTCACGGCGGGGTCNGCTGC
NNNGGGTTTCCCCCCA
ZZ13
TGCCTCNTTTNNNGTTATCTCNCAANNTGTGGGTNGNGNNNTCTCTCATGGNNNNNNGGGGGGTGNNNNNN
AGGCNNGGAAATATATANNNCGCNACNTTCTNNNTCGNNTATTCTAGAATCTCCNNCTTCGTGNAGGAGAN
TTTCACNCCACAANCCGNNNTGATATNGGTTTTATNGTTTTGCNCACTCTCGCGANGTTGNNTCTNATTGT
NCCCACCATTGTACCACGTGTGTAGCTCTAGATATAAGGGGTNTGATGATTTGACTTTATCCCCATTTTCC
62
TCCCGGNTACCCCGGGCAGTCTCGCTAGAGTGCTNNACTAANTGGTAGCAACTCACAATANGGGGTGCGCT
TGTTGGGGGTNTTNCCCCAATATCTCAAGACNCGATNTGAACANCACTATGCACCATCTGTCTTCGTGCCA
CCGAAGTGGCTNCCTNCATTANAGAGNAATTCANGATATGTNNNNNNNNTGTAANGCTCTTCGCTTNNNTT
CAAATTAAACCACANGCTCCNGTGNTTGTGNGGGCCCCCGTCAATTCCTTNGAGTTTTAATCTTGCNACCG
CNCTCCCCGGGNGGAATANTNAATGCGTTNGCGGNNGCACAGATGANNTGACCACCNCTACACCTANTATT
CATCGTTNACNGCGNNNACTNCCNNGGTNTNNANNNNTGTTTGCTCCCCACGCTTTCGAGCCTCAGTGTNT
GTTACAGTTCAGAAGGGCGTCTTCNCCTGTGGTATTTCTTTCTAATCTCTACNNATTTTCACCGCTACACT
ANGAANTTCTCCTTTCTCTCNNGCACTCTANATANNAGNNNGNANCGCAGCACCCAGGTTAAGCCCNAGTA
TTTCACATCCCAATTAAATATCCCNCTACNCCCCCTTTANCCCANAAATCCGNANAACTCGTGGCCACCNA
CNTATTAC
ZZ16
GGCTCCTTCCTTACGGTCAGGTCACCGACTTCGGGTGCTTCCAACTCCCATGGTGNNNNNGGNGGTGTGTA
CAAGGCCCGGGAACGTATTCACCGCGACATTCTGATTCGCGATTACTAGCGATTCCAGCTTCATGTAGTCG
AGTTGCAGACTACAATCCGAACTGGGACTGGGTTTTTGTGATTTGCTCGACGTCGCCGTTTTGCGTCACTT
TGTTACCAGCCATTGTAGCACGTGTGTAGCCCAAGACATAAGGGGCATGATGATTTGACGTCATCCCCACC
TTCCTCCGTGTTATCCACGGCAGTCTCCCTAGAGTGCCCAACTGAATGATGGCTACTAAGAATAGGGGTTG
CGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCTC
TGCTCCGAAGAGAAGGTGTATCTCTACACCGGTCAGAGGGATGTCAAGCCTTGGTAAGGTTCTTCGCGTTG
CTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAATCTTGCGA
TCGTACTCCCCAGGTGGAGTGCTTATTGCGTTAGCTGCGGCACCGAGGGTTCCCCCCCGACACCTAGCACT
CATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGAGCCTCAGCGTCA
GTTTCAGTCCAGTAAGTCGCCTTCGCCACTGGTGTTCTTCCTAATATCTACGCATTTCACCGCTACACTAG
GAATTCCACTTACCTCTCCTGTACTCTAGCTTGATAGTTTTAAATGCAATCCCGAGGNTAAGCCTCGGGCT
TTCACATCTAACTTACCATGCCGCCTACTCTCCCTTTACNCCCAGTAATTCCGGATAACGCTTGCCCCCTA
CGTATTACCGCGGCTGCTGGCACGTANNTAGCCGGGCTTTCTTATTCAGGGTACCGGTCATTTTTTTCGTC
CCTNTNGANAGAANNTTTACNANGCCGAAACCCTTCTTCCTTCACGCGNNGNTGCTGCATTCAGGCTTTTC
NCCCATTGGTG
ZZ17
CCGCCTANANTNNNNNANTTCNNGTTTTNTTTGNTCTCATGGTNCCNNTTNNNNNNGTGTGTACAAGGCCC
GGGAACGGATTCACCGCGGCNTGCTGATCCGCGATTACTAGCAATTCCGGCTTCATGGGGGCGGGATGCAN
ACCCCAATCCGAACTGAGACTATTTTTNTGAGGTTTGCTCCACCTTGCGGNNTCGCTTCTCTCTGTTAATA
