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Investigation of electron transfer mechanisms
in electrochemically active microbial biofilms
Von der Fakultät für Lebenswissenschaften
der Technischen Universität Carolo-Wilhelmina
zu Braunschweig
zur Erlangung des Grades eines
Doktors der Naturwissenschaften
(Dr. rer. nat.)
genehmigte
D i s s e r t a t i o n
Kumulative Arbeit
von
Alessandro Alfredo Carmona Martínez
aus
Oaxaca / Mexiko
1. Referentin oder Referent:
Prof. Dr. Uwe Schröder
2. Referentin oder Referent:
Prof. Dr. Rainer Meckenstock
eingereicht am:
30.05.2012
mündliche Prüfung (Disputation) am:
05.10.2012
Druckjahr 2012
Vorveröffentlichungen der Dissertation
Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für
Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab
veröffentlicht:
Publikationen
Chapter 2: A.A. Carmona-Martinez, F. Harnisch*, L.A. Fitzgerald, J.C. Biffinger, B.R.
Ringeisen, U. Schröder, Cyclic voltammetric analysis of the electron transfer of Shewanella
oneidensis MR-1 and nanofilament and cytochrome knock-out mutants, Bioelectrochemistry,
81 (2011) 74-80.
Chapter 3: A.A. Carmona-Martínez, F. Harnisch*, U. Kuhlicke, T.R. Neu, Uwe Schröder,
Electron transfer and biofilm formation of Shewanella putrefaciens as function of anode
potential, Bioelectrochemistry, (2012) Accepted.
Chapter 4: A.A. Carmona-Martinez, K.H. Ly, P. Hildebrandt, U. Schröder, F. Harnisch*, D.
Millo*, Spectroelectrochemical analysis of intact microbial biofilms of Shewanella species for
sustainable energy production, In preparation, (2012).
Chapter 5: S. Chen, H. Hou, F. Harnisch, S. A. Patil, A. A. Carmona-Martínez, S. Agarwal,
Y. Zhang, S. Sinha-Ray, A. L. Yarin*, A. Greiner*, U. Schröder*, Electrospun and solution
blown three-dimensional carbon fiber nonwovens for application as electrodes in microbial
fuel cells, Energy & Environmental Science, 4 (2011) 1417-1421.
Chapter 6: S. Chen, G. He, A.A. Carmona-Martínez, S. Agarwal, A. Greiner, H. Hou*, U.
Schröder*, Electrospun carbon fiber mat with layered architecture for anode in microbial fuel
cells, Electrochemistry Communications, 13 (2011) 1026–1029.
Chapter 7: S.A. Patil, F. Harnisch*, C. Koch, T. Hübschmann, I. Fetzer, A.A. Carmona-
Martínez, S. Müller*, U. Schröder, Electroactive mixed culture derived biofilms in microbial
bioelectrochemical systems: the role of pH on biofilm formation, performance and
composition, Bioresource Technology, 102 (2011) 9683–9690.
Chapter 8: F. Harnisch*, C. Koch, I, Fetzer, A. A. Carmona-Martínez, S. F. Hong, S. A.
Patil, T. Hübschman, U. Schröder, S. Müller*, Electroactive mixed culture derived biofilms in
microbial bioelectrochemical systems: the role of inoculum and substrate on biofilm
formation and performance, In preparation (2012).
*indicates authors of correspondence
Tagungsbeiträge
Oral presentations:
A.A. Carmona-Martínez, F. Harnisch, U. Kuhlicke, T.R. Neu, U. Schröder. 2012. Electron
Transfer and Biofilm Formation of Shewanella putrefaciens as Function of Anode Potential.
Submitted for oral presentation at the EU-ISMET meeting: From extracellular electron
transfer to innovative process development, Ghent (Belgium), September 27th – 28th, 2012.
A.A. Carmona-Martínez, 2009. Microbial fuel cells: an alternative for the production of
clean electricity. Abstract F128. Presented at the German Academic Exchange Service
Scholarship Holders Meeting. Hanover (Germany). June 19th – 21th, 2009.
Poster presentations:
A.A. Carmona-Martínez, S. Patil, F. Harnisch, U. Schröder, S. Chen, C. Greiner, A.
Agarwal, H. Hou, Y. Zhang, S. Sinha-Ray, A. Yarin. 2011. High Surface Area Electrospun
and Solution-blown Carbonized Nonwovens to Enhance the Current Density in
Bioelectrochemical Systems (BES). Abstract ELE 026. Presented at Wissenschaftsforum
Chemie 2011, Bremen (Germany), September 4th – 7th, 2011.
A.A. Carmona-Martínez, F. Harnisch, U. Schröder. 2010. Analysis of the electron transfer
and current production of Shewanella oneidensis MR-1 wild-type and derived mutants.
Abstract P058. Presented at Electrochemistry 2010: From microscopic understanding to
global impact, Bochum (Germany), September 13th – 15th, 2010.
A.A. Carmona-Martínez, F. Harnisch, U. Schröder. 2009. Cyclic voltammetry as a useful
technique to characterize electrochemically active microorganisms: Shewanella putrefaciens.
Abstract AE15. Presented at Wissenschaftsforum Chemie 2009, Frankfurt am Main
(Germany), August 30th – September 2nd, 2009. ISBN: 978-3-936028-59-1.
„Gedruckt mit Unterstützung des Deutschen Akademischen
Austauschdienstes“
To Yolanda, Jesús and Virginia,
for their love and support...
Acknowledgements
First and foremost, I express my gratitude towards my supervisor Prof. Dr. Uwe Schröder for
supporting me since the very first moment I applied for the scholarship to conduct Ph.D.
studies in Germany. Prof. Schröder encouraged me to pursue my own ideas while providing
me invaluable academic freedom and substantial support throughout my entire Ph.D.
I would like to thank as well Dr. Falk Harnisch for his supervision, critical suggestions and
academic inspiration. I want also to thank all the time he has invested in my thesis with
constant guidance during design, planning, data analysis and manuscript writing.
I deeply appreciate the financial and logistic support by the German Academic Exchange
Service providing me a Ph.D. scholarship that allowed me not only to conduct my thesis work
but also by procuring all necessary support to enjoy the academic German culture.
Furthermore, I thank the financial support by the Mexican Secretariat of Public Education for
providing me a complementary Ph.D. scholarship during my stay in Germany.
I am very much grateful to Dr. Sunil A. Patil and Dr. Siang-Fu Hong for valuable
experimental assistance, cooperation and fun time during my stay at the Technischen
Universität Carolo-Wilhelmina zu Braunschweig. Thanks to their hands-on experience, I was
able to solve in a successful way several experimental obstacles.
I would like to sincerely acknowledge the following people for their support and successful
collaboration: 1) Dr. B.R. Ringeisen, Dr. L.A. Fitzgerald and Dr. J.C. Biffinger at the Naval
Research Laboratory in Washington, USA; 2) Dr. T.R. Neu and Ute Kuhlicke at the
Helmholtz Centre in Magdeburg, Germany; and finally 3) Dr. D. Millo, K.H. Ly and Prof. Dr.
P. Hildebrandt at the TU Berlin.
I thank all former and current members of the Sustainable Chemistry and Energy Research
group at the TU Braunschweig for their individual contributions to a very friendly research
atmosphere full of respect and kind collaboration with its invaluable 10 am coffee break
together with the social activities in the group, key components of an enjoyable research.
I express my gratefulness towards my friend circle in Braunschweig.
-i-
Table of contents (brief)
Chapter 1 Extracellular electron transfer in Bioelectrochemical systems: bridge between
natural environments and applied technologies...................................................1
Part I Electron transfer mechanisms of pure culture biofilms of
Shewanella spp.
Chapter 2 Cyclic voltammetric analysis of the electron transfer of Shewanella oneidensis
MR-1 and nanofilament and cytochrome knock-out mutants...........................33
Chapter 3 Study of Shewanella putrefaciens biofilms grown at different applied potentials
using cyclic voltammetry and confocal laser scanning microscopy..................47
Chapter 4 Spectroelectrochemical analysis of intact microbial biofilms of Shewanella
putrefaciens for sustainable energy production.................................................61
Part II Porous 3D carbon as anode materials for performance of
electrochemically active mixed culture biofilms
Chapter 5 Electrospun and solution blown three-dimensional carbon fiber nonwovens for
application as electrodes in microbial fuel cells................................................71
Chapter 6 Electrospun carbon fiber mat with layered architecture for anode in microbial
fuel cells.............................................……………………………....................82
Part III The influence of external factors on electrochemically active
mixed culture biofilms
Chapter 7 Electroactive mixed culture derived biofilms in microbial bioelectrochemical
systems: the role of pH on biofilm formation, performance and
composition.......................................................................................................90
Chapter 8 Electroactive mixed culture derived biofilms in microbial bioelectrochemical
systems: the role of inoculum and substrate on biofilm formation and
performance.....................................................................................................108
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Table of contents (extended)
1 Extracellular electron transfer in Bioelectrochemical systems: bridge between
natural environments and applied technologies .......................................................................... 1
1.4.1.1 DET via membrane-bound redox-enzymes .............................................................................. 5
1.4.1.1.1 Shewanella oneidensis DET via membrane-bound redox-enzymes .................................... 5
1.4.1.1.2 Geobacter sulfurreducens DET via membrane-bound redox-enzymes ............................... 6
1.4.1.2 DET via self-produced microbial nanowires ............................................................................ 6
1.4.1.2.1 Geobacter sulfurreducens DET via self-produced microbial nanowires ............................. 7
1.4.1.2.2 Shewanella oneidensis DET via self-produced microbial nanowires .................................. 7
1.4.2.1 MET via artificial exogenous mediator molecules ................................................................... 9
1.4.2.2 MET via natural exogenous mediator molecules ..................................................................... 9
1.4.2.3 MET via self-produced mediator molecules ............................................................................. 9
1.5.1.1 Microbial fuel cells ..................................................................................................................15
1.5.1.2 Microbial electrolysis cells ......................................................................................................15
1.5.1.3 Microbial desalination cells .....................................................................................................15
1.5.1.4 Microbial solar cells ................................................................................................................16
1.5.1.5 Enzymatic fuel cells ................................................................................................................16
1.1 Prelude ................................................................................................................................................... 1
1.2 Ecological significance of insoluble metal electron acceptors: the example of iron ............................. 2
1.3 Electron transfer processes in the environment ..................................................................................... 3
1.4 Microbial extracellular electron transfer mechanisms ........................................................................... 4
1.4.1 Microbial direct extracellular electron transfer (DET) ...................................................................... 5
1.4.2 Microbial mediated extracellular electron transfer (MET) ................................................................ 8
1.5 Bioelectrochemical systems (BESs) .....................................................................................................11
1.5.1 Types of Bioelectrochemical systems ..............................................................................................13
1.6 Performance of Bioelectrochemical systems ........................................................................................16
1.6.1 Performance based on the improvement of electrode materials .......................................................18
1.6.2 Performance based on the study of environmental factors affecting biofilm formation ..................19
1.7 Aim of this Dissertation ........................................................................................................................21
1.8 Structure of the Thesis and personal contribution ................................................................................22
1.9 Comprehensive summary .....................................................................................................................26
-iii-
2 Cyclic voltammetric analysis of the electron transfer of Shewanella oneidensis MR-1
and nanofilament and cytochrome knock-out mutants ...................................................... 33
2.1.1.1 Direct electron transfer (DET) .................................................................................................34
2.1.1.2 Mediated electron transfer (MET) ...........................................................................................36
3 Study of Shewanella putrefaciens biofilms grown at different applied potentials
using cyclic voltammetry and confocal laser scanning microscopy.. ................................. 47
2.1 Introduction ..........................................................................................................................................33
2.1.1 Extracellular electron transfer mechanisms of S. oneidensis MR-1 wild type and mutants .............34
2.2 Materials and methods ..........................................................................................................................36
2.2.1 General conditions ...........................................................................................................................36
2.2.2 Cell cultures and media ....................................................................................................................36
2.2.3 Bioelectrochemical experiments ......................................................................................................37
2.2.4 Data processing ................................................................................................................................37
2.3 Results and discussion ..........................................................................................................................38
2.3.1 Bioelectrochemical current production ............................................................................................38
2.3.2 Cyclic voltammetric analysis and data processing ...........................................................................39
2.4 Conclusions ..........................................................................................................................................46
3.1 Introduction ..........................................................................................................................................47
3.1.1 Influence of the electrode potential on electroactive microbial biofilms .........................................49
3.2 Materials and methods ..........................................................................................................................50
3.2.1 General conditions ...........................................................................................................................50
3.2.2 Cell cultures and media ....................................................................................................................50
3.2.3 Bioelectrochemical set-up and experiments .....................................................................................51
3.2.4 Electrochemical data processing ......................................................................................................51
3.2.5 Confocal Laser Scanning Microscopy..............................................................................................52
3.3 Results and discussion ..........................................................................................................................52
3.3.1 Bioelectrochemical current production ............................................................................................52
3.3.2 Cyclic voltammetric analysis ...........................................................................................................54
3.3.3 Biofilm imaging using confocal laser scanning microscopy (CLSM) .............................................58
3.4 Conclusions ..........................................................................................................................................60
-iv-
4 Spectroelectrochemical analysis of intact microbial biofilms of Shewanella
putrefaciens for sustainable energy production ................................................................... 61
5 Electrospun and solution blown three-dimensional carbon fiber nonwovens for
application as electrodes in microbial fuel cells ................................................................... 71
5.2.1.1 Gas-assisted electrospinning carbon fiber mat (GES-CFM) ...................................................73
5.2.1.2 Electrospun carbon fiber mat (ES-CFM) .................................................................................74
5.2.1.3 Solution-blown carbon fiber mat (SB-CFM) ...........................................................................74
4.1 Introduction ..........................................................................................................................................61
4.2 Materials and methods ..........................................................................................................................64
4.2.1 Materials and methods .....................................................................................................................64
4.2.2 Cell cultures and media ....................................................................................................................64
4.2.3 Electrochemical set-up for the growth of anodic electrocatalytic biofilms ......................................65
