PhD Thesis Alessandro Carmona 2012

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.

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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

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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
3.2.3 Bioelectrochemical set-up and experiments .....................................................................................51
3.2.4 Electrochemical data processing ......................................................................................................51
3.2.5 Confocal Laser Scanning Microscopy..............................................................................................52
3.3.2 Cyclic voltammetric analysis ...........................................................................................................54
3.3.3 Biofilm imaging using confocal laser scanning microscopy (CLSM) .............................................58
3.4 Conclusions ..........................................................................................................................................60

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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
4.2.1 Materials and methods .....................................................................................................................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
4.4 Conclusions ..........................................................................................................................................70
5.1 Introduction ..........................................................................................................................................71
5.2.1 Carbon fiber preparation ..................................................................................................................73
5.2.2 Electrode preparation .......................................................................................................................75
5.2.3 Bioelectrochemical experiments ......................................................................................................75
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

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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
6.2.1 Carbon fiber preparation ..................................................................................................................83
6.2.2 Electrode preparation .......................................................................................................................84
6.2.3 Bioelectrochemical measurements ...................................................................................................84
6.2.4 SEM imaging ...................................................................................................................................84
6.3.1 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
7.2.2 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
7.3.1 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

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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

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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

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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

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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

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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.

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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.

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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).

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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).

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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

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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-

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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

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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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