GCCATTGTAGTACGTGTGTAGCCCAAGTCATAAAGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCG
TTTTGTCCACGGCAGTCTCACTAGAGTCCTCTTGCGAATNAACTANTAATAAGGATTGCGCTCGTTGCGGG
ACTTAACCCAACCNCTCANGACACGAGCTGACGANAACCATGCACCTCCTGNCTCAACNTCCCGAANGAAA
ACCTAATCTCTTNNNCGGNCGTTGGATGTTAAGANCTGNTAACGTTTTTCGNNTTGTTNNNAATTAAANCA
NNNACTCCACTGCTTGTGCGGGCCNNCGACAATTCCTTTGANNTTCAACCTTGCGGNNTNCTCCCCACTNN
ATTANTTANNTGTGTTAACTGCTNACGGAGAGGGTNANTC
ZZ18
CNTTAGGTCGCTGCCTCGCTTACGCGTTATCTCAAAAATTTTGGATATNGCNNTCTCTNGTGGTGNNANGG
GGGGTGTGTACNGNGCCNGGGAACGTATTCCCCGCCACTTTCTGATTCGNGATTACTAACATCTCCCTTTT
CGTGTGCGAGANTTTCCCCCTACTCTCCAAAGTGATATCGGTTTTATTTTTTTGCTCTCTCTCGCGGGGTT
GTCTCTCATTGTACCCACCATTGTAACACGTGTGTACCCCTACACATNGGGGGCGTGAAGATTTGACCTCC
TCCCCCCCTTCCTCCCGGATAACCCGGGCACTCTCGCTAGAGTGCTCAAATAAGTGGTAACAACTAAAAAT
AAGGGGTGCTCTCGGTGCGAGATTTAACCCAATATCTCAAGACACGATGTGAACACCACTATGCACCATGT
GTCTTCGTGCCCCAAAAGTGTTTTCCTCCTTTACAGAATATTTCGAGATATGTCAAGTATATGTAAGGTTT
TTCGTGTTGCTTCATATTAAAACATGTGCTCCTCTGCGTGTGCGGCCCCCCGAATATTCCTNNGATTTTTA
63
CTCGTGNNACCGCACTCCCCNNGNNNAATNNTTAGTGNNATAGCNGNNGCACAGANGNCGTGANNNCCTCT
ACATCTAATATTCATCGTATACNGTGTGNACTACNNNGATATCTACTCGTGNNTGCTCCCCACTCTCTCGA
GTCTCAGTGTCATATACACTCNANANAGGCNNCTTCNCCTCTGATATTCTTCCTAATCTCTACNCATTNCN
NCGCTACNCTANNNNNTCTCNNTNNCTCTCGTGCACTCTAGATATCCAGTGNNNNANGCANCNCCNGNGAT
AAGCNAGNNTATCTCACATCACANATAAATATNCNNCTACTCTCTCTNTACNCCNNNNANNTCGAGACAAC
TNGNGCCATCCTACATANTACCGCNNNCTGCTGGNCACANNANATANNNCGNGTCTTCCTNCNGTGGGANA
CCGNNNANTNNNNTCCNCNAAANANANANTTTTANNACCNAAAGCGCCGNTCANC
ZZ20
CGTGTANGAGAGTNTCANNNTACTCTCNNNTGTGATATCGGTTTTATAGTTGTGCTCTCTCTCGAGAGGGT
GTCTCTCAGTGTANCGACTAGTGTANCACGTGTGTACCNNTANACATNGGGGGNGTGAAGATTTGACNNCN
TCCCCNCCTTCCTCCNNGATAACCNGGGNAGTCTCTCTAGAGTGCTCAACTAAGTGGNANCAACTAANAAT
NNNGGGTGCTCTCGGTGCGAGACTTAACCCAATATCTCAAGACNNGATNTGAANANNACTATGCACCATGT
GTCTTCGTGCCANCAANGTGTCTTCCT
ZZ21
GGGTGTACAAGACCCGGGAACGTATTCACCGCGGCATTCTGATCCGCGATTACTAGCGATTCCAGCTTCAT
GTAGTCGAGTTGCAGACTACAATCCGAACTGAGACGTTATTTTTGGGGTTTGCTCCAGATCGCTCCTTTGC
TTCCCTTTGTTTACGCCATTGTAGCACGTGTGTAGCCCAAATCATAAGGGGCATGATGATTTGACGTCATC
CCCACCTTCCTCCAGGTTATCCCTGGCAGTCTCCCCAGAGTGCCCAACTTGACTTGCTGGCTACTAAGGAT
AAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCT
GTCTGGAATGCCCCGTAGGGAAGGGATCGTTACATCCCGGTCATTCCGATGTCAAGACTTGGTAAGGTTCT