4.2.4 Growth of anodic electrocatalytic biofilms ......................................................................................66
4.2.5 Cyclic voltammetry ..........................................................................................................................66
4.2.6 Electrochemical data processing ......................................................................................................66
4.2.7 Spectroelectrochemical set-up for SERRS measurements ...............................................................66
4.2.8 SERRS measurements ......................................................................................................................66
4.3 Results and discussion ..........................................................................................................................67
4.3.1 Bioelectrochemical current production ............................................................................................67
4.4 Conclusions ..........................................................................................................................................70
5.1 Introduction ..........................................................................................................................................71
5.2 Materials and methods ..........................................................................................................................73
5.2.1 Carbon fiber preparation ..................................................................................................................73
5.2.2 Electrode preparation .......................................................................................................................75
5.2.3 Bioelectrochemical experiments ......................................................................................................75
5.3 Results and discussion ..........................................................................................................................76
5.3.1 Biocatalytic current generation at modified carbon electrodes ........................................................76
5.3.2 Analysis of electroactive biofilms grown at modified carbon electrodes with Scanning electron
microscopy ....................................................................................................................................................77
5.3.3 Cyclic voltammetry of electroactive biofilms grown at modified carbon electrodes .......................79
5.4 Conclusions ..........................................................................................................................................81
-v-
6 Electrospun carbon fiber mat with layered architecture for anode in microbial fuel
cells...........................................................................................................................................82
7 Electroactive mixed culture derived biofilms in microbial bioelectrochemical
systems: the role of pH on biofilm formation, performance and composition ................. 90
7.2.8.1 Flow-cytometry .......................................................................................................................94
7.2.8.1.1 Sample fixation and DNA staining .................................................................................... 94
7.2.8.1.2 Multiparametric flow-cytometry ........................................................................................ 94
7.2.8.2 T-RFLP and Sequencing .........................................................................................................95
6.1 Introduction ..........................................................................................................................................82
6.2 Materials and methods ..........................................................................................................................83
6.2.1 Carbon fiber preparation ..................................................................................................................83
6.2.2 Electrode preparation .......................................................................................................................84
6.2.3 Bioelectrochemical measurements ...................................................................................................84
6.2.4 SEM imaging ...................................................................................................................................84
6.3 Results and discussion ..........................................................................................................................85
6.3.1 Properties and performance of carbon fiber mat electrode materials ...............................................85
6.3.2 Biocatalytic current generation at carbon fiber mat electrode materials ..........................................87
6.3.3 Analysis of electroactive biofilms grown at carbon fiber mat electrode materials with Scanning
electron microscopy ......................................................................................................................................87
6.4 Conclusions ..........................................................................................................................................89
7.1 Introduction ..........................................................................................................................................90
7.2 Materials and methods ..........................................................................................................................91
7.2.1 General conditions ...........................................................................................................................91
7.2.2 Electrochemical set-up .....................................................................................................................92
7.2.3 Microbial inoculum and growth medium .........................................................................................92
7.2.4 Biofilm growth (fed-batch experiments) ..........................................................................................92
7.2.5 Biomass determination .....................................................................................................................93
7.2.6 Metabolic analysis ............................................................................................................................93
7.2.7 Continuous flow mode operation and pH-regime studies ................................................................93
7.2.8 Microbiological analysis ..................................................................................................................94
7.3 Results and discussion ..........................................................................................................................96
7.3.1 Biofilm formation and performance at different constant pH ..........................................................96
7.3.2 Biofilm performance at varying pH-environment during operation .................................................97
7.3.3 Influence of the pH and buffer capacity on the electron transfer .....................................................99
7.3.4 Microbial biofilm analysis .............................................................................................................101
7.4 Conclusions ........................................................................................................................................107
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8 Electroactive mixed culture derived biofilms in microbial bioelectrochemical
systems: the role of inoculum and substrate on biofilm formation and performance ... 108
9 Supplementary information: Chapter II .................................................................... 120
10 Supplementary information: Chapter III ................................................................... 130
11 Supplementary information: Chapter VII ................................................................. 136
12 References ...................................................................................................................... 148
8.1 Introduction ........................................................................................................................................108
8.2 Materials and methods ........................................................................................................................111
8.2.1 General conditions .........................................................................................................................111
8.2.2 Electrochemical set-up ...................................................................................................................111
8.2.3 Microbial inoculum and growth medium .......................................................................................112
8.2.4 Biofilm growth in bioelectrochemical half-cells ............................................................................112
8.2.5 Cyclic voltammetry ........................................................................................................................113
8.2.6 Metabolic analysis for coulombic efficiency calculation ...............................................................113
8.3 Results and discussion ........................................................................................................................113
8.3.1 Current density production of enriched microbial electroactive biofilms as a function of microbial
inoculum and substrate ................................................................................................................................113
8.3.2 Bioelectrocatalytic activity of enriched microbial electroactive biofilms as a function of microbial
inoculum and substrate ................................................................................................................................115
8.4 Conclusions ........................................................................................................................................118
11.1 Influence of the buffer capacity ..........................................................................................................136
11.2 Terminal restriction fragment polymorphism (T-RFLP) analysis: Anode biofilm composition at the
different pH values determined by T-RFLP ...................................................................................................137
11.3 Terminal restriction fragment polymorphism analysis: Anode chamber community composition at pH
7 and 9 at different feeding cycles determined by T-RFLP ............................................................................140
11.4 Relationship of community composition when cultivated at different pH and under successive feeding
cycles determined by T-RFLP ........................................................................................................................140
11.5 Flow-cytometric analysis. ...................................................................................................................142
11.5.1 Community structure when cultivated at pH 9 at successive feeding cycles determined by flow
cytometry ....................................................................................................................................................142
11.5.2 Community structure when cultivated at pH 6 at successive feeding cycles determined by flow
cytometry ....................................................................................................................................................143
11.6 Relationship of community structure when cultivated at different pH and under successive feeding
cycles determined by flow cytometry .............................................................................................................144
11.7 Statistical Analysis of flow-cytometric data .......................................................................................145
11.8 Biofilm detachment ............................................................................................................................146
11.9 Multivariate statistical analysis of the flow-cytometric pattern using n-MDS-plots ..........................147
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Index of figures
Figure 1-1 Simplified iron cycle in aquatic environments. Figure drawn with modifications after (Luu and
Ramsay, 2003, Nealson and Saffarini, 1994). ........................................................................................... 3
Figure 1-2 Overall illustration of microbial ET mechanisms found in the literature. A) Direct extracellular
electron transfer via membrane bound cytochromes and conductive nanowires and B) Mediated
extracellular electron transfer via a mediator molecule (Medred or Medox) (see text). Here ET
mechanisms are represented in the field of BESs with electrode materials as final electron acceptors but
the same illustration could be applied for bacteria in natural environments using for instance iron oxides
as terminal electron acceptors. Figure drawn with modifications after (Schröder, 2007). ........................ 4
Figure 1-3 Roles of outer membrane cytochromes of A) Shewanella oneidensis and B) Geobacter sulfurreducens
in extracellular electron transfer. IM: inner membrane, OM: outer membrane and PS: periplasm. Figure
drawn with modifications after (Shi, et al., 2009). .................................................................................... 6
Figure 1-4 Scanning electron microscope micrographs of: A) Geobacter sulfurreducens (ATCC 51573)
(Malvankar, et al., 2011); B) Shewanella oneidensis MR-1 (Gorby, et al., 2006); C) Synechocystis sp.
PCC 6803 (Gorby, et al., 2006) and D) co-culture of Pelotomaculum thermopropionicum and
Methanothermobacter thermautotrophicus showing nanowires connecting the two genera (Gorby, et al.,
2006). ........................................................................................................................................................ 8
Figure 1-5 Number of publications reporting the use of “Bioelectrochemical systems” (Scopus data base, January
2012). Illustration based on (Schröder, 2011). ........................................................................................ 12
Figure 1-6 Overall view of Bioelectrochemical systems. Production of electricity and useful metabolites take
place in BESs. These microbial/ enzyme/ organelles based systems consist of an anode (oxidation
process), a cathode (reduction process) and typically a membrane separating both electrodes (see also
Table 1-2). Depending on the membrane specificity (Harnisch and Schröder, 2009), type of catalysts at
both electrodes (Franks, et al., 2010, Rosenbaum, et al., 2011), and the source of the reducing power
(Logan, et al., 2008, Logan, et al., 2006) a diverse spectrum of research and practical applications can
be found (see Section 1.5.1). Drawn with modifications after (Rabaey and Rozendal, 2010). ............... 13
Figure 1-7 Illustration of the enhancement of the anodic current density performance in BESs. Current density
values taken from representative literature data: (Aelterman, et al., 2006, Bond, et al., 2002, Catal, et al.,
2008a, Catal, et al., 2008b, Chen, et al., 2011, Gil, et al., 2003, He, et al., 2011, He, et al., 2005,
Holmes, et al., 2004b, Katuri, et al., 2010, Kim, et al., 1999b, Kim, et al., 1999d, Liu, et al., 2005, Liu,
et al., 2010c, Milliken and May, 2007, Min and Logan, 2004, Park and Zeikus, 2000, Park, et al., 2001,
Torres, et al., 2009, Zhao, et al., 2010b, Zuo, et al., 2006). Illustration based on Ref. (Schröder, 2011).
................................................................................................................................................................ 17
Figure 1-8 Schematic illustration of the research areas within the three chapter I, II and III. .............................. 22
Figure 2-1 Direct (DET) and mediated (MET) electron transfer pathways utilized by S. oneidensis wild type and
mutants. In every scheme it is indicated which strains can perform the respective electron transfer
mechanisms (Chang, et al., 2006, Nielsen, et al., 2010, Rabaey, et al., 2010). A) Electron transfer via the
cytochrome pool. Transmembrane pilus electron transfer via B) pil-type pilus and via C) msh-type
pilus, and D) biofilm formation behaviour. OM: Outer membrane and IM: Inner membrane................ 35
-viii-
Figure 2-2 A) and B) CVs for non-turnover conditions for S. oneidensis WT and mutants using a scan rate of 1
mV s−1; C and D) provide the respective baseline corrected curves. ...................................................... 39
Figure 2-3 A) and B) CVs for turnover conditions for S. oneidensis WT and mutants using a scan rate of 1 mV
s−1. ........................................................................................................................................................... 40
Figure 2-4 Plot of the base line corrected height of the oxidation peak of redox-system I (Δi−0.2) as function of
the maximum chronoamperometric current density of the respective microbial culture. ....................... 42
Figure 2-5 Plot of the corrected turnover CV signal and the performed analysis on the example of S. oneidensis
MR-1. (Similar plots of all strains can be found in Fig. S9-8 and Fig. S9-9 in the Supplementary
Information for Chapter 2). ..................................................................................................................... 43
Figure 3-1 Representative chronoamperometric fed-batch cycles of S. putrefaciens at graphite electrodes; applied
potentials: -0.1, 0, +0.1, +0.2, +0.3 and +0.4 V vs. Ag/AgCl; CV measurements during turn-over (A)
and non turn-over (B) conditions respectively. ....................................................................................... 53
Figure 3-2 Chronoamperometric current density of S. putrefaciens as function of the applied electrode potential.
................................................................................................................................................................ 53
Figure 3-3 A) Representative cyclic voltammograms of S. putrefaciens for turn-over conditions and B)
respective first derivatives of the voltammetric curves; scan rate: 1 mV s-1. .......................................... 55
Figure 3-4 A) Cyclic voltammograms for non turn-over conditions for S. putrefaciens using a scan rate of 1 mV
s−1; B provides the respective baseline corrected curves. ........................................................................ 56
Figure 3-5 Plot of the base line corrected height (○) and area (□) of the oxidation and reduction peaks of redox-
system shown in Fig. 3-4 as function of the applied potential. For visual convenience, reduction peak
areas are shown as negative values. ........................................................................................................ 57
Figure 3-6 Maximum intensity projection of confocal laser scanning microscopy data sets showing Shewanella
putrefaciens biofilms grown on electrode surfaces at different applied potentials. A) -0.1 V, B) 0 V, C)
+0.1 V, D) +0.2 V, E) +0.3 V and F) +0.4 V; (all vs. Ag/AgCl). Colour allocation: reflection of
electrode – grey, nucleic acid stained bacteria – green. .......................................................................... 58
Figure 3-7 Biofilm quantification of Shewanella putrefaciens biofilms grown on electrode surfaces at different
applied potentials. ................................................................................................................................... 59
Figure 4-1 Principle representation of a BES operating in the DET mode (see below). Electrons derived from the
oxidation of the organic substrate catalyzed by the bacterial cell are shuttled to the electrode via OMCs.
................................................................................................................................................................ 62
Figure 4-2 Electrochemical half cell set-up under potentiostatic control. Insert shows a photograph of the
nanostructured silver ring working electrode. ......................................................................................... 65
Figure 4-3 Chronoamperometric curve of a biofilm formation using a silver ring electrode poised at +0.05 V in a
batch experiment using 18 mM sodium lactate as the substrate and S. putrefaciens cells as biocatalyst.67
Figure 4-4 A) CV of the active biofilm formed on a silver ring electrode under non-turnover conditions (i.e. in
the absence of the substrate sodium lactate) at a scan rate of 1 mV s-1. B) Respective SOAS baseline
corrected curves. ..................................................................................................................................... 68
-ix-
Figure 4-5 SERR spectra of the reduced (upper spectrum) and oxidized (lower spectrum) OMCs, obtained at -
425 and 0 mV, respectively. The spectra were obtained with excitation at λ = 413 nm, laser power of 1
mW, and an acquisition time of 90 s. Potentials refer to the Ag/AgCl (KCl 3 M) reference electrode
(210 mV vs. SHE). .................................................................................................................................. 69
Figure 5-1 (A) Schematic drawing of an electrospinning setup (derived from ref. (Greiner and Wendorff, 2007)).
Solution blowing differs from electrospinning by the use of a high-speed nitrogen jet flow (230–250 m
s-1) instead of a high voltage electric field to accelerate and stretch the polymer solution into a fibrous
form (Sinha-Ray, et al., 2010). (B) Electrochemical cell for the simultaneous study of different
electrode materials. ................................................................................................................................. 73
Figure 5-2 Biocatalytic current generation at a GES-CFM modified carbon electrode in a model semi-batch
experiment. The GES-CFM electrode was modified by a wastewater-derived secondary biofilm grown
in a half-cell experiment under potentiostatic control. The electrode potential was 0.2 V. .................... 77
Figure 5-3 Scanning electron microscopic images of (A) carbon felt, (B) an electroactive biofilm grown at
carbon felt, (C) GES-CFM, (D) an electroactive biofilm grown at GES-CFM, (E) high resolution image
of GESCFM illustrating the occurrence of inter-fibre junctions, and (F) crosssectional view of GES-
CFM electrode. ....................................................................................................................................... 78
Figure 5-4 Cyclic voltammograms of an electroactive biofilm grown at GESCFM. The voltammograms were
recorded under turnover conditions [in the presence of substrate (10 mM acetate), curve A], as well as
nonturnover conditions (the absence of substrate, curve B). The biofilm was a wastewater-derived
secondary biofilm grown at a potential of 0.2 V under potentiostatic control. The scan rate was 1 mV s-
1. .............................................................................................................................................................. 80
Figure 6-1 A) Top view and B) cross-sectional view SEM images of carbon mat from TP; C) EDX spectra of
NCP-based carbon fiber; D) top view and E) cross-sectional view SEM images of layered-ECFM; F)
cross-sectional view SEM image of 2D-ECFM. ..................................................................................... 86
Figure 6-2 Biocatalytic current generation curves of carbon fiber mats in a half-cell experiment measured at
room temperature. Arrows represent replacement of medium. ............................................................... 87
Figure 6-3 SEM images of biofilms in: A-C belong to layered-CFM; D and E belong to commercial carbon felt;
and F belongs to 2D-ECFM. ................................................................................................................... 88
Figure 7-1 Performance of electroactive biofilms grown and operated at different pH-values: Maximum current
densities (filled circles; derived from chronoamperometric fed-batch experiments at 0.2 V vs. Ag/
AgCl) and coulombic efficiencies (open squares) of primary, wastewater derived biofilms are shown.
The substrate was 10 mM acetate. .......................................................................................................... 96
Figure 7-2 A) Chronoamperometric current density changes (at 0.2 V vs. Ag/ AgCl) for a biofilm initially grown
at pH 7.0 in relation to variations of the growth medium pH (numbers indicate the respective pH-value
of operation); B) Steady state current densities at 0.2 V vs. Ag/ AgCl of biofilms grown at pH 8 (circles)
and pH 7.0 (squares) at varying medium pH (derived from experiments similar as shown in A)). ........ 98
Figure 7-3 Influence of the operational pH: Cyclic voltammograms obtained at different operation pH (using a
constant ionic strength of 50 mM) at a scan rate of 1 mV s-1 during non-turnover conditions for
wastewater derived, acetate-fed biofilm formed at pH 7.0. (For pH 6 to pH 8 steady-state CVs are
shown, for pH 5 the 3rd CV-curve). ..................................................................................................... 100
-x-
Figure 7-4 Bacterial community profiles of the inoculum and the successive media of the anode chamber of a pH
7 grown biofilm (electrode-set 2). The profile of the community is cytometrically determined by the
cells’ DNA content labelled with the A-T specific fluorescent dye DAPI and the cells’ forward scatter
behaviour (FSC). As a result fingerprint-like cytometric patterns emerged as subsets of cells which
gather in numerous clusters of changing cell abundances therein. Up to 250000 cells were analysed and
the dominant sub-populations presented in yellow colour. The peak in the lower left corner of the
histograms represents the noise of the cytometer as well as unstained cell debris. ............................... 103
Figure 7-5 Dalmatian-n-MDS analysis with overlaid cytometric flow-plots derived from anode chamber
communities and anode biofilms when treated over several feeding cycles and different pH-values.
Black patches in flow-plots depict gate positions, cycle number is given with c 1–5 and pH-affiliation
with various grey/black labels (black: pH 7, grey: pH 9, light grey: pH 6, bold fringe around flow-plot:
electrodes; details see text and S11-2 to S11-10 for raw data). ............................................................. 106
Figure 8-1 A) Electrochemical half cell set-up under potentiostatic control and B) Exemplary established
bioelectrochemical active biofilm enriched from primary wastewater fed with acetate. The red color is
mainly caused by the hemes (Jensen, et al., 2010). ............................................................................... 112
Figure 8-2 Bioelectrocatalytic performance of electroactive microbial biofilms derived from different inocula
with fed batch operation in potentiostatically controlled half-cell experiments (+0.2 V vs. Ag/ AgCl) at
graphite rod electrodes. The substrate was 10 mM sodium acetate or sodium lactate respectively. ..... 114
Figure 8-3 Exemplary cyclic voltammograms. Electroactive microbial biofilms derived from different inocula
grown with Sodium acetate (10 mM) recorded during non-turnover (A, C, E and G) and turnover
conditions (B, D, F and H) conditions. The scan rate used was 1 mV s-1. ............................................ 116
Figure 8-4 Exemplary cyclic voltammograms. Electroactive microbial biofilms derived from different inocula
grown with Sodium lactate (10 mM) recorded during non-turnover (A, C, E and G) and turnover
conditions (B, D, F and H) conditions. The scan rate used was 1 mV s-1. ............................................ 117
Figure 8-5 Exemplary cyclic voltammograms from electroactive microbial biofilms derived from primary
wastewater grown with 10 mM sodium lactate (A) or 10 mM sodium acetate (B) recorded during
turnover conditions. First derivatives of biofilms grown with sodium lactate (C) or sodium acetate (D).
.............................................................................................................................................................. 118
Figure S9-1 Schematic drawing of an electrochemical cell for the study of the electron transfer mechanisms and
current production. The electrochemical cell consists of an anode, a cathode and, a membrane
separating both. An oxidation process occurs at the anode, in this case lactate oxidation, in which
electrons and protons are produced. The electrons flow to the cathode through an external circuit or
potentiostat in which the electrons can be can be quantified. Meanwhile the protons are released to the
media and lately they migrate to the cathode chamber to react with molecules of water and electrons
finally producing hydrogen for example. Figure drawn with modifications after (Rabaey and Verstraete,
2005, Schröder, 2008). .......................................................................................................................... 121
Figure S9-2 Electrochemical half cell set-up under potentiostatic control. Description: “Top view” shows the 5
necks of the 250 mL flask. In section A-A’ details of the working electrode, counter shielded electrode
and reference electrode are given. In section B-B’ the port for filtrated air, filtrated nitrogen and media
supply are detailed. ............................................................................................................................... 122
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Figure S9-3 Exemplary fed-batch chronoamperometric cycles (0.2 V vs Ag/AgCl) of Shewanella oneidensis
MR-1 Wild-type and knock-out mutants on equally-sized graphite rod anode electrodes, in half cells
utilizing lactate (18 mM) as the electron donor and anodes as electron acceptors. ............................... 123
Figure S9-4 Cyclic voltammetry at 1 mV s-1 (A, C and E) and First derivative plots of CV data (B, D and F) of S.
oneidensis Wild-type (E and F) and mutants (A and B: ΔpilM-Q/ΔmshH-Q; C and D: ΔpilM-Q) during
Turnover conditions. OxT states for oxidation turnover peak, RedT states for reduction turnover peak
and ET states for redox turnover system. Every time 4 exemplary CVs are shown. ............................. 124
Figure S9-5 Continuation of Fig. S9-4. Cyclic voltammetry at 1 mV s-1 (G, I and K) and First derivative plots of
CV data (H, J and L) of S. oneidensis mutants (G and H: ΔmshH-Q; I and J: Δflg; K and L:
ΔmtrC/ΔomcA) during Turnover conditions. OxT states for oxidation turnover peak, RedT states for
reduction turnover peak and ET states for redox turnover system. Every time 4 exemplary CVs are
shown. ................................................................................................................................................... 125
Figure S9-6 Cyclic voltammetry at 1 mV s-1 (A, C and E) and First derivative plots of CV data (B, D and F) of S.