TCGCGTTGCTTCCAATTAAACCACATGCTCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCAT
TCTTGCGAACGTACTCCCCAGGTGGAATACTTATTGCGTTAGCGGCGGCACCGAAGAGCTTTGCTCCCCAA
CACCTAGTATTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGAG
CCTCAACGTCAGTTACAGTCCAGTAAGCCGCCTTCGCCACTGGTGTTCCTCCTAATATCTACGCATTTCAC
CGCTACACTAGGAATTCCACTTACCTCTCCTGCACTCTAGCACCACAGTTTCCAAAGCAGTCCCGGGGTTG
AGCCCCGGGCTTTCACTCCAGACTTGCANNGCCGCCTACGCTCCCTTTTACACCCAGTAAATNNGGATAAC
GCTTTGCCCCCTACGTATTACCGCGGGCTGCTGGGNACGTANNTTANCCCGGGGCTTCTTANNCNGTACNG
TCATTTTCTTCCCNTGCTGNTAGAAGCTTTANNTACCGAAANNACTTTNNTCNNTCNNNGCGNNNCNCNTN
GANCNANGGNTTCCCCCCNTTGNCCCAA
ZZ22
CGGCAGCTCCCTCCTTACGGTTGGGTCACTGACTTCGGGCGTTACCAACTCCCATGGTGTGACGGGCGGTG
TGTACAAGACCCGGGAACGTATTCACCGCGGCATTCTGATCCGCGATTACTAGCGATTCCAGCTTCATGTA
GTCGAGTTGCAGACTACAATCCGAACTGAGACGTTATTTTTGGGGTTTGCTCCAGATCGCTCCTTTGCTTC
CCTTTGTTTACGCCATTGTAGCACGTGTGTAGCCCAAATCATAAGGGGCATGATGATTTGACGTCATCCCC
ACCTTCCTCCAGGTTATCCCTGGCAGTCTCCCCAGAGTGCCCAACTTGACTTGCTGGCTACTAAGGATAAG
GGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTC
TGGAATGCCCCGTAGGGAAGGGATCGTTACATCCCGGTCATTCCGATGTCAAGACTTGGTAAGGTTCTTCG
CGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCATTCT
TGCGAACGTACTCCCCAGGTGGAATACTTATTGCGTTAGCGGCGGCACCGAAGAGCTTTGCTCCCCAACAC
CTAGTATTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGAGCCT
CAACGTCAGTTACAGTCCAGTAAGCCGCCTTCGCCACTGGTGTTCCTCCTAATATCTACGCATTTCACCGC
TACACTAGGAATTCCACTTACCTCTCCTGCACTCTAGCACCACAGTTTCCAAAGCAGTCCCGGGGTTGAGC
CCCGGGCTTTCACTCCAGACTTGCAGTGCCGTCTACGCTCCCTTTACACCCAGTAAATCCGGATAACGCTT
GCCCCCTACGTATTACCGCGGCTGCTGGCACGTANTTAGCCGGGGCTTCTTANTCNGNTACCGNCATTTTC
TTCCCTGCTGATAGAGCTTTACATACCGAAATANTNCTTCACTCACGC
64
ZZ23
GCGCTNCCTCCTTACGGTTGGGTCACTGACTTCGGGCGTTACCAACTCCCATGGTGTGACGGGCGGTGTGT
ACAAGACCCGGGAACGTATTCACCGCGGCATTCTGATCCGCGATTACTAGCGATTCCAGCTTCATGTAGTC
GAGTTGCAGACTACAATCCGAACTGAGACGTTATTTTTGGGGTTTGCTCCAGATCGCTCCTTTGCTTCCCT
TTGTTTACGCCATTGTAGCACGTGTGTAGCCCAAATCATAAGGGGCATGATGATTTGACGTCATCCCCACC
TTCCTCCAGGTTATCCCTGGCAGTCTCCCCNNAGTGNCCAACTTGACTTGCTGGCTANNAANGATAAGGGT
TGCGCTCGTTGCGGGACTTAACCCAACATCTCACAACACGAGCTGACGACAACCATGCACCACCTGTCTGG
AATGCCCCGTAGGGAAGGGATCGTTACATCCCGGTCATTCCGATGTCAAGACTTGGTAAGGTTCTTCGCGT
TGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCATTCTTGC
GAACGTACTCCCCAGGTGGAATACTTATTGCGTTAGCGGCGGCACCGAAGAGCTTTGCTCCCCAACACCTA
GTATTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGAGCCTCAA
CGTCAGTTACAGTCCAGTAAGCCGCCTTCGCCACTGGTGTTCCTCCTAATATCTACGCATTTCACCGCTAC
ACTAGGAATTCCACTTTACCTCTCCTGCACTCTAGCACCACAGTTTCCAAAGCAGTCCCGGGGTTGANCCC
CCGGGNTTTCACTCCNGACTTGCAGTGNCGNCTACGCTTCCTTTACACCCAGTANATCCNGATAACGCTTG
CCCCCTACNTATTACCCGCGGNTGCTGGCACGTANTTAGCCNGGGGNCTTCTTAGTCAGGTACCGTCATTT
TCTTCCNNNNCTGANNNAGTTTTNNATACCNNANATACTTCTTCCANNNNACGCNGNNTNCNCNTGNATTC
AGGNTNNCCTCCNTTNGTCCAATATNNCCCNNCNNNCNNCCNNCCGNANGGAAGTTNG
ZZ25
TTCNNNGCTGCCTCCTTACGGTTAGCTCACTGGCTTCGGGTATTGCCAACTCCCATGGTGTGNNNGGCGGT
GTGTACAAGACCCGGGAACGCATTCACCGCGACATTCTGATTCGCGATTACTAGCAACTCCGACTTCATGC
AGGCGAGTTGCAGCCTGCAATCCGAACTGGGATCGGCTTTAAGAGATTGGCAACTTATCGCTAAGTAGCTA
CTCGTTGTACCGACCATTGTAGCACGTGTGTAGCCCAGGACATAAAGGGCATGATGATTTGACGTCATCCC
CACCTTCCTCCGATTTATCATCGGCAGTCCCTCTAGAGTGCTCAGCCGAACTGTTAGCAACTAAAGGCAAG
GGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTC
ACCTCAGTCCCCGAAGGGAAAGGATTATCTCTAATCCGGTCCGAGGGATGTCAAGCCCTGGTAAGGTTCTT
CGCGTTGCTTCGAATTAAACCACATGCTCCGCTGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCATA
CTTGCGTACGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCGGCACCGAGGTTTGACCCCCAACACC
TAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGTGCATC
AGCGTCAGTATAAGTCCAGAAAGTCGCCTTCGCCACTGGTATTCCTCCTAATATCTACGCATTTCACCGCT
ACACTAGGAATTCCACTTTCCTCTCCTTANCTCAAGCCTTCCAGTTTNAAATGCTTACCACGGTTGAGCCG
TGACCTTTCACATCTGACTTAAAAAGGCCGCCTACGCACCCTTTTACGCCCAATAANNCCNGACAACGCTT
GCTCCCCTACGTATTACCGCGGCTGCNNNCACGTAGTTAGCCCGGGCTTNNTNCNTANNGTACCGTCATTA
TCGTCCCTTTAGNANAGAAANTTTACNNACCNGNANGNCCNTNCNTCGTTCACG
ZZ26
TCTACTANNNGATGTGATATCGGTTNTATAGTTGTGCTCTCTCTCGAGANGNTGTATCTCAGTGTANCGAC
TAGTGTANCACGTGTGTACCCCTACACATNGGGNGCGTGAAGATNTGACNNCNTCCNCNCCTTCCTCCNGG
NNAACCCGGGCAGTCTCGCTAGAGTGCTCATCTAAGTGGNAGCAACTAANAATNNGGGGTGCNCTCGGTGC
GAGACTTANCCNAATATCTCAANACNNGATNTNANNACNACNATGCNNCATGTGTNTTCGTGNCNNANAAG
TGTNTTNCTCNNTTACAGANNATTTNGAGANATGTCAAGTNTANGANNNGCTCTTCGNNNTGNTTCAAATT
ANANCACGTGCTNCTGTGCGTGTGCGGNCCCGCGTCAATTCCTNAGAGTTNNANTCNNGAGACTGCACTCC
NNNNGNANNATACTNNGNGNNATAGNNNNNNNACANANGNNNNNANANNNTACNTA
65
Appendix D
Supplemental Materials – Chapter 4
Figure D.1 Current produced by sMFC inoculated with bog sediment and operated at 48°C.