oneidensis Wild-type (E and F) and mutants (A and B: ΔpilM-Q/ΔmshH-Q; C and D: ΔpilM-Q) during
Non-turnover conditions. OxT states for oxidation turnover peak, RedT states for reduction turnover
peak and ET states for redox turnover system. Every time 4 exemplary CVs are shown. ..................... 126
Figure S9-7 Continuation of Fig. S9-6. Cyclic voltammetry at 1 mV s-1 (G, I and K) and First derivative plots of
CV data (H, J and L) of S. oneidensis mutants (G and H: ΔmshH-Q; I and J: Δflg; K and L:
ΔmtrC/ΔomcA) during Non-turnover conditions. OxT states for oxidation turnover peak, RedT states for
reduction turnover peak and ET states for redox turnover system. Every time 4 exemplary CVs are
shown. ................................................................................................................................................... 127
Figure S9-8 Data analysis for each catalytic centre (redox-system I and II). On the left column an exemplary
turnover CV for each strain can be seen. In the center is its respective non-turnover CV. On the right
column the final subtracted CV is presented on which the signal height of each catalytic wave was
estimated at suitable fixed potentials. A-C) ΔpilM-Q/ΔmshH-Q. D-F) ΔpilM-Q. G-I) Wild-type. (see
also Fig. 2-5 in Chapter II for details) ................................................................................................... 128
Figure S9-9 Continuation of Fig. S9-8. Data analysis for each catalytic centre (redox-system I and II). On the left
column an exemplary turnover CV for each strain can be seen. In the center is its respective non-
turnover CV. On the right column the final subtracted CV is presented on which the signal height of
each catalytic wave was estimated at suitable fixed potentials. J-L) ΔmshH-Q. M-N) Δflg, P-R)
ΔmtrC/ΔomcA. (see also Fig. 2-5 in Chapter II for details) ................................................................. 129
Figure S10-1 Electrochemical cell set-up. A) Electrochemical cell hosting six potentiostatic controlled working
electrodes without S. putrefaciens cells. B) Electrochemical cell with M1 growth media inoculated with
whole cells of S. putrefaciens. Insert: photograph showing a reddish pellet of S. putrefaciens formed
when media was spinned down. ............................................................................................................ 133
Figure S10-2 Representative cyclic voltammograms for Shewanella putrefaciens biofilms grown in the presence
of (non-basal, e.g. 0.1 μM) higher levels of Riboflavin (1 μM). Respective first Derivatives of each
voltammogram are also shown, scan rate 1 mV s-1. .............................................................................. 134
-xii-
Figure S10-3 Effect of the Riboflavin concentration in the extracellular electron transfer. Representative cyclic
voltammogram of a Shewanella putrefaciens biofilm grown at a poised (+0.4 vs Ag/AgCl) graphite
electrode. The basal concentration of Riboflavin in the growth media was 0.1 μM as reported in the
Materials and Methods section (left panel). The voltammogram was recorded at maximum biofilm
activity after the start of the chronoamperometry with a scan rate of 1 mV s-1. Voltammetry of all
Shewanella biofilms grown at different applied potentials with no additional supplementation of
Riboflavin (0.1 μM) showed only one inflection point centered at 0 V (vs Ag/AgCl). After six semi
batch chronoamperometric cycles a pulse of fresh substrate containing 1 μM of Riboflavin was injected
into the electrochemical cell (right panel). For the experiment with additional Riboflavin (1 μM) not
only the inflection point at 0 V was observed but also an inflection point centered at -0.4 V
characteristic of the mediator molecule Riboflavin (Peng, et al., 2010b), indicating that this molecule
participated in the extracellular electron transfer process. Furthermore, from the pronounced sharp rise
of the inflection point centered at the midpoint potential of Riboflavin, provided an example of how this
mediator molecule increases the electron transfer (Marsili, et al., 2008a). ........................................... 135
Figure S11-1 Influence of the buffer capacity: Cyclic voltammogramms (1mV s-1) at pH 7, wastewater derived
and acetate–fed biofilms at varying buffer concentration, A) non-turn over B) turn over conditions. . 136
Figure S11-2 T-RFLP chromatograms (restriction digestion with RsaI and MspI) of the anode biofilms formed at
pH 7. The x axis represents the length of terminal restriction fragments and the y axis the relative
fluorescence units. On the right the area of every peak is shown as percentage of the total peak area. The
RsaI peak at 238 bp (503 bp with MspI) is shown in bright yellow and represents Geobacter
sulfurreducens (identified after sequencing). ........................................................................................ 137
Figure S11-3 T-RFLP chromatograms (restriction digestion with RsaI and MspI) of the anode biofilms formed at
pH 9. The x axis represents the length of terminal restriction fragments and the y axis the relative
fluorescence units. On the right the area of every peak is shown as percentage of the total peak area. The
peak at 238 bp (503 bp with MspI) is shown in bright yellow and represents Geobacter sulfurreducens
(identified after sequencing). In the sample of electrode-set 2 this organism could not be detected. This
biofilm comprised several phylotypes. ................................................................................................. 138
Figure S11-4 T-RFLP chromatograms (restriction digestion with RsaI and MspI) of the anode biofilms formed at
pH 6. The x axis represents the length of terminal restriction fragments and the y axis the relative
fluorescence units. On the right the area of every peak is shown as percentage of the total peak area. The
RsaI peak at 238 bp in the electrode-set 2 is shown in bright yellow and represents Geobacter
sulfurreducens (identified after sequencing the sample of electrode-set 2). In the small dashed window
the peak position is drawn to a larger scale to see that the peak position of the RsaI peak is different in
the sample of set 1 and set 2. The main MspI peak is found at 161 bp that is also different from what
was found for Geobacter sulfurreducens in the other samples (Figures S11-2 and S11-3 above). This
clearly shows that Geobacter sulfurreducens could not be detected in the sample of electrode-set 1. This
biofilm comprised several phylotypes. ................................................................................................. 139
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Figure S11-5 T-RFLP chromatograms (electrode-set 2, restriction digestion with RsaI) of the replenished
medium at the different feeding cycles. On the right the area of every peak is shown as percentage of
the total area. The peak at 238 bp is represented in bright yellow colour. It was only found in samples of
the feeding cycles at pH 7 and not in those at pH 9 (less than 1%). In this figure, in comparison to the
Fig. S11-2 above, a different resolution on the y axis was chosen to give a better overview of the present
diversity. Equal amounts of DNA were used for the analysis of all samples. ....................................... 140
Figure S11-6 Similarity analysis derived from anode chamber communities when treated over respective feeding
cycles at pH 7 and 9 (all electrode set 2). As can be observed, the T-RFLP derived composition of the
pH 7 and 9 communities was clearly different. Undoubtedly, the electrode biofilms were similar in T-
RFLP composition for pH 6 and 7 whereas the biofilm composition on the electrode treated at pH 9 was
different (Analysis: non-metric MDS, similarity measure: Bray-Curtis). ............................................. 141
Figure S11-7 Analysis of community structure by measuring the cells’ DNA contents and Forward scatter
behavior. Samples were harvested from the pH 9 anode chamber (electrode-set 2). ............................ 142
Figure S11-8 Analysis of community structure by measuring the cells’ DNA contents and Forward scatter
behavior. Samples were harvested from the pH 6 anode chamber (electrode set 2). ............................ 143
Figure S11-9 Cluster dendrogram derived from anode chamber communities when treated over several feeding
cycles and at different pH. Feeding cycle numbers and pH affiliation are given with c 1-5 and pH 6 to
pH 9 (shown for electrode-set 2). As can be observed, the structure of the inoculum community and that
of the pH 9 electrode are clearly different from all other samples. It is also obvious that distinct feeding
cycles cluster together such as pH 7 c1 to c3, pH 6 c2 to c4 and, pH 9 c2 to c4. It can be stated that
similar micro-environments like successive feeding cycles at a distinct pH value generated related
community structures. A few of the pH related communities clustered apart like pH 7 c4 to c5 or pH 6
c1 but are nevertheless close to each other if the similarity analysis of Figure S11-9 is included.
Undoubtedly, the electrode biofilms were similar in structure for pH 6 and pH 7. .............................. 144
Figure S11-10 Illustration of methodology used for estimating community similarities of cytometric flow plots
using a Dalmatian-plot. Areas of gates were estimated as sum of pixels for presence-absence when cell
abundances taken into account. Sums were calculated from plots of each of the samples separately and
for the overlap of two samples, respectively. For similarity estimation a modified Jaccard index was
used (Figure S11-10 taken from (Müller, et al., 2011). ......................................................................... 146
Figure S11-11 Photograph of the detachment of a pH 7 grown biofilm from an electrode due to extreme pH-
conditions (pH 11). ............................................................................................................................... 146
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Index of tables
Table 1-1 Representative microbially produced redox mediators. ........................................................................ 10
Table 1-2 Common terminology for the BES technology..................................................................................... 14
Table 2-1 Summary of the studied mutants and the achieved maximum current densities per projected electrode
surface area, the literature data are the reported maximum current densities in MFC experiments at
constant resistances. ................................................................................................................................ 38
Table 2-2 Result of the CV subtraction analysis (details in Fig. 5 and the text). .................................................. 45
Table 5-1 Cumulative data on electrocatalytic current densities obtained at different electrode materials. The
substrate was 10 mM Sodium acetate. .................................................................................................... 76
Table 6-1 Properties and anodic performance of carbon fiber mats. ..................................................................... 85
Table S9-1 Comparison of geometric current densities for Shewanella oneidensis Wild-type in different studies.
.............................................................................................................................................................. 120
Table S10-1 Comparison of geometric current densities for different strains of Shewanellaceae. ..................... 130
Table S10-2 Shewanella strains used as comparison in Table S10-1 and a description of their isolation
environment. ......................................................................................................................................... 132
Table S10-3 Cathodic and anodic peak positions, formal potential (vs. Ag/AgCl) and width of potential window,
ΔE, at a scan rate of 1 mV s-1 after SOAS baseline correction. ............................................................ 132
- 1 -
CHAPTER I
1 Extracellular electron transfer in Bioelectrochemical
systems: bridge between natural environments and
applied technologies
1.1 Prelude
In this introductory chapter a comprehensive description of microbial electron transfer
mechanisms in anoxic natural environments and the application of this natural process into a
promising, multi interdisciplinary -and still in continuing development technology- is given.
Section 1.2 illustrates the ecological significance of insoluble metal electron acceptors in nature.
Iron is taken as a model example to explain its bio-mobility in the environment. Here the
participation of some exemplary microorganisms capable of reducing iron is described. Section
1.3 provides a general definition of microbial extracellular electron transfer (ET) and describes
how microbiologists discovered this process in two model microorganisms now commonly used
as exemplary dissimilatory metal reducing bacteria. Later, one of the first applications for ET in
the field of bio-remediation and more recently in the field of Bioelectrochemical systems (BESs)
is provided. BESs not only have allowed the study of microbial ET but also permitted the
development of promising applications. Section 1.4 presents two known ET mechanisms
performed by bacteria, i.e., direct and mediated extracellular electron transfer (DET and MET
respectively). For DET, detailed descriptions on representative dissimilatory metal reducing
bacteria are given. In the case of MET, mediating redox species that transfer electrons between
the bacteria and the final electron acceptor are presented. Section 1.5 gives an overall
introduction to BESs. First, BESs represent an additional approach for the study of microbial ET
and second, they have emerged as an applied technology based on microbial ET. Finally Section
1.6 provides a comprehensive view on one of the main motivations in the development of BESs:
the improvement of current density production focused for near future applications. Different
aspects are exemplified with the case of 3D new electrode materials that improve the overall
performance of BESs. Finally, several environmental factors affecting the formation and
performance of electroactive biofilms are discussed.
-2-
1.2 Ecological significance of insoluble metal electron acceptors: the example of iron
Until the late 70s, reduction of Fe(III) to Fe(II) in sedimentary and subsurface environments was
believed to be the result of purely abiotic processes (Cornell and Schwertmann, 2007, Fenchel
and Blackburn, 1979). Now it is known that bacterial utilization of Fe(III) oxides as the terminal
electron acceptor is an important practice in anaerobic environments in which the reduction of
Fe(III) to Fe(II) is a enzymatically catalyzed bacterial process (Gralnick and Newman, 2007,
Lovley, 1993). Bacterial reduction of Fe(III) oxides has diverse significant ecological
repercussions, for example the quality of water can be modified by the increment of dissolved
Fe(II) that changes the taste of drinking water (Lovley, 2000) and furthermore Fe(III) is thought
to be the most abundant of all the available terminal electron acceptors in several subsurface
environments (Lovley, 1991). Some known representative microorganisms capable of utilizing
iron as final electron acceptor include: Geobacter metallireducens (Lovley, 1993),
Desulfuromonas acetoxidans (Roden and Lovley, 1993), Pelobacter carbinolicus (Lovley, et al.,
1995), members of the genus Desulfuromusa (Fredrickson and Gorby, 1996), Shewanella
oneidensis (Myers and Nealson, 1988), Ferrimonas balearica (Lovley, 2000), Geovibrio
ferrireducens (Caccavo Jr, et al., 1996) and Geothrix fermentans (Coates, et al., 1999).
The reduction of Fe(III) is considered as a predominant process due to the iron cycle reactions
(Lovley, et al., 1993), some of them with an important participation of bacteria (see below).
According to Luu and Ramsay (Luu and Ramsay, 2003), first solid oxides settle into the oxygen
transition zone called suboxic zone (Fig. 1-1). Simultaneously phosphate and metals are
removed via precipitation and complexation. In the suboxic zone carbon oxidation takes place
by bacteria via the utilization of iron as terminal electron acceptor. During iron reduction,
organic phosphate and metals are released into the oxic zone. From the oxidation of carbon,
Fe(II) forms insoluble precipitates in the suboxic zone such as siderite (FeCO3), pyrite (FeS2),
vivianite [Fe3(PO4)2] and magnetite (Fe3O4). Additionally some species of Fe(II) diffuse into the
oxic zone where finally reoxidation of Fe(II) occurs to form insoluble oxides and if no input of
organic carbon takes place, oxides accumulate in sediments of the suboxic zone, otherwise the
cycle continues again. Since the distribution of Fe(III) in the environment depends on the
amount of organic matter present (Pan, et al., 2011), Fe(III) oxides get retained in the sediment
when no organic matter is available diminishing the cycling of iron. Therefore the mobility of
certain compounds in the environment mainly depends on the biotransformation of organic
matter by microorganisms, making the study of these processes of great importance.
-3-
Figure 1-1 Simplified iron cycle in aquatic environments. Figure drawn with modifications after
(Luu and Ramsay, 2003, Nealson and Saffarini, 1994).
1.3 Electron transfer processes in the environment
Extracellular electron transfer (ET) is a general mechanism by which microorganisms generate
energy for cell growth and maintenance (Hernandez and Newman, 2001), i.e., bacteria transfer
electrons from their internal metabolism through a chain of trans-membrane proteins to finally
reduce insoluble metal electron acceptors. In the early 90s, environmental microbiologists
realized the importance of microbial ET to insoluble metal electron acceptors in several
biogeochemical cycles and progressively applied this extraordinary finding, e.g., on the
bioremediation of contaminated sites (Lovley, 1991, Nealson, et al., 1991). More recently this
finding has been used in an interdisciplinary way not only to study the fundamentals of
microbial ET but also to apply this concept in the so-called Bioelectrochemical systems (BESs)
(Rabaey, et al., 2010) (section 1.5). The basic and applied interest on microbial ET has rapidly
increased since the publication of two breakthrough papers introducing two of the first known
bacteria capable of reducing insoluble metal electron acceptors: Shewanella oneidensis MR-1
(Myers and Nealson, 1988) and Geobacter sulfurreducens PCA (Caccavo, et al., 1994).
-4-
Furthermore, the exploration of how microbes breathe minerals has been later stimulated by the
publication of both genomes (Heidelberg, et al., 2002, Methé, et al., 2003), making possible
genetic manipulations to study their respective ET pathways (see Chapter 2 for an example on
Shewanella oneidensis MR-1 knock-out mutants).
1.4 Microbial extracellular electron transfer mechanisms
To date mainly two microbial ET mechanisms have been recognized in the literature (Gralnick
and Newman, 2007, Hernandez and Newman, 2001, Lovley, 2011, Schröder, 2007, Watanabe,
et al., 2009). In one of those mechanisms named as direct extracellular electron transfer (DET),
electrons are transferred from the respiratory chain in the cell to an extracellular insoluble
compound or final electron acceptor (e.g., iron oxides or conductive electrode materials in
BESs) via a complex architecture involving several outer membrane cytochromes (Millo, et al.,
2011) (Fig 1-2A), an ability often conventionally awarded only to gram-negative bacteria
(Hernandez and Newman, 2001, Lovley, 2008a, Rosenbaum, et al., 2011, Shi, et al., 2009) with
some recent exceptions of gram-positive bacteria (Cournet, et al., 2010, Marshall and May,
2009, Wrighton, et al., 2011).
Figure 1-2 Overall illustration of microbial ET mechanisms found in the literature. A) Direct
extracellular electron transfer via membrane bound cytochromes and conductive nanowires and
B) Mediated extracellular electron transfer via a mediator molecule (Medred or Medox) (see text).
Here ET mechanisms are represented in the field of BESs with electrode materials as final
electron acceptors but the same illustration could be applied for bacteria in natural environments
using for instance iron oxides as terminal electron acceptors. Figure drawn with modifications
after (Schröder, 2007).
-5-
Another well-considered DET mechanism which is still under investigation is the ET via
cellular appendages facing the extracellular environment (i.e., microbial nanowires) found in
several bacteria (Bretschger, et al., 2010b) (Fig 1-2A) (see section 1.4.1). On the other side,
microorganisms are capable of ET via mediator molecules that, i) get reduced by outer
membrane cytochromes and later oxidized onto extracellular insoluble compounds or onto
conductive electrode materials as in the case of BESs; or ii) via periplasmatic or cytoplasmatic
redox couples that serve as reversible terminal electron acceptors, transferring electrons from the
bacterial cell to a final electron acceptor (Schröder, 2007). This mechanism is usually named as
mediated extracellular electron transfer (MET) (Marsili and Zhang, 2010) (Fig 1-2B) (see
section 1.4.2).
1.4.1 Microbial direct extracellular electron transfer (DET)
1.4.1.1 DET via membrane-bound redox-enzymes
As pointed out in section 1.2, diverse groups of microorganisms are now known to engage in
electron transfer to extracellular insoluble compounds. More recently with the use of conductive
electrode materials (anodes) in BESs, an additional number of microorganisms have joined to
the list of -recently named- exoelectrogenic bacteria capable of performing DET (Logan, 2009);
e.g., Desulfuromonas acetoxidans (Bond, et al., 2002), Escherichia coli K12 (Schröder, et al.,
2003), Rhodoferax ferrireducens (Chaudhuri and Lovley, 2003), Aeromonas hydrophila (Pham,
et al., 2003), Desulfobulbus propionicus (Holmes, et al., 2004a), Hansenula anomala (Prasad, et
al., 2007), Rhodopseudomonas palustris DX-1 (Xing, et al., 2008), Klebsiella pneumoniae L17
(Zhang, et al., 2008) and Proteus vulgaris (Rawson, et al., 2011) among others. While it is
commonly accepted that microbial ET occurs within complex communities found in BES
anodes (Logan and Regan, 2006a), the in-depth study of microbial ET mechanisms has revolved
around two model exoelectrogenic bacteria families: Shewanellaceae and Geobacteraceae
(Bretschger, et al., 2010b).
1.4.1.1.1 Shewanella oneidensis DET via membrane-bound redox-enzymes
As recently reported by Shi and co-workers (Shi, et al., 2009), DET performed by Shewanella
oneidensis depends on inner (IM) and outer membrane (OM) proteins that are known to be
directly involved in the reduction of insoluble metals that act as extracellular electron acceptors
(or in the case of BESs: electrode materials). These proteins include the inner membrane
tetrahaem c-Cyt CymA that is a homologue of NapC/NirT family of quinol dehydrogenases, the
-6-
periplasmic decahaem c-Cyt MtrA, the outer membrane protein MtrB and the OM decahaem c-
Cyts MtrC and OmcA (Fig. 1-3A).
All these proteins together form a pathway to transfer electrons from the quinone/quinol pool in
the inner membrane to the periplasm (PS) and then to the outer membrane where MtrC and
OmcA can transfer electrons directly to the surface of electrode materials.
1.4.1.1.2 Geobacter sulfurreducens DET via membrane-bound redox-enzymes
On the other side, DET performed by Geobacter sulfurreducens (as reported by Shi and co-
workers (Shi, et al., 2009)) relies on the outer membrane proteins tetrahaem c-Cyt OmcE and
hexahaem c-Cyt OmcS that are believed to be located on the cell surface where they are
suggested to transfer electrons to type IV pili. Type IV pili are hypothesized to transfer electrons
directly to Fe(III) oxides (or in the case of BESs: electrode materials). OmcE and OmcS also
receive the electrons from the quinone/quinol pool in the inner membrane (Fig. 1-3B).
Figure 1-3 Roles of outer membrane cytochromes of A) Shewanella oneidensis and B)
Geobacter sulfurreducens in extracellular electron transfer. IM: inner membrane, OM: outer
membrane and PS: periplasm. Figure drawn with modifications after (Shi, et al., 2009).
1.4.1.2 DET via self-produced microbial nanowires
The fundamental comprehension of microbial ET mechanisms is still in progress (Bretschger, et
al., 2010b) since non-conclusive and debatable experimental evidence of an additional DET
process via self-produced microbial nanowires has come to light (Lovley, 2011). This recently
found DET mechanism is not only expected to change the way scientists will look at microbial-
-7-
electrode interactions but also it could commence a new whole applied research field due to the
promising application of microbial nanowires as nano bio-conductive materials (Malvankar, et
al., 2011). In general, the information devoted to the analysis of conductive bacterial nanowires
is scarce. However experimental evidence of microbial-like nanowires has been reported for
some microorganisms as described below. There exists evidence showing the presence of
microbial-like nanowires in nutrient limited cultures of the cyanobacterium Synechocystis sp.
PCC 6803 (Fig. 1-4C) and in co-cultures of Pelotomaculum thermopropionicum and
Methanothermobacter thermautotrophicus (Fig. 1-4D) (Gorby, et al., 2006). Additionally,
putative nanowires have been observed in sulfate limiting cultures of Desulfovibrio vulgaris and
in environmental samples from hydrothermal vents. Nevertheless, only visual information in
this regard has been presented so far (Bretschger, et al., 2010b). Whereas microbial-like
nanowires structures have been observed in several bacterial cultures (Bretschger, et al., 2010b),
hitherto; to the best of my knowledge and beyond the optical description, only four works
devoted to the electrochemical and spectroscopical characterization of these structures have
been published (according to “Scopus”, February 2012) and all of them using either the model
exoelectrogenic bacterium G. sulfurreducens or S. oneidensis.
1.4.1.2.1 Geobacter sulfurreducens DET via self-produced microbial nanowires
One of the first observations on microbial nanowires was made by Reguera and co-workers
(Reguera, et al., 2005) on G. sulfurreducens. They have found that a nanowire-deficient mutant
of G. sulfurreducens could not reduce Fe(III). Additionally by using atomic force microscopy
they suggested that these G. sulfurreducens nanowires could be conductive. A few years later,
additional information on the possible conductivity of G. sulfurreducens nanowires was
provided by Malvankar and co-workers (Malvankar, et al., 2011). They have showed the
metallic-like conductivity (along centimeter-length scale) in microbial nanowires produced by
G. sulfurreducens. Moreover, they have even suggested that these structures could possess
similar properties to those of synthetic metallic nanostructures (Fig. 1-4A).
1.4.1.2.2 Shewanella oneidensis DET via self-produced microbial nanowires
On the other hand, only one year later to the first finding of nanowires in G. sulfurreducens,
Gorby and co-workers provided evidence on the conductivity of electrical microbial nanowires
produced by S. oneidensis in direct response to electron-acceptor limitations (Gorby, et al.,
2006). Four years later El-Naggar and co-workers (El-Naggar, et al., 2010) presented an
additional contribution in this regard confirming the conductivity of such microbial nanowires
produced by S. oneidensis MR-1 (Fig. 1-4B). Independent of the source of microbial nanowires,
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the experiments reported so far present the bacterial nanowires as a viable microbial strategy for
DET and more importantly represent a promising alternative for future nano bio-conductive
materials. Ultimately, although DET (via membrane-bound redox-enzymes or via microbial
nanowires) seems to be an imperative microbial ET mechanism in some species of
microorganisms, mediated electron transfer (MET, explained in the following section) via
mediator molecules has been proved as well to have an outstanding participation in the overall
ET process (see Chapter 2).
Figure 1-4 Scanning electron microscope micrographs of: A) Geobacter sulfurreducens (ATCC
51573) (Malvankar, et al., 2011); B) Shewanella oneidensis MR-1 (Gorby, et al., 2006); C)
Synechocystis sp. PCC 6803 (Gorby, et al., 2006) and D) co-culture of Pelotomaculum
thermopropionicum and Methanothermobacter thermautotrophicus showing nanowires
connecting the two genera (Gorby, et al., 2006).