66
Figure D.2 Current produced by duplicate sMFCs (a) sMFC1 and (b) sMFC2. A steady increase of
current produced over time was recorded.
67
Figure D.3 Current density uptake (a) and consumption of electrons (b) by BES operated at
applied potential of −0.4 V after inversion of sMFC anodes into MEC cathodes. After 30 days
applied voltage was decreased to −0.5 V. Sediment reactors and MECs were incubated at 30°C.
68
Figure D.4 HPLC chromatograms obtained from analysis of cathode chamber solutions from two-
chamber BES operated at 48°C with (a) biocathode obtained from former sMFC (b) biocathode
obtained from open circuit sMFC and (c) sterile control reactors to examine excreted metabolites.
Red arrows show to peak of acetic acid.
Appendix E
Pure Culture Test Using Trichococcus palustris DSM9172
Figure E.1 Current uptake by Trichococcus palustris DSM 9172, a strain that was shown to be
dominant in biocathode communities (see Chapter 3). A set potential of −0.5 V was applied at
30°C. TP1 (green) and TP2 (blue) are duplicate reactors. Red curve is a sterile control.
70
Appendix F
Pure Culture Test Using Desulfobacterium autotrophicum ATCC43914
Figure F.1 Desulfobacterium autotrophicum is a fully sequenced and well characterized obligately
anaerobic sulfate-reducing acetogen able to fix CO2 coupled with oxidation of hydrogen gas. In
this experiment hydrogen was replaced by a graphite electrode as source of energy and reducing
equivalents. Current uptake by BES inoculated with Db. autotrophicum, is shown during fourth
week of operation at an applied potential of −0.5 V and incubated at 30°C. Reactors R1 and R2
have electrodes that were acclimated to BES by addition of hydrogen and then transferred to new
reactors. Reactors R3 and R4 contain former acclimated culture of Db. autotrophicum but contain
new electrodes (recolonization test); reactor was degassed with CO2:N2 (20:80) mix to remove
residual hydrogen and to provide CO2 for autotrophic growth. Blue line is the control containing
sterile medium and sterile graphite electrodes.
71
Figure F.2 Decrease of mmoles of CO2 headspace gas over 21 days. It can be seen that greatest
decrease in CO2 content occurred in reactor R4, which also showed greatest current uptake. This
might indicate CO2 fixation coupled with consumption of electrons from cathode.
72
Figure F.3 HPLC chromatograms of control reactor (a) on day 0 and (b) on day 21.
73
Figure F.4 HPLC chromatograms of reactor R1 (a) on day 0 and (b) on day 21. Increase of acetate
is detected.
74
Figure F.5 HPLC chromatograms of reactor R2 (a) on day 0 and (b) on day 21. Increase of acetate
is detected.
75
Figure F.6 HPLC chromatograms of reactor R3 (a) on day 0 and (b) on day 21. Increase of acetate
is detected.
76
Figure F.7 HPLC chromatograms of reactor R4 (a) on day 0 and (b) on day 21. Increase of acetate
is detected.
77
Figure F.8 Results of GC measurements on day 21. Acetate peaks are detected in all cathode
solutions inoculated with Db.autotrophicum except that of the sterile control reactor. (Control)
Control reactor. (R1) Reactor R1. (R2) Reactor R2. (R3) Reactor R3. (R4) Reactor R4.