1.4.2 Microbial mediated extracellular electron transfer (MET)
Microbial mediated extracellular electron transfer (MET) requires transfer of electrons from the
respiratory chain in the cell to extracellular inorganic material via a redox mediator molecule.
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The known microbial MET occur via i) artificial exogenous mediator molecules; ii) natural
exogenous mediator molecules; and iii) self-produced mediator molecules.
1.4.2.1 MET via artificial exogenous mediator molecules
In early experiments with BESs, the need of exogenous mediator molecules was believed to be
crucial for bacteria to transfer electrons to electrodes immersed in bacterial solutions (Cohen,
1931). The approach of using these molecules was applied again in the 1980s mainly by
Bennetto and co-workers (Bennetto, et al., 1983). The majority of mediator molecules were
based on phenazines (Park and Zeikus, 2000), phenothiazines (Delaney, et al., 1984),
phenoxazines (Bennetto, et al., 1983) and quinones (Tanaka, et al., 1988) demonstrating their
suitability as redox mediators between certain bacteria and electrode materials. More recently,
additional compounds have been reported as well, e.g.: resazurin (Sund, et al., 2007), humate
analog anthraquinone 2-6-disulfonate (Milliken and May, 2007) and methyl viologen (Aulenta,
et al., 2007). Although exogenous mediator molecules are easy to dose and their redox potential
may be adjusted over a wide range by careful design of the molecule (Marsili and Zhang, 2010),
their main disadvantage is the necessity of a regular addition of these compounds, which from a
practical point of view is technologically unfeasible and environmentally questionable
(Schröder, 2007).
1.4.2.2 MET via natural exogenous mediator molecules
In MET, microbes can use natural exogenous (non self-produced) electron shuttling compounds
available in the subsurface environment such as humic acids (Fredrickson, et al., 2000a,
Fredrickson, et al., 2000b, Lovley, et al., 1996, Straub, et al., 2005), cysteine (Doong and
Schink, 2002, Kaden, et al., 2002) or sulfur-containing compounds (Straub and Schink, 2003).
The importance of such natural exogenous mediator molecules lies in the fact that this kind of
molecules have found to be responsible for MET in natural sediments (Nielsen, et al., 2010).
1.4.2.3 MET via self-produced mediator molecules
Finally and more importantly (from the ecological and applied point of view), it is assumed that
microorganisms due to environmental restriction use endogenous redox mediators (self-
produced by bacteria) to accomplish the production of energy for cell growth and maintenance
by the reduction of insoluble terminal electron acceptors. Initial experiments to produce and
characterize mediator molecules were done through insoluble metal reduction assays (Caccavo,
et al., 1994, Myers and Nealson, 1988). Only relatively recently, the use of BESs (see Section
-10-
1.5) has stimulated the general interest on externally microbial ET (Bond, et al., 2002, Kim, et
al., 1999a).
To date, mainly experiments with gram-negative bacteria have contributed with evidence that
microorganisms are able to perform MET mechanisms (Marsili and Zhang, 2010). Microbial
known mediators are listed in Table 1-1. In general, these molecules have provided experimental
evidence on the possibility to transfer electrons to electrode materials and according to
assumptions made by Marsili and Zhang (Marsili and Zhang, 2010), redox mediator molecules
would be able to transfer electrons between bacteria and final electron acceptors regardless of a
solid metal oxide or an electrode material. Such an ability in conjunction with the fact that self-
produced mediator molecules from one bacteria can be used further by a different bacteria (as in
the case of Pseudomonas sp. and Brevibacillus sp. PTH1 (Pham, et al., 2008)) increases the
applications of this specific MET mechanism.
Table 1-1 Representative microbially produced redox mediators.
Microoganism
Mediator molecule
Reference
Sphingomonas xenophaga
4-amino-1,2-naphthoquinone
(Keck, et al., 2002)
Pseudomonas aeruginosa
Phenazine-1-carboxylic acid
(Price-Whelan, et al., 2006)
Pseudomonas chlororaphis
Phenazine-1-carboxamide
(van Rij, et al., 2004)
Shewanella oneidensis
Flavin mononucleotide
(von Canstein, et al., 2008)
Shewanella algae
Melanin
(Turick, et al., 2002)
Bacillus pyocyaneus
Pyocyanine
(Friedheim and Michaelis,
1931)
Propionibacterium
freundenreichii
2-Amino-3-carboxy-1,4-
naphthoquinone
(Hernandez and Newman,
2001)
Shewanella alga
Cyanocobalamin
(Workman, et al., 1997)
Acetobacterium woodii
Hydroxycobalamin
(Hashsham and Freedman,
1999)
Pseudomonas stutzeri
Pyridine-2,6-bis
(Lewis, et al., 2001)
Methanosarcina thermophila
Porphorinogen-type molecules
(Koons, et al., 2001)
Geobacter metallireducens
Anthraquinone-2,6-disulfonate
(Cervantes, et al., 2004)
Shewanella oneidensis
1,4-Dihydroxy-2-naphthoate
derivative
(Ward, et al., 2004)
Klebsiella pneumoniae
Anthraquinone-2,6-disulfonate
(Li, et al., 2009b)
aMore detailed information can be found in the following references: (Hernandez and Newman,
2001, Marsili and Zhang, 2010, Schröder, 2007, Watanabe, et al., 2009).
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1.5 Bioelectrochemical systems (BESs)
From Section 1.1 there has been a constant reference on BESs since these systems have
represented a driving force in the elucidation of microbial electron transfer mechanisms.
Although it could be assumed that microbial BESs represent a novel research field, this is not
completely true. The technology in fact is quite old and just recently has been revisited
(Schröder, 2011). The ability of microorganisms to transfer electrons from the internal
metabolic chains to extracellular terminal acceptors (with the concomitant production of an
electric current) was discovered more than 100 years ago (Schröder, 2011). However, this
finding has attracted increasing attention only during the last decade (Hernandez and Newman,
2001, Schroder, 2007, Watanabe, et al., 2009). Michael C. Potter reported in the year 1911 the
electromotoric force between electrodes immersed in bacterial cultures in a battery (Potter,
1911). In Potter’s communication, he concluded that electric energy could be generated from the
microbial decomposition of organic compounds. With this unusual (at that time) combination of
microbiology and electrochemistry, Potter was a pioneer providing one clearer hint on the
consequences of the bacterial metabolism. As reviewed in previous sections, microbial ET has
received great attention not only for the basic knowledge of how electrons end at an electron
acceptor from the geochemistry point of view but also for the possible use of this extraordinary
process in bioremediation, in the production of bioenergy and/ or more recently in the
production of valuable products by the so called BESs (Rabaey, et al., 2009, Rabaey and
Rozendal, 2010). Additionally, this interest has been clearly reflected by the number of
publications including the use of BESs (Fig. 1-5).
In BESs, a plenitude of possible applications can be found (Fig. 1-6), from the original and
promising production of electricity (Logan, et al., 2006), to hydrogen as a clean fuel (Logan, et
al., 2008) and the production of useful chemicals (Rabaey and Rozendal, 2010) such as
hydrogen peroxide, extraordinarily from wastewater (Fu, et al., 2010, You , et al., 2010).
Nonetheless, the cited applications in this section would not be possible without the basic
research on the microbe-electrode interactions which inexorably turn out to contribute to the
betterment of the overall performance of this kind of systems by eliminating (or at least
diminishing) electrochemical losses of BESs (Schröder and Harnisch, 2010). Therefore, the
analysis of the microbe-electrode interactions would lead not only to a higher comprehension on
improving the overall performance of BESs (see section 1.5) from the power production point of
view but also on improving a more precise electron uptake by microorganisms for the
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production of useful and industrial demanded biochemicals (Nevin, et al., 2010, Ross, et al.,
2011).
Figure 1-5 Number of publications reporting the use of “Bioelectrochemical systems” (Scopus
data base, January 2012). Illustration based on (Schröder, 2011).
As shown in Fig. 1-6, microbial-electrode interactions can take place in both electrode chambers
depending on the application for which the BES has been designed. A simplified version of a
BES system as shown in the insert of Fig. 1-6 is a potentiostatic controlled electrochemical half-
cell in which an anode and a cathode are hosted within one vessel (LaBelle, et al., 2010). This
experimental approach assures similar biological and environmental conditions for both
electrodes and increases the reproducibility of the experiment by maintaining one of the
electrodes at a constant potential permanently controlled against a reference electrode (e.g., vs.
Ag/AgCl) (Bard, et al., 2008). This type of BES (with multiple modifications) is the one that has
been extensively used in this Thesis.
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Figure 1-6 Overall view of Bioelectrochemical systems. Production of electricity and useful
metabolites take place in BESs. These microbial/ enzyme/ organelles based systems consist of
an anode (oxidation process), a cathode (reduction process) and typically a membrane separating
both electrodes (see also Table 1-2). Depending on the membrane specificity (Harnisch and
Schröder, 2009), type of catalysts at both electrodes (Franks, et al., 2010, Rosenbaum, et al.,
2011), and the source of the reducing power (Logan, et al., 2008, Logan, et al., 2006) a diverse
spectrum of research and practical applications can be found (see Section 1.5.1). Drawn with
modifications after (Rabaey and Rozendal, 2010).
1.5.1 Types of Bioelectrochemical systems
Depending on the application, the BES receives a different name s as seen in Table 1-2. From
the different BESs that can be found in the literature, only a few of them have attracted most of
the scientific community’s attention, e.g.: microbial fuel cells (MFCs), microbial electrolysis
cells (MECs), microbial desalination cells (MDCs), microbial solar cells (MSC) and enzymatic
fuel cells (EFCs).
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Table 1-2 Common terminology for the BES technology.
Name
Abbrev.
Definition
Ref.*
Bioelectrochemical
system
BES
An electrochemical system in which biocatalysts
(microorganisms) perform oxidation and/ or reduction at
electrodes
[1]
Microbial fuel cell
MFC
A BES that produces net electrical power
[2]
Microbial electrolysis
cell
MEC
A BES to which net electrical power is provided to achieve
a certain process or product formation
[3]
Bioelectrochemically
assisted microbial
reactor
BEAMR
A BES to which net electrical power is provided to achieve
a certain process or product formation
[4]
Bio-electrical reactor
BER
A reactor in which current is provided to microorganisms
to stimulate their metabolism
[5]
Biocatalyzed
electrolysis cell
BEC
A BES to which net electrical power is provided to achieve
a certain process or product formation
[6]
Biochemical fuel cell
BFC
An electrochemical system in which biocatalysts function
as catalysts for oxidation and/ or reduction reaction at
electrodes
[7]
Biofuel cell
BFC
An electrochemical system that use biocatalysts to convert
chemical energy to electrical energy
[8]
Sediment microbial
fuel cell
SMFC
MFC operated at underwater sediment interface
[9]
Benthic unattended
generator
BUG
MFC operated at underwater sediment interface
[10]
Enzymatic fuel cell
EFC
An electrochemical system in which biocatalysts
(enzymes) perform oxidation and/ or reduction at
electrodes
[11]
Microbial desalination
cell
MDC
An MFC for desalinating water based on using the
electrical current generated by exoelectrogenic bacteria
[12]
Microbial solar cell
MSC
An MFC that exploits the energy of light and the activity
of phototrophic microorganisms to produce electricity
[13]
Mitochondrial biofuel
cell
MBFC
A new class of BES that uses whole organelles (e.g.,
mitochondria) as catalysts
[14]
Note: Table based on information available in (Rabaey, et al., 2010). *References in Table: 1: (Rabaey,
et al., 2007); 2: (Logan, et al., 2006); 3: (Logan, et al., 2008); 4: (Ditzig, et al., 2007); 5: (Thrash and
Coates, 2008); 6: (Rozendal, et al., 2006b); 7: (Lewis, 1966); 8: (Cooney, et al., 2008); 9: (Reimers, et
al., 2000); 10: (Lovley, 2006); 11: (Minteer, et al., 2007); 12: (Kim and Logan, 2011); 13: (Rosenbaum
and Schröder, 2010); 14: (Bhatnagar, et al., 2011).
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1.5.1.1 Microbial fuel cells
As a general definition, microbial fuel cells (MFCs) are devices that use bacteria as the catalysts
to oxidize organic and inorganic matter and generate current (Logan, et al., 2006). According to
Logan and co-workers (Logan, et al., 2006), in a MFC bacteria oxidize organic matter and
release carbon dioxide and protons into solution and electrons to an anode. Electrons are then
transferred by DET or MET to the anode (or working electrode) and flow to the cathode (or
counter electrode) linked by a conductive material containing a resistor, or operated under a load
(see Fig. 1-6). Finally, the electrons that are transferred from the anode to the cathode combine
with protons (that diffuse from the anode chamber through a physical separator) and oxygen
provided from air to produce water.
1.5.1.2 Microbial electrolysis cells
Unlike MFCs, Microbial electrolysis cells (MECs) use electrochemically active bacteria to
break down organic matter, combined with the addition of a small voltage that results in
production of hydrogen gas (Logan, et al., 2008). MECs used to produce hydrogen gas are
similar in design to MFCs that produce power, but there are important differences. According to
Logan and co-workers (Logan, et al., 2008) in a MFC, when oxygen is present at the cathode,
current can be produced, but without oxygen, current generation is not spontaneous. However, if
a small voltage is applied, current generation is forced between both electrodes and hydrogen
gas is produced at the cathode through the reduction of protons.
1.5.1.3 Microbial desalination cells
Microbial desalination cells (MDCs) are based on transfer of ionic species out of water in
proportion to current generated by bacteria (Luo, et al., 2012). Developed by Cao and co-
workers (Cao, et al., 2009), MDCs consist of three chambers, with an anion exchange membrane
next to the anode and a cation exchange membrane by the cathode, and a middle chamber
between the membranes filled with water that is being desalinated. When current is generated by
bacteria on the anode, and protons are released into solution, positively charged species are
prevented from leaving the anode by the anion exchange membrane and therefore negatively
charged species move from the middle chamber to the anode. In the cathode chamber protons
are consumed, resulting in positively charged species moving from the middle chamber to the
cathode chamber. This loss of ionic species from the middle chamber results in water
desalination.
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1.5.1.4 Microbial solar cells
When sunlight is converted into electricity within the metabolic reaction scheme of a MFC, this
system is described as photosynthetic MFC or microbial solar cell (MSC) (Rosenbaum, et al.,
2010b). MSCs are used to convert light into electricity by exploiting the photosynthetic activity
of living, phototrophic microorganisms (Rosenbaum and Schröder, 2010). These BESs have
been described in detail by Rosenbaum and co-workers (Rosenbaum, et al., 2010b). In their
publication they indentify five different approaches that integrate photosynthesis with MFCs: a)
photosynthetic bacteria at the anode with artificial mediating redox species, b) hydrogen-
generating photosynthetic bacteria with an electrocatalytic anode, c) photosynthesis coupled
with mixed heterotrophic bacteria at the anode, d) direct electron transfer between
photosynthetic bacteria and electrodes and e) photosynthesis at the cathode to provide oxygen.
1.5.1.5 Enzymatic fuel cells
Enzymatic fuel cells (EFCs) are energy conversion devices that use enzymes as biocatalysts to
convert chemical energy to electrical energy (Cooney, et al., 2008). According to Cooney and
co-workers (Cooney, et al., 2008), BESs are usually classified on the basis of the type of
biocatalyst employed. There are three types of biocatalyst used in BESs: microbes, organelles,
and enzymes, each of this type has advantages and disadvantages. While MFCs can operate for
years (Logan, 2010) and completely oxidize their fuel, MFCs have been limited by low current
and power densities. On the other hand, EFCs have been shown to have higher current and
power densities, but have been limited by incomplete oxidation of fuel and lower active lifetime
(Minteer, et al., 2007).
1.6 Performance of Bioelectrochemical systems
As one can see from the literature (Schröder, 2011), one of the motivations for the development
of the BES technology has been a competitive “race” to increase the current production and
trying to make this technology an affordable option for the treatment of wastewater with the
concomitant consequence production of sustainable electricity and biochemicals (Rabaey and
Rozendal, 2010).
Here, the understanding of microbial-electrode interactions has been part of the global effort to
accomplish BESs with an enhanced performance. Current density based on available anode
surface area has made a noticeable development (Fig 1-7). Since 1999, the experimental
biotransformation of substrate (fuel) to electric energy (Schröder, 2007) has been performed
with the utilization of dissimilatory metal reducing bacteria (e.g., from the Shewanellaceae
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family (Kim, et al., 1999b, Kim, et al., 1999d)). The performance of the current density
production has seen a considerable increment from only 0.013 μA cm-2 (Kim, et al., 1999d) to
more than 30 A m-2 (see Chapter 5 and 6).
Figure 1-7 Illustration of the enhancement of the anodic current density performance in BESs.
Current density values taken from representative literature data: (Aelterman, et al., 2006, Bond,
et al., 2002, Catal, et al., 2008a, Catal, et al., 2008b, Chen, et al., 2011, Gil, et al., 2003, He, et
al., 2011, He, et al., 2005, Holmes, et al., 2004b, Katuri, et al., 2010, Kim, et al., 1999b, Kim, et
al., 1999d, Liu, et al., 2005, Liu, et al., 2010c, Milliken and May, 2007, Min and Logan, 2004,
Park and Zeikus, 2000, Park, et al., 2001, Torres, et al., 2009, Zhao, et al., 2010b, Zuo, et al.,
2006). Illustration based on Ref. (Schröder, 2011).
The betterment of performance of BESs based on the current density is (among other factors)
due to: i. the fabrication of porous three dimensional materials that allow bacteria to take
advantage of higher electrode surface areas to release electrons (Katuri, et al., 2011,
Šefčovičová, et al., 2011, Xie, et al., 2011, Yu, et al., 2011) (see Chapter 5 and 6); ii. the
comprehension of how electrochemically active bacteria associate with some electrode materials
through improved anode enrichment processes (Kim, et al., 2004, Liu, et al., 2008, Rabaey, et
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al., 2004); and iii. through the study of the process of biofilm formation influenced by
environmental factors (see Chapter 7 and 8).
1.6.1 Performance based on the improvement of electrode materials
Current density production in BESs has been always one of the most attractive objectives to be
achieved with these type of systems (Schröder, 2011) and as one can see from Fig. 1-7, the race
for improving the performance and finally making BESs an -on field- applied technology will
still continue (Keller, et al., 2010). To achieve this, contributions of the design of new materials
will be invaluable since these materials will have the challenge to enhance the microbe-electrode
interaction either by increasing the surface of contact between electroactive biofilms and
electrode materials or by allowing new electrode materials to collect more electrons effectively
from the internal metabolism of bacteria.
To date many strategies have been used in order to enhance the performance of BESs. These
strategies could be summarized as below:
i. improvement in the architecture design of BESs (Cheng, et al., 2006);
ii. increment of the buffer capacity in cathodic and anodic chambers (Fan, et al., 2008);
iii. use of respiratory inhibitors (Chang, et al., 2005);
iv. improved enrichment and acclimatization procedures of electroactive microbial biofilms
(Liu, et al., 2008);
v. construction of conductive artificial biofilms by the immobilization of electroactive bacteria
(Yu, et al., 2011); and just recently
vi. use of carbon based three dimensional electrode materials (Katuri, et al., 2011, Logan, et al.,
2007, Šefčovičová, et al., 2011, Xie, et al., 2011, Zhao, et al., 2010b).
In fact, commercially available carbon based materials are considered to be the most widely
used materials for BESs anodes due to their biocompatibility, chemical stability, high
conductivity, and relatively low cost (Wei, et al., 2011). All of these advantages have been
exploited in some recent reports that have succeeded in modifying these materials to enhance
the production of anodic current density (see below some examples).
For instance, Zhao and co-workers (Zhao, et al., 2010b) used a conductive polyaniline nanowire
network with three-dimensional nanosized porous structures as BESs anodes. They reported
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substantial improvements (10 to 100 fold) in current and power densities in comparison to
conventional two-dimensional materials (Schröder, 2011). More recently Katuri and co-workers
(Katuri, et al., 2011) fabricated three-dimensional microchannelled nanocomposite electrodes.
These materials allowed the growth of Geobacter sulfurreducens biofilms over the three-
dimensional surface, providing acetate oxidation current densities of up to 25 A m−2. Xie and
co-workers (Xie, et al., 2011) reported a carbon nanotube sponge composite that provided a
three-dimensional scaffold that was favorable for microbial colonization. This nanotube sponge
allowed the increment of 2.5 times the previously reported maximum areal power density and 12
times the previously reported maximum volumetric power density.
Independently from the previous examples on carbon based three dimensional electrode
materials, Chapter 5 and 6 present two studies in this regard showing conditions that allowed the
production of the highest current density values reported so far by bio-electrochemically active
biofilms.
1.6.2 Performance based on the study of environmental factors affecting biofilm
formation
In the field of BESs, it has been assumed that the treatment of wastewater could be one of the
most appealing applications (Logan, et al., 2006). In fact, in order to make BESs a successful
technology in wastewater treatment, researchers have to pay special attention to the
environmental and external factors that influence the biofilm, considered to be “powerhouse” of
BESs (Franks, et al., 2010). In the literature one can find different approaches that have been
utilized in order to decipher the factors influencing the formation of anodic biofilms in BESs.
For instance, Patil and co-workers (Patil, et al., 2010) investigated the temperature dependence
and temperature limits of wastewater derived anodic microbial biofilms. They demonstrated that
these biofilms are active in a temperature range between 5 and 45°C. Additionally, they also
demonstrated that elevated temperatures during initial biofilm growth not only accelerated the
biofilm formation process but they also influenced the bioelectrocatalytic performance of these
biofilms when measured at identical operation temperatures. For example, the time required for
biofilm formation decreased from above 40 days at 15°C to 3.5 days at 35°C. On the other side,
Zhang and co-workers (Zhang, et al., 2011) investigated the effects of external resistance on
biofilm formation and electricity generation of microbial fuel cells. The morphology and
structure of the biofilms developed at 10, 50, 250 and 1000 Ω was characterized. They
demonstrated that the biofilm structure played a crucial role in the maximum power density and
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sustainable current generation of BESs. Their results showed that, maximum power density of
their BESs increased when the external resistance decreased. They have attributed their results
to the existence of void spaces beneficial for proton and buffer transport within the anode
biofilm, which maintains a suitable microenvironment for electrochemically active
microorganisms. Furthermore, Biffinger and co-workers (Biffinger, et al., 2009) used a high-
throughput voltage based screening assay to correlate current output from a BESs containing
Shewanella oneidensis MR-1 to biofilm coverage over 250 h (among other experimental
conditions). BESs operated by Biffinger and co-workers permitted data collection from nine
simultaneous S. oneidensis MR-1 BESs experiments in which each experiment was able to
demonstrate organic carbon source utilization and oxygen dependent biofilm formation on a
carbon electrode. Finally, Ieropoulos and co-workers (Ieropoulos, et al., 2010) have
hypothesized that the processing of large volumes of wastewater in BESs would require
anodophilic bacteria operating at high flow-rates. Therefore, they examined the effect of flow-
rate on different microbial consortia during anodic biofilm development using inocula designed
to enrich either aerobes/ facultative species anaerobes. By using scanning electron microscopy
they showed some variation in biofilm formation where clumpy growth was associated with
lower power. In a different category, experiments using genetic manipulations should be
mentioned. For example, the use of knocked mutants of bacteria in order to delete from their
genome the production of outer membrane surface structures needed to adhere to solid surfaces
and generate ticker and robust electroactive biofilms (Bouhenni, et al., 2010, Rollefson, et al.,
2009). Regardless of the previous examples on factors influencing the formation of anodic
biofilms in BESs, Chapter 7 and 8 present two more detailed examples on this aspect.
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1.7 Aim of this Dissertation
Because of the issues raised in the previous sections in this chapter, the aim of my dissertation
was to investigate different aspects of microbial-electrode interactions in BESs. The different
objectives of this Ph.D. Thesis are divided into the following chapters:
Part I Electron transfer mechanisms of pure culture biofilms of Shewanella spp.
Chapter 2 Cyclic voltammetric analysis of the electron transfer of Shewanella
oneidensis MR-1 and nanofilament and cytochrome knock-out mutants.
Chapter 3 Study of Shewanella putrefaciens biofilms grown at different applied
potentials using cyclic voltammetry and confocal laser scanning microscopy.
Chapter 4 Spectroelectrochemical analysis of intact microbial biofilms of Shewanella
putrefaciens for sustainable energy production.
Part II Porous 3D carbon as anode materials for performance of electrochemically active mixed
culture biofilms.
Chapter 5 Electrospun and solution blown three-dimensional carbon fiber nonwovens
for application as electrodes in microbial fuel cells.
Chapter 6 Electrospun carbon fiber mat with layered architecture for anode in microbial
fuel cells.
Part III The influence of external factors on electrochemically active mixed culture biofilms.
Chapter 7 Electroactive mixed culture biofilms in microbial bioelectrochemical
systems: the role of pH on biofilm formation, performance and composition.
Chapter 8 Electroactive mixed culture biofilms in microbial bioelectrochemical
systems: the role of the inoculum and substrate on biofilm formation, performance and
composition.
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1.8 Structure of the Thesis and personal contribution
This Ph.D. Thesis tackled various aspects within bioelectrochemical systems. For this reason,
several available experimental techniques were utilized. From electrochemical voltammetric
techniques, via confocal laser scanning microscopy, to surface-enhanced resonance Raman
scattering. The results of this Thesis are divided into three main parts regarding the respective
area of study:
Part I Electron transfer mechanisms of pure culture biofilms of Shewanella spp.
Part II Porous 3D carbon as anode materials for performance of electrochemically active mixed
culture biofilms.
Part III The influence of external factors on electrochemically active mixed culture biofilms.
From Fig. 1-8 one can see that the different parts of this Thesis were focused mainly on the
investigation of several processes occurring at the interface between the electrode material and
bioelectroactive biofilms. In the following lines, the chapters contained in this thesis are listed.
Furthermore an appreciation of my personal contribution to each is provided.
Figure 1-8 Schematic illustration of the research areas within the three chapter I, II and III.
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Part I. Electron transfer mechanisms of pure culture
biofilms of Shewanella spp.
CHAPTER 2
The idea of this project came as a continuation of previous experiments performed on
Shewanella at the University of Greifswald. The reason to use Shewanella oneidensis MR-1 as a
biological model came from a collaboration impelled by Dr. B.R. Ringeisen’s visit to our
Institute in November 2008. At that time Prof. Dr. U. Schröder and Dr. F. Harnisch motivated
me to work on electrochemical active biofilms of wild-type and mutant strains of S. oneidensis
kindly provided by Dr. B.R. Ringeisen’s team at the US Naval Research Laboratory. The
growth of Shewanella strains was performed in close collaboration with L.A. Fitzgerald and J.C.
Biffinger. I designed, planned and performed all experimental work in Braunschweig in close
collaboration with Dr. F. Harnisch. I analyzed/ interpreted data and wrote the manuscript
together with Dr. F. Harnisch. During the whole process of this project we maintained useful
discussions with Dr. B.R. Ringeisen’s team. Prof. Dr. U. Schröder gave useful advice.
CHAPTER 3
The lack of information on the electron transfer mechanisms of electrochemical active biofilms
of Shewanella initiated this study. Most of the previous studies were generally carried out with a
single applied potential or each study used different operational parameters, which makes it
difficult to compare among studies. I took care of the whole maintenance process and growth of
S. putrefaciens. I designed, planned and performed all experimental work in Braunschweig in
close collaboration with Dr. F. Harnisch and Prof. Dr. U. Schröder. CLSM measurements were
performed at the Helmholtz Centre, Magdeburg by U. Kuhlicke and me in close collaboration
with Dr. T.R. Neu. I analyzed/ interpreted data and wrote the manuscript together with Dr. F.
Harnisch. For the CLSM data we maintained invaluable discussions with Dr. T.R. Neu. Prof.
Dr. U. Schröder gave useful advice and guidance.
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CHAPTER 4
The idea of this project came as a continuation of previous spectroelectrochemical experiments
performed in our group on Geobacter biofilms. The idea to use Shewanella putrefaciens as a
biological model came from the total lack of information on the electron transfer mechanisms
using spectroelectrochemical tools such as surface-enhanced resonance Raman scattering. This
project was conducted in several phases at the TU Braunschweig and at the TU Berlin. At the
TU Braunschweig, I took care of the whole maintenance process and growth of S. putrefaciens
biofilms in electrochemical half-cells. In close collaboration with Dr. D. Millo, I designed,
planned and performed experimental work at the TU Berlin in the group of Prof. Dr. P.
Hildebrandt. Under the supervision of Dr. Millo (who performed the SERRS experiments and
analyzed the spectra with the assistance of Khoa H. Ly), and Dr. Harnisch, I analyzed and
interpreted data. During the whole process of this project, Prof. Dr. U. Schröder has given useful
advice and guidance.
Part II. Porous 3D carbon as anode materials for
performance of electrochemically active mixed culture
biofilms
CHAPTER 5
The motivation of the study can be assigned to the continuous efforts in the field of BESs to
improve the overall performance of the systems; especially in terms of electrode materials, as
the bioelectrocatalytic activity plays a key role. The motivation for this project came from S.
Chen who was finishing at the time his Ph.D. at the Philipps-Universität in Marburg in the group
of Prof. Greiner. This project was conducted in several phases at the Universty of Marburg and
the TU Braunschweig. At the TU Braunschweig we tested a series of 3D porous carbon fiber
based materialss, produced by gas-assisted electrospinning at the Philipps-Universität and a
series of electrospun and solution-blown carbon fibers fabricated by the group of Prof. Yarin at
the University Illinois at Chicago on the suitability to serve as electrode materials for BESs. At
all times I was deeply involved in the growth and maintenance of waste-water derived
electroactive biofilms and the test of the mentioned electrode materials as well as data analysis
and interpretation.
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CHAPTER 6
Encouraged by our previous work presented in detail in Chapter 5, this study was driven by the
continuous efforts in the field of BESs to develop high performance three-dimensional electrode
materials for electroactive biofilms. This project was conducted at the Philipps-Universität
Marburg in the group of Prof. Greiner (material development) and at the TU Braunschweig in
the group of Prof. Schröder (electrode characterization). We tested a series of electrospun
carbon fiber mats with layered architecture and investigated these materials on their suitability
for growth and performance of electroactive waste water derived anodic biofilms. At all times I
was deeply involved in the growth and maintenance of the waste-water derived electroactive
biofilms and the test of the mentioned electrode materials as well as data analysis and
interpretation.
Part III. The influence of external factors on
electrochemically active mixed culture biofilms
CHAPTER 7
The investigation of environmental parameters that affect the formation and performance of
electroactive biofilms stimulated this study since the majority of studies are restricted to a
neutral pH. Specifically how the pH value influences the biofilm growth (lag-time), steady state
anodic bioelectrocatalytic activity and microbial composition. I was involved in the replication
of fed-batch experiments at pH 6, 7 and 9 and also in the operation of continuous flow
experiments for pH-regime and buffer capacity studies.
CHAPTER 8
Most of the experiments designed to study electroactive biofilms in BESs are generally carried
out with one substrate or one microbial inoculum varying different operational parameters.
Therefore in order to exclude the influence of operational variables and to investigate only the
effect of an individual microbial inoculum source and an individual substrate, the experiments
here presented were conducted with half-cell set-ups under potentiostatic control with multiple
inocula and substrates. I was deeply involved in the recollection of inocula samples, preparation
of materials needed for half-cell experiments, later in the growth and maintenance of
electroactive biofilms and as well as in data collection, analysis and interpretation.
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1.9 Comprehensive summary
Shewanella is frequently used as a model microorganism for microbial bioelectrochemical
systems (BESs) such as microbial fuel cells (MFCs) or microbial electrolysis cells (MECs). In
chapter 2, we used cyclic voltammetry (CV) to investigate extracellular electron transfer
mechanisms from Shewanella oneidensis MR-1 (WT) and five deletion mutants: membrane
bound cytochrome (ΔmtrC/ΔomcA), transmembrane pili (ΔpilM-Q, ΔmshH-Q, and ΔpilM-
Q/ΔmshH-Q) and flagella (Δflg). We demonstrate that the formal potentials of mediated and
direct electron transfer sites of the derived biofilms can be gained from CVs of the respective
biofilms recorded at bioelectrocatlytic (i.e. turnover) and lactate depleted (i.e. nonturnover)
conditions. As the biofilms possess only a limited bioelectrocatalytic activity, an advanced data
processing procedure, using the open-source software SOAS, was applied. The obtained results
indicate that S. oneidensis mutants used in this study are able to bypass hindered direct electron
transfer by alternative redox proteins as well as self-mediated pathways.
Figure: How does Shewanella transfer its electrons to solid acceptors? Using cyclic
voltammetry direct and mediated electron transfer of S. oneidensis MR-1 and related mutants
were investigated. The subsequent analysis, based on an elaborate open source software data -
processing, indicates a correlation of the maximum current density (x-axes of the graph) of the
respective mutant and its mediated electron-transfer ability (respective CV- peak height on the
y-axes).
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It has been shown for anodic biofilms in MFCs that the microorganisms therein can be
influenced by the applied electrode potential. In chapter 3, we studied the influence of the
applied electrode potential on the anodic current production of Shewanella putrefaciens NCTC
10695. Furthermore, we used cyclic voltammetry (CV) and confocal laser scanning microscopy
(CLSM) to investigate the microbial electron transfer and biofilm formation. It is shown that the
chronoamperometric current density is increasing with increasing electrode potential from 3 µA
cm-2 at -0.1 V up to ~12 µA cm-2 at +0.4 V (vs. Ag/ AgCl), which is accompanied by an
increasing amount of biomass deposited on the electrode. By means of cyclic voltammetry we
demonstrate that direct electron transfer (DET) is dominating and the planktonic cells play only
a minor role.
Figure: Is the current generation, jmax, a function of the applied electrode potential?
Representative chronoamperometric fed-batch cycles of S. putrefaciens at graphite electrodes;
applied potentials: -0.1, 0, +0.1, +0.2, +0.3 and +0.4 V vs. Ag/AgCl; CV measurements during
turn-over (A) and non turn-over (B) conditions respectively.
0 1 2 3 4 5 6 7 8
-2
0
2
4
6
8
10
12
14
16
+0.4
+0.3
+0.2
+0.1
0.0
-0.1
A
A
A
jmax/ A cm-2
time/ days
B
B
B
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Crucial for the functioning of bioelectrochemical systems (such as MFCs and MECs) is the
complex protein architecture responsible for the electron transfer (ET) across the
bacteria/electrode interface. The ET pathway involves several multiheme redox proteins denoted
as outer membrane cytochromes (OMCs). In chapter 4 these OMCs were studied by a
combination of surface-enhanced resonance Raman scattering (SERRS) spectroscopy and
electrochemistry. The experiments presented in chapter 4 were performed on microbial biofilms
of S. putrefaciens. These have shown that OMCs do not contribute significantly to the
heterogeneous ET across bacteria/electrode interface. These studies have been performed on
biofilms grown on nanostructured Ag electrodes at the poised potential of +50 mV (vs.
Ag/AgCl). Although these conditions allow the formation of a biofilm on the Ag electrode, they
may have a negative impact on the amount of OMCs expressed by the bacteria (see chapter 3).
In fact, optimal biofilm growth requires pore positive potentials. However, these conditions
cannot be met by the Ag substrate, which undergoes oxidation at potentials higher than +150
mV (vs. Ag/AgCl).
Figure: Electrochemical measurements (A) performed in combination with SERRS (B)
allowed to control and monitor the activity of the microbial biofilm. This chapter aims at
providing the first spectroelectrochemical characterization of microbial biofilms of a strain of
the Shewanellaceae family by probing (i) structural information about the OMCs, (ii) the
participation of the OMCs to the ET, and (iii) the influence of soluble redox mediators
competing with OMCs. The experiments presented here contributed to elucidate the
function/structure relationship of OMCs in living cells, providing unique insight into the ET
across the bacteria/electrode interface. The development of novel analytical strategies to
overcome this limitation is presently under evaluation in our groups.
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In chapter 5 we exploited electroactive bacteria in bioelectrochemical systems like MFCs that
promise a great potential in the context of sustainable energy supply and handling. A major
challenge in this context is to increase the performance of such systems, a necessity for a future
success of this new technology. During the past decade the average current densities of biofilm
anodes have already increased significantly from milliampere per square metre level to between
7 and 10 A m-2. In this study it is demonstrated that by using three-dimensional carbon fiber
electrodes prepared by electrospinning and solution blowing the bioelectrocatalytic anode
current density reaches values of up to 30 A m-2, which represents the so far the highest reported